Browsing Theses and Dissertations School of Pharmacy by Author "Thohan, Sanjeev"
Liver slice technology: An in vitro model for integrated biotransformationThohan, Sanjeev; Weiner, Myron, Ph.D. (1998)The objective of this work was to examine the use of tissue slices as an in vitro model for the study of integrated drug metabolism (biotransformation). Liver slices are more representative of the in vivo situation with respect to cellular architecture and diversity than hepatocytes or subcellular fractions. This work has demonstrated the effects of model inducing agents on hepatic phase I-phase II integrated drug metabolism using alkoxycoumarin derivatives. Additionally, this work detailed the development of an in vitro model for novel compound screening using acetaminophen and paraquat as model toxicants. Dynamic incubation of liver slices was found to be superior to a linear incubation over a 2 hour incubation. Biotransformation was assessed with the use of 7-methoxy-, 7-ethoxy- and 7-hydroxycoumarin (7-HC). The major metabolite of O-dealkylase activities of cytochrome P 450, 7-HC, was conjugated with glucuronic acid or sulfate moieties in a capacity limited fashion. Total phase II activity was assessed using 7-HC as the primary substrate and found to be 7-fold higher than total phase I activity. Phenobarbital pretreatment induced liver slice O-demethylase and O-deethylase activities 3.1- and 3.6-fold over control values, respectively. Glucuronosyl transferase activity for 7-HC was found to be increased over sulfation in all metabolite profiles by pretreatment with phenobarbital. 3-methylcholanthrene pretreatment showed a novel induction profile with a 1.8-fold increase in O-demethylase activity and 9-fold increase in O-deethylase activity over control rat liver slices. Increased levels of free 7-HC from O-dealkylase activities indicated possible substrate competition from persistence of 3-methylcholanthrene or its hydroxylated metabolites in the liver slice. The utility of liver slices as a paradigm to investigate cytotoxicity by intracellular enzyme release was detailed with the use of acetaminophen and paraquat. Phenobarbital was shown to potentiate mitochondrial damage by oxidative mechanisms. These studies highlight the utility and versatility of liver slices in the analysis of biotransformation and bioactivation. Implementation of the tissue slice system as a routine screen may facilitate drug discovery and development.