Ochratoxin A (OTA) is a naturally occurring chlorophenolic mycotoxin that contaminants a wide range of food products and poses a human cancer threat. Studies on the effects of OTA in mammalian tissue have shown that it causes deletion mutations through the induction of double-strand DNA breaks. The toxin also undergoes metabolism to generate reactive intermediates that covalently attach to DNA to generate DNA adducts that serve as biomarkers for OTA exposure. Because DNA adduct formation is strongly correlated with mutagenicity and carcinogenicity, OTA-mediated DNA adduction may be at the root of its carcinogenic potential. Our studies have shown that OTA, like other chlorinated phenols, undergoes metabolism to generate radical species that attach to the C8-position of 2′-deoxyguanosine (dG) to form carbon-linked (C-linked) and oxygen-linked (O-linked) C8-dG adducts. To gain an understanding of the biological impact of such DNA adducts, model C- and O-linked C8-dG adducts have been incorporated site-specifically into DNA substrates and studied using primer-elongation assays with DNA polymerase enzymes. Our studies demonstrate that bulky C-linked and O-linked C8-dG lesions structurally related to the authentic OTA C8-dG adducts are strong blocks of DNA replication. The implications of these model studies to OTA-mediated mutagenicity will be discussed. C-Linked C8-dG adducts also exhibit fluorescent properties and act as effective G mimics in antiparallel G-quadruplex structures when placed in <i>syn</i>-G positions. An aptamer that binds OTA selectively folds into an antiparallel G-quadruplex. Our studies demonstrate how emissive C8-dG adducts can serve as probes for OTA detection. Selective placement of C-linked C8-dG adducts within the OTA aptamer can enhance aptamer affinity for the toxin and provide a fluorescent response to OTA binding.

DNA Adduction by ochratoxin A: Insight for mechanism of action and aptasensor development for mycotoxin detection

251st National Meeting (2016)

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