Onset of hepatocarcinogen-specific cell proliferation and cell cycle aberration during the early stage of repeated hepatocarcinogen administration in rats

We have previously reported that a 28-day treatment of carcinogens evoking target cell proliferation activates G(1)/S checkpoint function and apoptosis, as well as induction of aberrant ubiquitin D (Ubd) expression, suggesting disruptive spindle checkpoint function, in rats. The present study aimed to determine the onset time of rat liver cells to undergo carcinogen-specific cell cycle aberration and proliferation. Animals were treated orally with a hepatocarcinogenic dose of methyleugenol or thioacetamide for 3, 7 or 28 days. For comparison, some animals were subjected to partial hepatectomy or treated with noncarcinogenic hepatotoxicants (acetaminophen, a-naphthyl isothiocyanate or promethazine). Carcinogen-specific liver cell kinetics appeared at day 28 as evident by increases of cell proliferation, p21(Cip1+) cells, phosphorylated-Mdm2(+) cells and cleaved caspase 3(+) cells, and upregulation of DNA damage-related genes. Hepatocarcinogens also downregulated Rbl2 and upregulated Cdkn1a and Mdm2, and decreased Ubd(+) cells co-expressing phosphorylated-histone H3 (p-Histone H3) and p-Histone H3(+) cell ratio within the Ki-67(+) proliferating population. These results suggest that it takes 28 days to induce hepatocarcinogen-specific early withdrawal of proliferating cells from M phase due to disruptive spindle checkpoint function as evidenced by reduction of Ubd+ cells staying at M phase. Disruption of G(1)/S checkpoint function reflected by downregulation of Rbl2 as well as upregulation of Mdm2 suggestive of sequestration of retinoblastoma protein is responsible for the facilitation of carcinogen-induced cell proliferation at day 28. Accumulation of DNA damage probably in association with facilitation of p53 degradation by activation of Mdm2 may be a prerequisite for aberrant p21(Cip1) activation, which is responsible for apoptosis. Copyright (C) 2015 John Wiley & Sons, Ltd.