Development of RT h-CLAT, a Rapid Assessment Method for Skin Sensitizers Using THP-1 Cells as a Biosensor

Abstract

In recent years, in vitro skin sensitization assays have been recommended as animal-free alternatives for the safety assessment of cosmetics and topical drugs, and these methods have been adopted in OECD test guidelines. However, existing assays remain complex and costly. To address this, we recently developed a more efficient, cost-effective, and accurate method for evaluating skin sensitizers by using immune cell-derived THP-1 cells as a biosensor, coupled with an RT-PCR-based assay. In this study, we further refined this method to enable even faster assessment of skin sensitization. By performing comprehensive RNA sequencing (RNA-Seq) analysis, we examined gene expression profiles induced by sensitizers in THP-1 cells to identify potential sensitization markers, ultimately selecting the optimal markers and conditions for evaluation. Our findings indicate that after exposing a test chemical to THP-1 cells for 5 h, measuring the expression levels of the JUN and HMOX1 genes via real-time PCR allows for a reliable assessment of sensitization. A test compound is defined as a sensitizer if either gene shows a more than two-fold increase in its expression compared to the control. Applying this improved method, designated as RT h-CLAT, we evaluated the sensitization potential of 43 chemicals. The results demonstrated higher accuracy compared to the human cell line activation test (h-CLAT) listed in the OECD guidelines, while also reducing the required assessment time from two days to one.

Keywords: HMOX1; JUN; RNA-Seq analysis; alternative methods; biomarker; in vitro skin sensitization test.

Figure 1

The overall process of selecting candidate marker genes for sensitization assessment using THP-1 cells. After selecting genes whose expression levels were significantly increased or decreased compared to the control, 12 genes have remained as candidates. The expression levels of 10 genes were increased by the treatment of sensitizers, while those of 2 genes were decreased.

Figure 2

Principal component analysis of genes expressed in THP-1 cells exposed to sensitizers and non-sensitizers. PC1 and PC2 indicate the first and second principal component scores, respectively. The number shows the contributing ratio of each score. (a) Each circle shows the expression profile of genes in THP-1 cells exposed to different chemicals including the control. (b) Each circle shows the expression profile of genes in THP-1 cells exposed to sensitizers (T), non-sensitizers (C), and controls (N).

Figure 3

Changes in the expression levels of five candidate marker genes after chemical treatments to THP-1 cells. (a–i) The changes in the expression levels of HMOX1, JUN, PPP1R15A, BTG2, and PMAIP1 genes after treatment of THP-1 cells with p-benzoquinone, methyl pyruvate, 1-naphthol, butyl glycidyl ether, aniline, eugenol, cinnamyl alcohol, chlorobenzene, and vanillin, respectively. The results of LLNA [3,19] and h-CLAT [7,26] for each chemical are also shown. Dark gray bars indicate more than 2-fold increases in the gene expression levels compared to the control. If the expression level of the gene was increased more than 2-fold in two or more of the three trials, the chemical used in the treatment was evaluated as a sensitizer.

Figure 3

Changes in the expression levels of five candidate marker genes after chemical treatments to THP-1 cells. (a–i) The changes in the expression levels of HMOX1, JUN, PPP1R15A, BTG2, and PMAIP1 genes after treatment of THP-1 cells with p-benzoquinone, methyl pyruvate, 1-naphthol, butyl glycidyl ether, aniline, eugenol, cinnamyl alcohol, chlorobenzene, and vanillin, respectively. The results of LLNA [3,19] and h-CLAT [7,26] for each chemical are also shown. Dark gray bars indicate more than 2-fold increases in the gene expression levels compared to the control. If the expression level of the gene was increased more than 2-fold in two or more of the three trials, the chemical used in the treatment was evaluated as a sensitizer.