Negi Chander K, Bláhová Lucie, Phan Audrey, Bajard Lola, Blaha Ludek
RECETOX, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, Czech Republic.
Chem Res Toxicol. 2024 Sep 16;37(9):1549-1561. doi: 10.1021/acs.chemrestox.4c00223. Epub 2024 Aug 29.
Emerging environmental contaminants, organophosphate flame retardants (OPFRs), pose significant threats to ecosystems and human health. Despite numerous studies reporting the toxic effects of OPFRs, research on their epigenetic alterations remains limited. In this study, we investigated the effects of exposure to 2-ethylhexyl diphenyl phosphate (EHDPP), tricresyl phosphate (TMPP), and triphenyl phosphate (TPHP) on DNA methylation patterns during zebrafish embryonic development. We assessed general toxicity and morphological changes, measured global DNA methylation and hydroxymethylation levels, and evaluated DNA methyltransferase (DNMT) enzyme activity, as well as mRNA expression of DNMTs and ten-eleven translocation (TET) methylcytosine dioxygenase genes. Additionally, we analyzed genome-wide methylation patterns in zebrafish larvae using reduced-representation bisulfite sequencing. Our morphological assessment revealed no general toxicity, but a statistically significant yet subtle decrease in body length following exposure to TMPP and EHDPP, along with a reduction in head height after TPHP exposure, was observed. Eye diameter and head width were unaffected by any of the OPFRs. There were no significant changes in global DNA methylation levels in any exposure group, and TMPP showed no clear effect on DNMT expression. However, EHDPP significantly decreased only DNMT1 expression, while TPHP exposure reduced the expression of several DNMT orthologues and TETs in zebrafish larvae, leading to genome-wide aberrant DNA methylation. Differential methylation occurred primarily in introns (43%) and intergenic regions (37%), with 9% and 10% occurring in exons and promoter regions, respectively. Pathway enrichment analysis of differentially methylated region-associated genes indicated that TPHP exposure enhanced several biological and molecular functions corresponding to metabolism and neurological development. KEGG enrichment analysis further revealed TPHP-mediated potential effects on several signaling pathways including TGFβ, cytokine, and insulin signaling. This study identifies specific changes in DNA methylation in zebrafish larvae after TPHP exposure and brings novel insights into the epigenetic mode of action of TPHP.
新兴环境污染物有机磷酸酯阻燃剂（OPFRs）对生态系统和人类健康构成重大威胁。尽管有大量研究报道了OPFRs的毒性作用，但其表观遗传改变的研究仍然有限。在本研究中，我们调查了暴露于磷酸二（2-乙基己基）苯酯（EHDPP）、磷酸三甲苯酯（TMPP）和磷酸三苯酯（TPHP）对斑马鱼胚胎发育过程中DNA甲基化模式的影响。我们评估了一般毒性和形态变化，测量了整体DNA甲基化和羟甲基化水平，评估了DNA甲基转移酶（DNMT）的酶活性，以及DNMTs和十一-易位（TET）甲基胞嘧啶双加氧酶基因的mRNA表达。此外，我们使用简化代表性亚硫酸氢盐测序分析了斑马鱼幼虫的全基因组甲基化模式。我们的形态学评估显示没有一般毒性，但暴露于TMPP和EHDPP后体长出现了具有统计学意义但细微的下降，同时暴露于TPHP后头部高度降低，而眼直径和头部宽度不受任何一种OPFRs的影响。任何暴露组的整体DNA甲基化水平均无显著变化，TMPP对DNMT表达没有明显影响。然而，EHDPP仅显著降低了DNMT1的表达，而暴露于TPHP则降低了斑马鱼幼虫中几种DNMT同源物和TETs的表达，导致全基因组异常DNA甲基化。差异甲基化主要发生在内含子（43%）和基因间区域（37%），分别有9%和10%发生在外显子和启动子区域。差异甲基化区域相关基因的通路富集分析表明，暴露于TPHP增强了与代谢和神经发育相关的几种生物学和分子功能。KEGG富集分析进一步揭示了TPHP对包括TGFβ、细胞因子和胰岛素信号在内的几种信号通路的潜在影响。本研究确定了暴露于TPHP后斑马鱼幼虫DNA甲基化的特定变化，并为TPHP的表观遗传作用模式带来了新的见解。
