NITRIC OXIDE SYNTHESIS,NITROSOGLUTATHIONE FORMATION AND POSSIBLE GENERATION OF PEROXYNITRITE IN THE CULTURE OF ALVOLAR MACROPHAGES WITH ASBESTOS OR MAN-MADE MINERAL FIBERS.

When cultured with chrysotile asbestos or man-made mineral fibers(MMMF)，rat alveolar macrophages(AMs)concomitantly produced in a significant amount of nitric oxide(NO)and superoxide anion(O_2-)，indicating a possible formation of peroxynitrite(ONOO-)and its decomposition to hydroxyl radical(·OH)，which is very toxic to various cells and tissues.These findings strongly suggest that all of NO，O_2-，ONOO-and OH derived from decomposition of ONOO-are implicated in the development of asbestos-or MMMF-induced lung diseases.Moreover，such findings were also consistent with NO synthesis and histological pathologies observed in the previous studies of rat instilled asbestos into the trachea.NOsythesized by AMs cultured with asbestos or MMMF also readily reacts with thiol compounds，especially with reduced glutathione(GSH)in the cells to form nitrosocompounsuch as nitroslutathione(GS-NO)。As expected，GS-NO was detectable in the conditioned media from the culture of AMs with asbestos or MMMFS，but hardly detected in the cell layer。This finding indicates that GS-NO is released into extracellular space similarly to oxidized glutathione.The amount of GS-NO in the conditioned media from culture of AMs with asbestos or MMMFs was small and comparable to that from the control culture of AMs alone。However，AMs cultured with bacterial endotoxin，LPS remarkably enhanced NO synthesis to release a large amount of GS-NO in the conditioned media.Thus，mineral fibers added into the culture of AMs did not increase the amount of GS-NO.It therefore follows that as far as GSH synthesis systems of AMs are not affected in the culture with mineral fibers，intracellular GSH level is not significantly decreased。

When cultured with chrysotile asbestos or man-made mineral fibers(MMMF), rat alveolar macrophages (AMs) concomitantly produced in a significant amount of nitric oxide(NO) and superoxide anion (O_2-), indicating a possible formation of peroxynitrite (ONOO-) and its decomposition to hydroxyl radical (・OH), which is very toxic to various cells and tissues. These findings strongly suggest that all of NO, O_2- , ONOO- and OH derived from decomposition of ONOO- are implicated in the development of asbestos- or MMMF-induced lung diseases. Moreover, such findings were also consistent with NO synthesis and histological pathologies observed in the previous studies of rat instilled asbestos into the trachea.NOsythesized by AMs cultured with asbestos or MMMF also readily reacts with thiol compounds, especially with reduced glutathione (GSH) in the cells to form nitrosocompounds such as nitrosoglutathione (GS-NO). As expected, GS-NO was detectable in the conditioned media from the culture of AMs with asbestos or MMMFS, but hardly detected in the cell layer. This finding indicates that GS-NO is released into extracellular space similarly to oxidized glutathione. The amount of GS-NO in the conditioned media from culture of AMs with asbestos or MMMFs was small and comparable to that from the control culture of AMs alone. However, AMs cultured with bacterial endotoxin, LPS remarkably enhanced NO synthesis to release a large amount of GS-NO in the conditioned media. Thus, mineral fibers added into the culture of AMs did not increase the amount of GS-NO.It therefore follows that as far as GSH synthesis systems of AMs are not affected in the culture with mineral fibers, intracellular GSH level is not significantly decreased.