Abstract:
Two specific goals were addressed for this dissertation. First to investigate
and identify the mechanistic profile of ketoconazole (KT)-induced hepatotoxicity
by utilizing in vivo and in vitro approaches determining the mechanism of action
for the hepatotoxicity incurred. To date, there has not been a mechanistic
determination of the hepatotoxicity associated with KT in vivo. This dissertation
evaluates the possible metabolic bioactivation of KT by cytochrome-P450 (CYP)
or flavin-containing monooxygenases (FMO) resulting in covalent binding with
hepatic macromolecules. The hypothesis of this study was to reveal whether
covalent binding by the parent compound, KT, and/or reactive metabolites
produces hepatic damage associated with increased serum alanine
aminotransaminase (ALT) release and decreased hepatic glutathione (GSH). The
first objective was determination of in vivo covalent binding in a dose-time
response comparison in Sprague-Dawley (SD) rat ALT and GSH levels. Increased
ALT and reduced hepatic GSH levels occurred. The second objective was an in
vitro comparison of covalent binding with GSH levels utilizing SD microsomal
protein with incubations of KT. Covalent binding decreased with added GSH to
microsomal incubations. Thirdly, correlate in vivo with in vitro findings. Covalent
binding of KT in vivo and in vitro occurred with increased doses and time. The
final objective was to determine the bioactivation pathway utilizing heat
inactivation and no NADPH in vitro. Covalent binding of KT decreased in the
absence of NADPH and deactivation of FMO.
The second goal was to determine and quantitate in vitro the presence of
FMO isozymes in microsomes of the human intestinal duodenum, jejunum, ileum,
and colon as well as the Caco-2 (HTB-37), epithelial intestinal (CCL-241) and
colon (CRL1790) cell lines. The presence of FMO could result in a first-pass effect
decreasing the bioavailability of soft nucleophiles or a toxicity effect due to
inhibition or modulation of the enzyme from co-administration. To date, this is the
first evaluation of FMO isoforms in human intestine and cell lines. Western blot
techniques were utilized for detection of human FMO1, FMO3, and FMO5 using
human FMO-expressed recombinant cDNA from a baculovirus system.
