Borenfreund E, Puerner J A
Toxicology. 1986 May;39(2):121-34. doi: 10.1016/0300-483x(86)90130-7.
A simple, rapid assay, based on the lysosomal incorporation of neutral red by cells, conveniently carried out in 96-well microtiter plates, was used to evaluate the cytotoxic effect of cationic and anionic metal salts on BALB/c mouse 3T3 fibroblasts. Ranking of the metals according to their decreasing potency was based on spectrophotometrically determined absorbance of the neutral red, extracted from surviving viable cells. The rank order was Cd greater than Hg greater than Ag greater than Zn greater than Mn greater than Cu greater than Co greater than Ni greater than Cr(III) for the cationic metals and Cr2O7 greater than CrO4 greater than AsO2 greater than AsO4 greater than SeO3 greater than SeO4 greater than MnO4 for the anionic metals tested. Cationic metals incubated with cultures in medium containing 1% fetal bovine serum (FBS) were 3-4 times more toxic than in medium with 10% FBS. Cadmium served as a representative metal for the use of this assay not only for concentration, but also for time dependent exposures. Thus a 10% cytotoxic effect after 1 h of incubation with 60 microM cadmium was increased to 90% after 6 h. Examination of the effect of metal-metal interaction on cytotoxicity showed a marked reduction of cadmium toxicity by zinc and to a lesser degree, by nickel. The neutral red assay was also effectively used to investigate the effect of the chelators ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA) and 2,3-dimercaptosuccinic acid (DMSA) on cadmium-induced injury. Cytotoxicity by cadmium was completely inhibited by EDTA, and partially by NTA, but DMSA was ineffective. Reduction of copper toxicity by chelation was less efficient than for cadmium. Use of a chelator as therapy against metal poisoning was only partially effective and limited to administration within 2 h after incubation of cells with cadmium. It is believed that the neutral red assay can be a valuable tool for the screening of cytotoxic and potentially therapeutic agents under controlled in vitro conditions.
一种基于细胞对中性红的溶酶体摄取的简单、快速检测方法，可在96孔微量滴定板中方便地进行，用于评估阳离子和阴离子金属盐对BALB/c小鼠3T3成纤维细胞的细胞毒性作用。根据从存活的活细胞中提取的中性红的分光光度法测定吸光度，对金属按其效力递减进行排序。阳离子金属的排序为：镉＞汞＞银＞锌＞锰＞铜＞钴＞镍＞三价铬；所测试的阴离子金属的排序为：重铬酸根＞铬酸根＞亚砷酸根＞砷酸根＞亚硒酸根＞硒酸根＞高锰酸根。在含有1%胎牛血清（FBS）的培养基中与培养物一起孵育的阳离子金属的毒性比在含有10%FBS的培养基中高3至4倍。镉不仅作为该检测方法在浓度方面的代表性金属，而且在时间依赖性暴露方面也是如此。因此，与60微摩尔镉孵育1小时后产生10%的细胞毒性作用，在6小时后增加到90%。对金属-金属相互作用对细胞毒性的影响进行的研究表明，锌可显著降低镉的毒性，镍的降低程度较小。中性红检测方法还有效地用于研究螯合剂乙二胺四乙酸（EDTA）、次氮基三乙酸（NTA）和2,3-二巯基丁二酸（DMSA）对镉诱导损伤的影响。EDTA可完全抑制镉的细胞毒性，NTA可部分抑制，但DMSA无效。通过螯合降低铜毒性的效率低于镉。使用螯合剂作为金属中毒的治疗方法仅部分有效，且仅限于在细胞与镉孵育后2小时内给药。据信，中性红检测方法可成为在可控的体外条件下筛选细胞毒性和潜在治疗剂的有价值工具。
