Nepal Rahul Upadhyay, Jeong Tae Cheon
College of Pharmacy, Yeungnam University, Gyeongsan, Republic of Korea.
J Toxicol Environ Health A. 2025;88(15):589-603. doi: 10.1080/15287394.2025.2472224. Epub 2025 Mar 4.
The covalent binding of sensitizer to skin proteins is referred to as key event 1 of the adverse outcome pathway in skin sensitization. Recently, -acetyl-L-cysteine methyl ester (NACME) was demonstrated to react selectively with skin sensitizers , such that NACME might be applied as an electron donor in developing a spectrophotometric test for determining skin sensitization potential of chemicals. To avoid possible color interference by certain test chemicals, a fluorometric test method was developed using monobromobimane (mBBr), a thiol-reactive fluorescent probe. Similar to previous methods utilizing the reaction of NACME with sensitizers, unreacted NACME occurred which was then measured fluorometrically using mBBr, rather than 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). Following the optimization of test conditions, the same 64 test chemicals used in the previous study were tested to determine the predictive capacity of the current method. Results showed a predictive capacity of 81.1% sensitivity, 81.5% specificity, and 81.3% accuracy with a cutoff NACME depletion of 11.3%. Although these values were relatively lower than the previous test using DTNB, the results were still comparable to OECD-approved test methods and that color interference issues might be ruled out. Data demonstrated that NACME might be viewed as a candidate for identifying reactive skin sensitizers. Further, this method might be considered as a complementary or supportive method to the former DTNB assay as a screening tool for assessing the tendency of a chemical to initiate skin sensitization in case of test chemicals showing color interference.
致敏剂与皮肤蛋白质的共价结合被称为皮肤致敏不良结局途径中的关键事件1。最近，已证明N-乙酰-L-半胱氨酸甲酯（NACME）能与皮肤致敏剂发生选择性反应，因此NACME可作为电子供体，用于开发一种分光光度法来测定化学品的皮肤致敏潜力。为避免某些测试化学品可能产生的颜色干扰，开发了一种使用硫醇反应性荧光探针单溴联苯胺（mBBr）的荧光测试方法。与之前利用NACME与致敏剂反应的方法类似，会出现未反应的NACME，然后使用mBBr而非5,5'-二硫代双（2-硝基苯甲酸）（DTNB）进行荧光测定。在优化测试条件后，对之前研究中使用的相同64种测试化学品进行测试，以确定当前方法的预测能力。结果显示，当NACME消耗截止值为11.3%时，预测能力为灵敏度81.1%、特异性81.5%、准确度81.3%。尽管这些值相对低于之前使用DTNB的测试，但结果仍与经经合组织批准的测试方法相当，且颜色干扰问题可以排除。数据表明，NACME可被视为识别反应性皮肤致敏剂的候选物质。此外，对于显示颜色干扰的测试化学品，该方法可被视为前一种DTNB测定法的补充或支持方法，作为评估化学品引发皮肤致敏倾向的筛选工具。
