Oxidative stress triggers cytokinesis failure in hepatocytes upon isolation

Abstract

Primary hepatocytes are highly differentiated cells and proliferatively quiescent. However, the stress produced during liver digestion seems to activate cell cycle entry by proliferative/dedifferentiation programs that still remain unclear. The aim of this work was to assess whether the oxidative stress associated with hepatocyte isolation affects cell cycle and particularly cytokinesis, the final step of mitosis. Hepatocytes were isolated from C57BL/6 mice by collagenase perfusion in the absence and presence of N-acetyl cysteine (NAC). Polyploidy, cell cycle, and reactive oxygen species (ROS) were studied by flow cytometry (DNA, phospho-histone 3, and CellROX^® Deep Red) and Western blotting (cyclins B1 and D1, and proliferating cell nuclear antigen). mRNA expression of cyclins A1, B1, B2, D1, and F by reverse transcription (RT)-PCR was also assessed. Glutathione levels were measured by mass spectrometry. Here we show that hepatocyte isolation enhanced cell cycle entry, increased hepatocyte binucleation, and caused marked glutathione oxidation. Addition of 5 mM NAC to the hepatocyte isolation media prevented glutathione depletion, partially blocked ROS production and cell cycle entry of hepatocytes, and avoided the blockade of mitosis progression, abrogating defective cytokinesis and diminishing the formation of binucleated hepatocytes during isolation. Therefore, addition of NAC to the isolation media decreased the generation of polyploid hepatocytes confirming that oxidative stress occurs during hepatocyte isolation and it is responsible, at least in part, for cytokinesis failure and hepatocyte binucleation.

Keywords::

• hepatocyte isolation
• binucleation
• glutathione
• N-Acetyl cysteine
• cell cycle

Acknowledgements

The authors thank J. Markovich and G. Herrera for assistance in cytometry analysis. The authors also thank the Spanish Ministry of Economy and Competitiveness for the funds provided by SAF2012-39694 with FEDER funds and CSD-2007-00020 to J.S.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by SAF2012-39694 with FEDER funds and CSD-2007-00020 to J.S., from the Spanish Ministry of Economy and Competitiveness.