We propose to continue our studies on the characterization of cytochrome P-450 (P-450) isozymes in humans. Our interest is directed toward those isozymes known to be involved in genetic polymorphism of certain oxidative activities in humans and the roles of these enzymes in influencing the susceptibility of individuals to carcinogens and drugs. Four human liver P-450s have already been purified in our laboratory; these are termed P-450, P-450PA, P-450 MP, and P-450 NF and appear to be involved in the polymorphisms of oxidation debrisoquine (DB), phenacetin (PA), mephenytoin (MP), and nifedipine (NF), respectively. We propose to: (I) isolate specific cDNAs coding for the above four P-450s (and the rat homolog of P-450DB, P-450UT-H) and use these probes to (A) isolate and sequence cDNAs coding for the entire proteins (full-length cDNAs), also utilizing partial N-terminal protein sequence information derived from Edman degradation to identify full-length cDNAs and peptide information to match DNA sequences with proteins, (B) identify any quantitative differences in the levels of mRNA or protein for each P-450 related to the polymorphism in catalytic activity in humans, (C) identify any structural variations at the cDNA level which are involved in the polymorphisms, and (D) assign the gene for each P-450 to human chromosomes; (II) utilize inhibitory antibodies to each P-450 to elucidate substrate specificity, particularly with regard to potentially dangerous chemicals; (III) examine the levels of each P-450, its mRNA, and its catalytic activity in the extrahepatic tissues lung, kidney, and placenta; (IV) purify and characterize the human liver P-450 involved in the polymorphic metabolism of tolbutamide and develop cDNA probes (as well as identify the human liver P-450s involved in the oxidation of the model drugs antipyrine and hexobarbital to their various products); (V) utilize antibodies to rat liver P-450UT-F, P-450UT-A, and P-450PB-C to identify and purify homologous human liver P-450s, determine if their activities show variation in humans, and carry out the studies outlined under points I and II above; (IV) clone and sequence the full-length cDNA coding for human liver cytochrome b5, and (VII) localize these enzymes in human liver and other tissues using immunohistochemical techniques. The information derived from these studies may be of use in understanding host factors involved in risk assessment of carcinogenic and toxic chemicals, as the substrate specificity of known human P-450s can be elucidated in vitro and variations among individuals can be understood from blood cell DNA if this work proves to be successful.
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