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

Different methods for evaluating unscheduled DNA synthesis (UDS) and r eplicative DNA synthesis (RDS) were studied in hepatocytes of F344 rat s exposed in vivo to dimethylnitrosamine (DMN) or CCl4. Hepatocytes we re isolated and incubated in Willi ams' medium E supplemented with eit her [H-3]thymidine for autoradiography or 5-bromo-2'-deoxyuridine for immunoenzymatic staining, In the method of liquid scintillation counti ng, the cells were incubated with [H-3]thymidine with or without hydro xyurea. The nuclear fraction was isolated and the incorporation of [H- 3]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/mu g DNA in the presence of hydroxyurea me asured by a liquid scintillation counter. CCl4 at doses of 50-400 mg/k g 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 immuno enzymatic staining, respectively, and of 2991-24256 (0 dose; 324) dpm/ mu g DNA in the absence of hydroxyurea with the method of liquid scint illation counting. Similar dose-dependent induction of UDS and RDS was observed with these methods, These results suggest that the methods o f liquid scintillation counting and immunoenzymatic staining have almo st the same sensitivity for measuring UDS and RDS as that of autoradio graphy.