Human aldehyde reductase (AKR1A1), an abundantly expressed aldoketo reductase (AKR), oxidizes PAH-trans-dihydrodiol proximate carcinogens to highly reactive and redox active o-quinones which produce reactive oxygen species (ROS). Other pathways for PAH activation include: (1) the activation of PAH-trans-dihydrodiols by CYP1A1 and CYP1B1 to form anti-diol-epoxied; and (2) the activation of PAH by CYP peroxidases to form radical cations. However, the relative contribution of these competing pathways to PAH activation in humans is unknown and will be assessed in this Project.
In aim#1, the ability of human CYP1A1 or CYP1B1 expressed in bacterial membranes to compete with recombinant AKR1A1 for trans-dihydrodiols in a reconstituted in vitro system will be studied.
In aim#2, the activation of BP-7,8-diol, BA-3,4-diol and DMBA-3,4-diol to the corresponding o-quinones will be measured in MCF-7 and human bronchoalveolar cells stably transfected with AKR1A1. Transfectants will also be treated with the CYP1A1/CYP1B1 inducer TCDD and the formation of antidiol epoxied assessed. These experiments will determine the dominant transdihydrodiol metabolites produced in cells that express either AKR1A1 or AKR1A1 plus CYPs. Studies will be repeated with BP to determine the proportion of the parent hydrocarbon that is diverted down each of these two pathways.
In aim#3, AKR1A1 transfectants will be treated with the respective diols and the formation of extracellular and intracellular ROS will be measured by EPR and fluorescence methods. ROS formation will be attributed to the AKR pathway by showing that the effect is absent in mock-transfectants and blocked by AKR1A1 inhibitors.
In aim#4, AKR1A1 transfectants will be treated with BP-7,8-diol, BA-3,4-diol and DMBA-3,4-diol to determine whether DNA-adducts attributed to the AKR pathway are formed, e.g., stable and depurinating PAH o-quinoneadducts and those derived from ROS e.g., 8-oxo-dGuo and the decomposition of lipid hydroperoxides.
In aim#5, AKR1A1 transfectants will be incubated with BP-7,8-diol before and after induction with TCDD so that DNA-adducts that form in the presence of AKR1A1 or AKR1A1 plus CYP1A1/CYP1B1 can be quantified. By treating bronchoalveolar AKR1A1 transfectants with TCDD and exposing them to BP, the adducts that arise from o-quinones and ROS, diol-epoxied and radical cations will determine which of the three pathways of PAH activation dominates. Adduct levels will be quantified in the Bioanalytical Core (Core B) by LC/MS methods developed in Project 2. Relationships between PAHmetabolite profiles, ROS-formation and type of DNA adducts will be analyzed in the biostatistical component of Core A.
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