The elucidation of the regulation of heme synthesis is crucial for the understanding of hepatic cytochrome P450 synthesis, since the major portion (greater than or equal to 65%) of heme in the liver is used for the synthesis of this microsomal hemoprotein. Two of the principal reactions in the host defense mechanism against environmental results are (i) the acute-phase response, and (ii) the cytochrome P450-mediated biotransformation, however, their relationship has received little attention. Limited evidence suggests, however, that there may be a significant interaction between these two systems. In order to study their interaction, it is necessary to use human-liver derived cells, since there are substantial interspecies differences in P450-dependent biotransformation capacity, particularly between animals and man. Our goal in this study is to elucidate the mechanism of the regulation of synthesis of heme and cytochrome P450 in cultured human liver-derived cells, and the effects of the acute-phase reaction on gene expression of heme pathway enzymes and cytochrome P450. We will use HepG2 and Hep3B human hepatoma cells, which have the biochemical features of normal human liver, and HepG2f cells, which have less differentiated properties than HepG2 and Hep3B, for these studies. We have demonstrated that both HepG2 and Hep3B cells show an acute-phase reaction by treatment with interleukin-6 or DMSO; that these hepatoma cells contain heme biosynthetic enzymes (delta-aminolevulinate synthase[ALAS], delta-aminolevulinate dehydratase [ALAD], porphobilinogen deaminase and uroporphyrinogen decarboxylase), heme oxygenase (HO), and cytochrome P450, and that these enzymes show inducible responses by treatment with acute-phase inducers. We have extensively characterized the mode of the induction of ALAS and ALAD, the two major enzymes in the heme biosynthetic pathway which show an induction response to the acute-phase reaction, and HO, the rate-limiting enzymes in heme catabolism, as well as gene expression of acute-phase proteins. In order to further delineate the relationship between these systems, we propose in the current application to examine (I) effects of acute-phase inducers on the levels of mRNAs encoding ALAS, and HO, (II) constitutive expression of mRNAs for cytochrome P450 in untreated hepatoma cells, (III) the effects of the induction of the acute-phase reaction on mRNAs for inducibile cytochrome P450s, and (IV) the role of a nuclear factor specific to the IL-6 responsive element in the human HO gene. These studies should clarify the relationship between the acute-phase reaction and the gene activation of heme biosynthetic and catabolic enzymes and that of cytochrome P450s.
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