|Budget Amount *help
¥11,400,000 (Direct Cost : ¥11,400,000)
Fiscal Year 1995 : ¥1,800,000 (Direct Cost : ¥1,800,000)
Fiscal Year 1994 : ¥9,600,000 (Direct Cost : ¥9,600,000)
Pulsed-field gel electrophoresis (PFGE) has emerged as a powerful tool for the study of high-molecular weight DNA.PFGE is generally used for detection of cellular DNA double-strand breaks. In addition, we have designed an experimental protocol which allows the detection of DNA single-strand breaks plus alkali-labile sites by PFGE.In this study, we have investigated damage to cellular and isolated DNA by Ames-test negative carcinogens in the presence of metal ions using both PFGE and DNA sequencing techNique. Active species causing DNA damage were investigated by the ESR-spin trapping method. We have shown that in the presence of Cu (II), Ames-test negative carcinogens (benzene metabolites, o-phenylphenol metabolites, caffeic acid, pentachlorophenol metabolites, tryptophan metabolites) caused damage to isolated DNA through H2O2 formation. PFGE showed that Ames-test negative carcinogens induced DNA strand breaks in cultured human cells in the presence of Mn (II). With alkali treatment, DNA single-strand breaks were observed. The strand breakage was increased by 3-aminotriazol (a catalase inhibitor) and decreased by catalase, indicating the involvement of H2O2. The DNA damage was decreased by o-phenanthroline, indicating the involvement of transition metal ion. These results suggest that in the presence of Mn (II) or Cu (II), Ames-test negative carcinogens produce H2O2, which is activated by transition metals to cause damage to DNA in vitro and probably in cultured cells.