Embryonic exposure to di(2-ethylhexyl) phthalate disturbs DNA methylation status of the ovaries in adult female zebrafish and the early development of F1 generation

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer commonly associated with flexible polyvinyl chloride (PVC) and numerous consumer products. Due to its extensive presence, concerns have been raised regarding its long-term biological effects, particularly during early developmental stages.

In this study, we investigated whether early-life exposure to DEHP induces persistent epigenetic alterations in adult zebrafish ovaries and affects early development of F1 generation. Zebrafish embryos were exposed to DEHP (0, 10, 100, and 1000 nmol/L) from 5 hours post-fertilization (hpf) to 120 hpf, and then reared under standard conditions until adulthood (6 months). Ovaries were collected to assess global and gene-specific DNA methylation, as well as expression of DNA methyltransferases (dnmt). Global DNA methylation was evaluated using immunohistochemistry with a 5-methylcytosine (5mC) antibody, while gene-specific methylation was analyzed by the methylation-sensitive restriction enzyme qPCR (MSRE-qPCR). In parallel, developmental parameters of F1 embryos and larvae were assessed using morphometric analyses.

Global DNA methylation levels were significantly reduced in ovarian granulosa cells of DEHP-exposed groups, accompanied by downregulation of dnmt1 and dnmt3aa expression. Gene-specific analysis revealed increased methylation in promoter regions of lhr and esr1, while no changes were observed in fshr.

Parental embryonic exposure to DEHP also impaired F1 development, resulting in reduced embryo size and a higher incidence of non-inflated swim bladders at 120 hpf, along with decreased swim bladder size.

Overall, these findings demonstrate that early-life DEHP exposure induces persistent epigenetic alterations in adult ovaries and adversely affects early developmental competence of the next generation.

This research was funded by Ministry of Science, Technological Development and Innovation of the Republic of Serbia (Grants No. 451-03-33/2026-03/ 200125 & 451-03-34/2026-03/ 200125)