Ochratoxin A (OTA) is a renal carcinogen in rodents. Its human health significance is unclear. It likely depends upon the mechanism of carcinogenesis. In a previous microarray study a reduction in nuclear factor-erythroid 2 p45-related factor 2 (Nrf2)-dependent gene expression was observed in the kidney but not in the liver of rats fed OTA up to 12 months. Nrf2 regulates detoxification and antioxidant gene expression. The present report shows that OTA decreased the protein expression of several markers of the Nrf2-regulated gene battery in kidney in vivo indicating that the effects observed at mRNA level may be of biological significance. The OTA-mediated Nrf2 response could be reproduced in an NRK renal cell line and in primary hepatocyte cultures. In in vitro systems, an OTA-mediated inhibition of Nrf2 activity was demonstrated by electrophoretic mobility shift and Antioxidant Regulatory Element-driven luciferase reporter assays. The reduction of Nrf2-regulated gene expression resulted in oxidative DNA damage as evidenced by formation of abasic sites in vitro and confirmed in kidney in vivo. All OTA-mediated effects observed were prevented by pretreatment of cell cultures with inducers of Nrf2 activity. Our data suggest that reduction of cellular defense against oxidative stress by Nrf2 inhibition may be a plausible mechanism of OTA nephrotoxicity and carcinogenicity.

The diterpenes cafestol and kahweol (C&K) have been identified in animal models as two potentially chemoprotective agents present in green and roasted coffee beans. It has been postulated that these compounds may act as blocking agents by producing a co-ordinated modulation of multiple enzymes involved in carcinogen detoxification. In this study, we investigated the effects of C&K against the covalent binding of aflatoxin B1 (AFB 1 ) metabolites to DNA. Male Sprague-Dawley rats were treated with increasing amounts of a mixture of C&K in the diet (0-6200 p.p.m.) for 28 and 90 days. A dose-dependent inhibition of AFB 1 DNA-binding was observed using S9 and microsomal subcellular fractions from C&K-treated rat liver in an in vitro binding assay. Significant inhibition was detected at 2300 p.p.m. and maximal reduction of DNA adduct formation to nearly 50% of the control value was achieved with 6200 p.p.m. of dietary C&K. Two complementary mechanisms may account for the chemopreventive action of cafestol and kahweol against aflatoxin B1 in rats. A decrease in the expression of the rat activating cytochrome P450s (CYP2C11 and CYP3A2) was observed, as well as a strong induction of the expression of the glutathione-Stransferase (GST) subunit GST Yc2, which is known to detoxify highly the most genotoxic metabolite of AFB 1 . These data and the previously demonstrated effects of C&K against the development of 7,12-dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis at various tissue sites suggest the potential widespread effect of these coffee components against chemical carcinogenesis.

No abstract

Ochratoxin A (OTA) is a mycotoxin occurring in a variety of foods. OTA is nephrotoxic and nephrocarcinogenic in rodents. An OTA-mediated increase of the inducible nitric oxide synthase (iNOS) expression was observed in normal rat kidney renal cell line and in rat hepatocyte cultures, suggesting the induction of nitrosative stress. This was associated with an increased nuclear factor kappa-light chain enhancer of activated B cells activity. The potential consequences of iNOS induction were further investigated. A significant increase in the levels of protein nitrotyrosine residues was observed with OTA. In addition, OTA was found to increase the level of DNA abasic sites in both cell cultures system. This end point was used as an indirect measure of 8-nitroguanine formation. Treatment of the cells with L-N(6)-(1-iminoethyl) lysine, a specific inhibitor of iNOS activity, inhibited the OTA-mediated overnitration of proteins but did not reduce the level of DNA abasic sites. It was found previously that nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) activators were able to restore the cellular defense against oxidative stress and could prevent DNA abasic sites in cell cultures. In the present study, pretreatment of the cells with activators of Nrf2 prevented OTA-mediated increase in lipid peroxidation, confirming the potential of Nrf2 activators to confer protection against OTA-mediated oxidative stress. In addition, it was found that Nrf2 activators could also prevent OTA-induced protein nitration and cytotoxicity. In conclusion, the present data further confirm oxidative stress as a key source of OTA-induced DNA damage and provide additional evidence for a role of this mechanism in OTA carcinogenicity. The exact role of nitrosative stress still remains to be established.

No abstract