Elsevier

Toxicology

Volume 204, Issues 2–3, 15 November 2004, Pages 187-195
Toxicology

DNA damage and micronuclei induced in rat and human kidney cells by six chemicals carcinogenic to the rat kidney

https://doi.org/10.1016/j.tox.2004.06.057Get rights and content

Abstract

Six chemicals, known to induce kidney tumors in rats, were examined for their ability to induce DNA fragmentation and formation of micronuclei in primary cultures of rat and human kidney cells, and in the kidney of intact rats. Significant dose-dependent increases in the frequency of DNA single-strand breaks and alkali-labile sites, as measured by the Comet assay, and in micronuclei frequency, were obtained in primary kidney cells from both male rats and humans of both genders with the following subtoxic concentrations of five of the six test compounds: bromodichlorometane (BDCM) from 0.5 to 4 mM, captafol (CF) from 0.5 to 2 μM, nitrobenzene (NB) from 0.062 to 0.5 mM, ochratoxin A (OTA) from 0.015 to 1.215 μM, and trichloroethylene (TCE) from 1 to 4 mM. Benzofuran (BF), consistent with its carcinogenic activity for the kidney of female, but not of male rats, at concentrations from 0.125 to 0.5 mM gave positive responses in cells from females but did not induce DNA damage and increased the frequency of micronuclei in cells from males to a lower extent; in contrast, it was active in cells from humans of both genders. DNA-damaging and micronuclei-inducing potencies were similar in the two species. In agreement with these findings, statistically significant increases in the average frequency of both DNA breaks and micronucleated cells were obtained in the kidney of rats, given p.o. a single dose (1/2 LD50) of the six compounds, BF in this assay being more genotoxic in female than in male rats. Taken as a whole, these findings give further evidence that kidney carcinogens may be identified by short-term genotoxicity assays, using as target kidney cells, and show that the six chemicals tested produce, in primary cultures of kidney cells from human donors, effects similar to those observed in rats.

Introduction

In the preliminary screening phase, the standard battery of genotoxicity tests may be unable to identify tissue-specific carcinogens (Brambilla and Martelli, 2004); certainly it does not provide any useful information on the possible species-specificity of the test compound. Therefore, a reasonable approach should be the use of in vitro and/or in vivo assays capable of identifying chemicals genotoxic for a specific organ of rodents, and repeating the same assays in primary cultures of human cells from the same organ. We have previously demonstrated that five chemicals, 1,4-dichlorobenzene, lead acetate, nitrilotriacetic acid, phenacetin, and potassium bromate, known to induce kidney tumors in rats, induce DNA fragmentation and micronuclei formation in kidney cells from both rats and humans with similar potency (Robbiano et al., 1999). The present study was performed to better assess whether the same battery of in vitro and in vivo assays consistently provides results positively correlated with the induction of tumors in the rat kidney, and to examine whether the genotoxic effects induced by the test compounds in primary cultures of human kidney cells are quantitatively similar to those observed in rat kidney cells. The six chemicals tested were benzofuran, bromodichloromethane, captafol, nitrobenzene, ochratoxin A, and trichloroethylene; all these chemicals are carcinogenic to both mice and rats. Benzofuran (IARC, 1995) was found to increase the incidence of renal cell adenocarcinomas in female but not in male rats; on the basis of inadequate evidence for carcinogenicity in humans and sufficient evidence for carcinogenicity in experimental animals it was classified by IARC as possibly carcinogenic to humans (group 2B). Bromodichloromethane (IARC, 1991a, IARC, 1999) was found to increase the incidence of renal cell adenocarcinomas in both male and female rats; on the basis of inadequate evidence for carcinogenicity in humans and of sufficient evidence for carcinogenicity in experimental animals, it was classified by IARC as possibly carcinogenic to humans (group 2B). Captafol (IARC, 1991b) was found to cause a dose-related increase in the incidence of renal carcinomas in male rats and of benign renal tumors in female rats; despite the absence of data in humans, it was classified by IARC as probably carcinogenic to humans (group 2A) on the basis of a sufficient evidence for carcinogenicity in experimental animals and of the positive responses in a wide range of genotoxicity assays. Nitrobenzene (IARC, 1996) was found to induce in male rats, by inhalation exposure, the development of renal tubular-cell adenomas as well as of hepatocellular neoplasms and of thyroid follicular-cell adenomas and adenocarcinomas; on the basis of inadequate evidence for carcinogenicity in humans and of sufficient evidence for carcinogenicity in experimental animals, it was classified by IARC as possibly carcinogenic to humans (group 2B). Ochratoxin A (IARC, 1993) was found to cause, in rats of each sex, a dose-related increase in the incidence of renal-cell adenomas and carcinomas; on the basis of inadequate evidence for carcinogenicity in humans and of a sufficient evidence for carcinogenicity in experimental animals, it was classified by IARC as possibly carcinogenic to humans (group 2B). Trichloroethylene (IARC, 1995) was found to cause in male rats a significant increase in the incidence of uncommonly occurring renal tumors and of testicular tumors, when administered by the oral route, and a marginal incidence of renal tumors by inhalation; on the basis of limited evidence for carcinogenicity in humans and a sufficient evidence for carcinogenicity in experimental animals, it was classified by IARC as probably carcinogenic to humans (group 2A).

All the six chemicals were tested in vitro and in vivo for their ability to induce DNA fragmentation, as evaluated by the Comet assay, and increase the frequency of micronuclei in kidney cells. In vitro assays were performed in primary cultures of kidney cells from both rats and human donors; in vivo DNA fragmentation and micronuclei formation were examined in the kidney of rats treated by the oral route.

Section snippets

Chemicals and animals

Benzofuran (BF, >99% pure), bromodichloromethane (BDCM, >98% pure), captafol (CF, 99% pure), nitrobenzene (NB, 99% pure), and ochratoxin A (OTA, 98% pure) were purchased from Aldrich Chimica (Milan, Italy); N-nitrosodimethylamine (NDMA, 99.8% pure), and trichloroethylene (TCE, reagent grade) were purchased from E. Merck (Darmstadt, Germany); collagenase type IV, Williams’ medium E (WME), and epidermal growth factor (EGF) were purchased from Sigma (Milan, Italy); insulin and fetal bovine serum

Results

As already observed in previous studies (Robbiano et al., 1996, 1999), observations performed by light microscopy revealed that large majority of cells isolated from the cortex of both rat and human kidney were of proximal tubules. As pointed out by Bruggeman et al. (1989), xenobiotics and their metabolites may be concentrated to toxic levels especially in the epithelial cells of the proximal tubule, and relatively high levels of xenobiotic-metabolizing enzymes are present in these cells. A

Discussion

The results of this study indicate that six chemicals, BF (IARC, 1995), BDCM (IARC, 1991a, IARC, 1999), CF (IARC, 1991b), NB (IARC, 1996), OTA (IARC, 1993), and TCE (IARC, 1995), which are known to cause the development of kidney tumors in rats, induced, in primary cultures of rat kidney cells, a dose-dependent increase in the frequency of both DNA breaks and micronucleated cells. With BF, carcinogenic to kidney in female but not in male rats, the responses were concordant with its sex

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