Jong‐Heum Park1, Dipti Mangal2, Kirk A. Tacka3, Amy Quinn3, Ronald G. Harvey4, Ian A. Blair3,2, T.M. Penning3,2
1Center of Excellence in Environmental Toxicology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6084, USA
2Center for Cancer Pharmacology, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6084; and
3*Center of Excellence in Environmental Toxicology and
4The Ben May Institute for Cancer Research, University of Chicago, Chicago, IL 60637
Tóm tắt
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-9α,10β-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.
