María Elena Quintanilla

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. In F2 UChA × UChB rats (in which all genes are ‘shuffled’), a high acute tolerance of the offspring predicts higher drinking than a low acute tolerance. It is further shown that high‐drinker animals ‘learn’ to drink, starting from consumption levels that are one half of the maximum consumptions reached after 1 month of unrestricted access to 10% ethanol and water. It is likely that acquired tolerance is at the basis of the increases in ethanol consumption over time. (2) Non‐drinker rats carry a previously unreported allele of aldehyde dehydrogenase‐2 (Aldh2) that encodes an enzyme with a low affinity for Nicotinamide‐adenine‐dinuclectide (NAD+) (Aldh22), while drinker rats present two Aldh2 alleles (Aldh21 and Aldh23) with four‐ to fivefold higher affinities for NAD+. Further, the ALDH2 encoded by Aldh21 also shows a 33% higher Vmax than those encoded by Aldh22 and Aldh23. Maximal voluntary ethanol intakes are the following: UChA Aldh22/Aldh22 = 0.3–0.6 g/kg/day; UChB Aldh23/Aldh23 = 4.5–5.0 g/kg/day; UChB Aldh21/Aldh21 = 7.0–7.5 g/kg/day. In F2 offspring of UChA × UChB, the Aldh22/Aldh22 genotype predicts a 40–60% of the alcohol consumption. Studies also show that the low alcohol consumption phenotype of Aldh22/Aldh22 animals depends on the existence of a maternally derived low‐activity mitochondrial reduced form of nicotinamide‐adenine‐dinucleotide (NADH)‐ubiquinone complex I. The latter does not influence ethanol consumption of animals exhibiting an ALDH2 with a higher affinity for NAD+. An illuminating finding is the existence of an ‘acetaldehyde burst’ in animals with a low capacity to oxidize acetaldehyde, being fivefold higher in UChA than in UChB animals. We propose that such a burst results from a great generation of acetaldehyde by alcohol dehydrogenase in pre‐steady‐state conditions that is not met by the high rate of acetaldehyde oxidation in mitochondria. The acetaldehyde burst is seen despite the lack of differences between UChA and UChB rats in acetaldehyde levels or rates of alcohol metabolism in steady state. Inferences are drawn as to how these studies might explain the protection against alcoholism seen in humans that carry the high‐activity alcohol dehydrogenase but metabolize ethanol at about normal rates.

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.

Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model.

Humans who carry a point mutation in the gene coding for alcohol dehydrogenase‐lB (ADH1B*2; Arg47His) are markedly protected against alcoholism. Although this mutation results in a 100‐fold increase in enzyme activity, it has not been reported to cause higher levels of acetaldehyde, a metabolite of ethanol known to deter alcohol intake. Hence, the mechanism by which this mutation confers protection against alcoholism is unknown. To study this protective effect, the wild‐type rat cDNA encoding rADH‐47Arg was mutated to encode rADH‐47His, mimicking the human mutation. The mutated cDNA was incorporated into an adenoviral vector and administered to genetically selected alcohol‐preferring rats. The Vmax of rADH‐47His was 6‐fold higher (P<0.001) than that of the wild‐type rADH‐47Arg. Animals transduced with rAdh‐47His showed a 90% (P<0.01) increase in liver ADH activity and a 50% reduction (P< 0.001) in voluntary ethanol intake. In animals transduced with rAdh‐47His, administration of ethanol (1g/kg) produced a short‐lived increase of arterial blood acetaldehyde concentration to levels that were 3.5‐ to 5‐fold greater than those in animals transduced with the wild‐type rAdh‐47Arg vector or with a noncoding vector. This brief increase (burst) in arterial acetaldehyde concentration after ethanol ingestion may constitute the mechanism by which humans carrying the ADH1B*2 allele are protected against alcoholism.—Rivera‐Meza, M., Quin‐tanilla, M. E., Tampier, L., Mura, C. V., Sapag, A., Israel, Y. Mechanism of protection against alcoholism by an alcohol dehydrogenase polymorphism: development of an animal model. FASEB J. 24, 266–274 (2010). www.fasebj.org

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.

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 (Aldh22) of mitochondrial aldehyde dehydrogenase that does not exist in high-alcohol consumers, which mostly carry the Aldh21 allele. The enzyme coded by Aldh22 has a four- to five-fold lower affinity for NAD+ than that coded by Aldh21. The present study was designed to determine whether these polymorphisms account for differences in voluntary ethanol intake and to investigate the biological mechanisms involved. Low-drinker F0Aldh22/Aldh22 rats were crossed with high-drinker F0Aldh21/Aldh21 rats to obtain an F1 generation, which was intercrossed to obtain an F2 generation that segregates the Aldh2 alleles from other genes that may have been coselected in the breeding for each phenotype. Data show that, with a mixed genetic background, F2Aldh21/Aldh21 rats voluntarily consume 65% more alcohol (P<0.01) than F2Aldh22/Aldh22 rats. A major phenotypic difference was a five-fold higher (P<0.0025) peak blood acetaldehyde level following ethanol administration in the lower drinker F2Aldh22/Aldh22 compared to the higher drinker F2Aldh21/Aldh21 animals, despite the existence of identical steady-state levels of blood acetaldehyde in animals of both genotypes. Polymorphisms in Aldh2 play an important role in: (i) determining peak blood acetaldehyde levels and (ii) modulating voluntary ethanol consumption. We postulate that the markedly higher peak of blood acetaldehyde generated in Aldh22/Aldh22 animals is aversive, leading to a reduced alcohol intake in Aldh22/Aldh22 versus that in Aldh21/Aldh21 animals.

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 en‐ zyme with a low affinity for NAD+, whereas rats bred for high‐alcohol consumption do not present the Aldh22 al‐ lele. This enzyme is inserted into mitochondria, where NADH‐ubiquinone oxidoreductase (complex I) regener‐ ates NAD+. The possible influence of complex I on ALDH2 activity and voluntary ethanol intake was investi‐ gated. Homozygous Aldh22/Aldh22 rats derived from a line of high‐drinker F0 females (and low‐drinker F0 males) showed a markedly higher ethanol consumption (3.9±0.5 g•kg‐1•day‐1) than homozygous animals derived from a line of low‐drinker F0 females (and high‐drinker F0 males) (1.8±0.4 g•kg‐1•day‐1). Mitochondria of F2 rats derived from high alcohol‐consuming females were more active in oxidizing substrates that generate NADH for complex I than were mitochondria derived from low alcohol‐con‐ suming females, leading in the former to higher rates of acetaldehyde metabolism and to a reduced aversion to ethanol. This is the first demonstration that maternally derived genes can either allow or counteract the pheno‐ typic expression of a mutated gene in the context of alcohol abuse or alcoholism.—Quintanilla, M. E., Tampier, L., Valle‐Prieto, A., Sapag, A., Israel, Y. Com‐ plex I regulates mutant mitochondrial aldehyde dehydro‐ genase activity and voluntary ethanol consumption in rats. FASEBJ. 19, 36‐42 (2005)

Activated mesenchymal stem cell administration inhibits chronic alcohol drinking and suppresses relapse-like drinking in high-alcohol drinker rats

Neuroinflammation has been reported to follow chronic ethanol intake and may perpetuate alcohol consumption. Present studies determined the effect of human mesenchymal stem cells (hMSCs), known for their anti‐inflammatory action, on chronic ethanol intake and relapse‐like ethanol intake in a post‐deprivation condition. Rats were allowed 12–17 weeks of chronic voluntary ethanol (10% and 20% v/v) intake, after which a single dose of activated hMSCs (5 × 105) was injected into a brain lateral ventricle. Control animals were administered vehicle. After assessing the effect of hMSCs on chronic ethanol intake for 1 week, animals were deprived of ethanol for 2 weeks and thereafter an ethanol re‐access of 60 min was allowed to determine relapse‐like intake. A single administration of activated hMSCs inhibited chronic alcohol consumption by 70% (P < 0.001), an effect seen within the first 24 hours of hMSCs administration, and reduced relapse‐like drinking by 80% (P < 0.001). In the relapse‐like condition, control animals attain blood ethanol (‘binge‐like’) levels >80 mg/dl. The single hMSC administration reduced relapse‐like blood ethanol levels to 20 mg/dl. Chronic ethanol intake increased by 250% (P < 0.001) the levels of reactive oxygen species in hippocampus, which were markedly reduced by hMSC administration. Astrocyte glial acidic fibrillary protein immunoreactivity, a hallmark of neuroinflammation, was increased by 60–80% (P < 0.001) by chronic ethanol intake, an effect that was fully abolished by the administration of hMSCs. This study supports the neuroinflammation‐chronic ethanol intake hypothesis and suggest that mesenchymal stem cell administration may be considered in the treatment of alcohol use disorders.

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. The aim of this study was to identify the genetic basis for the difference in the reoxidation of NADH in these nondrinker (UChA) and drinker (UChB) rats. Methods Seven mitochondrial genes and two chromosome X genes encoding complex I subunits from rats of both lineages were amplified from liver DNA and sequenced. Results The UChA and UChB rat lines differ in their Nd2, Nd4, Nd5 and Nd6 mitochondrial genes and in the encoded proteins. Most noteworthy are ND2 and ND4 whose amino acid variations lead to changes in three-dimensional structure models. The ND2 proteins also differ in the number of predicted transmembrane domains. The Nd1 and Nd3 genes have silent substitutions, whereas Nd4L and the exonic sequences of the nuclear genes Ndufa1 and Ndufb11 show no differences between the UChA and UChB lines. Conclusion Amino acid variations in four complex I subunits encoded in the mitochondrial genome may contribute to explain the differences between UChA and UChB rats in their capacity to reoxidize NADH and in their alcohol intake, suggesting that mitochondrial genes may constitute maternal factors of alcoholism.