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

已证实自由基参与了多种生物现象，如突变、致癌、缺血再灌注损伤、辐射/紫外线损伤等。到目前为止，我们已经报道自由基在氮三乙酸铁(Fe-NTA)诱导的大鼠肾细胞癌模型的致癌过程中有很深的作用。本研究的目的是1)寻找铁-NTA诱导的肾癌发生的限速基因，并进一步探讨这些基因的突变和表达情况；2)通过分析自由基修饰的分子来阐明致癌机制。我们采用了两种策略来确定靶基因：1)评估与自由基代谢相关的基因；2)分析已知的癌基因或抑癌基因ras和p53。我们发现谷胱甘肽S转移酶pi在该致癌模型的早期阶段具有特异性的诱导作用。我们还发现，在诱导的肾细胞癌中，H、K和N-ras基因没有发生突变，而P53突变的发生率很低。自由基反应的主要目标是脂质、核酸和蛋白质，每年都有越来越多的产品被报道。然而，我们认为，具有生物学意义的分子数量有限。用气相色谱-质谱法测定了Fe-NTA处理的大鼠肾脏中C2-12饱和醛和不饱和醛的含量，发现4-羟基-2-壬烯醛(HNE)的增加幅度最大。HNE与蛋白质的半胱氨酸、组氨酸或赖氨酸残基发生反应。我们制备了针对HNE修饰蛋白的单抗，并对Fe-NTA处理后的肾脏标本进行了ELISA法和免疫组织化学分析。

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.