Detection of ochratoxin A-induced DNA damage in MDCK cells by alkaline single cell gel electrophoresis (comet assay)

Abstract

The mycotoxin ochratoxin A (OTA), a widespread contaminant of food and feedstuffs, is nephrotoxic, immunosuppressive and carcinogenic in domestic and laboratory animals. Additionally, it is suspected as being responsible for urinary tract tumours in patients suffering from Balkan endemic nephropathy. Moreover, evidence has accumulated that OTA is a genotoxic carcinogen, although the mechanism that results in DNA damage has not been fully resolved. In this study, the induction of DNA damage by OTA and the subsequent DNA repair was investigated by alkaline single cell gel electrophoresis (comet assay) in cells originally derived from the kidney, a target organ of OTA. With modifications of the method, the influence of OTA uptake into the cells and of DNA repair on the genotoxic effect of OTA should be investigated. In Madin-Darby canine kidney (MDCK) cells, OTA induced single-strand breaks in a concentration dependent manner. When an external metabolising enzyme system (S9-mix from rat liver) was added, this genotoxic effect was significantly stronger. By co-incubation with methotrexate or with the mycotoxin citrinin, a substrate of the organic anion transporter, the adverse effect of OTA was inhibited. When DNA repair was inhibited by addition of cytosine arabinoside and hydroxyurea, the tail length increased dramatically and all treated cells showed single-strand breaks. A further culture of the damaged cells in the absence of any supplement resulted in a complete repair of the DNA damage within 2 h. Adverse effects on the mechanisms of DNA repair, or exposure to OTA in periods of reduced DNA repair capacity may influence the genotoxic potency of OTA and have to be regarded as a further mechanism by which genotoxic effects of OTA can be performed.

Ochratoxin A induces oxidative DNA damage in liver and kidney after oral dosing to rats

“…Decreased concentrations of vitamin E in plasma of rats [30], and of glutathione in liver of mice, in primary hepatocytes and in CV-1 cells have also been reported after OTA treatment [22,36,37]. Moreover, OTA has been found to induce DNA strand breaks in different systems and to generate oxidized DNA bases in vitro [22,31,38,39].…”

Ochratoxin A Toxic Effect on Rat Kidneys and the Potential Protective Effect of Ginseng: Histopathologic, Histochemical, and Image Analysis Morphometric Studies

“…According to Wang and Groopman [36], mycotoxin produced chromosomal aberration, micronuclei, sister chromatid exchange and unscheduled DNA synthesis. Arlt et al [37], Lebrun and Fultmann [38] stated that ochratoxin induced DNA single strand breaks (SSB) and DNA adducts. In addition, Obrecht-Pflumio and Dirheimer [39] reported that ochratoxin is metabolized to genotoxic metabolites which interact with DNA may cause genetic damage in both target tissue independent of direct covalent binding to DNA.…”

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).…”