Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute, Japan; The United Graduate School of Veterinary Science, Yamaguchi University, Japan.
Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute, Japan.
Toxicology. 2020 Jun;439:152476. doi: 10.1016/j.tox.2020.152476. Epub 2020 Apr 23.
Two non-animal test methods, KeratinoSens™ and LuSens, have been approved by the Organization of Economic Cooperation and Development (OECD) test guidelines for evaluating the sensitization potential of chemicals, and been positioned as a method for appraising key event (KE)-2, namely, the keratinocyte response component of the Adverse Outcome Pathway (AOP) in sensitization process. However, these two methods require separate cytotoxicity tests to determine the concentrations to be tested in the main test. Therefore, we developed a simple and highly accurate KE-2 test method named α-Sens that uses the dual luciferase assay system and attempted a further application of luciferase-based determination of cell viability to calculate the normalized Antioxidant response element (ARE)-mediated transcriptional activity, named normalized ARE Activity (nAA), to evaluate the sensitizing potential of chemicals. A cell line carrying the ARE-inducible Firefly luciferase reporter gene and Thymidine kinase (TK) promoter-driven Renilla luciferase gene was established and used for the α-Sens. A total of 28 chemicals, consisting of 19 skin sensitizers and nine non-skin sensitizers were tested by this assay system. The α-Sens yielded an accuracy (%), sensitivity (%), and specificity (%) against corresponding values for local lymph node assay of 96.4 %, 95.0 %, and 100 %, respectively, and for human data of 100 % for all. The α-Sens gave clear positive results for phenyl benzoate and eugenol, chemicals for which KeratinoSens™ or LuSens yielded false-negative results, using a new parameter. Our results suggest that better prediction capacity could be achieved by using nAA as a classifier compared to other existing KE-2 test methods. In conclusion, the α-Sens is promising as a simple and highly accurate in vitro skin sensitization test method for evaluation of KE-2.
两种非动物测试方法，KeratinoSens™和 LuSens，已经被经济合作与发展组织（OECD）的测试指南批准用于评估化学品的致敏潜力，并被定位为评价关键事件（KE）-2的方法，即致敏过程中不良反应途径（AOP）的角质细胞反应成分。然而，这两种方法需要单独的细胞毒性测试来确定主测试中要测试的浓度。因此，我们开发了一种简单而高度准确的 KE-2 测试方法，称为α-Sens，该方法使用双荧光素酶测定系统，并尝试进一步应用基于荧光素的细胞活力测定来计算归一化抗氧化反应元件（ARE）介导的转录活性，称为归一化 ARE 活性（nAA），以评估化学品的致敏潜力。建立了一种携带 ARE 诱导萤火虫荧光素酶报告基因和胸苷激酶（TK）启动子驱动的海肾荧光素酶基因的细胞系，并将其用于α-Sens。使用该测定系统共测试了 28 种化学品，包括 19 种皮肤致敏剂和 9 种非皮肤致敏剂。α-Sens 的准确性（%）、灵敏度（%）和特异性（%）分别为局部淋巴结试验的 96.4%、95.0%和 100%，人类数据的 100%。对于苯佐卡因和丁香酚等化学品，KeratinoSens™或 LuSens 产生了假阴性结果，而α-Sens 使用新参数给出了明确的阳性结果。我们的结果表明，与其他现有的 KE-2 测试方法相比，使用 nAA 作为分类器可以获得更好的预测能力。总之，α-Sens 有望成为一种简单而高度准确的体外皮肤致敏测试方法，用于评价 KE-2。
