The metabolism and toxicity of furosemide in the Wistar rat and CD-1 mouse: a chemical and biochemical definition of the toxicophore.

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%), γ-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 γ-ketocarboxylic acid, identified by nuclear magnetic resonance spectroscopy, indicates bioactivation of the furan ring. Formation of γ-ketocarboxylic acid was P450-dependent. In mouse liver microsomes, a γ-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.