Dynamic Alterations in DNA Methylation Precede Tris(1,3-dichloro-2-propyl)phosphate-Induced Delays in Zebrafish Epiboly

Tris­(1,3-dichloro-2-propyl)­phosphate
(TDCIPP) is an organophosphate
flame retardant that impacts zebrafish epiboly, an effect that may
be associated with genomewide hypomethylation. Using zebrafish as
a model, the objectives of this study were (1) to quantify concentration-dependent
impacts of TDCIPP on epiboly, (2) to determine whether co-exposure
with folic acid (FA), a methyl donor, mitigates TDCIPP-induced impacts,
and (3) using 10 previously identified TDCIPP-susceptible loci, to
rely on bisulfite amplicon sequencing (BSAS) to monitor CpG methylation
dynamics across multiple TDCIPP concentrations in the presence or
absence of FA. Embryos were exposed to TDCIPP from 0.75 h postfertilization
(hpf) to 2, 4, 6, or 24 hpf in the presence or absence of 1 mM FA.
Although TDCIPP delayed epiboly by up to 3 h by 6 hpf and induced
malformations by 24 hpf, FA was unable to mitigate TDCIPP-induced
effects at any stage that was evaluated. Moreover, while no differences
in global methylation were detected using a 5-methylcytosine DNA enzyme-linked
immunosorbent assay, BSAS revealed that TDCIPP-induced effects on
CpG methylation were dependent on concentration and developmental
stage and that early effects on methylation do not persist despite
continuous exposure. Our findings demonstrate that TDCIPP delays zebrafish
epiboly, a phenotype that is preceded by complex, dynamic alterations
in DNA methylation.