Park Jong-Heum, Mangal Dipti, Tacka Kirk A, Quinn Amy M, Harvey Ronald G, Blair Ian A, Penning Trevor M
Center of Excellence in Environmental Toxicology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6084, USA.
Proc Natl Acad Sci U S A. 2008 May 13;105(19):6846-51. doi: 10.1073/pnas.0802776105. Epub 2008 May 12.
Polycyclic aromatic hydrocarbons (PAHs) are tobacco carcinogens implicated in the causation of human lung cancer. Metabolic activation is a key prerequisite for PAHs to cause their deleterious effects. Using human lung adenocarcinoma (A549) cells, we provide evidence for the metabolic activation of (+/-)-trans-7,8dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-trans-dihydrodiol) by aldo-keto reductases (AKRs) to yield benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione), a redox-active o-quinone. We show that B[a]P-7,8-trans-dihydrodiol (AKR substrate) and B[a]P-7,8-dione (AKR product) lead to the production of intracellular reactive oxygen species (ROS) (measured as an increase in dichlorofluorescin diacetate fluores-cence) and that similar changes were not observed with the regioisomer (+/-)-trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene or the diol-epoxide, (+/-)-anti-7,8-dihydroxy-9alpha,10beta-epoxy-7,8,9,10-tetrahydro-B[a]P. B[a]P-7,8-trans-dihydrodiol and B[a]P-7,8-dione also caused a decrease in glutathione levels and an increase in NADP(+)/NADPH ratios, with a concomitant increase in single-strand breaks (as measured by the comet assay) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo). The specificity of the comet assay was validated by coupling it to human 8-oxo-guanine glycosylase (hOGG1), which excises 8-oxo-Gua to yield single-strand breaks. The levels of 8-oxo-dGuo observed were confirmed by an immunoaffinity purification stable isotope dilution ([(15)N(5)]-8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry (LC-ESI/MRM/MS) assay. B[a]P-7,8-trans-dihydrodiol produced DNA strand breaks in the hOGG1-coupled comet assay as well as 8-oxo-dGuo (as measured by LC-ESI/MRM/MS) and was enhanced by a catechol O-methyl transferase (COMT) inhibitor, suggesting that COMT protects against o-quinone-mediated redox cycling. We conclude that activation of PAH-trans-dihydrodiols by AKRs in lung cells leads to ROS-mediated genotoxicity and contributes to lung carcinogenesis.
多环芳烃（PAHs）是烟草中的致癌物，与人类肺癌的发生有关。代谢活化是PAHs产生有害影响的关键前提条件。我们使用人肺腺癌（A549）细胞，提供了醛酮还原酶（AKRs）将（±）-反式-7,8-二羟基-7,8-二氢苯并[a]芘（B[a]P-7,8-反式-二氢二醇）代谢活化为苯并[a]芘-7,8-二酮（B[a]P-7,8-二酮）的证据，B[a]P-7,8-二酮是一种具有氧化还原活性的邻醌。我们发现B[a]P-7,8-反式-二氢二醇（AKR底物）和B[a]P-7,8-二酮（AKR产物）会导致细胞内活性氧（ROS）的产生（以二氯荧光素二乙酸酯荧光增加来衡量），并且区域异构体（±）-反式-4,5-二羟基-4,5-二氢苯并[a]芘或二醇环氧化物（±）-反式-7,8-二羟基-9α,10β-环氧-7,8,9,10-四氢-B[a]P未观察到类似变化。B[a]P-7,8-反式-二氢二醇和B[a]P-7,8-二酮还导致谷胱甘肽水平降低和NADP(+)/NADPH比值升高，同时单链断裂增加（通过彗星试验测量）以及7,8-二氢-8-氧代-2'-脱氧鸟苷（8-氧代-dGuo）增加。通过将彗星试验与人8-氧代鸟嘌呤糖基化酶（hOGG1）偶联来验证彗星试验的特异性，但hOGG1会切除8-氧代-Gua以产生单链断裂。通过免疫亲和纯化稳定同位素稀释（[(15)N(5)]-8-氧代-dGuo）液相色谱-电喷雾电离/多反应监测/质谱（LC-ESI/MRM/MS）测定法确认了所观察到的8-氧代-dGuo水平。在与hOGG1偶联的彗星试验中，B[a]P-7,8-反式-二氢二醇产生了DNA链断裂以及8-氧代-dGuo（通过LC-ESI/MRM/MS测量），并且儿茶酚-O-甲基转移酶（COMT）抑制剂增强了这种作用，这表明COMT可防止邻醌介导的氧化还原循环。我们得出结论，肺细胞中AKRs对PAH-反式-二氢二醇的活化导致ROS介导的遗传毒性，并促进肺癌发生。
