Skin sensitization assessment has progressed from the use of animal models towards the application of New Approach Methodologies (NAMs). Several skin sensitization NAMs are accepted for regulatory use, but a majority relies on submerged cell cultures that limit their applicability domain, posing challenges for testing hydrophobic chemicals and mixtures. A newly developed three-dimensional (3D) Nrf2 reporter epidermis model for skin sensitization assessment is reported. This NAM may help to overcome these limitations. The NAM combines the -like biology and exposure conditions of 3D epidermis models with the reliability, convenience, and cost-effectiveness of secreted reporter gene technology. The Keap1-Nrf2-ARE pathway was chosen as the reporter gene read-out, as it is induced by most skin sensitizers and already adopted in OECD Test guideline 442D. Immortalized human primary keratinocytes (Ker-CT) were stably transfected with the pIGB-Nrf2-SEAP vector to construct a Nrf2 reporter cell line. Ker-CT Nrf2 reporter cells showed negligible basal expression of the Secreted Embryonic Alkaline Phosphatase (SEAP) reporter, which was induced 13.5-fold by exposure to the skin sensitizer cinnamic aldehyde (CA). Co-exposure to CA and the Nrf2 inhibitor glucocorticoid clobetasol propionate significantly suppressed the CA-induced SEAP expression, confirming dependance of the SEAP expression on Nrf2 activation. Using air-liquid interface and animal constituent free culture conditions, the Ker-CT Nrf2 reporter cells differentiated to stratified 3D epidermis models with an like skin architecture and functional skin barrier. Evaluation of a Ker-CT Nrf2 reporter cell-based 2D assay by testing 10 conventional reference chemicals showed a predictive accuracy for skin sensitization potential of 80% and 70% compared to LLNA and human data in two independent laboratories and a high intra- and interlaboratory reproducibility. Moreover, the 3D epidermis models predicted 3 sensitizing and 2 non-sensitizing reference chemicals correctly in a first proof-of-concept study. Further investigations foresee the testing of additional chemicals, including hydrophobic compounds and mixtures to confirm the potential of the 3D epidermis models to broaden the applicability domain for NAM-based skin sensitization assessment.

中文翻译：

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一种用于皮肤致敏评估的新型三维 Nrf2 报告基因表皮模型

皮肤致敏评估已从使用动物模型发展到应用新方法（NAM）。几种皮肤致敏 NAM 已被监管使用，但大多数依赖于水下细胞培养，这限制了它们的适用范围，给疏水性化学品和混合物的测试带来了挑战。报道了一种新开发的用于皮肤致敏评估的三维（3D）Nrf2 报告基因表皮模型。该 NAM 可能有助于克服这些限制。 NAM 将 3D 表皮模型的类似生物学和暴露条件与分泌报告基因技术的可靠性、便利性和成本效益相结合。选择 Keap1-Nrf2-ARE 途径作为报告基因读数，因为它是由大多数皮肤致敏剂诱导的，并且已在 OECD 测试指南 442D 中采用。用pIGB-Nrf2-SEAP载体稳定转染永生化人原代角质形成细胞（Ker-CT），构建​​Nrf2报告细胞系。 Ker-CT Nrf2 报告细胞显示分泌型胚胎碱性磷酸酶 (SEAP) 报告基因的基础表达可忽略不计，该表达因暴露于皮肤致敏剂肉桂醛 (CA) 而诱导 13.5 倍。共同暴露于 CA 和 Nrf2 抑制剂糖皮质激素丙酸氯倍他索显着抑制了 CA 诱导的 SEAP 表达，证实了 SEAP 表达对 Nrf2 激活的依赖性。使用气液界面和无动物成分的培养条件，Ker-CT Nrf2 报告细胞分化为具有相似皮肤结构和功能性皮肤屏障的分层 3D 表皮模型。通过测试 10 种传统参考化学品，对 Ker-CT Nrf2 报告基因基于细胞的 2D 测定进行评估，结果显示，与两个独立实验室中的 LLNA 和人类数据以及实验室内和实验室间的高数据相比，皮肤致敏潜力的预测准确度分别为 80% 和 70%再现性。此外，3D 表皮模型在第一项概念验证研究中正确预测了 3 种致敏和 2 种非致敏参考化学品。进一步的研究预计将测试其他化学物质，包括疏水性化合物和混合物，以确认 3D 表皮模型扩大基于 NAM 的皮肤致敏评估的适用范围的潜力。