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基于C2C12细胞的2,7-二溴咔唑肌发育毒性效应

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郝迪, 谢群慧, 杨广磊, 陈旸升, 李云平, 夏英杰, 徐丽, 赵斌. 基于C2C12细胞的2,7-二溴咔唑肌发育毒性效应[J]. 生态毒理学报, 2023, 18(4): 293-303. doi: 10.7524/AJE.1673-5897.20230411001
引用本文: 郝迪, 谢群慧, 杨广磊, 陈旸升, 李云平, 夏英杰, 徐丽, 赵斌. 基于C2C12细胞的2,7-二溴咔唑肌发育毒性效应[J]. 生态毒理学报, 2023, 18(4): 293-303. doi: 10.7524/AJE.1673-5897.20230411001
shu
Hao Di, Xie Qunhui, Yang Guanglei, Chen Yangsheng, Li Yunping, Xia Yingjie, Xu Li, Zhao Bin. Toxicity of 2,7-dibromo-9H-carbazole on Myogenesis Based on C2C12 Cells[J]. Asian journal of ecotoxicology, 2023, 18(4): 293-303. doi: 10.7524/AJE.1673-5897.20230411001
Citation: Hao Di, Xie Qunhui, Yang Guanglei, Chen Yangsheng, Li Yunping, Xia Yingjie, Xu Li, Zhao Bin. Toxicity of 2,7-dibromo-9H-carbazole on Myogenesis Based on C2C12 Cells[J]. Asian journal of ecotoxicology, 2023, 18(4): 293-303. doi: 10.7524/AJE.1673-5897.20230411001
shu

基于C2C12细胞的2,7-二溴咔唑肌发育毒性效应

  • 1. 中国科学院生态环境研究中心, 环境化学与生态毒理学国家重点实验室, 北京 100085;

  • 2. 中国科学院大学中丹学院, 北京 100190;

  • 3. 中国科学院大学, 北京 100049;

  • 4. 国科大杭州高等研究院环境学院, 杭州 310024;

  • 5. 香港科技大学生命科学学院, 香港 999077

    • 作者简介: 郝迪(1998-),女,硕士研究生,研究方向为分子环境毒理学,E-mail:hd1031200182@163.com
    通讯作者: 谢群慧,E-mail:qhxie@rcees.ac.cn; 
  • 基金项目:

    国家自然科学基金面上项目(21976201)；国家自然科学基金重点项目(21836004)；国家重点研发计划项目(2018YFA0901100)

  • 中图分类号: X171.5

Toxicity of 2,7-dibromo-9H-carbazole on Myogenesis Based on C2C12 Cells

  • 1. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China;

  • 2. Sino-Danish College, University of Chinese Academy of Sciences, Beijing 100190, China;

  • 3. University of Chinese Academy of Sciences, Beijing 100049, China;

  • 4. College of Environment, Hangzhou Institute for Advanced Study, Hangzhou 310024, China;

  • 5. Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong 999077, China

    • Corresponding author: Xie Qunhui, qhxie@rcees.ac.cn ; 
  • Fund Project:

  • 摘要: 肌肉有执行机体运动和能量代谢的重要作用。据报道二噁英暴露可严重影响肌肉发育。但目前与多氯代二苯并呋喃(polychlorinated dibenzofurans, PCDFs)结构相似的卤代咔唑(polyhalogenated carbazoles, PHCZs)的肌肉发育毒性尚不明确。本研究旨在探究2,7-二溴咔唑(2,7-dibromo-9H-carbazole, 27-BCZ)的肌肉发育毒性效应。应用C2C12细胞成肌分化模型,在细胞水平上通过苏木素-伊红染色法检测融合指数和单条肌管内细胞核数2个指标评价细胞形态的变化,在分子水平上通过qRT-PCR来测定肌细胞分化调节分子和分化标志物的基因表达,探究27-BCZ对肌发育的影响及其分子机制。细胞水平实验结果显示,与对照组相比,27-BCZ 10-7mol·L-1暴露3 d后,肌管数量减少、形态变细变短,且分布杂乱;单条肌管内细胞核数减少、参与肌管融合的细胞核比例(融合指数)降低;27-BCZ 10-9~10-7 mol·L-1暴露6 d后,虽然单条肌管内细胞核数没有显著变化,但融合指数显著降低。分子水平实验结果显示,27-BCZ 10-7 mol·L-1在分化末期对肌纤维结构蛋白肌球蛋白重链(myosin heavy chain, MyHC)的2个编码基因Myh3和Myh4的表达具有显著抑制作用。另外,27-BCZ还影响肌生成调节因子家族(myogenic regulatory factors, MRFs)基因的表达,包括显著抑制分化早期调节因子MyoD和末期调节因子Mrf4的mRNA表达,显著促进Myogenin mRNA表达。此外,27-BCZ暴露能显著促进芳香烃受体(aryl hydrocarbon receptor, AhR)信号通路下游基因cyp1a1和cyp1b1的mRNA表达,提示27-BCZ在肌分化模型中具有类二噁英活性。总结上述结果,我们发现,在肌生成过程中,27-BCZ表现出与二噁英类似的AhR活性和肌发育干扰效应,为27-BCZ肌肉发育相关健康效应和AhR相关毒理机制研究提供了基础数据。
    Abstract: Muscles play an important role in executing body movements and energy metabolism. It has been reported that exposure to dioxins can affect muscle development seriously. However, the muscle developmental toxicity of polyhalogenated carbazoles (PHCZs), which are structurally similar to polychlorinated dibenzofurans, is currently unclear. This study aimed to investigate the muscular developmental toxicity of 2,7-dibromo-9H-carbazole (27-BCZ). We applied the C2C12 cell myogenic differentiation model and evaluated the changes in cell morphology by using hematoxylin eosin staining. Fusion index and the number of nuclei in a single muscle tube was used as main parameters for monitoring the myotube formation. At the molecular level, we used qRT-PCR to determine the gene expression of muscle cell differentiation regulatory molecules and differentiation markers, and explore the effects and molecular mechanisms of 27-BCZ on muscle development. The results at cell level showed that after 3 days of exposure to 27-BCZ at 10-7 mol·L-1, the myotubes formed were fewer, thinner, shorter and less organized than those of control group. The number of nuclei in the myotube and the fusion index were decreased. After 6 days of exposure to 27-BCZ at 10-9~10-7mol·L-1, although there was no significant change in the number of nuclei in the myotubes, the fusion index was significantly decreased. Moreover, 27-BCZ at 10-7 mol·L-1 significantly inhibited the expression of Myh3 and Myh4, two encoding genes of myosin heavy chain (MyHC), at the end stage of differentiation. Furthermore, 27-BCZ disturbed the expression of myogenic regulatory factors (MRFs), which play important roles in regulating the process of muscle differentiation. We found significantly down-regulation of the mRNA expression of the early differentiation MRF MyoD and the late differentiation MRF Mrf4, while promoting the expression of Myogenin. In addition, we found that 27-BCZ treatment can significantly induce the expression of cyp1a1 and cyp1b1, two classical downstream genes of aryl hydrocarbon receptor (AhR). These results indicate that 27-BCZ may have dioxin-like AhR activity during muscle differentiation. In summary, we found that 27-BCZ exhibited AhR activity and myogenesis interference effects similar to dioxins in C2C12 cells, providing fundamental data for further studies of 27-BCZ on muscle development-related health effects and AhR-related toxicological mechanisms.
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  • 收稿日期:  2023-04-11
郝迪, 谢群慧, 杨广磊, 陈旸升, 李云平, 夏英杰, 徐丽, 赵斌. 基于C2C12细胞的2,7-二溴咔唑肌发育毒性效应[J]. 生态毒理学报, 2023, 18(4): 293-303. doi: 10.7524/AJE.1673-5897.20230411001
引用本文: 郝迪, 谢群慧, 杨广磊, 陈旸升, 李云平, 夏英杰, 徐丽, 赵斌. 基于C2C12细胞的2,7-二溴咔唑肌发育毒性效应[J]. 生态毒理学报, 2023, 18(4): 293-303. doi: 10.7524/AJE.1673-5897.20230411001
shu
Hao Di, Xie Qunhui, Yang Guanglei, Chen Yangsheng, Li Yunping, Xia Yingjie, Xu Li, Zhao Bin. Toxicity of 2,7-dibromo-9H-carbazole on Myogenesis Based on C2C12 Cells[J]. Asian journal of ecotoxicology, 2023, 18(4): 293-303. doi: 10.7524/AJE.1673-5897.20230411001
Citation: Hao Di, Xie Qunhui, Yang Guanglei, Chen Yangsheng, Li Yunping, Xia Yingjie, Xu Li, Zhao Bin. Toxicity of 2,7-dibromo-9H-carbazole on Myogenesis Based on C2C12 Cells[J]. Asian journal of ecotoxicology, 2023, 18(4): 293-303. doi: 10.7524/AJE.1673-5897.20230411001
shu

基于C2C12细胞的2,7-二溴咔唑肌发育毒性效应

    通讯作者: 谢群慧,E-mail:qhxie@rcees.ac.cn; 
    作者简介: 郝迪(1998-),女,硕士研究生,研究方向为分子环境毒理学,E-mail:hd1031200182@163.com
  • 1. 中国科学院生态环境研究中心, 环境化学与生态毒理学国家重点实验室, 北京 100085;
  • 2. 中国科学院大学中丹学院, 北京 100190;
  • 3. 中国科学院大学, 北京 100049;
  • 4. 国科大杭州高等研究院环境学院, 杭州 310024;
  • 5. 香港科技大学生命科学学院, 香港 999077
  • 收稿日期:  2023-04-11
基金项目:

国家自然科学基金面上项目(21976201)；国家自然科学基金重点项目(21836004)；国家重点研发计划项目(2018YFA0901100)

摘要: 肌肉有执行机体运动和能量代谢的重要作用。据报道二噁英暴露可严重影响肌肉发育。但目前与多氯代二苯并呋喃(polychlorinated dibenzofurans, PCDFs)结构相似的卤代咔唑(polyhalogenated carbazoles, PHCZs)的肌肉发育毒性尚不明确。本研究旨在探究2,7-二溴咔唑(2,7-dibromo-9H-carbazole, 27-BCZ)的肌肉发育毒性效应。应用C2C12细胞成肌分化模型,在细胞水平上通过苏木素-伊红染色法检测融合指数和单条肌管内细胞核数2个指标评价细胞形态的变化,在分子水平上通过qRT-PCR来测定肌细胞分化调节分子和分化标志物的基因表达,探究27-BCZ对肌发育的影响及其分子机制。细胞水平实验结果显示,与对照组相比,27-BCZ 10-7mol·L-1暴露3 d后,肌管数量减少、形态变细变短,且分布杂乱;单条肌管内细胞核数减少、参与肌管融合的细胞核比例(融合指数)降低;27-BCZ 10-9~10-7 mol·L-1暴露6 d后,虽然单条肌管内细胞核数没有显著变化,但融合指数显著降低。分子水平实验结果显示,27-BCZ 10-7 mol·L-1在分化末期对肌纤维结构蛋白肌球蛋白重链(myosin heavy chain, MyHC)的2个编码基因Myh3和Myh4的表达具有显著抑制作用。另外,27-BCZ还影响肌生成调节因子家族(myogenic regulatory factors, MRFs)基因的表达,包括显著抑制分化早期调节因子MyoD和末期调节因子Mrf4的mRNA表达,显著促进Myogenin mRNA表达。此外,27-BCZ暴露能显著促进芳香烃受体(aryl hydrocarbon receptor, AhR)信号通路下游基因cyp1a1和cyp1b1的mRNA表达,提示27-BCZ在肌分化模型中具有类二噁英活性。总结上述结果,我们发现,在肌生成过程中,27-BCZ表现出与二噁英类似的AhR活性和肌发育干扰效应,为27-BCZ肌肉发育相关健康效应和AhR相关毒理机制研究提供了基础数据。

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