Detection of DNA damage in primary cultures of rat hepatocytes following in vivo and in vitro exposure to genotoxic agents.

Abstract

A simplified method for the quantitation of DNA damage in nonlabeled hepatocytes, using a fluorometric technique for the quantitation of DNA in conjunction with a modification of the alkaline elution technique of Kohn et al [1976], following chemical treatment in vitro and in vivo, is described. Freshly isolated hepatocytes were treated in vitro with 2-acetylaminofluorene, aflatoxin B1, and dimethylnitrosamine, then examined for DNA damage. Exposure to each of these compounds resulted in DNA damage. Hepatocytes isolated from rats treated with the hepatocarcinogens 2-acetylaminofluorene, benzidine, azoxymethane, dimethylhydrazine, dimethylnitrosamine, and diethylnitrosamine sustained DNA damage as evidenced by increased alkaline elution. DNA damage in hepatocytes was also observed as a result of treatment with methylmethanesulfonate and azaserine. The hepatotoxin carbon tetrachloride did not induce DNA damage in hepatocytes isolated from treated animals. A comparison of the induction of DNA damage and of unscheduled DNA synthesis in hepatocytes from the same animals revealed that in most cases the extent of elution of DNA from filters was proportional to the induction of DNA repair.