Influence of acetaminophen treatment and hydrogen peroxide treatment on the release of a CINC-related protein and TNF-α from rat hepatocyte cultures

Abstract Western blot analysis of conditioned media from hepatocytes exposed to H 2 O 2 revealed that a 28 kDa protein was released dose-dependently in response to 1–10 mM H 2 O 2 . The 28 kDa protein was present in freshly isolated hepatocytes and exhibited cross-reactivity towards an antibody against CINC/gro. The intracellular amount of the protein decreased in parallel to the H 2 O 2 -induced release into the medium. The CINC-related protein was absent in media harvested after 1 h of treatment. The delivery of CINC-related protein correlated with the extent of cell damage as judged from lactate dehydrogenase leakage. Likewise, exposure of hepatocytes to 10–50 mM acetaminophen resulted in a dose-dependent release of the CINC-related protein after 24 h of culture. In contrast, monomeric CINC (molecular weight approximately 6.5 kDa) but not the 28 kDa CINC-related protein was released by lipopolysaccharide (LPS)-stimulated Kupffer cells. The amount of monomeric CINC liberated by Kupffer cells was diminished upon acetaminophen-treatment. Also, the release of tumor necrosis factor- α by hepatocytes was reduced after exposure to high acetaminophen doses (40–50 mM). In contrast to this finding, TNF- α release from hepatocyte cultures was not affected after H 2 O 2 treatment. These data suggest that damaged hepatocytes release proinflammatory cytokines which may aggravate liver injury through activation of neutrophils and monocytes. The results indicate that the appearance of the CINC-related protein is due to impairment of plasma membrane integrity as the consequence of massive cell damage. In addition, APAP inhibited the release of monomeric CINC from LPS-activated Kupffer cells and of TNF- α from hepatocytes even at concentrations that were not sufficient to affect cell viability.