Oxidative Stress of Asbestos or its Substitute to the Lung

1.大鼠肺泡巨噬细胞、RAW264.7或J774巨噬细胞系是小鼠建立的巨噬细胞系，用石棉或人造矿物纤维(如玻璃纤维、陶瓷纤维、岩棉或石棉)替代石棉培养，可诱导一氧化氮合酶(NOS)的产生，并增加NO的产生。气管内注入石棉的大鼠肺泡巨噬细胞培养中NO合成增加。AM、RAW264.7或J774在石棉或MMMF.4的作用下，超氧阴离子、O_2^-和NO的产生显著增加。当与石棉或MMMF共同培养时，RAW264.7、AM或J774显著降低还原型和氧化型细胞内谷胱甘肽水平。综上所述，这些发现表明，AM、RAW264.7或J774的氧化应激在暴露于石棉或MMMF.6的情况下显著增强。众所周知，NO或过氧亚硝酸盐ONOO是NO和O_2^的产物，它与蛋白质中的游离硫基或还原型谷胱甘肽(GSH)发生硝化反应。这些反应产物为RS-NO或GS-NO。我们成功地建立了一种用荧光染料测定RS-NO或GS-NO的简便新方法。用这种方法，我们发现在加入石棉或MMMF的AM、RAW264.7或J774细胞的条件培养液中，GS-NO或RS-NO的含量增加。这一增加至少部分反映了培养细胞中谷胱甘肽水平的下降，表明氧化应激的增强或蛋白质功能的改变。此外，当与石棉或霉菌共同培养时，AM、RAW264.7或J774可促进肿瘤坏死因子-α、IL-1β、IL-6等前炎性细胞因子的产生。向气管内注入石棉的大鼠的BALF(支气管肺泡灌洗液)中发现了这种增加。这些发现表明，胶原合成增加，并将进展为尘肺的发展，即肺纤维化的迟早。

1. Alveolar macrophages of rats, RAW264.7 or J774 cells which is macrophage cell line established from mouse, induced nitric oxide(NO) Synthase(NOS), and increased the prduction of NO in the culture with asbestos or man-made mineral fiber(MMMF), such as glass fiber, ceramic fiber, rock wool or slug wool by way of substitute for asbestos.2. Increase in NO Synthesis was confirmed in the culture of AM from rats instilled asbestos into trachea.3. Generation of superoxide anion, O_2^- as well as NO was significantly enhanced in the culture of AM, RAW264.7 or J774 with asbestos or MMMF.4. When cultured with asbestos or MMMF, RAW264.7, AM or J774 decreased significantly intracellular glutathione levels in both reduced-and ozxdized-form.5. Taken altogether, these findings imply that oxidative stress in AM, RAW264.7 or J774 was strongly euhanced on exposure to asbestos or MMMF.6. It is well known that NO or peroxynitrite ONOO which is product of NO and O_2^- causes a nitration reaction with free thiol group in protein or wih reduced-form glutathione(GSH). These reaction products are RS-NO or GS-NO. We succeeded in developing a simple and new method for measuremert of RS-NO or GS-NO by using fluorescent dye. Using this method, we found an increased amount of GS-NO or RS-NO in conditioned media of AM, RAW264.7 or J774 cells cultured with asbestos or MMMF. This increase reflects at least in part a decrease in glutathione level in the cultured cells, suggesting the enhancement of oxidative stress or change of protein functions.7. Moreover, when cultured with asbestos or MMMF, AM, RAW264.7 or J774 responded to increase the production of proin- flammatory cytokines such as TNF-α, IL-1β, IL-6. The increases were found in BALF (Bronchoalveolar lavage fluid) from rats inntilled asbestos into trachea. These findings suggeet that collagen synthesis increases and will progress to the development of pneumoconiosis, that is, lung fibrosis soon or later.