The long-term objective of this proposal for extension of the research grant is to fully understand the role of the alcohol-inducible forms of cytochrome P-450, P-450IIE1 and P-450IIE2, in alcohol metabolism and in the chemical toxicities, mutagenesis, and carcinogenesis associated with alcohol abuse.
The specific aims are (a) to purify alcohol-inducible cytochrome P-450 IIE2 to homogeneity after heterologous expression and to determine whether P-450 IIE1 in kidney, nasal mucosa, and lung, and P-450 IIE2 in lung are different from their counterparts in liver, (b) to determine whether P-450 IIE1 and IIE2 and any other variants differ in their activities toward biologically occurring substrates such as glycerol and other alcohol, acetone and other carbonyl compounds, retinoids, and lipid hydroperoxides; (c) to compare the activities of P- 450 IIE1 and IIE2 toward a variety of xenobiotics and in the reduction of O2 to H2O2 and oxygen radicals in the absence of added substrates; and (d) to study differences in the developmental expression of the P- 450 IIE1 and IIE2 genes. Although we have purified the unstable P-450 IIE2 in small amounts from neonatal rabbit liver, the number of animals required to obtain enough of the enzyme for thorough characterization is prohibitive. A full-length CDNA for this cytochrome will be cloned and expressed in yeast and E. coli and the cytochrome will be isolated from large scale cultures. Catalytic activities will be measured in the reconstituted system toward various added substrates. Our recent finding that P-450 IIE1 is the most active of the cytochromes tested in the reductive Beta-scission of fatty acid hydroperoxides to oxoacids and hydrocarbons raises the important question of whether the chronic exposure of animals and humans to alcohol leads to a loss of membrane integrity because of lipid alterations. As time permits, we will explore the pathological effects of liquid peroxidation related to alcohol intake as well as the possibility that the products of lipid peroxidation may serve some useful biological functions. Although P-450 IIE1 is known to metabolize a large variety of foreign compounds, including ethanol and other alcohols, ketones, nitrosamines, and halogenated alkanes, alkanes, and ethers, as well as aromatic compounds, almost no information is yet available with P-450 IIE2. The substrates to be tested will include the prohepatotoxin acetaminophen, procarcinogens such as N-nitrosodimethylamine and N-nitrodiethylamine, and the drug chlorzoxazone. Preliminary results indicate that P-450 IIE2 MRNA and protein are detectable immediately after birth, whereas IIE1 MRNA (and,therefore, protein) are not detectable until day 14. The nature of this differential regulation will be studied, including the effects on the progeny of alcohol administration to pregnant or nursing rabbits, and attempts will be made to determine whether the two cytochromes have functional differences that are important during the first few weeks of life. The results may help elucidate some of the biochemical effects in the fetal alcohol syndrome. In addition, the mechanism by which IIE1 and IIE2 mRNA are elevated in the Watanabe hyperlipidemic rabbits will be determined.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA006221-10
Application #
3109426
Study Section
Biochemistry, Physiology and Medicine Subcommittee (ALCB)
Project Start
1983-12-01
Project End
1996-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
10
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Vatsis, Kostas P; Coon, Minor J (2005) Oxidative aldehyde deformylation catalyzed by NADPH-cytochrome P450 reductase and the flavoprotein domain of neuronal nitric oxide synthase. Biochem Biophys Res Commun 337:1107-11
Vatsis, Kostas P; Peng, Hwei-Ming; Coon, Minor J (2005) Abolition of oxygenase function, retention of NADPH oxidase activity, and emergence of peroxidase activity upon replacement of the axial cysteine-436 ligand by histidine in cytochrome P450 2B4. Arch Biochem Biophys 434:128-38
Vatsis, Kostas P; Peng, Hwei-Ming; Coon, Minor J (2002) Replacement of active-site cysteine-436 by serine converts cytochrome P450 2B4 into an NADPH oxidase with negligible monooxygenase activity. J Inorg Biochem 91:542-53
Peng, H M; Coon, M J (2000) Promoter function and the role of cytokines in the transcriptional regulation of rabbit CYP2E1 and CYP2E2. Arch Biochem Biophys 382:129-37
Vaz, A D; McGinnity, D F; Coon, M J (1998) Epoxidation of olefins by cytochrome P450: evidence from site-specific mutagenesis for hydroperoxo-iron as an electrophilic oxidant. Proc Natl Acad Sci U S A 95:3555-60
Coon, M J; Vaz, A D; McGinnity, D F et al. (1998) Multiple activated oxygen species in P450 catalysis: contributions To specificity in drug metabolism. Drug Metab Dispos 26:1190-3
Peng, H M; Coon, M J (1998) Regulation of rabbit cytochrome P450 2E1 expression in HepG2 cells by insulin and thyroid hormone. Mol Pharmacol 54:740-7
Jiang, Y; Kuo, C L; Pernecky, S J et al. (1998) The detection of cytochrome P450 2E1 and its catalytic activity in rat testis. Biochem Biophys Res Commun 246:578-83
Kuo, C L; Vaz, A D; Coon, M J (1997) Metabolic activation of trans-4-hydroxy-2-nonenal, a toxic product of membrane lipid peroxidation and inhibitor of P450 cytochromes. J Biol Chem 272:22611-6
Pernecky, S J; Coon, M J (1996) N-terminal modifications that alter P450 membrane targeting and function. Methods Enzymol 272:25-34

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