Using transcriptomics data and Adverse Outcome Pathway networks to explore endocrine disrupting properties of Cadmium and PCB-126. Using transcriptomics data and Adverse Outcome Pathway networks to explore endocrine disrupting properties of Cadmium and PCB-126

Omics-technologies such as transcriptomics offer valuable insights into toxicity mechanisms. However, integrating this type of data into regulatory frameworks remains challenging due to uncertainties regarding toxicological relevance and links to adverse outcomes. Current assessments of endocrine disruptors (EDs) relevant for human health require substantial amounts of data and primarily rely on standardized animal studies. Identifying EDs is a high priority in the EU, and so are efforts to replace and reduce animal testing. Alternative methods to investigate EDs are needed, but there is also a lack of health risk assessment methodology that supports regulatory uptake of novel mechanistic information. This study aims to utilize Adverse Outcome Pathways (AOPs) to integrate transcriptomics data for identifying EDs, by establishing a link between molecular data and adverse outcomes. Cadmium (Cd) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) were used as model compounds due to their observed effects on the endocrine system, but not yet being identified as EDs in the EU regulatory setting. Estrogen, androgen, thyroid and steroidogenesis (EATS)-related AOPs were extracted from AOPWiki to generate an AOP network. RNA sequencing (RNA-Seq) was conducted on zebrafish (Danio rerio) embryos exposed to Cd or PCB126 for 4 days. RNA-Seq data were then linked to the AOP network by connecting Gene Ontology biological process (GO BP) terms from the experimental data to key events in the network. Enrichment Maps in Cytoscape and the QIAGEN Ingenuity Pathway Analysis (IPA) software were also used to identify potential ED properties and support the assessment. Potentially EATS-related GO BP terms were identified for both compounds. A lack of accurate standardized terms in KEs of the AOP network hindered a data-driven mapping approach. Instead, manual mapping of GO BP terms onto the AOP network revealed more connections, underscoring the need for harmonizing AOP development for regulatory use. IPA results further supported potentially EATS-related effects of both compounds. Evidence from the Enrichment Maps revealed perturbations of several biological functions, which may be related to either EATS or non-EATS endocrine mechanisms. While AOP networks show promise in integrating RNA-Seq data, several challenges remain. Future research should focus on further developing this methodology to improve the use of mechanistic data in risk assessments. Standardizing AOP development to include accurate GO terms is crucial for enabling automatic integration of transcriptomics data through AOPs. Overall design: To investigate the effects of cadmium or 3,3',4,4',5-pentachlorobiphenyl (PCB126) on zebrafish embryos. The embryos were exposed to cadmium or PCB126 at three different doses each, from 1dpf to 5 dpf. At 5 dpf, RNA-seq was performed.

以转录组学（transcriptomics）为代表的组学技术，可为毒性机制研究提供极具价值的见解。然而，由于毒理学相关性及与不良结局关联的不确定性，将此类数据纳入监管框架仍面临挑战。当前针对与人类健康相关的内分泌干扰物（endocrine disruptors, EDs）的评估，需要大量数据且主要依赖标准化动物实验。在欧盟，识别内分泌干扰物是一项优先要务，替代并减少动物实验的相关工作亦是如此。当前亟需研发用于筛查内分泌干扰物的替代方法，但同时也缺乏能够支持监管机构采纳新型机制信息的健康风险评估方法体系。
本研究旨在通过建立分子数据与不良结局之间的关联，利用不良结局途径（Adverse Outcome Pathways, AOPs）整合转录组学数据以筛查内分泌干扰物。本研究选取镉（cadmium, Cd）与3,3',4,4',5-五氯联苯（3,3',4,4',5-pentachlorobiphenyl, PCB126）作为模型化合物，因其已被观测到可对内分泌系统产生影响，但尚未在欧盟监管框架中被认定为内分泌干扰物。
从AOPWiki数据库中提取与雌激素、雄激素、甲状腺及类固醇生成（estrogen, androgen, thyroid and steroidogenesis, EATS）相关的AOPs，构建AOP网络。对暴露于镉或PCB126并持续4天的斑马鱼（Danio rerio）胚胎开展RNA测序（RNA-Seq）实验。随后，通过将实验数据中的基因本体学生物过程（Gene Ontology biological process, GO BP）术语与AOP网络中的关键事件进行关联，将RNA-Seq数据与该网络进行对接。本研究还借助Cytoscape中的富集图谱（Enrichment Maps）以及QIAGEN Ingenuity Pathway Analysis（IPA）软件，识别潜在的内分泌干扰物特性并辅助评估工作。
两种模型化合物均被鉴定出存在潜在的与EATS相关的GO BP术语。AOP网络中关键事件（Key Events, KEs）缺乏精准的标准化术语，这阻碍了数据驱动的映射方法的应用。因此，通过手动将GO BP术语映射至AOP网络，可识别出更多关联，这凸显了为监管用途统一AOP开发标准的必要性。IPA的分析结果进一步证实了两种模型化合物潜在的EATS相关效应。富集图谱的分析结果显示，多种生物学功能发生扰动，这些扰动可能与EATS相关或非EATS类的内分泌机制有关。
尽管AOP网络在整合RNA-Seq数据方面展现出应用潜力，但仍存在诸多挑战。未来的研究应聚焦于进一步完善该方法体系，以提升机制数据在风险评估中的应用价值。统一AOP的开发标准并纳入精准的GO术语，对于实现通过AOP自动整合转录组学数据至关重要。
实验整体设计：探究镉与3,3',4,4',5-五氯联苯（PCB126）对斑马鱼胚胎的影响。将斑马鱼胚胎分别以三种不同剂量暴露于镉或PCB126中，暴露周期为受精后1天至5天（1dpf至5dpf），并于受精后5天（5dpf）开展RNA测序实验。