Ethanol as a prodrug: brain metabolism of ethanol mediates its reinforcing effects

Background: While the molecular entity responsible for the rewarding effects of virtually all drugs of abuse is known, that for ethanol remains uncertain. Some lines of evidence suggest that the rewarding effects of alcohol are mediated not by ethanol per se but by acetaldehyde generated by catalase in the brain. However, the lack of specific inhibitors of catalase has not allowed strong conclusions to be drawn about its role on the rewarding properties of ethanol. The present studies determined the effect on voluntary alcohol consumption of two gene vectors, one designed to inhibit catalase synthesis and one designed to synthesize alcohol dehydrogenase (ADH), to respectively inhibit or increase brain acetaldehyde synthesis. Methods: The lentiviral vectors, which incorporate the genes they carry into the cell genome, were (i) one encoding a shRNA anticatalase synthesis and (ii) one encoding alcohol dehydrogenase (rADH1). These were stereotaxically microinjected into the brain ventral tegmental area (VTA) of Wistar‐derived rats bred for generations for their high alcohol preference (UChB), which were allowed access to an ethanol solution and water. Results: Microinjection into the VTA of the lentiviral vector encoding the anticatalase shRNA virtually abolished (?94%p < 0.001) the voluntary consumption of alcohol by the rats. Conversely, injection into the VTA of the lentiviral vector coding for ADH greatly stimulated (2 to 3 fold p < 0.001) their voluntary ethanol consumption. Conclusions: The study strongly suggests that to generate reward and reinforcement, ethanol must be metabolized into acetaldehyde in the brain. Data suggest novel targets for interventions aimed at reducing chronic alcohol intake.     

Relationship of brain ethanol metabolism to the hypnotic effect of ethanol. II: Studies in selectively bred rats and mice

BACKGROUND: To clarify the role of brain acetaldehyde in the hypnotic effect of ethanol, we compared the ethanol-oxidizing capacity (rate of acetaldehyde accumulation) and catalase and aldehyde dehydrogenase activity in the brains of animals genetically selected for different sensitivities to the hypnotic effect of ethanol. METHODS: We used high, low, or control alcohol-sensitive rats (HAS, LAS, and CAS) and short- and long-sleep mice (SS and LS), as well as SS x LS recombinant inbred mice with known strain differences in mean duration of ethanol-induced sleep. We studied the rate of accumulation of acetaldehyde from ethanol in brain homogenates of these animals and correlated those values with their hypnotic sensitivity to ethanol. RESULTS: Acetaldehyde accumulation from ethanol was significantly higher in the brain homogenates from HAS rats and LS mice with high sensitivity to the hypnotic effect of ethanol in vivo, compared with LAS rats and SS mice with low sensitivity to ethanol. A correlation was found between the duration of ethanol-induced sleep and the in vitro rate of accumulation of ethanol-derived acetaldehyde in the brains of recombinant SS x LS mice strains. There was no correlation of sleep time with brain catalase levels. There were no line differences in brain catalase or aldehyde dehydrogenase or in alcohol or aldehyde dehydrogenase activity in livers of LAS, CAS, and HAS rats or in SS and LS mice. CONCLUSIONS: A correlation between the brain acetaldehyde accumulation, but not catalase levels, and the central effect of ethanol was demonstrated in animals genetically differing in initial sensitivity to the hypnotic effect of ethanol.     

Relationship of brain ethanol metabolism to the hypnotic effect of ethanol. I: Studies in outbred animals

BACKGROUND: This study was designed to investigate the relationship between the ethanol-oxidizing capacity of the brain, accumulation of acetaldehyde, and ethanol-induced hypnosis in animals in vivo. METHODS: Randomly outbred albino rats were treated with ethanol, and the duration of ethanol-induced loss of the righting response (sleep time) was measured. They were killed 2 weeks later (without further in vivo administration of ethanol), and brain homogenates were prepared to measure the accumulation of acetaldehyde from ethanol added in vitro. In a similar way, we determined the sleep time and, 5 days later, the rates of acetaldehyde accumulation in brains of heterogeneous mice. RESULTS: Significant correlations between the duration of ethanol-induced sleep and acetaldehyde accumulation in vitro were found. The Km value of the process of acetaldehyde accumulation was lower in long-sleeping, as compared with short-sleeping, rats. A similar result was also obtained in genetically heterogeneous mice. Animals with a longer duration of ethanol-induced sleep had a higher level of the accumulation of ethanol-derived acetaldehyde in brain homogenates, as compared with the short-sleeping mice. Rats and mice with the intermediate duration of ethanol-induced sleep had an intermediate value of acetaldehyde accumulation in brain homogenates. There was no correlation between brain catalase activity and ethanol-induced loss of the righting response in either the rats or the mice. CONCLUSIONS: This study is a direct demonstration of the positive correlation between ethanol-derived acetaldehyde accumulation in vitro in the brain and a central (behavioral) effect of alcohol in outbred rats and mice in vivo.     

Reward and relapse: complete gene-induced dissociation in an animal model of alcohol dependence

Background: In animal models of continuous alcohol self‐administration, in which physical dependence does not constitute the major factor of ethanol intake, 2 factors likely contribute to the perpetuation of alcohol self‐administration: (i) the rewarding effects of ethanol and (ii) the contextual conditioning cues that exist along with the process of self‐administration. Present studies are aimed at understanding the relative contribution of these factors on the perpetuation of heavy alcohol self‐administration, as an indication of relapse. Methods: Wistar‐derived UChB high ethanol drinker rats were allowed access to 10% ethanol and water on a 24‐hour basis. In initial studies, an anticatalase shRNA gene‐coding lentiviral vector aimed at inhibiting acetaldehyde generation was administered into the ventral tegmental area (VTA) of the animals prior to ethanol access. In subsequent studies, the lentiviral vector was administered to animals, which had consumed ethanol on a 24‐hour basis, or a 1‐hour basis, after the animals had reached high levels of ethanol intake for 60 to 80 days. In final studies, quinine (0.01%) was added to the ethanol solution to alter the conditioning taste/smell cues of alcohol that animals had chronically ingested. Results: Data indicate that the administration of an anticatalase vector into the VTA of naïve animals blocked reward and alcohol self‐administration, while it was, nevertheless, inactive in inhibiting alcohol self‐administration in rats that had been conditioned to ingest ethanol for over 2 months. The lack of inhibitory effect of the anticatalase vector on ethanol intake in animals that had chronically self‐administered ethanol was fully reversed when the contextual conditioning cues of the alcohol solution were changed. Conclusions: Data highlight the importance of conditioning factors in relapse and suggest that only abolishing or blunting it, along with long‐lasting pharmacological treatment to reduce ethanol reward, may have protracted effects in reducing alcohol self‐administration.     

Enzymatic mechanisms of ethanol oxidation in the brain

BACKGROUND: The exact enzymatic mechanisms of ethanol oxidation in the brain are still unclear. The catalase-mediated oxidation of ethanol was demonstrated in rat brain using incubation of brain homogenates with catalase inhibitors. The role of the alcohol dehydrogenase (ADH) or cytochrome P450-dependent system in this process is possible, but has not been confirmed. The objective of the study was to determine the contribution of the different enzymatic pathways to ethanol oxidation in brain homogenates from mice and rats. METHODS: Three approaches were used to investigate the enzymatic mechanisms of ethanol oxidation in the brain of rats and mice: (1) preincubation of brain homogenates with inhibitors of the ethanol-metabolizing enzymes (catalase, CYP2E1, ADH, and ALDH); (2) utilization of mice with genetic deficiency in ethanol-metabolizing enzymes (catalase, CYP2E1, or both enzymes); and (3) determination of ethanol oxidation in brain subcellular fractions known to have differential activity of ethanol-metabolizing enzymes. The ethanol-derived acetaldehyde (AC) and acetate were determined in brain samples by gas chromatography. RESULTS: The catalase inhibitors sodium azide (5 mM) and aminotriazole (5 mM) as well as CYP2E1 inhibitors diallyl sulfide (2 mM) and beta-phenethyl isothiocyanate (0.1 mM) lowered significantly the accumulation of the ethanol-derived AC and acetate in brain homogenates. The ADH inhibitor 4-methyl pyrazole (5 mM) significantly decreased the acetate but not the AC accumulation. Ethanol-derived AC accumulation in brain homogenates of acatalasemic mice was 47% of the control value, 91% in CYP2E1-null mice, and 24% in double mutants (with deficiency of both catalase and CYP2E1). The highest levels of ethanol oxidation were found in microsomal and peroxisomal subcellular brain fractions, where CYP2E1 and catalase are located, respectively. CONCLUSIONS: Catalase is the key enzyme of ethanol oxidation in the brain of rodents: it may be responsible for about 60% of the process. CYP2E1 plays an important role in ethanol oxidation in the rodent brains. Alcohol dehydrogenase plays a minor role, if any, in this process. Aldehyde dehydrogenase plays the crucial role in the further oxidation of ethanol-derived AC in the brain homogenates.     

A new assessment of the ability of oral ethanol to function as a reinforcing stimulus

BACKGROUND: In animal studies, the ability of ethanol to function as a reinforcer has been described as weak to moderate. This is in contrast with the human condition, where the consumption of alcoholic beverages can result in a variety of unwanted drinking behaviors. However, when the ethanol self-administration pattern is examined, animal studies over the last several years indicate that the ability of ethanol presentation to maintain behavior may be greater than originally assumed. METHODS: We reevaluated the ability of ethanol to function as a reinforcing stimulus in two paradigms by using an analysis of drinking bout characteristics. Data from previous studies that employed two self-administration models were analyzed. With the "dipper" model, small amounts of ethanol are presented after each completion of a response requirement; with the "sipper" model, the animal is allowed access to a drinking tube that contains ethanol for an extended period after completing a single response requirement. For both models, the consumption pattern could be characterized as occurring in a bout. Each drinking bout was divided into runs within the bout, and run rates and size were analyzed. As well, in the sipper model, the data on response requirement size were reviewed to demonstrate the ability of ethanol presentation to maintain high levels of responding in this model. RESULTS: From this assessment, we suggest that ethanol presentation in non-food- or non-water-restricted rats is as reinforcing as many other stimuli generally considered to be strong reinforcers (i.e., food in food-restricted rats). Using run size, we demonstrated that intake control appears to be regulated by shifts in run size during the bout and not run rate. CONCLUSIONS: Assessment of the pattern of ethanol consummatory bouts and the behaviors that precede them is critical in understanding how ethanol functions as a reinforcer. By using a drinking pattern analysis, the shifts in the momentary salience of the ethanol stimulus can be evaluated in these animal models. In addition, the separation of responding required to gain access to ethanol from consumption of ethanol demonstrated that ethanol presentation in this procedure can be a strong reinforcer for rats.     

The “First Hit” Toward Alcohol Reinforcement: Role of Ethanol Metabolites

This review analyzes literature that describes the behavioral effects of 2 metabolites of ethanol (EtOH): acetaldehyde and salsolinol (a condensation product of acetaldehyde and dopamine) generated in the brain. These metabolites are self‐administered into specific brain areas by animals, showing strong reinforcing effects. A wealth of evidence shows that EtOH, a drug consumed to attain millimolar concentrations, generates brain metabolites that are reinforcing at micromolar and nanomolar concentrations. Salsolinol administration leads to marked increases in voluntary EtOH intake, an effect inhibited by mu‐opioid receptor blockers. In animals that have ingested EtOH chronically, the maintenance of alcohol intake is no longer influenced by EtOH metabolites, as intake is taken over by other brain systems. However, after EtOH withdrawal brain acetaldehyde has a major role in promoting binge‐like drinking in the condition known as the “alcohol deprivation effect”; a condition seen in animals that have ingested alcohol chronically, are deprived of EtOH for extended periods, and are allowed EtOH re‐access. The review also analyzes the behavioral effects of acetate, a metabolite that enters the brain and is responsible for motor incoordination at low doses of EtOH. Also discussed are the paradoxical effects of systemic acetaldehyde. Overall, evidence strongly suggests that brain‐generated EtOH metabolites play a major role in the early (“first‐hit”) development of alcohol reinforcement and in the generation of relapse‐like drinking.     

Family History of Alcoholism and the Mediation of Alcohol Intake by Catalase: Further Evidence for Catalase as a Marker of the Propensity to Ingest Alcohol

Earlier studies have suggested that catalase activity (CA) may represent a biological marker of alcohol intake in animals and in humans. An initial study was designed to rule out the possibility that CA is induced as a function of acute alcohol intake. Subjects ( n = 80) were presented with either an alcohol (0.5 g/kg of body weight) or control solution, and asked to provide four 100-μl blood samples at 0.0, 0.5, 2.0, and 24.0 hr. Results showed no differences in CA between individuals who had received alcohol, and controls, even when the effects of previous drinking history were covaried out. This lack of effect of acute alcohol intake on the possible induction of CA further supported the notion that CA may be a viable marker of alcohol intake, rather than the converse. In the second study, the relation between CA and alcohol intake was investigated in individuals with a family history (FH) of alcoholism (FH+), and in those without a family history of alcoholism (FH-). Subjects ( n = 607) completed the Michigan Alcoholism Screening Questionnaire, the MacAndrew Scale, and the Concordia University Alcohol Screening Questionnaire; answered questions concerning their FH for alcoholism; and provided a 100-μl blood sample. Results showed that FH+ individuals had higher mean CA compared with FH- individuals. When individuals with FH+ were compared with those with FH-, differences in the pattern of relation between CA and alcohol intake were observed. Although a significant relation between CA and alcohol intake was obtained for both FH- and FH+ individuals, this relation was significantly higher ( p < 0.001) for individuals with FH+. Results from a multiple regression analyses suggested that CA in FH+ individuals made the highest single contribution to the variance, even after accounting for several additional variables. These results support the contention that CA may be a biological marker of the propensity of FH+ individuals to consume alcohol.     

Alcohol as a Risk Factor for Brain Damage: Neurologic Aspects

Alcohol consumption in excess affects the brain negatively, both immediately and in the long-term. Brain lesions in alcohol abusers are multiple and are multifactorial in origin. The toxic effect of ethanol, withdrawal from alcohol, nutritional deficits, and electrolyte disturbances, as well as liver damage, may contribute to the ethiopathogenesis of brain injury. The susceptibility of the brain to the negative effects of alcohol may be influenced by sex and age. The role of genetic factors and interactions of several licit and illicit drugs with alcohol needs further investigation.     

Sex differences in the neurotoxic effects of adenosine A1 receptor antagonism during ethanol withdrawal: reversal with an A1 receptor agonist or an NMDA receptor antagonist

Background: Neuronal adaptations that occur during chronic ethanol (EtOH) exposure have been observed to sensitize the brain to excitotoxic insult during withdrawal. The adenosine receptor system warrants further examination in this regard, as recent evidence has implicated adenosine receptor involvement in the behavioral effects of both EtOH exposure and withdrawal. Methods: The current studies examined effects of adenosine A₁ receptor manipulation on neuronal injury in EtOH‐naïve and EtOH‐withdrawn male and female rat hippocampal slice cultures. EtOH‐naïve and EtOH pretreated (43.1 to 26.9 mM from days 5 to 15 DIV) cultures were exposed to the A₁ receptor agonist 2‐Chloro‐N⁶‐cyclopentyladenosine (CCPA; 10 nM), the A₁ receptor antagonist 8‐Cyclopentyl‐1,3‐dipropylxanthine (DPCPX;10 nM), or the N‐methyl‐D‐aspartate (NMDA) receptor antagonist D,L,‐2‐amino‐5‐phosphovalerate (APV; 20 μM) at 15 days in vitro (DIV). Cytotoxicity was measured in the primary neuronal layers of the dentate gyrus, CA3 and CA1 hippocampal regions by quantification of propidium iodide (PI) fluorescence after 24 hours. Immunohistochemical analysis of A₁ receptor abundance was conducted in EtOH‐naïve and EtOH pretreated slice cultures at 15 DIV. Results: Twenty‐four hour exposure to DPCPX in EtOH‐naïve slice cultures did not produced neurotoxicity in any region of slice cultures. Though withdrawal from 10 day EtOH exposure produced no toxicity in either male or female slice cultures, exposure to DPCPX during 24 hours of EtOH withdrawal produced a marked increase in PI uptake in all hippocampal culture subregions in female cultures (to ～160% of control values). A significant effect for sex was observed in the CA1 region such that toxicity in females cultures exposed to the A₁ antagonist during withdrawal was greater than that observed in male cultures. These effects of DPCPX in EtOH withdrawn female and male slices were prevented by co‐exposure to either the A₁ agonist CCPA or the NMDA receptor antagonist APV for 24 hours. No differences in the abundance of A₁ receptors were observed in male and female EtOH‐naïve or EtOH pretreated cultures. Conclusions: The current findings suggest that the female hippocampus possesses an innate sensitivity to effects of EtOH exposure and withdrawal on neuronal excitability that is independent of hormonal influences. Further, this sex difference is not related to effects of EtOH exposure on A₁ receptor abundance, but likely reflects increased NMDA receptor‐mediated signaling downstream of A₁ inhibition in females.     

Ethanol, glutamate, and the ventral tegmental area--a commentary on: Ding, Engleman, Rodd, and McBride, "ethanol increases glutamate neurotransmission in the posterior ventral tegmental area of female wistar rats"

The recent study by Ding and colleagues (2012) utilized in vivo microdialysis to demonstrate that acute and repeated alcohol administration produces dose-dependent effects on extracellular levels of the glutamate in the posterior ventral tegmental area. These findings have important implications for interactions between glutamatergic and dopaminergic mediation of alcohol reinforcement in the ventral midbrain.     

Gene expression in brain: a window on ethanol dependence,neuroadaptation, and preference

This article represents the proceedings of a symposium at the 2002 joint RSA/ISBRA Conference in San Francisco, California. The organizer was Paula L. Hoffman and the co-chairs were Paula L. Hoffman and Michael Miles. The presentations were (1) Introduction and overview of the use of DNA microarrays, by Michael Miles; (2) DNA microarray analysis of gene expression in brains of P and NP rats, by Howard J. Edenberg; (3) Gene expression patterns in brain regions of AA and ANA rats, by Wolfgang Sommer; (4) Patterns of gene expression in brains of selected lines of mice that differ in ethanol tolerance, by Boris Tabakoff; (5) Gene expression profiling related to initial sensitivity and tolerance in gamma-protein kinase C mutants, by Jeanne Wehner; and (6) Gene expression patterns in human alcoholic brain: from microarrays to protein profiles, by Joanne Lewohl.     

Ethanol metabolism by HeLa cells transduced with human alcohol dehydrogenase isoenzymes: control of the pathway by acetaldehyde concentration

Background: Human class I alcohol dehydrogenase 2 isoenzymes (encoded by the ADH1B locus) have large differences in kinetic properties; however, individuals inheriting the alleles for the different isoenzymes exhibit only small differences in alcohol elimination rates. This suggests that other cellular factors must regulate the activity of the isoenzymes. Methods: The activity of the isoenzymes expressed from ADH1B*1, ADH1B*2, and ADH1B*3 cDNAs was examined in stably transduced HeLa cell lines, including lines which expressed human low K_m aldehyde dehydrogenase (ALDH2). The ability of the cells to metabolize ethanol was compared with that of HeLa cells expressing rat class I alcohol dehydrogenase (ADH) (HeLa‐rat ADH cells), rat hepatoma (H4IIEC3) cells, and rat hepatocytes. Results: The isoenzymes had similar protein half‐lives in the HeLa cells. Rat hepatocytes, H4IIEC3 cells, and HeLa‐rat ADH cells oxidized ethanol much faster than the cells expressing the ADH1B isoenzymes. This was not explained by high cellular NADH levels or endogenous inhibitors; but rather because the activity of the β1 and β2 ADHs was constrained by the accumulation of acetaldehyde, as shown by the increased rate of ethanol oxidation by cell lines expressing β2 ADH plus ALDH2. Conclusion: The activity of the human β2 ADH isoenzyme is sensitive to inhibition by acetaldehyde, which likely limits its activity in vivo. This study emphasizes the importance of maintaining a low steady‐state acetaldehyde concentration in hepatocytes during ethanol metabolism.     

Lateral asymmetries in the frontal brain: effects of depression and a family history of alcoholism in female adolescents

BACKGROUND: Subtle electroencephalographic (EEG) abnormalities have been detected among subjects with depressed affect. The present study attempted to discern whether these abnormalities reflect a main effect or an interaction between depression and either of two family history variables--a family history of alcoholism or a family history of depression. METHODS: The subjects were 151 adolescent females, aged 14 to 20 years, of whom 58 met DSM-III-R diagnostic criteria for a lifetime history of a major depressive episode. The electroencephalogram was recorded from 31 electrode sites while the subjects sat relaxed, with their eyes open, for 5 min. RESULTS: Analyses of EEG data revealed that a personal history of depression and a family history of alcoholism had opposite effects on the EEG power spectrum. Depression was associated with an increase in alpha power (7.5-12.5 Hz). In contrast, a family history of alcoholism was associated with an increase in fast beta power (19-30 Hz) and a decrease in theta power (4-7 Hz). There were no significant main or interactive effects of a family history of depression. Current source density topographic analyses of the significant group differences in alpha and fast beta power demonstrated that the effects of depression could be localized to the right frontal brain, whereas the effects of a family history of alcoholism were localized to the left frontal area. CONCLUSIONS: The laterally opposite effects of depression and a family history of alcoholism suggest a high level of functional differentiation of the frontal brain. They also suggest that the different neurophysiological substrates of depression and familial risk can be distinguished through the use of modern methods of EEG source localization.     

IgAs against acetaldehyde-modified red cell protein as a marker of ethanol consumption in male alcoholic subjects, moderate drinkers, and abstainers

BACKGROUND: Alcohol abuse has been shown to result in the production of antibodies against acetaldehyde-modified epitopes in proteins. However, as yet, only limited information has been available on the clinical usefulness of such responses as markers of hazardous drinking. METHODS: We developed an ELISA to measure specific IgAs against acetaldehyde-protein adducts. This method was evaluated in cross-sectional and follow-up studies on male heavy drinkers with a current ethanol consumption of 40 to 540 g/d (n=40), moderate drinkers consuming 1 to 40 g/d (n=25), and abstainers (n=16). The clinical assessments included detailed interviews on the amounts and patterns of ethanol consumption and various biochemical markers of alcohol abuse and liver function. RESULTS: The mean antiadduct IgAs (198+/-28 U/L) in the alcohol abusers were significantly higher than those in the moderate drinkers (58+/-11 U/L, p<0.001) or abstainers (28+/-8 U/L, p<0.001). The values of moderate drinkers were also higher than those in abstainers (p<0.05). The amount of ethanol consumed during the period of 1 month preceding blood sampling correlated strongly with antiadduct IgAs (r=0.67, p<0.001). The sensitivity (73%) and specificity (94%) of this marker were found to exceed those of the conventional laboratory markers of alcohol abuse in comparisons contrasting heavy drinkers with abstainers although not in comparisons contrasting heavy drinkers with moderate drinkers. During abstinence, antiadduct IgAs disappeared with a mean rate of 3% per day. In additional analyses of possible marker combinations, antiadduct IgAs, together with CDT, were found to provide the highest sensitivity and specificity. CONCLUSIONS: Measurements of antiadduct IgAs may provide a new clinically useful marker of alcohol abuse, providing a close relationship between marker levels and the actual amounts of recent ethanol ingestion.     

Blockade of cue-induced brain activation of abstinent alcoholics by a single administration of amisulpride as measured with fMRI

BACKGROUND: Once alcohol dependence is established, alcohol-associated cues may induce dopamine release in the reward system, which is accompanied by alcohol craving and may lead to relapse. In cocaine addicts, dopamine release in the thalamus was positively correlated with cocaine craving. We tested the effects of the atypical dopamine D(2/3) blocker amisulpride on cue-induced brain activation in a functional magnetic resonance imaging (fMRI) paradigm. METHODS: Alcohol-associated and neutral pictures were presented in a block design to 10 male abstinent alcoholics (1-3 weeks after detoxification) and 10 healthy men during fMRI. The fMRI scans were acquired before and 2 hours after the oral application of 400 mg amisulpride. Before and after each scan, alcohol craving was measured with visual analogue scales. RESULTS: Before the application of amisulpride, alcohol versus control cues elicited a higher blood oxygen level-dependent (BOLD) signal in the left frontal and orbitofrontal lobe, left cingulate gyrus, bilateral parietal lobe, and bilateral hippocampus in alcoholics compared with healthy controls. After amisulpride, alcoholics showed a reduced activation in the right thalamus compared with the first scan. Alcoholics no longer showed significant differences in their cue-elicited BOLD response after amisulpride medication compared with medication-free controls. Self-reported craving was not affected by amisulpride medication. CONCLUSIONS: Amisulpride medication was associated with reduced cue-induced activation of the thalamus, a brain region closely connected with frontostriatal circuits that regulate behavior and may influence relapse risk.