Figure 1.

FeCl₂ and CuCl₂ modulate the effects of fisetin on survival and GSH. (A) Fisetin reduces the potentiation of glutamate toxicity by FeCl₂ and CuCl₂. HT22 cells were treated with 2.5 mM glutamate in the presence of 5 µM fisetin and/or FeCl₂ or CuCl₂ at the indicated concentrations. Cell survival was measured after 24 h with the MTT assay. The experiments were done in quadruplicate and the results are the average of 5–6 independent experiments. (B) Fisetin reduces the potentiation of total GSH loss by FeCl₂ and CuCl₂. HT22 cells were treated with 2.5 mM glutamate in the presence of 5 µM fisetin and/or FeCl₂ or CuCl₂ at the indicated concentrations. Cells were harvested after 24 h and total GSH was measured. The results are the average of 5-6 independent experiments. * indicates p < 0.05 and *** indicates p < 0.001 relative to fisetin + glutamate alone and ^ indicates p < 0.05, ^^ indicates p < 0.01 and ^^^ indicates p < 0.001 relative to glutamate alone. (C) Fisetin similarly reduces the potentiation of total GSH loss by FeCl₂ and CuCl₂ in both cytoplasm and mitochondria. HT22 cells were treated with 2.5 mM glutamate in the presence of 10 µM fisetin and/or 5 µM FeCl₂ or CuCl₂ (0.5:1, metal:fisetin). Cells were harvested after 24 h, separated into cytoplasmic and mitochondrial fractions and total GSH was measured. The results are the average of 4–6 independent experiments. ** indicates p < 0.01 and *** indicates p < 0.001 relative to fisetin + glutamate alone. ^^ indicates p < 0.01 and ^^^ indicates p < 0.001 relative to glutamate alone. (D) Fisetin reduces free intracellular Fe²⁺ levels at Fe²⁺:fisetin ratios of 0.5:1 and below. Free intracellular Fe²⁺ was measured using the dye PhenGreen SK after 3 h of treatment with 10 µM fisetin and the indicated concentrations of FeCl₂. (E) Fisetin reduces free intracellular Cu²⁺ levels at Cu²⁺:fisetin ratios of 0.25:1 and below. Free intracellular Cu²⁺ was measured using the dye PhenGreen FL after 3 h of treatment with 10 µM fisetin and the indicated concentrations of CuCl₂. The experiments were done in sextuplicate and the results are the average of 4–6 independent experiments. * indicates p < 0.05 and ** indicates p < 0.01 relative to the metal alone.