The entire surface of the cercarial stage of the human blood fluke Schistosoma mansoni is covered by a 1-microns thick, highly immunogenic, fucose-rich glycocalyx (GCX). Using strategies based on enzymatic, chemical, and mass spectrometric analysis, we have defined the structures of the major glycans released by reductive elimination from GCX. They comprise a heterogeneous population of multifocosylated complex oligosaccharides with the following nonreducing terminal sequences: [formula: see text] Our structural data suggest that these tri- to pentafucosylated epitopes are carried on type 1, R-->Gal beta-1-->3GalNAc, and type 2, R-->Gal beta 1-->3(R-->GlcNAc beta-1-->6)GalNAc, core structures via repeat units of (3GalNAc beta 1-->4(Fuc alpha 1-->2Fuc alpha 1-->2Fuc alpha 1-->3)GlcNAc beta-1-->3Gal alpha-->)n, where n is mainly 0 and 1, and all sugars are in the pyranose form. The proposed structure represents the first instance where an alpha-galactosylated beta-GalNAc(1-->4)-beta-GlcNAc sequence occurs as a repeating unit in a glycoprotein. It is also unique in being substituted with oligofucosyl appendages. The unusual oligosaccharide structures described here, particularly the potentially immunodominant oligofucosyl moieties, are most likely responsible for the known potency of GCX in modulating various immune responses including complement activation, B cell mitogenesis, and delayed type hypersensitivity in schistosomiasis.

Drug toxicity to T-antigen-immortalized human liver epithelial (THLE) cells stably transfected with plasmid vectors that encoded human cytochrome P450s 1A2, 2C9, 2C19, 2D6, or 3A4, or an empty plasmid vector (THLE-Null), was investigated. An automated screening platform, which included 1% dimethyl sulfoxide (DMSO) vehicle, 2.7% bovine serum in the culture medium, and assessed 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium reduction, was used to evaluate the cytotoxicity of 103 drugs after 24h. Twenty-two drugs caused cytotoxicity to THLE-Null cells, with EC₅₀ ≤ 200 μM; 21 of these drugs (95%) have been reported to cause human liver injury. Eleven drugs exhibited lower EC₅₀ values in cells transfected with CYP3A4 (THLE-3A4 cells) than in THLE-Null cells; 10 of these drugs (91%) caused human liver injury. An additional 8 drugs, all of which caused human liver injury, exhibited potentiated THLE-3A4 cell toxicity when evaluated using a manual protocol that included 0.2% or 1% DMSO, but not bovine serum. Fourteen of the drugs that exhibited potentiated THLE-3A4 cell toxicity are known to be metabolized by P450s to reactive intermediates. These drugs included troglitazone, which was shown to undergo metabolic bioactivation and covalent binding to proteins in THLE-3A4 cells. A single drug (rimonabant) exhibited marked THLE cell toxicity but did not cause human liver injury; this drug had very low reported plasma exposure. These results indicate that evaluation of toxicity to THLE-Null and THLE-3A4 cell lines during drug discovery may aid selection of drugs with reduced propensity to cause drug-induced liver injury and that consideration of human exposure is required to enhance data interpretation.

ABSTRACT:The

Acyl glucuronides (AGs) are common, chemically reactive metabolites of acidic xenobiotics. Concerns about the potential of this class of conjugate to cause toxicity in man require efficient methods for the determination of reactivity, and this is commonly done by measuring transacylation kinetics. High-performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) spectroscopy were applied to the kinetic analysis of AG isomerization and hydrolysis for the 1-beta-O-AGs of ibufenac, (R)- and (S)-ibuprofen, and an alpha,alpha-dimethylated ibuprofen analogue. Each AG was incubated in either aqueous buffer at pH 7.4 or human plasma at 37 degrees C. Aliquots of these samples, taken throughout the reaction time course, were analysed by HPLC-MS and (1)H-NMR spectroscopy and the results compared. For identification of the AGs incubated in pH 7.4 buffer and for analysis of kinetic rates, (1)H-NMR spectroscopy generally gave the most complete set of data, but for human plasma the use of (1)H-NMR spectroscopy was impractical and HPLC-MS was more suitable. HPLC-MS was more sensitive than (1)H-NMR spectroscopy, but the lack of suitable stable-isotope labelled internal standards, together with differences in response between glucuronides and aglycones, made quantification problematic. Using HPLC-MS a specific 1-beta-O-AG-related ion at m/z 193 (the glucuronate fragment) was noted enabling selective determination of these isomers. In buffer, transacylation reactions predominated, with relatively little hydrolysis to the free aglycone observed. In human plasma incubations the observed rates of reaction were much faster than for buffer, and hydrolysis to the free aglycone was the major route. These results illustrate the strengths and weaknesses of each analytical approach for this class of analyte.

The metabolism of [(14)C]-diclofenac in mice was investigated following a single oral dose of 10 mg/kg. The majority of the drug-related material was excreted in the urine within 24 h of administration (49.7 %). Liquid chromatographic analyses of urine and faecal extracts revealed extensive metabolism to at least 37 components, with little unchanged diclofenac excreted. Metabolites were identified using a hybrid linear ion-trap mass spectrometer via exact mass determinations of molecular ions and subsequent multi-stage fragmentation. The major routes of metabolism identified included: 1) conjugation with taurine; and 2) hydroxylation (probably at the 4'-and 5-arene positions) followed by conjugation to taurine, glucuronic acid or glucose. Ether, rather than acyl glucuronidation, predominated. There was no evidence for p-benzoquinone-imine formation (i.e. no glutathione or mercapturic acid conjugates were detected). A myriad of novel minor drug-related metabolites were also detected, including ribose, glucose, sulfate and glucuronide ether-linked conjugates of hydroxylated diclofenac derivatives. Combinations of these hydroxylated derivatives with acyl conjugates (glucose, glucuronide and taurine) or N-linked sulfation or glucosidation were also observed. Acyl- or amide-linked-conjugates of benzoic acid metabolites and several indolinone derivatives with further hydroxylated and conjugated moieties were also evident. The mechanisms involved in the generation of benzoic acid and indolinone products indicate the formation reactive intermediates in vivo that may possibly contribute to hepatotoxicity.