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.
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 micrograms/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 Ca(2+)-influx, as absence of extracellular Ca2+ did not attenuate SDS-induced cytotoxicity while it completely blocked ionomycin-induced cytotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)
Yang
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Division of Pharmacology & Toxicology, College of Pharmacy, University of Texas, Austin 78712, USA.
Acosta
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D000244:Adenosine Diphosphate / Q000032:analysis; D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000032:analysis*; D002478:Cells, Cultured; D003315:Cornea / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D004847:Epithelial Cells; D004848:Epithelium / Q000737:chemistry / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007089:Image Enhancement; D015759:Ionomycin / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D011817:Rabbits; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D013501:Surface-Active Agents / Q000494:pharmacology*
D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D015759:Ionomycin; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D002118:Calcium