Identification of gene expression markers and development of evaluation method using cell-based and RT-PCR-based assay for skin sensitising potential of chemicals

Abstract

1. Recently, alternatives to animal testing have been used to evaluate skin sensitisers in cosmetic products. However, testing is still complicated and expensive. To develop a simpler, cost-effective and more accurate evaluation method for the skin sensitising chemicals, we employed cell-based and RT-PCR-based assay.

2. Representative sensitiser specific gene expression in THP-1 cells was analysed by microarray. Gene ontology (GO) analysis revealed that 26 genes induced by the sensitisers were associated with immune function.

3. First, seven of the 26 genes were chosen arbitrarily as candidate markers for our sensitisation assay. Then, THP-1 cells were exposed to 13 reference chemicals with known sensitising potential, and real-time RT-PCR assays targeting the candidate marker genes were performed. Among them, six markers were able to properly evaluate the sensitisation potential by classifying the gene induction rates with appropriate criteria. Especially, the results of the assay using TREM1 and TNFRSF12A gene markers showed 100% sensitivity and specificity.

4. An existing test method, h-CLAT, requires a flow cytometer and is complicated to operate. In contrast, our method is relatively simpler and more cost-effective. Therefore, our method is a promising one to evaluate sensitising chemicals.

Keywords:

• Skin sensitisation
• THP-1
• microarray
• TREM1
• TNFRSF12A
• FN14

Acknowledgements

We wish to thank Satoshi Kiyonobu, Nanami Masumoto and Noriko Muneuchi for their valuable comments and technical assistance.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported in part by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP 24590157.