Table_1_Associations Between Behavioral Effects of Bisphenol A and DNA Methylation in Zebrafish Embryos.XLSX

Endocrine-disrupting contaminants have been associated with aberrant changes in epigenetic pathways in animals. In this study, zebrafish embryos were exposed bisphenol A (BPA) to search for associations between behavior and epigenetic mechanisms in fish. For concentration-dependent responses, embryos were exposed to a range of BPA concentrations (0.1 nM to 30 μM). Embryos were analyzed for locomotor activity at 3-, 4-, and 5-days post fertilization (dpf) in response to changing light conditions. Based on concentration-dependent effects on behavior and gene expression, 10 μM BPA [from 24 to 96 hours post fertilization (hpf)] was used for a whole-genome bisulfite sequencing (WGBS) study searching for genome-wide impacts on DNA methylation. Over the examined concentration ranges, hyperactivity was demonstrated for exposures to 0.001 μM BPA in comparison to embryos exposed to lower or higher BPA concentrations. Transcriptional analysis showed significant effects at >0.01 μM BPA for two genes related to DNA methylation (dnmt1, cbs). BPA exposure did not significantly affect global DNA methylation, but 20,474 differentially methylated (DM) sites in 4,873 genes were identified by WGBS analysis. Most DM sites were identified within gene bodies. The genes with the most DM sites were all protocadherin 2 gamma subfamily genes, related to axon targeting, synaptic development and neuronal survival. KEGG pathways most significantly affected by BPA exposure were phosphatidylinositol signaling system, followed by VEGF and MAPK signaling pathways. This study shows that BPA can affect zebrafish embryo swimming activity at very low concentrations as well as affecting numerous methylated sites in genes which are overrepresented in functionally relevant metabolic pathways. In conclusion, altered methylation patterns of genes associated with nervous system development might lead to abnormal swimming activity.

内分泌干扰物（Endocrine-disrupting contaminants）已被证实与动物表观遗传通路的异常改变相关。本研究将斑马鱼胚胎暴露于双酚A（BPA）中，旨在探究鱼类行为与表观遗传机制之间的关联。为考察浓度依赖性应答，我们将胚胎置于浓度范围为0.1 nM至30 μM的BPA溶液中进行暴露处理。分别于受精后3、4、5天（dpf），检测胚胎在光照条件变化下的运动活性。基于行为与基因表达的浓度依赖性效应，我们选取10 μM BPA（暴露时段为受精后24至96小时，hpf）开展全基因组亚硫酸氢盐测序（WGBS）分析，以探究其对DNA甲基化的全基因组影响。在所考察的浓度范围内，相较于暴露于更低或更高浓度BPA的胚胎，0.001 μM BPA暴露组的胚胎表现出运动亢进。转录组分析显示，当BPA浓度高于0.01 μM时，两个与DNA甲基化相关的基因（dnmt1、cbs）的表达出现显著变化。BPA暴露并未显著影响整体基因组DNA甲基化水平，但通过WGBS分析共鉴定出4873个基因上的20474个差异甲基化（DM）位点。绝大多数DM位点位于基因本体区域内。差异甲基化位点富集最多的基因为原钙粘蛋白2γ亚家族基因，这类基因与轴突靶向、突触发育及神经元存活密切相关。受BPA暴露影响最为显著的KEGG通路为磷脂酰肌醇信号通路，其次为血管内皮生长因子（VEGF）与丝裂原活化蛋白激酶（MAPK）信号通路。本研究证实，极低浓度的BPA即可干扰斑马鱼胚胎的游泳行为，同时还会改变大量基因的甲基化位点，这些基因在功能相关的代谢通路中显著富集。综上，与神经系统发育相关的基因甲基化模式改变，可能会引发胚胎游泳行为异常。