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

Abstract

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is an organophosphate flame retardant that impacts zebrafish epiboly – an effect that may be associated with genome-wide hypomethylation. Using zebrafish as a model, the objectives of this study were to (1) quantify concentration-dependent impacts of TDCIPP on epiboly; (2) determine whether co-exposure with folic acid (FA) – a methyl donor – mitigates TDCIPP-induced impacts; and (3) using ten previously identified TDCIPP-susceptible loci, 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 post-fertilization (hpf) to 2, 4, 6, or 24 hpf in the presence or absence of 1 mM FA. Although TDCIPP delayed epiboly up to 3 h by 6 hpf and induced malformations by 24 hpf, FA was unable to mitigate TDCIPP-induced effects at all stages evaluated. Moreover, while no differences in global methylation were detected using a 5-methylcytosine (5-mC) DNA ELISA, 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.

Tris(1,3-dichloro-2-propyl) phosphate disrupts dorsoventral patterning in zebrafish embryos

“…Although internal embryonic doses of TDCIPP at 6 hpf ( Volz et al, 2016 ) are likely higher than TDCIPP levels detected in human placental samples ( Ding et al, 2016 ), our study suggests that, if concentrations are elevated in utero , TDCIPP has the potential to impact the normal progression and trajectory of early embryonic development. While neither phenotypic ( McGee et al, 2012 ) nor transcriptomic changes (this study) were detected at 2 hpf (pre-MZT), our microarray data revealed that significant changes in gene expression were induced by 6 hpf (post-MZT)—changes that are also associated with impacts on epiboly progression ( Kupsco et al, 2017 ).…”

“…The current study builds upon our previous work demonstrating that TDCIPP induces significant defects on epiboly and embryonic development ( Kupsco et al, 2017 ; McGee et al, 2012 ; Volz et al, 2016 ). Although internal embryonic doses of TDCIPP at 6 hpf ( Volz et al, 2016 ) are likely higher than TDCIPP levels detected in human placental samples ( Ding et al, 2016 ), our study suggests that, if concentrations are elevated in utero , TDCIPP has the potential to impact the normal progression and trajectory of early embryonic development.…”

“…While quantifying TDCIPP-induced epiboly defects in our previous study ( Kupsco et al, 2017 ), two distinct phenotypes emerged at both concentrations: (1) epiboly-delayed embryos at the dome-30% epiboly stage at 6 hpf, but eventually complete epiboly and (2) epiboly-arrested embryos at the high stage at 6 hpf, but continue developing in the absence of epiboly. Untreated embryos are normally at the germ ring or shield stage by 6 hpf.…”

“…However, no significant malformations were observed following initiation of 3 µM TDCIPP exposure at 5.25 hpf (50% epiboly), suggesting that cleavage and/or blastula stages of development represent sensitive windows for TDCIPP-induced developmental toxicity ( McGee et al, 2012 ). Indeed, initiation of TDCIPP exposure at 0.75 hpf resulted in a concentration-dependent delay or arrest in epiboly during blastula and gastrula (4–6 hpf) ( Fu et al, 2013 ; Kupsco et al, 2017 ). Epiboly delays at ∼6 hpf also occurred in unexposed F1 embryos following TDCIPP-exposed F0 parents, an effect that was likely due to maternal transfer into F1 embryos ( Yu et al, 2017 ).…”

Mass mortality of fish embryos in a lake ecosystem attributable to a flame retardant

“…Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), a chloro-organophosphorus flame retardant, is produced industrially in high volumes. TDCIPP has been reported to cause embryo mortality in a zebrafish embryo exposure ex vivo (Volz et al 2016;Kupsco et al 2017). Therefore, in the present study, we utilized reproductive characterization of crucian carp in natural population to address following questions.…”