title: The metabolism and toxicity of furosemide in the Wistar rat and CD-1 mouse: a chemical and biochemical definition of the toxicophore.
abstract: Furosemide, a loop diuretic, causes hepatic necrosis in mice. Previous evidence suggested hepatotoxicity arises from metabolic bioactivation to a chemically reactive metabolite that binds to hepatic proteins. To define the nature of the toxic metabolite, we examined the relationship between furosemide metabolism in CD-1 mice and Wistar rats. Furosemide (1.21 mmol/kg) was shown to cause toxicity in mice, but not rats, at 24 h, without resulting in glutathione depletion. In vivo covalent binding to hepatic protein was 6-fold higher in the mouse (1.57 +/- 0.98 nmol equivalent bound/mg protein) than rat (0.26 +/- 0.13 nmol equivalent bound/mg protein). In vivo covalent binding to mouse hepatic protein was reduced 14-fold by a predose of the cytochrome P450 (P450) inhibitor, 1-aminobenzotriazole (ABT; 0.11 +/- 0.04 nmol equivalent bound/mg protein), which also reduced hepatotoxicity. Administration of [(14)C]furosemide to bile duct-cannulated rats demonstrated turnover to glutathione conjugate (8.8 +/- 2.8%), gamma-ketocarboxylic acid metabolite (22.1 +/- 3.3%), N-dealkylated metabolite (21.1 +/- 2.9%), and furosemide glucuronide (12.8 +/- 1.8%). Furosemide-glutathione conjugate was not observed in bile from mice dosed with [(14)C]furosemide. The novel gamma-ketocarboxylic acid, identified by nuclear magnetic resonance spectroscopy, indicates bioactivation of the furan ring. Formation of gamma-ketocarboxylic acid was P450-dependent. In mouse liver microsomes, a gamma-ketoenal furosemide metabolite was trapped, forming an N-acetylcysteine/N-acetyl lysine furosemide adduct. Furosemide (1 mM, 6 h) became irreversibly bound to primary mouse and rat hepatocytes, 0.73 +/- 0.1 and 2.44 +/- 0.3 nmol equivalent bound/mg protein, respectively, which was significantly reduced in the presence of ABT, 0.11 +/- 0.03 and 0.21 +/- 0.1 nmol equivalent bound/mg protein, respectively. Furan rings are part of new chemical entities, and mechanisms underlying species differences in toxicity are important to understand to decrease the drug attrition rate.
authors.affiliation: Drug Safety Research Group, Department of Pharmacology and Therapeutics, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool, Merseyside L69 3GE, UK. dom@liv.ac.uk
authors.forename: Dominic P
authors.initials: DP
authors.lastname: Williams
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Antoine
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Butler
authors.forename: Russell
authors.initials: R
authors.lastname: Jones
authors.forename: Laura
authors.initials: L
authors.lastname: Randle
authors.forename: Anthony
authors.initials: A
authors.lastname: Payne
authors.forename: Martin
authors.initials: M
authors.lastname: Howard
authors.forename: Iain
authors.initials: I
authors.lastname: Gardner
authors.forename: Julian
authors.initials: J
authors.lastname: Blagg
authors.forename: B Kevin
authors.initials: BK
authors.lastname: Park
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D065607:Cytochrome P-450 Enzyme Inhibitors; D005665:Furosemide / Q000378:metabolism* / Q000633:toxicity*; D005978:Glutathione / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008862:Microsomes, Liver / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar; D013045:Species Specificity
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D005665:Furosemide; D005978:Glutathione