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品的急性毒性制定的，然而，迫切需要更多的分子水平的信息，以帮助了解与个别化合物和混合物的亚致死毒性相关的风险。这包括了解：毒性作用方式、生物累积、生物转化、排泄和体内污染物结合。"21世纪世纪的毒性测试"（EPA）报告强调了这一点，该报告指出，"新的范例应有助于评估不同生命阶段的敏感性，了解毒性发生的机制，并考虑同时累积接触多种和不同化学品的风险"。 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。全面的多相（CMP）NMR光谱（我的团队和布鲁克公司共同开发的一种新型NMR技术）允许对完整样品进行全方位的固体、凝胶和液体NMR实验。因此，CMP-NMR允许在未改变的样品中原位研究和区分所有相中的所有键。CMP-NMR尚未应用于体内，但它是解开体内污染物的命运，相互作用和反应性以及识别生物体中任何结构变化的理想方法。此外，静态NMR流动研究（即，在供应有食物和氧气的管中的生物体）提供了一个互补的低应力环境来研究生物体的代谢反应。 这项研究将结合联合收割机CMP-NMR和静态流动NMR，以提供一个理想的框架，允许污染物的体内命运/反应性和生物体对应激源的代谢反应之间的直接相关性。所得到的信息对于回答一些关键问题很重要，例如：污染物的毒性作用模式和受到干扰的生化途径是什么？污染物结合后是否仍然有毒？污染物是否在体内永久结合/隔离？是否会形成更多的毒性或高活性（例如自由基）生物转化产物？代谢紊乱是永久性的还是生物体完全恢复？许多污染物的亚致死水平能产生协同毒性效应吗？环境条件如何影响这些过程？这些问题的答案是至关重要的，制定有意义的和现实的环境政策的污染物水平，但在很大程度上是不可能的，或非常具有挑战性的答案使用标准的急性毒性测试，目前最常见的做法。 这项研究将影响许多领域。CMP-NMR将首次在体内（在任何领域）开发，由于它允许研究和区分所有阶段的所有键，因此它可能是了解一般生物过程的关键未来工具。其次，这项研究将提高对水生毒性的基本认识。通过同时测量污染物的行为和生物体的代谢应激反应，应该可以解释为什么某些化学品（和混合物）是有毒的，以及如何随着环境条件而变化。最后，将基于发现的NMR研究结果技术转让给质谱分析，为政府机构提供了基础，以制定未来的协议，旨在使用常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.