Interactive toxicity of inorganic mercury and trichloroethylene in rat and human proximal tubules: effects on apoptosis, necrosis, and glutathione status

Simultaneous or prior exposure to one chemical may alter the concurrent or subsequent response to another chemical, often in unexpected ways. This is particularly true when the two chemicals share common mechanisms of action. The present study uses the paradigm of prior exposure to study the interactive toxicity between inorganic mercury (Hg(2+)) and trichloroethylene (TRI) or its metabolite S-(1,2-dichlorovinyl)-l-cysteine (DCVC) in rat and human proximal tubule. Pretreatment of rats with a subtoxic dose of Hg(2+) increased expression of glutathione S-transferase-alpha1 (GSTalpha1) but decreased expression of GSTalpha2, increased activities of several GSH-dependent enzymes, and increased GSH conjugation of TRI. Primary cultures of rat proximal tubular (rPT) cells exhibited both necrosis and apoptosis after incubation with Hg(2+). Pretreatment of human proximal tubular (hPT) cells with Hg(2+) caused little or no changes in GST expression or activities of GSH-dependent enzymes, decreased apoptosis induced by TRI or DCVC, but increased necrosis induced by DCVC. In contrast, pretreatment of hPT cells with TRI or DCVC protected from Hg(2+) by decreasing necrosis and increasing apoptosis. Thus, whereas pretreatment of hPT cells with Hg(2+) exacerbated cellular injury due to TRI or DCVC by shifting the response from apoptosis to necrosis, pretreatment of hPT cells with either TRI or DCVC protected from Hg(2+)-induced cytotoxicity by shifting the response from necrosis to apoptosis. These results demonstrate that by altering processes related to GSH status, susceptibilities of rPT and hPT cells to acute injury from Hg(2+), TRI, or DCVC are markedly altered by prior exposures.     

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.     

Dichlorovinyl cysteine (DCVC) in the mouse kidney: tissue-binding and toxicity after glutathione depletion and probenecid treatment

The kidney binding of dichloro[¹⁴C]vinyl cysteine (¹⁴C-DCVC, 8 mg/kg body wt) and the kidney histopathology of DCVC (5 mg/kg body wt) were examined and compared in female C57BL mice subjected to various treatments. To evaluate the roles of organic anion transport and glutathione (GSH) status, mice were pretreated with probenecid (inhibitor of organic anion transport), l-buthionine-S,R-sulfoximine (BSO; inhibitor of GSH synthesis) or with diethyl maleate (DEM; GSH-depleting agent). In addition, the sites of ¹⁴C-DCVC binding in BSO-treated and control mice were monitored by microautoradiography. Probenecid was found to inhibit both kidney binding and toxicity of DCVC. In BSO-treated mice, DCVC binding remained roughly unchanged, whereas nephrotoxicity was severely increased and topographically extended to the subcapsular region. Microautoradiography showed that the site of DCVC binding in the straight portion of the proximal tubule was not changed by BSO. In DEM-treated mice, a clearly decreased DCVC binding was observed, while the effect on nephrotoxicity was minute. The effects of probenecid on DCVC binding and toxicity support a role for carriermediated transport of DCVC equivalents into the target cells. The BSO result suggests a protective function of GSH towards the nephrotoxicity of DCVC. Moreover, they support our previous contention that a primary lesion occurs at the site of DCVC binding, followed by a secondary, dose-dependent lesion localized outside the DCVC-binding region. In the case of DEM it is proposed that a DEM-GSH conjugate might compete for the uptake and/or activation of DCVC in the target cells.Part of this study was presented at the 10th European Drug Metabolism Workshop, Guildford, England, 6–11 July, 1986     

Renal cysteine conjugate beta-lyase-mediated toxicity studied with primary cultures of human proximal tubular cells

The beta-lyase pathway has been shown to mediate the nephrotoxicity of S-cysteine conjugates of a variety of haloalkenes in a number of animal models in vitro and in vivo. However, there is no information available concerning this mechanism of bioactivation in human tissues. In this investigation a well-characterized model of human proximal tubule epithelial cells, the presumed target cell, was used to investigate the toxicity of a series of glutathione and cysteine conjugates of nephrotoxic haloalkenes. Both S-(1,2-dichlorovinyl)-glutathione (DCVG) and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) caused dose-dependent toxicity over a range of 25 to 500 microM. DCVC was consistently found to be more toxic than DCVG, but the inclusion of gamma-glutamyltransferase (0.5 U/ml) increased the toxicity of DCVG to that observed with an equimolar concentration of DCVC, indicating that metabolism to the cysteine conjugate is an important rate-limiting step in this in vitro model. S-(1,2,3,4,4-Pentachlorobutadienyl)-L-cysteine, S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine, and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine were also found to be toxic to human proximal tubular cells. Incubation with [35S]DCVC resulted in covalent binding of 35S-label, which increased linearly to a final level of 1.05 nmol/mg protein at 6 hr. Aminooxyacetic acid (250 microM), an inhibitor of pyridoxal phosphate-dependent enzymes such as beta-lyase, protected the cells from the toxicity of all of the cysteine conjugates and inhibited the covalent binding of 35S-label from [35S]DCVC to cellular macromolecules. The results of the present study provide the first evidence that human proximal tubular cells are sensitive to the toxicity of glutathione and/or cysteine conjugates of a variety of chloro- and fluoroalkenes which are activated via the beta-lyase pathway. The implications for human health are discussed.     

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.     

Cellular energetics and glutathione status in NRK-52E cells: toxicological implications

Cellular energetics and redox status were evaluated in NRK-52E cells, a stable cell line derived from rat proximal tubules. To assess toxicological implications of these properties, susceptibility to apoptosis induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a well-known mitochondrial and renal cytotoxicant, was studied. Cells exhibited high activities of several glutathione (GSH)-dependent enzymes, including gamma-glutamylcysteine synthetase, GSH peroxidase, glutathione disulfide reductase, and GSH S-transferase, but very low activities of gamma-glutamyltransferase and alkaline phosphatase, consistent with a low content of brush-border microvilli. Uptake and total cellular accumulation of [14C]alpha-methylglucose was significantly higher when cells were exposed at the basolateral as compared to the brush-border membrane. Similarly, uptake of GSH was nearly 2-fold higher across the basolateral than the brush-border membrane. High activities of (Na(+)+K(+))-ATPase and malic dehydrogenase, but low activities of other mitochondrial enzymes, respiration, and transport of GSH and dicarboxylates into mitochondria were observed. Examination of mitochondrial density by confocal microscopy, using a fluorescent marker (MitoTracker Orange), indicated that NRK-52E cells contain a much lower content of mitochondria than rat renal proximal tubules in vivo. Incubation of cells with DCVC caused time- and concentration-dependent ATP depletion that was largely dependent on transport and bioactivation, as observed in the rat, on induction of apoptosis, and on morphological damage. Comparison with primary cultures of rat and human proximal tubular cells suggests that the NRK-52E cells are modestly less sensitive to DCVC. In most respects, however, NRK-52E cells exhibited functions similar to those of the rat renal proximal tubule in vivo.     

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.     

Renal cysteine conjugate C-S lyase mediated toxicity of halogenated alkenes in primary cultures of human and rat proximal tubular cells

Proximal tubular cells from human (HPT) and rat (RPT) kidneys were isolated, grown to confluence and incubated with S-(1,2-dichlorovinyl)- l-cysteine (DCVC), S-(1,2,2-trichlorovinyl)- l-cysteine (TCVC), S-(1,1,2,2-tetrafluoroethyl)- l-cysteine (TFEC) and S-(2-chloro-1,1-difluorethyl)- l-cysteine (CDFEC), the cysteine conjugates of nephrotoxicants. The cultures were exposed to the conjugates for 12, 24 and 48 h and the toxicity determined using the MTT assay. All four conjugates caused dose-dependent toxicity to RPT cells over the range 50-1,000 microM, the order of toxicity being DCVC>TCVC>TFEC=CDFEC. The inclusion of aminooxyacetic acid (AOAA; 250 microM), an inhibitor of pyridoxal phosphate-dependent enzymes such as C-S lyase, afforded protection, indicating that C-S lyase has a role in the bioactivation of these conjugates. In HPT cultures only DCVC caused significant time- and dose-dependent toxicity. Exposure to DCVC (500 microM) for 48 h decreased cell viability to 7% of control cell values, whereas co-incubation of DCVC (500 microM) with AOAA (250 microM) resulted in cell viability of 71%. Human cultures were also exposed to S-(1,2-dichlorovinyl)-glutathione (DCVG). DCVG was toxic to HPT cells, but the onset of toxicity was delayed compared with the corresponding cysteine conjugate. AOAA afforded almost complete protection from DCVG toxicity. Acivicin (250 microM), an inhibitor of gamma-glutamyl transferase (gamma-GT), partially protected against DCVG (500 microM)-induced toxicity at 48 h (5% viability and 53% viability in the absence and presence of acivicin, respectively). These results suggest that DCVG requires processing by gamma-GT prior to bioactivation by C-S lyase in HPT cells. The activity of C-S lyase, using TFEC as a substrate, and glutamine transaminase K (GTK) was measured in rat and human cells with time in culture. C-S lyase activity in RPT and HPT cells decreased to approximately 30% of fresh cell values by the time the cells reached confluence (120 h), whereas the decline in GTK activity was less marked (50% of the fresh cell values at confluence). Rat cells had threefold higher activity than human cells at each time point. This higher activity may partly explain the differences in toxicity between rat and human proximal tubular cells in culture.     

Cellular glutathione conjugation of aziridines in isolated rat hepatocytes

Glutathione conjugation of aziridines was found in isolated rat hepatocytes in experiments using the optical isomers of (1- and d-) aziridinecarboxylic acid (AZC) and (1- and d-) 1-methyl-2-beta-naphthylaziridine (NAZ). 1-AZC much more effectively consumed glutathione than d-AZC, and the yield of the glutathione conjugate during 2 hr of incubation exceeded 200% of the cellular glutathione detected at the initiation of the incubation. Such a high yield of 1-AZC-GSH conjugate would occur only when conjugation efficiently proceeds without interference against the GSH resynthesis route in the liver cells. The cytotoxicity of 1-AZC was very weak and did not affect cell viability of the isolated hepatocytes even after the formation of AZC-GSH conjugate. Consequently, we supposed that GSH is not essential for supporting the viability of the isolated hepatocytes. For very slow GSH conjugate formation of d-AZC, we envisaged poor membrane transport of the d-isomer resembling to the selective incorporation of d- and 1-proline observed in some plant cells. Both isomers of NAZ were markedly cytotoxic and depressed the cell viability. The yield of the glutathione conjugate from NAZ did not exceed the cellular GSH level detected at the initial stage of incubation. The highly cytotoxic compound nitrosomethane, generated in the first biotransformation step of the metabolism of NAZ, can obstruct the resynthesis route of GSH by inhibiting the ATP generation process as discussed previously (Ref. 3). Decreasing the cellular GSH by treatment with 1-AZC enhanced the susceptibility of the isolated hepatocytes to NAZ toxicity. d-AZC did not affect the viability of cells treated with NAZ.     

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     

Cytotoxicity of S-conjugates of the sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl) vinyl ether (Compound A) in a human proximal tubular cell line

Fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE) is a fluorinated alkene formed by degradation of the volatile anesthetic sevoflurane in anesthesia machines. FDVE is nephrotoxic in rats but not humans. Rat FDVE nephrotoxicity is attributed to FDVE glutathione conjugation and bioactivation of subsequent FDVE-cysteine S-conjugates, in part by renal beta-lyase. Although FDVE conjugation and metabolism occur in both rats and humans, the mechanism for selective toxicity in rats and lack of effect in humans is incompletely elucidated. This investigation measured FDVE S-conjugate cytotoxicity in cultured human proximal tubular HK-2 cells, and compared this with known cytotoxic S-conjugates. HK-2 cells were incubated with FDVE and its GSH, cysteine S-mercapturic acid, cysteine S-sulfoxide, and mercapturic acid sulfoxide conjugates (0.1-2.7 mM) for 24 h. Cytotoxicity was determined by lactate dehydrogenase (LDH) release, total LDH, and the ability of viable cells to reduce a tetrazolium-based compound (MTT). FDVE was cytotoxic only at concentrations >/=0.9 mM. No increase in LDH release was observed with either FDVE-GSH conjugate. The FDVE-cysteine conjugates S-(1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl) ethyl)-L-cysteine (DFEC) and (Z)-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl) vinyl)-L-cysteine ((Z)-FFVC) caused significant differences in LDH release and MTT reduction only at 2.7 mM; (Z)-FFVC was slightly more cytotoxic. Both S-(1,1-difluoro-2-fluoromethoxy-2-(trifluoromethyl) ethyl)-L-cysteine sulfoxide (DFEC-SO) and (Z)-N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl) vinyl)-L-cysteine sulfoxide ((Z)-N-Ac-FFVC-SO) caused slightly greater changes in LDH release or total LDH than the corresponding equimolar DFEC and (Z)-N-acetyl-S-(1-fluoro-2-fluoromethoxy-2-(trifluoromethyl) vinyl)-L-cysteine ((Z)-N-Ac-FFVC) conjugates. In contrast to FDVE S-conjugates, S-(1,2-dichlorovinyl)-L-cysteine was markedly cytotoxic, at concentrations as low as 0.1 mM. These results show that human proximal tubular cells are relatively resistant to FDVE and FDVE S-conjugate cytotoxicity. This may partially explain the lack of FDVE nephrotoxicity in humans.     

Role of cytochrome P450 and glutathione S-transferase alpha in the metabolism and cytotoxicity of trichloroethylene in rat kidney

The toxicity and metabolism of trichloroethylene (TRI) were studied in renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. TRI was slightly toxic to both PT and DT cells, and inhibition of cytochrome P450 (P450; substrate, reduced-flavoprotein:oxygen oxidoreductase [RH-hydroxylating or -epoxidizing]; EC 1.14.14.1) increased TRI toxicity only in DT cells. In untreated cells, glutathione (GSH) conjugation of TRI to form S-(1,2-dichlorovinyl)glutathione (DCVG) was detected only in PT cells. Inhibition of P450 transiently increased DCVG formation in PT cells and resulted in detection of DCVG formation in DT cells. Formation of DCVG in PT cells was described by a two-component model (apparent Vmax values of 0.65 and 0.47 nmol/min per mg protein and Km values of 2.91 and 0.46 mM). Cytosol isolated from rat renal cortical, PT, and DT cells expressed high levels of GSH S-transferase (GST; RX:glutathione R-transferase; EC 2.5.1.18) alpha (GSTalpha) but not GSTpi. Low levels of GSTmu were detected in cortical and DT cells. Purified rat GSTalpha2-2 exhibited markedly higher affinity for TRI than did GSTalpha1-1 or GSTalpha1-2, but each isoform exhibited similar VmaX values. Triethyltinbromide (TETB) (9 microM) inhibited DCVG formation by purified GSTalpha-1 and GSTalpha2-2, but not GSTalpha1-2. Bromosulfophthalein (BSP) (4 microM) only inhibited DCVG formation by GSTalpha2-2. TETB and BSP inhibited approximately 90% of DCVG formation in PT cytosol but had no effect in DT cytosol. This suggests that GSTalpha1-1 is the primary isoform in rat renal PT cells responsible for GSH conjugation of TRI. These data, for the first time, describe the metabolism of TRI by individual GST isoforms and suggest that DCVG feedback inhibits TRI metabolism by GSTs.     

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.     

Mycotoxin fumonisin B1 alters cellular redox balance and signalling pathways in rat liver and kidney

Mycotoxin fumonisin B(1) (FB(1)) is a frequent contaminant of grain, particularly maize, but the mechanism of its toxicity in the kidney and liver is not fully understood. FB(1)-stimulated oxidative stress might disturb cellular redox state and signal transduction pathways of the target cells. In this study we measured total intracellular glutathione (GSH), and assessed mitogen-activated protein kinases (MAPKs) activation and the expression of heat shock proteins (Hsps) Hsp25 and Hsp70 in the liver and kidney of male Wistar rats given 0.5 mg FB(1)/kg b.w. intraperitoneally for 2 or 7 days. The effect of FB(1) on GSH levels, MAPK activation and Hsp expression was found to be related to the type of tissue affected and the length of treatment. In rat liver, cellular GSH content increased, Hsp expression was up-regulated, and ERK and p38 were activated after the 7-day treatment, while even the 2-day treatment sufficed to produce phospho-JNK signal. In rat kidney, GSH levels decreased after the 2- and 7-day treatment with FB(1), while after the 7-day treatment all three MAPKs were activated, Hsp25 expression increased and Hsp70 expression decreased. In conclusion, FB(1) alters cellular redox balance, which leads to tissue-specific activation and expression of redox-sensitive signalling molecules. It seems that kidney cells are more sensitive to adverse effects of FB(1).     

Health risk assessment of environmental exposure to trichloroethylene

A review of the animal data showed trichloroethylene (TRI) to be of low acute toxicity. Repeated exposure showed that the target organs were the liver, and to a lesser extent, the kidney. TRI is not mutagenic or only marginally mutagenic. There is no evidence of fetotoxicity or teratogenicity. TRI is judged not to exhibit chronic neurotoxicity. Lifetime bioassays resulted in tumors in both the mouse and the rat. However, because of qualitative and quantitative metabolic differences between rodent and human, no one suitable tumor site can be chosen for human health risk assessment. In addition, of the several epidemiology studies, none has demonstrated a positive association for increased tumor incidence. A review of the health effects in humans shows TRI to be of low acute toxicity and, following chronic high doses, to be hepatotoxic. Environmental exposure to TRI is mainly via the atmosphere, while the contribution from exposure to drinking water and foodstuffs is negligible. The total body burden was calculated as 22 micrograms/day. The safety margin approach based on human health effects showed that TRI levels are well within the safety margin for the human no-observable-effect level (10,000 times lower). The total body burden represents a risk of 1.4 X 10(-5) by linearized multistage modeling. Therefore, by either methodological approach to risk assessment, the environmental occurrence of TRI does not represent a significant health risk to the general population or to the population in areas close to industrial activities.     

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.     

Consideration of the target organ toxicity of trichloroethylene in terms of metabolite toxicity and pharmacokinetics.

Trichloroethylene (TRI) is readily absorbed into the body through the lungs and gastrointestinal mucosa. Exposure to TRI can occur from contamination of air, water, and food; and this contamination may be sufficient to produce adverse effects in the exposed populations. Elimination of TRI involves two major processes: pulmonary excretion of unchanged TRI and relatively rapid hepatic biotransformation to urinary metabolites. The principal site of metabolism of TRI is the liver, but the lung and possibly other tissues also metabolize TRI, and dichlorovinyl-cysteine (DCVC) is formed in the kidney. Humans appear to metabolize TRI extensively. Both rats and mice also have a considerable capacity to metabolize TRI, and the maximal capacities of the rat versus the mouse appear to be more closely related to relative body surface areas than to body weights. Metabolism is almost linearly related to dose at lower doses, becoming dose dependent at higher doses, and is probably best described overall by Michaelis-Menten kinetics. Major end metabolites are trichloroethanol (TCE), trichloroethanol-glucuronide, and trichloroacetic acid (TCA). Metabolism also produces several possibly reactive intermediate metabolites, including chloral, TRI-epoxide, dichlorovinyl-cysteine (DCVC), dichloroacetyl chloride, dichloroacetic acid (DCA), and chloroform, which is further metabolized to phosgene that may covalently bind extensively to cellular lipids and proteins, and, to a much lesser degree, to DNA. The toxicities associated with TRI exposure are considered to reside in its reactive metabolites. The mutagenic and carcinogenic potential of TRI is also generally thought to be due to reactive intermediate biotransformation products rather than the parent molecule itself, although the biological mechanisms by which specific TRI metabolites exert their toxic activity observed in experimental animals and, in some cases, humans are not known. The binding intensity of TRI metabolites is greater in the liver than in the kidney. Comparative studies of biotransformation of TRI in rats and mice failed to detect any major species or strain differences in metabolism. Quantitative differences in metabolism across species probably result from differences in metabolic rate and enterohepatic recirculation of metabolites. Aging rats have less capacity for microsomal metabolism, as reflected by covalent binding of TRI, than either adult or young rats. This is likely to be the same in other species, including humans. The experimental evidence is consistent with the metabolic pathways for TRI being qualitatively similar in mice, rats, and humans. The formation of the major metabolites--TCE, TCE-glucuronide, and TCA--may be explained by the production of chloral as an intermediate after the initial oxidation of TRI to TRI-epoxide.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure-nephrotoxicity relationships of glutathione pathway intermediates derived from organic solvents

The nephrotoxicity of glutathione (GSH) pathway metabolites derived from toluene (TOL), styrene (STYR), bromobenzene (BB), acrylonitrile (ACLN) and 2-chloroacrylonitrile (CACLN) were compared with that of dichlorovinylcysteine (DCVC), using renal brush border and basal-lateral uptake parameters as indices. Cysteine conjugates and mercapturates of ACLN did not alter p-aminohippurate (PAH) uptake by renal tubule suspensions in contrast to its chlorinated homologue. O-, m- and p-conjugates of BB inhibited PAH uptake by 43-82%, the mercapturates showing more potency than corresponding cysteine conjugates. The TOL derivatives N-acetylbenzylcysteine curtailed PAH uptake but benzylcysteine was more effective. The GSH conjugate and mercapturate synthesized from STYR oxide were also active inhibitors but not its cysteine conjugate. Among all GSH pathway metabolites studied, only DCVC and phenylhydroxyethylglutathione, derived from STYR oxide, impeded the renal basal-lateral uptake of [14C]tetraethylammonium (TEA) while DCVC was the sole inhibitor of brush border transport events such as the uptakes of [3H]glutamate and [14C]alpha-methyl-D-glucoside. These data indicate that GSH conjugation represents a non-nephrotoxic detoxication pathway for ACLN. In contrast, GSH conjugation with 2-chloroacrylonitrile and with aromatic solvents like TOL, STYR, BB gives rise to nephrotoxic mercapturates which may be less potent but show more specificity for the organic anion transport system than DCVC.     

Reactions of the nitroso and hydroxylamine metabolites of sulfamethoxazole with reduced glutathione. Implications for idiosyncratic toxicity.

N4-oxidation of sulfonamides has been implicated in the pathogenesis of idiosyncratic reactions to these antimicrobials. In vitro toxicity assays employing mononuclear leukocytes as target cells have shown that the toxicity of sulfamethoxazole hydroxylamine (SMX-HA) is inhibited by exogenous glutathione, suggesting that conjugation with glutathione is an important detoxification pathway. However, in these experiments, significant depletion of cellular glutathione only occurred at concentrations of SMX-HA greater than or equal to 300 microM. At concentrations of SMX-HA which produce 50% toxicity in mononuclear leukocytes (approximately 100 microM), there was not a significant loss of glutathione. SMX-HA also caused a small but significant increase in oxidized glutathione concentrations. In cell-free experiments, reduced glutathione (GSH) prevented the autooxidation of SMX-HA to nitrososulfamethoxazole (nitroso-SMX). During this process, oxidized glutathione was formed. GSH rapidly reacted with nitroso-SMX to form a labile semimercaptal conjugate. Physiologically relevant concentrations of GSH (i.e. 1 mM) favored thiolytic cleavage of the semimercaptal to form SMX-HA. Isomerization of the semimercaptal to the more stable sulfinamide occurred at low GSH concentrations. Purified glutathione transferases had no effect on the reaction of SMX-HA with GSH. Therefore, glutathione is important in protecting cells from the toxicity of SMX-HA largely by preventing its further oxidation to nitroso-SMX. Stable glutathione conjugates are likely to be formed only in small quantities under physiological conditions. Conjugation with glutathione would not be expected to be a major pathway for clearance of the hydroxylamine and nitroso metabolites of sulfonamides.