Development of flow-based and magic angle spinning in-vivo NMR to understand environmental stress

目前的环境政策主要是根据个别化学物质的急性毒性制定的，然而，迫切需要更多的分子水平信息来帮助了解与个别化合物和混合物的亚致死毒性有关的风险。这包括了解：毒性作用模式，生物积累，生物转化，排泄和污染物体内结合。“21世纪的毒性测试”（EPA）报告强调了这一点，该报告指出“新范式应有助于评估不同生命阶段的易感性，了解毒性发生的机制，并考虑同时累积暴露于多种不同化学品的风险”。核磁共振（NMR）是现代研究中最强大的工具之一，与大多数分析方法不同，它可以在体内应用。综合多相（CMP）核磁共振波谱（我的团队和Bruker共同开发的一种新型核磁共振技术）允许在完整的样品上进行全方位的固体，凝胶和液体核磁共振实验。因此，CMP-NMR允许研究所有相中的所有键，并在未改变的样品中进行原位研究和区分。CMP-NMR尚未在体内应用，但对于揭示体内污染物的命运、相互作用和反应性以及识别生物体中的任何结构变化是理想的。此外，静态核磁共振流动研究（即生物体在提供食物和氧气的管中）为研究生物体的代谢反应提供了一个互补的低应激环境。本研究将结合CMP-NMR和静态流NMR，提供一个理想的框架，允许污染物的体内命运/反应性与生物体对应激源的代谢反应之间的直接关联。由此产生的信息对于回答以下关键问题非常重要：污染物的毒性作用模式和受干扰的生化途径是什么？当污染物被绑定时，它还有毒吗？在体内是否发生污染物的永久结合/隔离？是否会形成更多毒性或高活性（例如自由基）的生物转化产物？代谢紊乱是永久性的还是生物体能完全恢复？亚致死水平的众多污染物是否会产生协同毒性效应？环境条件如何影响这些过程？这些问题的答案对于制定有关污染物水平的有意义和现实的环境政策至关重要，但使用目前最常见的标准急性毒性测试在很大程度上是不可能或非常具有挑战性的。这项拟议的研究将影响许多领域。CMP-NMR将首次在体内（任何领域）开发，因为它允许研究和区分所有相的所有键，它可能是未来理解生物过程的关键工具。第二，本研究将提高对水生毒性的基本认识。通过同时测量污染物行为和生物体的代谢应激反应，应该有可能解释某些化学物质（和混合物）有毒的原因和方式，以及这种毒性如何随环境条件而变化。最后，将基于核磁共振发现的技术转移到质谱分析，为政府机构提供了开发未来方案的基础，旨在使用通用的分析平台检测亚致死毒性。

Present environmental policies are set primarily on the basis of acute toxicity of individual chemical species, however, additional molecular-level information is desperately needed to help understand risks associated with sub-lethal toxicity of individual compounds and mixtures. This includes understanding: the toxic mode of action, bioaccumulation, biotransformation, excretion and contaminant binding in-vivo. This is stressed in an report “Toxicity Testing in the 21st Century” (EPA) which states “The new paradigm should facilitate evaluating the susceptibility of different life-stages, understanding the mechanisms by which toxicity occurs, and considering the risks of concurrent, cumulative exposure to multiple and diverse chemicals”. Nuclear Magnetic Resonance (NMR) is one of the most powerful tools in modern research and unlike most analytical approaches can be applied in-vivo. Comprehensive Multiphase (CMP) NMR spectroscopy (a novel NMR technology co-developed between my group and Bruker) permits the full range of solid, gel and liquid NMR experiments to be performed on an intact samples. As such CMP-NMR permits the study of all bonds in all phases to be studied and differentiated in-situ in unaltered samples. CMP-NMR has yet to be applied in-vivo but is ideal for unravelling the fate, interactions and reactivity of contaminants in-vivo as well as identifying any structural changes in the organism. In addition, static NMR flow studies (i.e. organism in a tube supplied with food and oxygen) provide a complimentary low stress environment to study an organism’s metabolic response. This research will combine CMP-NMR and static flow NMR to provide an ideal framework that permits the direct correlation between the in-vivo fate/reactivity of the contaminant and the organism’s metabolic response to the stressor. The resulting information is important to answering key questions such as: What is the toxic-mode-of-action of the contaminant and the biochemical pathways disturbed?; When bound, is a contaminant still toxic?; Does permanent binding/sequestration of the contaminant occur in-vivo?; Do more toxic or highly reactive (for example radicals) biotransformation products form?; Are metabolic perturbations permanent or do the organisms fully recover?; Can sub-lethal levels of numerous contaminants produce a synergistic toxic effect?; How do environmental conditions affect these processes? Answers to these questions are critical for setting meaningful and realistic environmental policies regarding contaminant-levels but are in large part impossible or very challenging to answer using standard acute toxicity tests which are currently the most common practice. This proposed research will impact many areas. CMP-NMR will be developed for the first time in-vivo (in any field) and as it permits all bonds in all phases to be studied and differentiated it likely represents a key future tool to understand biological processes in general. Second, this research will improve the fundamental understanding of aquatic toxicity. By simultaneously measuring contaminant behavior and the metabolic stress response from the organism it should be possible to explain why and how certain chemicals (and mixtures) are toxic and how this varies with environmental conditions. Finally, technology transfer of the discovery-based NMR findings to Mass Spectrometry provides government agencies the basis to develop future protocols aimed at detecting sub-lethal toxicity using a commonly available analytical platform.