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. 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.