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Comparative analysis of hepatic ethanol metabolism in Fawn-Hooded and Wistar-Kyoto rats
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Alcohol 30 (2003) 75–79
Brief communication
Comparative analysis of hepatic ethanol metabolism in Fawn-Hooded and Wistar-Kyoto rats Daniel J. Lodge*, Andrew J. Lawrence Department of Pharmacology, Monash University, Box 13E, Clayton, Victoria 3800, Australia Received 7 March 2002; received in revised form 13 March 2003; accepted 22 March 2003
Abstract Results of a number of studies have supported the suggestion that a correlation exists between voluntary ethanol consumption and enhanced ethanol metabolism in some (but not all) rodent strains. However, as yet, the capacity for alcohol-preferring Fawn-Hooded (FH) rats to metabolize ethanol has not been investigated. Hence, the aim of the current study was to compare the activities of the major hepatic enzymes involved in ethanol metabolism—cytosolic alcohol dehydrogenase (ADH) and mitochondrial aldehyde dehydrogenase (ALDH)— in the FH rat and its alcohol-nonpreferring counterpart, the Wistar-Kyoto (WKY) rat. In addition, the effect of chronic (5 weeks in vivo) ethanol pretreatment on the activity of these enzymes was investigated. Alcohol-naive FH rats were found to have significantly higher ADH activity (⫹61%) and no significant change in ALDH activity when compared with findings for WKY rats. In addition, chronic ethanol self-administration produced a small increase in ADH activity (⫹14%) in WKY rats only. Taken as a whole, these findings are the first to demonstrate an increased in vitro hepatic ethanol metabolism in alcohol-preferring FH rats and further demonstrate an association between hepatic ethanol metabolism and voluntary ethanol self-administration in rodents. 쑖 2003 Elsevier Inc. All rights reserved. Keywords: ADH; ALDH; Ethanol metabolism; Liver; Fawn-Hooded rat
1. Introduction The major metabolic pathway for the elimination of ethanol in the rat occurs in the liver through a two-step process. The first, and rate-limiting, step is the oxidation of ethanol by alcohol dehydrogenase [alcohol: oxidized form of nicotinamide adenine dinucleotide (NAD⫹) oxidoreductase, EC 1.1.1.1 (ADH)] in a reaction that liberates acetaldehyde, a highly toxic intermediate that under normal conditions is immediately converted to acetate by the enzyme aldehyde dehydrogenase [aldehyde: NAD⫹ oxidoreductase, EC 1.2.1.3 (ALDH)] (Isselbacher & Greenberger, 1964; Westerfeld, 1961). Results of early studies, in which the genetic predisposition of rodents to consume ethanol was investigated, supported the suggestion of a correlation between voluntary ethanol consumption and enhanced ethanol metabolism (McClearn et al., 1964; Petrova, 1985; Sheppard et al., 1968). This has since been demonstrated to be true for some (but not all) alcohol-preferring rodent models. The majority of these studies have concentrated on the increased ADH
* Corresponding author. Tel.: ⫹61-3-99054688; fax: ⫹61-3-99055851. E-mail address: [email protected] (D.J. Lodge). Editor: T.R. Jerrells 0741-8329/03/$ – see front matter 쑖 2003 Elsevier Inc. All rights reserved. doi: 10.1016/S0741-8329(03)00097-1
activity of alcohol-preferring C57BL6 mice (compared with findings for alcohol-nonpreferring DBA mice) (McClearn et al., 1964; Sheppard et al., 1968), whereas results of other studies have demonstrated a correlation between ethanol preference and ADH activity in albino rats (Petrova, 1985). In addition, there have been a number of studies whose results support the suggestion that reduced ALDH activity may be associated with ethanol avoidance (Koivisto & Eriksson, 1994; Sheppard et al., 1968, 1970). Fawn-Hooded (FH) rats are an inbred strain that have a high preference (70%–80%) for ethanol over tap water in a two-bottle, free-choice paradigm (Rezvani et al., 2002), and, for this reason, there has been extensive research into the neurochemical aspects pertaining to ethanol reward in these animals (Chen et al., 1998; Cowen et al., 1998; Lodge et al., 2000). However, as yet the capacity for FH rats to metabolize ethanol has not been examined. Therefore, the aim of the current study was to investigate the activity of the two major components of ethanol metabolism (hepatic, cytosolic ADH and mitochondrial ALDH) in both alcoholnaive FH and Wistar-Kyoto (WKY) rats. The WKY rats are used routinely as a control for FH rats because they are both inbred strains derived from common ancestry (Chen et al., 1998; Lodge et al., 2000). In addition, the effect of chronic (5 weeks in vivo) ethanol consumption on the activity of these two enzymes in both rat strains was examined.
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D.J. Lodge, A.J. Lawrence / Alcohol 30 (2003) 75–79
2. Methods 2.1. Animals All experiments were performed in accordance with the Prevention of Cruelty to Animals Act 1986 under the guidelines of the National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia. A total of 22 rats were used in the current study. All rats were housed in the Department of Pharmacology, Monash University animal house and kept at constant temperature and humidity, with lights on from 0700 to 1900 and free access to food (standard rat chow) and water. The WKY rats were obtained from the Biological Research Laboratories, Austin Hospital (Australia), whereas the FH rats were obtained from a colony bred at Central Animal Services, Monash University (Australia) with the use of the breeding stock received from Dr. Amir Rezvani, University of North Carolina, Chapel Hill, North Carolina (USA). 2.2. Chronic ethanol consumption Alcohol-naive, male, WKY (n ⫽ 4; weight for each, 320 ⫾ 11 g; age, 109 ⫾ 2 days old) and FH (n ⫽ 4; weight for each, 306 ⫾ 21 g; age, 107 ⫾ 2 days old) rats received tap water ad libitum. A second group of WKY (n ⫽ 4; weight for each, 330 ⫾ 9 g; age, 113 ⫾ 3 days old) and FH (n ⫽ 4; weight for each, 344 ⫾ 10 g; age, 118 ⫾ 2 days old) rats were individually housed, and an artificially sweetened ethanol solution [10% volume/volume (vol./vol.)], rather than water, was provided ad libitum for 5 weeks. Additional rats (n ⫽ 3 per strain) received only artificially sweetened tap water for 5 weeks. 2.3. Liver preparation Rats were killed by decapitation at the same time of the day to avoid circadian variations (∼0900). Their livers were dissected out on ice and rinsed with a physiological saline solution [(PSS); composition in mM: NaCl, 118.0; KCl, 4.7; NaH2PO4, 1.0; MgCl2, 1.2; CaCl2, 1.3; NaHCO3, 25.0; and ethylenediaminetetraacetic acid (EDTA), 0.04] and weighed (wet). Each liver was divided into four pieces and incubated for 1 h at 37ºC in 20 ml of carbogenated PSS. Each portion was then chopped into 0.5 × 0.5-mm pieces with a McIlwain tissue chopper before being homogenized with a Potter-Eveljhem Teflon/glass homogenizer in 25 ml of ice-cold sucrose buffer (0.25 M sucrose, 5 mM TRIS, 0.5 mM EDTA, and 0.5 mM dithiothreitol; pH ⫽ 7.5). The homogenates were centrifuged (4ºC) at 700g for 10 min to remove cellular debris, and the resultant supernatant was then centrifuged (4ºC) at 10,000g for 30 min. The pellet (mitochondrial fraction) was resuspended in 10 ml of pyrophosphate buffer (50 mM sodium pyrophosphate; pH ⫽ 8.8). The supernatant was centrifuged (4ºC) at 48,000g for 1 h, with the resultant supernatant being the cytosolic fraction.
The protein concentration of each sample (mitochondrial and cytosolic) was then determined in duplicate by the method of Lowry et al. (1951) with the use of bovine serum albumin as a standard. Samples were stored at ⫺80ºC overnight before determination of enzyme activity. 2.4. Alcohol dehydrogenase assay The activity of hepatic ADH was determined by the reduced form of nicotinamide adenine dinucleotide (NADH)– induced increase in absorbance at 340 nm on a Novaspec spectrophotometer essentially as previously reported (Trave´s et al., 1995). In brief, the assay was performed in duplicate in a final volume of 2.5 ml at 37ºC in glycine buffer (0.1 M glycine; pH ⫽ 10.0) containing 2.4 mM β-NAD⫹ and cytosolic fraction corresponding to 1 mg of protein. After 5 min of equilibration, the reaction was initiated by the addition of ethanol (10 µM–100 mM) and incubated for 5 min before the increase in absorbance was measured relative to a blank reaction (no ethanol added). The effect of pyrazole (10 mM) on the response to 10 mM ethanol was examined to confirm ADH activity. 2.5. Aldehyde dehydrogenase assay The activity of hepatic ALDH was determined by the NADH-induced increase in absorbance at 340 nm on a Novaspec spectrophotometer, essentially as previously reported (Tottmar et al., 1973). In brief, the assay was performed in duplicate in a final volume of 2.5 ml at room temperature in pyrophosphate buffer (50 mM sodium pyrophosphate; pH ⫽ 8.8) containing 1.5 mM β-NAD⫹; pyrazole (0.1 mM), to inhibit ADH activity; rotenone (2 µM in dimethylsulfoxide: 0.2% of final volume), to inhibit mitochondrial NADH oxidase; sodium deoxycholate [0.01% weight/volume (wt./ vol.)], to release latent activity and to increase clarity of the solution for spectrophotometric analysis; and mitochondrial fraction corresponding to 1 mg of protein. After 5 min of equilibration, the reaction was initiated by the addition of acetaldehyde (100 µM–1 M) and incubated in a sealed spectrophotometric tube (to minimize acetaldehyde evaporation) for 15 min before the increase in absorbance was measured relative to a blank reaction (no acetaldehyde added). The effect of disulfiram (0.1 mM in ethanol: 0.2% of final volume) on the response to 10 mM acetaldehyde was examined to confirm ALDH activity. 2.6. Data analysis All increases in absorbance were converted to nanomoles of NADH formed per minute per milligram of protein relative to a β-NADH standard curve (β-NADH dissolved and diluted in 10 mM NaOH). All data are represented as mean ⫾ S.E.M. All curves were analyzed by a two-way analysis of variance (ANOVA), followed by either a Student– Newman–Keuls (for strain comparisons) or a Dunnett post
D.J. Lodge, A.J. Lawrence / Alcohol 30 (2003) 75–79
hoc test. All statistics were calculated by using the SigmaStat software program (SPSS, Chicago, IL, USA). 2.7. Materials β-Nicotinamide adenine dinucleotide (β-NAD⫹), βnicotinamide adenine dinucleotide reduced form (β-NADH), bovine serum albumin, Folin and Ciocalteu’s phenol reagent, rotenone, glycine, and dithiothreitol were purchased from Sigma (St. Louis, MO, USA), whereas pyrazole was obtained from Aldrich (Milwaukee, WI, USA). Deoxycholic acid was purchased from Calbiochem (San Diego, CA, USA), and ethanol was obtained from the Commonwealth Serum Laboratories (Parkville, Victoria, Australia). Acetaldehyde was obtained from MERCK (Munich, Bavaria, Germany), and disulfiram was a gift from Lederle Labs (Philadelphia, PA, USA).
3. Results Spectrophotometric analysis of cytosolic ADH and mitochondrial ALDH activity revealed a concentration-related increase in absorbance in both rat strains (Fig. 1). Comparison of these curves revealed significant differences between ethanol-naive FH and WKY rats, with FH rats having significantly increased ADH activity (⫹61%), but not ALDH activity, when compared with findings for WKY rats (Fig. 1). Chronic ethanol intake in vivo over a 5-week period was constant, with an ethanol intake of 8.8 ⫾ 1.0 g/kg/day for FH rats and 5.5 ⫾ 0.8 g/kg/day for WKY rats. This produced an increase in ADH activity in WKY rats only (⫹14%; P ⬍ .05, two-way ANOVA, followed by Student–Newman– Keuls test), whereas no significant effect on ALDH activity was observed (Fig. 2). To confirm the selectivity of each assay, the effect of pyrazole and disulfiram on the maximum effect of ethanol and acetaldehyde, respectively, was examined. Pyrazole
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(10 mM) was able to completely inhibit maximal ADH activity (WKY rats: ⫺99.5 ⫾ 0.3%; FH rats: ⫺99.2 ⫾ 0.4%; n ⫽ 11; P ⬍ .05, paired Student t test), whereas disulfiram (0.1 mM) significantly attenuated maximal ALDH activity (WKY rats: ⫺36.7 ⫾ 9.2%; FH rats: ⫺40.4 ⫾ 4.6%; n ⫽ 11; P ⬍ .05, paired Student t test). In addition, comparison of liver weight, relative to body weight, revealed no significant differences, either between rat strains or owing to chronic ethanol consumption (Table 1).
4. Discussion The current study is the first to examine the process of hepatic ethanol metabolism in the alcohol-preferring FH rat. The results have revealed significant strain differences, with ethanol-naive FH rats possessing significantly higher ADH activity than observed for WKY rats. Because it has been shown that ADH activity correlates well with ethanol elimination rates in vivo (Lumeng et al., 1979), these findings support the suggestion that FH rats have a higher metabolic capacity for ethanol than that of their alcohol-nonpreferring counterparts. These findings are in accord with results of earlier studies, demonstrating increased ADH activity in the livers of alcohol-preferring C57BL6 mice (when compared with findings for alcohol-nonpreferring DBA mice) (McClearn et al., 1964; Sheppard et al., 1968), and with results of more recent studies, demonstrating that albino rats with high ethanol preference display higher ADH activity than that for rats with low ethanol preference (Petrova, 1985). The correlation between increased ADH activity (ethanol metabolism) and voluntary ethanol consumption would be easy to explain if these rats were drinking to their metabolic capacity. However, this is not the case because it has been shown that voluntary ethanol intake in C57BL6 mice (Rodgers, 1966) and FH rats (unpublished observations,
Fig. 1. Effect of increasing substrate concentrations on the activity of cytosolic alcohol dehydrogenase (A) and mitochondrial aldehyde dehydrogenase (B) throughout the livers of alcohol-naive Fawn-Hooded (■) and Wistar-Kyoto (▲) rats. *Denotes statistically significant difference compared with findings for Wistar-Kyoto rats (P ⬍ .05, two-way analysis of variance, followed by Student–Newman–Keuls test; n ⫽ 4). NADH ⫽ Reduced form of nicotinamide adenine dinucleotide.
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Fig. 2. Activity of cytosolic alcohol dehydrogenase (ADH) and mitochondrial aldehyde dehydrogenase (ALDH) in Fawn-Hooded (A) and Wistar-Kyoto (B) rats chronically consuming sweetened ethanol (ADH ●, ALDH 䊐) or vehicle (ADH ■, ALDH ×). *Denotes statistically significant difference compared with findings for water-only group (P ⬍ .05, two-way analysis of variance, followed by Student–Newman–Keuls test; n ⫽ 3 or 4). NADH ⫽ Reduced form of nicotinamide adenine dinucleotide.
D.J. Lodge and A.J. Lawrence, 2002) can be significantly increased by artificially sweetening the ethanol solution. Therefore, FH rats are not simply drinking to their metabolic capacity, and the correlation that exists between ethanol intake and ADH activity is not likely to be causal. Hence, it seems that constant inbreeding and ethanol exposure (to previous generations) over a long period has resulted in genetic adaptation to ethanol consumption in the form of increased ADH activity. There has also been a suggestion that altered ALDH activity may play a role in ethanol preference because this enzyme is responsible for the removal of acetaldehyde (Isselbacher & Greenberger, 1964; Westerfeld, 1961). However, because ADH is the rate-limiting enzyme in ethanol metabolism (Isselbacher & Greenberger, 1964), a substantial decrease in ALDH activity is required to result in significant acetaldehyde accumulation. Because only a slight decrease was observed in the current study, it is unlikely that the alcohol-avoiding phenotype of the WKY rat is a result of acetaldehyde accumulation. In the current study, we also examined the effect of chronic (5 weeks in vivo) ethanol self-administration on the activity of both ADH and ALDH. Many investigators have reported an induction of ethanol metabolism after chronic ethanol consumption (Lieber & DeCarli, 1970, 1972; Matsuzaki et al., 1981). However, in the WKY rats in the current study, only a small increase in ADH activity (⫹14%) was observed. Another system that is involved in the elimination of ethanol in vivo is the microsomal ethanol oxidizing
Table 1 Relative liver weights (grams per kilogram of body weight) of Fawn-Hooded and Wistar-Kyoto rats receiving no treatment, chronic (5 weeks in vivo) ethanol pretreatment, or vehicle Rat strain
Control
Vehicle
Chronic ethanol
Wistar-Kyoto Fawn-Hooded
36.1 ⫾ 1.7 39.3 ⫾ 0.8
34.4 ⫾ 1.4 38.7 ⫾ 2.3
33.0 ⫾ 1.2 40.2 ⫾ 0.8
Data are presented as mean ⫾ S.E.M.; n ⫽ 3 or 4 per group.
system (MEOS). It is distinguished from ADH by co-factor requirements [reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) instead of NAD⫹) and, under normal conditions, has been suggested to account for 20%– 25% of total ethanol metabolism (Lieber & DeCarli, 1972; Matsuzaki et al., 1981). It has been documented that, although chronic ethanol administration causes a substantial increase in ethanol metabolism, the major changes are associated with an induction of the MEOS because alterations in ADH activity are relatively small (Lieber & DeCarli, 1970, 1972; Matsuzaki et al., 1981). Although results of the current study demonstrate that ADH activity is only marginally increased by chronic ethanol consumption, the effect on total ethanol metabolism cannot be determined because the activity of other (albeit relatively minor) pathways such as MEOS was not determined. In conclusion, results of the current study have demonstrated a significant difference between the alcohol-preferring FH and alcohol-nonpreferring WKY rat strains with regard to the capacity of hepatic ADH in vitro. More specifically, significantly increased ADH activity seen in the alcoholnaive FH rat parallels that observed in other alcohol-preferring rodent models, further suggesting a correlation between ADH activity and ethanol consumption. However, it seems unlikely that this is a causal relation. Rather, increased ADH activity is likely to be a genetic adaptation to ethanol consumption that has developed over a number of years of inbreeding. Acknowledgments This work was supported by the Australian Brewers’ Foundation and the National Health and Medical Research Council (Australia), of which A.J. Lawrence is a Senior Research Fellow. References Chen, F., Rezvani, A., Jarrott, B., & Lawrence, A. J. (1998). Distribution of GABAA receptors in the limbic system of alcohol-preferring and
D.J. Lodge, A.J. Lawrence / Alcohol 30 (2003) 75–79 non-preferring rats: in situ hybridisation histochemistry and receptor autoradiography. Neurochem Int 32, 143–151. Cowen, M., Chen, F., Jarrott, B., & Lawrence, A. J. (1998). Effects of acute ethanol on GABAA release and GABAA receptor density in the rat mesolimbic system. Pharmacol Biochem Behav 59, 51–57. Isselbacher, K. J., & Greenberger, N. J. (1964). Metabolic effect of alcohol on the liver. N Engl J Med 270, 351–356. Koivisto, T., & Eriksson, C. J. (1994). Hepatic aldehyde and alcohol dehydrogenases in alcohol-preferring and alcohol-avoiding rat lines. Biochem Pharmacol 48, 1551–1558. Lieber, C. S., & DeCarli, L. M. (1970). Hepatic microsomal ethanoloxidizing system. In vitro characteristics and adaptive properties in vivo. J Biol Chem 245, 2505–2512. Lieber, C. S., & DeCarli, L. M. (1972). The role of the hepatic microsomal ethanol oxidizing system (MEOS) for ethanol metabolism in vivo. J Pharmacol Exp Ther 181, 279–287. Lodge, D. J., Short, J. L., Mercer, L. D., Beart, P. M., & Lawrence, A. J. (2000). CCK/dopamine interactions in Fawn-Hooded and Wistar-Kyoto rat brain. Peptides 21, 379–386. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J Biol Chem 193, 265– 275. Lumeng, L., Bosron, W.F., & Li, T.-K. (1979). Quantitative correlation of ethanol elimination rates in vivo with liver alcohol dehydrogenase activities in fed, fasted and food-restricted rats. Biochem Pharmacol 28, 1547–1551.
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Matsuzaki, S., Gordon, E., & Lieber, C. S. (1981). Increased alcohol dehydrogenase independent ethanol oxidation at high ethanol concentrations in isolated rat hepatocytes: the effect of chronic ethanol feeding. J Pharmacol Exp Ther 217, 133–137. McClearn, G. E., Bennett, E. L., Hebert, M., Kakihana, R., & Schlesinger, K. (1964). Alcohol dehydrogenase activity and previous ethanol consumption in mice. Nature 203, 793–794. Petrova, M. A. (1985). Serum and liver alcohol dehydrogenase levels in rats differing in alcohol motivation. Byulleten’ Eksperimental’noi Biologii i Meditsiny 100, 583–584. Rezvani, A. H., Parsian, A., & Overstreet, D. H. (2002). The FawnHooded (FH/Wjd) rat: a genetic animal model of comorbid depression and alcoholism. Psychiatr Genet 12, 1–16. Rodgers, D. A. (1966). Factors underlying differences in alcohol preference among inbred strains of mice. Psychosom Med 28, 498–513. Sheppard, J. R., Albersheim, P., & McClearn, G. E. (1968). Enzyme activities and ethanol preference in mice. Biochem Genet 2, 205–212. Sheppard, J. R., Albersheim, P., & McClearn, G. (1970). Aldehyde dehydrogenase and ethanol preference in mice. J Biol Chem 245, 2876–2882. Tottmar, S. O., Pettersson, H., & Kiessling, K. H. (1973). The subcellular distribution and properties of aldehyde dehydrogenases in rat liver. Biochem J 135, 577–586. Trave´s, C., Camps, L., & Lo´pez-Tejero, D. (1995). Liver alcohol dehydrogenase activity and ethanol levels during chronic ethanol intake in pregnant rats and their offspring. Pharmacol Biochem Behav 52, 93–99. Westerfeld, W. W. (1961). The intermediary metabolism of alcohol. Am J Clin Nutr 9, 426–430.
Alcohol 30 (2003) 75–79
Brief communication
Comparative analysis of hepatic ethanol metabolism in Fawn-Hooded and Wistar-Kyoto rats Daniel J. Lodge*, Andrew J. Lawrence Department of Pharmacology, Monash University, Box 13E, Clayton, Victoria 3800, Australia Received 7 March 2002; received in revised form 13 March 2003; accepted 22 March 2003
Abstract Results of a number of studies have supported the suggestion that a correlation exists between voluntary ethanol consumption and enhanced ethanol metabolism in some (but not all) rodent strains. However, as yet, the capacity for alcohol-preferring Fawn-Hooded (FH) rats to metabolize ethanol has not been investigated. Hence, the aim of the current study was to compare the activities of the major hepatic enzymes involved in ethanol metabolism—cytosolic alcohol dehydrogenase (ADH) and mitochondrial aldehyde dehydrogenase (ALDH)— in the FH rat and its alcohol-nonpreferring counterpart, the Wistar-Kyoto (WKY) rat. In addition, the effect of chronic (5 weeks in vivo) ethanol pretreatment on the activity of these enzymes was investigated. Alcohol-naive FH rats were found to have significantly higher ADH activity (⫹61%) and no significant change in ALDH activity when compared with findings for WKY rats. In addition, chronic ethanol self-administration produced a small increase in ADH activity (⫹14%) in WKY rats only. Taken as a whole, these findings are the first to demonstrate an increased in vitro hepatic ethanol metabolism in alcohol-preferring FH rats and further demonstrate an association between hepatic ethanol metabolism and voluntary ethanol self-administration in rodents. 쑖 2003 Elsevier Inc. All rights reserved. Keywords: ADH; ALDH; Ethanol metabolism; Liver; Fawn-Hooded rat
1. Introduction The major metabolic pathway for the elimination of ethanol in the rat occurs in the liver through a two-step process. The first, and rate-limiting, step is the oxidation of ethanol by alcohol dehydrogenase [alcohol: oxidized form of nicotinamide adenine dinucleotide (NAD⫹) oxidoreductase, EC 1.1.1.1 (ADH)] in a reaction that liberates acetaldehyde, a highly toxic intermediate that under normal conditions is immediately converted to acetate by the enzyme aldehyde dehydrogenase [aldehyde: NAD⫹ oxidoreductase, EC 1.2.1.3 (ALDH)] (Isselbacher & Greenberger, 1964; Westerfeld, 1961). Results of early studies, in which the genetic predisposition of rodents to consume ethanol was investigated, supported the suggestion of a correlation between voluntary ethanol consumption and enhanced ethanol metabolism (McClearn et al., 1964; Petrova, 1985; Sheppard et al., 1968). This has since been demonstrated to be true for some (but not all) alcohol-preferring rodent models. The majority of these studies have concentrated on the increased ADH
* Corresponding author. Tel.: ⫹61-3-99054688; fax: ⫹61-3-99055851. E-mail address: [email protected] (D.J. Lodge). Editor: T.R. Jerrells 0741-8329/03/$ – see front matter 쑖 2003 Elsevier Inc. All rights reserved. doi: 10.1016/S0741-8329(03)00097-1
activity of alcohol-preferring C57BL6 mice (compared with findings for alcohol-nonpreferring DBA mice) (McClearn et al., 1964; Sheppard et al., 1968), whereas results of other studies have demonstrated a correlation between ethanol preference and ADH activity in albino rats (Petrova, 1985). In addition, there have been a number of studies whose results support the suggestion that reduced ALDH activity may be associated with ethanol avoidance (Koivisto & Eriksson, 1994; Sheppard et al., 1968, 1970). Fawn-Hooded (FH) rats are an inbred strain that have a high preference (70%–80%) for ethanol over tap water in a two-bottle, free-choice paradigm (Rezvani et al., 2002), and, for this reason, there has been extensive research into the neurochemical aspects pertaining to ethanol reward in these animals (Chen et al., 1998; Cowen et al., 1998; Lodge et al., 2000). However, as yet the capacity for FH rats to metabolize ethanol has not been examined. Therefore, the aim of the current study was to investigate the activity of the two major components of ethanol metabolism (hepatic, cytosolic ADH and mitochondrial ALDH) in both alcoholnaive FH and Wistar-Kyoto (WKY) rats. The WKY rats are used routinely as a control for FH rats because they are both inbred strains derived from common ancestry (Chen et al., 1998; Lodge et al., 2000). In addition, the effect of chronic (5 weeks in vivo) ethanol consumption on the activity of these two enzymes in both rat strains was examined.
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D.J. Lodge, A.J. Lawrence / Alcohol 30 (2003) 75–79
2. Methods 2.1. Animals All experiments were performed in accordance with the Prevention of Cruelty to Animals Act 1986 under the guidelines of the National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Experimental Purposes in Australia. A total of 22 rats were used in the current study. All rats were housed in the Department of Pharmacology, Monash University animal house and kept at constant temperature and humidity, with lights on from 0700 to 1900 and free access to food (standard rat chow) and water. The WKY rats were obtained from the Biological Research Laboratories, Austin Hospital (Australia), whereas the FH rats were obtained from a colony bred at Central Animal Services, Monash University (Australia) with the use of the breeding stock received from Dr. Amir Rezvani, University of North Carolina, Chapel Hill, North Carolina (USA). 2.2. Chronic ethanol consumption Alcohol-naive, male, WKY (n ⫽ 4; weight for each, 320 ⫾ 11 g; age, 109 ⫾ 2 days old) and FH (n ⫽ 4; weight for each, 306 ⫾ 21 g; age, 107 ⫾ 2 days old) rats received tap water ad libitum. A second group of WKY (n ⫽ 4; weight for each, 330 ⫾ 9 g; age, 113 ⫾ 3 days old) and FH (n ⫽ 4; weight for each, 344 ⫾ 10 g; age, 118 ⫾ 2 days old) rats were individually housed, and an artificially sweetened ethanol solution [10% volume/volume (vol./vol.)], rather than water, was provided ad libitum for 5 weeks. Additional rats (n ⫽ 3 per strain) received only artificially sweetened tap water for 5 weeks. 2.3. Liver preparation Rats were killed by decapitation at the same time of the day to avoid circadian variations (∼0900). Their livers were dissected out on ice and rinsed with a physiological saline solution [(PSS); composition in mM: NaCl, 118.0; KCl, 4.7; NaH2PO4, 1.0; MgCl2, 1.2; CaCl2, 1.3; NaHCO3, 25.0; and ethylenediaminetetraacetic acid (EDTA), 0.04] and weighed (wet). Each liver was divided into four pieces and incubated for 1 h at 37ºC in 20 ml of carbogenated PSS. Each portion was then chopped into 0.5 × 0.5-mm pieces with a McIlwain tissue chopper before being homogenized with a Potter-Eveljhem Teflon/glass homogenizer in 25 ml of ice-cold sucrose buffer (0.25 M sucrose, 5 mM TRIS, 0.5 mM EDTA, and 0.5 mM dithiothreitol; pH ⫽ 7.5). The homogenates were centrifuged (4ºC) at 700g for 10 min to remove cellular debris, and the resultant supernatant was then centrifuged (4ºC) at 10,000g for 30 min. The pellet (mitochondrial fraction) was resuspended in 10 ml of pyrophosphate buffer (50 mM sodium pyrophosphate; pH ⫽ 8.8). The supernatant was centrifuged (4ºC) at 48,000g for 1 h, with the resultant supernatant being the cytosolic fraction.
The protein concentration of each sample (mitochondrial and cytosolic) was then determined in duplicate by the method of Lowry et al. (1951) with the use of bovine serum albumin as a standard. Samples were stored at ⫺80ºC overnight before determination of enzyme activity. 2.4. Alcohol dehydrogenase assay The activity of hepatic ADH was determined by the reduced form of nicotinamide adenine dinucleotide (NADH)– induced increase in absorbance at 340 nm on a Novaspec spectrophotometer essentially as previously reported (Trave´s et al., 1995). In brief, the assay was performed in duplicate in a final volume of 2.5 ml at 37ºC in glycine buffer (0.1 M glycine; pH ⫽ 10.0) containing 2.4 mM β-NAD⫹ and cytosolic fraction corresponding to 1 mg of protein. After 5 min of equilibration, the reaction was initiated by the addition of ethanol (10 µM–100 mM) and incubated for 5 min before the increase in absorbance was measured relative to a blank reaction (no ethanol added). The effect of pyrazole (10 mM) on the response to 10 mM ethanol was examined to confirm ADH activity. 2.5. Aldehyde dehydrogenase assay The activity of hepatic ALDH was determined by the NADH-induced increase in absorbance at 340 nm on a Novaspec spectrophotometer, essentially as previously reported (Tottmar et al., 1973). In brief, the assay was performed in duplicate in a final volume of 2.5 ml at room temperature in pyrophosphate buffer (50 mM sodium pyrophosphate; pH ⫽ 8.8) containing 1.5 mM β-NAD⫹; pyrazole (0.1 mM), to inhibit ADH activity; rotenone (2 µM in dimethylsulfoxide: 0.2% of final volume), to inhibit mitochondrial NADH oxidase; sodium deoxycholate [0.01% weight/volume (wt./ vol.)], to release latent activity and to increase clarity of the solution for spectrophotometric analysis; and mitochondrial fraction corresponding to 1 mg of protein. After 5 min of equilibration, the reaction was initiated by the addition of acetaldehyde (100 µM–1 M) and incubated in a sealed spectrophotometric tube (to minimize acetaldehyde evaporation) for 15 min before the increase in absorbance was measured relative to a blank reaction (no acetaldehyde added). The effect of disulfiram (0.1 mM in ethanol: 0.2% of final volume) on the response to 10 mM acetaldehyde was examined to confirm ALDH activity. 2.6. Data analysis All increases in absorbance were converted to nanomoles of NADH formed per minute per milligram of protein relative to a β-NADH standard curve (β-NADH dissolved and diluted in 10 mM NaOH). All data are represented as mean ⫾ S.E.M. All curves were analyzed by a two-way analysis of variance (ANOVA), followed by either a Student– Newman–Keuls (for strain comparisons) or a Dunnett post
D.J. Lodge, A.J. Lawrence / Alcohol 30 (2003) 75–79
hoc test. All statistics were calculated by using the SigmaStat software program (SPSS, Chicago, IL, USA). 2.7. Materials β-Nicotinamide adenine dinucleotide (β-NAD⫹), βnicotinamide adenine dinucleotide reduced form (β-NADH), bovine serum albumin, Folin and Ciocalteu’s phenol reagent, rotenone, glycine, and dithiothreitol were purchased from Sigma (St. Louis, MO, USA), whereas pyrazole was obtained from Aldrich (Milwaukee, WI, USA). Deoxycholic acid was purchased from Calbiochem (San Diego, CA, USA), and ethanol was obtained from the Commonwealth Serum Laboratories (Parkville, Victoria, Australia). Acetaldehyde was obtained from MERCK (Munich, Bavaria, Germany), and disulfiram was a gift from Lederle Labs (Philadelphia, PA, USA).
3. Results Spectrophotometric analysis of cytosolic ADH and mitochondrial ALDH activity revealed a concentration-related increase in absorbance in both rat strains (Fig. 1). Comparison of these curves revealed significant differences between ethanol-naive FH and WKY rats, with FH rats having significantly increased ADH activity (⫹61%), but not ALDH activity, when compared with findings for WKY rats (Fig. 1). Chronic ethanol intake in vivo over a 5-week period was constant, with an ethanol intake of 8.8 ⫾ 1.0 g/kg/day for FH rats and 5.5 ⫾ 0.8 g/kg/day for WKY rats. This produced an increase in ADH activity in WKY rats only (⫹14%; P ⬍ .05, two-way ANOVA, followed by Student–Newman– Keuls test), whereas no significant effect on ALDH activity was observed (Fig. 2). To confirm the selectivity of each assay, the effect of pyrazole and disulfiram on the maximum effect of ethanol and acetaldehyde, respectively, was examined. Pyrazole
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(10 mM) was able to completely inhibit maximal ADH activity (WKY rats: ⫺99.5 ⫾ 0.3%; FH rats: ⫺99.2 ⫾ 0.4%; n ⫽ 11; P ⬍ .05, paired Student t test), whereas disulfiram (0.1 mM) significantly attenuated maximal ALDH activity (WKY rats: ⫺36.7 ⫾ 9.2%; FH rats: ⫺40.4 ⫾ 4.6%; n ⫽ 11; P ⬍ .05, paired Student t test). In addition, comparison of liver weight, relative to body weight, revealed no significant differences, either between rat strains or owing to chronic ethanol consumption (Table 1).
4. Discussion The current study is the first to examine the process of hepatic ethanol metabolism in the alcohol-preferring FH rat. The results have revealed significant strain differences, with ethanol-naive FH rats possessing significantly higher ADH activity than observed for WKY rats. Because it has been shown that ADH activity correlates well with ethanol elimination rates in vivo (Lumeng et al., 1979), these findings support the suggestion that FH rats have a higher metabolic capacity for ethanol than that of their alcohol-nonpreferring counterparts. These findings are in accord with results of earlier studies, demonstrating increased ADH activity in the livers of alcohol-preferring C57BL6 mice (when compared with findings for alcohol-nonpreferring DBA mice) (McClearn et al., 1964; Sheppard et al., 1968), and with results of more recent studies, demonstrating that albino rats with high ethanol preference display higher ADH activity than that for rats with low ethanol preference (Petrova, 1985). The correlation between increased ADH activity (ethanol metabolism) and voluntary ethanol consumption would be easy to explain if these rats were drinking to their metabolic capacity. However, this is not the case because it has been shown that voluntary ethanol intake in C57BL6 mice (Rodgers, 1966) and FH rats (unpublished observations,
Fig. 1. Effect of increasing substrate concentrations on the activity of cytosolic alcohol dehydrogenase (A) and mitochondrial aldehyde dehydrogenase (B) throughout the livers of alcohol-naive Fawn-Hooded (■) and Wistar-Kyoto (▲) rats. *Denotes statistically significant difference compared with findings for Wistar-Kyoto rats (P ⬍ .05, two-way analysis of variance, followed by Student–Newman–Keuls test; n ⫽ 4). NADH ⫽ Reduced form of nicotinamide adenine dinucleotide.
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Fig. 2. Activity of cytosolic alcohol dehydrogenase (ADH) and mitochondrial aldehyde dehydrogenase (ALDH) in Fawn-Hooded (A) and Wistar-Kyoto (B) rats chronically consuming sweetened ethanol (ADH ●, ALDH 䊐) or vehicle (ADH ■, ALDH ×). *Denotes statistically significant difference compared with findings for water-only group (P ⬍ .05, two-way analysis of variance, followed by Student–Newman–Keuls test; n ⫽ 3 or 4). NADH ⫽ Reduced form of nicotinamide adenine dinucleotide.
D.J. Lodge and A.J. Lawrence, 2002) can be significantly increased by artificially sweetening the ethanol solution. Therefore, FH rats are not simply drinking to their metabolic capacity, and the correlation that exists between ethanol intake and ADH activity is not likely to be causal. Hence, it seems that constant inbreeding and ethanol exposure (to previous generations) over a long period has resulted in genetic adaptation to ethanol consumption in the form of increased ADH activity. There has also been a suggestion that altered ALDH activity may play a role in ethanol preference because this enzyme is responsible for the removal of acetaldehyde (Isselbacher & Greenberger, 1964; Westerfeld, 1961). However, because ADH is the rate-limiting enzyme in ethanol metabolism (Isselbacher & Greenberger, 1964), a substantial decrease in ALDH activity is required to result in significant acetaldehyde accumulation. Because only a slight decrease was observed in the current study, it is unlikely that the alcohol-avoiding phenotype of the WKY rat is a result of acetaldehyde accumulation. In the current study, we also examined the effect of chronic (5 weeks in vivo) ethanol self-administration on the activity of both ADH and ALDH. Many investigators have reported an induction of ethanol metabolism after chronic ethanol consumption (Lieber & DeCarli, 1970, 1972; Matsuzaki et al., 1981). However, in the WKY rats in the current study, only a small increase in ADH activity (⫹14%) was observed. Another system that is involved in the elimination of ethanol in vivo is the microsomal ethanol oxidizing
Table 1 Relative liver weights (grams per kilogram of body weight) of Fawn-Hooded and Wistar-Kyoto rats receiving no treatment, chronic (5 weeks in vivo) ethanol pretreatment, or vehicle Rat strain
Control
Vehicle
Chronic ethanol
Wistar-Kyoto Fawn-Hooded
36.1 ⫾ 1.7 39.3 ⫾ 0.8
34.4 ⫾ 1.4 38.7 ⫾ 2.3
33.0 ⫾ 1.2 40.2 ⫾ 0.8
Data are presented as mean ⫾ S.E.M.; n ⫽ 3 or 4 per group.
system (MEOS). It is distinguished from ADH by co-factor requirements [reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) instead of NAD⫹) and, under normal conditions, has been suggested to account for 20%– 25% of total ethanol metabolism (Lieber & DeCarli, 1972; Matsuzaki et al., 1981). It has been documented that, although chronic ethanol administration causes a substantial increase in ethanol metabolism, the major changes are associated with an induction of the MEOS because alterations in ADH activity are relatively small (Lieber & DeCarli, 1970, 1972; Matsuzaki et al., 1981). Although results of the current study demonstrate that ADH activity is only marginally increased by chronic ethanol consumption, the effect on total ethanol metabolism cannot be determined because the activity of other (albeit relatively minor) pathways such as MEOS was not determined. In conclusion, results of the current study have demonstrated a significant difference between the alcohol-preferring FH and alcohol-nonpreferring WKY rat strains with regard to the capacity of hepatic ADH in vitro. More specifically, significantly increased ADH activity seen in the alcoholnaive FH rat parallels that observed in other alcohol-preferring rodent models, further suggesting a correlation between ADH activity and ethanol consumption. However, it seems unlikely that this is a causal relation. Rather, increased ADH activity is likely to be a genetic adaptation to ethanol consumption that has developed over a number of years of inbreeding. Acknowledgments This work was supported by the Australian Brewers’ Foundation and the National Health and Medical Research Council (Australia), of which A.J. Lawrence is a Senior Research Fellow. References Chen, F., Rezvani, A., Jarrott, B., & Lawrence, A. J. (1998). Distribution of GABAA receptors in the limbic system of alcohol-preferring and
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