Abstract:The efficacy of high-resolution 1 H nuclear magnetic resonance ( 1 H-NMR) spectroscopy-based metabonomics was studied in a model of rat liver toxicity. Hepatotoxicities were induced in male rats using methylene dianiline, clofibrate and galactosamine. Twenty-four-hr urine from days 1 to 5 after treatment were subjected to 1 H-NMR evaluation of the biochemical effects. Blood were also taken at Days 2, 3 and 5 to examine biochemical changes associated with hepatotoxicities, and histopathological changes were evaluated at termination. Increases in liver enzymes were observed in animals treated with methylene dianiline or galactosamine, and histopathological analysis revealed changes associated with hepatobiliary damage and hepatocellular necrosis in methylene dianiline-and galactosamine-treated animals, respectively. Principal component analysis and statistical Spotfire analyses were used to visualize similarities and differences in urine biochemical profiles produced by 1 H-NMR spectra. The biochemical effects of methylene dianiline and galactosamine were characterized by elevated levels of glucose, fructose, b-hydroxybutyrate, alanine, acetoacetate, lactate and creatine and decreased levels of hippurate, 2-oxoglutarate, citrate, succinate, trimethylamine-N-oxide, taurine and N-acetylglutamate in rat urine. Clofibrate treatment elevated the levels of N-methylnicotinamide and 3,4-dihydroxymandelate and decreased the levels of 2-oxoglutarate and N-acetylaspartate. This work shows that combinations of 1 H-NMR and pattern recognition are powerful tools in the evaluation of the biochemical effects of xenobiotics in liver.

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Immunogenic properties of bovine serum albumin (BSA) conjugates of various β-lactam antibiotics (ABs) and cross-reactivity of the produced antibodies were investigated in guinea pigs. Each AB-BSA conjugate was immunized with complete Freund’s adjuvant. Following the second injection, AB-specific antibodies were detected with high titers. Produced antibodies showed concentration-dependent inhibition by the corresponding AB. Cross-reactivity between the produced antibodies and ABs was evaluated with two methods, i.e. by testing for reactivity with the various AB-ovalbumin conjugates, and by assaying for inhibition of enzyme-linked immunosorbent assay (ELISA) by the various ABs. The latter method could detect minor cross-reactions more sensitively than the former one. The results suggested that similarity of the acyl side chain of a cephalosporin nucleus or a penicillin nucleus contributes to immunological cross-reactivity.

Skeletal changes induced by treatment of pregnant rats with four potent teratogens, busulfan, acetazolamide, vitamin A palmitate, and ketoconazole, were evaluated using Alizarin Red S and Alcian Blue double-staining to investigate the relationship between drug-induced skeletal malformations and cartilaginous changes in the fetuses. Pregnant rats (N = 8/group) were treated once or twice between gestation days (GDs) 10 to 13 with busulfan at doses of 3, 10, or 30 mg/kg; acetazolamide at 200, 400, or 800 mg/kg; vitamin A palmitate at 100,000, 300,000, or 1,000,000 IU/kg; or ketoconazole at doses of 10, 30, or 100 mg/kg. Uterine evaluations and fetal external and skeletal examinations were conducted on GD 20. Marked skeletal abnormalities in ribs and hand/forelimb bones such as absent/ short/bent ribs, fused rib cartilage, absent/fused forepaw phalanx, and misshapen carpal bones were induced at the mid- and high-doses of busulfan and acetazolamide and at the high-dose of vitamin A palmitate and ketoconazole. Increased incidences of discontinuous rib cartilage (DRC) and fused carpal bone (FCB) were observed from the low- or mid-dose in the busulfan and acetazolamide groups, and incidences of FCB were increased from the mid-dose in the vitamin A palmitate and ketoconazole groups. Therefore, DRC and FCB were detected at lower doses than those at which ribs and hand/forelimb malformations were observed in the four potent teratogens.

This 1 H nuclear magnetic resonance metabonomics study was aimed to determine urinary biomarkers of cholestasis resulting from inhibition of biliary secretion of bile or obstruction of bile flow. To inhibit biliary secretion of bile, cyclosporine A was administered to male Sprague-Dawley rats. Obstruction of bile flow was induced by administration of 4,4¢-methylene dianiline, a-naphthylisothiocyanate or bile duct ligation. Clinical pathological and histopathological examinations were performed to confirm cholestatic injury and 1 H nuclear magnetic resonance spectral data for urine samples were analysed to determine similarities and differences in profiles of metabolites using the Spotfire Ò . In cyclosporine A-treated groups, serum total bilirubin and bile acid were significantly increased but no remarkable hepatic histopathological-changes were observed. In 4,4¢-methylene dianiline-, a-naphthylisothiocyanate-and bile duct ligationtreated groups, serum alkaline phosphatase, c-glutamyltranspeptidase and total bilirubin levels increased significantly, and hepatic histopathological-changes were observed. On urinary 1 H nuclear magnetic resonance spectral analysis, area intensities derived from 0.66 to 1.90 ppm were decreased by cyclosporine A, whereas they were increased by other treatments. These metabolites were identified using the NMR suite Ò as bile acids, branched-chain amino acids, n-butyrate, propionate, methyl malonate and valerate. These metabolites were further investigated by K-means clustering analysis. The cluster of these metabolites is considered to be altered by cholestasis. We conclude that bile acids, valine and methyl malonate have a possibility to be urinary cholestatic biomarkers, which distinguish a difference in mechanism of toxicity.

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