The Role of Replication Stress in Ochratoxin A Genotoxicity

真菌毒素赭曲霉毒素A(OTA)是迄今已知的最有效的肾脏致癌物质之一，但其致癌机制，特别是OTA遗传毒性的分子机制仍需解决，以提高对饮食暴露于OTA的人类健康风险的科学评估。在回顾有关OTA的遗传毒性和致癌性作用模式的现有信息后，欧洲食品安全局最近得出结论，OTA引起的特定突变和染色体损伤谱，包括染色体高度凝聚、异常分离的染色单体、多极有丝分裂纺锤体、内复制、多倍体和非整倍体，可能是由于未解决的复制应激所致。复制应激被广泛定义为复制分叉进程的减慢或停滞，越来越多地被认为是基因组不稳定和癌症的主要来源。重要的是，轻度复制应激减缓复制分叉进程可能逃脱检查点控制，从而允许DNA复制不足或未解决DNA损伤的细胞继续进入有丝分裂，导致有丝分裂缺陷和染色体分离错误。使用DNA纤维分析在单分子分辨率下的复制分叉动力学，我们最近发现OTA显著延缓了人类肾脏细胞的复制分叉进程。这些数据支持这样的假设，即DNA复制的扰动可能是OTA致癌的关键事件。然而，还需要进一步的工作来确定OTA诱导的复制应激的原因和后果。本项目的总体目标是通过进一步阐明OTA诱发遗传损伤的机制来解决食品中OTA风险评估中的不确定性，本项目旨在(1)研究OTA存在下复制叉慢的分子原因，(2)表征细胞对OTA介导的复制应激的反应，(3)从实验上支持复制应激、有丝分裂异常和OTA诱导的遗传损伤之间的机制联系，以及(4)确认在体外肾细胞中确定的关键分子和细胞事件在OTA致癌的大鼠肾脏靶点，特别强调剂量-反应特征作为风险评估的基础。

The mycotoxin ochratoxin A (OTA) is one of the most potent renal carcinogens known to date, but its mechanism of carcinogenicity and in particular the molecular mechanism underlying OTA genotoxicity still needs to be resolved to improve science-based assessment of human health risks associated with dietary exposure to OTA. In reviewing the available information on the mode of action of OTA genotoxicity and carcinogenicity, the European Food Safety Authority recently concluded that the specific spectrum of mutations and chromosomal damage induced by OTA, consisting of chromosome hypercondensation, abnormally separated chromatids, multipolar mitotic spindes, endoreduplication, polyploidy and aneuploidy, may arise from unresolved replication stress. Replication stress, which is broadly defined as the slowing or stalling of replication fork progression is increasingly recognized as a major source of genomic instability and cancer. Importantly, mild levels of replication stress that slow replication fork progression may escape checkpoint control, thus allowing cells with under-replicated DNA or unresolved DNA damage to continue into mitosis, leading to mitotic defects and chromosome segregation errors. Using the DNA fiber assay to analyze replication fork dynamics at single molecule resolution, we recently showed that OTA significantly delays replication fork progression in human kidney cells. These data support the hypothesis that perturbation of DNA replication may be a critical event in OTA carcinogenicity. However, further work is needed to define the cause and consequences of replication stress induced by OTA. With the overall objective of addressing uncertainties in the risk assessment of OTA in food by further elucidating the mechanisms underlying OTA–induced genetic damage, this project aims (1) to investigate the molecular cause for replication fork slowing in the presence of OTA, (2) to characterize the cellular response to OTA-mediated replication stress, (3) to experimentally support a mechanistic link between replication stress, mitotic aberrations and genetic damage induced by OTA , and (4) to confirm key molecular and cellular events identified in kidney cells in vitro at the target site of OTA carcinogenicity in rat kidney, with particular emphasis on dose-response characeterization as a basis for risk assessment.