Lack of association between tumour necrosis factor receptor types 1 and 2 gene polymorphism and severe acute alcoholic hepatitis.

BACKGROUND/AIMS: Tumour necrosis factor-alpha (TNF-alpha) is involved in the physiopathology of severe acute alcoholic hepatitis (AAH) binding with TNF receptor types TNFR1 and TNFR2, whose serum concentrations are elevated. We studied the role of TNFR1 and TNFR2 gene polymorphism in AAH patients. METHODS: One hundred and ninety-two patients (58 AAH with Maddrey score >or=32, 44 non-AAH cirrhoses, 90 healthy individuals) were genotyped for A36G TNFR1 and T676G TNFR2 using polymerase chain reaction-restriction fragment length polymorphism technique. Serum sTNFR1 and sTNFR2 were assayed. RESULTS: The AAH and two control groups did not differ for genotype distribution. In three groups, A (36 TNFR1) and T (676 TNFR2) allelic frequencies were similar, at 0.47, 0.47, 0.44 and 0.78, 0.81, 0.80, respectively. The 36 TNFR1, 676 TFNR2 genotypes did not influence on prognostic scores (Maddrey, Child-Pugh), nor in response to corticosteroids or 6-month survival. sTNFR1 levels were higher in AAH than healthy group (3.07+/-1.14 vs. 1.17+/-0.27 ng/ml, P<0.001) and sTNFR2 levels were higher in AAH than cirrhosis (3.6+/-1.02 vs. 3.1+/-1.03, P<0.05) and healthy groups (3.6+/-1.02 vs. 1.91+/-0.54, P<0.001). However, sTNFR1 and sTNFR2 levels did not vary significantly according to genotypes. CONCLUSION: These results did not support an association between 36 TNFR1, 676 TNFR2 gene polymorphisms and AAH.

Predicting the effect of naltrexone and acamprosate in alcohol-dependent patients using genetic indicators.

Acamprosate and naltrexone are effective medications in the treatment of alcoholism. However, effect sizes are modest. Pharmacogenomics may improve patient-treatment-matching and effect sizes. It is hypothesized that naltrexone exerts its effect through genetic characteristics associated with the dopaminergic/opioidergic positive reinforcement system, whereas acamprosate works through the glutamatergic/GABAergic negative reinforcement system. Alcohol-dependent subjects were randomly assigned to either acamprosate or naltrexone. Subjects participated in a cue-exposure experiment at the day before and at the last day of medication. Reductions in cue-induced craving and physiological cue reactivity were measured. Differential effects of naltrexone and acamprosate on these outcomes were tested for different polymorphisms of the opioid, dopamine, glutamate and GABA-receptors. Significant matching effects were found for polymorphisms at the DRD2, GABRA6 and GABRB2 gene. In addition, a trend was found for the OPRM1 polymorphism. This provides evidence for the matching potential of genotypes. It is expected that more effective treatments can be offered when genetic information is used in patient-treatment-matching.

Ethanol-sensitive brain regions in rat and mouse: a cartographic review, using immediate early gene expression.

BACKGROUND: Ethanol addiction has been conceptualized as a progression from occasional, impulsive use to compulsive behavior. Ethanol-dependence is a chronic pathology with repeated cycles of withdrawal, craving, and relapse. Specific molecular and cellular mechanisms underlie these transition stages. METHODS: This review aimed at elucidating whether there are also adaptations in the pattern of brain regions responding to ethanol. This paper reviews the evidence in rodents for activation of specific brain regions, assessed by induction of IEG expression, following acute and chronic ethanol exposure. RESULTS: The review sheds light on the specific patterns of response in regions of the brain to different types of ethanol exposure and shows that activation of specific brain regions may occur in particular phases of the development of ethanol addiction. Some brain regions respond consistently following acute or chronic treatments or withdrawal: the prefrontal cortex; nucleus accumbens; lateral septum; hippocampus; perioculomotor urocortin-containing cells population (pIIIu), also known as Edinger-Westphal nucleus; central nucleus of the amygdala; and the paraventricular nucleus of hypothalamus. The two last brain areas are particularly activated by relapse-inducing stressors. It is of interest that the amygdala, hippocampus, and prefrontal cortex, which belong to the reward system, are activated by cue-induced relapse to ethanol self-administration in rodents and humans, while activation of these regions is reversed with anti-craving compounds. Following chronic exposure, IEG induction desensitizes while withdrawal reactivates these regions. DISCUSSION: Some responding regions are implicated in reward related processes (VTA, extended amygdala, hypothalamus, hippocampus, prelimbic cortex, ventral part of lateral septum) and some others in aversive-related processes (area postrema, nucleus of solitary tract). CONCLUSION: A better understanding of the neural circuits affected by ethanol and their adaptations during the development of ethanol addiction will provide new opportunities for developing appropriate therapies.

Perinatal alcohol exposure in rat induces long-term depression of respiration after episodic hypoxia.

RATIONALE: Little is known about the effects of alcohol exposure during pregnancy, which is responsible for fetal alcohol syndrome and the respiratory network functions, especially respiratory network plasticity (e.g., long-term facilitation) elicited after repeated short-lasting hypoxic episodes. The mechanism of induction of respiratory long-term facilitation involves 5-HT(2A/2C) receptors, which also participate in the response to hypoxia. Because fetal alcohol exposure is known to reduce serotonin centrally, and synaptic plasticity in the hippocampus, we hypothesized that alcohol exposure during gestation might impair respiratory long-term facilitation after hypoxic episodes. OBJECTIVES: To analyze the effects of prenatal and postnatal alcohol exposure on respiratory long-term facilitation in 5- to 7-day-old rats. METHODS: Respiratory frequency and amplitude were measured in vivo and in an in vitro rhythmic medullary slice before and after three hypoxia episodes or three applications of a 5-HT(2A/2C) receptor agonist in vitro. 5-HT(2A/2C) receptor mRNA was measured from the slice. MEASUREMENTS AND MAIN RESULTS: Alcohol exposure impaired respiratory long-term facilitation and induced long-term depression of respiration in both in vivo and in vitro models. Alcohol altered 5-HT(2A/2C) mRNA expression, although 5-HT(2A/2C) agonist efficacy was not altered in increasing rhythmic activity in slices. However, a higher concentration of 5-HT(2A/2C) agonist was necessary to induce transient facilitation in slices from ethanol-exposed animals, suggesting disturbances in induction and maintenance mechanisms of respiratory long-term facilitation. CONCLUSIONS: Respiratory facilitation after repeated hypoxia was converted to long-term depression in rats treated with alcohol in utero. Alcohol exposure during pregnancy may therefore induce long-term maladaptive behavior of the respiratory system in neonates.

Long-term alterations in vulnerability to addiction to drugs of abuse and in brain gene expression after early life ethanol exposure.

Exposure to ethanol early in life can have long-lasting implications on brain function and drug of abuse response later in life. The present study investigated in rats, the long-term consequences of pre- and postnatal (early life) ethanol exposure on drug consumption/reward and the molecular targets potentially associated with these behavioral alterations. Since a relationship has been demonstrated between heightened drugs intake and susceptibility to drugs-induced locomotor activity/sensitization, anxiolysis, we tested these behavioral responses, depending on the drug, in control and early life ethanol-exposed animals. Our results show that progeny exposed to early life ethanol displayed increased consumption of ethanol solutions and increased sensitivity to cocaine rewarding effects assessed in the conditioned place preference test. Offspring exposed to ethanol were more sensitive to the anxiolytic effect of ethanol and the increased sensitivity could, at least in part, explain the alteration in the consumption of ethanol for its anxiolytic effects. In addition, the sensitivity to hypothermic effects of ethanol and ethanol metabolism were not altered by early life ethanol exposure. The sensitization to cocaine (20 mg/kg) and to amphetamine (1.2 mg/kg) was increased after early life ethanol exposure and, could partly explain, an increase in the rewarding properties of psychostimulants. Gene expression analysis revealed that expression of a large number of genes was altered in brain regions involved in the reinforcing effects of drugs of abuse. Dopaminergic receptors and transporter binding sites were also down-regulated in the striatum of ethanol-exposed offspring. Such long-term neurochemical alterations in transmitter systems and in the behavioral responses to ethanol and other drugs of abuse may confer an increased liability for addiction in exposed offspring.

The -308 TNFalpha gene polymorphism in severe acute alcoholic hepatitis: identification of a new susceptibility marker.

BACKGROUND: This study investigated whether genetic polymorphisms in the tumor necrosis factor alpha (TNFalpha) gene's promoter region play a role in severe acute alcoholic hepatitis (AAH). METHODS: One-hundred and fifty patients (58 AAH patients, 45 cirrhosis group free-AAH, 47 healthy group) were genotyped for 3 TNFalpha polymorphisms (-238, -308, -863) using a polymerase chain reaction-restriction fragment length polymorphism technique. Serum TNFalpha levels were determined. RESULTS: The TNFalpha-308 allele A frequency was significantly lower in AAH group (0.09), than cirrhosis (0.28) (p < 0.001), and healthy groups (p < 0.001). The TNFalpha-308 A/G, A/A genotypes were significantly lower in AAH group, than cirrhosis (p = 0.005), and healthy groups (p < 0.001). For AAH group, there were no clinical, biological, and serum TNFalpha differences between the -308 G/G and A/G, A/A genotype patients, apart higher transaminase in the former group (p = 0.02). AAH and cirrhosis groups did not differ for the frequency of TNFalpha-238, -863 polymorphisms. The specific genotype did not appear to have any influence on the therapeutic response following corticotherapy or posttreatment 6-month survival. In the AAH group, nonsurvivors had higher TNFalpha levels than survivors (7.9 +/- 8.8 vs. 3.3 +/- 1.6 pg/ml, p = 0.01). CONCLUSIONS: These results attest to the involvement of TNFalpha-related genetic factors in susceptibility to AAH.

The lack of CB1 receptors prevents neuroadapatations of both NMDA and GABA(A) receptors after chronic ethanol exposure.

As the contribution of cannabinoid (CB1) receptors in the neuroadaptations following chronic alcohol exposure is unknown, we investigated the neuroadaptations induced by chronic alcohol exposure on both NMDA and GABA(A) receptors in CB1-/- mice. Our results show that basal levels of hippocampal [(3)H]MK-801 ((1)-5-methyl-10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine) binding sites were decreased in CB1-/- mice and that these mice were also less sensitive to the locomotor effects of MK-801. Basal level of both hippocampal and cerebellar [(3)H]muscimol binding was lower and sensitivity to the hypothermic effects of diazepam and pentobarbital was increased in CB1-/- mice. GABA(A)alpha1, beta2, and gamma2 and NMDA receptor (NR) 1 and 2B subunit mRNA levels were altered in striatum of CB1-/- mice. Our results also showed that [(3)H]MK-801 binding sites were increased in cerebral cortex and hippocampus after chronic ethanol ingestion only in wild-type mice. Chronic ethanol ingestion did not modify the sensitivity to the locomotor effects of MK-801 in both genotypes. Similarly, chronic ethanol ingestion reduced the number of [(3)H]muscimol binding sites in cerebral cortex, but not in cerebellum, only in CB1+/+ mice. We conclude that lifelong deletion of CB1 receptors impairs neuroadaptations of both NMDA and GABA(A) receptors after chronic ethanol exposure and that the endocannabinoid/CB1 receptor system is involved in alcohol dependence.

CB1 receptor knockout mice display reduced ethanol-induced conditioned place preference and increased striatal dopamine D2 receptors.

Cannabinoids and ethanol activate the same reward pathways, and recent advances in the understanding of the neurobiological basis of alcoholism suggest that the CB1 receptor system may play a key role in the reinforcing effects of ethanol and in modulating ethanol intake. In the present study, male CB1 receptors knockout mice generated on a CD1 background displayed decreased ethanol-induced conditioned place preference (CPP) compared to wild-type (CB1(+/+)) mice. Ethanol (0.5, 1.0, 1.5, and 2.0 g/kg) induced significant CPP in CB1(+/+) mice at all doses tested, whereas it induced significant CPP only at the highest dose of ethanol (2.0 g/kg) in CB1(-/-) mice. However, there was no genotypic difference in cocaine (20 mg/kg)-induced CPP. There was also no genotypic difference, neither in cocaine (10-50 mg/kg) nor in D-amphetamine (1.2-5 mg/kg)-induced locomotor effects. In addition, mutant and wild-type mice did not differ in sensitivity to the anxiolytic effects of ethanol (1.5 g/kg) when tested using the elevated plus maze. Interestingly, this decrease in ethanol efficacy to induce CPP in CB1(-/-) mice was correlated with an increase in D2/D3 receptors, as determined by [3H]raclopride binding, whereas there was no difference in D1-like receptors, as determined by [3H]SCH23390 binding, measured in the striatum from drug-naive mice. This increase in D2/D3 binding sites observed in CB1 knockout mice was associated with an altered locomotor response to the D2/D3 agonist quinpirole (low doses 0.02-0.1 mg/kg) but not to an alteration of quinpirole (0.1-1.0 mg/kg)-induced CPP compared to wild-type mice. Altogether, the present results indicate that lifelong deletion of CB1 receptors reduced ethanol-induced CPP and that these reduced rewarding effects of ethanol are correlated to an overexpression of striatal dopamine D2 receptors.

Biphasic effect of acamprosate on NMDA but not on GABAA receptors in spontaneous rhythmic activity from the isolated neonatal rat respiratory network.

Acamprosate (calcium acetylhomotaurinate) has been shown to be effective in attenuating relapse in human alcoholics. The precise mechanism for acamprosate has been yet to be determined as there may be multiple sites of action for this drug. We investigated the mechanism of action of acamprosate on a spontaneous rhythmic activity recorded from hypoglossal nerve rootlet (XII) in neonatal rat brainstem slices. At 30 microM, acamprosate reversibly increased burst amplitude and reduced burst frequency, whereas at higher concentrations (100-400 microM) it induced a reversible and concentration-dependent inhibition of this activity. Interestingly, acamprosate (30 microM) enhanced the effects of low NMDA-induced excitation (1.5 microM), but inhibited higher NMDA-induced excitation (2.5, 5 microM) by 50-70%, demonstrating a differential effect on NMDA-induced excitation. Blockade of GABAA receptors did not affect the increase in amplitude of 30 microM acamprosate and partially abolished the inhibitory effects of 200 microM acamprosate. At 200 microM, acamprosate reduced high NMDA-induced excitation and abolished NMDA-evoked excitatory tonic phase, suggesting that excitatory effect of low concentrations of acamprosate mainly involved NMDA receptors, while the inhibitory effects at higher concentration included an increase in GABAA-mediated inhibition with a reduction of NMDA-mediated excitation. Consequently, combined blockade of both receptors abolished all effects of acamprosate tested at all concentrations. These results show that the effects of acamprosate are mediated via both GABAA and NMDA receptors and suggest a partial co-agonist role on NMDA receptors, at the level of a spontaneously active network.

Decreased alcohol self-administration and increased alcohol sensitivity and withdrawal in CB1 receptor knockout mice.

Recent advances in the understanding of the neurobiological basis of alcohol dependence suggest that the endocannabinoid system may play a key role in the reinforcing effects of ethanol. In the present study, disruption of CB1 receptors in mice generated on a CD1 background decreased both ethanol consumption and preference. This decreased ethanol self-administration was associated with increased sensitivity to the acute intoxicating effects of ethanol. Mutant mice were more sensitive to the hypothermic and sedative/hypnotic effects of acute ethanol administration (1.5-4.0 g/kg), although plasma ethanol concentrations did not differ from those of controls. Moreover, wild-type mice exhibited normal locomotor activation caused by 1.0-2.5 g/kg injection of ethanol, whereas mutant mice displayed sedation in response to the injection of the same ethanol doses. The severity of alcohol withdrawal-induced convulsions was also increased in CB1(-/-) mice. Our results suggest that CB1 receptors participate in the regulation of ethanol drinking and demonstrate that their disruption lead to increased ethanol sensitivity and withdrawal severity.

Chronic ethanol exposure differentially regulates NOS1 mRNA levels depending on rat brain area.

Several works have suggested a potential role for nitric oxide in alcohol-seeking behavior and we have recently shown that the specific blockade of the expression of the neuronal nitric oxide synthase (NOS1) decreases rat ethanol intake. Our previous results have also shown that chronic ethanol exposure has differential effect on the brain NOS activity depending on rat brain area. In the present study, we examine the effects of chronic administration of ethanol on the NOS1-mRNA levels measured with the competitive reverse transcriptase-polymerase chain reaction technique. Chronic administration of ethanol differentially regulated NOS1-mRNA levels depending on rat brain area. Chronic ethanol exposure had no effect on the NOS1-mRNA levels in frontal cortex, but decreased the NOS1-mRNA levels in hippocampus (P<0.01, 39% decrease) and induced a strong increase in striatum (P<0.01, 92% increase). These effects of ethanol were not affected by 7-nitro indazole (25 mg/kg, i.p. daily for 1 week) treatment. These data further support that NOS1 is regulated by chronic exposure to ethanol and that these effects are related to modifications of mRNA levels.

Low ethanol sensitivity and increased ethanol consumption in mice lacking adenosine A2A receptors.

We have shown previously that the severity of handling-induced convulsions during ethanol withdrawal was reduced in A2A receptor knock-out (A2AR-/-) mice. In the present report, we further characterize the role of adenosine A(2A) receptors in ethanol consumption and neurobiological responses to this drug of abuse. Male A2AR-/- mice showed increased consumption of solutions containing 6 and 20% (v/v) ethanol compared with wild-type (A2AR+/+) control mice; female A2AR-/- mice showed increased consumption of solutions containing 6 and 10% ethanol. This slightly higher ethanol consumption was also related to increased ethanol preference. In contrast, A2AR-/- mice showed normal consumption of solutions containing either sucrose or quinine. Relative to A2AR+/+ mice, A2AR-/- mice were found to be less sensitive to the sedative effect of 3.0 gm/kg ethanol, as measured by more rapid recovery from ethanol-induced loss of righting reflex, and to the hypothermic effects of 1.5, 3.0, and 4.0 gm/kg ethanol, although plasma ethanol levels did not differ significantly between the two genotypes. The selective adenosine A2A receptor antagonist ZM 241385 (4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol) (10-30 mg/kg) significantly attenuated ethanol-induced (4.0 gm/kg) hypothermia in CD1 mice. To assess whether ethanol administration would induce differential tolerance in A2AR-/- and wild-type mice, we administered ethanol (3.0 gm/kg) over 4 consecutive days and found no difference in the development of tolerance; however, female A2AR-/- mice showed a lower tolerance-acquisition rate. These data suggest that activating the A2A receptors may play a role in suppressing alcohol-drinking behavior and is associated with the sensitivity to the intoxicating effects of acute ethanol administration.

Chronic ethanol consumption induces tolerance to the spatial memory impairing effects of acute ethanol administration in rats.

A large number of studies in rats have investigated the effects of acute and chronic ethanol administration on performance on many spatial learning and memory tasks. However, no study has addressed the problem of whether chronic ethanol consumption induces tolerance to acute ethanol-induced spatial memory deficits. In this study, we analyzed the behavioral effects of acute ethanol administration on spatial memory and locomotor activity in rats chronically intoxicated by ethanol. Male Sprague-Dawley rats were given as their only available liquid source a 10% (v/v) aqueous ethanol solution for 2 weeks before behavioral testing and during the 1-week behavioral testing period. They were treated intraperitoneally with 1.5 g/kg of ethanol 30 min before daily training in the Morris water maze, a spatial memory task sensitive to hippocampal damage. Our results demonstrate that learning and spatial memory of ethanol-consuming animals were not altered compared with control rats. Chronic ethanol consumption had no effect on spatial reference memory in terms of either the distance traveled to find the hidden platform during the acquisition phase of the experiment, or the time spent in the training quadrant during the retention trial. Acute ethanol administration impaired spatial memory in control rats and this impairment was reversed in chronic ethanol-consuming animals, revealing that chronic ethanol consumption did induce tolerance to the spatial memory deficits induced by acute ethanol injection, although plasma ethanol levels did not differ between the two groups. In contrast, chronic ethanol consumption did not induce tolerance to the acute ethanol-induced stimulatory locomotor activity measured in the same animals. Our results, therefore, indicate that chronic ethanol consumption induces tolerance to the cognitive impairing effects, but not to the locomotor stimulatory effects of acute ethanol administration in rats, suggesting that these two behavioral effects of ethanol do not share a common mechanism in the CNS.

Effect of prenatal and postnatal ethanol exposure on the developmental profile of mRNAs encoding NMDA receptor subunits in rat hippocampus.

It has been proposed that assembly of the final NMDA receptor complex may be modified by prenatal ethanol exposure, resulting in long-term alterations of NMDA receptor pharmacology. We investigated the effect of prenatal and postnatal ethanol exposure on the developmental profile of mRNAs encoding NMDA receptor subunits in rat hippocampus. Female Sprague-Dawley rats were chronically intoxicated for 4 weeks with a 10% (v/v) ethanol solution administered throughout pregnancy and lactation. Hippocampus and cerebellum were isolated from pups (postnatal days 1-28) of the ethanol-exposed and ad libitum groups. Our results, using a semiquantitative RT-PCR technique, showed a selective effect of ethanol exposure on the various NMDA receptor subunits. Ethanol exposure significantly increased the levels of NR1(1XX), NR1(X11) and NR2(D) mRNAs on postnatal days 7 and 14 and decreased the level of NR2(C) on postnatal day 1. Immunoblot analyses demonstrated that NR2(D) protein levels were increased on postnatal day 7 after ethanol exposure. However, the developmental profile of mRNAs encoding for NR2(A-B), NR3(L/S), GBP and Gly/TCP-BP subunits were not affected. Moreover, no significant effects of ethanol exposure were observed on the developmental transition from expression of NR1(0XX) to NR(1XX) splice variants occurring in the cerebellum on postnatal day 19. Unexpectedly, [(3) H]MK-801 binding experiments showed that ethanol exposure increased the B (max) values of high-affinity sites on postnatal days 14 and 28, with no change of K (d) values. These findings indicate that prenatal and/or postnatal ethanol exposure alters the hippocampal levels of mRNAs encoding for certain subunits and the density of high-affinity [(3) H]MK-801 binding sites. As these subunits have been shown to modulate the functional properties of NMDA receptors, these results suggest that this altered expression could be involved in the neurodevelopmental disorders associated with fetal ethanol exposure.