Effects of chronic ethanol administration on brain protein levels: A proteomic investigation using 2-D DIGE system
Introduction
The central nervous system (CNS) appears to be an especially sensitive target for ethanol with chronic alcohol administration inducing adaptive changes that are manifested as tolerance and physical dependence as well as resulting in neurotoxicity. The molecular mechanisms responsible for these changes have not been fully elucidated, but most likely involve alterations in protein activity, level of expression, intracellular localization or post-translational modification. Genomic studies have identified changes in the expression of a number of genes belonging to diverse functional groups after chronic ethanol exposure (Liu et al., 2004, Mayfield et al., 2002). Most pronounced and consistent changes were obtained in gene families encoding mitochondrial proteins, proteins involved in signal transduction and synaptic transmission, as well as myelin-related proteins (Liu et al., 2004, Mayfield et al., 2002, Sokolov et al., 2003). Identification of ethanol-sensitive genes is important for a complete understanding of the molecular effects of ethanol. However, differences in mRNA expression are not reliable predictors of differences in protein expression (Chen et al., 2002, Greenbaum et al., 2003, Gygi et al., 1999). Alcohol administration has been shown to alter protein translation without changing mRNA levels (Dodd and Lewohl, 1998). In addition, genomic studies do not provide insight into post-translational modifications of proteins. Thus, a necessary step in understanding the effects of chronic ethanol exposure requires characterizing the ethanol-induced protein changes.
Because of recent advances in electrophoresis and mass spectrometry, a more global investigation of the effects of chronic ethanol treatment on brain proteins (i.e., the brain proteome) is now technologically possible. In the present study, the Ettan 2-D fluorescence difference gel electrophoresis (DIGE) system and MALDI-TOF mass spectrometry were used to identify proteins whose level of expression was altered by chronic ethanol exposure. By incorporating the use of an internal standard and by allowing controls and experimental groups to be run on the same gel, the Ettan DIGE system reduces intergel variability and simplifies protein spot matching thus facilitating quantification of protein differences. Current studies were carried out using the long-fin striped strain of zebrafish Danio rerio. Zebrafish offer a number of benefits for studying the CNS effects of ethanol (Dlugos and Rabin, 2003, Gerlai et al., 2000). Besides advantages in cost and ease of breeding and maintenance, zebrafish are readily amenable to genetic manipulation. In addition, the DNA sequences of humans and zebrafish are similar (Barbazuk et al., 2000). Furthermore, zebrafish are sensitive to pharmacological concentrations of ethanol and exhibit strain differences in terms of sensitivity and tolerance to ethanol (Dlugos and Rabin, 2003).
Section snippets
Ethanol treatment and maintenance
The long-fin striped strain of zebrafish, D. rerio, were obtained from local suppliers (The Fish Place, North Tonawanda, NY, Blackwinds Pet Supply, Niagara Falls, NY, and Markheim Tropical Fish and Pet Store, Amherst, NY) and were housed in a 10-gal tank containing dechlorinated, filtered, tap water heated to 24 °C. Fish diet consisted of flake fish food supplemented with live brine shrimp. Chronic ethanol exposure was carried out by transferring the zebrafish to a 5-gal covered aquarium
Results
Normally, zebrafish demonstrate a swimming pattern in which they move in unison and appear clustered. However, after acute exposure to ethanol, they appeared less clustered with a larger distance between each fish and its nearest neighbor as well as occupying a greater area of the test tank (Dlugos and Rabin, 2003). After one week of ethanol treatment, there was a significant increase in the distance between each fish and its adjacent neighbor compared to the baseline (Fig. 1). However, with
Discussion
In the present study, LFS zebrafish were continually exposed to 0.5% (v/v) ethanol which has been shown to result in brain alcohol levels of 1.86 μg/mg wet weight (Dlugos and Rabin, 2003). This concentration of ethanol is well within the range of alcohol levels observed in humans (Redmond, 1983, Urso et al., 1981) and is at least comparable to the alcohol levels reported in several rodent studies (Carson and Pruett, 1996, Clemmesen and Hemmingsen, 1984, Lumeng et al., 1982, Majchrowicz, 1975,
Acknowledgements
We gratefully acknowledge the technical support provided by Maureen A. Adolf. This work was supported by the Interdisciplinary Creative Research Activities Fund from the University at Buffalo.
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