Preface This second issue of the Toxicologist is devoted exclusively to the abstracts of the platform and poster sessions of the 21st Annual Conference of the Society of Toxicology, held at the Sheraton-Boston Hotel, Boston, MA, February, 22-26, 1982. The abstracts are reproduced, without editing, as accepted by the Program Committee of the Society of Toxicology, and appear in the same numerical sequence as the papers appear in the Conference's official program. An alphabetical author index, listing the corresponding paper number, appears on pp. 194. Copies of this issue are available at $5 each (U.S. funds) from Society of Toxicology 475 Wolf Ledges Parkway Akron, OH 44311-1087

® 1982, Society of Toxicology

1 BUTYLATEDHYDROXYTOLUENE DOES NOT INHIBIT CIPRO­ ment of a 6-MC protein or amino acid adduct in FIBRATE AND DI-(2-ETHYLHEXYL)PHTHALATEINDUCED 6-MC photoallergenicity. (Supported in part by PEROXISOMEPROLIFERATION IN RAT LIVER. N.D. the Research Institute for Fragrance Materials). Lalwani, H. Hayashi, M.K. Reddy, C. Moehle, and ) J.K. Reddy, Dept. Pathol., Northwestern Univ. Med. Sch., Chicago, IL. 3 ALTERATIONOF CALCIUM-DEPENDENTGABARELEASE AND Evidence now suggests that hepatic peroxisome THEENZYME ACTIVITIES OF GLUTAMICACID DECARBOXY­ proliferators as a class are carcinogenic. It is LASE(GAD) AND GABA-y-KETOGLUTARATE TRANSAMINASE hypothesized that persistant proliferation of (GABA-T)IN RESPONSETO THEACUTE AND CHRONIC AD­ peroxisomes and increase in peroxisomal S-oxida­ MINISTRATIONOF PHENCYCLIDINE(PCP). J. C. Norris, tion initiates the neoplastic transformation of T. Nabeshima, S. P. Sivam and I. K. Ho, Dept. liver cells by increasing the intracellular pro­ Pharmacol. & Toxicol., Univ. MSMed. Ctr., Jack­ duction of DNA-damaging H202 and other reactive son, MS 39216. oxygen intermediates. In the present study we have investigated the effect of antioxidant buty­ The involvement of GABAergicsystem with acute lated hydroxytoluene (BHT) to determine whether and chronic PCP administration was investigated free radical scavanging antioxidant could inhibit by noting alterations in calcium-dependent GABA peroxisome proliferation induced by hypolipidemic release in mouse brain slices and the activity of drug ciprofibrate (2,-[4-{2,2-dichlorocyclopro­ the enzymes, GADand GABA-T,of the whole mouse pyl}-phenoxy]2-methyl propionic acid) and DEHP. brain. The chronic administration of PCP for the Ciprofibrate at 0.1% concentration in diet in­ release study was accomplished by osmotic mini­ creased peroxisomal enzyme activities in livers pumps at a rate of 1 mg/day. The GABArelease of male F344 rats. Simultaneous feeding of BHT for the chronic animal in both the striatal and at either 7000 ppm or 700 ppm in diet did not in­ cerebellar slices was decreased while the time of hibit the peroxisome proliferation and peroxisomal the change was different for the two discrete enzyme activities inducible by ciprofibrate. areas. The striatal slices returned to the con­ trol values after 4 and 6 days of continuous ad­ Similarly, BHT failed to inhibit DEHP induced per­ ministration whereas the cerebellum slices were oxisome proliferation in rat liver. Additional decreased only in the 4-day treatment group. The studies are required to ascertain whether BHT tissues of acutely treated mice responded in a inhibits the peroxisome proliferator induced manner dependent on the dosage level. The low carcinogenesis in rat liver. Supported by NIH dose (10 mg/kg) of PCP increased ca++-dependent grant GM 23750. GABArelease in striatum. The high dose (40 mg/ kg) of PCP, however, demonstrated a decrease in GABArelease in striatum. The enzyme activities of GADand GABA-Twere measured in animals which 2 A MECHANISMFOR 6-METHYLCOUMARINPHOTOALLER­ received 40 mg/kg, i.p., once daily for five days. GENICITY. M.W.Tabor, S. Kato, M. Ohno and R.R. The chronically PCP treated animals demonstrated Suskind, Dept. of Environ. Hlth, Univ. of Cin­ a significant increase in GADand GABA-Tlevels. cinnati Med. Ctr., Cincinnati, OH. Sponsor: The acutely PCP treated animals showed a decrease Dr. C. C. Smith. in the GADactivity and no change in the GABA-T activity. Thus, it seems that the GABAergicsys­ Photoallergic dermatitis and allergic hypersen­ tem is altered by the presence of PCP. (Supported sitivity have been reported in persons using by research grant DA-01310from NIDAand toxicol­ commercial products containing 6-methylcoumarin ogy training grant ES-07045 from NIEHS) (6-MC) as a fragrance material. The purpose of our study was to investigate the biochemical basis of the photoallergic reactions of 6-MC. We have examined the ultraviolet (UV) catalyzed 4 DRUGMETABOLIZING EN2YMES IN C3HlOT½ CELLS photochemical reactions of 6-MC with representa­ EXPOSEDTO POLYCHLORINATEDBIPHENYLS (PCBs). tive amino acids (lys, ser, gly, glu, cys, met, H.P. Cihla, G, Reddy, & D.R. Norback. Depart­ phe, tyr, and trp) and glutathione (GSH) under ments of Pathology, William S. Middleton Mem­ physiological conditions. These reactions were orial Veterans' Hospital and the University of used to model the photochemical reactions of Wisconsin, Madison, Wisconsin, 53705. 6-MC with proteins. The source of UV light We have shown the cytotoxic and oncogenic catalysis was either UVA of wavelengths from effects of certain technical PCBs and PCB an­ 310 nm to 400 nm or UVB of wavelengths from alogues on the C3Hl0T½ cell system. In order 280 nm to 370 nm. Samples were exposed for 1, to define possible mechanisms of pathologic 12 or 24 hrs, and then reaction products were change, we studied basal drug metabolizing en­ isolated by either silica gel TLC or sequential zyme activities of this cell line and th re­ extraction using a series of nonpolar to polar sponse to Aroclor 1260. Cells (1.5 x 10)5 were solvents followed by silica gel TLC of the ex­ plated in T-75 flasks and, 24 hours later, were tracts. UVA was a more potent catalyst than continuously exposed to either 10 ppm PCB or 1% UVB for all reactions studied. For 6-MC alone, acetone. On 6, 10 or 15 days, cells were col­ the UV reaction yielded three products at 12 lected and microsomal and cytosolic fractions and 24 hrs. The production of the least polar were prepared. The specific activities of mi­ of these products was quenched by cys and GSH. crosomal styrene oxide epoxide hydrolase (EH), For the reactions of 6-MC with amino acids and cytosolic glutathione S-epoxide transferase (GT) GSH, the UV reactions appeared to produce 6-MC and microsomal P-450-associated aldrin epoxidase photoconjugates with lys, cys, and GSH, and to a (AE) were assayed. Six day cultures de-111onstrated lesser degree with tyr, ser, trp and phe. Gly, basal activity for AE (4.3 pmole dieldrin/mg of glu and met appeared to be unreactive. The re­ protein/30 minutes). Basal levels of EH and GT sults of this study clearly suggest the involve- from preconfluent cultures (1.51 +/- 0.38 and 14.20 +/- 1.17 nrnole/mg/min, respectively) de­ reduced in the presence of the membrane­ clined approximately 50% after confluence had stabilizing agents triamcinolone and been reached (day 15). Exposure to PCB produced chlorpromazine. no change in EH activity at any of the time Supported by NIH Biomedical Grant 363183-4. points. However, GT activity increased approxi­ mately 50% in preconfluent cultures (days 6 and 10) and approximately 10% in postconfluent cul­ 7 ANAEROBICACTIVATION OF CHLORAMPHENICOLBY RAT tures. These enzymes may play a role in the LIVER MICROSOMES.P.L. Morris, T.R. Burke and metabolism of PCBs and, thus, may alter the L.R. Pohl, NHLBI, Bethesda, MD,: Sponsor: effects of PCBs on this cell culture system. J.R. Gillette. (Supported by the Veterans' Administration, NIH Chloramphenicol (CAP, RNHCOCHClz)has previous­ grant CA 22140 and ACS grant IN-35-4.) ly been shown to be activated by rat liver cyto­ chrome P-450 aerobically to an oxamyl chloride (RNHCOCOCl)that either covalently bonded to 5 THEEFFECT OF ASCORBICACID ON COPPER AND microsomal protein (RNHCOCO-protein) or reacted NITRITE-INDUCEDOXIDATIVE CHANGES IN with water to form CAP oxamic acid (RNHCOCOOH). ERYTHROCYTES:INTERSPECIES DIFFERENCES. In the present study we have found that rat E.J. Calabrese, G.S. Moore and M. McCarthy, liver cytochrome P-450 also activates CAP an­ Division of Public Health, University of aerobically by another mechanism. When a mix­ Massachusetts, Amherst, MA. ture of 3H-(benzylic position) and 14c(dichloro­ Ascorbic acid incubation with human acetamido carbons) labeled CAP (0,1 mM) were erythrocytes markedly reduced the occurrence incubated anaerobically with rat liver micro­ of sodium nitrite induced methemoglobin in a somes (2 mg/ml) from phenobarbital (PB) pre­ dose dependent fashion. In marked contrast, treated rats and a NADPHgenerating system, both ascorbic acid incubation with Dorset sheep labels were bound irreversibly to microsomal erythrocytes treated with sodium nitrite did protein to approximately the same amount (330 not have any effect on the occurrence of pmol/mg protein/30 min) and monodeschloro-CAP sodium nitrite induced methemoglobin (DCl-CAP, RNHCOCH2Cl, 1228 pmol/mg protein/30 formation. min) was formed, Under these reaction condi­ Using an identical in vitro experimental tions, no nitro-reduction products of CAP were protocol as with the nitrite experiments, detected. The formation of covalently bonded ascorbic acid was found to markedly enhance product and DCl-CAP were decreased by over 70% the occurrence of copper induced oxidative when the incubations were conducted in the pre­ stress (i.e. increasing methemoglobin levels) sence of SKF 525A (0.5 mM), CO (100%) or in the in both sheep and humans. absence of an NADPHgenerating system. Moreover, less than 10% as much covalently bound product and DCl-CAP was detected when microsomes from control rats or rats pretreated with s-naphtho­ 6 BIOCHEMICAL TOXICITY IN MICE OF SUB­ flavone were used, These results suggest that a STANCES PRODUCED BY ctostridium difficile. PB inducible form of cytochrome P-450 reductively M. Ehrich, Virginia-Maryland Regional dechlorinates CAP into a radical intermediate College of Veterinary Medicine, (RNHCOSHCl) that either reacts covalently with Blacksburg, VA. microsomal protein or abstracts a hydrogen atom Clostridium difficile toxin is re­ to form RNHCOCH2Cl. cognized as the etiological agent of im­ portance in antibiotic-induced diarrhea and pseudomembranous colitis. Adminis­ tration of toxin to mice causes grossly 8 HYPERMETHYLATIONIN THE MSO-EPILEPTOGENICBRAIN. observable liver damage, which can be REVERSALBY DILANTIN, PHENOBARBITALOR ADENOSINE quantitized biochemically by elevations PLUS HOMOCYSTEINE. R.A. Schatzl, T.E. Wilens2, in certain serum enzymes. For example, S.B. Tatter2 and O.Z. Sellinger 2 . lDivision Tox­ glutamic pyruvic transaminase (SGPT) and icology, Northeastern Univ., Boston, MA 02115 isocitrate dehydrogenase (ICD) levels and 2MHRI, Neurochem. Lab., University of Michi­ were raised to 291% + 20 and 297% + 20 gan, Ann Arbor, MI 48109. Administration of the of control levels, respectively (mean+ convulsant L-methionine-d,1-sulfoximine (MSO) re­ SEM, N = 5, p<0.01), using a semipurified sults in increases in the methylation of hista­ preparation containing both of the high mine, phospholipids and the carboxyl moieties of molecular weight toxins (500,000-600,000 some proteins. Co-administration of equimolar daltons) produced by this microorganism. doses of adenosine (Ado)+ homocysteine (Hey) Clearance of sulfobromophthalein (BSP) elevates brain levels of S-adenosyl-1-homocysteine was delayed following iv administration (AdoHcy), a potent endogenous inhibitor of trans­ of toxin, with only 51% of the dye cleared methylation reactions. This increase in AdoHcy in 30 minutes versus 93% for control mice. levels inhibits, to varying degrees, the afore­ The activity of liver microsomal enzymes, mentioned methylation reactions and prevents, at indicated by 0-demethylation of p-nitro­ least partially, the MSO-induced increases in anisole, was also reduced (to 69% + 6 of methylation as well as MSO seizures. More recent­ control). Toxic C. difficile cult~re ly we have found that chronic administration of supernatant inactivated by incubation the clinical anticonvulsants Dilantin (DPH) of with 0.4% formaldehyde had no effect on phenobarbital (PB) also increases brain AdoHcy any of these biochemical parameters. levels although to a lesser extent than Ado+Hcy Although a mechanism for the toxic action treatment. PB treatment decreased protein car­ of C. difficile products has not been boxymethylation (PCM) while DPH was without ef­ defined, we noted that toxic effects were fect. Both anticonvulsants, however, prevented

2 the MSO-induced increase in PCM. These agents difference in APAP-induced nephrotoxicity between also increased the latency to, and decreased the these two strains of rats may be differences in the incidence of, MSO seizures. In view of the pro­ generation of p-AP. Therefore, studies were carried posed integral role of PCM in membrane associated out to investigate the difference between strains in p­ phenomena (i.e. nerve signal transduction) it is AP generation from APAP both in vivo and in vitro. In likely that the MSO-induced increase in PCM is a vitro there was no significant difference between reflection of increased neuronal activity. Wheth­ strains in APAP deacetylation by kidney and liver er or not this increased PCM is actually causative homogenates. In vivo APAP and its metabolites were in MSO seizures remains to be determined, however, excreted at equal rates by both strains at doses of the findings that agents that inhibit methylation APAP (250 and 500 mg/kg) that did not produce also prevent MSO-induced seizures as well as in­ nephro-1:oxicity as measured by blood urea nitrogen creases in methylation, lend support, albeit in­ (BUN). However, at a dose of 900 mg/kg APAP, BUN direct, to this possibility. Supported by NINCDS, was increased (7x) and APAP excretion decreased in F grant No. 06294 to OZS. Sponsored by D. Brown. rats. In addition, F rats excreted more p-AP (free and conjugated) than SD rats when p-AP was expressed as percent of total APAP plus metabolites excreted (F, 9 PROBENECIDALTERS THE RENAL TRANSPORT OF CITRININ. 5,21 .±. 0.82% vs S, 2.50 _:1:_ 0.37%). These studies W. O. Berndt and A. W. Hayes, Dept. Pharmacol. & demonstrate that there is no difference between the Toxicol., Univ. MSMed. Ctr., Jackson, MS39216 two strains in the excretion of APAP to the kidney and Rohm& Haas Co., Spring House, PA 19477. after i.p. injection. However, the excretion of p-AP in relation to other metabolites in the F rat after a Citrinin is a nephrotoxic fungal toxin that nephrotoxic dose of APAP is greater than that in the may have an important role in various animal dis­ SD .. (Supported by USPHS Grant ES 00560.) eases and perhaps in some human nephropathies. Clearance experiments in anesthetized, Spraque­ 11 STUDIES ON THE INHIBITION BY L-ASCORBICACID Dawley rats indicate that citrinin (an organic (LAA) OF THE COVALENTBINDING OF ACETAMINOPHEN anion) is secreted. The citrinin:inulin clear­ METABOLITESTO HEPATIC MICROSOMESIN VITRO. ance ratio is approximately 3. Probenecid is Lake, B.G., Harris, R.A., Phillips, J.C. and known to block the renal secretion of many anion­ Gangolli, S.D., The British Industrial Biological ic chemicals. Tune and others used probenecid to Research Association, Carshalton, Surrey, SMS block cephaloridine transport and nephrotoxicity. 4DS. U.K. In the present experiments probenecid was tested for its effects on citrinin-induced mortality, Acetaminophen, a widely used nonnarcotic citrinin-induced changes in renal transport, and analgesic drug, is known to be hepatotoxic in the renal accumulation of citrinin. Rats were large doses. Previous studies have shown that pretreated with probenecid (30-120 mg/kg) 30 min acetaminophen is metabolised by cytochrome P-450 before the administration of 55 mg/kg of citri­ dependent mixed function oxidase (MFO) enzymes nin. Mortality measured at 72 hr was reduced to reactive electrophilic intermediates which significantly. Renal slice PAHand TEAtransport covalently bind to tissue macromolecules result­ was significantly enhanced at 72 hr by probenecid ing in cell necrosis and death. We have plus citrinin than with citrinin alone. Finally, investigated the effect of L-ascorbic acid (LAA) a large dose of probenecid permitted kidney ci­ on this process. The covalent binding of [14c] trinin levels to reach only 50 µg/g compared to acetaminophen metabolites to male and female almost 200 µg/g in animals given citrinin alone. mouse and male hamster hepatic microsomes was Finally, the urinary excretion of citrinin and inhibited by reduced glutathione, cysteamine, its metabolites was reduced dramatically during L-cysteine and LAA. Although the sulfhydryl the first 8 hr after citrinin administration to compounds were more effective inhibitors than probenecid-pretreated rats compared to animals LAA, the combination of LAA with any of the thiol receiving citrinin only. These data taken to­ agents resulted in additive inhibition of gether demonstrate that probenecid can block the covalent binding. Investigations into the renal transport of citrinin and that reduction of mechanism of' inhibition of covalent binding of this transport is a significant factor in reduc­ [14c]acetaminophen metabolites indicated that LAA ing nephrotoxicity. Hence to produce toxicity, probably acts by scavenging the reactive citrinin or a metabolite utilizes anion transport intermediates generated by MFO enzymes rather to enter renal tissue. (Supported by ES 01643.) than by the inhibition of their formation. These results suggest that LAA, at concentrations found in rodent and human liver, may supplement 10 ACETAMINOPHEN NEPHROTOXICITY AND METAB­ the endogenous protective mechanisms (e.g. OLISM IN FISHER 344 AND SPRAGUE-DAWLEY reduced glutathione) which operate in vivo to RATS. J.F. Newton, Jr., J.K. Howe-Baughman, D.E. prevent the covalent binding of reactive Erickson, W.E. Braselton, Jr., M.W. Gemborys, G.H. acetaminophen metabolites and hence hepatic Mudge and J.B. Hook. Department of Pharmacology necrosis. (Sponsored by Beecham Products and Toxicology, Center for Environmental Toxicology, Research Department, U.K.). Michigan State University, East Lansing, MI 48824 and Department of Pharmacology and Toxicology, Dartmouth College, Hanover, NH 037 55. 12ACETAMINOPHEN-INDUCEDHEPATIC GLYCOGENDEPLETION, Jack A, Hinson, Joann B. Mays and Alex M. Cameron, Acetaminophen (APAP) produces renal cortical NCTR, Jefferson, Arkansas 72079 necrosis in the Fisher 344 (F) rat but not in the Sprague Dawley (SD) rat. Recently, we have reported that the Acetaminophen(A)-induced hepatotoxicity is F rat can metabolize APAP to the nephrotoxic p­ mediated by a reactive metabolite. In microsomal aminophenol (p-AP). One possible explanation for the incubations the metabolite can react with gluta-

3 thione (GSH) to form an A-GSH conjugate or can be function may be due to its influence on the reduced to A by NADPHor ascorbate. Detoxifica­ pituitary-testes axis rather than on the testes tion by GSH occurs .in..m.o, however, detoxifica­ alone. tion by reduction has not been demonstrated.

Hepatic glycogen (GLY) depletion occurs following 14· DISPOSITIONAL 1 DIFFERENCES OF CADMIUMAND MERCURY hepatotoxic doses of A. Since GLY depletion may IN RAT HEPATOCYTEPRIMARY CULTURES. R.J. Gerson increase glycolysis and energy production result­ and Z.A. Shaikh, Division of Toxicology ing in increased reduction of the reactive metabo- Univ. of Rochester, Rochester, N.Y. 14642 .. lite, repair of damaged macromolecules or glucu­ The in vivo administration of inorganic Cd and ronidation of A, its role as a detoxification Hg has been shown to result in markedly different mechanism was investigated in mice. Following a metal concentrations in rat liver. Primary cult­ hepatotoxic dose of A, PAS stained liver sections ures of rat hepatocytes were utilized to gain in­ showed GLY depletion occurring initially in the sight into the dispositional differences between centrilobular areas. A time course following ad­ these chemically similar metals. Hepatocyte mono­ ministration of A (500 mg/ kg) showed GLY was de­ layer cultures were exposed to varying concentrat­ pleted by 25% at 0.5 hr, 60% at 1 hr and 80% from 1.5 to 4 hrs, whereas GSH was depleted by 70% at ions of Cd or Hg (3,10,30,lOOpM) in serum-contain­ 0.5 hr and 80% at 1 to 4 hrs. GLY depletion cor­ ing medium for 30 min at 37°c and 5% CO2, The responded to hyperglycemia (267 mg% at 2.5 hr and cells were then washed and incubated in fresh control values at 4 hrs.) An A dose response medium for the remainder of the experiment. In curve (50 to 500 mg/kg at 2.5 hr) demonstrated GSH cells exposed to 3µM Cd there was an initial eff­ lux of Cd from the hepatocytes when placed in and GLY were depleted by similar amounts. A non­ fresh medium, followed by a gradual reuptake of hepatotoxic dose of A (100 mg/kg) decreased GSH and GLY by 25% and significantly increased blood metal, concommitant with its binding to metallo­ glucose. Starvation overnight decreased GSH and thionein (MT). This reuptake could be blocked GLY by 40 and 75% respectively and dramatically with lOµg/ml cycloheximide. Efflux of Hg from Hg­ increased susceptibility to A toxicity as indica­ exposed hepatocytes was also observed upon add­ ition of fresh medium but there was no reuptake. ted by increase in SGPT values. Availability of glucose water (5%) overnight resulted in GSH lev­ Measurement of lactate dehydrogenase (LDH) re­ els not significantly different from starved ani­ leased into the medium demonstrated that Hg was mals and GLY levels similar to fed animals. Starv­ cytotoxic at lOOµM whereas Cd showed toxicity at ed animals and animals given glucose water were lOpM. Elevations in LDH correlated with morpho­ equally susceptible to A hepatotoxicity. These logical signs of toxicity in the cultures. Hepat­ data suggest that A induces glycogenolysis and ocytes exposed to 3µM Cd contained eight times hyperglycemia, but apparently this does not pro­ more Cd at 24 hrs than hepatocytes exposed to the tect the animal against the hepatotoxicity of A. same concentration of Hg. At a dose of 3µM metal, 70% of the Cd and less than 1% of the Hg contain­ ed in the hepatocytes was bound to MT. In hepat­ ocytes exposed to 30µM Hg a maximum of 10% of the 13 PREVENTIONOF TESTICULARDAMAGE BY DIETARY Hg was bound to MT. Hepatocytes exposed to 3µM Cd ALUMINUMIN RATS WITH DIETARY ZN ANDCU had four times more metal bound to MT than hepat­ DEFICIENCY- Jianye Liu, M.D. and Klaus L. ocytes exposed to 30µM Hg (336 vs. 84 pmoles/mg Stemmer, M.D., University of Cincinnati Medical protein in homogenate). These studies indicate Center, Kettering Laboratory, Cincinnati, Ohio that isolated hepatocytes differentiate between 45267 these two metals and preferentially accumulate The role of Al in body functions is still Cd. (Supported by NIH Grants ES01247 and ES01448) unknown. Previous studies demonstrated that the interactions between Al and certain essential metals may be responsible for the expression of 15 FLAVORAVERSIONS INDUCED BY CADMIUM: IMPORTANCE Al neurotoxicity in rats. To elucidate the OF ROUTEOF EXPOSURE.R. C. MacPhail, Neurotoxi­ effects of Al on the testicular damage caused by cology Division, U.S. Environmental Protection Zn or Cu deficiency, an experiment was performed Agency, Research Triangle Park, NC 27711. on rats fed purified diet containing low Zn, low {Sponsor: L. W. Reiter) Cu, or sufficient levels of Zn and Cu, with or These experiments further evaluated the utility without addition of Al. The control group of of conditioned flavor aversions for rapidly de­ animals were fed Purina Lab Chow. Observations tecting the effects of toxicants. Individual were noted after 120 days. rats were adapted to a daily restricted (30 min) Severe testicular atrophy was seen in rats water availability schedule. Whenintakes had fed either low Zn or low Cu diet, but the damage stabilized, a 0.1% (w/v) sodium sacch~rin solu­ was more pronounced in the low Zn group. The tion was substituted for water and was followed testicular lesions included seminiferous tubular approximately 20 min after fluid availability by cell degeneration and necrosis, as well as reduc­ administration of cadmium. Cadmium(CdCl ) was tion of spermatogenesis. All spermatogenic cell dissolved in normal saline solution and w~s admin­ types were affected including Sertoli cells, but istered intraperitoneally (i.p.), subcutaneously Leydig cells were spared. (s.c.) or by gastric intubation {p.o.). The WhenAl was added to the diet, testicular dosage ranges were: 0.125-1.0 mg/kg (i.p.); 0.33- destruction was reduced. This indicated that the 2.62 (s.c.); and 13.0-104.0 mg/kg {p.o.). Dosages presence of Al in the diet protected the testes were expressed as base, and represented 3.5, 7.0, against the damage caused by Zn or Cu deficiency. 14.0 and 28.0% of the LD50sreported by Kotsonis Pituitary glands were also examined. Hypertrophy and Klassen (TAP41 :667, 1977). Each dose-effect was more pronounced in Al treated rats with Zn or determination included groups of 5-6 rats that Cu deficient diet compared with those groups of received Cd, the saline vehicle or no treatment. rats fed Zn or Cu deficient diet alone. This Three days later each rat was given simultaneous suggests that the protection of Al on gonadal access to saccharin and water, and preferences

4 were determined. This procedure was repeated ization after wet digestion of the samples. A until Cd was administered, and flavor preference correlation was seen between the range of cad­ was assessed, on three occasions. Flavor mium levels and the incidence of hypertension. aversions induced by Cd (i .p.) were apparent only Also smokers showed higher levels of cadmium at the largest dosage and after repeated than non-smokers. Concentrations of cadmium in saccharin-cadmium pairings. Flavor aversions amniotic fluid ranged from 0.044 mcg/dl to induced by Cd (s.c.) were proportional to the 0.981 mcg/dl; with a mean of 0.243 mcg/dl and dosage and became more prominent with repeated a standard deviation of 0.198. Diabetes, an pairings. Flavor aversions induced by Cd (p.o.) additional parameter considered in the study, were maximal at the smallest dosage and did not show any correlation to the distribut­ after one pairing. These results extend recent ion of cadmium levels. findings of flavor aversions produced by several toxicants, including heavy metals, and suggest further that route of administration is an 18 THE EFFECTS OF METHYLMERCURYBINDING TO important determinant of the magnitude of toxi­ MICROTUBULES.D, G, Vogel, R, L, Margolis, and cant-induced flavor aversions. N, K. Mottet, Dept. of Pathology, Univ. of Wash, and Fred Hutchinson Cancer Research Center, 16 ALTERATIONOF MEMBRANEFLUIDITY AS A POSSIBLE Seattle, WA, Sponsor: M.R. Juchau MECHANISMOF CADMIUMTOXICITY, M.A. Amoruso, Methylmercury (MeHg) is known to affect the G, Witz,.and B,D, Goldstein, Dept, of Environ­ integrity of microtubules (MTs) which perform a mental and Community Medicine, CMDNJ-Rutgers multitude of cellular functions, The interaction Medical School/Rutgers Univ. Joint Graduate of MeHg with MTs is being investigated as one of Program in Toxicology, Piscataway, NJ many possible mechanisms for the observed human Preliminary studies from this laboratory and animal teratogenic effects of MeHg, The showed that exposure of rat pulmonary alveolar direct interaction of MeHg and MTs was examined macrophages and human granulocytes to cadmium in in vitro with tubulin purified from bovine brain vitro botg inhibits (l0- 2-io-3M)and enhances by three cycles of assembly-disassembly and a (10-S-10- M) superoxide anion radical (02 •) pro­ crude tubulin purified from rat brain, Polymeri­ duction, findings which may relate to the toxic zation was done at 30°C and monitored at 350 nm. effects of cadmium in vivo, o;• is formed by a After 30 min polymerization the bovine brain MTs plasma membrane bound oxidase; hence a possible were 100% inhibited at 30 uM MeHg, 50% inhibited mode of action of cadmium could involve alter­ at 14.5 uM, and less than 20% inhibited at con­ ations of membrane fluidity by this metal ion, centrations below 6 uM, After 30 min polymeri­ This possibility was explored in human erythro­ zation the crude rat brain MTs were 100% inhibit­ cyte ghosts using fluorescence polarization ed at 300 uM, 50% inhibited at 132 uM, and less techniques, Membranes labelled with the fluor­ than 30% inhibited at concentrations below 6 uM, escent lipid probe all-trans l,6-diphenyl-1,3,5- Total protein concentrations (umoles per ml) for hexatriene (DPH) had increased DPH polarization the bovine and crude rat tubulin were twice the 2 values when treated with 10- 5-10- M cadmium for MeHg concentration needed to produce 50% inhibi­ 1 hr at 25°c. The DPH polarization at 25° in­ tion of polymerization and approximately equal creased from 0.338+.002 at zero Cd++ to 0.361+ to the MeHg concentration needed to produce 100% .005 at 10- 2M ca++~ Exposure of ghosts to catt inhibition of polymerization. This suggests the also increased the native protein fluorescence binding of MeHg to one site per tubulin monomer polarization. At 25°c, control ghosts had a pro­ is sufficient to inhibit polymerization, Since tein polarization value of 0.154!.001 while MeHg binds to sulfhydryl groups of proteins, the ghosts treated with 10- 3 and 10- M ca++ had binding of MeHg to free sulfhydryl groups of values of 0.176+.001 and 0.182±,001 respectively. tubulin was examined using Ellman's reagent and Erythrocyte gho;ts treated with 10- 2 or 10-~ monitored at 412 nm. There were 7 free sulfhydryl Cd* had an increased native membrane emission groups per tubulin monomer and binding MeHg to compared to control ghosts. The increases were only one produced 100% inhibition of MT polymer­ observed at temperatures ranging from 15-40°c. ization, The results are consistant with the pro­ Temperature profiles with respect to polarization posed mechanism of MeHg interaction with MT to showed higher pr~tein and DPH polarization values produce cellular disfunctions. (NIEHS ES 00677, 2 for 10~ and 10-~ Cd++-treated ghosts compared ES 07032, and USPHS GM 28189), to control ghosts over the 15-40°c temperature range. These data suggest that interaction of cadmium with cellular membranes may alter mem­ 19 EFFECTOF SELENIUMON DISTRIBUTION, DEMETHYLATION brane lipid and protein fluidity which may lead ANDEXCRETION OF METHYLMERCURYBYTHE GUINEA PIG. to abnormal cellular function. Elzbieta Komsta-Szumska,K.R. Reuhl and D.R. Miller, Biological Sciences, National Research 17 CADMIUMCONCENTRATIONS IN AMNIOTICFLUIDS. Council of Canada, Ottawa, Ont. Canada KlA OR6. F.W. Fochtman, C.F. Kritko, and C.L. Winek, Sponsor: C.T. Miller. Dept. of Pharmacology-Toxicology, Duquesne The effect of sodium selenite on methyl­ mercury poisoning was studied in the guinea pig. University School of Pharmacy, Pittsburgh, PA. 203 The compounds: CH3 HgC1and Me2 Se03 were There have been reports linking cadmium to given separately or simultaneously (per os) as a both hypertension and cigarette smoking. This single equimolar dose (10 mg CH3Hg/kg and 3.8 mg study involved analysis of amniotic fluid from Se/kg). Tissue distribution, excretion, accumu­ women for which the history was known. A total lation of inorganic and organic mercury were of 85 individuals were evaluated. Cadmium con­ determined 1,3,7 and 13 days after dose. It centrations were measured utilizing atomic abs­ was found that sodium selenite decreased reten­ orption spectrophotometry with carbon rod atom- tion of MeHgin various organs {µg/g tissue),

5 mainly after 13 days (2.5 fold). Selenium Microsomal heme oxygenase (HO) activity was accelerated accumulation of MeHgin the cerebrum measured by gas chromatographic quantitation of CO and cerebellum specifically 1 day after adminis­ liberated in vitro by NADPH-dependentenzymatic tration, but on day 13, the level of methyl­ degradationof heme, Microsomes were prepared mercury was lower than for the group given MeHg from organs of male Fischer rats killed at speci­ alone. The concentration of MeHg in both kidney fied intervals after sc injection of NiC12; .and liver after simultaneous administration of control rats received similar injection of NaCl methylmercuric chloride and sodium selenite were vehicle, HO activity (nmol CO/hr/mg microsomal significantly lower than those for group adminis­ protein) is reported as x ± SD [with no. of rats tered only with methylmercury. Selenium had no in brackets]. Time-Course Study: At specified significant effect on subcellular mercury dis­ hours after Ni-treatment (O. 25 mM/kg), renal HO tribution in the liver, kidney and cerebrum, activity was: 3 h, 4.8 ± 1,3 [SJ; 6 h, 8.5 ± 1.8 other than that which could be accounted for by [S]; 9 h, 14 ± 4 [S]; 17 h, 30 ± 8 [SJ; 24 h, 26 ± its effect on the whole tissue uptake. Sodium 14 [S]; 48 h, 18 ± 9 [S]; 72 h, 20 ± 9 [SJ; selenite changed the biotransformation of methyl­ controls (3-72 h), 5,8 ± 1,2. [8]. Dose-Response mercury in kidney, liver, cerebrum , cerebellum, Study: At 17 h after Ni-treatment at specified red blood cells and plasma. Three days after dosages, renal HO activity averaged: 0 mM/kg dosing with MeHgand Se, it was found that the (controls), 5.6 ± 1,6 [15]; 0.06 mM/kg, 8,9 ± 2,8 relative amounts of inorganic mercury were lower [SJ; 0.15 mM/kg, 16 ± 4 [SJ; 0, 25 mM/kg, 30 ± 8 than for the group administered with MeHgalone. [ 5] ; o. 38 mM/kg, 42 ± 9 [4] ; o. 65 mM/kg, 92 ± 15 Studies on excretion of MeHgshowed the [6]: O.75 mM/kg, 35 ± 7 [SJ. Organ-Selectivity mercury level excreted by urine was increased by Study: At 17 h after Ni-treatment (0, 25 mM/kg), selenium only 24 hr after administration, however HOactivity in other organs averaged: liver, 28 ± mercury excretionvia faeces was decreased during 7 [6] vs 9.2 ± 1,8 [6] in controls; spleen, 18 ± 4 13 days of observation. vs 16 ± 4 [6] in controls; lungs, 5,0 ± 1,6 [6] vs 3.6 ± 1,2 in controls; brain, 12 ± 1 [6] vs 10 ± 1 [6] in controls, Since maximal Ni-induction of HO 20INDUCTIONOF DNASTRAND BREAKS BY NICKELCHLORIDE activity occured in renal microsomes at 17 h after ANDCRYSTALLINE NICKEL SULFIDE IN CHINESEHAMSTER sc injection of NiCl2 (0. 65 mM/kg), these OVARYCELLS. S.H. Robison and M. Costa, Div. of experimental conditions are currently being used Tox., Dept. of Pharm., Univ. of Texas Med. School, to determine the effects of (a) hypoxia, (b) Houston, Tx., 77025. chelating agents, (c) actinomycin, cyclohexamide, and puromycin, and (d) glutathione on Ni-induction The effect of NiCl2 and crystalline nickel of microsomal HO activity, (Supported by DOE sulfide (aNiS) on the DNAof Chinese hamster ovary grant EV-03140 and NIEHS grant ES-01337). eel Is was studied. Both compou11asl.aused DNA strand breaks when the UNAwas analyzed by the al­ Kal1ne sucrose gradient technique. Cel Is were prelabelled witn [3H] thymidine for 48 hrs and 22 NEUROPATHOLOGICALLESIONS IN MICEFOLLOWING NEO­ a 11owed to reach confluence. The ce 11s were then NATALEXPOSURE TO TRIMETHYLTIN.K.R. Reuhl, B. exposed to the nickel compoundsfor the requisite Mackenzie, and L.W. Chang. Ecotoxicol. Group, amount of time, washed with ice cold saline, and Div. Biol., NRC Canada, and Dept. Pathol., Univ. harvested. Cells were resuspended in saline and Ark., Little Rock, AR. Sponsor: C.T, Mj]Jer. gently layered onto a lysis solution which had been overlayered on a 5-20% alkaline sucrose gra­ Trimethyltin (TMT)is a potent neurotoxicant dient. When[3H] thymidine radiolabelled DNAfrom of increasing concern in the industrial environ­ cells exposed to NiCl2 at 1 µg/ml for only 2 hrs meot. Light microscopy of adult animals has de­ was analyzed, a high degree of DNAstrand breakage monstrated selective injury to the hippocampus, was observed. Treatment with crystalline aNiS for amygdala, and pyriform cortex. We examined the 24 hrs also induced a significant number of DNA neonatal mouse to determine whether TMThas simi­ strand breaks at concentrations of 1 µg/ml. The lar regional specificity in the developing brain. breakage of DNAstrands was dependent upon the Two day old BALB/cmice were injected s.c. concentration and time of exposure with respect to with either 3 mg TMT/kgb.w., or 0.9% saline, NiCl2 and crystalline aNiS. Strand breaks are in­ and sacrificed at 4 hour intervals for the first duced at nickel concentrations which normally had day post treatment, and daily until day 25 for no detectable effect upon cell division. A con­ morphological evaluation. The earliest ultra­ centration dependent effect upon size and number structural evidence to cell injury was observed average molecular weight was observed with both at 8 hours, and consisted of mild dilation of the NiCl2 and crystalline aNiS. Since DNAstrand endoplasmic reticulum. Between 24 hours, and 7 breakage occurred at such low concentrations, days, increasing numbers of neurons in the pyri­ these results suggest that nickel compoundswhich form cortex and hippocampus appeared dead. De­ cause cellular transformation have highly selec­ generative changes were observed in both axons tive and specific effects upon DNAstructure. and dendrites. Light microscopical changes (Supported by grant #R808048from the U.S. EPAand appeared first at 16-24 hours, and consisted of by contract #ER60016from the Dept. of Energy and increased eosinophilia and nuclear pyknosis. by PHSgrant #CA06570from the NCI.) Pathological alterations progressed in severity, peaking between days 2 and 7. The hippocampus and pyriform cortex were most extensively in­ 21 TIME-COURSE,DOSE-RESPONSE AND ORGAN-SELECTIVITY volved, although other cortical areas and the FORNi(II)-INDUCTION OF MICROSOMALHEME OXYGENASE caudate nucleus were involved to a lesser extent. IN RATS. F.W. Sunderman Jr,, M,C, Reid, L,M, After day 10, the number of degenerating neurons Bibeau, & J, Linden, Lab, Med, & Pharmacol, Depts, decreased rapidly, and by day 25, general decrease Univ, of Conn. Medical School, Farmington, CT, in neuronal density was seen in involved areas.

6 The present study suggests that the neonatal hrs interval to control and Zn-Drats. Even animal is significantly more sensitive to TMT though the distribution of Cd in liver, kidney than the adult. and pancreas in both groups was similar, the Mt concentration in pancreas was significantly decreased in Zn-D. The plasma and tissue levels 23 ABSORPTION AND TRANSPLACENTALTRANSFER OF TIN of Zn were also decreased in Zn-Drats following DERIVED FROMDIMETHYLTIN DICHLORIDE AND STANNOUS Cd injection. The decrease in both Zn and Mt CHLORIDE. E.A. Noland, P.T. McCauley, R.J. Bull levels was more prominent in pancreas of zn-D and K. Kelty, Health Effects Research Laboratory, rats than other organs. The results suggest U.S.E.P.A., Cincinnati, OH 45268 that of all the organs studied, the induction Dimethyltin dichloride (DMDC) is commonly of Mt in pancreas is most sensitive to Zn used as a stabilizer in PVC pipe used for trans­ status and zinc may be the primary inducer of Mt port of potable water. in various tissues. Learning deficiencies have been observed (Supported by grants from MRC, Canada and ILZRO). postnatally in pups from DMDCtreated dams (Tay­ lor, Noland and Bull, unpublished results, 1981). The present work was undertaken to determine 25 ABSENCEOF URINARYBLADDER AND KIDNEY TOXICITY IN whether these abnormalities could be reasonably RATS AND GUINEA PIGS EXPOSEDTO INHALEDTERE­ attributed to an increase in the tin content of PHTHALICACID AND DIMETHYLTEREPHTHALATE. the brain during gestation. T.R. Lewis, D.W. Lynch, and R.L. Schuler. Femal~ Sprague-Dawley rats were exposed to National Institute for Occupational Safety and distilled water as drinking water which contained Health, Division of Biomedical and Behavioral 40 mg tin/1 as either SnClz or DMDCfrom 2-weeks Science, Experimental Toxicology Branch, prior to breeding to parturition. Controls re­ Cincinnati, OH. ceived only distilled water. Before first nursing pups were sacrificed and blood was taken by Terephthalic acid (TPA) and dimethyl terephthal­ carotid artery bleeding and brains were removed. ate (DMI') are extensively used in the plastics industry for the production of polyester fibers Dams were bled by syringe and needle from the and films. The combined production of DMT and abdominal aorta. All tissues were analyzed for tin by flameless atomic absorption. Blood from TPA exceeds 4 billion lbs annually. Adult male dams and pups exposed to DMDCin drinking water Sprague-Dawley rats and adult male Hartley guinea demonstrated more than 20x as much tin as either pigs were divided into three groups and exposed to O (control), 10 mg/m3 TPA and 15 mg/m3 DMT control or SnClz exposed rats (P(0.0001). Simi­ (6 hr/day, 5 days/week for 6 months). The larly, in DMDCexposed groups the pup's brains had "respirable" dust concentration for both TPA and nearly 3x the tin content of the other two groups DMTwas 5 mg/m3 . Serial sacrifices were con­ (P(o.0001). ducted after 30, 60, and 140 exposures. Body These data demonstrate that tin introduced weights of exposed animals remained similar to as DMDCis absorbed much better than inorganic tin controls throughout the study. Organ weights and crosses the placenta to the fetus. This (lung, liver, kidney, and spleen) and organ to resulted in a significantly elevated tin concen­ body weight ratios similarly were not affected. tration in the pup's brain at birth. Therefore, Clinical chemistry parameters (SMA 12 screen) and it may be concluded that the learning deficits are routine urinalysis parameters indicated no dif­ accompanied by elevated tin concentrations in the ferences between exposed and control groups. brain. Gross and histopathologic evaluations of tissues from animals exposed to TPA and DMTwere within normal limits. These data suggest that TPA and 24 THE EFFECT OF ZINC STATUS ON THE SYNTHESIS OF DMTdo not induce renal or urinary bladder tox­ METALLOTHIONEININ VARIOUS TISSUES OF RATS. icity following low-level inhalation exposures at nuisance dust levels. This finding is in con­ s. Onosaka and M.G. Cherian; Depts. of Pathology, trast to the results of feeding studies with TPA Pharmacology and Toxicology; University of Western and DMT in which reduced weight gain, hematuria, Ontario, London, Ontario, Canada bladder hyperplasia, and bladder uroliths have been observed. Metallothioneins (Mt) of various tissues contain exclusively bound zinc and any change in zinc status can alter its synthesis and tissue 26DOSE-RESPONSE RELATIONSHIPS IN 2-HEXANONE-INDUCED deposition. In the present study the changes in POTENTIATIONOF CHCl3 NEPHROTOXICITY. W.R. Hewitt Mt levels and its inducibility in Zn injected and and E.M. Brown. Department of Veterinary Anatomy­ deficient rats are undertaken. Mt levels in Physiology, College of Veterinary Medicine, Uni­ eleven tissues of control and rats injected with three different doses of ZnS04 (20 mg Zn/kg for 2, versity of Missouri-Columbia, Columbia, MO 65211 4 or 7 times) were measured by Cd-hem method. A Previous studies have demonstrated that sev­ dose dependent increase in Mt levels was observed eral ketonic solvents, including 2-hexanone (Hx), in pancreas, liver, kidney and small intestine could potentiate the nephrotoxic action of CHCl3 after ZnS04 injection - the highest amount being in rats. The purpose of this study was to deter­ in the pancreas. A positive correlation was mine the dosage range over which Hx potentiated found between Zn and Mt concentrations and the CHCl3 kidney injury. CHCl3-induced nephrotox­ relative Mt inducibility was similar in these four icity was evaluated in male, Fischer 344 rats tissues (slopes of regression lines 12.6 to pretreated (po) with Hx in dosages ranging from 1 15.5). In order to study the effect of Mt to 15 mmol/kg. After 18 hr a challenging dosage induction in Zn deficiency (Zn-D), 1 mg Cd/kg as of CECl3 (0.5 ml/kg, ip) was given. Kidney dam­ CdClz was injected subcutaneously, 3 times at 48 age was determined 24 hr later, using blood urea

7 nitrogen (BUN) and creatinine (Cr) content and male Wistar rats for 7 days. Clearances of inulin and renal cortical slice p-aminohippurate (PAH) and cationic egg-white lysozyme were performed in ~e tetraethyl ammonium ion (TEA) uptake, Renal dam­ isolated perfused rat kidney. Unlabelled and 12 I­ age was confirmed histologically. Maximum poten­ lysozyme were added to the perfusate to achieve con­ tiation of CHCl3 nephrotoxicity was observed in centrations of 80-120 mg/liter. Trichloroacetic acid­ rats pretreated with 10 mmol/kg Hx. Although Cr soluble radioactivity (TCA) and high performance liquid content and slice PAH uptake were altered in rats chromatography (HPLC) were used to quantify the ·receiving 1 mmol/kg Hx + CHCl3, no effect on BUN release of tyrosine to the perfusate. Reabsorption of content or slice TEA uptake was observed with this lysozyme averaged about 70% of the filtered load in regimen. Thus the minimum effective dosage for control and netilmicin treated kidneys. Reabsorption of Rx-induced potentiation of CHCl3 renal injury was lysozyme decreased to about 50% and 57% of the approximately 1 mmol/kg. Hx (10 mmol/kg) did not filtered load in gentamicin and tobramycin treated alter the nephrotoxicity of K2Cr207 (5, 10 or kidneys, respectively. Tyrosine perfusate concentration 20 mg/kg, sc), suggesting that Hx did not poten­ increased to about 3 µg/ml in control and netilmicin tiate CHCl3 damage by nonspecifically increasing treated kidneys, and to about 2 µg/ml in tobromycin the susceptibility of the proximal tubule to toxic kidneys after 120 min perfusion time. No increase in insult. However 10 mmol/kg Hx + CHCl3 did not de­ tyrosine concentration was detected in the perfusates press renal cortical glutathione content. Thus it from gentamicin treated kidneys. remains unclear if Hx potentiates CHCl3 renal in­ This study reveals the greater nephrotoxic poten­ jury by increasing proximal tubular bioactivation tial of gentamicin which impairs the endocytic reab­ of CHCl3. sorption of lysozyme and almost completely inhibits (Supported in part by USPHS grant 1 R0l 0H00986 lysosomal proteolysis. (Supported in part by USPHS 02) grant ES00560. Dr. Cojocel is supported by a Fellow­ ship grant from Deutsche Forschungsgemeinschaft 13- 27 A COMPARISON OF THE EFFECTS OF VANADATE Co 114-/1-1.) ADMINISTERED IN VIVO AND IN VITRO ON ORGANIC ION ACCUMULATION BY RENAL CORTICAL SLICES. 29 MOLECULARMECHANISMS INVOLVED IN THE BLADDER J.H. Smith, W.E. Braselton, S.R. Tonsager and J.B. TOXICITY OF SODIUM0RTH0PHENYLPHENOL (SOPP). Hook, Department of Pharmacology and Toxicology, Reitz, R.H., Fox, T. R., Quast, J. F., Hermann, Center for Environmental Toxicology, Michigan State E. A., and Watanabe, P. G. Tax. Res. Lab., Dow University, East Lansing, MI 4-8821/-. Chemical USA, Midland, MI 48640. Vanadium is a ubiquitous metal that can be de­ tected in trace amounts in most mammalian tissues, SOPP has been reported to increase urinary primarily the renal cortex. Vanadate, an oxyanion tumors in rats (Hiraga & Fujii, Fd. Cosmet. Tax., derivative of vanadium, produces a profound diuresis 19, 303 (1981). However, Opp (free base) and SOPP and natriuresis when infused in rats that may be associ­ failed to produce mutations in 5 strains of S. ated with its properties as a potent Na-K-ATPase typhimurium (2Xl0-4M) and SOPP did not induc;-un­ inhibitor. Thus it was of interest to quantify the scheduled DNA synthesis in rat hepatocytes (10- effects of vanadate on other membrane transport func­ 4M). DNA alkylation was not detected after gavage tions. Accumulation of the organic ions p-amino­ with 500 mg/kg of OPP or SOPP (det. limit 1 alk/ hippurate (PAH) and tetraethylammonium (TEA) in rat 10 6 nucleotides). These studies suggest little or renal cortical slices was inhibited by vanadate in a no genotoxicity for these materials. dose-dependent g,anner a~medium vanadate concentra­ Animals consuming diets with 2% OPP or SOPP were tions from 10- to 10- M. The slice content of sacrificed after 3, 7, 14, 30 and 90 days. Pro­ vanadium was linear (7 .5 to 325 µM V/ g wet weight of gressive hyperplasia of bladder epithelium was tissue) a; medi~ vanadate concentrations ranging noted in the SOPP group, but was absent in the from 10- to 10- M as determined by plasma emission OPP group. Focal kidney lesions were observed in spectrometry. Inhibition of PAH and TEA accumulation the OPP group but were largely absent in the SOPP was r~versible at vanadate concentrations less than 1.5 group. x Io- M and corresponded to a reduction of slice The met~bolism of both OPP. and SOPP was dose dep­ vanadium content. Injection of 1 or 5 mg vanadium/kg endent. Below 150 mg/kg, 90-97% of the urinary me­ intraperitoneally produced a profound diuresis and tabolites were identified as sulfate and glucur­ natriuresis during the first hour. The ability of renal onide conjugates, with 3-6% oxidation products cortical slices from these rats to accumulate PAH was apparently derived from microsomal (MFO) pathways. related to tissue vanadium concentrations. Increasing Following 500 mg/kg (gavage) or dietary adminis­ medium K+ concentrations potentiated vanadate tration (2%) of OPP and SOPP 20-30% of the urinary inhibition of PAH accumulation which corresponded metabolites were oxidation products. Thus the pro­ wit~jn~ibition of sodium pump activity, as determined duction of reactive MFO metabolites may be corre­ by K uptake. These results suggest that vanadate lated with the bladder toxicity observed in rats acts on proximal tubule transport of PAH via inhibition consuming diets containing high levels of SOPP but of Na-K-A TPase. (Supported by USPHS Grant ES not observed after low levels of SOPP. The lack of 00560.) genotoxicity of OPP and SOPP coupled with the dose dependent production of an oxidative metabolite suggests that the toxicity and carcinogenicity may 28 EFFECTS OF AMINOGL YCOSIDES ON RENAL REAB­ be manifest only at high metabolically saturating SORPTION AND LYSOSOMAL DEGRADATION OF doses. LOW MOLECULAR WEIGHT PROTEINS. C. Cojocel and J.B. Hook, Department of Pharmacology/Toxi­ cology, Center for Environmental Toxicology, Michigan 30 PRECLINICALTOXICOLOGY OF NITROST AT, State University, East Lansing, MI 4-8824-. A STABILIZED INTRAVENOUSNITROGLYCERIN. J.A. Anderson, E.J. McGuire, J.R. Watkins, Aminoglycosides, gentamicin, netilmicin and to­ J.E. Fitzgerald, and F .A. de la Iglesia, bramycin, were each administered (30 mg/kg/day) to Dept. of Toxicology, Warner-Lambert/Parke-

8 Davis Pharmaceutical Res. Div., Ann Arbor, 32 THE EFFECT OF EXERCISE ON CARDIOTOXICPOTENTIAL Ml 48105 OF ADRIAMYCININ RHESUS MONKEYS. C.R. Hassler, The intravenous nitroglycerin, Nitrostat, con­ D.C. Thake, R.I. Hamlin*, and P. Feder, Battelle stitutes a stable formulation for hospital Columbus Labs., Columbus, OH 43201, *Ohio State use. Previous formulations were difficult to Univ. College of Vet. Med. Sponsor: G.L. Fisher evaluate since the nitroglycerin was quickly adsorbed to the internal surfaces of the An experiment was devised to determine the ef­ venoclysis equipment with subsequent loss of fects of post treatment exercise on the cardio­ efficacy. The acute and subacute intra­ toxic potential of adriamycin. Rhesus monkeys venous toxicology of Nitrostat was evaluated were utilized. Cardiac function was evaluated in in mice, rats, rabbits, and dogs. For acute these animals by echocardiography. They were studies, mice and rats were given single iv treated with adriamycin until a significant devia­ doses. LOSO values in mice were 17.3 mg/kg tion of the velocity of circumferential fiber for males and 18.2 mg/kg for females. Com­ shortening (VCF), shortening fraction (SF), or parable acute toxicity in rats was 24.4 systolic time interval (PEP/LVET) was observed. (males) and 23.2 mg/kg (females). Daily iv The average dose injected was 8.3 mg/kg., with a administration to rats for two weeks at dose range of 6-11 mg/kg. Following treatment, one levels of 2.5, 5.0, and 10.0 mg/kg were half of the monkeys, including both treated and wel I tolerated by most animals. A slight vehicle control animals, were trained to perform non-dose related suppression of weight gain vigorous exercise in response to a food reward. and food consumption was noted in both Computer controlled exercise devices were de­ treated and vehicle control animals. There signed to simultaneously exercise 24 monkeys. were no drug related clinical laboratory or There were no detectable differences in mortality pathologic findings. In an escalating dose or myocardial damage between exercised and seden­ range study in dogs, iv doses of 90 mg tary monkeys. Decrease in heart rate was posi­ (infusion: 2 mg/min) or higher resulted in tively correlated with exercise. SF and VCF emesis, increased heart rate and ECG generally decreased with drug treatment but de­ changes. Repeated iv administration to dogs creased more in the sedentary animals. The PEP/ at 1 and 3 mg/kg/day for two weeks did not LVET ratio was less sensitive. Microscopic induce electrocardiographic, clinical or patho­ evaluation indicated primarily vacuolation, loss logic changes. Venous irritation potential of myofibers, and myolysis in the most severly was evaluated by injections into the mar­ affected animals. Nearly all animals had some ginal ear vein of rabbits for five consecutive degree of myocardial damage. There was good days. Minor trauma at injection sites was correlation between histologic and echocardio­ found but there was no pathologic evidence graphic data with dose. Extensive damage was of significant vascular irritation. These data observed in monkeys given 10-11 mg/kg. At do­ indicate that Nitrostat solution induced no sages of 6-7 mg/kg there was generally minimal unusual toxic manifestations. damage. Results from monkeys given 8-9 mg/kg were extremely variable. Supported by NCI 31 STUDIESOF THEINTERACTION OF BETABLOCKING DRUGS Contract #NOl-CM-17365. ANDCALCIUM ANTAGONISTS. J.A. Vick, E.H. Herman and T. Balazs, Food and Drug Administration, Washington~ D.C. 33 ISOPROTERENOL-INDUCEDLETHALITY IN MALE MICE The increased use of beta blocking drugs and FOLLOWINGMANGANESE, SELENIUM, AND CADMIUM calcium antagonists in the treatment of cardio­ TREATMENT. M.J. Deimling, J.B. Modrak, and R.C. vascular disease prompted studies of their Schnell, Department of Pharmacodynamics and interactions. A series of 21 minipigs were used Toxicology, College of Pharm·acy, University of to study the possible toxic effects of one widely Nebraska Medical Center, Omaha, NE 68105. used beta blocker, propranolol, and 2 increas­ Male, Swiss-Webster mice were treated intraperi­ ingly popular calcium antagonists, verapamil and toneally with manganese chloride (2 or 5 mg nifedipine. The minipigs were anesthetized with Mn++/kg), sodium selenite (2.4 mg se++ /kg), Na pentobarbital and instrumented to record cadmium acetate (1 mg cd++;kg), or normal saline arterial blood pressure, heart rate, EKGand (10 ml/kg), either alone or following a prior respiration. In the first group of minipigs, injection of Mn, at various time periods prior to verapamil 0.5 mg/kg was given by i.v. infusion receiving i soproterenol (ISO, 400 mg/kg, i.p.) over 10 minutes following 0.5 mg/kg propranolol and the total number of deaths were recorded 72 i.v., and produced marked hypotension, severe hours later. Mn (5 mg/kg) increased lethality bradycardia, A-V blockade and death in 8 of 8 when administered alone either 24 (9/11) or 4 animals. In contrast, nifedipine 0.5 mg/kg i.v. (10/11), but not 72 (3/11), hours prior to ISO as after the same dose of propranolol produced only compared with saline treated animals (4/11), An modest decreases in blood pressure and heart additional study demonstrated that while Mn (5 rate, no A-V block and survival in 8 of 8 mg/kg) administration alone 24 hours prior minipigs. Lethal doses (5-10 mg/kg) of increased ISO lethality as compared to control nifedipine alone did produce profound decreases animals (6/10 vs. 2/10, respectively), admini­ in arterial blood pressure and heart rate but no stration of Mn (2 mg/kg) three days prior to the A-V block. All 5 minipigs died of what appeared higher dose resulted in protection against to be cardiovascular collapse. The lethal lethality (3/10). Mn (2 mg/kg) alone did not effects of verapamil and propranolol combination increase lethality (2/10). Se increased and of high doses of nifedipine could be reversed lethality when administered alone 24 (9/10), but with calcium chloride i.v. (150 mg/kg). These not 72 (4/10) or 4 (5/10), hours prior to ISO as studies indicate that verapamil but not compared to saline treated animals (5/10). nifedipine increases the toxicity of propranolol, Interestingly, the administration of Mn (5 mg/kg) an effect which can be reversed by calcium. 2 days prior to Se protected against the increase

9 in lethality observed 24 hours following (5/10). is of sufficient magnitude to indicate the Cd administered 4 (10/10), but not 72( 7/10) or possibility of cardiotoxic effects. 24 (5/10), hours prior to ISO increased lethality This study was undertaken to determine the as compared with control animals (6/10). The toxic effects of n-hexane on isolated rabbit results of these studies indicate that Mn, Se, heart when perfused with Chenoweth solution and Cd can enhance ISO-induced lethality in male· containing n-hexane and using Anderson's mice. These results also suggest that the prior coronary perfusion apparatus. Perfusate administration of Mn can block enhanced ISO­ concertration of n-hexane which produced greater induced lethality following Se or Mn. (Supported than 50% inhibition of inotropic activity after by NIEHS Grant ES-02425) 60 minutes of perfusion was found to be 9.6 mg/1 (0.11 mmol/1).

34 ELECTROCARDIOGRAPHIC FINDINGS IN DOGS WITH SUBCLINICAL PARVOVIRUS-RELA TED CARDIOMY - 36 l?LUORESCENCEPOLARIZATION STUDIES OF HUMAN OPATHY. S. A. Kutz, P. S. Struve, D. K. Detweiler, R. ERYTHROCYTEMEMBRANES EXPOSED TO OZONEIN VITRO. E. Cimprich, J. L. Robertson, P. J. DeBaecke, and C. G. Witz, B.D. Goldstein, L. Myers-Basting, and S. Streett, Stuart Pharmaceuticals, a Division of ICI M.A. Amoruso,Department of Environmental & Com­ Americas Inc., Biomedical Research Department, Toxi­ munity Medicine,CMDNJ-Rutgers Medical School/ cology and Pathology Sections, Wilmington, DE and the Rutgers University Joint Graduate Program in School of Veterinary Medicine, University of Pennsyl­ Toxicology, Piscataway, NJ. vania, Philadelphia, PA. Sponsor: L. E. Gangwer Previous studies from this laboratory on in­ tact red cells and red cell membranes showed that Canine parvovirus is currently a problem for com­ there is a dose-dependent relationship between mercial dog breeders supplying animals for safety eval­ ozone exposure and a number of red cell membrane uation and toxicology studies. Puppies that survive the parameters including lipid peroxidation and loss acute enteric and/or cardiac forms of the disease of native protein fluorescence.Studies on oxidant appear normal and later are sold by the breeder. These damage due to ozone were extended to investiga­ apparently healthy dogs may actually have irreversible tions on membrane fluidity as measured by subclinical sequelae from the acute disease. We found fluorescence polarization techniques.Ghosts three beagle dogs purchased from a commercial breeder labelled with the fluorescent lipid probe all to have abnormal electrocardiograms. These ab­ trans-l,6-diphenyl-1,3,5-hexatriene (DPH) had normalities included multifocal ventricular ectopic significantly higher DPH polarization values beats, coupled ventricular ectopic beats, and fusion (p(0.001) than control ghosts.The DPH polariza­ beats. The R/S amplitude ratios were less than in 0.5 tion values, which ranged from 0.3251.003 to lead rV and were less than 0.9 in leads V and • 2 2 v4 0.037t.001 for control ghosts at 250 increased Normal variants included slurred and notched" P waves by 0.017 to 0.023 p units for ozonized ghosts. in limb leads, and low amplitude R waves (less than 0.6 DPH bound to ozonized ghosts which had lost more mV) in leads rV , , or • Two of these three dogs 2 v2 v4 than 50% of their native fluorescence exhibited were successfully iaentified by their abnormal electro­ a small decrease (-10%) in the excited state cardiograms before treatment. The other dog had three lifetime and a major decrease (-40%) in sufficiently normal pretreatment electrocardiograms fluorescence intensity.Protein polarization that it was assigned to a safety evaluation study. After values which ranged from 0.170!.001 to 0.176!.001 oral intubation and gavage, almost all post-dose at 250 for control ghosts also increased signif­ electrocardiograms contained several ventricular icantly (p<0.001) after ozone exposure, the ectopic beats and other changes as described above. Serum was available from two of these three dogs and highest increase being 0.037 p units.In contrast both showed a titer of l: 1280 to canine parvovirus. At to the DPH polarization, the increases in protein necropsy, fine gray streaks were present on both ventri­ polarization correlated directly with decreases cular surfaces of the heart in all three dogs. Myo­ in native protein fluorescence.These data sug­ cardial fibrosis was confirmed by microscopic ex­ gest that oxidative damage due to ozone results in membrane structural changes and decreases in amination. If such affected animals are not identified membrane lipid fluidity, possibly due to cross­ and eliminated before treatment, the differentiation of compound-induced changes from pre-existent disease linking of membrane components by lipid peroxida­ tion products formed after ozone exposure.(This may be difficult. work was supported by NIH grant ES00673.)

35 EFFECT OF LOWLEVELS OF n-HEXANEON ISOLATED 37 SENSORYIRRITATION PROPERI'IES OF COMPOUNDSPRO­ PERFUSEDRABBIT HEART. Raje R. R., Arnold & DUCINGNASAL TUMORS IN THE RAT. G. L. Kennedy, Marie Scwartz College of Pharmacy & Health Jr., B. A. Burgess, R. J. Gardner, J. R. Gibson Sciences, Long Island University, Bklyn, NY. and H. J. Trochimowicz, E. I. du Pont de Nemours Sponsor: Lawrence W.R. and Company, Inc., Haskell Laboratory for Toxi­ cology and Industrial Medicine, Newark, DE 19711 n-Hexane is a widely used industrial solvent which is regarded as water immiscible. Its In recent years several important chemicals, volatility and well documented neurotoxicity including formaldehyde, 1,4-dichlorobutene-2, make it an occupational hazard. On determining dimethylcarbamoyl chloride, epichlorohydrin, the solubility of n-hexane in water by gas hexamethylphosphoramide, and others, have been chromatography, it was found that water becomes shown to cause a unique nasal tumor in rats. saturated with n-hexane at 12 mg/1. In our Many of these materials are known respiratory laboratory it was found that n-hexane dissolves irritants. This study attempted to determine to a considerable extent (20 mg/lOOml) in the what, if any, correlation exists between upper blood of the rats following acute inhalation respiratory tract irritation and nasal tumor for­ exposure. The level of solubility in blood mation in the rat. Male rats were restrained and

10 exposed head-only for 10-minute periods to atmos­ for H; pheres of each of the chemicals listed previously • transient, dose-related behavioral signs as well as the nasal tumorigens 2,3,4-trichloro­ during week 1 butene-l; bis chloromethyl ether; and 1,2- • no significant effect on body weight, ethoxy-3-phenoxypropane. The irritants methyl hematology, or clinical chemistry, acrylate; methyl methacrylate; hydrogen chloride; except, dose-related increase in serum titanium tetrachloride; and chlorine were also alkaline phosphatase at week 26 studied. Atmospheric concentrations causing a (females only) 50% decrease in respiratory rate (RDSO's) were for T; determined. The data indicate no direct correla­ • increase in body weight at 1,500 ppm tion between the sensory irritation endpoint and dose level (males only) nasal tumorigenic potential. • no significant effect on hematology or clinical chemistry, except, dose­ related decrease in blood glucose at 38DOSE COMPARISONBETWEEN B6C3Fl MICE AND F-344 week 26 (females only). RATS FOLLOWINGFORMALDEHYDE INHALATION. J. C. Conclusions: The absence of a pattern of toxic F. Chang, E. A. Gross, J. A. Swenberg and C. S. responses suggests that neither heptane nor Barrow. Chem. Ind. Inst. of Toxicology, Research toluene present a serious toxic hazard under Triangle Park, NC 27709. common use conditions. The significance of the weight gain (T-exposed males) and of the isolated We have previously shown that B6C3Fl mice were effects on alkaline phosphatase (H-exposed more sensitive than F-344 rats to the sensory ir­ females) and blood glucose (T-exposed females) ritation effect of formaldehyde (CH o). The con­ 2 remains obscure. centration at which the respiratory rate (f) de­ Note: These studies were conducted under creased by 50% was 4.9 for mice vs. 31.7 ppm for sponsorship by the American Petroleum Institute. rats. This species difference in sensory irrita­ tion may lead to a difference in the amount of CH 0 inhaled and its subsequent toxicity to the 2 respiratory tract. To investigate this possibil­ 40 ETHYLENEGLYCOL MONOMETHYL ETHER: A SUBCHRONIC ity, the CH o dose deposited in the nasal mucosa INHALATIONSTUDY IN RATS AND RABBITS. Miller, R. 2 of mice and rats was assessed. Naive or CH R., Ayres, J. A., Young, J. T., and McKenna, M. J. 2o­ pretreated (6 or 15 ppm, 6 hr/day, 4 days) mice Tox. Res. Lab., H&ES, Dow Chemical USA, Midland, and rats were exposed to 6 or 15 ppm CH o for 6 hr MI 48640. 2 and f and tidal volume (VT} were measured. From Male and female F344 rats and N.Z.W. rabbits were these, the minute volume lV) was calculated. exposed to 0, 30, 100 or 300 ppm ethylene glycol During exposure, the f of all groups was depressed monomethyl ether (EGME) vapor 6 hr/da, 5 da/wk without a compensatory increase.in VT. However, for 13 wks. Some rabbits in the 100 and 300 ppm while a near 20% depression in VE was seen in all exposure groups died during the course of the groups of rats, a greater reduction occurred in exposures. Body weight gains of male and female pretreated mice. The reduction was -20% (naive) rats in the 300 ppm group were depressed, and vs. 40% (pretreated) for the 6 ppm mice and -35% weights of thymus glands and testes of both rats (naive) vs. 50% (pretreated) for the 15 ppm mice. and rabbits exposed to 300 ppm were significantly The dose of CH o distributed over the surface area 2 lower than for controls. Hematologic parameters, of the nasal epitheliu2 was then calculated and particularly white blood cell counts, were depress expressed as µg/min/cm. Thus, the calculated ed in both rats and rabbits in the 300 ppm group. dose was similar for the naive and pretreated rats Histopathologic observations included diffuse, but lesser for the pretreated mice. The dose re­ severe bilateral degenerative changes of testicu­ ceived by the 15 ppm-pretreated mice was -so% less lar germinal epithelium in rats and rabbits expos­ than that of the pretreated rats and comparable to ed to 300 ppm. Less pronounced histopathologic the 6 ppm-pretreated rats. This calculated dose changes occurred in testes of 3 of 5 rabbits in difference bef~een mice and rats at 15 ppm was the 100 ppm group as well as in 1 of 5 rabbits supported by CH o whole-body autoradiographic 2 exposed to 30 ppm. No testicular effects were studies on similarly exposed rats and mice. Th~s, found in rats in the 100 or 30 ppm groups, by normalizing CH o dosimetry data to µg/min/cm, 2 A follow-up evaluation of EGME-induced testicular species difference in respiratory tract toxicity, damage in rabbits is currently in progress. including nasal tumor incidence, can be better Evaluation of the functional effect of EGMEon compared. fertility and dominant lethality in rats is discussed in a companion paper (Rao, et al.).

39 SUBCHRONICINHALATION TOXICITY STUDIES OF N-HEPTANE(H) AND TOLUENE(T) IN THE RAT. s. C. Lewis and C. E. Holdsworth*, Medicine 41 CHRONICINHALATION TOXICITY OF ETHYLENEOXIDE and Environmental Health Department, Research AND PROPYLENEOXIDE IN RATS AND MONKEYS--A and Environmental Health Division, E~on PRELIMINARYREPORT. D.W. Lynch, T.R. Lewis, and Corporation, East Millstone, NJ and *American W.J. Moorman. National Institute for Occupa­ Petroleum Institute, Washington, DC. tional Safety and Health, Division of Biomedical and Behavioral Science, Experimental Toxicology Male and female Sprague-Dawley rats were exposed Branch, Cincinnati, OH. to Hor T 6 hours/day, 5 days/week for 26 weeks. Approximate time-weighted-average concentrations The chronic toxicity of ethylene oxide (ETO) and were: for H; 0, 400, or 3,000 ppm and for T; propylene oxide (PO), two industrially important O, 100, or 1,500 ppm. No treatment-related epoxides, was evaluated in a two-year inhalation deaths were observed. Salient findings were: study (7 hr/day, 5 days/week). Male weanling

11 Fischer 344 rats and adult male cynomolgus ERYTHROCYTESIN HUMANSAND IN DORSETSHEEP, A monkeys were exposed to O (control), 50 and 100 MODELWITH AN ERYTHROCYTEG-6-PD DEFICIENCY. ppm ETO and 100 and 300 ppm PO. The purpose of E.J. Calabrese, G.S. Moore, and P. Williams, the rat exposures was to conduct a rodent bio­ Division of Public Health, University of assay while the monkeys were utilized to evaluate Massachusetts, Amherst, MA. effects 9f these epoxides on major organ systems. It has been hypothesized that individuals Multiple indices including pulmonary function, deficient in erythrocyte G-6-PD activity may electrocardiography, clinical chemistry, hematol­ be at increased risk to the effects of ogy, urinalysis, gross and histopathology, neuro­ inhalation of elevated levels of ambient physiology, neuropathology ana mutagenicity have ozone. This study evaluated the effects of been assessed to determine the toxic responses to three proposed toxic ozone intermediates (i.e. ETO and PO. Body weights from rats exposed to methyl oleate hydroperoxide, methyl linoeate ETO and PO at all exposure concentrations and hydroperoxide, and methyl oleate oxonide) on body weights from monkeys exposed to 100 ppm ETO red cell metabolism indicative of oxidant were significantly reduced (p < 0.05; :!:_-test) stress (i.e. increase in methemoglobin levels, compared to controls. A dose response effect was decreases in glutathione levels) in normal evident on rat survival with survival at 24 human erythtocytes and in human and sheep months ranging from 20% in the ETO 100 ppm group erythrocytes which are G-6-PD deficient. The to 50% in controls. Eye irritation, consisting results indicated that human G-6-PD deficient of tearing, and nasal discharge were evident in red cells were markedly more susceptible than monkeys exposed to 300 PO. Clinical chemistry, normal human red cells to each stressor agent hematology, and urinalysis results indicate no with respect to methemoglobin formation. The differences between exposed and control monkeys. results of the sheep responses were Other indices (including histopathology) are cur­ sufficiently different from the human rently being evaluated and statistically analyzed. deficient to discourage its usefulness as a model to predict responses of human G-6-PD deficient erythrocytes. 42 ETHYLENEOXIDE VAPORTWO-YEAR INHALATION STUDY ON RATS: W.M. Snellings, c.s. Weil and R.R. Maronpot, Bushy Run Research Center, Export, PA 44 THE EFFECT OF LOW-LEVELOZONE EXPOSURE UPON THE COURSEOF£· berghei INFECTION IN FEMALE Fischer 344 rats were exposed to 100, 33, 10 or 0 A/J STRAIN MICE. G.S. Moore, E.J. Calabrese ppm of ethylene oxide (EtO) for 6 hrs per day, 5 and K.H. Mottent. days per week for two years. Initially, 120 rats Nine-month old female A/J mice were per sex per group were exposed, and every 6 months inoculated intraperitoneally from the same a portion of the animals was sacrificed to deter­ suspension of Plasmodium berghei on day zero mine possible effects. For one or both sexes,in­ (0) of this study. The infected mice were halation of EtO resulted in a depression of body randomly allocated to one of two stainless weight gain and an increase in mortality at 100 steel exposure chambers maintained at 72 + 3°F and at 33 ppm of EtO but not at the 10 ppm level. and relative humidity of 34 + 1%. Mice were At the 6-, 12-, and 18-month sacrifice intervals, then exposed to either filtered air or filtered there were no consistent patterns of association air with 0.30 1 .01 ppm 03 for 3 hour intervals of histologically confirmed organ damage with any every third day for 18 days. After each alteration in urinalysis, hematology, serum clini­ exposure period blood smears were obtained from cal chemistry, or organ weight. Skeletal muscle tail blood and stained via Giemsa. Slides were atrophy was present in both sexes of the 100 ppm subsequently examined on a double blind basis exposure group at the 24-month sacrifice interval. for the number of parasitized cells. Mice in Also, at this interval, histologic findings con­ the treatment group exposed to 0.30 1 .01 ppm firmed hematologic evidence that exposure to EtO o3 showed statistically significant increases resulted in an increased prevalence of mononuclear in number of parasitized cells (p < 0.03 to cell leukemia. The prevalence of this neoplasm in p < 0,005) during the study. Further, the the females was treatment-related and increased treated mice died an average of 3.5 days earlier for each of the three EtO-exposure groups. The than control mice. prevalence of other neoplasms was increased as shown by the greater percentage of EtO-exposed rats with more than two neoplasms; this was noted 45 ALVEOLARPERMEABILITY TO SERUMPROTEIN AFTER FIVE also in each of the three exposure groups for the HOURSOF 2 PPM OZONEEXPOSURE IN RATS SELECTIVELY female rats. Furthermore, in both the 100 and 33 BREDFOR SUSCEPTIBILITY OR RESISTANCETO SALT­ ppm exposure groups, the percentage of female rats INDUCEDHYPERTENSION. S.N. Schafrank, D.L. Costa, with a malignant neoplasm was increased. The R.W. Wehner & E. Jellett, Medical Dept., Brook­ frequency of peritoneal mesothelioma was treatment­ haven National Laboratory, Upton, NY 11973. related in the male rats exposed to 100 or 33 ppm Sponsor: R.T. Drew of EtO. While the incidence of mononuclear cell Sprague-Dawley rats susceptible (S) to salt­ leukemia and peritoneal mesothelioma in the air­ induced hypertension suffered higher mortality control rats was similar to those reported in the when exposed daily to 2 ppm ozone than did hyper­ literature, the possible contribution of sialo­ tension-resistant (R) rats. To examine this dacryoadenitis viral outbreak (which occurred differential sensitivity to ozone, alveolar perme­ during the 15th exposure month) to the EtO expo­ abilities to serum albumin were measured in both sure-related tumors in unknown. (Sponsored by ozone-exposed and control Sand R rats. Five to consortium of worldwide EtO producers) seven week old female rats maintained on a low­ salt (0.4% NaCl) diet were injected intravenously with 1125 bovine serum albumin, (28 microcuries/ml, 43THE EFFECT OF PROPOSEDOZONE INTERMEDIATES (.:i.!!_ 10 mg albumin/ml) at 45 microcuries/kg of body vitro) ON NORMALAND G-6-PD DEFICIENT weight. The rats were then exposed to either 2

12 ppm ozone or air for five hours. After pentobarbi­ Sponsor: R.T. Drew tal anesthesia, they were exsanguinated and the A proven distributed function architecture 1- 3 has lungs were lavaged in situ with saline through a been used in the development and implementation of tracheal cannula. The lavage and blood samples a microprocessor-based network to meet the require­ were measured for radioactivity using a NaI-well ments as set by the Brookhaven National Laboratory gamma counter. Results indicated that Sand R Inhalation Toxicology Facility for control and control rats have similar pulmonary permeabilities monitoring of exposure chambers, monitoring of the to I 125 -albumin. As expected, ozone caused sig~ chamber room environment, alarm enunciation,cnimal nificant increases in permeability, 156% and 110% weighing, data acquisition and management. The for the Sand R rats, respectively. The S-ozone­ equipment is presently grouped into 6 clusters exposed animals have an alveolar permeability to each composed of up to 6 microprocessor, nodes as­ albumin that is 34.5% (p <.05) greater than that signed specific compatible functions. The system of R rats exposed to ozone. Pathological examina­ includes 30 LSI-11 microprocessors, 2 million tion was conducted on sim~ arly exposed Sand R bytes of semiconductor memory and 300 million rats not injected with I 1 5-albumin. Sloughing of bytes of disk space. The nodes communicate via epithelial tissue, mucous formation and an accumu­ cluster memory of a maximum size of several mil­ lation of macrophages were much more frequent lion bytes. Clusters communicate among each other among S-ozone-exposed animals than in R-ozone-ex­ via a one MHz frequency-modulated link using low­ posed rats. This increased damage among S rats cost coaxial cable. The clusters are located as correlates well with the increased protein perme­ close to the sources of data as feasible, which ability levels that were found. may be several thousand feet apart. Emphasis is 1

13 are available to the user either automaticilly or hrs post-dosing. Selected tissues (brain, spinal upon request. Designed to meet the requirements cord, sciatic nerve, lung, liver, kidney) were of the toxicology user, the system has allowed the sampled, oxidized to 14co2, and quantified by safe, reliable operation of a multichamber inha­ scintillation counting. (Counts obtained were lation facility with minimal personnel. converted to tissue concentration of ACMD, expressed as nmoles/gm, for statistical analy­ *Research supported by the U.S. Dept. of Energy ses.) Within dosing periods (5 or 7 days), con­ under contract #DE-AC02-76CH00016. centrations of ACMD(as 14c) were significantly greater (p <0.05, Student's one-tailed t-test) in 11-week old vs 5-week old animals, excepting 49 ASPECTS OF THE IN VITRO METABOLISMOF DIMETHYL­ sciatic nerves within the 5-day groups. Analysis NITROSAMINE(DMN) BY RAT LIVER PREPARATIONS, of data via two-way ANOVArevealed that 80% of Lake, B.G., Collins, M.A., Harris, R.A., Cottrell, the variation in brain and spinal cord ACMDcon­ R.C,, Phillips, J.C., and Gangolli, S,D., The centration, and 61% of the variation in sciatic British Industrial Biological Research Associa­ nerve ACMDconcentration could be explained tion, Woodmansterne Road, Carshalton, Surrey, using age as a factor and body weight as a co­ SM5 4DS, U.K. variate. Dosing on a per kg body weight basis The hepatocarcinogen dimethylnitrosamine (DMN) is exposed tissues of 11-week old rats to dis­ metabolised by rat liver preparations to methanol proportionately higher amounts of toxin relative and formaldehyde and recent evidence suggests the to 5-week old rats, possibly explaining the involvement of multiple enzymic pathways in this earlier onset of toxic signs seen by Kaplan and process. In this study we have compared the prop­ Murphy in the older animals. (Supported by erties of the enzyme(s) responsible for hepatic 5-T32-ES07091 and ES-82130.) DMNmetabolism with cytochrome P-450 dependent mixed function oxidase (MFO) enzymes, monoamine oxidase (MAO,EC 1.4.3.4) enzymes and the Ziegler 51 BIOTRANSFORMATIONENZYMES (CYTOCHROME P-450 mixed function amine oxidase enzyme (EC 1.14.13.8). DEPENDENTMONO-OXYGENASES) IN THE NASAL EPITHE­ The treatment of rat hepatic microsomal prepara­ LIALMEMBRANE ANDTURBINATES OF SEVENSPECIES, tions with the chaotropic agent KI progressively A. R. Dahl, W. M. Hadley and J. M. Benson, Love­ destroyed cytochrome P-450 and associated MFO lace Inhalation Toxicology Research-Institute, activities. However, the metabolism of DMNto P.O. Box 5890, Albuquerque, NM87185 formaldehyde and MAOactivity were less affected. Similarly, in storage stability studies with post­ The nose is a major point of entry for xeno­ mitochondrial supernatant fractions DMNmetabolism biotics and since the way in which xenobiotics and MAOenzymes were stable under conditions where are metabolized often influences their toxicity, MFO enzymes were not. In inhibitor studies the the biotransformation enzymes of the nasal cavity enzyme(s) responsible for DMNmetabolism had tissues are of great importance to understanding different sensitivities to MFO enzymes and the toxic effects to those tissues. We have isolated Ziegler enzyme catalysed N-oxidation of N,N-di­ the microsomes (endoplasmic reticulum) from the methylaniline. The treatment of rats with cobalt­ nasal epithelial tissue and turbinates of the ous chloride resulted in an inhibition of DMN dog, rat, mouse, Syrian hamster, guinea pig, and metabolism,MFO and MAOenzymes, but had little rabbit. The nasal cavities were opened by split­ effect on N,N-dimethylaniline N-oxidation. These ting the skinned head along the midline and the results indicate differences between the hepatic relevant tissues were removed using forceps. enzyme(s) catalysing the degradation of DMNand Assays included measurement of the reduced a) cytochrome P-450 dependent MFO enzymes and b) carboxy adduct at 450 nm, para-nitroanisole 0- the Ziegler mixed function amine oxidase. The demethylase, aryl hydrocarbon hydroxylase and apparent similarity between DMNmetabolism and aniline hydroxylase activities. Levels of mono­ MAOenzymes may suggest the involvement of an N­ oxygenase activities and cytochrome P-450 content oxidase enzyme in the metabolism of this nitros­ comparable to that of liver on a per mg protein amine. (Supported by the U.K. Ministry of basis were found in all species. Pigmented Agriculture, Fisheries and Food). animals were found to be particularly high in cytochrome P-450 and this may be related to the more sensitive sense of smell reported for this 50 TISSUE DEPOSITION OF ACRYLAMIDEIN MULTIPLY-DOSED phenotype. ( Research conducted under DOECon­ 5-WEEKVS 11-WEEK MALEHOLTZMAN RATS. L.A. tract No. DE-AC04-76EV01013.) Rylander Yueh and D.E. Carter. Dept. of Pharma­ col. and Tox., Univ. of Arizona, Tucson 85721. The onset of functional acrylamide (ACMD)­ 52 EFFECT OF STRUCTUREON THE METABOLICRELEASE OF induced neuropathy has been reported to occur CYANIDEFROM NITRILES. T. Hasan, M. Hassan, S. sooner in 11-week old male Holtzman rats vs 5- Kuttab, and E. H. Silver, Toxicology and Medic­ week old rats (Kaplan & Murphy, Toxicol. Appl. inal Chemistry Programs, College of Pharmacy and Pharmacol. 11_, 259-68, 1972). To determine if Allied Health Professions, Northeastern Univer­ changes in distribution of ACMDto tissues sity, Boston, MA. Sponsor: D.R. Brown (especially nervous tissue) could explain this Nitriles are extensively used in the manu­ observation, 5- and 11-week old male Holczman facture of plastics, synthetic fibers, dyes and rats (2 groups each age, n=5) were dosed once pharmaceuticals. Several reports indicate that daily with 50 mg/kg ACMDip. Length of dosing nitriles may metabolically release cyanide (CN""). period (5 or 7 days, one of each age group per We have conducted structure-activity studies to period) corresponded to times of onset of neural investigate the metabolism of saturated and deficit observed by Kaplan and Murphy. On day 5 unsaturated nitriles to CN-. Male Sprague­ or 7, animals were dosed with 50 mg/kg ACMD Dawley rats were given a nitrile (0. 75 mmol/kg) containing 50 µCi/kg 2,_3-14c-ACMD, then killed 20 po, ip, or iv. Urine was collected for a 24 hr

14 period and analyzed for thiocyanate (ScN-). J. H. Wedig*, SRI International, Menlo Park, CA; Administration(po) of the saturated nitriles, Olin Corporation, New Haven, CT*. aceto-,propio-,n-butyro-,n-pentane-,n-hexane-, The metabolic disposition of the MgSO4 adduct of isobutyro-, and trimethylaceto- resulted in dipyrithione (Omadine® MDS, a broad spectrum 11.5±2.4% (mean S.E.), 65.3±2.9%, 64.7±2.7%, antimicrobial for use in shampoos as an anti-sebor­ 43.5±2.5%, 32.4±1.2%, 74.0±2.6%, 0.1±0.04% of rheic agent and in cosmetics as a preservative) was the dose excreted as SCN- in the urine, studied in several species. respectively.Administration(ip) of the same After i.v. injection of 2-6- 14 C-Omadine® MDS, the nitriles resulted in 4.2±0.5%, 56.8±1.1%, radioactivity was excreted predominantly in urine. 55.0±1.3%, 27.8±2.7%, 33.7±1.6%, 49.1±1.4%, and Urinary metabolite patterns in rat, rabbit, rhesus 0.2±0.1%, respectively.The unsaturated nitriles, monkey and swine were quite similar. One major acrylo-,croto-, and 3-butene-, yielded 37.3±1.9%, metabolite accounted for over 80% of the metabo­ 5.6±0.6%, and 28.9±1.4% of the dose as SCN- after lites. This was identified as the S-glucuronide of 2- po and 4.5±0.4%, 4.6±0.8%, and 17.7±2.9% after ip mercaptopyridine N-oxide. administration, respectively.Administration(iv) Three transient and one, more persistent, metab­ of propionitrile resulted in 44.6±3.7% of the olite were detected in rat plasma by HPLC analysis. dose excreted as SCN- compared to 0.1±0,04% for The persistent metabolite is 2-methylsulfonyl­ acrylonitrile.The time course of SCN- excretion pyridine (2MSP). The three transient metabolites was similar for propionitrile and acrylonitrile. were tentatively identified as 2-methylthiopyridine These results suggest that length of the carbon N-oxide, 2-methylsulfinylpyridine N-oxide and 2- chain, presence of substituents at the a-carbon, methylsulfinylpyridine from their mass spectra. position of double bonds, and, for some compounds, Approximately 8 hours after injection of Omadine® route of administration, are important factors MDS, the only plasma metabolite present was 2MSP. in the release of cyanide from nitriles.(Sup­ The plasma half-life of 2MSP was approximately 68 ported by Northeastern University's Research and hours for rats, 37 hours for rabbits and 22 hours for Scholarship Development Fund.) rhesus monkeys. Twenty-four hours after dermal application of Omadine MDS in a shampoo vehicle on the back 53 QUANTITATIVEPRECIPITATION OF FORMICACID FROM (clipped, occluded) of rats twice +daily for 4 days°'" METHANOL.G.G. Berg, Toxicology Div., Dept. of the plasma levels of 2MSP were 33-7 ng/ml and 254- Radiation Biol. and Biophysics, University of 31 ng/ml for doses of 2 mg/kg and 20 mg/kg, Rochester Sch. of Medicine, Rochester, NY14642. respectively. These studies suggest that in the species compari­ A convenient method for the separation of radio­ son, (1) the major urinary metabolites are identical; actively labeled methanol from its metabolites (2) the persistent metabolite in blood is identical, was developed for tissue dosimetry in concurr­ with the half life: rats> rabbits> rhesus monkeys. ently reported toxicity tests. It consisted of fixation and extraction in a methanol-form­ aldehyde-formic acid fixative designed to 55 METABOLISMOF A MILKWEEDCARDENOLIDE, USCHARIDIN. stabilize labeled trace compounds, followed BY MONARCHBUTTERFLY LARVAE, DANAUSPLEXIPPU~. by selective precipitation of formic acid with M.A. Marty and R.I. Krieger, Department of barium from a mixture of the extract with Environmental Toxicology, University of Califor­ tetrahydrofuran. nia, Davis, CA, and Department of Veterinary Solution A:formic acid,37.5 ml;37%formaldehyde, Science, University of Idaho, Moscow, ID. 75 ml;methanol to make 750 ml. Larvae of the Monarch butterfly utilize Solution B:formic acid,12.5 ml;37% formaldehyde milkweeds, Asclepias spp., which contain car­ 25 ml;water to make 250 ml. denolides (CD) as their sole food source. Fixative:mix A and B;for standards,add C-14 labe­ Certain CD are retained in tissues of larvae, led methanol or formic acid at 10 nCi/ml. pupae, and adults. The CD may be a chemical Ba reagent:co11·oidal suspension of 0.20M defense against predation. We investigated the Ba(OH)28H20in methanol (mag. mixing). role of microsomal monooxygenases in the metabo­ Extraction:t,ssue sample into 2 ml of chilled lism of a milkweed cardenolide, uscharidin, fixative; add vol. of Solution A equal to isolated from Asclepias currassavica. Homogen­ tissue weight; homogenizewith Polytron; ates of larval gut and fat body demonstrated a rinse probe with 2 changes of 2 ml fixative; low level of monooxygenase activity as measured pool; let stand overnight; centrifuge. by p-chloro-N-methylaniline N-demethylation, Separation: to 1 vol. of supernatant or standard Uscharidin was used as a substrate with portions add 1.2 vol. Ba reagent (5 min.); add 4.4 vol. of these homogenates. Extraction and TLC (E.M. tetrahydrofuran, stir, chill (15 min.); Silica Gel 60 with concentrating zone; developed 3X CHC1 :Me0H:OCHNH 90:6:1 or 2X Ethyl Acetate: centrifuge. 3 2 Assay: by scintillation counting of last super- MeOH 97:3) revealed biotransformation of natant in Aquasol-2(NEN). uscharidin. The products cochromatographed Recoveries of methanol in the final supernatant with two cardenolide standards, calotropin, and averaged 97.6% (S.D. = 1.16%, n=7). Removal of calactin. HPLC also confirmed their identity. formic acid from supernatant averaged 91.7% (S.D. Uscharidin biotransformation was not inhibited = 1.26%, n=7) and remained complete up to a 6:1 by CO, sesamex, or piperonyl butoxide. Subse­ sto i chiometr i c ratio of formate to barium. quent subcellular fractionation revealed the (Research supported in part by NIEHSGrant highest activity is in the soluble fraction. ES01247to the Env. Health Scienc~Center). Larval monooxygenase is apparently not involved in uscharidin biotransformation. The reaction appears to be mediated by a pyrazole-insensitive S 4 METABOLIC DISPOSITION OF DIPYRITHIONE. C. NADPH-dependent dehydrogenase. Supported in Mitoma, T. Steeger, J. Rogers, D. Thomas and part by NSF Grant DEB 7514266.

15 56 DISPOSITION AND EXCRETIONOF 1,3-DIPHENYLGUANIDINE phase II reactions (conjugative pathways) involv­ IN MALEFISCHER RAT. Y.M. Ioannou and H.B. ing N-acetylation and conjugation with glucuronic Matthews, Nat. Inst. Env. Hlth. Sci., Research and sulfuric acids. There was no evidence for Triangle Park, NC. the formation of hydroxylated metabolites over the dose range studied. The absorption, distribution, metabolism and ex­ cretion of 1,3-diphenylguanidine (DPG), a chemical used extensively as an accelerator in the rubber 58SEX DIFFERENCES IN THE METABOLISMOF 2,6-DINITRO­ industry, was studied in the male Fischer rat. TOLUENEBY ISOLATEDPERFUSED RAT LIVERS. R.M. DPG was completely absorbed after oral doses of Long and D.E. Rickert, Chem. Ind. Inst. of Toxi­ 1.52, 15.2 and 152 µmol/·kg body weight. The cology, Res. Triangle Park, NC 27709 tissue distribution of DPG was not affected by the dose or route of administration, oral or intra­ Female rats are less susceptible than male rats venous (tail vein). Liver and muscle were the to the hepatocarcinogenic action of a commercial major depots of DPG and contained up to 60 to 70% mixture of dinitrotoluene (DNT) isomers (75.8% of the radioactivity in the body. Skin, kidneys 2,4-DNT, 19.5% 2,6-DNT). Sex differences in and adipose tissue were also sites for DPG de­ hepatic macromolecular covalent binding after position. DPG was excreted in approximately equal administration of 2,4-DNT are associated with amounts in the feces, via the bile, and the urine. decreased biliary excretion of the dose in female Total excretion accounted for approximately 75% of rats resulting in decreased availability for the total dose in 24 hours and close to 100% in intestinal microfloral metabolic activation. 2,6- 72 hours. Bile duct cannulations resulted in DNT is more genotoxic and shows greater activity around 50% excretion of DPG in the bile within 2 in hepatic initiation/prop~tion systems than 2,4- hours and up to 75% in 6 hours. Urine and bile DNT. Significantly more C is covalently bound were analyzed for DPG metabolites using High to hepatic macromolecules in male than female Pressure Liquid Chromatography. All DPG-derived 1~vers after a single, oral dose (10 mg/kg) of radioactivity in the bile was in the form of a C-2,6-DNT. This study examined sex differences single major metabolite whereas approximately 60% in hepatic metabolism and biliary excretion of of the radioactivity present in urine co-chromato­ 2,6-DNT by isolated perfused Fischer-344 rat livers. Livers were ~erfused with medium con­ graphed with the parent compound. The remainder 1 of the radioactivity excreted in urine consisted taining 70 or 20 µM C-2,6-DNT. Bile was col­ of one major (30%) and two minor metabolites. In lected and perfusate was srwpled over a 2 hr summary, DPG is readily absorbed, metabolized and period. Disappearance of C from the perfusate excreted by the rat. was biphasic; half-times at both concentrations for the initial and finf~ phases were 9 and 59 minutes. Excretion of· C in the bile was 5 and 2 57 METABOLISMOF 5-AMIN0-1-NAPHTHOLIN THE RAT. times greater in male than female livers at 70 µM P.M. Enriquez and G.D. DiVincenzo, Health, Safety, (137 vs 26 nmoles/g liver), and 20 µM (34 vs 19 and Human Factors Laboratory, Eastman Kodak Co., nmoles/g liver), respectively. The major com­ Rochester, NY 14650. pounds in the perfusate were 2,6-DNT and 2,6- dinitrobenzyl alcohol glucuronide (1~6-DNBAlcG). The metabolic fate of 5-amino-1-naphthol (5AlN) is The major metabolite (~ 95% of the C) _in the of interest because of the association of certain bile was 2,6-DNBAlcG. Thus, observations with aromatic amines with carcinogenesis in experimen­ 2,6-DNT parallel those made with 2,4-DNT. This tal animals. This association frequently involves suggests that the two isomers are metabolized by a hydroxylation step which leads to the formation the liver and excreted in the bile in a similar of electrophilic intermediates. Knowledge of the manner. metabolism of 5AlN is potentially of use in pre­ dicting its capacity to form active metabolites. [14c]5AlN was administered by gastric intubation 59 METABOLISMOF NITROTOLUENESBY RAT CECALMICRO­ to Charles River CD rats at doses 1, 37 and 135 FLORA. S.R. Schnell, M.J. Turner, D.E. Rickert mg/kg of body weight. In a separate experiment and J.G. Dent. Chemical Industry Institute of the rats were also dosed with 150 mg/kg of un­ Toxicology, Research Triangle Park, NC. labeled 5AlN daily for four consecutive days. Be­ tween 74 and 85% of the administered dose was ex­ An important step in the production of toxic creted in the urine. Over 98% of the urinary metabolites from aromatic nitro compounds may be radioactivity was characterized. In addition to reduction of the nitro group. The hepatocarcino­ unchanged 5AlN, 5-acetamido-1-naphthol (5AA1N) and genic dinitrotoluenes require reduction by rat conjugates of glucuronic and sulfuric acids were intestinal flora for optimum production of a identified as metabolites. Unchanged 5AlN and metabolite which covalently binds to macromole­ 5AA1N accounted for less than 3% of the dose. The cules. The formation of methemoglobin following amount of 5AlN converted to 5AA1N varied inversely exposure of rats to nitrobenzene is dependent on with the dose (36 to 64%) and both compounds were reduction by cecal microflora. The purpose of excreted in the urine predominantly as glucuronide this investigation was to determine the relative and sulfate conjugates. The ratio of glucuronide rates of reduction of three isomeric nitrotolu­ to sulfate conjugates for both compounds increased enes by cecal microflora. Cecal microflora were directly with the dose. Two minor metabolites collected under low oxygen tension and incubated were not characterized. Rats dosed repeatedly anaerobically with 100 µM 2-, 3- or 4-nitrotolu­ with 150 mg/kg of 5AlN showed no significant ene (2NT, 3NT or 4NT). Metabolites were separat­ change in the metabolite excretion pattern com­ ed by reverse phase HPLC using a C-X8 column and pared to rats dosed singly. These findings show a methanol/phosphate buffer gradient. 3NT was that in the rodent model the metabolism of 5AlN more rapidly metabolized than either 2NT or 4NT was dose dependent, and occurred predominantly by (620 versus 240 and 550 nmoles/g cecal con-

16 tents/min, respectively). The major metabolite tissues were removed and assayed for total 14c by produced in each case was the corresponding oxidation to 14co2. Parent PNT was determined aminotoluene (confirmed by GC/MS); 2 additional for certain tissues by an extraction procedure peaks, as yet unidentified, were detected with demonstrated to be selective for parent compound. each nitrotoluene. These results demonstrate 14c-PNT was rapidly metabolized and cleared from that the position of ring substituent substan­ whole blood with approximately three percent of tially affects the rate of metabolism of aromatic dose remaining at two hr, and less than 0.2% of nitro compounds. The markedly slower rate of dose as parent compound. Peak tissue levels reduction of 2-nitrotoluene is probably a reflec­ occurred at 15 min, then rapidly decayed by four tion of hydrogen bonding between nitro and methyl hr to approximately one percent in muscle and groups as well as steric effects of the methyl skin, and less than one percent in fat, kidney group. These results suggest that more unmetabo­ and liver. At four hr 73±13% (x±SD) of the total lized 2NT will be absorbed from the intestine and activity in fat was parent compound; no other that 2NT may have a different toxicological pro­ tissues assayed contained detectable amounts of file from 3NT and 4NT. parent PNT at this time point. 14c-PNT was rapidly excreted in the urine, with 57±2% appearing by one hr, 70±8% by four hr and 82±4% 60 METABOLISMOF TOLUENEIN RAT ISOLATED HEPATOCYTES. by 24 hr. At one hr the urine contained approximately three percent of the dose as parent D. Guest, P.J. Deisinger, and G.D. DiVincenzo, PNT. While 30±1% of dose appeared in the bile Health, Safety and Human Factors Lab., Eastman of anesthetized rats by three hr, only 4±1% was Kodak Co., Rochester, NY 14650. found in feces by 24 hr suggesting that PNT The in vitro metabolism of toluene was investi­ undergoes significant enterohepatic circulation, gatedto validate the use of isolated hepatocytes By seven days total fecal excretion was 6±1%. as a rapid metabolic screening technique for in­ These data demonstrate that after a single intra­ dustrial chemicals. Toluene metabolism is gener­ venous dose, PNT is rapidly metabolized and ally thought to occur via oxidation of the methyl excreted, and has no major tissue storage depots. substituent, but recent evidence suggests that (Supported by NIEHS NOl-ES-8-2130.) aromatic oxidation occurs to a small extent. This possibility was investigated using primary hepato­ cytes isolated from male Sprague-Dawley rats. Cells were incubated in sealed flasks with 0.5, 62INTERACTIONS OF NITROBENZENEWITH HEPATIC MICRO­ 5.0 or 25.0 µmol toluene for up to 1 hr. Analysis SOMES: EVIDENCEFOR CYTOCHROMEP450 UNCOUPLING. by HPLC showed that toluene is rapidly metabo­ Levin, A.A., Bus, J.S., and Dent, J.G. Chemical lized to benzoic acid (BA) and hippuric acid (HA). Industry Institute of Toxicology, Research Tri­ Benzoyl glucuronide (BG) was not formed to any angle Park, NC 27709. appreciable extent even when formation of HA was Nitrobenzene exposure (300 mg/kg) induces met­ saturated. In control cells the initial rates of hemoglobinemia, testicular necrosis and hepatic metabolism were (nmol toluene/106 cells/15 min), cellular change in rats. This study investigated for 0.5 µmol, 4.8 (3.4 HA, 1.4 BA); for 5 µmol, the interactions of nitrobenzene (NB) with he­ 14.0 (3.3 HA, 10.7 BA); for 25 µmol, 22.6 (0.7 HA, patic microsomes isolated from Fischer-344 rats. 21. 9 BA). In cells from phenobarbital (PB)­ NB (0.1 rnM) was metabolized aerobically by micro­ treated rats the corresponding initial rates somes to a rate of 0.02 nmol/min/mg protein as were; for 0.5 µmol, 12.5 (8.1 HA, 4.4 BA); for determined by HPLC analysis of products. By com­ 5 µmol, 61.1 (6.8 HA, 54.3 BA); for 25 µmol, 80.3 parison aniline was metabolized at 1.5 nmol/min/ (2.4 HA, 77.9 BA). Control or PB cells incubated mg prot. Addition of NB to microsomes produced a with BA (0.1 or 0.5 mM) did not form BG but did Type I P450 binding spectrum, indicating that the produce HA. Cells incubated with 4-nitroanisole, lack of extensive metabolism of NB was not due to however, rapidly produced 4-nitrophenol glucuro­ the absence of P450 binding. NB (O.l mM-1.0 mM) nide and this reaction was induced by PB treat­ stimulated microsomal oxygen consumption and ment. Release of alanine aminotransferase was NADPHoxidation. The V for the cofactor uti- not increased and glutathione concentrations were lization was in the ranfixof 12-16 nmol/min/mg only slightly depressed, suggesting that toluene prot., which was 600 to 800-fold greater than the is not hepatotoxic in vitro. No evidence was observed rate of metabolism. NB-stimulated oxygen found for other toluene metabolites. Since me­ consumption and NADPHutilization were inhibited tabolism in PB cells accounted for 98-100% of the by CO and SKF-525A, indicating a role for P450. toluene (0.5 or 5.0 µmol expts) in 60 min, aro­ NB stimulated production of superoxide (o -=-) by matic mtidation can only occur to a very minor microsomes as determined by monitoring adreno­2 extent. chrome formation. The rate of NB stimulated O..:... production was similar to the increases in NADPH and o utilization produced by NB and was inhibi­ 2 61 PARANITROTOLUENE:DISTRIBUTION AND EXCRETIONIN ted by CO. Nitrobenzene stimulated adrenochrome MALEFISCHER 344 RATS. C.L. Detlefs, D.F. Perry, formation was inhibited by superoxide dismutase. D.E. Carter, and I.G. Sipes. Toxicology Program, These data are consistent with a P450 mediated Univ. of Arizona, Tucson, AZ 85721. reduction of the nitrogroup to form a nitroanion radical which may be reoxidized to NB by molecu­ While little data is available regarding the lar oxygen, thus generating 0 ..:.... This mechanism 2 disposition and metabolism of p-nitrotoluene of futile redox cycling and 0 ..:...production dif­ 2 (PNT), its structural similarity to known hazard­ fers from that proposed for paraquat since the ous chemicals indicates the rieed for such infor­ latter is dependent only on P450 reductase. The mation. Male Fischer 344 rats were killed at uncoupling of cytochrome P450 and release of 0 ..:... 2 times ranging from 5 min to 7 days after a single elicited by NB in microsomes may be related to intravenous dose of 8 mg/kg 14c-PNT. Select the toxicity of the compound.

17 63 A COMPARATIVESTUDY OF HEPATICMIXED-FUNCTION WhenOTB was given daily (l mg/kg x 7 days) elim­ OXIDASESIN 3 RAT STRAINS FED DIETS CONTAINING ination kinetics and tissue distribution of the 20% CABBAGE(BRASSICA OLERACEA)K. W. Miller and 1st and 7th doses were similar. Also with con­ G. S. Stoewsand, Cornell University, Geneva, NY tinuous daily treatment there was an accumulation of 14c in all tissues except visceral fat. Tis­ Consumption of Brassica vegetables has bee:11shown sues that accumulated 14c to the greatest extent in this laboratory to inhibit tumor formation in were lung, stomach, thyroid, liver and skin. rats challenged with a hepatocarcinogen; possibly (Supported by NIH grant ES01906). by altering the activity of the hepatic microso­ mal mixed-function oxidase system (J. Envir. Path. Toxicol. 2, 399. 1978). This study focuses on 65 0 the activity of different hepatic mixed-function 6~I~£~A:~~N~5~I5i~~I~~i0~~~u1~~H~N~A~~TA~~i6~~CE oxidase enzymes, and organ weight changes in Fisher-344 (F), Long-Evans (LE), and Sprague­ FORDESULFURATION. K. D. Williams, W. R. Porter Dawley (SD) rats. Weanling male rats were fed and R. E. Peterson, School of Pharmacy, Univer­ either a purified or an isocaloric, isonitrogen­ sity of Wisconsin, Madison, WI. ous diet containing 20% cabbage. After 4 weeks, The objective was to compare 14c and 35s the LE and SD rats showed a significantly lower dithiobiuret (OTB)with respect to elimination weight gain, corresponding with decreased diet kinetics and tissue distributian in ~gts. Rats intake and diet efficiency compared to controls. were treated with l mg/kg of C or S OTBand Two-way analysis of variance linked cabbage diets killed 3, 6, 12 or 24 hr later. For both 14c and to a significant depression of NADPHcytochrome C 35s the T1;2 for plasma disappearance was about reductase activity in all strains. Aniline hy­ 10 hr and 70% of the doses were eliminated in droxylase and p-nitrophenol 0-demethylase levels urine in 24 hr. The concentration of OTB-derived varied markedly between strains, lowest levels radioactivity in the thyroid was 8-45x higher occurring in F and SD respectively. Aniline hy­ than plasma. Also the concentration of 14c and droxylase was significantly elevated at 3 weeks 35s OTBequivalents in the thyroid was similar and depressed at 4 weeks in LE cabbage-fed rats as was their thyroid elimination kinetics. The The LE rats showed a pattern of early elevation concentration of 14c and 35s OTBequivalents in and later depression for 0- and N-demethylase as brain, gastrocnemius, sciatic nerve, lung and well. Excepting LE, aminopyrine N-demethylase kidney was 0.5-l.2x that of plasma. Elimination activity tended to be elevated in the two other of radioactivity from these tissues paralleled strains consuming the cabbage diets. Hepatic that from plasma and within each tissue elimina­ microsomal protein was significantly increased tion of 14c paralleled 35s. The only tissue in cabbage-fed SD rats. Cabbage treatments in­ where elimination of 14c and 35S was not parallel creased testes weight in SD and thyroid weights was the liver. T1;2 for hepatic elimination of in LE. LE and SD strains showed a significantly 14c was slower (15 fir) than 35s (10 hr). Thin smaller thymus. This investigation shows that layer chromatography (TLC) of the 24 hr urine hepatic enzyme activities and organ changes due sample from rats treated with 14c OTBrevealed to consumption of cabbage are strain dependent. six peaks. One peak migrated with OTB(17% of dose), a second with monothiobiuret (11%), and a third with thiuret (8%). Of the three unknown 64 DOSEDEPENDENT CHANGES IN ELIMINATIONAND TISSUE peaks, two were more polar than OTB(9, 21%) and DISTRIBUTIONOF 14c DITHIOBIURETIN THERAT. one was less polar (2%). TLCof urine from 35s W. D. Atchison, J. Dickins, W. R. Porter, and OTB-treated rats demonstrated that sulfate was a R. E. Peterson, School of Pharmacy, University metabolite. The results demonstrate that OTB of Wisconsin, Madison, WI. undergoes metabolism in the rat and that desul­ furation is one of the processes involved. The aim was to compare elimination ki~etics (Supported by NIH grant ES01906). and tissue distribution of OTB-derived C in rats under various dosage regimens. To assess the effect of OTBdose rats were administered l 66 SYNTHESIS,STABILITY IN SOLUTION,AND SEPARATION or 25 mg/kg 14c OTBip and killed 3, 6, 12 or 24 OF 35s-OITHIOBIURETFROM DECOMPOSITION PRODUCTS hr later. T1;2 for plasma disappearance (8-10 hr), ANDPOTENTIAL METABOLITES. W. R. Porter, K. D. cumulative urinary excretion (65-75% of dose) and Williams and-R. E. Peterson, School of Pharmacy, cumulative fecal excretion (2-4%) were similar in University of Wisconsin, Madison, WI. the low and high dose grou~s. The highest con­ centration of OTB-derived 4c was in the thyroid Daily treatment of rats with 2,4-dithiobiuret where it was 13x higher than plasma in the l mg/kg (OTB) produces a delayed onset, flaccid, skeletal group but only 3x higher in the 25 mg/kg group. muscle weakness progressing from the hind limbs to This difference may be due to saturation of the the forelimbs, trunk, and head. OTBwas labeled thyroid' s ability to concentrate OTB. In other with 35s for metabolic studies. Exchange of 35s tissues the concentration of 14c OTBequivalents between OTBand hydrogen 35s-sulfide occurs within was 0.5-2.0x that of plasma and elimination of 2 hr in boiling 0.01 N HCl under a N2 atmosphere. radioactivity from most of these tissues para­ 40-75% of the initial OTBwas recovered in crys­ lleled plasma. Exceptions were thyroid, stomach, talline form. The OTBcontained 10-90% of the skeletal muscle and brain in the l mg/kg group initial radioactivity on a molar basis. Air oxi­ and brain in the 25 mg/kg group. At 25 mg/kg dation of OTBoccurs in solution at elevated tem­ relatively less radioactivity was distributed to peratures. OTBsolutions at neutral pH are stable the thyroid, visceral fat and plasma than l mq/kg, for up to 8 days at 4° in air. Degassed OTBsolu­ and more was distributed to other tissues par­ tions are stable for 24 hr at room temperature, ticularly the lung. The lung concentration of but solutions stored ~n air at room temperature 14C was 60x_higher in the 25 than l mg/kg group. lose greater than 5%of the initial OTBwithin 24

18 hr. Solutions stored for 5-8 days at room temper­ chlorodiane blue (CDB) and pigment yellow 12 (PY ature degrade to thiuret (oxidized OTB)monothio­ 12), was studied in adult male Fischer 344 rats to biuret (a OTBhydrolysis product) and other pro­ determine whether these compounds were absorbed ducts. OTBcan be separated from its oxidation and metabolized. [14c]CDB and [14c]PY12 were syn­ and hydrolysis products by thin-layer chroma­ thesized from uniformly labeled [14c] dichloro­ tography (TLC) on silica gel with acetonitrile: benzidine (11 µCi/µmole). [14c]CDB or [14c]PY12 carbon tetrachloride: formic acid (60: 5: 2) or by was administered via gastric intubation or dermal­ TLCon microcrystal line cellulose with ethanol: ly at doses of 1.24-2.65 µmole/kg of body weight l M ammoniumacetate (75: 30). TLCspotting and (2.73-5.82 µCi/rat). Serial blood samples were development must be done at 4° in a N2 atmosphere. taken during an 8 hr period after oral dosing; no Using these methods, the radiochemical purity of radioactivity was detected in the blood of rats the 35S-DTBwas greater than 96%. TLCspotting in which received either compound. A 24 hr balance air and development at room temperature in a N2 study indicated that orally administered [14C]CDB atmosphere may be accomplished if excess unlabeled was not absorbed from the gastrointestinal tract, OTBand decomposition product standards are added. with the total dose found in the feces. Less than Negligible exchange or decomposition of 35S-DTB 0.05% of the dose could be detected in the liver occurs under these conditions. (Supported by NIH 24 hr after oral administration of [14c]PY12; the grant ESOl906). balance of the unabsorbed radioactivity was in the feces. [14c]CDB or [14c]PY12 was applied dermally to the shaved backs of rats; no radioactivity was found in the blood or liver, with the entire dose 67 FATEOF 14c-CARBONTETRACHLORIDE IN RATS FOLLOW­ detected at the application site. In vitro stud­ ING INHALATIONDURING A NOVELAND STANDARD WORK­ ies of CDB, with and without liver enzyme metab­ WEEK.D. J. Paustenbach, G. P. Carlson, G. S. olic activation, in£.!_ typhimurium TAlOO, TA98, Born and J. E. Christian, Schools of Health Sci­ TA1535, TA1537, and TA1538 showed that it was not ence and Pharmacy and Pharmacal Science, Purdue mutagenic at all doses tested (25-1000 µg/plate). University, West Lafayette, IN 47907. Only chemical reduction of CDB with dithionite produced a compound, presumably dichlorobenzidine, Although the metabolism and toxic actions of CCl4 which was mutagenic with metabolic activation in have been well studied, the pharmacokinetic para­ strains TA1538 and TA98. Thus, the relatively in­ meters of excretion have not been thoroughly eval­ soluble pigments CDB and PY12 are neither absorbed uated for all 3 excretory pathways. Male Sprague­ nor metabolized in vivo, nor was CDB metabolized Dawley rats were exposed in a closed-loop inhala­ by liver enzymes in vitro. tion chamber to 100 ppm of 14C-CC14vapor for 8 hr/day for 10 of 12 consecutive days (standard workshift) or 12 hr/day for 7 (4+3) of 12 days 69 TOXICOKINETICSAND METABOLISM OF A SINGLE ORAL (novel workshift). Rates and routes of elimina­ DOSE OF 0-ETHYL 0-4-NITROPHENYL PHENYLPHOSPHONO­ tion of 14c activity were followed for over 100 THIOATE(EPN) IN-CHICKS. M.B. Abeu-Donia, Y.M. hr after termination of exposure. Following the Hernandez and N.S. Ahmed. Department of Pharma­ standard workshift exposure, expired CCl4 and cology, Duke University Medical Center, Durham, fecal 14c activity comprised 52%and 41%, respec­ NC 27710. tively of the total 14c excreted. Following the novel workshift exposure, the values were 32%and The toxicokinetics and metabolism of a single 1 mg (2.7 µCi)/kg oral dose of uniformly phen 1- 62%. In both cases the urine accounted for less 14 14 than 6% of the total 14C excreted and exhaled CO2 labeled [ C]EPN (0-ethyl 0-4-nitrophenyl [ C] for 2% or less. For both workshifts the excre­ phenylphosphonothi~ate) ha:;-e been studied in 1- tion in the breath could be described by a 2-com­ week old chicks. One control and three treated partment 1st order pharmacokinetic model (r2=o.98). chicks were killed at each of the following time In the breath, the Th for the 8 hr group for the intervals: 0.5, 2, 4, 8 , and 12 days. Radioacti­ vity was rapidly absorbed from the gastrointesti­ 1st phase was 110 min and for the 2nd phase, 490 14 min. For the 12 hr group, the T½'S for the 1st nal tract and distributed in all tissues. C in and 2nd phases were 80 min and 580 min. It is of tissues reached a peak of 16.9% of the dose after interest that the feces constituted a significant ½ day and decreased to 0.6% at 4 days. The pathway for the excretion of the highly volatile tissues of the gastrointestinal tract had the CC14and that 14c activity could still be detected highest concentration of radioactivity, followed in breath, urine and feces 104 hr after exposure. by urinary bladder, bile and liver. Among nervous This study suggests that slight changes in dosing tissues, concentration of 14 C was highest in the regimen may influence the rate and route of excre­ peripheral nerves. The spinal cord had the next tion of a toxicant and that this may need to be highest concentration, while the brain had the considered with respect to potential occupational least. By the end of 4 days 91.3% of the 14 c health hazards in industries with novel work­ had been eliminated in the combined urinary-fecal shifts. excreta. By the end of the experiment this per­ centage reached 93.1%. No 14 C was detected in

the expired CO2 • Following the oral administra­ tion of [ 14 C]EPN, a monophasic body level curve 68FATE OF DICHLOROBENZIDINE-BASEDPIGMENT CHLORODI­ was observed. The half-life for the elimination ANE BLUE AND PIGMENTYELLOW 12 IN VIVO & IN VITRO of 14 C from chick body was 16 hours, corresponding 1 to a rate constant of 0.04 hr- • EPN and metabo­ G.M. ~. C. Snyder, F. Joachim, International lites were identified by high-pressure liquid and Business Machines Corp., Materials Toxicology sequential thin-layer chromatographic systems. Dept., San Jose, CA 95193 and C. Mitoma, SRI Polar EPN metabolites accounted for most of the International, Menlo Park, CA 94025. radioactivity identified in the excreta. The fate of two dichlorobenzidine-based pigments, (Supported in part by NIEHS Grant No. ES02717).

19 70 EFFECTSOF INDUCTIONAND OF S0LUBILIZATI0NON IN compared to the rat, indicating a lower circulat­ VITROALTERATION OF MICROSOMAL CYTOCHROME P-45O ing level of the free monoester may be a possible BYALLYLIC TOXICANTS. J.M. Patel and K.C. Leib­ explanation for the resistance of this species to man. Dept. of Pharmacology, Univ. of Florida, DBP-induced testicular injury. (Supported by the Gainesville, Fla. U.K. Ministry of Agriculture, Fisheries and Food). Destruction of cytochrome P-450 (P-450) and of 14 heme, and formation of green pigment by several 72DISPOSITION OF [ c]HEXAMETHYLENEDIAMINEIN FIS­ allylic industrial toxicants has been shown in CHER-344 RATS AFTER ORAL, INTRAPERITONEAL,AND hepatic microsomes from phenobarbital (PB)-treat­ INTRAVENOUSADMINISTRATION. R.M. David and H.d'A. ed rats [Patel et al., Fed. Proc. 40:636 (1981)]. Heck, Chemical Industry Institute of Toxicology, In the present study, we examined the effects of Research Triangle Park, NC 27709. Sponsor: allyl chloride (Alcl) and allyl bromide (Albr) on these parameters in liver microsomes and solubil­ J.E. Gibson ized microsomes from control and PB- and 3-methyl­ Hexamethylenediamine (1,6-diaminohexane) cholanthrene (MC)-treated rats. The destruction (HMDA), a constituent of nylon and other polymers, of P-450, loss of heme, and formation of green is a respiratory and conjuctival irritant report­ pigment after incubation with Alcl and Albr were edly associated with the development of hemolytic 5- and 4-fold greater in microsomes from PB-treat­ anemia in man. HMDAis also an inhibitor of ed rats than in those from control and MC-treated ornithine decarboxylase, the key enzyme responsi­ rats, respectively. Whenmicrosomes were solubil­ ble for the biosynthesis of polyamines. Initial ized, the loss of P-450 and heme remained in the experiments were undertaken to investigate the same proportion as in intact microsomes, but the disposition of HMDAin rats after oral (p.o.), formation of green pigment after incubation with intraperitoneal (Lp.), and intravenous (i.v.) ad­ Alcl or Albr was 1/10 of that in intact microsomes T£nistration. Following administration of [1,6- from PB-treated rats, and completely disappeared C]HMDAby either oral or i.p. routes, approxi­ mately 20% of ihe dose was recovered in the ex­ in microsomes from MC-treated or control rats. 1 The effect of Alcl required NADPHin all prepar­ pired air as CO over a 72-hour period, while ations. Although Albr caused destruction of P-450 only trace(< 1.6%) amounts remained in the tis­ in the absence of NADPH,destruction was more sues and carcass. The principal excretory route extensive in the presence of NADPHin all pre­ was the urine, which accounted for 79.6% (i.p,) parations. Addition of glutathione or of proto­ and 49.4% (p.o.) of the dose, respectively, Fecal porphyrin IX did not protect P-450 or heme from excretion accounted for 1.4% (i.p.) and 27.6% destruction by Alcl or Albr in any preparation. (p.o.). Analysis of radioactivity in different (Supported in part by USPHSgrant 0H-00316) tissues showed that the prostate contained the highest concentration of radioactivity following either route of adminisf~ation. Intravenous administration of [1,6- C]HMDAfollowed by whole­ 71 COMPARISONOF URINARYMETABOLITES OF 14c DI-N­ body autoradiography demonstrated a high concen­ ANDMONO-N-BUTYL PHTHALATE IN HAMSTERAND RAT: tration of radioactivity in the thymus as well as POSSIBLE RELATIONSHIP TO EFFECTS ON TESTICULAR the prostate. High-performance liquid chroma­ TISSUE. Foster, P.M.D., Cook, M.W., Thomas, L.V., tography (HPLC) of urine indicates at least two Walters, D.G. and Gangolli, S.D., The British m Jor and two minor peaks of radioactivity. Industrial Biological Research Association, [ 1 C]HMDAdoes not appear to be a major urinary Woodmansterne Road, Carshalton, Surrey, SMS 4DS. constituent. It is not presently known if the U.K. tissue distribution of radioactivity indicates Previous studies have shown rats to be susceptible target organ toxicities of HMDA. to t'esticular injury produced by either di- (DBP) or mono-n-butyl phthalate (MBP), whereas hamsters appear resistant. A study was therefore under­ 73 MEASUREMENTOF 7-METHYLGUANINEIN URI~E AS AN taken to see if this difference in susceptibility ESTIMATEOF DMNFORMATION FROM THE REACTIONOF could be ascribed to any species differences in AMINOPYRINEAND NITRITE IN VIVO. C.T.Gombar the metabolism of these two compounds. 14C-DBP and P.N.Magee, Fels Res. Inst., Temple Univ. and 14c-MBP were administered with non-labelled School of Med., Phila., Pa. 19140 carrier at dose levels known to produce testicular A variety of secondary and tertiary amines are atrophy in the rat (2g/DBP/kg body wt - 10µCi/kg known to react with nitrous acid under simulated body wt; 800mgMBP/kg body wt - 10µCi/kg body wt). gastric conditions to form potentially carcinogen­ Urine was collected over a 48hr period and radio­ ic nitrosamines. One of the best examples of active metabolites analysed using reversed phase this type of reaction is the formation of di­ HPLC. In both species and using both compounds methylnitrosamine (DMN) from the reaction of the major metabolite was found to be MBP­ aminopyrine (AP) with sodium nitrite under glucuronide, and not MBP as previously reported. acidic conditions. In order to estimate the ex­ This metabolite accounted for approximately 70% tent of this reaction in vivo by noninvasive of dose found in urine. Interestingly the amounts means we have utilizedan assay for 7-methyl­ of unconjugated MBP were quite different with guanine (7meG) in urine after administration of values of approximately 18% and 6% respectively 14C-AP with sodium nitrite. The basis for this for the rat and hamster. All the metabolites assay is the fact that there is a dose dependent found after administration of 14C-DBP could be increase in the excretion of 14C-7meG in rats accounted for in the 14C-MBP experiments confirm­ treated with 14C-DMN. To standardize the assay ing the need for esterase activity prior to we administered various doses of 14C-DMN to rats, absorption. Gut esterase activity in the two collected urine for 24 hrs and measured the species was found to be comparable. The observed amount of 14C-7meG in the urine. As a check on lower urinary excretion of MBP in the hamster the assay we have also measured 14C-7meG in the 20 liver DNA of these same animals since it is and 20% of the dose, respectively. At 4 hr, known that this parameter increases linearly however, these tissues each contained <10% of the with dose. Rats were then treated with 14C-AP dose. Liver and kidney reached maximum values at or 14C-AP with nitrite. The rats treated with 1 hr of 8 and 3% of the dose, respectively. 14C-AP alone showed no 14C-7meG in their urine Except for fat, all tissues contained primarily or liver DNA. Rats treated with 14C-AP plus metabolites by 4 hr. Excretion was virtually nitrite did have measurable amounts of 14C-7meG complete after 2 days: 40% in urine, 20% in in their urine and methylation of liver DNA. The feces, and 25% in expired air. Direct measure­ doses of DMNestimated from each of these para­ ment of radioactivity excreted in bile showed 10% meters were in good agreement. The amount of of the dose excreted within 3 hr. A fraction of 14C-7meG in the urine should, therefore, be a the metabolites excreted in bile were reabsorbed, reasonable estimate of the amount of DMNformed since radioactivity excreted in the feces by 2 in vivo. Work supported by NIH, CA-09214, days was only 60% of that found in intestinal CA-12227,and CA-23451. contents at 2 hr. Urine and feces contained no detectable unmetabolized TCP. Small amounts of 74METABOLISMOF 4,4'-DICHLOROBIPHENYL (4,4'-DCB) BY unchanged TCP were found in intestinal contents HUMANLIVER MICROSOMES. R.G. Schnellmann, T.J. at 15 and 30 min, but these were reabsorbed, Gillespie, C.W. Putnam, and I.G. Sipes. Depts. since no unchanged TCP appeared in the feces. The of Pharmacol. and Surgery, Az. Hlth. Sci. Ctr., major urinary metabolites are probably mercap­ Tucson, AZ 85724. turic acids because 1) 85-100% of the radio­ Previous studies in our laboratory have shown activity in acid, neutral, or basic urine was not that human liver microsomes can metabolize 2,2', extracted by ethyl acetate, and 2) thin-layer 3,3',6,6'-hexachlorobiphenyl, but apparently not chromatography of urine revealed compounds that 2,2',4,4',5,5'-hexachlorobiphenyl, to hydrox­ gave positive reactions with ninhydrin. ylated metabolites. Since the para-position is (Supported by NIH Grants ES82130 and 5-T32- usually the preferred site for hydroxylation, we ES07091.) investigated if human hepatic microsomes could metabolize 4,4'-dichlorobiphenyl (4,4'-DCB), a 76 EFFECT OF SENESCENCEON THE BIOACTIVATIONOF congener of low chlorination, but substituted in ALIPHATIC HALIDES. A.J. Gandolfi, A.B. DiRenzo, both para positions. Human hepatic microsomes J.N. McDougal, and I,G. Sipes. Departments of were prepared from livers obtained during resec­ Anesthesiology and Pharmacology, University of tion and were characterized with respect to Arizona, Tucson, AZ 85724. cytochrome P-450 (0.35±0.13 nmoles/mg), NADPH­ The disposition and metabolism of drugs and cytochrome c reductase (137±32 nmoles/mg/min), other xenobiotics is known to be altered in the benzphetami~e-N-demethylase (0.73±0.3 nmoles/mg/ aged. Since the toxicities of xenobiotics are min) and biphenyl-4-hydroxylase (0.94±0.15 often linked to their metabolism, this study nmoles/mg/min). Aqueous extractable metabolites focused on the effect of senescence on the bio­ were formed when 14c-4,4'-DCB (58 mCi/mmole) was activation and covalent binding of aliphatic incubated at 37°C under air with these micro­ halides to microsomal protein and lipid. Studies somes. The reaction was linear with respect to were performed using hepatic microsomes isolated protein concentration (0.25 to 1.0 mg/ml) and from Fischer 344 rats aged 4, 11, and 27 mo. time (0-90 min). The apparent Km and Vmax for The 14c labeled aliphatic halides studied were formation of aqueous extractable metabolites was 1,2-dibromoethane, carbon tetrachloride, tri­ 0.4 µMand 1.2 pmoles/mg/min, respectively. HPLC chloroethylene, and 1,1,2-trichloroethane. The analysis (C1s reverse phase; eluted with 50:44: 27 mo old rats were found to have significantly 5:1 MeCN:H20:EtOH:acetic acid) of the metabolite less hepatic cytochrome P-450 (-35%), cytochrome fraction revealed at least five peaks of radio­ b5 (-32%), NADPHcytochrome C reductase (-31%), activity, which probably represent distinct and ethylmorphine N-demethylase activity (-43%) metabolites of 4,4'-DCB. The major peak of radio­ when compared to the younger age groups. The activity co-eluted with synthetic 4,4'-dichloro- hepatic glutathione transferase activity was not 3-hydroxybiphenyl, which is also the major different between the different age groups but metabolite produced by the monkey and dog in microsomal UDP-glucuronyltransferase activity vivo. These results indicate that an unsubsti­ was elevated (+136%) in the 27 mo group. The tuted para position is not essential for hydrox­ hepatic microsomal bioactivation and covalent ylation of PCBs by human hepatic microsomes. binding of the aliphatic halides increased (Supported by ES-82130 and 5-T32-ES07091.) slightly between 4 and 11 mo; however, the microsomal bioactivation and covalent binding 75 DISPOSITION OF 1,2,3-TRICHLOROPROPANE IN RATS. decreased 50-75% in the 27 mo group. As reported R.F. Volp, D.E. Carter, and I.G. Sipes. Toxicol. by others, we found senescence decreased the Program, Univ. of Arizona, Tucson, AZ 85724. reactivity of hepatic microsomal biotransforma­ Trichloropropane (TCP) is an intermediate in tion enzymes. In addition, the microsomal the manufacture of pesticides and rubber and bioactivation of xenobiotics appears to be exhibits structural resemblance to so~e pesti­ suppressed indicating a possible decreased poten­ cides of recognized toxicity, e.g., dibromo­ tial for the expression of toxicities requiring chloropropane. The tissue distribution, excre­ bioactivation. (Supported in part by NIH tion, and metabolism of 14c-TCP were studied in Grant AG01289.) male Fischer 344 rats following an i.v. dose of 3.2 mg/kg. Within 1 hr 65% of radioactivity in 77 CHARACTERIZATIONOF THE UPTAKEOF ESTRADIOL-178- the blood was metabolites, which disappeared with D-GLUCURONIDEAND TAUROCHOLICACID IN ISOLATED an initial half-life of 3 hr and a terminal half­ RAT HEPATOCYTES. W.J. Brock and M. Vore, Grad. life of 8 days. TCP distributed rapidly into fat Center for Toxicology and Dept. of Pharmacology, and muscle; at 15 min, these tissues contained 30 University of Kentucky, Lexington, KY.

21 Studies from this laboratory have demonstrated its possible in vivo significance. that estradiol-176-D-glucuronide (E217G), but not This work supported in part by NIEHS 5 T32 estradiol-176-3-D-glucuronide (E23G), causes a 07062 and 5 D04 AH01661. dose dependent, reversible cholestasis, and a decreased bile acid secretion after its iv admin­ istration in the rat (JPET 214:87, 1980). Infu­ 79 FUNCTIONALINTEGRITY OF ISOLATED RAT HEPATOCYTES. sions of taurocholate (TC; 80 µmol/hr) completely C. E. Green, J.E. Dabbs, and C. A. Tyson, SRI protected against the cholestasis induced by E2- International, Menlo Park, CA 94025. Sponsor: 17G (11 µmol/kg, iv). These studies suggest that D.C.L. Jones. E217G and TC compete for transport at a single organic anion carrier site. This investigation Although suspensions of isolated hepatocytes have was designed to: 1) characterize the uptake of been used in a variety of studies on the metabo­ E217G, and 2) determine if E217G or the non­ lism and toxicity of foreign chemicals, it is cholestatic E23G competitively inhibit the uptake generally believed that the viability and func­ of taurocholate using isolated rat hepatocytes as tional integrity of such cell suspensions decline a model system. Hepatocytes were prepared from within a few hours. These studies were under­ female Sprague-Dawley rats (200-260g) by a colla­ taken to assess the stability of several liver genase digestion of the liver and were judged to functions in hepatocytes isolated by collagenase be 85-95% viable by trypan blue exclusion. The perfusion. Hepatocytes were isolated from adult rate of [3H]-E217G uptake was linear for approxi­ male Sprague-Dawley rats and were incubated at mately 2 min at concentrations of 5,10,50,100 and a density of 1.0 x 106 viable cells/ml of hormone 150 µM; kinetic parameters were Vmax = 2.9+0.3 supplemented Waymouth's medium in 5% C02-95% 02. nmol/min/mg protein and Km= 314+27 µM. The up­ During a 6-hour incubation period, the viability take of [3H]-TC was linear for approximately 3 (trypan blue exclusion) decreased from 86% to 77%.. min at concentrations of 2,5,10,20,50 and 200 µM; However, rates of urea synthesis, gluconeo­ kinetic parameters were Vmax = 0.94±_0.2 nmol/min/ genesis, and protein synthesis did not change mg protein and~= 16.4:!::_l.4 µM. However, Dixon significantly. The cytochrome P-450 content of plots indicated that neither E217G nor E23G, at the hepatocytes also remained stable from the concentrations of 1-300 µM, inhibited TC uptake. start of the experiments to 6 hours, 28.3 ± 7.6 These data therefore indicate that although E217G pmoles/µg DNA and 24.3 ± 5.5 pmoles/µg DNA, + and TC are both taken up readily by hepatocytes, respectively. The addition of cofactors (NADP they appear to be transported by distinct organic and NADPHgenerating system) to the suspension anion carriers. (Supported by HD13250). medium did not stimulate the N-demethylation of p-chloro-N-methylaniline (PCMA), indicating 78INDANOL DEHYDROGENASE(ID): SOMEPROPERTIES OF that the majority of cells had intact membranes THE ENZYMEFROM HUMAN PLACENTA. A.P. Kulkarni, even immediately after isolation. The rate of B. Strom and W. Houser. Toxicology Program, Dept. metabolism of PCMAdid decrease about 30% during of Env. & Ind. Hlth, The University of Michigan, the incubation, but the rates of 0-demethylation Ann Arbor, MI and conjugation of p-nitroanisole and the metabolism of benzopyrene remained relatively Some characteristics of ID isolated from full constant. Preliminary data suggest that hepato­ term placenta of non-smoker women were investi­ cytes prepared by biopsy perfusion, a method gated under in vitro conditions. ID converts 1- which can be used to isolate cells from larger indanol to 1-indanone in the presence of either species, had similar characteristics. This NAD+ or NADP+. The rates of generation of reduced work was supported by NIH GM28158-0l. pyridine nucleotides was monitored spectrophoto­ metrically at 37° as an increase in absorbance at 340 nm with time. The amount of 1-indanone formed 80 COMPARATIVERATES OF BENZENEMETABOLISM IN PRIMARY in the incubation media was estimated by gas HEPATOCYTECULTURES. S.A. Knadle, C.B. Salocks, J. liquid chromatography. Preliminary experiments on Nakashima, and J.L. Byard, Department of Environ­ the subcellular localization indicated ID to be mental Toxicology, Univ. of California, Davis, CA exclusively associated with the microsomal frac­ tion. Low activity observed with mitochondrial Since benzene is thought to be metabolically acti­ and cytosolic fractions may be due to contami­ vated in the liver, a comparison of metabolism nating microsomes. Therefore, washed microsomes rates in primary hepatocyte cultures from humans were used in the subsequent experiments. Narrow and laboratory animals may explain species differ­ substrate specificity of ID is evident from the ences in susceptibility to bone marrow toxicity. fact that only 1-indanol but not 2-indanol can Hepatocytes were obtained by biopsy perfusion and serve as a substrate. Although ID can utilize cultured in chemically defined, hormone-supple­ either NAD+ or NADP+ as a cosubstrate, the acti­ mented media in collagen-coated glass culture ves­ vity is 20-60% higher in the presence of NAD+ than sels sealed with Teflon-silicone discs. These cul­ NADP+. The range of specific activity of ID tures were found to metabolize and covalently bind (nmol/min/mg protein) varied between 9 to 12 for a single dose of aflatoxin Bi to the same extent NAD+ and 4 to 7 for NADP+ in different samples as cultures in unsealed plastic dishes. Further­ exam~ned. The rates of NAD(P)H and 1-indanone more, these cultures oxidatively metabolized glu­ formation were linear with respect to time, pro­ cose at the same rate as unsealed cultures. Non­ tein and substrate concentration. The kinetics cytotoxic doses of [14c]benzene were injected of ID to establish Km values for 1-indanol, NAD+ after 24 hrs of culture. The dose partioned be­ and NADP+was found to be complex and the experi­ tween air and medium with a coefficient of 0.23 ments in progress may shed some light on this (v/v) at 37°C. At time points from 2-50 hrs after problem. The purification of ID from human pla­ benzene addition, aliquots of medium were removed, cental microsomes and evaluation of substrate adjusted to pH 12, and unmetabolized benzene was specificity is planned in the future to establish extracted with ethyl acetate. Hepatocytes from

22 male S-D rats converted 20% of 2.3 µg and 0.23 µg presently available. To obtain uniformly 14c­ doses(l2.5 µg DNA/culture) to water-soluble meta­ labelled macrocyclic PAs from~- vulgaris, a CO2 bolites in 48 hrs by first order kinetics. Cul­ plant growth chamber was constructed. The CO2 tures from a CD-1 mouse (4.3 µg DNA/culture) meta­ chamber has a capacity of 80 3" pots, each con­ bolized only 3.3% of these doses in 48 hrs, also taining 4 plants. At 4 weeks plants were placed by first order kinetics. Triplicate cultures in the CO2 plant growth chamber, allowed to 14 13 (7.9 µg DNA/culture) from one human liver biopsy acclimate for 1 week and exposed to co 2 . co 2 metabolized 36% of a 2.3 µg dose and 66% of a 0.46 was also provided, and the amount of incorporation µg dose of benzene to water soluble metabolites in of each carbon was determined using 13C-NMR. To 48 hrs. These preliminary results suggest that in determine the optimum PA production by~- vulgaris humans benzene is metabolized more rapidly and the a number of plants were removed at selected inter­ metabolism becomes saturated at a lower dose than vals over a 3½ week period. The PA concentration in mice and rats. (Supported by a Monsanto Fund appeared to increase as the plants matured. 14 Fellowship and NIEHS Training Grant PHS (ES07059- In a following experiment, 25 mCi of co 2 was 03). given to 4 week old plants over a 6 hour ~eriod. Pyrrolizidine alkaloid production, total 4c ac­ tivity and specific activity of each PA were de­ 14 81 PLACENTALAND MILK TRANSFER OF THEORGANOPHOSPHATE termined over a 3 week period. The highest c incorporation in the PAS occurred 4 to 5 days INSECTICIDE,PARATHION, AND ITS DISPOSITIONIN FE­ 14 TUSESAND NURSING OFFSPRING. S.D. Weitman, M.J. post exposure to C02. The greatest amount of Vodicnik, A.A.R. Hamid, and J.J. Lech, Dept. of PA produced is senecionine which exhibits a Pharm. & Tox., Med. Col. Wisconsin,Milwaukee, WI. specific activity between seneciphylline and retrorsine. Senecionine appears to be a possible Existing data on the transfer of parathion to fe­ precursor of retrorsine as its per cent in the tuses and nursing offspring are limited. Studies plants decreases as retrorsine increases. with pregnant guinea pigs failed to demonstrate Using the above data, 800 mCi of 14co2 were the transplacental transfer of parathion. Further­ used over a 12 day period to maximize specific more, other investigators were unable to demon­ activity and yield. The average specific activ­ strate the presence of parathion in the milk of ity of the isolated PAs was 3.5 mCi/mM. (Supported dairy cows exposed to this chemical. However, by NSF 7806924.) since neonatal rodents have been suggested to possess increased susceptibility to this compound, we have studied the transfer and disposition of 14c-parathion in pregnant and 1actating mice. Par­ 83 THE IN VITRO COVALENTBINDING OF SENECIONINE AND athion was administered IP to 18 day pregnant or SENECIPHYLLINE TO MOUSE LIVER MICROSOMESAND DNA. nursing Sprague-Dawley mice at a dose of 3mg/kg in D. F. Eastman and H. J. Segall, Dept. of V. M. corn oil. Animals were sacrificed at various times Physic. Sci., Univ. of Calif., Davis, CA 95616 posttreatment and 14c activity determined in ma­ Pyrrolizidine alkaloids (PAs) are natural plant ternal, fetal and suckling tissues. Maximal 14c hepatotoxins and carcinogens. Plants and food activity in fetuses was observed at 0.5h following stuffs containing PAs are consumed inadvertently maternal treatment, reaching levels of 0.075µg and intentionally by both humans and animals. 14C-equivalents/g fetal tissue. Activity was still This study examined the in vitro covalent binding measurable 24h posttreatment and was higher than mediated by the cytochrome P-450 mixed function maternal blood, brain or kidney levels. In the oxidase system of two PAs, 14c-senecionine (SN) stomach contents of 5-day old nursing offspring of and 14c-seneciphylline (SY) to female BALB/c parathion-treated mothers, measurable amounts of mouse liver microsomal macromolecules, added calf 14c were detected as early as 1hr following mater­ thymus DNA and deoxyguanosine. nal treatment with maximal levels achieved at 6h Microsomal incubations followed standard proce­ (0.599µg 14C-equivalents/g milk coagulate). Levels dures. Ti-,e covalent binding was measured as the at 12h in urine, liver, and kidney in offspring radioactivity remaining in the 105,000 x g pellet were 1.79, 0.0017 and 0.0049µg/g tissue, respec­ or the precipitated DNA after exhaustive washing tively. Maternal levels were 5.4, 0.039 and 0.055 with methanol, diethyl ether and chloroform. µg/g in the corresponding tissues. Transfer of Covalent binding as measured in the complete parathion or its metabolites to the mouse fetus or system (including microsomes, DNA, cofactors and via milk may be significant due to both acute tox­ room atmosphere) was 606 pmole SY per mg DNA, 802 icity and increased susceptability of newborns to pmole SY per mg microsomal macromolecules, 347 parathion as well as to its proposed effects on pmole SN per mg DNA, and 509 pmole SN per mg perinatal development. Supported by HD13591 and microsomal macromolecules. In incubations using MOD15-9. boiled microsomes, an absence of NADPH generating system or an N2 atmosphere the covalent binding to microsomal macromolecules and DNA was reduced to 6 to 22% of the complete system. Incubations 82 ISOLATION OF 14C-MACROCYCLICPYRROLIZIDINE with 10- 3 M KCN had no effect on the binding of ALKALOIDS. C.H. Brown and H.J. Segall, Dept. the PAs to ether DNA or microsomal macromolecules. V.M. Physic. Sci., Univ. of Calif. Davis, CA Incubations with 14C-PAs and [1',2'-3H]-deoxygua­ 95616. nosine substituted for DNA produced one peak and Senecio vulgaris is a common perennial in the a possible second peak on a C1a reverse phase western United States which contains 3 macrocyc­ HPLC system which could be adducts. lic pyrrolizidine alkaloids (PAs), all hepatoxins These experiments indicate that the covalent and suspected hepatocarcinogens. Unfortunately, binding of PAs to liver macromolecules seen in obtaining uniformly 14C-labelled macrocyclic PAs vivo is probably mediated through activation---;-f for metabolic studies has been difficult as syn­ the PAs by the cytochrome P-450 mixed function thetic methods for some macrocyclic PAs are not system. (Supported by NSF 7806924).

23 84 IRREVERSIBLE BINDING OF ISOLATED PYRROLIZIDINE ( 1 mM) was used as the cofactor. The ALKALOID (SENECIONINE) METABOLITESTO MOUSELIVER findings suggest that MDA is metabolized MICROSOMALPROTEINS. L. J. Willis, H.J. Segall in vitro by lower Km cytosolic aldehyde and D. F. Eastman, Dept. V.M. Physio. Sci., Univ. dehydrogenase(s) which require NAD+. of Calif., Davis, CA 95616 Supported by NIAAA grant-03257. Pyrrolizidine alkaloids (PAs) are natural plant toxins found in a wide variety of plant species. Many of these are consumed by both humans and domestic animals and are considered a public 86STUDIES ON PHENOL-0-METHYLTRANSFERASEAND THIOL­ health hazard. Senecionine, a PA native to S-METHYLTRANSFERASE. P. T. Lin and W. C. Dauterman Senecio vulgaris plus other plants, causes liver Toxicology Program, Dept. Entomology, N. C. State necrosis and is a suspected carcinogen. This University, Raleigh, NC study examined the in vitro covalent binding of senecionine to various microsomal proteins to Phenol-0-methyltransferase from rat liver was determine if PAs bound to specific proteins. studied with regard to the stabilization and In an in vitro experiment, 14 C-senecionine (Sp. solubilization of this enzyme. Enzyme induction act. 3.5--;;;-Ci/mM) was incubated with female BALB/c of phenol-0-methyltransferase by phenobarbital mouse liver microS';'ITles plus an NADPH generating was demonstrated. A substrate specificity study system. After centrifugation at 105,000 x g for using 22 monophenolic substrates showed no 1 hour, the pellet was washed Sx with methanol positive correlation between structure and and 3x with diethylether. The protein binding activity. patterns of the senecionine metabolites were ex­ amined by SDS-polyacrylarnide slab gel and disc A comparative study of thiol-S-methyltransferases gel electrophoresis. The protein binding was present in the microsomes and the soluble measured as the percent of radioactivity relative fraction of mouse liver was conducted. Enzyme to protein intensity. activity in both fractions was partially purified All results indicate that there was a uniform and some kinetic parameters were investigated. binding to microsomal proteins with a possible increase of radioactivity binding at lower mole­ cular weights (<10,000 M.W.). Using a variety of 87 SODIUM AZIDE: BIOAVAILABILITY AND METABO­ acrylamide disc gels (10%, 12%, 15%) or slab gels produced the same effect. Boiling of the samples LISM IN RATS E. W. Lee, Biomedical Science Depart­ for 5 minutes with glycerol, mercaptoethanol and ment, General Motors Research Laboratories, Warren, SDS prior to loading the sample on the gels did MI 48090 not appreciably alter the binding pattern. Our previous investigation (Toxicologist, 1:21-22, 1981) reported the absence of azide in blood and the lack of 85 METABOLISM OF MALONDIALDEHYDE BY RAT toxicity of the daily dose of 23 mg/kg sodium azide (NaN ) in drinking water for 90 or 147 days in labora­ LIVER ALDEHYDE DEHYDROGENASE. J. J. 3 tory rats. This contrasts sharply with the lethal effects Hj~lle, J .H. Grubbs and D.R. Petersen, of a single dose of 42 mg/kg administered perorally. Dept. of Pharmacology, School of The bioavailability and in vitro metabolism of NaN Pharmacy, Univ. Of Colorado, Boulder, CO 3 was therefore investigated to identify the mechanisms 80309. Sponsor: C.D. Klaassen. responsible for the absence of toxicity of azide in rats. The peroxidation of hepatic mitochondrial Although azide was detected in the plasma as early as 5 and microsomal membrane lipids produces min after the oral administration (40 mg/kg), no azide malondialdehyde ( MDA) which undergoes was identified in both plasma and tissues 24 hours later. metabolism in vivo and in mitochondrial During the same period, no azide was excreted in the preparationsin vitro. The purpose of feces or exhaled in the air and only 7 .9 µg of azide was this investigationTs to assess the role excreted in the urine. Rat liver was found as the organ of hepatic aldehyde dehydrogenases in the responsible for the deactivation of azide since (1) in the metabolism of MDA. The cytosolic frac­ in vitro study, the liver homogenate rapidly destroyed tion contained the highest MDA metaboli­ NaN , whereas homogenates of other tissues did not, zing capacity as determined by MDA and rhin the in vivo study, the azide absorbed from the disappearance and by the rate of MDA gastrointestinal tract was metabolized as soon as it stimulated production of NADH. Two cyto­ reached the liver. At the maximum reaction rate, one solic aldehyde dehydrogenases, one with a milliliter of 20% liver homogenate metabolized 1.13 µg Km for MDA of approximately 10 µM and NaN:l per min. A peak level of azide in plasma, 49 another with a Km value of approx. 800 µM µg/m--i,, was detected after a 40 mg/kg dose but no had Vmax values of O. 88 and O. 59 nMol azide was found in the plasma with a bolus dose of 0.38 NADH produced/min/mg cytosolic protein, mg/kg; trace amounts were detected with 0.75 mg/kg. respectively. Minimal activity of either This indicates that a single oral dose of 0.38 mg/kg does enzyme was observed using NADP+. MDA not exceed the metabolic capacity of the liver. Also, stimulated production of NADH from NAD+ since the oral administration required less than 10 sec was completely abolished by the addition for the appearance of azide in plasma, the calculated of the aldehyde dehydrogenase inhibitor rate of sodium azide absorption from the gastrointes­ disulfiram (10- 5M). Mitochondria con-­ tinal tract after intake of 23 mg/kg/day was equivalent tained an enzyme with Km values for MDA only to 0.0027 mg/kg/10 sec. This intake rate is well of 7 mM and 31 mM using the cofactors below the metabolizing capacity of the rat liver tissue, NAD+ and NADP+, respectively. The Vmax and it is therefore not surprising that no azide was activity per g liver of the mitochondrial detected in the plasma and no hematological or histo­ enzyme was approx. 70 times less than pathological abnormalities were observed in the experi­ either of the cytosolic enzymes when NAD+ mental animals with this dose level.

24 88 DISPOSITION OF DIMETHYLAMINE(DMA) IN F-344 RATS strain. Two additional regions of 14c activity AFTER INHALATIONEXPOSURE. M. J. McNulty and H. have been tentatively identified through HPLC d'A. Heck, Chem. Ind. Inst. of Tox. Res. Triangle and alumina column chromatography as conjugates Park, NC 27709 Sponsor: D.E. Rickert. of the free metabolites. In the non-extractable tissue residue from both strains, the 14c acti­ DMAis a gas used in a number of diverse vity was primarily associated with the lipid and industrial applications. The current recommended protein fractions, although some activity was threshold limit value for DMA (10 ppm) produced associated with RNA, DNA, glucosaminoglycan, and no measurable toxicity in F-344 rats (6 hr/day, 5 carbohydrate. Findings to date indicate that days), but focal ulceration and necrosis of the the major difference in the metabolic profiles of respiratory epithelium were induced at 175 ppm MC in C57BL/6 and DBA/2 mice is in the level of using the same treatment schedule (Swenberg, J. metabolism and not in the types of metabolites A., and Barrow, C. S.; unpublished observations). produced. (Supported by NIEHS Grant ES07073 and No information has been reported on the disposi­ The Ohio Coal Laboratory Research Association). tion and metabolism of DMAf!ter 'inhalation. Male F-344 rats were exposed to C-labeled DMAgas in head-only exposure chambers for 6 hours at concen­ 90 Effect of Increasing Levels of Dietary Protein on trations of either 10 ppm or 175 ppm. At each the Metabolic Activation and Covalent Binding of dose, the excretion of DMA-derived radioactivity Aflatoxin B1. J.R. Hayes, Co. McCall, D.M. into urine, feces, and co was determined during Bagley. Dept. Pharmacol., Med. College of Va., 2 a 72 hour post-exposure period. The disposition Va. Commonwealth Univ., Richmond, VA. 23298 of radioactivity into various tissues was also investigated. The majority of recovered radio­ Low dietary p_roteh, levels (5%) decrease the activity appeared in the urine (76% at 10 ppm; hepatocarcinogenicity of aflatoxin B1 (AFB1), 86% at 175 ppm) and feces (approximately 5 to 10% in part due to a decrease in hepatic microsomal for each group). Less than 4% of the recovered mixed function oxidase (MFO) activity, which ac­ radioactivity appeared in the expired air as CO 2 tivates AFB1 to its ultimate carcinogen. Al­ suggesting only slight oxidative metabolism. though low levels of dietary protein are consumed Approximately 7% of the label remained in the by certain populations, others may consume pro­ tissues and carcass 72 hours post-exposure. tein above that required for maintenance. This Concentrations of radioactivity were highest in study was designed to ascertain the effects of the tissues of the respiratory tract (nasal increasing levels of dietary protein on the meta­ mucosa, trachea, and lung) as well as the liver, bolic activation of AFB1. Male, weanling kidney, and intestine. The nasal mucosa contained Sprague-Dawley derived rats were fed the AIN-76 the highest deposition of radioactivity, most semi-purified diet with casein levels of 5, 10, probably due to the high water solubility of DMA 20, 30 and 40% for 10 days. Body weight in­ and the fact that the nasal mucosa is the initial creased until protein levels reached 20% and then point of entry of the gas into the rat. Results became asymptotic. Liver weights were higher suggest that in the rat saturation of metabolism only in the 5 and 10% groups due to lipid infil­ and excretion is not achieved at 175 ppm DMA, and tration. Microsomal protein increased with di­ that oxidative metabolism of DMAis minimal. etary protein up to 20% and then became asymp­ totictic. N-demethylation of ethyl-morphine (EM) increased until dietary protein levels reached 30% and then became constant. Cytochrome c re­ 89 IN VIVO METABOLISMOF 3-METHYLCHOLANTHRENE(MC) ductase activity and cytochrome P-450 levels IN PREGNANTC57BL/6 AND DBA/2 MICE. J.D. George, followed a trend identical to EM metabolism. In J.M. Manson, S. Vater, and D. Warshawsky, Dept. vitro metabolic activation of AFB1 to a pro­ Envir. Hlth., Univ. of Cincinnati, Cincinnati, duct(s) Cdpable of covalently binding DNA in­ OH. SPONSOR: P.B. Hammond creased until a level of 30% protein was reached and then became asymptotic. In vivo covalent MC has been linked to carcinogenesis in labora­ binding of an AFB1 metabolite(s) to hepatic tory animals and their transplacentally exposed DNA, RNA and protein followed the same trend as offspring. Activation of MC to reactive inter­ the in vitro studies. Inappropriately low levels mediates is catalyzed by microsomal aryl hydro­ of dietary protein may be protective in respect carbon hydroxylase (AHH), the activity of which to AFB1 carcinogenicity, but high levels may correlates positively with tumorigenesis. This increase risk. study compares the in vivo metabolic profiles of (This study was supported by Grant IN-105E from MC in the maternal and fetal tissues of C57BL/6 the American Cancer Society) (high AHH) and DBA/2 (low AHH) mice. Maternal liver, placenta, and whole fetuses from pregnant mice exposed to 14c-MC were evaluated for extrac­ table- and bound 14c activity. HPLC analyses of 91 IS THEREA LOW-DOSETHRESHOLD FOR HEPATIC MACRO­ ethylacetate extracts revealed no qualitative MOLECULARBINDING OF AFLATOXINB1? B. S. differences in the spectrum of metabolites,,.:\:otirid Appleton, M. P. Goetchius and T. C. Campbell, in the tissues of either strain of mouse; ·_se·ve'n Div. of Nutritional Sciences, Cornell Univer­ metabolites, in addition to MC, have been id,enti­ sity, Ithaca, NY. fied. In both strains, the placenta appear~ to be acting as a reservoir for the parent' 0 c~pound. The possibility of a glutathione dependent The free MC metabolite profiles of the twd '; dose threshold for rat liver macromolecular bind­ strains of mice differ quantitatively for all ing of aflatoxin B1 (AFB1) was investigated. In tissues studied. Tlssues from DBA/2 mice con­ Experiment I, matute rats were given 6 dosage tained higher relative levels of MC, which is levels of [ 3 H]-AFB to determine if a dose consistent with the lower AHH activity of this threshold for macr6molecular binding would be

25 observed. In Experiment II, immature rats were 93 TOXICOLOGICAND CARCINOGENICEVALUATION OF used to determine whether or not the extent of FENVALERATEIN THE B6C3Fl MOUSE. C. M. Parker macromolecular adduct formation at low doses and C. B. McCullough, Toxicology Research, Shell could be modified by depleting hepatic gluta­ Development Co., Houston, TX, J.B. M. Gellatly, thione with diethyl maleate pretreatment. Shell Toxicology Lab. (Tunstall), Sittingbourne, Nanogramper kilogram doses of radiolabeled AFB1 Kent, UK and C. D. Johnston, Litton Bionetics, produced measurable covalent binding of Inc., Kensington, MD Sponsor: G. A. Van Gelder aflatoxin to DNA,RNA and protein, and the extent of this binding increased linearly over a Fenvalerate is a synthetic pyrethroid used for dose range of 0.01-1.00 µg/kg. Macromolecular broad spectrum insect control on cotton, fruit, adduct formation was observed at the lowest dose and vegetable crops. Groups of SO male.and fe­ used (10 ng/kg) which is within the human male B6C3Fl mice were fed dietary concentrations exposure range. Rats whose liver glutathione of 10, SO, 250, or 1250 ppm Fenvalerate for two was decreased from 5 mg/g of liver to 2.3 mg/g years. Two groups of control mice, 50 per sex of liver by diethyl maleate pretreatment showed per group, received basal diet only. Mortality only a small and inconsistent increase in was increased and body weight was significantly macromolecular adduct formation. Moreover, this decreased in male and female mice in the 1250 ppm increase was independent of AFB1 dose and was treatment group. Mean body weight of female mice more associated with the amount of AFB1 found in in the 250 ppm group was also generally lower the liver homogenate rather than with the than controls after the 60th week of feeding. intracellular events presumably involved in Decreased ALB and increased GOT levels in mice glutathione conjugate formation. These results fed 1250 ppm Fenvalerate were the only effects indicate that macromolecular binding of AFB1 is observed in the hematology and serum chemis~ry essentially a linear function of dose at low parameters examined. Mean organ weights and exposures and that hepatic glutathione activity organ/body weight ratios of brain, heart, liver, has little or no role in that binding. kidneys, and adrenal glands were not affected by (Supported by American Cancer PT 104 and USPPHS treatment. The only non-neoplastic pathology POl CA26755.) observed in the study was multifocal granulomata in lymph nodes, liver and spleen of 1250 ppm male mice and 250 ppm and 1250 ppm female mice. Less 92 SUBCHRONICDIETARY TOXICITY STUDIES ON 2-CHLOR0- severe granulomatous changes were present in 6-(2-FURANYLMETHOXY)-4-(TRICHLOROMETHYL)PYRI­ mesenteric lymph nodes of 50 and 250 ppm male DINE, (DOWCO*444), A DEVELOPMENTALFUNGICIDE, mice. No statistically significant·differences IN RATSAND DOGS, Jersey, G.C., Gorzinski, were observed in either the number or type of S.J., Barna-Lloyd, T., Quast, J.F., Tollett, neoplasms in mice fed Fenvalverate diets when. J.T. and Schwetz, B.A., Health & Environmental compared to concurrent controls. Thus, Fenva­ Sciences, DowChemical U.S.A., Midland, MI lerate was found not to be carcinogenic in B6C3Fl 48640 and Lake Jackson, TX 77566, mice at a maximum tolerated dose under the conditions of the test. To assess systemic toxicity, groups of male and female rats were fed diets supplying DOWCO444 at dose levels of O (control), 1, 5, 15 or 30 94 THEASSOCIATION OF ORALLY-ADMINISTEREDCAPTAN mg/kg body weight/day for 13 weeks (12/sex/ WITHMOUSE DEOXYRIBONUCLEIC ACID. C. A. Sel sky group) or for 16 weeks (8/sex/group). Those fed and D. W. Matheson, Stauffer Chemical Company, for 16 weeks were held on recovery for an addi­ Environmental Health Center, Farmington, CT tional 8 weeks. No effects attributable to 06032. Sponsor: R. I. Freudenthal . treatment were found in rats fed 1 mg/kg/day for 13 weeks, or in any experimental group after the Captan increases the incidence of duodenal tu­ 8-week recovery period. In rats fed for 13 mors when chronically administered at relative­ weeks, microscopic changes (increased hepatocel­ ly high concentrations in the diet of CD-1 mice. lular cytoplasmic microvacuolation and centri­ DNAwas isolated from various organs of male lobular swelling) were found in males and CD-1 mice removed 24 hrs. after oral administra­ females at 15 and 30 mg/kg/day and in some tion of 14C-captan, to determine if a difference females at 5 mg/kg/day. The hepatic changes in binding to DNA occurred between target and were reversible based on the 8-week recovery nontarget organs. Captan was administered. at results. doses of 1600 mg/kg (specific activity 1.9 and 0.19 mCi/mmole) and 156 mg/kg (56 mCi/mmole). DOWCO444 was also fed to male and female dogs DNAwas quantitated by absorption at 254 nm. (6/sex/group) at dose levels of 0, Q.05, 0,5 or 14c-radioactivity was quantitated by scintilla­ 5 mg/kg body weight/day for 6 months. Effects tion spectrometry. No radioactivity was detec­ of treatment, found only in dogs fed 5 mg/kg/day ted associated with the DNAisolated from the were: increased liver weights in males and fe­ duodenumand testes of mice administered 1600 males, increased alkaline phosphatase levels in mg/kg at either specific activity. · Radioactiv­ males but not females, increased relative kidney ity was detected associated with DNA isolated weight in females, and decreased RBCcount in from the duodenum, nonduodenal intestine, liver, males·. No hi stopatho l ogi ca 1 1es ions c 1early stomach, kidneys, and testes of mice adminis­ corresponding to the increased liver or kidney tered 156 mg/kg (specific activity 56 mCi/ weights were found. Based on these studies, the mmole). The number of trichloromethyl carbon liver is considered to be the primary target atoms per DNAnucleotide was calculated and organ in rats and dogs. The no-observable­ ranged from 5 x l o-6 (testes) to 4. 2 x l o-5 effect level (NOEL)in rats was 1 mg/kg/day (liver). DNAwas subjected to neutral dialy­ and in dogs 0.5 mg/kg/day. sis. Up to 90% of the associated l 4C-radi oac­ *Trademark of The DowChemical Company. t i vi ty was dialyzable depending upon the DNA

26 sample. DNA samples were also centrifuged to first observed in significant numbers at 18 months. equilibrium in neutral CsCl buoyant density gra­ Cystic endometrial hyperplasia (78%) and splenic dients. The gradients indicate that some of the extramedullary hematopoieses (56%) were present associated 14c-radioactivity in testicular DNA from puberty onward. The tumor incidence for all is due to covalent interaction. No other tissue groups was within the range of published data. The DNAdemonstrated a covalent component. The ma­ time course of development. of these lesions was jor association in these other tissues appears similar in control and test groups. Based on the to be ionic. Therefore, except for binding in results of this study, it was determined that Etri­ the testes (nontarget) no differences were de­ diazol was not carcinogenic in CD-1® mice. tected between target and nontarget tissues.

97 THE ROLE OF HYDROGEN PEROXIDE IN MICROSO­ MAL LIPID PEROXIDA TION. L.A. Morehouse, M. 95 A DERIVEDCHRONIC T0XICITY-0NC0GENICITY STUDY Tien, J.R. Bucher and S.D. Aust, Dept. of Biochemis­ OF MYCELIALM0NENSIN SODIUM IN THE RAT. L.C. try, Michigan State University, East Lansing, MI 48824 Howard, M.N. Novilla and E.R. Adams (Sponsor, J.L. Emmerson). Toxicology Division, Lilly Aerobic organisms, during enzymatic hydroxyla­ tions and oxidations of numerous endogenous sub­ Research Laboratories, Greenfield, IN. strates and some xenobiotics, generate reactive and Groups of 100 male or female Wistar rats, potentially toxic, partially reduced forms of oxygen. derived from parents given diets containing Accumulation of these potentially toxic species, which monensin sodium (MS), were maintained for two could result in peroxidative damage of cellular mem­ years on diets containing 0, 33, 50 and 80 ppm branes, is prevented by enzymes such as superoxide dismutase, catalase, and g.'utathione peroxidase, which MS activity as mycelial MS (1.4-5.0 mg MS catalyze either the further reduction or the dismuta­ activity/kg/day). The reproductive capacity of tion of the potentially toxic biproducts. the parents, including fertility, litter size, Hydrogen peroxide can react with reduced transi­ gestation length and survival, and progeny tion metals generating the highly reactive hydroxyl survival and sex distribution was unaffected. radical (-OH), most often proposed as the predominant Body weight gain was decreased by MS treatment initiating species of microsomal lipid peroxidation. In for both parents and their offspring during order to assess the potential involvement of •OH, postpartum development. In the two year study, generated from hydrogen peroxide, in microsomal lipid body weight gain was decreased during the peroxidation, we have altered the concentration of initial two weeks of the study. Survival was microsomal hydrogen peroxide and measured the resul­ increased in a dose-related manner. Observa­ ting rates of lipid peroxidation. Hydrogen peroxide tions of physical signs of toxicity and concentration in microsomes was changed by adding evaluation of hematology, clinical chemistry, exogenous catalase, washing to reduce endogenous urinalysis and organ weight parameters did not catalase and glutathione peroxidase activities, and reveal effects related to MS administration. inhibiting endogenous catalase activtiy with azide in Inflammatory and degenerative conditions were either the presence or absence of exogenous hydrogen found to occur randomly in control and MS peroxide. In only one instance was the rate of lipid treated rats. The latency and prevalence of peroxidation, as measured by rnalondialdehyde forma­ benign and malignant neoplasms were similar in tion, affected; exogenous hydrogen peroxide added to control and MS treated rats. In conclusion, microsomes, previously incubated with azide, inhibited the continued exposure of rats to diets lipid peroxidation, the opposite effect from that containing up to 80 ppm MS activity during~ predicted if -OH, generated from hydrogen peroxide, is utero development and throughout their lifetime actually the major initiating species. Neither these produced neither chronic toxicity nor carcino­ results, nor the inability of known •OH traps to inhibit genicity. microsomcd lipid peroxidation, support the prevalent theory for the initiation of microsomal lipid peroxida­ tion. (Supported in part by NSF Grant No. PCM-79- 96 · ONCOGENIC BIOASSAY OF 3-TRICHLOROMETHYL- 15328) 5-ETHOXY-1,2,4, THIADIAZOLE, ETRIDIAZOL, IN CD-1® MICE. E. F. Erker, L. J. Slaughter, F. Sperling and W. L. West, Depts. of Pharmacology& 98 THE CYTOCHROMEP-450 DEPENDENTMONOOXYGENASE Pathology, Howard University, Med. Sch., Washing­ SYSTEMFROM THE LIVER OF UNINDUCEDMICE. ton, D.C. 20059 & J. Wedig, Olin Corp., New Haven, P. Levi, G. Bissette, and E. Hodgson. Toxicology CT 06511. Program, Department of Entomology, North Carolina The fungicide, 3-trichloromethyl-5-ethoxy-1,2,4, thia­ State University, Raleigh, NC. diazole, Etridiazol, was bioassayed using individually housed CD-1® mice. Approximately 60 post weaning Most previous studies of purified cytochrome mice of each sex were begun in each of the following P-450 used as starting material the livers of groups: Naive and Vehicle Control (Corn Oil added) animals pretreated with a variety of inducing and 320, 640, 1280 ppm Etridiazol in the diet. Six agents. The present study utilized the liver and 12 month protocol sacrifices were performed. At from uninduced mice. Liver microsomes were 18 months, one half of the mice were sacrificed. The solubilized with sodium cholate and purified by remaining mice were fed on naive control food for column chromatography using phenyl sepharose, three months and were sacrificed. GLP was DEAE cellulose, and hydroxylapatite. At least followed. Reversible dose related increases in Liver two forms were purified to a specific content of weight and organ/body weight ratios were observed in 18-20 nmoles/mg protein, and it is apparent from the absence of gross or microscopic lesions. Alveo­ ion exchange chromatography and SDS gel electro­ genic adenomas (11.4%) and cacinomas of the lungs phoresis that there are more than two forms. (28%), the most commonly occurring tumors, were NADPHcytochrome P-450 reductase, molecular

27 weight 79000, was purified by column chromato­ urea nitrogen, decreased accumulation of p-aminohip­ graphy using DEAEcellulose and 2'5'ADP Sepharose puric acid by kidney slices, and increased accumulation 4B. The cytochrome P-450 monooxygenase system of tetraethylammonium by kidney slices. Thus, lung, was reconstituted by combining these components liver and kidney are each affected by this MCT treat­ with phosphoiipid. Current studies involve the ment regimen, and functional effects on lung precede activity of the reconstituted system towards effects on other tissues. (Supported by NIH Grant various substrates, changes in the cytochrome ES01861.) P-450 profile after induction by specific inducers, e.g., isosafrole, and the inhibition of cytochrome P-450 by methylenedioxyphenyl 101 EFFECTSOF POSTNATALTRIETHYLTIN (TET) ON RADIAL­ compounds. ARM-~.AZEBEHAVIOR IN THE JUVENILERAT. D. B. Miller and L. W. Reiter, Neurotoxicology Division, Health Effects Research Laboratory, US Environ­ mental Protection Agency, Research Triangle Park, 99 A COMPARATIVEKINETICS STUDY OF MONOCHLORAMINE ANDCHLORINE IN RAT. M.S. Abdel-Rahman, D.M. North Carolina 27711. Waldron, and R. J. Bul1, CMDNJ-NewJersey Medical Postnatal exposure to TET results in permanent al­ School, Newark, N.J. 07103 teration in brain function as evidenced by hyper­ activity in both the juvenile and adult rat. In The problem of trihalomethane formation now the present experiment the effects of postnatal exists with the use of chlorine (HOCl) as a dis­ TET on the learning and memory capabilities of infectant in drinking water. Monochloramine the juvenile rat were assessed with an automated (NH2Cl) may be considered as an alternative to radial-arm-maze. The maze consists of a center HOClas a disinfectant in public water supplies. area from which 8 arms radiate; a food pellet is This study was conducted to compare the kinetics available at the end of each arm. The most eff­ between NH236c1and HQ36c1in rats. Radioactiv­ icient performance consists of entering an arm ity was absorbed from the gastrointestinal tract only once to obtain the pellet; any further arm following the administration of NH236c1(1.9 µCi) entries are considered as errors. Photocells in or HQ36cl (0.7 µCi) orally and the peak plasma various maze locations provide an index of act­ levels of 36c1 occurred at 4 and 2 hrs. for ivity. Long-Evans hooded rat pups were injected NH236c1and Ho36c1 resgectively. The distribu­ i.p. on postnatal day 5 with 0, 3, or 6 mg/kg of tion of NH236c1 and Ho36c1 was highest in plasma TET bromide; all 8 pups in a litter received the followed by whole blood, testes, skin, bone same dose. Beginning at 37 days of age rats were marrow, stomach and kidney, while the lowest .'given a daily 10-min session in the maze for 15 activity was observed in the fat. Subcellular consecutive days. Rats were allowed to make arm distribution in liver fractions after 24 hrs. selections throughout the session. TET-treated from the administration of NH236c1 and Ho36c1 rats initially made more errors but were at con­ revealed that about 72%of 36Cl was detected in trol levels by the end of the 15 days. On day 1 the cytosol fraction. The half-life for 36Cl of training the TET-treated rats did not differ absorption was 1.42 and 2.16 hr-1. while the from controls in the number of photocell counts elimination from plasma was 38.8 and 44.l hr-1 but by day 3 were more active; this hyperactivity after oral NH236c1and HQ36c1treatment respec­ persisted for the remainder of testing. This tively. Urinary excretion accounted for most hyperactivity is consistent with our previous of the 36cl eliminated in both treatments, how­ findings. In addition, the observed accuracy ever 7 and 29%of the total 36c1 excreted in differences suggest that postnatal TET adminis­ feces were observed after NH236c1and HQ36cl tration affects learning capability. administration. No 36Cl was detected in expired air throughout the 120 hrs. studied. (Supported by U.S.E.P.A.) 102 MICIOlAVE INDUCEDrnANGFS IN CANALICULARMEMBRANE FillCI'IOO OF THE RAT. D.G. Lange, M.E. D'Antuono, and J.M. Fuji.rroto. Research Services, VA Madical 100 INFLUENCE OF INGESTION OF MONOCROT ALINE Center, VKX:ld,WI, and Dept. of Pharmacology and BY RATS ON LUNG, LIVER AND KIDNEY FUNCTION. Toxicology, Madical College of Wis. , Milwaukee, R.A. Roth, B. Barayni, C-H. Kuo, L.A. Dotzlaf and J.B. wr. Hook. Department of Pharmacology and Toxicology Acute exposure of phenobarbital anesthetized nale ~Center for Environmental Toxicology, Michigan Sprague-Dawley rats (225-350g) to 2.45 GHz micro­ State University, E. Lansing, MI 48824. wave irradiation (120rrW/an2, 30 min) produced a 30 rise in core body t.errg;ierature, and signifi­ Young, male rats were treated with monocrota­ cantly increased (22.7%) the arrount of 3H-sucrose line (MCT) ad libitum in the drinking water (22 g/ml) recovered f:t:un the biliary tree following its in­ for up to 28days, and the development of toxicity in fusion into the biliary tree by a segmented retro­ lung, liver and kidney was examined. Clearance of grade intrabiliary injection (SRII). Similarly, perfused 5-hydroxytryptamine by isolated lungs of the recovery of 14c-mannitol administered by SRII treated rats was decreased as early as 14 days. This decreased by ll.7%. Animals exp::,sed to an equiva­ was a<:companied by increases in lung/body weight ratio lent thennal load fran a radiant heat source had and in lactate dehydrogenase activity in cell-free bron­ increases in both 3H-sucrose (70. 7%) and 14c-roan­ chopulmonary lavage fluid. Hypertrophy of the right nitol (31. 7%) recoveries following SRII. These heart and increased inflow perfusion pressure of isolat­ changes in SRII recoveries returned to control ed lungs were apparent by 21 days of MCT treatment. values when the animals returned to norrrothermia. After 28 days of treatment biliary indocyanine green Chronic exposure (4 days) to the above thermal excretion was decreased and plasma glutamic pyruvic loads, by micro.vave irradiation and radiant heat, transaminase activity was slightly elevated. Twenty­ resulted in irreversible changes in canalicular eight days of treatment also resulted in elevated blood membrane function, in norrrothermic animals. In

28 chronic microwave exposed anirnals, 3H-sucrose re­ The response could be eliminated by a rat liver covery increased 95.1% and 14c-mannitol recovery _microsomal preparation (S-9) independent of the increasa:l 50.0%. In anirnals chronically exposed addition of NADor NADP. To explore this inacti­ to radiant heat, only 3H-sucrose recovery in­ vating phenomenon the metabolism of DMP by rat creased (46.0%). Following acute exposure to l:oth 'liver was examined by means of a TLC-spectrophoto­ microwave and radiant thennal loads, bile flow metric assay. Since skin is a potential target rate increased in a tenq;Jerature dependent fashion. organ for this topically applied repellent, meta­ After chronic thennal exposure, bile flow rate bolism by this organ was also studied. Monomethyl­ decreased between 19.9 to 32.2%. Thus, chronic phthalate (MMP) was the predominant phthalate met­ exposure to thennal loads produced irreversible abolite formed from DMP incubation with liver S-9. alterations in the function of the hepatic cana­ MMPwas found to be non-mutagenic in the Ames licular membrane function, which were not a re­ assay. Thus, a mono-est erase activity of S-9 would sult of changes in bile flow rate. In addition, appear to be the cause of the inactivation of the the chronic microwave exposure produced greater mutagenic response of DMP. When the in vitro rates changes in canalicular membrane function than an of MMPformation by liver and skin were compared equivalent thennal load from a radiant heat on a mg wet weight basis, there was a 60-fold source. Supported by NIEHS Grant FS02006. greater rate in the hepatic tissue. Additionally, methanol was assayed by means of a modified Nash procedure and was found to be produced in equimo­ 103 HEPATORENALTOXICITY OF DBCP INGESTED IN DRINKING lar amounts to MMP. Many mutagens have been shown WATER. G. Kyle, R. Luthra, J.V. Bruckner, A.S. to bind readily to macromolecules. Since liver and Berkowitz, and J.J. Heindel, Dept. Pharmacol., epidermis have markedly different capacities to Div. Tox. and Dept. Repro. Med. & Biol., Univ. metabolize DMP to non-mutagenic forms, the in Vitro Texas Med. Sch., Houston, TX 77025 binding of carbonyl 14c-labelled DMPwas compared ·in these two tissues. DMP bound to epidermal It is now recognized that 1,2-dibromo-3-chloro­ ~acromolecules at a rate of 0.98 nmoles per mg TCA propane (DBCP) can produce liver and kidney injury precipitate, a rate that was 5 fold greater than_ in animals, as well as testicular damage and car­ that in liver. Thus, the lower rate of de-esteri­ cinogenesis. The following experiments were car­ fication of DMP in the skin relative to liver is ried out to assess the hepatorenal injury poten­ correlated with the higher rate of binding to DMP tial of the chemical when it is present as a water in the epidermis and thus suggests a mutagenic/ contaminant. DBCP was added to the drinking water carcinogenic hazard of this compound in skin. of adult male s.-D. rats in concentrations of 5, Supported by NIEHS grants 00454 and 07067. 50, 100 and 200 ppm for up to 60 days. Water con­ sumption and body weight were monitored weekly. Hepatic damage was assessed by measuring changes in morphology and in levels of the enzymes arginine succinate lyase, ornithine carbamyl transferase (OCT), sorbitol dehydrogenase, glutamic-pyruvic 105IDENTIFICATION, PURIFICATION, AND CHARACTERIZATION transaminase and glutamic-oxaloacetic transaminase OF NON-SELENIUMGLUTATHIONE PEROXIDASE FROM in serum. Nephrotoxicity was assessed by measuring GUINEA PIG LIVER MICROSOMES. J. R. Burgess, BUN, renal cortical slice transport of organic C. C. Reddy, C.-P. D. Tu, C. Y. Ho, R. W. Scholz, ions, and excretion of maltase and alkaline phos­ and E. J. Massaro, The Pennsylvania State phatase (AP) in urine. Substantial decreases in University, University Park, PA 16802. water consumption and body weight were seen in animals drinking water containing 200 ppm DBCP. Glutathione-s-transferases (GSH-Trs) comprise Depression in body weight gain was initially a group of multifunctional enzymes involved in the manifest 3 weeks into the study. Increases in biotransformation/detoxification of a broad levels of OCT in the serum were observed as early spectrum of xenobiotics. They are present in the as one week in animals consuming 100 or 200 ppm cytosol of most cells and in some cases microsomal DBCP. Significant changes in OCT were also ob­ fractions. Recently, attention has been drawn to served at the lower dosages after one month of the the peroxidase (Px) activity associated with the regimen. Kidney injury, as reflected by inhibition cytosolic GSH-Trs; this activity has been impli­ of transport of organic ions in renal cortical cated in the protection of membranes from damage slices, was seen within 1 week of consumption of due to lipid peroxidation. However, lipid peroxi­ water containing 100 or 200 ppm DBCP. No signifi­ dation occurs in the hydrophobic vicinity of cant changes in levels of AP or maltase in the membranes and these areas are not readily acces­ urine were detected during the initial two weeks. sible to the cytosolic enzymes. For this reason, Findings to date indicate that DBCP, even when in­ membrane associated GSH-Px activity may be of gested in very small amounts in divided doses, can special importance. We have monitored the GSH-Px produce adverse effects in the liver and kidneys. activity in pure microsomes isolated from rat and (Supported by EPA Contr. 68-01-4839 & NIEHS ES07090) guinea pig tissues. Of the various tissues screened, liver microsomes exhibited significant GSH-Px activity-guinea pig liver having approx­ imately 3 times greater activity-and only marginal 104COMPARISONOF THE METABOLISMAND BINDING OF DI­ activities were observed in kidney microsomes. A METHYLPHTHALATEIN SKIN ANDLIVER. W.J. Kozumbo, protein exhibiting both GSH-Tr and GSH-Px activity R.J. Rubin and R. Kroll. Dept. Env. Hlth. Sci., was isolated from guinea pig liver microsomes and Johns Hopkins Univ., Baltimore, MD 21205 purified to homogeneity by DEAE-cellulose ion ex­ change and s-hexyl glutathone-sepharose 6B affin­ Cbr laboratory has previously shown that di­ ity chromatography. Seperation of several forms methylphthalate (DMP), a widely used insect re­ was accomplished through the use of chromato­ pellent, produced a positive dose-related mutagen­ focusing and CM-cellulose chromatography. ic response in the Ames TAl00 bacterial strain. Different forms of GSH-Trs from guinea pig liver

29 were characterized by determining their substrate under these conditions. Using glucose oxi dase and specificity, isoelectric points and SDS gel glucose to generate H202 in the presence of electrophoretic pattern. (supported by EPA the heme proteins produces cross-linking and grant# R. 807746 and NIH grant# ROl ES02678-0l.) adduct fonnation. Free hemin has been found to catalyze the cross-linking and benzo(a)pyrene adduct formation of nonhemeproteins in the pres­ 106 CHARACTERIZATIONOF THE FLUOROACETATEDEFLUORI­ ence of H202. This work emphasizes the bio­ NATION ACTIVITY IN MOUSE LIVER. Andrew I. logical need for peroxidases and catalases to Soiefer and Paul J. Kostyniak. Department of Pharmacology and Therapeutic~, SUNY at Buffalo, protect oxygen carrying electron transport heme School of Medicine, Buffalo, N.Y. 14214 Sponsor: proteins and possibly non-hemeproteins from I w. Clarkson . interaction with peroxides of exogenous or Recent studies have identified a defluor­ ination system in the 105,000g fraction of rat endogenous origin. liver which required glutathione (GSH) for the liberation of free fluoride ion from fluoroacetate (FAc). The present investigation reports an analogous defluorinating system in 108\XENOB I OT I C-1 NDUCED HYPOFUNCT I ONAL PSEUDO­ the 105,000g supernatant fraction of mouse liver HYPERTROPH IC ROUGH ENDOPLASMIC RETICULUM with three to five times the specific activity seen in the rat. The defluorinating activity IN RAT LIVER. C. Difonzo, R.A. Martin, was dependent upon GSH concentration in the G. Feuer, J.M. Sturgess and F.A. de la assay, with peak activity observed above 5mM Iglesia, Warner-Lambert/Parke-Davis Pharm. GSH. The reaction was further characterized as having a pH optima of 8.2 and an apparant Km of Res. Sheridan Park, Miss., Ont. Canada, and 5mM with FAc as substrate. In Tris-HCl the Ann Arbor, Ml; Dept. of Clin. Biochemistry, defluorinating activity was found to be labile Univ. of Toronto, Toronto, Canada when stored at 4 degrees C. The addition of dithiothreitol, GSH, or 2-mercaptoethanol (2-ME) stabilized the activity, with the latter To explore the hypothesis that hypertrophy providing concentration dependent protection for of smooth endoplasmic reticulum (HHSER) is greater than 150 hours. In comparin!!j the time courses for sulfhydryl oxidation and a primary event in the pathogenesis of intra­ defluorination activity there was a loss in hepatic cholestasis, concomitant adminis­ defluorination when the concentration of reduced tration of cobalt chloride (CoCl2) and pheno­ sulfhydryl fell below 500 uM. Protection of the defluorination activity for up to 360 hours was barbital (PB) with or without lithocholic acid found when the Tris buffer was replaced by (LCA) has been studied in rats. Liver micro­ phosphate. This protection was enhanced by the somes were assayed and quantitative micro­ inclusion of 15mM 2-ME or 1 mM EDTA in the buffer. Initial purification steps demonstrated scopy was used to determine surface and that the defluorination activity separated out enzyme loads of endoplasmic reticulum (ER) with anionic proteins in DEAE-Sephadex membranes. Hepatotoxicity was observed with ion-exchange chromatography. This technique also separated the defluorination activity from a 11 CoC I 2 regimens. PB compensated CoC I,, 90% of the glutathione S-transferase activity dependent hepatotoxicity; CoCI 2 + PB with measurable with 1-chloro-2,4-dinitrobenzene. or without LCA, increased microsomal protein Defluorination activity did not bind to· a GSH affinity column which selectively separates it and phospholipid, and decreased glucose-6- from a highly purified anionic glutathione phosphatase (G6P) activity with no change S-transferase. This work was supported by a in cytochrome P-450. Aminopyrine-N­ grant from NIEHS # ES02001. demethylase (APDM) activity was decreased

by CoCl 2 + PB with LCA, but not by CoCl 2 + PB. The tot a I ER surface was unchanged

107 INTERACTIONSOF HEME PROTEINS WITH HYDROGEN PER­ by CoCI 2 + PB with and without LCA, but OXIDE: PROTEINPOLYMERIZATION ANDCOVALENT BIND­ RER values were significantly increased and

INGOF BENZO(A)PYRENE.R.H. Rice, Lab. of SER marginally decreased. Concurrent CoCl 2 Toxicol., Harvard Sch. Pub. Hlth., Boston, MA. + PB with or without LCA increased the Sponsor: J.L. Whittenberger ratios of microsomal protein and phosphol ipid Organisms must contend with highly toxic per­ per ER membrane area. G6P-ase activity/RER oxides and other activated oxygen species. ratio was reduced by the combined treat­ Illustrating potential toxicity, H202 has ments, but there were no changes in cyto­ been found to interact in a deleterious fashion chrome P-450 or APDM activity/SER ratios. with hemoglobins, myoglobins and cytochromes-c These morphofunctional correlations indicated

from a variety of organisms. Uponexposure to O.1 that CoCl 2 + PB with or without LCA, did to 1 mMH202 (or benzoyl peroxide) at room not induce HHSER but caused functional temperature and neutral pH, these proteins rapid­ impairment and resulted in an hypofunctional ly form covalent dimers and polymers detectable pseudo-hyperplastic RER. by detergent gel electrophoresis. Fonnation of cross-linked humanhemoglobin with H2D2does not occur in the presence of ascorbic acid, 109 METABOLISMSTUDIES ON WR-158,122, AN ANTIMALARIAL NaN3, thiourea, aniline or catalase. Nonheme DRUG, IN BILE DUCT-CANNULATEDRATS. C.C. Smith, proteins such as s-lactoglobulin do not show this M.W. Tabor, and G.J. Wolfe, Dept. Of Env. Hlth. cross-linking behavior alone upon exposure to Univ. of Cincinnati Med. Ctr., Cincinnati, OH. H202 but do so in the presence of hemepro- teins including humanhemoglobin or sperm whale The purpose of this study was to investigate the myoglobin. If the hemeproteins are treated with metabolic fate of the antifolate antimalarial H202 in the presence of benzo(a)pyrene, the compound WR-158,122, an amino-(naphthylsulfonyl)­ latter forms covalent adducts with the protein to quinazoline, in bile duct-cannulated and unoper­ a small extent. Under the standard conditions ated control (intact) rats and rhesus monkeys. employed, on the order of 1 benzo(a)pyrene adduct Previously we described the extraction of highly is formed per 100 humanhemoglobin polypeptide polar urinary metabolites of WR-158,122 from chains. Other oxidizable small molecules such as rodent and simian urine. Using a previously des­ 17s-estradiol are also bound to the heme proteins cribed method for preparing milled feces powders

30 we have developed a sequential extraction proced­ Med.; Lab. of Toxicol., Harvard Sch. Pub. Hlth.; ure for fractionating the 14c-WR-158,122 metabo­ Chem. Dept., Northeastern Univ., Boston, MA. lites in the feces of rats and monkeys given sin­ Sponsor: G. Wogan gle oral doses of the compound. For the solvent sequence of benzene (Bz), ethyl acetate (EtAc), This laboratory has previously found evidence acetonitrile (ACN) and tetrahydrofuran (THF) we that prostatic androgen inactivation and egress found that EtAc and Bz extracted> 90% of the 14c, involve cytochrome P450-mediated hydroxylation EtAc accounting for the larger portion. When this reactions. We here give the first report of a par­ extraction procedure was applied to feces from in­ tial analysis of the C1903-metabolites pro- tact rats considerably more 14c appeared in the duced by organ culture incubation of rat ventral ACN and THF extracts than was found with the feces prostate (RVP) with radioisotope-labeled testos­ from bile duct-cannulated rats. This finding terone. In two separate experiments, RVP explants was confirmed using a silica gel TLC procedure. (22 and 14 mg) placed on lens paper covering Next we developed an HPLC procedure for separating stainless-steel grids were incubated for 21 hr in WR-158,122 and its metabolites on Porasil using surface contact with serum-fre·e Trowell' s T8 medi­ a linear gradient from 100% methylene chloride to um containing 1.7 µM 4-1 4c-testosterone. Meta­ 100% methanol. The HPLC results showed: 1) the bolic disposition was determined autoradiographi­ EtAc extracts contained the unmetabolized parent cally after TLC. Of the total steroids recovered compound for both animal groups; 2) the Bz ex - from the organ-culture media, testosterone com­ tracts contained some parent compound for both prised 39% and 48%, total C1902-metabolites groups, and relatively nonpolar metabolites in 31% and 28% including 15% and 12% Sa-dihydro­ greater amounts in cannulated than in intact rats; testosterone, and C1903-steroids in four 3) the ACN and THF extracts contained polar meta­ chromatogram zones 30% and 24%, respectively. The bolites from intact rats and virtually no meta­ 2 most polar C1903-fractions were resolved by bolites from the cannulated rats. (Supported in 2-dimensional TLC and HPLC. 5a-Androstane-3S,6a,- part by Army Contract DAMD17-79-C-9196). 17S-triol and 3S,6a-dihydroxy-5a-androstan-17-one were identified as the major components of these fractions by reverse-isotope-dilution analysis; together, they accounted for 13% and 8% of the 2 110 DISPOSITION OF TOPICAL AND ORALLY ADMINIS­ radiosteroid patterns. The 3S-hydroxy-5a­ TERED INDOMETHACIN IN PATIENTS UNDERGOING androstane configuration of the identified CATARACT EXTRACTION SURGERY. M.S. Bernstein, C1903-metabolites supports our previously­ D.R. Sanders and M.A. Evans, Interdisciplinary Toxicol­ suggested pathway of prostatic testosterone ogy Program, Depts. of Pharmacology and Opthalmol­ ogy, University of Illinois Medical Ctr., Chicago, IL metabolism which effects activation by Sa-reduc­ 60680. tase and inactivation and egress by the coupled Sa-3-oxosteroid reductase/Sa-3S-hydroxysteroid Cystoid macular edema (CME), a postoperative hydroxylase reactions. complication of cataract surgery, is hypothesized to result from increased prostaglandin levels in the aque­ 112 ALLYL CHLORIDE: PHARMACOKINETICSAND METABOLISM ous humour due to surgical trauma. CME is associated FOLLOWINGADMINISTRATION TO CDF-FISCHER 344 RATS with ocular vascular incompetence which sometimes BY THREE ROUTES. J.M. Waechter, Jr., J.C. Ramsey, causes permanent macular damage. This study was B.E. Kropscott, J.F. Quast, T.D. Landry and designed to evaluate the effects and disposition of W.H. Braun, Toxicology Research Laboratory, indomethacin (I) in the reduction of CME based on its Health & Environmental Sciences, USA, The Dow inhibition of prostaglandin synthesis. Topical (I) was Chemical Company, Midland, MI. administered onto the cornea as drops of a 1% opthal­ mic suspension during the 24- hours preceding surgery. The pharmacokinetic fate of ally! chloride (AC) Patients given the drug orally, received 25 mg capsules when administered by three routes appeared to be within the preoperative period. Topical application of dose-dependent and route-de£~ndent. Following a (I) to the eye was shown to reduce the incidence of CME single oral dose of 100 mg C-AC/kg body weight, without clinical evidence of toxicity, as assessed by the majority of radioactivity was excreted via fluorescein angiography at 4- months postoperatively. the urine (polar metabolites) and expired air Aqueous humour and plasma samples were taken from (either as CO2 or parent compound). A one­ patients at the time of surgery for HPLC-fluorescence compartment bimodal absorption model adequately analysis of (I). Patients receiving oral (I) in the 24- H described the data for parent compound blood preoperative period, were found to have therapeutic levels following oral administration, while a amounts of drug in the plasma but no detectable levels two-compartment model was used to model the in aqueous humour. Patients given (I) topically were data from intravenous dosing and inhalation observed to have levels ranging from 77 to 666 ng/ml in exposures. The clearance half-lives of AC from aqueous humour but no detectable levels in the plasma. the blood following inhalation exposure (at These values of non-protein bound (I) in aqueous humour )100 ppm) increased with increasing exposure. reflect what normally would be considered toxic levels At 10 and 100 ppm, AC was cleared quickly from of drug. This study demonstrates the critical role which the blood (half-lives <30 minutes). IT'Jlllediately disposition plays in relation to varying tissue sensitivi­ following a single six hour exposure of rats to ties to indomethacin. AC, liver, kidney and lung non-protein sulfhy­ dryl (NPSH) were markedly depleted at 2000 or 1000 ppm, slightly depleted at 100 ppm, and not 111 METABOLISMOF RADIOTESTOSTERONETO 3S, fu-DIHY­ significantly different from control at 10 ppm. DROXYSa-STEROIDS BY RAT VENTRALPROSTATE IN Rats exposed to 1000 or 2000 ppm of AC vapor ORGANCULTURE. R.L. Sousa, p. Ofner, R.L. Vena, for six hours showed treatment-related kidney P. Krinksy-Feibush, & P.W. LeQuesne, Lemuel cytotoxicity when sacrificed 48 hours post­ Shattuck Hosp.; Urol. Dept., Tufts Univ. Sch. exposure. No light microscopic changes indica-

31 tive of a treatment-related effect were observed and their urine and feces collected daily. The in the kidneys of rats in the 10 or 100 ppm three-day total urinary excretion of activity exposure groups. Since the AC inhalation was 23.9% (S.D. 4.8%) of the administered dose concentration that causes both kidney toxicity and 40.8% (S.D. 3.7%) was excreted in the feces. and significant NPSH depletion in the rat are Tissue residues in the dams were highest in the similar, saturation of the. glutathione detoxi­ fat (23 ppm), kidney (1.6 ppm), uterus (5.0 ppm), fication mechanism may well be the critical adrenals (1.7 ppm), and liver (1.0 ppm). Com­ determination of the AC toxicity threshold. parable tissue levels in nursing pups (excluding fat and uterus) ranged from .5 - .9 ppm. The disposition of 14 C-toxaphene in lactating rats follows that in pregnant or virgin animals but 113 PHARMACOKINETICSOF ORALLYAND INTRAVENOUSLYAD­ tissue residues in the lactating young were much MINISTERED 1,1-DICHLOROETHYLENEIN THE R¾T. higher than in fetuses. L. Putcha, J.V. Bruckner, and S. Feldman , Dept. Pharmacol., Div. Toxicol., Univ. Texas Med. Sch.; Dept. Pharmaceutics, Univ. Houston*, Houston, TX. 115 RING-CHAINTAUTOMERISM OF THE ANTICOAGULANT RODENTICIDEHARFARIN AND ITS AROMATICHYDROXY­ Although a number of aliphatic halocarbons LATEDMETABOLITES. A. 0. Obaseki and W. R. have been identified as contaminants of drinking Porter (Sponsor: R. E. Peterson), School of water supplies, little definitive information is Pharmacy, University of Wisconsin, Madison, WI. available on the pharmacokinetics and toxic poten­ tial of these chemicals upon their ingestion. The Warfarin is metabolized by rat hepatic cyto­ present study of a representative halocarbon, 1,1- chrome P-450 monooxygenases in vitro at a rate of dichloroethylene (1,1-DCE), was undertaken to char­ about one mole/mole of enzyme/mm:---Homogenous acterize: (1) the kinetics of the intravenously­ P-450 isoenzymes yield multiple products from injected chemical; (2) the absorption, bioavaila­ warfarin, which exists in both polar and non­ bility, and elimination of the orally-administered polar solvents as a mixture of open-chain and chemical. Three dosage-levels of 1,1-DCE (25, SO, diastereomeric ring hemiketal tautomeric forms. and 100 mg/kg bw) in PEG 400 were administered in­ Nuclear magnetic resonance studies in dimethyl­ travenously (i.v.) and orally to fasted male Spra­ sulfoxide (DMSO)and acetonitrile revealed that gue-Dawley rats. Serial blood samples were taken the rate of tautomeric interconversion was grea­ from the tail artery of the lightly etherized ani­ ter than 16 sec-1. In DMSOsolution, 9% of the mals for 4 hours after dosing. 1,1-DCE concentra­ warfarin is in the open-chain form, while 64% tions in the whole blood were determined by gas exists as the R,R/S,S cyclic hemiketal and 27% as chromatographic head-space analysis. Evaluation of the R,S/S,R cyclTc-hemiketal. 5-Hydroxywarfarin, the i.v. data revealed that the disappearance of not a reported metabolite, exists entirely as the 1,1-DCE frvm the systemic circulation followed a open-chain form in DMSO;3'-, 4'-, 6-, 7-, and 8- triexponential pattern. The half-life of the ter­ hydroxywarfarin exist as mixtures of the open­ minal log-linear phase was comparable in animals chain form (9-13%), the R,R/S,S cyclic hemiketal receiving 25 and 50 mg/kg (i.e. approximately 40 (60-70%) and the R,S/S,R-cycTic hemiketal (24- min), but considerably longer in animals receiving 28%). The 13c chemTcal-shifts of aromatic ring 100 mg/kg (i.e. approximately 70 min). Evaluation carbon atoms at the sites of metabolic attack of data from the oral dosing experiments revealed on warfarin are unaffected by ring-chain tauto­ that 1,1-DCE was very rapidly absorbed from the merization. The knownmetabolites 4'-, 6'-, 7-, G.I. tract, with peak blood levels reached within and 8-hydroxywarfarin differ little from warfarin 8 to 10 min after 1,1-DCE administration. Bio­ in tautomeric composition, although 6- and 8- availability, as determined by comparing the areas hydroxywarfarin have the highest proportion of under the blood concentration-time curves, in­ open-chain form. The latter are formed prefer­ creased with the increasing oral dose-levels. Re­ entially by a cytochrome P-450 isoenzyme induced sults of the foregoing experiments demonstrate by polycyclic aromatic hydrocarbons. The rela­ that 1,1-DCE is readily absorbed from the G.I. tive amounts of multiple products formed from tract and elirr.inated from the bloodstream in a warfarin by homogeneouscytochrome P-450 iso­ dose-dependent manner. (Supported by EPA Grant enzymes appear to be thermodynamically controlled No. R808282) with respect to tautomerization. (Supported by U.W. Graduate School #110277).

114 THE DISPOSITION OF 14 C-TOXAPHENEIN THE LACTAT­ ING RAT. G.A. Pollock and J.F. Rogauskas, 116 CHROMATOGRAPHICTRANSLOBULAR MIGRATION OF XENO­ Veterinary and Comparative Anatomy, Pharmacology/ BIOTICS IN RAT LIVER. S. Tsuda and T. Nakat­ Toxicology, and Physiology, University of Idaho, sugawa, Department of Environmental and Forest Moscow, ID 83843. Biology, State University of New York, College of Environmental Science and Forestry, Syracuse, The environmental and physiological disposi­ NY 13210 tion of toxaphene has only recently been investi­ gated and is poorly understood. The present To investigate the significance of the chromato­ investigation was undertaken to better understand graphic translobular migration of xenobiotics, the disposition of toxaphene in the lactating rat a perfusion system was developed simulating in and the transfer to the young. vivo conditions. Blood somewhat diluted with Lactating Sprague-Dawley rats were orally Waymouth's medium was obtained from a 300-g male dosed with approximately 10xl0 6 dpm of 14 C­ Sprague-Dawley rat and recirculated through the toxaphene in olive oil. The dams and young were liver of the blood donor. A radiolabeled xeno­ housed in glass metabolism cages for three days biotic, such as parathion, dichlorobenzenes and

32 4-nitroanisole, was dissolved in the blood and was pretreated with vegetable oil i.p. daily a 0.4-ml pulse dose was infused into the liver for 3 days to serve as oil-treated control. On via a Teflon valve. Aliquots of the eluate frac­ the fourth day n-hexane was injected i.p., using tions were analyzed for the parent compound and 8 mice per dose, in six graded doses for each the concentration was plotted against the elu­ group. The 48 hour LD50 values were significant­ tion volume. Virtual absence of the compound at ly lower for toxaphene and cigarette smoke the void volume and subsequent appearance of a pretreated animals and higher for piperonyl peak gave evidence of chromatographic behavior butoxide pretreated animals than the controls. of these xenobiotics through the liver lobule in vivo. Factors affecting parameters of migration and implications of this phenomenon in assessing health hazards of xenobiotics were investigated. 119 BUTYLAND METHYL ACRYLATE: 13-WEEK ORAL TOXI­ CITYSTUDIES IN CDFFISCHER 344 RATS. S. J. Gorzinski, G. C. Jersey, C. E. Wade, E. A.· 117 RESPONSETO ETHANOLCONTAINING LIQUID DIETS IN Hermann, S. B. Mccollister and R. J. Kociba, THEFISCHER-344 (F-344) ANDSPRAGUE-DAWLEY (SD) Toxicology Research Laboratory, H&ES,Dow RAT. N. Misslbeck, D. Kim, I. Barbeau, T. C. Chemical U.S.A., Midland, MI 48640, Campbell and D. A. Roe, Div. of Nutr. sc-,.--;-­ Groups of male and female rats were administered Cornell University, Ithaca, NY. butyl acrylate in the drinking water at approxi­ mately 0, 12, 73 or 84 mg/kg/day for males and A study was designed to compare the effects 0, 15, 91 or 111 mg/kg/day for females. A sat­ of strain and diet on the response of the rat to ellite group of male and female rats was given ethanol-containing liquid diets. Six week old 150 mg butyl acrylate/kg/day via gavage for 13 male F-344 or SD rats were fed either a low fat weeks. In a separate study, rats of both sexes (LF) diet (8% of kcals) or high fat (HF) diet were given drinking water solutions of methyl (35%of kcals) containing ethanol (35%of acrylate at targeted dose levels of 0, 1, 5 or kcals). A control group for each strain and 20 mg/kg/day. diet was pair-fed a diet with dextrin-maltose replacing the ethanol, and an additional group Water intake was slightly decreased to a similar was offered ad libitum the non-ethanol diets. degree at all concentrations of butyl acrylate Animals were sacrificed after 3 wks. Although in both sexes, probably the result of unpalata­ food intake was similar for both diets, SD rats bility. Male rats given the highest dose level drank more per day (75 mls) than F-344 rats (60 had slightly decreased body-weight gain. Rats mls). Weight gain amongpair-fed animals was given 150 mg/kg/day via gavage had statistically not significantly different. In SD rats liver significantly increased relative liver weight triglyceride (TG) content was not significantly ratios. No morphological changes were seen upon altered by ethanol for the LF animals, but was light microscopic examination of tissues by increased for the HF animals. Pair-fed SD either route. Thus, only minimal indications of control animals had significantly lower liver TG toxicity were seen in this study for dose levels levels than did ad lib SD control rats (9.4 mg/g of 84-111 mg/kg/day in the drinking water or 150 vs 25.4 mg/g liver). F-344 rats showed no mg/kg/day via gavage. increase in liver TG on ethanol diets. Aniline The 20 mg/kg/day dose level of methyl acrylate hydroxylase activity was increased and ethyl­ produced decreased body-weight gain and water morphine N-demethylase activity remained unaf­ consumption in both sexes. Females at this fected by ethanol feeding. Both cytochrome dose level also had increases in urinary spe­ P-450 and cytochrome b5 levels were higher in cific gravity and relative kidney weight ratio. ethanol animals. SD animals tolerated the Both sexes given the highest dose had an in­ ethanol better than F-344 rats, but were more creased incidence of renal tubular dilatation sensitive to the fat content of the diet. In and eosinophilic cast formation. Based on the conclusion, the effects of ethanol on the rat is data generated, the no-observable-effect level strain and diet dependent. (Supported by USPHS (NOEL)of methyl acrylate for both sexes in this POl CA26755 and NIH-AM07158.) study was determined to be 5 mg/kg/day.

118 EFFECT OF METABOLICINDUCTION ON ACUTE TOXICITY OF n-HEXANE IN MICE. Raje R.R., Arnold & Marie 120 FACTORSINFLUENCING ACRYLONITRILE TOXICITY Schwartz College of Pharmacy & Health Sciences, R, Jaeger, I. Cote, A. Rogers, E. Silver ands. Long Island University, Bklyn, NY. Szabo. Inst. Env. Med,, NYU, New York, NY; Dept. Sponsor: Lawrence W.R. Nutr. & Food Sci. MIT, Cambridge, MA; Dept. Pathol. Harvard Medical School, Boston, MA. n-Hexane is widely used industrial solvent. Its volatility and well documented neurotoxicity The- acute toxicity of acry lonitrile (ACN), as make it an occupational hazard. It has been measured by lethality or decreased of tissue reported that the neurotoxicity is attributed to glutathione (non-protein sulfhydryl, NPSH) n-hexane itself as well as its toxic metabolites. concentration was measured in male or female The purpose of this study was to determine the Charles River SD rats at 6-7 weeks of age. When effect of metabolic induction on acute toxicity dosed during the day, the acute oral LD in of n-hexane in female mice.. Groups of 48 S/W Purina fed rats was: 124 mg/kg (male), 121 50mg/kg mice each were pretreated daily for 3 days with (female). When inhalation exposure was done toxaphene 50 mg/kg i.p., piperonyl butoxide during the day, the acute 4 hr inhalation LC 5 mg/kg i.p. and cigarette smoke inhalation for in Purina chow fed rats was: ca. 300 ppm (male1, 0 1 minute respectively. A fourth group of 48 >615 ppm (female). PCB enhanced lethality in mice was an untreated control and a fifth one females at 600 ppm. A lipotrope deficient, high

33 fat diet resulted in lower oral LD50 in both J.J. Barkley, U.S. Army Medical Bioengineering R & males and females. Inhalation values were D Laboratory, Fort Detrick, Frederick, MD. lowered in females. Males were affected to a lesser extent. As measured by hexobarbital The toxic effects of 2,4,6-trinitrotoluene (TNT) sleeping time, the snythetic diet decreased were examined in rats, dogs, and mice following mixed fun ct ion oxidase activity. In rats fed subchronic dosing. Fischer 344 rats and B6D2Fl Purina chow, NPSH concentration was higher in mice received TNT in the diet for 13 and 6 weeks, females than males (0.59 .±. .09 vs. 0.31 .±. .06 mg respectively. Beagle dogs were given daily doses NPSH/100 g body weight, respectively). The high of TNT in gelatin capsules for 26 weeks. Doses fat diet, irrespective of lipotrope deficiency, (mg/kg/day) ranged from 5-300 for rats,0.5-32 for increased these values in brain and liver. dogs, and 1-2000 for mice. Although experimental Values in females were still elevated above the designs were different (in part a function of concentration in males. 14C-ACN binding after their purpose, e.g. rodent studies were conducted oral 14C-ACN administration in males (females to select doses for chronic studies), the data not studied), while quantitatively different due allowed for qualitative interspecies comparisons. to diet, did not differ qualitatively. Whole In general, sensitivity to TNT toxicity increased blood had significant amounts of bound 14C as surface area/body weight decreased, i.e. dogs activity as well as the highest specific were most sensitive followed by rats then mice, activity. Liver had the highest tot a 1 activity Major toxic effects observed for all 3 species in­ as a % of oral dose. (Supported by NIEHS cluded ataxia, decreased food intake with subse­ ES-01871, ES-00002, and ES-00260. RJJ is the quent reductions in body weight gain, anemia (re­ recipient of an NIEHS RCDA. SS is the recipient duced hematocrit, hemoglobin, and RBCs), methemo­ if an NIAMDRCDA). • globinemia, hypercholesterolemia, splenomegaly with congestion and hemosiderosis, hepatomegaly with hepatocellular hypertrophy and Kupffer cell 121ACUTEAND SUBCHRONIC ORAL TOXICOLOGIC STUDIES OF hemosiderosis, and testicular atrophy with degen­ HEXAHYDRO-l,3,5-TRINITR0-1,3,5-TRIAZINE(ROX) IN eration of germinal epithelium. Compensatory re­ RATSAND MICE. L.C.K. Wong, J.M. Cholakis, C.B. sponses to the anemic state included reticulocyto­ Hong, H.V. Ellis, Ill, and J.C. Dacre, Pharma­ sis, macrocytosis, and an increase in nucleated cology/Toxicology Department, Midwest Research RBCs. Toxic manifestations limited to 2 species Institute, Kansas City, MO,and U.S. ArmyMedical included increased kidney weights with hemoglobin­ Bioengineering R&DLaboratory, Ft. Detrick, like casts, splenic extramedullary hematopoiesis, Frederick, MD. and neural tissue vacuolation. Additional toxic effects were species-specific, and reinforced the Despite widespread military use of the high explo­ determination of major target organs based on sive ROX,only limited toxicological data are results in at least 2 of the species studied. available. Some results of studies in progress (Supported by the U.S. Army Medical Bioengineering with Fischer 344 rats and B6C3Flhybrid mice are R & D Laboratory under Contract DAMD17-79-C-9120). reported here. The military grade ROXused had the following composition: 2.2 ± 0.1% water, 88.6 ± 0.9% ROX,ca. 9% HMX(octahydro-l,3,5,7- 123 MUTAGENICAND REPRODUCTIVE STUDIES OF HEXAHYDRO­ tetranitro-1,3,5,7-tetrazocine). The oral LD50 l,3,5-TRINITR0-1,3,5-TRIAZINE(ROX) IN RATSAND values (95% confidence limits) in mg/kg were: 119 RABBITS. J.L. Minor, R.D. Short, Jr., D.L. Van (110-128) in male rats, 119 (108-129) in female Goethem, L.C.K. Wong,and J.C. Dacre, Pharma­ rats, 97 (82-116) in male mice, and 59 (25-139) in cology/Toxicology Dept., Midwest Research Insti­ female mice. Ninety-day subchronic feeding stud­ tute, Kansas City, MO,and U.S. Army"Medical Bio­ ies were carried out as follows: six groups of engineering R&DLaboratory, Fort Detrick, both rats and mice (10 animals/sex/group) were fed Frederick, MD. ROXin the diet at levels of 0, 10, 14, 20, 28and 40 mg/kg/day. A supplemental study with mice ( 12/ Studies were conducted with ROXto evaluate the sex/group) had four groups of 0, 40, 60, and 80 mutagenic effects (AmesSalmonella/microsome test, mg/kg/day for 2 weeks followed by 0, 320, 160 and dominant lethal assay) and reproductive effects 80 mg/kg/day for the remaining 11 weeks. In rats, (teratology, two-generation reproduction) in the 40 mg/kg dose was toxic (decreased mean body Charles River CDrats and NewZealand white rab­ weights, and food consumption in male rats only) bits. Mutagenic studies. ROXproduced numbers while other specific toxic effects (e.g. decreased of revertants similar to those in vehicle-treated hematocrit and SGPT)were small and inconsistent. controls at doses up to l mg/plate in all five The 28 mg/kg dose produced no apparent toxic strains of~- typhimurium, both without and with effects. In mice, the 320 mg/kg dose produced activation with the S-9 fraction (Aroclor 1254). unscheduled deaths, hyperactivity, increased Groups of male rats from the F reproduction kidney weight with mild tubular nephrosis (males), study were fed ROXdiets containing0 0, 5, 16, or and increased liver weight accompanied by peri­ 50 mg/kg/day for 13 weeks and then mated with portal hepatocellular vacuolization (males) and virgin females. There was no evidence of either microgranulomas (females). No toxicologically a preimplantation loss of blastocysts or a post­ important effects were seen in mice fed the lower implantation loss of embryos. ROXtherefore is doses of ROX. (Supported by the U.S. ArmyMedical not mutagenic in the Amesand dominant lethal R&DCommand under Contract No. 17-78-C-8027.) tests. Reproductive studies. Animals were given ROXsuspensions by gavage at dose levels of 0, 0.2, 2.0 and 20 mg/kg/day; rats on days 6 to 19 122 COMPARATIVETOXICITY OF TNT IN RATS, DOGS, AND and rabbits on days 7 to 29 of gestation. ROX MICE. B.S. Levine, E.M. Furedi, D.E. Gordon, J.M. was not teratogenic to rats and rabbits although Burns, J.H. Rust, and P.M. Lish, Life Sciences maternal toxicity (neurotoxicity), lethality and Division, IIT Research Institute, Chicago,IL and embryotoxicity were seen in the high dose rats.

34 No effects were seen at the 2 mg/kg level. Four 12 months. High dose males had decreased body groups of rats (22/sex/group) were fed ROXdiets weights without decreased food consumption. No at levels of 0, 5, 16, and 50 mg/kg for 13 weeks treatment related effects on survival rates were and then mated. The F1 generations (26/sex/group) noted. No statistical differences were measured were similarly mated after 13 weeks on the diets. in hematological or clinical chemistry parameters Reproduction was adversely affected only in the determined at 3, 6, 12, 18, and 24 months. Organ high dose group; fertility, viability and lacta­ weight decreases were noted for liver' spleen, tion indexes ~ere reduced. (Supported by the U.S. and kidney from high dose males. However, no ArmyMedical Research & Developmentcorrmand under histopathologic alterations attributable to test Contract No. 17-78-C-8027.) material intake were observed in tissues from interim or terminal sacrifices. The incidence of neoplastic and non-neoplastic lesions ob­ 124 TOXICOLOGICASSESSMENT OF ISOPROPYLMETHYL­ served were similar in all test groups. PHOSPHONICACID. F.J. Meeler and J.C. Dacre, Dept. of Toxicology, Litton Bionetics-;- I~ A three-generation reproduction study was con­ Kensington, MD,and U.S. ArmyMedical Bioengineel"­ ducted with ATMP at dietary concentrations of ing R&DLaboratory, Fort Detrick, Frederick, MD. 300, 1000, and 3000 ppm. Each Fo group con­ Isopropyl methylphosphonic acid (IMPA)is the tained 12 male and 24 female Long-Evans rats. ultimate mammalianmetabolite of diisopropyl No adverse effects were observed on fetal, pup, methylphosphonate (DIMP). DIMPis a decomposition or adult survival, or on parental and pup body product of GB, a U.S. Armytoxic agent. DIMP weights. No adverse effects were noted in and IMPAhave been found in the groundwater of the mating or fertility parameters measured for any Rocky Mountain Arsenal. Since there are no pub­ test group during the study. Histopathologic lished toxicological data on IMPA,the following evaluation of selected tissues from F3b pups studies were made: Acute oral LD50in rats and indicated no treatment-related effects. mice; acute dermal toxicity in rabbits; primary occular irritation in rabbits; skin sensitization in guinea pigs; Amesmutagen assay, and subchronic 126 THECOMPARATIVE TOXICITIES AND CARCINOGENICITIES toxicity over a period of 90 days in rats. OF BENZIDINEAND DIRECT BLUE 6 IN RATS. J.H. Oral LD50values (95%confidence limits) in mg/kg Mennear and B.N. Gupta, National Toxicology Pro­ for sodium IMPAwere: 7650 (6560-8920) in male gram, NIEHS,P.O. Box 12233, Research Triangle rats, 6070 (4760-7740) in female rats, 5620 (4530- Park, NC 27709. 6990) in male mice, and 6550 (5140-8360) in fsnale Direct Blue 6, a bisazobiphenyl dye knownto mice. Application of sodium IMPAto the intact be metabolized to benzidine (B) produces hepato­ and abraded skin of rabbits at doses up to 2.0 cellular carcinoma when fed to rats at dietary g/kg produced no signs of systemic toxicity but concentrations of 1500 and 3000 ppm. The rapid did produce mild skin irritation; rabbit eyes onset of carcinoma (4 weeks) suggested that the similarly treated produced no signs of irritation. effect might be mediated through mechanismsother Sodium IMPAdid not induce dermal sensitization than simple metabolism to B. The objective of in the guinea pig nor did it exert a mutagenic the present study was to compare the potencies of effect in any of the five strains of Salmonella the dye and B with respect to general toxicity typhimurium in the Amesassay when tested with and carcinogenicity. Groups of 20 female F344 and without S9 rat liver microsomes. Rats which rats were exposed to drinking water containing received 300, 1000 or 3000 ppmof sodium IMPAin either B-2HC1(100-400 ppm) or the dye (500-2000 the drinking water for 90 days exhibited no signs ppm) for up to 90 days. The concentrations of B of toxicity when compared to controls. Clinical were selected to be equivalent to B exposure if hematology and chemistry, as well as histopatho­ dye treated rats completely metabolized the chem­ logic evaluation of tissues taken at necropsy, ical. Based on water consumption, the average revealed no adverse effects. Hence, IMPAhas a daily doses of B were 10, 21, 28 and 31 mg/kg low degree of toxicity and should present l i tt·1 e while the B equivalents of the dye consumptions if any hazard to those exposed to it. were 13, 22, 30 and 37 mg/kg/day. All rats ex­ (Research supported by the U.S. ArmyMedical R&D posed to the two highest doses of B died within Commandunder Contract No. 17-77-C-7003.) 9 weeks while only 6 rats exposed to the high dye concentration died during the study. Amongrats surviving to terminal sacrifice, both chemicals 125 CHRONIC TOXICITY AND REPRODUCTIONSTUDIES ON produced dose-related increases in serum gamma AMINOTRI(METHYLENEPHOSPHONIC ACID) [ATMP) glutamyl transpeptidase with the magnitude of M.W. Stevens, Dept. of Med. & Environ. Health, effect produced by the 21 mg/kg/day dose of B Monsanto Co., St. Louis, MO 63166 (G.J. being similar to that produced by the highest Levinskas) dose of the dye. Additional dose-related effects produced by both chemicals included: thymic invo­ Phosphonates are used industrially as chelating lution, increased liver weights, and grossly ob­ agents, with major application as scale and servable hepatic nodules. Hepatocellular car­ corrosion inhibitors in cooling or boiler waters. cinoma was diagnosed in some rats that died after These agents have benefit in replacing environ­ receiving 28 mg/kg/day of B for up to 9 weeks. mentally unacceptable products such as chromates. The results suggest that Direct Blue 6 is not A chronic toxicity study was conducted with ATMP more toxic than an equivalent dose of B. using groups of 70 male and 70 female Long-Evans rats. ATMP was administered at dietary concen­ trations equivalent to 50, 150, or 500 mg/kg/day 127 EVALUATION OF IMMUNOLOGIC AND HEMATOLOGIC EF­ for 2 years. Interim sacrifices of 10 male and FECTS OF FUSARIUM T-2 TOXIN IN THE BOVINE. B. 10 female rats per group were conducted at 6 and HOOK, G.D. OSWEILER, AND G. M. BUENING, COL-

35 LEGE OF VETERINARYMEDICINE, UNIVERSITY OF MIS­ Male F-344 rats and B6C3Fl mice were exposed by SOURI, COLUMBIA,MO., 65211. inhalation to 1500 ppm of methyl chloroform (MC) 6 hr/da, 5 da/wk for approximately 16 months. On Mycotoxins in animal feeds may affect the last day of repeated exposure the animals health, growth performance, reproduction and were exposed to 1500 ppm of 14 C-labeled MC for disease susceptibility in food animals. T-2 6 hours and the routes of elimination of 14 c toxin has been shown to be irrmunosupressive in activity followed for 72 hours. The fate of 14 c­ ·laboratory animals. A do_se response study was MC in the repeatedly exposed animals was compared conducted in young calves. Groups of three to a group of rats and mice which had been expos­ calves each were dosed with T-2 mycotoxin at ed concurrently for 16 months to chamber air (age­ O, 0.1, 0.3, and 0.6 mg/kg body weight in matched controls) prior to receiving the single gelatin capsules daily for six weeks. Calves 6 hour exposure to 1500 ppm of 14 C-MC. The routes were clinically evaluated daily and blood was of excretion and tissue concentration of 14 c monitored weekly for changes in clinical chem­ activity were similar between the singly and istries, hematology, coagulation and/or immuno­ repeatedly exposed rats and mice. The major route logical effects. Tests were conducted that mea­ of elimination of MC was exhalation of the parent sure both humoral and cell mediated irrmunity. chemical in the expired air and constituted Food consumption and weight gain were also approximately 97% of the total recovered radio­ monitored. Grain refusal was noted in the high activity in rats and 92-94% in mice. The remaining dose (0.6 mg/kg) group as ear I y as 36 hours radioactivity (~3-9%) was recovered as metabolized following initial administration of the toxin. MC in the expired air ( 14 C02) and as nonvolatile A lesser effect was seen at the 0.3 mg/kg radioactivity in the urine, feces, carcass and level. Three additional calves ( inanition con­ cage wash. Mice were found to eliminate MC more trols) were pair fed an equivalent amount as rapidly via the pulmonary route and to biotrans­ the high dose group. A 11 ca Ives were necrop­ form approximately 5-fold more MC on a body weight s i ed and evaluated for any morphological dif­ basis than rats. Repeated exposure to MC did not ferences which included histologic examination significantly affect the disposition of MC compar­ of most tissues. Organ weights and major lymph­ ed to the singly exposed rats and mice. Thus even oid tissues were compared. T-2 toxin produced after long-term repeated exposure to MC, its bio­ a dose related thyrnic involution. In addition, transformation remains limited. preliminary results suggest that a cell medi­ ated (mitogen stimulation) and humeral (IGM, globulin and total protein) responses were al­ 130 THE PHARMACOKINETICSAND MACROMOLECULARINTER­ so altered. (Supported by USDA SEA CR901-15- ACTIONS OF TRICHLOROETHYLENEIN MICE AND RATS AS 164). RELATEDTO ONCOGENICITY. Stott, W, T., Quast, J. F., and Watanabe, P. G. Toxicology Res. Lab., Dow Chemical USA, Midland, MI 48640

Male B6C3Fl mice were observed to metabolize in­ 128 CITRININMYCOTOXICOSIS IN THE RABBIT. C. haled 1,1,2-trichloroethylene (TRI) (600 ppm/6 hr) Hanika and W.W~Carlton, Dept. Vet. Microbial., to a greater extent (262%) than male Osborne­ Sch. of Vet. Med., and J. Tuite, Dept. of Bot. Mendel rats. Mice were also observed to metabolize and Plant Pathol., Sch. of Agri., Purdue Univer­ more (332%) inhaled TRI to a hepatic macromolecu­ sity, W. Lafayette, IN lar binding metabolite in vivo than rats. Oral administration of TRI resulted in treatment-relat­ In 3 trials, single or multiple doses of ed hepatocellular cytotoxicity in repeated dosing citrinin dissolved in 0.5N NaOHand adjusted trials in the mouse. Hepatic effects observed in to neutral pH with HCl were administered to rab­ mice treated with 2400 mg/kg/day TRI for 3 days bits by the oral or intraperitoneal route. The were primarily centrilobular swelling with focal 72-hour LD50was 50 mg/kg body weight by intra­ necrosis and increased DNA synthesis (220% of peritoneal administration and 134 mg/kg by the control). Treatment of mice with TRI for a 3 week oral route. The primary clinical sign was fluid period (5 days/wk) resulted in a dose-related diarrhea commencing8 hours after oral adminis­ increase in hepatocellular swelling with giant and tration. Pathologic alterations were confined mineralized cells present in 2400 mg/kg/day dosed primarily to the kidney and consisted of necro­ animals. Contrasting the mouse data, rats appear­ sis of proximal convoluted tubules and straight ed to be less sensitive to TRI, displaying enhanc­ segments. The earliest histopathologic change ed hepatic DNA synthesis levels (175% of control) was vacuolation of tubular epithelial cells and but no histopathology after a similar 3 week was seen as early as 8 hours post oral adminis­ treatment with 1100 mg/kg/day TRI. An estimate of tration. Seven days after citrinin administra­ the extent of TRI interaction with hepatic DNA in tion, regeneration of tubular epithelium with vivo was also determined. Only a very low levelof slight tubular necrosis were observed. Rabbits TRI-DNA interaction was observed in mice adminis­ given repeated sublethal doses of citrinin had tered a carcinogenic dose level of 1200 mg/kg TRI mild tubular degeneration and necrosis and (max. est.= 0.62 ± 0.43 alkylations/10 6 deoxy­ tubular regeneration. nucleotides). When coupled with the weak or nega­ tive responses of pure TRI in in vitro mutagene­ sis assays, these data indicatea relative lack of genotoxic potential. These data in toto sug­ 129 THE FATE OF INHALEDMETHYL CHLOROFORM (1,1,1-TRI­ gest an epigenetic mechanism of tumor formation CHLOROETHANE)FOLLOWING SINGLE OR REPEATEDEXPO­ in the B6C3Fl mouse, implying that a tumorigenic SURE IN RATS AND MICE. Schumann, A. M., Fox, T. response to TRI exposure in these animals would R., and Watanabe, P. G., Toxicology Research Lab., only be evident upon chronic administration of Dow Chemical USA, Midland, MI 48640 high, cytotoxic dose levels of TRI.

36 131 EFFECTOF CHLORDECONEONTHE HEPATOTOXICITY OF 1, enzymes. These results indicate that the poten­ 1,2-TRICHLOROETHYLENEANDBROMOBENZENE. H. M. tiation of CCl4 hepatotoxicity by CDcan be de­ Mehendale and V. G. Lockard, Depts. Pharmacol. & monstrated after a single oral dose of CDequi­ Toxicol. and Pathology, Univ. Miss. Med. Ctr., valent to that previously used in the 15 day di­ Jackson, MS39216, USA. etary protocol. Maximal potentiation of CCl4 hepatotoxicity is observed 24 hr after CD. These Previous studies from this laboratory have results are consistent with the CDinduction of shown that pretreatment with chlordecone (CD) re­ the CCl4 bioactivation system. (Supported by sults in potentiated hepatotoxicity and lethality ES-01369and ES-07045.) of CCl4 in rats. The objective of these studies was to investigate whether pretreatment with CD affected the hepatotoxicity of two agents struc­ turally and mechanistically dissimilar to CCl4. 133 POTENTIATIONOF CHLORINATED HYDROCARBON TOXICITY Male Sprague-Dawley rats (300-400 g} were exposed BY2,5-HEXANEDIONE IN PRIMARY CULTURES OF ADULT to O or 10 ppm CD, mirex (M), or 225 ppm pheno­ RATLIVER PARENCHYMAL CELLS. J.D. Jernigan, barbital (PB) in the diet for 15 days. On day 15 J.G. Pounds, R.D. Harbison, Vanderbilt University, the animals received ip injection of trichloro­ Nashville, TN. and U. of Arkansas for Medical ethylene (TCE; 5 or 10 mmole/kg) or bromobenzene Sciences, Little Rock, AR. (BB; 1, 3 or 5 mmole/kg) in corn oil vehicle. Potentiation of halocarbon-induced hepatotoxicity Controls received the vehicle alone. Twenty-four by 2,5-hexanedione (HD) in rats and mice is a hr later, animals were surgically prepared under well-recognized phenomenon. To investigate this pentobarbital anesthesia for hepatobiliary func­ potentiation in vitro, male Sprague-Dawley rats tion tests using phenolphthalein glucurontde (PG). were pretreated either with HD, 15 mmol/kg, p.o., Serum glutamic pyruvic transaminase (SGPT), serum in corn oil, or with corn oil, and the hepatocytes glutamic oxalic transaminase (SGOT)and isocitric isolated 18 hr. later by enzymatic perfusion. dehydrogenase (ICD) were determined from blood Following overnight adaptation to in vitro samples. Liver samples were processed for histo­ conditions, groups of cultures wereexposed to pathology. Hepatobiliary function tests, serum CHCl3(10 rnM), CDCl3(10 rnM)or 1,1,2-trichloro­ enzymes and histopathology results indicate that ethane (TRI) (5 mM). As a measure of toxicity, CDand M did not and PB did potentiate the hepa­ aliquots of media were removed at various times to totoxicity of TCE. BB hepatotoxicity was mildly determine levels of lactate dehydrogenase (LOH) potentiated by CDand M treatments. PB treatment released by the cells into the media. Two hours caused much greater potentiation of BB hepato­ following exposure to CHCl3or CDCl3, hepatocytes toxicity and the interaction was lethal at 3 and isolated from HD-treated rats exhibited LOH 5 mmole/kg doses. These results indicate that release twice that of corn oil treated rats. At the capacity of CDto markedly potentiate the 6 hr after treatment with the halocarbons, hepato­ hepatotoxicity and lethality of CCl4 is specific cytes from HD-treated rats showed a 4-fold in­ to halomethanes and that marked potentiation by crease in media LOHlevels over controls for CDis not a generalized phenomenonaffecting CHCl3and CDCl3-treated cells, and a 2.5-fold structurally and mechanistically divergent he­ increase for TRI-treated cells. A deuterium patotoxins. (Supported by ES 01369.} isotope effect for toxicity using CHCl3was observed in cells from both control and HD­ treated rats. WhenHD was added directly to hepatocytes isolated from untreated rats, there 132 TIMECOURSE OF THECHLORDECONE TREATMENT FOR THE was a concentration-dependent protection against POTENTIATIONOF CCl4 HEPATOTOXICITY.J. S. hepatotoxicity when, 18 hr later, CHCl3(10 mM) Klingensmith and H. M. Mehendale, Dept. Pharmacol. was added to the cells. These studies indicate & Toxicol., Univ. Miss. Med. Ctr., Jackson, MS that the isolated rat hepatocyte culture is a 39216. useful model to study the potentiation of halogenated hydrocarbon-induced liver damage. It has been demonstrated that dietary pre­ (Supported by USPHSGrant GM07628) treatment (10 ppm) with chlordecone (CD) for 15 days markedly enhances the hepatotoxicity and le­ thality of CCl4. The objectives of these experi­ ments were: to determine if potentiation of CCl4 134 METABOLISMOF TRICHLOROETHYLENEBY ISOLATEDRAT hepatotoxicity by CDcould be demonstrated by ad­ HEPATOCYTES. R.E. Miller and F,P. Guengerich, ministering a single oral dose of CDequivalent Depts. of Pharmacol. and Biochem. and Ctr. in to the previous dietary protocol; and to define Environmental Toxicology, Vanderbilt Univ., the time course of CD-induced sensitivity to Nashville, TN. 37232. CCl4. Groups of 4 male Sprague-Dawley rats (250- 350 g) were given a single oral dose of CD (10 The metabolism of trichloroethylene (TCE) was mg/kg) in corn oil. CCl4 (0.1 ~l/kg) was given studied using hepatocytes isolated by collagenase ip at 6, 12, 18, 24, 36, 48 and 72 hr after CD. perfusion of livers from Osborne-Mendel rats. Control animals received corn oil (1 ml/kg) alone Previous work from our laboratory using microsomal in place of the CDand CCl4 treatments. Rats and reconstituted cytochrome P-45O-containing were killed 24 hr later, liver sections were mixed function oxidase systems indicated that an fixed in buffered formalin and stained with H&E­ oxygenated TCE-cytochrome P-45O transition state PAS. Blood samples were taken and three serum and not the epoxide was probably an obligate enzymes (SGPT, SGOT,and ICD) were measured. A intermediate in TCE metabolism. TCE-oxide was rapid rise in all three enzymes occurred starting detected in microsomal but not in hepatocyte sys­ at 6 hr after exposure to CD, with a plateau at tems, TCE metabolites detected in hepatocytes approximately 24 hr. Histopathological assess­ included: chloral, trichloroethanol, trichloro­ ment of liver damage parallels the rise in serum ethanol glucuronide, trichloroacetic acid, glyoxy-

37 lie acid and carbon dioxide. Chloral, trichloro­ haloethyl)glutathione followed by a sulfhydryl­ ethanol and trichloroethanol glucuronide were the promoted E2 elimination of halogen from the con­ major-metabolites, Only chloral, glyoxylic acid jugate. Alternatively, GSHmay act as a base and and TCE-oxide were detected in rat liver micro­ form ethylene and the sulfenyl halide of GSHby a somal systems, Hepatocyte viability was greater direct E2 reaction. We have differentiated these than 80% for at least six hours with 1 rnM TCE. mechanisms by using meso-1,2-dideutero-1,2-di­ There was no detectable decrease in glutathione chloroethane as a substrate. The substrate (5mM) levels during this time period. A glutathione was incubated with GSH(20mM), sodium phosphate conjugate was tentatively identified at very low buffer (20mM,pH 7.4) and liver cytosol prepared levels. Kinetic studies in hepatocytes support from male Long-Evans rats . .9.2 -Ethylene was anal­ the proposed scheme that TCE is metabolized by the yzed for stereochemical configuration by FT-infra­ mixed function oxidase systems to chloral which is red spectroscopy. meso-1,2-Dideutero-1,2-di­ subsequently metabolized by the cytosolic dehydro­ chloroethane was metabolized exclusively to (Z)- genases to trichloroethanol and trichloroacetic 1,2-dideuteroethylene. Since E2 eliminations-in acid, A time lag exists between chloral and tri­ non-ordered systems require an anti-periplanar chloroethanol formation and between trichloro­ orientation of leaving groups, this result sug­ ethanol and formation of its glucuronide in hepa­ gests the involvement of a substitution-elimina­ tocytes. (Supported in part by USPHS Grants tion sequence and is consistent with the inter­ ES02205 and GM 07628). mediate formation of ethylene-S-glutathionyl­ episulfonium ion. Furthermore, this result sup­ ports the concept that this episulfonium ion is a reactive intermediate involved in the mutagenic 135 RELATIONSHIP BETWEENTRICHLOROETHYLENE METABOLISM action of 1,2-dihaloethanes. In contrast, pre­ AND ITS HEPATOTOXICITY. J. A. Buben and E. J. vious studies have shown that meso- and dl~2,3- O'Flaherty, Department of Environmental Health, dibromobutane undergo a direct-U-elimination re­ University of Cincinnati, Cincinnati, OH 45267 action to yield (E)- and (Z)-2-butene, respective­ SPONSOR: P. B. Hammond. ly. (Supported by-USPHSgrants ES01082&GM07397) This study was undertaken to establish the rela­ tionship between the metabolism of trichloro­ ethylene (TRI) and its hepatotoxicity when high doses of TRI are administered. Male Swiss-Cox 137 EFFECTOF HYPOXIAON CCl4 HEPATOTOXICITY.M.W. mice were treated by gavage with 100., 200, 400, Anders and E.S. Shen, Dept. of Pharmacology:univ­ 800, 1600, or 2400 mg/kg TRI per day for 6 weeks. of Minnesota, Minneapolis, MN 55455. Urine was collected at various times throughout Halothane undergoes reductive bioactivation, the dosing period and the urinary metabolites, and hypoxia is knownto enhance the hepatotoxic­ trichloroethanol and trichloroacetic acid, were ity of halothane. Since CCl4 also undergoes re­ quantitated by gas chromatography and used as an ductive bioactivation, the effect of hypoxia on index of the amount of TRI metabolized. The CC14 hepatotoxicity was studied. Male rats were following effects on the liver were seen: the exposed for 2 hr to CCl4 and differing 02 concen­ liver weight/body weight ratio increased with trations in Leach-type chambers. ChamberCC1 4 dose, and was 18%, 31% and 61% greater than con­ and 02 concentrations were monitored by gas chro­ trol at the 400, 800 and 1600 mg/kg doses res­ matography and polarography, respectively. SGPT pectively. Glucose-6-phosphatase activity was activities were measured 24 hr after exposure. decreased by less than 10% at the 800 mg/kg Exposure of rats to 4852 ppm CC14 and 100, 21, 14 level, but by 30 and 36% at the two highest dose and 6% 02 resulted in SGPTactivities of 489, 420, levels. Slight increases in SGPT were also seen 3768, and 1788 I.U./1, respectively. Exposure of at the highest two dose levels. The metabolism rats to air and 0, 1243, 2523, 4842, and 7565 ppm of TRI was found to be linear throughottt the dose CC14 resulted in SGPTactivities of 35, 32, 69, range examined, with about 30% of the dose ad­ 420, and 2188 I.U./1, respectively; when 12%oxy­ ministered being accounted for by the urinary gen was used, the corresponding SGPTactivities metabolites. Saturation of metabolism cannot were 32, 665, 691, 3768, and 4200 I.U./1, respec­ account for the increased toxic effects seen tively. Conjugated diene formation, measured above 800 mg/kg TRI. This work was supported by immediately after exposure to CC14, was not al­ NIEHS Toxicology Training Grant ES 07073 and by tered by hypoxia. Hepatic microsomal cytochrome funds from the Monsanto Company. P-450 concentrations were decreased immediately after exposure to CC14, but were the same when rats were exposed to CC14 in the presence of 12 and 21%0 • Hepatic chloroform concentrations 136 STEREOCHEMISTRYOF THE METABOLISM OF 1,2-0ICHLORO­ 2 1 1 were higher in animals exposed to CC14 (5095 ppm) ETHANETO ETHYLENE.J. Livesey , M. Anders , P. 1 under normoxic than under hypoxic conditions, Langvardt2 , C. Putzig 2 , and R. Reitz~, ( )Dept. of and hepatic CC1 concentrations were the same Pharmacology, Univ. of Minnesota, Minneapolis, MN 4 2 in animals exposed to 12 or 21%0 2 and 5095 ppm 55455 and ( )Health and Environmental Sciences, ffl 4. The covalent binding of metabolites of DowChemical Co. USA, Midland, MI 48640. CC14 to microsomal proteins and lipids was higher under hypoxic than under normoxic cond­ Recent studies have demonstrated that 1,2-dibromo­ itions. These results show that typoxia increases ethane and 1,2-dichloroethane are metabolized to markedly the hepatotoxicity of CC14 and that this ethylene (Drug Metab. Dispos.' 7 (1979)199). This increase in hepatotoxicity is accompanied by an biotransformation is catalyzed by hepatic cyto­ increase in the covalent bindin~ of CC1~metabo­ solic enzymes, requires glutathione (GSH)and is lites to hepatic microsomal lipids and ~roteins specific for vicinal-dihaloalkanes. Twomechanisms but not by an increase in lipid peroxidation or have been proposed for this reaction: the first the destruction of cytochrome P-450. Supported involves SN2 conjugation with GSHto form S-(2- by NIH Grant ES 00953.

38 138 PRODUCTIONOF ELECTROPHILICCHLORINE DURING RAT nol + CCl4 were 443%, 147%, and 2063% of control LIVER MICROSOMALMETABOLISM OF CARBONTETRA­ values, respectively. Thus, CCl4 metabolism ap­ CHLORIDE. SOURCEOF ELECTROPHILICCHLORINE. pears to inhibit microsomal and mitochondrialALDK B.A. Mico*, J.W. George and L.R. Pohl, NHLBI, In vivo, this may lead to high levels of aldehydes Bethesda, MD. Sponsor: J.R. Gillette. which may, in part, contribute to the potentiation We have recently reported (The Pharmacologist of CCl4 hepatotoxicity by ethanol. 23,113(1981)) the trapping of electrophilic chlorine or bromine with 2,6-dimethylphenol (DMP) to form 4-halo-2,6-dimethylphenol during microsomal metabolism of CCl4, CBrCl3 and 140 LIVER-PROTECTIVEACTIVITY OF AUCUBINAGAINST CBr4. As the first step in mechanistic studies HEPATIC DAMAGECAUSED BY CCl4 INTOXICATION. of this reaction, we have exam;ged the origin I-M. Chang, and H.S.Yun. (Spon:R.E. Peterson), Natural Prod. Res. Inst., Seoul Nat'nl Univ., of the trapped chlorine with C c1 4 (99 atom %) and Na37c1 (92 atom%). Liver microsomes Seoul 110, Korea (2 mg protein/ml) were incubated in the presence Previously, authors reported that the Plantago of NADPH(1 mM), DMP (1 mM) and either ~914 asiatica semen exhibited significant liver-prot­ (natural ab~ndance, 5 mM), C~!4 plus Na ~ ective activity against the hepatic damage in (100 mM), C 5c1 (5 mM) or C C1 plus Na 7Cl. 4 4 mice produced by CC14 intoxication. Aucubin, The incubation mixtures, after extraction with an iridoid glucoside compound, was isolated from ether and careful concentration, were analyzed the Plantago asiatica semen and its potential by capillary gas chromatography mass spectrometry liver-protective activity was evaluated in mice in the selective ion mode (m/z 156 and 158). intoxicated with CCl4. Analysis of the incubations with CCl4, with or without added Na37c1, revealed the natural 1) Effect of liver microsomal enzyme system; the abundance ratio of chlorine 35 and 37 isotopes administation of aucubin (4Smg/kg/day for 2 (3:1) in the trapped chlorine product. In con­ days) reduced markdly the duration of sleep t3!st, trapped chlorine from incu~1tions with induced by hexobarbital after CC14 intoxica­ tion(0.2ml/mouse/day for 2 days) in compari­ C c1 4 , with and without added Na Cl, showed better than 99% of the 35 c1 isotope in the son with those of CC14 alone treated mice. product. These studies show that CCl4 is the 2) Serum transaminase activities; source of the electrophilic chlorine. They the administration of aucubin prevented the also indicate that the electrophilic chlorine elevation of serum GOT and GPT activities in does not result from oxidation of chloride ion mice received CCl4. 3) Liver RNA and protein syntheses, through a chloroperoxidase mechanism nor does the compound appeared to inhibit the incorpo­ it rapidly exchange with chloride ion. *Pharmacology Resarch Associate, NIGMS. rdlio~Iates of 3u uridinc ~nd L~(4,5(n)-3H) isoleucine into liver RNA and protein. The mode of RNA inhibition appeared to be additive with Actinomycin D. 139 ETHANOLPOTENTIATION OF CCl HEPATOTOXICITY: A 4) Histological examination; liver in mice recei­ POSSIBLE ROLE OF ACETALDEHYBEDEHYDROGENASE (ALDH) ved aucubin after cc1 4 intoxication appeared INHIBITION. M. ·.Kenel and !• Kulkarni, Toxicology to be significantly improved in comparison Program, Sch. of Pub. Hlth, The University of with those of CCl4 alone treated group. Michigan, Ann Arbor, MI The results obtained from experimental criteria Liver microsomes of adult male Sprague-Dawley in the above indicated that the aucubin appeared rats were incubated at 37° in the presence of 1 to be one of biologically active ingredients mMNADPH and/or CCl4 (1 µ1/mg protein). NAD(P) responsible for liver-protective activity in dependent microsomal ALDH (relative) specific plantago asiatica semen. (Supported by Interna­ activities were then estimated after resedimenta­ tion. NADPHalone decreased NADdependent ALDH tional Foundation Grant #318R, Sweden) activity by 46%, 54%, and 70% after 5, 10 and 20 min incubations, respectively. CCl4 plus NADPH caused an even greater loss as NAD dependent ALDH decreased by 83%, 89%, and 94% after 5, 10 and 20 141 ASSESSMENTOF THE EMBRYOTOXICAND TERATOGENIC min incubations. Inclusion of 0.2 mMEDTA, a POTENTIALOF NONOXYNOL-9IN RATS UPONVAGINAL blocker of lipid peroxidation, prevented loss of ADMINISTRATION. H.S. Buttar, Bureau of Drug ALDHactivity due to NADPHalone, but only parti­ Research, Health Protection Branch, Ottawa, ally in the presence of NADPH+ CCl4. This sug­ Canada. gests that covalent binding of an active inter­ The effects of nonoxynol-9 (N-9), the active mediate is primarily responsible for decreased ingredient of vaginal spermicides, were studied ALDHactivity. At least equal depression of mito­ on the conceptus of Wistar rats. Th~ day of chondrial ALDH (I+ II) was noted when microsomes finding sperm in the vagina was counted as day and mitochondria were similarly incubated; however, zero of pregnancy. A single dose (2.5 mg/100 g) no inhibition of mitochondrial ALDHwas observed of aqueous solution of N-9 was administered when cytosol was then added. This may be due to intravaginally (O.l ml/100 g) only once to ether inhibition of lipid peroxidation. Microsomal and anesthetized rats on gestational days 1 to 10. mitochondrial NAD(P)+ dependent ALDH in ethanol­ Control rats were given an equal volume of treated (5 g/kg p.o.) rats remained unchanged. distilled water. The vulval metallic clips, The CC14-treated (1 ml/kg i.p.) group exhibited a used to prevent leakage of the solution, were decrease in activity. Rats receiving ethanol 18 hr removed 24 h post-treatment. The uterine prior to CCl4 had an even greater decrease. NAD(P)+ contents were examined on day 21 of gestation. dependent cytosolic ALDHactivities were not No signs of maternal toxicity were observed in changed. SGPT values for CCl4, ethanol, and etha- any of the N-9 treated dams. The incidences

39 of non-pregnancy and resorptions were high in intubation difficulties. Three 4050-3000 and one dams treated on days 3, 4, 5 or 6 of pregnancy. 1350 mg/kg/day rabbits died (2 to 10 or 12 dosages, The number of live fetuses was significantly respectively). One (1350 mg/kg/day) rabbit reduced in dams dosed on days 4, 5, 8 and 9, aborted (17 dosages). Tetany was observed in two whereas the average litter size of dams treated rabbits (4050-3000 mg/kg/day). In rabbits which on gestational day 10 was similar to that of the died, black discoloration of the skin, fur and controls. A significant reduction in fetal muscle tissue, including diaphragm, was observed weight was observed in dams treated 5 days grossly; the heart was not discolored. Antemortem post-conception. N-9 did not cause any agent-effects were anorexia, depressed maternal discernible visceral or skeletal malformations. body weight (450 to 4050 mg/kg/day), dark urine, The results suggest that single vaginal ungroomed coat, orange coloring of skin and application of N-9 is embryolethal and sclera, black skin and fur, thin appearance and fetocidal but nonteratogenic in the rat at a decreased spontaneous motor activity (1350 to 4050- dose approximately ten times higher than that 3000 mg/kg/day). Pregnancy occurred in 3, 4, 3, recommended for controlling conception in 3 and 1 rabbits in the respective dosage groups; women. 3, 4, 2, 1 and O of these pregnant rabbits sur­ vived to day 29 of gestation. Compared to con­ trols, pregnancy rates were slightly decreased 142 TERATOGENICPOTENTIAL OF SUSPENSIONSOF TRINITRO­ (450 to 4050-3000 mg/kg/day), and litter size FLUORENONE(TNF) ADMINISTEREDORALLY TO SPRAGUE­ slightly decreased with resorption rate slightly DAWLEYRATS. M.S.Christian, A.M.Hoberman, Argus increased (1350 mg/kg/day). No grossly malformed Research Laboratories, Inc., Horsham, PA, and fetus was observed. On the basis of this dosage­ T.H.F.Smith, IBM Corporation, White Plains, NY. range study, TNF does not appear to be a unique hazard to the rabbit conceptus at dosages which TNF was administered as aqueous Methocel® suspen­ are maternally toxic. sions at dosage volumes of 10 ml/kg orally via gavage to naturally-bred (day O=plug) Crl:COBs® CD®(SD)BR rats. In pilot studies, 8 female rats per group were administered dosages of O(vehicle), 150, 450, 1500 and 4500 mg/kg/day on days 6 through 19 of gestation. No deaths occurred, but 144 MICROSOMALENZYME INDUCTION AND REPRODUCTION IN brown urine was observed in rats administered all THREELINES OF JAPANESEQUAIL FED POLYBROMINATED TNF dosages and black discoloration of the skin BIPHENYLS. D. Polin, S. Bursian, L. Shull, Dept. and fur was observed in rats administered dosages Ani. Sci., MI State Univ., E. Lansing, MI 48824. of 450 to 4500 mg/kg/day dosages. Maternal Sponsor: S.D. Aust. weight gain was inhibited by dosages of 150 through 4500 mg/kg/day and an increased incidence of early resorptions occurred at 1500 and 4500 Japanese quail from lines* selected for low mg/kg/day dosages. In the teratology study (L) or high (H) response of plasma cholesterol to dosages of O(vehicle), 60, 300 and 1500 mg/kg/day ACTHwere compared to a random-bred (R) line for of TNF were administered on days 6 through 19 of sensitivity to polybrominated biphenyls (PBBs) gestation to 25 naturally-bred (day O=plug) and induction of hepatic mixed function oxidases female rats per dosage group. No deaths occurred. (MFOs). Pregnancy occurred in 23, 20, 19 and 22 rats ACTHhad no effect on either plasma cho­ Caesarean-sectioned on day 20. Dosage-related lesterol or hepatic benzo{a)pyrene hydroxylase inhibition of maternal weight gain and brown urine and hexabarbital hydroxylase activities in the L was observed in rats in all TNF-dosage groups. line. ACTHdid cause the expected marked in­ Black discoloration of the skin and fur occurred crease of plasma cholesterol in the H line, but in rats administered 1500 mg/kg/day dosages of did not alter the activities of hepatic MFOs. TNF. Fetal body weights were decreased in the PBBs at 80 ppm in the diet reduced hatchability high dosage group litters. No gross external to 60% of the control value for the L line as fetal malformations were observed. Based on these compared to 80% of the control value for the H data, TNF does not appear to be a unique hazard line. Thus, the L line appeared to be more to the rat conceptus at dosages which are sensitive to PBBs. maternally toxic. In a second experiment egg production, a re­ flection of the pituitary-gonadal axis, was re­ duced to 14% in the L line fed 80 ppm PBBs as compared to 38 and 49% in the Hand R lines, 143 DOSAGE-RANGESTUDY OF THE TERATOGENICPOTENTIAL respectively. Hatchability and subsequent growth OF SUSPENSIONSOF TRINITROFLUORENONE(TNF) ADMIN­ of F1 chicks were less in the Land H lines as ISTERED ORALLYTO NEWZEALAND WHITE RABBITS. compared to the R line when parents were fed 80 M.S.Christian, A.M.Hoberman, Argus Research ppm PBBs. Also MFOactivities (benzo(a)pyrene Laboratories, Inc., Horsham, PA, and T.H.F.Smith, hydroxylase and aminopyrine N-demethylase) and IBM Corporation, White Plains, NY. cytochrome P450 (448) concentrations were induced by 80 ppm PBBs more in Hand R parents than in Virgin female NZWrabbits (Dutchland Labs.) were those of the L line. artificially inseminated (insemination= day O), The data indicated that the L line of quail four assigned to each dosage group A_nd adminis­ was more sensitive to PBB's toxicity based on re­ tered aqueous suspensions (MethoceW, 10 ml/kg) of productive parameters while induction of hepatic TNF at dosages of O(vehicle), 150, 450, 1350 and MFOactivity was significantly less than in the H 4050-3000 mg/kg/day days 6 through 28 of presumed and R lines. gestation. The 4050 mg/kg/day dosage was reduced * USDA-SEA, Athens, GA: Ors. H. Siegel and H.L. to 3000 mg/kg/day after one treatment because of Marks

40 145 THEREPRODUCTIVE TOXICITY OF CITRINININ RATS. morphology, motility, and concentration. Elec­ R. V. Reddy, A. W. Hayes and W. 0. Berndt, Dept. tron micrographs indicate that the site of Pharmacol. & Toxtcol., Univ. Mississippi Med. action may involve the sperm plasma membrane. Ctr., Jackson, MS39216. Histological examination of the testes and epididymides revealed a slight increase in Living things are more sensitive to the ad­ degenerating spermatids in small portions of verse influences of the environment during early a few tubules. A clear dose-response relation­ developmental stages. Several fungal metabolites, ship was observed at 4, 12, and 30 mg/kg with produced in commonfood and feedstuffs, are tera­ 0.2 mg/kg being the no-effect dose level in togenic in experimental animals. Citrinin, a this study. Protein RIA kits were obtained fungal metabolite produced by molds infecting from NIAMDD,DR. A. F. Parlow. wheat, rye, oats and mixed barley, is nephrotox­ ic. Teratogenicity and embryo- and fetotoxicity of citrinin were investigated using pregnant CD-1 147 TESTICULAR DAMAGE IN THE RABBIT RESULTING FROM Charles River rats. The compoundwas administer­ ed subcutaneously on days 3 and 6-15 of pregnancy SIMPLE CHEMICAL CUTANEOUS IRRITATION in 5% NaHC03. A reduction in weight gain and in­ Wong, z. A., VonBurg, R., Spangler, W. L., and creased mortality of dams occurred with 35 mg/kg MacGregor, J. A. Chevron Environmental Heal th of citrinin. Treatment with citrinin during the Center, Richmond, California 94802. Sponsor: period of major organogenesis had no detectable J. K. Kodama adverse prenatal effects externally and also no significant increase in resorptions of implanted Although the albino rabbit has long been regarded embryos, but was associated with adverse effects as the species of choice for studying the cutane­ on fetal growth and to some extent on survival. ous irritation potential of substances, it has No qross, skeletal and visceral anomalies were also been used to evaluate systemic toxicity from observed. Although developmental defects were substances applied topically. Recent reports of not produced as a result of treatino the unborn testicular degeneration in rabbits treated topi­ rats with citrinin, it did exhibit fetotoxicity cally with a wide variety of agents have been by decreasing average fetal body weight. Judg­ surprising. The purpose of this study was to ing from the number of maternal deaths, 35 mg/kg determine whether testicular degeneration could of citrinin treatment on certain days of gesta­ have resulted simply from a response to cutaneous tion probably approac~ed the kD50: ~i~tr~bution, 1 irritation, Ten adult male rabbits (2.2-2.7 kg) excretion and metabolism of C-c1tr1n1n ,sunder were exposed daily to 2 ml/kg of a 2.5% (w/w) investigation in pregnant rats after subcutaneous solution of sodium hydroxide five days/week for administration of 35 mg/kg. The results of the four sucessive weeks. Ten sham control rabbits study indicated that citrinin is not embryocidal were run concurrently. Most of the treated rab­ or teratogenic but is fetotoxic when given to fe­ bits showed marked skin irritation, reduced food male CD-1 rats during pregnance. (Supported by consumption and decreased body weights during the USPHSresearch grant ES 02191.) study. No mortality or overt toxicity was observed. At necropsy, the testicular weights of the treated rabbi ts were significantly less {p 0,05) than those of the controls. Risto­ 146 THESITE OF ORDRAM®'SANTIFERTILITY EFFECT IN pathology revealed testicular degeneration in the MALERATS. J.M. Killinger, J.L. Minor, P.O. treated animals characterized by the increased Royal, R.A. Williams, J.R. Downs, and G.M. frequency of giant cell formation and the overall Zwicker, Env. Hlth. Ctr., Stauffer Chem. Co., decrease in the number of seminiferous tubules Farmington, CT Sponsor:. R.I. Freudenthal displaying spermatogenesis. Therefore, simple cutaneous irritation can induce degenerative The site of Ordram's antifertility effect in lesions in the testicles of adult rabbits. The male rats was investigated. In part I, male basis for this response may be stress-related; rats received either 0, 12, or 60 mg/kg of however, these results do indicate a limited Ordram for 5 days, then they were mated with usefulness of the rabbit in evaluating the sys­ 1 female per week for 10 weeks. There was a temic toxicity of many chemicals. substantial reduction in male fertility during the third post-treatment week, indicating the late spermatid stage was sensitive to Ordram exposure. The no-effect level was 12 mg/kg. In 148 ETHYLENEGLYCOL MONOMETHYL ETHER (EGME): INHALA­ part II, male rats were treated with either 0, TION REPRODUCTIONAND DOMINANT LETHAL STUDY IN 0.2, 4, 12, or 30 mg/kg of Ordram for 5 weeks, RATS. K.S. Rao, S.R. Cobel-Geard, J.T. Young, then they were mated with 2 females per week T.R. Hanley, Jr., W.C. Hayes, J.A. John, J,H. for 1 week. At terminal sacrifice, blood, Ouellette and R.R. Miller, Tox. Res. Lab., H&ES, sperm samples, and reproductive tissues were Dow Chemical U.S.A., Midland, MI 48640 taken for evaluation. A comparison was made between dose, fertility, changes in plasma Male and female Sprague-Dawley rats were exposed hormone levels (testosterone, FSH, LH, TSH, T3 by inhalation to 0, 30, 100 or 300 ppm EGME and T4), sperm morphology, motility and viabil­ vapor 6 hours/day, 5 days/week for 13 weeks. ity and morphologic changes in the testes and Following 13 weeks of exposure, each rat was epididymides. There were no measurable treat­ mated with an unexposed rat of the opposite sex. ment-related changes in serum hormone concen­ Exposure to 300 ppm EGMEresulted in complete trations which correlated with the reduction infertility in male rats. No adverse reproduc­ in male fertility. The dose-response relation­ tive effects were observed in males exposed to ship observed for male fertility could be 30 or 100 ppm nor in females exposed up to and correlated with changes in sperm viability, including 300 ppm EGME. There was no evid~nce

41 of a dominant lethal effect at 30 or 100 ppm; tects alterations of maternal blood flow to the the absence of litters (infertility) precluded placenta by detecting changes in the clearance of evaluation for dominant lethality at 300 ppm. tritiated water from mother to fetus. In order In order to assess the reversibility of infer­ to report the resulting data, maternal doses were tility, the males exposed to 300 ppm were rebred expressed relative to the maternal plasma concen­ with unexposed virgin females at. 13 and 19 weeks tration measured at 30 minutes after dosing and . post-exposure. At both of these mating intervals doses administered from the fetal side were ex­ 50% of the males previously exposed to 300 ppm pressed as the concentration measured in the per­ were fertile, indicating partial recovery. fusing fluid. Maternally administered benzo(a)­ Based on these results it is concluded that pyrene (7.7 µM), cadmium(1.0 µM), methylmercury exposure of rats to concentrations of EGMEas (200 nM), and inorganic mercury (40 nM) did not high as 100 ppm for 13-weeks resulted in no affect maternal blood flow to the placenta. Ma­ adverse effects on reproduction or on dominant ternal administration of inorganic lead (20 nM) lethality. caused sporadic changes in maternal blood flow to the placenta and monomericplutonium-239 (3 nM) reduced maternal blood flow to the placenta. Fe­ tally administered dinitrophenol (3 µM) did not affect maternal blood flow while ouabain (300 nM), 149 IMPACT OF PRENATALLEAD EXPOSUREON BIOCHEMICAL cadmium(37 µM), and fluorocitric acid (300 µM) MARKERSIN RAT LIVER AND HEART. K.L. Blackburn, reduced the maternal blood supply to the fetus. R.J.Bull, and M.K. Smith, Health Effects Research Based on data obtained in this laboratory and Laboratory, USEPA, Cincinnati, OH. others, it appears that toxic materials which disrupt the maternal supply of blood to the fetus The effects of prenatal lead exposure were tend to be toxic to the products of conception, evaluated in rat pups from dams treated with lead but not necessarily teratogenic. (Supported by nitrate during gestation. Dams were dosed by the U.S; Dept. of Energy Contracts DE-AC06-76RL0- gavage on days 6-17 of gestation with 50 or 500 1830 and E-40-1-GEN-242and NICHDGrant HD00727). mg/kg Pb(N03)z. One-half of the litters were cross-fostered at birth onto control dams. Bio­ chemical markers included cytochrome c oxidase activity in heart, liver and kidney and-monoamine 151 ONTOGENYOF CARDIAC ORNITHINE DECARBOXYLASE DOC oxidase activity towards three substrates (tryp­ ANDITS RESPONSIVENESSTO B-ADRENERGIC B-ADR tamine, phenylethylamine and 5-hydroxytryptamine) STIMULATIONIN THE NEONATAL RAT. s.P. Miska, in heart, liver and kidney. Biochemical markers J.R. Harmon, P. Webb, and G.L. Kimmel, Div. of were followed from three to 90 days post-partum. Teratogenesis Res., Nat. Center for Toxicol. In addition, dam weight gains during gestation, dam Res., Jefferson, AR. Sponsor: L. Fishbein. and pup blood leads, pup body and organ growth rates, litter size and sex ratio at birth and pup In the adult rat B-ADRstimulation of cardiac mortality were monitored. Pups were evaluated for growth parallels ODCactivation, and ODCre­ EKG abnormalities and changes in frequency of sponse to sympathetic nervous system (SNS) stim­ norepinephrine induced arrhythmias. ulation is used as an indicator of SNScontrol. In both dose groups a significant effect on To determine the effects of prenatal drug expos­ sexual differentiation of liver enzyme patterns ure on subsequent SNSdevelopment, it is neces­ was established. Lead exposed male offspring sary to define the normal postnatal alterations exhibited enzyme profiles which were statistically in basal ODCand responsiveness to direct s-ADR indistinguishable from female controls. These stimulation. DOCactivity was measured in rat effects occurred in the absence of effects on hearts on day 20 gestation (20DG)through day 22 litter size, pup body weight at birth or subsequent postnatal (22P.ND)under saturating conditions of growth rate, or increased mortality. In addition, L-ornithine and pyridoxal 5-phosphate. Basal ODC these effects occurred in both cross-fostered and activity fell from 4 U (nmol CO2/mgProt-hr) at noncross-fostered groups suggesting that they re­ 200Gto less than 0.5 U at 18PND,remaining low sulted from prenatal interference with liver en­ until after 21PND.The B-ADRagonist isoprotere­ zyme imprinting. Preliminary data indicate that nol (IPR) at lOmg/kg s.c. consistently resulted the serum steroid levels of the pups were signifi­ in peak ODCstimulation at 4 hr postinjection in cantly disturbed. postnatal hearts; however, at 200Gdeterminations were prevented by maternal mortality at 0.30mg/kg s.c. In dose-response studies IPR produced a maximal response at lOmg/kg s.c. in all postnatal ISOALTERATIONS OF THE MATERNAL BLOOD SUPPLY TO THE ages (increase from control: 6PND= 1.2 U; 14PND FETUSBY TOXIC MATERIALS. Bruce J. Kelman. = l.3U; and 21PND= 4.8U). With control ODCacti­ Pacific Northwest Laboratory, Richland, WA99352 vity at 6PND>>14 PND>21PND, these increases in the magnitude of IPR stimulation of ODCactivity Dynamicmeasurements were performed to evaluate between 2-3 weeks after birth result in even lar­ the effects of nine diverse toxic materials on ger increases in% change from control at older maternal blood flow to the placenta. The experi­ ages, indicating a greater sensitivity of the mental system uses a perfusion technique based on heart at these ages. Thus, this study defines the an isolation of the fetal circulation of the pla­ normal ontogenic patterns of ODCand responsive­ centa in situ and has been previously described. ness to direct B-ADRstimulation. It should now Materials were introduced into the maternal cir­ be possible to determine in prenatally exposed culation by injection into a carotid artery and animals whether alterations in ODCresponse to into the fetal circulation of the placenta by SNSstimulation reflect changes in neural addition to the p~rfusing fluid. This system de- development, or in heart ODCsensitivity.

42 152 EVALUATIONOF BEHAVIORAL TOXICITY IN NEONATES. 154 AQUATICTOXICITY AND BIODEGRADATIONOF CAPROLACTAM Robert Infurna & Bernard Weiss, Division of Loewengart, G., Allied Corporation, Morristown, Toxicology & Environmental Health Sciences N.J. 07960 Center, University of Rochester School of Medicine &Dentistry, Rochester, N.Y. 14642, Caprolactam, the monomer for the production of Sponsor: G.G. Berg Nylon-6, is a major industrial chemical. The literature contains little information about the Animal models in Teratology employing sen­ fate and effects of caprolactam in the aquatic sitive measures of the functional integrity of environment. Areas lacking adequate data the nervous system following perinatal exposure included toxicity to aquatic organisms, phytotoxi­ are required to unmask incipient behavioral tox­ city, and biodegradation of caprolactam in surface icity in neonates. The task is a challenging waters. one as the behavioral assessment of neural func­ Environmental tests that were incorporated into tion amongaltricial mammalianneonates is re­ the program were: static acute toxicity with three stricted by their limited behavioral repertoire fish and an invertebrate species, time-independent and underdeveloped sensory systems. Wereport flow-through toxicity to a fish species, seed ger­ the results of two behavioral paradigms employed mination and seedling growth phytotoxicity tests as part of our protocol for the evaluation of the with six terrestrial plants, growth inhibition ontogenesis of sensorimotor function in animals. tests with four aquatic plants, determination of The initial paradigm evaluates a response that the rate of biodegradation in natural waters mea­ is both characteristic and fundamental to all sured both by disappearance of caprolactam and mineralization to co , and determination of the mammalianspecies, Suckling Behavior. The diag­ 2 nostic significance of suckling has been made effects of temperature and substrate concentration apparent by the reports of suckling behavior on the biodegradation rate. anomalies amonghuman infants and suckling rats Based on the results of these tests, capro­ exposed to ethanol, methanol and caffeine. The lactam is characterized as a chemical that has a second paradigm studies the tendency of neonatal low toxit potential to fish, invertebrates, plants animals to orient and locomote to their nesting and microbes tested and can be efficiently site when removed from it. The Homingbehavior degraded by microbes in enriched surface waters. test has been shown to be sensitive to 1) the Primary degradation of caprolactam proceeded to an prenatal effects of methyl and ethyl alcohols, extent of more than 90% caprolactam disappearance 2) the acute effects of food additives such as within ten days. Under the same conditions, caffeine and tartrazine, 3) the psychopharma­ approximately 50% of the caprolactam underwent ultimate degradation to co . An induction period cological effects of amphetamineand, 4) the 2 influence of circadian rhythms. These paradigms observed for ultimate degradation suggests a rapid are relatively inexpensive to execute and many first step followed by slower, rate limiting steps be used in Teratological studies with various in the route to ultimate degradation. mammalianspecies. Supported in part by grant It is concluded that low level release of capro­ MH-11752from NIMH,grant ES-O1247from NIEHS, lactam does not present an environmental hazard and Contract No. DE-ACO2-76EVO349Owith the U.S. and the combination of physical, chemical, and Dept. of Energy. biological properties would mitigate adverse environmental effects of spills.

155 TISSUE DISTRIBUTION AND BINDING OF HEXACHLORO­ 153 THE PATHOLOGYOF SUBLETHALEXPOSURE OF FATHEAD CYCLOPENTADIENETO PROTEINS IN TROUT. P. Sinhaseni MINNOWSTO CADMIUM: A MODELFOR AQUATICTOXICITY and~. Hartung, Dept. of Env. & Ind. Hlth, Sch. of ASSESSMENT. Stromberg, P. C. and Ferrante, J. G., Pub. Hlth, and G.W. Jourdian, Rackham Arthritis Battelle Columbus Labs, 505 King Ave, Columbus, Res. Unit, The University of Michigan, Ann Arbor, OH 43201 Sponsor: G. L. Fisher MI Fathead minnows were exposed to an LC50 concentra­ tion of CdC12 for 96 hours then removed to clear Hexachlorocyclopentadiene (Hex) is relatively water for 21 days. Fish were removed at intervals toxic to fish and other aquatic organisms. The from Day 1 to Day 21 and evaluated for histopath­ high chemical reactivity of this compound suggests ological lesions. Lesions consisted of epithelial the possibility of its binding to biological necrosis in the skin, oral cavity, bronchial cham­ components. ber, and gastrointestinal tract. There was a Rainbow trout (Salmo fairdneri), approx. 400 g, patchy lifting of the gill epithelium interpreted were exposed to 120 ppb4C-Hex (specific activity as cellular degeneration and branchial edema. 5.3 mCi/mM) under static conditions until moribund Renal tubular necrosis was also noted. Epithelial (approx. 4 hr). Of the 14c added, 23.4% was lesions were most severe during the 96, hour ex­ recovered in 80.1% of the total fish wet weight. posure. Renal and gill lesions were observed up Ten organs were analyzed for 14c-activity. The to 7 days after exposure was terminated. It is highest concentration (dpm/mg wet weight) was proposed that histopathological evaluation of found in liver (169) and gills (81). Muscle which LC50 fish is an inexpensive and more sensitive comprised 59.5% of the total body weight contained method of assessing damage due to exposure to the highest amount of 14c (41.9% of total recov­ toxic substances. In addition, this model allows ered 14c-activity). assessment of reversibility and recovery. The When muscle was treated with chloroform­ protocol can be changed to include subchronic and methanol (2:1), 52.8% of 14c was found in dena­ chronic exposures. Microautoradiographic and tured protein, 9.6% was found in the lipid radiotracer studies may contribute data on tissue enriched chloroform fraction and 28.1% was found localization, metabolism, and elimination of the in the water phase containing water soluble com­ substance. ponents. After exhaustive digestion with pronase and gel mean TCB water concentration (0.1 µg/L), and mean filtration on columns of Sephadex G-25 and Sepha­ TCB uptake efficiency of the gills (60-86%) over dex G-15, a series of ~inhydrin-positive peaks the 48-hr exposure period. The total calculated were obtained coincident with 14c-activity. The µg of TCB absorbed by brook (17.8) and rainbow 14c ninhydrin-positive fractions were further trout (24.5) were then compared to the actual resolved by high voltage paper electrophoresis body burden measurements of TCB for brook (17.4) and paper chromatography. and rainbow trout (25.6)~ the latter measurements The results of these studies demonstrate that also included excretory losses through the urine, Hex is readily taken up by fish, and distributed feces, and across the gill surface. Approximate­ to all tissues. The highest amount is present in ly 1% of the total dose was excreted with 75% of muscle where most of the 14c label is bound to that lost being in the urine. The agreement protein rather than being extractable with lipids. between the whole body burden measurements of TCB This study is supported in part by the Rackham and the total calculated µg taken up was Grant from The University of Michigan. excellent in both species (within 10%). Species differences seen in total accumulated µg of TCB were due to a larger respiratory volume and to a 156 THEEFFECT OF B-NAPHTHOFLAVONE(BNF) TREATMENTON higher gill uptake efficiency in the rainbows. 35S-METHIONINEINCORPORATION INTO HEPATIC MICRO­ The mass-balance measurements were converted to SOMALPROTEIN OF RAINBOWTROUT. M.J. Vodicnik, dose by dividing by the fish weight. Calculated L.A. Rau, and J.J. Lech, Dept. of Pharmacology and and measured doses were 29.6 and 28.4 µg/kg/48 Toxicology, Med. Col. of Wisconsin, Milwaukee, WI. hr, respectively, for brook trout and 33.3 and 32.3 µg/kg/48 hr for rainbow trout. The ability While inducers of the 3-methylcholanthrene (3-MC)­ to accurately calculate a water dose for type have been shown to elevate the hepatic micro­ individual fish that could be directly compared somal metabolism of substrates specific for cyto­ to a mammalian dose was demonstrated. chrome P-448 in fish, it has not been demonstrated whether this process is a result of de novo syn­ thesis of hemoprotein(s) P-450 or anactivation of monooxygenase activity. Wetherefore studied the 158 A COMPARISONOF THE ACUTETOXICITY OF THREE effect of BNFon the in vivo incorporation of 355_ OXIMES TO AQUATICORGANISMS. R. J. Buccafusco, methionine into hepatic infcrosomal protein of rain­ M.A. Palmieri, and K. F. Pfeifer. Department of bow trout. Animals (60-70 g) were injected IP with Environmental Science, Allied Corporation, corn oil or 1gomg/kg BNF. They were each injected Morristown, N. J. Sponsor: G. V. Loewengart with 50 µCi S-methionine at 12 h and killed 36 h Acetaldehyde oxime (AAO), aldicarb oxime (ADO) following exposure to the amino acid. Microsomal and methylthioisobutyraldehyde oxime (MTAAO)are samples were taken for scintillation counting. intermediates used in the synthesis of agricultur­ Ethoxyresorufin-0-deethylation (EROD)was assessed al chemicals. The acute toxicity of these com~ and solubilized iiiicrosomes were electrophoresed. pounds to the rainbow trout, the bluegill sunfish Gel tracks were sliced into 0.5 cm segments and and the aquatic invertebrate, Daphnia magna, was counted. Duplicate gels were ~repared for fluoro­ determined under static conditions as part of an graphy. The incorporation of 35s-methionine into environmental hazard assessment. The results in­ protein was elevated in BNF-treated animals (22,732 dicate that there is no apparent or predictable +3652 vrs 42 ,643+5918 dpm/mgprotein) and ERODac­ pattern of species sensitivity to these chemicals. tivity in these-fish was 67-fold greater than that ADO was more acutely lethal to the rainbow trout in controls (0.801+0.253 vrs 0.012+.007 nmol/min/ (LC-50=28 mg/L) than the bluegill (LC-50=301 mg/L) mg protein). Densitometry showed that BNFtreatment or the daphnid (EC-50=343 mg/L). However, the resulted in the intensification of a band with a bluegill was the most sensitive species to AAO molecular weight of 57,000 and the 0.5 cm sections (LC-50=31 mg/L) and MTAAO(LC-50=120 mg/L). Addi­ of the gel containing this band had 4-fold greater tionally, at sublethal concentrations of ADO radioactivity than the corresponding region of gels (-158 mg/L) bluegill experienced an apparent cen­ from control animals. Fluorography of gels demon­ tral nervous system or neuromuscular effect that strated the intensification of several proteins, was characterized by a general loss of equilibrium the most pronounced of which appeared at 57,000. and swimming ability. This immobilization is These data suggest that the elevation in cyto­ similar to the response observed after exposing chrome P-448-type catalytic activity observed fol­ fish to a low concentration of a general anes­ lowing treatment of rainbow trout with 3-MC-type thetic such as tricaine methane sulfonate (MS-222). ADO had a similar effect on daphnids agents is a result of an increase in de novo pro­ but not on rainbow trout. The bluegill and daph­ tein synthesis. Supported by NIH Grants ES01985 nids which experienced this effect subsequently and ES 01080. regained their equilibrium and swimming ability when placed in water without ADO. The results 157 DOSE IETERMINATIONSFOR WATERBORNE2,5,2' ,5'­ demonstrate that chemicals with similar structures TETRACHLOROBIPHENYLIN TWOSPECIES OF TROUT:.A can have unique biological activity and that the MASS-BALANCEAPPROACH. J.M. McKim and E. M. sensitivity of aquatic organisms to closely re­ Heath. U.S. EPA, Environ. Res. Lab., Duluth, MN lated chemicals is not always predictable. 55804. Sponsor: W. H. Gingerich

In order to evaluate dose determinat~ons of 159 AN ACCURATE,WIDE RANGEHPLC METHODFOR THE DE­ 14c-TCB made on transected brook trout TERMINATIONOF TOXICANTOCTANOL:WATER PARTITION (Salvelinus fontinalis) and rainbow trout (Salmo COEFFICIENTS. J.E. Garst and W.C. Wilson, Dept. gairdneri) a mass-balance study was undertaken. of Animal Sci., Coll. of Agric., Univ. of Illin­ The total weight of TCB removed by the gills from ois, Urbana, IL 61801. SPONSOR: B.J. Wilson, the respiratory water flow was calculated by Dept. Biochem., Vanderbilt University Medical determining mean respiratory volume (5-8 L/hr), School, Nashville, TN 67232.

44 A major physical factor governing interactions 161 DOSAGELEVELS ASSOCIATEDWITH THE INDUCTION between xenobiotics and biological macromolecules OF RESPIRATORYTRACT INFLAMMATIONBY ACUTE is the hydrophobicity of the agent. A frequently EXPOSURESTO TITANIUMTETRACHLORIDE SMOKES. used measure of hydrophobicity is the logarithm ~- Ballantyne, Union Carbide Corporation, of the chemical's octanol:water partition coeffi­ Charleston, WV 25303. cient log P(o:w). However, shake-flask partition coefficients are subject to numerous errors. Mea­ Titanium tetrachloride (TT) vapor surement by high pressure liquid chromatographic reacts with atmospheric water, producing (HPLC) techniques may minimize certain problems, oxychlorides and hydrogen chloride with the but the available methods often have limited ran­ 1 iberation of heat. The resultant smoke is ge and reproducibility or require special columns. known to.cause injury to the respiratory tract, An HPLC method has been developed which affords but the pathology produced and its dose accurate and reproducible octanol:water partition dependency are not well documented. In this coefficients over the Oto 7 range. Correlations study rabbits, rats, guinea pigs and mice were between the literature octanol:water log P and the acutely exposed to TT smokes at the fol lowing corrected HPLC elution volumes are better than dosages, expressed as TT in mg min m-3; 0. 999 with high reproducibility for numerous or­ 14,800 - 58,800 (delivered over 30 minutes), ganic chemicals. Unlike other HPLC methods, this 5,000 - 10,000 (over 10 minutes) and 400 (over procedure uses commercial RP-8 columns, flow 1 or 10 minutes). Dosages in excess of 5,000 rates above 1 ml/min, and dual solvents to im­ mg min m-3 produced acute inflammatory effects prove peak shapes and speed elution. It may be as follows. Congestion, neutrophil completely computer-controlled, enabling rapid, infiltration and focal necrosis of laryngeal routine log P (o:w) determinations at costs com­ and tracheal mucosa; scattered foci of parable to the calculation of values, but with necrotizing bronchial itis; alveolar capillary better results. Variables affecting theprocedure congestion with scattered areas of intra­ and correlations of toxicological data with HPLC alveolar haemorrhage and edema. These log P(o:w) will be presented. histopathological features were most severe in Measurements of various methylated and halo­ the 24 hours following exposure, and resolved genated benzenes suggest that substituent con­ in the subsequent 7 to 14 rlays. In general the stants are not additive above 4 log P(o:w) units. severity of inflammatory change was dose-related. Other findings indicate that second components, No specifi~ histopathological features were formed in the shake-flask, may cause errors in seen in the respiratory tract of animals exposed traditional log P measurement. This work was to TT smoke at dosages of 400 mg min m-3 (over supported by ES 02193, BRSG S07 RR07030, HATCH a 1 or JO-minute exposure period). In 20-323, and the University of Illinois. contrast with the severe irritation seen following liquid or high vapor contamination of the eye, no adverse ophthalmic effects were observed at the dosages of TT employed in this 160 AN ASSAY FOR DETECTINGERYTHROPOIETIC DAMAGE IN study. · RAINBOWTROUT (SALMOGAIRDNERI) USING 59FE IN­ CORPORATION. K. Cooper and R. Snyder, Graduate Program in Toxicoloqy, Rutgers Univ., Piscataway, NJ ORB54 162HEXAMETHYLPHOSPHORAMIDE(HMPA) NASALCARCINO­ GENESIS: SELECTIVE RETENTIONOF 14 C-HMPARADIO­ Detection of erythropoietic damaqe in mice can be ACTIVITY IN TARGETTISSUES AND ITS INHIBITION BY determined by monitoring the level of 59Fe uptake METYRAPONE. R. W. Rickard and P. J. Gillies, E. into developinq red blood·cells (RBC) (Lee et al., I. du Pont de Nemours and Company, Inc,, Haskell 1974, Appl. Pharmacol. 27, 431-436). We developed Laboratory for Toxicology and Industrial Medicine, an assay for trout (R0-100 g) based on the above Newark, Delaware 19711. Sponsor: G. L. Kennedy, work and that of Walker (Dissertation, 1975, Mich, Jr. State Univ.). We injected trout ip for three consecutive ~ays with either benzene (440 mg/kg) The role of metabolism and disposition in the in­ in corn oil, or corn oil as a control. On day 4 duction of nasal turbinate squamous cell carci­ fish were injected ip with 0.5 uCi 59Fe and sac­ nomas was investigated in the rat. Rats were rificed 11 days later. 59Fe content was measured administered 14 C-HMPAvia nose only inhalation or in blood from the caudal vein, and in samples of oral gavage. The maxilloturbinate (MT), nasal liver, spleen, head kidney, pyloric caeca, in­ turbinate (NT), and ethmoid (ET) regions of the testine, gill and skeletal muscle. Compared to nose contained 7, 21, and 25-fold more radio­ control values, benzene pretreatment resulted in activity than other tissues in the body 72 hours significant decreases of 59Fe incorporation in after inhalation exposure; quantitatively similar RBC's (70%), and in the three organs involved in results were obtained at 168 hours. The reten­ hematopoiesis in fish: liver (71%), kidney (43%) tion of radioactivity in these regions may be a and spleen (23%) ( p< 0.02 for all values). Usina unique property of the nose since even after oral the same protocol, we injected toluene (440 mg/ka, administration the MT, NT and ET regions exhibi­ ip) and found no decrease of 59Fe uptake in blood ted 3- to 7-fold more radioactivity than other or in any of the tissues. Further studies are tissues. To in~estigate the possibility that currently being conducted into the comparative this retention depended on the metabolism of HMPA sensitivity for detection of hematopoietic damage to a reactive species, similar studies were con­ by this method versus standard cell counting tech­ ducted in rats pre-treated with metyrapone. Mety­ niques. On the basis of these preliminary data, rapone virtually eliminated the retention of this assay appears to be useful for determininq radioactivity in the MT, NT and ET regions. To erythrotoxic environmental pollutants. ascertain whether metyrapone inhibits HMPAmeta­ (Supported by NIH Grant F:S00322.) bolism in the nose as opposed to the liver, the

45 retention of radioactivity in the nose was deter­ was initially decreased but returned to normal mined in partially-hepatectomized rats. Partial within 1 year. In animals treated with BHTalone hepatectomy did not alter retention of radioacti­ the lung changes were similar but less severe. vity in the nose. These data indicate that the Preliminary data indicate that lung compliance is nose contains an enzyme system capable of metabo­ decreased in mice 3 months after treatment with lizing HMPA. Within the nose, tumors often BHTand oxygen. Thus long-lasting morphologic develop in the NT and ET regions. Thus, the pre­ and biochemical alterations were produced as a ferential uptake and metabolism of HMPAin these result of potentiation of lung injury by oxygen. regions may represent an etiological factor in Research sponsored jointly by Grants HL-17907and the pathogenesis of HMPA-induced tumors. RR-00169from NIHand Office of Health and Environ­ mental Research, U.S. Dept. of Energy, under contract W-7405-eng-26with the Union Carbide Corporation. 163 THE KINETICS OF URETHANEMETABOLISM IN SWISS-COX MICE. S. P. Sichak and E. J. O'Flaherty. Dept. of Envir. Hlth., Univ. of Cincinnati, College 165 STUDIES ON THE DEPOSITION AND DISTRIBUTION OF of Medicine, Cincinnati, OH 45267, Sponsor: CATECHOLFROM WHOLE CIGARETTE SMOKE IN BC3Fl/CUM P. B. Hammond. MICE. K.K. Hwang, O. Sonko, D.R. Dansie, R.E. A number of investigators have observed a quad­ Kouri, and C.J. Henry, Dept. of Experimentar-­ ratic relationship between acute urethane dose Oncology, Microbiological Associates, 5221 River and cumulative pulmonary adenoma incidence in Road, Bethesda, MD 2081.6-1493 mice. However, analyses relating cumulative Tritiated-catechol has been used to follow the adenoma incidence to total internal exposure to pharmacokinetics and metabolic fate of inhaled radiolabel from radiolabeled urethane indicate catechol in cigarette smoke in BC3Fl/Cum mice. that this relationship is linear. These obser­ The presence of 3H-catechol in the smoke was vations may be reconcilable by consideration of verified by silica gel chromatography, and gas the saturability of urethane elimination. Single chromatography/mass spectrometry. Mice were ex­ doses of from 0.4-1.4 mg urethane per g body posed to 10% (v/v) 2Rl cigarette smoke on the weight were given intraperitoneally to 7-week­ Walton Horizontal Smoking Machine under standard old Swiss-Cox mice. Urethane was monitored in conditions of 35 ml puff volume, 2 sec/puff, 10 the blood as a function of time by measuring puffs/cigarette. The deposition and distribution ethanol released from urethane by alkaline hydro­ of inhaled catechol were determined in all inter­ lysis and using thin layer chromatography to in­ nal tissues, urine and feces. Data showed that sure that any urethane metabolites that could clearance was occurring during the 10 minute generate ethanol by alkaline hydrolysis were not smoke exposure period. Immediately after this simultaneously being measured. The data demon­ exposure period, 56% of the radioactivity was strate that urethane elimination is saturable in found in the blood, with 8% found in the lung, this dose range and allow estimation of Vm and 12% in the respiratory tract, 13% in the liver, Km for the elimination process. (Supported by and 14% in the kidneys. By 120 minutes after PHS Grant CA 29917 awarded by the National exposure, less than 0.6% of the total dose was Cancer Institute, and by PHS Grant ES 07073. found in the lung and over 94% of the inhaled radioactivity was found in the urine. Less than 10% of these radioactive urinary metabolites were 164 EXPERIMENTALLUNG FIBROSIS IN THEMOUSE: LONG­ extractable by chloroform-methanol (CM) or ethyl­ TERMMORPHOLOGIC ANDBIOCHEMICAL FEATURES. W.M. acetate (EA) solutions. Upon treatment with S­ Haschek, A.J.P. Klein-Szanto, J.A. Last,* K.r glucuronidase and arylsulfatase, at least 95% of Rieser,*, S. Lock, W.E. Dalbey and H.P. Witschi. the radioactive urinary metabolites were extract­ Biology Division, Oak Ridge National Laboratory, ed by CM or EA solutions. These organic extract­ Oak Ridge, TN 37830 and *Department of Internal able fractions were purified, analyzed, and three Medicine, University of California, Davis, CA chemicals positively identified: catechol, 95616 hydroquinone and 4-methylcatechol. We conclude that catechol in smoke is rapidly Both the early inflammatory and the late pro­ absorbed, redistributed, conjugated, and excreted liferative phases of the-adult respiratory dis­ in urine from mice exposed to whole cigarette tress syndrome can be produced in mice with the smoke. antioxidant, BHT,and 70%oxygen. The present Supported by The Council for Tobacco Research - study followed the morphologic and biochemical USA, Inc. progression of these lung lesions for 1 year. Youngadult male Balb/c mice were injected ip with 400 mg/kg BHTdissolved in corn oil, or corn oil alone, and immediately exposed to 70%oxygen, 166AUTORADIOGRAPHICANALYSIS OF DNA SYNTHESIS IN PUL­ or air, for 6 days. Mice were killed at various MONARYTISSUES OF MICE EXPOSEDTO WHOLECIGARETTE time intervals up to 1 year after BHTtreatment SMOKE. K.K. Kanagalingam, S.M. Reed, D.R. Dansie, and the lung changes were evaluated by light and W.C. Hall, R.E. Kouri, and C.J. Henry, Dept. of electron microscopy, and by determination of total Experimental Oncology, Microbiological Associates, lung hydroxyproline levels and changes in collagen 5221 River Road, Bethesda, MD 20816-1493 types. In animals treated with BHTand oxygen there was initially a diffuse interstitial pneu­ DNA replication, as measured by estimation of monitis. With time the inflammatory changes sub­ Labeling Index (LI), was determined in BC3Fl/Cum sided but degenerative, emphysematouschanges mice after daily exposure to whole cigarette smoke became apparent. Elevated lung hydroxyproline over a 12 month period. Immediately foilowing the levels present 2 weeks after treatment persisted last smoke-exposure, lung DNAwas labeled with a up to 1 year. The percentage of type III collagen 1.0 hr. pulse of tritiated thymidine (3H-TdR) and

46 tissues processed for autoradiography. LI in 168 EFFECTS OF IN VIVO AND IN VITRO METALEXPOSURES ON smoke-exposed mice was increased over controls PULMONARYMACROPHAGES. K.L. McNeill, C.J. Democko, after 9 weeks of exposure. This increase per­ and G.L. Fisher, Battelle Columbus Laboratories sisted over the entire study period. Of the 4000- Columbus, Ohio 43201 6000 cells counted in both sham-exposed and shelf­ control mice, not more than 3 cells had any signi­ In vitro dose-response for dissolved zinc, sele­ ficant number of grains. The percent of total nium, vanadium, or nickel has been evaluated uti­ cells labeled after 13 weeks was about 20-fold and lizing functional assays with pulmonary macro­ 14-fold greater in mice exposed to smoke from 3Al phage from Balb/c male mice. Pulmonary macro­ and 2Rl cigarettes, respectively. In the smoke­ phages were lavaged and attached to glass cover­ exposed lung, the distribution of cells with slips to obtain a pure cell population. After grains was regular. No labeling of cells of the rinsing nonadherent cells, the attached cells were bronchial epithelium was found, rather the uptake exposed to the test material in media for twenty of 3H-TdR was confined to alveolar cells. Neither hours. At the end of the incubation period, via­ necrosis nor epithelial hyperplasia was observed bility was evaluated for each group, and phagocyt­ in any area of the lung that would account for the ic capacity was determined after challenge with increase in LI. A high percentage (~67%) of cells carbon-coated latex microspheres for one hour in in the smoke-exposed mice were labeled with less fresh media. Comparable in vitro exposures using than 30 grains per nucleus. In contrast, in the bovine alveolar macrophages (BAM) were also done. control mice only about 35% of the total cells In vivo dose-response with several compounds (Naz contained less than 30 grains per nucleus. This Se03, ZnO, Vz05, Asz03, or Ni3S2) was studied in low grain number is generally indicative of DNA mice that had been exposed by intratracheal in­ repair synthesis. Thus, these data suggest that stillation. Function of pulmonary macrophages was whole cigarette smoke, while stimulating normal measured on cells harvested from these animals two DNA replication may induce unscheduled or DNA re­ weeks after exposure. Comparison of the in vitro pair synthesis as well. cytoxicity with the in vivo toxicity datademon­ Supported by The Council for Tobacco Research-USA, strates that macrophage cytotoxicity is not pre­ Inc. dictive of the whole animal toxicity. This phenom­ enon is most clearly shown with selenium and zinc. In vitro dose-response and metal interaction stud­ ies with BAMusing relatively insoluble particles of nickel, manganese, zinc and soluble selenium 167 PULMONARYP-NITROANISOLE 0-DEMETHYLASE ACTIVITY and vanadium were also performed. Synergism was IN RATS FOLLOWINGEXPOSURE TO HEAVYMETAL AERO­ displayed with nickel and maganese. Selenium did SOLS. S.J. Williams 1, 2, J.Ai Graham3 , F.J. 3 2 not change the functional response of cells to Miller , and D.B. Menzel. Haskell Lab., E.I. nickel but was antagonistic when coexposed with du Pont de Nemours & Co., Inc., Newark, DE. 2 vanadium. Zinc also appears to be antagonistic to 19711; Depts. of Pharmacology and Medicine, nickel exposure. This work demonstrates the util~ Duke Univ. Med. Center, Durham, N.C. 27710; 3 ity of the BAMsystem in in vitro evaluation of HERL,USEPA, Research Triangle Park, N.C. 27711. metal interactions. Supported by the Electric Power Research Institute (Contract #RP1639-2) Heavy metals and polycyclic aromatic hydro­ carbons co-exist in polluted air in the respira­ ble particulate fraction. The metabolism of p-nitroanisole (NA), a model substrate for 169 POTENTIATIONOF PHAGOCYTIC UPTAKE OF NONCARCINO­ mixed function oxidases, was studied in a per­ GENICAMORPHOUS NICKEL SULFIDE PARTICLES RESULTS fused lung preparation following a single 2-hr IN MORPHOLOGICALTRANSFORMATION OF SHE CELLS. exposure of rats to aerosols of NiC12 (430 or J.D. Heck, M.P. Abbracchio and M. Costa, Div. of 150 µg Ni/m 3), CoClz (300 or 125 µg Co/m ), or Toxicol., Dept. Pharmacol., Univ. of Tx. Med. CdClz (450 or 200 µg Cd/m3). Three hr post­ School, Houston, TX. 77025 exposure to the high and low NiClz concentra­ tions, NA 0-demethylase activity was inhibited Particles of crystalline aNiS are avidly by 52 and 43% respectively. At 48 hr, the phagocyti zed by mammaliance 11s, are potently low dosed rats were nearly recovered, while carcinogenic and induce morphological transforma­ activity in the high dosed rats was still in­ tion in Syrian Hamster Embryo(SHE} ce 11s in hibited by 42%. Inhibition was related directly vitro; while particles of amorphousNiS ofsimi­ to the Ni content of the lungs. Inhibition of lar size and elemental composition and having si­ activity was also observed 3 hr following ex­ milar low water solubility are not phagocytized posure to CoC12 aerosols, but was maximal 24- and lack carcinogenic and transforming activity. 48 hr post-exposure (58 and 25% following ex­ Observations of NiS particle binding to charged posure to the high and low level, respectively). filters which indicated that there exists a Recovery was complete 72 hr post-~xposure. difference in particle surface charge between the Following exposure to 200 µg Cd/m no immediate amorphous and crystalline particles were con­ effect was measured, but at 72 hr 0-demethylase firmed by subsequent zeta potential determina­ activity was 54% greater than controls and tions: amorphous and crystalline NiS exhibit sur­ remained elevated by 25% 168 hr post-exposure. face charges of +9.17 and -27.01 mV, respectively Thus, the effects of individual metal salts on at pH 7.2. X-ray photoelectron spectroscopy also pulmonary NA 0-demethylase are unique and these revealed differences in surface composition bet­ results suggest that inhaled metals may alter ween the two particle types. Crystalline NiS the metabolic and distributional profiles of particle surfaces had a Ni/S ratio of 2.1 com­ respirable organic pollutants as well. (Sup­ pared to a ratio of 1.5 for the amorphous NiS ported by EPA Grant R806337, NIH Grant ES01859, particles. The sulfate/sulfide ratio was 0.5 for and a Fellowship from CIIT.) crystalline NiS and 2.0 for amorphousNiS. The

47 present study demonstrates that rendering the (TA-98, with and without rat liver S-9). Chemi­ surfaces of noncarcinogenic amorphousNiS parti­ cal analysis was chiefly by gas chromatography cles more negative by chemical reduction poten­ and mass spectrometry. All materials required tiates th~ir phagocytic uptake and their induc­ metabolic activation to be mutagenic. Rawgas tion of morphological transformation of SHEcells had a specific mutagenicity of 6.7 revertants/µg to levels similar to those seen with carcinogenic (50,000 revertants/1 process stream). Polycyclic crystalline aNiS. Thus it appears that the selec­ aromatic compounds, including methylnaphthalenes, tive phagocytosis of crystalline aNiS particles phenanthrene, fluoranthene and chrysene and by cells rather than a physicochemical feature nitrogen-containing compoundswere positively unique to their crystalline structure compared to identified in highly mutagenic fractions of raw the amorphous form accounts for their potent gas. Gas cleanup by humidifier-tar trap system transforming activity. (Supported by NIEHStrain­ and Venturi scrubber led to a small reduction in ing grant #ES07090,and EPAgrant #R808048,) specific mutagenicity of the gas (4.1 revertants/ µg) but, due to a substantial reduction in mass loading, a significant reduction in overall 170 RESPIRATORYTRACT DEPOSITION AND RETENTION OF mutagenicity was achieved (2200 revertants/1). INHALEDULTRAFINE PARTICLE ASSOCIATED BENZO(A)­ Chemical composition of this material was similar PYRENE,J. D. Sun, R. K. Wolff, G. M. Kanapilly to that of raw gas. Combustion products had a and R. 0. McClellan, Lovelace Inhalation Toxi­ specific mutagenicity of 1.1 revertant/µg (22 cology Research Institute, P.O. Box 5890, Albu­ revertants/1) and also contained several poly­ querque, NM87185 cyclic aromatic compounds. Results indicate that process stream material is potentially harmful to Manypolycyclic aromatic hydrocarbons (PAHs)are workers exposed through fugitive emissions or associated with ultrafine, airborne particles. during maintenance or repair of the gasifier. One example is diesel exhaust. This study in­ Combustion products have relatively low biologi­ vestigated the possible effect of particle asso­ cal activity compared to raw gas. (Research ciation on respiratory tract deposition and reten­ performed under DOEContract DE-AC04-76EV01013.) tion of PAHs. Surrogate diesel exhaust particles were generated by thermal degradation (1100°C) of vaporized (160°C) 67gallium tetramethylheptane­ dione to form insoluble 67Ga oxide particles. Vaporized 3H-benzo(a)pyrene (3H-BaP) was mixed 172 MUTAGENICITYOF PARTICULATE EXHAUST FROM DIESEL with the 67Ga oxide aerosols to obtain 3H-BaP ANDSPARK IGNITION ENGINE CARS. C. R. Clark, coated 67Ga oxide particles (15%3H-BaP by mass). Lovelace Inhalation Toxicology Research Insti­ The ultrafine, 3H-BaPcoated 67Ga oxide aerosol tute, P.O. Box 5890, Albuquerque, NM87185, had a mass median diameter of 0.1 µm with a con­ D. E. Seizinger and T. M. Naman,Bartlesville centration of 4.1 µg/1 of air. Pure 3H-BaPaero­ Energy Technology Center, Bartlesville, OK74003 sols were generated by self nucleation of a simi­ Extracts of particulate exhaust collected from lar 3H-BaPvapor (0.1 µm; concentration= 1.0 µg/ diesel and spark-ignition engine equipped cars 1). Fischer-344 rats were exposed (30 min) to are mutagenic in bacterial test systems. Differ­ each aerosol by nose-only inhalation. Animals ences in gasoline and diesel fuel composition and were sacrificed 30 min, 2, 6, 12 hr, 1, 2, 5, 8 exhaust particle characteristics warrant investi­ and 16 days post-exposure. Trachea and lungs were gation into the chemical characteristics of the taken for both gammaand scintillation counting. particle-associated mutagens. Exhaust particles The deposition 30 min post-exposure in trachea and were collected from four diesel and four spark­ lungs for 67Ga oxide associated, and pure 3H-BaP ignition engine cars operated on a climate-con­ aerosols was 10 ± 7 ng and 134 ± 59 ng, and 9 ± 2 trolled chassis dynomometer. Dichloromethane ng and 66 ± 17 ng, respectively. In the same (DCM)extracts of the exhaust particles were frac­ tissues, the time required to clear 90%of this tionated on silica gel to yield DCMand methanol deposited 3H was 1 day and 1 day, and 1 1/2 hr and (MEOH)eluates and the DCMeluate further frac­ 4 hr, respectively. In contrast, the half life of tionated by high pressure liquid chromatography 67Ga oxide retention was~ 65 days in lung. These (HPLC). Distribution of mass into various HPLC results indicate that particle association of BaP fractions was similar for the 4 diesel samples may facilitate lung deposition and extend the with 76-80%of the mass eluting in nonpolar, ali­ retention times of this PAHin the respiratory phatic and polycyclic aromatic hydrocarbon (PAH) tract. (Research performed under DOEContract DE­ fractions. These fractions demonstrated only AC04-76EV01013.) weak or no mutagenicity in Salmonella strain TA- 98. Most of the mutagenicity (75-95%) eluted in a moderately polar fraction which constituted 171 TOXICOLOGICALANDCHEMICAL CHARACTERIZATION OF 9-11% of the total mass of the extracts. This THEPROCESS STREAM OF ANEXPERIMENTAL LOWBTU fraction has been shown to contain oxygenated and COALGASIFIER. J.M. Benson, S. L. Gunsett, R. L. nitrated PAH. Mutagenic potencies ranged from 10- Hanson and G. J. Newton (Sponsor: R.O. McClellan) 50 revertants/µg compared to 1-7 rev/µg for crude Lovelace Inhalation Toxicology Research Insti­ extracts. The mutagenicity of the silica gel tute, P.O. Box 5890, Albuquerque, NM87185 MEOHe 1 ua te (10-19%of the mass) ranged from 1-3 The process stream of an experimental low rev/µg. In contrast, only 25-36%of the mass of Btu coal gasifier and combustion products of the spark-ignition engine exhaust particle extracts clean gas were characterized as to their muta­ eluted in HPLCnonpolar fractions while more po­ genic properties and chemical composition. lar constituents eluting in the MEOHfraction Samples were obtained at selected positions along comprised 38-49%of the mass. Moderately polar the gasifier process stream using a c_ondenser HPLCfractions had% mass values similar to the train. Mutagenicity was assessed using the diesel samples. (Research supported in part Salmonella mammalianmicrosome mutagenicity assay by DOEContract No. DE-AC04-76EV01013.)

48 173 MUTAGENIC PROPERTIES OF COKE OVEN EMIS­ dinitropyrenes in the presence of either NADH or SIONS AND DIESEL PARTICLES J-S. Siak and T. C. NADPH. The increased activity was partly attributable Pederson, Biomedical Science Department, General to cytosolic enzymes, but also included a cytosol­ Motors Research Laboratories, Warren, Ml 48090. independent activation by reduced pyridine nucleotides Sponsor: J. J. Vostal - presumably the result of a direct non-enzymatic reduction. The dinitropyrenes, as well as rnononitro­ Both coke oven emission and diesel exhaust particles P AH, are believed to be among the pacterial mutagens consist of a carbonaceous core with adsorbed hydrocar­ detected in extracts of diesel exhaust particulate, and bons and carcinogenic effects of coke oven emission their activation as well as deactivation might occur in have been proposed as a model to predict the health rnarn rnalian tissues. The liver microsomal enzyme-cata­ impact of inhaled diesel emissions. However, the lyzed increase of rnononitro-PAH rnutagenicity in the chemical composition of both adsorbed fractions is Salmonella assay is apparently dependent on further different and the benzo[a] pyrene content in coke oven bacterial enzyme activation. emission extract (COE) is much higher than in diesel particle extract (DPE). Nitro-PAH have been identified in diesel particles as the major contributors to the rnutagenic activity and are not detectable in coke oven 175 THE LATE SEQUELAE OF CHRONIC INHALATION OF emissions. In this study, we used the Ames assay and HIGH LEVELS OF DILUTED DIESEL EXHAUST IN THE thin-layer chromatography (TLC) to analyze and com­ RAT LUNG H. J. White, K. L. Smiler, and B. D. Garg, pare extracts of COE and DPE. Particulate samples Biomedical Science Department, General Motors Re­ were collected on Pallflex filters and extracted with search Laboratories, Warren, MI 48090. Sponsor: J. J. dichlorornethane. The extracts and their TLC fractions Vostal were assayed for rnutagenic activity by Salmonella In previous work [J. Appl. Tox., 1:104, 1981] we have typhimurium strain TA98. Of the two extracts, DPE reported the prompt response of the alveoig.r Type II elicited a direct-acting response from TA 98, whereas COE had weak direct-acting response and required rat pneurnocyte during inhalation of 6000 µg/rn of diesel liver S9 preparation to exhibit a stronger response. emission exhaust. The present investigation analyzes When chrornatographed, COE and DPE each displayed further the chronic changes in the rat lung seen afte 3 distinct UV fluorescent patterns. No distinct pattern exposure to diesel particulates at a level of 1500 µg/rn for up to 2 years. Again, the presence of increased was observed in the direct-acting rnutagenic activity of TLC fractions of COE. In contrast, DPE exhibited a numbers of Type II cells and striking increases of intra­ strong response in the rnonosubstituted PAH and polar alveolar phospholipids, a product of these cells, is fractions. For COE, most of the S9 activated rnuta­ clearly evident. This material, along with the high genic activity was found in the polar and P AH frac­ burden of particulate, is ingested by the alveolar mac­ tions. The data indicate that COE and DPE have rophages. As a result, macrophages become foamy in appearance and tend to accumulate and agglomerate at different biological and chemical profiles. The muta­ gens in DPE are mostly direct-acting and attributed to first in alveoli near terminal bronchioles, and later, nitrosubstituted aromatic compounds, whereas the more diffusely. By electron microscopy, needle-like structures probably representing cholesterol are seen mutagens of COE are polycyclic aromatics and polar within alveolar macrophages. At a later stage these compounds which require mammalian enzyme activa­ structures increase in size and can also be seen extra­ tion. Therefore, COE should not be used as a model for cellularly within alveolar spaces. With this change, the assessment of the diesel particle activity in the mast cells appear within alveolar septa in which also living organism. increased collagen is laid down. Occasionally, the focal accumulations of cholesterol and fibrous tissue form a so-called "cholesterol granulorna," a condition that de­ rives from many causes including exposure to other 174 MUTAGENIC ACTIVATION OF NITRO-PAH BY particulates such as Sb2o3, rnarihuana smoke, anti­ MICROSOMAL AND CYTOSOLIC ENZYMES T. C. neoplastic drugs, and even a supposed viral disease in Pederson and J-S. Siak, Biomedical Science Depart­ the feral mongoose. Collagen fibers, are generally ment, General Motors Research Laboratories, Warren, distributed diffusely within the alveolar septa, allowing MI 48090. Sponsor: J. J; Vostal for preservation of the alveolar architectural integrity. Such a benign fibrotic change is probably a corn rnon Rat liver S9 microsomal and cytosolic enzyme activa­ lung response to the long-term inhalation exposure to tion of nitro-PAH compounds in the Salmonella muta­ high levels of non-injurious particles, representing more tion assay was examined using the nitroreductase-defi­ a non-specific reaction of the defense system to the cient strains TA98NR and TA98NR/l,8-DNP 2• Mono­ presence of inert particles, rather than necessarily nitro-PAH compounds such as 2-nitrofluorene, 1-nitro­ related to the nature of the diesel particle per se. pyrene and 6-nitrochrysene were activated by NADPH­ dependent microsomal enzymes when using strain TA98NR, but not TA98NR/l,8-DNP • Other mutagens such as daunornycin and S9-activatea B[a] P were rnuta­ 176 EFFECT OF PROLONGED EXPOSURE TO DIESEL genic in both strains. The lack of rnutagenic activity by EXHAUST ON THE. PULMONARY CLEARANCE OF microsomal rnononitro-PAH metabolites in TA98NR/­ INHALED DIESEL PARTICLES, T. L. Chan, P. S. Lee, l,8-DNP is probably attributable to the absence of and W. E. Hering, Biomedical Science Department, bacteriai enzymes required for conversion of the micro­ General Motors Research Laboratories, Warren, MI somal metabolites to ultimate genotoxic forms. The 48090. Sponsor: J. J. Vostal effects of S9 enzymes on the dinitropyrenes are dis­ tinctly different than those observed with rnononitro­ The effect of diesel exhaust exposure on the pulmonary PAH. NADPH-dependent microsomal enzymes clearance of diesel particles was studied in Fischer 344 markedly reduced the rnutagenicity of 1,3-, 1,6-, and 1,8- rats. Test animals were first exposed to diluted diesel dinitropyrene. A similar decrease in activity was exhaust in exposure chambers at nornin131particulate observed using the unfractionated S9 preparation, but concentrations of O, 250, and 6000 µg/rn . The expo­ the cytosolic fraction increased the activity of all three sure regimen was 20 hrs/day, 7 days/week, lasting from

49 7 to 112 dayl'l Groups of 24 exposed animals were then investigated. Exposure of unanesthetized male exposed to C-tagged diesel particles in a nose-only or female rats to particulate concentrations inhalation chamber within two hours after they were ranging from 0.5 to 6 mg/1 resulted in concentra­ removed from the chronic exposure. The concentration tion-related decreases in breathing frequency and of the racyoactively labelled diesel particles was 6.4 ± minute volume during two hours of exposure. 0.3 mg/m • The exposure was continued for 45 min­ Following a single exposure to 4 mg/1, the num­ utes. In preselected t£meintervals after the radio­ ber of free cells obtained from the lung by active exposure, the C activity in the lung was saline lavage increased,reaching a maximum at measured to determine the retention of diesel particles. 1-2 days after exposure and returning to preex­ Experimental data collected so far for animals having posure value by one week. The number of alveo­ previous particulate lung burdens of approximately 10 lar macrophages lavaged was initially decreased mg of diesel particles, indicated an apparent decrease on days 1 and 2 post-exposure, increased by day in the lung clearance during the first 24 hours after 4, and normal by day 7. The decrease on day 2 exposure, and long-term retention of the radioactive was measured after exposure to several concentra­ diesel particles was significantly higher in the exposed tions and also found to be related to particulate groups even after 50 days. The increased retention may concentration, although no effect was seen after be caused either by the decreased mucociliary clear­ exposure to 0.5 or 1.3 mg/1. Histopathological ance or by phagocytosis of particles by particle-laden evaluation of the lung, the primary site of macrophages migrating to "sequestered" regions in the damage, and pulmonary function tests (including lung. Phagocytosis of radioactive particles by leuko- quasistatic pressure-volume curves and single­ ·cytes with slower clearance might also have been breath carbon monoxide diffusing capacity) were involved in the retention process. The transport of the also performed. radioactive particles into the sequestered regions con­ Sponsored by the U.S. Army Medical Bioengi­ taining "aggregated" macrophages remains to be deter­ neering Research and Development Laboratory and mined; if excluded, the prolonged retention must be the Office of Health and Environmental Research, interpreted as a sign of impaired clearance, which can U.S. Department of Energy, under contract W-7405- potentiate the accumulation of diesel particles in the eng-26 with the Union Carbide Corporation. lungs of animals exposed to high concentrations in chronic inhalation studies.

179 PUIM'.)NARYRESPONSE OF FISCHER 344 RATSAND CD-1 177 EFFECTS OF REPEATEDEXPOSURES TO AEROSOLIZED MICE 'ID EXPOSURE 'ID DIESEL EXHAUST,R.F. DIESEL FUEL. W. Dalbey*, S. Lock*, S. Garfinkel*, R. Jenkins**, R. Holmberg**, and M. Guerin**· Henderson, J. L. Mauderly, R. 0. McCTeTTan,B. V. *Biology Division and **Analytical Chemistry Mokler and H. C. Redman,Lovelace Inhalation Division, Oak Ridge National Laboratory, Oak Toxicology Research Institute, P.O. Box 5890, Ridge, TN. Albuquerque, NM87185 To evaluate the effect on the lung of inhala­ High concentrations of aerosolized diesel fuel tion of diesel exhaust, Fischer 344 rats and CD- are used as visual obscurrants by the military. 1 mice were exposed to diluted, whole diesel The effects of repeated exposure on 'Sprague Daw­ exhaust 7 hr/da, 5 da/wk for 6 months. Animals ley rats of both sexes is being investigated. were exposed to a1r concentrations of 0, 300, Exposure variables include concentration (1.33 to 3300 or 6500 µg/m of particulate matter. Pul­ 6 mg/1), duration of exposure (2 or 6 hours), and monary response was evaluated by analysis of frequency of exposure ·(1/wk or 3/wk). Extensive lung airway washings and enzymatic activity of chemical and physical characterization of the the lung tissue. Parameters measured in the la­ aerosol was performed during the exposures. Ani­ vage fluid included total and differential cell mals received nine exposures and were then tested counts, soluble protein and sialic acid content 1-2 days and 2 weeks post-exposure for a variety and the followin9 enzyme activities: lactate de­ of endpoints. These measurements included number hydrogenase (LOH), glucose-6P-dehydrogenase and phaeocytic activity of lavaged pulmonary (G6PDH),glutathione reductase (GR) and peroxi­ cells, pulmonary function tests, neurotoxicity dase, alkaline and acid phosphatase and s-glu­ screening assays, blood cell counts, body and curonidase. The enzyme activities were also organ weights, clinical chemistry, and histopath­ measured in the lung tissues. Lavage fluid anal­ ology. The most pronounced effects occurred in yses indicated an inflammatory response in the the lungs and included dose-related increases in mice at the mediumand high exposure levels and the number of alveolar macrophages, atelectasis, in the rats at the high exposure level. Largest and changes in pulmonary function. elevations were in GRand s-glucuronidase activi­ Sponsored by the U.S. Army Medical Bioengi­ ties followed by lesser increases in LOHand acid neering Research and Development Laboratory and phosphatase activities, sialic acid and protein the Office of Health and E~vironmental Research, content and leukocyte count. Tissue responses U.S. Department of Energy, under contract W-7405- were smaller in magnitude than those seen in the eng-26 with the Union Carbide Corporation. lavage fluid but indicated an increase in lysoso­ mal enzymes and in GRactivities. The mice also showed an increase in G6PDHactivity. For all 178 EFFECTS OF SINGLE EXPOSURESTO AEROSOLIZED parameters, the mice showed a greater effect at a DIESEL FUEL. W. Dalbey, S. Lock, R. Rice, T. lower exposure level than did the rats. Addi­ Ross, S. Garfinkel, and L. Balogh. Biology tional animals will continue to be exposed and Division, Oak Ridge National Laboratory, Oak examined at six month intervals throughout the Ridge, TN. life span of the rodents to evaluate exposure time-effect relationships for diesel exhaust. The effects of single exposures of Sprague­ (Research supported by DOEContract DE-AC04- Dawley rats to aerosolized diesel fuel were 76EV01013.)

50 180 COMPARISONOF TISSUE BURDENS FOLLOWING WHOLE-BODY of individual experiments to take place simul­ ANDNOSE-ONLY EXPOSURES OF FISCHER-344RATS TO taneously while minimizing personnel time and AEROSOLS.R. K. Wolff, L. C. Griffis, C. H. Hobbs computer costs. (Research performed under an and R. 0. McClellan, Lovelace Inhalation Toxi­ interagency agreement between EPA(79-D-X5033) cology Research Institute, P.O. Box 5890, Albu­ and DOEunder Contract No. DE-AC04-76EV01013.) querque, NM87185 Twogroups of Fischer-344 rats were exposed to 67Ga203aerosols (0.1 µm-volumemedian diam­ 182 CARBONMONOXIDE - NaCl INTERACTIONIN THE eter) either in a whole body or nose-only exposure DEVELOPMENTOF SYSTEMICHYPERTENSION, mode. Lung deposition was very similar for the R. N. Shiotsuka and R. T. Drew, Medical Dept., two exposure modes: 15 ± 3%for whole body and 15 Brookhaven National Laboratory, Upton, N.Y. 11973 ± 7%for nose-only. Pelt deposition was quite The influence of carbon monoxide (CO) on the high for whole-body exposures: 2.1 times the development of systemic hypertension was studied initial lung burden as compared to 0.3 times for using Dahl rats selectively bred for susceptibility nose-only exposures. Pelt activity was primarily (S) and resistance (R) to NaCl induced hypertension. confined to headskin for nose-only exposures. Forty "S" and 40 "R" female rats were maintained on Since the Ga203 particles were relatively insol­ a low (0.4%) salt diet until 5 weeks of age when 20 uble very little was absorbed systemically. Most "S" and 20 "R" rats were transferred to a high (8%) of the excreted radioactivity was in feces and it salt diet. At 6 weeks of age, groups of 10 "S" and represented material initially deposited in the 10 "R" rats from the low and high salt groups were upper respiratory tract and on the pelt. Total exposed to 500 ppm CO, 21 hours/day for 62-63 activity excreted in feces expressed as percent of consecutive days. This exposure resulted in an initial lung burden was 1.6 times greater follow­ equilibrium carboxyhemoglobin concentration of 42%. ing whole-body exposures than for nose-only Carbon monoxide exposure and a high salt diet pro­ exposures. Subtracting amounts expected to be moted the development of hypertension in "S" rats cleared from the respiratory tract as determined when compared to "S" rats fed an identical salt from initial deposition measurements suggested diet and exposed to air. Three of the.10 CO that approximately 70%of pelt activity was in­ exposed "S" rats on the high salt diet died during gested following whole-body exposures as compared the last week of exposures. All air exposed "S" to approximately 90%for nose-only exposures. The rats on the high salt diet survived. No "R" rats high gastrointestinal intakes of material and nor "S" rats on low salt diet developed hyper­ possible absorption for more soluble materials tension and all rats in these groups survived the must be considered in inhalation exposures, par­ study. Carbon monoxide induced a cardiomegaly of ticularly whole-body exposure, when assessing 36% in "S" rats on both high and low salt diets. toxicological effects. (Research performed The "R" rats exposed to CO exhibited a 29% and a under DOEContract NumberDE-AC04-76EV01013.) 22% cardiomegaly on high and low salt diets, respectively. CO exposure also produced varying degrees of splenomegaly. There were no changes in whole body, brain, or adrenal weights attributable 181 A SIMPLECOMPUTER INTERFACED INHALATION EXPOSURE to CO exposure. The consistent hematologic response SYSTEMFOR TOXIC GASES. R. E. Gregory, L. R. to this hypoxic challenge was elevated total hemo­ Wilson and J. A. Pickrell (Sponsor: R. ·O. globin (~77%) and hematocrit (~57%), irrespective McClellan), Lovelace Inhalation Toxicology Re­ of diet or line of rat. Thus, CO in conjunction search Institute, P.O. Box 5890, Albuquerque, NM with a high salt diet enhances the development of 87185 systemic hypertension in the susceptible line of A simple computer-controlled and monitored Dahl rats. small animal inhalation exposure system has been *Research supported by the U.S. Dept. of Energy designed and constructed. Unlike other computer under contract DE-AC02-76CH00016 and NIEHS Training interfaced exposure systems which use individual Grant No. 5T-32ES07088-03. stand-alone minicomputers, our system operates in a time-sharing configuration with the Institute's main computer system. The exposure system is 183 EFFECTS OF CADMIUMON THE HEPATOTOXICITYOF BROMO­ interfaced through a specially designed serial BENZENEIN RATS. S. Chakrabarti and J. Brodeur. control unit (SCU)which uses a simple prepro­ Dep. med. travail et hyg. milieu, Fae. Med., Uni¼ grammedmicroprocessor in a multidrop configura­ de Montreal, Montreal, Quebec, Canada H3C 3J7. tion. This device allows on-line, real-time com­ The hepatotoxicity of bromobenzene (BB) is due puter control and data acquisition without full­ to its metabolic activation to bromobenzene epoxi­ time computer commitment. The exposure system de mediated by hepatic microsomal mixed function was designed to expose groups of rats to O ppm, oxidase (MFO) system. Cadmium is known to inhibit 1 ppm, 5 ppmof nitrogen dioxide (N02) and 1 ppm the hepatic MFO system and hence, could affect the with two spikes to 5 ppmper day, 7 hours/day, hepatotoxicity of BB. When male rats were treated 5 days/week for the animals' life span. The with 0.2, 0.5 and 1 mg/kg (i.p.) of cadmium chlo­ computer, after initial operator interaction, ride (CdC12) 24 h prior to the i.p. injection of controls the initiation of exposure, the adminis­ 2.5 mmole/kg of BB and sacrificed 48 h after the tration of N02 spikes, chamber gas sampling and BB dose, a significant decrease in the SGPT acti­ analysis and termination of exposure. The com­ vity and a decrease (but not significant) in SGOT puter also continuously monitors and records activity as well as a significant decrease inSGPT (24 hours/day) chamber flow, pressure, tempera­ activity were observed in 0.2 mg/kg and 0.5 mg/kg ture and relative humidity and sets alarms when of CdCl2-treated rats respectively. Similarly a these parameters exceed established limits. The reduction in the activities of SGOT and SGPT (al­ use of the SCUin computer-controlled and moni­ though not significant) from those of BB-alone­ tored exposure systems will allow a large number treated rats was noticed when rats were given 1

51 mg/kg of CdC12 6 h prior to BB (1 mmole/kg) or We reported previously _that metallothionein (MT) simultaneously with BB and sacrificed 24 h after levels, measured by radioimmunoassay (RIA),increase the BB dose. In a separate study, different groups in plasma and urine of rats upon Cd exposure of rats were fed 25, 100 and 200 ppm of CdCl2 in (Fundam.Appl. Toxicol.,1:1, 1981).The purpose drinking water daily for four weeks prior to the of this study was to investigate whether exposure administration of a single 2.5 mmole/kg dose of to some other divalant metals will also result in BB and were sacrificed 48 h after the BB dose. No similar elevation in MT levels. Female Sprague­ reductions in the activities of SGOT and SGPTwere Dawley rats (248±9 g) were injected subcutaneously noticed except that of SGPT was decreased close to with either saline, 5 umoles/Kg/day of CdCli,HgCl~ significance in 200 ppm CdC12-pretreated rats. PbCl2, CuS04 or ZnCl2 for 5 days. Blood and urine· However the liver microsomal system in such chro­ samples were collected on days 0,1,3 and 5 and nic Cd-treated rats showed a significant decrease analyzed for MT by the RIA. The plasma MT levels in a) liver weights, b) microsomal protein concen­ were low and ranged from less than the detection trations, and c) the activities of aniline hydro­ limit (150ng/ml) to 320ng/ml. Rats injected with xylase and aminopyrine N-demethylase, but the le­ Cd or Hg had higher levels of MT in their plasma vels of cytochrome P-450 did not change. Thus, than rats injected with other metals or saline. among other factors, the depression of MFO system The urinary MT level in control rats on day O was due to different Cd-pretreatments may not be suf­ 0.85±0.17mg/g creatinine. There was no significant ficient enough to reduce significantly themetabo­ change in urinary MT level in rats given up to 5 lic activation of BB to its epoxide and hence its injections of Pb or Cu or a single injection of hepatotoxicity. (Supported by MRC Grant, MA-6159) Cd or Zn. Only Hg-injected rats showed a five-fold increase in MT level on day 1. In rats given 3 injections of Zn or Cd the urinary MT concentracion 184 DEXAMETHASONE-INDUCEDALTERATION OF THE DISPO­ increased about two- and four-fold, respectively. SITION OF Cd AND TRACEMETALS IN DEVELOPINGRATS. While the mean MT level in Hg-injected rats in­ E.M.K. Lui, Dept. of Pharmacol. and Tox., The creased steadily during the five days to almost Univ. of Western Ontario, London, Ont., Canada 21 times of the mean control value, those of the Sponsor:· G.M. Cherian Cd- or Zn- injected rats did not change signif­ icantly after three days. In conclusion, it Recent studies have shown that glucocorticoid appears that the urinary MT levels are affected by stimulates the uptake of Zn and Cd in hepatic exposure to not only Cd but also Hg and Zn. Also, tissues of adult rats. Since Zn contents as well the elevation in urinary MT level appears to be as metallothionein levels are much higher in the greater after Hg treatment than after Cd or Zn newborn period than in adulthood, we have investi­ treatment. (Supported by NIH Grants ES 01247, gated the effect of dexamethasone (Dx) treatment ES 01448 and ES 07026). on hepatic trace metal metabolism as well as the disposition of Cd in the neonatal rat. To assess the effect of Dx on trace metal contents in the liver, Dx (1 ug/gm) was administered twice daily 186 EFFECT OF CHRONICEXPOSURE AND WITHDRAWALOF CAD­ by sc. injection to newborn rats on days 3,4,5, MIUMON HEPATIC AND RENALGLUCONEOGENIC ENZYMES and 6 post-partum and killed 72 hr later. Dx IN RATS. B. Rajanna, K. D. Chapatwala, M. Hobson, treatment reduced the hepatic Zn levels by 50% but and D. Desaiah. Div. Nat. Appl. Sci., Selma Univ., increased the hepatic Fe contents by 2-3 fold. In Selma, AL 36701 and Dept. Neurol. Univ. Miss. contrast, Dx treatment did not alter these two Med. Ctr., Jackson, MS 39216. parameters in the kidney. Moreover, the hormone Adult male Sprague-Dawley rats were exposed to 25, treatment resulted in a reduction of the increase 50 and 75 ppm cadmium (C) mixed in their diet for in body weight as well as an increase in both the 6 months in order to study the chronic effects of liver and kidney to body weight ratios in the neo­ Con gluconeogenesis. Experiments also were con­ nates. To examine the disposition of Cd in neo­ ducted to determine the reversibility of the ef­ natal rats, CaC12 (1 mg/kg, sc.) was administered fects in the metal withdrawn rats. Body weight on days 7 and 8 postpartum and the neonates were gain, serum protein (SP), serum glucose (SG), se­ killed 24 hr following the second injection. High rum enzymes (SGOT, SGPT), hepatic and renal glu­ Cd concentrations were attained in the liver; the cose -6, phosphatase (G-6,Pase), fructose-1,6, total hepatic Cd contents represented 17% of the diphosphatase (FDPase) and phosphoenol-pyruvate total dose of Cd. The Cd concentrations in extra­ carboxykinase (PEPCK) were determined at 2,4 and hepatic tissues (kidney, heart, lung, gastro­ 6 months after treatment and 4 and 6 months after intestinal tract) were approximately 8-12% of cessation of the treatment. A significant 20-30% those detected in the liver. However, brain reduction in body weight gain was observed in ra­ tissues contained small amounts of Cd. Dx pre­ ts receiving 50 and 75 ppm C. Rats withdrawn treatment (days 3 to 6) reduced the hepatic Cd from C showed normal weight gains. SG levels we­ concentrations by 25%. This was accompanied by an re significantly increased in rats fed on 50 and increase in the Cd concentrations in extrahepatic 75 ppm C. SP was not significantly changed from tissues. These data suggest that Dx treatment controls. Three-four fold increase in SGOT and i) altered metabolism of Zn 1and Fe in the liver SGPT levels were seen in rats fed on Cat all ti­ and ii) reduced hepatic uptake of Cd in neonatal me points. SGOT levels returned to near normal rats. in rats withdrawn from C but SGPT levels stayed higher than normal in 50 and 75 ppm groups. All 3 gluconeogenic enzymes both in kidney and liver 185 EFFECT OF SUBACUTEEXPOSURES TO CADMIUM,MERCURY, were significantly elevated in C treated rats ex­ LEAD, COPPER AND ZINC ON THE PLASMAAND URINARY cept in 25 ppm at 60 days after treatment. The METALLOTHIONEINLEVELS IN RATS. Y.H. Lee, C. increase in enzyme activities was dose and time Tohyama and Z.A. Shaikh, Division of Toxicology, dependent. Rats withdrawn from C showed a rever­ University of Rochester, Rochester, NY. sal of effects with all 3 enzymes. G-6,Pase was,

52 however, significantly reduced 6 months after two hours after administration. The metabolite, withdrawal.' The significant increase in SG and dimethylarsinic acid, appeared in the urine by gluconeogenic enzymes with C exposure and rever­ one hour after administration of either form. sal of these parameters after withdrawal of the Thus, there appeared to be a quantitative differ­ metal suggest that C may be altering the carbohy­ ence in the uptake of the two forms by the liver; drate metabolism in rats. (Supported by PHS this question and the possibility of a metabolic Grant RR-08169). difference were examined by using isolated hepa­ tocytes. The hepatocytes took up 14-33% of a dose of 2.4 nmole arsenite but less than 2% of a 187 HEPATOTOXICITY AFTER ACUTE EXPOSURE OF RATS dose of arsenate. Furthermore, analysis of the TO CADMIUM.R.E. Dudley and C.D. Klaassen, Dept. medium showed that the hepatocytes rapidly meta­ bolized arsenite, but not arsenate, to dimethyl­ of Pharmacology, Univ. of Kansas Medical Center, arsinic acid. When the dose of arsenite was in­ Kansas City, KS. creased to 12 or 24 nmole, the amount of meta­ bolite also increased; at 120 nmole arsenite, the While the liver is generally not considered a amount of metabolite dropped. Thus, the liver target organ of Cd toxicity, we observed severe appears to be more important in the metabolism injury after acute administration of Cd to rats. of arsenite than in the metabolism of arsenate. Therefore, we examined the effect of a high dose of Cd ( 3. 9 mg Cd/kg) on the liver. Rats were (Supported by NIH Grants ES01247, ES01448, injected iv with Cd and sacrificed 0, 1, 2, 4, 6, ES07026) 8 and 10 hrs later. Plasma activities of alanine amino transferase (ALT), aspartate amino trans­ ferase (AST), and alkaline phosphatase (AP) were 189 ABSORPTION,CIRCULAR DICHROISM AND MAGNETIC CIR­ determined as were plasma concentrations of CULARDICHROISM STUDIES OF BISMUTHCONTAINING bilirubin (BIL) and glucose (GLU). In addition, METALLOTHIONEINS.Martin J. Stillman and Jadwiga heart, lung, duodenum, kidney, spleen, liver, A. Szymanska, The University of Western Ontario, skeletal muscle and testis were removed for light Department of Chemistry, London, Ontario, Canada microscopic evaluation. ALT and AST activity N6A 5B7. Sponsor: G.M. Cherian rose from control levels of 58 and 47 SF units/ml to peak activities of 2500 and 2233 at 10 hrs. Metallothionein (MT) induction by variety of ions AP activity rose from 10.5 to 37.1 BL units/1 (Cd, Zn, Cu, Hg, Ag) is well known•. However, while BIL concentrations increased From 0.20 to recently Bi has been shown to induce MT, and Bi 0.81 mg/dl. These changes are similar to those is subsequently found incorporated in the MT. observed with high doses of CC1 ( 1 ml/kg, ip). 4 Absorption, circular dichroism (CD) and magnetic In addition, plasma GLU levels decreased signifi­ CD spectra of Bi-containing MT isolated from rat cantly after Cd. Histopatho logical changes were livers and kidneys have been measured. These not observed in tissues other than liver and data are compared with spectra observed for C'd, testis. Pathological changes were already Zn-MT 1 and 2 from rat livers. The CD and MCD evident in liver 1 hr after Cd exposure and spectra of the renal equine and hepatic rat included parenchymal cell enlargement, eosino­ metallothionein are remarkably alike. Both sets philia, increased numbers of mitotic cells of samples exhibit a derivative-like CD with a and occasional necrosis. At 4, 6, and 8 hrs, positive lobe at 250 nm, and a positive, deriva­ isolated parenchymal cell enlargements and tive-shaped MCDwith a negative lobe at 251 nm. eosinophilia persisted and the extent of necrosis The Bi-containing protein samples do not exhibit was increased. Ten hrs after Cd, parenchymal such features. The Bi, Zn-MT appears quite cell necrosis was more extensive and was accompa­ similar to Zn-MT. Bi model compound data, pH nied by fatty vacuoliz~tion. These changes were titration and Bi loading experiments indicate diffuse and not localized to a specific region that absorption near 290 nm most probably arises ( i.e. periportal vs centrolobular). These data from the Bi in the protein. indicate the liver is a major target organ of acute Cd toxicity. (Supported by USPHS Grants ES-07079 and ES-01142) 190 EFFECT OF IRON CHELATORS ON THE MECHANISM 188 A POSSIBLE DIFFERENCEIN THE SITE OF METABOLISM OF NADPH-CYTOCHROME Pt+50 REDUCTASE­ OF ARSENITEAND ARSENATE IN RATS. S.A. Lerman DEPENDENT LIPID PEROXIDATION. J.R. Bucher, M. and T.W.'Clarkson, Dept. of Rad. Biol. & Biophys. Tien, L.A. Morehouse, and S.D. Aust, Dept. of Univ. of Rochester, Rochester, NY. Biochem., Michigan State Univ., E. Lansing, MI t+882t+ The initiation of NADPH-dependent lipid peroxidation Differences in the kinetics and metabolism of (LP) by NADPH-cytochrome Pt+50 reductase is thought arsenite and arsenate were studied in rats. Five to occur either through an iron catalyzed Haber-Weiss minutes after IV administration of 4.8 nmole ar­ reaction resulting in hydroxyl radical ( •OH) formation, senite or arsenate to male Sprague-Dawley rats or through formation of a reactive ADP-Fe-oxygen blood levels of arsenic were only 10% of the ini­ complex. The two possible mechanisms were assessed tial dose. Blood arsenic levels then rose; by in two model LP systems. The reductase, NADPH and four hours about 60% of the initial dose of ar­ the iron chelates were incubated with liposomes of senite and 30% of the initial dose of arsenate extracted rat liver microsomal phospholipids, or with was in the blood compartment. The liver contained linoleic acid dispersed by the addition of 1% lubrol. LP, 16% of the dose five minutes after arsenite admin­ NADPH oxidation, and O consumption by the reductase istration but this dropped to less than 3% by all require added EDTA-belated iron (EDTA-Fe) and do two hours. The liver contained less than 2% of not occur when all the iron present is chelated with the initial dose of arsenate at five minutes and ADP or in the absence of iron chelates. LP with EDTA-

53 Fe does not occur in the liposomal system, but does Toxicity resulting from exposure to inorganic occur with linoleate dispersed with detergent. EDTA- · salts of manganese (Mn) is frequently character­ Fe-dependent LP is inhibited by •OH traps (100 mM ized by extrapyramidal neurologic symptoms. In mannitol, 42%; 100 mM ethanol, 40%) and catalase (1 animals, numerous studies with Mn have reported unit/ml, 37% inhibition), and formation of the •OH• changes in brain neurotransmitters, especially DMPO adduct is seen with EPR spectroscopy. In dopamine (DA), which may be related to these .contrast, reductase-dependent LP with EDTA-Fe and symptoms. ADP-chelated iron (ADP-Fe) is not sensitive to Injecting mice with the organic fuel-additive, inhibition by •OH traps in the liposomal system (0-9% methylcyclopentadienyl-Mn-tricarbonyl (MMT)on a inhibition by concentrations of • OH traps sufficient to subchronic schedule (80 mg/kg,sc, on alternate inhibit purely • OH dependent LP by 88-94%), but is days for 3 weeks) resulted in a small, but signi­ somewhat sensitive in the detergent dispersed system ficant increase in brain Mn and an accompanying (100 mM mannitol, 22%; 100 mM ethanol, 14%). In 18-23% decrease in striatal and limbic DA con~en­ addition, LP is stimulated (390%) by the addition of trations. Acute injections failed to produce ADP-Fe to EDTA-Fe. EPR spectroscopy shows no these changes. The DA metabolite, dihydroxyphenyl •OH-DMPO adduct when the reductase is incubated acetic acid, was also decreased after MMT. In with NADPH, EDTA-Fe and ADP-Fe, the system which addition, the pharmacologic effects of amphetamine catalyzes maximum rates of LP. The data indicate which depends on the release of presynaptic DA reductase-dependent LP with EDTA-Fe may occur for its effects, was also altered by MMT. The though an • OH-dependent mechanism, but in the dose-response curve for amphetamine-induced stim­ presence of ADP-Fe, the mechanism likely involves an ulation of motor activity was shifted to the left ADP-Fe-oxygen complex. Supported by NSF PCM-79- in the subchronic MMTgroup, while the stimulant 15328. effects of the direct DA receptor agonist, apo­ morphine was not reduced. These results suggest that MMTproduces a biochemical and functional 191 AMELIORATIONOF MANGANESE-INDUCEDALTERATIONS OF deficit in brain DA synaptic activity, similar to DRUGRESPONSE AND METABOLISM BY PRETREATMENTWITH the effects reported for inorganic Mn. (Supported SUBTHRESHOLDMANGANESE. M.J. Deimling and R.C. by NIH Grant# ES02511.) Schnell, Department of Pharmacodynamics and Toxicology, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68105 193 EFFECTSOF DIETARYPROTEIN CONTENT ON LOCOMOTOR Acute treatment with manganese chloride at a ACTIVITYDURING CHRONIC LEAD EXPOSURE IN MALEAND threshold dose of 5 mg Mn++/kg (i.p.) produces a FEMALERATS. A.J. Verlangieri, J.J. Meyer, T.B. significant prolongation of barbiturate-induced Barnes and J.C. Kapeghian, Dept. of Pharmacology, hypnosis in the male rat as a result of decreased Sch. of Pharmacy, Univ. of Mississippi, Univ., MS hepatic metabolism by the microsomal mixed 38677. Sponsor: W.M. Davis. function oxidase (MFO) system (Pharmacologist 22, Nutrition has been reported to be a factor in 199, 1980). Additional experiments showed this lead-induced toxicity in rats. To examine this was attributable to decreased levels of more fully, male and female Wistar rats were main­ cytochrome P-450. Recent experiments have shown tained on either a low, normal, or high protein­ that the administration of a subthreshold dose of to-carbohydrate diet and received either 75. ppm of 1 mg Mn++/kg (i.p.) to male rats three days prior lead acetate in the drinking water or deionized to treatment with a threshold dose of Mn produces water for 52 weeks. Locomotor activity was mea­ a significant protection against enhanced drug sured routinely in Wahman LC-33 running whee1 response as measured by the duration of cages. Mean food and water consumption, body hexobarbi tal hypnosis. Rats treated with a weights, and urinary delta-aminolevulinic acid threshold dose of Mn slept significantly longer (ALA) levels were recorded for each group. By than the control group while animals treated with week 52, the low protein diet alone resulted in subthreshold Mn alone, or prior to receiving a significant reductions in body weights of both threshhold dose of Mn, awoke at time periods sexes while high protein produced no effect. Chron­ which did not differ significantly from the ic lead ingestion further reduced body weights of control group. Examination of aniline, both sexes in the low protein group but resulted ethylmorphine, and hexobarbital metabolism by the in an elevation in this parameter in males on the hepatic microsomal fraction in vitro revealed normal diet. Water consumption was unaffected by that pretreatment with subthreshold Mn could diet or lead treatment. Food consumption was un­ protect against decreased hepatic MFOactivity changed by diet but was significantly elevated by which occurs following treatment with a threshold chronic lead ingestion in both sexes on low pro­ dose of Mn alone. Adsci ti tious experiments tein. Urinary ALA was reduced by low protein revealed that amelioration of manganese-induced while lead ingestion produced significant increas­ decreases in hepatic MFO activity could result es in this parameter by week 52 in all groups. The from protection against decreased levels of low protein diet produced significant increases in hepatic cytochrome P-450 which were observed in locomotor activity in both sexes while high pro­ animals treated with a threshold dose of Mn alone tein decreased activity in males. Under a normal but not in animals treated with subthreshold Mn diet, females demonstrated a higher locomotor ac­ alone, or in combination with threshold Mn. tivity than males, however this was transient by (Supported by NIEHS Grant ES-02425) week 52. Chronic lead ingestion significantly in­ creased locomotor activity of both sexes on low protein. This response was also observed in males on high protein. Under normal dietary conditions 192 ORGANO-MANGANESEINDUCED ALTERATIONS IN BRAIN lead exposure lowered locomotor activity in males DOPAMINE. G. Gianutsos and M. T. M11,:-ray,Sect. and increased activity in females. Thus, the nu­ Pharmacol. & Toxicol., U. Conn., St~~rs, CT tritional component in chronic lead exposure has 06268. Sponsor: _§_.~- Cohen. geen shown to interact in it's toxicity profile.

54 194 LEAD EXPOSUREAND RENALFUNCTION IN RATS: IS THE 196 THE EFFECT OF LEAD ON THE RABBIT RETINA D.M. RAT A GOODMODEL FOR LEAD INDUCEDNEPHROPATHY? Talsma, K.L. Stemmer, Univ. of Cincinnati Medical W.D. Adams, E.J. O'Flaherty, and E. Taylor. Center, Kettering Lab., Cincinnati, OH 45267 Dept. of Environmental Health, University of Evidence exists that the toxicity of lead may Cincinnati, College of Medicine, Cincinnati, OH. be related to its pro-oxidant effect on cellular 45267 SPONSOR: P.B. Hanunond. components. The retina is susceptible to this Lead nephropathy, characterized functionally by type of stress, and the present study was per­ depression of glomerular filtration rate (GFR) formed to determine the ocular effects of sys­ and effective renal plasma flow (ERPF), is temic lead administration. associated with prolonged occupational exposure Rabbits were given drinking water with zero to lead. Production of comparable lead-related or 2000 ppm of added lead for periods of eight renal functional deficits in rats has been diffi­ or twelve weeks. They were sacrificed following cult to achieve. Recently, Aviv et al. (Kidney lead administration. Their retinas were examined International 17, 430-437 (1980) reported re­ ultrastructurally. Retinal catalase activity and ductions in GFRand in ERPF in weanling rats malondialdehyde (MA) levels were also assayed. given large amounts of lead in their drinking Structural changes after 8 and 12 weeks in­ water. We have examined in rats some of the cluded swelling of the retinal pigment epithelium factors that might be expected to influence the (RPE), thickening of the RPE basal lamina, and likelihood of developing lead-induced renal func­ an increase in lipofuscin within the RPE of ex­ tional damage. These factors include magnitude posed animals. Thinning of the photoreceptor of lead exposure, age of the rat, diet, and nuclear layer also occurred. occurrence of generalized lead toxicity as re­ Catalase activity was decreased in the exposed flected in body weight loss. The relative im­ animals and MA levels were increased. portance of these factors has been evaluated us­ These findings point to an effect of lead on ing GFR (as inulin clearance) and ERPF (as para­ the oxidant status of the retina and further in­ aminohippurate clearance) measurements as indices dicate the sensitivity of these organ to pro­ of renal functional competence. Diets examined pxidant. stresses. were the NIH-07, AIN, and standard Purina Rodent Chow diets. (Supported by USPHS grants ES 07073 and ES 01872). 197 LEAD UPTAKEIN BRUSH-BORDERVESICLES FROMRAT KIDNEY CORTEX. W. Victery and B.A. Fowler, Lab­ 195 CELLULARMETABOLISM OF LEAD: A KINETIC ANALYSIS oratory of Pharmacology, NIEHS, Research Triangle IN THE ISOLATED RAT HEPATOCYTE. J.G. Pounds and Park, NC 27709 R. Wright, Natl. Cntr. for Toxicol. Res., Jefferson, AR 72079. Sponsor. L. Fishbein Lead accumulates rapidly and quite specifically Much effort has been directed toward understand­ in renal proximal tubule cells but the mechanism ing the pharmacokinetics of lead in humans and in of this process is not well understood. In this experimental animals, particularly to the simi­ study, isolated plasma membrane vesicles from rat larities and interactions with calcium metabolism. kidney cells were used to evaluate the nature of Relatively little attention has been given to the the lead transport process. Vesicles prepared subcellular distribution and kinetic behavior of according to the technique of Beck and Sacktor lead. Experiments were conducted to characterize were incubated with Pb-203 with carrier Pb ace­ the steady-state kinetic behavior of Pb++ and to tate under a variety of conditions. At timed in­ identify the biological structures associated tervals (0-90 min), the reaction was stopped by with kinetic compartments. Hepatocytes were iso­ addition of cold buffer and rapid Millipore fil­ lated from Sprague-Dawley rats by enzymatic per­ tration. Washing the vesicles with buffer con­ fusion, placed into primary cell culture, labeled taining 1 mMPb acetate or 1 mMCaC1 showed that 2 with 210pb as 3uM Pb(N03)2 for 3 or 24 hours, and Pb was more effective in displacing nonspecifical­ the kinetic parameters determined by analysis of ly bound label and hence all experiments were per­ desaturation curves. Extracellular compartments formed with 1 mMPb acetate in the stop bath. were defined by sensitivity to EGTA and LaCl3 and Lead uptake was found to be time dependent and intracellular compartments identified by their appeared to saturate at 100 µM Pb in the incuba­ behavior following exposure of the cells to KCN, tion mixture. To differentiate between Pb uptake Ouabain, 2,4-dinitrophenol, cAMP, Antimycin A, or a.nd binding, Pb uptake was studied in vesicles phosphate. The kinetic behavior of lead can be whose volume was osmotically altered by sucrose described by a three compartment system. Since addition to the incubation media. It was found no kineti'c pool was labile to chelation by EGTA that Pb concentration in the vesicles varied di­ or to displacement by LaCl3 ,· it is concluded that rectly with vesicle volume indicating uptake. unlike ca++, hepatocytes do not contain a signi­ Vesicle incubation in either a NaCl or KCl gradi­ ficant pool of Pb bound to the cell surface. The ent (extravesicular > intravesicular concentra­ intracellular Pb is characterized by two small tion) increased the initial rate of Pb uptake by compartments (42 and 46 pmol/mg cell protein) with approximately 50% but not the final equilibrium desaturation halftimes of 2 and 20 minutes value. Incubation of vesicles at 0°C did not al­ respectively. The bulk (80%) of the cellular ter Pb uptake; however, incubation with either 210pb is found in the kinetically deep compart­ 5 mMEDTA or EGTA reduced Pb uptake to approxi­ ment which contains 330 pmol/mg and exchanges with mately 60% of control values. The data indicate a halftime of 600 minutes. This compartment is that Pb is extensively bound to the brush-border believed to include mitochondrial 210pb, based on membrane under in vitro conditions but also demon­ its sensitivity to KCN, dinitrophenol, and phos­ strate that accumulation occurs within the mem­ phate. These findings indicate that the cellular brane vesicles and that the rate of this process metabolism of lead is very similar to the cellu­ can be influenced by extravesicular ion composi- lar metabolism of calcium in hepatocytes. tion. (Supported by NIEHS 1 F32 ES05213).

55 198 INCREASEDNEURON SPECIFIC ENOLASE (NSE) LEVELS IN were again exposed to Pb-Glu (10-5M to 10-2M Pb) RATBRAINSTEM FOLLOWING PERINATAL LEAD TREATMENT for 30 min. The cellular toxicity of Pb was E.T. Roginski, F.C. Beuthin, R.T. Louis-Ferdinand measured using an inverted phase contrast and P.J. Marangos; College of Pharmacy and Allied microscope or by membrane leakage test using 3H Health Professions, WayneState University, Deoxy Glucose loading. The Pb pretreated cells Detroit, MI 48202 and Clinical Psychobiology showed significantly less toxic effects of Branch, NIMH,Bethesda, MD20205 further exposure to Pb than the control cells both in morphology and in the membrane leakage Increases in NSEare observed during post­ test. The results suggest that INIB may have a natal neuronal maturation and following treatments protective role in Pb toxicity. which increase dopaminergic activity. Since peri­ (Supported by NIH, ES 01535) natal lead treatment has been associated with de­ layed CNSdevelopment and altered dopaminergic function, the objective of this study.was to de­ 200 TESTOSTERONE-MEDIATEDSEXUAL DIMORPHISM IN THE termine the effect of lead on NSElevels and METABOLISMOF INORGANICMERCURY IN MICE. total enolase activity in the weanling rat. Z.A. Shaikh, Div. of Toxicology, University of NewbornSprague Dawley rats received lead Rochester, Rochester, NY. acetate (10 mg/kg/day, i.p.) and littermate con­ trols received equimolar sodium acetate from day Mice exposed to Hg vapor exhibit sexual dimorphism of birth to postnatal day 20. Pups were sacri-­ in its metabolism (Toxicoligist, 1:438, 1981). ficed on day 21 by decapitation, trunk blood Whether this sex difference is due to differential collected, and brains dissected into four areas; uptake and retention of Hg after its oxidation to cerebrum, cerebellum, hippocampus and the rest of Hg2 + was examined in the present study. In 2 the brain (brainstem). addition, the role of testosterone in Hg + metabo­ A slight but significant decrease (14.5%) in lism was also evaluated. Adult male mice of C3H 203 body weight in the lead treated group was associ­ strain were injected i.p. with 1 µmole HgC12 /Kg ated with increased blood lead levels (control= and sacrificed 24 hr later. As deter~ined by 5.7+ 1.9 ug/dl; lead treated= 521.0+ 42.0 ug/dl). whole-body counting, the retention of 203 Hg was There was no change in the specific activity of significantly lower in females as compared to the total enolase in any brain region while NSE levels males; the female mice lost about 40% of the were unaltered except in the brainstem where an injected dose, whereas, the male. mice lost less increase (18.8% p< 0.05) was seen. The ratio, µg than half of this amount. The greater retention NSE/unit enolase, was also increased (26% p< 0.05) of Hg in males was due to the accumulation of the in brainstem only. metal in their kidneys; the kidneys of these mice These date indicate: (1) neuronal maturation contained about 40% of the dose as compared to as measured by brain NSElevels is not decreased those of the female mice which contained only 25% at postnatal day 21 by perinatal lead treatment at of the dose. Six percent of the dose was found in doses which produce a decrease in body weight and livers of both male and female mice. No signifi­ (2) perinatal lead exposure increases the fraction cant difference in the intracellular distribution of NSE isozyme in the brainstem. (Supported by of Hg was noted. Metallothionein accounted for USPHSNIH Grant No. ES 01638) up to one-fourth of the tissue Hg in both liver and kidney tissues of male and female mice. In orchidectomized males the metabolism of Hg was 199 THE PROTECTIVE ROLE OF INTRANUCLEARINCLUSION very similar to that in the females. Similarly, BODIES IN LEAD TOXICITY IN RAT KIDNEY PRIMARY pretreatment of the female mice with testosterone CELLS. abolished the differences in Hg metabolism between males and females. Thus, it seems that there are T.A. Twarog, and M.G. Cherian; Dept. of Pathology sex differences in Hg2 + metabolism and that the university of Western Ontario, London, Ontario, renal uptake and retention of Hg2 + is affected by Canada. testosterone levels in the animals. The presence of ultrastructurally (Supported by NIH Grants ES 01247 and ES 01248) discernible intranuclear inclusion bodies (INIB) in certain tissues is considered to be a characteristic feature of lead poisoning. INIB 201POSTNATAL ONTOGENYOF THE NEPHROTOXICRESPONSE have been identified as lead bound to non-histone TO MERCURICCHLORIDE. G.P. Daston and R,J, acidic proteins (Lab. Invest. 29: 488, 1973). In Kavlock, HERL, USEPA, Research Triangle Park, a previous study from our laboratory, we have NC (Sponsor: N, Chernoff) shown the formation of characteristic INIB (Toxicol. Appl. Pharmacol. 56: 418, 1980) in rat The toxicity of mercuric chloride (MC) to kidney epithelial cells in primary cultures, on the adult rat kidney is well documented; however, exposure to an equimolar complex of lead-glutamic the renal response in the developing animal is acid (PbGlu). Even though the induced formation not known. The effects of MC on excretory func­ of INIB due to lead exposure has been known for tion during postnatal maturation of the kidney some time, its role in Pb induced nephropathy is were studied, Sprague-Dawley rats were injected not yet fully understood. The presence of Pb s.c. with 5 mg/kg MC at 1, 8, 15, 22 or 29 days induced INIB could result in either cellular after birth. Renal function was assessed 24, 48 resistance or susceptibility to further exposure and 120 hours after exposure. Functional measure­ to Pb. In order to.elucidate their function in ments included urine volume, pH, osmotic pressure Pb toxicity, rat kidney cells were isolated and and urinary content of chloride, protein, glucose maintained in MEM-D-Val. to confluency. The and blood, For every parameter, the renal sens­ resulting epithelial cell colonies were treated itivity to MC increased with increasing age at with 10- 5M PbGlu for 2 days repeatedly to form the time of treatment. Body weight was unaffected INIB. These pretreated cells and control cells by the exposure in .1. day old~, b1,1_twas. reduced

56 by 120 hours post-injection (p.i.) in the next The clinical syndrome of methylmercury intoxica­ four age groups. Kidney weights were unaffected tion frequently includes skeletal muscle weakness. at the younger ages, but were 15-50% higher than The purpose of the present study was to determine controls at the older ages. The treatment pro­ if, and how, acute exposure with methylmercury· duced a transient polyuria by 48 hours p.i. at altered neuromuscular transmission in vitro in a all ages. Urinary osmolality was lowered in mammalianskeletal muscle preparation.--mects all rats except those treated on day 1. This of mE!'thylmercury(4,20, and 100 µM) on post­ effect disappeared by 120 hours at the early synaptic potentials were assessed in the rat ages, but persisted in the more mature animals. phrenic nerve-hemidiaphragm using conventional Urine pH was lowered in animals treated on days microelectrode recording techniques. At concen­ 8, 15 and 22, but was raised in those injected trations of 20 and 100 µM, methylmercury increased on day 29. Urine chloride content was reduced the frequency of miniature end-plate potentials in MC exposed rats. Urinary protein content (MEPPs)from control values of 0.3-0.6/sec to 1.5- was elevated at all ages except the earliest, 10/sec. Increase in MEPPfrequency occurred after but the degree of proteinuria was more severe 15-40 min of exposure to 20 µM, and 5-15 min after in the older animals. Glucosuria was detected 100 µMmethylmercury. At a concentration of 4 µM, in animals in the three oldest age groups at methylmercury did not increase MEPPfrequency, 24 and 48 hours p.i., and was most severe at and, in fact, slightly decreased it. MEPPampli­ the oldest age. Hence, the neonatal kidney is tude was not significantly altered by any of the largely insensitive to MC toxicity. The renal above concentrations of methylmercury. End-plate response to MC increases sharply during the potentials (EPPs) evoked under diminished quantal preweaning period. content were decreased in amplitude, and finally blocked by 20 and 100 µM, but not by 4 µMmethyl­ mercury. EPP block occurred after 30-40 min with 202LOSS OF CUTANEOUSSENSITIVITY, AND HYPERREACTIVI­ 20 µMand after 4-5 min with 100 µMmethylw.ercury. TY FOLLOWINGACUTE METHYL MERCURY POISONING IN At the time of EPP block, MEPPs~ere still obser­ THE RAT. M. F. Wu, J. R. Ison, J. R. Wecker, & ved. Resting membranepotential of muscle fibers J. A. Foss, Dept. of Psychology, Dept. of Radia­ was not affected significantly by any concentra­ tion Biology and Biophysics, and Envir. Hlth. tion of methylmercury. The observed effects of Sci. Ctr., University of Rochester, Rochester, methylmercury were not reversible upon washing it NY. Sponsor: R. W. Wood out. These results are consistent with the hypothesis that methylmercury alters prejunctional A common early symptom of human methyl function. Supported by NIH grants ES02330and mercury poisoning is cutaneous paresthesia-­ ES05207. distal hypaesthesia, numbness and tingling. These effects have not been studied in animals. Here we characterize the cutaneous deficits which 204 CALMODULININHIBITORS PROTECTRATS AGAINSTMER­ follow acute exposure to this neurotoxic sub­ CURIC CHLORIDE-INDUCEDNEPHROTOXICITY. Jana L. stance in rats using the method of reflex modula­ Cox, R. C. Giles*, and S. D. Harrison, Jr., tion to measure sensory dysfunction. Adult rats Graduate Center for Toxicology and Animal Disease received a total body burden of 40 mg/kg(n=25); Diagnostic Center*, Univ. of Kentucky, Lexington, 13 mg/kg(n=6); or O mg/kg(vehicle; n=l7) methyl KY. mercury chloride in 5 days. At various times after poisoning (+l day to +20 weeks) cutaneous Because prochlorperazine protects mice sensitivity and reactivity were assessed. Weak against nitrosourea nephrotoxicity (Pharmacolo­ shocks (60-220).lA) were delivered to the tail 80 gist 19: 236, 1977), we have studied a series of msec before an intense t'one(l20db) which elicited similar compounds in a different model. Young, a startle reflex, and their sensory impact de­ adult male Sprague-Dawley rats received a single fined by reflex inhibition. On other days tail ip dose of HgC12 (1 mg/kg) alone or in combina­ shocks of higher intensities (100-500)-lA) were tion with one of 5 calmodulin inhibitors (CI, 4 given to elicit flinch responses. High dose rats or 18 µmol/kg/day ip daily for 5 days, day 1 = showed a steady loss of cutaneous sensitivity for day of HgCl2): Prochlorperazine (PCP), trifluo­ about 4 weeks, with substantial recovery at 20 perazine (TFP), perphenazine (PPA), fluphenazine weeks. ~igh dose rats became hyperreactive at (FPA), or chlorprothixene (CPT). On days 1, 3, 6 about4-5 weeks after poisoning as did low dose and 15, 24-hr urine samples were collected for animals at 20 weeks. Hyperreactivity ~ust be measurement of creatinine (Cr), leucine amino­ peptidase (LAP), and N-acetyl-glucosaminidase based on ,a different structural deficit than cu­ taneous loss because it had a different temporal (NAG). Rats were killed on day 16, and their course and appeared in different animals. Struc­ kidneys were weighed and fixed in 10% formalin (pH 7) for histopatho1ogic evaluation. HgCl2 tural damage will be assessed at the end of be­ havioral testing. These data demonstrated that produced elevated urinary enzyme activity on day 1. Histopathologic evaluation of kidneys from reflex modification methods are useful in the ana­ representative rats confirmed tubular dilatation lysis of sensory dysfunction following toxic ex­ posure and that the rat model of methyl mercury and necrosis, Rats treated with HgCl2 + CI poisoning is a reasonable analog for human expo­ exhibited less elevation of one or both of LAP and NAG on day 1. On day 1, NAG activity (U/mg sure. Supported by NIEHS grant ES01248. Cr) after HgCl2 alone was 40 ± 7 (x ± SEM); NAG activity after HgCl2 + CI (4 µmol/kg/day) was: PCP, 33 ± 13; TFP, 13 ± 3; PPA, 17 ± 2; FPA, 32 ± 203METHYLMERCURY-INDUCEDNEUROMUSCULAR DEPRESSION IN 4; CPT, 19 ± 2. The higher CI dose (18 µmol/kg/ THERAT. W.D. Atchison and T. Narahashi, Dept. day) resulted in more dramatic attenuation of of Pharmacology, Northwestern Univ. Med. Sch., enzyme activity by PCP, TFP, and PPA, but data Chicago, IL 60611. for FPA and CPT suggested additive toxicity.

57 Cytoprotection by CI was confirmed histopatholo­ maximum of 11.2 mV less than control on day 28. gically. The possible role of calmodulin in TET caused a progressive hindlimb paralysis HgCl2-induced nephrotoxicity seems to warrant that paralleled the reduction of RMPs; both further study. were completely reversible. l'hree weeks after rats were withdrawn from TET RMPs were restored to control values and the animals appeared to 205THEEFFECT OF TRIETHYLTINON SPECIFIC BRAIN PRO­ be motorically normal. TET had no effect on TEINSIN THEDEVELOPING RAT. J.P. O'Callaghan, the frequency, amplitude or incidence of min­ D.B. Miller and L.W. Reiter, Neurotoxicology iature endplate potentials. Spontaneous. ACh Division, Health Effects Research Laboratory, US release and its action on the postsynaptic EPA, Research Triangle Park, NC 27711. membrane were not affected by TET suggesting that TET reduces RMPs by inhibiting the bio~ The effects of triethyltin (TET) on the content energetic capacity of the muscle and that this of specific proteins from subcellular fractions myogenic toxicity is a significant factor in of the central nervous system were assessed in the development of muscle weakness followiµg the developing rat. Myelin (M), synaptic plasma exposure. membrane(SPM) and synaptosomal cytosol (SC) Supported, in part, by NIEHS ES-02645 & ES-07094 fractions were prepared from 22 day old Long­ Evans rats that had received either saline or TET (3.0 & 6.0 mg/kg, i.p.) at 5 days of age. The 207EFFECTS OF ORGANOTINSON MAXIMALELECTROSHOCK day of birth was taken as day zero. In order SEIZURE(MES) RESPONSIVENESSIN MICE. S. V. Doctor to detect the presence of specific phosphoprotein and D. A. Fox. Div. of Tox., Univ. of Texas Med. markers associated with Mand SPM,the endogenous Sch., Houston, Texas. phosphorylation 3~f these fractions was assayed Organot.in compounds are gaining connnercial in vitro using P ATPas a phosphate donor. The importance as biocides, catalyst~ and as polymer protein composition of each fraction was examined stabilizers. In this study the neurobehavioral· by sodium dodecyl sulfate polyacrylamide slab effects of structurally related organotins were gel electrophoresis. Specific protein phosphor­ studied using the MES test. Male mice were inject­ lation was detected by autoradiography and ed (ip) with either O, 3.5 or 17 .5 _µmoles/kg of specific protein and phosphoprotein content of trimethyltin(TMT) bromide, triethyltin(TET) bro­ each fraction was quantified by microdensito­ mide, tri-n-propyltin(TPT) chloride, tri-n-butyl­ metry. The postnatal day-5 administration of tin(TBT) bromide, tricyclohexyltin(TCT) bromide TETcaused a dose-related decrease in the con­ or triphenyltin(TPhT) acetate. Additional groups centration of two proteins associated with the of mice were injected with 26.25 µmoles/kg of TCT M-enriched subfraction. At a dose of 6.0 mg/kg or TPhT, 17.5 or 70 µmoles/kg of sodium bromide of TET, these proteins were depleted approxi­ (NaBr) or 17.5 µmoles/kg of stannic bromide (Sn mately 68%relative to control values. The af­ Br4). Mice were seizure tested at 0.5, 4, 21-24 fected proteins were of molecular weights (M) and 96 hrs following the injections. Mice exposed 20,000 & 25,000, values that closely approxi~ate to TMT, TET, TPT or TBT exhibited dose dependent those associated with the M basic and M proteo­ decreases in MES severity, assessed by seizure lipid protein, respectively. The electrophoretic grade distributions and seizure phase durations. patterns of SPMand SC proteins were not affected The tri-n-alkyltins, in order of decreasing abi­ by TET. Specific phosphoprotein markers asso­ lity in reducing MES responsiveness, were: TMT> ciated with SPMwere also not affected by TET, TET>TPT>TBT. In contrast to the tri-n-alkyltins, however, a phosphoprotein corresponding to M TCT and TPhT did not produce a significant change basic protein was markedly reduced in the M frac­ in seizure grade distributions. However, mice tion. These results suggest that early postnatal treated with the two higher dose levels of TCT or administration of TETmay interfere with myelino­ TPhT exhibited an increased seizure severity, ass­ genesis. essed by phase durations, at 30 min post-treat­ ment. At 4 and 24 hrs post-treatment, exposure to the two higher doses of TPhT caused a decrease in 206 TRIETHYLTINREDUCES THE RESTING MEMBRANEPOTEN­ MES severity followed by a recovery of normal TIAL OF RAT SOLEUSMUSCLE IN SITU. seizure severity by 96 hrs. Conversely, mice ex­ W.R. Millington and G.G. Bierkamper, Laboratory posed to the higher doses of TCT exhibited an in­ of Neuromuscular Toxicology, The Johns Hopkins creased MES severity at 4, 24 and 96 hrs post­ treatment. No changes in seizure severity were University, Baltimore, Md. (Sponsor;~- Fechter) observed in groups of mice treated with NaBr or Muscle weakness is a prominent component of SnBr4. (Supported by NIEHS Trg. Grt. #ES07090, the toxic syndrome which results from prolonged NIOSH Grt. #OH07085 and U. T. Bio. Med. Res. exposure to triethyltin (TET). The etiology of Supp.) this phenomenon includes, in part, an alteration of acetylcholine (ACh) release from motoneurons but there are also indications that TET causes 208 BEHAVIORALCONSEQUENCES OF TRIMETHYLTIN EXPOSURE a primary myopathy. We have found that chronic IN GRASSHOPPERMICE. Hulebak, K.L., Z. Annau. TET exposure (30mg/L. drinking water) causes a Dept. Environ. Hlth. Sci., Div. Toxicology, The time-dependent reduction of resting membrane Johns Hopkins Univ., 615 N. Wolfe St., Baltimore, potentials (RMP) recorded from the soleus mus­ MD 21205 cles of intact anesthetized rats. The RMPs of TET-exposed rats were significantly lower than Trimethyltin (TMT)is an organometallic neuro­ those of paired control animals after four days toxin which produces specific neuronal lesions in (5.4 mV). They were further reduced by contin­ discrete brain regions. Damagedareas include uous TET exposure for 8, 14 and 21 days to a the hippocampus, pyriform cortex, septal nucleus,

58 and amygdala. These lesions have been shown in 210DEPOSITIONOF INHALEDFIBERS IN THEBEAGLE DOGS, the rat to be accompanied by hyperirritability L. C. Griffis, S. J. McAllen and J. A. Pickrell and possibly increased agonistic behaviors. (Sponsor: R. 0. McClellan), Lovelace Inhalation The present study examines the toxicity of TMT Toxicology Research Institute, P.O. Box 5890, in a wild-type rodent, Onychom¥storridus, the Albuquerque, NM87185 grasshopper mouse. These carnivorous rodents not The pulmonary deposition of inhaled asbestos and only display a diverse repertoire of social be­ glass fibers has been predicted by others using haviors, but also show predatory behavior towards models which incorporated the aerodynamic be­ prey such as crickets. havior of these materials. The purpose of these Same-sex pairs of male and female Onychomys studies was to measure the deposition of very were exposed (p.o.) to 0, 0.5, and 1.0 mg/kg TMT small asbestos and glass fibers inhaled by the weekly, for 3 weeks. The performance of the ani­ Beagle dog. Crocidolite asbestos were radio­ mals was monitored weekly on a variety of behav­ labeled with 59Fe by neutron activation. Young ioral tasks both before, during, and following adult Beagle dogs from our colony were given 60- exposure. These tasks consisted of a measure of minute nose-only exposures to an aerosol of activity in running wheels, of the auditory star­ either asbestos or glass fibers which were gen­ tle response, cricket-killing as a measure of erated in a fluid bed aerosol generator. Dogs predatory aggression, as well as complex social were killed and tissue samples taken either 4 behaviors in paired animals. days or immediately after exposure. The mass of Results indicate sex- and dose-related disrup­ fibers in a sample were determined from the radio­ tions of complex social, agonistic, and general activity in the sample and the specific activity activity behaviors of the mice, independent of of the fibers in the exposure aerosol. Four dogs effects of the toxin in activity and reactivity exposed to a mean asbestos concentration of 71 µg/ as measured by the tests of auditory startle res­ L deposited a total of 64 ± 6% (mean± SD) of the ponse and running wheel performance. inhaled fibers. Alveolar deposition was esti­ Supported by ES 02277 and ES 07094. mated from the activity remaining in the lung 4 days after exposure and was 17 ± 3%. Deposition normalized for tissue weight in the right apical and left diaphragmatic lung lobes was 20%higher and 13%lower than the mean lobar deposition with 209THE PULMONARYRESPONSE TO SIZED GLASS FIBERS IN the other lobes between these values. The meas­ RATS. D.M. Bernstein, R.T. Drew, M. Kuschner, ured alveolar deposition for crocidolite asbestos Medical Dept., Brookhaven National Laboratory, in the dog is in excellent agreement with the predicted value (~ 17%) for deposition of fibers Upton, NY 11973 of this size in the humanlung. (Research sup­ A number of studies have suggested that both ported under DOEContract No. DE-AC04-76EV01013.) length and diameter of glass fibers are important parameters in determining their deposition and translocation in the lung and in the occurrence of 211 ACUTEINHALATION STUDIES WITH AN AMORPHOUS a pathological response in the lung. To character­ SILICONDIOXIDE DUST. J. B. Scott, S. M. ize the biological response to glass fibers, a MacAskill, P. J. Belizi, B. O. Stuart, and study has been conducted to determine the trans­ G. M. Zwicker. Stauffer Chemical Co., Environ. location and ultimate fate of fibers of defined Health Center, Farmington, CT 06032. Sponsor: sizes after introduction into the respiratory R. I. Freudenthal. tract of rats by instillation and inhalation. The Acute inhalation studies were performed using a fibers have geometric mean diameters of 1.5 µm low density (.051 g/ml) amorphous silicon diox­ (crg = 1.1) and lengths of either 5 µm (cr = 1.49) ide dust which is used as extending filler. A or 60 µm (crg = 3.76). g modified version of a NBSdust generator was Serial sacrifices following intratracheal used to aerosolize the material. Chambercon­ instillation of either 2 mg or 20 mg doses have centrations using membranefilters and particle shown the short fibers to lie primarily within size analyses were determined gravimetrically. mononuclear phagocytes in both the lung and lymph Male and female Sprague-Dawley rats were exposed nodes. The majority of long fibers, however, can­ to 1.12 mg/1 for 4 hours which resulted in 55% not be totally engulfed by macrophages, nor are mortality by one day after the exposure. Values they cleared to the lymph nodes, although smaller for mass median aerodynamic diameters (MMAD) fragments accompanying the long fibers may be so were 7.8 and 5.8 um, with geometric standard cleared., The long fibers produce a striking deviations (og) of 3.25 and 2.16, respectively. foreign body reaction in the lung, particularly Although this level is less than 1/4 of the when impacted in the bronchi. Over 90% of the EPA-prescribed concentration for acute inhala­ fibers deposited in the lung were cleared from tion testing, the cause of death was apparently the animals. Both long fibers and short fibers suffocation by the dust and not due to any were found in the pleural cavity. At 18 months intrinsic toxicity. The surviving rats showed post exposure the surface area of both the long temporary clinical effects, but necropsy after and short fibers were found to have been reduced 14 days showed no apparent adverse effects of by 25 to 50 percent through dissolution. The dis­ the exposure. Rats were also exposed for 1 solution of these fibers may be an important hour to 1.05 mg/1 with a MMADof 3.45 um and og factor in the lack of any observed fibrogenic or of 2.72. The rats experienced temporary clinical carcinogenic response to these fibers in the effects, but there were no deaths and no adverse animals. effects were found at necropsy 14 days later. *Research supported by the Thermal Insulation Extensive modifications were made to the genera­ Manufacturers Association and the U.S. Dept. of tion and sampling systems, and rats were exposed Energy under contract DE-AC02-76CH00016. for 1 hour to 2.57 mg/1 with a MMADof 3.95 um

59 and o9 of 1.80. Again, the rats experienced µg/m3) and ZnOaerosols (250 µg/m3). Two groups some temporary clincal effects, but there served as unexposed contra ls for the experimental occurred no deaths,and no persistent adverse groups. After 6 and 5 weeks of continuous effects. The implications of acute inhalation exposure, a 1 hr nose-only exposure to 59Fe203 in exposures to required very high levels of low all groups was performed where the inert iron density dusts are discussed. oxide particles served as testaerosol for measuring lung clearance. This clearance was measured up to 70 days. It was found that 1) CdO inhalation increased long-term lung clearance of 212PULMONARY FUNCTION MEASUREMENTS IN GUINEA Fe203 significantly, 2) Combinedexposure to PIGS AFTER EXPOSURE TO SUBMICRON AEROSOLS CdO+ ZnOdid not influence this lung clearance. OF ZINC. OXIDE AND SULFUR DIOXIDE, H.F. The biological half lives for the slow phase of Lam, R. Peisch and M.O. Amdur, Dept. of Fe203 clearance from the lung in the control Nutr. and Food Sci., Mass. In~t. Tech., groups was 37 and 40 days, respectively. After Cambridge, MA 02139. Cd exposure, this was 62 days and after combined We studied the effect of zinc oxide Zn and Cd exposure it was 32 days. The fast phase aerosols, ZnO (CMD 0.05 µm, crg 2.0), gen­ of Fe203 lung clearance was not affected by erated by the condensation of supersatu­ CdOor by the combined exposure. rated vapors, in the presence and absence Thus, simultaneous CdO-ZnOinhalation counter­ of moisture and sulfur dioxide (S02), We acts the impaired lung clearance following CdO evaluated lung volumes, single breath inhalation alone. In the lung, too, Zn shows a diffusing capacity of the lungs for car­ protective effect on Cd toxicity. bon monoxide (DLC 0 ), dynamic compliance (Cdyn) and fl~w resistance (Rf) in male Charles River Hartley guinea pigs imme­ 214IBE EFFECTSOF CAIJ'1IUMON PHAGOCYTOSIS BY diately after exposure. Animals were ex­ ALVEOLARMACROPHAGES ANDON Ti02 CLEARANCEFROM posed for 3 hrs to 1 ppm so 2 and 0,7,1.4, THELUNGS OF RATS. B.J. Greenspan, Bushy Run 2.3, or 6.0 mg/m 3 ZnO mixed in a furnace Research Center, Export, PA and P.E. Morrow, at 480°C, Water vapor was added either Dept. Radiation Biology and Biophysics, Univ. to the exposure chamber or to the furnace, Rochester, Rochester, NY so and 1.4 mg/m 3 ZnO mixed in a dry 2 This study deals with the hypothesis that the furnace and administered to animals in a lymphatic uptake of particles from the lung humid chamber caused a 10% decrease below parenchyma increases when phagocytosis by pul­ control values in vital capacity (VC), monary macrophages is inhibited. An in vitro functional residual capacity (FRC) and assay for phagocytosis of latex particles was total lung capacity (TLC) with no changes used to quantify macrophage function. Lung in the alveolar volume (VA) and DLCO' clearance, lymphatic uptake, and macrophage Cdyn was increased and Rf was decreased. function in vivo were assayed by exposing the In animals exposed to so and 2,3 and 6.0 2 animals totitanium dioxide (Ti02) dust. mg/m3 ZnO generated in a humid furnace, Cadmiumchloride (CdCl2) was chosen as the lung volumes (VC, TLC, FRC) decreased by toxicant to inhibit phagocytosis. Rats were 12% and DLco by 23% and 53% respectivel 3. exposed to CdCl2 aerosols at 2.5 mg Cd/m3, VA was decreased by 11% in the 6,0 mg/m MMAD=0.35µm, crq=l.43, or 5.0 mg Cd/m3, MMAD=0.42 ZnO exposure group. C and Rf values µm, cr =1.60, for 30 minutes. Control animals did not differ from tR6~e of controls in receiied a saline aerosol. At various times post both these groups. The nature of the res­ exposure, the lavageable macrophages were purified ponse, decreases in lung volumes, VA and on an albumin gradient and assayed, in vitro, for DLCO suggests an effect in the periphery phagocytic activity. Phagocytosis was depre~sed of the lung probably involving the clo­ for 5 days following exposure to 5.0 mg Cd/m3. sure or narrowing of small airways. Sup­ Macrophagesfrom animals exposed to 1.5 mg Cd/m3 ported by NIEHS grant ES 02429-01), appeared to be activated immediately and at one day post exposure. To examine the effects on clearance and lymphatic uptake, rats were exposed 213 PREVENTIONOF CdO INDUCED IMPAIRED LUNG CLEARANCE to CdCl2 aerosols and Ti02 dust, 13-15 mg/m3, BYSIMULTANEOUS ZnO INHALATION. Oberdorster, G., MMAD=l.03µm, cr =2.29. Pre-exposure to 5.0 mg Hochrainer, D. and Oldiges, H. Dept. RBB,Medical Cd/m3decreased 9the initial deposition of Ti02 Center, University of Rochester, Rochester, NY by 40 percent when compared to a saline aerosol 14642 and Fraunhofer Institut for Toxicology and preexposure. Although the overall clearance of Aerosol Research, 5948 Sc~mallenberg, W.-Germany. the dust was not different in the cadmium-exposed (Sponsor: Morrow, P .E.) animals, the lymph node burden was 2.7 times higher in the CdCl2-exposed animals than in the It has been shown in several studies that Zn controls. Exposures to 1.5 mg Cd/m3had no can protect the organism against certain effects effect on lung clearance or lymphatic uptake. of Cd. Similarly, deficiency of Zn enhances the WhenTi02 exposure preceded a 5.0 mg Cd/m3 toxicity of Cd. In our study we wanted to find exposure, the results were similar. out if and to what extent the effect of CdO inhalation on lung clearance is prevented by ZnO inhalation. Four groups of 30 rats each were 215 PHAGOCYTOSISANDDISSOLUTION OF CARCINOGENICaNiS used: one group was continuously exposed to CdO PARTICLES;VISUALIZATION BYVIDEO INTENSIFICATION aerosols, 10 µg/m3 (mass median aerodynamic MICROSCOPY.R.M. Evans, P.J.A. Davies, and M. diameter 0.46 µm, crq=l.5). A second group was Costa, Div. of Tox., Dept. of Pharm., U. Texas continuously exposea to a combination of CdO (10 Med. School at Houston, Tx. 77025.

60 Ins?luble crystalline aNiS particles produce Numbersof peroxidase positve macrophages severe inflammatory reactions at sites of injec­ increased in the urban air dust, Si02 and PbO tion with the subsequent development of sarcomas. group. Although macrophage response may also These particles have been shown to cause morpho­ depend on lung dust burden, the inactivity of PbO logical transformation of Syrian Hamster Embryo and Si02 in the bacterial challenge model may (SHE) cells by a mechanismdependent on particle partly 5e explained by an influx of pulmonary phagocytosis (Costa, M. and Mollenhauer, H.H., macrophages. Science 209, 616, 1980). The purpose of this study was to further elucidate the subcellular events associated with aNiS-induced transforma­ 217 tion through the use of time-lapse Video Intensi­ COMPARISONOF THE IRRITANCY OF COAL,OIL, VOLCANIC fication Microscopy (VIM). Alternate use of ASHAND ASH CONSTITUENTS. G.E. Hatch, E. Boykin fluorescent phase, and bright field VIMallowed F.J. Miller, J.A. Graham, and D.E. Gardner, ' for visualization of both NiS particles (bright Envir. Tox. Div., Health Effects Res. Lab., EPA, field) and intracellular components such as lyso­ Research Tri. Park, NC. somes (phase microscopy). Cultivation of SHEand Sensory irritation of fly ash from oil-fired Chinese hamster ovary cells in a Dvorak chamber (OF), fluidized bed coal (FB), and conventional improved resolution of intracellular organelles coal (CC) combustion power plants, and from Mt. at high magnification. Particle phagocytosis St. Helens volcano (VA)was studied in mice. The occurred primarily in regions of active cellular irritating sensation due to contact with ash was ruffling without the noticeable appearance of a quantitated by observation of a characteristic vacuole. Following endocytosis, lysosomes moved contraction of the dorsal musculature of the toward and aggregated around the internalized mouse following intraperitoneal injection of the particle. Lysosomeswere repeatedly observed to ash suspension. Dose-response curves and estim­ contact the particle-vacuole complex. This lyso­ ation of the 50%effective dose were based on the somal contact may participate in the dissolution percentage of animals showing a positive response of crystalline aNiS particles. After 24 hours at various ash concentrations. Procedural modi­ most particles were surrounded by lysosomes, and fications improved reproducibility; validation aggregated around the nucleus. It is possible studies using 10 environmental chemicals confirmed that dissolution products Ni(2+) from endocytized that a positive correlation exists between this NiS, released in proximity to the nucleus may be ~rr~tation test and the upper respiratory tract the ultimate agent causing cellular transforma­ ~rr~tancy test of Alarie. The relative ranking of tion. ( Supported by grant #ES-02487and #ES-070 1rr1tant potency was: Of and OF leachate>>FB>CC> 90 from NIEHSand by grant #R808048from the U.S. VA. Other than OF fly ash, saline leachates showed no irritating effects. OF fly ash was EPA). about 160Xmore irritating than VAand 5X more irritating than a reference compound,sodium dodecyl sulfate. Study of chemicals present in fly ash or similar to those present indicated that 216 TOXICITYOF AIR CONTAMINANTSDETERMINED BY acidic or basic compounds, heavy metal ions and LAVAGABLELUNG CELLS. Oberdorster, G., Ferin, metal oxides, and insoluble particles could all J., Pott, F. and Morehouse, B. Dept. RBB, contribute to the irritancy of an ash. Leachable Medical Center, University of Rochester, heavy metals appeared to be mainly responsible for Rochester, NY14642 and Med. Institute for Air the irritant effects of OF ash, while insoluble Hygiene and Silicosis Research, University of particles, including metal oxides, appeared to ac­ Dusseldorf, 4 Dusseldorf, W.-Germany. (Sponsor: count for the irritancy of the other fly ash sam­ Shaikh, Z.) ples: The mouse peritoneal irritancy test, shown previously to correlate with controlled humaneye It has been observed that some pollutants with sting studies on shampoos, appears to be also knownpulmonary toxicity (e.g., PbO, Si02) do not suppress pulmonary defense against bacteria relevant to study of environmental chemicals. It when tested by the "infectivity model". Using a may be useful for samples difficult to aerosolize numberof compoundswith various toxicity, · or for those available only in small quantities. including PbO and Si02, we studied their effect on pulmonary cells. Peroxidase positivity has been linked to young alveolar macrophages (AM), 218 COMPARISONOF THE UTILITY OF INCAPACITATIONDATA making it possible to distinguish between old and VERSUS LETHALITY DATA IN THE DETERMINATIONOF THE new populations of macrophages. Peroxidase TOXICITY OF COMBUSTIONPRODUCTS OF MATERIALS. staining was used, therefore, to assess AM B.C. Levin, M.M. Birky, Center for Fire Research response. The following substances were instilled National Bureau of Standards, Wash., D.C. 20234 ' intratracheally into rats, after suspension or and D. Malek, Dept. of Physiology, The George Washington University, Wash., D.C. 20037 dissolution in water: CdS04 (100 µg), PbO (1 mg), Si02 (500 µg), Ti02 (500 µg), During the development of a test method for the urban air dust sample of the Ruhr valley (5 mg) toxicological assessment of inhaled combustion H20 (0.3 ml). The instilled volume was kept at' products, we measured both time-to-incapacitation 0.3 ml: Twenty-four hours later, the lungs of (hind-leg flexure model) and lethality in Fischer the animals were lavaged and a differential cell 344 rats. Both time-concentration curves (time­ count was performed. Comparedto the control to-incapacitation vs. mg/1*) and EC values (H20) rats, urban air dust, PbO and Si02 led (mg/1* which cause 50% of the rats ~2become in­ to an increase in total cell numbers, whereas capacitated) were used to classify materials. CdS04and Ti02 did not. Numbersof PMN's Most of the animal exposures were 30 min; a few were increasea in the urban air dust, PbO SiO were allowed to continue until 100% were inca­ and CdS04~roup, whereas m~crophagenumbe;s ha~ pacitated. In those 30 min exposures where 2/6 decreased in CdS04, PbO, air dust and Si02. - S/6 animals were incapacitated, a statistical

61 analysis called the best linear unbiased esti­ Boron Trifluoride forms a liquid dihydrate in mate (BLUE) was performed to estimate the mean air containing as little as 50 ppm of water. It and standard deviation of the time-to-incapaci­ was, therefore, decided to evaluate the inhala­ tation of all 6 animals tested. The LC was tion toxicity of boron trifluoride using an 50 calculated for the 30 min exposure and for the aerosol of the dihydrate. Using Fischer 344 rats, 30 min exposure plus 14 day post-exposure the four hour inhalation Lc was 1.21 mg/L. The 50 period. Eight materials evaluated in both signs elicited by the exposures included symptoms flaming and non-flaming modes were classified of respiratory irritation as well as body weight according to the incapacitation and lethality depression. For the surviving rats, there was an data. The results showed that the classification apparent recovery. Increases in both liver and of materials did not change with the method of kidney weights were found in all treatment groups analysis. In addition, delayed toxicological ef­ when compared to the control. Subsequently, a two fects leading to post-exposure deaths cannot be week inhalation toxicity study was performed. evaluated by the incapacitation model. Based on Four groups of 10 rats were exposed to liquid these results, it was decided that the LCSO was aerosols 6 hours/day, 5 days/week to expjsure sufficient to toxicologically classify materials concentrations of O, 24, 66 and 180 mg/m. By with regard to their combustion products. An the end ~f the first week, all rats in the additional test of a 10 min exposure to 30 mg/1* 180 mg/m group had died. There was no mortality of material was included to identify those in the two lower-level exposure groups although materials whose combustion products act rapidly. both groups showed symptoms of respiratory irri­ tation and body weight gain depression Lung *mass loading/chamber volume weights and lung/body weight ratios were elevated in all exposed groups. Following the two-week study, a thirteen-week study was initiated. 219 TOXICITY OF THERMALDEGRADATION PRODUCTS OF Groups of 40 rats were exposed 6 hours/day, POLYTETRAFLUOROETHYLENE(PTFE). D.E. Malek, R.A. 5 days/~eek to concentrations of 0, 2, 6 and Kenney, B.C. Zook, M.S. Lasser. Dept. of Physiol­ 18 mg/m. Observations through the first 6 weeks ogy, The George Washington Univ., Wash., D.C. of the study have not demonstrated overt signs of 20037 and M.M. Birky, Ctr. for Fire Research, a toxic response; hematological and clinical NBS, Wash., D.C., 20234. Sponsor: B.C. Levin laboratory parameters measured in all groups The LC [30 min exposure and 14 day post-exposure during Week 5 appeared normal. Measurements of 50 (PE)] of PTFE thermal degradation products was urinary and serum fluoride levels showed established to be 0.045 mg/1* as compared to increases in all treatment groups when compared 22.8 mg/1* for Douglas fir in m:ale rats. Because to control values. Final results will be dis­ of the much higher acute toxicity of PTFE, a cussed. study was initiated to evaluate the'J)hysiological and pathological changes induced by PTFE thermal decomposition products. Groups of six rats were 221 Paper Withdrawn exposed for 30 min to a single concentration of products produced by decomposing PTFE at 595°c. The concentrations ranged from 0.005 to 5.025 mg/1*. Arterial blood, heart rate (HR) and 222 Carcinogenicity study of the pesticide Maleic Hydrazide in direct arterial blood pressure (ABP) were mea­ Mice. J.R.P. Cabral, V. Ponomarkov, and L. Tomatis, sured within the exposure and at 12, 24, and 36 International Agency for Research on Cancer, Lyon, France hrs PE from rats exposed to 0.045 mg/1*. Necrop­ sies followed the PE monitoring. Respiratory impairment seen in rats exposed to 0.045 mg/1* The carcinogenicity of the pesticide Maleic Hydrazide during the exposure became more severe in the PE (MH) was studied in C57BL/B6 mice. The free acid of MH period as indicated by changes in blood %02Hb, was used (98.5% pure; containing 0.6 ppm of a hydrazine Po Pco , and pH. Normal mean values± SE for 2 2 impurity). The carcinogenicity experiments with MH the~e parameters were 93.7 + 0.7, 91.8 + 2.7, included: a) subcutaneous (s.c.) administration to 163 30.4 + 1.0, and 7.448 + 0.011, respectively. By 36 hrs PE, the %0?Hb, P02' and pH decreased to infant mice (5, 10, 20 and 20 mg MH per mouse on days 1, 7, 14 and 21 after birth, respectively); and b) oral adminis­ 69.9 ± 8.5, 58.7 f 5.9 and 7.347 ± 0.018, Pco 2 increased slightly to 32.9 + 2.2. Bradycardia tration (gavage) to 82 male and female mice, from weaning and depression of ABP were evident within the for life, at a weekly dose of 510 mg/kg bw MH. Groups of exposure and PE. By 36 hrs PE, HR decreased from untreated and solvent controls were also available. The 513 beats/min to 287 + 59; normal systolic ABP experiments were terminated at 120 weeks. The s.c. adminis­ decreased from 158 + 2 mmHg to 94 + 7 and dias­ tolic ABP declined from 129 + 1 to-80 + 6. His­ tration to infant mice resulted in high preweaning mortality tologic evaluation of tissues revealed-pulmonary when compared with the respective controls. Liver-cell lesions and evidence of disseminated intravascu­ tumours were observed in 7 males treated s.c. with MH. In lar coagulation. The observed histopathology was the solvent-treated (tricaprylin) males, 3 liver-cell tumours both concentration and time related. were also found. One liver-cell tumour was seen in untreated *mass loading/exposure chamber volume male controls. No liver-cell tumours were observed in treated or control females. The difference in the incidence of liver tumours among the various groups was not significant. When 220Inhalation Toxicology Studies with Boron MH was administered orally (weekly for life), liver-cell Trifluoride Dihydrate. G. M. Rusch, G. M. Hoffman, W. E. Rinehart tumours were observed in 4 males and in 2 females of the Corporate Medical Affairs, Allied Corporation, treated group, and in 1 male and in 1 female of the solvent P.O. Box 1021R, Morristown, NJ 07960 (olive oil) control group. In the respective untreated controls

62 1 male had a liver-cell tumour. The incidence of tumours at treated with B(a)P alone or TPA alone, developed papillomas. sites other than the liver was similar in all groups. These These results confirm that C57BL/86 mice are reasonably results partially confirm the negative finding of a recent sensitive to a conventional two-stage carcinogenesis protocol. parallel study in rats (Van Der Heijden, Toxicology, 19: 139- However, when the same amount of B(a)P was injected sub­ 150, 1981). cutaneously to pregnant mothers and their descendants were painted on skin of the back with 5 µg of TPA twice a week from weaning for 93 weeks, no persistent papilloma was 223 RESULTS OF A CHRONICTOXICITY AND ONCOGENICITY observed. Similarly, the transplacentally exposed mice were STUDYIN RATS FOLLOWING24 MONTHSOF ACRYLO­ injected intraperitoneally with 5 to 10 µg of TPA twice a NITRILE INHALATIONEXPOSURE. J. F. Quast, T. S. week. After 100 weeks, the number of mice that died with Gushow, D. J. Schuetz, M. F. Balmer, C. N. Park, tumour (4/63) was not significantly different from that in the and M. J. McKenna. Health & Environmental group without TPA injection (3/64). Taken together, these Sciences, USA, Dow Chemical, U.S.A., Midland, results suggest that under the experimental conditions used, MI 48640. a conventional mouse skin initiation-promotion carcinogenesis A 24-month industry supported study was conducted is demonstrable, but transplacental initiation-postnatal in Sprague-Dawley, Spartan substrain rats to promotion of skin and internal organs has not been clearly assess the chronic toxicity and oncogenicity of observed so far in C57BL/B6. The experiment is being acrylonitrile (AN) following 24 months of inhal­ ation exposure. Groups of 100 rats per sex were continued and all the surviving mice are scheduled to be exposed to either O, 20 or 80 ppm AN for 6 hours sacrificed at 120 weeks of age. per day, 5 days per week for 24 months. Evaluation of hematology, urinalysis, and clini­ cal chemistry parameters did not reveal any 225 DERMAL CARCINOGENESIS BIOASSAYS OF DIESEL alterations indicative of an adverse effect on PARTICULATES AND THEIR DICHLOROMETHANE bone marrow, kidney, or liver function. Decreased EXTRACT, L. R. DePass, K. C. Chen*, L. G. Peterson, body weight, early mortality, unthrifty clinical and C. S. Weil, Bushy Run Research Center, Export, PA appearance, and increased incidence of palpable and *Biomedical Science Department, General Motors tumors were noted in exposed rats, especially at Research Laboratories, Warren, MI 48090. Sponsor: the 80 ppm level. Early mortality in the exposed J. J. Vostal rats resulted in a decreased incidence of patho­ logic changes usually seen in aged rats. Diesel particulates and dichloromethane extract of diesel particulates were tested to assess their potential A statistically significant (p<0.05) increased as complete carcinogens and as initiators or promoters incidence of tumors in the central nervous system of carcinogenesis. The test agents were applied as (CNS), ear canal gland (Zymbal gland), tongue, suspensions to the dorsal skin of male C3H mice at the and small intestine was observed in the 80 ppm highest doses that were non-irritating and sufficiently group of males. In the 20 and 80 ppm groups of flowable for application. Lower concentrations were female rats the tumor incidence was statistically also used to obtain information on the dose-response significantly increased in the CNS and the relationships. Acetone was used as the vehicle in all mammary gland. In addition, in the 80 ppm female studies; diesel particles were applied in 10% or 5% group the incidence of ear canal gland (Zymbal concentrations, and dichloromethane extract . was gland) tumors was increased. Tumors considered administered as 50%, 25%, 10%, or 5% suspension. treatment related but not occurring at a statis­ Dosing was performed 3 times per week in the initiation tically significant increased incidence were also and complete carcinogenesis studies, and 5 times per noted in the stomach of 80 ppm male rats, the week in the promotion studies. Positive control groups brain of 20 ppm male rats, and the nasal turbi­ received repeated application of 0.2% benzo[a] pyrene nates of 80 ppm female rats. An apparent decrease (B[a] P) for complete carcinogenesis, or a single appli­ in tumors of the pituitary, adrenals, thyroid, cation of 1.5% B[a] P, or of 10% particulates or 50% of pancreas, and testes was_observed in exposed rats. particulate extract suspensions, followed by repeated application of 0.0001% phorbol myristate acetate (PMA) for the initiation studies. In promotion studies, repeated application of PMA was replaced by 10% 224 TWO-STAGE CARCINOGENFSIS ON SKIN AND IN suspension of diesel particulates or 50% and 25% of INTERNAL ORGANS OF C57BL/B6 MICE. H. Yamasaki, extract suspensions. The test agents were applied J.R.P. Cabral, D. Galendo, and L. Tomatis, International similarly in place of B[a) P in the complete carcino­ Agency for Researchon Cancer, Lyon, France. genesis and initiation studies and in place of PMA in the promotion studies. At the end of two years of treat­ Promoting effect of phorbol esters, as represented by ment, there was no substantial evidence for a positive 12-0-tetradecanoyl phorbol-13-acetate (TPA), has been effect in complete carcinogenic, initiator or promotor extensively studied on mouse skin, using a conventional activity of either diesel particulate suspension or initiation-promotion protocol. Since it has been shown that dichloromethane extract in any of the test groups. these tumour promoters exert a variety of biological and bio­ Statistically significant increases in tumor yield have chemi~al effects on the cells derived from various tissuesother been observed in both positive control groups, thus establishing the validity of the test system. than mouse skin, it was of our interest to examine the promoting activity of TPA on internal organs as well as on skin. When fifty C57BL/B6 mice, 6 week old, were painted 226 PROMOTIONOF LIVER TUMORSIN B C F MALEMICE BY 6 once with 3 mg of benzo(a)pyrene (B(a)P) and painted with PARTIAL HEPATECTOMYOR DIETARYcnotINE DEFICIENC~ 5 µg of TPA twice a week, 64% of mice developed papillomas P.M. Newberne, and A.J. Clark, Dept. of Nutr. and by 30 weeks of treatment, whereas none of. control groups, Fd. Sci., M.I.T. Cambridge, MA

63 Groups of male weanling B c F strain of bladder. Since other tumor promoters are also 6 mice were fed a control semisyntlietic diet (C) or cocarcinogens, we evaluated the possibility that the same diet devoid of choline (CD). Some mice saccharin is also a cocarcinogen. Male Fisher 344 from each group were subjected to partial hepa­ rats simultaneously received 0.02% dibutylnitro­ tectomy (HepX) after two weeks on diet. Sequen­ samine (DBN)in their drinking water and 5%sodium tial sacrifices of mice from each group revealed saccharin in their diet for a total of 26 weeks. the following: after three months, no tumors in The liver of rats that received only DBNcontained any mice although the CD mice had enzyme defi­ 16 hyperplastic nodules among 5 of 29 (17%) cient and fat-free islands of proliferating hepa­ animals and 2 hepatocellular carcinomas in 1 of 29 tocytes; after six months choline deficient mice (3%) of the animals. The simultaneous admin­ only had liver tumors; after nine months control istration of sodium saccharin to the animals that mice, 0% tumors, C + HepX mice 20% tumors, CD received DBN, produced an enhanced hepatocar­ mice 100% tumors and CD+ HepX mice 40% tumors. cinogenic response of 106 hyperplastic nodules in Autoradiographic studies indicated that both HepX 17 of 21 ( 81%) animals and 81 hepatoce 11u l ar and CD increased DNA synthesis and liver cell carcinomas in 17 of 21 (81%) animals. The number proliferation at all periods examined. These of preneoplastic focal alterations in the liver data suggest that the liver of BC F mice is that were induced by DBN,was al so enhanced by sensitive to agents or condition~ Jhich are not sodium saccharin. The livers of untreated control tumorigens themselves but which may promote tu­ rats and rats that received only sodium saccharin mors. were free of preneoplastic foci, hyperplastic (Supported in part by USPHS grant ES00597 and a nodules and hepatocellular carcinomas. In the grant from Eli Lilly Co.) urinary bladder, treatment with DBN produced simple and nodule hyperplasia that was enhanced by 227 INITIATING AND PROMOTINGEFFECTS OF METHAPYRILENE simultaneous treatment with saccharin. Sodium ASSESSED BY THE ENZYME-ALTEREDFOCI BIOASSAY. D. saccharin did not result in either a preneoplastic Couri, S.R. Wilt, and M.M. Milks, Division of hyperplasia or a neoplastic response in the uri­ Toxicology, Dept. of Pharmacology, College of nary bladder of the rats. Sodium saccharin ad­ Medicine, Ohio State University, Columbus, OH. ministered as 5% of the diet was demonstrated to be The once commonly used antihistamine methapyri­ £ocarcinogenic toward DBNcarcinogenicity in the lene was recalled by the FDA in 1979 after lab­ liver and urinary bladder of rats. oratory findings that the drug produced liver tu­ mors when fed to rats. However methapyrilene was not mutagenic in the Ames assay nor was the drug 229 DRUGMETABOLIZING ENZYMES IN THE LIVERS AND active in transforming hamster embryo cells in HEPATIC TUMORSOF RATS TREATEDWITH POLY­ culture. For these reasons, the initiating and CHLORINATEDBIPHENYLS. G. Reddy, H.P. Cihla, promoting effects of methapyrilene were evaluated R.H. Weltman, & D.H. Norback, Wm. s. Middleton using the rat hepatic enzyme-altered foci bio­ Mem. VA Hospital, Univ. of Wis., Madison, WI, assay. In this study, putative preneoplastic is­ 53705. lands were identified by the focal occurence of Hepatic drug metabolizing enzyme activities the enzyme y-glutamyl-transpeptidase (GGT) in in the livers of rats were investigated after chronic exposure to Aroclor 1260(PCB) or 2 3 6 hepatic tissue. Male Sprague-Dawley rats were , ' ' , , , partially hepatectomized and twenty-four hours 2 ,3 ,6 -hexachlorobiphenyl (HCB). Sprague- later were administered either methapyrilene (50 Dawley rats of both sexes were fed diets con­ mg/kg), nitrosodiethylamine (30 mg/kg), or dis­ taining PCB (100 ppm for 16 mo followed by 50 tilled water by oral gavage. Subsequently, the ppm) or HCB (100 ppm for 9 mo followed by 5 mo animals received drinking water containing either period of intermittent exposure followed by 100 500 ppm phenobarbital (a known promoter of hepa­ ppm). After 24 mo they wer·e fed a normal diet tocarcinogenesis), 200 ppm methapyrilene, or con­ until sacrifice. The hepatic microsomal enzyme trol tap water ad libitum for eight weeks. Fresh activities of aldrin epoxidase (AE), styrene frozen sectionsof liver were then prepared, oxide epoxide hydrolase (EH), and cytosolic stained, and scored for GGT-positive foci. Meth­ glutathione S-epoxide transferase (GT) and the apyrilene, when tested for initiating activity quantity of cytochrome P-450 were measured. and promoted by phenobarbital did not produce Livers from male rats fed HCB contained enzyme foci above background levels. However, when sub­ levels equal to or decreased from that of con­ stituted for phenobarbital as the promoting agent trols. Livers from PCB-fed male rats showed following nitrosodiethylamine initiation, metha­ enzyme levels 1.3 to 4 fold higher than that of pyrilene enhanced enzyme-altered foci formation controls. Livers from female rats exposed to to an equal or greater extent than did phenobar­ HCB showed enzyme levels equal to or decreased bital. These results suggest that the carcino­ from control levels. Liver tumors or surrounding genic effects of methapyrilene are related to its tissues of female rats fed PCB showed 2 to 10 ability to promote liver cells already initiated fold higher enzyme activities and cytochrome by dietary or other environmental carcinogens. P-450 content than controls. AE activity was slightly higher in surrounding tissues than in tumors, while EH activity was 1.5 times higher 228 COCARCINOGENICITYOF SACCHARIN TOWARD N-DIBUTYL­ in the tumors than in the surrounding tissues. NITROSAMINECARCINOGENICITY IN RAT LIVER AND The levels of P-450 and GT in tumors and in sur­ URINARYBLADDER. M.A. Pereira, Health Effects rounding tissue were nearly equal. In conclus­ Research Laboratory, U.S. EPA, Cincinnati, OHand ion, increased enzyme activities and P-450 con­ Alfred L. Britt, Department of Laboratory Animal tent persisted 2 to 5 months after discontinu­ Medicine, University of Cincinnati Medical Cen­ ation of PCB exposure. These enzymes in tumor ter, Cincinnati, OH. Sponsor: R.J. Bull and in the surrounding tissue likely play a role Saccharin has been demonstrated to be a promoter in the metabolism of PCBs. (Supported by the VA of chemical carcinogenesis in the rat urinary and NIH grants CA22140 & 5-T32-ES07015,)

64 230 HEPATICEFFECTS OF a-HEXACHLOROCYCLOHEXANEIN THE At 100 mg/kg, the levels of binding at 0.5, 1.5, MOUSE.F. Iverson, L. Tryphonas, C. Miller, and 4.5 hrs were 3.1 + 0.3, 7.4 + 0.4, and 43.9 Toxicology Research Division, Foods Directorate, + 0.8 micromoles adduct/mole DNA-phosphorus, Health Protection Branch, Tunney's Pasture, r"espectively. Thus, the magnitude of adduct Ottawa, Canada, KlA OL2. formation tends to increase with both time and dose. Furthermore, analysis of this DNA by Manycyclic chlorinated hydrocarbon compounds bouyant density equilibrium centrifugation showed produce increased hepatic microsomal enzyme the radioactivity bound to the isolated DNA activity, enhanced DNAsynthesis and hepatic co-migrated with the unlabeled DNA at 1.7 g/mL. nodules in mice. Pathological classification of These results suggest that 2,4-TDA initiates these lesions is often disputed but several hepatocarcinogenesis in rats by directly inter­ studies with rat liver systems suggest that acting with DNA. hepatic nodules are precursors of hepatocellular carcinoma (HCC). Wegave hexachlorocyclohexane (HCH,a isomer) 232 PHORBOLESTERS MODULATETHE BINDING OF SEVERAL in the diet to male HPBblack mice. 500 ppm PEPTIDE HORMONESTO PITUITARY CELLS. R. Osborne a-HCHfor 9 months or 250 ppma-HCH for 23 months and A.H. Tashjian, Jr., Lab. of Toxicol., Harvard produced a 100%incidence of hepatic nodules. Sch. Pub. Hlth. and Dept. of Pharmacol., Harvard There was no evidence of HCCand control animals Med. Sch., Boston, MA. Sponsor: J,L. Whittenberger showed no spontaneous tumor formation. Foci of Phorbol esters (PE) are potent tumor promoters cellular alteration, commonlyfound in carcinogen which have been shown to alter specifically the exposed rat liver, were not observed in the mice. binding of epidermal growth factor (EGF) to Additional studies showed that 250 ppmand 500 diverse cell types in culture. In G84C1 cells, ppma-HCH at 4 weeks produced a significant but a clonal strain of rat pituitary cells, PE stimu­ similar increase in liver weight and mixed late the synthesis and release of prolactin and function oxidase activities associated with an inhibit growth hormone production after binding increase in the cytochrome with a CObinding peak to specific PE receptors. G84C1 cells also at 450 nm. DNAsynthesis was increased 2-fold have specific membrane receptors for EGF, thyro­ with 250 ppma-HCH and 4-fold with 500 ppma-HCH. tropin-releasing hormone (TRH), and somatostatin Enzymeactivity in nodules from 9 month a-500 (SRIH). Treatment with PE decreased the binding dosed animals was within 20%of the actt.vity in of EGF, TRH and SRIH to GH4C1 cells. 12-0- non-nodular areas. Tetradecanoyl-13-phorbol acetate (TPA) at 100 These studies suggest that a) hepatic nodules ng/ml decreased 125I-EGF binding to 15% of con­ formed in response to a-HCHin this species of trol within 1 h. By Scatchard analysis of 125I­ mouse do not progress to HCCb) that P450 depend­ EGF binding data, a 1-h pretreatment with TPA de­ ent enzyme activity in nodules is not decreased creased both the apparent affinity (2- to 6-fold) over that in surrounding areas and c) that the and the number of 125I-EGF receptors (by 25 to early appearance of nodules with 500 ppma-HCH is 50%). After 4 h of treatment, TPA decreased 3H­ associated with enhanced DNAsynthesis and not TRH binding to 35% of control due to a decrease in increased microsomal enzyme activity. affinity (2- to 5-fold) with no change in the num­ ber of TRH receptors. After 24 h, TPA also de­ creased 125I-Tyrl-SRIH binding to 40% of con­ trol. Effects of TPA on peptide binding were 231 BINDING OF 2,4-TOLUENEDIAMINETO RAT LIVER DNA, temperature dependent, occurring at 37°but not RNA, AND PROTEIN. Anthony J. Salerno,~. 4°C, and reversible. The potency order for de­ Unger, F. J. McMahon, and C. S. Aaron, Allied creased peptide binding was TPA > phorbol-12,13- Corporation, Morristown, NJ. didecanoate (PDD) > phorbol-12,13-dibenzoate, The aromatic amine 2,4-toluenedi:amine while phorbol and 4a -PDD were inactive, which (2,4-TDA) is a proven hepatocarcinogen in the parallels the activity of these compounds as tumor rat. Previous studies (Dybing et al, Arch. promoters. Thus, unlike findings reported in cer­ Toxicol., 1:213-218 (1978) demonstrated binding tain other cells, the actions of PE on peptide of 2,4-TDA to liver protein and RNA, but failed ligand binding in GH4C1 cells were not speci- to detect binding to liver DNA. The data sug­ fic for EGF. Modulation of receptors for multiple gested that 2,4-TDA may initiate hepatocaricino­ regulatory signals may play a role in the mecha­ genesis by an indirect mechanism, rather than by nism by which PE promote neoplastic transforma­ interacting directly with DNA. The purpose of tion. this study was to further investigate 2,4 TDA and the mechanism by which it initiates hepato­ carcinogenesis. ~~le Fischer 344 rats were ex­ posed to (methyl- C)-2,4-TDA by i.p. injection 233 BIOCHEMICALTOXICOLOGY OF PROPYLENE. T.G. Osimitz at 10 mg/kg and 100 mg/kg in groups of nine per and R.B. Conolly. Toxicology Program, Sch. of exposure level. The animals were sacrificed in Pub. Hlth, The University of Michigan, Ann Arbor, groups of three each at 0.5, 1.5; and 4.5 hours MI. Sponsor: R.H. Cornish following exposure. Immediately after sacrifice We have previously shown that propylene is the livers were excised, homogenized, and frozen acutely hepatotoxic in male Charles River COBS (-80°C) for subsequent extraction and analysis Sprague-Dawley rats pretreated with polychlorimted of DNA, ribosomal R..~A, and protein. In addition biphenyl (PCB; Aroclor 1254, 100 mg/kg for 3 days). to confirming the previously published level of We now report that pretreatment of rats withpheno­ 2,4-TDA binding to RNA and protein, binding to barbitol (PB; 80 mg/kg for 4 days), B-napthoflavone DNAwas also observed. At 10 mg/kg, the levels (BNF; 60 mg/kg for 4 days) or a mixture of the two of binding at 0.5, 1.5, and 4.5 hrs were 3.5 + at the above doses failed to result in the elevation 1.2, 3.9 + 0.8, and 2.6 + 0.1 micromoles adduct/ of serum sorbitol dehydrogenase (SDH) or liver/body mole DNAphosphorous, respectively (mean_:!: SEM). weight ratios observed in PCB-pretreated exposed

65 animals. Likewise, there was no change in cyto­ G. Witz and B.D. Goldstein, Dept. of Environmen­ chrome P-450 levels from not-exposed controls. tal and Community Medicine, CMDNJ-Rutgers Medical Pretreatment with isosafrole (75 mg/kg for 3 days) School/Rutgers University Joint Graduate Program followed by a 4-hour exposure to 50,000 ppm propy­ in Toxicology, Piscataway, NJ. lene also failed to result in increases in SDH or In the present study we have evaluated the liver/body weight ratios. No measure of cytochrome effects of certain of the proposed toxic benzene P-450 levels was made. The unique ability of PCB metabolites on mouse hepatic ALAD activity, which pretreatment to elicit hepatotoxicity from propy­ has previously been shown to be decreased follow­ lene suggests that PCB induces an enzyme or enzyme ing injection of benzene in rats, and on hepatic system distinct from those that PB, BNF, and iso­ SH content in six weeks old male CD-1 mice. In safrole induce and that this system is responsible vitro studies revealed a dose-responsive inhibi­ for propylene activation. In vitro incubation of tion of ALAD activity by 10- 3-10-5M benzoqui­ hepatic microsomes from PCB-pretreated rats with none, muconaldehyde and hydroquinone, but not by 40% propylene and a NADPH-generating system re­ catechol or benzene. Following incubation for 1 sulted in a decrease in cytochrome P-450 ((+)pro­ hour, benzoquinone, hydroquinone, muconaldehyde, pylene~ 1.98 + 0.06 nmole P-450/mg protein vs. and benzenetriol decreased both hepatic protein (-)propylene:-2.43 .± 0.06 nmole P-450/mg protein). and free SH by_jt least 50% at the highest con­ This effect was NADPHdependent. There was no de­ centration (10 M), while phenol, catechol and crease in P-450 in anaerobic incubations contain:ing benzene produced only a 13-22% decrease at 10-3M. microsomes, NADPH-generating system, propylene and However, there were no statistically significant a glucose oxidase system (anaerobic: 1.93 + 0.02 decreases in hepatic or red cell ALAD activity nmole P-450/mg protein vs. aerobic: 1.53 +-0.07 in vivo after intraperitoneal administration of nmole P-450/mg protein). This implies that oxida­ 0.45.f'moles of these compounds. Certain of the tive metabolism of propylene is necessary for in benzene metabolites did produce statistically vitro destruction of P-450 to occur. significant changes in hepatic SH content fol­ Supported, in part, by NIEHS 5 T32 ES07062 train­ lowing injection. Benzenetriol and muconalde­ ing grant. hyde decreased free SH by 11% and 12% respec­ tively but there was no change in protein SH con­ tent. Benzenetriol produced a 12% decrease in 234 EFFECT OF FLUORIDE ON MICROSOMALBENZENE METABO­ protein SH but did not affect the free SH while LISM. G. Post and R. Snyder, nept. of Pharma­ benzene caused a 21% decrease in protein SH and cology, Thomas Jefferson Univ., Philadelphia, PA a 13% increase in free SH. The toxicological 19107 and Graduate Program in ~oxicology, Rutgers significance of these small changes in hepatic Univ., Piscataway, NJ 08854. sulfhydryl levels is questionable. The lack of ~he stimulation of benzene (B) metabolism by confirmation of an effect of benzene on mouse fluoride (F) was studied in rat liver microsomes. hepatic ALAD activity in vivo previously re­ Metabolites measured were phenol (Ph) and an un­ ported for the rat may reflect species differ­ identified aqueous component (Ao). In microsomes ence. (Supported by NIH grant ES 02558) from control (I), benzene (II) and beta-naphtho­ flavone (III) induced rats, in the absence of fluoride, Ph formation increased from 0.002-0.08 236 mM Band plateaued from 0.8-2 mM B; in phenobar­ STIMULATIONOF LIPID PEROXIDATIONBYFERROUS bital induced microsomes (IV), no plateau was IRONINDUCED DECOMPOSITION OF LINOLEIC ACID reached by 2 mM B. Ph per mg protein was great­ HYDROPEROXIDE.H.W. Leung, M.J. Vang and R.D. est in II, intermediate in I and III, and least Mavis, Environ. Secretariat, National Research in IV below 0.6 mM B. At 2 mM B, the rate in IV Council, Ottawa, Ont., and Dept. of Radiation was higher than in I and III. F stimulation was Biol. &Biophysics, Sch. of Med. &Dent., Univ. dependent on F concentration; other halides had of Rochester, Rochester, NY. no effect. In I, II, and III, F stimulated at all Sponsor: P.E. Morrow B concentrations above 0.08 mM; in IV, stimulation Peroxidation of the membranephospholipids occurred from 0.03-0.6 mM B,but not at 2 mM B. F is believed to be the mechanismof toxicity for did not affect the proportions of Ph and Aq. In many environmental chemicals. Lipid IV, Ph was always less than 60% of the total, hydroperoxide, a major intermediary product of while in I, Ph was over 70%. In II and III, the lipid peroxidation, is beleived to undergo percent Ph increased as B concentration increased. decomposition to generate free radicals capable The percent Aq was greatest in IV. Thus the char­ of initiating further lipid peroxidation. acteristics of B metabolism appeared to differ Linoleic acid hydroperoxide (LOOH)was prepared among the microsomal preparations. In I, II and enzymatically from linoleic acid with soybean I·II, metabolism was stimulated by F and saturated lipoxygenase. Although pure LOOHalone was above 0.8 mM B; the rate in II was much higher remarkable stable at 37°c, it was rapidly than in I or III. Less of this activity was seen decomposedby FeSO4at both pH 5 and 7.4. in IV. Another activity, not affected by F and However, Fe2~ completely chelated with EDTA not saturated at 2 mM B, occurred in IV. Forma­ did not break downLOOH at either pH. tion of Aq also appeared induced in IV. These Furthermore, EDTA-Fe2+with or without LOOH data suggest that the array of microsomal benzene produced no malondialdehyde when incubated with metabolizing enzymes was modified by these induc­ liposomes of extracted microsomal lipid. ing agents. However, while Fe2+ could stimulate liposomal (Supported by NIH Grant ES00322.) peroxidation, the initial rate of peroxidation was higher when LOOHwas also present. This potentiation of lipid peroxidation by LOOHwas 235 ALTEREDHEPATICS AMINOLEVULINICACID DEHYDRATASE observed at pH 7.4, when Fe2+ was rapidly (ALAD) AND SULFHYDRYL(SH) CONTENTDUE TO PRE­ autoxidized, but not in acidic conditions when SUMEDBENZENE METABOLITES. B.S. Lallian, G.S. Rao, Fe2+ was stabilized. This data suggest that

66 Fe2+ is able to break downLOOH.to a free status on in vitro lipid peroxidation of liver radical intermediate. At low effective Fe2+ microsomalpreparations obtained from male Long­ concentrations, this free-radical species may Evans Hooded rats fed chemically defined diets attack polyunsaturated fatty acids, thus containing adequate or documented deficiencies of enhancing the rate of lipid peroxidation. E and/or Se (+E, +Se; -E, + Se; +E, -Se; -E, However, Fe2+ at high effective -Se;). In both enzymatic and non-enzymatic concentrations probably reduce this reactive systems, lipid peroxidation, as determined by the intermediate directly to the relatively stable measurement of thiobarbituric acid reactive pro­ hydroxy product. ducts, was markedly inhibited by reduced glutathione (GSH) in E supplemented groups. However, the inhibitory effect of GSH was not observed in a non-enzymatic system containing heat 237 FREE RADICALFORMATION, LIPID PEROXIDATION,ENERGY treated microsomes. It was also found that micro­ METABOLISMAND INTRACELLULARCa2+, Na+ AND~ IN somal E levels were not affected by heat treatment. RAT CEREBRALCORTEX SLICES EXPOSEDTO OXYGENAT This suggests that GSH may be inhibiting lipid HIGH PRESSURE. R.C. Dirks and M.D. Faiman, Dept. peroxidation via enzyme dependent regeneration of of Pharmacol. and Toxicol., Univ. of Kansas, microsomal E. Several other sulfhydryl compounds Lawrence, KS 66045 were tested in place of GSH and found to have no It is proposed that during hyperbaric oxygen E dependent inhibitory effect. Kinetic properties (OHP) exposure, free radicals are formed leading of inhibition of lipid peroxidation by GSH was to the peroxidation of membrane lipids, altered found to be complex. In addition, the effects of membrane permeability, and the disruption of cell­ diethylhydroxylamine, a potent free radical ular homeostatic mechanisms necessary for the re­ scavenger as well as anti-tumorogenic agent, was establishment of normal ionic gradients. It is tested and found to markedly inhibit lipid further proposed that these abnormal ion movements peroxidation in both enzymatic and non-enzymatic play an important role in the initiation of 02-in­ systems. (supported by EPA Grant# R807746). duced convulsions. In the studies to be reported,. the effects of OHP on free radical formation, lip­ id peroxidation, ion movement, and factors affec­ ting ion transport were investiated in rat cere­ 239 IN VITRO HEPATOTOXICITY OF THE GLUTATHIONE bral cortex slices. Rat brain slices, approximate­ DEPLETORS DIETHYLMALEATE, IODOACETAMIDE, ly 0,5mm thick, were cut with a Stadie-Riggs tis­ AND ACETAMINOPHEN. D.B. Mitchell and D. Acosta, sue slicer and placed in a bicarbonate-buffered College of Pharmacy, The University of Texas, Austin, balanced salt solution containing 10 mMglucose. TX 7871Z. The brain slices were incubated at 37oc for 90 min. with continuous oxygenation und.er oxygen We have developed a model of primary cultures of pressures from 1 to 14 ata. Using spin-trapping postnatal rat hepatocytes to investigate the mecha­ techniques, free radical formation was demonstra­ nisms by which xenobiotics produce cellular injury. ted beginning at 6 ata. At 9 ata, tissue malonal­ Glutathione (GSH) is a unique tripeptide that has a dehyde levels (indicator of lipid peroxidation) well-known role in the hepatic conjugation and detoxi­ were significantly increased when compared to non­ cation of xenobiotics and is associated with the main­ exposed control slices. Also at 9 ata, an increase tenance of normal cellular structure and function. In in intracellular Na+ and ca+ and a decrease in order to test the hypothesis that GSH depletion may be intracellular K+ was observed. No change in brain a primary pathophysiologic event in cell injury, we slice Na+r ATPase activity was observed; however, chose to compare the toxic effects of 3 different brain slice ATP content dropped significantly be­ chemicals known to deplete cellular GSH pools: di­ ginning at 9 ata. It is concluded that OHP causes ethylmaleate (D), iodoacet~ide (I), and acetaminophen changes in ion movement which may be due to de­ (A). Treatment with I (10 M) depleted GSH levels by creased cellular energy production or, possibly, 90% within 1 hr. Cell viability decreased significantly, altered membrane permeability as a result of free LDH leakage was 500% of control, and lipid peroxi­ radical-initiated lipid peroxidation. The altered datio~ was Z times control values. A higher dose of ion movements could then be responsible for the D(lO M) was needed to produce similar toxic effects 02-induced convulsions. Supported in part by NIH as I. Whereas I and D caused significant GSH depletion grant# T32-GM 07775 and NS-07797, and toxicity by 1 hr, A required higher doses and longer treatments to deplete GSH (Z4 hr, 65%). Toxicity produced by A was not as pronounced as that observed 238 INHIBITION OF LIPID PEROXIDATIONBY REDUCED with I or D. The degree of toxicity observed with the GLUTATHIONE: EVIDENCEFOR THE ENZYMATICREGEN­ three agents may reflect their individual interactions ERATIONOF VITAMIN E IN LIVER MICROSOMES, with GSH and cellular constituents. The concurrent C. E. Thomas, C. C. Reddy, C-Y. Ho, R. W, Scholz, treatment of cells with A and D had an additive effect and E, J. Massaro,* The Pennsylvania State on toxicity. The time lag prior to toxic effects caused University, University Park, PA.· by A may be the result of cytochrome P-450 mediated metabolism to the toxic intermediate that binds to Lipid peroxidation, initiate.d by a number of GSH. In general, GSH depletion that reached approxi­ atmospheric oxidants such as ozone and nitrogen mately 70% of control values resulted in irreversible dioxide as well as endogeneously generated hepatocellular injury. reactive oxygen species, has been implicated as a causative factor in oxidative cellular damage. However, vitamin E (E) and selenium (Se) function synergistically to constitute an important anti­ 240 MECHANISTICSTUDIES ON THE IN VITRO METABOLISM oxidant defense mechanism to protect the cell from OF THIOACETAMIDE-S-OXIDEBY RAT LIVER MICRO­ peroxidative damage. In these studies, we have SOMES. M.C. Dyroff, and R.A. Neal. Center in investigated the effects of dietary E and Se Toxicology, Department of Biochemistry,

67 Vanderbilt University, Nashville, TN 37232, during hepatotoxicity-induced disruption of cellular and Chemical Industry Institute of Toxicology, homeostasis,, SAM may react non-enzymatically with Research Triangle Park, NC 27709, nucleophilic sites in DNA. Whatever mechanism is ultimately found to be responsible for the methylation Thioacetamide is a much studied hepato­ of DNA during hepatotoxicity, the consequence, carcinogen which binds covalently to cellular genetic damage, may relate cytotoxicity to macromolecules in vivo and after microsomal carcinogenicity. (Supported by U.S. Air Force metabolism in vitr~ Previous work in this Contracts 33615-76-C-5005 and 33615-80-C-0512). laboratory has identified the major hepatic protein covalent adduct formed in vivo as being N- E: -acetyllysine. Greater than 90% of the radioac ti vi ty which is bound to protein in 242 EVIDENCE FOR A PUTATIVE N-NITROSOPYRRO­ vitro during microsomal metabolism of thio­ LIDINE-DERIVED DNA ADDUCT. E.J. Hunt and R.C. acetamide-S-oxide has been identified as the Shank. Dept. Comm. &: Env. Med., Univ. of Calif. same adduct, N- s-acetyllysine. Acetamide, Southern Occup. Health Ctr., Univ. of Calif., Irvine, another product of thioacetamide-S-oxide CA. 92717. (Supported by PHS Grant CA 21955) metabolism by rat liver microsomes, and the Many carcinogens, including dialkylnitrosamines, protein adduct, N- s -acetyllysine, appear to are thought to induce cancer through covalent binding be derived from a common intermediate postu­ of derivatives to target organ DNA. Some evidence lated to be thioacetamide-S-dioxide. Addition suggests that N-nitrosopyyrolidine (NP) and other of thiol-containing compounds to the in vitro cyclic nitrosamines are at variance with this mechan­ incubation media decreased the levels of radio­ ism (Cancer Res. 33:1634, 1974). Liver DNA was activity covalently bound to protein to a small isolated 3, 6, 12, ls,'""24, 30, or 36 hr after rats were extent. Aliphatic amines decreased the level given 900 mg NP/kg body wt po. The DNA was sub­ of binding to a much greater extent. Studies jected to neutral thermal hydrolysis in pH 7 buffer for with 18-0 enriched water or gas indicated that 1 hr at 100°, and hydrolysates were fractionated by the oxygen atoms of acetamide and the acetyl strong cation exchange high pressure liquid chroma­ group of N- s-acetyllysine are derived solely tography. Elution of fractions was monitored by from water. Chemical oxidation of thio­ fluorescence spectrophotometry (excitation, 286 nm; acetamide-S-oxide in the presence of protein emission interference filter, 343 nm). An unidentified results in covalent binding of radioactivity to fluorescent material was detected in all DNA hydroly­ the protein. The adducts which are formed are sates from NP-treated rats, but not in hydrolysates highly pH dependent with N- E: -acetyllysine from control animals. The putative adduct formed being the major adduct formed at pH 7 .4 and rapidly; the concentration of this material in liver N- s -acetamidinolysine being the predominant DNA increased more than 3-fold from 3 to 12 hr adduct formed at pH 10.4. (Supported by NIH following NP treatment, then began to gradually grants ES 00075, ES 00267 and ES 07028.) decrease over later times. The fluorescent material did not co-chromatograph with standard guanine derii­ atives methylated or ethylated in either the 7- or O - 6 position. The concentration of fluorescent material in 241 7-METHYLGUANINE AND 0 -METHYLGUANINE liver DNA hydrolysates was dose-dependent in rats FORMATION IN LIVER DNA OF RATS, MICE, AND killed 12 hr after receiving 14, 28, 57, 113, 225, 450, or HAMSTERS TREATED WITH HEPATOTOXINS. R.A. 900 mg NP/kg body wt. Neutral thermal hydrolysis is Becker and R.C. Shank, Dept. Comm. &: Env. Med., known to release 3-alkyladenine and 7-alkylguanine University of California, Irvine, CA 92717 from DNA; alkylguanines fluoresce well, while alkyl­ Previous studies have demonstrated 7-methyl­ adenines do not. Hence, the putative DNA adduct in 6 guanine (7-MG) and 0 -methylguanine (0 6 -MG) in liver NP-treated rats may be a guanine derivative substi­ DNA from rats treated with hydrazine. We have now tuted in the 7-position. This evidence is consistent quantitated 7-MG and in some cases 0 6 -MG in liver with the generalization that NP, as other chemical DNA from rats treated with hepatotoxic doses of carcinogens, binds covalently to target organ DNA. ethanol, carbon tetrachloride, phosphorus, bromo­ benzene, and puromycin. Neither 7-MG nor 0 6 -MG was detected in comparable amounts of liver DNA from control animals. Kinetics of formation and ·243 D(+)GALACTOSAMINE CYTOTOXICITY IN PRIMARY removal of 7-MG and 0 6 -MG in liver DNA were CULTURES OF POSTNATAL RAT HEPATOCYTES. investigated in rats treated with 90 mg hydrazine/kg K.S. Santone and D. Acosta, College of Pharmacy, The body wt. and killed 0.25-96 hr later. 7-MG and 0 6 -MG University of Texas, Austin, TX 78712. formed rapidly and concomitantly; half-maximum We have developed an in vitro system of primary methylation occurred within 1 hr after treatment. At cultures of postnatal rat hepatocytes to serve as an maximum methylation (6-24 hr after exposure) liver experimental model of chemical toxicity. Because the DNA contained 870 µ mol 7-MG and 80 µ mol 0 6 -MG hepatotoxicity of galactosamine has been investigated per mol guanine. 7-MG was removed from DNA at a extensively in vivo, we chose it as a model hepatotoxin rate of approximately 50% in 47 hr, while the half-life to evaluate the specificity and sensitivity of our in of 0 6 -MG in liver DNA was approximately 13 hr. vitro system for hepatotoxicity studies. To evaluate its Similar levels of 7-MG and 0 6 -MG were found in liver potential cytotoxicity, we utilized commonly used para­ DNA from mice and hamsters killed 1-24 hr after meters of in vitro toxicity, trypan blue viability test exposure to hepatotoxic doses of hydrazine. The and cell protein levels, as well as more sensitive indices molecular mechanisms responsible for liver DNA of toxicity developed in our laboratory-leakage of methylation during hepatotoxicity are under investi­ cytoplasmic enzymes and lactate to pyruvate ratios gation. S-Adenosylmethionine (SAM) has been tenta­ (L/P). Liver parenchymal cells were isolated by an in tively identified as the proximal methylating species. situ collagenase perfusion technique and cultured 24 hr The ratio of 7-MG to 0 6 -MG at maximum methylation prior to experimental treatments. The cultured hepa­ is consistent with an SNl reaction, suggesting that tocytes were exposed to 0.25, 0.50, and 1.0 mM galac-

68 tosamine for durations of 2, 6, 12, and 24 hr. A small this drug. Four groups of day 15 Sprague-Dawley rat fetuses but significant decrease in viability (ZO%) was observed were directly injected with 2 µI dimethylsulfoxide containing only after the highest dose and longest period of 0, 25, 50 or 200 µg Th. Females were sacrificed on day 20 of treatment. No significant changes in total cell protein gestation (positive vaginal smear day 'O'). The percentage of were observed at any of the concentrations or time frames studied. A 33% increase in leakage of cyto­ dead or resorbed fetuses was 14% in control and in low and plasmic lactate dehydrogenase (LDH) over control lev­ middle dose and 74% in high dose Th. Among the living els was observed as early as 6 hr with the highest dose young 0/48(0%) control, 1/64(1.6%) low-, 5/65(7.7%) middle­ of galactosamine. Longer exposures did not increase and 4/10 (40%) high-dose groups had limb deformities, the leakage of LDH. Decreases in L/P were shown to including hemimelia, amelia and ectrodactylia. Since be dose- and time-dependent, with a maximum decrease metabolic products, rather than the parental Th, are believed of 50% occurring by 12 hr. Our findings indicate that LDH leakage and L/P are sensitive and reliable in­ to be involved in the teratogenic process in sensitive species, dicators of galactosamine cytotoxicity in a primary an attempt was made to test the teratogenic activity of blood culture system of postnatal rat hepatocytes. plasma from female cynomolgus (Macaca irus) previously treated with Th. Blood plasma samples were taken from one untreated and one Th-treated monkey and injected into the fetal rat bodies, 10 µI/fetus. Of 56 fetuses that received blood 244 DECREASEDRESPONSE TO PHORBOLESTERS INVOLVESA plasma from the treated monkey 66% were dead or resorbed TWO-STEPPROCESS INCLUDING RECEPTOR MODULATION. while 10/19 (53%) living young had malformations (limb S. Jaken, M. Phillips, and A.H. Tashjian, Jr., Interdisciplinary Prog. in Hlth. and Lab. of deformities as above). The figure in control fetuses was 32% Toxicol., Harvard Sch. Pub. Hlth., Boston, MA. dead or resorbed and 0/73(0%) malformed. Given the close Sponsor: J.L. Whittenberger similarity between monkeys and humans in terms of response to teratogenic stimuli, these results suggest an interesting Tumor-promoting phorbol esters (PE) act on approach for testing drugs that may adversely affect human cells via interaction with specific membrane conceptuses. receptors. Escape from PE-mediated ~ctions, pos­ sibly including tumor promotion, have been docu­ mented. We have studied the mechanisms by which 246 THE TERATOGENICITYOF THE AMINOPHENOLS(AP) IN cells escape from PE effects using as a model SYRIAN GOLDENHAMSTER (SGH). J.V. Rutkowski system a clonal strain of mouse pituitary cells and V.H. Ferm, Dept. of Pharmacol./Toxicol. and (D16) which synthesize and secrete ACTH. PE en­ Dept. of Anat./Cytol., Dartmouth Med. Sch., hance ACTH release from D16 cells 2- to 5-fold Hanover, NH. Sponsor: _!!.Q_. Roebuck above unstimulated control levels. ED5os for this response, -16 nM for phorbol dibutyrate p-AP has been recently demonstrated to be a (PDBu), -2 nM for phor bol myri state acetate (PMA) metabolic product of the analgesic-antipyretic agree closely with the apparent 1

245 TERATOGENICITY OF THALIDOMIDE BY DIRECT INJECTION INTO FETAL RATS. L. Rossi, M. Ferro, 247 A COMPUTERIZED TEST FORM GENERATION A. Verardi, 0. Barbieri and A. Mondino, 1st. Ricerche Biomed. SYSTEM FOR BEHAVIORAL TERATOLOGY STUDIES 'RBM', lvrea, and 1st. Scientifico per lo Studio e la Cura WITH RATS. S. A. Kutz, N. J. Troise, P. D. Sweigart, A. J. George, P. J. Rugen, P. J. DeBaecke dei Tumori, Genova - Italy. Sponsor: J.R.P. Cabral. and R. Teichman, Stuart Pharmaceuticals, a Division of ICI Americas Inc., Biomedical and Clinical Re­ The teratogenic effects of thalidomide (Th) were studied in search D~partments, Toxicology and Biostatistics rats, a species usually resistant to malformations induced by Sections, Wilmington, DE. Sponsor: L. E. Gangwer

69 We designed a computerized form generation system 249 FETALDEVE~0PMENT IN NEWZEALAND WHITE {NZW) utilizing SAS programming via CMS terminals to: (1) RABBITSTREATED IV WITHETHYLENE OXIDE (ET0) generate preprinted data recording forms for various DURINGPREGNANCY. C.A. Kimmel, J.B. LaBorde, C. behavioral teratology tests; (2) calculate testing days; Jones-Price, T.A. Ledoux and T.A. Marks, Div. of and (3) serve as data submission forms for statistical Teratogenesis Research, NCTR/FDA/DHHS,Jeffer­ analyses. We use these computer-generated forms for son, AR, and Research Triangle Inst., Research the following behavioral/developmen.tal tests for rats: Triangle Park, NC. ptp weights, pinna detachment, surface righting, cliff avoidance, incisor eruption, eye opening, negative geo­ Previous studies on the iv administration of taxis, swimming development, open field activity, ET0 to pregnant mice (LaBorde and Kimmel, TAP swimming maze, and operant visual discrimination 56:16-22, 1980} indicated a teratogenic effect learning. The major advantages of this system following 150 mg/kg on days 6-8 of gestation. include: (1) elimination of manual data transcription; Studies were therefore conducted in rabbits to (2) elimination of human error in calculating testing assess the teratogenic potential in a non-rodent dates; (3) the ability to spread parturition dates over species. NZWrabbits were administered 0, 9, 18 several weeks, thereby equalizing the work load; and or 36 mg/kg ET0 iv (in 5% dextrose) on days 6-14 (//-)providing more consistent data recording and scor­ of gestation or 0, 18 or 36 mg/kg iv on days 6-9 ing conditions. We have conducted two major behav­ of gestation. Preliminary studies had indicated ioral teratology studies using the computer-generated the maximum tolerated dose (MTD) to be approxi­ forms. In the first study, 1 computer-calculated test mately 40 mg/kg. Maternal toxicity was assessed date was overlooked. This resulted in the loss of 8 of throughout pregnancy and on day 30, dams were approximately 5,696 test results and represents a killed and uterine contents examined. A gross human error rate of approximately 0.14 percent. In examination of surviving fetuses included exter­ the second study, 8 test dates were missed. This nal, visceral and skeletal evaluations. A sig­ resulted in the loss of 59 of approximately 11,680 test nificant trend toward decreased maternal weight results and represents a human error rate of approxi­ gain was seen during treatment and throughout mately 0.51 percent. We are currently conducting a gestation after treatment either on days 6-9 or third behavioral teratology study and have added a 6-14. No significant effects were seen in the dynamic computer-generated daily activity schedule feta 1 parameters examined after treatment on for use by the laboratory to prevent overlooking test days 6-9 of gestation. However, a significant dates. We also added an automatically-generated OS increase in mean number and% resorptions/litter data set (database type file) for use by data entry was noted in the 36 mg/kg dose group treated on personnel. days 6-14 of gestation. No other effects on fetal viability, weights or morphology were seen after the 1anger treatment period. Thus, ETO administration to pregnant rabbits does increase 248 EVALUATION OF THE DESIGN AND SCORING embryotoxicity, but only after treatment METHOD OF SEVERAL BEHAVIORAL TERATOLOGY throughout organogenesis and at a dose that also TESTS FOR RATS. N. J. Troise, S. A. Kutz, P. J. produces maternal toxicity. Unlike the effect Rugen, P. D. Sweigart, A. J. George, P. J. DeBaecke of ETD in mice, no teratogenic effects were and R. Teichman, Stuart Pharmaceuticals, a Division detected in rabbits in this study. of ICI Americas Inc., Biomedical and Clinical Re­ search Departments, Toxicology and Biostatistics Sections, Wilmington, DE. Sponsor: L. E. Gongwer 250 EFFECTOF A SINGLEORAL DOSE OF CAFFEINEDURING PREGNANCYON BLOODFLOW AND SUBSEQUENT EMBRYO/ We conducted a uniformity study to evaluate the FETALDEVELOPMENT IN THE RAT. C.A. Kimmel, C.G. design and scoring method of several behavioral tera­ White, T.F. Grafton, C.J. Nelson and G.L. Kimmel, tology tests. Ten pregnant HLA-SD rats approxi­ Div. of Teratogenesis Research and Div. of Biome­ mately 13 weeks of age were dosed by intubation at 5 try, NCTR/FDA/DHHS,Jefferson, AR. ml/kg with distilled deionized water on days 6-19 of gestation. On postnatal day 3, a maximum of nine Alterations in blood flow to the uterus and pups from each of the ten litters were randomly its contents during pregnancy have been suggested selected for behavioral testing. The following be­ to account for the teratogenicity and/or embryo­ havioral/developmental tests were evaluated: pinna toxicity of several agents, including caffeine. detachment, incisor eruption, eye opening, surface Using the radioactive microsphere technique righting, cliff avoidance, grasp/holding, negative geo­ (Buelke-Sam and Holson, 1980), blood flow to taxis, swimming development, and open field activity. several maternal organs, including ovary, uterus, Interpretation of statistical analyses of these data decidua and chorioallantoic placenta (CAP), was yielded the following recommendations and results: measured following a single dose of 0 or 120 (1) pups should be randomly selected for testing by sex mg/kg caffeine by gavage to pregnant CD rats on on postnatal day l; (2) the use of more litters with day 12 of gestation. At 1 and 4 hrs after treat­ fewer pups is preferred for analysis of treatment ment, animals were anesthetized and sr85_labeled responses; (3) an all or none scoring method is microspheres {25u diam.) were infused via the acceptable for pinna detachment, incisor eruption, and right carotid artery. Who1 e body and tis sue eye opening; (//-) female pups develop pinna detach­ radioactivity were determined. Maternal cardiac ment, incisor eruption, and eye opening faster than output (CO) and absolute flow (fl; ml/min), rela­ male pups; (5) surface righting, cliff avoidance, and tive flow (f2; ml/min/g tissue) and flow as %CO grasp/holding should be timed for test completion and {f3) to each tissue were calculated. Maternal CO not timed to meet an arbitrary criteria for success; was not altered. Ovarian fl was reduced at 4 hrs (6) the negative geotaxis test should be completed and f2 and f3 at 1 and 4 hrs after caffeine prior to any ocular light perception; and (7) female treatment. Uterine fl, f2 and f3 were reduced at rats are more active than male rats in the open field both times. Decidual weight was reduced at 1 hr, test. f2 at 4 hrs, and fl and f3 at both times. How-

70 ever, CAPweight and blood flow were not altered and eventually some spermatogenic arrest, by caffeine treatment. Examination of embryos there is no evidence that levels of these from these litters and from other animals at 24 PAEs that might be leached from plastic hrs after treatment and fetuses at term did not medical devices can exert such toxic reveal any effect of this dose of caffeine on actions. Using a series of dose regimens viability, growth or physical development. Thus, of DEHP or MEHP (ranging from 1 mg/kg to a single dose of caffeine significantly reduced 100 mg/kg daily x 5-ip) that were con­ blood flow to the ovary, total uterus and de­ firmed not to decrease the total of cidua. In spite of this effect, conceptal devel­ endogenous zinc in rat testes or prostate opment was normal. This may be attributable to glands, investigations were undertaken to maintenance of normal blood flow to the CAP, a study a number of zinc-related events. functioning placental unit by day 12 of gesta­ Gonadal alkaline phosphatase, an enzyme tion. affected by androgens as well as being zinc-dependent, was not altered by either DEHP or MEHP in sexually mature rats. 251 TERATOGENICEVALUATION OF ETHYLENE CHLOROHYDRIN Likewise, no PAE-induced changes in pros­ (ECH)IN MICEAND RABBITS. J.B. LaBorde, C.A. tate alkaline phosphatase activity were Kimmel, C. Jones-Price, T.A. Marks and T.A. noted. Neither RNA nor DNA levels in Ledoux, Div. of Teratogenesis Research, NCTR/ reproductive organs were affected by FDA/DHHS,Jefferson, AR, and Research Triangle levels of DEHP/MEHP that would be compar­ Inst., Research Triangle Park, NC. able to those encountered as a result of Earlier teratology studies with ethylene their being leached from plastic medical oxide (ETO) in mice (LaBorde and Kimmel, TAP devices. The lack of changes in gonadal 56:16-22, 1980) reported teratogenicity of ETO nucleic acids was correlated with normal at 150 mg/kg iv on days 6-8 of gestation. ECH, morphology in the seminiferous tubules. a reaction product of ETOand chloride ion, may The present findings reveal a wide range be a residue in poorly degassed medical devices of no-effect levels of DEHP or MEHP in following sterilization with ETO. This study reproductive organs of the rat .. assessed the teratogenic potential of ECH(in 5% dextrose) administered iv to CD-1 mice (O, 60 or 120 mg/kg) on days 4-6, 6-8, 8-10 or 10-12 of 253 FAILURE OF PHTHALATE ACID ESTERS (PAE) gestation, and to NZWrabbits (0, 9, 18 or 36 TO STIMULATE LIPID PEROXIDATION IN RAT mg/kg) on days 6-14 of gestation. The highest REPRODUCTIVE ORGANS. Walter C. Brogan, dose levels approximated the MTDin each spe­ III, Karen A. Curto and John A. Thomas. cies. Litters were examined on day 17 (mouse) Dept. Pharmaco1. & Toxicol., West Virgin­ or day 30 (rabbit) of gestation. No significant ia University Medical Center, Morgantown, maternal or embryo-fetal effects were noted in West Virginia 26506. the rabbit. In mice, a significant reduction Although dietary zinc deficiency and was noted in maternal weight gain during treat­ PAE-induced zinc deficiency reportedly en­ ment at all time periods. Maternal weight gain hance hepatic lipid peroxidation, no throughout gestation was significantly reduced studies have examined this relationship following 120 mg/kg on days 8-10 and 10-12 of in the gonads despite the fact that the gestation. A significant increase was seen in testes are a primary site of toxic ac­ mean resorptions/litter after treatment on days tions of diethylhexyl phthalate (DEHP) 4-6 and 10-12 at 120 mg/kg. Meanfetal weight/ and/or monoethylhexyl phthalate (MEHP). litter was significantly decreased at the high When rat testes or prostate were incu­ dose for all treatment periods. A significant bated (e.g. 60 min) with varying concen­ increase in the mean number of malformed fetuses trations (viz., 10-5 to 5 x 10-2M) of was observed only on days 8-10 at 120 mg/kg. either DEHP or MEHP, there was no increase Malformations were mainly skeletal, although a in the production of a thiobarbituric low incidence of cleft palate, open eye, micro­ acid-reacting chromogen, malonaldehyde melia, and adactyly was recorded. Thus, ECH (MDA). Further, MDA levels were not in­ increased malformations and resorptions, but creased when NADPH was added to the in only following treatment that was toxic to ma­ vitro system. Testes obtained from rats ternal mice. Additionally, ECHwas not terato­ previously injected with DEHP or MEHP genic in the rabbit with the dosing regimen (e.g. 48 or 100 mg/kg daily x 5-ip) fail­ employed in this study. ed to reveal any significant decrease in cytochrome P-450 content or aryl hydro­ carbon hydroxylase activity. While addi­ 252 FURTHER STUDIES ON THE EFFECTS OF THE tional studies are underway to determine PHTHALATE ACID ESTERS (PAE) ON RAT MALE whether these PAEs can affect gonadal REPRODUCTIVE ORGANS. Karen A. Curto, steroidogenesis, the present findings do Robert E. Mccafferty, Michael P. Donovan not indicate that lipid peroxidation is involved in DEHP/MEHP-induced gonadal and John A. Thomas. Dept. Pharmacol. & toxicity. Toxicol., West Virginia University Medi­ cal Center, Morgantown, West Virginia 26506. 254 ACUTE OR CHRONIC DIETARY EXPOSURE TO While high doses of diethylhexyl POL YCHLORINA TED BIPHENYLS (PCBs): EFFECTS phthalate (DEHP), and its principal me­ ON MATERNAL METABOLIC HOMEOSTASIS AND ON tabolite monoethylhexyl phthalate (MEHP), EMBRYOTOXICITY CAUSED BY 5-FLUORODEOXY­ reportedly cause gonadal zinc depletion URIDINE (FUdR) IN MICE. F. Welsch, K.S. Hardyniec

71 and D.B. Stedman (Sponsor: J.I. Goodman), Dept. 20 of each sex, from each treatment will be pre­ Pharmacol./Toxicol. and Ctr. Env. Toxicol., Mich. sented. Completion of these studies will provide State Univ., E. Lansing, MI 48824. basic information on toxicity produced by the si­ multaneous exposure of several environmental con­ The potential consequences of multiple low level taminates and will help determine what effect MeHg exposure to chemicals on embryonal/fetal development has on potentiation of cancer induced by nitroso are of great concern in prenatal toxicology. In this compounds. (Supported in part by USPHSGrants contribution we describe the effects of PCBs Aroclor ES-00210 and ES-00040). 1254; 0,1,10 and 100 ppm for 90 days prior to breeding (=chronic) or beginning on day 6 of gestation (=acute) fed to ICR Swiss mice on the metabolism of xenobi­ otics whose fate is coupled to cytochromes P450 and 256 EARLYPOSTNATAL DEVELOPMENT OF PRENATALLY P448. We have also measured the fetal body burden of IRRADIATEDRATS. M.B. Newby-Schmidt, J.A. PCBs and correlated all observations with embryotox­ Donoso, L.A. Neighbors and S. Norton. Dept. of ici ty induced by a single i.p. injection of FUdR (7.5 or Pharmacology, University of Kansas College of 20 mg/kg; 260-265 hrs after mating). This teratogen Health Sciences, Kansas City, KS 66103. can be activated in the embryo/fetus. The effects will be compared in ongoing experiments to those caused by A series of developmental behavioral tests was cyclophosphamide which in mice relies solely on mater­ used to assess the performance of prenatally nal activation. Liver P450 content was increased by 100 irradiated rat pups. Pregnant rats were ppm PCBs both in acute and chronic mice. Aminopy­ irradiated on gestational day 15 with doses of rine demethylase activity was elevated in chronic 100 0, 25, 50, 75 and 125 R. Neurobehavioral and 10 ppm animals, but acutely only at 100 ppm. testing was begun on postnatal day 1. Pre­ Among the P448 mediated reactions aryl hydrocarbon liminary data indicate that pups exposed to hydroxylase and ethoxyresorufin-0-deethylase activity 125 R show a marked developmental lag with were induced by chronic and acute 100 ppm PCBs and progressively less delay at the lower doses of also by chronic 10 ppm, but by none of the other X-ray. This dose response was evident in the exposure levels. While PCBs alone had no teratogenic pups' performance on the the righting reflex action, FUdR alone was very effective (Welsch and . (days 1 to 14), reflex suspension (days 5 to Hardyniec, Teratology 23, 68A, 1981). The differing 14) and spatial maze exploration (day 35) tests PCBs regimens caused a variety of alterations in the as well as a test in which the rats are placed expression of teratogenicity including changes in fre­ on a hole board and the observer counts the quency of malformations at specific sites. One ex­ number of times the rat pokes its nose into the ample is an increase in polydactyly of the hind limbs holes (day 35). In addition, a marked effect among 7 .5 mg/kg FUdR plus chronic PCBs exposed was noted on the time of eye-opening with fetuses in comparison to FUdR alone. (Supported by controls averaging 14.6 days of age and the NIH Grant ES 02461.) pups exposed to 125 R averaging 16.2 days of age. There are two possible explanations for the development delay produced by X-irradiation; 1. the effects on behavioral development are 255METHYLMERCURYEFFECT ON TRANSPLACENTALTOXICITY reflecting the gross morphological damage to INDUCEDBY NITRITEAND ETHYLUREA IN RATS. the CNS that is produced by X-irradiation on J.E. Nixon and J.P. Luebke, Dept. of Food Sci. gestational day 15 (Mullenix et al., Exp. & Tech. and Env. Hlth. Sci. Cntr., Oregon Neurol., 48: 310, 1975) or 2. since developmen­ State University, Corvallis, OR 97331 tal delayis known to occur with thyroid In our lab io ppm methylmercury chloride (MeHg) deficiency (Eayrs and Lishman, Brit. J. Anim. increased the transplacental toxicity of sodium Behav., 3: 17, 1955), possibly the x-=-irradiation nitrite (N0 ) and ethylurea (EU) for reproduction is damaging the thyroid and producing functional and latency 2of neurogenic tumor development in hypothyroidism. (Supported in part by USPHS rats. To determine whether the MeHgeffect was grants NS 16694 and ES 07079.) dose dependent and effective with different levels of NO -EU exposure, 4 levels of MeHg (0, 5, 10 and 15 pp~) were fed from weanling age until parturi­ 257 ASSESSMENTOF PERINATALLYINDUCED RENAL DYSFUNC­ tion to female Wister rats in combination with 4 TION. R.J. Kavlock and J.A. Gray. HERL, USEPA, levels of N02 in the drinking water (0, 0.75, 1.00 Research Triangle Park, NC (Sponsor: N. Chernoff) and 1.50 g/lJ and EU in the diet (0, 2.4, 3.2 and 4.8 g/kg) during gestation. Litter rate was not In order to ascertain the sensitivity of a affected by either the level of MeHgor NO -EU series of tests we developed for the evaluation and ranged from 88 to 100% for all treatmefits with of renal function in neonatal rodents (The 16 females per group. The combination of the Toxicologist 1:82 (1981)), we administered four highest level of MeHgand N0?-EU produced the most known or suspected renal toxicants to Sprague severe effects on litter size, birth weight and Dawley rats at critical periods of development survival (ca. 56, 90 and 60% of positive controls) and applied a diuresis test with and without but the effects did not consistently increase anti-diuretic hormone on postnatal day 3 (PD 3), linearly with increasing level of MeHgfor all a hydropenia test on PD 6, and determined parameters. Survival to weanling age decreased kidney weights, glomerular counts in mid-hilar with increasing MeHglevel with the 2 highest le­ cross sections and the specific activity of vels of NO -EU. A reproduction efficiency index renal alkaline phosphatase on PD 3 ~nd 6, computed f~om litter rate, litter size and survi­ Chlorambucil (CHL) was given i.p. on day 11 of val with units of number of pups weaned/female/ gestation at 0, 3 and 6 mg/kg, Nitrofen (NIT) treatment showed the best dose-response for MeHg was given p.o. on days 7-16 of gestation at 0, at each level of N02-EU. Mortality and neurologic 4.17, 12.5 and 25 mg/kg/day, Benomyl (BEN) was tumor incidence and latent period for 40 progeny, given p.o. on PD 1 at 0, 37.5, 75, 150 and 300

72 µg/pup and mercuric chloride (MER) was given 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), s.c. on PD 1 at O, 2 and 4 mg/kg. CHL produced a widespread envirorunental contaminant, is a morphological defects of the urogenital system known teratogen and potent inducer of mixed­ and impaired growth, morphological and bio­ function oxidase activity in rodents. This chemical differentiation, and the diuretic study was designed to examine the effects of response at 6 mg/kg. NIT impaired growth at TCDDon development in the ferret. Animals 25 mg/kg and altered renal differentiation as received 1,6,13.5,20,30 or 60 µg TCDD/kgbody low as 12.5 mg/kg and physiological responses weight subcutaneously as a single dose on day 18 at the lowest dose tested. BEN retarded growth or as½ the dose on days 18 and 20 of gestation. above 75 µg/pup and affected renal differentia­ Laparotomies were perfonned on days 28,29 and 30 tion at that dose. MERfailed to alter any of gestation due to toxicity observed at the two parameter, indicating the very young animal highest doses. The average number of implant differs markedly from the adult in response to sites, live, dead, and resorbed fetuses, as well that compound. The relative sensitivity of the as fetal weight, crown-rump length, and specific various tests was rated by rank ordering the malformations was recorded. In addition, S-9 F-values obtained from ANOVAacross the four homogenates were obtained from maternal and fetal compounds. Overall, organ weight was the most liver and placenta, and levels of aryl-hydro­ sensitive indicator followed by the water carbon hydroxylase (.AHH)activity and cytochrome diuresis tests, body weight, biochemical and P-450 were determined. morphological parameters of differentiation, Increases in the number of dead and resorbed and hydropenic parameters. fetuses, as well as retarded growth of viable fetuses was fol.ll1din all test groups. In addi­ tion, several malformations, including unilateral and bilateral palotoschisis, open eyelids, 258 TERATOLOGYSTUDIES ON BENZOTHIAZOLESULFENAMIDES anasarca, and brachygnatha, were observed in M.W. Stevens, Dept. of Med. & Environ. Health, fetuses which were exposed to TCDD,whereas none Monsanto Co., St. Louis, MO 63166 (G.J. were present in control animals. Maternal and Levinskas) fetal .AHHactivity and cytochrome P-450 levels Amine substituted mercaptobenzothiazoles (sul­ showed significant individual variation in both fenamides) are used in the rubber industry as ac­ the liver and placenta. The data obtained in­ celerators for vulcanization processes. Three dicate that ferrets may be "non-responsive" to commercial sulfenamides, N-cyclohexyl- (I), N-t­ heaptic MFOinduction by TCDD. butyl- (II), N-morpholino- (III), were tested for In conclusion, TCDDwas teratogenic and teratogenic activity in Charles River CD® rats. embryotoxic to ferrets at all doses tested with Groups of 25 pregnant female rats were treated no demonstrated no-effect level. orally on days 6 through 15 of gestation with corn oil suspensions of test material at concen­ trations of: I - 100, 300, 500 mg/kg/day, II - SO, 150, 500 mg/kg/day, 260 DISTRIBUTIONOF METHANOLAND METABOLITES IN III - 100, 300, 1000 mg/kg/day. MATERNALAND FETAL TISSUES. R. Infurna and G.G. Surviving animals were sacrificed on day 20 and Berg, Div. of Toxicology, Dept. of Radiation-­ uterine horns were examined for number of implan­ Biol. and Biophysics, Univ. of Rochester School tations, viable or nonviable fetuses, or resorp­ of Medicine & Dentistry, Rochester, NY14642 tions. One-half of the total fetuses were exam­ Prima gravida rats in the 17th day of gestation ined for internal anomalies after preservation were exposed to 2%methanol in drinking water, in Bouins solution; one-half were examined for and sacrificed on the following day after consum­ skeletal anomalies after clearing and staining. ing approx. 2g of methanol per kg. C-14 methanol Mean fetal body weight reduction at the high was administered either overnight in the same wa­ dosage of I was associated with a significant de­ ter, or by gavage with equivalent activity (50 crease in maternal weight gains. Mean fetal uCi) 1 hr before sacrifice. Animals anesthetized weights were comparable to controls at all with pentobarbital were made hypothermic in an levels for II and III. No significant differ­ ice pack and dissected on ice; tissue samples ences in mean numbers of implantations, resorp­ were preserved in ice-cold fixative: 57%meth­ tions, or viable fetuses were observed after anol, 6%formic acid, 3.7% formaldehyde (w/v), treatment. No significant difference between to preserve volatile trace compounds. The control groups and groups treated with I, II or supernatant extract of homogenizedsamples was III were noted in external malformations, soft assayed for total activity and for partition of tissue malformations, or skeletal anomalies. label between methanol and formate. One hour All incidences for observed anomalies were after intake of label, concentrations of extract­ within ranges expected from historical controls. able methanol and metabolites averaged 45 umoles/g No teratogenic response was observed in rats wet wt. in maternal tissues; fetal bodies and after treatment with these 3 substituted livers averaged 50 umoles/g, fetal heads and mercaptobenzothiazoles during the period of brains 55 umoles/g. After an overnight exposure, organogenesis. maternal tissues averaged 20 umoles/g, fetal tis­ sues 25 umoles/g, and fetal brains 30 umoles/g. Formic acid constituted a large fraction (aver-· aging 20%) of label extracted from maternal liv­ 259 TI-IEEFFECTS OF 2, 3, 7, 8-TETRAQ-ILORODIBENZO-p­ ers. In fetal livers it averaged 3% at 1 hr and DIOXIN ON FETAL DEVELOPMENTIN TI-IEFERRET. D. more than doubled overnight. In maternal kidneys Muscarella, D. Dennett, J.G. Babish and D. Noden. it averaged 10%in 1 hr, dropping to 1%over­ Depts. of Preventive Medicine and Anatomy, NYS night. The results confirmed a rapid equilibra­ College of Veterinary Medicine, Cornell Univer­ tion of methanol across placental and blood-brain sity, Ithaca, NY. barriers and showed a more active production of

73 fonnate in adult livers than in other tissues. nonviable fetuses, early and late resorptions and Techniques reported here yield a rapid and con­ corpora lutea were recorded. The fetuses were venient quantitation of tissue doses of methanol removed, individually weighed, sexed and examined for and its extractable and tissue-bound metabo­ external, visceral and skeletal malformations and varia­ lites. Supported in part by Contract No. tions. DE-AC02-76EV03490with the U.S. Dept. of Energy. A statistically significant decrease in maternal body weight gain was observed in the Triamcinolone Acetonide treated group when compared to the control group. One of 12 treated rats aborted on gestation day 261 TRANSPLACENTALTOXICITY ASSOCIATEDWITH GENETIC 15 and another died of maternal toxicity on gestation DIFFERENCESAT THE MURIJ:IEAH LOCUS. R.G. York day 18. Skin irritation was minimal. Significant and J.M. Manson, Kettering Lab., Univ. of increases in post-implantation losses and a Cincinnati, Cincinnati, OH Sponsor: P.B. Hammond corresponding decrease in mean number of viable The Ah locus controls the synthesis and inducibi­ fetuses were observed in the treated group as well as lity of aryl hydrocarbon hydroxylase (AHH), one decreased fetal weight. The number of litters with of the xenobiotic metabolizing enzymes of the P- malformed fetuses was significantly increased following 448 monooxygenase system. Based on the induci­ treatment. Cleft palate and omphalocele were the bility of the Ah locus i.nbred strains of mice most frequently observed anomalies. Triamcinolone have been shown to be highly sensitive or resis­ Acetonide Cream is a suitable positive teratogen when tant to toxicity when exposed as adults to poly­ administered topically to rats. cyclic aromatic hydrocarbons (PAH). This study is an attempt to determine whether toxicity (car­ cinogenicity) caused by transplacental PAH expo­ sure can be correlated with inducibility of the 263TERATOGENICITYOF OCHRATOXIN A IN RATS. K. Ah locus. C57BL/6J (Ah inducible) and DBA/2J Mayura, A. W. Hayes and W. 0. Berndt, Dept. Phar­ (Ah noninducible) mice were appropriately crossed macol. &Toxicol., Univ. of Mississippi Med. Ctr., so that fetuses with both phenotypes were found Jackson, MS39216 in the same litter. The~e fetuses were exposed Teratogenic potential of ochratoxin A (a po­ in utero on days 15,16 and 17 of gestation to 3- tent nephrotoxin and a teratogen, produced by methylcholanthrene (3-MC) given orally to the dam various species of Aseergillus and Penicillium) at doses of 7,21, or 63 mg/kg/day. Analysis of in rats has been studied by determining dose re­ variance on 90 dams and their litters showed the sponse and time course studies. Crystalline high dose group significantly (p<.03) different ochratoxin A was injected single dose subcutane­ from controls for percent pups surviving day 4, ously to the rats on one of gestation days 4, 5, mean pup ~eight on days 7,14 and 21, and number 6, 8, 9 and 10. The dose levels tested were 5, of pups weaned per litter. This weight depres­ 2.5, 1.75, 1.0 and 0.5 mg/kg. There was complete sion in the high group was still significant fetal resorption in rats given 5 mg/kg, whereas (p<.05) at 90 days of age at which time the off­ in rats given 1.0 and 0.5 mg/kg, much less fetal spring were phenotyped by zoxazolamine paralysis resorption occurred and the fetuses did not show time. The high dose group also contained stat­ any abnormalities with regard to gross, skeletal istically fewer inducible than noninducible pups. and internal soft tissue. There was an tncreased This may be due to the higher levels of toxic 3- incidence of fetal resorption in rats given 2.5 MC metabolites in the inducible fetuses, result­ mg/kg. Amongthe dose levels tried, 1.75 mg/kg ing in more resorptions, stillbirths and neonatal ochratoxin A seems to be the minimumteratogenic deaths. At twenty weeks of age carcinogenicity dose in rats which resulted in decreased fetal and AHH activity in the lung and liver will be weight and various fetal malformations like om­ measured to determine if differences at the Ah phalocele, ectopia cordis, anophthalmia and in­ locus did result in differences in transplacental ternal hydrocephalus. Skeletal defects also carcinogenesis. (Supported by NIEHS Grant ES- were noted involving ribs, vertebrae, sternebrae 07073 and the Ohio Coal Lab Research Association). and skull. Largest number of resorptions, great­ est depression of fetal weights, and largest number of malformations occurred when ochratoxin A was injected on either day 5 or day 6. 262TRIAMCINOLONE ACETONIDE (ARISTOCOR-re) (Supported by USPHSresearch grant ES 02191.) ADMINISTEREDTOPICALLY TO RATS AS A POSITIVE TERATOGEN. D. E. Rodwell, K. M. Werchowski and G. B. Briggs, WIL Research Laboratories, Inc.,- Cincinnati, OH 45241 Sponsor: G.N. Mir 264 THE EFFECT OF URANIUMON THE FEMALEREPRODUCTIVE This study was conducted to determine the suit­ SYSTEMOF THE RAT. H.K. Gardner, F.A. Smith, ability of 0.1 % Triamcinolone Acetonide Cream R.B. Baggs, Departments of Radiation Biology and (Aristocort4D) as a positive teratogen, when applied Biophysics, and Laboratory Animal Medicine, Uni­ topically to shaved backs of pregnant Charles River versity of Rochester, Rochester, NY. COBS"' CD"'rats. Each rat was administered 0.03 ml of Triamcinolone Acetonide Cream or control material, Although uranium is an extensively studied toxin, Hydrophilic Plastibase, from gestation days 6 through it has been studied predominantly in males. The 15. Each rat was fitted with an Agar (acetate) collar increasing environmental appearance of uranium during the treatment period to prevent oral ingestion of hexafluoride and its hydrolysis products, uranyl the test or control materials. Prior to mating rats were fluoride and hydrofluoric acid, has necessitated conditioned with the collars. Each female was weighed a study in the female. A pilot project was periodically throughout gestation and examined daily therefore undertaken. Adult virgin Sprague-Daw­ for signs of systemic toxicity and skin irritation. Dams ley rats were intratracheally instilled with ei­ were sacrificed on gestation day 20 by carbon dioxide ther 2.5 mg uranyl fluoride in tris buffer per inhalation and the location and number of viable and kg body weight or with equal volumes of buffer,

74 and sacrificed on day 1, day 4 or days 5-7 post Omadine MDS, a broad spectrum anti-microbial pro­ instillation. Evidence for histopathological posed for use as an anti-seborrheic agent in sham­ damage was sought in sections of ovary, uterus, poos and a preservative in cosmetics, was evaluated vagina, lung, kidney and trachea. At least two in a series of studies to assess fertility and general of the three rats in each dosed group exhibited reproduction, perinatal and postnatal function and asynchrony between estrous cycle phases of the teratogenic potential. ovary and uterus. The ovaries of all dosed rats In one set of experiments, Omadine MDS was given appeared to be functional and at or near estrous orally to COBS-CD Rats by gastric intubation at however, some uteri were in diestrus. In addi­ dosage levels of 1, 3 and 7 .5 mg/kg to males for 60 tion, some uteri had features characteristic of days prior to mating (with untreated females), to several divergent phases. Vaginitis was also females for lt+ days prior to mating (with untreated noted in some treated rats. The inability of males) through lactation day 21, or to females from the uteri of effected rats to synchronize their gestation day 15 through lactation day 20. Controls activities with those of the ovaries casts doubt received distilled water on comparable regimen. No on the ability of these uteri to accept ova. adverse effects were noted at l mg/kg. Dosage Further, the vaginitis may reduce the recepti­ levels of 7.5 mg/kg were toxic in these experiments. vity of the effected females to males. Direct There were clinical signs of intolerance, reduced effects on the ovary may cause a change in hor­ body weight gain and high mortality of parental mone production whereas direct effects on the animals. Further, marked motor impairment was uterus or vagina may alter their ability to re­ observed in the animals, manifested by ataxia and spond to hormones, or may cause irritation. hind limb paralysis. Offspring parameters of Whether the effects are a direct res~lt of the treated animals in the fertility study were com­ agent on the reproductive tract, or are an in­ parable to controls at all dosage levels, however, in direct result of the generally debilitating ef­ the perinatal study, neonatal body weights and fects of this toxin, it is likely that the re­ survival during lactation were affected in the 7.5 productive capacity of these rats was impaired. mg/kg group. In experiments assessing teratogenic potential, Omadine MDS incorporated in a shampoo base vehicle, was applied dermally to shaved intra­ 265 ABSCRPTION, DISTRIBUTION, EXCRETION AND COVALENT scapular skin for two hours/day on days 6-18 of BINDINGOF 14C-TRIFLUOROETHANOL.R.M. gestation to New Zealand white rabbits at dosage Wilkenfeld, F.A. Smith. Dept. Rad. Biol. and levels of 0.t+5, 1.5 and 5 mg/kg or on days 6-15 of Biophysics. Univ. of Rochester, Rochester, NY. gestation to COBS-CD Rats at dosage levels of 1, 10 Previous studies in our laboratory have shown and 30 mg/kg. Omadine MDS was not teratogenic in that the volatile solvent 2,2,2-trifluoroethanol the rat. Preliminary examination of the rabbit data rapidly produces testicular damagefollowing i.p. indicated no teratogenic capabilities. administration or inhalation. The f9te of car­ bon-14 labeled trifluoroethanol (2-1 C-TFE) in the male rat was investigated over the inter­ 267 A REPRODUCTIVEASSESSMENT OF MALEAND FEMALE HAM­ val of 15 minutes to 48 hours after intraperi­ STERS ANDRATS EXPOSEDTO TOXIC CHEMICALSDURING toneal injection of 100 or 5 mg/kg. Findings CRITICAL DEVELOPMENTALPERIODS. Gray, L.E.,Jr., were similar for· each dose. The disappearance of J.W. Laskey, J. Ostby, J. Ferrell, J. McLamb. 14c activity from the plasma was biphasic, with Health Effects Reserach Laboratory, USEPA, RTP, half times of 2 hours and 48 hours. Testicular NC 27711· (Sponsor Robert W. Chadwick). levels of radioactivity 15 minutes to 48 hours Presently chemicals are tested for their po­ postinjection were found to be comparable to tential to damage the reproductive system by ob­ those in the liver, kidney, and lung. Thus, serving the chemical's effects on fertility, lit­ there is no evidence of testicular concentration ter size and survival over 2 or 3 generations. of TFE, and the fact that this organ can be Alternatives to these protocols are needed be­ severely damagedby the compoundreflects a cause these studies are time consuming, expensive, genuine sensitivity of the testis to trifluoro­ and insensitive. The majority of the protocols ethanol. The pattern of 14c excret1·on was: suggested as replacements are focused on adult urine » expired air » feces. No 4co2 males with little attention given towards females was detected in expired air. or developmental susceptibility. The present One hour following the administration of study was an attempt to develop a more comprehen­ 14c-TFEto control rats, 3%of the radioactivity sive protocol. In this study male (M) and female in the testis (on a mg protein basis) was bound (F) hamsters (H) and rats (R) were exposed to to proteins, while 5%of the radioactivity in the known reproductive toxicants (including DBP, liver was associated with proteins. Pretreatment Kepone, MSG, Cd) during sexual differentiation of of animals with phenobarbital did not signifi­ the brain, hypothalamic maturation, puberty and cantly increase the levels of apparent protein as young adults. The age at puberty was deter­ binding in either organ. The levels of protein mined by vaginal opening in FR, behavioral estrus binding observed in these experiments are so low in FH, preputial separation in MR and flank gland as to be of questionable significance as a growth in MH. Estrus cyclicity was observed in possible mechanismof trifluoroethanol's toxicity. FR and FH. Sexual behavior was observed in FH and MH. The animals were bred, dosed through gestation, and fertility, litter sizes, offspring 266 REPRODUCTIVE TOXf COLOGY OF O¥ADINE growth and survival were measured. Following MDS, D. E. Johnson , J L. Schardein , breeding MR and MHwere necropsied and reproduc­ 1 1 W:­ Goldenthal , F. X. Wazeter and J. H. Wedigl' tive tissues were measured (including the testes, International Research and Develo~ment Corp. , epididymis, seminal vesicle, flank gland) sperm Mattawan, MI 1+9071 and Olin Corp. , New Haven, concentration in the vas deferens was measured, CT 06511. testicular histology was evaluated and hCG stim-

75 ulated serum testosterone was measured. Results Reproductive assessment was not conducted in demonstrate that exposure to known reproductive the groups receiving 74 and 152 µM CD/kg due to toxicant9 using this protocol can be easily iden­ the mortality of 10 and 60 percent, respectively. tified and the effects of exposure during sensi­ Testes, seminal vesicle and epididymes weights tive developmental periods are often more severe were reduced at least 40 to 50 percent at doses and persistent than adult exposure. of either 16 or 33 µM Cd/kg, and sperm concentra­ tion in the vas deferens and hCG stimulated serum [T] were undetectable. 268EVALUATIONOF THE MALEREPRODUCTION SYSTEM IN Doses of 1.6 to 7.4 µM Cd/kg did not produce YOUNGAND ADULT RATS AFTER EXPOSURETO BENOMYL. tissue weight decrements, however, significant Carter, S.D., J.F. Hein, and J.W. Laskey. Health dose related decrements in vas deferens sperm Effects Research Laboratory, USEPA, Research Tri­ concentration (p < 0.0001) and hCG stimulated angle Park, NC 27711 (Sponsored by Robert W. serum [T] (p < 0.003) were observed. In vitro Chadwick). androgen production following in vivo hCG stimu­ lation was unaltered in the twolower Cd doses. Adult Sprague-Dawley male rats (65 days of These results demonstrate that acute Cd ex­ age) received 10 daily treatments of 0, 200 or posure at doses reported to have no observable 400 mg Benomyl/kg/d by gavage. Body weight, tis­ effect result in a decrease of both spermatogenic sue weights, total epididymal sperm count and potential and androgen secretory potential, and sperm concentration from the vas deferens were demonstrate the utility of a multivariate ap­ measured 14 days after the last treatment. Tes­ proach to reproductive assessment. ticular histology was evaluated in O and 400 mg/ kg/d groups. A second study used prepuberal Sprague-Dawley male rats (33 days of age) which 270 THEEFFECT OF ORDRAM®ONTHEFERTILITY OF MALE received 10 treatments of O or 200 mg/kg/d by MICEAND RABBITS. J.M. Killinger, P.O. Royal and gavage. At 3, 17, 31, 45 and 59 days after the G.M. Zwicker, Env. Hlth. Ctr., Stauffer Chem. last treatment, 8 animals per treatment group Co., Farmington, CT Sponsor: R.I. Freudenthal were killed and measurements similar to those in the first study were made. Ordram, a rice herbicide, was tested for effects Significant findings included 40-50% de­ on the fertility of male mice and rabbits. In pressions in the total epididymal sperm counts study I, 100 male CD-1mice of proven fertility and in the vas deferen sperm concentrations in were randomized into 5 dose groups; vehicle con­ adult animals treated with 200 or 400 mg/kg/d. trol, 2, 20, 100, and 200 mg/kg/day. The males Histological evaluations of testicular sections were dosed daily by oral gavage for 7 weeks. from 6 adult animals in the 400 mg/kg/d group Fertility was determined by mating each treated indicated a slight to moderately severe hypo­ and control male with 2 untreated females during spermatocytogenesis in 2 animals and a slight to treatment and again after a 4-week recovery severe generalized hypospermatogenesis in 2 ani­ period. An interim sacrifice was performed on 5 mals. No treatment effects were found in body males/group at the completion of dosing and a weight, liver, kidneys, testes, seminal vesicles, final sacrifice was performed on the remaining or epididymides weights in the adult animal. Re­ males after the end of the recovery period. sults from the study using the younger animal in­ Treatment-related antifertility effects were dicated no significant treatment effects in body observed in the 100 and 200 mg/kg dose groups weight, tissue weights, total epididymal sperm during treatment. There were reductions in the count, vas deferen sperm concentration or his­ number of pregnancies but no increase in resorp­ tology at any of the observation times. tions. The study clearly shows a no-effect lev­ This data suggests that younger animals are el of 20 mg/kg for the observed anti fertility less sensitive to 200 mg Benomyl/kg/d than adults effects in male mice. No antifertility effects receiving the same dose. were observed in the fertility test after the 4-week recovery period, demonstrating complete reversibility. There were no histological or macroscopic changes indicative of a compound­ 269 EFFECTS OF LOWACUTE DOSES OF CADMIUMCHLORIDE ON related effect on the testes, epididymides, thy­ THE REPRODUCTIVESYSTEM OF MALERATS. Laskey, roids, or pituitaries in any treatment group. J.W., G.L. Rehnberg, S.D. Carter, J. Hein. In study II, 37 male Dutch-Belted rabbits of Experimental Biology Division, Health Effects Re­ proven fertility were assigned to 4 dose groups. search Laboratory, U.S. Environmental Protection There were 10 males in the vehicle control group Agency, Research Triangle Park, NC 27711 and 9 males each in the 2, 20, and 200 mg/kg (Sponsored by Robert W. Chadwick) dose groups. A similar study design was em­ Eighty 70 day old male rats were injected ployed as described above for the mice. No evi­ (sc) with either o, 1.6, 3.1, 7.4, 16, 33, 74, dence of impaired fertility was found. Also, or 152 µM Cd (as CdCl2)/kg. Typically studies of there were no histological or macroscopic . this nature use Cd doses of 10 to 50 µM/kg or changes found in the testes, epididymides, pitu­ greater and it has been reported that testicular itaries, thyroids, or adrenals which could be toxicity does not occur at 6 µMor below. Four­ related to the administration of Ordram. teen days post dosing all animals were killed and testes, seminal vesicles and epididymes removed and weighed. Sperm concentration was determined 271 REPRODUCTIVETOXICITY OF METHYL-(l-BUTYLCAR­ in semen samples from the vas deferens. Serum BAMOYL)-2-BENZIMIDAZOLECARBAMATE (BENOMYL) IN [T] was measured following maximal in V'ivo hCG MALERATS. T.B. Barnes, K.M. Hayward, A.J. stimulation. In selected groups decapsulated Verlangieri, and M.C. Wilson, Dept. of Pharmacol. testes were incubated in vitro and T production Sch. of Pharmacy, Univ. of Mississippi, Univer­ assessed. sity, MS. Sponsor: W.M. Davis The present investigation into the reproductive The method employed allowed for repeated collec­ toxicity of Benomyl was undertaken to determine tion of ejaculated semen samples and for the the biochemical basis and functional significance correlation of sperm count, mating behavior and of the damage previously reported by this lab­ hormone levels within the same an±mal. (Supported oratory (presented at SOT, 1980) to the male by ERC Grant OH-07091-05). reproductive system. The experiment was divided into two phases, a 70 day feeding phase followed by a 70 day recovery phase. Adult male Wistar 273THE ROLE ·OF GUT MICROFLORAIN NITROBENZENE­ rats were fed chow mixed with 0.02, 2.0, or 200 INDUCEDTESTICULAR NECROSIS. Levin, A.A., Popp, ppm Benomyl. Control animals received powdered J.A., and Dent, J.G. Chemical Industry Insti­ chow for the 70 day treatment period. Following tute of Toxicology, Res. Triangle Park, NC the feeding study, one-half of the animals per 27709. group entered the recovery phase. In order to determine the effects of this fungicide on the Nitrobenzene (NB) produces a number of toxic testes, mutagenic, behavioral, and reproductive effects in rats including methemoglobinemia tests were performed during both the feeding and (MetHb). Intestinal microflora metabolize nitro­ recovery phases. At the termination of both benzene to nitrosobenzene and phenylhydroxyl­ phases sperm counts were evaluated and a amine, both potent inducers of MetHb. Induction complete necropsy was performed on all animals. of MetHb is dependent on the presence of gut At the termination of the feeding phase sperm flora, since NB fails to induce MetHb in axenic counts were depressed in the highest dosage group or antibiotic treated rats (Reddy et al., Bio­ (200 ppm). A bilateral decrease in the relative chem. Pharm. 25). The current study investigated testicular weights of all dosage groups was also the role of gut microflora in the etiology of observed. This damage to the testes was not NB-induced testicular necrosis. Fischer-344 permanent however, since following the recovery rats were treated p.o. with neomycin sulfate phase no differences in the sperm counts or in (100 mg), bacitracin (50 mg) and tetracyline (50 relative testicular weights were observed mg) in 1% carboxymethylcellulose or vehicle between any of the dosage groups. The functional alone twice daily for 2 days prior to gastric and biochemical significance of this damage is intubation with 300 mg/kg NB in corn oil. The under further investigation. antibiotic regime was continued twice daily for 3 days and once daily for 2 additional days. The Supported in part by the Research Institute of rats were killed 5 days after exposure to NB. Pharmaceutical Sciences. The dose of nitrobenzene used was the lowest single dose which was previously shown to induce testicular necrosis. Antibiotic treatment pre­ 272 ASSESSMENTOF MALEREPRODUCTIVE TOXICITY DUE TO vented NB-induced MetHb. At sacrifice NB-antib­ CARBONDISULFIDE: USE OF A NEWTECHNIQUE. S.J. iotic treated rats had 2+2% vs 2o+8% methemoglo­ Tepe and H. Zenick, Dept. of Envir. Hlth. Col. bin in NB treated rats. -In vitro-analysis of of Med., University of Cincinnati, Cincinnati, the metabolic activity ofthe cecal contents OH. Sponsor: P.B. Hammond showed that antibiotic treatment completely hibited the metabolism of NB while the vehicle A new method for evaluating male reproductive treated rats metabolized NB at greater than 1 function in the rat was developed and applied to µmole/min/g cecal contents. Despite the inhibi­ assessing the reproductive toxicity of carbon di­ tion of cecal metabolism, the seminiferous sulfide (CS2). Experienced, mature, male, Long tubules of the testis from antibiotic treated Evans Hooded rats were mated weekly and observed had a similar incidence and severity of necrosis for a pre-exposure baseline and after 1,4,7 and as vehicle treated controls. Therefore the 10 weeks of exposure to 600 ppm CS2 or filtered induction of testicular necrosis by nitrobenzene air. At those times the male rat was placed in appears to be independent of gut microflora a plexiglass cage with an ovariectomized, hor-· metabolism. mane-primed estrus female. The mating behavior was observed and scored for mount latency (ML), numbers of mounts (M) and intromissions (I), and 274 EFFECTS OF 2,5-HEXANEDIONEON REPRODUCTIVEHOR­ for ejaculation latency (EL). The female was MONESAND TESTICULARENZYME ACTIVITIES IN THE sacrificed within 30 min. of ejaculation, her F-344 RAT. R. E. Chapin, R. M. Norton, J. A. reproductive tract excised, opened and rinsed of Popp, and J. S. Bus. Chem. Ind. Inst. Toxicol., ejaculate, and an ejaculated sperm count deter­ Res. Triangle Park, NC mined. Ninety hours after observed matings, male Chronic administration of 2,.5-hexanedione rats had 2 ml of blood drawn via cardiac puncture (2,5-HD) to animals causes azoospermia and histo­ for radioimmunoassay of plasma LH, FSH and tes­ pathology in some central nervous system (CNS) tosterone. By week 4, animals exposed to cs2 had areas. These studies were designed to assess the decreased ML and EL, and by week 7 also had degree of CNS involvement in the testicular decreased sperm counts when compared to control lesions seen in the rat after 6 wks of 2,5-HD animals experiencing the same mating regimen but consumption. Additionally, activity measurements exposed to filtered air (p < 0.05, ANOVA). Num­ were made of some enzymes found in specific tes­ bers of mounts and intro~issions remained un­ ticular cell types. 2,5-HD was fed to adult F- changed, as did plasma LH and FSH concentrations. 344 rats as a 1% solution in the drinking water. Plasma testosterone was reduced in exposed ani­ After 1, 3, and 6 wks of treatment, 6 treated mals during weeks 1,4 and 7 but not during week rats, their pair-fed controls, and 6 ad lib con­ 10 (p < 0.05, ANOVA). These data suggest a trols were sacrificed. Serum levels of luteiniz­ direct-testicular effect of CS2, causing de­ ing hormone (LH), follicle stimulating hormone creased sperm count and testosterone concentra­ (FSH) and testosterone were not depressed at any tions with the correlated changes in ML and EL. point examined, indicating that 2,5-HD does not

77 cause testicular atrophy by decreasing release of Sertoli cell-germ cell interrelationships, with LH and FSH. At 6 wks, the testes were azoosper­ shedding of spermatids and spermatocytes from the mic. This coincided with a rise in LH (mean± SE germinal epithelium. We have therefore examined difference, treated value minus pair fed value= the effects of some phthalates on Sertoli cell 47 + 13 ng/ml) and FSH (mean+ SE difference, 280 function. This was assessed in vivo by measuring + 30 ng/ml), while testostero;e levels were secretion of seminiferous tubular fluid and ;-nchanged. Hormonal values from pair fed rats androgen binding protein (ABP) after unilateral were not different from ad lib group values. ligation of the testicular efferent ducts. For After 3 wks, the testes ~re histologically in vitro studies, mixed cultures of Sertoli and normal. However, the specific activity of the germ cells were prepared by trypsin and Sertoli cell enzymes S-glucuronidase and y­ collagenase digestion of testes from 4 wk old glutamyl transpeptidase was decreased by 68 ± 3% rats. Fluid secretion and ABP production were and 20 + 7%, respectively (mean+ SE, n = 6). markedly inhibited within 3 hr after a single Liver, ;hich is not affected histologically by dose of dipentyl- (2200 mg/kg) or mono-(2-ethyl­ 2,5-HD, showed slightly decreased lysosomal hexyl)phthalate (MEHP) (1000 mg/kg) but were un­ enzyme activities (S-glucuronidase and acid affected even after 3 doses of diethylphthalate, phosphatase) at all times. The significant an ester not causing testicular injury. Addition changes in Sertoli cell localized enzymes before of MEHP to Sertoli-germ cell cultures accelerated the onset of azoospermia, and the lack of effect detachment of germ cells from the Sertoli cell on LH and FSH, suggest a direct toxic effect on monolayer at concentrations as low as 10-6M. the testis. Studies with other phthalate monoesters showed a correlation between production of germ cell 275 MORPHOLOGICALMATURATION OF RAT TESTIS detachment in vitro and testicular toxicity in R.H. Matter and H. W. Tvedten, Dept. of Path., vivo. These results suggest that Sertoli ceITs Mich. State Univ., E. Lansing, MI 48824 may be a primary target of phthalates causing Sponsor: J.B. Hook testicular injury and that testicular cell cultures may be useful for investigating The immature testis has only recently been recog­ mechanisms of action and for screening compounds nized as a potential selective target of a likely to cause testicular injury by similar variety of chemical toxicants. Since this organ mechanisms to phthalate esters. changes markedly during maturation, the response (Supported by the U.K. Ministry of Agriculture, to toxicants may vary depending on age at time Fisheries and Food). of exposure. It is therefore important to de­ fine specifically the morphology at differing stages of cellular development in testis. The 277 BEHAVIOR OF OFFSPRING OF' DAMS GIV.c.N object of this investigation was to identify ETHANOLDURING GESTATION. C, Kruger­ critical stages of development at which effects McDermott*, and I, Rosenblum, I,E.P.T., of toxicants might be most profound. Testes Albany Medical College, Albany, N,Y, from male Sprague-Dawley rats at 1, 3, 6, 9, 12, This study has been conducted to de­ 15, 18, 24, 30, 36 and 45 days of age were fixed termine whether ethanol, given to preg­ in Bouin's, embedded in glycol methacrylate and nant dams at a critical time during sectioned at 2 µ for morphologic analysis. organogenesis of the central nervous Based on the data obtained and structural and system, will result in behavioral abber­ functional events observed by others, 4 critical ations in the offspring. Ethanol (20 ages were identified: day 6, both Sertoli cell mg/kg of 20% ethanol) p.o. was given to precursors and gonocytes, primordial germ cells, dams on day 8 of gestation; control rats were actively dividing and Leydig cells were of received normal saline. Rate of gain in fetal origin; day 15, Sertoli cells were forming body weight of dams during gestation and tight junctional complex}s, a critical part of reproductive performance was observed. the blood-testis barrier, spermatogonia were Offspring were examined postnatally for dividing mitotically to produce primary spermato­ gross evidence of teratology. They were cytes and Leydig cell numbers were at their also examined at the time they were nadir; day 24, spermatids had formed, thus killed, Growth rates were recorded. reflecting both mitosis and meiosis and adult Behavior was measured by a shuttle box Leydig cells were beginning to differentiate; in a passive avoidance situation. day 45, the time of puberty, Sertoli cells had The first behavioral test was done on attained all adult characteristics, mature the 25th post natal day. Analyses of spermatozoa had developed and there was a full data indicate that offspring of dams re­ complement of adult Leydig cells. Thus, exposure ceiving ethanol required more trials to of animals during these 4 time periods should learn the task than did controls. By allow detection of any differential suscepti­ the second test (day J2), the differ­ bility of the immature rat testis to chemical ence was no longer evident. Offspring toxicants. of ethanol treated dams showed noter­ atology but the rate of weight gain was 276EFFECTS OF PHTHALATEESTERS ON RAT TESTICULAR slower when compared to controls. CELL CULTURESAND ON SERTOLI CELL FUNCTIONIN THE It is concluded that ingestion of INTACT TESTIS. Gray, T.J.B., Beamand, J.A. and ethanol by pregnant dams results in a Gangolli, S.D., The British Industrial Biological decrement in behavior of the offspring Research Association, Woodmansterne Road, which is negated with time. The decre­ Carshalton, Surrey SM5 4DS. U.K. ment may be due to l~delayed learning capacity 2)motor deficiency. Testicular injury produced by phthalate esters is characterised by an early disruption of normal *Shell Fellow

78 278 THEOBROMINEMETABOLISM AND PHARMACOKINETICSIN this tolerance are reduced absorption and a shift PREGNANTAND NON-PREGNANTFEMALE RATS. S,M. Tarka in the tissue distribution of Cd. Male Sprague­ Jr. and C.A. Shively, Hershey Foods Corporation, Dawley rats received a single sc pretreatment Hershey, PA. Sponsor: J.F. Borzelleca (2.0 mg Cd/kg) either 1, 2, 4, 8, or 16 days prior to iv administration of the challenge dose The metabolism and plasma kinetics of orally ad­ (3.9 mg Cd/kg). Mortality was 100% in control ministered theobromine (TB) were studied in preg­ rats. Tolerance was evident following injection nant (P) and non-pregnant (NP) female Sprague­ of the lethal dose in Cd-pretreated rats since no Dawley rats at doses of 5, 10, 50, 100 and 200 mg 14 mortality was observed in any of the pretreated /kg using TB sodium acetate and [8- C] TB as a rats. Since Cd pretreatment induces sy nth es is radioactive tracer. No statistically significant of hepatic metal lothionein, a low molecular differences were observed in mean TB plasma half­ weight Cd-binding protein, its induction might life, apparent volume of distribution and clear­ enable a greater percentage of the challenge dose ance in either P- or NP-rats after TB doses of 5- to be sequestered 1n liver of pretreated animals 200 mg/kg. Plasma kinetic parameters of P-rats and less distributed to target organs of toxicity were also similar to NP-rats. Plasma radioacti­ (kidney, testes). To determine if a challenge vity was >99% TB and <1%6-amino-5[N-methylformyl­ dose of Cd distributes differently after Cd amino]-1-methyluracil as shown by HPLC analysis. pretreatment, rats were dosed as follows: Group Analysis of urinary metabolites by HPLC and a 1 (control) received a saline pretreatment and an radioactivity monitoring system (after oral TB 14 iv challenge dose ( 2.0 mg Cd/kg), Group 2 re­ doses of 5 mg/kg TB with 10 µCi [8- C] TB) re­ ceived a sc pretreatment (2.0 mg Cd/kg) and the vealed more extensive TB metabolism in P- than in challenge dose, and Group 3 was pretreated with NP-rats with 38% and 55% administered TB excreted Cd and given a saline challenge, The distribu­ as unchanged TB, respectively. Urinary meta­ tion of the challenqe dose of Cd in the Cd-pre­ bolites identified were 6-amino-5[N-methylformyl­ treated group was estimated as the difference in amino]-1-methyluracil (18-24%), 3-methylxantnine tissue concentrations between Group 2 and Group (5-9%), 7-methylxanthine (3%), 3,7-dimethyluric 3. At 2 and 24 hrs fol lowing Cd challenge, the acid (2-5%) and 7-methyluric acid (1%). The content of radioactive Cd was determined. No percent of the TB dose recovered in urine (0-48 marked shift in the distribution of Cd due to Cd hours) was 76.7% for NP-rats and 76.2% for P-rats. pretreatment was observed in control and pre­ treated rats. We conclude that differences in absorption or tissue distribution of Cd are 279 EFFECTS OF BERYLLIUMIN THE PRESENCEAND AB­ unlikely explanations for development of toler­ SENCEOF MAGNESIUMIN RATS. K. Chetty, M. Alam, ance to Cd. (Supported by USPHS Grants ES-07079 *E. Johnson, S. Henry, E. Mann, and E. Beteet and ES-01142). Division of Natural Science and Mathematics, LeMoyne-Owen College, Memphis, TN 38126. Sponsor: J. Autian 281EFFECTS OF CADMIUMAND ZINC IN VITRO ON TESTICULAR AND HEPATIC MICROSOMALBENZO(A)PYRENE AND STEROID Male Sprague-Dawley rats were fed with METABOLISM. L.T. Wetzel and H.D, Colby, West beryllium as sulfate (0,5,50,500 and 1000 ppm) Virginia Univ. Med. Ctr., Morgantown, WV 26506 in their diets, with and without magnesium. A number of heavy metals have previously been Changes in body weights and mortality rate were shown to alter the rates of metabolism of various determined periodically up to 8 weeks. On the xenobiotic substrates by hepatic microsomal en­ 4th and 8th week, rats were sacrificed to obtain zymes. Studies were carried out to compare the internal organs, each organ weighed and then pre­ effects of cadmium (Cd) and zinc (Zn) in vitro on served for future histological studies. Rats the metabolism of benzo(a)pyrene (BP) and steroids fed on 5 ppm beryllium and above showed growth by rat testicular or hepatic microsomes. Liver inhibition by the 2nd week in the absence of and testis microsomes were p~ incubated ~!th vary­ magnesium. Such inhibitory effects were overcome ing concentrations(l.95 x 10 7 -2.0 x 10 M) of in the presence of Mg++ in the diet. Rats fed on each metal and then assayed for enzyme activities. beryllium showed higher mortality rates in the In liver microsomes, both Cd and Zn produced dose­ absence of magnesium. Rats receiving 1000 ppm dependent decreases in BP hydroxylase activity as beryllium showed a significant reduction in measured by the production of fluorescent metabo­ thymus and spleen weights, both in the presence lites (phenols). HPLC analyses further demonstra­ and the absence of magnesium. Beryllium caused ted decreases in the production of BP phenols and an increase in the weights of liver and kidney. dihydrodiols by hepatic_~icrosomes at metal con­ Histological, biochemical and immunological centrations> 1.25 x 10 M. Cd also produced a studies are in progress. dose-depende;t decrease in hepatic epoxide hydra­ *Visiting Professor, Dept. of Anatomy, UTCHS, tase activity, but Zn had little effect on this Memphis, TN enzyme. I~ testis microsomes, preincubation with (Minority BioMedical Support Program, NIH RR- 1.25 x 10- M Cd enhanced BP hydroxylase activity, 08179). increasing the production of all metabolites. The same concentration of Zn did not af!~ct BP metabo­ lism. At concentrations > 1. 0 x 10 M, both metals 280 STUDIES ON THE MECHANISMOf TOLERANCETD CADMIUM inhibited the production of BP dihydrodiols and TOXICITY. P.L. Goering and C.D. Klaassen, Dept. phenols by testicular microsomes. Testicular of Pharmacology, Univ. of Kansas Medical Center, epoxide hydratase was inhibited by Cd in a dose­ Kansas City, KS. dependent manner but Zn had little or no effect. One of the testicular microsomal enzymes required Animals pretreated with a sub-lethal dose of for steroidogenesis, 17a-hydroxylase, was also af­ cadmium (Cd) subsequently develop tolerance to a fected by Cd. Cd decreased 17a-hydroxylase acti­ lethal dose of Cd. Two possible mechanisms for vity at all concentrations of metal tested, inclu-

79 ding those which enhanced BP metabolism. The re­ chelator intended for use in the gut or blood sults indicate that both Cd and Zn influence micro­ stream; and combinations of these. DF at 25 mM somal BP and steroid metabolism and that the ef­ decreased Fe content in the cells by 23%. For fects vary with the metal, tissue, and enzyme sensitivity in detecting synergism, DF and DHBP studied. (Supported by DOE/Mete DE-AT21-79MC11284 were tested at 5.0 mMand ionophores at~ 10-fold and NIH GM 07039). lower concentrations. Results were(% Fe release relative to control cells, based on 10 6 of each): DF, O; DHBP, O; FC (0.2 mM), 2; FC + DF, 19; 282MUCOSAL UPTAKE OF CADMIUMIN JEJUNUM OF IMMATURE FC + DHBP, 34. CHA (0.5 mM) with or without DHBP RATS. K.K. Gibson and D.R. Johnson (spon: P.B. was the most effective in releasing cellular Fe Hammond), Depts. Env. Health & Physiol., Univ. of but cell viability was reduced to 3-4%, indicating Cincinnati Med. Cen., Cincinnati, OH 45267. significant cytotoxicity. These results suggest Absorption of metals by the small intestine of that properly chosen ionophore-polymer combina­ newborn rats has been observed to be greater than tions may be more effective than DF for reducing absorption by adult intestine. Factors that in­ excess Fe load in target organs. Exneriments to fluence mucosal uptake and whole body retention optimize CHA-chelator mediated Fe release at lower have been identified; however, the mechanism in­ noncytotoxic levels and to examine other ionophore volved in the elevated uptake has not been eluci­ chelate comibinations are in progress. (Supported dated. Experiments have been performed to mea­ by NIH Grant AM 25647-03). sure the removal of Cd from proximal jejunum of newborn rats. Sprague-Dawley rat pups of either sex were examined on days 14, 20, 22, and 28 after 284 ACCUMULATIONOF MANGANESEIN BRAINS OF RATS FOL­ birth. Intestinal cell uptake was measured using LOWINGPRENATAL OR NEONATALEXPOSURE. P. Kontur single pass perfusion of 0.02, 0.05, 0.1 and 0.2 and L.D. Fechter, Dept. Environm. Hlth. Sci., mM 109cdc1 2'through 2-7 cm segments of proximal School of Hyg. and Pub. Hlth., The Johns Hopkins jejunum. The perfusate was collected over 5 min­ Univ. ute intervals, samples drawn, and the radioactiv­ The consequences of chronic Mn exposure in ity compared with the pre-perfusion solution. Re­ young organisms whose brains are undergoing rapid ducing the perfusion flow rate from 0.4 to 0.1 development has received little study. There is ml/min resulted in an increase in Cd uptake in all evidence, however, that neonatal animals maybe age groups and concentrations. Both increasing more susceptible to Mn toxicity because Mn is not concentration and age reduced Cd uptake. At each excreted prior to weaning and because it shows en­ concentration, uptake was highest in 14 day old hanced relative accumulation in the brain of imm­ pups compared to all other age groups. At 0.02 ature organisms. A series of experiments were de­ mM CdC12 , 0.50±0.13%Cd/mg (mean±S.D.) was removed signed to examine the accumulation of Mn in the from the intestinal lumen of 14 day old pups while brains of animals exposed either prenatally or 0.08% Cd/mg was removed by intestine of 28 day old neonatally to MnCl2. animals. At each age, uptake was highest at 0.02 Pregnant long Evans rats when given MnCl2 in compared to 0.2 mM CdC12 . On day 14, 0.50±0.13% their drinking water (5,10 and 20 mg/ml) show de­ Cd/mg tissue was removed during perfusion with pressed maternal body weight gain only at the 10 0.02 mM Cdcl 2 ; while 0.20±0.08%Cd/mg tissue was and 20 mg/ml doses. Birthweight and litter size removed at 0.2 mM CdC12 . Thus, flow rate, Cd con­ were depressed at the 20 mg/ml dosage but not at centration, and age influence Cd uptake from solu­ the 5 and 10 mg/ml doses. Measurement of brain Mn tions perfusing the jejunum of the newborn. This by flameless atomic absorption did not show any information may prove useful in elucidating the significant elevations in experimental subjects. mechanism responsible for the high Cd uptake and Further studies with the radionuclide 54Mn indi­ absorption by immature intestine. (Supported by cate that only small amounts (1-3%) of an acutely NIH grants ES00159 and ES07073, and the Natural administered dose crosses the placenta. Sciences and Engineering Research Council of Direct administration of MnCl2 (0, 25 and 50 Canada.) µg/g/day) to rats by intubation during lactation does not alter body weight gain. The highest dose leads to an increase in Mn levels in several brain 283 EFFICACY/TOXICITY SCREENINGWITH FE-LOADED regions as measured by flameless atomic absorption HEPATOCYTESYSTEMS. C. A. Tyson and S. LeValley, These results suggest that the placenta is an SRI International, Menlo Park, CA. Sponsor: effective barrier for the prevention of Mn accum­ D.C.L. Jones. ulation in the rat fetus and that the fetus is unlikely to be particularly susceptible to mater­ Although various cell systems are used or have nal manganese exposure. Neonatal exposure, results been proposed as prescreens for efficacy/toxicity in accumulation of manganese in the brains of ex­ assessments of new chelating agents, no one is perimental animals and can therefore be used as a using freshly isolated Fe-loaded cells for that model for studying the interaction of Mn with the purpose. Male Sprague-Dawley rats (200-300 g) catecholamines in developing animals. Supported were administered ip 50 mg Fe dextran/injection in part by NIH grants ES00454 and ES07067. on 3 alternate days. Liver cells were isolated 48 hr later by a perfusion method. Loads of 6 7-14 µg Fe/10 total cells (>95% hepatocytes), 285THE INFLUENCE OF CHRONICMANGANESE ADMINISTRATION 15-fold higher than normal, were achieved. The AND EDTA TREATMENTON THE TISSUE LEVELS AND cells were cultured in hormone-supplemented URINARYEXCRETION OF Mn, Zn, Fe, and Cu IN RATS. Waymouth's medium for 22 hr± cholylhydroxamic acid (CHA) and ferrichrome (FC), ionophores for A.M. Scheuhammer and M.G. Cherian, Dept. of Fe; deferioxamine (DF), the test standard; 2,3- Pathology, university of Western Ontario, dihydroxybenzoyl-substituted poly(vinylamine­ London, Ontario, Canada N6A 5Cl vinylsulfonate) (DHBP), a new synthetic Fe A neurological syndrome resembling Parkinson's

80 disease has been observed to develop in humans Tox. & Env. Hlth. Sci. Ctr., Dept. of Rad. Biol. chronically exposed to excessive levels of & Biophy., Univ. of Rochester Sch. of Med., Ro­ manganese (Mn) (Neurology 17: 128-136, 1967). chester, NY. Sponsor: Ronald W. Wood. While some researchers haveconsidered chelation Little work has been directed towards lead therapy to be ineffective in the treatment of Mn exposure beyond weaning. We report a systematic intoxication (Neurology 18: 376-382, 1968) others replication of an earlier study of chronic post­ have reported partial success using EDTA (Arch. weaning lead exposure on schedule-controlled be­ Neurol. 30: 59-64, 1974; Br. J. Ind. Med. 28: havior including biological measures of lead body 78-82, 19'71). In the present study, male rats burden. Exposure of male rats to 500 ppm sodium were exposed ip to 3.5 mg Mn/kg/day as MnC12 for acetate or 50, 100 or 500 ppm lead acetate in 30 days. Treatment was then discontinued and the drinking water began at 21 days of age. The lev­ urinary excretion of Mn, Zn, Fe and Cu was er press response was shaped in a standard oper­ monitored with or without EDTA treatment (CaNa2 ant chamber at 55 days of age. Next, a fixed-in­ EDTA, 50 mg/kg/day for 10 days. Kidney, testes, terval (FI) 60 sec schedule of food reinforcement brain and muscle, but not liver, revealed elevated was imposed. Mn levels due to Mn exposure alone. Of the 3 Despite changes in the strain of rats, diet, brain regions examined, the tegmentum and striatum FI value, and experimental laboratory, results accumulated more Mn than the hippocampus. EDTA clearly resembled those of the previous study. treated rats excreted 12 times as much Mn/day in All three exposure concentrations again increased urine as those not treated with EDTA: excretion of response rates. The higher the exposure concen­ Zn was increased 6x, Fe 2x, and Cu was decreased tration, the greater the number of sessions to 18%. However, EDTA administration did not hasten maximal rate-increasing effect. Rate increases the natural elimination of excess tissue Mn except of 200-300% of control values occurred after 30, from skeletal muscle. Mn treatment was accompanied 60 and 90 sessions for the 50, 100 and 500 ppm by decreased hepatic Fe levels, and increased Cu groups, respectively. Such delayed effects at levels in the testes and the corpus striatum and higher concentrations suggest the presence of hippocampus of the brain. Except for the physiological or behavioral compensatory mechan­ elevated brain Cu, these effects were reversible isms. Blood lead values of treated groups increa­ if Mn treatment was discontinued. A highly sed over the first 100 exposure days, then de­ significant positive correlation (r= +O. 74, clined slightly, averaging 2, 12, 19.5 and 55 ug/ p < 0.01), was noted between the urinary excretion dl for the 0, 50, 100 and 500 ppm groups, respec­ of Mn and Fe under the influence of EDTA treatment tively, after 150 exposure days. These data fur­ suggesting a relationship between the availability ther confirm the vulnerability of rats to lead in­ of chelatable Mn and Fe. toxication_, even from moderate dose levels, beyond (Supported by Ontario graduate scholarship) the nursing period and may require some revision of current notions about sensitive periods. Sup­ ported hy ES01247, ES01248 and ES05177 (NIEHS) and 286 TISSUE DISTRIBUTION OF LEAD IN NEONATALRATS DOE contract No. DE-AC02-76EV03490. G, D, Miller, C, C, Reddy, T, F. Massaro, and E. J, Massaro. The Pennsylvania State University, University Park, PA 16802. 288 THE EFFECTS OF DIETARY LACTOSEON THE ABSORPTION Although there are efforts to control the AND RETENTIONOF INORGANICLEAD IN SUCKLINGAND dissemination o:f lead (Pb) into the environment, WEANLINGRATS. P.J. Bushnell and H.F. DeLuca, Pb toxicity continues to be a major public health Dept. Biochem., Univ. of Wisconsin, Madison, WI. hazard, particularly with children, who have the (Sponsor: H.L. Evans). tendency to absorb a higher percentage (as high The ability of the milk sugar lactose to as 80%) of their Pb intake than adults (3-10%). increase the absorption and retention of many However, little is known about the retention and divalent cations, including Ca, Fe, Zn, Mg, Cu, tissue distribution of Pb in the newborn Mn, Mo, and V, suggests a nonspecificity of especially during chronic exposure, Accordingly, action which should not exclude toxic elements we have investigated the pharmacokinetics of such as Pb. The present studies assessed the Pb metabolism in neonatal rats chronically effects of lactose on ingested Pb in suckling exposed to Pb, Starting from day 6 of parturition and weanling rats. In tracer studies fasted rats on every third day, the pups were administered were intubated with 4 µCi of 210pb or 2 µCi of intragastrically with either 0, SO, 100, 150, or 203Pb, and 0, 1-, 3, or 6 mg lactose per gram body 200 mg Pb/kg as Pb acetate containing 100 µci of weight at 14, 18, 22, 26, 34, or 52 days of age,. radioactive 210pb. On day 21, animals were After a second overnight fast, the rats were sacrificed and tissue levels of Pb were measured, killed and the radioactivity in blood, GI tract The highest concentrations of Pb were observed in (including feces), liver, kidneys, and femur was the bone, stomach, kidney, intestine, and measured by gamma emission spectroscopy. Re­ intestinal contents, with lesser amounts in blood, tention in each organ was determined as a percent liver, and brain. There was no appreciable Pb in of dose, while absorption was calculated by either heart, lung or spleen. A dose related difference from the radioactivity remaining in depression in body weight gain was observed in Pb the GI tract. Under these conditions, lactose treated animals. It was also observed that the increased the absorption and retention of Pb at major target organ effected by chronic Pb exposure 3 and 6 mg/g, and at 22 and 26 days of age, is the spleen as evidenced by a significant compared to water and glucose controls. Maltose decrease in weight. and galactose did not affect the absorption or retention of Pb; nor did lactose affect excretion of intravenous 203Pb. In chronic feeding studies 287 DELAYEDBEHAVIORAL TOXICITY OF LEAD WITH INCREAS­ with nonradioactive Pb, suckling rats were offer­ ING EXPOSURECONCENTRATION: A SYSTEMATICREPLICA­ ed lactose or glucose at 0, 80, or 160 mMand Pb TION. D.A. Cory-Slechta and B. Weiss, Div. of at 0, 10, or·100 ppm in drinking water, and a

81 semipurified diet containing either adequate day s.c.)or water from day 6 to 10. A blood lead (.43%) or low (.02%) Ca. Rats were killed as increase (66%) at day 7 was paralleled by increased before and femur and kidneys were collected. bone Pb (45%) with no change in brain Pb level. Tissue Pb levels were assayed by flameless atomic No significant effects of T on Pb level in these absorption spectroscopy to assess retention of tissues were observed on days. 9 and 11. Concomi­ ingested Pb. Under these conditions, lactose tant Pb (10 mg/kg i.p.) and T (20 mq/kq s.c.) ad­ reduced the retention of dietary Pb and protected ministration did not alter 24 hour fecal excretion against the increaseof Pb retention caused by · of Pb while a 70% increase in urinary Pb was the low-Ca diet. (Supported by NIEHSGrant observed. ES-05147-02). Weanling SDrats .received Pb (10 mq/kq/day i.p.) or Pb+ T (2 mq/kq/day s.c.) for 20 days and tissue Pb levels were determined o-n day 21. 289CHARACTERIZATIONOF NEONATAL TRIETHYL LEAD NEURO­ Blood, brain, bone, kidney and liver Pb levels TOXICITYIN RATPUPS. R.M. Booze, C.F. Mactutus, were 26-37%lower in Pb+ T treated animals. H.A. Til~1n, and Z. Annau. Lab. Behav. Neurol. Results suggest: (1) acute Pb lethality is Toxicol., NIEHS,Research Triangle Park, NC 27709 not affected by concomitant T administration; and Johns Hopkins Univ., Baltimore, MD. (2) T treatment of lead-burdened animals increases blood Pb initially, perhaps by alteration of soft Although alterations in development due to in tissue distribution; (3) the tissue Pb lowering organic lead poisoning have been intensely inves­ effect of T administered concomitantly is not the tigated, little is knownabout the early toxicity result of decreased Pb absorption. (Supported of organic lead compounds. Assessment of develop­ by USPHSNIH Grant No. ES 01638) mental consequences due to triethyl lead (TEL) intoxication included (1) determination of the acute LD50as 12.8 + .9 mg/kg, and (2) detailed 291CROPDYSFUNCTION AND MOTOR COORDINATION IN examination of early neurobehavioral sequelae. PIGEONSEXPOSED TO LEADINTRAMUSCULARLY. The offspring of 12 Fischer 344 dams were pooled I.J. Boyer, D.A. Cory~Slechta, R.B. Baggs, Div. on postpartum day 3 with 4 pups of each sex of Tox., Dept. of Rad. Biol. & Biophys., U. Roch. assigned per litter. On day 5 pups were adminis­ Sch. Med.,Roch., NY14642. Sponsor:George G. Berg tered either a sham-injection, 15%ethanol, 3 Toxicity of orally administered lead acetate is mg/kg or 6 mg/kg TELvia s.c. injection (20 µl). manifested by crop dysfunction in pigeons. The Small, but significant, weight reductions for 3 mechanismcould be_local or systemic, since crop (6%) and 6(13%) mg/kg dosed pups were observed stasis (c.s.) is also produced by lesions in the (days 14-30). Early sensory deficits of TEL pups cochlea or cerebellum. This problem was resolved indicated by decreased homing ability (day 7) and by comparing pharmacokinetics and toxicity of nipple attachment (day 9) accompanied alterations lead acetate after injection (i .m.) and ingestion in motor control (day 10). While these initial (crop intubation). After a single injected dose, effects were transitory in nature, weekly activity blood lead levels peaked in 10-12 days, and evaluations demonstrated progressive development remained above 30%of peak level in 60 days.After of dose-related hypoactivity (days 14, 22, 29). ingestion, blood levels peaked within 24 hrs., During 72 and 144 hr tests of passive avoidance declined to 50% in 4 days, and less than 3% retention (days 21, 25) performance effects­ remained in 30 days. In tests of repeated expo­ hypoactivity-were noted in TELmales in opposition sures, c.s. was determined radiographically in 3 to retention loss in low dose female pups. No of 5 birds injected with 3 priming doses of 24 mg differences in startle responsiveness occurred on lead acetate per kg and maintained with the same initial trials (day 20), however, over 10 trials dose every 4 days to give blood lead levels of TELanimals displayed increased habituation. In 450-1100 µg%. Local necrosis was observed at the summarythe TELneurotoxic profile indicates an site of injection (pectoralis) but not in the alteration in arousal mechanisms, manifested in crop, grossly or by light microscopy. Three birds an increased rate of habituation and hypoactivity. exposed orally to 81 mg/kg and maintained with 45 mg/kg every 5 days showed blood lead levels as Supported in part by NIEHSGrant #ES07094and high as 2250 µg% without showing c.s. Crop stasis ES02277. was found in one bird exposed orally to 3 doses · of 121 mg/kg at 5 day intervals. This stasis was reversed by treatment with CaNa2EDTA(i.m.). 290ALTEREDLEAD DISTRIBUTION IN THIAMINE-TREATED Seven of the 8 pigeons were given a behavioral RODENTS test of balance and coordination before and dur­ R.T. Louis-Ferdinand, L. Glass, R. Sharp and ing the exposure period. Indications of impair­ ·F.C. Beuthin; College of Pharmacy and Allied ment were seen only in the 4 birds without c.s. Health Professions, WayneState University, The results confirm a systemic and reversible Detroit, MI 48202 mechanismof lead-induced c.s., but at a site distinct from the CNStarget of behavioral lead Bratton et al. [Tox. Appl. Pharmac. 59. 164 toxicity. (Supported in part by grants ES-01247 (1981)] reportedthat co-administration of thia­ and ES-01248 from NIEHS). mine (T) decreased lethality and tissue lead (Pb) levels in calves receiving lead orally for 20 days. The objective of this study was to evalu­ 292 EFFECTSOF INORGANICLEAD ON CARBONIC ANHYDRASE ate the effect of Ton i.p. Pb lethality and ACTIVITYAND REGIONAL BRAIN LEAD AND ZINC LEVELS. tissue distribution in rodents. D.A. Fox and R. Ku. Div. of Tox. ,Dept. of Pharm., In adult male SWmice, daily T (20 mg/kg Univ. of Texas Med. Sehl., Houston, Texas 77025 s.c.) had no effect on the acute Pb LD (5 days). Mice preloaded with Pb (10 ma9kg/day Experimental findings reveal that exposure to i.p.) for five days received either T (20 mg/kg/ inorganic lead (Pb) produces both convulsant and

82 anticonvulsant effects: the response being both of the gut microflora which, in turn, affects the dose and age dependent. Similarly, acetazolamide, elimination rate and body burden of Hg. a carbonic anhydrase (CA) inhibitor, produces both (Supported by NIEHS ES01247, ES01248, US/DOE a dose and age dependent alteration in seizure re­ EY76C0223490). sponsiveness. To determine if the effects of Pb were due to alterations in CAactivity, in vitro CA activity was examined using PbC12 and NaCl. CA 294DISTRIBUTION ANDDEMETHYLATION OF METHYLMERCURY activity was inhibited by 10-9 and l0- 8M PbC1 IN NEONATALRATS. Thomas, D.J. 1 , H.L. Fisher2, 7 3 2 L.L. Ha112, P. Mushak3, M. Sumler4. Sch. Hyg. while 10- to l0- M PbCl increased CAactivity Johns Hopkins Univ., Balt. MD1 , USEPA/HERL, RTP, relative to NaCl control~. These results suggest NC2, UNC Sch, Med., Chapel Hill, NC3 , NSI, RTP, that alterations in CA activity may partially ac­ NC4 • count for the dose dependent convulsant and anti­ Seven day old Long Evans rats received a convulsant effects of Pb. In addition, regional-­ single dose of l µmole of methyl (20 3Hg) mercury cortex, hippocampus (HIPP), and cerebellum--levels per kg sc and were sacrificed 1,2,4,10 and 32 of zinc (Zn) and Pb were analyzed at 21 and 100 days post dosing (dpd). Whole body radioassay days of age in neonatally Pb-exposed rats pre­ indicated little if any whole body clearance of viously demonstrated to exhibit dose and age de­ Hg until 17 days of age when there was definite pendent alterations in seizure responsiveness (Fox initiation of a single exponential clearance pro­ et al. Neurotoxicology l :149,1979). At 21 .days of cess. At 32 dpd about 70% of the dose of Hg was age a dose dependent increase and decrease in the retained. At all time points, greater than 80% Pb and Zn concentrations, repectively, was observ­ of the Hg in blood was in an organic form. Hg in ed in all brain regions. These brains, regardless muscle ranged from 79 to 100% organic, except at of treatment, showed a selective accumulation of 32 dpd when it had decreased to 66% organic. At Pb in the HIPP. At 100 days of age, almost three 10 dpd about 36% of the Hg in kidney was in an months post-exposure, a dose dependent increase organic form but by 32 dpd it was only 8%. Thus, and decrease in Pb and Zn concentrations, respect­ the kidney is a major depot for inorganic Hg pro­ ively still existed. However, only in the control duced by metabolism of methyl-Hg. Organic Hg ac­ group'was there a selective accumulation of Zn in counted for 48% of the Hg in liver at 10 dpd and the HIPP. This latter effect may partially account 38% at 32 dpd. In the brain, the organic frac­ for the demonstrated age dependent alterations in tion was 95% at l dpd and declined to 60% by 32 seizure responsiveness in neonatally Pb-exposed dpd. The percentage of Hg body burden in pelt rats. (Supported by NIEHSTrng. Grant #ES07090, rose from 30 to 85%. At all time points, about NIOSHGrant #0H07090and U.T. Biomedical Research 90% of the Hg in pelt was organic Hg. Starter Grant). These results confirm the initial high re­ tention of Hg in methyl-Hg treated neonatal rats. 293 DEMETHYLATIONOF METHYLMERCURYBY MOUSE GUT FLORA During this high retention period, a large part IN VITRO - EFFECT OF AGE AND DIET. I.R. Rowland* of the initial dose of Hg entered the pelt. In R.D. Robinson, R.A. Doherty,* B.I.B.R.A., Woodman-. all tissues except pelt, Hg content declined rap­ sterne Rd. Carshalton, Surrey, U.K. and Environ. idly from a total of 30% of the dose at 10 dpd to Health Sciences Center, Univ. of Rochester, 10% at 32 dpd. These results show that demethy­ Rochester, N.Y. Spon. T.W. Clarkson. lation of methyl-Hg begins by l dpd and occurs The rate of fecal Hg excretion after oral admin­ throughout the interval of slow Hg excretion. istration of methylmercuric chloride (MeHg) is Thus, demethylation of methyl-Hg and initiation much slower in pre-weanling mice (T!2>20 days) than of clearance of Hg appear to be independent pro­ in post-weanling mice, which excrete Hg at the cesses. (Supported in part by USPHS Grant adult rate (T½ about 10 days). In addition, adult ES-01104) mice maintained on an evaporated whole milk diet excrete Hg slowly (T½ about 20 days) after a single p.o. dose of MeHg. Differences in the in 29SsEX DIFFERENCESIN MERCURYDISTRIBUTION AND EX­ vitro rates of demethylation of MeH!(>90%absorbed CRETIONBY METHYLMERCURYTREATED RATS. Fisher, in the gut) to inorganic Hg (about 10% absorbed) H.L,l, L.L. Hall 1 , D.J. Thomas2, P. Mushak3 , M. were investigated as a possible explanation for Sumler 4 . USEPA/HERL, RTP, NC1 , Sch. Hyg., Johns the age and diet effects. Hopkins Univ., Balt., MD2 , UNC Sch. Med,, Chapel In cultured cecal and colon contents from 10 day Hill, Nc3, NSI, RTP, NC4 . old (pre-weanling) mice, radiolabelled MeHg was Male and female Long Evans rats (56 days demethylated slowly (8% of added MeHg demethylated old) received l µmole of methyl (203Hg) mercury/ in 48 hours) whereas the cultured cecal contents kg sc and were sacrificed l to 98 days post from 20 day old (post-weanling) mice converted dosing (dpd). Total and organic Hg contents of over 50% of added MeHg to inorganic Hg in 48 hour~ tissues and excreta were determined. a rate comparable to that in adult animals. Cecal At 98 dpd, total body organic Hg body burden contents from adult mice fed an evaporated whole (BB) was 7.5% of the methyl-Hg dose in males and milk diet or a pelleted rodent diet (RMH3000) 5.8% in females. Percentages of total tissue Hg differed in their capacity to demethylate MeHg contents present as organic Hg ranked from high­ in vitro and possessed markedly different bacterial est to lowest as pelt, muscle, blood, kidney, floras. Cecal contents from mice fed the pelleted liver, and brain. Total organic Hg BB was dif­ diet metabolized MeHg rapidly (about 37% de­ ferent in male and female due to difference in methylated in 24 hours), while those from milk .fed pelt and muscle. Total inorganic Hg BB peaked at mice demethylated only 8% of added MeHg in 24 hours. about 8% of the dose of methyl-Hg. Pelt and kid­ These results indicate the importance of the neys were the largest depots for inorganic Hg. effects of diet and age on the metabolic activity Inorganic Hg content of pelt declined more slowly

83 than that of kidneys. Inorganic Hg content of transfer were studied in vitro. Concentrations other tissues ranked from highest to lowest as necessary for half maximal inhibition of oxygen muscle, blood, liver, and brain. No male-female uptake (EC5ol were determined polarographically. difference in total inorganic Hg BB was found. The EC50 f.or a NADH-linked substrate system Between 32 and 98 dpd, the average ratio of the (glutamate, malate, and maleate) was 18 µm while total organic Hg BB to the total inorganic Hg BB the EC50 using succinate as substrate was 15 µm. was 3.7 for males and 3.0 for females. Although At 64 µm, NADH-linked substrate oxidation was fecal excretion of inorganic Hg was about equal inhibited 60% while succinate oxidation was in males and females, females excreted about 1.27 inhibited 80%. At concentrations of Na2PdCl4 times as much organic Hg in feces than did males. sufficient to inhibit oxygen uptake, there was Females excreted 2.9 times as much organic Hg and a concomitant decrease in the rate of ADP 1.6 times as much inorganic Hg in urine than did phosphorylation measured by proton absorption. males. Additions of uncoupling agents (carbonyl These results confirm an earlier observation cyanide-m-chlorophenol phenylhydrazone, CCCP) of sexual differences in retention of Hg. Dif­ had no effect on Na2PdC14 inhibition. State IV ferences huve been found in the retention of or­ respiration was not increased at low Na2Pdc1 4 ganic Hg in muscle and pelt and in the rate and concentrations and there was no activation of form of Hg excreted in urine and feces. (Sup­ the Mg-ATPase. Data from these experiments ported in part by USPHS Grant ES-01104). indicate that Na2PdC14 is a mitochondrial respiratory chain inhibitor in vitro.

296ROLE OF GUT FLORA IN MERCURYEXCRETION AFTER METHYLMERCURYADMINISTRATION IN THE MOUSE. I.R. ROWLAND*,R.D. ROBINSON& R.A. DOHERTY., 298 FACTORSAFFECTING NICKEL SUBSULFIDETOXICITY. *BIBRA, Woodmansterne Rd., Carshalton Surrey U.K. D.A. McNeill, C.L. Chrisp, and G.L. Fisher, and Environ. Health Sciences Center, Univ. of Battelle Columbus Laboratories, Columbus OH 43201 Rochester, Rochester, N.Y. Spon. T.W. Clarkson. The toxicity of intratracheally instilled Ni3S 2 Adult mice fed a pelleted rodent diet (RMH 3000), particles of different size distributions was in­ evaporated whole milk, or a semi-synthetic liquid vestigated in two mouse strain·s. The LDso for diet (GIBCO 116 EC), exhibit different rates of strain A/J mice exposed to coarse (maximum whole body Hg elimination (T½'s for GIBCO, RMH, diameter of 20 µm) particles of Ni 3s2 was 50 mg/ and milk were 6, 10, and 20 days, respectively) kg. In contrast, the LD50 for both strain A/J and and fecal Hg elimination after a single oral dose B6C3Fl mice exposed to fine (maximum diameter of of methylmercuric chloride (MeHg). After 6 days, 2µm) particles was 4 mg/kg, i.e. the fine parti­ 36%, 22%, and 9%,respectively of the initial dose cles were about 12 times as toxic as the larger was excreted in the feces. The proportion of or­ particles. The results of multiple exposure of ganic Hg in the feces (5%, 21%, and 34%, respec­ these two different sized particles were compared tively) indicates that more demethylation occurred in another experiment. Strain A/J mice exposed in the first two groups and that this may affect once per week for four weeks to coarser particles fetal elimination. To assess the contribution of had an LD50 of 2 mg/kg. Both strains of mice the gut flora to in vivo demethylation and excre­ similarly exposed to the finer material resulted tion of Hg, we gave antibiotics to mice on each in an LDso of l mg kg, i.e. fine particles were diet to eliminate their intestinal flora before only twice as toxic as coarse particles of Ni3S 2 MeHg administration. Antibiotic treatment reduced with that mode of exposure. When those same two fecal elimination to nearly zero in the GIBCO and sized particles were incubated in horse serum at milk groups, and the RMHgroup excreted only 6% of 37°c for 3 days, the fine particles were three the initial dose in the feces after 6 days. Anti­ times more soluble then the coarse material. These biotic-treated mice on the GIBCO and RMHdiets had in vitro and in vivo studies indicate that both higher tissue Hg concentrations and higher propor­ particle size and mode of exposure may effect the tions of organic Hg in the feces, cecal contents, biological availability and therefore toxicity of liver, kidney, and gut wall than their non-treated intratracheally administered Ni3S2- Supported by eohorts. The antibiotic treatment had little ef­ the Electric Power Research Institute (Contract fect on these parameters in the milk-fed mice. Number RP1639-2) These results are consistent with the theory that demethylation of MeHg by intestinal bacteria is a major factor determining the rate of excretion of 299 EFFECT OF INADEQUATEVITAMIN E AND/OR SELENIUM Hg and that modification of the gut flora by diet is at least partly responsible for the differences NUTRITION ON ARACHIDONICACID METABOLISMIN RAT in whole body retention and excretion of Hg by mice TISSUES. T. L, Grasso, T, J, Labosh, C, E. Thomas, C. C, Reddy, R. W, Scholz and on different diets. (NIEHS ES 01247, ES 01248, US/DOE EY76C023490) E, J, Massaro. The Pennsylvania State University, University Park, PA 16802,

297 EFFECTS OF DISODIUMTETRACHLOROPALLADATE ON Vitamin E (Vit E), as a free radical ISOLATED RAT LIVER MITOCHONDRIA. R.E. Biagini scavenger, and selenium (Se), as an essential com­ and W.J. Moorman, National Institute for Occupa­ ponent of glutathione peroxidase (EC 1,11,1.9.) tional Safety and Health, Division of Biomedical (GSH-Px) function synergistically to modulate free and Behavioral Science, Experimental Toxicology radical production and hydroperoxide tone of the Branch, Cincinnati, OH; and G.W. Winston, Mt. cell, Thus, they have the potential to affect the Sinai School of Medicine, Department of Bio­ enzymes of prostanoid biosynthesis and also the chemistry, New York, NY. Sponsor: T.R. Lewis. profile of products formed in the arachidonic acid The effects of Na2PdCl4 on isolated rat liver (A.A,) cascade, Accordingly, we investigated the mitochondrial electron transport and energy influence of altered Vit E and Se nutrition on

84 A. A. metabolism in tissues of Long-Evans Hooded (MES) severity in adult rats (Fox et al.,The Tox­ rats fed chemically defined purified diets con­ icologist 1 :45,1981) and mice (Doctor and Fox, The taining adequate or documented deficiencies of Toxicologist 2:this issue,1982). The mechanisms Vit E, Se or both (diets: +E, +Se; -E, +Se; +E, responsible for these changes are not known. This -Se; -E, -Se). Cyclooxygenase activity of lung study was undertaken to examine the neuropharma­ and liver microsomal preparations was significant­ cological basis of this anticonvulsant effect. ly increased in Vit E deficiency whereas PG Male mice were injected (ip) with specific post­ dehydrogenase activity (a key enzyme in PG cata­ synaptic antagonists of the noradrenergic (NA), bolism) was increased two-fold in Se deficiency. dopaminergic (DM), serotonergic (5HT) and muscar­ Similarly, the activity of GSH-s-transferases inic cholinergic ~ACh) systems or with a presyn­ (a group of related enzymes involved in leuko­ aptic depletor of NA, DMand 5HT prior to dosing triene biosynthesis) was markedly increased in with TET. Mice were MES tested at 0.5 hr post TET liver, kidney and lung cytosolic preparations injection. The MES pattern is characterized by 5 of Se deficient animals. The Se-independent grades of severity: 1 (minimal) to 5 (maximal). GSH-Px activity of GSH-s-transferases was Control and all drug treated mice had grade 5 concomitantly increased in Se-deficient states, seizures. 1 mg/kg of TET (TET-1) produced 20% while Se-dependent GSH-Px activity was completely grade 4 and 80% grade 5 seizures, while 5 mg/kg abolished. In addition, epoxide hydrolase (an TET (TET-5) produced 90% grade 2 and 10% grade 3 enzyme that competes with GSH-s-transferases for seizures. Reserpine (RESRP), yohimbine (YOH), me­ leukotriene A4) was slightly increased in liver, tergoline, haloperidol and scopolamine protected but not in the lung. (supported by EPA Grant against the effect of TET-1, while propraolol did II R807746), not. However, only RESRP and YOHafforded any pro­ tection against TET-5. This suggests that there is a dose related effect of TET on various neurotrans­ 300METAL ION TOXICITY IN DROSOPHILA. Nelwyn T. mitter systems: at low doses of TET the alpha NA, Christie* and K. Bruce Jacobson, Univ. of Tu.- DM, 5HT and MAChsystems modify the anticonvul­ Oak Ridge Grad. Sch. of Biomed. Sci., Biol. Div., sant effect,while at high doses of TET only the ORNLt, Oak Ridge, TN 37830 Sponsor: H.P. Witschi alpha NA system is involved. Correlative neuro­ Our studies of metal ion toxicity have focused on chemical data will also be presented. (Supported in vivo macromolecular alterations, especially by NIEHS Trng. Grant #ES07090, NIOSH Grant effects on Q(+)tRNAs. (Q=7-deazaguanosine with a #OH07085 and Univ.Texas Biomedical Research Start­ cyclopentenediol at C-7 through a -CH2-NH2- er Grant). linkage.) As an aid in evaluating mechanisms of action, 13 metal ions with diverse physicochemical parameters have been examined: the IIb ions Zn2+, Cd2+ and Hg2+. the Ila ions Be2+ Mg2+ Sr 2+ and 302 EFFECTS OF TRIETHYLTINON THE POSTNATALDEVELOP­ + , ' l ' MENTOF THE CEREBRALPENTOSE PHOSPHATE PATHWAY. Ba2 ; the transition elements, NiL+, Cu2+ Co2+, R.M.Rocco, J.B.Blumberg, D.R.Brown. ,Program in and Mn2+; and trivalent ions, y 3+ and Cr 3 For +. Toxicology, Northeastern University, Boston, MA quantitative estimates of toxicity for these ions 02115. we utilized statistical parameters of LC50 and R, the change in concentration between the LC2.5 and The exact mechanism whereby triethyltin (TET) the LC97.5. A standard assay procedure using 30 causes decreased myelin synthesis in postnatal animals was devised to quantitate the effect of rat brain is unknown. TET is a useful neurotoxic sex, genotype, temperature, seasonal variations, tool for studying hypomyelinating conditions. The and developmental stage on the response of Droso­ purpose of these experiments was to examine the phila to metal ions in the diet. Statistically effect of TET on the development of selected en­ reliable estimates of sensitivity under a variety zymes in the cerebral pentose phosphate pathway of conditions have provided a basis for interpret­ (PPP). TET has been shown to cause a selective ing the biochemical alterations observed in re­ 2 effect on myelinogenesis (Beaker, et al J. Neuro­ sponse to metal ions, notably Cd +. Six divalent chem 36:44, 1981). Histochemical studies have cations were tested for thei~ effect on Q(+)tRNA. indicated that many PPP enzymes are concentrated Cd2+-treatment of adults causes an increase in 2 within myelinated tracks (Kauffman et al, J.Neuro­ the levels of Q(+)tRNA. Sr + will either prevent chem __!1:1, 1972). The PPP is a major source of age-related increases in Q(+)tRNA or cause an in­ NADPHwhich is required for lipid synthesis and crease, depending on the concentration in the appears particularly active in oligodendrocytes,15 diet. In addition both the toxic ion Hg2+ and the 2 mg/1 TET S04 was added to the drinking water of essential ion Zn + cause an increase in Q(+)tRNA Sprague-Dawley dams from day 1 postpartum. Whole in adults at sublethal concentrations. These ob­ pup brains were taken on days 3,5 and 15, per­ servations suggest a correlation between a toxic fused with cold saline and homogenized in 0.32 M response and alterations of levels of Q(+)tRNA. sucrose. On day 15, a period of active myelina­ *supported by Health and Safety Res. Div. of ORNL. tion, the total protein per brain of test ~ups toperated by Union Carbide Corp. under contract (N=5) was 63% and cyclic nucleotide phosphodies­ W-7405-eng-26 with the U.S. DOE. terase (EC 3.1.4.37), an index of myelin forma­ tion, was 26% of control values. Glucose-6-phos­ phate dehydrogenase (EC 1.1.1.49) and 6-phospho­ 301 NEUROPHARMACOLOGICALANALYSIS OF THE ANTICONVUL­ gluconate dehydrogenase (EC 1.1.1.44), the first SANT EFFECTS OF TRIETHYLTIN(TET) IN ADULTMICE. two PPP steps were 126% of control. Transketo­ D.A. Fox. Div. of Tox.,Dept. of Pharm., Univ. of lase (EC 2.2.1.1), the PPP rate limiting step, was Teaxs Med. Sehl . , Houston, Texas, 77025. 63% of controls and has been reported to corre­ late with degree of myelination in developing Acute treatment with TET causes dose depend­ rabbit spinal cord (Lunine et al, J.Neurochem 18: ent decreases in maximal electroshock seizure 1113, 1971). Lacate dehydrogenase (EC 1.1.1.27),

85 a soluble enzyme in the glycolytic pathway, was birds. The third bird that received 1.75 mg/kg unchanged. Thus, TET disrupts the PPP at the TMTdid not recover during a 2 month period, but transketolase control point and this effect is was successfully retrained under the same schedule associated with rnyelin membrane synthesis. at that time. A dose of 1 mg/kg TMTproduced no effects in any of the four birds that received the dose for the first time, but weekly or biweekly 303 STUDIES ON THE NEUROTOXICITYOF TRIMETHYLTIN(TMT) administration of this dose in two of these birds S,V, Doctor, L,G, Costa, D. Kendall, S,J. Enna produced a gradua 1 decline in rates of responding and S.D. Murphy, Div, of Tox, and Dept. of Neur­ under the fixed-ratio schedule of food presenta­ obiology and Anatomy, Univ. of Texas Med, Sch, tion. Under light microscopy, pathological changes Houston, Texas.77025. of nerve cells in the hippocampus, the pyriform cortex, and the cerebral cortex could be identified TMT, produced industrially as an intermediate 24 hrs after exposure to 3 mg/kg TMT. These path­ in synthetic processes, has been involved in sev­ ological changes included chromatolytic changes eral cases of human intoxication. However little (disappearance of Nissl substance and eccentric is known on its mechanism of neurotoxicity. The nuclei), accumulation of eosinophilic hyalinoid acute i,p. LDSO in male mice was found to be 2,9 material in neuronal cytoplasm, and neuronal + 0,1 mg/kg. Following administration of 4.26 mg/ shrinkage. kg the animals appear sedated for 2-4 hrs and then recover until, at 14-16 hrs, tremors occur foll­ owed by convulsions and death, The duration of the 305 TWOACUTE HUMAN POISONING CASES DUE TO EXPOSURE sedation and time to death vary depending on the TO DIAZINON TRANSFORMATIONPRODUCTS IN EGYPT. dose, TMT also causes a dose dependent antinocic­ S.A. Soliman, G.W. Sovocool, A. Curley, N.S. eptive effect which is maximal at lh and is block­ Ahmed, S. El-Fiki and A. El-Sebae. US Environ­ ed only by atro~ine, TMT, however, is ineffective mental Protection Agency, Research Triangle Park, in displacing ( H)-QNB binding in vitro and does NC and Alexandria University, Alexandria, Egypt. not affect (3H)-QNB binding and acetylcholinester­ This is a report of two cases of high acute toxi­ ase in vivo. Since the apparent cholinergic med­ city of diazinon in spraymen working in public iation of antinociception resembles that found health occupations in Alexandria, Egypt. Sympto­ with different GABAagonists (Kendall et al,, J.P. matology was similar to that previously reported ~., in press) we investigated the effect of TMT for exposure to parathion or other organophos­ on the GABAergic system. TMT inhibits the in vitro phorus insecticides. Plasma and RBC cholines­ uptake of (3H)-GABA into brain synaptosornes with terase (ChE) activity values were determined in an ICSO of 90 µMas compared with a value of 45 µM blood samples obtained from both cases at dif­ for the specific GABAuptake inhibitor, nipecotic ferent times after the incident. Cholinesterase acid. Fourteen hours after administration of TMT activity showed a marked reduction up to 18 days (4.26 mg/kg) to mice, synaptosornal GABAuptake is after exposure. In case 1, blood cholinesterase inhibited 25% and GABAlevels are 23% decreased. 3 activity was recovered to about 90% of the normal At this dose ( H)-norepinephrine uptake was inhi­ level of activity 28 days after the poisoning bited, TMT does not appear to act directly on the incident. This activity recovered to about the GABAergic receptor, since it is inactive in dis­ same level in case 2 but after only 20 days from placing (3H)-GABA binding in brain in vitro, These the poisoning date. Experimental results sugges­ data suggest the involvement of GABAin TMT neuro­ ted that this acute toxicity was due to unsuit­ toxicity, although other neurotransmitters could able storing conditions of the emulsifiable be involvedo (Supported by NIEHS Research and Tr­ concentrate (EC) formulation of diazinon. The aining grants ES01831 and ES07090 and grant NS- used sample of diazinon that was applied was 13803). stored in "tin" containers (made of tin plated sheet steel). The EC (60%) was not in compliance with the WHOstandard specifications regarding 304 EFFECTSOF TRIMETHYLTIN INJECTIONSIN PIGEONS. the emulsion stability tests and due to the pre­ D.E. McMillan, M. Brocco, G.R. Wenger, and L.W. sence of crystals in the EC. A sample of this Chang. Dept. of Pharmacologyand Interdisciplinary crystalline material was subjected for analysis. Toxicology and Dept. of Pathology, University of Gas chromatographic analysis combined with mass Arkansas for Medical Sciences, Little Rock, AR. spectrometric techniques failed to identify in­ tact diazinon in samples of that material. The Male white Carneaux pigeons recei_ved intramus­ sample represented virtually complete .conversion cular injections of trimethyl tin hydrochloride of diazinon into transformation products. Sulfo­ (TMT). Two birds receiving 3 mg/kg doses died tepp, dithionotetraethylpyrophosphate and mono­ within 48 hrs. Two others, given 3 mg/kg of TMT thionotetramethyl pyrophosphate were two of the were alive 24 hrs after injection, at which time identified products in the sample. These pro­ they were sacrificed. Three birds receivin~ 3 ducts are highly toxic compared with diazinon. mg/kg TMTin divided doses two weeks apart (1 and 2 mg/kg doses) survived, but exhibited impaired motor coordination, abnormal body posture, and 306 TRIMETHYLTININDUCED PATHOLOGICAL CHANGES IN THE decreased food intake. One of these birds slowly BRAIN STEM NEURONS. L.W. Chang, T.M. Tiemeyer, recovered over several weeks time, but the other K.R. Reuhl*, D.E. McMillan and G.R. Wenger. De­ two birds remained impaired for two months before partments of Pathology and Pharmacology, University sacrifice. Three birds receiving 1.75 mg/kg TMT of Arkansas for Medical Sciences, Little Rock, AR showed similar symptoms lasting 1 week to several and Research Council of Canada*, Ottawa, Canada. months in duration. Responding maintained under a Trirnethyltin (TMT) has been reported to be a po­ fixed-ratio schedule of food presentation by these tent neurotoxicant. Besides the hippocarnpal birds was markedly decreased, but gradually in­ neurons, some large neurons in the brain stern creased over a period of 2 to 3 weeks in two of the were also found to be affected by this toxic com-

86 pound. The purpose of this report is to provide Because of the projected increase in the use of light and electron microscopic observations on diesel passenger cars, the possible health the pathological effects of TMT on these nerve effects of their exhaust emission are being in­ cells. BABL/c mice with average body weight of vestigated. The exhaust particles from diesel 25 gms were injected (i.p.) with trimethyltin passenger cars were collected, and the associated chloride at a dosage of 3.0 mg/kg b.w. Animals organic chemicals were extracted using dichloro­ were sacrificed at 48 hours by means of intracar­ methane as solvent. The resulting extract had dial perfusion of 2.5% buffered glutaraldehyde. low mutagenicity toward Chinese hamster ovary The medulla-brain stem area was sampled for light (CHO)cells in culture (~ 1/200 of benzo(a) and electron microscopy studies. Light micro­ pyrene). The extracts, however, had definite scopic examination revealed that many nerve cells comutagenic effects. WhenCHO cells were treated in the brain stem were affected. These nerve with a diesel exhaust particle extract in combi­ cells appeared to have eccentric nuclei and a nation with a muta~en [N-methyl, N'nitro, N­ hyalinization of their cytoplasm. Many neurons nitroso-guanidine (MNNG)or benzo(a)pyrene also displayed a laced or foamy appearance of (B(a)P)l the mutant frequency induced was 200- their cytoplasm with eventual vacuolation and dis­ 300%of that expected from the mutagenicity of integration of the entire nerve cell. Electron each agent alone. This co-mutagenicity was ob­ microscopy also demonstrated a loss of the rough served for all diesel exhaust particle extracts endoplasmic reticulum. Extensive fragmentation (from cars of 5 different manufacturers) tested, and dilatation of the cytoplasmic membranes (endo­ using mutation at the hypoxanthine-guanine phos­ plasmic reticulum and Golgi apparatus) also oc­ phoribosyl transferase gene locus, mutation at curred giving rise to the foamy appearance and the Na+-K+-ATPasegene locus, and induction of eventual vacuolation of these nerve cells. No sister-chromatid-exchange as endpoints. The significant degenerative change was observed in interaction between diesel exhaust extract and the myelin sheaths. It can be concluded in our B(a)P is of particular importance since B(a)P is study that, unlike triethyltin (TET) which is my­ a knownenvironmental pollutant, and experimen­ elinotoxic, TMT is neuronotoxic producing direct tal mutagen and carcinogen. Our data therefore toxic damage on the neuronal bodies. Ourobserva­ suggest the possible enhancement of the activi­ tions also suggest that TMT may be neurotoxic on ties of environmental mutagens/carcinogens by the biological membrane level producing severe the chemicals associated with diesel exhaust intracellular edematous and vacuolation conditions. particles. This enhancement effect should be one of the factors considered in the health- risk analysis of the diesel exhaust emission. 307 MANGANESECHLORIDE EXPOSURE ALTERS HIGH AFFINITY (Research performed under DOEContract No. DE­ RECEPTORBINDING AND DRUG-INDUCED ACTIVITY IN AC04-76EV01013.) MALERATS. J.M. Gerhart and H.A. Tilson, Labora­ tory of Behavioral and Neurological Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709. 309 BINDING OF CACADYLICACID TO MACROMOL.ECULESIN (SPON: Richard Mailman). RAT TISSUES. J. T. Stevens, CIBA-GEIGY Cor­ Adult male Fischer-344 rats were exposed for poration, Greensboro, NC, and J. D. Farmer, up to 30 days via their drinking water to either U.S. EPA, Research Triangle Park, NC 0, 10, 20, or 40 mg MnCl2/mldH20. The average daily dose for 10 mg MnCl2/mldH20-exposed rats Pharmacokinetics studies have shown that was 166 mg MnC12 per day resulting in a cumulative cacadylic acid administered as a single dose dose of 5.0 g MnCl2over 30 days. No significant orally, intravenously or intratracheally was changes in body weight or water consumption still present in rat tissues at 105 days post­ occurred at this dose level. Rats exposed to administration (Stevens et al., Environ. either 20 or 40 mg MnCl2/mldH20 had significantly Hlth. Perspect., 19, 15~7). As a result decreased body weights within a 7 day exposure and of these findings,studies were undertaken to were excluded from further testing. Exposure to evaluate the potential of this organic arseni­ 10 mg MnCl2/mldH20 produced slight, but not cal and/or its metabolites to bind covalently statistically significant increases in whole brain to macromolecules of the lung, liver and kid­ Mn levels, as determined by atomic absorption ney of male and female Sherman rats after a spectroscopy. However, Mnlevels were significant­ single intravenous dose of 200 mg/kg of ca­ ly increased in sera and adrenal samples. Activity cadylic acid (8.8 µCi/mg of c1'+ label). responsiveness to a pharmacological challenge with Approximately 3 to 9% of the radiolabel de­ harmine, but not apomorphine, was altered in Mn­ tected in the homongenates of the lung, liver exposed rats. Neurochemical alterations were seen and kidney (expressed per mg of protein) was in neurotransmitter high-affinity receptor binding found to be covalently bound when evaluated at assays. [3H]-spiroperidol binding was significant­ 1 and 72 hours post-administration; a slightly ly increased in the striatum, but not in the higher level of binding was noted at 72 hours. frontal cortex. [3H]-muscimol binding was signifi­ Strong binding to nuclear RNA was greater in cantly decreased in the hippocampus but not these three tissues at the early time point cerebellum. [3H]-diazepam binding was not altered than at 72 hours; whereas, binding to nuclear in the hippocampus. DNA level remained relatively constant during this period. The significance of the binding of cacadylic acid to nuclear DNA is not clear, 308 CO-MUTAGENICEFFECTS OF DIESELEXHAUST PARTICLE since previous metabolism studies did not EXTRACTSIN CULTUREDMAMMALIAN CELLS. A. P. Li, suggest the formation of an electrophilic R. E. Royer, A. L. Brooks and R. 0. McClellan, metabolite(s). An alternate possibility based Lovelace Inhalation Toxicology Research Institute, on spectrometric evidence with purified calf P. 0. Box 5890, Albuquerque, NM87185 thymus DNA, will be considered.

87 310 COMPARISONOF THE MUTAGENICAND CARCINOGENIC ± 20.4]; L = [666.1 ± 14.3]; M'lR = [734.1 ± 19.8]. ACTIVITIES OF ASPHALTAND COALTAR PAINTS USED IN Thus, M-vRincreaserl the disposition of PB to the . CONTACTWITH POTABLE WATER. M. Robinson, R.J. brain, thereby decreasing its onset of action • Bull, D. Cmehil, R. Slater and J.R. Meier, Health Ambient temperature affecterl the times of onset Effects Research Laboratory, U.S.E.P.A., Cin­ for control vs. M-vRgroups: at 19oC, 27.5 = 3.0 cinnati, OH 45268 vs. 15.5 ± 2.6; at 27oC, 14.0 ± 1.5 vs. 11.7 ± 15 min. Supporterl by ES02006 and 5T32 ES07043. Tanks and pipes used for storage and trans­ port of drinking water are often coated with paints which contain either coal tar or asphalt pitch. We have begun a comparison of the potencJ 312 BIOAVAILABILITYSTUDIES OF SOOT CONTAMINATEDWITH of a series of products containing these materials POLYCHLORINATEDDIBENZO DIOXINS (PCDD) AND DIBEN­ ZOFURANS(PCDF) FORMEDIN A FIRE IN A PCB-FILLED utilizing the Salmonella/microsome assay (Ames TRANSFORMER. J.B. Silk.worth, L. Kaminsky, P. test) and mouse skin initiation/promotion stud­ O'Keefe, D. McMartin and R. Smith, Div. of Labs. ies. Eight enamels were chosen for test; 4 and Res., NYS Dept. of Health, Albany, NY asphalt-based (AP) and 4 coal tar-based ( CTP). Initiation/promotion studies were conducted in A fire in a PCB-filled transformer produced wide­ the SENCARmouse (40 females per group) utilizing spread contamination of an 18-story building in single topical applications. A 20-week promotion Binghamton, NY, with soot-like material containing schedule was used applying 1 ug of TPA (12-0- 2 ppm 2,3,7,8-tetrachlorodibenzo-p-dioxin, >47 ppm tetradecanoyl phorbol-13-acetate) 3 times weekly. 2,3,7,8-tetrachlorodibenzofuran, 0.5% total PCBs Ames test results with AP were negative at up to in addition to numerous other PCDF and PCDD con­ 10 ul/plate. A very high mutagenic activity was geners. The bioavailability of the highly toxic found with CTP with rat liver S-9 fraction. components of the soot was investigated because of Significant tumor initiating activity was ob­ the possible high adsorbancy of the soot matrix. served with AP as well as CTP. The potency of AP Female Hartley guinea pigs (350-450g), n=6, were was considerably less, requiring approximately administered a single oral dose of either l-1250 200 ul/mouse to obtain an average of 0.5 tumors/­ mg crude soot in 0.75% acqueous methyl cellulose animal at 20 weeks vs. 0.2 ul/mouse required of (MC)/kg body weight or a benzene Soxlet extract in the CTP. These data correspond to a potency MC equivalent to 4-1000 mg crude soot/kg. Activa­ equivalent to approximately 15 to 60 mg of benzo­ ted carbon served as control. Body and organ (a)pyrene [B(a)P] per ml of coal tar-based prod­ weights were determined and histopathology and h~­ uct. Estimates of B(a)P equivalence in AP would matology were conducted only for animals which re­ be 3 orders of magnitude lower. Total B(a)P and ceived up to 500 mg soot/kg and survived 40 days. B(e)P content of CTP ranged from 1.9 to 3.4 mg/ml; A significant (p<0.05) increase in the absolute although total identified polyaromatic hydro­ and relative (to brain) spleen weights was obser­ carbon concentrations ranged from 29 to 60 mg/ml. ved only at 10 mg soot/kg. A decrease in the ab­ Since the bulk of these compounds are weak or solute and relative thymus weights was observed at inactive as carcinogens, the analyses to date 100 and 500 mg soot/kg and a decrease in the abso­ account for a relatively small fraction of the lute and relative kidney weights at 500 mg soot/M. biological activity present. A decrease in the WBCcount occurred only at 100 mg soot/kg. Body weight loss was observed at 500 mg soot/kg and above. The 14- and 21-day LD50s 311 DECREASED TIME OF CNSEI' AND IOCREASED BRAIN DIS­ for crude soot were 1029 and 692 mg/kg, respect­ POSITIOO OF PHENOBARBITAL (PB) AFTER MICID'JAVE ively, and the 14-and 21-day LD50s for soot ex­ IRRADIATICN (Mm). E.B. Benson, J.M. Fuj:inoto tract were 625 and 282 mg soot-equivalent/kg, re­ and D.G. Lange. Research Services, V.A. Center, pectively. These results indicate that in an acute woad, WI, and Ilept. of Pharnacology and Toxico­ exposure to guinea pigs, the organic solvent ex­ logy, M:rlical College of Wisconsin, Milwaukee, WI. tractable toxic components of the soot are approx­ M-vRhas been reporterl to increase cerebral blood imately 40-60% bioavailable. flav and blood-brain barrier penreability. PB is a drug whose phannacodynarnics would likely be affecterl by these pararreters. Male swiss Cox 313 TECHNICALPENTACHLOROPHENOL (PCP-T)-INDUCED SUP­ mice, maintainerl at an arooient terperature of 23° PRESSIONOF MURINEHUMORAL IMMUNE RESPONSE. N. I. c, ~e exposerl for 10 min to near-field MvR at Kerkvliet, Sch. of Vet. Med., Oregon State Univ., 2.45 GHz power density 10 rrW/cm2, to give a 1.2° Corvallis, OR. C increase in rectal terperature. An incandes­ PCP-Tis a widely used wood preservative and gen­ cent bulb (L) was adjusted to give an equivalent eral biocide. We previously reported highly sig­ increase in rectal terperature. Within 1 min nificant enhancement of tumor growth and depres­ after irradiation, sodium PB, 150 II¥3"/kgwas given sion of cell~mediated innnunity in mice chronical­ iv. Onset of anesthesia was the time fran injec­ ly exposed to PCP-T contaminated diets. The pre­ tion to when the mice could no longer right then­ sent studies examined the humeral innnune response selves for a 5 min period. Exp. I: Cnset of an­ in mice exposed to 50, 100, 250 or 500 ppm PCP-T esthesia (min) and corresponding brain concentra­ in the diet for 9-11 weeks. No overt toxicity tion of [PB, nrrol/g] ± S.E. ~e for control (C) = was apparent at these exposure levels. The T­ 17.4 ± 1.6 [926.4 ± 7.5]; L = 13.3 ± 0.6 [905.4 ± cell dependent antiSRBC splenic antibody response 29.6]; Mm= 8.3 ± 0.3 [900.2 ± 11.2]. Mmde­ was quantitated in Swiss-Webster mice using the creaserl onset relative to C and L groups. Since Hemolytic Antibody Isotope Release (HAIR) assay. all 3 groups had the sarre brain [PB] at the onset The T-cell independent antiDNP-Ficoll response of anesthesia, the brain sensitivity was not was quantitated in C57Bl/6 mice by Cunningham's changerl. Exp. II: Brain concentrations of [PB, plaque assay. Serum antiDNP antibody levels were nrrol/g] measurerl 5 min after PB, iv, were C = [603 measured by passive hemagglutination. Following

88 exposure to 250 or 500 ppm PCP-T, the primary IgM (Sponsor: H. W. Dorough) response to SRBC was reduced to 30 and 19% of the In recent years much work has been performed control response and the secondary 'IgM response using hypoxic radiosensitization as an aid to was delayed by 1 day and suppressed to 54 and 45% curative and palliative radiotherapy in the of control, respectively. The secondary IgG re­ treatment of cancer. Misonidazole (Ro-07-0582) sponse was also delayed 1 day and the peak respon­ has been shown to be an effective radiosensitizer se suppressed to 82 and 68% of control. Exposure in non-human systems and apparently mimics oxygen to 50 ppm PCP-T slightly delayed the IgM response in its mode of action when irradiated in the pre­ but did not affect its magnitude. The IgG re­ sence of hypoxic cells. Using misonidazole, a sponse was not altered by 50 ppm PCP-T. A direct system was examined which would ascertain the suppressive effect of PCP-Ton the number of anti­ effectiveness of new drugs at enhancing DNA body-forming cells (AFC) in the spleen was obser­ strand breakage and viability loss after irradia­ ved following immunization with DNP-Ficoll. The tion. Purified ¢X-174 RF DNAwas exposed to gam­ number of IgM AFC/106 spleen cells was reduced to ma radiation under anoxic and oxic conditions in 57, 54, and 33% of control in animals exposed to the presence and absence of misonidazole. DNA 50, 100, and 250 ppm PCP-T. In addition, peak strand breakage was determined using ultracentri­ serum antibody titers to DNP were delayed and dose fugation while DNAviability was measured using dependently suppressed in PCP-T exposed mice. Com­ three calcium treated isogenic Escherichia coli parable spleen weights and cell recoveries follow­ host strains having differential DNA repair ca­ ing immunization suggested that cells from PCP-T pabilities. The test system was most useful in animals proliferate but are inhibited in the pro­ a) indicating radiosensitization capability, b) duction of antibody, particularly IgM antibody. suggesting the concentration dependence of the chemical dose-effect relationship, c) measuring dose modification factors, and d) indirectly 314 COMPARATIVEHAZARDS OF METHANOL,ETHANOL, ISOPROP­ relating biological damage to clear molecular ANOLAND GASOLINE ALONE AND IN MIXTUREAS FUELS endpoints. Oxygen effect values measured by the FORVEHICLES. H.S. Posner, NIEHS,NIH, P.O. Box test system were somewhat smaller than those 12233, Research Triangle Park, N.C. 27709 observed in vivo. (Support by NCI grant POl-CA- Sponsor: J.A. Moore 17786.) - -- It is important that severe health problems not be created in switching to new fuels. Though gas­ oline has been the predominant fuel for vehicles in the U.S.A., plans are in progress to substitute 316 DETECTIONOF SHORT-PATCHDNA REPAIR IN SUSPENSION methanol, ethanol or mixtures of gasoline or diesel CULTURESOF HEPATOCYTES.M.J.Olson, D.A.Casciano fuel with lower alcohols. and J.G.Pounds, Natl. Cntr. for Toxicol. Res., Review of the literature shows that gasoline Jefferson, AR 72079. Sponsor L.Fishbein toxicity is slight after ingestion during siphon­ The detection of short-patch DNA repair by ing mainly by adults or inadvertent swallowing by scintillation spectrophotometry (SS) in the pri­ children, unless inspiration into the lungs occurs mary rat hepatocyte/DNA repair system has been (serious and mainly seen in children); after difficult due to the small number of bases insert­ inhalation in confined spaces by children and ad­ ed, variability in the level of scheduled DNA syn­ ults, and sniffing or huffing mainly by children thesis and nonspecific thymidine binding. This re­ and teenagers, which have resulted on occasion in port demonstrates that suspension cultures of hep­ death by sedation or sudden death believed to be atocytes are suitable for detection of short­ by effect on the heart. In most cases recovery patch repair if steps are taken to reduce the occurs with rest and supportive care. Alkyl lead level of semi-conservative DNA replication and to has added additional toxicity. remove nonspecifically bound thymidine. Isolated Between methanol (M), ethanol (E) and isoprop­ hepatocytes from Sprague-Dawley rats were placed anol (I), M has the highest vapor pressure (100, into suspension culture with hydroxyurea (HU) and 40 and 33 mmHg, at about 20°C, resp.), the slowest after preincubation were exposed to dimethylnitro­ metabolism (0.3 and 27 mg/100 ml/hr for Ma2d E, samine (DMN), ultraviolet irradiation (UV) or resp.), a high skin absorption (0.145 mg/cm/min methylmethanesulfonate (MMS). 3H-thymidine was after 15 min on the forearm, i~creasing to 0.220 added to each culture and incubation was contin­ then decreasing to 0. 185 mg/cm/min after 35 and ued. Cells were processed for SS after preparation 60 min, resp.); the lowest minimal oral lethal of whote cell homogenates, an 800xg. fraction or dose (75, 300-400 and 240 ml, resp.); and the nuclei isolated by discontinuous sucrose gradient longest latent period before symptoms(up to 72 hr, centrifugation. Specific activity of DNA obtained an hour and several hours, resp.). The Min a from each step was determined and comparisons were mixture of 15%Min diesel fuel was also absorbed made between treatments. 10 mMHU reduced 3H in­ 3 times as fast on the forearm as pure M. Treat­ corporation in control cultures by 40%-50% at all ment for M poisoning, on the whole, has been the points in the nuclear purification procedure. DMN most intensive and most prolonged. Mand less so produced dose-dependent UDS at concentrations of I have been abuse problems as substitutes for E. 0.1 - 1. 5 mM. This repair was apparent only in M could be more safely used in closed systems the 800xg. fraction and isolated nuclei. Incuba­ such as turbines, fuel cells, etc. rather than tion for 4 hrs was necessary to detect UDS at DMN in large quantities in the open environment. concentrations less than 1.0 mM. Repair of UV-in­ (Sponsored by Dr. John A. Moore. duced damage was apparent in all three steps of nuclear purification. In all cases the fold dif­ ference in 3H incorporation was greatest when com­ 315 RADIOSENSITIZATIONOF ¢X-174 REPLICATIVE FORM paring isolated nuclei of DNA damaged cells to DNABY MISONIDAZOLE(Ro-07-0582). D. W. controls. Preparation of an 800xg. fraction did Brewster and R. C. Christensen, Graduate Center not increase the sensitivity of UDS detection for for Toxicology, Univ. of Kentucky, Lexington, KY. UV compared to whole cell homogenates.

89 317 A SIMPLE, SENSITIVE AND QUANTITATIVEASSAY FOR Parameters monitored included body METALLOTHIONEIN.. D .. L. Eaton and B. Toal, Dept. ,of weight, feed consumption, some absolute Environmental Health and Inst. for Environmental and relative thyroid weights and histo­ Studies, Univ. Washington, Seattle, WA 98195. pathology. A scoring system for the (Sponsor: J.S. Woods). pathological lesions was established and Methods currently utilized to measure tissue a proposed general statistical procedure metallothoineins (MT) either rely on non-routinely for assessing reversibility was applied available substances (RIA), or lack the sensitiv­ to the results. The severity and extent ity to measure low levels of MT. Onosaka et al. of reversibility of the histopathological (Eisei Kagaki 24: 128, 1978) described a method for lesions were found to be a function of measuring MT intissues which utilized guinea pig the duration of exposure to ETU. blood hemolysate to scavenge exogenous 109cd not bound by MT in the sample. We have modified and evaluated this method for estimating MT. Aliquots of heat-denatured 15000 x g homogenate supernatant 319 NEONATALEXPOSURE TO STEROLANALOGS, Ul8, 666A and are incubated with 109cd at a final Cd concentra­ AY9944, INDUCESSPONTANEOUS EPILEPTIFORM ACTIVITY tion of 1.0 µg Cd/ml. Cd not bound to MT is re­ IN ADULTRATS. G.G. Bierkamper, C.P. Sarkar* and moved by addition of 2% hemoglobin solution fol­ R.J. Cenedella*, Dept. Environmental Health Sci., lowed by heat denaturation and centrifugation to The Johns Hopkins Univ., Baltimore, MD and *JDept. remove the precipitate and excess Cd. The super­ Biochemistry, Kirksville College of Osteopathic natant is then quantitated for Cd by gamma count­ Medicine, Kirksville, MO. (Sponsor: L. Fechter) ing. Utilizing this procedure on Sephadex G-75 Earlier work in our laboratories suggests that and DEAE A25 column chromatography fractions of interference with brain lipid metabolism during liver homogenates from Cd and Zn treated rats, Cd the critical period of rapid brain growth in the remained in the supernatant only in fractions neonatal rat can result in the development of where MT elutes. Tissue levels of MT were eval­ chronic, epileptiform activity later in life uated with this assay in Zn (200 µmol/kg), Cd (10 (Brain Res. 150:343,1978). Administration of Ul8, µmol/kg), Hg (5 µmol/kg) and non-treated rats. 666A (lOmg/kg,s.c.), an inhibitor of cholesterol MT levels in non-treated rats were low but measur­ biosynthesis, to neonatal rats induces a recurrent able (1.6 and 10.1 nmol Cd bound/ g of liver and spontaneous epileptic condition by 10 weeks of age kidney, respectively), whereas Zn, Cd and Hg in­ as assessed by flurothyl seizure threshold test­ duced liver MT was 441, 252 and 10 nmol Cd bound/ ing and electrocorticography. The objective of g tissue, respectively, The limit of sensitivity the present study was to re-examine the character­ for this assay is 0.5 nmol Cd bound/ g tissue. istics and time-course of Ul8,666A-induced seiz­ Coefficient of variation (COV) of replicate assays ures and to test similar analogs, AY9944 and 20, of a given supernatant dilution are less than .02. 25-diazacholesterol for potential epileptogenici­ However, measurement over several dilutions re­ ty. Rats treated with Ul8,666A from birth (but sults in greater variation, with a COV of about not from 30-60 days of age) exhibited recurrent, .10. The sensitivity of this assay allows quanti­ stereotypic seizure patterns consisting of 3-5s tative determination of MT in small amounts of bursts of high voltage spiking identical to our tissue from organs other than liver and kidney, such previous report. Administration of AY9944 (lOmg/ as lung, spleen, testis, placenta and fetal liver. kg,s.c.) to neonatal rats every 6 days for 7 wks resulted in spontaneous epileptiform activity in the adult similar, though more frequent than the Ul8,666A seizures. Epileptiform activity was an­ 318 ASSESSING THE REVERSIBILITY OF THYROID alyzed by computer and plotted as the power spec­ LESIONS THAT RESULT FROM FEEDING ETU. tral density for comparisons. Neither acute ad­ D.L. Arnold, M.G. Bickis, E.A. Nera, P.F. ministration of Ul8,666A, AY9944 nor chronic ex­ McGuire and I.e. Munro, Food Directorate, posure to 20,25-diazacholesterol (lOmg/kg,s.c., Health Protection Branch, Ottawa, Canada. q6d for 5 wks) caused any evidence of recurrent Ethylenethiourea (ETU) has been shown seizures. Induction of epileptiform activity by to produce various teratological, toxico­ Ul8,666A and AY9944 may be correlated to changes logical and tumorigenic effects in rats in brain lipids. It is concluded that neonatal and mice. The objective of the present exposure to neurotoxic substances may lead to per­ study was to ascertain whether thyroid manent alterations in brain function in the adult. changes elicited by the inclusion of ETU in the diet of rats were reversible. Groups of four randomly selected, weanling, Caesarian-derived, Sprague­ 320 3-METHYLCHOLANTHRENE-IRONDEXTRAN INTERACTIONS, Dawley male littermates (obtained from L, Brooks and M,A, Gallo, Dept. of Environmental Biobreeding, Ottawa) were fed diets and Community Medicine, CMDNJ-Rutgers Medical containing O (32 litters), 75 (64 School/Rutgers Univ. Joint Graduate Program in litters) or 150 (64 litters) ppm ETU for Toxicology, Piscataway, New Jersey periods of 7 to 82 weeks. Upon disconti­ nuation of the ETU diet, one littermate The combined effects of two hepatic enzyme from each of 4 control and 16 treated inducers on subcellular hepatic systems were litters were killed to assess the studied. 3-Methylcholanthrene(3MC) is a potent extent of thyroid lesions elicited by inducer of mixed function oxidase activity(MFO) ETU. The remaining three littermates and Iron Dextran(ID) has been shown to induce were then fed control diet for 9 to lysosomal systems. 42 weeks during which time the second, Sprague-Dawley rats were injected i.p. with third and fourth littermates were 3MC (20 mg/kg/day) for three consecutive days, killed to assess the thyroid lesions. followed by i.p. injection of either ID (125 mg/

90 kg/day) or saline for three consecutive days. response of murine lymphocytes to T-cell mitogens Appropriate control groups were maintained. but has very little mitogenic activity alone in­ Treatment with 3MC resulted in significant vitro. In order to examine the effects of TPA increases (p 0.05) in MFO activity in terms of in vivo , its interaction with Staph. enterotoxin production of 4-0H Biphenyl (approximately A(SEA), a recently reported in vivo T-lymphocyte 300%),production of 2-0H Biphenyl (approximately mitogen, was investigated. Back skin of Balb/c 750%) and measurement of cytochrome P450 mice was painted with 2, 6, or 10 ug of TPA in (approximately 200%). When treatment with 3MC 100 ul of acetone twice weekly for two weeks. was followed by administration of ID this Twenty-four hours after the last dose 20 ug of induction was attenuated. Administration of SEA was administered retro-orbitally. Forty­ iron, regardless of pretreatment, resulted in eight hours after the SEA injection spleen significant increases in membrane lipid lymphocyte mitogenic activity was increased in a peroxidation. Induction by iron of lysosomal dose-related manner, a 60% increase occurring at enzymes in the post-mitochondrial supernatant the highest (10 ug) dose. However, unlike its and post-lysosomal supernatant was observed to effects on lymphocytes in vitro, skin painting be greater in the presence of 3MC. with TPA stimulated mitogenesis.even when no Microscopic examination of stained liver other mitogen was present. Mitogenic activity in preparations suggested that iron exacerbates mice treated with TPA, but not with SEA, was previously reported sinusoidal distention approximately 50% of the levels induced in ace­ induced by 3MC. tone-treated mice by injection of SEA. One application of 6 ug of TPA was sufficient to induce a two-fold enhancement of mitogenesis 321 CROSS-LINKEDENVELOPE FORMATION IN CULTUREDHUMAN within 24 hours. Peak stimulation was reached KERATINOCYTES:MODULATION BY HYDROCORTISONEAND between 48 and 72 hours after dosing. When mice VITAMIN A. P.R. Cline, J.G. Rheinwald & R.H. received two weeks of TPA treatments, the maximum Rice, Lab. of Toxicol., Harvard Sch. Pub. Hlth. stimulation reached 48 hours after the last ap­ and Sidney Farber Cancer Inst., Boston, MA. plication was more than twice the maximum level Sponsor: J.L. Whittenberger achieved after one TPA application. An 8-fold It has been found that 2,3,7,8-tetrachlorodi­ enhancement of mitogenic activity was observed benzo-p-dioxin (TCDD) inhibits growth in culture at that time. of certain human keratinocyte lines derived from Supported by NIEHS Toxicology Training Grant squamous cell carcinomas and that this inhibition ES-07073 and NCI grant CA-24022. is antagonized by hydrocortisone. In the present work, we have examined modulation by hydrocorti­ sone and vitamin A of cross-linked envelope forma­ tion, a distinctive marker of keratinocyte differ­ 323 EFFECTS OF CHLORINATEDPHENOLS ON IMMUNITYIN entiation. Cells of the carcinoma line SCC 13 were RATS. J.H. Exon and L.D. Koller, Veterinary grown on a lethally irradiated 3T3 feeder layer in Science, University of Idaho, Moscow, ID. Dulbecco-Vogt Eagle's medium supplemented with 5% Female Sprague-Dawiey rats were exposed to 0, 5, fetal calf serum and examined soon after reaching 50 or 500 ppm 2-chlorophenol (2-CP) or penta­ confluence for competence to form ionophore­ chlorophenol (PCP) from weaning to 3 weeks post­ inducible envelopes. Under these conditions, 10% parturition after breeding at 90 days of age. or fewer of the cells were competent to form enve­ Progeny were weaned at 3 weeks of age and con­ lopes, whereas 40% to 60% of the cells grown with tinued on chlorophenol treatment for 10 weeks at added hydrocortisone (0.4 µg/ml) were competent. which time major immune functions were tested. When the cells were grown and passaged in medium Humoral immunity was measured by an indirect supplemented with charcoal extracted fetal calf ELISA, cell-mediated immunity was monitored by serum, however, 60% of the cells were competent delayed-type hypersensitivity (DTH) to oxazolone, to form envelopes even without added hydrocorti­ and macrophage function was tested by phagocyto­ sone; when grown in the presence of treated serum sis of sheep red blood cells. Rats treated with and retinyl acetate (0.1 µg/ml), only 10% of the cyclophosphamide were included as a positive cells were competent, but with addition of hydro­ immunosuppressed control. PCP-treated rats had cortisone as well, 60% were competent. These re­ significantly decreased antibody titers and DTH sults are interpreted to mean that low concentra­ response and increased induced peritoneal macro­ tions of retinyl acetate in untreated fetal calf phage numbers which displayed hyperphagocytic serum suppress cellular competence to form cross­ activity. Immune responses in rats treated with linked envelopes, and that hydrocortisone is 2-CP were not significantly different from · capable of antagonizing this effect. Since TCDD controls. The data indicate that l) the immune ~as not inhibitory to the growth of cells compe­ system may be a sensitive target for PCP toxicity tent to form envelopes, this work emphasizes that but not for 2-CP, 2) closely related chloro­ target cell physiology is a critical factor in phenolic chemical isomers may exert different response to toxic agents and must be considered in toxic effects on the immune system, and 3) PCP exploration of TCDD toxic mechanisms. can exert depressive effects on some major immune parameters while enhancing others.

322 STIMULATIONOF MURINESPLENIC LYMPHOCYTESAFTER SKIN PAINTING WITH A TUMORPROMOTER. 324 EFFECTSOF MONOETHYLHEXYLPHTHALATE (MEHP) ON R.A. Schoof and c.s. Baxter, Dept. of Environ~. MOUSEANTIBODY-PRODUCING CELLSIN VITRO. Daniel mental Health, Univ. of Cincinnati Medical Center Wierda. Dept. Pharmacology& Toxicology, West Cincinnati, OH. Sponsor: P.B. Hammond. Virginia University Medical Center, Morgantown, The tumor promoter 12-o-tetradecanoyl pho~­ West Virginia 26506. Sponsor: John A. Thomas. bol-13-acetate (TPA) synergistically enhances the MEHPis a metabolite of diethylhexyl phthalate,

91 a widely used plasticizer. Although MEHPcan be 326 IMMUNOTOXICITYOF MERCURICCHLORIDE IN B6C3F detected in blood stored in plastic bags, little MICE. M. P. Dieter, M. I. Luster, J. H. Dean,1 G. is knownabout its immunotoxicity. This study A. Boorman, and C. W. Jameson. NIEHS/NTP, Box examined the effect of MEHPon the development 12233, Research Triangle Park, NC. Sponsor:~ of mouse (B6C3Fl) spleen and bone marrow anti­ H. Mennear. body-producing cells (APC). The development of A panel of humoral and cell-mediated immune APCwas induced by the addition of mitogens function tests were utilized to determine the (dextran sulfate and lipopolysaccharide) or by immunotoxicity of mercuric chloride, since prior sheep red blood cells to marrow or spleen cul­ studies have not completely assessed the nature of tures, respectively. After 4-5 days incubation the immune defects. Six week old female B6C3F in the presence of 10-6-10-3M concentrations of mice were provided with drinking water with O, 1 3, MEHP,the number of APCgenerated in each cul­ 15 and 75 ppm mercury (as mercuric chloride) for 7 ture was assessed with standard hemolysin-in-gel weeks. At termination there were 4 and 22 ppm assays. No significant decreases in spleen or mercury in spleen from the two highest dose groups, marrow APCfrequencies were observed with 10-6 and no mercury accumulation in bone marrow or thy­ and 1Q-5Mconcentrations of MEHPwhen compared mus. The 75 ppm mercury dose caused general immu­ with controls. However, with 10-4Mconcentra­ nosuppression but also resulted in non-specific tions of MEHP,the APCfrequency was reduced to toxicity, as indicated by depressed body~ thymus, 60%of control in spleen cultures and 76%of and liver weights plus hematological changes. How­ control in marrow cultures. Total inhibition of ever, several cell-mediated immune responses were APCformation was seen in all cultures incubated also depressed in the 15 ppm dose group, where no with 10-3Mconcentrations of MEHP. These re­ overt toxic responses occurred. Lymphoproliferative sults demonstrate that MEHPis immunotoxic in responses to T-cell mitogens were <50% of controls vitro only at relatively high concentrationsand and decreases in the mixed leukocyte response to suggest that similar concentrations of MEHP allogeneic leukocytes were observed. Host resist­ would need to be attained in vivo before any re­ ance assays also suggested these were cell-mediated duction in immunocompetencewouldoccur. Sup­ immune defects in mercury-treated mice. Dose ported by W.V.U.Medical Corporation and N.I.H. dependent depression of pentose shunt, glycolytic, Biomedical Research Grant #5-S07-RR05433-18. and Krebs cycle metabolism in bone marrow, thymus, and spleen were seen and believed to have been associated with some of the cellular immune defects. Neither lymphoproliferative responses to B-cell mitogens nor immunoglobulin concentrations were 325 METHODSFOR INDUCINGAND ELICITING DELAYEDCON­ altered in mercury-treated mice. The only B-cell TACT HYPERSENSITIVITYBY VAGINALOR SKIN EXPOSURES defect we observed was a decrease in the spleen IN FEMALEGUINEA PIGS. Newmann, E.A., Buehler, E. PFC response to the T-dependent antigen, SRBC, at V., The Procter & Gamble Company, Miami Valley the 75 ppm dose level, but there was none to the Laboratories, Cincinnati, OH 45247 T-independent, B-cell mitogen, LPS. The primary immunotoxicity of mercuric chloride appears to be A technique is described that permits vaginal­ exerted on cell-mediated immunity since several only exposure to test materials without incidental defects occurred at a lower dose than that of skin (perianal) contamination. We have used it to humoral-mediated immunosuppression. determine the efficacy of the vaginal route for the induction and elicitation of delayed hypersen­ sitivity. 327 ALTERATIONSIN THE NATURAL KILLER (NK) CELL ACTIVITYOF MICEFOLLOWING EXPOSURE TO NICKEL, 2,4-dinitrochlorobenzene(DNCB) or 2,4-dinitroflor­ CADMIUMORMANGANESE. R.J. Smialowicz, R.R. obenzene(DNFB) will induce and elicit delayed con­ Rogers, R.J. Garner and M.M.Riddle. Health tact hypersensitivity in female guinea pigs after Effects Research Laboratory, Research Triangle vaginal or skin exposures. Female guinea pigs Park, NC 27711. (Sponsored by N. Chernoff). sensitized by skin exposures to DNCB cross-reacted in the vagina and on the skin to DNFB. Moreover, Natural killer (NK)cells have been impli­ animals sensitized to DNFB cross-reacted in the cated as being an important effector mechanismin vagina and on the skin to 2,4-dinitrobenzensulfon­ tumor cell cytolysis in vitro. NKcells display ic acid, sodium salt (DNBS0 ). a spontaneous cytotoxic reactivity against synge­ 3 neic and allogeneic tumor cells in the absence Tissue changes produced in the vagina by DNFB in­ of immunological priming and as such have been dicated that innnune responses of the delayed type suggested to play a significant role as a first were present and could be discriminated from the line of defense against newly arising malignant primary irritation present. Vaginal challenges cells. We present evidence that several metals with DNBS0 produced a more dramatic delayed con~ of environmental significance alter NKcell 3 tact hypersensitivity response with little or no activity in mice. ~f cell activity was determined primary irritation. Microscopically, the major using the in vitro Cr release cytotoxicity differences in sensitized animals challenged by assay and an in vivo tumor cell clearance assay. vaginal exposure were submucosal perivascular lym­ A single intramuscular injection of NiC12 (18.3 phocytic cuffing and an overall increase in the mg/kg) caused a significant suppression Tn NK inflannnatory response, activity which returned to normal within a few days. This suppression was not related to the These data suggest that guinea pigs produce delay­ generation of suppressor cells. A single injec­ ed contact hypersensitivity by vaginal exposure to tion of CdCl (6.25 mg/kg) caused a similar compounds that have been reported to produce simi­ suppression ~n NKactivity. On the other hand, lar reactions in the skin of both guinea pigs and a single intramuscular injection of MnCl?.(80 mg/ humans. kg) caused a significant enhancementof AK

92 activity which persisted for several days. The halogenated hydrocarbon that binds to the TCDD injection of A/J mice, which have low NKcell binding protein, also enhanced carrageenan-induced activity, with MnCl?caused a significant increase paw edema, but 2,4,5,2' ,4',5'-hexabromobiphenyl in NKactivity as measured by both the in vitro (90 mg/kg) which does not bind had no proinflamma­ cytotoxicity assay and the in vivo tumorc~ tory effect. (Both compoundssubstantially in­ clearance assay. These results indicate that duced liver microsomal enzymes in these rats.) assessment of NKcell activity using these The results suggest that the effect of TCDDon sensitive assays can be of significant utility carrageenan-induced paw edema may be mediated by in assessing immunotoxiceffects of environmental the TCDDbinding protein. (Supported by NIH pollutants. Whether alterations in NKcell grant ES01332.) activity play a significant role in the develop­ ment of tumors following exposure to certain toxic agents remains to be determined. 330 THEEFFECT OF 12-0-TETRADECANOYLPHORBOL-13-ACE­ TATE (TPA) ON THE ANTIBODYPRODUCING CAPABILI­ TIES OF IN VITRO SENSITIZEDSPLEEN CELLS. 328 THE ELISA: APPROACHTO ASSESS HUMORALIMMUNITY Shopp, G., White;K., and Munson, A. Dept. of FOR IMMUNOTOXICOLOGY.L.D. Koller and J.H. Exon, Pharmacology, Medical College of Virginia, Veterinary Medicine, University of Idaho, Moscow, Richmond, VA. ID. TPA affects several elements of the immunesys­ Irnmunotoxicology is a relatively new field of tem and the study of the mechanisms of these ef­ investigation that is becoming recognized and fects may offer insight concerning its mechanism used by toxicologists involved in drug and chemi­ of action as a tumor promotor. This study exa­ cal evaluations. The purpose of this study was mines the effect of TPAon the in vitro immuni­ to develop a highly sensitive, quantitative zation of BDFl mouse spleen cells to sheep red immune assay procedure that could assess humoral blood cells, as measured by the number of IgM immune responses in rats and mice used in chemi­ cal testing procedures. The enzyme-linked antibody forming cells (AFC). The ED50 that immunosorbent assay (ELISA) proved to be not only suppressed AFC was between 7.7 x 10-lOM and highly sensitive and quantitative, but also 7.7 x 10-9M; 7.7 x 10-BM resulted in com­ simple to perform, reliable, economically fea­ plete inhibition. TPAdid not significantly af­ sible, extremely accurate, required few test fect cell viability at these concentrations animals and could be automated. The antigens (trypan blue exclusion test). A time course tested were bovine sera albumin and ovalbumin study showed that the suppression seen was not and the chemicals evaluated to validate the due to a shift of the peak response day. When system were lead, polychlorinated biphenyl, TPA (7.7 x 10-BM) was added on consecutive cyclophosphamide and dexamethasone, all of which days (day O = immunization; day 5 = assay for were immunosuppressive at the dosages tested. AFC), it i nhi bi ted AFCwhen added on days O and Practical application of the procedure appropri­ 1. Addition on day 2 resulted in 50 O/o inhi­ ate to immunotoxicology will be discussed. bition, whereas days 3 and 4 resulted in a 2.2 and 1.3 fold increase, respectively. These re­ sults suggest that TPA may be affecting the macrophage early in the immuneresponse, consi­ 329 A PROINFLAMMATORYEFFECT OF TCDDON CARRAGEENAN­ dering the macrophage is important in the i ni­ INDUCEDINFLAMMATION IN THE RAT PAW. H. M. ti al 48 hours. However, because of the enhanced Theobald, R. W. Moore and R. E. Peterson, School response on day 3, other cell types such as the of Pharmacy, University of Wisconsin, Madison, WI. suppressor T-cell may also be affected. (Sup­ ported by NIEHSgrant USPHIT32ES07087.) As previously reported (The Pharmacologist 22: 197, 1980) a single oral dose of 2,3,7,8-tetra­ chlorodibenzo-p-dioxin (TCDD)enhanced carra­ geenan-induced paw edema in male rats assayed 5 331 FAT QUALITYAND QUANTITY, DIMETHYLHYDRAZINE(DMH) days after treatment. Wehave now further char­ AND IMMUNEFUNCTION. M. Locniskar, K.M. Nauss acterized this response. The increase in paw and P.M. Newberne, Dept. of Nutr. and Fd. Sci., edema volume was detectable when assayed 2 days M.I.T. Cambridge, MA after 10 µg TCDD/kg,maximal after 5 days and still present at 29 days. Five days after TCDD Male Sprague-Dawley rats were fed either a (10 µg/kg), edema volumes in TCDDand vehicle low (I) mixed fat (5%) diet or a high fat diet treated rats were similar during the first 60 min (24%). The high fat diets contained either sa­ after hind paw injection of carrageenan, but turated (II), unsaturated (III) or partially sa­ after 90 min the edema volumes were greater in turated (IV) fats. Half of each group received TCDDtreated rats and the difference was maximal DMH15 mg/kg 5X. The toxic effect of DMH, ex­ between 3 and 4.5 hr. Thus, the effect of TCDD pressed as a depression in the splenic lymphocyte on paw edema occurs during the delayed phase of transformation response to Concanavalin A (Con A) the inflammatory response to carrageenan. Also was observed only in the low fat group. The re­ at 5 days, the effect of TCDDon carrageenan­ sponse of DMHtreated rats was 4,452 ± 1.025* as induced paw edemawas dose related from 0.1 µg/kg compared with the untreated rats 17,813 ± 2,824* (no effect) to 100 µg/kg (near maximal effect) (P <.01). In comparing the three high fat diets with an approximate ED50of 6 µg/kg. Thus, the (II, III, IV) to the low fat (I) diet, only diet enhancement of carrageenan paw edema is a new III, the unsaturated fat, had a significant dose related effect of TCDDthat is as sensitive (P <.05) effect in depressing the transformation as the well knowneffect on thymus weight. response to Con A, Phytohernmagglutinin and Poke­ 3,4,5,3' ,4' ,5'-Hexabromobiphenyl (5 mg/kg), another weed Mitogen. These results indicate that alter-

93 ation in the quality and quantity of dietary fat mice, were suppressed (75-80%control) by MALbut has selective and varying effects on the local not by DDVP. Brain cholinesterase (CHE)activi­ and systemic immune responses. These factors ties in the same mice were 57%and 80% control, must be considered in immunotoxicology studies. respectively. The IgM suppression by PAR& MAL but not DDVPmay be a function of the duration and *CPM stimulated~ SEM intensity of OP-induced cholinergic crisis during Supported in part by NIH Grant CA26917. the post-immunization period. Mice were dosed as above or with PAR(16mg/kg) and killed at selected times. Brain CHEwas decreased to 20-25%control within 3hr by each OP. With DDVP,CHE returned to 332 THEEFFECTS OF NICKELCHLORIDE ON CELLULAR AND 70% in 5hr after dosing; yet with both PAR& MAL, HUMORALIMMUNITY. R.J. Smialowicz, R.R. Rogers, CHEremained at 15-25%control for more than 12hr. M.M.Riddle, G.A. Stott. Health Effects The cholinomimetic, arecoline (65mg/kg) caused a Research Laboratory, RTP, NC 27711 (Sponsored short-lived cholinergic crisis but no IgM suppres­ by N Chernoff). sion. Whencholinergic crisis was extended over Nickel has been implicated as a potential 4hr after sustained-release arecoline, IgM PFCwas toxicant in a number of clinically significant suppressed (59%control). Results suggest that pathological findings in man. There is also IgM PFC suppression by OP is secondary to choli­ evidence for significant alterations in host nergic stress, rather than a result of direct OP defense mechanisms of experimental animals action on the immunesystem. (Supported by NIEHS treated with nickel. In the present study the grants 05205 and 02524) effects of nickel chloride on the cellular and humoral immuneresponse of mice was examined. A single intramuscular injection of nickel chloride (18.3 mg/kg) caused a significant 334 EFFECTSOF PARATHIONONTHE IgM AND IgG RESPONSES involution of the thymus within two days follow­ TO SHEEPRED CELLS (SRC) IN TWOMOUSE STRAINS. ing treatment. Thymic involution was not corre­ G.P.Casale, R.A.DiCapuaand S.D.Cohen. Sch. of lated with an increase in serum corticosterone Pharmacy, Univ. Conn., Storrs, CT 06268 levels. Significant reductions in the in vitro Organophosphates have been reported to suppress mitogen stimulated response of lymphocytes from the primary IgM response. The present study was nickel chloride treated mice (single injection undertaken to determine the effects of parathion of 36.6 mg/kg) were observed for the T-cell on both primary IgM and IgG responses, as deter­ mitogens phytohema9glutinin (PHA)and con­ mined by modified Jerne plaque assay, in inbred canavalin A (Con A) but not the B-cell mitogen (C57Bl/6) and outbred (CD-1) male mice. For the lipopolysaccharide (LPS). Significant suppres­ IgM studies, mice were immunizedwith SRC, dosed sion of the primary antibody response to the 2 days later with parathion (16mg/kg,po) and T-cell dependent antigen sheep red blood cells killed on day 4 post-immunization. Brain and was observed following a single injection of plasma cholinesterase activities were about 50%of 18.3 mg/kg NiCl . In all cases the inmuno­ control in both strains. In 3 separate experi­ suppressive eff~cts of NiCl were found to be ments with Bl/6 mice, splenic IgMplaque forming transient with responses returning to normal cells(PFC), expressed either per 106 nucleated within a few days. No alteration in the cells or per spleen, were suppressed (10-30%of response of mice immunizedwith the T-cell control). With CD-1mice, the PFCresponse was independent antigen polyvinylpyrrolidone was less consistently and less severely suppressed observed following treatment with nickel. (30-75%control). In spite of the high dose of Furthermore, the phagocytic capacity of resident parathion used (40%mortality in less than 2 days) peritoneal macrophages from nickel treated the results suggest a selective effect on splenic (18.3 mg/kg) mice was not significantly differ­ IgM PFC. ent from saline-injected mice. The results For the IgG studies, PFCwere enumerated 8 or 10 suggest that NiCl? predominantly affects T-cell days post-immunization. Parathion (16mg/kg,po) mediated inmune responses. was administered to Bl/6 mice either 2 days post SRCor 2 days prior to splenectomy. Additional mice of both strains were given parathion (5mg/kg) on alternate days after immunization for a total 333 ORGANOPHOSPHATE(OP) POISONING AND SUPPRESSION Of of 4 doses. IgG PFC in treated and control mice THEPRIMARY IgM RESPONSETO SHEEPRED CELLS (SRC) were not significantly different. The lack of an IN C57Bl/6 MICE.G.P.Casale, S.D.Cohen and R. A. effect on IgG PFC further supports the hypothesis Dicapua. Sch. Pharmacy, Univ.Conn.,Storrs,CT 06268 of a selective effect of parathion on IgMPFC. The basis for this demonstrated selectivity is not In a previous study we demonstrated that parathion known. (Supported by NIEHSgrants 05205 and (PAR), at doses which caused 40% lethality, selec­ 02524). tively suppressed the IgM response to SRCin sur­ viving mice. This study was undertaken to deter­ mine if other OP insecticides produced a similar effect at doses which produce overt signs of chol­ 335 IMMUNOSUPPRESSANTANDANTIINFLAMMATORY EFFECTS OF inergic poisoning. Male mice were immunizedwith DIETHYLSTILBESTROL(DES) • Holsapple, M.P., Mora­ SRCand dosed, po, 2 days later with malathion han, P., Bick, P., and Munson, A.E. Medical Col­ (MAL),720mg/kg, or dichlorvos (DDVP),120mg/kg. lege of Virginia, Richmond, VA 23298 They were killed on day 4 post-immunization for determination of the primary IgM response by a Female adult B3C6Flmice were administered DES modified Jerne plaque assay. DDVPkilled 17%and (s.c.) in corn oil at dosages of 0.2, 1.0, 2.0, MALkilled 50%of the mice within 2 days. IgM and 4.0 mg/kg body weight per day for 14 consecu­ plaque forming cells, from spleens of surviving tive days. Exposure to DESproduced a dose-rela-

94 ted decrease in the delayed hypersensitivity res­ sults of their corresponding oxygen analogues. ponse (OHR)and the proliferation of the popliteal These oxons are produced in vivo by oxidative de­ lymph nodes following challenge with sheep ery­ sulfuration of the parentcompounds. Therefore, throcytes, and in the acute inflammatory response for in vitro studies, one must have the oxon to carrageenan. The effects of the OHRto chal­ analogues:--Treatment of the sulfur analogues lenge with keyhole limpet hemocyanin were less with bromine provides a simple method for con­ dramatic following exposure to DESby two sche­ version to the corresponding oxons. Our results dules. In other groups of mice, body weights, or­ indicated that treatment of leptophos and EPN, as gan weights, and differential cell counts were de­ phosphonothioates, and ethyl-parathion and termined. DES-treated mice gained more weight methyl-parathion, as phosphorothioates, with an than the controls. There was a dose-dependent in­ aqueous saturated bromine solution can convert crease in liver weight, a slight, non-dose-related these compoundsinto their corresponding oxons increase in spleen weight, and a remarkable thymic in situ. These reactions are rapid with yields involution with a significant reduction noted at approaching 100%. The resultant oxon solutions 0.2 mg/kg DES. Exposure to only 4.0 mg/kg DES may be used for biochemical studies after mini­ produced significant alterations in the differen­ mal further treatment. In vitro inhibition of tial counts as the lymphocytes were increased and AChEor NTE(indicated byI~lues) was found the polymorphonuclear leukocytes were decreased. to be identical when standa~9 oxons were compared In parallel studies, mice were treated with DES to those obtained using this method. Further for 14 days as before, but were allowed to recover verification of the oxon products of this re­ for 30 days prior to testing. In these animals, action was provided by HPLCand GLCtechniques. there were no effects on either the OHRto sheep erythrocytes or on the acute inflammatory response to carrageenan, and all organ weight changes were 338 HEINZ BODYPRODUCTION AND HEMATOLOl.lCAL CHANGES reversed. These results indicate that a suppres­ IN THE HEN AFTERADMINISTRATION OF A SINGLE ORAL sion of cell-mediated immunity with a correspond­ DOSE OF B-BUTYLMERCAPTAN AND B-BUTYL DISULFIDE. ing involution of the thymus occurs following sub­ K.M. Abdo, P.R. Timmons and M.B. Abeu-Donia, chronic exposure to DESand that both effects are Department of Pharmacology, Duke University Medi­ reversed within 30 days. The results with the cal Center, Durham, NC 27710 and National Toxico­ carrageenan test suggest that at least part of the logy Program, NIEHS, Research Triangle Park, NC immunosuppressant activity by DESis mediated by 27709, non-specific anti-inflammatory effects. (Suppor­ n-Butyl mercaptan (nBH) is a breakdown product of ted by NIEHScontract ES-1-5001.) S,S,S-tri-n-butyl phosphorotrithioate (DEF) and ]:,]:,]:-tri-~-butyl Phosphorotrithioite (merphos) in hens and in the environment. n-Butyl disulf:lde is an oxidati0n product of nBM. A single oral 336 IN VITRO INHIBITION OF HUMAN LYMPHOCYTETRANS­ dose of each compound (nBM o"r n-butyl disulfide) FORMATIONBY NABILONE. G. A. W. Waterhouse, was administered in gelatin capsules to groups of L. D. Brandt, S. B. Leapman, and R. B. Forney, five 12-month old laying hens. A third group of Department of Pharmacology and Toxicology and five hens was given gelatin capsules. One day Department of Surgery, Indiana University School after administration, the dosed hens exhibited of Medicine, Indianapolis, IN. weakness which progressed to unsteadiness and Lymphocytes obtained from normal, healthy, drug­ inability to stand by the third day. In addition, free individuals were incubated alone or in the their combs turned pale 18-24 hours after dosing, presence of 1.0 µg/ml to 100 µg/ml phytohem­ and then became dark at 48 hours. After these agglutinin (PHA), lxlo-7 M to lxlo-3 M Nabilone*, initial reactions, the condition of the hens im­ or a combination of the two compounds. The in­ proved. Heinz bodies and extensive erythrocyte corporation of 3H-thymidine was used as a measure deformation and lysis were observed in blood of lymphocyte responsiveness. lxlo-3 M Nabilone smears taken from hens 48 hours after treatment. completely abolished the response to all concen­ Hemoglobin concentration, hematocrit, erythro­ trations of PHA, while lxio-4 M Nabilone inhibi­ cytes and glucose-6-phosphate dehydrogenase acti­ ted 90% of the stimulation induced by 10 µg/ml to vity were significantly lower than in the control 100 µg/ml PHA. Stimulation by concentrations of hens, while methemoglobin was significantly PHA lower than 10 µg/ml were completely inhibited higher. As the clinical condition of these hens by lxlo-4 M Nabilone. At concentrations lower improved, these hematologic changes became normal. than lxlo- 4 M, Nabilone had little or no effect _g_BMcaused an initial increase in plasma butyryl­ on the PHA induced responses. Nabilone by itself cholinesterase activity which returned to normal had no effect on lymphocytes, while PHA alone il­ by the end of the 28-day experiment. Also, brain lustrated a dose dependent increase in 3H-thymi­ acetylcholinesterase activity was not different dine incorporation. from that of the control at termination. (Supported in part by NIEHS Grant No. ES02717). *A synthetic cannabinoid, courtesy of Eli Lilly and Company, 'Indianapolis, IN

339 THEEFFECTS OF DELTAMETHRIN, · A SYNTHETIC PYRE­ THROIDINSECTICIDE ON LOCOMOTOR ACTIVITY IN RATS. 337 A QUANTITATIVEIN SITU OXIDATION METHOD FOR K. M. Crofton and L. W. Reiter, Curriculum in ORGANOPHOSPHONO:-ANDPHOSPHOROTHIOATES. S. A. Toxicology, University of North Carolina, Chapel Soliman and Robert D. Zehr, Neurotoxicology Divi­ Hill, NCand Neurotoxicology Division, Health sion, US EPA, Research Triangle Park, NC 27711. Effects Research Laboratory, US EPA, Research Triangle Park, NC 27711. Most of the well knownbiological effects of Deltamethrin is a synthetic pyrethroid insecti­ organophosphono-and phosphorothioates are re- cide that is currently experiencing a vast in-

95 crease in worldwide usage. A report is made here This IgE antibody-mediated hypersensitivity may of the effects of deltamethrin exposure on loco­ provide the basis for hypersensitivity occasional­ motor activity. Three experiments were per­ ly observed in exposed humans. (Supported by EPA formed to determine: (1) the dose-effect function Cooperative Agreement CR-807295). for changes in locomotor activity, (2) the time course of effects over a 24 hr period, and (3) the effects of 5 daily exposures. Long Evans hooded rats approximately 70 days old received 341 DISPOSITION AND TOXICITY OF PARAQUATDELIVERED deltamethrin in 0.2 ml/kg corn oil. For all SUBCUTANEOUSLYBY INJECTION AND OSMOTICPUMP IN experiments, activity testing was conducted for THE RAT. M.S. Dey, R.G. Breeze,* R.A. Dey, R.I. 1 hr in a figure-eight maze (Reiter, L. W. et Krieger, L.J. Naser, and B.E. Renzi, Dept. al., Environ. Hlth. Perspect. 12:119-123, 1975). Veterinary Science, Univ. of Idaho, Moscow, ID In the dose-effect study, animals received and *College of Veterinary Medicine, Washington either 0, 2, 4 or 8 mg/kg p.o. 2 hr before State Univ., Pullman, WA. testing. In the time course study 4 mg/kg p.o. Quantitative measurements of blood and tissue was administered either 1, 2, 4, 8 or 24 hr paraquat (PQ) burdens are necessary to establish before testing. In the repeated dosing and the in vivo importance of active PQ accumulation repeated testing study, animals received 4 mg/kg in the production of lung disease. Subcutaneous · p.o. either 2 hr before or 2 hr after testing for weekly (4x) administration of PQ (72 µmoles/kg) five days. Significant decreases in activity effectively and reproducibly produce lung disease were found for the 2, 4 and 8 mg/kg groups in in Sprague-Dawley rats. After a single sc dose, the dose response study. The ED50was 3.59 mg/kg peak blood levels (ca. 55 ~oles/ml) were 1 and the estimated behavioral toxicity index obtained 20-30 min after CH -PQ. After 7 d the (LD50/ED50)was 14.5. For the time course study, blood level was ca. ~.04 nmoles/ml3 and tissues· 1 significant decreases in activity were found at ranked as.follows ( CH •PQ/g): carcass, lung, 1 and 2 hr after dosing, with recovery by 4 hr injection site, brain, small3 intestine, large after dosing. The repeated dosing/testing study intestif~ and feces, heart, liver, kidney, adre- revealed no cummulative effect of 4 mg/kg delta­ nals. CH -PQ in carcass accounted for >90% methrin administered for 5 days. These results (ca. 4.0 nmoles/g)3 of the total body burden. indicate that deltamethrin is an acute behav­ Rapid PQ clearance complicates the design of ioral toxicant with rapid onset and recovery studies of active lung ac~ulation. To achieve of effects on locomotor activity. a stable blood level Alze~osmotic pumps were implanted in the left thigh. Blood drawn using an indwelling jugular cannula contained 0.6-1.6 nmoles PQ/ml at steady state. Highest tissue 340 ALLERGICHYPERSENSITIVITY TO THE INSECTICIDE burdens after 19 d were seen in intestines, kid­ MALATHIONIN MICE. J. R. Cushman and J.C. neys, and lung (6-11 nmoles). Carcass contained Street. Toxicology Program, Utah State Univ., ca. 6.0 nmoles/g which was 54-65% of the total Logan, UT. body burden. Anorexia, weight loss, and pilo­ erection were observed after 18 d. At virtually The widely used insecticide malathion ([(di­ the same dosage, rats given PQ using the osmotic methoxyphosphinothioyl)thio]butanedioic acid, di­ pumps had more severe lung damage than rats ethyl ester) has been reported to cause allergic injected sc. PQ is rapidly absorbed using responses iri some exposed people. The following osmotic pumps which permit establishment of a studies were performed to determine whether lung disease model without compromising the malathion would cause cell-mediated or antibody­ function of other organs, particularly the kid­ mediated hypersensitivity in female BALB/c mice. ney. (Supported in part by NIH Biomedical Delayed-type hypersensitivity was determined by Research Development Grants 1 SOS RR09073-01A2 change in ear thickness, 5-[125I]iodo-2'deoxy­ and 2 S07 RR07170.) uridine incorporation in the ear, and histology of the ear following ear challenge of mice previously treated on the abdomen. Half of the mice were pretreated with cyclophosphamide to 342 SUBCHRONICTOXICITY OF ETHYLENETHIOUREAIN MICE enhance any response. No differences were AND RATS. P. Kurtz, A. Peters, D. Donofrio and observed between malathion-treated and control R. Chhabra, Battelle Columbus Laboratories, Col­ mice. umbus OH 43201 and National Institute of Environ­ To elicit malathion-specific antibodies of the mental Health Sciences, Research Triangle Park, NC IgE class, a conjugate of the anhydride of the 27709. metabolite [(dimethoxyphosphinothioyl)thio]­ butanedioic acid with keyhole limpet hemocyanin Ethylenethiourea (ETU) is a decomposition product was administered intraperitoneally with aluminum of ethylenebisdithiocarbamate agricultural fungi­ hydroxide as adjuvant. The degree of conjugation cides and has been previously reported to produce was 8.7 haptens per 100,000 daltons of protein. thyroid carcinoma in the rat. This study examined Sera were collected following three sequential possible differences between rat and mouse in sus­ sensitizations and tested for specific IgE using ceptibility to the effects of subchronic exposure the passive cutaneous anaphylaxis (PCA) test in to ETU in dosed feed, In a preliminary study Sprague-Dawley rats. The anhydride of the mala­ groups of 10 rats and 10 mice were continuously thion metabolite coupled to bovine serum albumin exposed to ETU concentrations between 500 and 8000 was used as the challenge antigen (degree of con­ ppm for 14 days. Exposure to the three highest jugation was 12. 4 haptens per molecule of protein). concentrations produced total (8000 and 4000 ppm) Specific IgE was produced following the second and or partial (2000 ppm) group mortality in rats but third sensitizations in the mice receiving 10 and no mortality in mice. The greater toxicity of ETU 100 ug of conjugate. in rats is consistent with reports that this com-

96 pound has a longer half-life in rats than in mice. adenomatous and carcinomatous mouse liver lesions In the 13 week subchronic study, 10 rats per sex grafted into syngeneic hosts and to determine the were exposed to ETU concentrations ranging from effect of treatment type, donor age and meta­ 60 to 750 ppm and ten mice per sex were exposed static potential on this parameter. to doses ranging from 125 to 2000 ppm. There Lesions were obtained from male B6/C3F1 mice fed were no compound-related deaths in rats or mice. chow or chow mixed with a chlorinated hydrocarbon, Growth depression was evident in both male and pesticide (DCB). Animals were sacrificed at 18 or female rats exposed to 750 ppm and in male rats 24 months of age. Hosts were .. sacri.J;iced at 8 . exposed to 500 ppm. Weight gain was mildly de­ months, or when grafts reached 2cm in diameter. pressed in mice exposed to 2000 ppm. Thyroid Lesions diagnosed as non-malignant (hyperplastic follicular hyperplasia was seen in all the rats or benign) were more likely to grow after trans­ of the 750 and 500 ppm groups and in 16/20 rats plantation if obtained after 24 months, rather of the 250 ppm group. Thyroid adenomas occurred than after 18 months (0.025

97 of this enzyme activity by the ductal ~lements planimetry must be performed in a darkened room, of cholangiofibrosis remains to be elucidated. and the operator cannot have access to the (Supported by the VA and NIH grant CA22140.) CRT/keyboard terminal, the digitizer surface has been divided into regions of distinct command and alphanumeric data entry through which the operator controls the data flow. Processing errors are 346 IRON EXCLUSIONFROM AFLATOXIN Bl-INDUCED signalled to the operator by simple audio patterns HEPATOCEU.ULARCARCINOMA IN RATS. M.A. Gallo, generated by the computer logic. Algorithms for L. Brooks, J. McCoy, I. Clark, Dept. Environ­ data storage, and retrieval by experiment, treat­ mental and Community Medicine, Dept of Pathology, ment, animal, and slide numbers in random access Dept of Surgery, CMDNJ-Rutgers Medical School/ files for statistical manipulations have also been Rutgers Univ. Joint Graduate Program in devised. Toxicology, Piscataway, N.J. Previous studies have shown that partial 348 hepatectomy followed by an hepatic carcinogen ENHANCEMENTOF AFLATOXIN B1 INDUCEDELEVATION OF induces foci which exclude iron. The purpose of BLOODa-FETOPROTEIN AND EMERGENCE OF y-GLUTAMYL this experiment was to test the hypothesis that TRANSPEPTIDASEHEPATIC FOCI BY DIETARY VEGE­ altered foci induced by Aflatoxin Bl (AFBl) are TABLES. J.N. Boyd, N. Misslbeck, T.C. Campbell, resistant to iron accumulation without any other and G.S. Stoewsand,* Div. of Nutritional manipulation, i.e.,partial hepatectomy or Sciences and *Dept. of Food Science, Cornell altered diet. University, Ithaca and *Geneva, N.Y. Male weanling Sprague-Dawley rats received This investigation was undertaken to deter­ 50 ug/rat/day of AFBl in DMSOby stomach mine if dietary vegetables would influence intubation to a total dose of 1.10 mg AFBl/rat. response of rats to aflatoxin B1. Diets con­ Age- and sex-matched controls received similar taining 25%freeze-dried ground green beans, red volumes of DMSO. Eighteen months after admin­ table beets, or butternut squash were formulated istration of the last dose of the toxin half to be isocaloric and isonitrogenous to the the animals in each group received iron dextran AIN-76 basal diet. Youngmale Fischer rats were intraperitoneally (125 mg/kg/day) for three fed one of the diets with or without l ppm days and the other half of each group received aflatoxin B (AB1) for 8 weeks. Plasma a­ no iron treatment. fetoprotein 1(AFPJ was determined weekly. Preserved liver sections stained with Without AB there were no differences in AFP hematoxylin and eosin (H&E) indicated the levels at Any time. AB1 treated groups always presence of mixed cell hepatocellular carcinomas had significantly higher (p < .05) levels than in AFBl-treated rats. No hepatic lesions were non-AB1 groups, and amongthe AB1-treated groups observed in control rats. Iron staining by each ot the vegetable diets resulted in signif­ the Gomori method showed exclusion of iron icantly higher levels than did the AIN basal dextran from the tumor sites. Areas of normal diet. Liver sections were histochemically tissue were deeply stained for iron, both in stained for y-glutamyl transpeptidase (GGT). Kupffer cells and hepatocytes. Animals receiving no AB had no GGT-positive foci. Amongthe AB1 tr~ated groups, the basal, bean, beet, and squash diets, respectively, resulted in 17.9, 33.0, 28.5, and 30.0% of liver 347 METHODAND ALGORITHMFOR RAPID QUANTITATIONAND section area positive for GGT,and3.6, ~2.8, MASS-ARCHIVALOF HEPATIC FOCI USING A MINICOMPUTER 16.9, and 12.8 hepatic cell foci per cm. These COUPLEDDIGITIZER ANDPROJECTING MICROSCOPE. J.P. data suggest that the vegetable diets enhanced Berez, Health Effects Research Laboratory, U.S. AB-induced preneoplastic transformation in EPA, Cincinnati OH. Sponsor: R.J. Bull young1 rats. (Supported in part by NIH ES 07052.) Quantitation of enzymatically altered liver lesions (foci) induced by chemical carcinogens re­ quires accurate planimetry of histologic slides and microscopic areas. By today's state-of-the­ 349 DOSE-RELATEDINDUCTION OF PLASMAa-FETOPROTEIN art in automation, discrimination of histochemi­ ELEVATIONAND EMERGENCE OF y-GLUTAMYL TRANSPEP­ cally phenotypic GGTase rich lesions from diffuse TIDASE-POSITIVEHEPATIC FOCI IN RATSFED non-specific artifacts can be only done by the AFLATOXINB1 • N. Misslbeck, J.N. Boyd, T.C. trained eye. It is not probable that inexpensive, Campbell, and G.S. Stoewsand,* Div. of Nutri­ fully computerized color-pattern processing will tional Sciences and *Dept. of Food Science, be available for this purpose. On the other hand, Cornell University, Ithaca and *Geneva, N.Y. the vast numbers of slides generated in one study, and the tedium and inaccuracy of manual integration The purpose of this study was to develop a methods presents a major obstacle to the routine model system employing oncodevelopmental use of the liver foci assay for carcinogenic proteins for rapid in vivo detection of preneo­ assessment of chemicals. We developed an inexpen­ plastic transformation. Youngmale Fischer rats sive, accurate and rapid semi-automated method for were fed 0, 0.1, 0.5, 1.0, or 2.0 ppmaflatoxin processing large numbers of liver slides. In this B1 (AB1) in the standard AIN-76 diet beginning procedure the histologic slide is projected onto one week post weaning. Blood a-fetoprotein the calibrated surface of a flat bed touch digi­ (AFP) concentrations were determined weekly from tizer by means of a projecting microscope. A weeks four through seven. At all times plasma trained operator delineates foci boundaries by AFP increased linearly with increasing dose of tracing at any desired magnification with a stylus. AB1. Animals were killed by decapitation after The digital output is integrated and stored in a eight weeks. Liver sections were stained for mini-computer - disk storage device. Since the y-glutamyl transpeptidase (GGT)activity, and

98 number of GGT-positive hepatic· foci per sq. cm. and characteristic pattern of toxic and histolo­ and% of section area positive for GGTwere gic responses, TCDDhas been shown to be a pro­ determined. Increasing the doses of AB1 re- 2 moter of hepatic cancer in rats. To test the- tu­ sulted in 0, 0.25 mm2) by size and by compression of the surrounding parenchymal cells. In the DMN& PB group, livers 352 ASSESSMENTOF THE CAPACITYOF POLYBROMINATED of affected male mice at 6,8,12 and 16 wk exhibited 6,10,17 and 12 iron resistant F/cm2 and BIPHENYLSTO SERVEAS PROMOTERSOF HEPATOCARCINO­ 10,13,9 and 9 GGT-F/cm2, respectively. At these GENESIS. R.K. Jensen, S.D. Sleight, J.I. Good­ same time periods iron resistant-Fin affected ~' S.D. Aust and J.E. Trosko, Depts. of Path., females ranged from 5-11 F/cm2, while GGT-F Pharmacol. & Tax. , Biochem. and Ped. & Hum. De­ ranged from 9-16 F/cm2. Iron resistant HN were vel., Mich. State Univ., E. Lansing, MI 48824. first observed in male mice at 8 wk; however, Large doses of Firemaster (FM), a mixture of poly­ GGT-HNwere not noted until 12 wk. By 12 and 16 brominated biphenyls (PBB), cause hepatic cancer wk affected males had 6 and 4 iron resistant in rats. At noncytotoxic concentrations, FM and HN/cm2 while affected femaies had 4 and 6, its major congener, 2,2',4,4',5,5'-hexabromobi­ respectively. At these same time points, males phenyl (HBB) inhibit intercellular communication had 6 and 3 GGT-HN/cm2 while females only dis­ in vitro, a property associated with known tumor played GGT-HNat 16 wk (6 HN/cm2). The DMNalone promoters. The purposes of this study were to group contained an average of 4 F/cm2 (iron assess the in vivo capacity of PBB to promote he­ resistant or GGT) while no HN were noted in any patocarcinogenesis and to evaluate the reliabili­ group except the DMNand PB. Lung nodules were ty of the in vitro assays. FM BP-6 and HBB were apparent as early as 6 wk in both sexes of the compared with phenobarbital (Pb), in a two-stage DMNalone group and at 12 wk in the DMNand PB model for hepatocarcinogenesis devised by Pitot group. This model could provide a practical and associates. Female Sprague-Dawley rats short-term in vivo tool for the detection of weighing 200-250 g were injected ip with 10 mg/kg early sequential cellular alterations produced by of diethylnitrosamine (DEN) 24 hours after a 2/3 initiators and promoters of carcinogenesis. partial hepatectomy (PH). Thirty days after PH, (Supported in part by BSRG funds from the College groups of 6 rats were fed a control diet or diets of Pharmacy and ES-ff2130.) containing Pb (500 ppm), FM BP-6 (10 or 100 ppm), or HBB (10 or 100 ppm). Rats not partially he­ patectomized or given DEN were fed Pb, FM BP-6 or HBB at the same levels. At 6 months rats were 351 ASSESSMENTOF THE CAPACITYOF 3,3',4,4',5,5'­ killed and tissue samples were collected for his­ HEXABROMOBIPHENYLTO SERVEAS A PROMOTEROF tologic and histochemical studies. Enzyme alter­ HEPATOCARCINOGENESIS.R.K. Jensen, S.D. Sleight, ed foci were identified by using y-glutamyl J.I. Goodman, C.D. Millis, S.D. Aust and J.E. transpeptidase. The average number of enzyme Trosko, Depts. of Pathology, Pharmacology & Toxi­ altered foci per cm2 were: control, 4; Pb, 58; cology, Biochemistry and Pediatrics & Human Devel­ opment, Mich. State Univ., E. Lansing, MI 48824. FM BP-6 (10 ppm), 201; FM BP-6 (100 ppm), 86; HBB (10 ppm), 34; HBB (100 ppm), 84. Values in rats The objective of this study was to determine if not hepatectomized or given DEN were: control, 3,3',4,4',5,5 1-hexabromobiphenyl (HBB) can act as O; FM BP-6 (10 ppm), 7; FM BP-6 (100 ppm), 14; a promoter of hepatocarcinogenesis. This congen­ HBB (10 ppm), O; HBB (100 ppm), 1. FM BP-6 er and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) caused decreased weight gain, thymic atrophy and are similar in that they both cause 3-methylchol­ enlarged and vacuolated hepatocytes. These re­ anthrene type induction of hepatic microsomal sults indicate that FM BP-6 and HBB act as pro­ drug-metabolizing enzymes and produce a similar moters of hepatocarcinogenesis.

99 353 THE PATHOGENESISOF COUMARININDUCED CHOLANGIO­ within cholangiofibrosis. Possibly proliferating FIBROSIS IN THE RAT. Evans, J.G., Lake, B.G. and ducts exposed to a carcinogen may be susceptible Conning, D.M., The British Industrial Biological to neoplastic change. It is concluded that most Research Association, Woodmansterne Road, of tumours (with mucus production and an adeno­ Carshalton, Surrey, SM5 4DS. U.K. matous pattern), induced by 3'MeDAB, even those are variants of hepatocellular carcinoma. Chronic administration of coumarin to rats Cholangiofibrosis is not a prequisite in the results in proliferation of bile ducts considered development of such tumours and this lesion should to be either cholangiofibrosis or cholangio­ not be taken as indicative of a carcinogenic carcinoma. The.development of the lesion obtain­ process. (Supported by the U.K. Ministry of ed in rats fed coumarin 5,000 ppm may be divided Agriculture, Fisheries and Food). into four phases: 1) fine ductal proliferation extending between portal tracts and central veins; 2) proliferation of regular ducts around branches 355 THE LIGHT AND ELECTRONMICROSCOPIC EVALUATION of the hepatic vein; 3) proliferation of OF CHOLANGIOCELLULARNEOPLASMS IN THE RAT LIVER irregular ducts containing mucus, polymorphs FOLLOWINGCHRONIC AROCLOR 1260 EXPOSURE. cellular debris and embedded in a fibrous matrix; 4) the development of extensive intestinal R.H. Weltman and D.R. Norback, Wm. S. Middleton metaplasia. These changes were preceded by hepa­ Mem. VA Hosp. Univ. of Wis., Dept. of Path., Madison, Wisconsin, 53705. tocyte damage, in particular damage to the canalicular membrane. Once established, ducts in Simple cholangioma (C), cystic C, cholan­ centre of the lesion degenerate and were replaced giofibrosis (CF), and mixed hepatocholangio­ cellular carcinoma (MHC) were observed in the by fibrous tissue. This process was associated livers of 93 Sprague-Dawley rats fed Aroclor with the accumulation of mucus, cellular debris (lOOppm for 16 months followed by 50ppm for 8 within the duct, distension and finally rupture months) at an incidence of 38, 7, 9, and 26%, of the duct wall. In general, reversal to control respectively. C(8%), CF(4%), and MHC(O%) diets, leads to almost complete atrophy of the occurred in 81 rats fed a control diet. The lesion and replacement by scar tissue. However cholangiocyte, a pale-staining epithelial cell in one animal fed coumarin for nine months and in the bile duct of the portal triad, possessed killed at eighteen months an active lesion was the following subcellular morphology: A clefted still present. Concurrent histochemical and bio­ nucleus; few cisternae of rough endo- chemical studies showed an initial reduction in plasmic reticulum (ER), tubules of smooth ER, the activity of certain enzymes. The activity of Golgi complexes, and mitochondria; many ribo­ ~-glutamyl transpeptidase was enhanced and hepatic somes, cytoplasmic filaments, and apical surface glutathione was raised. Cholangiocarcinoma was microvilli; lateral junctions and membrane un­ not seen in any animal and no foci of altered dulations; and a smooth basal surface resting hepatocyte was observed. It is concluded that on a basal lamina. A cholangiocyte subcellular the lesion induced in rats is best considered to morphology was observed in C cells. CF pos­ be cholangiofibrosis with no evidence of progress sessed cuboidal to columnar simple and pseudo­ to neoplasia. (Supported by the U.K. Ministry of stratified epithelium, numerous mitoses, and Agriculture, Fisheries and Food). few PAS-positive goblet cells. Cells of CF de­ monstrated features of cholangiocytes; diff­ erences included a prominent nucleolus, in­ 354 THE SIGNIFICANCE OF CHOLANGIOFIBROSISAS A "PRE­ creased organelles, and numerous desmosomes. NEOPLASTICLESION". Evans, J.G., Conning, D.M. In the MHC, the ductal cell was PAS negative and Applebey, E.G., The British Industrial Bio­ and the features varied. Some cells possessed logical Research Association, Woodmansterne Road, a cholangiocyte morphology. Other cells con­ Carshalton, Surrey, SM5 4DS. U.K. tained subcellular features typical of hepato­ cytes: A round nucleus, prominent nucleolus, The histogenesi; of carcinoma of liver having an abundant rough ER, lamellar smooth ER, lyso­ adenomatous or ductal pattern is uncertain. Many somes, smooth intercellular surfaces, absent consider that such tumours arise from bile ducts basal lamina, and canaliculi between ductal and is preceded by cholangiofibrosis; others cells and hepatocytes. Aroclor 1260 induces consider that they are histological variants of lesions of both hepatocellular (Toxicologist 1, hepatocellular carcinoma. The changes produced in 65, 1981) and cholangiocyte differentiation. the liver of rats by 3'Methyl-4-diaminoazobenzene (3'MeDAB) and by coumarin are compared. 3'MeDAB administration led to extensive oval cell prolif­ eration and then to cholangiofibrosis. This was 356 DINITROTOLUENEPROMOTION OF DIETHYLNITROSAMINE followed by hepatic nodules with variable enzyme (DEN) INITIATED HEPATOCYTESIN VIVO. T.B. patterns and later, trabecular and adenomatous or Leonard and J.A. Popp, Chemical Industry Insti­ duct carcinoma. Coumarin induced cholangio­ tute of Toxicology, Research Triangle Park, NC fibrosis in which four distinct stages could be 27709. Sponsor: J.A. Swenberg recognised. No altered foci, areas or nodules were seen, and no hepatocellular tumours developed. Technical grade DNT (tDNT) is a potent hepato­ The ultrastructure appearance of cholangiofibrosis carcinogen when fed to F-344 male rats for one was compared with that of adenomatous areas of year (100% incidence of hepatocellular carcino­ carcinoma. Cells forming the lining epithelium mas). However, tDNT is only a weak initiator in both were similar. Carcinomas with ductal or when evaluated in hepatic initiation-promotion adenomatous pattern were deep within unequivocal systems suggesting that the promoting activity hepatocellular carcinoma and it is considered that of tDNT may be a major determinant of its car­ they represent variants of such tumours. cinogenic potency. The present studies were Occasionally ductal tumours appeared to arise designed to ascertain whether tDNT had promot-

100 ing activity and to investigate the mechanism 358 2-ACETYLAMINOFLUORENE(AAF) DEPENDENTINCREASES of tDNT promotion. Male F-344 rats were treated IN EPOXIDEHYDROLASE: SPECIES, STRAINAND SEX with a single dose of DEN (150 mg/kg), permitted DIFFERENCES. M.E. Graichen and J.G. Dent. to recover for 2 wk, and then fed tDNT in the Chemical Industry Institute of Toxicology, Re­ diet for 3 wk. Promotion activity was quanti­ search Triangle Park, NC 27709. tated by determining the incidence of histo­ chemically identifiable hepatic y-glutamyl Exposure of rats to hepatocarcinogens causes transpeptidase positive (GGT+) foci. Feeding substantial increases in hepatic microsomal 14 mg/kg/day and 35 mg/kg/day of tDNT caused epoxide hydrolase (EH) activity. The potency of a dose-dependen increase in GGT+ foci (2. 5 compounds in increasing EH is related to their and 6.5 foci/cm, 2 respectively) relative carcinogenic potency. The objective of this to initiation control (DEN alone, 0.6 foci/ study was to investigate the relationship be­ 2 cm ) an2 promotion control (tDNT alone, O. 3 tween hepatocarcinogenicity and increases in foci/cm) values. To investigate the mechanism epoxide hydrolase using the known species, of tDNT promotion, the effectiveness of tDNT strain and sex differences in susceptibility to (35 mg/kg/day) as a replacement for 2-acetyl­ AAF carcinogenicity. Following administration aminofluorene (2-AAF) in a hepatic "growth of AAF in the diet or via ip injections, liver selection" protocol and its ability to alter microsomes or 10,000 g supernatent was prepared. DNA synthesis in hepatocytes following partial EH activity was measured with benzo(a)pyrene-4,5- hepatectomy (PH) were determined. tDNT was active oxide as substrate. Three daily ip injections of in the growth selection protocol (15.8 GGT+ foci/ AAF, at doses between 5'and 80 mg/kg/day, caused 2 cm) buz not as effective as 2-AAF (40.1 GGT+ dose-related increases in EH in male F-344 and CD foci/cm). When tDNT was fed (35 mg/kg/day) for rats. The log-linear dose response obtained had 2 wk prior to PH, it had no effect on the the same slope in both strains and the ED was 50 kinetics of DNA synthesis following PH. In 13 mg/kg/day. The maximum increase observed was conclusion, tDNT is effective in promoting DEN 350% of control activity in both strains. Doses initiated hepatocytes but does not significantly of 2-AAF up to 160 mg/kg/day failed to elicit alter DNA synthesis in hepatocytes following PH. significant increases in EH activity in CD female rats and caused only a 40% increase in female F-344 rats. No increase in EH activity was 357 DNA ALKYLATIONDURING CHRONICDMN EXPOSURE OF detected in guinea pigs treated with AAF at doses MALEAND FEMALEC3H MICE. C. Lindamood III, between 40 and 240 mg/kg/day for 3 days. In DBA2 K.C. Billings, M.A. Bedell and J.A. Swenberg, and C57BL6 mice fed 0.02% AAF for 2 weeks, Chem. Ind. Inst. Tax., Res. Triangle Park, NC. decreases (45 and 55%, respectively) in EH were· observed. By comparison, F-344 male rats fed this The hepatocarcinogenicity of dimethylnitros­ diet exhibited a 460% increase in EH activity. amine (DMN) is sex-dependent in C3H mice follow­ Female rats, mice and guinea pigs are poor N­ ing chronic oral exposure. DMNcauses predomi­ hydroxylators of AAF and are resistant to the nently hepatomas in males and capillary endo­ carcinogenic action of AAF. These results theliomas in females (Den Engelse et al.; Europ. support the hypothesis that increases in EH J. Cancer. 10:129, 1974). The purpos-;-of this activity are related to the carcinogenic action study was to tssess the role of the promutagenic of AAF and to the metabolic activation of AAF. DNA lesion, 0 -methylguanine (06MG), in the cel­ lular specificity of DMN's carcinogenicity in C3H mice. Male and female mice were given O or 359 EFFECTS OF ALPHA-NAPHTHYLISOTHIOCYANATE,LITHO­ 10 ppm DMNad libitum in the drinking H o for 2, 2 CHOLIC ACID AND SELECTEDHEPATOCARCINOGENS ON 4 or 16 days. The DMNdosage varied from 1.74- EPOXIDE HYDROLASE,DT-DIAPHORASE AND SERUMINDI­ 2.14 mg/kg/day among the treatment groups. Livers CATORSOF CHOLESTASIS. Thompson, M.B., Dent, J. were perfused in situ with collagenase and hepato­ ~. Graichen, M.E., Neptun, D.A., Scortichini, cytes and nonparenchymal cells (NPCs) separated by differential centrifugation. DNAwas isolated B.H., and Popp, J.A. Chemical Industry Institute of Toxicology, Research Triangle Park, NC 27709. by hydroxyapatite chromatography and purine bases separated by HPLC and quantitated from UV-absorb­ The classic cholestatic agent alpha-naphthy­ ing and fluorescing peaks. Concentrations of 7- lisothiocyanate (ANIT) elevates the hepatic pre­ methylguanine (7-MG) in the 2 liver cell popula­ neoplastic marker epoxide hydrolase (EH). Abili­ tions of both sexes' increased between 2 and 4 ties of 3 hepatocarcinogens and 2 cholestatic days of exposure and remained relatively constant agents to produce cholestasis and enhance hepatic thereafter. NPCs of both sexes accumulated 06MG EH activity were compared. Hepatocarcinogens 2- over the time course of DMNadministration, such acetylaminofluorene (AAF) at 100 ppm or 3 1-methyl- that increasing 06MG/7MGratios were observed. 4-dimethylaminoazobenzene (3'-MeDAB) at 600 ppm Male and female hepatocytes demonstrated increased in the diets or diethylnitrosamine (DEN) at 40 removal of 06MG after exposure to DMN. Concen­ ppm in the drinking water were given to male, trations of hepatocellular 06MG in both sexes Fischer-344 rats (200-270 g}. The cholestatic were 5 to 7 fold less at 16 days than at 4 days, agents ANIT at 1000 ppm or lithocholic acid (LA) resulting in decreased 06/7MG ratios. While this at 10,000 ppm in the diets were fed to comparable represents the first demonstration of cell specif­ groups. Concentrations of total bile acids (BA), ic alkylation in the mouse liver following chronic total cholesterol (CH) and total bilirubin (BIL) exposure to DMN, no correlation between cell and the activity of gamma-glutamyltranspeptidase specificity of 06MG accumulation and specificity (GGT) were measured in serum at 2,4 and 6 weeks. for carcinogenesis was observed. Factors such Activities of hepatic microsomal epoxide hydro­ as cell replication and differential repair of lase (EH) and cytosolic DT-diaphorase (DTD) were other promutagenic DNA adducts must also be determined. Serum BA concentrations were in­ considered. creased by all treatments. ANIT treatments pro-

101 duced the greatest increases (7,000% relative to cultures were washed and exposed to the controls). AAF and 3'-MeDAB caused intermediate test chemical and 3H-thymidine. DNA increases (600 to 1000%) and DEN and LA the repair was determined by autoradiogra­ small~st (: 300%). CH and BIL concentrations and phy. Carcinogens and their noncarcino­ activities of GGT and DTD were only increased by genic analogues from the structural treatment with ANIT (: 300 to 2000%). EH was in­ classes of alkylating agents, aromatic creased (150 to 600%) by all treatments except amines, mycotoxins, polycyclic aromatic LA. At 2 and 6 weeks animals exposed to AAF, DEN hydrocarbons, nitrosamines, and amino­ and 3'-MeDAB had a direct correlation (p = 0.001) azo dyes were tested. In both species, between serum BA and EH activities. In conclu­ DNA repair was elicited by all the sion, selected hepatocarcinogens are cholestatic carcinogens. No noncarcinogens were as determined by increased BA. An association positive in the mouse HPC/DNA repair was also noted between BA concentrations and EH test. However, DNA repair was seen in activities. hamster hepatocytes exposed to pyrene or aflatoxin Gz, which are not carcino­ genic in rats or mice, but have not 360 VARIATION IN GENOTOXIC RESPONSE TO been tested in hamsters. The mouse, 3,2'-DIMETHYL-4-AMINOBIPHENYL (DMAB) IN which is not sensitive to the effects HEPATOCYTE PRIMARY CULTURES (HPC) of aflatoxin Bi, required a dose of DERIVED FROM FOUR MAMMALIAN SPECIES. 10-4M to elicit maximum repair while C.J. Maslansky and G.M. Williams, only 10- 6 M was required for hamster American Health Foundation and New York hepatocytes. These findings demon­ Medical College, Valhalla, N.Y. strate that genotoxic chemicals can be The rat HPC/DNA repair assay detects identified using mouse or hamster hepa­ the genotoxic effects of chemicals. tocytes and that species differences in The development of HPCs from other sensitivity exist. species facilitates study of patterns of xenobiotic metabolism and genome 362 INHIBITIONOF AFLATOXINB1 CARCINOGENESISIN RAIN­ interaction as they may relate to BOWTROUT BY DIETARY s-NAPHTHOFLAVONE (s-NF), AND differences in in vivo susceptibility INDOLE-3-CARBINOL(I-3-C). J.D. Hendricks, J.E. to carcinogenesJ.s. Variations in the Nixon, G.S. Bailey, and R.O. Sinnhuber, Dept--:---or induction of DNA repair in HPCs derived Food Sci. & Tech., Oregon State University, from different species have been Corvallis, OR97331. detected in response to the carcinogen Several compoundssuch as flavonoids, selenium, DMAB. HPCs were initiated from the antioxidants and certain vitamins reportedly re­ rat, mouse, hamster and rabbit. duce the induction of cancer in experimental ani­ Hepatocytes were exposed to DMAB and mals, and many function by affecting the mixed­ 3H-thymidine for 18 hrs. DNA repair function oxidase (MFO)system. The following was determined au torad iographi ca 1 ly. compounds: 50 and 500 ppm s-NF, 1000 ppm flavone, DNA repair induced by DMAB varied 1000 ppm of a tangeretin-nobilitin mixture, 1000 considerably between the species ppm s-ionone, 1000 ppm I-3-C and 2000 ppmquerce­ investigated. Although DNA repair was tin were examined for protection against afla­ maximal in hamster hepatocytes, rat toxin B1 (AFB1)hepatocarcinogenesis and induc­ hepatocytes also displayed appreciable tion of the MFOsystem in rainbow trout. These levels. A dose dependent response was compoundswere fed to fingerling rainbow trout elicited from both species. Rabbit and for 2 months. At that time the activity of mouse hepatocytes displayed lesser several MFOenzymes and cytochrome P450 content levels of repair. These differences in were measured and the trout were exposed to 20 repair induction were reflected in ppb AFB1for 10 days in the same diets. After variations in DMAB metabolism in HPCs feeding the test diets without AFB1for another derived from each species. These month and basal diet for 11 months, the tumor finding demonstate the utility of HPCs incidence was determined. s-NF induced the trout de rived f r om o the r s p e c i es .in s tu dies MFOsystem in a dose-dependent manner while involving xenobiotic metabolism and tangeretin-nobilitin was less effective. Flavone, genotoxicity testing. quercetin, s-ionone and I-3-C did not induce the MFOsystem. I-3-C ands-NF provided marked pro­ tection against AFB1-inducedhepatocarcinogenesis, 361 THE USE OF MOUSE AND HAMSTER CELLS IN while the other compoundswere ineffective. THE HEPATOCYTE PRIMARY CULTURE ( HPC) / Tumor incidences were 5.1% for 1000 ppm I-3-C, DNA REPAIR TEST. C.A. McQueen, D.M. 7.5% for 500 ppm s-NF and 19.6% for 50 ppm s-NF, Kreiser and G.M. Williams, American compared to 44.9% for the AFB1-positive control. Health Foundation, Valhalla, N.Y. These data are interesting because induction of The HPC/DNA repair test using rat hepa­ the MFOsystem was not required for protection tocytes detects the genotoxic potential against AFB1carcinogenesis. Both s-NF and I-3-C of chemicals. Since species differ­ provided marked protection but only s-NF induced ences in susceptibility to chemical the MFOsystem. (Supported in part by U.S.P.H.S. carcinogens exist, the test was Contract NOl CP85660and Grant ES00541) extended to other species. Hepatocytes were isolated from either Syrian golden hamsters or B6C3F1 mice by in situ 363 DAMAGETO RAT LIVER DNA FROM IN VIVO EXPOSURE TO perfusion of the liver with collagen­ MONOCROTALINE,A PYRROLIZIDINEALKALOID. T.W. ase. After cell attachment,the primary Petry, G.T. Bowden, R.J. Huxtable, and I.G. Sipes.

102 Departments of Pharmacology and Radiation present remained in the DNA of both cell types follow­ Oncology, University of Arizona College of ing administration of either AAF or N-OH-AAF. These Medicine, Tucson, AZ 85724. results suggest that AAF preferentially damages DNA of PC due to an increased capacity of these cells to Pyrrolizidine alkaloids (PAs) are known to damage carry out the N-hydroxylation of AAF. The ability of DNA in vitro, to be hepatocarcinogens in animal PC and NPC to metabolize N-OH-AAF to a reactive models, and have been linked to an increased species appears similar. The binding of C to specific incidence of hepatic carcinoma among South regions of the genome of PC and NPC is under investi­ African Bantus. We have employed alkaline elu­ gation. (Supp. by the MSU Agric. Exp. Station.) tion to detect and characterize DNAdamage resulting from in vivo exposure of male Sprague­ Dawley rats (150-280g) to single intraperitoneal doses of the PA, monocrotaline. Hepatic nuclei 365 ACRYLAMIDEAS A TUMORINITIATOR IN THEMOUSE SKIN. were isolated, and served as the source of DNA in R.J. Bull, M. Robinson and D. Cmehil, Health all experiments. Histological evaluation of Effects Research Lab., U.S.E.P.A., Cincinnati, OH liver sections taken at the time of nuclei prep­ 45268 aration revealed no signs of cytotoxicity. No Acrylamide is a common monomeric unit of a evidence of single-strand breaks, as would be variety of commeFcial products. It has been indicated by an increased rate of DNA elution recognized for sometime as a neurotoxin. However, from the filter as compared to controls, was its structural resemblance to ethyl carbamate detected at either 4 or 8 hr after doses of up to (urethane), an established tumor initiator, led 120mg/kg of monocrotaline. As monocrotaline may us to examine the carcinogenic properties of this be bioactivated to bifunctional alkylating agents, chemical in the mouse skin. A small scale experiments were conducted to detect DNAcross­ experiment was carried out to test the hypothesis links. In contrast to the results of the single­ that acrylamide may act as a tumor initiator. The strand break experiments, evidence for DNA cross­ study involved administration of a single dose of links was observed at doses as low as 5mg/kg, 4 30 mg/kg by each of 3 routes of exposure (oral, hr after administration of the PA. DNAcross­ intraperitoneal and topical) to female SENCAR linking was detected by an apparent decrease in mice. These initiating doses were followed in 2 the number of X-ray induced single strand breaks, weeks by repeated application of 1 ug 12-0-tetra­ and consequently a decreased rate of DNA elution decanoyl phorbol-13-acetate (TPA) 3 times weekly from the filter as compared to controls, when for 20 weeks. At 32 weeks following the start of nuclei from both groups were X-irradiated. Pre­ promotion the following cumulative tumor counts liminary experiments to differentiate between (papillomas) were noted: Oral gavage, acrylamide DNA-protein crosslinks, and DNA-DNAinterstrand 8 animals in 40 (8/40) with tumors and average of crosslinks suggest that a mixture of the two 0.25 tumors per animal; distilled deionized Hz0 types is induced. (Supported by an AFPE Fellow­ (DHz0) 6/20 and 0.35; I.P., acrylamide 18/40 and ship and NIH Grants ES-8-2130 and HL-25258.) 0.55, DH20 3/20 and 0.20; topical acrylamide 6/40 and 0.15 and acetone 3/20 and 0.25. It appears from this data that acrylamide via the I.P. route is capable of initiating tumors. 364 QUANTITATIVE ASSESSMENT OF CARCINOGEN­ As in previous studies with urethane, the potency MODIFIED DNA IN SPECIFIC LIVER CELL POPULA­ of acrylamide is greater by a systemic route than TIONS FOLLOWING A SINGLE DOSE OF N-2- the topical route of exposure. Experiments de­ ACETYLAMINOFLUORENE (AAF) OR ITS N-HY - signed to develop the dose-response relationships DROXY DERIVATIVE (N-OH-AAF). B.L. Baranyi, involved in acrylamide-induced tumorigenesis are J.A. Swenberg and J.I. Goodman. Dept. of Pharma­ underway. cology and Toxicology, Mich. State University, East Lansing, MI 48824, and (JAS) Chem. Industry Inst. of Toxicology, Research Triangle Park, NC 27709. AAF-induced hepatocarcinogenesis appears to be 366 DERMALCARCINOGENESIS BIOASSAYS OF PYROLYSISFUEL targeted to parenchymal cells (PC). Our objective was OIL (PF0) ANDPYROLYSIS GASOLINES (PG) IN C3H to determine whether or not there is preferential MICE. L.R. DePass, L.G, Peterson, C.S. Weil, binding of carcinogen (C) residues to the DNA of these Bushy Run Research Center, Export, PA. target cells and, if so, the possible basis for the selectivity. AAF · and its N-hydroxy derivative Pyrolysis fuel oils result from high temperature (N-OH-AAF), the first product in the metabolic activa­ cracking of hydrocarbon components of naphtha or tion of AAF, were employed for these studies. Male liquefied petroleum gas (LPG) feedstocks. These Sprague-Dawley ats weighing 200-250 g were injected, hydrocarbons are pyrolyzed at temperatures up to i.p., with (ring- 3H)AAF (64 µ Ci/ 1.8 µ moles/ 100 g) or 950°C after which temperatures are reduced to 3 (ring H)-N-OH-AAF (100 µ Ci/ 1.8 µ moles/ 100 g). 400°C. Further temperature reductions are accom­ Animals were sacrificed at either the time of peak plished by either injecting water into the pro­ binding of C to DNA, 2 hr or 18 hr post-injection, when cess (water quench) or using a gasoline fraction­ N-OH-AAF or AAF, respectively, were used, and at 3 ator (oil quench) system. In this study 3 sam­ days after maximum binding. PC and non-parenchymal ples of PFO, 3 samples of pyrolysis gasoline (a (NPC) cells were isolated by centrifugal elutriation. C c5+ fraction condensed during compression and modified DNA was purified by hydroxyapatite column cooling of the cracked gas) and a c9+ fraction of chromatography. Following AAF administration, the the pyrolysis gasoline were painted on the clipped µ moles of adducts formed/mg DNA of PC was 2.7 times skin of groups of 40 male C3H/HeJ mice 3 times that of the NPC. When N-OH-AAF was employed, weekly for the lifetime of the animals. Methyl­ equal amounts of adducts were found associated with cholanthrene, distilled water and acetone were DNA of PC and NPC. By 3 days after the period of painted as the positive and 2 negative control maximum DNA binding 50-59% of the adducts initially substances respectively. All 3 PFO samples were

103 highly oncogenic to mouse skin resulting in tu­ (SRC-II), is a complete carcinogen for mouse skin mor incidences of greater than 50%, The PFO sam­ and therefore should elevate ODCactivity in a ple derived from a naphtha feedstock by an oil manner similar to other promoters. To test this quench process showed significantly greater onco­ hypothesis, ODCinduction by HDwas compared with genic activity than both samples from LPG feed­ the activation of this enzyme by the potent skin stocks by water quench processes. Among the tumor promoter 12-O-tetradecanoylphorbol-13-ace­ groups receiving PG samples, 4 skin tumors oc­ tate (TPA). A single application of 23 mg of HD curred in the group that received the naphtha­ or 17 nmoles of TPAto the dorsal skin of female derived oil quench sample compared to 1 and 0 Charles River CD-1mice resulted in 75-fold and tumors respectively in the groups that received 27O-fold increases respectively, in epidermal ODC the LPG-derived, water quench samples. No skin activity. Twice-weekly applications for 4 weeks tumors were observed in the solvent controls. of the same doses elevated ODClevels 2O-fold in These results suggest a relationship between onco­ those animals receiving HDand 135O-fold in the genic activity and either the feedstock (and re­ mice treated with TPA. The kinetics of induction lated reaction conditions) or the cooling process with both HDand TPAwere similar, exhibiting used in the production of PFO and PG. Published peak activity 3-5 hours after application and re­ data suggest that the feedstock may be the more turning to near-basal levels within 12 hours; important determinant of oncogenic activity, (Weil however, with multiple applications HDdid not and Condra, Am. Ind. Hyg. J., 38:730, 1977). exhibit the amplification- ·of induction in ODC activity seen with TPA. Hepatic ODCactivity was also found to be elevated following dermal appli­ 367 INITIATION-PROMOTIONSTUDIES WITH COAL LIQUEFAC­ cation of both HDand TPA. The similarity in the TIONMATERIALS. D. Dennis Mahlum. Pacific pattern of ODCinduction after a single applica­ Northwest Laboratory, Richland, WA99352. tion of HDand TPAsuggests that this assay may High boiling (>55O°F) coal liquids such as heavy be useful for screening SRCmaterials as poten­ distillate (HD) from the solvent refined coal-II tial promoters. (Worksupported by the U.S. Dept. (SRC-II) process are mutagenic in the Amesassay of Energy Contract No. DE-ACO6-76RLO-183Oand and carcinogenic for mouse skin. Chemical char­ National Science Foundation Grant No. SPI-816O230) acterization studies have identified a number of mutagenic constituents in HDbut similar efforts to identify carcinogenic components have been hampered by the long times and high costs re­ 369 A NEW SYRIAN GOLDE'NHAMSTER ( SGH ) HYBRID quired for standard skin-painting assays. An UNIQUELYSUITABLE FOR LIFETIME TOXICITY AND initiation-promotion assay was therefore used to CARCINOGENESISSTUDIES. R.A. Adams, P. Bernfeld, study the carcinogenicity of coal-derived li­ F. Homburger, E. Soto, C.G. Van Dongen, Bio­ quids. Basic (BF), basic tar (BTF), neutral tar Research Institute, Cambridge, MA. (NTF), and polynuclear aromatic (PNA)fractions Long-term toxicity or car­ were prepared from HDby solvent extraction. cinogenesis studies employing SGH's have often These fractions were tested for their initiating suffered from the re1atively short life span of activities by applying a single dose to the some of the commercially available SGH's. A shaved back of Charles River male CD-1mice. first generation hybrid of two carcinogen­ Positive control groups were initiated with susceptible inbred lines of SGH's is now avail­ either dimethylbenzanthracene or benz(a)pyrene. able, designated BIO® FlD Alexander. Reproduc­ Beginning two weeks after initiation, all mice tive parameters derived from 1570 matings are: received twice weekly applications of 5 µg of the fertility 70%; litter size at birth 9.4 with 8.3 promoter, phorbol myristate acetate (PMA). Pap­ surviving at weaning. These hybrids are as illomas were noted and recorded for each animal susceptible to cancer induction by s.c. dimethyl­ at time of PMAapplication. Heavy distillate and benzanthracene or gavaged methylcholanthrene as its fractions all showed initiating activity. the parental strains, BIO® 15.16 (sire) and However, the incidence of mice with tumors, the BIO® 87 .20 (dam) (Homburger, Adams Soto and rate of tumor appearance, and the total number of Van Dongen, 1979 in Progr. Exp.Tumor Research, tumors varied with the test material. Considera­ pp. 215-222, Vol. 24). tion of these parameters suggests the following Lifetime studies show 50% order of activity: DMBA>HD= NTF> BaP > BTF survival of males at 108 weeks and 50% of the >BF> PNA. These data are in good agreement females at 86 weeks. 38% of the males are still with the results of long-term skin painting living at 113 weeks and 32% of females survived assays and suggest that initiation-promotion may 90 weeks. The incidence of spontaneous tumors provide .a relatively rapid and inexpensive method was extremely low in both sexes, and none were for studyi~g the tumorigenicity of coal-derived of the type usually induced by standard car­ materials. (Work supported by the U.S. Dept. of dinogens. Energy Contract No. DE-ACO6-76RLO-183O). The earlier mortality of females appears to be due to nephrosis which is more severe than the mild to moderate 368 MOUSEEPIDERMAL ORNITHINE DECARBOXYLASE INDUCTION tubular dilatation observed in males. BYSOLVENT REFINED COAL MATERIALS. D.L. Springer, D. Swanger, and D.D. Mahlum. Pacific Northwest Laboratory, Richland, WA 99352. The induction of the enzyme ornithine decarboxy­ 370 TIME TO INDUCTIONBY DIETHYLSTILBESTROL(DES) OF lase (ODC)has been proposed as an early essen­ PALPABLEMAMMARY TUMORS IN C3H MICE. tial phenotypic change observable in mouse epi­ D.L. Greenman, J.H. Farmer, NCTR, Jefferson, AR; dermal cells exposed to skin tumor promoters. and B. Highman, U. Ark. Med. Sci., Little Rock,AR Heavy distillate (HD), the high boiling coal To clearly define the dose response relationship liquid from the solvent refined coal process for the DES induction of mammary tumors and to

104 relate this to non-neoplastic responses to DES, International Agency for Research on Cancer, Lyon, France, over 2800 C3H/HeN(MTV+) female mice were fed DES and Medical Research Council, Toxicology Unit, Carshalton, for their lifetime at O, 2.5, 5, 10, 20, 40, 80, U.K. 160, 320, and 640 ppb. Mice were palpated weekly for tumors and removed from study when masses reached a 1 cm diameter. Sacrifices were also The incidence of testicular mesotheliomas due to the scheduled at 3 and 26 weeks. The crude inciden­ carcinogen FAA was studied in four-week-old male Fischer ces of histologically verified mammary tumors 344 rats. The experimental animals were fed the carcino­ for the dose-order listed above were 78, 84, 83, genic diet (0.06% FAA) during 3 weeks and then the control 83, 80, 80, 84, 86, 90, and 92%. These estimates diet for 1 week. This schedule was carried out for 3 were refined by simultaneously estimating non­ parametrically the distributions of time-to­ complete cycles (12 weeks). A smaller group of rats was removal for 1 cm tumors (considered lethal) and treated for 1 complete cycle only (4 weeks) of FAA. One for histologically verified tumors. Time adjusted group of untreated controls was also available. The surviving estimates of mammary tumor incidence for each rats were sacrificed at 59 weeks of age. The administration dose at 273 days were 75, 68, 67, 66, 67, 74, 81, of FAA for 3 complete cycles resulted in a high incidence of 89, 99, and 100%, in dose order. The estimated liver, testis and Zymbal gland tumours. The testicular mean times to removal for 1 cm tumors were 265, tumours were mesotheliomas and occurred in 9/25 rats. No 273, 273, 267, 268, 260, 250, 230, 194, and 169 days. The mean times for histologically verified such tumours were observed in animals treated for 1 cycle mammary tumors were 246, 253, 263, 257, 257, 239, only or in untreated controls. Macroscopically, these 245, 220, 177, and 152 days. The Peto test for tumours appeared as flat, nodular or papillary, greyish or dose related trends was significant for both pinkish growths on the surface of the testis and/or distributions. Mice sacrificed at 3 and 26 weeks epididymis. Microscopically, the tumours were seen to arise showed an apparent dose response for certain non­ from the surface of the peritesticular mesothelium. The first neoplastic endpoints. At both sacrifices, the prevalence of moderate to severe uterine glandu­ testicular mesothelioma was observed 30 weeks after end of lar hyperplasia was not dose dependent and was treatment. No metastases from these tumours were found. less than 10% at 40 ppb DES or less, but the The high incidence of testicular mesotheliomas, a rare type prevalence rose from 10-12% at 80 ppb to 100% at of tumour in this and other rat strains, suggestsan association 320 or 640 ppb. These results show the importance with treatment. The present experimental model may be of making time adjustments on crude incidence useful in elucidating the mechanisms involved in the estimates and suggest that neoplastic and non­ neoplastic responses to DES occur over the same production of mesothelial tumours of the testis by chemical dose range. carcinogens.

371 ZINC DEFICIENCY, METHYLBENZYL NITROSAMINE CAR­ 373 Phorbol Diester Receptors on HL-60 Human CINOGENESIS AND DNA SYNTHESIS IN ESOPHAGEALMUCO­ Promyelocytic Leukemia Cells. R.W. Lane, R.B. SA OF THE RAT. T. Schrager and P.M. Newberne, Coles, R.A. Carchman, J. F. Borzelleca. Dept. Dept. Nutr. and Fd. Sci., M.I.T. Cambridge, MA Pharmacol., Medical College of Va., Virginia Zinc deficiency causes hyperplasia and para­ Commonwealth University, Richmond, Va. 23298 keratosis in the esophageal epithelium of many animal species, and increases susceptibility of Experimentally-induced carcinogenesis in­ this tissue to chemical carcinogens. Male volves two separate steps, initiation and promo­ Sprague-Dawl·ey rats were given either a control tion. Promotion can be caused by a number of diet (60 ppm) or a diet deficient in Zn (3 ppm). chemicals, the classic compounds being the phor­ After one month on these diets, some rats received bol diesters. It has recently been demonstrated 2.5 mg/kg body weight methyl-benzyl nitrosamine that there are specific binding sites for the (MBN), twice a week for three weeks. At the end phorbol diesters on the plasma membranes of many of the dosing period, one group of control ad different types of cells. Such specific binding libitum animals was switched to zinc deficient sites have also been characterized in our labo­ diet and one group of zinc deficient was switched ratory using HL-60 cells, a continuous cell line to control diet. Zinc deficiency throughout the of human promyelocytes, with tritiated phorbol experiment significantly enhanced tumor incidence dibutyrate (3H-PDBu). These specific binding (88% vs. 31% contra~ diet ad lib and 14.7% con­ sites were characterized by both their kinetic trol pair fed, p < .0005).-Switching diets gave and equilibrium properties. Binding was satur­ equivocal results. After one month on diets the able and reversible. The Ko was determined to zinc deficient diet only,led to much greater DNA be approximately 50nM with 7.5 x 105 sites/­ synthetic activity than the control diet. Follow­ cell. Competition for binding sites by other ing the first dose of MBN, DNA synthetic activity phorbol diesters was directly related to the dropped to background levels for both groups but ability of the compound to promote tumor forma­ exceeded background at end of dosing. Ten days tion; compounds not related to the phorbol di­ after the last dose of MBN, DNA synthesis was esters did not compete. The effect of PDBu on significantly greater in the control groups. cellular function was also examined, using the These results suggest that zinc deficiency acts release of the enzyme N-acetylglucosaminidase during both the initiation and promotion stages (NAGA) as a marker. The release of NAGAin­ of the carcinogenic process. (Supported in part creased in both time and concentration. The by USPHS Grant CA 25382). curves were similar to those obtained for 3H-PDBu binding, exhibiting similar values for the EC50 of NAGArelease and Ko for bind- 372 Testicular mesotheliomas in rats exposed to N-2-Fluorenyl- ing. Nonpromoting phorbol diesters did not acetamide (FAA). J.R.P. Cabral and G.E. Neal. cause NAGAto be released. The correlation be-

105 tween binding and biological function, the com­ alcohol phorbol had no effect on either response. petition for binding sites only between active The PGE2 response therefore appears to be medi­ phorbol diesters, and the low concentrations em­ ated via the PDBu receptor. Ability of cells to ployed indicate that the binding sites for phor­ increase PGE2 production in response to PE cor­ bol diesters on HT,-60 cells are receptors. related with increased phospholipase activity (Supported in part by EPA Grant R-808861010 and which liberates arachidonate, the fatty acid pre­ NIEHS Training Grant IT 32E507087) cursor of PGE2• In responsive cells, PE treat­ ment increased by 50% [3H]arachidonate release from biosythetically labeled cell membranes. In­ 374 CARCINOGENICITYOF FLUOROCARBONS: ASSESSMENTBY creased lipase activity appeared to be substrate specific, since there was no increase in the re­ SHORT-TERMIN VITRO TESTS AND CHRONICEXPOSURE IN 14 RATS. E. Longstaff, M. Robinson, C. Bradbrook, lease of either [ c]oleate or [3H]palmitate. J.A. Styles and I.F.H. Purchase. ICI, Central All 4 cell lines, however, responded to PE as Toxicology Lab., Alderley Park, Nr. Macclesfield, shown by decreased binding of epidermal growth factor, with ED5os of 5-15 nM. Thus, the inabi­ Cheshire. SK10 4TJ, UK. lity of SaOS cells to respond to PE with increased Twenty-one fluorocarbons were evaluated for production of PGEz does not result from defec­ mutagenic activity using S.typhimurium. Twelve tive PE binding and may reflect an abnormality in of these were also assessed in the BHK21 cell the generation of a secondary signal which medi­ transformation assay. Monochlorodifluoromethane ates this response. (FC-22), chlorofluoromethane (FC-31), 1-chloro-1, 1-difluoroethane (FC-142b), 1,1-difluoro-2- chloroethane (FC-142) and 1,1,1,-trifluoroethane (FC-143a), showed mutagenic activity towards 376 THE TOXICITY OF METHYLCARBAMATE IN FISCHER 344 Salmonella and FC-31 and FC-142b were positive in RATS AND B6C3Fl MICE. Hall, w.c.; Hwang, K.K.; the BHK21 test. The in vivo carcinogenicity of Kovatch, R.M. *; Dinowitz, M.; Microbiological some of these chemicals was also assessed by a Associates, 5221 River Road, Bethesda, MD. *Tracor long-term bioassay. Wistar rats (40/sex/group) Jitco, Inc.i. 1776 E. Jefferson Street, Rockville MD Sponsor: K. E. Kouri ' • were dosed by gavage with 300mg/kg bodyweight of 3% w/v in corn oil FC-22, FC-31, FC-133a (2- Male and female Fischer 344 rats and B6C3Fl mice chloro-1,1,1-trifluoroethane), FC-134a (1,1,1,2- were administered methyl carbamate in water by tetrafluoroethane) and FC-143a, five days a week gavage in a 90 day subchronic toxicity study. for one year. One unclosed and two dosed control The animals were necropsied and given a complete groups were employed. The study was terminated microscopic examination. In rats, compound­ at 126 wks. Rats dosed with FC-31 showed a high related histopathologic lesions were found in the incidence of stomach squamous cell carcinomas and liver, bone marrow, spleen, and salivary gland of fibrosarcoffias. Female rats dosed with FC-133a both sexes and in the testes of the males. The had a markedly increased incidence of uterine most significant lesions occurred in the liver and adenocarcinomas whilst the males of the group had were characterized by proliferative changes of a greatly increased incidence of testicular hepatocytes consisting of basophilic and other interstitial cell tumours and all had marked foci of cellular alteration, frequent mitoses with testicular atrophy. Carcinogenicity was not seen atypical forms, hepatocellular necrosis of a focal with any of the other fluorocarbons. These nature, and pigmentation of Kupffer's cells. results correlate favourably with the short-term Other toxic changes observed in rats were diffuse tests except for FC-133a. This discrepancy can atrophy of the bone marrow, and acinar atrophy, be accounted for by proposing that FC-133a is an duct hyperplasia and inflammation of salivary epigenetic carcinogen. glands of both sexes, and excessive pigmentation of the spleen of males. In male but not female mice, minimal to mild hepatocellular necrosis and/ or increased mitotic rate of hepatocytes were observed in animals receiving 375 to 1500 mg/kg. 375 PHQRBQLESTERS INCREASE PROSTAGLANDIN PRODUCTION An hepatocellular adenoma was also observed in a IN HUMANOSTEOSARCOMA CELLS. M.A. Shupnik and A.H. high dose male mouse. Because of the patchy Tashjian, Jr., Lab. of Toxicol., Harvard Sch. Pub. Bl.th. and Dept. of Pharmacol., Harvard Med. Sch., nature of the former lesion, it is not known Boston, MA. Sponsor: J.L. Whittenberger whether it is directly attributable to the test substance. Phorbol esters (PE) are potent tumor-promoting The proliferative nature of the hepatic lesions and inflammatory agents in many tissues. suggest that methyl carbamate may be biologically 20-[JHJ-phorbol 12,13-dibutyrate (PDBu) binds to active in rodents. Chronic studies to determine specific high affinity sites on several clonal the carcinogenicity of this compound are in lines of human osteosarcoma cells (G-292, TX-4, progress. MG--63 and SaOS) with apparent Kas of 8-20 nM. This study was performed under the Carcinogenesis Treatment with PDBu increased prostaglandin E2 Bioassay Program of the National Toxicology (PGE2) production in 3 of the 4 cell lines; SaOS Program t~rough a contract with Tracor Jitco, Inc. cells were unresponsive. Maximal increase in PGEz production was 3- to 5-fold above control values, and ED5os were 10-20 nM, in agreement with the Kas for binding to the PDBu receptor. Phorbol 377 A THREEMONTH SUBCHRONIC DIETARY STUDY WITH 12-0-myristate-13-acetate (PMA) the most potent TERT-BUTYLPHENYLDIPHENYL PHOSPHATE (TBP) IN phorbol ester in terms of tumor promotion, was 6 IN RATS. S.E. Hastings, R.W. Thomassen, times more potent than PDBu in increasing produc­ M.W.Sauerhoff and G.M. Zwicker. tion of PGE2 and in competing for [3H]PDBu Stauffer Chemical Company,Environmental Health binding, while the biologically inactive parent Center, Farmington, CT

106 TBP, a mixed tri-aryl phosphate fire resistant of a-oxo non-quaternary thiohydroxamates of value hydraulic fluid, was administered in the diet in the treatment of poisoning due to cholinester­ to Sprague-Dawley derived rats for 90 days. ase inhibition. Rats {20/sex/dose) were administered diet con­ taining 100, 400 or 1600 ppm TBP. Rats were examined daily and given physical exams weekly. Individual body weights and food con­ 379 KINETICANALYSIS OF SPECIESDIFFERENCE rn ACETYL­ sumption were recorded weekly. Parameters eval­ CHOLINESTERASESENSITIVITY TO ORGANOPHOSPHATEIN­ uated at the mid-point of the study and at ter­ SECTICIDES.C. Wangand S.D. Murphy, Div. of Tox. mination included hematology, blood chemistry Univ. of Texas Med. Sch., Houston, TX. and urinalysis. All animals were examined com­ Previous studies showed that acetylcholinester­ pletely at necropsy with selected organs weighed ase (AChE)from different species had different Tissues were fixed for light microscopic examin­ sensitivity to inhibition by paraoxon and the oxy­ ation. Body weights and food consumption in all gen analog of azinphos methyl (Gutoxon) (Murphy, treated groups were comparable to the controls. et al, TAP, 12:22, 1968). In this study, the sen­ The daily average consumption of TBPwas sitivity of brain tissue AChEfrom rat, chicken, 6.6, 26.6 and 107.5 mg/kg at 100, 400 and and catfish to inhibition by methyl paraoxon, 1600 ppm respectively. The appearance, behavior paraoxon, Gutoxon, and ethyl Gutoxon were studied. and mortality of treated males and females were The most striking differences were: 1) Catfish similar to their respective controls. There brain AChEwas 25X less sensitive to methyl para­ were no toxicologically significant changes in oxen than chicken brain AChE,and 29X less sensi­ the hematology or blood chemistry parameters. tive to paraoxon. 2) Chicken brain AChEwas 28X While brain and RBCcholinesterase were un­ less sensitive to Gutoxon than rat brain AChE,and changed, plasma cholinesterase was decreased up llX less sensitive to ethyl Gutoxon. The kinetic to 41%in the 1600 ppm males and females. Urin­ parameters, affinity constant (Ka) and phosphory­ alysis did not reveal any toxicologically signi­ lation constant (Kp), were determined by the me­ ficant findings. Gross necropsy examination re­ thod of t1ain and Iverson (Biochem. J., 100:525, vealed no significant lesions. The mean abso­ 1966). For methyl paraoxon, Ka was 242 µMand Kp lute liver weight was increased in the 1600 ppm was 14.7 min-1 for catfish brain AChE;for chicken males and females. There were no histopathol­ brain AChE,Ka was 25 µMand Kp was 37.9 min-1. ogically significant findings in any tissues Catfish brain AChEhad 9.7X less affinity for examined. These data demonstrate the relatively methyl paraoxon and 2.6X slower rate of phosphory­ low subchronic toxic potential of TBP. lation than chicken brain AChE.The overall dif­ ference is 25X, which agrees ~ith the difference in ~C5o's (chicken 1.48 X 10- M; Catfish 3.73 X 10- MJ. For paraoxon, Ka=202,8 µMand Kp=22.2min-l for catfish brain AChE;for chicken brain AChE, 378 SR-3018, A NEWNON-QUATERNARY THIOHYDROXIMATE Ka=6.3 µMand Kp=25.2min-1, For Gutoxon, Ka=88.7 CHOLINESTERASEREACTIVATOR. R. Howd, R. Kenley, µMand Kp=8.3 min-1 for chicken brain AChE; for and C. Mosher. SRI International, Menlo Park, CA. rat brain AChE, Ka=31.5 µMand Kp=56,8min-1. For SPONSOR: D.C.L. Jones. ethyl Gutoxon, Ka=58.4 µMand Kp=24.1min-1 for In the search for improved reactivators of acetyl­ chicken brain AChE;for rat brain AChE, Ka=108.1 cholinesterase (AChE) inhibited by organophosphor­ µMand Kp=365,3min-1, These kinetic studies sug­ us compounds{ we synthesized pBrC6 H4 C(O)C(NOH)S­ gest that the active site of AChEdiffers between (CH2)2N(C2H5J2·HCl, (SR-3018), and evaluated its species. (Supported by a SOTFellowship sponsored ability to reactivate AChE inhibited by ethyl p­ by The Procter and Gamble Companyto C.W. and by nitrophenyl methylphosphonate (ENMP). In vitro, Research Grant ES01831from NIEHS.) SR-3018 was about l/30th as potent in reactivating eel AChE as 2-PAM. However, due to the non-quater­ nary nitrogen of SR-3018 it appeared likely to pass the blood-brain barrier, unlike 2-PAM, thus being of potential use in reactivating inhibited 380 EFFECTS OF CHLORDECONEAND ITS METABOLICAND brain AChE. Tests of SR-3018 in male Swiss-Wel:ster PHOTODEGRADATIONPRODUCTS ON ISOLATEDRAT LIVER mice showed a SC LD50 of about 460 mg/kg and, at MITOCHONDRIA. S. D. Soileau and D. E. Moreland, 300 mg/kg, a protective ratio against ENMPof 1.8 Toxicol. Frog., Dept. Crop Sci., N.C. State alone, and 5.6 with 25 mg/kg of atropine sulfate. Univ., Raleigh, NC CE.,._Hodison) Striking behavioral effects, including stereotyp­ ies, tremors, and Straub tail occurred at this Partitioning of chlordecone, chlordecone alco­ high dose of SR-3018, suggesting penetration into hol, monohydrochlordecone, and dihydrochlordecone the brain. Against 500 µg/kg of ENMP( 1.5 LD50), (4 to 100 µM) into isolated rat liver mitochon­ 250 or 300 mg/kg of SR-3018 had a less protective dria increased the permeability properties of the effect and a shorter duration of action than a inner membrane as evidenced by: inhibition of high dose, 100 mg/kg, of 2-PAM. However, at both valinomycin-induced swelling, induction of pas­ 250 and 125 mg/kg, SR-3018 reactivated peripheral sive swelling, oxidation of exogenous NADH, and AChE as well as 100 mg/kg of 2-PAM in mice killed induction of lysis. Associated with the increase 5 hrs after being given these drugs 30 sec before in permeability was stimulation of state 4 and 500 µg/kg ENMP. SR-3018 significantly reactivated inhibition of state 3 respiration for the oxida­ brain AChE at 125 mg/kg, but reactivation was less tion of succinate and glutamate. Except for the at 250 or 300 mg/kg, suggesting the possibility inhibition of valinomycin-induced swelling, the that effects on AChE might contribute to its tox­ order of potency for all assays was chlordecone icity at high doses, although it does not inhibit alcohol~ chlordecone > monohydrochlordecone > eel AChE in vitro. The ability to reactivate brain dihydrochlordecone. Mirex, which has the same AChE might make SR-3018 or another of our series carbon skeleton as the above compounds, but con-

107 tains no oxygen, did not affect any of the edema. Cerebrospinal fluid pressure (CSFP) in reactions at a saturating concentration of 40 µM. rats 24 hours after a 1 mg/kg dose of The hydrated ketone or hydroxyl moiety and the branethalin was 251 + 22.7 mmof water compared chlorine content appear to be responsible for to a control value or 68 + 5.0. CSFPreturned lytic and inhibitory potency. Lysis was associ­ to control levels 7 days after treatment. ated with leakage of water-soluble matrix enzymes, Daily treatment of bromethalin intoxicated rats but not solubilization of integral proteins. with dexamethasone s.c. reduced the time in \..tlich CSFPreturned to control levels to 4 days. Intravenous infusion of hypertonic urea 24 hours after bromethalin treatment reduced 381 ASCORBICACID AND PARAQUAT:OXYGEN DEPLETION CSFPto near control levels after 30 minutes. WITH CONCURRENTOXYGEN ACTIVATION, M.R. Montgom­ Ho1Ever, \..tleninfusion was discontinued, CSFP ~' J, Furry, S.J. Gee, and R.I. Krieger. VA rose to near starting levels at approximately Hospital and Depts. of Phann. and Comprehensive the same rate. These data suggest that the Med., Univ. of S. Florida, Tampa, FL and Dept. cerebral edema produced by sub-lethal doses of of Veterinary Med., Univ. of Idaho, Moscow, ID bromethalin can be ameliorated by treatment Ascorbic acid and paraquat produce an efficient with steroid therapy and/or an osmotic diuretic. redox pair which will deplete oxygen from physi­ ological buffer systems. The reaction rate is three fold more rapid in sucrose buffer than in Krebs-Ringer-phosphate; in distilled water the 383 DIFFERENTIAL TOXIC RESPONSESOF RATS AND GERBILS rate is very slow but measurable. This reaction TO THE INSECTICIDE CARBARYL. I. S. Silver and is partially blocked by superoxide dismutase H. W. Dorough, Graduate Center for Toxicology, (38% decrease) or catalase (36% decrease) and is University of Kentucky, Lexington, KY. potentiated by the hydroxyl radical scavenger, In the present study, the toxicity, anticho­ dimethyl sulfoxide (86% increase). Mitochondria linesterase activity, and ester hydrolysis rates isolated af~er incubation of rat lung slices of carbaryl (1-naphthyl N-methylcarbamate) in with 1.0mM paraquat and lOmM ascorbate were female rats (R) and gerbils (G) were compared. unresponsivi to addition if ADP. Also, metabo­ Carbary! was more toxic to gerbils than rats. A 1 lism of (1- 4c)- and (6- C)-glucose was inhib­ 300 mg/kg oral dose killed 100% (20/20) and 70% ited by 50% in the same lung slice preparations. (14/20), respectively, and the 7-day oral dose These results suggest a synergistic interaction LDso was approximately 80 mg/kg/day for the ger­ of ascorbate and paraquat which results in dis­ bil and 250 mg/kg/day for the rat. When carbaryl­ ruption of subcellular energy metabolism. Para­ naphthyl-14C was given orally (50 mg/kg), maximum quat accumulation, an active transport process 14C-carbaryl equivalents occurred in the blood of the pulmonary cell membrane, was also inhib­ after 15 min (R, 14 µg/ml; G, 9 µg/ml) causing ited 40% by the addition of ascorbate. These re­ 46% blood ChE inhibition in the rat and 25% in sults suggest that the previously reported in the gerbil. Brain ChE was similarly inhibited vivo potentiation of paraquat toxicity by ascor­ (R, 56%; G, 24%). In vitro assays revealed no bate may be related to either: 1) a decreased inherent differences in the sensitivity of blood subcellular oxygen availability, or 2) the pre­ and brain ChE to carbaryl. Carbamate ester hy­ sence of activated oxygen species, or 3) both. drolysis, determined by measuring respiratory 14 Supported by grants from VA Medical Research 14C02 from carbaryl-carbonyl- C, was much great­ and NIH Biomedical Research Development Grant er in the gerbil. After 4 and 24 hrs, the per­ 1 S08 RR09073-01A2. cent of dose hydrolyzed was 48 and 65 for the gerbil, and 17 and 28 for the rat. Since ester hydrolysis destroys the antiChE activity, these data might suggest the gerbil to be more tolerant 382 THEMETABOLISM OF BROMETHALIN AND ITS EFFECTS to carbaryl poisoning than the rat. The more ONOXIDATIVE PHOSPHORYLATION ANDCEREBROSPINAL rapid hydrolysis rate may contribute to the lower FLUIDPRESSURE. L. D. Cherry, M. D. Gunnoe and levels of carbamate in gerbil blood and the cor­ R. B. L. van Lier (D. G. Hoffman), Toxicology responding lower level of ChE inhibition in the Division, Lilly Research Laboratories, Eli blood and brain. Nonetheless, the toxicity data Lilly and Co., Greenfield, IN. clearly show the gerbil to be the more sensitive of the two species to carbaryl, and it is assumed Branethalin, N-methyl-2,4-dinitro-N­ that the toxicological consequences of the ChE (2,4,6-tribroroophenyl)-6-(trifluoromethyl)benzen­ inhibition in the gerbil is far greater than that amine is being developed as a new single feeding in the rat. Symptoms of poisoning were the same rodenticide. Previous data from this laboratory in both species, thus reducing the possibility (The Toxicologist 1:114, 1981) suggests that that carbaryl acted other than to inhibit ChE in bromethalin is a potent uncoupler of oxidative the gerbil. (Supported by EPA Grant No. R805143). phosphorylation in liver and brain mitochondria in vitro. Treatment of rats with SKF-525Acan substantially reduce or delay acute bromethalin ·toxicity suggesting that the compoundmust be 384 THE INFLUENCEOF DIETARY FIBER ON THE ABSORPTION metabolized to the active toxicant. In vitro AND DISPOSITION OF MIREX. J.D. deBethizy and J.C. studies of demethylated bromethalin have shown Street, Toxicology Graduate Program, Utah State this compoundto be 500-1000 times more potent University, Logan, UT 84322. than bromethalin as an uncoupler. Using malate as substrate, a 50 percent decrease in the P/0 Selected xenobiotics are being utilized as models ratio was observed at 5 pmol/mg protein. of putative colon carcinogens in a study of the The microscopically detectable toxic effect influence of major types of dietary fiber upon of bromethalin is characterized by intramyelinic xenobiotic toxicokinetics. Adult, male Wistar ma rats were fed standardized, isocaloric diets con­ with the rapid biotransformation of lindane in taining no fiber or 15% (w/w) cellulose, lignin, these mice, leads to impaired conjugation of hemicellulose(Metamucil), or pectin for 30 days, polar metabolites, elevated tissue binding of An additional group was fed lab chow ad libitum these metabolites with reduced metabolism and/or as a reference control, Mirex (l0mg/kg) was ad­ excretion. ministered by gavage and plasma samples collected over 15 days, Plasma samples were analyzed for the parent compound by GC-ECDfollowing hexane ex­ traction, The toxicokinetic interpretation was 386 CORRELATIONOF THE ESTR0GENICACTIVITY OF o,p'­ performed with the aid of the computer program, DDTWITH NUCLEAR LEVELS OF ESTROGENRECEPTOR. AUTOAN2,generating models for each fiber type. A. K. Robison, V. R. Mukku, J. S. Ireland, and Equilibrium dialysis binding studies were conduc­ G. M. Stancel {Sponsor: S. D. Murphy), Dept. of ted with mirex and each dietary fiber type. Hemi­ Pharmacology, Univ. Texas Medical School, cellulose and pectin-feeding produced lower peak Houston, TX. plasma concentrations of mirex than control and o,p'-DDT and related pesticides produce estro­ cellulose-fed animals, Lignin produced higher genic effects in vivo and in vitro. To deter­ peak plasma concentrations than control(no fiber) mine if these effects resuTt from the interac­ or any other fiber type. The disposition of mirex tion of the pesticide with the classical estro­ was best described by a two-compartment open model gen receptor system we have measured estrogenic with first order absorption for all groups. Lig­ responses of the rat uterus and the nuclear trans­ nin pro~yced a faster rate of elimination (k = location of estrogen receptors as a function of 0.043hr -i of mirex than no fiber control e 1 the dose of o,p'-DDT administered both in vivo (0.011hr ), The absorption of mirex from the and in vitro. Nuclear levels of estrogen recep­ GIT was similar for all groups fed the experimen­ tor were measured by an exchange assay using tal diets (ka=0.340), which wa~ lower than the isolated nuclei. After in vivo administration lab chow-fed group (k =0.500hr 1), Lignin produc­ of o,p'-DDT, nuclear leveTs~estrogen receptor ed a faster rate of t~ansfer of mirex from the reach a maximum in 3 hours, and increases in uter­ ine weight and DNAsynthesis reach a maximum in deep compartment into the central compartment(k 21= 0.0145) than the other fiber groups(k =0,005). 24 hours. In the dose range of 10-1000 mg/kg These effects can be partially attributed2 to dif­ o,p'-DDT there is a high degree of correlation ferential binding to the fibers. Fiber type ef­ {r=0.98} between these two .growth responses and fects the disposition of mirex in rats. (Supported levels of nuclear estrogen receptor. Following a 1 hour incubation of uteri with o,p'-DDT in In part by NCI grant CA-25580,) vitro we also measured nuclear levels of estrogen receptor and the induction of a specific estrogen inducible protein (IP) using a double label iso­ tope procedure. In the concentration range of 385 THE EFFECTOF PHENOTYPICDIFFERENCES AND SUB­ 30-1000 uM there is also a high degree of corre­ CHRONICPRETREATMENT ON THE METABOLISMOF lation (r=0.96} between these two parameters. LINDANEBY INBREDMICE, R.W. Chadwick, M.F, These results provide strong support for the Copeland, R. Froehlich, J.D. Perry and G.L. hypothesis that the estrogenic effects of o,p'­ Wolff. HERL, USEPA, Research Triangle Park, NC DDTare indeed mediated by interaction of the In a collaborative effort with NCTR, the pesticide with the classical estrogen receptor comparative effect of phenotypic differences on system in target tissues for this hormone. (Sup­ the metabolism of lindane by female (YS x VY) ported by NIH grants HD-08615, HD-00099 and F hybrid mice fed 160 ppm lindane in their diet ES-07090.) for1 13, 26, 52, 78, and 104 weeks is being studied to determine whether enzyme activity and/or metabolite profiles can be correlated with the incidence of liver neoplasms. Twenty 387 ORGANOPHOSPHORUS - INDUCED NEURITE DE­ four hours prior to sacrifice, both controls GENERATION IN HUMAN NEUROBLASTOMA and mice, fed 160 ppm lindane for 13 weeks, CELLS. D.G. Graham and M.B. Abou-Donia, Depts. were d£~ed p.a. with 18.2 mg (containing 60.2 of Pathology and Pharmacology, Duke University µCi (U C)lindane)/kg. Total excreted radio­ Medical Center, Durham, N.C. 2771 0. activity ~as significantly reduced in pretreated yellow (A Y;a) and black (a/a) phenotypes when Human neuroblastoma cells have been developed as compared with controls. The treated yellow an in vitro model for organophosphorus-induced de­ mouse excreted significantly reduced levels of layed neurotoxicity assuming ( l) that neurite degen­ radioactivity in urinary fractions representing eration in vitro is analogous to axonal degeneration in the conjugated alcohol metabolites, the conju­ vivo, and {2}that reductions in neurotransmitter gated chlorophenols and the polar metabolites uptake can serve as quantifiable indices of neurite when compared to controls. On the other hand, degeneration. Evidence for neurite degeneration the treated yellow phenotype excreted signi­ after exposure of LA-N-1 cells to agar-adsorbed ficantly more free alcohols and had signifi­ organophosphorus esters (OP's) has been obtained by cantly higher tissue levels of radioactivity electron microscopy. Homogenization and varifi­ than controls. Treated black (a/a) mice had cation of neurite cytosol and swelling of neurite significantly higher dechlorinase activity mitochondria were apparent when compared to the than controls and phenotypic variations in the cytosol and mitochondria of adjacent cell bodies or excretion of 9 lindane metabolites were ob­ with neuri tes from agar-exposE_r;f controls. Re­ served. Results from this study indicate that ductions in cocaine-inhibi table [ H] norepinephrine subchronic lindane pretreatment of the yellow uptake occurs after 3 hour exposures of these cells to (Avy/a) mouse, which is the phenotype most a variety of OP's, and the reductions follow pseudo­ susceptible to hepatoma formation, together first order kinetics. That the hydrophobic OP's enter

109 the neuroblastoma cells adsorbed to agar was shown antiviral activity and teratogenic potential. in the demonstration that leptophos oxon at 0.5 f:M The studies provided animal safety data for resulted in 16.9 ±6.9% (p>0.05) inhibition of [ HJ drug evaluation in neonatal herpes, a life­ norepinephrine uptake when adsorbed to 6.7 µg of threatening condition. Groups of 2-day old agar/ml, increasing to 31.5 ±4.6% (p<0.01) with 67 rats were given 5 consecutive daily ip doses µg agar/ml and 89.8±1.8% (p<0.001) with 333 µg of 400, 200, 100 and 25 mg of Ara AMP/kg agar/ml. Leptophos oxon was alone among leptophos or 4 mg of Ara-C/kg. Observations were made and its phosphorus-containing degredative P:f.ducts in daily for clinical toxicity and animals from its ability to result in reductions in [ H] nor­ each group were necropsied at 7, 14, 21 epinephrine uptake, pointing to this metabolite as the and 35 days of age. Clinical, pathologic or ultimate neurotoxin in leptophos-induced delayed developmental abnormalities were absent in neurotoxicity. Initial experiments have shown that v1 darab i ne phosphate-treated groups and the in vitro effect of OP's is not shared by phenyl organogenesis was comparable to controls. benzylcarbamate. (Supported by EPA Grant Ara-C caused significant delay in fur and R806400030). body growth with impairment in organo­ genesis. These phenomena were characterized by cerebellar cortical hypoplasia and dys­ plasia of th~ retina and renal cortex. 388 THE SUBACUTEORAL TOXICITY OF A 5: 1 COMBINATION Delayed cerebellar development was evident OF SULFADIMETHOXINEANDORMETHOPRIM IN THE DOG with Ara-C by a marked reduction in size of T. J. Hayes, R. M. McClain, and H. Westen, De­ cerebellar folia and hypo-cellularity of the partment of Toxicology and Pathology, Hoffmann­ internal and external granular layer with La Roche Inc., Nutley, N. J. delayed involution of the latter. Retinal dys­ Tablets of sulfadimethoxine and ormethoprim plasia was bi lateral and extensive and char­ (5:1) were given orally to beagle dogs at dosages acterized by rosette formation of photo­ of O (control), 0 (excipients), 27.5, 82.5 and receptor eel Is. Immature renal tubular and 137. 5 mg/kg/day. Necropsi es were done after 8 glomerular structures were indicative of weeks of treatment to assess the effects of impaired nephrogenesis. In the absence of treatment, and after a further 12 weeks without reference histopathological changes with treatment to assess the reversibility of effects. vidarabine phosphate, the magnitude of dose Treatment re 1ated effects were principally levels provide an adequate margin of safety. those of hypothyroidism. High-dose treatment was associated with: marked thyroid hyperplasia, enlarged thyroid stimulating cells in the pitui­ tary, decreased serum T3 and T4 , e 1evated serum 390 INTRAVENOUSTOXICITY OF WR-2721 (NSC-296961) cholesterol, decreased hemoglobin and hematocrit, IN CDF MICE AND BEAGLEDOGS. G.W.Thompson, 1 reduced heart rate and R wave amp1 i tudes (ECG). G.N.Rao, B.G.Boysen, M.W.Balk, A.E.Fekete, Other high-dose findings were increased liver B.C.Dickie, S.M.Glaza, Raltech Scientific Servi­ weights and multifocal vacuolation of hepato­ ces, A Division of Ralston Purina Company, Madi­ cytes, thymic involution, marginal testicular son, WI. and K.S.Greenspun, Battelle Toxicology atrophy, decreased serum folate, and minimal eye Program Office, Vienna, VA. (Spon: D.D.Dietz) changes. After 12 weeks without treatment, the WR-2721 ((Ethanethiol,2-[(3-aminopropyl)amino]-; thyroids showed functional recovery but were dihydrogen phosphate ester)) is a radioprotector somewhat enlarged due to abundant colloid drug that enhances normal cells' defenses against storage. Minimal eye change continued to be radiation while not protecting tumor cells. It is present in 1 dog, but not in another. All other the first radioprotective drug approved by FDA changes were reversible. for use in humans. Mid-dose treatment produced no changes in The lethality of single intravenous doses of eyes, hemoglobin, thymus, liver or testes. Other WR-2721 was determined in mice. The defined LD1o, changes 1i sted above were observed in reduced LD50, and LDgo in mice were: males-539.8, 604.6, incidence and magnitude. Changes at the low-dose and 677.3 mg7kg; females-471.5, 557.1, and 658.2 were limited to elevated cholesterol, increased mg/kg, respectively. Mice were given the 0.5 LD10, thyroid and liver weights, enlarged thyroid LD10, and LD5o doses and evaluated for toxicity. stimulating cells in the pituitary, and mild Dogs were given 0.1 mouse LD10 to 1.3 mouse LD10 follicular thyroid hyperplasia. doses adjusted to the body surface area of dogs. These data further characterize the syndrome Clinical signs seen in the mice included hypoac­ of sulfonamide-induced hypothyroidism in dogs and tivity, ataxia, decreased limb tone, impaired demonstrate the reversibility of these effects. righting reflex, dyspnea, bradypnea, and prostra­ tion. Surviving mice at termination (29 days) ap­ peared normal and had normal weight gains. Clini­ cal signs seen in dogs included emesis, hypo­ thermia, hypoactivity, and ataxia. High BUN and 389 SYSTEMIC TOXICITY STUDY OF NUCLEOTIDE SG0T values in treated mice at Day 2 indicated ANALOGANTIVIRAL AGENTS IN NEONATAL possible nephrotoxicity. Treatment related hema­ RATS. A. Gough, N. Barsoum, S. Gracon and tology and clinical chemistry changes were not J.M. Sturgess Warner-Lambert/Parke-Davis observed in dogs. Pathological changes caused by Res. Inst., Sheridan Park, Miss., Canada WR-2721 in mice were lymphoid necrosis of lymph­ (Sponsor: F. de la Iglesia) oid organs and renal tubular epithelial degenera­ The effects of a new antiviral agent, tion at interim sacrifice, but no drug induced vidarabine phosphate on postnatal growth lesions were seen at termination. Drug induced and development of the rat were evaluated lesions seen in dogs included renal tubular epi­ and compared to 1- 13 -D-arab i nofuranosy 1- thelial necrosis; congestion, hemorrhage and ede­ cytosi ne (Ara-C), an agent with established ma of lungs, liver, kidney, gastrointestinal

110 tract and lymph nodes. The 0.1 mouse LD10 dose tions 2 hr after administration of the daily dose (males-8.0 mg/kg, 160 mg/M2; females-7.0 mg/kg, when compared to predose concentrations. Elec­ 140 mg/M2) was nontoxic in dogs. trocardiograms taken at that time showed slight Supported by NCI Contract No. NOl-CM-17365. increases in QT intervals in high-dose dogs throughout the dosing period and in intermediate­ dose dogs after 6 months of dosing. Pathologic evaluation of animals after 1 year of dosing did 391DIFFERENTIAL TOXICITY OF N-(2-CARBOXYPHENYL) not reveal any compound-related gross or histo­ RETINAMIDEAND N-(4-CARBOXYPHENYL)RETINAMIDE IN pathologic changes in the heart, skeletal muscle, RATS. D.Emmerling and P.Kurtz. Battelle Memorial liver, or any other organ. No serious adverse Institute, Columbus, OH. Sponsor: G. Fisher effects were noted in dogs given the low dose of The toxicity of two synthetic retinoid analogs was tiamulin for 1 year. Results of this study examined and compared in 91-day gavage studies support the use of tiamulin in food-producing with Sprague-Dawley rats. Ten male and ten female animals. rats were given either N-(2-carboxyphenyl) retina­ mide (2CPR) or N-(4-carboxyphenyl) retinamide (4CPR) at dosages of 4, 14, 50, or 150 mg/kg. Treatment with 4CPR produced typical manifesta­ 393 TOXICOLOGICALEVALUATION OF MARCELLOMYCIN- AN tions of retinoid intoxication (bone fracture, ANTINEOPLASTICANTHRACYCLINE ANTIBIOTIC. R. A. dermal lesions, and anemia) but the required dose Buroker, D. F. Johnson, C. R. Comereski, G. H. levels were at least three times higher than those Hottendorf and H. Madissoo, Department of Toxi­ required in positive control studies with all­ cology, Bristol Laboratories, Syracuse, NY. trans retinoic acid. Furthermore, the two car­ Marcellomycin, a trisaccharide anthracycline an­ boxyphenyl retinamides were not equally toxic. tibiotic possessing antitumor activity, was iso­ Administration of 4CPR produced a statistically lated from Actinosporangium ~- The toxic ef­ significant growth depression in male rats at the fects of marcellomycin observed in single-dose 50 and 150 mg/kg dose levels while no effect on toxicologic studies in mice iv (14.35-22,55 mg/ growth was observed for either sex receiving 2CPR. kg) and dogs iv (2.05-4.51 mg/kg), and in multi­ Significant reductions in HGB, HCT, and RBC values ple-dose studies in dogs iv (12 doses 0.49-1.48 were seen in both sexes receiving the same two mg/kg 2X/week) and rats sc (9 weekly doses 4.4- high doses of 4CPR but the hemograms of the 2CPR 13.2 mg/kg), were dose-related and primarily man­ animals showed no significant effects. Radio­ ifested by suppressive action on myeloid, espe­ graphic examination of the long bones indicated cially the erythrocytic and thrombocytic series, fractures in the 4CPR-treated females at 50 and and on lymphoid tissues. However, initially a 150 mg/kg but no fractures in either sex given the neutrophilic leukocytosis was observed in all 2CPR analog. The microscopic evaluation of tissue species, which was considered to be due to mobi­ from high dose animals in each study did not indi­ lization of the marginal and bone marrow neutro­ cate treatment-related changes. It therefore phil pools. Subsequently, suppression of leuko­ appears that N-(2-carboxyphenyl) retinamide is cytes was seen in dogs and in rats prior to death. less toxic than the close structural analog, Other toxicities observed included enteropathy, N-(4-carboxyphenyl) retinamide. (Supported thyroid follicular proliferation, atrophy of the by NCI Contract No. NOl-CP-85650.) prostate and seminal vesicles, uterine hypoplasia, testicular and pancreatic degeneration, inflamma­ tion and necrosis at injection sites, hemorrhage in various organs, and serofibrinous edema in the sc rat study. In general, these toxicities were 392 TIAMULIN: ONE-YEARORAL TOXICOLOGIC AND PATHO­ reversible in surviving animals during 4 to 6.5 LOGIC STUDYIN YOUNGDOGS. A. C. Stevens, week recovery periods. Significant cardiotoxi­ Y. H. Yoon, P. L. Sibley, and G. R. Keim, city was not demonstrated with marcellomycin. Departments of Toxicology and Pathology, The Squibb Institute for Medical Research, New Brunswick, N. J. Sponsor: J. S. Kulesza Tiamulin is a semisynthetic derivative of the 394 SPECIES DIFFERENCESIN ANEMIACAUSED BY PHTHALA­ antibiotic, pleuromutilin. It is active against mycoplasma organisms and Treponema hyodysen­ ZINE DERIVATIVEANTIHYPERTENSIVE DRUGS. teriae, infectious agents in food-producing ani­ S. Takayama, Y. Akiyama, T. Onodera, and T. mals. A 1-year oral toxicologic and pathologic study in young dogs was undertaken to determine Akimoto, Res. Inst, of Daiichi Seiyaku, Tokyo, the toxic potential of tiamulin and its suita­ Japan. bility for use in food-producing animals. Tia­ mulin hydrogen fumarate was administered orally This experiment was performed to clarify the once daily to groups of eight beagle dogs each at daily doses of 0, 3, 10, and 30 mg/kg. The dogs mechanism of species difference between rats were 3 to 4 months old at the start of dosing. and beagles in anemia caused by hydralazine, One high-dose male died after 5 months of dosing and showed congestion or hemorrhage in most or­ ecarazine, and budralazine, These drugs produced gans, slight focal myocardial necrosis, and gas­ anemia in rats receiving oral doses of 120mg/kg tric mucosal erosions. The remaining high-dose and more, and in beagles given oral doses of 10 dogs survived the 1-year dosing period. High­ and intermediate-dose dogs showed slight to mg/kg and more. Hematological examination revea­ moderate increases in serum LOH, specifically led normochromia, activation of erythropoiesis, isoenzymes LDH4 and LDH5. These dogs also showed slight decreases in serum potassium concentra- and shortening of erythrocyte life span in both

111 animals, and appearance of Heinz bodies in the of radical/peroxide damage. ADRgenerates 02, which can be detoxified to H202by superoxide erythrocytes of beagles. In vitro experiments dismutases (SODs). The essential trace metals, indicated that the erythrocytes of beagles were copper, zinc, and manganese, are associated with SODsand were examined for relationships to ADR more susceptible to ferrihemoglobin formation cardi otoxi city. by 1-hydrazinophthalazine, one of metabolites Weanling male Sprague-DawleyCD rats were given ADRi.p. 2 mg/kg b.w. x 9 days. Diet and of the drugs, than those of rats. environment were trace metal controlled. Cardiac These findings suggest that the ferrihemoglobin CuZn-SOD,total SOD,Cu, Zn, and Mnwere assayed. EKGsand histopathology were scored. SODwas formation by 1-hydrazinophthalazine may play an assayed in solution and in heart homogenate, with important role in species difference in anemia or without ADR. Whole heart Mnwas significantly decreased. caused by hydralazine, ecarazine, and budrala­ There were significant non-specific EKGchanges, zine. but not QRSwidening. Significant histopatho­ logical changes were seen in heart, kidney, and liver. Trends indicate CuZn-SODwas increased 395 FURTHERSTUDIES ON MUSCULAR DEGENERATION. IN RATS in the heart, but total SODwas decreased. 188 AFTERPOSTNATAL TREATMENT WITH 6-MERCAPTOPURINE. x 10-6MADR gave 50%inhibition of CuZn-SOD F.R. Alleva, T. Balazs, and L.J. Slaughter, Food standard in aqueous solution. 3.33 x 10-8MADR and Drug Administration and HowardUniversity, completely inhibited total SODin heart homog­ Washington, D.C. , enate. The results suggest that changes in Mnand Daily subcutaneous injections with 6-mercapto­ SODsmay be related to ADRcardiotoxicity. purine monohydrate (6-MP) at 2 mg base/kg from 2 to 22 days of age has been reported to induce Supported by NIEHSToxicology Training Grant atrophic muscular degeneration with fat replace­ ES-07073. ment in thigh and sublumbar muscles of rats beginning at four months of age (Alleva, et~., The Toxicologist, p. 115, 1981). The present study was done to determine in one experiment whether offspring of male and female rats treated 397Pulmonary Responsiveness to Chlorcyclizine: with 6-MPas above display fatty atrophy of their Comparison Between Newborn and Adult Rats. muscles while a second experiment was aimed at S. Kacew, M.R. Parulekar and R. Narbaitz, Depart­ determining whether treatment of rats with 6-MP, ments of Pharmacology and Anatomy, Univ. of 2 mg/kg sc, daily from 25 to 45 days of age_also Ottawa, Ottawa, Ontario. results in such muscular changes. In the first Chronic ingestion of dietary chlorcyclizine experiment 40 (20 females, 20 males) randomly from 3 to 6 weeks has been reported to produce selected rat offspring of treated parents and 14 an accumulation of foam cells in pulmonary alveo­ (seven females, seven males) offspring (controls) li of adult rats. Recent studies have shown that of saline treated parents displayed normal in newborn rats treated with chlorphentermine muscles when killed at seven months of age. less accumulation of foam cells is apparent in Eight (four females, four males) of the trea~ed pulmonary alveoli as compared to that seen in parents when killed at seven months of age dis­ adults. Experiments were thus undertaken to (a) played, as expected, fatty atrophy of their determine the effects of chlorcyclizine on new­ muscles while eight controls had normal muscles. born rat lung and (b) to compare pulmonary res­ Blood vessels and sciatic nerves taken from the ponsiveness of newborn with adult. Daily, oral hind legs of these treated parents were normal, administration of 30 mg/kg chlorcyclizine for 1 adding further evidence (Alleva, et~., Drug week produced 1-2 foam cells in a few peripheral and Chemical Toxicology, in pressJthat the fatty alveoli of newborn lungs. Treatment with 10 mg/ atrophy results from a primary myopathy. In the kg/day drug for 7 days produced no apparent second experiment all 13 (eight females, five change in newborns. Surprisingly, in adult lungs males) treated as well as 13 saline-control rats of 10 or 30 mg/kg/day chlorcyclizine-treated rats had normal muscles when killed at nine months of no foam cells were present. The 30 mg/kg chlor­ age. Mechanistic studies involving biochemical cyclizine-induced rise in foam cells of newborn and electron microscopic examination of muscle lungs was associated with an increase in incor­ taken from rats treated daily with 6-MP, 2 mg/kg poration of thymidine into DNA and elevation in sc, from 2 to 22 days of age are in progress. ratio of phosphatidylcholine to sphingomyelin con­ tent in newborn lung. In contrast, the absence of apparent foam cells in adults was accompanied by either no change or decreased incorporation of 396 THEEFFECT OF ADRIAMYCINCARDIOTOXICITY ON thymidine into DNA. The concentration of lung ESSENTIALTRACE METAL METABOLISM IN THE RAT phosphatidylcholine to sphingomyelin ratio was HEART- Brian C. Lee & Klaus L. Stenmer. Univ. significantly decreased from control. Our data Cincinnati Medical Ctr., Kettering Lab. show that the pulmonary responsiveness of newborns Cincinnati, OH45267 to chlorcyclizine differs from adults. The decrease in the ratio of phosphatidylcholine to Adriamycin (ADR,doxorubicin HCl) is a sphingomyelin is indicative of respiratory dis­ cardiotoxic anthracycline cancer chemotherapy tress to adults suggesting that newborns may adapt agent that is suspected of interfering with more readily to chlorcyclizine, a finding pre­ essential trace metal metabolism in the heart. viously reported for chlorphenterrnine. (Supported ADRcardiotoxicity is theorized to be a result by the Medical Research Council of Canada).

112 398 COMPARATIVERESPONSE TO NABILONEAND DELTA-9- hormone, nicotine, reserpine, thiouracil TETRAHYDROCANNABINOLIN THE ASSESSMENTOF ABUSE and degenerative lesions by cysteamine. A LIABILITY.' P. J. Pence, L. Lemberger, number of compounds, especially amphiphilic B. J. Cerimele, and R. B. Forney, Department of ones, produce whorls of smooth endoplasmic Pharmacology and Toxicology, Indiana University reticulum detectable microscopically as School of Medicine and the Lilly Laboratory for cytoplasmic inclusions without other cellular Clinical Research, Indianapoli~, IN. changes. Nabilone is a synthetic compound similar in chem­ ical structure to delta-9-tetrahydrocannabinol (THC), the main active ingredient in marihuana. 400 INHIBITION OF RETROGRADEAXOPLASMIC TRANSPORT BY It is a centrally acting cannabinoid which has ACRYLAMIDE. M.S. Miller, T.F. Burks and I.G. been studied clinically because it reduces the Sipes, Program in Pharmacology and Toxicology, nausea and vomiting associated with cancer chemo­ Arizona Health Sciences Center, Tucson, AZ 85724. therapy, even in those patients who are refrac­ Monomeric acrylamide (ACMD) produces central­ tory to conventional antiemetics. Since no di­ peripheral distal axonopathy in laboratory animals rect quantitation of nabilone's effects compared and humans following repeated exposure. The mech­ with THC had been made, a study was designed to anisms by which ACMDinduces axonopathy are make qualitative and quantitative comparisons of currently unknown. However, alterations in axonal certain physiological, psychometric, and psycho­ transport have been suggested to be involved in motor parameters of these drugs: to evaluate (1) the production of axonopathy. Fast and slow abuse liability, (2) effects on psychomotor per­ anterograde axonal transport have been reported to formance of such skills as driving, and (3) ef­ be largely unchanged in ACMDintoxicated animals. fects on heart rate and blood pressure. Treat­ This study addresses the effects of ACMDon retro­ ments were administered both orally (po) and in­ grade axonal transport mechanisms. Retrograde travenously (iv) to 6 subjects in a double-blind axonal transport was measured with purified nerve manner according to a repeated measures design. growth factor (NGF) iodinated by the lactoperoxi­ Subjects were monitored for 8 hours following dase method(~ 80 µCi/mmole). Male Sprague-Dawley treatment administration. Subjects rated the 5 rats (250 g) received 10 consecutive daily doses treatments on a preference scale from 1 (least of ACMD(SO mg/kg/day ip) or saline. One day liked) to 5 (best liked). The mean scores± S.E. after the last dose each animal received a sin~le were (1) 1.17 ± 0.17 for placebo, (2) 2.92 ± 0.38 unilateral injection (20 µl) of a solution contain­ for THC po, (3) 3.17 ± 0.60 for nabilone po, (4) ing 125I-NGF (1 µCi) in the right front footpad. 3.42 ± 0.46 for nabilone iv, and (5) 4.33 ± 0.42 At various times thereafter (4,8,12,24 hours) for THC iv. All 6 subjects stated they would use animals were killed and accumulation of 1251 in THC again either iv or po for its euphorigenic dorsal root ganglia (DRG) C4-C7 was determined by effect; 5 out of 6 subjects stated they would use gamma counting. Retrograde axonal transport of nabilone either iv or po for its euphorigenic ef­ 125I-NGF was also measured in rats immediately fect. No subject would use placebo again. Phar­ following single doses of ACMD(0.5-100 mg/kg ip). macological effects seen and psychomotor data Repeated ACMDadministration (10 x 50 mg/kg) sig­ will be presented. nificantly delayed the appearance of 1251 in DRG C4-C7. Single doses of ACMDinhibited the accumu­ 125 lation of 1 in DRG C4-C7 by up to 90% in a 399 THE EFFECTS OF DRUGSAND CHEMICALSUPON THE dose-dependent manner (ED50 ~ 25 mg/kg ip) at STRUCTUREOF THE ADRENALGLAND. W. E. Ribelin, times corresponding with maximum accumulation in Monsanto Co., Env. Health Lab., St. Louis, MO. saline treated control animals. These data Surveys of the relevant literature indicate that suggest that alterations in retrograde axonal the susceptibility of the endocrine tissues to transport systems may be involved in the etiology of ACMD-induced axonopathy. compound-induced lesions may be ranked in the (Supported by a Graduate Student Development Award following decreasing order of frequency: adrenal, testis, thyroid, ovary, pancreatic to M.S.M. and USPHS Grants DA02163 and ES82130.) islets, parathyroid and pituitary. The first two are by far the most frequently affected. Pathologists not studying endocrine effects are 401 VISUALINDICES OF ACRYLAMIDENEUROTOXICITY IN often unaware of the number of compounds pro­ PRIMATES.W. H. Merigan, E. Barkdo 11, and ducing specific lesions in the adrenal. Most J.P.J. Maurissen, Environmental Health Sciences frequently affected is the zona fasciculata and Center, Division of Toxicology, Department of reticularis. Compounds producing either degen­ Radiation Biology and Biophysics, University of erative or proliferative lesions there are Rochester Medical Center, Rochester, NY. acrylonitrile, aminoglutethimide, aniline, Sponsor: R. Wood chenodeoxycholic acid, cysteamine, dibromo­ While peripheral neuropathy is the most chloropropane, DDD, dilantin, dimethylbenzan­ marked sign of acrylamide exposure, recent thracene, ethanol, fluphenazine, hexadimethrine reports have demonstrated concurrent central bromide, nitrogen oxides, polyglutamic acids, nervous system damage. Weexplored central polyanetholsulfonate plus aminocaprionic acid, nervous system effects by measuring visual poly-(l,5-dimethyl-1,5-diazaundecamethlene thresholds and visual evoked cortical responses dimethobromide), suramin, thioacetamide and in macaque monkeys during-acrylamide thioguanine. Lesions in the zone glomerulosa administration. Thre~ monkeys were given 10 are produced by aminoglutethimide, aniline, mg/kg/day acrylamide orally 5 days/week until ethanol, hexadimethrine bromide and Ponceau they were markedly ataxic (330 to 470 mg/kg SX (F.D. and C. Red #4). Proliferative or total dose). One monkeywas then sacrificed hypertrophic changes in the medulla are produced and the remaining two monkeys were studied by o-chlorobenzylidene malononitrile, growth during a 140-day recovery period. A fourth

113 monkey, a time matched control, received S. Joynes, Division of Drug Biology, Food and vehicle. Drug Administration, Washington, D.C. The three experimental monkeys showed Phenylpropanolamine (PPA), an appetite supres­ altered visual thresholds and evoked potentials sant, decreased spontaneous motor activity (SMA) well before overt signs of toxicity. Visual in adult and aged hamsters (L.R. We~ss, Toxi­ acuity and flicker-fusion thresholds were cologist 1, 20, 1981). This report compares the reduced and the latency of pattern-reversal effects of ephedrine (EPH), a chemically related evoked potentials increased. After dosing was sympathomimetic, and d-amphetamine (DAP), a CNS terminated in two monkeys, the latency of stimulant-anorexiant with abusive potential, with evoked potentials and the flicker-fusion PPA on SMA measured in BRS activity chambers. thresholds returned to control values within a The drugs were administered by gavage in a volume few weeks. Visual acuity of both monkeys, on of 10 ml of water/kg to adult hamsters 30 min the other hand, showed some recovery and then prior to testing. Activity was recorded for 15, stabilized below control values. 30, 45, and 60 min and analyzed as cumulative and These results indicate that visual measures noncumulative counts for each time period. PPA are appropriate for assessing effects of doses were 25, 50, 100, and 200 mg/kg compared to acrylamide on the central nervous system. 12.5, 25, 50, and 100 mg/kg for EPH and 1.6, 3.1, (Supported by grants ES-01885, ES-01248, 6.3, 12.5, and 25 mg/kg for DAP. These were run ES-01247 and in part under Contract No. with water-treated controls. A depression of SMA DE-AC02-76EV03490with the U.S. Department of was noted at all doses of PPA being significant Energy.) over the entire 60 min period for cumulative activity and for noncumulative counts at 15, 30 and 45 min. In contrast, EPH caused pronounced depression of SMA at 12.5 and 25 mg/kg doses of 402 EFFECTSOF ACRYLAMIDEMONOMER ONSENSORY both types of activity but no significant changes THRESHOLDSIN MONKEYS. Maurissen, J.P.J., and at 50 and 100 mg/kg. DAP showed stimulation of Weiss, B., Div. Toxicol., Env. Hlth. Sci. Ctr., SMA in doses above 3.1 and these were signifi­ Dept. of Radiat. Biol. & Biophys., Univ. of cant at 45 and 60 min for noncumulative counts. Rochester School of Medicine and Dentistry, At the 6.3, 12.5, and 25 mg/kg doses, all Rochester, NY. Sponsor: G.G. Be~g. hamsters showed stereotyped amphetamine Sensory disturbance characterizes acrylamide­ behavior. The results of this comparative study induced peripheral neuropathy in humans. Quan­ indicates that PPA seems to act on the brain in a titative evaluation of vibratory and electrical manner that is pharmacologically different from sensitivity has been carried in four monkeys. DAP but similar to EPH and suggests that the Each monkey served as its own control. Monkeys possible appetite suppressant effects of PPA may were trained to report detection of vibratory not be associated with stimulation or an and electrical stimuli. Sensitivity thresholds amphetamine-like action in the CNS. were determined with a tracking procedure be­ fore, during and after dosing. Monomeric acrylamide was dissolved in water and mixed 404 TIME-COURSEAND DOSE-RESPONSE ASSESSMENT OF with fruit juice just before oral administra­ CHLORDECONE(C)-INDUCED TREMOR IN RATS. H. A. tion. The monkeys received 10 mg/kg of acryla­ Tilson and J. M. Gerhart, Laboratory of Behavioral mide, 5 days a week until the appearance of and Neurological Toxicology, National Institute toxic signs (total administered dose from 320 of Environmental Health Sciences, Research to 450 mg/kg). They were observed 5 days a Triangle Park, NC 27709. (SPON: R. B. Mailman). week. Twice a week, they were weighed and pre­ A procedure based on spectral analysis of whole sented with a visuomotor task. They had to body movementswas developed to quantify C-induced retrieve small baits placed in recessed wells tremor in rats. Male, Fischer-344 rats were given under a grid. Weight loss usually preceded the single or repeated i.p. doses of C and the fre­ onset of gross behavioral disturbance, such as quency (cps} over 2.5-20 Hz bands at 2.5 Hz loss of balance. Impaired coordination paral­ intervals and intensity (-dbv) at each Hz interval leled decreased body weights. Vibration sensi­ were measured. The most prevalent Hz (Peps) and tivity decreased during and remained affected intensity (Pdbv) were also recorded. Spectral for several weeks after cessation of dosing. analysis of the tremor produced by single doses of Vibration sensitivity impairment outlasted all C could be differentiated from the spectral the other effects. Electrical sensitivity was analysis of movementproduced by various doses of not clearly affected. These data illustrate harmine (10-20 mg/kg, i.p.) and apomorphine (0.5-2 the usefulness of vibration sensitivity as a mg/kg, i.p.). The administration of 10-100 mg/kg means of characterizing the time course of in­ C produced measurable tremor at 1, 5, and 12 hrs toxication and recovery in toxic peripheral postdosing. The peak effect of Con tremor neuropathies. (Supported by a Contract with occurred between 5-12 hrs postdosing. The Peps the DowChemical Co., Nalco Chemical Co. and was approximately 12-13 cps following 50-100 mg/kg SOHIO,by Grants ES-01247 and ES-01248 from C 5-12 hr postdosing. Tremor was also quantified NIEHS,and in part under Contract No. in rats receiving 5-10 mg/kg C for 10 consecutive DE-AC02-76EV03490with the U.S. Dept. of days. Twenty-four hrs after the last dose of C, Energy.) spectral analysis indicated a Peps that was approximately the same as that obtained following acute administration of higher doses (~12 cps). Subsequent experiments with trihexphenidyl 403 COMPARATIVEEFFECTS OF PHENYLPROPANOLAMINEWITH (Artane) and pizotifen (BC-105) suggest that the EPHEDRINE AND DEXTROAMPHETAMINEON SPONTANEOUS tremor produced by C may have a cholinergic and MOTORACTIVITY IN HAMSTERS. L.R. Weiss and serotonergic component.

114 405 HYPOTHALAMICNEUROTOXIC POTENTIAL OF ORAL positive trials. Injection of 3 mg/kg(½ E.D. MONOSODIUML GLUTAMATEIN THE WATERDEPRIVED for controls) of pentobarbital in experimental YOUNGMOUSE. R.F. Mankes, K.-F. Benitz, rats dosed with 2.5 g/kg of ethanol produced I. Rosenblum, R. LeFevre, N. Wilson-Martino, a significant prolongation of ataxia measured V. Shah, K. Barronl and R. Abraham. Institute of on the Roto-Rod. These observations suggest a Experimental Pathology and Toxicology and Dept. potential method for studying other acute inter­ of Neurologyl, Albany Medical College, Albany, actions. (Supported by NIEHS Training Grant N.Y. 12208. IT32-ES-07058.) Acidic amino acids cause hypothalamic lyso­ somal damage and neuronal necrosis in neonatal rodents when given by non-dietary means. The capacity of Monosodium Glutamate (MSG) to induce 407 NEUROTOXICEVALUATION OF TETRAKISHYDROXYMETHYL similar neuronal damage was investigated under PHOSPHONIUMCHLORIDE IN FISCHER 344 RATS. free feeding and after a period of water D. Thake, P. Kurtz, and S. Carter, Battelle deprivation. Weanling ICR strain mice (120) of Columbus Labs., 505 King Ave., Columbus, OH 43201 both sexes were used 10 Mand 10 F per group: Sponsor: G.L. Fisher Group I-Control; Group II-free ingestion of 5.0% A previous study had indicated that the flame re­ Aq. MSG; Group III-14 hr. water deprivation~ tardant chemical tetrakis hydroxymethyl phosphon­ Group IV-water deprivation and 2.0% MSG; Group V­ ium chloride is capable of inducing peripheral water deprivation and 5.0% MSG; and Group VI-Sg neuropathy in rodents. This study was designed MSG/kg given by gavage (positive control). Water to verify and elaborate upon those results. deprivation caused depressed body weights. Food Female Fischer 344 rats were exposed daily via consumption was decreased in male mice of Groups oral gavage to 40 or 80 mg/kg/day of tetrakis III-V but test solution consumption was unchanged hydroxymethyl phosphonium chloride for 45 or 90 by water deprivation (Groups III-V). Mice of days. Neurobehavioral evaluations were conducted Group VI exhibited prostration, lethargy and 7 after 45 and again after 90 days of exposure. animals died (26%) after dosing. Histologic Rats were terminated on days 45 and 90 following examination of hypothalami from all mice revealed initial exposures and were perfused with five· no abnormalities in structure in Groups I-III. percent glutaraldehyde through the ascending a­ In hypothalami of Group IV mice, minimal orta. Brains and spinal cords were evaluated histologic changes were observed in 62% of histologically by light microscopy and peripheral neuronal cells and in 73% from Group V. The nerves were evaluated histologically through the hypothalamic lesion involving neuronal necrosis use of plastic embedded sections and electron was observed in the Arcuate-Median Eminence microscopy. Clinical signs associated with THPC region of all mice from Group VI. The data administration occurred only at the higher dosage presented suggest that the toxicity of MSG is level and included decreased weight gain, lack of potentiated by water deprivation in a dose­ response to external stimuli, stiff gait, and related manner and the method of intake paresis. Mild to severe degenerative lesions were influences the localization and severity of the observed in peripheral nerves (sciatic, tibial, lesion. Supported in part by NIEHS Training muscle branches, and plantar) in rats given 80 Grant ST32ES07058-03. mg/kg and terminated at 90 days. Mild degenera­ tive lesions were observed in peripheral nerves of rats given 80 mg/kg and terminated at 45 days. 406 ROTO-RODPERFORMANCE EVALUATION OF PENTOBARBITAL Spontaneous motor activity, forelimb grip -ETHANOLINTERACTION IN CHRONICALLYETHANOL strength, and hindlimbgrip strength, were signifi­ TREATEDRATS. P.E. Kish, and I. Rosenblum, cantly reduced at both 45 and 90 days in the rats Inst. of Exp. Path. and Tox., Albany Medical exposed to 80 mg/kg but not in those exposed to College, Albany, NY. 40 mg/kg. No significant chemical effects were seen on rectal temperature or landing-foot spread There are few interaction studies in animals at either test interval. These results indicate that are chronically exposed, and potentially that neurotoxicity results from repeated exposure tolerant to the effects of a chemical. to this chemical in Fischer 344 Rats. Pentobarbitals interaction with ethanol using performance on a Roto-Rod, was studied in male Sprague-Dawley albino rats weighing from 200- 310 g. All rats we're pre-trained to remain on 408 MODIFICATIONOF CENTRALNERVOUS SYSTEM ENDOGENOUS a 3 inch diameter rod rotating at 10 r.p.m. for PHOSPHORYLATIONIN ORGANOPHOSPHORUSDELAYED NEURO­ periods of two minutes. Experimental animals PATHY. D. J. Huggins and R. J. Richardson, Toxi­ were injected i.p. daily with 1.5, 1.75, 2.0, cology ·Research Lab; Dept. of Environ. & lndust. 2.25, or 2.5 g/kg of ethanol (15.1% v/v in Hlth., Sch. of Pub. Hlth., The Univ. of Michigan, normal saline). After injection the animals Ann Arbor, MI were tested at 10 minute intervals for 90 min­ utes on the Rote-Rod. Tolerance to ethanol's Some organophosphorus compounds (OPs) can pro­ effects were observed to develop in animals duce a characteristic peripheral axonopathy ap­ injected with doses up to 2.0 g/kg, generally proximately two weeks after a single dose. To by day 14. No tolerance was observed in rats evaluate whether a disruption in endogenous phos­ at higher doses, even at the end of testing at phorylation is involved in the toxic mechanism, 21 days. Dose-response curves were prepared protein kinase-mediated phosphorylation was stu­ for pentobarbital in both control and experi­ died in subcellular fractions from brainstem of mental rats. In control rats doses of 6 mg/kg control and OP-treated White Leghorn hens. Pro­ or greater gave positive trials (falling off), teins phosphorylated after incubation with y-[32p] on the Rote-Rod. In experimental animals doses - ATP were separated and visualized with SDS­ of 10 mg/kg or greater were required to produce polyacrylamide gel electrophoresis and autoradio-

115 graphy. Amongthe four major phosphoproteins pre­ later. At this time the cells of the external sent in axolemmal fractions, phosphorylation was granule layer (EGL) are rapidly dividina. Morpho­ decreased in a single phosphoprotein (MW=33K)from metric analysis of the EGLfrom MeHgand buffer hens sacrificed 15 days after dosing with neuro­ treated animals showed differences in several toxic doses of dibutyl dichlorvos (1.5 and 2.8 mg/ parameters. The number of cells in the EGL/unit kg, sc). Equimolar doses of the homolog, dichlor­ area was counted in 5 regions of the cortex. The vos did not produce this decreased phosphoryla­ total number of cells was significantly reduced by tion; nor did a subneurotoxic dose of di butyl di­ MeHgtreatment (Analysis of Variance o<.025). The chlorvos (0.28 mg/kg, sc). The effect was also mitotic activity of the EGLwas evaluated as a absent at time points prior to onset of clinical possible mechanism for the decrease in cell number, signs of paralysis (1 ,3,7, and 10 days) and could but the total numbers of mitotic figures/section not be demonstrated in synaptosomal or microsomal and the mitotic indices in the 5 regions counted fractions of brainstem. These results suggest were not significantly altered by MeHq. Rather, that endogenous phosphorylation in the CNS is dis­ cells appeared to be injured or dying in the rupted in a specific way by dibutyl dichlorvos treated animals, as indicated by condensed nuclfti. in vivo and that the effect correlates temporally The number of cells with nuclei measuring < 3 µn with the frank degenerative phase of the neuro­ in diameter was increased after MeHgtreatment pathy. (Supported in part by research grant (p<.002). The thickness of the EGLin the same 5 ROI ES01611-03/04, NIEHS; D. J. H. was recipient area was not significantly decreased. The percent­ of a Stauffer Chemical Co. Scholarship in Toxico­ age of late mitotic figures (ananhase/mitotic logy.) figures) was reduced in the MeHganimals (p<.002). The incomplete mitosis may contribute to the decreased cell number by causing cell death. This metaphase arrest, presumably due to loss of spindle 409 SIX MONTHDAILY TREATMENTS OF SHEEPWITH NEURO­ microtubules, may be an important mechanism in TOXICORGANOPHOSPHORUS COMPOUNDS. S.A. Soliman, MeHq-causedneurotoxicity in developing animals. J. Farmer, A. Curley and W.F. Durham. Neurotoxi­ (Supported by ES 01247, ES 01248, ES 07026, DOE cology and Environmental Toxicology Divisions, EY 76C023490). Health Effects Research Laboratory, U.S. Environ­ mental Protection Agency, Research Triangle Park, NC 27711. 411 IDENTIFICATIONOF NEUROTOXICESTERASE IN HUMAN The delayed neurotoxic effects of TOCP, lepto­ PLACENTA., P. E. Gurba and R. J. Richardson, phos, EPNand DEFon male sheep were studied Toxicology Research Lab., Dept. of Environmental during 6 months of daily treatment under field & Industrial !-!1th., Sch. of Pub. Hlth. , The conditions. Sheep were given daily oral doses of Univ. of Michigan, Ann Arbor, MI the tested compounds at 5, 5, 1 and 5 mg/kg/day, Neurotoxic esterase (NTE) has been implicated as respectively, for 180 days. The neurotoxic ef­ the primary event in the development of delayed fects were studied on clinical, histological and organophosphorus (OP) neuropathy. NTE has been biochemical bases. The results were compared found in the nervous tissue of all vertebrate with a vehicle-control group of sheep given corn species examined, and it has been found in oil (0.1 ml/kg/day) only. All the DEF-treated non-neural tissue such as lymphocytes and ileum. sheep died during the experiment following 77 to We recently found that human placenta also 92 successive daily doses. All other sheep were contains NTEactivity. The activity in the crude sacrificed 24 hrs after the 180th daily treatment. tissue is 16,1 nmoles/min/mg protein (units) when Blood, brain, spinal cord and sciatic nerve phenylvalerate is used as the substrate. tissues were taken for histopathological and/or Subcellular fractionation yielded mitochondria, biochemical examinations. The results indicated microsomes, and post-microsomal supernatant with that leptophos induced ataxia, ievere ataxia activities of 38.1, 36.6 and 0.6 units and paralysis in sheep following about 4 months respectively indicating that the activity is of treatment. TOCPproduced only mild ataxia in membrane bound as in nervous tissue. Since the one out of 4 sheep during the last week of ex­ specific activitiies of the mitochondria and periment. On the other hand none of the EPN­ microsomes were approximately equal, they were treated sheep showed clinical signs of neurotoxi­ pooled for other studies (referred to as P2P3). city during.the course of experiment at the The P2P3 activity could be solubilized with tested dose level. These clinical results were Triton X-100 when the ratio of detergent to supported by histopathological findings and also protein was adjusted to 0.30. Titration of the by biochemical results using neurotoxic esterase crude, P2P3, and Triton-solubilized material with measurements. paraoxon (PO) and mipafox gave mipafox pl-50 values of 4.7, 5.0 and 5.0 respectively. The activity of the solubilized enzyme is also 410 MORPHOMETRICANALYSIS OF THEEFFECT OF METMYL inhibited by low levels of mercuric acetate MERCURYON DEVELOPING MOUSE CEREBELLAR CORTEX. (pl-SO of 5.8) suggesting that a sulfhydryl group as well as a serine residue is required for the P.R. Sager, R.A. Doherty, P.M. Rodier*, Div. of ester hydrolysis. The discovery of NTE in human Toxicol.; *Dept. Anatomy, Univ. of Rochester placenta is valuable because it is an easily Medical Center, Rochester, NY. Soon: F.A. Smith obtainable tissue source of human origin. It is Methylmercury (MeHg) is known to interfere with also present in sufficient quantities to allow CNSdevelopment in humans and in several animal complete purification and characterization models. The effect of MeHgon developing mouse studies. cerebellar cortex is beino investiqated. MeHg (Supported in part by research and training (8 mg/kg body weight) was orally administered to 2 grants SR01ES01611-03/04 and ST32ES07062-02/03, day old BALB/cmice which were sacrificed 24 hours NIEHS.)

116 412 SOLVENTTOXICITY AND WATERINSOLUABLE COMPOUND dose of amygdalin, but an identical intravenous DELIVERY SYSTEMFOR IN VITRO WHOLEEMBRYO CULTURE dose failed to cause significant changes in the K.T. Kitchin and M.T-:-Ebron, ETD, USEPA, Research concentrations of the metabolites, The biotrans­ Triangle Park, NC (Sponsor: William F. Durham) formation patterns of amygdalin in hamsters were similar to those in cancer patients (JAMA 13: 591, In order to study the embryotoxicity and 1981), The data suggest that the teratogenic teratogenicity of water insoluable chemicals, activity was not an exclusive property of the solvents or chemical delivery systems of low amygdalin molecule and that the parent drug toxicity and teratogenicity to the developing itself was not responsible for interference with embryo must be found. Therefore, day 11 rat the rapid cell multiplication that is character­ embryos were cultured for 2 days in whole rat istic of embryonic growth, The embryopathic serum containing 0.1, 0,5 and 2,5 volume per cent effects of laetrile appear due to cyanide of ethyl alcohol, dimethylsulfoxide, acetone, released in vivo by the activity of gut bacterial tween 80, corn oil and 10% acetone/90% corn oil. S-glucosidase:- No adverse effects occurred at 0,1% of any solvent. At 0,5% ethyl alcohol and tween 80 significantly reduced embryonic growth and in­ creased the incidence of embryonic abnormalities. 414 INHALATIONTERATOLOGY STUDY ON MONOCHLOROBENZENE With the exception of corn oil and acetone/ IN RATS AND RABBITS, J. A. John, W. C. Hayes, corn oil, embryos cultured in media containing T. R. Hanley, Jr., K. A. Johnson, T. S. Gushow, 2.5% of various solvents failed to grow (only and K. S. Rao. Toxicology Research Laboratory, 10-20% of final control embryonic DNA and pro­ Health and Environmental Sciences, USA, Dow tein) did not differentiate, and did not have a Chemical U.S.A., Midland, MI 48640. beating heart. Corn oil suspended in rat serum by use of ultrasound was extremely non­ An inhalation teratology study on monochloroben­ toxic even at concentrations of 2,5 and 10%. zene (MCB) was conducted using Fischer 344 rats Growth parameters of embryos cultured in and New Zealand white rabbits. Groups of bred serum containing corn oil were indistinguishable rats and artificially inseminated rabbits were from controls and overall morphogenesis was exposed to O, 75, 210, or 590 ppm of MCB, for 6 good (particularly at 2.5%). Ethyl alcohol, hr/day during days 6 through 15 (rats) or 6 acetone and tween 80 showed dysmorphogenic through 18 (rabbits) of gestation. Pregnant effects at doses below those found to be rats exposed to 590 ppm MCB showed evidence of embryolethal. The order of increasing toxicity, but no evidence of embryolethality or embryo toxicity and teratogenicity of the teratogenicity was apparent at any of the studied liquids was corn oil

117 toxicity in the azaserine treated dams were not 417 TERATOGENICEVALUATION OF ALIPHATIC NITRILES IN observed. Control hamsters received injections RATS. P.E. Berteau, G. J. Levinskas, AND D.E. of saline. The hamsters were sacrificed on day Rodwell. Monsanto Co. St. Louis, MO and Interna­ 12 of the 16 day gestation period and a count tional Research and Development Corp., Mattawan, made in each hamster of viable and resorbing MI. (dead) fetuses. Live fetuses were examined for The teratogenic potential of .fcetonitrile growth retardation and gross external malforma­ (I), propionitrile (II) and adiponitrile (III) tions. Azaserine was found to have both embryo­ were evaluated in rats. Aqueous solutions of I lethal and teratogenic effects in the hamster. or II or of a corn oil solution of III were Fetal mortality in azaserine treated dams ranged administered by gavage to separate groups of 25 from 17 to 100% varying with the dosage, route of mated Charles River rats on gestation days 6 to administration and day of gestation. For fetal 19 inclusive. Daily dosage levels (mg/kg body mortality, a dose-response relationship was shown. weight) were: (I) 125, 190 and 275; (II) 20, 40 Fetal mortality among the controls did not exceed and 80 and (III) 30, SO and 80. Control groups 5%. The incidence of fetal abnormalities ranged of 25 mated rats received each of the vehicles from 9 to 100% in treated hamsters, versus 3% in alone. On gestation day 20, all females were controls. The most common fetal abnormalities sacrificed and the number and location of viable were runting (9% incidence in treated versus 3% and non-viable fetuses and early and late resorp­ in controls) and limb defects (10% incidence in tions and the number of total implantations and treated versus 0% in controls). These results, corpora lutea were recorded. All fetuses were compared with previous studies on rats, suggest individually weighed, and examined for external that azaserine is equally embryolethal in the and visceral or skeletal malformations and hamster and the rat. The hamster is also more variations. resistant to the teratogenic actions of azaserine There were some maternal deaths in each of than the rat. Therefore, azaserine is both less the high dose groups with all three compounds and teratogenic and carcinogenic in the hamster than one death at the mid-dose level of those dosed the rat. Supported by NIH Grants RR-05392 and with III. Reduced maternal body-weights occurred CA-26594. in the high dose groups with I and II but not III. There were no other distinct adverse maternal clinical observations in any treated groups. Embryotoxic effects occurred in the high dose group with I and II as evidenced by increases in 416 LACK OF TERATOGENICITY IN RATS WITH early resorptions and post-implantation losses. AMSACRINE. J.A. Anderson, J.R. Watkins, No teratogenic responses were observed at any dose J. Petrere, J.E. Fitzgerald and F.A. level with any of these three nitriles. de la Iglesia, Dept. of Toxicolo~Warner­ Lambert/Parke-Dav is Pharmaceu ti ca I Res. Div. Ann Arbor, Ml 48105 418 EMBRYOLETHALITYAND MALFORMATIONCAUSED BY PATERNALETHANOL IN MALELONG EVANS RATS. The acridinylamino derivative amsacrine, an R.F. Mankes, W.P. Rockwoodl, R. Lefevre. anti neoplastic agent for refractory leu­ G. Rockwood,_ H. _Bates, K.F. Benitz, T. Reffinan, kemias, was studied for embryotoxic or tera­ A.LT._ Walker, and_ R; Abraham. Institute of togenic potential in pregnant rats. The com­ Experimental Pathology and Toxicology, Albany pound was given ip on Days 6 to 9 of Medical College, Albany, N.Y. and lRussell Sage gestation to groups of 20 female CD rats at College, Troy, N.Y. levels of 2, 1, and 0.5 mg/kg/day. Sham Despite wide recognition of the teratogenic and untreated control groups were used also. and genotoxic effects of ethanol (ETOH) During the treatment period, high dose (2.0 consumption, few studies have examined the mg/kg) dams lost weight, whereas other effects of paternal ethanol exposure on fetal groups were unaffected. Weight gain sup­ development. 20 male Long Evans rats were pression continued in the post-dosing period. divided into two equal groups and given either Food consumption during treatment was com­ tap water (group I) or 20% ETOH (group II) as parable with control groups. Litter sizes, the sole source.of drinking water for 60 days. post-implantation losses, and fetal weights Five of 30 matings (group II) as compared to were adversely affected at 2.0 mg/kg/day; Group I proved infertile. Implantation ~ites, 1 .0 and 0.5 mg/kg/day had no significant live birth rates, total litter weights and pup effects on litter and fetal parameters al­ weights were- decreased while fetal deaths were though - there was reduced body weight of increased two-fold over controls. Malformations fetuses from dams given 0.5 mg/kg day and (Microcephalus, Microptthalmia, Cranial fissure, stunting at 1.0 mg/kg/day. Four fetuses from and Hydronephosis) were noted in 55% of the the treated groups were grossly abnormal: offspring of Group II compare~ to 12% in two at 2.0 mg/kg, one at 1.0 mg/kg, and controls. At necropsy, one rat of Group II had one at 0.5 mg/kg. Two vehicle control fetuses gross testicular atrophy. Absolute and relative were also abnormal. No malformations testicular weights were significantly decreased occurred in the untreated control group. in the alcohol treated group compared to Osteogenesis inhibition and minor skeletal ab­ Group I. These results suggest that paternal normalities were manifestations of fetotoxicity ethanol consumption causes dominant lethal among treated groups. The results indicate mutations and testicular atrophy, and therefore, that amsacrine in rats induces marked should be considered a significant risk factor embryotoxicity (2.0 mg/kg) and fetotoxicity in reproduction. ( 1.0 and 0.5 mg/kg) but frank teratogenicity Supported by NIEHS Training Grant 5T32ES07058-03 was not evident even when a few ma Ifor­ and a Basic Medical Research Grant from Royal mations were seen at 1.0 and 0.5 mg/kg/day. Shell, LTD.

118 419 COMBINEDORAL ADMINISTRATION OF ETHANOLAND corticosterone levels were essentially normal when ACETAMINOPHENIN PREGNANTMICE: POSSIBLE measured at 400 or 800 days of age. No differ­ ANTAGONISMIN TERAT(X;ENESIS?A. Loeb1, ences were found between control offspring and R, Mankes, R, LeFevre, and H. Bates (Sponsor offspring exposed to the higher dose of either in­ R, Abraham). Institute of Experimental secticide. This non-linear dose response is con­ Pathology and Toxicology, Albany Medical sistent with other reports. Results suggest that College, Albany, N.Y. prenatal exposure to these low levels of Carbo­ Ethanol is a teratogen in human and furan or Diazinon has only a minimal effect on laboratory rodents, frequently ingested in endocrine function and that this effect is combination with other agents. Acetaminophen is transitory. a widely used analgesic, whose effects on the developing embryo remain unclear. Pregnant female !CR strain mice were used to evaluate the possible interaction between ethanol (ETOH) and 421 IMMUNOLOGICALEFFECTS OF COMPLEXCHEMICALS ON THE Acetaminophen (APAP)1 Group 1- control, group DEVELOPINGIMMUNE SYSTEM OF RATS EXPOSEDIN 2- 0,4ml ETOH/kg, group 3- 4,0ml ETOH/kg, group UTERO. J.E. Morris and T.M. Graham, Battelle, 4- 25mg APAP/kg, group 5- 250mg APAP/kg, group Biology Department, Pacific Northwest Laboratory, 6- 0.4ml ETOHand 25mg APAP/kg, and group 7- Richland, WAand F.D. Andrew, Syntex Research, 4. 0ml ETOHand 250mg APAP/kg. Equal volume Palo Al to, CA. doses were given by gavage from day 6 to 15 of gestation. Sponsor: D. D. Mahlum Fetal death rate and visceral malformation were increased 160 to 180% in mice given ETOH Responses of the developing immune system to (group 2) as compared to controls (group 1). Aroclor 1254 and Solvent Refined Coal I (SRC-I) APAP (group 4) caused both visceral and skeletal and II (SRC-II) materials were measured in rats. anomalies without embryolethality. __ Rats were exposed in utero by daily gavage of the Combinations of ETOHand APAP (groups 6 and 7), dam on days 12-16 of pregnancy, and were killed at caused lower fetal death rates and fewer 40 d of age. The dose groups included: 0.7 malformations than either ethanol or APAPalone, ml/kg/day process solvent (PS, SRC-I), 0.3 ml/kg/ The data suggest that combinations of ETOH day heavy distillate (HD, SRC-II), 0.8 ml/kg/day and APAP, rather than interacting 2.5% Aroclor 1254 (Ar, positive control), corn oil synergestically, may act as antagonists, possibly (vehicle control), and untreated rats (shelf through altered microsomal metabolism. control). Body, spleen, thymus, liver and brain 1B,S,-M,D. Program, Union College, weights were recorded. Lymphocyte function was Schenectady, N.Y. Supported in part by NIEHS measured by mitogen-induced lymphocyte activation Training Grant 5T32ES0?058-03 and a Basic assays (in vitro correlate of cell-mediated Medical Research Grant from Royal Dutch immunity) using cells from the peripheral blood, Shell, Ltd. spleen and thymus. The distribution of T-lympho­ cytes in spleen cell preparations was based on 3H-Uridine uptake. PS-exposed offspring had reduced body (10%) and thymus (10%) weights while 420 ADRENAL FUNCTION AND HEPATIC METABOLISM HD-exposed rats had weight reductions of the body IN ADULT MICE EXPOSED IN UTERO TO CARBO­ (10%), thymus (22%) and liver (14%). Ar-exposed FURAN OR DIAZINON: A LONGITUDINAL EVAL­ rats showed weight decreases in body (50%), thymus UATION. J. Spyker Cranmer, D.L. Avery and (50%), liver (28%) and brain (7%). HD- and Ar­ M. F. Cranmer, Div. of Interdisciplinary Toxicol­ exposed animals had significantly decreased thymus ogy, University of Arkansas for Medical Sciences cell responses to phytohemagglutinin (35% and 44% and Jefferson Professional Services, Little Rock, respectively); AR-exposed rats also had decreased AR. peripheral blood response to the mitogens Con­ canavalin-A (50%) and pokeweed mitogen (50%). T­ The functional status of the endocrine system of lymphocyte populations in spleen cell preparations mice prenatally exposed to Ca rbofuran or Dia zinon arenot altered in exposed rats. was evaluated by assessing adrenal function and hepatic metabolism at three stages in the lifespan. Gravid dihybrid mice were fed 0, O. 01 or O. 50 mg/kg Carbofuran or 0, 0.18 or 9.00 mg/kg 422 IMMUNOTERATOLOGY OF CHLORDANE. I. CELL­ Dia zinon daily throughout gestation. Mothers of MEDIATED AND HUMORAL IMMUNE RESPONSES. all pesticide and vehicle-control groups gave birth J.M. Cranmer, J.B. Barnett, D.L. Avery, and to approximately equal numbers (48/group) of M.F. Cranmer, Div. of lnterdiscip. Tox., Univ. viable, overtly normal offspring. Adrenal produc­ of AR Med. Sci. and Jefferson Professional tion and liver reduction capacity for corticosterone Services, Little Rock, AR. were measured in these offspring at 101 days of age; plasma concentrations of corticosterone were The functional status of the immune system of assayed at 101, 400 and 800 days of age. Expo­ mice prenatally exposed to a chlorinated hydro­ sure to the lower doses of both anticholinesterase carbon pesticide was evaluated by assessing cell­ compounds resulted in impairment of hepatic me­ mediated and humeral immune responses. Gravid tabolism of corticosterone in vitro due to a loss in Balb/c mice fed 0, 0.16 or 8.0 mg/kg chlordane reductive capacity per unltliver weight. Plasma daily throughout gestation produced viable, levels of corticosterone were correspondingly overtly normal offspring. Cell-mediated immune elevated in these offspring but without a con­ (CMI) responses were measured in 101-day-old comitant increase in adrenal steroidogenesis in offspring by contact hypersensitivity responses vitro. No evidence for persistence of these - to oxazolone. Mice were sensitized by application effects was observed among older animals; plasma of 25 µI of 8.0% solution of oxazolone to the de-

119 nuded flank. Five days later, offspring were microsomal phosphatidylcholine (PC) and phospha­ challenged by percutaneous application of 10 µ I tidylethanolamine (PE) fractions were studied. of an 0. 1% oxazolone solution to the dorsal surface 16a-Hydroxyprogesterone significantly increased of the ear. Degree of induration was determined the fatty acid content of both PC and PE fractions by daily micrometer measurement of ear thickness of total liver and microsomes. Both saturated and for 3 days after challenge. Subjects exposed to unsaturated components were raised, unsaturated 8.0 mg/kg/day chlordane had a significantly (p < to greater extent, so sat/unsat ratio was decrea­ 0.01) depressed CMI response when compared to sed. In contrast, pregnanolone significantly de­ offspring of the control or lower dose groups. creased total fatty acid, saturated and unsatur­ The primary humoral response to sheep red blood ated fractions both in PC and PE components. The cell (SRBC) innoculation was determined at 101 reduction of unsaturated fatty acid was greater days of age using the hemolytic plaque assay. resulting in an increased sat/unsat ratio. The Subjects were immunized with 0.1 ml of 1% washed contrasting action of these test compounds were SRBC. lgM antibody released from spleen cells observed in 16:0, 16:1, 18:0, 18:1, 18:2, 20:3, was detected by addition of complement which 20:4, 22:5 and 22:6 fatty acids in PC and 16:0, caused lysis of the SRBCs. There were no dif­ 18:1, 18:2, 20:4 and 22:6 in PE. 16a-Hydroxy­ ferences between treated and control groups in the progesterone significantly increased, while preg­ number of plaque-forming cells (PFC) produced nanolone decreased mono- and polyunsaturated fatty by spleen cells. Results of this study revealed acids. Drug metabolism is also increased or de­ two significant findings: ( 1) a severe depression creased by these test compounds respectively. of the functional CMI response in apparently Changes in unsaturation of fatty acids are con­ normal offspring, and (2) no effect on the T-cell nected with phase equilibria changes of lipid dependent humoral immune response. membranes. Thus, it may be that the regulatory action of progesterone metabolites on the function of the endoplasmic reticulum is associated with intrinsic structural changes connected with 423 A TERATOGENICEVALUATION OF DIAZINON®IN THE modifications of the phospholipid moiety. RABBIT. W.R.Campbell, S.B.Harris & J.E.Holson, CIBA-GEIGY Corporation, Greensboro, NC and Science Applications, Inc., LaJolla, CA. Diazinon, an extensively used organophosphate 425 THEINFLUENCE OF CAPTANON LEVELS OF REDUCED insecticide, was administered to pregnant New SULFHYDRYLSIN THE DUODENUM OF MICE. M.W. Zealand rabbits to evaluate its prenatal Sauerhoff, J. DeBaun, J.B. Miaullis, R.l. toxicity. The compound was orally adminis­ Freudenthal, Stauffer Chemical Company, Environ­ tered by gavage at O (control), 7, 25, and mental Health Center, Farmington, CT and Mt. 100 mg/kg from days 6-18 of gestation. All View Research Lab., Mt. View, CA does were weighed throughout the study and toxic signs evaluated. Fetuses were weighed, sexed, and evaluated for external, visceral, Chemically reactive metabolites have been impli­ and skeletal abnormalities and variations. cated as mediators of toxicity, including car­ Significant doe mortality (40.9 percent) with cinogenicity. Glutathione is conjugated with concomitant pharmacologic signs were observed reactive metabolites resulting in detoxification at 100 mg/kg. Neither embryotoxicity and excretion.Numerous studies have shown that (decreased fetal weight and increased ernbryo­ thiols are important in the degradation of Cap­ lethality) nor frank teratogenicity (exter­ tan. The objective of this study was to deter­ nal, visceral or skeletal anomalies) were mine the effect of Captan Technical in vivo on observed at 100 mg/kg. Furthermore, lower duodenal sulfhydryls.Male and female B6C3Flmice dose levels of 7 and 25 mg/kg produced no were administered Captan as a single oral gavage significant maternal or fetal toxicity. The in corn oil at doses of 0,20,200 or 2000 mg/kg. findings suggest that maternal toxicity was Mice were sacrificed at 2,4,24 and 72 hrs. Cap­ achieved at dose levels below those which tan administration resulted in an increase in cause fetal weight reduction and increases in duodenal sulfhydryls. Significant increases were either embryolethality or malformations. observed as early as 2 hrs. following adminis­ There was no evidence of a teratogenic effect tration of Captan and persisted for at least 72 at any of the dose levels tested. hrs. The mean value of reduced duodenal sulfhy­ dryls (+s.d.) for control males and females was 1595+11TIug/g and 1524+ 152 ug/g, respectively. In a-separate study to-evaluate the effects of subchronic inqestion on duodenal sulfhydryls, 424 EFFECT OF PROGESTERONEMETABOLITES ON FATTY ACID Simonsen mice ingested diet containing Captan CONTENTAND COMPOSITIONOF PHOSPHOLIPIDS FROMTHE Technical at levels of 500,1000,2000,4000,8000 ENDOPLASMICRETICULUM OF RAT LIVER. M.S.I.Dhami or 16000 ppm for up to 30 weeks. Mice were sac­ and G. Feuer, Dept. of Clin. Biochem., University rificed on days 1,2,5,9,14,21 ,35,49,84 and 213 of Toronto and Can. Mem. Chirop. Coll., Toronto, days of treatment. Duodenal sulfhydryls were Ontario, Canada. elevated at the 500 ppm level and above at all It was shown earlier that progesterone meta­ sacrifice intervals. Maximal increases were bolites elicited contrasting action on hepatic about 160%of control values. Contrary to a drug metabolism (Dhami et al., Toxicology, 14, 99, proposed genetic mechanism for captan-induced 1979). This raised the question whether this ac­ small intestinal tumors, the results of this tion is connected with membrane-bound phospholip­ study do not show a depression of sulfhydryls. id. To test this, the effects of 16a-hydroxypro­ Perhaps captan-induced tumors are produced gesterone and 5S-pregnane-3a-ol-20-one on fatty through an epigenetic mechanism. Recent obser­ acid content and composition of total liver and vations lend credibility to this hypothesis.

120 426 THE INTERACTIONOF ANTHRAQUINONEDERIVATIVES levels was complete by day 7. With both these WITH DNAAND PROTEIN IN VITRO. II. COVALENT compounds there was a rebound increase in the BINDING. B.M. Elliott and J. Ashby, ICI, Central incorporation of 2-14c glycine into newly formed Toxicology Lab., Alderley Park, Nr. Macclesfield, erythrocytes. This occurred at day 10 forbenzene Cheshire, SK10 4TJ, UK. Sponsor: I.F.H. Purchase. (40% increased) and day 13 for cyclophosphamide (100% increased). These data suggest that the To evaluate the possible contribution of covalent initial reduction in erythrocyte production is binding to the Ames positive resp~nses reported caused by the depletion of nucleated cells in the for many 9,10-anthraquinone (AQ) derivatives, 14 bone marrow, whilst the delayed recovery and studies with [ C] - labelled 1,4-dihydroxy, 1- rebound increase in erythrocyte production must be amino-2-methyl and 1-nitro-2-methyl AQ have been the result of prolonged effects on more immature performed to examine their binding to DNA and bone marrow cells. protein in vitro. The chemicals were incubated with calf-thymus DNA in Tris buffer pH 7.0 at 37°C for varying times in the presence or absence of a rat liver S9 fraction. DNA was isolated by 428 EFFECTS OF BENZENEAND OTHERCHEMICALS ON 72 HR phenol/cresol extraction and precipitation, and 59FE UTILIZATION IN SWISS ALBINO MICE. R. Snyder subjected to ribonuclease and protease treatment and B.N. Engelsberg, Dept. of Pharmacology, before assessment of bound radiolabel. All three Thomas Jefferson Univ., Philadelphia, PA 191()7 AQs bound to DNA, with 1,4-dihydroxy and 1-nitro- and Graduate Program in Toxicology, Rutgers 2-methyl AQ requiring S9 (0.5 and 0.7 pmol/mg DNA Univ., Piscataway, NJ 08854. respectively after 60 min. incubation) while 1- The report (Sammett et al., Toxicol. Environ. amino-2-methyl AQ bound in the presence and Hlth. 5, 785, 1979) thatpartial hepatectomy pro­ absence of S9 (3.2 and 1.7 pmol/mg DNA respect­ tected-rats against benzene toxicity and our re­ ively). Binding to protein was measured with S9 cent observation that mice were similarly pro­ fraction as above, precipitating the protein with tected suggested that a toxic metabolite of ben­ TCA, and exhaustively extracting the unbound AQ zene travels from liver to bone marrow. We with solvents. 1,4-dihydroxy, 1-amino-2-methyl attempted to characterize the metabolite by ad­ and 1-nitro-2-methyl AQ all bound to protein with ministering each of a series of known and sus­ values after 45 min. incubation of 1050, 790 and pected benzene metabolites to Swiss Albino mice, 270 pmol/mg protein respectively. Such binding We measured their effect on red cell production was not seen when albumin was used, suggesting a using the 59Fe uptake technique of Lee et al. requirement for enzymic activation of the (Toxicol. Appl. Pharmacol. 27, 411, 1974).~ice compounds to enable binding to protein. were treated twice per day for three days with These data suggest that covalent interactions saline, a negative control, benzene (1760 mg/kg, with DNAmay be responsible for the Ames test sc) in corn oil, a positive control, or the test results with certain nitro and amino AQs. The compound. On day 4 59Fe was injected via tail role of covalent binding in the activity of 1,4- vein and 72 hr later the blood was sampled for dihydroxy AQ and other AQs which were more active 59Fe uptake. The following compounds were admin­ in the absence of S9 in the Ames test, is not istered scat the designated doses expressed in however clear. mg/kg: phenol, 172; hydroouinone, 47.5; resorci­ nol, 25(); catechol, 31 and 62; p-benzoauinone, 17; a combination of p-benzoquinone, 17, with hydroouinone, 47.5. The ip route was used for 427 A COMPARISONOF THE ACTION OF BENZENEAND CYCLO­ hydroauinone, 50; 1,2,3-benzenetriol, 150; 1,2,4- PHOSPHAMIDEON THE ERYTHROIDSYSTEM OF THE RAT. benzenetriol, 20; 1,3,5-benzenetriol, 350: diben­ AF Wright and MS Rose*, ICI, Central Toxicology zo-p-dioxin, 100; muconic dialdehyde, 2; ?.,5-di­ Lab., Alderley Park, Nr. Macclesfield, Cheshire, hydroxy-l, 4-benzoquinone, 8. 5 and J 7. llnlike SK10 4TJ. UK. *ICI Corporate Biosciences Group, benzene, none of the comoounds tested markedly Runcorn Heath, Cheshire, UK. Sponsor: I.H.F. reduced 59Fe uptake. The reactive metabolite of Purchase benzene has vet to be identified. Benzene and cyclophosphamide both cause a wide Supported by NIH grant FS00322. variety of disorders of the haemopoietic system. Both compounds can affect the proliferating and non-proliferating cells of the bone marrow, and thus have the potential to alter erythropoiesis. 429 RELATIVE TOXICITY OF FIVE BENZENEMETABOLITES ON Rats have been treated with benzene or cyclophos­ CFU-GMCULTURES. R. Boyd, J, Griffiths*, V, phamide and the erythroid activity of the bone Kindt*, R, Snyder*, J. Caro and A. Erslev, Car­ marrow in vitro compared with the production of deza Fnd., Thomas Jefferson Univ., Philadelphia, erythrocytes in vivo. Benzene (two s.c. doses of PA 19107 and *Graduate Program in Toxicology, 2. 2 g/kg) causedamarked reduction (70% at day 3) Rutgers Univ., Piscataway, NJ 08854. in the amount of 2-14c glycine incorporation (over 24 hrs) into newly formed erythrocyte_s. This Benzene metabolites (bm) are responsible for ben­ reduction in erythrocyte production was confirmed zene-induced bone marrow depression (Snyder et by a reduction (80% at day 3) in the number of al., Rev. Biochem. Tox. 3, 123, 1981). To deter­ reticulocytes present in the peripheral blood, and mine relative toxicity of the metabolite(s), was coincident with a depletion of nucleated mouse granulocyte-monocyte colony-forming units cells (50% at day 3) and haem synthesis (70% at (CFU-GM) were exposed to five bm. CFU-GMcul­ day 3) in the bone marrow. Cyclophosphamide, tures were grown in agar suspension (Metcalf, (50 mg/kg) caused a greater reduction inerythro­ Hemopoietic Colonies, New York; Springer Verlag, cyte production (96%), reticulocytes (90%), bone 15, 1977). Medium (..<- MEM) was supplemented with marrow nucleated cells (90%) and haem synthesis 20% FBS and a 1:2 dilution of Salmonella typhimu­ (90%). The recovery of these parameters to control rium endotoxin-induced murine colony stimulating

121 factor. Phenol (P), catechol (C), p-benzoquinone metabolism in rats, we have explored whether re­ (PBO), hydroquinone (HO), or 1,2,4-benzenetriol tinoic acid (RA) affects the ability of platelets (BT) was added. Concentrations of bm ranged from to metabolize AA. Young, adult male Sprague­ 10- 8 to 10-2M; zero-concentrations were verified Dawley rats were treated once per day for 14 days by HPLC analysis. Granulocyte and monocyte col­ by oral gavage with RA (20 mg/kg/day) or RA (20 onies per plate were determined on day 7 and ex­ mg/kg/day)+ indomethacin (INDO, 5 mg/kg/day). pressed as% of control. P was the least toxic Control rats received diluent (0.2 ml/100 gm of bm, producing no suppression of colony formation body wt) consisting of aqueous propylene glycol below a concentration of 10-3M. PBO and HQ began (10%) and Cremophor EL (8%). On Day 12 (first to suppress colony formation at 10-GM and were the treatment= Day 1) all rats received 0.5-1.0 µCi most toxic bm tested. C was less toxic than PBQ of 14C-AA sc. Pooled 24-hr urine samples w~re and HQ. BT was intermediate between C and P. The collected for Days 12-14. Rats were killed 24 hr combined concentrations of bm in bone marrow of after last treatment, and blood was collected for benzene-treated mice (Longacre et al., J. Toxicol. preparation of platelet-rich plasma (PRP). Mix­ Environ. Health 7, 223, 1981) were similar to tures of PRP (0.5 ml), 14C-AA (12 nCi), and throm­ those which inhibited growth in CFU-GM. Although bin (2.5 U) were incubated 10 min at 37° and then the data from the CFU-GM studies suggest that frozen. The following AA metabolites were sepa­ these compounds are potentially hemotoxic, it has rated by TLC and quantitated by liquid scintilla­ yet to be demonstrated that they are active in tion: prostaglandin (PG)F2a, 6-keto-PGF1a, and vivo. thromboxane (TX)B2. The data, expressed as fmoles (Supported by NIH Grant ES00322.) of 14C-AA converted to each metabolite, indicate that the ability of rat platelets to metabolize 14c-AA is not affected by 14 days of oral RA. RA 430 THE REACTIVITY OF TRANS,TRANS-MUCONALDEHYDE,A + INDO produced a significant decrease in the pro­ POSSIBLE TOXIC METABOLITE.OF BENZENETOWARDS duction of PGF2a (144 ± 12 fmoles/10 min, x ± SEM) GLUTATHIONE. G.S. Rao, G. Witz, and B.D. vs control (201 ± 12 fmoles/10 min) in this assay. Goldstein, Dept. of Environmental and Community The data suggest that repeated doses of retinoids Medicine, CMDNJ-Rutgers Medical School/Rutgers may not alter the capacity of platelets to sup­ University Joint Graduate Program in Toxicology, port oxygenation processes critical for the regu­ Piscataway, NJ lation of aggregation, but the possibility of At present the metabolite(s) responsible for effects on endogenous AA pools warrants further the leukemogenic action of benzene is not known. study. (Supported by McDowell Cancer Network and We have hypothesized that muconaldehyde, an alpha, Amer. Cancer Soc. grant IN 106F.) beta-unsaturated six-carbon diene dialdehyde, may play a role in benzene hematotoxicity.Alpha,beta­ unsaturated aldehydes·are known to react with SH­ 432 EVALUATIONOF EXPOSURELEVELS TO PHTHALATEACID containing compounds. In the present studies, we ESTERS DURING BLOODTRANSFUSION. R. Kardish, investigated the kinetics for the reaction of M. Tocchi, *G. Posen, +R.K. Smiley, tJ.M. Bowman, trans,trans-muconaldehyde with glutathione. The and +G. Rock, Canadian Red Cross, B.T.s., Ottawa rate of the reaction was followed by measuring Ontario, *Renal Dialysis Unit, Ottawa Civic the decrease in optical density at 272 nm, the Hospital, +Detartment of Medicine, Ottawa General major absorption band of muconaldehyde which dis­ Hospital and Canadian Red Cross, B.T.S., appears after 1,2-addition of glutathione. First Winnipeg, Manitoba. Sponsor: J.S. Woods order reaction rates with respect to muconalde­ hyde were observed in borate, pH 8.1, and tris, The risk of exposure to plasticizers such as di- 2-ethylhexyl phthalate (DEHP) which leach from pH 8.1 and pH 7.4. The apparent secon~ or~Ir rate 1 plastic medical devices is currently being re­ constants were 79.3, 43.2, and 29.1 H s in assessed in view of the recent documentation from borate, pH 8.1, tris, pH 8.1 and tris pH 7.4 re­ N.I.H. of the carcinogenicity of DEHP. This sub­ spectively. These data show that the reaction is ject is of concern not only in the multi-trans­ pH-dependent and varies with the type of buffer fused patient whose plasticizer intake is re­ used. The stoichiometry of the reaction obtained latively large but also to the blood donor by measuring the amount of muconaldehyde and glu­ population, who may be exposed to plasticizer tathione consumed at t=30 min was 1:1 on a during routine pheresis procedures. To assess molar basis in tris buffer. It has been suggested the exposure level in these various groups, blood that the rate of reaction between alpha, beta­ samples were analyzed for DEHP and its more toxic unsaturated aldehydes with sulfhydryl compounds metabolite mono-2-ethylhexyl phthalate (MEHP) is directly proportional to their toxicity. In using HPLC. Of the 28 hemodialysis patients, .comparison with literature data on toxic alpha DEHP was detected in 9 pre-dialysis blood samples beta-unsaturated aldehydes, our studies show that (1.0 to 14.0 µg/ml) and in 9 post-dialysis blood muconaldehyde is less reactive than acrolein but samples (l.O to 14.5 µg/ml). MEHP was detected far more reactive than presumed toxic lipid per­ in 7 of the pre- and 14 of the post-dialysis oxide deomposition products of this nature. samples. However, no phthalate acid esters were (Supported by NIH grant ES 002296). detected in the blood of 7 severe Factor VIII deficient hemophiliacs either before or after cryoprecipitate administration, although the 431 TOXICOLOGYOF RETINOIDS: METABOLISMOF ARACHI­ total intake of DEHP and MEHP per hemophiliac was DONIC ACID BY PLATELETS AFTER 14-DAY RETINOIC as much as 5.0 mg and 0.3 mg per week respective­ ACID TREATMENTIN RATS. S. D. Harrison, Jr. and ly. No detectable levels of DEHP or MEHPwere C. J. Viau, Graduate Center for Toxicology, Uni­ observed in the pre- or post-blood samples of versity of Kentucky, Lexington, KY. donors undergoing plasmapheresis or cytapheresis. In the course of experiments to determine Patients who have been multiply plasmapheresed the effects of retinoids on arachidonic acid (AA) for many yeard did not show detectable levels of

122 MEHPor DEHP. The data suggests that while there and the lowest significant doses of all test is exposure to phthalate esters during blood compounds were calculated. For both species, transfusion the accumulative dosage and there­ the most favorable dose-responses were observed fore the risk is relatively small. at S-9 levels of 25 to 100 ul per plate (500 ul) for 2-acetamidofluorene, cyclophosphamide, and aflatoxin B . 7,12-Dimethylbenz(a)anthracene 1 and hydrazine effected similar responses regard­ 433 PROMUTAGENACTIVATION BY Schistosoma mansoni less of the S-9 level, especially at their higher INFECTEDMICE. S.A. Lacey, J.G. Babish, and doses. Response to benzo(a)pyrene was equivocal. J.R. Georgi. Dept. of Preventive Medicine, N.Y. Mutagenic activity was determined per ul S-9 as S. College of Veterinary Medicine, Cornell well as per nmole Cytochrome P450. Overall University, Ithaca, NY. responses were qualitatively similar between the Hepatic cytochrome P-450 mediated promutagen species, although some quantitative differences activation was examined in Schistosoma mansoni were shown to exist. infected, female, CD-1 mice. Animals were ex­ posed by tail immersion to eith~r 108, 254, or 438 cercariae/well; the duration of infection was 30, 60, or 90 days prior to killing. Liver 435 L-AZASERINEINDUCTION OF A PROPHAGE:ENHANCING homogenates (25% w/v) from control and parasite ACTIVITY OF TISSUE EXTRACTSFROM NORMAL AND DIET­ infected animals were centrifuged at 9000 x g. ARYMETHYL GROUP DEFICIENT RATS. J.L. Suit, B. Supernatant fractions from the 9000 x g centri­ Cruz, B.D. Roebuck and A.E. Rogers, Depts. Biol., fugation (S-9) were used to examine the metabolic and Nutr. and Fd. Sci., M.I.T. Cambridge, MAand activation of 2-aminoanthracene (M), 2-aceta­ Dept. Pharm. and Tox., Dartmouth Med. Sch., Hano­ midofluorene (AAF), aflatoxin B (AFB ) and benzo ver, NH. 1 1 (a)pyrene(BP). Salmonella typh~lmAX'iumTAlOO was L-azaserine is a potent carcinogen that, in used to detect mutagenic metabolites. Fraction­ bacteria, can cause mutation and bacteriophage in­ al log doses of S-9 protein (5) were assayed duction without prior activation by mammalian en­ using a promutagen concentration giving 75% max­ zymes. However, we found that rat tissue extracts imal mutagenic response of control S-9. markedly enhanced azaserine phage inducing activi­ S-9 from S. mansoni infected mice, in general, ty. In the Inductest of Moreau, et al, prophage produced significantly fewer revertants per mg induction in Escherichia coli (A)byazaserine S-9 protein for all compounds. Cercarial dose alone began only after a 40-60 minute lag. Addi­ and duration of infection showed no correlation tion of rat hepatic post mitochondrial supernatant with depression of mutagenic activity. However, (S9) enhanced induction by reducing the lag; more at 30 days post infection, BP mutagenicity was than 50% of the population was induced by 40 min­ greater in infected animals than controls. Plots utes. Preparations from rats fed a diet deficient of revertants/nmole P-450 vs nmole P-450 showed in methyl groups were about twice as effective in (1) decreasing mutagenicity of all compounds the enhancement of induction at a given time as with increasing P-450 concentration and (2) a S9 fractions from control animals. (The methyl decrease in revertants/nmole P-450 in infected group deficient diet greatly enhances hepatocar­ animals at all concentrations of P-450 for M, cinogenesis by azaserine). Enhancing activity AAF, and AFB1. was found also in pancreatic extracts, but in les­ The decreased production of mutagenic meta­ ser amount than in liver and was unaffected by bolites per nmole P-450 indicates a parasite­ diet. Most of the S9 activity was found to be in mediated effect on a specific form(s) of P-450 or the cytosol; it was destroyed by trypsin. Hepatic a change in ratio of other enzymatic processes cytosol enhanced also azaserine mutagenesis in involved in deactivation of reactive metabolites Salmonella typhimurium, at short times of ex­ of the P-450 system. posure. Under Inductest conditions cytosol pro­ moted incorporation of label from 14c-azaserine into the bacteria, most of it into acid insoluble material, usually after a 15-20 minute lag. This 434 COMPARATIVEMETABOLIC ACTIVATION OF PROMUTAGENS pattern differed from the rapid active transport BY FERRETAND RAT HEPATIC S-9 FRACTIONS. and incorporation of the amino acids tryptophan K.A. Frederick, J.G. Babish, and B.E. Johnson, and praline into bacteria that was not affected by Department of Preventive Medicine, New York cytosol. The results suggest that cytosolic fac­ State College of Veterinary Medicine, Cornell tor(s) that are influenced by diet can act on aza­ University, Ithaca, New York. serine so that it enters bacteria more readily and reacts with macromolecules. Investigation of biotransformation reactions in the ferret is requisite for .its development as a toxicologic research morlel. Several chemi­ cal promutagens subject to metabolic activation 436 DETERMINATIONOF CYTOCHROMEP-450 SUBPOPULATIONS by the hepatic microsomal mixed-function .._oxidase IN CONTROLAND PHENOBARBITAL INDUCED RAT LIVER system were.assayed by Salmonella typhimurium MICROSOMES. J. R. Gumbrecht and M.R. Franklin, reversion to histidine prototrophy, using the Dept. of Biochem. Pharmacol. and Toxicol., Univ. 9,000 xg. supernatant fraction (S-9) of consti­ of Utah, Salt Lake City, UT 84112. tutive ferret and rat hepatic tissue homogenates. Previous investigators have employed the forma­ Amounts of S-9 were varied in an effort to deter­ tion of metabolic-intermediate complexes from nor­ mine the optimal activating concentration in each benzphetamine in conjunction with other ligand species for those xenobiotic compounds tested. interactions to characterize 4 different subpopula­ The slope of the linear portion of the dose­ tions of cytochrome P-450 in microsomes from pheno­ response curve, the potency (revertants per ug), barbital induced rat livers. The use of piperonyl

123 butoxide, which can also produce a metabolic-inter­ sistent with the liver as a primary organ of DPH mediate complex, enables 2 of those 4 subpopula­ metabolism. The capacity of the fetus and neo­ tions to be further differentiated. A subpopula­ nate to metabolize DPH was less than that of the tion of cytochrome P-450 capable of forming a meta­ darns, (Supported by NIEHS Contract No, bolic-intermediate from norbenzphetamine but not NOl-ES-8-2151). interacting with metyrapone can be separated into 2 different types, one that can and one that can­ not form a metabolic-intermediate complex from pip­ eronyl butoxide. Another subpopulation that forms 438 EFFECT OF DOSE ON GAFFEINE METABOLISMBY THE a metabolic-intermediate complex with norbenzpheta­ ISOLATEDPERFUSED RAT LIVER. Satu M. Somani, mine and interacts with metyrapone can be similarly Dept. Pharmacology, Southern Illinois Univers­ subdivided into 2 types; one that also forms a ity School of Medicine, Springfield, IL 62708, metabolic-intermediate complex from piperonyl bu­ U.S.A. toxide and one that cannot. Of the 6 types present Metabolism of caffeine has been extensively in induced microsomes, 3 appear novel to pheno­ studied in animal and man. However, there is barbital induction. They comprise 2 types that can paucity of data on the effect of dose on its form metabolic-intermediate complexes from nor­ metabolism and its uptake by the isolated per­ benzphetamine and not piperonyl butoxide, but only fused rat liver. The perfusion studies were one interacts with metyrapone, and a third type carried out at four different doses of caffeine; which interacts with metyrapone but is unable to 2(5.6)/(5), 77.3(15) 154(30) and 309(60) umole form metabolic-intermediate complexes from either mg kg rat body weight. 14C-caffeine solu- substrate. Of the 3 types existing in both induced tion (1 ml) for each dose was added to the per­ and non-induced microsomes, only 1 (which interacts fusate in the reservoir after equilibration of with metyrapone and forms metabolic-intermediate liver for about 30 min. At least four livers complexes from both substrates) occurs at a higher were used at each dose level. 0.2 to 0.5 ml of concentration in induced microsomes. perfusate sample was taken out at different time The use of metabolic-intermediate complexes in intervals. Radioactivity was measured in each conjunction with conventional heme ligands has perfusate sample and the amount of radioactivity enabled the characterization of 3 types of cyto­ retained in the liver was calculated by mass chrome P-450 in non-induced microsomes and 6 types balance equation at each time interval. Caf­ in phenobarbital-induced rat liver microsomes. feine and its metabolites were determined in .1 Supported by USPHS Grant #CA 15760. ml perfusate by HPLC. The uptake of caffeine by the liver appeared to be dependent on the dose. The amount of drug taken up ranged form 8 to 35 percent. A linear increase in the uptake of 437 THE DISPOSITION AND METABOLISMOF DIPHENYLHYDAN­ caffeine by the liver on a per gm basis was seen TOIN IN MATERNAL,FETAL AND NEONATALTISSUES with the increase in dose, at the end of the AFTER PERINATAL EXPOSUREOF RAT DAMS. A.E, Chin, experiment. The uptake was not a saturable P.J. Kurtz, B.D.Carlton, A.C.Peters, and R.S. process at the doses studied. Major metabolites Chhabra, Battelle Memorial Institute, Columbus, (M) of caffeine viz. theophylline, theobromine, OH. and National Institute of Environmental 3-methylxanthine, 1-methylxanthine and 3-methyl­ Health Sciences, Research Triangle Park, NC, uric acid were determined. The metabolites (M) in the perfusate were 15.4% at 5 mg/kg which Female Fischer 344 rats were fed 800, 240, 80 and consistently decreased to 4.6% at 60 mg/kg dose, O ppm diphenylhydantoin (DPH) from two weeks pri­ whereas the percent of metabolites (M) remained or to breeding until treatment to evaluate the almost the same ("' 5%) in the liver at the end effect of perinatal exposure to DPH on the dispo­ of the experiment irrespective of dose. sition and metabolism of a subsequent single i.v. dose of DPH (50 mg/kg) given during gestation or postpartum. In rats administered radiolabeled DPH on Day 18 of gestation the levels of radio­ 439 DRUGMETABOLISM IN RODENTSINFECTED WITH TRYPAN­ active DPH equivalents in placenta and fetal liv­ OSOMESOR TREATEDWITH TRYPANOCIDES. H.G. Sher­ er were found to be in equilibrium with the DPH tzer, Kettering Laboratory, Univ. of Cincinnati, pool in maternal blood, while maternal liver lev­ Cincinnati, OH 45267, and J. Ed. Hall and J.R. els were 3-4 x greater than maternal blood levels Seed, Dept. Parasit., Sch. Pub. Hlth., Univ. NC, in all dose groups studied, A transplacental Chapel Hill, NC 27514. SPONSOR: P.B. Hammond transport of DPH is evident. Residual concentra­ tions of.DPH equivalents in maternal and fetal We used two animal models to evaluate the acute tissues four hours after injection of radiola­ and chronic effects of infection with Trypano­ beled DPH were dependent upon the level of prior soma brucei gambiense on hepatic drug metabolism. maternal exposure indicating a dose-dependent The mousewas used as a model for the acute in­ difference in clearance rates, These results can fection, since it exhibits fulminating parasit­ be explained in terms of a balance between an in­ emia about 3 days post inoculation. The field duction of DPH metabolizing enzymes resulting in vole Microtus montanus was used to evaluate the an increased DPH elimination and a saturation of chronic infection, since after inoculation it these enzymes due to an accumulation of DPH dur­ undergoes waves of parasitemia for over 4 weeks. ing the perinatal exposure period, In lactating Three days after inoculation in mice, there was females administered radiolabeled DPH on Day 12 hepatic edema and decreases in tissue DNA, cyto­ postpartum, dam blood levels of DPH equivalents chrome P-450 and capacity for metabolizing sev­ correlated with milk levels and blood levels in eral foreign compounds. In Microtus, after 28 the pups, indicative of neonate exposure via the days there was tissue edema and a large decrease milk. Accumulation of DPH equivalents was obser­ in hepatic cytochrome P-450 content and related ved to occur in maternal and neonatal livers con- enzyme activities. However, there was little

124 change in other endoplasmic reticulum enzyme synthesis of cytochrome P-450. Since induction markers not relating to cytochrome P-450. These of phase II metabolism has not been thoroughly markers include glucose-6-phosphatase, cytochrome studied, effect of the following xenobiotics on ~5 and NAD(P)H-cytochrome £ reductases. The data phase I and phase II biotransformation were eval­ indicate that there is selective toxicity for uated: phenobarbital (PB), 3-methy lcho lanthrene hepatic cytochrome P-450 during trypanosome in­ ( 3-MC), pregnenolone-160(-carbonitrile (PCN), 2,- fection in Microtus. In both the mouse and 3, 7 ,8 ,-tetrachlorodibenzo-p-dioxin, isosafrole Microtus, common trypanocides such as Surarnin, ( ISF), trans-stilbene oxide (TSO), butylated Melarsoprol B, ethidiurn bromide and Stilbam, were hydroxyanisole ( BHA). These inducers produced themselves found to be potent inhibitors of drug the expected effects on hepatic cytochrome P-450, metabolism in vitro. Furthermore, after chronic ethy lmorphine and benzphetamine N-demethy lat ion, treatment of mice with Suramin or Melarsoprol B, benzo-[a]pyrene hydroxylation, ethoxyresorufin there was a significant increase in pentobarbital 0-deethylation, and styrene oxide hydration. PCN sleeping times, indicative of an impaired capa­ and 3-MC were the only chemicals that increased city for pentobarbital metabolism and detoxifica­ N-acetylation of ;:l-naphthylamine (60 and 4m.s, tion in vivo. We suggest that during trypano­ respectively). Glutathione conjugation of di­ somiasis or with associated chemotherapy, there chloronitrobenzene, dinitrochlorobenzene and is a reduced capacity to metabolize foreign com­ sulfobromophthalein was induced from 50 to 1709.S pounds. (Supported by UNDG/World Bank/WHO and by TSO, BHA, ISF, and PB. Glucuronidation was NIH-GM 27928). also enhanced by treatment with these inducers. For example, PB increased glucuronidation of chloramphenicol (250%) and morphine (115%); 3-MC 440 THE EFFECT OF PREGNANCYAND ESTRADIOL-176 TREAT­ increased conjugation of 1-naphthol and p-nitro­ MENTON THE BILIARY TRANSPORTMAXIMUM OF ORGANIC phenol ( 19mo and 809.S, respectively); PCN enhanced ANIONS IN THE ISOLATED PERFUSED RAT LIVER. A.C. chloramphenicol ( 135~.S) and morphine ( 5~.S) conju­ Auansakul and M. Vore, Grad. Center for Toxicology gation but had no effect on that of 1-naphthol or and Dept. of Pharmacology, Univ. of Kentucky, p-nitrophenol. The most dramatic effect was the Lexington, KY. . 110~0 increase in glucuronidation of digitoxi­ The purpose of this study was to investigate the genin monodigitoxoside by PCN. PCN was the only effect of pregnancy and estrogen on the biliary inducer to enhance sulfation of 2-naphthol (28%), transport maximum (Tm) of three organic anions in taurolithocholate (11Ho), and dehydroepiandro­ the isolated perfused liver system. Livers from sterone ( 140~0). In conclusion, PCN induces a untreated female (control), estradiol-17S (Ez) wider spectrum of phase II react ions than the treated female (1 mg/kg/day, s.c. for 14 days) and other microsomal enzyme inducers. (Supported by pregnant (19-21 days of gestations) rats were per­ USPHS Grants AM 14513 and ES 07079). fused with 20% male rat blood in a Krebs-Ringer bicarbonate buffer equilibrated with 95% Oz/5% CO2 at 37°C. Dibromosulfophthalein (DBSP), [14c]5- phenyl-5-parahydroxyphenylhydantoin (HPPH) and 442 ALCOHOL-STRESSINHIBITION OF MIXED FUNCTIONOXI­ [14c]morphine were infused for 90 min into the DASE METABOLISMIN THE RAT. D. Brown, J. Ning, perfusate for a total dose of 41.2, 18 or 40.5 M. Bogdanffy, I. Krull, S. Kuttab, and L. Shargel. µmol respectively. The concentration of [14c]HPPH Toxicology Program, Col. Pharrn. & Allied Hlth Prof and [14c]morphine declined in the perfusatewhereas Northeastern Univ. Boston, MA. the concentrations of [14C]HPPH-glucuronide (HPPH­ Stress and Alcohol are environmental factors G) and [14C]morphine-glucuronide (MG) increased which alter the metabolism of xenobiotics. Both during the 90 min experiment indicating that the acute stress and acute alcohol inhibited in vitro rate of formation of the glucuronide exceeded its and in vivo metabolism of Hexobarbital inthe rat. rate of excretion in bile. The Tm (nmol/min/g (Chung and Brown, 1976 Life Sci. 18 123). This liver) of DBSP was 26.8+1.4, 30.1+2.7, 18.6+0.8; investigation gives further evidence that acute of HPPH-G was 25.0_:t::l.4,-15.6_:t::1.8,-18.0_:t::1.6;-and of alcohol po and/or acute stress inhibit other hepa­ MG was 20.9+2.2, 9.4+1.2, 10.6+1.8 for control, tic MFO pathways. Ez-treated and pregnant rats respectively. This Rats were given 1 to 4 g/kg Alcohol (ETOH) and/ indicated that Ez treatment significantly decreas­ or stressed by hind leg ligature (HLL). The rats ed (P<.05) the Tm for HPPH and MG but not for DBSP were sacrificed (1 hr) by decapitation, livers ex­ where-;;:s pregnancy decreased (P<0.5) the Tm for all cised and homogenized in cold 1.15% KCL. The three organic anions. Bile flow (µ1/min/g liver) 9000 x g fraction was used to determine Aminopy­ was significantly decreased in Ez-treated and rine N-demethylase (AP) and Aniline hydroxylase pregnant rats only in the experiments using mor­ (AH) activities. phine, thus the effects of pregnancy and estrogen AP activity was inhibited 26% by 3 g/kg ETOH on bile flow do not appear to be responsible for 28% by HLL and 46% by ETOH and HLL. ETOH (1, 2 & the decrease in biliary excretion. These data 4 g/kg) inhibited AP but inhibition is not dose suggest the presence of multiple carriers for related. HLL for 30 and 60 min. inhibited AP ac­ organic anions which are differentially affected tivity 20 and 28% respectively (P<0.05). by estrogen treatment and pregnancy. (HD13250). AH activity was inhibited 12 to 22% by ETOH (1, 2 & 4 g/kg). HLL (30 min) inhibits AH 17%. HLL and ETOH inhibits AH 26 to 37%. (P-0.05) Benzpyrene (BP) metabolism was measured by dis­ 441 INDUCTION OF HEPATIC PHASE II BIDTRANSFORMATION appearance using 100,000 x g fractions from rats IN THE RAT. T.N. Thompson, J.B. Watkins, 7. pre-treated with 3-methylcholantrene (30 mg/kg}. Gregus, and C.D. Klaassen, Dept. of Pharmacology, Acute ETOH 5 g/kg 1 hr prior to sacrifice inhibit­ Univ. of Kansas Medical Center, Kansas City, KS. ed BP metabolism 16.7% and corticosterone (12.7mg/ Numerous xenobiotics and endogenous ch~micals kg ip) 15 minutes before sacrifice inhibits 31.2%. induce phase I biotransformations by enhancing In adrenalectomized rats neither treatment inhib-

125 ited BP metabolism. Blood ETOH (1 hr) were 0.17 91, 78 and 56%of control, respectively, at 2hr and 0.29 g/100 g for the 3 and 5 g/kg doses. after FEN. It declined further at 5hr and by 12hr These findings demonstrate that several MFO liver CE was at 4% of control after 500mg/kg. pathways are inhibited by acute ETOH and stress Hepatic GSHwas decreased maximally at 5hr after and suggest that the mechanism of the inhibition each dose (32%control after 500mg/kg}, and some may be mediated by the adrenals. (Supported by recovery occurred by 12hr (45%control after 500 NU Research and Scholarship Development Fund). mg/kg). In contrast, hepatic MFO(aniline hydrox­ ylase and .2_-nitroanisole 0-demethylation) activ­ ities were reduced to 15-20%of control as early as 2hr after 500mg/kgand remained at that level 443 INFLUENCEOF CHRONICETHANOL CONSUMPTION ON through 12hr. MFOactivity 2, 5 or 12hr after 50 COCAINE-INDUCEDHEPATOTOXICITY AND METABOLISM. mg/kg was 45, 63 and 75%of control. Results F.J. Peterson, N.J. Lindemann and P.H. Duquette. indicate that the MFOand brain CHEresponses to V.A.M.C. Research Service, Mpls. Mn. 55417 FENare similar, with maximal inhibition occurring (sponsor: M.W. Anders). by 2hr. In contrast, maximal inhibition of liver CE does not occur until 12hr after FEN. The long­ Chronic ethanol (E) consumption (10% E in the lasting biochemical effects of FEN, in the absence drinking water) potentiates the hepatotoxicity of cholinergic symptoms, may be of toxicologic of acetaminophen. Because cocaine and importance. acetaminophen produce hepatic damage and are metabobized to reactive intermediates, the effect of chronic E consumption on cocaine­ induced heptatotoxicity and metabobism was 445 EFFECTOF FENITROTHION(FEN) EXPOSUREON ACET­ studied. Cocaine, given i.p. to mice, produces AMINOPHEN(APAP)-INDUCED HEPATOTOXICITY. G.L. hepatic damage (elevation in SGPT activities) Ginsberg, M.Placke and S.D.Cohen. Sect. Pharmacol in a dose dependent manner. Chronic E intake & Toxicol. ,Sch. Pharmacy, and Dept. Pathobiol., for 3 or 12 weeks decreases cocaine-induced_ Coll. Agric., Univ. Conn., Storrs, CT 06268 hepatotoxicity while acetaminophen hepatic Depletion of hepatic glutathione(GSH) prior to damage is strikingly enhanced. The in vitro APAPadministration enhances APAPhepatotoxicity. N-demethylation of cocaine is not altered by GSHis involved in the detoxification of the in­ E intake at 3 weeks but is increased by E secticide FENand the possibility of simultaneous intake for 12 weeks. The in vitro covalent exposure to APAPand FENexists. This study was binding of actaminophen t~microsomal protien undertaken to determine if interactions of toxic­ is increased with E intake for 3 and 12 weeks. ologic importance might occur during such exposur~ Total hepatic cytochrome P-450 is unchanged by Liver GSHlevels were maximally depressed to 58 3 or 12 week E consumption. Phenobarbital and 30%control 5 hr after 50 or 500 mg FEN/kg,ip, pretreatment increases cocaine-induced respectively. Similarly pretreated mice were hepatotoxicity, the in vitro metabolism of challenged with APAP(400 or 600mg/kg,po) and cocaine and hepatic cytochrome P-450 content. plasma sorbitol dehydrogenase (SDH)activity was Cocaine (lmM) stimulates the NADPH- supported measured 18hr later as an index of hepatic damage. consumption of 02 Two fold. This cocaine­ No SDHelevation was detected in the FENpretreat­ induced increase in Oz consumption is decreased ed mice, yet in 2 of 23 vehicle-pretreated mice by SOD and catalase. The results suggest that SDHwas elevated after 400mgAPAP/kg; and after although the N-demethylation of cocaine is 600mgAPAP/kg, 15/19 mice had elevated SDH(x=775% increased, the chronic consumption of E, when control). The apparent FENprotection was con­ not inducing an increase in total cytohrome firmed microscopically. The massive necrosis and P-450 content, may interfer with the activation 70%lethality after 800mgAPAP/kg was prevented of norcocaine to the proximal toxin responsible by 5 hr pretreatment with 50mg FEN/kg. To deter­ for hepatic injury. mine if the protection might be due to FENpre­ vention of APAPconversion to an electrophilic intermediate, we measured aniline hydroxylase(AOH) and p-nitroanisole 0-demethylase(PNA) as indica­ 444 FENITROTHIONDEPLETION OF GLUTATHIONEAND INHIB­ tors of mixed function oxidase(MFO)activity. ITIONOF TISSUEESTERASES AND HEPATIC MIXED Five hr after FEN50 or 500mg/kg, AOHactivity FUNCTIONOXIDASES. G.L.Ginsberg and S.D.Cohen, was 64 and 18%and PNAwas reduced to 39 and 22% Sect. Pharmacol. & Toxicol., Sch. Pharmacy, Univ. control, respectively. With respect to inter­ Conn., Storrs, CT 06268 actions with APAP,these results suggest that FEN inhibition of MFOpredominates over FENdepletion Fenitrothion[0,0-dimethyl-0-(3-methyl-4-nitro­ of GSH. Thus, even though GSHstores were lower­ phenyl) phosphorothioate] is a widely used organo­ ed, less APAPelectrophile was generated and phosphate insecticide. In addition to the expect­ critical macromolecules were spared. ed inhibition of tissue esterases, inhibition of mixed function oxidases(MFO) and depletion of hep­ atic glutathione(GSH) have also been demonstrated after Fenitrothion(FEN) exposure. This study was undertaken to investigate the dose and time re­ 446 SCANNINGELECTRON MICROSCOPY OF ACETAMINOPHEN­ sponse relationships of these FENeffects. At 2hr INDUCEDHEPATOTOXICITY IN MICE. R.M, Walker, after ip injection of 50, 200 or 500mgFEN/kg, in W.J. Racz, and T.F. McElligott, Departments of mice, brain cholinesterase(CHE) was reduced to 52, Pathology and Pharmacology, Hotel Dieu Hospital 52 and 36%of control activity respectively. With­ and Queen's University, Kingston, Ontario, in 12hr, activity had returned to 75%of control Canada. Sponsor: G.C. Becking. after 50mg/kg, but had not changed after 500mg/kg. We have previously shown that acetaminophen­ Liver carboxylesterase(CE) activity was reduced to induced hepatotoxicity involves alterations of

126 cell surface morphology and the development of interactive effects of exposure to mixtures of massive centrilobular congestion due to these two chemicals were evaluated. entrapment of red blood cells (RBC) before the Supported, in part, by training grants T32 GM07767 appearance of necrosis. These phenomena have NIH, and T32 ES07062, NIEHS. been investigated by scanning electron microscopy (SEM). Within 1.5 hr of acetaminophen administration (750 mg/Kg by gavage), SEM of centrilobular areas reveals 448 TifE EFFECT OF UBIQUINONE-10 ANTAGONISMUPON TifE endocytic vacuoles at the sinusoidal poles of ACUTETOXICITY OF DOXORUBICIN. A.B. Combs, E. hepatocytes, reduced numbers of microvilli in Lewandowski, and *K. Folkers, Dept. of Pharmacol­ the Disse space, loss of the stud-like ogy of the College of Pharmacy and *Inst. for projections from the lateral surfaces of Biomedical Repearch, Univ. of Texas, Austin, TX. hepatocytes, dilation of bile canaliculi, and Sponsor: D. ficosta enlargement of the sinusoidal lining cell Previous work in this laboratory has shown that fenestrations. By 3 hr after acetaminophen ubiquinone-10 (CoQ) can reduce the acute toxicity RBC's are in the Disse space, between of doxorubicin (DOX) in rats and mice. It is not hepatocytes, and within the endocytic vacuoles clear whether that protection is due to the non­ of many hepatocytes. The number of RBC's within specific antioxidant actions of CoQ, or to its the Disse space is sufficient to collapse the very specific coenzyme functions. The present original sinusoidal lumen by 6 hr. Sinusoidal study was designed to help differentiate between lining cells are not lost, but apparently held these mechanisms. The in vitro CoQ-antagonist, in position by cytoplasmic projections from 2-hydroxy-3-n-dodecylmercapto-1,4-naphthoquinone hepatocytes, preservation of intercellular (I), or its-vehicle, 10% Tween-20, was injected junctions, and anchorage by fat storing cells ip at a dose of 50 mg/kg in female CD-1 mice. It within the Disse space. We suggest that was injected two hours following a 10 mg/kg ip hepatotoxic congestion develops as a result of dose of phytonadione. One hour following I, alterations in the relationship between single acute doses of 15 or 20 mg/kg of DOX or hepatocytes and sinusoidal lining cells which its vehicle, water, were given ip. Animal sur­ then separate and thus produce an expanded Disse vival was determined on a daily basis. After 12 space. RBC's enter the Disse space through the days, the survival frequencies were: 1) untouched enlarged fenestrations in the sinusoidal lining controls and vehicle controls, 100% each (10/10 cells. each), 2) DOX, 15 mg/kg, + vehicle for I, 92% (Supported by the Canadian Liver Foundation). (23/25), 3) DOX, 20 mg/kg, + vehicle for I, 65% (17/26), 4) water, 10 ml/kg,+ I, 96% (25/26), 5) DOX, 15 mg/kg,+ I, 36% (9/25), and 6) DOX, 447 THE NEPHROTOXICITYOF 1,2-DICHLOROETHANEAND 1,1- 20 mg/kg,+ I, 12% (3/26). The data indicate that DICHLOROETHYLENEIN MALESPRAGUE-DAWLEY RATS. an antagonist to CoQ can enhance the toxicity of N.M. Jackson, W.J. Al-Hassan, and R.B. Conolly, DOX. This, in turn, provides evidence that a Toxicology Program, Sch. of Pub. Hlth, The Univer­ more specific action than antioxidation may be sity of Michigan, Ann Arbor, MI involved in the reduction of DOX toxicity by CoQ. Sponsor: H.H. Cornish Sponsored by Daniel Acosta.

1,2-Dichloroethane and 1,1-dichloroethylene are important industrial chemicals. Male Charles River Sprague-Dawley rats were exposed by inhala­ 449 EFFECT OF A LOW-PROTEINDIET ON CONTRACEPTIVE tion to various concentrations of these chemicals STEROID-INDUCEDCHOLESTASIS IN FEMALERATS. for four hours and killed 24 hours after the A. Idris, U.A. Boelsterli, H. Kotik, and start of exposure. In normally fed rats exposed T. Balazs, Division of Drug Biology, Food and to 2000 ppm 1,2-dichloroethane, there was a signi­ Drug Administration, Washington, D.C. ficant (p <0.05) increase in kidney to body weight ratios (gm kidney/100 gm body weight; exposed: Due to the wide use of oral contraceptives (OC) 1.73 + 0.12 vs. control: 0.97 + 0.08), BUN (mg in human populations suffering from malnutrition, urea "ii"itrogen/100 ml; exposed:-50.61 .± 4.35 vs. an increasing number of women might be exposed control: 8.37 + 0.61) and serum creatinine (mg to the hepatotoxic effect of OC when the liver is creatinine/100 -;;:;1; exposed: 3.46 + 0.46 vs. con­ compromised due to protein deficiency. To study trol: 0.83 + 0.02). Rats fasted-18 hours prior such a possible interaction in the rat model, to exposure to 2000 ppm 1,2-dichloroethane had groups of female Sprague-Dawley rats (6 to 10 BUN and serum creatinine values significantly per group) were given daily oral doses of a higher than fed exposed rats. These values were: combination of OC, 17a-ethinyl estradiol (EE) and BUN (fasted: 65.37 + 9.73 vs. fed: 50.61 + 4.35) 19-norethisterone (NE), for a maximum of 8 weeks. and serum creatinine (fasted: 4.51 .± 0.35-vs. fed: A low-dose group received 0.25 mg/kg EE and 3.46 + 0.46). Rats exposed to 1000 ppm 1,2-di­ 2.5 mg/kg NE. A high-dose group was given weekly chlor~ethane for four hours showed slight but sig­ doubled doses until 4 mg/kg EE and 40 mg/kg NE nificant increases in serum creatinine values at were reached. The rats were fed a low-protein both 24 and 48 hours post-exposure. Rats exposed (LP) diet (8%), starting 8 weeks before treatment to 5000 ppm for four hours all died between 2 and with OC, and continuing throughout the study. 18 hours after the end of the exposure. The Cholestatic liver injury, as measured by basal acute nephrotoxicity of a structurally related bile flow, bile acid secretion, and BSP excre­ chemical, 1,1-dichloroethylene was also investi­ tion, could be induced in control rats receiving gated. Since in addition to their structural the LP diet alone. In control animals on a similarities, 1,2-dichloroethane and 1,1-dichloro­ normal diet, OC administration alone also pro­ ethylene share common metabolic pathways, the duced a marked dose-dependent cholestatic effect.

127 However, the combination of a chronic LP diet tories. For the dominant lethal study, male rats with OC resulted in an ameliorating effect on were fed a diet containing EDA·2HC1 at 0.5, the pathophysiology of bile secretion and hepatic 0.15, 0.05 or O g/kg/day for 23 weeks prior to excretory function. Thus, in the rat, a mild mating. A positive control group of male rats protein malnutrition does not enhance the was treated with a single ip injection of 0.25 cholestatic effect of the steroids but rather mg/kg triethylenemelamine. Each male rat was protects the liver from cholestatic liver damage mated with 1 naive female/week for three caused by an LP diet or OC administration alone. consecutive weeks. Approximately 13 days after conception, female rats were sacrificed and uteri were examined. The criteria studied included: fertility, corpora lutea count, 450 FEASIBILITY OF USING HEPATOCYTESIN A CYTOTOXIC implantations/female and late and early fetal PRESCREEN. D. L. Story, S. J. Gee, K. Hawk­ deaths/female. There were no dose-related Prather, C. A. Tyson, SRI International, Menlo responses in any of the criteria examined in the Park, CA, and D. H. Gould, USEPA, Washington, EDA-treated rats; however, marked responses were D.C. 20460. Sponsor: D.C.L. Jones. noted in the· positive control group. We conclude that there is no evidence in these studies A potential prescreen cytotoxicity assay is being implicating EDA as a mutagen. evaluated. Isolated hepatocytes (1.5 x 10 6 cells per ml) were incubated for up to 6 hr at concen­ trations of 10, 1 or 0.1 mMin 3 ml of hormone­ supplemented Waymouth's medium. Using glutamate­ 452 MUT AGENIC ACTIVITY OF COCAINE NITROXIDE oxaloacetate transaminase (GOT) release as the RADICAL IN THE AMES SALMONELLA TEST SYSTEM. test criterion, the lowest concentrations elici­ M.A. Evans and Joseph Jankauskis. Interdisciplinary ting GOT release were: 0.1 mMfor cadmium ToX1cology Program, Dept. Pharmacology, University of acetate, chlorhexidine diacetate, 2-chlorobi­ Illinois Medical Center., Chicago, IL 60680. phenyl, 4-chlorobiphenyl, mercuric acetate; 1 mM Cocaine (C) produces an acute hepatic necrosis in for 3-amino-1,2,4-triazole, sodium selenate, mice pretreated with agents which enhance microsomal 2,4-dinitrophenol; iodoacetate, sodium arsenite, metabolism. The necrosis is species specific for the 2,4,2' ,4'-tetrachlorobiphenyl, tris(2,3-dibromo­ mouse and is associated with decreased glutathione propyl)phosphate; and 10 mMfor diethanolamine, concentration and increased covalent binding of radio­ hydrazine hydrate, paraquat, phosphonoacetic acid, labelled cocaine to microsomal protein. Incubation of styrene, and styrene oxide. CBrCl3, CCl4, 1,1,1- cocaine or norcocaine with mouse Ii ver microsomal and 1,1,2-trichloroethane and CHCl3 were active suspensions and a NADPH generating system produces a in the 1-10 mMrange, with CBrCl3 and CCl4 being the most cytotoxic of these haloalkanes in the nitroxide radical based on the appearance of the prescreen. Dimethylnitrosamine did not release characteristic electron spin resonance spectra for nitr­ oxides. The half life for the nitroxide in the micro­ GOT even at a concentration of 10 mM. To deter­ mine if cytotoxicity is occurring at lower con­ somal suspension at 4°C is less than four minutes. centrations than indicated by GOT release, other Norcocaine (IO µg) incubated with mouse hepatic viability indices such as ATP level and urea S-9 fraction showed weak mutagenicity (31 revertants/ synthetic capacity of hepatocytes are being plate) with TA 98 and strong mutagenici ty (836 rever­ assessed. This type of assay may be useful as tants/plate) with TA 1538. No mutagenicity was a rapid, economical prescreen to identify chemi­ observed with hepatic S-9 fraction from rat or rabbit or cals warranting further animal studies. This with heat-treated mouse hepatic S-9 fraction. work was supported by EPA Contract 68-01-5079. Further studies . demonstrated that chemically synthesized cocaine nitroxide was directly mutagenic with TA 1538 but other known metabolites of cocaine were inactive without activation. The apparent greater 451 ASSESSMENTOF THE MUTAGENICPOTENTIAL OF mutagenicity of cocaine nitroxide in TA 1538 vs. TA 98 ETHYLENEDIAMINE: IN VITRO AND IN VIVO STUDIES. suggests that the nitroxide directly alters DNA struc­ R. S. Slesinski, P. J. Guzzie, w. C. Hengler, ture and is not dependant on errors during DNA repair. M. D. Woodside, R. s. H. Yang and P. G. Watanabe. This is consistent with results from in vitro studies Bushy Run Research Center, Mellon Institute-Union which demonstrated that the nitroxide reacts irreversi­ Carbide Corporation, Export, PA and Toxicology bly with guanine, cysteine and reduced glutathione to Research Laboratory, Dow Chemical Co., Midland, form a covalently-bound adduct. ML

Ethylenediamine (EDA) was evaluated for potential genotoxic activity using a battery of 453 MUTATIONPROMOTED BYACTIVATED OXYGEN SPECIES. in vitro and an in vivo mammalian tests: Chinese H. P. Misra and C. I. Wei, Lab. for Energy-Rel. Hamster Ovary (CHO) gene mutation, Sister Hlth. Res., Univ. of Calif., Davis, CA (Spon: Chromatid Exchange (SCE) with CHO cells, S. N. Giri). Unscheduled DNA Synthesis (UDS) in primary rat hepatocytes and a Dominant Lethal study using Benzo(a)pyrene [B(a)P] can be activated by Fischer 344 rats. In vitro tests evaluated a cytochrome P450 containing monooxygenasesystem minimum of five doses spanning a wide range of to potent mutagens and highly reactive DNAbinding concentrations. EDA did not produce a dose­ species. Since the microsomal drug-metabolizing related mutagenic effect in either the CHO system requires the presence of both reducing mutation test or in the SCE test with or with­ agent and molecular oxygen, the role of activated out a rat liver S9 activation system. With oxygen species in the activation of B(a)P to an hepatocytes, no dose-related effect was produced active mutagen was investigated. In these studies by EDA in UDS tests at two different labora- we have examined the antimutagenic effects of

128 several knownscavengers of different activated 455 PHENOBARBITALALTERATION OF 1-NITRONAPHTHALENE oxygen species in the AmesSalmonella typhimurium LEVELS IN TARGETORGANS OF TOXICITY. O. Dankovic test system in the presence of B(a)P with and and H.H. Cornish. Toxicology Program, Sch. of without liver microsomal (S-9) fraction. In the Pub. Hlth, The University of Michigan, Ann Arbor, presence of S-9, the polycyclic aromatic hydro­ Ml carbon B(a)P, was confirmed mutagenic using tester 1-Nitronaphthalene (1-N.N) is acutely toxic to strains TA 98 and TA 100. Superoxide dismutase, both Jung and liver; pretreatment with phenobar­ which removes 02- radicals, at 10 -> 100 µg/ml bital prevents Jung toxicity, but potientiates on agar plates had no significant effect on the I iver damage. It was previously shown that pheno­ number of revertants produced by activated B(a)P. barbital Jowers the total level of 1-NN ~ metabo­ Catalase, which removes H202, at 10 -> 100 µg/ml lites by more than 50% in liver, and 80% in Jung. or mannitol, which removes OH•, at 3 -> 12 rrM had The levels of unchanged J-NN in Jung, I iver and trivial effect. Dimethylfuran (DMF),an effective blood have now been measured, using a chloroform/ singlet oxygen scavenger, however, inhibited the methanol/water system for extraction, and reverse mutagenic response of activated B(a)P in a dose­ isotope dilution for quantification. dependent manner. Thus, in TA 100 strain using Fol lowing phenobarbital pretreatment l~NN levels 0.5 µg/plate B(a)P, the inhibition of number of in Jung, I iver, and blood were 7.9, 5.5, and 6.7%, revertants by 3, 6 and 9 mMDMF was 40, 60 and respectively, of the levels in the 1-NN treated 75%, respectively. In TA 98 strain, using 4 µg/ control animals. These data suggest that an plate B(a)P, 6 mMDMF inhibited 50%of the number increased rat of metabolism and excretion prevents of revertants. These levels of DMFwere neither 1-NN from reaching the lung in sufficient quanti­ toxic to the cells nor were they found to have any ties to produce lung damage. The excretory rate mutagenic activity in the absence of B(a)P. The and metabolic profile of 1-NN in control and antimutagenic activity of DMFwas dependent upon phenobarbital pretreated animals is now being the activation of B(a)P by S-9 system. These data investigated. indicate a major role for singlet oxygen in pro­ (Supported in part by NIEHS training grant moting mutagenesis (Supported by U.S. Dept. of ES07062.) Energy).

456 SPECTROSCOPICSTUDIES OF Cd, Zn-METALLOTHIONEIN. 454 IN VIVO GENOTOXICEFFECT OF 1,2-DICHLOROETHANEIN A.Y.C. Law and M.J. Stillman, Dept. of Chem., MALEB6C3Fl MICE. R.D. Storer, P.A. Bank, and Univ. of Western Ontario, London, Ontario, Canada R.B. Conolly, Toxicology Program, Sch. of Pub. N6A 5B7. Sponsor: G.M. Cherian Hlth, The University of Michigan, Ann Arbor, MI Following injections of aqueous solutions of the Sponsor: R.H. Cornish B group metals zinc, cadmium and copper, a low molecular weight, sulfur-rich protein named met­ Results from the N.C.I. Bioassay Program have allothionein containing these metals at various shown that 1,2-dichloroethane (1,2-DCE) is carcin­ mole ratios can be isolated. The absorption, cir­ ogenic to the liver of male B6C3Fl mice when cular dichroism (CD) and magnetic circular di­ administered chronically by gavage. Our objective chroism (MCD) recorded for Cd, Zn-MT isolated is 1) to characterize the extent of DNAdamage from rat livers, from crab and guinea pig livers, produced in vivo in the liver of male B6C3Fl mice are all very similar. Comparison of the absorp­ following single oral doses of 1,2-DCE and 2) to tion and MCDspectra measured for thiolatocadmate identify what, if any, role glutathione conjugation model compounds confirms that the signal in the has in the in vivo activation of 1,2-DCE to a DNA­ 250 nm region arise from S->-Cdcharge transfer. damaging agent~ale B6C3Fl mice were gavaged The effect of changing the pH, loading the pro­ with a single 200 mg/kg dose of 1,2-DCE dissolved tein with Cd2+ and extensive dialysis were fol­ in corn oil and sacrificed 4 hours later. Suspen­ lowed using all three techniques. Analysis of sions of liver nuclei were prepared and analyzed the spectral changes suggests that while both the for evidence of DNAdamage by alkaline sucrose zn 2+ and Cd2+ may be displaced at low pH values, density gradient centrifugation. Animals dosed only the Cd2+ is rebound when the pH is returned orally with corn oil or by I.P. injection with 10 to 7. In addition, when excess Cd2+ is added mg/kg dimethylnitrosamine (DMN) served as negative In v,l;tJr,o the energies of the charge transfer and positive controls respectively. In a series transitions change significantly, which implies of three experiments, gradient profiles from 1,2- that the effect of added Cd2+ is to alter the DCE treated animals consistently showed slower protein conformation, which in turn results in a sedimentation relative to controls. The percent change in the stereochemistry around the original, of the total DNArecovered in the first five frac­ protein-bound Cd2+ ions. The metal-free protein tions of the gradients was significantly decreased; prepared by exhaustive dialysis at low pH does from 68.1 9.3% (control) to 52.4 12.3% + + not rebind Cd2+. (treated) 6;;:ean of three experiments-.± std. dev., p <0.02). By contrast, DMN(10 mg/kg) reduced the percent of the DNArecovered in the first five fractions from 68.1 + 9.3% to 7.0 + 3.0%. Liver/ 457 RESISTANCEOF DIPLOID HEPATOCYTESIN THE body weight ratios, ierum sorbitol-dehydrogenase NEONATALRAT TO HALOGENATEDHYDROCARBONS. levels, and light microscopic evaluation at 24 J. Carlson and R, Abrabam, Institute of hours after 1,2-DCE treatment (200 mg/kg) did not Experimental Pathology and Toxicology, Albany reveal any evidence of liver necrosis. Liver Medical College. Albany, N. Y. 12208 glutathione levels (as non-protein sulfhydryl, 1 Previous studies from this laboratory have hour post dose) were significantly depleted (59.1%) indicated that rodent polyploid hepatocytes in 1,2-DCE (200 mg/kg) treated animals relative to selectively synthesize DNAand divide in response controls. to chemicals that are carcinogenic, leading to

129 the hypothesis that tetraploid and octaploid Research Laboratory, United States Environmental hepatocytes, but not diploids, are predisposed to Protection Agency, Cincinnati, OH 45268 and Ohio tumor formation. Unlike the adult rodent liver, University College of Osteopathic Medicine, Ath­ neonatal rats have a single class of diploid ens, OH 45710. Sponsor: R.J. Bull hepatocytes, and it was of interest to investi­ gate whether mirex, which is carcinogenic, would We have previously reported that CHCl3 is a very induce premature development of polyploidy. To potent stimulator of rat hepatic Ornithine De­ investigate this hypothesis further, neonatal carboxylase (ODC)activity. The ability of sev­ rats (5 or 15 day old) were given the chloro­ eral trihalomethanes to effect either hepatic or carbon, mirex (4.5 mg/kg) daily for 7 days. renal ODCactivity in male and female Fischer 344 Hepatic nuclei wire isolated and analyzed on rats was investigated. Chlorodibromomethane was the Coulter Counter· for alterations in volumes the most potent stimulator of hepatic ODCin both and frequencies. No significant differences were males and females while having a·unique inhibitory found between the nuclei of experimental and effect on male renal ODC. The other compounds control groups. These observations lead us to tested were all stimulatory in male and female believe that diploid hepatocytes are resistant livers, slightly stimulatory in male rat kidney to transformation by carcinogenic agents, and may but al 1 were virtually without any effect in explain why primates and humans, whose livers female kidney. are prepon~erently diploid, are not susceptible to these agents To further investigate the mechanism of tri­ halomethane stimulation of hepatic ODC, animals Supported by 5T32ES07058-03, and a Research Grant were pretreated with Diethyl Maleate (DEM,325 from the Royal Dutch Shell Company. mg/Kg) 2 hrs. prior to treatment with saline (control) or CHCl3. The level of hepatic ODCin DEMtreated rats was not different from control. However, the effect of DEMon CHCl3 induced ODC 458 DEPLETIONOF GLUTATHIONEBY METHYLCHLORIDE AND stimulation was a 3-fold enhancement of the stim­ RELATIONSHIP TO LIPID PEROXIDATION. D.J. ulation observed by CHCl3itself. Although, the Kornbrust and J.S. Bus, Chemical Industry Inst. effect of CDCl3was not statistically different of Toxicology, Research Triangle Park, NC, 27709. from CHCl3, the effect of DEMon CDCl3induced ODC Inhalation of methyl chloride by male B C F 6 3 1 stimulation was not nearly as dramatic as its mice results in a concentration-dependent deple­ effect on CHCl3. Furthermore, the direct addition tion of gluthathione (GSH) in liver, kidney and of CHCl3to in vitro ODCincubations from control brain. · Exposure for 6 hr to 100 ppm CH Cl (the animals had no effect on ODCactivity. These current TLV) lowered liver GSH by over ~0%, while findings suggest that the induction of ODCby exposure to 2500 ppm for 6 hr reduced liver GSH CHCl3is related to its metabolism. to 2% of control levels. For those exposures which decreased liver GSH to le·ss than 20% of con­ trol levels, the extent of liver GSH depletion was closely correlated with the capacity of a 9000 x g supernatant fraction from the liver to undergo 460 EFFECTOF TCDDON THE RESPONSE OF RATLIVER TO lipid peroxidation (LP), quantitated by measuring PARTIALHEPATECTOMY. B. J. Christian and R. E. the amount of thiobarbituric acid-reactive mater­ Peterson, Univ. of Wisconsin, Madison, WI. ial formed during a 20 min. incubation. Addition of GSH to the incubation prevented the LP. Also, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) surviving mice exhibited a rapid recovery of liver enhanced 3H-thymidine (Tdr) incorporation into GSH such that by 4 hr post-exposure LP was no rat hepatic DNAafter 1/3 hepatectomy (TAP 58: longer demonstrable. GSH was depleted to a lesser 398, 1981). Our aim was to further describethe extent in mouse brain and kidney, compared to response. It was found that this response to a liver, and no relationship to peroxidation was ob­ 1/3 hepatectomy performed 5 days after TCDD,was served for single exposures to CH Cl. A dose­ dose related from 1 µg/kg (no effect) to 30 µg/kg 3 dependent decrease in liver GSH was also produced (maximum)with an apparent ED50of 4-6 µg/kg. by diethylmaleate, although a nearly lethal amount The effect was greatest if rats were 1/3 hepatec­ (2 ml/kg) was required to lower liver GSH to less tomized 5-10 days after 5 µg/kg of TCDD. If rats than 10% of control levels under which conditions treated with TCDD(5 µg/kg, 5 days) were not the amount of LP was 3.S-fold less than in mice hepatectomized, or were given a 2/3 hepatectomy, exposed to 2000 ppm CH Cl. GSH was depleted to 3H Tdr incorporation was similar to control. a lesser extent by cH)~1 in the liver, kidney and However, if rats were laparotomized 3H Tdr incor­ brain of Fischer-344 rats, a species less sensi­ poration was 2-3x higher than control. Hepatic tive to the toxic effects of CH Cl. Exposure of enzyme activities and 3H Tdr incorporation into rats to 2000 ppm resulted in a ~-fold higher level liver DNAwere also compared at designated times of GSH remaining in the liver and a 30-fold lower from 2 to 32 hr after 1/3 hepatectomy. Activities amount of LP compared to mice similarly exposed.· of ornithine decarboxylase (a marker of G1 pro­ These findings suggest that depletion of GSH by gression), tyrosine aminotransferase, and o­ CH Cl to levels insufficient to prevent the occur­ glutamyl transpeotidase were altered by 1/3 reAce of lipid peroxidation may underlie the hepatectomy. However, all changes in enzyme hepatotoxicity of the compound. activities were similar in the two groups. In contrast, 3H Tdr incorporation was 2-3x higher in the TCDDgroup 24-28 hr after the operation. To determine if. the enhanced incorporation into DNA 459 TRIHALOMETHANESANDRAT RENAL AND HEPATIC ORNITH­ was caused by more hepatocytes entering S phase, INE DECARBOXYLASEACTIVITY. R.E. Savage, Jr., P. autoradiography was done. Labeling indices for Wiehl, C. Guion and M.A. Pereira, Health Effects periportal, midzonal and centrilobular hepatocytes

130 in control and TCDD-treated rats were similar. total and resting oxygen consu~ption before and Thus, TCDDenhances 3H Tdr incorporation into after 0, 15 and 50 µg TCDD/kg. Motor activity hepatic DNAof 1/3 hepatectomized rats by a mech­ was depressed by both doses of TCDD. Total anism that does not involve more hepatocytes oxygen consumption was reduced 13 and 22% below entering S phase. (Supported by NIH grant control levels in the 15 and 50 µg/kg groups ES01332.) respectively, one week after TCDD. This effect was reversible at the low dose but not at the high dose. Similar depressions in resting oxygen consumption were also found. Rats whose weight 461 COMPARISONOF BODY WEIGHT LOSS AND OXYGEN CON­ had been lowered before treatment (by food re­ SUMPTIONIN RATSTREATED WITH 2,3,7,8-TETRACHLORO­ striction) exhibited hyperphagia immediately DIBENZO-P-DIOXIN(TCDD) AND PAIR-FED CONTROL after dosing (when food was provided ad lib) RATS. M. D. Seefeld, S. ~I. Corbett, R. E. Keesey demonstrating that the ability to eat,s---i:etained and R. E. Peterson, Univ. of Wisconsin, Madison, after treatment. These studies indicate that WI. TCDD-inducedweight loss is not due to an increase Male rats were given a sinole oral dose of in metabolic rate. Rather, we suggest that TCDD TCDD(15, 25 or 50 µg/kg) and.paired mates, may lower a "set-point" for regulated body weight matched on a body weight basis, received an in a dose-dependent manner. If true, the reduced equivalent volume of acetone/corn oil. Daily food intake would be secondary to the rat's effort food intakes were corrected for spilled food to reduce its body weight to a lower maintenance since TCDDtreatment caused a progressive dose­ level. (Supported by NIH Grant ESOl332). related increase in spillage. Failure to account for spillage results in overestimating t~e amount of food consumed by TCDD-treated rats with con­ sequent overfeeding of pair-fed controls. Young 463 INDUCTIONOF 7-ETHOXYCOUMARIN0-DEETHYLASE rats (80-100 g) given 25 µg TCDD/kgdisplayed an ACTIVITY IN HUMANEPITHELIAL CELLS IN CULTURE: immediate reduction in food intake and weight gain EVIDENCEFOR DIOXIN RECEPTOR. L.G. Hudson and which persisted over the 35 day post-treatment W.F. Greenlee, Lab, of Toxicol., Harvard Sch. Pub. period, and 65%of the rats in this group died. Hlth., Boston, MA 02115. Sponsor: John G, Dent Pair-fed controls exhibited an identical weight 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a response but only 13%of the rats died. Experi­ potent inducer of cytochrome Pi-450-mediated ments using adult rats (275-300 g) demonstrated monooxygenase activities, including aryl hydro­ that pair-fed controls display identical weight carbon hydroxylase (AHR) and 7-ethoxycoumarin loss during the first 10 days after treatment as 0-deethylase (ECOD). In certain inbred strains of rats given 25 and 50 µg TCDD/kg. After day 10, mice this response is regulated by a single gene­ pair-fed rats exhibited a greater ability to main­ tic locus coding for a cytosolic receptor protein. tain their body weight on the reduced food intake In the present study the induction of ECODacti­ than TCDD-treated rats. In the 25 and 50 µg/kg vity by TCDD and several halogenated analogues dosage groups, 33 and 75% of the rats died re­ was examined in 5 human cell lines derived from spectively, whereas in the respective pair-fed squamous cell carcinomas (SCC) of epidermal and groups O and 15%of the rats died. In rats housed oral epithelia. The cells were grown in the pres­ in a thermoneutral environment, doses of 15 and 50 ence of a lethally irradiated feeder layer of µg TCDD/kgdepressed both total and resting oxygen mouse 3T3 cells in Dulbecco-Vogt modified Eagle's consumption, however, oxygen consumption was medium supplemented with 5% fetal calf serum, similarly reduced in pair-fed controls. These Addition of varying concentrations of TCDD studies demonstrate that reduced food intake is (lo-11 - 10-7 M) to confluent cultures of sec the primary cause of body weight loss and de­ 4, 9, 13 and 15 cells resulted in a dose-dependent pressed oxygen consumption in TCDD-treated rats. increase in ECODactivity with an ED50 ranging (Supported by NIH Grant ES01332). from 1 to 3 nM. Near maximal induction was ob­ served within 24 h. Under the same culture condi­ tions, addition of TCDDto SCC 12F2 cells at con­ centrations up to 1 µM produced no significant increase in ECODactivity. The structure-activity 462 EFFECTOF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN relationship for the induction of ECODactivity (TCDD)ON FOOD INTAKE, BODY WEIGHT AND ENERGY in the four responsive lines (SCC 4, 9, 13 and EXPENDITUREIN THERAT. M. D. Seefeld, S. W. 15) showed the same stereospecificity required Corbett, R. E. Keesey and R. E. Peterson, Univ. for binding to the TCDD receptor in murine liver. of Wisconsin, Madison, WI. Using sucrose density gradient analysis, specific binding of radiolabeled TCDDwas detected in the Food intake, body weight and energy expendi­ cytosol fractions from the responsive cell lines, ture were studied in male rats (275-300 g) whereas no specific binding could be detected in treated with a single oral dose of TCDD(5, 15, the SCC 12F2 cells. These data indicate the pres­ 25 or 50 µg/kg) or acetone/corn oil. Immediately ence of a putative receptor for TCDDin cultured after TCDDtreatment a progressive dose-related human epithelial cells that appears to regulate decrease in food intake and body weight was at least one inducible enzyme activity, observed. At 5 and 15 µg TCDD/kgthe effect was reversible in that these groups were consuming appropriate amounts of food for their reduced weights by days 15 and 25 respectively, whereas 464 EFFECTS OF CHLORDECONEAND ITS STRUCTURALANA­ the reductions in intake at the 25 and 50 µg/kg LOGS ON rct-P-NITROPHEpYL PHOSPHATASEIN RAT BRAIN doses were more persistent. Energy expenditure SYNAPTOSOMES.S. K. Bansal and D. Desaiah. Dept. was assessed in rats housed in a thermoneutral Neurology, Veterans Administration Med, Ctr. and environment by monitoring motor activity and Univ. Miss. Med. Ctr., Jackson, MS 39216.

131 The effects of chlordeconf, mirex, and four photo depletion. However, the greater susceptibility derivatives of mirex on K -stimulated p-nitro­ of kidney and testis to DBCPinjury following DM phenyl phosphatase in rat brain synaptosomes were pretreatment suggests that hepatic NPSmay protect determined. Additionally, the effect of chlorde­ against DBCPtoxicity in a general sense. cone on the substrate and K+ activation kinetics was also determined. Rat brain synaptosomes.were prepared by Ficol-Sucrose gradient centrifugation and the enzyme activity was determined by calori­ 466 EFFECTOF CHLORDECONEONCCl4 MEDIATEDDESTRUC­ metric method. Dose-response curves were deter­ TIONOF CYTOCHROMEP-450 ANDASSOCIATED PARAME­ mined by assaying the enzyme activity in the ab­ TERS. J. S. Klingensmith and H. M. Mehendale, sence and presence of several concentrations of Univ. Miss. Med. Ctr., Jackson, MS 39216. the test chemicals. The sensitivity of the enzy­ Wehave suggested that chlordecone (CD) en­ me to the various chemicals was highly variable. hances the hepatotoxicity of CCl4 by altering the Chlordecone was the most effective inhibitor with metabolism of CCl4. If altered metabolism of an ID50 of 7 µM. All other chemicals showed a CCl4 is indeed the mechanismof CD-potentiation, maximum inhibition of 20% at 20 µM concentration. then administration of sublethal doses of CCl4 The order of inhibition at 20 µM concentration was would be expected to lower parameters associated chlordecone (100%) > 8-monohydro mirex (20%) > 2, with the hepatic MFOsystem to a proportionately 8-dihydromirex (10%) > mirex (10%) > 10-monohydro greater extent in CD-treated rats than in control mirex (9%) > 5,10-dihydro mirex (2%). Double re­ rats. Male Sprague-Dawley rats (250-350 g) were ciprocal plots of the data obtained on the effect treated with CD, po (10 mg/kg) in corn oil (c.o.) of chlordecone on the substrate and K+ activation and control received c.o. vehicle alone. Twenty­ kinetics showed that the Vmax and Km were decrea­ four hr later, rats received an ip injection of sed with respect to paranitrophenyl phosphate, CCl4 or c.o. vehicle. Rats fed c.o. initially while it was classical non-competitive inhibition received 250 µl CCl4/kg, while rats fed CD ini­ with respect to K+ activation. These results tially, received 25 µl CCl4/kg. Twenty-four hr suggest that chlordecone is the most effective later, the animals were used to measure hepatic inhibitor among its structural analogs tested on microsomal protein, cytochrome P-450, microsomal paranitrophenyl phosphatase in rat brain synapto­ glucose-6-phosphatase, and aminopyrine-N-demethy­ somes. (Supported by PHS Grant ES-02443 of the lase activity. Serum enzymes (SGPT, SGOT,!CD) National Institute of Environmental Health were quantitated to serve as indices of hepatic Sciences). damage. Our findings show a significant increase in cytochrome P-450 content, G-6-Pase, and amino­ pyr.ine-N-demethyl ase activity in rats fed CDthan in the control group and a parallel decrease in 465 RELATIONSHIPOF TISSUENON-PROTEIN SULFHYDRYL microsomal protein, cytochrome P-450, G-6-Pase, GROUPS(NPS) TO THETOXIC EFFECTS OF l ,2-DIBROM0- and aminopyrine-N-demethylase activity in both 3-CHLOROPROPANE(DBCP). W.M. Kluwe, A. Greenwell CDand control animals which received CCl4. and F. Harrington (J. A. Moore, sponsor), Nation­ These results are supportive of the primary work­ al Toxicology Program, P.O. Box 12233, Research ing hypothesis that CDpotentiates CCl4 hepato­ Triangle Park, NC. toxicity by altering the metabolism of CCl4, and suggest that the mechanismof this alteration in DBCPis a nematocide causally associated with metabolism involves induction of a P-450 species infertility in male pesticide formulators and responsible for the bioactivation of CCl4. (Sup­ shown to produce testicular, epididymal, renal ported by ES-01369 and ES-07045). and hepatic injury in rats. Subcutaneous admin­ istration of DBCPto adult, male rats transiently depleted hepatic and caput (head) epididymal, but not renal or testicular NPScontents. The liver, kidney and testis _exhibited increases in tissue· 467 EFFECTOF CHLOROFORMAND DRINKING WATER CON­ NPSwithin 48 hr of treatment. The glutathione­ CENTRATESONMOUSE RENAL CORTICAL SLICE UPTAKE OF depletor diethyl maleate (DM)transiently lowered P-AMINOHIPPURATE.L.W. Condie and C.L. Smallwood, hepatic, renal and caput epididymal NPSin a dose­ Health Effects Research Laboratory, U.S. Environ­ and time-dependent manner. mental Protection Agency, Cincinnati, OH. Spon­ Single injections of DBCPproduced dose-depen­ sor: R.J. Bul1 dent lesions in the kidney, testis, caput epididy­ mis and liver. DMprior to DBCPenhanced the A recent research thrust of our laboratory has nephrotoxic potency of DBCP(greater elevation of been the evaluation of target organ toxicities BUNand more severe renal tubular necrosis in DM from exposure to environmental chemicals. The pretreated animals). Serum glutamic pyruvic effect of environmental chemicals on active up­ transaminase activity was greater in DMpretreated take of p-aminohippurate (PAH) by mouse renal than in non-pretreated animals after DBCP. cortical slices was utilized as a potential screen However, DMalone appeared to be hepatotoxic, sug­ for nephrotoxicity. Other renal function tests gesting additive effects on the liver. Seminif­ included blood urea nitrogen (BUN)and serum cre­ erous tubular degeneration was greater in rats atinine while serum glutamate pyru_vate trans­ pretreated with DMthan in non-pretreated aminase (SGPT) activity was measured as an in­ controls. dicator of 1iver function. Male CD-1mice were These results indicate that DBCPis a deple­ administered chloroform by daily oral gavage for tor of hepatic and caput epididymal NPSin the 14 days prior to sacrifice at levels of 150 mg/kg, acutely-toxic dose range. Since NPSconcentra­ 75 mg/kg and 38 mg/kg. PAHuptake was inhibited by tions were not lowered in two of the major target 64% and 17% in kidney slices taken from the 150 organs, kidney and testis, acute DBCPinjury would mg/kg and 75 mg/kg dose groups, respectively. BUN not appear to be dependent on local glutathione and SGPTvalues were only elevated in the high

132 dose group. Organic chemicals were concentrated synthesis on and transfer from the microsomes. from drinking water of two cities by reverse The major route of synthesis of PC in rat liver osmosis. Reverse osmosis extracts were daily is the sequential methylation of PE; therefore the injected intraperitoneally (200 mg/kg) for three in vivo incorporation of label from 3H-ethanol­ days prior to sacrifice. PAHuptake into kidney amine occurs in both PC and PE. The PC:PE ratio slices was inhibited 25%by this treatment while of counts decreases in the microsomal and mito­ BUNand serum creatinine levels were not altered. chondrial outer membranes and increases in the These studies suggest that the measurementof PAH inner membrane 44 hours after CCl4 exposure - the uptake in mouserenal cortical slices may serve as same time that mitochondrial function and PL com­ a useful tool for screening nephrotoxins that are position are most greatly altered. Two microsomal present in complex environmental mixtures. methyltransferases are responsible for the con­ version of PE to PC. The in vitro activities of both enzymes decrease in the 10 to 40 hour period after cc1 4 exposure. A soluble liver protein 468 PROTECTIVEEFFECT OF AUCUBAJAPONICA AGAINST that functions in vitro to transfer PL's between CARBONTETRACHLORIDE-INl)UCED LIVER DAMAGE. K. H. membranes has been described. The activity of 1 Yang, T. J. Kwon, and I. M. Chang (Span:~ this protein increases after CCl4 exposure, sug­ Peterson). Dept. of Biological Science and gesting that in in vivo it is part of the repair Engineering, Korea Advanced Institute of Science mechanism working to restore membrane PL composi­ and Technology, and Natural Products Research tion, 1 Institute, Seoul National University , Seoul, (Supported by PHS grant ES01919 and NIEHS training Korea grant ES07062.) Many plants have been used as herbs in oriental medicine for thousand years, but in most cases their effectiveness has never been proved. Among them Aucuba j aponica has be~n known to have protec­ 470 THE EFFECT OF 1,3-BUTANEDIOL ON THE HEPATOTOXIC

tive effect against liver diseases. Our object ACTION OF CC14 IN RATS. L.A. Hewitt, W.R. Hewitt was to determine if extracts from Aucuba j aponica and G.L. Plaa, Dept. de pharmacologie, Universite protects CCl4-induced liver damage and to isolate de Montreal, Montreal, Quebec, Canada. active compound for possible therapeutic use. 1,3-Butanediol (BD) has several potential uses Leaves of the Aucuba japonica were extracted with in the processing of human and animal diets, in­ methanol (90%, v/v) for 6 hrs. The methanol cluding use as a source of calories. BD can in­ extract was dried under reduced pressure and then crease circulating ketone bodies. Evidence sug­ dissolved in saline. Rats were pretreated with gests that ketosis can potentiate the hepatic the extract for two days (600 mg/kg/day, po) and action of haloalkanes (Hewitt et al.; Fed. Proc. CCl4 (0.5 ml/kg, ip) was administered 30 min after ~: 3118, 1980), BD given po potentiates CC14 the second dose. Pretreatment of the extract hepatotoxicity (Hewitt, Plaa; TAP 47: 177, 1979). effectively protected CCl4-induced depression in The present study characterizes thehepatotoxic plasma disappearance and biliary excretion of response of male Sprague-Dawley rats to CC14 fol­ bromosulfophthalein (BSP) determined 24 hr after lowing pretreatment with BD, Animals were given the CCl4 challenge. Percent recovery of BSP in free access to drinking solutions containing 0, bile in 60 min for control, CCl4, extract+ CCl4 0.1, 1, 5, and 10% (w/v) BD, During the 7-day treated rats was 66.8 ± 1.9, 56.2 ± 1.4, and 71.2 pretreatment period, body weight,ketone excretion ± 1.8, respectively. Pretreatment of the extract and solution consumption were monitored daily. also protected CCl4-induced increased SGPT activi­ Collateral experiments determined plasma ketones ty and liver triglyceride content. Further puri­ and liver GSH on Days 1, 3 and 7. On Day 7 one­ fication of the extract suggested that an iridoid half of the rats in each group received CC14 (O.l glycoside, aucubin is the most probable active rnl/kg,ip) in corn oil; the balance received corn compound. (Supported by 1981 Korea Science and oil alone. On Day 8 the liver damage was assess­ Engineering Foundation Research Grant) ed by histological and biochemical parameters (plasma bilirubin, GPT and OCT), BD reduced GSH and increased plasma ketones on Days 3 and 7, CC1 toxicity was enhanced in a dose-related man­ 4 469 THE EFFECT OF CCl4 EXPOSUREON MITOCHONDRIAL ner, with a BD threshold between 0.1 and 1,0%. MEMBRANEPHOSPHOLIPIDS. A. Wisner-Gebhart, R.A. 'Possible mechanisnsinclude induction of MFO en­ Head, and M.J. Brabec. Toxicology Program, Sch. zymes. BD was a relatively weak inducer of the of Pub. Hlth, The University of Michigan, Ann usual MFO parameters: 10% BD increased cyt. P-450 Arbor, MI (~ 57%) and aniline hydroxylase activity(~ 85%); aminopyrine N-demethylase was not altered, How­ Over a period of 10 to 66 hours following CCl4 ever BD greatly increased the ln vUJr.obinding of exposure, alterations in the phospholipid (PL) 14 cc1 4 to microsomal protein (aerobic~ 9-fold, composition of rat hepatocyte mitochondrial mem­ anaerobic~ 3-fold). These results show that BD branes parallel the loss of function of this or­ enhances the hepatotoxicity of CC14 and that this ganelle. The percentage of phosphatidylcholine is, in part, the result of increased biotransfor­ (PC) decreases and that of phosphatidylethanola­ mation, (Supported by grants from NSERC, NHRDP, mine (PE) increases in the microsomal and mito­ and IRSSTQ), chondrial outer membranes; while the inner mem­ brane shows changes in the opposite direction for each of these PL' s. Many authors have demonstrated the direct free radical effect of CCl4 on the PL's 471 EFFECT OF CALCIUM ANTAGONISTS ON CARBON of the microsomes but neve~ of the mitochondrial TETRACHLORIDE-INDUCED LIVER NECROSIS. L.D. membranes. Alterations to the mitochondrial PL's Schuman and M.A. Evans, Interdisciplinary Toxicology may therefore occur as a secondary effect of PL Program, Environmental and Occupational Health

133 Sciences, School of Public Health and Department of significant decrease in hepatic secretion of TG at 2 hrs Pharmacology, College of Medicine, Univ. of Illinois with an increased hepatic TG content observed at 6 hrs. Medical Center, Chicago, IL 60680. The increased accumulation of hepatic lipids was consistent with a 25% decrease in Ii ver MT produced by The present studies were designed to evaluate the methylene chloride. These results indicate that disrup­ effects of calcium antagonists on carbon tetrachloride­ tion of the tubulin assembly-disassembly process is induced necrosis. Mice received a single intraperitoneal associated with the development of acute hepatic dose of CCl (0.2 ml/kg) and then administered at 4 steatosis produced by the chlorinated hydrocarbons. selected time schedules the calcium chelator ethylene­ diamine tetraacetate (EDT A) (200 mg/kg) or the organic calcium antagonist verapamil (10 mg/kg). Both EDTA and verapamil significantly decreased CC\-induced 473 PCBs: EFFECTSOF STRUCTUREONTHE INDUCTION OF hepatic necrosis at 24 hrs. as determined by liistopath­ CYTOCHROMEP-448 DEPENDENTMONOOXYGENASES IN ological examination. Treatment with EDTA or verapa­ HEPATOMACELLS. T. Sawyer and S. Safe, Guelph mil alone produced no significant changes from control Waterloo Centre for Graduate Work in Chemistry, in Ii ver histology. Animals receiving EDT A and CCI 4 Chemistry Department, University of Guelph, also showed significantly lower serum glutamic pyruvic Guelph, Ontario, and Department of Veterinary transaminase (SGPT) levels, but only if administration Physiology and Pharmacology, College of Veter­ began 30 minutes prior to injection of CCI . Adminis­ inary Medicine, Texas A&MUniversity, College tration of verapamil to animals receiving ct1 failed to 4 Station, TX. Sponsor: L. W. Robertson lower elevated SGPT 1Jevels. Thus, SGPT levels in this study does not appear to be a sensitive index of hepato­ The relative activities of polychlorinated protection by the calcium antagonist. This may be due biphenyls as inducers of cytochrome P-448 depen­ to separation of the reversible plasma membrane dent monooxygenaseswere determined by measuring damage from irreversible cellular necrosis. Both the dose effecting half-maximal (EC50) induction calcium antagonists failed to significantly block the of benzo[a]pyrene hydroxylase and ethoxyresorufin increased Ii ver calcium at 24 hours. These results may 0-deethylase activities in rat hepatoma H-4-II-E reflect the massive influx of calcium subsequent to cells in culture. There was an excellent corre­ removal of the antagonists at 12 hours and/ or the lation between the relative potencies of the in­ presence of different intracellular pools of calcium. dividual polychlorinated biphenylsusing both en­ These studies indicate that co-administration of cal­ zyme assays and the most active congener, 3,3', cium antagonists significantly reduces CC1 -induced 4 4,4' ,5-pentachlorobiphenyl, was only 3-4 times hepatic necrosis in mice. However, the serum trans­ less active than 2,3,7,8-tetrachlorodibenzo-p__­ aminase values and Ii ver calcium content were not dioxin. In addition there was an excellent cor­ consistent with the observed reduction in cellular relation between the in vivo and in vitro activ­ necrosis as evidenced by histopathology. ities of the polychlorinated biphenyls and their relative affinities for the cytosolic Ah receptor protein. (Sponser: National Cancer Institute of Canada). 472 ROLE OF HEPATIC MICROTUBULE SYSTEM IN CHLORINATED HYDROCARBON INDUCED HEPATIC STEATOSIS. F.M. Selan and M.A. Evans, Interdisciplin­ ary Toxicology Program, Dept. Pharmacology, Uni ver­ 474 EFFECTSOF STRUCTUREONTHE ACTIVITY OF POLY­ si ty of Illinois Med. Ctr., P.O. Box 6998, Chicago, IL BROMINATEDBIPHENYLS (PBBs) AS MICROSOMALENZYME 60680. INDUCERS.L. W. Robertson, A. Parkinson and S. Recent studies have demonstrated that inhibitors Safe, Veterinary Physiology and Pharmacology, of the tubulin assembly-disassembly process can also College of Veterinary Medicine, Texas A&MUniv­ produce a decreased lipoprotein secretion and intracell­ ersity, College Station, TX. and Department of ular accumulation of triglycerides in the Ii ver. The Chemistry, University of Guelph, Guelph, Ontario, purpose of this investigation was to examine the role of NlG 2Wl Canada. the hepatic microtubular (MT) system in chlorinated hydrocarbon-induced hepatic steatosis. Microtubules The effects of structure on the activity of were separated from free tubulin by differential centra­ synthetic polybrominated biphenyl (PBB) congen­ fugation and measured by a modified radiolabelled ers as microsomal enzyme inducers has been stud­ colchicine binding assay which was shown to be selec­ ied in the immature male Wistar rat. Using tive and quantitative for hepatic tubulin protein. Pro­ enzymic assays and ligand-binding measurements, duction of steatosis was assessed by measurement of PBBcongeners were classified as phenobarbitone total li-ver lipids recovered by the method of Folch. The (PB)-or 3-methylcholanthrene (MC)-type inducers release of triglycerides (TG) from the Ii ver was esti­ of cytochrome P-450 dependent monooxygenases. mated in vivo by the method of Byers and Friedman Important qualitative differences in the modes of (Amer. J. Physiol. 198: 629-631, 1960) using Tri ton WR induction exist between PBBsand their chloro 1339. At doses consistent with the production of fatty analogs. Where analogy exists, PBBsare in gen­ liver carbon tetrachloride (CCI ) produced a significant 4 eral the more potent inducers. The structure­ decr~ase in hepatic MT in male mice. The decrease in activity rules for PBBsas PB-type inducers are MT was time--dependent and restoration of hepatic MT as yet unresolved. PBBswith at least l meta and at 72 hrs. was associated with a ·decrease in total liver 2 para substituents are MC-type inducers of cyto­ lipids to control values. Disassembly of MT and hepatic chrome P-448, whereas PCBsrequire at least 2 steatosis was achieved with doses of CCI as low as meta and 2 para substituents for this activity. 4 0.025 ml/kg and maximal disassembly was observed at PBBswhich are mono or di ortho derivatives of the 0.1 ml/kg. These results were consistent with observed MC-type inducers may be mixed (PB+ MC)-type CCI -induced inhibition of hepatic triglyceride secre­ inducers. (Sponsored in part by the Natural 4 tion. Further studies conducted with methylene chlor­ Sciences and Engineering Research Council of ide (CH c1 ), (2 ml/kg, P.O.) also demonstrated a Canada). 2 2 13-4 475 2,3,4,4',5-PENTACHL0R0BIPHENYL:SEGREGATION OF Current data suggests that aldehydic pro­ ACTIVITYWITH THE Ah LOCUS. A. Parkinson, L. ducts of lipid peroxidation possess W. Robertson, M. A-:-campbell, L. Uhlig, S. substantial cytotoxic properties. Carbon Bandiera and S. Safe, Guelph-Waterloo Centre tetrachloride, a potent stimulator of for Graduate Work in Chemistry, Department of hepatic microsomal lipid peroxidation Chemistry, University of Guelph, Guelph, Ontario was tested for possible effects on hepa­ and Department of Veterinary Physiology and tocellular aldehyde metabolism. Carbon Pharmacology, College of Veterinary Medicine, tetrachloride administered intragastri­ Texas A&MUniversity, College Station, TX. cally at a dose of 1 ml/kg 24 hr before sacrifice produced an elevation in serum 2,3,4,4' ,5-Pentachlorobiphenyl is repre­ alanine aminotransferase activity, hepa­ sentative of a subclass of halogenated biphenyls tic fatty infiltration, centrilobular which include a pattern of hepatic drug-metab­ necrosis and significant decreases in the olizing enzymes similar to that produced by content of hepatic microsomal cytochrome coadministration of phenobarbitone plus 3- P-450. Concurrently, the aldehyde dehy­ methylcholanthrene. Administration of 2,3,4,4', drogenase (ALDH) activity (E.C. 1.2.1.3) 5-pentachlorobiphenyl to C57BL/6J responsive of mitochondrial and cytosol ic fractions mice leads to a dose-response relationship for was significantly depressed. The lower the induction of benzo[a]pyrene hydroxylase ac­ Km ALDH located in the mitochondria show­ tivity and the development of thymic atrophy. ed a 43% lower specific activity compared In contrast this halogenated compounddid not to control values. Cytosolic ALDH speci­ exert comparable biologic and toxic effects fic activity was decreased 33% by carbon in the nonresponsive DBA/2Jmice. It is con­ tetrachloride treatment. In addition to cluded that the effects of mixed-type halogen­ these in vivo findings the low Km ALDH of ated aromatic inducers also segregate with the mitochondria was shown to be inhibited Ah locus. (Sponsored by the National Insti­ during in vitro lipid peroxidation under tutes of Health, Grant No. 1 R0l ES02798 01). a variety~conditions. These data suggest that aldehyde metabolism is impaired following CC14 exposure, an effect that may prolong the intracellular 476 HEPTA-AND0CTACHL0R0NAPHTHALENE CONGENERS ARE half life of aldehydic products of lipid POTENTINDUCERS OF RATHEPATIC MICROSOMAL BENZ0 peroxidation. Supported by NIAAA grant - [a]PYR~NEHYDR0XYLASE. M.A. Campbell, L. Safe, 03527. S. Bandiera, L. W. Robertson and S. Safe, De­ partment of Chemistry, University of Guelph, Guelph, Ontario, NlG 2Wl Canada and Veterinary Physiology and Pharmacology, College of Veter­ 478 ELEVATIONOF HEPATICCONJUGATED DIENES IN THEAB­ inary Medicine, Texas A&MUniversity, College SENCEOF CE1LULARINJURY IN 1 DAY-OLDRATS AFTER Station, TX. CARBONTETRACHLORIDE (CC1 ) TREATMENT.A.T. Shehata and~- Hitchcock, 4Yale University and Induction of rat liver microsomal enzymes John B. Pierce Foundation, 290 Congress Ave., New by three polychlorinated naphthalene congeners Haven, CT. 06519 octachloronaphthalene, 1,2,3,4,5,6,7- and 1 ,2, 3,4,5,6,8-heptach1 oronaphtha 1ene was investigated. We investigated the time course of onset of The congeners were prepared by repeated re­ cc14-induced hepatotoxicity in 1- to 3-day-old crystalization from Halowax 1051 in chloroform/ rats. Activities of hepatic aspartate amino­ petroleum spirits (octachloronaphthalene) semi­ transferase (AST) and alanine aminotransferase preparative HPLC(1,2,3,4,5,6,8-heptachloronaph­ (ALT) in control animals were identical in all thalene) and lithium aluminum hydride reduction age groups and were, respectively, 218±22 and of octachloronaphthalene (1 ,2,3,4,5,6,7-hepta­ 20±2,7 Sigma units per g liver. Twenty-four hrs. following CC1 treatment (1 ml/kg, i.p.) plasma chloronaphthalene). The compounds were admin­ 4 istered to immature male Wistar rats in doses activity of AST in rats injected at age 1, 2 and 1 3 days were increased, respectively, by 1.2%t3%, ranging from 12 to 1200 µ-nol/kg- • On the ba­ sis of enzymic activities as well as ligand 810%±20%and 2380%±45%:plasma activity of ALTin binding (CO and EIC) measurements and SOS poly­ the same g~oups were, respectively, 0.5%±2%, acrylamide gel electrophoresis, the results in­ 340%±25%and 613%±20%. These data suggest age dicated that the heptachloronaphthalenes were difference in susceptibility to CC14 rather than potent MC-type inducers (maximumactivity< 60 lack of hepatic enzymes to be released. Gas 1 chromatographic analysis of heptane-extracts of µ-nol/kg- ). 0ctachloronaphthalene, also an MC­ type inducer was much less potent (maximumac­ liver, kidney, lung and fat homogenates from rats 1 of all age groups did not detect the presence of tivity at 300 µnol/kg- ) and a number of isomer­ ic dichloronaphthalenes did not induce microso­ cc14 metabolites. In 1-day-old rats, conjugated mal benzo[a]pyrene hydroxylase. dienes, a measure of lipid peroxidation, were found to be significantly elevated only in liver homogenates 90 mins following cc1 treatment (1 ml/kg, i.p.)(absorbance at 2434 nm of lipid per g liver: 0.123±0.0147 for cc1 -treated rats and 477 INHIBITION OF RAT LIVER ALDEHYDEDEHYDRO­ 0.085±0,006 for corn oil-treated4 rats; p(0.05). GENASE BY CARBON TETRACHLORIDE. J. J. Under these conditions, hepatic activities of AST Hjelle, J.H. Grubbs, D.G. Beer and D.R. and ALTwere unchanged. The results suggest that Petersen, Dept. of Pharmacology, School lipid peroxidation can occur without hepatic in­ of Pharmacy, Univ. of Co~orado, Boulder, jury in CC1-treated newborn rats. Supported by CO 80309. Sponsor: C.D. Klaassen. Grant #5T32-ES-07086.4

135 479 THETRANSFER OF 3,4,5,3' ,4' ,5'-HEXACHLOROBIPHENYL 12.4%; liver 87.8 ± 9.6%; brain 62.6 ± 2.6%; kidney (6-CB) FROMMOTHERS TO FETUSESAND NURSING OFF­ 53.4 ± 2.4%. As recovery of 1,1-DCE was comparable SPRING. M.J. Vodicnik and J.J. Lech, Dept. of to that of 1,2-DCE, each could be used as an in­ Pharm. & Tox., Med. Col. Wiscons~Milwaukee, WI ternal standard for the other when measuring tis­ sue concentrations. 1,1-DCE levels measured in The pharmacokinetic behavior of 14c-6-CB in virgin male S.-D. rats 15 min. after i.p. injection of and pregnant/lactating Sprague-Dawley mice was 15 mg/kg 1,1-DCE (mean values in ng/mg tissue± SE assessed. Mice were injected IP with 3mg/kg 6-CB for triplicate analyses on tissues from 4 rats) on Day 2 of pregnancy and sacrificed at various were: adipose tissue 1.35 ± 0.37; liver 1.53 + 0.35 times along with matched virgin controls. Tissues brain 0.16 ± 0.03; kidney 0.73 ± 0.21. This purge were prepared for liquid scintillation counting and trap technique appears well suited for kinetic and hepatic microsomal ethoxyresorufin-0-deethyla­ studies of halocarbons in target tissues. tion (EROD)determined. Suckling offsprTng from (Supported in part by EPA Grant R808282). 6-CB-treated mothers were sacrificed at selected times, tissues analyzed for 14c activity and hepatic microsomal ERODassessed. No significant differences were noted in the tissue distribution of 6-CB between virgin and pregnant mice on Days 481 CHOLESTYRAMINE ENHANCES FECAL EXCRETION OF 6,12 or 19 of gestation. ERODactivity between PENTACHLOROPHENOL. K. Rozman, L. Ballhorn, T. the two groups did not differ at Days 6 or 12 of Rozman, C. Klaassen, and H. Greim. Gesellschaft pregnancy but was significantly depressed in pre~­ fur Strahlen-und Umweltforschung mbH Muenchen, nant animals sacrificed on Day 19 (2.118±0.231 vs 8042-Neuherberg, FRG and Dept. of Pharmacology, 0.552±0. 105 nmol/min/mg protein). ERODactivity in University of Kansas Medical Center, Kansas City, mothers remained below that in virgins through Day KS. 15 of lactation. On Day 19 of gestation, fetal content of 6-CB was approximately O.l5µg/g tissue, Three male rhesus monkeys were-provided with a representing less than 1% of that concentration in 9Jle duct bypass and were dosed twice with maternal adipose tissue. During lactation, 6-CB C-pentachlorophenol (50 mg/kg, po) four weeks was rapidly eliminated in milk. While no detect­ apart. During the seven days following the first able elimination of 6-CB from the adipose tissue dose, the monkeys excreted 56 and 3~, of the dose of virgins occurred, the t 1/2 of elimination of into urine and feces, respectively. Up to 30% of 6-CB from the fat of lactating mothers was 2.5 the dose was excreted into b1 le in one day days. These greatly enhanced rates of elimination indicating a considerable enterohepatic circula­ were observed in all maternal tissues examined. tion of pentachlorophenol. Since cholestyramine Nursing offspring accumulated the PCBin liver and binds acidic compounds such as carboxylic acids skin and ERODactivity was positively correlated and phenols, it was of interest to determine if with hepatic 6-CB content. These data support this resin would enhance the elimination of pentachlorophenol. There~are, twenty-four hr those which suggest that minimal transplacental after the second dose of C-pentachlorophenol, transfer of highly chlorinated PCBsoccurs, but the monkeys were placed on a diet containing 4~, that they are readily transferred to offspring cholestyramine for six consecutive days. During through milk. This appears to be the case with this time 28 and 55~, of the dose was excreted both coplanar and non-coplanar isomers. Supported into urine and feces, respectively. The body by HD13591 and MOD15-9. burden of pentachlorophenol was markedly reduced by cholestyramine treatment as its total excre­ tion (urinary plus fecal) was enhanced by 4a'L 480 QUANTITATIONOF 1,1- and 1,2-DICHLOROETHYLENEIN While cholestyramine increased the plasma dis­ TISSUES BY PURGE AND TRAP GAS CHROMATOGRAPHY. appearance of pentachlorophenol, it produced a s.-N. Lin, F.W.Y Fu, and J.V. Bruckner, Anal. Chern. more marked decrease in its urinary excretion. Center and Div. Toxicol., Univ. Texas Med. Sch., This effect suggests that a considerable amount Houston, TX 77025. of pentachlorophenol in urine probably originates from the enterohepatic cycle. Cholestyramine Characterization of the uptake and disposition enhanced the fecal excretion of pentachlorophenol of volatile halocarbons in animals is complicated from 8 to 183 mg. Since only about 104 mg was by the lack of suitable methods for analysis of excreted into bile, cholestyramine probably also the parent compounds in body tissues. Our objec­ increases elimination of pent~chlorophenol tive was to develop a sensitive and accurate meth­ directly across the intestinal wall. od for extraction and quantitation of two repre­ sentative halocarbons, 1,1- and 1,2-dichloroethy­ lene (1,1-DCE & 1,2-DCE). Six-mg portions of tis­ sues were frozen at -40°C, minced and combined with 200 ul of purified water in a 3.5-rnl vial im­ 482 IN VITROOCULAR TOXICITY TESTING. S.D. Spilman mersed in a 60°C stirred water bath. To avoid and W.H.J. Douglas, Dept. of Anatomyand Cellular foaming, the stream of purging gas (i.e. helium) Biology, Tufts Univ. Sch. of Med., 136 Harrison was applied 2 mm above, rather than below the sam­ Ave., Boston, MA. 02111. Sponsored by D.R. Brown ple surface. The purged 1,1- and 1,2-DCE were re­ tained on Tenax GC 180-100 rnes·h within a Tekrnar An in vitro toxicity test is being developed sample concentrator, then desorbed by heating and to compare cytotoxicity of chemical substances vented into a Tracor 560 gas chromatograph equip­ toward cultured corneal endothelial cells with ped with a Hall detector in the halogen mode. The ocular irritancy in vivo. Confluent monolayers detection limit for 1,1-DCE in this system was of corneal endothelial cells are established in 50 pg. Recoveries of 1,2-DCE from tissues spiked 24-well dishes from primary cultures, incubated in vitro (expressed as the mean± SE of 5 determi­ with various concentrations of test substances nations per sample) were: adipose tissue 93.3 ± and positive and negative control substances,

136 then evaluated for a variety of biochemical and 484 OCULARMANIFESTATIONS PRODUCED BY6-AMINONICO­ morphological indices of cytotoxicity. Preliminary TINAME.J.A. Render and W.W.Carlton. Dept. results indicate that benzalkonium chloride, of Vet. Microbial. and Pathol., Sch. of Vet. silver nitrate, hydrogen peroxide, and lauryl Med., Purdue University, West Lafayette, IN sulfate, mediate half-maximal 51Cr release at 4 X 47907. 10-5M, 8 X 10-4M, 2 X 10-5M, and 4 X 10-4Mdoses, 6-Aminonicotinamide (6-AN), an antagonist respectively, during 1 hour incubations. Rela­ of nicotinamide, has been used as a cancer chemo­ tively non-toxic substances, propylene glycol, therapeutic agent and in studies of nicotinic dimethylsulfoxide, and ethanol, werr not cytotoxic acid deficiency. Whengiven to rabbits, by ip at concentrations less than 1 M. 5 Cr release was injection it produced ocular alterations in demonstrated to be dependent on duration of the both Dutch Belted and NewZealand White breeds. exposure to a test substance as well as concen­ Ocular lesions of 6-ANtoxicosis consisted clin­ tration. At 10, 30, 60, and 90 minutes, benzal­ ically of iridal congestion with occasional hem­ konium chloride at 10-4 M elicited 48%, 49%, 56%, orrhages. The principal histopathological alter­ and 60%of the maximum51cr release. Other assays ations were swelling and loss of staining affin­ entail quantifying LOHrelease, morphometric ity of the peripheral pigment epithelium and analysis of endothelial cells, lymphocyte activa­ epithelial cells of the posterior iris and cil­ tion, and vital dye or dye exclusion methods of iary body. The cells initially and, in time, measuring cytotoxicity in vitro. These studies most severely affected were the juxtastromal will be extended to include a variety of known cells of the ciliary web. Inner epithelial cells ocular irritants and non-irritant substances, to of the pars plicata and inner iridal epithelial afford a broad data base with which to confirm or cells were moderately affected while the peri­ invalidate the in vitro assays as a model system pheral pigment epithelial cells were occasionally for screening potential ocular irritants. and minimally affected. Alterations were present in rabbits given 6-ANat approximate daily doses This work was supported by a grant from the New of 3 mg/kg, 5 mg/kg and 7.5 mg/kg for 5 days but England Anti-Vivisection Society. not at 1 mg/kg body weight for 9 days. After 4 daily injections at an approximate daily dose of 3 mg/kg, the alterations were present, but slight. At this dose, ocular alterations be­ 483 EVALUATIONOF EYE IRRITATION TESTING. K.J. came increasingly more severe as the number of Falahee, C.S. Rose, S.S. Olin, H.E. Seifried injections increased. Death occurred approxi­ (Tracor Jitco, Inc., Rockville, MD), N.P. Page mately after 8 days of dosing. (National Library of Medicine, Bethesda, MD) and D. Sawhney (US EPA, Washington, DC). The ocular safety of materials is determined 485 PULMONARYAND SENSORYIRRITATION IN MICE DURING primarily by observing the irritation produced by INHALATIONOF POLYMERICISOCYANATES. D. Weyel test agents insti_lled directly into the rabbit and Y. Alarie, Dept. of Ind. Environ. Hlth. Sci., eye. The object of this study was to review the Grad. Sch. of Pub. Hlth., Univ. of Pittsburgh, scientific basis for published guidelines, par­ Pittsburgh, PA 15261 ticularly those recently developed by the Organ­ The use of monomeric and polymeric isocyanates in isation for Economic Cooperation and Development a wide variety of industries has been increasing. (OECD) and the Interagency Regulatory Liaison Little is known about the toxicity of polymeric Group (IRLG). These guidelines are essentially isocyanates. The pulmonary and sensory irrita­ the same and recommend instillation of 0.1 ml tion of an aliphatic polyisocyanate (DES-N, Mobay material into the rabbit eye with observation for Chemical Corporation), based on hexamethylene di­ at least 72 hr. To increase cost-effectiveness isocyanate (HDI) were studied in Swiss-Webster and optimize use of animals, the guidelines reduce male mice during aerosol exposures in the range the number of animals and permit exclusions based 25-130 mg/m3. The aerodynamic equivalent dia­ on very high(~ 11.5) or low(~ 2) pH, and demon­ meter (AED) and geometric standard deviation for strated dermal irritation. This is based on the the aerosol were 0.58 µm and 2.35, respectively. high probability that agents meeting these criter­ High speed liquid chromatography was used to ia will be severe eye irritants. Data reviewed determine both free HDI and DES-Nin the ex­ support excluding strong alkalies (pH~ 11.5). posure chamber. Each exposure was for 4 hours Exclusions of acias below pH 2 is not as well sup­ during which the tidal volume pattern and re­ ported. Data indicate that the majority of severe spiratory rate of groups of 4 mice were recorded. dermal irritants would also be eye irritants. The Unlike the monomeric isocyanates, DES-N acted monkey appears the most predictive animal model predominantly as a pulmonary irritant, evoking for human response; however, the rabbit still re­ little sensory irritation. The concentration re­ mains the species of choice due to practical con­ quired to reduce the respiratory rate 50% due to siderations and the large data base which is use­ pulmonary irritation was found to be 57.1 mg/m3. ful for comparative purposes. Generally greater The LC50, determined by counting the number of sensitivity of the rabbit eye compared to the deaths within the 24 hour period following the human allows a conservative extrapolation to human 4 hour exposure was found to be 91.2 mg/m3. On risk. The development of alternative methodolo­ groups of animals sacrificed 2 hours after the 4 gies (e.g. in vitro tests) and the use of topical hour exposure, the concentration to increase anesthetics is discussed. A tier strategy to eye lung weight by 50% was found to be 45 mg/m3. irritation testing is proposed which screens sub­ Based on comparisons with other pulmonary ir­ stances on the basis of pH, dermal irritation and ritants, the maximum concentration permitted results from other tests. (Supported by the EPA, in industry should be 1 mg/m3 with a time Contract No. 68-01-6176, C. Auer, Project Officer. weighted average for an 8 hour period of 0.6 Contents do not_ reflect agency policy.) mg/m3.

137 486 CONCENTRATION-DEPENDENTINDUCTION OF THE PULMONARY Benzophenonecompounds such as oxybenzone reduce HYPERSENSITIVITYRESPONSE TO TOLUENEDIISOCYANATE. or prevent phototoxicity due to their UVA M.H. Karol, Dept. of Indus. Env. Hlth, Sci., Grad. absorbance. Oxybenzone(3 percent in ethanol) Sch. of Pub. Hlth., Univ. of Pittsburgh, Pitts­ application to the tail completely inhibited the burgh, PA 15261 phototoxic effect of 100 mg/kg benoxaprofen. The sunscreen .e.-aminobenzoicacid does not absorb Many organic industrial chemicals, including appreciable UVAand did not confer protection. toluene diisocyanate (TDI) are known to cause These results provide experimental evidence for astlnnatic reactions in sensitized workers. In the application of benzophenone-containing order to prevent sensitization, it is necessary to sunscreens to reduce or prevent clinical understand the relationship between exposure con­ benoxaprofen-induced phototoxicity. centration and development of pulmonary sensiti­ vity. An animal model was used to evaluate this relationship. Groups of guinea pigs were exposed via inhalation to concentrations of TDI ranging 488 DERMALSENSITIZATION BY TOLUENE from 0.1 to 0.5 ppm. Animals were exposed to the DIISOCYANATE (TOI). F,J. Koschier, E.J. TDI vapors for 3 hours per day on 5 consecutive Burden, c.s. Brunkhorst, and M.A. days. On day 21 blood was drawn from animals for Friedman. American Cyanamid, Wayne, NJ. evaluation of TDI-specific antibodies. TDI­ TDI is known to cause pulmonary specific respiratory sensitivity was determined by sensitization and has rec,ently been shown bronchial provocation challenge (BPC) using TDI to cause allergic skin reactions. The antigens. No antibodies were detected in animals present study was designed to determine exposed to 0.1 ppm TDI. However, animals exposed the threshold dose for dermal to 0.25 ppm or greater displayed TDI-specific anti­ sensitization. Young adult guinea pigs bodies in their sera and antibody titers increased received an open, epicutaneous induction in a logaritlnnic relationship with TDI exposure dose (50mcl) of 8,20 or 40% TOI in concentration. Similarly, bronchial sensitivity n-butyl ether on day O. On day 5, was not apparent in animals exposed to 0.1 ppm TDI animals were challenged with 0,025, 0,05, but was evident in animals exposed to higher TDI 0.1, 0,2 and 0.4% TDI (25mcl). concentrations. However, exposure of animals to Twenty-four hours after the day 5 concentrations of 2 ppm or greater resulted in pul­ application, the mean Draize irritation monary damage and animals were less able to respond scores for the challenge doses of the 8% upon BPC with TDI antigens. Recognition of the group were 2.4 and 3.4 for 0,025 and 0,4% concentration-response and threshold concentration TOI, respectively, and from the 40% relationship governing inhalation sensitization to group, 2,4 and 4,2, respectively. The TDI should permit establislnnent of safe airborne ve11icle control anrl challenge doses exposure levels for industrial workers to prevent produced scores of 0,6-1.0, and since all sensitization. Supported by NIEHS Grant flES01532 the challenge doses produced scores and NIOSH #OH00865. greater than 2.0, no threshold was apparent at these induction and challenge levels. An additional experiment was performed at lower doses where animals 487 EVALUATIONOF BENOXAPROFEN PHOTOTOXICITY IN AN received an induction dose (50mcl) of AN!MAL MODEL. G. T. Brophy ( SPON: J2...... G.. either 4 or 8~ of TOI and were challenged Hoffman). Toxicology Division, Lilly Research with n.o, 0.006, 0.012, 0,025, 0.05, and Laboratories, Greenfield, IN 46140 0.1% (25mcl). Twenty-four hours after the challenge applications, the mean A wide variety of drugs induce phototoxic effects Draize irritation scores for the clinically, viz. phenothiazines, sulfonamides, chal1enge doses of the 4% groups were psoralens, tetracyclines. Phototoxicity has been 1.0, 1,0, 0.9, 1.6, 1.4 and 1.8, and the associated with the clinical use of benoxaprofen 8% groups were 1,0, 1.1, 1.1, 2.1, 2.4 in some cases. Benoxaprofen phototoxicity was anc'l 3.0, respectively. The data from the investigated using an in vivo mouse tail model second expe'riment suggest that TOI has an (Acta Dermatovener (StockhoTrii)56:373, 1976). apparent no effect challenge level of Briefly, test compoundswere adiiiTnistered to ICR 0,012% (3 mcg) when 4% (2,000mcg) and 8% mice, the intact tails were irradiated with UVA (4,000mcg) induction doses were used. for 5 hours and at 24 hours the evaporable water content .of a tail segment was determined·to quantitate edema formation resulting from photo­ toxic inflammation in the tail. The classical 489 INITIAL RESULTSOF THE DERMALEFFECTS OF OIL phototoxic agent chlorpromazine had a minimum FROMCOAL GASIFICATION. R.E. Jones, F.R. phototoxic dose (MPD)of 2.5 mg/kg (i.p.) Kirchner, F.J. Tremmel, and C.A. Reilly, Jr., whereas the MPDof benoxaprofen was 10 mg/kg Div. Bio. Med. Res., Argonne Nat. Lab., Argonne, (p.o.); these doses are comparable to those used IL. Sponsor: W.E. Dalbey clinically. Benoxaprofen is not significantly metabolized in the mouse, rat, monkeyor man and The chronic toxicity and carcinogenicity of a metabolic induction or inhibition with pheno­ process-derived recycle oil (RO) from a high-BTU barbital or SKF525A did not alter benoxaprofen coal gasification pilot plant is being deter­ phototoxicity. The two major benoxaprofen UV mined. RO collected during several experimental degradation products were comparatively less runs was pooled to form a representative sample. phototoxic than the parent compound(MPD's 100 Both the RO and its nonvolatile organic compo­ mg/kg). These derivatives do not appear to be nents (NVO) are being tested. Treatment groups . responsible for benoxaprofen phototoxicity. consist of 50 SKH hairless mice either untreated

138 or painted 3 times/week with RO, NVO, benzo(a)­ 491 EVIDENCE FOR PYRROLE FORMATION IN THE pyrene (+ control), or acetone (solvent control). PATHOGENESIS OF HEXANE NEUROPATHY. D.C. Mice are examined for gross evidence of infla.Dlllla­ Anthony and D.G. Graham, Dept. of Pathology, Duke tory, hyperplastic, or neoplastic dermal effects University Medical Center, Durham, N.C. 27710. and for systemic changes through analysis of blood and urine. Dermatitis, scaling, and scab The hypothesis that covalent binding of 2, 5-hexane­ formation were seen following oil treatment, dione (HD) to neurofilament proteins is the initial injury being most severe in the NV0 group. Urine and in the molecular pathogenesis of hexane neuropathy was serum were collected during weeks 22, 26, 35, and studied using the structural analogue 3, 4-dimethyl-2, 39. Urine was normal in all groups except that 5-hexanedione (DMHD). Due to steric properties, with NVO, which displayed a transient reduction DMHD should form a pyrrole ring more rapidly than HD in volume had a darker color. The NV0-treated after imine formation and be less likely to form di­ mice had elevated serum glutamate oxaloacetate imines, two possible mechanisms of neurofilament transaminase (GOT) and glutamate pyruvate trans­ aggregation. Twenty-four Sprague-Dawley rats (170 aminase (GPT), decreased urine GOT and increased gm) were divided into four equal groups and injected urine and serum urea nitrogen (UN) levels, and i.p. five times a week with HD (4 mmoles/kg), HD (0.25 creatinine (C) excretion. The RO-treated mice mmoles/kg), DMHD (0.25 mmoles/kg) or water. When were normal except for increased serum GPT and hind limb paralysis occurred, the animals were perfused urine C levels. Although results are incomplete, with 4% glutaraldehyde. The DMHD-treated animals they indicate pathological changes taking place showed a more rapid clinical course characterized by in the liver (GPT), kidneys (UN), and muscle (GOT) weight loss and progressive fore-and-hind-limb weak­ & C). Benzo(a)pyrene and both oil-treated groups ness, reaching end-point after 19.6 ± 1.2 days. Rats developed skin tumors starting 10-12 weeks into receiving HD at 16 times the dose given the DMHD rats the experiment. The NV0-treated mice displayed reached end-point after 35.8 ±2.6 days, with no a more severe tumor response with a shorter llrluc­ appreciable fore-limb weakness. After 8 weeks of tion time. Histologic examination of the tumors treatment, the low-dose HD-treated animals showed no is in progress. (Work supported by US DOE under signs of toxicity. Morphologic studies revealed the contract No. W-31-109-ENG-38.) characteristic giant axonal swellings with proximal paranodal neurofilament accumulations in the high-dose HD-treated and DMHD-treated animals, although in the latter group the changes occurred much more proxi­ 490 SHORT-LATENCY SOMATOSENSORY EVOKED mally with severe Wallerian-like degeneration distally. POTENTIALS IN PRIMA TES INTOXICATED WITH In vitro formation of pyrrole was more rapid when ACRYLAMIDE: IMPLICATIONS . FOR TOXIC ethanolamine was reacted at pH6 with DMHD than with NEUROPA THIES IN MAN. H. Schaumburg, J. Arezzo HD, occurring approximately seven-fold more rapidly at and P.S. Spencer, Inst. of Neurotoxicology, Albert 37°C. These findings suggest that pyrrole deriviti­ Einstein Coll. of Med., Bronx, N.Y. 10461. zation of neurofilament lysinyl residues follows imine formation and that more rapid irreversible alterations Toxic axonal peripheral neuropathies are accompanied lead to more proximal aggregations of neurofilaments. by early degeneration of the distal ends of long (Supported by NIEHS grant ES 02611-01). ascending sensory axons in the dorsal columns and gracile nuclei (central-peripheral distal axonopathy). This study sought to determine whether detectable electrophysiological changes preceded axonal degeneration in this system, and whether such changes 492 IMMUNOLOGICEVALUATION OF PATIENTS WITH POLY- are reversible. Monkeys were daily injected with levels CHLORINATEDBIPHENYL POISONING. K.J. Chang,1 of either 10, 3, 2 or I mg/kg of acrylamide monomer. K.H. Hsieh,. 2 T.P. Lee,3 S.Y. Tang,4 and T.C. Tung,4 Brainstem somatosensory evoked petentials (SEPs) and peripheral nerve conduction velocities were recorded Dept. of Surg~, Ped.~ and Biochem~, College of weekly from surface electrodes prior to and during Med., National Taiwan Univ., Taipei, Taiwan 3 • intoxication. The earliest abnormality detected ( 1-2 R.O.C., and Dept. of Ped., Children's Hosp., weeks in the 10 mg/kg animals) was a latency shift of approximately 200 usec in the onset of near-field SEP State Univ. of New York at Buffalo, NY 14222 activity overlying the gracile nucleus following Sponsor: G.L. Plaa unilateral electrical stimulation of the peroneal nerve. This CNS effect preceded observed morphological or In March 1979, an endemic disease due to chronic behavioral abnormality, or alteration in the conduction poisoning from polychlorinated biphenyl (PCB) velocity recorded overlying distal sural nerve, cauda contamination in cooking rice oil occurred in equina, or spinal cord below C5. The SEP profile central Taiwan. Exposure to PCB in human recorded overlying the cuneate nucleus remained subjects and experimental animals cause various unchanged following median nerve stimulation. When metabolic and physiologic abnormalities including intoxication was stopped at this stage, the immune suppression. We have studied cellular and configuration of the SEP gradually returned to normal. humoral immunity among 30 PCB-poisoned patients. Progressively longer delay before onset of latency shift and 23 normal human subjects. The results and occurred in animals intoxicated at lower levels. conclusions are summarized as follows: 1) PCB Animals intoxicated with I mg/kg first displayed poisoning caused decreased concentrations of IgA physiological abnormalities only after 18 months of and IgM but not that of IgG. We also found that daily dosage. This study suggests that the non-invasive the percentages of total T-cells, active T-cells brainstE¥YJ SEP is a sensitive tool for the early detection and Tµ-cells were decreased, while the percentages of peripheral neuropathies of the distal-axonopathy of B-cells and Tr-cells were not affected. 2) type, that these conditions are potentially reversible Monocytes and polymorphonuclear leukocytes ob­ and that extremely prolonged delay of onset can occur tained from patients have lower percentages of with low-level intoxication. cells bearing immunoglobulin and complement re-

139 ceptors. 3) The size of induration after strep­ however, after a 6 hr exposure to 600 or 1200 tokinase/streptodornase intradermal injection had ppm, styrene was eliminated according to a non­ a negative correlation with whole blood PCB linear saturable model. In addition to the concentration (r=-0.54,p

These studies demonstrate that the fate of 493 MUTATION,DNA LABELING, ANDINDUCTION OF GROWTH styrene in the B6C3Fl mouse, as in the rat, is IN SOFT AGAROF BHK-21/CL 13 CELLS BY MNNG,DMN, dependent on exposure concentration. Therefore, ANDNITROSOCIMETIDINE. L.R.Barrows, C.T.Gombar, at high saturating doses, toxicological informa­ and E.....N_~, Fels Res. Inst., Temple Univ. tion will be of limited value for hazard assess­ School of Med., Phila., Pa. ment purposes. (Supported by the Chemical Manufacturers Association). Dimethylnitrosamine (DMN) has been reported to induce BHK-21 Cl 13 cell growth in soft agar, in the absence of an added activation system. To determine if DMNis mutagenic to BHK cells, an 495 KINETIC ANALYSESOF CHLORPYRIFOSAND PARATHION ouabain resistance mutation (or+) assay was METABOLISMBY MOUSEHEPATIC MICROSOMES. L.G. established. BHKcell growth was inhibited by Sultatos and S.D. Murphy, Div. of Tax., Univ. of 0.5 mMouabain in the growth medium; this inhibi­ TX Medical School at Houston, Houston, TX 77025 tion was overcome by the addition of K+. DMNwas Chlorpyrifos (0,0-diethyl 0-3,5,6-trichloro- then compared to the mutagenic methylating agents 2-pyridyl phosphorothionate) (CPS) was metabo­ MNNGand nitrosocimetidine (NC). MNNGand NC lized to chlorpyrifos axon (CPO) and 3,5,6-tri­ yielded dose dependant induction of or+ clones, chloro-2-pyridinol (TCP) by mouse hepatic micro­ DMNwas not mutagenic. Optimal phenotypic expres­ somes. Formation of both CPO and TCP required sion time for ouabain resistance was between 3 NADPH,and was inhibited by CO. Kinetic analyses and 6 days. or+ clones retained their phenotype using Cornish-Bowden plots determined the appKm's after 3 weeks growth in nonselective medium. The for formation of CPO and TCP to be 20.9µM + 3.3 ability of the test compounds to methylate DNA in and 16.1 µM + 3.4 respectively, while the - BHKcells was determined; MNNGand NC yielded de­ appVmax's for the same reactions were 3.9nmols/ tectable levels of 7-methylguanine, DMNdid not. min/100 mg liver~ 0.2 and 8.lnmols/min/lOOmg li­ The compounds were tested for the ability to in­ ver~ 0.3 respectively. Incubation of parathion duce BHKcell growth in soft agar, and MNNG,NC, (0,0-diethyl 0-4-nitrophenyl phosphorothionate) and DMNdid at luM, 20uM, and 338uM respectively. (PS) with mouse hepatic microsomes produce para­ The results suggest that BHK cells are not able axon (PO) and p-nitrophenol (PNP). The appkm's to activate DMNto a mutagenic intermediate. for the formation of PO and PNP were 29.6 µM + Therefore, DMNappears to induce BHK cell growth 4.2 and 26.5 µM + 3.8 respectively, with - in soft agar by a non-genotoxic mechanism. NC appVmax's of 5.8nmols/min/100mg liver+ 0.6 and was found to be as effective a mutagen and in­ 6.7nmoles/min/100mg liver+ 0.5 respectively. In­ ducer of BHK cell growth in soft agar as MNNG cubation of both PS and CPS at various concentra­ at equitoxic concentrations, approximately 4 tions with mouse hepatic microsomes resulted in fold less effective at equimolar concentrations. inhibition of production of PO, PNP, CPO, and TCP This work was supported by the NIEHS and NIH characteristic of mixed-type inhibition. After grants F32-ES0519401, CA-09214, CA-12227, and consideration of all possible kinetic models which CA-23451. could explain these data, we concluded that true mixed inhibition did not occur. Kinetic analyses using preparations containing multiple forms of the same enzyme which catalyze the same reaction, 494 THE PHARMACOKINETICSOF STYRENEIN THE B6C3Fl but are unequally sensitive to a competitive in­ MOUSE. F. A. Smith, J.C. Ramsey, P. E. Kastl, hibitor, will give results that can erroneously K. D. Nitschke, J. F. Quast, E. A. Hermann and be interpreted as mixed inhibition. If both PS W. H. Braun. Toxicology Research Laboratory, and CPS are metabolized by more than one form of The Dow Chemical Company, Midland, MI 48640. cytochrome P-450, and each differentially compe­ titively inhibits the other's metabolism by mul­ The pharmacokinetics of styrene was defined in tiple forms, plots of the inhibition kinetics will B6C3Fl mice exposed by inhalation to either 80, falsely suggest mixed inhibition. (Supported by 200, 600 or 1200 ppm vapor for up to 12 hr. grants ESOlti31 and ES05223 from NIEHS) Continuous 12 hr exposure to styrene resulted in plateau blood levels of approximately 0.71, 1.69, 39.3 and 84 µg/ml for the 80, 200, 600 and 1200 ppm exposures respectively. The plateau blood concentrations increased proportionately 496 PHARMACOKINETICSAND METABOLISM OF DIETHYLENE­ with exposure between 80 and 200 ppm, but dis­ TRIAMINEIN THE RAT. T. R. Tyler, J. W. Lento, proportionately between 200 and 1200 ppm. The J. A. McKelvey, M. J. Tallant and R. S. H. Yang. 3-fold increase from 200 to 600 ppm caused a Bushy Rtm Research Center, Mellon Institute­ 23-fol

140 absorbed from the GI tract and lung. Radio­ 498 PHARMACOKINETICSTUDIES of 3,3',4,4'-TETRA­ activity from the compound was distributed in a CHLOROAZOBENZENEAND 3,3',4,4'-TETRACHLOROAZOXY­ fairly uniform manner throughout the tissues of BENZENE IN RAT. M. T. Stephen Hsia and Charles the rat. Urine and feces were the primary F. Burant, Environmental Toxicology Center and routes of excretion with less than 2% of the Department of Entomology, University of administered dose being expired as 14co 2 • Wisconsin, Madison, Wisconsin 53706. More than 96% of the administered dose was 3,3',4,4'-tetrachloroazobenzene (TCAB) and eliminated within 48 hours after dosing. These 3,3' ,4,4'-tetrachloroazoxybenzene {TCAOB) were data indicate that the route of administration, recenty found to pose occupational health hazards oral or endotracheal, had little effect on the among chemical workers handling contaminated distribution of radioactivity within the body chloroanilide herbicides. Both compounds are or on the pattern of elimination. In comparing genotoxic and can cause dramatic atrophic changes results from animals receiving DETA at 500 in the lymphoid organs, particularly in the mg/kg with those receiving the compound at SO thymus of laboratory rodents. The pharmaco­ mg/kg there was a significant increase in kinetic profiles of TCAB and TCAOBwere investi.­ urinary excretion and a significant decrease in gated in male Sprague-Dawley rats. TCAB was 14co elimination at the higher dosage 2 found to be cleared from the body more rapidly level. There was also a shift in the percent­ than TCAOBwhen a single oral does of [14c]­ age of DETA and metabolites recovered under the labeled TCAB or TCAOBwas administered to rats. peaks of urinary chromatographic profiles for While 66% of the administered TCAB dose was the two dosage levels. These observations excreted via the urine and feces within the first suggest that saturation processes may be 24 hr, TCAOB-treated animals were only able to involved at the high dosage level. Three clear 37% of the administered dose by the same pharmacokinetic parameters (bioavailability, elimination route. Examination of the tissue total clearance and terminal half-life) of DETA distribution of the remaining radioactivity 5 in the rat were compared at the dosage level of days after dosing indicated that, for both SO mg/kg following oral, endotracheal and iv compounds, the adipose tissue as represented by dosing. Irrespective of the route of adminis­ the epididymal fat pad contained the highest tration, the pharmacokinetic parameters are levels of radioactivity. High concentrations comparable. wer~ also found in various excretory organs. The rapid clearance of TCAB and TCAOBby rat as 497 MEPERIDINE CARBOXYLESTERASEACTIVITY IN MOUSE observed in the present study is significant LIVER AND HUMANLIVER. M. Lotti, A. Ketterman, since these agents are isosteric to 2,3,7,8- and R.E. Talcott. Northern California Occupation­ tetrachlorodibenzo-p-dioxin, and all three mole­ al Health Center, Univ. of CA, San Francisco, cules bind reversibly ·with high affinity to the San Francisco, CA. same cytosolic receptor. These pharmacokinetic Meperidine carboxylesterase activity can be meas­ data may explain the differences between binding ured in liver subcellular fractions by coupling affinity and biological potency seen with these the release of ethanol to the reduction of a tet­ compounds in previous studies. (Supported in razolium dye. This particular study was underta­ part by USPHS Grants, R01-ES01737 and R23- ken to compare and contrast the activities pre­ ES01737 from NIEHS,& Grant #110066, from the sent in Swiss mouse liver and in human liver.The Graduate School, Univ. of Wisconsin-Madison. latter samples were obtained from seven patients undergoing abdominal surgery and were normal in histologic appearance. In mouse liver, the meperidine carboxylesterase activity was distri­ 499 SPECIES DIFFERENCES IN THE METABOLISMAND DIS­ buted between the mitochondrial and microsomal POSITION OF MORPHOLINE. Sohn, O.S., Fiala, fractions, with.approximately 2/3 occurring in E.S., Conaway*, C.C. and Weisburger, J.H. the mitochondrial fraction. In contrast, human Naylor Dana Institute for Disease Prevention, liver meperidine carboxylesterase activity was American Health Foundation, Valhalla, N.Y. detected only in the microsomal fraction. The 10595 and *Texaco Inc., Beacon, N.Y. 12508. meperidine carboxylesterase activities in mouse liver mitochondria, mouse liver microsomes, and Morpholine (tetrahydro-1,4-oxazine) is an impor­ human liver microsomes were characterized by de­ tant industrial chemical with a wide variety of termining Michaelis·constants and organophosphate applications. Thus, it is essential that its sensitivities. The Km and Vmax values (T=37°) metabolism and disposition be thoroughly under­ for the mouse liver mitochondrial activity, the s toad. We examined the b load plasma levels and mouse liver microsomal activity, and the human urinary metabolites of morpholine in three ro­ liver microsomal activity were estimated, respec­ dent species: the Sprague Dawley rat, the Syrian tively, as 87 uM, 1,62 nmol/min/mg protein; 460uM, golden hamster and the strain II guinea pig. 1.4 nmol/min/mg protein; and 830 uM, 0.8 nmol/mir/ Marked differences were found between the guinea mg protein. With paroxon as the inactivator, the pig and the other two species with respect to corresponding IC50 values (T=37°, pH=8.0, incuba­ plasma levels as well as the metabolism of mor­ tion time=20 min) were 0.3 uM, 1.2 uM, and 4 uM, pholine. After the i.p. administration of 125 and with DFP, 0.15 uM, 0.5 uM, and 0.5 uM. These mg/kg of morpholine- 14c (SO µCi) per animal, the results suggest a similarity between the liver blood plasma half-lives in the rat, hamster and microsomal meperidine carboxylesterases of mice guinea pig were: 115 min., 120 min. and 300 min. and humans, but also show that the mouse has at respectively. In all three species, approx. 80% least one liver mitochondrial esterase with a of the radioactivity was excreted in the urine relatively high affinity for meperidine and a in 24 hrs. However, while non-metabolized mor­ relatively high sensitivity to organophosphate pholine-14c constituted up to 99% of the urinary inactivators. Supported by NIEHS. Grant#E~02616. radioactivity in the rat and hamster, a signifi-

141 cant portion of the dose (approx. 20%) appeared produce brain and mammary tumors in the Fischer in guinea pig urine as N-methylmorpholine-N­ 344 rat. The liver is not a target organ for oxide. This new metabolite of morpholine was this compound, but it is likely to be a major isolated by Sephadex LH-20 chromatography and site of metabolism of the compound and we have confirmed by TLC, HPLC and mass spectrometry. investigated the ability of isolated Fischer Additionally, HPLC analysis of guinea pig livers 344 hepatocytes to metabolize the compound. We and other organs indicated the presence of N­ have found that these cells transform AN by methylmorpholine and possibly N-hydroxymorpho­ both conjugative and oxidative pathways. The line as well as morpholine and N-methylmorpho­ major metabolite of AN is a conjugate of line-N-oxide. Hitherto, morpholine was not con­ reduced glutathione (GSH), S-(2-cyanoethyl)­ sidered to be extensively metabolized. However, GSH. However, we have found that hepatocytes such conclusions were based on work done only in will oxidize the double bond of AN to form the the rat. The present report underscores the epoxide of AN (2-cyanoethylene oxide). This value of using multiple species in such studies. material has been identified by co-chroma­ tography with an authentic standard on an HPLC system. After .2 hr of incubation with 400 uM AN, the ratio of epoxide _to GSH conjugate was 500 PHARMACOKINETICS OF INHALED METHYL CHLORIDE found to be 0.074. SCN, a known urinary (CH Cl) IN HUMANVOLUNTEERS. R. J. Nolan, metabolite of AN, has also been established as 3 T. D. Landry, D. L. Rick, L. P. McCarty, G. L. a hepatocyte metabolite of AN through_the use Agin and J. H. Saunders, Biomedical Research of mass spectrometry. The ratio of SCN to GSH Lab., The Dow Chemical Co., Midland, MI 48640. adducts was found to be 0.169 after 4 hr of Sponsor: M. B. Chenoweth. incubation with 250 uM AN. It has been hypgthesized by numerous investigators that Six normal healthy male volunteers 25-41 years SCN arises from an unstable cyanohydrin which of age were exposed for 6 hr on two separate is formed by AN epoxide. The demonstration of days to 50 and 10 ppm of CH Cl. Concentrations AN epoxide production by hepatocytes provides 3 of CH Cl in the venous blood and expired air experimental support for this hypothesis. 3 increased rapidly and reached an apparent plateau during the first 30-60 min of the exposure. Venous blood and expired air CH Cl 3 concentrations for each volunteer were propor­ 502 PENTACHLOROPHENOL(PCP) IN BLOOD AND URINE OF tional to the exposure concentration. There OCCUPATONALLY-EXPQSEDMEN. J. ij. Saunders, D. L. were two biologically discrete groups of volun­ Rick and R. J. Nolan, Biomedical Research Lab, teers based on significant differences in the The Dow Chemical Co., Midland, MI 48640. concentrations of CH Cl in their blood and ex­ Sponsor: M. B. Chenoweth. haled air. During t~e 50 ppm exposure, plateau PCP concentrations in casual blood and urine blood and exhaled air CH Cl concentrations were specimens from occupationally-exposed men have 3 110 ng/ml and 39 ppm in two volunteers, and 39 been reported, but previous reports have not ng/ml and 25 ppm in the four remaining volunteers. specified the temporal relation between specimen Following the exposure, CH Cl was eliminated in a collection and exposure. We collected serial biphasic manner. Those wifh the higher blood blood and urine specimens from each of 5 volun­ and exhaled CH Cl concentrations eliminated this teers, occupationally-exposed for at least 3 3 material at a slower rate than the others. How­ weeks. Specimens were collected before, during ever CH c1 was rapidly eliminated by all volu­ and after work on the last of 7 consecutive work­ 3 nteers and thus has a low potential to accumulate days, and thereafter for about 72 hours away from during prolonged or repeated exposures. Blood work. · Pre and post work plasma and urine concen­ non-protein sulfhydryls were not affected by trations were similar for each of the individuals. exposure to CH c1. Urinary S-methyl cysteine The averages ranged from 0.8 to 12.0 µg/mg in 3 excretion was extremely variable, not altered plasma, while casual urines obtained during the by exposure to CH Cl, and should not be used same day ranged from 0.25 to 1.76 mg PCP/mg cre­ to monitor occupafional exposure to CH Cl. Blood atinine. These values are comparable to those 3 and expired air CH Cl concentrations were ade­ reported by others. Since Braun et al., (1979, 3 quately described using a two-compartment physi­ in Toxicology and Occupational Medicine, Elsevier, ological model and the differences between the N.Y.) have shown that 86% of ingested PCP was two groups can be explained by a two-fold dif­ eliminated in volunteers' urine within a week of ference in the rate at which CH Cl was dosing, 24 hr urinary excretion of PCP should be 3 metabolized. a good index of occupational exposure. We found that individuals excreted 0.8 to 5.9 mg/day in the urine with an apparent elimination half-life of about 175 hrs; the amounts excreted in urine 501 METABOLISM OF ACRYLONITRILE BY ISOLATED were consistent with estimated pulmonary uptake FISCHER 344 RAT HEPATOCYTES. L.E. Geiger, at or below the current ACGIH-recommended TLV®. L.L. Hogy, F.P. Guengerich, and R.A. Neal. Concurrent measurements of PCP in the breathing Center in Toxicology, Department of Bio­ zone showed considerable variability, but pro­ chemistry, Vanderbilt University School of vided the same relative estimate of individual Medicine, Nashville, TN 37232 and Chemical exposure with greater convenience. Industry Institute of Toxicology, Research Triangle Park, NC 27709. Acrylonitrile (AN), with 1980 U.S. production

of 1.8 billion pounds I is used in the 503 COMPARISON OF THE ACUTE TOXICITY OF TETRAMETHYL­ production of plastics and elastomers. Among SUCCINONITRILE (TMSN) AND SUCCINONITRILE (SN). its toxic effects, AN has been found to P.A. Doherty, R.P. Smith and V.H. Ferm, Dept. of

142 Pharmacol,/Toxicol. and Dept. of Anat./Cytol., observed after exposure to diethylether, while Dartmouth Medical School, Hanover, NH. the other anesthetics reduced UDPGAabout 25~•• Borneo! and galactosamine decreased UDPGA by The acute toxicity of several aliphatic ni­ 8 5-9mo. Thus, numerous xenobiot ics alter the triles including SN has been attributed to cyan­ concentration of UDPGA in rat liver which may ide (CN) liberation in vivo (TAP 59: 589, 1981). influence the rate of. glucuronidation. (Sup­ Male CD-1 mice were used and compounds given ip. ported by USPHS grants AM 14513 and ES 07079). Although equivalent with respect to CN content, the LD50 of SN (0.78 mmol/kg) and mean time to death (308 min) differed substantially from TMSN (LD50, 0.13 mmol/kg, mean time to death, 10 min). 505 PRESYSTEMIC ELIMINATION OF DIETHYLSTILBESTROL Signs of toxicity were dissimilar as well. The BY RAT LIVER. T.N. Thompson and C.D. Klaassen, classic CN antidote, thiosulfate, protected Dept. of Pharmacology, Univ. of Kansas Medical against SN toxicity but had no effect on TMSN Center, Kansas City, KS. toxicity. Cyanide was present in tissues of SN Some chemicals do not ·effectively enter the treated animals but not in those given TMSN. In general systemic circulation even though they are hamsters SN produced teratogenic effects similar readily absorbed by the gastrointestinal tract. to those produced by CN and other nitriles, while This phenomenon is known as the first-pass effect TMSNwas not teratogenic. Anesthetic doses of or presystemic elimination. Hepatic and/or phenobarbital and barbital were able to protect intestinal biotransformation and hepatic extrac­ against TMSNinduced convulsions and death but not tion are primarily responsible for this phenome­ against SN toxicity. Administration of CC1 sc 24 4 non. Therefore, any xenobiotic which is effi­ hours prior to nitrile treatment prevented the ciently excreted by the liver into bile might be toxic effects of SN but not those of TMSNalthough expected to exhibit presystemic elimination. death appeared to be delayed. Phenobarbital en­ Accordingly, the synthetic estrogen d iethy lsti 1- zyme induction potentiated the toxicity of low b estrol (DES) was examined to determine if the doses of either nitrile. 3-Methylcholanthrene liver decreases its systemic availability. pretreatment had no effect on TMSNbut slightly ~ale Sprague-Dawley rats were administered potentiated the toxic effects of SN. Pretreat­ H-labeled DES (0.005, 0.05 and 0.5 mg/kg) into ment with 4-methylpyrazole delayed death in SN either the i leocolic ( portal administration) or treated animals by at least 6 hours. Beta-carbon hydroxylation to an unstable cyanohydrin which de­ femora 1 ( systemic administration) vein. Plasma and bile samples were collected for 90 min and composes to release cyanide may account for the the concentration of total DES, unchanged DES and toxicity of SN. The high toxicity of TMSNdoes not appear to depend on metabolic release of cyan­ its glucuronide were determined. Both total and ide, but seems to be primarily an effect of the unchanged DES disappeared from the plasma more parent compound. (Supported by Grants HL 14127, rapidly after portal than systemic administra­ ES 00697, ES 07104 and the Ryan Foundation.) tion. In contrast, DES glucuronide constituted a greater percentage of total plasma DES after portal than systemic administration. Comparison of the area under the plasma concentration vs time curve (AUC) indicated that the systemic 504 CHEMICALALTERATION OF RAT LIVER UDP-GLUCURONIC availability of DES after portal administration ACID (UDPGA) CONTENT. J.B. Watkins and C.D. was less than 40% of that after systemic admini­ Klaassen, Department of Pharmacology, University stration. The rate of biliary excretion of total of Kansas Medical Center, Kansas City,KS 66103. DES during the first 15 min was higher following Glucuronidation, a major phase II reaction, is portal administration than by the systemic involved in metabolic conversion of xenobiot ics route. These results indicate that the liver can to more water soluble compounds which are then reduce the systemic availability of DES by excyeted into urine and/or bile. This process is 60-7a'o, (Supported by USPHS Grant AM-14513). dependent upon enzymatic activity of UDP-glucur­ ony !trans ferase and intracellular concentrations of UDPGA. Many xenobiotics alter hepatic glucur­ onyltransferase activity, but their effect on 506 HEPATICEFFECTS OF 1,2,4,5-TETRACHLOROBENZENEIN UDPGAcontent is unknown. Male Sprague-Dawley THE PREGNANTRAT. K.T. Kitchin, ETD, USEPA, rats were pretreated with: 1) microsomal enzyme Research Triangle Park, NC (Sponsor: William inducers ( 7 ,8-benzoflavone, benzo( a)pyrene, F. Durham) butylated hydroxyanisole, isosafrole ( ISF), 3-methylcholanthrene, phenobarbital, pregnenolone 1,2,4,5-Tetrachlorobenzene (TCB) is an 16- -carbonitrile (PCN), 3,4,7 ,8-tetrachlorodi­ industri·a1 intermediate used in the production of benzo-p-dioxin, trans-stilbene oxide); 2) hepato­ 2,4,5-trichlorophenoxyacetic acid, a herbicide toxins (ally! alcohol (AA), aflatoxin (ATX), - which contains trace quantities of TCDD. Because nap h thy 1 i sothiocyanate, bromobenzene, cadmium of possible maternal hepatic or reproductive (Cd), carbon tetrachloride, 1,2-dichloroethylene) effects of this uncharged, low molecular weight, 3) commonly used anesthetics ( urethane, d1ethy !­ lipophilic compound 0, 30, 100, 300 and 1,000 ether, pentobarbital) and 4) inhibitors of glu­ mg/kg/day of unpurified TCB was orally admini­ curonidat ion ( galactosamine and borneol). Rats stered to pregnant rats on days 9, 10, 11, 12 were decapitated prior to removal of the liver. and 13 and the animals were sacrificed on day All inducers except PCN and I SF increased UCl'GA 14 of pregnancy. No maternal deaths were content 36 to 85% above control (410 nmoles recorded and body weight gain was significantly UDPGA/g liver). The hepatotoxins AA, ATX, and decreased only in the 1,000 mg/kg/day group, Cd reduced UDPGA approximately 20-35~.. Greater Maternal liver weight, liver to body weight ra­ than 9m. depletion of hepatic UDPGAcontent_ was tio and hepatic microsomal protein content were

143 unaffected by TCB treatment. Although day 14 for Environmental Toxicology and Department of NADPH-cytochrome c reductase activity was not Animal Science, Michigan State University, East affected, the maternal hepatic microsomal Lansing, MI 48824. cytochrome P-450 content was significantly increased by administration of 1,000 mg/kg/day Hexachlorobenzene (HCB) is a ubiquitous environ­ of TCB. Microsomal N-demethylation of amino­ mental contaminant that has been shown to induce pyrine was slightly increased from 2.4 to 3.4 cytochrome P-450 and a variety of microsomal mixed and 3.5 ~moles/mg protein/min at doses of 300 function oxidases (MFO). Minks were administered and 1,000 mg/kg TCB. However, maternal hepatic either O ppm, l ppm or 5 ppm HCB in the diet 21 weeks microsomal ethoxyresorufin 0-deethylase prior to mating and were continued on HCB supple­ activity was greatly increased from 14 to mented diet throughout gestation. Kits were weaned 30, 40, 50 and 49 pmole/mg protein/min and placed on a regular diet containing no HCB and at in pregnant rats administered 0, 30, 100, 16 weeks of age were killed. Prenatal HCB treatment 300 and 1,000 mg/kg/day TCB. The microsomal caused a dose dependent increase in hepatic cyto­ rates of p-nitrophenol and phenolphthalein chrome P-450 (0.09+0.0l, 0.12+0.0l, and 0.17+0.0l glucuronidation were not increased by TCB nmoles/mg protein in -0 ppm, l ppm and 5 ppm treat­ administration. The maternal hepatic micro­ ment groups respectively). In contrast, microsomal somal enzY]lle induction observed after TCB ethoxyresorufin-o-deethylase (EROD) ethoxycoumarin­ administration to pregnant rats suggests the o-deethylase (ECOD) and benzphetamine-n-demethylase presence of both cytochrome P-450 and P-448 (BND) were not significantly different in any treatment inducers in the sample of 1,2,4,5-tetrachloro­ groups. Renal EROD, ECOD, BND and cytochrome P- benzene used. 450 were not altered in any treatment groups. There were no pathological changes evident in either kidney or liver. HCB treatment had no effect on renal function as determined by blood urea nitrogen and accumulation 507 FREE RADICALACTIVITY INCREASE IN SUBCELLULAR of p-aminohippurate (PAH) and tetraethylammonium FRACTIONSFROM PCB INDUCEDRAT LIVER. D. (TEA) by renal cortical slices. It is interesting to note Havkin-Frenkel, J.D. Rosen and M.A. Gallo, Dept. however, that kidney slices from mink demonstrated of Food Science, Cook College, Rutgers Univ.; very little ability to accumulate PAH (slice to medium Dept. of Environmental and Corrnnunity Medicine, (S/M) ratio=2,46 + 0.12) while TEA transport was much CMDNJ-Rutgers Medical School, Rutgers Univ./ greater (S/M TEA ratio=l3.13 + 0.96). These data Rutgers Medical School Joint Toxicology Graduate indicate that prenatal exposure of minks to HCB will Program, Piscataway, NJ have residual effects on components of the hepatic Chlorinated hydrocarbons have been shown to be MFO system. (Supported by USPHS Grant ES00560.) tumorigens in rodent species. Several of these compounds, including PCBs, do not induce muta­ tions in in vitro systems and must be considered as possible epigenetic tumorigens. Arochlor 1254 509 DOSE-DEPENDENTBIOCHEMICAL RESPONSE TO ANDMETABO­ induces mixed function oxidases in rodents and LIC EXCRETIONOF BROMOBENZENEIN RATS. S. Chakra­ this induction may lead to the increased produc­ barti and J. Brodeur. Dep. med. trav. et hyg. mil., tion of oxygen radical species. Studies were Fae. med., Univ. Montreal, Montreal, Que., Canada. conducted in subcellular fractions to determine Adult male rats were treated (i.p.) with diffe­ the ability Arochlor 1254 induced livers to form rent doses of bromobenzene (BB) (0.1, 0.5, 1.0, hydroxy radical ex vivo. 2.5 and 5 rrnnole/kg) in corn oil and sacrificed 24 Male Sprague-Dawley rats (100-125g) were ad­ h after each dose. While there was no significant ministered Arochlor 1254, I.P., in corn oil at O, increase in such biochemical response at 0.1 and 10,30,100 mg/kg/day for four (4) days. Livers 0.5 rrnnole/kg BB dose, the activities of serum were homogenized in 5 volumes of 0.15M KCl and transaminases (SGOT and SGPT) attained their maxi­ fractions separated by differential centrifuga­ mum values at 1 rrnnole/kg BB dose and remained tion. fairly at the same value at higher doses of BB. Relative liver ·weight was increased (p 0.01) In metabolic excretion studies, various urinary at lOOmg/kg/day as were aniline hydroxylase and metabolites of BB were measured at 0-7½ h, 7½-24 o-demethylase in the post-lysosome/peroxisome hand 0-24 h periods. At each of these time in­ supernatant fraction (approximately 7-fold and tervals, neither the volume of urine nor the con­ 2-fold respectively). Hydroxy radical measured centration of creatinine was affected by different by ethylene production per gram liver was in~ doses of BB. Total nonprotein sulfhydryl contents creased 270% in the supernatant fraction of the started to increase from 0.5 mmole/kg BB and rea­ high dose group. There was a dose dependent in­ ched a fairly constant value at higher doses of crease in lipid peroxidation in the whole homo­ BB at each time interval. Among four hydroxymeta­ genate (p=0.07). These data suggest that the bolites of BB, the percent of o-bromophenol in­ increase in oxygen radical species from PCB is creased with increasing doses of BB and reached a the result of the induction of mixed function maximum of~ 35% at 5 mmole/kg dose at 0-7½ h but oxidases. decreased to~ 22% during 0-24 h. The m-bromophe­ (Sponsored in part by N.J. Agricultural nol initially increased to~ 31% at 2.5 mmole/kg Experiment Station) BB but decreased to~ 15% during 0-24 h. However, the percent of p-bromophenol remained fairly cons­ tant c~40%) even with increasing doses of BB du­ ring different time periods. The percent of p­ 508 THE EFFECTS OF PREN AT AL EXPOSURE OF HEXA­ bromocatechol decreased from 22% at 0.1 mmole BB CHLOROBENZENE ON MIXED FUNCTION OXIDASES to nearly zero at 2.5 or 5 mmole BB during 0-7½ h AND RENAL FUNCTION IN THE MINK. G.F. Rush, V. and then increased to a fairly constant 25% level Adler, M. Bleavins, R. Aulerich and J.B. Hook. at higher doses. The amount of total hydroxymeta­ Department of Pharmacology and Toxicology, Center bolites excreted per dose of BB injected decreased

144 with increasing doses of BB. Thus, as the bioche­ cant elevation of SGPT activity only, but no such mical response to BB hepatotoxicity increased and effect was observed when these were administered attained a maximum, the% dose excreted with re­ simultaneously. But treatment with 2 mg/kg ZnC12 gard to total urinary metabolites decreased with 6 h before the administration of 1 mmole/kg BB or increasing doses of BB. (Supported by MRC Grant, simultaneously with BB produced a reduction in the MA-6159). activities of the transaminases (although not significant) from those of BB-alone-treated rats. When rats were fed 50, 250 and 500 ppm of ZnC1 2 in drinking water daily for four weeks prior to 510 COMPARISONOF THE BINDING PROPERTIES OF CYTOSOLIC the injection of 2.5 mmole/kg BB and sacrificed RECEPTORSFOR 2,3,7,8-TETRACHLORODIBE~ZO-.E_-DIOXIN 48 h after the BB dose, a reduction in the SGOT (TCDD) IN VARIOUSMAMMALIAN SPECIES. T.A. activity and a tendency towards reduction in the Gasiewicz and G. Rucci, Env. Hlth. Sci. Ctr., SGPT activity were noticed in 250 ppm treated rats Univ. of Rochester, Rochester, NY, 14642. Sponsor: only. Liver microsomal system showed an increase T.W. Clarkson. in the concentration of microsomal proteins and the activity of aminopyrine N-demethylase in such The binding of TCDD and its congeners to a cy­ chronically treated rats. Thus changes in the tosolic receptor protein appears to correlate with hepatotoxicity of BB depend not only on the dose their ability to elicit toxic effects in mammals. of zinc but also on the time of its exposition. Differences in species susceptibility to TCDDmay (Supported by MRC Grant, MA-6159). be related to alterations in the binding proper­ ties of these receptors. Utilizing a hydroxylap­ atite assay system, we determined the number of receptors, apparent equilibrium dissociation con­ 512 HEXACHLOROBUTADIENENEPHROTOXICITY IN PIPERONYL stants (K ), and the ability of TCDD congeners to BUTOXIDETREATED RATS. M.E. Davis, Dept. of bind to h~patic and thymic cytosolic receptors Pharmacol. and Toxicol., West Virginia Univ. from male guinea pigs, rats, hamsters, C57BL/6J, Medical Center, Morgantown, WV 26506 (Sponsor: and B6D2F /J mice. Hepatic receptor concentrations M.J. Reasor) ranged fr!m 23 to 74 fmol/mg cytosolic protein. There appears to be no relation between hepatic Hexachlorobutadiene (HCBD) is a by-product of receptor number and the acute LD50 values for chlorinated ethylene synthesis. Treatment with these species. However, those species (guinea pig, HCBD causes acute renal failure and degeneration rat) which have been found to be most sensitive to of the straight portion of the proximal tubule. TCDD-induced thymic atrophy showed the highest HCBD is metabolized by rats, possibly by oxida­ levels (47-140 fmol/mg) of thymic receptors as tion and probably by conjugation with glutathione compared to B6D2F /J mice and hamsters which show­ 1 (GSH). The present studies examined the effects ed the lowest levels (6-10 fmol/mg). In the liver of blocking mixed function oxidase (MFO) activity there appeared to be a general relation between on the development of nephrotoxicity. Male, receptor affinity towards TCDD and the acute LD50 Sprague-Dawley derived rats were maintained in values in .ltihese species .Hepatic receptors from the stainless steel metabolism cages for daily urine guinea pig showed the highest affinity(~= .06nM) collection. Piperonyl butoxide (PIP; 50 mg/kg) while those from B6D2F /J mice and hamsters demon­ 1 was administered i.p. 60 min before HCBD (300 strated lower affinities (0.32-0.42 nM). A similar mg/kg). Renal function was assessed after 5 hr, relationship was observed in the thymus. Also, the 1 and 2 d. Renal blood flow (RBF) was decreased ability of various TCDD congeners to bind to he­ in both groups at 5 hr and then returned to con­ patic receptors generally decreased with increas­ trol values by 2 d. At each time RBF was de­ ing acute LD50 values. These data s~ggest differ­ creased slightly, but not significantly, more in ences in properties of the receptor molecules may the HCBD group compared to PIP+HCBD. Glomerular contribute to species differences in TCDD-induced filtration rate was concomitantly reduced and was toxicity. (Supported by NIH Grant ES02515 and a not affected by pretreatment with PIP. Blood Grant from the Pharmaceutical Manufacturers Assoc­ urea nitrogen was significantly elevated on both iation.) days and was not influenced by PIP pretreatment. HCBD treatment decreased urine osmolality and caused glucosuria equally in control and PIP pre­ treated rats. Hepatic GSH concentration was not 511 EFFECT OF ZINC ON BROMOBENZENE-INDUCEDACUTE HEPA­ decreased by PIP and PIP pretreatment did not TOTOXICITY IN RATS. S. Chakrabarti andJ. Brodeur block the HCBD-induced decrease of GSH concentra­ Dep. med. du travail et hyg. du milieu, Fae. Med., tion. These data suggest that activation of HCBD Univ. de Montreal, Montreal, Que., Canada H3C 3J7 by MFO is not necessary for development of neph­ When adult male rats were treated with 0.5, 2, rotoxicity. (Supported by WVUMedical Corp. and and 10 mg/kg (i.p.) of zinc chloride (ZnC12) 24 h NIH Biomedical Research Grant 5 S07-RR05433-18) prior to the i.p. injection of 2.5 mmole/kg of bromobenzene (BB) and sacrificed 48 h after the BB dose, significant increases in the levels of activities of the serum transaminases (SGOT and 513 HEXACHLOROCYCLOPENTADIENE(HEX) DISPOSITION AND SGPT) from those of bromobenzene-alone-treated METABOLISMIN RATS. C. C. Yu, and Y. H. Atallah, Research and Development Department, Velsicol rats were observed at 0.5 mg/kg of ZnC12 whereas an increase (but not significant) in such activi­ Chemical Corporation, Chicago, IL 60611 ties were noticed at 2 mg/kg of ZnC12 • Pretreat­ Hex is an intermediate for the synthesis of ments of rats for 48 h with the above doses of flame retardants· and cyclodiene insecticides. ZnC12 however produced no such effect. Treatment 14c-Hex was administered to both male an~ female of rats with 2 mg/kg ZnC1 6 h prior to the admi­ Sprague-Dawley rats by oral intubation (po, 10-25 2 nistration of 2.5 mmole/kg BB resulted in signifi- mg/kg) and to female rats by intravenous injection

145 (iv, 0.7mg/kg). Blood, tissues, and excreta were against BB hepatotoxicity may be attributed to an collected and analyzed for radioactivity and induction of increased hepatic glutathione levels metabolite(s) characterization. In vitro degra­ by selenium. (Supported by MRC Grant, MA-6159). dation of 14c-hex by gut content, feces, and liver homogenate from rats was also carried out. Orally-administered 14c-hex was mainly eliminated in feces (70%) and urine (16%) in 48 hr. Over the 515 EFFECTS OF 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN ON same period, an equal amount (20%) of the iv­ RNA POLYMERASEI INDUCTIONIN RAT LIVER. C.L. administered radiocarbon was eliminated in feces Potter, I.G. Sipes and D.H. Russell, Toxicol. and urine. In po-administered rats, the radio­ Frog. and Depts. of Pharmacol. and Anesthesiol., carbon level in various tissues, arranged in Univ. of Arizona, Tucson, AZ 85724. descending order, was kidney, liver, fat, blood, lung, spleen, heart, brain, and muscle. In Previously, we have reported that pretreatment iv-dosed rats, the 14c level, arranged in de­ of rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin scending order, was kidney, blood, spleen, liver, (TCDD) inhibits the extent of hepatic induction lung, heart, fat, brain, and muscle. The phar­ of ornithine decarboxylase, the rate limiting macokinetics of iv-administered hex in rats could enzyme in biosynthesis of the polyamines (putres­ be described by a two-compartment open model cine, spermidine and spermine). Since increased system. The biological half life of hex in rats hepatic ornithine decarboxylase activity has been following iv administration was 32 hr. The body positively correlated to a subsequent increase in burden and the area under the curve for blood in RNA polymerase I activity, we examined the effects the po-administered rats was about 1/10 and 1/70, of TCDD on RNA polymerase I induction. Male respectively, that of the iv-administered rats. Sprague-Dawley rats (100-150 g) received a single These results indicated that only a fraction of i.p. injection of 5 µg/kg TCDD in acetone:corn the po-administered hex was absorbed in the gas­ oil (1:17). Controls received vehicle only. trointestinal tract. Hex was rapidly degraded in Hepatic RNA polymerase I activity was subsequently the rat body. No unchanged hex was detected in stimulated 25-50% by partial hepatectomy or by excreta and tissues by GC, TLC, and HPLC analyses. administration of dexamethasone (0.5 mg/kg) in Most of the degradation products were polar as normal saline. At 5 hr or 16 hr after partial characterized by solvent extraction, TLC, and hepatectomy, or 4 hr after dexamethasone, times HPLC analyses. The in vitro study confirmed that at which TCDD has been shown to strongly inhibit hex was rapidly metabolized to polar compounds. ornithine decarboxylase induction, animals pre­ treated one week earlier with T~DD exhibited RNA polymerase I activity 20-50% lower than controls; i.e. TCDD-treated rats exhibited 0-50% as much 514 EFFECTS OF CHRONICTREATMENTS WITH MERCURYOR stimulation of RNA polymerase I activity as did SELENIUMON BROMOBENZENE-INDUCEDACUTE HEPATO­ the controls. In unstimulated liver, RNA polymer­ TOXICITY IN RATS. S. Chakrabarti and J. Brodeur. ase I activity, as well as protein DNA, RNA and Dep. Med. du travail et hyg. du milieu, Fae. Med. spermine levels, remained essentially the same in Univ. de Montreal, Montreal, Que., Canada H3C 3J7 control and TCDD-treated rats one week after Our previous studies have shown that acute treatment. However, TCDD-treated rats had sig­ treatment with mercury or selenium could protect nificantly lower putrescine and spermidine the bromobenzene (BB)-induce

146 in the lines sec 9, 12F2, 12B2 and 13 was in­ genie seizure susceptible rats. Subsequently, sev­ hibited. The ED50 for this response was approxi­ eral studies have been done to investigate this mately 0.1 nM, with maximal inhibition at 10 nM. interaction. In one study (Henley et al. NSRF:28, Similar values were obtained for the induction of '81), 58 female rats were placed in4groups: Gl, cytochrome Pi-450 mediated monooxygenase activi­ controls; G2, CHL in drinking H20 (800 µg/ml) for ties in these cells, SCC 4 cells, a line exhibit­ 10d then 10d H20; G3, 124 dB SPL noise exposure ing the least differentiated character, were not (90sec, 1/wk for 3 wks); & G4, G3 followed by G2 inhibited in colony expansion by TCDD, Addition of protocols. Since otitis media (OM) hampers ossicu­ hydrocortisone (0.4 µg/ml) antagonized the inhi bi­ lar chain conduction & round window recordings tory effect of TCDDwith an accompanying shift in (RWR), only 9,6,3 & 9 rats were recorded from Gl- the dose-response curves. In two lines (SCC 9 and 4. Input-output functions were obtained from RWR SCC 13) a dose-related stimulatory response to of N1 responses to clicks & cochlear microphonics TCDDwas noted in the presence of hydrocortisone. (CM) to 4,8,12 & 15 KHz pure tones. These data We conclude that the response to TCDDdepends on were reported by Henley et al., however, detailed both the cell line and the presence of hydrocorti­ cochlear morphology has notbeen described. Scan­ sone in the culture medium. The dose-response ning electron microscopy (SEM) examination of the parameters suggest that the observed effects are organ of Corti (OC) of Gl & G2 were normal. Two mediated by the TCDD receptor. G3 rats had a small "acoustic lesion" in the middle cochlear turn. Missing inner (IHC) & outer (OHC) hair cells characterized the lesion. The site was that portion of the OC sensitive to 4-8 517 THE TOXICITY OF 2,3,7,8-TETRACHLORODIBENZO-p­ KHz. Power spectrum analysis of the noise (2 ring­ DIOXIN AND PERFLUORODECANOICACID IN L5178Y MOUSE ing school bells) showed the greatest dB intensity LYMPHOMACELLS. A.M.Rogers,* M.E. Andersen and at 4-16 KHz, which is a reasonable match of the K. C. Back, AFA1'1RL/THT,Wright-Patterson AFB, OH lesion site. SEM of the OC of G4 rats revealed 45433 progressive damage from the middle turn extending Perfluorinated fatty acids of chain length 10 or thru the hook. The upper middle turn had sporadic greater cause toxic effects in vivo similar to OHC loss & intact IHC's with damaged stereocilia. those caused by 2,3,7,8-tetrachlordibenzo-p­ The mid-middle turn contained supporting cells & dioxin (TCDD). We have studied the effects of only sporadic IHC's; no OHC's. The lower middle these compounds in vitro using L5178Y mouse turn contained only headplates &/or other support­ lymphoma cells. Cells were treated in suspension ing cells. The basal turn & hook had no identifi­ for 24-96 hr time periods. Suspension growth was able cellular elements. Thus, SEM data supports determined by haemocytometer counts. At the end the functional data of Henley et al. of the treatment period, cells were plated in soft agar to determine clone formation. No effect on suspension growth was noted for dioxin treatment (highest dose, 0.05 µg/ml). In soft 519 IMPAIRMENTIN COCHLEARFUNCTION PRODUCED BY OTO­ agar, levels of TCDD greater than 0.01 µg/ml TOXIC INTERACTIONOF CHLORAMPHENICOL(CHL) AND caused a reversible change in clone formation and NOISE, C.M.Henley, R.D.Brown, S.Kupetz, J.E.Penny, reduced plating efficiency. Clones were larger, K.Hodges, and P.C.Jobe, Depts of Pharmacol, Anat, less discrete and lacked a normal morphology. and Psych, L.S.U. Med Sch, Shreveport, LA. This effect was expressed after a treatment time of 36 hr (ce3 cell generations). Treated cells Ototoxic interaction between CHL and noise was recovered their normal clone morphology after first seen by D.W. Glenn (M.S.thesis,'79) while growth iu control medium for 48 ht (ce4 cell performing audiogenic seizure studies. In a study generation). Perfluorodecano.ic aL:id (PFDA) by Henley et al. (NSRF:28, '81), 58 female rats treatment had uo effect on suspension growth were assignedto 4 groups: Gl, controls; G2, 10d below lytic levels (500 µg/ml). PFDA levels of CHL in drinking water (800 µg/ml) then 10d rest; 1 µg/ml for 24 hr (2 cell divisions) markedly G3, 124 dB SPL wide band noise exposure (90sec, reduced plating efficiency without affecting 1/wk for 3wks); and G4, G3 followed by G2 proto­ growth in suspension. The effect was reversible cols. Because of otitis media (OM), only 9,6,3, & after growth in fresh medium for 48 hr (4 cell 9 rats were recorded from Gl-4. Input-output (I/0) divisions). We have also studied a number of functions were obtained from round window record­ other perfluorina_ted acids. Similar results ings (RWR) of N1 responses to clicks and cochlear were obtained for acids with chain length greater microphonics (CH) to 4,8,12, and 15 KHz pure than 10. Perfluorinated acids of chain length 8 tones. The I/O's of N1 , CM at 4KHz, and the high­ or less had no effect on clone formation. Pre­ est intensities of CM at 8KHz were lowest in G4; liminary results of mutagenicity experiments with however, the 8KHz CM was not significantly dif­ PFDA and TCDDwill be discussed. PFDA does not ferent in G3 & G4. At 12 and 15 KHz there was a induce mutation in 4 selective systems. TCDD nonsignificant trend for G4 to be lower than Gl-3. induces mutation in the thymidine and thioguanine SEM evaluations are reported by Penny, et al. at systems. *NRC Resident Research Associate this meeting. Due to OM, the presently reported study was done. In this study, no OM occurred (7, 7,16 & 18 rats were recorded in Gl-4). The 1st 5 days of RWRmatched the first study; however, 518 COCHLEARDAMAGE PRODUCED BY OTOTOXICINTERACTION post 5d, no differences were observed in Gl-4; OF CHLORAMPHENICOL(CHL) AND NOISE. J.E. Penny, i.e., the CHL/noise interaction was transient (SEM K. Hodges, S. Kupetz, C.M. Henley, R.D. Brown and studies are being completed). Differences in the P.C. Jobe. Depts of Anat, Pharmacol, and Psych, 2 studies may have been due to OM &/or use of dif­ L.S.U. Med Sch, Shreveport, LA. ferent preparations (preps) of CHL (1st study - Ototoxic interaction of CHL and noise was first VETICOL®, 2nd-TEVCOCIN®). Preliminary results of observed by D.W. Glenn (M.S.thesis,'79) in audio- a 3rd study, which include HPLC CHL serum levels,

147 indicate a difference in bioavailability in the appropriate LD50all mice died within a few days preps. Thus, low dose oral CHL following brief of exposure. At dosages that were 37.5% of the noise exposure results in transient or permanent appropriate LD50both T and P substantially re­ ototoxicity. Bioavailability of CHL preps may duced motor activity; these effects were maximal account for the observed difference in duration on the 10th day of dosing and did not change over of ototoxicity. the next 20 days of dosing. The effects of T and P were also proportionately comparable on both ambulation and rearing. C produced no notice­ able effects on motor activity throughout the 520 A MATHEMATICALMODEL TO STUDYTHE TIME COURSE AND dosing regimen. Moreover at 37.5% of the LD50, DOSE-DEPENDENTIN VIVO EFFECT OF TOCPON CHICKEN T, P and C inhibited brain AChEby 22, 87 and BRAINNEUROTOXICESTERASE ACTIVITY. S.A. Soliman, 65%, and NTEby 58, 0 and 49%, respectively. D. Svendsgaard, and L.W. Reiter, Neurotoxicology These results suggest that certain OP insecti­ and Biometry Divisions, Health Effects Research cides produce enduring effects on the motor acti­ Laboratory, US EPA, Research Triangle Park, NC vity of mice which appear to be unrelated to AChE 27711. or NTElevels in brain. In addition, the pro­ portionate constancy of effects on ambulation and The time course and dose-dependent in vivo effect rearing suggests that motor impairment produced of TOCPon hen brain neurotoxic esteraseTNTE) by OPs in mice is not related to a hindlimb weak­ were studied. Hens were given a single oral dose ness similar to that observed in many other of O (corn oil only), 50, 100, 250, 500 or 750 species. mg/kg of TOCP.On days 1, 2, 3, 7, 10, 14, 21 and 28 after treatment 3 hens per group were sacri­ ficed and brain NTEactivity levels were deter­ mined. Hens were also observed daily for abnor­ 522 PLASMAACETYLCHOLINESTERASE ACTIVITY--A POTENTIAL malities in gait. Results indicated that dose MARKEROF PERIPHERALNEUROTOXICITY. B.F.Bass and levels of 250 mg/kg or greater induced signs of A.M.Goldberg, Division of Toxicology, The Johns delayed neuropathy. For each dose of TOCPpeak Hopkins University, Baltimore, Md. inhibition of brain NTEactivity, computed as percentage of each corresponding control mean, Increases in plasma acetylcholinesterase(AChE) occurred on day 2 and the effect was dose-depen­ activity have been associated with various natu­ dent up to 10 days after treatment. By 21 days rally occurring neuromuscular disorders. The fol- post-treatment all treatment groups had returned . lowing studies were undertaken to examine whether to greater than 90%of control. Mathematical chemically induced disorders wou~d result in a analysis of these data indicate that the level similar increase and, if so, would this increase of NTEactivity following TOCPadministration can precede overt toxicity. Two well known neuro­ be described by the following equation: toxic compounds, 2,5-hexanediol and acrylamide, were chosen. Sprague-Dawley rats were exposed via their E=lOO(1+,\ 1:') ,:=.. (exp-k,t-exp-keo/ c'::c,] drinking water to 0.5% 2,5-hexanediol for up to a e e 2 nine weeks. By week 6, 12% of the animals ex­ where Eis the percent NTEactivity, tis the hibited signs of the neuropathy and by week 8, time (in days) following exposure, Dis the dose all treated animals had developed peripheral in mg/kg and K, k, C and C are constants motor disturbances. Plasma AChE activity was detennined froM th~ data. Jo~nson (J. Environ. found to differ significantly between control and Sci. Hlth Bl5:823-841, 1980) has reported that treated groups (p<.01). At week 4, plasma AChE OP-induced delayed neuropathy develops only after activity in the treated group was elevated. This 80% inhibition of NTEhas occurred; accordingly, upward trend continued, reaching statistical sig­ the minimumataxia dose of TOCPin the hen is nificance at week 10 (p<.01). In the second estimated to be 143 mg/kg. study Sprague-Dawley rats were initially adminis­ tered 3 daily doses of acrylamide (SOmg/kg). Two days later, daily injections were resumed for six days, but at half the dose (2Smg/kg). By day 6 serum AChE activity was found to be significantly 521 NEUROBEHAVIORALEFFECTS 0~ REPEATEDEXPOSURE OF higher in the treated animals than in controls MICETO ORGANOPHOSPHATEINSECTICIDES. R.C. Mac­ (p<.01), at a time when only 29% of the treated Phail and S. A. Soliman, Neurotoxicology Divi­ animals exhibited overt signs of peripheral nerve sion, US EPA, Research Triangle Park, NC 27711. toxicity. Not until day 12 did all of the treat­ ed group show motor dysfunction. These studies evaluated the effect of 30-day In conclusion, the above studies are further exposures to TOCP(T), parathion (P) and cyano­ evidence that plasma AChE activity is a marker of fenphos (C) on the motor activity of mice and on neuromuscular disorders, whether chemically in­ brain levels of acetylcholinesterase (AChE)and duced or naturally occurring. The data further neurotoxic esterase (NTE). Mice were tested at suggest that plasma AChE activity is a potential five-day intervals and received all treatments early indicator of peripheral neurotoxicity and p.o. after the test session. In different ex­ would be a useful tool in toxicity testing. periments, mice (N=5-10) received OP dosages that Supported in part by NIEHS 07067 & 00454 were 4%, 10%, 25%, 37.5% or 50%of the acute LD50spreviously determined in our laboratory. Each experiment also included mice that received either no treatment or the corn-oil vehicle. 523 ANTICHOLINESTERASEACTIVITY OF CARBOFURANAND ITS None of the OPs produced an appreciable effect on DIBUTYLAMINOSULFENYLDERIVATIV!, CARBOSULFANIN motor activity at dosages that were 25%or less RATS, R.I. Krieger, M.S. Dey, L.J. Naser, B.E. of the appropriate LD50, whereas at 50%of the Renzi, and N. Umetsu.* Dept. of Veterinary

148 Science, Univ. of Idaho, Moscow, ID, and *Otsuka homologous to the rat amygdala (15.1 ±. o.6), hip­ Chemical Co. Ltd., Naruto, Japan. pocampus (15.0 ±. 1.3), cerebellum (13.9 ±. 1.3), 2,3-Dihydro-2,2-dimethyl-7-benzofuranyl (di-n­ hypothalamus (9.7 ±. 0.5) and brainstem (8.8 ±. butylaminosulfenyl)(methyl)carbamate (DBSC) 0.4). These differences in distribution of NTE retains insecticidal activity but is 5- to 10- activity in the nervous system of the two species fold less toxic than carbofuran (CF: rat oral may be related to their differential sensitivity· LDSOca. 10 mg/kg). Umetsu and Fukuto (1981) to the neurotoxic actions of organophosphorus suggest that derivatization allows DBSCto be compounds. (Supported by NIEHS grant ES01611-03) metabolized to nontoxic products before intoxi­ cation can take place. Red blood cell ~cetyl­ cholinesterase activity (ACh)was measured using a radiometric procedure and ( 3H-acetyl) choline 525 ACUTEAND CHRONIC TOLERANCE TO ORGANOPHOSPHORUS iodide substrate. Female Sprague-Dawley rats INSECTICIDES.L.G. Costa, B.W. Schwab and S.D. (172-278g) with jugular cannulas were given CF Murphy, Div. of Toxicology, Univ. of TexasMed. or DBSC. Blood samples (0.2 ml) were collected School, P.O. Box 20708, Houston, TX, 77025. serially during test periods of up to Sh. Equi­ molar i.v. dosages (CF 50 µg/kg or DBSC86 µg/kg) Male mice, treated for two weeks with the rapidly produced ACh inhibition. The only overt insecticide disulfoton (0,0-diethyl 2-ethylthio­ signs of toxicity were minor muscle fascicula­ ethyl phosphorodithioate; 10 mg/kg/day) became tions. Maximuminhibition was observed after 2 tolerant to the hypothermic and antinociceptive min and control levels of ACh were measured after effects of a single injection of disulfoton and 2h. Neither the extents of inhibition nor recov­ the muscarinic agonist oxotremorine. Tolerant ery times of the two groups were statistically animals presented a reduced binding of the speci­ different. The basis for the lower peroral toxi­ fic musc1rinic antagonist (3H)-quinuclidinyl ben­ city of DBSCcompared to CF was investigated zilate ( H-QNB)in central and peripheral tissues. using EtOH and corn oil solutions. CF (SO µg/kg) In forebrain, the number of receptors (Bmax) was in either vehicle reduced ACh about 40%within decreased 40%with no change in the affinity con­ 10 min. DBSC(86 µg/kg) was inhibitory in EtOH stant. Acetylcholinesterase (AChE) activity was but not in corn oil. An 8-fold higher DBSC 15% of control. 48h after a single injection of dosage (690 µg/kg) in corn oil produced about 45% disulfoton (10 mg/kg) mice were more resistant ACh inhibition after 60-180 min. The apparent than control to the hypothermic and antinocicep­ lower peroral toxicity of DBSCcompared to CF tive effect of a second administration of the probably results from slower absorption as well same insecticide and to oxotremorine. Tolerance as more extensive metabolism to nontoxic products. was not present 96h after a single administration (Supported in part by NIH Biomedical Research of disulfoton. AChEactivity was 65 and 27% inhi­ Development Grant 1 S08 RR 09073-01A2). bited after 48 and 96h, respectively. The first injection of disulfoton gave a 74% inhibition of AChEactivity after 4h. Similar degrees of inhi­ bition (73 and 72%) were found after a second in­ 524 DISTRIBUTIONOF NEUROTOXICESTERASE ACTIVITY IN jection at 48 or 96h. (3H)-QNBbinding did not RATAND HEN NERVOUS SYSTEM. P.J. Hollingsworth, differ from control at either time point while B.R. Dudek, c.B. Smith and R.J. Richardson. Toxi­ h1gh affinity binding of the muscarinic agonist cology Research Lab., Dept. of Envir. & Ind. ( H)-oxotremorine-M \'las significantly decreased Hlth. and Dept. of Pharmacol., Univ. of Mich., (-37%) 48h after disulfoton. These results show Ann Arbor 48109. that in chronic tolerance there is a decrease of Inhibition and aging of neurotoxic esterase (3H)-QNBbinding, possibly indicating a loss of (NTE), a phenyl valerate (PV) hydrolase, by or­ central muscarinic receptors. In acute tolerance ganophosphorus compounds results in a delayed antagonist binding is unaffected and only a de­ neuropathy in sensitive species. Rats and hens crease in agonist binding was found, indicating differ in their sensitivity to the neurotoxic ac­ a differential regulation of muscarinic receptors tions of these compounds. The purpose of the pre­ depending on duration of exposure. (Supported by s~t study was to compare the distribution of NTE NIEHSgrant# ES 01831 and Training Grant GM in discrete homologous areas of the rat and hen 07405). nervous system. Male, Sprague-Dawley rats and adult, white Leghorn hens were decapitated and specific areas of the nervous system were dis­ sected and homogenized. NTE activity was deter­ 526 EVIDENCEFOR LEPTOPHOS-INDUCED NEUROPATHY IN mined by the differential titration of PV ester­ CHICKSEXPOSED ON INCUBATIONDAY 14. L. P. ases using paraoxon and mipafox. In the rat, Sheets and S. Norton, Dept. of Pharmacology, highest activity (in nmol/min/mg protein±. SEM, Univ. of Kansas Medical Center, Kansas City, KS n L 7) was in the hypothalamus (9.1 ±. 0.9) and 66103. lowest in the hippocampus (5.0 ±. 0.5). Intermed­ iate values were found for the brainstem (8.0 ±. It has been reported that the young of all 0.6), lumbar cord (7.1 ±. 1.1), amygdala (7.0 ±. sensitive species are resistant to organophos­ 0.5), thoracic cord (6.9 ±. 0.2), cervical cord phate-induced delayed neuropathy (OPIDN)and (6.3 ±. 0.3), cerebral cortex (5.8 ±. 0.6), caudate that chicks become sensitive around 60 to 70 nucleus (5.5 ±. 0.3) and cerebellum (5.2 ±. 0.7). days of age. OPIDNis characterized as a distal In the hen highest activity was in the cerebral axonopathy with symptoms first becoming apparent cortex (19.6 ±. 1.1) and the area homologous to 7 to 14 days following treatment. A single 250 the rat caudate nucleus (19.5 ±. 1.5) whereas low­ mg/kg oral dose of leptophos is approximately a est activity was in the lumbar (4.2 ±. 0.4), thor­ threshold dose for causing delayed neuropathy in acic (2.8 ±.0.2) and cervical (2.2 ±. 0.1) spinal adult chickens. We injected this dose in propy­ cord. Intermediate values were found in the area lene glycol into chicken eggs on incubation day

149 14. Following hatching, both functional and cology Division, US EPA, Research Triangle Park, morphological evidence of hind! i~b motor neuro­ NC 27711 and Department of Pesticide Chemistry, pathy were observed. Deficits in function of Alexandria University, Alexandria, Egypt. other muscle groups were not apparent and choli­ nergic symptoms were neg] igible. Functional Three phenylphosphonate derivatives have been impairment was seen on hatching as staggering, synthesized and tested for toxicity in hens. Two wide-based gait, splay leg and occasionally of the compounds,RO-ethyl S-benzyl phenylphospho­ complete ataxia. At higher doses, or with corn nothioate (Inezin) and 0-methyl 0-benzylidenyl­ oil as the vehicle, curled toes were also seen. phenylhydrazone phenylphosphonothioate, which Except for the curled toes, which worsened, are used as fungicides, had low acute toxic these signs generally diminished during the effects in hens. The LD50values were greater first few days. These animals effectively than 2000 and 1000 mg/kg, respectively. No signs competed for food, with no difference in weight of delayed neur~pathy were detected in hens that gain, but some unsteadiness was retained. Fifty received Inezin or the hydrazone compoundafter days after hatching, when blindfolded, these single oral doses of 2000 or 1000 mg/kg were chicks were significantly less able to remain administered. On the other hand, 0-n-propyl 0- perched on a rod than were control chicks. (2-propynyl) phenylphosphonate (also-known as Histological studies showed no clear evidence of NIA-16388) which has been recommendedfor use spinal cord or cerebellar tissue damage. However, as an insecticide synergist, was found to be pre] iminary examination of hind] imb tissue showed relatively toxic in hens; its 24-hr LD50value di~tal axonal loss with associated muscle atrophy. was approximately 340 mg/kg. This compoundpro­ Whether this neuropathy represents the character­ duced delayed neuropathy in hens given an oral istic delayed neuropathy seen in adult animals dose of 400 mg/kg and pretreated with atropine is being investigated. (Supported in part by sulfate in order to minimize cholinergic effects. USPHSgrants ES 07079, MH17279 and HD02528.) The clinical signs of neurotoxicity were apparent 12-17 days after treatment and were similar to those produced by tri-o-cresylphosphate (TOCP). 527 ALTERATIONOF TISSUECOENZYME-A LEVELS DURING Degenerative lesions of axons were observed in ACRYLAMIDEINTOXICATION. M.J. Miller, M.S. the sciatic nerve and spinal cord of hens sacri­ Miller, I.G. Sipes and D.E. Carter. Toxicology ficed 30 days after treatment with the NIA-16388 Program and Dept. of Pharmacology, University of and resembled those produced by TOCPand similar Arizona, Tucson, AZ 85721. compounds. In addition, administration of NIA- Chronic low-level exposure to the neurotoxin 16388 inhibited hen brain neurotoxic esterase by acrylamide (ACMD)produces central-peripheral 90% at 24 hrs after administration. These re­ distal axonopathy. The mechanism of ACMD~induced sults indicate that the organophosphorus insecti­ neuropathy is currently unknown, but has been cide synergists should also be tested for their postulated to occur through inhibition of the ability to produce delayed neuropathy in hens. glycolytic-TCA pathway. Schoental and Cavanagh (1977) suggested that ACMDmay bind directly to coenzyme-A (CoA) and thus decrease the available CoAcontent required for neuronal metabolism and 529 A METHODFOR MEASURING IN VITRONEUROTOXIC ESTER­ maintenance of axon integrity. To test this ASEACTIVITY WITH METABOLICACTIVATION. G. L. hypothesis, CoAcontent of neural and non-neural Sprague and M. J. Hendricks (Spon: T. R. Castles) tissues during ACMDintoxication has been Stauffer Chemical Company, Toxicology Department, measured. Sprague-Dawley rats (300 g) received Richmond, California 94804 daily doses of ACMD(SO mg/kg/day ip) or saline. Rats were sacrificed after cumulative ACMDdoses Neurotoxic esterase (NTE) activity has been mea­ of 50,150,250 and 350 mg/kg. Cerebral cortex, sured in vitro to predict organophosphorus ester cerebellum, spinal cord, heart and liver were neurotoxicity. The in vitro NTEtest cannot be removed immediately and assayed for CoAcontent used for compounds tnatriiusf be metabolized to by the radioenzymatic assay of Chan and Ebadi produce neurotoxicity. Metabolic activation has (1981). Neuropathy was assessed by measuring the not previously been used with this test because duration of response to a 1% zingerone solution extraneous protein would interfere with it. This applied to the eye. Prolonged response to study describes an activation system that is com­ zingerone was detected following cumulative ACMD patible with the in vitro NTEtest. The general doses of 150 mg/kg or greater. No depletion of procedures are asroTTows'; (l) prepare liver tissue CoA content was observed at times corre­ microsomes from an adult, male rat, (2) preincu­ sponding with the development of neuropathy. bate microsomes, with or without NADPH,with Tissues demonstrated a significant (p <.OS) diethyl-p-nitrophenyl phosphorothionate (parath­ increase in CoAcontent after a cumulative ACMD ion), tri-o-tolyl phosphate (TOCP)or 4-bromo-2, dose of 250 mg/kg. These data demonstrate that 5-dichlorophenyl methyl phenylphosphonothionate the mechanism by which ACMDinduces axonopathy (leptophos), (3) remove microsomes by ultracen­ does not involve depletion of CoA. Elevated trifugation and (4) determine the effect of the levels of CoA detected suggest impaired utiliza­ supernatant (contain_ing test material, metabo­ tion of CoA during the development of axonopathy. lites and no interfering protein) on the in vitro (Supported by a Graduate Student Development NTEtest. Parathion (0.12-12 uM, non-neurotoxic), Award to M.J.M. and NIEHSES82130.) failed to inhibit NTEactivity either with or without metabolic activation. TOCP(0:12-12 uM) had no effect on NTEactivity without metabolic activation but complete NTEinhibition occurred 528 DELAYEDNEUROPATHY IN HEN BY THE INSECTICIDE when activation was used with 12 uMTOCP. In SYNERGISTNIA-16388 (0-n-PROPYL0-(2-PROPYNYL) the absence of metabolic activation, 0.12-12 uM PHENYLPHOSPHONATE).SaTah A. Soliman, Neurotoxi- leptophos had no effect on NTEactivity. How-

150 ever, concentration-dependent NTEinhibition or hexane (2000 ppm) 14 hr/day, 7 days/week for occurred (0.12-12 uM) when metabolic activation 14 weeks. Both solvents inhibited weight gain. was used (35% inhibition at 12 uMleptophos). Hexane caused a neurotoxic syndrome characterized The procedure described has usefulness for (1) by reduced grip strength (esp. hindlimb), motor screening delayed neurotoxicity of small quan­ activity, and startle responses. Initial acqui­ tities of research organophosphorus esters and sition of a conditioned avoidance response (CAR) (2) studying metabolic mechanismsfor delayed was also impaired, but subsequent performance was neurotoxicity. Preliminary results suggest intact. Toluene did not cause the peripheral leptophos requires metabolic activation to motor symptoms associated with exposure to hexane, produce delayed neurotoxicity. However, CAR acquisition and performance was impaired along with a tone intensity discrimina­ tion test. The latter effect was still evident several weeks after the last exposure to toluene. 530 ASSESSMENTOF FENITROTHIONNEUROTOXICITY IN THE These results can be interpreted to suggest that HEN, H,D, Durham and D,J, Ecobichon, Dept. of toluene may cause a persistent cognitive dysfunc­ Pharmacol. Ther., McGill U,, Montreal, CANADA tion without the peripheral symptoms caused by hexane. (Supported by NIDA Contract A report of human poisoning by fenitrothion, No. 271-80-3712) (0,0-dimethyl 0-(4-nitro-m-tolyl)phosphorothioatey suggested that this agent caused a persistent neuropathy, To test this hypothesis, groups of 532 STARTLEREFLEX AND GAP DETECTIONFOLLOWING ACUTE fasted adult White Leghorn hens were treated with EXPOSURETO ETHYLALCOHOL IN CONTROLAND METHYL single, toxic doses of fenitrothion (500 mg/kg), MERCURYPOISONED RATS. J. R. Wecker, J. R. Ison, TOTP mg/kg) or trichlorfon (100 mg/kg) in (500 J, A. Foss, and M. F. Wu, Dept. of Psychology, peanut oil or aqueous solution by oral gavage, Dept. of Radiation Biology and Biophysics, and At post-treatment intervals of 1,7,14,28,42 and Envir. Hlth, Sci. Ctr., Univ. of Rochester, 56 days, birds were anesthetized with chloralose Rochester, NY. Sponsor: R. W. Wood. (100 mg/kg). Maximal conduction velocity of the Although the most apparent deficit accompany­ sciatic nerve and post-tetanic potentiation (PTP) ing alcohol exposure is in motor control, sensory of the soleus muscle contraction to nerve stimu­ disturbances have been reported. For example, lation at 0.4Hz were measured as indices of peri­ methyl alcohol affects persistence in the audi­ pheral nerve and muscle function, To elicit te­ tory system, disrupting a rat's ability to detect tani, the sciatic nerve was stimulated at fre­ gaps in white noise, as measured by reflex modi­ quencies of 50-200Hz, The birds were killed by fication. The present experiments examined gap exsanguination and brain and spinal cord AChE and detection following ethyl alcohol poisoning, and, neurotoxic esterase (NTE) were assayed as well as further-, its interaction with organic mercury hepatic and renal carboxylesterase (CE) and plas­ poisoning. Rats (n=S) were given progressive ma pseudocholinesterase (PChE), No change in doses of 0.00, 0.25, 1.00, and 2.00 g/kg (IP, 16% maximal conduction velocity was observed in any w/v solution) at one hour intervals. Thirty treatment group although TOTP-treated birds were minutes after each injection, gap detection was clinically neuropathic, No inhibition of PTP was assessed by presenting gaps of 0,2,4,6,8,12, and observed after treatment with fenitrothion or 20 msec in white noise, 190 msec before a startle trichlorfon but results with TOTP were variable, eliciting tone burst. As with methyl alcohol, TOTP caused marked reductions in NTE, little ef­ the effect of the gap progressively declined with fect on AChE but inhibited PChE and tissue CE at dose level. In contrast to methyl alcohol, the days 1 and 7 of post-treatment, Fenitrothion and size of the control startle reflex was signifi­ trichlorfon had no effect on NTE at any post­ cantly decreased by 85% (twice the effect of treatment interval but markedly reduced brain and methanol). To examine the interaction between spinal cord AChE, plasma PChE and hepatic and re­ methyl alcohol and methyl mercury poisoning, 15 nal CE at days 1 and 7, The results demonstrated that, unlike TOTP, fenitrothion (and trichlorfon) rats (6 control, 6 with 13 mg/kg, and 3 with 40 administered as single toxic doses did not cause mg/kg, methyl mercury chloride) received the a persistent peripheral or central neuropathy dosing regimen. All animals showed a similar detectable by the parameters measured. decline in gap detection with increasing dose, and no interactions were found. Differential behavioral responding under these two compounds may reflect differences in metabolism and com­ partmentalization resulting from the differences 531 NEUROBEHAVIORALEFFECTS OF SUBCHRONICEXPOSURE OF in molecular structure. These results are part WEANLINGRATS TO TOLUENEOR HEXANE. G, Pryor, of an ongoing effort to characterize behavioral J, Dickinson, R. Howd, and C. Rebert. SRI performance following exposure to alcohols of International, Menlo Park, CA. Sponsored by: different carbon length. S,1pported by NIEHS grant D.C.L. Jones, ES01248. Clinical and epidemiological evidence has impli­ cated toluene as a potential neurotoxicant associated with solvent abuse by humans, 533 IN VIVOPOTENTIATION OF HEROIN'S ANALGETIC ACTION especially adolescents, but controlled laboratory AND"!'NVITROINHIBITION OF HEROINBINDING TO verification in animals is lacking. Therefore, OPIATERECEPTORS FOLLOWING ESTERASE INHIBITION. we assessed the neurotoxic potential of toluene G.M.Carlson, G.Gianutsos, S.D.Cohen,R.Heyman,Sect compared with that of the known neurotoxicant Phannacol. & Toxicol., P.Salva, G.Hite, Sect. Med. hexane, which is also present in abused solvent Chem., Sch. Phannacy, Univ. Conn., Storrs,CT 06268 mixtures. Male weanling rats (Fischer-344) were This study was undertaken to assess the role of exposed by inhalation to toluene (900or 1400 ppm) esterase activity on the action of heroin in the

151 mouse and on the binding of heroin to opiaterecep­ 535 IMPAIRMENTOF VISUAL SYSTEMDEVELOPMENT FOLLOWING tor~ in rat brain homog~n~te. Prior inhibition of ORGANOTINEXPOSURE. W.E. Howell, D. Miller and peripheral esterase act1v1ty by triorthotolylphos­ R.S. Dyer, US EPA, Neurotoxicology Division, Re­ phate(TOTP),125mg/kg,ip 18 hr before test, in male search Triangle Park, NC 27711 Sponsor: L.W. mice, increased the analgetic potency of heroin Reiter --- but not of morphine. The potency of 6-mono-acetyl morphine(6-MAM)was only marginally increased. Adult exposure to triethyltin (TET) produces de­ The ED50of heroin, by the acetic acid-induced generation of white matter, edema, vacuolation o~ writhing test, without esterase inhibition was myelin and histotoxic hypoxia (Magee et al., 0.24mg/kg sc(0.20-0.28,95%CI) and following peri­ 1957) and visual evoked response (VER) disturb­ pheral (but not central) esterase inhibition was ances (Dyer et al., 1981). Perinatal TET expo­ ~.042mg/kg (0.024-0.074). Peripheral plasma chol- sure also produces changes in the adult VER (Dyer 1nesterase(CHE) and carboxylesterase activities et al., 1981). Acute adult trimethyltin (TMT) were inhibited 80 and 93%respectively, while exposure produces selective lesions within the brain CHEwas unaffected by TOTPpretreatment. limbic system as well as VER changes (Dyer et al., 1981). In this study, the VER was used to This suggests that peripheral esterases hydrolyze study visual system development following peri­ the acetyl group at position #3 of heroin while natal exposure to either TET or TMT. On post­ prevention of this hydrolysis by TOTPincreased natal day 5, Long-Evans hooded rat pups were in­ the lipophilicity, CNS-penetrability and potency jected i.p. with either 0, 3 or 6 mg/kg TET bro­ of heroin. mide in saline or intubated with O, 5, 6 or 7 mg/ In opiate receptor binding studies, heroin dis­ kg TMT chloride in saline. On postnatal days 12 placed 3H-etorphine(ICso=3xl0-7M). The addition of and 21, pups were anesthetized (Chloropent) and paraoxon to the incubation media resulted in a tested using the VER. Averaged responses to lower potency(Ic 50=1.6xlo-6M). Morphine or 6-MAM single and paired-flashes (500 msec flash separa­ without paraoxon yielded IC50 values,~ etorphine, tion) were derived. In the single-flash para­ of 2xlo-7M and 9.2x10-8M respectively; the digm, TET exposure increased day 12 Pl latency addition of paraoxon failed to significantly alter and day 21 PlNl peak-to-peak amplitude. TMT ex­ these binding constants. Results suggest that posure on the other hand increased day 12 N3 la­ esterase activity in the binding preparation de­ tency and day 21 PlNl and N1P2 amplitudes. No acetylates heroin at position #3 and that this significant TET or TMT differences were observed hydrolysis is necessary for optimal opiate bind­ for responses recorded during the paired-flash ing. (Supported by NIDAGrant #DA-016112). paradigm, suggesting that visual system recovery function was not impaired. The VER changes ob­ served on days 12 and 21 in this study are differ­ ent from those seen in adult animals following day 5 TET exposure (Dyer et al., 1981), suggesting a 534 CONDUCTIONVELOCITIES OF SENSORYAND MOTOR NERVES progression in the nature of the TET visual system IN GALACTOSENEUROTOXICITY.. D.A. Delio and H.E. toxicity during postnatal development. The quali­ Lowndes, Dept. Pharmacology, CMDNJ, NJ Medic~ tative differences in the VER changes following School, Newark, NJ. day 5 TET and TMT exposure suggest different sites and mechanisms of action for these structurally Abnormalities of sensory nerve conduction are similar organotin compounds. an early feature of diabetic neuropathy; such changes are consistent indicators of subclinical neuropathy. Galactosemic diabetic neuropathy is characterized by an accumulation of galactitol, accompanied by osmotic swelling in sciatic nerves. 536 EFFECT OF CYCLODIENEPESTICIDES ON BEEF HEARTAND Although impaired motor nerve conduction velocity RAT BRAIN ATPase ACTIVITIES. B. D. Mehrotra, S.• (MNCV) has been detected in this experimental K. Bansal, and D. Desaiah, Chem. Dept., Tougaloo diabetic neuropathy, a corresponding defect in College, and Dept. Neural., V.A. and Univ. Miss. sensory nerve conduction velocity (SNCV) has not Med. Ctr., Jackson, MS 39216. been investigated. Sprague-Dawley rats (250-300 gms) were fed a 40% galactose diet for 3 weeks. Comparative effects of aldrin, dieldrin, telodrin, Conduction velocities of single sensory and motor endrin and isodrin on different ATPase activities axons between sciatic or tibial nerves and dorsal in beef heart mitochondrial (BHM) and rat brain or ventral roots were recorded and compared to synaptosomal (RBS) fractions were determined in age and weight matched controls. No significant vitro. BHMfractions were prepared by the con­ differences between age matched control and ventional centrifugation method and the RBS were galactosemic animals were observed. However, prepared by Ficol-Sucrose gradient centrifugatioo when compared to weight matched controls, both method. Na+-K+ ATPase, oligomycin-sensitive (O.S) sensory and motor conduction velocities in whole and insensitive (O.I) Mg2+ ATPases, and K+- par­ nerve trunks and single fibers were decreased anitrophenyl phosphatase (KPNPPase) were deter­ (p<0.05); the decrease in whole root SNCV was mined in RBS and only the Mg2+ ATPase activities more pronounced for both nerves (p<0.05). were determined in BHM. Dose-response curves After terminating the galactose diet for one were determined by assaying the enzyme activities week, sensory and motor conduction velocities in the absence and presence of 10-120 µM concen­ recovered in sciatic and tibial nerves, with trations of each test chemical. BHM (O.S) Mg2+ values approaching or exceeding those of con­ ATPase activity was inhibited by all 5 chemicals trols. These data suggest that galactose feed­ tested. Aldrin and telodrin were the most potent ing induces a reversible peripheral neuropathy inhibitors-with an ID50 of 40 and 80 µM respect­ which encompasses sensory and motor fibers, both ively. About 30% inhibition was observed with proximally and distally. dieldrin, endrin and isodrin and the inhibition

152 , was not dose-dependent. O.I Mg2+ATPase was not effects of a single injection of chlordecone significantly inhibited by any chemical except administered at the time of hypothalamic sexual aldrin. RBSATPases were also sensitive to these differentiation (day 4) on brain neuropeptides. compounds. Aldrin and telodrin were more effec­ Offspring of Fischer rats were used in this study. tive than others. A 50% inhibition of O.S Mg2+ They were divided into two groups of 80 rats each ATPase activity was obtained at 80 µMof aldrin (40 males and 40 females). One group received and telodrin. Na+-K+ATPase and O.I Mg2+ATP­ 1.0 mg/pup chlordecone in DMSO;the other received ase activities showed a maximuminhibition of only DMSO. Both were administered in a volume of 40%at the highest concentration tested for al­ 10 µl. They were sacrificed by decapitation at drin and telodrin. KPNPPasewas not inhibited the age of 21, 70, 120, and 180 days. Brain significantly by any compoundtested. These re­ regions (striatum, hippocampus, hypothalamus, sults suggest that ATPase system in heart and and pituitary) were di~sected out and the content CNSmay be selectively inhibited by aldrin and of neuropeptides, [Met J-enkephalin (ME), s­ telodrin but not by their structural analogs. endorphin (s-E), neurotensin (NT), and substance (Supported by PHSGrants ES-02443 awarded to D.U P (SP), were determined by radioimmunoassay (RIA). and RR-08110awarded to B.D.M.) For each time period there were 10 males and 10 females per treatment group. Chlordecone treat­ ment showed a selective reduction in s-E in the hypothalamus of both sexes at the age of 21 days. 537PREWEANINGEVALUATION OF KEPONE NEUROTOXICITY. In DMSO-treatedanimals, the MEcontent in the C.F. Mactutus, K.L. Unger, and H.A. Tilson. Lab. pituitary was equal for both sexes at 21 days of Behav. Neurol. Toxicol., NIEHS,Research Triangle age. However, at 70 through 180 days, the level Park, NC 27709. (SPON: H.B. Matthews). of this peptide in the male was twice that for the Neonatal exposure of rats to Kepone, during a female. Chlordecone reduced the MEconcentration period of sexual differentiation of the brain, in the male but not the female at the age of 70 impairs learning/retention abilities prior to and 120 days. Other brain regions showed no weaning (Neurosci. Abst., 1981). The present study significant changes in peptide levels following provides complimentary assessments of sensory, chlordecone treatment. Thus, these results in­ motor, and arousal systems during the preweaning dicate that chlordecone has a specific effect on period following neonatal Kepone intoxication. hypothalamic and pituitary peptides. Offspring of Fischer 344 rats were pooled and randomly assigned to dams three days after birth. On postnatal day 4, pups received a s.c. injection (20 µl) of either dimethylsulfoxide (OMSO)or 1 539 NITROIMIDAZOLENEUROTOXICITY IN THE MOUSE: CLIN­ mg/pup of Keponedissolved in DMSO. Body weights ICALRELEVANCE AND POSSIBLE MECHANISM(S). P.J. on days 14 and 21 were slightly (9%-14%), but Conroy and T.H. McNeill, Univ. of Rochester significantly, depressed in both sexes by the Cancer Center, Div. of Exptl. Therapeutic~ Depts. neonatal Keponeexposure. Whole body movements, osf Radiology and Neuro]oqy, Rochester, NY. monitored via a spectral analyzer, indicated a ponsored oy T.W. Clarkson. significant tremor (12.4-13.0 cps) in the Kepone­ The clinical effectiveness of nitroimidazole hy­ treated pups on postnatal days 10, 14, and 18. poxic cell radiosensitizers such as misonidazole The development of sensory reactivity to a 4 kHz, (MIS) and metronidazole (METRO)is considerably 110 db tone was examined on days 12, 16, and 20. reduced by the dose limitation imposed by neuro­ Significantly enhanced responsiveness was noted toxic side effects. The purpose of this investi­ in the Kepone-treated pups relative to their DMSO gation was (i) to determine if data on the rela­ littermates (19%males, 39% females). Evaluation tive neurotoxicity of nitroimidazole sensitizers of generalized activity/arousal, as assessed by in mice using different quantitative functional testing pups in isolation but in the presence of endpoints were similar, (ii) to compare misonida­ homecage shavings, indicated a significant Kepone­ zole (MIS) with desmethylmisonidazole (DESMIS)in induced hypoactivity in both sexes on day 15 (32%- relation to central nervous system (CNS) versus 33%). On day 21, hypoactivity was evidenced by peripheral nervous system (PNS) effect~ and, (iii) accelerated habituation in the Kepone-intoxicated to determine potential mechanism(s) of neurotoxi­ females. These neurobehavioral alterations in city. The data indicate good agreement between early life are similar to those observed in our the functional endpoints used to determine rela­ adult Kepone toxicity studies, and thus provide tive neurotoxicity (rotorod performance and high another avenue td pursue the mechanisms of Kepone­ frequency hearing loss) and the incidence of CNS induced tremor and altered responsiveness to and PNSpathology in mice. In general, CNSand novel/stressful environments. PNSlesion development coincided suggesting these effects are separate. MIS is 2 times more toxic per administered dose and equitoxic per brain ex­ posure dose compared with DESMISin BALB/ckamice. 538CHLORDECONE(KEPONER) EXPOSURE ALTERED RAT BRAIN DESMISis slightly more toxic than MIS to the CNS PEPTIDES. S.F. Ali, and J.S. Hong. Laboratory of C3Hmice. DESMISgiven i.v. resulted in more of Behavioral and Neurological Toxicology, NIEHS, severe PNSlesions than either i.p. or oral DESMIS Research Triangle Park, NC 27709. Sponsor: R.B. or i.p. or oral MIS. Significant decreases in the Mailman lactate/pyruvate ratio of the brain stem of MIS Chlordecone (KeponeR) is an organochlorine treated mice indicates that inhibition of glycoly­ insecticide causing neurological disorders such sis is a potential mechanism of neurotoxicity. as tremor, ataxia, hyperkinesis, anxiety, irrita­ Results are evaluated in relation to their predic­ bility, and short-term memoryloss in humans. tive significance to clinical testing of sensiti­ Therefore, the central nervous system is one of zers. (Supported by National Institute of Health the major sites of act~on affected by chlordecone Grants NS-16147and CA-11051and the United toxicity. The present study investigated the Cancer Council, Rochester, NewYork.

153 540 NEUROTOXICITYPRODUCED BY SUBCHRONIC(90 DAYS) observed but was not clearly identifiable as para­ SIMULTANEOUSINHALATION OF N-HEXANEAND METHYL nodal. Hens treated with 10 ppm MBK: 1 mg/kg EFN ISO-BUTYL KETONEIN HENS. M.B. Abou-Donia, D.G. had minimal unequivocal lesions with rare axonal Graham and P.R. Timmons. Departments of Pharmaco­ swellings. These were not as large as those seen logy and Pathology, Duke University Medical Genta:, in MBKneurotoxicity, but instead resembled the Durham, NC 27710. histopathologic lesions caused by EPN. (Supported in part by NIOSH Grant No. OH00832). Groups of 5 adult hens were exposed to vapors of n-hexane, methyl iso-butyl ketone (MiBK) or mix­ tures of n-hexane and MiBK. Inhalation exposure of either-1,000 ppm n-hexane, 2,000 ppm n-hexane 542 A COMPARISONOF HEXANENEUROTOXIC POTENTIAL IN or 1,000 ppm M_!BKca~sed leg weakness during the WEANLINGAND ADULTRATS. R. Howd, T. Steeger, 90-day exposure. The clinical condition of these J. Dickinson, and G. Pryor, SRI International, birds improved during the 30-day observation pe,.. Menlo Park, CA. Sponsor: D.C,L. Jones. riod after their exposure to the vapors. These hens lost weight during treatment, but regained Hexane inhalation can cause neurotoxicity, appar­ most of the lost weight after cessation of expo­ ently through formation of the metabolite 2,5- sure. By contrast, simultaneous exposure to mix­ hexanedione (HD). In previous work, we had shown tures of 1,000 ppm Ee-hexane and 1,000 ppm or 500 (J. Neurotox. Terat., in press) that in adult ppm MiBK caused leg weakness and ataxia which rats, sub-chronic hexane exposure led to increas­ progressed to paralysis. Although the clinical ing HD blood levels over several days, apparently condition of these hens slightly improved during due to induction of liver microsomal enzymes. the 30-day observation period, more of these hens When blood HD levels were maintained at 50 µg/ml remained paralyzed at the end of the experiment. or more by chronic hexane exposure, detectable The neurologic dysfunction was accompanied by neuropathological changes occurred within 4-8 swollen axons and by degeneration of axons and weeks. A further question remained as to the myelin of the spinal.cord and peripheral nerves. neurotoxic potential of hexane in young, prepuber­ These hens lost weight during exposure but regain­ tal animals, since liver metabolic enzymes often ed some of the lost weight during the observation increase during development (and adolescents are period. Hens simultaneously exposed to 1,000 ppm a primary human population of solvent abusers). n-hexane and 100 ppm MiBK were not different from We exposed weanling male Fischer 344 rats to 1000- those exposed only to 1,000 ppm Ee-hexane. The 2000 ppm of hexane on various schedules, in acute mechanism of the joint neurotoxic action of n-hex­ and chronic experiments, including a direct com­ ane and MiBK seems to be related to liver micro­ parison to adult males. HD was synthesized well somal induction in treated animals. (Supported in in weanling rats, and its levels increased in part by NIOSH Grant No. OH00823). blood for several days of constant exposure to hexane at 1000 or 2000 ppm, as in adult males. However, HD levels in the blood of weanlings were about 50 to SO% of those in adults with the same 541 INTERACTIONBETWEEN SUBCHRONIC (90 DAYS) DERMAL exposure. The subacute hexane toxicity appeared APPLICATION OF 0-ETHYL 0-4-NITROPHENYL PHENYL­ correspondingly less in the weanlings than in the PHOSPHONOTHIOATE(EPN) ANDCONTINUOUS INHALATION adults. In contrast, in a chronic exposure, rats OF TECHNICALGRADE METHYL BUTYL KETONE (MBK) IN exposed to hexane from weaning to adulthood de­ PRODUCINGNEUROTOXICITY IN HENS. M.B. Abou-Donia, veloped neuropathology at a rate consistent with D. G. Graham, P.R. Timmons and K.M. Abdo. Depart­ that in rats exposed first as adults, with an in­ ments of Pharmacology and Pathology, Duke Univer­ crease in liver and decrease in testicular weights, sity Medical Center, Durham, NC 27710. and decrease in weight gain similar to those ob­ served in young adults. (Supported by NIDA Con­ Four groups of hens (5 hens/group) were treated tract No. 271-80-3712.) for 90 days with a daily dermal dose of 1.0 mg/kg of EPN (85%) in 0.1 ml of acetone on the unpro­ tected back of the neck. Three of these groups were exposed simultaneously to 100, 50, or 10 ppm of MBK (methyl n-butyl ketone : methyl iso-butyl 543 EVOKEDPOTENTIALS IN RATS EXPOSEDAS WEANLINGS ketone 7:3) -in inhalation chambers. One group was TO TOLUENEOR HEXANE, C.S, Rebert, G, T. treated only with the dose of EPN. Three other Pryor and S,S, Sorenson, SRI International, groups of hens were exposed continually in inhala­ Menlo Park, CA. Sponsor: D.C.L. Jones tion chambers to the same concentrations of MBK without EPN treatment. The treatment period was Clinical signs of neuropathology associated followed by a 30-day observation period. Combined with the abuse of volatile solvents contain­ exposure to both EPN and MBK caused more severe ing toluene, hexane and other compounds indi­ signs of neurotoxicity than exposure to either cates a need to independently determine the individual compound. The severity of histopatholo­ neurotoxic effects of components of solvent gical lesions in hens given daily dermal doses of mixtures. Hexane has been extensively studied 1 mg/kg EPN in combination with inhaled MBKde­ in the laboratory but rigorous, experimental pended on the MBKconcentration. There clearly verification of the neurotoxicity of toluene were more lesions in the 100 ppm MBK: 1 mg/kg EPN is lacking. We compared the effects of tol­ group than in the others. In most hens in this uene and hexane on several electrophysiologic group, Wallerian degeneration was seen along with indicants of peripheral and central nervous large paranodal axonal swellings. The morphology system activity. Male weanling rats (Fischer and distribution of histopathologic lesions were 344) were exposed by inhalation to toluene characteristic of MBK-induced lesions. In the 50 (900 or 1400 ppm) or hexane (2000 ppm) 14 hr/ ppm MBK: 1 mg/kg EPN group axonal swelling was day, 7 days/week for 14 weeks. Hexane caused

154 a mild neuropathy indicated by increased lat­ Ozone causes changes in behavior at con­ encies of the ventral caudal nerve action centrations as low as 0.12 ppm. To determine potential and components of the somatosensory, if its irritant properties are of behavioral visual and auditory cortical evoked responses. significance, we used a behavioral test in Hexane affected only the amplitude of the 5th which mice respond to turn off, or escape an component of the brainstem auditory evoked irritant. Initially, mice were trained to turn response. This finding was in contrast to off 1000 ppm arrrnonia. Each mouse was required previous results showing an effect on latency to poke its nose five times into one of two of that component with exposure of rats to conical recesses. Completion of this require­ 1000 ppm hexane for 24 hrs/day. Only the ment terminated delivery of the irritant for effects on the ventral caudal nerve, and de­ one minute, and produced a facial shower of creased amplitude of cortical and brain- clean humidified air from the conical recesses. stem auditory components persisted for one Irritant delivery was also terminated after 60 week after termination of exposures. Toluene seconds if the animal did not respond. Control had an effect only on the amplitude of the procedures demonstrated that this behavior is fifth component of the brainstem response. not due to a general increase in activity These findings are consistent with the results resulting from irritant exposure, nor is it of behavioral measures indicating a general maintained by the presence of the facial shower lack of neurotoxicity of toluene, except for alone. After the concentration-effect curves tests of cognitive function which indicate for ammoniawere determined, each mouse was deficits of learning and discrimination. exposed to ozone in an ascending and descending (Supported by NIDA Contract No. 271-80-3712) sequence of concentrations (0.25-24.0 ppm). Ozone exposures alternated every other day with exposures to 1000 ppmammonia as a control for changes in baseline behavior. As the concen­ tration of ozone increased, the numberof escape 544 THE EFFECT OF TRACEHALOTHANE ON 2' 3' CYCLIC responses increased and the latency decreased. NUCLEOTIDE3'PHOSPHODIESTERASE (CNPASE) IN THE Thus ozone, a lower airway irritant, will DEVELOPINGRAT BRAIN. J,J. Nagelhout, F.C. maintain escape behavior. Supported by Beuthin, R.T. Louis-Ferdinand, Coll. Phcy. & All. DA00623,ES01247, and in part under contract Hlth. Prof., Wayne State University, Det. MI 48202 with DOEDE-AC02-76EV03490.

On subcellular fractionation, CNPase activity is primarily localized in the myelin fraction of rat brain and has been used as an enzyme marker 546 MACROCYCLIC(CROWN) ETHERS: THEIR TOXICITY NEURO­ of myelination. Chronic exposure to subanesthetic MUSCULARPHARMACOLOGY AND STRUCTUREACTIVITY RE­ LATIO~SHIPS. S. C. Gad, F. A. Gavigan, W. D. concentrations of the volatile anesthetic 3 Halothane inhibits leucine incorporation into Adams and G. Schieman, Allied Corporation, Mor­ myelin in rats exposed during the prenatal and risto~, NJ; University of Cincinnati, Cincinnati, early postnatal period (Wiggins et al. 1979). OH and CMDNJ- Rutgers Medical School, Piscata­ In order to determine the effect of trace way, NJ. concentrations of Halothane on the developmental The six compounds currently comprising profile of CNPase, 2 groups of pregnant Sprague the commercially available Crown Ethers and Crown Dawley rats were exposed to 500 p.p.m. or 250 Ether derivatives (12-Crown-4, 15-Crown-5, 18- p.p.m. Halothane in air, 8 hours per day, 5 days Crown-6, Dibenzo-18-Crown-6, Dicyclohexano-18- per week from the third day of conception through Crown-6, and Hexacyclen trisulfate) were evalu­ postnatal day 10. Control animals received air ted for acute lethality in mice (i.p. and oral), alone. The specific activity of CNPase was de­ rabbits (dermal), and rats (oral, i.p. and der­ mal). In all species, the LD by any single creased by 500 p.p.m. Halothane 34% (p<,05) and 5 15% at postnatal days 17 and 20 respectively. route were bound to be 18-C-6 ~ 15-C-5 < 12-V-4 Body weight was decreased in the treated group with the relative lethality of the substituted 15%, 20% and 12% at days 17, 20 and 25. In the compounds depending on route. 250 p.p.m. group, CNPase activity was decreased Associated with the three parent Crown at 17 days (p<.01), and body weight decreased at Ethers have been reports of marked acute and in­ 14, 17 and 20 days. Brain/body weight ratios termediate term neuromuscular, cardiovascular, were unaffected at either dose. and behavioral effects, which has been alternately The data indicate that pre & postnatal hypothesized to be due to either ion trapping/ Halothane exposure delays the development of the chelating (in the ring structure) or to a neuro­ myelin marker enzyme, CNPase. This suggests that logically active metabolite. The neural effects exposure to trace concentrations of Halothane were instigated in rats via the i.p. route. The during neurodevelopment inhibits myelination in neurologic effects of the unsubstituted Crown the rat. (Supported by University Anesthesiologists Ethers were found to parallel the lethal effects, of Detroit) but this was not the case for the substituted derivatives. From this and other observations, it is established that the unusual neurologic and cardiovascular effects are due to a metabolite 545 BEHAVIORALEVALUATION OF SENSORY IRRITATION BY and not due to an ion trapping mechanism. OZONE,J. L. Tepper, R. Wood, B. Weiss. Depts. of Pharmacology and Radiation Biology & Biophysics, Div. of Toxicology, Environmental 547 CHANGFS IN HIGH AFFINITY 3H GABA BINDING IN Health Sciences Center, University of RAT CEREBRAL CORTEX AND HIPPOCAMPUS Rochester, School of Medicine and Dentistry, AFTER ELECTROSHOCK. S. M. Ross I and C.R. Craig, Rochester, NY14642 Department of Pharmacology and Toxicology, West

155 Virgina University Medical Center, Morgantown, West not altered. Thus while altered MN excitability Virgina 26505. I Present address: Institute of might contribute to increased excitability of Neurotoxicology, Rose Kennedy Center, Albert Einstein spinal reflexes, the fasciculations are more College of Medicine, Bronx, N. Y. I 0461. Sponsor: likely to be related to the presence of the ad­ P.S. Spencer jacent giant axon formations. Post synaptic 3H GABA binding was assessed in Supported by the Amyotrophic Lateral Sclerosis rat cerebral cortex and hippocarrµus after electroshock Society of America. to determine a possible role of GABA in electroshock induced seizures. After seizure induction, a marked increase in specific 3H GABA binding to crude cortical synaptic membranes was recorded. The binding increased by 37% and 45% at 15 and 30 minutes, 549 INTRACELLULARRECORDINGS FROM GIANT AXONFORMAT­ respectively. GABA binding returned to preseizure IONS IN IDPN NEUR0PATHY. H.E. Lowndes and B.G. values by I hour and remained there for at least 3 Gold, Dept. Pharmacology, CMDNJ, NJ Medical hours. Scatchard analysis of the binding isotherms School, Newark, NJ. indicated that the increase in 3H GABA binding at 30 minutes was due to a rapid increase (39%) in the Giant axon formations (GAF) are large swellings number of available post synaptic GABA receptor resulting from accumulation of neurofilaments in binding sites (Bmax) rather than an alteration in the first proximal internodes of motor axons receptor affinity (Kd). In contrast to the results (Griffin et al, Science, 202,633,1978). The obtained from the rat cerebral cortex, only minimal and purpose of this study was to determine the effect non-significant increases in 3H GABA specific binding of the GAF on action potentials (AP) conducted were present in the hippocarrµus of experimental and along individual axons. Cats were given S,S'­ control animals Y2 and I hour after electroshock iminodipropionitrile (IDPN) 50 mg/kg/week and treatment. Moreover, Scatchard analysis on data investigated 5 weeks after the first injection. derived from controls and animals subject to Action potentials evoked by antidromic stimula­ electroshock demonstrated no change in receptor tion of motor axons or orthodromically by dorsal affinity (Kd) or the maximum number of binding sites root stimulation were recorded in GAF with micro­ (Bmax) after 1/2to 2 hours. electrodes. Impalements in GAF were discrimi­ The increase in the number of cortical GABA nated from those in motoneurons by the absence of binding sites (Bmax) may indicate an attempt by the EPSPs and IPSPs. APs indistingushable from those cortex to enhance the physiological eftect of GABA at in normal axons were occasionally seen. Others the level of high affinity post synaptic GABA receptor displayed double hump potentials which occurred site. This proposed mechanism could have important randomly during the course of the AP. Hump implications for the control of neuronal excitability as potentials occurring on the rising phase of the it relates to seizure suppression. AP on antidromic stimulation shifted to the fall­ This research was supported in part by the West ing phase upon orthodromic stimulation during the Virgina Medical Corporation and NIH Biomedical same impalement. The characteristics of these Research Grant No. 5 SO7-RR05433-18. action potentials are strikingly similar to the reflection potentials predicted to result from sudden increases in diameter of axons (Parnas et al, J. Neurophysiol. 39,909,1976). Since 548 INCREASEDEXCITABILITY OF MOT0NEUR0NSIN S,S'­ motoneurons in IDPN treated cats often generate IMIN0DIPR0PIONITRILE (IDPN) NEUROPATHY.B.G. repetitive APs upon single stimulation, it is Gold and H.E. Lowndes, Dept. Pharmacology, CMDNJ, conjectured that the repetition might arise in NJ Medical School, Newark, NJ. part from reflection of APs from GAF and subse­ quent re-activation of the motoneuron. GAF occur IDPN induces giant axon formations (GAF) in in some cases of amyotrophic lateral sclerosis the first proximal internode of motor axons. and this phenomenon may contribute to the elec­ These GAF are similar to those seen in some cases tromyographic observations in this disorder. of motor neuron disease (MND) (Carpenter, Neural. 18,841,1968). !1NDis characterized by increased Supported by the Amyotrophic Lateral Sclerosis excitability of spinal reflexes and fasciculation Society of America. potentials which may be of central origin. The present study investigated the electrophysiology of motoneurons (MN) in IDPN neuropathy. Cats were given IDPN (50 mg/kg/wk) and examined 5 550 IN VIVO FORMATIONOF SUBSTITUTEDPYRROLE ADDUCTS weeks·after the first injection. Action poten­ ANDNEUROTOXICITY OF 2,5-HEXANEDIONEIN THE tials in MNwere recorded with microelectrodes, HEN. A. P. Decaprio, P. Weber, and .ll,.. Abraham, evoked by antidromic or orthodromic stimulation Inst. Exp. Pathol. and Toxicol. and Dept. of and verified by the presence of EPSPs and IPSPs. Biocheaistryl, Albany Medical College, Albany, Initial segment (IS) conduction time was decreas­ N.Y. 12208. ed (p<0.05) from a normal value of 0.23+0.008 to In vitro studies in this laboratory have dem­ 0.19+0.007 ms. Somatic-dendritic (SD) thresholds onstrated the formation of 2,5-dimethylpyrrole were-decreased from 31.6+0.84 to 26.9+0.96 mV adducts from the reaction of the neurotoxic n­ (p<0.05). Multiple acti;-n potentials-occurred hexane metabolite 2,5-hexanedione (2,5-HD) with in response to a single stimulation in 16% of primary amines. Such a reaction accounts for both the recordings in IDPN vs 1.3% in normal cats. the observed in vivo tissue binding of 2,5-HD and The results sugge·st that there is an increased the specific requirement for they -diketone excitability of the MNmembrane in IDPN neuro­ structure in this neuropathy. Purified CNS myelin, pathy. However, the repetitive firing does not axolennna-enriched,.and axonal protein fractions appear to originate in the MN ~ince the amplitude were obtained from adult, White Leghorn hens giv­ and time course of the afterhyperpolarization was en daily, oral doses of 20 or 70 mg/kg 2,5-HD for

156 up to 20 weeks or 200 mg/kg 2,5-HD for up to 7 cute response to the neuroexcitant properties of weeks. Electrophoretic analysis ang protein MSG. MSG presumably excerts its neurotoxicity staining using a modified Ehrlich's reagent re­ via excessive stimulation of excitatory receptors vealed conversion of CNS protein amino groups to in select brain regions that lack blood brain substituted pyrrole adducts in 70 and 200 mg/kg barriers. Recently, it has been reported that exposure groups, but not at 20 mg/kg. Although neonatal administration of MSG results in in­ binding was detected in many CNS proteins, the creased susceptibility to pentylenetetrazol-induc­ myelin basic protein exhibited particularly ed seizures in adult mice, however this finding strong affinity for 2,5-HD. Hydrolysis and amino has not gone unchallenged. The aim of the pres­ acid analysis of myelin basic protein from treat­ ent study was to ascertain if neonatal adminis­ ed hens revealed conversion of lysine £-amino tration of MSG produces permanent alterations in groups to pyrrole derivatives. Cessation of dosing CNS excitability. Male CF-1 mice were injected resulted in eventual clearance of the adduct from (s.c.) on post-natal day 4 with either 4 mg/g MSG spinal cord myelin protein. Electrophoretic stud­ (n=6), 1 mg/g MSG (n=6) or saline (n=l2). In ies provided no evidence for simple neurofilament addition, a fourth group (n=4) received 4 mg/g protein cross-linking as the mechanism of action MSG (i.p.) on post-natal day 30. Seizures were of 2,5-HD. Hens at the two upper dose levels dis­ chemically-induced with flurothyl ether on post­ played neurotoxic signs and pathologic changes natal days 60, 90 and 120 and time (sec.·+ SEM) similar to those seen in rodents and man, demon­ to full clonic-tonic seizure was recorded:- The strating the susceptibility of this species. These seizure threshold for the saline-treated mice was results provide evidence for pyrrole formation as 305 + 12.7 and did not vary over time. Mice the mode of in vivo binding of 2,5-HD, and suggest treated on post-natal day 30 had elevated seizure a possible direct action of the compound upon mye­ thresholds (451 ± 23.1) on all 3 test days. Mice lin protein. (Supported by a Society of Toxicology that received MSG neonatally exhibited a signifi­ Predoctoral Fellowship sponsored by the Procter cant increase in time to seizure on the day 60 and Gamble Co.) . · measure (4 mg/g 408 + 54.4; 1 mg/g 442 + 41.8) which reverted to control thresholds on-days 90 and 120. In conclusion, MSG treatment increases flurothyl ether seizure threshold and the perman­ 551 EPHAPTIC TRANSMISSIONBETWEEN MOTONEURONS IN ency of this effect appears to be dependent on CATS WITH S,S'-IMINODIPROPIONITRILE NEUROPATHY. the age at which MSG-induced neuronal damage H.E. Lowndes and B.G. Gold, Dept. Pharmacology, occurs. (Supported by NIEHS grants ES07094 and CMDNJ, NJ Medical School, Newark, NJ. ES02277).

Cats given S,S'-iminodipropionitrile (IDPN) develop giant axon formations (GAF) in the first proximal internodes of motor axons (Gold et al, Neuropathol Appl. Neurobiol, 1981). GAF become 553 INHALATIONTOXICITY OF DECALIN AFTER 20, 28 AND demyelinated as they enlarge, giving rise to pos­ 35 DAYS EXPOSURE. L. C. Stone, G. T. Lawhorn, sible electrical 'short-circuiting' or ephaptic J. C. McKinney, M. S. McCracken and R. L. transmission to adjacent motoneurons, dendrites Kanerva. The Procter & Gamble Company, Miami or GAF. Cats were given IDPN, 50 mg/kg once Valley Laboratories, Cincinnati, OH. weekly for 5 weeks. Action potentials were re­ Sponsor E. V. Buehler corded in lumbar motoneurons using micropipettes Decalin (decahydronaphthalene) is a saturated and evoked by antidromic stimulation of ventral cyclic hydrocarbon which has a wide variety of root filaments. In normal cats, an action industrial uses. Published data indicate this potential can be evoked only by a single motor chemical produces a specific type of nephro­ axon. Ventral roots were divided into two or toxici ty in male rats after 90 days in a more filaments which were stimulated sequenti­ continuous inhalation exposure regimen. The ally after successful impalement of a motoneuron. purposes of our study were 1) to determine Of those cells tested in the IDPN-treated cats, whether or not a similar type of renal toxicity action potentials could be evoked in 12% of the could be induced in a shorter time period in motoneurons by stimulation of more than one fila­ male rats and 2) to determine if male mice were ment. Thus ephaptic transmission occurs in IDPN also sensitive to the nephrotoxic effects of neuropathy and most likely results from the pres­ decalin inhalation exposure. Fischer 344 male ence of the GAF. ·GAF are also seen in some cases rats and C57B1/6 male mice were exposed of amyotrophic lateral sclerosis (ALS) (Carpenter, continuously (22 hours/day, 7 days/week) for Neurol. 18,841,1968). Ephaptic transmission may 20, 28 or 35 days to decalin vapors at O, 25, thus underlie the doublet and synchronous action 62 .5 and 125 ppm. Kidney lesions were observed potentials seen electromyographically in this in all 3 test groups of rats after either 20, disorder. 28, or 35 days exposure. The nephrotoxici ty was characterized by 1) hyaline droplet Supported by the ALS Society of America. formation in the cytoplasm of the proximal convoluted tubule epithelium, and 2) the presence of granular casts at the corti­ comedullary junction. These changes were time 552 MONOSODIUMGLUTAMATE-INDUCED ALTERATIONS IN FLUR­ and dose dependent and are similar to the renal OTHYLSEIZURE THRESHOLD. R. Dawson and G.G. toxicity reported in male rats after 90 days of Bierkamper, Dept. Env. Hlth. Sci., Div. Toxicol., continuous inhalation. No pathologic effects The Johns Hopkins Univ., Baltimore, MD (Sponsor: were observed in other tissues of rats nor in Z. Annau). the kidneys of mice exposed to the high dose of Parenteral administration of monosodium gluta­ decalin. Our results show that the nephro­ mate (MSG) to rats produces convulsions as an a- toxici ty can be produced by decalin in

157 less than 90 days, a situation which will make activity measurements represent upper limits to it more feasible to conduct studies designed to attainable CH20 concentrations, which may be much evaluate the functional significance of the greater than the actual concentrations, a method kidney damage observed in the male rat. was developed to assay CH o directly using gas 2 chromatography-mass spectrometry. Free and (acid-labile) bound CH but not irreversibly­ 2o, bound CH are detectable. Rats were exposed to 2o, 554 A SIX MONTHMULTI-SPECIES INHALATIONSTUDY WITH CHO (6 ppm, 6 hr/day, 10 days) and sacrificed MALEIC ANHYDRIDE. R. D. Short, F. R. Johannsen, within 10 minutes of the last exposure. Concen­ C. E. Ulrich*, Monsanto Co., St. Louis, MO 63166 trations of CHO in the nasal mucosa were appar­ and *I.R.D.C., Mattawan, MI 49071 ently unchange~ from control (endogenous) values (0.5 µmole/g), suggesting that absorbed CH 0 in 2 This study was initiated to assess the safety of the mucosa must be rapidly metabolized andTor atmospheres that contained maleic anhydride. bound irreversibly. Rapid metabolism is possible Accordingly, rats (15/sex/group), hamsters since homogenates of the nasal mucosa were as (15/sex/group), and monkeys (3/sex/group) were efffctive as liver homogenates in catalyzing the treated 6 hours a day 5 days a week for 6 months. NAD- dependent oxidation of CH o to formate, Atmospheres were generated by subliming maleic 2 anhydride and were monitored using Tenax collec­ tion columns and gas chromatography to detect total maleic; i.e. maleic anhydride plus maleic acid. Hematology, clinical chemistry and urin­ 556 PATHOLOGICALEFFECTS OF CHRONICFORMALDEHYDE alysis were performed in all species at 3 and INHALATIONIN RATS ANDMICE. W. D. Kerns, 6 months. Pulmonary function tests (respiratory K, L. Pavkov, and D, J, Donofrio, Battelle rate, tidal volume, resistance and compliance) Columbus Laboratories, Columbus, OH 43201 and were performed in monkeys at O, 3 and 6 months. .§.:._!L_Gralla, CIIT, RTP, NC. 27709 In addition, a histopathological examination was performed in 30 tissues from both sexes of an­ Male and female Fischer 344 rats and B6C3Fl mice imals in control and high dose groups. The were exposed to formaldehyde vapor concentrations mean analytical concentrations were O, 1.1, of 2, 6, or 15 ppm for 24 months. Selected ani­ 3.3 and 9.8 mg/m3 of total maleic. Dose related mals from the study were maintained for an addi­ signs of nasal and occular irritation were ob­ tional six months without formaldehyde exposure. served. These signs included discharge, sneezing, Nasal carcinomas were observed in rats exposed gasping and coughing. No significant treatment­ to 6 and 15 ppm and in mice exposed to 15 ppm. related mortality was observed in any species. Neoplasia was preceeded by dysplastic and meta­ Reduced weight gains were observed only in mid plastic alterations of the respiratory epithelium and high dose rats, which had terminal body in the anterior portion of the nasal cavity in weights greater than 90% of control values. No rats and mice. In the 2-ppm exposure group treatment-related effects were observed in (rats), squamous metaplasia was the only change hematology, clinical chemistry, urinalysis, present. In the 6-ppm (rats) and 15-ppm (rats and pulmonary function. Microscopic evaluation and mice) exposure groups, there was continued of tissues revealed no histopathologic changes pr.ogr.ession of the metaplastic epithelium to related to treatment. Therefore, no effect squamous epithelial hyperplasia with increased levels, which disregard overt signs of irrita­ keratin production and then to squamous papillary tion, were considered to be 1. 1 mg/m3 of total hy1,erplasia with foci of cellular atypia, These maleic for rats and 9. 8 mg/m3 of total maleic lesions then progressed to carcinomas "in situ" for hamsters and monkeys. and finally to invasive squamous cell carcinomas of the nasal cavity. At 27 months (3 months post-exposure), there was a decrease in the fre­ quency of squamous metaplasia in the rat (2 and 555 DISPOSITION ANDANALYSIS OF FORMALDEHYDEIN F-344 6 ppm) and mice (6 and 15 ppm). In the 15-ppm RATS AFTER INHALATIONEXPOSURE. H. d'A. Heck, M. exposure group (rats), there was no regression C. Schmitz, and E. L. White, Chemical Industry of squamous metaplasia, Bone marrow hyperplasia Institute of Toxicology, Research Triangle Park, was also associated with formaldehyde inhalation NC 27709. Sponsor: D.E. Rickert. in male and female rats (15 ppm). In summary, formaldehyde vapor was shown to be a nasal car­ Formaldehyde (CH 0) induced squamous cell car­ cinogen in rats (6 and 15 ppm) and mice (15 ppm). cinomas in the nasai turbinates of F-344 rats Supported by Chemical Industry Institute of exposed to 6 or 15 ppm of CH o (6 hr/day, 5 aays/ Toxlcology 2 1 week, 2 years). Disposition studies using CH 0 2 showed that a principal site of absorption was the nasal cavity. The concentration of radio­ activity in the nasal mucosa was approximately 557NASALMUCOSAL RESPONSE INDUCED BY A RESPIRATORY proportional to both the airborne concentration IRRITANT(METHYL CHLOROACETATE). D. G. Keyes, and the time of exposure An equivalent concen­ 14 R. J. Kociba, J. Henck. Toxicology Research tration of 0.7 µmole of CH 0/g of tissue is w. 2 Laboratory, Health and Environmental Sciences, attained ftter 6 hr of exposure to 6 ppm, and 1.7 USA, Dow Chemical U.S.A., Midland, MI 48640. µmole of CH 0/g of tissue after 6 hr of ex­ 2 £Ssure to 15 ppm. Equivalent concentrations of Groups of male and female Fischer 344 rats were. CH o in other tissues (liver, lung, heart, exposed to O, 10, 25 or 75 ppm methyl chloroace­ kidney,2 spleen, small intestine, brain, testes) tate vapor 6 hours/day for 11 consecutive days. were typically one or two orders of magnitude Parameters monitored included appearance and less than in the nasal mucosa. As the radio- demeanor, body weight, organ weights, clinical

158 chemistry, hematology, urinalysis and gross and 559 GUINEAPIG RESPIRATORYRESPONSES TO ISOCYANATO­ histopathological examination. ETHYLMETHACRYLATE- ANDISOCYANATOETHYL PRO­ Rats exposed to 75 ppm MCACshowed definite tox­ PIONATE-PROTEINCONJUGATES L. S. Mullin, C. K. icity noted as eye irritation, decreased body Wood and N, D. Krivanek, E. I. du Pont de Nemours and Company, Inc., Haskell Laboratory, Newark, DE weights, decreased alkaline phosphatase activity secondary to inanition, and hemoconcentration Exposure to certain isocyanates has been asso­ secondary to general toxicity. There was also ciated with development of respiratory sensitiza­ evidence of increased liver weights (females), tion in some people, In this study, guinea pig decreased BUNvalues and increased SGPT and SGOT respiratory response to protein conjugates of activities. In addition, there was gross and his­ 'isocyanatoethyl methacrylate (IEM) and isocya­ topathologic evidence of upper and lower respira­ tory tract reaction.characterized as epithelial natoethyl propionate (IEP) was evaluated by the hyperplasia, squamous metaplasia and inflanunation. method of Karol (AIHA J. 39:546-556, 1978). Guinea pigs were exposed daily up to two weeks to Rats exposed to 25 ppm MCACalso exhibited toxici­ an aerosol of bovine serum albumin (BSA) or BSA ty noted as very slight eye irritation, very conjugated with IEM or IEP. After the initial slight elevations of SGOTand SGPT activities and exposures, the guinea pigs were challenged with reaction of the upper and to some extent mid­ BSA, isocyanate conjugated to guinea pig serum respiratory tract characterized as epithelial albumin (GSA), or isocyanate monomer or polymer. hyperplasia and squamous metaplasia. Cross response to IEM and IEP conjugates was also tested. After a two week induction, guinea pigs' Rats exposed to 10 ppm MCACexhibited a lesser degree of toxicity noted as minimal eye irritation exposed to BSA-IEM conjugated from 24% to 98% and epithelial hyperplastic changes in the nasal exhibited a marked increase in respiratory rate turbinates. and in some cases bronchospasms. Response was related to degree of conjugation. Some guinea Thus, this study indicates that morphologic pigs exhibiting a response to BSA-IEM cross­ changes in the upper respiratory tract are the reacted to BSA-IEP. No responses to unconjugated most sensitive index of exposure of the rat to BSA were observed. In guinea pigs responding to vapors of methyl chloroacetate. As with all BSA-IEM, >0.1 ppm IEM monomer vapor elicited inhalation studies in rats, these data must be similar b'ii°t delayed responses; 0.01 ppm IEM did interpreted in view of the fact that the rat is not, An IEM polymer aerosol with <0.0004% mono­ an obligatory nasal breather. mer did not elicit a response. These data sug­ gest a response threshold. Guinea pigs exposed to BSA-IEP (62% conjugated) had sensitization­ type responses and cross reacted to BSA-IEM and 558 INHALATIONTOXICITY OF A FLUOROTELOMER- ZONYr.® GSA-IEM. Application of BSA-IEP or IEP monomer TELA. B. A. Burgess, s. D. Nash, and G. L. to the scratched skin of guinea pigs that re­ Kennedy, Jr., E. I. du Pont de Nemours and sponded by inhalation to BSA-IEP resulted in Company, Inc., Haskell Laboratory for Toxicology immediate wheal and flare response not seen in and Industrial Medicine, Newark, Delaware 19711 unexposed animals. All of these responses sug­ Zonyl® TELA (CAS #25398-32-7) is a mixture of per­ gest immediate hypersensitivity. fluoroalkyl iodides consisting mainly of C-6 to c- 12 telomers and is a raw material used in the manufacture of a number of functional fluorochemi­ cals such as surfactants and repellents. The 560 ETHYLENEGLYCOL MONOBUTY( ETHER (EGBE) VAPOR9- material is a paste or liquid at room temperature DAYAND 90-DAY INHALATIONSTUDIES IN FISCHER-344 and boils around ll6°C. Male rats were exposed RATS. D.E. Dodd, W.M. Snellings and B. Ballantyne, for single 4-hour periods to vapor/aerosol evolved Union Carbide Corporation, Bushy Run Research from spraying the material onto a heated surface Center, Export, PA. prior to being swept into the exposure chamber. Exposed rats showed labored breathing, salivation, The toxicity of EGBEvapor was studied in male and and reduced response to sound. The LC50 was esti­ female rats in order to assess possible hazards mated to be 107 mg/L. In a second study, groups' from short-term repeated exposures to high concen­ of male rats were exposed to either 0, 1.5, or 9.6 trations and longer-term repeated exposures to mg Zonyl® TELA/Lair for 10 6-hour periods (5 lower concentrations. In the shorter-term study exposure days, 2 rest days, 5 exposure days). rats were exposed for 9 days (6 hrs/day) to EGBE This was followed by a 14-day ob~ervation period. concentrations of 245, 86, 20 or O (control) ppm. At the higher concentration, 2 rats died during The most noteworthy finding was in blood, where a the exposures, rats demonstrated rapid breathing .. statistically significant depression of red blood and reduced response to sound, had slightly lower \cell (RBC) count (approximately 20% below control), serum urea nitrogen and total protein levels, and hemoglobin (Hgb), mean corpuscular Hgb concentra­ had initial body weight losses during exposure tion with increased nucleated RBC's, retlculocytes followed by recovery. No tissue pathology was and mean corpuscular volume were observed in males seen in surviving rats with the deaths observed and females at 245 ppm. Less marked, but statis­ attributable to severe pulmonary edema. All signs tically significant, effects on erythroid para­ of a response to the chemical except the lowered m~ters were found in rats exposed to 86 ppm EGBE. serum protein concentrations abated during a 14- An additional group of rats allowed 14-days post­ day recovery (no further treatment) period. At a e*posure recovery had a substantial, but not com­ concentration of 1.5 mg/L, Zonyl® TELA-was well­ plete, reversal of the affected blood parameters. tolerated and rats showed only a slightly reduced In the subsequent longer-term study, rats were response to sound. Body weights, clinical chemi­ ·exposed to EGBEat concentrations of 77, 25, 5 or stry, and pathology data from these treated rats 0 ppm for 13 weeks (6 hrs/day, 5 days/wk). Slight, compared favorably to that from the controls. but statistically significant, decreases in RBC

159 count (13% below control) and Hgb, accompanied by level) were exposed for 1-hour to the following an increase in mean corpuscular Hgb, were observed chloroformates (CF): methyl-CF, ethyl-CF, sec­ in the 77-ppm-treated female rats after 6 weeks of butyl-CF, isobutyl-CF and phenyl-CF. CF vapors exposures. Females of the 77 ppm group also had a were generated using a Battelle-developed genera­ transient decrease in body weight gain. However, a: tor and concentrations were monitored by IR spec­ the conclusion of the 90-day study, there were no trophotometry, Toxic signs for all CF's included significant effects at any of the EGBEconcentra­ ocular and nasal discharge, dyspnea, lethargy and tions on body weight, organ weights, hematology, weight loss; most deaths occurred within 24 hours and urine or clinical chemistries. Furthermore, no of exposure, Rats that died generally had lung gross or microscopic lesions related to EGBEexpo­ hemorrhage and edema. In the methyl-CF study addi­ sure were found. These findings confirm the known tional rats were sacrificed for histopathology. RBCtarget toxicity of EGBEand demonstrate th~ Rats at 0.36 and 0.47 mg/1 dose levels sacrificed occurance of this effect above the current TLV of 25 ppm. within 24 hours had alveolar hemorrhage and mucus epithelial necrosis and degeneration in the nasal turbinate, trachea, bronchi and bronchioles. Re­ generative changes indicated resolution of pre­ 561NITROETHANE: A 13-WEEK INHALATIONTOXICITY STUDY viously observed lesions by days 9-10. Calculated IN RATS AND MICE. Gushow, T. S., Bell, T. J., .LC50's (mg/1) for both sexes combined are: methyl­ Burek, J. D., Potts, W. J., and McKenna, M. J., CF, 0.45; ethyl-CF, 0.87; sec-butyl-CF, 1.82; Tox. Res. Lab., Dow Chemical USA, Midland, MI isobutyl-CF, 1.81; phenyl-CF, >l.24. Correlation 48640 of these LC50's with molecular weight (MW) fit a linear model; correlation coefficient 0.9996. A 13-week inhalation study was conducted to assess Slopes from regression analyses of male and female the toxicity of nitroethane(NE) in F-344 rats and data are 0.025 and 0.041, respectively, supporting B6C3Fl mice. In the 13-week study each species was increased male sensitivity. Phenyl-CF did not exposed to 0, 100, 350, or 1000 ppm (0, 0.3, 1.0 cause death at the highest achievable exposure or 3.0 mg/1) of NE for 6 hr/ da, 5 da/wk for a level(l.24 mg/1) in agreement with the linear mod­ total of 64-65 exposures with an interim sacrifice el prediction (2.5 mg/1). This work indicates that of rats and mice after 20-21 exposures. the toxicity of chloroformates may be predicted Exposure of rats to 1000 ppm NE resulted in de­ creased body weight gain, elevated methemoglobin from their molecular weights. Supported by PPG Industries, Inc. levels (MetHb) with cyanosis, increased reticulo­ cytes and Heinz bodies in peripheral blood and associated splenic extramedullary hematopoiesis. Other major target organ effects in these animals included degenerative and inflammatory changes in 563 DISPOSITION OF METHYLETHYL KETONEIN F-344 RATS the olfactory nasal epithelium, hepatocellular AFTER INHALATIONEXPOSURE. J. S. Bus, D. Deyo, vacuolization, decreased cytoplasmic granularity M. Cox and E. L. White, Chemical Industry Insti­ of renal cortical tubular epithelium and ductal tute of Toxicology, Research Triangle Park, NC epithelial cells of the salivary glands. Rats 27709 exposed to 350 ppm NE showed similar but less severe changes in MetHb, spleen, nasal turbinates The purpose of this study was to characterize and salivary glands. Minimal changes in MetHb, the disposition of methyl ethyl ketone (MEK) in spleen and salivary glands were observed in rats Fischer-344 rats after a single inhalation expo­ sure to 500, 1500 and 5000 ppm *EK. Male rats exposed to 100 ppm NE. 1 Mice exposed to 1000 ppm NE showed increased Met­ were exposed for 6 hr to (1,2- C)-MEK, aftn: which total excreta (urine, feces, expired CO Hb, evidence of toxicity in the salivary glands, 2 liver, olfactory nasal epithelium and multinuclea­ and MEK) were collected for 72 hr. In other ted spermatids in the testes. Overall these experiments various tissues were analyzed for changes were less severe than those observed in MEKand the metabolites 2-butanol, 3-hydroxy- 2- rats. Less extensive toxicity was observed in mice butanone (3H2B) and 2,3-butanediol (2,3BD) by exposed to 350 ppm and only MetHb, liver, salivary gas chromatography-mass spectrometry at selected glands and nasal turbinates were affected. Mice times after exposure to nonradioactive MEK. exposed to 100 ppm NE showed minimal changes in Radioactivity collected in the various excreta the nasal turbinates and transient (even during fractions plus tissues and carcass (expressed as continued exposure) effects on salivary gland epi­ percent of total radioactivity collecte1 was as follows: at 500 ppm, exhaled MEK 16%, 4co thelium only. The 100 ppm NE exposure concentra­ 2 tion was judged to be a minimal effect level under 46%, urine 32%, feces 1% and tissues and carcass the conditions of this study. 5%; at 1500 ppm the distribution of the respec­ tive fractions was 31%, 26%, 38%, 1% and 3%; and at 5000 ppm the distribution was 50%, 14%, 33%, 1% and 2%, respectively. Blood NEK concentrations 0.5 hr after exposure were 40, 189 and 739 µg/ml 562 DIFFERENTIAL TOXICITY OF INHALEDCHLOROFORMATES for the respective groups and had declined to IN FISCHER 344 RATS. fi.E.Wilkinscm, B.A.l"rentice, low concentrations in all tissues (blood, brain, A.T. Mosberg and G.L. Fisher, Battelle Columbus liver, kidney, lung, and sciatic nerve) by 12 hr Laboratories, Columbus,OH, and A.P. Leber, PPG after exposure._ Both 2-butanol and 3H2B were Industries, Inc., Pittsburg, PA. detected in all tissues examined, but at concen­ trations generally less than 50% of MEK. 2,3BD Carbonochloridic acids (chloroformates) comprise was observed only in blood. These results a class of chemical reactants widely used as in­ indicate that MEK is extensively metabolized dustrial intermediates. They are corrosive by skin after inhalation exposure and that saturation of or eye contact and may be irritating and toxic MEKmetabolic pathway(s) occurred over the when inhaled. Fischer 344 rats (5/sex/exposure exposure range studied.

160 564 EFFECTOF CHLORPHENTERMINE(CP) ON THE PULMONARY increases in cytotoxicity, decreased protein syn­ CLEARANCEOF (14C)-5-HYDROXYTRYPTAMINE (5-HT) IN thesis, and morphological damage. Increasing RABBITIN VIVO. T. Morita, L. S. Angevine and time of exposure also decreased cell viability. H. M. Mehendale, Dept. Pharmacol. &Toxicol., The morphological damage after 24 hours included Univ. Miss. Med. Ctr., Jackson, MS 39216, USA. cell lysis, detachment of cells from the basement membrane, and the appearance of cytoplasmic Previous studies from this laboratory have de­ vacuoles, distended nuclei, and cells with de­ monstrated that the pulmonary clearance of 5-HT graded nuclear membranes. This exposure model is perturbed by CP, an observation possibly me­ provides quick, reproducible information on sev­ chanistically related to drug-induced pulmonary eral cellular endpoints and may be useful in hypertension. The objective of these experiments short-term toxicological testing of toxic gases. was to determine if the effects of CP could be The system is currently being tested with a vari­ demonstrated in vivo. Pulmonary uptake and me­ ety of toxic gases and will be expanded to in­ tabolism of (RC)-5-HT were examined in single­ clude assays on genotoxicity. (Research supported pass pulmonary perfusion using reference indica­ under DOEContract No. DE-AC04-76EV01013.) tor-radioisotope dilution technique (Catravas and Gillis, JPET 213: 120, 1980). Male NewZealand albino rabbitsTl.9 ± 0.6 kg) were surgically prepared under pentobarbital anesthesia (Nembu­ 566 MAJORFINDINGS IN A TWENTY-FOURMONTH INHALATION talR, 40 mg/kg). The right jugular vein and left TOXICITY STUDYOF METHYLCHLORIDE IN MICE AND carotid artery were then cannulated to the level RATS, K.L. Pavkov, W.D. Kerns, C.E. Chrisp, D.C. of the right atrium and ascending aorta, respec­ Thake, R.L. Persing, H.H. Harroff, Battelle tively. Arterial BP, blood pH, rectal tempera­ Columbus Laboratories, Columbus, OH; E.J. ture, blood hematocrit were monitored during the Gralla, CIIT, RTP,NC. experiment. After 25 min stabilization, controls received injection of saline vehicle and CP Male and female B6C3Fl mice and Fischer 344 rats treated animals received CP iv (jugular vein, were exposed to O, 51, 224 or 997 ppm methyl 0.5, 1 or 3 mg/kg). Ten min later, 1 ml fresh chloride (MeCl) 6hr/da, 5 da/wk, for 24 mo. saline solution containing 500 µg cardiogreen Sacrifices in randomly selected animals occurred (CG) and 0.5 µCi (14C)-5-HTwas injected into ju­ after 6, 12, 18, and 24 mo. Parameters evaluated gular vein and simultaneously blood samples from included: body weight, organ weights, pathology, the artery catheter were collected at 1 sec in­ hematology and urinalyses. Survival was adversely tervals for 31 sec (dead space 6 sec) and ana­ affected in both species and sexes at the 997-ppm lyzed for 5-HT and 5-HIAA. % Extraction of 5-HT exposure concentration. Growth rate was signifi­ for controls was 79.2 ± 2.3 (n = 6), which was cantly(pO.O5) with increased Type II cell origin on collagen gels and subse­ exposure time. Methyl chloride induced patho­ quently, incubating the cultures at an air/water logic lesions were not observed in female interface. The cells can be maintained for up to rats. Supported by CIIT. 2 weeks at the interface and the cultures provide a system to expose lung cells directly to toxic fumes and gases. Nitrogen dioxide (N02) was used to test the responsiveness of the cultures to 567 INTERACTIVEEFFECTS OF JP-5 VAPOREXPOSURE AND toxic gases. Exposure to N02 resulted in cyto­ ELEVATEDTEMPERATURE ON RENALLESION INDUCTION. toxicity, decreases in protein synthesis, and 1.1. Pitts II, A.P. D'Addario, M.J. Cowan, Jr., morphological alterations similar to those found and R, H. Brunert. Naval Medical Research Insti­ in vivo, but at lower doses. Dose-response rela­ tute/Toxicology Detachment and tAir Force Aero­ tionships were determined for one-hour exposures space Medical Research Laboratory, Wright­ at room temperature to 0.5-6.0 ppm N02 in 95% Patterson AFB OH 45433. Sponsor: M.E. Andersen. air - 5% CO2 (flow rate= 2.6 liter/ minute; cham­ ber volume 5.4 liters). Time-response was deter­ Interactions between chemical toxicity and ele­ mined at 6 ppm N02 for 15-60 minutes. The cul­ vated temperatures are incompletely undP.rstood. tures were analyzed for trypan blue dye-exclusion, Exposures to fuel vapors or chemicals often occur clonogenicity, and 3H-lysine incorporation. In­ at temperatures greater than 25°C (eg. ship creasing concentrations of N02 caused linear engine rooms), We have studied the effects of

161 repeated exposure to JP-5 vapors at elevated are immediate and unequivocal, and are not ascrib­ temperatures. Male F344 rats were exposed to able to migration of stem cells between femur 2900 mg/m3 JP-5 for 6 hrs/ day, 5 day/wk, for 60 and spleen. exposure days in 30 t Leach chambers at 38°C. The 4 exposure groups were control, fuel, heat­ control, and fuel+ heat. 24 hr urine, blood and selected tissues were obtained at termination of 569 CHANGESIN PHOSPHOLIPIDBIOSYNTHETIC ENZYMES IN exposures. Urine and serum assays were divided TYPE II CELLS ANDMACROPHAGES (PAM) ISOLATEDFROM into three groups for multivariate analysis of RAT LUNGSAFTER NO EXPOSURE. E.S. Wright, M.J. variance (MANOVA): (I) Serum: CPK, AST, ALT Vang, J.N. Finkelstein and R.D. Mavis, Univ. of and glucose; (II) Urine: LDH, AST, Alk. Phos., Rochester Sch. of Med. & Dent., Rochester, NY and GGT activities;~(III) Urine volume, Sponsor: P.E. Morrow urine GGT, serum alb/ glob, creatinine clearance, and fractional urea clearance. MANOVA(I) was The activities of phospholipid biosynthesizing significant for temperature (T) and for fuel (F) enzymes and subcellular marker enzymes were meas­ effects with no interactive effect. MANOVA(II) ured in Type II cells and PAM isolated from rat and MANOVA(III) were significant for the T, F, lungs 48 hours after exposure to air or 40 ppm and interactive effects. The kidney was the only N0 • DNA and protein increased in cell fractions 2 organ affected by exposure. Kidneys from the from N0 -exposed lungs, but N0 exposure had no 2 2 rats exposed to room air both at 25 and 38°C were effect on the percent of Type II cells in isolated unremarkable. Kidneys from the rats exposed to cell fractions. A general increase in biosynthet­ the fuel at 25°C had the usual proximal tubular ic enzyme activities (units/mg DNA) was observed necrosis, but the kidneys from rats exposed to in Type II cells from NO -exposed rats, but no fuel at 38°C evidenced minimal cellular altera­ change was detected in tie activity of the micro­ tions. This lack of pathologic response with somal marker enzyme, NADPHcytochrome..£ reductase. combined exposure is consistent with the inter­ G-3-P acyltransferase and choline phosphotransfer­ active effects found in the biochemistry data. By ase increased 171 and 168%, and phosphatidate both pathologic and biochemical criteria the phosphohydrolase increased 69%. Glycerolphosphate toxic response to JP-5 may be significantly phosphatidyltransferase increased 143% and succin­ altered according to environmental conditions. ate cytochrome c reductase, the mitochondrial marker enzyme, increased 111%. PAM recovered by lavage from normal rat lungs were enriched in enzymes of PC synthesis relative to Type II cells, 568 THE EFFECT OF BENZENEINHALATION ON MURINEHEMATO­ but contained three-fold less glycerolphosphate phosphatidyltransferase activity. Exposure to N0 POIETIC PRECURSORCELLS (CFU-e, BFU-e, CfU-gm). 2 R.L. Hilderbrand, L.L. Pitts, M. Kohsaki, M.J. resulted in a significant increase in NADPHcyto­ Murphy, Jr t NMRI/TD, Wright-Patterson AFB OH chrome_£ reductase activity (46%) in PAM, but no 45433 and +The Bob Hipple Laboratory for Cancer change in any biosynthetic enzymes. The increases Research, 3525 Southern Boulevard, Dayton, OH in activity of phospholipid biosynthetic enzymes 45429. Sponsor: V.L. Carter, Jr. in Type II cells, but not PAM, is consistent with a specific stimulation of surfactant phospholipid The inhalation of benzene has been shown to pro­ biosynthesis in Type II cells 2 days after expos­ duce severe blood dyscrasias which may ultimately ure to N0 , when Type II cell proliferation is 2 include pancytopenia or leukemia. To investigate occurring. these effects assays reflecting committed stem Supported by contract with USDOEand NIH cell functions [erythrocytic colony forming units Training Grant 5T32 HL07216. (CFU-e), erythrocytic burst forming units (BFU-e), and granulocytic/macrophagic colony forming units (CFU/gm)] were used. Male BsD2F1 mice were ex­ posed by inhalation to 4000 ppm of benzene for 8 570 A SUBCHRONICACROLEIN INHALATION STUDY IN RATS: hrs a day for either 0, 1, 2, 3, 5, 7 or 14 days. I. BIOCHEMICALAND PATHOLOGICAL CHANGES. Groups of mice were sacrificed at 0900 hrs on the R.S. Kutzman, E.A. Popenoe, B.Y. Cockrell, M.A. morning following exposure for the given period. Schmaeler and R.T. Drew, Medical Dept., Brookhaven Femoral marrow was sterilely collected from 6 National Laboratory, Upton, NY and Experimental 5 femurs and pooled. Nucleated cells (2 x 10 ) as Pathology Laboratories, Herndon, VA. a single cell suspension were plated in 1 ml of a-medium with methylcellulose in quadruplicate Groups of Fischer 344 rats were exposed to cultur·es. Counting of colonies was after incuba­ either filtered air, 0.4, 1.4, or 4.0 ppm acrolein tion of 2 days for CFU-e and 9 days for BFU-e and for 62 days (6 hrs/d, 5 d/wk). Mortality was ob­ CFU-gm. Following one day of exposure, femoral served only in the 4.0 ppm chamber where 32 of 57 cellularity, CFU-e, BFU-e, and CFU-gm/plate were males died, but none of the 8 exposed females died. significantly depressed. The cellularity remained Both male and female rats in the 4.0 ppm group depressed for the duration of the exposure. On gained weight at a significantly slower rate than the other hand, CFU-e and BFU-e/plat·e were de­ the control groups. Exposure to 4.0 ppm acrolein pressed for 3 or 2 days, respectively, and then resulted in bronchiolar epithelial necrosis and rebounded to significantly higher concentrations sloughing, bronchiolar edema with macrophages, and than controls. By day 7 the concentrations of focal pulmonary edema. These lesions were on CFU-e and BFU-e were so elevated that the total occasion associated with edema of the trachea and number of CFU-e or BFU-e/femur had actually re­ peribronchiol lymph nodes and acute rhinitis. Only bounded to control values. CFU-gm/femur remained 3 of 31 rats exposed to 1.4 ppm acrolein had expo­ depressed at all exposure periods. CFU-e, BFU-e sure related lesions. The lungs of the 4.0 ppm or CFU-gm/spleen did not increase. The toxic group were heavier than those of the larger control effects of benzene on hematopoietic stem cells animals. Relative to controls there was a 20% in-

162 crease in dry lung weight, accompanied by a 1.5% Stepwise discriminant analysis was used to increase in water content. This increased dry identify those pulmonary function and biochemical weight and the absence of a significant change in parameters which best distinguished four groups of the amount of DNA and protein per unit dry weight acrolein exposed rats. This technique selected indicated that the increased lung weight of this and linearly combined a set of significant dis­ group was due to increased cellularity. Lung criminating variables which forced the exposure connective tissue content increased as a result of groups to be as distinct as possible. When com­ acrolein exposure. As a function of dry weight, pleted the effectiveness of a discriminating elastin levels were 177% of control values in ani­ function, based on these variables, was checked by mals exposed to 4.0 ppm acrolein. Significant in­ using it to classify each animal originally studied. creases were observed in the collagen concentration Groups of male Fischer 344 rats were exposed to O, in both the 1.4 and 4.0 ppm group where it was 0.4, 1.4 or 4.0 ppm acrolein for 62 days. Pulmon­ 111% and 133%, respectively, of the control group. ary function tests and connective tissue measure­ ments provided more than 25 variables used for *Research supported by U.S. Dept. of Energy under discriminant analysis. Collagen, elastin and the contract DE-AC02-76CH00016 and the National expiratory flow rate at 25% vital capacity explain­ Toxicology Program under Interagency Agreement ed 89% of the dispersion among the 4 exposure Number 222-YOl-ES-9-0043. groups. A discriminant function based on these variables correctly classified 69% of the animals. Because the 0.'4 and 1.4 ppm animals were poorly classified, each exposure group was assessed in­ 571 A SUBCHRONICACROLEIN INHALATION STUDY IN RATS: dividually with the control group. Analyses were II: ASSESSMENTOF PULMONARYFUNCTION. D.L. Costa conducted using either the biochemical measure­ and R.S. Kutzman, Medical Dept., Brookhaven Nation­ ments, the respiratory physiology data, or all of al Laboratory, Upton, NY 11973. Span: R.T. Drew the parameters. Collagen most effectively dis­ Groups of 24 male Fischer 344 rats (SPF) were criminated the individual exposure groups(with DNA exposed 62 days (6 h/d, 5 d/wk), to O, 0.4, 1.4 or in the 1.4 ppm group) if biochemical v.ariables were 4.0 ppm acrolein (ACR). Mortality was observed used. The most discriminating physiology variable only in the 4.0 ppm group (59%). Seven days post­ was different for each exposure group regardless exposure, a series of functional tests to assess of analysis used. This variation of the most sig­ pulmonary mechanics was performed on each animal. nificant parameter may be the result of distinct These tests included analyses of breathing pat­ lesions at each exposure concentration. terns, quasi-static compliance (QSC), diffusion capacity (DLC ), flow volume (FV) dynamics, and *Research supported by the U.S. Dept. of Energy 0 distribution of ventilation. At 4.0 ppm ACR, the under contract #DE-AC02-76CH00016 and NTP contract markedly depressed FV effort, the leftward shift #222-YOl-ES-9-0043. of the QSC curve, and the enlarged lung volumes suggest the predominance of obstructive airway disease. However, dynamic compliance and volume adjusted DLCOwere unaltered. Also, the unexpect­ 573 A SUBCHRONICACROLEIN INHALATION STUDY IN RATS:IV ed expeditious washout of lung N2 challenges a simple classification of the functional lesion. IMPACT ON HYPERTENSION-SENSITIVEAND RESISTANT These findings were corroborated by the focally ANIMALS. R.S. Kutzman*, R.W. Wehner and S.B. severe airway/parenchymal damage and significant Haber, Medical Dept., Brookhaven National increases in total elastin and collagen. At 0.4 Laboratory, Upton, NY 11973. Sponsor: R.T. Drew ppm, FV dynamics were enhanced without apparent histological and tissue compositional alternation. The Dahl selected rat lines, one susceptible Compared to controls, the effort-independent ex­ to salt-induced hypertension (DS) and the other piratory flow rates at 25% and 10% vital capacity resistant to salt-induced hypertension (DR) were were elevated 37% and 44%, respectively (p<0.001). subchronically exposed to filtered air, 0.4, 1.4, This restrictive lung behavior was supported by or 4.0 ppm acrolein. All of the DS rats exposed the slight (12%, ns) decrease in the tangent slope to 4.0 ppm acrolein died within the first 11 days, of the QSC curve. Those rats exposed to 1.4 ppm while 60% of the DR animals survived. Neither did not differ functionally from controls. How­ dose dependent blood pressure changes nor altered ever, occasional bronchiolar pathology and an 18% behavioral characteristics were evident following increase (p<0.001) in total collagen were observed. acrolein exposure. Surviving DR animals exposed These data suggest the development of two distinct to 4.0 ppm acrolein had increased serum alkaline dose dependent functional lesions which exhibit phosphatase (71%), SGOT (42%), and SGPT (59%) opposing effects on the measured functional levels when compared to control animals. Exposure parameters. to 4.0 ppm acrolein also led to pulmonary edema and significant increases in collagen and elastin *Research supported by the U.S. Dept. of Energy content of lung. There was a marked difference in under contract #DE-AC02-76CH00016 and NTP contract the pulmonary pathology observed in DS and DR #222-YOl-ES-9-0043. rats exposed to 4.0 ppm acrolein. The lungs of the DS rats exhibited severe airway epithelial necrosis with edema and hemorrhage, while surviv­ ing DR rats primarily showed a proliferative 572 A SUBCHRONICACROLEIN INHALATION STUDY IN RATS: change. Following exposure to 0.4 and 1.4 ppm III. APPLICATION OF DISCRIMINANTANALYSIS TO acrolein, both rat lines displayed similar patho­ IDENTIFY KEY INDICES OF PULMONARYTOXICITY. logic change. Epithelial hyperplasia and/or P. Hu, D.L. Costa and R.S. Kutzman, Medical Dept., clusters of macrophages were usually found near Brookhaven National Laboratory, Upton, NY 11973 terminal bronchiolar areas. These findings Sponsor: R.T. Drew suggest that further investigation of the physio-

163 pathologic sensitivity of the DS rat line may parameters. Significant decreases in erythrocyte elucidate a model for investigating the underlying count, mean cell hemoglobin and hematocrit and an characteristics of stress susceptible populations. increase in immature erythrocytes were observed after 10 exposures. Exposure to 87 ppm aniline *Research supported by U.S. Dept. of Energy under depressed erythrocyte, neutrophil, and platelet contract DE-AC02-76CH00016 and the National counts throughout the recovery. The organ to Toxicology Program (Interagency Agreement Number body weight ratios for the liver, spleen and 222-YOl-ES-9-0043). thymus were also significantly altered. A 14% increase for liver (87 ppm) and 48 and 272% in­ creases for spleen ratios were observed after 10 exposures to 45 and 87 ppm aniline, respectively. 574 EFFECTSOF INHALEDTOLUENE AND m-XYLENE ON These values returned to normal during the reco­ CONDITIONEDBEHAVIOR. G. David Gentry, Ronald very period. Thymus ratios were elevated 9 and W. Wood,Department of Radiation Biology""""'ari'd 30% above controls in the 87 ppm group following Biophysics, Environmental Health Sciences the exposure and recovery periods, respectively. Center, Division of Toxicology, University of These results suggest a relationship between Rochester Medical Center, Rochester, NY 14642. MetHb levels and several toxicity parameters in rats exposed to aniline. Four rats performed five days per week on a fixed interval 5 minute (FI 5) schedule of food reinforcement (i.e., the rats received sweetened condensed milk for the first lever press after 576 SUBCHRONIC TOXICITY OF MORPHOLINE IN five minutes had elapsed since the previous RATS. C. C. Conaway*, W. B. Coate, and reinforcer). After behavioral indices were R. W. Voelker. Sponsor: R.· D. Harbison. stable, the rats inhaled toluene or m-xylene *Texaco Inc., Beacon, NY 12508 and Hazle­ for two hours immediately preceding the ton Laboratories America, Inc., Vienna, behavioral sessions on Tuesdays and Fridays; VA 22180 Thursdays were sham-exposure days. The toluene The toxicity of morpholine, an important and m-xylene concentrations were ~60, 1000, industrial chemical, was investigated by 1780, and 3000 ppmgiven first in ascending, the inhalation route of exposure. Ex­ then in descending order; two rats received perimental exposures for the subchronic toluene first and the other two m-xylene study were 25, 100 and 250 ppm six hours/ first. Changes in post-reinforcement pause, day, five days/week for a total of 13 response rate, and index of curvature were weeks. Rats exposed to these doses concentration-related. Across the 100 minute demonstrated shallow, rapid breathing behavioral session, decreasing blood levels patterns. Irritant effects of morpholine were associated with both increases and exposures were evident mostly in the high decreases in response rate. The direction of exposure group, and a bloody discharge change was a function of exposure concentration: was observed around the nose and mouth in for lower concentrations there was an initial the 250 ppm dose rats after the first increase in rates; for 3000 ppm there was an week of exposure; salivation was also ob­ initial decrease followed by an increase. served. Ten rats/sex/dose level were Index of curvature showed the largest magnitude sacrificed after 7 weeks exposure. Ex­ effect for the three measures, and post-rein­ posure to 250 ppm morpholine resulted in forcement pause showed the slowest recovery. focal erosion of the maxillary turbinates This research was supported by grants and focal SQuamous metaplasia, which was DA00623, ES01247, ES05106 and DE-AC02-76EV03490 observed in 6/10 male rats and 2/10 fe­ with the Departmentof Energy. male ra~s. A sporadic increase in por­ phyrins in the Harderian gland sections was noted. Rats sacrificed at 13 weeks confirmed results observed at week 7; 575 SUBACUTE INHALATION TOXICITY OF ANILINE; THE the lesions were more advanced and in­ RELATIONSHIP BETWEENMETHEMOGLOBIN FORMATION AND volved the nasal septum and anterior TOXICITY. F. O. O'Neal, E. I. du Pont de Nemours nasal cavities. Evidence of progressive a~d Company, Inc., Haskell Laboratory for Toxico­ chronic murine pneumonia was observed in logy and Industrial Medicine, Newark, DE 19711 the 250 ppm dose groups and lower dose Sponsor: G. L. Kennedy, Jr. groups. No other compound-related histo­ morphologic alterations were observed in Male rats were exposed head-only to 0, 17, 45 and 25 or 100 ppm exposure groups. There 87 ppm aniline 6 hrs/d, 5 d/wk for 2 wks. Methe­ were no compound-related effects on body moglobin (MetHb) levels were monitored throughout weight, clinical chemistry, gross necrop­ exposure and a subsequent 13-day recovery period. sy, or organ weight data. Rats were divided into two groups and clinical and histopathological examinations performed following the last exposure and the 13th recovery day. Similar MetHb levels were observed in rats 577 REVERSIBLE ACTIVE-SITE SPECIFIC INHIBITION OF exposed to O and 17 ppm aniline. During exposure CHOLINESTERASES BY ISOCYANATES. W.E. Brown, A. to 45 and 87 ppm aniline, MetHb levels ranged H. Green, M.H. Karol and Y. Alarie, Dept. Biol. from 1.7- to 6-fold and from 7.5- to 26-fold Sci. , Carnegie-Mellon Univ. , Dept. Ind. Env. H1th. greater than controls, respectively. These Sci., Grad. Sch. of Pub. Hlth., Univ. of Pitts­ values gradually decreased to control levels by burgh, Pittsburgh, PA 15261 the 11th day of recovery. Aniline exposures of Isocyanates are highly reactive chemicals used in 45 and 87 ppm also affected other hematopoietic the manufacture of polyurethanes. Exposure to·

164 small amounts of isocyanates produces respiratory two cell types in the testis, there is not only a symptoms in innnunologically sensitized persons. difference in biochemical capability to activate Exposure to higher concentrations of isocyanates chemical agents but a structural specificity for produces similar respiratory symptoms in all selective conjugation of cytotoxic and transform­ individuals as a result of irritation. The ing reactive intermediates. [*Operated by UCC ability of isocyanates to function as inhibitors Corp. with the U.S. DOE] of serine proteases (Brown and Wold, Biochern, 12, 828, 1973) prompted investigation of the effects of industrial isocyanates on cholinesterase. Hexa­ methylene diisocyanate (HDI), 2,6 toluene diiso­ 579 IN VIVO METABOLISMOF THE ANTI-JUVENILE HORMONE cyanate (2,6 TDI) and hexyl isocyanate (HI) com­ pRECOCENEII AND THE GENERATIONOF DIHYDRODIOL pletely inhibited purified human serum cholines­ METABOLITES. M. T. Stephen Hsia and Scott J. terase at molar ratios of 4:1 to 8:1 (isocyanate Grossman, Environmental Toxicology Center and added: enzynie). Isocyanate inhibition was active­ Department of Entomology, University of site directed since known competitive cholines­ Wisconsin, Madison, Wisconsin, 53706. terase inhibitors protected the enzyme. 2,4 TDI, phenyl isocyanate and o-tolyl isocyanate showed Precocene II (6,7-dimethoxy-2,2-dimethyl-2H­ little enzyme inhibition. With these isocyanates, benzo-[b]pyran), a potent anti-juvenile hormone, ,molar ratios of 50:1 or greater were required for induces precocious metamorphosis and steriliza­ 50% inhibition. Enzyme inhibition was also tion in several insect species, and has been achieved by in vitro exposure of cholinesterase to proposed as a prototype fourth-generation 1 ppm atmospheres of isocyanates. Under these insect control agent. This compound was conditions HDI and HI were the most potent inhi­ recently found to be acutely toxic in male bitors. The inhibition of cholinesterase by iso­ Sprague-Dawley rats at intraperitoneally admin­ cyanates was reversible with a half-time of istered doses of 300 mg/kg and above. The approximately 12 hours. Maximum reversibility primary lesion was severe centrolobular necrosis occurred at pH 7.5. These results indicate that of hepatic parenchymal cells. Liver damage was isocyanates are potent inhibitors of cholines­ evidenced by the massive release of glutamic­ terase. The reversibility may explain the re­ oxalacetic transaminase and glutamic-pyruvic covery from respiratory rate depression observed transaminase into the serum. When a 100 mg/kg in animal models of sensory irritation where dose of [3H]-precocene II was administered to animals are exposed to vapors of isocyanates. rats, it was found to be rapidly metabolized Supported by the Winters Foundation and NIOSH and excreted from the body. Approximately 77% /IOH00865. of the recovered dose was cleared within the first 24 hours. Identification of the various urinary metabolites was accomplished via the use of: a) Amberlite XAD-2 column chromatography, 578 CELL SPECIFIC ACTIVATIONAND DETOXIFICATIONOF bl high pressure liquid chromatography, and c) BENZO(a)PYRENEBY TWOCLONED MURINE TESTICULAR gas chromatography-mass spectrometry. One of CELL LINES. R. Filler and L. S. Woods, Biology the most significant findings is that precocene Division, Oak Ridge National Laboratory*, P. O. II is metabolized to a stereoisomeric cis/trans Box Y, Oak Ridge, TN. Sponsor: W.M. Haschek mixture of 3,4-dihydrodiol in vivo as well as in vitro. It is thus postulated that hepato­ The interaction of environmental toxins toxicity occurs as a result of an overload of a within the male reproductive system is compli­ highly reactive epoxide intermediate formed cated by the presence of several distinct testi­ during metabolism. (Supported in part by a cular cell types. To define more clearly the USDA Hatch Grant, #2407, from the College of potential relationship between chemically-induced Agricultural and Life Sciences, Univ. of toxic affects and endogenous metabolic capability Wisconsin-Madison, and a Biomedical Research of specific testicular components to activate and Grant, #181532, from NIH.) detoxify chemical agents, we have examined two cloned cell lines identified as of the Leydig (TM3) and Sertoli (TM4) cell types. The poly­ cyclic aromatic hydrocarbon, benzo(a)pyrene 580 N-VINYL-2-PYRROLIDINONE: DISPOSITION AND METABO­ [B(a)P], was used as a probe for defining cellu­ LISM STUDIES. G.A. Digenis and J.S. Mcclanahan, lar metabolic activity. TM3 cells metabolized College of Pharmacy, University of Kentucky, B(a)P 160x more effectively than did TM4 cells. Lexington, Kentucky 40506. This difference in chemical activation was corre­ lated with major differences in the formation of N-Vinyl-2-pyrrolidinone (NVP) is the monomer 9,10-,4,5-,7,8-dihydrodiols, quinones and 9-0H­ from which polyvinylpyrrolidinone preparations B(a)P derivatives as determined by HPLC. No sig­ are made. The latter are of great pharmaceutical nificant amounts of 3-0H-B(a)P were detected. value and contain a small percentage of NVPwhose Long-term (72 hrs.) incubation of TM3 cells with metabolism and disposition is unknown. Elimina­ 5 µg/ml B(a)P did not produce cytotoxic affects. tion studies of 14c(vinyl)-NVP showed that blood Therefore, the role of two detoxification reac­ levels of the intact compound drop rapidly after tions was assessed by determining the presence of i.v. administration with an elimi~ation half life glucuronic acid conjugates and sulfate ester of 1.5 hours. Following administration (i.v.) of derivatives. Treatment of water-soluble meta­ 14c (vinyl)-NVP, total radioactivity.in the urine bolites with B-glucuronidase resulted in the of rats after 6 hours ranged from 40-65% of the hydrolysis of 4,5- and 7,8-dihydrodiols and 3 and dose, while that excreted in the bile ranged 9 phenol metabolites. Similar treatment with from 17-20% of the dose. Intact NVP in these arylsulfatase released only 4,5- dihydrodiol. cases was found to be less than 0.2~ of the dose These observations demonstrate that, for at least in the urine and 2.3% in the bile·. Of the total

165 14c-activity administered, 69-75% appeared in studies employed a novel sandwiched barrier de­ the urine in the first 24 hours. Distribution vice to prevent animal access to the site and 14 studies of c-activity in vital tissues and permit air exposure. Significant absorptio~ 4 organs of rats 6 hours after i.v. administration occurred over a 7-day period when 200 ugm [ C]­ of 14c(vinyl)-NVP showed that the liver (4.3- TNF/kg was applied to 4cm2 of skin in acetone, a 6.5% of the dose) and small intestines (3.5-5.7% trichloroethane-based cleaning fluid and water as of the dose) are the organs with the highest indicated by urinary excretion of 12%, 15%and accumulation of 14c-activity. Preliminary 3% of the applied doses, respectively. Maximum attempts to elucidate the metabolic fate of 14c­ excretion in urine occurred within the first 24 (vinyl)-NVP from urine samples showed 12% of the hr. Fecal excretion of radioactivity was about radioactivity was incorporated into acetate and 30%greater than urinary excretion in all cases, the major remaining portion of the 14c-activity with a maximal rate at 48 hr. No radioactivity into a highly water soluble compound(s). Protein was detected in expired CO. 4 2 binding studies with rat iiver homogenates indi­ [1 c]-TNF was rapidly cTeared from blood (half­ cated that no alkylating activity was associated life < 1 hr) following a single i.v. dose in DMSO. with NVP due to epoxidation of its vinyl moiety. Nearly 20%of the dose was excreted in the urine It was concluded that 14c(vinyl)-NVP ~s rapidly within 24 hr. followed by an additional 3% over metabolized and eliminated in the rat. The liver the next 6 days. High levels of radioactivity and small intestines are the organs with the were detected 1 hr after treatment in the liver, highest accumulation of 14c-activity and the kidney, abdominal fat, small intestine, and lung urine and bile are the major routes of elimina­ with lesser amounts in the stomach, large intes­ tion. tine, spleen, and testes. Supported by a contract from the IBMCorporation.

581 ENZYMATICREDUCTION OF 1-NITROPYRENEBY RAT LIVER FRACTIONS. J.P. Nachtman, Chevron Env. 583 ~tal::x:>lism of 2-rrethylnaphthalene in vitro: Hlth. Ctr., Richmond, CA 94802, and E.T. Wei, rrethyl group oxidation. M.J .~lancon &J':J.I.ech, Dept. of Env. Hlth. Sci. Sehl. Puhl. Hlth., r.ept. of Phann. & Tox., M:rl. Coll. of Wisa:msin, Univ. of California, Berkeley, CA 94720. Milwaukee, WI. Previous studies have shcMn 2-naphthoyl glycine Nitroarenes have been detected in photocopy to­ to be a major rretabolite of 2-rrethylnaphthalene ners and diesel exhaust particulates and are (2~N) in rats. Ring oxidation of ~ by hepa­ potent mutagens without metabolic activation tic rnicrosones from several species and fonnation in the Ames Test. Certain nitroarenes show S-9 of 2-naphthoic acid (2NA) from ~N by rat liver Mix dependent activity in mammalian cell cul­ rnicrosorres have been reported. In order to gain ture assays, which indicates that S-9 contains insight into the methyl group oxidation of rreth­ some capacity to activate these compounds. Rat ylated PAHs the oxidation of ~ to 2NAwas liver functions such as cytosol, microsomes and studied in rat an::l rainl::.ow trout liver horrogen­ S-9 supermatant were found to reduce anerobi­ ates(LH) and subcellular fractions via the use of cally 1-nitropyrene (1-NP) to 1-aminopyrene ~N, 2-hydroxynethylnaphthalene(2MaNOH) and 2- (1-AP). The reaction was stopped by addition naphthaldehyde (20CHO)as substrates. The harog­ of HCl. Substrate (1-NP) was extracted with enates ( 4ml .25M sucrose/g liver) were fraction­ cyclohexane, the product extracted from the ated by centrifugation. The tissue preparations alkalinized aqueous fraction in cyclohexane. were incubated with substrate(generally lrnM), 1-AP and 1-NP concentrations were determined 0.05rrM NAD, 0.05rrM NADP,15mM MgCl2, 12mMG-6-P, by spectrofluorimetry. 0.04M ro4 (pH 7.4) and 0.125M sucrose at 37°C for rat preparations an::l 22°C for trout preparations. The rate of reduction was much greater upon ad­ After incubation, analysis for ~, 2NCHOand 2- dition of NADPHand was dependent upon the NA was done by HPLCusing a C-18 colunm and elu­ amount of S-9 Mix added. Not all 1-NP added is tion with H20•CH3CN·CH3COOH(65•35·0.l) Rat Ll:I recoverable as 1-NP plus 1-AP, indicating that forrred 2NA from all 3 substrates approx. 6 times some material may exist as intermediates (-NHOH as rapidly as did trout Ll:I. The ability to me­ or -NO). These results imply that mammalian tabolize 2NCHOto 2NAwas present in the various enzymes can reduce 1-NP to potentially more fractions of rat and trout liver. With 2MaNOHas reactive species. substrate, cytosol was the rrost active fraction from trout liver an::l the activities of individual fractions accounted for total harogenate activi­ ty, while mitochondria was the nost active frac­ 14 582 THEEXCRETION AND TISSUE DISTRIBUTION OF [ cJ- tion fran rat liver an::l theactivities of individ­ 2,4,7-TRINITR0-9-FLUORENONEIN THE RAT. ual fractions did not account for total harogen­ ate activity. Treatment of rats and trout with William F. Busby Jr., Mark E. Goldman, and Gerald l0Orng/kg Aroclor 1254 ip in corn oil increased N. Wogan;Dept. of Nutrition and Food Science, the rretabolism of ~ and 2MeNOHby rat Ll:I but Massachusetts Institute of Technology, Cambridge, not by trout Ll:I. The pattern of rretal::x:>lites MA.02139 and Frank Aldrich and Thomas Smith; forrred with rat Ll:I and fractions also changed. IBMCorporation, White Plains, NY 10601. Supported by NIH Grants ES01985 & ES01080. Experiments have been performed to measure urinary fecal excretion and tissue distribution following administration of the photoconducting 584 DISPOSITION OF DIISODECYL PHTHALATE FOLLOW­ compound[14c]-2,4,7-trinitro-9-fluorenone (TNF) ING ORAL EXPOSURE IN RATS D. G. Pegg, in 275 g male Fischer rats. The skin absorption Biomedical Science Department, General Motors

166 Research Laboratories, Warren, MI 48090. Sponsor: activity had been excreted by 8 hr. With in­ J. J. Vostal creasing dose, however, a nonlinear increr~e in the maximum rate of excretion as exhaled CO 2 Diisodecyl phthalate (DIDP) is a plasticizer used in was observed. At the highest dose level, only polyvinyl chloride materials. Though extremely low in 20% of the a1~inistered radioactivity had been volatility and aqueous solubility, leaching of trace excreted as co by 8 hr. Pretreatment of 2 amounts of the ester from vinyl materials may occur · animals with disulfiram (an aldehyde dehydrogenase under certain conditions. The purpose of this study was inhibitor) resulted in a significr~t decrease in to investigate the disposition of DIDP administered the initial rate of excretion of CO , indicat­ 2 perorally, as an aid in evaluation of potential exposure ing that the two-carbon metabolism of AAO is via ~¼1:ects. Adult male Sprague-Dawley rats were used. the tricarboxylic acid cycle. ( C) DIDP dissolved in corn oil was administered by gavage in doses of 25 µmoles/kg. Within 72 hrs after dosing, 99% of the administered radioactivity was re­ covered in excreta, 66% in feces and 33% in urine. 586 METABOLICFATE OF ETHYLENEDIAMINEIN THE RAT Elimination of radioactivity _fn urine followed first PARALLELTO A TWO-YEARCHRONIC TOXICITY STUDY: order kinetics (k = 0.064 hr ). Using high pressure (I) PHARMACOKINETICSTUDY. Raymond S. H. Yang, liquid chromatog~phy (HPLC), 30% of the radioactivity M. J. Tallant, J. W. Lento and J. A. McKelvey. in urine during the 24 hr period immediately following Bushy Run Research Center, Mellon Institute­ exposure co-e1fkted with the phthalic acid standard. Union Carbide Corporation, Export, PA. The remaining C in urine eluted in a broad peak more polar than the monoester phthala te, suggesting the As part of a two-year chronic toxicity presence of oxidative metabolites of the monoester. study, the pharmacokinetics of ethylenediamine DIDP was not detected. In contrast, 56% of radio­ (EDA) was studied in Fischer 344 rats of both activity was associated with the parent compound in sexes at day zero (naive animals), 6 months feces, only traces of phthalic acid were present, and (controls and high level animals) and 18 months the remainder eluted as apparent metabolites of the (controls and high level animals). The rats, monoester phthalate. In tissues, radioactivity was which were randomized along with the rest of the detected in highest concentration in the gastrointes­ animals on the toxicity study, were taken for tinal tract, liver and kidney (85, 6.1, 0.6% of the body pharmacokinetic and metabolic studies at the burden, respectively), and was present in only trace specified time. A single po dose of 50 mg amounts in other organs within the first 24 hrs after 14c-EDA•2HC1/kg was given to each·rat and the exposure. After 48 hrs, radioactivity was below detec­ plasma kinetics was followed for a 24-hr period. tion in fll tissues except intestines, liver and kidney. Four pharmacokinetic parameters (ahsorption rate When (1 C) DIDP was administered daily for 5 days, constant, terminal half-life, area under the plateau levels in major organ tissues were attained curve and apparent metabolic rate constant) were within two days, but declined rapidly once treatment compared with respect to age, sex and dose. With was discontinued. There was no evidence of prolonged absorption rate constant, there appeared to be a retention of DIDP in tissues. dose-related difference. The rats subjected to chronic dietary EDA·2HC1 at 0.35 g/kg/day (high level animals) had a slower rate of absorption. In the case of the terminal half-life, the 585 DISPOSITION OF ACETALDEHYDEOXIME IN THE RAT. differences, if any, were not obvious. This was P. D. Unger and A. J. Salerno, Corporate Medical probably due to the variability among the Affairs, Allied Corporation, Morristown, NJ. animals. With area under the curve, there was a strong indication for age-related changes. The The disposition of acetaldehyde oxime (AAO), a older rats had higher values for area under the chemical intermediate used in the production of curve than the younger rats. The apparent insecticides, was determined in th male Fischer metabolic rate constant was derived from the rate 14 344 rat. Animals were given (1,2- C)AAO, of formation of 14co2 as a result of orally, at dose levels of 0.75, 7.5, 45, 375 14c-EDA·2HC1 dosing. The comparison of this 1 and 750 mg/kg, and the excretion of co in the constant under the various experimental con­ exhaled air, and excretion of radioactivity 2 in ditions revealed sex-related differences. The the urine and feces for up to 96 hr following female appeared to be more active metabolically. dosing was determined. Radioactivity remaining in the liver, kidneys, testes, muscle, spleen, fat and blood at the end of the experiment was also determined. Fecal and urinary excretion 587 METABOLICFATE OF ETHYLENEDIAMINEIN THE RAT accounted for 1-2.9% and 11.7 - 14.7% of the PARALLELTO A TWO-YEARCHRONIC TOXICITY STUDY: administered radioactivity, respectively, by (II) MATERIALBALANCE STUDY. Raymond S. H. Yang, 96 hr. No dose-related differences were noted M. J. Tallant, J. W. Lento and J. A. McKelvey. in the amount of radioactivity excreted in the Bushy Run Research Center, Mellon Institute­ urine and feces. Reversed-phas HPLC analysis Union Carbide Corporation, Export, PA. 14 of the urine of animals given ( C)AAO indicated the presence of at least 4 metabolites. On As part of a two-year chronic toxicity the basis of chromatographic characteristics, one study, the metabolic fate of ethylenediamine of the minor metabolites has been identified as (EDA) was evaluated in Fischer 344 rats of both a ~tic acid. The primary route of excretion of sexes at day zero (naive animals), 6 months ( 1 C)AAO-derived radioactivity was as exhaled co (controls and high level animals) and 18 months 2 accounting for 63-68% of the administered radio­ (controls and high level animals). A single po activity by 96 hr. Elimination by this route at dose of 50 mg 14C-EDA•2HC1/kg was given to the lower dose levels was rapid; at a level of each rat and material balance studies were 7.5 mg/kg nearly 60% of the administered radio- conducted for a 48-hr period. The recovery of

167 radiochemical(s) in the following parameters was dogs. Animals were exposed via head-only chambers . canpared with respect to age sex and dose: to 50 or 1000 ppm MeCl for 6 hr (rats) or 3 hr excreta (urine and feces), 14coz, major organs (dogs). Blood was sampled during and after expo­ (liver, kidney, lung and brain) and the carcass. sure, and MeCl was measured by gas chromatography. There was no apparent sex-related difference in Also, a procedure for measuring vapor uptake (in the amount of radiochemical(s) eliminated via the rats) was developed and used for measurements of excreta. However, there were indications of MeCl uptake at apparent steady state. age-related limitation of excretory capability. In both species a linear 2-compartment open The dose-related difference in excretion is model accurately described parent compound pharma­ apparently limited to the 18-month old animals in cokinetics of inhaled MeCl. Blood MeCl concentra­ which the EDA-treated rats (both sexes) had a tions (at apparent steady-state) were proportion­ higher rate of excretion in both the first and ate to exposure concentration. These values aver­ the second 24-hr periods. The expiration of aged 190, 3900 ng/g (rats), and 160, 3700 ng/g 14co 2 , an indicator of EDA biotransformation, (dogs) in animals exposed to 50 and 1000 ppm res­ may be age-, sex- and dose-dependent. In general, pectively. Post-exposure elimination of MeCl from a higher rate was observed in older, female, blood was unaffected by exposure concentration in control rats. The relatively low 14 co 2- either species, but was slightly slower in dogs expiration rate and small differences among the than rats. Uptake of MeCl at steady state (measur­ EDA-treated rats are interpreted as the result of ed in rats only) was approximately proportionate competition from non-labeled EDA in the dietary to exposure concentration; the values obtained intake. A few age-, sex- an

168 tion test, exhibited edema on histological ob­ were performed 1, 2, and 4 hrs after treatment. servation, and were found in lesser concentra­ 14c-Carbaryl equivalents in the muscle, brain, tion in lung. 3-Methylthiophene was not toxic lung, trachea, fat, and liver were essentially the in any of the tests. The metabolic reasons for. same for both routes of exposure, but residues af­ these differences remain to be elucidated. Sup­ ter 1 hr in the blood and kidney of animals ex- ported by GM-26,366. posed by inhalation were 3X the level of those from rats receiving an oral dose. After 4 hrs, the level of radiocarbon in the blood was similar for both groups, although the kidney maintained the differential observed after 1 hr. A markedly 591 EFFECT OF AMMONIAON HEPATIC MICROSOMALENZYME different pattern in disposition was evident when ACTIVITY IN THE MOUSE. J.C. Kapeghian, A.B. the remaining carcass was homogenized and aliquots Jones and I.W. Waters, Depts. of Pharmacology and radioassayed. The carcass of animals treated or­ Pharmaceutics, Sch. of Pharmacy, Univ. of Missis­ ally contained 22, 20 and 5% of the dose after 1, sippi, University, MS 38677. Sponsor: W.M. 2 and 4 hrs, while corresponding values for ani- Davis. mals inhaling the carbamate were 80, 55 and 50% ---It has been suggested that NH3 liberated of the dose. Urinary elimination of radiocarbon from urine and feces could be responsible for de­ after 24 hrs accounted for approximatley 90% of creased hepatic microsomal parameters seen in the oral or inhaled dose and, consequently, no rats housed in a dirty environment. This hypoth­ difference was noted in total body burden. How­ esis was tested following both NH4Cl administra­ ever, the data clearly show that inhalation of tion and NH3 gas exposure (dynamic) in male ICR carbaryl results in a much greater exposure of the mice. All hepatic microsomal enzyme assays were total body to the residues than does the same dose performed on the 12,500 g supernatant fraction. consumed orally. (Supported by EPA Grant# R805143). Neither acute nor repeated (6 d) NH4Cl adminis­ tration (250 mg/kg, i.p.) had any effect on hexo­ barbital sleep time, aminopyrine N-demethylase (type I), or aniline hydroxylase (type II) acti­ vity. Acute NH3 gas exposure at non-lethal levels (1350 ppm; 4 hr) had no effect on hexobar­ bital hypnosis; however, lethal concentrations 593 COMPARATIVEHYDROLYSIS AND DISPOSITION OF INHALF.J) (4380 ppm; 4 hr) increased this parameter. Acute AND ORALLYADMINISTERED CARBOFURAN-CARBONYL- 14c exposure to lethal NH3 levels (4700 ppm; 4 hr) IN RATS. M. Trono and H. W. Dorough, Graduate increased total microsomal protein and type I en­ Center for Toxicology, University of Kentucky, zyme while type II activity was unchanged. There Lexington, KY. was no inhibition of either enzyme when hepatic microsomal fractions were incubated in NH3/02 at­ Rats treated with a single oral dose of carbofur­ mospheres. Repeated NH3 (4 hr/d; 4 d) at 522, an hydrolyzed 66% of the administered radiocarbon 350 or 115 ppm reduced microsomal protein and in 12 hrs as determined by quantitation of ex­ type I enzyme but not type II; body weights were pired 14C02. Animals exposed to carbofuran va­ depressed after 522 or 350 ppm. When mice ex­ pors for 1 hr at approximately the same dosage posed to air only were pair-fed to control for level (175 µg/kg) hydrolyzed 12 times less of the body weight reductions following repeated NH3, carbamate. Approximately 18% of the single oral the microsomal effects were indistinguishable dose was eliminated in the urine in 12 hrs, while from NH3-treated animals. In no instance did non­ rats exposed to carbofuran vapors eliminated only lethal NH3 exposures cause inhibition of type I 7%. Fecal elimination of radiocarbon for both or type II enzymes (per mg microsomal protein); routes of exposure was less than 6% of the dose. therefore, our results indicate that NH3 is not Tissues from both groups were assayed for radio­ a hepatic microsomal enzyme inhibitor in mice. carbon content at 1, 3, 12, and 24 hrs after (Supported in part by NIH Grant 5T32 GM07099-05 treatment. Most tissues contained higher levels and by the University of Mississippi Research In­ of residues in the inhalation treated rats, with stitute of Pharmaceutical Sciences). the difference decreasing with time. The radio­ carbon content of the GI tract was greater in the orally treated rats at 1 and 3 hrs after expo­ sure, but at 12 hrs the GI tract of inhalation 592 DISPOSITION OF CARBARYL-14c RESIDUES FOLLOWING treated rats contained a significantly larger INHALATIONAND ORAL EXPOSURE. B. L. Roberts and (p < .OS) quantity of the administered dose (11%) H. W. Dorough, Graduate Center for Toxicology, than those treated orally (7%). Radiocarbon in University of Kentucky, Lexington, KY. the blood of inhalation treated rats decreased Preliminary studies in our laboratory showed only slightly during the 24 hrs after exposure, that nose-only exposure of rats to carbaryl (l- with 7% of the dose in the blood at 1 hr and 6% 14c-naphthyl-N-methylcarbamate) vapors for 1 hr at 24 hrs. Conversely, the levels in orally resulted in over 80% of the inhaied dose being treated rats declined from an initial 6% of the retained in the body and that the distribution dose at 1 hr to 1% at 24 hrs. The ratio of brain and excretion after 24 hrs did not differ from to blood radiocarbon was significantly higher in that of an oral dose. The present investigation inhalation than in orally treated rats, while the was conducted to determine the distribution of reverse situation occurred in the liver. Since inhaled residues shortly after exposure when body carbofuran is a potent anticholinesterase agent, burdens are greatest, and to compare the finding the greater transfer of residues from the blood with those of orally treated animals. Female to the brain suggests that an inhalation exposure Sprague-Dawley rats were administered 6 µg/kg dur­ would be more significant toxicologically than an ing a 1-hr exposure to the vapors and the same equivalent oral dose. (Supported by EPA Grant dose was given orally to other animals. Analyses No. R805143).

169 594RETENTION AND FATE OF INHALEDALDICARB IN RATS. fleeted by significant increases, following an H. H. Lysy and H. W. Dorough, Graduate Center for initial plateau, in exhaled 1,1-DCE levels after Toxicology, University of Kentucky, Lexington, KY. the first hour of exposure. Periodic blood samp­ ling from the femoral vein, upon cessation of the Aldicarb [2-methyl-2-( 14 C-methylthio)-propionalde­ 150 ppm exposures, yielded data consistent with a hyde 0-methylcarbamoyl oxime] vapors were admin­ two compartment open pharmacokinetic model. The istered to rats via nose-only exposure for 1 hr half-life during the initial, rapid redistribution and the retention, distribution, and excretion of phase was approximately 2 min, while that for the the insecticide determined. The mean retention elimination phase was 17 min. These results indi­ of the inhaled vapors (0.1 and 0.2 ppm in the air) cate that 1,1-DCE is readily absorbed systemically was 83.4 ± 6.6% and was independent of the ppm in and eliminated via the lung, and that these pro­ the air and of the minute volume of the animals, cesses are dependent upon time and intensity of which ranged from 118 to 290 ml/min. Urinary exposure. (Supported by EPA Grant No. R808282) elimination over a 24-hr period accounted for 81% of the retained radioactive dose and was the same as that of an equivalent oral dose. With excep­ 596 EFFECTOF CHLORPHENTERMINEPRETREATMENT ONTHE tion of the liver where residue levels were simi­ DISTRIBUTIONOF CHLORPHENTERMINEIN ISOLATED lar, the tissues of animals exposed to aldicarb PERFUSEDRAT LUNGS. L. S. Angevine, V. G. vapors contained higher levels of radiocarbon Lockard, H. M. Mehendale, Dept. Pharmacol. & during the first 12 hrs than those from rats Toxicol., Univ. MSMed. Ctr., Jackson, MS39126, treated orally but little difference was noted USA. after 24 hrs. The blood of the inhalation treated animals contained 5, 4, 2 and 1% of the dose after Our previous studies have shown that subacute 1, 3, 12 and 24 hrs, while for the orally dosed treatment of rats which chlorphentermine (CP) animals the maximum levels did not exceed 2% of (50 mg/kg/day, po, 7d) roughly doubles the total the dose and were undetected after the 3-hr pulmonary phospholipids and significantly en­ sampling period. Approximately 50% of the radio­ hances the accumulation of CP in perfused lungs. carbon in the urine of inhalation treated rats It was of interest to determine if the accumula­ was unextractable with organic solvent, whereas tion of CP was associated with a particular frac­ 80% could not be extracted from urine of those tion of the lung, and if the enhanced uptake was dosed orally. Although there were a number of correlated with the increased phospholipids. quantitative differences among the organoextrac­ Male Sprague-Dawley rats were treated with CP in table metabolites, the greatest difference was saline as indicated above the controls received that 5 to 10 times more of the aldicarb sulfone the vehicle only. Artificially ventilated iso­ derivative was present in this urine fraction lated rat lungs were perfused with Krebs Ringer from animals treated with the aldicarb vapors. bicarbonate buffer containing bovine serum albu­ The tissue and excretory data indicate that the min. After perfusion, lung homogenatewas sub­ detoxification of aldicarb is much less efficient jected to standard fractionation procedures. when inhaled than when ingested. (Supported by Some lungs were examined histopathologically. No EPA Grant No. R801543.) correlation was observed between increased CP up­ take and increased lung phospholipids. After 60 min of perfusion, CP was distributed uniform­ ly amongthe subcellular fractions and CP treat­ 595 THE PHARMACOKINETICSOF 1,1-DICHLOROETHYLENEIN ment did not alter this. However, pulmonary RATS DURINGAND AFTER INHALATIONEXPOSURE. C.E. macrophages obtained from CP treated animals con­ tained 8 times more CP than in controls. The in­ Dallas, F,W. Weir, J.V. Bruckner*, L. Putcha*, creased CP uptake by the lung following treat­ and S. Feldman**, Environ. Science Disc., Univ. ment coincides with the increased CP in the lung Texas Sch. of Public Hlth.; Dept. Pharmacol., Div. lavage of treated rats. The macrophages in the Toxicol., Univ, Texas Med. Sch*.; Dept. Pharmaceu­ lung tissue and lavage fluid from rats treated tics, Univ. Houston**; Houston, TX 77025 with CP were more numerous and were larger than The scientific validity of using inhalation those observed in control lungs. These results data to predict risks of toxic injury upon inges­ indicate that a closer stoichiometric associa­ tion of halocarbon contaminants in drinking water tion may be found between accumulated CP and is uncertain. In the first of a series of compara­ macrophage uptake rather than with the pulmonary tive oral/inhalation studies, the pharmacokinetics phospholipids as previously thought. (Supported · of inhaled 1, 1-dichloroethylene (I..HlCE) was studied. by HL-20622and ES-07045) Anesthetized male Sprague-Dawley rats inhaled 25, 75, 150, or 300 ppm 1,1-DCE for 4 hrs. from a my­ lar bag via a tracheostomy with a miniaturized one-way breathing valve. Periodic samples were 597 EFFECTSOF CHLORPHENTERMINETREATMENT ONRAT taken adjacent to the valve from the separate in­ ALVEOLARMACROPHAGE PHAGOCYTOSIS Lehnert, fluent air and exhaled breath streams and analyz­ B.E., Ferin, J., Dept. RBB,University of ed for 1,1-DCE content by gas chromatography. Pul­ Rochester Medical Center, Rochester, NY14642. monary retention, or% uptake, decreased over time (Sponsor: Morrow, P.E.) following initiation of exposures until equilibria Recently, one of us (J.F.) found that the were established. Percent uptake, as measured phospholipidosis-inducing agent chlorphenter­ after reaching initial equilibrium, varied inver­ mine (CPT) markedly suppressed in vivo titanium sely with the 1,1-DCE vapor concentration. Moni­ dioxide particle clearance fromthelung when toring of respiratory flow rates with integration admini'stered to rats during the first week to yield minute volumes enabled the measurement of after Ti02 aerosol exposure. In that al- cumulative body burdens of 1,1-DCE at each samp­ veal ar macrophages (AM)are held to play an ling point. Apparent saturation kinetics were re- important role in Ti02 clearance, we eval-

170 uated the effects of CPTtreatment in rats on 599COMPARISONOF THE GENOTOXICITYOF PARTICULATE AMphagocytic function in vitro using sheep red EXTRACTSOF SMOKE-FREEAND FOREST FIRE SMOKE­ blood cells opsonized with~(EA) as test POLLUTEDAIR. C. J. Viau, J.M. Lockard,* H. G. particles. Rats were administered 25 mg/kg Enoch,+ and P. S. Sabharwal,* Grad. Ctr. for B.W. and 30 mg/kg B.W. CPTon the second and Toxicol. and T. H. Morgan Sch. of Biol. Sci.,* first days, respectively, prior to AMharvest­ Univ. of Ky., and Ky. Dept. of Energy,+ Lexington, ing by lung lavage. In all cases (CPT treated KY (SPON: S. D, Harrison, Jr.). and controls), >95 % of AMretrieved excluded The genotoxicity of particulate organic mat­ trypan blue. Comparedto controls, CPT treat­ ter collected from air polluted with forest fire ment significantly decreased the percent of smoke was compared to that from air collected at cells as monolayers that were phagocytic the same site in Lexington, KY on days without (CPT-AM:35 + 8.5% vs. Cont-AM:59.7 + 5.6%; forest fire smoke. The benzene/acetone extracts p<0.001). The mean numbers of EAper-phago­ from these samples were assayed using the Salmo­ cytic cell were no different in both the CPT-AM nella reversion assay and the sister chromatid and the Cont-AM. In a newly developed EA-AM exchange (SCE) assay in cultured human lympho­ phagocytic assay system allowing particle-AM cytes. Salmonella strains TA98 and TAlOO were interaction in suspension, the percent of phago­ used with and without the addition of Arochlor­ cytic cells was significantly less for CPT-AM induced rat liver homogenate (S9). Each sample than Cont-AM: (CPT-AM:44 + 3.9% vs. induced dose-related increases in mutagenicity Cont-AM: 72 + 5.4%, P0.01). Our findings that CPTcauses smoke-free air. Similarly, on a volume basis a depression in AMphagocytic function in vitro smoky air induced 43 times more SCE in human is consistent with the notion that Ti02- -­ lymphocytes than did smoke-free air. Furthermore, clearance suppression by CPTresults, at least on the basis of particulate weight the smoky air in part, from AMfunctional impairment. extract induced 5 to 16 times more bacterial re­ versions and 21 times more SCE than did the ex­ tract from smoke-free air. These results indi­ cate that the higher mutagenicity of the smoky 598 REPEATEDINHALATION CHALLENGE OF GUINEA PIGS DIS­ air was due not only to the heavier particulate PLAYINGDELAYED RESPIRATORY SENSITIVITY. J, load of the air but also to increased mutagenici­ Stadler and M.H. Karol, Dept. of Ind. Env. Hlth. Sci., Grad. Sch. of Pub. Hlth., Univ. of Pitts­ ty of the particles. This is probably a conse­ burgh, Pittsburgh, PA 15261 quence of the higher fraction of organic-extracta­ ble matter in the smoke particles: 53% vs 5%. Delayed-onset sensitivity reactions, charac­ (Supported by contract S4529 of the Institute of Minerals Research, Corrnn. of Kentucky). terized by symptomatology commencing several hours following exposure, have been noted to occur in industrial workers. Frequently repeated episodes result in earlier onset of symptoms. The underlying pathogenesis of this allergic response 600 THE BEHAVIOROF INHALEDCARBON DISULFIDE (CS2) IN is unknown. An animal model of delayed-onset RAT BLOOD. C.W. Lam and V. DiStefano, Division pulmonary sensitivity was employed to evaluate of Toxicology, Univ. of Rochester, School of Medicine, Rochester NY. Sponsor: D. Taves these responses. To induce delayed-onset sen­ sitivity, guinea pigs were injected with Freund's It has been reported that the correlation Complete Adjuvant, then challenged three weeks between cs2 concentrations in blood and in air is later with an aerosol of tuberculin PPD. Upon poor, and that blood CS2 cannot be used as an first challenge, reactions were apparent after 6 indicator of exposure. One of the reasons for hours and reached maximum response 9-12 hours this statement may be that the CS2 is present in following inhalation of aerosol. Animals were the blood in two forms, free and bound. challenged at regular 2 week intervals with PPD Our studies were undertaken to characterize aerosol for a total of five challenges. Pul­ these two forms of CS2 in the blood of exposed monary sensitivity responses were elicited by rats. Both forms of cs2 in the blood increased each of the challenges. However, the onset of linearly with inhalation concentration. The response was earlier with repeated inhalation bound CS2 in the blood also increased linearly challenge and maximal response was frequently with time when rats were exposed to 2 mg/1 cs2 up observed 4-5 hours following exposure. The to 8 hours, Free CS2 was eliminated rapidly by a earlier response was not a result of development two exponential first order process (half-times of non-specific pulmonary hyperreactivity since 8.5 and 54.1 min); bound cs2 was eliminated control animals repeatedly exposed to PPD aerosol similarly but more slowly (half-times 2,2 and failed to demonstrate any pulmonary response. 42.5 hours). The direct proportionality of blood Serologic evaluation of experimental an:il!lals at bound cs2 concentration to the inhalation concen­ the height of responsiveness failed to indicate tration and exposure time coupled with the slow the presence of cytophilic antibodies. However, elimination of blood-bound CS2 suggest that it histologic examination of pulmonary tissue re­ may be used as an indicator for cs2 exposure. vealed mononuclear cell infiltration consistent In in vitro studies, cs2 bound to both rat and with delayed innnunologic reaction. Evaluation of human blood. The majority (85%) of blood CS2 was this animal model may provide insight into the found in the red blood cells. Plasma and hemo­ innnunologic mechanisms governing delayed-onset lysate of RBC from cs2-exposed rats were dialyzed pulmonary reactions in sensitized industrial at 4°C, There was no detectable loss of bound workers. Supported by NIEHS #ES01532. cs2 from the plasma and only a slight loss from

171 the hemolysate (9%) after 24 hours. The elution TA100,TA1535,TA1537, and TA1538 were profile of the bound cs 2 was coincident with the exposed to melamine in the presence and spectrophotometric curve of hemoglobin (540 nm). absence of metabolic activation at doses In in vitro studies, cs 2 bound to commercial hemo­ up to 5000ug/plate. Under conditions of globin, albumin and myoglobin. cs 2 also bound to the test no increase in the number of glycine, cysteine and S-methylcysteine, but not revertants/plate was observed relative to to N-acetylcysteine. This indicates that cs 2 control in any treatment group. Melamine binds to amino groups but not sulfhydryl groups. was then tested in the CHO/HGPRT forward These studies are supported by NIEHS Grants mutation assay (O'Neill, J.P.et.al.Mut 5 ES02039 and 5 ES07026. Res,45:91,1977) and in the in vitro CHO sister chromatid exchange assay (Perry, P.et.al. Nature,251:156(1974). In both of these assays, CHO cells were exposed to 601 A NON-INVASIVEMETHOD FOR REPEATEDEVALUATION OF concentrations of melamine ranging from PULMONARYPERFORMANCE IN UNANESTHETIZEDAND 600 to 1000 ug/ml with and without liver UNRESTRAINEDGUINEA PIGS. K.L. Wong and Y. Alarie S-9 activating fraction. Results of the Dept. of Ind. Envir. Hlth. Sci., Grad. Sch. Pub. CHO/HGPRT assay indicate melamine failed Hlth., Univ of Pittsburgh, Pittsburgh, PA 15261 to induce an increase in the mutation frequency above that of controls. A new method for testing pulmonary function with­ Similarily, no increase in the number of out the drawbacks of using restraint, anesthesia sister chromatid exchanges were observed or surgery was used. The effects of pneumotoxi­ in the CHO cells exposed to melamine. cants on tidal volume (VT) and respiratory rate These data clearly suggest that melamine (f) of· unrestrained and unanesthetized guinea­ does not represent a genetic risk under pigs were measured non-invasively with a whole­ conditions of exposure and use. body plethysrnograph. Since it is known that in­ creased lung flow resistance can produce a reduced ventilatory response to CO2, the co1-induced in­ 603 THE EFFECT OF SUBSTRATESAND INHIBITORS OF MONO­ crease in VT & f was used to evaluate pneumotoxi­ AMINEOXIDASE (MAO) ON HEPATIC DIMETHYLNITROS­ city. Female, English smooth haired guinea pigs, AMINE (DMN) METABOLISMAND MUTAGENICITY IN THE weighing 250-350 g and a mixture of 10% CO2, 20% AMESTEST. Lake, B.G., Rowland, I.R., Collins, 02 and 70% N2 were used. To test the applicabi­ M.A., Phillips, J.C., Cottrell, R.C., and lity of the-method, the effects of H2S04 mist on Gangolli, S.D., The British Industrial Biological VT & f and the CO2-induced increase in them, were Research Association, Carshalton, Surrey, SM5 4DS. measured in guinea-pigs before and for 5 days U.K. after an 1-hr inhalation exposure to H2S04 (MMD= Dimethylnitrosamine (DMN), a potent hepato­ 1 um) at 0, 23.5, 33.1, 40.4, 49.l or 72.7 mg/m3. carcinogen and mutagen, requires metabolic H2S04 mists did not produce any significant activation to exert its biological effects. changes in VT & f, except at 72.7 mg/m3 which in­ Although it has been suggested that hepatic DMN creased VT 2· folds 0.5 hr after exposure. How­ metabolism is catalysed by cytochrome P-450 ever, there was a concentration-related reduction dependent mixed function oxidase (MFO) enzymes, in CO2-induced increase in VT & f after H2S04 ex­ recent studies have provided evidence for several posure, with the effect inf being less dramatic pathways of DMNmetabolism. We have investigated and long lasting than VT. The effect of H2S04 on the effect of a number of substrates and the CO2-induced increase in VT also showed a con­ inhibitors of monoamine oxidase (MAO, EC 1.4.3.4) centration-response relationship with the curves on DMNmetabolism and DMN-induced mutagenicity. heing linear and slopes significantly different The metabolism of DMNto formaldehyde by rat from zero at all times in 5 days post-exposure. hepatic postmitochondrial supernatant fractions The slopes of these curves showed a significant was markedly inhibited by a number of MAO trend of decrease with time suggesting that the inhibitors (e.g. aminoacetonitrile, benzyl animals were recovering. In conclusion, this cyanide, indazole and pargyline) and by MAOsub­ method is useful in studying the development and strates (e.g. benzylamine, 2-phenylethylamine, recovery from pneurnotoxicity caused by chemicals. tryptamine and tyramine), but not by the deamina­ ted aldehyde, acid and alcohol products of the latter class of compounds. At the concentrations which inhibited DMNmetabolism the MAOsubstrates 602 Mutagenicity Testing of Melamine. and inhibitors had little effect on a range of R.W.Mast, M.A.Friedman and R.A.Finch, MFO enzyme activities. Whilst the mutagenicity American Cyanamid, Wayne,N.J. and Raltech of DMNin the Ames test was markedly inhibited by Scientific Services,Madison,WI. MAOsubstrates and inhibitors, these compounds Melamine (l,3,5-triazine-2,4,6- had little effect on the mutagenicity of either triamine) is an important industrial aflatoxin B1 or benzo(a)pyrene. These results monomer used in the production of high suggest the participation of an enzyme(s) with pressure laminate resins, molding MAO-like properties, unrelated to cytochrome compounds and resins. The only note­ P450, in the hepatic metabolism/bioactivation of worthy biological responses to melamine this nitrosamine. (Supported by the U.K. include crystalluria and bladderstone Ministry of Agriculture, Fisheries and Food). formation in chronic studies and convul­ sions in acute studies. In order to further understand its biological activity melamine was tested in a battery 604 EFFECT OF DIFFERENT EXPOSUREPROTOCOLS OF ETHYL of short term mutagenicity assays. In the CARBAMATEON SISTER CHROMATIDEXCHANGE (SCE) Ames Salmonella assay, strains TA98, FREQUENCIESIN VIVO. M. Cheng and M.K. Conner,

172 Dept. of Ind. Env. Hlth. Sci. and Bios., Grad. cycle or between the first and second cycles, Sch. of Pub. Hlth., Univ. of Pittsburgh, Pitts­ suggest that DEB's activity is confined to a burgh, PA 15261, Sponsor: Y. Alarie single cell cycle. Examination of third division metaphases following DEB treatment concurrent with The present study was undertaken to evaluate BrdU revealed near baseline reciprocal and lower the effect of timing of ethyl carl:famate (EC) ad­ than expected nonreciprocal SCE. Since an ministration relative to 5-bromodeoxyuridine approximate 3 fold elevation remaining after an (BrdU) infusion on SCE induction and persistence 8.5 hour delay between DEB and BrdU infusion of SCE inducing lesions in bone marrow (BM) and suggests that SCE inducing lesions may persist for alveolar macrophage (AM) cells of BDF1 mice. When more than one cycle, additional protocols are EC was given immediately prior to BrdU infusion, presently being evaluated. i.e. at the beginning of the 1st cell cycle of BrdU incorporation, SCE frequencies observed in 2nd division metaphases of AM and BM were 32.8 ±. 4.8 and 28.9 + 4.2, respectively. When BrdU in­ 606MAMMALIAN CELL POINT MUTATION ASSAY fusion was allowed to progress for a third cell WITH THE ANTINEOPLASTIC AMSACRINE. cycle, non-reciprocal frequencies in AM and BM R.5. Lake, E.J. McGuire, and E.:_A: were comparable to those previously described (AM, de la Iglesia Dept. of Toxicology Warner­ 32,3 + 3.3; BM, 23.0 + 2.8) and reciprocal ex­ Lambert/Parke-Davis Pharmaceutical Res. changes were elevated-(AM, 6.1 ±_ 0.7; BM, 5.8 ±_ Div., Ann Arbor, Ml 48105 0.6) over controls (AM, 1.5 + 0.07). Persistence A battery of short-term in vitro tests was of SCE inducing lesions as suggested by elevated designed for evaluating carcinogenesis poten­ reciprocal frequencies was confirmed by experi­ tial of the antineoplastic agent amsacrine, mental data following approximately a 2 cell cycle an acridinylamino anisidine derivative. Prev­ delay prior to BrdU infusion (AM, 12.2 + 1.0; BM, ious genotoxicity assays showed frameshift 9.7 + 1.2). However, SCE frequencies observed in mutagenesis at the hisC3076 locus in 2nd division cells following EC injection between Salmonella TA-1537 but no confirmed mutagen­ the 1st and 2nd cycles of BrdU infusion was higher esis in TA-98, TA-100, TA-1535, and TA-1538. than expected (AM, 39.3 + 3.1; BM, 42.3 + 3.0). In Chinese hamster ovary eel I assays, Unlike previously studied mutagens our observa­ amsacrine induced sister-chromatid exchanges tions with EC are in contrast to the general and hyperdiploid chromosomes. There are no assumption that SCE in 2nd division cells are a data available on mammalian point mutation cumulative total of those induced during both 1st with amsacrine and a V-79 Chinese hamster and 2nd cycles of BrdU incorporation. Additional I ung eel Is assay was conducted to detect experiments are in progress in an attempt to HGPRT locus mutation. Cytotoxicity was estab­ clarify these inconsistencies. (This researchwas lished at 2 µg/ml and one hour exposures supported in part by EPA Grant CR806-815-0l-2/680/ were used with 2, 1, 0.5 and 0.25 µg 10/07 and BRS 2507RR05451 from Division of drug/ml in duplicate flasks. Positive control Research Resources, DHHS). was ethylmethane sulfonate at 800 µg/ml. Lethality occurred at 2 and 1 µg/ml. A significant number of 6-thioguanine resistant mutant colonies were induced at 0.5 and 605 MULTICELLULARSISTER CHROMATIDEXCHANGE (SCE) 0.25 µg/ml. The results indicate high INDUCTIONBY DIEPOXYBUTANE(DEB) J.E. Luo, O.G. mutagen sensitivity of this assay when com­ Gotera, M.K. Conner, Depts. of Ind. Env. Hlth. pared to the dose required to induce reverse Sci. and Biostat., Grad. Sch. of Pub. Hlth. Univ. mutation in prokaryotes with a 1,000-fold of Pittsburgh, Pittsburgh, PA Sponsor: Y. Alarie separation in m1n1mum effective dose. The evaluation of the battery of tests conducted A multicellular in vivo assay was employed unequivocally render a genotoxic potential. to evaluate DEB as anSCE inducer in bone marrow Further, the mammalian eel I assays demon­ (BM) and alveolar macrophage (AM) cells of intact strate a differential effect on various genetic and in BM, AM and regenerating liver cells of endpoints and that cytogenetic lesions ob­ hepatectomized SWmice. Relative to baseline SCE served in Chinese hamster eel Is are capable levels, significant increases were induced over a of leading to specific locus mutation. The DEB concentration range of 9.7 uM/kg (1.5 x base­ correlation of this mutagenic potential in line) to 291 uM/kg ·(7. 5 x baseline). No cellular somatic and germinal eel Is in vivo has not specificities were apparent as similar dose­ been established. response relationships were obtained in all cell types. An acute i.v. injection was approximately 1.5 times more effective in inducing SCE than a 607 PRELIMINARYCHARACTERIZATION OF MUTAGENSPRODUCED comparable i.p. dose. Regardless of the route of AS A RESULT OF CHLORINATIONOF HUMICACIDS. J.R. injection, 193.8 uM/kg DEB produced similar cellu­ Meier, R.D. Lingg, K.L. Baker, W.H. Kaylor and lar responses in SW and BDF1 mice. The effect of R. J. Bull, Heal th Effects Research Laboratory, acute i.v. administration of 193.8 uM/kg DEB to U.S.E.P.A., Cincinnati, OH 45268 intact BDF1 mice prior to, at the same time as, and after initiation of bromodeoxyuridine (BrdU) Chlorination of drinking water has been infusion was examined. If SCE are a cumulative shown to produce mutagcnic byproducts in addition total of exchanges induced during the first and to the trihalomethanes. Much of the precursor second division~cell cycles of BrdU incorporation, material of these products is thought to be humic comparable mean SCE/cell levels (5-6 x baseline) and fulvic acids. Consequently, we are exploring induced in second division cells following admini­ the use of humic acids as model precursors of stration of DEB at the beginning of the first BrdU mutagcnic chemicals formed by water chlorination.

173 Determinations of mutagcnic activity were per­ 609 MUTAGENESISOF NITRO-DIBENZO-P-DIOXINS. William formed by use of a bacterial mutation assay (Ames E. White, Jr. and S. Grace Rock, Div. Molec. test) with five Salmonella tester strains. Muta­ Biol., Dept. of Biochem., University of Alabama genie activity was detectable in humic solutions in Birmingham, Birmingham, AL 35294. Sponsor: after chlorination with HOCl with strains TA98 and R.L. Mundy Halogenated dibenzo-p-dioxins are very toxic TAlOO. The addition of S-9 caused a 70% decrease compounds which are produced in side reactions in act1.v1.ty. The formation of mutagens was in the preparation of herbicides and also by in­ linearly related to humic concentration in the complete combustion. The 2,3,7,8 tetrachloro range of 0.2-1.6 mg/ml TOC and to chlorine con­ isomer has been extensively studied and found to centration in the range of 0.1-1.0 chlorinc:car­ induce certain mixed function oxidases. Reports bon molar ratios. Using a humic TOC concentration of its mutagenicity conflict, but the best evi­ of 1 g/1, a chlorinc:carbon ratio of 1:1, and an dence today indicates that it is not mutagenic. initial reaction pH of 7.0, the level of mutagen 2-Nitrodibenzo-p-dioxin was prepared by formation was measured at 2,323 net rcvcrtants/mg heating the dipotassium salt of catachol with TOC for TAlOO, and 309 net rcvcrtants/mg TOC for 2,3 dichlorobenzene in DMSO. Likewise, 2,3 di­ TA98. When different sources of the humic ma­ chloro-7-nitrodibenzo-p-dioxin was prepared by terials were used, the levels of mutagens formed heating the dipotassium salt of 4,5 dichloro­ were similar. Mutagcnic activity was not detected catachol with 3,4 dichloronitrobenzene. This when chlorination was carried out at alkaline pH compound was identical to that prepared by nitra­ (11.5). Little effect on activity was observed tion of 2,3 dichlorodibenzo-p-dioxin. after heating the chlorinated solutions to 37°C or Both nitrodioxins were mutagenic for Ames after purging the solutions to remove volatile tester strain TA1538. No S-9 fraction was re­ compounds. Raising the pH of the chlorinated quired because the bacterial nitroreductase can solutions to pH 10 resulted in a 35% loss of reduce the nitrodioxins to the active species. act1.v1.ty. These results indicate that primarily Neither dioxin was mutagenic for strains TA1535 non-volatile mutagens arc formed from the chlori­ nor TA1537. nation of humic substances which arc similar in These results indicate that the dibenzo-p­ several respects to the mutagens found in drinking dioxin ring system can bind to DNA or other water concentrates. critical targets and suggests that the lack of mutagenicity of 2,3,7,8 TCDD and other halo­ genated compounds may result from lack of meta­ 608 DETECTION OF MUTAGENICITYIN HUMANURINE. bolic activation and not from improper binding. L. Recio, H.G. Enoch, M.A. Hannan. Graduate Cent­ er for Toxicology, Ky. D.O.E:, McDowell Cancer Network, Lexington, Ky. Sponsor S.D. Harrison. 610 GENOTOXICITYEVALUATION OF A PYRIDOQUINA­ Yamasaki and Ames(PNAS 74:3555,1977) reported the ZOLINE TOPICAL ANTIALLERGICAGENT. mutagenic activity of XAD-2 urine concentrates R.S. Lake, D. Brusick, and F.A. de la (U.C.) from cigarette smokers in Salmonella .!.Y.E.!:!l:_­ Iglesia Dept. of Toxicology, Warner-Lambert/ murium. The same techniques may be used to monitor Parke-Davis Pharm. Res., Ann Arbor, human exposure to various mutagenic/carcinogenic MI and Dept. of Molec. Toxicology, Litton substances. In this connection we felt that fur­ Bionetics, Kensington, MD ther studies on the following would be useful: 1, Pyridoquinazolines may be useful in pre­ Dose-response relationship (number of cigarettes venting local allergic reactions due to mast­ smoked versus mutagenic activity), 2. Persisten­ eel I mediator release. In vitro bioassays ce of mutagenic activity in smokers' U.C. after were applied to evaluate the genotoxic poten­ cessation of smoking,3. Possible interaction(s) tial of (2-methy 1-11-oxo-11 H-pyri do [2, 1-b ]­ of smoke components with other substances, 4. qui nazol ine-8-carboxyl ic acid), the archetype Possible geographical variations in urine mutagen­ compound of this series. Salmonella icity. We studied the above using cigarette smok­ typhimurium plate incorporation assays run ers and nonsmokers from Lexington,Ky. and nonsmok­ at 1 to 1000 µ g/plate in the 5 standard ers from Ashland, Ky. A standardized protocol was tester strains and ·mitotic gene conversion used to assay the mutagenic activity of cigarette assay in yeast strain D4 revealed no micro­ smokers' and nonsmokers' XAD-2 U.C.s in Salmonella bial mutagenicity. Mice rece1v1ng 500 mg/kg typhimurium TA98. We found that there was no po exhibited no significant mutagenic activ­ dose-response relationship in a population of 16 ity of urinary metabolites. Sister-chromatid different smokers while a clearly postive dose­ exchange (SCE) in mouse lymphoma L5178Y response was observed in an individual smoker and in hamster eel Is was increased in the studied. Secondly, we found that mutagenicity metabolic activation phase and nonactivation though gradually decreasing can be detected in a phase, respective I y. The magnitude of these smoker's urine even a week after the person stop­ effects was small (less than 1.6 fold over ped smoking. In combination with the aromatic a­ controls) and not consistently dose related. mine,2-aminoanthracene, cigarette smokers' U.C. The consequences of this SCE inducing­ showed a synergistic effect on mutagenicity. In capacity was studied in V79 hamster lung addition, we observed a significant difference in eel Is for mutation at the HGPRT locus. At the mutagenicity of non-smokers' U.C.s (control) toxicity limited doses ranging from 18 to 75 from the two different areas. The significance µg/ml, which induced SCE in the previous of these findings will be discussed in relation to assays, there was no i nducfion of 6-th io­ the application of body fluid assays for detecting guan i ne resistant mutants. Since antiallergy occupational exposure to mutagens/carcinogens.This compounds exert effects on the eel I work was supported by ARC contract No. 79-207, C0- membrane, the weak SCE-inducing capacity 7162-79-1-302-0917 may be non-specific and attributable to var-

174 ious degrees of bromodeoxyuridine interaction (Hales and Jain, Biochem. Pharmacol. 29,256, 1980). with DNA in the presence of drug. Semi­ Since our studies of chloroallyl oomp"mii:tds as quantitative probabilistic analyses indicate Salmonella mutagens have demonstrated PB-induced minimal intrinsic genotoxic potential comple­ microsomal activation (Loper et al, EMS 12th Meet­ mented by the lack of systemic toxicity in ing, Pl2, p.56, 1981; manuscriptin prepn.), we experimental animals. tested metyrapone as an inhibitor using these and several control carcinogens. Metyrapone alone was neither toxic nor mutagenic (TA98, TAlOO, TA1535); it gave a dose-dependent inhibition of mutagenesis 611 GENOTOXICITYEVALUATION OF AMSACRINE. of aflatoxin B1 (up to 4-fold in TAlOO), triallate R.S. Lake, C.L. Heifetz and F.A. de la (6-fold, TA1535, and sulfallate (7-fold, TA1535), Iglesia, Dept. of Toxicology, Warner-Lambert/ and a dose-dependent enhancement of mutagenesis of Parke-Davis Res. Div., Ann Arbor, Ml 48105 benzo (a) pyrene (5-fold, TA98), 2-acetylamino­ fluorene (3-fold, TA98)., and 2-aminoanthracene Acridinylamino derivatives constitute com­ (8-fold, TA98). The S-chloroallyl thiocarbamates, pounds with antineoplastic properties. Compar­ triallate and diallate, have been proposed to ative genotoxicity and hence carcinogenic form proximate mutagens by a similar mechanism potential of 4'-(9-acridinylamino) methane­ initiated by sulfoxidation (Schuphan et al, Sci­ sulphon-m-anisidide (amsacrine) was studied ence 205, 1013, 1979). we have shown both to be in several bacterial and mammalian eel I in preferentially activated by PB-induced S9. How­ vitro assays. In a Salmonella plate incorpo­ ever, while metyrapone inhibited triallate muta­ ration assay, reverse mutation was observed genesis, diallate mutagenesis was neither inhib­ only at 269 µ g/plate in the nonactivation ited nor enhanced. Supported in part by EPA grant phase in frameshift tester strain TA-1537. CR806872. L. o. is supported by NIEHS Toxicology In a preincubation modification of this Training Grant ES07073. assay, significantly increased revertant counts were obtained in TA-1537, TA-1535, and TA-100 at a cytotoxic dose of 672 µ g/ plate without 59. In the presence of 59, mutagenic activity was manifest only with TA-1537, indicating that potential base sub­ 613 Time Course of In Vivo Induction of Sister stitution activity of amsacrine was prevented Chromatid Exchange by Ethylnitrosourea and more readily than its frameshift activity Methylnitrosourea. James L. Charles, Richard A. specific for TA-1537. At these high cytotoxic Carchman, David Kram and Joseph F. Borzelleca, doses, a significant portion of presumed Dept. Pharmacol., Medical College of Va., revertants did not grow in histidine-deficient Richmond, VA 23298 medium and could have represented pheno­ copies. By contrast, doses as low as 0.02 The time course for induction of sister µg/ml in culture medium without 59 induced chromatid exchanges ( SCE) in bone marrow cells a greater than fivefold increase in sister­ was studied in mice administered either methyl­ chromatid exchange (SCE) rate and chroma­ nitrosourea (MNU)or ethylnitrosourea (ENU). soma I damage frequency in Chinese hamster Intraperitoneal injection of MNU, 50 mg/kg, at ovary eel Is in vitro. SCE response was more intervals up to 38 hours prior to and 19.5 hours sensitive to 59 detoxification than the chromo­ after 5-Bromo-2'-dioxyuridine (BrdU) pellet im­ somal aberration frequency. The wide differ­ plantation resulted in an induction of 10.1 (SEM ences between the effective dose of amsacri ne = 0.6) SCE per cell at 2 hours post BrdU implan­ in Salmonella and mammalian cell assays il­ tation with a reduced response at time points lustrated the relative insensitivity and before or after BrdU implantation. Baseline SCE perhaps lack of utility of bacterial muta­ was 3.6 (SEM = 0.4). These results indicate genesis assays in predicting the mutagenic/ that the DNA damage induced by MNUas reflected carcinogenic potential of acridinylamino com­ by an increased incidence of SCE is repaired by pounds. bone marrow cells in approximately 2.8 cell cycles and the biolgocial half-life of the re­ pair is 18 hours. Based on the repair phase data one can predict the SCE results for MNU ad­ ministered post BrdU implantation. 612 METYRAPONEENHANCES MICROSOMAL-DEPENDENT MUTAGENE­ The time course of S~E induction following SIS. L.M. Distlerath, J.C. Loper, and M.W. Tabor. ENU (50 mg/kg) administration is more complex Dept. of Environ. Hlth. and Dept. of Microbiology, with at least two distinct repair phases appar­ Univ. of Cincinnati Med. Ctr., Cincinnati, Ohio ent. The maximum induction of SCE at 2 hours 45267. Sponsor: c.c. Smith after BrdU implantation is 10.9 (SEM = 1.2). A Metyrapone ( 2-methyl-l, 2-di-3-pyridyl-l-pro­ relatively slow repair phase is evident from 4 anone), an inhibitor of adrenal steroid hydroxy­ to 0.5 cell cycles prior to BrdU jmplantation. lation, is used clinically in assessment of pi­ A second and faster repair phase is apparent tuitary-adrenal function and in treatment of cush­ from 0.5 to O cell cycles prior to BrdU implan­ ing' s syndrome. Metyrapone has been shown to in­ tation. ~he biological half-life for fast re­ hibit several microsomal enzyme activities and to pair is approximately 3 hours and that of the stimulate the hydroxylation of acetanilide and slow repair is approximately 39 hours. aniline. The presumed mechanism of inhibition of These results imply that a protocol utiliz­ drug exidation is binding to one or more pheno­ ing a fixed time point for compound administra­ barbital (PB)-inducible forms of cytochrome P450. tion post ErdU may give inadequate and/or mis­ Metyrapone has been used as an inhibitor in micro­ leading results in SCE assays. somal-mediated Salmonella mutagenesis assays ( (Supported by EPA Grant 808861010)

175 614 TALINPROTEIN SWEETENER: TOXICOLOGY AND SAFETY at low doses. Also shown is that the absorption ASSESSMENT:J.D.Higginbotham 1 , D.J.Snodin 1 , and of methanol is significantly increased in the J.W.Daniel 2 . 1Tate and Lyle Group Research and presence of 5% gasoline by almost a factor of 3, Development, Reading and 2 Life Science Research, indicating an increased possibility of methanol Stock, Essex, U.K. toxicity in the presence of gasoline. Talin protein is obtained by aqueous extraction of the fruit of Thaumatococcus daniellii indigenous to West Africa. It is 2500 x sweeter than sucrose, is non-cariogenic and has the 616 THE EFFECT OF DIISOPROPYLFLUOROPHOSPHATE(DFP) ON property of enhancing and extending many SERUMCALCIUM AND MAGNESIUM. W. E. Ridder and flavours. The extract contains several related R. B. Forney, Department of Pharmacology and sweet proteins (Thaumatins) of known amino-acid Toxicology, Indiana University School of Medicine, composition and sequence. They contain a normal Indianapolis, IN. complement of amino-acids apart from histidine and there are no unusual peptide linkages or end­ Three doses (s.c.) of DFP which caused convul­ groups. The digestibility of Talin protein in sions in a group of male Wistar rats were selec­ rats is equal to that of egg-albumen but has a ted. Each dose was administered to paired groups lower biological value reflecting the absence of of fed and fasted animals. Serum magnesium and histidine. Sub-acute studies in rats and dogs calcium concentrations were determined in animals have shown the 'no-effect level' to be in excess that convulsed. Mg increased but Ca did not. of 2 g/kg/day. This dose was non-teratogenic The increase was not dose related. In another when administered to pregnant rats from Days 6-15 experiment, a convulsant dose of strychnine was of pregnancy, and did not induce dominant lethal administered. Both Mg and Ca increased but Mg/Ca mutations in male mice treated on 5 consecutive did not. This different response may be related days. to the different mechanisms of convulsant action A double-blind cross-over study in which human of the two drugs. volunteers received either 100 mg Talin protein/ day or lactose for 14 days showed no immunologi­ cal reaction as judged by the absence of an ana­ phyllactic antibody response. The absence of sensitisation was confirmed in a double-blind 617 ANALYSISOF HEMOPERFUSIONFOR THE TREATMENTOF study in which 50 volunteers consumed 15 g chewing PARATHIONTOXICITY. D. Eigenberg, T. Pazdernik gum/day containing either 150 ppm Talin protein or and J. Doull, Dept. of Pharmacology, Univ. of placebo for 28 days. Clinical examination further Kansas Med. Ctr., Kansas City, KS 66103. revealed that the protein was non-irritant to the buccal mucosa. It may be concluded, therefore, Organophosphate (OP) insecticides are frequently that the use of Talin orotein either as a high­ involved in human poisonings. Hemoperfusion is density sweetener or a flavour enhancer in food a purported option for treatment of severe and pharmaceutical products will not be hazard­ poisoning cases when conventional atropine and ous. pral idoxime therapy fails. Rats dosed with 3 mg/kg of parathion I .V. were hemoperfused with a cartridge containing 10 g of XAD-4 resin; heme­ perfusion was initiated 30 minutes after injec­ tion. Ninety minutes and five hours after 615 SKIN ABSORPTIONOF METHANOL.R. Lindenschmidt, dosing, blood was taken from the inlet and M. Elbeera, and R. B. Forney, Department of outlet sides of the cartridge. Five hours after Pharmacology and Toxicology, Indiana University dosing, rats were sacrificed and the brains and School of Medicine, Indianapolis, IN. livers were excised and frozen until analyzed by Recently, methyl alcohol has received substantial gas chromatography. Tissue clearance of para­ attention as a fuel source, either alone or in thion and the total amount of parathion removed combination with petroleum products. The scope were calculated. Parathion was effectively of this study was to determine the skin penetrat­ cleared from the blood, but the level of para­ ing ability of methanol alone and in combination thion in the brain and liver was not altered by with gasoline. Male albino rats had a 4 x 10 cm hemoperfusion. The total amount of parathion area of skin on the dorsal and mid-lumbar region removed was less than 1% of the dose administered. clipped free of hair. The exposed area was cov­ The ability of hemoperfusion to protect against ered by.a gauze pad and the test substance ap­ parathion lethality was examined. After orally plied. This area was covered by a polyethylene administering an LO 99 dose of parathion to sheet to prevent evaporation. Each test animal rats, hemoperfusion with a cartridge containing was caged with a control animal to detect the 10 g of XA0-4 was begun. Hemoperfusion neither possibility of methanol absorption by inhalation. protected against lethality nor delayed the time Methanol doses ranging from 1.442 to 13.606 gm/kg to death. Kinetic studies indicate that para­ were administered with blood samples taken every thion is rapidly distributed to tissues (t, = 30 min for the first 4 hr and then at 24 hr. The 14 min.) and the volume of distribution is 2 ~arge blood was analyzed for methanol by head space gas (VD= 45 1/kg). Because of the kinetic proper­ chromatography. The effect gasoline has on the ties of parathion, hemoperfusion is unable to skin absorption of methanol was also examined. significantly reduce the body burden of para­ Various concentrations of leaded regular gasoline thion. It is concluded, at least in rats, that were added to methanol and then applied to the hemoperfusion is not an effective mode of skin, with blood samples being taken every 30 min therapy for parathion toxicity. (Supported by for 4 hr. This study shows that methanol was de­ NIH Grant ES-07079 and U.S. Army DAMD17-78-C- tected in systemic circulat.ion very rapidly, even 8039)

176 618 FACTORSCONTRIBUTING TO THE DIFFERENCE IN ACUTE 620 EVALUATIONOF THE RABBIT AS ANALTERNATE MODEL TOXICITYOF CHLORPYRIFOSAND METHYL CHLORPYRIFOS. FORMEASURING THE ACUTE ORAL TOXICITY OF PESTI­ L.G. Sultatos, L.G. Costa, C. Wang, and S.D. CIDEGRANULAR FORMULATIONS. S. E. Hastings, G. ~. Div. of Tox., Univ. of Texas Medical P. Zwicker, M.w. Sauerhoff. Stauffer Chemical ~ at Houston, Houston, TX 77025. Company,Environmental Health Center, Our previous studies have established that Farminqton, CT the 12-fold greater toxicity of chlorpyrifos (0,0- The rabbit was investigated as an alternate diethyl 0-3,5,6-trichloro-2-pyridinol phosphoro­ animal model for evaluating the acute oral thionate) (CPS) compared to its dimethyl analog, toxicity of intact pesticide granular formul­ methyl chlorpyrifos (MCPS),can only partially be ations. Trithion® Technical (Fig 1) is an explained by extensive glutathione-mediated detox­ organophosphorous insecticide-acaracide which ification of MCPS.The present study identifies may be prepared in granular formulation for additional factors which contribute to this dif­ crop-field application. While the technical ference. Recovery of brain acetylcholinesterase grade Trithion has a purity of 90.5%, the 20G (AChE)and carboxylesterase (CarE) from inhibition formulation product contains 20.9% active in vivo was faster in mice that received CPS (70 ingredient. In contrast Trithion Technical, a mg/kgf°"than in mice given an equitoxic dose of liquid, is easily administered to animals by MCPS(lOOOmg/kg). This faster recovery following gavage to determine toxicity, but reliable CPS probably reflects the more rapjd elimination toxicity testing of the intact granular and lower tissue levels of CPS, since brain levels formulation is more difficult since for many of MCPSwere 40-50 times higher than those of CPS. technical reasons it cannot be readily Furthermore, no CPSwas present 48hr following administered directly to rats. Also, if one administration whereas MCPSwas measurable until considers administration of granular material 7 days after administration. Neither oxon could in gelatin capsules to rats there are definite be detected at any time point. Additionally, the limitations on the amount of granular material tl/2 for mouse brain AChEreactivation in vitro that can be administered conveniently. This was 30 min fo 11owing i nhi bi ti on by MCPS-ox~ study was divided into three parts. A (MCPO),but was 8hr after inhibition by CPS-oxon feasibility study was first conducted in which (CPO)oMoreover, after inhibition by MCPOAChE it was shown that intact qranules in gelatin eventually returned to control values, while CPO­ capsules were administered to rabbits. The inhibited AChEonly returned to 50-55%of control second part involved a comparison of the levels. The concentration of CPOrequired to in­ sensivity of both rats and rabbits to oral hibit 50%of the AChEactivity of mouse brain doses of the technical material. In the third homogenate (IC50) was 3.6nM, while that for MCPO part of the study, data were evaluated to was 1733,5nM. This substantial difference in IC50s compare the toxicity of the qranular formulation and the rates-of reactivation of inhibited AChE and the non-granular technical material in probably contribute to the marked difference in rabbits to quantitate the reliability of pre­ acute mammaliantoxicity of CPSand MCPS.(Sup­ dicting granular product toxicity based on ported by grants ES01831and ES05223from NIEHS.) technical material toxicity.

619 RIBAVIRIN: A SUBACUTETOXICOLOGICAL EVALUATION IN RATS AND DOGS. F.E. R:eno, J.A. Trutter, and 621 THE EFFECT OF TIME OF DOSING ON CHEMICALTOXICITY. R.W. Voelker, Jr., Hazleton Laboratories America, R. Luthra, G. Kyle, and J.V. Bruckner, Dept. Vienna, VA., and G.L. Wannarka, P.G. Canonico, Pharmacol., Div. Toxicol., Univ. Texas Med. Sch., and M. Kastello, U.S. Army Med. Res. Inst. of Houston, TX 77025 Inf. Dis., Ft. Detrick, Frederick, MD. Although it has been well established that many Ribavirin (l-S-D-ribofuranosyl-1,2,4-triazole-3- physiological processes exhibit diurnal rhythms in carboxamide) is a nucleoside that has shown man and animals, this is rarely considered when efficacy against high hazard fevers. As part of assessing toxicity. These studies therefore were a general safety testing program, ribavirin.was initiated to determine whether the time of dosing evaluated for subacute toxicity in rats and dogs significantly influences the actions of a classic following oral administration to rats and dogs hepatotoxin,carbon tetrachloride (CCl4) and a for 28 days. Dosage levels were 30, 60, and 120 nephrotoxin, mercuric chloride (HgCl2), Adult male mg/kg/day in rats, ·and 15, 30, and 60 mg/kg/day S.-D. rats were dosed orally with CCl4 (0.05, 0.25, in dogs. In the rat the principal effect noted 0.5 ml/kg) and by ip injection with HgCl2 (1 mg/kg) was a decrease in circulating red blood cell mass during their active cycle (6 pm-6 am) and sleep that was observed to some degree in all dose cycle (6 am-6 pm). Hepatotoxicity was measured by groups. Histopathologically, lymphoid depletion increases in the enzymes ornithine carbamyl trans­ was observed among the high dose animals. In the ferase, arginine succinate lyase and sorbitol de­ dog mortality was produced among the high dose hydrogenase in serum. Significant increases over animals. All dose groups exhibited degrees of controls in the levels of these enzymes were ob­ enteritis; anorexia, body weight losses, and served in animals treated with 0.05 ml CCl4/kg at physical deterioriation were apparent at the two 8 pm. When CC14 was given at 8 am, 0,25 ml/kg was highest doses. Clinical laboratory studies required to produce significant increases in levels revealed evidence of non-regenerative anemia in of the enzymes. Similarly, dosing with HgC12 at the two highest dose groups, and leukopenia in 8 am produced maximal decreases in renal cortical the high dose group. Histopathological evalua­ transport of organic ions at 24 hr; after HgC12 tion of tissues revealed bone marrow hypoplasia, dosing at 8 pm, maximal decreases in transport lymphoid depletion in the thymus, gastric were not seen until 48 hr post-dosage. Alkaline cytoplasmic vacuolation, and increased pigment phosphatase and maltase appeared in greater quan­ in the spleen in the two highest dose groups. tities in the urine sooner when HgC12 was given at

177 8 pm. Rhythmic changes intoxicant tissue disposi­ with hematology, blood biochemistry, absolute tion and metabolism, as well as glutathione (GSH) and relative organ weights and pathology para­ levels may explain these observations. Maximal meters. No overt signs of toxicity or behav­ levels of GSH were found at around 6 am in the ioral abnormalities were observed among the liver, and at 3 am and 3 pm in the kidney. The rabbits. There were no mortalities and no findings in this study indicate that animals may significant adverse effects in the low dose and be more sensitive to chemicals when exposed during control rabbits. Two female rabbits from the their active span. This is most representative of high dose group and two female rabbits in the human exposure situations, since man commonly en­ 50 mg/kg/day group died during the course of the counters chemicals during his waking hours. study. Males treated with the highest dose (Supported by EPA Grants R808282 and CR807449 and (100 mg/kg/day) exhibited reduced rate of NIEHS Training Grant ES07090) weight gain and lower food consumption. No significant treatment related effects were seen in the blood biochemistry, urine and hematolo­ gical parameters of control and treated animals. 622 HEAT PRECIPITATION OF ACID SOLUBLECOLLAGEN AS Histopathologic examination of organs or tissues A SCREEN FOR SEVERE CORNEALOPACIFACIENTS, S. J. from both control and AMA treated animals did Williams, E. I. du Pont de Nemours & Co., Inc., not reveal any significant chemical related Haskell Laboratory for Toxicology & Industrial microscopic alterations. The animals of the Medicine, Newark, DE 19711 Sponsor: G. L. satellite group appeared fully recovered Kennedy, Jr. following the 28-day exposure to AMA. The data of this study suggest that the normal industrial An in vitro screen to detect severe eye irritants handling of AMAshould not present any signifi­ prior to in vivo testing could avoid production cant health hazard, however, proper care must of irreparable damage to the rabbit eye. Since be taken to avoid direct contact with the skin permanent corneal injury is the most insidious because of the high rate of AMAabsorption form of chemically induced eye damage, a model through the skin of the rabbit. matrix of collagen and glycosaminoglycan, the most abundant biochemical entities in the cornea, was employed to detect chemical damage. Twelve water soluble chemicals, 7 with known ability to produce corneal opacity and 5 inactive in the 624EFFECT OF AGE OF HENS ON THEIR SENSITIVITY TO cornea, were prepared at 10% concentrations. DELAYEDNEUROTOXICITY INDUCED BY A SINGLE ORAL Zero, 0.01, 0.05 or 10.0 mL of test solution was DOSE OF TRI-0-TOLYL PHOSPHATE. M.B. Abou-Donia, mixed with 3 mL of collagen solution (0.1% acid­ H.M. Makkawy, A.E. Salama and D.G. Graham. Depart­ soluble calf skin collagen, 0.2% chrondroitin ments of Pharmacology and Pathology, Duke Univer­ sulfate in 0.05Mtrisbuffer with 0.2M NaCl) in sity Medical Center, Durham, NC 27710. separate cuvettes. The test mixtures were A single oral dose of 750 mg/kg tri-o-tolyl phos­ warmed for 2 minutes at 37° and transferred to an phate (TOTP) was administered in gel~tin capsules isothermal spectrophotometer with% transmittance to six groups, (five each) of leghorn hens at dif­ (540 mm) read every 2 min for up to 80 min. In ferent ages i.e., 2, 3, 6, 12, 18, and 30 months. controls an opaque, semi-solid gel forms with a Controls consisted of five untreated hens from T-1/2 to maximum of 20 min. With test compounds, each age group. After dosing, the hens were ob­ the gelation kinetics may be altered. All 5 served for 21 days. The results showed that de­ compo1,111dsknown to be without effect in vivo had velopment of TOTP-induced delayed neurotoxicity no effect on gelation. Of the remaining 7 was age dependent. While the youngest group (2 chemicals known to be opacifacients, 5 exhibited months) did not differ from control hens, all positive effects while 2 were inactive. Thus, other age groups developed delayed neurotoxicity. the results from this in vitro test concurred ---- Four hens of the 3-month group developed gross in 83% of the cases with the known in vivo --- ataxia, while the fifth hen progressed to paraly­ toxicity of each compound. These preliminary sis. Among the 6-month hens, three showed severe results will be expanded by broadening the range ataxia and two developed paralysis. The groups of of chemicals studied and by prospective studies 12 month old and 18 month old hens each had 3 hens of new compounds. with paralysis and two with ataxia with near para­ lysis. All 30-month old hens developed paralysis. Histopathological examination showed degeneration of the axons and myelin in the spinal cord and 623 SUBCHRONICDERMAL TOXICITY OF ALLYL peripheral nerves. Severity of histopathological METHACRYLATEIN RABBITS. W. H. Siddiqui, and changes in nervous tissues paralleled that of the E. J. Hobbs, Toxicology Dept., Health and Env. clinical condition. Plasma butyrylcholinesterase Sci. , Dow Corning Corp. , Midland, MI 48640. (BuChE) activity in control hens varied inversely with age. Control hens in the 2-month age group This study was designed to assess the toxicity had the highest plasma BuChE activity while the30- potential of Allyl Methacrylate (AMA) follow­ month old hens had the lowest BuChE activity (43% ing repeated dermal applications in rabbits of the first group). This pattern also held true for 28 days. Four groups of rabbits were for the 10TP-treated hens. In addition,the young­ treated with the test material at dose levels er treated hens recovered plasma BuChE activity, of 0, 25, 50 and 100 mg/kg/day. One satellite while the older hens had a partial recovery. These group of six male and six female rabbits was results suggest that plasma BuChE may play, at also treated with 100 mg/kg/day of AMA. Mor­ least in part, an important role in the insensiti­ tality, behavioral reactions, growth and food vity of young chickens to delayed- neurotoxicity. consumption were observed and measured along (Supported in part by NIEHS Grant No. ES02717).

178 625 EFFECTOF LUNG,LIVER AND KIDNEY TOXICANTS ON at 18 months did not show any bladder calculi and RESPIRATORYRATES OF MICE. P.J. Hakkinen*, R. treatment related gross lesions. Histopathologi­ Frankel+, M. Harden, L.A. Balogh*, and H.P. cal evaluation of tissues of 10 animals of each Witschi. *univ. of Tenn-Oak Ridge Grad:-Sch Bio­ sex at the control and high dose groups necropsied medical Sciences and Biology Division, Oak Ridge at 18 months showed changes of nephrosis in kid­ National Laboratory, Oak Ridge, TN 37830 neys of control and treated groups, and a few thy­ Studies by Travis et al (Radiat.Res. 84: 133, roid and pituitary tumors in the control as well 1980), and Collis et al (Br.J.Cancer 41: 901, as the treated animals. The incidence and severi­ 1980) have shown that changes in the respiratory ty of these lesions in the high dose males and fe­ rate (RR) of mice correlated well with histologi­ males are not statistically different from the re­ spective control groups. Clinical and pathologi­ cal changes for both early and latex-ray- and cal evaluation of all animals which died during cyclophosphamide-induced lung damage. In the the course of the study or necropsied at termina­ present studies, mice were exposed i.p., i.t. or tion of the treatment period is in progress. by inhalation to the lung toxicants, butylated ·hydroxytoluene (BHT)± 70% oxygen (02) ± 200 rad thorax x-irradiation, bleomycin ± 80% 02, cyclo­ phosphamide ± 80% 02, cadmiumchloride aerosol, 627 EFFECTS OF RIBAVIRIN ON BLOODAND BONEMARROW OF 3-methylfuran, methylcyclopentadienyl manganese MONKEYS. M.D. Kastello, T.M. Cosgriff, P,G. tricarbonyl, beryllium sulfate; the kidney toxi­ Canonico, G.L. Wannarka and C.T. Spears. Dept. of cant, mercuric chloride; the liver toxicant, car­ Antiviral Studies, U.S. Army Medical Res. Inst. of bon tetrachloride; or were starved. Time course Inf. Dis., Ft. Detrick, Frederick, MD. 21701 studies of the RR changes measured with a total Sponsor: F. Reno. body plethysmograph found that each lung toxicant produced a distinctive change in RRwith differ­ Ribavirin (Virazole, l-S-D-ribofuranosyl-1,2,4- ent days for onset of changes and for peak RR. triazole-3-carboxamide) is a potent antiviral drug Studies with BHT± 200 rad found that the RR that is effective against Lassa fever. Rhesus right before sacrifice (2 weeks after BHT)corre­ monkeys and man develop anemia during high-dose lated well (r = 0.97) with the degree of pulmon­ treatment with ribavirin. The mechanism by which ary fibrosis as measured by changes in hydroxy­ the anemia occurs may involve RBC destruction, proline content. Liver and kidney toxicants and suppression of hematopoiesis or both. The effect starvation on the other hand produced decreases of ribavirin on blood and bone marrow was measured or slight increases only in RR. These studies in monkeys injected IM with ribavirin for 10 days suggest that RRmeasurements in mice allow non­ at a dose of 30 or 100 mg/kg/day. Both groups invasive quantitation of the development and res­ developed a normochromic, normocytic anemia by day olution of acute injury following lung toxicants. 10 that was mild in the low-dose group and severe Research sponsored jointly by the Office of Health in the high-dose group. Recovery from anemia was & Environ. Res., U.S. Dept. of Energy under con­ complete by day 42. Total and differential leuko­ tract W-7405-eng-26with Union Carbide Corp. & cyte counts, MCHand MCHCdid not change from con­ subcontract 3322 from Biol. Div to the Univ. of trol levels. Reticulocyte counts and MCV in­ Tennessee. +oak Ridge Assoc. Univ. undergraduate creased after treatment and returned to control student, Summer1981 from SUNY,Stony Brook, NY. levels by day 65. Osmotic fragility of erythro­ cytes was not changed. Dose-related thrombocyto­ sis occurred on days 15-22; in both groups; plate­ 626 CHRONICTOXICITY OF MELAMINEIN FISCHER 344 RATS. let counts returned to control levels on day 42. G.N.Rao, P.J.Giesler, T,E.Palmer, R.W.Mast, M.A. Dose-related erythroid hypoplasia occurred in bone Friedman, and C.B.Shaffer, Raltech, Division of marrow by day 10 followed by hyperplasia on day 22 Ralston Purina Company, Madison, WI. and American and a return to control level by day 42. Myeloid Cyanamid Company, Chemicals Group, Wayne, NJ. precursors were not affected. Differential Melamine is an intermediate in the manufacture counts of erythroid precursors showed a signifi­ of resins, plastics, and chemical compounds. cant (p < 0.05) decrease in late erythroid forms; Chronic exposure of rats to diets containing 0.1 early forms were either unchanged or increased. and 1.0% melamine in the diet for two years resul­ Megakaryocytes were significantly increased ted in melamine phosphate calculi, epithelial hy­ (p < 0.05) on day 10 in both groups. Vacuolization perplasia, and benign papillomas of the bladder at was observed in erythroid precursors in both the 1.0% level. The papillomas probably resulted groups on day 10 only. Blood and bone marrow from irritation due to calculi. The objective of effects of ribavirin were reversible when treat­ the present study is to evaluate inherent tumor­ ment was discontinued. genicity of melamine, if any, in the absence of bladder calculi. Fischer 344 rats, males at 0.01, 0.05, and 0.1% melamine in the diet and females at 0.01, 0.1, and 0.2% melamine in the diet are being 628 MORPHOLOGICCHANGES IN THE GASTRIC MUCOSAOF treated for a minimum of 24 months and a maximum RATS AND DOGS TREATEDWITH AN ANALOGOF of 30 months. Statistically significant differ­ PROSTAGLANDINE. A. W. Kramer, Jr., ences in body weights, feed consumptions and phar­ W. J. Dougherty, A. R. Belson, B. M. Henderson, macotoxic signs were not evident between the American Cyanamid Company, Medical Research Div.,. treated and control groups during the first 24 Wilbur G. Malcolm Toxicology Laboratories, months. Hematology, clinical chemistry and urin­ Pearl River, N.Y. 10965 Sponsor: M.J.Iatropoulos alysis determinations on 10 animals of each sex at each dose level and control, done at 6, 12, 18, American Cyanamid Company compound CL 115,574, and 24 months did not show any differences between an analog of prostaglandin Eis active ~rally in the treated and control groups. Necropsy of 10 inhibiting gastric acid secretion and in pro­ animals of each sex of each dose level and control tecting against gastric.ulcers induced by stress,

179 ethanol and non-steroidal anti-inflannnatory and S.S. Sorenson, Neuroscience Program, SRI drugs. Sprague Dawley rats were given doses up International, Menlo Park, CA, Sponsored bv: to 0.2, 2.0 and 20.0 mg/kg/day for 6 months. D.C.L, Jones. Beagle dogs were given doses up to 0.4, 0,8 and 1.6 mg/kg/day fbr a year. The only toxic The effects of CS2 on several physiological mea­ clinical sign was diarrhea, which occurred at all sures were studied to determine the adequacy of a dose levels in both species. In the rats, gross battery of tests, consisting primarily of evoked findings at necropsy were limited to the gastric potentials from peripheral nerve and brain, for mucosa. They consisted of a dose related widen­ assessing neurotoxicity in rats, Measures inclu­ ing of the cuticular ridge in the mid and high ded body weight, rectal temperature, tail temp­ dose groups. Microscopically, it consisted of erature, forelimb and hindlimb grip strengths, a simple hyperplasic of the cuticular ridges; latency of the compound action potential of the stratified squamous epithelium. The dogs showed ventral caudal ne1ve, parameters of the somato­ a multifocal hyperplasia of the foveolar sensory, auditory, and visual cortical evoked epithelium in the pyloric antrum. Neither responses, and of the brainstem auditory evoked species had atypical cellular changes in their response (BAER). The rats had chronic electrode hyperplastic lesions. Furthermore, the hyper­ implants and were tested weekly while awake and plasia in the dog was well differentiated and restrained, Twelve rats each were dosed with 0.0, without pseudostratification of cells and 171, 286, or 400 mg/kg of CS2 in sesame oil (i.p., increased mitotic activity. These hyperplastic 2 ml/kg) once per day, five days per week for 12 changes are possibly adaptive reflecting a weeks. Preliminary results after four weeks of pharmacologic effect of a prostaglandin possibly injections were as follows: temperatures remained related to "cytoprotection." constant and equivalent among the groups; body weight increases were observed in all groups but weight gain was slowed in medium and high dose groups; forelimb and hindlimb grip strengths were 629 PERCUTANEOUSACUTE AND CHRONICRENAL TOXICITY reduced in the medium and high dose groups; latency INDUCEDBY SHALE OIL AND PETROLEUMDERIVED JET of the caudal nerve action potential was unaffected AND DIESEL FUEL. J.R. Easley and J.M. Holland, but latency of the fifth component of the BAERwas Biol, Div., Oak Ridge National Lab., Oak Ridge,TN lengthened in proportion to the dose of CS2 Sponsor: W.M. Haschek · administered. There are at least 4 reported instances of Supported by EPA Grant R807150020 acute renal failure in humans associated with : UIJSsive skin exposure to diesel fuel. This sug­ gests that middle distillates can penetrate the skin and, under certain conditions, are poten­ 631 AGE-RELATEDSUSCEPTIBILITY OF RAT ENDOCRINEPAN­ tially nephrotoxic. In the course of a chronic CREAS TO CYPROHEPTADINE. S.A. Chow and L.J. dermal carcinogenesis bioassay of shale oil de­ Fischer, Dept. of Pharmacology, ToxicologyCenter, rived jet and diesel fuels, we have obtained University of Iowa, Iowa City, IA. clinical and histopathologic evidence which indi­ cates that these materials, while possessing a Cyproheptadine (CPH), an antiserotonin-anti­ very low skin carcinogenic potency, have the histaminic drug, produces pancreatic S-cell dam­ potential to induce both acute and chronic renal age after repeated oral doses to adult rats. d~mage following prolonged skin exposure. Characteristics of this effect are a reduction in Cltnical findings included elevated water insulin content and vacuolization of the endo­ · consumption and urine production and low urine plasmic reticulum. The purpose of this study was osmolality. Acute lesions were microscopic and to examine possible age-related changes in CPR­ were restricted to the cortex. There was dilata­ induced pancreotoxicity. CPR at doses of 5, 11, tion of tubules in the corticomedullary zone. 22.5 or 45 mg/kg was given orally once daily for The distended tubules were filled with protein­ two days to 10-, 15-, 25- or SO-day-old Sprague­ aceous precipitates. There was focal necrosis Dawley rats. Serum and pancreatic insulin (IRI) and degeneration of the tubular epithelium. and serum glucose were measured 24 hrs after the After 68 weeks of three times weekly dermal last dose. In all age groups, CPR caused a dose­ exposure, severe renal pathology was evident, dependent decrease in pancreatic and serum IRI both grossly and microscopically. Again, the levels. These effects of CPH, however, were cortex was most severely involved. Lesions were greater in young rats. In SO-day-old rats, a sig­ focal, as well as diffuse. The overall frequency nificant decrease in pancreatic and serum IRI was and severity of lesions was both dose and treat­ detected only at high doses (22.5 and 45 mg/kg). ment related. Lesions were also more severe in However, in 10- and 15-day-old rats, the effects female than in male mice. Histologically, the were observed after the 5 mg/kg dose. No signifi­ chronic lesion consisted of a loss of tubular cant difference in serum glucose level was parenchyma. Fibrosis was not present, nor were detected among various doses and age groups. there any inflammatory infiltrates. Glomeruli Electron microscopic examination of pancreatic did not show changes other than those related to S-cells from young animals indicated a loss of senescence. (Research sponsored by the Office of insulin-containing granules and changes in the Health and Environmental Research, US DOE, under endoplasmic reticulum. contract W-7405-eng-26 with the Union Carbide The time course of CPR-induced changes in the Corporation.) pancreatic S-cell was studied in 15-day-old rats. Pancreatic IRI in CPR-treated rats was signifi­ cantly lower than control at 24 hrs after the first dose. The maximum effect of CPR on pancre­ 630 EFFECTS OF CARBONDISULFIDE ON ELECTRICAL atic and serum IRI was reached at 24 hrs after INDICANTS OF NEUROTOXICITYIN RATS, C.S. Rebert the second dose and thereafter returned to con-

180 trol levels within 24 hrs. These results indicate control values. The recovery of grip-strength that the CPR-induced alterations in the $-cell was complete 4 weeks after dosing stopped. In are age-related and reversible upon drug with­ contrast, the milk-licking of some mice had not drawal. (Supported by GM12675.) recovered even 9 weeks after dosing. The 20 mg/kg dose of acrylamide produced a decrease in grip-strength after 5 weeks but no change in milk-licking or body weight even after 7 weeks 632 ALLURARED DYE STUDY IN MICE. A.T. Modak and of dosing. The new appetitive behavioral proce­ V.K. Berry, Div. of Toxicology and Dept. of dure used in this study has documented a robust Anaton~, U.T. Health Science Center, San behavioral effect of acrylamide which occurred Antonio, Tx 78284. before other signs of toxicity. (Supported in part by PHS Grant OH-00973 from NIOSH, by A six week toxicity study on red dye #40 training Grant ES-07065 and Center Grant was carried out to investigate histopathologic ES-00260 from N.I.E.H.S.) effects in male Sprague Dawley mice, weighing 18-22 g. Twenty four mice were divided into 4 equal groups. All mice were conditioned to drink water between 10:00 AMto 10:30 AM. 634 CYTOTOXICITYSTUDIES OF HEATEDCHRYSOTILE ASBESTOS Group I received only drinking water, Group II R. Valentine, M.W. Chang*, R.W. Hart*, and G.L. received drinking water containing 50 mg of Fisher, Battelle Columbus Laboratories, Columbus, bouillon per 100 ml of water, and Group III OH 43201 and *National Center for Toxicological and IV received red dye /140, 0.25 mg and 0.05 Research, Jefferson, Arkansas 72079 mg/ml, respectively, in water containing bouillon. The Hater intake was 111easuredafter The concern of asbestos carcinogenesis has inten­ each period and mice were weighed once a week. sified from an occupational hazard to a general At the end of six weeks, mice were sacrificed and public health hazard. In our attempt to develop a tissue slices from brain, kidney, liver and simple and accurate analytical technique for as­ spleen were fixed by immersion in a fixative bestiform minerals, we observed that many of these containing 2% glutaraldehyde, 1% formalderiyde, naturally occurring minerals were thermolumines­ and 11~aero 1ei n in O.05 M cacodyl ate buffer at cent. Asbestos thermoluminescence presumably re­ pH 7.2. Tl1e samples were rinsed in 0.07 M caco­ sults from the thermally-induced release of elec­ dylate buffer with 10% sucrose. Post fixation was trons "trapped" in lattice imperfections. We hy­ done in 2¼ osmium prepared in 0.05 M cacodylate pothesized that asbestos thermoluminescence is re­ buffer containing 10% sucrose. The samples were lated to the presence of electron traps and is re­ rinsed in cacodylate buffer, delzydrated in sponsible for its unique biological toxicity. We acetone series and were embedded in Spurr's have found that the thermoluminescence produced plastic. Thin sections were stained with uranyl upon heating chrysotile asbestos is distinctive acetate and lead citrate and examined in Siemens and that heated asbestos exhibits decreased bio­ Elirniskop 101 at 80kV accelerating voltage. logical toxicity. Bovine alveolar macrophages, Chronic ingestation of dye #40 had no significant used to assess asbestos cytotoxicity, were exposed effect on water consu,nption, growth and develop­ to varying concentrations of untreated, heat­ ment of 11iice. Also no rnaJor histopathologic treated and acid-leached Canadian chrysotile as­ alterations were oberved in the brain, kidney, bestos. Compared to untreated asbestos, macrophage liver and spleen following chronic ingestation viability was significantly higher in both the of red dye #40. heat-treated and acid-leached asbestos fiber groups. Macrophage elastase activity, indicative of cell secretion, was increased in all groups compared to unexposed, control macrophages. Addi­ 633 BEHAVIORALEFFECTS OF ACRYLAMIDEIN MICE. J.J. tionally, elastase activity from the heated and Teal AND H.L. Evans, Inst. of Environmental acid-leached asbestos groups were higher than the Medicine, N.Y.U. Med. Center, 550 First Ave., untreated asbestos groups. Concurrent with de­ N.Y., N.Y. 10016. creased macrophage cytotoxicity, heat-treament of Although toxicants are generally reported chrysotile asbestos (~ 4000C) reproducibly de­ to decrease homecage food and water consumption, creased the amount and rate of bovine serum albu­ subchronic administration of acrylamide was min bound to asbestos. These observations indicate found to increase episodic milk-licking even in that asbestos interactions with biological mate­ severely intoxicated mice. Two strains of mice, rials were reduced after heat treatment and are C57BL/6J and CD-1, were injected i.p. 5 times consistent with the hypothesis that exoelectrons weekly with either saline, 20 mg/kg or 60 mg/kg are involved in asbestos fiber toxicology. of acrylamide. Mice were weighed daily and tested for milk-licking (10% sweetened milk) during a 15-min session. Hindlimb grip­ strength, a measure of neuropathy, was measured 635 GOSSYPOLINHIBITION OF SOLUBLEADENYLATE CYCLASE weekly. Mice receiving 60 mg/kg of acrylamide FROMBOVINE TESTIS. K. L. Olgiati, D. L. Toscano showed a significant increase in milk-licking by and W. A. Toscano, Jr. Dept. of Physiol. Lab of day 2 which persisted throughout the 16 day Toxicology, Harvard University Sehl. of Puhl. dosing period. There were no qualitative differ­ Health, Boston, MA 02115. Sponsor: John G. Dent ences in this effect between the two strains of mice and this effect was replicated in two Gossypol is a polyphenolic naphthyldialdehyde studies using CD-1 mice. The increase in milk­ found in the meal and oil of cotton plants licking occurred before the decrease in hindlimb (Gossypium 3:.) which has been shown to be a grip-strength (week 3) or the slight decrease in potent male contraceptive. It has been postula­ body weight which actually oscillated around ted that gossypol acts through inhibition of

181 sperm maturation and motility. We have examined 637 AN INVESTIGATIONOF 35 S-SULPHATE INCORPORATION the effects of gossypol on adenylate cyclase, a INTO RAT BONEMARROW CELL SUSPENSIONSAS A MEANS key regulatory enzyme in spermatogenesis. A sol­ OF QUANTITATINGCELLS WITHIN THE MYELOIDSERIES. uable form of adenylate cyclase was isolated from AF Wright, NM Blackett*, MS Rose+, ICI,Central the seminiferous tubule of mature bovine testis. Toxicology Lab., Alderley Park, Nr. Macclesfield, The enzyme shows a strict requirement for Mn2+­ Cheshire, SK10 4TJ. *Institute of Cancer ATP as a substrate. The Km value for ATP in the Research, Chester Beatty Institute, Fulham Road, presence of saturating Mn2+ ion is 580 µM. The London SW3 6JB. +rcr Corporate Biosciences enzyme is insensitive to calmodulin and only Group, Runcorn Heath, Cheshire, UK. slightly activated by sodium fluoride or the non­ hydrolyzable GTP analog Gpp(NH)p. Unlike the The se 1 ective. incorporation. . o f 35 S-sulphate, as a membrane associated form of adenylate cyclase result of mucopolysaccharide synthesis in certain found in mature sperm, the soluble enzyme is bone marrow cells has been identified as a process insensitive or inhibited by spermine and prot­ that may be utilized for quantitation of specific amine. The soluble form of adenylate cyclase is cells. A method for measuring the rate of 35s­ inhibited by gossypol in a concentration sulphate incorporation in bone marrow cell dependant manner. Half-maximal inhibition occurs suspensions has been developed. The amount of at a gossypol concentration of 100 µM. In con­ radiolabel associated with perchloric acid trast, adenylate cyclase isolated from bovine precipitates of bone marrow cells increased with cerebral cortex and solubilized with Lubrol PX time of incubation, was dependent on energy exhibits an EC5Q at 500 µM gossypol. The inhib­ production and protein synthesis, and represented ition of soluble adenylate cyclase from testis is incorporation into macromolecules.Autoradiography not reversed by sodium fluoride, calmodulin or of cells incubated with 35S-sulphate has demonst­ Gpp(NH)p, however inhibition is reversed by high rated that the radiolabel was associated specific­ concentrations of ATP (5 mM). Inhibition kinetic ally with bone marrow cells within the myeloid data for adenylate cyclase in the presence of series. When bone marrow cell suspensions were gossypol revealed competitive inhibition with an prepared from rats treated with 120µg endotoxin apparent Kr= 100 µM. We conclude that gossypol (Salmonella typhosa), the nucleated cell number inhibits testicular adenylate cyclase by forma­ was reduced (30% at day 1), whilst the rate of tion of a Schiff base in the active site. 35s-sulphate incorporation was increased (100% at Supported by NIH grant GM 22897. day 2). Determination of the cellularity of the bone marrow by conventional histopathology confirmed that endotoxin predominantly affected 636THE INTERACTIONOF ANTHRAQUINONEDERIVATIVES WITH the myeloid cell series. Intermediate and late DNA AND PROTEIN IN VITRO. I. INTERCALATIONWITH myeloid precursors were depleted (60% at day 1) DNA. B.M. Elliott and J. Ashby, ICI, Central whilst early myeloid cells increased by 50%at day Toxicology Lab., Alderley Park, Nr. Macclesfield, 3. These data suggest that the incorporation of Cheshire, SK10 4TJ, UK. Sponsor: I.F.H. Purchase. 35s-sulphate into rat bone marrow suspensions is able to reflect specific myeloid cells. Such Over 100 9,10-anthraquinone (AQ) derivatives have methods may provide additional quantitation of previously been examined in the Ames test, and chemically-induced myeloid dyscrasias within the many found positive in S.typhimurium strain bone marrow. TA1537 in the absence of any added metabolising fraction. This activity profile is suggestive of intercalation, and 12 AQs with hydroxy, amino or nitr·o groups were therefore studied for their ability to bind reversibly to DNA in vitro. 638 FORMATION OF 7-METHYLGUANINEIN LIVER DNA All the AQs showed a bathochromic shift in their FROM RATS TREATED IN VIVO WITH N-NITROSO­ visible spectra with calf-thymus DNA at pH 7.2, PYRROLIDINE. E.J. Hunt and R.C. Shank. Dept. suggesting a change to a more hydrophobic envir­ Comm. & Env. Med., Univ. of Calif. Southern Occup. onment. The ionisation of 1,4-dihydroxy AQ (1.1x Health Ctr., Univ. of Calif., Irvine, CA 92717 10- 6 M) on increasing the pH, was prevented by DNA (4x10- 3 M(P)) indicating removal from contact with 7-Methylguanine (7-MG) is formed in liver DNA the aqueous environment. These data suggest an during hepatotoxicity induced by a variety of association of the AQs with DNA that may be a chemicals that cannot directly methylate DNA (L.R. stacking along the wide groove, or an insertion Barrows and R.C. Shank, Toxicol. Appl. Pharmacol. 60, within the base pairs (intercalation). Only 1,4- in press). In a study using radiolabeled N-nitroso­ diamino AQ however showed competition for the pyrrolidine (NP), it was shown this hepatotoxin and binding sites of ethidium (a known intercalating hepatocarcinogen is not metabolized in the rat to an agent) on DNA and none of the AQs showed any agent which methylates liver DNA guanine (Topics in stabilisation of the double helix to thermal de­ Chemical Carcinogenesis, 213, 1972). 7-MG was naturation even at a chemical:DNA(P) concentration detected in liver DNA of adult male Sprague-Dawley ratio of 1:3. Equilibrium dialysis of [ 14 C]­ rats after administration of a hepatotoxic dose of NP labelled 1,4-dihydroxy, 1-amino-2-methyl and 1- using a highly sensitive high pressure liquid nitro-2-methyl AQ, showed the greatest affinity chromatography method which optically detects for DNA with 1,4-dihydroxy AQ, with an affinity fluorescent alkylated purines in DNA hydrolysates 3 1 constant of 4.0x10 W , 100-1000 times less than without the need for radiolabel. Rats were given 14, that for reference intercalating agents. 28, 57, 113, 225, 450, or 900 mg NP/kg body wt and It is concluded that the reversible interaction decapitated 12 hr later; liver DNA was isolated, of AQ derivatives with calf-thymus DNA is of low purified, and hydrolyzed at pH 7 .0 or 1.0. The amount 2 3 1 affinity (4x10 -4x10 M- ) and that intercalation of 7-MG in liver DNA obtained at each dose level was is unlikely on its own to account for the Ames 25, 64, 87, 58, 97, 112, and 57 µ mol per mol guanine, positive responses. respectively. No 7-MG was detected in comparable

182 amounts of liver DNA from rats 3 or 12 hr after coefficient (Km), The compounds selected were: treatment with vehicle alone. The detection of 7-MG p-phenylenediamine (p-PDA, I), o-PDA, 2-nitro-p­ in liver DNA of rats treated with NP is consistent with PDA, 2-amino-4-nitrophenol, 4-chloro-m-PDA, and similar observations in liver DNA from rats treated 4-amino-2-nitrophenol (II). with such diverse hepatotoxins as hydrazine, carbon Permeability studies were ccnducted by stand­ tetrachloride, ethanol, phosphorus, bromobenzene, and ard diffusion cell techniques using an aqueous puromycin (Shank, unpublished data). Although vehicle. Skin absorption was expressed in terms mechanisms for the indirect methylation of DNA of a permeability constant (kp). Canpounds I and guanine during hepatotoxicity remain undetermined, II exhibited the lowest and highest permeability, 7-MG produced in liver DNA following treatment with respectively (kp I=2.4xl0-4 cm/hr; kp II=2.8xlo-3 NP may be related to the hepatotoxicity and carcino­ cm/hr). genicity of this compound. (Supported by PHS Grant Ko values ranged fran 0.5 to 13.5, and with CA 21955 and Air Force Contract F33615-80-C-0512). one exception (2 amino-4 nitrophenol, K0 =13.S) they increased in the same order as the kp val­ ues. In general, changes in structure which increased the lipophilicity of the molecule were 639 OXIDATIONOF TETRAMETHYLHYDRAZINE BY PEROXIDIZ­ found to result in a corresponding enhancement of ING ENZYMES: AN ELECTRONSPIN RESONANCESTUDY, permeability. The moderate lipophilicity of the K. Sivarajah, B. Kalyanaraman, R. P. Mason, and canpounds as evidenced by the K0 values is in T. E. Eling. Laboratory of Pulmonary Function agreement with the moderate percutaneous absorp­ and Toxicology, National Institute of Environmen­ tion observed, tal Health Sciences, Research Triangle Park, NC Kmvalues were determined for 3 canpounds fran 27709. the decrease in concentration of dye in distilled water during a 3 day incubation. The values did Hydrazine derivatives are used in medicine, agri­ not rank the canpounds in the same order as did culture, and the aerospace industry. Some of their kp and Ko values. It appears that binding them are carcinogenic or toxic. In view of such to the stratum corneum may occur since a concen­ trati9Il dependence was observed on the resultant widespread application, a knowledge of their Km values. metabolism is of importance in furthering our understanding of detoxification/toxification mechanisms. The oxidation of tetramethylhydra­ zine (TMH) was carried out with horseradish peroxidase and H o . Electron spin resonance 641 DEPLETIONOF MOUSE HEPATIC GLUTATHIONEBY 2 2 (esr) spectroscopy identified the one-electron SELECTEDENDOGENOUS COMPOUNDS. R.C. James, S.M. oxidation intermediate as the TMH radical cation. Roberts and R.D. Harbison. Division of We propose a tentative mechanism involving the Interdisciplinary Toxicology, University of TMHradical cation and dication as possible Arkansas for Medical Sciences, Little Rock, AR intermediates during the oxidation of TMH. Sub­ Narcotics deplete hepatic glutathione (GSH), sequently, prostaglandin synthetase (PGS) was induce hepatocellular injury and potentiate the used to carry out a similar peroxidatic reaction. hepatotoxicity of other agents. These effects Aerobic incubation of ram seminal vesicle (RSV) may be regulated by CNS receptor control or microsomes (a source of PGS), arachidonic acid result from a direct chemical interaction of and TMHyielded the typical esr spectrum of the these compounds. The purpose of this study was TMHradical cation, which was indomethacin sensi­ to determine if other compoundsaffecting the CNS tive. Incubation of RSV, TMHand H o under N 2 2 2 or hepatic receptors might also produce similar yielded the same esr spectrum which was indome­ effects. Epinephrine (Epi), 0.5 mg/kg; glucagon, thacin insensitive. Thus, a peroxidative 1.0 mg/kg; dexamethasone, 1.0 mg/kg; and ethanol, reaction mechanism was inferred. Demethylation of 2.0 mg/kg, significantly lowered GSHby 20-50% TMHwas measured by measuring the formaldehyde within the first 3 hours following treatment. (HCHO) formed from aerobic incubations containing Epi increased serum glutamic-pyruvic transaminase RSV, TMHand arachidonic acid. The HCHOformation (SGPT)levels at least 100%,which was similar to increased with increasing enzyme and substrate the elevation produced by morphine. Glucagon had concentrations. The cation radical or the decay no effect on SGPT. The adrenergic blocker product formed may covalently bind to the tissue propranolol did not alter Epi-induced lowering of macromolecules and result in toxicity. hepatic GSH, but phentolamine completely antagonized this effect. Similarly, the a 1 blockers, prazosin and phenoxybenzamine had no effect on the Epi induced lowering of GSH,while 640 EFFECT OF CHEMICALSTRUCTURE ON THE PERCUTANECXJS the a 2 antagonist, yohimbine, caused complete ABSORPTIONAND PARTITION COEFFICIENTS OF HAIR blockade. Correspondingly, the a 2 agonists DYES. R.L. Bronaugh, E.R. Congdon and K.D. White, guanabenz and clonidine also lowered GSH. Divisicn of Toxicology, FDA, Washingtcn, DC Interestingly, phentolamine significantly 20204. Sponsor: T.F.X. Collins. antagonized the morphine depletion of GSH and An homologous series of hair dyes was selected conversely naltrexone significantly antagonized for percutaneous absorpticn studies with excised Epi, but neither inhibition was complete. These human skin. It was of interest to observe the studies suggest that several important endogenous effect of small changes in structure on skin compoundscan lower hepatic GSH and therefore absorption as well as to attempt to correlate the potentiate other hepatotoxic agents. This effect permeability of the canpamds with solubility may be receptor mediated with some sterochemical properties. These properties were expressed as specificity and overlap between the narcotic and (i) the octanol/water partition coefficient (K 0 ) adrenergic effects. (Supported by USPHS Grant and (ii) the stratum corneum/water partition ES00782)

183 642 UTILIZATIONOF IN VITROERYTHROCYTE FRAGILITY were still apparent following the 14-day recovery TO PREDICTTOXICITY OF A GROUPOF ANTIFUNGAL period. Depletion of lymphocytes in the spleen ECHINOCANDINB ANALOGS. L.C. Howard, M.D. and thymus along with hypocellularity and hypo­ Gunnoe, M. Debono, B.J. Abbott and J.R. Turner plasia of the bone marrow were also detected, (Sponsor, J.L. Emmerson). Toxicology Division, both lesions being dose-related and reversible. Lilly Research Laboratories, Greenfield, IN. Administration of intravenous doses of 200 mg/ kg/day of echinocandin B for 3 days to beagle 644 HYPOGLYCEMICEFFECT OF THE VENOMFROM THE SNAKE dogs resulted in signs of toxicity related to P,u.,~ivo~uJ.ip,u.,~ivo~uJ.i. S.A. Taha, Department of an intravascular hemolytic crisis. In vitro Pharmacology, College of Pharmacy, University of incubation of echinocandin Bat concentrations Riyadh, Riyadh, Saudi Arabia. Sponsor: K.N. achieved in vivo with canine and human erythro­ Salman cytes producedlysis and a shift in the fragil­ Venom from the snake P. p,u.,uvo~uJ.icaused ity curve. Analogs of echinocandin B prepared a significant reduction in the blood glucose by microbial deacylation of the linoleic acid level of both rats and rabbits. In normal rats moiety followed by selective N-acylation were the hypoglycemic effect was of short duration, tested fo~ their effects on erythrocytes. The reaching a peak 30 minutes after injection, and following relationships between structure and lasting for 1 hour. The effect was produced by erythrocyte fragility were determined. doses as low as 10 µg/kg; doses higher than Fragility increased with 1) length of fatty 20 µg/kg, although causing more pronounced hypo­ acid side chain, 2) saturated vs unsaturated glycemia, were lethal to the rats. No hypogly­ fatty acids, 3) trans vs cis configuration of cemia was produced in alloxan diabetic rats. In the fatty acid, 4flesser degree of moderately and severely diabetic rats, the com­ unsaturation, and 5) position of unsaturation bined administration of insulin and the venom (611>69>66). Evaluation of erythrocyte did not potentiate the hypoglycemia. Dose­ fragility and antifungal activity led to the response curves for the blood glucose level of selection of the n-tridecanoyl, n-lauroyl­ insulin-treated and insulin+ venom treated p-aminobenzoyl, and .E_-.!'.!_-octyloxybenzoylanalogs animals were not parallel. Measurement of plasma of echinocandin B for further toxicity studies insulin showed about 50% increase in insulin in dogs (100 mg/kg/day, i.v., 5 days). As level 30-60 minutes after venom injection. It is predicted by the in vitro erythrocyte fragility postulated that the venom acts partly through re­ testing, all compounaswere less toxic than lease of insulin and partly by a direct mechanism. echinocandin B.

645 INTRAVENOUS(IV) TOXICITY STUDY OF HOMOHARRING­ 643 DERMALTOXICITY OF HEXAFLUOROACETONE.G. L. TONINE (HH) (NSC 141633) IN CDFl MICE AND BEAGLE Kennedy, Jr., J.E. Henry, H. C. Chen and o. L. DOGS. K.N. Newman, R.G. Meeks, Southern Research Dashiell, E. I. du Pont de Nemours and Company, Institute, Birmingham, AL;· J.F. Ferrell, Experi­ Inc., Haskell Laboratory for Toxicology and mental Pathology Laboratories, Inc., Herndon, VA; Industrial Medicine, Newark, Delaware 19711. and K.S. Greenspun, Battelle TPO, Vienna, VA. Hexafluoroacetone (1,1,1,3,3,3-hexafluoro-2-pro­ panone sesquihydrate, HFA) is used as an organic HH, a drug derived from Cephalotaxus harring­ solvent, as an intermediate for organic synthesis, tonia, an evergreen native to China, has been and as a process intermediate. The material has shown to have marked anticancer activity against been shown to be both toxic and teratogenic to transplanted tumors of mice and is in clinical use pregnant rats following dermal exposures. In in China. Following single (xl) and five daily this study, male Crl-Cr® rats, 3 groups, 10 rats dose (x5) lethality studies in mice, xl and x5 per group, were clipped free of hair over the studies were performed in mice and dogs to deter­ back area and fitted with Teflon® collars to re­ mine target organ toxicity. duce grooming and ingestion. The material was Calculated estimates of the LDlO, 1f50, LD90 in applied dermally, 5 days/week for 2 weeks at mice were 12. 8, 18. 0, 2-nd 25. 3 mg/m (xl) and doses of either 65, 130, or 260 mg/kg. Another 5.8, 8.6, and 12.8 mg/m /day (x5). group of 10 rats treated with water served as In the mouse toxicity studies an anemia, reti­ controls. Clinical signs and body weights were culocytopenia, thrombocytopenia, and leukopenia recorded daily. Five rats from each group were were histopathologically confirmed by degenera­ sacrificed 4 hours following the 10th exposure tion and depletion of the lymphoid and hematopoie­ and given a complete pathologic examination, as tic tissues in mice sacrificed on day 4 (xl). In were the remaining rats following a 14-day reco­ the x5 study, lymphoid depletion of the thymus was very period. Rats treated at 260 mg/kg showed observed at the day 8 sacrifice, but increased severe clinical signs including the 2 deaths hematopoiesis in the bone marrow, liver, and following the 5th and 1 death following the 10th spleen was indicative of recovery. dose. Lacrimation, rapid breathing, lethargy, In dogs given xl and x5 HH IV, gastrointestinal and weight loss were seen. Rats given 130 mg/kg (GI) (emesis, hemorrhagic diarrhea), cardiac showed weakness and moderate to slight weight (tachycardia, premature ventricular contractions, loss with incomplete recovery post-exposure. ventricular arrhythmias), bone marrow (granulocy­ Rats treated with 65 mg/kg showed slight weight topenia), and lymphatic (lymphocytopenia) toxi­ loss which was not apparent during the recovery city were observed. Liver and kidney damage period. Testicular degeneration and atrophy (elevated SOOT, SGPT, SAP, and BUN) and electro­ were seen in all HFA groups with the damage most lyte imbalances were seen. Histopathological obvious at the 2 higher levels. These changes changes were seen in the lymphoid system, marrow,

184 GI tract, and kidneys. Myocardial degeneration evidence of cholestasis was evident. None of was seen in dogs given xl HH v at the mouse equi­ the clinical chemical and hematologic parameters valent (ME) LDl0 ( 12. 8 mg/m2 ) . Seven of eight showed any deviation from the controls. dogs (xl and xS) that received the ME LDl0 died Metabolic studies showed that the compound is from treatment. ME LDl0/2 and ME LDl0/10 were not rapidly metabolised and not present in plasma lethal. Supported by NCI _Contract NOl-CM-17365. in measurable amounts 4 hours after drug administration. Furthermore, the glycine conjugate.of FOBPN, which is formed by the loss of the cyanide group, in only accounting for 646 RED CELL KINETICS IN RIBAVIRIN-TREATEDMONKEYS. 4% in the urine, with significant cyanide P.G. Canonico, M.D. Kastello, and G. Wannarka. containing metabolite present in plasma and Dept. of Antiviral Studies, U.S. Army Med. Res. urine. It is therefore postulated that the Inst. Inf. Dis., Frederick, MD. Sponsor: F. Reno. cyanide group present, is responsible for the Man and rhesus monkeys may develop anemia dur­ biliary changes. ing treatment with the broad sp.ectrum antiviral ribavirin (Virazole, l-B-D-Ribofuranosyl-1,2,4- triazole-3-Carboximide). To assess whether the anemia is due to decreased production of erythro­ 648 NEPHROTOXICITYAND KIDNEY CONCENTRATIONOF cytes, increased destruction, or a comb~nation of SELECTEDFURAN DERIVATIVES IN MICE. L. Gammal, both factors, the disappear1nce of 1,3- H-di­ R. Wiley, G. Traiger, and S. Baraban, Depart­ isopropyl fluorophosphate ( H-DFP) labeled ery­ mentsof Pharmacology & Toxicology and Medicinal throcytes was measured in ribavirin-treated mon­ Chemistry, The University of Kansas, Lawrence, keys. KS 66045. Monkeys were divided into 3 groups, th ir red blood cells (RBC) labeled in vitro with 3H-DFP The furans are a class of compounds which and reinjected into each donoGThe first two have widespread occurrence in the environment groups were treated with either 15 or 60 mg/kg of and produce toxic effects in the liver, lung, ribavirin (IM) for ten days. The third group and kidney of rodents. The purpose of this served as saline treated controls. Hemograms study was to determine if a_corre 1at ion exists showed that by day 10 of treatment there were 12- between renal concentration of selected furan 26% and 53-60% decreases in RBC, hematocrit and derivatives and nephrotoxicity. Renal tissue hemoglobin in the low and high dose groups, re­ concentrations after 1,2, and 5 hr and spectively. All values returned to normal levels nephrotoxicity were determined in mice for the by day 42. A dose-related decrease in RBC sur­ following furan derivatives: furan, vival was observed from day Oto 28. Thereafter, 3-ethylfuran, 3-pentylfuran, 2-ethylfuran, red cell half-lives were comparable to control 3-methylthiophene, and 2-furamide. Three in­ values. These data indicate that ribavirin at a dices were used to assess nephrotoxicity: dose as low as 15 mg/kg decrease the half-life of histological observation, blood urea nitrogen RBC. This effect is reversible upon discontinua­ (BUN} concentration and renal urine concen­ tion of the drug. At 60 mg/kg, ribavirin also trating ability in water-deprived mice. The inhibited the release of RBC from the bone marrow. results demonstrated that 3-ethylfuran (150-300 Termination of treatment was accompanied by re­ mg/kg), 2-ethylfuran (150-250 mg/kg} and lease of RBC from the bone marrow a indicated by 3-pentylfuran (250 mg/kg) caused massive prox­ a drop in the specific activity of 3H-DFP-labeled imal tubular necrosis, elevated BUNlevels and red cells and marked reticulocytosis. No inhibi­ decreased urine concentrating ability. Furan tion of RBC-""release from the bone marrow was seen (175-350 mg/kg) caused proximal tubular necrosis in the low-dose group. We conclude that ribavirin but did not affect either BUN levels or urine can decrease red cell survival as well as inhibit concentration. 2-Furamide (150 mg/kg) and RBC release from the bone marrow. Both effects, 3-methylthiophene (600-800 mg/kg} caused no appear fully reversible when treatment is with­ change in any of the parameters measured. All drawn. compounds were shown to reach the kidney in reasonable concentrations, but there did not ap­ pear to be a simple correlation between kidney concentration and extent of damage among these substances. Supported by GM-26,366. 647 BILE DUCT PROLIFERATIONAFTER 4-FLUOR0-8- 0XOBENZENEPROPANENITRILEADMINISTRATION IN MONKEYS. R. J. Arceo, M. R. Irwin, M. J. Iatropoulos, American Cyanamid Company, Medical Research Division, Wilbur G. Malcolm 649 CARDIOTOXICITYOF BETA-ADRENERGIC AGONISTS IN Teii:icology Laboratories, Pearl River, NY 10965 MICEOF VARIOUSAGE GROUPS. X. Joseph, V. Whitehurst and T. Balazs, Food and Drug 4-Fluoro-B-oxobenzenepropanenitrile (FOBPN), a Administration, Washington, D.C. non-steroidal anti-inflammatory compound, when administered by nasogastric intubation at 10, To examine the influence of age on the sensi­ 60 and 100 mg/kg, continuously for up to 12 tivity to the cardiotoxicity of beta adrenoceptor months to cynomolgus monkeys, induced hepato­ agonists, we studied the acute toxicity and the biliary changes. These changes consisted myocardial lesion-inducing effects of isopro­ mainly of biliary duct hyperplasia with terenol and terbutaline in 3 groups of 4-, 8- and periductal inflammatory response. Associated 16-week-old CD-1 mice with average body weights were also varying degrees of fibroplasia with of 26, 33 and 38 g, respectively. The LD50 focal necroses of hepatocytes bordering the values for isoproterenol were 342.5, 317.7, and fibroplasia. No morphologic or clinical 325. 1 mg/kg for 4-, 8- and 16-week-old mice,

185 respectively; for terbutaline the values were 651 SUBCHRONICTOXICITY OF OCTACHLOROSTYRENEIN THE 206.3, 213.9 and 191.7 mg/kg for the respective RAT. D.C. Villeneuve, I. Chu, V. Secours, groups. Each group was injected ip with 1/10 V.E.O. Valli and G.C. Becking. Environmental and 1/100 of the LD50isoproterenol or terbuta­ and Occupational Toxicology Division, line with appropriate controls, and the hearts Environmental Health Directorate, Ottawa, were examined histologically. Lesions were Ontario and Biopath Analysts, Guelph, Ontario. graded on a scale of Oto 3 with 3 being the most severe. Isoproterenol at 1/10 of the LD50 Previous short-term studies with produced an average lesion score of 1.85 in octachlorostyrene (OCS), a demonstrated 16-week-old mice as compared to scores of 0.62 environmental contaminant indicated that it and 0.6 for 8- and 4-week-old mice, respectively. could produce hepatomegaly, induce mixed Similarly, 1/100 of the LD50dosage produced a function oxidases and cause histological changes greater score in 16-week-old mice. Terbutaline in thyroid and liver. The present study was did not.produce myocardial necroses in any age undertaken to determine the effects of group. These data indicate that even though subchronic exposure to this chemical. Male and the LD50values do not vary significantly among female rats were fed diets containing O, 0.05, the varigus age groups, the 16-week-old mice are 0 .5, 5.0, 50 or 500 ppm OCS in the diet for 13 more sensitive to the lesion-inducing effects of weeks. No mortality occurred at any dose level. isoproterenol than are younger mice and that Hepatomegaly was observed in both males and terbutaline, a predominantly S2 agonist, is much females at 50 and 500 ppm OCS. Increased kidney less cardiotoxic than isoproterenol, a s1 and s2 and spleen weights were observed at 500 ppm OCS. agonist, in mice. The significance of age vs Decreased hemoglobin values occurred with 50 and body weight in the sensitivity to isoproterenol 500 ppm OCS in male rats and with 500 ppm in is being investigated. females. Decreased hematocrit values were observed at 500 ppm in both males and females whereas decrease red blood cells were observed only in males receiving 500 ppm OCS. Induction of mixed function oxidases was observed in males 650 APPLICATIONOF COMPUTER-ASSISTEDELECTROCARDIO­ at 5. O ppm and higher and in females at 50 ppm GRAPHICANALYSIS TO CARDIOVASCULARTOXICOLOGY. and higher. Histological changes in thyroid, W.P. Watkinson, K.S. Robinson, M.A. Brice. liver, and kidney were noted in all treatment USEPA, RTP, NC 27711 (Sponsor: N. Chernoff) groups of both sexes. Octachlorostyrene accumulated in fat and liver in a dose-dependent manner. The data generated in this study There is a need to develop more efficient suggest that prolonged feeding of OCS can and cost-effective methods for identifying po­ produce biochemical, hematological and tentially toxic and/or teratogenic agents. histological changes at low levels and that Analysis of electrocardiographic waveforms (ECG) males tend to be more susceptible to the toxic may provide a sensitive index of general systemic effects of this chemical than females. as well as specific cardiovascular responses to toxic substances. Thus, an automated procedure based on a precise one-dimensional analysis of features within a generalized computer-enhanced 652 CARDIOVASCULARAND RESPIRATORY EFFECTS OF METHYL ECG waveform has been developed in our labora­ METHACRYLATEINFUSION IN THE ANESTHETIZEDDOG. tory, K.C. Baran, K.L. Marquis, H.H. Mincer and I.W. ECG signals are monitored, amplified, and Waters, Dept. of Pharmacology, Sch. of Pharmacy, recorded using standard techniques. The recorder Univ. of Mississippi, University, MS 38677. output signal is distributed to a microcomputer Sponsor: W.M. Davis system (CPU). Software developed for the CPU Methyl methacrylate monomer (MMA), a consti­ permits slow-motion review of the signal for tuent of orthopedic bone cement, has been re­ arrhythmia analysis. The CPU identifies and ported to cause severe bradycardia and hypoten­ superimposes 10 - 40 individual ECG complexes tion in humans undergoing orthopedic surgery. As and generates an "ensembled" waveform. Operator a model of this clinical situation MMAwas admin­ interaction permits delineation of specific istered as a slow infusion to anesthetized dogs. points on the displayed waveform and CPU calcu­ Eight mongrel dogs of either sex (13.4 + 1.3 kg) lation of heart rate and duration (msec) of were ·anesthetized with pentobarbital and main­ components within the ECG complex. tained in a stable anesthetic state. MMAwas in­ The primary advantages of this system in­ fused for approximately 10 minutes at either 3.1 clude: 1) enhance sensitivity - the use of func­ ml/hr (31.2 mg/kg, N=4) or 6.2 ml/hr (62.4 mg/kg, tional parameters should provide a more sensitive N=4). Pre-drug control levels were established index of toxicity than morphological parameters; and then measurements of both cardiovascular and 2) extensive automation - computer support de­ respiratory parameters were made at intervals creases operator time, increases precision, and from 5-180 minutes following the termination of permits rapid. screening of large numbers of ani­ MMAinfusion. There were significant increases mals; 3) versitility - this system provides the in arterial pC02, respiratory rate, oxygen uptake, capability to utilize a variety of animals, both and minute volume and significant decreases in anesthetized and unanesthetized, ranging in age arterial pH, systolic pressure, mean arterial from fetuses to geriatrics, and permits studies pressure, heart rate, and tidal volume (ANOVA, of block as well as longitudinal design; and 4) p < 0.05). Arterial p02 and diastolic pressure ease of replication -standardization of equip­ did not differ significantly from control. Lungs ment and techniques facilitates replication by appeared grossly edematous with large areas of other laboratories. hemorrhage. Histopathological examination re-

186 vealed severe focal lung edema (31.2 mg/kg) and a single dose of DPP and at 1, 2, 3 and 4 days edema with alveolar hemorrhage (62.4 mg/kg). following multiple doses. The testes were Swelling, vacuolization and congestion of hepa­ excised and fixed and sections made for examina­ tocytes occurred at both doses. No pathological tion by both light and electron microscopy. After changes were seen in the spleen, pancreas, kidney 6hr the lumen of the seminiferous tubules (ST) or heart. Humans exposed to MMAshould be moni­ had disappeared and by 24hr a marked degeneration tored for lung and liver damage in addition to of spermatids was prevalent, spermatocytes were changes in cardiovascular parameters. (Supported also affected but to a lesser degree. At 4 days in part by the Research Institute of Pharmaceu­ almost all ST showed atrophy with complete tical Sciences, University, MS and the Amer. disruption of the ST cell organisation. Found. for Pharmaceut. Ed.). Ultrastructural studies showed an early effect on Sertoli cells, with an accumulation of autophagic vacuoles·. The Sertoli cell cytoplasm in the central area of the ST showed degeneration 653 IN VITRO RATES OF COLLAGEN SYNTHESIS IN and detachment from the immature spermatids. At MOUSE LUNG TISSUE FOLLOWING THE ADMINI­ the same time spermatids showed changes in Golgi STRATION OF BUTYLATED HYDROXYTOLUENE. structure, with mitochondrial damage and a J.P. Kehrer, Dept. of Pharmacology, College of Phar­ decrease in rough ER. Later stages showed that macy, The University of Texas at Austin, Austin, TX. Sertoli cell cytoplasm continued to atrophy (sponsored by D. Acosta) accompanied by an accumulation of lipid. Thus it Treatment of mice with butylated hydroxytoluene would seem that DPP-induced damage to the testis (BHT) results in the formation of a dose-dependent is rapid in onset and that this may be due to a lung lesion. At doses of 300 mg/kg or greater pulmo­ disruption of the interactions between Sertoli nary fibrosis and the deposition of excess collagen cell cytoplasm and developing germinal cells. became evident within 14 days as shown by an eleva­ (Supported by the U.K. Ministry of Agriculture, tion of total lung hydroxyproline. The in vitro rate of Fisheries and Food). collagen synthesis was ~easured in minced lung t~ue as the formation of [ H] hydroxyproline from [ H] proline at 1, 2, 3, and 4 hours of incubation at 37° in 655 ACRYLAMIDEEFFECTS ON ACTIVITY OF GLYCERALDEHYDE- Dulbecco's Modified Eagle's Medium. The rate of syn­ 3-PHOSPHATEDEHYDROGENASE IN RAT BRAIN HOMOGENATE. thesis was elevated two days after BHT 400 mg/kg and I.L. Vyas, R.D. Howland and H.E. Lowndes, CMDNJ, reached a maximal rate of 150 pmol/mg dry wt/hr at NJ Medical School, Newark, NJ. day 7. The rate then declined but was still significant­ ly elevated at day 14. Expressing these data as a percentage of total protein synthesis committed to Acrylamide inhibits several glycolytic enzymes collagen demonstrated a specific stimulation of col­ in vivo and in vitro, including glyceraldehyde-3- lagen synthesis to a level of 1.5% at 7 days after BHT phosphate dehydrogenase (GAPDH)and neuron-spec­ ific enolase (NSE). We investigated the kinetics compared to control levels of 0.6%. Both the maxi­ mum and the control levels were the same as that of acrylamide inhibition of GAPDHto determine reported in vivo following BHT 400 mg/kg. Doses of if in vivo inhibition could occur via a direct BHT as low as 200 mg/kg produced a significant interaction of acrylamide with the enzyme. Glu­ tathione binds to acrylamide both directly and increase in both the collagen synthetic rate and the percentage of collagen synthesis. While DNA synthesis by an enzyme-catalyzed mechanism. Its ability was not elevated at BHT doses less than 200 mg/kg, to antagonize inhibition in tissue homogenates doses greater than this produced a dramatic increase. was also investigated. The rate of collagen synthesis exhibited a more linear Enzymatic activities were determined spectro­ dose-response relationship. These data show that the photometrically at 340 nm. Rat brain homogenate rate of collagen synthesis is elevated at levels of lung was incubated at 37°c ± acrylamide. Inhibition damage which do not result in the deposition of excess is via an irreversible, noncompetitive mechanism collagen. This study also shows that percentage of with Ki= l.12mM. Acrylamide at l.25mM inhibited protein synthesis devoted to collagen is the same in 50% of GAPDHactivity. Addition of glutathione vivo and in vitro in both normal and damaged lung (5mM) resulted in a 48% increase in the concen­ tissue andthat in vitro rates of collagen synthesis are tration of acrylamide required for 50% inhibition a sensitive indexof acute lung damage. (Supported by of activity, Cysteine was also capable of antag­ NIH-BRSG grant no. RR-07091-15 awarded to the Univ. onizing acrylamide inhibition of GAPDHbut was of Texas at Austin.) only one-tenth as potent. These results indicate that acrylamide can inhibit GAPDHdirectly in tissue homogenates and therefore the in vivo inhibition may also be a di­ rect inhibitioi-:--- 654 MORPHOLOGICALCHANGES PRODUCED IN THE TESTES OF YOUNGMALE RATS FOLLOWINGORAL ADMINISTRATION OF Supported by PHS Grant ES-02405. DI-N-PENTYL PHTHALATE. Foster, P.M.D., Creasy, D.M:-, Foster, J.R. and Gangolli, S.D., The British Industrial Biological Research 656 EFFECT OF PHTHALATEESTERS ON SUCCINATEOXIDATION Association, Woodmansterne Road, Carshalton, SM5 ANDENERGY COUPLING IN RAT LIVER MITOCHONDRIA. 4DS. U.K. R.L. Melnick and C.M. Schiller, National Toxicol­ Di-n-pentyl phthalate (DPP) has previously been ogy Program and Laboratory of Pharmacology, NIEHS, shown by us to induce testicular atrophy in Research Triangle Park, NC 27709 young male rats within 4 days when adminis2ered orally at 2.2 g/kg/day. DPP, at this dose level, A comparative study was conducted on the effects was given to young animals by oral intubation, of monobutyl phthalate (MBP), dibutyl phthalate animals were killed at O, 3, 6 and 24hr following (DBP), mono(2-ethylhexyl)phthalate (MEHP), and

187 di(2-ethylhexyl)phthalate (DEHP) on energy coup­ of hepatic peroxisomes and induce peroxisomal ling and electron transport activities of isolated enzymes in rats and mice. Since these compounds rat liver mitochondria. Energy coupling was also induce liver tumours in rodents, a relation­ eialuated by 2 approaches: 1) active transport of ship between peroxisome proliferation and K (plus valinomycin) driven by the oxidation of carcinogenesis has been suggested. We descrioe two respiratory chain substrates, succinate and here the induction of peroxisome proliferation in ascorbate + TMPD, and by the hydrolysis of ATP; primary hepatocyte cultures. and 2) succinate respiration rates in the absence Hepatocytes were isolated from male rats and presence of ADP (respiratory control ratios). (180-250g) by a collagenase perfusion technique Energy-linked processes were uncoupled most by DBP and were cultured in supplemented RPM1 1640 and MEHP. MBP had a moderate effect on energy medium containing the test compounds or DMSO. coupling and DEHP had no apparent effect. The Electron microscopy of hepatocytes cultured for potency of inhibition of succinate cytochrome c 48 hours in the presence of clofibrate, mono­ reductase activity followed the order MEHP > DBP > (2-ethylhexyl)phthalate or 2-ethylhexanol MBP = DEHP. MEHPwas found to be a noncompetitive (10- 4-10- 3M) showed markedly increased numbers of in~¼bitor of succinate dehydrogenase (Ki= 2.1 x peroxisomes. There was a time- and dose­ 10 M). It is concluded that phthalate esters dependent induction of palmityl CoA oxidation and affect mitochondrial activities by altering carnitine acetyltransferase (CAT), two peroxisomal . permeability properties of the inner membrane and markers. CAT activity reached 15 times control by inhibiting succinate dehydrogenase activity. levels in cells cultured for 72hr with Sx10-4M clofibrate, whereas the mitochondrial marker carnitine palmityltransferase was only increased by 3-fold at this point. No effects on peroxisome 657 EFFECTSOF ACRYLAMIDEPRETREATMENT ONTHE number or enzyme activity were produced by n­ BEHAVIORALSUPPRESSION OF PSYCHOACTIVEAGENTS IN hexanol or by two microsomal enzyme inducers, RATS. R.E. Squibb and H.A. Tilson. Lab. Behav. phenobarbital and 1,2-benzanthracene. These Neural. Toxicol., NIEHS,Research Triangle Park, findings indicate that primary hepatocyte cultures NC 27709 (SPON.: B. Fowler). may be useful for studying the mechanisms under­ lying, and the consequences of, peroxisome Rats were trained to lever touch for food on a proliferation. (Supported by the U.K. Ministry Variable Interval (VI) 15 sec schedule of rein­ of Agriculture, Fisheries and Food). forcement. Dose-response alteration of VI re­ sponding by apomorphine, ct-amphetamine, clonidine, and chlordiazepoxide was studied alone and following an acrylamide treatment, which, by itself, did not 659 TEN YEAR CHRONICTOXICOLOGY OF NORLESTRIN alter VI responding. Male Fischer 344 rats were IN FEMALE MONKEYS. J.E. Fitzgerald and given 12.5 mg/kg of acrylamide by gavage 24 hrs F .A. de la Iglesia, Dept of Toxicology, prior to challenge with the psychoactive drugs. Warner-Lambert/Parke-Davis Pharmaceu ti ca I In the absence of acrylamide pretreatment, apo­ Res., Ann Arbor, Ml 48105 morphine produced responding that was 71, 52, and 20%of baseline after i.p. injections of 0.05, 0.1, For the evaluation of long term effects of and 0.2 mg/kg, respectively. Corresponding values the oral contraceptive Norlestrin, groups of after acrylamide pretreatment were 42, 26, and 15% 16 sexually mature female rhesus (Macaca of baseline, which is a significant shift in the mulatta) monkeys were studied over 10 years. dose-response curve to the left. Likewise, ct­ Norlestrin, a combination of norethindrone amphetamine given i.p. resulted in values that acetate and ethinylestradiol (50: 1) was given were 81, 53, and 14%of baseline after 0.25, 0.5, continuously on a cyclic regimen (21 days and l mg/kg, respectively. Following acrylamide on drug, 7 days off) at levels of 0.05, pretreatment, responding was 59, 42, and 11%of 0.51, and 2.55 mg/kg, representing 1, 10, control, respectively. No significant effect of and 50 multi pies of the human dose. Selected acrylamide pretreatment on the behavioral effects clinical and laboratory parameters were moni­ of clonidine and chlordiazepoxide was observed. tored throughout the study at established These data suggest that acute exposure to acryla­ intervals. Al I dose levels were wel I toler­ mide increas..es responsiveness to catecholaminergic ated, survival was not affected and there agents. The change in responsiveness to apomor­ were no alterations in coagulation or other phine and ct-amphetaminemay be related to the laboratory parameters. Ophthalmologically effects of-acrylamide on the affinity or density macu I ar pi gmen tary anoma Ii es were observed of dopamine receptors reported elsewhere (Agrawal in al I groups. Treatment associated path­ et al., Pharmacol. Biochem. and Behav., 14:527, ologic findings included ovarian and uterine 1981T. -- - atrophy and dilatation of ac1n1 and ducts in the mammary gland. These findings consti­ tuted exaggerated pharmacologic responses to­ gether with superimposed advanced senile 658 CHARACTERISTICSOF PEROXISOMEPROLIFERATION changes. Periodic vaginal cytologic examina­ INDUCEDIN ISOLATED HEPATOCYTESBY HYPOLIPIDEMIC tions and mammary gland palpations did not DRUGSAND PHTHALATEESTERS. Gray, T.J.B., demonstrate drug-related changes. The neo­ Beamand, J.A., Lake, B.G., Foster, J.R. and plasms found were three cutaneous papi I lamas Gangolli, S.D., The British Industrial Biological (1 low dose, 2 high dose); an uterine leio­ Research Association, Woodmansterne Road, myoma (high dose); a pancreatic duct Carshalton, Surrey, SMS 4DS. U.K. adenoma ( low dose) and a granulosa eel I carcinom~ of the ovary (control). The overal I Several hypolipidemic drugs and the plasticizer evaluation of the study indicated an ade­ -0i-(2-ethylhexyl)-phthalate cause proliferation quate long term safety margin since no mal ig-

188 nacies were elicited and 0.51 mg/kg or 10 determined for all groups. Examination of a times the human dose, was considered to be semilogarithmic plot of the time course of the IV the no-effect dose due to the absence of serum hydrazine concentration supports the use of significant toxicity or pathology. a one compartment model to describe hydrazine elimination. The apparent volume of distribution, serum half-life, and elimination rate constant for hydEfzine were 0.63 liter/kg, 2.3 hours, and 660 SYSTEMIC TOXICITY OF AMETANTRONEACETATE 0.29 hr respectively. Moderate depression was IN MICE AND DOGS. J.R. Watkins, S.N. Kim, observed in the IV group rabbits and severe U. Jayasekar~, J.L. Sanyer, J.A. Anderson, chemical burns were observed in all the percutane­ J.E. Fitzgerald and F .A. de la Iglesia, Dept. ously exposed groups. Selected sections of of Toxicology, Warner-Lambert/Parke-Davis hydrazine exposed skin from eac:h group of rabbits Pharmaceutical Res. Div., Ann Arbor, Ml 48105 were examined histopatllologically to correlate burn severity with duration of hydrazine exposure. Ametantrone acetate (NSC 287513) is an experi­ Bioavailability was determined by comparing the mental antineoplastic agent with activity areas under the serum hydrazine-time curves of against a comprehensive panel of solid trans­ the percutaneously exposed groups with the IV plantable tumors in mice. Studies in mice group. Anhydrous hy

189 This study investigates the sources of vari­ day 4 and 38(3)% on day 5 post induction, while ability in scoring ocular lesions and the sen­ acetone controls ran in parallel averaged sitivity and reproducibility of the test using a 19(0.2)% B(a)P metabolism. (Research sponsored by dose volume of 0.01 mL instead of the standard the Office of Health and Environmental Research, dose of 0.10 mL of test material. A series of 7 US DOE, under contract W-7405-eng-26 with the UCC) previously tested compounds were chosen and assigned letter codes A-H (Band G were the same). Thirteen rabbits were used each week for a total of 6 weeks (72 rabbits). Each week, 7 665 A COMPARATIVESTUDY OF IRRITATION RESPONSESWITH rabbits were dosed with 0.01 mL of test materials A-H (excluding G) by direct application to the ANDWITHOUT ANESTHETICS IN THE ALBINO RABBIT EYE. cornea. Compound G, used as a standard to C.A. Hoheisel, D.K. Lowther, G.W. Bierbower, R.L. measure test variability, was applied to each of Harris, U.S. Cons. Prod. Safety Comm., Direc. for Hlth. Sci., Washington, D.C. (R.E. Osterberg) the remaining 6 rabbits at a dose of 0.10 mL. Eyes were scored independently by 2 experienced readers at 1 and 4 hours and at 1, 2, 3, 7, 14 The reduction of pain and stress on laboratory and 21 days post-dosing. Statistical analysis animals is always a desirable goal. This can demonstra~ed that the greatest source of vari­ be accomplished only to the extent that the meth­ ability was rabbit to rabbit difference. Test od used to relieve pain does not jeopardize test group (one week to the other) and reader bias results. This study investigated the effects of (except at 4 hours) did not contribute to the treatment of the rabbit eye with anesthetics observed variability. Also, when comparing 0.10 prior to performing testing for eye irritation. Control rabbits were dosed with one of five model mL of G with 0.01 mL of B, a dose-effect re­ lationship was clearly discernable. When the irritants. Test rabbits were dosed with an anes­ thetic in both eyes and an irritant in only one compounds tested at 0.01 mL were ranked from of the eyes. Eyes were examined and scored with severest to mildest based on maximal score and and without an ocular slit lamp. All animals duration of effect, the order was the same as that obtained from previous studies using the were sacrificed on the 21st day post dosing and the eyes were retained for histopathological higher volume, but the effects were lessened. evaluation. Of the four anesthetics tested, Thus, reducing the dose volume from 0.10 to 0.01 tetracaine HCl, given in two equal doses 10-20 mL has little effect on the sensitivity of the minutes apart, was found to be the anesthetic of test (some very mild irritants may not be de­ choice for use in the rabbit eye. This drug tected), yet the extent and duration of damage to the rabbit eye is lessened. showed the combination of pain relief and the least alteration in ocular test scores.

664 A NOVELMETHOD FOR QUANTITATIVECUTANEOUS TOXICO­ KINETICS. J.M. Holland and J. Kao, Biology Div., 666 TOXICITY IN MONKEYSOF A RIBAVIRIN REGIMENFOR Oak Ridge National Lab., Oak Ridge, T~. USE IN LASSA FEVER PATIENTS. G.L. Wannarka, E.L. The skin is a primary route of exposure to Stephen, E.F. Jackson and P.G. Canonico, U.S. many occupational hazards as well as a surface Army Med. Res. Inst. of Inf. Dis., Ft. Detrick, upon which drugs and cosmetics are intentionally Frederick, MD 21701. Sponsor: F. Reno. applied. In spite of this there is little syste­ matic information dealing with the translocation The nucleoside, ribavirin (l-S-D-ribofuranosyl- and coupled bioconversion of surface applied 1,2,4-triazole-3-carboxamide), effective against materials by mammalian skin. high hazard viruses such as Lassa fever may pro­ To permit accurate quantitative comparisons duce hematological changes in some species. The across species, while preserving metabolic via­ present study determined the extent of these bility and the unit skin structure, we have de­ changes in monkeys given a ribavirin regimen to be signed a compact, multisample, skin permeability used in Lassa fever-infected patients. Rhesus mon­ chamber. One inch diameter skin discs form the keys were randomly assigned to either a ribavirin upper seal of a 1 ml chamber, through which fresh or saline control group. Drug was administered IV oxygenated tissue culture media is continuously in a 33 mg/kg loading dose, followed with 17 mg/kg pumped. The effluent from each chamber passes to q6h for 4 days, then 8 mg/kg q8h for an additional a fraction collector. The chamber is water jack­ 6 days; control monkeys received an equivalent eted, permitting incubation at any temperature. volume· of saline. Blood was drawn from the femoral Studies with benzo(a)pyrene on mouse skin re­ vein at selected intervals. Red blood cell counts veal that metabolism is linear through 5 micro­ decreased significantly by day 6 but returned to grams. The rate of benzo(a)pyrene metabolism, as pretreatment levels by the third week post-treat­ judged by recovery of radioactivity in the cham­ ment. Hematocrits were reduced 30% by day 7, ber effluent, is sigmoid, with the exponential reached their nadir on day 11 with a mean value phase occurring approximately two hours after ap­ of 22%. Hemoglobin values dropped to 7-8 mg/dl, plication. The overall extent of metabolism is remained at that level for 10 days, and gradually influenced by strain, sex and status of the mixed returned to pretreatment levels. Three days after function oxida.se system. Average % B(a)P metabo­ discontinuance of drug, reticulocytes increased lized in 18 hours (±SE) was 28(4) and 26(3) in over 400% and returned to control levels by day C3H female and male, respectively, while in 44. On day 5 of treatment, platelet levels in­ C57BL/6, a strain more sensitive to chemical skin creased, peaked on day 14 and returned to base­ carcinogenesis, the values were 29(2) and 35(1) line levels. No significant changes were observed in female and male, respectively. Following in in total and differential WBCcounts. Haptoglobin, vivo topical induction with 2 µg TCDD in 200 µl SGOT, SGPT, total and direct bilirubin, BUN, blood oj acetone, male C3H skin metabolized 28(1)% on glucose and body weight were not altered. These

190 data indicate that the proposed therapeutic regi­ ture on the response of platelets to phenol, and men of ribavirin modifies the normal hemogram of (3) the ability to overcome the inhibitory effect rhesus monkeys. Observed effects appear to be of phenol on platelet aggregation. fully reversible with hemograms returning to nor­ Platelet aggregation was measured by the mal limits upon discontinuance of ribavirin. This loss of turbidity in a Chronolog Aggregometer at is an important consideration in the treatment of 37°C, with the secondary wave quantified as the Lassa fever in man. percent change in light transmittance from 1-5 min after ADP induction. A one minute exposure to phenol was found to inhibit the secondary wave of ADP induced platelet aggregation in a dose de­ pendent fashion. When studied with relation to 667 SUBCHRONICTOXICITY OF LEVAIR®IN BEAGLEDOGS. the time and temperature of incubation of phenol A.C. Katz, D.W. Frank, M.W. Sauerhoff, and G.M. with platelet rich plasma, the inhibition was Zwicker. Stauffer Chemical Company, Environmen­ found to decrease with increasing pre-incubation tal Health Center, Farmington, CT. time at 37°C but not at 0°C. It was also found that the inhibitory effect of phenol can be over­ The purpose of this study was to determine the come by the addition of arachidonic acid. Thus, toxic effects of Levair® [NaAl3H14(P04)34H20J these findings confirm inhibition of secondary in beagle dogs when administered in the diet at aggregation by phenol. Furthermore, they suggest concentrations of 0, 0.3, 1.0 or 3.0 percent for the site of this action of phenol is at the in­ 6 months. Levair is widely used commercially as itiation of the secondary wave of aggregation. a leavening acid. Six male and 6 female beagle· This work was supported in part by grant 5T- dogs were assigned to each of the four treatment 32ES07026 from the National Institute of Environ­ groups. All animals were observed twice daily mental Health Sciences. for physical and behavioral changes. Body weights and food consumption were reported each week. Selected hematological and serum chemis­ 669 ACUTEAND SUBACUTE TOXICITY OF OCTACHLOROSTYRENE try parameters were measured during the pre-test IN THE RAT. I. Chu, D.C. Villeneuve, V. acclimation period and at 6-week intervals dur­ Secours, V.E.O. Valli and G.C. Becking. ing the study. Urinalyses were performed on· Environmental and Occupational Toxicology samples collected prior to initiation of the Division, Environmental Health Directorate, study, and at 8, 17 and 25 weeks. After 27 Ottawa, Ontario and Biopath Analysts, Guelph, weeks of treatment, all animals were necropsied. Ontario. Vital organs were weighed and tissues were exam­ Octachlorostyrene (OCS) is an environmental ined microscopically. No adverse treatment re­ contaminant found in the Great Lakes region of lated clinical signs were observed. Mean body North America. In view of the lack of toxicity weights of all groups of animals fed Levair were data on this compound, acute and subacu te comparable to those of the controls at all week­ experiments were carried out. For the acute ly determinations. Weekly mean food consumption study, groups of 10 male were administered by values of all male groups given Levair were com­ gavage single oral doses of OCS at levels of parable to those of the control group throughout 1300, 1690, 2190, 2850 or 3710 mg/kg and killed the study. Statistically significant (p<0.05) 14 days later. No deaths occurred at any dose food consumption depression was found sporadic­ level. OCS at a level of 1690 mg/kg and higher ally in all groups of female dogs given Levair. caused increased liver weights, mixed function No significant absolute or relative organ weight oxidase activity and serum cholesterol and uric differences were found between any of the Levair acid levels. In the subacute study, groups of treated groups and their respective controls. ten male and female rats were fed diets Evaluation of hematological, blood chemistry and containing 0.5, 5.0, 50 or 500 ppm of OCS for 28 urinalysis data revealed no toxicologically sig­ days. Growth rate and food consumption were not nificant trends. Microscopic examination showed affected by treatment. Liver hypertrophy and no histomorphologic changes considered to be hepatic microsomal enzyme induction were related to treatment. observed in animals fed 50 ppm OCS or higher. Elevations in serum cholesterol, total protein, potassium and SDH occurred in rats fed 500 ppm OCS. Histological changes occurred in the liver 668 PLATELETAGGREGATION: INHIBITION OF SECONDARY and thyroid of rats exposed to levels as low as AGGREGATIONBY PHENOL. M.L. Coan, K.R. Case, and 5.0 ppm of OCS. OCS residues accumulated in the H.B. Bosmann, Toxicology Training Program, Depts fat and liver in a dose-dependent manner. These of Radiation Biology and Biophysics and Pharma­ data suggest that although OCS has a very low cology and Toxicology, University of Rochester, acute toxicity, the administration of low levels Rochester, NY. Sponsor: T.W. CLarkson. in the diet for 28 days can produce both biochemical and histological changes in the rat Abnormal platelet function in patients with similar to other organohalogens. chronic renal failure has been associated with elevated levels of phenol and phenolic acids in serum. In vitro studies have demonstrated the 670 SUBACUTETOXICITY OF DIETARYCADINENE IN FISCHER inhibition --- of secondary aggregation by phenol, 344 RATS. D.E. Johnson, L.C. Uraih, W.R. Richter, suggesting the site of action to be at the re­ M.B. Powers, and D.C. Jessup, Int. Res. Dev. Corp. lease reaction. The studies discussed in this Mattawan, MI and Nat. Tox. Prog., Bethesda, MD. paper explored: (1) the dose related effect of one minute exposure to phenol on human platelet Cadinene, a mixture of isomers with partially un­ aggregation, (2) the effect of time and tempera- saturated sesquiterpenoid structures, occurs nat-

191 urally in essential oils from Junipers and Cedars. 672 IDENTIFICATION OF LABORATORYANIMALS. C.K.Wood, It is used as a flavoring agent in foods and as a C.E.Cover, W.A.Cook and J.R.Gibson. Haskell fragrance in cosmetics and soaps. Cadinene was Laboratory for Toxicology and Industrial Medicine. selected for the NTP Bioassay program because of Newark, Delaware 19711. the lack of toxicity or metabolism data in the literature and its human use pattern. In the 13- Federal guidelines regarding the conduct of tox­ week toxicity study, F344 rats (IO/sex/group) were icity studies specify that test animals be un­ given cadinene mixed in the diet at O (I), 3125 iquely identified. Means for identification have (II), 6250 (III), 12500 (IV), 25000 (V) and 50000 included ear tags, ear-/toe-clips and tatooes. (VI) ppm. Significant decreases in mean body Another method utilizing a radio transponder non­ wei.ght and/or food consumption were seen in all surgically implanted in each animal and a non­ ~reated groups except the II males. Mortalities contacting electronic interrogator has been de­ (four) occurred only in the VI group. Organ veloped. The transponder, which is implanted sub­ weight changes included increased liver (III - VI cutaneously in unanaesthetized animals, has cir­ groups) and decreased testicular (VI males), and cuitry for utilizing an external power source to thymic weights (V and VI groups). Histopathologi­ transmit one of a possible thousand different cal findings included toxic hepatitis: biliary identification codes, thus providing accuratever­ hyperplasia, hepatocellular degeneration, necrosis, ification of animal identification and assuring regeneration, and intracellular pigment occurred compliance to GLPs. The system also allows simul­ in various degrees among V, VI males and III - VI taneous collection of body weight data when coupl­ females. Toxic nephrosis: tubular degeneration ed to an electronic balance. In a preliminary (and cortical necrosis in animals that died) and evaluation of the system's potential utility in long-term bioassay programs, "dummy" implants of regeneration and pigmentation in tubular epithe­ the same size and consisting of transponder encap­ leal cells occurred in various degrees in all male sulation material were inserted into 10 weanling groups and IV - VI females. Testicular degenera­ tion occurred to a trace degree in IV, V males rats. After 14 months, a grossly-apparent thick­ and a severe degree in VI males; there was also ening of tissue surrounding the implants was seen atrophy of preputial and prostate glands. VI in all rats. The thickenings exhibited slow grow­ females revealed ovarian, endometrial and clitoral th and achieved relatively constant size 1-2 a trophy. Lymphoid depletion was evident in thymus months after first being observed. Gross patholo­ gical examination of 4 rats that died within 16 and mesenteric lymph nodes in the VI group. The findings in liver and kidney were considered to be months after implantation revealed the presence direct test article effects, whereas, effects on of fat tissue accumulation around the implants. reproductive and hematopoietic tissues may have Except for mild, transient dermatitis in the been secondary to inanition. region of the implant observed in 8 rats early after implantation, no gross abnormalities have been observed. Weight gain and blood chemistries have been normal. Tissue and systemic responses of 671 THE LEDTOXPROTOCOL SYSTEM: AN AUTOMATED, the rats to the implants presently appears to be INTERACTIVEPROTOCOL INFORMATION SYSTEM. favorable. Further progress in evaluating the R. S. Wasserman, J. F. Noble, M. Mandel, responses to these implants will be presented. Med. Res. Div./Information Services Div., Wilbur G. Malcolm Toxicology Labs., American Cyanamid Co., Pearl River, NY. 673 ACUTETOXICITY OF URANIUMEXTRACTANT (UE) AND UE An automated protocol information system has CO-SOLVENT. W.J. Powers, S.C. Gad and B.J. Dunn, been developed at the Medical Research Division Corporate Medical Affairs, Allied Corp., Morris­ of American Cyanamid as part of an interactive town, NJ real time data acquisition and reporting system Uranium extractant, an organophosphorus, was de­ supporting the operations of Toxicology Research. veloped with a dibutyl butyphosphonate co-solvent system for the recovery of low concentrations of The Toxicology Project Management Group enters uranium from phosphoric acid waste streams. The planning information via the planning subsystem. UE co-solvent was designed to be used with UE in The Study Director then interactively builds the the recovery process. A Tier I acute battery of remainder of the protocol using standard .menus tests were employed to establish a data base for and paragraphs to select the parameters, these compounds. UE and UE co-solvent produced activities and operating procedures required for· irreversible ocular damage in rabbits thus being the study. User specified parameters, schedules classified as corrosive eye irritants. Both UE and specific operating instructions may be used and UE co-solvent were also irritating and cor­ to override the standard defaults in special rosive to intact non-abraded rabbit skin. UE cases. A compound dictionary provides all test was a dermal nonsensitizer in guinea pigs. In substance related data. the acute dermal toxicity test in rabbits at 2 The elements and activities of the protocol then mL/kg, UE caused necrosis of the application site. drive the data acquisition work routines and Body weight was significantly (p < 0.05) de­ table generation programs. Schedules are pressed in treated animals for at-least one week provided for individual tasks such as animal post dosing. The testes showed evidence of weighing, clinical pathology and necropsy. germinal epithelial atrophy. UE was also tested Ordering information is made available to the for acute oral toxicity in F-344 rats at dose Animal Colony Management System. levels of 1.25, 3.0, 5.0 and 10.0 mL/kg in both Once the protocol is reviewed and approved, the sexes. The oral LD50 was 3.9 mL/kg in males and file is locked and all protocol amendments must 4.4 mL/kg in females. Females in the 3.0 mL/kg be entered via an edit program which maintains group showed a significant (p < 0.05) depression a disc based audit trail of all changes. of body weight. The rate of b;dy weight gain for

192 both males and females was significantly (p < 0.05) synthesis. Covalent binding of B to mtDNAresults depressed on days 1,4,7 and 11 post-dosing. - in an inability of mtRNApolymerase to transcribe Clinical observations deemed compound related in­ the genome which subsequently results in an in­ cluded: mydriasis, lacrimation, cyanosis, pros­ ability of translation. Since an inhibition of tration, diminished respiration, hemsturia and mt protein synthesis can result in a loss of cel­ salivation. Necropsy observations associated lular energy derived from oxidative phosphoryla­ with UE treatment included: liver discoloration, tion, the consequence in BM cells might be an in­ urogenital staining, hemorrhagic lung, intra­ hibition of the maturation of the precursors of cranial hemorrhage and porphyrins out of mouth, the circulating blood cells leading to aplastic nares and eyes. In sunnnary, acute exposure to UE anemia. (Supported by ES00322). resulted in severe ocular and dermal irritation and both oral and dermal systemic toxicity. The co-solvent is similarly a severe ocular and dermal irritant. 675 THEEFFECT OF ANEXPERIMENTAL REDUCTION OF SODIUMIN DRINKINGWATER ON THE BLOOD PRESSURE DISTRIBUTIONPATTERNS OF ELEMENTARYSTUDENTS. 674 BENZENEINHIBITS MITOCHONDRIALRNA AND PROTEIN E.J. Calabrese and R.W. Tuthill, Division of SYNTHESIS G. Kalf, T. Rushmore, and R. Snyder*, Public Health, University of Massachusetts, Thomas Jefferson Univ., Phi la., PA and *Rutgers Amherst, MA. Univ., New Brunswick, N.J. An experimental bottled water study Chronic exposure to benzene (B) causes progres­ assessed the effect on blood pressure (BP) of sive degeneration of bone marrow (BM) and aplas­ lowering sodium concentration in the water of tic anemia or leukemia. B-induced anemia might some fourth graders of a "high" sodium result from covalent binding of a toxic metabolite communitywho had previously been shown to to BM mitochondrial (mt) DNAand subsequent inhi­ have significantly higher blood pressure than bition of mt transcription and translation. Mt comparable students in an adjacent "low" from the livers of partially hepatectomized rats sodium communityclosely matched for injected ip with B (2200 mg/kg) 10 hr post-hepa­ socio-demographic variables of concern. For tectomy show an inhibition of mtRNA synthesis three months, trios of children matched by when incubated in vitro. Regenerating rat liver sex, school, and baseline BP, used different mt, free of microsomal contamination, incubated water for all cooking and drinking purposes, with B in vitro show a dose-dependent inhibition with BP monitored bi-weekly. Pupils were of all three types of RNAsynthesis and an inhi­ randomly allocated to the three water bition of protein synthesis. The effects of conditions: (l) high sodium water bottled benzene on mt macromolecular synthesis do not from their own communitydistribution system, appear to result from solvent effects since (2) low sodium water bottled from the equivalent concentrations of toluene do not in­ distribution system of the comparison hibit. Inhibition of mtRNA synthesis by B requi­ communitywith sodium added to the level of res NADPHsuggesting that B is activated by a the high sodium communitywater, and (3) low mono-oxygenase system in the organelle. Mt from sodium water bottled from the distribution rat liver, incubated in vitro with 1 mMB, metab­ system of the low sodium communitybut with no olize the B to hydroquinone, catechol, phenol and sodium added. two unknown compounds as determined by HPLC; one Preliminary findings indicated that BP of these compound may represent the active spec­ levels amongthe girls but not the boys on the ies which covalently binds to mtDNA. Most impor­ low sodium water exhibited marked decreases in tantly, mt from rabbit BM cells incubated in BP over the test period when compared to the vitro with l mMB also show an inhibition of RNA other two "high" sodium groups.

193 Author Index

A Barkdoll, E. 401 Boyer, I.J. 291 Aaron, C.S. 231 Barkley, J.J. 122 Boykin, E. 217 Abbott, B.J. 642 Barna-Lloyd, T. 92 Boysen, B.G. 390 Abbracchio, M.P. 169 Barnes, T.B. 193,271 Brabec, M.J. 469 Abdo, K.M. 338,541 Barnett, J.B. 422 Bradbrook, C. 374 Abra"ham, R. 405,418,457,550 Barron, K. 405 Brandt, L.D. 336 Adams, E.R. 95 Barrow, C.S. 38 Braselton, W.E. 10,27 Adams, R.A. 369,533 Barrows, LR. 493 Braun, W.H. 112,494 Adams, W.D. 546 Barsoum, M. 389 Breeze, R.G. 341 Bass, B.F. 522 Adams, W.D. 194 Brewster, D.W. 315 Bates, H. 418,419 Adler, V. 508 Brice, M.A. 650 Baxter, C.S. 322 Agin, G.L. 500 Briggs, G.B. 262 Beamand, J.A. 276,658 Ahmed, A.E. 588 Britt, A.L. 228 Ahmed, N. 69 Becker, R.A. 241 Bracco, M. 304 Alam,M. 279 Becking, G.C. 651,669 Brock, W.J. 77 Aldrich, F. 582 Bedell, M.A. 357 Brodeur, J. 183,509,511,514 Al-Hassan, W.J. 447 Beer, D.G. 477 Brogan, W.C. 253 Ali, S.F. 538 Belizi, P.J. 211 Bronaugh, R.L. 640 Amdur, M.0. 212 Bell, T.J. 561 Brooks, A.L. 308,565 Amoruso, M.A. 16,36 Belson, A.R. 628 Andersen, M.E. 517,661 Benitz, K.F. 405,418 Brooks, L. 320,346 Anderson, J.A. 30,416,660 Benson, E.B. 311 Brophy, G.T. 487 Andrew, F.D. 421 Benson, J.M. 51,171 Brown, C.H. 82 Angevine, L.S. 564,596 Berez, J.P. 347 Brown, E.M. 26 Annau, Z. 208,289 Berkowitz, A.S. 103 Brown, R.D. 518,519 Anthony, D.C. 491 Berndt, W.0. 9,145,263 Brown, W.E. 577 Applebey, E.C. 354 Bernfeld, P. 369 Bruckner, J.V. 103,113,480,595,621 Appleton, B.S. 91 Bernstein, D.M. 47,48,209 Bruner, R.H. 567,661 Arceo, R.J. 647 Bernstein, M.S. 110 Brunkhorst, C.S. 488 Arezzo, J.C. 490 Berry, V.K. 632 Brusick, D. 610 Ashby, J. 426,636 Berteau, P.E. 417 Buben, J.A. 135 Atchison, W.D. 64,203 Beteet, E. 279 Buccafusco, R.J. 158 Auansakul, A.C. 440 Beuthin, F.C. 198,290,544 Bucher, J.R. 97,190 Aulerich, R. 508 Biagini, R.E. 297 Buening, G.M. 127 Aust, S.D. 97,190,351,352 Bibeau, L.M. 21 Burant, C.F. 498 Avery, D.L. 420,422 Bick, P. 335 Burden, E.J. 488 Ayres, J.A. 40 Bickis, M.G. 318 Burek, J.D. 561 Bierbower, G.W. 665 Burgess, B.A. 37,558 B Bierkamper, G.G. 206,319,552 Burgess, J.R. 105 Babish, J.G. 259,433,434 Billings, K.C. 357 Burke, T.R. 7 Back, K.C. 517,661 Birky, M.M. 218,219 Burks, T.F. 400 Baggs, R.B. 264,291 Bissette, G. 98 Burns, J.M. 122 Bagley, D.M. 90 Blackburn, K.L. 149 Buroker, R.A. 393 Bailey, G.S. 362 Blackett, N.M. 637 Baker, K.L. 607 Bursian, S. 144 Bleavins, M. 508 Balazs, T. 31,395,449,649 Busby, W.F. 582 Blumberg, J.B. 302 Balk, M.W. 390 Bushnell, P.J. 288 Boelsterli, U.A. 449 Ballhorn, L. 481 Byard, J.L. 80 Bogdanffy, M. 442 Balmer, M.F. 223 C Balogh, L.A. 178,625 Boorman, G.A. 326 Bandiera, S. 475,476 Booze, R. 289 Calkins, J.E. 662 Bank, P.A. 454 Born, G.S. 67 Cameron, A.M. 12 Bansal, S.K. 464,536 Borzelleca, J.F. 373,613 Campbell, M.A. 475,476 Baraban, S. 590,648 Bosmann, H.B. 668 Campbell, T.C. 91,117,348,349 Baran, K.C. 652 Bowden, G.T. 363 Campbell, W.R. 423 Baranyi, B.L. 100,364 Bowman, J.M. 432 Canonico, P.G. 619,627,646,666 Barbeau, I. 117 Boyd, J.N. 348,349 Carchman, R.A. 373,613 Barbieri, 0. 245 Boyd, R. 429 Carlson, G.M. 533

The numerals following author's name refer to abstract numbers.

194 Carlson, G.P. 67 Cook, A.W. 672 Dhami, M.S.I. 424 Carlson, J. 457 Cook, M.W. 71 DiCapua, R.A. 333,334 Carlton, B.D. 437 Cooper, K. 160 Dickie, B.C. 390 Carlton, W.W. 128,484 Copeland, M.F. 385 Dickins, J. 64 Caro, J. 429 Corbett, S.W. 461,462 Dickinson, J. 531,542 Carpenter, S.J. 415 Cornish, H.H. 455 Dieter, M.P. 326 Carter, S. 407 Cory-Slechta, D.A. 287,291 Difonzo, C. 108 Carter, S.D. 268,269 Cosgriff, T.M. 627 Digenis, G.A. 580 45,571,572 Casale, G.P. 333,334 Costa, D.L. Dimmler, D.G. 47,48 Casciano, D.A. 316 Costa, L.G. 303,525,618 Dinowitz, M. 376 Case, K.R. 668 Costa, M. 20,169,215 DiRenzo, A.B. 76 Cater, K.C. 350 Cote, I. 120 Dirks, R.C. 237 Cenedella, R.J. 319 Cottrell, R.C. 49,603 DiStefano, V. 600 Cerimele, B.J. 398 Cover, CE. 672 Distlerath, L.M. 612 Chan, T.L. 176 Cowan, M.J., Jr. 567 DiVincenzo, G.D. 57,60 Chang, J.M. 140,468 Cox, J.L. 204 Doctor, S.V. Chang, J.C.F. 38 Cox, M. 563 207,303 Chang, K.J. 492 Craig, C. 547 Dodd, D.E. 560 Chang, L.W. 22,304,306 Cranmer, J.M. 420,422 Doherty, P.A. 503 Chang, M.W. 634 Cranmer, M.F. 420,422 Doherty, R.A. 293,296,410 Chapatwala, K.D. 186 Creasy, D.M. 654 Donofrio, D.J. 342,556 Chapin, R.E. 274 Crofton, K. 339 Donoso, J.A. 256 Charles, J.L. 613 Cruz, B.D.O. 435 Donovan, M.P. 252 Chen, H.H.C. 643 Curley, A. 305,409 Dotzlaf, L.A. 100 Chen, K.-C. 225 Curto, K.A. 252,253 Dougherty, W.J. 628 Cheng, M. 604 Cushman, J.R. 340 Douglas, W.H.J. 482 Cherry, L.D. 382 D Doull, J. 617 Chetty, K. 279 Downs, J.R. 146 Dabbs, J.E. 79 Chhabra, R.S. 342,437 Dudek, B.R. 524 Dacre, J.C. 121,123,124 Chin, A.E. 437 Dudley, R.E. 187 D'Addario, A.P. 567 Cholakis, J.M. 121 Dunn, B.J. 673 Dallas, CE. 595 Choo, H. 590 Duquette, P.H. 443 Chow, S.A. 631 Daniel, J.W. 614 Durham, H.D. 530 Chrisp, CE. 566 Dankovic, D. 455 Durham, W.F. 409 Chrisp, CL. 298 Dansie, D.R. 165,166 Dyer, R.S. 535 Christensen, R.C. 315 D'Antuono, M.E. 102 Dyroff, M.C. 240 Christian, B.J. 460 Dasheill, O.L. 643 Christian, J.E. 67 Daston, G.P. 201 E 142,143 Dauterman, W.C. 86 Christian, M.S. Easley, J.R. 629 300 David, R.M. 72 Christie, N.T. Eastman, D.F. 83,84 Chu, I. 651,669 Davies, P.J.A. 213 Eaton, D.L. 317 Cihla, H.P. 4,229,345 Davis, M.E. 512 Ebron, M.T. 412 Cimprich, R.E. 34 Davis, T.E. 662 Edwards, D.F. 343 Clark, A.J. 226 Dawson, R., Jr. 552 Ehrich, M. 6 Clark, I. 346 Dean, J.H. 326 Eigenberg, D. 617 Clarkson, T.W. 188 DeBaecke, P.J. 34,247,248 Elbeera, M.A. 615 Cline, P.R. 321 DeBaun, J. 425 Eling, T. 639 Cmehil, D.W. 310,365 deBethizy, J.D. 384 Elliott, B.M. 426,636 Coan, M.L. 668 Debona, M. 642 Ellis, H.V. 121 Coate, W.B. 576 DeCaprio, A.P. 550 El-Sebae 305 Cobel-Geard, S.R. 148 Deimling, M.J. 33,191 Elshabrawy, 0. 588 Cockrell, B.Y. 570 Deisinger, P.J. 60 Emmerling, D. 391 Cojocel, C. 28 Delio, D.A. 534 Engelsberg, B.N. 428 Coles, R.B. 373 Deluca, H.F. 288 Enna, S.J. 303 Collins, M.A. 49,603 Democko, CJ. 168 Enoch, H.G. Combs, A.B. 448 Dennet, D. 259 599,608 Comereski, C.R. 393 Dent, J.C. 59,62,2 73,358,359 Erickson, D.E. 10 Conaway, CC. 499,576 DePass, L.R. 225,366 Enriquez, P.M. 57 Condie, L. W. 467 Desaiah, D. 186,464,536 Erker, E.F. 96 Congdon, E.R. 640 Detlefs, CL. 61 Erslev, A. 429 Conner, M.K. 604,605 Detweiler, D.K. 34 Essigmann, E.M. 344 Conning, D.M. 353,354 Dey, M.S. 341,523 Evans, J.C. 353,354 Conolly, R.B. 233,447,454 Dey, R.A. 341 Evans, R.M. 215 Conroy, P.J. 539 Deyo, D. 563 Exon, J.H. 323,328

195 F Gerson, R.J. 14 H Falahee, K.J. 483 Gianutsos, G. 533 Haber, S.B. 573 Farmer, J.D. 309 Gianutsos, G. 192 Hadley, W.M. 51 Farmer, J.H. 370 Gibson, J.R. 37,672 Hakkinen, P.J. 625 Farmer, J. 409 Gibson, K.K. 282 Hall, J.E. 439 Farooqui, M.Y.H. 588 Giesler, P.J. 626 Hall, W.C. 166 Feder, P. 32 Giles, R.C. 204 Hall, W.C. 376 Fekete, A.E. 390 Gillespie, T.J. 74 Hamid, R. 81 Feldman, S. 113,595 Gillies, P.J. 162 Hamlin, R.I. 32 216,597 .Ferin, J. Ginsberg, G.L. 444,445 Hanika-Rebar, C. 128 Ferm; V.H. 246,503 Glass, L. 290 Hanley, T.R., Jr. 148,414 Ferrante, J.G. 153 Glaza, S.M. 390 Hannan, M.A. 608 Ferrell, J. 267 Goering, P.L. 280 Hanson, R.L. 171 645 Ferrell, J.F. Goetchius, M.P. 91 Harbison, R.D. 133,641 Ferro, M. 245 Gold, B.G. 548,549,551 Harden, M. 625 Feuer, G. 108,424 Goldenthal, E.I. 266 Hardyniec, K.S. 254 Fiala, E.S. 499 Goldman, M.E. 582 Harkin-Frenkel, D. 507 Filler, R. 578 Goldstein, B.D. 16,36,235,430 Harmon, J.R. 151 Finch, R.A. 602 Gombar, C.T. 73,493 Harrington, F. 465 Finkelstein, J.N. 569 Goodman, J.I. 351,352,364 Harris, R.A. 11,49 Fisher, H.L. 294,295 Harris, R.L. Gordon, D.E. 122 665 Fisher, G.L. 168,298,562,634 Harris, S.B. 423 Gorzinski, S.J. 92,119 Fochtman, F.W. 17 Harroff, H.H. 566 Gotera, 0.G. 605 Folkers, K. 448 Hart, R.W. 634 Gough, A. 389 Forney, R.B. 336,398,615,616 Hartung, R. 155 Gould, D.H. 450 Foss, J.A. 202,532 Hassan, M. 52 Gracon, S. 389 Foster, J.R. 654,658 Hasan, T. 52 Foster, P.M.D. 71,654 Graepel, GJ 663 Hassler, C.R. 32 Fox, T.R. 29,129 Grafton, T.F. 250 Hastings, S.E. 377,620 Frank, D.W. 667 Graham, J.A. 167,217 Hatch, G.E. 217 Frankel, R. 625 Graham, T.M. 421 Havkin-Frenkel, D. 507 Franklin, M.R. 436 Graichen, M.E. 358,359 Hawk-Prather, K. 450 Frederick,. K.A. 434 Gralla, E.J. 556 Hayashi, H. 1 Freudenthal, R.I. 425 Grasso, T.L. 299 Hayes, A.W. 145,263 Friedman, M.A. 488,602,626 Gray, J.A. 257 Hayes, A.W. 9,145,263 Froehlich, R. 385 Gray, L.E., Jr. 267 Hayes, T.J. 388 Fu, F.W.Y. 480 Gray, T.J.B. 276,658 Hayes, W.C. 148,414 Fujimoto, J.M. 102,311 Green, A.H. 577 Hayward, K.M. 271 Furedi, E.M. 122 Green, C.E. 79 Head, R.A. 469 Furry, J. 381 Greenlee, W.F. 463,516 Heath, E.M. 157 G Greenman, D.L. 370 Heck, H. d' A. 72,88,555 Greenspan, B.J. 214 Heck, J.D. 169 Galendo, D. 224 Greenspun, K.S. 390,648 Heifetz, C.L. 611 Gamma!, L. 590,648 Greenwell, A. Hein, J.F. 268,269 Gangolli, S.D. ll,49,71,267,603,654,658 465 Gregory, R.E. 181,565 Heindel, J.J. 103 Gardner, D.E. 217 Gregus, Z. 441 Henck, J.W. 557 Gardner, H.K. 264 Greim, H. 481 Henderson, B.M. 628 Gardner, R.J. 37 Griffis, L.C. 180,210 Hendricks, J.D. 362 Garfinkel, S. 177,178 Griffiths, J. 429 Hendricks, M.J. 529 Garg, B.D. 175 Gross, E.A. Hengler, W.C. 451 Garner, R.J. 327 38 Grossman, S.J. 579 Henley, C.M. 518,519 Garst, J.E. 159 85,477 Henry, C.J. 165,166 Gasiewicz, T.A. 510 Grubbs, J.H. Guengerich, F.P. 134,501 Henry, J.E. 643 Gavigan, F.A. 546 Guerin, M. Henry, S. Gee, S.J. 381,450 177 279 Guest, D. 60 Hering, W.E. 176 Geiger, L.E. 501 Guion, C. Hermann, E.A. 29,119,494 Gellatly, J.B.M. 93 459 Gemborys, M.W. 10 Gumbrecht, J.R. 436 Herman, E. 31 Gentry, G.D. 574 Gunnoe, M.D. 382,642 Hernandez, Y.M. 69 George, A.J. 247,248 Gunsett, S.L. 171 Hewitt, L.A. 470 George, J.D. 89 Gupta, B.N. 126 Hewitt, W.R. 26,470 George, J.W. 138 Gurba, P.E. 411 Heyman, R. 533 Georgi, J.R. 433 Gushow, T.S. 223,414,561,589 Higginbotham, J.D. 614 Gerhart, J.M. 307,404 Guzzie, P.J. 451 Highman, B. 370

196 Hilderbrand, R.L. 568 Jenkins, R. 177 Kohsaki, M. 568 Hinson, J.A. 12 Jensen, R.K. 351,352 Komsta-Szumska, E. 19 Hitchcock, M. 478 Jernigan, J.D. 133 Kontur, P. 284 Hite, G. 533 Jersey, G.C. 92,119 Kornbrust, D.]. 458 Hjelle, J.J. 85,477 Jessup, D.C. 670 Kostyniak, P.J. 106 Ho,C.Y. 105,238 Joachim, F. 68 Kotik, H. 449 Ho, I.K. 3 Jobe, P.C. 518,519 Kouri, R.E. 165,166 Hobbs, C.H. 180 John, J.A. 148,414 Kovatch, R.M. 376 Hobbs, E.J. 623 Johnson, B.E. 434 Kozumbo, W.J. 104 Haberman, A.M. 142,143 Johnson, D.R. 282 Kram, D. 613 Hobson, M. 186 Johnson, D.E. 266,670 Kramer, A.W. 628 Hochrainer, D. 213 Johnson, D.F. 393 Kreiser; D.M. 361 Hodges, K. 518,519 Johnson, E. 279 Krinsky-Feibush, P. 111 Hodgson, E. 98 Johnson, K.A. 414 Kritko, C.F. 17 Hoffman, G.M. 220 Johnston, C.D. 93 Krivanek, N.D. 559 Hoffman, T. 418 Jones, A.B. 591 Kroll, R. 104 Hogy, LL. 501 Jones, R.E. 489 Kropscott, B.E. 112 Hoheisel, C.A. 665 Jones-Price, C. 249,251 Kruger-McDermott, C. 277 Holdsworth, C.E. 39 Joseph, X. 649 Krull, I. 442 Holland, J.M. 629,664 Jourdian, G.W. 155 Ku, R. 292 Hollingsworth, P.J. 524 Joynes, S. 403 Kuo, C.-H. 100 Kupetz, S. 518,519 Holmberg, R. 177 K Holsapple, M.P. 335 Kurtz, P.J. 342,391,407,437 Kalf, G. 674 Holson, J.E. 423 Kuschner, M. 209 Kalyanaraman, B. 639 Hamburger, F. 369 Kuttab, S. 52,442 Kaminsky, L. 312 Hong, C.B. 121 Kutz, S.A. 34,247,248 Kanagalingam, K.K. 166 Hong, J.-S. 538 Kutzman, R.S. 570,571,572,573 Kanapilly, G.M. 170 Hook, B.S. 127 Kwon, T.J. 468 Kanerva, R.L. 553 Hook, J.B. 10,27,28,100,508 Kyle, G. 103,621 Kao, J. 664 Hottendorf, G.H. 393 Kapeghian, J.C. 193,591 L Houser, W. 78 Kardish, R. 432 LaBorde, J.B. 249,251 Howard, LC. 95,642 Kastello, M.D. 619,627,646 Labosh, T.J. 299 Howd, R. 378,531,542 Kastl, P.E. 494 Lacey, S.A. 433 Howe-Baughman, J.K. 10 Kato, S. 2 Lake, B.G. 1 l,49,353,603,658 Howell, W.E. 535 Katz, A.C. 667 Lake, R.S. 606,610,611 Howland, R.D. 655 Kavlock, R.J. 201,257 Lallian, B.S. 235 Hsieh, K.H. 492 Kaylor, W.H. 607 Lalwani, N. 1 Hu, P. 572 Keesey, R.E. 461,462 Lam, C.-W. 600 Hudson, L.G. 463 Kehrer, J.P. 653 Lam, H.F. 212 Huggins, D.J. 408 Keim, G.R. 392 Landry, T.D. 112,500,589 Hulebak, K.L. 208 Keller, W.C. 661 Lane, R.W. 373 Hunt, E.J. 242,638 Kelty, K. 23 Lange, D.G. 102,311 Huxtable, R.J. 363 Kendall, D. 303 Langvardt, P. 136 Hwang, K.K. 165,376 Kenel, M. 139 Laskey, J.W. 267,268,269 I Kenley, R. 378 Lasser, M.S. 219 Iatropoulos, M.J. 647 Kenney, R.A. 219 Last, J.A. 164 Idris, A. 449 Kerns, W.D. 566 Law, A.Y.C. 456 Infurna, R. 152,260 Kerns, W.D. 556 Lawhorn, G.T. 553 Ioannou, Y.M. 56 Ketterman, A. 497 Leapman, S.B. 336 Ireland, J.S. 386 Keyes, D.G. 557 Ledoux, T.A. 249,251 Irwin, M.J. 647 Killinger, J.M. 146,270 Lee, B.C. 396 Ison, J.R. 202,532 Kim,D. 117 Lee, P.S. 176 Kim,S.N. 660 Lee, T.P. 492 J Kimmel, G.L. 151,250 Lee, Y.H. 185 Jackson, E.F. 666 Kindt, V. 429 Lefevre, R. 405,418,419 Jackson, N.M. 447 Kirchner, F.R. 489 Lehnert, B.E. 597 Jacobson, K.B. 300 Kish, P. 406 Leibman, K.C. 70 Jaken, S. 244 Kitchin, K.T. 412,506 Lemberger, L. 398 James, R.C. 641 Klein-Szanto, A.J.P. 164 Lento, J.W. 496.586,587 Jameson, C.W. 326 Klingensmith, J.S. 132,466 Leonard, T.B. 356 Jankauskis, J. 452 Kluwe, W.M. 465 LeQuesne, P.W. Ill Jayasekara, U. 660 Knadle, S.A. 80 Lerman, S.A. 188 Jellett, E. 45 Kociba, R.J. 119,557 Leslie, R. 608

197 Leung, H.W. 236 McNeill, K.L. 168 Mincer, H.H. 652 Le Valley, S. 283 McNeill, T.H. 539 Minor, J.L. 123 Levi, P. 98 McNulty, M.J. 88 Minor, J.L. 146 Levin, A.A. 273,621 McQueen, C.A. 361 Miska, S.P. 151 Misra, H.P. 453 Levinskas, G.J. 417 M Lewandowski, E. 448 Misslbeck, N. 117,348,349 MacAskill, S.M. 211 Lewis, S.C. 39 Mitchell, D.B. 239 MacGregor, J.A. 147 Mitoma, C. (i,A.P. 308,565 54,68 Mackenzie, B. 22 Modrak, J.B. 33 Lin, P.T. 86 MacPhail, R.C. 521 Moehle, C. 1 "Lin, 5.-N. 480 Mactutus, C.F. 289,537 Mokler, B.V. 179 Lindamood, C., III 357 Madissoo, H. 393 Mondino, A. 245 Lindemann, N.J. 443 Magee, P.N. 73,493 Moore, G.S. 5,43,44 21 Linden, J. Makkawy, H.M. 624 Moore, R.W. 329 Lindenschmidt, R.C. 615 Malek, D.E. 218,219 Moorman, W.J. 41,297 Lingg, R.D. 607 Mandel, M. 671 Morahan, P. 335 Lish, P.M. 122 Mankes, R. 405,418,419 Morehouse, B. 216 Liu, J. 13 Mann, E. 279 Morehouse, L.A. 97,190 Livesey, J. 136 Manson, J.M. 89,261 Moreland, D.E. 380 Lock, S. 164,177,178 Marangos, P.J. 198 Morita, T. 564 Lockard, J.M. 599 Margolis, R.L. 18 Morris, J.E. 421 Lockard, V.G. 131,596 Marks, T.A. 249,251 Morris, P.L. 7 Locniskar, M. 331 Maronpot, R.R. 42 Morrow, P.E. 214 Loeb, A. 419 Marquis, K. L. 652 Mosberg, A.T. 562 Long, R.M. 58 Martin, R.A. 108 Mosher, C. 378 Marty, M.A. 55 Longstaff, E. 374 Mottent, K.H. 44 Mason, R. 639 Loper, J.C. 612 Mottet, N.K. 18 Maslansky, C.J. 360 Lotti, M. 497 Mudge, G.H. 10 Massaro, E.J. 105,238,286,299 Lowther, D.K. 665 Mukku, V.R. 386 Massaro, T.F. 286 Luebke, J.P. 255 Munro, J.C. 318 Mast, R.W. 602,626 Munson, A.E. Lui, E.M.K. 184 330,335 Matheson, D.W. 94 Murphy, J.P. 661 Luo, J.E. 605 Matter, R.H. 275 Murphy, M.J. 568 Luster, M.I. 326 Matthews, H.B. 56 Murphy, S.D. 303,379,495,525,618 Luthra, R. 103,621 Mauderly, J.L. 179 Murray, M.T. 192 Lynch, D.W. 25,41 Maurissen, J.P.J. 401,402 Muscarella, D. 259 Lysy, H.H. 594 Mavis, R.D. 236,569 Mushak, P. 294,295 Mayrua, K. 263 Mc Myers-Basting, L. 36 Mays, J.B. 12 McAllen, S.J. 210 Meeler, F.J. 124 N McCafferty, R.E. 252 Meeks, R.G. 645 Nabeshima, T. 3 McCall, C.0. 90 Mehrotra, B.D. 536 Nachtman, J.P. 581 McCarthy, M. 5 Meier, J.R. 310,607 Nakashima, J. 80 McCarty, L.P. 500 Melancon, M.J. 583 Nakatsugawa, T. 116 McCauley, P.T. 23 Melnick, R.L. 656 Naman, T.M. 172 McClain, R.M. 388 Menzel, D.B. 167 Narahasi, T. 203 McClanahan, J.S. 580 Merigan, W.H. 401 Narbaitz, R. 397 McClellan, R.O. 170,179,180,308 Meyer, J.J. 193 Naser, L.J. 341,523 McCollister, S.B. 119 Miaullis, J.B. 425 Nash, Stephen D. 558 McCoy, J. 346 Mico, B.A. 138 Nauss, K.M. 331 McCracken, M.S. 553 Milks, M. 227 Neal, G.E. 372 McCullough, C.B. 93 Miller, C. 230 Neighbors, L.A. 256 McDougal, J.N. 76 Miller, D.B. 101,205,535 Nelson, C.J. 250 McElligott, T.F. 446 Miller, D.R. 19 Neptun, D.A. 359 McGuire, E.J. 30,606 Miller, F.J. 167 Nera, E.A. 318 McGuire, P.F. 318 Miller, F.J. 217 Newman, K.N. 645 McKelvey, J.A. 496,586,587 Miller, G.D. 286 Newmann, E.A. 325 McKenna, M.J. 40,223,561,589 Miller, K.W. 63 Newton, G.J. 171 McKim,J.M. 157 Miller, M.J. 527 Newton, J.F. 10 McKinney, J.C. 553 Miller, M.S. 400,527 Ning, J. 442 Mclamb, J. 267 Miller, R.E. 134 Nitschke, K.D. 494 McMahon, F.J. 231 Miller, R.R. 40,148 Noble, J.F. 671 McMartin, D.N. 312 Millington, W.R. 206 Noden, D. 259 McNeill, D.A. 298 Millis, C.D. 351 Nolan, R.J. 500,502

198 Noland, E.A. 23 Ponomarkov, V. 222 Robinson, K.S. 650 Norback, D.H. 4,229,345,355 Popenoe, E.A. 570 Robinson, M. 374 Norris, J.C. 3 Popp, J.A. 273,274,356,359 Robinson, M. 310,365 Norton, R.M. 274 Porter, W.R. 64,65,66,115 Robinson, R.D. 293,296 Norton, S. 256,526 Posen, G. 432 Robison, A.K. 386 Novilla, M.N. 95 Posner, H.S. 314 Robison, S.H. 20 Post, G. 234 Rocco, R.M. 302 0 Pott, F. 216 Rodwell, D.E. 262,417 Obaseki, A.O. 115 Potter, C.L. 515 Rock, G.A. 432 Oberdorster, G. 213,216 Potts, W.J. 561 Rock, S.G. 609 O'Callaghan, J.P. 205 Pounds, J.G. 133,195,316 Rockwood, G. 418 O'Flaherty, E.J. 135,163,194 Powers, M.B. 670 Rockwood, W.P. 418 Ofner, P. 111 Powers, W.J. 673 Rodier, P .M. 410 Ohno, M. 2 Prentice, B.A. 562 Roe, D.A. 117 O'Keefe, P. 312 Pryor, G. 531,542,543 Rogauskas, J.F. 114 Oldiges, H. 213 Purchase, I.F.H. 374 Rogers, A.E. 120,435 Olgiati, K.L. 635 Putcha, L. 113,598 Rogers, A.M. 517 Olin, S.S. 483 Putnam, C.W. 74 Rogers, J. 54 Olson, M.J. 316 Putzig, C. 136 Rogers, R.R. 327,332 O'Neal, F.O. 575 Roginski, E.T. 198 Onosaka, S. 24 Q Rose, C.S. 483 Osborne, R. 232 Quast, J.F. 29,92,112,130,223,494 Rose, M.S. 427,637 Osimitz, T.G. 233 Rosen, J.D. 507 R Ostby, J. 267 Rosenblum, I. 277,405,406 Osweiler, G.D. 127 Racz, W.J. 446 Ross, S.M. 547 Ouellette, J.H. 148 Rajanna, B. 186 Ross, T. 178 Raje, R.R. 35,118 p Rossi, L. 245 Ramsey, J.C. 112,494 Roth, R.A. 100 Page, N.P. 483 Rao, G.N. 390,626 Rowland, LR. 293,296,603 Palmer, T.E. 626 Rao, G.S. 235,430 Royal, P.D. 146,270 Palmieri, M.A. 158 Rao, K.S. 148,414 Royer, R.E. 308 Park, C.N. 223 Rau, L.A. 156 Rozman, T. 481 Parker, C.M. 93 Rebert, ClS. 531,543,630 Rubin, R.J. 104 Parkinson, A. 474,475 Reddy, C.C. 105,238,286,299 Rucci, G. 510 Parulekar, M.R. 397 Reddy, G. 4,229 Rugen, P.J. 247,248 Paustenbach, D.J. 67 Reddy, J. 1 Rushmore, T. 674 Pavkov, K.L. 556,566 Reddy, M.K. 1 Russell, D.H. 515 Pazdernik, T. 617 Reddy, R.V. 145 Rust, J.H. 122 Pederson, T.C. 173,174 Redman, H.C. 179 Rutkowski, J.V. 246 166 Pegg, D.G. 584 Reed, S.M. Rozman, K. 481 269 Peisch, R. 212 Rehnberg, G.L. Rusch, G.M. 220 21 Pence, P.J. 398 Reid, M.C. Rush, G.F. 508 489 Penny, J.E. 518 Reilly, C.A., Jr. Rylander Yueh, L.A. 50 Pereira, M.A. 228,459 Reiser, K.M. 164 Perry, D.F. 61 Reitz, R.H. 29,136 s Perry, J.D. 385 Render, J.A. 484 Sabharwal, P.S. 599 Persing, R.L. 566 Reno, F.E. 619 Safe, L. 476 Peters, A.C. 342,437 Renzi, B.E. 341,523 Safe, S. 473,474,475,476 Petersen, D.R. 85,477 Reuhl, K.R. 19,22,306 Sager, P.R. 410 Peterson, F .J. 443 Rheinwald, J. 321 Salama, A.E. 624 Peterson, LG. 225,366 Rice, R. 178 Salerno, A.J. 231,585 Peterson, R.E. 64,65,66,329,460,461,462 Rice, R.H. 107,321,516 Salocks, C.B. 80 Petrere, J. 416 Richardson, R.J. 408,411,524 Salva, P. 533 Petry, T.W. 363 Richter, W.R. 670 Sanders, D.R. 110 Pfeifer, K.F. 158 Rick, D.L. 500,502 Santone, K.S. 243 Phillips, J.C. 11,49,603 Rickard, R.W. 162 Sanyer, J.L. 660 Phillips, M. 244 Rickert, D.E. 58,59 Sarkar, C.P. 319 Pickrell, J.A. 181,210 Ridder, W.E. 616 Satoshi, T. 394 Pitot, H.C. 345 Riddle, M.M. 327,332 Saunders, J.H. 500,502 Pitts, L.L., II 567,568 Rinehart, W.E. 220 Savage, R.E., Jr. 459 Plaa, G.L. 470 Roberts, B.L. 592 Sawhney, D. 483 Placke, M. 445 Roberts, S.M. 641 Sawyer, T. 473 Pohl, L.R. 7,138 Robertson, J.L. 34 Schafrank, S.N. 45 Polin, D. 144 Robertson, L.W. 474,475,476 Schardein, J.L. 266

199 Schatz, R.A. 8 Smiley, R.K. 432 T Schaumburg, H.H. 490 Smith, C.B. 524 Tabor, M.W. 2,109,612 · Scheuhammer, A.M. 285 Smith, J.H. 27 Taha, S.A. 644 Schieman, G. 546 Smith, M.K. 149 Takeshi, A. 394 Schiller, C.M. 656 Smith, R. 312 Takeshi, 0. 394 Schmaeler, M.A. 570 Smith, R.P. 503 Talcott, R.E. 497 Schmidt, M.B. 256 Smith, T.H.F. 142,143,582 Talsma, D.M. 196 5chmitz, M.C. 555 Snellings, W.M. 42,560 Tallant, M.J. 496,586,587 Schnell, S.R. 59 Snodin, D.J. 614 Tang, S.Y. 492 74 .Schnellmann, R.G. Snyder, C. 68 Tarka, S.M., Jr. 278 Schofz, R.W. 105,238,299 Sohn, O.S. 499 Tashjian, A.H., Jr. 232,244,375 Schoof, R.A. 322 Soiefer, A.I. 106 Tatter, S.B. 8 Schrager, T.A. 371 Taylor, E. 194 Soileau, S.D. 380 Schuetz, D.J. 223 Teal, J.J. 633 Sonko, 0. 165 Schuler, R.L. 25 Teichman, R. 247,248 Sorenson, S.S. 543,630 Schuman, L.D. 471 Tepe, S.J. 272 Soto, E. 369 Schumann, A.M. 129 Tepper, J.L. 545 Sousa, R.L. 111 Schwab, B.W. 525 Thackrara, J.W. 662 Schwetz, B.A. 92 Sovocool, G.W. 305 Thake, D.C. 32,407,566 Scortichini, B.H. 359 Spangler, W.L. 147 Theobald, H.M. 329 Scott, Joy B. 211 Spears, C.T. 627 Thomas, C.E. 238,299 Secours, V.E.O. 651,669 Spencer, P.S. 490 Thomas, D. 54 Seed, J.R. 439 Sperling, F. 96 Thomas, D.J. 294,295 Seefeld, M.D. 461,462 Spilman, S.D. 482 Thomas, J.A. 252,253 Segall, H.J. 82,83,84 Sprague, G.L. 529 Thomas, L.V. 71 Seifried, H.E. 483 Springer, D.L. 368 Thomassen, R.W. 377 Seizinger, D.E. 172 Squibb, R.E. 657 Thompson, G.W. 390 Selan, F.M. 472 Stadler, J. 598 Thompson, M.B. 359 Sellinger, O.Z. 8 Stance!, G.M. 386 Thompson, T.N. 441,505 Selsky, C.A. 94 Stedman, D.B. 254 Tiemeyer, T.M. 306 Shaffer, C.B. 626 Steeger, T. 54,542 Tien, M. 97,190 Shah, V. 405 Stemmer, K.L. 13,196,396 Tilson, H.A. 289,307,404,537,657 442 Shargel, L. Stephen, E.L. 666 Timmons, P.R. 338,540,541 Sharp, R. 290 Stevens, A.C. 392 Toal, B. 317 Sheets, L.P. 526 Tocchi, M. 432 Stevens, M.W. 125,258 Shehata, A.T. 478 Tohyama, C. 185 Stevens, J.T. 309 Shen, E.S. 137 Tollett, J.T. 92 Stillman, M.J. 189,456 Shertzer, H.G. 439 Tomatis, L. 222,224 Stoewsand, G.S. 63,348,349 Shiotsuka, R.N. 182 Tonsager, S.R. 27 Stone, L.C. 553 Shively, C.A. 278 Toscano, D.L. 635 Shopp, G. 330 Storer, R.D. 454 Toscano, W.A., Jr. 635 Short, R.D., Jr. 123 Story, D.L. 450 Traiger, G. 590,648 Shull, L. 144 Stott, G.A. 332 Tremmel, F.J. 489 Trochimowicz, H.J. Shupnik, M. 375 Stott, W.T. 130 37 Troise, N.J. 247,248 Siak, J.-S. 173,174 Streett, C.S. 34 Trono, M. 593 Sibley, P.L. 392 Strom, B. 78 Trosko, J.E. 351,352 Sichak, S.P. 163 Stromberg, P.C. 153 Trutter, J.A. 619 Siddiqui, W.H. 623 Struve, P.S. 34 Tryphonas, L. 230 312 Silkworth, J.B. Stuart, B.O. 211 Tsuda, S. 116 Silver, E. 120 Sturgess, J.M. 108,389 Tu, C.-P.D. 105 Silver, E.H. 52 Styles, J.A. 374 Tuite, J. 128 Silver, I.S. 383 Suit, J.L. 435 Tung, T.C. 492 Sinhaseni, P. 155 Sultatos, L.G. 495,618 Turner, J.R. 642 Sinnhuber, R.O 362 Sumlar, M. 294,295 Turner, M.J. 59 Sipes, I.G. 61,74,75,76,350,363, Sun, J.D. 170 Tuthill, R.W. 675 400,515,527 Sunderman, F.W., Jr. 21 Tvedten, H.W. 275 Sivam, S.P. 3 Twarog, T.A. 199 Suskind, R.R. 2 Slater, R.W. 310 Tyler, T.R. 496 Svendsgaard, D. 520 Slaughter, L.J. 96,395 Tyson, C.A. 79,283,450 Sleight, S.D. 351,352 Swanger, D. 368 Slesinski, R.S. 451 Sweigart, P.O. 247,248 u Smallwood, C.L. 467 Swen berg, J .A. 38,357,364 Uhlig, A. 475 Smialowicz, R.J. 327,332 Szabo, S. 120 Ulrich, C.E. 554 Smiler, K.L. 175 Szymanska, J.A. 189 Umetsu, N. 523

200 Unger, K.L. 537 Waters, I.W. 591,652 Williams, R.A. 146 Upreti, R.K. 588 Watkins, J.B. 441,504 Williams, S.J. 167,622,663 Uraih, L.C. 670 Watkins, J.R. 30,416,660 Willis, L.J. 84 Watkinson, W.P. 271,650 Wilson, L.R. 181 V Webb, P.J. 151 Wilson, M.C. 271 Valentine, R. 634 Weber, P. 550 Wilson, W.C. 159 Valli, V.E.O. 651,669 Wecker, J.R. 202,532 Wilson-Martino, N. 405 Van Dongen, C.G. 369 Wedig, J.H. 54,96,266 Wilt, S. 227 Winek, C.L. Vang, M.J. 236,569 Wehner, R.W. 45,573 17 Winston, G.W. 297 Van Goethem, D.L. 123 Wei, C.-I. 453 Wisner-Gebhart, A.M. 469 van Lier, R.B.L. 382 Wei, E.T. 581 Witschi, H.P. 164,625 Yater, S. 89 Weihl, P. 459 Wogan, G.N. 582 Vena, R.L. Ill Weil,C.S. 42,225,366 Wolfe, G.J. 109 Verardi, A. 245 Weir, F.W. 595 Wolff, G.L. 385 Verlangieri, A.J. 193,271 Weisburger, J.H. 499 Wolff, R.K. 170,180 Viau, C.J. 431,599 Weiss, B. 152,287,402,545 Wong, K.L. 601 Vick, J. 31 Weitman, S.D. 81 Wong, Z.A. 147 Victery, W. 197 Welsch, F. 254 Wood, C.K. 559,672 Villeneuve, D.C. 651,669 Weitman, R.H. 229,345,355 Wood, R.W. 545,574 Vodicnik, M.J. 81,156,479 Wenger, G.R. 304,306 Woods, L.S. 578 Voelker, R.W., Jr. 576,619 Werchowski, K.M. 262 Woodside, M.D. 451 Vogel, D.G. 18 West, W.L. 96 Wright, A.F. 427,637 Volp, R.F. 75 Westen, H. 388 Wright, E.S. 569 VonBurg, R. 147 Wetzel, LT. 281 Wright, R. 195 Vore, M. 77,440 Weyel, D.A. 485 Wu,M.F. 202,532 Vyas, I.L. 655 White, C.G. 250 w White, E.L. 555,563 y White, H.J. 175 Wade, C.E. 119 Yamasaki, H. 224 White, K.D. 640 Waechter, J.M., Jr. 112 Yang, K.H. 468 White, K.L., Jr. 330 Waldron, D.M. 99 Yoon, Y.H. 392 White, W.E., Jr. 609 York, R.G. Walker, A.I.T. 418 261 Whitehurst, V. 649 Yoshihiro, A. 394 Walker, R.M. 446 Wierda, D. 324 Young, J.T. 40,148 Wall,J.M. 589 Wilens, T.E. 8 Yun, H.S. 140 Walters, D.G. 71 Wiley, B. 648 Wannarka, G.L. 619,627,646,666 Wilkenfeld, R.M. 265 z Wang, C. 379,618 Wilkinson, G.E. 562 Zamora, P.O. 565 Warshawsky, D. 89 Willhite, C.C. 413 Zehr, R.D. 337 Wasserman, R.S. 671 Williams, G.M. 360,361 Zenick, H. 272 Watanabe, P.G. 29,129,130,451 Williams, K.D. 65,66 Zook, B.C. 219 Waterhouse, G.A.W. 336 Williams, P. 43 Zwicker, G.M. 146,211,270,377,620,667

201