Aldo-Keto Reductases and PAH Metabolism/Activation

醛酮还原酶和 PAH 代谢/激活

基本信息

批准号：
8627119
负责人：
Trevor M Penning
金额：
$ 21.68万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
1986
资助国家：
美国
起止时间：
1986-03-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8627119
关键词：
8-Oxo-2&apos -Deoxyguanosine A549 AKR1C1 ARNT gene Affect Aromatic Polycyclic Hydrocarbons Aryl Hydrocarbon Receptor Base Excision Repairs Benzo(a)pyrene Biological Assay CYP1A1 gene Carcinogens Catechols Cell Line Cell Nucleus Cells Comet Assay Consumption Coupled DNA DNA Adducts DNA lesion Environmental Pollutants Enzymatic Biochemistry Enzymes Epithelial Epoxy Compounds Etiology Exposure to Funding Gene Expression Gene Expression Profile Genes Genetic Genomics Genotype Glycols Grant Guanine Health Human Hydrogen Peroxide In Vitro Individual International Agency for Research on Cancer Lead Lesion Lung Lung Adenocarcinoma Malignant Neoplasms Malignant Squamous Cell Neoplasm Malignant neoplasm of lung Measurement Measures Mediating Metabolic Metabolic Activation Metabolism Molecular Mus Mutation NAD(P)H dehydrogenase (quinone) 1, human NQO1 gene Natural regeneration Non-Small-Cell Lung Carcinoma Oral Oxidation-Reduction Oxidoreductase Oxygen Pathway interactions Phase Phenotype Play Predisposition Production Protein Isoforms Quinone Reductases Quinones RNA Rattus Reactive Oxygen Species Recombinants Reporting Risk Assessment Role Smoker Specificity Superoxides Testing Time Tobacco Tobacco smoke Variant adduct aryl hydrocarbon receptor ligand benzo(a)pyrene-DNA adduct cancer cell cancer initiation cigarette smoke-induced cigarette smoking improved lung carcinogenesis oral dysplasia oxidative DNA damage repair enzyme response stable isotope tobacco exposure

项目摘要

DESCRIPTION (provided by applicant): Human Aldo-Keto Reductases (AKR1A1, AKR1C1-AKR1C4) catalyze the activation of polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols to yield catechols which autooxidize to electrophilic and redox-active PAH o-quinones. In human lung adenocarcinoma (A549) cells this pathway results in the production of reactive oxygen species (ROS) and formation of the mutagenic lesion 8-oxo-2'-deoxyguanosine (8-oxo-dGuo). Thus, in addition to PAH diol-epoxide-DNA adducts, oxidative DNA lesions may contribute to lung cancer initiation. Recently, AKR1C1-1C3 and AKR1B10 genes were identified as tobacco exposure and response genes in human bronchial epithelial and buccal cells. Four aims will elaborate the role of the AKR pathway in cancer of the airway. In Aim#1, the phase I [quinone reductases (QR)] enzymes that redox-cycle the PAH o-quinones will be identified. Unexpectedly, AKRs have substantial PAH o-quinone reductase activity and we will compare their catalytic efficiency and ability to generate ROS with NQO1. The phase II enzymes that remove the PAH- catechols (COMT, SULTs and UGTs) and PAH o-quinones (GSTs) from this redox-cycle will also be identified in vitro. In Aim#2, the metabolic fate of the PAH o-quinone ([3H]-benzo[a]pyrene-(B[a]P)-7,8-dione) will be elucidated in human bronchoalveolar (H358), A549 and immortalized bronchial epithelial (HBEC-tk) cells that produce this metabolite. Catechol and o-quinone conjugates will be identified and enzymes responsible phenotyped using real-time qRT-PCR. Aims 1 and 2 will identify enzymes whose polymorphic variants may affect individual susceptibility to PAH exposure and response. Aim #3, will determine whether the aryl hydrocarbon receptor (AhR) delivers PAH o-quinones to the nucleus to cause oxidative DNA damage. Studies will be performed in murine Hepa1 cells and their genetic AhR and ARNT negative variants and in HBEC-tk cells in which either AhR or ARNT have been silenced with si-RNA. The formation of 8-oxo-dGuo will be measured by an immunoaffinity capture LC-MS stable isotope dilution assay. Aim#4 will determine whether cigarette smoke condensate alters the transcriptome in HBEC-tk cells and oral dsyplasia cells with consequential elevation of AKR expression and 8-oxo-dGuo formation upon exposure to B[a]P or B[a]P-7,8- trans-dihydrodiol. These studies will inform genotyping and validate genomic studies in lung and buccal cancer. They will demonstrate that by acting as cigarette smoke and response genes, AKRs increase oxidative DNA damage and the mutagenic burden.

描述（由申请人提供）：人醛酮还原酶（AKR1A1、AKR1C1-AKR1C4）催化多环芳烃（PAH）反式二氢二醇的活化，产生儿茶酚，儿茶酚自动氧化为亲电子且具有氧化还原活性的PAH邻醌。在人肺腺癌细胞 (A549) 细胞中，该途径导致活性氧 (ROS) 的产生并形成诱变病变 8-oxo-2'-脱氧鸟苷 (8-oxo-dGuo)。因此，除了 PAH 二醇-环氧化物-DNA 加合物外，氧化性 DNA 损伤也可能导致肺癌的发生。最近，AKR1C1-1C3和AKR1B10基因被鉴定为人支气管上皮和口腔细胞中的烟草暴露和反应基因。四个目标将详细阐述 AKR 通路在气道癌中的作用。在目标#1 中，将鉴定对 PAH 邻醌进行氧化还原循环的 I 相 [醌还原酶 (QR)] 酶。出乎意料的是，AKR 具有显着的 PAH 邻醌还原酶活性，我们将比较它们与 NQO1 的催化效率和产生 ROS 的能力。还将在体外鉴定从该氧化还原循环中去除 PAH-儿茶酚（COMT、SULT 和 UGT）和 PAH 邻醌（GST）的 II 相酶。在目标#2中，将阐明多环芳烃邻醌（[3H]-苯并[a]芘-(B[a]P)-7,8-二酮）在人支气管肺泡（H358）、A549中的代谢命运和产生这种代谢物的永生化支气管上皮（HBEC-tk）细胞。将使用实时 qRT-PCR 鉴定儿茶酚和邻醌缀合物，并对负责的酶进行表型分析。目标 1 和 2 将鉴定其多态性变异可能影响个体对 PAH 暴露和反应的易感性的酶。目标#3，将确定芳基碳氢化合物受体 (AhR) 是否将 PAH 邻醌传递到细胞核以引起氧化 DNA 损伤。研究将在小鼠 Hepa1 细胞及其遗传 AhR 和 ARNT 阴性变体以及 AhR 或 ARNT 已被 si-RNA 沉默的 HBEC-tk 细胞中进行。 8-oxo-dGuo 的形成将通过免疫亲和捕获 LC-MS 稳定同位素稀释测定来测量。目标 #4 将确定香烟烟雾冷凝物是否会改变 HBEC-tk 细胞和口腔发育不良细胞中的转录组，从而在接触 B[a]P 或 B[a]P-7 后导致 AKR 表达升高和 8-oxo-dGuo 形成,8-反式二氢二醇。这些研究将为肺癌和口腔癌的基因分型提供信息并验证基因组研究。他们将证明，通过充当香烟烟雾和反应基因，AKR 会增加氧化 DNA 损伤和诱变负担。