1H NMR-based metabonomic investigation of tributyl phosphate exposure in rats

Tributyl phosphate (TBP) is a toxic organophosphorous compound widely used in many industrial applications, 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 metabonomics 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 24h 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 corresponding 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).