An improved method of mouse liver micronucleus analysis: an application to age-related genetic alteration and polyploidy study.

ABSTRACT

The performance of a micronucleus test in liver cells in vivo requires two laborious procedures stimulation of hepatocytes to division and dissociation of liver tissue into a single-cell suspension. We propose the method of inhalation treatment of mice with carbon tetrachloride to induce cell proliferation and alkaline dissociation of previously fixed tissue. The micronucleus incidence and ploidy classes in terms of cytophotometric DNA content were determined in liver of mice of three age groups (around 2.5, 5.0 and 7.0 months old) after CCl4 treatment or partial hepatectomy. The data obtained show that both methods give the same results. The fraction of micronucleated hepatocytes was 0.69% at the age of 2.5 months; it increased to 8.5% and then to 13.5% at 5.0 and 7.0 months respectively. Simultaneously, the ploidy classes changed both with the aging of the animal and after induced liver regeneration. The percentage distribution of micronucleated cells by ploidy class showed that cells carrying micronuclei were the higher ploidies rather than the population in general. Since polyploid cells contain multiple molecular targets for genetic damage, the micronucleation index per genome unit was estimated. Then the real rate of accumulation of both intrinsic endogenous (and probably the exogenously induced) preclastogenic genetic alterations in hepatocytes during the adulthood of mice was evaluated to be 0.03% per diploid genome per day. This seems to be the first description of the phenomenon of liver cell aging in terms of micronuclear aberrations.