* 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
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ABSTRACT |
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Key Words: trichloroethylene; kidney; metabolism; cytochrome P450; proximal tubular cells; enzyme induction.
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INTRODUCTION |
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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
).
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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.
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MATERIALS AND METHODS |
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Animals.
Male Fischer 344 (F344) rats (200300 g; 1215 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 x g for 20 min. The supernatant was then centrifuged for 60 min at 105,000 x g. The resulting pellets were resuspended in buffer and were recentrifuged an additional 60 min at 105,000 x 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., 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 8595%, and yield of PT and DT cells from two rat kidneys was typically
30 x 106 cells and
12 x 106 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 x 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 x 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 0.51.0 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 (020 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 µl of 1,3-dibromopropane [DBP, Aldrich Chemicals, Milwaukee, WI, diluted 1000-fold 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 x 0.25 mm ID, 0.5 µm 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 representing a single determination from an individual tissue preparation. Significant differences between means for data were first assessed by a one-way 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.
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RESULTS |
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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.
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DISCUSSION |
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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 = 0.13 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 process was distinguished, with a Km value of 0.900.98 mM Tri and a Vmax 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 Km and a 15- to 30-fold lower Vmax. 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 suggest 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 SKF-525A 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 typedependent 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 ß-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, 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 P450-inducing 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 P450-dependent 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.
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NOTES |
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1 To whom correspondence should be addressed. Fax: (313) 577-6739. E-mail: l.h.lash{at}wayne.edu.
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