title: An in vitro skin sensitization assay termed EpiSensA for broad sets of chemicals including lipophilic chemicals and pre/pro-haptens.
abstract: 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 (P2X7for 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 P2X7specific 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.
authors.affiliation: Kao Corporation, R&D, Safety Science Research, 2606 Akabane Ichikai-Machi Haga-Gun, Tochigi 321-3497, Japan. Electronic address: saitou.kazutoshi@kao.co.jp.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Saito
authors.affiliation: Kao Corporation, R&D, Safety Science Research, 2606 Akabane Ichikai-Machi Haga-Gun, Tochigi 321-3497, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Takenouchi
authors.affiliation: Kao Corporation, R&D, Safety Science Research, 2606 Akabane Ichikai-Machi Haga-Gun, Tochigi 321-3497, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nukada
authors.affiliation: Kao Corporation, R&D, Safety Science Research, 2606 Akabane Ichikai-Machi Haga-Gun, Tochigi 321-3497, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Miyazawa
authors.affiliation: Kao Corporation, R&D, Safety Science Research, 2606 Akabane Ichikai-Machi Haga-Gun, Tochigi 321-3497, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sakaguchi
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D051700:Activating Transcription Factor 3 / Q000235:genetics; D000485:Allergens / Q000633:toxicity*; D000826:Animal Testing Alternatives; D001681:Biological Assay; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D004137:Dinitrochlorobenzene; D005721:Glutamate-Cysteine Ligase / Q000235:genetics; D050956:HSP40 Heat-Shock Proteins / Q000235:genetics; D006241:Haptens / Q000633:toxicity*; D006801:Humans; D006967:Hypersensitivity; D016209:Interleukin-8 / Q000235:genetics; D015603:Keratinocytes / Q000187:drug effects* / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D023441:Local Lymph Node Assay; D051267:NF-E2-Related Factor 2 / Q000235:genetics; D058920:Purinergic P2X Receptor Antagonists / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: C490449:ATF3 protein, human; D051700:Activating Transcription Factor 3; D000485:Allergens; C494650:DNAJB4 protein, human; D004137:Dinitrochlorobenzene; D050956:HSP40 Heat-Shock Proteins; D006241:Haptens; D016209:Interleukin-8; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D058920:Purinergic P2X Receptor Antagonists; D034741:RNA, Small Interfering; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D007770:L-Lactate Dehydrogenase; C546365:GCLM protein, human; D005721:Glutamate-Cysteine Ligase
keywords: Alternative method; Gene expression; Lipophilic chemicals; Pre/pro-haptens; Reconstructed human epidermis (RhE) model; Skin sensitization