Department of Toxicogenomics, GROW School for Oncology and Reproduction, Maastricht University, Maastricht, Netherlands.
Institute of Interdisciplinary Research in Molecular Human Biology (IRIBHM), Université Libre de Bruxelles, Brussels, Belgium.
Front Endocrinol (Lausanne). 2023 Sep 22;14:1200211. doi: 10.3389/fendo.2023.1200211. eCollection 2023.
Phthalates are a class of endocrine-disrupting chemicals that have been shown to negatively correlate with thyroid hormone serum levels in humans and to cause a state of hyperactivity in the thyroid. However, their mechanism of action is not well described at the molecular level.
We analyzed the response of mouse thyroid organoids to the exposure to a biologically relevant dose range of the phthalates bis(2-ethylhexyl) phthalate (DEHP), di-iso-decylphthalate (DIDP), di-iso-nonylphthalate (DINP), and di-n-octylphthalate (DnOP) for 24 h and simultaneously analyzed mRNA and miRNA expression via RNA sequencing. Using the expression data, we performed differential expression and gene set enrichment analysis. We also exposed the human thyroid follicular epithelial cell line Nthy-ori 3-1 to 1 µM of DEHP or DINP for 5 days and analyzed changes in chromatin accessibility via ATAC-Seq.
Dose-series analysis showed how the expression of several genes increased or decreased at the highest dose tested. As expected with the low dosing scheme, the compounds induced a modest response on the transcriptome, as we identified changes in only mmu-miR-143-3p after DINP treatment and very few differentially expressed genes. No effect was observed on thyroid markers. Ing5, a component of histones H3 and H4 acetylation complexes, was consistently upregulated in three out of four conditions compared to control, and we observed a partial overlap among the genes differentially expressed by the treatments. Gene set enrichment analysis showed enrichment in the treatment samples of the fatty acid metabolism pathway and in the control of pathways related to the receptor signalling and extracellular matrix organization. ATAC-Seq analysis showed a general increase in accessibility compared to the control, however we did not identify significant changes in accessibility in the identified regions.
With this work, we showed that despite having only a few differentially expressed genes, diverse analysis methods could be applied to retrieve relevant information on phthalates, showing the potential of in vitro thyroid-relevant systems for the analysis of endocrine disruptors.
邻苯二甲酸酯是一类内分泌干扰化学物质，已被证明与人类血清甲状腺激素水平呈负相关，并导致甲状腺功能亢进状态。然而，其作用机制在分子水平上尚未得到很好的描述。
我们分析了小鼠甲状腺类器官对生物相关剂量范围内的邻苯二甲酸二（2-乙基己基）酯（DEHP）、邻苯二甲酸二异葵酯（DIDP）、邻苯二甲酸二异壬酯（DINP）和邻苯二甲酸二正辛酯（DnOP）暴露 24 小时的反应，并通过 RNA 测序同时分析了 mRNA 和 miRNA 的表达。利用表达数据，我们进行了差异表达和基因集富集分析。我们还将人甲状腺滤泡上皮细胞系 Nthy-ori 3-1 暴露于 1µM 的 DEHP 或 DINP 中 5 天，并通过 ATAC-Seq 分析染色质可及性的变化。
剂量系列分析显示了在测试的最高剂量下，几个基因的表达如何增加或减少。正如预期的那样，由于低剂量方案，化合物在转录组上仅引起适度的反应，因为我们仅在 DINP 处理后鉴定到 mmu-miR-143-3p 的变化，并且很少有差异表达的基因。对甲状腺标志物没有影响。Ing5 是组蛋白 H3 和 H4 乙酰化复合物的一个组成部分，与对照相比，在三种情况下都持续上调，并且我们观察到处理之间差异表达的基因之间存在部分重叠。基因集富集分析显示，在处理样本中，脂肪酸代谢途径富集，而在受体信号和细胞外基质组织相关途径的控制中富集。ATAC-Seq 分析显示与对照相比，可及性普遍增加，然而，我们没有在鉴定的区域中发现可及性的显著变化。
通过这项工作，我们表明，尽管只有少数差异表达的基因，但可以应用多种分析方法来获取有关邻苯二甲酸酯的相关信息，这表明体外甲状腺相关系统在分析内分泌干扰物方面具有潜力。
