A DNA double strand breakage assay was developed and used to measure in vitro effects of free radicals derived from crystalline silica in aqueous suspension. DNA damage by silica alone was found to occur during a 3-week incubation, whereas control DNA was stable in buffer. Seven silica samples were tested and found to differ in their ability to damage DNA in vitro. DNA damage was blocked by catalase. The hydroxyl radical scavengers, DMSO and sodium benzoate, had an inhibitory effect on DNA damage as did most metal chelators. Hydrogen peroxide, superoxide dismutase and deferoxamine were found to accelerate DNA damage. Hydrogen peroxide-accelerated damage, however, was inhibited by deferoxamine. Dissolved oxygen concentration (measured with an oxygen electrode) was found to decrease immediately after exposure of silica to an aqueous environment. This effect could be enhanced by the addition of deferoxamine and was associated with a decrease in ferrous iron (measured by spectrophotometric quantitation of the chelate to bathophenanthroline sulfonate). DNA damage was blocked under anoxic conditions (argon atmosphere). A collaboration with the Laboratory of Clinical Science, NIMH, was initiated in order to measure thymine glycol levels by mass spectrometry in DNA samples exposed to silica. Preliminary results show increased levels of thymine glycol in silica-treated samples and correlate well with oxidative damage as measured by the DNA strand breakage assay. Efforts to measure DNA strand breakage in silica-treated cells were begun, using a previously described pulsed field gel electrophoresis technique. Efforts to determine the possible mutagenic effects of altered DNA bases produced by silica in vitro are also underway using a shuttle vector plasmid containing the reporter gene supF. Measurement of the infrared spectra of DNA and silica (alone and in combination) was undertaken in order to identify a possible chemical interaction between silica and isolated DNA. Surface binding of the cationic dye Janus green B to silica in suspension was found to show a linear correlation with the surface area of silica, measured by the nitrogen adsorption method.
