芳香烃受体介导双酚A致斑马鱼胚胎发育毒性
Aryl hydrocarbon receptor-mediated developmental toxicity in zebrafish embryos induced by bisphenol A
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摘要:
背景 双酚A(BPA)在生命早期暴露会导致发育毒性,芳香烃受体(AhR)能够调控各种发育和生理功能,研究发现BPA可使其激活。
目的 以斑马鱼作为模式动物,研究AhR在BPA所致斑马鱼胚胎发育毒性中的作用。
方法 将斑马鱼胚胎随机分为8组进行染毒,分别为DMSO对照组、AhR抑制剂CH223191(CH)对照组、BPA暴露组(1、5、25 μmol·L-1)、CH+BPA(1、5、25 μmol·L-1)组。染毒时间为受精后2 h(2 hpf)至受精后96 h(96 hpf),分别观察斑马鱼胚胎的死亡率、孵化率、畸形率、心率、体长及运动能力。通过荧光定量PCR方法检测AhR相关基因(
ahr2、cyp1a1、cyp1b1 )、心脏发育关键基因(nkx2.5、sox9b )、神经发育关键基因(elavl3、gfap、mbp、syn2a )、氧化应激相关基因(nrf2、sod1/2、ho1、nqo1 )的表达。结果 与DMSO对照组相比,25 μmol·L-1 BPA暴露组斑马鱼胚胎出现孵化延迟、心率下降、畸形率升高,5 μmol·L-1和25 μmol·L-1 BPA暴露组出现死亡率增加、体长缩短以及运动距离下降的现象(
P < 0.05)。与相应BPA暴露组相比,CH+25 μmol·L-1 BPA组斑马鱼的心率、畸形率、运动能力和CH+5 μmol·L-1 BPA组及CH+25 μmol·L-1 BPA组的体长均有所恢复(P < 0.05)。但孵化率和死亡率并未受CH影响。荧光定量PCR结果表明,BPA组AhR相关基因(ahr2、cyp1a1 和cyp1b1 )的表达上调(P < 0.05);CH联合处理后能降低各BPA组ahr2、cyp1a1 基因的过表达及25 μmol·L-1 BPA组cyp1b1 的过表达(P < 0.05)。心脏发育相关基因(nkx2.5 和sox9b )的表达分别在25 μmol·L-1和5、25 μmol·L-1 BPA组出现下调(P < 0.05);CH联合处理后上述基因的低表达恢复(P < 0.05)。神经发育相关基因elavl3 在25 μmol·L-1 BPA组出现下调,gfap 和syn2a 在5、25 μmol·L-1 BPA组出现下调,mbp 在各BPA组均出现下调(P < 0.05);CH联合处理后上述基因的低表达恢复(P < 0.05)。BPA暴露后氧化应激相关基因的表达也出现改变,其中nrf2 在25 μmol·L-1 BPA组出现上调,sod1、ho1 和nqo1 在5、25 μmol·L-1 BPA组出现上调,sod2 在各BPA组均出现上调(P < 0.05);CH干预后这些基因的表达均恢复到正常水平(P < 0.05)。结论 BPA可能通过激活AhR,上调
cyp1a1 和cyp1b1 基因的表达,增强活性氧的产生,从而导致斑马鱼胚胎的发育毒性。Abstract:Background Exposure to bisphenol A (BPA) in the early life period can induce developmental toxicity. Aryl hydrocarbon receptor (AhR) can regulate various developmental and physiological functions, and can be activated by BPA.
Objective This experiment uses zebrafish as a model animal to study the role of AhR in the developmental toxicity of zebrafish embryos induced by BPA.
Methods Zebrafish embryos were randomly divided into eight groups: DMSO control group, AhR inhibitor CH223191 (CH) control group, BPA exposure groups (1, 5, and 25 μmol·L-1), and CH+BPA (1, 5, and 25 μmol·L-1) groups. The exposure period was from 2 h post-fertilization (2 hpf) to 96 h post-fertilization (96 hpf). The mortality, hatching rate, malformation rate, heart rate, body length, and locomotor ability of zebrafish embryos were evaluated. The expressions of AhR-related genes (
ahr2, cyp1a1 , andcyp1b1 ), cardiac development-related genes (nkx2.5 andsox9b ), and neurodevelopment-related genes (elavl3, gfap, mbp , andsyn2a ), and reactive oxygen species-related genes (nrf2, sod1, sod2, ho1 , andnqo1 ) were detected by fluorescence quantitative PCR.Results Compared with the DMSO control group, the zebrafish embryos in the 25 μmol·L-1 BPA group had delayed hatching, decreased heart rate, and elevated malformation rate, and the 5 μmol·L-1 and 25 μmol·L-1 groups showed shortened body length, increased mortality, and decreased movement distance (
P < 0.05). Compared with corresponding BPA exposure groups, the heart rate, malformation rate, and locomotor ability of the zebrafish in the CH+25μmol·L-1 BPA group, and the body length of the CH+5μmol·L-1 BPA group and the CH+25μmol·L-1 BPA group all recovered (P < 0.05). However, the hatching rate and mortality rate were not affected by CH. The results of fluorescence quantitative PCR showed that AhR-related genes (ahr2, cyp1a1 , andcyp1b1 ) were up-regulated in the BPA groups (P < 0.05), and CH combined treatment reduced the overexpression ofahr2 andcyp1a1 in each BPA group andcyp1b1 in the 25 μmol·L-1 BPA group (P < 0.05). The expression of cardiac development-related genenkx2.5 was down-regulated in the 25 μmol·L-1 BPA group and that ofsox9b was down-regulated in the 5 and 25 μmol·L-1 BPA groups, respectively (P < 0.05), and the expressions were recovered after the CH combined treatment (P < 0.05). The neurodevelopment-related geneelavl3 was down-regulated in the 25 μmol·L-1 BPA group (P < 0.05),gfap andsyn2a were down-regulated in the 5 and 25 μmol·L-1 BPA groups (P < 0.05), andmbp was down-regulated in all BPA groups (P < 0.05); the above genes were recovered after the CH combined treatment (P < 0.05). The expressions of reactive oxygen species-related genenrf2 was up-regulated in the 25μmol·L-1 BPA group,sod1, ho1 , andnqo1 were up-regulated in the 5 and 25μmol·L-1 BPA groups, andsod2 was up-regulated in all BPA groups (P < 0.05); after the CH treatment, the expressions of these genes returned to normal levels (P < 0.05).Conclusion BPA may activate AhR, up-regulate the expressions of
cyp1a1 andcyp1b1 genes, and enhance the production of ROS, leading to the developmental toxicity in zebrafish embryos.