Measurement of DNA damage by terminal deoxynucleotidyl transferase reaction

An improved terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling method for the quantification of DNA damage in tissues and cultured cells was developed. Many reports have revealed that histochemistry of DNA damage can be visualized using immunohistochemistry for the terminal deoxynucleotidyl transferase reaction in tissue sections. However, few reports have described quantification of DNA damage in tissues or cells. In this study, to estimate the degree of DNA damage, the confirmed method for histochemistry using biotinylated dUTP and deoxynucleotidyl transferase was applied to label the cleaved DNA ends caused by DNA damage in tissues or cells. After end-labeling, avidin-conjugated peroxidase was reacted. A significant correlation was observed between numbers of cleaved DNA ends and peroxidase activity after the reaction. The obtained signals for presented method showed higher than those for ordinary method, and correlate with degree of DNA damage caused by serum deprivation and chemical dose. In addition, DNA damage caused by apoptosis in cells treated with 6-hydroxydopamine or Cu and in the tissues of rats administered a diet containing no Zn could be evaluated quantitatively using the present method.