Strong binding of silica to DNA in vitro was detected using the new technique of attenuated reflectance Fourier transform infrared spectroscopy. Hydrogen bonding between the surface silanol groups and the phosphate backbone of DNA was demonstrated. Substitution of deuterium oxide for water in the aqueous buffer allowed for more accurate assessment of the conformational region of the DNA spectrum and revealed some as yet uncharacterized subtle changes in the DNA bases associated with silica binding. In collaboration with Dr. Guthrie, large thin silica plates with a defined crystal surface orientation were prepared for use in infrared spectroscopy. Initial results showed that the quality of silica spectra was superior to previous methods. The technique proved feasible for studying DNA interactions with defined silica surfaces. Modification of a previously developed in vitro DNA strand breakage assay was undertaken to distinguish silica-induced single strand breaks from double strand breaks. Using a circular plasmid DNA, strand breaks were detected at earlier times after exposure to silica. Strand breakage was found to occur by a sequence of single strand nicking followed by eventual overlapping double strand breaks. Densitometry was used with this new assay to compare relative DNA strand breakage activity among seven silica samples. Experiments were undertaken in order to detect possible DNA strand breakage inside cells exposed to silica. By pulsed field gel electrophoresis, no double strand breaks were found in BALB/3T3 cells exposed to silica from one to seven days. An alkaline unwinding assay for intracellular single strand breaks was set up with silica exposure for 24 hr followed by incubation at 4 degrees C for 16 hr to block DNA repair. Preliminary results suggest that single strand breaks are detectable. A newly developed technique for measuring surface charge on silica preparations by binding to the cationic dye, Janus Green B, was shown to correlate well with PVPNO binding to silanol groups and to surface area as measured by the nitrogen adsorption technique. This Janus Green B technique now appears to be the most sensitive technique available for measurement of silica surface areas.
