Abstract
Ethanol is metabolized to acetaldehyde mainly by the alcohol dehydrogenase pathway and, to a lesser extent, through microsomal oxidation (CYP2E1) and the catalase-H2O2 system. Acetaldehyde, which is responsible for some of the deleterious effects of ethanol, is further oxidized to acetic acid by aldehyde dehydrogenases (ALDHs), of which mitochondrial ALDH2 is the most efficient. The aim of this study was to evaluate zebrafish (Danio rerio) as a model for ethanol metabolism by cloning, expressing, and characterizing the zebrafish ALDH2. The zebrafish ALDH2 cDNA was cloned and found to be 1892 bp in length and encoding a protein of 516 amino acids (Mr = 56,562), approximately 75% identical to mammalian ALDH2 proteins. Recombinant zebrafish ALDH2 protein was expressed using the baculovirus expression system and purified to homogeneity by affinity chromatography. We found that zebrafish ALDH2 is catalytically active and efficiently oxidizes acetaldehyde (Km = 11.5 μM) and propionaldehyde (Km = 6.1 μM). Similar kinetic properties were observed with the recombinant human ALDH2 protein, which was expressed and purified using comparable experimental conditions. Western blot analysis revealed that ALDH2 is highly expressed in the heart, skeletal muscle, and brain with moderate expression in liver, eye, and swim bladder of the zebrafish. These results are the first reported on the cloning, expression, and characterization of a zebrafish ALDH, and indicate that zebrafish is a suitable model for studying ethanol metabolism and, therefore, toxicity.
Footnotes
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This work was supported in part by National Institutes of Health Grants AA11885 and EY14390 (V.V.), AA12783 (R.L.T.), A T32 AA 07464 (A.P.), and a FPE Pre-doctoral Fellowship (T.E.).
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N.L. and T.E. equally contributed to this work.
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The GenBank accession numbers for the zebrafish and human ALDH2 cDNA are AAM19352 and AY621070, respectively.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.104.002964.
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ABBREVIATIONS: ADH, alcohol dehydrogenase; ALDH2, aldehyde dehydrogenase 2; FAS, fetal alcohol syndrome; 2-ME, 2-mercaptoethanol; PVDF, polyvinylidene difluoride; Sf9, Spodoptera frugiperda; RACE, rapid amplification of cDNA ends; BCA, bicinchoninic acid; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.
- Received November 10, 2004.
- Accepted February 7, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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MOLECULAR CLONING, BACULOVIRUS EXPRESSION, AND TISSUE DISTRIBUTION OF THE ZEBRAFISH ALDEHYDE DEHYDROGENASE 2
