Acute and chronic alcohol dose: population differences in behavior and neurochemistry of zebrafish

The zebrafish has been in the forefront of developmental genetics for decades and has also been gaining attention in neurobehavioral genetics. It has been proposed to model alcohol-induced changes in human brain function and behavior. Here, adult zebrafish populations, AB and SF (short-fin wild type), were exposed to chronic treatment (several days in 0.00% or 0.50% alcohol v/v) and a subsequent acute treatment (1 h in 0.00%, 0.25%, 0.50% or 1.00% alcohol). Behavioral responses of zebrafish to computer-animated images, including a zebrafish shoal and a predator, were quantified using videotracking. Neurochemical changes in the dopaminergic and serotoninergic systems in the brain of the fish were measured using high-precision liquid chromatography with electrochemical detection. The results showed genetic differences in numerous aspects of alcohol-induced changes, including, for the first time, the behavioral effects of withdrawal from alcohol and neurochemical responses to alcohol. For example, withdrawal from alcohol abolished shoaling and increased dopamine and 3,4-dihydroxyphenylacetic acid in AB but not in SF fish. The findings show that, first, acute and chronic alcohol induced changes are quantifiable with automated behavioral paradigms; second, robust neurochemical changes are also detectable; and third, genetic factors influence both alcohol-induced behavioral and neurotransmitter level changes. Although the causal relationship underlying the alcohol-induced changes in behavior and neurochemistry is speculative at this point, the results suggest that zebrafish will be a useful tool for the analysis of the biological mechanisms of alcohol-induced functional changes in the adult brain.     

Early development of neuronal hypothalamic thermosensitivity in birds: influence of epigenetic temperature adaptation

The aim of the study was to investigate the prenatal influence of different incubation temperatures on the early postnatal development of neuronal hypothalamic thermosensitivity in birds. The experiments were carried out in brain slices of 1-, 5- and 10-days-old Muscovy ducklings incubated at 35, 37.5 (control) or 38.5 degrees C during the last week of incubation. Firing rate of neuronal activity was recorded extracellularly during sinusoidal temperature changes. The results show that the temperature experienced prenatally has a clear influence on postnatal neuronal hypothalamic thermosensitivity. For instance, at the 10th day post-hatching, exposure to the cooler prenatal incubation temperature resulted in elevated neuronal hypothalamic warm sensitivity through an increased proportion of warm sensitive neurons and a reduced proportion of cold sensitive neurons in comparison with the control group. Exposure to the warmer prenatal incubation temperature induced the opposite effect. In these age group changes in neuronal hypothalamic thermosensitivity occur in relation to the prenatal temperature experienced (proximate adaptive). During the first days of life, prenatal temperature load induced a significant change in the thermosensitivity of hypothalamic neurons which was independent of the direction of change in incubation temperature in comparison with control conditions (proximate non-adaptive). Changes in the thermosensitivity of hypothalamic neurons after prenatal temperature experiences observed in all age groups may be the result of epigenetic temperature adaptation.     

Sexually dimorphic effects of maternal alcohol intake and adrenalectomy on left ventricular hypertrophy in rat offspring

In humans, low birth weight and increased placental weight can be associated with cardiovascular disease in adulthood. Low birth weight and increased placental size are known to occur after fetal alcohol exposure or prenatal glucocorticoid administration. Thus the effects of removing the alcohol-induced increase in maternal corticosterone by maternal adrenalectomy on predictors of cardiovascular disease in adulthood were examined in rats. Alcohol exposure of dams during the last 2 wk of gestation resulted in significantly decreased fetal weight and increased placental weight on gestational day 21. Adult female, but not male, offspring of alcohol-consuming mothers exhibited left ventricular hypertrophy. Placental 11beta-hydroxysteroid dehydrogenase-2 (11beta-HSD-2) mRNA levels, measured by Northern blot, were decreased in females but not males. Adrenalectomy of alcohol-consuming dams reversed the increase in placental weight and the decrease in female placental 11beta-HSD-2 expression and eliminated the left ventricular hypertrophy of adult female offspring. These data suggest that alcohol-induced changes in placental 11beta-HSD-2 mRNA levels and left ventricular weight are coupled in female offspring only and depend on maternal adrenal status.     

Effect of alcoholic intoxication of female mice prior to pregnancy on the ultrastructure of neurons in the sensorimotor cortex of the progeny

Sensorimotor cortex in the offspring of female rats alcoholized in the prepregnancy period revealed signs of delayed neuronal development and dystrophic changes in the neurons especially on the 14th day after birth. In 21-day-old animals the reparative changes increased, but normalization of neuronal ultrastructure was not observed. The dystrophic changes suggest that prenatal brain hypoxia plays an important role in the pathogenesis of alcohol-induced neuronal lesions in the offspring.     

Metallothionein protection against alcoholic liver injury through inhibition of oxidative stress

Antioxidants are likely potential pharmaceutical agents for the treatment of alcoholic liver disease. Metallothionein (MT) is a cysteine-rich protein and functions as an antioxidant. This study was designed to determine whether MT confers resistance to acute alcohol-induced hepatotoxicity and to explore the mechanistic link between oxidative stress and alcoholic liver injury. MT-overexpressing transgenic and wild-type mice were administrated three gastric doses of alcohol at 5 g/kg. Liver injury, oxidative stress, and ethanol metabolism-associated changes were determined. Acute ethanol administration in the wild-type mice caused prominent microvesicular steatosis, along with necrosis and elevation of serum alanine aminotransferase. Ultrastructural changes of the hepatocytes include glycogen and fat accumulation, organelle abnormality, and focal cytoplasmic degeneration. This acute alcohol hepatotoxicity was significantly inhibited in the MT-transgenic mice. Furthermore, ethanol treatment decreased hepatic-reduced glutathione, but increased oxidized glutathione along with lipid peroxidation, protein oxidation, and superoxide generation in the wild-type mice. This hepatic oxidative stress was significantly suppressed in the MT-transgenic mice. However, MT did not affect the ethanol metabolism-associated decrease in NAD(+)/NADH ratio or increase in cytochrome P450 2E1. In conclusion, MT is an effective agent in cytoprotection against alcohol-induced liver injury, and hepatic protection by MT is likely through inhibition of alcohol-induced oxidative stress.     

Effect of alcohols on the structure of caseins: circular dichroism studies of κ-casein A

The alcohols methanol, ethanol, propan-1-ol and 2,2,2-trifluoroethanol were added to aqueous solution of κ-casein A. Far u.v. circular dichroism spectroscopy revealed a greater proportion of -helix structure in the presence of alcohol; the effectiveness of the alcohol in causing this structural change increased in the order methanol to 2,2,2-trifluoroethanol. No alterations in secondary or tertiary structure were found in κ-casein A on reduction and S-carboxymethylation using near and far u.v.c.d. spectroscopy. The isolated macropeptide section of κ-casein was less sensitive to the addition of alcohols; it appeared that the linkage of macropeptide to the para-κ-casein section affected the case with which solvent induced conformational changes occurred. The conformational changes found in the presence of alcohols could not be correlated simply with the changes in the dielectric constant or with the alcohol-induced collapse of the hairy macropeptide layer of casein micelles or ther precipitation.     

Leptin deficiency enhances sensitivity of rats to alcoholic steatohepatitis through suppression of metallothionein

Oxidative stress is stated to be a central mechanism of hepatocellular injury in alcohol-induced liver injury. Recent reports have shown that Kupffer cell dysfunction in the leptin-deficient state contributes partly to the increased sensitivity to endotoxin liver injury. Here we report that leptin also plays a key role in the development of alcoholic liver injury and that leptin signaling in hepatocytes is involved in cellular mechanisms that mediate ethanol-induced oxidative stress. We found that chronic ethanol feeding in leptin receptor-deficient Zucker (fa/fa) rats for 6 wk resulted in a much more severe liver injury and augmented accumulation of hepatic lipid peroxidation compared with control littermates. The hepatic induction of stress-response and antioxidant proteins, such as metallothionein (MT)-1 and -2, was significantly suppressed in fa/fa rats after chronic ethanol feeding. Zinc concentration in liver was also decreased in fa/fa rats, compared with control littermates. In primary cultured hepatocytes from fa/fa rats, incubation with ethanol significantly suppressed MT-1 and -2 expressions. Addition of leptin to leptin-deficient ob/ob mouse primary hepatocytes led to an increase in MT-1 and -2 mRNA levels and a decrease in oxidative stress after incubation with ethanol. In conclusion, leptin deficiency enhances sensitivity of rats to alcohol-induced steatohepatitis through hepatocyte-specific interaction of MT-1 and -2 and resultant exaggeration of oxidative stress in hepatocytes. These findings suggest that leptin resistance in hepatocytes is an important mechanism of alcohol-induced liver injury.     

Interleukin-22 Protects against Acute Alcohol-Induced Hepatotoxicity in Mice

The protective effects of interleukin-22 (IL-22) on acute alcohol-induced liver injury were investigated. Mice were gavaged with 7 doses of alcohol (56% wt/vol, 15.2 mL/kg of body weight for each dose) over the 24 h, and IL-22 (0.5 mg/kg BW) was given to the mice by injection into the tail vein 1 h after alcohol administration. The results indicated that acute alcohol administration caused prominent hepatic microvesicular steatosis and an elevation of serum transaminase activities, induced a significant decrease in hepatic glutathione in conjunction with enhanced lipid peroxidation, and increased hepatocyte apoptosis as well as hepatic TNF-alpha production. IL-22 treatment attenuated these adverse changes induced by acute alcohol administration. The protective effects of IL-22 on alcohol-induced hepatotoxicity were due mainly to its anti-inflammatory, anti-oxidant, and anti-apoptotic features.     

IGF-I/IGFBP-3 ameliorates alterations in protein synthesis, eIF4E availability, and myostatin in alcohol-fed rats

Chronic alcohol consumption decreases the concentration of the anabolic hormone IGF-I, and this change is associated with impaired muscle protein synthesis. The present study evaluated the ability of IGF-I complexed with IGF-binding protein (IGFBP)-3 to modulate the alcohol-induced inhibition of muscle protein synthesis in gastrocnemius. After 16 wk on an alcohol-containing diet, either the IGF-I/IGFBP-3 binary complex (BC) or saline was injected two times daily for three consecutive days. After the final injection of BC (3 h), plasma IGF-I concentrations were elevated in alcohol-fed rats to values not different from those of similarly treated control animals. Alcohol feeding decreased the basal rate of muscle protein synthesis by limiting translational efficiency. BC treatment of alcohol-fed rats increased protein synthesis back to basal control values, but the rate remained lower than that of BC-injected control rats. The BC partially reversed the alcohol-induced decrease in the binding of eukaryotic initiation factor (eIF)4E with eIF4G. This change was associated with reversal of the alcohol-induced dephosphorylation of eIF4G but was independent of changes in the phosphorylation of either 4E-BP1 or eIF4E. However, BC reversed the alcohol-induced increase in IGFBP-1 and muscle myostatin, known negative regulators of IGF-I action and muscle mass. Hence, exogenous IGF-I, administered as part of a BC to increase its circulating half-life, can in part reverse the decreased protein synthesis observed in muscle from chronic alcohol-fed rats by stimulating selected components of translation initiation. The data support the role of IGF-I as a mediator of chronic alcohol myopathy in rats.     

Positional and species analysis of membrane phospholipids extracted from goldfish adapted to different environmental temperatures.

1. The proportion of ethanolamine phosphoglycerides in microsomal fractions of goldfish intestine increases at low environmental temperatures. The fatty acyl composition also changes, the proportion of C22:6 and C20:4 fatty acids increasing in positions 1 and 2 and position 2 respectively. The proportion of C16:0 and C18:0 fatty acids falls in position 1 and there is an apparent switch of C18:1 and C20:1 fatty acids from position 2 to position 1. 2. The proportion of choline phosphoglycerides does not depend on the previous environmental temperature of the fish. Temperature-dependent changes in fatty acyl composition in positions 1 and 2 take place in a way similar to that described for ethanolamine phosphoglycerides, but in this case C22:6 substitution is confined to position 2. 3. Choline phosphoglycerides have been further separated into 7 different molecular species. The amounts of species 3 to 7 increase and the amount of species 2 decreases at low adaptation temperature. These changes only account for part of total change in fatty acyl composition. The remaining changes occur by chain substitution within species. 4. Present results show temperature adaptation to be highly complex, involving both quantitative and qualitative changes in different phospholipids. The possible physiological significance of these changes are discussed together with the effects these changes might have on cholesterol-phospholipid interactions.     

Interleukin-22 protects against acute alcohol-induced hepatotoxicity in mice

The protective effects of interleukin-22 (IL-22) on acute alcohol-induced liver injury were investigated. Mice were gavaged with 7 doses of alcohol (56% wt/vol, 15.2 mL/kg of body weight for each dose) over the 24 h, and IL-22 (0.5 mg/kg BW) was given to the mice by injection into the tail vein 1 h after alcohol administration. The results indicated that acute alcohol administration caused prominent hepatic microvesicular steatosis and an elevation of serum transaminase activities, induced a significant decrease in hepatic glutathione in conjunction with enhanced lipid peroxidation, and increased hepatocyte apoptosis as well as hepatic TNF-alpha production. IL-22 treatment attenuated these adverse changes induced by acute alcohol administration. The protective effects of IL-22 on alcohol-induced hepatotoxicity were due mainly to its anti-inflammatory, anti-oxidant, and anti-apoptotic features.     

Amphora margalefii Tomàs var. lacustris P. Sánchez var. nova,a new brackish water diatom

The rotifer Brachionus plicatilis can grow in a wide range of salinities and temperatures, but rapid shifts in both salinity and temperature may result in immobilized, non-swimming rotifers. The goal of this study was to examine the effect of perturbations in temperature and salinity on the swimming pattern of the rotifer.Only slight changes in mobility were observed when rotifers were exposed to changes in temperature (from 20 °C to 8–30 °C) and to an increase in salinity (from 20% to 30%). When the salinity was reduced to 15% and 5%, the proportion of mobile rotifers was reduced to 50% and 5%, respectively. The rotifers were throughout more resistant to perturbations in temperature than to those of salinity.Combined temperature and salinity perturbations compared to perturbations in each factor separately suggested a synergetic effect of temperature and salinity on the rotifers locomotion. Transfer from cultivation conditions to low salinity (5%) and high temperature (28 °C) resulted in very low percent of mobile rotifers (0–10%). However, if the temperature was reduced to 8 °C concomitant with the changes in salinity, the percent of mobile rotifers was 85%.Rotifers use a high share of their metabolic energy for locomotion, and it is therefore not surprising that perturbations in salinity and temperature may result in partial or complete immobilization.     

Protective effects of sodium nitrite preconditioning against alcohol-induced acute liver injury in mice

The purpose of the present study was to investigate the effect of sodium nitrite (SN) on alcohol-induced acute liver injury in mice. Forty male C57bL/6 mice were randomly divided into 4 groups. Acute alcohol-induced liver injury group were injected intraperitoneal (ip) with alcohol (4.5 g/kg); SN preconditioning group were pretreated with SN (16 mg/kg, ip) for 12 h, and received alcohol (4.5 g/kg, ip) injection; Control and SN groups were treated with saline and SN, respectively. After the treatments, liver index (liver/body weight ratio) was determined. Colorimetric technique was performed to measure the serum alanine transaminase (ALT), aspartate transaminase (AST), liver superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activities, as well as malondialdehyde (MDA) content. The pathological index of liver tissue was assayed by HE and TUNEL fluorometric staining. Using Western blot and immunohistochemistry staining, the expression of hypoxia-inducible factor-1α (HIF-1α) protein was detected. The results showed that, compared with acute alcohol-induced liver injury group, pretreatment with low doses of SN decreased liver index and serum levels of ALT and AST, weakened acute alcohol-induced hepatocyte necrosis, improved pathological changes in liver tissue, increased live tissue SOD, GSH-Px and CAT activities, reduced MDA content and apoptosis index of hepatocytes, and up-regulated HIF-1α protein level in liver tissue. These results suggest that the pretreatment of SN can protect hepatocytes against alcohol-induced acute injury, and the protective mechanism involves inhibition of oxidative stress and up-regulation of HIF-1α protein level.     

Protective effect of glycine supplementation on the levels of lipid peroxidation and antioxidant enzymes in the erythrocyte of rats with alcohol-induced liver injury

We studied the effect of glycine supplementation on lipid peroxidation and antioxidants in the erythrocyte membrane, plasma and hepatocytes of rats with alcohol-induced hepatotoxicity. Administering ethanol (20%) for 60 days to male Wistar rats resulted in significantly elevated levels of erythrocyte membrane, plasma and hepatocyte thiobarbituric acid reactive substances (TBARS) as compared with those of the experimental control rats. Decreased activities of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GPx) and glutathione reductase (GR) were also observed on alcohol supplementation as compared with those of the experimental control rats. Glycine was administered at a dose of 0.6 g kg(-1) body weight to rats with alcohol-induced liver injury, which significantly decreased the levels of TBARS and significantly elevated the activities of SOD, CAT, GSH, GPx and GR in the erythrocyte membrane, plasma and hepatocytes as compared to that of untreated alcohol supplemented rats. Thus, our data indicate that supplementation with glycine offers protection against free radical-mediated oxidative stress in the erythrocyte membrane, plasma and hepatocytes of animals with alcohol-induced liver injury.     

The effects of growth temperature on the methyl sterol and phospholipid fatty acid composition of Methylococcus capsulatus (Bath)

Growth of Methylococcus capsulatus (Bath) at temperatures ranging from 30 to 50 degrees C resulted in changes to the whole cell lipid constituents. As temperature was lowered, the overall proportion of hexadecenoic acid (C16:1) increased, and the relative proportions of the delta 9, delta 10 and delta 11 C16:1 double bond positional isomers changed. Methyl sterol content also increased as the growth temperature was lowered. The highest amounts of methyl sterol were found in 30 degrees C cells and the lowest in 50 degrees C cells (sterol-phospholipid ratios of 0.077 and 0.013, respectively). The data are consistent with a membrane modulating role for the sterol produced by this prokaryotic organism.     

Sites in TM2 and 3 are critical for alcohol-induced conformational changes in GABA receptors

Abstract gamma-Aminobutyric acid type A (GABA(A)) receptors are molecular targets for alcohols. Previous work suggests that S270 and A291 residues in the transmembrane (TM) 2 and 3 domains of the GABA(A) receptor alpha subunit are components of an alcohol-binding pocket, and S270I and A291W mutants abolished ethanol potentiation. Our results showed that A295C and F296C residues in the TM3 of the GABA(A) receptor alpha1 subunit are accessible to hexylmethanethiosulfonate (HMTS) in the alcohol-bound state, but not in the resting state. Thus, the A295C and F296C sites become water-accessible as a result of alcohol-induced conformational changes. If S270 or A291 residues are sites of alcohol binding, then S270I or A291W mutations should prevent alcohol-induced conformational movements within the TM3 domain. To investigate this question, the accessibility of HMTS reagent to double mutants (A291W/A295C, A291W/F296C, S270I/A295C or S270I/F296C) in the presence of ethanol or hexanol was tested. The A291W or S270I mutations markedly reduced the accessibility of HMTS to all the double mutants in the ethanol-bound state, and to S270I/F296C, A291W/A295C and A291W/F296C double mutants in the hexanol-bound state, suggesting that the A291 or S270 residues are critical sites for alcohol binding and alcohol-induced conformational changes.     

Changes in desaturase activity and the fatty acid composition of microsomal membranes from liver tissue of thermally-acclimating rainbow trout

Summary Rainbow trout (Salmo gairdneri) were acclimated to either 5 or 20°C, and then transferred to the opposite temperature, and changes in the fatty acid composition of liver microsomal membranes and the activities of the hepatic Δ9, Δ6, and Δ5 desaturases were measured at intervals of up to one month post-transfer. Inital changes (days 0–3) in fatty acid composition were: (1) an increase in the proportion of saturates and a decrease in the proportion of polyunsaturates during warm acclimation, and (2) a decrease in the proportion of saturates during cold acclimation. The activity of the Δ6 desaturase approximately doubled immediately following the changes in temperature, but alterations in Δ9 and Δ5 desaturase activities required at least 3 days to occur. The results indicate that desaturase enzymes do not play a major role in the initial adaptation of membrane fatty acid composition to changes in temperature. However, the desaturase enzymes may be involved in the later stages (3–28 days) of the acclimatory process. The proportion of monoenes was well correlated with Δ9 desaturase activity during both transfers, and appeared to be adjusted as required to offset changes in the proportion of polyunsaturates. Supported by National Science Foundation Grant PCM-8301757 to J.R.H.     

FGF21 mediates alcohol-induced adipose tissue lipolysis by activation of systemic release of catecholamine in mice

Alcohol consumption leads to adipose tissue lipoatrophy and mobilization of FFAs, which contributes to hepatic fat accumulation in alcoholic liver disease. This study aimed to investigate the role of fibroblast growth factor (FGF)21, a metabolic regulator, in the regulation of chronic-binge alcohol-induced adipose tissue lipolysis. FGF21 KO mice were subjected to chronic-binge alcohol exposure, and epididymal white adipose tissue lipolysis and liver steatosis were investigated. Alcohol exposure caused adipose intracellular cAMP elevation and activation of lipolytic enzymes, leading to FFA mobilization in both WT and FGF21 KO mice. However, alcohol-induced systemic elevation of catecholamine, which is known to be a major player in adipose lipolysis by binding to the β-adrenergic receptor, was markedly inhibited in KO mice. Supplementation with recombinant human FGF21 to alcohol-exposed FGF21 KO mice resulted in an increase in fat loss in parallel with an increase of circulating norepinephrine concentration. Furthermore, alcohol consumption-induced fatty liver was blunted in the KO mice, indicating an inhibition of fatty acid reverse transport from adipose to the liver in the KO mice. Taken together, our studies demonstrate that FGF21 KO mice are protected from alcohol-induced adipose tissue excess-lipolysis through a mechanism involving systemic catecholamine release.     

Nimodipine's interactions with other drugs: I. Ethanol

Adult mice (Binghamton Heterogeneous stock) received different doses of ethanol (0.5, 1.0, or 2.0 g/kg) administered alone or in combination with the voltage-sensitive calcium channel antagonist, nimodipine (Bay e 9736). Both 20 and 60 minutes later, sensitivity to ethanol was assessed in terms of rotorod activity and changes in rectal temperatures. Nimodipine (5 mg/kg) alone did not alter rectal temperature or motor coordination, but at both observation periods nimodipine potentiated the hypothermia induced by the highest dose of alcohol (2.0 g/kg) and exaggerated alcohol-induced motor incoordination at all doses. The present set of results indicates that the inhibition of voltage-dependent calcium channels can exaggerate ethanol-induced effects.