Elsevier

Toxicology

Volume 171, Issues 2–3, 28 February 2002, Pages 95-103
Toxicology

DNA damage in tissues of rat treated with potassium canrenoate

https://doi.org/10.1016/S0300-483X(01)00562-5Get rights and content

Abstract

Potassium canrenoate (PC), a competitive aldosterone antagonist previously found to increase tumor incidence in rats and to produce genotoxic effects in in vitro systems, was examined in rats to acquire information on its genotoxic activity in vivo. Intragastric administration of 1/2 LD50 produced, as revealed by the Comet assay, a modest but statistically significant increase in the frequency of DNA lesions in liver but not in thyroid and bone marrow of male rats, and in thyroid and bone marrow but not in liver of female rats. In contrast with the frankly positive responses observed in primary cultures of rat hepatocytes (Martelli et al., Mutagenesis 14 (1999) 463–472) any evidence of DNA repair and micronuclei formation was absent in liver of rats treated with 1/2 LD50, and initiation of enzyme-altered liver preneoplastic lesions did not occur in the liver of rats given 100 mg/kg PC once a week for 6 successive weeks. A high and dose-dependent frequency of DNA lesions was found to occur in testes and ovaries of rats given single doses ranging from 1/8 to 1/2 LD50.

Introduction

Potassium canrenoate (PC), a competitive aldosterone antagonist widely used as a diuretic and in the treatment of hypertension, has been shown to cause in rats a dose-dependent increase in myelogenous leukemia and statistically significant increases in malignant tumor of the liver, thyroid, brain and mammary gland (Cook et al., 1988). Experimental evidence indicates that this steroid is a genotoxic carcinogen, and suggests a possible carcinogenic risk for humans. PC was found to be metabolized by rat hepatic S9 to 3α- and 3β-hydroxy-6β,7β-epoxycanrenoate acting as direct mutagens in the mouse lymphoma assay (Cook et al., 1988). Subsequent in vitro studies revealed that the cytochrome P-450IIIA is responsible for the formation of the epoxide (Cook et al., 1993). More recently a dose-dependent degree of DNA fragmentation and of DNA repair synthesis and a modest but statistically significant increase in micronucleated cells were observed in primary cultures of hepatocytes from rats of both genders. In this study the response was more evident in females than in males (Martelli et al., 1999). In primary human hepatocytes from one male and two female donors PC caused a similar effect in terms of DNA fragmentation, whereas DNA repair was less marked than in rat hepatocytes and micronucleus formation was absent. Taking into account all these findings, we deemed that information on the genotoxic activity of PC in vivo would be useful for a better knowledge of its carcinogenic activity. In this study, in order to investigate the influence of pharmacokinetic events (absorption, distribution and elimination) not reproducible in in vitro systems on the genotoxic activity of PC, we examined in rats of both genders its capability of inducing DNA fragmentation and DNA repair, and of causing formation of micronuclei and of enzyme altered preneoplastic foci in the liver. In addition, the occurrence of a PC-induced DNA fragmentation was examined in the thyroid and the bone marrow which are targets of its carcinogenic activity, as well as in organs expressing enzymes involved in the metabolism of steroids, i.e. ovaries and testes.

Section snippets

Chemicals

PC, collagenase type IV, Williams' medium E (WME), N-nitrosodimethylamine (NDMA), were purchased from Sigma Chimica (Milan, Italy); N-nitrosodiethylamine (NDEA) and 2-acetylaminofluorene (2-AAF) from Merck (Darmstadt, Germany); [methyl-3H]thymidine (specific activity, 23–25 Ci/mmol) from Amersham International (Amersham Italia, Milan, Italy). All the other chemicals were of the purest grade available.

Sprague–Dawley male (200–250 g) and female (150–200 g) rats were purchased from Harlan

Results

Data provided by the DNA damage/Comet assay performed in liver thyroid and bone marrow cells from rats of both sexes given p.o. a single dose of 325 mg/kg (1/2 LD50) PC are listed in Table 1. None of these rats died or showed marked signs of toxicity. DNA migration was measured for 50 nuclei per rat per organ and the statistical analysis was carried out using the pooled 150 nuclei of three rats. Both tail length and tail moment of DNA migration are shown as indexes of DNA fragmentation. In the

Discussion

The distinction between the two primary mechanistic categories of carcinogens, genotoxic and non-genotoxic, is of critical importance in quantitative risk assessment, namely to identify relevant levels of human exposure; linear non-threshold dose–response models are used for extrapolation involving genotoxic chemicals and threshold models for non-genotoxic chemicals.

PC, which has been shown to cause in rats a dose-dependent increase in the incidence of various tumors (Cook et al., 1988), has

References (17)

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