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he Opioid Peptides Enkephalin and �-Endorphin inlcohol Dependence

ldiko Racz, Britta Schürmann, Anna Karpushova, Martin Reuter, Sven Cichon, Christian Montag,obert Fürst, Christian Schütz, Petra E. Franke, Jana Strohmaier, Thomas F. Wienker, Lars Terenius,rban Ösby, Agneta Gunnar, Wolfgang Maier, Andras Bilkei-Gorzó, Markus Nöthen, andndreas Zimmer

ackground: Experimental evidence indicates that the endogenous opioid system influences stress responses as well as reinforces effectsf addictive drugs. Because stress is an important factor contributing to drug dependence and relapse, we have now studied ethanolreference in enkephalin– and �-endorphin– deficient mice under baseline conditions and after stress exposure.

ethods: In the present study we used a two-bottle choice paradigm to study ethanol consumption and stress-induced ethanol prefer-nce. To examine alcohol withdrawal symptoms the forced drinking procedure was employed. We performed an association analysis in twoase-control samples of alcohol addicts to determine whether these opioid peptides also contribute to ethanol dependence in humans.

esults: Ethanol consumption was significantly reduced in the absence of �-endorphins, particularly in female knockout animals. Stressxposure results in an increased ethanol consumption in wild-type mice but did not influence ethanol-drinking in �-endorphin knockouts.nkephalin-deficient mice showed no difference from wild-type mice in baseline ethanol preference but also showed no stress-inducedlevation of ethanol consumption. Interestingly, we found a two-marker haplotype in the POMC gene that was associated with alcoholependence in females in both cohorts.

onclusions: Together these results indicate a contribution of �-endorphin to ethanol consumption and dependence.

ey Words: Addiction, alcohol, �-endorphin, case-control studies,nkephalin

enetic risk factors contribute to addiction disorders,including alcoholism (1,2). A recent survey has shownthat nearly 100 genes have been studied for contribution

o ethanol responses in genetically modified mice (3). Theseeports support pharmacological studies suggesting the involve-ent of various neurotransmitter systems as modulators of

thanol effects (glutamate, �-aminobutyric acid [GABA], dopa-ine, serotonin, cannabinoids and opioids) (4).The endogenous opioid system consists of �-, �-, and �-opi-

id receptors (MOP, DOP, and KOP) and the opioid peptidesnkephalin, �-endorphin, and dynorphin. These peptides areroduced from specific precursors (enkephalin/preproenkepha-

in [PENK], �-endorphin/proopiomelanocortin [POMC], dynor-hin/preprodynorphin [PDYN]) (5). Enkephalins and �-endor-hin are the endogenous ligands at MOP and DOP receptors.he �-endorphin binds MOP and DOP receptors with approxi-ately equal affinity, whereas enkephalin has a more than

enfold higher affinity for the DOP receptor. Dynorphins selec-ively activate KOP receptors and additionally several non-opioideceptors (6).

rom the Institute of Molecular Psychiatry (IR, BS, AB-G, AZ), Department ofMedical Genetics (AK, SC, MN), Department of Psychology (MR, CM),Institute of Medical Biometry, Informatics and Epidemiology (RF, TFW),Department of Psychiatry (CS, PEF, WM), University of Bonn, Germany;Central Institute of Mental Health (JS), University of Heidelberg, Mann-heim, Department of Psychiatry (PEF), University of Düsseldorf, Düssel-dorf, Germany and the Department of Clinical Neuroscience (LT, UO,AG), Karolinska Institute, Stockholm, Sweden.

ddress reprint requests to Ildiko Racz, Ph.D., Institute of Molecular Psychi-atry, Life & Brain Center, University of Bonn, Sigmund-Freud-Str. 25,53105 Bonn, Germany; E-mail: iracz@uni-bonn.de; neuro@uni-bonn.de..

eceived November 16, 2007; revised May 14, 2008; accepted May 14, 2008.

006-3223/08/$34.00oi:10.1016/j.biopsych.2008.05.008

Agonists for MOP and DOP receptors are rewarding forhumans and animals. They stimulate the activity of the mesolim-bic reward system and dopamine release in the nucleus accum-bens. In contrast, KOP agonists can produce dysphoria and placeaversion and decrease dopamine levels through activation ofpresynaptic KOP receptors in the nucleus accumbens (7). Be-cause ethanol exposure increases �-endorphin and enkephalinlevels, the endogenous opioid system might mediate some of therewarding effects of alcohol (8). In fact, pharmacological block-ade of opioid receptors with unselective antagonists naloxoneand naltrexone reduces ethanol consumption, similar to moreselective DOP and MOP antagonists (9–11). Knockout (KO)mouse studies confirmed the role of MOP and DOP receptors inethanol reward. Thus, MOP KO mice show less ethanol-inducedplace preference and little or no ethanol self-administration, witha stronger effect in females compared with males. In contrastDOP KOs drank more alcohol than wild-type (WT) mice (12–14).Surprisingly, these results were not yet supported by the analysisof mice with a genetic deletion of enkephalin (ENK-KO) or�-endorphin (END-KO). Thus, ENK-KO mice showed a similaroperant ethanol self-administration as WT animals, whereasEND-KO mice displayed a somewhat increased ethanol self-administration or preference. Even double KO mice lacking bothenkephalin and �-endorphin learned to self-administer ethanolsimilarly to WT animals (15). Because the deletion of both ofthese peptides should result in a much-reduced MOP tone, it isdifficult to understand why the peptide and the receptor KOphenotypes do not match.

A number of studies in humans have evaluated the role ofpolymorphisms in opioid receptors in samples of alcoholicpatients from diverse ethnic origin. Many of those have focusedon a MOP coding variant, A118G. The results from these studiesremain, by and large, inconclusive, because positive and nega-tive results have been reported (16–29). Whereas two coding

variants of the DOP receptor, G80T and T921C, were analyzed in

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lcohol-dependent Taiwanese Hans (21) and heroin- and alco-ol-dependent Caucasians of German origin (30). Neither ofhese two studies found evidence for an association. One studyas analyzed a CA repeat at the 3= end of PENK but found nossociation with alcohol dependence in populations of Asian,frican-American, and Caucasian origin (31). A recent analysis ofingle nucleotide polymorphisms (SNPs) in POMC and PENK inultiplex alcohol-dependent Caucasian American families alsorovided no support for the association of PENK and POMColymorphisms with alcohol dependence. However, allelic vari-nts in the PENK and POMC gene were associated with thearrower phenotype of opioid dependence in these families29).

In the present study we first evaluate END-KO and ENK-KOice in paradigms for ethanol preference, somatic dependence,

nd stress-induced alcohol drinking, followed by genetic analy-es of cohorts of alcoholic patients. Because previous studiesave demonstrated a gender-specific effect of Oprm1 deletion onthanol preference, we analyzed males and females separately.ur results provide evidence for a gender-specific role of-endorphin in ethanol preference and dependence.

ethods and Materials

nimalsThe generation of enkephalin-deficient and �-endorphin–

eficient mice has been described previously (32,33). The tar-eted mutation of the preproenkephalin Penk1 locus completelyisrupted the gene, thus resulting in a null allele. In contrast,-endorphin–deficient mice were generated by introducing atop codon just upstream of the �-endorphin peptide sequence,onsequently leaving all other POMC peptides intact (34). Allnimals were crossed for more than 10 generations to C57BL/6Jice and were therefore congenic for this genetic background.ere we refer to preproenkephalin KO mice as ENK-KO and to-endorphin KO animals as END-KO.

Animals were housed individually under reversed light-darkonditions (lights on: 7:00 PM; lights off: 9:00 AM) with free accesso food and drinking solutions. Experiments were conductedith 8–10-week-old mice.

cute Alcohol Effect and ToleranceTo determine ethanol-induced hypothermia, we first mea-

ured rectal body temperature of ethanol-naïve mice—with atainless steel rectal probe designed for mice and connected to aortable thermocouple thermometer (BAT-12, Harvard Instru-ents, Millis, Massachusetts). This was immediately followed by

n intraperitoneal injection of ethanol (saline, 1 g/kg, 2 g/kg, andg/kg). Body temperature was determined again 30 min after the

njection.Ethanol-induced hypothermia was expressed as the differ-

nce in body temperature before and after the injection. Toetermine the development of alcohol tolerance to the hypother-ic effects of ethanol, we performed the same experiment after

he animals ran through a forced drinking period of 22 days. Inhis period animals obtained an ethanol solution as their onlyluid source. The ethanol concentration was gradually increaseds follows: day 1–3, 4% ethanol; day 4–6, 8% ethanol; and fromay 7 onward, 16% ethanol. Tolerance was assessed as aecrease in the hypothermic effect of acute alcohol treatment

fter the forced drinking procedure.

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Alcohol Preference and Stress-Induced Ethanol DrinkingEthanol preference was assessed as previously described

(35,36). Briefly, two drinking bottles with 8% v/v alcohol (EtOH)or drinking water were available to the animals ad libitum. Thepositions of the bottles were changed daily. The ratio of alcoholto total fluid consumption, the amount of consumed ethanol(g/kg), the body weight (g), and food consumption (g) weredetermined twice/week. These data were used to calculate theaverage daily consumption.

Animals that have been studied in the two bottle choiceparadigm for 2 months were then exposed to a mild foot shock(Supplement 1). Animals were then returned to their home cageswith ethanol and water bottles. To assess the effect of stress onethanol preference alcohol and water consumption were deter-mined 24 hours and 96 hours after the shock and calculated asthe average daily consumption.

Physical Signs of WithdrawalTo determine physical signs of dependence animals received

16% ethanol solution in the forced drinking procedure as previ-ously described. After 4 weeks it was replaced with water.Scoring of handling-induced convulsion (HIC-score) was per-formed at first in ethanol-naïve animals, at the end of thedrinking phase to obtain a baseline, as well as 3 hours afterwithdrawal as previously described (37). The animals were liftedby the tail, held for 3 sec, and rotated gently, and their behaviorwas scored on a scale between zero and three as follows: 0 � notremor or convulsion, 1 � mild tremor on lifting and turning, 2 �continuous severe tremor on lifting and turning, 3 � clonicforelimb extensor spasm on lifting. All scorings were performedin a blinded manner by the same investigator.

Human SubjectsIn our exploratory study the group comprised 247 patients

with a definite current DSM-IV diagnosis of alcohol dependence(180 men, mean age � 42.0 � 9.0; 67 women, mean age � 44.3 �8.5) and 247 population-based control subjects (180 men, meanage � 41.7 � 8.9; 67 women, mean age � 44.2 � 8.4). Thesample size of the female subgroup is different for markerrs934778 and rs3769671 (patients: 83 women, mean age � 44.4 �8.6; control subjects: 140 women, mean age � 45.8 � 9.6).Substance abuse and dependence (including alcohol and illicitdrugs) as well as psychiatric disorders were assessed with thewhole Structured Clinical Interview for DSM-IV Axis I Disorders(SCID). An experienced psychiatrist made the DSM-IV diagnosisof alcoholism on the basis of the SCID-interview and all availableclinical information and records. Subjects that additionally metDSM-IV criteria for dependence on an illicit drug or had recently(1 year) any major psychiatric disorder were excluded. A historyof psychiatric disorders, such as a history of a depressive episodedid not lead to exclusion. Patients were recruited at the Univer-sity of Bonn, Department of Psychiatry, or collaborating psychi-atric hospitals in Düsseldorf, Mainz, and Essen. The population-based control sample of Caucasians of German origin wascollected with the support of the local Census Bureau of the cityBonn. The sample was established within the framework of theGerman National Genome Research Network I (NGFN I; http://www.ngfn.de) of the Federal Ministry of Education and Researchbetween the years 2000 and 2003 to serve as an epidemiologicalcontrol sample for complex genetic studies within the NGFN. Tofurther verify our findings, a group of healthy control womenconsisting of German blood donors (120 individuals, mean age �

29.5 � 10.1) and a group of female alcoholic subjects (n � 72;

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ean age � 47.3 � 10.6) were genotyped for marker rs934778nd rs3769671. The female patients were recruited in the Clinicschenburg, a hospital specializing in treatment of alcohol abusend alcoholism. They met ICD-10 criteria for alcohol depen-ence (F10.25) of the World Health Organization (WHO) andame to the rehabilitation clinic after alcohol detoxification andarticipated in the study directly after admission to the Clinicschenburg. The individuals in this group had no clinical diag-osis of dependence on an illicit drug. All patients and controlubjects were Caucasians of German origin.

In our replication study, affected individuals (240 men, meange � 47.25 � 10.65; 114 women, mean age � 46.09 � 10.39)nd control subjects (202 men, 113 women) of Scandinavianncestry served as a replication cohort. The alcoholic patientsere collected within the Swedish Alcoholism in Siblings Study.lcoholism was defined according to DSM-IV criteria after atructural psychiatric interview with the Schedules for Clinicalssessment in Neuropsychiatry (38). The comorbidity with anyther psychiatric disorder was � 1%. A group of healthy controlndividuals consisted of Swedish blood donors. There was nonformation available concerning the age of the control subjects.

NP Selection, Genotyping, and Statistical AnalysisAccording to HapMap project data for Caucasians, the human

ENK gene is located in a single 92-kb haplotype block. Theredicted tagging SNPs for this block were analyzed. In contrastarkers of the human POMC gene locus show weak linkageisequilibrium (LD). We therefore randomly selected SNPs withn average spacing of 3 kb. The DNA was isolated from wholelood or permanent cell lines derived from Epstein–Barr virus-ransformed lymphocytes. Matrix-assisted laser desorption/ion-zation time-of-flight (MALDI-TOF) mass spectrometry-basedNP genotyping was performed on a MassARRAY platform withPLEX chemistry (Sequenom, San Diego, California; http://www.equenom.com/). The genotyping success rates were 98% origher. Additional probes were genotyped with the ABI Assays-n-Demand SNP Genotyping products (Applied Biosystems,oster City, California). Statistical analysis was performed withhe program FAMHAP version 17 (39–41). The option “singlecc”as used to calculate unadjusted p values for association of

ingle markers. For haplotype analysis FAMHAP estimates hap-otype frequencies with the expectation-maximization (EM)-lgorithm (39). With the option “hapcc”, p values were computedy a permutational 2-based test.

The statistical analysis of the animal experiments is described

able 1. Development of Tolerance to Ethanol After Chronic Alcohol Expo

WT

Naïve Chronic Naïv

aline .13 � .24 .55 � .38 .54 �g/kg .25 � .24 .25 � .23 .35 �g/kg 2.05 � .24a .68 � .35b 1.59 �g/kg 4.44 � .25a 4.57 � .45a 3.75 �

Intraperitoneal ethanol (EtOH) treatment caused dose-dependent hypog/kg. Wild-type (WT) and enkephalin knockout (ENK-KO) animals develope

ignificant hypothermia (n � 5). END-KO, �-endorphin knockout.aSignificant difference compared with saline-treated group.bSignificant difference compared with ethanol-naïve group.

n Methods and Materials in Supplement 1.

Results

Acute Alcohol Effect and ToleranceAs shown in Table 1, alcohol treatment of drug-naïve WT,

ENK-KO, and END-KO mice caused dose-dependent hypother-mia, with significant effects at a dose of 2 g/kg and 4 g/kgethanol [F (3,168) � 272.09, p � .001]. The WT and ENK-KOanimals developed tolerance to alcohol after 1-month of ethanol-drinking, because only 4 g/kg but not 2 g/kg induced significanthypothermia [F (1,168) � 5.107, p � .025]. In contrast, END-KOmice developed no tolerance during the forced drinking regi-men. We did not find gender-specific differences.

Alcohol Preference and Stress-Induced Ethanol DrinkingTo examine ethanol consumption, the animals had a free

choice between an alcohol solution and water. The preferenceand the amount of ethanol consumed were correlated (r �.85–.9) in WT and ENK-KO animals. In END-KO mice there wereno correlations, but preference and ethanol consumptionpointed to the same direction (data not shown).

Because, female mice generally showed a higher preferencefor EtOH than males [F (1,216) p � .0001], we examined the twogenders separately (Figure 1). The END-KO female animalsconsumed significantly less EtOH and showed significantly lowerpreference for alcohol compared with WT and ENK-KO females[F (2,822) p � .0001], whereas in the two latter groups we foundno difference [F (1,588) p � .77] (Figure 1). The EtOH consump-tion and preference remained stable during the 7-week testingperiod.

Ethanol consumption was also the lowest in male END-KOmice compared with WT and ENK-KO males [F (2,894) p �.0001], although END-KO and WT animals showed the samepreference for alcohol. Thus, the combined liquid consumptionwas higher in WT compared with END-KO mice. We found nosignificant difference between WT and ENK-KO animals in EtOHconsumption [F (1,678) p � .14] (Figure 1), but in the first 3 weeksthe ENK-KO mice showed significantly higher preference forEtOH. All strains showed a small but significant decrease of EtOHconsumption during the examined period [F (6,894) p � .0001],but this effect was more pronounced in the ENK-KO animals.

To assess the effect of stress on ethanol preference, weexposed the animals to a foot shock. We routinely use only malemice for these experiments, because female C57BL/6J alreadyhave a high EtOH preference that is not further elevated after afoot shock. Stress caused a significant elevation in EtOH con-sumption of WT mice 24 hours after the exposition [F (3,211) p �.03]. This effect was transient and normalized after 96 hours

ENK-KO END-KO

Chronic Naïve Chronic

.42 � .35 .11 � .27 .32 � .29

.02 � .35 .83 � .25 .87 � .31.84 � .35b 3.50 � .26a 3.00 � .22a

3.02 � .35a 4.50 � .23a 5.01 � .28a

ia in alcohol-naïve animals, which was significant at a dose of 2 g/kg anderance during chronic EtOH treatment; only 4 g/kg alcohol injection caused

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ess EtOH 24 hours and 96 hours after foot shock [F (3,115) p �025]. Thus the stressor produced not an increase but a paradox-cal decrease in EtOH consumption (Figure 2A). We found noffect of stress on alcohol preference in END-KO animalsF (3,116) p � .46] (Figure 2B).

igure 1. Ethanol (EtOH) consumption and preference of preproenkepha-in-deficient (ENK-KO) and �-endorphin– deficient (END-KO) and wild-typeWT) mice. In all strains females consumed more ethanol than males. Alcoholonsumption and preference of female END-KO animals was significantly

ower than in the other two strains. The male END-KO mice consumed theowest amount of EtOH compared with WT and ENK-KO males, althoughND-KO and WT animals showed the same preference for alcohol. Thus, theombined liquid consumption was higher in WT compared with END-KOice. The male ENK-KO mice showed significantly higher preference for

lcohol in the first 3 weeks of the two bottle-choice sessions (n � 20/gender/train).

igure 2. Stress-induced alcohol drinking. (A) The WT animals consumedignificantly more ethanol and showed significantly higher preference forlcohol 24 hours after exposure to a mild foot shock. This effect was tran-ient. Ninety-six hours later they consumed the same amount and showedhe same preference as before the shock procedure (n � 20). The ENK-KO

ice consumed significantly lower alcohol 24 and 96 hours after the shockn � 20). (B) The END-KO mice showed no changes in ethanol preferencend consumption after the mild foot shock (n � 20). *p � .05; **p � .01.

bbreviations as in Figure 1.

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Physical Signs of Ethanol DependencePhysical signs of dependence were analyzed by evaluation of

HIC-scores in ethanol-naïve animals, at the end of EtOH drinkingphase and 3 hours after withdrawal from alcohol. The HIC-scoresshowed no gender difference; thus we combined the results fromboth genders. In all examined strains there was a clear increasein HIC-scores after withdrawal (p � .001). In END-KO mice wefound a slight but not significant elevation of HIC-scores in theEtOH-drinking group (Table 2).

SNP GenotypingHaving established that deficiency of enkephalin and endor-

phin affects behaviors relevant to ethanol dependence in mice,we hypothesized that these genes also contribute to ethanoldependence in humans. We therefore performed a case-controlstudy in a German sample of alcohol-dependent patients andcontrol subjects, followed by a replication study in a Swedishcohort.

We genotyped all five predicted tagging SNPs covering ahaplotype block of 92 kb (HapMap) around the human PENKgene encompassing the two exons. We had to exclude markerrs10108778 due to assay problems. The SNP rs12545109 islocated 40 kb downstream from the 3’ end, and rs2670029 26 kbis upstream of the transcription initiation site (Table 3; Figure 3).The indicated haplotype block for markers rs2576581,rs12548084, and rs2670029 on the basis of the LD structure ofGerman control subjects (Figure 3) is part of the correspondinghaplotype block predicted by HapMap data for Caucasian pop-ulations. In contrast, markers at the POMC gene locus showedweak LD in Caucasians (HapMap data). We therefore selectednine SNPs with an even spacing of approximately 3 kb extendingfrom 11 kb upstream of exon 1 up to 6 kb downstream from exon3 in German control subjects (Figure 3). In accordance with datafrom HapMap, we also found weak LD between markers.

Because we observed gender-specific phenotype differencesin our KO mice, we evaluated the allele and genotype distribu-tions in the entire samples as well as in the female and male

Table 2. Physical Withdrawal Symptoms After 1 Month of ForcedDrinking After 3 Hours of Withdrawal

HIC-Score Frequencies

0 1 2 3

WTNaïve 50 50 0 0EtOH 42.5 56.3 1.25 0Withdrawal 1.25 17.5 45 36.25

ENK-KONaïve 50 50 0 0EtOH 75 22.5 2.5 0Withdrawal 25 37.5 35 0

END-KONaïve 78 22 0 0EtOH 28 60 12 0Withdrawal 8 44 44 4

The frequencies of higher handling-induced convulsion (HIC-score)were significantly increased 3 hours after withdrawal compared with theEtOH-naïve and EtOH-drinking animals. Naïve: HIC-scores of EtOH naïveanimals (n � 10/strain); EtOH: HIC-scores during the drinking phase (WT: n �80, ENK-KO: n � 40, END-KO: n � 26); Withdrawal: 3 hours after replacingethanol with water (WT: n � 80, ENK-KO: n � 40, END-KO: n � 26). Abbrevi-ations as in Table 1.

subgroups. In the German sample, all markers showed expected

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llele frequencies (Table 3) and revealed no significant devia-ions from Hardy-Weinberg-equilibrium (HWE) (p � .05).

The unadjusted p values for the PENK locus for allele frequen-ies of rs12545109 (p � .031) and rs2576581 in women (p � .007)Table 3) suggested association with the disease phenotype.oth SNPs also showed low p values for genotype distributionsetween the overall group of alcoholic patients and controlubjects (Table 4). After Bonferroni correction for multipleesting, only marker rs2576581 remained below the 5% signifi-ance threshold (allele distribution corrected p � .028; Armit-ge’s Trend Test corrected p � .036). However, we were not ableo confirm this in the Swedish replication sample (Tables 1 and 2n Supplement 1).

For rs2164808 and rs934778, located upstream of the POMC

able 3. SNPs Major Allele Frequency and Association with Alcohol Depen

ene Marker SNPa Genomic Locationb Sample

ENK rs12545109e C\A 57476460 Alle

C\A FemaleC\A Male

ENK rs2576581e G\A 57528504 AllG\A FemaleG\A Male

ENK rs12548084 C\T 57540810 AllC\T FemaleC\T Male

ENK rs2670029 C\A 57547550 AllC\A FemaleC\A Male

OMC rs2164808e G\A 25230680 AllG\A FemaleG\A Male

OMC rs7589318 G\A 25231876 AllG\A FemaleG\A Male

OMC rs1866146 T\C 25234077 AllT\C FemaleT\C Male

OMC rs6713532 T\C 25238337 AllT\C FemaleT\C Male

OMC rs934778ef T\C 25242728 AllT\C FemaleT\C Male

OMC rs3769671f A\C 25243657 AllA\C FemaleA\C Male

OMC rs6545976 G\T 25248154 AllG\T FemaleG\T Male

OMC rs6719226 C\G 25249516 AllC\G FemaleC\G Male

OMC rs874401 C\T 25255784 AllC\T FemaleC\T Male

SNPs, single nucleotide polymorphisms; OR, odds ratio; CI, confidence inaThe allele frequency is shown for the left allele.bGenomic location is based on National Center for Biotechnology InformcUncorrected p value of FAMHAP statistic for association between SNPsd95% confidence interval.eSignificant association with alcohol dependence.fNote that the sample size is different for these markers.

ene and in intron 1, respectively, we found differences for allele

and genotype distributions between German female alcoholicpatients and control subjects (Tables 3 and 4). In the Swedishreplication sample, we found evidence for an association ofmarker rs934778 in the entire sample and of marker rs874401 inwomen (Tables 1 and 2 in Supplement 1). However, afterBonferroni correction, only marker rs934778 showed signifi-cance in German women (allele distribution corrected p � .036;Armitage’s Trend Test corrected p � .027).

We next performed a haplotype analysis with sliding win-dows and found no PENK haplotypes with significant frequencydifferences between cases and control subjects (data not shown).Interestingly, for POMC we detected an association with the T-Ahaplotype of marker rs934778 and rs3769671 in the group ofalcohol-dependent women in both the German (p � .12) and the

e in the German Sample

Control Subjects Cases pc OR CId

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.769 .708 .260 .729 [.420–1.265]

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.515 .568 .094 1.242 [.963–1.602]

.418 .586 .007e 1.971 [1.206–3.222]

.552 .565 .729 1.054 [.782–1.422]

.946 .945 .924 .973 [.560–1.694]

.948 .954 .820 1.139 [.372–3.485]

.946 .941 .800 .921 [.486–1.745]

.569 .606 .239 1.165 [.903–1.503]

.478 .585 .082 1.539 [.946–2.504]

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.612 .462 .014e 1.840 [1.128–3.002]

.536 .506 .413 1.130 [.843–1.515]

.678 .651 .368 .885 [.679–1.154]

.701 .600 .084 .638 [.383–1.063]

.669 .668 .956 .992 [.727–1.353]

.670 .669 1.000 .997 [.764–1.301]

.664 .636 .635 .885 [.534–1.465]

.672 .683 .767 1.049 [.766–1.435]

.771 .798 .308 1.171 [.864–1.588]

.739 .754 .779 1.083 [.622–1.886]

.783 .816 .280 1.224 [.848–1.765]

.692 .732 .088 1.217 [.971–1.525]

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.967 .974 .404 1.298 [.701–2.403]

.974 .970 .725 3.969 [.487–2.812]

.957 .978 .124 1.959 [.820–4.681]

.967 .959 .489 .79 [.404–1.543]

.970 .961 .683 .757 [.199–2.884]

.966 .958 .566 .798 [.368–1.729]

.966 .959 .571 .827 [.428–1.598]

.970 .961 .683 .757 [.199–2.884]

.964 .958 .666 .847 [.397–1.806]

.812 .837 .303 1.189 [.855–1.652]

.791 .800 .856 1.057 [.581–1.922]

.819 .852 .240 1.268 [.853–1.885]

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iscussion

Endogenous opioid peptides modulate the effects of manyrugs of abuse. Here we have analyzed the role of enkephalinsnd �-endorphin in animal models of ethanol preference, etha-ol preference after stress exposure, ethanol withdrawal symp-oms, and in a human case-control study with alcoholic patients.ur results indicate that �-endorphin modulates ethanol prefer-nce and stress-induced relapse in animals. In humans, polymor-hisms in the �-endorphin encoding POMC gene are associatedith alcoholism. Interestingly, we found gender-specific differ-nces in animal and in human studies.

Our findings are entirely consistent with the previous dem-nstration that voluntary ethanol intake was decreased in MOPO animals (i.e., in animals lacking the receptor with the highestffinity for �-endorphin) (12,13,42). They also agree with thehenotype of DOP KOs, which showed increased ethanolonsumption. Together these data suggest that endorphin/MOPnd enkephalin/DOP signaling modulates ethanol preferencend consumption in mice. This is indeed one of the fewxamples showing a perfect match of receptor/ligand KO phe-otypes, thus lending further confidence to the relevance ofhese findings. Although enkephalins can also bind and activateOP, it is unlikely that they contribute much to the MOP-ediated modulation of ethanol reward, because we and others

ound no difference between WT and ENK-KO mice in awo-bottle-choice and a conditioned place-preference paradigm43). Together these findings suggest that �-endorphin acting onOP plays a critical role in ethanol reinforcement, whereas bothpioid peptides seem to be involved in stress-induced relapse.

Our results do not agree with previous studies reporting onperant intravenous and oral self-administration of ethanol as

igure 3. Location and linkage disequilibrium (LD) of genotyped single nuclenes and the location of the analyzed SNPs are indicated. The coding regiontrand. The LD on the basis of the German control group reveals one Haploty5: 4 kb). The LD-plots were generated with Haploview (http://www.broadere indicated.

ell as ethanol consumption in a two-bottle–choice paradigm in

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�-endorphin–deficient mice. We show here that mice lacking�-endorphin consumed less of an 8% ethanol solution than WTcontrol subjects. In contrast Grisel et al. (44) found a higherethanol preference in �-endorphin KO mice compared with WTanimals with an ethanol concentration of 7% but not with 10%. Astudy by Grahame et al. (45) supported these findings, becausethey found no significant genotype effect in a free accessparadigm with an ethanol concentration of 10% but increasedethanol consumption in END-deficient mice in a scheduledaccess paradigm where EtOH was only available for 2 hours/day.They suggested that consumption of 10% alcohol during limitedaccess is opioid-dependent, because naltrexone treatment re-duced drinking in WT and in KO mice (46). In a paradigm forintravenous operant self-administration, END-deficient micereadily acquired self-administration behaviors, whereas WT micedid not. No genotype effects were observed when ethanol wasdelivered orally in the operant paradigm (45). The reason forthese disparate results is not clear. Because the genetic back-ground of the animals was very similar in all studies (C57BL/6),procedural or environmental effects most likely account for thesedifferences. One obvious environmental factor is the ambientstress level to which the animals are exposed. Environmentalstressors include odorants, noise, vibrations and other factorsthat differ between animal facilities. Indeed, we found a signifi-cant genotype effect after stress exposure, which increased EtOHconsumption in WT but not in END-KO mice. Endogenousopioids and their receptors are expressed in neuronal stress-response circuits including the paraventricular nucleus (PVN),the nucleus of the solitary tract, and raphe nuclei (47–50), whichare also involved in the modulation of drug reward (51). It is wellknown that stress increases ethanol consumption in mice (36)

e polymorphisms (SNPs). The gene structure for the human PENK and POMCplotted in orange. The transcription template for both genes is the negativeck for PENK (marker 2 3 4: 19 kb) and two for POMC (marker 2 3: 2 kb; marker

edu/mpg/haploview/) with the Gold-heatmap color scheme, and r2 values

eotids are

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and contributes to ethanol-seeking behavior in humans (52).

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hus endogenous opioids might modulate ethanol consumptionn stress conditions.

Because several previous studies have shown that femaleice have a higher ethanol preference than males, we analyzedoth genders independently. We found that the difference inthanol consumption between WT and END-KO mice was mucharger in females than in males. Thus, the lack of �-endorphinffected female mice more strongly than males.

Important gender differences have also been observed forubstance abuse disorders, including alcoholism, in humans (53).hese differences relate to the age of onset, motivation for drugse, comorbidities, and numerous other factors. For example, ineavy drinkers adrenocorticotropic hormone and pituitary �-en-orphin were significantly lower compared with control subjects,

able 4. SNPs Genotype Distribution and Association with Alcohol Depend

ene MarkerGenomicLocationa Sample Control Subje

ENK rs12545109d 57476460 Alld CC � 148 AC � 81Female CC � 39 AC � 25Male CC � 109 AC � 56

ENK rs2576581d 57528504 All GG � 63 AG � 120Femaled GG � 11 AG � 34Male GG � 52 AG � 86

ENK rs12548084 57540810 All CC � 218 CT � 22Female CC � 60 CT � 7Male CC � 158 CT � 15

ENK rs2670029 57547550 All CC � 78 AC � 124Female CC � 13 AC � 38Male CC � 65 AC � 86

OMC rs2164808d 25230680 All GG � 75 AG � 125Femaled GG � 24 AG � 34Male GG � 51 AG � 91

OMC rs7589318 25231876 All GG � 117 AG � 101Female GG � 33 AG � 28Male GG � 84 AG � 73

OMC rs1866146 25234077 All TT � 117 CT � 97Female TT � 28 CT � 33Male TT � 89 CT � 64

OMC rs6713532 25238337 All TT � 151 CT � 79Female TT � 37 CT � 25Male TT � 114 CT � 54

OMC rs934778de 25242728 All TT � 195 CT � 191Femaled TT � 107 CT � 120Male TT � 88 CT � 71

OMC rs3769671e 25243657 All AA � 382 AC � 25Female AA � 221 AC � 12Male AA � 161 AC � 13

OMC rs6545976 25248154 All GG � 228 GT � 16Female GG � 63 GT � 4Male GG � 165 GT � 12

OMC rs6719226 25249516 All CC � 231 CG � 15Female CC � 63 CG � 4Male CC � 168 CG � 11

OMC rs874401 25255784 All CC � 165 CT � 71Female CC � 41 CT � 24Male CC � 124 CT � 47

Abbreviations as in Table 3.aGenomic location is based on NCBI human genome assembly B36.bUncorrected p value of FAMHAP statistic for association between SNPscp value of the Armitage‘s trend test.dSignificant association with alcohol dependence.eNote that the sample size is different for these markers.

hereas plasma cortisol levels were higher. These differences in

hormone levels were more pronounced in female than in malesubjects. It was therefore interesting to find that the POMCtwo-marker haplotype (rs934778, rs3769671) was associated withalcoholism in women, but not in men. Because the femalesubgroup was relatively small, the effect size (odds ratio) of thepolymorphism might have been overestimated. Nevertheless, wefound the same association in the Swedish replication sample. Incontrast, the association of markers rs2576581 and rs12545109(PENK) with the alcohol dependence phenotype in Germanwomen was not replicated in the Swedish cohort. Together ourfindings in humans and animals strongly support a contributionof �-endorphin to ethanol dependence in female subjects.

Because previous studies in MOP receptor KOs have alsoshown a stronger effect in female subjects (12), our finding

in the German Sample

Casesp

(Global)bp

(Armitage)c

A � 15 CC � 126 AC � 97 AA � 22 .104 .035d

A � 3 CC � 33 AC � 26 AA � 6 .475 .260A � 12 CC � 92 AC � 71 AA � 16 .152 .068A � 56 GG � 85 AG � 104 AA � 52 .103 .104A � 22 GG � 24 AG � 27 AA � 13 .019d .009d

A � 34 GG � 61 AG � 77 AA � 38 .499 .736T � 2 CC � 218 CT � 27 TT � 0 .288 .924T � 0 CC � 59 CT � 6 TT � 0 .815 .815T � 2 CC � 158 CT � 21 TT � 0 .228 .805A � 44 CC � 93 AC � 111 AA � 41 .343 .242A � 16 CC � 22 AC � 32 AA � 11 .155 .072A � 28 CC � 71 AC � 78 AA � 30 .696 .764A � 47 GG � 65 AG � 113 AA � 67 .090 .060A � 9 GG � 15 AG � 30 AA � 20 .039d .015d

A � 38 GG � 49 AG � 83 AA � 47 .507 .420A � 29 GG � 110 AG � 99 AA � 36 .612 .388A � 6 GG � 26 AG � 26 AA � 13 .178 .099A � 23 GG � 83 AG � 73 AA � 23 .999 .956C � 33 TT � 113 CT � 102 CC � 30 .848 1.000C � 6 TT � 26 CT � 32 CC � 8 .832 .621C � 27 TT � 87 CT � 69 CC � 22 .701 .783C � 17 TT � 158 CT � 75 CC � 12 .572 .326C � 5 TT � 36 CT � 26 CC � 3 .778 .777C � 12 TT � 122 CT � 48 CC � 9 .592 .306C � 34 TT � 178 CT � 130 CC � 24 .146 .082C � 14 TT � 97 CT � 45 CC � 10 .0004d .003d

C � 20 TT � 80 CT � 84 CC � 14 .283 .896C � 1 AA � 314 AC � 17 CC � 0 .561 .409C � 0 AA � 141 AC � 9 CC � 0 .722 .722C � 1 AA � 171 AC � 8 CC � 0 .294 .134T � 0 GG � 223 GT � 20 TT � 0 .480 .480T � 0 GG � 59 GT � 5 TT � 0 .677 .677T � 0 GG � 163 GT � 15 TT � 0 .558 .558G � 1 CC � 222 CG � 20 GG � 0 .398 .575G � 0 CC � 59 CG � 5 GG � 0 .677 .677G � 1 CC � 162 CG � 15 GG � 0 .428 .671T � 11 CC � 173 CT � 64 TT � 8 .601 .316T � 2 CC � 43 CT � 18 TT � 4 .463 .858T � 9 CC � 130 CT � 45 TT � 4 .349 .255

lcohol dependence.

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ystem in alcohol dependence. Although alcohol consumption isigher in men compared with woman, this is probably culturallyiased. Nevertheless, our results further contribute to the ideahat the genetic regulation of drug reward and dependenceiffers between genders.

A recent study using a diverse cohort of European Americansrom alcohol-dependent families found no association betweenENK or POMC polymorphisms and alcoholism (29). For PENK,he study used different sets of markers. For POMC, Xuei et al.lso analyzed rs934778 (SNP of the haplotype linked withlcoholism in our sample) but did not find a significant associa-ion of this SNP with alcohol dependence. Nevertheless, thisarker showed a significant association with a more severe

ubtype of alcoholism, which is characterized by comorbidityith opioid dependence (29). Unfortunately, Xuei et al. did notifferentiate between female and male gender in their sample,hich encompassed predominantly male subjects.Taken together these data suggest a contribution of endoge-

ous opioid system to behaviors associated with alcoholism. The-endorphin seems to be important for ethanol preference, andnkephalin and �-endorphin modulate the effects of stress onthanol consumption. A significant role of POMC in femaleubjects was confirmed in our human association studies. Thus,he correlation of distinct alcohol-related behaviors with thendogenous opioid peptides deserves further studies in humans.

Ildiko Racz and Britta Schürmann contributed equally to thisork.

This work was supported by grants from the Federal Ministryf Education and Research (NGFN2, to AB-G, WM, and AZ), theational Institute of Drug Abuse, United States (RO1, DA016768

o AZ), the Suchtforschungsverbund NRW (BMBF, to WM), theuropean Commission (Framework VI, PL005166 to AZ), thetockholm County Council, the Swedish Alcohol Retail Monopo-y’s Council for Alcoholic Research, and the Bristol-Myers Squibbto UÖ).

Dr Ösby reports having received fees as a member of Speakers’ureau and consulting fees from AstraZeneca, Bristol-Myersquibb, Eli Lilly, and Pfizer.

The other authors report no biomedical financial interests orotential conflicts of interest.

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