Single cell analysis in toxicity testing: the mitogenic activity of thioacetamide in cultured rat hepatocytes...

Rat hepatocytes were cultured at 4% O2 and 13% O2 and exposed to the nongenotoxic rodent carcinogen thioacetamide (TA) from 24 to 72 h after isolation at exposure levels between 0.01 and 0.33 mM. Hepatocytes and isolated nuclei were analyzed by DNA-protein flow cytometry. An aggregate correction procedure was applied and the proportion of S-phase, diploid, tetraploid or octoploid hepatocytes as well as binucleated cells, were measured or calculated. The proportion of S-phase cells within the diploid hepatocytes increased with increasing concentration of TA up to 3.9-fold, whereas the corresponding increase in S-phase mononucleated tetraploid cells was only 1.8-fold. S-phase binucleate tetraploid cells showed no increase. In the tetraploid hepatocytes, the mitogenic stimuli was detectable only in cultures maintained at 4% O2. The relative contribution of binuclear cells was increased 1.5-fold in the octoploid cells. It is concluded that the mitogenic activity of TA initiates DNA synthesis in diploid hepatocytes in the G1 and in the following G2 cell-cycle phase, omitting karyogenesis. The cellular protein content is not affected which indicates that the mitogenic activity of the chemical is not necessarily associated with an increase in cellular protein content. The results obtained correspond well with data of in vivo studies. The method applied therefore allows the mitogenic activity of nongenotoxic carcinogens to be detected in vitro within 48 h and their mode of action to be elucidated.