Transcriptomic Responses of Zebrafish Embryos to Environmentally Relevant, Low-Dose (2-Ethylhexyl) Phthalate Exposure...

Di(2-ethylhexyl) phthalate (DEHP) is a high-production-volume plasticizer and ubiquitous environ-mental contaminant with established endocrine-disrupting potential. While zebrafish transcriptomic studies have typically used high concentrations and long exposure windows, less is known about genome-wide responses during late embryogenesis/early larval maturation under environmentally relevant exposures. Here we profiled whole-organism transcriptomic responses to a short DEHP exposure during a developmentally sensitive transition (96-120) hours post-fertilization, hpf) and interpreted responses using differential expression, enrichment analyses, and endocrine-focused protein-protein interaction (PPI) network modeling.

Wild-type AB zebrafish lar-vae (96 hpf) were exposed to DEHP at [10^-9 M] or solvent control for 24 h. Larvae were pooled per replicate (25 lar-vae/pool) and processed for poly(A)-selected RNA-seq. Reads were quality-controlled, aligned to the Danio rerio reference genome, and quantified at gene- level. Differential expression was performed using DESeq2. Functional enrichment used KEGG over-representation analysis (ORA) and gene set enrichment analysis (GSEA). Zebrafish genes were mapped to human orthologs for GO/KEGG and STRING-based endocrine subnetworks, which were visualized and interrogated using STRINGdb and visNetwork.

Low-dose, short-term exposure does not produce large gene-level effects but induces coordinated, pathway-level transcriptional remodeling. KEGG ORA showed significant enrichment of MAPK signaling and regulation of actin cytoskeleton with additional enrichment of axon guidance and neuroactive ligand-receptor interaction. GSEA detected coordinated downregulation of KEGG neurodegeneration collections with negative normalized enrichment scores reflecting shared gene sets re-lated to mitochondrial function, proteostasis, cytoskeletal organization, and stress-response pathways. Endo-crine-focused STRING subnetworks indicated consistent downregulation of CYP19A1 within estrogen metabo-lism/biosynthesis modules and downregulation of upstream androgen biosynthetic enzymes HSD3B2 and CYP17A1, alongside upregulation of HSD17B3 and proteostasis-associated factors including DNAJA1. Endocrine network to-pology highlighted regulatory and cofactor nodes affecting receptor-linked transcription, consistent with indirect endocrine modulation rather than large receptor-transcript changes.

In summary, this study demon-strates that exposure to low-dose DEHP during a critical period of zebrafish embryonic development is associated with modest but coordinated transcriptomic changes across multiple biological pathways. Pathway enrichment and network-based analyses highlight estrogen- and androgen-associated processes, along with broader signaling, met-abolic, and structural pathways, as transcriptionally responsive during this window. Importantly, these findings reflect molecular-level associations rather than direct evidence of functional or physiological endocrine disruption. Instead, they identify candidate pathways and regulatory networks that may be sensitive to low-level environmen-tal exposure and warrant further investigation. Collectively, this work underscores the value of systems-level tran-scriptomic approaches for detecting subtle, pathway-wide responses to environmentally relevant exposures during development.