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.