Causes and consequences of replication stress induced by Ochratoxin A and its role in Ochratoxin A carcinogenicity

Ochratoxin A (OTA), a mycotoxin frequently found in food, is considered a significant health concern due to its potent renal carcinogenicity. Since the MoA underlying OTA tumorigenicity remains largely unknown and recent studies provided evidence for a weak mutagenicity of OTA in rat kidney (Kuroda et al. 2014), EFSA in its 2020 risk evaluation applied the MOE approach (Schrenk et al. 2020). However, the panel emphasized that the evaluation involves considerable uncertainties, given the inconclusive evidence for a direct interaction of OTA with the DNA (Schrenk et al. 2020). To support human risk assessment, the present study aimed to further elucidate the primary molecular events involved in OTA carcinogenicity. Results obtained in vitro revealed that OTA significantly delays replication fork progression in HK-2 cells through a global mechanism, but fails to elicit a full DDR capable of inducing a checkpoint-mediated cell cycle arrest in intra S or G2/M. As a consequence, cells with under-replicated or damaged chromatin may proceed into mitosis, jeopardizing cell division and in turn genomic stability. Effects of OTA on DNA replication were evident after only 1 h of exposure and at concentrations relevant to tumorigenesis by OTA in vivo (Rached et al. 2007). These findings support the hypothesis that OTA-induced mitotic aberrations, genetic damage and mutagenicity may derive from unresolved replication stress (Schrenk et al. 2020). IPOND and extended chromatin fiber analyses indicated that alterations in the chromatin environment may be linked to OTA-induced replication stress, as demonstrated by the absence of HDAC1 and reduced H3K9ac on nascent chromatin following 1 h OTA exposure, although a causal link remains to be experimentally established. The molecular mechanism underlying OTA-induced replication fork slowing is therefore still unclear. Dose-response characterization of events considered critical to OTA tumorigenicity in kidneys of male F344/DuCrl rats exposed to OTA (21, 35, 50, 70 and 210 µg/kg bw) for 90 days (5 days/week), revealed a dose-dependent increase in karyomegaly (suggestive of failed cytokinesis) and enhanced cell proliferation (indicated by PCNA) in the OSOM of rats exposed to OTA doses ≥ 35 µg OTA/kg bw and ≥ 50 µg OTA/kg bw (based on statistical significance), respectively. Likewise, a statistically significant upregulation of Avil and Hist1h1t/H1f6 was observed in rat kidney at OTA doses ≥ 35 µg OTA/kg bw. In contrast, significantly elevated levels of yH2AX indicative of DNA DSBs, were only observed in the OSOM of rats treated with 210 µg OTA/kg bw, but not at the lower carcinogenic dose of 70 µg OTA/kg bw, suggesting that DNA damage is unlikely to be an essential, primary event in OTA carcinogenicity and may rather occur secondary to mitotic disruption and cell proliferation, which are proposed to act together to promote genomic instability and tumor formation by OTA. Taken together, results of this study point toward replication stress as a critical early event in OTA genotoxicity, support a non-DNA reactive MoA of OTA and identify enhanced cell proliferation as the most sensitive readout of OTA toxicity in rat kidney, with a BMDL10 of 2.85 µg OTA/kg bw/day, potentially serving as a suitable POD for human risk assessment.