Document zzkjdBeppXkBBj5MJvQkN684z

(Reprinted from Nature, Vol, 255, No. 5510, pp. 641-643, June 19, 19751 Tissue-mediated mutagenicity of vinylidene chloride and 2-chlorobutadiene in Salmonella typhimurium Chlorinated hydrocarbons, such as vinyl chloride or vinylidene chloride (1,1-dichloroethylene), are produced in large quantities and are present in the environment'. Recently, vinyl chloride monomer has been shown to be carcinogenic in animals and man2-4 and mutagenic in microbial systems5 Vinylidene chloride (VDC). a structurally related substance and co-polymer of vinyl chloride, is used in the manufacture of plastics"; it could also occur as a decomposition product of 1,1,1-trichloroethane1. Another chemically related compound, 2-chlorobutadiene (chloroprenc), has been used in the manu facture of synthetic rubber since 1930. We have examined the mutagenicity of VDC and 2-chloro butadiene in Salmonella typhimurium strains, using a tissuemediated assay which has been found effective in detecting the mutagenicity of various carcinogens, such as nitrosamines8, vinyl chloride1 and many others1011. Tests for mutagenicity were carried out according to the procedure developed by Ames er al.n and modified by Bartsch el al.5 using the histidine-auxotroph strains of 5 typhimurium TAI530 and TAI00, provided by Professor B N. Ames. In this procedure, Petri dishes containing 9,000g tissue supernatant, an NADPH-generating system and the bacteria in a soft agar layer were exposed to various concentrations of VDC or 2-chlorobutadiene vapour in a desiccator at 37 C in the dark. The compounds were then replaced by air, and, after further incubation for up to 48 h at 37 C, the number of histidinerevertant colonies per plate was counted. The concentration of VDC or 2-chlorobutadiene in the incubation medium was determined by gas-liquid chromatography5. In the experimental conditions used, the concentrations of VDC in the aqueous phase after 2 h of exposure to 0.2, 2 or 20% VDC in air (v/v) were 3.3 x 10-* M, 3.3 x 10-3 M or 3.3 x 10M, respectively. The concentrations of 2-chlorobutadiene in the aqueous phase after 2 h of exposure to 0.5, 2 or 8% vapour in air (v/v) were 7xlO~`M, 3.7xlO~JM or 1.65x10"* M, respectively. No further increases were observed after up to 7 h of exposuie. Fig. 1 shows the mutagenic effect on 5. typhimurium TA1530 and TA100 of exposure to 0.2, 2 or 20% VDC in air in the presence of 9,000^ liver supernatant of phenobarbitonepretreated mice (0.1% in the drinking water for 7d). When the cofactors (NADP+ and glucose-6-phosphate), required for activity of microsomal mixed-function oxidase, were omitted from the incubation mixture, no mutagenic elfect was observed. The mutagenic response, which was greater in the TAI00 strain, increased in both strains after exposure to up to 2% VDC in air. The lower mutagenic response observed with a concentration of 20% VDC may result from an inhibitory action of VDC and (or) its metabolite(s) on the microsomal enzymes responsible for the metabolic activation of VDC. 4-Methoxyphenol, which is used as a stabiliser of VDC. when applied at up to 500 pg per plate, was not mutagenic for either strain, whether in the presence or absence of microsomal liver fraction from phenobarbitone-pretreated mice. Exposure of TA100 strain to 2% VDC in air in the presence of a hepatic microsomal fraction from untreated or pheno barbitone-pretreated mice, caused a linear increase in mutagenic response up to 4 h. Thus, exposure to 2 or 20% VDC in air for 4 h was used to assay rat and mouse liver, kidney and lung fractions (9,000g- supernatant) for their ability to convert VDC into mutagenic metabolites (Table 1). Mouse liver, kidney and lung fractions efficiently converted r600 % Vinylidene chloride/air (v/v) Fig. 1 VDC concentration-dependent induction of reverse mutations in 5. lyphumiriuin strains TAI530 and TAI00. I'ctn dishes containing 9,000g liver supernatant from phenobarbi tone-pretreated male OF-1 mice, NADP* (2 pmol per plate), gluco$e-6*phosphate (2.5 pmol per plate) and the bacteria in a histidine-deficient medium were exposed to 0, 0.2, 2 Or 20% VDC in air (by volume) for 4 h in a desiccator at 37 C in the dark. VDC was removed under vacuum and replaced by air, and the incubation was continued up to 48 h at 37 C. Bacterial survival was estimated by seeding TAI530 (10"; dilution) on a histidine-enriched medium. Control assays were carried out by omitting the cofactors (NADP*. glucosc-6-phosphate). Mean values s.e from 1 -4 scries of experiments, each on a pool of five mouse livers, are plotted. Bacteria were plated in triplicate. VDC, containing 0 3% 4-mcthoxyphenol as antioxidant, was obtained from Mcrck-Schuchardt (Darmstadt, Federal Re public of Germany). Black' control, stippled, TA 1530, cross- hatched, TA100. VDC into mutagenic metabolites in vitro, activity being greatest in the liver. Phenobarbitone pretreatment of the mice produced an increase in the mutagenic response in assays with all three organ fractions. A much lower mutagenic response was observed in rats than in mice with liver and kidney fractions, and there was only minimal activity with a lung fraction. Rats have been reported to show hepatic and renal damage following inhalation of VDC (ref. 12). When the concentration of VDC in air was raised from 2 to 20%, an increased mutagenic effect was noted only with mouse kidney and lung fractions As has been shown previously for vinyl chloride, these results demonstrate that the mutagenic effect of VDC is mediated by microsomal enzymes from various organs, in the presence of an NADPH-generating system and oxygen; these enzymes probably form the alkylating intermediate(s), since 5. typhimurium TAI530 strain is specifically reverted by monofunctional alkylating agents11. Evidence for the parti cipation of a microsomal mixed-function oxidase is further supported by the observation of changes in mutagenicity in SL 095937 Experiment no. i 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Table 1 Mouse and rat tissue mediated mutagenicity of VDC in S. typhimunutn TA100* Species Phenobarbitone pretreatment Tissue (9,000# supernatantt) Cofactorsj 2% VDC in air flt\S + Relative revel uints activity i' per plate OF-I mouseJ BDVI rat ? Yes Ves No No Yes Yes No No Yes Yes No No No No No No No No Liver Kidney Lung Liver Kidney Lung 500 - 23 150 23 + 10 5 - 330 + 49 100 -- 163- 4 5 -L 147-15 45 31+ 7 9 * 67 * 2 20 -- 20 + 3 6 -i- 34 4 10 - 5 4 1 * 21 + 5 6 6r 9 2 -i- 95-L 7 30 --0 0 -- 18+ 4 5 - I8 2 5 -r 9-fc 2 3 - 9+ 7 3 20% VDC in air /'* Relative revertants activity i| per platelj 330 -.29 7+ 5 435+46 1+ 3 173+ 5 17+ 2 125+ 5 16 - 1 48* 5 10* 1 37* 3 I4 8 77 -t 5 2 2 16+ 2 21+4 11 -t 2 12 1-6 75 2 100 0 40 4 29 4 11 2 8 3 18 0 4 5 3 3 *Assays carried Out as described in Fig. I. + Equivalent to 38 mg wet tissue per plate tNADP+ (2.0 nmol per plate) and glucose-6-phosphate (2.5 pmo! per plate) !)Mean values - s.e. from 1-4 experiments, each using pooled tissues from 4 mice or 3 rats. The number of spontaneous mutations per plate (49 2) has been subtracted from each value Relative mutagenic activity was expressed by taking the value obtained in experiment 3 as 100, vitro and (or) the toxicity of VDC following pretreatment of rats or mice with various drugs which are known to modify the activity of drug-metabolising enzymes. As shown in Table I, phenobarbitone pretreatment of mice caused an increased mutagenic response. In another series of experiments, we examined the effects of pretreating female BD-VI rats with ihenobarbitone (0.1 % in the drinking water for 7d), pregNfcrnenolone-lba-carbonitrile (PCN; 50 mg kg-1; 5 times orally at 12 h intervals) or aminoacetonitrile (AAN: 500 mg kg-1, one subcutaneous injection 24 h before the assay) on the mutagenic effect of exposure to 2 % VDC in air on S. typhtmurium TA1530 strain with liver fractions in assays as described in Fig. I. Phenobarbitone caused a twofold increase in the mutagenic response, whereas PCN or AAN resulted in reductions of 40 or 60%, respectively, when compared with the corresponding values obtained from untreated rats. Carlson and Fuller13 reported an increased mortality by VDC in rats following phenobarbitone pretreatment. The addition of sulphur-containing compounds to in vitro mutagenicity assays containing mouse-liver fractions reduced the mutagenic effect of exposure to 2% VDC in air on the 5. typhtmurium TAI530 strain. Addition of N-acetyl-cysteine and N-acetyl-methionine (12 pmol of each per ml soft agar layei) caused an 80% reduction of mutagenic response com pared with the appropriate control. These results indicate that the mutagenic VDC metabolites) is trapped by nucleophilic sulphur groups, thus competing for binding to bacterial DNA. This observation parallels the findings of Jaeger et at." who observed that the toxicity of VDC in rats is correlated with hepatic glutathione concentration, and that cysteine has a protective effect. Chloroethylene oxide, the suggested primary metabolite of vinyl chloride, has been shown to be an alklylating and muta genic agent". Since epoxides are now recognised as obligatory intermediates in the metabolism of olefinic compounds by hepatic microsomal mixed function oxidase15, it can be icistulated that 1,1-dichloroethylene oxide may be a primary eactive metabolite of VDC. It is also possible that partial dechlorination1" of VDC by microsomal enzymes results in vinyl chloride and its metabolic products. Exposure of S- typhimurittm TA 100 strain to 0.5 % up to 8 % of 2-chlorobutadiene in air in the absence of any metabolic activation system caused a linear increasing mutagenic response, as a function of the 2-chlorobutadiene concentration, which, at a concentration of 8%, reached three times the spontaneous mutation rate (Table 2). Exposure to higher concentration (20%) caused a strong toxicity in the bacteria. This mutagenic and (or) toxic effect could be caused by a direct action of 2-chlorobutadiene or, more likely, by one of its enzymic (bactena) or non-enzymic breakdown products. A three or twofold increased mutagenic response, respectively, was observed when a fortified 9,000# liver supernatant from either phenobarbitone or untreated mice was added to such assays (Table 2). These results support an enzymic formation of a mutagenic metabolite(s) from 2-chlorobutadiene, probably an epoxide, as it was strongly implicated for vinyl chloride5. Most chemical carcinogens have now been found to be mutagens, when assayed in one of the mutagenicity tests that combine microbial or mammalian cell systems as genetic targets with an in vitro or in vivo metabolic activation system1'. The increasing evidence of a possible correlation between mutageni city and carcinogenicity does not mean that one biological effect may be equated with another. Thus, mutagenicity in micro organisms cannot be automatically assumed to imply a possible carcinogenic effect in man. Mutagenicity tests are at present an excellent method for screening environmental chemicals to be submitted to a more careful set of bioassays. While there are no criteria at present for extrapolation from experimental data to man. rhe available evidence indicates that precautions are justifiable on the basis of experimental evidence of carcinogeni city. There is, in fact good indication that experimental evidence of carcinogenicity can in cerlain cases predict a similar effect in man: as was the case ofdiethylstilboestrol, Ws-chloromethylether and vinyl chloride1*. Recenlly, VDC has been found lo be carcinogenic in the rat, (Viola, Bigotti and Caputo, personal communication) and 2chlorobutadiene has been suggested as being responsible for an increased incidence of skin and Jung cancer among exposed workers1*. A high incidence of chromosome aberration in lymphocytes from the peripheral blood of workers exposed to 2-chlorobutadiene has been reported50. These findings, com bined with the present results, indicate that an epidemiological survey of workers exposed to these substances may be necessary, as well as measures to prevent, or greatly reduce, exposure of humans. We thank Mrs C. Gabet and Mr A. Barbin for technical SL 095938 Experiment no. 1 2 3 4 5 6 7 Table 2 Mutagenicity of 2-chlorobutadiene in 5- lypht/nurium TA100* 2-chlorobutadienet in air (%) Phenobarbitone pretreatment his* revertants/plate 9,000g liver supernantant +cofactorsi 0 Yes or no 35 + 3 0.5 Yes No 175*15 101 * 4 2 Yes No 232 - 17 154 15 8 Yes No 331 *30 306-25 KCI 352 49 3 70 4 117 + 6 Bacteria were exposed to 0.5, 2 and 8,, 2-chlorobutadiene in air (v/v) for 4 h at 37 C in presence of a NADPH generating system and a 9,000g liver supernatant from either phenobarbitone-treated or untreated male OF-I mice as described in Fig. I. f2'Chlorobutadiene (purity 98.94%) was provided by Distugil, Le Pont de C'laix. France, contaminated with 0.98% 1-chlorobutadiene, 370 p.p.m. butadiene and 280 p.p m. vinylacetylene Tertiary butylcatechole which was present in 2-chlorobutadiene as an antioxidant (200 p.p.m.) was not mutagenic up to 500 pg per plate for the TA100 strain either in the presence or absence of a microsomal liver fraction of phenobarbuone pretreated mice. tWhen the cofactors (NADP+ and glucose-6-phosphate) were omitted, the number of his*' revertants per plate was identical with assays containing KCI only. Mean values +s.e. from 2 series of experiments, each on pooled livers from 4 mice, 9,000g- supernatant + cofactors was replaced by a 0.9% KCI solution. assistance. This research was partially supported by a contract with the US National Cancer Institute. Helmut Bartsch Christian Malaveille Ruggero Montesano Lorenzo Tomatis international Agency for Research on Cancer, Unit of Chemical Carcinogenesis, 50, Cours Albert Thomas, 69008 Lyon, France Received April 11; accepted May 7, 1975. 1 McConnell, G , Ferguson, O, M,, and Pearson, C R., Endeavour, 34, 13 (1975). 2 Viola, P L , BigotO. A,, and Caputo. A,. Cancer Res , 31, 516 (J97j). 3 Maltom. C . and Lefemine, G., Environ, Ret, 7, 387, (1974), 4 Creech, J L , and Johnson, M N ./ orcup Med., 16, 150 (1974) 5 Bartsch. H , Malaveille, C , and Montesano, R , Ini. J. Cancer, 15, 429 (1975), * Malaveille. C . ei a/, Btochem htophys. Res. Cowman,, 63, 363 (1975). 1 Rannug, U , Johansson, A., Ramel, C., and Wachtmoster. C, A., AMBIO, 3, 194(1974) ! Levinson C. in Vinyl chloride a iase study of the new occupational health hazard (International Chemical Federation, Geneva, 1974) 9 Bartsch, H.. Malaveille, C , and Montesano, R., Cancer Res., 35, 644(1975). '0 McCann, J.. Spingarn, N E , Kobon, J,, and Ames, B. N., Proc, natn. Acad. Sei. U $ A.. 72, 979 (1975). 11 Ames, B, N., Durston, W, E., Yamasaki, E., and Lee, F. D., Proc, natn. Acad. Sci. USA . 8, 2281 (1973). 12 Prendergast. J A,. Jones R A . Jenkins. L, J., and Siegal, J., Toxicol appl. Pharmacol.. 10, 270 (1967), 1 ' Carlson. G P , and Fuller. G. C.. Res, Comun. ehem. Path. Pharm,, 4, 553 (1972), 14 Jaeger, R J . Conolly, R B , and Murphv. S, D,, Erp mnler Path., 20. 187 (19741 1 ' Dalv, J W , Jenna, D, M., and W ilk tip. R . Experirntia. 28, t 129 (1972). 1 * Gandolfi, A. J , ami Van Dyke, R A , littu hem. htnphvi Ret Comun., 53.3(1977) ,T Mliter. J, A., and Miller. E, C , J. natn Caneer Inst . 47, 5 (1971). International Agency for Research on Cancer. Monographs On the Evaluation of Carcinogenic Risk of Chemicals to Man. 4, 6 and 7 (WHO, Lyons, 19743 19 Khachatryan, E. A,, Gig Tr Prof Zohol., 18, 54 (1972): Problems in Oncology, 18, 85 (1972) 20 Katosova, L. D , Gigiena truda t professional 'nye zabolevamja, 10, 30 (1973). 7 SL 095939