title: Identification of the Additional Mitochondrial Liabilities of 2-Hydroxyflutamide When Compared With its Parent Compound, Flutamide in HepG2 Cells.
abstract: The androgen receptor antagonist, flutamide, is strongly associated with idiosyncratic drug-induced liver injury (DILI). Following administration, flutamide undergoes extensive first-pass metabolism to its primary metabolite, 2-hydroxyflutamide. Flutamide is a known mitochondrial toxicant; however there has been limited investigation into the potential mitochondrial toxicity of 2-hydroxyflutamide and its contribution to flutamide-induced liver injury. In this study we have used the acute glucose or galactose-conditioning of HepG2 cells to compare the mitochondrial toxicity of flutamide, 2-hydroxyflutamide and the structurally-related, non-hepatotoxic androgen receptor antagonist, bicalutamide. Compound-induced changes in mitochondrial oxygen consumption rate were assessed using Seahorse technology. Permeabilization of cells and delivery of specific substrates and inhibitors of the various respiratory complexes provided more detailed information on the origin of mitochondrial perturbations. These analyses were supported by assessment of downstream impacts including changes in cellular NAD(+)/NADH ratio. Bicalutamide was not found to be a mitochondrial toxicant, yet flutamide and 2-hydroxyflutamide significantly reduced basal and maximal respiration. Both flutamide and 2-hydroxyflutamide significantly reduced respiratory complex I-linked respiration, though 2-hydroxyflutamide also significantly decreased complex II and V-linked respiration; liabilities not demonstrated by the parent compound. This study has identified for the first time, the additional mitochondrial liabilities of the major metabolite, 2-hydroxyflutamide compared with its parent drug, flutamide. Given the rapid production of this metabolite upon administration of flutamide, but not bicalutamide, we propose that the additional mitochondrial toxicity of 2-hydroxyflutamide may fundamentally contribute to the idiosyncratic DILI seen in flutamide-treated, but not bicalutamide-treated patients.
authors.affiliation: *Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, University of Liverpool, Liverpool L69 3GE, UK a.ball3@liverpool.ac.uk.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Ball
authors.affiliation: *Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, University of Liverpool, Liverpool L69 3GE, UK.
authors.forename: Laleh
authors.initials: L
authors.lastname: Kamalian
authors.affiliation: Department of Molecular and Clinical Pharmacology, The Wolfson Centre for Personalised Medicine, University of Liverpool, Liverpool L69 3GL, UK.
authors.forename: Ana
authors.initials: A
authors.lastname: Alfirevic
authors.affiliation: GlaxoSmithKline, Safety Assessment, Ware SG12 0DP, UK.
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Lyon
authors.affiliation: *Department of Molecular and Clinical Pharmacology, MRC Centre for Drug Safety Science, University of Liverpool, Liverpool L69 3GE, UK.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Chadwick
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D003470:Culture Media; D005485:Flutamide / Q000031:analogs & derivatives* / Q000633:toxicity*; D005690:Galactose; D005947:Glucose; D056945:Hep G2 Cells; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010101:Oxygen Consumption; D013481:Superoxides / Q000378:metabolism
chemical_list: D003470:Culture Media; D009243:NAD; D013481:Superoxides; C014290:hydroxyflutamide; D005485:Flutamide; D000255:Adenosine Triphosphate; D005947:Glucose; D005690:Galactose
keywords: 2-hydroxyflutamide; DILI; flutamide; mitochondria.