Molekulare Mechanismen der Toxizität und Kanzerogenität des Mykotoxins Ochratoxin A

霉菌毒素和食品污染物赭曲霉毒素A(OTA)是迄今为止研究过的最强的肾脏致癌物质之一。OTA导致肾脏肿瘤形成的分子事件仍然知之甚少，但越来越多的证据表明OTA不是一种诱变的、DNA反应的致癌物。我们实验室最近的工作为OTA致癌机制提供了重要的新证据，该机制涉及通过干扰有丝分裂中染色体分离和进展的关键调控因子来干扰有丝分裂，导致细胞分裂受阻或不对称，并伴随着非整倍体获得和后续肿瘤形成的风险增加。在OTA处理的细胞培养中，经常观察到密集和异常分离的中期染色体，这表明在OTA存在的情况下姐妹染色单体的过早分离可能是有丝分裂时细胞异常分裂和细胞死亡的初始触发因素。目前已知姐妹染色单体之间的凝聚力是由几种有丝分裂酶控制的，如CDK1、Aurora或Polo-like-kinase(PLK)，这些有丝分裂的关键调控因子的异常表达与恶性转化有关。OTA处理大鼠肾脏的基因表达分析支持CDK1、Aurora B和Plk1在OTA介导的鸵鸟毒素A毒性和致癌性3致癌的分子机制中的作用，体外初步结果也表明OTA确实可能改变了已知的参与染色体凝聚和分离的有丝分裂酶的活性。然而，为了全面了解OTA的毒性机制，OTA的时间序列和主要细胞靶点仍有待阐明。在这个项目中，我们的目标是通过结合现代细胞和分子生物学技术，如免疫印迹、免疫荧光/共聚焦显微镜和流式细胞仪分析，通过测定其细胞靶标(例如，组蛋白H1、组蛋白H3和粘附素)在体外和体内处理OTA的大鼠肾脏中的磷酸化状态，来确定OTA对CDK1/Cyclin B1、Plk1和Auora B活性的时间和浓度依赖性的影响。预计这项研究的结果不仅有助于阐明OTA致癌的机制，作为改进风险评估的基础，而且还将进一步加深我们对非诱变、非DNA活性化学物质致癌作用的理解。

The mycotoxin and food contaminant ochratoxin A (OTA) is one of the most potent renal carcinogens studied to date. The molecular events leading to renal tumor formation by OTA are still poorly understood, but increasing evidence suggests that OTA is not a mutagenic, DNA-reactive carcinogen. Recent work in our laboratory provide important new evidence in support of a mechanism of OTA carciogenicity involving disruption of mitosis through interference with key regulators of chromosome separation and progression through mitosis, resulting in blocked or asymmetric cell division, accompanied by an increased risk of aneuploidy acquisition and subsequent tumor formation. In OTA treated cell cultures, condensed and abnormally separated metaphase chromosomes were frequently observed, suggesting that premature separation of sister chromatids in the presence of OTA may provide the initial trigger for aberrant cell division and cell death at mitosis. It is now known that cohesion between sister chromatids is controlled by several mitotic kinases, e.g. Cdk1, Aurora, or Polo-like-kinases (Plk), and aberrant expression of these key regulators of mitosis has been associated with malignant transformation. Gene expression analyses in kidneys of OTA treated rats support a role of Cdk1, Aurora B and Plk1 in OTA mediated Molecular Mechanism of Ochratoxin A Toxicity and Carcinogenicity 3 carcinogenicity, and preliminary results in vitro also suggest that OTA may indeed alter the activity of mitotic kinases known to be involved in chromosome condensation and segregation. However, for a comprehensive understanding of the mechanism of OTA toxicity, the temporal sequence of events and primary cellular targets of OTA remain to be elucidated. Within this project, we aim to determine the time- and concentration-dependent effects of OTA on the acitivty of Cdk1/Cyclin B1, Plk1 and aurora B through determination of the phosphorylation status of their cellular targets (e.g. histon H1, histon H3 and cohesin) both in renal epithelial cells in vitro and in kidneys of OTA treated rats in vivo using a combination of modern cell & molecular biology techniques, such as immunoblotting, immunofluorescence/confocal microscopy and FACS analysis. It is expected that results from this study will not only help to elucidate the mechanism of OTA carcinogenicity as a basis for improved risk assessment, but will also further our understanding of the carcinogenic action of non-mutagenic, non-DNA reactive chemicals.