Document 0qO7YbpedZkz5wYZOqM8DaX9J

i -* k iTf973 Int. J. Cancer: 15, 429-437 (1975) nr .// HUMAN, RAT AND MOUSE LIVER-MEDIATED MUTAGENICITY *e OF VINYL CHLORIDE IN S. TYPHIMUR1UM STRAINS by I H. Bartsch, C. Malaveille and R. Moktesano a International Agency for Research an Cancer, Unit of Chemical Carcinogenesis, ISO Cours Albert Thomas, 69008 Lyons, France Exposure ofS. typhimurium strains TA 1530, TA 1535 ami G-46 to vinyl chloride increased the number of His1- .reverumts/plate 16. 12 or 5 times over the spontaneous .it !' mutation rate. After 6h of exposure to vinyl chloride, the mutagenic response for '7 TA 1530 strain was enhanced 7-, 4- or 5-fold when fortified postmiiochnndrial liver fractions from humans, rats or mice moty added. The enzyme-mediated vinyl chloride a mutagenicity was dependent on an \'ADPH generating system and the enzyme activity i >t was localized in o liver microsomal fraaion; 9,0(XIg liver supernatant was three times more active than iiicrosames, while liver cytasol or alcohol dehydrogenase did not affect j, the mutagenicity. Phcnobarhitune pretieeitmenr ofrats and mice increased the mutagenic response by up I > 15-40% as compared to untreated controls. The relative mutagenic j: activities of VCM, taking the value front mouse liver as 100, for TA 1530 strain mediated by 9,000 >: g tissue fractions uw: rut liver, 80; mouse and rat kidney, 20 ` and 16; mouse and rat lung, less than 7; human liver (from Jour biopsy specimens), 170, 64, 70 and 46. Ch/oroacera/dehyde and chloroacetic acid, a urinary metabolite of ' VCM. showed toxic effects, white cldoroethanol imj weakly mutagenic for TA 1530 , e i Strain. l: r i Vinyl chloride monomer (VCM) is extensively 1974). The systemic action of this carcinogen and used for the production of polyvinyl chloride and its low chemical reactivity suggest that the other plastic material, and also as a propellant biological effects of VCM arc dependent upon for aerosols. Carcinoid-ninny m._r.-n .An its metabolic activation. In these studies, we exposed to 30.000 ppm VCM in air, \*as first report the mutagenicity of VCM and its u reported by Viola er at. (1971), Subsequently, presumed metabolites in S. typhimuriutn strains, Matiom and Lcfcminc "(1974) showed that angio mediated by liver and other tissue fractions of sarcomas of the liver, as well as other tumours, rut, mouse and human might. i were induced in rats and mice exposed to various doses of VCM by inhalation. Recently, cases of angiosarcoma of the liver have been found in workers exposed to VCM during the polymeriza MATERIAL AND METHODS Chemicals tion process and a causal relationship between VCM (purity 99.9%) was generously provided VCM exposure and this type of tumour in man by Rhonc-Progil, Lyons. France, contaminated has been established (Creech and Johnson. 1974; with cihariol (30 ppm), water (20 ppm), methyl- Heath et a!., 1974; Lee and Harry, 1974; IARC, chloride (-.20 ppm) and non-volatile substances Received: October 16. 1974. \ I 429 RSV 001648 i;rHAH ISOI AL. ( '5 ppm). Chloroaecluldehyde (50 "' aqueous under identical conditions, and stored in liquid solution). cliloroaeclic add Imp 61-63" C) were nitrogen for 3 weeks (Sample A and D), and for obtained from Fluka Co.. Uuchs. Switzerland 1 week (Sample B and C). The total and micro and chloroclhanol (bp 30 C) from Merck- Sohuchardl. Darmstadt. Fed. Rep. of Germany. Alcohol dehydrogenase (200U/mg of protein) and glucosc-6-phosphaic dehydrogenase (450 U/ somal protein content per ml of the 9.000 g supernatants from IMMreated or untreated animals were: rat and mouse liver. 29.7 I 3 (20), 5.1 1 1.1 (IX); rat and mouse lung. 21.4 3 (9), mg of protein) were purchased from Bochringcr 1.7 | 0.3 (X); rat and mouse kidney, 17.4 : 1.7 (7). Mannheim, Fed. Rep. of Germany, or Sigma 2.1 : 0.4 (7). ( '.=*st>; number of animal pools Chemical Co.. St. Louis, Miss.. USA. All other analyzed shown in parentheses). S. typhhnurium products were obtained commercially and were of the purest grade available. strains TA 1530. TA 1535, G-46 and TA 1538. kindly provided by Professor B. Ames. University of California. Berkeley. USA, were grown over Gas clnawatngraphy night in nutrient broth and resuspended in saline. Mutagenicity assays were performed in two ways. VCM concentrations were determined in a Packard model 417 gas chromatograph, fitted Afrihoti A with a glass column (0.? ' 400 cm), which was packed with Porapack 0. 100-120 mesh (Packard If not otherwise specified. 9.000 g tissue S.A.. Paris. France). A flame ionization detector supernatants, fortified with the appropriate with hi. as carrier gas-was used at a flow rate of cofaciors (NADP , 2/moles; glucosc-6-phos- 45 ml/min.' For routine use. the column, the phatc. 2.5/rmolcs; MeCK. 4 pmolcs). phosphate detector and the injection pon were maintained buffer pit 7.4 <50mnoics) and 3-6 10s bacteria at 100. 160 and 140 C. whereby VCM had a per plate were combined in a soft-agar layer and retention time of 2* mm. A freshly prepared solution of VCM in methanol was used as a standard. plated in duplicate or triplicate on Petri dishes with Davis minimal agar, as described (Ames ft at.. 1973). When microsomal fractions were utilized, the medium was supplemented with Animals amt prctreajnienl 1.0 U of glucosc-6-phosphate dehydrogenase. Bacterial survival was determined in parallel by Adult male BD-1V rats (100-130 g). bred in seeding bacteria (10 M0 ` dihuions) on a our laboratories, and male OF-1 mice (30-35 el, histidine-enriched medium. Mutagenicity tests obtained from (Ha-Credo. St. Gernmin-scir- with VCM wore performed hv evoosme the Petri L'Arbresle. France, were fed on a Charles Riser dishes to VCM in an in dcssicators (volume: ORF diet. Some rats and mice received pheno- 70-15 H for no to 4K h at 37 ` C in the dark. For barbtione sodium (PU) in their drinking water (I mg/ml) for 7 days prior to the preparation oT the tissue fractions. Human liver samples, with shorter treatments, the VCM was removed under vacuum and replaced by air. and the incubation was continued up to 4$ h at 37' C. The concen no pathologic lesions, obtained by biopsy from tration of VCM io the Vogcl-JJonncr medium was four adult patients, were kindly provided by determined by gas liquid chromatography of Dis. G. Della Porta and U. Veronesi {Islhuto 6 and 4K h following exposure at 37' C to 0.2, 2 Na/ion.ilc per lo Studio c la Cura dci Tumori, and 20VCM in air (v/v). Milan, Italy). XT ir u ie at sc ft 1 i, <kv i 6 -al V \ I Mftinn! 8 A Inhiyi-airily as.sen s This was carried out by incubation in liquid The 9.000 g supernatants or microsomal and sus|vcnsion (Burtsch et at.. 1975). The medium soluble Auctions were freshly prepated from (final volume 220 /<!) contained 60 pi of 9.000 g tissue pools from 3-5 animals, by centrifugation of liver supernatant from PB-pretreated. mice, it homogenate* (3 ml of 0 15 m KO/g wet tissue), !.6pmol MgCI... 1 pmol glucosc-6-phosphatc. as described elsewhere (B.irtseh ft a!.. 1975). and 0.X pmol NADP . 20 pmol Sorensen phosphate utifi/ed in the assay* 3-5 li later. The 9.000 g hull'er pit 7 4, 20 pi bacteria! suspension G-46 or . liver suj>ernataui from lour humans was prepared TA 1530 (2-4 I0` ceils) and an initial cunccn- 430  in liquid I >1. and for .mil micro* 9.000 unircatcd >.7-l3 (20). 1.4.1:2 (9). 4 >1.7(7). imal pools xphinuoiunt 1 TA I53S. University >w ii ovcrd in saline, ii io ways. i v l issue ijipropriate O'.e-fi-plios* phosphate t>* bacteria r layer and Vtri dishes I (Ames et uons were .nted with >drogenase. parallel by us) on J nicily tests hi: the Petri n (volume: djik. For oved under incubation : lie conccnicdium was graphy of l' to 0.2, 2 `it in liquid nc medium ..fy.ooo a ited mice, phosphate, i phosphate n G-46 or i.d concen tration in the medium of t' OS.t m WM. I he iiu'uhaiion n,i> earned out at .'7 C tor 30 mm under O. and was Mopped by dilution with ice-cold saline, The number of His revoriants and survivors was determined by plating on selective media as previously described (Uarlseh et a!., 1975). RESULTS Mutagenic response us a function ofdose and time of VCAf exposure S. typiiinnninni strains TA 1530. TA 1535 and G-46, which arc sensitive to monofunctional alkylating agents, were specifically reverted by VOM to His protoirophy. following exposure to various concentrations of VCKf in air. in the presence of a 9,000 x mouse liver supernatant and cofactors as listed under Method A (Table I). Among the strains tested, the TA 1530 showed the highest mutagenic response and the number of His' rcvcrtanis/plate increased approximately six. 12 or 28 times mcr the spontaneous imitation rate following exposure to-0.2. 2 or 20% VCM in air for 4M h. The G-46 and TA 1535 strains also showed a xniiil.il mm.iyvmc doc response, apivaicil lcs> mmimiiu- ilum t.\ l5.M>su,im W uli TA 153k* .strain, which is specifically ieverted by frumeshift mutagens, no significant increase in the number of His! revertams after an exposure to 20% VCM in air for 48 h was noticed. Under these conditions, the concentrations of VCM in the incubation medium after 6h of exposure to 0.2, 2 or 20% VCM in air were 4 -10 ;m, 40:-I0`m and 400x10 3 m, and no further increase was observed up to 4S h. Under these experimental conditions and for all S. typhinmrinoi strains utilized. VCM showed no cytotoxic cllects. Hacleriul survival, even at the concen tration of 20% VCM in air, was between 86 and 131 "'(Table I). In another scries of experiments. TA 1530 strain was exposed to 20% VCM in air for various lengths of time (Fig. I). In the absence of a 9.000 y x mouse liver supernatant, VCM exerted a mutagenic action per sc in TA 1530 strain. The number of His rcvcrtanls increased us a linear function of the lime of exposure to VCM reaching, after 48 h, 20 times the level of the spontaneous mutation rale. In the presence table t VCM CONtTVTRATION.ort*LNorKT INHUCTlON OF REVERSE MUTATIONS EN*on- *,, vest t.OCKI t Mip. icnl.ieiorx * rhcooh.trhilAnc l>rt( remmpnt Kfl. of 11k * rrvrrtani* phtie ( TA 15.1(1 Sirjiln 7A 1535 haeierial turwval ') G-4r, TA 151* 10 Yes or no 10 : 3 MOO) 8 = 2 MOO) 6 ` 3 MOO) 23 ` 1 ()00) 2 -) Yes 3 0.2 - Yes 4 -f No 5 - No 62 : 7 41 ; 2 (131) 45 ` 10 27 =. 15 44 . 4 ` 23 4 (86) 39 i 0 21 '. 9 20 s. 15 10 L 1 (1 II) 11 *> M! 8 6 Yes 118 ^ 23 76 < II 25 1 2 7 -- Yes 39 26(116) 40 i 12(86) 13 1 1 (103) 8 No 106 t 19 64 ` 22 19 - 1 9 - No 70 < R 33_' 2 15.1: 1 10 II 20 12 13 -t- Yes -- Yes r No No 278 i 16 131 L17 (123) 232 1 K 155 11 179 98 i 36(129) 161 60 98 ' 31 62 i. 3 40 !- 0 (M9) 56 `i- 0 30 !1 Id 328 (118) Sir,1.11 7 A 15.10 ...... I.ue in rarciuhv;l <(. K 10*1. TA 1*15 <5.1 UH: C.-U* <5 K 10') and TA 151K |H.l . 10*1. rre etpnvM in 0 J, 2 an.I nr ^ir . VCM in air I v.x lor 4* Ii ul ' t C m I lie |>iv>cih'( of ail N AIll'll general my vysicm and a lMKN' t liver wipi-rn-iiam Irniii culirr i'livni>lM<Aiinne.irv.iiiil nr umrc.iltxl nine. ' U.icien.il Miixn.il l.tr t.icli rvi'fimuni .n ali-urrniiiii-xJ >n parallel: mean value' for each exposure finup arc liticil. `Cefaclor*. NAI7J** .nnl rlucn\e-i>>>i<i(io'phaie. ` Mean 'aliicaisii irnm l wo -eric* of npcrinienn. each uHli/my a pnol or fixe ninuve 1 nets. 431 RSV 0016486 IIAKISCN IT At.. ( "5 ppm). Chlornacetuklehydc (5()",, aqueous under identical conditions, and stored in liquid solution), chloroacetic acid (mp 61-63 O were nitrogen for 3 weeks (Sample A and D). and for obtained from Fluka Co., Bucks, Switzerland I week (Sample B and C). The total and micro aiul chlorocthanol (bp I2S-I30C) from Merck* somal protein content per ml of the 9.000'- g Selntchurdt. Darmstadt. Fcii. Rep. of Ciermany. supernatants from PR-treated or untreated Alcohol dehydrogenase (200 U/mg of protein) animals were: rat and mouse liver. 29.7 ' 3 (20), and glueoso-6-phosphnic dehydrogenase (450 U/ 5.1 ! 1.1 (18): rat and mouse lung. 21.4 *3 (9), mg of protein) were purchased from Bochringer 1.7 0.3 (8); rat and mouse kidney, 17.4 ! 1.7(7). Mannheim, Fed. Rep. of Germany, or Sigma 2.1-l 0.4 (7), ( t -- so; number of animal pools Chemical Co.. St. Louis. Miss.. USA. AH other analyzed shown in parentheses). S. typhimnrinm products were obtained commercially and were strains TA 1530. TA 1535. C.-4G and TA 1538. of the purest grade available. kindly provided by Professor U. Ames. University of California. Berkeley. USA. were grown over Gas chromatography night in nutrient broth and resuspended in saline. Mutagenicity assays were performed in two ways. VCM concentrations were determined in a Packard model 417 gas chcomtitograph. fitted Method A with a glass column (0.3 '400 cm), which was packed with Porapack 0. 100-130 mesh (Packard If not otherwise specified, 9.000- ^ tissue S.A., Paris, France). A Dame ionization detector supernatants, fortified with the appropriate with Nj us carrier gas was used al a How rate of eofactors (NADP . 2/molcs: glucosc-6-plios- 45 ntl/min. For routine use. Hie column, the phatc, 2 5/'moles: MgCL. 4/'moles), phosphate detector and the injection port were maintained buffer pit 7.4 (50."moles) and 3-6-TO* bacteria at 100. 160 and 140" C. whereby VCM had a per plate were combined in a soft-agar layer and retention time of 2Smin. A freshly prepared plated in duplicate or triplicate on Petri dishes solution of VCM in methanol was used ns a with Davis minimal agar, as described (Ames er standard. al.. 1973). When microsomal fractions were utilized, the medium was supplemented with Animatv nut! prcncanttcnt 1.0 U of glueosc-6-phosphatc dehydrogen ise. Bacterial survival was determined in parallel by Adult male BD-IV rats (100-I30g), bred in seeding bacteria (10 MO'1 dilutions) on a our laboratories, and male OF-I mice (30-35 gt. histidine-enriched medium. Mutagenicity tests obtained from Illa-Crcdo. St. Germain-sut* with VCM were performed by exposing the Petri L'ArbresIc. France, were fed on a OJiailes River dishes It' VCM in air in dcssicalurs (volume: CRF diet. Some rats and mice received phenn- 10-15 I) for up lo 48 h at 37 ' C in the dark. For barbitonc sodium (PR) in their drinking water shorter treatments, the VCM was removed under (t mg.'ml) for 7 days prior to the prcpaialton of vacuum and replaced by air. and the incubation the tissue fractions. Human liver samples, with was continued up to 4X h at 37' C. The concen no pathologic lesions, obtained by biopsy Troni tration of VCM in the Vogef-Ronner medium was four adult patients, were kindly provided by determined by gas liquid chromatography of Dr.s G. Della Porta and U. Vemnosi (Istiiuto 6 aiul 4X h following exposure at 37'C to 0.2, 2 Nu/iotude per lo Studio e la Cura dot Tinnori. and 20% VCM in air (v/v). Milan, Italy). Mutagenicity assays Tlic 9,000 g supernatants or microsomal and soluble fractions were freshly prcpaicd from tissue pools from 3-5 animals, by centrifugation of a homogenate (3 ml of 0.1 5 m KOl/g wet tissue), as described eKewheic (Rartscb ct al., 1935). and utilized in the assays 3-5 h later. The 9.0tH> g liver siipcrnaljnt from four humans was prepared Method li This was carried out by incubation in liquid suspension (Bartseh <7 al.. 1975). The medium (final volume 220 /<l) contained 60 /l of 9,000 - .e liver supernatant from PR-prclrcalcd mice, 1.6/miol McCI;. 1 /miol glucnsc-6-phosphatc. 0,S .nmol NADI* . 2d/mini Sorensen phosphate hullcr |'ii 7 4. 20 /d bacterial sus|>ensioti G-46 or TA 1530 (2-4 IU: cells) and an initial eonccn- 430 RSV 0016487 T m liquid i. and for -.d micro9.000 imt rested ? 3 (20). 4 ! 3 (9). : 1.7(7), Mill pools hiiiniriain I A 1538. niversity *w n overI in saline. mo ways. a tissue propriatc e-6-phos-fiosphatc bacteria layer and ii r dishes (Ames et mis were ted with rngenase. uallel by o on a % iiy tests the Petri (volume: laik. For >ed under M^ii halion veonccnJiutn was uapliy of to 0.2, 2 in liquid medium 9.000 yK vd mice, fiosphalc, .'hosphatc (i-46 or I concen MuiAtw-Nicirv m vinvi. niu>Kit>r tration in the medium of 0.0X3 m VCM. Tlic incubation was carried out at 37 C for 30 min under O. and was stopped by dilution with ice-cold saline. Tlic number or His' revertants and survivors was determined by plaiing on selective media as previously described (Bartsch o/(i/,, 1975). RESULTS Mutagenic response as a Junction ofebse otal filin' of VCM exposure S. typfrinuaiuni strains TA 1530. TA 1535 and G-46. which arc sensitive to monofunctional alkylating agents, were specifically reverted by VCM to His prototrophy, following exposure to various concentrations of VC'M fit air. in the presence of a 9.000 g mouse liver supernatant and cefaclors as listed under Method A (Table I). Among the strains tested, the TA 1530 showed the highest mutagenic response and the number of His revcrtants/plaic increased approximately six. 12 or 28 times over llic spontaneous mutation rate following exposure to 0.2. 2 or 20",; VCM in air for 48 h. The G46 and TA 1535 strains also showed a similar mutagenic dose response, but appeared less sensitive than TA IS30 strain. With TA I53K strain, which is specifically reverted by frameshift mutagens, no significant increase in the number of His- revertants after an exposure to 20".p VCM in air for 48 h was noticed. Under these conditions, the concentrations of VCM in the incubation medium after 6h of exposure to 0.2. 2 or 20"^ VCM in air were 4^ 10-!m, 40 10 iM and 400.!0'iM. and no further increase was observed up to 48 h. Under these experimental conditions and for all 3T. typhinmritun strains utilized. VCM showed no cytotoxic effects. Bacterial survival, even at the concen tration of 20,/n VCM in air, was between 86 and 131 (Table I). In another series of experiments, TA 1530 strain was exposed to 20% VCM in air for various lengths of lime (Fig. 1). In the absence of a 9.000:-#,' mouse liver supernatant. VCM exerted a mutagenic action per .\e in TA 1530 strain. The number of His' revertants increased as tt linear function of the time of exposure to VCM reaching, after 4S h, 20 times the level of the spontaneous mutation rale. In the presence TABLE I VCM CONCFNTRATION-DF.PCNDENT INDUCTION OF REVERSE MUTATIONS Evp. *; vcm 9.000 * MJp. PhcMohar hiiftrK No. prdrcaimenl No. of H iv* icvcrianl* '-pLaitc ("' bacicri.nl survisal >> TA IS JO Si rain TA IS.15 C-4fi TA 12.18 10 Yc% nr no 10 - 3 (100) K - 2 (100) 6 ; 3(100) 23 1 {100) 2 -i. Yes 3 0.2 _ Yes 4 -i No 5 - No 62 7 41 2 (131) 45 ' 10 27 : 15 44 h 4 ' 23 ' 4 (86) 39 J- 0 21 !. 9 20 J 15 10 )- 1 (III) 11 -J 2 11 8 r> 72 8 9 i Yes .. Yes *r No - No 118 13 39 ; 26 (116) 106 1 19 70 i 8 76 i It 40 ' 32 (86) 64 - 22 33 i 2 25 * 2 13 < l (|03) 19 f 1 154: 1 10 11 20 12 13 4- Yes - Yes *i No No 278 : 16 131J 17 (123) 232 8 155 * M 179 98 j 36(129) 161 60 98 * 31 62 ] 3 40 l 0(119) 56 0 30 1 10 32 t8 Ot8) * Strain 1 A I MO [Kkuti.i i-I.iic hi p.iro.i In v.O l(it IBM: 1 A IMS (VI I0*>: C.-JO <V* HIM and TA IJJ8 (JO (O'). . *'iTncd M-i <1.2. 2 .iml ir 20'.. VCM iii .nr <i.i> l.>r JKh .K .O C iii till' presence of an N A Dl'l I peneraunj sjtiem and a fl.OllOj g liver Mipi-rn.ilanl Jrnrn cnlu-j rIli-ilidv.rdn<i<h*.I rc.tKil or milrcau'.l nil..* ' ll.icicri.il iunn,il for ca.li c\pcriii'i dvte mimed in par.ill.l: mean \alue* lor eiich exposure croup are tided. * Cnf.-iclors: NADI'* and fckico\c-A- phn.|ili.ifc * Mean uuluc*i jii Irom io *cric of experiments. each iiiilifing : pool of five mouse livers. 431 RSV 0016488 RAKTSTII FT AL. of a 9.000 yg liver supcrnalanl of PB-pretreated mice, Ihc number of His' revcriants increased, after 3. 6, 9 or 48 h of exposure to VCM, 4. 3. 2.5 and 2-fold as compared to the assays without the liver fractions (Fig. I). When the number of His* revertants obtained with the liver fractions were corrected by those observed without any metabolic activation system, a plateau was reached after 9 h of exposure. No increase over the spontaneous nuilulion rate with strains TA 1530. TA 1535 and G-46 was detected when the incubation was performed for 30 min in liquid suspension (Method B) in the presence of a fortified 9.000> g liver supernatant from PB-pretreated mice and at an initial co iccniration of 0.083 m VCM in the medium, A/magenic response os a function ofanimal species and tissues, including human tissues S.fyp/?.TA1530+MOUSE LIVER in vitro TO 20% VCM IN AIR Figure 1 Mutagenic response of TA 1530 strain after VCM exposure for various lengths of time. Mutagenicity assays (procedure A) were performed with TA 1530 and pooled liver fractions from five phcnobarbuonc-prctrcntcd mice, following exposure to 20"; VCM in air ai 37" C in ihc presence of a 9.000 <g supernatant and an NADl'H generating system (o). or a 9.000 g supernatant, an NAIOJ'H generating system. NAP' (4 pmotcs/platc) and alcohol dehydrogenase (5.9 Units/platc) (A), or a 100.000 - supernatant.an NADPII generating system, NAD' and alcohol dehydrogenase (x),or KC1 and NADP\ only (). Ral or mouse liver, kidney and lung fractions (9.000 -<g supernatant) were assayed, following exposure to 20% VCM for 6 h at 37f C, for their capability to generate VCM metabolites muta genic for TA 1530 strain (Table II). Fortified liver fractions front PB-pretreated or untreated mice and rats showed an enzymatic capacity to convert VCM into mutagenic metabolites in vitro, which was equally high in both species (Table II, exp. Nos. 2 and 14). The mutagenic response in the liver of PB-prelreatcd or untreated animals was, respectively, four and three times higher than that of (lie appropriate control (Table II. exp. Nos. 2, 14, 4 and 16 versus No. 1). With kidney and lung fractions from mice or rats, only a marginal mutagenic response, or none at all. was observed with either PB-pretreated or untreated animals (Table If, exp. Nos. 6-13 and 18-25 versus No. I). Poslmitochondrial liver supernatants from four adult humans showed a great variation in (heir capacity to convert VCM into mutagenic meta bolites (Tables II, exp. Nos. 26-33). Sample A caused a mutagenic response which was seven times higher than that of the appropriate control (Table II, exp. No. 26 versus No. I) and twice as high us that obtained with rat or mouse liver. Samples B, C and D showed moderate activity (Table II, exp. Nos. 28. 30 and 32) and samples C and D caused a mutagenic response which appeared to be independent of the added cofactors, NADP1 and glucose-6-phosphatc (Table II, exp. Nos. 30-33). Attempts to characterize enzyme (s) and meiabtihtc(s) involved in VCM mutagenicity Fortified postnnlnchondrial (9,000 xy) liver supernatant from PH-prctrcalcd mice, blit not the soluble proteins (100,000\g supernatant), signi ficantly increased the number of His' revertants of TA 1530 strain by 7. 4, 3, 2.5 and 2 times, following exposure to VCM for 1.5, 3, 6, 9 or 432 48 h, lainu of 5. of h I00,( mu ta genet m tn. 3 4 5 6 7 8 9 10 It 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 t h ftom t.* >h K /ail or RSV 0016489 T OlUlillion ..ml G-46 vi formed l Illin the ivrnaiant il concert- ml species : fractions following . for their ncs mutanlicd liver .ucd mice to convert nv, which lc II, exp. use in the mats was, than that p. Nos. 2, and lung marginal . observed tl animals *ru No. J). from four m in their nic metaSample A a <ts seven ic control 'it twice as use liver, to activity dimples C we which added CO- phosphate mtl mere ly) liver itt not the me), signiicvcrtants l 2 times, V 6, 9 or MUTAGENICITY OF VINYL CHLORIDE 48 h, respectively, us compared to assays con taining KCI and NADP* only (Fig. I). Addition of 5.9 U of alcohol dehydrogenase and 4/<mol of NAD' to either the fortified 9.000 'P or 100.000 <p liver supernatant did not increase the mutation rate (Fig. IV When the naDph generating system (NADP1 and glucosc-6- phosphatc) was omitted from the 9,000 liver supernatants, the number of His1 rcvcrianis was reduced to control levels (Table II, exp. Nos. 2-5 and 14-17 versus No. I). In another series of experiments, the subccllular localization of enzyme activities), which converts VCM into mutagenic metabolites, was examined. A hepatic TABLE II HUMAN, RAT AND MOUSE TISSUE*MEDIaTED MUTAGENICITY OF VCM IN S. TYMIMUfttUkl TA ISM E*p. Nf*. Speeks Phenrtharbiinne prclrcaimcnl Organ 1 9.000 * tup. cefaclors No. nr (Mil* - llivi*)1 rcvcrtanri/plale Relative acitmy i-- 2 3 Mouse 4 5 6 7 8 9 10 tl 12 13 14 IS Rat 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Human 31 sample 32 33 A B C D -- Yes Yes No No Yes Yes No No Yes Yes No No \cs Yes No No Yes Yes No No Yes Yes No No No None Liver Lung Kidney Liver Lung Kidney Liver 4* + -- + -- + -- + -- + -- + -- 4-- + -- 1 - + - + - 1 -- + -- + -- 4-- -1- 5332 (4) * 242 1 61 (3) * 129^88 (3) 150143 (4) 46i2 ` 67 1 62i 9 63115 55+ 7 90 | 8 93 !- 3 67111 80 + 21 203113 761 7 1831 6 47+18 66 |-2I 66 I 11 75 h 7 49 i 9 84+20 f6-t23 90+-I3 92+ 19 380119 9J 6 174141 99 4 185 1-20 234115 157-fc 4 115129 (100) 457 243 283 87 126 117 117 104 170 175 126 151 383 143 345 87 124 124 141 92 158 162 170 173 717 175 328 J87 349 441 266 217 ' Eaimaleiu If* JS inp of wet I iviue/piale. 1 Muiapcnieii) (procedure A). C h of eipoturc lo 20\ VCM in nir .11 37* C. Number nfipoiitancout niuiaiiom/pl.iic(hno) from each viiluc juhu.icicii. Mean suluci tmi ami tiuiMltee Afililt'ereni experiment* in parenthesis. each iiiili/niv, pnokiJ tissue* from five mite. Mean values *-ei* from ilircc mutagenicity -juaj-i for cepcrimcmi Nns. J lo 33, utilizing pooled miuei from five mice or three ralt or iriitividual human samples. 433 RSV 0016490 n \i< isct i rx ai.. microsomal fraction From PB-prctrcatcd mice increased the number of His reverttmts in the presence of an NADPH generating system (Tithlc III, exp. Nos. 4-5). The mutagenic response, however, was lower when compared to the 9.000 f> sui>crnatant or to the recombined microsomal and cytosol Fraction (Tables III, exp. Nos. 2. 3. 8 and 9). Liver cytosol did not increase the mutagenic response of VCM (Table III, exp. Nos. 6 and 7 and Fig. I). At various VCM concentrations and in all three bacterial strains (TA 1530. TA 1535 and G-46), a 15*40 % enhancement of the mutagenic response was observed when liver fractions (9.000 - /> supernatant) from PH-prctrcatcd mice were compared to untreated controls (Table I). in rats. PH pretreatment caused a "similar increase of the mutation rale (Table II, exp. Nos. 14 and 16). Chloroacctaldcliyde and chloroeihanol, two presumed metabolites, and chloroacctic acid, a urinary metabolite of VCM (Hefner ci /., 1974; Grigorcseu and Toba. 1966). were assayed in equimolar concentrations For their mutagenic action in TA 1530 strain (Table III). Chloroacetaldehyde and chloroacctic acid caused a Inch toxicity on the bacteria. Chloroeihanol. at the highest concentration utilized (I0S/unolcs/platc) increased the number of His' rcvcrianls 10 times over the spontaneous mutation rate (Table IV, exp. Nos. 26-28 w.vw.T No. I). A live-fold increase of the number of His rcvcriants over the spon taneous level was observed at 10.8 mmoles of the chlofiNicclaldehydc/plate(Table IV.cxp. No. 14). Chloroacctic acid was toxic at all concentrations, but did not cause a direct or tissue-mediated mutagenic response in TA 1530 strain (Table IV, exp. Nos. 2-10). DISCUSSION S. typhhnuriiun strains TA 1530, TA 1535 and G-46 were reverted by VCM to His prototrophy (Fig. I). which indicates that the mutagenic cITcct is induced by base-pair substitution. For this type of mutation, a covalent interaction of VCM or its mctabolilc(s) with the bacterial DNA is required. The inability ofTA 1538 strain to revert in the presence of VCM to His1 confirms that VCM or its mclaboiitc(s) do not act as frameshift mutagens. The highest mutagenic response was seen with TA 1530 strain, which is cxcisionrcpair-dcficicnt (mrU*) and a much lower response was noticed with G*46, which is mrB` (Ames </ ai.. 1973). During 48 h of exposure to 0.2-20% VCM in air, no cytotoxicity was noted for any of the four strains (Table I). Exposure of TA 1530 strain to 20% VCM in atr in the absence of any metabolic activation TAiu.n in HFrtXT OF VARIOUS SUHCLLl UI.AK TISSUE 1 RAOTIONS AND COI ACTORS ON Tin: MllAdlMC LI IIXT Ol VCM lisp. No. 1 2 3 4 5 6 7 8 9 Mouse l<%rr 1 fi.iclion None (KC!> 9.000 * x sup. Microsomal fraction 100,000 x sup. (cytosol) Microsomal fraction cytosol d:tof.>Cli>r$ -4 -- -- t+ -- No. of lllii' Uiso 4 1 * revcM.ynu/pl.nc 96 r.lS 310-18 128 J: 88 175 i 20 86 t 2 138 -r 12 90 16 501 i25 95 -10 Relume neiiviiy (100) 323 133 182 90 J44 94 522 100 ' T.<|<ut;ili'nl li' >K i-iik ol mu ii.mk i-1.ui-. ' Miii.iccniriiy ihv.im <pmci'dme A}: (< li of exposure m from i-.ii.li i.ilnr Mihiucicd. VC'M in nir nl .1?" C; number of -ipoiil.iiHiim muuiinnUpli'le (Iik0> * Mi-jn t.iluo J.*i> o' three rnui.ipcnicny aisaii. ejeh uiili/mp ponied iir iiiiuc from fixe phcnoh.-irbiicinc-prclreiitcd mice. 434 .sy re* wa 48 cat lik cn oh mi nu 9, co. I I I I I I 2 2 2 2 2 2 RSV 0016491  1535 and mnlropliy :enic effect For ihi$ n of VCM .1 DNA is in lo revert .firms that frameshift pome was . excisionncIi lower .li is trBfc \posurc to was noted VCM in activation jcit' ay '<10) ! 13 1.0 `0 >44 '14 : vi nt/plaie thup) MUTAGENICITY l>l VINYL CHLORIDE system caused a linear increasing mutagenic response, as a function of incubation time, which was 20 limes the spontaneous mutaiion rale at 48 h (Fig. I). This mutagenic c licet could he caused by a direct action of VCM or. more likely, by one of its enzymic (bacteria) or noncnzymic break-down products. A much higher mutagenic response was observed when a 9.000 'g liver supernatant from mice or rats was added to such an assay. The number of His- revertants increased, after 3, 6. 9, 12 or 48 h. by 4. 3, 2.5 or 2 limes when compared to the assays without a tissue fraction (Fig. I). These results strongly support an enzymic formation of VCM intermediates which are mutagenic for TA 1530 strain. To study the subcciluiur localization of these enzymes, various liver fractions from PB-pretreated animals were assayed. A microsomal fraction in the p csence of an NAI3PH generating system significantly increased the number of His' revertants over controls, where (he co-factors had been omitted (Table III). A 100.000liver supernatant, in the presence or absence of NADP', did not modify the mutagenic response. The recombined microsomal and cytosol fractions gave a response table tv MUTAGENIC EFFECT OF CHLOROACET1C ACIO. CHLOROACETALDEKYDE AND CHLOROETHANOL IN STRAIN TA 15)0 Eip. No. ^ Compound (oMolcifplatcl 9,000 tup. 1 cofacton No. of Hit4 revcrianit/plair % bacterial wrvivat 1 I None H- -- |44 (100) 2 Chloroacetic acid (1.1) + 3 -f- 4- + - 13 -h2 1 I36 16`:4 100 5 6 7 8. 9. 10 (10.8) (108) + + - + -- - .L 14 + 2 IJ-fck 82 0 <0.004 0 1 ChloroncclaldchyOc (1.1) 4 12 + 13 -- 4-- -- 16 I 3 23 4 6 3018 <0.004 14 15, 16 17, !8. 19 (10.8) (108) + * + -- 604-8 0 0 --<0.004 0 20 Chlorocthrmol (1.1) 4* 21 + 22 -- -- -- I44 I7l J8 + 6 100 23 00.8) + + 264-2 24 +-- 28 6 25 ---- 27*1 100 26 008) + + 1554:8 27 + -- 120*7 28 ---- 72*0 100 1 1.univalent in l`J mu of pnnled >ei liver iivmic from five ph.-noh.ihiinnt--pctrcaieiJ n.ice rcr plaie. Muusviticit) avvjvviprnccvJurc A), following incubation for 4(1 h at .17' C. Survival wii dclcrminctl rih l.fiy 10 1 Direel plutinn <cm in 2.1 ml n( inf. ajar. 4 M*an valuc$S(> nf mo mutagenicity nss.iyv hacuria/plite. 435 - 7 . .v 6 ? RSV 0016492 UAKTM'H liT AL. similar (o that observed with the original 9.000 -'g His1 revertants in TA 1530 strain (Table IV). supernatant (Table ill). Thc.se results strongly Rosenkranz ct al. (1974) also reported a weak imply that the microsomal mixed function direct mutagenic action of chlorocthnnol in the oxidase plays a role in VCM bioiransformation. same strain. Clilnrocihylcnc oxide caused a This is further supported by Use requirement of mutagenic effect in the TA 1530 strain and it was an NADPH generating system for the enzymic shown to he a strong alkylating agent (Malavcillc formation of mutagenic metabolites by liver <//., 1975). fractions (Table Hi. as well as by the increase of His* revertants, when mice and rats were preIrcatcd with PB. The addition of soluble liver proteins to a microsomal fraction significantly increased the mutagenic response (Table III). Cons:qucntly. a two-step activation mechanism, each being localized in a different cell compart ment, could be involved. On the other hand, the protective cfTccl of liver cytosol against lipid peroxidation (Kamutaki ct/., 1974) may prolong the viability of the microsomal enzymes and thus increase the yield of mutagenic VCM metabolites. Among the various tissues of mice and rats, only hepatic post mitochondrial supernatants were highly efficient in the presence of an NADPH generating system in converting VCM into metabolites mutagenic for TA 1530 strain. One human liver sample showed an activity twice as high ns that of rat or mouse liver in converting VCM into mutagenic metabolites. The three other samples showed a moderate activity (Table II). The extrapolation of the results obtained in our in vitro mutagenicity studies to the mechanism of tumour induction by VCM in vivo is difficult When the li^er-mcdiated mutagenic response to make at present. Although it becomes more was corrected' by the corresponding values evident that the majority of chemical carcinogens, obtained without any tissue fractions (Fig. I). after metabolic activation, show a mutagenic the resulting curve indicated a viability of the effect (Miller and Miller, 1971), it is not yet clear enzyme system involved in VCM biotransforma- which of the VCM mctabolitc(s) is(arc) respon lion up to 9 h after incubation. Although the VCM concentration was not rate-limiting, the number of His* revertants caused by enzymic formation sible for the mutagenic action and whether it(thcy) is(rc) identical with those responsible for the carcinogenicity of VCM. However, our did not increase further (Fig. I). studies have indicated that human, mouse and Our inability to find a mutagenic effect, when rat liver, which arc the major target organs for bacteria strains G-46 and TA 1530 were incubated with an initial concentration of 0.083 m of VCM in liquid suspension (Method B), may be due to the carcinogenicity of VCM in humans as well as in animals, efficiently convert this carcinogen into mutagenic metabolites. We have applied this mu the rapid loss of VCM in the liquid phase by diffusion into the atmosphere, as well as to the slowness of the enzyme-mediated formation of mutagenic mclahofilcs within the first hour of incubation. Chlorocthylcnc oxide, a possible reactive inter mediate, which may be formed from VCM by tagenicity test to a structural analogue of VCM. vinylidcnc chloride (1, l-dichloroclhylcnc), which is used in the production or plastic materials. Our results show that the mutagenic effect is higher than (hat observed with VCM. when the com pound is mctabolically activated by rat or mouse microsomal liver enzymes (unpublished data). the hepatic microsomal mixed-function oxidase, rearranges spontaneously to chloroacctaldchyde (Zief and Schramm. 1964). Thus, two conceivable metabolites, chloroacctaldchyde and chlorocthnnol. and a urinary excretion product, chloroacetic acid (Grigorcscu and Toba, 1966; Hefner ct til., 1974). were assayed for thetr mutagenic ACKNOWLEDGEMENTS This study was partially supported by the National Cancer Institute, USA, Contract NOICP-55630. The authors arc greatly indebted to Drs. G. action at equimolar concentrations. At the Della Porta. U. Veronesi and M. Boiocchi for concentration of 10.8 /unolcs/platc. ehloroacet- providing the samples of human tissues. The nldchydc and citloroacctic acid showed a strong excellent technical assistance of Mis. C. Gabet, toxicity in the TA 1530 strain. Only chloroclhanol. Miss A. M. Camus and Mr. A, Harbin is grate at a 40 him concentration (I OX /unolcs/platc). fully acknowledged. The authurs wish to thank caused a significant incicasc in the number of Miss A. Pickett for typing this manuscript. 436 l AmeS. Lit. test vati* Sri. Bartsin nun and Creep liver /. 0` Grigo Asp17, < HiatiCha liver Stai HErM1 Gei1 inhr. Ann I NTfH * hue WOfi RSV 0016493 f\ . , wf.lk m the :.*-d a ,l it was j U.lljVCille i .md rats, |;.in(swcre SADPH i'M into -rain. One \ twice as '(inverting hrcc other i Table II). htained in ncchanism is difficult `incs more irdnogens, mutagenic >t yet dear ip) respon.1 whciher nnsible for ever, our nnusc and organs Tor as well as igcn into this mu* of VCM, >c), which .-rials. Our i is higher i the com* r or mouse >t data). d by the nact NOI- 0 Drs. G. locchi for *ucs. The C. Gabet, 'it is grate* 1 to thank upt. MUTAUCNICITY <W VINYL CMLOH(n MUTAGI-Nirm- DU CHLORURK DE VINYLL* DANS DES SOUCHES Dli 5. TYPHIMURIUM EN PRESENCE DE FOIE HUMAIN, DE RAT OU DE SOURIS L'exposition an chlorttre de rinyle ties sonehes TA 1530, TA 1535 et G-46 tic S. lyphimurium fait augmenter ie notnbre tic rcvcrtattls fits' lluiitc tic Petri respeetirement tie 16, 12 et 5 fois par rapport att taux tie mutation spontanee. Apres six hettres tTcxposition tic la souehe TA 1530 att chlorttre tie rinyle, Teffet mutagenc est multip/ie par 7, 4 et 5, forsque Ton a/oute lafraction postmitoeitondriale tiefoie hiimain, tie rat ou tie sottris et les eofaetcurs enzymatii/ues reqais. L'enzyme imp/iquee thus la transformation tlu chlorttre tie rinyle ett mutagene requiert on systeme generoteur tic NA DPH, et se sitae thus la fraction mierosontale. Le surnageant it 9,000 g presente tine activite vnzyntatique trots fois plus importante que eetlc ties microsomes, tantlis tfttc le cytosol on I'olrool tleshytlrogenase'n'afieete pas la mufapettieite. Le prciraitcinrnt tie rats et tie souris att phenolxtrbital augmente tie IS it 40% Veffet tiittingene par comparaison avee les animaux temoins. Les activites nuttughnes relatives tlu chlorttre tie rinyle, exprimecs cn poureentage (foie tic sottris -- 100%), determiners par les fractions tissulaires jrbtennes it 9,000 g et revefees avee la souehe TA 1530 so/it: foie tie rat, 80%; rein tic sottris et de rat, 20 et I6%\ pontoon dc sottris et tie rat, <7%; foie htnnain (quatre biopsies), 170, 64, 70 et 46%. La chloroaeetaldehyde et I'acitle chloroacetiquc, an metabolite du cMorure de rinyle excrete dans l'urine, se rtfrelent ires toxiques, tandis que le chlorocthanol est faiblement mutagene pour la souehe TA IS30. REFERENCES Ames, B. N., Dukston. W. E., Yamasaki, E.. and Lee. F. D., Carcinogens are mutagens: a simple test system combining liver homogenates for acti vation and bacteria for detection. Proc. not. Acad, Set. (IVash.), 8. 2281-2285 (1973). Bartsch. H., Malavmlle. 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