纳米银和银离子对斑马鱼胚胎早期生长发育的影响及作用机制

为探究纳米银对水生生物的毒性作用,选取斑马鱼胚胎为受试生物,考察了纳米银对斑马鱼胚胎早期生长发育的影响,同时比较了纳米银与银离子对斑马鱼胚胎的毒性作用和机理。实验将受精后4小时(4 hpf)的斑马鱼胚胎分别暴露于不同浓度的纳米银和银离子溶液中至96 hpf,观察并记录了胚胎的死亡、孵化和畸形等指标。应用吖啶橙(AO)染色实验研究了胚胎暴露之后的细胞凋亡情况,并且应用荧光定量PCR技术分析了相关基因的表达水平。研究结果表明,随着暴露浓度的增加,纳米银和银离子均能导致斑马鱼胚胎的死亡率增加和孵化率降低,并且引起孵化延迟。纳米银和银离子的96 h半数致死浓度(96 h-LC50)分别为11.75 mg·L-1和0.054 mg·L-1。银离子毒性远大于纳米银毒性。暴露的斑马鱼胚胎均表现出体长变短和卵黄囊肿大的畸形。AO染色结果表明,纳米银和银离子处理组胚胎的躯干和卵黄囊部位存在细胞凋亡信号。基因表达分析结果显示,1.93 mg·L-1纳米银显著提高了斑马鱼胚胎caspase9的表达(P0.05),而0.006 mg·L-1的银离子就能显著上调COX-2a(P0.01)和COX-17(P0.05)基因的表达,同时0.036 mg·L-1银离子增加了斑马鱼体内p53基因的表达(P0.05)。以上研究结果说明,纳米银可能通过caspase通路诱导细胞凋亡进而影响斑马鱼胚胎的生长发育;而银离子不但影响氧化系统基因通路,还能通过p53诱导凋亡进而阻滞斑马鱼胚胎的生长发育。

Effects of Silver Nanoparticles and Silver Ions on the Early Development of Zebrafish Embryos and Toxicity Mechanisms

To investigate the effects of silver nanoparticles (Ag NPs) on the early development of aquatic organisms and compare with that of silver ions, zebrafish embryos were selected and exposed to different concentrations of Ag NPs or Ag+ from 4 hours post fertilization (hpf) to 96 hpf. The survival, hatching and malformations of treated zebrafish embryos were examined. The cell apoptosis in treated zebrafish embryos were observed by the acridine orange (AO) staining. Related genes and their expression profiles were analyzed with fluorescence quantitative PCR. Results showed that exposure to Ag NPs or Ag+ induced increased mortality, decreased hatching rate and delayed hatching in zebrafish embryos in a concentration-dependent manner. The 96 h half lethal concentration (96 h-LC50) of Ag NPs and Ag+ on zebrafish embryos were 11.75 mg?L-1 and 0.054 mg?L-1 respectively. The toxicity of Ag+ was greater than that of Ag NPs. Exposed zebrafish embryos exhibited shorter body length and enlarged yolk sac. The AO staining results showed cell apoptosis in the trunk and yolk sac of zebrafish were observed respectively after treatment with Ag NPs and Ag+. The gene expression analysis showed that Ag NPs significantly up-regulated the expression of caspase9 in zebrafish embryos at the concentration of 1.93 mg?L-1 (P<0.05). Exposure to 0.006 mg?L-1 of Ag+ significantly up-regulated the expressions of COX-2a (P<0.01) and COX-17 (P<0.05) in zebrafish embryos. The expression level of p53 gene were also significantly increased in zebrafish embryos after exposed to 0.036 mg?L-1 Ag+ (P<0.05). This study suggested Ag NPs could induce apoptosis in zebrafish embryos via the caspase-dependent pathway and thus affected the development of exposed zebrafish embryos. However, exposure to Ag+ not only affected the oxidation-related gene pathway, but also induced apoptosis via p53 pathway and inhibited the development of exposed zebrafish embryos.