NMR-based metabonomic study on the subacute toxicity of aristolochic acid in rats

Abstract

The subacute toxicity of aristolochic acid (AA) was investigated by ¹H NMR spectroscopic and pattern recognition (PR)-based metabonomic methods. Model toxins were used to enable comparisons of the urinary profiles from rats treated with known toxicants and AA at various time intervals. Urinary ¹H NMR spectra were data-processed and analyzed by pattern recognition method. The result of visual comparison of the spectra showed that AA caused a renal proximal tubular and papillary lesion and a slight hepatic impair. Pattern recognition analysis indicated that the renal proximal tubule lesion was the main damage induced by AA, and the renal toxicity induced by AA was a progressive course with the accumulation of dosage by monitoring the toxicological processes from onset, development and part-recovery. These results were also supported by the conventional clinical biochemical parameters.

Introduction

NMR spectroscopy of biofluids has brought a new chemical method to help identify the site and possible mechanism of toxicity and offered much information of endogenous metabolites and their variation in pathological states (Nicholson and Wilson, 1989, Lindon et al., 1999, De Graaf and Behar, 2003, Constantinou et al., 2004). Numerous studies have demonstrated the utility of NMR spectroscopy in toxicological fields, especially in the study of nephrotoxins (Holmes et al., 1992, Anthony et al., 1994, Gartland et al., 1989, Nicholson et al., 1989) and hepatotoxins (Beckwith-Hall et al., 2003, Waters et al., 2001, Singh et al., 2003). It provides a method of monitoring the toxicological processes of xenobiotics and clinical toxicology (Maschke et al., 1995, Bales et al., 1988, Wu et al., 2004). With the improvements in instrument sensitivity and relative resolution of NMR spectrometers at higher magnetic field strengths which gives a greater sensitivity to the methodology increasing the amount of biochemical information from ¹H NMR spectra. Pattern recognition (PR) analysis is performed in a multidimensional parameter space using dimension–reduction techniques to maximize information from NMR profile (Xu, 2004). Several methods, such as principal component analysis (PCA) and non-linear mapping (NLM) techniques (Lindon et al., 1999) have been used to classify toxins on the basis of ¹H NMR spectra of urine. The application of ¹H NMR spectroscopy to the study of metabolites in biofluids combined with pattern recognition to classify NMR-derived data has led to a ‘metabonomic’ approach to biochemical assessment (Holmes et al., 1998b). As a method of monitoring the progression of toxicity and recovery caused by toxins or xenobiotics, metabonomics represents a systemic approach for measuring time-related biochemical responses of metabolic composition variance (Keun et al., 2002, Lindon et al., 2003, Wu et al., 2005). Principal components analysis (PCA) is a widely used dimension–reduction method, in which the NMR spectra can be reduced to a set of peak intensity descriptors, and used to detect toxicity and disease based on NMR biomarkers as well as identify similarities or differences between the samples from control and toxin-treated animals.

Aristolochic acid (AA) is an active component derived from Aristolochia species, which is used to treat arthritis, gout and rheumatism. In addition, its anti-fungal, anti-bacterial, anti-viral, and even anti-tumor pharmacological effects have been reported (Zhang et al., 2004). However, AA was considered to be related to a progressive renal fibrosis, which was so-called ‘Chinese-herb nephropathy’ (CHN) (Vanherweghem et al., 1993). The histological findings in renal superficial cortex from ‘CHN’ patients were interstitial fibrosis and tubular atrophy (Cosyns et al., 1994, Depierreux et al., 1994). AA is metabolized into aristolactam in liver and DNA adducts have been detected in kidney and liver tissues (Debelle et al., 2003). The mutations were also detected in p53 and H-ras genes which were associated with carcinogenesis process (Arlt et al., 2000, Arlt et al., 2001). AA-induced renal fibrosis and chronic renal failure model was established in salt-depleted Wistar rats (Debelle et al., 2002). However, the subacute toxicological process caused by AA is still uncertain. As the characteristic toxicity of AA is severe, the toxic process and biochemical effect of AA should be carefully studied. In the present paper, the onset, development and recovery toxicological processes of AA to the rats were investigated by NMR-based metabonomic methods. Model toxins causing hepatic or renal lesion, which were well documented by NMR spectroscopy, were used to enable comparisons of the urinary NMR profiles to be made with ones obtained from animals treated with known toxicants.