Department of Forensic Medicine, Faculty of Medicine, Kagawa Medical University, 1750-1, Ikenobe, Miki, Kita, Kagawa 761-0793, Japan
Received 12 March 2002; in revised form 12 August 2002; accepted 28 October 2002
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ABSTRACT |
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INTRODUCTION |
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AcH, the first metabolite of EtOH, is formed by the oxidation of EtOH, which is catalysed primarily by alcohol dehydrogenase (ADH) in the liver. AcH is a highly reactive compound, which can produce several neurochemical, behavioural and neurotoxic effects. It can adversely alter normal brain function by: (1) altering cellular function (Tabakoff et al., 1976); (2) altering the metabolism of biogenic amines (Truitt and Walsh, 1971
); (3) forming adducts with neurotransmitters (Israel et al., 1986
); (4) generating bioactive derivatives, such as tetrahydroisoquinolones and tetrahydropapaverine, after interaction with catecholamines (Bardsley and Tipton, 1980
). Genetically, a high accumulation of AcH may occur in the blood of some Asian populations with lower ALDH activity following EtOH ingestion, which may lead to individual discomfort and aversion to alcohol (Enomoto et al., 1991
).
Several in vitro and in vivo studies have suggested that AcH is formed by EtOH in the brain. Tabakoff et al.(1976) reported that all AcH that enters the brain is metabolized by ALDH when blood levels are below 70 µM. Sippel (1974)
noted that very high AcH concentrations (>50 µM) are needed in the blood before it can be detected in the brain. Another study by Westcott et al.(1980)
demonstrated that blood and brain levels of AcH were significantly correlated in rats treated with an ALDH inhibitor followed by EtOH. Heap et al.(1995)
argued that the circulating blood concentration of AcH was similar to that in the brain, and that AcH readily crosses the bloodbrain barrier. In contrast, Zimatkin (1991)
and Zimatkin et al.(1998)
found that blood AcH derived from the peripheral metabolism of EtOH penetrates into the brain with difficulty, due to the presence of a high concentration of ALDH at the bloodbrain barrier. Furthermore, it has been reported that EtOH is metabolized in vitro to AcH primarily by catalase in the brain (Gill et al., 1992
; Zimatkin et al., 1998
).
We investigated the in vivo formation of salsolinol in the rat striatum under conditions involving high AcH concentrations and the effects of EtOH and AcH on striatal dialysates of DA and also 5-hydroxytryptamine (5-HT) in freely moving rats after various treatments.
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MATERIALS AND METHODS |
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Reagents
Cyanamide (CY) and DA were purchased from Wako Pure Chemical Industries (Japan). Salsolinol, 5-HT and 4-methylpyrazole (4-MP) were purchased from Sigma Chemical Gmbh (Germany), and all other reagents used were the highest pure-grades available.
Microdialysis technique
After anaesthetizing each rat with pentobarbital-Na (50 mg/kg, intraperitoneally), the skull was exposed and a small hole was drilled to allow implantation of a guide cannula (8 mm long; AG-8, Eicom, Japan) into the rat striatum. Stereotaxic coordinates with respect to the microdialysis probe tip were as follows: anterior, 0.2 mm to the bregma; lateral, 3 mm; height, 3 mm below the dura (Paxinos and Watson, 1986). The guide cannula was anchored firmly to the skull surface in each rat by dental cement, and then a dummy cannula was inserted through the guide cannula for 1 day. A concentric dialysis probe (8 mm long, A-I-83, Eicom) with an active dialysis membrane (3 mm long, i.d. 0.20 mm, o.d. 0.22 mm, cut-off value 50 kDa) constructed from hemicellulose dialysis tubing was inserted into the guide cannula on the day of experimentation in unanaesthetized and freely moving rats. Ringers solution (147 mM NaCl, 4 mM KCl, 2.25 mM CaCl2) was perfused continually through the probes inlet at a constant flow rate of 0.8 µl/min during microdialysis. The animals were deprived of food throughout microdialysis.
Experimental groups
Six experimental groups were used in the present study: (1) a control group (EtOH, 1 g/kg); (2) CY alone; (3) CY + EtOH; (4) CY + 4-MP + EtOH; (5) 4-MP + EtOH; (6) 4-MP alone. Rats received an intraperitoneal injection of EtOH [20% (v/v), 1 g/kg] 1 h after administration of CY (50 mg/kg), 4-MP (82 mg/kg), or both.
High-performance liquid chromatographic (HPLC) conditions
Simultaneous quantification of DA, 5-HT and salsolinol in dialysate samples was performed using Eicom HPLC systems equipped with an electrochemical detector (ECD-300, Eicom) and an autosampler (EAS-20, Eicom). The perfused dialysates were collected every 5 min. The main operative conditions of HPLC were as follows: column (Eicom-PAK PP-ODS; 4.6 x 30 mm), oven temperature 25°C, detector, oxidation potential (+400 mV versus an Ag/AgCl reference analytical electrode), mobile phase: 0.1 M phosphate buffer [mixture of NaH2PO4 (15.6 g/l) and Na2HPO4 (14.2 g/l) solution, 100:16, pH 6], methanol (1%, v/v), EDTA-Na2 (50 mg/l), and decanesulphonate-Na (500 mg/l) at a flow rate of 0.5 ml/min. Injections were automatically performed onto the analytical column during the experimental period. The perfused dialysates were collected every 5 min by an autoinjector connected to an automated HPLC-ECD, and chromatograms were recorded with a chromatocorder 21 (Tosoh, Japan).
Quantification of EtOH and AcH
Quantification of EtOH and AcH in the blood were performed by the head-space GC method, according to our previous report (Kinoshita et al., 1995).
Histology
At the end of microdialysis, the rats were deeply anaesthetized by pentobarbital-Na and decapitated. The brains were removed for histological verification of the tip location of the probes by visual inspection under the dissecting microscope.
Statistical analysis
A statistical program of StatView (J-4.5; USA) was used for data analysis. Values are expressed as means ± SD. Statistical analysis of data was performed using Students t-test. Values of P < 0.05 were accepted as representing significant differences.
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RESULTS |
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DISCUSSION |
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In order to confirm our results, an additional series of experiments was designed, using CY + 4-MP or 4-MP with EtOH, 4-MP and CY, to investigate the release of salsolinol in the brain. In these experiments, an additional agent, 4-MP (a selective ADH inhibitor), was given to the animals and was necessary in order to alter certain behavioural and neurotoxic effects due to elevated AcH concentrations. Hence, most of the adverse effects that characterize alcohol sensitivity were efficiently attenuated by 4-MP (Kupari et al., 1983); this was demonstrated in cases of ALDH inhibition by CY as well as in individuals who carried the ALDH2*2 allele (Wilkin and Fortner, 1985
). However, we did observe that the accumulation of AcH was low in rats treated with CY + 4-MP following EtOH, whereas ALDH activity was inhibited by CY.
Acute intake of EtOH had no effect on salsolinol formation in the striatum (Sjoquist et al., 1983). One possible explanation may be that there was not a sufficient delay following ingestion, which would have led to a disturbance in the DA status and subsequent rise in salsolinol concentrations (Myers et al., 1985a
). A previous study has shown that the striatal salsolinol concentration increased significantly in rats exposed to AcH, whereas the DA concentrations did not differ remarkably after AcH treatment (Myers et al., 1985a
). These data suggest that AcH is the agent responsible for salsolinol formation in the brain. Therefore, any elevation in brain salsolinol concentrations might be due to an increase in brain AcH concentration. The results of our study supports this notion. To our knowledge, the present study represents the first report on in vivo salsolinol formation in freely moving rats.
ALDH is responsible not only for the metabolism of exogenous EtOH, but also for the oxidation of biogenic aldehydes in the central nervous system and in the periphery. These aldehydes are formed through oxidative deamination of biogenic amines by monoamine oxidase. CY, a potent ALDH inhibitor in the liver, as well as in the brain (Hellstrom and Tottmar, 1982), was used in the present experiments. The brain inhibition may alter the metabolism of biogenic amines by promoting the formation of condensation products or by increasing the levels of biogenic aldehydes. A previous in vivo report showed that extracellular concentration of both DA and 5-HT significantly decreased in the nucleus accumbens after acute intraperitoneal injection of AcH to rats (Ward et al., 1997
). Other authors have demonstrated, using rat models, that the DA levels in the striatum remained almost unchanged following chronic treatment with EtOH and AcH (Myers et al., 1985a
; Matsubara et al., 1987
) and that 5-HT levels also remained largely the same in the nucleus accumbens following acute exposure to EtOH (Heidbreder and De Witte, 1993
). On the other hand, salsolinol itself was shown to have no effect on the levels of DA and 5-HT in the striatum following acute exogenous administration (Antkiewicz-Michaluk et al., 2000
). In the present study, DA and 5-HT concentrations in the striatum did not significantly change. It is known that two opposing factors maintain extracellular DA concentration: (1) neuronal release of DA; and (2) subsequent uptake of DA via the DA-specific transporters. Salsolinol promotes the release of catecholamines into the nerve terminal, inhibition of reuptake of catecholamines, and inhibition of the enzymes MAO and COMT (Heikkila et al., 1971
; Giovine et al., 1976
; Weiner and Collins, 1978
). Our results do not support data supporting any of these mechanisms.
In conclusion, DA-derived salsolinol concentration in the striatum correlated well with the concentration of blood AcH in the present study. Changes in salsolinol concentrations following exposure to AcH do not alter DA and 5-HT concentrations in the rat striatum.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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