[CANCER RESEARCH 35, 2500-2505, September 1975] Inhibition of the Acute Toxicity and Adrenocorticolytic Effect of 7, 12-Dimethylbenz( a)anthracene by Isopropylvaleramide and Allylisopropylacetamide in the Rat'

Arpad Somogyi, Wayne Levin, Sipra Banerjee, Ronald Kuntzman, and Allan H. Conney

Epplev Institute for Research in Cancer, University ofNebraska Medical Center. Omaha, Nebraska 68/05 [A . S.], and Department ofBiochemistrv and Drug Metabolism, Hoffmann-La Roche Inc., Nutley, New Jersey 071 10 [ W. L.. S. B.. R. K., A . H. C.]

SUMMARY large variety of chemicals, among others polycyclic aro matic hydrocarbons. This destruction of cytochrome P-450 Isopropylvalenamide (IVA) and allylisopropylacetamide is brought about by a metabolite ofAlA (1, 4, 12) as well as (AlA) inhibit hemorrhagic adrenocortical necrosis and of several barbiturates (e.g., secobarbital, aprobarbital, mortality caused by 7, 12-dimethylbenz(a)anthracene allobarbital) ( 12) and requires the presence of an allyl group (DMBA) in female Sprague-Dawley rats. Unlike their in the molecules of the parent compounds. On the other effect on hepatic microsomal cytochrome P-450, the anti hand, a structural analog of AlA, IVA, which contains a DMBA action of these compounds does not depend on the propyl (instead of the reactive allyl) group (Chart I), has no presence of the reactive allyl group in the molecule. effect on cytochrome P-450 levels in liver microsomes (I, Similarly, related barbiturates, regardless of whether they 12). In the course of our investigations on the relationship contain, like AlA, an allyl group and consequently destroy between the metabolism and toxicity of polycyclic aromatic cytochrome P-450 (secobarbital and aprobarbital) or have, hydrocarbons the question arose as to whether AlA could like IVA, saturated side chains and therefore do not affect alter various biological actions of carcinogens (e.g., the microsomal hemoprotein (pentobarbital and phenobar DM BA) that are believed to require metabolic activation by bital), proved ineffective in preventing both adrenal damage microsomal enzymes (5, 6, 14, 15). and death caused by DMBA. Hence, the protective action of This paper deals with the effect of AlA and IVA on IVA and AlA cannot be attributed to the destruction of the hemorrhagic adrenocortical necrosis and mortality elicited microsomal enzyme system responsible for the activation of by DMBA in the rat. The influence of IVA on the clearance DMBA. The toxicity ofanother carcinogen, dimethylnitros of [3H]DMBA and its metabolites from the adrenals, the amine, which also requires metabolic activation by micro liver, and the blood and on the in vitro metabolism of somal enzymes, isnot influenced by either IVA or AlA. DMBA and benzo(a)pyrene was also studied. IVA, which counteracts the adrenocorticolytic action of DMBA when given prior to, simultaneously with, or even after this carcinogen, has no discernible effect on hydrocar MATERIALS AND METHODS bon metabolism in vivo or in vitro. IVA is one of the most powerful inhibitors of the acute toxicity of DMBA. It has Female Sprague-Dawley rats (Carworth Farms, New the simplest aliphatic structure and the smallest molecule City, N. Y.), weighing 150 to 180 g, housed in stainless among protectors of the adrenals against hydrocarbon steel cages, and maintained on Purina laboratory chow induced damage; its mechanism of action awaits further elucidation. 0

CFI2=CF1—CH2 CH—(' —-NH2

INTRODUCTION CH3---CH

Administration of AlA,2 a porphyrogenic compound, CH3 causes a rapid and transient decrease in hepatic microsomal cytochrome P-450 (4, 11), the substrate-binding site and terminal oxidase of the enzyme system that metabolizes a 0 I This work was supported in part by Contract PH-43-68-959 from the National Cancer Institute, NIH. CH3—CH2-----CH2-—CH---C—NH2 2 The abbreviations used are: AlA, allylisopropylacetamide; IVA, isopropylvaleramide; DMBA, 7,12-dimethylbenz(a)anthracene; DMSO, CH3—CH dimethyl sulfoxide; 3-MC, 3-methylcholanthrene; SKF 525A, @-di ethylaminoethyldiphenyl-n-propyl acetate. CH3 Received February 5, 1975; accepted June 5, 1975. Chart 1. Chemical structures of AlA (upper) and IVA (lower).

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1975 American Association for Cancer Research. Inhibition of DMBA Toxicity by I VA and A IA and tap water ad libitum, were used in most experiments. Table I To test the effect of IVA and AlA on dimethylnitrosamine The effect ofAlA, IVA, secobarbital, aprobarbital, pentobarbital, and toxicity, female Sprague-Dawley rats (Holtzman Farms, phenobarbital on DMBA-induced adrenal necrosis and toxicity Madison, Wis.) with an average weight of 100 g were Female Sprague-Dawley rats received iv. 5 mg (for adrenal necrosis) or used. All i.v. injections were given into the jugular vein 12 mg (for acute toxicity) of DMBA dissolved in 125 or 300 @lDMSO, respectively. Other compounds (equimolar amounts to AlA, 25 mg/kg) under light ether anesthesia. DMBA (40 mg/mI) dissolved were dissolved in 0.9% NaCI solution containing 2.5% of DM50: these in DMSO, as described elsewhere (17), and dimethyl were injected i.p. I hr prior to DM BA. Control animals received the vehicle nitrosamine (8 mg/ml) in 0.9% NaC1 solution were in (NaCI solution with 2.5% DMSO) rather than any of the potential jected i.v. AlA, IVA (Hoffmann-LaRoche Inc., Nutley, protectors. Adrenal necrosis was appraised 3 days after DM BA injection, N. J .), secobarbital, aprobarbital, pentobarbital, and and cumulative mortality was judged after 30 days. phenobarbital (Gane's Chemical Works, New York, N. Y) ofAdrenal were injected i.p. or i.v. in 0.9% NaCI solution containing 2.5 or 5% DMSO as specified in the corresponding Potential protectorsDose necrosisMortality(5mg(12mgDMBA)DMBA)None5/55/5AlA250/50/10IVA250/50/10Secobarbital°462/2Aprobarbital375/55/5Pentobarbital445/56/6Phenobarbital455/56/6(mg/kg)Incidence tables. The concentration of these compounds was chosen to require, at every dose level, the injection of 10 ml/kg. 3-MC (Sigma Chemical Co., St. Louis, Mo.) dissolved in corn oil (10 mg/mI) and SKF 525A (Smith, Kline and French, Philadelphia, Pa.) in 0.9% NaCl solution (4 mg/mI) were injected i.p. All solutions were prepared immediately prior to injection. Experiments on adrenal necrosis were terminated 3 days after the i.v. administration of5 mg of DMBA. In studies in which lethality served as the end point, the animals were @2Since the strong hypnotic action of secobarbital (46 mg/kg) used in observed for 30 days following the injection of 12 mg these studies killed 3 of 5 animals within a few hr of its injection, i.e.. before adrenal necrosis could have developed, this compound was excluded from DMBA or 14 days after the administration of dimethylni the toxicity experiments in which a substantially higher dose of DM BA was trosamine (80 mg/kg), and the cumulative mortality was administered. recorded. The incidence of adrenal necrosis or mortality (number of positive animals per total number of animals in Table 2 the group) is given in the tables. Benzo(a)pyrene hydroxyl The role of dose and time of the administration of I VA on its protective ase activity was determined by the method of Wattenberg et action against DMBA -induced adrenal necrosis a!. (19) as modified by Nebert and Gelboin (16). Further All animals received iv. S mg DMBA dissolved in 125 M1of DMSO (considered as 0 hr). IVA was injected iv. in 0.9% NaCI solution details are given in the legends to the tables and to Chart 2. containing 2.5% DMSO. The 1st group was given the vehicle of IVA. Adrenal necrosis was appraised 3 days following the administration of DMBA. RESULTS Administration of IVAIncidence In preliminary experiments, we found that the adminis of tration of 12.5 to 400 mg of AlA per kg body weight 5 hr or necrosisDoseTimeNone13/1325.00(mg/kg) adrenal 1 mm prior to an i.v. dose of DMBA protected rats against adrenal necrosis induced by this carcinogen. As indicated by —Shr0/525.00 the results of Table 1, in addition to AlA, IVA also hr0/525.00—1 prevented both adrenal damage and mortality caused by —1mm0/312.50 mm0/S10.00—I DMBA, while the 4 barbiturates used, regardless of whether —1mm0/36.25 they contained an allyl group (secobarbital, aprobarbital) —Shr5/56.25 or not (pentobarbital, phenobarbital), failed to show a pro —2hr0/S6.25 tective effect. —Imm0/95.00 Table 2 reveals that IVA, administered under a variety of —Imm3/S2.50 mmS/S25.00—I conditions, can prevent damage inflicted by DMBA to the +30mm0/525.00 adrenals of rats. The minimal but fully protective dose of +lhr4/525.00 IVA (6.25 mg/kg) is one-halfofthat ofAlA (12.5 mg/kg). +2hrS/S IVA appears to be, on a molar basis, one of the most effective inhibitors of DMBA toxicity and cellular damage. administration of DMBA. On the other hand, IVA adminis The protective action of IVA seems to depend both on the tered later than 30 mm following the hydrocarbon cannot dose and the time of its administration. While IVA (6.25 arrest and reverse the initiated process of cell destruction. mg/kg) given 2 hr or 1 mm before an adrenocorticolytic Apparently, damage caused by DMBA within the 1st hr in dose of DMBA offers full protection, this amount of IVA the adrenals is sufficient to result in adrenocorticolysis. fails to affect the incidence of adrenal lesions when given 5 Data presented in Table 2 strongly suggest that, for its hr prior to the carcinogen. However, a sufficiently high dose protective effect, the presence of IVA (or some of its (e.g., 25 mg/kg) is effective regardless of whether it is metabolites) is essential. Thus, IVA appears to participate injected as early as 5 hr before or as late as 0.5 hr after the directly in its anti-DMBA action rather than through the

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A. Somogyi et a!.

0,000 . —-@--——@-----—@-@-___ induction of DMBA-metabolizing enzymes. It was conceiv CONTROL VA 3MC able, therefore, that IVA might impede the metabolism of A 4 ‘ @:@oIs 0 . DMBA, i.e., inhibit the production ofthe adrenocorticolytic 5000- and toxic metabolites of DMBA, particularly as such a mode of action has been suggested to explain the abolition 4 \: by SKF 525A (20) and by liver injury (21) of DMBA •0 0 0 induced adrenal lesions. To investigate this possibility, @ 20C0- [3H]DMBA was injected i.v. into rats pretreated 1 hr earlier with IVA (25 mg/kg) or with the solvent of this amide. As 0 shown in Chart 2, IVA did not influence the concentration @ 000- of DMBA and its metabolites in the adrenals (the target I- organ), in the liver (a nontarget organ), or in the blood E @ -\S during an observation period of 6 hr following the adminis E 500— @0 tration of the carcinogen. On the other hand, pretreatment with 3-MC, a stimulator of DMBA metabolism, resulted in substantially reduced levels of radioactivity in the liver, the 9 200- adrenals, and the blood 1 hr after [3H]DMBA administra tion. These observations were further supported by data, shown in Table 3, revealing no substantial differences between control animals and those pretreated with IVA as regards the total radioactivity or the ratio of water-soluble Hours After DMRA Admtn,stration to organic solvent-extractable radioactivity in the adrenals I Chart 2. The effect of IVA on the distribution and clearance of hr following the administration of [3H]DMBA. Under [‘H]DMBA and its metabolites. Female Sprague-Dawley rats received an similar conditions, 3-MC, an inhibitor of DMBA-induced iv. injection of 4 mg of DMBA (containing 58 zCi of [3H]DMBA; adrenocorticolysis (2, 7, 8) and a potent inducer of the Amersham-Searle, Arlington Heights, Ill.) dissolved in 100 M1Of DMSO. microsomal enzyme system that metabolizes aromatic IVA (25 mg/kg) was given iv. 1 hr before the administration of DMBA. hydrocarbons (3, 10), substantially decreased the total Control animals received 0.9% NaCl solution containing 2.5% of DMSO radioactivity while increasing the amount of water-soluble (the solvent of IVA) I hr prior to DMBA. Groups of 3 rats each were sacrificed by abdominal aorta puncture IS, 30, 60, 180, and 360 mm later. metabolites of DMBA in the adrenal glands, as previously To induce high levels of hydrocarbon-metabolizing enzymes, 3 animals reported (10). On the other hand, SKF 525A, a powerful were given 3-MC (30 mg/kg) i.p. dissolved in 0.5 ml ofcorn oil 24 hr before inhibitor of both DMBA-induced adrenal necrosis (20) and the administration of DMBA. These animals served as positive controls for microsomal aryl hydrocarbon hydroxylase (9), exerted an induction of DMBA metabolism and were sacrificed I hr after the DMBA opposite effect. injection. A 65- to 100-mg piece ofthe liver and both adrenals (30 to 60 mg) Similarly, the chromatographic profile of DMBA me were removed. These organs and 100 .@lof heparinized blood were tabolites extracted from the adrenals did not indicate an solubilized overnight at S0@ in I ml of Soluene. The blood samples were effect of IVA on the metabolism of DMBA. The combined then treated with 0.5 ml of a 20% solution of benzoyl peroxide in toluene organic solvent extracts of the adrenal glands, harvested in and kept at 50°for an additional 30 mm. All samples were cooled, 15 ml of Scintisol and 0.2 ml ofglacial acetic acid were added, and the radioactivity was counted for 10 mm in a Packard Tri-Carb spectrometer. Mean of SE. is not apparent, it was smaller than the size of the symbols used to dpm/mg tissue or @lof blood ±SE. is shown. In those cases when the designate the means.

Table 3 The effect of various compounds on the level of [3H]DMBA in the adrenals Female Sprague-Dawley rats received [3H]DMBA (4 mg; 100 MCi) iv. in 100 @zlofDMSO I hr after IVA or SKF S25A or 24 hr after 3-MC. Animals were sacrificed under light ether anesthesia by exsanguination (aorta puncture) 1 hr following the administration of DM BA. Adrenal glands from 5 rats were pooled and homogenized in 4 volume of cold water. Two volumes of cold acetone and 6 volumes ofhexane were added, and the mixture was shaken for 20 mm and centrifuged. The organic solvent phase was removed, and the aqueous phase was extracted a 2nd time with acetone-hexane. The tissue was finally extracted with 10 volumes ofethyl acetate. All ofthe organic solvent extracts were pooled for quantitation of organic solvent-extractable DMBA and its metabolites. Then. the nonextractable radioactivity in the aqueous phase was determined.

(%)Non Totalradioactivity in adrenalsRadioactivity Organic solvent Treatmenttissue)extractableextractableNoneI with protectors(dpm/mg

1,6644.795.3IVA, iv.12,0664.495.63-MC,25 mg/kg, i.p.5,4769.590.5SKF30 mg/kg, S25A, 12 mg/kg, i.p.17,8602.997.1

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DMBA animals treated with 10 or 25 mg IVA per kg were similar to those of control rats. Furthermore, IVA, present at I mM °—° CONTROL x—x ISOPROPYLVALERAMIDE concentration in the incubation mixture, which contained the enzyme, cofactors, and substrate, resulted in only a small decrease in the metabolism of DMBA (Metabolite B), while 0. 1 mM and 0.01 mM concentrations appeared to have no effect on the in vitro metabolism of either benzo(a)pyrene or DMBA. Since dimethylnitrosamine also requires metabolic acti vation by the microsomal mixed-function oxidase system

( 13, 1 5), it appeared of interest to investigate whether the 0. C-) toxicity ofthis nitroso carcinogen would be affected by IVA or AlA. As shown in Table 5, cdntrary to their powerful anti-DMBA action, these 2 amides failed to influence the toxicity of dimethylnitrosamine, even at dose levels as high as 50 mg/kg.

DISCUSSION

CENTIMETERS from ORIGIN The data presented here indicate that AlA and particu Chart 3. Thin-layer chromatographic profile of DMBA metabolites. larly IVA are powerful inhibitors of DMBA-induced death Details on the experimental procedure are referred to in the text. Authentic. Table S standards were used as markers. The influence of IVA and AlA on the acute toxicits of dimetkvlnitrosamine experiments described in Table 3, were evaporated to Female Sprague-Dawley rats averaging 100 g received iv. dimethylni dryness, and a portion was chromatographed on Gelman trosaminedissolvedin (80 mg/kg) in 0.9% NaCI solution. IVA and AlA were SA thin-layer sheets with 2.5% ethanol in benzene as the compoundswere0.9% NaCI solution containing 5% of DMSO. The latter ofdimethylnitrosamine.Incidenceinjected (50 mg/kg) iv. 30 mm before the administration developing solvent (10). The chromatograms were cut into 0.5-cm strips and the radioactivity was quantitated. The ofPretreatment profile of DMBA metabolites present in the adrenal glands mortality@'None of rats treated with IVA was similar to that seen in control rats (Chart 3). 6/10IVA The lack of influence of IVA on hydrocarbon metabolism 4/9AlA was further confirmed in experiments summarized in Table 5/10

4. The amounts of fluorescent phenolic metabolites of (1 Cumulative mortality as judged 14 days following the administration benzo(a)pyrene and DM BA formed by liver homogenates of of dimethylnitrosamine.

Table 4 The effect of I VA on the in vitro metabolism of benzo(a)pvrene and DMBA In Experiment A, animals were given iv. injections of IVA I hr before sacrifice. Whole liver homogenates were used. The incubation mixture contained 90@zmoles ofpotassium phosphate buffer (pH 7.4), 3 @molesof MgCl2 , 0.5 Mmole of NADPH, and I or IS mg of liver (wet weight) for the benzo(a)pyrene or DMBA assay, respectively. The hydrocarbons were added (25 zg in 50 @lof acetone)just before the incubation, which was performed at 37°.Fluorescent DM BA metabolite A fraction was measured at 382 nm (excitation) and 532 nm (emission), and the metabolite B fraction was measured at 324 nm (excitation) and 400 nm (emission). Further details of the assay procedure for DMBA and benzo(a)pyrene metabolism have been described elsewhere (10, 18). In those cx periments where IVA was added directly to the incubation mixture (Experiment B), the liver of an untreatedratwas used.

metabolites (arbitrary units/IS mg (a)pyrene formed liver/IS mm) (ng/mg BA.IVAExperimentIVA3-Hydroxybenzo liver/S mm)DMBA A

injected into ratsNone 46 10mg/kg 10.6 31 49 51B.IVA 25mg/kg10.8 10.829 34

added directly to 46 incubation mixtureNone IOzM 10.3 36 39 l00@M 10.3 39 39 I000MM10.8 9.229 35 21

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1975 American Association for Cancer Research. A. Somogyi et a!. and adrenal damage in rats. AlA has been shown to inhibit REFERENCES the metabolism of several drugs by destruction of cyto chrome P-450 (1, 12), and therefore it was thought that AlA 1. Abbritti, G., and Dc Matteis, F. Decreased Levels of Cytochrome inhibited the toxicity of DMBA by decreasing -its metabo P-450 and Catalase in Hepatic Porphyria Caused by Substituted lism. We were surprised to find that secobarbital and Acetamides and Barbiturates. Chem.-Biol. Interactions. 4: 281 -286, 1972. aprobarbital, 2 barbiturates that also destroy cytochrome 2. Dao, T. L., and Tanaka, Y. Inhibitory Effect of 3-Methylcholanthrene P-450 and decrease drug metabolism, had no effect on on Induction of Massive Necrosis of Adrenal Cortex by 7,12-Di DMBA toxicity. All 3 of the above compounds possess an methylbenz(a)anthracene. Proc. Soc. Exptl. Biol. 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Z., 338: 106-1 13, 1963. 8. Huggins, C., and Fukunishi, R. Induced Protection of Adrenal Cortex with 4 times the minimal fully protective dose of IVA (25 Against 7, 12-Dimethylbenz(a)anthracene. Influence of Ethionine. In mg/kg) prior to the administration of [3H]DMBA failed to duction of Menadione Reductase. Incorporation of Thymidine-H'. J. influence the level of DM BA and its metabolites in the Exptl. Med., 119: 923-942, 1964. adrenals. Also, the presence of high concentrations of IVA 9. Kuntzman,R.,Levin,W.,Schilling,G.,andAlvares,A.TheEffectsof in an in vitro incubation system (as high as 1 mM) failed to 3-Methylcholanthrene and Phenobarbital on Liver Microsomal Hemo substantially alter the activity of hepatic enzymes that proteins and on the Hydroxylation of Benzpyrene. In: J. R. Gillette, A. metabolize benzo(a)pyrene and DMBA. It is noteworthy H. Conney, G. J. Cosmides, R. W. Estabrook, J. R. Fouts, and G. J. that the toxicity of dimethylnitrosamine, another carcino Mannering (eds.), Microsomes and Drug Oxidations, pp. 349-369. gen requiring metabolic activation by microsomal enzymes, New York: Academic Press, Inc., 1969. is not influenced by either IVA or ALA. 10.. Levin, W., and Conney, A. H. Stimulatory Effect of Polycyclic Hydrocarbons and Aromatic Azo Derivatives on the Metabolism of While at the present time we cannot provide a definite 7, l2-Dimethylbenz(a)anthracene. Cencer Res., 27: 193 1- 1938, 1967. explanation for the protective effect of IVA and AlA, it 11. Levin, W., Jacobson, M., and Kuntzman, R. Incorporation of appears that IVA has little or no influence on the metabo Radioactive-ô-Aminolevulinic Acid into Microsomal Cytochrome lism of DMBA in vitro or in vivo. Nonetheless, the P-4S0: Selective Breakdown of the Hemoprotein by Allyliso possibility that IVA inhibits the formation of a quantita propylacetamide and Carbon Tetrachloride. Arch. Biochem. 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Influence of “FeederCells―and independent of its action on the adrenals, such an interac Inducers and Inhibitors of Microsomal Mixed-Function Oxidases on tion would be expected to occur in several other organs (e.g., Hydrocarbon-induced Malignant Transformation of Cells Derived bone marrow, intestinal mucosa, etc.). from C3H Mouse Prostate. Cancer Res., 32: 721-725, 1972. IVA is a remarkably nontoxicsubstance. It is the smallest 15. Miller, J. A. Carcinogenesis by Chemicals: An Overview—G. H. A. molecule and has the simplest aliphatic structure of the Clowes Memorial Lecture. Cancer Res., 30: 559-576, 1970. numerous compounds known to inhibit DMBA-induced 16. Nebert, D. W., and Gelboin, H. V. Substrate-Inducible Microsomal toxicity. These properties, along with its failure to influence Aryl Hydroxylase in Mammalian Cell Culture. I. Assay and Proper the metabolism of DMBA, might enable IVA to become a ties of Induced Enzyme. J. Biol. Chem., 243: 6242-6249, 1968. tool in studies directed at nonmetabolic aspects of DM BA 17. Somogyi, A., and Kovacs, K. Dimethyl Sulfoxide, a Convenient Solvent of 7, l2,-Dimethylbenz(a)anthracene for Intravenous Injec carcinogenesis. Investigations are presently under way in tion. Cancer Res., 30: 1958- 1962, 1970. our laboratory to determine whether IVA and AlA affect 18. Somogyi, A., Kovacs, K., Solymoss, B., Kuntzman, R., and Conney, the carcinogenicity of DMBA. We are currently exploring A. H. Suppression of 7,l2-Dimethylbenz(a)anthracene-produced Ad the effects of these amides on the biological actions of other renal Necrosis by Steroids Capable of Inducing Aryl Hydrocarbon types of chemical carcinogens as well. Hydroxylase. Life Sci., JO: 1261-1271, 1971.

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19. Wattenberg, L. W., Leong, J. L., and Strand, P. J. Benzpyrene benz(a)anthracene by @9-Diethylaminoethyldiphenyl-n-propyl Acetate Hydroxylase Activity in the Gastrointestinal Tract. Cancer Res., 22: (SKF 525-A). Brit. J. Exptl Pathol., 49: 44-51, 1968. 1120—1125,1962. 1. Wheatley, D. N., Kernohan, I. R., and Currie, A. R. Liver Injury and 20. Wheatley, D. N. Prevention of the Adrenocorticolytic Actions of the Prevention of Massive Adrenal Necrosis from 9,l0-Dimethyl-l,2- 7,12-Dimethylbenz(a)anthracene and 7-Hydroxymethyl-l2-methyl benzanthracene in Rats. Nature, 211: 387-389, 1966.

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Arpad Somogyi, Wayne Levin, Sipra Banerjee, et al.

Cancer Res 1975;35:2500-2505.

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