Document oMmnmkKLn02O45Xqm3RaQG6n7

ToncoL 41 159-169 (1979) TOXICOLOGY C Springer- Verlag 1979 Original Investigations Molecular Mechanism of 1,1-Dichloroethylene Toxicity: Excreted Metabolites Reveal Different Pathways 0f Reactive Intermediates p. Reichert, H. W. Werner, M. Mettler, and D. Henschler litfdrui fur Pharmakoiogi* und Toxikolofie. Univenitit Wurzburg. y,ribacber Landstrasie 9. D-8700 Wurzburg. Federal Republic of Germany Abftract- The excretion and biotransformation of [,4C] 1,1-dichloroethylene (vinyiidene chloride, VDC) after administration of a single oral dose has been investigated in female rats. Seventy-two hours after a dose of 0.5, 5.0, and 50.0 mg/kg, 1.26, 9.70, 16.47%. respectively, are exhaled as unchanged VDC, and 13.64. 11.35,6.13% as uCOr The main pathway of etimination is through renal excretion with 43.55, 53.88. 42.11% of the administered radioactivity. Through the biliary system, 15.74, 14.54, 7.65% of the activity are ehminated. The isolation of the main metabolites of VDC from 24 h urine is accom plished through the combined application of solvent extraction, ion exchange chromatography and thin layer chromatography. Then gas chromatography and mass spectrometry are used for their identification. Three metabolites have been identified: thiodiglycolic acid, N-aeetyl-S-(2-carboxymethyI)cysteine and methyl* thio-acetylaminoethanol. In addition, three smaller unidentified radioactive peaks have been found. Thiodiglycolic acid is the main metabolite in VDC metabolism. The simultaneous formation of an ethanolamine- and a cysteineconjugation product points to different reaction pathways of the postulated in termediate reactive epoxide; ethanolamine probably originates from membrane lipids, which react with VDC-epoxide and/or its derivatives. This pathway could explain, in part, the parenchyma damaging effect of VDC. Key words: 1,1-Dichloroethylene (Vinylidene chloride) -- Pharmacokinetics -- Biotransformation -- Mercapturic acid -- Molecular toxicity. Introduction 1,1-Dichloroethylene (vinylidene chloride, VDC) has been employed for decades as i monoroere in the manufacture of plastic materials. Not until 1977 was the carci nogenic effect of this substance demonstrated (Viola and Caputo, 1977). The initial suspicion of such a toxic property has been expressed with the recognition of the nructurally closely related vinyl chloride as a carcinogen in humans (Creech and Johnson, 1974; Lee and Harry, 1974) and animals (Mahoni and Lefemine, 1975). SL 066269 0340-5761/79/0042/0159/S 02.20 wo Since dm, numerous reports hive appeared concerning the possible CMci*>len,e potential of all chi mated ethytenes. In bacterial ten nans, after metabolic activation, a mutagenic effect has w. proven for vinyl chlo .de, VDC and trichloroethylene. However, tetrachloroethvi and 1,2-diebloiroethylenes (cis and trans) are not mutagenic (Baruch a ^ jn?* Oreini a aL, 1975). Previously unknown reactive metabolites are responsible'fTM. l mutagenic and carcinogenic effect of VDC (Bonse a al., 1975). The rate of me.ty' oKsm of this substance is dose-dependent and saturable, as shown in experimen with isolated perfused rat livers (Reichert and Henschler, 1978) and whole uhm !* (Jones and Hathway, 1978). However, it is suppressed by inhibitors of microsom 1 mixed-function-oxidases and through a lowered glutathione concentration in ^ liver (Reichert and Henschler. 1978; Reichert a aL, 1978). Studies concerning the fate of VDC in rats have recently been reported (j0n and Hathway, 1978: McKenna a al.. 1978). Although we largely confirm the result of McKenna a al (1978) with regard to pharmacokinetic data obtained after orj application (Reichert and Werner, 1978), differences continue to exist in the qualita tive and quantitative characterization of the metabolites appearing in the wine McKenna a aL (1978) have isolated four main metabolites from ret urine, two 0f which they have identified: thiodiglycolic acid and the mercapturie acid N-acety|-s. (2-hydroxyethyl)cyneine. Jones and Hathway (1978) have found two main meubo- lites: besides thiodiglycolic acid, they postulate a cyclic product as an mn metabolite. In addition, they have shown ehloroacetic add, ditfaioglycoiic tod and thioglycolic add in tester concentrations. With regard to these discrepancies, further studies concerning the metabolite # VDC appear to be necessary. In this paper, we report on the metf.boiisre of VDc after oral administration in rats, which together with the discovery of previously undescribed metabolites, expands knowledge of the behavior of this substance tn organisms and demonstrates new possibilities of reaction mechamsmfs). \ crG were u**d m ll)^P ^'%la__scmboiamc J,ou*h the case- St , were collected at 1**C1 VDC L*,sbolism cage thro ,,-oved with Dnentt Jpi et al. (19'6). *< f(?0tsined 50 ml toluer -h* remaining two trt fw "c: wert Samples from the ,,ucn medium (C. Rot Rested in 20% KOH Meh vial was measur SiparaiiO* of Urinary wTextracted with me The extract was evep. elution was analysed ,0.25 mm layers of siiic ,40 : 15 : 15. v/v/v). C b,vaknt sulfur eompot g<fthold radioehroman methanol (50 ml). Thes ,11 evaporated drvr 200 x 6 mm column ehiate was collects: UNamSMnlasla Methods Chtmkab. U-[`*C) VDC was jwrehaaed from New England Nuclear Carp, end had a specific ti dioactmry of 0.475 mCi/mmok. Its radiochemical purity exretrtrri 91%,-at shown by radwfis-curo matosraphy- The **C-labeled VDC was sohibtiired in tricapriline. Thiodiglycolic acid was purchased from E. Merck. Darmstadt. FRG. S42<arboxymeth>IVi-oi Mine from EGA-Chemie, Stambmm. FRG. N-acetyl-S-(2<arboxymethyl)-t--cysteine u prepaiel tn acetylation of $-(2-carborymcthylH-cysttmt with acetic anhydride according to Ozawa (19631. In identity was confirmed by GC/MS. Methylthio-acetylaminoethanol (MAAE) was prepared as nwrcaptoacetic acid (EGA-Chamic. Steinheim. FRG) (3 mmol 276.3 mg) was methylated with tr ethereal rotation of diazomethane. The reaction mixture was then treated with 2-imtnoeiiuro (3 mmol 183 ms) m a water bath (80* C) far 20 mis. The reaction product was chanctenzsd tn GC/MS. Further darivatitatiani ofMAAE were achieved by reaction with propionyl chloride end tmt trifluoroacetic anhydride: 50 ul of Ay MAAE was reacted with an excess of propionyt chionde e i water bnih at 80* C far 13 mm and subsequently evaporated to drynesj in a stream of dry tenotet This syntbeeu in analogy to a similar reaction between coiamine and propionyl chloride, dmenbrt by Jandan at aL (1972). Triflanroaeetyiarion was perforated by aetabSshad methods (Kaoe' e L 1974). For terifleassan of carboaylc adds, an excess of freshly prepared n-butanol-3N HO ammd to the --Rutyiaboo was than petformed in a water both (80* C/15 min) as demnM * Kata* at aL (1974). SL 066^70 Otttetion ai>~ ' layer plates and fractior dart of Kaiacr at al (19 with a Vartan 2700 gas at 70 eV. Fractions cor using a Vartan 2700 GC detector. The scparatioi ChromQ, 100/120 me 4*C/min, injector 25CF Results Pharmacokinetics p, In Table 1, the elimi compiled. The m 50.0 mg/kg, is recov v**tile radioactivity D. Reichert m. carcinogenic ic effect has been trichloroethylene uch et aL, 1975; sponsible for the he rate of metabn in experiments sd whole animals rs of microsomal centration in the i reported (Jones onfirro the results stained after oral dst in the quaiitaing in the urine, rat urine, two of arid N-acetyl-Swo main metabo as an additional ithioglycolic acid he metabolism of laboUsm of VDC sry of previously this substance in ^jpi(s). nd had a specific ra ws by radiotaa-chro- erboxymethylH-cys one was prepared by to Oxawa (1963). Its s prepared aa follow] i methylated with an with 2-amiooethsnol was characterized by jnyl chloride and with opionyl chloride tn a -earn of dry nitrogen, yl chloride, described sthoda (Kaiser et aL butano)-3N HC1 was min) at dcacribed by l.l.pichloroechyletK Macabofam 161 and Traaamna. Female Wtaar rats (180--220 g), Inatinit ftir Verauchsticrzucbt, Hannover, ware uaed m all Kndiai, Fain of rata were adminittarad single doecs of 1"C] VDC (as a solution . gicapfline, 3 nd/kg) by stomach tube between 9 and 10 *.m. The animals were transferred into an metabobam cage immediately after doting. A constant rate of dry air (300 ml/nun) was drawn (jtf0Ugh the cage. Standard dim (Aftromm*) and water were supplied ad libitum. c^nfplinf and Measurement qfRadioactivity. Excreted ['*C1 activity was followed for 72 h. Urine and were collected at 6 h intervals, the cage being rinsed with t total volume of 100 ml water m several -jrDons- ("Cl VDC and l4COj were collected in 0.3 h and 3 h intervals by drawing the air from the J^bobsm cage through a series of traps. Before entering the first trap, the moisture of the nr was -H&iwi with Drieme (W. H. Hammond, Dncrite Co.). Following the procedure described by Wata^ et a). (1926), we separated the [14C] VDC and "CO, in four consecutive traps. The first two jjiotained 30 ml toluene/2-metboxyethanol solution (8 : 2. v/v) each, and were cooled in a dry ice bath, -pg remaining two traps contained 100 ml of diethanolamine/2-methoxyethanol (1:1, v/v) each. The gjpt for l4C03 were maintained at room temperature. Samples from the traps and urine samples were transferred directly in Rodszint 22* liquid scintilmedium (C, Roth. Karlsruhe. FRG) for counting the radioactivity. The carcass was weighed and jjpjMni in 20% KOH- Portions of the digests were added to scintillation counting vials. The activity in ^ vial was measured in t Packard liquid scintillation counter. 5tperation of Urinary Metabolites. The collected 24 b urine of two rats was freeze-dried, the residue .a extracted with methanol at room temperature until it contained less then 1% of the radioactivity, pw extract was evaporated to a small volume at 40* C under reduced pressure. One pert of tins gluoon was analysed by thin-layer chromatography. Aliquots were qxXtad on silica gel plates (&2J mm layers at silica gel, F 234, E. Merck and developed in e-butanol: acetic acid : water solution ,40:15:13. v/v/v). Compounds were detected by spraying with nmhydrin or with a reagent for Isnlsnt sulfur compounds (Knight and Yeung, 1938). The radioactive zonae were located with a imhoid radiochromatofram scanner, and removed by mraping oft end cm action of the aiUea gel with Mhasol (30 ml)- Tbeac fractions were analysed by GC/M5. Another pan at the mmhanoiic solution ms evaporated to dryness, dissolved in pyridine-formate buffer (0.03 M. pH 3) and passed through a 3)0* 6 mm ootumn of Annex A 3 (Bio Rad Lab.). Operating praasnre was 10-13 bar at 30* C. The m 1 ml fractions. Single fractiocu wen evaporated and danvetizad aa described Dtacrion and Identification qf Urinary Metabolites- Radioactive substances from zones on the thinteser pistes and fractions eluted from the cation exchange eoturnn were butylated following the proceart of Kaisrr et aL (1974). OC/MS was performed with a Varian CH 7 mass spectrometer combined with s Venn 2700 gas chromatograph, and Varian 5S 100 MS dan system. Mass spectra were taken a 70 eV. Fraction* containing radioactive metabolites were analyzed by radio gas chromatography uni a Varian 2700 GC equipped with a flame ionization datacter and a Bcrthold RGC 170 radiogas detector. The separation was carried out on a 6 ft glass column packed with 3% OV 223 on Gas Ctaom Q, 100/120 mesh. Carrier gas He 30 mi/min. Temperatures: column oven 80--270* with ' C/imn. injector 250* C, detector 270* C. Results fhemacokinttics of P*C] VDC after Oral Administration (a Table 1, the damnation pathways of [,4C] activity after a single oral application * compiled. The main portion of the l'*C) (42--53%), in a dose-range from 0.5 to rag/kf, it recovered from the urine. Both, in urine end in feces, only the non'*dle radioactivity has been determined. SL 066271 v 162 TM 1. Excretm of radioactivity foOowin* oral Hn*l-do adnumstrarion of l,4C) VDC -- _____^________TM mt Sfnmon Time (h) Don ------------------------------------- 0.5 mj/kf 5.0 mg/ki % of administered activity Exhaled sir VDC CO: Total Urine Total Fens Total Carcass and tissues Cage rinse Total 0-72 0-72 0- 6 6-12 12-24 24-48 48-72 0-12 12-24 24-48 48-72 1.26 0.39 13.64 + 4.72 14.90 i 4.76 18.40 3.93 14.59 + 1.73 7.97 4 0.55 1.99 4 0.69 0.60 4 0.02 43.55 4 4.59 0.11 0.07 832 4 2.68 5.86 4 0.52 1.25 4 0.47 15.74 4 3.32 537 4 0.60 9.80 4 3.13 8936 4 3.74 9.70 4 1.26 11.35 4 2-65 21.05 4 3.69 16.39 4 2.20 22.79 4 1.87 11.14 t 2.25 3.00 4 1.52 0.56 4 0.36 53.88 4 4.14 1.68 4 1-23 6.24 4 3.02 6.24 4 1.89 038 4 0.06 14.54 4 1.18 233 4 0.42 731 4 031 99J1 4 131 16.47 ; 4 6-13 4 0.33 22.60 4 4.37 4.76 1.39 32-83 = 2.20 19.65 4 24| 4.06 4 0.83 0.81 4 0.2J 42.1J 4 343 0.75 4 048 2.71 4 1.17 3.69 4 047 0.50 4 0.1| 7.65 + 1.47 2.77 4 044 5.91 + 079 8134 4 3.90 ( [.DteWow*1 10.01 ft aii* In the portion, unchanged [14C1 VDC and `*C02 are identified. Afar i application of 0.5 mg/kg, only 1.2% of the total radioactivity it exhaled u vn- changed l,4C] VDC, 13.6% as ,4COj. A reversal in the proportion of VDC to CO. has occurred after a dote of 50.0 mg/kg. In this case, about three times more VDC Am CO, it excreted. The elimination of [**C1 VDC as a function of time after 0.i. , 5.0. and 50.0 mg/kg it shown in Fig. 1. Figure 2 represents the biphase pattern of 1 ,4CO} after a dose of 0J and 50.0 mg/kg over a period of 72 h. Is . Table 2 are the calculated elimination halMivet of radioactivity listed, excreted is [,4CJ VDC, l4C0p and urinary activity. From both, figures (1 and 2) and tables (l and 2), the biphasie elimination is evident at all dose levels by pulmonary ud \ urinary excretion. Ft*- I Ft*. 1. Expire versus time ( Ftt> 2. Setnili 50.0 mg/k| I TaMe 2. Half liv VDC Isolation and Identification of Urinary Metabolites The isolation of the "*" metabolites of VDC in the urine of rats is accomplished through the successive procedures of solvent extraction, ion exchange chromatog raphy and thin layer chromatography. Finally the metabolites are separated isd identified by *-- of combined gas chromatography and mass spectrometry. Tbs layer chromatography has revealed 5 radioactive, clearly resolved zones (A, B. C. D, and E). The least polar fraction E (Rf 034, about 12% of the activity) on the oo Phase or dtminstion D -- 0. VDC Urine 41 4 SL 066272 D. Reichert ,, ^ of ("CJ VDC to ^ *1 50.0 mg/kg ~~ 163 t 1.26 i 2.65 t 3.69 t 2.20 t 1-87 i 2.25 i 1.52 i 0.36 t 4.14 t 1.23 i 3.02 i 1.89 t 0.06 i 1.18 t 0.42 :0JI t 1.21 16.47 4.24 6.13 ; 0.33 22.60 - 4.37 4.76 * 1.39 1183 = 2.20 19.65 2.41 4.06 z 0.83 0-81 r 0.25 4111 3.53 0.75 0.4* 171 1.17 3.69 0.47 0.J0 0.1* 7.65 1.47 177 0J4 5.91 0.79 *1.04 3.9o C02 are identified. After ty is exhaled as un- don of VDC to CO, Jt three times more VDC unction of time after 0.5, ts the biphasic pattern of FK-1. Expired I '*C1 VDC expressed as percentile of the administered dose (0.5, 5.0, tod 50.0 mg/kg) virsui tune (h). In this figure end Fig. 2 each value is the mean S.D. from three experiments ver a period of 72 h. In ctivity listed, excreted as -*s (1 and 2) and tables (1 Rg. 2. Semilogarithrruc concentration-01116 curves of "CO, in the expired air following doses of 0.5 and 50.0 mg/kl [,4C1 VDC orally eveis by pulmonary and TsHe 1 Half lives of elimination of [`*C1 VDC. "CO, and activity in unne after oral edministrauon of ! "Cl VDC i of rats is accomplished on exchange chromatogolites are separated and mass spectrometry. Thin resolved zones (A, B. C. % of the activity) on the Muse Dose d -------------------------------------------------------------------------------------- donation 0.5 mg/kg 5.0 mg/kg 50.0 mg/kg Rapid Slow Rapid Slow Rapid VDC 43 min - 18 mm 2 h 54 min 27 nun CO, 4 h 30 min 35 h 4 h 45 min 24 h 2 h 45 min Qtat 4h 30 min IS h 30 mm 3 b 45 min 14 h 5 b 15 min Slow 6 h 21 min 20 h 45 min 25 h SL 066273 164 D.l***' plate has been scraped off and eluted with methanoL An initial analysis with GC/u has indicated that the active material could be methyhhio-acetylaminoeth/^ (MAAE). Therefore, we have synthesized this substance. The synthetic product ^ metabolic fraction E have shown identical retention times on columns with diffe^ I stationary phases (SE 30 and OV 225). The mass spectra are in agreement J"' respect to the molecular ion and the typical fragments (Fig. 3). For further chv ** tenzadon, the metabolite and synthetic product were then converted into two difi^ WO' CMj-S-CM2-C -NH-CHj-CMjOM MaaE SO' 1 90 100 . " a% pO_ lit * T9D 200 it MvnboLit* 1 k .JL 90 * 1 _M^ ! i* Ul 1 1U---------------------------------------------------------------------------------------------------------------------------------------KD 190 200 CH^-CHj-CCHj-O-Cu-0^ M tt MW 2tS Mate *TFAt / CHjdj 11 toa * * I _L__ |M n _______ ________________ wo wo too - lit * M i_____________ 2S0 nM MtObotl.TftkA /CHjOj `J <> Ul ______ L wo______________w_o______________201 0 - -J________________ 2SO m/. , ,.Diewowethyw pSo?sifer *3)*' *^** The main 0-37). The co butyl ester wi mor r acid- M of the r; -jycolic aac. 9 In additic c0mpo"nds v umn aft bu be a mercapt identical witl (fig. 4). Coc material has that N-acetyl A compansr from the thii turic acid. A in a further c captunc aci The mas The spectra the type of fiat of lOOben^p indicate tha\ equivocal st ,100 SO LiM. so 100 ICO) CHj*S-eMj-C- NM-CMyCHj-O-C * CMj-CHj MW 209 MAAE pmptowyi dor** 1W .M JhL_----------------------------- -------------------- 190 Li. M L m 1 '** WO mmoMIiw- propwiyt eMenM 111 l__________________________________________________________________ WO 200 no mit j. Mass apacca of dw matabofe in TLC loee E and rymhwitad meflndthio-acatylainiiiowiiaaot Trifluoroaoscyi- sad proptucyl-ilamamw ot awtahnHtr and smhwirie substsnct are compand 100-1 % so> 1. c * so- JU Fh 4. Mat SL 66274 D.IUichnWi #"* <*/* -cetyitminoetiun. nthetic product ta. lunrns with differ^ in agreement win For further ch*2 rted into two differ tH-CHj-CMjOH V. -CHj-CHr-ol-e,-c^ Va " 0 m,, o J-C-CHj-CMj 0 c*Hond 3 m/. no-ocotylvniDoctbsaoi. net we compered I.*4jiehkway* Moobota 165 ^ derivative* by esterification of the primary alcohol group with trifluoroacetic hydride and propionyl chloride. The conversion has proceeded smoothly in both catey The triftuoroacetyi and propionyl esters from the synthetic product and meta- fr*ct*on E *hw the same retention times, and their mass spectra are identical , ^ig. 3)- This indicates that the fraction E metabolite is identical with MAA. The main activity (approx. 40%) on the thin layer plate is found in zone D (Rf 0 37). The column chromatographically purified acid fraction is converted to the ^uty) ester with n-butanol-3N HC1. Radio GC of this deriv&tized sample reveals a mlJor peak cochromatographing with the dibutylester of authentic thiodiglycolic | Moreover, the mass spectrum of the butylated metabolite is identical with that of the reference compound, thus proving that the metabolite in fraction D is thiodiglycolic acid. In addition to MAAE and to thiodiglycolic acid, three additional radioactive compounds were demonstrated by radio GC in the eluate of the ion exchange col umn after butylation. There is reason to suggest that one of the components might | oe mercapturic add. The mass spectra of one of the metabolites proved to be , identical with that of the butyl ester of N-acetyl-S-(2-carboxymethyl)cysteme (pig. 4). Coehromatography of the synthetic products with radioactive biological material has verified the identity of both substances. At this point it has been certain that N*acetyl-S-(2-carboxyinethyl)cysteine is a metabolite of TLC zone A, B, or C. A comparison of the RF-value of the synthetic product and radioactive material I from the thin layer plate is interpreted to mean that fraction B contains this mercap- tnric add. A lack of agreement in retention time and mass spectrum has been found in a further comparison to other mercapturic adds (hydroxyethyi- and methyl-mereapturic add). The mass spectra of the remaining two urinary metabolites are shown in Fig. 5. The spectra imply that the metabolites may have closely related structures, because the type of fragmentation appears to be similar. The ion at m/e 57 and the difference of 100 between the ions with the highest mass (presumably the molecular ion) may indicate that the metabolites are mono- and dicarboxyiic adds. However, no un equivocal structure could be assigned to these metabolites so far. 0 C^Mj-O-C -CHj -S-CHj-CM NM*C0-CM3 MW 333 in re _L I DO 250 30C M mlt Mrtaooiit*. C^HjOH HCl m it* J-L m m n _J___ I . ,t..... I.... too ISO 200 250 300 IV A Mom tptett* of the butyl atm of N-aeetyl-&<2-evbacyindiyl)cyttiM tod m m/t SL 066275 r" j 166 T! D` R**hn n i.i p,chioroethyfcjif % f*4 In ienmity Mats ipecirm or unidentified VDC metabolites after butvlation Discussion The results of this investigation confirm the report by McKenna et aL (1978) con. ! censing the pharmacokinetics of VDC after a single oral application in rats. In t dose range of 0.5--30.0 mg/kg the orally administered VDC is quickly and completely absorbed. This is substantiated on the one hand by the initial part of the j i I pulmonary excretion of VDC and C02 and on the other hud through a rapid and i extensive renal and biliary excretion of metabolic end products. | However, cfiffsences exist in the qualitative and quantitative interpretation of the metabolic end products of VDC metabolism. The reason for this disparity could stem from different analytical techniques. McKenna et al (1978) have employed high pressure Squid chromatography, Jones and Hathway (1978) thin layer and gm chromatography. Thiodiglycoiic acid has been confirmed as the main metabolite m urine. Starting from chloroacetic add as an intermediate in VDC metabolism, the formation of thiodiglycoiic add can be explained (Fig. 6): chloroacetic add has been identified is a metabolite from VDC in fiver tissue (Reichert and Bashti, 1976) and in urine (Jones and Hathway, 1978). Yllner (1970) has shown thiodiglycoiic add in the urine of mice after application of monochloroacetate. Chloroacetic add reacts with glutathione in an enzyme-catalyzed reaction, to form carboxymethyl glutathione (Fig. 6). Cleavage of the glycine and glutamine fragments yields carboxymethylcysteine. Apparently, this compound is preferentially dominated and subsequently decarboxylated. The main metabolite thiodiglycoiic add is the end product of this reaction sequence. Up to now, the intermediate product, carboxymethylcysteine has not been shown in VDC metabolism. However, the identification of the N-teety! derivative of this compound can be considered is indirect evidence. N-aeetyl-S-(2-carboxymethyl)cysteine is the only mercaptunc add we have found in VDC metabolism. In our studies, there was no indication of the formation of the hydroxyetbylmercapturic add (McKenna et aL, 1978). We Mnwv account for this pathway, which would involve a reductive dechlorination of | j VDC as a major sap in metabolism. The gnat importance of glutathione in the metaboiization rate and the toxicity of VDC has been etiesaad previously (Reichert et aL, 1978; Jaeger it ai, 1974). H0 ci- C-C-nh- CH-` 1 Ct-CMj-CO- HjC-S-CWj-CO-K 4. Metabolic However, in addi. gation and the eli reaction mechani dves. The formal step could be the t intermediate prodi chloride shift. T scribed above, c remains bound to t nolamine. an obi cleavage of the j. acetic add. Addin acetic add chlorid functional SH-gr recently been pc SL 066276 **** m/f 300 piehlofecthylene Metabolism i.i Cl H vc-c" o' ''h c--c . a'" % c HI 0il Ct-C-C-Cl I 167 HC t l (1978) conion in rmts. In a uickiy and comlitial pan of the >ugh a rapid and trpretation of the disparity could have employed tin layer and gas line. Starting formation of teen identified as '6) and in urine acid in the urine zed reaction, to : and glutamine i is preferentially ite thiodiglycolic the intermediate tolism. However, be considered as 'nly mercapturic no indication of al, 1978). We iechlorination of and the toxicity er et aL, 1974). Cl-CHj- CO-NH-CHj-CKj-OH MOOC-CMj-S-CHj-CM-COOH HjC-S-CMy-CO-HH-CHr-CMj-OH H0CC-Chr*-O^-C00H HOOC-CMj-S-CHj-CM-COCH MH-CO-CM] Ftf. 6. Meubolic pathways of VDC. Identified metabolites underlined However, in addition to the described reaction sequence involving glutathione conju gation and the elimination of S-containing end products, another totally different reaction mechanism must exist which leads to the formation of ethanolamine deriva tives. The formation of this unusual metabolite is not yet clarified in detail An initial step could be the formation of chloroacedc add chloride (Fig. 6). This electrophilic intermediate product arises from the very unstable epoxide by an intramolecular chloride shift The largest part is hydrolyzed to chloroacetic add, which, as de scribed above, conjugates with glutathione. A smaller portion, however, evidently remains bound to the membrane and can directly react there with phosphatidyletba- nolamine, an obligatory constituent of lipid membranes. The subsequent enzymatic cleavage of the phosphatidyl residue leads to the ethanolamine derivative of chloro- scetic add. Additional support for this meubolic pathway is-the fact that chloro acetic chloride is electrophilic enough to react directly with nucleophiles, e.g., functional SH-groups (Reichert and Werner, unpublished). A similar mechanism has recently been postulated by Cohen et al. (1975) in halothane metabolism. They SL 066277 " *"*.* presume Am trifluorotceti': acid, a major halothane metabolite, can react a with pboaphatidylethanolamine.The enzymatic cleavage then results in N-Jn^ acetytetbanolandne. Uor&- Now the question remains as to how the methylthio-group is introduced intermediary product chloroacetyl aminoethanol Two ways are conceivable*?!? *** nucleophilic attack of a methylthio-group (Lr, of methionine), or formation ^ glutathione conjugate. To conclusively answer this question, further investit f ` are required. The increasing number of metabolites with the insertion of the m*1?"5 thio-group on an electrophilic carbon atom have been compiled recently by j ' I and Testa (1978). They conclude from the present data that, in most ^ ^ner nucleophilic attack is theprobable pathway. Irtct The results of this study show that the inactivation of VDC in vivo occurs bv large number of reactions, some of which could be demonstrated. The identify.,' 1 of the main metabolites from VDC in rats leads to an improved understanding of^ toxic properties of this substance. Consequently, the covalent binding of chlor^ acetic acid chloride on membrane constituents (It, phosphatidylethanolantan* could explain, in part, the parenchyma damaging effect of VDC. 1 l Acknowitdftmtnti. This work vu supported in pen by BC Chcmk. Hdddbag. We wish to tt,.^ ^ E. Sdrihz. Phynoiotiseh-chenusebes lnstmiu Wurzburg. for providini the column chromatotruh, The skilful techntcai assistance provided by Miss Ch. HSttertioff (animal work) sod Mrs. J. Coihtn (mass spectrometry) is grasduily acknowledged. ujwoi. C~ Ufemine. M Atrn. N Y. Acad. ! ' KeBfle.M^:2np M 0( vinylideoe chkm (1978) H.: The dehyd 0 cysteine. Bull Che aeicben. D. Bashu, h -effused liver of th Reichert. D-. Henschler perfused ret liver. I Re,chert. D.. Werner. I olistt! end hepatotoi (1978) Reichert. D.. Werner. H administration m rr Viole- p u- Ceputo. A 45-47 (1977) Wstenebe. P. C.. McG< inf inhalation expo; yltner. S.: Metabolism. (1970) Received February 26, References Banach, fL, MalavnBc. C. Mootrtann. R_ Tomatis. L: Tinas osediand tnutsssmcuy of vmyk** chloride and 2-driorobutadiaat m Sabnondla typhimurium. Natan 235, 641-643 (1975) Bones. G, Urban. Ttu Rdchsn, D_ Hanachisr, D.: Chemical reactivity, metabolic adnm fanenm and biological metmry of chlorinated ethylene* in the isoiaud perfbead rat Svw prspanmi Biochta. Pharmacol 24, 1829-1834 (197J) Cohen, E- N. TrudeH 2. R_ Edmonds, H. N., Wasson, E.; Urinary metabolites of hio-- is tm. AaesthaeMogy 43, 392-401 (1975) Creech, J. I_ Johnson, M. N.; Angiosarcoma of Hvar in tbs manufacture of polyvinyl chinks. j Occnp. Mad. 16, 130-131 (1974) Graim. H-. Bones, G-. Rndwnn. 7-- Racbcrt, D, Hanachkr, D.: Mammary in vitro end pottsui cardnofmicicy of chlorinated athyimes as s function of metabolic oanrane fbrmeooc. Btodm Pharmacol 24, 2013-2017 (1975) Jaeger, R. J. Conofly. R. B,, Morphy, S. D-; Effect of 18 h fast and flutathicmc depletion on l.l dichloroethykne-indtmed hepnmtomcity md lethality in rats. Exp. Mol. PathoL 26, 1B7--191 (1974) Jendta, D. L, Booth, R. A-, Rock, M.: Simultaneous tnicroestimation of choline and acetylcholine b> gas chromatography. Anal Cham. 44, 1879--18*1 (1972) Jeaaar, P, Team, R: Novel pathways in drug metabolism. Xnobicbca >, 1--23 (1978) Jonm, B. K-, Hsshway, D. E.: Tbs biological fate ofvinyiidene chloride in rats. Cbem.-BioL lnttnet 26, 27-41 (1978) Raiser. F. E, GArfcc, C. W,, Zamwalt, R. W,, Kuo. K C.: Amino add analysis. Hydrolyvs. ieasi ,chaga dan-up, thritatiiatinn and quantitaaoe by gas-liquid chromatography. J. Chrcmstop 94 113-133 (1974) XMflhL H. R, Yang. L.: Btachsmical studies of tube agata. Btocham. J. 76, 111-119 (19581 Lae, F. J, Harry, D. S.; Angiosarcoma of the liver in an vinyl Alcalde worker. Lancet t, 1316-1311 (1*74) XT *"*! t in N-trifluoJ educed into u,. nceivable: direct formation 0f a er investigations * of the methyl =tly by Jenner cases, the direct rivo occurs by a "he identification ^standing 0fthe wiiug of chiorolyiethanolamine) ' wish to thank Dr m chromatograph, "** Mrs. J. Colbert (j.pjcbMoethytatt Metabofcem 169 jjdk, C. Lafamine. G.: Carcinogenicity bioanayt of vinyl cidande. Research plan and early results. M AfML N.Y. Asad Set 144. 195-218 (1975) jtsf*- M. L Zawipai. J. A- Madrid, E 0,, Braun. W. H.: Metaboiisnt and pharmacokinetic profile ^of.iuyMwe chloride in rat* followini oral administration. Toxicol AppL Pharmacol. 45, 821--835 (1978) M#a- M,: The dehydratint cydilation of S-carboxymethvl-cyiteine and N-acetyi-S-carboxymethyl^jystnne- Bull Chem. Soc. Japan 34, 920-922 (1963) spctrert. D- Bashtl N.: Metabolism and disposnion of 1.1 -dichloroethylene in the isolated bloodperfused liver of the rat. Naunyn-Schmiedeberg's Arch. Pharmacol. [Suppi.] 293, R25J (1976) epcheru D- Henschler. D.: Uptake and hepatotoxidtv of 1.1-dichloroethylene by the isolated bloodperfused rat liver. Ini Arch. Occup. Environ. Health 41, 169-178 (1978) tetebert- D-, Werner, H. W,, Henschler. D.: Role of liver glutathione in 1.1-dichloroethylene metab olism and hcpatotoxicity in intact rats and isolated perfused rat liver. Arch. Toxicol 41, 169-178 (1978) RoCt,en D,, Werner. H. W,: Disposiuon and metabolism of I'*C1 1.1-dichloroethylene after single oral ^ministration in rats. Naunyn-Schmiedeberg's Arch. Pharmacol tSuppl] 302, R22 (1978) Viols. P 1-- Caputo. A.: Carcinogenicity studies on vmyiidcne chloride. Environ. Health Perspeci 21, 45--47 (1977) Watiaabc, P. C.. McGowan, G. R,, Madrid. E. 0.. Gehhng, P. J.: Fate of [UC1 vinyl chloride followmg inhalation exposure in rats. Toxicol. Appl. Pharmacol. 37, 49-59 (1976) vsner. S.: Metabolism of chloroaeetat*-l-"C in the mouse. Acta PharraacoL (Kbh.) 30, 69--80 (1970) Received February 26, 1979 i -maty of vinylidene (1!975) formation it^Br ppireparation. f halothaae in man. ilyvinyl chloride. J. vitro and potential wmation. Biochem. ie depletion on 1.1 JthoL 20, 187-198 md acetylcholine b> :5 (1978) 'bem.-Biol Interact. Hydrolysis, ionexhy. J. Chromategr. 111-119 (1958) meet I, 1316-1318 SL 066279