Genome-wide Association Study and Meta-analysis on Alcohol-Associated Liver Cirrhosis Identifies Genetic Risk Factors.

BACKGROUND AND AIMS: Only a minority of heavy drinkers progress to alcohol-associated cirrhosis (ALC). The aim of this study was to identify common genetic variants that underlie risk for ALC. APPROACH AND RESULTS: We analyzed data from 1,128 subjects of European ancestry with ALC and 614 heavy-drinking subjects without known liver disease from Australia, the United States, the United Kingdom, and three countries in Europe. A genome-wide association study (GWAS) was performed, adjusting for principal components and clinical covariates (alcohol use, age, sex, body mass index, and diabetes). We validated our GWAS findings using UK Biobank. We then performed a meta-analysis combining data from our study, the UK Biobank, and a previously published GWAS. Our GWAS found genome-wide significant risk association of rs738409 in patatin-like phospholipase domain containing 3 (PNPLA3) (odds ratio [OR] = 2.19 [G allele], P = 4.93 × 10-17 ) and rs4607179 near HSD17B13 (OR = 0.57 [C allele], P = 1.09 × 10-10 ) with ALC. Conditional analysis accounting for the PNPLA3 and HSD17B13 loci identified a protective association at rs374702773 in Fas-associated factor family member 2 (FAF2) (OR = 0.61 [del(T) allele], P = 2.56 × 10-8 ) for ALC. This association was replicated in the UK Biobank using conditional analysis (OR = 0.79, P = 0.001). Meta-analysis (without conditioning) confirmed genome-wide significance for the identified FAF2 locus as well as PNPLA3 and HSD17B13. Two other previously known loci (SERPINA1 and SUGP1/TM6SF2) were also genome-wide significant in the meta-analysis. GeneOntology pathway analysis identified lipid droplets as the target for several identified genes. In conclusion, our GWAS identified a locus at FAF2 associated with reduced risk of ALC among heavy drinkers. Like the PNPLA3 and HSD17B13 gene products, the FAF2 product has been localized to fat droplets in hepatocytes. CONCLUSIONS: Our genetic findings implicate lipid droplets in the biological pathway(s) underlying ALC.

Obesity, Diabetes, Coffee, Tea, and Cannabis Use Alter Risk for Alcohol-Related Cirrhosis in 2 Large Cohorts of High-Risk Drinkers.

INTRODUCTION: Sustained high alcohol intake is necessary but not sufficient to produce alcohol-related cirrhosis. Identification of risk factors, apart from lifetime alcohol exposure, would assist in discovery of mechanisms and prediction of risk. METHODS: We conducted a multicenter case-control study (GenomALC) comparing 1,293 cases (with alcohol-related cirrhosis, 75.6% male) and 754 controls (with equivalent alcohol exposure but no evidence of liver disease, 73.6% male). Information confirming or excluding cirrhosis, and on alcohol intake and other potential risk factors, was obtained from clinical records and by interview. Case-control differences in risk factors discovered in the GenomALC participants were validated using similar data from 407 cases and 6,573 controls from UK Biobank. RESULTS: The GenomALC case and control groups reported similar lifetime alcohol intake (1,374 vs 1,412 kg). Cases had a higher prevalence of diabetes (20.5% (262/1,288) vs 6.5% (48/734), P = 2.27 × 10-18) and higher premorbid body mass index (26.37 ± 0.16 kg/m2) than controls (24.44 ± 0.18 kg/m2, P = 5.77 × 10-15). Controls were significantly more likely to have been wine drinkers, coffee drinkers, smokers, and cannabis users than cases. Cases reported a higher proportion of parents who died of liver disease than controls (odds ratio 2.25 95% confidence interval 1.55-3.26). Data from UK Biobank confirmed these findings for diabetes, body mass index, proportion of alcohol as wine, and coffee consumption. DISCUSSION: If these relationships are causal, measures such as weight loss, intensive treatment of diabetes or prediabetic states, and coffee consumption should reduce the risk of alcohol-related cirrhosis.

High Resolution Genomic Scans Reveal Genetic Architecture Controlling Alcohol Preference in Bidirectionally Selected Rat Model.

Investigations on the influence of nature vs. nurture on Alcoholism (Alcohol Use Disorder) in human have yet to provide a clear view on potential genomic etiologies. To address this issue, we sequenced a replicated animal model system bidirectionally-selected for alcohol preference (AP). This model is uniquely suited to map genetic effects with high reproducibility, and resolution. The origin of the rat lines (an 8-way cross) resulted in small haplotype blocks (HB) with a corresponding high level of resolution. We sequenced DNAs from 40 samples (10 per line of each replicate) to determine allele frequencies and HB. We achieved ~46X coverage per line and replicate. Excessive differentiation in the genomic architecture between lines, across replicates, termed signatures of selection (SS), were classified according to gene and region. We identified SS in 930 genes associated with AP. The majority (50%) of the SS were confined to single gene regions, the greatest numbers of which were in promoters (284) and intronic regions (169) with the least in exon's (4), suggesting that differences in AP were primarily due to alterations in regulatory regions. We confirmed previously identified genes and found many new genes associated with AP. Of those newly identified genes, several demonstrated neuronal function involved in synaptic memory and reward behavior, e.g. ion channels (Kcnf1, Kcnn3, Scn5a), excitatory receptors (Grin2a, Gria3, Grip1), neurotransmitters (Pomc), and synapses (Snap29). This study not only reveals the polygenic architecture of AP, but also emphasizes the importance of regulatory elements, consistent with other complex traits.

CIS-Acting Allele-Specific Expression Differences Induced by Alcohol and Impacted by Sex as Well as Parental Genotype of Origin.

BACKGROUND: Alcohol use disorders (AUDs) are influenced by complex interactions between the genetics of the individual and their environment. We have previously identified hundreds of polygenic genetic variants between the selectively bred high- and low-alcohol drinking (HAD and LAD) rat lines. Here, we report allele-specific expression (ASE) differences, between the HAD2 and LAD2 rat lines. METHODS: The HAD2 and LAD2 rats, which have been sequenced, were reciprocally crossed to generate 10 litters of F1 progeny. For 5 of these litters, the sire was HAD2, and for the other 5 litters, the sire was a LAD2. From these 10 litters, 2 males and 2 females were picked from each F1 litter (N = 40 total). The F1 pups were divided, balancing for sex and direction of cross, into an alcohol (15%) versus a water control group. Alcohol drinking started in the middle of adolescence (~postnatal day 35) and lasted 9 weeks. At the end of these treatments, rats were euthanized, the nucleus accumbens was dissected, and RNA was processed for RNA-sequencing and ASE analyses. RESULTS: Analyses revealed that adolescent ethanol (EtOH) drinking, individual EtOH drinking levels, parentage, and sex-of-animal affected ASEs of about 300 genes. The identified genes included those associated with EtOH metabolism (e.g., Aldh2); neuromodulatory function (e.g., Cckbr, Slc6a7, and Slc1a1); ion channel activity (e.g., Kcnc3); and other synaptic and epigenetic functions. CONCLUSIONS: These data indicate that EtOH drinking differentially amplified paternal versus maternal allelic contribution to the transcriptome. We hypothesize that this was due, at least in part, to EtOH-induced changes in cis-regulation of polymorphisms previously identified between the HAD2 and LAD2 rat lines. This report highlights the complexity of gene-by-environment interactions mediating a genetic predisposition for, and/or the active development of, AUDs.

Brief report: genetics of alcoholic cirrhosis-GenomALC multinational study.

BACKGROUND: The risk of alcohol-related liver cirrhosis increases with increasing alcohol consumption, but many people with very high intake escape from liver disease. We postulate that susceptibility to alcoholic cirrhosis has a complex genetic component and propose that this can be dissected through a large and sufficiently powered genomewide association study (GWAS). METHODS: The GenomALC Consortium comprises researchers from Australia, France, Germany, Switzerland, United Kingdom, and United States, with a joint aim of exploring the genetic and genomic basis of alcoholic cirrhosis. For this National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism funded study, we are recruiting high-risk drinkers who are either cases (with alcoholic cirrhosis) or controls (drinking comparable amounts over similar time, but free of significant liver disease). Extensive phenotypic data are obtained using semistructured interviews and patient records, and blood samples are collected. RESULTS: We have successfully recruited 859 participants including 538 matched case-control samples as of September 2014, using study-specific inclusion-exclusion criteria and data collection protocols. Of these, 580 are cases (442 men and 138 women) and 279 are controls (205 men and 74 women). Duration of excessive drinking was slightly greater in cases than controls and was significantly less in women than men. Cases had significantly lower lifetime alcohol intake than controls. Both cases and controls had a high prevalence of reported parental alcohol problems, but cases were significantly more likely to report that a father with alcohol problems had died from liver disease (odds ratio 2.53, 95% confidence interval 1.31 to 4.87, p = 0.0055). CONCLUSIONS: Recruitment of participants for a GWAS of alcoholic cirrhosis has proved feasible across countries with multiple sites. Affected patients often consume less alcohol than unaffected ones, emphasizing the existence of individual vulnerability factors. Cases are more likely to report liver disease in a father with alcohol problems than controls, consistent with a potential genetic component to the risk of alcoholic cirrhosis.

Comparison of the effect of the GABAΒ receptor agonist, baclofen, and the positive allosteric modulator of the GABAB receptor, GS39783, on alcohol self-administration in 3 different lines of alcohol-preferring rats.

BACKGROUND: Administration of the GABA(B) receptor agonist, baclofen, and positive allosteric modulator, GS39783, has been repeatedly reported to suppress multiple alcohol-related behaviors, including operant oral alcohol self-administration, in rats. This study was designed to compare the effect of baclofen and GS39783 on alcohol self-administration in 3 lines of selectively bred, alcohol-preferring rats: Indiana alcohol-preferring (P), Sardinian alcohol-preferring (sP), and Alko Alcohol (AA). METHODS: Rats of each line were initially trained to respond on a lever, on a fixed ratio (FR) 4 (FR4) schedule of reinforcement, to orally self-administer alcohol (15%, v/v) in daily 30-minute sessions. Once responding reached stable levels, rats were exposed to a sequence of experiments testing baclofen (0, 1, 1.7, and 3 mg/kg; i.p.) and GS39783 (0, 25, 50, and 100 mg/kg; i.g.) on FR4 and progressive ratio (PR) schedules of reinforcement. Finally, to assess the specificity of baclofen and GS39783 action, rats were slightly food-deprived and trained to lever-respond for food pellets. RESULTS: The rank of order of the reinforcing and motivational properties of alcohol was P>sP>AA rats. Under both FR and PR schedules of reinforcement, the rank of order of potency and efficacy of baclofen and GS39783 in suppressing alcohol self-administration was P>sP>AA rats. Only the highest dose of baclofen reduced lever-responding for food pellets; this effect was common to all 3 rat lines. Conversely, no dose of GS39783 altered lever-responding for food in any rat line. CONCLUSIONS: These results suggest that: (i) the strength of the reinforcing and motivational properties of alcohol differ among P, sP, and AA rats; (ii) the reinforcing and motivational properties of alcohol in P, sP, and AA rats are differentially sensitive to treatment with baclofen and GS39783; (iii) the heterogeneity in sensitivity to baclofen and GS39783 of alcohol self-administration in P, sP, and AA rats may resemble the differential effectiveness of pharmacotherapies among the different typologies of human alcoholics; and (iv) the GABA(B) receptor is part of the neural substrate mediating the reinforcing and motivational properties of alcohol.

Behavioral profiling of multiple pairs of rats selectively bred for high and low alcohol intake using the MCSF test.

Genetic aspects of alcoholism have been modeled using rats selectively bred for extremes of alcohol preference and voluntary alcohol intake. These lines show similar alcohol drinking phenotypes but have different genetic and environmental backgrounds and may therefore display diverse behavioral traits as seen in human alcoholics. The multivariate concentric square field™ (MCSF) test is designed to provoke exploration and behaviors associated with risk assessment, risk taking and shelter seeking in a novel environment. The aim was to use the MCSF to characterize behavioral profiles in rat lines from selective breeding programs in the United States (P/NP, HAD1/LAD1, HAD2/LAD2), Italy (sP/sNP) and Finland (AA/ANA). The open field and elevated plus maze tests were used as reference tests. There were substantial differences within some of the pairs of selectively bred rat lines as well as between all alcohol-preferring rats. The most pronounced differences within the pairs of lines were between AA and ANA rats and between sP and sNP rats followed by intermediate differences between P and NP rats and minor differences comparing HAD and LAD rats. Among all preferring lines, P, HAD1 and HAD2 rats shared similar behavioral profiles, while AA and sP rats were quite different from each other and the others. No single trait appeared to form a common 'pathway' associated with a high alcohol drinking phenotype among all of the alcohol-preferring lines of rats. The marked behavioral differences found in the different alcohol-preferring lines may mimic the heterogeneity observed among human alcoholic subtypes.

Hepatic and extrahepatic cancer in cirrhosis: a longitudinal cohort study.

OBJECTIVES: We conducted a retrospective cohort study in cirrhotic patients to understand (i) the risk of developing hepatocellular carcinoma (HCC) after an initial negative screening computed tomography (CT) scan and its relationship with underlying etiology and (ii) the risk of extrahepatic cancers (EHCs). METHODS: Our cohort consisted of 952 cirrhotics who had at least one contrast-enhanced CT scan over a 5-year period from 1997 to 2002. We assessed their risk of HCC and EHC until the study closure (31 December 2007). Using data from the Indiana State Cancer Registry (ISCR), standardized incidence ratios (SIRs) were calculated for HCC and EHC. RESULTS: The cohort's follow-up was 4.7±3.0 years. The frequency of HCC at baseline and during follow-up was 6.9 and 7.2%, respectively. The 1-, 3-, and 5-year HCC incidence after an initial negative CT scan was 1.2, 4.4, and 7.8%, respectively. The 1-, 3-, and 5-year EHC incidence was 2.2, 4.5, and 6.8%, respectively. The most common EHCs were breast, lung, and lymphoma. Incidence of both HCC (P=0.016) and EHC (P=0.004) varied significantly by the etiology of underlying cirrhosis. The SIRs for HCC and EHC were 186 (95% confidence interval (CI) 140-238) and 1.83 (95% CI 1.36-2.36), respectively. Compared with adjusted ISCR data, cirrhosis due to alcohol (SIR 2.73, 95% CI 1.14-4.33) but not other etiologies had significantly higher incidence of EHC. CONCLUSIONS: This study furthers our understanding of HCC and EHC risk in cirrhosis. If confirmed by other studies, these data will assist in developing optimal strategies for monitoring of cancer in individuals with cirrhosis.

Genomewide SNP screen to detect quantitative trait loci for alcohol preference in the high alcohol preferring and low alcohol preferring mice.

BACKGROUND: The high and low alcohol preferring (HAP1 and LAP1) mouse lines were selectively bred for differences in alcohol intake. The HAP1 and LAP1 mice are essentially noninbred lines that originated from the outbred colony of HS/Ibg mice, a heterogeneous stock developed from intercrossing 8 inbred strains of mice. METHODS: A total of 867 informative SNPs were genotyped in 989 HAP1 x LAP1 F2, 68 F1s, 14 parents (6 LAP1, 8 HAP1), as well as the 8 inbred strains of mice crossed to generate the HS/Ibg colony. Multipoint genome wide analyses were performed to simultaneously detect linked QTLs and also fine map these regions using the ancestral haplotypes. RESULTS: QTL analysis detected significant evidence of association on 4 chromosomes: 1, 3, 5, and 9. The region on chromosome 9 was previously found linked in a subset of these F2 animals using a whole genome microsatellite screen. CONCLUSIONS: We have detected strong evidence of association to multiple chromosomal regions in the mouse. Several of these regions include candidate genes previously associated with alcohol dependence in humans or other animal models.

Effects of concurrent access to multiple ethanol concentrations and repeated deprivations on alcohol intake of high-alcohol-drinking (HAD) rats.

High-alcohol-drinking rats, given access to 10% ethanol, expressed an alcohol deprivation effect (ADE) only after multiple deprivations. In alcohol-preferring (P) rats, concurrent access to multiple ethanol concentrations combined with repeated cycles of EtOH access and deprivation produced excessive ethanol drinking. The current study was undertaken to examine the effects of repeated alcohol deprivations with concurrent access to multiple concentrations of ethanol on ethanol intake of HAD replicate lines of rats. HAD-1 and HAD-2 rats received access to 10, 20 and 30% (v/v) ethanol for 6 weeks. Rats from each replicate line were assigned to: (1) a non-deprived group; (2) a group initially deprived of ethanol for 2 weeks; or (3) a group initially deprived for 8 weeks. Following the restoration of the ethanol solutions, cycle of 2 weeks of ethanol exposure and 2 weeks of alcohol deprivation was repeated three times for a total of four deprivations. Following the initial ethanol deprivation period, deprived groups significantly increased ethanol intakes during the initial 24-hour re-exposure period. Multiple deprivations increased ethanol intakes, shifted preference to higher ethanol concentrations and prolonged the duration of the elevated ethanol intakes for up to 5 days. In addition, repeated deprivations increased ethanol intake in the first 2-hour re-exposure period as high as 5-7 g/kg (which are equivalent to amounts consumed in 24 hours by HAD rats), and produced blood ethanol levels in excess of 150 mg%. The results indicate that HAD rats exhibit 'loss-of-control' of alcohol drinking with repeated deprivations when multiple ethanol concentrations are available.

Acute ethanol effects on local cerebral glucose utilization in select central nervous system regions of adolescent alcohol-preferring (P) and alcohol-nonpreferring (NP) rats.

BACKGROUND: Alcohol abuse among adolescents is a major health and developmental problem. The 2-[(14)C]deoxyglucose (2-DG) technique allows for the in vivo quantification of local cerebral glucose utilization (LCGU) as a measure of functional neuronal activity. METHODS: Local cerebral glucose utilization rates were examined after acute ethanol administration within selected brain regions of adolescent alcohol-preferring (P) and -nonpreferring (NP) rats. Postnatal day 45 male P and NP rats were injected with saline or 1.0 g/kg ethanol, i.p., 10 minutes prior to an intravenous bolus of [(14)C]-2-deoxyglucose (125 microCi/kg). Image densities were determined using quantitative autoradiography and LCGU values calculated. RESULTS: Acute ethanol injection significantly decreased LCGU rates in select brain regions including the olfactory tubercles, the frontal cortex (Fr), and subregions of the posterior hippocampus (pCA1 and pCA3). Acute ethanol had no significant effects on LCGU rates in any region of the adolescent NP rats. Significant basal LCGU rate differences were apparent between the rat lines in a nearly global fashion with adolescent P rats having much higher basal LCGU rates compared with adolescent NP rats. CONCLUSIONS: These findings suggest that the adolescent P and NP rats are less sensitive to the effects of acute ethanol than their adult counterparts. The adolescent P rat is relatively more sensitive to the initial effects of acute ethanol in select brain regions as compared with the adolescent NP rat. Additionally, the innate hyper-excited state of the adolescent P central nervous system is a likely factor in the development of their high alcohol drinking behaviors.

Autonomic activation associated with ethanol self-administration in adult female P rats.

The present study examined changes in heart rate (HR) prior to and during limited access ethanol drinking in adult female P rats. P rats were implanted with radio-telemetric transmitters to measure HR. Daily testing involved a 90-min pre-test period (water only available) and a subsequent 90-min test period [either water (W) or ethanol available]. After a week of habituation, one ethanol group had access to ethanol for 7 weeks (CE), and another ethanol group had access for 4 weeks, was deprived for 2 weeks and then had access for a final week (DEP). Analyses of HR revealed that CE and DEP rats had significantly higher HR than W rats during test periods that ethanol was present and that DEP rats displayed higher HR during the early test period of the ethanol deprivation interval, as well. These data indicate that ethanol drinking induces HR activation in adult female P rats, and that this activation can be conditioned to the test cage environment, paralleling reports on contextual conditioning and cue-reactivity in alcoholics exposed to alcohol-associated stimuli. Therefore, this behavioral test may prove advantageous in screening pharmacotherapies for reducing craving and relapse, which are associated with cue-reactivity in abstinent alcoholics.

Effects of alcohol and saccharin deprivations on concurrent ethanol and saccharin operant self-administration by alcohol-preferring (P) rats.

Consumption of sweet solutions has been associated with a reduction in withdrawal symptoms and alcohol craving in humans. The objective of the present study was to determine the effects of ethanol and saccharin (SACC) deprivations on operant oral self-administration. Alcohol-preferring (P) rats were allowed to lever press concurrently self-administer ethanol (15% vol/vol) and SACC (0.0125% g/vol) for 8 weeks. Rats were then maintained on daily operant access (nondeprived), deprived of both fluids (2 weeks), deprived of SACC and given 2 ml of ethanol daily, or deprived of ethanol and given 2 ml of SACC daily. All groups were then given 2 weeks of daily operant access to ethanol and SACC, followed by an identical second deprivation period. P rats responded more for ethanol than SACC. All deprived groups increased responding on the ethanol lever, but not on the SACC lever. Daily consumption of 2 ml ethanol decreased the duration of the alcohol deprivation effect (ADE). Home cage access to 2 ml of SACC also decreased the ADE but to a lesser extent than access to ethanol. A second deprivation period further increased and prolonged the expression of an ADE. These results show ethanol is a more salient reinforcer than SACC. With concurrent access to ethanol and SACC, P rats do not display a saccharin deprivation effect. Depriving P rats of both ethanol and SACC had the most pronounced effect on the magnitude and duration of the ADE, suggesting that there may be some interactions between ethanol and SACC in their CNS reinforcing effects.

Effects of short deprivation and re-exposure intervals on the ethanol drinking behavior of selectively bred high alcohol-consuming rats.

Alcoholics generally display cycles of excessive ethanol intake, abstinence and relapse behavior. Using an animal model of relapse-like drinking, the alcohol deprivation effect (ADE), our laboratory has shown that repeated 2-week cycles of ethanol deprivation and re-exposure, following an initial 6-week access period, result in a robust ADE by alcohol-preferring (P) and high alcohol-drinking (HAD-1 and HAD-2) rats. These rat lines have been selectively bred to prefer a 10% ethanol solution over water. The present study examined whether P and HAD rats would display an ADE using much shorter ethanol deprivation and re-exposure intervals. Rats were given either continuous or periodic concurrent access to multiple concentrations (10%, 20%, and 30% [vol/vol]) of ethanol. The periodic protocol involved access to ethanol for 12 days followed by four cycles of 4 days of deprivation and 4 days of re-exposure to ethanol access. High-alcohol-drinking rats displayed a robust 24-h ADE upon first re-exposure (HAD-1: approximately 5 vs. 8g/kg/day; HAD-2: approximately 6 vs. 9g/kg/day, baseline vs. re-exposure), whereas P rats ( approximately 7 vs. 8g/kg/day) displayed a modest, nonsignificant, increase in 24-h intake. In a separate group of rats, ethanol intake and blood alcohol concentrations after the first hour of the fourth re-exposure cycle were HAD-1: 2.0g/kg and 97 mg%, HAD-2: 2.3g/kg and 73 mg%, and P: 1.2g/kg and 71 mg%; with all three lines displaying a robust first hour ADE. These findings suggest that (a) an ADE may be observed with short ethanol deprivation and re-exposure intervals in HAD rats, and (b) the genetic make-up of the P and HAD rats influences the expression of this ADE.

Evaluation of laboratory tests for cirrhosis and for alcohol use, in the context of alcoholic cirrhosis.

Laboratory tests can play an important role in assessment of alcoholic patients, including for evaluation of liver damage and as markers of alcohol intake. Evidence on test performance should lead to better selection of appropriate tests and improved interpretation of results. We compared laboratory test results from 1578 patients between cases (with alcoholic cirrhosis; 753 men, 243 women) and controls (with equivalent lifetime alcohol intake but no liver disease; 439 men, 143 women). Comparisons were also made between 631 cases who had reportedly been abstinent from alcohol for over 60 days and 364 who had not. ROC curve analysis was used to estimate and compare tests' ability to distinguish patients with and without cirrhosis, and abstinent and drinking cases. The best tests for presence of cirrhosis were INR and bilirubin, with areas under the ROC curve (AUCs) of 0.91 ± 0.01 and 0.88 ± 0.01, respectively. Confining analysis to patients with no current or previous ascites gave AUCs of 0.88 ± 0.01 for INR and 0.85 ± 0.01 for bilirubin. GGT and AST showed discrimination between abstinence and recent drinking in patients with cirrhosis, including those without ascites, when appropriate (and for GGT, sex-specific) limits were used. For AST, a cut-off limit of 85 units/L gave 90% specificity and 37% sensitivity. For GGT, cut-off limits of 288 units/L in men and 138 units/L in women gave 90% specificity for both and 40% sensitivity in men, 63% sensitivity in women. INR and bilirubin show the best separation between patients with alcoholic cirrhosis (with or without ascites) and control patients with similar lifetime alcohol exposure. Although AST and GGT are substantially increased by liver disease, they can give useful information on recent alcohol intake in patients with alcoholic cirrhosis when appropriate cut-off limits are used.

Daily patterns of ethanol drinking in peri-adolescent and adult alcohol-preferring (P) rats.

Alcohol abuse among adolescents continues to be a major health problem for our society. Our laboratory has used the peri-adolescent alcohol-preferring, P, rat as an animal model of adolescent alcohol abuse. Even though peri-adolescent P rats consume more alcohol (g/kg/day) than their adult counterparts, it is uncertain whether their drinking is sufficiently aggregated to result in measurable blood ethanol concentrations (BECs). The objectives of this study were to examine daily alcohol drinking patterns of adolescent and adult, male and female P rats, and to determine whether alcohol drinking episodes were sufficiently aggregated to result in meaningful BECs. Male and female P rats were given 30 days of 24 h free-choice access to alcohol (15%, v/v) and water, with ad lib access to food, starting at the beginning of adolescence (PND 30) or adulthood (PND 90). Water and alcohol drinking patterns were monitored 22 h/day with a "lickometer" set-up. The results indicated that (a) peri-adolescent P rats consumed more water and total fluids than adult P rats, (b) female P rats consumed more water and total fluids than male P rats, (c) there were differences in alcohol, and water, licking patterns between peri-adolescent and adult and female and male P rats, (d) individual licking patterns revealed that alcohol was consumed in bouts often exceeding the amount required to self-administer 1 g/kg of alcohol, and (e) BECs at the end of the dark cycle, on the 30th day of alcohol access, averaged 50 mg%, with alcohol intakes during the last 1 to 2 h averaging 1.2 g/kg. Overall, these findings indicate that alcohol drinking patterns differ across the age and sex of P rats. This suggests that the effectiveness of treatments for reducing excessive alcohol intake may vary depending upon the age and/or sex of the subjects being tested.

Extracellular dopamine levels are lower in the medial prefrontal cortex of alcohol-preferring rats compared to Wistar rats.

Previous studies have identified deficiencies in the mesocorticolimbic dopamine (DA) systems of alcohol-preferring (P) rats. This study uses quantitative microdialysis to compare the extracellular levels of DA in the medial prefrontal cortex (MPF) of P rats and outbred Wistar rats and also compares the effects of systemic ethanol administration on DA levels in the MPF using traditional microdialysis. In experiment 1, male Wistar and P rats were implanted with loop-style microdialysis probes and later perfused at 0.5 microl/min with artificial cerebrospinal fluid for 120 min prior to five baseline (20-min) sample collections. Three concentrations (5, 10, and 20 nM) of DA were then perfused in random order for 100 min each. Samples (20-min) were collected and stored at -70 degrees C until assayed using high performance liquid chromatography/electrochemical detection (HPLC/EC), and the data were analyzed using the quantitative no-net-flux (NNF) method. In experiment 2, male Wistar and P rats were implanted with dialysis probes aimed at the MPF. After collecting four baseline samples, all rats were injected (i.p.) with one dose of either 0.9% saline or 2.0 g/kg ethanol. Microdialysis samples were collected at 20-min intervals and stored at -70 degrees C until analyzed by HPLC/EC. NNF microdialysis yielded significantly (P<.05) lower extracellular DA concentrations in the MPF of P rats compared to Wistar rats (2.0+/-0.4 vs. 4.8+/-0.4 nM, respectively). The extraction fractions were not different between the P and Wistar groups (69+/-3 vs. 65+/-3%, respectively). No significant change in extracellular DA levels was observed in P rats or Wistar rats after either saline or 2g/kg ethanol. The lower extracellular concentrations of DA in the MPF of P rats compared to Wistar rats, without a difference in the extraction fraction, suggest that DA neurotransmission is lower in the MPF of the P rat. This lower DA neurotransmission could be a result of reduced activity of the DA neurons projecting to the MPF, reduced excitatory or increased inhibitory tone occurring locally within the MPF, and/or reduced DA innervation to the MPF. The lack of effect of systemic EtOH administration on extracellular DA levels in the MPF suggests that unlike the mesolimbic DA system, the mesocortical DA system is not responsive to acute EtOH administration.

Effects of multiple alcohol deprivations on operant ethanol self-administration by high-alcohol-drinking replicate rat lines.

Previously, we reported that the expression of an alcohol deprivation effect (ADE) under 24-h free-choice alcohol-drinking access in high-alcohol-drinking (HAD) replicate lines of rats is dependent upon repeated cycles of alcohol access and forced abstinence. In the present study, operant techniques (including progressive ratio measures) were used to examine the effects of initial deprivation length and number of deprivation cycles on the magnitude and duration of the ADE in HAD rats to test the hypothesis that repeated deprivations increase the reinforcing effects of ethanol. Adult male HAD-1 and HAD-2 rats were trained in two-lever operant chambers to concurrently self-administer 15% ethanol (v/v) on a fixed-ratio (FR)-5 schedule and water on an FR-1 schedule of reinforcement in daily 1-h sessions. Following 10 weeks of daily 1-h sessions, the HAD-1 and HAD-2 rats were randomly assigned to one of four groups (n=6-8/group/line): nondeprived, or deprived of alcohol for 2, 5, or 8 weeks. Following this initial period, the deprived groups were given 15% ethanol again in the operant chambers for a 2-week period, following which they were deprived again for 2 weeks (all three deprived groups). Following the fifth deprivation, the rats underwent a progressive ratio test to determine the breakpoints for the nondeprived and deprived groups. The expression of an ADE under operant conditions in HAD rats was dependent upon exposure to repeated cycles of ethanol access and abstinence. Additionally, repeated deprivations increased both the magnitude and the duration of the ADE as indicated by increased responding on the ethanol lever for more sessions. Breakpoint values for the deprived groups were 1.5-fold and twofold higher than the value for the nondeprived group for the HAD-1 and HAD-2 rats, respectively. The results suggest that repeated alcohol deprivations increased the expression of an ADE and the reinforcing effects of ethanol in both HAD replicate lines of rats, and these effects were more pronounced in the HAD-2 line than the HAD-1 line.

Npy deletion in an alcohol non-preferring rat model elicits differential effects on alcohol consumption and body weight.

Neuropeptide Y (NPY) is widely expressed in the central nervous system and influences many physiological processes. It is located within the rat quantitative trait locus (QTL) for alcohol preference on chromosome 4. Alcohol-nonpreferring (NP) rats consume very little alcohol, but have significantly higher NPY expression in the brain than alcohol-preferring (P) rats. We capitalized on this phenotypic difference by creating an Npy knockout (KO) rat using the inbred NP background to evaluate NPY effects on alcohol consumption. Zinc finger nuclease (ZNF) technology was applied, resulting in a 26-bp deletion in the Npy gene. RT-PCR, Western blotting and immunohistochemistry confirmed the absence of Npy mRNA and protein in KO rats. Alcohol consumption was increased in Npy(+/-) but not Npy(-/-) rats, while Npy(-/-) rats displayed significantly lower body weight when compared to Npy(+/+) rats. In whole brain tissue, expression levels of Npy-related and other alcohol-associated genes, Npy1r, Npy2r, Npy5r, Agrp, Mc3r, Mc4r, Crh and Crh1r, were significantly greater in Npy(-/-) rats, whereas Pomc and Crhr2 expressions were highest in Npy(+/-) rats. These findings suggest that the NPY-system works in close coordination with the melanocortin (MC) and corticotropin-releasing hormone (CRH) systems to modulate alcohol intake and body weight.

Dopamine and serotonin content in select brain regions of weanling and adult alcohol drinking rat lines.

The objective of the present study was to examine innate differences in the tissue content of dopamine (DA), serotonin (5-HT) and their metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) in five brain regions of weanling and adult alcohol-preferring (P), alcohol-nonpreferring (NP), high-alcohol-drinking (HAD) and low-alcohol-drinking (LAD) selected rat lines. Adult male and weanling (postnatal day 25) male rats were killed by decapitation and brains were rapidly dissected for the following regions: olfactory tubercles (OTU), nucleus accumbens (ACB), septum (SEP), anterior cerebral cortex (ACTX) and amygdala (AMYG). Tissue extracts were assayed by HPLC with electrochemical detection. Due to significantly higher content levels in the adults, adult and weanling animals were analyzed separately. Significant differences were found in the ACB and OTU between the adult lines in both DA and 5-HT content, with P and HAD rats having lower levels than NP and LAD rats, respectively. Significant differences in DA content between the weanling lines were also found in the OTU, with P and HAD rats having lower DA levels than NP and LAD rats, respectively. These results confirm previous findings of an association between innate low DA content in select limbic regions and high alcohol drinking behavior.