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A. J. Ondricek, J. W. Simpkins, J. K. Vishwanatha; Phytoestrogens as Neuroprotectants in Optic Neuropathies. Invest. Ophthalmol. Vis. Sci. 2008;49(13):867.
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Phytoestrogens are a class of plant flavenoids that act as free radical scavengers utilizing hydroxyl groups on a conjugated ring structure. In this study we characterized fisetin hydrate, apigenin, kaempferol, genistein and genistin. We hypothesize that mitochondrial distress results in retinal ganglion cell death in optic neuropathies and that phytoestrogens are a plausible treatment in normal pressure neuropathies as well as when co-administered with intraocular pressure (IOP) lowering drugs. Iodoacetic acid (IAA) induced oxidative stress and mitochondrial dysfunction in cultured RGC-5 cells and was therefore used as a model to test the efficacy of phytoestrgens as antioxidants and neuroprotectants.
Transformed rat retinal ganglion cells (RGC-5) were treated with varying concentrations of IAA from 2-10 µM. Antioxidants were administered at concentrations of 5-50 µM along with IAA. Cell viability was assessed by Calcein/AM assay. Involvement of reactive oxygen species (ROS) was determined by H2DCF (dichlorofluorescein)-DA assay, caspase-3 activity by fluorescent assay, and changes in mitochondrial membrane potential (MMP) were determined by JC-1 fluorescent dye.
IAA is cytotoxic to RGC-5 cells and induces the generation of ROS in vitro. 17-β estradiol rescues RGC-5 cells from death in the presence of IAA, as well as generation of ROS. Apigenin and fisetin hydrate do not rescue IAA induced cytotoxicity to RGC-5 cells. Genistein rescues RGC-5 cells in the presence of IAA, however, it also increases caspase activation and does not inhibit the generation of ROS. Kaempferol and genistin rescue RGC-5 cells from IAA induced cell death as well as reduce caspase activation and ROS generation.
IAA is cytotoxic to retinal ganglion cells. IAA cytotoxicity was reversed in the presence of exogenous antioxidants, kaempferol, genistein and genistin. IAA treatment results in a loss of MMP of RGC-5 cells. DCF assay suggested generation of ROS by IAA treatment, which was reversed by the phytoestrogens kaempferol and genistin. Caspases were activated in the presence of IAA, which was rescued by kaempferol and genistin. Taken together these results suggest that though these compounds are structurally very similar, functional differences do exist.
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