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 co nstructed for use in in vivo mutagenesis assays. After chemical treatm ent of mice carrying the lacI target gene, genomic DNA is isolated and the shuttle vector is recovered by exposing the DNA to lambda phage p ackaging extracts in vitro. Mutations in the lacI target gene that ina ctivate the repressor gene allow expression of the alphalacZ reporter gene, resulting in blue mutant plaques. We have examined the ability o f two genotoxic agents, dimethylnitrosamine (DMN) and methymethane sul fonate (MMS), to induce mutations in these transgenic mice. Both compo unds induce a variety of DNA adducts in mouse liver; DMN is a hepato-c arcinogen that induces significant hepatic cell proliferation, but NMS is not hepatocarcinogenic and does not induce hepatic cell proliferat ion. The effects of animal age, differences in strain and dosing regim en, and length of expression time were evaluated. Mice were treated fo r 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 f requency in liver. In 3 week old mice, DMN (2 mg/kg/day) produced 10- to 20-fold elevations in mutant frequency that increased with expressi on time and the number of treatments. In contrast, MMS (20 mg/kg/day) failed to increase the mutant frequency. DMN failed to induce mutation s in 6 week old mice at 2 mg/kg/day, but 4 mg/kg/day yielded significa nt elevations in hepatic mutations. Sequencing results indicate that t reatment of mice with DMN produced predominantly C:G --> T:A transitio ns. At either 4 or 6 mg/kg DMN per day, both C57BL/6 and B6C3F1 mice y ielded comparable mutation frequencies. These results suggest that enh anced cell proliferation and/or mutational spectra may contribute to t he hepatocarcinogenic potency of these chemicals.