Induction of unscheduled DNA synthesis in rat hepatocytes following in vivo treatment with dinitrotoluene

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Abstract
The induction of unscheduled DNA synthesis (UDS) by the potent hepatocarcinogen technical grade dinitrotoluene (tgDNT; 76% 2,4-DNT, 19% 2,6-DNT) was examined by using the in vivo-in vitro hepatocyte DNA repair assay. Male Fischer-344 rats were treated by gavage and hepatocytes were isolated by liver perfusion and cultured with [3H]thymidine. UDS was measured by quantitative autoradiography as net grains per nucleus (NG); .gtoreq. 5 NG was considered positive. Controls consistently had -3 to -6 NG. A dose-related increase in UDS was observed 12 h after treatment, with 200 mg/kg tgDNT producing 26 NG. A 50-fold increase in the number of cells in S-phase was observed at 48 h after treatment. This increase in S-phase cells could be suppressed in the presence of 10-20 mM hydroxyurea (HU), while the same levels of HU did not affect the level of UDS at 12 h after treatment. 2,4-DNT produced only a weak response, in contrast to 2,6-DNT which was a potent inducer of UDS. Treatment of female rats with tgDNT yielded only modest increases in UDS and DNA replication relative to males. These results are consistent with the carcinogenicity studies and indicate that tgDNT is a potent genotoxic agent, with 2,6-DNT contributing the major portion of the effect.