Do Silicon-Oxygen Radicals Play a Role in the Quartz-Induced Hemolysis and Fibrogenicity?

"INTRODOCTIONIn an earlier communication 1 from our laboratory it was reported that mechanical crushing of coal and quartz under normal air atmosphere generates of free radicals on the particle surfaces, and that these radicals decay with time, hence pointing to a higher toxicity of fresh dusts in relationship to pneumoconiosis and silicosis. More recently Fubini et al.2 have also reported the detection by electron spin resonance (ESR) of the formation of SiO. and Si-type of radicals from quartz particles crushed under atmospheric conditions. In agreement with earlier ESR studies on single crystals of quartz crushed under high vacuum ( -10-10 torr>3 and subsequent exposure to air,3 and to other gases,4 these radicals were identified2 as being formed by the homolytic cleavage of the Si-0-Si bonds and the reactions of the Si• and SiO- radical with atmosphere. Fubini et al.2 also suggested that these radicals might be involved in the mechanism of the fibrotic action by silica, either by transforming the particle surface into a selective oxidating agent or as an initiator of a sequence of reactions leading to fibrosis. Earlier Gabor and Anca5 bad reported that lipid ·peroxidation caused by free radicals on the silica surface might be involved in the red blood cell membrane damage. Thus far, however, no parallel cytotoxicity, fibrogenicity, and free radica1 studies on a given quartz dust sample have been reported, except for some earlier work from our laboratory.1·6·7 We now present more recent results obtained from parallel cytotoxicity, fibrogenicity, and free radica1 measurements on a freshly made quartz dust. The dust's free radica1 content was measured using ESR spectroscopy while its cytotoxicity potential was estimated via hemolysis. Hemolysis was employed as the toxicity test because it is a widely used method for estimating the potential of a dust for disrupting the cell membrane. 8 The fibrotic potential was followed by measuring the dust-induced lipid peroxidation, using linoleic acid as a model lipid. As discussed below, the results obtained suggest new clues to the mechanism of the quartzrelated cytotoxicity and fibrogenicity. MATERIALS AND METHODSReagentsCrystalline quartz particles with a size range of 0.2-2.5 mm were obtained from the Generic Respirable Dust Technology Center, Pennsylvania State University, University Park, Pennsylvania. These particles were crushed in air to obtain quartz dust samples with particle sizes smaller than 20 microns. We chose to work with a dust with mixed particle sizes, rather than a specific range, as an effort to simulate the mining atmosphere. An agate mortar-pestle arrangement was used for the crushing and grinding because of the close similarity of the structure of agate to that of quartz. Diethylenetriaminepentaacetic acid (DETAPAC) were purchased from Sigma. All other chemica1s were purchased from Fisher or Aldrich.Hemolysls ExperimentsHemolytic activity of silica was measured, following an established procedure,9 as the amount of hemoglobin released from a 2 % suspension of sheep erythrocytes after incubation with 10 mg of silica dust for one hour at 37 °C. The hemoglobin release was estimated via the absorbance at 540 nm using a Gioford spectrophotometer. The procedure was calibrated by substituting the silica dust by a phosphate buffer solution as a negative control (background) and O.S % Triton-X-100 as a positive control (100% hemolysis). The percentage of hemolysis was calculated as follows:"