Polycyclic aromatic hydrocarbons (PAH) are tobacco carcinogens implicated in the causation of human lung cancer. Human aldo-keto reductases (AKRs) convert PAH trans-dihydrodiols to highly reactive and redoxactive PAH-o-quinones which may contribute to the carcinogenic process. AKR1C isoforms that may be involved are highly overexpressed in human lung adenocarcinoma cells (A-549) and in patients with non-small cell lung carcinoma. Whether the AKR pathway can generate PAH o-quinones, reactive oxygen species (ROS), DNA-lesions, and mutate the tumor suppressor gene p53 in human lung cells is unknown.
Aim # 1 will determine whether human cells that overexpress AKR1C isoforms produce PAH o-quinones and ROS using LC/MS and EPR detection methods, respectively. Each cell-line will be incubated with a trans-dihydrodiol precursor that is preferentially oxidized by the AKR overexpressed, e.g., MCF-7-AKR 1C9 transfectants, lung adenocarcinoma cells (A-549) and H-358-AKR1C2 transfectants (human bronchoalveolar cells) will be exposed to benzo[g]chrysene(B[g]C)-11,12-diol (fjord-region), dimethylbenz[a]anthracene(DMBA)-3,4-diol (methylated-bay-region), and benzo[a]pyrene(BP)-7,8-diol (bay-region), respectively. Where tumor potency is often fjord region > methylated-bay-region > bay-region PAH.
Aim # 2, will determine whether these cells produce DNA lesions following exposure to these trans-dihydrodiols. Rapid preliminary analysis will use [32p]-post-labeling, an aldehyde-reactive probe and EC-HPLC methods to detect stable adducts, abasic sites, and 8-oxo-2-deoxyguanosine (8-oxo-dGuo), respectively. LC/MS/MS methods with stable isotope dilution will be ultimately used to quantify stable and depurinating PAH o-quinone-DNA adducts and 8-oxo-dGuo using [15N5]-stable analogs.
Aim # 3 will determine whether functional expression of the aryl hydrocarbon receptor (AhR) is required to translocate PAH o-quinones to the nucleus to cause DNA-lesions. Human lung AhR +ive and defective cells will be exposed to B[g]C-11,12-dione, DMBA-3,4-dione and BP-7,8-dione and levels of 8-oxo-dGuo will be measured as a marker of PAH o-quinone DNA lesions.
Aim # 4 will adapt an in vitro yeast reporter gene system that scores the transcriptional competency of p53 to determine whether B[g]C-11,12- dione, DMBA-3,4-dione and BP-7,8-dione or the ROS they generate mutate WT p53 in human bronchial epithelial cells (BEAS-2B cells). AKR1C overexpressing cells that contain WT p53 will be challenged with trans-dihydrodiols to determine whether p53 mutation is AKR mediated. Validation of a role for AKRs in PAH-induced lung carcinogenesis may target this pathway for prevention and intervention of this disease.
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