INDUCTION OF HEPATIC MUTATIONS IN LACI TRANSGENIC MICE

Transgenic B6C3F1 and C57BL/6 mice containing a lambda shuttle vector that carries a lacI target and an alphalacZ reporter gene have been constructed for use in in vivo mutagenesis assays. After chemical treatment of mice carrying the lacI target gene, genomic DNA is isolated and the shuttle vector is recovered by exposing the DNA to lambda phage packaging extracts in vitro. Mutations in the lacI target gene that inactivate the repressor gene allow expression of the alphalacZ reporter gene, resulting in blue mutant plaques. We have examined the ability of two genotoxic agents, dimethylnitrosamine (DMN) and methymethane sulfonate (MMS), to induce mutations in these transgenic mice. Both compounds induce a variety of DNA adducts in mouse liver; DMN is a hepato-carcinogen that induces significant hepatic cell proliferation, but NMS is not hepatocarcinogenic and does not induce hepatic cell proliferation. The effects of animal age, differences in strain and dosing regimen, and length of expression time were evaluated. Mice were treated for 5, 14 or 21 days and were sacrificed 1, 8 or 22 days after the final dose to evaluate the effects of increased expression time on mutant frequency in liver. In 3 week old mice, DMN (2 mg/kg/day) produced 10- to 20-fold elevations in mutant frequency that increased with expression time and the number of treatments. In contrast, MMS (20 mg/kg/day) failed to increase the mutant frequency. DMN failed to induce mutations in 6 week old mice at 2 mg/kg/day, but 4 mg/kg/day yielded significant elevations in hepatic mutations. Sequencing results indicate that treatment of mice with DMN produced predominantly C:G --> T:A transitions. At either 4 or 6 mg/kg DMN per day, both C57BL/6 and B6C3F1 mice yielded comparable mutation frequencies. These results suggest that enhanced cell proliferation and/or mutational spectra may contribute to the hepatocarcinogenic potency of these chemicals.