Simultaneous Measurement of Unscheduled and Replicating DNA Synthesis by Means of a New Cell Culture Insert DNA Retention Method: Rapid Induction of Replicating DNA Synthesis in Response to Genotoxic Carcinogens

In order to measure simultaneously replicating DNA synthesis (RDS) and unscheduled DNA synthesis (UDS) in rat hepatocytes responding to exposure to carcinogens, a new method, namely the “cell culture insert DNA retention (CDR)” method, was developed. All CDR procedures for cell culture, digestion of cytoplasm and retention of DNA were performed on membranes attached to cell culture containers. Four subgroups of primary cultures of hepatocytes prepared from rats were exposed to a genotoxic or non‐genotoxic carcinogen with or without 10 mM hydroxyurea and incubated for 4 h with 10 μCi/ml [(3)H]thymidine. The membranes were then processed for both liquid scintillation and autoradiography. Among seven tested chemicals, three genotoxic agents, 3,2′‐dimethyl‐4‐aminobiphenyl, 2‐acetylaminofluorene and diethylnitrosamine, and two non‐genotoxic carcinogens, nafenopin and phenobarbital, induced RDS within 4 h after the exposure, indicating that these carcinogenic agents induce cell proliferation in non‐proliferating rat hepatocytes prior to the emergence of genotoxic changes. Several indices were devised to characterize the genotoxicity of the tested chemicals. The induction patterns obtained showed a wide variation in the individual characteristics of carcinogen‐induced genotoxicity and mitogenicity in the early phase of initiation. This is the first report of simultaneous measurement, by using a combination of autoradiography and liquid scintillation, of UDS and RDS induced in rat hepatocytes. The described CDR approach will be useful for risk assessment and characterization of carcinogenic and tumor‐promoting agents.