PAHs can be activated by three major pathways. First, CYP1A1 and CYP1B1 can convert trans-dihydrodiols to (+)-anti-diol-epoxides, which yield stable cis- and trans-N2-2'-deoxyguanosine (dGuo) and N6-2'-deoxyadenosine (dAdo) adducts. Second, CYP peroxidase can convert the parent PAH to radical cations that can form depurinating N7- and C8-guanine (Gua) and N7-adenine (Ade) adducts. Third, AKRs can convert trans-dihydrodiols to o-quinones, which redox cycle to generate reactive oxygen species (ROS). The PAH oquinones can give rise to a series of stable N2-dGuo and N6-dAdo adducts, as well as depurinating N7-Gua and N7-Ade adducts that arise by 1,4-Michael addition to the o-quinone. By contrast, ROS directly modify DNA to form 7,8- dihydro-8-oxo-2'-deoxyguanosine as well as inducing the formation of lipid hydroperoxides. Lipid hydroperoxides undergo homolytic decomposition to form the bifunctional electrophiles malondialdehyde, 4-hydroxy-2-nonenal, 4-oxo-2- nonenal, and 4,5-epoxy-2(E)-decenal. These electrophiles then covalently modify DNA to form a series of propano and etheno adducts derived from dGuo and dAdo. In this project, stable isotope dilution LC/MS/MS methods will be developed to quantify covalent modifications to calf thymus DNA by benzo[a]pyrene (BP), anti-BPDE, and BP-7,8-dione as well as by BA-3,4-dione, and DMBA-3,4-dione. LC/MS/MS methods will also be developed for the quantitation of DNA-adducts derived from ROS and lipid hydroperoxides during redox cycling of BP-7,8-dione, BA-3,4-dione and DMBA-3,4-dione. Rigorous validation of the analytical methodology will be conducted so that further in vitro and in vivo studies can be conducted with high specificity. In collaboration with Project 1, we will determine whether AKR1A1 transfectants produce o-quinone or ROS derived adducts in cells. We will determine the predominant pathway of BP and BP-7,8-diol activation in these transfectants following induction of CYP1A1 and CYP1B1 by TCDD. The relative importance of the major pathways of BP activation will also be identified by quantifying DNA-adducts in SENCAR mouse skin a site of PAH carcinogenesis using the procedures established for DNA and cell culture systems. Urinary DNA-adducts will also be quantified. DNA-adduct profiles will then be compared with BP- 7,8-dione-, BA-3,4-dione-, and DMBA-3,4-dione-treated mice. To achieve these goals, efficient synthesis of the major PAH-DNA adducts will be achieved in the Bioanalytical Core. The [15N5]-analogs for use as internal standards will be synthesized by Dr. Harvey in this project using novel synthetic strategies. Adduct levels will be subjected to biostatistical analysis in Core A.
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