Mally Angela, Pepe Gaetano, Ravoori Srivani, Fiore Mario, Gupta Ramesh C, Dekant Wolfgang, Mosesso Pasquale
Department of Toxicology, University of Würzburg, Germany.
Chem Res Toxicol. 2005 Aug;18(8):1253-61. doi: 10.1021/tx049650x.
Ochratoxin A (OTA) is a potent nephrotoxin and renal carcinogen in rats, but the mechanism of OTA tumorigenicity is unknown. Ochratoxin A has been shown to be negative in many genetic toxicology test in vitro. However, the potential of OTA to induce genotoxic effects has not been investigated in male rats, the most sensitive species for OTA-induced tumor formation. In this study, male F344 rats were repeatedly administered OTA (0, 250, 500, 1000, and 2000 microg/kg of body wt) or the non-chlorinated analogue ochratoxin B (OTB; 2000 microg/kg of body wt) for 2 weeks (5 days/week), and DNA breakage was analyzed in target and nontarget tissues using the comet assay both in the absence and presence of formamidopyrimidine-DNA (Fpg) glycosylase. Potential DNA-adduct formation was also analyzed in the target organ kidney by 32P-postlabeling using two different solvent systems. DNA-strand breaks were evident in liver, kidney, and spleen of animals treated with OTA, and a similar degree of DNA damage was observed in rats treated with OTB, despite the lower toxicity of OTB. Moreover, the presence of DNA damage did not correlate with histopathological alterations, which were evident in the kidney but not in the liver. In liver and kidney, the extent of DNA damage was further enhanced in the presence of Fpg glycosylase, which is known to convert oxidative DNA damage into strand breaks, suggesting the presence of oxidative DNA damage. Oxidative DNA damage as a mechanism of OTA-dependent DNA damage is consistent with the absence of lipophilic DNA adducts as assessed by 32P-postlabeling analysis. No spots indicative of OTA-related DNA adducts were observed in kidney DNA extracted from OTA-treated animals by 32P-postlabeling analysis, despite the use of synthetic standard for postulated adducts. A small, but not significant, increase in the incidence of chromosomal aberrations (essentially chromatid and chromosome-type deletions) was observed in splenocytes from rats treated with OTA in vivo and subsequently cultured in vitro to express chromosomal damage. These aberrations are also compatible with oxidative DNA lesions since they are not typically caused by chemical carcinogens which form covalent DNA adducts. Together, with the lack of evidence for formation of lipophilic DNA adducts as assessed by postlabeling, these data suggest that OTA may cause genetic damage in both target and nontarget tissues independent of direct covalent binding to DNA.
赭曲霉毒素A（OTA）是一种对大鼠有强效的肾毒素和肾致癌物，但其致瘤机制尚不清楚。赭曲霉毒素A在许多体外遗传毒理学试验中呈阴性。然而，OTA诱导遗传毒性效应的潜力在雄性大鼠中尚未得到研究，而雄性大鼠是OTA诱导肿瘤形成最敏感的物种。在本研究中，雄性F344大鼠连续2周（每周5天）重复给予OTA（0、250、500、1000和2000微克/千克体重）或非氯化类似物赭曲霉毒素B（OTB；2000微克/千克体重），并在有无甲酰胺嘧啶-DNA（Fpg）糖基化酶的情况下，使用彗星试验分析靶组织和非靶组织中的DNA断裂情况。还通过使用两种不同溶剂系统的32P后标记法分析靶器官肾脏中潜在的DNA加合物形成情况。在接受OTA处理的动物的肝脏、肾脏和脾脏中明显存在DNA链断裂，在用OTB处理的大鼠中也观察到了类似程度的DNA损伤，尽管OTB的毒性较低。此外，DNA损伤的存在与组织病理学改变不相关，组织病理学改变在肾脏中明显，但在肝脏中不明显。在肝脏和肾脏中，在Fpg糖基化酶存在的情况下，DNA损伤程度进一步增强，已知Fpg糖基化酶可将氧化性DNA损伤转化为链断裂，这表明存在氧化性DNA损伤。氧化DNA损伤作为OTA依赖性DNA损伤的一种机制，与通过32P后标记分析评估的缺乏亲脂性DNA加合物是一致的。通过32P后标记分析，在从接受OTA处理的动物提取的肾脏DNA中未观察到指示OTA相关DNA加合物的斑点，尽管使用了假定加合物的合成标准品。在用OTA体内处理并随后在体外培养以表达染色体损伤的大鼠的脾细胞中，观察到染色体畸变（主要是染色单体和染色体型缺失）的发生率有小幅但不显著的增加。这些畸变也与氧化性DNA损伤相符，因为它们通常不是由形成共价DNA加合物的化学致癌物引起的。综合来看，以及通过后标记评估缺乏亲脂性DNA加合物形成的证据，这些数据表明OTA可能在靶组织和非靶组织中引起遗传损伤，而与直接与DNA的共价结合无关。
