Defining PAH-mediated carcinogenesis in lung: CYP metabolism and B[a]P transport

描述(申请人提供)：在这项拟议的研究中，苯并[a]芘(B[a]P)及其代谢物将被用作模型化合物，以确定多环芳烃在肺癌发生中的主要代谢途径。一般认为，细胞色素P4501A1，即细胞色素P4501A1，主要负责将B[a]P代谢成7，8-二羟基-9，10-环氧基-7，8，9，10-四氢苯并[a]芘(B[a]PDE)，最终形成DNA加合物，是致癌的反应性代谢产物，从而将细胞色素P4501A1与这种环境致癌物质的毒理效应联系起来。位于肿瘤抑制基因P53启动子区域的CpG岛上已检测到DNA加合物的形成。在所有癌症中，超过40%的癌症通过点突变使P53失活。然而，在对人肺泡细胞H358的初步研究中，发现未经处理的细胞色素P1A1/1B1缺失细胞的DNA加合物水平高于经2，3，7，8-四氯二苯并对二恶英(TCDD)诱导表达的H358细胞。这一数据与CYP1A1 KO小鼠研究的文献结果相关联，在这些研究中，确定了CYP1A1具有保护作用而不是毒性作用。这表明了B[a]P代谢过程中另一种CYP的代谢活性。其他几种CYP酶也能够催化这种代谢。初步数据还表明，TCDD可能对B[a]P反应性代谢物的运输有影响。众所周知，TCDD能够诱导ABCC药物转运蛋白家族的几个成员；因此，解释了为什么经TCDD处理的肺细胞中观察到较少的DNA加合物形成。这项建议有两个目的，将确定在肺中负责B[a]P代谢的主要CYP，并确定B[a]PDE转运体在肺细胞DNA加合物形成中的作用。稳定的同位素稀释LC-MRM/MS方法将用于DNA加合物的定量。DNA加合物水平将与RT-PCR和一种新的定量蛋白质组学方法SI LAC测定的mRNA和蛋白质表达相关。将研究包括1A1、1B1、1A2、2B6、2E1、2J2、3A4和3A5在内的Cyps。在药物转运体表达被siRNA抑制的肺细胞中，DNA加合物的形成也将被监测。这一领域的持续研究将提供令人振奋的新治疗策略(药物靶点)，可用于预防多环芳烃介导的DNA损伤。与公众健康相关：众所周知，吸烟会导致90%的肺癌，但只有10%的吸烟者会患上肺癌。对肺细胞中CYP代谢和B[a]PDE转运的研究将提供一个实验模型，以探索CYP代谢和转运的个体差异是否会导致这些现象。

DESCRIPTION (provided by applicant): In this proposed research, benzo[a]pyrene (B[a]P) and it's metabolites, will be used as model compounds to determine the major metabolic pathways of PAHs in lung carcinogenesis. It is generally accepted that cytochrome P4501A1, CYP1A1, is primarily responsible for the metabolism of B[a]P to 7,8-dihydroxy-9,10- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE), the ultimate DNA-adduct forming, carcinogenic reactive metabolite; thus connecting CYP1A1 to the toxicological effects of this environmental carcinogen. DNA-adduct formation has been detected at CpG islands located in the promoter region of the tumor suppressor p53. Inactivation of p53 by point mutations is seen in over 40% of all cancers. However, in preliminary research using H358, human bronchoalveolar lung cells, it was found that DNA-adduct levels were higher in non-treated CYP1A1/1B1 null cells compared to H358 cells in which CYP1A1 expression was induced with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). This data correlates with literature results of CYP1A1 KO mice stuides in which it was determined that CYP1A1 conferred a protective rather than a toxic effect. This is indicative of the metabolic activity of another CYP in the metabolism of B[a]P. Several other CYP enzymes are capable of catalyzing this metabolism. Preliminary data has also indicated that TCDD may have an effect on the transport of B[a]P-reactive metabolites. It is known that TCDD is able to induce several of the ABCC drug transporter family members; therefore, explaining why less DNA-adduct formation is observed in lung cells pretreated with TCDD . This proposal, which has two aims, will identify the predominate CYP responsible for B[a]P metabolism in the lung and determine the role of B[a]PDE transporters in DNA-adduct formation in lung cells. A stable isotope dilution LC-MRM/MS method, previously developed, will be used for DNA-adduct quantification. DNA-adduct levels will be correlated to mRNA and protein expression determined by RT-PCR and a novel quantitative proteomics method, SI LAC. CYPs including 1A1, 1B1, 1A2, 2B6, 2E1, 2J2, 3A4, and 3A5 will be investigated. DNA-adduct formation will also be monitored in lung cells in which drug transporter expression has been inhibited by siRNA. Continuing research in this area will provide exciting new therapeutic strategies (drug targets) that can be employed to prevent PAH-mediated DNA damage. Relevance to public health: Cigarette smoke is known to cause 90% of lung cancer, but only 10% of smokers develop lung cancer. Examination of alternative CYP metabolism and B[a]PDE transport in lung cells will provide an experimental model to explore whether inter-individual differences in CYP metabolism and transport contribute to these phenomenon.