McMillian M K, Li L, Parker J B, Patel L, Zhong Z, Gunnett J W, Powers W J, Johnson M D
Department of Molecular Toxicology, The R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ 08869, USA.
Cell Biol Toxicol. 2002;18(3):157-73. doi: 10.1023/a:1015559603643.
A simple resazurin-based cytotoxicity assay is presented for screening of cytotoxicity in hepatocytes and liver cell lines. Human hepatoma (HepG2) cells in 96-well culture plates were exposed to known toxic (cisplatin, 5-fluorouracil, ethionine, flufenamic acid, and diflunisal) and control (transplatin, 5-chlorouracil, methionine, and acetylsalicylic acid) compounds for 1-3 days, and resazurin (5 micromol/L) was added. A conventional short-term (1 h) assay was first performed, where cytotoxicity is indicated by decreased reduction of resazurin to its fluorescent product resorufin. Our improved assay consists of additionally measuring fluorescence 2-4 days later, when cytotoxicity is indicated by a striking increase in the concentration of resorufin, resulting from two distinct processes. First, viable liver-derived cells slowly convert resorufin to nonfluorescent metabolites. Fluorescence of control cell wells decreased to background during a 2- to 4-day exposure to resazurin. This metabolism of resorufin was largely blocked by dicumarol and to lesser extents by disulfiram and SKF525a. Second, dead or dying cells slowly convert resazurin to resorufin but do not further metabolize resorufin; thus this fluorescent metabolite accumulates to high levels in wells with dead cells by 2 to 4 days. A similar increase in fluorescence associated with cytotoxicity was observed in primary cultures of rat hepatocytes using the long-term resazurin-based assay. In addition to an improved signal relative to the short-term assay, the inversion of the fluorescent signal from high = alive short-term to high = dead long-term allows determination of two independent cytotoxicity endpoints after addition of one innocuous vital dye.
本文介绍了一种基于刃天青的简单细胞毒性检测方法，用于筛选肝细胞和肝癌细胞系的细胞毒性。将96孔培养板中的人肝癌（HepG2）细胞暴露于已知毒性化合物（顺铂、5-氟尿嘧啶、乙硫氨酸、氟芬那酸和二氟尼柳）和对照化合物（反铂、5-氯尿嘧啶、蛋氨酸和乙酰水杨酸）中1至3天，然后加入刃天青（5微摩尔/升）。首先进行传统的短期（1小时）检测，刃天青还原为荧光产物试卤灵的减少表明细胞毒性。我们改进的检测方法还包括在2至4天后额外测量荧光，此时细胞毒性由试卤灵浓度的显著增加表明，这是由两个不同过程导致的。首先，有活力的肝源性细胞会将试卤灵缓慢转化为非荧光代谢物。在暴露于刃天青2至4天的过程中，对照细胞孔的荧光降至背景水平。双香豆素在很大程度上阻断了试卤灵的这种代谢，双硫仑和SKF525a的阻断作用较小。其次，死亡或濒死的细胞会将刃天青缓慢转化为试卤灵，但不会进一步代谢试卤灵；因此，到2至4天时，这种荧光代谢物在含有死亡细胞的孔中会积累到高水平。使用基于刃天青的长期检测方法，在大鼠原代肝细胞培养中也观察到了与细胞毒性相关的类似荧光增加。除了相对于短期检测有改善的信号外，荧光信号从短期的高 = 存活反转到长期的高 = 死亡，使得在加入一种无害的活性染料后能够确定两个独立的细胞毒性终点。
