Replication stress: an early key event in ochratoxin a genotoxicity?

Ochratoxin A (OTA), a widespread food contaminant and potent renal carcinogen in rodents, is weakly genotoxic in mammalian cells. The mechanisms underlying OTA-induced genetic damage are still poorly understood. In its recent risk assessment, the European food safety authority (EFSA) considered that the specific spectrum of mutations and chromosomal damage induced by OTA may derive from unresolved replication stress. The aim of the present work was to experimentally test the hypothesis that OTA interferes with DNA replication and to characterize the cellular response to OTA-mediated replication stress. Using the DNA fiber assay to study replication fork dynamics at single molecule resolution, a small but statistically significant global delay in replication fork progression was observed in human kidney (HK-2) cells exposed to OTA at ≥ 10 µM. OTA-mediated interference with DNA replication was confirmed by a concentration-related decrease in incorporation of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) into newly replicating DNA in HK-2 cells arrested in late G(1)/S via double thymidine block and treated with OTA during S phase. Western blot and immunofluorescence analyses revealed a significant concentration-related increase in γH2AX in cells exposed to OTA. Co-localization of γH2AX foci with 5-chloro-2'-deoxyuridine (CldU) incorporated into cells during S phase and increased γH2AX labeling along newly replicating chromatin fibers visualized using the extended chromatin fiber assay support a replication-coupled mechanism of OTA-induced DNA damage. Experiments with cells synchronized in late G(1)/S or late G(2) demonstrated that exposure of cells to OTA during S phase, but not mitosis, leads to a significant concentration-related increase in H2AX, providing further evidence that OTA may act primarily during S phase of the cell cycle. However, OTA did not appear to efficiently activate ATR-Chk1 and ATM-Chk2 DNA damage response pathways, suggesting that cells with under-replicated DNA or unresolved DNA damage may escape checkpoint control and may continue into mitosis, with potentially deleterious consequences for genomic integrity. Overall, results from this study provide first experimental evidence for perturbation of the S phase replisome machinery by OTA and point toward replication stress as an early key event in OTA genotoxicity.