Department of Legal Medicine and
3 Department of Ophthalmology, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo, 663-8501,
1 Department of Forensic Medicine, Kagawa Medical University, 1750-1, Miki, Kita, Kagawa, 761-0793,
2 Department of Legal Medicine, Nara Medical University, 840, Shijo-cho, Kashihara, Nara, 634-8521 and
4 Forensic Science Laboratory, Hyogo Prefectural Police Headquarters, 4-1, Shimoyamate-dori 5-chome, chuo-ku, kobe, 650-8510, Japan
Received 21 May 2001; in revised form 23 August 2001; accepted 22 September 2001
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ABSTRACT |
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INTRODUCTION |
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High and low alcohol preference rodents, developed by selective breeding, are widely used as animal models in the study of alcoholism (Li et al., 1994). There have been many studies performed to assess differences in alcohol preference in these rodents. Previously, there was a report that acetaldehyde concentration following ethanol injection in an alcohol avoidance strain is higher than that of an alcohol preference strain of mice (Sheppard et al., 1970
). In the AA/ANA rat model developed in Alko by Eriksson (1968), it has been reported that the blood concentration of acetaldehyde following ethanol administration is significantly higher in the ANA rat than in the AA rat (Eriksson, 1973
; Koivisto et al., 1993
). It has been reported that liver ALDH activity in the ANA rat is significantly lower than that of the AA rat (Eriksson, 1973
; Koivula et al., 1975
; Koivisto and Eriksson, 1994
). However, the polymorphism of ALDH2 was not associated with alcohol preference in the AA/ANA model (Koivisto et al., 1993
).
Recently, high alcohol-preferring rat lines (HAP) and low alcohol-preferring rat lines (LAP) were newly developed from the Wistar rat colony by Hishida (1996), and differences in ALDH activity and in cytosolic ALDH (ALDH1) polymorphism have been observed between these two lines. The activity of liver ALDH in HAP rats was higher than that of LAP rats (Hishida, 1996). Three phenotypes of ALDH1, termed AA, AC and CC type, have been revealed by the band pattern of isoelectric focusing (Negoro et al., 1997
). The AA type was observed in almost all HAP rats, and almost all LAP rats were CC type (Hishida, 1996
). It has also been reported that pharmacological effects of methamphetamine may be influenced by ethanol preference in this line. The HAP rats showed significantly lower dopamine and serotonin release in the striatum and nucleus accumbens than LAP rats following methamphetamine administration (Yamauchi et al., 2000
). However, there is little information on possible differences in acetaldehyde concentration between these two rat lines following ethanol administration (Hishida, 1996
). The present study was designed to investigate differences in ethanol metabolism in vivo between the HAP and LAP lines.
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MATERIALS AND METHODS |
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The alcohol-naive male rats used in this experiment aged from the 22nd24th generation of the HAP line and the 26th28th generation of the LAP line, with a weight range of 350400 g. All animals were housed with a 12-h light/12-h darkness cycle in a temperature- and humidity-controlled environment with free access to food and water. A venous cannula was implanted in the jugular vein in each animal 1 day prior to the experiment, under pentobarbital anaesthesia (50 mg/kg, intraperitoneally). Three experimental groups were employed in each of the rat lines. Rats received an intravenous (i.v.) injection of ethanol at three treatment levels, i.e. 2.0, 3.5 and 5.0 g/kg [in the form of a 20% (v/v) ethanol solution in 0.15 M NaCl]. Ethanol was infused for 10 min at a steady rate. Blood samples (500 µl) were taken immediately prior to i.v. injection of ethanol at t = 0, 30, 60 and 120 min following administration of ethanol. After each blood sampling, an equal volume of heparinized saline was infused. The concentrations of blood ethanol and acetaldehyde were measured by headspace gas chromatography, as previously described (Okada and Mizoi, 1982). Acetaldehyde was purchased from Merck (Munich, Germany). All other reagents were purchased from Wako Pure Chemicals (Osaka, Japan). Following treatment, rats were killed by decapitation and their livers were removed immediately. The preparation of subcellular fractions and isoelectric focusing were performed, as previously described (Negoro et al., 1997
), to identify the typing of the liver cytosolic ALDH1 polymorphism.
Data are expressed as mean ± SD. Statistical analysis of the data was performed by one-way analysis of variance followed by the Fisher PLSD test. P < 0.05 was considered statistically significant. This study was approved by the Animal Investigation Committee, Hyogo College of Medicine.
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RESULTS |
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DISCUSSION |
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The difference in acetaldehyde levels following ethanol administration has also been observed in other alcohol-preferring lines, such as AA and ANA following 1.5 g/kg ethanol administration (Eriksson, 1973; Koivisto et al., 1993
). In our experiments, three different doses of ethanol were employed, including 2.0, 3.5 and 5.0 g/kg, and a difference was noted in lineages in the latter two treatment groups. In comparing the HAP and LAP lines, differences in ALDH activity have already been reported (Hishida, 1996
), and genetic differences associated with the ALDH1 phenotype have been demonstrated previously (Negoro et al., 1997
). In humans and rats, a mitochondrial low Km ALDH (ALDH2) plays a major role in acetaldehyde metabolism in vivo (Eriksson et al., 1975
; Svanas and Weiner, 1985
; Eckardt et al., 1998
). However, the cytosolic ALDH1 in the rat significantly differs from that in the human. The reported Km value of rat ALDH1 for acetaldehyde is 15 ± 3 µM, which is relatively lower than that in humans (Klyosov et al., 1996
), suggesting that in the human liver, mitochondrial ALDH2 mainly oxidizes acetaldehyde at physiological concentrations, whereas in the rat liver, both mitochondrial and cytosolic ALDH are functional (Klyosov et al., 1996
).
In our experiments, the line difference in blood acetaldehyde concentration was clearly observed. As this difference was observed at a high dose of ethanol, it may reflect the difference in the high Km ALDH (ALDH1) activities due to the different phenotypes in HAP/LAP lines. However, the doses of ethanol used were relatively higher than that consumed by voluntary drinking, and the peak concentrations of acetaldehyde following i.v. administration of ethanol were therefore higher than that after voluntary drinking. From the present and previous results, we conclude that ALDH1 polymorphism may be a major factor in acetaldehyde accumulation in HAP/ LAP lines, but is unlikely to explain their different alcohol preferences.
Although there are several reports that alcohol preference may correlate with ALDH activity more in the brain than in the liver (Amir, 1978; Socaransky et al., 1984
), this mechanism is still relatively unknown. There is a report that ALDH is involved in biogenic amine metabolism (Berger and Weiner, 1977
). However, oxidation of biogenic aldehydes occurs in mitochondria, and the physiological role of cytosolic ALDH1 is still unknown (Tank et al., 1986
). Under basal conditions, there was no difference in the extracellular contents of biogenic amines, such as dopamine and serotonin, in striatum and nucleus accumbens between HAP and LAP lines (Yamauchi et al., 2000
). As acetaldehyde itself has many pharmacological actions (Brien and Loomis, 1983
), it may act on the central nervous system (Kinoshita et al., 2001
), where differences in acetaldehyde elimination may contribute to ethanol preference. However, as we have not yet investigated brain ALDH activity nor metabolites of biogenic amines in both rat lines, further studies are needed to clarify this hypothesis.
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FOOTNOTES |
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