1 Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107,
2 Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206, USA and
3 Center for Gene Pharmacotherapy, Millennium Institute, University of Chile, Santiago, Chile
Received 17 January 2000; in revised form 19 April 2000; accepted 5 May 2000
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ABSTRACT |
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INTRODUCTION |
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Disulfiram has serious disadvantages, including a number of side-effects, such as severe hypotension, paraesthesias, and motor neuropathies (Gallant, 1987; Peachey and Annis, 1989
; Dupuy et al., 1995
), which markedly reduce compliance with self-administration. Brewer (1984) and Christensen et al. (1991) showed that when disulfiram is administered in tolerable doses, only 50% of patients develop the disulfiramethanol reaction.
In 7090% of humans, a single gene mutation protects against abuse of alcohol or alcoholism (Harada et al., 1982; Thomasson et al., 1991
; Higuchi, 1994
; Tu and Israel, 1995
). Such a gene encodes high-affinity ALDH2 (Yoshida et al., 1985
), a mitochondrial enzyme that metabolizes acetaldehyde, the first product of ethanol oxidation (Svanas and Weiner, 1985
; Cao et al., 1988
; Klyosov et al., 1996
). Means to specifically inhibit the transcription or translation of this gene on a long-term basis could be used as potential agents for the treatment of alcoholism.
The development of new aversive means to reduce alcohol consumption requires an experimental paradigm to test the drug-induced aversion to ethanol. We describe here studies designed to test the effect of an aversive medication.
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MATERIALS AND METHODS |
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Animals were administered disulfiram (100 mg/kg) or vehicle (between 09:00 and 10:00) for 4 days. On the evening of day 3, water was removed for 18 h, whereas food was provided ad libitum. On the morning of day 4, animals were dosed as described above, and 2 h later the animals were allowed access to a 6% (v/v) ethanol solution as the only drinking fluid. Consumption of the ethanol solution was measured at 1, 2, 3, 4, and 5 h. In some experiments, plasma acetaldehyde was determined at 0, 1, 3, 5, and 24 h after the animals were given access to 6% ethanol. For the latter studies, blood samples were removed from femoral catheters while the animals were freely consuming the ethanol solution.
Plasma acetaldehyde levels
Blood was collected in heparin, placed immediately on ice and centrifuged at 10 000 g for 5 min to harvest plasma for subsequent acetaldehyde determinations. Acetaldehyde was determined by high-pressure liquid chromatography (HPLC) using a modification of the method described by Lucas et al. (1986). Plasma (250 µl) was mixed with 4 ml of ice-cold perchloric acid (0.6 M in 0.15 M sodium chloride) and centrifuged at 10 000 g for 10 min at 4°C. Acetaldehyde present in the supernatant was derivatized by the addition of 2,4-dinitrophenylhydrazine. The absorbance of the eluted derivatives was monitored at 365 nm. A five-point standard curve with an acetaldehyde concentration range of 020 µM was prepared for HPLC analysis. All samples were quantified from a standard curve prepared on the same day the samples were extracted into iso-octane. The linear correlation of each of the standard curves was 0.991.
Determination of mitochondrial ALDH2 activity
Mitochondria from the livers collected from control and disulfiram-treated animals were isolated as described by Tank et al. (1981). Mitochondria were resuspended in 0.1 M sodium phosphate buffer, pH 7.4 and stored at 70°C until protein content and ALDH activities were determined. Prior to enzyme activity determinations, the thawed mitochondrial samples were incubated with Triton X-100 (2% v/v) at 37°C for 15 min to ensure complete lysis and release of ALDH.
The low Km ALDH activity in the isolated mitochondria was assayed as previously described by Tank et al. (1981) with minor modifications. The assay was performed at 37°C in 1.0 ml of reaction mixture, and was initiated by the addition of acetaldehyde to a final concentration of 10 µM. ALDH2 activity was determined by recording the change in absorption at 340 nm, due to NADH formation, with a Beckman DU®640 spectrophotometer. Protein concentration in the resuspended mitochondria was measured by the Micro BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA) as described by the manufacturer, with bovine serum albumin as the standard.
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RESULTS |
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DISCUSSION |
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The use of the limited access paradigm to assess the effect of aversive medication has resulted in conflicting data which also limits its use. In this paradigm, rats were trained for about 60 days to consume alcohol solutions which were available to the animal for 10 min/day. Cyanamide, an inhibitor of ALDH, increased, rather than decreased ethanol consumption in this model (Aragon et al., 1993).
The method used in the present study was to pair the putative dysphoric effects of the disulfiramethanol reaction with the taste of ethanol, a variant of the conditioned taste aversion paradigm in which a novel taste is paired with the administration of a noxious substance or a noxious condition (the ethanoldisulfiram reaction). It is well established (Nolan, et al., 1997; Scalera et al., 1997
; Barber et al., 1998
; Yasoshima and Yamamoto, 1998
) that a robust conditioned taste aversion quickly develops after the first pairing of the new taste with a noxious agent. In our studies, the effect was fully demonstrable in as little as 5 h following ethanol presentation, which allows fluid deprivation of the animals for slightly less than 24 h. Such a paradigm requires that: (i) ethanol be consumed for the first time by animals that have been pretreated with disulfiram; (2) enough ethanol be consumed by the animal in a short time to generate high blood acetaldehyde levels which lead to the malaise of the disulfiramethanol reaction; (3) ideally, an inbred strain be used to reduce variability and increase inter-laboratory reproducibility.
Data obtained after the disulfiram dosing regimen described here indicate that a strong aversion to ethanol is observed when plasma acetaldehyde levels are in the range of 4060 µM resulting from a reduction in mitochondrial ALDH activity of 87%. This reduction in activity is in line with the reduction of ALDH activity observed in human ALDH2-2/ALDH2-1 heterozygotes (Enomoto et al., 1991; Xiao et al., 1995
) who are markedly protected against alcohol abuse and alcoholism (Harada et al., 1982
; Thomasson et al., 1991
; Higuchi, 1994
; Tu and Israel, 1995
). In ALDH2-deficient individuals, the oral consumption of 0.5 g/kg ethanol yielded plasma acetaldehyde concentrations of 35.4 µM as compared to 2.1 µM in controls (Harada et al., 1981
).
Overall, studies presented here show that a simple animal model can be used to determine the aversion against ethanol elicited by drugs that reduce ALDH activity and which result in increases in acetaldehyde levels comparable to those seen in humans who consume alcohol and who carry an inactive ALDH allele.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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