Studies on the effects of vitamin E on neuroblastoma N1E 115

The effects of DL-alpha-tocopherol and DL-alpha-tocopherol succinate on neuroblastoma N1E 115 cells were studied. Tocopherol had no growth-arresting properties, whereas its succinate ester derivative inhibited growth at concentrations greater than or equal to 20 microM. The succinate derivative was taken up somewhat more readily than free tocopherol; however, for any equal uptake of both forms of vitamin E, only the succinate derivative could affect growth. Tocopherol succinate was taken up without marked conversion to tocopherol. Following uptake, plasma membrane and organelle fractions contained most of the vitamin E derivatives; however, the particulate and membrane fractions were about twice as enriched in the succinate derivative as in free tocopherol. On the other hand, a proportionally higher amount of unconjugated vitamin E was recovered in the cytosol fraction. Fluorescence polarization studies indicated no differences in the overall fluidity of the plasma membranes treated or not treated with either form of vitamin E. The data point to the functionality of the free carboxyl group of the succinate derivative as a basis for the difference in potency of the two forms of vitamin E.     

Effects of adolescent ethanol exposure on ethanol consumption in adult rats.

Ethanol exposure during early development could predispose an individual to increased ethanol consumption. Given the high prevalence of adolescent ethanol abuse, it is important to assess the potential impact of adolescent ethanol exposure on the development of alcohol drinking. The following study was designed to assess the initiation of ethanol consumption in adult rats after exposure to ethanol vapors during adolescence. Male Sprague-Dawley rats (n = 23) were exposed to ethanol vapor for 12 h per day for 10 consecutive days between postnatal days 30 and 40. Ethanol vapor exposure maintained blood ethanol levels averaging 250 mg/dl. All rats were subsequently trained to self-administer ethanol after a 52-day withdrawal period. When ethanol consumption was assessed in the adult rats (>3 months old) there were no significant differences in initiation or maintenance of ethanol self-administration between ethanol-exposed and control rats. In addition, there were no group differences in the ability of a noise stressor presented before the drinking session to transiently decrease ethanol intake. Overall, these findings indicate that forced exposure to ethanol vapor during adolescence does not seem to be sufficient to alter initiation or maintenance of limited-access ethanol self-administration.     

Effects of adolescent ethanol exposure on sleep in adult rats

Although adolescent ethanol (EtOH) exposure has been associated with long-lasting changes in brain function, little is known as to whether EtOH exposure during adolescence alters sleep and cortical arousal. This study examined protracted alterations in sleep in adult rats exposed to EtOH during adolescence. Adolescent male Wistar rats were exposed to EtOH vapor for 12 h/day for 5 weeks. Cortical electroencephalograms were obtained during 4-h recording sessions after 5 weeks of withdrawal from EtOH. Adolescent EtOH exposure significantly reduced the mean duration of slow-wave sleep (SWS) episodes and the total amount of time spent in SWS in EtOH-exposed rats, compared to controls. Spectral analysis revealed that adolescent EtOH exposure significantly increased cortical peak frequencies during SWS in the 2-4, 4-6, and 6-8 Hz bands. Taken together, our findings suggest that chronic EtOH exposure in adolescent rats reduces measures of SWS, an effect also seen as part of normal aging. Although the cellular and molecular mechanisms mediating the consequences of EtOH exposure on the aging process are not known, the similarities between adolescent EtOH exposure and aging merits further investigation.     

Effects of chronic ethanol exposure on sleep in rats.

Sleep disturbance is a common complaint in alcoholics. When polysomnographic studies are performed in alcoholics, reductions in slow wave sleep are a common finding; however, few studies have evaluated the effects of chronic alcohol exposure on sleep in animal models. In the present study, the sleep EEG was evaluated in 40 Wistar rats who were exposed to chronic alcohol or control conditions in vapor chambers. Rats were exposed to ethanol vapors or control chambers for 6 weeks and then withdrawn. Sleep EEG was recorded before exposure (baseline), immediately following exposure, and 5 weeks after withdrawal from the ethanol/control chambers. In the ethanol-exposed animals, blood ethanol levels averaged 192 mg/dL over 6 weeks of exposure. Chronic ethanol exposure and withdrawal was not found to affect either slow wave sleep latency or slow wave sleep duration; however, overall spectral power as well as power in the delta, theta, and beta frequencies were significantly reduced following chronic exposure (2-4 Hz, [F(1, 17) = 18.11, p = 0.001], 4-6 Hz, [F(1, 17) = 15.98, p = 0.001], 6-8 Hz [F(1, 17) = 15.52, p = 0.001], 8-16 Hz band [F(1, 17) = 18.73, p < 0.0001], 16-32 Hz [F(1, 17) = 10.13, p = 0.005], and 1-50 Hz [F(1, 17) = 17.03, p = 0.001]. After 5 weeks of withdrawal, significant decreases still persisted in the delta and theta frequencies (2-4 Hz [F(1, 16) = 6.21, 0.024], 4-6 Hz [F(1, 16) = 6.26, 0.024], and 6-8 Hz [F(1, 16) = 4.84, p = 0.043]). These findings suggest that spectral analysis of the EEG is a highly sensitive measure of the effects of ethanol on sleep. These findings additionally demonstrate that chronic ethanol exposure can produce persistent diminution in the systems that generate cortical slow waves in the rat and thus may provide a model for understanding the mechanisms underlying sleep disturbances associated with alcoholism.     

Effects of prenatal ethanol exposure on iron utilization in the rat

The effects of prenatal ethanol exposure on iron metabolism in rat dams and pups were studied. Sperm-positive nulliparous dams were assigned to groups on the third day of gestation (G3): ET rats were fed a liquid diet containing 9% ethanol (v/v); PC rats were pair-fed a non-ethanol, isocaloric liquid diet; FC rats were fed the same nonethanol diet adlibitum. All animals were individually housed in stainless steel metabolic cages from G3 to G18 and transferred to polypropylene cages to await delivery. Food intake and dam body weight were significantly less in the ET and PC groups compared to the FC control group. Water intake was significantly greater in the ET dams than in controls. Gestation length was significantly increased in the ET rats only. Pup body weight was significantly decreased in the ET rats only compared to controls. Apparent absorption of iron in the ET dams was significantly greater than in the PC and FC dams. The inutero ethanol exposure resulted in a significant increase in liver and femur iron concentrations in the newborn pups when compared to the PC and FC control pups. The marked increase clue to understanding presence of liver pathology that has been reported to occur in children with fetal alcohol syndrome.     

Effects of prenatal ethanol exposure on ethanol-induced locomotor activity in rats

In adulthood, offspring with Fetal Alcohol Effects exhibit different responses to ethanol than nonexposed offspring; however, the majority of this research has utilized high levels of ethanol prenatally with resultant behavioural and/or physical anomalies. The present research focused on moderate prenatal ethanol exposure and subsequent challenge with low doses of ethanol. Following impregnation, female rats were exposed to between 3-4 g/kg of ethanol in a saccharin solution daily in a voluntary free-choice with water during one of each trimester or during all trimesters. There was a no-ethanol control group which received saccharin only as well as a group of foster dams who drank only water. At 30 days of age, male offspring were tested in an open-field after injection of either 1 g/kg ethanol or a comparable volume of saline. Rats exposed prenatally to ethanol in the second trimester showed enhanced locomotor activity. However, rats exposed prenatally to ethanol in either the first, third or during all trimesters showed no significant augmentation in activity. The results are discussed in terms of the adverse effects of moderate levels of prenatal ethanol exposure on fetal neurochemical development, specifically, the excitatory and inhibitory systems.     

Effects of in utero ethanol exposure on serotonin uptake in cortical regions

Previously, this laboratory found that the 19- and 35- to 37-day-old offspring of rats that consumed ethanol on a chronic basis prior to parturition had a decreased cortical content of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) as well as a decreased number of cortical 5-HT1 binding sites. These results emphasized the sensitivity of the developing cortical serotonergic nerves to the effects of in utero ethanol exposure. In the present study, we examined the effects of in utero ethanol exposure on an additional component of the developing cortical serotonergic systems. Specifically, we examined the uptake of [3H]-5-HT by synaptosomes which were isolated from the motor or somatosensory regions of the cerebral cortex. The results demonstrated that the Vmax for serotonin uptake was significantly decreased (p less than 0.025) by approximately 15-20% in the motor cortices of the 19- and 35-day-old offspring of rats that consumed ethanol on a chronic basis prior to parturition. In addition, there was a significantly (p less than 0.025) approximately 30% decrease in the Km for serotonin uptake in the motor cortex of 35-day-old offspring of ethanol-fed rats. In contrast, neither the (Km) nor the Vmax for serotonin uptake were significantly altered (p greater than 0.05) in the somatosensory cortices in 19- or 35-day-old offspring of ethanol-fed rats. These results emphasize the selective sensitivity of developing cortical projections of the serotonergic system.     

Chronic intermittent ethanol exposure during adolescence: Effects on social behavior and ethanol sensitivity in adulthood

This study assessed long-lasting consequences of repeated ethanol exposure during two different periods of adolescence on 1) baseline levels of social investigation, play fighting, and social preference and 2) sensitivity to the social consequences of acute ethanol challenge. Adult male and female Sprague-Dawley rats were tested 25 days after repeated exposure to ethanol (3.5 g/kg intragastrically [i.g.], every other day for a total of 11 exposures) in a modified social interaction test. Early-mid adolescent intermittent exposure (e-AIE) occurred between postnatal days (P) 25 and 45, whereas late adolescent intermittent exposure (l-AIE) was conducted between P45 and P65. Significant decreases in social investigation and social preference were evident in adult male rats, but not their female counterparts following e-AIE, whereas neither males nor females demonstrated these alterations following l-AIE. In contrast, both e-AIE and l-AIE produced alterations in sensitivity to acute ethanol challenge in males tested 25 days after adolescent exposure. Ethanol-induced facilitation of social investigation and play fighting, reminiscent of that normally seen during adolescence, was evident in adult males after e-AIE, whereas control males showed an age-typical inhibition of social behavior. Males after l-AIE were found to be insensitive to the socially suppressing effects of acute ethanol challenge, suggesting the development of chronic tolerance in these animals. In contrast, females showed little evidence for alterations in sensitivity to acute ethanol challenge following either early or late AIE. The results of the present study demonstrate a particular vulnerability of young adolescent males to long-lasting detrimental effects of repeated ethanol. Retention of adolescent-typical sensitivity to the socially facilitating effects of ethanol could potentially make ethanol especially appealing to these males, therefore promoting relatively high levels of ethanol intake later in life.     

Effects of calcium channel inhibitors on ethanol effects and pharmacokinetics in healthy volunteers

Previous studies have shown that calcium channel antagonists alter the effects of alcohol in animals and humans. We selected a phenylalkylamine, verapamil, and a dihydropyridine, nimodipine, to determine whether these drugs would affect the subjective or psychomotor effects of ethanol in humans. Subjects ingested verapamil (80 mg, PO), nimodipine (30 and 60 mg, PO), or placebo 60 min before drinking an alcohol (0.7 g/kg) or placebo beverage. Subjects' mood, psychomotor performance, physiological status, and blood alcohol levels were assessed up to 3 h after beverage ingestion. Alcohol increased “drunk” ratings and impaired psychomotor performance (p < 0.05). Blood alcohol levels were decreased by nimodipine pretreatment, but not by verapamil pretreatment. Subjective and psychomotor effects of alcohol were not altered as a function of nimodipine or verapamil pretreatment. Nimodipine, verapamil, and alcohol, either alone or in combination, had no effect on blood pressure or heart rate.     

Effects of in utero ethanol exposure on cortical 5-HT2 binding sites

Previous studies by this laboratory found that in utero ethanol exposure results in a decrease of several presynaptic components of serotonin (5-HT) containing neurons in the cerebral cortex and cortical regions. The present study examined whether in utero ethanol exposure similarly affects the development of the cortical targets of serotonergic projections. Specifically, we examined the influence of in utero ethanol exposure on 5-HT2 binding sites in the motor and somatosensory cortices of the offspring of rats that consumed a control or 6.6% (v/v) ethanol liquid diet on a chronic basis prior to parturition. These studies demonstrated that neither the Kd nor Bmax for the binding of [3H]-ketanserin to membranes from the motor and somatosensory cortices of 19- and 35-day-old rats were significantly altered by in utero ethanol exposure. These results suggest that the development of the postsynaptic target areas of cortical serotonergic projections are more resistant to the effects of in utero ethanol exposure than the presynaptic components of these projections.     

Effects of periadolescent ethanol exposure on alcohol preference in two BALB substrains.

Ethanol exposure during adolescence is a rite of passage in many societies, but only a subset of individuals exposed to ethanol becomes dependent on alcohol. To explore individual differences in response to ethanol exposure, we compared the effects of periadolescent ethanol exposure on alcohol drinking in an animal model. Male and female mice of two BALB substrains were exposed to ethanol in one of three forms--choice [water vs. 10% (volume/volume) ethanol], forced (10% ethanol in a single bottle), or gradual (single bottle exposure, starting with 0.5% ethanol and increasing at 2-day intervals to 10% ethanol)--from the 6th through the 12th week of age and administered two-bottle alcohol preference tests (10% ethanol vs. water) for 15 days immediately thereafter. All three forms of ethanol exposure increased alcohol preference in male and female BALB/cByJ mice, relative to findings for ethanol-naive control animals. Only gradual ethanol exposure produced an increase in alcohol preference in BALB/cJ mice. During extended alcohol preference testing (for a total of 39 days) of mice in the gradual ethanol exposure group, the higher alcohol preference of the gradual ethanol-exposed BALB/cByJ male mice persisted, but alcohol preference of control group female mice in this strain--formerly ethanol naive, but at this point having received 10% ethanol in the two-bottle paradigm for 15 days--rose to the level of alcohol preference of female mice in the gradual ethanol exposure group. This finding demonstrated that both adolescent and adult ethanol exposure stimulated alcohol preference in female mice of this strain. Across days of testing in adulthood, alcohol preference of the gradual ethanol-exposed BALB/cJ mice decreased, resulting in a lack of effect of gradual exposure to ethanol on alcohol preference in both male and female mice of this strain during the period of extended testing. These strain differences support a genetic basis for the effects of ethanol exposure on alcohol preference and fit within a body of literature, showing substantial individual differences in the effects of ethanol exposure among genetically undefined rats and differences in response to ethanol exposure among inbred rat strains. Exploration of the mechanisms underlying this gene by environment interaction in a mouse model may help elucidate individual differences in the effects of ethanol exposure in human beings and contribute to the understanding of the causes of alcoholism.     

Effects of prior ethanol exposure on ethanol self-administration in a continuous access situation using retractable drinking tubes.

To examine whether exposure to ethanol influences subsequent ethanol consumption using a continuous access procedure, two groups of rats were given differing initial exposure to ethanol. One group underwent a sucrose-substitution initiation procedure. The second group received abbreviated initiation consisting of one-session exposure to each ethanol/sucrose combination used in standard initiation. The animals were then provided with 23 h/day access to ethanol (10%, v/v) from a retractable drinking tube. Food pellets were available following a single-lever press, and water was available from a sipper tube. After 5 weeks, the data indicated that few significant differences existed between the groups on total ethanol (g/kg), food or water consumed. The overall intake (g/kg/day), number of ethanol bouts per day, and amount consumed per bout (g/kg/bout) were substantially lower than observed in previous research using ethanol presented in a dipper. However, differences in g/kg per ethanol bout did differ significantly between the two groups with the group receiving standard initiation showing more ethanol consumed per bout. These data agree with our previous work indicating that initiation results in larger drinking bouts.     

丙烯酰胺对NB-1细胞钙稳态的影响

目的研究丙烯酰胺(AM)对神经细胞钙稳态的影响,探讨AM致神经损伤的可能机制。方法将人神经母细胞瘤(NB-1)细胞诱导分化成熟后,以不同浓度AM进行体外染毒,采用噻唑蓝(MTT)比色法检测神经元损伤程度,并应用ATP酶检测试剂盒测定不同损伤程度的细胞Ca2+-ATP酶和Na+-K+-ATP酶活力的变化。以Fluo-3/AM为荧光指示剂,用激光共聚焦方法检测细胞内游离钙离子浓度的瞬时变化情况。结果 AM染毒组成熟NB-1细胞存活率有明显下降,提示AM对神经细胞有毒性作用;AM染毒组Ca2+-ATP酶和Na+-K+-ATP酶活力值也均下降,差异具有统计学意义(P<0.05);AM作用于成熟NB-1细胞可立即引起细胞内钙离子浓度升高。结论 AM可能通过影响神经细胞钙稳态产生毒性作用。     

Effects of chronic ethanol exposure on fatty acids of rat brain glycerophospholipids

The lipid composition was analysed in forebrain subcellular fractions from rats treated with ethanol for three weeks and control rats. Increased proportions of oleic acid and a decrease in palmitic acid were consistently found in total glycerophospholipid fractions after ethanol exposure. The fatty acid compositions of individual phospholipids were also significantly changed. The proportion of docosahexaenoic acid was decreased in brain phosphatidylserine. In contrast to the decrease in the degree of unsaturation in phosphatidylserine, there was an opposite change in phosphatidylcholine wherein the degree of unsaturation was increased. No changes were produced in total cholesterol or phospholipid concentrations. These results point to a high degree of complexity of the mechanisms behind ethanol-induced changes in membrane lipid composition. The decrease in unsaturation in phosphatidylserine is probably an adaptive effect in order to counteract the fluidizing effect of ethanol. There are two possible explanations for the increase in unsaturation in brain phosphatidylcholine. The change may be due to adaptation to other biophysical effects, e.g., expansion of the membrane surface or be secondary to a change in liver lipid metabolism.     

Effects of ethanol and calcium on lipid order of membranes from mice selected for genetic differences in ethanol intoxication

Fluorescent probes were used to compare the physical properties of membranes from mice selected for sensitivity (LS) and insensitivity (SS) to the hypnotic action of ethanol. Brain synaptic plasma membranes (SPM) from LS mice were more sensitive to the disordering action of ethanol than those from LS mice when probes were located near the membrane surface. However, the membrane core of membranes from the two lines was equally sensitive to ethanol. The genetic differences in ethanol sensitivity of the membrane surface were eliminated when fluorescence measurements were carried out in the presence of 2-3 mM CaCl2. Consistent with behavioral data, differential genetic sensitivity to the disordering action was not obtained with longer chain alcohols. The genetic difference in ethanol sensitivity was not detected with erythrocyte membranes or lipids extracted from SPM. These results indicate that there is a structural difference in the surface of brain membranes of LS and SS mice than may influence their sensitivity to ethanol.     

Effects of prenatal ethanol exposure on postnatal renal function and structure in the rat

Effects of prenatal ethanol exposure on postnatal renal function and structure in the rat. Renal function and morphology were studied in 90-day-old offspring of ethanol-fed (E) rats and were compared to pair-fed control (C) animals. Compared to C rats, E rats were smaller at birth, had higher fractional sodium excretion (p less than 0.01) and lower fractional potassium excretion (p less than 0.01). In E rats, sodium (Na) restriction resulted in a significant increase in urine flow and Na wastage, whereas C rats remained in Na balance. E rats developed hyperkalemia, when potassium (K) intake was increased from 2.8 to 14 mEq/day. Baseline creatinine clearance, urine and blood osmolalities and pH, plasma electrolytes and aldosterone concentrations were similar in both groups. There was no significant difference in wet or dry kidney weight, renal water content, or renal tissue concentrations of Na or K between the two groups. No difference was found in gross morphology or light microscopic appearances of the kidneys between E and C rats. Thus rats exposed to ethanol during fetal life have a defect in urine concentration and Na conservation when fed a low Na diet and a defect in K excretion when given a K load without evidence of any gross or light microscopic renal structural abnormalities at 90 days of age.     

Neuronal differentiation modifies the effect of ethanol exposure on voltage-dependent calcium channels in NG 108-15 cells

The effect of prolonged (72 h) ethanol (200 mM) exposure on the labeling of L-type (using tritiated PN 200-110) and N-type (using iodinated ω-conotoxin) voltage-dependent calcium channels was investigated in cultured NG 108-15 cells. In undifferentiated cells ethanol produced an 80% increase in PN 200-110 B_max and no changes in ω-conotoxin binding. Differentiation had a profound effect on the response of cells to ethanol, which in differentiated neuron-like cells decreased w-conotoxin binding (−53.5%) leaving PN 200-110 labeling of L-type channels unaffected. The effect was time dependent and reversible upon ethanol withdrawal. The decreased ω-conotoxin binding was accompanied by a reduced ability of ω-conotoxin to inhibit K⁺-stimulated calcium uptake. The results demonstrate that in cultured NG 108-15 cells ethanol differentially affects DHP and ω-conotoxin-sensitive, voltage-dependent calcium channels and that the effect is also modulated by differentiation of the cell to a neuronal phenotype.     

急性乙醇暴露对人原代肝细胞血红素氧化酶的影响

目的　研究急性乙醇暴露对人原代肝细胞血红素氧化酶活力及蛋白水平的影响。方法　经体外灌流、分离培养人原代肝细胞 ,观察乙醇对人原代肝细胞上清液中天冬氨酸氨基转移酶 (AST)的释放及谷胱甘肽 (GSH)含量的变化 ,用westernblot方法检测乙醇对人原代肝细胞血红素氧化酶活力及蛋白水平的影响。结果　急性乙醇暴露导致人原代肝细胞上清液中释放的AST增加 ,并呈明显的剂量效应和时间效应关系 ;此外 ,在 10 0mmol L乙醇 2 4h暴露下 ,肝细胞中的GSH明显降低 ,而HO 1酶活力在 0 5～ 12h之间明显升高 ,随后开始降低 ,且HO 1蛋白水平变化趋势与此一致。结论　HO 1酶活力的升高可能与乙醇暴露下人原代肝细胞氧化损伤的保护有关