The aim is to describe the relationship between maintenance and inducibility of isozymes of cytochrome P450 and heme metabolism in the liver. The model systems to be studied are hepatocyte tissue cultures of chicken embryo or adult rat. A long-term objective is that these culture systems should be as representative as possible of the intact liver for studies of metabolism of endogenous and exogenous compounds (particularly carcinogens and hepatotoxins). These model culture systems differ in their abilities to maintain basal and inducible levels of enzymes of the heme biosynthetic pathway and various isozymes of cytochrome P450. At this time it seems that the chick system maintains the properties of the adult chicken liver whereas the rat liver culture is deficient in inducibility of ALA synthase, the first enzyme of the heme biosynthetic pathway and varies in its abilities to synthesize and maintain the different isozymes of cytochrome P450. We intend to use recently available antibodies to the P450 isozymes to study the effects of depleting or expanding available heme supply on maintenance of individual P450 isozymes in both these culture systems. Heme metabolism in both culture types will be studied by an HPLC procedure already established in the laboratory. We plan to use cDNA's for chicken ALA synthase and P450's to gain further understanding of how drugs induce both these activities. The following aspects of particular P450 isozymes in rat liver cultures will be studied: for P450p, the accumulation of apoprotein lacking heme in cultures treated with dexamethasone and macrolide antibiotics; for P450d, the mechanism of isosafrole stabilization of the protein; and for P450b, the apparent rapid loss of heme once cells are placed in culture. In each of these cases the effects of addition of heme on metabolic stability of the apoproteins will be studied.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA025012-12
Application #
3166646
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1978-06-01
Project End
1991-12-31
Budget Start
1990-01-01
Budget End
1990-12-31
Support Year
12
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Dartmouth College
Department
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
Sinclair, P R; Bement, W J; Healey, J F et al. (1994) Effects of hemopexin on heme-mediated repression of 5-aminolevulinate synthase and induction of heme oxygenase in cultured hepatocytes. Hepatology 20:741-6
LeDuc, B W; Sinclair, P R; Walton, H S et al. (1994) Cocaine toxicity in cultured chicken hepatocytes: role of cytochrome P450. Toxicol Appl Pharmacol 125:322-32
Sinclair, P R; Gorman, N; Walton, H S et al. (1994) Protoporphyrinogen accumulation in cultured hepatocytes treated with the diphenyl ether herbicide, acifluorfen. Cell Mol Biol (Noisy-le-grand) 40:891-7
Nims, R W; Sinclair, P R; Sinclair, J F et al. (1993) Dose-response relationships for the induction of P450 2B by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzne (TCPOBOP) in rat and cultured rat hepatocytes. Xenobiotica 23:1411-26
Xu, L C; Sinclair, P R; Bresnick, E (1993) Induction of cytochrome P450IA1 and its recombinant construct in H4IIE rat hepatoma cells. Int J Biochem 25:13-21
Potter, D; Chroneos, Z C; Baynes, J W et al. (1993) In vivo fate of hemopexin and heme-hemopexin complexes in the rat. Arch Biochem Biophys 300:98-104
Louis, C A; Sinclair, J F; Wood, S G et al. (1993) Synergistic induction of cytochrome P450 by ethanol and isopentanol in cultures of chick embryo and rat hepatocytes. Toxicol Appl Pharmacol 118:169-76
LeDuc, B W; Sinclair, P R; Shuster, L et al. (1993) Norcocaine and N-hydroxynorcocaine formation in human liver microsomes: role of cytochrome P-450 3A4. Pharmacology 46:294-300
Lindenthal, J; Sinclair, J F; Howell, S et al. (1993) Toxicity of paracetamol in cultured chick hepatocytes treated with methotrexate. Eur J Pharmacol 228:289-98
Sinclair, P R; Gorman, N; Walton, H S et al. (1993) Ascorbic acid inhibition of cytochrome P450-catalyzed uroporphyrin accumulation. Arch Biochem Biophys 304:464-70

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