Abstract A45: Kupffer cell and EtOH DNA synthesis
Authors: Owumi Solomon E. 
Published: May 2012

The Kupffer cells (KC) are the resident hepatic macrophage located in the sinusoidal space of the liver. KC plays an important role in hepatic homeostasis and in the response of the liver to xenobiotics. It phagocytizes foreign bodies, old red blood cell and interacts with endotoxin resulting in its activation. Activated KC releases cytokines such as tumor necrosis factor alpha (TNF-α) and reactive oxygen species (ROS) like superoxide. TNF-α and superoxide have been implicated in signalling transduction involved in cell growth, gene expression and apoptosis. Ethanol (EtOH) consumption has been causally linked to the etiology of primary hepatocellular cancer (HCC), although EtOH is not a known carcinogen. One such mechanism of KC activation is via EtOH-induced endotoxemia, which leads to an increase in cytokine and ROS production by the KC, consequently impacting on and potentially induces hepatocyte growth. Here we propose that depletion of Kupffer cells will attenuate the effect of EtOH induced endotoxemia, decreasing hepatocyte deoxyribonucleic acid (DNA) synthesis required for cell growth.

Rate of hepatocyte DNA synthesis was examined as a marker of cell growth in C57BL6 mice depleted of KC (KC−) with clodronate-liposome and in KC competent mice (KC+). Both groups of mice were fed an isocaloric EtOH-liquid diet (3%w/v EtOH). Control mice were fed a liquid diet without EtOH for 1 week. DNA synthesis was assessed by 5-bromo-2′-deoxyuridine (BrdU) incorporation into hepatocytes undergoing replicative DNA synthesis by BrdU-immunohistochemistry. Apoptotic bodies' formation was examined by the TUNEL assay and Extracellular Regulated Kinases (ERK 1/2) protein phosphorylation believed to be involved in growth signalling pathway was evaluated by western blotting. TNF-α released was assessed by total mRNA transcript via RT-PCR. Toxicity was assessed by the presence of liver transaminases aspartate amino transferase (AST) and alanine amino transferase (ALT) in serum.

Serum AST and ALT were within normal reference range in control and treated mice indicative of insignificant EtOH or clodronate induced toxicity. In KC+ mice fed EtOH hepatic DNA synthesis increased 183% compared to control KC+ mice. Hepatocyte DNA synthesis in KC− remain at the same level with control KC+ mice. In KC− mice fed EtOH there was a 50% decrease in hepatic DNA synthesis when compared to control KC+ mice and a 74% decrease compared to (KC+) mice fed EtOH-liquid diet. TNF-α released was 148% in KC+ mice fed EtOH, 69% in KC− and 72% in KC− fed EtOH compared to control mice KC+. However, there was a slight increase in TNF-α released in KC− fed EtOH-liquid diet when compared to KC− control. An increase in the phosphorylation of protein kinases p42/44 (ERK1/2) in the control mice KC+ fed EtOH was observed when compared to KC− mice fed ethanol. There was a slight increase in apoptotic bodies' accumulations in KC− fed EtOH-liquid diet and in KC− control mice.

Taken together these observations suggest that EtOH induces hepatocyte DNA synthesis in KC+ mice and to a lesser extent in KC− mice indicative of a role for the KC in hepatocyte DNA synthesis and may be involved in the development of hepatocarcinogenesis. Depletion of KC attenuates the downstream effect of ethanol induced endotoxemia, potentially by a mechanism involving reduced TNF-α and ROS production with its concomitant effect on ERK 1/2 signaling pathway on hepatocyte DNA synthesis thereby supporting our hypothesis.

Citation Information: Cancer Prev Res 2010;3(1 Suppl):A45.