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暴露于烟草烟雾致癌物和DNA修复基因多态性。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）需要通过N，O-乙酰转移酶（NAT 2）的额外活化。特定的细胞色素P450多态性和NAT 2多态性有助于特定癌症的风险，如乳腺癌，胰腺癌和结肠癌。然而，确定肝细胞癌中CYP 450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CYP 450多态性本身是否足以增加致癌物的遗传毒性，我们在缺乏类似解毒酶的酿酒酵母（酵母）中表达了CYP 450基因。我们先前观察到，酵母中特定CYP 450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相关的检查点蛋白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多态性的子集是否影响遗传重组和突变的频率以及对AFB 1和肝癌物质的DNA损伤反应。在第二个具体目标中，我们将确定CYP 1A 2和NAT 2多态性是否影响酵母中HA的代谢活化。在第三个具体的目标，我们将开发一个表达系统的CYP 3A 4和CYP 3A 5多态性，这将使我们能够代谢活化的致癌物质。因此，在酵母中的这些研究将为确定肝癌中细胞色素P450多态性的潜在风险提供一种新的策略，并作为可以在高等真核生物中进行的其他实验的模板。

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.