EFFECTS OF ETHANOL AND ACETALDEHYDE ON THE ENZYMES OF GLYCOGEN METABOLISM

This report concerns a study of the effect of ethanol and acetaldehydeon the regulatory enzymes of glycogen metabolism. It demonstratesan inhibition of glycogen phosphorylase kinase at pH 6.8 ata very low concentration of ethanol. There was no effect ofacetaldehyde on this enzyme. Neither ethanol not acetaldehydehas been shown to have any effect on glycogen synthase, glycogenphosphorylase, protein phosphatase or independent and cAMP-dependentprotein kinases. This inhibition could explain the high concentrationof glycogen in the muscle tissue of chronic alcoholics thatis found when ethanol is present in skeletal muscle.     

ACUTE EFFECTS OF ETHANOL ON PROTEIN SYNTHESIS IN THE RAT

The effect of ethanol, administered acutely (75 mmol kg^–1),on protein synthesis in the whole body and in the tissues ofthe rat was investigated. Ethanol decreased whole-body proteinsynthesis by 41% and also that in individual tissues (liver:60%; muscle: 75%; heart: 45%; kidney: 59%; spleen: 73%; plasmaprotein: 44%; lung: 64%). These data indicate a generalisedeffect upon protein synthesis and are discussed in relationto the possible mode of action of ethanol.     

COMPARISON OF THE ACUTE EFFECTS OF ETHANOL ON LIVER AND SKELETAL MUSCLE PROTEIN SYNTHESIS IN THE RAT

(1) The acute effects of ethanol on protein synthesis by liverand skeletal muscle were investigated in young (95–100g) rats. Rats were injected intraperitoneally with ethanol,75 mmol/kg body wt; controls were injected with isovolumetric0.15 M NaCl. After 140 min rates of protein synthesis were measuredby injection of a large dose of L[4³H]phenylalanine and at 150min rats were killed. (2) Fractional rates of protein synthesisin control animals were approximately four to five times greaterin liver than muscle. Absolute rates were, however, comparablein liver and skeletal muscle. Ethanol reduced the fractionalrate of liver protein synthesis by 5–20%; the responsefor muscle was relatively greater (25–30%). The decreasein the amount of protein synthesised by muscle was also greaterthan that by liver. (3) After 150 min, plasma gamma-glutamyltransferase, alanine aminotransferase, alkaline phosphatase,lactate dehydro-genase and creatine kinase activities were alldecreased by 25–60%. Aspartate aminotransferase activitywas increased by 42%, though this was not statistically significant.(4) Increased plasma glucose and triglycerides in ethanol-dosedrats indicated that limitations in substrate supply were notmediating factors in reducing protein synthesis. Ethanol wasalso able to exert its effects in the presence of elevated insulinlevels. A direct effect of ethanol, or its metabolites, on proteinsynthesis, is therefore implied.     

EFFECT OF ACUTE ETHANOL DOSAGE ON NUCLEOTIDE LEVELS IN THE RAT JEJUNUM: RELATIONSHIP TO PROTEIN SYNTHESIS

In previous studies we have shown that ethanol-fed rats generateantibodies reactive with proteins modified by acetaldehyde invitro and that their livers contain proteins modified by acetaldehyde.In this study we demonstrate that the antibodies from theseanimals react with the modified proteins found in their livers.Furthermore, when the antibodies reactive with specific proteinswere isolated, they were found to react with all of the modifiedproteins detected by the whole serum. This suggests that allof the proteins modified by acetaldehyde in vivo carry the sameor similar epitope(s). In addition, antibodies from alcoholicsand a rabbit immunised with proteins modified by acetaldehydein vitro also reacted with the liver cytosolic proteins fromethanol-fed rats. Therefore it appears that similar epitopesare generated in alcoholics as a result of ethanol misuse, inrat liver due to prolonged ethanol feeding and by the in vitromodification procedure used to produce the immunogen for therabbit.     

EFFECTS OF DIETS DECREASING ETHANOL CONSUMPTION ON ACETALDEHYDE METABOLISM IN UChA AND UChB RATS

It has previously been reported that the introduction of a newissue (D1) of a stock diet (D0) caused a significant decreasein voluntary ethanol consumption in rats of our UChA (low ethanolconsumer) and UChB (high ethanol consumer) strains, and that,after changing to a diet lacking in animal products (D3), ethanolconsumption reached the previous level attained in UChB ratsand exhibited a bimodal distribution in UChA rats in such away that about one-third of these consumed more than 2 ml ofa 10% (v/v) ethanol solution per 100 g of body weight, as didUChB rats. When UChA rats exhibiting high ethanol consumptionand also UChB rats were fed on the D3 diet with a brewer's yeastsupplement, a significant decrease in ethanol intake was observed.Since significant correlations of voluntary ethanol intake toblood acetaldehyde levels, and to hepatic and brain aldehydedehydrogenase (AldDH) activities, have been reported, the effectson these parameters of diet D1 and of supplements of brewer'syeast or disulfiram were studied. Results showed that rats ofboth strains fed on a D1 diet exhibited a significant increasein blood acetaldehyde level after ethanol administration, concomitantwith an inhibition of hepatic and brain AldDH activities anda significant decrease in ethanol intake. Thus, this effectcould be related to a disulfiram-hke activity. The decreasein ethanol intake induced by brewer's yeast was not accompaniedby changes of blood acetaldehyde level after ethanol, nor byinhibition of hepatic AldDH activity in either strain. Nevertheless,a significant inhibition of brain AldDH activity in UChB, butnot in UChA, rats was observed. Thus, the effect of brewer'syeast on ethanol consumption could not be ascribed to a disulfiram-likeactivity of this supplement.the beginning of pregnancy, couldbe toxic for the human placenta and development of the fetus.     

THE EFFECTS OF THE NITRIC OXIDE SYNTHASE INHIBITOR 7-NITROINDAZOLE ON ETHANOL PHARMACOKINETICS IN RATS AFTER ACUTE AND CHRONIC ETHANOL ADMINISTRATION

The aim of this work was to study the effects of the nitricoxide synthase (NOS) inhibitors 7-n,troindazole (7-NI) and N^G-nitro-L-arginine(L-NOARG) on the effects and pharmacokinetics of ethanol inrats. Ethanol at a dose of 4 g/kg, i.p. induced sleep in rats(sleep time: 117.2 ± 30.7 min). Administration of theNOS inhibitors 7-NI (20 mg/kg, i.p.) and L-NOARG (20 mg/kg,i.p.) 30 min before ethanol significantly increased the durationof ethanol-induced sleep. L-NOARG also significantly increasedthe toxicity of ethanol as evidenced by increased post-experimentallethality Ethanol at a dose of 2 g/kg (i.p.) did not inducesleep in vehicle-treated rats; however, the combined administrationof ethanol (2 g/kg) and 7-NI at doses of 40, 80, and 120 mg/kgcaused sleep, for 49.4 ± 3.7, 204.0 ± 13.3, and447.5 ± 62.8 min, respectively. L-NOARG (20 mg/kg) hadno effect on ethanol concentrations in blood after acute ethanoladministration (4 g/kg). 7-NI in lower doses (20 and 40 mg/kg)had no effect and in higher doses (80 and 120 mg/kg) significantlyslowed ethanol clearance during the 12 h after ethanol administration.The effect of 7-NI (20 mg/kg) on ethanol pharmacokinetics afterchronic ethanol administration (inhalation for 18 days) wasalso studied. The administration of 7-NI immediately after theend of ethanol exposure had a pronounced effect on ethanol pharmacokinetics;in 7-NI-treated rats the fall in ethanol concentrations wassignificantly slower as compared with vehicle-treated rats.In 7-NI- treated rats, blood-ethanol levels were higher at 3,6, 9, and 12 h after the end of ethanol exposure.     

INFLUENCE OF CGS 8216 ON SOME ACUTE EFFECTS OF ETHANOL

The central pharmacological effects of ethanol are well knownand resemble those of the benzodiazepines (BZD). In additionBZD may interact with ethanol, resulting in enhanced cerebraldepression. Ro 15-4513, BZD inverse agonist, potentially antagonizesa lot of effects of ethanol but not all. In our study, anotherBZD inverse agonist, CGS 8216, reverses the hypnotic effectof ethanol and hypoactivity in mice and rats. CGS 8216 increasedalso ethanol's locomotor stimulation in mice. This supportsthe hypothesis that some effects of ethanol are mediated bythe GABA-BZD-chloride channel receptor complex. Our behaviouralexperiments described in this report suggest that CGS 8216,like Ro 15-4513, may act on the alpha-6 subunit of this receptorcomplex.     

THE KINETICS OF THE ADDITION OF GLUCOSE AND ACETALDEHYDE TO HISTONE PROTEINS

The time-course of the reaction of H1 and total histone withglucose, acetaldehyde or both has been studied using the NBTreduction test and fluorescence. With both methods, purifiedH1 histone gave higher absorbance with acetaldehyde than witha 1:1 combination of glucose and acetaldehyde. For total histone,the opposite was found; a 1:1 combination of the above two aldehydeshad the higher absorbance. As an explanation, the possibilityof different reactivity of the amino groups with glucose andacetaldehyde is proposed. A possible simultaneous interactionbetween glucose, acetaldehyde and serum protein, mainly albumin,may alter the results of the diagnostic protein glycation methods,e.g. of the fructosamine test; and, therefore, also the monitoringof diabetes.     

EXCRETION OF MALONDIALDEHYDE, FORMALDEHYDE, ACETALDEHYDE AND ACETONE IN THE URINE OF RATS FOLLOWING ACUTE AND CHRONIC ADMINISTRATION OF ETHANOL

Recent studies have shown that xenobiotics which induce oxidativeStress result in an increased production and excretion of acetaldehyde(ACT), formaldehyde (FA), acetone (ACON) and malondialdehyde(MDA) in the urine of rats. We have therefore examined the effectof acute and chronic ethanol administration on the excretionof these four lipid metabolites in female Sprague-Dawley rats.Urine samples were collected over dry ice for 6 hr time periods.Aliquots of urine were derivatized with 2,4-dinitrophenylhydrazineHCl, and extracted with n-pentane. High pressure liquid chromatography(HPLC) was used to quantitate and the hydrazones of the fourlipid metabolite products. Following a single, oral, acute doseof 5 g ethanol/kg, urinary excretion of ACT increased approximately5.8-fold from 6 to 12 hr post-treatment, and decreased thereafter.FA excretion decreased by approximately 50% from 0 to 12 hr,returned to control values in the 18–24 hr urine samples,and was 1.3-fold greater than control values at 42–48hr. ACON increased 3.1-fold over control values from 0 to 30hr and remained elevated throughout the remaining 18 hr of thestudy. The excretion of MDA increased approximately 1.5-foldfrom 18 to 36 hr, then remained constant through the 48 hr timepoint. In a separate series of experiments, a chronic oral doseof 0.5 g ethanol/kg was administered to rats for 10 consecutivedays and the urinary excretion of the lipid metabolites MDA,FA, ACT and ACON was examined for 11 days, beginning with thefirst day of ethanol administration. During the chronic administrationof ethanol, a significant increase in the urinary excretionof ACT began on day 4. An increase in ACON excretion was firstobserved on day 6, and increases in MDA and FAexcretion werefirst observed on days 8 and 10, respectively. The results clearlydemonstrate that both acute and chronic alcohol consumptionmarkedly afier lipid metabolism and the excretion of lipid metabolites.     

DISTRIBUTION OF ACETALDEHYDE IN HUMAN BLOOD: EFFECTS OF ETHANOL AND TREATMENT WITH DISULFIRAM

The distribution of free and bound acetaldehyde in human bloodwas studied. Fresh whole blood was precipitated with a perchloricacid (PCA) in saline solution and an aliquot of the crude samplewas taken for determination of ‘total’ acetaldehyde.The remaining sample was centrifuged and the clear supernatanttaken for analysis of ‘soluble’ acetaldehyde. ‘Bound’acetaldehyde was calculated by subtracting soluble from totalamounts. In samples collected from healthy control subjects,the acetaldehyde level in separated plasma was usually belowthe limit of detection of the method (0.2 µM), while muchhigher concentrations (> 2.5 µM) were detected whenanalyses were carried out on whole blood. In whole blood, about70% was recovered as bound (i.e. PCA-insoluble) acetaldehyde.The soluble (i.e. free + PCA-soluble) level was higher thanthat found in separated plasma, suggesting that some acetaldehydewas liberated from the blood cells by PCA treatment. In bloodspiked with ethanol, a spontaneous formation of acetaldehydeoccurred during the analytical procedure. The artefactual formationincreased only the soluble amount, while the bound level remainedunchanged. Likewise, in samples drawn from intoxicated subjects,artefactual formation of acetaldehyde was observed in the solublefraction, while the bound amount was not significantly increased.No significant differences in acetaldehyde levels were foundbetween males and females, nor between healthy control subjectsand alcoholic patients undergoing treatment with the aldehydedehydrogenase inhibitor disulfiram (Antabuse() However, someof the Antabuse patients possessed elevated levels of boundacetaldehyde.     

THE EFFECT OF NALOXONE ON THE HEPATOCELLULAR REDOX STATE AND SERUM ETHANOL CONCENTRATIONS FOLLOWING ACUTE ETHANOL ADMINISTRATION

Naloxone hydrochloride (2.0 mg/kg) has been found to reversethe significant decreases in the hepatic cytosolic and mitochondria1[NAD⁺]/[NADH] ratios observed after acute ethanol administrationin rats. This correction of the ethanol-induced changes in thehepatocellular redox state by naloxone was, however, not aswiatedwith any lowering of serum ethanol concentrations or an observablereduction in the extent of intoxicatlon. Thls lack of antagonismof alcohol intoxication by naloxone was not affected by thefeeding status of the animals, the time point after naloxoneadministration at which serum ethanol concentration was determinedor the method used for ethanol analysis. Thus this study hasfailed to confirm that naloxone antagonises acute alcohol intoxication,in spite of 11s potent abllity to reverse the ethanol-inducedchanges in the hepatic redox state.     

EFFECTS OF LIFELONG ETHANOL CONSUMPTION ON THE ULTRASTRUCTURE AND LIPOPIGMENTATION OF RAT HEART

The myocardial interactions of ageing and lifelong ethanol ingestionwere studied in the ethanol-preferring AA (Alko Alcohol) lineof rats. Samples of the left ventricle from young control rats(3-month), old control rats (30-month) and rats exposed to ethanolfrom 3 to 30 months of age, were studied in terms of myocardialultrastructure and lipopigmentation. Electron microscopic morphometryshowed an age-related increase in the volumetric densities oflipofuscin and unspecified sarcoplasm, while the proportionsof mitochondria, myofibrils and tubular structures remainedunaltered in the left ventricular myocardium. Lifelong ethanolexposure increased the proportion of sarcoplasmic reticulumand transverse tubules, apparently due to dilation of the tubularstructures. Mitochondria were significantly larger in the ethanol-exposedrats compared to the control rats of the same age, while thevolumetric proportion of mitochondria tended to decrease inthe ethanol-exposed group. Fluorescence microscopic image analysisshowed that myocardial lipopigmentation (proportion of myocardialarea covered by autofluorescent lipopigrnents) was about two-foldin the old ethanol-exposed rats compared to the old controlrats, and 13-fold compared to the young controls. It is concludedthat ageing and chronic ethanol ingestion produce rather differentpatterns of alteration in myocardial ultrastructure, which doesnot support the concept of ethanol-induced accelerated ageing.The enhancement of myocardial lipofuscin accumulation is thoughtto reflect chronic oxidative stress in the hearts exposed toethanol.     

ANALYSIS OF ETHANOL AND ACETALDEHYDE RECOVERY FROM PERCHLORIC ACID-TREATED BLOOD

Recovery of acetaldehyde (0–20 µM) or ethanol (0–50mM) added to blood and subsequently treated with perchloricacid (PCA) was evaluated using head-space gas chromatographyand compared with controls. Using blood from five dogs, <100%of acetaldehyde and ethanol was recovered from PCA-treated samples.Mathematical models of putative binding mechanisms indicatedacetaldehyde partitioned simply between supernatant and PCA-inducedprecipitate and occupied <1% of acetaldehyde binding siteson precipitate; ethanol partitioned simply between supernatantand precipitate and occupied >62% of ethanol binding sites.The mathematical model also indicated acetaldehyde binding is2500-fold stronger than ethanol binding. These results indicateas much as 46.4% of acetaldehyde may be bound to PCA-inducedprecipitate formed in whole blood. This loss of acetaldehydeis 3- to 4-fold greater than acetaldehyde loss caused by evaporationfrom PCA-treated blood.     

乙醇对铁过载大鼠肝细胞脂质过氧化和胶原代谢的影响

目的：研究乙醇对铁过载大鼠肝原代细胞脂质过氧化反应和胶原合成的影响。探讨铁和乙醇在诱导肝细胞脂质过氧化反应时的相互关系，以及脂质过氧化反应对胶原合成的影响。方法：在饲料中添加３％（ｗ／ｗ）的Ｃａｒｂｏｎｙｌｉｒｏｎ造成铁过载动物模型，从铁过载大鼠和正常大鼠中分离来的肝原代细胞与不同浓度的乙醇（０、２５、５０、１００ｍｍｏｌ／Ｌ）及０．５ｍｍｏｌ／Ｌ去铁敏（ｄｅｓｆｅｒｏｘａｍｉｎｅ）共同培养２４小时。测定肝组织和细胞中铁含量，肝细胞成活率，肝细胞中脂质过氧化产物丙二醛（ＭＤＡ），还原型谷胱甘肽（ＧＳＨ）、羟脯氨酸含量。结果：铁过载组肝细胞铁含量明显高于对照组，而从铁过载组分离来的肝细胞随乙醇剂量的增加细胞中ＭＤＡ、羟脯氨酸含量增加、ＧＳＨ含量下降，加入０．５ｍｍｏｌ／Ｌ去铁敏可有效地抑制此变化。结论：铁和乙醇在诱导肝细胞脂质过氧化反应时存在协同作用，肝细胞内铁含量增高可增加乙醇的肝细胞毒性，铁和乙醇诱导的脂质过氧化反应／产物可刺激肝细胞胶原的合成     

乙醇急、慢性摄入对外周血及红细胞膜钠泵活性的影响

[目的]观察乙醇急性和慢性摄入对外周血细胞和红细胞膜Na -K -ATP酶活力的影响.[方法]健康家兔16只,其中8只从静脉注射500ml·L-1的乙醇溶液(按0.5g·kg-1体重);另8只注射等积的生理盐水作对照.另取健康SD大鼠18只,其中9只以500ml·L-1的乙醇溶液(按0.5g·Kg-1·day-1)连续灌胃15d;另9只,以等积的生理盐水灌胃作对照.最后实验动物取血,观察血常规和红细胞膜Na -K -ATP酶活力.[结果]乙醇急性静脉注射可引起家兔外周血的红细胞计数、血小板计数明显减少,而红细胞平均容积、红细胞平均血红蛋白含量明显增加;对红细胞膜Na -K -ATP酶活力没有影响.相反,乙醇慢性灌胃后大鼠外周血的白细胞计数、血红蛋白含量、红细胞平均血红蛋白浓度、红细胞膜Na -K -ATP酶活力明显增高,而血小板计数明显减少.[结论]乙醇摄入时间、累积次数不同对外周血有不同的影响.     

EFFECT OF ACUTE AND CHRONIC ETHANOL EXPOSURE ON THE RAT BRAIN OPIATE RECEPTOR FUNCTION

A reduction of the number and a decrease in the dissociationconstant of low-affinity opiate receptor binding sites for metenkephalinin brain membranes was noted in rats after chronic ethanol treatment.These changes were reversed with time, and at 48 hr after withdrawal,tissue from ethanol and pair-fed groups demonstrated similarbinding characteristics. Preincubation of the control rat brainmembrane fraction with the ultrafiltrate of the supernatantobtained from the brain membrane fraction of rats chronicallytreated with ethanol resulted in changes of affinity and numberof opiate receptor binding sites which resembled the changesobserved in rats after long-term treatment with ethanol. Acuteethanol administration (4 g/kg) was shown to eliminate the biphasiccharacter of Scatchard plots usually seen in control animals.Addition of ethanol to control brain membranes was also foundto after significantly the binding of ‘opiate’ peptidesto such membrane fractions.     

DIFFERING EFFECTS OF CARBOHYDRATE, FAT AND PROTEIN ON THE RATE OF ETHANOL METABOLISM

The rate of metabolism of ethanol in humans has been assessedby intravenous infusion of ethanol/saline under feedback controlto maintain a constant blood alcohol concentration. After equilibration,meals consisting predominantly of carbohydrate, fat or proteinwere eaten and changes in ethanol metabolic rate were found.Carbohydrate caused a significant increase in this rate andfat or protein caused small but non-significant decreases. Infusionof ethanol/saline resulted in a temporary fall in plasma freefatty acid levels and a steady rise in plasma triglycerides.The changes in alcohol metabolism following carbohydrate cannotbe accounted for by changes in insulin, free fatty acid or lactate/pyruvatelevels.     

EFFECTS OF ETHANOL ON TESTICULAR STEROIDOGENESIS IN THE RAT

The effects of ethanol and NAD⁺ on the pregnenolone-to-testosteronepathway of testicular steroidogenesis in lysed Leydig cell preparationswere investigated. Testosterone and four precursor steroidswere determined by gas chromatography/mass spectrometry afterincubation of the cell preparations with 100 µM of pregnenoloneand appropriate concentrations of ethanol (1–100mM). Concentrationsof all 4-ene-steroids measured were significantly decreasedeven at the lowest concentration of ethanol. When NAD⁺ (0.1mM) was added to the incubation medium, the levels of progesteroneand 17-hydroxyprogesterone returned to control values, whereasthose of androstenedione and testosterone remained decreased.These results suggest that ethanol may inhibit testicular steroidogenesisby suppressing at least two steps in the pregnenolone-to-testosteronepathway, the pregnenolone-to-progesterone step catalysed byNAD⁺-dependent 5-ene-3ß-hydroxysteriod dehydrogenase/isomeraseand the 17-hydroxyprogesterone-to-androstenedione step catalysedby the NAD⁺-independent C_17–20 lyase.     

电磁辐照对海马病理形态学及相关因子表达的影响

目的对比性研究3种波段电磁辐射、对大鼠海马脑区的损伤病理及相关因子表达情况,为防治措施提供生物学依据。方法126只大鼠随机分为对照组(C)、高功率S波段微波辐照组(S)、高功率X波段微波辐照组(X)、电磁脉冲辐照组(E),C组给予伪照射、余3组分别给予100 mW.cm-2、100 mW.cm-2及6×104V.m-1,辐照后6 h、1 d、3 d、7 d、14 d、28 d活杀,海马组织经HE染色及甲苯胺兰染色后光镜观察病理学改变;SP免疫组织化学染色法检测GFAP、IL-1β、iN-OS的表达;应用图像分析仪进行定量检测。结果辐照后6 h~7 d各辐照组海马神经元变性和凋亡。辐照后3 d,E、S、X组尼氏体积分光密度(IOD)分别为4.44±0.83、2.70±0.76、2.22±0.69,明显低于C组(13.76±4.88,P<0.01)。辐照后6 h各照射组海马神经元超微结构呈缺血性改变、辐照后3 d出现空泡和次级溶酶体,血管间隙增宽水肿;辐照后3 d,S、X组的IL-1β(IOD)分别为1.29±0.71、1.22±0.43,明显高于C组(0.96±0.29,P<0.01)。iNOS分别为1.44±0.60、1.87±0.52、1.69±0.43,明显高于C组(0.65±0.25,P<0.01)。GFAP分别为0.14±0.07、0.16±0.07、0.16±0.07,明显低于C组(0.19±0.09,P<0.05或p<0.01)。结论本实验条件下,3种波段电磁辐照皆可导致大鼠海马组织损伤,GFAP、IL-1β、iNOS因子参与了电磁辐照所致损伤的病理过程;高功率微波作用较电磁脉冲明显,,而X的作用又较S明显。