title: Development of an in vitro skin sensitization test based on ROS production in THP-1 cells.
abstract: Recently, it has been reported that reactive oxygen species (ROS) produced by contact allergens can affect dendritic cell migration and contact hypersensitivity. The aim of the present study was to develop a new in vitro assay that could predict the skin sensitizing potential of chemicals by measuring ROS production in THP-1 (human monocytic leukemia cell line) cells. THP-1 cells were pre-loaded with a ROS sensitive fluorescent dye, 5-(and 6-)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), for 15min, then incubated with test chemicals for 30min. The fluorescence intensity was measured by flow cytometry. For the skin sensitizers, 25 out of 30 induced over a 2-fold ROS production at more than 90% of cell viability. In contrast, increases were only seen in 4 out of 20 non-sensitizers. The overall accuracy for the local lymph node assay (LLNA) was 82% for 50 chemicals tested. A correlation was found between the estimated concentration showing 2-fold ROS production in the ROS assay and the EC3 values (estimated concentration required to induce positive response) of the LLNA. These results indicated that the THP-1 cell-based ROS assay was a rapid and highly sensitive detection system able to predict skin sensitizing potentials and potency of chemicals.
authors.affiliation: Safety Science Research Laboratories, Kao Corporation, 2606 Akabane, Ichikai-Machi, Haga-Gun, Tochigi 321-3497, Japan. saitou.kazutoshi@kao.co.jp
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Saito
authors.forename: Masaaki
authors.initials: M
authors.lastname: Miyazawa
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nukada
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Sakaguchi
authors.forename: Naohiro
authors.initials: N
authors.lastname: Nishiyama
mesh_terms: D000485:Allergens / Q000633:toxicity*; D000826:Animal Testing Alternatives; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017449:Dermatitis, Allergic Contact / Q000209:etiology*; D006241:Haptens / Q000633:toxicity*; D006801:Humans; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D000485:Allergens; D006241:Haptens; D017382:Reactive Oxygen Species