Tris(1,3-dichloro-2-propyl)phosphate Induces Genome-Wide Hypomethylation within Early Zebrafish Embryos

Abstract

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is a high-production volume organophosphate-based plasticizer and flame retardant widely used within the United States. Using zebrafish as a model, the objectives of this study were to determine whether (1) TDCIPP inhibits DNA methyltransferase (DNMT) within embryonic nuclear extracts; (2) uptake of TDCIPP from 0.75 h postfertilization (hpf, 2-cell) to 2 hpf (64-cell) or 6 hpf (shield stage) leads to impacts on the early embryonic DNA methylome; and (3) TDCIPP-induced impacts on cytosine methylation are localized to CpG islands within intergenic regions. Within this study, 5-azacytidine (5-azaC, a DNMT inhibitor) was used as a positive control. Although 5-azaC significantly inhibited zebrafish DNMT, TDCIPP did not affect DNMT activity in vitro at concentrations as high as 500 μM. However, rapid embryonic uptake of 5-azaC and TDCIPP from 0.75 to 2 hpf resulted in chemical- and chromosome-specific alterations in cytosine methylation at 2 hpf. Moreover, TDCIPP exposure predominantly resulted in hypomethylation of positions outside of CpG islands and within intragenic (exon) regions of the zebrafish genome. Overall, these findings provide the foundation for monitoring DNA methylation dynamics within zebrafish as well as identifying potential associations among TDCIPP exposure, adverse health outcomes, and DNA methylation status within human populations.

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

“…Interestingly, clustered heat maps revealed variable impacts of TDCIPP and FA at different stages (Figure 2A; Table S4); raw percent methylation data for all treatments (including vehicle controls) and stages are available within Tables S5 (2 hpf), S6 (4 hpf), and S7 (6 hpf). Relative to vehicle controls, the magnitude of the effect on methylation decreased from 2 to 6 hpf despite being continuously immersed in TDCIPP, suggesting that TDCIPP-effects on methylation are reversible within a 4-h exposure period even though internal TDCIPP doses increase from 2 to 6 hpf 17 . In addition, the direction of response (hyper- vs. hypo-methylation) was not consistent from 2 to 6 hpf within each treatment.…”

“…Genomic DNA samples (50 ng for 2 hpf; 150 ng for 4 hpf; 150 ng for 6 hpf) were bisulfite-treated using an EZ DNA Methylation-Lightning Kit (Zymo). Ten regions of interest (ROIs) were identified based on TDCIPP-susceptible loci identified from our previous WGBS study (SI Table S1) 17 . These 10 ROIs were selected based on five CpGs that were also impacted by FA within humans 21 , while the remaining five ROIs were selected to represent exonic and intergenic regions with a range of methylation differences relative to vehicle controls.…”

“…Interestingly, TDCIPP did not inhibit DNA methyltransferase activity in vitro up to 500 μM 17 , suggesting that TDCIPP may induce CpG hypomethylation via depletion of methyl donors (e.g., S -adenosyl-L-methionine). Methyl supplementation has the potential to reverse chemically-induced DNA methylation 18,19 and maternal supplementation with methyl donors (such as folic acid) has been shown to alter methylation profiles in offspring 20,21,22 .…”

“…Therefore, we conducted whole-genome bisulfite sequencing (WGBS) to identify the magnitude and extent, if any, of 5-azaC- and TDCPP-induced effects on the entire DNA methylome at 2 hpf (following exposure from 0.75–2 hpf) at single base-pair resolution 17 . Within this study, we found that rapid embryonic uptake of 250 μM 5-azaC or 2 μM TDCIPP from 0.75 to 2 hpf resulted in chemical- and chromosome-specific alterations in cytosine methylation at 2 hpf, and that TDCIPP-induced impacts to the zebrafish DNA methylome at 2 hpf are complex and primarily localized to regions outside of classical CpG islands 17 . Importantly, this study yielded positional information about altered CpG methylation within TDCIPP-susceptible loci – data that may be useful for identifying future biomarkers of TDCIPP exposure within zebrafish and humans.…”

Structural basis for DNMT3A-mediated de novo DNA methylation

“…The measurement procedures for 5-mdC and dG in genomic DNA were described previously 36, 37 . Briefly, 1 μg of genomic DNA prepared from cells was enzymatically digested into nucleoside mixtures.…”