Toxicology Letters 199 (2010) 10–16 Contents lists available at ScienceDirect Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet 1 H NMR-based metabonomic investigation of tributyl phosphate exposure in rats Muniasamy Neerathilingam a,b,1,2 , David E. Volk a,1,2 , Swapna Sarkar a , Todd M. Alam c , M. Kathleen Alam d , G.A. Shakeel Ansari a , Bruce A. Luxon a,b,∗ a Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555-1157, United States b UTMB Bioinformatics Program, University of Texas Medical Branch, Galveston, TX 77555-1157, United States c Department of Electronic and Nanostructured Materials, Sandia National Laboratories, Albuquerque, NM 87185-0886, United States d Energetics Characterization Department, Sandia National Laboratories, Albuquerque, NM 87185-1455, United States article info Article history: Received 18 April 2010 Received in revised form 24 July 2010 Accepted 26 July 2010 Available online 3 August 2010 Keywords: Metabonomics Tributyl phosphate (TBP) Dibutyl phosphate (DBP) Nuclear magnetic resonance (NMR) Urine Rat abstract Tributyl phosphate (TBP) is a toxic organophosphorous compound widely used in many industrial appli- cations, including significant usage in nuclear processing. The industrial application of this chemical is responsible for occupational exposure and environmental pollution. In this study, 1 H NMR-based metabo- nomics has been applied to investigate the metabolic response to TBP exposure. Male Sprague-Dawley rats were given a TBP-dose of 15 mg/kg body weight, followed by 24 h urine collection, as was previously demonstrated for finding most of the intermediates of TBP. High-resolution 1 H NMR spectroscopy of urine samples in conjunction with statistical pattern recognition and compound identification allowed for the metabolic changes associated with TBP treatment to be identified. Discerning NMR spectral regions cor- responding to three TBP metabolites, dibutyl phosphate (DBP), N-acetyl-(S-3-hydroxybutyl)-l-cysteine and N-acetyl-(S-3-oxobutyl)-l-cysteine, were identified in TBP-treated rats. In addition, the 1 H NMR spectra revealed TBP-induced variations of endogenous urinary metabolites including benzoate, urea, and trigonelline along with metabolites involved in the Krebs cycle including citrate, cis-aconitate, trans- aconitate, 2-oxoglutarate, succinate, and fumarate. These findings indicate that TBP induces a disturbance to the Krebs cycle energy metabolism and provides a biomarker signature of TBP exposure. We show that three metabolites of TBP, dibutylphosphate, N-acetyl-(S-3-hydroxybutyl)-l-cysteine and N-acetyl-(S-3- oxobutyl)-l-cysteine, which are not present in the control groups, are the most important factors in separating the TBP and control groups (p < 0.0023), while the endogenous compounds 2-oxoglutarate, benzoate, fumarate, trigonelline, and cis-aconetate were also important (p < 0.01). © 2010 Elsevier Ireland Ltd. All rights reserved. Tributyl phosphate (TBP) is a toxic organophosphorous com- pound widely used in nuclear processing (Demina et al., 1990; Muller et al., 1987; Stradling et al., 1985), chemical industries (Berne et al., 2007) and the formulation of fire-resistant aircraft hydraulic fluids (Berne et al., 2007). As a consequence, environmen- tal pollution and occupational exposure involving TBP is a major public health concern due to cholinergic toxicity and neurotoxic- ity effects (Berne et al., 2007; Raushel, 2002). Workers exposed to TBP concentrations of 15 mg/m 3 in air have complained of nau- sea and headache (Abou-Donia and Nomeir, 1986), while direct ∗ Corresponding author at: Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555-1157, United States. Fax: +409 747 6850. E-mail address: baluxon@utmb.edu (B.A. Luxon). 1 Authors equally contributed to this work. 2 Current address: Institute for Molecular Medicine for the Prevention of Human Diseases, University of Texas Health Science Center Houston, 1825 Pressler, Houston, TX 77030, United States. administration of TBP to rats produced urinary bladder hyperplasia, papillomas and transitional cell carcinomas at high doses (Auletta et al., 1998). TBP also presents an acute toxicity hazard to freshwa- ter living organisms, even at low concentrations (Hernadez, 2002; Michel et al., 2004; Nakamura, 1991a,b). Recently, we (Alam et al., 2010) analyzed the differences between control and TBP-fed rats using O-PLSDA and VIP scores, and in this report, we compare those past results with those obtained using other analytical meth- ods, including chemical shift binning, spectral deconvolution and principal components analysis. Metabonomics is a multi-parametric approach that allows detection of the metabolic response due to chemical exposure (Feng et al., 2002; Wei et al., 2008) and has previously been used for environmental (Viant et al., 2003), pharmaceutical (Coen et al., 2004), biomarker discovery (Jordan and Cheng, 2007) and toxi- cology (Shockcor and Holmes, 2002) investigations. Metabonomics can combine multivariate pattern recognition techniques with the metabolic profiling capabilities of a wide range of technologies, including gas chromatography–mass spectrometry (GC–MS), liquid 0378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2010.07.013