Oxidant stress is critically involved in various
liver diseases.
Superoxide formation causes c-Jun NH2-terminal
kinase (JNK)- and caspase-dependent apoptosis in cultured hepatocytes. To verify these findings in vivo, male Fisher rats were treated with
diquat and
menadione. The
oxidant stress induced by both compounds was confirmed by increased formation of
glutathione disulfide and
4-hydroxynonenal protein adducts. Plasma
alanine aminotransferase activities increased from 46+/-4 U/l in controls to 955+/-90 U/l at 6 h after
diquat treatment.
Hematoxylin and
eosin staining of liver sections revealed large areas of necrotic cells at 3 and 6 h.
DNA strandbreaks, evaluated with the
terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, showed clusters of TUNEL-positive cells, where the staining was predominantly cytosolic and the cells were swollen, indicating oncotic
necrosis. There was no significant increase in
caspase-3 activities or relevant release of
DNA fragments into the cytosol at any time between 0 and 6 h after
diquat treatment. Despite the activation of JNK after high doses of
diquat, the JNK inhibitor SP-600125 did not protect against
diquat-induced
necrosis.
Menadione alone did not cause liver injury, but, in combination with
phorone and FeSO4, induced moderate oncotic
necrosis. On the other hand, if animals were treated with
galactosamine/
endotoxin as positive control for apoptosis,
caspase-3 activities were increased by 259%, the number of TUNEL-positive cells with apoptotic morphology was increased 103-fold, and DNA fragmentation was enhanced 6-fold. The data indicate that liver cell death initiated by
diquat-induced
superoxide formation in vivo is mediated predominantly by oncotic
necrosis and is independent of JNK activation.