Mutations in mitochondrial aldehyde dehydrogenase (ALDH2) change cofactor affinity and segregate with voluntary alcohol consumption in rats.

Genetic factors influence alcohol consumption and alcoholism. A number of groups have bred alcohol drinker and non drinker rat strains, but genetic determinants remain unknown. The University of Chile rat lines UChA (low drinkers) and UChB (high drinkers) display differences in the relative K(m) for NAD+ of mitochondrial aldehyde dehydrogenase (ALDH2) but no V(max) differences. The relative K(m) differences may be due to mitochondrial changes or to genetic differences coding for ALDH2. We investigated whether there are differences in the coding regions of ALDH2 cDNA in these lines and whether the Aldh2 genotype predicts the phenotype of alcohol consumption and the K(m) of ALDH2 for NAD+. Liver cDNA was prepared, and the Aldh2 transcript was amplified, cloned and sequenced. Genotyping was conducted by DNA amplification and restriction enzyme digestion. When compared to Aldh21 of Sprague-Dawley, 94% of the UChA (low drinker) rats (n = 61), presented a mutation that changes Gln67 to Arg in the mature enzyme (allele referred to as Aldh22). In UChB (high drinker) rats (n = 69), 58% presented the Aldh21 allele, while 42% presented the Gln67Arg change plus a second mutation that changed Glu479 to Lys (allele Aldh23). The Aldh22 allele was absent in high drinker rats. Rats of different Aldh2 genotypes displayed marked phenotypic differences in both ethanol consumption (g/kg/day; means +/- SE): (Aldh21/Aldh21) = 5.7 +/- 0.2, (Aldh22/Aldh22) = 0.9 +/- 0.2 and (Aldh23/Aldh23) = 4.6 +/- 0.2; and K(m)s for NAD+ of 43 +/- 3 microm, 132 +/- 13 microm and 41 +/- 2 microm, respectively (Aldh22 versus Aldh21 or Aldh23; P < 0.0001 for both phenotypes). Overall, the data show that alleles of Aldh2 strongly segregate with the phenotype of ethanol consumption and the relative K(m) for NAD+ of ALDH2. Bases mutated suggest that non drinker Aldh22 is ancestral with regard to the coding changes in either Aldh21 or Aldh23, variants which would allow ethanol consumption and may provide an evolutionary advantage by promoting calorie intake from fermented products along with carbohydrates.

Differences in sensitivity to the aversive effects of ethanol in low-alcohol drinking (UChA) and high-alcohol drinking (UChB) rats.

A conditioned taste aversion paradigm was used to determine whether aversion to the pharmacological effects of ethanol, apart from orosensory cues, can contribute to differences in voluntary ethanol consumption in rats of the low-alcohol drinking (UChA) and the high-alcohol drinking (UChB) strains. "Alcohol-naive" UChA and UChB rats were injected intraperitoneally with ethanol (0.5, 1.0, 1.5, or 2.0 g/kg) or saline, paired with consumption of a banana-flavored solution during five conditioning trials. Repeated pairings of banana-flavored solution and ethanol at a dose of 1.5 g/kg produced aversion to the banana-flavored solution in UChA rats, but not in UChB rats, at comparable blood ethanol levels. In addition, the highest dose of ethanol tested (2.0 g/kg) produced stronger aversion to the banana-flavored solution in UChA rats, compared with findings in UChB rats. From these results it is suggested that rats of the UChA strain find the postingestional effects of high-dose ethanol more aversive than do UChB rats. Differences in voluntary ethanol consumption seem to be associated with differences in sensitivity to the aversive effects of ethanol.

Effect of naltrexone on acute tolerance to ethanol in UChB rats.

The effect of naltrexone (a non-selective opioid receptor antagonist) on both alcohol consumption in a voluntary selection situation and acute tolerance to the motor impairment effect of ethanol was examined in female high alcohol-drinking (UChB) rats using the tilting plane test. In experiment 1, the effect of naltrexone on alcohol consumption was studied in UChB rats which were given a daily 1-h period access to a 10% (v/v) ethanol solution with food and water always available. Naltrexone in doses of 5 or 10 mg/kg intaperitoneal (i.p.) caused dose-dependent reduction in voluntary alcohol intake by 45% and 66%, respectively, without altering daily water intake. In experiment 2, the effect of naltrexone (5 mg/kg i.p.) on acute tolerance to motor impairment effect of a dose of 2.3 g ethanol/kg i.p. was examined. A comparison of control (C) and naltrexone (Nal) UChB groups indicated that naltrexone slowed the recovery of the motor activity and reduce acute tolerance development at comparable ethanol levels in cerebral blood. These results suggest a contribution of the opioid system to acute tolerance to ethanol.

Acute tolerance, alcohol sensitivity and drinking pattern in the F2 generation of UChA and UChB rats.

OBJECTIVE: Studies in mice and rats bred for their behavioral response to ethanol have revealed that there may be a relationship between acute tolerance development, sensitivity to ethanol and alcohol preference. We saw a need to obtain more genetically meaningful correlations between ethanol consumption and acute tolerance to motor impairment or sensitivity to hypothermia induced by ethanol. In the present article, we provide results on acute tolerance, sensitivity to hypothermia and voluntary ethanol consumption in an F2 generation of a cross between inbred UChA (low) and UChB (high) ethanol-consuming rats. METHOD: Naive UChA and UChB rats were tested for acute tolerance development to motor impairment and sensitivity to hypothermia induced by the intraperitoneal administration of a dose of ethanol of 2.3 g/kg body weight. Rats were then offered ad libitum a 10% v/v ethanol solution, distilled water and rat food, and classified according to their drinking scores. Next, one female UChA (low consumer) was mated with one male UChB (high consumer) and one female UChB (high consumer) mated with one male UChA (low consumer). The F2 generation of these inbred rats was used to test acute tolerance, sensitivity and voluntary ethanol consumption. RESULTS: UChB rats developed acute tolerance more rapidly and were less sensitive to the ethanol dose than UChA rats. The F2 generation was designated Hybrid A (HA) for those offspring from the UChA female grandparent and Hybrid B (HB) for the offspring from the UChB female. Results show clearly that there is a relationship between the ability of the rat to acquire acute tolerance to motor impairment, the sensitivity to hypothermia induced by ethanol, and the ethanol preference of the rat. The multiple correlation analysis between the three behaviors showed a significant value in HA rats (r = -0.76, p < .01) and in HB rats (r = -0.83, p < .01). CONCLUSIONS: This study of a cross between UChA and UChB rats showed that the three parameters are related since they clustered together in the F2 generation. This result leads us to speculate that if we could control acute tolerance development in the rats we might also control their voluntary ethanol consumption.

Differences in ethanol sensitivity and acute tolerance between UChA and UChB rats.

OBJECTIVE: In order to learn more about the genetic factors that determine the different responses to ethanol that contribute to the voluntary consumption of ethanol, this study examines the sensitivity to a nonnarcotic dose of ethanol (2.3 g/kg IP) of rats genetically selected for their low (UChA) and high (UChB) ethanol voluntary consumption. METHOD: Sensitivity was evaluated by studying both ethanol-induced (2.3 g/kg) motor impairment in a modified tilting-plane test and hypothermia. Blood ethanol concentration obtained after the ethanol dose and the correlation between ethanol sensitivity and voluntary ethanol consumption were also studied. RESULTS: Results obtained with both tests revealed that UChB rats were less sensitive to ethanol than UChA ones. The genetic difference in motor impairment appeared not to be the result of different blood ethanol levels. Furthermore, rats of both strains recovered motor activity when blood ethanol was at the highest level, indicating the development of acute tolerance. The acute tolerance appeared to develop in shorter time in UChB than in UChA rats. In contradistinction, time course of hypothermia was significantly related to that of blood ethanol. A significant correlation between motor impairment and ethanol voluntary consumption (p<.001) was obtained. CONCLUSIONS: The difference in motor impairment reported here might be related to differences between the strains in the ability to develop acute tolerance to ethanol. Acute tolerance development appears to be positively correlated to voluntary ethanol consumption by the rat.

Oxidation of acetaldehyde by isolated aortic rings of UChA and UChB rats.

The rate of acetaldehyde metabolism was measured in aortic rings from rat strains genetically bred for high (UChB) and low (UChA) voluntary ethanol consumption. The results show that in aortic rings from naive UChB rats, acetaldehyde oxidation rates were significantly greater than the rates observed in aortic rings from naive UChA rats. These strain differences are explained by different activity of vascular low-Km aldehyde dehydrogenase (ALDH). Chronic feeding of ethanol to UChA rats did not alter their aortic ALDH activity. The results of the present study provides additional evidence that the activity variation for the low-Km ALDH between both rat strains exists in various organs and tissues.

Effect of nicotinamide administration on ethanol consumption and on liver and brain acetaldehyde oxidation rate, by UChB rats.

The activities of liver and brain aldehyde dehydrogenase, an NAD(+) dependent enzyme, which controls acetaldehyde oxidation have been reported to play a role in voluntary ethanol consumption. It has been reported that nicotinamide administration to rats increases NAD(+) levels, that may increase acetaldehyde oxidation rates if basal NAD(+) levels are not saturating for the enzyme. In the present paper the effect of nicotinamide administration on voluntary ethanol consumption by genetically high ethanol consumer UChB rats and brain and liver mitochondrial acetaldehyde oxidation were studied. Administration of nicotinamide 250 or 500 mg/kg i.p. to UChB rats, produced a significant reduction in their voluntary ethanol consumption and increased brain acetaldehyde oxidation in brain but not liver homogenates.These results suggest that basal NAD(+) levels are not saturating for brain aldehyde dehydrogenase and that the reduction of ethanol consumption by UChB rats may be the consequence of a change in the brain redox state, rather than the local level of acetaldehyde.

Effect of bromocriptine on acute ethanol tolerance in UChB rats.

It has been suggested that a higher capacity to develop acute tolerance during a single dose of ethanol may promote higher ethanol consumption in alcohol-preferring rodents. Several studies have shown that the dopaminergic system may be involved in voluntary ethanol consumption. In the present paper we studied the effect of bromocriptine, a dopaminergic agonist drug, that is known to reduce voluntary consumption of ethanol, on acute tolerance in high (UChB) ethanol consumer rats. Acute tolerance was evaluated in bromocriptine and saline-treated rats by motor impairment induced by a subnarcotic dose of ethanol of 2.3 g/kg IP using a modified tilting plane test. Results showed a highly significant positive correlation between acute tolerance and the voluntary ethanol consumption by the rat. Bromocriptine treatment decreased ethanol consumption and also decreased acute tolerance development. This adds further support to the postulate that the acquisition of acute tolerance to ethanol may promote increased alcohol consumption. Moreover, these results also suggest that dopaminergic receptors involved in ethanol voluntary consumption may also be in acute tolerance development.

Effect of calcium channel blocker diltiazem on some depressant actions of ethanol in UChA and UChB rats.

Rats genetically selected for their different ethanol voluntary consumption, UChA (low consumer) and UChB (high consumer) were used. Naive UChA and UChB rats or submitted to ethanol chronic exposure, received an i.p. dose of ethanol (2.76 g/kg) alone or 30 min after an oral dose of diltiazem (10 mg/kg), a calcium channel blocker. A significant potentiation of the narcosis and hypothermia induced by the dose of ethanol was observed in UChA diltiazem-pretreated rats not previously exposed to ethanol, while no potentiation in narcosis time appears in UChA rats chronically exposed to ethanol that acquire tolerance. In the UChB line of rats, diltiazem did not potentiate ethanol depressant actions in naive or chronic ethanol-exposed rats. Diltiazem did not modify ethanol blood levels. These results indicate that the inhibition of voltage-dependent calcium channels can exaggerate ethanol-induced effects in naive rats but not when tolerance was developed. Results suggest that UChB rats may have some innate tolerance that may be due to genetic difference in calcium channel function.

Effect of captopril on voluntary consumption of ethanol, water and solid food by UChA and UChB rats.

Angiotensin converting enzyme inhibitors (ACEI) have been reported to reduce ethanol consumption in rats, but it is unclear whether this effect is specific for ethanol or secondary to effects on appetite or satiation for calories or water. In the present study we assessed the effect of captopril, an ACEI, on the voluntary consumption of 10% ethanol solution, water and solid food in our strain of rats genetically selected for their voluntary consumption of ethanol, namely UChA (low consumer) and UChB (high consumer). Captopril (30 mg/kg) was injected intraperitoneally for 3 consecutive days to UChA and UChB rats and ethanol, water and food intake were measured before, during and after captopril treatment; these results were compared with those produced by a control saline solution. Results showed that captopril produced a significant reduction of alcohol voluntary consumption in UChB but not in UChA rats. However, this effect was not specific for ethanol since captopril also induced a significant decrease in food intake leading to a loss of weight in both rat strains, suggesting that it seems to be secondary to changes in appetite for calories.

Acetaldehyde metabolism by liver mitochondrial ALDH from UChA and UChB rats: effect of inhibitors.

We have observed that blood acetaldehyde (AcH) levels after an ethanol dose were significantly higher in disulfiram-pre-treated UChA (low ethanol consumer) than in UChB (high ethanol consumer) rats. In order to explore these results further, we studied the effect of disulfiram (300 mg/kg i.p.) and chlorpropamide (80) mg/kg i.p.) pre-treatment on blood AcH levels after oral ethanol (60 mmol/kg) and on AcH metabolism by liver mitochondrial aldehyde(s) dehydrogenase(s) from UChA and UChB rats. AcH metabolism by liver mitochondrial aldehyde dehydrogenase (ALDH) was studied by following AcH disappearance rate and the formation of NADH at 340 nm in the incubation medium. The results showed that chlorpropamide, like disulfiram, produced a higher blood AcH level consistent with a greater inhibition of the low-Km mitochondrial ALDH in the UChA rats than in the UChB rats. These drugs did not inhibit the high Km mitochondrial ALDH. Kinetic studies of mitochondrial ALDH show that low-Km mitochondrial ALDH from UChB rats exhibits a higher affinity for NAD than UChA rats. This observation could explain the different inhibition of ALDH by both drugs, assuming that the inhibitors reduce NAD availability, the rate limiting step in the mitochondrial ALDH oxidation.

Acetaldehyde metabolism by brain mitochondria from UChA and UChB rats.

The acetaldehyde (AcH) oxidizing capacity of total brain homogenates from the genetically high-ethanol consumer (UChB) appeared to be greater than that of the low-ethanol consumer (UChA) rats. To gain further information about this strain difference, the activity of aldehyde dehydrogenase (AIDH) in different subcellular fractions of whole brain homogenates from naive UChA and UChB rat strains of both sexes has been studied by measuring the rate of AcH disappearance and by following the reduction of NAD to NADH. The results demonstrated that the higher capacity of brain homogenates from UChB rats to oxidize AcH when compared to UChA ones was because the UChB mitochondrial low Km AIDH exhibits a much greater affinity for NAD than that of the UChA rats, as evidenced by four-to fivefold differences in the Km values for NAD. But the dehydrogenases from both strains exhibited a similar maximum rate at saturating NAD concentrations. Because intact brain mitochondria isolated from UChB rats oxidized AcH at a higher rate than did mitochondria from UChA rats only in state 4, but not in state 3, this strain difference in AIDH activity might be restricted in vivo to NAD disposition.

Effects of aminotriazole on ethanol, water, and food intake and on brain catalase in UChA and UChB rats.

Aminotriazole (AT), a catalase inhibitor, was administered to UChA (low ethanol consumer) and UChB (high ethanol consumer) rats. Ethanol, water, and solid food intake were measured during basic, treatment, and posttreatment periods. The effects of AT on brain catalase activity and acetaldehyde recovered during incubation of brain homogenates with ethanol were also studied in rats of both strains. Results showed that AT decreased voluntary ethanol intake in UChB rats, and also diminished the consumption of food by rats of both strains. No strain difference in brain catalase activity and acetaldehyde recovered during ethanol incubation was observed. The results suggest that AT effect on ethanol consumption is secondary to a reduction in the appetite for calories and not related to its catalase blocking effect.

Acetaldehyde metabolism: differences between UChA and UChB rats.

In order to explore the influence of acetaldehyde (AcH) metabolism on the voluntary ethanol intake of genetically low (UChA) and high (UChB) ethanol consumer rats, the AcH disappearance rate (ADR) after incubation with homogenates and subcellular fractions from liver and brain was determined. In addition, the effect of disulfiram pretreatment on AcH metabolism was studied. Male adult rats of both strains were used. ADR was assayed in total homogenates, and in mitochondrial as well as 9000 g supernatant fractions of liver and brain. AcH was measured by gas chromatography. In some experiments, rats were pretreated with disulfiram (300 mg/kg po) 24 hr before the studies. The result showed no strain difference in ADR in homogenates or subcellular fractions of liver from untreated rats, but for disulfiram pretreated rats a significantly lower decrease of ADR in samples from UChB compared to UChA rats was observed. This result is consistent with a lower peak AcH level in UChB compared to UChA rats after a load of ethanol (60 mmole/kg ip). Concerning brain homogenates, a higher ADR was observed in homogenates and crude mitochondrial fractions of UChB than of UChA rats. This difference was not observed when the incubation was performed without adding NAD or in the absence of oxygen. These results provide evidence of strain differences in mitochondrial AcH metabolism, the nature and origin of which deserve further study.

Investigations on the ethanol-induced flushing reaction: effects of propranolol and dipyridamole on acetaldehyde and prostacyclin metabolism.

Disulfiram, an aldehyde dehydrogenase (ALDH) inhibitor, induces a flushing reaction upon the ingestion of ethanol, exerting aversion against alcohol that has been used in the treatment of alcoholism. This unpleasant response has been associated with an accumulation of acetaldehyde, and more recently, with an increase in vascular prostacyclin (PGI2) production. To evaluate the possibility of evoking the flushing reaction with drugs less toxic than disulfiram, we studied the effects of propranolol and dipyridamole on ALDH and PGI2. Acetaldehyde oxidation rate was assessed by gas chromatography in mitochondria from rats treated with these drugs for seven days. Prostacyclin generation was determined in rat aortic rings incubated in Krebs-Ringer with these drugs separately and associated to acetaldehyde, and measured by radioimmunoassay of 6-keto-PGF1 alpha. Propranolol inhibited acetaldehyde oxidation rate whereas dipyridamole did not. Furthermore, propranolol increased blood acetaldehyde levels without affecting ethanol elimination rate. Both drugs stimulated prostacyclin synthesis but only dipyridamole enhanced the stimulatory effect of acetaldehyde on vascular prostacyclin production. These results strongly suggest the possibility of producing a deterrent effect on the consumption of alcohol by using propranolol or dipyridamole. In contrast to disulfiram, these drugs could potentially induce the flushing reaction in humans in the presence of low acetaldehyde concentrations; this new therapeutic approach might have an important clinical and toxicological relevance.

Effect of diet and disulfiram on acetaldehyde blood levels after ethanol in UChA and UChB rats.

Acetaldehyde (AcH) levels in blood samples taken from different zones of the vascular system 2 h after a p.o. dose of ethanol (2.76 g/kg) were studied in UChA (low ethanol consumer) and UChB (high ethanol consumer) rats fed a diet devoid of animal products, diet 1 (D1), and a diet containing fish meal, diet 2 (D2), and in rats pretreated with disulfiram (600 mg/kg p.o.). The results showed that, while there is no significant difference between UChA and UChB rats fed D1 with respect to blood AcH levels and the basal activity of the hepatic mitochondrial high-affinity aldehyde dehydrogenase (AIDH), a significant strain difference was observed in rats fed D2, which induced high blood AcH levels in UChA rats but not in UChB ones. No strain differences were observed in blood ethanol levels in the two groups of rats. When rats fed D1 were pretreated with disulfiram, the raising of AcH blood levels induced by ethanol after disulfiram was significantly higher in UChA than in UChB rats in suprahepatic vein, femoral vein, and tail blood. This difference was concomitant with a greater inhibition of the hepatic mitochondrial high-affinity ADH activity in UChA rats than in UChB ones, whether disulfiram was administered in vivo or in vitro, which excluded the possibility that the strain difference would be caused by a different bioavailability of disulfiram.

Metabolism of acetaldehyde by rat isolated aortic rings: does endothelial tissue contribute to its extrahepatic metabolism?

Acetaldehyde (AcH) metabolism in isolated aortic rings was studied by assessing in vitro-added AcH disappearing rate by head space gas chromatography. It was found that AcH was metabolized by aortic rings or by homogenates prepared in 0.1 M phosphate buffer containing Triton X-100, by an NAD-dependent enzyme with characteristics similar to those of aldehyde dehydrogenase (AIDH) present in mitochondria from rat liver and brain. This enzyme appears to be present in the vascular endothelium, since the action of aortic rings showed a remarkable decrease by its removal. Extrahepatic metabolism of AcH was assessed by the differences between AcH levels found in samples of blood obtained from the suprahepatic vein, carotid artery, femoral vein, and tail cut of rats. The in vitro activity of aortic rings, as well as the extrahepatic AcH metabolism, were significantly decreased by pretreatment of rats with disulfiram. The wide distribution of vascular endothelium throughout the body suggests that this tissue could contribute to AcH extrahepatic metabolism.

Ethanol intake: effect on liver and brain mitochondrial function and acetaldehyde oxidation.

The effect of a chronic ethanol consumption by forcing rats to drink a 20% v/v ethanol solution as sole drinking fluid, for 3 months, was evaluated on: liver and brain mitochondrial function, the capacity of isolated mitochondria to oxidize acetaldehyde, as well as on the low Km mitochondrial AlDH activity, in rats. The O2 uptake by liver and brain mitochondria in the presence of glutamate + malate, succinate or ascorbate + TMPD, was measured polarographically with a Clark electrode. Acetaldehyde oxidation was measured by the disappearance rate in presence of the intact or disrupted mitochondria (AlDH activity) by gas chromatography. Results indicate that an ethanol intake of 11 g/kg b.wt. per day produce a significant reduction of the liver mitochondrial respiration tested with all the substrates used, including acetaldehyde. In contrast, the activity of AlDH in disrupted mitochondria remained unchanged. These results are in accord with the idea that a progressive deterioration of liver mitochondrial function appears with the increase in amount of ethanol consumed, and that alterations of acetaldehyde oxidation by intact mitochondria can be detected before an alteration of the AlDH activity. Concerning the brain, this ethanol consumption regimen did not affect the brain mitochondrial respiration tested with glutamate + malate, succinate or ascorbate + TMPD, but it induces an increase in acetaldehyde oxidation rate by intact brain mitochondria. The imposed increase in the cerebral aldehyde oxidizing capacity could reflect a principal biochemical mechanism underlying neural adaptation to ethanol.

Effect of 3-amino-1,2,4-triazole on the hypothermic effect of ethanol and on ethanol tolerance development.

The effect of aminotriazole (AT), inhibitor of catalase activity, on hypothermia and narcosis induced by ethanol and on the acquisition of tolerance to the ethanol hypothermic and narcotic effect was studied. Rats were pretreated with AT (1 g/kg IP) 1 hour before the test dose of ethanol (2.76 g/kg IP) and narcosis time, hypothermia and ethanol blood levels evaluated (first test). For studies on tolerance to ethanol, rats of the first test received daily (for 7 days) a dose of AT (1 g/kg IP) 1 hour before ethanol (2.76 g/kg) given by gavage, and the same parameters evaluated (8th day test). Results were compared to similar groups of rats without AT pretreatment (controls, 1st and 8th day test). Rats pretreated with AT exhibited a shorter narcosis time induced by ethanol but this treatment did not alter the hypothermic effect of ethanol nor ethanol disposal rate. Chronic ethanol treatment induced tolerance to the narcotic and hypothermic effect of ethanol as well as a metabolic tolerance. AT administered daily before the dose of ethanol produced a partial blockade of the development of tolerance to the narcotic effect of ethanol, but did not alter the development of hypothermic or metabolic tolerance. The brain catalase system seems to play a role in narcosis and on the development of tolerance to this effect of ethanol, but not in the hypothermic effect or in the development of tolerance to this ethanol effect. Since the inhibition of liver catalase activity by AT treatment was not correlated with changes in ethanol disposal rate, the liver catalase system appears not to play a role in the metabolic tolerance.

Acetaldehyde metabolism by isolated hepatic mitochondria from rats consuming different amounts of ethanol.

We have previously reported that in rats, ethanol intake as low as 4 g/kg per day induced mitochondrial alterations, detected by a decrease in mitochondrial O2 uptake supported by substrates entering at the three sites of coupled phosphorylation. Since it has been reported that acetaldehyde oxidation occurs mainly inside the mitochondria, linked to the respiration chain, the effect of daily previous administration of low doses of ethanol during a month, on the acetaldehyde oxidation rate by intact rat liver mitochondria was studied. Determination of acetaldehyde oxidation rate and O2 uptake accompanying acetaldehyde removal by liver mitochondria indicates that the mean value of these parameters studied in rats consuming low amount of ethanol (0.5-3.0 g/kg per day) was significantly higher than that of controls drinking only water (P less than 0.001, in state 3, and P less than 0.05, in state 4). This enhancing effect of ethanol cannot be ascribed to an uncoupling of oxidative phosphorylation and also not to a change in aldehyde dehydrogenase activity which was measured in disrupted mitochondria. On the other hand, high ethanol consumption (4-7 g/kg per day), which alters mitochondrial function, did not decrease mitochondrial acetaldehyde oxidation, as compared to rats drinking water only. This result differs from the decrease induced by higher levels of previous ethanol intake (12-14 g/kg per day) as reported by several authors, showing that possibly this effect is dose dependent.