Document N2n6LJY9kOgOLy14E6ndByV9y

Chem.-Biol. Interactions, 20 (1973) 27--41 6 Elsevier/North-Holland Scientific Publishers Ltd. 27 THE BIOLOGICAL FATE OF VINYLIDENE CHLORIDE IN RATS B.K. JONES and D.E. HATHWaY Imperial Chemical Industries Ltd., Central Toxicology Laboratory, Alderiey Park, Cheshire SK1Q -ITJ (U.K.) (Received July 4th, 1977) (Accepted October 14th, 1977) SUMMARY The main eliminative route for [14C] vinylidene chloride ([14C]DCE) after intragastric, i.v. or i,p. administration to rats is pulmonary; both unchanged DCE and DCE-related CO: are excretediy that route and other DCE meta bolites via the kidneys. Part of the urinary 14C is of biliary origin. After intragastric dosing, the plot of the pulmonary output of unchanged DCE against the logarithm of reciprocal doses in biphasic. Pulmonary elimination of DCE and C02 and urinary excretion of DCE metabolites after an intra gastric dose occupy 3 days. In comparison, 80% of a small i.v. dose is excret ed unchanged within 1 h of injection; more than 60% within 5 min. Biotransformation of DCE affords thiodiglycollic acid, and an .V-acetylS-cysteinyl-acetyl derivative as major urinary metabolites together with substantial amounts of chloroacetic acid, dithiogiycollic acid and thioglycollic acid. It is probable that chloroacetic acid, which is a DCE metabolite per se, lies on a main metabolic pathway for DCE, since it affords several meta bolites in common with DCE. Furthermore, electrolysis of one molecular proportion of the [14C] thiodiglycollate metabolite from [1-|4C]DCE or [1-I4C1 chloroacetic acid gives 1 equivalent of l4CO;, and this evidence is consistent with the transformation of DCE into chloroacetic acid by a mechanism involving the migration of one Cl atom and the loss of the other one. C02 (and hence urea) may be produced through the action of epoxide hydratase on 1,1-dichloroethylene oxide or by a minor oxidative pathway for chloroacetic acid. The N-acetyl-S-cysteinyl-acetyl derivative is probably formed via the reaction of 1,1-dichloroethylene oxide and glutathione S-epo.\ide transferase. INTRODUCTION The widespread usage of the polymer of vinylidene chloride (1,1-dichloro- Abbrevnations: DCE, vinylidene chloride. TFAA, trifluoroacetic anhydride. CMA 050611 . i h i i e n g i M H i m i r i i t j f . a ii. ir r a _] 2S ethylene, DCE) for packaging film and for coating other packaging materials, and the fact that the structurally-related vinyl chloride is a frank carcinogen in animals and in man (see ref. 1, which contains a summary of the position of this subject), make an understanding of the biological fate of DCE in mammals desirable. This paper describes the results of such ah investigation and their possible interpretation. The scientific background to this problem has grown during the time that the present experiments were being made. Thus, in vitro, DCE was shown to be mutagenic in the tissue-mediated Ames test [2,3]. A much higher mutagenic response was observed in mice than in rats with kidney and liver fractions [2], but the mutagenicity of DCE was very much lower than that of vinyl chloride [3]. A high incidence of an unusual kidney tumour, the kidney adenocarcinoma, has now been found [4] in the Swiss mice, but not in the Sprague--Dawley rats, that had been exposed chronically to DCE. However, so far no case of DCE carcinogenicity has been reported in man. --r MATERIALS AND METHODS Chemicals Vinylidene chloride was supplied by Imperial Chemical Industries Ltd.. Mond Division, Runcorn, Cheshire. Before each experiment, stabiliser was removed by washing with alkali and water. Dithioglycollic acid (Aldrich Chemical Co.), and thiodiglycollic acid and thioglycollic acid (both obtained from the Sigma Chemical Co.) were all of grade 1 quality with a purity exceeding 99.5%. S-(2-Carboxymetliyl)-L-cysteine was prepared (5] from chloroacetic acid plus L-cysteine hydrochloride, and the structure of the crystalline product (m.p. 175--17GJ C, decomp.) was confirmed by mass spectrometry. 4-Acctyl-5-carboxy-3-oxo-tetrahydro-l,4-thiazine was prepared from yV-acrtyl-S-(2-carboxymethyl)-L-cystcine by the method of Ozawa [6], All reagents and solvents were of AnalaR grade or of the next best quality available. Radioactive chemicals ( [1-'JC]DCE and [2-'JC]DC. with a specific activity of 1.0 mCi mo! and with a chemical and radiochemical purity exceeding 99.0%. were synthesized from IJCO; by our colleagues, Mr. D.C. Greenslatle and Dr, J.A. Heslop, ol Imperial Chemical Industries Ltd., Petrochemicals Division. Billitigham. Cleveland. Whereas in the case of [1-I-1C|DCE. '""CO; was converted into [ 1 ''`Cl acetic acid by the Grignard reaction with methyl iodide, m that of [2-lllC|DCE, MCO: was reduced to [ ,JC] methanol, which was transformed by a reaction sequence involving ] 'JC| methyl iodide and ( 2-l'*C| acetonitrile into 12-,'*C] acetic acid. Whence [1-1JC] and [2-IJC] acetic acids respectively were transformed through the variously labelled forms of the reaction sequence, dichloro- . CMA. 050612 I 4*,..i-^w>xJww 29 acetic acid. 2.2-dichloroethanol and 1,12-trichloroethane, into the pure liquids [1-MC]DCE and [2-l4C]DCE. b.p. 37JC, which were stabilized by addition of 10 ppm of hydroquinone. (1,2-Elimination of 1,1,2-trichloroethane was specific, and gave DCE exclusively, but the attempted 1,2eliminanon of 2,2-dichloroethanol furnished intractable polymer.) On account of the risk of polymerization, it was convenient to store [MC]DCE as a solution in peroxide-free com oil at --203C. (Preparative details will be included in a forthcoming paper by D.C. Greenslade and J.A. Heslop, on the synthesis of these substances.) [1*IJC]Chloroacetic acid (52 mCi'mmol). [2-l4C]chloroacetic acid (42 mCi/mmol). L-(U-I4C] cysteine hydrochloride 124.5 mCi mmol) and (U-,JC] glycine (10 mCi/mmol) were obtained from the Radiochemical Centre. Amersnam. Bucks. Experiments with animals Adult male rats (approx. 2 months old. 200 g body wt.) were used [Alderley Park strain (Wistar-derived), specific pathogen-free). and kept on a standard pellet diet. (a) For the purpose of excretion-retention experiments, groups of 4 animals were administered single doses of (2-iJC]DCE (either 500 gg.kg or 350 mg/kg: 1 gCi) as a corn-oil solution by the intracastric, i.v. and i.p. routes. The animals were housed singly in glass metabolism cages for 72 h after dosing, and l4C was measured in the urine, faeces and exhaled air. which was drawn successively through trichloroethylene at -7Q;C and CO; absorbers (for details, see ref. 1). (b) 3 rats, equipped with a biliary fistula, were each given an mtragastric dose of (1-IJC]DCE (350 mg/kg: 15 gCi) as a corn-oil solution, and they were maintained in restraining cages for 4S h, during which time bile and urine were collected. The 4S-h urine was also collected from control animals, which had been given the same dose. For the identification of urinary metabolites [1,7], groups of 4 animals were administered intragastrically; (c) [MC]DCE (350 mg/kg; lOaCi per animal) m corn-oil solution. (d) [ 1 -1-1C] chloroacetic acid 150 mg/kg; G.5 aCi) in aq. solution. (e) [2-"*C] chloroacetic acid (50 mg/kg; 11.4 uCi) in aq. solution. (f) 2 rats were each given 2 i.p. injections of an aq solution of L-[L'-IJC] cysteine hydrochloride with a 17-h interval (total dose, approx. 50 uCi). 2 h after the second injection, each animal was given a single mtragastric dose .of DCE (350 mg/kg) in corn-oil solution, and the 4S*h urine was collected. (g) 2 rats were each administered 2 i.p. injections of an aq. solution of 20 uCi of L-[t.'-l4C)eysiPine hydrochloride and 20 gCi of [l:-`JC]glycine with an lS-h interval. 1 h after the second injection, each animal was given a single mtragastric dose of DCE (350 mg/kg) in corn-oil solution, and the 43-ii urine was collected. (h) To eacn young rat (approx. SO g body wt.i, there was administered an CMA 050613 intragastric dose of 20 ^Ci of [l4C]DCE. 30 and 60 min, and 2 days after dosing, the animals were deeply anaesthetised and were rapidly frozen by immersion in solid CO:. Longitudinal sagittal sections, 20 y in thickness, were cut with a mechanically operated Cambridge microtome, in a Bright cryostat at --20*0; some were cut through the vertebrae and others through a kidney of each animal. Apposition autoradiograms were prepared from the freeze-dried sections [1]. Measurement of radioactivity An automated and computerized Intertechnique Model SL30 liquid scintillation spectrometer was used for measurement of 14C, making use of standard channels-ratio quench-correction curves. Liquid samples were admixed with standard scintillator and radio-assayed direct, and samples of faeces were burnt in an Intertechnique "Oxymat" solid-sample oxidizer. Systematic separation of the urinary jnetabolites into fractions of chemically similar substances Urine from each group of rats, (c) to (g), was separated into 3 fractions by anion-exchange chromatography in a manner analogous to that described by Green and Hathway [1]. The 3 N HC1 fractions were evaporated separately to dryness under reduced pressure, and the various residues were methylated with an ethereal solution of diazomethane. Methanolic solutions of the' resulting O-methyl esters were analysed on GC columns and in the GC*mass spectrometer system. The neutral fractions and the 3 N acetic acid fractions were also evaporated separately to dryness under reduced pressure, and the different residues were examined by TLC. After esterification with diazo methane or butan-l-ol` (Fischer-Speier) and acylation with trifluoroacetic anhydride (TFAA) [1,7], the individual compounds, in the residues resulting from the evaporation of the acid hydrolyates of the last two fractions, were analysed on gas chromatography (GC) columns and in the GC-mass spectro meter system. Thin-layer chromatography The material eluted from the anion exchange column with 3 X acetic acid was applied as bands on 500 ag SiO; gel GF thin-layer plates which were developed with butan-l-ol--acetic acid--water (4:1: 2). Zones of 1JC were located with a Panax TLC scanner, excised and eluted with methanol. Gas chromatography Fractions containing radioactive metabolites were examined with a Pvc Model 104 instrument that was equipped with flame-ionization detection and that was coupled to an E.S.I. Nuclear 504 Radiogas detector. The column effluent was split in the ratio of 10:1 between the Radiogas detector and the flame-ionization detector. This gas chromatograph was fitted wnh glass columns (2.1 m long X 4 mm int. diam.), which were packed with 07 CMA 050614 (w/w) 0V-1Q1 on Supelcoport (80--100 mesh size), and run at 100'C and 180C. GC analysis was also used for the identification of unchanged DCE. which had been collected from the exhaled air in cold traps containing 1,1,2trichloroethylene [1]. In that case, the gas chromatograph was fitted with glass columns (1.5 m long X 4 mm int. diam.), which were packed with 10% (w/w) squalene on Chromosorb P (SO--100 mesh size), and run at SO'C. All of the columns were operated at a 40 ml/min flow-rate of a (95 : 5 v/v) Ar--CO; mixture. Radioactive peaks were located, and the corresponding samples were analysed by GC-mass spectrometry, using the same columns under identical operating conditions. GC-mass spectrometry An LKB2091 gas chromatograph-mass spectrometer system was used for the E.I. spectra of the compounds described. RESULTS For much of the work, either of the labelled forms of DCE, viz. l.idichloro-[l-14C] and [2-wC]ethylenes ([1-`4C]DCE and (2-'4C]DCE) was used, since CO: and urea were the only DCE metabolites with a single Catom, and they were produced in similar low proportions from the 2 labelled forms. Excretion of radioactivity The total excretion data obtained from rats after 500-gg and 350-mg intragastric, i.v. and i.p. doses of [2-'4C]DCE per kg are detailed m Table 1. In every case except one, almost all of the radioactivity was recovered during the first 72 h after dosing, but after low intragastric administration, small amounts of 14C were still being excreted during 72--103 h after dosing. At the different dose levels, there is a marked change in excretion pattern after intragastric administration (Table I). Whereas with the higher dose, nearly 70% as unchanged DCE and 1% of CO; are excreted via the lungs, with the lower dose, urinary excretion accounts for S0%. and less than 1% of un changed DCE together with 4--6% of CO; are eliminated by the pulmonary route. However, about 210 times more DCE was metabolized at the higher dose level than at the lower one. The change in excretion pattern appears to be due to a saturable drug metabolism and to an efficient arterial-alveolar transfer of unchanged DCE from systemic blood that leaves a relatively low concentration of material for biotransformation in successive passes through the liver. Thus, 30% of a small i.v. dose of DCE is excreted unchanged from systemic blood within an hour of injection; more than 60% within 5 min. Predictably, the excretion pattern after a small i.p. injection is intermediaie between that resulting from i.v. injection and that from intragastric adminis tration (Table I); some of the DCE is taken up into systemic blood and is excreted unchanged via the lungs and some is absorbed into the hepatic- CMA 050615 9T90S0 W O EXCRETION OF ,4C IN THE URINE AND BILE AFTER AN INTRAGASTRIC DOSE OF(l-'*C)DCE 3 normal rats and 3 animals equipped with a biliary fistula were each dosed mtragsstrically with 350 mg of [ 1-'*C )DCE/kg in corn-oil solution; unne and urine and bile were collected as appropriate Over a 48-h period. Normal rais Urine Rais equipped with biliary fistula Urine Bile Recovery of ,4C ( of dose) 0--3 h 3--6 h 6-24 h 0 2.9 0.9 15.3 ; l.S 0 0.2 0.1 0.9 ; 0.7 -J. 2.1 - 0.3 '6.5 * 0.5 5.9 : 1.7 24-43 h 3.1 : 1.0 2.4 . 0.4 2.4 t 0.6 portal system and is metabolized by the liver. After intragastric dosing, the rate of pulmonary excretion of unchanged DCE and COj parallels that of the 3-day excretion of urinary metabolites. Hence, the matching formation of CO: probably belongs to the same metabolic pathway as that for the urinary metabolites. GC analysis established that, apart from COunchanged DCE was the only product, related to the original substance, which was excreted via the lungs. D04** 1000 Kit* I plot ni lh puimonarv til' uticlumuril IX*K a4tum?a tht- ivvipm cal :> jroup* I rat* wit*- du-M-il i *, with DCfcl at ito-** U*vcl* m liu- rjncu *I 0 ft to GaO me; kb!, rach point n-prt-vMii* t imi*4p*tult,ni valur* CMA. 050617 34 The fact that the cumulative urinary excretion of 14 C in whole animals after a single intragastric dose of [1-,4C]DCE is roughly equal to the sum of the separate cumulative biliary and urinary eliminations, in animals equipped with a biliary fistula, suggests that the origin of at any rate part of the urin ary radioactivity was biliary (Table II). This finding and its possible enterohepatic implication are consistent with the relatively slow excretion of the water-soluble [l4C] urinary metabolites from intact animals. For groups of rats that had been administered various dose levels within a 1300-fold dose range, the plot of the pulmonary excretion of unchanged DCE (expressed in terms of percentage of the dose) against the logarithm of reciprocal doses is biphasic (Fig. 1), and this relationship reflects a "pool phenomenon" associated with a saturable drug metabolism. Whereas the initial steep slope represents the fast release of DCE, which had not been absorbed by the body organs and tissues or which exceeded the saturation of the body pool, the subsequent gentle slope relates to the slow release of DCE from the body pool, which in the case of this lipid-soluble, waterinsoluble substance is presumably adip'ose tissue plus liver. Whole-animal autoradiography Sequential autoradiograms of longitudinal sagittal sections through whole animals, which had been dosed intragastrically with [14C]DCE show large 14C concentrations in the kidneys and liter after 30 min and a more general distribution of 14C throughout the soft organs of the body at 1 h. The kid neys and liver retain 14C for the longest times after dosing. 3S3 Separation and identification of urinary DCE metabolites The fraction of the bulked urines of treated animals, which was displaced TABLE III CAS CHROMATOGRAPHIC CHARACTERISTICS OP THE METHYL AND /i-BUTYL ESTERS OF SOME DCE METABOLITES AND RELATED SUBSTANCES Esterified DCE metabolites Methvl chloroacetate Methyl thioclycollate Dimethyl thioduOycollate Dimethyl dithioelycollate Methyl S-( 2 -earboxymethyl (cysteine n-Butyl chloroacetate /l-Butyl thiuitlycullate Di n hutyl thiod idly collate Di n 'butyl dithioutycollate n Butyl .i`-(C-carbnxymethyl (cysteine GC-retentlon times (mini on columns coated with QV-101 Run .it 100"C Run at 1 SO'C 2.0 3.9 8.3 6.3 10.2 10 T 2.9 5.9 0.1 13.5 GMA. 050618 by 3 N HC1 from anion-exchange resin in the acetate form, was esterified with diazomethane, and an aliquot portion was analysed on GC columns of 6% OV-101 for peaks containing appreciable amounts of radioactivity. (GC retention times are given in Table III). Mass spectra of the substances in those peaks were obtained with another aliquot portion, which had been injected on to the similar column of a GC-mass spectrometer system. Chloroacetic acid, dithioglycollie acid, thioglycollic acid and thiodiglycollic acid were found to be the principal DCE metabolites in this fraction; conventional mass spectra of the corresponding methyl and n-butyl esters were identical with those of authentic materials. (Mass spectra of methyl esters of the first three substances are shown in Figs. 2 (a--c), whereas that of di-n-butylthiodiglycollate was illustrated previously [1].) A single major DCE metabolite was weakly absorbed on anion-exchange resin in the acetate form (v. supra), but not on cation-exchange resin, and was displaced from the former either by 3 N acetic acid or by deionised water that had been allowed to become acidic in thf.atmosphere. Its identification was exacerbated by difficulties of denvative formation and by the unamen ability uf the compound or its (possible) derivatives to GC methods. Thus, the fraction, which was displaced by 3 N acetic acid, was shown by TLC to contain a single l4C-compound, which gave a yellow colour reaction with ninhydnn. The product from attempted esterification of this compound with diazomethane did not afford an interpretable mass spectrum, when it was injected on to the probe of the mass spectrometer. However, hydrolysis of this uC-compound with boiling 3 N HC1 for 1 h, followed by esterifica tion of the product with butan-l-ol and acylation with trifluoroaeetic anhydride (TF.AA), gave the pure di-n-butyl ester, .V-TFA derivative of S-j2earboxymethyl) cysteine, the mass spectrum of which was identical with that of authentic material. Moreover, a few minutes acid hydrolysis of the l4C-compound with boiling N HC1 gave (V-acetyl-S-(2-carboxymethyl)cystcine; the mass spectrum [Fig. 2(d)] of the dimethyl ester was identical in all respects with that of authentic material. All of the radioactivity assoc iated with the DCE metabolite was accountable in terms of a single hydroly tic product [1JC];V-acetyl-S-(2-carboxymcthyl)cysteine. In addition, the GC and mass spectrometnc properties of 4-acetyl-5-carboxy-3-oxo-tetrahydro-l, 4- thiazine, prepared from iV-acctyl-S-(2-cnrboxymethyl)cysteinu [G], were shown to differ from those of the DCE metabolite. The absence of glycine from the DCE metabolite was found in experiments with unlabelled DCE in rats in which the cysteine-cystine and glycine pools had been labelled adequately with 14C, and in fact S-( uC](2-carboxymethyl)cysteine. but not [''Clglycine, was identified in the acid hydrolysate of the resulting [|,4C]DCE metabolite by mass spectrometry. Since these experiments exclude the possibility of internally-compensated lactam or glutathione-derived S-(2-eajboxymethyl)eysteinylglycine dipeptide structures for the major DCE metabolite concerned, there is a reasonable supposition that it may have been formed via glutathione-derived .V-aeetyl5- cystemyl-acetyl chloride. However, on that assumption, an A'-acetyl-S- CMA 050620 1 5*3 3S& S eg? m M iF as? TABLE IV RELATIVE PROPORTIONS OF THE URINARY METABOLITES OF DCE AND CHLOROACETIC ACID OF RATS, DOSED INTRAGASTRICALLY EITHER WITH [l-l4C]DCE OR WITH ['*C)CHLOROACETIC ACID DCE and chloroacetic acid metabolites Thiodiglycollic acid A'-Acetyl-S-cystemyl-acetyl derivative Dithioglycollie acid Thioglycollic acid Chloroacetic acid Urea S-(Carboxymethyl)cy*teine % of Urinary radioactivity DCE Chloroacetic acid 37.0 48.0 90,0 0 5.0 3.0 3.0 3.0 3.0 0 0.5 0.5 0 2.0 cysteinyl-aretyl-S-cysteine structure would be unacceptable, because experiments with unlabelled DCE in rats, in which the cysteine-cystine pool had been labelled adequately with HC, yielded the di-n-butylester, .V-TFA deriva tive of S-[l4C](2-carboxymethyl) cysteine, but not of [l4C]eysteine, in the hydrolysate of the resuiting (l4C]metabolite. Hence, while the pertinent structural features of the .V-acetyl-S-cysteinyl acetyl (DCE) metabolite are now known, more work is required to establish its exact structure. [l4ClUrea was shown by isotope-dilution analysis to be a relatively minor [l4C]DCE metabolite in the neutral fraction from the urine of treated rats. The iV-TFA derivative was crystallized (with the naturally-occurring un labelled urea) to a constant specific activity, and the purified product was shown by mass spectrometry to be identical with that of authentic material. Urinary chloroacetic acid metabolites The fact that more than 100 times as much chloroacetic acid was excreted from rats dosed with DCE than had been found m a similar investigation with vinyl chloride, where chloroacetic acid was a minor or negligible met abolite [7], and that in the present work chloroacetic acid may have been formed in vivo through spontaneous rearrangement of 1,1-dichlorocthvlene oxide into chloroacetyl chloride [S], suggested that the metabolism of DCE and chloroacetic acid may be interrelated. This supposition is strongly supported by the fact that several of the urinary metabolites of DCE were found by the previously-described methods to be excreted from animals dosed with chloroacetic acid per se (Table IV). DISCUSSION The foregoing evidence suggests a scheme (Fig. 3). which is economical in 3 ; ; j CMA 050622 39 I s(ch2co2h)2 I HSCHjCOjH 1 (sch2co2h)2 (g) (h) (j) Fig. 3. Scheme for the metabolism of vinyiidene chloride in rats. respect of primary biotransformations, for the metabolism ofDCE(a) (Fig. 3), in which chloroacetic acid (b) occupies a key position. There is a strong supposition that detoxification of chloroacetic acid is effected through the enzyme-catalysed reaction with glutathione and ensuingdegradativesequence for the resulting carboxymethylglutathione (d), and that this represents the principal metabolic pathway for chloroacetic acid and a major one for DCE. Thiodiglyeollic acid (g) is the ultimate detoxification product, and previous work (7j has established the biotransformation of S-<2-earboxymethyl)cysteine (f) into thiodiglyeollic acid (g) by a pathway involving transamina tion and oxidative decarboxylation. (In the present investigation, some evidence was found for the formation of the intermediary lactic and pyruvic acids.) Since thioglycolhc acid (h) may be formed through CS bond fission CMA 050623 40 of the symmetrical thiodiglycollic acid (g), (h) and dithioglycollic acid ij) might be appended from (g). A feasible metabolic pathway of thiodlglycollic acid from chloroaeetie acid and involving cysteine desulphhydrase [EC 4.4.1.1] has now been shown to be unacceptable in experiments with unlabelled DCE in rats, in which the cysteine-cystine pools had been labelled adequately with l4C. Since labelled thiodiglycollic acid resulted, a part of the C-skeleton must be derived in fact from cysteine. Whilst the formation of a small amount of [l4C]dithioglycoIlic acid (and hence of the intermediate [14CJthioglycollie acid) is reconcilable with the predictable action of an unspecific thionase [5], which would lyse a small proportion of the preponderating [IJC]thiodiglycollic acid to give some ['4C]thioglycollic acid, these substances were undetected in a similar experiment with vinyl chloride [7], in which thiodiglycollic acid was also the major metabolite. However, had unlabelled thioglycollic acid and diihiogivcollic acid been produced from DCE via the cysteine desulphhydrase pathway, they would not have been identifiable in the presence of their labelled counterparts. Irrespective of whether [1-UC]DCE or [l-'4C]chloroacetic acid was administered to rats, one molecular proportion of the resulting [ l4C]thiodiglvcollie acid gave 1 equivalent of |4CO; in the micro-scale [9] Kolbe electrolysis reaction [10]. This evidence is utterly consistent with the transformation of (a) into (b) (Fig. 3), which involves migration of one Cl atom and the loss of the other one. Hence, the metabolic pathway (Fig. 3), which was tentatively * proposed for the biotransformation of vinylidene chloride into thiodiglycollie acid, does in fact operate in rats. In view of the established metabolic pathway for thiodiglycollic acid, the formation of the A'-aeetyl-S-cysteinyl-acetyl derivative (el (Fig. 3) by the metabolism of DCE, but not of chloroaeetie acid (Table IV). is rather puzzlmg. However, this evidence must mean that this compound. ie) arises from an earlier metabolite of DCE than chloroaeetie acid. Present work suggests. but does not prove, that this compound may be formed in fact from 1,1- wdichloroethylene oxide through the agency of glutathione S-cpoxide-trans* ferase [EC 4 .7]. to afford S-glutathionyl acetyl chloride tc) (Fig. 3), and its subsequent reactions (v. supra). This supposition is important, since the reactivity displayed by 1.1-diehloropthyleno oxide may be particularly relev ant to the possible interaction of reactive DCE metabolue(s) with mousekidney D.N'A (v. supra). If this should prove to be the case, then such inter action would be analogous to that of vinyl chloride with rat-liver DX.\ m vivo winch, we have found [11], leads to the production of Gu-D-nbofuranosyl-5-oxo-5.G-diliydroimidazo-[ 1,2-c]pyrimidine and SJ-D-nhofuranosyl-imidazo-[2.1-i Jpunne residues and subsequent depurination [11], Another point about the scheme (Fig. 3) concerns the origin of up to <w of CO; in DCE metabolism. Whether this CO; anses(i) through the action of epoxide hydratase (EC 4.2.1.G3] on 1.1-dichlorocthylone oxide and subse quent degradation of resulting intermediates or (ii 1 through the oxidation of chloroaeetie acid is equivocal. Metabolic studies have provided evidenee ol a minor oxidative pathway for chloroaeetie acid, which leads to CO; I-T7 of \ j j = f : 5 f jj jj g j g i ;i i 2 ; J j ! j Qvjft. 050624 the dose) via glycollic acid and oxalic acid; both intermediates have been found in mice [12] and a trace of oxalic acid was detected in rat material in the present work. REFERENCES 1 T. Green and D.E. Hath way, The biological fate in rats of vinyl chloride in relation to its oncogenicity. Chem.-Biol. Interact. 11 (1975) 545--562. 2 H. Baruch. C- Malaveille, R. Montesano and L- Tomatis, Tissue-mediated mutagenicity of vinvlidene chloride and 2-chlorobutadiene in Salmonella tvphimurium. Nature, 255 (1975) 641-643. 3 H. Greim, G. Bonse, Z. Radwan, D. Reichert and D. Henschier. Mutagenicity in vitro and potential carcinogenicity of chlorinated ethylene* as a function of metabolic oxirane formation, Biochem. Pharmacol. 24 (1975) 2013--2017. 4 C. 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