Activating liver carcinogens in yeast by expressing CYP450 polymorphisms

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DESCRIPTION (provided by applicant): Hepatocellular carcinoma (HCC) has been correlated with specific p53 mutations and exposure to aflatoxin B1 (AFB1). Individual response to liver carcinogens shows great variability. High risk factors include chronic infection with hepatitis B (HBV) and C viruses (HCV), while additional risk factors include exposure to tobacco smoke carcinogens and polymorphisms in DNA repair genes. Cytochrome P450 genes, such as CYP1A1, CYP1A2, CYP3A4, and CYP3A5 encode proteins that activate potent liver carcinogens into genotoxic epoxides. Heterocyclic aromatic amines (HAs) require additional activation by N,O- acetyltransferase (NAT2). Specific cytochrome P450 polymorphisms and NAT2 polymorphisms contribute to risk of specific cancers, such as breast, pancreatic and colon. However, determining the risk of CYP450 and NAT polymorphisms in HCC is complicated by many modifying factors that can lead to detoxification of metabolites. To determine whether CYP450 polymorphisms per se are sufficient to increase the genotoxicity of carcinogens, we have expressed the CYP450 genes in Saccharomyces cerevisiae (yeast), which lacks similar detoxification enzymes. We previously observed that expression of specific CYP450 genes in yeast is sufficient to stimulate carcinogen-associated mutation and recombination, the transcriptional induction of DNA repair genes after AFB1 exposure, and AFB1-associated activation of checkpoint protein Rad53 (CHK2). We propose to further screen CYP1A1, CYP1A2, CYP3A4 and CYP3A5 polymorphisms that are associated with increased cancer risk. In the first specific aim, we will determine whether a subset of CYP1A1 and CYP1A2 polymorphisms affect frequencies of genetic recombination and mutation and the DNA damage response to AFB1 and liver carcinogens. In the second specific aim, we will determine whether CYP1A2 and NAT2 polymorphisms affect the metabolic activation of HAs in yeast. In the third specific aim, we will develop an expression system for CYP3A4 and CYP3A5 polymorphisms that will enable us to metabolic activation of carcinogens. These studies in yeast will thus provide a new strategy for determining the potential risk of cytochrome P450 polymorphisms in liver cancer, and serve as templates for additional experiments that can be conducted in higher eukaryotic organisms.