The purpose of this project is to provide an understanding of DNA damage and mutagenicity of ochratoxin A (OTA). OTA is a mycotoxin produced by several species of Aspergillus and Penicillium fungi. It is widely found in food products, particularly in northern climates of Europe and North America. In humans, OTA has been implicated in a fatal kidney disease in which patients suffer from urinary tract tumors. Studies on the effects of OTA in mammalian tissue have shown that it facilitates DNA cleavage and DNA adduction. Although this data provides a basis for its genotoxicity, the mode of DNA damage and mutagenicity by OTA is currently not known. In the present proposal, experiments are designed to test the hypothesis that DNA damage and mutagenicity by OTA is initiated through its oxidation to a reactive guinone intermediate (OTQ). This pathway also generates reactive oxygen species that facilitate DNA cleavage. Utilizing an Fe-porphyrin/hydrogen peroxide oxidation system, copper(II) and liver microsomes to model the metabolic activation of OTA, the ability of OTA/OTQ to mediate DNA alkylation will be characterized utilizing duplex DNAs and individual DNA nucleosides. These experiments are anticipated to provide critical data on the nature of DNA adducts mediated by OTA upon oxidation. The specificity of DNA cleavage by OTA will be characterized using human genomic DNA as substrate, and the data obtained will be compared to the damage induced by OTA in vivo in the DNA of intact cultured normal human fibroblasts. The mutagenicity of OTA-exposed DNA in human cells will then be assessed using the mutation-reporting shuttle plasmid pSP189. The damaged pSP189 plasmid will be transfected into human Ad293 cells, and then the DNA will be recovered and the OTA-induced mutation spectrum will be characterized. These experiments are expected to provide new information of the specificity of DNA damage caused by OTA and the mutations that result from that damage. Completion of the studies outlined in this proposal will provide the first comprehensive analysis of the interaction of OTA with DNA at the molecular level, and will demonstrate the mutation spectrum resulting from OTA-mediated DNA damage.
