DIHYDRODIOL DEHYDROGENASE AND PAH METABOLISM/ACTIVATION

二氢二醇脱氢酶和 PAH 代谢/激活

• 批准号: 3178561
• 负责人: Trevor M Penning
• 金额: $ 20.92万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-03-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3178561
• 关键词: DNA damage; HTC cell; MCF7 cell; adduct; benzopyrenediol epoxide; benzopyrenes; biotransformation; carbopolycyclic compound; cell free system; chemical carcinogen; chemical carcinogenesis; complementary DNA; electron spin resonance spectroscopy; embryo /fetus cell /tissue; enzyme mechanism; epoxides; fibroblasts; free radical oxygen; hamsters; high performance liquid chromatography; hydrogen peroxide; laboratory rat; liver cells; mutagen testing; oxidation reduction reaction; oxidoreductase; quinones; superoxides; tissue /cell culture; transfection; transfection /expression vector

Dihydrodiol dehydrogenase (DD;EC 1.3.1.20) can suppress the formation of anti-diol epoxides of polycyclic aromatic hydrocarbons (PAH), which are ultimate carcinogens, by oxidizing their precursor transdihydrodiols to yield PAH o-quinones. For example, DD will catalyze the oxidation of trans-7,8-dihydroxy-7,8-dihydrobenzoa[a]pyrene (B[a]P-diol) to yield 7,8- dihydroxybenzo[a]pyrene which then autooxidizes to benzo[a]pyrene-7,8- dione (BPQ. BPQ and other PAH o-quinones produced by this enzyme form conjugates will cellular nucleophiles, enter le minus redox-cycles and generate presumptive superoxide anion and o-semiquinone radicals and are cytotoxic in hepatoma cells. BPQ can also form BPQ-deoxyguanosine (dG) adducts with DNA. Since PAH o-quinones may be cyto- and genotoxic, DD may initiate a new pathway of PAH metabolism and carcinogen activation. This pathway of PAH metabolism will be assessed in isolated rat hepatocytes and hamster embryo fibroblasts by measuring the conversion of [3H]-b[a]P-diol into BPQ and its conjugates. These metabolites will be identified by co-chromatography with authentic synthetic standard on RP-HPLC. To verify that DD catalyzes the formation of these metabolites studies will be replicated in the presence of selective inhibitors for this enzyme. To amplify this new pathway of PAH metabolism, mammalian expression vectors containing the cDNA for DD in the sense (pRcCMV/DD) and anti-sense (pRcCMV/DD anti-sense) direction, will be stably transfected into cells which metabolize PAH but display only basal levels of DD expression (Hep-G2 and MCF7 cells). As a result stable transfectants should divert [3H]-B[a]P-diol into 7,8- dihydroxybenzo[a]pyrene and BPQ. Metabolites that arise from the intermediate hydroquinone and quinone can then be identified. The metabolic fate of [1,3-3H-BPQ will also be elucidated in hepatoma cells and isolated rat hepatocytes. To determine if DD initiates a pathway of PAH activation, the formation of transient superoxide anion radicals during the enzyme catalyzed oxidation of PAH trans-dihydrodiols will be inferred by measuring oxygen uptake and H2O2 formation. Intermediate o-semiquinone radicals will be spin-trapped with 5,5-dimethyl-1-pyrroline N-oxide for ESR spectroscopy. o-Semiquinone radicals produced during the activation of PAH o-quinones by enzymatic le minus redox-cycling will be detected by spin- stabilization. The genotoxicity of BPQ will be measured by incubating hepatoma cells with [3H]-BPQ and detecting BPQ-dG adducts. The genotoxicity of ten unlabeled PAH o-quinones generated by DD will be examined in cell free systems (calf thymus DNA in the presence and absence of le minus redox-cycling), and in hepatoma cells. The formation of covalent adducts will be measured by [32P]-post-labeling and oxidative DNA damage will be measured by detecting increases in 8-hydroxy-2'-dG levels. The mutagenicity of these PAH o-quinones will be assessed using Salmonella tester strains (Ta102 and TA104) which are sensitive to mutagens that cause oxidative DNA damage.

二氢二醇脱氢酶（DD; EC 1.3.1.20）可以抑制形成 多环芳烃的抗二醇环氧化物（PAH）， 最终致癌物，通过将其前体转二醇氧化为 产生pah o-quinones。 例如，DD将催化氧化 Trans-7,8-二羟基-7,8-二氢苯并[A] pyrene（B [a] p-Diol）产生7,8-- 二羟基苯甲[a] pyrene，然后自氧化为苯并[a] pyrene-7,8-- Dione（BPQ。BPQ和其他由这种酶形式产生的PAH O-Quinones 结合物将细胞亲核试剂，进入le氧化还原循环，并 产生推定的超氧化阴离子和O-链氨基氨基酮自由基，是 肝癌细胞中的细胞毒性。 BPQ还可以形成BPQ-脱氧鸟苷（DG） DNA加合物。 由于PAH O- Quinones可能是细胞和遗传毒性的DD 可以启动PAH代谢和致癌激活的新途径。 PAH代谢的这种途径将在孤立的大鼠中评估 肝细胞和仓鼠胚胎成纤维细胞通过测量转化率 [3H] -b [a] p-二醇及其偶联物。 这些代谢产物会 可以通过具有正宗合成标准的共染色体来识别 RP-HPLC。 为了验证DD催化这些代谢产物的形成 在存在选择性抑制剂的情况下，将重复研究 这种酶。 为了扩大这种新代谢的新途径，哺乳动物 在含义上包含DD cDNA的表达向量（PRCCMV/DD） 和反义（PRCCMV/DD反义）方向，将是稳定的 转染到代谢PAH但仅显示基础水平的细胞中 DD表达（HEP-G2和MCF7细胞）。 结果稳定 转染剂应将[3H] -b [A] p-Diol转移到7,8-- 二羟基苯甲[a] pyrene和bpq。 由 然后可以鉴定中间氢酮和喹酮。 这 [1,3-3H-BPQ的代谢命运也将在肝癌细胞中阐明 和孤立的大鼠肝细胞。 为了确定DD是否启动了PAH激活的途径，则形成 酶催化过程中瞬时超氧阴离子自由基 通过测量氧气将推断出PAH反二氢二醇的氧化 吸收和H2O2形成。 中间O-链氨基氨基酮自由基将是 用5,5-二甲基-1-吡咯氨酸N-氧化物进行自旋捕获，用于ESR光谱。 o激活PAH O-Quinones产生的O-链氨基氨基酮自由基 通过酶促氧化还原循环，将通过自旋检测 稳定。 BPQ的遗传毒性将通过孵化来测量 具有[3H] -BPQ并检测BPQ-DG加合物的肝癌细胞。 这 DD产生的十个未标记的PAH O-Quinones的遗传毒性将是 在无细胞系统中检查（在存在的小牛胸腺DNA和 缺乏LE减去氧化还原循环）和肝癌细胞。 形成 共价加合物将通过[32p]标记和氧化测量 DNA损伤将通过检测8-羟基-2'-DG的增加来衡量 水平。 这些PAH O- Quinones的诱变性将使用 沙门氏菌测试仪菌株（TA102和TA104），对 引起氧化DNA损伤的诱变剂。