Vinyl Carbamate As a Promutagen and a More Carcinogenic Analog of Ethyl Carbamate1

Home , Carbamic acid, Ethyl carbamate

[CANCER RESEARCH 38, 3793-3804, November 1978] 0008-5472/78/0038-OOOOS02.00 Vinyl Carbamate as a Promutagen and a More Carcinogenic Analog of Ethyl Carbamate1

Gary A. Dahl, James A. Miller,2 and Elizabeth C. Miller

McArd/e Laboratory for Cancer Research, University of Wisconsin Center for Health Sciences, Madison, Wisconsin 53706

ABSTRACT /V,A/-dimethylcarbamate, or methyl-, 2-chloroethyl-, 1- or 2- propyl-, or 1-butyl carbamate (2, 17, 18). Ethyl /V-hydroxy- Vinyl carbamate was much more active (10 to 50 times) carbamate, however, was nearly as carcinogenic as ethyl than ethyl carbamate for the initiation of skin tumors and carbamate (2, 25). In view of the metabolic formation of for the induction of lung adenomas in mice. Vinyl carba ethyl /V-hydroxycarbamate from ethyl carbamate (3, 29) and mate was also mutagenic to Salmonella typhimurium TA the role of A/-hydroxylation in the activation of carcinogenic 1535 and TA 100 in the presence of reduced nicotinamide aromatic amines (6, 26), the N-hydroxy derivative was adenine dinucleotide phosphate-fortified rat or mouse suggested as a possible proximate carcinogenic metabolite liver mitochondria! supernatant fractions. This mutagenic (2, 3, 25). However, later results were not consistent with activity was inhibited strongly by cytochrome P-450 inhib this suggestion. Thus, ethyl N-hydroxycarbamate was read itors. No mutagenic activity was observed for vinyl carba ily reduced in vivo to ethyl carbamate (28, 29). Further, the mate in the absence of added liver preparations or for number of lung adenomas induced by ethyl A/-hydroxycar- ethyl carbamate in the presence or absence of liver bamate, but not by ethyl carbamate, was decreased in mice fractions. treated simultaneously with SKF 525A4 (16). The latter Extensive tests with sensitive methods failed to detect compound inhibits the reduction of ethyl A/-hydroxycarba- vinyl carbamate as a metabolite of ethyl carbamate in the mate, but it does not affect the /V-hydroxylation of ethyl mouse in vivo. However, on administration of [efhyM- 14C;1,2-3H]ethylcarbamate to adult mice the 3H/14Cratios carbamate in vivo (29, 30). Residues of ethyl carbamate are bound covalently to of the hepatic DNA-, rRNA-, and protein-adducts were macromolecules in vivo in tissues in which it is carcino similar to each other and much lower than the ratio of the genic (4, 5, 19, 35), as has been noted previously for many administered ethyl carbamate. These data are consistent chemical carcinogens (12, 24, 27). Early studies suggested with the presence of desaturated and/or oxidized ethyl that the entire ethoxycarbonyl residue was bound to RNA groups in the macromolecular adducts. The qualitatively (4). More recent studies by Prodi ef al. (35) and by Lawson similar, but much stronger, carcinogenic activity of vinyl and Pound (20, 33) indicated that 14C or 3H from 1- or 2- carbamate as compared to that of ethyl carbamate sug labeled ethyl carbamate was bound to DNA, while very little gests that the metabolic pathways of these two carba- or no isotope from [carbon//-14C]- or [efrtoxy-180]ethyl car mates may converge in the formation of similar or identi bamate was bound to this macromolecule. cal electrophilic reactants that bind covalently to macro- The above structure-activity relationships, the data on the molecules m vivo and initiate carcinogenesis. nucleic acid binding of residues from ethyl carbamate, and the carcinogenicity of vinyl chloride (14) suggested that INTRODUCTION vinyl carbamate, CH,=CHâ€”Oâ€”COâ€”NH.,, might be a prox imate carcinogenic metabolite of ethyl carbamate. If Ethyl carbamate (urethan3), CH3â€”CH2â€”Oâ€”COâ€”NH2, formed, vinyl carbamate might be enzymatically epoxidized first shown to be carcinogenic in the lungs of mice by to yield an ultimate carcinogen. The present paper reports Nettleship et al. (31) in 1943, induces tumors in a variety of our attempts to test this hypothesis. Some of these results tissues and in several species (reviewed in Refs. 13 and 30). have been reported in an abstract (7). Extensive studies by Larsen (17, 18) and by Berenblum ef al. (2) showed that structural modifications of ethyl carba MATERIALS AND METHODS mate generally result in a dramatic loss of activity for the initiation of skin papillomas or for the induction of lung Instrumentation and General Procedures adenomas in mice. For example, ethyl carbamate was much more carcinogenic than ethyl A/-methylcarbamate, ethyl General. IR and UV spectra were obtained using a Beck- man IR-10 spectrophotometer (Beckman Instruments, Ful- 1 In honor of the 70th birthday of Dr. Harold P. Rusch, M. D., founder and lerton, Calif.) and a GCA/McPherson EU-707-11 double- long-time Director of the McArdle Laboratory for Cancer Research and creator of its productive research atmosphere. beam spectrophotometer (GCA/Precision Scientific, Chi This work was supported by Grants CA-07175, CA-15785, CA-22484, and cago, III.), respectively. NMR spectra were determined on a CA-09135 from the National Cancer Institute, USPHS. 60-MHz Perkin-Elmer R-12 spectrometer (Perkin-Elmer, 2 To whom requests for reprints should be addressed. 3 Confusion is caused by continued reference to ethyl carbamate as "urethan(e)." The terms "urethan(e)" and "polyurethan(e)" are group names "The abbreviations used are: SKF 525A, 2-diethylaminoethyl-2,2-diphen- for monomeric and polymeric esters of carbamic acid or substituted car- ylvalerate hydrochloride; NMR, nuclear magnetic resonance; HPLC, high bamic acids, respectively. The authors propose that the name "urethan(e)" performance liquid chromatography; DPEA, 2-[(2,4-dichloro-6-phenyl)phe- no longer be used as a synonym for ethyl carbamate. noxyjethylamme: S-13, microsomes plus cytosol obtained by centrifugation Received June 26, 1978; accepted August 14, 1978. at 13,000 x g for 10 min.

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Norwalk, Conn.) or a 90-MHz Bruker NMR spectrometer was purchased from Aldrich Chemical Co. Ethyl /V-hydrox (Broker Instruments, Billerca, Mass.) with tetramethyl silane ycarbamate was prepared in our laboratory (9). NAD+, (Aldrich Chemical Co., Inc., Milwaukee, Wis.) as an internal NADP+, glucose 6-phosphate, and Tris were purchased standard. A Varian CH-7 mass spectrometer (Varian Asso from Sigma Chemical Co., St. Louis, Mo. Glucose-6-phos- ciates, Palo Alto, Calif.), equipped with an electron impact phate dehydrogenase was obtained from Worthington Bio source, a digital mass marker which was standardized with chemical Corp., Freehold, N. J. DPEA was kindly provided perfluorokerosene (Peninsular Chem Research, Inc., by Dr. R. E. McMahon of the Eli Lilly Co., Indianapolis, Ind., Gainesville, Fla.), and a direct-insertion probe (Variset and SKF 525A was a gift of Smith, Kline, and French Corp., Madison, Wis.), was used to obtain mass spectra. 3H Laboratories, Philadelphia, Pa. and 14C were quantitated in a Nuclear-Chicago Isocap 300 Synthesis of Vinyl Carbamate. Vinyl chloroformate was or a Mark III Model 6880 liquid scintillation counter (both synthesized in a chemical hood by pyrolysis of ethylene products of Searle Analytic, Inc., Des Plaines, III.); the glycol bis(chloroformate) according to the general method samples were dissolved in either RIA-Solve II or a toluene/ of Lee (22). The pyrolysis was accomplished in a 30- x 3-cm PPO/POPOP scintillation cocktail (both purchased from Vycor silica tube packed with 3/16-in diameter Pyrex glass Research Products International, Elk Grove Village, III.). helices. Heating was effected by two 12-ft 400-watt beaded Melting points were determined with the Accumelt appara heaters (Marsh Beaded Heaters, Lake Jackson, Texas) con tus (American Instrument Co., Silver Springs, Md.). Solid nected in parallel and wrapped around the pyrolysis tube. ethyl and vinyl carbamate were handled in chemical hoods The temperature, measured by a thermocouple inside a since both compounds sublime readily, even at room tem silica finger that extended into the center of the pyrolysis perature and atmospheric pressure. Latex gloves were tube, was maintained at about 500Â°with a Thermolyne worn for all studies with these compounds. Temcometer (Scientific Products, McGaw Park, III.). Ethyl Gas Chromatography. Gas chromatography was per ene glycol bis(chloroformate) (kindly supplied by Mr. P. R. formed on a Varian 2800 Chromatograph (Varian Aero Heinze, BASF Wyandotte Corp., Parsippany, N. J.) was graph, Walnut Creek, Calif.) equipped with a hydrogen added to the pyrolysis tube (80 to 120 drops/min) from a flame ionization detector and a 6-ft x 0.25-in glass column dropping funnel equipped with a side arm through which packed with 15% diethylene glycol Buccinate on 60 to 80 N2 was passed at a rate of 12 to 14 ml/min. The gas exited mesh acid-washed Chromosorb W (Anspec Co., Inc., Ann from the pyrolysis tube to a 300-ml 2-necked round-bottom Arbor, Mich.). Injector and detector temperatures were flask which was connected in series to two additional traps. 250Â°,the column oven was set at 100-125Â° to obtain the All three receivers were submerged in Dry Ice-acetone, and desired retention time, and the nitrogen carrier gas flow gases from the last trap were bubbled through 10% NaOH. was 48 ml/min. The retention times for ethyl and vinyl The pyrolysate collected in the three receivers was pooled carbamates were 4.4 and 7.3 min, respectively, at 125Â°. and stored at -20Â° in the dark. The crude pyrolysate was Ethyl N-hydroxycarbamate produced a peak with the same distilled under reduced pressure (b.p. 21-22Â° at 10 cm retention time as ethyl carbamate; it was probably con mercury) through a 20-cm glass wool-insulated Vigreaux verted in part to ethyl carbamate under the chromatography column and water-cooled condenser. The crude chlorofor conditions. The limits of detection for ethyl or vinyl carba mate was collected in a receiver submerged in Dry Ice- mate were less than 20 ng/2-^l injection. acetone. HPLC. HPLC of the carbamates was accomplished using Vinyl carbamate was synthesized from the crude vinyl an ALC 201 liquid Chromatograph system equipped with a chloroformate by the general method of Schaefgen (36). 3.9-mm x 30-cm /Â¿Porasil column and a R-401 refractome- One volume of the crude vinyl chloroformate in 4 volumes ter (all products of Waters Associates, Milford, Mass.). All of CH2CI2and 2 volumes of cyclohexane at 0Â°were saturated samples and solvents were filtered through 0.2-fj.m Milli- for 5 to 10 min with NH., gas. Most of the solvent was then pore filters (Millipore Corp., Bedford, Mass.) and degassed removed with a slow stream of N2, CH2CI2 was added to before use. When the Â¿iPorasil column was eluted at 4 ml/ dissolve the vinyl carbamate, and the NH4CI was removed min with a mixture of redistilled n-hexane (Skelly Solve B) by filtration under reduced pressure through a sintered- and ethyl acetate (6/1, v/v), the retention times (center of glass filter. The vinyl carbamate was crystallized from peaks) were 3, 4.5, and 10.8 min for ethyl /V-hydroxycarba- CH2CI, at -20Â°, and the crystals were collected on a cold mate, vinyl carbamate, and ethyl carbamate, respectively. sintered-glass filter by rapid filtration at reduced pressure. At the attenuation used (8x), a 1-cm peak was obtained In some cases, sublimation at a pressure of 100 to 200 urn with the refractive index detector with approximately 100 mercury was necessary to obtain a pure product. /Â¿gof vinyl carbamate or approximately 300 /Â¿gof either The yields from the various steps, especially the pyrolysis, ethyl carbamate or ethyl /V-hydroxycarbamate. Although the were variable. In an average experiment, 90 ml of ethylene peaks were fairly broad and showed some tailing, there was glycol bis(chloroformate) yielded 14 ml of pyrolysate which no overlap between them. Samples were prepared and gave 6 ml of crude vinyl chloroformate on distillation. The injected in dry ethyl acetate or CHCL,, since the injection of yield of pure vinyl carbamate [m.p. 53-54Â°corrected; litera even small volumes of polar solvents (methanol or water) ture, 52-54Â°(36)] was 575 mg [about 1% based on ethylene caused the carbamates to elute at the solvent front. glycol bis(chloroformate)]. Since the full characterization of vinyl carbamate has not Chemicals been published, our data are reported here. On elementary analysis (Huffman Laboratories, Wheatridge, Colo.), all val Ethyl carbamate (>99% purity by gas chromatography) ues were within 0.4% of theory for C:,H5NO2. The IR spec-

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trum of vinyl carbamate showed the following absorption volatility, the solvent was removed as follows. The ethyl car maxima: 3420(s), 3340(s), 3290(s), 3195(m), 3100(w), bamate solution was transferred to a 50-ml round-bottom 2000(w), 1905(w), 1710(s, broad), 1647(m), 1611(m), flask that had a long neck with a side-arm outlet tube and a 1602(s), 1490(w), 1400(s), 1343(s), 1288(w), 1148(s), 1120(s). glass finger at the bottom (equipped with a 24/35 standard 1049(s), 950(s), 874(s), 859(s), 777(s), 680(w), 615(m), taper outer joint about Vs of the distance from the top). The 525(m), 435(m), 425(m). The mass spectrum revealed a side-arm outlet tube was connected to a series of four traps prominent molecular ion at mie = 87 and fragments at mie cooled with Dry Ice and a fifth trap cooled with liquid N2; = 71, 44, and 43. The vinyl protons of vinyl carbamate gave the exiting gas was finally passed through 1 N KOH. N2 was an ABX pattern in the NMR spectrum which was nearly directed onto the surface of the methanolic solution of identical with that of the vinyl protons of vinyl acetate. The tritiated ethyl carbamate through a long, narrow-gauge sharp AB protons were centered at S = 4.6 ppm (range 4.3 syringe needle inserted through a serum cap while the flask to 4.9 ppm); the X proton peaks were centered at 8 = 7.2 was partially submerged in a water bath at 60Â°.Within 3.5 ppm (range 7.0 to 7.43 ppm); the amide protons produced hr, the evaporation was complete; most of the methanol a broad peak under the AB portion of the spectrum. As a condensed in the first 3 Dry Ice traps. The finger containing further confirmation of structure, 10 mg of vinyl carbamate the solid ethyl carbamate was broken off above the standard in 2 ml of ethanol containing 2 mg of 5% palladium on taper joint, closed with a standard taper cap, and stored at charcoal (Engelhard Industries, Newark, N. J.) was quanti -20Â°. Additional tritiated carbamate was recovered by re tatively hydrogenated to ethyl carbamate within 1 hr at 25Â°, peating the above process with the methanol from the as determined by gas chromatography. traps. HPLC of aliquots of the purified ethyl carbamate [et/>yM-14C]- and [et/iyM,2-3H]Ethyl Carbamate. [ethyl- showed that its radiochemical purity was >99%; less than 1-14C]Ethyl carbamate (4.8 mCi/mmol; New England Nu 0.05% of the total 3H chromatographed with added unla- clear, Boston, Mass.) was determined to be approximately beled vinyl carbamate. Ethyl carbamate was the only com 95% radiochemically pure by liquid scintillation analysis of ponent observed by gas chromatography. The specific 30-sec fractions obtained by HPLC. activity was 486 mCi/mmol. [efhy/-1,2-3H]Ethyl carbamate was synthesized by Amer- sham Corporation (Arlington Heights, III.) by reductive tri- Toxicity and Carcinogenicity Studies tiation of 100 mg of vinyl carbamate. We were not informed of the exact procedure used by Amersham, but we supplied CD-1 female mice (Charles River Breeding Laboratory, them with the materials and the following information. A Wilmington, Mass.) or A/Jax female mice (Jackson Labora solution of 100 mg of vinyl carbamate in 20 ml of spectro- tory, Bar Harbor, Maine), both 6 to 8 weeks old, were grade dioxane containing 30 mg of 5% palladium on char housed in hanging wire-mesh cages with 5 mice/cage. All coal was quantitatively (>99%) reduced when shaken at 25Â° mice were fed Wayne Breeder Blox (Allied Mills, Inc., under 55 to 60 psi of H2 for 90 min (Parr hydrogÃ©nation Chicago, III.) and water ad libitum; they were weighed at apparatus, Parr Instrument Co., Moline, III.). Since both the beginning of each experiment and monthly thereafter. ethyl and vinyl carbamate sublime readily, the carbamate When administered i.p., the carbamates were injected in solutions cannot be taken to dryness without appreciable 0.005 ml of sterile 0.87% NaCI per g of body weight; losses. Exchangeable 3H can be removed by addition and negative controls received only the saline solution. Mice distillation of several successive aliquots of methanol to the treated topically were shaved with electric clippers before dioxane solution, if the evaporation of each aliquot of the first application and thereafter as needed. The topical methanol is stopped before any of the dioxane is removed. doses of the carbamates were applied inside a chemical When three successive 45-ml aliquots of methanol were hood, and the animals were then kept in hooded racks for 2 added to 25 mg of ethyl carbamate in 5 ml of dioxane, and days. each methanol aliquot was removed at 20 to 30 cm Hg and Skin papillomas were counted at 2-week intervals begin 25Â°,77% of the carbamate was recovered in the dioxane. ning at their first appearance. The lungs were fixed in 10% Dioxane and methanol do not form azeotropes. buffered formalin and adenomas visible on the surface (a1 HPLC of an aliquot of the [eÃAjy/-1,2-3H]ethyl carbamate mm diameter) were enumerated. On death or at the termi (total 3H = 4.57 Ci) from Amersham, together with ethyl and nation of the experiments, all mice were subjected to gross vinyl carbamate as refractive index markers, showed that routine autopsies, which included inspection of the external vinyl carbamate was the only appreciable radioactive impu and s.c. tissues and the organs of the abdominal and rity (11%). Very little radioactivity (<0.02%) eluted at the thoracic cavities. Representative tumors were fixed in 10% solvent front; thus, any labile tritium had been almost buffered formalin, stained with hematoxylin and eosin, and completely exchanged. The vinyl carbamate impurity was examined histologically. removed by further reduction. Palladium on charcoal (5%, Acute Toxicity. The toxicity by 10 days for vinyl carba 4 mg) in 3 ml of dry methanol was prehydrogenated with 50 mate in 2-month-old CD-1 female mice was determined by psi of H, for 1 hr at 25Â°with shaking. About 0.7 Ci of the i.p. injection of single doses of 0.062, 0.125, 0.25, or 0.5 impure carbamate in 7 ml of methanol was added, and mg/g of body weight (4 mice/group). In a second experi hydrogÃ©nation was continued for 2 hr. The H2 was ex ment, groups of 9 CD-1 female mice, 2 to 3 months old, hausted through a trap cooled with Dry Ice. The solution received i.p. injections of either ethyl carbamate (0.05, 0.2, was filtered through a glass frit, and 70 mg of nonradioac- 0.5, or 1.0 mg/g of body weight) or vinyl carbamate (0.05, tive ethyl carbamate were added. 0.1, or 0.2 mg/g). On Days 1, 3, and 7, 3 mice from each To minimize losses of the carbamate because of its group were killed, and the liver, heart, lungs, spleen, bone,

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1978 American Association for Cancer Research. G. A. Dahl et al. kidney, and gastrointestinal tract were fixed in 10% buffered duced male CD-1 rats (Charles River Breeding Lab) or from formalin for histological examination. female CD-1 or A/Jax mice (8). S-13 preparations were also Skin and Lung Tumorigenesis in CD-1 Mice. Each mouse made from livers of female CD-1 mice that had been given in the skin tumorigenesis studies was pretreated topically i.p. injections of 0.1 mg of 3-methylcholanthrene (Sigma with 1.2 mg of croton oil (Amend Drug and Chemical Co., Chemical Co.) in 0.01 ml of trioctanoin (Sigma) per g of New York, N. Y.) in 0.2 ml of redistilled acetone 18 to 24 hr body weight 24 hr previously or had received 0.05% sodium before each topical or i.p. administration of a carbamate.5 phÃ©nobarbital (Mallinckrodt Chemical Co., Inc., St. Louis, Each mouse in the first experiment (40 mice/group) was Mo.) in their drinking water for 7 days. Rats were fasted 12 given two topical applications of ethyl (5 or 60 mg/mouse) to 24 hr before decapitation, and the livers were perfused or vinyl (5 mg/mouse) carbamate or the solvent only. In a with 0.87% NaCI solution before removal. Mice were fasted second experiment, each mouse (about 30 mice/group) for 12 hr before being decapitated; their livers were not received 2 topical applications of ethyl (2.5, 5, or 60 mg/ perfused. S-13 preparations were prepared in the same mouse) or vinyl (2.5 or 5 mg/mouse) carbamate. In both manner from lungs of female CD-1 mice. experiments, the two doses of the carbamate were given 1 The following subcellular fractions from CD-1 female week apart. In a third experiment, each CD-1 mouse re mouse livers were obtained from 20% homogenates in 5 ceived a single i.p. injection of 0.065 mg of ethyl or vinyl mM Tris-HCI-0.25 M sucrose, pH 7.8: nuclear, 600 x g (10 carbamate per g of body weight or 2 i.p. injections 1 week min) pellet; mitochondrial, 13,000 x g (10 min) pellet; apart of either 1 mg of ethyl carbamate per g of body weight microsomal, 105,000 x g (60 min) pellet; and cytosolic, or the saline vehicle only. Beginning 1 week after the last 105,000 x g supernatant. Each subcellular pellet was carbamate application, each mouse was treated topically washed but, to avoid dilution, the washings were not added with 0.9 mg of croton oil in 0.15 ml of acetone twice weekly. to the supernatants. The overall recovery of protein (11) The negative controls received the croton oil pre- and post- was 65%. The percentages of the recovered protein in each treatments, but they were otherwise given only the vehicle fraction were: nuclear, 27; mitochondrial, 18; microsomal, instead of a carbamate. Experiments 1, 2, and 3 were 19; and cytosolic, 35. terminated at 32, 35, and 28 weeks, respectively, after the first treatment. Analyses of Mouse Tissues and Urines for Ethyl and Vinyl Lung Tumorigenesis in A/Jax Mice. In the first experi Carbamates ment, mice given a single i.p. injection of 0.032, 0.065, or Extraction and Purification Procedure for Mouse Tis 0.5 mg of ethyl carbamate per g of body weight or 0.032 or sues. Whole 5-day-old CD-1 mice or tissues from adult A/ 0.065 mg of vinyl carbamate per g were killed after 22 Jax mice injected i.p. with ethyl and/or vinyl carbamate weeks. In a second experiment, which was terminated after were frozen in liquid nitrogen and stored at -70Â° until 28 weeks, vinyl carbamate was administered as a single i.p. assayed. dose of 0.016, 0.032, or 0.065 mg/g. Mice in the third The tissue (6 to 100 g) was extracted with 3 volumes (w/v) experiment received the following i.p. doses (mg/g of body of CHCI:i in a Waring Blender (Waring Products, New weight x no. of doses): ethyl carbamate, 0.01 x 5 or 0.5 x Hartford, Conn.). The extract was filtered through Whatman 1; vinyl carbamate, 0.016 x 1, 0.01 x 5, or 0.005 x 10. The No. 1 filter paper (Whatman Inc., Clifton, N. J.) under multiple doses were given at 1-week intervals, and the reduced pressure (10 cm mercury), and the residue was re- experiment was terminated 20 weeks after the initial treat extracted 3 times in the same manner. Based on partition ment. coefficients in CHCI3-H20 (determined to be 0.61 and 0.65 Mutagenesis Assays for ethyl and vinyl carbamate, respectively) and the assump tion that CHCI,., extracts the carbamates as readily from The mutagenicities of ethyl and vinyl carbamate for S. tissue as it does from water, four extractions should have typhimurium missense mutants TA 1535 or TA 100 and for recovered about 98% of each carbamate. the frameshift mutant TA 98 (kindly provided by Dr. Bruce The combined CHCI., extracts were reduced to 100 ml on a rotary evaporator (25 cm mercury, 37Â°water bath) ap Ames, University of California, Berkeley, Calif.) were as sayed by the top agar method (1). plied to a 70 to 230 mesh dry silica (EM Laboratories, Inc., The bacterial culture (approximately 10e bacteria/0.1 ml), Elmsford, N. Y.) column (3 x 34 cm), and drawn just below 0.5 ml of an NADPH- and NADH-generating system (8), 0.5 the top of the column bed. The column was then eluted to 4.5 mg of S-13 protein [biuret assay (11)] in 0.02 to 0.18 under reduced pressure (10 cm mercury) with 1 liter of ml (or buffer only), and 0.01 ml of an aqueous solution of a water. The water and CHCI, moved as immiscible layers; the carbamate were added sequentially to an assay tube that boundary was evident if the column outlet was closed for a contained 2.0 ml of top agar (45Â°);the entire contents were short time. The CHCI3 was discarded, since it contained immediately poured onto a minimal agar plate (1). In a few very little or no ethyl or vinyl carbamate. The first 400 ml of experiments, bacterial survival was determined (1). the water eluate were extracted 4 times with 3 volumes of In most cases, the S-13 preparations were 13,000 x g (10 CHCI3. This extract was reduced to 1 to 10 ml on a rotary min) supernatants of 40% liver homogenates from unin- evaporator (25 cm mercury, 37Â°).Aliquots were analyzed by gas chromatography (nonradioactive carbamates) or by â€¢¿Thecroton oil pretreatment was based on the studies of Pound and Bell HPLC ([ef/?y/-1,2-3H]ethyl carbamate). The recoveries of (32) and Pound and Withers (34), who showed that this treatment increased ethyl and vinyl carbamate from whole 5-day-old mice that the yield of skin tumors in mice initiated with ethyl carbamate and promoted with croton oil. were frozen immediately after i.p. injection were 80 to 95%

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1978 American Association for Cancer Research. Carcinogenic/ties of Vinyl and Ethyl Carbamates and 30 to 40%, respectively, as compared to theoretical each injected i.p. with 5.62 /^mol (0.5 mg) of values of about 95%. 14C;1,2-3H]ethyl carbamate per g of body weight in 0.01 ml The above procedure was devised to minimize losses due of sterile 0.87% NaCI solution per g. The mice were killed to the volatility of the carbamates. Procedures that included 12 hr after injection, and the livers were immediately re evaporation to dryness with a rotary evaporator or with a moved, frozen in liquid N2, and stored for up to 1 month at stream of nitrogen gave low and variable recoveries. Large -20Â°.DMA and rRNA were isolated from pools of 3 livers by losses also occurred during evaporation of solvents such the method of Irving and Veazey (15). Protein was precip as ethanol and methanol even if the solutions were not itated from the phenol layer with 2 volumes of acetone, taken to dryness. collected by centrifugation, and washed by successive Extracts of untreated mice did not contain any compo suspension and recentrifugation in acetone, ether, and 95% nents with retention times on gas chromatography or HPLC ethanol. The isolated macromolecules were dried under 10 similar to those of ethyl or vinyl carbamate that could be cm mercury continuous vacuum for at least 48 hr before detected by the flame ionization or refractive index detec determination of radioactivity. tors. The DMA was quantitated from its absorbance at 260 nm Analysis of Whole 5-Day-Old CD-1 Mice. In some experi in dilute saline-citrate (15 rriM NaCI-1.5 mM sodium citrate, ments 5-day-old mice of both sexes were given i.p. injec pH 7.0), in which E\^ = 160 (15). The A2(i,,/A280ratiowas tions of 0.56 or 5.62 /*mol of nonradioactive ethyl carba 1.70 to 1.79; the A2ti0/A280ratiofor pure DMA is about 1.85 mate per g of body weight and were killed 1, 2, 4, or 8 hr (15). rRNA was quantitated by its absorbance at 260 nm in later. When extracts of these mice were analyzed by gas dilute saline-citrate [Ejif = 200 (15)]; its A2fi(l/A2((0ratiowas chromatography, the limit of detection of vinyl carbamate 1.93 to 2.06, as compared to literature values of about 2.0 was about 0.1 nmol/2-/Al injection. Thus, as little as 1 to 2 (15). The protein was quantitated gravimetrically. nmol of vinyl carbamate per g of tissue could be detected For determination of 14Cor 3H, 0.2 to 3 mg of DNA were when the extract from 50 g of mice was reduced to a volume incubated with bovine spleen phosphodiesterase (Worth- of 1 to 2 ml. ington Biochemical Corp.); 1 to 6 mg of rRNA were digested In other experiments 5-day-old mice were injected i.p. with RNase A (Worthington). Dry protein (10 mg) was with 0.56 or 5.62 jumol of [efhy/-1,2-3H]ethyl carbamate per solubilized with 1.0 ml of Soluene 100 (Packard Instrument g of body weight (7 to 9 /nCi/g of body weight). Some mice Company, Inc., Downers Grove, III.) containing 5 to 10% also received injections of 0.11 or 1.15 /umol of vinyl water. Aliquots of these solutions were added to RIA Solve carbamate per g and/or 0.1 to 0.2 /umol of DPEA per g at II scintillation cocktail (10 ml). specified times to reduce the metabolism of any radioactive vinyl carbamate formed. The 2Q-/j.\ aliquots of the final RESULTS purified extracts (1 to 3 ml), together with nonradioactive ethyl (300 ^g) or vinyl (100 /Â¿g)carbamate as refractive Acute Toxicity of the Carbamates index markers, were assayed by HPLC and determination of 3H in the fractions eluted at the retention time of vinyl None of the female CD-1 mice given i.p. injections of carbamate. Calculations of yields of "vinyl carbamate" were 0.062 mg of vinyl carbamate per g of body weight died corrected for the expected 42% loss of tritium during within 10 days, but 3 of 4 mice injected with 0.125 mg/g did. dehydrogenation in vivo of ethyl carbamate (assuming no No gross tissue lesions were seen in the latter animals. The isotope effect). mice injected with 0.25 or 0.5 mg/g died within 24 hr with blood-congested lungs and, in some cases, general edema. Extraction of Viscera from Adult A/Jax Mice. Female mice (7 months old) were injected i.p. with 0.56 Â¿imol(15 The cause of death was not evident from the histological piCi) of tritiated ethyl carbamate per g of body weight. After samples studied, but some of the mice showed degenera 3 hr, the mice were injected with 1.15 Â¿imolof vinyl tion of the intestinal epithelium, hypoplasia of the bone carbamate per g; they were killed by cervical dislocation marrow, or hepatic degeneration, none of which were seen 0.25, 0.5, or 1.0 hr later. The combined organs from both in control mice. Ethyl carbamate at levels of 0.05 to 1 mg/g the thoracic and peritoneal cavities (6 to 12 g) were ex of body weight caused no apparent gross or histologically tracted and assayed for vinyl carbamate by HPLC and detectable toxicity. determination of 3Has just described. The limit of detection Comparative Carcinogenic Activities of Vinyl and Ethyl was <0.1 nmol/g of tissue. Carbamate Extraction of Urine. Beginning 2 hr after i.p. injection of tritiated ethyl carbamate, urine was collected in Dry Ice- In agreement with many previous studies (13, 30), ethyl cooled containers for 24 hr from groups of seven 10-week- carbamate initiated skin papillomas and induced lung ade old female CD-1 mice. The urine was diluted with water to a nomas on topical or i.p. administration to mice. Under the volume of 100 ml and extracted 4 times with 3 volumes of same conditions, vinyl carbamate induced the same types CHCI3. The CHCI..Jextracts were reduced to 3 to 5 ml on a of tumors, but it was 10 to 50 times more active than ethyl rotary evaporator (25 cm mercury, 37Â°),and 20-jnl aliquots carbamate. were assayed by HPLC. Tumor Induction by Topical or I.P. Treatment of CD-1 Mice. Topical applications of vinyl carbamate, after pre Binding of Ethyl Carbamate to Hepatic Macromolecules in treatment with croton oil and followed by repetitive twice Vivo weekly doses of croton oil, yielded 10 to 20 times as many A/Jax male or female mice (2 to 3 months, 25 g) were skin papillomas and lung adenomas as were obtained from

NOVEMBER 1978 3797

Table 1 Incidences ol sk/n papillomas and of lung adenomas in female CD-1 mice given topical or i.p. doses of ethyl carbamate or vinyl carbamate. The shaved backs of CD-1 female mice were treated topically with 1.2 mg of croton oil in 0.2 ml of redistilled acetone 18 to 24 hr before each topical (Experiments 1 and 2) or i.p. (Experiment 3) dose of ethyl or vinyl carbamate. The topical doses of the carbamates were applied in 0.2 ml of acetone to the croton oil-treated area of the skin; i.p. doses were administered in 0.87% sterile NaCI solution. Beginning 1 week after the last dose of a carbamate, all of the mice in each experiment were twice weekly treated topically with 0.9 mg of croton oil in 0.15 ml of acetone. Incidence of skin papillomas Incidence of lung adenomas

19wk 29 wk 32 wk

of of of micewithpapilno.papil micewithpapilno.papil micewithadenono.adeno

No.Total micestartNo.ofmiceNo. lomas/mouseNo.ofmiceNo. lomas/mouseNo.ofmiceNo. mas/mouse dose" atof lomasAv. lomasAv. masAv. Experiment 1 Ethyl carbamate 60 mg/mouse x 2 40 40 15 1.6 40 19 3.4 40 33 8.8 5 mg/mouse x 2 40 40 3 0.1 40 10 0.3 40 17 1.0 Vinyl carbamate 5 mg/mouse x 2 40 39 19 3.5 35 23 4.5 34 18.9 Acetone only 40 40 0 0 40 1 0 40 7 0.4

27 wk 32 wk 35 wk

Experiment 2 Ethyl carbamate 60 mg/mouse x 2 30 2930303231309229901.10.10.11.10.802930303230301143121401.80.20.12.01.802930283230272416133227157.31.00.912.04.50.9 5 mg/mouse x 2 31 2.5 mg/mouse x 2 30 Vinyl carbamate 5 mg/mouse x 2 33 2.5 mg/mouse x 2 31 Acetone only 31

17 wk 25 wk 28 wk

Experiment3Ethyl carbamate1.0 20.065mg/g x 1Vinyl mg/g x carbamate0.065 10.87%mg/g x NaCIsolution only40414142394130411921103.20.11.70374126412451515.40.23.9032392641301424428.30.619.20.2 " All topical treatments (Experiments 1 and 2) were divided into 2 equal doses given 1 week apart. In Experiment 3, the 0.065-mg/g .treatments were each given as a single i.p. injection; the 2.0 mg/g dose was divided into 2 equal parts given i.p. with an interval of 1 week.

the same doses of ethyl carbamate under the same condi The topically administered compounds caused little gross tions (Table 1). For instance, in Experiment 1 a total dose of toxicity and no gross skin lesions (other than tumors) at the 10 mg of vinyl or ethyl carbamate initiated an average of 4.5 doses used, but the i.p. injection of 0.065 mg of vinyl or 0.3 skin papillomas/mouse, respectively, by 29 weeks. At carbamate per g of body weight resulted in 37% mortality 32 weeks, the average numbers of lung adenomas were 19 by the 28th week. and 1, respectively. Mice that received a total topical dose Lung Tumors in A/Jax Mice. As expected from studies of 120 mg of ethyl carbamate had approximately the same with ethyl carbamate (37), A/Jax mice were more sensitive incidence of skin papillomas and about one-half as many than CD-1 mice to the induction of lung adenomas by the lung adenomas as mice that received only 10 mg of vinyl carbamates (Table 2). In Experiment 1, vinyl carbamate was carbamate. Similar differences between the tumor inci about 50 times more carcinogenic than ethyl carbamate dences induced by the 2 carbamates were observed in the when both compounds were administered at a dose of second experiment, although the overall incidences of both 0.032 mg/g of body weight. This dose of vinyl carbamate skin and lung tumors were somewhat lower than in Experi induced an average of 42 adenomas/mouse as compared ment 1. I.p. doses of the carbamates were generally 2 to 3 to only 0.8 adenomas/mouse for mice treated with the same times more effective than topical doses for the initiation of amount of ethyl carbamate. The toxicity of vinyl carbamate skin papillomas and the induction of lung adenomas, but administered at a level of 0.065 mg/g of body weight was their relative activities were similar to those just noted evident from the lower incidences of tumors and the poorer (Experiments). survivals as compared to those for mice that received only

3798 CANCER RESEARCH VOL. 38

Table 2 Incidences of lung adenomas in female A/Jax mice given i.p. injections of ethyl or vinyl carbamate The carbamates were injected in 0.005 ml of 0.87% NaCI solution per g of body weight. Multiple injections were given at 1-week intervals. of ofmice no. of no. ofexp.(wk")22222222222228282828202020202020No.mice with adenomas Dose(mg/g)Experiment of mice2020202033203020202092020201914Terminationautopsied192020133320819201792020201914No.adenomas191715133338192059152020193Av./mouse17.41.70.819.142.30.221.435.220.00.419.31.225.253.225.20.4 1Ethyl carbamate0.5 x10.065 x10.032 x1Vinyl carbamate0.065 x10.032 x10.87% NaCIsolution onlyExperiment 2Vinyl carbamate0.065 x10.032 x10.016 x10.87% NaCIsolutiononlyExperiment

3Ethyl carbamate0.5 x10.01 x5Vinyl carbamate0.016 x10.01 x50.005 +100.87% NaCIsolutiononly

x 10Initial " Weeks after the 1st dose.

one-half this dose. and NADH-generating system (Chart 1). With strain TA 1535 Other Tumors in Mice and Rats. In unfinished experi and optimal conditions about 300 to 400 revertants were ments modeled after those of Vesselinovitch and Mihailo- obtained per /Â¿molof vinyl carbamate. No mutagenicity of vich (39, 40), multiple injections of vinyl and ethyl carba vinyl carbamate for strain TA 98 was detected either in the mates into (C57BI/6/Jax x C3H/He/Jax)F, mice and into absence or the presence of fortified liver S-13. Fischer rats over the first 3 to 5 weeks after birth have in With strain TA 1535, the mutagenic response increased duced similar spectra of tumors. In each case, vinyl carba with the level of vinyl carbamate up to about 500 /Â¿g/plate mate appears to be much more active than ethyl carbamate. and with the amount of mouse or rat liver S-13 up to about The mice have developed lung adenomas, hepatomas, 2 mg of S-13 protein per plate. With the S-13-supplemented thymic lymphomas, and Harderian gland adenomas, while system, omission of the NADP+ or heating the liver fraction hepatomas, neurofibrosarcomas of the ear lobe, and seba at 80Â°for 3 min before adding it to the assay tubes led to ceous gland carcinomas of Zymbal's gland have developed complete loss of the mutagenic activity (Table 3). Omission in Fischer rats. The latter two tumor types were observed of NAD+ or the glucose 6-phosphate-glucose-6-phosphate by Tannenbaum ef al. (38) in inbred Sprague-Dawley rats dehydrogenase system caused only small losses in muta treated with ethyl carbamate. genic activity; the cytosol fraction is a good source of the Mutagenicity of Vinyl and Ethyl Carbamate dehydrogenase and oxidizable substrate (10). Inhibition of mutagenesis was also observed on addition of inhibitors of In agreement with previously reported results (23), ethyl the cytochrome P-450 or P-448 monoxygenases (Table 4). carbamate was not mutagenic for S. typhimurium strains SKF 525A (0.5 and 1.0 /Â¿mol/plate) caused a 21% or 72% TA 1535, TA 100, or TA 98 either alone or with the addition reduction in the number of revertants. DPEA was more of NADPH- and NADH-supplemented rat or mouse liver effective; inhibitions of 47% and 71% were observed with microsome-cytosol preparations (S-13). Vinyl carbamate 31 and 62 nmol/plate. In the presence of DPEA or SKF 525A was also not mutagenic for these strains without metabolic at the concentrations tested, there was no decrease in the activation, but it was mutagenic for strains TA 1535 and TA number of bacterial colonies on nutrient agar plates com 100, which detect missense mutations, when the system pared to similar plates without these compounds. was fortified with rat or mouse liver S-13 and an NADPH- The endoplasmic reticulum appears to be the primary

NOVEMBER 1978 3799

600 Table4 Inhibition by DPEA and SKF 525A of liver S-13-mediated mutagenicity of vinyl carbamate for Salmonella typhimurium TA |500 1535 E Mutagenicity was measured by the top agar method (1). Each Â«400 assay tube contained approximately 10s bacteria, 200 /*g of vinyl Ã carbamate, and 1 mg of S-13 protein from CD-1 female mouse liver; o other components were as detailed in Chart 1. DPEA and SKF 525A Â«300 were added in 10 Â¿Â¿gofsterile double-distilled water. A background â€¢¿ of 20 revertants/plate was subtracted from the values given. or 200 InhibitorNoneDPEASKF

525Anmol/plate1631621252502505001000Revertants/plate5104002701507040430340120 100 200 300 400 ,ug Carbamate /Piate Chart 1. Comparison of the mutagemc effects of ethyl and vinyl carbamate for S. typhimurium TA 1535. The mutagenicity was measured by the top agar method (1) with about 10' bacteria/plate. When a carbamate was old female CD-1 mice and those that had received either an tested with metabolic activation, the complete metabolizing system was i.p. injection of 0.1 mg of 3-methylcholanthrene per g of contained in each assay tube. This consisted of 2 mg of S-13 protein from CD-1 female mouse (6 to 8 weeks old) liver, 1.5 fimol of NADP*, 0.75 /imol of body weight 24 hr before being killed or 0.05% of phÃ©no NAD*, 10 (imol of glucose 6-phosphate. 0.44 unit of glucose-6-phosphate barbital in the drinking water for 7 days. No mutagenicity dehydrogenase. 5 nmo\ of EDTA, and 6.25 /imol of MgCL in 125 mM was detected for vinyl carbamate when fortified S-13 prep phosphate buffer, pM 7.8. The carbamates were added to the assay tubes in 10 Â¿ilofsterile, distilled water. A background of 20 revertants/plate has been arations from mouse lungs (2 to 3 mg of protein per plate) subtracted from the values reported. Upper curve, vinyl carbamate with were substituted for the liver preparations. metabolic activation; bottom line, vinyl carbamate without metabolic activa tion and ethyl carbamate with or without metabolic activation; bars, ranges Vinyl carbamate (11.5 /Â¿mol/ml)was incubated for 30 min of values. with a fortified CD-1 mouse liver S-13 (2 mg of protein per ml), and the mixture was then centrifuged at 105,000 x g Table 3 for 45 min. No mutant colonies of strain TA 1535 were induced when 0.5-ml aliquots of the resulting supernatant Cofactor and enzyme requirements for the mutagenicity of vinyl carbamate for Salmonella typhimurium TA 1535 were added immediately to the bacteria in the agar overlay. Mutagenicity was measured by the top agar method (1) with About 400 to 500 revertant colonies should have been approximately 10" bacteria, 100 Â¿igofvinyl carbamate, and 2 mg of observed if the mutagenic metabolite was released into the S-13 protein (female CD-1 mouse liver) per plate; other components supernatant fraction and was stable for the 1-hr period. were as described in Chart 1. A background of 35 revertants/plate Vinyl carbamate was stable in double-distilled water for at has been subtracted from the values given. least 1 week at room temperature in the dark. No. of revert Mutagenicity of Related Compounds. Vinyl A/./V-di- Modifications ants/plate methylcarbamate (synthesized from vinyl chloroformate None 450 - S-13 and cofactors and dimethylamine) was mutagenic to S. typhimurium TA 0 1535 or TA 100 in the presence of mouse or rat liver S-13 - Glucose-6-phosphate and glucose- 330 6-phosphate dehydrogenase preparations, but it was about 20 times less active than - NAD* 330 vinyl carbamate. Thus, 28 Â¿Â¿molofvinyl A/,N-dimethylcar- - NADP- 0 S-13 heated at 80Â°for3 min bamate induced about 420 revertants of TA 1535 above 0 background with 2 mg of protein from CD-1 male rat liver S- 13 per plate. No mutations were induced in the absence of the S-13. As previously found by other workers (23), we did subcellular site in the liver for metabolism of vinyl carba not observe any mutagenicity of vinyl acetate (Aldrich mate to a mutagen. After subtraction of the spontaneous Chemical Co.) at levels up to 260 jumol/plate for these S. mutants, the average number of revertants per mg of typhimurium missense strains either with or without meta protein was 115 for the microsomal fraction as compared to bolic activation by fortified CD-1 female mouse liver S-13. 15, 35, 20, and 0 for the whole homogenate or the nuclear, Vinyl formate (Polysciences, Inc., Warrington, Pa.) at mitochondrial, or cytosolic fractions, respectively. Since amounts up to 135 /xmol/plate, whether or not the latter S- these fractions were not purified, it is likely that some or all of the activities of the nuclear and mitochondrial fractions 13 was present, also did not mutate S. typhimurium TA were due to contaminating endoplasmic reticulum. 1535. In limited studies, liver S-13 preparations from male and Attempts to Find Vinyl Carbamate as a Metabolite of Ethyl female 2- to 3-month-old CD-1 and A/Jax mice had similar Carbamate in Vivo activities in eliciting the mutagenic activity of vinyl carba mate. Furthermore, no significant difference was observed Vinyl carbamate was not detected as a metabolite of ethyl between the activities of the livers of control 2- to 3-month- carbamate by gas chromatography of extracts from 5-day-

3800 CANCER RESEARCH VOL. 38

old CD-1 mice that had been injected with 0.56 or 5.62 Â¿Â¿molnonradioactive vinyl carbamate (Table 5), even though 0.5 of nonradioactive ethyl carbamate per g of body weight, to 6 Â¿iCiof 3H (1 to 2% of that in the total extract) was even though as little as 0.1 nmol/2 Â¿Â¿Iofextract, or about 1 applied to the column. More than 99% of the 3H in the to 2 nmol/g of body weight, could be detected by our extracts (3Hrecoveries from the mice were 11 to 90% of the methodology. About 70% and 35% of a 0.56 ptmol/g dose of dose) eluted at the retention time of ethyl carbamate. As ethyl carbamate were recovered unchanged after 4 and 8 determined by gas chromatography, at least 5 to 10% and hr, respectively. At least 40 and 20% of a dose of vinyl 45% of nonradioactive vinyl carbamate (1.15 Â¿imol/g of carbamate (0.56 /Â¿mol/gof body weight) were present 1 and body weight, i.p.) injected into 5-day-old CD-1 or adult A/ 2 hr after injection, respectively. Of the injected vinyl Jax mice, respectively, was present in extracts of mice carbamate, 50 to 75% was recovered in concentrated ex killed 1 hr later. Vinyl carbamate was administered in an tracts (1 to 2 ml) from two 50-g samples of 5-day-old mice effort to reduce the metabolic destruction of any radioactive that were killed 15 min after injection of 4.6 nmol/g of body vinyl carbamate that might have been formed. DPEA, given weight. Thus, this level of vinyl carbamate should have to some mice in another attempt to inhibit the destruction been detectable, if it was present. of any metabolically formed vinyl carbamate, also did not No 3H-containing peak with the retention time of vinyl permit detection of a radioactive peak with the retention carbamate was detected on HPLC of extracts from whole 5- time of vinyl carbamate. After correction for the theoretical day-old mice or from viscera of adult mice that had been 42% loss of 3H on dehydrogenation of [efft//-1 ,2-3H]ethyl injected with [ef/iy/-1,2-3H]ethyl carbamate with or without carbamate, the baseline levels of 3H in the three fractions

Table 5 Failure to detect tritiated vinyl carbamate in tissues from 5-day-old CD-1 and adult A/Jax mice injected with [ethyl-1,2-3H]ethyl carbamate Whole 5-day-old CD-1 mice or tissues from adult female A/Jax mice were given injections of tritiated ethyl carbamate and, in some cases, vinyl carbamate and/or DPEA as indicated in the table. The whole baby mice and abdominal or thoracic tissues from the adult mice were extracted and analyzed by HPLC for tritiated vinyl carbamate as described under "Materials and Methods." eluted at vinyl retention time of carbamatenmol/gbodywt.1151150115011501150115115115115115011501150Timeinjected(hr)3,4,53.53.53.53.544443.753.753.75DPEAnmol/gbodywt.100100200200100200100100100100Timeinjected(hr)000004 ethyl vinylcarbamate''nmol/gbody carbamate(Ohr),nmol/gbody 3Hfromcolumn Tissuesource5-Day-old micekilled (%of wt.5620r5620''560"560"5620e560^seo'SKf56(/5620''560"560"560"560"560"560"560"560"560"560a560"Nonradioactive(hr)363663.7544.54.533366777744.254.75Recoveryofdose")565327734840404038666268556990574752111712'Hwt1.161.710.310.250.520.140.090.070.061.730.280.770.670.510.580.040.130.120.060.070.02%ofdose0.020.030.060.040.010.020.020.010.010.030.050.140.120.090.100.010.020.020.010.010.004 CD-1 miceViscera,

]5,6J4,5,64,5,64,5,6Time

adultA/Jax miceTritiated

" More than 99% of the 3H recovered from the HPLC column eluted as unchanged ethyl carbamate. * No 3H-containing peak with the retention time of vinyl carbamate was detected in any of the extracts. The values given are the maximum amounts of tritiated vinyl carbamate (after correction for the theoretical 42% loss of 3H expected on dehydrogenation of [ethyl- 1,2-3H]ethyl carbamate) that could have been present if the total 3H in the three fractions (6 ml of eluate) eluting at the retention time of vinyl carbamate were in vinyl carbamate. However, because of the very large amounts (0.5 to 6 ^iCi) of 3H injected onto the HPLC column, low levels of 3H eluted continuously. Since the base-line levels of 3H have not been subtracted from the values given and since there was no peak of 3H in the vinyl carbamate region (even though at least 5 to 10% and 45% of the nonradioactive carrier vinyl carbamate injected into 5-day-old CD-1 and adult A/Jax mice, respectively, was recovered in the extracts 1 hr later), the data provided no evidence for the formation of vinyl carbamate in vivo. c Specific activity, 1.4 mCi/mmol. d Specific activity, 14.1 mCi/mmol. e Specific activity, 1.6 mCi/mmol. f Specific activity, 12.8 mCi/mmol. " Specific activity, 26.8 mCi/mmol.

NOVEMBER 1978 3801

that contained vinyl carbamate accounted for sO.005 to (Chart 2). 0.15% of the total radioactivity applied to the HPLC column. As would be expected for a proximate carcinogen, vinyl This radioactivity was probably mainly or entirely due to the carbamate was much more potent in inducing tumors in the continual elution of low levels of 3H as a result of the high skin or lungs of mice than was ethyl carbamate. Only vinyl levels applied to the column. carbamate, of all the carbamate analogs which have been Vinyl carbamate also could not be detected as a urinary tested, exceeds ethyl carbamate in carcinogenic potency. metabolite of [efhy/-1,2-3H]ethyl carbamate (0.6 to 5.6 The hypothesis that vinyl carbamate is not carcinogenic /nmol/g of body weight, specific activities from 1.2 to 13 directly, but requires metabolism to a reactive electrophile, mCi/mmol) injected i.p. into adult CD-1 mice in three was supported by the mutagenicity data. Thus, vinyl carba experiments. About 1 to 3% of the injected 3H was re mate was not mutagenic per se, but it became mutagenic to covered in the urine excreted 2 to 26 hr after injection. Of S. typhimurium strains TA 1535 or TA 100 on supplementa the 3H in the urine extracts, more than 99% had the tion with a rat or mouse liver microsome plus cytosol retention time of ethyl carbamate on HPLC. No peak of system. NADPH was required for mutagenicity in this sys tritiated product eluted from the Chromatographie column tem. Cytochrome P-450 appears to be responsible for this with the retention time of vinyl carbamate, and the baseline conversion of vinyl carbamate to a mutagen, since the levels of 3H in these fractions could have accounted for addition of DPEA or SKF 525A to the liver metabolizing urinary excretion of no more than 0.5 nmol of vinyl carba system strongly inhibited the formation of revertants of TA mate per mouse. 1535. These data suggest that vinyl carbamate epoxide is Hepatic Macromolecule-bound Derivatives of Ethyl Car bamate in Vivo Table 6 Hepatic nucleic acid- and protein-bound radioactivity in A/Jax When A/Jax male or female mice were injected i.p. with mice given [ethyl-1-"C;ethyl-1,2-3H]ethyl carbamate solutions containing [effty/-1-14C;1,2-3H]ethyl carbamate In each experiment, three A/Jax mice were given i.p. injections and were killed 12 hr later, the ratios of 3H/14C bound to of 0.5 mg of [ef/7y/-1-14C;etf7y/-1,2-3H]ethyl carbamate per g of body hepatic DNA, rRNA, and protein were much lower than the weight 12 hr before being killed. The liver macromolecules were isolated and the radioactivity was quantitated as described under 3H/14Cratios of the injected ethyl carbamate (Table 6). The "Materialsand Methods." average decreases in the 3H/'"C ratios were 62%, 75%, and 62%, respectively, for hepatic DNA, rRNA, and protein from Female mice Male mice the female mice. The average decreases for the male mice 3H/14C were: DNA, 67%, rRNA, 76%; and protein, 73%. 51*13 On the basis of the specific activity of the injected [ethyl- EthylinjectedDNArRNAProteinDNArRNAProtein32"11812nmold0.42.932Â° carbamate 1-'4C]ethyl carbamate, 2 to 11 x 1014residues from ethyl 128 1212 carbamate were bound per mg of DNA. This high level of 1451Â»221314ethyl binding, corresponding to 1 to 6 ethyl carbamate residues residuesbound/mgc1.9carbamate per 10" nucleotides, is in agreement with some values in the literature for another mouse strain (20, 33). The incor 0.40.6 poration of 14Cinto DNA was apparently not a consequence 0.62.1 of incorporation of 14C-containing small molecules during 6.00.40.47.1 synthesis of new DNA. Thus, very little, if any, 14Cor 3Hwas " Specific activity: 3H = 3.9 mCi/mmol; "C = 0.12 mCi/mmol. b Specific activity: 3H = 9.2 mCi/mmol; 14C = 0.18 mCi/mmol. contained in the adenine and guanine recovered after '' The levels of bound residues were calculated from the incor hydrolysis of the DNA with 1 N MCI for 1 hr at 100Â°(M. poration of 14C; the specific activities were 170 to 500 dpm/mg of Ribovich, J. A. Miller, and E. C. Miller, unpublished data). DNA, 100 to 200 dpm/mg of rRNA, and 550 to 2800 dpm/mg of The levels of binding of residues of ethyl carbamate to rRNA protein. and protein (equivalent to 2 to 4 x 10'" and 1 to 5 x 1015 rf Sample for UV analysis lost. residues per mg, respectively, based on 14C) are more CHvCHo-0-C-NH, difficult to interpret, because it was not excluded that , 2 M 2 incorporation of radioactivity occurred as a result of a new synthesis of these macromolecules subsequent to oxidation of ethanol derived by hydrolysis of the ethyl carbamate (30). Some 14C and 3H from the rRNA hydrolysates chromatographed on ion exchange columns with adenine and gua nine.

CH2-CH-0-C-NH2 DISCUSSION Several lines of evidence obtained in these studies are consistent with our initial hypothesis that ethyl carbamate is dehydrogenated in vivo to the proximate carcinogen vinyl COVALENT ADOUCIS WITH NUCLEOPHILES carbamate and that the latter metabolite undergoes epoxi- IN CRITICAL CELLULAR MACROMOLECULES dation to form a reactive electrophilic ultimate carcinogen Chart 2. Some possible initial steps in carcinogenesis by ethyl carbamate.

3802 CANCER RESEARCH VOL. 38

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1978 American Association for Cancer Research. Carcinogenic/ties of Vinyl and Ethyl Carbamates an electrophilic metabolite of vinyl carbamate in this sys support the conclusion of Lawson and Pound (21) and tem. Pound et al. (33) that all of the DNA-bound adducts can be No increase was observed in the ability of liver prepara degraded to ethanol. tions from 3-methylcholanthrene- or phenobarbital-induced A possible alternate explanation of our data is that vinyl CD-1 mice to metabolize vinyl carbamate to a mutagen for carbamate is not a metabolite of ethyl carbamate and that TA 1535 relative to similar preparations from uninduced both compounds are metabolized in vivo to intermediates mice. It is possible that the enzyme responsible for metab in common or parallel pathways to an ultimate reactive olism of vinyl carbamate to its putative epoxide is not metabolite(s) that binds covalently to macromolecules inducible, or at least not inducible in the CD-1 mice tested. (Chart 2). Alternatively, enzymes that metabolize vinyl carbamate via Further studies on the metabolism and carcinogenicity of competitive pathways or enzymes that detoxify vinyl carba these carbamates and related compounds are in progress mate epoxide may have also been induced so that no net in this laboratory. increase in mutagenicity resulted. Our hypothesis, however, was not supported by the failure to detect vinyl carbamate as an in vivo metabolite of ACKNOWLEDGMENTS ethyl carbamate in the mouse under a variety of conditions. At the McArdle Laboratory, we are indebted to Priscilla Kimberly and Mary In spite of being able to detect as little as 1 to 2 nmol of Kolstad for assistance with the animal tests, to Judith Blomquist and Diane vinyl carbamate per g of body weight by gas chromatogra- Chambliss for the mutagenicity tests, and to Lona Barsness and Drs. Henry Pilot and George Michalopoulos for the histolÃ³gica!examinations. Dr. Philip phy and less than 1 nmol/g by scintillation counting of Hart and James Blackburn of the School of Pharmacy, University of Wiscon- HPLC fractions derived from [efA?y/-1,2-3H]ethylcarbamate- son-Madison, kindly assisted us with the NMR determinations. Details on the injected mice, we were unable to detect vinyl carbamate as synthesis of vinyl carbamate were graciously supplied by Dr. J. R. Schaef- a metabolite of ethyl carbamate in extracts of whole 5-day- gen, Pioneering Research Division. Experimental Station, E. I. du Pont de Nemours and Co., Wilmington, Delaware. old CD-1 mice, in extracts of viscera obtained from adult A/ Jax mice, or in extracts of urine from CD-1 adult mice. In some experiments, carrier nonradioactive vinyl carbamate REFERENCES and/or DPEA were also injected to protect any labeled vinyl carbamate, which might have been formed, from rapid 1. Ames, B. N., McCann, J., and Yamasaki. E. Methods for Detecting Carcinogens and Mutagenswith theSa/mone//a/mammalian-microsome metabolic destruction. Even though the nonradioactive Mutagenicity Test. Mutation Res.,3J: 347-363. 1975. carrier vinyl carbamate was detected in the extracts by gas 2. Berenblum, I., Ben-lshai, D., Haran-Ghera. N., Lapidot, A., Simon. E., chromatography, no tritiated vinyl carbamate was found. It and Trainin, N. Skin Initiating Action and Lung Carcmogenesis by Derivatives of Urethane (Ethyl Carbamate) and Related Compounds. is possible that the injection of vinyl carbamate and/or Biochem. Pharmacol., 2: 168-176, 1959. DPEA inhibited or destroyed a putative enzyme involved in 3. Boyland, E., and Nery, R. The Metabolism of Urethane and Related Compounds. Biochem. J., 94: 198-208,1965. the formation of vinyl carbamate from ethyl carbamate. 4. Boyland, E., and Williams, K. Reaction of Urethane with Nucleic Acids in While these collective data indicate vinyl carbamate is not a Vivo. Biochem. J..111: 121-127, 1969. metabolite of ethyl carbamate, the possibility remains that 5. Chavan, B. G., and Bhide. S. V. Interaction of Urethan with Macromole cules in Male and Female Newborn, Adult, and Tumor-bearing Mice. J. vinyl carbamate is formed at an enzymatic site in vivo and Nati. Cancer Inst., 49: 1019-1025, 1972. immediately further metabolized. 6. Clayson, D. B., and Garner, R. C. Carcinogenic Aromatic Amines and Related Compounds, in: C. E. Searle (ed.), Chemical Carcinogens. ACS Although we were not able to find vinyl carbamate as a Monograph 173, pp. 366-461. Washington, D. 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3804 CANCER RESEARCH VOL. 38

Gary A. Dahl, James A. Miller and Elizabeth C. Miller

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