Donald G. Robertson

Promoting coherent minimum reporting guidelines for biological and biomedical investigations: the MIBBI project

The Minimum Information for Biological and Biomedical Investigations (MIBBI) project aims to foster the coordinated development of minimum-information checklists and provide a resource for those exploring the range of extant checklists.

Contemporary issues in toxicology - The role of metabonomics in toxicology and its evaluation by the COMET project

The role that metabonomics has in the evaluation of xenobiotic toxicity studies is presented here together with a brief summary of published studies. To provide a comprehensive assessment of this approach, the Consortium for Metabonomic Toxicology (COMET) has been formed between six pharmaceutical companies and Imperial College of Science, Technology and Medicine (IC), London, UK. The objective of this group is to define methodologies and to apply metabonomic data generated using (1)H NMR spectroscopy of urine and blood serum for preclinical toxicological screening of candidate drugs. This is being achieved by generating databases of results for a wide range of model toxins which serve as the raw material for computer-based expert systems for toxicity prediction. The project progress on the generation of comprehensive metabonomic databases and multivariate statistical models for prediction of toxicity, initially for liver and kidney toxicity in the rat and mouse, is reported. Additionally, both the analytical and biological variation which might arise through the use of metabonomics has been evaluated. An evaluation of intersite NMR analytical reproducibility has revealed a high degree of robustness. Second, a detailed comparison has been made of the ability of the six companies to provide consistent urine and serum samples using a study of the toxicity of hydrazine at two doses in the male rat, this study showing a high degree of consistency between samples from the various companies in terms of spectral patterns and biochemical composition. Differences between samples from the various companies were small compared to the biochemical effects of the toxin. A metabonomic model has been constructed for urine from control rats, enabling identification of outlier samples and the metabolic reasons for the deviation. Building on this success, and with the completion of studies on approximately 80 model toxins, first expert systems for prediction of liver and kidney toxicity have been generated.

Summary recommendations for standardization and reporting of metabolic analyses.

The Standard Metabolic Reporting Structures (SMRS) working group outlines its vision for an open,community-driven specification for the standardization and reporting of metabolic studies.The Standard Metabolic Reporting Structures (SMRS) working group outlines its vision for an open,community-driven specification for the standardization and reporting of metabolic studies.

Metabolomics in Toxicology: Preclinical and Clinical Applications

Metabolomics first burst into the toxicology arena approximately 10–12 years ago, experiencing rapid growth as a science in the intervening period (Fig. 1). Though metabolomics still trails the publication rates of its sister technologies, genomics and proteomics, it is closing the gap in terms of both the numbers of publications and the quality of those publications. One continuing source of confusion is differentiating the terms metabolomics, metabonomics, and metabolic profiling. Although metabonomics was the first term formally defined (Nicholson et al., 1999), the term metabolomics came into usage shortly thereafter (Fiehn, 2002). Metabolic profiling is used by some as a generic term to avoid confusion of the two aforementioned ‘‘omic’’ terms, but that phrase itself can be confused with the comprehensive metabolite analysis of xenobiotics, so it is debatable whether its use adds any clarity to the situation. Whatever the case, it is now clear that metabolomics is the term preferred by most practitioners (Fig. 1). Therefore, metabolomics will be used in this review with the understanding that it represents all three terms. For most people, the names can be used interchangeably, but the reader is still advised to utilize all three terms when performing literature searches. Now that we have determined what to call it, we need to define what it is. The literature is replete with fine-tuned definitions, but the most succinct definition still appears to be most appropriate, and that is that metabolomics is ‘‘the comprehensive and quantitative analysis of all metabolites’’ (Fiehn, 2001). Every word in that phrase could be subject to debate as no analytical technique can measure ‘‘all’’ metabolites and what exactly qualifies as a metabolite? Do dietaryderived metabolites qualify, what about gut flora–derived metabolites? Rather than wade into this academic argument, we will leave it to the reader to define metabolomics as he or she chooses. For the toxicologist, the important point is that a metabolomics approach has the potential to reveal novel biochemical sequelae of toxicant administration that can lead to mechanistic insights and identification of biomarkers of cause and/or effect. Furthermore, the technology has the potential to characterize models or disease states to provide biochemical bases for observed interactions with xenobiotics. Given the explosion of recent literature, this review will not revisit the origins of metabolomics to any extent but will focus on recent advances in the field. Although botanical, environmental, and nutritional applications of the technology have expanded with the field, there is now such a wealth of literature this review will not be able to cover those burgeoning areas of metabolomic research. After providing some historical perspective, this review focuses on the biomedical applications of metabolomic technology with emphasis on new analytical approaches, preclinical, and clinical applications of the technology, particularly those areas relevant to practicing toxicologists.

NMR of biofluids and pattern recognition: assessing the impact of NMR parameters on the principal component analysis of urine from rat and mouse

The ability to interpret metabolic responses to toxic insult as expressed in altered urine composition and measured by NMR spectroscopy is dependent upon a database of proton NMR spectra of urine collected from both control and treated animals. Pattern recognition techniques, such as principal component analysis (PCA), can be used to establish whether the spectral data cluster according to a dose response. However, PCA will be sensitive to other variables that might exist in the data, such as those arising from the NMR instrument itself. Thus, studies were conducted to determine the impact that NMR-related variables might impart on the data, with a view towards understanding and minimizing variables that could interfere with the interpretation of a biological effect. This study has focused on solvent suppression methods, as well as instrument-to-instrument variability, including field strength. The magnitude of the NMR-induced variability was assessed in the presence of an established response to the nephrotoxin bromoethanamine. Changes caused by the model toxin were larger and easily distinguished from those caused by using different solvent suppression methods and field strengths.

Ultra Performance Liquid Chromatography-Mass Spectrometry Profiling of Bile Acid Metabolites in Biofluids: Application to Experimental Toxicology Studies

We have developed an ultra performance liquid chromatography-mass spectrometry (UPLC-MS(E)) method to measure bile acids (BAs) reproducibly and reliably in biological fluids and have applied this approach for indications of hepatic damage in experimental toxicity studies. BAs were extracted from serum using methanol, and an Acquity HSS column coupled to a Q-ToF mass spectrometer was used to separate and identify 25 individual BAs within 5 min. Employing a gradient elution of water and acetonitrile over 21 min enabled the detection of a wide range of endogenous metabolites, including the BAs. The utilization of MS(E) allowed for characteristic fragmentation information to be obtained in a single analytical run, easily distinguishing glycine and taurine BA conjugates. The proportions of these conjugates were altered markedly in an experimental toxic state induced by galactosamine exposure in rats. Principally, taurine-conjugated BAs were greatly elevated ( approximately 50-fold from control levels), and were highly correlated to liver damage severity as assessed by histopathological scoring (r = 0.83), indicating their potential as a sensitive measure of hepatic damage. The UPLC-MS approach to BA analysis offers a sensitive and reproducible tool that will be of great value in exploring both markers and mechanisms of hepatotoxicity and can readily be extended to clinical studies of liver damage.

Metabonomic assessment of vasculitis in rats.

The vasculitides are a heterogeneous group of lesions, characterized by inflammation and necrosis of the vascular wall and have proven to be a disconcerting dilemma in the development of several classes, of therapeutics. Metabonomics is an emerging technology having great potential for rapid noninvasive assessment of toxicity in vivo and providing identification of peripheral surrogate markers of toxicity. Metabonomic evaluation of CI-1018, a selective type 4 phosphodiesterase inhibitor associated with vasculitis in rats, was undertaken. Two experiments were performed in which CI-1018 was administered for up to 4 d to groups of male Wistar rats at doses up to 3000 mg/kg. Urine was collected from all animals pretest and daily for metabonomic analysis. Eleven of 38 CI-1018-treatment animals were found to have vascular injury of varying severity at doses ≥750 mg/kg. Principal component analysis produced a clear pattern separation among 8 of 11 animals with lesions and 36 of 37 animals without lesions in samples collected on d 3 or 4. These data demonstrate that the metabonomics approach has significant potential for developing a noninvasive method for identifying, vasculitis in rats. It remains to be seen if urinary analyte patterns identified in this study are reproducible and wheter a biomarker pattern for vasculitis can be established.

Standard reporting requirements for biological samples in metabolomics experiments: mammalian/in vivo experiments

With the increasing production of metabolomic data there is an awareness of a need for a standardised description of this data to aid assessment, exchange, storage and curation of information from metabolomic studies. In this manuscript the first draft of a minimum requirement for the description of the biological context of a metabolomic study involving mammalian subjects is described. This recommendation has been produced by the Metabolomics Standards Initiative–Mammalian Context Working Sub-Group (MSI-MCWSG) as part of the wider standardisation initiative led by the Metabolomics society. The experiments considered include functional genomic studies, drug toxicology, nutrigenomics, clinical trials, and other human studies. Two reporting requirements are described for pre-clinical (e.g. functional genomics, toxicology) and clinical (e.g. clinical trials, nutrigenomics) studies. It is planned that this will lead to the development of a tool for the description of metabolomic experiments that enables storage, retrieval and manipulation of large amounts of data. This will benefit the assessment and dissemination of metabolomic data from mammalian studies.

Mechanisms and Biomarkers of Cardiovascular Injury Induced by Phosphodiesterase Inhibitor III SK&F 95654 in the Spontaneously Hypertensive Rat

The cardiovascular injury of the type III selective PDE inhibitor SK&F 95654 was investigated in SHR. Twenty-four hours after a single sc injection of 100 or 200 mg/kg of the drug, rats exhibited cardiomyocyte necrosis and apoptosis, interstitial inflammation, hemorrhage and edema, as well as mesenteric arterial hemorrhage and necrosis, periarteritis, EC and VSMC apoptosis, EC activation, and MC activation and degranulation. Elevated serum levels of cTnT and decreased cTnT immunoperoxidase staining on cardiomyocytes were detected in the drug-treated rats. Serum levels of α 2-macroglobulin and IL-6 were significantly elevated following drug treatment. NMR spectral patterns of urine samples are significantly different between the drug-treated and control rats. These results indicate that measurement of serum cTnT, acute phase proteins, and cytokines as well as metabonomic urine profiles may serve as potential biomarkers for drug-induced cardiovascular injury in rats. Increased expression of CD63 on MC (tissue biomarker of MC), of nitrotyrosine on MC and EC (an indirect indicator of NO in vivo), and of iNOS on MC and EC (source of NO) suggest that NO produced by activated and degranulated MC as well as activated EC play an important role in SK&F 95654-induced mesenteric vascular injury.

Metabonomics in preclinical drug development.

Metabonomics has emerged as a key technology in preclinical drug discovery and development. The technology enables noninvasive systems assessment of untoward effects induced by candidate compounds characterising a broad spectrum of biological responses on an individual animal basis in a relatively rapid-throughput fashion, thus making it an ideal addition to early preclinical safety assessment. However, the implementation and interpretation of the technology and data it generates is not something that should be trivialised. Proper expertise in biological sciences, analytical sciences (nuclear magnetic resonance and/or mass spectrometry) and chemometrics should all be considered necessary prerequisites. If these factors are properly considered, the technology can add significant value as a tool for preclinical toxicologists.