DNA damage by ochratoxin A in rat kidney assessed by the alkaline comet assay

Abstract

There are few studies of ochratoxin A (OTA) genotoxicity in experimental animals and the results obtained with cell cultures are inconsistent, although the carcinogenic potential of OTA for the kidney of experimental animals has been well established. We studied the genotoxic potential of OTA in the kidney of adult female Wistar rats (5 in each group) treated intraperitoneally with OTA (0.5 mg kg body weight -1 day -1 for 7, 14, and 21 days) measuring DNA mobility on agarose gel stained with ethidium-bromide using standard alkaline single-cell gel electrophoresis (comet assay). Negative control animals were treated with solvent (Tris buffer, 1.0 mg/kg) and positive control animals were treated with methyl methanesulfonate (40 mg/ kg) according to the same schedule. OTA concentrations in plasma and kidney homogenates in 7-, 14-, and 21-day treated animals were 4.86 ± 0.53, 7.52 ± 3.32, 7.85 ± 2.24 µg/mL, and 0.87 ± 0.09, 0.99 ± 0.06, 1.09 ± 0.15 µg/g, respectively. In all OTA-treated groups, the tail length, tail intensity, and tail moment in kidney tissue were significantly higher than in controls (P < 0.05). The tail length and tail moment were higher after 14 days than after 7 days of treatment (P < 0.05), and still higher after 21 days (P < 0.05). The highest tail intensity was observed in animals treated for 21 days, and it differed significantly from animals treated for 7 and 14 days (P < 0.05). OTA concentrations in plasma and kidney tissue increased steadily and OTA concentration in kidney tissue strongly correlated with tail intensity and tail moment values. These results confirm the genotoxic potential of OTA, and show that the severity of DNA lesions in kidney correlates with OTA concentration. CorrespondenceA.-M. Domijan

Possible Carcinogenic Mechanisms Underlying Renal Carcinogens in Food

“…In addition, immunohistochemical analysis clearly demonstrated the site-specific localization of γ-H2AX-positive cells in the OS tubular epithelium of OM 73) . Comet assays using OMs of gpt delta rats treated with OTA yielded positive results 73) that were suggestive of DNA strand breaks, as was previously reported [85][86][87][88] . Concerning the detrimental effects of OTA on steric DNA structures, the formation of OTA-specific DNA adducts was observed in human tissues by 32 P postlabeling methods, and was confirmed by mass spectrum analysis in animal tissues [89][90][91][92] .…”

Quercetin protects human peripheral blood mononuclear cells from OTA‐induced oxidative stress, genotoxicity, and inflammation

“…OTA was added to the cells at a final concentration of 10–50 μM/mL dissolved in 50% (v/v) ethanol (solvent concentration not exceeding 0.1%) and treated for 24 and 48 h. Based on the dose response, effective OTA concentration were fixed at 20 µM and effective treatment time was fixed at 24 h, which was followed for further studies. The concentrations of OTA were chosen (10–50 μM) based on previous reports, which reflect concentrations found in plasma and other tissues in rats after OTA was fed orally at 0.5 mg/kg (Zeljezic et al, ). Quercetin dissolved in dimethylsulfoxide (DMSO) was added to the cells to reach a final concentration of 5 and 10 μM (final solvent concentration did not exceed 0.1% v/v) to determine pre and post treatment efficacy.…”

Molecular mechanisms underlying ochratoxin A-induced genotoxicity: global gene expression analysis suggests induction of DNA double-strand breaks and cell cycle progression

“…Additional function that OTA inhibited topoisomerase II activity (Cosimi et al, 2009) might contribute to the disorder of the normal DNA repair. Thus, although the mechanisms underlying OTA-induced DNA strand breaks including DSBs remain uncertain, positive results in the comet assay (Zeljezić et al, 2006;Klarić et al, 2010) support the hypothesis that OTA has the potential to cause DNA strand breaks.…”

“…In addition to the fact that OTA-modified bases lead to the formation of apurinic sites (Cavin et al, 2007), the positive outcome in the alkaline comet assay allowed us to speculate that OTA induces DNA strand breaks followed by deletion mutations (Zeljezić et al, 2006). Otherwise, although there were no changes in 8-hydroxydeoxyguanine (8-OHdG) levels in kidney DNA extracted from the OM in the above-mentioned study, several lines of evidence, such as the generation of reactive oxygen species (ROS) (Schaaf et al, 2002), the inhibition of Nrf-2 activity (Cavin et al, 2007), and the positive results in formamidopyrimidine-DNA glycosylase (Fpg)-modified comet assays (Kamp et al, 2005), indicate the possibility that oxidative DNA damage triggers these mutations.…”