Lucinda G. Carr

Phenotypic and genotypic characterization of the Indiana university rat lines selectively bred for high and low alcohol preference

The Indiana lines of selected rats, the HAD and LAD replicates and the P and NP lines, were bred for high and low alcohol preference. The P and HAD lines have met criteria for an animal model of alcoholism in that they voluntarily consume sufficient ethanol to achieve significant blood alcohol concentrations, and their alcohol-seeking behavior is reinforced by the pharmacological effects of ethanol rather than its taste, caloric content, or other properties. These lines have been characterized extensively for associated behavioral and physiological phenotypes. The P and HAD rats show an enhanced responsiveness to the stimulatory effects of ethanol and reduced sensitivity to the aversive sedative effects of ethanol. Consistent findings with the selected lines include differences in the mesolimbic dopamine reward system, as well as differences in serotonin, GABA, endogenous opioid, and neuropeptide Y systems. Genetic mapping studies have identified quantitative trait loci influencing alcohol preference on chromosomes 3, 4, and 8 in the inbred P/NP rats and on chromosomes 5, 10, 12, and 16 in the noninbred HAD1/LAD1 rats. The elucidation of the genotypes and phenotypes that result in excessive alcohol intake may lead to a better understanding of alcohol abuse and alcoholism and could guide strategies for potential treatment and prevention.

Genotyping of human alcohol dehydrogenases at the ADH2 and ADH3 loci following DNA sequence amplification

Humans are polymorphic at two of the alcohol dehydrogenase (ADH) loci important in ethanol metabolism, ADH2 and ADH3. Although the coding regions of these genes are 94% identical, they produce subunits that differ greatly in kinetic properties in vitro. These differences are likely to be reflected in the pharmacokinetics of alcohol metabolism, but studies have been hampered by the need to use liver biopsy specimens to determine the ADH phenotype. This problem has now been overcome by determining the genotype at these loci using DNA that has been amplified in vitro by the polymerase chain reaction. We report here the identification of all three of the ADH2 alleles and both of the ADH3 alleles. Any pair of ADH2 or ADH3 alleles can be distinguished using allele-specific oligonucleotide probes directed at their single base pair difference. In addition, ADH2(2) can be distinguished from ADH2(1) and ADH2(3) by detecting a new MaeIII site created in the third exon by the single base pair alteration in ADH2(2).

Cloning and expression of the isopenicillin N synthetase gene from Penicillium chrysogenum

The isopenicillin N synthetase (IPS) gene from Penicillium chrysogenum was isolated from a recombinant bacteriophage lambda library using the Cephalosporium acremonium IPS (cIPS) gene as a heterologous hybridization probe. The protein coding region of the P. chrysogenum IPS (pIPS) gene was about 74% homologous to the cIPS gene, and the predicted amino acid sequences of the encoded proteins were about 73% homologous. Escherichia coli cells with the pIPS gene contained IPS activity whereas untransformed cells were completely devoid of this enzymatic activity. The transformed cells were also shown to contain an abundant protein accounting for about 10% of total cell protein which reacted strongly with anti-cIPS antiserum.

Gender differences in alcohol metabolism: Relationship to liver volume and effect of adjusting for body mass

BACKGROUND & AIMS Alcoholic liver disease purportedly develops more readily in women than in men. Some studies have demonstrated faster rates of alcohol elimination in women. This study examined whether gender differences in alcohol metabolism are related to differences in liver volume and/or differences in lean body mass. METHODS Ten men and 10 women had alcohol elimination rates determined by clamping of the breath alcohol concentration at 50 mg/dL by means of a constant rate of intravenous infusion of 6% ethanol. Liver volume was determined by computed tomography. RESULTS Mean alcohol elimination rate and mean computed liver volume were not significantly different in men and women. Lean body mass was 42% greater in men than in women. Consequently, the calculated alcohol elimination rate and liver volume per kilogram of lean body mass were 33% and 38% higher in women than in men, respectively. When the alcohol elimination rate was calculated per unit liver volume, no gender-related difference was found. CONCLUSIONS Women have greater clearance of ethanol per unit lean body mass, confirming previous oral alcohol administration studies. Women have approximately the same liver volume as men, explaining the equivalent alcohol elimination rates seen when men and women are compared on the basis of liver size.

α-Synuclein maps to a quantitative trait locus for alcohol preference and is differentially expressed in alcohol-preferring and -nonpreferring rats

Total gene expression analysis (TOGA) was used to identify genes that are differentially expressed in brain regions between the alcohol-naïve, inbred alcohol-preferring (iP), and -nonpreferring (iNP) rats. α-Synuclein, expressed at >2-fold higher levels in the hippocampus of the iP than the iNP rat, was prioritized for further study. In situ hybridization was used to determine specific brain regions and cells expressing α-synuclein in the iP and iNP rats. Similar to α-synuclein mRNA levels, protein levels in the hippocampus were higher in iP rats than iNP rats. Higher protein levels were also observed in the caudate putamen of iP rats compared with iNP rats. Sequence analysis identified two single nucleotide polymorphisms in the 3′ UTR of the cDNA. The polymorphism was used to map the gene, by using recombination-based methods, to chromosome 4, within a quantitative trait locus for alcohol consumption that was identified in the iP and iNP rats. A nucleotide exchange in the iNP 3′ UTR reduced expression of the luciferase reporter gene in SK-N-SH neuroblastoma cells. These results suggest that differential expression of the α-synuclein gene may contribute to alcohol preference in the iP rats.

Genomic screen for QTLs underlying alcohol consumption in the P and NP rat lines

Abstract. Selective breeding for voluntary alcohol consumption was utilized to establish the alcohol-preferring (P) and alcohol-nonpreferring (NP) rat lines. Inbreeding was initiated after 30 generations of selection and, after 19 generations of inbreeding, 384 F2 intercross progeny were created to identify quantitative trait loci (QTLs) influencing alcohol consumption. We had reported previously a QTL on Chromosome (Chr) 4; additional markers genotyped on Chr 4 have increased the maximum lod score from 8.6 to 9.2. This QTL acts in an additive fashion and continues to account for approximately 11% of the phenotypic variability. The 95% confidence interval is 12.5 cM and includes the candidate gene, neuropeptide Y. Subsequent to the identification of the QTL on Chr 4, a genome scan was completed to identify additional QTLs influencing alcohol consumption. A lod score of 2.5 was obtained on Chr 3, syntenic to a region previously reported for alcohol preference in mice. Analysis of Chr 8 produced a lod score of 2.2 near the dopamine D2 and serotonin 1b receptors, which have been previously reported as candidate genes for alcohol preference. Evidence for linkage to alcohol consumption was not found on any other chromosome. It therefore appears likely that, in addition to the QTL on Chr 4, multiple loci of small to moderate effect, such as those on Chrs 3 and 8, underlie the difference in alcohol consumption in the P/NP lines.

Efficient integrative transformation of Cephalosporium acremonium

SummaryA hybrid gene, IPNSp/HPTorf, was constructed by placing an 850 by sequence of Cephalosporium acremonium DNA next to the 5′ end of a bacterial open reading frame, HPTorf. The sequence was obtained as an 850 by NcoI restriction fragment from the 5′ noncoding region of the C. acremonium isopenicillin N synthetase (IPNS) gene. The HPTorf was obtained from a bacterial gene that coded for a hygromycin B phosphotransferase (HPT). Plasmids that contained IPNSp/HPTorf transformed C. acremonium to a stably maintained hygromycin B resistant phenotype. Southern analysis of total DNA from transformants demonstrated multiple integrations of the transforming DNA in the high molecular weight DNA of most transformants, but single integrations were observed in a few transformants. The number of transformants per μg of DNA was about 100 times greater than for plasmids that contained the HPTorf without any juxtaposed eucaryotic promoter sequence. Plasmids with the promoterless HPTnorf and plasmids with a truncated S. cerevisiae phosphoglycerate kinase promoter juxtaposed to the HPTorf transformed C. acremonium at equivalent low frequencies.Transformation of C. acremonium with linearized plasmid DNA produced at least 2–3 fold more transformants than the corresponding circular molecule. Several observations were made concerning protoplast formation and handling which made the transformation procedure more efficient and allowed a greater proportion of protoplasts to regenerate to viable walled cells.Plasmids were constructed that contained both the IPNSp/HPTorf and additional elements: fragments of C. acremonium ribosomal DNA (rDNA), or a fragment of C. acremonium mitochondrial DNA possessing activity as an autonomous replication sequence (ARS) in S. cerevisiae, or putative transcriptional tennination/polyadenylation signals from the IPNS gene. These plasmids transformed C. acremonium at frequencies experimentally equivalent to those containing IPNSp/HPTorf without any of these additional elements.

Gene expression changes in the nucleus accumbens of alcohol-preferring rats following chronic ethanol consumption.

The objective of this study was to determine the effects of binge-like alcohol drinking on gene expression changes in the nucleus accumbens (ACB) of alcohol-preferring (P) rats. Adult male P rats were given ethanol under multiple scheduled access (MSA; three 1-h dark cycle sessions/day) conditions for 8 weeks. For comparison purposes, a second ethanol drinking group was given continuous/daily alcohol access (CA; 24h/day). A third group was ethanol-naïve (W group). Average ethanol intakes for the CA and MSA groups were approximately 9.5 and 6.5 g/kg/day, respectively. Fifteen hours after the last drinking episode, rats were euthanized, the brains extracted, and the ACB dissected. RNA was extracted and purified for microarray analysis. The only significant differences were between the CA and W groups (p<0.01; Storey false discovery rate=0.15); there were 374 differences in named genes between these 2 groups. There were 20 significant Gene Ontology (GO) categories, which included negative regulation of protein kinase activity, anti-apoptosis, and regulation of G-protein coupled receptor signaling. Ingenuity analysis indicated a network of transcription factors, involving oncogenes (Fos, Jun, Junb had higher expression in the ACB of the CA group), suggesting increased neuronal activity. There were 43 genes located within rat QTLs for alcohol consumption and preference; 4 of these genes (Tgfa, Hspa5, Mtus1 and Creb3l2) are involved in anti-apoptosis and increased transcription, suggesting that they may be contributing to cellular protection and maintaining high alcohol intakes. Overall, these findings suggest that chronic CA drinking results in genomic changes that can be observed during the early acute phase of ethanol withdrawal. Conversely, chronic MSA drinking, with its associated protracted withdrawal periods, results in genomic changes that may be masked by tight regulation of these genes following repeated experiences of ethanol withdrawal.

Genetic associations of alcohol dehydrogenase with alcohol use disorders and endophenotypes in white college students.

Associations of alcohol dehydrogenase (ADH) gene polymorphisms (ADH1B*2 and ADH1C*1) with a lifetime alcohol use disorder (AUD) were examined in White college students. Alcohol-related endophenotypes likely to be influenced by elevations in acetaldehyde were also assessed. Individuals with an ADH1B*2 allele had lower rates of AUDs, consumed a lower maximum number of drinks in a 24-hr period, reported a greater level of response to alcohol, were more likely to have experienced alcohol-induced headaches following 1 or 2 drinks, and reported more severe hangovers than those lacking this allele. These findings are consistent with the hypothesis that enhanced sensitivity to alcohol and lower levels of alcohol use reflect the mechanism by which ADH1B*2 protects against developing an AUD.

Association of ALDH1 promoter polymorphisms with alcohol-related phenotypes in southwest California Indians.

BACKGROUND Cytosolic aldehyde dehydrogenase (ALDH1A1) is an important enzyme in the metabolism of acetaldehyde and the synthesis of retinoic acid. Two polymorphisms in the promoter region of ALDH1A1-ALDH1A1*2 and ALDH1A1*3-have recently been identified and described in small samples of Asian, Caucasian, and African individuals. The aim of this study was to determine the prevalence of these polymorphisms in a sample of Southwest California Indians and to test for associations with alcohol dependence and other substance-related behaviors. METHODS The participants in this study were 463 adult men and women recruited from 8 contiguous Indian reservations. A structured interview was used to gather information on demographics, psychiatric diagnoses, and personal drinking and drug use history. A blood sample was obtained from each participant, and leukocyte DNA was extracted and used to genotype for the presence of the ALDH1A1 promoter polymorphisms. RESULTS Twenty-seven participants (6%) possessed ALDH1A1*2 (frequency, 0.03), two participants possessed ALDH1A1*3, and one participant displayed both of these alleles. Individuals with an ALDH1A1*2 allele had lower rates of alcohol dependence and regular tobacco use than those without this allele. Individuals with ALDH1A1*2 also reported a significantly lower maximum number of drinks ever consumed in a 24-hr period, reported drinking fewer drinks per occasion when they first started drinking regularly, and reported lower expectations of alcohols effects compared with individuals without this allele. CONCLUSIONS Results from this study suggest that ALDH1A1*2 may be associated with protection from the development of alcohol and other substance use disorders.