Genetics and differential expression of NADH:ubiquinone oxidoreductase B8 subunit in brains of genetic strains of mice differing in voluntary alcohol consumption

Inbred strains of mice remain a valuable resource for genetic dissection of complex traits including responses to drugs and chemicals, particularly alcohol. As a novel source of candidate genes for further analysis, we have used mRNA differential displays to identify genes with differential expression in the brains of ethanol-preferring (C57BL/6J) vs. ethanol-avoiding (A/J, BALB/c, and DBA/2J) strains, with and without ethanol i.p. treatments (4 g/kg). We report on one such gene, NADH:ubiquinone oxidoreductase B8 subunit, that has a higher expression in the C57BL/6J. Further, its expression also increases following ethanol treatment as compared to the three alcohol-avoiding strains. This regulatory feature follows three single nucleotide polymorphisms (SNPs) in the promoter region across the four strains studied. The four strains represent only two haplotypes, one C57BL/6J-specific and the other found in the three alcohol-avoiding strains. Interestingly, one of the observed SNPs (-687 A/G) is located in the putative TFIID binding site with potential to regulate the expression of this gene and contribute to genotype-specific alcohol responses and effects involving reactive oxygen species (ROS).     

Genetic regulation of gene-specific mRNA by ethanolin vivo and its possible role in ethanol preference in a cross with RI lines in mice

Relative ethanol preference is a well-recognized phenotype in a number of species, including mice, but the molecular basis for this phenotype remains speculative. We generated novel recombinant inbred (RI) mouse lines from C57BL/6J (ethanol preferring) and BALB/c (ethanol avoiding) strains and evaluated the effect of ethanol feeding on the mRNA levels of three genes (Adh-1, Adh-2, andCas-1) of alcohol metabolism. Ethanol feeding affects the mRNA levels of all three genes in both a gene- and a genotype-specific manner. The effect of ethanol feeding onAhd-2 mRNA, in particular, is highly correlated with the relative ethanol acceptance of the genotypes. DNA sequencing of ∼500 bp of the 5′ upstream region of theAhd-2 gene has yielded identical sequence for the two strains and the genetically determined associated factors are hypothesized to be regulatory proteins. Quantitative trait locus analysis on the RI lines should lead to the molecular characterization and mapping of such gene-specific regulatory factors.     

High genetic susceptibility to ethanol withdrawal predicts low ethanol consumption

C57BL/6J (B6) inbred mice are well known to drink large amounts of alcohol (ethanol) voluntarily and to have only modest ethanol-induced withdrawal under fixed dose conditions. In contrast, DBA/2J (D2) mice are ``teetotallers' and exhibit severe ethanol withdrawal. Speculation that an inverse genetic relationship existed between these two traits was substantiated by meta-analysis of existing data collected in multiple genetic models, including large panels of standard and recombinant inbred strains, their crosses, and selectively bred mouse lines. Despite methodological differences among laboratories in measurement of both preference drinking and withdrawal, a nearly universal finding was that genotypes consuming large amounts of 10% ethanol (calculated as g/kg/day) during two-bottle choice preference drinking were genetically predisposed to low withdrawal scores in independent studies after either acute or chronic ethanol treatment. Conversely, low-drinking genotypes had higher withdrawal severity scores. The genetic relationship appears to be strongest in populations derived from B6 and D2, where data from more genotypes (BXD RIs, B6D2F₂s, BXD RI F₁s, and B6D2F₂-derived selectively bred lines) were available for analysis. Gene mapping studies in these populations identified four chromosome regions [on Chromosomes (Chrs) 1, 2, 4, and 15] where genes might potentially influence both traits. Among genotypes with greater genetic diversity (for example, a panel of standard inbred strains or selectively bred lines), the relationship was less pronounced. Thus, reduced susceptibility to the development of high alcohol use may be supported by increased genetic susceptibility to ethanol withdrawal symptoms. Received: 15 September 1998 / Accepted: 8 October 1998     

Differential effects of pentobarbital and ethanol in mice

The duration of the loss of righting reflex (RR) after ethanol, 4 g/kg, intraperitoneally (i.p.), was significantly longer in “long-sleep” (LS) than in “short-sleep” (SS) mice. This effect was shown to be correlated with differences in brain sensitivities to ethanol. In contrast, pentobarbital sodium (PB), 50 mg/kg, i.p., produced a significantly longer loss of RR in SS than in LS mice. The PB concentrations in the brain were the same in both mouse strains at the time of RR recovery suggesting equal sensitivities of the central nervous systems to PB. The rates of disappearance of PB from the blood were the same in both strains, but the apparent volume of distribution of PB in the LS strain was greater than in SS mice.In addition, C57BL/6J mice were found to be more sensitive than DBA/2J mice to PB, 50 mg/kg. In contrast, C57BL mice are known to be less sensitive than the DBA strain to ethanol. The PB concentration in the brain of DBA mice at the recovery of the RR was significantly greater than in C57BL mice. The apparent volumes of distribution of PB were not different in the two strains, but the rate of disappearance of PB from the blood of C57BL mice was significantly greater than for the DBA strain. In conclusion, factors which govern the brain sensitivities of selected mouse strains to ethanol and pentobarbital may not be equivalent.     

Gamma aminobutyric acid in different strains of mice effect of ethanol

Four strains of mice (C57BL/6J, DBA/2J, LS and SS), which differ in their voluntary intake and/or neural sensitivity to ethanol, were examined for the contents of γ-aminobutyric acid (GABA) in various brain regions. The effect of an acute dose of ethanol (4 g/kg) on the contents of this amino acid neurotransmitter in these mice were studied. The objective is to determine if the differences in sensitivity to ethanol may be reflected in differences in brain GABA contents in these animals.Results indicate that a previously documented ethanol-induced elevation of GABA in the whole mouse brain is a phenomenon observed in a variety of brain regions. No correlation was observed between GABA contents in the brain and neural sensitivity to ethanol.     

Acamprosate-responsive brain sites for suppression of ethanol intake and preference

Acamprosate suppresses alcohol intake and craving in recovering alcoholics; however, the central sites of its action are unclear. To approach this question, brain regions responsive to acamprosate were mapped using acamprosate microimplants targeted to brain reward and circadian areas implicated in alcohol dependence. mPer2 mutant mice with nonfunctional mPer2, a circadian clock gene that gates endogenous timekeeping, were included, owing to their high levels of ethanol intake and preference. Male wild-type (WT) and mPer2 mutant mice received free-choice (15%) ethanol/water for 3 wk. The ethanol was withdrawn for 3 wk and then reintroduced to facilitate relapse. Four days before ethanol reintroduction, mice received bilateral blank or acamprosate-containing microimplants releasing ～50 ng/day into reward [ventral tegmental (VTA), peduculopontine tegmentum (PPT), and nucleus accumbens (NA)] and circadian [intergeniculate leaflet (IGL) and suprachiasmatic nucleus (SCN)] areas. The hippocampus was also targeted. Circadian locomotor activity was measured throughout. Ethanol intake and preference were greater in mPer2 mutants than in wild-type (WT) mice (27 g·kg(-1)·day(-1) vs. 13 g·kg(-1)·day(-1) and 70% vs. 50%, respectively; both, P < 0.05). In WTs, acamprosate in all areas, except hippocampus, suppressed ethanol intake and preference (by 40-60%) during ethanol reintroduction. In mPer2 mutants, acamprosate in the VTA, PPT, and SCN suppressed ethanol intake and preference by 20-30%. These data are evidence that acamprosate's suppression of ethanol intake and preference are manifest through actions within major reward and circadian sites.     

Rapid tolerance to ethanol and high voluntary alcohol consumption in mice selected for brain weight

Mice of two strains selected for small and large brain weight (SB and LB, respectively) had free access to 10% alcohol and water within three months. At the end of this period, they consumed alcohol in daily dose of 6.9 +/- 0.9 and 7.5 +/- 0.8 g/kg, respectively. After a period of imposed three-day abstinence, the alcohol consumption by the mice of these strains increased by 68.6 and 49.3%, respectively. Exploratory behavior of independent groups of mice from these strains was studied in the closed cross-maze. The animals were injected with ethanol (2.4 g/kg, i.p.) or vehicle twice with a weekly interval. In SB mice, the first ethanol administration increased the total time of maze exploration and the number of stereotyped visits. The second ethanol administration did not increase the time of exploration but increased the number of stereotyped visits even to the greater extent. The latter indicates the development of rapid tolerance and sensitization of these behaviors to the drug, respectively. The ethanol administration inhibited exploratory patrolling behavior and defecations. In LB mice, both the first and second ethanol administrations increased the number of stereotyped visits and decreased the exploration time and the number of defecations. The results do not support the psychomotor stimulant hypothesis of alcohol addiction. It is proposed that SB and LB mice may serve as models for Cloninger's types 1 and 2 alcoholics and may be useful for investigation of neuropharmacological mechanisms of stimulatory and inhibitory effects of ethanol.     

Genetic considerations in the effects of ethanol in mice. I. Genotype-dependent alterations in alcohol dehydrogenase activity

Most genetic studies on individual and racial differences in sensitivity to alcohol intoxication have concentrated on genetic variations associated with structural genes for the enzymes involved in alcohol metabolism, including alcohol dehydrogenase (ADH; E.C. 1.1.1.1). We studied the ethanol-induced regulation of ADH following chronic administration of ethanol in mice. Newly weaned males from six inbred strains (BALB/c, C3H/HeSnJ, C3H/S, C57BL/6J, S.W., and 129/ReJ) were subjected to ethanol administration. Alterations in the level of liver ADH activity, relative to matched littermate controls, were evaluated. The change in ADH activity was found to be strain (genotype) specific, which may explain the contradictory results in the literature. Strains which showed induction of ADH activity, in general, reflected a strain-specific time-dependent profile. Strains which showed repression, however, were independent in the degree of repression to the duration of ethanol exposure. Such variable, ethanol-induced regulatory responses (induction/repression) in ADH activity of different genotypes may account for individual and population variations in response to alcohol. Additional work, however, is needed to establish the molecular bases of ADH inducibility and its specific role in relative susceptibility to alcohols.     

Enzyme activities and ethanol preference in mice

Alcohol dehydrogenase and aldehyde dehydrogenase, the two principal enzymes of alcohol metabolism, were assayed in the livers of the inbred mouse strains C57BL/6J and DBA/2J. Previous work has shown that animals of various C57BL substrains prefer a 10% ethanol solution to water in a two-bottle preference test, and that animals of various DBA/2 substrains avoid alcohol. In the present study, C57BL/6J mice were found to have 300% more aldehyde dehydrogenase activity than DBA/2J mice and 30% more alcohol dehydrogenase activity. The F₁ generation is intermediate to the parents in preference for the 10% alcohol solution and is also found to possess intermediate levels of alcohol and aldehyde dehydrogenase activity. These experiments suggest a systematic relationship between the behavioral trait of ethanol preference and the activity of aldehyde dehydrogenase and a similar but much less pronounced relationship with alcohol dehydrogenase.This research was supported by grant GM14547 from the National Institute of General Medical Sciences.     

Retinol/ethanol drug interaction during acute alcohol intoxication in mice involves inhibition of retinol metabolism to retinoic acid by alcohol dehydrogenase

Substantial evidence indicates that one consequence of alcohol intoxication is a reduction in retinoic acid (RA) levels. Studies on the mechanism have shown that chronic ethanol consumption induces P450 enzymes that increase RA degradation, thus accounting for much but not all of the observed decrease in RA. A reduction in RA synthesis may also be involved as ethanol competitively inhibits retinol oxidation catalyzed by alcohol dehydrogenase (ADH) in vitro. This may be important during acute ethanol intoxication and may contribute to adverse retinol/ethanol drug interactions. Here we have examined mice for the effect of either acute ethanol intoxication or Adh1 gene disruption on RA synthesis and degradation. RA produced following a dose of retinol (50 mg/kg) was reduced 87% by pretreatment with an intoxicating dose of ethanol (3.5 g/kg). RA produced in Adh1-null mutant mice following a 50-mg/kg dose of retinol was reduced 82% relative to wild-type mice, thus similar to wild-type mice pretreated with ethanol. Reduced RA production was associated with increased retinol levels in both ethanol-treated wild-type mice and Adh1-null mutant mice, indicating reduced clearance of the retinol dose. RA degradation following a dose of RA (10 mg/kg) was increased only 42% by ethanol pretreatment (3.5 g/kg) and only 26% in Adh1-null mutant mice relative to wild-type mice. These findings demonstrate that the reduced RA levels observed during acute retinol/ethanol drug interaction are due primarily to a decrease in ADH-catalyzed RA synthesis and secondarily to an increase in RA degradation.     

A strain-specific alteration of proteomic expression in mouse liver fructose 1,6-bisphosphatase isoforms by alcohol

To study alcohol-related metabolism across inbred mouse strains, liver tissues from C57BL/6J (B6, an alcohol-preferring mouse) and DBA/2J (D2, an alcohol-avoiding strain) mice were analyzed for proteomic expression patterns over time after a single-dose of alcohol (1.5 g/kg ingestion). Despite no significant difference in the elimination rate of blood ethanol, two-dimensional electrophoresis gel images of liver proteins showed that proteins in B6 mice exhibited faster response and more quantitative (spot numbers) and qualitative (spot densities) changes than in D2 mice. Among the differentially expressed metabolic enzymes, four variants (alpha, beta, gamma and delta) of fructose 1,6-bisphosphatase (FBPase), a key regulatory gluconeogenic enzyme, showed remarkable changes in expression with time across the strains. The degree of spot alteration in alpha- and gamma-variants of FBPase in B6 mice was much higher than in D2 mice, while the beta- and delta-forms were not changed as much. Mass spectrometry (MS) analysis showed that the 1714.9 +/- 1 mass peak from the alpha- and gamma-variants of FBPase was much stronger than that of the beta- and delta-variants in both strains regardless of spot density. This MS peak contains 2-ANHAPFETDISTLTR-16, located at the N-terminal of FBPase, where the N-terminal alanine was found to be trimethylated. Thus, we propose this N-terminal fragment as a potential site for enzyme modification in response to ethanol, allowing for differences in two-dimensional gel spot intensity of variants of FBPase in the two mouse strains.     

Genetically determined differences in ethanol sensitivity influenced by body temperature during intoxication

The present study investigated the importance of body temperature during intoxication in mediating differences between five inbred strains of mice (C57BL/6J; BALB/cJ; DBA/2J; A/HeJ; 129/J) in their acute sensitivity to the hypnotic effects of ethanol. Mice exposed to 22 degrees C after ethanol injection became hypothermic and exhibited statistically significant differences between strains in rectal temperatures at the return of the righting reflex (RORR), duration of loss of the righting reflex (LORR), and blood and brain ethanol concentrations at RORR. Exposure to 34 degrees C after injection offset ethanol-hypothermia and markedly reduced strain-related differences in rectal temperatures and blood and brain ethanol concentrations at RORR. Brain ethanol concentrations at RORR were significantly lower in C57, BALB, DBA and A/He mice exposed to 34 degrees C compared to mice exposed to 22 degrees C during intoxication suggesting that offsetting hypothermia increased ethanol sensitivity in these strains. Taken with previous in vitro studies, these results suggest that genetically determined differences in acute sensitivity to the behavioral effects of ethanol reflect differences in body temperature during intoxication as well as differences in sensitivity to the initial actions of ethanol at the cellular level.     

Regulation of heme oxygenase expression by alcohol, hypoxia and oxidative stress

AIM:To study the effect of both acute and chronic alcohol exposure on heme oxygenases(HOs) in the brain,liver and duodenum.METHODS:Wild-type C57BL/6 mice,heterozygous Sod2 knockout mice,which exhibit attenuated manganese superoxide dismutase activity,and liver-specific ARNT knockout mice were used to investigate the role of alcohol-induced oxidative stress and hypoxia.For acute alcohol exposure,ethanol was administered in the drinking water for 1 wk.Mice were pair-fed with regular or ethanol-containing Lieber De Carli liquid diets for 4 wk for chronic alcohol studies.HO expression was analyzed by real-time quantitative polymerase chain reaction and Western blotting.RESULTS:Chronic alcohol exposure downregulated HO-1 expression in the brain but upregulated it in the duodenum of wild-type mice.It did not alter liver HO-1 expression,nor HO-2 expression in the brain,liver or duodenum.In contrast,acute alcohol exposure decreased both liver HO-1 and HO-2 expression,and HO-2 expression in the duodenum of wild-type mice.The decrease in liver HO-1 expression was abolished in ARNT+/-mice.Sod2+/-mice with acute alcohol exposure did not exhibit any changes in liver HO-1 and HO-2 expression or in brain HO-2 expression.However,alcohol inhibited brain HO-1 and duodenal HO-2 but increased duodenal HO-1 expression in Sod2+/-mice.Collectively,these findings indicate that acute and chronic alcohol exposure regulates HO expression in a tissue-specific manner.Chronic alcohol exposure alters brain and duodenal,but not liver HO expression.However,acute alcohol exposure inhibits liver HO-1 and HO-2,and also duodenal HO-2 expression.CONCLUSION:The inhibition of liver HO expression by acute alcohol-induced hypoxia may play a role in the early phases of alcoholic liver disease progression.     

Sensitivity to the locomotor-stimulant effects of ethanol and allopregnanolone: a quantitative trait locus study of common genetic influence

Previous studies have suggested that common genetic mechanisms influence sensitivity to the locomotor-stimulant effects of ethanol and allopregnanolone. We conducted two quantitative trait locus (QTL) studies to identify chromosomal regions that harbor genes that influence locomotor response to ethanol (2 g/kg) and allopregnanolone (17 mg/kg) using F2 crosses between C57BL/6J and DBA/2J mice. Because our previous data from the BXD recombinant inbred strains had indicated that chromosome 2 contained QTL for sensitivity to the locomotor-stimulant effects of both ethanol and allopregnanolone, we also tested reciprocal chromosome 2 congenic strains for sensitivity to the locomotor-stimulant effects of both drugs. The F2 analysis for ethanol sensitivity identified significant QTL on chromosomes 1 and 2 and suggestive QTL on chromosomes 5 and 9. The analysis of the allopregnanolone F2 study identified suggestive QTL on chromosomes 3, 5 and 12. Suggestive evidence for a female-specific QTL on chromosome 2 was also found. The studies of congenic mouse strains indicated that both the congenic strains captured one or more QTL for sensitivity to the locomotor-stimulant effects of both ethanol (2 g/kg) and allopregnanolone (17 mg/kg). When Fisher's method was used to combine the P values for the RI, F2 and congenic studies of the chromosome 2 QTL, cumulative probability scores of 9.6 x 10(-15) for ethanol and 7.7 x 10(-7) for allopregnanolone were obtained. These results confirm the presence of QTL for ethanol and allopregnanolone sensitivity in a common region of chromosome 2 and suggest possible pleiotropic genetic influence on sensitivity to these drugs.     

Increased ethanol intake and preference in cyclin D2 knockout mice

Inhibitory effects of passive ethanol exposure on brain neurogenesis have been extensively documented in animal models. In contrast, a role of brain neurogenesis in ethanol self-administration has not been addressed, as yet. The aim of this study was to assess intake of, and preference for, ethanol solutions [2-16% (v/v)] in a mouse model of adult neurogenesis deficiency based on permanent knockout (KO) of cyclin D2 (Ccnd2). Wild type (WT) and Ccnd2 KO mice did not differ in 2% and 4% ethanol intake. The KO group consumed significantly more ethanol in g/kg when offered with 8% or 16% ethanol as compared with the WT controls. The WT and KO mice did not differ in 2% ethanol preference, but the KO group showed a significantly higher preference for 4-16% ethanol. Animal and human studies have suggested that the low level of response to the sedative/hypnotic effects of alcohol is genetically associated with enhanced alcohol consumption. However, in this study, there were no between-genotype differences in ethanol-induced loss of righting reflex. Previous reports have also suggested that high ethanol intake is genetically associated with the avidity for sweets and better acceptance of bitter solutions. However, the KO and WT mice consumed similar amounts of saccharin solutions and the KOs consumed less quinine (i.e. bitter) solutions as compared with the WTs. In conclusion, these results may indicate that Ccnd2 and, possibly, brain neurogenesis are involved in central regulation of ethanol intake in mice.     

Analysis of behavior using genetical genomics in mice as a model: from alcohol preferences to gene expression differences.

Most familial behavioral phenotypes result from the complex interaction of multiple genes. Studies of such phenotypes involving human subjects are often inconclusive owing to complexity of causation and experimental limitations. Studies of animal models argue for the use of established genetic strains as a powerful tool for genetic dissection of behavioral disorders and have led to the identification of rare genes and genetic mechanisms implicated in such phenotypes. We have used microarrays to study global gene expression in adult brains of four genetic strains of mice (C57BL/6J, DBA/2J, A/J, and BALB/c). Our results demonstrate that different strains show expression differences for a number of genes in the brain, and that closely related strains have similar patterns of gene expression as compared with distantly related strains. In addition, among the 24 000 genes and ESTs on the microarray, 77 showed at least a 1.5-fold increase in the brains of C57BL/6J mice as compared with those of DBA/2J mice. These genes fall into such functional categories as gene regulation, metabolism, cell signaling, neurotransmitter transport, and DNA/RNA binding. The importance of these findings as a novel genetic resource and their use and application in the genetic analysis of complex behavioral phenotypes, susceptibilities, and responses to drugs and chemicals are discussed.