Cytochrome P450 3A (CYP3A) levels are important determinants of aflatoxin and acetaminophen toxicity and the uptake and elimination of numerous therapeutic agents. CYP3A protein has been reported to be stabilized by some CYP3A substrates. The PI reported that several structurally and functionally distinct agents that are not known CYP3A substrates alter CYP3A protein stability in primary cultured rat hepatocytes. The changes in CYP3A stability are consistent with effects of these agents on phospholipase C or D (PLC or PLD, respectively). Studies in microsomes have indicated that the PLD metabolite, phosphatidic acid (PA), is important in determining CYP3A stability. We propose to test this hypothesis that CYP3A protein stability is dependent upon microsomal PA levels and to further elucidate the molecular mechanism by which phospholipases and other enzymes that regulate PA levels contribute to CYP3A protein stability.
The specific aims are: 1) To further establish the role of PLD and PLC in the regulation of CYP3A protein stability. 2) To determine if agents that alter CYP3A stability in primary cultured hepatocytes also function in in vitro incubations. 3.) To determine if PA analogs with different fatty acid moieties have distinct effects on CYP3A protein stability in microsomal samples. 4) To determine whether agents that regulate rat CYP3A stability act in a like manner in human primary cultured hepatocytes and microsomes. These studies will provide information that can be used to predict drug interactions by providing an understanding of the underlying molecular mechanisms involved in the post-translational regulation of CYP3A, and will test a new hypothesis regarding the interactions of phospholipase activities and a key drug-metabolizing enzyme.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054812-04
Application #
6523754
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Serrano, Jose
Project Start
1999-09-30
Project End
2004-08-31
Budget Start
2002-09-01
Budget End
2004-08-31
Support Year
4
Fiscal Year
2002
Total Cost
$200,301
Indirect Cost
Name
Battelle Pacific Northwest Laboratories
Department
Type
DUNS #
032987476
City
Richland
State
WA
Country
United States
Zip Code
99352
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Zangar, Richard C; Fan, Yang-Yi; Chapkin, Robert S (2004) Interactions of phospholipase D and cytochrome P450 protein stability. Biochem Pharmacol 68:503-12
Kimzey, Amy L; Weitz, Karl K; Guengerich, F Peter et al. (2003) Hydroperoxy-10,12-octadecadienoic acid stimulates cytochrome P450 3A protein aggregation by a mechanism that is inhibited by substrate. Biochemistry 42:12691-9
Zangar, Richard C; Kocarek, Thomas A; Shen, Shang et al. (2003) Suppression of cytochrome P450 3A protein levels by proteasome inhibitors. J Pharmacol Exp Ther 305:872-9
Zangar, R C; Kimzey, A L; Okita, J R et al. (2002) Cytochrome P450 3A conjugation to ubiquitin in a process distinct from classical ubiquitination pathway. Mol Pharmacol 61:892-904