Application of high-throughput Fourier-transform infrared spectroscopy in toxicology studies: contribution to a study on the development of an animal model for idiosyncratic toxicity
Introduction
Metabonomics is increasingly utilized in toxicological evaluations of candidate drugs and offers numerous advantages over conventional histopathological or enzymatic protocols (Nicholson et al., 1999, Nicholson et al., 2002). Metabonomics has been defined as “the quantitative measurement of the time-related multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification” (Nicholson et al., 1999). Operationally, it involves systematic spectral analysis of biofluid (typically urine) composition in order to associate organ toxicity with specific spectral patterns and to identify surrogate markers of toxicity.
The discipline has seen increasing acceptance in the pharmaceutical industry as evidenced by the formation of Consortium on Metabonomics in Toxicology (COMET) (Breau and Cantor, 2003) and is exemplified by a proven track record of biomarker discovery and early diagnosis of toxicological indications (Nicholls et al., 2000). The measurement technology adopted by COMET is nuclear magnetic resonance (NMR). NMR has considerable merit in such an approach as it is non-invasive, reasonably rapid (<5 min is typical), quantitative, and highly reproducible. This reproducibility allows the development of a stable database that can facilitate comparative assessments of the effect of different drug candidates on urinary metabolic profiles. Metabonomic investigations are now being extended into the phenotyping of transgenic animal models (Nicholson et al., 2002, Gavaghan et al., 2000) and clinical investigations (Brindle et al., 2002).
The increasing interest in extending metabonomics applications has coincided with a concomitant interest in pursuing alternative measurement technologies as complementary options to NMR. Thus, electrospray mass spectrometry (MS), either in direct injection mode (Allen et al., 2003, Goodacre et al., 2002, Vaidyanathan et al., 2001, Vaidyanathan et al., 2002) or coupled to liquid chromatography (Pham-Tuan et al., 2003, Plumb et al., 2002) has attracted increasing attention. The successful application of electrospray MS coupled to HPLC has now been described for a metabonomic analysis of rat urine from a toxicological study (Plumb et al., 2002).
We were interested in the application of Fourier-transform infrared spectroscopy (FT-IR) in metabonomics since it offers potential advantages in cost, simplicity and low sample volume requirements. Typical acquisition times are 5–10 s per sample. Furthermore, high-throughput instruments interfaced with a motorized stage that allows sampling from 96-well plates are now commercially available.
FT-IR offers considerable potential in contributing to biomedical studies (Petrich, 2001, Naumann, 2001, Ellis et al., 2003). Its application outside of cancer diagnostics is still limited, but recent literature examples of the use of FT-IR in biomedical research include the analyses of body fluids from diabetes (Petrich et al., 2000) and arthritis patients (Eysel et al., 1997, Staib et al., 2001) brain material infected with transmissible spongiform encephalopathies (Kneipp et al., 2002) and follicular fluid for investigating oocyte development (Thomas et al., 2000). Reports on applications to toxicology remain few but include evaluations of the effects of toxic agents such as carrageenan (Perromat et al., 2001, Melin et al., 2001) and carbon tetrachloride (Melin et al., 2000, Melin et al., 2001) on internal organs. FT-IR analyses of urine have focused primarily on multi-analyte measurements of specific urinary components (Heise et al., 2001) although there is one report on the use of FT-IR-based urine analysis to distinguish normal from rejecting renal allografts (Somorjai et al., 2002). We report now on a pilot study into the suitability of FT-IR as a technology that could be employed in metabonomic investigations in toxicology and on its contribution to characterizing a new model for idiosyncratic susceptibility. This model is based on the concept that mild inflammation induced by bacterial lipopolysaccharide (LPS) enhances the hepatotoxicity of pharmaceuticals and other xenobiotics (Ganey and Roth, 2001). Preliminary publication on this model with ranitidine describes it in more detail (Luyendyk et al., in press).
In our pilot study, 29 rats were fasted for 24 h pre-dose. Urine was collected at 6, 18, and 24 h. For the purposes of developing a model for idiosyncratic susceptibility (Ganey and Roth, 2001, Luyendyk et al., in press) sets of rats were treated with LPS, ranitidine, and with ranitidine/LPS co-administrations. The premise of the idiosyncratic susceptibility model is that LPS, a potent inflammatory agent, would act as a “sensitizer” to adverse side effects and thereby uncover toxicological responses to drug candidates. Ranitidine (Vial et al., 1991) a H-2 receptor antagonist used in the treatment of gastroesophageal reflux, has been associated with episodes of idiosyncratic hepatotoxicity.
For the purposes of our pilot study, we were focused on determining whether high-throughput FT-IR could discriminate (i) the three pre-dose sample populations (as a first assessment of the discriminating power of FT-IR-based urine analysis), (ii) inflammatory effects of LPS from control vehicle treatments, and (iii) ranitidine-treated subjects from subjects where LPS was co-administered with ranitidine. Our goal was to evaluate FT-IR as a potential metabonomic tool and to assess whether it had the potential to contribute to toxicological studies and to support characterization of the idiosyncratic susceptibility model.
Section snippets
Animals
Male, Sprague-Dawley rats (Crl:CD (SD)IGS BR; Charles River, Portage, MI) weighing 250–300 g were housed individually in plastic metabolism cages for these studies. Animals were fed powdered certified rat diet (Labdiet 5002, Purina Mills Inc., St. Louis, MO) and allowed access to water ad libitum. They were allowed to acclimate for 5 days in standard group rat laboratory cages and for 2 days in metabolism cages in a 12-h light/dark cycle prior to treatment.
Experimental protocol
Rats fasted for 24 h were given 44.4×106
Results and discussion
Urine collected at three different time-points from the pre-dose rat population represented an opportunity to assess the discriminatory potential use of high-throughput FT-IR in evaluating closely related populations. It offered a much larger data set (n=29) than that for the drug and LPS treated subsets (n=5). Discriminant function analysis (Fig. 1) showed good separation of all three pre-dose urine collection periods and clearly boded well for our investigations into the drug and LPS treated
Acknowledgements
Funding was provided by a grant from Pharmacia Corporation. J.P.L. was supported, in part, by NIEHS Training Grant 5 T32 ES07255.
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