Assessment of ochratoxin A exposure-related micronucleus formation in rat proximal tubular epithelial cells and expression profiling of related genes

Abstract

Ochratoxin A (OTA) is a renal carcinogen that characteristically induces karyomegaly as a precursor lesion for carcinogenesis in the proximal tubular cells of the outer stripe of the outer medulla (OSOM) in rats; however, the carcinogenic mechanism remains unknown. To elucidate the possible involvement of micronucleus (MN) formation on renal carcinogenesis by OTA, we performed in vitro MN assay using rat proximal tubular NRK-52E cells. Next, we administered carcinogenic doses of 1,2,3-trichloropropane (TCP), as a karyomegaly-inducing renal carcinogen, in addition to OTA, and 3-chloropropane-1,2-diol (3-MCPD), as a karyomegaly-non-inducing renal carcinogen, for 4 or 13 weeks to rats, and performed RNA-Seq analysis in the OSOM in 13-week cases of OTA and 3-MCPD. We also assessed the number of γH2AX⁺ MNs in the OSOM in all groups. In vitro assay revealed increased numbers of MNs from OTA concentrations without showing cytotoxicity. Enrichment analysis for genes showing altered expression specific to OTA revealed gene ontology terms such as spindle formation associated with chromosomal instability, and qRT-PCR validation analysis revealed upregulation of genes contributing to DNA double-strand break repair, spindle formation, and cell proliferation and cell cycle regulation, in an OTA-specific manner. Furthermore, TCP and OTA groups showed an increasing trend in the number of γH2AX⁺ MNs. These results suggest that OTA induces MNs in the OSOM tubular cells in association with karyomegaly formation and induces DNA double-strand breaks via disruption of mitotic regulation.