Elsevier

Toxicology

Volume 224, Issues 1–2, 5 July 2006, Pages 108-118
Toxicology

Analysis of renal cell transformation following exposure to trichloroethene in vivo and its metabolite S-(dichlorovinyl)-l-cysteine in vitro

https://doi.org/10.1016/j.tox.2006.04.036Get rights and content

Abstract

Trichloroethene (TCE) is classified as a potential human carcinogen although there is a significant debate regarding the mechanism of TCE induced renal tumor formation. This controversy stems in part from the extremely high doses of TCE required to induce renal tumors and the potential contribution of the associated nephrotoxicity to tumorigenesis. We have used Eker rats, which are uniquely susceptible to renal carcinogens, to determine if exposures to TCE in vivo or exposure to its metabolite S-(dichlorovinyl)-l-cysteine (DCVC) in vitro can transform kidney epithelial cells in the absence of cytotoxicity. Treatment with TCE (0, 100, 250, 500, 1000 mg/kg bw by gavage, 5 days a week) for 13 weeks resulted in a significant increase in cell proliferation in kidney tubule cells, but did not enhance formation of preneoplastic lesions or tumor incidence in Eker rat kidneys as compared to controls. In vitro, concentrations of DCVC, which reduced cell survival to 50%, were able to transform rat kidney epithelial cells. However, no carcinogen-specific mutations were identified in the VHL or Tsc-2 tumor suppressor genes in the transformants. Taken together, the inability of TCE to enhance formation of preneoplastic changes or neoplasia and the absence of carcinogen-specific alteration of genes accepted to be critical for renal tumor development suggest that TCE mediated carcinogenicity may occur secondary to continuous toxic injury and sustained regenerative cell proliferation.

Introduction

The carcinogenicity of trichloroethylene (TCE) has been extensively debated over the past 15 years. TCE has been widely used in cleaning and degreasing because of it favorable solvent characteristics, chemical stability, and relatively low acute toxicity. Several epidemiological studies on the mortality of trichloroethene-exposed workers and one study on mortality/morbidity did not reveal an association between exposure and renal tumors (Spirtas et al., 1991, Axelson et al., 1994, Anttila et al., 1995). However, an increased incidence of renal cell tumors was reported in a retrospective study of cardboard workers exposed to very high concentrations of trichloroethene over a prolonged period of time (Henschler et al., 1995). In NTP bioassays, high doses of TCE caused a small but sometimes significant increase in the incidence of renal tumors in male rats (NTP, 1988, NTP, 1990); the renal tumors were always accompanied by massive nephrotoxicity and cell death. However, the predictive power of the above bioassays was limited by poor survival indicating that the maximum tolerated dose was exceeded.

Bioactivation reactions are most likely responsible for TCE nephrotoxicity. Glutathione conjugation of TCE followed by metabolism by the enzymes of mercapturic acid pathway produces S-(dichlorovinyl)-l-cysteine (DCVC), which is accumulated in the proximal tubule cells of the nephron. Cleavage of DCVC by β-lyases, which is present at high concentrations in this part of the nephron, produces a highly reactive thioketene (Lash et al., 1986, Dekant et al., 1988, MacFarlane et al., 1989). DCVC is mutagenic in bacteria and induces mitochondrial toxicity, perturbation of intracellular calcium homeostasis, DNA-repair and DNA strand-breaks in cultured renal cells and in the renal cortex after in vivo administration (Dekant et al., 1986, Vamvakas et al., 1988, Vamvakas et al., 1989, Vamvakas et al., 1990, van de Water et al., 1993, McLaren et al., 1994). In addition, DCVC exerts marked specific nephrotoxicity in the kidney cortex in vivo, which is in line with the massive nephrotoxicity observed with trichloroethene in the long-term NTP study (Gandolfi et al., 1981, Darnerud et al., 1989, Lash et al., 1994, Vaidya et al., 2003).

The Eker rat is a unique animal model for renal cell carcinoma (RCC) (Everitt et al., 1992). These rats carry a germline alteration of the tuberous sclerosis 2 (Tsc-2) tumor suppressor gene (Yeung et al., 1994, Kobayashi et al., 1995), which results in an increased susceptibility to develop spontaneous and carcinogen-induced RCC (Walker et al., 1992a). Tumorigenesis in these animals proceeds via a well-characterized progression sequence from early preneoplastic lesions, identified as atypical tubules and hyperplasias, through adenomas and carcinomas. This progression sequence is identical to that which occurs in other rat strains following exposure to chemical carcinogens, but occurs in Eker rats with a higher frequency, both spontaneously and in response to chemical carcinogens. Many of the molecular events involved in RCC development in the Eker rat have been characterized and, with few exceptions, these tumors exhibit significant similarities to their cognate human disease (Walker et al., 1991, Walker et al., 1992b, Yeung et al., 1995).

Tumors arise in the Eker rat model as a result of inactivation of the remaining normal allele of the Tsc-2 gene following carcinogen exposure. The Tsc-2 gene is targeted by renal carcinogens in other rats strains as well and alterations in this gene have been reported in RCC arising as a result of exposure to diverse renal carcinogens (Kubo et al., 1994, Urakami et al., 1997, Satake et al., 1998). In addition to the Tsc-2 gene, the Von-Hippel-Lindau (VHL) tumor suppressor gene has been identified as a gene critical for renal tumor development and inactivation of this gene by mutation has been shown to occur in the majority of renal cell carcinomas in humans (Gnarra et al., 1994). Moreover, increased VHL mutation frequencies have been reported in renal cell tumors obtained from patients occupationally exposed to TCE and have been interpreted as evidence for a direct genotoxic effect of TCE (Brauch et al., 1999, Brauch et al., 2004).

An in vitro assay for transforming rat kidney epithelial (RKE) cells has been developed in which primary RKE cells are plated at clonal density and exposed to potential carcinogens (Walker and Ginsler, 1992). Transformants that arise are initially recognized by their extended growth potential, giving rise to transformed colonies that progress to become immortal and ultimately tumorigenic. RKE cells carrying the Eker mutation exhibit an increased susceptibility to transformation in vitro as measured by this assay (Horesovsky et al., 1994). Exposure to 2,3,4-tris(glutathion-S-yl)-hydroquinone, the active metabolite of hydroquinone, for example, induces morphological transformation of Tsc-2Ek/+ renal epithelial cells in vitro and numerous preneoplastic lesions in Eker rat kidneys as early as 4 months after initiation of treatment. These lesions were shown to exhibit loss of Tsc-2 gene function (Lau et al., 2001, Yoon et al., 2001, Yoon et al., 2002), demonstrating concordance of the in vitro assay with carcinogenicity in vivo. In this study, we exploited the increased sensitivity of the Eker rat to renal carcinogenesis to investigate the potential of TCE and its metabolite DCVC to induce renal cell transformation in vivo or in vitro, and to determine if genes critical for renal tumor development are targeted by this nephrotoxicant.

Section snippets

Chemicals

Unless otherwise indicated, all chemical and reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA and Deisenhofen, Germany).

Animals

Eker rats (Tsc-2Ek/+) were obtained from a closed breeding colony maintained at Science Park-Research Division at The University of Texas M.D. Anderson Cancer Center. Neonates were genotyped to identify rats carrying the Eker mutation by PCR. DNA was isolated from ear clippings by using the QIAmp tissue kit (Qiagen, Chatsworth, CA). The PCR reaction mixture

Nephrotoxicity of TCE

Eker rats are at increased risk for the development of RCC as a result of carcinogen exposure, thus making them a sensitive animal model for detection of chemicals with carcinogenic potential, especially in the kidney. Thus, we hypothesized that a low dose carcinogen exposure over a relatively short time interval (13 weeks) in these animals might be capable of separating nephrotoxicity from carcinogenicity. Rats were gavaged 5 days a week with doses of TCE from 0 to 1000 mg/kg body weight and at

Discussion

The Eker rat is a unique animal model for susceptibility to RCC (Everitt et al., 1992). Heterozygous carriers of the Eker mutation are at increased risk for the developement of both spontaneous and chemically induced renal cell carcinoma (Walker et al., 1992a, Yeung et al., 1994, Kobayashi et al., 1995). We have used the Eker rat as a sensitive model to assess the carcinogenic potential of TCE. Although high doses of TCE have been shown to induce kidney tumors in the rat (NTP, 1988, NTP, 1990),

Acknowledgments

Parts of this work were supported by the Deutsche Forschungsgemeinschaft (SFB 172/A13), the NATO Collaborative Research Grants Programme (CRG 960148), and grants from the National Institutes of Health to CLW (CA 63613 and ES 07784).

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