Elsevier

Biochemical Pharmacology

Volume 59, Issue 5, 1 March 2000, Pages 531-543
Biochemical Pharmacology

Molecular and Cellular Pharmacology
Role of cytochrome P450 and glutathione S-transferase α in the metabolism and cytotoxicity of trichloroethylene in rat kidney

https://doi.org/10.1016/S0006-2952(99)00374-3Get rights and content

Abstract

The toxicity and metabolism of trichloroethylene (TRI) were studied in renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. TRI was slightly toxic to both PT and DT cells, and inhibition of cytochrome P450 (P450; substrate, reduced-flavoprotein:oxygen oxidoreductase [RH-hydroxylating or -epoxidizing]; EC 1.14.14.1) increased TRI toxicity only in DT cells. In untreated cells, glutathione (GSH) conjugation of TRI to form S-(1,2-dichlorovinyl)glutathione (DCVG) was detected only in PT cells. Inhibition of P450 transiently increased DCVG formation in PT cells and resulted in detection of DCVG formation in DT cells. Formation of DCVG in PT cells was described by a two-component model (apparent Vmax values of 0.65 and 0.47 nmol/min per mg protein and Km values of 2.91 and 0.46 mM). Cytosol isolated from rat renal cortical, PT, and DT cells expressed high levels of GSH S-transferase (GST; RX:glutathione R-transferase; EC 2.5.1.18) α (GSTα) but not GSTπ. Low levels of GSTμ were detected in cortical and DT cells. Purified rat GSTα2–2 exhibited markedly higher affinity for TRI than did GSTα1–1 or GSTα1–2, but each isoform exhibited similar Vmax values. Triethyltinbromide (TETB) (9 μM) inhibited DCVG formation by purified GSTα1–1 and GSTα2–2, but not GSTα1–2. Bromosulfophthalein (BSP) (4 μM) only inhibited DCVG formation by GSTα2–2. TETB and BSP inhibited approximately 90% of DCVG formation in PT cytosol but had no effect in DT cytosol. This suggests that GSTα1–1 is the primary isoform in rat renal PT cells responsible for GSH conjugation of TRI. These data, for the first time, describe the metabolism of TRI by individual GST isoforms and suggest that DCVG feedback inhibits TRI metabolism by GSTs.

Section snippets

Chemicals

TRI (reported to be 99.9% pure, as judged by electron ionization mass spectrometry), collagenase type IV, bovine serum albumin (fraction V), acivicin [l-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid], BSP, l-γ-glutamyl-l-glutamate, and CDNB were purchased from the Sigma Chemical Co. DCVG was synthesized as previously described [34]. Purity (>95%) was determined by HPLC analysis, and identity was confirmed by proton NMR spectroscopy. TETB was purchased from the Aldrich Chemical Co.

Cytotoxicity of TRI in freshly isolated rat renal PT and DT cells

Freshly isolated rat renal PT and DT cells were incubated in the presence of various concentrations of TRI, ranging from 0.1 to 10 mM (data not shown). TRI was modestly cytotoxic to PT and DT cells only at the highest concentration tested (i.e. 10 mM), causing approximately a 20% increase in LDH release, whereas concentrations of TRI of 5 mM or below had no effect on LDH release.

Preincubation of PT and DT cells with metyrapone (0.25 mM), a general P450 inhibitor, increased TRI cytotoxicity in

Discussion

The objective of the present study was to determine differences in TRI cytotoxicity and metabolism between rat renal PT and DT cells and to use these differences to assess factors that are determinants of susceptibility to TRI. Freshly isolated cells were used so that susceptibility and enzyme expression and activity would be similar to that found in the kidneys in vivo. With knowledge of how renal metabolism and handling of TRI and its GSH-derived metabolites differ in rats and humans, these

Acknowledgements

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.

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