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

The ability of coumarin to induce UDS in male Spague-Dawley CD rat hepatocy tes bt vivo was assessed using the unscheduled DNA synthesis (UDS) assay. F rom a preliminary toxicity study the oral maximum tolerated dose (MTD) of c oumarin was determined to be 320 mg/kg body weight. For the UDS studies, ra ts were treated with 0 (corn oil control), 32 tone-tenth the MTD), 107 tone -third the MTD) and 320 (MTD) mg/kg coumarin via oral gavage. Rats were als o treated with 20 mg/kg body weight dimethylnitrosamine (DMN) or 50 mg/kg b ody weight 2-acetylaminofluorene (2-AAF) as positive controls for the 2-4 h r and 12-16 hr expression of UDS, respectively. Hepatocytes were isolated b y Liver perfusion either 2-4 hr or 12-16 hr after treatment and cultured in medium containing [methyl-H-3]-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 sign ificant increases in UDS assessed as the net nuclear grain count. Both geno toxins 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 replicat ive DNA synthesis. In summary, this study demonstrates that coumarin does n ot induce UDS in hepatocytes of male Sprague-Dawley CD rats after oral admi nistration at doses up to the MTD of 320 mg/kg. The responsiveness of the a nimals used in this study to genotoxic agents was demonstrated by the clear induction of DNA repair after treatment with DMN and 2-AAF. (C) 2000 Elsev ier Science. Ltd. All rights reserved.