Pathways of PAH activation in human lung cells

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DESCRIPTION (provided by applicant): Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that have been upgraded to human carcinogens by the WHO, International Agency for Research on Cancer (IARC). PAH are airborne pollutants found in smoke from fossil-fuel combustion, car-exhaust, fine-particulate matter (PM2.5), and first and second hand tobacco smoke. Inhalation toxicity likely results in cancer of the lung and airway. PAH require metabolic activation to exert their deleterious effects, however, the major pathway of PAH activation in the carcinogen target cell, normal human bronchial epithelial cells (NHBE) is uncertain. Three pathways have been proposed: the radical cation pathway (P450-peroxidase dependent) which produces depurinating-DNA adducts; the diol-epoxide pathway (P450-dependent) which produces stable-DMA adducts; and the formation of reactive and redox-active o-quinones (aldo-keto reductase (AKR) dependent), which can give rise to covalent DMA adducts and oxidative damage of DMA. Based on the consistent over-expression of AKR isoforms and the loss of one allele of hOGG1 (human oxoguanine glycosylase the base excision repair enzyme specific for removal 8-oxo-dGuo from DMA) in patients with lung cancer, we hypothesize that AKRs activate PAHs in human bronchial epithelial (HBE) cells and this results in increased oxidative DNA damage. Four aims are proposed to test this hypothesis. In Aim #1, the metabolism of benzo[a]pyrene (BP) will be measured in parental H358 cells (transformed HBE cells) in the presence and absence of (P450 and AKR) induction and the levels of radical cation metabolites (BP-1,6-, 3,6- and 6,12-diones), diol-epoxide metabolites (BP-tetrols) and o-quinone metabolites (BP-7,8-dione) will be quantified and identified by LC- MS. In Aim#2, a stable isotope dilution method using [13C]-BP-metabolites as internal standards to monitor the BP-metabolome will be established. In Aim#3, NHBE cells immortalized with cdk4 and human telomerase will be used to monitor the BP-metabolome by LC-MS stable isotope dilution methodology in the presence and absence of P450 and AKR inducers. In Aim#4, the ability of BP, BP-7,8-dihydrodiol (AKR substrate) and BP-7,8-dione (AKR product) to cause AKR-dependent 8-oxo-dGuo formation will be examined in NHBE cells. These studies will identify major BP-metabolites in NHBE cells which could be used to biomonitor human PAH exposure, and validate candidate genes for genetic predisposition studies.