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Papers by David Putt

Antioxidant defense in renal proximal tubular cells from normal and diabetic rats

The FASEB Journal

Primary cultures of proximal tubular (PT) cells from streptozotocin (STZ) diabetic rats had highe... more Primary cultures of proximal tubular (PT) cells from streptozotocin (STZ) diabetic rats had higher basal and toxicant‐stimulated reactive oxygen species and mitochondrial membrane potential and were more susceptible to oxidant injury as compared to those from age‐matched control rats. Both N‐acetyl‐L‐cysteine (NAC) and a cell‐permeable catalase derivative (Cat‐SKL) protected. Despite higher basal oxidant stress, mitochondria of diabetic PT cells had higher GSH content. Protein levels of mitochondrial GSH transporters were slightly higher in diabetes, that of superoxide dismutase 2 (SOD2) was modestly elevated, and that of total thioredoxin 2 (Trx2) was decreased at 30 days and increased at 90 days. Levels of 3‐nitrotyrosine‐modified proteins in mitochondria were somewhat higher at both times in diabetic rats. 4‐Hydroxy‐2‐nonenal (HNE)‐modified proteins were mostly increased at 30 days but decreased at 90 days. Thus, redox processes and mitochondrial energetics are markedly altered d...

Toxicology and Applied Pharmacology, Jun 1, 2007

Simultaneous or prior exposure to one chemical may alter the concurrent or subsequent response to... more 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-α1 (GSTα1) but decreased expression of GSTα2, 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.

Toxicology, Dec 1, 2006

To further develop primary cultures of human proximal tubular (hPT) cells for study of drug dispo... more To further develop primary cultures of human proximal tubular (hPT) cells for study of drug disposition, we determined kinetics and protein expression of several key transporters for organic anions and cations, peptides, and neutral amino acids. p-Aminohippurate uptake exhibited similar kinetics as published values, was inhibited by cephaloridine, cimetidine, methotrexate, and urate, consistent with function of both organic anion transporter 1 (OAT1) and OAT3. Transport rates by organic cation transporters (OCTs) were up to three-fold higher than those of OATs. Of the OCT substrates tested, triethanolamine exhibited the highest transport rates across the basolateral membrane (BLM). OCTN1 exhibited high-affinity, low-capacity BLM transport of l-carnitine. Glycylsarcosine transport by PepT2 was rapid and comparable to that of OCTs. Amino acid System L on the BLM exhibited comparable kinetic parameters for transport of l-leucine as the OATs. Efflux of verapamil across the brush-border membrane by P-glycoprotein was very rapid. Expression of carriers was generally maintained throughout 5 days of culture. Of the four OAT proteins studied (OAT1-4), expression of OAT1 and OAT3 was the most readily detected and exhibited interindividual variation. OCTN2 was the major OCT in hPT cells. Expression was also quantified for multidrug resistance-associated proteins 2 and 5 and P-glycoprotein. These results show that primary cultures of hPT cells express a diverse array of transporters for major classes of important drugs and are suitable for study of drug transport and disposition and assessment of potential drug-drug interactions in human kidney.

Journal of Controlled Release, Jan 4, 2010

Redox-responsive polyplexes represent a promising class of non-viral gene delivery vectors. The r... more Redox-responsive polyplexes represent a promising class of non-viral gene delivery vectors. The reducible disulfide bonds in the polyplexes undergo intracellular reduction owing to the presence of high concentrations of reduced glutathione (GSH). Available evidence suggests improved transfection activity of redox-sensitive polyplexes upon artificial modulation of intracellular GSH. This study investigates the effect of innate differences in GSH concentration in a panel of human pancreatic cancer cell lines on activity of reducible polyplexes of the four major classes of nucleic acid therapeutics: plasmid DNA (pDNA), messenger RNA (mRNA), antisense oligodeoxynucleotides (AON) and siRNA. In general, reducible polyplexes of linear poly(amido amines) (PAA) show improved activity compared to non-reducible polyplexes of PAA. Results demonstrate that increased GSH levels are associated with improved transfection of mRNA polyplexes but no clear trend is observed for pDNA, AON and siRNA polyplexes.

Mitochondrial GSH status in compensatory renal hypertrophy due to uninephrectomy and diabetic nephropathy

The FASEB Journal, Apr 1, 2011

Adaptive changes in renal mitochondrial redox status in diabetic nephropathy

Toxicology and Applied Pharmacology, 2012

Nephropathy is a serious and common complication of diabetes. In the streptozotocin (STZ)-treated... more Nephropathy is a serious and common complication of diabetes. In the streptozotocin (STZ)-treated rat model of diabetes, nephropathy does not typically develop until 30 to 45days post-injection, although hyperglycemia occurs within 24h. We tested the hypothesis that chronic hyperglycemia results in a modest degree of oxidative stress that is accompanied by compensatory changes in certain antioxidants and mitochondrial redox status. We propose that as kidneys progress to a state of diabetic nephropathy, further adaptations occur in mitochondrial redox status. Basic parameters of renal function in vivo and several parameters of mitochondrial function and glutathione (GSH) and redox status in isolated renal cortical mitochondria from STZ-treated and age-matched control rats were examined at 30days and 90days post-injection. While there was no effect of diabetes on blood urea nitrogen, measurement of other, more sensitive parameters, such as urinary albumin and protein, and histopathology showed significant and progressive worsening in diabetic rats. Thus, renal function is compromised even prior to the onset of frank nephropathy. Changes in mitochondrial respiration and enzyme activities indicated existence of a hypermetabolic state. Higher mitochondrial GSH content and rates of GSH transport into mitochondria in kidneys from diabetic rats were only partially due to changes in expression of mitochondrial GSH carriers and were mostly due to higher substrate supply. Although there are few clear indicators of oxidative stress, there are several redox changes that occur early and change further as nephropathy progresses, highlighting the complexity of the disease.

Journal of Pharmacology and Experimental Therapeutics, Oct 6, 2005

This study was undertaken to test the hypothesis that bronchiolar damage induced by trichloroethy... more This study was undertaken to test the hypothesis that bronchiolar damage induced by trichloroethylene (TCE) is associated with bioactivation within the Clara cells with the involvement of CYP2E1 and CYP2F2. Histopathology confirmed dose-dependent Clara cell injury and disintegration of the bronchiolar epithelium in CD-1 mice treated with TCE doses of 500 to 1000 mg/kg i.p. Immunohistochemical studies, using an antibody that recognizes dichloroacetyl lysine adducts, revealed dose-dependent formation of adducts in the bronchiolar epithelium. Localization of dichloroacetyl adducts in the Clara cells coincided with damage to this cell type in TCE-treated mice. Pretreatment of CD-1 mice with diallyl sulfone, an inhibitor of CYP2E1 and CYP2F2, abrogated the formation of the dichloroacetyl adducts and protected against TCE-induced bronchiolar cytotoxicity. Treatment of wild-type and CYP2E1-null mice with TCE (750 mg/kg i.p.) also elicited bron-chiolar damage that correlated with the formation of adducts in the Clara cells. Immunoblotting, using lung microsomes from TCE-treated CD-1 mice, showed dose-dependent production of dichloroacetyl adducts that comigrated with CYP2E1 and CYP2F2. However, TCE treatment resulted in a loss of immunoreactive CYP2E1 and CYP2F2 proteins and p-nitrophenol hydroxylation, a catalytic activity associated with both cytochrome P450 enzymes. The TCE metabolite, chloral hydrate, was formed in incubations of TCE with lung microsomes from CD-1, wild-type, and CYP2E1-null mice. The levels were higher in CD-1 than in either wild-type or CYP2E1-null mice, although levels were higher in CYP2E1-null than in wild-type mice. These findings supported the contention that TCE bioactivation within the Clara cells, predominantly involving CYP2F2, correlated with bronchiolar cytotoxicity in mice.

Toxicology and Applied Pharmacology, Mar 1, 2002

A marked sex dependence in the acute cytotoxicity of both Perc and TCVG was observed: Perc caused... more A marked sex dependence in the acute cytotoxicity of both Perc and TCVG was observed: Perc caused significant release of lactate dehydrogenase (LDH) in isolated kidney cells from male but not female rats, and TCVG caused much more LDH release from male than female rat kidney cells. Assessment of toxicity in suspensions of isolated mitochondria from kidneys of male and female rats revealed a generally similar pattern of sensitivity, with mitochondria from males exhibiting significantly more inhibition of State 3 respiration and decrease of respiratory control ratio than mitochondria from females. Respiratory function in mitochondria from male and female mice, however, was also significantly inhibited by Perc or TCVG but exhibited little sex dependence in the degree of inhibition. Comparison with results from similar studies using the congener trichloroethylene and its glutathione conjugate suggested that Perc and TCVG are more potent nephrotoxicants. Neither Perc nor TCVG produced any significant effects on cytotoxicity or mitochondrial function in isolated hepatocytes from rats or in isolated liver mitochondria from rats or mice, suggesting that the liver is not a major acute target for Perc or its glutathione conjugate. Thus, many of the species-, sex-, and tissue-dependent differences in toxicity of Perc and TCVG that are observed in vivo are also observed in these in vitro models.

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

Biochemical Pharmacology, Nov 1, 2002

Cellular energetics and redox status were evaluated in NRK-52E cells, a stable cell line derived ... more 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.

Biochemical Pharmacology, Aug 1, 2008

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

Toxicology, Jun 3, 2007

The relative importance of metabolism of trichloroethylene (Tri) and perchloroethylene (Perc) by ... more The relative importance of metabolism of trichloroethylene (Tri) and perchloroethylene (Perc) by the cytochrome P450 (P450) and glutathione (GSH) conjugation pathways in their acute renal and hepatic toxicity was studied in isolated cells and microsomes from rat kidney and liver after various treatments to modulate P450 activity/expression or GSH status. Inhibitors of P450 stimulated GSH conjugation of Tri and, to a lesser extent, Perc, in both kidney cells and hepatocytes. Perc was a more potent, acute cytotoxic agent in isolated kidney cells than Tri but Perc-induced toxicity was less responsive than Tri-induced toxicity to modulation of P450 status. These observations are consistent with P450-dependent bioactivation being more important for Tri than for Perc. Incubation of isolated rat hepatocytes with Tri produced no acute cytotoxicity in isolated hepatocytes while Perc produced comparable cytotoxicity as in kidney cells. Modulation of P450 status in hepatocytes produced larger changes in Tri-and Perc-induced cytotoxicity than in kidney cells, with non-selective P450 inhibitors increasing toxicity. Induction of CYP2E1 with pyridine also markedly increased sensitivity of hepatocytes to Tri but had little effect on Perc-induced cytotoxicity. Increases in cellular GSH concentrations increased Tri-and Perc-induced cytotoxicity in kidney cells but not in hepatocytes, consistent with the role of GSH conjugation in Tri-and Perc-induced nephrotoxicity. In contrast, depletion of cellular GSH concentrations moderately decreased Tri-and Perc-induced cytotoxicity in kidney cells but increased cytotoxicity in hepatocytes, again pointing to the importance of different bioactivation pathways and modes of action in kidney and liver.

Journal of toxicology and environmental health, Aug 1, 2006

Male and female Fischer 344 rats were administered trichloroethylene (TRI) (2, 5, or 15 mmol/kg b... more Male and female Fischer 344 rats were administered trichloroethylene (TRI) (2, 5, or 15 mmol/kg body weight) in corn oil by oral gavage and TRI and its metabolites were measured at times up to 48 hr in liver, kidney, blood, and urine. We tested the hypothesis that sex-dependent differences in distribution and metabolism of TRI could help explain differences in toxicity. Higher levels of TRI were generally observed in tissues of males. A biphasic pattern of TRI concentration was observed in liver, kidney, and blood of both males and females, consistent with enterohepatic recirculation. Higher concentrations of cytochrome P450 (P450)-derived metabolites (chloral hydrate, trichloroacetate, trichloroethanol) were observed in livers of males than in livers of females whereas the opposite pattern was observed in kidneys. Chloral hydrate was the primary P450-derived metabolite in blood and urine of males whereas trichloroacetate was the primary P450-derived metabolite in blood and urine of females. S-(1,2-Dichlorovinyl)glutathione (DCVG) was recovered in liver and kidney of female rats only and in blood of both male and female rats, with generally higher amounts found in females. S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), the penultimate nephrotoxic metabolite, was recovered in male and female liver, female kidney, male blood, and in urine of both males and females. The results demonstrate sex-dependent differences in recovery of key metabolites of TRI that may help explain differences in susceptibility to TRI-induced toxicity with both the liver and kidney as target organs.

Archives of Biochemistry and Biophysics, Jun 1, 2008

Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and... more Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and 2-oxo-glutarate carriers (OGC; Slc25a11). To determine whether these carriers function similarly in liver mitochondria, we assessed the effect of competition with specific substrates or inhibitors on GSH uptake in isolated rat liver mitochondria. GSH uptake was uniphasic, independent of ATP hydrolysis, and exhibited K m and V max values of 4.08 mM and 3.06 nmol/ min per mg protein, respectively. Incubation with butylmalonate and phenylsuccinate inhibited GSH uptake by 45-50%, although the individual inhibitors had no effect, suggesting in rat liver mitochondria, the DIC and OGC are only partially responsible for GSH uptake. H4IIE cells, a rat hepatoma cell line, were stably transfected with the cDNA for the OGC, and exhibited increased uptake of GSH and 2-oxoglutarate and were protected from cytotoxicity induced by H 2 O 2 , methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport in the liver.

Toxicology and Applied Pharmacology, Nov 1, 2001

dichlorovinyl)-L-cysteine (DCVC), the cysteine conjugate of the environmental and occupational co... more 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 M) 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 <100 M 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 M) 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 M 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.

Mitochondrial dysfunction in S‐(1,2‐dichlorovinyl)‐L‐cysteine (DCVC)‐induced apoptosis and necrosis in human proximal tubular cells

The FASEB Journal, Mar 1, 2006

Drug Metabolism and Disposition, Jun 29, 2005

Pulmonary cytotoxicity induced by trichloroethylene (TCE) is associated with cytochrome P450-depe... more Pulmonary cytotoxicity induced by trichloroethylene (TCE) is associated with cytochrome P450-dependent bioactivation to reactive metabolites. In this investigation, studies were undertaken to test the hypothesis that TCE metabolism to chloral hydrate (CH) is mediated by cytochrome P450 enzymes including CYP2E1, CYP2F and CYP2B1. Recombinant rat CYP2E1 catalyzed TCE metabolism to CH with greater affinity than did the recombinant P450 enzymes, rat CYP2F4, mouse CYP2F2, rat CYP2B1 and human CYP2E1. The catalytic efficiencies of recombinant rat CYP2E1 (V max /K m = 0.79) for generating CH was greater than those of recombinant CYP2F4 (V max /K m = 0.27), recombinant mouse CYP2F2 (V max /K m = 0.11), recombinant rat CYP2B1 (V max /K m = 0.07) or recombinant human CYP2E1 (V max /K m = 0.02). Decreases in lung microsomal immunoreactive CYP2E1, CYP2F2 and CYP2B1 were manifested at varying time-points after TCE treatment. The loss of immunoreactive CYP2F2 occurred prior to those of immunoreactive CYP2E1 and CYP2B1. These protein decreases coincided with marked reduction of lung microsomal p-nitrophenol hydroxylation and pentoxyresorufin O-dealkylation. Rates of CH formation in the microsomal incubations were time-dependent and were incremental from 5 to 45 min. The production of CH was also determined in human lung microsomal incubations: the rates were low and were detected in only three out of eight subjects. These results showed that, although CYP2E1, CYP2F and CYP2B1 are all capable of generating CH, TCE metabolism is mediated with greater affinity by recombinant rat CYP2E1 than by recombinant CYP2F, CYP2B1 or human CYP2E1. Moreover, the rates of CH production were substantially higher in murine than in human lung.

Toxicology and Applied Pharmacology, May 1, 1998

Per and GSH with isolated renal cortical cells and hepatocytes from male and female Fischer 344 r... more 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.

Journal of Pharmacology and Experimental Therapeutics, Mar 6, 2003

S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) is the penultimate nephrotoxic metabolite of the environm... more S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) is the penultimate nephrotoxic metabolite of the environmental contaminant trichloroethylene. Although metabolism of DCVC by the cysteine conjugate ␤-lyase is the most studied bioactivation pathway, DCVC may also be metabolized by the flavin-containing monooxygenase (FMO) to yield DCVC sulfoxide (DCVCS). Renal cellular injury induced by DCVCS was investigated in primary cultures of human proximal tubular (hPT) cells by assessment of time-and concentration-dependent effects on cellular morphology, acute cellular necrosis, apoptosis, mitochondrial function, and cellular glutathione (GSH) status. Confluent hPT cells incubated with as little as 10 M DCVCS for 24 h exhibited morphological changes, although at least 100 M DCVCS was required to produce marked changes. Acute cellular necrosis did not occur until 48 h with at least 200 M DCVCS, indicating that this is a high-dose, late response. The extent of necrosis was similar to that with DCVC. In contrast, apoptosis occurred as early as 1 h with as little as 10 M DCVCS and the extent of apoptosis was much less than that with DCVC. Mitochondrial function was maintained with DCVCS concentrations up to 100 M, consistent with hPT cells only being competent to undergo apoptosis at early time points and relatively low concentrations. Marked depletion (Ͼ50%) of cellular GSH content was only observed with 500 M DCVCS. These results, combined with previous studies showing protection from DCVC-induced necrosis and apoptosis by the FMO inhibitor methimazole, suggest that formation of DCVCS plays a significant role in trichloroethylene-induced renal cellular injury in hPT cells. Trichloroethylene (TRI; also known as trichloroethene) is a major environmental contaminant that is of concern for both workers who use it, primarily in metal degreasing, and for the general population, because of widespread contamination of soil and surface and groundwater. TRI produces acute toxicity or tumors in several tissues, with the target organ specificity and sensitivity exhibiting species-, strain-, and sex-dependent differences. Based on the overall weight of evidence, the International Agency for Research on Cancer (IARC, 1995) designated TRI as a "probable human carcinogen" and the National Toxicology Program (NTP, 2001) listed TRI in its Ninth Report on Carcinogens as "reasonably anticipated to be a human carcinogen". Most TRI toxicity is associated with its metabolism, which occurs by either cytochrome P450-dependent oxidation or glutathione (GSH) conjugation (Lash et al., 2000a). One of the established target organs for TRI is the kidneys, and renal toxicity is associated with metabolism by the GSH conjugation pathway . S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), the cysteine conjugate of TRI, was initially considered to be the primary, if not sole, penultimate nephrotoxic metabolite that produces renal cellular injury after bioactivation by the cysteine conjugate ␤-lyase (␤lyase). However, DCVC can also be metabolized by the flavincontaining monooxygenase (FMO) to produce DCVC sulfoxide (DCVCS) , which is highly reactive and is a potent nephrotoxicant in rats and is cytotoxic in isolated rat proximal tubular (rPT) cells .

Toxicology, Feb 1, 2008

We previously catalogued expression and activity of organic anion and cation, amino acid, and pep... more We previously catalogued expression and activity of organic anion and cation, amino acid, and peptide transporters in primary cultures of human proximal tubular (hPT) cells to establish them as a cellular model to study drug transport in the human kidney Membrane transport function in primary cultures of human proximal tubular cells. Toxicology 228,[200][201][202][203][204][205][206][207][208][209][210][211][212][213][214][215][216][217][218]. Here, we extend our analysis to drug metabolism enzymes. Expression of 11 cytochrome P450 (CYP) enzymes was determined with specific antibodies. CYP1B1, CYP3A4, and CYP4A11 were the only CYP enzymes readily detected in total cell extracts. These same CYP enzymes, as well as CYP3A5 and possibly CYP2D6, were detected in microsomes from confluent hPT cells, although expression levels varied among kidney samples. In agreement with Western blot data, only activity of CYP3A4/5 was detected among the enzyme activities measured. Expression of all three glutathione S-transferases (GSTs) known to be found in hPT cells, GSTA, GSTP, and GSTT, was readily detected. Variable expression of three sulfotransferases (SULTs), SULT1A3, SULT1E, and SULT2A1, and three UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A6, and UGT2B7, was also detected. When examined over the course of cell growth to confluence, expression of all enzymes was generally maintained at readily measurable levels, although they were often lower than in fresh tissue. These results indicate that primary cultures of hPT cells possess significant capacity to metabolize many classes of drugs, and can be used as an effective model to study drug metabolism.

Archives of Biochemistry and Biophysics, 2000

In previous studies, we provided evidence for uptake of glutathione (GSH) by the dicarboxylate an... more 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 dicarboxy-lates 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.