title: An in vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8.
abstract: Several studies have suggested that interleukin (IL)-8 can serve as a biomarker for discrimination of skin sensitizers from nonsensitizers. We established a stable THP-1-derived IL-8 reporter cell line, THP-G8, which harbors SLO and SLR luciferase genes under the control of IL-8 and glyceraldehyde 3-phosphate dehydrogenase promoters, respectively. After 6 h treatment with chemicals, normalized SLO luciferase activity (nSLO-LA) was calculated by dividing SLO-LA by SLR-LA, and the fold induction of nSLO-LA (FInSLO-LA) was calculated by dividing nSLO-LA of chemically treated cells by that of nontreated cells. The nSLO-LA of THP-G8 cells increased in response to lipopolysaccharide (LPS) and several sensitizers. The FInSLO-LA in THP-G8 cells induced by LPS or sensitizers positively correlated with their induction of IL-8 messenger RNA in THP-1 cells. The nSLO-LA value of THP-G8 cells was significantly increased (FInSLO-LA ≥ 1.4) by 13 of the 15 sensitizers as well as by 5 of the 7 nonsensitizers. Interestingly, pretreatment with N-acetylcysteine suppressed the increase in FInSLO-LA induced by all sensitizers (inhibition index (II) ≤ 0.8) but did not suppress that induced by most of the nonsensitizers. We then evaluated the performance of this assay using values of FInSLO-LA ≥ 1.4 and II ≤ 0.8 in at least two of three independent experiments as the criteria of a sensitizer, which resulted in test accuracies of 82% for the 22 chemicals used and of 88% for the chemicals proposed by European Center for the Validation of Alternative Methods. This newly developed assay is a candidate replacement for animal tests of skin sensitization because of its accuracy, convenience, and high throughput performance.
authors.affiliation: Department of Dermatology, Tohoku University Graduate School of Medicine, Sendai, Miyagi 980-8574, Japan.
authors.forename: Toshiya
authors.initials: T
authors.lastname: Takahashi
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Kimura
authors.forename: Rumiko
authors.initials: R
authors.lastname: Saito
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nakajima
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ohmiya
authors.forename: Kenshi
authors.initials: K
authors.lastname: Yamasaki
authors.forename: Setsuya
authors.initials: S
authors.lastname: Aiba
mesh_terms: D000485:Allergens / Q000633:toxicity*; D000826:Animal Testing Alternatives; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003877:Dermatitis, Contact / Q000209:etiology; D004790:Enzyme Induction; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000276:immunology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000096:biosynthesis* / Q000235:genetics; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D012882:Skin Tests / Q000379:methods*
chemical_list: D000485:Allergens; D016209:Interleukin-8; D008070:Lipopolysaccharides; D008156:Luciferases