Date on Master's Thesis/Doctoral Dissertation

5-2008

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Committee Chair

Prough, Russell A.

Author's Keywords

Cytochrome P450; 4-hydroxy-2-nonenal; Activator protein 1; Microsomes; Lipid aldehydes

Subject

Cytochrome P-450; Aldehydes

Abstract

4-hydroxy-2-nonenal (4-HNE) and propene-2-al (acrolein) are highly reactive á,â-unsaturated aldehydes. 4-HNE and acrolein are generated in vivo as products of lipid peroxidation. These aldehydes are implicated in the onset of several diseases including atherosclerosis and neurodegenerative disease. They also react with nucleophilic cellular macromolecules including proteins, DNA and phospholipids. Some of these reactions in vivo lead to inhibition of enzyme activities, depletion of glutathione and oxidative stress. Because of their pathophysiological relevance, attention has focused during the last decade on the ability of mammals to metabolize these lipid aldehydes. The purpose of this study was therefore to determine the role of cytochrome P450s in the metabolism of lipid aldehydes and how exposure to lipid aldehydes influences the regulation of the expression of xenobiotic metabolizing enzymes. Cytochrome P450s are a superfamily of heme proteins that catalyze the NADPH and O 2 -dependent monooxygenation of a wide variety of compounds. In addition, P450s are capable of catalyzing the biosynthesis of several molecules including the synthesis of sterols, fatty acids, and eicosanoids. In this study used a fluorescence spectroscopy and high performance lipid chromatography technique, coupled with mass spectrometry to demonstrate that CYPs metabolize lipid aldehydes. This metabolism involves monooxygenation by mouse, rat and human P450s, of 4-HNE to 4-hydroxynonenoic acid and the reduction of 4-HNE to 1,4-dihydroxynonene by specific P450s. By using general and specific P450 inhibitors, we have determined that hepatic P450s differentially contribute to 4-HNE metabolism. While Cyp2c29 catalyzes oxidative metabolism, Cyp3a predominantly catalyzes the reduction of 4-HNE. We demonstrated by microarray analysis and quantitative real-time PCR that expression of Cyp2c29, Cyp2c37 and Cyp3a25 is induced by acrolein and butylated hydroxyanisole, both activators of AP-1 in mouse liver. These P450s include Cyp2c29, Cyp2a5 and Cyp1A2. BHA and acrolein also induced other xenobiotic metabolizing enzymes such as GSTm1, heme oxygenase-1 and NADHP quinone oxidoreductase. Certain transcription factors function to regulate gene expression in response to xenobiotic challenge. Lipid aldehydes in particular regulate gene expression through the action of two transcription factors, namely AP-1 and Nrf2. We determined that Cyp2c29 is regulated by activator protein 1 (AP-l) transcription factor. Nrf2 was not involved in the induction of Cyp2c29 expression. The results from this study show that there is lipid aldehyde-dependent regulation of P450 gene expression. The induced Cyp2c29 goes on to metabolize substrate in a manner reminiscent of a self-regulatory loop of biological activity.

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