(The) use of primary hepatocytes culture to detect DNA single-strand breaks and unscheduled DNA synthesis induced by procarcinogens

Abstract

Procarcinogen induced DNA single-strand breaks and unscheduled DNA synthesis were measured in primary rat hepatocytes culture. For single-strand breaks assay, rat liver DNA was prelabeled by injecting $^3$H-thymidine during the peak of DNA synthesis following partial hepatectomy. Hepatocytes were isolated from the rat 2 week after surgery by collagenase perfusion technique and maintained in the hormonal medium AB. DNA single-strand breaks were measured by the alkaline elution technique. Aflatoxin B , 2-acetylaminofluorene and diethylnitrosamine made doseand time-related increases in the number of single-strand breaks. Unscheduled DNA synthesis was measured on the basis of incorporation of $^3$H-thymidine into DNA for carcinoge treated hepatocytes. DNA was purified by treating the cells with SDS-EDTA-glycine solution and proteinase K on 2 $\mu{m}$ pore size PVC filter. Aflatoxin B , 2-acetylaminofluorene and diethylnitrosamine also showed a dose-dependent unscheduled DNA synthesis response. These results showed a good correlation between two methods and alkaline elution was slightly more sensitive than was unscheduled DNA synthesis.