Non-invasive detection of drug toxicity in rats by solid-phase extraction and MALDI-TOF analysis of urine samples.

An access to fast and non-invasive techniques to infer or predict the drug-induced injury caused by newly developed drugs and to monitor therapeutic efficacy of established drugs during treatment are of the outmost importance in pharmaceutical industry and clinical diagnosis. Peptidome and low molecular weight proteome profiling is an emerging technique that allows the recognition of distinctive patterns and differentiation among diverse physiopathological conditions. In this article, we evaluated the utility of peptide/small protein profiling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled with WCX magnetic bead-based solid-phase extraction as a screening tool for drug toxicity assessment in urine samples. Given that drug-induced injury is primarily reflected in liver, three different, well-described hepatotoxic drugs were chosen for this work. These were: carbon tetrachloride (CCl(4)) which induces liver fibrosis, D(+)-galactosamine as a model for acute liver injury, and Escherichia coli-derived lipopolysaccharide to study the damage caused by endotoxins. The profiles obtained with a correct clustering analysis show that this methodology can be used as a non-invasive and straightforward approach to test for potential drug toxicity. Pharmaceutical research and drug development studies could benefit from this methodology as liver injury inducer compounds could be easily detected in vivo by non-invasive means, accelerating the launch of safer drugs to the market.

Liquid chromatography-mass spectrometric analysis of urinary metabolites and their pattern recognition for the prediction of drug-induced hepatotoxicity.

Multivariate pattern recognition (PR) analysis combined with LC/MS was utilized to evaluate the feasibility of predicting chemical-induced toxicity in rats. Urine samples were collected from rats treated with vehicles or four hepatotoxins, alpha-naphthyl isothiocyanate (ANIT), carbontetrachloride (CCl4), acetaminophen, and diclofenac, and analyzed by HPLC coupled with electrospray mass spectrometry. Chromatographic data were normalized using modified Z score transformation with those of the control group, to remove the vehicle effects for a further enhanced multivariate analysis. The LC/MS-based profiles of the urine samples showed different levels of endogenous metabolites, which were characteristic of each hepatotoxin. In the principal component (PC) map of the urinary spectra from rats treated with ANIT, the metabolic trajectory moved away from the predose position, reaching a maximum separation at the 32-48 h time period. The metabolic profiles partially recovered to the basal conditions on 7 days postdose. A principal component analysis was performed on the urinary spectra of rats treated with the vehicles or four hepatotoxins. Each group formed a distinct and isolated cluster in the PC map, indicating drug-induced perturbation in the urine profiles. To construct mathematical models for predicting drug-induced hepatotoxicity, supervised analyses, such as linear discriminant analysis and soft independent modeling of class analogy with residual distance (SIMCA-RD), were performed. The SIMCA-RD showed high predictability, over 95%, in the results of cross-validation using the leaving-one-out method. The developed LC/MS-PR approach might be a useful tool for the prediction of drug-induced hepatotoxicity and for the understanding of hepatotoxic mechanisms.

Uptake, tissue distribution, and fate of inhaled carbon tetrachloride: comparison of rat, mouse, and hamster.

Carbon tetrachloride is hepatotoxic in rats, mice, and hamsters. However, rats are less sensitive to the hepatotoxic effects of CCl(4) than the other two species. The purpose of this study was to compare the uptake, tissue distribution, and elimination of CCl(4) by these three rodent species. Groups of 20 F344/Crl BR rats, B6C3F(1) mice, and Syrian hamsters were exposed by nose-only inhalation for 4 h to 20 ppm (14)C-labeled CCl(4). The fate of (14)C was followed in tissues, excreta, and exhaled breath for 48 h after the exposure. At the end of the exposure, concentrations of CCl(4) equivalents (CE) in tissue were highest in liver of rats and mice, but highest in fat for rats. The liver received the highest dose of CCl(4) equivalents with the following species ranking: mouse > hamster > rat. Patterns of CE elimination were species and tissue dependent, with the majority of elimination occurring within 48 h after exposure. Rats eliminated less radioactivity associated with metabolism ((14)CO(2), urine and feces) and more radioactivity associated with parent compound (exhaled activity trapped on charcoal) than did mice or hamsters. The results indicate that ranking of species sensitivity to the hepatotoxic effects of inhaled CCl(4) correlates with CE dose to liver and with the ability to metabolize CCl(4).

Carbon tetrachloride metabolism in sheep and in Fasciola hepatica.

1. The excretion of carbon tetrachloride and its metabolites in bile and urine were studied.2. Liver flukes in vitro metabolized carbon tetrachloride and hexachloroethane by dechlorination.3. Carbon tetrachloride, liver lipid from rabbits which received carbon tetrachloride and a carbon tetrachloride methyl oleate complex were toxic to liver flukes in vitro, in the presence of sheep bile.4. A direct fasciocidal action of carbon tetrachloride may contribute to the therapeutic effect of the drug.