Groh, K. J. | Search bioRxiv

bioRxiv2026

A high-throughput method to computationally develop candidate adverse outcome pathways in humans: a proof of concept with insecticides and Parkinson Disease

Adverse outcome pathways (AOPs) describe stressor non-specific sequences of events between a first molecular trigger (molecular initiating event, MIE), causally linked key events (KEs), and an adverse outcome (AO). AOPs are intended to aid in chemical toxicity testing as a new approach methodology. However, commonly used AOP development methods depend on manual curation, which is labor intensive. As a result, there are still relatively few AOPs and a huge number of toxicity mechanisms and possible adverse outcomes remain undescribed. Therefore, systematic and high-throughput approaches to predict new AOPs are needed. Here, we developed and implemented a data integration-based framework to generate new candidate AOPs using insecticides and Parkinson Disease as a proof of concept. We integrated and statistically linked disconnected databases (e.g., Comparative Toxicogenomics Database, Human Protein Atlas, and Gene Ontology) to form MIE - KE (cell level) - KE (tissue level) - AO candidate AOPs. Through this systematic process, we generated 562,117 candidate AOPs, which we then scored using a weight of evidence (WoE) approach and prioritized 12,756 AOPs with a WoE >0.5. Through random sampling of 100 prioritized AOPs, we found 70% had external literature supporting their biological plausibility, and only 15% represented identifiably implausible associations. The prioritized AOPs describe varied mechanisms of toxicity related to e.g., MAPK, PTEN, and FGFR signaling pathways, with "increases phosphorylation of MAPK1" as the most frequent MIE. Our AOP generating approach yields consistently structured AOPs and can complement existing and emerging development methods to expand AOP coverage across different stressors and outcomes.

pharmacology and toxicology↗

bioRxiv2025

Multiomics profiling of zebrafish embryonic cell line PAC2 across growth phases to assess its relevance for toxicological studies

Permanent fish cell lines offer promising alternatives to traditional animal models for environmental risk assessment of chemicals. However, to facilitate their broader uptake into toxicity testing practice, a better understanding of functional capacities and expression of toxicologically relevant molecular targets is needed. Here, we present an extensive molecular profiling of the zebrafish embryonic cell line PAC2, combining global proteomics across cell population growth phases (over 7300 protein groups) with matched transcriptomics at exponential and stationary phase (over 14500 transcripts). Proteome coverage was sufficiently deep to reveal functional insights consistent with those derived from transcriptomics data, despite differences in the total number of measured genes. Major gene expression shifts detected upon transition from exponential to stationary cell population growth phase indicated reduction in DNA replication, translation, metabolism, and cell cycle regulation, along with increased stress responses, immune system responses, and extracellular matrix remodeling. Functional annotation revealed expression of core cellular processes along with a number of toxicologically relevant pathways, including xenobiotic metabolism, stress signaling, and nuclear receptors responsive to important chemical classes, such as steroids (e.g., estrogens, glucocorticoids) and chemicals known to disrupt lipid metabolism, e.g., through interaction with peroxisome proliferation activating receptors. These findings reinforce the potential of PAC2 cells to offer a versatile in vitro model for studying fish cell biology and omics-enhanced exploration of chemical toxicity mechanisms, aided by the well-developed molecular annotation in zebrafish. Moreover, the analysis approaches developed in this work offer a blueprint for molecular baseline characterization of other fish cell lines. This work thus strengthens the mechanistic foundation supporting the use of fish cell lines as alternative models in aquatic toxicity testing and risk assessment.

molecular biology↗