ASBESTOS CAUSES DNA STRAND BREAKS IN CULTURED PULMONARY EPITHELIAL-CELLS - ROLE OF IRON-CATALYZED FREE-RADICALS

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 Cr-51 release and that an iron chelator, phytic acid (500 mu M), 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 (ED(50)): 5 mu M] 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 mu g/ml or 0.25-2.5 mu g/cm(2)) caused significant WI-26 cell DNA-SB after prolonged exposure periods greater than or equal to 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.