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.

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.

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.

Differential induction of rat hepatic cytochromes P450 3A1, 3A2, 2B1, 2B2, and 2E1 in response to pyridine treatment.

Pyridine (PY) effects on rat hepatic cytochromes P450 (CYP) 3A1 and 3A2 expression were examined at the levels of metabolic activity, protein, and mRNA and were compared with those of CYP2B1/2 and CYP2E1. CYP3A metabolic activity as well as CYP3A protein and mRNA levels increased following treatment of rats with PY. CYP3A1 and CYP3A2 were differentially affected by PY treatment in terms of induction levels, dose dependence, and stability of mRNA. CYP3A1 mRNA levels maximally increased ~42-fold after PY treatment, whereas CYP3A2 mRNA level increased ~4-fold. Moreover, CYP3A1 mRNA levels decreased more rapidly than those of CYP3A2 as determined following inhibition of transcription with actinomycin D or cordycepin. Treatment of rats with PY resulted in a dose-dependent increase in CYP3A1, CYP3A2, and CYP2B1/2B2 protein levels. In contrast to the effects of PY treatment on CYP3A1 and 2B, CYP2E1 protein levels increased in the absence of a concomitant increase in CYP2E1 mRNA levels. Treatment of rats with PY at 200 mg/kg/day for 3 days increased both protein and mRNA levels of CYP3A2, whereas treatment with higher than 200 mg/kg/day for 3 days increased CYP3A2 protein levels without an increase in CYP3A2 mRNA levels. These data demonstrated that PY regulates the various CYPs examined in this study at different levels of expression and that PY regulates CYP3A1 expression through transcriptional activation and CYP3A2 expression through transcriptional and post-transcriptional activation at a low- and high-dose PY treatment, respectively.

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.

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.

Enrichment and functional reconstitution of glutathione transport activity from rabbit kidney mitochondria: further evidence for the role of the dicarboxylate and 2-oxoglutarate carriers in mitochondrial glutathione transport.

In previous studies, we provided evidence for uptake of glutathione (GSH) by the dicarboxylate and the 2-oxoglutarate carriers in rat kidney mitochondria. To investigate further the role of these two carriers, GSH transport activity was enriched from rabbit kidney mitochondria and functionally reconstituted into phospholipid vesicles. Starting with 200 mg of mitoplast protein, 2 mg of partially enriched proteins were obtained after Triton X-114 solubilization and hydroxyapatite chromatography. The reconstituted proteoliposomes catalyzed butylmalonate-sensitive uptake of [(14)C]malonate, phenylsuccinate-sensitive uptake of [(14)C]2-oxoglutarate, and transport activity with [(3)H]GSH. The initial rate of uptake of 5 mM GSH was approximately 170 nmol/min per mg protein, with a first-order rate constant of 0.3 min(-1), which is very close to that previously determined in freshly isolated rat kidney mitochondria. The enrichment procedure resulted in an approximately 60-fold increase in the specific activity of GSH transport. Substrates and inhibitors for the dicarboxylate and the 2-oxoglutarate carriers (i.e., malate, malonate, 2-oxoglutarate, butylmalonate, phenylsuccinate) significantly inhibited the uptake of [(3)H]GSH, whereas most substrates for the tricarboxylate and monocarboxylate carriers had no effect. GSH uptake exhibited an apparent K(m) of 2.8 mM and a V(max) of 260 nmol/min per mg protein. Analysis of mutual inhibition between GSH and the dicarboxylates suggested that the dicarboxylate carrier contributes a somewhat higher proportion to overall GSH uptake and that both carriers account for 70 to 80% of total GSH uptake. These results provide further evidence for the function of the dicarboxylate and 2-oxoglutarate carriers in the mitochondrial transport of GSH.

Influence of exogenous thiols on inorganic mercury-induced injury in renal proximal and distal tubular cells from normal and uninephrectomized rats.

Inorganic mercury (Hg(2+)) induced time- and concentration-dependent cellular injury in freshly isolated proximal tubular (PT) and distal tubular (DT) cells from normal (control) rats or uninephrectomized (NPX) rats. PT cells from NPX rats were more susceptible than PT cells from control rats, and DT cells were slightly more susceptible than PT cells to cellular injury induced by Hg(2+) (not bound to a thiol). Preloading cells with glutathione increased Hg(2+)-induced cellular injury in PT cells from control rats. However, coincubation of PT or DT cells from control or NPX rats with Hg(2+) and glutathione (1:4) provided significant protection relative to incubations with Hg(2+) alone. No support was obtained for a role for gamma-glutamyltransferase in glutathione-dependent protection. However, the organic anion carrier does appear to play a role in accumulation and toxicity of mercuric conjugates of cysteine in PT cells from control, but not NPX, rats. Coincubation with Hg(2+) and cysteine (1:4) had little effect on, or slightly enhanced, Hg(2+)-induced cellular injury at low concentrations of Hg(2+) in all cells studied. Coincubation with Hg(2+) and albumin (1:4) markedly protected PT and DT cells from control and NPX rats at all concentrations except the highest concentration of Hg(2+) in DT cells from NPX rats. 2,3-Dimercapto-1-propanesulfonic acid protected cells both when preloaded or added simultaneously with Hg(2+). Thus, renal cells from NPX rats are more susceptible to Hg(2+)-induced injury, PT and DT cells respond differently to exposure to Hg(2+), and thiols can significantly modulate the toxic response to Hg(2+).

Renal cellular transport of exogenous glutathione: heterogeneity at physiological and pharmacological concentrations.

Properties and kinetics of GSH transport into proximal tubular (PT) and distal tubular (DT) cells from rat kidney were determined to validate further the hypothesis that cellular differences in handling of GSH contribute to the greater susceptibility of DT cells to oxidant injury. PT and DT cells were incubated with a broad range of GSH concentrations, encompassing physiologically relevant (0.001 to 0.1 mM) to pharmacological (0.25 to 5 mM) levels of GSH. GSH uptake in PT cells was rapid, exhibiting an overshoot with a maximum at 1-min incubation. GSH uptake in DT cells reached maximal intracellular levels at 2- to 5-min incubations. GSH uptake in PT cells was resolved into two kinetically distinct processes, with Km values of 41.7 and 540 microM and Vmax values of 183 and 4885 pmol/min per 10(6) cells. In contrast, GSH uptake in DT cells was best described by one process, with Km and Vmax values of 1480 microM and 2094 pmol/min per 10(6) cells, respectively. Rates of GSH synthesis from 1 mM precursor amino acids were approximately 3-fold faster in PT cells, but rates of cysteine accumulation were 3.5-fold faster in DT cells. Accumulation of intracellular GSH in PT cells was 8-fold faster after incubation with 1 mM GSH than after incubation with 1 mM precursor amino acids. At both a physiological (10 microM) and a pharmacological (5 mM) GSH concentration, uptake exhibited marked Na+ and energy dependence, sensitivity to substrates for the organic anion and dicarboxylate carriers, and sensitivity to various gamma-glutamyl amino acids in PT cells only. Na+-dependent GSH uptake in PT cells was accounted for completely by activity of the organic anion and dicarboxylate carriers. These results indicate that DT cells possess limited capacity to transport GSH and suggest that exogenous GSH may not be effective in protecting other segments of the nephron besides the PT region from oxidants or other agents that alter GSH status.

Glutathione conjugation of trichloroethylene in human liver and kidney: kinetics and individual variation.

Isolated human hepatocytes exhibited time-, trichloroethylene (Tri) concentration-, and cell concentration-dependent formation of S-(1, 2-dichlorovinyl)glutathione (DCVG) in incubations in sealed flasks with 25 to 10,000 ppm Tri in the headspace, corresponding to 0.011 to 4.4 mM in hepatocytes. Maximal formation of DCVG (22.5 +/- 8.3 nmol/120 min per 10(6) cells) occurred with 500 ppm Tri. Time-, protein concentration-, and both Tri and GSH concentration-dependent formation of DCVG were observed in liver and kidney subcellular fractions. Two kinetically distinct systems were observed in both cytosol and microsomes from pooled liver samples, whereas only one system was observed in subcellular fractions from pooled kidney samples. Liver cytosol exhibited apparent Km values (microM Tri) of 333 and 22.7 and Vmax values (nmol DCVG formed/min per mg protein) of 8.77 and 4.27; liver microsomes exhibited apparent Km values of 250 and 29.4 and Vmax values of 3.10 and 1.42; kidney cytosol and microsomes exhibited apparent Km values of 26.3 and 167, respectively, and Vmax values of 0.81 and 6.29, respectively. DCVG formation in samples of liver cytosol and microsomes from 20 individual donors exhibited a 6.5-fold variation in microsomes but only a 2.4-fold variation in cytosol. In coincubations of pooled liver cytosol and microsomes, addition of an NADPH-regenerating system produced marked inhibition of DCVG formation, but addition of GSH had no effect on cytochrome P-450-catalyzed formation of chloral hydrate. These results indicate that both human kidney and liver have significant capacity to catalyze DCVG formation, indicating that the initial step of the GSH-dependent pathway is not limiting in the formation of nephrotoxic and nephrocarcinogenic metabolites.

Identification of S-(1,2-dichlorovinyl)glutathione in the blood of human volunteers exposed to trichloroethylene.

Healthy male and female human volunteers were exposed to 50 ppm or 100 ppm trichloroethylene (Tri) by inhalation for 4 h. Blood and urine samples were taken at various times before, during, and after the exposure period for analysis of glutathione (GSH), related thiols and disulfides, and GSH-derived metabolites of Tri. The GSH conjugate of Tri, S-(1,2-dichlorovinyl)glutathione (DCVG), was found in the blood of all subjects from 30 min after the start of the 4-h exposure to Tri to 1 to 8 h after the end of the exposure period, depending on the dose of Tri and the sex of the subject. Male subjects exposed to 100 ppm Tri exhibited a maximal content of DCVG in the blood at 2 h after the start of the exposure of 46.1 +/- 14.2 nmol/ml (n = 8), whereas female subjects exposed to 100 ppm Tri exhibited a maximal content of DCVG in the blood at 4 h after the start of the exposure of only 13.4 /- 6.6 nmol/ml (n = 8). Pharmacokinetic analysis of blood DCVG concentrations showed that the area under the curve value was 3.4-fold greater in males than in females, while the t1/2 values for systemic clearance of DCVG were similar in the two sexes. Analysis of the distribution of individual values indicated a possible sorting, irrespective of gender, into a high- and a low-activity population, which suggests the possibility of a polymorphism. The mercapturates N-acetyl-1,2-DCVC and N-acetyl-2,2-DCVC were only observed in the urine of 1 male subject exposed to 100 ppm Tri. Higher contents of glutamate were generally found in the blood of females, but no marked differences between sexes were observed in contents of cyst(e)ine or GSH or in GSH redox status in the blood. Urinary GSH output exhibited a diurnal variation with no apparent sex- or Tri exposure-dependent differences. These results provide direct, in vivo evidence of GSH conjugation of Tri in humans exposed to Tri and demonstrate markedly higher amounts of DCVG formation in males, suggesting that their potential risk to Tri-induced renal toxicity may be greater than that of females.

Role of extracellular thiols in accumulation and distribution of inorganic mercury in rat renal proximal and distal tubular cells.

Distribution of inorganic mercury (Hg) into both acid-soluble and protein-bound fractions of proximal tubular (PT) cells from the rat increased with increasing concentrations of Hg up to 10 microM. Little correlation was found between subcellular distribution of Hg and dose in distal tubular (DT) cells. Cellular accumulation of Hg was rapid, reaching equilibrium values by 10 to 15 min. Cellular content of Hg was significantly higher in PT cells than in DT cells at 1 microM Hg. To assess the effect of extracellular thiols on the intracellular accumulation of Hg, PT and DT cells were coincubated with Hg and cysteine, glutathione (GSH), bovine serum albumin (BSA) or 2,3-dimercapto-1-propanesulfonic acid (DMPS) in a 4:1 thiol:Hg molar ratio. Coexposure with Hg and cysteine increased intracellular accumulation of Hg in PT cells at 0.1 microM Hg relative to exposure to Hg alone, consistent with an Hg-cysteine conjugate being a transport form of Hg. In contrast, coexposure with Hg and BSA or DMPS markedly decreased accumulation of Hg relative to cells exposed to Hg alone in both cell types. Coexposure with Hg and GSH also decreased accumulation of Hg relative to exposure to Hg alone, but the decrease was less than coexposure with either BSA or DMPS, suggesting that either an Hg-GSH complex may be a transport form or that some of the Hg-GSH complexes were degraded to Hg-cysteine by the action of brush-border membrane enzymes. These results demonstrate that extracellular thiols markedly alter the renal accumulation of Hg and suggest that some Hg-thiol conjugates may be important physiological transport forms of Hg in the kidney.

Glutathione conjugation of perchloroethylene in rats and mice in vitro: sex-, species-, and tissue-dependent differences.

Perchloroethylene (Per)-induced nephrotoxicity and nephrocarcinogenicity have been associated with metabolism by the glutathione (GSH) conjugation pathway to form S-(1,2,2-trichlorovinyl)glutathione (TCVG). Formation of TCVG was determined in incubations of Per and GSH with isolated renal cortical cells and hepatocytes from male and female Fischer 344 rats and with renal and hepatic cytosol and microsomes from male and female Fischer 344 rats and B6C3F1 mice. The goal was to assess the role of metabolism in the sex and species dependence of susceptibility to Per-induced toxicity. A key finding was that GSH conjugation of Per occurs in kidney as well as in liver. Although amounts of TCVG formation in isolated kidney cells and hepatocytes from male and female rats were generally similar, TCVG formation in subcellular fractions showed marked sex, species, and tissue dependence. This may be due to the presence of multiple pathways for metabolism in intact cells, whereas only the GSH conjugation pathway is active in the subcellular fractions under the present assay conditions. TCVG formation in kidney and liver subcellular fractions from both male rats and mice were invariably higher than corresponding values in female rats and mice. Amounts of TCVG formation in rat liver subcellular fractions were approximately 10-fold higher than in corresponding fractions from rat kidney. Although rats are more susceptible to Per-induced renal tumors than mice, amounts of TCVG formation were 7- to 10-fold higher in mouse kidney subcellular fractions and 2- to 5-fold higher in mouse liver subcellular fractions of both sexes compared to corresponding fractions from the rat. Hence, although the higher amounts of TCVG formation in liver and kidney from male rats correspond to their higher susceptibility to Per-induced renal tumors compared with female rats, the markedly higher amounts of TCVG formation in mice compared with rats suggest that other enzymatic or transport steps in the handling of Per in mice contribute to their relatively low susceptibility to Per-induced renal tumors

Glutathione conjugation of trichloroethylene in rats and mice: sex-, species-, and tissue-dependent differences.

Glutathione (GSH) conjugation of trichloroethylene (Tri) to form S-(1,2-dichlorovinyl)glutathione (DCVG) has been implicated in the nephrotoxicity and nephrocarcinogenicity of Tri. Marked sex- and species-dependent differences exist, however, in the susceptibility to Tri-induced renal toxicity, with the male rat being the most susceptible. The present study, therefore, focuses on potential differences in the initial step of the GSH pathway. Rates of DCVG formation were measured in suspensions of isolated renal cortical cells and isolated hepatocytes from male and female Fischer 344 rats and in kidney and liver microsomes and cytosol from male and female Fischer 344 rats and B6C3F1 mice to determine if sex- and species-dependent differences in GSH conjugation correlate with susceptibility to renal toxicity from Tri. Rates of gamma-glutamyltransferase (GGT) with gamma-glutamyl-p-nitroanilide and glycylglycine as substrates and GSH S-transferase (GST) with 1-chloro-2,4-dinitrobenzene as substrate were also measured in liver and kidney subcellular fractions to provide further information on the biochemical basis of susceptibility to Tri. Rates of DCVG formation in rat kidney cells and kidney subcellular fractions were 5- to 20-fold lower than those in rat hepatocytes and liver subcellular fractions. Rates of DCVG formation in kidney cells and subcellular fractions were comparable in male and female rats with the exception of male rat kidney microsomes, where DCVG formation was below the limit of detection, and those in liver cells and subcellular fractions were >3-fold higher in male rats than in female rats. Rates of DCVG formation in mouse kidney subcellular fractions were approximately 10-fold higher than in corresponding fractions from the rat, whereas those in mouse liver subcellular fractions were 4- to 8-fold higher than in corresponding rat tissues, with rates in male mouse liver cytosol and microsomes being modestly higher than in corresponding fractions from female mice. GGT activity was barely detectable in livers, was about 20-fold higher in rat kidneys than in mouse kidneys, and was slightly higher in female rat kidneys than in male rat kidneys. GST activity with 1-chloro-2,4-dinitrobenzene as substrate exhibited tissue-, sex-, and species-dependent patterns that were generally similar to those with Tri as the substrate. These results suggest that the higher susceptibility to Tri-induced renal toxicity of male rats as compared with female rats correlates with rates of DCVG formation. The high rates of DCVG formation in mice, however, indicate that other factors, possibly including differences in activities of cysteine conjugate beta-lyase or N-acetyltransferase, may also be important determinants of the susceptibility to Tri.

Pathways of glutathione metabolism and transport in isolated proximal tubular cells from rat kidney.

Cellular uptake and metabolism of exogenous glutathione (GSH) in freshly isolated proximal tubular (PT) cells from rat kidney were examined in the absence and presence of inhibitors of GSH turnover [acivicin, L-buthionine-S,R-sulfoximine (BSO)] to quantify and assess the role of different pathways in the handling of GSH in this renal cell population. Incubation of PT cells with 2 or 5 mM GSH in the presence of acivicin/BSO produced 3- to 4-fold increases in intracellular GSH within 10-15 min. These significantly higher intracellular concentrations were maintained for up to 60 min. At lower concentrations of extracellular GSH, an initial increase in intracellular GSH concentrations was observed, but this was not maintained for the 60-min time course. In the absence of inhibitors, intracellular concentrations of GSH increased to levels that were 2- to 3-fold higher than initial values in the first 10-15 min, but these dropped below initial levels thereafter. In both the absence and presence of acivicin/BSO, PT cells catalyzed oxidation of GSH to glutathione disulfide (GSSG) and degradation of GSH to glutamate and cyst(e)ine. Exogenous tert-butyl hydroperoxide oxidized intracellular GSH to GSSG in a concentration-dependent manner and extracellular GSSG was transported into PT cells, but limited intracellular reduction of GSSG to GSH occurred. Furthermore, incubation of cells with precursor amino acids produced little intracellular synthesis of GSH, suggesting that PT cells have limited biosynthetic capacity for GSH under these conditions. Hence, direct uptake of GSH, rather than reduction of GSSG or resynthesis from precursors, may be the primary mechanism to maintain intracellular thiol redox status under toxicological conditions. Since PT cells are a primary target for toxicants, the ability of these cells to rapidly take up and metabolize GSH may serve as a defensive mechanism to protect against chemical injury.

Metabolism of aflatoxin B1 by rabbit and rat nasal mucosa microsomes and purified cytochrome P450, including isoforms 2A10 and 2A11.

The nasal mucosa of some mammalian species are susceptible to the toxicity of aflatoxin B1 (AFB1), a potent hepatocarcinogen, but little is known about the nasal enzymes involved in the metabolic activation of AFB1 or the metabolites produced. In the present study, the metabolism of AFB1 was studied with nasal microsomes from rats and rabbits and with several purified isozymes of rabbit P450 in a reconstituted enzyme system. The rates of AFB1-N7-guanine DNA adduct formation with rabbit and rat nasal microsomes are over 3- and 10-fold higher, respectively, than with liver microsomes from the same species. On the other hand, the rates of formation of AFM1 (9a-hydroxy-AFB1) and AFQ1 (3-hydroxy-AFB1) products known to be less toxic, are lower with nasal than with liver microsomes. Of particular interest, nasal microsomes produce high levels of six unidentified polar metabolites that are not formed by microsomes from liver or several other tissues. These same products are also generated by P450 NMa purified from rabbit nasal microsomes in a reconstituted system, but not by five other isozymes of cytochrome P450 (1A2, 2B4, 2E1, 2G1, 3A6) that are known to be present in nasal microsomes. AFB1-DNA adducts are formed by P450 NMa at a rate 3-fold higher than that by nasal microsomes. The DNA adducts are formed at much slower rates by P450s 2G1, 2B4, and 1A2, and adducts are not formed at measurable rates by P450s 2E1 and 3A6. Moreover, AFB1-DNA adduct formation is also catalyzed by cDNA-derived, heterologously expressed P450s 2A10 and 2A11, both of which are known to be present in the purified P450 NMa preparation. The Km and Vmax values of the two isozymes for DNA adduct formation are comparable to those for nasal microsomes. Furthermore, the formation of AFB1-DNA adducts by nasal microsomes is decreased by nicotine, a known inhibitor of P450 NMa. These data indicate that members of the P450 2A gene subfamily play an important role in the metabolic activation of AFB1 in rabbit and rat nasal mucosa and suggest a molecular basis for assessing the health risk associated with inhalation exposure to this procarcinogen in humans.

Cytochrome P450-catalyzed hydroxylation of hydrocarbons: kinetic deuterium isotope effects for the hydroxylation of an ultrafast radical clock.

The ultrafast radical clock probe trans-1-methyl-2-phenylcyclopropane (1CH3) and its mono-, di-, and trideuteriomethyl analogues were oxidized by phenobarbital-induced rat liver microsomal enzymes. This cytochrome P450-catalyzed hydroxylation of 1CH3 gave three products: the alcohol trans-(2-phenylcyclopropyl)methanol (2), the rearranged alcohol 1-phenylbut-3-en-1-ol (3), and the phenol trans-2-(p-hydroxyphenyl)-1-methylcyclopropane (4). The identification of both the unrearranged and rearranged products of oxidation, 2 and 3, is consistent with the formation of a radical intermediate via a hydrogen atom abstraction from the methyl group by the catalytically active iron-oxo center. Hydroxylation of three deuteriomethyl forms of 1CH3 produced the analogous deuterated products, although in different amounts of each. Perdeuteration of the methyl group (1CD3) disfavored oxidation at the methyl group and caused an increase in the oxidation of the phenyl ring (metabolic switching). By comparing the amounts of alcohols and phenol formed from the individual, noncompetitive oxidation of 1CH3 and 1CD3 the overall (i.e., combined primary and secondary) deuterium kinetic isotope effect (DKIE) was found to be 12.5. Intramolecular DKIEs for 1CHD2 and 1CH2D were 2.9 and 13.2, respectively. From these results, the primary and secondary DKIEs were calculated to be 7.87 and 1.26, respectively, values that indicate that there is extensive C--H bond stretching in the transition state for the rate-controlling step in P450-catalyzed hydroxylation of 1CH3.(ABSTRACT TRUNCATED AT 250 WORDS)