Collaborative Research: Linking metal nanoparticle chemical modifications at the luminal/intestinal epithelia interface to intracellular alterations of essential metal homeostasis

由于金属纳米颗粒的广泛应用及其带来的技术进步，全球金属纳米颗粒产量的增加提出了它们对人类健康和环境的潜在影响的问题。细胞已经进化到严格调节必需和有毒金属离子的摄取和细胞内浓度；然而，金属纳米颗粒似乎绕过这些正常的摄取系统，以一种不太受控制的方式在细胞中积累。由于缺乏适当的生物学模型，确定增强或减少金属纳米颗粒摄取、生物反应性和毒性的暴露情景往往很困难。为了在细胞水平上进行这样的研究，这个研究项目使用了虹鳟（Oncorhynchus mykiss）的鱼肠模型。一旦进入细胞，纳米颗粒的生物反应性和毒性取决于它们的化学转化，这一过程目前尚不清楚。本研究项目试图将细胞内外金属纳米颗粒的动态化学修饰与细胞内基本金属稳态的改变联系起来。在这个项目中产生的机械知识被设想为支持关于金属离子和金属纳米颗粒暴露的人类和环境风险评估。此外，鱼肠模型的发展将有利于监管科学，因为它有可能降低测试成本，并有助于在毒理学风险评估中建立无动物替代品。这个多学科项目为两名研究生研究助理和多名本科生研究助理提供了宝贵的合作培训机会。本研究项目旨在评估金属纳米颗粒对人体必需微量金属体内平衡的影响。必需金属和非必需金属的时空分布与细胞内金属生物利用度的生物标志物相关，建立了细胞内化学修饰和生物反应之间的联系。虽然使用简化的细胞培养基和化学平衡模型可以确定细胞外溶解的金属形态，但细胞内金属形态更难确定。细胞内金属形态的准确评估需要一种技术，允许在原位测量，如x射线光谱和显微镜。本研究项目研究如何将RTgutGC中细胞外银和二氧化钛纳米颗粒的行为（溶解金属离子的聚集、溶解和形态形成）与金属纳米颗粒诱导的细胞内化学修饰（必需微量金属的水平和定位）与分子和细胞反应（金属转运体的mRNA和蛋白质水平以及细胞器成像）联系起来。RTgutGC是一种独特的肠鱼细胞系，当在transwell插入物上生长时，在体内发育出肠上皮的几个特征。从毒理学/药理学的角度来看，研究金属纳米颗粒的细胞定位和对必需金属随时间分布的局部影响将是有价值的，而且在水产养殖中也是有价值的，水产养殖目前正在研究使用纳米技术来改善营养和药物的吸收。

The increase in global metal nanoparticle production, due to the vast array of applications they can be used in and the technological advancements they bring about, raises questions about their potential impact on human health and the environment. The cell has evolved to tightly regulate the uptake and intracellular concentration of essential and toxic metal ions; however, metal nanoparticles seem to bypass these normal uptake systems and accumulate in the cell in a less controlled manner. Determining the exposure scenarios that enhance or reduce metal nanoparticle uptake, bio-reactivity, and toxicity is often difficult due to the lack of appropriate biological models. To allow for such investigations at the cellular level, this research project uses a model of the fish intestine derived from rainbow trout (Oncorhynchus mykiss). Once inside the cell, the bio-reactivity and toxicity of nanoparticles depends on their chemical transformation, a process which is currently poorly understood. This research project endeavors to link the dynamic chemical modifications of metal nanoparticles outside and inside the cell and intracellular alterations of essential metal homeostasis. The mechanistic knowledge generated in this project is envisioned to support human and environmental risk assessment with regard to metal ions and metal nanoparticle exposure. Moreover, the development of the fish intestine model will benefit regulatory science by potentially reducing the cost of testing and contributing to the efforts to establish animal-free alternatives in toxicology risk assessment. This multidisciplinary project is an opportunity for valuable collaborative training for two graduate research assistants and multiple undergraduate research assistants. This research project is designed to evaluate the impact of metal nanoparticles on the homeostasis of essential trace metals. Temporal and spatial distributions of essential and non-essential metals are being correlated to intracellular biomarker of metal bioavailability, establishing a link between intracellular chemical modifications and biological responses. While it is possible to determine dissolved metal speciation outside the cell using simplified cell culture media and chemical equilibrium models, intracellular metal speciation is more difficult to ascertain. Accurate evaluation of intracellular metal speciation requires a technique that allows for in situ measurement such as X-ray spectroscopy and microscopy. This research project studies how to link extracellular silver and titania nanoparticles behavior (agglomeration, dissolution, and speciation of dissolved metal ions) to intracellular chemical modifications induced by metal nanoparticles (levels and localization of essential trace metals) to molecular and cellular responses (mRNA and protein levels of metal transporters and imaging of cellular organelles) in RTgutGC. RTgutGC is a unique intestinal fish cell line that when grown on transwell inserts, develops several of the features found in intestinal epithelia in vivo. Study of cellular localization of metal nanoparticles and local influence on essential metal distribution over time will be valuable from a toxicology/pharmacology standpoint but also in aquaculture, which is now looking into the use of nanotechnologies to improve uptake of nutrients and drugs.