Elsevier

Toxicology

Volume 150, Issues 1–3, 7 September 2000, Pages 83-98
Toxicology

Cytotoxicity of trichloroethylene and S-(1,2-dichlorovinyl)-l-cysteine in primary cultures of rat renal proximal tubular and distal tubular cells

https://doi.org/10.1016/S0300-483X(00)00252-3Get rights and content

Abstract

Activities of several glutathione-dependent enzymes, expression of cytochrome P450 isoenzymes, and time- and concentration-dependent cytotoxicity of trichloroethylene (TRI) and S-(1,2-dichlorovinyl)-l-cysteine (DCVC) were evaluated in primary cultures of proximal tubular (PT) and distal tubular (DT) cells from rat kidney. These cells exhibited cytokeratin staining and maintained activities of all glutathione-dependent enzymes measured. Of the cytochrome P450 isoenzymes studied, only CYP4A expression was detected. CYP4A mRNA and protein expression were higher in primary cultures of DT cells than in PT cells and were increased in DT cells by ciprofibrate treatment. Incubation of cells for 6 h with concentrations of TRI as high as 10 mM resulted in minimal cytotoxicity, as determined by release of lactate dehydrogenase (LDH). In contrast, marked cytotoxicity resulted from incubation of PT or DT cells with DCVC. Addition to cultures of TRI (2–10 mM) for 24 or 72 h resulted in modest, but significant time- and concentration-dependent increases in LDH release. Treatment of cells with DCVC (0.1–1 mM) for 24 h caused significant increases in LDH release and alterations in cellular protein and DNA content. Finally, exposure of primary cultures to TRI or DCVC for 72 h followed by 3 h of recovery caused a slight increase in the expression of vimentin, consistent with cellular regeneration. These studies demonstrate the utility of the primary renal cell cultures for the study of CYP4A expression and mechanisms of TRI-induced cellular injury.

Introduction

Trichloroethylene (TRI) is a colorless, volatile liquid and alkenyl halide (Manahan, 1992). The lipophilic character, non-flammability, and high boiling point of TRI make it useful in a variety of industrial processes, including metal degreasing and dry cleaning, and TRI was also once used as an anesthetic (Davidson and Beliles, 1991). Because of these uses, high amounts of TRI have evaporated into the atmosphere and contaminate ground and surface water and food, raising the possibility of both occupational and general exposure of humans to TRI. The symptoms of toxic TRI exposure include central nervous system depression, abnormal liver function and irritating effects to the skin and mucous membranes of the respiratory tract (Reichert, 1983). TRI is also a putative human carcinogen and causes kidney cancer in male rats and possibly in humans (Davidson and Beliles, 1991, Henschler et al., 1995).

TRI is metabolized by two separate pathways. Oxidative metabolism of TRI by cytochrome P450 (P450) results in the formation of chloral hydrate, which can be further metabolized to trichloroacetic acid, dichloroacetic acid, trichloroethanol, monochloroacetic acid, and oxalic acid (Miller and Guengerich, 1983). TRI can also be metabolized by conjugation with glutathione (GSH) to form S-(1,2-dichlorovinyl)glutathione (DCVG). DCVG can be further metabolized by γ-glutamyltransferase (GGT; EC 2.3.2.1) and cysteinylglycine dipeptidase (EC 3.4.13.12) to form S-(1,2-dichlorovinyl)-l-cysteine (DCVC). DCVC can be either N-acetylated to form N-acetyl-DCVC, which can be deacetylated back to DCVC, or metabolized by cysteine-conjugate β-lyase (β-lyase; EC 4.4.1.13) to form a reactive thiol. The renal effects of TRI have been primarily attributed to the formation of DCVC, whose metabolites can rearrange to form potent acylating species. Subsequent acylation of proteins and DNA may lead to cytotoxicity and mutagenesis (Anders et al., 1988, Goeptar et al., 1995).

The toxic effects of TRI are believed to be a result of repeated exposures to TRI over an extended period of time. This is true of several chemicals, and chemical-induced injury to not only the kidney, but to many other organs, is often the result of repeated or chronic exposure to a chemical over a longer period of time. Because of this, efforts have been made to develop models that can be used for the study of long-term chemical-induced injury. An in vitro model that mimics the in vivo state would greatly facilitate the study of chemical-induced renal injury. A model has been previously developed using primary cultures of proximal tubular (PT) and distal tubular (DT) cells from rat kidney (Lash et al., 1995). These cultured cells maintain similar biochemical function as the freshly isolated cells. For example, levels of marker enzymes, such as GGT and hexokinase (EC 2.7.1.1), in cultured PT and DT cells and the activities of alkaline phosphatase, cellular energy metabolism enzymes, and the expression of cytokeratins, are maintained over a period of at least 5 days. Furthermore, the same cell-specific patterns of susceptibility to chemical toxicants such as methyl vinyl ketone and tert-butyl hydroperoxide, monitored with freshly isolated cells, were also observed in cultures of PT and DT cells.

The goal of the work presented in this study is to use these previously validated cultures to study the cytotoxicity of TRI and DCVC over an extended period of time (days vs. hours). Previous work showed that both TRI and DCVC cause cytotoxicity in freshly isolated PT and DT cells, but these studies only examined the effects of these chemicals after 2-h incubations (Lash et al., 1994, Cummings et al., 2000). Although the PT cells are the major in vivo target cell population for TRI and DCVC, examination of cytotoxicity in a well-characterized non-target cell population (i.e. DT cells) will provide additional insight into factors that are responsible for the toxicity. It has also been demonstrated that freshly isolated PT and DT cells express CYP2E1, CYP2B1/2, CYP2C11, and CYP4A2/3 but not CYP3A1/2 (Cummings et al., 1999). The expression of these enzymes in primary cultures of PT and DT cells, however, has not been determined. Examination of the cytotoxicity of TRI and its metabolite DCVC in these cultures will advance the knowledge of the mechanism(s) by which these agents cause cellular injury. Preliminary results of this work have been presented.3

Section snippets

Materials

Percoll, collagenase (Type I; EC 3.4.24.3), powdered 1:1 mixture of Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12), HEPES, bovine serum albumin (BSA; fraction V), γ-glutamyl-p-nitroanilide, p-nitrophenylphosphate, penicillin G, streptomycin sulfate, amphotericin B, insulin (from bovine pancreas), human transferrin, sodium selenite, hydrocortisone, 3,3′,5-triiodo-dl-thyronine, thyrocalcitonin (from bovine thyroid gland), and ciprofibrate were purchased from Sigma (St. Louis, MO).

Activity of GSH-dependent enzymes in primary cultures of PT and DT cells

Freshly isolated PT and DT cells were placed in primary culture and reached confluency in 5–7 days (Fig. 1). Cells formed a monolayer and exhibited epithelial morphology, as demonstrated previously (Lash et al., 1995). Activities of GGT, GRD, GPX, GST, GCS, and hexokinase were measured in freshly isolated PT and DT cells (Day 0=freshly isolated cells), and in cells from 3 and 5-day-old cultures. For the most part, the activity of all these enzymes did not change significantly over the time

Discussion

This investigation describes for the first time, studies in primary cultures of PT and DT cells from rat kidney of both acute and longer-term incubations with TRI and its metabolite DCVC and data on activity and expression of several drug-metabolizing enzymes. Data from this study showed that primary cultures of PT and DT cells are capable of maintaining most of their activities of GSH-dependent enzymes and hexokinase, thus mimicking closely freshly isolated cells. More importantly, the ratio

Acknowledgements

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK40725 (to L.H.L.) and through the use of the Imaging and Cytometry Core of the EHS Center (Grant P30-ES02526), provided by the National Institute of Environmental Health Sciences.

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  • Cited by (0)

    1

    Present address: University of Arkansas Medical Sciences, Division of Pharmacology & Toxicology, Slot 638, 4301 Markham Street, Little Rock, AR 72205, USA.

    2

    Present address: Department of Molecular Biosciences, Pacific Northwest National Laboratory, P.O. Box 999, P7-56, Richland, WA 99352, USA.

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