Hairpin Ribozyme Genes Curtail Alcohol Drinking: from Rational Design to in vivo Effects in the Rat.

Ribozyme genes were designed to reduce voluntary alcohol drinking in a rat model of alcohol dependence. Acetaldehyde generated from alcohol in the liver is metabolized by the mitochondrial aldehyde dehydrogenase (ALDH2) such that diminishing ALDH2 activity leads to the aversive effects of blood acetaldehyde upon alcohol intake. A stepwise approach was followed to design genes encoding ribozymes targeted to the rat ALDH2 mRNA.

The sequenced rat brain transcriptome--its use in identifying networks predisposing alcohol consumption.

Unlabelled: A quantitative genetic approach, which involves correlation of transcriptional networks with the phenotype in a recombinant inbred (RI) population and in selectively bred lines of rats, and determination of coinciding quantitative trait loci for gene expression and the trait of interest, has been applied in the present study. In this analysis, a novel approach was used that combined DNA-Seq data, data from brain exon array analysis of HXB/BXH RI rat strains and six pairs of rat lines selectively bred for high and low alcohol preference, and RNA-Seq data (including rat brain transcriptome reconstruction) to quantify transcript expression levels, generate co-expression modules and identify biological functions that contribute to the predisposition of consuming varying amounts of alcohol. A gene co-expression module was identified in the RI rat strains that contained both annotated and unannotated transcripts expressed in the brain, and was associated with alcohol consumption in the RI panel.

Long-term inhibition of ethanol intake by the administration of an aldehyde dehydrogenase-2 (ALDH2)-coding lentiviral vector into the ventral tegmental area of rats.

Previous studies suggest that acetaldehyde generated from ethanol in the brain is reinforcing. The present studies tested the feasibility of achieving a long-term reduction of chronic and post-deprivation binge ethanol drinking by a single administration into the brain ventral tegmental area (VTA) of a lentiviral vector that codes for aldehyde dehydrogenase-2 (ALDH2), which degrades acetaldehyde. The ALDH2 gene coding vector or a control lentiviral vector were microinjected into the VTA of rats bred for their alcohol preference.

Gene specific modifications unravel ethanol and acetaldehyde actions.

Ethanol is metabolized into acetaldehyde mainly by the action of alcohol dehydrogenase in the liver, while mainly by the action of catalase in the brain. Aldehyde dehydrogenase-2 metabolizes acetaldehyde into acetate in both organs. Gene specific modifications reviewed here show that an increased liver generation of acetaldehyde (by transduction of a gene coding for a high-activity liver alcohol dehydrogenase ADH1(*)B2) leads to increased blood acetaldehyde levels and aversion to ethanol in animals.

The alcohol deprivation effect: marked inhibition by anticatalase gene administration into the ventral tegmental area in rats.

Background: Animals that have chronically consumed alcohol and are subsequently deprived of it markedly increase their intake above basal levels when access to alcohol is reinstated. Such an effect, termed the alcohol deprivation effect (ADE), has been proposed to reflect (i) an obsessive-compulsive behavior, (ii) craving, or (iii) an increased reinforcing value of ethanol (EtOH). It has been reported that acetaldehyde, a highly reinforcing metabolite of EtOH, is generated in the brain by the action of catalase.

Varenicline and cytisine: two nicotinic acetylcholine receptor ligands reduce ethanol intake in University of Chile bibulous rats.

Rationale: Neuronal nicotinic acetylcholine receptors (nAChRs) are pharmacological targets that have recently been implicated in the reinforcing effects of many drugs of abuse, including ethanol. Varenicline and cytisine are nAChR partial agonists in clinical use as smoking cessation aids. However, their efficacies to reduce alcohol consumption have not been fully studied.

Reduction of ethanol consumption in alcohol-preferring rats by dual expression gene transfer.

Aims: To mimic, in an animal model of alcoholism, the protective phenotype against alcohol consumption observed in humans carrying a fast alcohol dehydrogenase (ADH1B*2) and an inactive aldehyde dehydrogenase (ALDH2*2).

Methods: We developed a multiple expression cassette adenoviral vector (AdV-ADH/asALDH2) encoding both a fast rat ADH and an antisense RNA against rat ALDH2. A control adenoviral vector (AdV-C) containing intronic non-coding DNA was also developed.

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.

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 (1g/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 (~6-7g/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.

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.

Acetaldehyde burst protection of ADH1B*2 against alcoholism: an additional hormesis protection against esophageal cancers following alcohol consumption?

This account of recent work presented at the 4th International Symposium on Alcohol Pancreatitis and Cirrhosis reports animal studies aimed at determining the role of the "acetaldehyde burst," generated shortly upon ethanol intake, as the mechanism of protection against alcoholism conferred by the ADH1B*2 polymorphism. Literature studies discussed suggest an additional role of the acetaldehyde burst on the paradoxical (hormesis) protection of the ADH1B*2 polymorphism against esophageal cancers in alcoholics.

Polymorphisms in mitochondrial genes encoding complex I subunits are maternal factors of voluntary alcohol consumption in the rat.

Objective: Alcohol is detoxified in the liver by oxidizing enzymes that require nicotinamide adenine dinucleotide (NAD+) such that, in the rat, the availability of NAD+ contributes to control voluntary ethanol intake. The UChA and UChB lines of Wistar rats drink low and high amounts of ethanol respectively and differ in the capacity of their mitochondria to oxidize NADH into NAD+. This function resides in complex I of the respiratory chain and its variation is linked to genes transmitted through the maternal line.

Effect of concurrent saccharin intake on ethanol consumption by high-alcohol-drinking (UChB) rats.

This study examined the effect of concurrent presentation of a highly palatable saccharin solution on ethanol consumption during the acquisition or maintenance of ethanol drinking by high-alcohol-drinking (UChB) rats. Rats were exposed to ethanol (10% v/v) and water under a home cage, two-bottle, free-choice regimen with unlimited access for 24 hours/day. After 7 days (acquisition) of ethanol exposure, a third bottle containing saccharin (0.

Ethanol induces stronger dopamine release in nucleus accumbens (shell) of alcohol-preferring (bibulous) than in alcohol-avoiding (abstainer) rats.

Several studies on the differences between ethanol-preferring versus non-preferring rat lines suggest an innate deficit in the mesolimbic dopaminergic system as an underlying factor for ethanol volition. Rats would try to overcome such deficit by engaging in a drug-seeking behaviour, when available, to drink an ethanol solution over water. Thus, in the present study we compared the effect of a single dose of ethanol (1 g/kg, i.

Tolerance to disulfiram induced by chronic alcohol intake in the rat.

Background: Disulfiram, an inhibitor of aldehyde dehydrogenase used in the treatment of alcoholism, is an effective medication when its intake is supervised by a third person. However, its therapeutic efficacy varies widely, in part due to the fact that disulfiram is a pro-drug that requires its transformation into an active form and because it shows a wide range of secondary effects which often prevent the use of doses that ensure full therapeutic effectiveness. In this preclinical study in rats we report the development of tolerance to disulfiram induced by the chronic ingestion of ethanol, an additional source of variation for the actions of disulfiram with possible therapeutic significance, We also addresses the likely mechanism of this effect.

Baclofen reduces ethanol intake in high-alcohol-drinking University of Chile bibulous rats.

ABSTRACT Treatment with gamma-aminobutiric acid (GABA(B)) receptor agonist, +/-baclofen, has been shown to reduce ethanol intake in selectively bred Sardinian alcohol-preferring rats. The general goal of the present study was to characterize the high ethanol consumption high-alcohol-drinking University of Chile bibulous (UChB) rats with regard to the anti-alcohol effect of GABA(B) receptor stimulation. UChB rats were treated with the more active enantiomer of baclofen [R(+)-baclofen; at a dose of 1.

Gene therapy reduces ethanol intake in an animal model of alcohol dependence.

Background: Some gene polymorphisms strongly protect against the development of alcoholism. A large proportion of East Asians carry a protective inactivating mutation in aldehyde dehydrogenase (ALDH2*2). These subjects display high levels of blood acetaldehyde when consuming alcohol, a condition that exerts a 66 to 99% protection against alcohol abuse and alcoholism.

Combined effects of aldehyde dehydrogenase variants and maternal mitochondrial genes on alcohol consumption.

Two lines of rats bred to differ in their voluntary alcohol consumption--the alcohol-abstaining UChA rats and the alcohol-drinking UChB rats--differ in how effectively toxic acetaldehyde is removed during alcohol metabolism. UChB animals carry efficient variants of the aldehyde dehydrogenase 2 (ALDH2) genes and have active mitochondria, resulting in fast removal of acetaldehyde. UChA animals, in contrast, carry less efficient ALDH2 variants and less active mitochondria, which result in transient elevations of acetaldehyde levels after alcohol ingestion.

Dopamine release in the nucleus accumbens (shell) of two lines of rats selectively bred to prefer or avoid ethanol.

Lower tissue levels of dopamine and 5-hydroxytryptamine (5-HT) have been found in the nucleus accumbens of alcohol-naïve rats selectively bred to prefer ethanol than in rats bred to avoid it. These findings have led to the hypothesis that differences in the dopamine and 5-HT tone may be linked to ethanol preference. In the present study we used the in vivo microdialysis technique to determine the actual extracellular levels of dopamine, its metabolites 3,4-dihydroxyphenyl acetaldehyde (DOPALD), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-HT and 5-hydroxyindolacetic acid (5-HIAA) in the shell of nucleus accumbens of rat lines selectively bred as either high-ethanol (UChB) or low-ethanol (UChA) drinkers.

Sex differences, alcohol dehydrogenase, acetaldehyde burst, and aversion to ethanol in the rat: a systems perspective.

Individuals who carry the most active alcohol dehydrogenase (ADH) isoforms are protected against alcoholism. This work addresses the mechanism by which a high ADH activity leads to low ethanol intake in animals. Male and female ethanol drinker rats (UChB) were allowed access to 10% ethanol for 1 h.

The UChA and UChB rat lines: metabolic and genetic differences influencing ethanol intake.

Ethanol non-drinker (UChA) and drinker (UChB) rat lines derived from an original Wistar colony have been selectively bred at the University of Chile for over 70 generations. Two main differences between these lines are clear. (1) Drinker rats display a markedly faster acute tolerance than non-drinker rats.

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.

Polymorphisms in the mitochondrial aldehyde dehydrogenase gene (Aldh2) determine peak blood acetaldehyde levels and voluntary ethanol consumption in rats.

Dependence on alcohol, a most widely used drug, has a heritability of 50-60%. Wistar-derived rats selectively bred as low-alcohol consumers for many generations present an allele (Aldh2(2)) of mitochondrial aldehyde dehydrogenase that does not exist in high-alcohol consumers, which mostly carry the Aldh2(1) allele. The enzyme coded by Aldh2(2) has a four- to five-fold lower affinity for NAD than that coded by Aldh2(1).

Complex I regulates mutant mitochondrial aldehyde dehydrogenase activity and voluntary ethanol consumption in rats.

Animals selectively bred for a desirable trait retain wanted genes but exclude genes that may counteract the expression of the former. The possible interactions between selected and excluded genes cannot be readily studied in transgenic or knockout animals but may be addressed by crossing animals bred for opposite traits and studying the F2 offspring. Ninety-seven percent of Wistar-derived rats selectively bred for their voluntary low-alcohol consumption display a mutated nuclear allele of aldehyde dehydrogenase Aldh22 that encodes an enzyme with a low affinity for NAD+, whereas rats bred for high-alcohol consumption do not present the Aldh22 allele.