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

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.