Kamp D W, Israbian V A, Preusen S E, Zhang C X, Weitzman S A
Department of Medicine, Northwestern University Medical School, Chicago, Illinois.
Am J Physiol. 1995 Mar;268(3 Pt 1):L471-80. doi: 10.1152/ajplung.1995.268.3.L471.
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.
石棉通过尚不确定的机制导致肺纤维化和多种恶性肿瘤。活性氧在一定程度上导致石棉毒性。然而，尚不清楚石棉诱导的自由基产生是否通过诱导DNA链断裂（DNA-SB）导致肺泡上皮细胞（AEC）细胞毒性。我们检验了这样一个假设，即体外石棉诱导的AEC损伤是由于铁催化的自由基生成，进而导致DNA-SB。我们发现，通过51Cr释放评估，铁石棉会损伤培养的人肺上皮样细胞（WI-26细胞），并且一种铁螯合剂植酸（500微摩尔）可减轻这些影响。氯化铁处理的植酸不能减轻WI-26细胞损伤这一观察结果支持了铁在导致这些影响中所起的作用。基于在二甲基亚砜存在下甲醛（HCHO）的羟基自由基样物质（·OH）依赖性形成来评估·OH的产生。多种矿物粉尘诱导了显著水平的·OH形成（30分钟时的HCHO纳摩尔数：羰基铁，85±21；铁石棉，14±2；温石棉，7±1；二氧化钛，2.5±0.5）。植酸显著减少了石棉诱导的·OH产生。通过碱性解旋、溴化乙锭荧光技术评估对AEC的DNA损伤。过氧化氢在30分钟暴露期后在WI-26细胞中引起剂量依赖性DNA-SB [50%有效剂量（ED50）：5微摩尔]，这与其他细胞系相似。铁石棉在培养7至10天（I型样肺泡）的WI-26、A549和原代分离的大鼠II型肺泡细胞中诱导剂量依赖性DNA-SB。较低剂量的铁石棉（0.5 - 5微克/毫升或0.25 - 2.5微克/平方厘米）在延长暴露期（≥2天）后导致显著的WI-26细胞DNA-SB。植酸改善了所有三种培养的AEC中的DNA损伤。30分钟时矿物粉尘诱导的·OH产生与4小时时WI-26细胞中的DNA-SB之间存在直接相关性（P < 0.0005）。这些数据表明，矿物粉尘可对石棉的相关靶细胞AEC直接产生基因毒性。此外，这些结果为铁催化的自由基介导石棉诱导的肺毒性这一前提提供了额外支持。
