In vivo short-term assays of repair and replication of rat liver DNA.
A short-term in vivo method for assay of repair and replication of rat liver DNA has been developed, by which possible hepatocarcinogens could be identified in a few days. F344 rats were treated orally with two genotoxic hepatocarcinogens, dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2AAF), or a nongenotoxic hepatocarcinogen, carbon tetrachloride (CCl4). Then at suitable times after treatment, their hepatocytes were isolated by a two-step collagenase perfusion technique in situ and incubated with [3H]dThd with or without hydroxyurea, which inhibits DNA replication. Their nuclear DNA was then extracted and the incorporation of [3H]dThd into nuclear DNA was determined in a liquid scintillation counter. Unscheduled DNA synthesis (DNA repair), induced by DMN at doses of 2.5-10 mg/kg body weight and by 2AAF at doses of 12.5-50 mg/kg body weight, could be detected 2 h and 4 h after their administration as an increase of DNA synthesis of up to 5.8-fold and 6.0-fold, respectively, in the presence of hydroxyurea. Replicative DNA synthesis, induced by CCl4 at a dose of 200 mg/kg body weight, could be detected 48 h after its administration as a 23-fold increase of DNA synthesis in the absence of hydroxyurea and was inhibited approximately 97%-99% by hydroxyurea. Replicative DNA synthesis induced by 2AAF at a dose of 25 mg/kg body weight 16 h after its administration could be detected as a 6.8-fold increase of DNA synthesis in the absence of hydroxyurea. These results show that unscheduled and replicative DNA synthesis can be clearly distinguished by simultaneous measurements of the incorporation of [3H]dThd into nuclear DNA in the presence and absence of hydroxyurea.
Department of Molecular Oncology, University of Tokyo, Japan.
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Sawada
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Furihata
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Matsushima
D015073:2-Acetylaminofluorene; D000818:Animals; D002251:Carbon Tetrachloride / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004260:DNA Repair / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects*; D004128:Dimethylnitrosamine; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051381:Rats; D011916:Rats, Inbred F344
D004247:DNA; D015073:2-Acetylaminofluorene; D002251:Carbon Tetrachloride; D004128:Dimethylnitrosamine