HEPATOTOXIN METABOLISM AND ITS REGULATION WITHIN LIVER

该协议的基本前提是微分

The basic premise of this protocol is that the differential susceptibilities of hepatocytes as well as nonparenchymal cells to toxicities resulting from the bioactivation of xenobiotics are primarily due to cell-specific differences in xenobiotic activation and detoxication. Although marked intrahepatic and intralobular heterogeneity occur during xenobiotic metabolism and its induction, many fundamental questions remain to be answered regarding why such heterogeneity exists. Thus, our objectives in this project are: [a] to more completely characterize differences in the abilities to hepatocytes across the lobule to enzymatically activate and detoxicate xenobiotics; [b] to more conclusively identify all liver cells which can metabolize xenobiotics; and [c] to gain insight into how the expression of xenobiotic-metabolizing enzymes in these cells are regulated normally, by inducers, and ontogeny. Specifically, we propose: [1] to further define intralobular heterogeneity in the distributions of different forms of cytochrome P-450, determine which xenobiotic-metabolizing enzymes are present in nonparenchymal cells, and ascertain if the expression of enzymes in hepatocytes and nonparenchymal cells correlate with those of their mRNAs in untreated adult rats; [2] to determine if xenobiotic-metabolizing enzymes can be induced in nonparenchymal cells, if there are intrazonal differences in the kinetics of induction of enzymes, and if increases in holoenzyme and mRNA levels in cells correlate; and [3] to investigate the postnatal ontogeny of xenobiotic- metabolizing enzymes and of heterogeneity in their intralobular distributions. To accomplish this work, semiquantitative immunohistochemistry will be employed for determining the intrahepatic localizations and intralobular distributions of cytochromes P-450, NADPH-cytochrome P-450 reductase, epoxide hydrolase, gluthione S- transferases, and UDP-glucuronosyltransferases, in situ hybridization will be utilized for studying the expressions of mRNAs encoding these enzymes, and histochemistry will be used for examining xenobiotic monooxygenase activities. Histologic findings on enzymes, their mRNAs, and monooxygenase activities will be correlated with in vitro biochemical and immunochemical determinations. The use of this histologic approach will allow us to study gene expression and its regulation within individual cells at both the transcriptional and protein levels. This, in turn, will enable us to gain fundamental, new insight into both the presence of and explanation for cell-specific differences in xenobiotic metabolism and its regulation in liver and will aid in the attainment of our long-term goal to elucidate the mechanism(s) responsible for cell-specific differences in the metabolism of hepatotoxins and other xenobiotics.