DNA damage in tissues of rat treated with potassium canrenoate
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
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Optimization of upcyte<sup>®</sup> human hepatocytes for the in vitro micronucleus assay
2013, Mutation Research - Genetic Toxicology and Environmental MutagenesisCitation Excerpt :The high %MN rate in these cells is unlikely to be related to the age of the donors (as reported for human lymphocytes [22]) since cells were all from young donors. Like upcyte® hepatocytes, the %MN in control human and rat primary hepatocytes is reported to be donor-dependent and varies between 0% and 17.5% in rat hepatocytes and, between 0% and 9.3% in human hepatocytes from donors older than 60 [23,24]. Once optimised conditions were achieved using EGF and OSM supplements, the %MN in control upcyte® hepatocytes from donors 740, 422A, 653 and 721B were 7%, 17%, 9% and 23%, respectively.
A core in vitro genotoxicity battery comprising the Ames test plus the in vitro micronucleus test is sufficient to detect rodent carcinogens and in vivo genotoxins
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