Cytochrome P450-Dependent Metabolism of Trichloroethylene in Rat ...

60, 11–19 (2001) Copyright © 2001 by the Society of Toxicology

TOXICOLOGICAL SCIENCES

Cytochrome P450-Dependent Metabolism of Trichloroethylene in Rat Kidney Brian S. Cummings,* Jean C. Parker,† and Lawrence H. Lash* ,1 *Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, Michigan 48201; and †National Center for Environmental Assessment, U.S. Environmental Protection Agency, Washington, DC 20460 Received July 27, 2000; accepted November 7, 2000

the possibility of both occupational and general human exposure (Davidson and Beliles, 1991). Tri produces both acute and chronic toxicity with multiple target organs, and its effects have been studied in several species, including humans (NTP, 1982, 1990). There are significant species-dependent differences in the toxic responses to Tri (Brown et al., 1990; Lash et al., 1995; NCI, 1976; NTP, 1982, 1990; Weiss, 1996). The kidneys are one target organ (Lash et al., 2000a), and data from exposed human workers indicate a possible increased risk of kidney cancer (Brown et al., 1990; David et al., 1989; Henschler et al., 1995a; Nagaya et al., 1989; Weiss, 1996). Such claims have been disputed, however, and controversy has arisen surrounding the correlation of human kidney cancer with Tri exposure (Bloemen and Tomenson, 1995; Henschler et al, 1995b; Swaen, 1995). A central point of this controversy involves the differences in metabolism pathways for Tri in male rats and humans. Tri is metabolized by two separate pathways, glutathione (GSH) conjugation and cytochrome P450 (P450)-dependent oxidation (see Lash et al., 2000b for a review of Tri metabolism; Fig. 1). The former pathway produces S-(1,2-dichlorovinyl)glutathione (DCVG), which can be metabolized further to the cysteine conjugate S-(1,2-dichlorovinyl)-L-cysteine (DCVC). The initial step of the pathway, GSH conjugation of Tri, can occur in both the kidneys and the liver of both rodents and humans (Lash et al., 1995, 1998, 1999), although the liver has a much higher capacity than the kidneys. Subsequent steps occur in the kidneys, and metabolites from this pathway are selectively associated with nephrotoxicity and possibly with nephrocarcinogenicity (Lash et al., 2000a,b). The second metabolic pathway involved in the metabolism of Tri is oxidative metabolism by P450. Tri is believed to be first metabolized to either Tri-epoxide, dichloroacetyl chloride, or chloral, which is in equilibrium with chloral hydrate (CH) (Fig. 1). Each of these initial metabolites are further metabolized to yield various products, including mono-, di-, and trichloroacetate, trichloroethanol and its glucuronide, glyoxylate, glycolate, and oxalate. Four different P450 enzymes have been identified as playing a role in hepatic Tri metabolism: CYP1A1/2, CYP2B1/2, CYP2C11, and CYP2E1 (Guengerich,

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. Key Words: trichloroethylene; kidney; metabolism; cytochrome P450; proximal tubular cells; enzyme induction.

Trichloroethylene (Tri) is a colorless, volatile liquid and alkenyl halide. The lipophilicity, nonflammability, and high boiling point of Tri make it useful in several industrial processes, including metal degreasing and dry cleaning. Tri was also once used as an anesthetic. Because of these uses, high amounts of Tri contaminate ground and surface water, raising This work was supported by National Institutes of Diabetes and Digestive and Kidney Diseases grant R01-DK40725 and by a cooperative agreement with the U.S. Environmental Protection Agency (CR-824183). The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. 1 To whom correspondence should be addressed. Fax: (313) 577-6739. E-mail: [email protected]. 11

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CUMMINGS, PARKER, AND LASH

FIG. 1. Proposed schematic of P450-dependent oxidative metabolism of trichloroethylene. Tri is thought to be oxidized by P450 enzymes to either Tri-epoxide, dichloroacylchloride, or chloral, which is in rapid equilibrium with CH. P450 enzymes responsible for Tri metabolism in the liver are listed, while those responsible for Tri metabolism in the kidney have not been determined previously. The three initial metabolites undergo additional reactions to yield several products, including glyoxylate, glycolate, mono-, di-, and trichloroacetate, and trichloroethanol and its glucuronide. Tri can also be metabolized by GSH-dependent conjugation to DCVG, which is metabolized further to DCVC.

1991; Guengerich et al., 1991; Koop et al., 1985; Nakajima et al., 1988, 1990, 1992a,b, 1993). There remains some controversy about the nature and identity of the initial metabolites generated by P450. Miller and Guengerich (1983) had concluded that Tri-epoxide was not an obligate intermediate in the formation of CH and that Tri-epoxide cannot be the intermediate responsible for the irreversible binding of metabolites of radiolabeled Tri to proteins and DNA. They showed further that metabolism of Tri by P450 can inactivate P450 by destruction of the heme group. This study was supported by Green and Prout (1985), who showed that the major products of an epoxide intermediate of Tri should be CO, CO 2, dichloroacetic acid (DCA), and monochloroacetic acid. In contrast, CH, trichloroacetic acid (TCA), and trichloroethanol (TCOH) were the major metabolites recovered in both in vivo and in vitro studies. Assessments that the intermediate of Tri and P450 must have some degree of chemical stability (unlike an epoxide) led to the hypothesis that the majority of Tri undergoes a chlorine migration in an oxygenated Tri-P450 transition state. This chlorine migration would then lead to formation of CH. Forkert and colleagues have unequivocally demonstrated the existence of an epoxide for 1,1-dichloroethylene in mouse lung and liver (Forkert, 1999a,b; Forkert et al., 1999), and Guengerich and coworkers (Cai and Guengerich, 1999, 2000) have recently shown that Tri-epoxide can exist in aqueous solution and can react directly with nucleophiles, producing protein adducts. Hence, Tri-epoxide is one of the initial products of the catalytic action of P450 enzymes on Tri.

Elfarra et al. (1998) showed that Tri was oxidized in rat, mouse, and human liver microsomes to CH and TCOH. CH and TCOH were the only metabolites consistently detected, and only small amounts of DCA, TCA, oxalic acid, or TCOH glucuronide were detected in some samples. Tri metabolism to CH followed biphasic kinetics in both rat and human liver microsomes, but only monophasic kinetics in mouse liver microsomes. The P450 enzymes involved in Tri metabolism in either rat or human liver microsomes were not determined in that study, however, nor was the extent of oxidative metabolism of Tri in the kidney studied. Because we observed previously that preincubation of rat kidney cells with P450 inhibitors increased the amount of GSH conjugation of Tri (Cummings et al., 2000a), we hypothesized that oxidative metabolism of Tri by P450 occurs in these cells. Furthermore, we recently showed that freshly isolated PT and DT cells from rat kidney express several P450 enzymes, including CYP2E1, CYP2C11, CYP2B1/2, and CYP4A2/3 (Cummings et al., 1999). The goal of this study was, therefore, to determine if Tri is metabolized to any of its oxidative metabolites (i.e., CH, DCA, TCA, TCOH) in the rat kidney and the specific P450 enzymes involved in this metabolism. Data from this study are the first to show that Tri can be metabolized to CH in the rat kidney. These data thus provide additional insight into the renal handling of Tri and should help in assessing the role of the kidneys as a target organ for Tri. MATERIALS AND METHODS Chemicals. Tri (reported to be 99.9% pure, as judged by electron ionization mass spectrometry), collagenase type IV, bovine serum albumin (fraction V), CH, DCA, TCA, and TCOH were purchased from Sigma Chemical Co. (St. Louis, MO). Antibodies for CYP2E1 and CYP2C11 were purchased from Oxford Biomedical (Rochester Hills, MI). All other chemicals were purchased from commercial sources and were of the highest purity available. Animals. Male Fischer 344 (F344) rats (200 –300 g; 12–15 weeks of age; Charles River Breeding Laboratories, Inc., Wilmington, MA) were used. Rats were housed in a controlled room on a 12-h light/dark cycle and were given commercial rat chow and water ad libitum. For clofibrate induction, rats were given clofibrate (0.2 g/kg/day) or corn oil (⫽ control) by ip injection for 3 days. For pyridine induction, rats were given pyridine (0.1 g/kg/day) or saline (⫽ control) by ip injection for 3 days. Isolation of rat hepatic and renal microsomes and cytosol. Liver and kidney microsomes were isolated as described previously (Lash et al., 1998). Rats were anesthetized with ip injections of pentobarbital (0.11 ml/100 g body weight) and were then injected with 0.2 ml of 0.2% (w/v) heparin in 0.9% NaCl (saline) into the tail vein. The abdomen was opened, and the liver or kidneys were removed, rinsed with buffer (250 mM sucrose, 10 mM triethanolamine/HCl, 1 mM EDTA, pH 7.6), and homogenized in 3 ml of buffer per gram tissue. Homogenates were initially centrifuged at 9000 ⫻ g for 20 min. The supernatant was then centrifuged for 60 min at 105,000 ⫻ g. The resulting pellets were resuspended in buffer and were recentrifuged an additional 60 min at 105,000 ⫻ g to produced washed microsomes. The microsomal pellets were resuspended in buffer containing 10% (v/v) glycerol and were stored at – 80°C until used. Enzymatic activities were normalized to protein concentrations, which were determined as described below. Preparation of isolated PT and DT cells. Isolated renal cortical cells were obtained by collagenase perfusion from kidneys of male F344 rats (Jones et al.,

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RENAL METABOLISM OF TRICHLOROETHYLENE 1979). To obtain enriched populations of PT and DT cells, cortical cells were subjected to density-gradient centrifugation in Percoll as described previously (Lash and Tokarz, 1989). Briefly, after induction of anesthesia with pentobarbital (0.11 ml/100 g body weight), rats were injected with 0.2 ml of 0.2% (w/v) heparin in 0.9% (w/v) saline. Marker enzyme activities and functional assays were used to confirm the identity and purity of the two cell populations (Lash and Tokarz, 1989). Cell concentrations were determined in the presence of 0.2% (w/v) trypan blue with a hemacytometer, and cell viability was estimated by measuring the fraction of cells that excluded trypan blue. Initial cell viabilities were 85–95%, and yield of PT and DT cells from two rat kidneys was typically ⬃30 ⫻ 10 6 cells and ⬃12 ⫻ 10 6 cells, respectively. Preparation of microsomes from isolated PT and DT cells. Microsomes were prepared from PT and DT cells for Western blots by homogenization of the cells in a Polytron ultrasonic homogenizer followed by centrifugation at 11,000 ⫻ g for 20 min to spin down nuclei, mitochondria, and cellular debris (Cummings et al., 1999). Supernatant from this step was centrifuged in a tabletop ultracentrifuge at 105,000 ⫻ g for 90 min at 4°C. The resulting pellet was washed with microsomal buffer (100 mM potassium phosphate, pH 7.4, 1 mM EDTA, and 20% glycerol) and was resuspended by sonication using a microprobe. Assay of Tri metabolism by P450 oxidation pathway. Measurement of P450-derived metabolites of Tri was done essentially as described by Elfarra et al. (1998). All incubations were carried out in 1-ml glass vials at 37°C for the indicated amounts of time (typically 15 or 30 min, depending on tissue type). Microsomes or cell extracts (1.0 mg protein/ml) were resuspended in 0.5–1.0 ml of 50 mM Tris-HCl buffer (pH 7.4). In measurements made with PT and DT cells, detergent (0.1%, v/v, Triton X-100) was added to lyse the tissue. Tri (0 –20 mM in acetonitrile, 0.5%, v/v) was then added, the mixture was incubated at 37°C for 3 min, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) (1 mM final) was added to initiate the reaction. For experiments testing the effect of P450 inhibitors, reaction mixtures were incubated with either 0.25 mM SKF-525A or 1 mM chlorzoxazone for 15 min prior to the addition of Tri. These concentrations of inhibitors have previously been established to yield maximal inhibition of P450 activity without significant cytotoxicity (Cummings et al., 1999; Lash and Tokarz, 1995; Lash et al., 1994a). Reactions were allowed to proceed for the indicated amounts of time, stopped by flash-freezing in a dry-ice acetone bath, thawed, and 1 ␮l of 1,3-dibromopropane [DBP, Aldrich Chemicals, Milwaukee, WI, diluted 1000fold in 0.5% (v/v) aqueous acetonitrile] was added as an internal standard. Samples were then extracted with 0.25 ml ethyl acetate. Sample analysis was carried out with a Perkin-Elmer Autosystem XL gas chromatograph fitted with a PE-210 30 m ⫻ 0.25 mm ID, 0.5 ␮m thickness column (Perkin-Elmer, Norwalk, CT) and an electron-capture detector. Metabolites were analyzed by injection of the ethyl acetate extracts into a split injector set at 200°C with a detector temperature of 300°C and an He flow rate of 24.8 cm/sec. The initial oven temperature was 35°C, and this was maintained for 11 min. Temperature was then increased at 10°/min to 120°C, where it was held for 19 min. Retention times for Tri and CH were ⬃3.9 and 6 min, respectively. Retention times for DCA, TCOH, DBP, and TCA were ⬃2.5, 14, 16, and 19 min, respectively. Assay limits of detection for Tri, CH, DCA, TCOH, and TCA were approximately 0.4, 0.1, 3, 0.1, and 3 fmol/mg protein, respectively. Western blot analysis of individual P450 enzymes. The expression of individual P450 enzymes in microsomes was determined by Western blot analysis, essentially as described previously (Cummings et al., 1999, 2000a,b). Microsomes isolated from tissue homogenates or isolated renal cells were subjected to SDS-PAGE on a 7.5% gel. This was followed by transfer of the gel to nitrocellulose. Nitrocellulose membranes were then exposed to the indicated antibodies. Alkaline phosphatase-conjugated secondary enzymes and substrate were used to detect protein bands. Band density was determined by scanning laser densitometry. Protein determination and data analysis. Protein determination was done using the bicinchoninic acid protein determination kit from Sigma, using bovine serum albumin as a standard. All values are means ⫾ SD of measurements made on the indicated number of experiments, with each experiment

FIG. 2. Kinetic analysis of CH formation in rat kidney microsomes. Rat kidney microsomes (1.0 mg protein/ml) were incubated with 0.4 –5 mM Tri for 3 min at 37°C prior to addition of 1 mM NADPH. After 15 min, reactions were terminated by flash-cooling, thawed, and extracted with 0.25 ml ethyl acetate. Sample analysis was carried out as explained in the Materials and Methods section. Lines were obtained by linear regression. Results are the means ⫾ SD of three experiments, each performed with microsomes isolated from separate animals. (A) y ⫽ 5.995x ⫹ 6.647, r 2 ⫽ 0.952. (B) y ⫽ – 0.978x ⫹ 0.158, r 2 ⫽ 0.833.

representing a single determination from an individual tissue preparation. Significant differences between means for data were first assessed by a oneway analysis of variance. When significant F values were obtained, the Fisher’s protected least significance t test was performed to determine which means were significantly different from one another, with two-tail probabilities ⬍ 0.05 considered significant.

RESULTS

Metabolism of Tri to CH in Freshly Isolated Rat Liver and Kidney Microsomes The rate of CH formation in rat liver microsomes incubated with 2 mM Tri and 1 mM NADPH was 2.55 nmol/min/mg protein, which is similar to that reported by Elfarra et al. (1998). Tri metabolism by P450-dependent oxidation was then measured in freshly isolated rat kidney microsomes (Fig. 2). CH was the only metabolite detected consistently in rat kidney microsomes. It was not possible to determine how much of the Tri that was metabolized could be accounted for by CH formation, as the Tri was present in great excess, and the amounts of CH formed were (depending on the concentration of Tri used as substrate) anywhere from ⬍ 0.05% to ⬍ 1% of the total amount of Tri present. Previous work by Elfarra et al. (1998) showed that CH can be used as a valid indicator of initial P450-dependent metabolism of Tri. CH formation was NADPH dependent and linear for up to 15 min in both liver and kidney microsomes; rates in kidney microsomes were approximately 10% of those in liver microsomes (data not shown). Lineweaver-Burk and Eadie-Hofstee analyses gave similar kinetic parameters (K m values were 0.90 and 0.98 mM Tri and V max values were 150 and 158 pmol CH formed/ min/mg protein by Lineweaver-Burk and Eadie-Hofstee analyses, respectively). Although a single enzymatic process is suggested by the linear regression analysis performed on these

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increased 2.6-fold, while renal CYP2C11 expression was increased ⬃2-fold. Pyridine treatment significantly increased CH formation in liver microsomes, causing a 2.5-fold increase, whereas clofibrate treatment did not significantly increase CH formation in rat liver microsomes (Table 1). In contrast, kidney microsomes isolated from rats treated with pyridine or clofibrate had significantly higher rates of CH formation. The increase in CH formation was greater in kidney microsomes isolated from rats treated with pyridine (5-fold) than kidney microsomes isolated from rats treated with clofibrate (2.2-fold). Effect of Inhibition of P450 on CH Formation in Liver and Kidney Microsomes

FIG. 3. Effect of pyridine on CYP2E1 expression in rat liver and kidney microsomes. (A) Representative Western blot using a polyclonal rabbit antihuman CYP2E1 antibody, showing expression of CYP2E1 in 10 ␮g of rat liver microsomes (lanes 1 and 2) and 20 ␮g of rat kidney microsomes (lane 3 and 4). Lanes 1 and 3 are microsomes from saline-treated rats, while lanes 2 and 4 are microsomes from pyridine-treated rats (0.10 g/kg/day; 3 days). (B) Densitometric analysis of CYP2E1 protein expression.

data, and the regression coefficients are very good, there is some apparent curvature in data points, suggesting the possibility that multiple enzymes are involved. Further, a nonlinear regression analysis of these plots also gave very good regression coefficients (r 2 ⬎ 0.9; data not shown). However, the limited number of data points (i.e., six) precludes a more detailed analysis of the kinetics.

Rat liver and kidney microsomes were isolated from either control rats or from rats pretreated with either pyridine or clofibrate, and the formation of CH in the presence of either buffer, SKF-525A (0.25 mM), or chlorzoxazone (1 mM) was measured (Table 1). In both liver and kidney microsomes from control rats, SKF-525A and chlorzoxazone significantly decreased CH formation. The level of inhibition (⬃50%) in liver microsomes caused by SKF-525A was equal to that caused by chlorzoxazone, while preincubation of kidney microsomes with SKF-525A resulted in significantly lower levels of CH than in kidney microsomes preincubated with chlorzoxazone (80% inhibition vs. 55% inhibition). In studies with liver and kidney microsomes isolated from rats pretreated with one of

Effect of P450 Induction on CH Formation in Rat Liver and Kidney Microsomes The effect of two P450 inducers, pyridine and clofibrate, on the oxidative metabolism of Tri was studied next. Pyridine induces CYP2E1 in both rat liver and kidneys (Hotchkiss et al., 1995). Pyridine treatment (0.10 g/kg/day; 3 days ip injection) significantly increased CYP2E1 expression in both liver and kidney microsomes (Figs. 3A and 3B), resulting in a 5-fold and a 2-fold increase in CYP2E1 expression in liver and kidney microsomes, respectively. We previously showed that clofibrate treatment (0.20 g/kg/ day; 3 days) significantly increases CYP4A and CYP2B expression in both rat liver and kidney microsomes (Cummings et al., 1999), suggesting that expression of several P450 enzymes is altered by clofibrate. Consequently, the effect of clofibrate treatment on the expression of CYP2C11 and CYP2E1 in both rat liver and kidney microsomes was studied. Clofibrate treatment did not increase CYP2E1 or CYP2C11 expression in liver microsomes, but did increase the expression of these enzymes in kidney microsomes (Fig. 4). Renal CYP2E1 expression was

FIG. 4. Effect of clofibrate on CYP2E1 and CYP2C11 expression in rat liver and kidney microsomes. (A) Representative Western blot using a polyclonal rabbit anti-human CYP2E1 antibody, showing expression of CYP2E1 in 10 ␮g of rat liver microsomes (lanes 1 and 2) and 20 ␮g of rat kidney microsomes (lane 3 and 4). Lanes 1 and 3 are microsomes from saline-treated rats, while lanes 2 and 4 are microsomes from clofibrate-treated rats (0.20 g/kg/day; 3 days). (B) Densitometric analysis of CYP2E1 protein expression. (C) Representative Western blot using a monoclonal mouse anti-rat CYP2C11 antibody, showing expression of CYP2C11 in 10 ␮g of rat liver microsomes (lanes 1 and 2) and 20 ␮g of kidney microsomes (lane 3 and 4). Lanes 1 and 3 are microsomes from corn oil–treated rats, while lanes 2 and 4 are microsomes from clofibrate-treated rats (0.20 mg/kg/day; 3 days). (D) Densitometric analysis of CYP2C11 protein expression.

RENAL METABOLISM OF TRICHLOROETHYLENE

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TABLE 1 Effect of Inducers and Inhibitors of Cytochrome P450 on Chloral Hydrate Formation in Rat Liver and Kidney Microsomes CH formation (nmol/min/mg protein) Treatment Control rats Buffer SKF-525A Chlorzoxazone Pyridine-treated rats Buffer SKF-525A Chlorzoxazone Clofibrate-treated rats Buffer SKF-525A Chlorzoxazone

Liver microsomes

Kidney microsomes

1.70 ⫾ 0.37 0.79 ⫾ 0.01* 0.71 ⫾ 0.01*

0.172 ⫾ 0.053 0.026 ⫾ 0.005* 0.068 ⫾ 0.024*

4.49 ⫾ 0.16** 2.09 ⫾ 1.27* 0.72 ⫾ 0.16*

0.338 ⫾ 0.061** 0.052 ⫾ 0.019* ,** 0.048 ⫾ 0.019*

1.21 ⫾ 0.83 0.73 ⫾ 0.15* 0.51 ⫾ 0.09* ,**

0.142 ⫾ 0.047 0.060 ⫾ 0.017* ,** 0.054 ⫾ 0.005*

Note. Sample analysis was carried out by gas chromatography, as described in the Materials and Methods section. Results are the means ⫾ SD of single measurements from three separate microsome preparations. All statistical comparisons were made between measurements in microsomes from the same tissue. *Significantly different from buffer-treated control from the corresponding treatment group (p ⬍ 0.05). **Significantly different from identically treated samples from control rats (p ⬍ 0.05).

the two inducers, SKF-525A and chlorzoxazone significantly decreased CH formation only in liver or kidney microsomes isolated from pyridine-treated rats; neither compound significantly inhibited P450-dependent metabolism of Tri in liver or kidney microsomes from clofibrate-treated rats. SKF-525A was only able to decrease CH formation to control levels, but not below, in liver microsomes from pyridine-treated rats.

FIG. 5. Time and NADPH dependence of CH formation in PT and DT cells. Time (A) and NADPH (B) dependence of CH formation in PT (filled circle or bar) and DT (open circle or bar) cells were determined by incubating the detergent-solubilized cells in the presence or absence of 2 mM Tri for 3 min at 37°C prior to the addition of NADPH (1 mM) or solvent control. After the indicated time, reactions were terminated by flash-cooling, thawed, and extracted with 0.25 ml of ethyl acetate. Sample analysis was carried out as explained in the Materials and Methods section. Results are the means ⫾ SD of three experiments, each with cells isolated from separate animals.

formation in PT cells was not detectable at concentrations of Tri below 0.5 mM, reached a maximum at 7.5 mM Tri, and was lower at higher concentrations (10 and 12 mM) of Tri (Fig. 6A). This behavior is similar to previous results on the kinetics of GSH conjugation of Tri, which showed inhibition of catalytic activity by product at high substrate concentrations (Cummings et al., 2000a). Attempts at analysis of kinetics of CH formation in PT cells by either Lineweaver-Burk or EadieHofstee analysis were unsuccessful due to the unusual concentration dependence. In contrast, CH formation in DT cells exhibited Michaelis-Menten kinetics (Fig. 6B), and analysis by an Eadie-Hofstee plot showed P450-dependent metabolism of Tri occurred by a low-affinity, low-capacity process (K m ⫽ 13.9 mM, V max ⫽ 110 pmol/min/mg protein; regression line: Y ⫽ –13.867X ⫹ 109.598; r 2 ⫽ 0.967).

P450 Oxidative Metabolism of Tri in PT and DT Cells We have previously studied GSH-dependent metabolism and cytotoxicity of Tri in isolated renal PT and DT cells to explore nephron heterogeneity and to better characterize factors responsible for the PT cell specificity of Tri-induced renal damage (Cummings et al., 2000a; Lash et al., 1994b). Oxidative metabolism of Tri in both PT and DT cells was measured by analysis of CH formation, and was found to be NADPH dependent and linear through 30 min of incubation (Figs. 5A and 5B). Amounts of CH formation in detergent extracts from PT cells incubated with 2 mM Tri and 1 mM NADPH were approximately 3-fold higher than those in extracts from DT cells similarly incubated with Tri and NADPH. Omission of NADPH from the incubation mixture completely eliminated any detectable CH formation. Kinetic analysis of CH formation in PT and DT cells indicated markedly different behavior in the two cell types. CH

FIG. 6. Kinetic analysis of CH formation in PT and DT cells. Detergentsolubilized PT cells (A) and DT cells (B) were incubated in the presence of various concentrations of Tri for 3 min at 37°C prior to the addition of 1 mM NADPH. After 30 min, reactions were terminated by flash-cooling, thawed, and extracted with 0.25 ml ethyl acetate. Sample analysis was carried out as explained in the Materials and Methods section. Results are the means ⫾ SD of three experiments, each with cells isolated from separate animals. Curves were obtained by regression (third-order and second-order polynomial for panels A and B, respectively). (A) y ⫽ – 0.450x 3 ⫹ 7.013x 2 - 14.417x - 0.047, r 2 ⫽ 0.986. (B) y ⫽ – 0.252x 2 ⫹ 7.152x ⫹ 0.038, r 2 ⫽ 0.999.

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SKF-525A significantly inhibited CH formation in both PT and DT cells by ⬎ 70%. In contrast, chlorzoxazone had no effect on CH formation in PT cells, but modestly inhibited CH formation in DT cells by ⬃40%. DISCUSSION

FIG. 7. Effect of clofibrate on CYP2E1 and CYP2C11 expression in PT and DT cells. (A) Representative Western blot using a polyclonal rabbit anti-human CYP2E1 antibody, showing expression of CYP2E1 in 20 ␮g of microsomes isolated from kidney homogenate (lane 1) or 60 ␮g of microsomes isolated from PT cells (lanes 2 and 3) or DT cells (lane 4 and 5). Lanes 3 and 5 are microsomes from saline-treated rats, while lanes 2 and 4 are microsomes from pyridine-treated rats (0.10 mg/kg/day; 3 days). (B) Representative Western blot using a monoclonal mouse anti-rat CYP2C11 antibody, showing expression of CYP2C11 in 20 ␮g of microsomes isolated from kidney homogenate (lane 1) or 60 ␮g of microsomes isolated from PT cells (lanes 2 and 3). Lane 2 is microsomes from corn oil–treated rats, while lane 3 is microsomes from clofibrate-treated rats (0.20 mg/kg/day; 3 days).

Effect of Pyridine and Clofibrate Treatment on CH Formation in PT and DT Cells We have shown previously that pretreatment of rats with clofibrate did not increase expression of CYP4A2, CYP4A3, or CYP2B1/2 in microsomes isolated from PT or DT cells (Cummings et al., 1999). Similar to that study, neither pyridine nor clofibrate treatment caused significant increases in the expression of either CYP2C11 or CYP2E1 in microsomes isolated from PT or DT cells (Figs. 7A and 7B). CH formation was also not significantly increased in PT or DT cells isolated from rats treated with either pyridine or clofibrate as compared to that in cells isolated from control rats (Table 2). Effect of P450 Inhibition on CH Formation in PT and DT Cells The formation of CH in the presence or absence of SKF525A (0.25 mM) or chlorzoxazone (1 mM) was determined in PT and DT cells that were isolated from control rats (Table 2).

The present studies were conducted to characterize P450dependent metabolism of Tri in rat kidney. Although the P450 pathway for Tri metabolism is not considered to be directly associated with the kidneys as a target organ for Tri, there is evidence that intrarenal activity of the oxidative pathway influences intrarenal activity of the GSH conjugation pathway (Lash et al., 1999). The mechanism by which this occurs is presumably by competition for substrate. We have recently characterized the expression of several P450 enzymes in rat kidney (Cummings et al., 1999), many of which are also present in the liver and have been demonstrated to use Tri as a substrate. Hence, enzymes capable of catalyzing the oxidative metabolism of Tri are likely present in the kidneys. Interestingly, however, the key P450-derived metabolites of Tri, such as CH, TCA, and DCA, are not toxic in in vitro renal preparations (Lash et al., 1995), suggesting that any role that the P450 pathway would have in the kidneys would be one in which its function would modulate the activity of the primary pathway that leads to intoxication of the kidney cells. Although our gas chromatography method allowed for detection of all the major P450-derived metabolites of Tri (i.e., CH, TCA, TCOH, DCA, oxalate) with a high degree of sensitivity (limit of detection ⫽ 0.1–3 fmol/mg protein), CH was the only metabolite consistently detected in incubations of rat kidney microsomes with Tri and NADPH. Therefore, we used CH formation as an index of P450-dependent metabolism of Tri. As shown in Figure 2, kinetic analysis by either of the two common linear transformation methods gave a reasonably good fit of the data and nearly identical results; only a single

TABLE 2 Effect of Inducers and Inhibitors of Cytochrome P450 on Chloral Hydrate Formation in Rat Renal PT and DT Cells CH formation (nmol/min/mg protein) Pretreatment/treatment

PT cells

DT cells

Buffer/buffer Pyridine/buffer Clofibrate/buffer Buffer/SKF-525A Buffer/chlorzoxazone

0.257 ⫾ 0.068 0.185 ⫾ 0.075 0.177 ⫾ 0.159 0.047 ⫾ 0.021* 0.298 ⫾ 0.058

0.116 ⫾ 0.036 0.172 ⫾ 0.022 0.053 ⫾ 0.046 0.017 ⫾ 0.004* 0.063 ⫾ 0.015*

Note. Sample analysis was carried out by gas chromatography, as described in the Materials and Methods section. Results are the means ⫾ SD of single measurements from three separate cell preparations. All statistical comparisons were made between measurements in one cell type. *Significantly different from buffer/buffer (p ⬍ 0.05).


process was distinguished, with a K m value of 0.90 – 0.98 mM Tri and a V max value of ⬃150 pmol CH formed/min/mg protein. Elfarra et al. (1998) reported two kinetically distinct processes for Tri oxidation to CH in rat liver microsomes. The present results for P450-dependent metabolism of Tri in rat kidney microsomes indicate that the process is markedly less efficient, with both a nearly 10-fold higher K m and a 15- to 30-fold lower V max. As noted in the Results section, some curvature may exist in these plots, which would indicate that multiple enzymes are involved in the metabolism. The limited number of data points precludes a more detailed analysis of the kinetics. Hence, we can conclude that there is at least one enzymatic process responsible and that the overall kinetics of the process are those described by the linear transformations. It is likely that multiple P450 enzymes are involved, as the data on CYP induction by pyridine and clofibrate, and inhibition with SKF-525A and chlorzoxazone suggest that both CYP2E1 and CYP2C11 contribute to CH formation with Tri as substrate in rat kidney microsomes. The conclusion that CYP2E1 and CYP2C11 are involved in Tri metabolism must be made with the caveat that the data supporting this represent only correlations. Thus, the increased protein levels on Western blots and the increased formation of CH represent correlates because the immunoblots and enzyme activity measurements were done on separate microsomal or cell preparations. Our data agree with those of Miller and Guengerich (1983), who showed that treatment of rats with either pyridine or phenobarbital increased CH formation from Tri in rat liver microsomes. As is typical, effects of P450 enzyme inducers, when they occur in both liver and kidneys, are markedly greater in the liver. Thus, pyridine treatment increased expression of CYP2E1 protein approximately 5-fold in liver microsomes, but only 2-fold in kidney microsomes. Surprisingly, pretreatment of rats with pyridine produced a larger increase in enzyme activity in kidney microsomes (⬃5-fold) than in liver microsomes (⬃2.5-fold). The explanation for this reversed order for activity stimulation is unclear, but may relate to tissue-specific regulatory mechanisms or effects on multiple P450 enzymes. To our knowledge, this is the first time that clofibrate, or any similar compound belonging to the group of chemicals known as peroxisome proliferators, has been shown to increase CYP2C11 expression in vivo. An increase in CYP2E1 expression by ciprofibrate was reported in primary cultures of hepatocytes and renal cortical cells and in kidneys of rats (Zangar et al., 1995, 1996). Data from the present study demonstrated that clofibrate causes a kidney-selective induction of several P450 enzymes. Although the cause of this tissue selectivity is unknown, it is important to note that basal levels of expression of CYP2E1, CYP2B, and CYP2C11 in the kidneys are very low compared with those in the liver, which could account, in part, for the observed positive response in the kidneys and negative response in the liver. These relatively widespread increases in renal P450 expression induced by clofibrate sug-

17

gest that a signaling pathway is present that causes an overall increase in renal P450 expression. This up-regulation may be due to a specific transcription factor or, because of the effects of clofibrate on fatty acid metabolism, to increased levels of certain fatty acids or fatty acid metabolites. The latter suggestion seems more likely, because clofibrate and similar compounds increase fatty acid metabolism in vivo (Ronnis et al., 1998). Furthermore, some of the products of increased fatty acid oxidation (e.g., ketone bodies) have been shown to increase CYP2E1 in primary cultures of rat hepatocytes (Ronnis et al., 1998). Additional study is needed to clarify the mechanism of action of clofibrate in the rat kidney. Both SKF-525A, a relatively nonspecific P450 inhibitor, and chlorzoxazone, a competitive inhibitor of CYP2E1, were effective inhibitors of CH formation in microsomes from both liver and kidney. In kidney microsomes, SKF-525A produced significantly more inhibition than chlorzoxazone, consistent with the suggestion that other P450 enzymes besides CYP2E1 are involved in Tri metabolism. In liver or kidney microsomes from rats pretreated with pyridine, however, chlorzoxazone was a better or comparable inhibitor, respectively, of CH formation. This observation is consistent with the induction of CYP2E1 by pyridine. In contrast to these findings, neither inhibitor significantly affected rates of CH formation in liver or kidney microsomes isolated from clofibrate-pretreated rats, indicating that in clofibrate-pretreated rats, Tri oxidation occurs primarily by a P450 enzyme that is insensitive to SKF525A and is distinct from CYP2E1. Studies on P450 expression and activity were also conducted in suspensions of freshly isolated PT and DT cells from the rat, as these cells have been used as a model for studying cell-type specific patterns of cell injury and Tri metabolism and cytotoxicity (Cummings et al., 2000a; Lash and Tokarz, 1989). The rate of CH formation from 2 mM Tri was approximately 4-fold higher in PT cells than in DT cells. The toxicological significance of this cell type– dependent difference is unclear. However, we showed previously that the level of renal P450 activity influences GSH conjugation of Tri (Cummings et al., 2000a; Lash et al., 1999) and, presumably, the rate of generation of reactive metabolites from the cysteine conjugate ␤-lyase pathway. The higher P450 activity in PT cells suggests that Tri metabolism and cytotoxicity will be influenced by P450 status to a greater extent in these cells, which is consistent with the cell-type specificity in the kidney for Tri- and DCVC-induced nephrotoxicity. Metabolism was shown to be absolutely dependent on NADPH. Similar to findings with GSH conjugation of Tri (Cummings et al., 2000a), oxidation of Tri was inhibited at high substrate concentrations in PT cells but not in DT cells, suggesting that different P450 enzymes are responsible for Tri metabolism in the two cell types. Unlike the results with microsomes derived from renal cortical homogenates, expression of CYP2E1 after pretreatment with pyridine or expression of CYP2C11 after pretreatment with clofibrate was not altered in microsomes derived from isolated PT or DT cells. However,

18


SKF-525A produced nearly 85% inhibition of CH formation in both cell types, and chlorzoxazone produced approximately 50% inhibition of CH formation in DT, but not PT, cells. These results are still consistent with a role for CYP2E1 in renal metabolism of Tri. It is surprising that no induction due to pyridine or clofibrate was detected in the isolated cells. This is not due to a lack of capacity of the cells to respond to P450inducing agents, as we have shown that CYP4A2 and CYP4A3 protein are induced in these cells by pretreatment of rats with clofibrate (Cummings et al., 1999). Further studies are needed to resolve this apparent discrepancy. In summary, this is the first study to characterize P450dependent metabolism of Tri in the rat kidney and provide evidence from the use of inducers and inhibitors that CYP2E1 primarily, and CYP2C11 secondarily, are responsible for metabolism of Tri to CH in rat kidney. The various P450-derived metabolites of Tri do not appear to play a role in any renal toxicity, although we have shown that modulation of renal P450 activity does influence both GSH conjugation of Tri (Lash et al., 1999) and acute cytotoxicity induced by Tri in rat renal PT cells (Cummings et al., 1999). Applicability of these findings to humans is complicated by the fact that human kidney apparently does not express CYP2E1 (Amet et al., 1997; Cummings et al., 2000b), and metabolism of Tri to CH in isolated human PT cells was either barely detectable or completely undetectable (Cummings and Lash, 2000; Cummings et al., 2000a). These considerations suggest that the modulating effect of renal P450 activity on renal toxicity of Tri, which is mediated by metabolism via the GSH conjugation pathway, will be less significant in humans than in rats. REFERENCES Amet, Y., Berthou, F., Fournier, G., Dreano, Y., Bardou, L., Cledes, J., and Menez, J.-F. (1997). Cytochrome P450 4A and 2E1 expression in human kidney microsomes. Biochem. Pharmacol. 53, 765–771. Bloemen, L. J., and Tomenson, J. (1995). Increased incidence of renal cell tumors in a cohort of cardboard workers exposed to trichloroethene. Arch. Toxicol. 70, 129 –133. Brown, L. P., Rarrar, D. G., and de Rooij, C. G. (1990). Health risk assessment of environmental exposure to trichloroethylene. Regul. Toxicol. Pharmacol. 11, 24 – 41. Cai, H., and Guengerich, F. P. (1999). Mechanism of aqueous decomposition of trichloroethylene oxide. J. Am. Chem. Soc. 121, 11656 –11663. Cai, H., and Guengerich, F. P. (2000). Acylation of protein lysines by trichloroethylene oxide. Chem. Res. Toxicol. 13, 327–335. Cummings, B. S., and Lash, L. H. (2000). Metabolism and toxicity of trichloroethylene and S-(1,2-dichlorovinyl)-L-cysteine in freshly isolated human proximal tubular cells. Toxicol. Sci. 53, 458 – 466. Cummings, B. S., Zangar, R.C., Novak, R. F., and Lash, L. H. (1999). Cellular distribution of cytochromes P-450 in the rat kidney. Drug Metab. Dispos. 27, 542–548. Cummings, B. S., Parker, J. C., and Lash, L. H. (2000a). Role of cytochrome P450 and glutathione S-transferase ␣ in the metabolism and cytotoxicity of trichloroethylene in rat kidney. Biochem. Pharmacol. 59, 531–543. Cummings, B. S., Lasker, J. M., and Lash, L. H. (2000b). Expression of

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