Saito Kazutoshi, Takenouchi Osamu, Nukada Yuko, Miyazawa Masaaki, Sakaguchi Hitoshi
Kao Corporation, R&D, Safety Science Research, 2606 Akabane Ichikai-Machi Haga-Gun, Tochigi 321-3497, Japan.
Kao Corporation, R&D, Safety Science Research, 2606 Akabane Ichikai-Machi Haga-Gun, Tochigi 321-3497, Japan.
Toxicol In Vitro. 2017 Apr;40:11-25. doi: 10.1016/j.tiv.2016.12.005. Epub 2016 Dec 10.
To evaluate chemicals (e.g. lipophilic chemicals, pre/pro-haptens) that are difficult to correctly evaluate using in vitro skin sensitization tests (e.g. DPRA, KeratinoSens or h-CLAT), we developed a novel in vitro test termed "Epidermal Sensitization Assay: EpiSensA" that uses reconstructed human epidermis. This assay is based on the induction of multiple marker genes (ATF3, IL-8, DNAJB4 and GCLM) related to two keratinocyte responses (inflammatory or cytoprotective) in the induction of skin sensitization. Here, we first confirmed the mechanistic relevance of these marker genes by focusing on key molecules that regulate keratinocyte responses in vivo (P2X for inflammatory and Nrf2 for cytoprotective responses). The up-regulation of ATF3 and IL-8, or DNAJB4 and GCLM induced by the representative sensitizer 2,4-dinitrochlorobenzene in human keratinocytes was significantly suppressed by a P2X specific antagonist KN-62, or by Nrf2 siRNA, respectively, which supported mechanistic relevance of marker genes. Moreover, the EpiSensA had sensitivity, specificity and accuracy of 93%, 100% and 93% for 29 lipophilic chemicals (logKow≥3.5), and of 96%, 75% and 88% for 43 hydrophilic chemicals including 11 pre/pro-haptens, compared with the LLNA. These results suggested that the EpiSensA could be a mechanism-based test applicable to broad sets of chemicals including lipophilic chemicals and pre/pro-haptens.
为了评估那些难以通过体外皮肤致敏试验（如DPRA、KeratinoSens或h-CLAT）进行正确评估的化学物质（如亲脂性化学物质、前/原半抗原），我们开发了一种名为“表皮致敏试验：EpiSensA”的新型体外试验，该试验使用重建的人表皮。该试验基于在皮肤致敏诱导过程中与两种角质形成细胞反应（炎症或细胞保护）相关的多个标记基因（ATF3、IL-8、DNAJB4和GCLM）的诱导。在此，我们首先通过关注体内调节角质形成细胞反应的关键分子（炎症反应的P2X和细胞保护反应的Nrf2）来确认这些标记基因的机制相关性。代表性致敏剂2,4-二硝基氯苯在人角质形成细胞中诱导的ATF3和IL-8或DNAJB4和GCLM的上调分别被P2X特异性拮抗剂KN-62或Nrf2 siRNA显著抑制，这支持了标记基因的机制相关性。此外，与LLNA相比，EpiSensA对29种亲脂性化学物质（logKow≥3.5）的敏感性、特异性和准确性分别为93%、100%和93%，对43种亲水性化学物质（包括11种前/原半抗原）的敏感性、特异性和准确性分别为96%、75%和88%。这些结果表明，EpiSensA可能是一种基于机制的试验，适用于包括亲脂性化学物质和前/原半抗原在内的广泛化学物质。
