Oxidative DNA damage induced by Ochratoxin A in the HK-2 human kidney cell line: evidence of the relationship with cytotoxicity.

Ochratoxin A (OTA) is a mycotoxin produced by species of the genera Aspergillus and Penicillium. The kidneys are the target organ of this mycotoxin and it is considered a potent renal carcinogen in male rats. The mechanisms of its genotoxicity and carcinogenicity have been studied thoroughly, but controversial results have been published. The aim of this study was to evaluate the ability of OTA to produce single-strand DNA breaks and oxidative DNA damage in the human renal proximal tubular epithelial cell line (HK-2), due to the fact that there is no study on human kidney cells as the toxic target. In addition, we attempted to determine if biotransformation processes mediate OTA genotoxicity. Therefore, single-cell gel electrophoresis assay (comet assay) was performed after 3h- and 6h-treatments using different OTA concentrations, both cytotoxic and non-cytotoxic, in order to be able to distinguish a genotoxic effect of the mycotoxin from an indirect effect derived from its general cellular toxicity. No effect was shown where no cytotoxicity was found, both in the presence and in the absence of metabolic activation (10% rat liver S9-mix). However, oxidative DNA damage was shown at cytotoxic concentrations when formamidopyrimidine DNA glycosylase (FPG) and endonucleaseIII (EndoIII) were introduced in the assay with or without metabolic activation. Furthermore, at these concentrations, an elevation of reactive oxygen species was measured and pre-incubation with N-acetyl-L-cysteine was able to produce a slight protective effect on OTA-induced oxidative DNA damage as well as cytotoxicity. These data suggest that OTA is not acting as a direct genotoxic carcinogen and that oxidative stress is implicated in the genotoxicity and cytotoxicity observed in these human renal cells.

1, 2, 4-Triazine N-oxide derivatives: studies as potential hypoxic cytotoxins. Part II.

New 1, 2, 4-Triazine N-oxide and N, N'-dioxide derivatives were synthesized in order to obtain compounds as selective hypoxic cell cytotoxins. The starting heterocycles have been prepared using a standard microwave oven in a clean and good-yielded process. The reactivity of methyl-1, 2, 4-triazine N(4)-oxide and N(1), N(4)-dioxide with different electrophilic agents has been studied. The desired products were obtained only when iminium electrophiles were employed. The regioselectivity of this process has been studied by means of experimental and theoretical (at ab initio level) procedures. Theoretically was expected that the most stable intermediates where the benzylic-like anion from position 5. A fact which agreed with the experimental observed regioselectivity. The new compounds were tested for their cytotoxicity in oxia and hypoxia. Some of them proved to be less active in hypoxic conditions than tirapazamine, 3-amino-benzo[1, 2-e]1, 2, 4-triazine N(1), N(4)-dioxide. Derivative 19, 6-methyl-5-[2-(5-nitrothienyl)ethenyl)-1, 2, 4-triazine N(4)-oxide, was the most cytotoxic compound, but it was non-selective.