Department of Chemistry, Mass Spectrometry Center, QOPNA, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal.
Chem Res Toxicol. 2013 Mar 18;26(3):368-78. doi: 10.1021/tx300472d. Epub 2013 Mar 7.
The sensitizing potential of chemicals is currently assessed using animal models. However, ethical and economic concerns and the recent European legislative framework triggered intensive research efforts in the development and validation of alternative methods. Therefore, the aim of this study was to develop an in vitro predictive test based on the analysis and integration of gene expression and intracellular signaling profiles of chemical-exposed skin-derived dendritic cells. Cells were treated with four known sensitizers and two nonsensitizers, and the effects on the expression of 20 candidate genes and the activation of MAPK, PI3K/Akt, and NF-κB signaling pathways were analyzed by real-time reverse transcription polymerase chain reaction and Western blotting, respectively. Genes Trxr1, Hmox1, Nqo1, and Cxcl10 and the p38 MAPK and JNK signaling pathways were identified as good predictor variables and used to construct a dichotomous classifier. For validation of the model, 12 new chemicals were then analyzed in a blind assay, and from these, 11 were correctly classified. Considering the total of 18 compounds tested here, 17 were correctly classified, representing a concordance of 94%, with a sensitivity of 92% (12 of 13 sensitizers identified) and a specificity of 100% (5 of 5 nonsensitizers identified). Additionally, we tested the ability of our model to discriminate sensitizers from nonallergenic but immunogenic compounds such as lipopolysaccharide (LPS). LPS was correctly classified as a nonsensitizer. Overall, our results indicate that the analysis of proposed gene and signaling pathway signatures in a mouse fetal skin-derived dendritic cell line represents a valuable model to be integrated in a future in vitro test platform.
目前，化学物质的致敏潜力是通过动物模型来评估的。然而，出于伦理和经济方面的考虑，以及最近的欧洲立法框架，人们大力研究开发和验证替代方法。因此，本研究的目的是开发一种基于分析和整合化学暴露皮肤衍生树突状细胞的基因表达和细胞内信号谱的体外预测性测试。用四种已知的致敏剂和两种非致敏剂处理细胞，分别通过实时逆转录聚合酶链反应和 Western blot 分析对 20 个候选基因的表达和 MAPK、PI3K/Akt 和 NF-κB 信号通路的激活的影响。确定基因 Trxr1、Hmox1、Nqo1 和 Cxcl10 以及 p38 MAPK 和 JNK 信号通路为良好的预测变量，并用于构建二项分类器。然后，为了验证模型，在盲法检测中分析了 12 种新的化学物质，其中 11 种被正确分类。考虑到这里测试的 18 种化合物的总数，17 种被正确分类，一致性为 94%，敏感性为 92%（13 种致敏剂中有 12 种被识别），特异性为 100%（5 种非致敏剂中有 5 种被识别）。此外，我们还测试了我们的模型区分致敏剂和非变应原但免疫原性化合物（如脂多糖 [LPS]）的能力。LPS 被正确地归类为非致敏剂。总的来说，我们的结果表明，在小鼠胎儿皮肤衍生树突状细胞系中分析拟议的基因和信号通路特征代表了一种有价值的模型，可以整合到未来的体外测试平台中。
