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Integrated Metabonomic Analysis of Bromobenzene-Induced Hepatotoxicity: Novel Induction of 5-Oxoprolinosis

Nigel J. Waters,*^† Catherine J. Waterfield,^‡ R. Duncan Farrant,^§ Elaine Holmes,^† and Jeremy K. Nicholson^†

Biological Chemistry, Division of Biomedical Sciences, Sir Alexander Fleming Building, Imperial College, London, SW7 2AZ, United Kingdom, Preclinical Safety Sciences, GlaxoSmithKline Research & Development, Ware, Herts., SG12 0DP, United Kingdom, Physical Sciences, GlaxoSmithKline Research & Development, Stevenage, Herts., SG1 2NY, United Kingdom

J. Proteome Res., 2006, 5 (6), pp 1448–1459

DOI: 10.1021/pr060024q

Publication Date (Web): May 11, 2006

To whom correspondence should be addressed. Current Address: Physical & Metabolic Sciences, AstraZeneca R&D Charnwood, Loughborough, Leics. United Kingdom. Tel: +44 (0)1509 647034. Fax: +44 (0)1509 645576. E-mail: nigel.waters@astrazeneca.com.

†

Imperial College.

‡

Preclinical Safety Sciences, GlaxoSmithKline Research & Development.

Current Address: Health Assessment, Syngenta CTL, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, United Kingdom.

Physical Sciences, GlaxoSmithKline Research & Development.

Abstract

We present here a definitive metabonomic analysis in order to detect novel biomarker and metabolite information, implicating specific putative protein targets in the toxicological mechanism of bromobenzene-induced centrilobular hepatic necrosis. Male Han-Wistar rats were dosed with bromobenzene (1.5 g/kg, n = 25) and blood plasma, urine and liver samples were collected for NMR and magic angle spinning (MAS) NMR spectroscopy at various time-points postdose, with histopathology and clinical pathology performed in parallel. Liver samples were analyzed by 600 MHz ¹H MAS NMR techniques and the resultant spectra were correlated to sequential ¹H NMR measurements in urine and blood plasma using pattern recognition methods. 1D ¹H NMR spectra were data-reduced and analyzed using principal components analysis (PCA) to show the time-dependent biochemical variations induced by bromobenzene toxicity. In addition to a holistic view of the effect of hepatic toxicity on the metabolome, a number of putative protein targets of bromobenzene and its metabolites were identified including those enzymes of the glutathione cycle, exemplified by the presence of a novel biomarker, 5-oxoproline, in liver tissue, blood plasma, and urine. As such, this work establishes the importance of metabonomics technology in resolving the mechanistic complexity of drug toxicity as well as the benefits of frontloading this approach in drug safety evaluation and biomarker discovery.

Keywords: bromobenzene • 5-oxoproline • magic angle spinning • MAS ¹H NMR spectroscopy • liver • Han-Wistar rat • metabolomics • hepatocellular necrosis

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