title: Asbestos causes DNA strand breaks in cultured pulmonary epithelial cells: role of iron-catalyzed free radicals.
abstract: Asbestos causes pulmonary fibrosis and various malignancies by mechanisms that remain uncertain. Reactive oxygen species in part cause asbestos toxicity. However, it is not known whether asbestos-induced free radical production causes alveolar epithelial cell (AEC) cytotoxicity by inducing DNA strand breaks (DNA-SB). We tested the hypothesis that asbestos-induced AEC injury in vitro is due to iron-catalyzed free radical generation, which in turn causes DNA-SB. We found that amosite asbestos damages cultured human pulmonary epithelial-like cells (WI-26 cells) as assessed by 51Cr release and that an iron chelator, phytic acid (500 microM), attenuates these effects. A role for iron causing these effects was supported by the observation that ferric chloride-treated phytic acid did not diminish WI-26 cell injury. Production of hydroxyl radical-like species (.OH) was assessed based upon the .OH-dependent formation of formaldehyde (HCHO) in the presence of dimethyl sulfoxide. A variety of mineral dusts induced significant levels of .OH formation (nmol HCHO at 30 min: carbonyl iron, 85 +/- 21; amosite asbestos, 14 +/- 2; chrysotile asbestos, 7 +/- 1; titanium dioxide, 2.5 +/- 0.5). Phytic acid significantly diminished the asbestos-induced .OH production. DNA damage to AEC was assessed by the alkaline unwinding, ethidium bromide fluorometric technique. Hydrogen peroxide caused dose-dependent DNA-SB in WI-26 cells after a 30-min exposure period [50% effective dose (ED50): 5 microM] that was similar to other cell lines. Amosite asbestos induced dose-dependent DNA-SB in WI-26, A549, and primary isolated rat alveolar type II cells maintained in culture for 7-10 days (alveolar type I-like). Lower doses of amosite (0.5-5 micrograms/ml or 0.25-2.5 micrograms/cm2) caused significant WI-26 cell DNA-SB after prolonged exposure periods (> or = 2 days). Phytic acid ameliorated DNA damage in all three cultured AEC. There was a direct correlation between mineral dust-induced .OH production at 30 min and DNA-SB in WI-26 cells at 4 h (P < 0.0005). These data suggest that mineral dusts can be directly genotoxic to relevant target cells of asbestos, AEC. Furthermore, these results provide additional support for the premise that iron-catalyzed free radicals mediate asbestos-induced pulmonary toxicity.
authors.affiliation: Department of Medicine, Northwestern University Medical School, Chicago, Illinois.
authors.forename: D W
authors.initials: DW
authors.lastname: Kamp
authors.forename: V A
authors.initials: VA
authors.lastname: Israbian
authors.forename: S E
authors.initials: SE
authors.lastname: Preusen
authors.forename: C X
authors.initials: CX
authors.lastname: Zhang
authors.forename: S A
authors.initials: SA
authors.lastname: Weitzman
mesh_terms: D001194:Asbestos / Q000494:pharmacology*; D017639:Asbestos, Amosite / Q000494:pharmacology; D017632:Asbestos, Serpentine / Q000494:pharmacology; D002461:Cell Line, Transformed; D002860:Chromium Radioisotopes / Q000378:metabolism; D004247:DNA / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects*; D004622:Embryo, Mammalian; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D017665:Hydroxyl Radical / Q000378:metabolism; D008168:Lung; D010833:Phytic Acid / Q000494:pharmacology
chemical_list: D017632:Asbestos, Serpentine; D002860:Chromium Radioisotopes; D017639:Asbestos, Amosite; D001194:Asbestos; D017665:Hydroxyl Radical; D010833:Phytic Acid; D004247:DNA; D006861:Hydrogen Peroxide