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Copper induces expression and methylation changes of early development genes in Crassostrea gigas embryos
Aquatic Toxicology ( IF 4.5 ) Pub Date : 2018-01-03 , DOI: 10.1016/j.aquatox.2018.01.001
Rossana Sussarellu , Morgane Lebreton , Julien Rouxel , Farida Akcha , Guillaume Rivière

Copper contamination is widespread along coastal areas and exerts adverse effects on marine organisms such as mollusks. In the Pacific oyster, copper induces severe developmental abnormalities during early life stages; however, the underlying molecular mechanisms are largely unknown. This study aims to better understand whether the embryotoxic effects of copper in Crassostrea gigas could be mediated by alterations in gene expression, and the putative role of DNA methylation, which is known to contribute to gene regulation in early embryo development.

For that purpose, oyster embryos were exposed to 4 nominal copper concentrations (0.1, 1, 10 and 20 μg L−1 Cu2+) during early development assays. Embryotoxicity was monitored through the oyster embryo-larval bioassay at the D-larva stage 24 h post fertilization (hpf) and genotoxicity at gastrulation 7 hpf. In parallel, the relative expression of 15 genes encoding putative homeotic, biomineralization and DNA methylation proteins was measured at three developmental stages (3 hpf morula stage, 7 hpf gastrula stage, 24 hpf D-larvae stage) using RT-qPCR. Global DNA content in methylcytosine and hydroxymethylcytosine were measured by HPLC and gene-specific DNA methylation levels were monitored using MeDIP-qPCR.

A significant increase in larval abnormalities was observed from copper concentrations of 10 μg L−1, while significant genotoxic effects were detected at 1 μg L−1 and above. All the selected genes presented a stage-dependent expression pattern, which was impaired for some homeobox and DNA methylation genes (Notochord, HOXA1, HOX2, Lox5, DNMT3b and CXXC-1) after copper exposure. While global DNA methylation (5-methylcytosine) at gastrula stage didn’t show significant changes between experimental conditions, 5-hydroxymethylcytosine, its degradation product, decreased upon copper treatment. The DNA methylation of exons and the transcript levels were correlated in control samples for HOXA1 but such a correlation was diminished following copper exposure. The methylation level of some specific gene regions (HoxA1, Hox2, Engrailed2 and Notochord) displayed changes upon copper exposure. Such changes were gene and exon-specific and no obvious global trends could be identified. Our study suggests that the embryotoxic effects of copper in oysters could involve homeotic gene expression impairment possibly by changing DNA methylation levels.



中文翻译:

铜诱导Crassostrea gigas胚胎早期发育基因的表达和甲基化变化

铜污染在沿海地区很普遍,并对软体动物等海洋生物产生不利影响。在太平洋牡蛎中,铜会在生命的早期阶段引起严重的发育异常。然而,基本的分子机制尚不清楚。这项研究旨在更好地了解铜在Crassostrea gigas中的胚胎毒性作用是否可以通过基因表达的改变来介导,以及DNA甲基化的推定作用,众所周知,DNA甲基化有助于早期胚胎发育中的基因调控。

为此,在早期发育试验期间,牡蛎胚胎暴露于4个名义铜浓度(0.1、1、10和20μgL -1 Cu 2+)。受精后24小时通过D-幼虫阶段的牡蛎胚胎-幼虫生物测定法监测胚胎毒性(hpf),并在7 hpf进行排卵时进行遗传毒性监测。平行地,使用RT-qPCR在三个发育阶段(桑树3 hpf桑,虫阶段,7 hpf胃tru虫阶段,24 hpf D-幼虫阶段)测量了编码假定的同源,生物矿化和DNA甲基化蛋白的15个基因的相对表达。通过HPLC测量甲基胞嘧啶和羟甲基胞嘧啶中的总体DNA含量,并使用MeDIP-qPCR监测基因特异性DNA甲基化水平。

从10μgL -1的铜浓度观察到幼虫异常的显着增加,而在1μgL -1及更高的浓度下检测到显着的遗传毒性作用。所有选择的基因均呈现出阶段依赖性的表达模式,该模式对于某些同源盒和DNA甲基化基因(Notochord,HOXA1,HOX2,Lox5,DNMT3b和CXXC-1受损。虽然在胃胚阶段的总体DNA甲基化(5-甲基胞嘧啶)在实验条件之间没有显示出显着变化,但其降解产物5-羟甲基胞嘧啶在铜处理后却降低了。HOXA1对照样品中外显子的DNA甲基化与转录水平相关但是铜暴露后这种相关性减弱了。铜暴露后,某些特定基因区域(HoxA1,Hox2,Engrailed2Notochord)的甲基化水平显示出变化。这种变化是基因和外显子特有的,无法确定明显的全球趋势。我们的研究表明,铜对牡蛎的胚胎毒性作用可能通过改变DNA甲基化水平而导致同源基因表达受损。

更新日期:2018-01-03
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