The modified IL-8 Luc assay, an in vitro skin sensitisation test, can significantly improve the false-negative judgment
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The modified IL‑8 Luc assay, an in vitro skin sensitisation test, can significantly improve the false‑negative judgment of lipophilic sensitizers with logKow values > 3.5 Yutaka Kimura1 · Chizu Fujimura1 · Setsuya Aiba1 Received: 14 July 2020 / Accepted: 8 October 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract False-negative judgment due to poor chemical solubility is a problem with in vitro skin sensitisation tests. Water-insoluble chemicals are typically dissolved in DMSO in most sensitisation tests but precipitate when diluted with medium beyond their solubility in water. Such tests lack procedures to rule out false-negative judgments due to poor solubility. The IL-8 Luc assay (OECD442E) is unique in that if chemicals do not dissolve at 20 mg/mL in medium and have no effect on IL-8 luciferase activity (IL8LA), they are classified as indeterminate. The purpose of the present study was to reduce the number of indeterminate chemicals and improve assay performance. The IL-8 Luc assay can simultaneously examine glyceraldehyde 3-phosphate dehydrogenase luciferase activity (GAPLA) and IL8LA, and thus we examined the correlation between the reduction of GAPLA (defined as Inh-GAPLA) and the reduction of propidium iodide (PI)-excluding cells for three sensitizers and three non-sensitizers. We observed a significant correlation between luciferase activity driven by the GAPDH promoter of THP-G8 cells and the number of viable cells. Furthermore, chemicals providing an Inh-GAPLA value below 0.8 always reduced the ratio of PI-excluding cells to less than 0.6. Using the modified criteria, indeterminate chemicals are judged as negative if they provide Inh-GAPLA values below 0.8. This modification reduced the number of indeterminate chemicals and increased specificity, highlighting the unique advantage of the IL-8 Luc assay. Keywords Skin sensitisation test · Luciferase assay · Lipophilic chemicals · Reporter assay
Introduction There are three reasons for false-negative judgments in in vitro skin sensitisation tests (IVSSTs). Reason 1 is that chemicals classified as prohaptens must be metabolized to acquire biological activity as sensitizers. Reason 2 is that sensitisation may be induced by a unique biological process different from that described in the adverse outcome pathway (AOP) for skin sensitisation, as may be the case with urushiol (Kim et al. 2016). Reason 3 is perhaps the Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00204-020-02934-9) contains supplementary material, which is available to authorized users. * Setsuya Aiba [email protected] 1
Department of Dermatology, Tohoku University Graduate School of Medicine, 1‑1 Seiryo‑machi, Aobaku, Sendai 980‑8574, Japan
most critical: sensitizers dissolve poorly in culture medium, resulting in concentrations too low to stimulate the target cells. Most IVSSTs use DMSO to dissolve chemicals, but the chemicals often precipitate when diluted with medium to beyond their solubili
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