Antioxidant effect of flavonoids after ascorbate/Fe(2+)-induced oxidative stress in cultured retinal cells.

In this study, we investigated the structure-activity relationship of four flavonoids, i.e. eriodictyol, luteolin, quercetin, and taxifolin, in cultured retinal cells after ascorbate/Fe(2+)-induced oxidative stress. The relative order of antioxidant efficacy, determined by the thiobarbituric acid method, was the following: eriodictyol > quercetin > luteolin > taxifolin. Upon preincubation, the flavonoids were also effective in reducing the extent of lipid peroxidation. Oxidative stress, determined by the changes in fluorescence of 2',7'-dichlorodihydrofluorescein, was also decreased in the presence of the flavonoids, showing the following order of antioxidant efficacy: eriodictyol > taxifolin approximately quercetin > luteolin. Ascorbate/Fe(2+)-induced oxidative stress or incubation in the presence of the flavonoids did not significantly affect the viability of retinal cells. We also evaluated the degree of membrane partition of the flavonoids. In this system, the results strongly suggest that the higher antioxidant activity of the flavonoids is not correlated with the presence of a double bond at C(2)-C(3) and/or a hydroxyl group at C(3) on the C ring, but rather may depend on the capacity to inhibit the production of reactive oxygen species to interact hydrophobically with membranes. Eriodictyol was shown to be the most efficient antioxidant in protecting against oxidative stress induced by ascorbate/Fe(2+) in the retinal cells.

Distinct glycolysis inhibitors determine retinal cell sensitivity to glutamate-mediated injury.

In this study, we analyzed how distinct glycolysis inhibitors influenced the redox status of retinal cells, used as a neuronal model. Three different approaches were used to inhibit glycolysis: the cells were submitted to iodoacetic acid (IAA), an inhibitor of glyceraldehyde 3-phosphate dehydrogenase, to 2-deoxy-glucose (DG) in glucose-free medium, which was used as a substitute of glucose, or in the absence of glucose. The redox status of the cells was evaluated by determining the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). By the analysis of dose-response curves of MTT reduction, IAA showed values of IC50 = 7.02 x 10(-5) M, whereas DG showed values of IC50 = 7.42 x 10(-4) M. Upon 30 min-incubation, glucose deprivation, per se, did not significantly affect MTT reduction. We also evaluated the reduction of MTT as an indicator of cell injury by exposing the cells to 100 microM glutamate during the decrement of glycolysis function. In the presence of glutamate, for 2 h, there was a decrease in MTT reduction, which was potentiated in the presence of DG (10-20% decrease), in the presence of IAA (about 30% decrease) or in glucose-free medium (about 30% decrease). Major changes observed by the MTT assay, upon exposure to glutamate, indicative of changes in the redox status of retinal cells, were concomitant with variations in intracellular ATP. Under glucose deprivation, endogenous ATP decreased significantly from 38.9+/-4.4 to 13.3+/-0.7 nmol/mg protein after exposure to 100 microM glutamate. The results support a different vulnerability of retinal cells after being exposed to distinct forms of glycolysis inhibition.

Effect of glucose deprivation and acute glutamate exposure in cultured retinal cells.

The relationship between bioenergetics and the glutamate system was analyzed in a neuronal model of retinal cells in culture, submitted to glucose deprivation and exposed to glutamate for 2 h, and compared with exposure to glutamate in the presence of glucose. Under glucose deprivation, a reduction (about 1.1-fold) in the energy charge of the cells occurred, probably as a result of a decrement (by about 75%) in the cellular redox efficacy, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. In the absence of glucose, exposure of retinal cells to 10 microM glutamate potentiated the reduction in the energy charge (by about 1.2-fold) and induced a significant increase in the uptake of 45Ca2+ by the cells (1.3-fold), although no significant changes were observed in the presence of glucose. Under glucose deprivation, 100 microM glutamate caused an irreversible cell membrane damage, as shown by the significant increase in lactate dehydrogenase (LDH) leakage (about 1.8-fold). A significant increase in membrane depolarization, measured by the reduction of [3H]tetraphenylphosphonium+ ([3H]TPP+) uptake, was also observed after glutamate exposure in the absence of glucose. In the presence of glucose, high glutamate concentrations (10 mM) induced a major increase in Ca2+ entry into the cells and membrane depolarization, without affecting the energy charge or cell survival. In contrast, in the absence of glucose, 10 mM glutamate did not alter Ca2+ accumulation by the cells and a smaller decrease in membrane potential occurred, as compared to 100 microM glutamate exposure. Data shown in this study suggest that during a prolonged (2 h) and acute exposure to high glutamate (10 mM), under glucose deprivation conditions, the neuronal systems have "adaptive" mechanisms that allow the survival of cells. These findings may have implications in neuronal degeneration occurring during brain ischemia.