Ethanol self-administration and alterations in the livers of the cynomolgus monkey, Macaca fascicularis

Background: Most of the studies of alcoholic liver disease use models in which animals undergo involuntary administration of high amounts of ethanol and consume diets that are often high in polyunsaturated fatty acids. The objectives of this study were (1) to evaluate whether cynomolgus monkeys (Macaca fascicularis) drinking ethanol voluntarily and consuming a diet with moderate amounts of lipid would demonstrate any indices of alcoholic liver disease past the fatty liver stage and (2) to determine whether these alterations were accompanied by oxidative stress. Methods: Six adult male and 6 adult female cynomolgus monkeys were allowed to consume ethanol voluntarily for 18 to 19 months. Additional monkeys were maintained on the same consumption protocol, but were not provided with ethanol. During the course of the study, liver biopsy samples were monitored for lipid deposition and inflammation, serum for levels of liver enzymes, and urine for concentrations of the isoprostane (IsoP) metabolite, 2,3‐dinor‐5,6‐dihydro‐15‐F_2t‐IsoP, a biomarker for oxidative stress. Liver mitochondria were monitored for respiratory control and liver for concentrations of neutral lipids, adenine nucleotides, esterified F₂ isoprostanes, oxidized proteins, 4‐hydroxynonenal (HNE)‐protein adducts, and protein levels of cytochrome P‐450 2E1 and 3A4. Results: Ethanol consumption ranged from 0.9 to 4.05 g/kg/d over the period of the study. Serum levels of aspartate amino transferase were elevated in heavy‐consuming animals compared with those in ethanol‐naïve or moderate drinkers. Many of the ethanol consumers developed fatty liver and most showed loci of inflammation. Both hepatic energy charge and phosphorylation potential were decreased and NADH‐linked respiration was slightly, but significantly depressed in coupled mitochondria as a result of heavy ethanol consumption. The urinary concentrations of 2,3‐dinor‐5,6‐dihydro‐15‐F_2t‐IsoP increased as high as 33‐fold over that observed in ethanol‐abstinent animals. Liver cytochrome P‐450 2E1 concentrations increased in ethanol consumers, but there were no ethanol‐elicited increases in hepatic concentrations of the esterified F₂ isoprostanes, oxidized proteins, or HNE‐protein adducts. Conclusion: Our studies show that cynomolgus monkeys undergoing voluntary ethanol consumption for 1.5 years exhibit many of the features observed in the early stages of human alcoholic liver disease. Ethanol‐elicited fatty liver, inflammation, and elevated serum aspartate amino transferase were evident with a diet that contained modest amounts of polyunsaturated lipids. The dramatic increases in urinary IsoP demonstrated that the animals were being subjected to significant oxidative stress that correlated with their level of ethanol consumption.     

维生素E对小鼠慢性乙醇性肝损害的保护作用

目的 探讨乙醇性肝损害的发病机制,并研究维生素E对乙醇性肝损害的保护作用。方法 昆明种小鼠30只,分为三组：正常对照组（自由饮水,不做其他处理）、乙醇组（自由饮用2．5％乙醇8周）、乙醇＋维生素E组（自由饮用2．5％乙醇8周,同时经口给予维生素E200mg/kg,每天1次,共8周）。分别测定血清丙氨酸转氨酶、肝匀浆蛋白含量、肝匀浆丙二酰乙醛含量及肝匀浆超氧化物歧化酶、过氧化氢酶、谷胱甘肽、谷胱甘肽－S－转移酶、谷胱甘肽还原酶、谷胱甘肽过氧化酶的活性。结果 摄入乙醇8周后多种抗氧化酶及抗氧化物发生改变,维生素E可拮抗乙醇的上述作用,使指标的改变发生逆转。结论 维生素E对慢性乙醇性肝损害的保护作用。     

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.     

Subjective and objective responses to ethanol in moderate/heavy and light social drinkers

BACKGROUND: Individuals who drink heavily are at an increased risk for adverse consequences of drinking and progression of their drinking habits to abuse or dependence. Therefore, it is important to delineate factors associated with their heavy drinking. METHODS: We examined individual differences in subjective and objective responses to ethanol associated with level of consumption by reanalyzing data from the nine heaviest and nine lightest social drinkers from each of two independently collected subject samples: Holdstock and de Wit (1998) and King et al. (1997). The light drinkers in both samples consumed five or less alcoholic drinks per week, whereas the moderate/heavy drinkers consumed eight or more drinks per week with frequent binge episodes. Acute subjective and objective responses to ethanol (0.6 or 0.8 g/kg) or placebo were compared in the two groups at baseline and during rising and falling blood alcohol concentrations. RESULTS: Moderate/heavy drinkers reported greater stimulant-like and fewer sedative-like and aversive subjective effects after ethanol than did lighter drinkers. These differences occurred in the absence of any group differences in breath alcohol levels, performance effects, or neuroendocrine changes or in overall reports of feeling any drug effects. CONCLUSIONS: These data indicate that habitual moderate/heavy ethanol use was associated with greater stimulant-like effects after an acute dose of alcohol. This finding is consistent with the idea (Newlin and Thomson, 1990, 1999) that individuals who experience greater stimulant-like effects during the ascending limb and lesser sedative-like effects on the descending limb of the blood alcohol concentration curve may be at greater risk for developing ethanol use disorders. Although we cannot determine the causality of this association, sensitivity to the stimulant effects of ethanol may play an important role in the continuation of heavy ethanol use and the increased risk of negative consequences from this use.     

Epigenetic effects of ethanol on liver and gastrointestinal injury

INTRODUCTIONEthanol actions are diverse and fascinatingly complex. Chronic ethanol causes injury to almost all organ systems including liver and gastrointestine (GI)[1] and has serious medical and public health implications[2]. Alcohol increases the risk …     

乙醇预处理诱导增强肝脏对缺血再灌流的耐受性

目的 探讨胃饲乙醇预处理对肝脏缺血再灌流损伤的影响，确定适当预处理方案，并作初步评价。方法 雄性成年Wistar大鼠36只，随机分6组，胃饲乙醇浓度40％，A组8g／kg、B组7g／kg、C组6g／kg、D组5g／kg、E组4g／kg、正常组0g／kg；以中毒症状及肝组织病理为指标，判定大鼠急性乙醇胃饲中毒剂量；选定剂量范围继续本实验。大鼠78只，随机分为4组：正常对照组、胃饲乙醇组、缺血组(IR)、胃饲乙醇预处理组(EP)；采用尾叶转流下的肝缺血模型，于再灌流3、6、12、24h留取标本；采用正交设计，以乙醇浓度、剂量、胃饲时机为因素，分设3个水平，大鼠54只，肝缺血90min，于再灌流24h采样检测。结果 急性胃饲乙醇≤5g／kg预处理后，动物中毒症状轻，无死亡；乙醇预处理可以在一定程度上减轻肝脏90min的缺血再灌流损伤；在A1B1C3预处理模式下，即40％乙醇5g／kg胃饲后24h行肝缺血手术对大鼠肝脏缺血再灌流损伤的保护作用最强。结论 适当剂量的乙醇胃饲预处理是一种安全的预处理措施，有望成为增强肝脏对缺血再灌流损伤耐受性的一种较好的预处理方式。     

The effects of ethanol consumption on vasculogenesis potential in nonhuman primates

Background:  Vasculogenesis is essential to the preservation and repair of damaged or diseased vessels. Alcohol is the most commonly abused drug among young adults, but its effects on vessel growth and repair are unknown. The basis of vascular repair is endothelial progenitor cell (EPC) recruitment to assist in the formation of new vascular network (vasculogenesis). Therefore, the objective of this study was to measure the effects of ethanol consumption on the production, mobilization and vasculogenesis potential EPCs in nonhuman primates.
Methods:  Four to five year-old (young adult) male rhesus monkeys consumed monkey chow and water (Control, n  = 7), or chow and water + ethanol (Alcohol, 2.45 g/d, n  = 7) for 12 months. Peripheral blood (PB) and bone marrow (BM) samples were collected for fluorescence-activated cell-sorting analysis of cell surface antigens (CD45, CD31, CD44, CD133, VEGF-R2 – or KDR); and for capillary formation on Matrigel-coated plates.
Results:  There were greater numbers of nonhematopoeitic stromal cells (CD45−) and putative mesenchymal progenitor cells (CD45−/CD44+) in the PB and BM of Alcohol versus Control monkeys ( p  < 0.05). Additionally, there were greater numbers of EPCs (CD45−/CD133+/KDR+) in the BM and PB of Alcohol versus Control monkeys ( p  < 0.05). However, the EPCs of Alcohol monkeys were less likely to form capillaries on matrigel-coated plates than Control monkeys ( p  < 0.05).
Conclusions:  Ethanol consumption in monkeys markedly increased the production and mobilization of EPCs, but decreased their ability to form capillaries. The pathophysiologic consequences of such effects are unclear, but may represent an ethanol-induced chronic stress on the BM, resulting in EPC.     

Myocardial effects of ethanol consumption in the rat with streptozotocin-induced diabetes

BACKGROUND: Rats with streptozotocin (STZ)-induced diabetes exhibit alterations in cardiac function, ventricular remodeling, and changes in cell signaling, which includes protein kinase C (PKC) isoforms. Moderate consumption of ethanol has a beneficial effect on cardiovascular outcomes in the general population, an effect that has recently been found to extend to patients with diabetes mellitus. We studied the effect of low-dose ethanol consumption on cardiac function, geometry, and PKC isoforms in the rat with STZ-induced diabetes. METHODS: Four groups of rats were studied over 8 to 10 weeks: control, STZ-induced diabetes, 12% (v/v) ethanol consumption, and STZ-induced diabetes plus 4% (v/v) ethanol consumption. Invasive hemodynamic measurements were performed; myocardial tissue was obtained for analysis for total PKC and cytosolic and membrane protein content of PKC-alpha, PKC-delta, and PKC-epsilon, and two-dimensional and M-mode echocardiograms were obtained. RESULTS: Compared with rats with diabetes alone, consumption of 4% ethanol prevented the decrease in left ventricular dP/dt seen with diabetes alone, as well as the increase in left ventricular internal dimension. Up-regulation of PKC-alpha, -delta, and -epsilon occurring in the diabetic animals was also prevented by ethanol consumption, whereas ethanol alone had no effect on PKC isoform pattern. CONCLUSIONS: These data suggest that STZ-induced cardiac remodeling and dysfunction are associated with increases in PKC activity, particularly PKC-alpha, -delta, and -epsilon, and that consumption of ethanol can prevent these changes.     

The effects of gonadectomy on age- and sex-typical patterns of ethanol consumption in Sprague-Dawley rats

Background: Ethanol intake levels characteristic of adult males and females emerge postpubertally. The present set of experiments examined the consequences of prepubertal and adult gonadectomies to explore whether the presence of gonadal hormones at puberty exerts organizational influences and/or plays an activational role in age‐ and sex‐typical patterns of ethanol consumption. Methods: Male and female Sprague–Dawley rats were gonadectomized (GX), received sham gonadectomy (SH), or were left nonmanipulated (NM) at 1 of 2 ages, either prepubertally on postnatal day (P) 23 (early) or postpubertally in adulthood on P70 (late). Early surgery animals were tested for ethanol consumption either during adolescence (P28 to 39) or in adulthood at the same age that late surgery animals were tested (P75 to 86). Voluntary ethanol consumption was indexed using a 2‐hour limited‐access paradigm, with access to 2 bottles: one containing water and the other a sweetened ethanol solution. Results: Age of GX did not impact patterns of ethanol consumption. Removal of testicular hormones in males, regardless of age of removal, elevated consumption levels in adulthood to female‐typical levels. Ovariectomy did not have notable effects on ethanol drinking in females. Ethanol intake and preference of early SH males were significantly greater than those of both late SH and NM males. Removal of the gonads prior to puberty did not influence ethanol drinking or preference during adolescence in either males or females. Conclusions: These results suggest that testicular hormones play an activational role in lowering ethanol intake and preference of adult male rats. Pubertal hormones, in contrast, were found to exert little influence on ethanol drinking or preference during adolescence, although the effect of surgical manipulation itself during development was found to exert a long‐lasting facilitatory effect on ethanol consumption in adulthood.     

Lack of effects of recombinant human GH on spermatogenesis in the adult cynomolgus monkey (Macaca fascicularis)

OBJECTIVE: The effects on male reproductive parameters after 1 year of treatment with recombinant human GH to the cynomolgus monkey were investigated. DESIGN: Twenty-four male cynomolgus monkeys were given daily subcutaneous doses of 0 (vehicle) (n=7), 0.4 (n=5), 2.0 (n=5) and 10.0 (n=7) IU/kg bodyweight for 52 weeks. At completion of the treatment period two control and two high-dose animals were left for a 12-week treatment-free period. METHODS: Before and during the treatment period and during the recovery period, sperm analyses, testicular volume measurements and hormone analyses of prolactin (PRL), LH, FSH, testosterone and IGF-I in serum, and analysis of serum antibodies against human GH were performed. Testicular morphology was monitored by biopsies, predose and on day 15 of the study, and with light microscopy on organ samples collected at time of death, at the end of the treatment, and during recovery periods respectively. RESULTS: Of all studied parameters, alterations were observed only in serum levels of IGF-I and PRL. IGF-I showed a dose-dependent increase throughout the treatment, with a normalisation during the treatment-free period. PRL decreased significantly in animals given 10.0IU/kg per day from week 14 of treatment and throughout the study but with a normalisation upon cessation of treatment. Spermatogenesis, as judged from semen analysis, testicular volume measurements and testicular morphology was not affected. CONCLUSION: This controlled preclinical study demonstrates that high doses of human GH do not alter male reproductive parameters in a non-human primate model.     

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.     

IgAs against acetaldehyde-modified red cell protein as a marker of ethanol consumption in male alcoholic subjects, moderate drinkers, and abstainers

BACKGROUND: Alcohol abuse has been shown to result in the production of antibodies against acetaldehyde-modified epitopes in proteins. However, as yet, only limited information has been available on the clinical usefulness of such responses as markers of hazardous drinking. METHODS: We developed an ELISA to measure specific IgAs against acetaldehyde-protein adducts. This method was evaluated in cross-sectional and follow-up studies on male heavy drinkers with a current ethanol consumption of 40 to 540 g/d (n=40), moderate drinkers consuming 1 to 40 g/d (n=25), and abstainers (n=16). The clinical assessments included detailed interviews on the amounts and patterns of ethanol consumption and various biochemical markers of alcohol abuse and liver function. RESULTS: The mean antiadduct IgAs (198+/-28 U/L) in the alcohol abusers were significantly higher than those in the moderate drinkers (58+/-11 U/L, p<0.001) or abstainers (28+/-8 U/L, p<0.001). The values of moderate drinkers were also higher than those in abstainers (p<0.05). The amount of ethanol consumed during the period of 1 month preceding blood sampling correlated strongly with antiadduct IgAs (r=0.67, p<0.001). The sensitivity (73%) and specificity (94%) of this marker were found to exceed those of the conventional laboratory markers of alcohol abuse in comparisons contrasting heavy drinkers with abstainers although not in comparisons contrasting heavy drinkers with moderate drinkers. During abstinence, antiadduct IgAs disappeared with a mean rate of 3% per day. In additional analyses of possible marker combinations, antiadduct IgAs, together with CDT, were found to provide the highest sensitivity and specificity. CONCLUSIONS: Measurements of antiadduct IgAs may provide a new clinically useful marker of alcohol abuse, providing a close relationship between marker levels and the actual amounts of recent ethanol ingestion.     

微导管碘油阿霉素乳剂联合无水乙醇肝段栓塞术治疗小肝癌

探讨经微导管碘油阿霉素乳剂联合无水乙醇肝段栓塞治疗小肝癌的可行性及临床疗效。联合肝段超选择和非超选择插管组各22例,前者以微导管“嵌入”肿瘤血管,后者以“低压流控法”进行栓塞化疗,比较两组疗效、副作用并做应用价值分析。结果表明;经微导管碘油阿霉素乳剂联合无水乙醇肝段栓塞与非超选择插管治疗组相比,疗效更佳,应用价值更加合理。本治疗方法操作简单方便、超选择成功率高、副反应少,可以明显提高存活率。具有较重要的临床应用价值。     

非酒精性脂肪性肝病大鼠肝X受体α基因表达变化及意义

目的研究NAFLD大鼠肝X受体α(LXRα)基因表达变化及意义。方法建立高脂饮食诱导NAFLD大鼠模型后,采用RT-RCR和Western blot法动态观察NAFLD大鼠肝组织中LXRα表达变化。结果4周时模型组大鼠血清游离脂肪酸含量达(0.33±0.03)mmol/L,对照组为(0.24±0.03)mmol/L,差异有统计学意义(P<0.05),随喂养时间延长逐渐升高,12周时模型组大鼠血清游离脂肪酸含量达(0.61±0.06)mmol/L,对照组为(0.25±0.01)mmol/L,差异有统计学意义(P<0.01)。血清ALT和AST含量在8周时模型组分别为75.8 U/L和138.9 U/L,对照组分别为54.8 U/L和81.4 U/L,差异有统计学意义(P<0.01),12周时模型组分别为102.3 U/L和179.1 U/L,对照组分别为54.3 U/L和79.2 U/L,差异有统计学意义(P<0.01)。2周时模型组大鼠肝组织中LXRα基因表达相对含量为0.62,对照组为0.33,差异有统计学意义(P<0.01),随着高脂饮食喂养时间的延长表达进一步增强,12周时模型组大鼠高达1.31,对照组为0.34,差异有统计学意义(P<0.01)。模型组大鼠肝组织中LXRα蛋白表达与基因表达趋势相同,与脂肪肝进展程度一致。结论LXRα基因表达变化与NAFLD的形成密切相关。     

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.     

Serum sialic acid as a marker of alcohol consumption: effect of liver disease and heavy drinking

BACKGROUND: Serum sialic acid (SA) has been suggested as a new marker of alcohol consumption. There are, however, only a few studies on the clinical value of such measurements. The relationship between serum SA and liver disease is also unknown. METHODS: We determined serum SA concentrations in a sample of 51 alcoholics and 20 healthy controls by using high-performance liquid chromatography with an anion-exchange column and pulsed amperometric detection. The alcoholic sample included 32 patients with liver disease, the severity of which was assessed by previously established combined clinical, laboratory, and morphological indices. In addition, there were 19 heavy drinkers without significant liver disease despite a well documented history of excessive alcohol consumption. RESULTS: The (mean +/- SD) SA concentrations (1.449 +/- 0.3019 mmol/liter) were significantly higher in the alcoholics than in the healthy controls (1.154 +/- 0.1702 mmol/liter). With the optimal cutoff limit for serum SA (1.425 mmol/liter), a specificity of 1.00 and sensitivity of 0.51 were obtained. The diagnostic accuracy of serum SA according to receiver operating characteristic analysis was good, with the area under the curve being 0.805 (0.052). Unlike the traditional serum markers of alcohol consumption (gamma-glutamyl transferase, carbohydrate-deficient transferrin, and aspartate amino transferase), serum SA was not found to be different between the alcoholics with or without liver disease. CONCLUSIONS: Our study suggests that serum SA is a sensitive marker of excessive alcohol consumption. Such measurements may also prove to be of value in conditions in which the results of the traditional markers reflect the severity of liver disease rather than alcohol consumption.