Triphenyl Phosphate Alters Methyltransferase Expression and Induces Genome-Wide Aberrant DNA Methylation in Zebrafish Larvae

Emerging environmental
contaminants, organophosphate
flame retardants
(OPFRs), pose significant threats to ecosystems and human health.
Despite numerous studies reporting the toxic effects of OPFRs, research
on their epigenetic alterations remains limited. In this study, we
investigated the effects of exposure to 2-ethylhexyl diphenyl phosphate
(EHDPP), tricresyl phosphate (TMPP), and triphenyl phosphate (TPHP)
on DNA methylation patterns during zebrafish embryonic development.
We assessed general toxicity and morphological changes, measured global
DNA methylation and hydroxymethylation levels, and evaluated DNA methyltransferase
(DNMT) enzyme activity, as well as mRNA expression of DNMTs and ten-eleven
translocation (TET) methylcytosine dioxygenase genes. Additionally,
we analyzed genome-wide methylation patterns in zebrafish larvae using
reduced-representation bisulfite sequencing. Our morphological assessment
revealed no general toxicity, but a statistically significant yet
subtle decrease in body length following exposure to TMPP and EHDPP,
along with a reduction in head height after TPHP exposure, was observed.
Eye diameter and head width were unaffected by any of the OPFRs. There
were no significant changes in global DNA methylation levels in any
exposure group, and TMPP showed no clear effect on DNMT expression.
However, EHDPP significantly decreased only DNMT1 expression, while
TPHP exposure reduced the expression of several DNMT orthologues and
TETs in zebrafish larvae, leading to genome-wide aberrant DNA methylation.
Differential methylation occurred primarily in introns (43%) and intergenic
regions (37%), with 9% and 10% occurring in exons and promoter regions,
respectively. Pathway enrichment analysis of differentially methylated
region-associated genes indicated that TPHP exposure enhanced several
biological and molecular functions corresponding to metabolism and
neurological development. KEGG enrichment analysis further revealed
TPHP-mediated potential effects on several signaling pathways including
TGFβ, cytokine, and insulin signaling. This study identifies
specific changes in DNA methylation in zebrafish larvae after TPHP
exposure and brings novel insights into the epigenetic mode of action
of TPHP.