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.
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