Evidence of an Oxidative Mechanism for the Hemolytic Activity of Silica Particles

"The formation of reactive oxygen species resulting from the interaction of silica dust particles with red blood cell membranes was investigated; particularly, the effect of surface hydroxyl (silanol) group concentration on the rate of formation of such reactive oxygen species was investigated. The rate of formation was measured indirectly through the effect of catalase, a hemoprotein peroxidase, on silica-induced hemolysis. It was found that the addition of exogenous catalase to erythrocytes markedly reduces the hemolysis caused by silica particles. Furthermore, the amount of catalase required for deactivation of silica per unit area of particle surface is lower for fumed silica particles and calcined crystalline particles than for uncalcined, crystalline silica, suggesting a correlation between the concentration of OH groups at the silica particle surface and its potential for generation of H2O 2• The addition of albumin, a copper chelator, also decreases hemolysis. These results suggest that the hemolysis caused by silica particles is at least partly related to the formation of H2O2 at the particle surface and its subsequent reaction with Cu+ ions. The relationship between the concentration of surface silanol groups on the silica surface and the amount of catalase required to decrease hemolysis may also provide a method for testing potential fibrogenicity of respirable dusts. IntroductionReactive hydroxyl groups on silica surfaces are known to be cytotoxic and are believed to play a major role in silicosis (1-9). It has been proposed that this cytotoxicity results from hydrogen bonding to cell membranes and a resulting increase in membrane fluidity and permeability (4). Surface hydroxyl groups on silica surfaces have also been shown to interact with carbonyl oxygens in cell membranes (1,5). An extensive study by Nolan et al. (6) of the relationship between the negative surface charge resulting from ionized silanol groups and the hemolytic activity of crystalline a-quartz (Min-U-Sil) has shown a positive correlation between hemolytic activity and the surface concentration of silanol groups. These authors demonstrated that the hydrogen bonding of polyvinyl-N-oxide (PVPNO) with silanol groups, as well as the bonding of metal cations to the ionized sm- groups, results in significant decreases in hemolytic activity. Furthermore, it has been found that the complete removal of surface hydroxyl groups from the surface of respirable silica particles nearly eliminates the hemolytic potential of silica (7), suggesting that hydroxyl groups might be involved in other cytotoxic processes associated with silica-induced lung injury. Reactive oxygen species have been .implicated in cellular damage caused by respirable silica particles. Evidence for the presence .of oxygen radical species (02, OH°, H2O2) generated by silica, especially on freshly formed surfaces, has been produced by various investigators. Kalbanev et al. (8) detected H2O 2 in aqueous suspensions of quartz. Electron spin resonance and spin trapping by Shi et al. (9) confirmed the presence of OH"" radicals in aqueous suspensions of quartz, and Gulumian and van Wyk (10) suggested that both glass and quartz produce hydroxyl radicals in the presence of H2O 2 • Gabor et al. (11) detected the presence of malondialdehydereactive lipid peroxides (i.e., lipid-0°) in cultures of macrophages and respirable silica particles using the thiobarbituric acid (TBA) reaction test. Will (12), however, reported that iron enhances the TBA value of peroxidized lipids, and Scheulen and Kappus (19) found that catalase augmented malondialdehyde formation, thereby increasing the TBA values."