Comparative Carcinogenicities and Mutagenicities of Vinyl Carbamate, Ethyl Carbamate, and Ethyl N-Hydroxycarbamate

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[CANCER RESEARCH 40, 1194-1203, April 1980) 0008-5472/80/0040-0000$02.00 Comparative Carcinogenicities and Mutagenicities of Vinyl Carbamate, Ethyl Carbamate, and Ethyl N-Hydroxycarbamate

Gary A. DahI,2 Elizabeth C. Miller,3 and James A. Miller

McArdle Laboratory for Cancer Research, University of Wisconsin Center for Health Sciences, Madison, Wisconsin 53706

ABSTRACT developed independently by Olofson et a!. for some N-substi tuted enol carbamates, is presented. When administered during the first S weeks after birth, vinyl and ethyl carbamates each induced ear duct and hepatic carcinomas and neurofibrosarcomas of the ear lobe in Fischer INTRODUCTION rats. Vinyl carbamate was more active than ethyl carbamate Structural modifications of the multipotential carcinogen for the induction of the latter two types of tumors. Similarly, ethyl carbamate (urethan), CH3CH2â€”O---COâ€”-NH2,generally vinyl carbamate administered in the first 3.5 weeks after birth result in dramatic losses of carcinogenic activity, which usually induced more liver tumors, thymomas, lung adenomas, and have been assayed by lung adenoma induction and by initiation Hardenian gland tumors in CS7BL/6J x C3H/HeJ F1 mice of skin papillomas in mice (reviewed in Refs. 12 and 20). than did ethyl carbamate. These data extend our earlier data One exception is ethyl N-hydroxycanbamate, CH3CH2â€” on the much greater potency of vinyl carbamate than of ethyl Oâ€”COâ€”-NHOH,ametabolite of ethyl carbamate (4, 19) with carbamate for the induction of lung adenomas in A/J and CD approximately one-half of the tumor-inducing ability of the 1 mice and for the initiation of skin tumors in CD-l mice. parent compound (2, 17). This metabolite has greater terato CD3CD2â€”Oâ€”COâ€”-NH2did not differ significantly from genic and chromosome-damaging activities than does ethyl CH3CH2â€”Oâ€”â€”CO----NH2forthe induction of lung tumors in Al carbamate (3, 5, 8). The N-hydroxy derivative reacts with J mice; tert-butyl carbamate had no significant activity in the cysteine derivatives (4) and with cytosine (21 , 23); ethyl car latter mouse strain. bamate does not show these chemical reactivities. Because of High doses of the mixed-function oxidase inhibitor 2-(2,4- its somewhat lower carcinogenic activity and its facile reduction dichloro-6-phenyl)phenoxyethylamine inhibited the induction to ethyl carbamate in vivo (18, 19), Minvish(19) suggested that of lung adenomas in A/J mice by ethyl N-hydroxycarbamate; ethyl carbamate may be a proximate carcinogen of ethyl N- lung tumor induction by ethyl on vinyl carbamate was not hydroxycarbamate. In support of this idea, Kaye and Trainin inhibited. A lower dose of the above inhibitor or of 2-diethyl (14) reported that administration of SKF 525A,4 which inhibits aminoethyl-2,2-diphenylvalerate did not significantly inhibit the reduction of the N-hydroxy derivative in vivo (19), inhibited lung adenoma induction by any of these canbamates. In confin the induction of lung adenomas in mice by ethyl N-hydroxycar mation of previous reports by other investigators, administra bamate but not by ethyl carbamate. Likewise, Nomura (25) tion of caffeine concurrently with ethyl carbamate inhibited the reported that caffeine administered during the first 36 hr after formation of lung adenomas in A/J mice; no consistent inhibi treatment with the carbamate inhibited induction of these ade tion was obtained under the same conditions when vinyl can nomas by ethyl canbamate but not by ethyl N-hydnoxycarba bamate or ethyl N-hydroxycanbamate was the carcinogen. mate. Since reduced nicotinamide adenine dinucleotide phos Recently, we reported that vinyl carbamate, CH2= phate-fortified duck liver microsome-cytosol preparations can CHâ€”Oâ€”COâ€”NH2,@5muchmore active than ethyl carbamate dehydrogenate aflatoxin B2 (2,3-CH2-â€”-CH2â€”)toaflatoxin B1 in initiating skin papillomas and in inducing lung adenomas in (2,3â€”CH=CHâ€”), the ability of this system to convert ethyl mice (7). Vinyl and ethyl canbamatewere not directly mutagenic carbamate to vinyl carbamate was examined. Vinyl carbamate for Salmonella typhimurium strains TA 1535 and TA 100. was not detected as a metabolite.Furthermore,ethyl carba However, vinyl carbamate, but not ethyl carbamate, was mu mate was not mutagenic for Salmonella typhimurium TA 100 in tagenic when the assay system was supplemented with a the presence of a rat and duck liver system that metabolized NADPH- and NADH-fortified rat or mouse liver S-i 3 system (7). both vinyl carbamate and aflatoxin B2 to mutagens. Ethyl N- This mutagenic activity was decreased by the cytochrome P hydroxycarbamate showed very weak direct mutagenic activity 450 inhibitors DPEA on SKF 525A. Vinyl carbamate was not (2 to 3 revertants4&mol) for S. typhimurium strains TA 98, TA detected as an in vivo metabolite of ethyl carbamate in mice, 1535, andTA 100; bothduckandrat liverpreparationsfortified although the low 3H:'4C ratio of hepatic DNA isolated from mice with reduced nicotinamide adenine dinucleotide phosphate and given injections of [ethyl-l-14C;l ,2-3H]ethyl carbamate sug reduced nicotinamide adenine dinucleotide reduced the muta gested that the major bound residue was not an intact ethyl genic activity. group (7). An improved synthesis of vinyl canbamate, similar to that This paper presents comparative data on the carcinogenic and mutagenic activities of vinyl carbamate, ethyl carbamate,

1 This work was supported by Grants CA-071 75, CA-22484, and CA-091 35 and ethyl N-hydroxycarbamate. from the National Cancer Institute, USPHS. 2 Present address: Centre National de Recherches Agronomlques, Route de 4 The abbreviations used are: SKF 525A, 2-dlethylaminoethyl-2,2-diphenyl Saint-Cyr, 78000 versaIlles, France. valerate hydrochloride; S-i 3, microsomes plus cytosol obtained by centrifugation 3 To whom requests for reprints should be addressed. of homogenate at 13,000 x g for I 0 mm; DPEA. 2-(2,4.dlchloro-6- Received October 22, 1979; accepted January 7, 1980. phenyl)ptienoxyethylamlne; HPLC. high-performance liquid chromatography.

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MATERIALS AND METHODS tube and a 1-liter trap) into a stirred, ice-cooled suspension of 200 mmol of Hg(CH2CHO)2in 200 ml of CH2CI2in a 2-necked Instrumentation and General Procedures 1-liter round-bottomed flask. The excess CI2CO exited the reaction vessel through a CaSO4drying tube, a 1-liter trap, and Instruments and/or methods for characterization (IA, nuclear 3 gas scrubberscontaining20% KOH. The reaction mixture magnetic resonance, melting point, and mass spectrometny) as was stirred for 1 hr more on ice and then for 2 hr at room well as for purification and assay (HPLC, gas chromatography, temperature. The suspension was filtered through a glass fnit, and liquid scintillation counting) of the carbamates were de after which the filtrate was maintained at 35Â°with stirring until scribed previously (7). no more CI2CO exited, as detected by phosgene indicator Chemicals paper (15). The vinyl chloroformate and CH2CI2were rapidly distilled (at 55â€”70Â°andabout 200 mm Hg) to separate the Ethyl carbamate, tert-butyl canbamate, and vinyl butyl ether liquid from solid impurities. werepurchasedfromAldrichChemicalCo.,Milwaukee,Wis. Reaction of the vinyl chloroformate with NH3(7, 28) yielded Ethyl N-hydroxycarbamate was prepared in our laboratory (10). 140 mmol of vinyl carbamate [about 70% based on NAD@, NADP@, glucose 6-phosphate, Tnis, and caffeine were Hg(CH2CHO)2,assuming that it reacts with 1 molar equivalent from Sigma Chemical Co., St. Louis, Mo. Glucose-6-phosphate of CI2CO];the physical (melting point, IA, and mass spectrum), dehydrogenase was obtained from Worthington Biochemical chromatographic (HPLC and gas chromatography), and muta Corp., Freehold, N. J. DPEA was kindly provided by Dr. A. E. genic properties were identical to those previously described McMahon, Eli Lilly Co., Indianapolis, Ind., and SKF 525A was (7). Olofson et a!. (26) independently reported the syntheses a gift of Smith, Kline, and French Laboratories, Philadelphia, of several enol chloroformates and N-substituted enol carba Pa. (carbonyl-14C]Ethyl carbamate (3 mCi/mmol) was pun mates by similar methods after the work reported above was chased from New England Nuclear, Boston, Mass., and was completed. determined to be about 98% radiochemically pure by liquid Synthesis of CD3CD2â€”O----CO-â€”NH2.Perdeuterated-ethyl scintillation analysis of 30-sec fractions obtained by HPLC on canbamate (CD3CD2â€”O--â€”CO-â€”NH2),correctedm.p. 46â€”47Â°, @Porasil(7).Phosgene and NH3were obtained from the Mathe was synthesized (>95% yield) by reaction of equimolar son Co., Joliet, Ill. HgO was from the J. T. Baker Co., Phillips amounts of CD3CD2ODand CI2COin dry ethyl ether and sub burg, N. J., and mercuric acetate was from Allied Chemical sequent addition of NH3.The product was sublimed to a liquid Co., Morristown, N. J. Perdeuterated ethanol, CD3CD2OD(nu N2-cooled cold finger at 100 mm Hg and 45Â°.Elementary clear magnetic resonance grade), was purchased from Norell analyses (Huffman Laboratories, Wheatnidge, Cob.) for C, H Chemical Co., Landisville, N. J. Aroclor 1254 was a gift from + D, N, and 0 were within 0.4% of theoretical. The mass the Monsanto Chemical Co., St. Louis, Mo. spectrum had an M'@peak at 94 but no peak at 89; thus, no All syntheses were carried out in an efficient chemical hood, impurity containing protium in the ethyl group was detected. and solvent-resistant neoprene gloves were worn. Solid ethyl and vinyl carbamate were handled in chemical hoods since Carcinogenicity Studies both compounds sublime readily at room temperature and atmospheric pressure. Methods (15) for the safe handling of The offspring of Fischer rats (Charles River Breeding Labo phosgene were followed. ratory, Wilmington, Mass.) and of female C57BL/6J (The Jack Synthesis of VInyl Carbamate. A new synthetic route to son Laboratory, Bar Harbor, Maine) and male C3H/HeJ (The vinyl carbamate was devised since the previous procedure (7) Jackson Laboratory) mice were obtained by breeding in this gave low and variable yields. Mencunidiacetaldehyde [Hg laboratory. Female A/J mice (6 to 8 weeks old) were purchased (CH2CHO)2, m.p. 82â€”84Â°corrected; literature 82â€”83Â°(22)] from The Jackson Laboratory. The breeding and nursing rats waspreparedin60%yieldfromvinylbutyletherviatheacetal and mice and the C57BL/6J x C3H/HeJ F1mice were main [(CH3O)@CHCH2â€”HgCH2CH(OCH3)OH]according to the tamed on wood-chip bedding (Northeastern Products Corp., method of Nesmeyanov et a!. (24). Since Ref. 24 is not readily Wannensbung,N. V.) in plastic cages. The A/J mice and the accessible and gives a relatively brief description of the syn rats after weaning were maintained in screen-bottomed cages. thesis, the following details are presented. Dry red HgO (108 The rats were maintained individually, and the mice were 9, 0.5 mol) and mercuric acetate (4 g, 0.01 3 mol) in 70 ml of housed 5 to a cage. All animals were fed Wayne Breeder Blox dry methanol were cooled on ice. To this mixture, vinyl butyl (Allied Mills, Inc., Chicago, Ill.) and water ad libitum. The ether (110 g, 1.1 mol) was added dropwise with vigorous animals were weighed at the beginning of each experiment and stirring and cooling by ice. After 2 hr of stirring, the reaction monthly thereafter. Unless indicated otherwise, the test com mixturewas allowedto warmto roomtemperature.After22 hr pounds were injected in sterile 0.9% NaCI solution (0.01 and of further stirring, the orange colon of the HgO disappeared. 0.005 ml/g body weight for rats and mice, respectively). Con The solution, containing some gray residue, was filtered trol animals always received injections of the solvent on the through a glass frit to give a clear, colorless filtrate. Fifty ml of same schedule as did the treated animals. dry ether were added, and the solution was cooled on ice. On death or at the termination of an experiment, all of the Addition of 15 ml of 0.1 N H2SO4 yielded a silvery-white animals were subjected to gross routine autopsies, which in precipitate, which was collected on a glass fnit under 100 mm cluded inspection of the external and s.c. tissues and the Hg vacuum, washed with 25 ml of dry methanol and 25 ml of organs of the abdominal and thonacic cavities. The lungs of all dry ether, and dried in a vacuum desiccator over CaSO4. A/J mice were fixed in 10% buffered formalin, and the lung For synthesis of vinyl chlonoformate, about 400 mmol of adenomas on the surface (@l mm in diameter) were enumer CI2CO were bubbled (after passing through a CaSO4 drying ated. All other tumors and other grossly abnormal tissues were

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1980 American Association for Cancer Research. G. A. DahI et a!. fixed in 10% buffered fonmalin, cut at 5 to 6 @m,andstained mediately after the injection of the carbamate. On the basis of with hematoxylin and eosin. the results of Theiss and Shimkin (30), the female A/J mice in Tumors in Fischer Rats. Starting within 24 hr of birth, male Experiment 2 (15 to 19 mice pengroup) were each given only and female Fischer rats were given 5 once-weekly i.p. injec 2 i.p. injections of caffeine (each 206 nmol per g body weight) tions of 92 nmol of vinyl canbamate pen g body weight on 10 at 3 hr before and 3 hr after a single i.p. injection of 2000 or twice-weekly injections of 92 nmol of ethyl or vinyl canbamate 4000 nmol of ethyl carbamate or ethyl N-hydnoxycarbamate per g, 3370 nmol of ethyl carbamate per g, or 0.9% NaCI per g body weight or of 75 or 150 nmol of vinyl carbamate per solution only. Almost all of the rats survived these treatments, g. In both experiments, control mice received 0.9% NaCI and 17 to 20 of each sex and dose were weaned. The rats in solution in place of the carbamate or caffeine. Both expeni several other litters were given 5 once-weekly i.p. injections of ments were terminated at 6.5 months. 380 nmol of vinyl carbamate per g body weight. Most of the latter rats died within 3 weeks, but those that survived were Statistics entered into the experiment. The experiment was terminated The significance levels for tumor incidences and tumor mul when the rats were 22 to 23 months old. This experiment was tiplicities were determined with Fischer's exact test (Ref. 6, p. similar to those carried out by Vesselinovitch and Mihailovich 195) and the Wilcoxontest (Ref. 11, p. 68), respectively. (32) with ethyl carbamate. Tumors in C57BL/6J x C3H/HeJ F1Mice. Beginning within Mutagenicity Assays 24 hr of birth, male and female C57BL/6J x C3H/HeJ F1mice were given 8 twice-weekly i.p. injections of ethyl carbamate Mutagenicity for S. typhimurium missense mutants TA 100 (46, 91 , 136, or 5625 nmol/g body weight) or vinyl carbamate or TA 1535 and for the frame-shift mutant TA 98 (kindly (46, 91 , or 136 nmol/g body weight) or 0.9% NaCI solution provided by Dr. Bruce Ames, University of California, Berkeley, only; >90% of the mice survived to weaning. At weaning, each Calif.) was assayed by the top agar method of Ames et a!. (1), group contained 18 to 25 mice of each sex. The surviving mice as modified by Swanson et a!. (29). 5-1 3 fractions were pre were killed at 15 to 16 months. Vesselinovitch and Mihailovich pared (7) from livers from Aroclon 1254-induced adult Fischer (31) used a similar protocol to induce tumors in mice of this rats (killed 5 days after the i.p. injection of 500 mg/kg body hybrid with ethyl carbamate. weight) or from uninduced 7-day-old Peking ducks (Sunnyside Lung Adenomas in A/J Mice. In Experiment 1, 6- to 8- Hatchery, Oregon, Wis.). week-old female A/J mice (30/group) were each given a single i.p. injection (3000 or 6000 nmol/g body weight) of an ethyl Attempts to Find Vinyl Carbamate as an in Vitro Metabolite carbamate which contained only protium or only deutenium in ofEthylCarbamate the ethyl group. The animals were killed 5 months after treat Each 3-mI assay mixture (in open 25-mI Erlenmeyen flasks) ment. contained 9000 nmol glucose 6-phosphate, 0.6 unit glucose In Experiment 2, the mice in one group were each given one 6-phosphate dehydrogenase, 1500 nmol NADP@,750 nmol i.p. dose of 2800 nmol of tert-butyl carbamate per g body NAD@,300 nmol MgCI2,and S-l 3 (13,000 x g, 10-mm super weight; then, since 8 of 18 mice died within 5 days, the level natant) or microsomes (105,000 x g, 60-mm pellet) from 250 was reduced to 1400 nmol per g for 5 more doses beginning mg of liven (in 1 ml of 50 m@potassium phosphate-250 m@ 1 week later. Other mice (1 8 per group) were each given 6 sucrose, pH 7.4, at 37Â°C)from 7-day-old Peking ducks in 50 twice-weekly i.p. doses of 700 nmol of tert-butyl carbamate or mM potassium phosphate buffer, pH 7.4. After 5 mm preincu ethyl carbamate per g body weight in dimethyl sulfoxide:waten bation at 37Â°with shaking, 1 @Ci(90nmol) of [carbonyl-14C]- (2:1 , by volume; 0.005 ml/g body weight). All mice were killed ethyl carbamate (in 0.005 ml dimethyl sulfoxide) and, in some 8.5 months after the first injection. cases, 3000 nmol of nonradioactive vinyl carbamate (in 0.05 To study the effects of cytochrome P-450 mixed-function ml H2O)were added. The flasks were then incubated at 37Â°C oxidase inhibitors on lung tumonigenesis by the carbamates, with shaking for 0 to 60 mm, at which times the reactions were female A/J mice (17 to 20 pengroup) were given i.p. injections stopped by chilling the flasks on ice. The contents of each of 40 nmol of DPEA or 130 nmol of SKF 525A per g body assay flask were extracted 4 times with 9 ml of CHCI3. The weight immediately before a single i.p. injection of ethyl car CHCI3extracts from each assay were pooled and evaporated bamate (4000 nmol per g), ethyl N-hydroxycarbamate (4000 to 0.5 to 1.3 ml on a rotary evaporator. The residual CHCI3 nmol per g), or vinyl carbamate (150 nmol per g). In addition, was filtered through a 0.2-@tmMilliponefilter (Millipore Corp., beginning 2 hr after the initial treatment, the mice in some of Bedford, Mass.), and 0.05- or 0.1-ml aliquots were fractionated the DPEA-treated groups received 7 more i.p. doses of DPEA by HPLC as described previously (7) for determination of at the same level at 2-hr intervals. All mice were killed 7 months radioactivity in the 30-sec fractions in which vinyl carbamate after treatment. and ethyl canbamate eluted. In an experimental design similar to that of Nomura (25) for studying the effect of caffeine on the development of lung RESULTS adenomas, the mice (15/group) were each given a single i.p. dose of ethyl carbamate (1100 or 5600 nmol/g body weight), Carcinogenlcity Studies ethyl N-hydroxycarbamate (950 or 4800 nmol/g), on vinyl canbamate (57 or 115 nmol/g). At each dose, the mice of one Comparative Carcinogenic Activities of Ethyl and Vinyl group were given 7 s.c. injections of 258 nmol of caffeine per Carbamates In Rats and Mice Treated Early In Life. Except g body weight at 6-hr intervals; the first dose was given im for the highest dose of vinyl carbamate injected into Fischer

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rats (380 nmol/g for each of 5 injections), the mortality among 92 nmol of vinyl carbamate pen g body weight than in those the ethyl and vinyl carbamate-treated Fischer rats and C57BL/ that received ten 92-nmol doses of ethyl canbamate per g 6 x C3H/He F1mice during the injection periods was less than during the first 5 weeks after birth. Thus, 33 and 50%, respec 10%. Likewise, after the injections were completed, the rats tively, of the male and female rats given 10 injections of 92 and mice treated with the carbamates gained weight at levels nmol of vinyl canbamate per g developed hepatic carcinomas, similar to those of the solvent-treated controls. Most of the while only 15 and 9%, respectively, of those given this dose of animals that did not develop tumors survived to the end of the ethyl carbamate had hepatic tumors. In these same groups, 5 experiments. of the 38 male and female rats treated with vinyl carbamate In agreement with earlier studies with ethyl canbamate (32), and none of those treated with ethyl carbamate had neurofi male and female rats treated with either ethyl or vinyl carbamate brosarcomas by the end of the experiment. There was no developed hepatic carcinomas, carcinomas of the ear duct statistically significant difference between the incidences of gland (Zymbal's gland), and neunofibrosarcomas of the ear squamous cell carcinomas of the ear duct gland in the rats lobe (Table 1); except for 1 ear duct gland carcinoma, none of given this dose of ethyl or vinyl carbamate. There was some these tumors were found in the control rats. The hepatic increase in incidence of hepatic tumors with increasing doses, carcinomas,first found in a rat that died at 15 months,devel especially for the rats that received vinyl carbamate; however, oped in higher frequency in rats that received 5 or 10 doses of the incidences of the other tumors showed no dose depend

Table 1 Tumors in Fischer rats treated with ethyl or vinyl carbamate BegInning within 24 hr of birth, Fischer rats were given i.p. injections of 5 once-weekly or 10 twice-weekly doses of vinyl carbamate, ethyl carbamate,oronlythesolvent(0.9%NaCIsolution).Theexperimentwasterminatedat22to23months.Forfurtherdetails,seeâ€˜â€˜Materials and Methods.â€•No. of ratswithNeurofi

No. of Hepatic Ear duct brosar- Testicu Dose (nmol/g body Av. age at rats au- carcino- carcino- comas of lar tu- Other tu Groupmors1 Carbamate wt x no. of doses) Sex death (mos.) topsied mas8 masb ear lobeC mors'@ Ethyl 92x10 M 2i.7Â±i.ie 20 3 2 0 2 2@ 02 Ethyl 92 x 10 F 21.8 Â±4.0 20 0 0

Ethyl 3370 x 10 M 19.6 Â±3.0 18 3â€• 4 1 3 1' @ 9'3 Ethyl 3370 x 10 F 20.4 Â±2.4 17 6h o

Vinyl 92 x 5 M 19.0 Â±3.6 19 6â€• 1 5 4 3' 7â€•4 Vinyl 92 x 5 F 20.4 Â±2.5 19 4m 2 2

Vinyl 92 x 10 M 17.8 Â±3.9 18 6 4 4 1 7Â° l@5 Vinyl 92x10 F 20.0Â±2.2 20 10 2 1

Vinyl 380 x 5 M 17.2 Â±3.8 10 8 4 0 0 1'6 Vinyl 380 x 5 F 15.2 Â±3.8 3 2 1 1

(0.9%NaCI (0.01 ml x 10) M 22.2 Â±1.2 20 0 1 0 9 3$ only) (0.9% NaCI F 21.5 Â±1.7 19 0 0 0 2' only)a

Moet were mixed nepatoceiiu@ar-cnoiangioceiuuuar. carcinomas; a iew were aiagnoseo as hepatocellular or cholangiocellular carcinomas. Groups 2, 3, 4, and 5 (males and females combined) had incidences of hepatic carcinomas which were significantly (p < 0.001 ) above those of the controls. Group 4 had a significantly higher incidence of hepatic carcinomas than did Group 1 ( p < 0.001). 6 some groups had significant or marginally significant incidences of ear duct tumors compared to 0.9% NaCI solution-treated controls (Group 6): Group 2, p < 0.08; Group 4, p < 0.05; Group 5, p < 0.003. C The significance levels for the incidences of neurofibrosarcomas of the ear lobe for Groups 3 and 4 compared to Group 6 were p <0.005 and/ < 0.003. respectively. Group 4 had a higher incidence of this tumor than did Group 1 (p < 0.03). These were interstitial cell tumors of the testis, which occur spontaneously in aged male Fischer rats. MeanÂ±S.D. I One fibroma and 1 papillary neoplasm of mesothelial origin. 9 One mammary adenocarcinoma. h Two more rats had microscopic hepatic carcinomas. I Neurofibrosarcoma of the rib cage. I One hepatic hemangioendothelioma, 1 mammary adenocarcinoma, 3 mammary adenomas, 1 adrenocortical carcinoma, 1 renal cell car@lnoma,1 adenocarclnoma on the tail, and 1 s.c. sebaceous gland carcinoma. Three more rats had microscopic hepatic carcinomas. I One islet cell acienoma of the pancreas, 1 s.c. mammary gland carcinoma, and 1 unclassified tumor of the small intestine. @ m more rat had microscopic hepatic carcinomas. n Two mammary adenomas, 2 lymphomas, 1 granulosa theca cell tumor of the ovary, 1 adenocarcinoma of the intestine, and 1 mammary carcinoma. 0 Two angiosarcomas of the liver, 2 pulmonary adenocarcinomas, 2 s.c. sarcomas, and 1 basosquamous papilloma of the lip. p One mammary fibroadenoma. 0 One epidermoid carcinoma of the lung, 1 mesothelloma, 1 s.c. fibrosarcoma, 2 s.c. fibromas, 1 leukemia, and 1 squamous cell papilloma oftheskin. r One mammary adenocarcinoma. aOneadrenocorticalcarcinoma,1osteogenictumoroftheupperjaw,and1s.c.fibroma. t One leukemia and 1 s.c. fibroadenoma.

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Table 2 Tumors other than liver tumors in C57BL/6J x C3H/HeJF, mice treated with ethyl or vinyl carbamate Beginning within 24 hr of birth, mice were given twice-weekly i.p. Injections of 8 doses of a carbamate or only the injection medium. The experiment was terminated at 15 to 16 months. For further details, see â€œMaterialsandMethods.â€• No. of with lung ad with thymomasMice miceDose miceMice enomasNo. of (nmol/galive atwith Harder wt X no. of of age at of Av. no., gland hi wIth GroupCarbamatebodytumors1 doses)Sex3.5 mos.No. mlcebAv.death (mos.)No.miceC mouseian mor?Mice other x 8 0 2 Ethyl 46x8 F 24 0 2 0.08 1(lf None â€˜43EthylEthyl46 91x8M M25 250 00 4 0.20 0d x8 0.2 g65EthylEthyl91 136x8F M22 250 04 2 0.080 2(2)None x 8 0.4 â€˜ 7 Ethyl 5625 X 8 M 17 5 Â±0.4â€• 5 0.7 3 (3) 8 Ethyl 5625x8 F 20 9 5.3Â±1.0 9 1.7 3(5) â€˜ 9 Vinyl 46x8 M 19 0 10 1.3 4(4) @â€˜ â€˜ 10 Vinyl 46 x 8 F 19 0 15 1.8 6 (7) 11 Vinyl 91 x8 M 19 3 7.2Â±4.2 15 2.1 0 m â€œ13 12EthylVinyl136 91 x8F F23 210 44.7 7.4Â±2.36 16 2.83(3) 5(5)None 2.1 Â°1 14VinylVinyl136x8 136 x 8M F23 199 67.3Â±3.15.6Â±2.210 10 1.91(2) 4 (7)None 5 NaCI only) ml X 8) 0.04 16(0.9%(0.9% NaCI only)(0.005(0.005 ml x 8)M F25 250 01 0 00 0None

a Only vinyl carbamate induced incidences of Harderian gland tumors which were significantly above those of the controls: Group 6, p < 0.1 1; Group 7, p < 0.06; Group 8, p < 0.08; Group 9, p < 0.03; Group 10, p < 0.005; Group 12, p < 0.02; Group 14, p < 0.03. The incidences of Hardenan gland tumors in some of the vinyl carbamate groups were significantly greater than the incidences in the groups of mice treated with the same doses of ethyl carbamate: Group 9 versus Group 1, p < 0.03; Group 10 versus Group 2, p < 0.03; and Group 12 versus Group 4, p < 0.03. b The following significance levels were obtained compared to the male (Group 1 5) or female (Group 1 6) confrol group: Group 7. p < 0.01: Group 8, p < 0.001 ; Group 11, p < 0.075; Group 12, p < 0.005; Group 13, p < 0.001 ; and Group 14, p < 0.005. For the groups treated with eight 136-nmol/g doses, vinyl carbamate was significantly more active than ethyl carbamate in inducing thymomas: Group 13 versus Group 5, p < 0.0005; Group 14 versus Group 6, p < 0.005. The difference was less significant for those given 8 doses of 91 nmol/g: Group II versus Group 3, p < 0.075; Group 12 versus Group 4, p < 0.05. c Only the highest dose of ethyl carbamate (Groups 7 and 8 combIned), but all of the doses of vinyl carbamate (male and female groups given each dose combined), induced significant Incidences of lung adenomas compared to controls (Groups 15 and 16) (p < 0.001 In each case). At each dose, vinyl carbamate induced significantly higher incidences of lung adenomas than did ethyl carbamate (p < 0.001 in each case). cI One dermal fibroma. aNumbersinparentheses,totalnumberoftumors. f One lymphoblastic lymphoma. g One epidermal papilloma. h Mean Â± S.D.

I Three epidermal papillomas. I Two granulosa theca cell tumors of the ovary. k One epidermal papilloma. I One granulosa theca cell tumor of the ovary. m Two epidermal papillomas, 1 epldermal carcinoma, and 2 keratoacanthomas. n One mammary adenocarcinoma.

0 Two hemangloendotheliomas and 1 renal cell carcinoma. p One mammary adenocarcinoma. ence. In addition, a variety of other tumors occurred sporadi mice with hepatomas was greater for each treated group than cally in both the treated and control rats, and a high proportion for the control group and, at least for the lower doses, the of the rats treated with the canbamates developed cataracts hepatic tumor multiplicities increased with the dose of each late in life. carbamate. For each dose level, the average number of hepa The male and female C57BL/6 x C3H/He F1 mice that tomas per mouse was greater for the vinyl carbamate- than for received either ethyl carbamate or vinyl carbamate during the the ethyl canbamate-treated mice. These differences were more first 3.5 wk after birth also developed a variety of tumors (Table marked for the female mice than for the male mice. For all of 2); these tumors have previously been reported in mice treated the doses of vinyl carbamate studied and for the highest dose with ethyl canbamate (31). Thymic lymphomas, first noted at of ethyl carbamate, there was no significant difference between 3.5 months, developed at average ages of 5 to 7 months. The males and females in the incidences of tumor-bearing animals incidences were dose related and, at equivalent doses, were or in the multiplicities of hepatomas per animal. The male mice higher for vinyl carbamate- than for ethyl carbamate-treated treated with the 3 lower doses of ethyl canbamate developed mice. Since at the higher doses as many as 45% of the mice more hepatomasthan did the female mice given the same died with thymomas and since hepatic tumors were not ob doses. served earlier than 7 months, the data for the latter tumor were Since lung adenomas were found in mice that died as early tabulated in relation to the number of mice alive in each group as 5 months, the data for the incidences and multiplicities are at 7 months (Table 3). The hepatic tumors were, with few not strictly comparable (Table 2). Nevertheless, there was a exceptions, diagnosed as well-differentiated hepatocellulan tendency for higher lung adenoma multiplicities in mice that carcinomas [i.e., type A, type B, on mixed type A-type B received the higher doses of either ethyl or vinyl carbamate hepatomas (9, 13)]. The percentage of both male and female (even though more of the latter mice died early with thymomas),

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Table 3 Hepatic tumors in C57BL/6J x C3H/HeJF, mice treated with ethyl or vinyl carbamate See Table 2 for experImental details.No. ofNo. ofmiceDose ofAv. age ofGroupCarbamateg (nmol/micemice dead atwithAv. no. body wt x at mos. mouse@'1Ethyl46 no. of doses)Sexalive 7 mos.a@7 (mos.)hepato mashepatomas/ 0.92Ethyl46 x 8M2514.8 Â±0.8c140.8 Â± 0.33Ethyl91 x 8F2315.0 Â±0.620.1 Â± 1.44Ethyl91 x8M2515.3Â±1.8222.5Â± 0.95Ethyl1 x 8F2215.7 Â±0.360.4 Â± 1.96Ethyl136 36 x 8M251 4.7 Â±0.8222.5 Â± 1.67Ethyl5625x8M912.4Â±2.293.1Â±1.48Ethyl5625x 8F2313.9 Â±1.280.8 Â±

5.19Vinyl46 x 8F1012.0 Â±3.474.8 Â± 3.21 x 8M1914.0 Â±2.3153.6 Â± 3.911VInyl910Vinyl46 x 8F1 91 3.8 Â±1.21 65.9 Â± 9.61 x 8M1412.0 Â±3.0137.9 Â± 1.613Vinyl136x8M1810.3Â±2.5146.6Â±5.814VInyl1362Vinyl91 x 8F1 91 1.7 Â±3.61 72.5 Â±

6.01 x 8F1212.4 Â±2.0105.6 Â± 0.415(0.9% NaCI Only)M251 5.0 Â±0.660.2 Â± 6(0.9% NaCI Only)F241 5.4 Â±0.200 aThefirsthepatomaswerefoundinamousethatdiedatabout7months. b All treatment groups except Group 2 had significantly more hepatomas than did the corresponding control groups. Group 15 (control) versus: Group 1, p < 0.005: Groups 3, 5, 7, 9, 11, and 13, p < 0.001. Group 16 (control) versus: Group 2, p < 0.08: Group 4, p < 0.005; Groups 6, 8, 10, and 12, p < 0.001. Also, vinyl carbamate induced significantly more hepatomas than did the same dose of ethyl carbamate: Group 9 versus Group 1, p < 0.001 ; Group 10 versus Group 2, p < 0.001 ; Group 11 versus Group 3, p < 0.05; Group 1 2 versus Group 4, p < 0.001 ; Group 1 3 versus Group 5, p < 0.03; and Group 1 4 versus Group 6, p < 0.001. C Mean Â± S.D.

and at any dose vinyl canbamate induced more lung adenomas Table 4 than did ethyl carbamate. Both ethyl and vinyl carbamate Lung adenomas in female A/J mice treated with (ethyl-H5]- or [ethyl-05]ethyl treated mice developed tumors of the Hardenian gland; the carbamate or with tert-butyl carbamate In Experiment 1, the mice (30/group) were each given a single i.p. injection incidences were generally significantly greater in the vinyl of [ethyl-H5]- or [ethyl-05)ethyl carbamate or only 0.9% NaCI solution. The mice carbamate-treatÃ©dthan in the ethyl carbamate-treated mice. were killed 5 months later. In Experiment 2, the mice (18/group) were adminis Harderlan gland tumors were generally noted in mice at least tered 6 equal twice-weekly doses of a carbamate or only the vehicle, except that the mice in Group 6 were given 1 dose of 2.8 @moIoftert-butyl carbamate per g 1 year old, but one tumor of this type was diagnosed in a body weight and then, beginning 1 week later, 5 more twice-weekly i.p. doses of mouse that died at 5 months. 1.4 @@molperg. tert-Butyl carbamate and ethyl carbamate were given in dimethyl Lung Adenomas In A/J Mice. Owing to the greaten strength sulfoxide:H2O or 0.9% NaCI solution, respectively. The mice In Experiment 2 were killed 8.5 months after the first treatment. of Câ€”Dthan of Câ€”Hbonds, CD3CD2â€”Oâ€”-COâ€”NH2wouldbe No. ofmiceAt expected to be less carcinogenic than CH3CH2â€”Oâ€”-COâ€”NH2 if dehydrogenation of the ethyl group is a nate-limiting step in endWithDoseof the metabolic activation of ethyl carbamate. However, the ex lungAv. no. of ad Experienomas/mentCarbamatebody (@tmol/gpen adeno induction of lung adenomas by the deutenium-containing ethyl wt)mentmasmouse1[ethyl-H,[Ethyl carbamate was not significantly different from that by the Â±2.4a protium-containing ethyl canbamate (Table 4). [ethyl-D5JEthyl 3.0 26 26 4.7 Â±2,6â€• tert-Butyl carbamate [(1,1-dimethylethyl)carbamate] cannot [ethyl-H5]Ethyl 6.0 29 29 10.9 Â±6.8 [ethyl-D5]Ethyl 6.030 30 30 9.6 Â±4.4â€• be dehydrogenated to yield a substituted vinyl carbamate. 0.12tert-Butyl(0.9% NaCI Only)3.0 3030 85.3 0.3 Â± Therefore, it would not be expected to be carcinogenic if 1,2- unsaturation was an important feature of proximate metabolites Â±0.8'@ tert-Butyl 4.2 14 4 0.4 Â±O.8@ of the carcinogenic alkyl carbamates. In agreement with this Ethyl 4.28 18 17 6.2 Â±2,6â€• hypothesis, mice treated with total doses of 4.2 on9.8 @imoIof (Dimethyl sulf 17 7 0.6 Â±1.0 oxide-water tert-butyl carbamate per g of body weight did not develop lung only) â€˜adenomasInincidences or multiplicities which were signifi (0.9% NaCI only)9.8 163 90.5 0.8 Â±0.9 candy different than those of the vehicle controls (Table 4). a Mean Â± S.D. Ethyl carbamate (4.2 @smol/g)induced an average of 6.2 lung b Not significantly different from tumor multiplicity for mice treated with same adenomas/mouse. dose of [ethy!-H5]ethyl carbamate. C Not significantly different from tumor multiplicity for mice treated with Attempts to Modify Lung Adenoma Induction by Cyto dimethyl sulfoxide:water only. chrome P.450 MIxed-Function Oxidase Inhibitors or Cat d Significantly above (p < 0.001 ) tumor multiplicity for mice treated with 0.9% fame. Neither SKF 525A nor DPEA given immediately before NaCI solution. the carbamate had a significant effect on lung tumorigenesis tumor in A/J mice (Table 5). These results confirm and extend by ethyl carbamate (4000 nmol/g body weight) or vinyl car the observations of Kaye and Trainin (14) and Yamamoto et a!. bamate (150 nmol/g) on on the spontaneous incidence of this (33) on the lack of effect of SKF 525A on the induction of lung

APRIL 1980 1199

Table 5 Effects of SKF 525Aethylcarbamate, and DPEA on lung adenoma formation in female A/J mice treated with carbamateBeginning ethyl N-hydroxycarbamate, or vinyl of1 immediately before a single i.p. dose of a carbamate, the mice were given i.p. injections inGroups30 nmol of SKF 525A per g body wt, 40 nmol of DPEA per g, or only 0.9% NaCI (0.005 ml per g); mice were1 4, 8, 12, and 16 received 7 more i.p. doses of 40 nmol of DPEA per g at 2-hr intervals. There 7 to 20 mice/group. The mice were killed 7 monthslater.Inhibitor miceTotalDose No. of

of(nmol/g dose With Av. no. (nmol/g At 7 lung ad- lung adeno Groupmas/mousea1 Carbamate body wt) Name body wt) mos. enomas 3,7b2 Ethyl 4000 None 18 18 7.1 Â± 3.93 Ethyl 4000 SKF 525A 130 19 19 8.4 Â± 2.54 Ethyl 4000 DPEA 40 18 18 7.4 Â± 2.65 Ethyl 4000 DPEA 320 19 19 7.4 Â± 2.36 Ethyl N-hydroxy- 4000 None 19 17 4.0 Â± Â±7 Ethyl N-hydroxy- 4000 SKF 525A 130 19 17 3.2 Ethyl N-hydroxy- 4000 DPEA 40 18 18 4.3 Â±2.0 819 Ethyl N-hydroxy- 4000 DPEA 320 19 18 2.4 Â± 3.410 Vinyl 150 None 15 15 11.3 Â± 5.Oe11 Vinyl 150 SKF 525A 130 15 15 14.0 Â± 11.2Â±3.71Vinyl 150 DPEA 40 17 17 6.2132 Vinyl 150 DPEA 320 19 19 10.7 Â± 0.314 None None 19 2 0.1 Â± 0.715 None SKF 525A 130 18 3 0.3 Â± Â±0.316None DPEA 40 19 2 0.1 None DPEA 320 18 3 0.2 Â±0.6 aUnlessotherwiseindicated,pvaluesare>0.1oncomparisonwiththemicegiventhesamecarbamate without an inhibitor. b Mean Â± S.D. @ C < 0.09 compared to Group 5. @ d < o.oi compared to Group 5. @ e < o.@ compared to Group 9.

adenomas by ethyl canbamate. However, although Kaye and (7), attempts to demonstrate the metabolism of ethyl carbamate Trainin (14) reported an inhibition of the formation of these to vinyl canbamate in preweaning mice and to activate ethyl adenomas by SKF 525A (130 nmol/g body weight) in ethyl N- carbamate for mutagenic activity for S. typhimurium strain TA hydroxycarbamate-treated SWR mice, we observed only a 100 with NADPH- and NADH-fortified rat and mouse liven S-i 3 marginally significant ( p < 0.09) inhibition with the same dose. preparations were unsuccessful. As an approach to finding a A single i.p. dose of 40 nmol of DPEA per g did not significantly model system for the metabolism of ethyl to vinyl carbamate, affect adenoma formation in A/J mice by ethyl N-hydnoxycar we investigated the capacity of duck liven microsomes plus bamate in our experiments, but 8 i.p. doses of 40 nmol of cytosol to carry out this reaction. This approach was prompted DPEA per g at 2-hr intervals reduced the multiplicity of lung by the high capacity of NADPH-fortified duck liver preparations adenomas in A/J mice treated with 4000 nmol of ethyl N- to dehydrogenate aflatoxin B2 to aflatoxin B1 (27). Ethyl can hydroxycarbamate from 4.0 to 2.4 (p < 0.01). These multiple bamate can be considered to be an analog in part of afla doses of DPEA did not inhibit lung adenoma induction by ethyl toxin B2, since both compounds contain the structure or vinyl carbamate. â€”CH2CH2â€”Oâ€”. In agreement with the results of Nomura (25) and Theiss and Neither ethyl carbamate (30 to 125 @mol/pIate)nonvinyl Shimkin (30) with Swiss mice, all of the A/J mice that received carbamate (1 to 8 @moI/plate)showeddetectable mutagenicity caffeine in addition to an i.p. dose of ethyl carbamate had fewer for strain TA 100 in the presence of the fortified S-i 3 prepa lung adenomas than did the control mice which received only ration (2 mg protein) from duck liver (Chart 1). Since vinyl ethyl carbamate (Table 6). These reductions of 19 to 38% on carbamate at this level is metabolized to a mutagen for strain treatment with caffeine had p values of <0.05 for both doses TA 100 by an S-i 3 fraction from rat or mouse liver (7), in one of ethyl carbamate in Experiment 1 and for the higher dose in experiment the S-i 3 preparations from both Aroclor 1254- Experiment 2. In contrast, these doses of caffeine yielded a induced rat liver and 7-day-old duck liver were added together statistically significant inhibition of lung adenoma formation by to the assay tubes (2 mg protein each). Under these conditions, ethyl N-hydnoxycarbamate in only one of 4 groups; in one case, the mutagenicity of vinyl carbamate was similar to that ob the caffeine-treated mice showed a 50% increase in adenoma served in assays supplemented with only the rat liver S-i 3 multiplicity (p < 0.04). In his experiment, Nomura (25) also preparation; as in earlier experiments (7, 16), ethyl carbamate obtained no statistically significant inhibition of ethyl N-hydrox (12 to 150 @imoI/pIate)showedno detectable mutagenicity. In ycarbamate-induced lung adenomas under these conditions. control studies, aflatoxin B2(up to 1.5 nmol/plate) showed no Similarly, lung adenoma induction by vinyl carbamate showed detectable mutagenicity for S. typhimurium strains TA 98 or TA a statistically significant inhibition by caffeine in only one of 4 100 in the presence of Aroclon 1254-induced rat liven S-i 3 cases. fraction (2 mg protein per plate). However, it induced about 4,000 and 15,000 revertants/nmol, respectively, with each of Attempts to Metabolize Ethyl Carbamate to Vinyl Carba these 2 strains when the assay was supplemented with the matewithDuckLiverPreparations.Inourpreviousstudieshepatic S-i 3 (2 mg protein) from 7-day-old ducks. Addition of

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Table 6 Effects of caffeine on lung adenoma formation in A/J mice treated with ethyl carbamate, ethyl N- hydroxycarbamate, or vinyl carbamate pergFemale A/J mice (6 to 8 weeks old) in Experiment 1 were given 7 s.c. doses of 0.05 mg of caffeine ofabody weight in 0.9% NaCI solution (0.005 ml per g) at 6-hr intervals from 0 to 36 hr after an i.p. dose bodyweight;carbamate. Female A/J mice in Experiment 2 were given 2 i.p. doses of 0.04 mg of caffeine per g acarbamate.the first dose was given 3 hr before and the second dose was given 3 hr after an i.p. dose of The mice were killed at 6.5months.Carbamate No. of mice

WithDose lung(nmol/g) At lung Av. no. of adenomas/Group At 6.5 adeno- mouseExperiment Structure body wt) Caffeine start mos. mas 1 Â±21 Ethyl 1120 + 15 15 12 2.3 Ethyl 1120 â€” 15 15 14 3.7 Â±2.4 47C43 Ethyl 5620 + 15 14 14 13.9 Â± 4.35 Ethyl 5620 â€” 15 15 15 17.9 Â± .76 Ethyl N-hydroxy- 950 + 15 15 10 1.9 Â±1 Ethyl N-hydroxy- 950 â€” 15 15 12 1.5 Â±1.0 37d87 Ethyl N-hydroxy- 4760 Â± 15 15 14 5.3 Â± 3.89 Ethyl N-hydroxy- 4760 â€” 15 14 14 7.8 Â± 1.510 Vinyl 57 + 20 20 19 3.0 Â± Vinyl 57 â€” 10 10 9 3.7 Â±3.6 1149012 Vinyl 115 + 15 14 14 8.8 Â± 3.113 Vinyl 115 â€” 15 15 14 6.4 Â± Â±0.114 (NaClonly) + 15 15 6 0.6 (NaCI only) â€” 15 15 7 0.7 Â±0.1

Experiment 2 15 Ethyl 2000+1613133.5Â±1.12000 16 Ethyl 17 Ethyl 4000â€”6.5Â±2.5@4000+15 1915 1815 184.3Â±2.1 18 Ethyl Â±3.6 19 Ethyl N-hydroxy 2000â€”l.SÂ±0.9@2000+15 1814 1714 149.5 20 Ethyl N-hydroxy Â±1.1 21 Ethyl N-hydroxy 26h4000â€”1919183.24000â€” +16 1515 1510 151.1 4.9 Â± 22 Ethyl N-hydroxy 2.275+1818173.2 Â± 23 Vinyl 2.075 Â± 24 Vinyl Â±2.2. 25 Vinyl 4.0'150â€”150â€” +20 2019 2019 203.8 9.8 Â± 26 Vinyl Â±4.0 27 (NaCI only) + 13 12 2 0.2Â±0.4' â€”19 â€”@- (NaCI only) 1619 1619 712.1 0.7Â±1.0

@ a < o.o@ compared to Group 2. b Mean Â± S.D.

@ C < 0.02 compared to Group 4. @ d < 0.05 compared to Group 8. @ e < 0.09 compared to Group 12. f p < 0.01 compared to Group 18. @ g < o.@ compared to Group 20. @ h < 0.04 compared to Group 22. @ S < compared to Group 26. â€˜p< 0.06 compared to Group 28.

an equal level of rat liver S-i 3 preparation did not greatly Mutagenicity of Ethyl N-Hydroxycarbamate modify the mutagenicity of aflatoxin B2 in the duck liver-me diated system. Ethyl N-hydroxycarbamate had very weak mutagenic activity In another experimental approach, vinyl carbamate was (2 to 3 revertants/@imolwith up to 50 @smol/plate)forboth S. sought as a metabolite of [carbonyl-'4C]ethyl carbamate when typhimurium TA 100 and TA 98 (Chart 2). The mutagenicity the latter was incubated with the duck liver microsome or S-i 3 decreased about 40% when the incubation mixture contained system that was supplemented with an NADH- and NADPH a fortified liver S-i 3 preparation. Hydroxylamine, a possible generating system. After incubation periods of i 0 to 60 mm, minor contaminant or metabolite of ethyl N-hydroxycarbamate, more than 90% of the 14Cwas recovered in CHCI3extracts of was toxic to these bacterial strains at levels above 0.5 @@mol/ the incubation mixtures. Of the nonradioactive vinyl carbamate plate; no mutagenicity was detected (Ref. i 6 and our data). (added to some incubation mixtures to protect any [14C]vmnyl carbamate formed from further metabolism), 70 to 75% was DISCUSSION recovered after a i -hr incubation. No peak of 14Cwasobserved at the retention time of vinyl carbamate on HPLC ofthe extracts, Although the carcinogenic action of ethyl carbamate has whether or not nonradioactive vinyl carbamate was added to received considerable study (see references in Refs 7 and 20), the incubation mixture. Thus, these experiments provide no its proximate and ultimate carcinogenic metabolites have not evidence for the formation of vinyl carbamate under conditions been identified. Recently, vinyl canbamate was suggested as a that permit the dehydrogenation of aflatoxin B2to aflatoxin B1. possible proximate carcinogen (7) by analogy to the dehydro

APRIL 1980 1201

1800 /x AFB2 (D+R) I â€”DUCK(D) 1600 I â€”--RAT(R)

AFB2(D).1 x AFB2 1400 I, aVC I @EC 1200 w Iâ€” < 1000 -J I 800 0 20 40 ETHYLN-HYDROXYCARBAMATE @ 600 I ,â€˜ @.,OVC(D+R) ,umol/plote w I ,@ @â€”â€¢-â€œ..,.@-DVC(R) Chart 2. The mutagenicity of ethyl N-hydroxycarbamate in S. typhimurium @ 400 :@f â€¢@p'@',Dâ€”---@@ AFB2 CR) strains TA 100 and TA 98 In the presence and absence of a NADPH-fortified 5- I â€¢/.â€˜ â€œ EC(D+R) 13 preparation from Aroclor 1254-induced rat liver. @ 200 .1,1' EC(D) .I@f @,LE EC CR) carbamate in the carcinogenicity of ethyl carbamate. However, I, -â€˜ the possibility that vinyl carbamate is tightly bound at the @ V0. @â€”_@@[email protected]_ @,@@_.oVC,CD)I enzymatic site where it is formed and converted to an ultimate @@ :j@@ :-1;LT::@@4@@: @) carcinogen cannot be ruled out. The higher activity of vinyl â€”@ â€”@ â€”@ carbamate is also explicable if ethyl and vinyl carbamate yield 0.1 0.2 0.3 nmol AFB2 3 6 9pmolVC common or related proximate carcinogenic metabolites (7). 50 100 l5Ojjmol EC Ethyl N-hydroxycarbamate, an in vivo metabolite of ethyl Chart 1. The mutagenicities of vinyl carbamate (VC), ethyl carbamate (EC), carbamate (4, i 9), is another proximate on ultimate carcino and afiatoxin B2 (AFB2) in 5. typhimurium strain TA 100 in the presence of genic metabolite of ethyl carbamate. Although the N-hydroxy NADPH-fortified S-i 3 preparations from Aroclor 1254-induced rat liver and uninduced duck liver. derivative has only about 50% of the carcinogenicity of the parent compound (Refs. 2 and 17; this paper), the excretion, genation by rat liver preparations of aflatoxin B2 to the much distribution, and/or metabolism of the relatively large amounts more potent carcinogen aflatoxin B1 (27). The much higher of ethyl N-hydroxycarbamate given for carcinogenicity tests carcinogenic activity of vinyl carbamate as compared to that of may differ from those for the smaller amounts formed in vivo at ethyl carbamate for a number of tissues of rats and mice (Ref. any one time from ethyl carbamate. Likewise, metabolically 7 and this paper) is consistent with this suggestion, especially formed ethyl N-hydnoxycarbamate may be utilized more effi since all of the other carbamate analogs of ethyl carbamate ciently for carcinogenesis if it is formed close to the sites where that have been tested were less active than ethyl carbamate it exerts its carcinogenic activity. The mutagenic activity of the (20). Likewise, the considerable loss of 3Hin the hepatic DNA N-hydroxy derivative, although weak, and its direct reaction bound residues from [ethyl-i -â€˜4C;1,2-3H]ethylcarbamate (mix with cytosine and cysteine derivatives (4, 21, 23) are consistent tune of 14@and 3H-Iabeled compounds) is consistent with the with a possible role of the N-hydroxy derivative in the induction occurrence of a dehydrogenated intermediate (7),5 The low 3H: of tumors by ethyl canbamate. 14Cratio in the DNA-bound product appears not to have been Administration of mixed-function oxidase inhibitors or caf due to a preferential activation of [ethyl-i -14CJethylcarbamate feine have been used by other investigators (14, 25, 30) as as compared to that of the tnitiated compound; thus, ethyl tools for probing the carcinogenicity of ethyl canbamate and carbamate substituted completely with deutenium in the ethyl ethyl N-hydroxycarbamate. Kaye and Trainin (14) found that group had essentially the same activity for the induction of lung SKF 525A decreased the number of ethyl N-hydroxycarba adenomas as did ethyl canbamate containing only protium mate-induced lung adenomas in their mice. On the basis of this (Table 4). Lastly, vinyl carbamate, but not ethyl carbamate, is finding and the report of Minvish (19) that SKF 525A inhibited metabolized by NADPH- and NADH-fortified rat on mouse liver the in vivo reduction of the N-hydroxy derivative, ethyl carba microsome-cytosol preparations to a mutagen, presumably the mate was suggested as a proximate carcinogen of ethyl N- epoxide, for S. typhimurium strains TA 100 and TA 1535 (7). hydroxycarbamate. We obtained no significant inhibition by Our inability to detect vinyl carbamate as an in vivo metabolite SKF 525A of lung adenoma induction by ethyl N-hydroxycar of ethyl carbamate in mice (7) or under in vitro metabolic bamate in A/J mice. On the other hand, repeated doses of @.Unditionssuitablefor dehydnogenation of aflatoxin B2to afla another mixed-function oxidase inhibitor (DPEA) caused a sta tistically significant reduction in the number of lung adenomas toxin B1(this paper) does not support the intermediacy of vinyl induced by the N-hydroxy derivative; the effect of DPEA on the in vivo reduction of ethyl N-hydroxycarbamate is not known. 5 Subsequent studies have shown that ethyl carbamate recovered from tissues of adult female A/J mice killed 12 hr after i.p. injection of [ethyl-1-'4C:1 .2- Neither SKF 525A non DPEA affected induction of these ade 3H)ethylcarbamate had the same 3H:'4Cratio as did the injected ethyl carbamate. nomas by vinyl carbamate, even though both of these inhibitors Thus, the lower 3H:'4Cratio of the ethyl carbamate residues bound to the hepatic macromolecules in vivo as compared to that of the injected carcinogen was not decreased the hepatic microsome-cytosol-mediated mutagen due to exchange of 3Hfrom the ethyl carbamate in vivo. icity of vinyl carbamate for S. typhimurium TA i 535 (7). Thus,

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1980 American Association for Cancer Research. Carcinogenicity and Mutagenicity of Carbamates these data do not provide support for either vinyl carbamate or 11. Hollander, M., and Wolfe, D. Nonparametric Statistical Methods, 503 pp. New York: John Wiley and Sons, Inc., 1973. ethyl N-hydroxycarbamate as a proximate carcinogen of ethyl 12. International Agency for Research on Cancer. Urethane. In: IARC mono carbamate or for vinyl carbamate epoxide as a carcinogenic graphs on the evaluation of carcinogenic risk of chemicals to man. Vol. 7, derivative of vinyl carbamate. pp. 111â€”140.Lyon, France: International Agency for Research on Cancer, 1974. As reported by Nomuna (25) and Theiss and Shimkin (30), 13. Jones, G., and Butler, W. H. Morphology of spontaneous and Induced administration of caffeine at about the same time as ethyl neoplasia. In: W. H. Butler and P. M. Newberne (eds.), Mouse Hepatic carbamate inhibited lung adenoma induction. Under the same Neoplasia, pp. 21â€”60.NewYork: Elsevier, 1975. 14. Kaye, A. M., and Trainin, N. Urethan carcinogenesis and nucleic acid conditions, there was no consistent inhibition of lung adenoma metabolism: factors influencing lung adenoma induction. Cancer Res., 26: induction by either vinyl carbamate or ethyl N-hydroxycarba 2206â€”2212,1966. 15. Matheson Gas Data Book. Phosgene, pp. 361â€”366.EastRutherford, N. J.: mate(Ref.25; this paper).Nomura(25)hassuggestedthatthe The Matheson Co., Inc., 1961. inhibition by caffeine of tumor induction by ethyl carbamate 16. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N. Detection of carcino may be related to its inhibitionof error-pronepostreplication gens as mutagens in the Salmonella/microsome test: assay of 300 chemi cals. Proc. NatI. Acad. Sd. U. S. A., 72: 5135â€”5139,1975. DNA repair and that ethyl carbamate and its N-hydnoxy metab 17. Miller, J. A., Cramer, J. W., and Miller, E. C. The N- and ring-hydroxylation olite are Independently carcinogenic. However, the metabolic of 2-acetylaminofluorene during carcinogenesis in the rat. Cancer Res., 20: interconvertibility of these 2 carcinogens makes the latter de 950-962, 1960. 18. Mlrvish, S. S. The conversion of N-hydroxyurethan to urethan in the mouse. duction open to question. Blochim. Biophys. Acta, 93: 673â€”674,1964. 19. Mlrvish, S. S. The metabolism of N-hydroxyurethane in relation to its carci nogenic action: conversion into urethane and an N-hydroxyurethane glucu ACKNOWLEDGMENTS ronide. Biochim. Biophys. Acta, 117: 1â€”12,1966. 20. Mirvish, S. S. The carcinogenic action and metabolism of urethan and N- We are Indebted to Norman Drlnkwater for the tests on statistical significance; hydroxyurethan. Adv. Cancer Res., 11: 1â€”42,1968. to Randall Knlbbs, James MacDonald, and Colleen McDermott for assistance 21. Mullinix, K. P., Rosenkranz, S., Carr, H. S., and Rosenkranz, H. 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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1980 American Association for Cancer Research. Comparative Carcinogenicities and Mutagenicities of Vinyl Carbamate, Ethyl Carbamate, and Ethyl N-Hydroxycarbamate

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

Cancer Res 1980;40:1194-1203.

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