The long-term objective of the proposed research is to elucidate the molecular basis for the unique ability of dogs to metabolize and eliminate certain highly chlorinated polychlorinated biphenyls (PCBs). PCBs are a class of compounds characterized by a biphenyl nucleus with varying degrees of chlorination on one or both phenyl rings. PCBs have been widely used in industry due to their resistance to thermal, chemical, and biological degradation. Although their production was terminated in the 1970s, PCBs are still in wide use in electrical capacitors and transformers. The high stability of PCBs be led to their persistence in the environment, and because of the serious toxicity associated with many PCBs, they pose a significant human health hazard. One of the key factors that determines the persistence of PCBs in the environment as well as in animal and human tissues is their metabolism. In general, highly chlorinated PCBs, especially those lacking two adjacent unsubstituted sites on one of the phenyl rings, are metabolized slowly by micro-organisms and mammals. One such compound which is highly persistent in the environment and in human tissues and breast milk is 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB). 245-HCB and the related polybrominated compound 2,2',4,4',5,5'-hexabromobiphenyl are both tumor promoters in rats. Of all species tested to date, only the dog appear to be able to efficiently metabolize and eliminate 245-HCB. Recent results from this laboratory have shown that this unique metabolic capability of the dog is due to the presence of a form of cytochrome P-450 called PBD-2 which has a higher hepatic concentration and intrinsic activity towards 245-HCB than the corresponding cytochromes P-450 in other species the proposed research will focus on determining the molecular basis for the unique properties of PBD-2. The individual specific aims are: to determine the nucleotide sequence of cDNA coding for PBD-2, to determine whether the PBD-2 gene is unique or a member of a multigene family, to determine the structure of and the presence of potential regulator elements in the gene(s) coding. for PBD-2, and to determine which domain of the protein is responsible for its unusual ability to metabolize 245-HCB. These studies should provide an explanation for the inability of humans to metabolize certain environmentally important PCBs and may also provide the basis for creating genetically altered bacteria or plants, specifically tailored to detoxify PCBs in the environment.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
1R01ES004995-01
Application #
3253197
Study Section
Toxicology Study Section (TOX)
Project Start
1989-02-01
Project End
1992-01-31
Budget Start
1989-02-01
Budget End
1990-01-31
Support Year
1
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Arizona
Department
Type
Schools of Pharmacy
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85722
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Szklarz, G D; Halpert, J R (1997) Use of homology modeling in conjunction with site-directed mutagenesis for analysis of structure-function relationships of mammalian cytochromes P450. Life Sci 61:2507-20
Harlow, G R; He, Y A; Halpert, J R (1997) Functional interaction between amino-acid residues 242 and 290 in cytochromes P-450 2B1 and 2B11. Biochim Biophys Acta 1338:259-66
Liu, J; He, Y A; Halpert, J R (1996) Role of residue 480 in substrate specificity of cytochrome P450 2B5 and 2B11. Arch Biochem Biophys 327:167-73
Harlow, G R; Halpert, J R (1996) Mutagenesis study of Asp-290 in cytochrome P450 2B11 using a fusion protein with rat NADPH-cytochrome P450 reductase. Arch Biochem Biophys 326:85-92
Fang, X; Halpert, J R (1996) Dithionite-supported hydroxylation of palmitic acid by cytochrome P450BM-3. Drug Metab Dispos 24:1282-5
Born, S L; John, G H; Harlow, G R et al. (1995) Characterization of the progesterone 21-hydroxylase activity of canine cytochrome P450 PBD-2/P450 2B11 through reconstitution, heterologous expression, and site-directed mutagenesis. Drug Metab Dispos 23:702-7
Klekotka, P A; Halpert, J R (1995) Benzyloxyresorufin as a specific substrate for the major phenobarbital-inducible dog liver cytochrome P450 (P4502B11). Drug Metab Dispos 23:1434-5
John, G H; Hasler, J A; He, Y A et al. (1994) Escherichia coli expression and characterization of cytochromes P450 2B11, 2B1, and 2B5. Arch Biochem Biophys 314:367-75
Hasler, J A; Harlow, G R; Szklarz, G D et al. (1994) Site-directed mutagenesis of putative substrate recognition sites in cytochrome P450 2B11: importance of amino acid residues 114, 290, and 363 for substrate specificity. Mol Pharmacol 46:338-45

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