Nephrotoxic and genotoxic N-acetyl-S-dichlorovinyl-L-cysteine is a urinary metabolite after occupational 1,1,2-trichloroethene exposure in humans: implications for the risk of trichloroethene exposure.

Excretion of mercapturic acids in the urine is indicative of the formation of electrophiles in the metabolism of xenobiotics. The determination of these mercapturic acids thus may be a useful method to estimate the exposure. We identified the nephrotoxic and mutagenic mercapturic acids N-acetyl-S-(1,2-dichlorovinyl)-L- cysteine and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine in the urine of workers exposed to 1,1,2-trichloroethene. A method to quantify these mercapturic acids by gas chromatography-mass spectrometry-selected ion monitoring was developed and appreciable amounts (2.8-3.8 mumole/L were found in human urine samples. Because deacetylation determines notably the amount of the excreted mercapturic acids, the formation of the resulting cysteine S-conjugates was comparably measured in subcellular fractions of rodent and human kidneys; significant species differences in acylase activity were found. The formation of mutagenic and nephrotoxic metabolites during 1,1,2-trichloroethene metabolism mandates a revision of the risk assessment of trichloroethene exposure.


Introduction
1,1,2-Trichloroethene (trichloroethene) is an important industrial chemical which is widely used because ofits favorable solvent characteristics, chemical stability, and relatively low acute toxicity. The carcinogenicity of trichloroethene has been extensively debated over the past 15 years. High doses of trichloroethene increase the rate of hepatocellular carcinomas in B6C3F, mice and induce renal tubular adenocarcinoma in male Fischer F344 rats (1). Bioactivation reactions are likely responsible for trichloroethene carcinogenicity. In both rats and mice, trichloroethene is metabolized by two pathways, oxidation by cytochrome P-450 and conjugation with glutathione by glutathione-S-transferases (Fig. 1). Glutathione (GSH) conjugation to S-(1,2-dichlorovinyl)glutathione (DCVG) might initiate trichloroethene nephrocarcinogenicity. DCVG is cytotoxic, mu-'Institut fir Toxikologie, Universitat Wurzburg, Versbacher Strasse 9, D-8700 Wurzburg, Federal Republic of Gernany.
tagenic, and nephrotoxic. The formed DCVG is cleaved by the enzymes ofmercapturic acid formation to S-(1,2-dichlorovinyl)-L-cysteine (DCVC). After accumulation in the kidney by active transport mechanisms, DCVC is a substrate for renal cysteine conjugate (3-lyase and is cleaved to yield pyruvate, ammonia, and the electrophile chlorothioketene (2). Evidence for the occurrence ofthis pathway in trichloroethene metabolism in vivo has been obtained by the identification in rat urine of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, the excretory product of the processing of DCVG by the enzymes ofmercapturic acid formation (3,4). DCVG was also identified as a biliary metabolite of trichloroethene in rats in vivo and as the product oftrichloroethene biotransformation in rat liver microsomes in the presence of GSH.
Trichloroacetic acid, a metabolite formed by oxidative metabolism, which is the major pathway oftrichloroethene metabolism in vivo, induced peroxisome proliferation selectively in mouse liver, a mechanism that may explain the hepatocarcinogenicity oftrichloroethene in mice (5). This process may not be relevant for human carcinogenic risk of trichloroethene exposure at lower doses, as oxidative metabolism does not result in mutagenic products. The experiments reported here demon-

Urine Collection
Urine was collected from individuals exposed to varying amounts of technical trichloroethene (purity not specified) during an 8-hr work shift when cleaning metal parts in a trichloroethene bath. Urine was collected for 16 hr after exposure and pooled.

Determination of Urinary Metabolites
Urinary trichloroacetic acid concentrations were determined according to the method of Tanaka and Ikeda (6). N-acetyl-Sdichlorovinyl-L-cysteine was quantified by GC-MS with selected ion monitoring (SIM) after a two-step concentration procedure. Briefly, the internal standard N-d3-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (125 nmole) was added to 2 mL of the urine. The urine was then adjusted to pH 1 with concentrated HCI and extracted twice with ether. The ether extracts were evaporated and the residue further purified with a C18 column (Millipore). The obtained solution was dried and treated with borontrichloride/methanol for esterification. GC-MS was performed with a HP5890 gas chromatograph with mass selective detector (MSD) (3). For selected ion monitoring, the fragments m/z 144 and 147 were monitored during the gas chromatographic separation. For identification of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine. and N-acetyl-S-(2,2-dichlovinyl)-L-cysteine, the pooled urine samples were adjusted to pH 1 with HCI and extracted with ether. The extract wars then fractionated by highperformance liquid chromatography (Partisil ODS 11, 80 x 250 mm, 5 Im, solvent A: TFA/H20, pH 2, B: methanol; A to B in
Quantification of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(2,2-dichlorovinyl)-Lcysteine By purification of urine and the use of the highly sensitive selected ion monitoring mode for GC-MS, the method used permitted the detection of 10 pmole of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine and the quantification of amounts between 50 and 10,000 nmole/L ofhuman urine. The trideuterated mercapturic acid was used as internal standard to compensate for the loss of mercapturic acid during sample work-up. As shown in Table 1, humans exposed to trichloroethene excreted appreciable amounts of the two isomers ofN-acetyl-S-dichlorovinyl-L-cysteine in urine. The SIM method used did not permit us to discriminate between Nacetyl-S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine due to different separation conditions. Relative to the excreted amounts of trichloroacetic acid in mice and rat urine, human urine contained higher concentrations of the mercapturates (Tables 1 and 2).
Mercapturic acid biosynthesis and urinary excretion are multistep pathways that may be influenced by species-dependent differences in the activities ofthe individual enzymes involved. The concentration of mercapturates in urine may be dependent on the balance between deacetylation of the mercapturic acid and acetylation of the cysteine S-conjugates (4). Deacetylation OHI 'OH generates substrates for the final bioactivation step, the cysteine conjugate (3-lyase-catalyzed reaction, in trichloroethene metabolism by glutathione conjugation. It may thus influence the excretion rates ofmercapturic acids. Wetherefore studiedthedeacetylation ofN-acetyl-S-(l,2-dichlorovinyl)-L-cysteine in kidney cytosol from different species (Table 3). Cytosol from NMRI mice showed the highest activity for N-acetyl-S-(1,2-dichlorovinyl)-Lcysteine followed by the kidney cytosol from Fischer 344 rats. Human kidney cytosol and cytosol from the Wistar strain of rats revealed comparable rates, suggesting that the high excretion rates ofN-acetyl-S-(1,2-dichlorovinyl)-L-cysteine in humans may be due to more intensive metabolism oftrichloroethene by GSH conjugation in humans than in Wistar rats. Our results demonstrate that N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine is a urinary metabolite of trichloroethene in occupationally exposed humans, which is excreted in substantial concentrations. The identification of this metabolite in human urine mandates a revision ofthe risk assessment oftrichloroethene exposure. The precursor of N-acetyl-S-(1,2-dichlorovinyl)-Lcysteine, S-(1,2-dichlorovinyl)-L-cysteine, is a potent mutagen in the Ames test; its mutagenicity depends on bioactivation by ,Blyase (7). Both S-dichlorovinyl-L-cysteine isomers are substrates for the j-lyase in rat kidney cytosol (Bimer et al., unpublished data). Enzymes with ,B-lyase activity are present in human kidney, thus, formation of mutagenic metabolites is expected to occur in the kidney during trichloroethene metabolism. S-(1,2-dichlorovinyl)-L-cysteine is a weak inducer of DNA repair in mammalian kidney cells (8). In these cells it also induces cell proliferation, indicating the potential ofthis metabolite to exert both initiating and promoting activities in the renal tissue. Our results suggest that a mutagenic and nephrotoxic metabolite is formed in human trichloroethene metabolism and therefore a risk of nephrocarcinogenesis is associated with trichloroethene exposure.