Huang Yujie, Tao Yizhou, Cai Chang, Chen Jin, Ji Cheng, Aniagu Stanley, Jiang Yan, Chen Tao
School of Public Health, Soochow University.
Toxicology, Risk Assessment and Research Division, Texas Commission on Environmental Quality.
J Vis Exp. 2021 Feb 15(168). doi: 10.3791/62021.
Ambient fine particulate matter (PM2.5) exposure can lead to cardiac developmental toxicity but the underlying molecular mechanisms are still unclear. 8-hydroxy-2'deoxygenase (8-OHdG) is a marker of oxidative DNA damage and γH2AX is a sensitive marker for DNA double strand breaks. In this study, we aimed to detect PM2.5-induced 8-OHdG and γH2AX changes in the heart of zebrafish embryos using an immunofluorescence assay. Zebrafish embryos were treated with extractable organic matters (EOM) from PM2.5 at 5 μg/mL in the presence or absence of antioxidant N-acetyl-L-cysteine (NAC, 0.25 μM) at 2 h post fertilization (hpf). DMSO was used as a vehicle control. At 72 hpf, hearts were dissected from embryos using a syringe needle and fixed and permeabilized. After being blocked, samples were probed with primary antibodies against 8-OHdG and γH2AX. Samples were then washed and incubated with secondary antibodies. The resulting images were observed under fluorescence microscopy and quantified using ImageJ. The results show that EOM from PM2.5 significantly enhanced 8-OHdG and γH2AX signals in the heart of zebrafish embryos. However, NAC, acting as a reactive oxygen species (ROS) scavenger, partially counteracted the EOM-induced DNA damage. Here, we present an immunofluorescence protocol for investigating the role of DNA damage in PM2.5-induced heart defects that can be applied to the detection of environmental chemical-induced protein expression changes in the hearts of zebrafish embryos.
暴露于环境细颗粒物（PM2.5）会导致心脏发育毒性，但其潜在的分子机制仍不清楚。8-羟基-2'-脱氧鸟苷（8-OHdG）是氧化DNA损伤的标志物，而γH2AX是DNA双链断裂的敏感标志物。在本研究中，我们旨在使用免疫荧光测定法检测PM2.5诱导的斑马鱼胚胎心脏中8-OHdG和γH2AX的变化。在受精后2小时（hpf），将斑马鱼胚胎用5μg/mL的PM2.5可提取有机物（EOM）处理，同时存在或不存在抗氧化剂N-乙酰-L-半胱氨酸（NAC，0.25μM）。二甲基亚砜用作溶剂对照。在72 hpf时，使用注射器针头从胚胎中取出心脏并固定和通透化。封闭后，样品用抗8-OHdG和γH2AX的一抗进行检测。然后洗涤样品并与二抗孵育。在荧光显微镜下观察所得图像并使用ImageJ进行定量。结果表明，PM2.5的EOM显著增强了斑马鱼胚胎心脏中的8-OHdG和γH2AX信号。然而，作为活性氧（ROS）清除剂的NAC部分抵消了EOM诱导的DNA损伤。在此，我们提出了一种免疫荧光方案，用于研究DNA损伤在PM2.5诱导的心脏缺陷中的作用，该方案可用于检测环境化学物质诱导的斑马鱼胚胎心脏中蛋白质表达的变化。
