N-ACETYLATION OF THE GLUTAMATE RESIDUE OF INTACT GLUTATHIONE CONJUGATES IN RATS: A NOVEL PATHWAY FOR THE METABOLIC PROCESSING OF THIOL ADDUCTS OF XENOBIOTICS

This article is available online at http://dmd.aspetjournals.org ABSTRACT: We report herein the identification of a novel metabolic pathway that involves acetylation of the amino group of the glutamic acid residue of intact glutathione (GSH) conjugates of a series of compounds in rat hepatocytes and in rats in vivo. The "nonacetylated" as well as the "acetylated" GSH conjugates of the compounds in question were detected in rat hepatocyte incubations and in rat bile. These conjugates were characterized by online liquid chromatography-mass spectrometry on an ion-trap mass spectrometer as well as accurate mass measurements using a high-resolution quadrupole time-of-flight instrument. The accurate mass measurements on the molecular ions of nonacetylated and acetylated GSH adducts clearly revealed the addition of a mass equivalent to C 2 H 2 O in the latter conjugates. Furthermore, the collisioninduced dissociation of the molecular ions of nonacetylated GSH adducts yielded fragment ions involving the loss of pyroglutamate (129 Da), which are typical of many GSH conjugates. For acetylated adducts, however, fragment ions resulting from a loss of 171 Da (equivalent to N-acetyl-pyroglutamate) were observed, indicating that acetylation had occurred on the glutamic acid residue of the GSH conjugates. An enzyme-catalyzed transacetylation process that utilized acetyl CoA as the acetyl donor, and resulted in the formation of the same acetylated adducts that were detected in rat hepatocytes and in rat bile, was identified in rat liver microsomes. This appears to be the first reported instance of N-acetylation of intact GSH conjugates in any species and represents a novel pathway of metabolic processing of thiol adducts of xenobiotics

Metabolic Activation of Fluoropyrrolidine Dipeptidyl Peptidase-IV Inhibitors by Rat Liver Microsomes

Abstract The current study evaluated the potential for two dipeptidyl peptidase-IV (DPP-IV) inhibitor analogues (MRL-A and MRL-B), containing a fluoropyrrolidine moiety in the structure, to undergo metabolic activation. The irreversible binding of these tritiumlabeled compounds to rat liver microsomal protein was time-and NADPH-dependent, and was attenuated by the addition of reduced glutathione (GSH) or N-acetylcysteine (NAC) to the incubation, indicating that chemically reactive intermediates were formed and trapped by these nucleophiles. Mass spectrometric analyses and further trapping experiments with semicarbazide indicated that the fluoropyrrolidine ring had undergone sequential oxidation and defluorination events resulting in the formation of GSH or NAC conjugates of the pyrrolidine moiety. The bioactivation of MRL-A was catalyzed primarily by rat recombinant cytochrome (CYP) 3A1 and 3A2. Pretreatment of rats with prototypic CYP3A1 and 3A2 inducers (pregnenolone-16alpha-carbonitrile (PCN) and dexamethasone) enhanced the extent of bioactivation, which in turn, led to a higher degree of in vitro irreversible binding to microsomal proteins (5-and 9-fold increase, respectively). Herein, we describe studies which demonstrate that the fluoropyrrolidine ring is prone to metabolic activation, and that GSH or NAC can trap the reactive intermediates to form adducts that provide insight into the mechanisms of bioactivation

‘It's all the way you look at it, you know’: reading Bill ‘Bojangles’ Robinson's film career

This paper engages with a major paradox in African American tap dancer Bill ‘Bojangles’ Robinson's film image – namely, its concurrent adherences to and contestations of dehumanising racial iconography – to reveal the complex and often ambivalent ways in which identity is staged and enacted. Although Robinson is often understood as an embodiment of popular cultural imagery historically designed to dehumanise African Americans, this paper shows that Robinson's artistry displaces these readings by providing viewing pleasure for black, as much as white, audiences. Robinson's racially segregated scenes in Dixiana (1930) and Hooray for Love (1935) illuminate classical Hollywood's racial codes, whilst also showing how his inclusion within these otherwise all-white films provides grounding for creative and self-reflexive artistry. The films' references to Robinson's stage image and artistry overlap with minstrelsy-derived constructions of ‘blackness’, with the effect that they heighten possible interpretations of his cinematic persona by evading representational conclusion. Ultimately, Robinson's films should be read as sites of representational struggle that help to uncover the slipperiness of performances of African American identities in 1930s Hollywood