A digitized fluorescence imaging study of intracellular Ca²⁺, pH, and mitochondrial function in primary cultures of rabbit corneal epithelial cells exposed to sodium dodecyl sulfate

Summary

Primary cultures of rabbit corneal epithelial cells have been developed as an in vitro system to predict irritancy potential and delayed cytotoxicity of surfactants in our laboratory. The objective of this study was to evaluate the effects of the surfactant sodium dodecyl sulfate (SDS), a common ingredient in consumer products, on intracellular Ca²⁺, pH, and mitochondrial function in this culture system. Ca²⁺ and pH were measured in single living corneal epithelial cells by ratio imaging of fura-2 and 2,′7′-bis(carboxyethyl)-5(6)-carboxyfluorescein fluorescence, respectively. Mitochondrial function was examined by probing mitochondrial membrane potential with the fluorescent dye rhodamine 123 and by measuring the ratio of ATP to ADP with an HPLC method. Cell viability was determined by fluorescence imaging of propidium iodide in single cells and LDH leakage assay in populations of cells. SDS (40 µg/ml) increased intracellular Ca²⁺ from 180±28nM to 453±86 nM within 2 min, and induced intracellular acidification (pHi dropped 0.3 units in 15 min). Treatment of the cultures with SDS also resulted in dissipation of the mitochondrial membrane potential and decrease of intracellular ATP/ADP. SDS-induced Ca²⁺ elevation and intracellular acidification preceded the loss of cell viability observed 20 min after exposure. However, SDS-induced cell injury does not appear to be triggered by extracellular Ca²⁺-influx, as absence of extracellular Ca²⁺ did not attenuate SDS-induced cytotoxicity while it completely blocked ionomycin-induced cytotoxicity. In summary, we observed a series of intracellular events that occurred temporally after exposure to the surfactant: elevation of intracellular Ca²⁺ and intracellular acidification, dissipation of mitochondrial membrane potential, decrease of ATP/ADP ratio, and subsequent cell injury.