There is increasing evidence that ethanol toxicity may be associated with elevated production of reactive oxygen intermediates. Among the mechanisms suggested by which ethanol produces oxidative stress, ethanol induction of the microsomal mixed-function oxidase system and cytochrome P450 2E1, and ethanol-derived NADH are of special interest. The specific objective of this application is to employ electron spin resonance (ESR) spectroscopy to determine production of radicals such as superoxide, hydroxyl and carbon center radicals such as the hydroxyethyl radical by liver cell organelles, particularly microsomes, and assess the influence of ethanol treatment on this production of reactive radical intermediates. The major advantages of ESR are that it provides unambiguous, direct determination of radicals, is highly sensitive, and is the only method to detect reactive intermediates such as HER. Experiments will be carried out which compare the effectiveness of NADH with that of NADPH in promoting radical generation and in interacting with a variety of iron complexes to catalyze radical generation by liver cell organelles such as microsomes, mitochondria, nuclei and plasma membranes. The role of P450 2E1 will be assessed using substrates, inhibitors and antibodies. The effects of anti-oxidants and redox cycling agents, and comparisons of results obtained by ESR and chemical detection, will be made. The effect of chronic ethanol treatment on ESR- detectable rates of oxygen and hydroxy radicals and HER production will be evaluated. Since ethanol toxicity originates in the perivenous zone, microsomes and other organelles will be isolated from periportal hepatocytes prepared from control and ethanol-treated rats, and production of reactive intermediates determined by ESR. To identify microsomal enzymes which play a role in the NADH-and NADPH-dependent generation of reactive radical intermediates, experiments with purified NADH-b5 reductase, NADPH-P450 reductase, b5 and P450 (especially P450 2E1) will be carried out, as will selected experiments with human liver microsomes and human liver P450 2E1.
A final aim will be to use ESR to evaluate production of glycerol and other polyhydroxylated alcohol radicals. It is anticipated that direct and specific ESR studies will provide new information on the generation of, and the role of, reactive radical intermediates in alcohol toxicity.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA009460-03
Application #
2045678
Study Section
Biochemistry, Physiology and Medicine Subcommittee (ALCB)
Project Start
1993-02-01
Project End
1997-01-31
Budget Start
1995-02-01
Budget End
1996-01-31
Support Year
3
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
New York
State
NY
Country
United States
Zip Code
10029
Perez, M J; Cederbaum, A I (2001) Spin trapping agents (Tempol and POBN) protect HepG2 cells overexpressing CYP2E1 against arachidonic acid toxicity. Free Radic Biol Med 30:734-46
Clancy, R; Cederbaum, A I; Stoyanovsky, D A (2001) Preparation and properties of S-nitroso-L-cysteine ethyl ester, an intracellular nitrosating agent. J Med Chem 44:2035-8
Novakov, C P; Feierman, D; Cederbaum, A I et al. (2001) An ESR and HPLC-EC assay for the detection of alkyl radicals. Chem Res Toxicol 14:1239-46
Caro, A A; Cederbaum, A I; Stoyanovsky, D A (2001) Oxidation of the ketoxime acetoxime to nitric oxide by oxygen radical-generating systems. Nitric Oxide 5:413-24
Sakurai, K; Stoyanovsky, D A; Fujimoto, Y et al. (2000) Mitochondrial permeability transition induced by 1-hydroxyethyl radical. Free Radic Biol Med 28:273-80
Puntarulo, S; Stoyanovsky, D A; Cederbaum, A I (1999) Interaction of 1-hydroxyethyl radical with antioxidant enzymes. Arch Biochem Biophys 372:355-9
Stoyanovsky, D A; Cederbaum, A I (1999) Metabolism of carbon tetrachloride to trichloromethyl radical: An ESR and HPLC-EC study. Chem Res Toxicol 12:730-6
Stoyanovsky, D A; Melnikov, Z; Cederbaum, A I (1999) ESR and HPLC-EC analysis of the interaction of hydroxyl radical with DMSO: rapid reduction and quantification of POBN and PBN nitroxides. Anal Chem 71:715-21
Stoyanovsky, D A; Wu, D; Cederbaum, A I (1998) Interaction of 1-hydroxyethyl radical with glutathione, ascorbic acid and alpha-tocopherol. Free Radic Biol Med 24:132-8
Puntarulo, S; Cederbaum, A I (1998) Production of reactive oxygen species by microsomes enriched in specific human cytochrome P450 enzymes. Free Radic Biol Med 24:1324-30

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