The adverse biological effects of most chemical carcinogens, including polycyclic aromatic hydrocarbons (PAH's), are dependent on the formation of reactive metabolites. The oxidative metabolism of such compounds are dependent upon induction of a specific group of cytochromes P-450. In the rabbit, cytochrome P-450 form 4 and cytochrome P-450 form 6 are iduced differentially as a function of age by PAH's in various tissues. By the use of recombinant DNA techniques, DNA sequences coding for rabbit liver form 4 and form 6 mRNA will be isolated and used to investigate regulatory events involved in the induction process. Clones ds-cDNAs complimentary to mouse cytochrome P1-450 and cytochrome P-448 mRNA, postulated to be equivalent to rabbit forms 4 and 6, will be used as probes to hybridize to rabbit liver ds-cDNA libraries. The recombinants that anneal will be subjected to hybrid selection-translation experiments to verify that the sequences are immunologically associated with form 4 and form 6. Following treatment with PAH's at different times during development, form 4 and form 6 mRNA and protein levels will be concommitantly examined in various tissues. The rates of invitro transcription, accumulation of intranuclear pre-mRNA forms, cytoplasmic mRNA and synthesis of membrane associated protein will be quantitated. These determinations will help to establish at what level the induction process of form 4 and form 6 is regulated in the various tissues as a function of age. The ds-cDNA clones will also be used to isolate specific genomic DNA sequences from a cosmid rabbit genomic library. The DNA sequences will be extensively characterized to determine the organization of coding sequences and important 5' flanking sequences responsible for the initiation of transcription. Cloned DNA fragments representative of 5' and 3' portions of the genes, will be employed to investigate if alterations in the DNA methylation status of the genes plays a role in modulating the expression of form 4 and form 6 genes during development and induction in various tissues. These studies, in combination with other ancillary recombinant DNA techniques, will aid in our understanding of the regulatory events involved in the expression of these cytochromes, and those events that contribute to the onset of chemical carcinogenesis.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA037139-03
Application #
3174859
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1984-04-01
Project End
1987-03-31
Budget Start
1986-04-01
Budget End
1987-03-31
Support Year
3
Fiscal Year
1986
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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