Grant R L, Yao C, Gabaldon D, Acosta D
College of Pharmacy, University of Texas, Austin 78712.
Toxicology. 1992 Nov 30;76(2):153-76. doi: 10.1016/0300-483x(92)90162-8.
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.
本研究旨在开发细胞毒性检测系统，该系统以兔角膜上皮细胞原代培养物作为实验模型，用于评估眼毒性药物以及这些体外检测系统预测刺激潜力和延迟毒性的能力。我们通过用抗角蛋白抗体（AE1/AE3）鉴定角蛋白以及证明对细胞完整性重要的代谢酶：乳酸脱氢酶、葡萄糖6-磷酸脱氢酶和醛缩酶，来表征培养物的上皮性质。比较了八种表面活性剂，并根据它们的细胞毒性潜力进行排序。我们通过测量胞质酶乳酸脱氢酶泄漏到培养基中的情况、进行形态学观察以及评估溶酶体中性红摄取和线粒体3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四氮唑（MTT）还原，来评估细胞毒性。用表面活性剂处理细胞1小时，并在去除表面活性剂24小时后评估延迟毒性的可能性。所有测试显示，不同类型表面活性剂的细胞毒性为阳离子型＞阴离子型 = 两性离子型＞非离子型。Triton X-100是一种非离子型表面活性剂，但却是一种强刺激物，其排名与阴离子型表面活性剂相似。体外排名与报道的体内Draize兔眼试验数据非常吻合。乳酸脱氢酶泄漏的24小时试验表明，轻度和无刺激性的表面活性剂在暴露1小时后未显示任何后续损伤，但极端和强刺激性的表面活性剂在暴露1小时后继续显示出进一步损伤。这些体外研究结果与报道的体内结果相似。如乳酸脱氢酶泄漏试验所示，中性红和MTT试验不能充分预测刺激性较强的表面活性剂的延长毒性。我们得出结论，使用兔角膜上皮细胞原代培养物进行的体外细胞毒性试验可用于对表面活性剂的细胞毒性潜力进行排序，但只有乳酸脱氢酶泄漏试验能够评估延长的细胞损伤。
