Evaluation of surfactant cytotoxicity potential by primary cultures of ocular tissues: I. Characterization of rabbit corneal epithelial cells and initial injury and delayed toxicity studies.
This investigation was undertaken to develop cytotoxicity assay systems using primary cultures of rabbit corneal epithelial cells as an experimental model to evaluate oculotoxic agents and the ability of these in vitro assay systems to predict irritancy potential and delayed toxicity. We have characterized the epithelial nature of the cultures by identifying keratins with antikeratin antibodies (AE1/AE3) and by demonstrating metabolic enzymes important to the integrity of the cells: lactate dehydrogenase, glucose 6-phosphate dehydrogenase and aldolase. Eight surfactants were compared and ranked according to their cytotoxic potential. We evaluated cytotoxicity by measuring leakage of the cytosolic enzyme, lactate dehydrogenase, into the medium, by making morphological observations and by assessing lysosomal neutral red uptake and mitochondrial 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction. The cells were treated for 1 h with the surfactants and the possibility of delayed toxicity was evaluated 24 h after removal of the surfactant. The cytotoxicity of the different types of surfactants as shown by all the tests was cationic > anionic = amphoteric > non-ionic. Triton X-100, a non-ionic surfactant but a severe irritant, had a ranking similar to anionic surfactants. The in vitro rankings corresponded well to reported in vivo Draize rabbit eye test data. The 24-h test for lactate dehydrogenase leakage showed that mild and non-irritating surfactants did not demonstrate any subsequent damage after a 1-h exposure, but the extreme and severe surfactants continued to show further damage after the 1-h exposure. These in vitro findings were similar to reported in vivo results. The neutral red and MTT tests did not adequately predict the prolonged toxicity of the more irritating surfactants, as was demonstrated by the lactate dehydrogenase leakage test. We conclude that in vitro cytotoxicity assays using primary cultures of rabbit corneal epithelial cells may be used to rank the cytotoxic potential of surfactants, but only the lactate dehydrogenase leakage test was able to assess prolonged cell injury.
D000826:Animal Testing Alternatives; D000818:Animals; D002478:Cells, Cultured; D004396:Coloring Agents; D003315:Cornea / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D005634:Fructose-Bisphosphate Aldolase / Q000032:analysis; D005954:Glucosephosphate Dehydrogenase / Q000032:analysis; D007633:Keratins / Q000032:analysis; D007770:L-Lactate Dehydrogenase / Q000032:analysis / Q000378:metabolism; D008297:Male; D009499:Neutral Red; D011817:Rabbits; D013501:Surface-Active Agents / Q000633:toxicity*; D013778:Tetrazolium Salts; D013844:Thiazoles
D004396:Coloring Agents; D013501:Surface-Active Agents; D013778:Tetrazolium Salts; D013844:Thiazoles; D009499:Neutral Red; D007633:Keratins; D007770:L-Lactate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D005634:Fructose-Bisphosphate Aldolase; C022616:thiazolyl blue