使用免疫荧光检测斑马鱼胚胎心脏PM2.5诱发DNA损伤
Summary
该协议使用免疫荧光检测检测斑马鱼胚胎解剖心脏中PM2.5诱发的DNA损伤。
Abstract
环境细颗粒物（PM2.5）暴露可导致心脏发育毒性，但潜在的分子机制尚不清楚。8-羟基-2’脱氧酶（8-OHdG）是氧化DNA损伤的标记，+H2AX是DNA双链断裂的敏感标记。在这项研究中，我们旨在通过免疫荧光检测斑马鱼胚胎心脏的PM2.5诱导8-OHDG和+H2AX变化。斑马鱼胚胎在受精后2小时（hpf）2小时时，以5微克/mL的PM2.5 处理可提取的有机物质（EOM）。DMSO 被用作车辆控制。在72马力，心脏被解剖从胚胎使用注射器针和固定和渗透。被阻塞后，用原样抗体对8-OHdG和+H2AX进行探测。然后用二次抗体清洗和孵育样品。由此产生的图像在荧光显微镜下观察，并使用 ImageJ 进行量化。结果表明，EOM从下午2.5 显著增强8-OHDG和+H2AX信号在斑马鱼胚胎的心脏。然而，NAC作为活性氧物种（ROS）的清除剂，部分抵消了EOM引起的DNA损伤。在这里，我们提出了一个免疫荧光方案，用于研究DNA损伤在PM2.5诱发的心脏缺陷中的作用，可用于检测斑马鱼胚胎心脏中环境化学诱发的蛋白质表达变化。
Introduction
空气污染是目前世界面临的一个严重的环境问题。环境细颗粒物（PM2.5）是空气质量最重要的指标之一，可携带大量有害物质进入血液循环系统，对人体健康造成严重危害。流行病学研究表明，PM2.5暴露可导致先天性心脏缺陷（CHDs）2，3的风险增加。动物实验的证据表明，PM2.5可导致斑马鱼胚胎和小鼠后代心脏发育异常，但PM2.5心脏发育毒性的分子机制仍鲜为人知。
DNA损伤可引起细胞周期骤停并诱发凋亡，从而可能广泛破坏祖细胞的潜力，进而损害心脏发育。有充分证据表明，环境污染物，包括PM2.5，有可能通过氧化应激机制8，9攻击DNA。人类和斑马鱼心脏发育都对氧化应激10、11、12敏感。8-OHdG 是一种氧化性 DNA 损伤标记，+H2AX 信号是 DNA 双链断裂的标志。N-乙酰-L-半胱氨酸（NAC）是细胞内半胱氨酸和谷胱甘肽的合成前体，被广泛用作抗氧化化合物。在这项研究中，我们使用NAC来研究氧化应激在下午2.5诱发DNA损伤13中的作用。
斑马鱼作为脊椎动物的模型，已被广泛用于研究心脏发育和人类心血管疾病，因为心脏发育机制在脊椎动物中保存高度。使用斑马鱼作为模型的优势包括它们体积小、繁殖能力强和饲养成本低。这些研究特别感兴趣的是，斑马鱼胚胎在早期发育过程中不依赖于循环系统，并且能够存活下来，严重心脏畸形14。此外，它们的透明度允许在显微镜下直接观察整个身体。因此，斑马鱼胚胎提供了一个绝佳的机会，以评估参与诱导心脏发育毒性的分子机制，由于接触各种环境化学品5，16，17。我们之前曾报道，PM2.5引起的氧化应激导致DNA损伤和凋亡，导致斑马鱼18的心脏畸形。在这项研究中，我们提供了一个详细的协议，用于调查PM2.5引起的斑马鱼胚胎心脏DNA损伤。
Protocol
Representative Results
Discussion
虽然斑马鱼是研究环境化学物质心脏发育毒性的极好的脊椎动物模型，但由于胚胎心脏体积小，很难获得足够的蛋白质进行西方污点分析。因此，我们提出了一种敏感的免疫荧光方法，用于量化暴露在PM2.5下的斑马鱼胚胎心脏中DNA损伤生物标志物的蛋白质表达水平。
在解剖过程中，保持心脏的完整性是很重要的。根据我们的经验，在 72 hpf 下执行隔离相对容易。此外?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国自然科学基金会（资助编号：81870239、81741005、81972999）和江苏省高等学校优先学术项目发展的支持。
Materials
| 8-OHdG Antibody | Santa Cruz Biotechnology, USA | sc-66036 | Primary antibody |
| Analytical balance | Sartorius,China | BSA124S | |
| BSA | Solarbio,Beijing,China | SW3015 | For blocking |
| DAPI | Abcam, USA | ab104139 | For nuclear counterstain. |
| DMSO | Solarbio,Beijing,China | D8371 | |
| Fluorescence microscope | Olympus, Japan | IX73 | For imaging fluorescence signals/ |
| Goat Anti-Rabbit IgG Cy3 | Carlsbad,USA | CW0159 | Secondary antibody |
| Goat Anti-Rabbit IgG FITC | Carlsbad,USA | RS0003 | Secondary antibody |
| N-Acetyl-L-cysteine(NAC) | Adamas-Beta, Shanghai, China | 616-91-1 | |
| Orbital shaker | QILINBEIER,China | TS-1 | |
| Paraformaldehyde | Sigma,China | P6148 | Make 4% paraformaldehyde for fixation. |
| Phosphate Buffered Saline | HyClone,USA | SH30256.01 | Prepare 0.1% Tween in PBS for washing. |
| PM2.5 sampler | TianHong,Wuhan, China | TH-150C | For 24-hr uninterrupted PM2.5 sampling. |
| Re-circulating aquaculture system | HaiSheng,Shanghai,China | The zebrafish was maintained in it. | |
| Soxhlet extractor | ZhengQiao,Shanghai, China | BSXT-02 | For organic components extraction. |
| Stereomicroscope | Nikon,Canada | SMZ645 | For heart dissection from zebrafish embryos. |
| Tricaine methanesulfonate (MS222) | Sigma,China | E10521 | To anesthetize zebrafish embryos |
| Tween 20 | Sigma,China | P1379 | |
| γH2AX Antibody | Abcam, USA | ab26350 | Primary antibody |
Referenzen
- Zhang, B., et al. Maternal Exposure to Air Pollution and Risk of Congenital Heart Defects. European Journal of Pediatrics. 175, 1520 (2016).
- Huang, C. C., Chen, B. Y., Pan, S. C., Ho, Y. L., Guo, Y. L. Prenatal exposure to PM2.5 and Congenital Heart Diseases in Taiwan. The Science of the Total Environment. 655, 880-886 (2019).
- Mesquita, S. R., et al. Toxic assessment of urban atmospheric particle-bound PAHs: relevance of composition and particle size in Barcelona (Spain). Environmental Pollution. 184, 555-562 (2014).
- Zhang, H., et al. Crosstalk between AhR and wnt/beta-catenin signal pathways in the cardiac developmental toxicity of PM2.5 in zebrafish embryos. Toxicology. 355-356, 31-38 (2016).
- Duan, J., et al. Multi-organ toxicity induced by fine particulate matter PM2.5 in zebrafish (Danio rerio) model. Chemosphere. 180, 24-32 (2017).
- Lorda-Diez, C. I., et al. Cell senescence, apoptosis and DNA damage cooperate in the remodeling processes accounting for heart morphogenesis. Journal of Anatomy. 234, 815-829 (2019).
- Kouassi, K. S., et al. Oxidative damage induced in A549 cells by physically and chemically characterized air particulate matter (PM2.5) collected in Abidjan, Cote d’Ivoire. Journal of Applied Toxicology. 30, 310-320 (2010).
- Gualtieri, M., et al. Gene expression profiling of A549 cells exposed to Milan PM2.5. Toxicology Letters. 209, 136-145 (2012).
- Li, S. Y., Sigmon, V. K., Babcock, S. A., Ren, J. Advanced glycation endproduct induces ROS accumulation, apoptosis, MAP kinase activation and nuclear O-GlcNAcylation in human cardiac myocytes. Life Sciences. 80, 1051-1056 (2007).
- Yamashita, M. Apoptosis in zebrafish development. Comparative biochemistry and physiology. Part B, Biochemistry & Molecular Biology. 136, 731-742 (2003).
- Moazzen, H., et al. N-Acetylcysteine prevents congenital heart defects induced by pregestational diabetes. Cardiovascular Diabetology. 13, 46 (2014).
- Sun, S. Y. N-acetylcysteine, reactive oxygen species and beyond. Cancer Biology & Therapy. 9, 109-110 (2010).
- Tu, S., Chi, N. C. Zebrafish models in cardiac development and congenital heart birth defects. Differentiation. 84, 4-16 (2012).
- Asnani, A., Peterson, R. T. The zebrafish as a tool to identify novel therapies for human cardiovascular disease. Disease Models & Mechanisms. 7, 763-767 (2014).
- Li, M., et al. Toxic effects of polychlorinated biphenyls on cardiac development in zebrafish. Molecular Biology Reports. 41, 7973-7983 (2014).
- Massarsky, A., Prasad, G. L., Di Giulio, R. T. Total particulate matter from cigarette smoke disrupts vascular development in zebrafish brain (Danio rerio). Toxicology and Applied Pharmacology. 339, 85-96 (2018).
- Ren, F., et al. AHR-mediated ROS production contributes to the cardiac developmental toxicity of PM2.5 in zebrafish embryos. The Science of the Total Environment. 719, 135097 (2020).
- van Berlo, J. H., Molkentin, J. D. An emerging consensus on cardiac regeneration. Nature Medicine. 20, 1386-1393 (2014).
- Yue, C., et al. Protective effects of folic acid on PM2.5-induced cardiac developmental toxicity in zebrafish embryos by targeting AhR and Wnt/beta-catenin signal pathways. Environmental Toxicology. 32, 2316-2322 (2017).
- Zhao, X., Ren, X., Zhu, R., Luo, Z., Ren, B. Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria-mediated apoptosis in zebrafish embryos. Aquatic Toxicology. 180, 56-70 (2016).
- Zhao, X., Wang, S., Wu, Y., You, H., Lv, L. Acute ZnO nanoparticles exposure induces developmental toxicity, oxidative stress and DNA damage in embryo-larval zebrafish. Aquatic Toxicology. 136-137, 49-59 (2013).
- Zhu, L., et al. DNA damage and effects on glutathione-S-transferase activity induced by atrazine exposure in zebrafish (Danio rerio). Environmental Toxicology. 26, 480-488 (2011).
