Comparison of autoradiography, liquid scintillation counting and immunoenzymatic staining of 5-bromo-2′-deoxyuridine for measurement of unscheduled DNA synthesis and replicative DNA synthesis in rat liver

Abstract

Different methods for evaluating unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) were studied in hepatocytes of F344 rats exposed in vivo to dimethylnitrosamine (DMN) or CCl 4. Hepatocytes were isolated and incubated in Williams' medium E supplemented with either [ 3H]thymidine for autoradiography or 5-bromo-2′-deoxyuridine for immunoenzymatic staining. In the method of liquid scintillation counting, the cells were incubated with [ 3H]thymidine with or without hydroxyurea. The nuclear fraction was isolated and the incorporation of [ 3H]thymidine into nuclear DNA was determined by a liquid scintillation counter. DMN at doses of 0.625–5 mg/kg body weight induced UDS of 1.6–37.9 (0 dose; −6.9) net grains/nucleus measured by autoradiography and 337–1377 (0 dose; 177) dpm/μg DNA in the presence of hydroxyurea measured by a liquid scintillation counter. CCl 4 at doses of 50–400 mg/kg body weight induced RDS in 1.5–12.1% (0 dose; 0.12%) and 1.8–14.6% (0 dose; 0.16%) of cells with the methods of autoradiography and immunoenzymatic staining, respectively, and of 2991–24256 (0 dose; 324) dpm/μg DNA in the absence of hydroxyurea with the method of liquid scintillation counting. Similar dose-dependent induction of UDS and RDS was observed with these methods. These results suggest that the methods of liquid scintillation counting and immunoenzymatic staining have almost the same sencitivity for measuring UDS and RDS as that of autoradiography.