Metabolism and Toxicity of Trichloroethylene and S-(1,2-Dichlorovinyl)-L-Cysteine in Freshly Isolated Human Proximal Tubular Cells

Brian S. Cummings and Lawrence H. Lash1

Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, Michigan 48201

Received June 1, 1999; accepted September 2, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trichloroethylene (Tri) caused modest cytotoxicity in freshly isolated human proximal tubular (hPT) cells, as assessed by significant decreases in lactate dehydrogenase (LDH) activity after 1 h of exposure to 500 µM Tri. Oxidative metabolism of Tri by cytochrome P-450 to form chloral hydrate (CH) was only detectable in kidney microsomes from one patient out of four tested and was not detected in hPT cells. In contrast, GSH conjugation of Tri was detected in cells from every patient tested. The kinetics of Tri metabolism to its GSH conjugate S-(1,2-dichlorovinyl)glutathione (DCVG) followed biphasic kinetics, with apparent Km and Vmax values of 0.51 and 24.9 mM and 0.10 and 1.0 nmol/min per mg protein, respectively. S-(1,2-dichlorovinyl)-L-cysteine (DCVC), the cysteine conjugate metabolite of Tri that is considered the penultimate nephrotoxic species, caused both time- and concentration-dependent increases in LDH release in freshly isolated hPT cells. Preincubation of hPT cells with 0.1 mM aminooxyacetic acid did not protect hPT cells from DCVC-induced cellular injury, suggesting that another enzyme besides the cysteine conjugate ß-lyase may be important in DCVC bioactivation. This study is the first to measure the cytotoxicity and metabolism of Tri and DCVC in freshly isolated cells from the human kidney. These data indicate that the pathway involved in the cytotoxicity and metabolism of Tri in hPT cells is the GSH conjugation pathway and that the cytochrome P-450–dependent pathway has little direct role in renal Tri metabolism in humans.

Key Words: trichloroethylene; human kidney; proximal tubular cells; glutathione conjugation; cytochrome P-450..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Trichloroethylene (Tri) is a common groundwater contaminant and putative human carcinogen (Davidson and Beliles, 1991Go). The low boiling point and lipophilic character of Tri makes it useful in a variety of industrial processes including metal degreasing and dry cleaning (Weiss, 1996Go). Tri causes hepatocarcinogenesis in mice (Forkert et al., 1985Go; Fukuda et al., 1983Go), and renal toxicity and carcinoma in rats (Maltoni et al., 1988Go), and studies have suggested that Tri can cause kidney cancer in humans (Davidson and Beliles, 1991Go; Henschler et al., 1995aGo). The ability of Tri to cause kidney cancer in humans is controversial and has been questioned (Bloemen and Tomenson, 1995Go; Henschler et al., 1995bGo; Swaen, 1995Go). Much of the controversy about the nephrotoxicity of Tri in humans exists because most of the studies with Tri have been performed in rats or mice. The renal effects of Tri in the human kidney have been attributed to its metabolism by glutathione (GSH)-dependent enzymes as opposed to metabolism of Tri by cytochrome P-450 monooxygenases (P-450) (Dekant et al., 1986Go, 1990Go). One aspect of this controversy involves the amount of Tri metabolized by the GSH-dependent pathway in humans as opposed to that in the rats. The relevance of this pathway in humans has been questioned, as much of the data about Tri metabolism have been determined in rats (Abelson, 1993Go). Tri produces both acute and chronic toxicity with multiple target organs (NTP, 1982NTP, 1990). There are also species-dependent differences in toxic responses to Tri (Brown et al., 1990Go; Lash et al., 1998Go), which further hampers studies on the mechanism of Tri-induced toxicity.

Tri is metabolized by at least two separate pathways, P-450 oxidative metabolism and GSH-dependent metabolism (Figs. 1 and 2GoGo). P-450 oxidative metabolism of Tri results in the formation of a Tri–P-450-oxygen intermediate that is believed to form chloral, which readily equilibrates with chloral hydrate (CH), via a chlorine migration (Miller and Guengerich, 1983Go). Tri can also be metabolized to a diacylchloride, which can be further metabolized to dichloroacetic acid (DCA) and monochloroacetic acid. Both of these metabolites can be further metabolized to oxalic acid. CH can be metabolized to trichloroacetic acid (TCA), which can be further metabolized to DCA. Finally, CH can also be metabolized to trichloroethanol (TCOH), which can be metabolized to TCA or TCOH glucuronide. Elfarra and colleagues (1998) showed that in rat and human liver microsomes, the only metabolites consistently detected were CH and TCOH. We found that in rat kidney microsomes, the only metabolite consistently detected was CH (B. S. Cummings, J. C. Parker, and L. H. Lash, manuscript submitted). Elfarra et al. (1998) showed that formation of CH in rat and human liver microsomes followed biphasic kinetics, but the P-450 isoforms responsible for the formation of CH were not determined. Our studies showed that in rat liver and kidney microsomes, CYP2E1 is one P-450 isoform involved in the metabolism of Tri to CH (Cummings et al., 2000Go). Other rat P-450 isoforms capable of metabolizing Tri to CH include those of the CYP2C family (Miller and Guengerich, 1983Go). Both CYP2E1 and CYP2C11 are expressed in our preparation of freshly isolated rat kidney cells (Cummings et al., 1999). The extent of P-450 oxidative metabolism of Tri in the human kidney has never determined.



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FIG. 1. Metabolism of Tri by the P-450 oxidative metabolism pathway. Tri is oxidized by P-450 isoforms to a Tri-O-P-450 intermediate. The Tri-O-P-450 intermediate can be metabolized further to dichloroacylchloride, which immediately rearranges to form dichloroacetic acid. Dichloroacetic acid can be further processed to monochloroacetic acid or oxalic acid. The Tri P-450-O-intermediate can also form chloral hydrate or oxalic acid. Chloral hydrate can be metabolized further to trichloroethanol (which can be metabolized further to trichloroethanol glucuronide) or trichloroacetic acid. Trichloroacetic acid can be processed further to dichloroacetic acid or trichloroethanol.

 


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FIG. 2. Metabolism of Tri by the GSH-dependent conjugation pathway. Tri is conjugated to GSH to form DCVG in both the liver and kidneys. DCVG is metabolized by GGT (inhibited by acivicin) and dipeptidase to form DCVC. DCVC can be metabolized by either cysteine-S-conjugate ß-lyase to form a reactive sulfhydryl-intermediate (DCVSH) or by N-acetyl-S-transferase, in a reversible process, to form N-acetyl-DCVC (NAcDCVC). NAcDCVC is then either excreted in the urine or it can be deacetylated to reform DCVC.

 
Tri is also metabolized by GSH conjugation to form S-(1,2-dichlorovinyl)glutathione (DCVG) (Lash et al., 1995Go, 1998Go) (Fig. 2Go). DCVG can be further metabolized by {gamma}-glutamyltransferase (GGT) and dipeptidase to form S-(1,2-dichlorovinly)-L-cysteine (DCVC). DCVC can then be metabolized by either cysteine S-conjugate N-acetyl-S-transferase (NAT), to form N-acetyl-DCVC (NAcDCVC), or by the cysteine conjugate ß-lyase (ß-lyase) or an S-oxidase activity of one or more isoforms of flavin-containing monooxygenase (FMO) (Ripp et al., 1997Go), to form a reactive thiol compound. NAcDCVC is excreted into the urine in both rats and humans after exposure to Tri (Birner et al, 1993Go; Commandeur and Vermeulen, 1990Go). NAcDCVC can also be deacetylated to reform DCVC. We have shown that in both rat and human kidney cytosol, Tri is metabolized to DCVG in a GSH-dependent process that follows biphasic kinetics (Lash et al., 1999aGo,bGo). We have also shown that the target site of Tri and DCVC within the rat kidney is primarily the proximal tubular (PT) cells (Lash et al., 1994Go). Tri metabolism and cytotoxicity in rat renal PT cells are GSH-dependent but are influenced by P-450 activity (Cummings et al., 2000Go). Rat renal PT cells are protected against DCVC-induced cytotoxicity by pretreatment with the ß-lyase inhibitor aminooxyacetic acid (AOAA) (Lash et al., 1994Go).

Little is known about the cytotoxicity of Tri or DCVC in human renal proximal tubular (hPT) cells. Chen et al. (1990) showed that DCVC was acutely cytotoxic in primary cultures of hPT cells and provided data that were consistent with a role for the ß-lyase in DCVC bioactivation. Effects of the parent compound, Tri, however, have not been assessed. The rates of Tri metabolism to either DCVG or CH in hPT cells have also never been determined. A recent report (Brüning et al., 1998Go) of a case involving acute Tri poisoning demonstrated that the products of the GSH-conjugation pathway can be formed in humans and correspond with increases in markers for renal damage (e.g., increases in ß2-microglobulin, albumin, etc.). Determination of the cytotoxicity of Tri in freshly isolated hPT cells will aid in the assessment of the human health hazard of Tri. Furthermore, analysis of the enzymes involved in both Tri and DCVC toxicity and metabolism will increase our understanding of the role of and capacity of the human kidney for drug metabolism.

The goal of this study was to determine the cytotoxicity and metabolism of Tri and DCVC in freshly isolated hPT cells. Data from this study show for the first time the cytotoxicity and metabolism of Tri in freshly isolated hPT cells. CH formation was only measured in one out of four samples of human kidney microsomes and was not detected in hPT cells. In contrast, DCVG formation was detectable in all patients tested and followed biphasic kinetics. The cytotoxicity of Tri was not altered by preincubation of hPT cells with P-450 inhibitors. Thus, these data suggest that the metabolism and cytotoxicity of Tri in hPT cells does not depend on or is not influenced by P-450 oxidative metabolism and that the toxicity of Tri is a result of its metabolism by GSH conjugation. Furthermore, data from this study show that, unlike rat renal PT cells, an enzyme(s) other than the ß-lyase plays a role in the cytotoxicity of DCVC in hPT cells, suggesting that the toxicity of DCVC is occurring by mechanisms that are distinct from those shown to occur in the rat.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Tri (reported to be 99.9% pure, as judged by electron impact ionization mass spectrometry), collagenase type I (EC 3.4.24.3), bovine serum albumin (BSA) (fraction V), acivicin, and L-{gamma}-glutamyl-L-glutamate were purchased from Sigma Chemical Co. (St. Louis, MO). DCVG and DCVC were synthesized as described previously (Elfarra et al., 1986Go). Purity (> 95%) was determined by HPLC analysis, and identity was confirmed by proton NMR spectroscopy.

Isolation and characterization of hPT cells from human kidney cortical slices.
Freshly isolated human kidney cortical slices were obtained from the Human Tissue Resources Core in the Department of Pathology at Harper Hospital (Detroit, MI). Individual slices were analyzed by a pathologist and all slices were determined to be normal (i.e., noncancerous, nondiseased) tissue. The age and gender of the subjects from whom the tissues were isolated are listed in Table 1Go. Cause of death is not listed in Table 1Go because in all cases just one kidney was removed for either biopsy or other purposes.


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TABLE 1 Data on Human Kidney Samples
 
Isolation of human kidney microsomes and cytosol.
Kidney microsomes were isolated as described by Sharer et al. (1992). Freshly isolated human renal cortical slices were obtained from the tissue received from the Human Tissue Resources Core from the Department of Pathology, Harper Hospital (Detroit, MI). Further information about the human renal cortical slices are presented in Table 1Go. Each slice was weighed, rinsed with buffer (250 mM sucrose, 10 mM triethanolamine/HCl, 1 mM EDTA, pH 7.6), and homogenized in 3 ml of buffer/g tissue. Homogenates were initially centrifuged at 9,000 x g for 20 min. The supernatant was filtered through cheesecloth and then centrifuged for 60 min at 105,000 x g. The resulting supernatant (cytosolic fraction) was used for enzymatic analysis and was stored at –80°C until use. The resulting pellets were resuspended in buffer and were centrifuged an additional 60 min at 105,000 x g to produce "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 explained below.

Isolation of hPT cells.
The method of Todd et al. (1995) was used to isolate and culture hPT cells from human renal cortical slices. This method results in kidney cells primarily of proximal tubular origin and involves digestion of minced cortical tissue with collagenase, a filtering step to remove tissue fragments, and a low-speed centrifugation step (Detrisac et al., 1984Go; Kempson et al., 1989Go). The fibrous renal capsule was removed from human renal cortical slices and the slice was weighed. The slices were then washed with sterile PBS, minced, and the pieces were placed in a trypsinization flask filled with 30 ml of Hanks buffer containing 25 mM NaHCO3, 25 mM HEPES, pH 7.4, 0.5 mM EGTA, 0.2% (w/v) BSA, gentamicin (50 µg/ml), collagenase (1.3 mg/ml), and CaCl2 (0.59 mg/ml). The solution was filtered prior to use. All buffers were continuously bubbled with 95% O2/5% CO2 and were maintained at 37°C. Minced cortical pieces were subjected to enzymatic dissociation for 15 min, after which the supernatant was filtered through 70-µm mesh to remove tissue fragments, centrifuged at 150 x g for 7 min, and the pellet resuspended in Krebs-Henseleit Buffer I (118 mM NaCl, 4.8 mM KCl, 0.96 mM KH2PO4, 0.12 mM MgSO4.7H2O, 25 mM NaHCO3, 25 mM HEPES, and 2% (w/v) BSA). These steps were repeated until complete digestion of the tissue was achieved (usually 4–5 cycles). Resuspensions were combined and centrifuged at 150 x g for 7 min. Pellets were washed with Krebs-Henseleit Buffer I and centrifuged at 150 x g for 7 min. The final pellet (hPT cells) was resuspended in Krebs-Henseleit Buffer II (same as Krebs-Henseleit Buffer I except no BSA was added). Approximately 70 x 106 cells were obtained from 1g of human renal cortical tissue.

Assay of Tri metabolism by the P-450 oxidation pathway.
Measurement of P-450 metabolites of Tri was done according to Elfarra et al. (1998) and EPA method number 151. All incubations were carried out in 1-ml glass vials at 37°C for the indicated amount of time (typically 15 or 30 min, depending on tissue type). Tissue (0.5 to 2.0 mg protein/ml) was resuspended in 0.5–1.0 ml of 50 mM Tris–HCl buffer (pH 7.4). Triton X-100 was then added to lyse the cells (not done with microsomes) at a concentration of 0.1% (v/v). Tri (0–2 mM) in acetonitrile (0.5%, v/v) was then added and the reaction was incubated at 37°C for 3 min, after which NADPH (1 mM final) was added to initiate the reactions. Reactions were allowed to proceed for the indicated amount of time, stopped by flash-freezing in a dry-ice acetone bath, and thawed; 1 µl of a 1000-fold diluted solution of dibromopropanol (DBP) (Aldrich Chemicals, St. Louis, MO) 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) 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 a He flow rate of 24.8 cm/sec. The initial oven temperature was 35°C for 11 min. Temperature was increased at 10°/min to 120°C, where it was held for 19 min. Retention times for Tri and CH were approximately 3.9 and 6 min, respectively. Retention times for DCA, TCOH, DBP, and TCA were approximately 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.

Assay of Tri metabolism by GSH conjugation pathway.
All incubations were performed in 25-ml polypropylene Erlenmeyer flasks on a Dubnoff metabolic shaking incubator (60 cycles/min) at 37°C. Isolated hPT cells were preincubated for 15 min with 0.25 mM acivicin to inhibit GGT activity before performing incubations to measure DCVG formation. Cells were then incubated with 0–10 mM concentrations of Tri. With isolated cells, Triton X-100 (0.1%, v/v, final concentration) was added to solubilize the plasma membrane. After incubations, reactions were terminated by addition of perchloric acid (10%, w/v, final concentration), and samples were derivatized with 1-fluoro-2,4-dinitrobenzene and processed for analysis of DCVG by HPLC as described previously (Lash et al., 1995Go). Reactions were performed in Krebs-Henseleit Buffer II (pH 7.35) minus BSA, as this was the buffer used for the cytotoxicity assays. Assay limit of detection for DCVG was approximately 50 pmol per 0.1-ml sample (containing approximately 0.05 mg tissue protein). The HPLC method does not distinguish between the 1,2- and 2,2-dichlorovinyl isomers of DCVG. However, only the 1,2-dichlorovinyl isomer is toxicologically relevant and is the predominant isomer formed from GST-catalyzed metabolism (Commandeur and Vermeulen, 1990Go; Commandeur et al., 1991Go; Ilinskaja and Vamvakas, 1996Go).

Cytotoxicity of Tri and DCVC in freshly isolated hPT cells.
The cytotoxicity of Tri or DCVC in isolated hPT cells was determined by release of lactate dehydrogenase (LDH) activity into the media or changes in total LDH activity. Results for incubations with DCVC are reported as percent LDH release; those for incubations with Tri are reported as decreases in total LDH activity because Tri was found to inhibit LDH activity in hPT cells (data not shown). Decreases in LDH activity were used previously to monitor the cytotoxicity of inorganic mercury, which also inhibits LDH activity (Lash and Zalups, 1992Go). LDH activity was determined spectrophotometrically by measuring the oxidation of NADH by the decrease in absorbance at 340 nm ({epsilon} = 6220 M–1cm–1). For experiments testing the effect of metyrapone and AOAA, cells were preincubated with 0.25 mM metyrapone or 0.1 mM or 1 mM AOAA for 15 min prior to the addition of Tri or DCVC.

Protein determination and data analysis.
Protein determination was done using the BCA protein determination kit from Sigma. All values are means ± SD of measurements made on the indicated number of separate preparations. 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxicity of Tri in Freshly Isolated hPT Cells
The cytotoxicity of Tri in hPT cells was assessed by measurement of the LDH activity in duplicate samples from two separate patient samples (98–015 and 99–026, n = 4 total). Testing a range of concentrations similar to those tested previously in rat PT cells (Cummings et al., 2000Go) was not done due to the lack of a sufficient amount of tissue. Tri at both 0.5 mM and 10 mM was cytotoxic to hPT cells, as determined by decreases in LDH activity after 2 h (Fig. 3Go). Cytotoxicity was modest, with 0.5 mM and 10 mM Tri producing approximately 20% and 30% decreases in LDH activity, respectively, after 2-h incubations. Pretreatment of cells with metyrapone (0.25 mM) did not alter the cytotoxicity of Tri at either concentration.



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FIG. 3. Effect of P-450 inhibition on the cytotoxicity of Tri in freshly isolated hPT cells. Freshly isolated hPT cells (0.5. to 10 x 106 cells/ml) were preincubated in the presence of either solvent control (i.e., 1.0%, v/v, acetone) or metyrapone (0.25 mM) for 15 min prior to the addition of Tri. At the indicated time points, the activity of LDH was determined. Results are the means ± SD of four measurements made on hPT cells from two separate kidney samples. *Significant difference (p < 0.05) from control.

 
GSH Conjugation of Tri in Freshly Isolated hPT Cells
GSH conjugation of Tri was measured in three separate patient samples (patients no. 98–415, 98–057, and 98–058) by determining the formation of DCVG after incubation with 0, 0.25, 0.5, 1, 2, 4, 8, or 10 mM Tri for 30 min. Lineweaver-Burk analysis of DCVG formation in these three samples resulted in a regression line yielding an r2 of 0.934 (Fig. 4AGo). This line gave apparent Km and Vmax values of 1.3 mM and 0.20 nmol/min per mg protein, respectively. Eadie-Scatchard analysis of DCVG formation in hPT cells resulted in two distinct regression lines with r2 values of 0.937 and 0.963 (Fig. 4BGo, lines 1 and 2, respectively). Line 1 gave apparent Km and Vmax values of 0.58 mM and 0.11 nmol/min per mg protein, respectively. Line 2 gave apparent Km and Vmax values of 29.4 mM and 1.35 nmol/min per mg protein, respectively.



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FIG. 4. Kinetic analysis of GSH conjugation of Tri in freshly isolated hPT cells. Freshly isolated hPT cells (0.5–2 mg protein/ml) were lysed and incubated in the presence of 0–10 mM Tri and 5 mM GSH at 37°C for 30 min. Metabolism was measured by quantitation of DCVG formation by HPLC after derivatization. Controls included the absence of Tri. Results were analyzed by both Lineweaver-Burk (A) or Eadie-Hofstee (B) analysis. Results are the means ± SD of three separate samples. Lines were obtained by linear regression.

 
Oxidative Metabolism of Tri by P-450 in Human Kidney Microsomes and Freshly Isolated hPT Cells
Oxidative metabolism of Tri was studied in human renal cortical microsomes from 2 male and 2 female patients, ranging in age from 40 to 63 years (patients no. 98–077, 98–088, 98–0179, and 98–244). CH formation was detected in only one patient (female, patient no. 98–077) at the highest concentration of Tri tested (2 mM). The rate of formation of CH was 0.13 ± 0.01 nmol/min per mg protein (mean ± SD of triplicate measurements made on this patient). No other P-450-derived metabolites of Tri were detected. CH formation was not detected in hPT cells from any patient tested (data not shown).

Cytotoxicity of DCVC in Freshly Isolated hPT Cells
Freshly isolated hPT cells (patients no. 98–001, 98–002, and 98–026) were preincubated with AOAA (0.1 mM), an inhibitor of the cysteine S-conjugate ß-lyase (Lash et al., 1990Go, 1994Go), or solvent control ([EtOH] < 1.0 % v/v) for 15 min prior to the addition of 0.5 or 1 mM DCVC. These concentrations were used as they had been shown previously to be cytotoxic in freshly isolated rat renal PT cells (Lash et al., 1994Go). Cytotoxicity was assessed by measurement of release of intracellular LDH activity. DCVC caused both time- and concentration-dependent cell death of hPT cells, causing approximately 60–75% LDH release after 2-h exposure to 1 mM DCVC (Fig. 5Go). Unlike Tri, DCVC did not cause any decrease in total LDH activity. Pretreatment of cells with AOAA did not alter the cytotoxicity of DCVC at any time point or with any concentration used. Pretreatment of hPT cells with higher concentrations of AOAA (i.e., 0.5 or 1 mM) also failed to alter DCVC-induced LDH release (data not shown).



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FIG. 5. Cytotoxicity of DCVC in freshly isolated hPT cells. Freshly isolated hPT cells (0.5 to 10 x 106 cells/ml) were preincubated in the presence of either solvent control (i.e., 1.0%, v/v acetone) or AOAA (0.1 mM) for 15 min prior to the addition of DCVC. At the indicated time points, the percent LDH release was determined. Results are the means ± SD of at least three separate kidney samples. *Significant difference (p < 0.05) from control.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although the function of the kidneys in humans in various physiologic processes, such as maintenance of fluid and electrolyte balance, is reasonably well characterized, the role of the kidneys in humans in xenobiotic metabolism is not well understood. The results presented in this study describe the cellular toxicity and metabolism of a common environmental pollutant and one of its metabolites in freshly isolated hPT cells Data on the toxicity and metabolism of Tri and DCVC, along with data on the ability of inhibitors to alter these processes, will aid in the determination of the human health risk of Tri along with that of other chemicals metabolized by these pathways.

Tri Cytotoxicity and Metabolism in Freshly Isolated hPT Cells
Previous data from our laboratory showed that the differences in the cytotoxicity of Tri between rat renal PT and DT cells correlated with differences in DCVG formation (Cummings et al., 2000Go). These data also showed that in the rat kidney, P-450 metabolism of Tri appears to be a route of detoxification. In rat kidney cells under normal conditions, Tri was only cytotoxic at the highest concentrations tested (10 mM) after 2 h of exposure. Preincubation of rat kidney cells with metyrapone increased the toxicity of Tri in rat renal DT cells but not PT cells. In this study, Tri appeared to be slightly more cytotoxic to hPT cells than to either rat renal PT or DT cells, causing significant increases in cytotoxicity at both 0.5 and 10 mM after just 1 h incubation (cf. Fig. 3Go), although the cytotoxicity was still relatively modest. It should be noted that decrease in total LDH activity, rather than LDH release, was used as an index of cytotoxicity in the present studies, because Tri was found to inhibit LDH activity in hPT cells. Because of this assay difference with cells from the two species, only a qualitative comparison, rather than a direct comparison, of results from rat PT cells and hPT cells is possible. Preincubation of hPT cells with metyrapone did not alter the cytotoxicity of Tri at any time point (cf. Fig. 3Go). These data suggest that P-450 oxidative metabolism does not play a major role in Tri-induced, acute cellular injury in hPT cells. This possibility was supported further by the fact that P-450-dependent, oxidative metabolism of Tri could not be measured in 3 out of the 4 human kidney microsome samples tested.

Because preincubation of hPT cells with metyrapone did not alter the cytotoxicity of Tri and because CH formation was measured in only one patient tested, it was hypothesized that the cytotoxicity of Tri is a consequence of metabolism by the GSH-dependent pathway. Data shown in Fig. 4Go demonstrate that DCVG, the first metabolite formed in this pathway, was detected in all patients tested. Furthermore, kinetic analysis of DCVG formation in hPT cells suggests that GSH conjugation of Tri in hPT cells may be a result of at least two separate enzymes or isoenzymes (cf. Fig. 4BGo). The apparent Km and Vmax values for these regression lines are distinctly different (0.51 mM and 0.10 nmol/min per mg protein vs. 24.9 mM and 1.0 nmol/min per mg protein), suggesting the presence of a relatively high-affinity, low-capacity enzyme system and a low-affinity, high-capacity enzyme system. At the concentrations tested in this study, the low-affinity, high-capacity enzyme system would not be saturated. In a recent study of Tri metabolism to DCVG in human kidney and liver (Lash et al., 1999aGo), we reported a significant degree of interindividual variability (3- to 8-fold) in the rate of GSH conjugation of Tri in subcellular fractions from individual human liver samples. Green et al. (1997) also reported a nearly 10-fold variation in cytosol from individual human liver samples. Due to the limited availability of human kidney tissue for our previous study, Tri metabolism in kidney subcellular fractions was measured only in pooled tissue samples. It is possible, therefore, that some degree of interindividual variability also exists in rates of renal GSH conjugation of Tri. However, interindividual variability for a given metabolic pathway in the kidneys is typically less than that for the liver. Additionally, error bars for velocity for the values shown in Fig. 4Go were modest, indicating that a high degree of interindividual variability (at least in the three individual kidneys used here) does not appear to exist for renal GSH conjugation of Tri.

DCVG formation at high concentrations of Tri (8 and 10 mM) decreased in a manner similar to that reported in studies with rat renal PT cells (Cummings et al., 1999a). We suggested that inhibition of Tri metabolism by either feedback inhibition or conjugation of DCVG with GST may be occurring in both rat renal PT cells and with purified GST{alpha} isoforms. If this same phenomenon is occurring in hPT cells, it could result in the biphasic pattern shown in Fig. 4BGo. Whether the two distinct slopes measured in Fig. 4BGo are a result of two distinct enzyme systems or a decrease in DCVG formation needs to be determined. Another possible explanation for the biphasic kinetics in the intact cells is that the enzymatic processes being measured in these cells reflect those occurring in the cytoplasm and microsomes.

Comparison of the kinetics of DCVG formation in hPT cells with those we determined previously in human kidney cytosol (Lash et al., 1999aGo) reveals several similarities and differences. First, both models displayed biphasic kinetics for Tri metabolism. The apparent Km values for Tri metabolism in human kidney cytosol (0.026 and 0.16 mM), however, were significantly lower than the apparent Km values determined in this study with hPT cells (0.51 and 24.9 mM). In contrast, the apparent Vmax values for Tri metabolism in human kidney cytosol (0.81 nmol/min per mg protein) were higher than the apparent Vmax value determined by Lineweaver-Burk analysis for Tri metabolism in hPT cells (0.20 nmol/min per mg protein). The Vmax values for GSH conjugation of Tri in hPT cells (as determined by Eadie-Scatchard analysis: 0.50 and 1.0 nmol/min per mg protein) are within the range that we reported previously for GSH conjugation of Tri in human kidney cytosol (Lash et al., 1999aGo). Differences in these values can be attributed to either the concentration of GST isoforms in human kidney cytosol as compared to that in hPT cells or the presence of other enzymes besides GSTs that can metabolize Tri.

From the discussion above, one may conclude that measurements of Tri metabolism in lysates from intact cells do not provide valid kinetic parameters. However, it is important to characterize metabolism of a chemical in the same in vitro model system in which its toxicity will be studied. A potential limitation of the measurement of GSH conjugation rates in intact cells that is not relevant for measurements in subcellular fractions (at least not in cytoplasm) is the presence of GGT activity, which may degrade the GSH conjugate, thereby leading to underestimation of the rate of product formation. The approach that we have used is to irreversibly inhibit GGT with acivicin (Cummings et al., 2000Go; Lash et al., 1995Go, 1998Go, 1999aGo). The difficulty that may arise is that GGT is present in renal tissue at extremely high activities, so that anything less than complete inhibition may leave enough residual activity to produce significant degradation of the GSH conjugate. Our use of an HPLC method to measure DCVG allows us to assess the extent of degradation products. Indeed, this issue is at most a minor problem in rat kidney cells (Cummings et al., 2000Go; Lash et al., 1995Go, 1998Go). Because GGT activity is approximately 5- to 10-fold higher in rat kidney than in human kidney (Hinchman and Ballatori, 1990Go), the problem of residual GGT activity would be expected to be less of a concern in human kidney cells than in rat kidney cells. Indeed, measurements of glutamate formation (data not shown), whose N-dinitrophenyl derivative is also detected by the HPLC method used to detect DCVG, suggested that little DCVG degradation occurred in the acivicin-pretreated human PT cells.

As discussed in some detail in a recent publication (Lash et al., 1999aGo), rates of GSH conjugation of Tri that we have reported are up to an order of magnitude greater than those reported by Green et al. (1997). Similarly, the present rates of GSH conjugation of Tri in intact hPT cells are also higher than those that Green and colleagues have measured in human kidney cytosol. An additional major qualitative difference between our present and previous studies and those of Green and colleagues is that we have obtained similar rates of GSH conjugation of Tri in rat and human PT cells and in rat and human kidney subcellular fractions, whereas Green et al. (1997) report markedly higher rates in rodent kidney subcellular fractions as compared with human kidney subcellular fractions. As noted previously (Lash et al., 1999aGo), no resolution to this discrepancy has been determined, although the use of different analytical methods may play some role. Additionally, we noted that our methodology for analysis of DCVG allows for correction for any nonenzymatic formation, and that quantitation is performed with respect to authentic standard so that we are certain of the identity of the HPLC peak being measured.

Another major difference in our studies of Tri metabolism and toxicity in rat renal PT cells (Cummings et al., 2000Go) and hPT cells is that renal P-450 activity in the rat appears to play a significant role in modulating the flux of Tri through the GSH-conjugation pathway so that alteration of renal P-450 activity by either induction or the presence of inhibitors leads to an alteration in the acute cytotoxicity of Tri or DCVC. In contrast, a role for P-450 oxidation of Tri in hPT cells cannot be proposed, as Tri did not undergo significant metabolism to CH in most patients tested, and alteration of P-450 activity had no effect on Tri toxicity or GSH-dependent metabolism. Finally, in rat renal PT and DT cells from male Fischer 344 rats, GST{alpha} is one of the main GST isoforms expressed (Cummings et al., 2000Go). We showed that three isoforms of rat GST{alpha} (1–1, 1–2, and 2–2) are capable of conjugating GSH with Tri. The human kidney expresses GST{alpha} among other GST isoforms (Hiley et al., 1994Go; Rodilla et al, 1998Go), but whether human GST{alpha} is capable of conjugating GSH to Tri is not known. The minimal level or absence of detectable P-450–dependent metabolism of Tri in hPT cells is consistent with the reported absence of CYP2E1 expression in human kidney microsomes (Amet et al., 1997Go). We have confirmed that CYP2E1 protein is undetectable in hPT cells and that total P-450 activity in human kidney is significantly lower than that in human liver or in rodent kidney (Cummings, B.S., Lasker, J.M., and Lash, L.H., manuscript in preparation).

Cytotoxicity of DCVC in hPT Cells
DCVC was highly cytotoxic in hPT cells at concentrations as low as 0.5 mM after just 1-h incubation (cf. Fig. 5Go). Pretreatment of cells with AOAA did not alter DCVC cytotoxicity as it had been previously shown to do in rat renal PT and DT cells (Lash et al., 1994Go). This also contrasts with the study of Chen et al. (1990), who showed that AOAA almost completely protected confluent primary cultures of hPT cells from DCVC-induced acute cytotoxicity. A major difference between the experimental design of the present study and that of Chen et al. is the use of freshly isolated hPT cells as compared with confluent primary cultures. A more detailed accounting of the expression and activity of enzymes in the hPT cells that are relevant to Tri and DCVC metabolism and toxicity is needed to clarify these findings.

Human kidney cytosol possesses ß-lyase activity, although the activity level is severalfold lower than that reported for both the rat and mouse kidney (Hawksworth et al., 1996Go; Lash et al., 1990Go; McCarthy et al., 1994Go). AOAA was able to inhibit activity in all three species equally. Thus, the data from the present study suggest that other pathways for DCVC-induced toxicity, besides metabolism by the ß-lyase, may be occurring in hPT cells. These alternative pathways should be investigated, as they might help to explain the greater susceptibility of rodents to Tri-induced kidney cancer as compared with the susceptibility in humans. One possible bioactivation enzyme is FMO, which converts DCVC to a reactive sulfoxide (Lash et al., 1994Go; Ripp et al., 1997Go, 1999Go). FMO-dependent metabolism of DCVC is also significantly greater in kidneys of rodents than in those of humans (Ripp et al., 1999Go), which may also help explain some of the species differences in susceptibility to Tri and DCVC.

Data from this study demonstrate that Tri can be cytotoxic to a distinct cell population of the human kidney and, once again, that metabolism of Tri by the GSH-dependent pathway can occur in human kidney tissue. To the best of our knowledge, this is the first time that the cytotoxicity of Tri has been studied at the cellular level in the human kidney. Although it had been shown that Tri could be metabolized to DCVG by human kidney cytosol (Lash et al., 1999aGo), the cytotoxicity of Tri was not studied previously. Furthermore, results from studies using populations of rat renal PT and DT cells suggested a role for renal P-450 in the metabolism of Tri (Cummings et al., 1999a). The present study showed that in freshly isolated hPT cells, P-450 metabolism of Tri does not significantly contribute to the overall metabolism or nephrotoxicity of Tri. Thus, although this study further proves the relevance of the GSH-dependent pathway for Tri metabolism in humans, it brings into question the relevance of the rat kidney as a model system for the study of toxicity and metabolism of Tri.

In summary, the cellular toxicity and metabolism of Tri and DCVC in freshly isolated hPT cells was determined for the first time. Data on the toxicity and metabolism of Tri and DCVC will be extremely useful in assessment of the human health risk of Tri or other similar chemicals that are metabolized by these enzymes. These data will also aid in the determination of the role of renal processes in drug metabolism in humans.


    ACKNOWLEDGMENTS
 
This research was supported in part from National Institute of Diabetes and Digestive and Kidney Diseases grant R01-DK40725.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (313) 577-6739. E-mail: l.h.lash{at}wayne.edu. Back


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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