SIGNIFICANCE OF FREE RADICAL-INDUCED DNA AND PROTEIN DAMAGE AND MODIFICATIONS ON CARCINOGENESIS

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It is established that free radicals are involved in a diversity of biological phenomena such as mutation, carcinogenesis, ischemia-reperfusion injury, and irradiation/ultraviolet damage. We have thus far reported that free radicals are deeply involved in the carcinogenic process of ferric nitrilotriacetate (Fe-NTA) -induced rat renal cell carcinoma model. The purpose of this study is 1) to identify rate-limiting genes for Fe-NTA-induced renal carcinogenesis and further explore the presence of mutations and the expression of the identified genes, and 2) to clarify carcinogenic mechanisms by analyzing modified molecules by free radicals.We used two strategies for identifying the target genes : 1) evaluation of genes associated with free radical metabolism ; 2) the analysis of known oncogene or oncosuppressor gene, ras and p53. We have found the specific induction of glutathione S-transferase pi in the early stage of this carcinogenesis model. We also found no incidence of mutations in H,K,and N-ras genes and a low incidence of p53 mutations in the induced renal cell carcinomas.Then, we analyzed the products modified by free radical reaction. Main targets for free radicals reactions are lipids, nucleic acids and proteins, and an increasing number of products are reported every year. We suppose, however, limited number of molecules have biological meaning. We have measured using gas chromatography/mass spectrometry the amount of C2-12 saturated and unsaturated aldehydes in the kidney of rats treated with Fe-NTA,and found that 4-hydroxy-2-nonenal (HNE) shows the highest increase. HNE reacts with cysteine, histidine or lysine residues of proteins. We made monoclonal antibodies for HNE-modified proteins, and analyzed renal samples after Fe-NTA treatment by ELISA and immunohistochemistry.