何梦婷, 王伟, 许洁, 张洁. 氧化应激参与溴乙酰胺致斑马鱼胚胎神经发育毒性[J]. 环境与职业医学, 2021, 38(6): 586-592. DOI: 10.13213/j.cnki.jeom.2021.20580
引用本文: 何梦婷, 王伟, 许洁, 张洁. 氧化应激参与溴乙酰胺致斑马鱼胚胎神经发育毒性[J]. 环境与职业医学, 2021, 38(6): 586-592. DOI: 10.13213/j.cnki.jeom.2021.20580
HE Mengting, WANG Wei, XU Jie, ZHANG Jie. Role of oxidative stress in neurodevelopmental toxicity of bromoacetamide in zebrafish embryos[J]. Journal of Environmental and Occupational Medicine, 2021, 38(6): 586-592. DOI: 10.13213/j.cnki.jeom.2021.20580
Citation: HE Mengting, WANG Wei, XU Jie, ZHANG Jie. Role of oxidative stress in neurodevelopmental toxicity of bromoacetamide in zebrafish embryos[J]. Journal of Environmental and Occupational Medicine, 2021, 38(6): 586-592. DOI: 10.13213/j.cnki.jeom.2021.20580

氧化应激参与溴乙酰胺致斑马鱼胚胎神经发育毒性

Role of oxidative stress in neurodevelopmental toxicity of bromoacetamide in zebrafish embryos

  • 摘要: 背景

    溴乙酰胺是一种饮用水消毒副产物,研究证实其具有细胞毒性和遗传毒性,但对于神经发育的毒性作用尚不明确。

    目的

    探究溴乙酰胺在斑马鱼胚胎期暴露所致神经发育毒性及可能机制。

    方法

    将野生型斑马鱼胚胎随机分为6组(每组n=30)进行染毒,分别为:对照组、溴乙酰胺各质量浓度(后称:浓度)(0.625、1.25、2.5、5、10 mg·L-1)染毒组。染毒时间为受精后2 h(2 hpf)至96 hpf。每天观察斑马鱼胚胎的死亡情况并更换培养液,最终统计72 hpf的孵化率和96 hpf的死亡率、畸形率、体长。于120 hpf时检测斑马鱼幼鱼的运动行为能力,并进行结果统计。用二氯二氢荧光素二乙酸酯(DCFH-DA)为探针检测斑马鱼体内活性氧(ROS)水平。通过荧光定量PCR方法检测神经发育相关基因(dlx2ngn1elavl3shhambpsyn2a)及氧化应激相关基因(Cu/Zn sodgpxcatnrf2ho-1)的表达情况。

    结果

    72hpf时,10mg·L-1溴乙酰胺染毒组斑马鱼胚胎的孵化率为88.40%,较对照组(100%)降低(P < 0.05)。96 hpf时,5、10 mg·L-1溴乙酰胺染毒组斑马鱼胚胎的死亡率为48.10%和96.06%,较对照组(15.92%)升高(P < 0.05);2.5、5 mg·L-1溴乙酰胺染毒组胚胎畸形率为21.38%和43.43%,也较对照组(1.94%)升高(P < 0.05);2.5、5 mg·L-1溴乙酰胺染毒组胚胎体长分别为5.21、4.94 mm,较对照组(5.40 mm)下降(P < 0.05)。运动检测发现:与对照组相比,溴乙酰胺染毒组均出现运动距离下降和运动时间缩短的现象,差异具有统计学意义(P < 0.05)。ROS染色发现,1.25、2.5、5 mg·L-1溴乙酰胺染毒组斑马鱼胚胎头部区域ROS表达水平高于对照组,差异具有统计学意义(P < 0.05)。定量PCR检测结果发现:与对照组相比,神经发育相关基因dlx2在2.5、5 mg·L-1染毒组表达下调,ngn1在5 mg·L-11染毒组下调,elavl3shha在1.25、2.5和5 mg·L-1染毒组下调,mbp在1.25、5 mg·L-1染毒组下调,syn2a在各染毒组均下调,差异具有统计学意义(P < 0.05);氧化应激相关基因Cu/Zn sod在2.5、5 mg·L-1染毒组上调,gpx在5 mg·L-1染毒组上调,catnrf2在1.25、2.5、5 mg·L-1染毒组上调,ho-1在各染毒组均上调(P < 0.05)。

    结论

    溴乙酰胺暴露后可能通过诱导氧化应激,抑制神经发育相关基因的表达,从而导致斑马鱼胚胎神经发育毒性。

     

    Abstract: Background

    Bromoacetamide (BAcAm) is a kind of disinfection by-products, and has been confirmed to have cytotoxicity and genetic toxicity, but its toxic effect on neurodevelopment is still unclear.

    Objective

    This experiment aims to study the neurodevelopmental toxicity and its mechanism caused by BAcAm on zebrafish embryos.

    Methods

    Wild-type zebrafish embryos were randomly divided into six groups (30 embryos in each group), namely a control group and five BAcAm exposure groups (0.625, 1.25, 2.5, 5, and 10 mg·L-1). The exposure period was from 2 h post-fertilization (hpf) to 96 hpf. The mortality of zebrafish embryos was detected every day, and the 72 hpf hatching rate and the 96 hpf mortality, deformity rate, and body length were calculated. The locomotor ability of zebrafish was tested at 120hpf. Reactive oxygen species (ROS) levels in zebrafish were detected by 2, 7-dichlorodi -hydrofluorescein diacetate (DCFH-DA) probe. The expression levels of neurodevelopment-related genes (dlx2, ngn1, elavl3, shha, mbp, and syn2a) and oxidative stress-related genes (Cu/Zn sod, gpx, cat, nrf2, and ho-1) were detected by fluorescence quantitative PCR.

    Results

    At 72 hpf, the hatching rate of zebrafish embryos in the 10 mg·L-1 BAcAm exposure group was 88.40%, lower than the 100% hatching rate in the control group (P < 0.05). At 96 hpf, the mortality rates of zebrafish embryos in the 5 and 10 mg·L-1 BAcAm exposure groups were 48.10% and 96.06%, respectively, higher than 15.92% in the control group (P < 0.05); the malformation rates of embryos in the 2.5 and 5 mg·L-1 BAcAm exposure groups were 21.38% and 43.43%, respectively, which were higher than 1.94% in the control group (P < 0.05); the body lengths of embryos in the 2.5 and 5mg·L-1 BAcAm exposure groups were 5.21 and 4.94mm, respectively, lower than 5.40mm in the control group (P < 0.05). The locomotor ability test results found that compared with the control group, the BAcAm exposure groups showed decreasing movement distance and time (P < 0.05). ROS staining results revealed that the ROS expression levels in the head region of zebrafish embryos in the 1.25, 2.5, and 5 mg·L-1 BAcAm groups were significantly higher than those in the control group (P < 0.05). Quantitative PCR results showed that the expression levels of neurodevelopment-related genes dlx2 were down-regulated in the 2.5 and 5 mg·L-1 BAcAm groups, ngn1 was down-regulated in the 5 mg·L-1 BAcAm group, elavl3 and shha were down-regulated in the 1.25, 2.5, and 5mg·L-1 BAcAm groups, mbp was down-regulated in the 1.25 and 5mg·L-1 BAcAm groups, syn2a was down-regulated in all BAcAm groups (P < 0.05); the expression levels of oxidative stress-related genes Cu/Zn sod was up-regulated in the 2.5 and 5 mg·L-1 BAcAm groups, gpx was up-regulated in the 5 mg·L-1 BAcAm group, cat and nrf2 were up-regulated in the 1.25, 2.5, and 5 mg·L-1 BAcAm groups, and ho-1 was upregulated in all BAcAm groups (P < 0.05).

    Conclusion

    BAcAm exposure may induce oxidative stress and inhibit the expression of neurodevelopment-related genes, thereby induce neurodevelopmental toxicity in zebrafish embryos.

     

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