title: Air pollution particles mediated oxidative DNA base damage in a cell free system and in human airway epithelial cells in relation to particulate metal content and bioreactivity.
abstract: Epidemiological studies demonstrate an association between increased human morbidity and mortality with exposure to air pollution particulate matter. We hypothesized that such effects may be associated with the ability of the particles to mediate generation of reactive oxygen species (ROS), either directly, via interaction with ambient oxygen or indirectly through initiation of an oxidative burst in phagocytes. To test this hypothesis, we determined 8-oxo-dG formation as a measure of direct generation of ROS, in response to particulate exposures to 2'-deoxyguanosine (dG), free and in calf thymus DNA in aerated solutions as the target molecule and cell culture, to assess the relationship between induction of oxidative damage, particulate metal content and metal bioreactivity. The HPLC-ECD technique was employed for separation and quantification of 8-oxo-dG, the most widely recognized marker of DNA oxidation. Particles used in this study include: Arizona desert dust (AZDD), coal fly ash (CFA and ECFA), oil fly ash (OFA and ROFA), and ambient air [SRM 1649 and Dusseldorf (DUSS), Germany]. The major difference between these particles is the concentration of water-soluble metals. The fly ash particulates OFA and ROFA showed a significant dose-dependent increase in dG hydroxylation to 8-oxo-dG formation over the control dG (p < 0.05), with yields 0.03 and 1.25% at the highest particulate concentration (1 mg/mL). Metal ion chelators and DMSO, a hydroxyl radical scavenger, inhibited this hydroxylation. In contrast, desert dust, coal fly ash and urban air particles induced 8-oxo-dG with yields ranging from 0.003 to 0.006%, respectively, with levels unaffected by pretreatment of the particles with metal ion chelators or addition of DMSO to the incubation mixture. When calf thymus DNA was used as a substrate, all the particles induced 8-oxo-dG in a pattern similar to that observed for dG hydroxylation, but with relatively less yield. Treatment of the particles with metal ion chelator before reacting with DNA or addition of catalase in the incubation mixture, suppressed 8-oxo-dG formation significantly (p < 0.05) in oil-derived fly ash particles only. To determine whether the oxidative responses of these particulates as shown in cell-free systems were consistent with responses using a more biologically relevant environment, human airway epithelial cells were treated with the particulates and induction of 8-oxo-dG was determined. All particles induced 8-oxo-dG in the DNA of cells above culture control, except CFA. Cells exposed to 10-400 mg/mL of ROFA for 2 h induced a dose-dependent increase in 8-oxo-dG formation. Treatment of ROFA with metal ion chelator attenuated these effects. Overall, damage enhancement by particulates in dG, calf thymus, and cellular DNA as determined by 8-oxo-dG formation under aerobic conditions is consistent with the concentration of water-soluble, not the total metal content of the particle.
authors.affiliation: Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
authors.forename: A K
authors.initials: AK
authors.lastname: Prahalad
authors.forename: J
authors.initials: J
authors.lastname: Inmon
authors.forename: L A
authors.initials: LA
authors.lastname: Dailey
authors.forename: M C
authors.initials: MC
authors.lastname: Madden
authors.forename: A J
authors.initials: AJ
authors.lastname: Ghio
authors.forename: J E
authors.initials: JE
authors.lastname: Gallagher
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D000388:Air / Q000032:analysis; D000393:Air Pollutants / Q000633:toxicity*; D000397:Air Pollution / Q000009:adverse effects*; D000818:Animals; D002244:Carbon / Q000032:analysis / Q000633:toxicity; D002417:Cattle; D002474:Cell-Free System; D002614:Chelating Agents / Q000494:pharmacology; D003031:Coal / Q000032:analysis / Q000633:toxicity; D060729:Coal Ash; D004247:DNA / Q000032:analysis / Q000187:drug effects; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004391:Dust / Q000032:analysis; D006801:Humans; D008670:Metals / Q000032:analysis / Q000633:toxicity*; D010084:Oxidation-Reduction; D052638:Particulate Matter; D017382:Reactive Oxygen Species / Q000378:metabolism; D020545:Respiratory Mucosa / Q000187:drug effects
chemical_list: D000393:Air Pollutants; D002614:Chelating Agents; D003031:Coal; D060729:Coal Ash; D004391:Dust; D008670:Metals; D052638:Particulate Matter; D017382:Reactive Oxygen Species; D002244:Carbon; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; C427203:calf thymus DNA; D003849:Deoxyguanosine