Development of DNA damage-analyzing method for estimating genotoxicity of chemical substances

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This research project was to develop a DNA damage-analyzing method for estimating genotoxicity of chemical substances. The DNA damage-analyzing method is based on both the DNA sequencing technique, using ^<32>P 5'-end-labeled DNA fragments obtained from human c-Ha-ras-1 protooncogene, and eledtron spin resonance-spin trapping methods. The analyzing method revealed that not only Ames test-positive carcinogens but also some Ames-test negative carcinogens caused DNA damage under certain conditions as follows. (1) Ames-test positive carcinogens react with DNA and mainly cause base modification. (2) Carcinogenic benzene and o-phenylphenol have not been shown to be mutagenic in bacterial test systems. 1,2,4-Benzenetriol (a benzene metabolite), and 2,5-dibydroxybiplienyl (an o-phenylphenol metabolite) caused DNA damage in the presence of Cu(II). These active species causing DNA damage are suspected to be copper-oxygen complexes rather than hydroxyl radicals. (3) Chromium(VI) is an Ames test-positive carcinogen, whereas iron(III) nitrilotriacetate cobalt(II) and nickel(II) are Ames test-negative carcinogens. Chromium(VI), iron(III) nitrilotriacetate, cobalt(II) and nickel(II) react with hydrogen peroxide leading to the production of active species such as hydroxyl radical, singlet oxygen and metal-oxygen complex, all of which cause DNA damage. (4) Recent observations have suggested that some tumor promoters cause DNA damage through the formation of free radicals. Sulfite (SO^2-_), which is thought to be a co-carcinogen or promoter, was rapidly autoxidized in the presence of Co(II) to produce SO^-_ radical, causing site-specific DNA damage.