Hepatotoxicity of ketoconazole in Sprague-Dawley rats: glutathione depletion, flavin-containing monooxygenases-mediated bioactivation and hepatic covalent binding.

1. This study has examined ketoconazole (KT)-induced hepatotoxicity in vivo and in vitro, using male Sprague-Dawley rats with [(3)H]KT (1.5 micro Ci mg(-1)) at 40 and 90 mg KT kg(-1) doses. Blood and liver samples were collected from 0 to 24 h for alanine aminotransaminase (ALT), glutathione (GSH) and covalent binding analyses. 2. Covalent binding occurred as early as 0.5 h, peaked at 2 h (0.026 +/- 0.01 nmol KT mg(-1) protein) and 8 h (0.088 +/- 0.04 nmol KT mg(-1) protein) for 40 and 90 mg KT kg(-1) doses, respectively. ALT levels increased at 0.5 h for the 40 and 90 mg KT kg(-1) doses (44.3 and 56.4 U ml(-1), respectively) relative to control, 22.7 U ml(-1). At 24 h, the 90 mg KT kg(-1) dose reduced hepatic GSH levels from 9.92 +/- 1.1 to 4.76 +/- 0.3 nmol GSH mg(-1) protein. 3. The role of the flavin-containing monooxygenases (FMO) utilized Sprague-Dawley microsomes with 1, 10 and 100 micro M [(3)H]KT. Maximum covalent binding occurring at 100 micro M KT. Heat inactivation of microsomal FMO significantly decreased covalent binding by 75%, whereas 1 mM GSH significantly reduced covalent binding by 65%. 4. Thus, KT-induced hepatotoxicity is dose- and time-dependent and appears to be FMO mediated, in part, to metabolites that may react with protein and, possibly, GSH.

Flavin-containing monooxygenase-mediated metabolism of N-deacetyl ketoconazole by rat hepatic microsomes.

Although ketoconazole is extensively metabolized by hepatic microsomal enzymes, the route of formation and toxicity of suspected metabolites are largely unknown. Reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice. DAK may be susceptible to successive oxidative attacks on the N-1 position by flavin-containing monooxygenases (FMO) producing potentially toxic metabolites. Previous laboratory findings have demonstrated that postnatal rat hepatic microsomes metabolize DAK by NADPH-dependent monooxygenases to two metabolites as determined by HPLC. Our current investigation evaluated DAK's metabolism in adult male and female rats and identified metabolites that may be responsible for ketoconazole's hepatotoxicity. DAK was extensively metabolized by rat liver microsomal monooxygenases at pH 8.8 in pyrophosphate buffer containing the glucose 6-phosphate NADPH-generating system to three metabolites as determined by HPLC. The initial metabolite of DAK was a secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole, which was confirmed by liquid chromatography/mass spectrometry and NMR spectroscopy. Extensive metabolism of DAK occurred at pH 8.8 in pyrophosphate buffer (female 29% and male 53% at 0.25 h; female 55% and male 57% at 0.5 h; and female 62% and male 66% at 1.0 h). Significantly less metabolism of DAK occurred at pH 7.4 in phosphate buffer (female 11%, male 17% at 0.25 h; female 20%, male 31% at 0.5 h; and female 27%, male 37% at 1 h). Heat inactivation of microsomal-FMO abolished the formation of these metabolites from DAK. SKF-525A did not inhibit this reaction. These results suggest that DAK appears to be extensively metabolized by adult FMO-mediated monooxygenation.