Document wKBBj65J9RQb7jmKyNvpkn736

R&S 114872 Mutation Itcseorch, 77 (1980) 175--179 DKIsevicr/No.'th-Hollanrl Biomedical Pres1; VC 6 *<? &-/ 9 175 Short Communication MUTAGENICITY OF VINYL CHLORIDE IN THE AMES TEST POSSIBLE ARTIFACTS RELATED TO EXPERIMENTAL CONDITIONS n. do MEESTER. M. DUViiRO ER-van B00AER7. M. EAMBOTTE-VANDEPAER, M. ROBERFROID, F. POXCEEET * and M. MEECIER Lnboralory of Biotoxicoiogy, University of I,ow:::n, School of Pharmacy, U.C.L.-73.69, B. 1200 Brussels (Belgium) (Received 11 .June 1979) (Revision received 11 September 1979) (Accepted 17 September 1979) The association between exposure to vinyl chloride (VCM) anti the occur rence of cancer in man has stimulated numerous investigations on the possible mutagenicity of VCM in several test systems. Exposure to vinyl chloride vapour in the presence of tin enzymatically active liver subcellular fraction has induced reverse mutations in Salmonella typhimurium [2,5,11,12] and Escherichia coli K12 [6], forward in mutations in Schizosacchcromyces pornbe [9] and V79 Chinese hamster cells [4], and mitotic gene conversions in Saccharomyccs cere visiae [9]. However, some studies have indicated that vinyl chloride is able to induce mutations towards S. typhimurium strain TA1535 in the absence of a meta bolic activating system. Some authors have reported that a much higher mutegenic response was obtained when liver post-mitochondrial fractions were added [2,12]. Another study [11], however, has shown that the reversion rates were only slightly different when the experiments were performed either in the presence or in the absence of a 9000 X p supernatant from the rat liver. More over, it has been reported that the addition of an NAD?K-generating system to the liver extract was not required for vinyl chloride to induce mutations and that a mutagenic effect could even be observed when the liver extracts were previously heated [5], These results arc in disagreement with those reported by Bartsch e.t al. [2] which clearly show that the enzymatically mediated mutagenic activity of vinyl chloride is significantly enhanced in the presence of an NADPH-generating system and in assays where the liver supernatants arc obtained from rats and mice that have been pretreated with phenobaibitonc. * To whom reprint rofiupsts should addroisrtl R&S 114873 TABLE 1 * DIRECT MUTAGENIC ACTIVITY OF VCM TOWARDS TA 1530 Concentrations of VCM in the atmosphere (%) Number of hit* rev./plate 2 331 e (7) 7 48 i 5 (10) 12 54 t 9 (7) 20 101 i 16 (10) * Moan values S.E..M. of 2 wwys performed in tripCicHte. Spontaneous reversion rates ore in parentheses. TABLE 2 MUTAGENICITY OF VCM TOWARDS TA1530 AS OBSERVED IN SIMULTANEOUSLY INCUBATED PLATES CONTAINING EITHER BACTERIA ALONE OR BACTERIA AND THE SO MIX OBTAINED EITHER FROM CONTROL MICE OR FRO.'.! MICE PRETREATED WITH AROCLOR 1254 The VCM concentration in the atmosphere was 2%. Mice j>rctre:irti**i Control Aroclor 1251 Fortified SD t; jetton --+ -- Number of In*4 rev./plate 206 t 18 (14) 52 9 (16) 562 t 71 (12) 102 i E (15) Mean values * S.E.M. of 2 assays performed in triplicate. Spontaneous reversion :s are in parentheses. table 3 MUTAGENIC ACTIVITY OF VCM TOWARDS TA1530 Plates containing either the bacteria alone or the S9 mix from control or pretreated mice alone were simultaneously incubated in an atmosphere of 2ft- VCM. Mice pretreatment Control Aroclor 1254 Number of kis* rev./plat* 74 t 8 (17) 261 t 32 (18) Mean values i S.E.M. of 2 assays performed in triplicate. Spontaneous reversion rates are In parentheses. TABLE 4 MUTAGENIC ACTIVITY OF VCM (2% IN THE ATMOSPHERE) TOWARDS TA1530 IN THE PRES ENCE OK VARIOUS PROTEIN FRACTIONS Kr-jWin fraction Boiled S9 mice Atoclor 1254 no cofactors $9 control mice no cofactors BSA + cofactors Number of his* rcv./plate 40 t 5 (19) 37 i 5 (13) 18 t 3 (14) Mean value,, r S.':. M. of 2 assays performed in triplicate. Spontaneous reversion rates are in parentheses. Among the sever; pounds, chlqtoethy t several test system*, 4 system [2,7,8,10,1: intermediates respo .} of this report used to evalufl^hc discrepancies obsorv Plates of minima TA1530 (2-7 X 10"' .1 described by Ames adult male NMRI ir. conditions. The pla ucts, Belgium) atmc trol device and its o exposure in the VC and further incubate had elapsed. The nu: In a preliminary * absence of any r. TA1530 increase phere. In a subsequent lI incubated in the d. Certain plates conta -i ! while the remainin* ; post-mitochondrial j pretreated with Arc ; trol and pretreated a -] The incorporate reversion rate.^d t the S9 liver fdBron (Table 2). These ob: ever, if one compare sion rate observed i plates containing b; exposed to VCM in enhanced reversion i an S9 mix obtain- d f This observation taining the bacteria 7 cator together with Comparison of the presence of plates < genicity of VCM t enhancement was e . from mice that had 1 Comparison of Tal R&S 114874 rAl'KMC ACi. ) OK VCM TOWARDS TA 3530 is of VCM hctt ('.>.) * rev./plate 2 33 i 6 (7) 7 48 t 5 (10) 1? 54 9 (7) 20 101 * IS (10) S.r ' -.f rfonoed in tripf. Spoof s.'-a* reversion rates are in pirintK.'iii. crrv O" VCM TOWARDS TA1530 AS OBSERVED IS SIMDLTANEOL'SLY 1SCC3ATED STAINING EITHER BACTERIA ALONE OR BACTERIA AND Til:. S9 MIX OBTAINED MICKOM CONTHOI. OK 1 ROM .MICE PRETKEATED WITH ARCELOR 1254 neinfiition in the atmosphere was 27r. tmont Control Aroclor 1254 fraction + -+ is* ifv./pIju 20S IS (14) 52 i 9 (16) 562 t 71 (12) 102 i 8 (15) t s.r ,M, of 2 perfr-rtned i; trirLT. '-'.'. Spontaneous tvvvr.v.on liter ere In parentheses. C ACT IVITY OK VCM TOWARDS TA1 530 inlni; either the bacteria alone or the S9 mix from control or pretreated mice alone were sis ir.r,.b?.ted in an atmosphere of 2;F VCM. inter* t Control Aroclor 1254 is* rev, /plate 74 8 (17) 261 32 (IS) t S.E.M. of 2 -stays perfor.neil in triplicate. Spontaneous reversion rates are in parenthes-j. 1C ACTIVITY OK VCM (2% IN THE ATMOSPHERE) TOWARDS T.M530 IN Tilt fitESTAiAOCS PRO I F IN FRACTIONS (ion Doileil Kp mice Arncior 25I no enfactors S!> v-mliol mice no eof.v.furs BSA * mfaator} ):L' rev.1'' .re r S r.M of 2 ass. 40 r 5 (19) 37 t 5 (13) 18 r 3 (14) performed j.n triplicate. Sp-miIh.r-ous reversion ran a are in p.trenihrsea. Among tho several possible hie pounds, c'rdorcethylene oxide a several lost systems in the abser system [2,7,8,10,11] and have intermediates responsible for the of this report is to demonstrate used to evaluate the mutagenicit; discrepancies observed between II Plates of minimal glucose aga: TA1530 (2-7 X 107 viable bacte: described by Ames and coworladult male NMRI mouse liver SD conditions. The plates were expc uets, Belgium) atmosphere in a c trol device and its operation hav exposure in the VCM atmosphen and further incubated in the dar had elapsed. The numbers of his' In a preliminary' step, the dire, absence of any metabolic activ; TA1530 increased proportional! phere. In a subsequent experiment, incubated in the desiccator, th Certain plates contained the bac while the remaining plates con post-mitochondrial fraction obt pretreated with Aroclor 1254. 'I trol and pretreated animals were The incorporation of the $9 reversion rate, and the number t the S9 liver fraction was obtainc (Table 2). These observations a< ever, if one compares the results sion rate observed in the plates plates containing both the bac exposed to VCM in the same ienhaneed reversion rates were o! an S9 mix obtained from mice pi This observation prompted a taining the bacteria but no liver calor together with plates cont Comparison of the results from presence of plates containing 5 genieity of VCM towards the enhancement was even more pt\ from mice that had been pretrea' Comparison of Tables 2 and 3 R&S 114875 CLT>AT`D U!iTAtNKn :i8 ..':.ih?scs. Atnnc vcre ''jntbeses. I Ur i-HKS- jjithcsei. 177 Among the several possible metabolites of vinyl chloride, two volatile com pounds, chloroethylene oxide and 2-chloroacetaldehyde, were mutagenic in several test systems in the absence of a liver microsomal metabolic activating system [2,7,8,10,11) and have therefoxe been considered as the metabolic intermediates responsible for the mutagenicity of vinyl chloride. The purpose of this report is to demonstrate that variations in the experimental conditions used to evaluate the mutagenicity of vinyl chloride could explain some of the discrepancies observed between the previously reported results. Plates of minima] glucose agar were inoculated with S. typhimarium strain TA1530 (2^7 X 107 viable bacteria/plate) in top agar according to the method described by Arnes and coworkers [1). S9 mix [1) (concentration, 150 //I adult male NMRI mouse liver S9/p!ate) was added to certain plates in various conditions. The plates were exposed to a controlled VCM (Matheson Gas Prod ucts, Belgium) atmosphere in a desiccator kept in the dark at 37 C. The con trol device and its operation have been previously described [3). After a 16-h exposure in the VCM atmosphere, the plates were removed from the desiccator and fmeher incubated in the dark at 373C until a total incubation time of 4S h had elapsed. The numbers of his* revertants were counted. In a preliminary step, the direct mutagenic activity of VCM was tested in the absence of any metabolic activating system (Table 1). The reversion rate of TA1530 increased proportionally to the concentration of VCM in the atmos phere. In a subsequent experiment, two sets of Petri plates were simultaneously incubated in the desiccator, the atmosphere of which contained 2% VCM. Certain plates contained the bacteria without any metabolic activating system, while the remaining plates contained both the bacteria and a fortified liver post-mitochondrial fraction obtained from either control mice or from mice pretreated with Aroclor 1254. The plates containing liver fractions from con trol and pretreated animals were used in separate experiments. The incorporation of the S9 mix into the plates significantly increased the reversion rate, and the number of his* revertants per plate was enhanced when the S9 liver fraction was obtained from animals pretreated with Aroclor 1254 (Table 2). These observations agree with previously reported data [2). How- ever, if one compares the results from Tables 1 and 2, one sees that the rever sion rate observed in the plates without S9 mix was slightly increased when plates containing both the bacteria and an S9 mix from control mice were exposed to VCM in the same incubation desiccator. Even more significantly" enhanced reversion rates were observed when those additional plates contained an S9 mix obtained from mice pretreated with Aroclor 1254. This observation prompted a third set of experiments in which plates con taining the bacteria but no liver fraction were exposed to 2% VCM in the desic cator together with plates containing the S9 mix but no bacteria (Table 3). Comparison of the results from Tables 1 and 3 indicates that the simultaneous presence of plates containing $9 mix in the desiccator enhanced the muta genicity of VC'ri towards the bacteria inoculated in separate plates. The enhancement was even more pronounced when the S9 mix had been obtained from mice that had been prclreated with the inducing agent, Aroclor 1254. Comparison of Tables 2 and 3 indicates that VCM metabolites were preferen- ^> ,* * i i tially trapped by bacteria that were in close contact with the S9 mix. Thus, when plates contain bacteria plus S9 mix, the amount of reactive metabolites which can migrate to the neighbouring plate appears to be lower than when bacteria are omitted. Finally, assays were performed to investigate the possible effects of the pro teins of the S9 mix on the mutagenicity of VCM. To plates of bacteria were added various protein fractions: S9 from mice pretreated with Aroclor 1254, heated in hailing water for 5 min (150 pl/plate); S9 from control mice (150 pi/ plate); and bovine serum albumin (3.8 mg (''150 pi S9)/p!atc) supplemented with salts and cofactors. These plates were successively incubated in an atmos phere of 2% VCM (Table 4). The results indicate that, under the present con ditions, addition of proteins to the plates had no significant effect on the muta genicity of VCM. In conclusion, and in agreement with previously reported studies [2,11,12], VCM has been shown to exert a direct mutagenic activity towards 5. typhimurium TA1530 which is related to its concentration in the atmosphere. The origin of the direct mutagenic effect remains unclear. The mu tagenicity of VCM is strongly enhanced in the presence of a fortified metabolizing liver extract. However, co-incubation of Petri plates containing either bacteria alone or only S9 subcellular fractions from liver tissue in an atmosphere of VCM enhances the mutation frequency as compared with fre quencies observed when f>. typhhnurium alone is exposed to the same atmos phere. The increase is more pronounced when the liver homogenates are derived from animals that have been pretreated with a metabolizing enzyme inducer. However, it must be pointed out that the results presented here could be affected by numerous experimental conditions, e.g. the amount of volatile metabolites, the volume of the desiccator, the number of plates, the composi tion of plates. The results suggest that the observed enhancement of the mutagenicity of VCM is correlated with the formation, under the influence of the liver-meta bolizing enzymes, of proximate mutagens such as chloroethylene oxide and 2chloroaceialdehyde. These two compounds are volatile intermediates and are, therefore, able to exert their direct mutagenic action towards the neighbouring bacteria. Iieferc-nces J Arnes W.IJ. Durston, E. Yamasaki ami f`.D. Lee, Carcinogens arc a simple test system combining liver homottcsiaU** (or activation and bacteria for detection, 2roc. Natl. Acad. Sci. (U.S.A.), S (107J; .*28! 22*15. 2 i'art'ich. H., C, VatavciMe, A. Harbin and G. Flanehc, Mutagenic am! alkyJaiir.A metabolites of halo- ethyirnvs, <ah!o';bu'udicncs am! dicb'nrobutcncv produced by rodent or human liver tissues. Evidence lor oxirPTie formation bv P*450*linktM xnterostmtaj monooxy^enascs. Arch. Toxicol., *41 (1979) 249-- 27$. 3 l)e Vccstcr, C`,, F. Por.colet. M. Unbctfroid and M. Mercter, Mutagenicity of acrylonitrile. Toxicology* II (I97JO 19-27. 4 i).-von. C\, T. Kurokt and It. Montu.-uno, nnitajieneiis of Chinese hamster eell- imf by various eh'/ntPMh-, 7iivl l:it. Cnnf. Environ. Mutagen*. Edinburgh, 1977, p. 150. 5 G.irro, A -1.. J.B. Guuoniih'.n and !\ Milvy, Vinyl chloride dependent inJ;^nes:s: efiert of liver tXtr.vVb: free raditvK, * i: y.itjon kVa., 33 (ti)7G) 81 --ht . 0 Cireirw. 'J. L'onse, 7. butlv/an, t;. Hviehert and 11. l.'Ouschler, Muta$enie:ty in vitro and putvntial carri*u>i,Mtici:y r.f ''hliniiuitocl el: * Vues as n function .* metabolic oxiram? formation. Hiorhem. Piiax* mil'24 (197:* ' !;* Wi. i i i i } ;i ) i }\ \ ;; S I i * : { ,! I ! ;; 3 ci i i i 7 llubcrman, E. II. L chloride metabolite 644. S Hussain, S., and S. < bolites, Chirm. -Rio] 0 Loprieno, N,, R. B.- C. Gcrvusi, C. Lcpiu of vinyl chloride m K5--yf.. 10 l.oprieno, N.. R. R. R. Xieci, C. Leporir by vinyl chloride m< 11 McCann, J,,- V. Sir possible metabolic chlotohydrin), viny 31UO--3193. 12 Kannux. If., R. GSL taidchyde, 2-chloro< Biol. Interact., 12 (1 33 R W JCbO> c-ra> v. TllllS. 'boiiU;.-; vvh>.'r; '.he pro* ria were r 1254, 150 fi\/ mented i :*.lmosr.t con' e Jiuita- '11,12], . phinuh ..re. The fortified ntaining -ie in an with frc* r' atmos.ates ate n JJ to _n -u 00 si si 179 7 Huberman. E.. tl. Barticli and I.. Sacha, Mutation induction in Chinese hamster V79 cells by uvo vinyl chloride metabolites. chloroathylene oxide and 2-ohloroscetaldehydr, Int. J. Cancer. IS (197a) 639-- e-u. 8 1'v.v 'in. S., and S. Ostermsn-Golkar. Comment or, the mutagenic effectiveness of vinyl chloride meta bolites, Chem. -Biol. Interact., 12 (1976) 265--267, 9 Lopricno. K., R. Barale, S. Baroncclli, C. Bauer. G, Bronzetti. A. CammeMini. G. Certignanl, C. Cnrsi. G. Gervasi. C. Leporirri. ft, Nieri, A.M. Rossi. G. Stret'i and G. Tnrehi, Evaluation of the genetic effect of vinyl chloride monomer (VCM) under the influence of liver micro5oir.es. Mutation Re*.. 40 (1976> 65 -96. 10 Eoprieno, ft., R. Barale S. Baroncelli. H. Bartsch, G. Eronretti, A. Catnmellir.i, C. Corsi, L>. Erorra, ft. Nieri, C. Lrporitri, D. Roscliini and A.M. Rossi, Induction of gene nutations and gene conversions be vinyl chloride metabolites in yeast. Cancer Res., 36 (1977) 253-1*257. 11 McCann, !.,* V. Simmon, D. Strcitwcfser and B.N. Ames, Mutagenicity of chloroacetaldehydr, a possible metabolic product of 1,2-dichloroethane (ethylene diehloridc), chloroethanol (ethylene chlorobydrin), vinyl chloride and cyclophosphamide. Prof. Natl. Acad. Sci. (U.S.A.), 72 (1975) 3190-3193. 12 Rnnug. V., ft. Gotlte and C.A. Waehtineister. The mutagenicity o! ehloroethylene oxide, chloroaeetal.lchyde, 2-chIoioethanol and ehloroacetic acid, conceivable metabolites of vinyl chloride. Chem.Biol. Interact., 12 (1976) 251--263. >"- V i i wiiwihr i isst system , i. (O.S.A.). .'s of tiaio;s, Evidence , uVU) 249-- 1 oyicologv. '.ai-.ttr crll- Wct of li.er i.d potential ,chem. I'har- t : liKi iS-.Tifci.i* // ^ T/777dAy / S#*1 C/y IIJ It; //7- ft<n<rj The obtained results reveal dose dependence for both lethality and terato ous study f> gcui-: outcome. Types of teratomas indicate that most of them are chondro- pathic and affect skeleton causing different malformations, evisceration and runting in somatic development. Further experimentation with some com pounds derived from erythromycin point to the fact that carbamide induce? A.G. Searle skeletal malformations and isoniazide leads to evisceration. Cytogenetic A cT 21 #/33 I.L. Hansteeii, L. Hilleslad and K. Thiis-Evensen, St. Joseph Hospital, Porsgrunn (Norway) Although, ties have be(Nature, 2b concentrate Chromosome studies in workers exposed to vinyl-chloride genetically - have therefc. Chromosome analysis have been done on 48-h lymphocyte cultures from 39 mosome ahe workers in the I'VC factory in Norway, and on 16 control persons matched to for long pen sex and age. Hone marrow samples were studied in four ca-es. so far sugge- Measurements of exposure are unfortunately not available before 1974- tive for tran- Hundred cells per individual were scored for breaks, gaps rings, dicentrics, They arc . fragments, chromatide exchanges, and "stable" rearrangements. Breaks and gaps were the predominant aberrations in the ce'ls both in the controls and in of the magn tion of chi' the worker.-. "Stable" rearrangements were found in more cells than was ex tained. pected. Caps are not included in the results. The results are presented as number of cells with aberrations, and also con sidered as total breaking events per 100 cells. The number of cells with aberra tions and total breaking events are not greatly increased for the workers com pare*] with the controls. The results were tested statistically by a Wileoxon two- 24 U. Rannug Laboratory. sample test, which showed that workers scored significantly higher on a 2..v* level. The results are discussed. The mutagen 22 Gh. Deknudt and A. Leonard, Dept, of Radiobiology, S.C.K.-C.E.N.. Mol (Belgium) Cytogenetic investigations on leucocytes of workers from a cadmium plant Chromosome analysis has been performed on workers of a cadmium plant, who were exposed to fumes and dust of cadmium and lead. The 35 workers were classified, according to the type and duration of exposure, into a group (cadmium service) of 23 imople exposed to high levels of lead and cadmium in the absence of zinc and into a group of 12 rolling-mill workers subjected mostly to zinc but also to lower levels of lead and cadmium. A higher yield of severe chromosome anomalies (chromatid exchange, disturbance of spiralisation, chromosome translocation, ring and dicentric chromosomes) together with a total lower number of structural aberrations was observed in the cad mium workers when compared to the rolling-mill group. These data will be discussed together with the results obtained in a previ Vinyl chi ously been trial process carcinogenii The manmixture of containing i uct has been EDC-tar Salmonella t substitution used to dissf was approx i i When, ho ing system v. effect. The componenl.