Renal membrane transport of glutathione in toxicology and disease.

Membrane transport processes, at both the plasma membranes and intracellular membranes, play critical roles in renal function and are a determining factor in the susceptibility of renal epithelial cells to blood-borne drugs and toxic chemicals. Proximal tubular epithelial cells possess a large array of transport proteins for organic anions, organic cations, and peptides on both basolateral and brush-border plasma membranes. Although these transporters function in excretion of waste products and reabsorption of nutrients, they also play a role in the susceptibility of the kidneys to drugs and other toxicants in the blood. The proximal tubules are typically the primary target cells because they are the first epithelial cell population exposed to such chemicals in either the renal plasma or glomerular filtrate and because of their large array of membrane transporters. Besides transport across the basolateral and brush-border plasma membranes, transport across intracellular membranes such as the mitochondrial inner membrane is a critical determinant of metabolite distribution. To illustrate the function of these transporters, carrier-mediated processes for transport of the tripeptide and antioxidant glutathione across the basolateral, brush-border, and mitochondrial inner membranes of the renal proximal tubule are reviewed. Studies are summarized that have identified the involvement of specific carrier proteins and characterized the role of these transporters in glutathione metabolism and turnover, susceptibility of the proximal tubules to oxidative and other stresses, and modulation in disease and other pathological processes.

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.

Acute, subacute, and subchronic oral toxicity studies of 1,1-dichloroethane in rats: application to risk evaluation.

1,1-Dichloroethane (DCE) is a solvent that is often found as a contaminant of drinking water and a pollutant at hazardous waste sites. Information on its short- and long-term toxicity is so limited that the U.S. EPA and ATSDR have not established oral reference doses or minimal risk levels for the volatile organic chemical (VOC). The acute oral LD(50) in male Sprague-Dawley (S-D) rats was estimated in the present study to be 8.2 g/kg of body weight (bw). Deaths appeared to be due to CNS depression and respiratory failure. In an acute/subacute experiment, male S-D rats were given 0, 1, 2, 4, or 8 g DCE/kg in corn oil by gavage for 1, 5, or 10 consecutive days. The animals were housed in metabolism cages for collection of urine and sacrificed for blood and tissue sampling 24 h after their last dose. There were decreases in body weight gain and relative liver weight at all dosage levels, as well as increased renal nonprotein sulfhydryl levels at 2 and 4 g/kg after 5 and 10 days. Elevated serum enzyme levels, histopathological changes, and abnormal urinalyses were not manifest. For the subchronic study, adult male S-D rats were gavaged with 0.5, 1, 2, or 4 g DCE/kg 5 times weekly for up to 13 weeks. Animals receiving 4 g/kg exhibited pronounced CNS depression, with more than one-half dying by week 11. The 2-g/kg rats exhibited moderate CNS depression. One 2-g/kg rat died during week 6. There were very few manifestations of organ damage in animals that succumbed or in survivors at any dosage level. Decreases in bw gain and transient increases in enzymuria were noted at 2 and 4 g/kg. Serum enzyme levels and blood urea nitrogen were not elevated, nor were glycosuria or proteinuria present. Chemically induced histological changes were not seen in the liver, kidney, lung, brain, adrenal, spleen, stomach, epididymis, or testis. Hepatic microsomal cytochrome P450 experiments revealed that single, high oral doses of DCE did not alter total P450 levels, but did induce CYP2E1 levels and activity and inhibit CYP1A1 activity. These effects were reversible and regressed with repeated DCE exposure. There was no apparent progression of organ damage during the 13-week subchronic study, nor appearance of adverse effects not seen in the short-term exposures. One g/kg orally (po) was found to be the acute, subacute, and subchronic LOAEL for DCE, under the conditions of this investigation. In each instance, 0.5 g/kg was the NOAEL.

In vitro nephrotoxicity induced by N-(3,5-dichlorophenyl)succinimide (NDPS) metabolites in isolated renal cortical cells from male and female Fischer 344 rats: evidence for a nephrotoxic sulfate conjugate metabolite.

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) induces nephrotoxicity in vivo that is characterized as acute polyuric renal failure and proximal tubular necrosis. However, earlier in vitro studies have failed to reproduce the in vivo nephrotoxicity seen with NDPS or its nephrotoxic metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA). The purpose of this study was to examine the nephrotoxic potential of NDPS, its known non-conjugated metabolites, the O-sulfate conjugate of NDHS (NSC), and the putative metabolite N-(3,5-dichlorophenyl)maleimide (NDPM) and its hydrolysis product N-(3,5-dichlorophenyl)maleamic acid (NDPMA) using freshly isolated renal cortical cells (IRCC). IRCC were obtained from untreated male or female Fischer 344 rats following collagenase perfusion of the kidneys. Cells (approximately 4 million per ml) (N=4) were incubated with up to 1.0 mM NDPS or an NDPS metabolite or vehicle for up to 120 min. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release into the medium. Only NSC (>0.5 mM) and NDPM (> or =0.5 mM) exposure increased LDH release from IRCC. NSC 1.0 mM or NDPM 0.5 mM increased LDH release from IRCC within 15--30 min of exposure. NDPS or the remaining NDPS metabolites did not increase LDH release at bath concentrations of 1.0 mM for exposures of 120 min. IRCC from male and female rats responded similarly to the toxic effects of NDPS and its metabolites. These results demonstrate that sulfate conjugates of NDPS metabolites can be fast acting nephrotoxicants and could contribute to NDPS nephrotoxicity in vivo. These results also suggest that the kidney probably accumulates toxic sulfate conjugates of NDPS metabolites rather than forming the conjugates. In addition, mechanisms responsible for gender differences in nephrotoxicity seen with NDPS and NDPS metabolites in vivo either occur prior to renal accumulation of sulfate conjugates and/or represent biochemical/physiological differences between the genders.

Functional and toxicological characteristics of isolated renal mitochondria: impact of compensatory renal growth.

Mitochondria were isolated from renal cortical homogenates from control rats and rats that had undergone uninephrectomy and compensatory renal growth (NPX rats). Activities of selected mitochondrial processes, including key enzymes of intermediary metabolism, glutathione-dependent enzymes, and glutathione transport, were measured, and the effects of three mitochondrial toxicants were assessed to test the hypothesis that compensatory renal growth is accompanied by increases in mitochondrial metabolism and that this is associated with increased susceptibility to injury from oxidants or other mitochondrial toxicants. Activities of malic and succinic dehydrogenases were significantly higher in mitochondria from NPX rats than in mitochondria from control rats. Although the rates of state 3 respiration were significantly higher in mitochondria from NPX rats, the rates of state 4 respiration and respiratory control ratios were not different between mitochondria from control and NPX rats. Activities of glutathione redox cycle enzymes did not differ significantly between mitochondria from control and NPX rats. However, the rates of uptake of glutathione into mitochondria were approximately 2.5-fold higher in tissue from NPX rats than in tissue from control rats. Incubation of mitochondria from NPX rats with three mitochondrial toxicants [tert-butyl hydroperoxide, methyl vinyl ketone, and S-(1,2-dichlorovinyl)-L-cysteine] caused greater inhibition of state 3 respiration and larger increases in malondialdehyde formation than similar incubations of mitochondria from control rats. These results indicate that mitochondria from hypertrophied renal cells are more sensitive to oxidants or mitochondrial toxicants. Baseline levels of malondialdehyde were also significantly higher in mitochondria from NPX rats, suggesting that a basal oxidant stress exists in mitochondria from hypertrophied cells.

Hepatic and renal toxicities associated with perchloroethylene.

Metabolism of perchloroethylene (Perc) occurs by cytochrome P450-dependent oxidation and glutathione (GSH) conjugation. The cytochrome P450 pathway generates tri- and dichloroacetate as metabolites of Perc, and these are associated with hepatic toxicity and carcinogenicity. The GSH conjugation pathway is associated with generation of reactive metabolites selectively in the kidneys and with Perc-induced renal toxicity and carcinogenicity. Physiologically based pharmacokinetic models have been developed for Perc in rodents and in humans. We propose the addition of a submodel that incorporates the GSH conjugation pathway and the kidneys as a target organ. Long-term bioassays of Perc exposure in laboratory animals have identified liver tumors in male and female mice, kidney tumors in male rats, and mononuclear cell leukemia in male and female rats. Increases in incidence of non-Hodgkin's lymphoma and of cervical, esophageal, and urinary bladder cancer have been observed for workers exposed to Perc. Limited, and not always consistent, evidence is available concerning the kidneys as a target organ for Perc in humans. Three potential modes of action for Perc-induced liver tumorigenesis are: 1) modification of signaling pathways; 2) cytotoxicity, cell death, and reparative hyperplasia; and 3) direct DNA damage. Four potential modes of action for Perc-induced renal tumorigenesis are: 1) peroxisome proliferation, 2) alpha-2u-globulin nephropathy, 3) genotoxicity leading to somatic mutation, and 4) acute cytotoxicity and necrosis leading to cell proliferation. Finally, the epidemiological and experimental data are assessed and use of toxicity information in the development of a reference dose and a reference concentration for human Perc exposure are presented.

Renal and hepatic toxicity of trichloroethylene and its glutathione-derived metabolites in rats and mice: sex-, species-, and tissue-dependent differences.

Acute cytotoxicity (lactate dehydrogenase release) of trichloroethylene (TRI), S-(1,2-dichlorovinyl)glutathione (DCVG), and S-(1,2-dichlorovinyl)-L-cysteine (DCVC) in freshly isolated renal cortical cells and hepatocytes from male and female rats was evaluated to test the hypothesis that the assay provides a valid indicator of sex- and tissue-dependent differences in sensitivity to TRI and its metabolites. We then determined mitochondrial toxicity (inhibition of state-3 and/or stimulation of state-4 respiration) in renal cortical and hepatic mitochondria from male and female rats and mice to assess sex-, tissue-, and species-dependent susceptibility. TRI was moderately cytotoxic in renal cells from male rats but was nontoxic in renal cells from female rats or hepatocytes from male or female rats. Acute cytotoxicity of both DCVG and DCVC was greater in renal cells from male rats than in renal cells from female rats. Although DCVC does not target the liver in vivo, it was a very potent hepatotoxicant in vitro. Mitochondrial toxicity in kidney and liver showed similar patterns, with mitochondria from male rats being more sensitive than mitochondria from female rats; order of potency was DCVC > DCVG >> TRI. State-3 respiration in mitochondria from mice was also inhibited, but the patterns and relative sensitivities differed from those in mitochondria from rats. Renal and hepatic mitochondria from mice were less sensitive than corresponding mitochondria from rats and renal mitochondria from female mice were significantly more sensitive than renal mitochondria from male mice. Thus, many of the species-, sex-, and tissue-dependent differences in toxicity observed in vivo are also observed in vitro.

Cytochrome p450-dependent metabolism of trichloroethylene in rat kidney.

The metabolism of trichloroethylene (Tri) by cytochrome P450 (P450) was studied in microsomes from liver and kidney homogenates and from isolated renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. Chloral hydrate (CH) was the only metabolite consistently detected and was used as a measurement of P450-dependent metabolism of Tri. Pretreatment of rats with pyridine increased CH formation in both liver and kidney microsomes, whereas pretreatment of rats with clofibrate increased CH formation only in kidney microsomes. Pyridine increased CYP2E1 expression in both liver and kidney microsomes, whereas clofibrate had no effect on hepatic but increased renal CYP2E1 and CYP2C11 protein levels. These results suggest a role for CYP2E1 in both the hepatic and renal metabolism of Tri and a role for CYP2C11 in the renal metabolism of Tri. Studies with the general P450 inhibitor SKF-525A and the CYP2E1 competitive substrate chlorzoxazone provided additional support for the role of CYP2E1 in both tissues. CH formation was higher in PT cells than in DT cells and was time and reduced nicotinamide adenine dinucleotide phosphate (NADPH) dependent. However, pretreatment of rats with either pyridine or clofibrate had no effect on CYP2E1 or CYP2C11 protein levels or on CH formation in isolated cells. These data show for the first time that Tri can be metabolized to at least one of its P450 metabolites in the kidneys and quantitate the effect of P450 induction on Tri metabolism in the rat kidney.

Biochemical and functional characteristics of cultured renal epithelial cells from uninephrectomized rats: factors influencing nephrotoxicity.

Primary cultures of renal proximal (PT) and distal tubular (DT) cells from control and uninephrectomized (NPX) Sprague-Dawley rats were established to characterize factors that are responsible for the altered susceptibility to nephrotoxicants that occurs after compensatory renal cellular hypertrophy. Cells were grown in serum-free, hormonally defined medium and parameters were measured on days 1, 3, and 5 of primary culture. PT and DT cells from control and NPX rats appeared to maintain epithelial characteristics in culture, as shown by cytokeratin staining, morphology, protein and DNA content, and enzyme activities. Activities of several glutathione-dependent enzymes, including gamma-glutamyltransferase, glutathione S-transferase, glutathione peroxidase, and gamma-glutamylcysteine synthetase, were significantly greater in PT cells from NPX rats than in PT cells from control rats when factored by protein content. Rates of alpha-methylglucose uptake across the basolateral and brush-border membranes and sodium-dependent uptake of glutathione across the basolateral membrane were 2- to 3-fold higher in PT cells from NPX rats than in PT cells from control rats. These results are consistent with the hypertrophied phenotype being maintained in primary cultures of PT cells from NPX rats. The marked alterations in transport may play central roles in the delivery of nephrotoxicants to the target cell, and thus, increases the probability of chemically induced injury or death. These findings also suggest that these cell cultures may be useful for the study of biochemical processes associated with compensatory renal cellular hypertrophy.

Measurement of glutathione transport.

This unit provides methods for analyzing uptake in rat kidney proximal tubule cells and rat kidney cortical mitochondria, preparation of proximal tubule cells and cortical mitochondria, and HPLC analysis of glutathione and related compounds.

Role of voltage-dependent anion channels in glutathione transport into yeast mitochondria.

Glutathione (GSH) is imported into mitochondria from the extra-mitochondrial cytoplasm. Translocation across the inner membrane of mitochondria is thought to occur via the dicarboxylate and 2-oxoglutarate carriers; however, the means by which GSH passes through the outer membrane is unknown. Disruption of the outer membrane of yeast mitochondria using either digitonin or osmotic shock did not alter GSH accumulation as compared with accumulation in intact mitochondria. These results suggested that passage across the outer membrane was not the rate-limiting step in GSH accumulation. Mitochondria isolated from yeast strains with a disruption in the major pore-forming protein of the outer membrane, VDAC1, accumulated GSH to a greater extent than mitochondria isolated from a wild-type strain. Disruption of the gene for VDAC2 did not affect GSH import. Thus, neither VDAC form is essential for GSH translocation into mitochondria, and the participation of another outer membrane channel in GSH import is possible.

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.

A role for bioactivation and covalent binding within epidermal keratinocytes in sulfonamide-induced cutaneous drug reactions.

Cutaneous reactions are the most common manifestation of delayed-type hypersensitivity caused by sulfamethoxazole and dapsone. In light of the recognized metabolic and immunologic activity of the skin, we investigated the potential role of normal human epidermal keratinocytes in the development of these reactions. Adult and neonatal normal human epidermal keratinocytes metabolized sulfamethoxazole and dapsone to N-4-hydroxylamine and N-acetyl derivatives in a time-dependent manner. The latter was catalyzed by N-acetyltransferase 1 alone as normal human epidermal keratinocytes did not express mRNA for N-acetyltransferase 2. Investigation of metabolism-dependent toxicity of sulfamethoxazole and dapsone, and subsequent incubation of normal human epidermal keratinocytes with the respective hydroxylamine metabolites, demonstrated that these cells were resistant to the cytotoxic effects of sulfamethoxazole hydroxylamine but not dapsone hydroxylamine. With prior depletion of glutathione, however, normal human epidermal keratinocytes became susceptible to the toxicity of sulfamethoxazole hydroxylamine. Covalent adduct formation by sulfamethoxazole hydroxylamine was detected in normal human epidermal keratinocytes, even in the absence of cell death, and was increased with glutathione depletion. Major protein targets of sulfamethoxazole hydroxylamine were observed in the region of 160, 125, 95, and 57 kDa. Dapsone hydroxylamine also caused covalent adduct formation in normal human epidermal keratinocytes. Together, these observations provide a basis for our hypothesis that normal human epidermal keratinocytes are involved in the initiation and propagation of a cutaneous hypersensitivity response to these drugs.

Metabolism of trichloroethylene.

A major focus in the study of metabolism and disposition of trichloroethylene (TCE) is to identify metabolites that can be used reliably to assess flux through the various pathways of TCE metabolism and to identify those metabolites that are causally associated with toxic responses. Another important issue involves delineation of sex- and species-dependent differences in biotransformation pathways. Defining these differences can play an important role in the utility of laboratory animal data for understanding the pharmacokinetics and pharmacodynamics of TCE in humans. Sex-, species-, and strain-dependent differences in absorption and distribution of TCE may play some role in explaining differences in metabolism and susceptibility to toxicity from TCE exposure. The majority of differences in susceptibility, however, are likely due to sex-, species-, and strain-dependent differences in activities of the various enzymes that can metabolize TCE and its subsequent metabolites. An additional factor that plays a role in human health risk assessment for TCE is the high degree of variability in the activity of certain enzymes. TCE undergoes metabolism by two major pathways, cytochrome P450 (P450)-dependent oxidation and conjugation with glutathione (GSH). Key P450-derived metabolites of TCE that have been associated with specific target organs, such as the liver and lungs, include chloral hydrate, trichloroacetate, and dichloroacetate. Metabolites derived from the GSH conjugate of TCE, in contrast, have been associated with the kidney as a target organ. Specifically, metabolism of the cysteine conjugate of TCE by the cysteine conjugate ss-lyase generates a reactive metabolite that is nephrotoxic and may be nephrocarcinogenic. Although the P450 pathway is a higher activity and higher affinity pathway than the GSH conjugation pathway, one should not automatically conclude that the latter pathway is only important at very high doses. A synthesis of this information is then presented to assess how experimental data, from either animals or from (italic)in vitro (/italic)studies, can be extrapolated to humans for risk assessment. (italic)Key words(/italic): conjugate beta-lyase, cysteine glutathione, cytochrome P450, glutathione (italic)S(/italic)-transferases, metabolism, sex dependence, species dependence, tissue dependence, trichloroethylene.

Modes of action of trichloroethylene for kidney tumorigenesis.

This article focuses on the various models for kidney toxicity due to trichloroethylene (TCE) and its glutathione-dependent metabolites, in particular S-(1,2-dichlorovinyl)-l-cysteine. Areas of controversy regarding the relative importance of metabolic pathways, species differences in toxic responses, rates of generation of reactive metabolites, and dose-dependent phenomena are highlighted. The first section briefly reviews information on the incidence and risk factors of kidney cancer in the general U.S. population. Epidemiological data on incidence of kidney cancer in male workers exposed occupationally to TCE are also summarized. This is contrasted with cancer bioassay data from laboratory animals, that highlights sex and species differences and, consequently, the difficulties in making risk assessments for humans based on animal data. The major section of the article considers proposed modes of action for TCE or its metabolites in kidney, including peroxisome proliferation, alpha(2u)-globulin nephropathy, genotoxicity, and acute and chronic toxicity mechanisms. The latter comprise oxidative stress, alterations in calcium ion homeostasis, mitochondrial dysfunction, protein alkylation, cellular repair processes, and alterations in gene expression and cell proliferation. Finally, the status of risk assessment for TCE based on the kidneys as a target organ and remaining questions and research needs are discussed.

Depletion of cellular glutathione by conditions used for the passaging of adherent cultured cells.

Cultured cells are commonly exposed to trypsin-containing solutions in order to prepare cell suspensions suitable for subculture. Conditions used to release and disperse monolayers of cultured murine hepatoma 1c1c7 and human breast epithelial MCF10A cells caused the loss (40-95%) of cellular glutathione (GSH), but did not affect viability. Glutathione contents returned to pretrypsinization values within 24 h of replating. In contrast, the GSH contents of cultured rat hepatoma 5L cells were not affected by trypsinization. Exposure of 1c1c7 cultures to H(2)O(2) or etoposide 1 or 24 h after replating resulted in concentration-dependent cytostatic and cytotoxic effects. The concentration-response curves defining the cytostatic and cytotoxic effects of etoposide, and the cytostatic effects of H(2)O(2) were not influenced by the timing of toxicant addition. However, 1c1c7 cultures treated with H(2)O(2) 1 h after replating were more susceptible to the cytotoxic actions of the peroxide than cultures treated 24 h after plating. These studies show that conditions commonly used for the passaging of cultured cells can lead to a transient, but profound loss of GSH in some cell lines. Furthermore, the outcome of cytotoxicity analyses can be influenced by the time elapsed between the plating of cultures and the addition of toxicant.

Expression of glutathione-dependent enzymes and cytochrome P450s in freshly isolated and primary cultures of proximal tubular cells from human kidney.

The expression of glutathione (GSH)-dependent enzymes and cytochrome P450 (P450) proteins in freshly isolated proximal tubular cells from human kidney (hPT), and the effect of primary culture on these enzymes, were determined. Freshly isolated hPT cells had relatively high activities of gamma-glutamyltransferase, gamma-glutamylcysteine synthetase, glutathione S-transferase (GST), glutathione disulfide reductase, and GSH peroxidase. Cytochrome P450 4A11 was detected in freshly isolated hPT cells, whereas CYP2E1 was not. Freshly isolated hPT cells also expressed GSTA, GSTP, and GSTT but not GSTM. Primary cultures of hPT cells maintained their epithelial-like nature and diploid status, based on measurements of morphology, cytokeratin expression, and flow cytometric analysis. hPT cells retained GSH-dependent enzyme activities during primary culture, whereas cells that had undergone subsequent passage exhibited a loss of activities of most GSH-dependent enzymes and no longer expressed P450s or GSTs. CYP4A11 expression in primary cultures of hPT cells was significantly increased after treatment for 48 h with either ethanol (50 mM) or dexamethasone (7 nM). GSTA, GSTP, and GSTT contents, although still detectable, were decreased compared with those of freshly isolated hPT cells. Our data show that hPT cells express enzymes involved in xenobiotic disposition, and that they thus provide a model suitable for studies of human renal drug metabolism. Furthermore, primary cultures of hPT cells may afford the opportunity to study factors regulating P450 enzyme expression in human kidney.

Dopamine toxicity in neuroblastoma cells: role of glutathione depletion by L-BSO and apoptosis.

Dopamine (DA), while an essential neurotransmitter, is also a known neurotoxin that potentially plays an etiologic role in several neurodegenerative diseases. DA metabolism and oxidation readily produce reactive oxygen species (ROS) and DA can also be oxidized to a reactive quinone via spontaneous, enzyme-catalyzed or metal-enhanced reactions. A number of these reactions are cytotoxic, yet the precise mechanisms by which DA leads to cell death remain unknown. In this study, the neuroblastoma cell line, SK-N-SH, was utilized to examine DA toxicity under varying oxidant states. Cells pretreated with the glutathione (GSH)-depleting compound, L-buthionine sulfoximine (L-BSO), exhibited enhanced sensitivity to DA compared to controls (non-GSH-depleted cells). Furthermore, in cells pretreated with L-BSO, the addition of ascorbate (250 microM) afforded significant protection against DA-induced toxicity, while pyruvate (500 microM) had no protective effect. To further characterize the possibility that DA is associated with oxidative stress, additional studies were carried out with manganese (30 microM) as a pro-oxidant. Manganese and DA (200 microM), although not cytotoxic when individually administered to SK-N-SH cells, had a synergistic action on cytotoxicity. Finally, morphological and molecular markers of programmed cell death (apoptosis) were observed in cells treated with DA and L-BSO. These markers included membrane blebbing and internucleosomal DNA fragmentation. These results suggest that DA toxicity is tightly linked to intracellular oxidant/antioxidant levels, and that environmental factors, such as excessive Mn exposure, may modulate cellular sensitivity to DA.

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.

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.