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     

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.     

Bone marrow and renal injury associated with haloalkene cysteine conjugates in calves

 Almost 40 years ago, it was reported that cattle-feed which had been extracted with hot trichloroethylene and then fed to calves produced renal injury and a fatal aplastic anaemia. The toxic factor was subsequently identified as S-(1,2-dichlorovinyl)-L-cysteine (DCVC). These original findings have been confirmed, a single intravenous dose of DCVC at 4 mg/kg, or 0.4 mg/kg intravenously per day administered for 10 days to calves produced aplastic anaemia, and renal injury after a single dose of 4 mg/kg. The toxicity to calves of a number of other haloalkene cysteine conjugates has been examined to ascertain whether, like DCVC, they produce bone marrow and renal injury. Intravenous administration of the N-acetyl cysteine conjugate of DCVC produced renal but not bone marrow injury at a molar equivalent dose to DCVC, indicating that the calf can deacetylate the mercapturic acid and further that sufficient chemical had reached the kidney to be a substrate for the enzyme cysteine conjugate β-lyase. However, intravenous administration of the α-methyl analogue of DCVC, which cannot undergo metabolism via the enzyme cysteine conjugate β-lyase, was without toxicity at doses about five-fold higher than DCVC. These latter findings provide strong evidence that metabolism of DCVC via the enzyme β-lyase is necessary for bone marrow and renal injury to occur. The cysteine conjugates of perchloro ethylene and hexachloro-1,3-butadiene(HCBD) when given intravenously to calves at molar equivalent doses to DCVC, or above, did not produce either bone marrow or renal injury. In contrast, intravenous administration of the cysteine conjugate of tetrafluoroethylene (TFEC) produced severe renal tubular injury in calves without affecting the bone marrow. In vitro studies with these haloalkene cysteine conjugates showed, like DCVC, that they were good substrates for calf renal cysteine conjugate β-lyase and toxic to renal cells as judged by their ability to reduce organic anion and cation transport by slices of calf renal cortex and inhibit the renal enzyme glutathione reductase. Calves were also dosed either orally or intravenously with HCBD to assess its toxicity. HCBD at higher molar equivalent doses than DCVC produced mid-zonal necrosis in the liver, renal tubular necrosis but no bone marrow injury in calves. The key findings emerging from these studies are (1) that none of the other cysteine conjugates, at molar equivalent doses to DCVC and above, produce bone marrow injury in calves, (2) TFEC produced only renal injury, suggesting that sufficient of the other conjugates had not reached the kidney for metabolism by β-lyase to produce cytotoxicity and (3) that HCBD itself is more toxic than its cysteine or mercapturic acid conjugate, suggesting that pharmacokinetics and disposition are important factors in determining the toxicity of these conjugates to calves. Further studies are needed to understand the basis for the selective toxicity of DCVC to the bone marrow of calves. Received: 16 October 1995/Accepted: 9 January 1996     

Cellular effects of reactive intermediates: Nephrotoxicity of S-conjugates of amino acids

Several cysteine S-conjugates are potent nephrotoxins and require enzymatic activation to produce cytotoxicity. Strategies based on the knowledge that renal cysteine conjugate -lyase is apparently a pyridoxal phosphate (PLP)-dependent enzyme have been exploited to test the hypothesis that a -lyase-dependent activation is required for the expression of cysteine S-conjugate-induced toxicity. First, the toxicity of the model conjugate S-(1,2-dichlorovinyl)-L-cysteine (DCVC) is blocked both in vivo and in isolated, renal proximal tubular cells by aminooxyacetic acid, an inhibitor of PLP-dependent enzymes. Second, the nonmetabolizable -methyl analogue S-(1,2-dichlorovinyl)-DL--methylcysteine is not toxic. Third, to test the hypothesis that the toxicity of DCVC is associated with the metabolic formation of a reactive thiol, S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC), which may undergo a PLP-dependent -elimination reaction to produce an identical thiol, was studied. DCVHC is a potent nephrotoxin, and, similar to DCVC, its toxicity was blocked by aminooxyacetic acid and the -methyl analogue S-(1,2-dichlorovinyl)-DL--methylhomocysteine was not toxic. Moreover, exposure of renal proximal tubular cells to propargylglycine, a suicide substrate for PLP-dependent enzymes that catalyze -elimination reactions, blocked the toxicity of DCVHC. Fourth, the renal mitochondrial -lyase is localized in the outer membrane; therefore, although DCVC was toxic to mitochondria, no toxicity was produced in mitoplasts, which shows that a suborganelle site of activation is involved in the mitochondrial toxicity of DCVC. Finally, the toxicity of both DCVC and DCVHC was blocked by probenecid, indicating a role for the anion transport system. DCVC and DCVHC inhibit cellular and mitochondrial respiration, indicating that mitochondria are primary intracellular targets for nephrotoxic S-conjugates. Thus, the nephrotoxicity of cysteine and homocysteine S-conjugates is dependent on enzymatic activation to produce a reactive thiol, which is involved in the production of cytotoxicity.Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthdayThis research was supported by National Institute of Environmental Health Sciences grant ES03127 to M. W. A.L. H. L. was supported by N. I. E. H. S. Institutional Research Service Award ES07026     

Transport of N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2)

N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-l-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study, we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mouse proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC.     

Transport and activation of S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine in rat kidney proximal tubules

An important step in understanding the mechanism underlying the tubular specificity of the nephrotoxicity of toxic cysteine conjugates is to identify the rate-limiting steps in their activation. The rate-limiting steps in the activation of toxic cysteine conjugates were characterized using isolated proximal tubules from the rat and 35S-labeled S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine (NAC-DCVC) as model compounds. The accumulation by tubules of 35S radiolabel from both DCVC and NAC-DCVC was time and temperature dependent and was mediated by both Na+-dependent and independent processes. Kinetic studies with DCVC in the presence of sodium revealed the presence of two components with apparent Km and Vmax values of (1) 46 microM and 0.21 nmol/mg min and (2) 2080 microM and 7.3 nmol/mg.min. NAC-DVVC uptake was via a single system with apparent Km and Vmax values of 157 microM and 0.65 nmol/mg.min, respectively. Probenecid, an inhibitor of the renal organic anion transport system, inhibited accumulation of radiolabel from NAC-DCVC, but not from DCVC. The covalent binding of 35S label to cellular macromolecules was much greater from [35S]DCVC than from NAC-[35S]DCVC. Analysis of metabolites showed that a substantial amount of the cellular NAC-[35S]DCVC was unmetabolized while [35S]DCVC was rapidly metabolized to bound 35S-labeled material and unidentified products. The data suggest that DCVC is rapidly metabolized following transport, but that activation of NAC-DCVC depends on a slower rate of deacetylation. The results are discussed with regard to the segment specificity of cysteine conjugate toxicity and the role of disposition in vivo in the nephrotoxicity of glutathione conjugates.     

Pharmacokinetic analysis of trichloroethylene metabolism in male B6C3F1 mice: Formation and disposition of trichloroacetic acid, dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-l-cysteine

Trichloroethylene (TCE) is a well-known carcinogen in rodents and concerns exist regarding its potential carcinogenicity in humans. Oxidative metabolites of TCE, such as dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are thought to be hepatotoxic and carcinogenic in mice. The reactive products of glutathione conjugation, such as S-(1,2-dichlorovinyl)-l-cysteine (DCVC), and S-(1,2-dichlorovinyl) glutathione (DCVG), are associated with renal toxicity in rats. Recently, we developed a new analytical method for simultaneous assessment of these TCE metabolites in small-volume biological samples. Since important gaps remain in our understanding of the pharmacokinetics of TCE and its metabolites, we studied a time-course of DCA, TCA, DCVG and DCVG formation and elimination after a single oral dose of 2100 mg/kg TCE in male B6C3F1 mice. Based on systemic concentration-time data, we constructed multi-compartment models to explore the kinetic properties of the formation and disposition of TCE metabolites, as well as the source of DCA formation. We conclude that TCE-oxide is the most likely source of DCA. According to the best-fit model, bioavailability of oral TCE was ∼ 74%, and the half-life and clearance of each metabolite in the mouse were as follows: DCA: 0.6 h, 0.081 ml/h; TCA: 12 h, 3.80 ml/h; DCVG: 1.4 h, 16.8 ml/h; DCVC: 1.2 h, 176 ml/h. In B6C3F1 mice, oxidative metabolites are formed in much greater quantities (∼ 3600 fold difference) than glutathione-conjugative metabolites. In addition, DCA is produced to a very limited extent relative to TCA, while most of DCVG is converted into DCVC. These pharmacokinetic studies provide insight into the kinetic properties of four key biomarkers of TCE toxicity in the mouse, representing novel information that can be used in risk assessment.     

Species differences in the nephrotoxic response to S-(1,2-dichlorovinyl)glutathione.

The present study was carried out to investigate the species differences in the nephrotoxic response to S-(1,2-dichlorovinyl)glutathione (DCVG) using rats, hamsters and guinea-pigs. DCVG was given intraperitoneally in physiological saline to groups of 5 animals at doses 0, 165 and 330 μmol/kg. Urine was collected for 24 h and the animals were then sacrificed. Significantly increased levels of urinary glucose, , γ-glutamyl transpeptidase, proteins and blood urea nitrogen were observed in rats at both dose levels of DCVG. An increase, but not of similar magnitude, of these biochemical parameters was noted in hamsters only at the higher dose of DCVG. Guinea-pigs showed significant increases in these biochemical parameters at the lower dose, but not at the higher dose. Light-microscopic studies showed increasing proximal tubular necrosis (PTN) in rats with increasing dose of DCVG, but PTN involving straight tubules only was observed at the higher dose in hamsters. PTN was again observed in guinea-pigs at the lower dose, but not at the higher dose of DCVG.     

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.     

Colchicine antimitosis causes progression of S-(1,2-dichlorovinyl)-L-cysteine-induced injury leading to acute renal failure and death in mice

Objective of the present study was to test the importance of tissue repair in the final outcome of S-(1,2-dichlorovinyl)-L-cysteine (DCVC)-induced nephrotoxicity using colchicine (CLC) intervention. Male Swiss Webster (SW) mice were administered a normally nonlethal dose of DCVC (30 mg/kg, i.p.) on day 0 and CLC (2 mg/kg, i.p.) at 42 and 66 h after administration of DCVC. The mice were observed for mortality and various renal injury and repair parameters were studied during a time course of 0-14 days. Administration of 30 mg DCVC/kg led to loss of renal architecture by day 1, which sustained until day 5, and regressed thereafter to reach normal architecture by day 10 resulting in 100% survival. Renal dysfunction as assessed by increases in plasma BUN and creatinine levels was concordant during this time course. Urinary volume increased significantly between days 10 and 14 with significant increases in urinary glucose concentrations on days 1-4. Calpain leakage increased from day 1 and remained so until day 5 before declining at later time points. In contrast, CLC intervention led to marked inhibition of S-phase DNA synthesis and 100% mortality by 120 h. H&E sections of kidneys revealed loss of renal architecture on day 1 which progressively worsened from day 2 to 4. Polyuria and glycosuria were evident during the first 2 and 3 days, respectively. Calpain immunohistochemistry revealed progressive leakage of calpain in the extracellular space during 2-4 days which lead to increased renal injury as evident from significant increases in calpain specific breakdown products (CSBPs) of alpha-fodrin during the same period of time. The group of mice receiving 2 mg CLC/kg alone showed a significant increase in urinary creatinine concentration on day 5. Neither the expression nor localization of aquaporin 1 was altered in any of the treatment groups. These results show that antimitotic intervention after DCVC-initiated renal injury leads to expansion and progression of that injury, which appears to be due to proteolytic destruction of neighboring cells mediated by calpain leaking out of necrosed renal tubular epithelial cells.     

Apoptotic responses stimulated by the trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine depend on cell differentiation state in BeWo human trophoblast cells

During pregnancy, the placental villous cytotrophoblasts differentiate via cell fusion and multinucleation to create syncytiotrophoblasts, a cell type at the maternal-fetal interface. Apoptosis of syncytiotrophoblasts is associated with adverse pregnancy outcomes. The human trophoblast BeWo cell line has been used as an in vitro model for this differentiation process, also known as syncytialization. In the current study, we exposed unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells to S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a metabolite of the industrial chemical trichloroethylene (TCE). DCVC exposure at 50 μM for 48 h decreased cell viability, increased cytotoxicity, increased caspase 3/7 activity, and increased nuclear condensation or fragmentation in BeWo cells regardless of their differentiation status. Investigating mechanisms of apoptosis, DCVC increased H₂O₂ abundance and decreased PRDX2 mRNA in all three BeWo cell models. DCVC decreased tumor necrosis factor-receptor 1 (TNF-R1) concentration in media and decreased NFKB1 and PRDX1 mRNA expression in syncytialized BeWo cells only. DCVC decreased BCL2 mRNA expression in syncytializing BeWo cells and in syncytialized BeWo cells only. Decreased LGALS3 mRNA was seen in unsyncytialized BeWo cells only. Together, these data suggest roles for oxidative stress and pro-inflammatory mechanisms underlying apoptosis in BeWo cells with differences depending on differentiation state.     

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.     

Species differences in the nephrotoxic response to S-(1,2-dichlorovinyl)glutathione

The present study was carried out to investigate the species differences in the nephrotoxic response to S-(1,2-dichlorovinyl)glutathione (DCVG) using rats, hamsters and guinea-pigs. DCVG was given intraperitoneally in physiological saline to groups of 5 animals at doses 0, 165 and 330 . Urine was collected for 24 h and the animals were then sacrificed. Significantly increased levels of urinary glucose, N-acetyl-β-d-glucosaminidase, γ-glutamyl transpeptidase, proteins and blood urea nitrogen were observed in rats at both dose levels of DCVG. An increase, but not of similar magnitude, of these biochemical parameters was noted in hamsters only at the higher dose of DCVG. Guinea-pigs showed significant increases in these biochemical parameters at the lower dose, but not at the higher dose. Light-microscopic studies showed increasing proximal tubular necrosis (PTN) in rats with increasing dose of DCVG, but PTN involving straight tubules only was observed at the higher dose in hamsters. PTN was again observed in guinea-pigs at the lower dose, but not at the higher dose of DCVG.     

Early Biological Indicators of S-(1,2-Dichlorovinyl)-L-Cysteine Nephrotoxicity in the Rabbit

ABSTRACT

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.     

Liquid chromatography electrospray ionization tandem mass spectrometry analysis method for simultaneous detection of trichloroacetic acid,dichloroacetic acid, S-(1,2-dichlorovinyl)glutathione and S-(1,2-dichlorovinyl)-L-cysteine

Trichloroethylene (TCE, CAS 79-01-6) is a widely used industrial chemical, and a common environmental pollutant. TCE is a well-known carcinogen in rodents and is classified as “probably carcinogenic to humans”. Several analytical methods have been proposed for detection of TCE metabolites in biological media utilizing derivatization-free techniques; however, none of them is suitable for simultaneous detection of both oxidative metabolites and glutathione conjugates of TCE in small volume biological samples. Here, we report a new combination of methods for assessment of major TCE metabolites: dichloroacetic acid (DCA), trichloroacetic acid (TCA), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), and S-(1,2-dichlorovinyl) glutathione (DCVG). First, DCA and TCA were extracted with ether. Second, the remaining aqueous fraction underwent solid phase extraction for DCVC and DCVG. Then, DCA and TCA were measured by hydrophilic interaction liquid chromatography ion exchange negative electrospray ionization tandem mass spectrometry, while DCVC and DCVG were measured by reverse phase positive electrospray ionization tandem mass spectrometry. This method was applied successfully to measure all 4 TCE metabolites in as little as 50 μl of serum from mice orally exposed to TCE (2100 mg/kg, 2 h). Serum concentrations (mean ± standard deviation) of the TCE metabolites obtained with this method are comparable or equivalent to those previously reported in the literature: DCA, 0.122 ± 0.014 nmol/ml (limit of detection: 0.01 nmol/ml); TCA, 256 ± 30 nmol/ml (0.4 nmol/ml); DCVG, 0.037 ± 0.015 nmol/ml (0.001 nmol/ml); DCVC, 0.0024 ± 0.0009 nmol/ml (0.001 nmol/ml). This method opens new opportunities to increase throughput and decrease number of animals required for mechanistic studies on TCE in rodents.     

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.     

Studies of the biochemical toxicology of uranyl nitrate in the rat

High resolution ¹H NMR spectroscopy of urine and plasma, conventional clinical chemical methods and histopathology have been applied to investigate the effects of uranyl nitrate (UN) on renal function and biochemistry in the Fischer 344 (F344) rat. Administration of UN (5 – 20 mg/kg) to male F344 rats resulted in a dose-related proximal nephropathy assessed conventionally by histopathology and urinary excretion of N-acetyl-β-d-glucosaminidase (NAG), and related to changes in the patterns of low MW metabolites observed in 400 MHz ¹H NMR spectra of urine. The changes in urinary metabolite profiles included elevations in glucose accompanied by minor elevations in certain amino acids (alanine, valine and glutamate). ¹H NMR urinalysis also revealed altered excretion of low MW metabolites which are not routinely measured, such as l-lactate, acetate, citrate, succinate and 2-oxoglutarate (2-OG). In addition, the striking appearance of high concentrations of 3-d-hydroxybutyrate (HB) in the urine was noted, in the absence of acetoacetate or acetone, and it is suggested that this may provide a new marker of proximal tubular damage for certain types of nephrotoxic mechanism. Broadening of the ¹H NMR signals of citrate following 10 mg/kg UN was shown to be due to a dynamic exchange process involving chelation with urinary Ca²⁺ and Mg²⁺ ions. Conventional biochemical analysis of plasma from UN-treated rats revealed dose-related increases in creatinine, urea and HB concentrations. ¹H NMR-detected evidence of raised alanine aminotransferase (ALT) levels in rats administered the highest dose of UN was indicated by the partial deuteration of alanine in lyophilised plasma reconstituted in ²H₂O. The degree of ¹H NMR-detected abnormalities agreed well with histopathological observations and conventional biochemical indices of nephrotoxicity and more fully characterised the renal changes produced by UN. The significance of HB-uria in UN-induced proximal nephropathy is discussed in relation to biochemical observations on other proximal nephrotoxins. Received 7 June 1993/Accepted 23 August 1993     

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.