Zinc oxide Nanoparticles (ZnO NPs) are widely used as emerging materials in agricultural and food-related fields, which exists potential safety hazards to public health and environment while bringing an added level of convenience to our original life. It has been proved that ZnO NPs could be taken up by pregnant women and passed through human placental barrier. However, the toxic potential for embryo development remains largely unanswered. In this study, we discovered that ZnO NPs caused the cytotoxicity in vitro. Inhibition of free Zn²⁺ ions in solution by EDTA or inhibition of Zn²⁺ ions absorption by CaCl₂ could partially eliminate ZnO NPs-mediated cell toxicity, though not redeem completely. This indicated that both nanoparticles and the release of Zn²⁺ ions were involved in ZnO NPs-mediated cytotoxicity. In addition, we also found that both nanoparticles and Zn²⁺ ion release triggered reactive oxygen species (ROS) production, which further induced cell toxicity, inflammation and apoptosis, which are mediated by NF-κB signaling cascades and the mitochondria dysfunction, respectively. Eventually, these events lead to the suppressed production and migration of cranial neural crest cells (CNCCs), which subsequently prompts the craniofacial defects in chicken embryos. The application of the antioxidant N-Acetyl-L-cysteine (NAC) rescued the ZnO NPs-induced cell toxicity and malformation of the CNCCs, which further verified our hypothesis. Our results revealed the relevant mechanism of ZnO NPs exposure-inhibited the development of CNCCs, which absolutely contribute to assess the risk of nanoparticles application.

氧化锌纳米颗粒（ZnO NPs）被广泛用作农业和食品相关领域的新兴材料，在对公众健康和环境造成潜在安全隐患的同时，为我们的原始生活带来更多便利。事实证明，ZnO NPs可以被孕妇吸收并通过人胎盘屏障。但是，胚胎发育的潜在毒性仍未得到解决。在这项研究中，我们发现ZnO NPs在体外引起细胞毒性。EDTA抑制溶液中游离Zn ²⁺离子或抑制CaCl ₂吸收Zn ²⁺离子可以部分消除ZnO NPs介导的细胞毒性，尽管不能完全赎回。这表明纳米颗粒和Zn ²⁺离子的释放均与ZnO NPs介导的细胞毒性有关。此外，我们还发现纳米颗粒和Zn ²⁺离子释放触发了活性氧（ROS）的产生，从而进一步诱导了细胞毒性，炎症和细胞凋亡，分别由NF-κB信号级联和线粒体功能障碍介导。最终，这些事件导致颅神经rest细胞（CNCC）的产生和迁移受到抑制，这随后提示了鸡胚的颅面缺陷。抗氧化剂N-乙酰基-L-半胱氨酸（NAC）的应用挽救了ZnO NPs诱导的细胞毒性和CNCCs的畸形，从而进一步证实了我们的假设。我们的研究结果揭示了ZnO NPs暴露抑制CNCCs发展的相关机制，这绝对有助于评估纳米颗粒应用的风险。