Prospective studies have not previously examined whether a family history of alcoholism and drinking motives conjointly predict a diagnosed DSM-IV alcohol abuse or dependence in adults, despite a large literature that each is associated with alcohol consumption. The focus of this study is the conjoint, prospective examination of these risk factors in a 10-year longitudinal study of adults who were at-risk drinkers at baseline.
Prospective, population-based cohort of drinkers aged 18 or older from a Northeastern U.S. area initially evaluated for history of alcohol use disorders and drinking motives in 1991 to 1992. New onset dependence was studied in those who never met the criteria for alcohol dependence at baseline (n = 423), and new onset abuse was studied in those who never met the criteria for alcohol abuse at baseline (n = 301) and who did not develop dependence during the follow-up.
Family history significantly interacted with 2 baseline drinking motives in predicting new onsets of DSM-IV alcohol dependence: drinking to reduce negative affect (OR 3.38; 95% CI 1.05, 10.9) and drinking for social facilitation (OR 3.88; CI 1.21, 12.5). Effects were stronger after conditioning the drinking motives on having a positive family history of alcoholism. In contrast, in predicting new onsets of alcohol abuse, drinking motives did not have direct effects or interact with family history.
Those who drank to reduce negative affect or for social facilitation at baseline were at greater risk of alcohol dependence 10 years later if they also had a family history of alcoholism. These results suggest an at-risk group that can be identified prior to the development of alcohol dependence. Further, the findings suggest utility in investigating the interaction of drinking motives with measured genetic polymorphisms in predicting alcohol dependence.
Alcohol Dependence; Alcohol Abuse; Reasons for Drinking; Drinking Motives; Family History of Alcoholism
Background and Aims
Previously, we reported that exposure of hepatitis C virus (HCV) core expressing ethanol-metabolizing cells to ethanol significantly suppresses proteasome activity which exists as 26S (20S and 19S) and as an unassociated 20S particle. The replacement of the constitutive proteasomal subunits with immunoproteasome (IPR) favors antigen processing. Here, we examined the effects of ethanol consumption by HCV core transgenic mice on proteasome activity in hepatocytic lysates and in partially purified 26S proteasome and the impact of these changes on antigen presentation.
HCV- and HCV+ core transgenic mice were fed chow diet with or without 20% (v/v) ethanol in water for 4 weeks. Following the feeding regimen, hepatocytes were isolated and examined for chymotrypsin-like proteasome activity, oxidative stress and the presentation of SIINFEKL-H2Kb complex. Additionally the constitutive proteasome and IPR were purified for further analysis and identification of proteasome-interacting proteins (PIPs).
Ethanol significantly decreased proteasome activity in hepatocytes of HCV+ mice and this finding correlated with oxidative stress and dysregulated methylation reactions. In isolated 26S proteasome, ethanol suppressed proteasome activity equally in HCV+ and HCV− mice. Ethanol feeding caused proteasome instability and lowered the content of both constitutive and IPR subunits in the 20S proteasome. In addition, the level of other PIPs, PA28 and UCHL5, were also suppressed after ethanol exposure. Furthermore, in ethanol-fed mice and especially, in HCV+ mice, the presentation of SIINFEKL-H2Kb complex in hepatocytes was also decreased.
Proteasomal dysfunction induced by ethanol feeding and exacerbated by the presence of HCV structural proteins led to suppression of SIINFEKL-H2Kb presentation in hepatocytes.
Hepatitis C virus; proteasome interacting proteins; methylation reactions; oxidative stress; antigen presentation
Alcohol abuse is a risk factor for bone damage and fracture-related complications. Through precise β-catenin signaling, canonical Wnt signaling plays a key role in fracture repair by promoting the differentiation of new bone and cartilage cells. In this study, we examined the effects of alcohol on the Wnt pathway in injured bone using a murine model of alcohol-induced impaired fracture healing.
Male C57Bl/6 or TCF-transgenic mice were administered 3 daily intraperitoneal doses of alcohol or saline. One hour following the final injection, mice were subjected to a stabilized, mid-shaft tibial fracture. Injured and contralateral tibias were harvested at 6, 9, or 14 days post-fracture for analysis of biomechanical strength, callus tissue composition, and Wnt/β-catenin signaling.
Acute alcohol treatment was associated with a significant decrease in fracture callus volume, diameter, and biomechanical strength at day 14 post-fracture. Histology revealed an alcohol-related reduction in cartilage and bone formation at the fracture site, and that alcohol inhibited normal cartilage maturation. Acute alcohol exposure caused a significant 2.3-fold increase in total β-catenin protein at day 6 and a significant decrease of 53% and 56% at days 9 and 14 respectively. LacZ staining in β-galactosidase-expressing TCF-transgenic mice revealed spatial and quantitative differences in Wnt-specific transcriptional activation at day 6 in the alcohol group. Days 9 and 14 post-fracture showed that acute alcohol exposure decreased Wnt transcriptional activation, which correlates with the modulation of total β-catenin protein levels observed at these time points.
Acute alcohol exposure resulted in significant impairment of fracture callus tissue formation, perturbation of the key Wnt pathway protein β-catenin, and disruption of normal Wnt-mediated transcription. These data suggest that the canonical Wnt pathway is a target for alcohol in bone, and may partially explain why impaired fracture healing is observed in alcohol-abusing individuals.
Acute alcohol; Wnt; β-catenin; fracture repair
AUDIT-C alcohol screening scores are associated with mortality, but whether or how associations vary across race/ethnicity is unknown.
Self-reported black (n=13,068), Hispanic (n=9,466), and white (n=182,688) male VA outpatients completed the AUDIT-C via mailed survey. Logistic regression models evaluated whether race/ethnicity modified the association between AUDIT-C scores (0, 1–4, 5–8, and 9–12) and mortality after 24 months, adjusting for demographics, smoking, and comorbidity.
Adjusted mortality rates were 0.036, 0.033, and 0.054, for black, Hispanic, and white patients with AUDIT-C scores of 1–4, respectively. Race/ethnicity modified the association between AUDIT-C scores and mortality (p=0.0022). Hispanic and white patients with scores of 0, 5–8, and 9–12 had significantly increased risk of death compared to those with scores of 1–4; Hispanic ORs: 1.93, 95% CI 1.50–2.49; 1.57, 1.07–2.30; 1.82, 1.04–3.17, respectively; white ORs: 1.34, 95% CI 1.29–1.40; 1.12, 1.03–1.21; 1.81, 1.59–2.07, respectively. Black patients with scores of 0 and 5–8 had increased risk relative to scores of 1–4 (ORs 1.28, 1.06–1.56 and 1.50, 1.13–1.99), but there was no significant increased risk for scores of 9–12 (ORs 1.27, 0.77–2.09). Post-hoc exploratory analyses suggested an interaction between smoking and AUDIT-C scores might account for some of the observed differences across race/ethnicity.
Among male VA outpatients, associations between alcohol screening scores and mortality varied significantly depending on race/ethnicity. Findings could be integrated into systems with automated risk calculators to provide demographically-tailored feedback regarding medical consequences of drinking.
alcohol; race; ethnicity; mortality; AUDIT-C
Studies of the adverse neurobehavioural effects of maternal alcohol consumption on the fetus have been largely confined to the postnatal period, after exposure to alcohol has finished. This study explored the brain function of the fetus, at the time of exposure to alcohol, to examine its effect on information processing and stability of performance.
Five groups of fetuses, defined by maternal alcohol consumption patterns, were examined: control (no alcohol); moderate (5-10 units/week either drunk evenly across the week, or as a binge, in 2-3 days); heavy (20+units/week drunk evenly, or as a binge). Fetal habituation performance was examined on three occasions, separated by seven days, beginning at 35 weeks gestation. The number of trials required to habituate on each test session and the difference in performance across test sessions was recorded.
Fetuses exposed to heavy binge drinking required significantly more trials to habituate and exhibited a greater variability in performance across all test sessions than the other groups. Maternal drinking, either heavily but evenly, or moderately as a binge, resulted in poorer habituation and moderate binge drinking resulted in greater variability compared to no, or even, drinking.
Decreased information processing, reflected by poorer habituation, and increased variability in performance may reflect the initial manifestations of structural damage caused by alcohol to the brain. These results will lead to a greater understanding of the effects of alcohol on the fetus's brain, enable the antenatal identification of FASD, and lead to the early implementation of better management strategies.
Fetal alcohol spectrum disorders; binge drinking; habituation; brain function; neurobehavioural development
Genome-wide association (GWA) studies have led to a paradigm shift in how researchers study the genetics underlying disease. Many GWA studies are now publicly available and can be used to examine whether or not previously proposed candidate genes are supported by GWA data. This approach is particularly important for the field of alcoholism because the contribution of many candidate genes remains controversial.
Using the Human Genome Epidemiology (HuGE) Navigator, we selected candidate genes for alcoholism that have been frequently examined in scientific articles in the past decade. Specific candidate loci as well as all the reported SNPs in candidate genes were examined in the Study of Alcohol Addiction: Genetics and Addiction (SAGE), a GWA study comparing alcohol dependent and non-dependent subjects.
Several commonly reported candidate loci, including rs1800497 in DRD2, rs698 in ADH1C, rs1799971 in OPRM1 and rs4680 in COMT, are not replicated in SAGE (p> .05). Among candidate loci available for analysis, only rs279858 in GABRA2 (p=0.0052, OR=1.16) demonstrated a modest association. Examination of all SNPs reported in SAGE in over 50 candidate genes revealed no SNPs with large frequency differences between cases and controls and the lowest p value of any SNP was .0006.
We provide evidence that several extensively studied candidate loci do not have a strong contribution to risk of developing alcohol dependence in European and African Ancestry populations. Due to lack of coverage, we were unable to rule out the contribution of other variants and these genes and particular loci warrant further investigation. Our analysis demonstrates that publicly available GWA results can be used to better understand which if any of previously proposed candidate genes contribute to disease. Furthermore, we illustrate how examining the convergence of candidate gene and GWA studies can help elucidate the genetic architecture of alcoholism and more generally complex diseases.
Alcohol dependence; Candidate genes; GWAS; Genetics
Alcohol is a widely abused substance and is responsible for significant morbidity and mortality worldwide. The precise mechanisms underlying ethanol's actions in the CNS remain elusive. In vitro studies suggest that GABAergic interneurons are important targets of ethanol action in the CNS. Although ethanol generally acts to inhibit CNS neurons, it appears to cause an increase in GABAergic interneuron excitability. However, it has yet to be demonstrated that ethanol produces this effect in the brain of behaving animals. Here, we demonstrate for the first time that acute ethanol exposure excites a subtype of GABAergic interneuron (cerebellar Golgi cell) in a freely moving animal.
Electrophysiological recordings were made from microwire arrays implanted in the anterior cerebellum of freely moving rats.
Cerebellar Golgi cells display a slow, irregular, spontaneous action potential firing pattern under control conditions. Ethanol caused dramatic and consistent increases in the rate and regularity of Golgi cell discharges, including a re-distribution of the power in the Golgi cell spike train, such that power became concentrated in the 26.7±7.3Hz region.
Taken together with our previous findings, these data suggest that a major mechanism of ethanol actions on cerebellar function is an ethanol-induced de-afferentation at the input stage of the cerebellar cortex in the form of granule cell inhibition, and that this inhibition is caused by an increase in Golgi cell firing. It is likely that Golgi cells may play a significant role both in the gating of information transmission to granule cells and in the modulation of the overall excitability of the cerebellum by tonically controlling granule cell activity.
alcohol; cerebellum; Golgi; granule; interneuron
Several lines of evidence link cannabinoid (CB) type 1 (CB1) receptor-mediated endogenous CB (eCB) signaling to the etiology of alcohol dependence (AD). However, to date, only peripheral measures of eCB function have been collected in living humans with AD and no human in vivo data on the potentially critical role of the brain CB1 receptor in AD have been published. This is an important gap in the literature, because recent therapeutic developments suggest that these receptors could be targeted for the treatment of AD.
Medication-free participants were scanned during early abstinence 4 weeks after their last drink. Using positron emission tomography (PET) with a high resolution research tomograph and the CB1 receptor selective radiotracer [11C]OMAR, we determined [11C]OMAR volume of distribution (VT) values, a measure of CB1 receptor density, in a-priori selected brain regions in men with AD (N=8, age 37.4± 7.9 years; 5 smokers) and healthy control (HC) men (N=8, age 32.5± 6.9 years; all non-smokers). PET images reconstructed using the MOLAR algorithm with hardware motion correction were rigidly aligned to the subject-specific magnetic resonance (MR) image, which in turn was warped to an MR template. Time-activity curves (TACs) were extracted from the dynamic PET data using a priori selected regions of interest delineated in the MR template space.
In AD relative to HC, [11C]OMAR VT values were elevated by ~20% (p=.023) in a circuit, including the amygdala, hippocampus, putamen, insula, anterior and posterior cingulate cortices and orbitofrontal cortex. Age, body mass index or smoking status did not influence the outcome.
These findings agree with preclinical evidence and provide the first, albeit still preliminary in vivo evidence suggesting a role for brain CB1 receptors in AD. The current study design does not answer the important question of whether elevated CB1 receptors are a pre-existing vulnerability factor for AD or whether elevations develop as a consequence of AD.
alcohol dependence; brain imaging; positron emission tomography; endogenous cannabinoids; CB1 receptor
Alcohol use disorders (AUDs) are clinically heterogeneous and strongly influenced by familial/genetic factors. Can we identify specific clinical features of AUDs that index familial liability to illness?
In twins from the Virginia Adult Twin Study of Psychiatric and Substance Use Disorders meeting DSM-IV criteria for lifetime AUDs, we examined whether clinical features of AUDs, including individual DSM-IV criteria for alcohol dependence (AD) and alcohol abuse (AA), predicted risk for AUDs in cotwins and/or parents. Analyses of individual criterion were repeated controlling for the total number of endorsed criteria.
Across these analyses, examining narrowly and broadly defined AUDs, risk of AUDs in relatives was more consistently predicted by abuse criteria than by dependence criteria, and by criteria reflecting negative psychosocial consequences rather than pharmacologic/biological criteria. Age at onset (AAO) poorly predicted risk in relatives. AUD associated legal problems, the one criterion slated for removal in DSM-5, was the most consistent single predictor of familial risk. Associations observed between individual criteria and risks of illness in relatives were generally stronger in monozygotic than dizygotic twin pairs, suggesting that these symptoms reflect a genetic risk for AUDs.
Individual DSM-IV criteria for AA and AD differ meaningfully in the degree to which they reflect the familial/genetic liability to AUDs. Contrary to expectation, the familial/genetic risk to AUDs was better reflected by symptoms of abuse and negative psychosocial consequences of AUD than by early AAO, or symptoms of tolerance and withdrawal.
Alcohol Abuse; Alcohol Dependence; Twin Studies; Heritability; Symptoms
Studies exploring differential effects of acute alcohol consumption on younger and older adults are lacking within the field of alcohol research, especially those using moderate doses. Previous studies addressing this question have tended to use complex behavioral tasks too broad to isolate specific neurocognitive processes affected by both alcohol and aging. Compromises in cognitive efficiency (i.e. the ability to respond both quickly and accurately) have previously been identified in both elderly and acutely intoxicated individuals.
The present study employed a visual-spatial, two-choice reaction time task to evaluate the interactive effects of aging and alcohol on cognitive efficiency. Our primary outcome measure was an efficiency ratio derived from each participant’s response accuracy (ACC) and mean reaction time (RT) (%correct/RT). Younger (25 – 35; n=22) and older (55 – 74; n=37) participants were randomly assigned to receive either a placebo or moderate alcohol dose intended to produce a peak BrAC of 0.04%. Participants performed the task at peak alcohol levels.
A significant interaction between age group and dose assignment was observed (F3,55=4.86, p=.03) for the efficiency ratio. Younger participants who received alcohol performed significantly better than did their older counterparts regardless of alcohol condition and despite no differences in performance between the two age groups in the placebo condition. Additional correlation analyses between ACC and RT suggested that moderately intoxicated older adults become more accurate as response times increase. This relationship was not observed in older adults in the placebo condition.
These data suggest that healthy individuals exhibit a differential susceptibility to the effects of alcohol depending on their age. Unfortunately, due to the presumed safety of moderate alcohol doses and a lack of studies investigating the interactive effects of acute alcohol consumption and aging, most individuals are unlikely to be aware of this relationship between alcohol consumption and age.
Alcohol; aging; cognition; speed-accuracy trade-off
Clinical trials for alcoholism have historically regarded alcohol consumption as the primary outcome. In a subset of trials, quality of life has been considered as a secondary outcome. Joint latent-variable modeling techniques may provide a more accurate and powerful simultaneous analysis of primary and secondary outcomes in clinical trials. The goal of the present study was to evaluate longitudinal associations between treatment status, alcohol consumption, and quality of life in the Combined Pharmacotherapies and Behavioral Interventions for Alcohol Dependence (COMBINE) study.
1,383 alcohol-dependent patients were randomized to nine treatment groups. Percent heavy drinking days (PHDD) and health-related QOL from the 30 days preceding baseline, week 16, and week 52 were calculated using the Form 90 and the Medical Outcomes Study Health Survey Short Form-12 (SF-12), respectively. Latent profile analysis (LPA) was conducted to determine an appropriate number of latent states to represent PHDD and QOL. Subsequently, univariate and coupled Hidden Markov Model (HMM)s (for PHDD f& SF-12 mental health and PHDD & SF-12 physical) were fit to the data.
LPA suggested that PHDD should be represented by 3 latent states and that each SF-12 scale should be represented by 2 states. Joint modeling results suggested that: (i) naltrexone significantly predicted decreased PHDD (p<0.05), and marginally predicted improved mental health QOL via decreased PHDD (p<0.10), and (ii) that the combinations of naltrexone and combined behavioral intervention (CBI), and acamprosate and CBI, each predicted significantly improved physical QOL (p<0.05), and marginally predicted decreased PHDD via improved physical QOL (p<0.10).
The present study illustrates a powerful and novel statistical approach for simultaneously evaluating the impact of treatments on primary and secondary outcomes in clinical trials. The present study also suggests that behavioral interventions may impact drinking behavior through their ameliorative effects on QOL.
hidden Markov model; COMBINE; joint analysis; alcoholism; alcohol dependence; naltrexone; acamprosate; behavioral; latent state
Variation in alcohol metabolism affects the duration of intoxication
and alcohol use. While the majority of genetic association studies
investigating variation in alcohol metabolism have focused on polymorphisms
in alcohol or aldehyde dehydrogenases, we have now tested for association
with genes in alternative metabolic pathways that catalyze the carbon
skeleton of ethanol and NADH reoxidation.
950 single nucleotide polymorphisms (SNPs) spanning 14 genes
(ACN9, ACSS1, ACSS2,
ALDH1A1, CAT, CYP2E1, GOT1,
GOT2, MDH1, MDH2,
SLC25A12, SLC25A13) were genotyped in
352 young adults who participated in an alcohol challenge study. Traits
tested were blood and breath alcohol concentration, peak alcohol
concentration and rates of alcohol absorption and elimination. Allelic
association was tested using quantitative univariate and multivariate
A CYP2E1 promoter SNP (rs4838767, minor allele
frequency 0.008) exceeded the threshold for study-wide significance (4.01
× 10−5) for two early blood alcohol concentration
(BAC), eight breath alcohol concentration (BrAC) measures and the peak BrAC.
For each phenotype the minor C-allele was related to a lower alcohol
concentration, most strongly for the fourth BrAC (P = 2.07 ×
10−7) explaining ~8% of the phenotypic
variance. We also observed suggestive patterns of association with variants
in ALDH1A1 and on chromosome 17 near
SLC25A11 for aspects of blood and breath alcohol
metabolism. A SNP upstream of GOT1 (rs2490286) reached
study-wide significance for multivariate BAC metabolism (P =
Overall, we did not find strong evidence that variation in genes
coding for proteins that further metabolize the carbon backbone of
acetaldehyde, or contribute to mechanisms for regenerating NAD from NADH,
affects alcohol metabolism in our European-descent subjects. However, based
on the breath alcohol data, variation in the promoter of
CYP2E1 may play a role in pre-absorptive or early
hepatic alcohol metabolism, but more samples are required to validate this
alcohol metabolism; association; genetics; CYP2E1; alcohol challenge
The current study tested age of onset as a moderator of intervention efficacy on drinking and consequence outcomes among a high-risk population of college students (i.e., former high school athletes).
Students were randomized to one of four conditions: assessment only control, combined parent-based intervention (PBI) and brief motivational intervention (BMI), PBI alone, and BMI alone. Participants (n = 1,275) completed web-administered measures at baseline (summer before starting college) and 10-month follow-up.
Overall, the combined intervention demonstrated the strongest and most consistent reductions across all outcomes, particularly with the youngest initiators. Participants who initiated drinking at the youngest ages had significantly lower peak drinking, typical weekly drinking, and reported consequences at follow-up when they received the combined intervention when compared to the control group. The BMI and PBI groups, when examined independently, demonstrated significant effects across outcomes but were inconsistent across the different age groups.
Results suggest the combination of a PBI and a peer-delivered BMI is an appropriate and efficacious way to reduce drinking and related consequences among individuals who initiated drinking earlier in adolescence and are at an increased risk of experiencing alcohol problems.
Age of Drinking Onset; Prevention; College Students; Parent
Alcohol has been shown to have a number of harmful effects on the lung, including increasing the risk of pneumonia and bronchitis. How alcohol increases the risk of these diseases is poorly defined. RhoA is a small guanosine triphosphate (GTP)ase that plays an integral role in many basic functions of airway epithelial cells. It is not known how alcohol affects RhoA activity in the airway epithelium. We hypothesized that brief alcohol exposure modulates RhoA activity in the airway epithelium through a nitric oxide (NO)/Cyclic GMP (cGMP)/Protein Kinase G (PKG) dependent pathway.
Primary airway epithelial cells were cultured and exposed to ethanol at various concentrations and times. The cell layers were harvested and RhoA activity was measured.
Alcohol induced a time- and concentration-dependent decrease in RhoA activity in airway epithelial cells. We were able to block this decrease in activity using Nω-Nitro-l-arginine methyl ester hydrochloride (L-NAME), a nitric oxide synthase (NOS) inhibitor. Likewise, we were able to demonstrate the same decrease in RhoA activation using 0.1µM sodium nitroprusside (SNP), an NO donor. To determine the role of cGMP/PKG, we pretreated the cells with a cGMP antagonist analogue, Rp-8Br-cGMPS. This blocked the decrease in RhoA activity caused by alcohol, suggesting that alcohol exerts its effect on RhoA activity through cGMP/PKG.
Alcohol decreases airway epithelial RhoA activity through an NO/cGMP/PKG- dependent pathway. RhoA activity controls many aspects of basic cellular function, including cell morphology, tight junction formation, and cell cycle progression and gene regulation. Dysregulation of RhoA activity can potentially have several consequences, including dysregulation of inflammation. This may partially explain how alcohol increases the risk of pneumonia and bronchitis.
ethanol; nitric oxide; PKG; cGMP; airway epithelium
Ethanol is metabolized by a two-step process in which alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde, which is further oxidized to acetate by aldehyde dehydrogenase (ALDH). Although variation in ethanol metabolism in humans strongly influences the propensity to chronically abuse alcohol, few data exist on the behavioral effects of altered ethanol metabolism. Here, we used the nematode C. elegans to directly examine how changes in ethanol metabolism alter behavioral responses to alcohol during an acute exposure. Additionally, we investigated ethanol solution osmolarity as a potential explanation for contrasting published data on C. elegans ethanol sensitivity.
We developed a gas chromatography assay and validated a spectrophotometric method to measure internal ethanol in ethanol-exposed worms. Further, we tested the effects of mutations in ADH and ALDH genes on ethanol tissue accumulation and behavioral sensitivity to the drug. Finally, we tested the effects of ethanol solution osmolarity on behavioral responses and tissue ethanol accumulation.
Only a small amount of exogenously applied ethanol accumulated in the tissues of C. elegans and consequently their tissue concentrations were similar to those that intoxicate humans. Independent inactivation of an ADH-encoding gene (sodh-1) or an ALDH-encoding gene (alh-6 or alh-13) increased the ethanol concentration in worms and caused hypersensitivity to the acute sedative effects of ethanol on locomotion. We also found that the sensitivity to the depressive effects of ethanol on locomotion is strongly influenced by the osmolarity of the exogenous ethanol solution.
Our results indicate that ethanol metabolism via ADH and ALDH has a statistically discernable but surprisingly minor influence on ethanol sedation and internal ethanol accumulation in worms. In contrast, the osmolarity of the medium in which ethanol is delivered to the animals has a more substantial effect on the observed sensitivity to ethanol.
Background & Rationale
Induction of reactive oxygen species (ROS) is a central mechanism in alcohol hepatotoxicity. Krüppel-like factor 6 (KLF6), a transcription factor and a tumor-suppressor gene, is an early responsive gene to injury; however, the impact of ROS and alcohol on KLF6 induction is unknown.
To investigate the contribution of two sources of ROS, cytochrome P450 2E1 (CYP2E1) and NAD(P)H quinone oxidoreductase (NQO1) and alcohol to the modulation of KLF6Full expression, splicing to KLF6_V1 and KLF6_V2 and the effect on TNFα, a downstream target.
Methods & Results
Endogenous ROS production in CYP2E1-expressing HepG2 cells induced mRNA and protein expression of KLF6Full and its splice variants compared to control cells. Incubation with pro-oxidants such as arachidonic acid (AA), β-naphtoflavone and H2O2, further enhanced KLF6Full and itssplice variants. The AA effects on KLF6Full and its splice forms were blocked by vitamin E -which prevents lipid peroxidation- and by diallylsulfide -a CYP2E1 inhibitor-. Menadione and paraquat –two pro-oxidants metabolized via NQO1- induced KLF6Full mRNA in a thiol-dependent manner. Antioxidants and a NQO1 inhibitor suppressed the menadione-dependent increase in KLF6Full and its splice variants mRNA. Furthermore, primary hepatocytes and livers from chronic alcohol-fed rats, with elevated lipid peroxidation, H2O2 and CYP2E1 but with low GSH, showed a ~2-fold increase in KLF6Full mRNA compared to controls. Inhibition of p38 phosphorylation further up-regulated the CYP2E1 and the AA effects on KLF6Full mRNA, whereas inhibition JNK and ERK1/2 phosphorylation decreased both. KLF6_V1 but not KLF6Full ablation markedly increased TNFα levels in macrophages; thus, TNFα emerges as a downstream target of KLF6_V1.
The novel effect of ROS on modulating KLF6Full expression and its splice variants could play a relevant role in liver injury and in TNFα regulation.
cytochrome P450 2E1; NAD(P)H quinone oxido-reductase; reactive oxygen species; TNFα
Alcohol abuse is frequently associated with nicotine use. The current experiments were conducted to establish an oral operant ethanol + nicotine (EtOH + Nic) co-use model, and to characterize some aspects of EtOH + Nic co-use. Methods: Rats were allowed to choose between EtOH alone or EtOH + Nic solutions. Additionally, P rats were allowed to concurrently self-administer 3 distinct EtOH solutions (10, 20, and 30%) with varying amounts of nicotine (0.07, 0.14, or 0.21 mg/ml) under operant conditions. P rats were also allowed to concurrently self-administer 2 distinct amounts of nicotine (0.07 and 0.14 mg/ml) added to saccharin (0.025%) solutions.
During acquisition, P rats responded for the EtOH + Nic solutions at the same level as for EtOH alone, and responding for EtOH + Nic solutions was present throughout all drinking conditions. P rats also readily maintained stable self-administration behaviors for Nic + Sacc solutions. The results demonstrated that P rats readily acquired and maintained stable self-administration behaviors for EtOH + 0.07 and EtOH + 0.14 mg/ml Nic solutions. Self-administration of EtOH+ 0.21 mg/ml Nic was established in only 50% of the subjects. P rats readily expressed seeking behaviors for the EtOH + Nic solutions, and reacquired EtOH + Nic self-administration during relapse testing. In addition, tailblood samples indicated that EtOH + Nic co-use resulted in pharmacologically relevant levels of both EtOH and Nic in the blood.
Overall, the results indicate that P rats readily consume EtOH + Nic solutions concurrently in the presence of EtOH alone, express drug-seeking behaviors, and will concurrently consume physiologically relevant levels of both drugs. These results support the idea that this oral operant EtOH + Nic co-use model would be suitable for studying the development of co-abuse and the consequences of long-term chronic co-abuse.
Co-use; Co-Abuse; Ethanol; Nicotine; EtOH + Nicotine-seeking; Relapse; Pavlovian Spontaneous Recovery; Alcohol Preferring P rat
Naltrexone is moderately effective for the treatment of alcohol dependence but there is great individual variability. The opioid receptor (OPRM1) SNP asn40asp has been shown to alter alcohol and naltrexone response in animals and humans. In addition, the brain opioid and dopamine systems interact and might underlie drinking and craving. This study investigated the effects of the OPRM1 SNP and dopamine transporter VNTR genetic differences on drinking, alcohol effects, and naltrexone response under controlled conditions in non-treatment seeking alcoholics.
265 non-treatment seeking individuals with alcohol dependence were genotyped a priori for the OPRM1 asn40asp SNP and post-hoc for DAT (SCL6A3) 9 and 10 VNTR’s. Asp40 carriers (n=43) and matched asn40 homozygotes (n= 40) were randomized to naltrexone or placebo for 7 days before receiving a priming drink and limited-access alcohol consumption in a bar-lab setting. Effects of genotypes on natural drinking as well as drinking, alcohol effects, and response to naltrexone in the bar-lab setting were examined by genotype.
There were no significant main effects of naltrexone or OPRM1 genotype, or any medication by OPRM1 interaction, on drinking variables. However, in individuals who had at least one DAT 9 VNTR, and who were also OPRM1 asn40 homozygotes, naltrexone reduced drinks/day consumed under natural conditions (p=0.006) but not in the bar-lab. OPRM1 asn40 homozygotes (p=0.028) and DAT 9 VNTR carriers (p=0.032) had more stimulation to alcohol after the priming drink.
This study does not support a salient role for the OPRM1 asp40 alone in predicting drinking or naltrexone effects. However, although exploratory and in need of replication, it introduces the possibility that epistasis between the OPRM1 gene and DAT gene might need to be taken into account when examining differential genetic response to alcohol or medication treatment, especially in early-stage alcoholics.
Alcoholism; Genetics; Medication; Drinking; Opioid Receptors
Perhaps the most difficult thing to ascertain concerning the behavior of another animal is its motivation. The motivation underlying the preference of Drosophila melanogaster for ethanol-rich food has long been ascribed to its value as a food. A recently introduced idea is that, as in humans, the pharmacological effects of ethanol also motivate the fly to choose ethanol-rich food over non-alcoholic food.
Flies are given a choice between pipets that contain liquid food and liquid food supplemented with ethanol. In some experiments, carbohydrates are added to the non-ethanol-containing food to balance the calories for ethanol.
We confirm that Drosophila melanogaster indeed prefer food that is supplemented with ethanol. However, if the alternative food choice is isocaloric, Drosophila melanogaster usually do not show any preference for a 10% ethanol solution. Even after ethanol preference has been established, it can be completely reversed if the alternative food is calorically supplemented. This occurs even when the carbohydrate solution used to balance calories is not gustatorily attractive. Furthermore, if the alternative food contains more calories than the ethanol food, the flies will prefer the non-ethanol food. We go on to show that during the preference assay that ethanol in the fly does not exceed 4 mM, which in mammals is a non-intoxicating dose.
We conclude that preference for ethanol in this assay arises not from the pharmacological effects of ethanol but rather because of its nutritive value.
preference; Drosophila; calorie; nutrition
Previous studies have shown that chronic ethanol ingestion results in impaired alveolar macrophage function, increased TGF-β1 production, and decreased antioxidant availability. Similarly, alternative activation (M2 activation) of alveolar macrophages also induces TGF-β1 production and impairs macrophage function. However, the potential links between ethanol-induced alveolar macrophage derangements, M2 activation, TGF-β1 production signaling, and oxidant stress has yet to be examined.
We hypothesized that ethanol-induced oxidant stress and induction of TGF-β1 signaling results in alternative activation which subsequently impairs the phagocytic capacity of alveolar macrophages.
Primary rat alveolar macrophages and the alveolar macrophages cell line NR8383 was treated with 0.08% ethanol ± the antioxidant glutathione (GSH) or a TGF-β1 neutralizing antibody for 5 days. Outcome measures included TGF-β1 production, reactive oxygen species (ROS) production, phagocytic capacity, and expression of markers of M2 activation.
Chronic ethanol treatment greatly decreased alveolar macrophage phagocytic function, increased ROS production, increased TGF-β1, and increased expression of markers of M2 activation. Glutathione supplementation and inhibition of TGF-β1 signaling during ethanol treatment prevented these alterations.
Ethanol treatment increased oxidant stress, TGF-β1 production, and alternative activation in NR8383 cells. However, GSH supplementation and ablation of TGF-β1 signaling prevented these effects. This suggested the ethanol-induced switch to a M2 phenotype was a result of decreased antioxidant availability and increased TGF-β1 signaling. Preventing ethanol-induced induction of alternative activation may improve alveolar macrophage function in alcoholic subjects and decrease the risk of respiratory infections.
macrophage; TGF-β1; oxidative stress; glutathione; alternative activation
In the aldehyde dehydrogenase 2 (ALDH2) gene, the ALDH2*2 allele, prevalent in East Asian populations, encodes an enzyme with severely reduced activity, thereby disrupting the normal metabolism of alcohol. Possession of the ALDH2*2 allele has been repeatedly shown to be associated with lower risk for alcohol dependence, and reduced alcohol use. However, relatively few studies have considered whether the magnitude of the effect of ALDH2 polymorphism upon drinking is related to developmental stage, or varies by environmental context.
In a longitudinally assessed sample of 356 adopted adolescents and young adults of East Asian descent, we examined the progression over time of the relationship between ALDH2 genotype and multiple measures of drinking behavior. We also sought to determine whether the environmental influences of non-biological parent and elder sibling alcohol use and misuse, as well as deviant peer behavior, moderated the effect of ALDH2 genotype upon alcohol use.
Across all measures of alcohol use, the association between ALDH2*2 allele possession and reduced drinking went from negligible to moderate between mid-adolescence and early adulthood. A combined index of adoptive parent alcohol use and misuse consistently moderated the protective effect of the ALDH2*2 allele across measures of quantity and frequency of alcohol use, and symptomology, such that high parental alcohol use and misuse reduced the protective effect of the ALDH2*2 allele, while low parental alcohol use and misuse enhanced the effect of the allele. Neither a combined index of elder sibling alcohol use and misuse, nor deviant peer behavior were consistently related to the effect of ALDH2 genotype.
The protective effect of the ALDH2*2 allele increases over the course of adolescence and young adulthood and is modified by the environmental influence of parental alcohol use and misuse. As such, ALDH2 provides a model system for exploring the nature of gene-environment interplay across development.
Gene-environment Interplay; Aldehyde Dehydrogenase; ALDH2; Adoption; Asian-Americans
Many children with heavy exposure to alcohol in utero display characteristic alterations in brain size and structure. However, the long-term effects of low-to-moderate alcohol exposure on these outcomes are unknown.
Using voxel-based morphometry and region-of-interest analyses, we examined the influence of lower doses of alcohol on gray and white matter composition in a prospectively recruited, homogeneous, well-characterized cohort of alcohol-exposed (n=11, age 19.5±0.3 yr) and control (n=9, age 19.6±0.5 yr) young adults. A large proportion of the exposed individuals were born to mothers whose alcohol consumption during pregnancy was in the low-to-moderate range.
There were no differences in total brain volume or total gray or white matter volume between the exposed and control groups. However, gray matter volume was reduced in alcohol-exposed individuals in several areas previously reported to be affected by high levels of exposure, including the left cingulate gyrus, bilateral middle frontal gyri, right middle temporal gyrus, and right caudate nucleus. Notably, this gray matter loss was dose dependent, with higher exposure producing more substantial losses.
These results indicate that even at low doses, alcohol exposure during pregnancy impacts brain development and that these effects persist into young adulthood.
prenatal alcohol exposure; voxel based morphometry; fetal alcohol spectrum disorders; brain volume
Prenatal alcohol exposure has been associated with pre- and postnatal growth restriction, but little is known about the natural history of this restriction throughout childhood or the effects of prenatal alcohol on body composition.
To examine the effects of heavy prenatal alcohol exposure on longitudinal growth and body composition.
85 heavy drinking pregnant women (≥ 2 drinks/day or ≥ 4 drinks/occasion) and 63 abstaining and light-drinking controls (< 1 drink/day, no binging) were recruited at initiation of prenatal care in an urban obstetrical clinic in Cape Town, South Africa, and prospectively interviewed during pregnancy about alcohol, smoking, drug use, and demographics. Among their children, length/height, weight, and head circumference were measured at 6.5 and 12 months and at 5 and 9 years. Percent body fat was estimated at age 9 years using bioelectric impedance analysis.
In multiple regression models with repeated measures (adjusted for confounders), heavy alcohol exposure was associated with reductions in weight (0.6 SD), length/height (0.5 SD), and head circumference (0.9 cm) from 6.5 months to 9 years that were largely determined at birth. These effects were exacerbated by iron deficiency in infancy but were not modified by iron deficiency or measures of food security at 5 years. An alcohol-related postnatal delay in weight gain was seen at 12 months. Effects on head circumference were greater at age 9 than at other age points. Although heavy alcohol exposure was not associated with changes in body composition, children with fetal alcohol syndrome (FAS) and partial FAS (PFAS) had lower % body fat than heavy exposed nonsyndromal and control children.
Heavy prenatal alcohol exposure is related to prenatal growth restriction that persists through age 9 years and an additional delay in weight gain during infancy. FAS and PFAS diagnoses are associated with leaner body composition in later childhood.
fetal alcohol syndrome; intrauterine growth retardation; postnatal growth; body composition; bioelectric impedance analysis; prenatal alcohol exposure; iron deficiency; food security
Fetal alcohol spectrum disorders (FASD) result from heavy prenatal alcohol exposure, and are characterized, in some cases, by CNS anomalies and cognitive impairment. Regional patterns of neuroanatomical abnormalities suggest that alcohol exerts selective damage on the developing fetal brain. This study assessed brain-behavior relationships in a sample of youth with histories of heavy prenatal alcohol exposure. The aim was to characterize how structural brain alterations observed in our previous studies relate to cognitive deficits commonly reported in individuals with histories of heavy prenatal alcohol exposure.
Twenty-one youth (mean age 13 years) with histories of heavy prenatal alcohol exposure and seven non-exposed healthy comparison subjects underwent structural magnetic resonance imaging (MRI) and neurobehavioral testing. Regional brain volumes within the alcohol-exposed group were correlated with neuropsychological measures of cognitive control and verbal learning/recall, as these aspects of cognition have previously been shown to be vulnerable to alcohol teratogenesis.
Between-group effect sizes revealed moderate to large cognitive performance and brain volume decrements in alcohol-exposed subjects, compared to typically developing peers. Within the alcohol-exposed group, volume of the caudate nuclei was the most consistent predictor of neuropsychological performance, after controlling for potentially confounding variables including total brain volume, IQ, and age.
These data are consistent with previous research associating gestational alcohol exposure with structural and functional changes of the caudate nucleus. Our findings extend this previous work by demonstrating that volume reductions of the caudate have behavioral relevance for this population, in relation to cognitive control and verbal learning and recall abilities.
fetal alcohol spectrum disorders (FASD); fetal alcohol syndrome (FAS); brain-behavior correlations; verbal learning/recall; cognitive control