Triphenyl phosphate induces coordinated DNA methylation and gene expression changes in aquatic embryonic cells

Abstract Triphenyl phosphate (TPHP) is a high-production-volume flame retardant and plasticizer that is widely detected in the environment and in biomonitoring studies. TPHP exposure has been linked to endocrine disruption, metabolic disruption, genotoxicity, and neurodevelopmental effects in vitro and in vivo. The diverse toxicological outcomes across studies suggest disruption of fundamental regulatory processes, such as epigenetic control of gene expression. Here, we used an immortalized embryonic cell line derived from steelhead trout (STE-137) to investigate coordinated transcriptional and epigenetic responses to TPHP exposure. Cells were exposed to 0 or 80 μM TPHP for 24 h, followed by RNA sequencing (RNA-seq) or whole-genome bisulfite sequencing (WGBS). Differential expression analysis identified 1622 significant genes, with significant enrichment of DNA replication and repair, cell cycle regulation, and endocrine signaling pathways and prominent lipid metabolism genes. Weighted gene co-expression network analysis revealed modules highly correlated with exposure, including those enriched for protein and amino acid metabolism, ion transport, and genomic stability. WGBS methylome analysis detected 382 differentially methylated regions (DMRs), the majority hypermethylated and within gene bodies. Notable alterations included a DMR in the htr2cl1 gene, encoding a serotonin receptor, and the brca2 gene, a key DNA damage enzyme. Integration of RNA-seq and WGBS datasets identified nine genes with both expression and methylation changes, alongside altered gene expression of several key epigenetic regulators. Our results provide molecular evidence for early initiating events relevant to adverse outcome pathways and highlight the importance of epigenetic endpoints in developmental toxicity assessment.