Iron Overload Induced Apoptotic Cell Death in Isolated Rat Hepatocytes Mediated by Reactive Oxygen Species

Isolated rat hepatocytes in culture were incubated with different concentrations of ironsorbitol(50, 100, 150, and 200 )lm) to assess the changes in reactive oxygen species (ros) andlipid peroxidation leading to apoptotic hepatocyte cell death. the viability ofhepatocytes wasdeclined depending on the iron concentration. one hour incubation of the cells with 100 )lm ironresulted in decreased of the hepatocyte viability down to 50% (ecso )lm). cellular glutathione(gsh) was depleted depending on the concentration of iron added to the hepatocytes in culture.decline in cellular gsh was associated with elevation in reactive oxygen species (ros)generation and formation ofthiobarbituric acid reactive substances (tbars) as index oflipidperoxidation. tbars concentration was elevated in hepatocytes exposed to >100 )lm of ironfor 40 min. a significant increase in ros formation was also observed in cells incubated with75 )lm of iron for 60 and 120 min. the consequences of ros-mediated damages to hepatocyteswere observed by dna fragmentation, nuclear staining by propidium iddide and finally withinduction of apoptotic hepatocyte cell death. terminal deoxynucleotie transferase-mediateddutp nick end labeling i.e. tunel assay (in situ- cell death-detection kit) and nuclear stainingwere also used to confirm apoptosis. these data clearly show that iron overload can causeapoptotic cell death in isolated hepatocytes and generation of ros precedes other changesrelated to oxidative stress.