Document zdZM0OjBGNDGG2JGDJJvgYgLz

Vol.4 No.II pp.!4*3- 14M, 19#3 Evidence of chloroethylene oxide being the reactive metabolite of vinyl chloride towards DNA: comparative studies with 2,2'-dichlorodiethylether LM.Gwteav, RJ.Lteb>, J.GJte and H-MJtoh Pharmnkoiogisches Inslicuc, UmveniMU Mainz, Mainz and Institut for Arbeiuphysiologu, UniversitSt Dortmund, Dortmund, FRG. (Received on 26 July 1983; accepted on 6 September 1983) Abstract The role* of cMoroethyteae oxide (CEO) and cUoroocetiMehyde (CAA) in atdMgeafcity of vinyl eWorld# (VC) bare been studied by eoapariug bkdogfcte effect* of VC expoenre wtth tboec of 2 -dkMorodtotfeykfber (bte(cUoroMtiyl)Mber, BCEE) as a mtaboic proenrsor of CAA. Btologfcul endpninti irutlgsiiil rrira rmrelnut pmtten hteftteg sudeleadd (RNA and DNA) attcylndon end tee potency of tee two cbemleate in indaee pranrcple*tlc AlPtee rteffckut fad in ral liver. After ntpoenra of rite to [l-^BCEE, BCEE derived radioactivity was bound to Iter prottene. Aniyte* of hydrolysate* of Uv*r RNA and DNA gave ao Indkntton for the fnrmartnn of rite* 7-N-<2-oxoetfey0fMHine, lj**iteeuoedeiitee or 3^f*.itb*nnrytmdne ratednm wdbln tfeo aodeie add*. Alter appBcadoa of VC, BCEE or chloroethanol <(CE), ateo a proenrsor of CAA) to young rate, only animate wpaeed to VC devdoped preueoptaede bepatoedlular ATP--a iklliiint fod. From these inverfnadons It le coadaded. that CEO (which is not formed darinf metab olism of BCEE and CE). not CAA, b the ultimate cardnogeaic prlndpte in VC cardnoteaidty. Introduction It is generally accepted that vinyl chloride (VO*, a proven liver carcinogen in man and laboratory animals (1), is metabolized by microsomal mooooxygcnaaes to the bifuactlonal alkylating agents chloroethylene oxide (CEO) and chloroacetaldcfaydc (CAA) (2-6). Experiments on the chang*> and biologicai behaviour of CEO and CAA (3,4) have suggested that CEO is the ultimate carcinogenic metabolite of VC. la other experiments CEO, in contrast to CAA, induced local tumors after repeated s.c. ad ministration and skin turnon in a classical initiationpromotion experiment in mice (5). Model studies with both rat liver microsotncs anti a reconstituted cytochrome P-430 system as metabolizing systems for VC and vinyl bromide (4) led to the conclusion, chat the haloethylene oxides wen the major alkylating agents bound to DNA whereas the 2-haloacetaldehydes were the major alkylating agents bound to pro tein. In che intact animal the major site of metabolic activation of VC is the endoplasmatic reticulum of the hepatocyte (6) from where according to Guengerich at at, (4) CEO should be To whom rcprini requests should be sent at: Institut for Arbriuphydoioste, an der UhlveniUR Dortmuod, Ardeystraase *7, D-4600 Dortmund I. FRG. Mbbteriutera: VC. vinyl chloride: CEO. chtoroethytane oxide: CAA. chloroacetaidchyde; BCEE. 2.2'-dichtorodieihyieUier; CE, chloroethaaoi. T ERL Press Ltd., Oxford. England. released to alkylate DNA whereas CAA generated by intra molecular rearrangement of CEO should be responsible for alkylation of liver proteins. A direct validation of this - hypothesis for conditions in vivo is not possible because of the spontaneous rearrangement of CEO into CAA. Due to its direct chemical reactivity, CAA when given to experimental by either route of application would most likely not reach the hepatic targets (e.g., proteins of the endoplasmatic reticulum) as it is when it is formed in the liver as a metabolic intermediate. Indirect evidence can therefore only be obtain ed by using a precursor of CAA other than CEO and com paring iu alkylating and oncogenic potencies with those of VC. 2^'-Dfchiocodiethytether (*bis(chloroethyOether', BCEE) is such a precursor which is transformed to CAA. The avail able evidence shows (7,8), that BCEE is nriitoaai in ccposition which leads to fonnation of equimolar amounts of CAA and 2-chloroechanol (CE). CE is subsequently com pletely mrrahoHrrrl to CAA ((9), see Figure 1). Oxidation to chloroacedc add and conjugation with glutathione follow, and after further modifications at the glutathione moiety thiodiglycoilie add is formed. Quantitatively > 100ft of the administered doee of BCEE is excreted as thiodigtycoflic add in the urine of rats after application of BCEE (dose 100ft, (8)). The studies described here were designed to assess the roles of CEO and CAA in carcinogenicity of VC by compar ing biological effects of VC exposure with those of BCEE as a metabolic precursor of CAA. Bktiogicai endpoints investi gated were covalent protein binding, audric add (RNA and DNA) alkylation and the potency of the two chemical* to in- duce preneopiuac ATPasetiefideot fod In rat fiver. In addi tion, the potency of CE (also a precursor of CAA) for induc tion of those fod was investigated. Mteariale and Methods Anenetr MateWtaar mca (200 s> Ivanovas, Make, FRG) woe used for the studia on mains bfcding, ante sad tank Whtar m (sun* bnedar) for the lev --r ae ATPisnkfidm fod. They ttewl e standard pete da (Alnoarin 1330. Lin. FRG) ead dteidng wane ad Ubitw*. L/OTBCflB M----- *I-W----QaQ- -IP-f-B-- C*-D-ia- ((-"Cm* DfcfaiorocSathyfatha (sp. ag. l.C mCt/nraol) was eyathariaed by NEN Onmcak (Boston, MA). NotwadkecriveU'-dlchlorodiwiiylether (chonkai purity 99*) ns obtained fans Maek-Sdtudiard (Hohenbruan. FRG), vfnyi chloride (chsmfcal purity 99*%) from Unde (Uaxasefalasshem. FRG). chloraetfcaaol (ctuoical purity 99*k> from EGA-Cbonic (Stknheim. FRO), hydroxyapatite (DNA-fride) from Bio-Rad Laboratories (Mttnehen. FRG), audanddas, radaobaan, i.FAahcaoadenesfric, 3,N-ethenocytidin*. rc. from SIGMA (Mfincbea, FRG). 7-Nm-OMMdiyh|uaaiaai l>N*-edjenodeo*yadancine and 3,N<etheoodeodtycytidine were symhedad u prerioudy described (13). Cbwknr binding cgwfeimts Exposureof 3 rats to [ l-wrjdfchlMOdkthytefterwas canted out in a dosed afl-iiasi mposuie syoam (18). To tatilkat* evaporation BCEE was applied to a niter paper inside the exposure chamber. The destine of Bt'Rft vapor in the inhalation chamber was measured by gas chromaxocnphy. After IS h exposura >93* of the adndnawred BCEE was talesn up by the animals. Up take wai 0.33 mCl/aninieL Twenty-four hours after Stan of etposure the animals wae removed from che exposure chamber and sacrificed. Liver, kidney, spleen, lung, small intestine and muscle tissues were removed end 1483 AP00010020  UM.GtaMr<rL acMoroeatnyiattwr CMj.CHCl iAyi ehiana* pron-qlfcjrlqtiqn Jf<nary wtaartifti MOOC-CH,'S-CH,-COOH thiaaiiycMlieae HOCH,-CK,-S-CH,-CH-COt3H ItH-CO-CH, M-acatyUS^3-yiyHy<lcyHa^ia Hg. 1. COttpandvt mcotatim of VC (I) and dkhlorodlabyiaiwr (7--9. CEO b aoa formed dwfaf maeballaa of dkbtaodWtyiathv. mnd at -B*C umS prnnwriri, UianutlMa MDdnt of redtoanlw maatoottwr of I1-"C]BC8I dra* fimwini was laemlual mookUds to 8ak # at (19. SNA wn bel--d from the Iher impta by a aodllhd phaool agractica (II). XMAampta of 3 Swan wqte eombfaad tad aubjaend to bydralrd*. Liver OKA wee prepared from pwtiM audei. isolated ooeeidai to L mao r oL (3&T1m DMA wateannad star tyiisof tbe oudd ad puriM by ctanmtopipay oo IbdcayiaidH aa daaoribad by Maado a at (31X Ator tUa purifioatan dn DNA <iwtain dut was diatyad at 4*C Cor 40 It apaiBK aq. disc. and sufauqiMxiy hafihltod DNA wnOw of 3 Sm wn canbaoad and hydiolyad aa rfrriMrl 6tioq. . Earynfc hydrotyrii of 1NA and DNA janptai commies 2 rag aucWc aeid ana parfomad aa pmtowaty daacribad (13). Tba nmUnc bydrolymai wen maioai by h.p^e. oo aa Andnoi Ad cofaum (10 x 330 one dutioa i wm* 45*C, 3 ad/mis dudea buffar A.03M aanoBnai fornuafOLOB M famie add and adjiaad to pH 4.33 wtn to M fbnaic add: data butte S, 0.4 M awontao fanmt/OM M farads add and ad* jutted to pH 7 abb ceoe. awmowk. Purio* sndta: 0--30 nfci (00b Bt, 30-13ado (Uapr iacnst 0-400S B), IS--lOOndn (Uosr Inenaat 40--1004% B), 100-120 ado (1004% B). Frocdooaaf3 fldadtwaeeaBatataadceundfarradioacbvfeyiitlO tal of AouaaoM(NENCbankdi). lMtaiom gfATTimiM/Um/xt Oreopa of4-10 noloor farad* Wtearran (tea Table 0) were mated far 3 weds, xardafScaapofT dayi.S)90wMtoVC(2CiOOp.p.sL.I b/day, 5 dtyi/week) a cawiad our anlrearty dtecrtbtd (16). Eapoaura coocomodoo waa conroOad by *le. BCEB waa aapaodad by sooindoo la commardal coffin ana and doaad orally wfth a tnfcroliur pipme. Two groupa of admals roedwad 30 ng or 23 mi BCEE/1t body weiibu napecdwty (doife doaa par day; 3 daya/waek). C2 waa appttad in tba same raaaaar in daily does of 30 rag or 25 ns/kf bodywdghtCoaroiacInials receivedthe oeam only. Afltr aa interval of 10 works Ahoui any manat the aasnait wart satriflead and cryoatat sodom of the ttwn wot prepared. Th section! <wt rained fer ATPue ascoidnc us Wackstda tt at (22). Tba irtta of ATP*sdeficient fad were quantitated in 5 cryoatat sections pet iivn using the Zds lattice piatrU*lOQ/23. Roonltt Covaient binding to tissue proteins After exposure of raw to [l-l*C]BCE, a considerable por* lion of BCEH derived radioactivity was bound to liver pro teins. Smaller amounts of irreversibly protein bound met- 1484 TbMa L lunnuible tawin lAaJtua in dlffbiein tm dnun aftn npoaunof d inimala to |**CIBCEB. in wpariacn wd ilafa nteiinert after analopow (UQVC erpn--w.(upakepnaai--fc 0.11 aG pqvc (tai9). Tbftw % of nOiomaMrr inwantbiy bound pw f time pratda (n 4) 9CEE vc Liter Luae Spiral KUaey SSNUUaMRK Muaeto 0J2 a 0.016 O.0T * 0415 0.06 a 0414 0.17 0.04 0.12 a 0.017 041 a 0.003 042 a 0.064 0.01 a 0411 0.08 a 0423 0.11 a 0411 a0 a 0423 041 a 0410 B munflerof umalr vara down are aneta nSD. abodtes were found in kidney, small intestine, lung and spleen (see Table I). Irreversible protein binding extends of reactive metabolites of [l-t4C]BCEE in differait tissues of rats are shown in Table I, given in percent of metabolized compound per g tissue. Covaient binding to DNA and RNA A test chromatogram (h.p.l.c. on Aminex-A6) of RNA and DNA nucleosides and presumed nucleic acid alkylation pro ducts of VC is demonstrated in Figure 2 (top). When liver DNA from rats exposed to I^CJVC (uptake per rat: 0.25 mCi) and isolated as already described (13) was hydrolyzed to the nucleosides and then separated by h.p.l.c. chromatography on Aminex-A6 (2 mg DNA/sample) a radioactive pmk was eluted representing the main alkylation product T-N-tt-axoetftyQguanine ((13), Figure 2, bottom). Analysis of hydrolysates of rat liver DNA isoiaied after ex posure to [MQBCEE (2 mg DNA/samplet Figure 3) showed no radioactivity where 7-N-<2-oxoethyi)guanine was expected to be eluted (26 min). Also, no significant radioactivity was eluted at 60 min and at 79 min, the retention times of AP0001002I WHfMKI ^*HND n*W- -i V- W - V-^ m-1M- MM. - 01 .J-i.yAi QOTO (HVtUltM Fig. 2. (*>Took tast ebra*o*pta (h.pJ.c.) ofmriowaudoodiiB mi baa of RNA art ONA or Aan-A6, tcfJwr with mvkcr subnoca of puWfchrt audde U aJkylMUB nmiinm oI VC (13,15). (*) Portomi UK ehroMUPQWm (dtrioa of rrfattivky) of hyrtmJy o12 me Ihm ONA from no ccpowd to (`CJVC (uptake 0.25 atO/wBiiaf). pm/3n>4 ON* Fig. 3. CfaroMnfim(durian of ndiaoGdvtty) of a hyAoljaiat of2 mg livar DNA from ma spend ["CWkMoreiiaUiyiah* (uptake 0.25 mCl/ubuaO. 1 ,N*-ethoiodeoxycytidine and 3,N4-ethenodcoxyadenosine (15). The only significant radiowsivity peak wai eluted with the void volume and represented moat probably some protein contamination of the ONA. Analysis of hydrolysates of rat liver RNA isolated after exposure to ("CIBCEE (Figure 4) also showed a main radioactivity peak attributable to con* laminating protein. Quantitative differences in protein con taminations between RNA and DNA are probably due to the different isolation procedures (modified phenol extraction procedure for RNA and hydroxyapatite purification of DNA). Two minor peaks of radioactivity in the RNA hydro lysates were eluted at 14 min and 36,5 min (guanosinc and adenosine), apparently due to incorporation of radioactive Irtte IL fuAwtian of ATPm krtrdn fad kt trt Bw of ns mpamd to vc, ifMr dome* of Bektacdddiyddn or driotortanol (at - sale, l fwddi ATPam-drtdmc ted m ri an of 4 mood* (* of Owr in) vc (Monteeof aubofcm. 1 k p tme/d) 0.2J * 029 0M * 0.34 Nuabm of *"> 4a 7f DkkknSsbiMw (50 Bf/kp No Bonm * fod arm SB 5f CUnomhmd (50 marts) No damn in ted aim 6m 8f la oomrol aainab (9 au * h ^(oombs old) Um ted nudd not atBHd oxos * 0.002*. Cl-fngnaaus into these natural nucleosides. No significant radioactivity could be demoted it dutioa times (47.5 mm and 60 min) where the two pnhHahed alkylation products of VC in RNA, 3,N<ethenocytidne and l.N'-etbenoadenosine (11.12) appearad from the cotusm. Induction ofATPw*d*fid*Mfod The potency of the CEO and CAA precursor VC and of the CAA precursors BCEE and CE to induce preaecplastic ATFise deficient hepetie fed was investigated. During the period most sensitive for fed induction (day 7 to day 28 (23)) newborn rats were exposed to doses of VC, BCEE or CE which generated comparable quantimes of CAA. When BCEE was given at doses of 50 mg/kg/day (350 pmol/kg'day) or 25 mg/kg/day (175 ^moi/kg/day), which corresponded to the endogenous CAA production of daily 8 h exposures to 250 p.p.m. or 125 p.p.m. VC, respec tively (24), no ATPase-deflden fed were observed (Table ID. Application of CE at doses of 50 mg/kg/day or 25 mg/ kg/day, theoretically equivalent to the endogenous CAA production at 8 h exposures to 250 p.p.m. or 125 p.pjn. VC, did also not induce ATPase-defieient fed. When rats are exposed to VC (8 h/day) a large number of preneoplastic ATPase-defident fod is induced, as already reported (23). Dfamstion The activation of VC (25) with subsequent covalent bind ing of reactive VC metabolites to macromolecules (25) has 14*5 AP000t0022 r L-M-Giriamr tt aL raised the question e* to which of the reactive metabolites, CEO or its rearrangement product CAA, were responsible for the obvious carctoogctuc effect of VC. Comparison of die chemical and biological activities of CEO with those of CAA (3) has led to the assumption of CEO as the ultimate carcinogenic metabolite of VC. How ever, most of the investigations supporting this view were done in vitro. In addition, in different test systems also CAA displayed some mutagenic activity (3). Comparative investigations in vitro on the role of 2-halo ethylene oxides and 2-haJoacctaJdehydes hi irreversible bind* mg of VC metabolites to protein and DNA* have been carried out by Guengerich tt at. (4). These authors concluded that 2-haloethytene oxides were the major alkylating agents responsible for DNA binding whereas the 2-haioncctaklehydes were major protein alkylating agents. This view is now confirmed by our present results on covalent binding of endogenously produced CAA to proteins, but not to nucleic adds of the liver (aaannis receiving (**C]BCEE). Application of comparable amounts of radioactive p*ClBCEE and (UC]VC resulted in about the same extents of covalonly protrin-bound radioactivity, mostly confined to proteins of die liver. Analysis of hydrolysates of liver DNA isolated after ex posure of the aT*""al* to [KCjBCEE gave no indication for the formation of ether 7-N-(2-oxocthy0gvianinc, l,N*-etbenodeoxyadcDosiBf or 3^<echenodecycyt}dine> DNA adducts which have been discussed (13 -- 15) to be responsible for the biological effects of VC. Analysis of hydrolysates of liver RNA belated after exposure of the animal* to ("CIBCEE showed one major radioactivity peak with the void volume, which must be attributed to protein contaminants; no specific alkylation product could be detected. This must lead to the conclusion that alkylation products observed after VC exposure in RNA (3,N*-ethenocytidine, lN*-thenoadenosiiie, 7-N-{2-oxothyl> guanoane) are fanned exctustvely of CEO. The assumption that CEO is the ultimately carcinogenic metabolite of VC in vivo is further supported by the present results on formation of hepatoedhUar ATPase-defldent foci after application of VC BCEB or CE to young rats: only exposed to VC developed pteneoptoric hepatocellu lar ATPase-defitiem fod. ATPase-deficiem fod in rats have been viewed as an **> for the oncogenic effect of various hepatocardnogsos (27), including VC (16) and other haloethyteneg (17). Lack of potency of BCEE and CE to in duce such fod is a further proof that CEO, in contrast to CAA, is responsible for ihejaidnogenic effects of VC. In ac cordance with our results a recent long-term toxicity study in rats with BCEE (28) revealed no carcinogenicity of this com pound. Acknowledgements The fbtaadri support of the `Deutsche Fonchunssgemenmschaft', pant No. Bo 491/M, is fiattfUUy acknowledged. References 1. International Agency for Reseudi on Cancer (1979). Vinyl chloride, polyvinyl chloride and vinyl chloride-vinyl acetate copolymers, in IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, VoL It. IARC. Lyon. pp. 377-138. 2. Lafc.RJ. (1912). Spedflc covaknr binding and toxicity of aliphatic hatogenared xettoUocio. Ret. Drug Mttob. Dmg Interactions, 3, l-t|. 3. Baroch.H., Malaveille,C.. Barbin.A. anti FlancM.G. (1979). Mutaeenic and alkylating metabolite of halo-ethylene, tWorebutadienes and diehlot^ 1486 butenes prockjcxd by rartria or homan live- deuea, Arch. Toxicol., 41, 249-277. 4. Cu*nerieh,F.P., Mmen.P.S.. Stott.W.T.. FosuT.JL and Watanabe, P.G. (1911). Rotes of 2-haloeftyteaa aside and 2-halmmildehydo derived from viay bromide and vinyl chloride ia irrevenftk binding to proton and SNA. Cancer Has., 41. 4391-4391. 5. Zajdria.P.. CroityA. BarMnA.. MalavetSa.^-. TomatbX. and Bart sch.H. (1990k. Cardan--tidty of chloroetiryime oxide, aa utemw reactive metaboite of vinyl chloride, and btdcUorMMChyOtther after subcutaneous administration and in attation-promorion ctperimne in ntice, Carer Re.. 49. 352-336. 6. Otteswaider,H. and Bok,H.M. (1990). Metabolic aedvatien of vinyl chloride and vinyl bromide by Inhered hepuocyta and hepeoc celts. J. Environ. Pathol ToxkoL, 4, 411-417. 7. Un**,RJ>., Kaylor.W.H., Pyie.M. and TardiiT,R.Q. (1979), ThfedigtycoUc arid: a major numboSte of bid-2-dik>reediyl)eher. TccocoL Appl PhomonL, 47, 23-34. S. MUterA and NorpochJC (1979), Identification of S<carboxyni((hylVL<yttein and thioeBgiyeotic arid, urinary metabolite of ^-bitichioroetfayDethtt' in the rax. Cancer leu. 7, 299-303. 9. Grunow.W. and Altmaan. HJ. (1992), TotteotiMrics of cboteoethanoi fat the rat after tingle oral adntiniaaaiion. Arch. Toabcol.. 49, 275-2S4. 20. Safc,H.M. sad TUsarJ.O. (1977), laewniNf tending of chlorinated etbyteae to macroraoteaites, /. Environ. Htolih Penpeet., 21,107-112. 11. Uib.lU. and Bok.KM. (1977), Aikyiadoa of RNA by veyl chloride metibog-- tn thro and is vtvot formation of l,N*iMOoadaootine. Toxicoiotr, s. isj-199. 12. Latb,RJ. and Bok.RM. (1971). Formarion of 3J4VdMMcyt)diae tnotettea in RNA by vinyl chloride i--ahotima in vitro and is vivo. Arch. ToxkoL 39,233-240. 13. LaftJU.. Gwta--.L-M. and BeitHJri. (19SD. DNA aUeyterioB by vtayi cfcknWt luataboi--r ethenodoriveovea or 7-aOtyinriOQ of guanine? Qwru-Bbl InmocOom, 37, 319-231. 14. SriMnrA, Van dar l.aitan.CJ.. Gwtiu--J-M., LaibJLJ. and SanelouP. (19SI), Modification of deoxyguanociae by chtofoethytene oxide, CorunOfSiWJH 2, S71-677. 15. GnauT. and HarwiyJXE. (1978), Imeractiona of vinyl chloride with re liver DNA M vivo, Ornn.-Bk>i. Immersions, 22. 211-224. 16. LaftJU., StOeUa.C.. BofcJ4Jri. and Kuaa,W. (1979), Vinyl chionte and triddoroediytenet conparirion of alkylatingefface ofmetabedtw and in duction of pcenaoplTfe enzyme rkfleteade in ni Ever, J. Conor Rm. CBn. Oncol, 94, 139-147. (7. faih,R..f. and FtieerJ.Q. (1992), Reactive nwaboftet anti caerino--riricy of bale--iatad etitjdaaei, Btochem. Phsemacoi., 31. 1-4. IS. BnkJfJ4.. KappoUL, BuchteA. and Bofe.W. (1976). Dbporitkm of [U-^Qvinyi chloride in the rat. Arch. Ttakol, X, 133-162. 19. BokJUd., KappuaJL, XauftnannJL, AppeUCJB., Bach--A. and Bok,W. (1976), MttaboS-- of ["Qvinyl chloride in vitro and Si vivo, in IAPC SOenti/k hMkmkm. No. 13.13M63. 20. LanuJ4X. Abakumova.O.Y., KucencoN.G., CorbecfaevaJ.A, Kuku*ldna.G.V. and SerebryasyiA-M. (1974). DtiferaKdagradadanm--of sOcyiated RNA, praodnand Dpkb in oonnaland tumor oeOi, GtiKerAe^ 34, 1534-1341. 21. Mriaahe.w.. Goktettin.OA. a-- HalLMJL (1974). Rapid isolation of mouse RNA from ceBi ia tissue culture, Anal Skrchm., SI, 8241. 22. Wadunjri.. MriseLB. and NtederiedxA. (I960), Histochemkal damensoatton of mhoe--ndrial adenosine trtphosphaas# with the tod adenosine triphosphatase teriuuqua. J. Hbtodtem. Cytochem., $, 387-388. 23. LaftAJ. and Boit,HJd. (1982), Induction of bepaioceHuiar micleotide-3'-triphoaphatase ATTase)-defictem fod by vinyl chloride (VO. Proc. Am. Assoc. Cancer Res., 23, Nr. 23t, 59. 24. FH--.J.G. and BeiuH.M. (1981). twhaia^ phaifnacokinatics bated on gas uptake studio. L Improveraen ofidsetic models. Arch. Taxxoi.,-*7, 279-292. 25. BaritiaA.. Bresfi,H., CroisyA-. Jacquignow.P.. Malavilte.C.. Vfon--aoe,iL and Bartschjf. (1973), Liver mlcrosoine-mediated formation of aikylatins acenta horn vinyl bromide and vinyl chloride, Blochsm. Biophys. Ret, Commun., 67, 596403. 26. KappuaJi., Bok.H.M., BuchtcrA and Boit.W. (1975). Rat liver atiaosooMi catalyse covalent Undine of ("Clvinyl chloride to macromoteeute, Harare, 297, 134,135. 27. EmmeiO(.P. and SchenrX (1990). The dm relevant cefl state in me liver catrinogenerit: a quantitative appnm.li. Blochsm. Biophvs. Acte, 60S. 247-304. 28. Wessburter.E.K., UBand.B.M., Natn.J., Gan.J.5, and Weisbutger. J.H. (1961), CarctnofKudty tests of certain environmental and industrial chemicals, J. Soil Cancer inst, 67, 7548. AP00010023