Alterations of the renal function in the isolated perfused rat kidney system after in vivo and in vitro application of S-(1,2-dichlorovinyl)-L-cysteine and S-(2,2-dichlorovinyl)-L-cysteine

 The nephrotoxic effects of the two isomers S-(1,2-dichlorovinyl)-L-cysteine (1,2-DCVC) and S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCVC) were investigated comparatively in the isolated perfused rat kidney with two different treatment regimens. In the first approach, the kidneys were exposed to the test compounds dissolved in the perfusion media after removal from the animal. In the second approach the test compounds were administered to rats in vivo and the nephrotoxicity was assessed in the isolated perfused kidney 6 h and 18 h post-treatment. The vicinal isomer 1,2-DCVC produced concentration- and time-dependent nephrotoxicity with both treatment regimens, as indicated by the impairment of glucose reabsorption, the increase of protein excretion and of γ-glutamyltransferase and alkaline phosphatase activities in urine. In contrast to the marked toxicity observed after in vivo and in vitro administration of 1,2-DCVC, the geminal isomer, 2,2-DCVC, was not nephrotoxic at all concentrations (0.5 and 2.5 mM in vitro, 40 and 70 mg/kg in vivo) investigated. Received: 31 March 1995 / Accepted: 17 July 1995     

N-acetyl S-(1,2-dichlorovinyl)-L-cysteine produces a similar toxicity to S-(1,2-dichlorovinyl)-L-cysteine in rabbit renal slices: differential transport and metabolism

Renal cortical slices were used to determine the toxicity of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (N-acetyl-DCVC) as well as to investigate the transport and metabolism of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and the N-acetyl derivative. N-Acetyl-DCVC produced dose- and time-dependent decreases in intracellular K+ content and lactate dehydrogenase activity. Histopathology demonstrated an initial S3 lesion followed by a lesion inclusive of all proximal tubules. N-Acetyl-DCVC was shown to be transported via the organic anion system by its ability to inhibit PAH transport by the cells and the ability of probenecid to decrease uptake (80%) and toxicity of N-acetyl-DCVC. DCVC, in contrast, was not transported by the organic anion system, but may be transported by one or more amino acid systems. N-Acetyl-DCVC must be deacetylated before undergoing metabolism by beta-lyase. This process must occur since covalent binding of a 35S-labeled reactive product from N-acetyl [35S]DCVC is observed within 1 hr. Both the uptake inhibitor, probenecid, and aminooxyacetic acid (AOAA), a beta-lyase inhibitor, decreased the covalent binding from N-acetyl [35S]DCVC (80 and 50%, respectively), but only AOAA inhibited the covalent binding of DCVC. AOAA also partially inhibited the toxicity of DCVC and N-acetyl-DCVC as determined by intracellular K+ content, lactate dehydrogenase activity, and histopathology. Despite the fact that a separate transport system and an additional enzymatic step (deacetylation) are required, N-acetyl-DCVC produces a lesion with similar intratubular specificity to that seen with DCVC. Therefore, the S3 specificity seen in vivo could be produced by either compound.     

Chronic toxicity of S-(trans-1,2-dichlorovinyl)-L-cysteine in mice

S-(trans-1,2-Dichlorovinyl)-L-cysteine (DCVC) exposure causes acute renal tubular cytotoxicity. To further characterize the effects of DCVC, a chronic study was undertaken. Male Swiss-Webster mice received DCVC dissolved in their drinking water at 0.01, 0.05 and 0.1 mg ml-1. At 4, 8, 21 and 37 weeks, animals were terminated. Bladders, spleens, livers, kidneys and eyes were removed for histopathological examination. At 0.05 and 0.1 mg ml-1 DCVC, growth retardation was evident by 21 weeks. By 26 weeks, all animals in the 0.1 mg ml-1 group had developed cortical cataracts. Cytomegaly, nuclear hyperchromatism and multiple nucleoli were noted in the cells of the pars recta region of the kidney by 4 weeks and correlated to time and dose. At later time points, renal tubular atrophy and early interstitial fibrosis were evident. The epithelial cytological cellular abnormalities appear to be dose-related. Minor pathological changes were noted in the spleen, while there was no effect on the liver or bladder. Chronic ingestion of DCVC results in severe kidney injury.     

N-biotinyl-S-(1,2-dichlorovinyl)-L-cysteine sulfoxide as a potential model for S-(1,2-dichlorovinyl)-L-cysteine sulfoxide: characterization of stability and reactivity with glutathione and kidney proteins in vitro

S-(1,2-Dichlorovinyl)-L-cysteine sulfoxide (DCVCS) is a reactive and potent nephrotoxic metabolite of the human trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine (DCVC). Because DCVCS covalent binding to kidney proteins likely plays a role in its nephrotoxicity, in this study biotin-tagged DCVCS, N-biotinyl-DCVCS (NB-DCVCS), was synthesized, and its stability in buffer alone and in the presence of rat blood or plasma was characterized in vitro. In addition, reactivity toward GSH and covalent binding to selected model enzymes and isolated kidney proteins were characterized. The half-lives of NB-DCVCS (39.6 min) and the DCVCS (diastereomer 1, 14.4 min; diastereomer 2, 6 min) in the presence of GSH were comparable. Incubating the model enzymes glutathione reductase and malate dehydrogenase with 10 μM NB-DCVCS for 3 h at 37 °C followed by immunoblotting using antibiotin antibodies demonstrated that glutathione reductase and malate dehydrogenase were extensively modified by NB-DCVCS. When rat kidney cytosol (6 μg/μL) was incubated with NB-DCVCS (312.5 nM to 5 μM) for 3 h at 37 °C followed by immunoblotting, a concentration-dependent increase in signal with multiple proteins with different molecular weights was observed, suggesting that NB-DCVCS binds to multiple kidney proteins with different selectivity. Incubating rat kidney cytosol with DCVCS (10-100 μM) prior to the addition of NB-DCVCS (2.5 μM) reduced the immunoblotting signal, suggesting that NB-DCVCS and DCVCS compete for the same binding sites. A comparison of the stability of NB-DCVCS and DCVCS in rat blood and plasma was determined in vitro, and NB-DCVCS exhibited higher stability than DCVCS in both media. Collectively, these results suggest that NB-DCVCS shows sufficient stability, reactivity, and selectivity to warrant further investigations into its possible use as a tool for future characterization of the role of covalent modification of renal proteins by DCVCS in nephrotoxicity.     

Detection of multiple globin monoadducts and cross-links after in vitro exposure of rat erythrocytes to S-(1,2-dichlorovinyl)-L-cysteine sulfoxide and after in vivo treatment of rats with S-(1,2-dichlorovinyl)-L-cysteine sulfoxide

S-(1,2-dichlorovinyl)- L-cysteine sulfoxide (DCVCS), a Michael acceptor produced by an FMO3-mediated oxidation of the trichloroethylene metabolite S-(1,2-dichlorovinyl)- L-cysteine (DCVC), is a more potent nephrotoxicant than DCVC. Because DCVCS incubations with N-acetyl- L-cysteine at pH 7.4, 37 degrees C resulted in the formation of three diastereomeric monoadducts and one diadduct, globin monoadducts and cross-links formed after in vitro incubations of rat erythrocytes with DCVCS (0.9-450 microM) for 2 h and those present at 30 min after in vivo treatment of rats with DCVCS (23 and 230 micromol/kg) were characterized. ESI/MS of intact globin chains revealed adduction of 1 DCVCS moiety on the beta2 chain at the three lowest DCVCS concentrations and on the beta1 chain after the in vivo treatment with 230 micromol/kg DCVCS. Interestingly, intact globin dimers and trimers were detectable by ESI/MS with all DCVCS concentrations in vitro (also by SDS-PAGE) and in vivo. LC/MS and MALDI/FTICR of trypsin digested peptides from globin samples obtained after in vitro (450 microM DCVCS) or in vivo exposure to DCVCS (230 micromol/kg) suggested the formation of DCVCS monoadducts not only with Cys93 and Cys125 of the beta chains but also with Cys13 of the alpha chains, whereas no monoadducted peptides were detected at lower DCVCS concentrations in vitro or in vivo. However, LC/MS and MALDI-TOF/TOF suggested the presence of several DCVCS-derived peptide cross-links both in vivo and in vitro at all DCVCS exposure levels. Collectively, the results indicate at least 4 out of the 5 cysteine moieties of the rat hemoglobin heterodimer may be alkylated by DCVCS, in reactions that could also lead to the formation of multiple cross-links. DCVCS- and N-acetyl-DCVCS (NA-DCVCS)-derived globin cross-links containing GSH and Cys were also detected by mass spectrometry, providing strong evidence for the reactivity and/or cross-linking ability of DCVCS, NA-DCVCS, and their GSH or Cys conjugates in both the in vitro and the in vivo. Thus, hemoglobin adducts and cross-links may be useful biomarkers to investigate the possible presence of DCVCS in circulation after DCVC or trichloroethylene exposure.     

Metabolism and toxicity of trichloroethylene and S-(1,2-dichlorovinyl)-L-cysteine in freshly isolated human proximal tubular cells.

Trichloroethylene (Tri) caused modest cytotoxicity in freshly isolated human proximal tubular (hPT) cells, as assessed by significant decreases in lactate dehydrogenase (LDH) activity after 1 h of exposure to 500 microM Tri. Oxidative metabolism of Tri by cytochrome P-450 to form chloral hydrate (CH) was only detectable in kidney microsomes from one patient out of four tested and was not detected in hPT cells. In contrast, GSH conjugation of Tri was detected in cells from every patient tested. The kinetics of Tri metabolism to its GSH conjugate S-(1,2-dichlorovinyl)glutathione (DCVG) followed biphasic kinetics, with apparent Km and Vmax values of 0.51 and 24.9 mM and 0.10 and 1.0 nmol/min per mg protein, respectively. S-(1,2-dichlorovinyl)-L-cysteine (DCVC), the cysteine conjugate metabolite of Tri that is considered the penultimate nephrotoxic species, caused both time- and concentration-dependent increases in LDH release in freshly isolated hPT cells. Preincubation of hPT cells with 0.1 mM aminooxyacetic acid did not protect hPT cells from DCVC-induced cellular injury, suggesting that another enzyme besides the cysteine conjugate beta-lyase may be important in DCVC bioactivation. This study is the first to measure the cytotoxicity and metabolism of Tri and DCVC in freshly isolated cells from the human kidney. These data indicate that the pathway involved in the cytotoxicity and metabolism of Tri in hPT cells is the GSH conjugation pathway and that the cytochrome P-450-dependent pathway has little direct role in renal Tri metabolism in humans.     

Thioacylating intermediates as metabolites of S-(1,2-dichlorovinyl)-L-cysteine and S-(1,2,2-trichlorovinyl)-L-cysteine formed by cysteine conjugate beta-lyase

The bioactivation mechanism of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) was studied with cysteine conjugate beta-lyase (beta-lyase) from Salmonella typhimurium and with the pyridoxal phosphate model N-dodecylpyridoxal bromide (PL-Br) as catalysts and with GC/MS to identify the metabolites formed. PL-Br converted S-2-benzothiazolyl-L-cysteine to 2-mercaptobenzothiazole and S-benzyl-L-cysteine to benzyl mercaptan, demonstrating the ability of PL-Br to serve as a model for beta-lyase. PL-Br and bacterial beta-lyase converted DCVC to chloroacetic acid and chlorothionoacetic acid and TCVC to dichloroacetic acid. Incubations of PL-Br with the S-conjugates in the presence of diethylamine resulted in the formation of N,N-diethylchlorothioacetamide from DCVC and of N,N-diethyldichlorothioacetamide from TCVC. Attempts to trap the enethiols, which are the expected initial products formed by beta-elimination, by reaction with methyl iodide in incubations with the beta-lyase model were not successful. The formation of thioacylating agents from the enethiols may contribute to the cytotoxic and mutagenic effects of DCVC and TCVC.     

Early biological indicators of S-(1,2-dichlorovinyl)-L-cysteine nephrotoxicity in the rabbit

The progression of changes in rabbit kidney function following dosing with the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVC, 20-50 mg/kg) was determined. Proteinuria was observed 0.5-1 hr after administration of DCVC at doses of 20-50 mg/kg. Blood urea nitrogen levels, glomerular filtration rates, urinary glucose excretion, and urine volume were also altered following DCVC dosing; however, these parameters were less sensitive than proteinuria as markers of early renal dysfunction. None of these latter four indicators were affected by low DCVC doses, nor were they altered by high DCVC doses until 1.5-2.5 hr after dosing. Dose-dependent morphological changes to kidney structure were also observed 5 hr after DCVC administration. Low doses caused damage restricted to brush border membranes in the pars recta, while higher doses produced a necrotic lesion encompassing all regions of the proximal tubule. This study indicates that DCVC can cause rapid renal dysfunctional changes which are first detected by elevated urinary protein excretion.     

Studies on the comparative toxicity of S-(1,2-dichlorovinyl)-L-cysteine,S-(1,2-dichlorovinyl)-L-homocysteine and 1,1,2-trichloro-3,3,3-trifluoro-1-propene in the Fischer 344 rat

The renal tubular toxicity of various halogenated xenobiotics has been attributed to their enzymatic bioactivation to reactive intermediates by S-conjugation. A combination of high resolution proton nuclear magnetic resonance (¹H NMR) spectroscopy of urine, renal histopathology and more routinely used clinical chemistry methods has been used to explore the acute toxic and biochemical effects of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) and 1,1,2-trichloro-3,3,3-trifluoro-1-propene (TCTFP) up to 48 h following their administration to male Fischer 344 (F344) rats. In the absence of gross renal pathology, ¹H NMR urinalysis revealed increased excretion of the tricarboxylic acid cycle intermediates citrate and succinate following DCVC administration. In contrast, both DCVHC and TCTFP produced functional defects in the S₂ and S₃ segments of the proximal tubule that were confirmed histologically. In these cases, ¹H NMR urinalysis revealed increased excretion of glucose, L-lactate, acetate and 3-D-hydroxybutyrate (HB) as well as selective amino aciduria (alanine, valine, glutamate and glutamine). The significance of the proximal nephropathies induced by DCVHC and TCTFP is discussed in relation to biochemical observations on other xenobiotics that are toxic by similar mechanisms. Received: 25 April 1994 / Accepted: 13 June 1994     

Alpha-ketoacids stimulate rat renal cysteine conjugate beta-lyase activity and potentiate the cytotoxicity of S-(1,2-dichlorovinyl)-L-cysteine

Renal cysteine conjugate beta-lyase (beta-lyase) catalyzes the bioactivation of nephrotoxic cysteine S-conjugates. beta-Lyase activity is present in both renal cytosolic and mitochondrial fractions, and, although the cytosolic beta-lyase is identical to glutamine transaminase K, the mitochondrial beta-lyase has not been characterized. Because beta-lyase is a pyridoxal phosphate (PLP)-dependent enzyme, pyridoxamine phosphate (PMP) formation may occur during the metabolism of cysteine S-conjugates. In this study, the effects of alpha-ketoacids, which may convert the PMP form of the enzyme to the pyridoxal phosphate form, on the metabolism and cytotoxicity of cysteine S-conjugates were examined; the PMP enzyme is catalytically inactive in beta-elimination reactions, but is catalytically active in transamination reactions. Both alpha-keto-gamma-methiolbutyrate (KMB) and alpha-ketobutyrate enhanced the metabolism of S-(2-benzothiazolyl)-L-cysteine (BTC) to 2-mercaptobenzothiazole by rat renal cytosol or mitochondria. KMB and phenylpyruvate potentiated both the cytotoxicity of S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in isolated rat renal proximal tubular cells and the inhibition of mitochondrial respiration produced by DCVC. These results are consistent with the formation of PMP during the renal cytosolic or mitochondrial metabolism of cysteine S-conjugates. Mitochondrial beta-lyase was previously localized in the outer membrane. To examine whether beta-lyase activity is present in mitoplasts, but in the PMP form, the effects of KMB on the metabolism of BTC to 2-mercaptobenzothiazole and on the DCVC-induced inhibition of state 3 respiration in mitoplasts were studied. The majority of the mitochondrial beta-lyase activity was present in the outer membrane, and the specific activity of the outer membrane beta-lyase was greater than that of the mitoplast beta-lyase. KMB produced equivalent stimulation of beta-lyase activity in intact mitochondria, in mitochondrial outer membranes, and in mitoplasts and potentiated DCVC-induced inhibition of respiration in intact mitochondria, but not in mitoplasts. These results provide additional evidence for the central role of beta-lyase in the bioactivation of nephrotoxic cysteine S-conjugates.     

Correlation of the in vivo and in vitro renal toxicity of S-(1,2-dichlorovinyl)-L-cysteine

The major site at which vinyl cysteine conjugates exert nephrotoxicity is the proximal tubule. Since this is the site of all active anion and cation transport, tubule transport integrity was used to assess nephrotoxicity. Tubules were isolated from young rabbits to study the in vivo and in vitro nephrotoxicity of the conjugate, dichlorovinyl cysteine (DCVC). In vivo exposure to DCVC caused necrosis in the pars recta region of the proximal tubules (20-100 mg/kg ip) and a dose-dependent decrease in tubular active transport. Addition of DCVC to the perfused kidney and tubule suspensions resulted in similar decreases in tubular organic ion transport. At 0.01 mM DCVC, transport was similar to controls while 1 mM DCVC completely inhibited active accumulation of the organic ions. Thus kidney tubule active transport is similarly inhibited in vivo and in vitro by DCVC indicating that bioactivation of DCVC and inhibition of active transport occur directly in the renal tubule.     

Role of mitochondrial dysfunction in cellular responses to S-(1,2-dichlorovinyl)-L-cysteine in primary cultures of human proximal tubular cells

The nephrotoxic metabolite of the environmental contaminant trichloroethylene, S-(1,2-dichlorovinyl)-l-cysteine (DCVC), is known to elicit cytotoxicity in rat and human proximal tubular (rPT and hPT, respectively) cells that involves inhibition of mitochondrial function. DCVC produces a range of cytotoxic and compensatory responses in hPT cells, depending on dose and exposure time, including necrosis, apoptosis, repair, and enhanced cell proliferation. The present study tested the hypothesis that induction of mitochondrial dysfunction is an obligatory step in the cytotoxicity caused by DCVC in primary cultures of hPT cells. DCVC-induced necrosis was primarily a high concentration (> or =50 microM) and late (> or =24h) response whereas apoptosis and increased proliferation occurred at relatively low concentrations (<50 microM) and early time points (< or =24h). Decreases in cellular DNA content, indicative of cell loss, were observed at DCVC concentrations as low as 1 microM. Involvement of mitochondrial dysfunction in DCVC-induced cytotoxicity was supported by showing that DCVC caused modest depletion of cellular ATP, inhibition of respiration, and activation of caspase-3/7. Cyclosporin A protected cells against DCVC-induced apoptosis and both cyclosporin A and ruthenium red protected cells against DCVC-induced loss of mitochondrial membrane potential. DCVC caused little or no activation of caspase-8 and did not significantly induce expression of Fas receptor, consistent with apoptosis occurring only by the mitochondrial pathway. These results support the conclusion that mitochondrial dysfunction is an early and obligatory step in DCVC-induced cytotoxicity in hPT cells.     

Mechanism of S-(1,2-dichlorovinyl)-L-cysteine- and S-(1,2-dichlorovinyl)-L-homocysteine-induced renal mitochondrial toxicity

The mechanism by which the nephrotoxic S-conjugates S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) produce toxicity in rat kidney mitochondria was studied by examining their effects on mitochondrial function, structural integrity, and metabolism. Both S-conjugates inhibited succinate-linked state 3 respiration and impaired the ability of mitochondria to retain Ca2+ and to generate a membrane potential; 30-60 min were required for maximal expression of these functional changes. Mitochondrial structure was damaged, as indicated by enhanced polyethylene glycol-induced shrinkage of matrix volume and by leakage of protein and malic dehydrogenase from the matrix; 60-120 min were required for maximal expression of these structural changes. Much shorter incubation times (15-30 min) were required for DCVC and DCVHC to decrease ATP concentrations, to alter the concentrations of several citric acid cycle intermediates, and to inhibit succinate:cytochrome c oxidoreductase and isocitrate dehydrogenase activities. Lipid peroxidation and oxidation of glutathione to glutathione disulfide also occurred. The relative time courses of these pathological changes indicate that the initial effects of DCVC and DCVHC in renal mitochondria are the inhibition of energy metabolism and the oxidation of glutathione. These changes then lead to alterations in mitochondrial function and ultimately to irreversible damage to mitochondrial structure.     

Apoptosis, necrosis, and cell proliferation induced by S-(1,2-dichlorovinyl)-L-cysteine in primary cultures of human proximal tubular cells.

Apoptosis, necrosis, and cell proliferation induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), the cysteine conjugate of the environmental and occupational contaminant trichloroethylene, were studied in primary cultures of human proximal tubular (hPT) cells. Cells from male and female donors were incubated with a range of concentrations of DCVC (10 to 1000 microM) for up to 48 h, and assessments of cellular morphology (phase-contrast microscopy), necrosis (lactate dehydrogenase (LDH) release), apoptosis(cell cycle analysis, annexin V staining, and caspase activation), and proliferation (cell cycle analysis and DNA synthesis) were made. Time- and concentration-dependent changes in cellular morphology, including elongation of cell shape, formation of intracellular vesicles, and formation of apoptotic bodies, were observed. Significant increases in LDH release occurred in hPT cells incubated with < or =100 microM DCVC for at least 24 h. hPT cells from males were modestly more sensitive to DCVC than those from females, with maximal LDH release of 78 and 65% in cells from males and females, respectively. Flow cytometry analysis of propidium iodide-stained and DCVC-treated hPT cells showed that apoptosis occurred at markedly lower concentrations (10 microM) and at much earlier incubation times (2 h) than necrosis. A small increase was also noted in the percentage of cells in S-phase after a 4-h treatment with as little as 10 microM DCVC, suggesting that cell proliferation was stimulated. This was supported further by increased DNA synthesis. These results show that DCVC causes apoptosis and enhances cell proliferation in hPT cells at environmentally relevant doses and at earlier time points and lower concentrations than necrosis.     

Cytotoxicity of trichloroethylene and S-(1, 2-dichlorovinyl)-L-cysteine in primary cultures of rat renal proximal tubular and distal tubular cells

Activities of several glutathione-dependent enzymes, expression of cytochrome P450 isoenzymes, and time- and concentration-dependent cytotoxicity of trichloroethylene (TRI) and S-(1, 2-dichlorovinyl)-L-cysteine (DCVC) were evaluated in primary cultures of proximal tubular (PT) and distal tubular (DT) cells from rat kidney. These cells exhibited cytokeratin staining and maintained activities of all glutathione-dependent enzymes measured. Of the cytochrome P450 isoenzymes studied, only CYP4A expression was detected. CYP4A mRNA and protein expression were higher in primary cultures of DT cells than in PT cells and were increased in DT cells by ciprofibrate treatment. Incubation of cells for 6 h with concentrations of TRI as high as 10 mM resulted in minimal cytotoxicity, as determined by release of lactate dehydrogenase (LDH). In contrast, marked cytotoxicity resulted from incubation of PT or DT cells with DCVC. Addition to cultures of TRI (2-10 mM) for 24 or 72 h resulted in modest, but significant time- and concentration-dependent increases in LDH release. Treatment of cells with DCVC (0.1-1 mM) for 24 h caused significant increases in LDH release and alterations in cellular protein and DNA content. Finally, exposure of primary cultures to TRI or DCVC for 72 h followed by 3 h of recovery caused a slight increase in the expression of vimentin, consistent with cellular regeneration. These studies demonstrate the utility of the primary renal cell cultures for the study of CYP4A expression and mechanisms of TRI-induced cellular injury.     

Mechanism of S-(1,2-dichlorovinyl)glutathione-induced nephrotoxicity

S-(1,2-Dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-DL-cysteine are potent nephrotoxins. Agents that inhibit gamma-glutamyl transpeptidase, cysteine conjugate beta-lyase, and renal organic anion transport systems, namely L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125), aminooxyacetic acid, and probenecid, respectively, protected against S-conjugate-induced nephrotoxicity. Furthermore, S-(1,2-dichlorovinyl)-DL-alpha-methylcysteine, which cannot be cleaved by cysteine conjugate beta-lyase, was not nephrotoxic. These results strongly support a role for renal gamma-glutamyl transpeptidase, cysteine conjugate beta-lyase, and organic anion transport systems in S-(1,2-dichlorovinyl)glutathione- and S-(1,2-dichlorovinyl)cysteine-induced nephrotoxicity.     

Identification of N-acetyl(2,2-dichlorovinyl)- and N-acetyl(1,2-dichlorovinyl)-L-cysteine as two regioisomeric mercapturic acids of trichloroethylene in the rat

The regioselectivity of glutathione conjugation to trichloroethylene (TRI) and the metabolism of its cysteine and N-acetyl-L-cysteine conjugates were investigated in the rat. Intraperitoneal (ip) administration of TRI to rats at a dose of 400 mg/kg resulted in excretion in urine of small amounts of the two distinct regioisomers N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (1,2-DCV-NAC) and N-acetyl-S-(2,2-dichlorovinyl)-L-cysteine (2,2-DCV-NAC). The vicinal (vic) isomer was excreted in a 2 times higher amount (16 nmol) than the geminal (gem) isomer (8 nmol). Intraperitoneal administration of a 1:1 mixture (2.5 mumol/kg each) of the two regioisomers of S-(dichlorovinyl)-L-cysteine (DCVC) to the rat resulted in excretion of the corresponding mercapturic acids in urine, the main fractions being excreted within 8 h after administration. The gem-dichlorovinyl isomer appeared to be acetylated to a higher extend than the 1,2-dichlorovinyl isomer; 73% vs 50% of the dose administered. Intraperitoneal administration of a 1:1 mixture (12.5 mumol/kg each) of the two regioisomers of N-(trideuterioacetyl)-S-(dichlorovinyl)-L-cysteine (DCV-NAC-d3) resulted in excretion of both deuterium-labeled and unlabeled mercapturic acids in urine. The vic isomer was excreted unchanged at a significantly higher percentage, 34% of dose (i.e., still deuterium labeled), than the gem isomer, 17% of the dose. This suggests less efficient metabolism of the vic isomer when compared to the gem isomer. Both regioisomers of DCV-NAC-d3 were excreted in urine unlabeled at 40% of the dose, which indicates that for both isomers deacetylation, followed by reacetylation (resulting in unlabeled DCV-NAC), is an important metabolic pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

A mechanism of S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L-cysteine toxicity to rabbit renal proximal tubules

S-(1,2,3,4,4-Pentachloro-1,3-butadienyl)-L-cysteine (PCBC) has been identified as the penultimate compound responsible for hexachlorobutadiene-induced nephrotoxicity. The primary goal of these studies was to determine the mechanism of PCBC-induced toxicity in rabbit renal proximal tubules by examining the early changes in tubular physiology. PCBC (20-500 microM) induced a specific sequence of toxic events. Following 15 min of exposure, 200 microM PCBC increased basal (25%) and ouabain-insensitive (78%) respiration. This was followed by a decrease in basal (46%), nystatin-stimulated (54%), and ouabain-insensitive (21%) respiration and a decrease in glutathione content (79%). Finally, there was a decrease in cell viability as measured by a decrease in LDH retention at 60 min. Direct probing of mitochondrial function revealed that the initial increase in respiration resulted from the uncoupling of oxidative phosphorylation, while the late changes in respiration appeared to result from gross mitochondrial damage characterized by inhibited state 3 respiration, inhibited cytochrome c-cytochrome oxidase, and inhibited electron transport. Studies utilizing tubules with decreased glutathione content revealed that glutathione plays little if any role in the early events of PCBC-induced toxicity. These results suggest that PCBC-induced mitochondrial dysfunction may initiate the renal proximal tubule injury.