College of Pharmacy, Yeungnam University, Gyeongsan, South Korea.
J Toxicol Environ Health A. 2021 Oct 2;84(19):783-799. doi: 10.1080/15287394.2021.1944939. Epub 2021 Jul 1.
A convenient fluorometrical test method to identify skin sensitizers was developed using reactivity with glutathione (GSH), a low molecular weight endogenous substance. Following incubation of test chemicals with GSH, the remaining GSH was quantitated fluorometrically by using monobromobimane (mBBr), a thiol-detecting agent, for determining % depletion of this endogenous substance by test chemicals. The experimental conditions optimized were: (1) reactivity of thiol compounds including GSH with mBBr, (2) effects of vehicles on reactivity, (3) molar ratios of GSH to test chemicals, and (4) reactivity of endogenous substance with test substances under different incubation times. When an optimized condition with DMSO as a vehicle for test chemicals and in 1:60 ratio for 24 hr at 4°C was applied to classify 48 well-known skin sensitizers and non-sensitizers, the predictive capacity was as follows: 88.2% sensitivity, 78.6% specificity, and 85.4% accuracy with 95.8% consistency of three trials when 10.3% depletion of GSH was used as a cutoff value. Because the present method employed relatively simple GSH as an acceptor for sensitizers and/or a relatively convenient fluorometric detection system in 96-well plates for a high throughput test, it would be a useful test tool for screening skin sensitization potential of test chemicals.
开发了一种方便的荧光检测方法，用于鉴定皮肤致敏剂，该方法利用与谷胱甘肽（GSH）的反应性，GSH 是一种低分子量的内源性物质。将受试化学物质与 GSH 孵育后，用单溴代丁二酰亚胺（mBBr），一种硫醇检测试剂，定量荧光检测剩余的 GSH，用于确定受试化学物质对这种内源性物质的耗竭%。优化的实验条件为：（1）包括 GSH 的巯基化合物与 mBBr 的反应性，（2）载体对反应性的影响，（3）GSH 与受试化学物质的摩尔比，以及（4）不同孵育时间下内源性物质与受试物质的反应性。当应用优化的条件，即 DMSO 作为受试化学物质的载体，在 4°C 下以 1:60 的比例孵育 24 小时，并将 GSH 耗竭 10.3%作为截断值，用于分类 48 种已知的皮肤致敏剂和非致敏剂时，预测能力如下：当使用 10.3%的 GSH 耗竭作为截断值时，88.2%的敏感性、78.6%的特异性和 85.4%的准确性，三次试验的一致性为 95.8%。由于本方法采用相对简单的 GSH 作为接受体，以及相对方便的 96 孔板荧光检测系统用于高通量测试，因此它将成为筛选受试化学物质皮肤致敏潜力的有用测试工具。
