title: Oxidative bioactivation of abacavir in subcellular fractions of human antigen presenting cells.
abstract: Human exposure to abacavir, a primary alcohol antiretroviral, is associated with the development of immunological drug reactions in individuals carrying the HLA risk allele B*57:01. Interaction of abacavir with antigen presenting cells results in cell activation through an Hsp70-mediated Toll-like receptor pathway and the provision of T-cell antigenic determinants. Abacavir's electrophilic aldehyde metabolites are potential precursors of neoantigens. Herein, we have used mass spectrometry to study the oxidative metabolism of abacavir in EBV-transformed human B-cells. RNA and protein were isolated from the cells and subjected to transcriptomic and mass spectrometric analyses to identify the redox enzymes expressed. Low levels of isomeric abacavir carboxylic acids were detected in subcellular fractions of EBV-transformed human B-cells incubated with abacavir. Metabolite formation was time-dependent but was not reduced by an inhibitor of Class I alcohol dehydrogenases. Relatively high levels of mRNA were detected for several redox enzymes, including alcohol dehydrogenase 5 (Class III), aldehyde dehydrogenases (ALDH3A2, ALDH6A1, and ALDH9A1), CYP1B1, CYP2R1, CYP7B1, and hydroxysteroid dehydrogenase 10. Over 2600 proteins were identified by mass spectrometry. More than 1000 of these proteins exhibited catalytic activity, and 80 were oxido-reductases. This is the first proteomic inventory of enzymes in antigen presenting cells. However, neither of the hepatic alcohol dehydrogenases of Class I which metabolize abacavir in vitro was expressed at the protein level. Nevertheless the metabolic production of abacavir carboxylic acids by B-cell fractions implies abacavir-treated immune cells might be exposed to the drug's protein-reactive aldehyde metabolites in vivo.
authors.affiliation: MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, University of Liverpool , Liverpool L69 3GE, United Kingdom.
authors.forename: Catherine C
authors.initials: CC
authors.lastname: Bell
authors.forename: Anahi
authors.initials: A
authors.lastname: Santoyo Castelazo
authors.forename: Emma L
authors.initials: EL
authors.lastname: Yang
authors.forename: James L
authors.initials: JL
authors.lastname: Maggs
authors.forename: Rosalind E
authors.initials: RE
authors.lastname: Jenkins
authors.forename: Jonathan
authors.initials: J
authors.lastname: Tugwood
authors.forename: Paul M
authors.initials: PM
authors.lastname: O'Neill
authors.forename: Dean J
authors.initials: DJ
authors.lastname: Naisbitt
authors.forename: B Kevin
authors.initials: BK
authors.lastname: Park
mesh_terms: D000938:Antigen-Presenting Cells / Q000166:cytology* / Q000378:metabolism*; D001711:Biotransformation; D002461:Cell Line, Transformed; D003600:Cytosol / Q000378:metabolism; D015224:Dideoxynucleosides / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000166:cytology; D013058:Mass Spectrometry; D008968:Molecular Conformation; D010084:Oxidation-Reduction; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D000099227:Abacavir
chemical_list: D015224:Dideoxynucleosides; D000099227:Abacavir