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

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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.