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

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 d irectly, via interaction with ambient oxygen or indirectly through initiati on of an oxidative burst in phagocytes. To test this hypothesis, we determi ned 8-oxo-dG formation as a measure of direct generation of ROS, in respons e to particulate exposures to 2 ' -deoxyguanosine (dG), free and in calf th ymus DNA in aerated solutions as the target molecule and cell culture, to a ssess the relationship between induction of oxidative damage, particulate m etal content and metal bioreactivity. The HPLC-ECD technique was employed f or separation and quantification of 8-oxo-dG, the most widely recognized ma rker 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 f ly ash particulates OFA and ROFA showed a significant dose-dependent increa se 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 p articles induced 8-oxo-dG with yields ranging from 0.003 to 0.006%, respect ively, with levels unaffected by pretreatment of the particles with metal i on chelators or addition of DMSO to the incubation mixture. When calf thymu s DNA was used as a substrate, all the particles induced 8-oxo-dG in a patt ern 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-ox o-dG formation significantly(p < 0.05) in oil-derived fly ash particles onl y. To determine whether the oxidative responses of these particulates as sh own in cell-free systems were consistent with responses using a more biolog ically relevant environment, human airway epithelial cells were treated wit h the particulates and induction of 8-oxo-dG was determined. Al particles i nduced 8-oxo-dG in the DNA of cells above culture control, except CFA. Cell s 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 attenuate d these effects. Overall, damage enhancement by particulates in dG, calf th ymus, and cellular DNA as determined by 8-oxo-dG formation under aerobic co nditions is consistent with the concentration of water-soluble, not the tot al metal content of the particle.