Activating liver carcinogens in yeast by expressing CYP450 polymorphisms

描述（由申请人提供）：肝细胞癌（HCC）与特定的p53突变和暴露于黄曲霉毒素B1（AFB 1）相关。个体对肝脏致癌物的反应有很大的差异。高风险因素包括慢性感染B型肝炎（HBV）和丙型肝炎病毒（HCV），而其他风险因素包括接触烟草烟雾。细胞色素P450基因，如CYP 1A 1、CYP 1A 2、CYP 3A 4和CYP 3A 5编码的蛋白质可将强效肝致癌物活化为遗传毒性环氧化物。杂环芳香胺（HA）需要额外的N，O-乙酰转移酶（NAT 2）的激活。特异性细胞色素P450多态性和NAT 2多态性 导致患乳腺癌、胰腺癌和结肠癌等特定癌症的风险。然而，确定HCC中P450和NAT多态性的风险是复杂的，许多修饰因素可以导致代谢物的解毒。为了确定P450多态性本身是否足以增加致癌物的遗传毒性，我们在缺乏解毒酶的酿酒酵母（酵母）中表达了P450基因。我们先前观察到，酵母中特异性P450基因的表达足以刺激致癌物相关的突变和重组，AFB 1暴露后DNA修复基因的转录诱导，以及AFB 1相关的检查点基因Rad 53（CHK 2）的激活。我们建议进一步筛选与癌症风险增加相关的CYP 1A 1、CYP 1A 2、CYP 3A 4和CYP 3A 5多态性。在第一个具体目标中，我们将确定CYP 1A 1和CYP 1A 2多态性是否影响基因重组和突变的频率以及对AFB 1和肝脏的DNA损伤反应 致癌物质。在第二个具体的目标，我们将确定是否CYP 1A 2和NAT 2 多态性影响酵母中HA的代谢活化。在第三个具体目标中，我们将 在酵母中开发CYP 3A 4和CYP 3A 5的表达系统， CYP 3A 3和CYP 3A 5多态性介导的致癌物代谢活化。这些 因此，研究将为确定细胞色素P450的潜在风险提供新的策略。 肝癌的基因多态性最终，这些信息将帮助公共卫生从业人员， 临床医生确定哪些人患肝癌的风险最高，并指导预防 和早期发现的努力。

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. 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 P450 and NAT polymorphisms in HCC is complicated by many modifying factors that can lead to detoxification of metabolites. To determine whether P450 polymorphisms per se are sufficient to increase the genotoxicity of carcinogens, we have expressed the P450 genes in Saccharomyces cerevisiae (yeast), which lacks the detoxification enzymes. We previously observed that expression of specific P450 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 the checkpoint gene 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 in yeast that will enable us to detect metabolic activation of carcinogens mediated by CYP3A3 and CYP3A5 polymorphisms. These studies will thus provide a new strategy for determining the potential risk of cytochrome P450 polymorphisms in liver cancer. Ultimately, the information will aid public health practitioners and clinicians to identify which individuals are at highest risk for liver cancer, and guide prevention and early detection efforts.