REGULATION OF MICROSOMAL HEMOPROTEINS

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Biotransformation of foreign substances in the liver plays an important role in the interaction between man and his chemical environment. The liver cytochromes P-450, a group of inducible hemoproteins prominently located in the endoplasmic reticulum of the hepatocyte, serve as an important biochemical locus for this interaction. Because the amounts and types of the cytochromes P-450 may be important for determining interindividual susceptibility to toxic effects of environmental agents, our laboratory will continue an ongoing project to define the basic biochemical and molecular details of how environmental agents induce these hemoproteins. Focusing on P-450p, a unique group of liver cytochromes in the rat, and homologous proteins, LM3c in the rabbit, and MLp in the mouse, we will define the basic mechanism by which the three known classes of inducers of these cytochromes, namely, glucocorticoids and antiglutocorticoids (like cyanopregnenolone, PCN) phenobarbital and related compounds, and macrolide antibiotics selectively induce this gene family. Our approach will be to isolate, purify, and characterize these proteins and the genes which encode these proteins. Using specific antibodies and cDNAs as molecular probes, we will define whether transcriptional or post-transcriptional mechanisms are involved in increased de novo synthesis of these proteins both in primary nonproliferating monolayer cultures of adult rat, rabbit, and mouse hepatocytes, and in living animals. We will test the hypothesis that there exists a unique "PCN receptor" or "PCN recognition system" that mediates transcriptional activation of the P-450p genes by ascertaining whether there are binding sites on these genes for the classic glucocorticoid receptor. Established techniques will be used to identify DNAse hypersensitive sites, receptor binding sites, and regulatory elements of the P-450 genes cloned as chimeric genes and transvected into homologous hepatocytes in culture. Finally, the same approach will be applied to digitoxigenin-monodigitoxoside UDP-glucuronyltransferase, a liver microsomal enzyme which appears to be controlled by factors similar to those operative in the P-450p system. It is expected that the results of these experiments will produce important new information in understanding molecular specificity for regulation of biotransformation reactions in the liver.