Regular article
Contemporary issues in toxicology the role of metabonomics in toxicology and its evaluation by the COMET project

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Abstract

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 1H 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.

Introduction

The importance of postgenomic technologies for improving the understanding of drug adverse effects has been highlighted recently Aardema and MacGregor 2002, Cockerell et al 2002 and these approaches have been recognized to include metabonomics. While there is a comprehensive literature on the use of metabonomics to investigate xenobiotic toxicity and this has been reviewed recently (Nicholson et al., 2002), a rigorous and comprehensive evaluation would be of considerable value. To this end, a consortium has been formed to investigate the utility of NMR-based metabonomic approaches to the toxicological assessment of drug candidates. The main aim of the consortium is to use 1H NMR spectroscopy of biofluids (and, in selected cases, tissues), with the application of computer-based pattern recognition and expert system methods to classify the biofluids in terms of known pathological effects caused by administration of substances causing toxic effects. The project is hosted at Imperial College of Science, Technology and Medicine (IC), University of London, UK, and involves funding by six pharmaceutical companies, namely, Bristol-Myers-Squibb, Eli Lilly and Co., Hoffman–La Roche, NovoNordisk, Pfizer Incorporated, and The Pharmacia Corporation.

The main objectives of the project are (1) provision of a detailed multivariate description of normal physiological and biochemical variation of metabolites in urine, blood serum, and selected tissues, for primarily selected male rat and mouse strains, based on 1H NMR spectra; (2) development of a database of 1H NMR spectra from animals dosed with model toxins, initially concentrating on liver and kidney effects; (3) development of expert systems for the detection of the toxic effects of xenobiotics based on a chemometric analysis of their NMR-detected changes in biofluid metabolite profiles; (4) identification of combination biomarkers of the various defined classes; (5) testing of the methods to assess the ability of metabonomics to distinguish between toxic and nontoxic analogues and to assess the specificity of the predictive expert systems. The classes of chemicals used and the types of toxicity investigated are as diverse as possible to assist the validation of NMR methods for use in early “broad” screening of candidates for toxicity.

In this concise review, the background to metabonomics is presented together with a survey of literature results where metabonomics has been used to probe xenobiotic toxicity based on target organ, regions within target organs, and biochemical mechanisms of action. The methods used in COMET are briefly summarized and results are used to exemplify the approach. Finally, a perspective on the future uses of metabonomics in drug safety assessment is given.

Section snippets

NMR spectroscopy of biofluids

When toxins interact with cells and tissues they disturb the ratios, concentrations, and fluxes of endogenous biochemicals in key intermediary cellular metabolic pathways. Under mild toxic stress, cells attempt to maintain homeostasis and metabolic control by varying the composition of the body fluids that either perfuse them or are secreted by them. In more severe toxicity states, cell death leads to loss of organ function and more marked biochemical changes occur in biofluids due to loss of

The COMET project

The pharmaceutical industry is increasingly interested in the possibility of metabonomics being useful for toxicity screening and so an initial meeting of interested parties was held in January 1999 and a Steering Committee to oversee the project was formed from selected and agreed personnel from each company and IC. The consortium operation commenced in January 2001 and is funded for 3 years.

The consortium is generating databases of 1H NMR spectra of rat and mouse urine and blood serum

Animal experiments and sample collection

The organization of the animal experiments is subject to a standard protocol. All animal studies are carried out in accordance with relevant national legislation and are subject to appropriate local review. Histopathology and clinical chemistry results are also included in the database. Tissues are being saved and toxicity assessment will be selected as appropriate according to the animal model used, compound pharmacology, pharmacokinetics, and toxicity.

Ten Sprague–Dawley rats or 8 B6C3F mice

Critical issues requiring resolution

It was clear from the outset that there would be a number of complex and critical issues to be resolved if this project were to be successful. Thus, considerable time was spent in ensuring that the group derived robust protocols and operating procedures which could be used at all of the laboratories involved.

First, it was important to define a set of compounds which gave reproducible and well-defined toxic effects and that the observed effects could be described in a common terminology across

The future: a perspective on metabonomics

Single markers of toxicity are unlikely to be of value in the detection of subtle lesions that are the most problematic in primary toxicology studies on novel drugs. However, such lesions would cause multiple low-level disruptions of intermediary metabolites in biofluids that would closely reflect the site and mechanisms of damage. Pattern recognition and multivariate statistical analysis of NMR spectra offers a realistic prospect of identifying these novel combination biomarkers of toxic

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  • Cited by (0)

    1

    Current address: Metabometrix Ltd., RSM, Prince Consort Road, London SW7 2BP, UK.

    2

    Current address: Lilly Research Laboratories, Greenfield IN 46163, USA.

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