Document Zn6N0nndDxpB5z8NDBdgbO0R8

CMpirH j value* a elnlmsii noticed, and 111 was N. This any of the of mutant ir dosas of ipports tha first stap of general enielty of ><as shown ieal series, nation that ;** expert.ituition > DEN. a lass y Index for amparativ- >1). ). M>., 16, ( r.vL i 1 Iff5, PdMd la Gm 1 MUTAGENICITY IN VITRO AND POTENTIAL CARCINOGENICITY OF CHLORINATED ETHYLENES AS A FUNCTION OF METABOLIC ' OXIRANE FORMATION H.Grain, G.Bonse, Z.Radwan, D.Reichert, D.Hanschler Departments of Toxicology of Gesellschaft fiir Strahlenund umweltforschung, 8042 Mtinehen-Neuherberg, and the University 8700 WUrzburg, Versbacher Landstr.9, West-Cermany IRtetntd 10 July 1971; tcapttd IfAutuu 197S1 #"V"C WU- All chlorinated ethylanes undergo biotransformation in mammalian organisms, the main pathway being an oxidation to oxiranes, as the first step. The stability of these oxiranes varies widely and depends on the number of chlorine substitu te rations and on the relative position of the substituents in the molecule. Symmetr- rg^f^rically substituted oxiranes from the tetrachloro- and the isomeric 1,2-diohloro- j&d ethylene a seem to be rather stable 1) ,2) In the case of vinyl chloride and tri- ' ehloroethylene the polarity of the unsynunetrlcal oxiranes exerts lesser stability land induces intramolecular rearrangement ' '. Attempts to prepare the oxirene iith the highest polarity from 1.1-dichloroethy`lene (1.1-DCE) remained unsuc- cessful till now. In the reaction of 1.1-DCE with m-chloroperoxybenzoic acid,the r '-corresponding oxirane could not be detected; Instead, the chloroacetyl chloride t e\ lws isolated as the product of oxirane-rearrangement However, no direct relationship exists between chemical reactivity and 4,biological effacts because the thermal rearrangement of the oxiranes leads to * Fdifferent chemical species: either acyl chlorides or aldehydes which are inspect of causing quite different biological effects. Besides rearrangement, ****>* may raact directly with biologic nucleophiles. Thus, the mutagenic 6). 2013 SL 066674 fzmm 2014 It1,.: ' if! W\ f. !f:' fry :i > HH [!' l activity of vinyl chloride (VCM) has been claimed to be exerted by a direct alkylating action of the oxirane6',7'. t VCM has been demonstrated to be carcinogenic in animals 8) ' 9) and in man (for references, see101).Quite recently, a carcinogenic activity of trichloro ethylene (TKI) has been reported in mice after long-term administration of rather high daily oral doses (O.S or 1.0 g/kg). This report11' in connection with the above considerations prompted us to determine the mutagenicity of the whole series of chlorinated ethylenes with a matabolic activating microsomal enzyme system in vitro. Materials and Methods. Tetrachloroethylene, trichloroethylene, cis- and trans-l,2-diehloroethylene, 1,1-dichloroethylane were obtained from Merck a Co., Darmstadt, as a.g. reagents; vinylchloride as a purified gas (> 99,9%) from BASF, Ludwigshafen. Mutagenic activity of the derivatives formed during micro- 12) aomal activation was tested in a metabolizing In vitro system with E coll K 12. One can use this bioauxotrophic strain in four mutation systems to test .mitagenic agents: In the three back mutation systems gal'*', arg+, and nad*, and In the mTR system, where forward mutation leads to resistance to 5-methyl-DLtryptophane13^. For the experiments 6 to 9 x 10 cells of an overnight culture were suspended in 1.5 ml Incubate containing 5 mg microsomal protein, isolated from mousa livers, and the NADPH generating system 5 mM MgClj, 16 mM DL-lsocltrate-Naj, 0.66 mM HADF-Na^, 20 yl isocitrate-dehydrogenase (20 milliunits/uli in 0.1 M phosphate buffer pH 7.4, as well as different concentrations of the test compounds. These concentrations were selected from preliminary experiments so that they did not reduce cell survival by more than 20 per cent (Table 1). After 2 hours of incubation in a shaking water bath at 37, the reaction was terminated in ice. The incubete was diluted In saline and plated on appropriate selective media ae described previously1*'. Survival of the E coli X 12 strain wes determined by plating on the complete medium. Mutagenicity is expressed as colony-forming units (efu) that wera counted on the appropriate selective media per efu counted on the complete medium. Liver mierosomes were isolated from male mice pretreated for 10 days with 0.1 per cent phenobarbital in the drinking 12) water to increase microsomal enzyme activity (: SL 066675 2015 direct 1 in man trichl roion of section ity of the :rosomal :is- and lerck a Co., .) from .ng micr i E coll ; to test nad+, and ;thyl-DLit culture isolated DL-is liunits/ul) . of the xperiments able 1). .ion was ppropriate 12 strain pressed as tive media d from male inking Tab.1i Mutagenicity of chlorinated ethylenes after incubation in a metabolic activating microsomal system concentre- tlon in_the medium** at 37 C %survival of bacteria % of spontaneous mutation rate in different operons of coli K, 12 gal+ arg' MTR nad' C12C CC12 Tetrachloroethylene 0.9 99-1 100 lOO 100 100 .CljC * CHC1 Trlchloroethylene 3.3 76-4 123-23 232-36 114-18 100 C12C - CH2 1.1-Dichloroethylene Cl .Cl V-c' H^ cis-1.2-0ichloroethylene :ir Cl H 2.5 2.9 74-7 88-5 120-14 229-26 100 100 100 100 100 100 'Nu trans-1.2-Dlchloro- ethylene .C1CH - CH2 fViny 1 chloride 2.3 10. S 90-3 72-3 100 100 100 100 t 231-20 663-141172^35 148-24 v? Ec- determined by GC analyses after injection of S pi of the liquid compounds; except vinyl chloride where the gas was introduced by bubbling through the liquid at 15 C. , ' Results and Discussion. The results of the experiments are listed in Table 5?T1- Cytotoxicity of the chlorinated ethylenes varies widely. To obtain 80-100 per cent- survival of the tester strain, only 1 mM of trichloroethylene could be used but 10 mM of vinyl chloride. No mutagenic activity of the chlorinated ethylenes was detected in the test ^system without microsomal enzyme activity. When the complete incubate with btabollcally active microsomes was used, conversion of VCM, 1.1-OCE ss well as W'. ^.?"TRI induced mutations, the latter compounds being less mutagenic than VCM. The f'V'highest mutation rates were detected in the arginine genes, whereas reversibill- SL 066676 ty in the gal* and ned* systems and tha forward mutation to MTB raalatanca wcra lass sensitive to tha mutagenic affects of the metabolites. Tetrachloroethylene and tha cis- and trana-iacners of dichloroethylane were not metabolized to mutagens at all. Direct comparison of the mutagenic activity of the chlorinated ethylenes is not poesible since different substrate concentrations had to be used to minimize cell death of tha tester strain. However it is evident that mutage nicity of VCM is several times higher than that of 1.1-DCE and TRI. Our results are indicative of a conspicuous correlation between the stability of ~>xiranesf as outlined earlier, and the mutagenicity of all six chlorinated e~ nes: those forming very unstable oxiranes (VCM, 1.1-DCE,TRI) induce mutations in the test system, whereas the others (Per, cis- and trans- 1.2-OCE) forming much mor stable oxiranes, do not. The mutagenicity of trichloroethylene, though only slight in extent in the gal* system, which is known as very sensitive, has not been anticipated. Tri chloroethylene is metabolized in vitro and in vivo to tha scarcely reactive chloral hydrate and furtheron to trlchloroethanol and trichloracetic ecid1^'*6^ Tha latter compounds are not known to induce cytotoxic or genetic effects. Con version of TBX-oxlrane to chloral in vivo is a quite unexpected reaction because thermal rearrangement In vitro entirely forms dichloroacetyl chloride17^. This different behaviour should be further investigated. The results of such ex periments may contribute to tha batter understanding of tha mutagenic and potentially carcinogenic properties of trlchloroethylsne. Acknowledgement! The skilful technical assistance of Mrs. Hesse and 5teinhllber ia gratefully acknowledged. slstance wars hloroethylene ilized to :d ethylene* > used to :at rautage- sen the Lty of all six 1.1-DCE,TRI) - and trans extent in the ipated. Triy reactive tic acid15),16) effects. Coneaction because iide17)'. This f such ex;enic and . --------- V ''YYtl il rnltaiiiii) runum ' ifhwn * * 2017 REFERENCES 7.M.Frank*1, C.E.Johnson, H.M.Pitt, J.org.Chem. 22, 1119 (1957) K.Griesbaum, R.Kibar, B.Pfeffer, Liebigs Ann.Chem. 1975, 214 H.Gross, J.Freiberg, Journ.f.Prakt.Chem. 311. 506 (1969) G.Bonse, Th.Urban, D.Reichert, P.Henschler, Biochem.Pharmacol.(1975) in press G. Bonse, D.Henschler, unpublished results U.Rannug, A.Johansson, C.Ramel, C.A.Wachtmeister, Ambio 3, 194 (1974) H. 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