title: Metabolic activation of hepatotoxic drug (benzbromarone) induced mitochondrial membrane permeability transition.
abstract: The risk of drug-induced liver injury (DILI) is of great concern to the pharmaceutical industry. It is well-known that metabolic activation of drugs to form toxic metabolites (TMs) is strongly associated with DILI onset. Drug-induced mitochondrial dysfunction is also strongly associated with increased risk of DILI. However, it is difficult to determine the target of TMs associated with exacerbation of DILI because of difficulties in identifying and purifying TMs. In this study, we propose a sequential in vitro assay system to assess TM formation and their ability to induce mitochondrial permeability transition (MPT) in a one-pot process. In this assay system, freshly-isolated rat liver mitochondria were incubated with reaction solutions of 44 test drugs preincubated with liver microsomes in the presence or absence of NADPH; then, NADPH-dependent MPT pore opening was assessed as mitochondrial swelling. In this assay system, several hepatotoxic drugs, including benzbromarone (BBR), significantly induced MPT in a NADPH-dependent manner. We investigated the rationality of using BBR as a model drug, since it showed the most prominent MPT in our assay system. Both the production of a candidate toxic metabolite of BBR (1',6-(OH)2 BBR) and NADPH-dependent MPT were inhibited by several cytochrome P450 (CYP) inhibitors (clotrimazole and SKF-525A, 100μM). In summary, this assay system can be used to evaluate comprehensive metabolite-dependent MPT without identification or purification of metabolites.
authors.affiliation: The Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
authors.forename: Maho
authors.initials: M
authors.lastname: Shirakawa
authors.affiliation: The Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Sekine
authors.affiliation: The Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
authors.forename: Ayaka
authors.initials: A
authors.lastname: Tanaka
authors.affiliation: Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Tokyo, Japan.
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Horie
authors.affiliation: The Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan. Electronic address: itokousei@chiba-u.jp.
authors.forename: Kousei
authors.initials: K
authors.lastname: Ito
mesh_terms: D065767:Activation, Metabolic; D000818:Animals; D001553:Benzbromarone / Q000378:metabolism / Q000633:toxicity*; D056486:Chemical and Drug Induced Liver Injury / Q000209:etiology* / Q000378:metabolism / Q000473:pathology; D065688:Cytochrome P-450 CYP2C9 Inhibitors / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D065692:Cytochrome P-450 CYP3A Inhibitors / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects*; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008933:Mitochondrial Swelling / Q000187:drug effects; D009249:NADP / Q000378:metabolism; D017207:Rats, Sprague-Dawley
chemical_list: D065688:Cytochrome P-450 CYP2C9 Inhibitors; D065692:Cytochrome P-450 CYP3A Inhibitors; D001553:Benzbromarone; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; C507848:CYP2C9 protein, rat; C465260:Cyp3a2 protein, rat; D051544:Cytochrome P-450 CYP3A
keywords: Benzbromarone; Drug-induced liver toxicity; Membrane permeability transition; Mitochondria; Reactive metabolite