Genetic Correlations Between Initial Sensitivity to Ethanol and Brain cAMP Signaling in Inbred and Selectively Bred Mice

BACKGROUND: Several lines of evidence have suggested a role for cAMP (adenosine 3',5'-cyclic monophosphate) signaling in the acute and chronic effects of ethanol. This study investigated whether there is a genetic correlation between cAMP synthesis in the brain and the acute effects of ethanol [alcohol sensitivity or acute functional tolerance (AFT)]. METHODS: By using nine inbred strains of mice, we measured initial sensitivity and AFT to ethanol with a test of balance on a dowel. Initial sensitivity was defined by the blood ethanol concentration (BEC0) at the loss of balance on a dowel after an ethanol injection [1.75 g/kg intraperitoneally (ip)]. When mice were able to regain balance on the dowel, BEC1 was determined, and a second ethanol injection was given (2 g/kg ip). Upon final regaining of balance, BEC2 was determined. AFT was defined by the difference between BEC1 and BEC2 (AFT = DeltaBEC = BEC2 - BEC1). Cyclic AMP synthesis was measured in whole-cell preparations in the cerebellum and other brain areas of mice of the nine inbred strains. RESULTS: Significant differences in BEC0 and AFT were seen among the mice of the nine inbred strains. Cerebellar basal and forskolin- and isoproterenol-stimulated cAMP production differed significantly between the strains, and BEC0 was found to correlate significantly with forskolin- and isoproterenol-stimulated cAMP accumulation in the cerebellum (r = 0.70 and 0.94, respectively). When we measured cAMP production in mesencephalic and telencephalic tissue in three strains of mice that differed significantly in isoproterenol-stimulated cAMP accumulation in the cerebellum, significant differences between strains were found only in telencephalic tissue. The relative relationship between the rank order of the three strains for cAMP accumulation in the telencephalon and initial sensitivity to ethanol was identical to that seen with the cerebellum. However, AFT did not correlate with cAMP accumulation in the cerebellum or any other brain area tested. CONCLUSIONS: These results suggest that cAMP-generating systems of the cerebellum and possibly the brain areas contained in telencephalic tissues (e.g., basal ganglia) may have an important relationship to an animal's initial sensitivity to the incoordinating effects of ethanol.     

The Effect of Age on Ethanol Metabolism and on the Hypothermic and Hypnotic Responses to Ethanol in the Fischer 344 Rat

The hypothesis that the central nervous system (CNS) of aged rats is more sensitive to acute ethanol effects was examined by measuring ethanol elimination rates, and the hypothermic and hypnotic responses to ethanol in four age groups (3, 12, 21, and 30 months) of Fischer 344 rats. Following a 2.5 g/kg intraperitoneal dose of ethanol, rectal temperature and blood ethanol concentration (BEC) were measured at regular intervals for 8 hr. The 30-month group's hypothermic response to ethanol was equivalent to the 3-month group's despite lower peak BEC. Response to a hypnotic dose (3.0 g/kg) of ethanol was evaluated by measuring the time for regain of righting reflex (RORR) and the BEC at RORR. The 3-month group slept for a shorter time, and awoke at a higher BEC, than the 12- or 21-month groups. The response of the 30-month group was more complex. Although this group had the lowest BEC at RORR, their sleep time was equivalent to the 3-month group. The shortened sleep time of the 30-month group is consistent with the observation of significantly lower peak BEC and higher metabolic rate (calculated from Widmark r values) in this group. Thus, aged rats of the Fischer 344 strain may be more sensitive to acute ethanol, but these CNS effects may be partially offset by enhanced ethanol disposition at older ages.     

Comparison of ethanol metabolism in male and female cynomolgus macaques (Macaca fascicularis)

The physiological consequences of drinking ethanol differ among men and women; however, the biological basis of this gender difference is unknown. Our study characterized sex-related blood ethanol concentration (BEC) 60 min postethanol administration and ethanol elimination rates in male and female monkeys and across the phases of the menstrual cycle. Subjects were male (n = 4) and female (n = 4) cynomolgus monkeys (Macaca fascicularis) with a history of ethanol exposure and maintained at a lean body weight by food restriction. On three separate occasions, each monkey was administered 1.0 g/kg ethanol intragastrically and blood samples (20 microl) were collected every 60 min over a 5-hr period. For females, three phases of the menstrual cycle were determined by the presence of menses and plasma progesterone levels. There was no effect of menstrual cycle on mean 60 min BECs or mean rates of elimination. Mean BECs 60 min after 1.0 g/kg ethanol were: males = 86 mg/dl (+/- 2; n = 4) and females = 82 mg/dl (+/- 5; n = 4). There was no effect of sex on the highest BEC measured, which occurred at the 60 min time point in all subjects. Female monkeys did have faster average rates of ethanol elimination [34 +/- 2 (mg/dl)/hr] compared with males [23 +/- 1 (mg/dl)/hr]. The sex differences in metabolism of ethanol found with the macaque monkey model correlates well with human subject studies and suggests this is an appropriate model to further explore gender differences in response to ethanol.     

The genetics of acute functional tolerance and initial sensitivity to ethanol for an ataxia test in the LSxSS RI strains

BACKGROUND: It has been proposed that development of tolerance to the behavioral effects of ethanol depends on the degree of impairment produced by the drug; that is, more sensitive individuals should develop greater tolerance. Tests of this hypothesis with respect to acute functional tolerance have produced contradictory results. We tested the hypothesis by examining the genetic relationship between initial sensitivity and acute functional tolerance in the LSXSS recombinant inbred mice. METHODS: We tested mice for initial sensitivity to the ataxic effects of 1.75 g/kg of ethanol in a stationary dowel balance test by determining blood and brain ethanol concentrations at fall. Acute tolerance to the ataxic effects of ethanol was determined by measuring blood ethanol concentration (BEC) at regain of dowel balance ability after the first injection (BEC1RB) and after a second ethanol injection of 2.0 g/kg (BEC2RB). Acute tolerance was quantified by the difference in ethanol concentration at the two regains of balance (BEC2RB - BEC1RB) or by the difference between the second regain and one of the initial sensitivity measures (BEC2RB - initial sensitivity). RESULTS: Four different measures of initial sensitivity were taken: two that used BEC values and two that used forebrain or hindbrain ethanol concentrations. We calculated acute tolerance values by using each of these initial sensitivity measures plus BEC2RB. No evidence of a genetic relationship between initial sensitivity and acute tolerance was found, which suggests that these are two independent phenomena with respect to stationary dowel balance. CONCLUSIONS: Three conclusions can be drawn from this work: (1) Orbital sinus BEC at early time points (<5 min postinjection) may or may not accurately reflect brain EC in mice, dependent on genotype; (2) there is no genetic relationship between initial sensitivity and acute tolerance to stationary dowel ataxia in the LSXSS RIs; and (3) sex-specific factors affect low-dose ethanol responses on the stationary dowel.     

Characterization of an Automated Apparatus for Precise Control of Inhalation Chamber Ethanol Vapor and Blood Ethanol Concentrations

Inhalation chambers with a monitoring and control apparatus for ethanol vapor exposure of small animals were constructed. A thermal conductivity detector was employed for continuous measurement of inhalation chamber ethanol vapor concentration. The concentration was maintained within a very narrow range (+/- 1 mg/liter) by incorporating into the design a feedback loop which controls the ethanol pump. As expected, the blood ethanol concentrations (BEC) of male Sprague-Dawley rats were positively and linearly correlated to the chamber ethanol concentration. When rats were exposed for 24 hr to a chamber ethanol concentration of 17, 25, or 32 mg/liter, correspondingly low, moderate, or high mean blood ethanol levels were obtained. When a large population of this strain of rats (n = 121) was exposed to a constant ethanol vapor concentration for 14 days (25 mg/liter) considerable interindividual variation in blood levels occurred. There was also individual variation over time in the BEC of animals monitored. The mean +/- SD BEC was 189 +/- 90 mg/100 ml for this population and a gaussian-like distribution was obtained with regard to BEC. Behavior characteristic of alcohol withdrawal was observed in rats with BEC greater than 120 mg/100 ml after 3.5, 7, or 14 days of exposure. This apparatus and inhalation paradigm make possible the precise control of chamber ethanol concentration which markedly enhances control over both intra- and intersubject fluctuation in blood ethanol levels during alcohol exposure and the comprehensive examination of relationships between a wide range of blood ethanol concentrations and their physiological and biochemical effects.     

Effect of the selective NMDA NR2B antagonist, ifenprodil, on acute tolerance to ethanol-induced motor impairment in adolescent and adult rats

Background: Adolescent rats have been observed to be less sensitive than adults to a number of acute ethanol effects, including ethanol‐induced motor impairment. These adolescent insensitivities may be related in part to the more rapid emergence of within session (acute) tolerance in adolescents than adults. Adolescent‐related alterations in neural systems that serve as ethanol target sites, including changes in NMDA receptor subunit expression, may influence the responsiveness of adolescents to acute ethanol effects. This study explored the role of NMDA NR2B receptors in the development of acute tolerance to ethanol‐induced motor impairment in male adolescent [postnatal day (P)28–30] and adult (P68–70) Sprague–Dawley rats. Methods: Motor‐impairing effects of ethanol on the stationary inclined plane and blood ethanol concentrations (BECs) were examined following challenge at each age with a functionally equivalent ethanol dose (adolescents: 2.25 g/kg; adults: 1.5 g/kg). Data were collected at two postinjection intervals (10 or 60 minutes) to compare rate of recovery from ethanol intoxication with BEC declines using the Radlow approach ( Radlow, 1994 ) and changes in motor impairment/BEC ratios over time for assessing acute tolerance. Results: Both vehicle‐treated adolescent and adult animals showed similar acute tolerance development to the motor‐impairing effects of ethanol at these functionally equivalent doses on the stationary inclined plane, as indexed by an increasing time‐dependent dissociation between BECs and ethanol‐induced motor impairment, with motor impairment declining faster than BECs, as well as by significant declines in motor impairment/BEC ratios over time. Acute tolerance development was reliably blocked by administration of the NR2B antagonist, ifenprodil, (5.0 mg/kg), in adult rats, whereas adolescents were affected by a higher dose (10.0 mg/kg). Conclusions: These data support the suggestion that alterations in NMDA receptor systems occurring during adolescence may contribute to reduced sensitivity to ethanol by enhancing the expression of acute tolerance development in adolescents relative to adults.     

Chronic Ethanol Consumption Alters Effects of Ethanol In Vitro on Brain Membrane Structure of High Alcohol Sensitivity and Low Alcohol Sensitivity Rats

In this study, we examined if differences in initial membrane sensitivity to ethanol were associated with development of membrane tolerance to ethanol. High Alcohol Sensitivity (HAS) and Low Alcohol Sensitivity (US) rats were administered a 15% ethanol solution in water as the sole source of fluid for 30 days. The amount of ethanol consumed per day did not significantly differ between the HAS and LAS rats. Development of membrane tolerance to in vitro effects of ethanol has been previously reported for bulk membrane fluidity and protein-lipid interaction. Our data expands the understanding of "membrane tolerance" phenomenon to protein distribution and bi-layer interdigitation. We also introduce genotype-dependent and genotype-independent properties of the membrane tolerance to ethanol. Chronic ethanol treatment produced genotype-dependent and genotype-independent membrane tolerance to ethanol. The in vitro effects of ethanol on synaptic plasma membrane (SPM) protein distribution and lipid bilayer interdigitation were abolished or decreased in the SPM of chronic ethanol-treated HAS rats, as compared with the SPM of HAS control rats (genotype-dependent tolerance). Protein distribution and bilayer interdigitation were not affected by ethanol in vitro in either chronic ethanol-treated or control LAS rats. Genotype-independent tolerance to ethanol in vitro was observed for SPM annular and bulk bilayer fluidity in chronic ethanol-treated HAS and LAS rats. It is concluded that initial sensitivity to ethanol contributes to the development of membrane tolerance to ethanol in HAS and LAS rats.     

Effect of hydrofluoroalkane–ethanol inhalers on estimated alcohol levels in asthmatic subjects

Background and objective:   Inhaled medication administered via a metered dose inhaler (MDI) is often used to treat asthma. Hydrofluoroalkane (HFA) has replaced chlorofluorocarbons (CFC) as the propellant and these new MDI may contain alcohol. This raises concerns that their use may transiently increase breath ethanol concentration (BEC), thereby interfering with random breath testing. It was hypothesized that HFA–ethanol MDI may contribute to raising BEC above the legal limit.
Methods:   The effect of a HFA–ethanol MDI on BEC was compared with that of CFC and placebo MDI and the effect of ingesting a standard amount of alcohol was also investigated. Asthmatic ( n  = 16) and normal control subjects ( n  = 15) were recruited for the double-blind, placebo-controlled study. Each subject used the three MDI in random sequence. BEC was measured at baseline and at 2, 5 and 15 min after inhalation of each MDI, using the Lion Alcometer SD-400TM. Spirometry was performed at baseline and 20 min after the last inhalation. An identical procedure was followed after alcohol consumption.
Results:   Use of the HFA–ethanol MDI resulted in a significant increase in BEC from 0.002 to 0.0138 mg/100 mL (28 mg/100 mL of blood, P  = 0.001) in asthmatics, and from 0.001 mg/100 mL to 0.016 mg/100 mL (33 mg/100 mL of blood, P  = 0.002) in normal subjects. By 5 min, there were no significant differences in BEC of asthmatics (0.0031 mg/100 mL) and normal subjects (0.003 mg/100 mL), when compared with baseline ( P  > 0.2).
Conclusions:   BEC are transiently elevated after inhalation of HFA–ethanol MDI; however, by 5 min, BEC had already returned to baseline levels. Thus the effect of HFA–ethanol MDI on BEC is transient and would be negligible after 5 min.     

Effect of Chronic Dietary Ethanol Consumption on Colonic Cancer in Rats Induced by 1,1-Dimethylhydrazine

Background: The pathogenetic correlation between chronic alcohol consumption and development of colon cancer is not clear. The role of alcohol abuse in the carcinogenic action of 1,1-dimethylhydrazine (DMH), which induces tumors in the colon, was evaluated.
Methods: Twenty male rats were fed liquid diets containing ethanol or carbohydrates for 39 weeks. DMH (20 mg/kg body weight, once a week) was injected subcutaneously from the 5th to the 20th week. Pair feeding was stopped at 10:00 am and DMH was administered at 02:00 pm . Ethanol was not detected in the blood at the time of injection. Liquid diets were provided again at 05:00 pm until 10:00 am next day. The animals were killed at the end of the 39th week, and the colons were removed for examination for the number of aberrant crypt foci (ACF) by methylene blue staining. Tissue sections were stained for histology and cytochrome P4502E1 (CYP2E1) expression.
Results: The number of ACF in colons obtained from ethanol-fed rats with DMH was 24 ( n =5, 4.4±2.5/rat), which was significantly ( p <0.001) more than that of the other treated rats: only 3 ( n =5, 0.6±0.5/rat) in the pair-fed control rats with DMH, and none in the ethanol-fed or control-fed rats without DMH. Cytochrome P4502E1 staining demonstrated marked expression in the colon mucosa from ethanol-fed rats, but not in the pair-fed control rats.
Conclusions: The increased expression of CYP2E1 induced by chronic ethanol consumption promotes the development of DMH-induced colon cancer.     

Intensity and Duration of Chronic Ethanol Exposure Is Critical for Subsequent Escalation of Voluntary Ethanol Drinking in Mice

Background: Excessive alcohol drinking continues to be an important health problem. Recent studies from our laboratory and others have demonstrated that animal models of ethanol dependence and relapse can contribute to understanding factors that contribute to excessive drinking. In this study, we tested the hypothesis that the amount and duration of ethanol exposure is critical for promoting the escalation in drinking by mice given access to ethanol in a limited access paradigm. Methods: We used several methods of chronic intermittent ethanol exposure in male C57BL/6J mice that would vary in the amount and duration of exposure to ethanol as indicated by blood ethanol concentrations (BEC). After establishing baseline drinking in the mice using a 2 hours, 2 bottle choice drinking paradigm, each study involved alternating between periods of ethanol exposure and periods of limited access to ethanol (1 cycle) for a total of 3 cycles. In Study 1, mice were allowed extended access (16 hours) to ethanol for oral consumption or remained in the home cage. In Study 2, the ethanol exposure consisted of intragastric gavage of increasing doses of ethanol or isocaloric sucrose as the control. Study 3 compared intragastric gavage combined with pyrazole, an alcohol dehydrogenase inhibitor, with vapor inhalation of ethanol using procedures known to lead to increased drinking in mice. Finally, Study 4 was a retrospective review of several studies conducted in our laboratory using inhalation procedures. The retrospective review encompassed a range of postvapor chamber BEC values and ethanol intakes that would allow a relationship between increased drinking and BEC to be examined. Results: Allowing mice to drink for longer periods of time did not cause increased drinking in subsequent limited access sessions. Likewise, gastric intubation of ethanol which produced high BEC (>300 mg/dl) with or without pyrazole did not increase drinking. Only the vapor inhalation procedure, which was associated with sustained BEC above 175 mg/dl for the entire exposure period resulted in increased drinking. The retrospective study provided further evidence that sustained BEC levels above 175 mg/dl was critical to the escalation in drinking. Conclusions: We found that the intensity (amount) and duration of ethanol exposure, indexed by BEC, is critical to produce increased drinking in mice. Specifically, BEC must regularly exceed 175 mg/dl for the escalation in drinking to occur. Future studies will examine neurobiological adaptations that may underlie the increased drinking behavior caused by chronic intermittent ethanol exposure.     

Indexing Withdrawal in Mice: Matching Genotypes for Exposure in Studies Using Ethanol Vapor Inhalation

Withdrawal Seizure-Prone (WSP) and -Resistant (WSR) mice have been bidirectionally selected for severity of handling-induced convulsions (HIC) following withdrawal from 72 hr of chronic ethanol vapor inhalation. During selection, daily injections of the alcohol dehydrogenase inhibitor, pyrazole, were used to enhance and stabilize blood ethanol concentrations (BEC). After 26 generations of selection, WSR mice show lower withdrawal BEC than WSP mice exposed to the same ethanol vapor concentrations. Because it is desirable to compare mice maintained at the same BEC to assess correlated responses to selection, this has necessitated exposing WSR mice to higher ethanol vapor concentrations than WSP mice to achieve matched chronic BEC. The experiments reported herein demonstrate two methods for producing matched withdrawal BEC: (1) by exposing mice to the same ethanol vapor concentration and varying the pyrazole dose; and (2) by administering only ethanol at different vapor concentrations and selecting some mice with approximately the same BEC. When exposed to the same ethanol vapor concentration, WSR mice given 1.0 mmol/kg pyrazole had withdrawal BEC equivalent to WSP mice given 0.75 mmol/kg pyrazole. However, WSP mice had much more severe withdrawal HIC than WSR mice. WSP and WSR mice metabolized ethanol at the same rate following withdrawal. The basis for the differential effectiveness of pyrazole is unknown. We also exposed mice to higher ethanol vapor concentrations in the absence of pyrazole. By exposing WSR mice to higher concentrations than WSP, roughly equivalent BEC on withdrawal was achieved. Because BEC are more variable in the absence of pyrazole, it was necessary to select animals of each genotype to achieve relatively matched BEC. Again, WSP mice had much more severe HIC on withdrawal than WSR.     

Quantitative trait locus mapping for acute functional tolerance to ethanol in the L x S recombinant inbred panel

BACKGROUND: Acute functional tolerance (AFT) develops shortly after ethanol administration, and is determined as the change in brain or blood ethanol concentration (BEC) measured at 2 behavioral or physiological endpoints. Acute functional tolerance studies in some rodent strains support a long-held hypothesis that more sensitive strains develop more within-session tolerance. We used the new, 74-strain L x S recombinant inbred (RI) panel, developed from inbred long-sleep (ILS) and inbred short-sleep (ISS) strains, to revisit this hypothesis and to map quantitative trait loci (QTLs) for AFT. We report replication of QTL regions reported by earlier studies of AFT and preliminary application of a coarse single nucleotide polymorphism map analysis to limit QTL intervals for subsequent candidate gene hypotheses. METHODS: Acute functional tolerance was assayed using a test of ataxia: loss and regain of balance on a stationary wooden dowel. Following an initial dose of 1.75 g/kg, BEC was measured at initial loss (BEC(0)) and regain of balance (BEC(1)). A second injection (2.0 g/kg) was administered and blood taken at the second regain of balance (BEC(2)). Acute functional tolerance was calculated as a difference score in 2 ways: (1) between BEC at the 2 successive regains of balance (AFT(1)), or (2) as the difference in BEC at final regain and at initial loss of balance (AFT(2)). We mapped QTLs for BEC(0), a measure of initial sensitivity, and both AFT scores. RESULTS: All 4 parental strains (LS, SS, ILS, and ISS) developed tolerance, replicating previous published reports. There were significant sex effects for 3 of these strains. The L x S panel showed a 128-fold range in tolerance, with a few strains showing negative tolerance (sensitization). The ISS surpassed the next highest RI strain by 55% and was more than 4 times greater than SS. Heritability estimates for both AFT measures were close to 0.25 for both sexes. One significant QTL accounting for approximately 18% of phenotypic variance (V(P)), on chromosome 12 (AFT(1)), and 1 suggestive QTL (16% V(P)), on chromosome 16 (AFT(2)), were identified. These QTLs replicated regions reported in other studies. A multiple QTL model incorporating the effects of all significant interacting QTLs was developed, explaining almost 60% of V(P). The chromosome 12 region was further investigated by haplotype analysis, which identified many nonpolymorphic regions within the confidence interval, and possible candidate genes in the polymorphic regions. CONCLUSIONS: Both SS and ISS developed greater AFT, assessed by both methods, than LS and ILS; this difference was significant in virtually all sex by strain comparisons. In the L x S RI, there was no correlation between initial sensitivity, measured by BEC at initial loss of balance, and either measure of AFT, on a stationary dowel. These results indicate that in this model system, initial sensitivity does not predict tolerance. Several QTLs for tolerance were identified; candidates in the narrowed chromosome 12 region, which has been reported in 2 other mapping studies, merit additional study.     

Cardiolipin from ethanol-fed rats confers tolerance to ethanol in liver mitochondrial membranes

In rats chronically consuming ethanol, the liver mitochondrial membranes develop resistance to the disordering effects of ethanol in vitro, so-called "membrane tolerance". To investigate the molecular basis of this tolerance in the inner mitochondrial membrane, multilamellar vesicles were produced by recombining the mitoplast phospholipids (quantitatively separated by preparative HPLC) from control and ethanol-fed animals in various combinations. The effect of in vitro ethanol on the physical properties of these vesicles was determined by electron spin resonance. Vesicles composed of all mitoplast phospholipids from control rats were disordered by 50-100 mM ethanol, whereas those made of the phospholipids from ethanol-fed animals were resistant. When phosphatidylcholine (46 mol %) or phosphatidylethanolamine (42 mol %) from ethanol-fed rats replaced the corresponding phospholipids of control rats, the vesicles were disordered by ethanol. By contrast, when as little as 2.5 mol % of cardiolipin (one-fourth the naturally occurring amount) from ethanol-fed rats replaced that phospholipid from control rats, vesicles were rendered entirely resistant to disordering by ethanol. The same amount of cardiolipin from ethanol-fed rats also conferred membrane tolerance to vesicles composed of bovine phospholipids, demonstrating that this effect is not restricted to rat mitoplast phospholipids. In vesicles composed of a single mitoplast-phospholipid class, only vesicles composed of cardiolipin from ethanol-fed rats resisted disordering. Phosphatidylinositol from liver microsomes of ethanol-fed rats also confers membrane tolerance and was the only microsomal phospholipid that formed tolerant vesicles. Thus, in livers of rats chronically fed ethanol, anionic phospholipids are selectively converted into potent promoters of membrane tolerance in both mitochondrial and microsomal membranes.     

The effect of ethanol on survival time in hemorrhagic shock in an unanesthetized swine model

Controversy exists as to whether ethanol intoxication causes exaggerated hypotension or increased mortality during hemorrhagic shock. Previous studies have used anesthetized animals. This limits data interpretation as anesthetic agents, particularly pentobarbital, have well-documented effects on hemodynamics and the response to hemorrhage. We studied the effects of moderate ethanol intoxication on blood pressure and survival time during fatal hemorrhagic shock in unanesthetized swine. Immature female swine weighing 15 to 20 kg were splenectomized and instrumented with chronic indwelling aortic catheters, right atrial catheters, and gastrostomy tubes. Four to seven days later the unanesthetized animals underwent hemorrhagic shock. Thirty minutes prior to the start of hemorrhage, the experimental group (n = 8) received 3 mL/kg of 100% ethanol mixed as a 1:3 solution with water through a gastrostomy tube. The control group (n = 8) received an equal amount of water. The distal aortic catheter was connected to a roller pump and blood was removed at a rate of 1 mL/kg/min until the animal died. Arterial pressure, heart rate, lactate ethanol and glucose levels, hematocrit, and arterial blood gases were measured in both groups at baseline and every 15 minutes thereafter. A mean ethanol level of 1,500 to 1,700 micrograms/mL was produced in the experimental group from baseline through 60 minutes. Data were analyzed using Student's two-tailed t test, and analysis of variance for repeated measures. There was no significant difference in survival time between the control (63.1 +/- 2.8 min) and ethanol (59.9 +/- 5.9 min) groups. Systolic blood pressure was significantly lower in the ethanol group after 15 minutes of hemorrhage (81 +/- 22 to 59 +/- 14 mm Hg, P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic intermittent ethanol exposure during adolescence blocks ethanol-induced inhibition of spontaneously active hippocampal pyramidal neurons

BACKGROUND: Binge alcohol drinking among adolescents has been a serious public health problem. A model of binge alcohol, chronic intermittent ethanol exposure (CIEE), during adolescence significantly attenuates ethanol-induced spatial memory deficits in rats. However, the attenuation was absent following a 12-day ethanol-free period. Since spatial memory is hippocampal dependent, a reduction in ethanol-induced spatial memory impairments may be due to a reduction in the ability of ethanol to inhibit the firing rate of single hippocampal pyramidal neurons following CIEE. METHODS: Beginning on postnatal day 30 (P30), male adolescent Sprague-Dawley rats (Harlan) were administered 5.0 g/kg ethanol (n = 10, CIEE-treated group) or an equivolume saline (n = 10, CISE-treated group) every 48 hours for 20 days. Single hippocampal pyramidal neurons from 5 CIEE-treated rats and 5 CISE-treated rats were recorded on the day following completion of the chronic intermittent exposure procedure (animals now P50). Additionally, neurons from 5 CIEE-treated rats and 5 CISE-treated rats were recorded 12 days after the completion of the chronic intermittent exposure procedure (animals now P62). RESULTS: Ethanol exposure during adolescence completely blocked ethanol-induced inhibition of hippocampal pyramidal neurons in rats that were CIEE exposed. However, the effect of CIEE on hippocampal neurophysiology was time dependent. Specifically, neurons recorded from CIEE-treated rats after a 12-day ethanol-free period had similar maximal inhibition as neurons from CISE-treated animals, although the time to reach inhibition was significantly greater in neurons from CIEE-treated rats. CONCLUSION: Chronic ethanol exposure during adolescence produces a reduction, or tolerance, to ethanol-induced inhibition of hippocampal pyramidal neural activity. Although the tolerance was greatly reversed after a 12-day ethanol-free period, neurons from CIEE animals inhibited slower than neurons from CISE animals. Since the hippocampus is known to be involved not only in spatial memory, but also in many other types of memory formation, the altered hippocampal functions because of CIEE during adolescence should be taken as a serious warning for society.     

Effect of Age on Metabolic Tolerance and Hepatomegaly following Chronic Ethanol Administration

Chronic consumption of ethanol often results in an increased rate of ethanol metabolism (metabolic tolerance) and in hepatomegaly. However, the extent of these changes is highly variable. We have found that these two phenomena are greatly influenced by age. We studied the effect of age on the development of metabolic tolerance and hepatomegaly and on the increase in hepatic oxygen consumption produced by chronic ethanol administration. The latter has been proposed to contribute to metabolic tolerance to ethanol. Ethanol was administered to female Sprague-Dawley rats with different initial ages (4, 6, 8, 11, and 17 weeks) for a 4-week period in a high-fat liquid diet. Control animals were pair-fed an isocaloric liquid diet in which ethanol was replaced with carbohydrate. Metabolic tolerance and hepatomegaly following chronic ethanol consumption were markedly dependent on the initial age of the animal, with young animals showing the largest increases. Although showing a similar general trend with age, the degree of metabolic tolerance was not linked proportionally with the degree of hepatomegaly. Perfused livers from young rats fed chronically with ethanol showed increases in ethanol metabolism and oxygen consumption, whereas no increase were observed in those from older animals. These findings support the hypothesis that an elevated rate of hepatic oxygen consumption contributes to metabolic tolerance. Total hepatic alcohol dehydrogenase activity was not increased by chronic ethanol consumption in any age group, demonstrating that an increase in the levels of this enzyme is not obligatory for metabolic tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphatidylinositol from ethanol-fed rats confers membrane tolerance to ethanol.

The presence of ethanol disorders (fluidizes) biological membranes, but its chronic administration confers resistance to this perturbation (membrane tolerance). The latter effect has been invoked as an explanation for behavioral tolerance in alcoholics, but the molecular basis for membrane tolerance is obscure. To study the molecular mechanisms of this acquired resistance to disordering, we fed rats ethanol (36% of total calories) for 35 days, after which we quantitatively separated the phospholipids of hepatic microsomal membranes by high-performance liquid chromatography. Multilamellar vesicles were prepared from the recombined phospholipid classes, and their physical properties were examined by electron spin resonance. Vesicles composed of phospholipids from untreated rats were disordered (fluidized) in the presence of ethanol, whereas those made from phospholipids of ethanol-fed rats were resistant to this effect. When phosphatidylcholine (66.5 mol %), phosphatidylethanolamine (21 mol %), or phosphatidylserine (4.0 mol %) from ethanol-fed rats replaced their corresponding phospholipids in vesicles prepared from microsomal phospholipids from untreated rats, the membranes were still disordered by ethanol. In contrast, when 2.5-8.5 mol % phosphatidylinositol from ethanol-fed rats replaced phosphatidylinositol from untreated rats, the reconstituted membranes were rendered resistant to ethanol-induced disordering. Liver microsomal phosphatidylinositol (2.5-8.5 mol %) from ethanol-fed rats also conferred membrane tolerance to vesicles composed of bovine liver and brain phospholipids, an effect which demonstrates that the ability of phosphatidylinositol to confer membrane tolerance is not restricted to the microsomal membrane.     

Effects of ethanol and ethanol withdrawal on nociception in rats

The effect of acute and chronic administration of ethanol and ethanol withdrawal on a radiant heat tail-flick assay of nociception was examined in rats. Acute administration of ethanol (2.0 g/kg, i.p.) produced peak antinociception (68% of maximum) by 30 min, and effects were gone by 120 min. Cumulative doses of ethanol (0.5-2.0 g/kg, i.p.) produced dose-dependent increases in latencies to 49% of maximum. During chronic administration, a liquid diet containing ethanol (6.5%) was given for 10 days. Tail-flick latencies were measured on day 0 (baseline), day 2, 4, 6, 8, and 10 of chronic ethanol and at 3, 6, 12, and 36 hr after removal of ethanol. To test for behavioral tolerance, both between- and within-group designs were used. In both between- and within-group experiments, the antinociceptive effects of chronic ethanol peaked by day 4 of exposure to the liquid diet, and tolerance developed by day 10. When the liquid diet was removed, hyperalgesia was detected at 6 and 12 hr after withdrawal, and was gone by 36 hr after withdrawal. When cumulative doses of ethanol (0.5-2.0 g/kg) were administered starting 12 hr after withdrawal, ethanol (0.5 g/kg) fully reversed the hyperalgesia induced by ethanol withdrawal, even though this dose was without antinociceptive effect in the absence of withdrawal. Higher doses of ethanol during ethanol withdrawal did not increase tail-flick latencies over baseline. In summary: (1) ethanol produces antinociception when administered acutely or chronically; (2) tolerance to the antinociceptive effects develops during chronic administration; (3) ethanol withdrawal induced hyperalgesia, which was reversed by ethanol; and (4) repeated testing did not produce behavioral tolerance.     

Effect of aging on in vivo and in vitro ethanol metabolism and its toxicity in F344 rats

To investigate the effect of aging on ethanol metabolism, 24 male and female F344 rats aged 2 and 12 mo that were fed a laboratory diet received ethanol (1.2 and 2.5 g/kg body wt) intraperitoneally. In male rats, in vivo ethanol elimination significantly decreased according to age both at high (436 +/- 38 vs. 294 +/- 27 mg/kg.h; p less than 0.01) and low (365 +/- 19 vs. 261 +/- 8 mg/kg.h; p less than 0.01) blood ethanol concentrations. Age did not influence the specific activity of hepatic or gastric alcohol dehydrogenase, whereas the activity was significantly decreased with age in the liver (p less than 0.05) and in the stomach (p less than 0.001) when related to body weight. In addition, the activity of the hepatic microsomal ethanol oxidizing system decreased significantly according to age (8.7 +/- 0.5 vs. 6.00 +/- 0.3 nmol/min.mg micr. protein; p less than 0.001). To study the response of ethanol-metabolizing enzymes to chronic ethanol ingestion, 2- and 19-mo-old male F344 rats were pair-fed nutritionally adequate liquid diets containing 36% of total calories either as ethanol or isocaloric carbohydrate for 3 wk. In this experiment specific alcohol dehydrogenase activity was not significantly affected by age, whereas the hepatic microsomal function estimated by the determination of cytochrome P450, microsomal ethanol oxidizing system, and aniline hydroxylation as well as hepatic mitochondrial low Km-acetaldehyde dehydrogenase activity was found to be markedly depressed with age (p less than 0.01). Chronic ethanol consumption increased microsomal enzyme activities in older rats to levels comparable to those observed in young animals prior to ethanol administration. Chronic ethanol feeding also resulted in an increased hepatic fat accumulation, which was significantly enhanced in older rats. In contrast to male rats, in vivo ethanol metabolism was practically identical for 2- and 12-mo-old female rats. These data demonstrate an enhanced toxicity of alcohol in older compared to younger male but not female rats associated with a delay in alcohol elimination both at high and low ethanol blood concentrations and a decrease in ethanol- and acetaldehyde-metabolizing enzyme activities.