PerspectiVeNMR-Based Metabolic Profiling and Metabonomic Approaches toProblems in Molecular ToxicologyMuireann Coen,* Elaine Holmes, John C. Lindon, and Jeremy K. Nicholson*Department of Biomolecular Medicine, Sir Alexander Fleming Building, Surgery, Oncology, ReproductiVeBiology and Anesthetics DiVision, Faculty of Medicine, Imperial College London, London SW7 2AZ, United KingdomReceiVed September 13, 2007We have reviewed the main contributions to the development of NMR-based metabonomic and metabolicprofiling approaches for toxicological assessment, biomarker discovery, and studies on toxic mechanisms.The metabonomic approach, (defined as the quantitative measurement of the multiparametric metabolicresponse of living systems to pathophysiological stimuli or genetic modification) was originally developedto assist interpretation in NMR-based toxicological studies. However, in recent years there has beenextensive fusion with metabolomic and other metabolic profiling approaches developed in plant biology,and there is much wider coverage of the biomedical and environmental fields. Specifically, metabonomicsinvolves the use of spectroscopic techniques with statistical and mathematical tools to elucidate dominantpatterns and trends directly correlated with time-related metabolic fluctuations within spectral data setsusually derived from biofluids or tissue samples. Temporal multivariate metabolic signatures can be usedto discover biomarkers of toxic effect, as general toxicity screening aids, or to provide novel mechanisticinformation. This approach is complementary to proteomics and genomics and is applicable to a widerange of problems, including disease diagnosis, evaluation of xenobiotic toxicity, functional genomics,and nutritional studies. The use of biological fluids as a source of whole organism metabolic informationenhances the use of this approach in minimally invasive longitudinal studies.Contents1. Introduction to Metabolic Profiling92. Background: The Early Days of Metabolic NMRspectroscopy103. Pattern Recognition for Sample Classificationand Biomarker Discovery104. Metabonomic and Integrated MetabonomicApplications in Toxicology135. Metabolic Information from Intact Tissues:Magic Angle Spinning (MAS) NMR146. Ultrahigh Field NMR Spectroscopy of BiologicalSamples157. Improved Analytical Technologies for MetaboliteIdentification: Solid-Phase ExtractionChromatography, Liquid Chromatography, andMass Spectrometry168. Statistical Spectroscopy and BiomarkerDiscovery179. Recent Consortium Projects Using NMR/MSDriven Metabonomics and Top-Down SystemsBiology in Toxicology1810. Clinical Metabonomics1911. Molecular Epidemiology1912. Integrated -Omic Applications2013. Pharmacometabonomics and Implications of theExtended Genome2014. Concluding Remarks211. Introduction to Metabolic ProfilingBroad spectrum metabolic profiling is now recognized as apowerful top-down systems biology tool that can provide a realworld link to other omics sciences (1–4). The terms metabolomics(5–7) and metabonomics (8) are widely applied to these types ofstudies, and the terminology is often used interchangeably. Meta-bonomics provides a whole-organism biological description of time-related multivariate metabolic response to a treatment. It facilitatesthe study of the metabolic products and interactions of hundredsof cellular metabolomes (metabolic complements) and fluidcompartments, which are unique to each cell type in the body butare coordinated in space and time, and this concept of the interactingmetabolomes has been termed the metabonome (9). A variety ofanalytical technologies have been applied to metabolic profilingin toxicology, but most approaches utilize NMR spectroscopy ormass spectrometry as these instrumentalities can capture informa-tion on hundreds or even thousands of metabolites in a sample ina single analytical run. To date, there have been more publicationsreflecting the application of NMR spectroscopy in metabolictoxicology (excluding drug metabolism applications), but modernLC-MS methods are now being successfully utilized in this area(10–12), and the balance will change although we consider thatthere will always be a role for NMR in rapid multivariate metabolicprofiling and in metabolic structural elucidation. As NMR spec-troscopy (in its own right) comprises a wide range of analyticaltechniques (and can be applied to biofluids as well as intact tissues),* To whom correspondence should addressed.Chem. Res. Toxicol. 2008, 21, 9–27 910.1021/tx700335d CCC: $40.75 2008 American Chemical SocietyPublished on Web 01/03/2008we have concentrated our review on the role of NMR spectroscopyin the development of toxicological metabonomics, but we havealso considered cognate biomedical applications as they will be ofincreasing importance in the future.2. Background: The Early Days of Metabolic NMRspectroscopyThe development of Fourier transform NMR spectroscopyin the late 1960s, the introduction of superconducting magnetsin the 1970s, and the consequent sensitivity increases resultedin the first applications of NMR spectroscopy for the metabolicprofiling of biofluids and cells. Since then, numerous studieshave concentrated on the use of1H NMR spectroscopy tocharacterize toxic response to drugs, as reflected in biofluidspectral signatures, and many novel metabolic markers of organ-specific toxicity have been discovered (13). The role ofmetabonomics in particular and magnetic resonance in generalin the toxicological evaluation of drugs has developed exten-sively (14).1H NMR spectroscopy is well suited to the studyof toxic events, as a biofluid fingerprint that reflects toxicresponse can be rapidly achieved without bias imposed byexpectations of the type of toxin-induced metabolic changes.Moreover, many of the NMR-detectable metabolites are presentat moderate to high concentrations and represent the productsand intermediates of many important or hub pathways that areaffected by many toxic or disease processes. Early applicationsof NMR spectroscopy found that quantitative changes inmetabolite patterns gave information on the location and severityof toxic lesions, together with insights into the underlyingmolecular mechanisms of toxicity (13, 15–17).Examples of early NMR studies of toxins include the effectsof exposure to cadmium and mercury salts, which are bothpotent nephrotoxins (18), with acute cadmium exposure alsocausing profound testicular toxicity. NMR methods were
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