Superfund Research Program
Stress Gene Induction in Mammalian Cells
Experiments were completed to evaluate oxidative stress by paraquat and copper (Cu) in neural cells and physiological consequences of interactions of heavy metals with the stress gene 78 kD glucose regulated protein (GRP 78) in glial cells.
Oxidative stress by paraquat. The objective of the first set of experiments was to study the toxicity of paraquat as an environmental risk factor involved in the incidence of neurodegenerative disease. Paraquat was found to induce oxidative stress in dopaminergic SH-SY5Y cells as evidenced by the elevation of reactive oxygen species, depletion of GSH, dissipation of mitochondrial membrane potential, increase in lipid peroxidation and protein oxidation, and DNA fragmentation. Therefore, these data support the hypothesis that the bipyridyl herbicide paraquat is an environmental risk factor implicated in the incidence of neurodegenerative disease.
Interactions of GRP78 with metals. Experiments were also conducted to directly evaluate the interactions of GRP78 with metals. GRP78 is molecular chaperone located in the endoplasmic reticulum (ER), functioning in protein folding, assembly and trafficking, and protecting against oxidative stress. In these experiments, it was demonstrated that heavy metals can target GRP78 in living cells. CCF-STTG1 astrocytoma cells were induced to express a chimeric protein for rat GRP78 with enhanced green fluorescence protein (EGFP) by transcient transfection with an engineered construct. Transfected cells were exposed to 50 µM CuSO4, 100 µM MnCl2 or 5 µM Pb acetate for 5 and 10 hrs and GRP78-EGFP distribution was examined by image analysis. In the GRP78-depleted cells, apoptosis-related gene expression was analyzed by a cDNA array. In all 96 apoptosis-related genes, only two anti-apoptotic gene expressions were modulated after 24hrs of GRP78 inhibition. Bfl-1 was down-regulated and Traf1 was up-regulated, suggesting GRP78 is associated with apoptosis. These results indicate that heavy metals can target GRP78 and induce its aggregation in vivo. Loss of GRP78 function may result in activation of apoptosis.