Induction and maintenance of ethanol self-administration in cynomolgus monkeys (Macaca fascicularis): long-term characterization of sex and individual differences

BACKGROUND: Investigations of oral ethanol self-administration in nonhuman primates have revealed important parallels with human alcohol use and abuse, yet many fundamental questions concerning the individual risk to, and the biological basis of, excessive ethanol consumption remain unanswered. Moreover, many conditions of access to ethanol in nonhuman primate research are largely unexplored. This set of experiments extends within- and across-session exposure to ethanol to more fully characterize individual differences in oral ethanol self-administration. METHODS: Eight male and eight female adult cynomolgus monkeys (Macaca fascicularis) were exposed to daily oral ethanol self-administration sessions for approximately 9 months. During the first 3 months, a fixed-time (FT) schedule of food delivery was used to induce the consumption of an allotted dose of ethanol in 16-hr sessions. Subsequently, the FT schedule was suspended, and ethanol was available ad libitum for 6 months in 16- or 22-hr sessions. RESULTS: Cynomolgus monkeys varied greatly in their propensity to self-administer ethanol, with sex and individual differences apparent within 10 days of ethanol exposure. Over the last 3 months of ethanol access, individual average ethanol intakes ranged from 0.6 to 4.0 g/kg/day, resulting in blood ethanol concentrations from 5 to 235 mg/dl. Males drank approximately 1.5-fold more than females. In addition, heavy-, moderate-, and light-drinking phenotypes were identified by using daily ethanol intake and the percentage of daily calories obtained from ethanol as criteria. CONCLUSIONS: Cynomolgus monkeys displayed a wide intersubject range of oral ethanol self-administration with a procedure that used a uniform and prolonged induction that restricted early exposure to ethanol and subsequently allowed unlimited access to ethanol. There were sex and stable individual differences in the propensity of monkeys to consume ethanol, indicating that this species will be important in characterizing risk factors associated with heavy-drinking phenotypes.     

Individual differences in hyperlipidemia and vitamin E status in response to chronic alcohol self-administration in cynomolgus monkeys

Background: Chronic ethanol self‐administration induces oxidative stress and exacerbates lipid peroxidation. α‐Tocopherol is a potent lipid antioxidant and vitamin that is dependent upon lipoprotein transport for tissue delivery. Methods: To evaluate the extent to which vitamin E status is deranged by excessive alcohol consumption, monkeys voluntarily drinking ethanol (1.36 to 3.98 g/kg/d for 19 months, n = 11) were compared with nondrinkers (n = 5, control). Results: Three alcohol‐drinking animals developed hyperlipidemia with plasma triglyceride levels (1.8 ± 0.9 mM) double those of normolipidemic (NL) drinkers (0.6 ± 0.2) and controls (0.6 ± 0.3, p < 0.05); elevated plasma cholesterol (3.6 ± 0.5 mM) compared with NL drinkers (2.3 ± 0.2, p < 0.05) and controls (2.9 ± 0.3); and lower plasma α‐tocopherol per triglycerides (14 ± 6 mmol/mol) than controls (27 ± 8) and NL drinkers (23 ± 6, p < 0.05). Hyperlipidemic monkey liver α‐tocopherol (47 ± 15 nmol/g) was lower than NL drinkers (65 ± 13) and controls (70 ± 15, p = 0.080), as was adipose α‐tocopherol (84 ± 37 nmol/g) compared with controls (224 ± 118) and NL drinkers (285 ± 234, p < 0.05). Plasma apolipoprotein (apo) CIII increased compared to baseline at both 12 and 19 months in the normolipidemic (p = 0.0016 and p = 0.0028, respectively) and in the hyperlipidemic drinkers (p < 0.05 and p < 0.05, respectively). Plasma apo H concentrations at 19 months were elevated hyperlipidemics (p < 0.05) relative to concentrations in control animals. C‐reactive protein (CRP), a marker of inflammation, was increased compared to baseline at both the 12‐ and 19‐month time points in the normolipidemic (p = 0.005 and p = 0.0153, respectively) and hyperlipidemic drinkers (p = 0.016 and p = 0.0201, respectively). Conclusion: A subset of alcohol‐drinking monkeys showed a predisposition to alcohol‐induced hyperlipidemia. The defect in lipid metabolism resulted in lower plasma α‐tocopherol per triglycerides and depleted adipose tissue α‐tocopherol, and thus decreased vitamin E status.     

The effects of moderate ethanol consumption on the liver of the monkey, Macaca fascicularis

BACKGROUND: Although evidence has accumulated for the cardioprotective effects of moderate ethanol consumption, little is known about the effects on the liver of consuming the equivalent of two drinks per day. The objective of this study was to determine the effects of moderate ethanol administration on the hepatic content of enzymes involved in ethanol oxidation, on hepatic lipid accumulation, and on serum markers of liver function/damage in the monkey, Macaca fascicularis. METHODS: Ovariectomized, adult monkeys were maintained for 34 months on an atherogenic diet containing cholesterol 1.21 mg/kJ. They were trained to drink ethanol plus vehicle at a dose of 0.5 g/kg body weight, which was administered 5 days a week for 2 years. Blood was collected for ethanol concentrations (1 hr after ethanol administration) and was also assayed for gamma-glutamyltransferase, alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities. Liver obtained at necropsy was analyzed for triglyceride and cholesterol contents and for alcohol dehydrogenase, cytochrome P450 2E1, and cytochrome P450 3A4 by Western blots. RESULTS: The blood ethanol concentrations measured 1 hr after ethanol administration were relatively constant over the 2-year dosing period. Hepatic levels of alcohol dehydrogenase and the cytochrome P450s were not significantly different between ethanol-consuming animals and control animals. Ethanol-associated increases in liver triglyceride were not significant due to high variability in hepatic lipid content in both the controls and ethanol consumers. However, covariance analyses using pretreatment concentrations of plasma cholesterol and apolipoprotein A-I suggested that the ethanol-related increase in hepatic free cholesterol was significant. Relative to controls, alcohol consumers had higher levels of serum ALT and a transient increase in ALP at 5 months. CONCLUSIONS: The observations made in this study on primates administered an atherogenic diet suggest that moderate ethanol ingestion has modest effects on the liver, including slightly increased ALT and ALP values. However, additional studies will be required to verify that this level of consumption is hepatotoxic when ingested over extended periods. This is still a concern because some human studies suggest that levels of ethanol considered to be cardioprotective cause liver injury when consumed over a lifetime.     

Effects of Acute and Chronic Doses of Naltrexone on Ethanol Self-Administration in Rhesus Monkeys

The effects of acute and chronic administration of intramuscular naltrexone (0.1, 0.3, 1.0, and 3.0 mg/kg) on oral ethanol (8%) self-administration were examined. Naltrexone (1.0 mg/kg) effects on the self-administration of ethanol concentrations ranging from 0.5 to 8% (w/v) were also investigated. Rhesus monkeys with substantial histories of drug and ethanol drinking served as subjects. During daily 3-hr sessions, monkeys were presented with ethanol solutions, concurrently available with water, under fixed-ratio reinforcement schedules. Naltrexone decreased the consumption of ethanol (g/kg). Biphasic temporal effects were observed within sessions. Naltrexone dose-dependently decreased the number of ethanol deliveries by a maximum of 56% ( n = 18; 3 monkeys × 6 sessions) during the first hour of the session. During the second and third hours, however, ethanol intake recovered such that maximum decreases over the 3-hr session were ∼27% ( n = 18), and the mean decrease was 16% ( n = 18). Often marked tolerance was observed, such that the effects of acute naltrexone administration were greater than effects after chronic administration. The self-administration of low ethanol concentrations (≤2% w/v) was increased in several monkeys, by up to 340%, after naltrexone pretreatment. In summary, the effects of naltrexone on ethanol self-administration, in drug- and alcohol-experienced rhesus monkeys, are not characterized by unitary decreases in measures of ethanol self-administration. Rather, differential naltrexone effects were a function of experimental parameters, including the dose and number of naltrexone injections, the ethanol concentration, and the time point of measurement.     

Ethanol operant self-administration in rats is regulated by adenosine A2 receptors

BACKGROUND: Recent findings suggest that adenosine is involved in the neural and behavioral effects of ethanol (EtOH). Studies in neural cell culture show that EtOH, via activation of adenosine A2 receptors, triggers cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling and CRE (cAMP regulatory element)-mediated gene expression and that this effect is blocked by inhibiting G-protein betagamma subunits. Recently, we reported that expression of a betagamma inhibitor in the nucleus accumbens (NAc) reduces EtOH drinking in rats. The NAc expresses high levels of the adenosine A2A receptor in GABAergic medium spiny neurons. If the reinforcing effects of EtOH are mediated through an A2 activation of cAMP/PKA signaling via betagamma, then A2 receptor blockade should attenuate EtOH consumption. Here we tested this hypothesis. Because adenosine A2 and dopamine D2 receptors are coexpressed in neurons of the NAc, we compared the effects of A2 blockade with those of D2 receptor blockade. METHODS: Male Long-Evans rats were trained to self-administer 10% EtOH in daily 30-min sessions with an active and an inactive lever. Separate groups of rats were given the D2 antagonist eticlopride (0.005, 0.007, and 0.01 mg/kg), the A2 antagonist 3,7-dimethyl-1-propargylxanthine (DMPX; 1, 3, 5, 7, 10, and 20 mg/kg), and the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.125, 0.25, and 0.5 mg/kg) by systemic injection. RESULTS: Eticlopride dose-dependently reduced EtOH drinking. DMPX showed a bimodal effect: 10 and 20 mg/kg decreased, but 1 mg/kg increased, EtOH consumption. DPCPX was without effect. CONCLUSIONS: In support of our hypothesis, the A2 antagonist DMPX attenuated EtOH self-administration. Low doses of the A2 antagonist enhanced EtOH drinking, consistent with the possibility that rats increase EtOH self-administration to overcome partial A2 blockade. The D2 antagonist eticlopride also decreased EtOH self-administration. These data provide the first evidence that pharmacological modulation of adenosine A2 receptors can regulate EtOH consumption in rats.     

Divergence of Ethanol and Acetaldehyde Kinetics and of the Disulfiram-Alcohol Reaction between Subjects with and without Alcoholic Liver Disease

Despite standardization, marked interindividual variation in the severity of the disulfiram-alcohol reaction (DAR) has been observed. We studied the DAR in 51 consecutive alcoholics with ( n = 16) and without ( n = 35) significant alcoholic liver disease. Clinical signs of the DAR were much weaker in the patients with compared with those patients without liver disease. Because acetaldehyde is thought to be the main cause of the DAR, we studied ethanol and acetaldehyde kinetics in 13 patients (6 females, 7 males) with alcoholic liver disease (documented by biopsy, clinical and/or radiological findings, and by quantitative liver function) [galactose elimination capacity (GEC) 4.2 ± SD 1.0 mg/min/kg; aminopyrine breath test (ABT) 0.14 ± 0.10% dose × kg/mmol CO₂] and 13 age- and sex-matched controls (alcoholics without significant liver disease, GEC 7.1 ± 0.7; ABT 0.81 ± 0.35). Clinical signs of acetaldehyde toxicity during the DAR (flush, nausea, tachycardia, and blood pressure drop) were absent in alcoholic liver disease, but clearly evident in controls. Blood ethanol kinetics were similar in both groups, C_max and area under the concentration-time curve (AUC) being 6.27 ± 1.82 and 368.9 ± 72.9 mmol × min/liter in alcoholic liver disease, and 6.62 ± 1.71 and 377.6 ± 124.5 in controls, respectively. In contrast, there was a strong ( p < 0.001) difference in C_max and AUC of acetaldehyde, respective values being 33.46 ± 21.52 and 1463.8 ± 762.5 μmol × min/liter in alcoholic liver disease, and 110.87 ± 56.00 and 4162.0 ± 2424.6 in controls. We hypothesize that the lack of disulfiram-induced acetaldehyde retention in the alcoholic liver disease group may be due to decreased formation of disulfiram metabolites.     

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.     

Ovarian oxytocin and neurophysin concentrations in the cynomolgus monkey (Macaca fascicularis)

The neurohypophysial hormone oxytocin has previously been found in the ovaries of several animal species. In ruminants ovarian oxytocin is postulated to have a luteolytic function, because of its high concentrations in the corpus luteum. In primates the role of ovarian oxytocin is not known. In the present study we measured the immunoreactive oxytocin and oxytocin-neurophysin content in paired ovaries removed from cynomolgus monkeys (Macaca fascicularis) during the late luteal phase of the cycle (Days 12-14 of the luteal phase or Days 26-28 of a menstrual cycle). Each animal was pulsed with synthetic gonadotropin-releasing hormone to maintain normal menstrual cyclicity. The concentration of oxytocin and its neurophysin during the late luteal phase was greater in the non-corpus luteum than corpus luteum-bearing ovary. By high pressure liquid chromatography and bioassay the oxytocin in both the corpus luteal and non-corpus luteal ovaries was similar to synthetic and posterior pituitary oxytocin. The finding of high concentrations of immunoreactive oxytocin in the non-corpus luteum-bearing ovary suggests that the function of ovarian oxytocin in primates may not be confined specifically to the corpus luteum.     

Oxytocin in the corpus luteum of the cynomolgus monkey (Macaca fascicularis)

To determine if oxytocin (OT) is present in cynomolgus monkey corpus luteum, OT was measured by a specific and sensitive RIA in 13 corpora lutea, ovarian venous plasma on the ipsilateral side and peripheral venous plasma at different stages of the luteal phase. Serial dilution of acetic acid extract of the corpus luteum showed parallelism with standard OT in the RIA. Total content of OT in corpus luteum was 1.9 +/- 0.5 ng (mean +/- SEM) with a content of 0.4-0.8 ng in early luteal phase, 1.0-6.2 ng in midluteal phase, and 0.4-0.7 ng in late luteal phase. OT concentrations in corpus luteum were 21.0-75.2 ng/g wet wt in early luteal phase, increasing to 34.4-602.5 ng/g in midluteal phase; and declining to 3.4-117.4 ng/g in late luteal phase. OT concentrations per mg protein in the corpus luteum were 0.05-19.6 ng with peak concentrations of 14.7-19.6 ng/mg protein on day 22. Sephadex G-25 column chromatography of the corpus luteum extract revealed a single peak for binding activity similar to that of synthetic OT on the RIA. Ovarian vein blood from the same side as the corpus luteum had a significantly higher OT concentrations of 161.2 +/- 29.7 pg/ml on days 15-24 than 16.8 +/- 3.6 pg/ml on days 25-28 (P less than 0.01) and peripheral plasma OT levels of 23.2 +/- 3.4 pg/ml (P less than 0.025). Our findings indicate that OT is present and probably produced by monkey corpus luteum with peak OT concentrations found in midluteal phase. Thus OT may play a role in primate corpus luteum function.     

Zolpidem generalization and antagonism in male and female cynomolgus monkeys trained to discriminate 1.0 or 2.0 g/kg ethanol

Background: The subtypes of γ‐aminobutyric acid (GABA)_A receptors mediating the discriminative stimulus effects of ethanol in nonhuman primates are not completely identified. The GABA_A receptor positive modulator zolpidem has high, intermediate, and low activity at receptors containing α₁, α_2/3, and α₅ subunits, respectively, and partially generalizes from ethanol in several species. The partial inverse agonist Ro15‐4513 has the greatest affinity for α_4/6‐containing receptors, higher affinity for α₅‐ and lower, but equal, affinity for α₁‐ and α_2/3‐, containing GABA_A receptors, and antagonizes the discriminative stimulus effects of ethanol. Methods: This study assessed Ro15‐4513 antagonism of the generalization of zolpidem from ethanol in male (n = 9) and female (n = 8) cynomolgus monkeys (Macaca fascicularis) trained to discriminate 1.0 g/kg (n = 10) or 2.0 g/kg (n = 7) ethanol (i.g.) from water with a 30‐minute pretreatment interval. Results: Zolpidem (0.017 to 5.6 mg/kg, i.m.) completely generalized from ethanol (≥80% of total session responses on the ethanol‐appropriate lever) for 6/7 monkeys trained to discriminate 2.0 g/kg and 4/10 monkeys trained to discriminate 1.0 g/kg ethanol. Zolpidem partially generalized from 1.0 or 2.0 g/kg ethanol in 6/7 remaining monkeys. Ro15‐4513 (0.003 to 0.30 mg/kg, i.m., 5‐minute pretreatment) shifted the zolpidem dose–response curve to the right in all monkeys showing generalization. Analysis of apparent pK_B from antagonism tests suggested that the discriminative stimulus effects of ethanol common with zolpidem are mediated by low‐affinity Ro15‐4513 binding sites. Main effects of sex and training dose indicated greater potency of Ro15‐4513 in males and in monkeys trained to discriminate 1.0 g/kg ethanol. Conclusions: Ethanol and zolpidem share similar discriminative stimulus effects most likely through GABA_A receptors that contain α₁ subunits, however, antagonism by Ro15‐4513 of zolpidem generalization from the lower training dose of ethanol (1.0 g/kg) may involve additional zolpidem‐sensitive GABA_A receptor subtypes (e.g., α_2/3 and α₅).     

Pavlovian conditioning with ethanol: sign-tracking (autoshaping), conditioned incentive, and ethanol self-administration

BACKGROUND: Conditioned incentive theories of addictive behavior propose that cues signaling a drug's reinforcing effects activate a central motivational state. Incentive motivation enhances drug-taking and drug-seeking behavior. We investigated the behavioral response to cues associated with ethanol and their interaction with operant self-administration of ethanol. METHODS: In two experiments, rats received operant training to press a lever for a sweetened ethanol solution. After operant training, the animals were given Pavlovian pairings of a brief and localized cue light with the sweetened ethanol solution (no lever present). Lever pressing for ethanol was then re-established, and the behavioral effects of the cue light were tested during an ethanol self-administration session. RESULTS: The conditioned responses resulting from pairing cue lights with the opportunity to ingest ethanol had three main effects: (1) induction of operant behavior reinforced by ethanol, (2) stimulation of ethanol-seeking behavior (magazine entries), and (3) signal-directed behavior (i.e., autoshaping, or sign-tracking). Signal-directed behavior interacted with the other two effects in a manner predicted by the location of the cue light. These conditioned responses interact with operant responding for ethanol reinforcement. CONCLUSIONS: These findings demonstrate the importance of Pavlovian conditioning effects on ethanol self-administration and are consistent with conditioned incentive theories of addictive behavior.     

Noncontingent and response-contingent intravenous ethanol attenuates the effect of naltrexone on hypothalamic-pituitary-adrenal activity in rhesus monkeys

BACKGROUND: The mechanism by which the opioid antagonist naltrexone suppresses overconsumption of ethanol is unclear. Oral ethanol consumption in humans increases hypothalamic-pituitary-adrenal (HPA) activity, and recent studies suggest that naltrexone may reduce ethanol consumption by modifying the HPA-stimulating effects of ethanol. The purpose of this study was to measure in rhesus monkeys the effects of ethanol and naltrexone, alone and in combination, on plasma levels of adrenocorticotropin hormone (ACTH). METHODS: Nine adult male and female rhesus monkeys with chronic, indwelling intravenous catheters were maintained on tethers that allowed ethanol delivery and blood sampling. In one study, the monkeys received intramuscular injections of saline or 0.32 mg/kg naltrexone followed by noncontingent intravenous bolus infusions of saline or 0.3 to 1.8 g/kg ethanol. In a second study, other monkeys were given intramuscular injections of saline or 0.01 to 0.3 mg/kg naltrexone and subsequently responded on levers to receive intravenous saline or ethanol 0.03 g/kg per injection. RESULTS: Ethanol, delivered either response contingently or noncontingently, did not produce systematic changes in ACTH plasma levels. Naltrexone alone produced increases in plasma ACTH that were attenuated by the subsequent administration of noncontingent or response-contingent ethanol. Naltrexone also produced dose-dependent reductions in intravenous ethanol self-administration. Linear regression analysis indicated that ethanol intake was negatively correlated with the plasma levels of ACTH over time. CONCLUSIONS: The route of administration may modulate ethanol's effects on HPA activity. Ethanol may attenuate naltrexone's effect on the HPA axis by impairing HPA axis sensitivity to other stimuli. The negative correlation between ethanol intake and ACTH levels supports the notion that naltrexone's effect of increasing HPA axis activity may be related to its ability to suppress ethanol consumption.     

Polymorphism of N-acetyltransferase 2 gene and the susceptibility to alcoholic liver cirrhosis: interaction with smoking

Background: Polymorphism of N‐acetyltransferase 2 gene was reported to be associated with the susceptibility of various cancers and liver diseases. However, its relationship to alcoholic liver disease is controversial and open to debate. The aim of this study was to evaluate the relationship of NAT2 genetic polymorphisms and the susceptibility to alcoholic liver cirrhosis (ALC) in Chinese, with special emphasis on the interaction of smoking. Methods: Peripheral white blood cell DNA from 148 patients with ALC, 104 patients with long‐term alcoholic drinking but without cirrhosis (ANC) and 209 healthy controls were genotyped for NAT2 using a polymerase chain reaction–restriction fragment length polymorphism method. The possible confounding factors were included for analysis. Results: There was no statistical difference in the frequency of NAT2 genotype or NAT2 acetylator status among the 3 groups. However, among the chronic alcoholic drinkers, the rapid acetylators with smoking habits had higher percentage of ALC than those without smoking habit (18.9% vs. 9.5%, p = 0.002). The adjusted odds ratio for rapid acetylator smoker to have ALC was 3.45 (95% CI = 1.53 to 7.76, p = 0.003). Conclusions: The genetic factor, NAT2 polymorphism, may interact with environmental factor, smoking, to confer different susceptibilities to ALC. NAT2 rapid acetylators with smoking habit may increase the risk of ALC in Chinese.     

Chronic Ethanol Feeding Alters Hepatocyte Memory Which is not Altered by Acute Feeding

Background:  Gene expression changes in the liver after acute binge drinking may differ from the changes seen in chronic ethanol feeding in the rat. The changes in gene expression after chronic ethanol feeding may sensitize the liver to alcohol-induced liver damage, which is not seen after acute binge drinking.
Methods:  To test this hypothesis, gene microarray analysis was performed on the livers of rats ( n  = 3) fed an acute binge dose of ethanol (6 g/kg body wt) and killed at 3 and 12 hours after ethanol by gavage. The gene microarrays were compared with those made on the liver of rats from a previous study, in which the rats were fed ethanol by intragastric tube for 1 month (36% of calories derived from ethanol).
Results:  Microarray analysis data varied between the acute and chronic models in several important respects. Growth factors increased mainly in the chronic alcohol fed rat. Changes in enzymes involved in oxidative stress were noted only with chronic ethanol feeding. Gene expression of fat metabolism was increased only with chronic ethanol feeding. Most importantly, epigenetic related enzymes and acetylation and methylation of histones changed only after chronic ethanol feeding.
Conclusions:  The results support the concept that chronic ethanol ingestion induces altered gene expression as a result of changes in epigenetic mechanisms, where acetylation and methylation of histones were altered.     

Circulating Neutrophils and Liver Injury in Rat Models of Experimental Alcoholic Liver Disease

The present study examined the relationship between circulating neutrophils and liver injury in two widely used rat models of chronic ethanol administration. Hematological alterations, liver histopathology, and biochemical indices of liver injury were assessed in rats receiving chronic ethanol by oral liquid diet feeding (Lieber-DeCarli method) or by continuous intragastric infusion (Tsukamoto-French method). Oral administration of ethanol did not affect circulating neutrophil counts, but resulted in minimal liver injury characterized by elevated serum alanine aminotransferase (79%), increased liver mass (15%), and moderate steatosis. In contrast, rats receiving ethanol by continuous intragastric infusion showed an ∼ 2-fold increase in circulating neutrophils, and a moderate degree of liver injury, indicated by a 169% elevation of serum alanine aminotransferase and a 2-fold increase in liver mass. Liver biopsies from these rats showed severe steatosis and scattered necrotic hepatocytes, and some neutrophil infiltrates. To determine whether an increase in the number of circulating neutrophils could potentiate liver injury induced by oral ethanol feeding, rats were treated with human recombinant granulocyte colony-stimulating factor at a dose of 100 μg/kg/day (sc) for 4 days. Treatment with granulocyte colony-stimulating factor resulted in a 6- to 9-fold increase in circulating neutrophil counts. Nevertheless, this change did not enhance the minor degree of ethanol-induced liver injury in this model. Our results indicate that, whereas neutrophil leukocytosis accompanies more severe manifestations of ethanol hepatotoxicity in rats, this condition per se does not directly induce or exacerbate ethanol-induced liver injury.     

Alcohol Mediates Increases in Hepatic and Serum Nonheme Iron Stores in a Rat Model for Alcohol-Induced Liver Injury

The notion that prolonged ethanol consumption promotes hepatocellular damage through interactions with iron was evaluated in rats fed ethanol with or without supplemental dietary carbonyl iron. The individual and combined pro-oxidant potential of these agents was evaluated in terms of their ability to perturb iron homeostasis and initiate hepatocellular injury. Sprague-Dawely rats received a high fat liquid diet for 8 weeks supplemented with 35% ethanol-derived calories (Alcohol group), 0.02 to 0.04% (w/v) carbonyl iron (Iron group), ethanol plus carbonyl iron (Alcohol + Iron group), or a diet containing carbohydrate-derived isocaloric calories (Control group). Hepatic and serum nonheme iron stores were significantly elevated (p < 0.05) in all treatment groups, compared with the Controls. Catalytically active low-molecular weight iron was detected in rats consuming alcohol and was markedly elevated (p < 0.05) in rats ingesting iron alone or iron in combination with alcohol. Elevations in serum ALT indicated significant hepatocellular injury in rats ingesting only alcohol, but was most prominent in the rats consuming ethanol in combination with iron (p < 0.05). Significant hepatic fatty infiltration, increased hydroxyproline content, and perturbations in reduced glutathione were also observed in the Alcohol and Iron treatment groups. Histochemical assessment of hepatic iron sequestration revealed that alcohol feeding resulted in deposition of ferric iron in the centrilobular area of the liver lobule. This unique alcohol-mediated iron deposition was histologically graded above Control group and was observed in both hepatocytes and Kupffer cells. Data presented herein suggest that alcohol alone or in combination with iron results in rather specific lobular patterns of hepatic iron deposition relevant to iron overload observed in human alcoholics. Furthermore, data suggest that alcohol- and iron-initiated prefibrotic events occur before extensive hepatocellular necrosis.     

Developmental changes in testicular inhibin and androgen-binding protein during sexual maturation in the cynomolgus monkey, Macaca fascicularis

Inhibin was measured by RIA in testicular extracts and plasma of cynomolgus monkeys during four stages of sexual maturation. Immunoactive inhibin levels were compared to those of another Sertoli cell secreted protein, androgen-binding protein (ABP). ABP steroid-binding (bioactive) activity was measured in testes and epididymal segments using the radiolabeled ligand [3H]dihydrotestosterone (DHT). Testicular immunoreactive inhibin concentrations were maximal in late prepubertal monkeys, 2.5-3.5 yr old, while the total testicular content of inhibin progressively increased with age into adulthood. Bioactive testicular ABP concentrations were maximal during the pubertal period of the cynomolgus monkey (3.5-4.0 yr old), while the total ABP content of the testes also increased with sexual maturation. Mean (+/- SE) plasma concentrations of inhibin and testosterone (T) in adults, 6-8 yr old (17.72 +/- 3.5 microliters inhibin equivalents/ml and 7.07 +/- 2.45 ng/ml T, respectively), were significantly higher (P less than 0.05 and P less than 0.001, respectively) than those in early prepubertal, juvenile monkeys, aged 1.5-2.5 yr (5.85 +/- 2.1 microliters inhibin equivalents/ml and 0.27 +/- 0.02 ng/ml T). The increased plasma levels of inhibin and T in adults were associated, respectively, with the increased inhibin and androgen contents of the testes in these same animals. The developmental changes in testicular steady state mRNA concentrations for the inhibin alpha-, beta A-, and beta B-subunits as well as ABP were examined during sexual maturation by Northern blot analysis using heterologous human cDNA probes. Densitometric analysis of the autoradiograms revealed that the inhibin alpha-subunit mRNA concentrations were higher than those of inhibin-beta A and -beta B and ABP mRNA during all stages of pubertal development. Although the relative concentrations of each inhibin subunit mRNA were decreased in the adult animals relative to those in the juvenile monkeys, the total amount of steady state mRNA for the subunits was greater than that in the immature animals. A similar situation existed for the ABP mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Effects of Ethanol Initiation Procedures on Ethanol Reinforced Behavior in the Alcohol-Preferring Rat

Rats from the alcohol preferring (P) line developed at Indiana University were initiated to self-administer ethanol orally without food or water restriction using either a sucrose-fading or a secondary-conditioning procedure. Following initiation, they were tested under a variety of operant conditions to examine the ability of ethanol to reinforce lever pressing behavior. Regardless of initiation procedure, the animals maintained lever pressing behavior with ethanol reinforcement, even at ethanol concentrations as high as 40% (v/v). Slightly higher daily session intakes (g/kg) were found at the higher ethanol concentrations following the secondary-conditioning initiation procedure compared with the sucrose-fading technique. When both ethanol and water were concurrently available, the rats showed a high preference for ethanol reinforcement. When varying concentrations of sucrose were substituted for water, the amount of ethanol ingested decreased as the concentration of the alternative sucrose increased. However, if the response requirement for the sucrose was substantially greater than that for ethanol, the rats shifted their responding to the lever associated with ethanol presentation. The results are discussed in relation to prior work using similar procedures with Long-Evans non-selected rats and with the alcohol non-preferring (NP) rat line.