Enhancing effects of carbon tetrachloride on in vivo mutagenicity in the liver of mice fed 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx)

Chronic stimulus subsequent to cell injury plays an important role in cancer development, but the precise mechanisms remain unknown partly because appropriate animal models are lacking. In the present study, the effects of hepatotoxicant carbon tetrachloride (CCl4) on in vivo mutagenicity were investigated using gpt delta mice with or without p53. Female B6C3F, p53-proficient or -deficient gpt delta mice were given a diet containing 300 ppm of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) for 13 weeks, concurrently with intraperitoneal injection of 1 ml/kg CCl4 solution once a week. Mutant frequencies of gpt and red/gam in p53-proficient mice fed MelQx were both significantly elevated by CCl4 co-treatment. Enhancing effects of CCl4 treatment were also noted in p53-deficient mice. In the mutation spectra analysis of gpt mutant colonies, G:C to T:A transversions were predominantly observed regardless of CCl4 injection, and clonal expansion of gpt colonies were increased in the co-treated group as compared with MelQx alone group. The present data showing no significant changes in mRNA expression levels of CYP1A2 and GSTa4 between MeIQx-treated groups with and without CCl4. In the Western blotting analysis, CYP1A2 protein levels were significantly decreased in the co-treated group as compared to MelQx alone group, and GST alpha protein levels were not changed among any groups. It is suggested that the mutant frequency by co-treatment with CCl4 might result from some factors other than p53 or MelQx metabolism/excretion. Thus, our data clearly demonstrate that this model could be a powerful tool for identifying the mechanisms underlying combinatorial effects on carcinogenesis.