title: Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) by PAH o-quinones: involvement of reactive oxygen species and copper(II)/copper(I) redox cycling.
abstract: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and procarcinogens that require activation by host metabolism. Metabolic activation of PAHs by aldo-keto reductases (AKRs) leads to formation of reactive and redox active o-quinones, which may cause oxidatively generated DNA damage. Spectrophotometric assays showed that NADPH caused PAH o-quinones to enter futile redox cycles, which result in the depletion of excess cofactor. Copper(II) amplified NADPH-dependent redox cycling of the o-quinones. Concurrent with NADPH oxidation, molecular oxygen was consumed, indicating the production of ROS. To determine whether PAH o-quinones can cause 8-oxo-dGuo formation in salmon testis DNA, three prerequisite experimental conditions were satisfied. Quantitative complete enzymatic hydrolysis of DNA was achieved, adventitious oxidation of dGuo was eliminated by the use of chelex and desferal, and basal levels of less than 2.0 8-oxo-dGuo/10(5) dGuo were obtained. The HPLC-ECD analytical method was validated by spiking the DNA with standard 8-oxo-dGuo and demonstrating quantitative recovery. HPLC-ECD analysis revealed that in the presence of NADPH and Cu(II), submicromolar concentrations of PAH o-quinones generated >60.0 8-oxo-dGuo adducts/10(5) dGuo. The rank order of 8-oxo-dGuo generated in isolated DNA was NP-1,2-dione > BA-3,4-dione > 7,12-DMBA-3,4-dione > BP-7,8-dione. The formation of 8-oxo-dGuo by PAH o-quinones was concentration-dependent. It was completely or partially inhibited when catalase, tiron, or a Cu(I) specific chelator, bathocuproine, was added, indicating the requirement for H(2)O(2), O(2)(-), and Cu(I), respectively. Methional, which is a copper-hydroperoxo complex [Cu(I)OOH] scavenger, also suppressed 8-oxo-dGuo formation. By contrast, mannitol, sodium benzoate, and sodium formate, which act as hydroxyl radical scavengers, did not block its formation. Sodium azide, which can act as both a hydroxyl radical and a (1)O(2) scavenger, abolished the formation of 8-oxo-dGuo. These data showed that the production of 8-oxo-dGuo was dependent on Cu(II)/Cu(I) catalyzed redox cycling of PAH o-quinones to produce ROS and that the immediate oxidant was not hydroxyl radical or Cu(I)OOH and that it is more likely (1)O(2), which can produce a 4,8-endoperoxide-dGuo intermediate.
authors.affiliation: Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Jong-Heum
authors.initials: JH
authors.lastname: Park
authors.forename: Sridhar
authors.initials: S
authors.lastname: Gopishetty
authors.forename: Lawrence M
authors.initials: LM
authors.lastname: Szewczuk
authors.forename: Andrea B
authors.initials: AB
authors.lastname: Troxel
authors.forename: Ronald G
authors.initials: RG
authors.lastname: Harvey
authors.forename: Trevor M
authors.initials: TM
authors.lastname: Penning
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D008297:Male; D010084:Oxidation-Reduction; D011084:Polycyclic Aromatic Hydrocarbons / Q000737:chemistry / Q000378:metabolism*; D011809:Quinones / Q000737:chemistry / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000378:metabolism*; D012474:Salmon
chemical_list: D018736:DNA Adducts; D011084:Polycyclic Aromatic Hydrocarbons; D011809:Quinones; D017382:Reactive Oxygen Species; D003300:Copper; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine