Double stranded DNA (phage lambda Hind III digest) was exposed in vitro to test particulate materials (see Project ZOICPO4491-15 LEP), either directly or through a dialysis membrane. No DNA damage was induced by dusts alone, or by hydrogen peroxide alone. In the presence of hydrogen peroxide, however, exposure to several dusts induced marked DNA damage. The effect was highest for ferric oxide, followed by cristobalite and various samples of quartz, low for tridymite and nearly absent for Chinese standard quartz. The mechanism of DNA damage was mediated by reactive oxygen species catalyzed by iron cations, as shown by its inhibition by the iron chelator deferoxamine and by the hydroxyl radical scavenger dimethylsulfoxide. Other modifying agents were studied. Spectrophotometric analysis of the adsorbance of the cationic dye Janus Green B on silica particles was found to be appropriate for the measurement of free negative charges (or iron coordination sites) on silica surfaces. The role of divalent and trivalent iron cations was studied to characterize the reactivity of silica surfaces. In the tested samples, Janus Green B adsorption was found to be roughly inversely proportional to the induction of DNA double strand breaks. Plasmid DNA from the shuttle vector pSI89 (which contains the supF reporter gene) was transfected into the human epithelial cell line 293. The cells were later exposed to test silica samples and pSI89 DNA reextracted and digested with Dpnl (to destroy all bacterially derived plasmid DNA). E. coli MBM 7070 strain, transformed with the mammalian-cell replicated plasmid DNA, was tested for mutant colony frequency in selective medium. The altered colonies were restreaked and their DNA sequenced to identify changes in the supF gene. Preliminary results showed a marked increase over background mutation levels following exposure to F600 quartz. Two other quartz samples, MQZ and CSQZ, showed a much lower effect. Characterization of the induced mutations by sequence analysis is currently under way.
