Cellular metabolism of dopamine (DA) generates H2O2, which is further reduced to hydroxyl radicals in the presence of iron. Cellular damage inflicted by DA-derived hydroxyl radicals is thought to contribute to Parkinsons disease. Previously we proposed that the oxidation of proteins that contain reactive Cys residues by DA-derived H2O2 might be largely responsible for DA-induced apoptosis in neuronal cells and play an important role in the pathogenesis of Parkinsons disease. Here, we investigated the correlation between protein oxidation by hydroxyl radicals and apoptosis of PC12 cells. One of the most common modification of proteins caused by hydroxyl radicals is the introduction of carbonyl groups into the side chains. Carbonyl group- containing proteins can be specifically modified by dinitrophenylhydrazine and detected by immunoblot analysis with antibodies to dinitrophenyl residues. The progress of DA-induced apoptosis, measured by cell viability as well as the cleavage of CPP32, a member of the caspase family, was compared with protein carbonyl formation. Exposure of PC12 cells to 3 mM DA resulted in time-dependent cell death with half maximal effect at 6 h. Treatment with 3 mM DA also caused the cleavage of precursor CPP32, which was not obvious for the first 3 h but nearly completed 6 h after treatment with DA. Both the cell death and CPP32 activation were partially prevented by the presence of NAC. However, carbonyl group formation detected by immunoblot analysis remained at background levels over the 6 h period of DA treatment and was not affected by NAC. It appears that the intracellular concentration of free iron was very low in PC12 cells and without iron the conversion of H2O2 to hydroxyl radicals apoptosis did not proceed to a significant extent. In support of this premise, protein carbonyl formation occurred when the intracellular iron level was increased by treating PC12 cellss with a membrane permeable complex of iron and 8-hydroxyquinoline. These results indicate that damage caused by hydroxyl radicals is minimal when PC12 cells were treated with DA alone and is not likely to be the immediate cause of apoptosis. - dopamine, apoptosis, hydroxyl radicals, PC 12 cells, iron
