Triphenyl\nPhosphate Alters Methyltransferase Expression\nand Induces Genome-Wide Aberrant DNA Methylation in Zebrafish Larvae

Emerging environmental\ncontaminants, organophosphate\nflame retardants\n(OPFRs), pose significant threats to ecosystems and human health.\nDespite numerous studies reporting the toxic effects of OPFRs, research\non their epigenetic alterations remains limited. In this study, we\ninvestigated the effects of exposure to 2-ethylhexyl diphenyl phosphate\n(EHDPP), tricresyl phosphate (TMPP), and triphenyl phosphate (TPHP)\non DNA methylation patterns during zebrafish embryonic development.\nWe assessed general toxicity and morphological changes, measured global\nDNA methylation and hydroxymethylation levels, and evaluated DNA methyltransferase\n(DNMT) enzyme activity, as well as mRNA expression of DNMTs and ten-eleven\ntranslocation (TET) methylcytosine dioxygenase genes. Additionally,\nwe analyzed genome-wide methylation patterns in zebrafish larvae using\nreduced-representation bisulfite sequencing. Our morphological assessment\nrevealed no general toxicity, but a statistically significant yet\nsubtle decrease in body length following exposure to TMPP and EHDPP,\nalong with a reduction in head height after TPHP exposure, was observed.\nEye diameter and head width were unaffected by any of the OPFRs. There\nwere no significant changes in global DNA methylation levels in any\nexposure group, and TMPP showed no clear effect on DNMT expression.\nHowever, EHDPP significantly decreased only DNMT1 expression, while\nTPHP exposure reduced the expression of several DNMT orthologues and\nTETs in zebrafish larvae, leading to genome-wide aberrant DNA methylation.\nDifferential methylation occurred primarily in introns (43%) and intergenic\nregions (37%), with 9% and 10% occurring in exons and promoter regions,\nrespectively. Pathway enrichment analysis of differentially methylated\nregion-associated genes indicated that TPHP exposure enhanced several\nbiological and molecular functions corresponding to metabolism and\nneurological development. KEGG enrichment analysis further revealed\nTPHP-mediated potential effects on several signaling pathways including\nTGFβ, cytokine, and insulin signaling. This study identifies\nspecific changes in DNA methylation in zebrafish larvae after TPHP\nexposure and brings novel insights into the epigenetic mode of action\nof TPHP.