Drug bioactivation leading to the formation of reactive species capable of covalent binding to proteins represents an important cause of drug-induced toxicity. Reactive metabolite detection using in vitro microsomal incubations is a crucial step in assessing potential toxicity of pharmaceutical compounds. The most common method for screening the formation of these unstable, electrophilic species is by trapping them with glutathione (GSH) followed by liquid chromatography/mass spectrometry (LC/MS) analysis. The present work describes the use of a brominated analog of glutathione, N-(2-bromocarbobenzyloxy)-GSH (GSH-Br), for the in vitro screening of reactive metabolites by LC/MS. This novel trapping agent was tested with four drug compounds known to form reactive metabolites, acetaminophen, fipexide, trimethoprim and clozapine. In vitro rat microsomal incubations were performed with GSH and GSH-Br for each drug with subsequent analysis by liquid chromatography/high-resolution mass spectrometry on an electrospray time-of-flight (ESI-TOF) instrument. A generic LC/MS method was used for data acquisition, followed by drug-specific processing of accurate mass data based on mass defect filtering and isotope pattern matching. GSH and GSH-Br incubations were compared to control samples using differential analysis (Mass Profiler) software to identify adducts formed via the formation of reactive metabolites. In all four cases, GSH-Br yielded improved results, with a decreased false positive rate, increased sensitivity and new adducts being identified in contrast to GSH alone. The combination of using this novel trapping agent with powerful processing routines for filtering accurate mass data and differential analysis represents a very reliable method for the identification of reactive metabolites formed in microsomal incubations.
The molecular structure of the blue mussel Mytilus edulis whole anchoring threads was studied by two-dimensional (13)C solid-state NMR on fully labeled fibers. This unique material proves to be well ordered at a molecular level despite its heterogeneous composition as evidenced by the narrow measured linewidths below 1.5 ppm. The spectra are dominated by residues in collagen environments, as determined from chemical shift analysis, and a complete two-dimensional assignment (including minor amino acids) was possible. The best agreement between predicted and experimental backbone chemical shifts was obtained for collagen helices with torsion angles (-75°, +150°). The abundant glycine and alanine residues can be resolved in up to five different structural environments. Alanine peaks could be assigned to collagen triple-helices, β-sheets (parallel and antiparallel), β-turns, and unordered structures. The use of ATR-FTIR microscopy confirmed the presence of these structural environments and enabled their location in the core of the thread (collagen helices and antiparallel β-sheets) or its cuticle (unordered structures). The approach should enable characterization at the molecular level of a wide range of byssus macroscopic properties.
Acetaminophen is known to cause hepatoxicity via the formation of a reactive metabolite, N-acetyl p-benzoquinone imine (NAPQI), as a result of covalent binding to liver proteins. Serum albumin (SA) is known to be covalently modified by NAPQI and is present at high concentrations in the bloodstream and is therefore a potential biomarker to assess the levels of protein modification by NAPQI. A newly developed method for the absolute quantitation of serum albumin containing NAPQI covalently bound to its active site cysteine (Cys34) is described. This optimized assay represents the first absolute quantitation of a modified protein, with very low stoichiometric abundance, using a protein-level standard combined with isotope dilution. The LC-MS/MS assay is based on a protein standard modified with a custom-designed reagent, yielding a surrogate peptide (following digestion) that is a positional isomer to the target peptide modified by NAPQI. To illustrate the potential of this approach, the method was applied to quantify NAPQI-modified SA in plasma from rats dosed with acetaminophen. The resulting method is highly sensitive (capable of quantifying down to 0.0006% of total RSA in its NAPQI-modified form) and yields excellent precision and accuracy statistics. A time-course pharmacokinetic study was performed to test the usefulness of this method for following acetaminophen-induced covalent binding at four dosing levels (75-600 mg/kg IP), showing the viability of this approach to directly monitor in vivo samples. This approach can reliably quantify NAPQI-modified albumin, allowing direct monitoring of acetaminophen-related covalent binding.
Xenobiotic metabolism in the liver can give rise to reactive metabolites that covalently bind to proteins, and determining which proteins are targeted is important in drug discovery and molecular toxicology. However, there are difficulties in the analysis of these modified proteins in complex biological matrices due to their low abundance. In this study, an analytical approach was developed to systematically identify target proteins of acetaminophen (APAP) in rat liver microsomes (RLM) using two-dimensional chromatography and high-resolution tandem mass spectrometry. In vitro microsomal incubations, with and without APAP, were digested and subjected to strong cation exchange (SCX) fractionation prior to reverse-phase UHPLC-MS/MS. Four data processing strategies were combined into an efficient label-free workflow meant to eliminate potential false positives, using peptide spectral matching, statistical differential analysis, product ion screening, and a custom-built delta-mass filtering tool to pinpoint potential modified peptides. This study revealed four proteins, involved in important cellular processes, to be covalently modified by APAP. Data are available via ProteomeXchange with identifier PXD002590.
Atrazine (ATZ), one of the most widely used herbicides worldwide, has been the subject of several scientific studies associated with its human and ecological risks. In order to study atrazine's toxicity, the formation of its metabolites and the result of their exposure must be assessed. This relies on our ability to detect and identify all of atrazine's metabolites; however, no previous untargeted screening method has reported the detection of all known metabolites and glutathione conjugates at once. In this study, a compound-specific, postacquisition metabolic screening method was employed following a generic HPLC separation coupled with high resolution time-of-flight mass spectrometry (TOF-MS) to detect Phase I metabolites and glutathione conjugates generated by in vitro human liver microsomal incubations. Our method was designed to be unbiased and applicable to a wide variety of compounds since methods that can detect a broad range of metabolites with high sensitivity are of great importance for many types of experiments requiring thorough metabolite screening. On the basis of incubations with atrazine and three closely related analogues (simazine, propazine, and cyanazine), we have proposed a new Phase I metabolism scheme. All known Phase I transformations of atrazine were successfully detected, as well as a new N-oxidation product. Novel reactive metabolites were also detected as well as their glutathione conjugates. These newly detected species were produced via imine formation on the N-ethyl group, a biotransformation not previously observed for atrazine or its analogues.
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