Profiling the immunotoxicity of chemicals based on in vitro evaluation by a combination of the Multi-ImmunoTox assay and the IL-8 Luc assay
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We established a luciferase reporter assay system, the Multi-ImmunoTox Assay (MITA), which can evaluate the effects of chemicals on the promoter activities of four cytokines: IL-2, IFN-γ, IL-1β, and IL-8. We previously reported that MITA correctly reflected the change in mRNA of human whole-blood cells treated with dexamethasone, cyclosporine, FK506, or several other immunosuppressive drugs. In this study, we combined MITA with the IL-8 Luc assay to detect skin sensitization chemicals (OECD 442E) (modified MITA: mMITA) and established a data set of 60 chemicals examined by mMITA. Using the mMITA results, chemicals can be classified based on the lowest observed effect level (LOEL) of chemicals in suppressing or augmenting the promoter activities of the four cytokines. Moreover, we demonstrated that K-means clustering and hierarchical clustering of the 60 chemicals based on the LOEL for their effects on IL-2 and IL-8 promoter activities and the judgment by the IL-8 Luc assay resulted in the same 6-cluster solution: cluster 1 with preferential suppression of IL-8, cluster 2 with suppression of IL-2 and a positive IL-8 Luc assay result, cluster 3 with suppression of both IL-2 and IL-8, cluster 4 with no effects on IL-2 or IL-8 and a negative IL-8 Luc assay result, cluster 5 with suppression of both IL-2 and IL-8 and a negative IL-8 Luc assay result, and cluster 6 with preferential suppression of IL-8. These data suggest that mMITA is a promising novel high-throughput approach for detecting unrecognized immunological effects of chemicals and for profiling their immunotoxic effects.
KeywordsImmunotoxicity Reporter assay Alternatives to animals
This work was supported in part by the New Energy and Industrial Development Organization (NEDO), by a Grant-in-Aid for Challenging Exploratory Research from the Japan Society for the Promotion of Science (KAKENHI Grant no. 26670473), by Health Labor Sciences Research in Japan, by the Ministry of Economy, Science and Industry in Japan, and by the Japanese Society for Alternatives to Animal Experiments. We are grateful to Mika Watanabe, Miwako Kobayashi, and Kohji Yamakage, PhD (Hatano Research Institute, Food and Drug Safety Center, Hadano, Japan), Rie Yasuno, PhD and Yoshihiro Ohmiya, MD, PhD (Biomedical Research Institute, Advanced Industrial Science and Technology, Tsukuba, Japan), Tomoko Iwaki and Yoshihiro Nakajima, PhD (Health Research Institute, Advanced Industrial Science and technology, Takamatsu, Japan), Mayumi Kobayashi and Takashi Omori, PhD (Division of Biostatistics, Department of Social/Community Medicine and Health Science, Kobe University School of Medicine, Japan), and Hajime Kojima (Japanese Center for the Validation of Alternative Methods, Tokyo, Japan) for their kind advise in developing MITA.
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Conflict of interest
The authors declare that there are no conflicts of interest.
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