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

Abstract

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.

Genotoxicity of Aflatoxin B1 and Ochratoxin A after simultaneous application of the in vivo micronucleus and comet assay

“…OTA mechanisms of action are not clearly determined but the ability to generate reactive oxygen species (ROS) may explain the lipid, protein and DNA damage (Ringot et al, 2006). The test battery for evaluating OTA genotoxicity gave negative results (IARC, 1993), but some positive results are found in some in vitro and in vivo studies such as DNA breaks in mammalian cell lines (Ehrlich et al, 2002;Lebrun and Follmann, 2002;Arbillaga et al, 2007), DNA damage and micronuclei in primary cultures of human and rat kidney cells (Robbiano et al, 2004) and cytogenetic damage and DNA adducts in rats treated with OTA (Mally et al, 2005;Pfohl-Leszkowicz and Manderville, 2007).…”

In vitro genotoxicity of mycotoxins ochratoxin A and fumonisin B1 could be prevented by sodium copper chlorophyllin – Implication to their genotoxic mechanism

“…Based on findings of OTA-related DNA adducts in cell cultures as well as in animal and human kidneys, some authors support the notion of direct OTA genotoxic action after metabolic activation (Pfohl-Leskowicz & Manderville, 2007). However, some studies indicate that OTA is not able to form reactive intermediates capable of interacting with DNA (Mally et al, 2005), and it has been suggested that OTA can induce DNA damage through oxidative stress (Arbillaga, Azqueta, Ezpeleta, & Lopez de Cerain, 2007;Domijan, Želježić , Kopjar, & Peraica, 2006;Kamp, Eisenbrand, Schlatter, Würth, & Janzowski, 2005;Liu et al, 2012). Similarly, some authors have proposed that oxidative stress may be the underlying mechanism of FB 1 genotoxicity Mary, Theumer, Arias, & Rubinstein, 2012;Mobio et al, 2000Mobio et al, , 2003, while others have failed to find an increase in ROS production together with DNA damage after FB 1 exposure, suggesting oxidative stress is not involved in FB 1 -induced DNA damage (Galvano et al, 2002).…”

Low doses of ochratoxin A induce micronucleus formation and delay DNA repair in human lymphocytes

“…Although the mechanism that leads to OTA genotoxicity, as measured through the comet assay, is not fully understood, evidence from in vitro (with different cell types) and in vivo studies suggests the role of oxidative stress (Ali et al, 2014;Arbillaga et al, 2007;Aydin et al, 2014;Hadjeba-Medjdoub et al, 2012;Hibi et al, 2013aHibi et al, , 2013bLebrun and Föllmann, 2002;Yang et al, 2014). It has also been reported that OTA biotransformation produces metabolites that are more genotoxic than the original toxin.…”