Antioxidants and ocular cell type differences in cytoprotection from formic acid toxicity in vitro.

Retinal photoreceptors and retinal pigment epithelial (RPE) cells are among the cell types that are sensitive to poisoning with methanol and its toxic metabolite formic acid. When exposed to formic acid in vitro, cultured cell lines from photoreceptors (661W) and the RPE (ARPE-19) were previously shown to accumulate similar levels of formate, but cytotoxic effects are greater in 661W cells. Here catalase and glutathione were analyzed in the two retinal cell lines to determine whether differences in these antioxidant systems contributed to cell-type-specific differences in cytotoxicity. Cells were exposed to formic acid (pH 6.8) in the culture medium in the presence or absence of a catalase activity inhibitor, 3-amino-1,2,4-triazole (AT), or a glutathione synthesis inhibitor, buthionine L-sulfoximine (BSO). Catalase protein, catalase enzyme activity, glutathione, glutathione peroxidase activity, cellular ATP, and cytotoxicity were analyzed. Compared to ARPE-19, 661W cells show lower antioxidant levels: 50% less glutathione, glutathione peroxidase and catalase protein, and 90% less catalase enzyme activity. In both cell types, formic acid treatment produced decreases in glutathione and glutathione peroxidase, and glutathione synthesis inhibition with BSO produced greater ATP depletion and cytotoxicity than formic acid treatment alone. In contrast, formate exposure produced decreases in catalase protein and activity in 661W cells, but increases in activity in ARPE-19. Treatment with the catalase inhibitor AT increased the formate sensitivity only of the ARPE-19 cells. ARPE-19 cells, therefore, may be less susceptible to formate toxicity due to higher levels of antioxidants, especially catalase, which increases on formate treatment and which has a significant cytoprotective effect for the RPE cell line.

Formate, the toxic metabolite of methanol, in cultured ocular cells.

Methanol has neurotoxic actions on the human retina due to its metabolite, formic acid, which is a mitochondrial toxin. In methanol poisoned animals, morphologic changes were seen both in retinal photoreceptors and in cells of the underlying retinal pigment epithelium (RPE). Here the effects of formate exposure on the two retinal cell types were analyzed in more detail in vitro using photoreceptor (661W) and RPE (ARPE-19) cell lines. Cells were exposed for time courses from minutes to days to sodium formate at pH 7.4 or to formic acid at pH 6.8, to simulate the metabolic acidosis that accompanies methanol poisoning. Formate accumulation, cellular ATP, cytotoxicity (lactate dehydrogenase (LDH) release) and cell phenotype were analyzed. Formate accumulated with a similar biphasic pattern in both cell types, and to similar levels whether delivered as sodium formate or as formic acid. ATP changes with sodium formate treatment differed between cell types with only 661W cells showing a rapid (within minutes), transient ATP increase. The subsequent ATP decrease was earlier in 661W cells (6 h) than the ATP decrease in ARPE-19 cells (24 h), and although both cell types showed evidence of cytotoxicity, the effects were greater for 661W cells. Both cell types showed enhanced morphologic and biochemical changes with formic acid treatment including earlier and/or greater effects on ATP depletion and cytotoxicity; again effects were more pronounced in 661W cells. Formate therefore is toxic for both cell lines, with 661W cells exhibiting greater sensitivity. Medium pH also appears to play a significant role in formate toxicity in vitro.