Document 7MXOvEeXE8YxynX6kaOR4rqDB

R&S 107498 in 310. JUDICAL SZSEARCH poa, ttikx D2SCR1PTI0H FORM V 63______________________ ** Duplicate in ell cards:--)> j /?SQ 68 69 1 76 year as-1961 "lie nunber [Right justify [ftuneric only] Author(s), as Last Kane 7S (Ho ruactuatioa) and codes for jour al as JAH1 preeeeaed-by. one blank space 1 | Gn'(^, a/. L '20 21 1 H2o/!j C~7- ____1 UMtL'ii'.ftJL*#___ >. 1 40 41 1 /?ADlC/i/V 1 1 77' 78 nzn Sub-Index Code 60 61 62 Li 12 13 Title of Retort; end with snace-hyphen-hyphecyspace. Tollov vizh Index Tems. separated fron each other with -coma-space. Avoid other, punctuation* do 'not abbreviate. * . 61 '62 ? |c.i^r In ]/l4^j ^'W "f /a- 1 f^ar'Cin^ 1 T~l i-> Cb^o^irio^-fC_+'nv Un^ + 22 77 /'lyfaJco/iV- O X./ 'Lfri'(_: i an -- -- /f`: 24 ource (Journal, Vol., Ku-ber, rages, Tate) nn I ~W-' r r jCq 1 0 y y yS3L 2-0 / ^ -- io <7 61 62.- or --< p di v--* 2 10 " -- ** - * 61 2 61 62 63 64 Bhcnd VoL 24. pp, 2013 5P17. IV,,. ,*75. ,, Cl,,, t\ 0000132 :1 V \ MUTAGENICITY IN VITRO AND POTENTIAL CARCINOGENICITY c cMWj ,, OF CHLORINATED ETHYLENES AS A FUNCTION OF METABOLIC [}> i. OXIRANK FORMATION fO H.Greim, G.Bonse, Z.Radwan, D.Reichert, D.Henschler Departments of Toxicology of Gesellschaft fur Strahlenund Umweltforschung, 80-32 Miinchen-Neuherber g, and the University 8700 Wurzburg, Versbacher Landstr.9, West-Germany L . 5j4^Jl lReceived 10 July 1975; accepted 19 A ugust 19 75) R&S 107499 Ali chlorinated ethylenes und-. rgo biotransformation in mamma 1 ian 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 tions and on the relative position of the substituents in the molecule. Symmetr ically substituted oxiranes from the tetrechloro- and the isomeric 1,2-dichloro- *1 \ > ethylenes seem to be rather stable . In the case of vinyl chloride and tri- chloroethylene the polarity of the unsymmetrical oxiranes exerts lesser stability and induces intramolecular rearrangement 3)'4) . Attempts to prepare the oxirane with the highest polarity from 1. 1-dichloroethylene M.l-DCE) remained unsuc cessful till now. In the reaction of 1.1-DCE with m-chloroperoxybenzoic acid,the corresponding oxirane could not be detected; instead, the chloroacetyi chloride was isolated as the product of oxirane-rearrangement 5) . However, no direct relationship exists between chemical reactivity and biological effects because the thermal rearrangement of the oxiranes leads to different chemical species; either acyl chlorides or aldehydes4' which are suspect of causing elite different biological effects. Besides rearrangement, oxiranes may react dnectly with biologic nuclcophi1es. Thus, the mutagenic 2013 R&S 107500 2014 Preliminary Communication* Activity of vinyl chloride (VCM) has been claimed to be exerted by a direct alkylating action of the oxirone6',7'. VCM has been demonstrated to be carcinoycnic in animals'1' and in man (for references, see70').Quite recently, a carcinogenic activity of trichloro ethylene (TRI) lias been reported in mice after long-term administration of rather high daily oral doses (0.5 or 1.0 g/kg). This report'7' in connection with the above considerations prompted us to determine the mutagenicity of the whole series of chlorinated ethylenes with a metabolic activating microsomal enzyme system in vitro. Materials ana Methods. Tetrachloroethylene, trichloroethylene, cis- and trans-1,2-dichloroethylone, 1,1-dichloroethy1ene were obtained from Merck 4 Co., Darmstadt, as a.g. reagents; vinylchloride as a purified gas (> 99,9%) from BASF, Ludwigshafen. Mutagenic activity of the derivatives formed during microsomal activation was tested in a metabolizing in vitro system 12 1 with E coli K 12. One can use this bioauxotrophic strain in four mutation systems to test nutagenic 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-DLtryptophane1'. 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 mouse livers, and the NADPH generating system 5 jrj-; MgCl^, 16 mM DL-isocitrate-Na^, 0.66 mM NADP-Na^, 20 pi isocitrate-dehydrogenase (20 milliunits/pl) in 0.1 M phosphate buffer pH 7.4, as well as different concentrations of the test compounds. These concentrations were selected from preliminary c-xperiments 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 incubate was diluted in saline and plated on appropriate selective media as described previously1^'. Survival of the E coli K 12 strain was determined by plating on the complete medium. Mutagenicity is expressed as colony-forming units (efu) that were counted on the appropriate selective media per efu counted on the complete medium. Liver microsomes were isolated from male mice pretreated for 10 days with 0.1 per cent phenobarbila 1 in the drinking water to increase microsomal enzyme activity 12) , I I I Trcliminary Communications 2015 R&S 107501 Tab.1: Mutagenicity of chlorinated ethylenes after incubation in a metabolic activating microsomal system C12C = CC12 Tetrachlcroethylene C12C = CHC1 Trichloroethylene ei2c = ch2 1.1-Dichloroet.hylene Cl Cl concent ra tion in the mod i umx at 37 C |"nJ isurviva1 of bacteria % of spontaneous mutation rate in different operons of E coli K12 gal+ arg+ mtr nad + 0.9 99-1 100 lOO 100 100 3.3 76-4 123-23 232-36 114*18 100 2.5 74-7 120-14 229*26 lOO 100 H*^ cis-1.2-Dichloroethylene Cl H = </ v/ ^Cl trans-1.2-Dichloro ethylene 2.9 2.3 88-5 90-3 100 lOO lOO 100 100 100 100 100 ClCH = CH2 Vinyl chloride 10.6 72-3 231-20 663-141 172*35 148-24 v* j determined by GC analyses after injection of 5 yl of the liquid compounds; except vinyl chloride -..here the gas vas introduced by bubbling through the liquid at 15 C. Re^u_H s_arid Oiscussion, The results of the experiments are listed in Table 1. Cytotoxicity of the chlorinated ethylenes varies widely. To obtain 80-100 per cent survival of the tester strain, only 1 M of trichloroethylene could be used but lo mM of vinyl chlonde. No mutagenic activity of the chlorinated ethylenes was detected in the test system without microsomal enzyme activity. When the complete incubate with metabolically active micresomes was used, conversion of VCM, 1.1-PCE as well as TRI induced mutations, the latter compounds being less mutagenic than VCM. The highest mutation latcs we:e detected in the aiginine genes, whereas reversibili- 2316 Preliminary Communication! ty in the gal+ and nad+ systems and the forward mutation to MTR resistance were less sensitive to the mutagenic effects of the metabolites. Tctrachloroethylene and the cis- and trans-isomers of dichloroethylene were not metabolized to mutagens at all. Direct comparison of the mutagenic activity of the chlorinated othylencs is not possible since different substrate concentrations had to be usc-d to minimize cell death of the 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 the oxiranes, as outlined earlier, and the mutagenicity of all six chlorinated ethylenes: those forming very unstable oxiranes (VCM, 1.1-DCE,TRI) induce mutations in the test system, whereas the others (Per, cis- and trans1.2-DCE) 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 the scarcely reactive chloral hydrate and furtheron to trichloroethanol and trichloracetic acid1^'^^ The latter compounds are not known to induce cytotoxic or genetic effects. Con version of TRI-oxi T-a.ne to chloral in vivo is a quite unexpected reaction because thermal rearrangement in vitro entirely forms dichloroacetyl chloride 17) . This different behaviour should be further investigated. The results of such ex periments may contribute to the better understanding of the mutagenic and potentially carcinogenic properties of trichloroethylene. Acknowledgement: The skilful technical assistence of Mrs. Hesse and Eteinhilber is gratefully acknowledged. G.Bonse, D.Henschler, unpublished results U.Rannug, A.Johansson, C.Ramel, C.A.Wachtmeister, Ambio 2, 194 (1974) H. Bartsch, C.Kalaveille, R.Montesano, Int.J.Cancer 1_5, 429 ( 1975) P.L.Viola, A.Bigotti, A.Caputo, Cancer Res. 31_, 516 (1971) C.Maltoni, G.Lefemine, Environm.Res. 387 (1974) J.W.Lloyd, J.occup.Ked. 16, 809 (1974); 17, 333 (1975) Memorandum, Dept.of Health, Education & Welfare, Washington, 20.3.1975 P.Czygan, H.Greim, AJ Garro, F.Hutterer, F.Schaffner, H.Popper, 0.Rosenthal, CY Cooper: Cancer Res. 2983 (1973) J. Ellenberger, G.Mohn, Arch.Toxicol. 3_3, 225 (1975) G.Mohn, J.Ellenbercer, D.McGregor, Mutation Res: 25, 187 (1974) K. C.Leibman, "o1-Pharmacol. 247 (1965) J.W.Daniel, Bi ochem.Pharmacol. J_2, 795 ( 1963) J.Dcrkosch, personal communication (1974) R&S 107503 f