The Role of Replication Stress in Ochratoxin A Genotoxicity

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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.