Koyama Hiroki, Maeda Ayami, Zhai Peiqi, Koiwai Keiichiro, Kurose Kouichi
Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo 108-8477, Japan.
Biosensors (Basel). 2024 Dec 20;14(12):632. doi: 10.3390/bios14120632.
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 and 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.
近年来，体外皮肤致敏试验已被推荐作为化妆品和局部用药物安全性评估的无动物替代方法，并且这些方法已被纳入经合组织测试指南。然而，现有的试验仍然复杂且成本高昂。为了解决这个问题，我们最近开发了一种更高效、经济且准确的方法，通过使用免疫细胞来源的THP-1细胞作为生物传感器，并结合基于逆转录聚合酶链反应（RT-PCR）的检测方法来评估皮肤致敏剂。在本研究中，我们进一步优化了该方法，以实现对皮肤致敏更快的评估。通过进行全面的RNA测序（RNA-Seq）分析，我们检测了致敏剂在THP-1细胞中诱导的基因表达谱，以识别潜在的致敏标记物，最终选择用于评估的最佳标记物和条件。我们的研究结果表明，将受试化学品暴露于THP-1细胞5小时后，通过实时PCR测量 和 基因的表达水平能够可靠地评估致敏情况。如果任一基因的表达水平相对于对照显示出两倍以上的增加，则受试化合物被定义为致敏剂。应用这种改进后的方法，即RT h-CLAT，我们评估了43种化学品的致敏潜力。结果表明，与经合组织指南中列出的人细胞系激活试验（h-CLAT）相比，该方法具有更高的准确性，同时将所需的评估时间从两天缩短至一天。
