ACETALDEHYDE METABOLISM: DIFFERENCES BETWEEN UChA AND UChB RATS

In order to explore the influence of acetaldehyde (AcH) metabolismon the voluntary ethanol intake of genetically low (UChA) andhigh (UChB) ethanol consumer rats, the AcH disappearance rate(ADR) after incubation with homogenates and subcellular fractionsfrom liver and brain was determined. In addition, the effectof disulfiram pretreatment on AcH metabolism was studied. Maleadult rats of both strains were used. ADR was assayed in totalhomogenates, and in mitochondrial as well as 9000 g supernatantfractions of liver and brain. AcH was measured by gas chromatography.In some experiments, rats were pretreated with disulfiram (300mg/kg po) 24 hr before the studies. The result showed no straindifference in ADR in homogenates or subcellular fractions ofliver from untreated rats, but for disulfiram pretreated ratsa significantly lower decrease of ADR in samples from UChB comparedto UChA rats was observed. This result is consistent with alower peak AcH level in UChB compared to UChA rats after a loadof ethanol (60 mmole/kg ip). Concerning brain homogenates, ahigher ADR was observed in homogenates and crude mitochondrialfractions of UChB than of UChA rats. This difference was notobserved when the incubation was performed without adding NADor in the absence of oxygen. These results provide evidenceof strain differences in mitochondrial AcH metabolism, the natureand origin of which deserve further study.     

Aversion to acetaldehyde: differences in low-alcohol-drinking (UChA) and high-alcohol-drinking (UChB) rats.

We have previously found the existence of a relation between activity of the brain mitochondrial aldehyde dehydrogenase (ALDH2) and consumption of ethanol in rats of the low-alcohol-drinking (UChA) and the high-alcohol-drinking (UChB) strains. The aim of the present study was to determine whether UChA and UChB rats also differed in sensitivity to the aversive effects of acetaldehyde (AcH). Aversion to AcH was studied by using a conditioned taste aversion (CTA) paradigm. Ethanol naive UChA and UChB rats were administered AcH intraperitoneally (50, 100, or 150 mg/kg) or saline and exposed to a banana-flavored solution during five conditioning trials. A strong dose-dependent CTA to AcH was found in UChA rats, whereas UChB rats did not show a CTA to any dose of AcH. At equal doses of AcH, cerebral venous blood AcH levels in UChA rats were consistently higher than in UChB rats, a finding that may reflect the previously observed differences in the activity of ALDH2 between these strains. However, this observation is unlikely to explain fully the differences observed because aversion to AcH was developed in the UChA strain at blood levels of AcH that did not produce any aversion in the UChB strain. These results support the suggestion that, for the first time, differences in central or systemic effects of AcH per se may play a major role in determining the aversion to AcH in drinker and nondrinker animals.     

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 (AlDH), 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 AlDH 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.     

The effects of alpha-methyl-p-tyrosine pretreatment on ethanol-induced narcosis and hypothermia, as well as in the development of tolerance to these effects in UChA and UChB rats

We have previously reported that UChA rats (genetically low ethanol consumer) develop tolerance to narcosis time easier than UChB rats (genetically high ethanol consumer). We also have reported that UChA rats develop tolerance to the hypothermic effect of ethanol, while in UChB rats the repeated administration of ethanol induces sensitization towards this effect. In the present paper the effects of alpha-methyl-p-tyrosine (AMPT)--a competitive inhibitor of norepinephrine synthesis--on ethanol-induced narcosis and hypothermia, as well as in the development of tolerance to these effects, were studied in both strains of rats. Results obtained show that AMPT pretreatment induced a significantly higher increase in narcosis time and hypothermia, as well as, greater susceptibility to ethanol toxicity in UChB than UChA rats. Furthermore, the simultaneous treatment with AMPT and ethanol did not change the development of tolerance to narcosis time in both strains and to hypothermia and sensitization in UChA and UChB rats respectively.     

BIOLOGICAL SIMILARITIES AND DIFFERENCES BETWEEN RATS GENETICALLY DIFFERENT IN ALCOHOL PREFERENCE

This paper is an inventory of some behavioural, biochemicaland pharmacological similarities and differences between twoinbred strains of Wistar rats differing in voluntary consumptionof ethanol, namely: UChA and UChB with low and high preferencesrespectively for ethanol under conditions involving free choicebetween a 10% (v/v) ethanol solution and distilled water. Thefollowing strain differences were observed: ethanol consumption(UChA<UChB); total water consumption (UChA<UChB); solidfood consumption (UChA>UChB); rate of recovery of ethanollabel in expired CO₂ (UChA<UChB); oxidation of ethanol toacetaldehyde by brain homogenates (UChA>UChB); acetaldehydedisposal by brain homogenates (UChA<UChB); ethanol (90 mmol/kg,i.p.) sleeping-time (UChA<UChB); chronic and acute toleranceto ethanol (UChA developed it, whereas UChB did not); lethaldoses of ethanol (UChA>UChB); recovery rate of the labelof gluconate in expired CO₂ (UChA<UChB); recovery rate ofthe label of fructose in expired CO₂ (UChA<UChB); blood-glucoselevel after glucose (1g/kg, i.p.) load (UChA<UChB). No straindifferences were observed in the rate of recovery in expiredCO₂ of the label of the following substrates: acetate, pyruvate,butyrate, citrate, ribose, glycerol, sorbitol, glucose and galactose.     

EFFECTS OF DIETS DECREASING ETHANOL CONSUMPTION ON ACETALDEHYDE METABOLISM IN UChA AND UChB RATS

It has previously been reported that the introduction of a newissue (D1) of a stock diet (D0) caused a significant decreasein voluntary ethanol consumption in rats of our UChA (low ethanolconsumer) and UChB (high ethanol consumer) strains, and that,after changing to a diet lacking in animal products (D3), ethanolconsumption reached the previous level attained in UChB ratsand exhibited a bimodal distribution in UChA rats in such away that about one-third of these consumed more than 2 ml ofa 10% (v/v) ethanol solution per 100 g of body weight, as didUChB rats. When UChA rats exhibiting high ethanol consumptionand also UChB rats were fed on the D3 diet with a brewer's yeastsupplement, a significant decrease in ethanol intake was observed.Since significant correlations of voluntary ethanol intake toblood acetaldehyde levels, and to hepatic and brain aldehydedehydrogenase (AldDH) activities, have been reported, the effectson these parameters of diet D1 and of supplements of brewer'syeast or disulfiram were studied. Results showed that rats ofboth strains fed on a D1 diet exhibited a significant increasein blood acetaldehyde level after ethanol administration, concomitantwith an inhibition of hepatic and brain AldDH activities anda significant decrease in ethanol intake. Thus, this effectcould be related to a disulfiram-hke activity. The decreasein ethanol intake induced by brewer's yeast was not accompaniedby changes of blood acetaldehyde level after ethanol, nor byinhibition of hepatic AldDH activity in either strain. Nevertheless,a significant inhibition of brain AldDH activity in UChB, butnot in UChA, rats was observed. Thus, the effect of brewer'syeast on ethanol consumption could not be ascribed to a disulfiram-likeactivity of this supplement.the beginning of pregnancy, couldbe toxic for the human placenta and development of the fetus.     

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.     

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.     

Tolerance development in UChA and UChB rats by ethanol inhalation

The development of tolerance by exposure to ethanol vapor in rats of the UChA and UChB strains has been studied. The exposure to ethanol vapor (6-7 mg per liter of air) during 46 hours developed clear tolerance to ethanol narcosis time in rats of both strains and sexes, while no tolerance to latency time was developed in any of these strains. Regarding the hypothermic effect of ethanol, only male rats of the UChB strains showed a decrease of rectal temperature significantly lower (p less than 0.05) than its respective control group. Blood alcohol levels (BALs) at awakening after the injection of ethanol (60 mmole/kg IP) were also determined. The results showed only a significantly lower BAL (p less than 0.05) in the females of the UChA strain subjected to ethanol inhalation.     

Structure vs. activity in the sulfonylurea-mediated disulfiram-ethanol reaction

The oral hypoglycemic agents, chlorpropamide (CP) and tolbutamide (TB) are known to elicit a clinical disulfiram-ethanol reaction (DER) when consumed with alcohol. In rats, this DER is manifested in vivo by the elevation of blood acetaldehyde (AcH) levels, a consequence of the inhibition of hepatic aldehyde dehydrogenase (AIDH). Administration of CP or TB to rats (1.0 mmol/kg, IP), followed by ethanol one hour before sacrifice, raised blood AcH levels 12- and 2-times that of control animals, respectively for CP and TB when measured at 3 hours, and 20-fold and 8-fold at 16 hours post drug administration. CP and TB had no effect on AIDH activity when incubated with either intact or osmotically disrupted rat liver mitochondria, indicating that a metabolite of CP or TB is responsible for the inhibition of AIDH in vivo. Hydrolysis products of CP, the 2'-hydroxylated products of CP, tolpropamide and tolethamide, or the 3'-hydroxylated analogs of CP and tolpropamide, were uniformly inactive in elevating ethanol-derived blood AcH. Pretreatment of rats with 3-amino-1,2,4-triazole or SKF-525A had no effect on the elevation of blood AcH mediated by CP or TB, while phenobarbital pretreatment decreased blood AcH by 69%. Although our results clearly indicated that side chain hydroxylation and subsequent oxidation do not play a role in AIDH inhibition by CP or TB, the nature of the side chain attached to the sulfonylurea moiety appears to influence this inhibitory activity in vivo. Thus, the order of activity in the homologous series was, chlorpropamide greater than chlorbutamide greater than chlorethamide much greater than chlormethamide, chlorisopropamide = 0.     

Acetaldehyde-reinforcing effects: differences in low-alcohol-drinking (UChA) and high-alcohol-drinking (UChB) rats.

It has been suggested that acetaldehyde has a biphasic effect on voluntary alcohol consumption. At low brain concentration, it might exert reinforcing effects, whereas high acetaldehyde levels would be predominantly aversive. The objective of the current study was to compare the effect of an intraperitoneal dose of acetaldehyde (50 mg/kg) in high-alcohol-drinking (UChB) and low-alcohol-drinking (UChA) rat lines, which differ in the activity of the brain mitochondrial class 2 aldehyde dehydrogenase (ALDH2) as a consequence of differences in their ALDH2 genotypes. A classical place-conditioning procedure was used to determine the reinforcing or aversive (or both) effects of acetaldehyde in ethanol-naive UChB and UChA rats. Environmental cues were paired with an intraperitoneal 50-mg/kg injection of acetaldehyde. On 10 consecutive days, each rat received one place conditioning per day; the acetaldehyde-pairing was alternated with saline-pairing. Results showed that conditioning with the 50-mg/kg dose of acetaldehyde induced place preference in UChB rats and place aversion in UChA rats. In a second experiment, UChB and UChA rats, pretested for ethanol preference, were injected with one 50-mg/kg dose of acetaldehyde or saline and tested for their voluntary ethanol consumption during 4 weeks. Results showed that the acetaldehyde dose induced a persistent and long-lasting enhancement of ethanol intake in UChB rats, but not in UChA rats. These results, together with the finding that after administration of a 50-mg/kg dose of acetaldehyde cerebral venous blood acetaldehyde levels in UChA rats were consistently higher than levels in UChB rats, support the suggestion that differential acetaldehyde levels, differential brain ALDH2 activity, or both were responsible for the different effects of acetaldehyde in the two rat lines.     

Effects of p-chlorophenylalanine on the voluntary consumption of ethanol, water and solid food by UChA and UChB rats

The immediate and the long-lasting effects of PCPA (126 mg/kg IP for 3 days) on the voluntary consumption of 10% v/v ethanol solution, water and solid food were studied in genetically low (UChA) and high (UChB) ethanol consumer rats. Data were analysed according to the method proposed by the authors for recognizing specific effects on ethanol consumption. Results confirmed immediate specific decrease of ethanol consumption in UChB rats, while a nonspecific decrease of it was observed in UChA rats. In UChB rats ethanol consumption recovered the basic level about nine days after the first dose. By contradistincion, in UChA rats a significant specific increase of ethanol consumption, starting at the first week after the treatment, was observed. During this period 12 out of 21 UChA rats reached an ethanol consumption level commonly observed in UChB rats, and 10 of them recovered the pretreatment level in 15 to 30 weeks. The other 2 maintained the high consumption until 38 weeks of observation. At that time the serotonin content of cortex and hippocampus of these 2 rats was normal.     

Effects of pyrazole on the voluntary consumption of ethanol, water and solid food in UChA and UChB rats

The effect of pyrazole (35 or 70 mg/kg IP) on the voluntary consumption of ethanol, water and solid food was studied in UChA (genetically low ethanol consumer) and UChB (genetically high ethanol consumer) rats of both sexes, under a free choice of 10% v/v ethanol solution, distilled water and solid food. the data were analyzed according to the method previously proposed for recognizing the specific effect on appetite-satiety of ethanol. The effect of pyrazole in UChB rats correlated with the pattern of specific effects on appetite-satiety for ethanol. The only effect observed in UChA rats was a decrease in the intake of solid food and total water. No significant sex differences were observed.     

Saccharin consumption and the effect of a long-term exposure to a sweetened alcoholic solution in high- (UChB) and low- (UChA) alcohol-drinking rats.

An association between saccharin consumption and alcohol intake has been observed in rodent lines genetically developed for alcohol preference or alcohol avoidance. It has also been proposed that a sweetened alcohol solution can condition rats to consume high amounts of alcohol. This work had two aims. First, to study the relationship between saccharin and alcohol intake in both high-alcohol-drinking UChB rats and low-alcohol-drinking UChA rats and, second, to determine whether a long-term exposure to a sweetened alcohol solution can increase their voluntary alcohol consumption. For the first purpose, UChB and UChA rats were tested under a free-choice paradigm between two graduated bottles, one containing a saccharin solution (0.1, 0.2, or 0.4% [wt/vol]) and the other water. For the second purpose, UChB and UChA rats that were under free choice between 10% alcohol and water, were offered a 10% alcohol solution containing 0.2% saccharin, instead of 10% alcohol for 1 month and were then returned to free choice between 10% alcohol and water. The first experiment showed that both lines have a high preference for saccharin at any concentration, but UChB rats drank twice as much saccharin solution as UChA rats and consequently they increased significantly their total daily fluid intake. This fact has been suggested to be an animal analogue of the clinical phenomenon known as "loss of control." In the second experiment a long-term exposure to a 10% alcohol solution containing 0.2% saccharin induced a significant increase in alcohol consumption in UChB rats once saccharin was faded out, whereas the alcohol consumption in UChA rats returned to the previous low value. This result indicates that UChA rats have a genetic predisposition to avoid alcohol. In conclusion, the results reported here for UChB and UChA rats show an association between saccharin and alcohol preference, and suggest that their different genotypes are probably involved in alcohol aversion.     

Effect of different treatment regimens with ethanol and p-chlorophenylalanine on tolerance development in UChA and UChB rats

Since we have previously observed a genetic difference in the development of tolerance to the narcotic effect of ethanol in UChA and UChB rats when providing them with a 10% v/v ethanol solution as sole drinking fluid, experiments were performed in order to know whether the resistance to development of tolerance to ethanol in UChB rats was also exhibited after other regimens of ethanol administration, namely, a 2.76 g/kg ethanol IP injection 24 hr before the experiment, only 10% v/v ethanol solution as sole drinking fluid for 21 days, or receiving acutely a daily dose of 2.76 g/kg ethanol by gavage for seven days. Participation of serotoninergic neurons was tested by treating rats with p-chlorophenylalanine (p-CPA), a known serotonin depletor. Results show that UChA rats developed tolerance to ethanol-induced narcosis and hypothermia, while UChB rats developed it to narcosis only when they received acute oral doses of ethanol for 7 days and did not develop tolerance to hypothermia with any of the treatment regimens. p-CPA pretreatment did prevent the development of tolerance in both strains of rats, confirming the participation of serotoninergic neurons in ethanol tolerance in rats.     

Place conditioning with ethanol in rats bred for high (UChB) and low (UChA) voluntary alcohol drinking

The main goal of this study was to investigate the ability of an ethanol dose (1 g/kg) administered intraperitoneally to induce conditioned place preference (CPP) and/or conditioned place aversion (CPA) in two lines of rats selectively bred for their high (UChB) or low (UChA) voluntary ethanol intake. It was found that five pairings with ethanol induced CPA in ethanol-naïve rats of both lines, but the magnitude of avoidance was lower in the UChB relative to the UChA rats, indicating that ethanol was less aversive to naïve rats bred for high alcohol drinking. After 2 months of high voluntary ethanol drinking (∼6–7 g/kg/day), in free choice between 10% ethanol and water, ethanol produced CPP in UChB rats, reflecting that ethanol had become rewarding to these rats. By contrast, the low voluntary ethanol intake (<1 g/kg/day) displayed by UChA rats preexposed for 2 months in free choice did not change ethanol-induced CPA. However, preexposure of UChA rats to forced ethanol drinking (∼5.7 g/kg/day) and the later inhibition of ethanol-derived acetaldehyde by 4-methylpyrazole (10 mg/kg intraperitoneal), an inhibitor of the enzyme alcohol dehydrogenase, not only increased their voluntary ethanol intake in free choice, but also had a facilitating effect on the development of CPP. Taken together, these results show that the expression of the reinforcing effects of ethanol required a period of voluntary ethanol intake in UChB rats, whereas in UChA rats, both prior exposure to forced ethanol drinking and reduction of high blood ethanol-derived acetaldehyde were required.     

Effects of serotonin uptake blockers and of 5-hydroxytryptophan on the voluntary consumption of ethanol, water and solid food by UChA and UChB rats

The effects of zimelidine, fluvoxamine, and citalopram (serotonin uptake blockers), as well as those of 5-hydroxytryptophan (serotonin precursor), on the voluntary consumption of 10% ethanol solution, distilled water and solid food were tested in UChA (genetically low ethanol consumer) and UChB (genetically high ethanol consumer) rats. Since it is well known that drugs which stimulate central serotonergic synapses decrease food and water intake, the data concerning the difference of the respective consumption during the treatment period and the pretreatment one were analysed with a method previously proposed (Alcohol 5:15-19; 1988) to recognize specific effects on ethanol intake. The results showed that while the decrease of ethanol consumption induced by the three serotonin uptake blockers appeared not to be specific of ethanol, the effects of 5-hydroxytryptophan in UChB rats satisfy the criteria for being considered as an expression of a decrease of the specific appetite--or increase satiety--for ethanol. Experimental results cannot help in the explanation of this difference.     

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.     

A method for recognizing specific effects on ethanol intake by experimental animals

A method of mathematical treatment of data concerning changes in voluntary consumption of ethanol solution, water and solid food, induced by experimental treatments in animals, in order to recognize effects on mechanisms involved in specific appetite and satiety for calories, water and ethanol is proposed. The need of such method arises from the fact that several experimental treatments tested by the effects on ethanol consumption alter at the same time the appetite or satiety for calories and/or for water, as well as ingestive behavior. The results of testing the method with the data obtained by treatment of UChA and UChB rats with disulfiram or cyanamide were consistent with the expected ones.