Indiana University-Purdue University Indianapolis (IUPUI) / The class IV alcohol dehydrogenase (ADH7, µ-ADH, σ-ADH) efficiently metabolizes ethanol and retinol. ADH7 is expressed mainly in the upper gastrointestinal tract with no expression in the liver unlike the other ADHs, and is implicated in various diseases including alcoholism, cancer and fetal alcohol syndrome. Genome wide studies have identified significant associations between ADH7 variants and alcoholism and cancer, but the causative variants have not been identified. Due to its association with two important metabolic pathways and various diseases, this dissertation is focused on studying ADH7 regulation and the effects of variants on this regulation using cell systems that replicate endogenous ADH7 expression. We identified elements regulating ADH7 transcription and observed differences in the effects of variants on gene expression. A7P-G and A7P-A, two promoter haplotypes differing in a single nucleotide at rs2851028, had different transcriptional activities and interacted with variants further upstream. A sequence located 12.5 kb upstream (7P10) can function as an enhancer. These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context, and should be considered in interpretation of the association of variants with alcoholism and cancer. The mechanisms governing the tissue-specific expression of ADH7 remain unexplained however. We identified an intergenic region (iA1C), located between ADH7 and ADH1C, having enhancer blocking activity in liver-derived HepG2 cells. This enhancer blocking function was cell- and position- dependent with no activity seen in CP-A esophageal cells. iA1C had a similar effect on the ectopic SV40 enhancer. The CCCTC-binding factor (CTCF) bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of downstream ADH enhancers and thereby contributes to the cell specificity of ADH7 expression. Thus, while genetic factors determine level of ADH7 transcriptional activity, iA1C helps determine the cell specificity of transcription.

Indiana University-Purdue University Indianapolis (IUPUI) / Smoke-free legislation in the United States has unintentionally resulted in a decline in alcohol consumption. However, more recently electronic-cigarettes (e-cigs), which are associated with alcohol use, are reportedly being used to circumvent smoking bans. The present study surveyed community dwelling individuals in the United States reporting e-cigs may be used where they drink (N=365, mean age=33.63, SD=9.91, 53.2% female, 78.9% Caucasian) to examine how e-cig use and alcohol consumption varies by the presence of smoking prohibition where one consumes alcohol. Results indicated that smoking prohibition was associated with a greater likelihood of being an ecig user than a cigarette user (OR=3.40, p<.001) and a higher likelihood of being an e-cig user than a dual user (OR=3.37, p<.001). Smoking prohibition was not associated with AUDIT scores (B=-0.06, p=.21), total drinks (B=-.07, p=.19), or average drinks (B=-0.02, p=.76). E-cig users reported significantly fewer average drinks when smoking is prohibited as compared to allowed, t(55)=3.26, p=.002. Overall, current results suggest smoking prohibition is associated with a greater likelihood of being an e-cig user; however, smoking prohibitions are not associated with alcohol consumption and related problems in the current participants, who all reported being able to use e-cigs where they consume alcohol. Future research should address potential conceptual, methodological, and sample limitations in order to better discern this relationship, as this line of research could have important implications for e-cig policy and alcohol use treatment

Indiana University-Purdue University Indianapolis (IUPUI) / Abuse of alcohol during adolescence continues to be a problem, and it has been shown that earlier onset of drinking predicts increased alcohol abuse problems later in life. High levels of impulsivity have been demonstrated to be characteristic of alcoholics, and impulsivity has also been shown to predict later alcohol use in teenage subjects, showing that impulsivity may be an inherent underlying biological process that precedes the development of alcohol use disorders. These experiments examined adolescent drinking in a high-drinking, relatively impulsive mouse population, and assessed its effects on adult drinking and adult impulsivity. Experiment 1: Selectively bred High-Alcohol Preferring (HAP II) mice, which are shown to be highly impulsive, were given either alcohol (free choice access) or water only for two weeks during middle adolescence or adulthood. All mice were given free choice access to alcohol following 30 days without access, in adulthood. Experiment 2: Adolescent HAP II mice drank alcohol and water, or water alone, for two weeks, and were then trained to perform a delay discounting task as adults to measure impulsivity. In each experiment, effects of volitional ethanol consumption on later behavior were assessed. We expected adolescent alcohol exposure to increase subsequent drinking and impulsivity. Adolescent mice consumed significant quantities of ethanol, reaching average blood ethanol concentrations (BECs) of 142 mg/dl in Experiment 1 and 108 mg/dl in Experiment 2. Adult mice reached average BECs of 154 mg/dl in Experiment 2. Mice pre-exposed to alcohol in either adolescence or adulthood showed a transient increase in ethanol consumption, but we observed no differences in impulsivity in adult mice as a function of whether mice drank alcohol during adolescence. These findings indicate that HAP II mice drink intoxicating levels of alcohol during both adolescence and adulthood, and that this volitional intake has long-term effects on subsequent drinking behavior. Nonetheless, this profound exposure to alcohol during adolescence does not increase impulsivity in adulthood, indicating that long-term changes in drinking are mediated by mechanisms other than impulsivity. Importantly, this research demonstrates that the HAP II mouse is a good candidate for a model of heavy adolescent alcohol consumption.

Indiana University-Purdue University Indianapolis (IUPUI) / Alcohol consumption produces a complex array of effects that can be divided into two types: the explicit pharmacological effects of ethanol (which can be quite separate temporally from time of intake) and the more temporally “relevant” effects (primarily olfactory and taste) that bridge the time from intake to the onset of the pharmacological effects. Dissociating these effects is essential to untangling the neurologic underpinnings of alcohol abuse and dependence. Intravenous self-administration of ethanol allows for controlled and precise dosing, bypasses first order absorption kinetics allowing for a faster onset of pharmacologic effects, and eliminates the confounding “non-pharmacological” effects associated with oral consumption. Intravenous self-administration of ethanol has been reliably demonstrated in both mouse and human experimental models; however, consistent intravenous self-administration of pharmacologically relevant levels of ethanol remains elusive in the rat. Previous work has demonstrated reliable elevated intravenous ethanol self administration using a compound reinforcer of oral sucrose and intravenous ethanol. The present study sought to elucidate the role of each component of this reinforcer complex using a multiple schedule study design. Male P rats had free access to both food and water during all intravenous self-administration sessions and all testing was performed in conjunction with the onset of the dark cycle. Once animals achieved stable operant responding on both levers for an orally delivered 1% sucrose solution (1S) on a FR4 schedule, surgery was conducted to implant an indwelling jugular catheter. Animals were habituated to the attachment of infusion apparatus and received twice daily sessions for four days to condition each lever to its associated schedule. Animals were then trained to respond on a multiple FR4-FR4 schedule composed of alternating 2.5 minute components. During one component only oral 1S was presented, while in the second component a compound reinforcer of oral 1S + IV 20% ethanol was presented (25 mg/kg/injection). Both levers were extended into the chamber during the session, with the active lever/schedule alternating as the session progressed across components. Average ethanol intake was 0.47 ± 0.04 g/kg. A significant increase in sucrose only reinforcers and sucrose lever error responding was found suggesting that sucrose not ethanol is responsible for driving overall responding. The current findings suggest that the existing intravenous ethanol self-administration methodology remains aversive in the rat.