A Comparison of the Effects of Ocular Preservatives on Mammalian and Microbial ATP and Glutathione Levels

Abstract

The aim of this study was to investigate the mechanism of action of the preservative sodium chlorite (NaClO2), and the relationship with intracellular glutathione depletion. A detailed comparison of the dose responses of two cultured ocular epithelial cell types and four species of microorganism was carried out, and comparisons were also made with the quaternary ammonium compound benzalkonium chloride (BAK), and the oxidant hydrogen peroxide (H2O2). The viability of mammalian and microbial cells was assessed in the same way, by the measurement of intracellular ATP using a bioluminescence method. Intracellular total glutathione was measured by reaction with 5,5′-dithiobis-2-nitrobenzoic acid in a glutathione reductase-dependent recycling assay. BAK and H2O2 caused complete toxicity to conjunctival and corneal epithelial cells at ∼25 ppm, in contrast to NaClO2, where >100 ppm was required. The fungi Candida albicans and Alternaria alternata had a higher resistance to NaClO2 than the bacteria Staphyloccus aureus and Pseudomonas aeruginosa, but the bacteria were extremely resistant to H2O2. NaClO2 caused substantial depletion of intracellular glutathione in all cell types, at concentrations ranging from <10 ppm in Pseudomonas, 25–100 ppm in epithelial cells, to >500 ppm in fungal cells. The mechanisms of cytotoxicity of NaClO2, H2O2 and BAK all appeared to differ. NaClO2 was found to have the best balance of high antibacterial toxicity with low ocular toxicity. The lower toxicity of NaClO2 to the ocular cells, compared with BAK and H2O2, is in agreement with fewer reported adverse effects of application in the eye.