The microsomal monooxygenase system, composed of the cytochrome P-450s as the terminal oxidase, play a central role in the metabolism of thousands of compounds including drugs, xenobiotics and carcinogens. The capacity of this system to handle such a diverse array of compounds reflects both the large number of distinct P-450s as well as the broad and overlapping substrate selectivity. These enzymes usually provide a defense against undesirable chemicals that gain access to the cell. However, certain forms are also capable metabolizing compounds such as polycyclic aromatic hydrocarbons to highly reactive intermediates that are capable of initiating a carcinogenic event. Many of the different forms are regulated differentially according age, tissue, inheritance and their ability to be induced in response to different types of agents. Therefore, variations in the expression of different P-450s depending on factors such as age, the level of induction following exposure to certain agents, or the genetic events that contribute to heritable differences, can alter the balance between the formation of toxic and non-toxic products. Differences in the expression of the P-450s can most be likely attributed to transcriptional or post transcriptional events. The critical aspects of the process are the trans acting factors that interact with cis acting regulatory sites in a manner which modulates the expression of the various genes. To understand the molecular events that control the expression of the different P- 450s, we have developed cDNA and genomic clones to a number of differentially regulated rabbit P-450s. Clones have been developed to rabbit P-450 4 and P-450 6. Both of these P-450s are inducible by polycyclic aromatic hydrocarbons and are regulated differentially with respect to age and tissue. We have also identified cDNA and genomic clones to rabbit P-450 1, which is expressed constitutively but reflects heritable differences governing the amount of P-450 1 that is expressed in various inbred rabbits and genetic crosses. In addition, a cDNA clone that encodes rifampicin inducible P-450 3c has also been identified. Molecular approaches will be pursued to determine if cis and trans acting factors can be identified that contribute to the differential expression of the P-450 genes. The employment of nuclear extracts will be used in DNA footprint and in vitro transcription analysis to determine the potential role of negative and positive regulators of transcription in response to induction, tissue specificity and temporal control. In addition, experiments will be outlined to determine the events that underlie the heritable differences responsible for the regulation of P-450 1. Combined, we anticipate that a clear understanding of the regulatory events as they relate to tissue specificity, age, inducibility and inheritance of the different P-450 genes will evolve from these studies.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA037139-04
Application #
3174854
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1984-04-01
Project End
1992-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
4
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Lamb, J G; Straub, P; Tukey, R H (1994) Cloning and characterization of cDNAs encoding mouse Ugt1.6 and rabbit UGT1.6: differential induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochemistry 33:10513-20
Okino, S T; Pendurthi, U R; Tukey, R H (1993) 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces the nuclear translocation of two XRE binding proteins in mice. Pharmacogenetics 3:101-9
Tukey, R H; Pendurthi, U R; Nguyen, N T et al. (1993) Cloning and characterization of rabbit liver UDP-glucuronosyltransferase cDNAs. Developmental and inducible expression of 4-hydroxybiphenyl UGT2B13. J Biol Chem 268:15260-6
Pendurthi, U R; Okino, S T; Tukey, R H (1993) Accumulation of the nuclear dioxin (Ah) receptor and transcriptional activation of the mouse Cyp1a-1 and Cyp1a-2 genes. Arch Biochem Biophys 306:65-9
Okino, S T; Pendurthi, U R; Tukey, R H (1992) Phorbol esters inhibit the dioxin receptor-mediated transcriptional activation of the mouse Cyp1a-1 and Cyp1a-2 genes by 2,3,7,8-tetrachlorodibenzo-p-dioxin. J Biol Chem 267:6991-8
Strom, D K; Postlind, H; Tukey, R H (1992) Characterization of the rabbit CYP1A1 and CYP1A2 genes: developmental and dioxin-inducible expression of rabbit liver P4501A1 and P4501A2. Arch Biochem Biophys 294:707-16
Tukey, R H; Okino, S T (1991) Quantitation of related gene products by nuclear run-on and northern blot analysis. Methods Enzymol 206:284-90
Hapgood, J; Cuthill, S; Soderkvist, P et al. (1991) Liver cells contain constitutive DNase I-hypersensitive sites at the xenobiotic response elements 1 and 2 (XRE1 and -2) of the rat cytochrome P-450IA1 gene and a constitutive, nuclear XRE-binding factor that is distinct from the dioxin receptor. Mol Cell Biol 11:4314-23
Pendurthi, U R; Lamb, J G; Nguyen, N et al. (1990) Characterization of the CYP2C5 gene in 21L III/J rabbits. Allelic variation affects the expression of P450IIC5. J Biol Chem 265:14662-8
Potenza, C L; Pendurthi, U R; Strom, D K et al. (1989) Regulation of the rabbit cytochrome P-450 3c gene. Age-dependent expression and transcriptional activation by rifampicin. J Biol Chem 264:16222-8

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