Abstract

The development and production of engineered 2D nanomaterials are expanding exponentially, increasing the risk of their release into the aquatic environment. A recent study showed 2D MnO₂ nanosheets, under development for energy and biomedical applications, dissolve upon interaction with biological reducing agents, resulting in depletion of intracellular glutathione levels within fish gill cells. However, little is known concerning their toxicity and interactions with subcellular organelles. To address this gap, we examined cellular uptake, cytotoxicity and mitochondrial effects of 2D MnO₂ nanosheets using an in vitro fish gill cell line to represent a target tissue of rainbow trout, a freshwater indicator species. The data demonstrate cellular uptake of MnO₂ nanosheets into lysosomes and potential mechanisms of dissolution within the lysosomal compartment. MnO₂ nanosheets induced severe mitochondrial dysfunction at sub-cytotoxic doses. Quantitative, single cell fluorescent imaging revealed mitochondrial fission and impaired mitochondrial membrane potential following MnO₂ nanosheet exposure. Seahorse analyses for cellular respiration revealed that MnO₂ nanosheets inhibited basal respiration, maximal respiration and the spare respiratory capacity of gill cells, indicating mitochondrial dysfunction and reduced cellular respiratory activity. MnO₂ nanosheet exposure also inhibited ATP production, further supporting the suppression of mitochondrial function and cellular respiration. Together, these observations indicate that 2D MnO₂ nanosheets impair the ability of gill cells to respond to energy demands or prolonged stress. Finally, our data demonstrate significant differences in the toxicity of the 2D MnO₂ nanosheets and their microparticle counterparts. This exemplifies the importance of considering the unique physical characteristics of 2D nanomaterials when conducting safety assessments.

摘要

工程二维纳米材料的开发和生产正呈指数增长，这增加了将其释放到水生环境中的风险。最近的一项研究表明，正在为能源和生物医学应用开发的二维MnO ₂纳米片在与生物还原剂相互作用时溶解，从而导致鱼g细胞内细胞内谷胱甘肽水平的消耗。然而，关于它们的毒性和与亚细胞器相互作用的了解甚少。为了解决这个差距，我们使用体外鱼g细胞系来代表虹鳟鱼（淡水指示物种）的目标组织，研究了二维MnO ₂纳米片的细胞摄取，细胞毒性和线粒体作用。数据表明细胞吸收MnO₂纳米片溶酶体和溶酶体区室中溶解的潜在机制。MnO ₂纳米片以亚细胞毒性剂量诱导了严重的线粒体功能障碍。MnO ₂纳米片暴露后，定量单细胞荧光成像显示线粒体裂变和线粒体膜电位受损。海马对细胞呼吸的分析表明，MnO ₂纳米片抑制了基础呼吸，最大呼吸和g细胞的备用呼吸能力，表明线粒体功能障碍和细胞呼吸活性降低。的MnO ₂纳米片的暴露也抑制了ATP的产生，进一步支持了线粒体功能和细胞呼吸的抑制。总之，这些观察结果表明2D MnO ₂纳米片会削弱cells细胞对能量需求或长时间应激的反应能力。最后，我们的数据证明了二维MnO ₂纳米片及其微粒对应物在毒性方面的显着差异。这体现了进行安全评估时考虑2D纳米材料独特物理特性的重要性。