Lack of effect of coumarin on unscheduled DNA synthesis in the in vivo rat hepatocyte DNA repair assay

Abstract

The ability of coumarin to induce UDS in male Sprague–Dawley CD rat hepatocytes in vivo was assessed using the unscheduled DNA synthesis (UDS) assay. From a preliminary toxicity study the oral maximum tolerated dose (MTD) of coumarin was determined to be 320 mg/kg body weight. For the UDS studies, rats were treated with 0 (corn oil control), 32 (one-tenth the MTD), 107 (one-third the MTD) and 320 (MTD) mg/kg coumarin via oral gavage. Rats were also treated with 20 mg/kg body weight dimethylnitrosamine (DMN) or 50 mg/kg body weight 2-acetylaminofluorene (2-AAF) as positive controls for the 2–4 hr and 12–16 hr expression of UDS, respectively. Hepatocytes were isolated by liver perfusion either 2–4 hr or 12–16 hr after treatment and cultured in medium containing [ methyl- 3H]thymidine for 4 hr and assessed for UDS by grain counting of autoradiographs. Coumarin treatment at doses of 32–320 mg/kg body weight had no statistically significant or dose-related effect on UDS in rat hepatocytes either 2–4 hr or 12–16 hr after dosing. In contrast, both DMN 2–4 hr after dosing and 2-AAF 12–16 hr after dosing produced significant increases in UDS assessed as the net nuclear grain count. Both genotoxins also increased the percentage of hepatocyte nuclei with greater than 5 net grains. Treatment with coumarin, DMN and 2-AAF had no statistically significant effect on the proportion of rat hepatocytes undergoing replicative DNA synthesis. In summary, this study demonstrates that coumarin does not induce UDS in hepatocytes of male Sprague–Dawley CD rats after oral administration at doses up to the MTD of 320 mg/kg. The responsiveness of the animals used in this study to genotoxic agents was demonstrated by the clear induction of DNA repair after treatment with DMN and 2-AAF.