Increase of proliferation and DNA damage in mouse hepatocytes treated with diethylnitrosamine

Abstract

As diethylnitrosamine (DEN) effect on cell proliferation, DNA damage and stem cell marker(s) expression have been largely unknown in mouse normal hepatocytes (AML-12 cells) cultured over a short-term period, this study was conducted to examine the cell proliferation, Ataxia telangiectasia mutated (ATM) and epithelial cell adhesion molecule (EpCAM) and Neighbor of Punc E 11 (Nope) expression in AML-12 cells treated with DEN for 24 and 48 h. Cells were treated with DEN (25-800 μg/mL) and cell phenotype was determined, and the MTT assay was used to quantify the proliferation of cells treated with DEN. Expression and distribution of ATM in AML-12 cells were determined by indirect immunofluorescence microscopy. And Western blot analysis of EpCAM and Nope was performed. Cell viability was significantly increased in response to all doses of DEN treatment compared to control at 24 h (p<0.05 or p<0.01). However, there was no significant increase at 48 h, even though it showed increased trend. Immunofluorescence staining of ATM showed that there was an increase of ATM expression at doses of 50, 100 and 200 μg/mL of DEN treatment, showing strong nuclear staining. Furthermore, Western blot analysis showed that DEN treatment showed increased trend of EpCAM and Nope expression. Taken together, DEN treatment increased cell proliferation in AML- 12 cells, and it was associated with increased ATM expression.