Influence of acetaminophen treatment and hydrogen peroxide treatment on the release of a CINC-related protein and TNF-alpha from rat hepatocyte cultures.
Western blot analysis of conditioned media from hepatocytes exposed to H2O2 revealed that a 28 kDa protein was released dose-dependently in response to 1-10 mM H2O2. 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 H2O2-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-alpha by hepatocytes was reduced after exposure to high acetaminophen doses (40-50 mM). In contrast to this finding, TNF-alpha release from hepatocyte cultures was not affected after H2O2 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-alpha from hepatocytes even at concentrations that were not sufficient to affect cell viability.
Horbach
M
M
Institute of Toxicology, University of Düsseldorf, Germany.
Gerber
E
E
Kahl
R
R
D000082:Acetaminophen / Q000633:toxicity*; D018712:Analgesics, Non-Narcotic / Q000633:toxicity*; D000818:Animals; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000378:metabolism*; D003429:Cross Reactions; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006131:Growth Inhibitors / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000633:toxicity*; D036341:Intercellular Signaling Peptides and Proteins; D007728:Kupffer Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000633:toxicity; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D009000:Monocytes / Q000166:cytology / Q000187:drug effects; D009363:Neoplasm Proteins / Q000378:metabolism; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects; D016877:Oxidants / Q000633:toxicity*; D051381:Rats; D017208:Rats, Wistar; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D018712:Analgesics, Non-Narcotic; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; C513297:Cxcl1 protein, rat; D006131:Growth Inhibitors; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D009363:Neoplasm Proteins; D016877:Oxidants; D014409:Tumor Necrosis Factor-alpha; D000082:Acetaminophen; D006861:Hydrogen Peroxide