Effects Of Chronic Ethanol Consumption Research Articles

Efeitos do alcoolismo e da desintoxicação alcoólica sobre o reparo e biomecânica óssea

OBJETIVO: Avaliar os efeitos do consumo crônico de etanol e da desintoxicação alcoólica sobre a resistência mecânica do osso e neoformação óssea junto a implantes de hidroxiapatita densa (HAD) realizados em ratos. MÉTODOS: Foram utilizados 15 ratos divididos em três grupos, sendo controle (CT), alcoolista crônico (AC) e desintoxicado (DE). Após quatro semanas, foi realizada implantação de HAD na tíbia e produzida falha no osso parietal, em seguida o grupo AC continuaram a consumir etanol e o grupo DE iniciaram a desintoxicação. Ao completar 13 semanas os animais sofreram eutanásia, os ossos foram coletados para o processamento histomorfométrico e os fêmures encaminhados ao teste mecânico de resistência. RESULTADOS: Os animais do grupo AC apresentaram menores valores de neoformação óssea, de calcemia e resistência mecânica, quando comparado aos grupos CT e DE. Os animais dos grupos DE apresentaram valores superiores em todas as variáveis avaliadas em relação ao grupo AC. CONCLUSÃO: O consumo de etanol interferiu na osteogênese ao redor de implante de HAD, nos níveis de cálcio e na resistência mecânica óssea. A desintoxicação alcoólica se mostrou eficaz, pois aumentou à osteogênese e osseointegração da HAD, a calcemia e resistência mecânica óssea. Nivel de Evidência I, estudo terapeutico.

Chronic Ethanol Consumption-induced Pancreatic β-Cell Dysfunction and Apoptosis through Glucokinase Nitration and Its Down-regulation

Chronic ethanol consumption is known as an independent risk factor for type 2 diabetes, which is characterized by impaired glucose homeostasis and insulin resistance; however, there is a great deal of controversy concerning the relationships between alcohol consumption and the development of type 2 diabetes. We investigated the effects of chronic ethanol consumption on pancreatic β-cell dysfunction and whether generated peroxynitrite participates in the impaired glucose homeostasis. Here we show that chronic ethanol feeding decreases the ability of pancreatic β-cells to mediate insulin secretion and ATP production in coordination with the decrease of glucokinase, Glut2, and insulin expression. Specific blockade of ATF3 using siRNA or C-terminally deleted ATF3(ΔC) attenuated ethanol-induced pancreatic β-cell apoptosis or dysfunction and restored the down-regulation of glucokinase (GCK), insulin, and pancreatic duodenal homeobox-1 induced by ethanol. GCK inactivation and down-regulation were predominantly mediated by ethanol metabolism-generated peroxynitrite, which were suppressed by the peroxynitrite scavengers N(γ)-monomethyl-L-arginine, uric acid, and deferoxamine but not by the S-nitrosylation inhibitor DTT, indicating that tyrosine nitration is the predominant modification associated with GCK down-regulation and inactivation rather than S-nitrosylation of cysteine. Tyrosine nitration of GCK prevented its association with pBad, and GCK translocation into the mitochondria results in subsequent proteasomal degradation of GCK following ubiquitination. This study identified a novel and efficient pathway by which chronic ethanol consumption may induce GCK down-regulation and inactivation by inducing tyrosine nitration of GCK, resulting in pancreatic β-cell apoptosis and dysfunction. Peroxynitrite-induced ATF3 may also serve as a potent upstream regulator of GCK down-regulation and β-cell apoptosis.

Interleukin-6 is an important mediator for mitochondrial DNA repair after alcoholic liver injury in mice

We investigated the hypothesis that a prominent effect of chronic ethanol consumption is mitochondrial DNA (mtDNA) injury and compared this injury in IL-6 knockout (KO) and wild-type (WT) mice. Ethanol feeding for 4 weeks resulted in steatosis and oxidative mtDNA damage (8-OHdG) in both IL-6KO and WT mice. However, the WT mice were able to repair the injury by increased production of mtDNA repair enzymes (OGG-1, Neil 1) and check point (p21, p53) proteins and avoid the mtDNA mutations. By contrast the IL-6 KO mice were unable to repair mtDNA resulting in deletions and diminished transcription of the mtDNA encoded protein cytochrome c oxidase subunit-I (COI). The mitochondrial injury was reflected by decreased membrane potential, reduced levels of ATP, and apoptosis-inducing factor (AIF)-induced apoptosis. IL-6 plays a critical role in allowing the liver to recover from significant mtDNA oxidation caused by alcohol. The data suggests that IL-6 activates mtDNA repair enzymes and induces cell cycle arrest allowing time for mtDNA repair.

O efeito do consumo crônico de etanol na absorção duodenal de ferro em camundongos

Alcoholists present an increase of iron hepatic concentration, although the responsible mechanisms for this deposition are still unknown. Despite the extensive literature related on the iron absorption in different pathological conditions, the effect of chronic ethanol consumption are still not conclusive and not completely understood. To verify the effect of chronic ethanol ingestion on duodenal absorption of iron. Ten male Swiss mice were divided into two groups: group 1 (n = 5) - control, and group 2 (n = 5) - water consumption with ethanol, as only water source. The animals were followed during 120 days. After this period, the duodenum was isolated and saline solution containing ascorbate of iron II in the 0,016 concentration of mg of iron element was infused. The effluent was collected in times 20, 40, 60, 80, 100 and 120 minutes. The results were analyzed by Mann-Whitney and Kruskal-Wallis tests. The significance was set for P<0.05. No difference was found between iron absorption as well as iron absorption curves in groups 1 and 2. The chronic consumption of ethanol did not alter iron absorption.

Chronic alcoholism associated with diabetes impairs erectile function in rats

To investigate the effects of chronic ethanol consumption and diabetes on nitric oxide (NO)-mediated relaxation of cavernosal smooth muscle (CSM). Male Wistar rats were divided into four groups: control, isocaloric, diabetic and ethanol-diabetic. The CSMs were mounted in organ chambers for measurement of isometric tension. Contraction of the strips was induced by electrical field stimulation (EFS, 1-32 Hz) and phenylephrine. We also evaluated the effect of ethanol consumption on the relaxation induced by acetylcholine (ACh; 0.01-1000 micromol/L), sodium nitroprusside (SNP, 0.01-1000 micromol/L) or EFS (1-32 Hz) in strips pre-contracted with phenylephrine (10 micromol/L). Immunoexpression of endothelial NO synthase (eNOS) and inducible NOS (iNOS) was also accessed. The endothelium-dependent relaxation induced by ACh was decreased in CSM from ethanol-diabetic rats when compared with the controls, with a mean (sem) of 21 (4) vs 37 (2)%. Similarly, the potency and maximal responses induced by SNP were reduced in the ethanol-diabetic [3.97 (0.38) and 85 (1)%, respectively] and diabetic groups [3.78 (0.56) and 81 (2)%, respectively] when compared with the controls [5.3 (0.22) and 90 (3)%, respectively] and isocaloric [5.3 (0.19) and 92 (1)%, respectively] groups. Noradrenergic nerve-mediated contractions of CSM in response to EFS were increased in rats from ethanol-diabetic and diabetic groups when compared with the control and isocaloric groups. Conversely, there were no differences in EFS-induced relaxation among the groups. The immunostaining assays showed overexpression of eNOS and iNOS in the CSM from diabetic and ethanol-diabetic rats when compared with the control and isocaloric rats. There was an impairment of relaxation of CSM from ethanol-diabetic and diabetic rats that involved a decrease in the NO-cyclic guanosine monophosphate signalling pathway by endothelium-dependent mechanisms accompanied by a change in the CSM contractile sensitivity.

Effects of chronic ethanol consumption on levels of adipokines in visceral adipose tissues and sera of rats

To investigate the effects of ethanol on adipokines (leptin, adiponectin, resistin, visfatin and cartonectin) levels in visceral adipose tissue (VAT) and sera, and explore the correlation between VAT and serum adipokine levels. Forty-eight Wistar rats were randomly divided into control, low, middle and high ethanol treatment groups that received 0, 0.5, 2.5, or 5.0 g of ethanol x kg(-1) x d(-1), respectively, via gastric tubes for 22 weeks. The levels of fasting blood glucose (FBG) and fasting serum insulin (FINS) were measured and homeostasis model assessment of insulin resistance (HOMA-IR) values were calculated. Adipokines in perirenal and epididymal VAT and sera were measured by enzyme-linked immunosorbent assays (ELISAs). High-dose treatments of ethanol (vs control group) significantly increased FINS (eg 37.86%) and HOMA-IR values (eg 40.63%). In VAT, levels of leptin, resistin and visfatin in the middle- and high-dose groups were significantly elevated, whereas adiponectin and cartonectin levels decreased. In sera, changes in adipokine levels were similar to that observed in VAT, with the exception of cartonectin. These ethanol-induced effects were dose-dependent. A positive correlation existed between VAT and serum adipokine levels, except for cartonectin. Chronic ethanol consumption affects adipokine levels in VAT and sera in a dose-dependent manner, with the exception of serum cartonectin. The altered levels of adipokines in VAT and sera are positively correlated.

Is alcohol beneficial or harmful for cardioprotection?

While the effects of chronic ethanol consumption on liver have been well studied and documented, its effect on the cardiovascular system is bimodal. Thus, moderate drinking in many population studies is related to lower prevalence of coronary artery disease (CAD). In contrast, heavy drinking correlates with higher prevalence of CAD. In several other studies of cardiovascular mortalities, abstainers and heavy drinkers are at higher risk than light or moderate drinkers. The composite of this disparate relation in several population studies of cardiovascular mortality has been a "U-" or "J-"shaped curve. Apart from its ability to eliminate cholesterol from the intima of the arteries by reverse cholesterol transport, another major mechanism by which HDL may have this cardioprotective property is by virtue of the ability of its component enzyme paraoxonase1 (PON1) to inhibit LDL oxidation and/or inactivate OxLDL. Therefore, PON1 plays a central role in the disposal of OxLDL and thus is antiatherogenic. Furthermore, PON1 is a multifunctional antioxidant enzyme that can also detoxify the homocysteine metabolite, homocysteine thiolactone (HTL), which can pathologically cause protein damage by homocysteinylation of the lysine residues, thereby leading to atherosclerosis. We demonstrated that moderate alcohol up regulates liver PON1 gene expression and serum activity, whereas heavy alcohol consumption had the opposite effects in both animal models and in humans. The increase in PON1 activity in light drinkers was not due to preferential distribution of high PON1 genotype in this group. It is well known that wine consumption in several countries shows a remarkable inverse correlation to local rates of CAD mortality. Significantly, apart from its alcohol content, red wine also has polyphenols such as quercetin and resveratrol that are also known to have cardioprotective effects. We have shown that quercetin also up regulates PON1 gene in rats and in human liver cells. The action of quercetin seems to be mediated via the active form of the nuclear lipogenic transcription factor, sterol-regulatory element-binding protein 2 (SREBP2) that is translocated from endoplasmic reticulum to the nucleus. However, the mechanism of action of ethanol-mediated up-regulation of PON1 gene remains to be elucidated. We conclude that both moderate ethanol and quercetin, the two major components of red wine, exhibit cardioprotective properties via the up-regulation of the antiatherogenic gene PON1.

Differential Effects of Chronic Ethanol Consumption and Withdrawal on Homer/Glutamate Receptor Expression in Subregions of the Accumbens and Amygdala of P Rats

Homer proteins are constituents of scaffolding complexes that regulate the trafficking and function of central Group1 metabotropic glutamate receptors (mGluRs) and N-methyl-d-aspartate (NMDA) receptors. Research supports the involvement of these proteins in ethanol-induced neuroplasticity in mouse. In this study, we examined the effects of short versus long-term withdrawal from chronic ethanol consumption on Homer and glutamate receptor protein expression within striatal and amygdala subregions of selectively bred, alcohol-preferring P rats. For 6 months, male P rats had concurrent access to 15% and 30% ethanol solutions under intermittent (IA: 4 d/wk) or continuous (CA: 7 d/wk) access conditions in their home cage. Rats were killed 24 hours (short withdrawal: SW) or 4 weeks (long withdrawal: LW) after termination of ethanol access, subregions of interest were micropunched and tissue processed for detection of Group1 mGluRs, NR2 subunits of the NMDA receptor and Homer protein expression. Within the nucleus accumbens (NAC), limited changes in NR2a and NR2b expression were detected in the shell (NACsh), whereas substantial changes were observed for Homer2a/b, mGluRs as well as NR2a and NR2b subunits in the core (NACc). Within the amygdala, no changes were detected in the basolateral subregion, whereas substantial changes, many paralleling those observed in the NACc, were detected in the central nucleus (CeA) subregion. In addition, most of the changes observed in the CeA, but not NACc, were present in both SW and LW rats. Overall, these subregion specific, ethanol-induced increases in mGluR/Homer2/NR2 expression within the NAC and amygdala suggest changes in glutamatergic plasticity had taken place. This may be a result of learning and subsequent memory formation of ethanol's rewarding effects in these brain structures, which may, in part, mediate the chronic relapsing nature of alcohol abuse.

Chronic Ethanol Consumption Induces Cavernosal Smooth Muscle Dysfunction in Rats

To investigate the effects of chronic ethanol consumption on nitric oxide (NO)-mediated relaxation in rat cavernosal smooth muscle (CSM). Male wistar rats were divided into 2 groups: control and ethanol. CSM obtained from both groups were mounted in organ chambers for measurement of isometric tension. Contraction of the strips was induced by electrical field stimulation (EFS, 1-32 Hertz) and phenylephrine. We also evaluated the effect of ethanol consumption on the relaxation induced by acetylcholine (0.01-1000 micromol L(-1)), sodium nitroprusside (SNP, 0.01-1000 micromol L(-1)), or EFS (1-32 Hz) in strips precontracted with phenylephrine (10 micromol L(-1)). Blood ethanol, serum testosterone levels, and basal nitrate generation were determined. Immunoexpression of endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) was also accessed. Ethanol intake for 4 weeks significantly increased noradrenergic nerve-mediated contractions of CSM in response to EFS. The endothelium-dependent relaxation induced by acetylcholine decreased after the ethanol treatment. Ethanol consumption decreased serum testosterone levels but did not affect the nitrate levels on rat CSM. The mRNA and protein levels for eNOS and iNOS receptors were increased in CSM from ethanol-treated rats. Ethanol consumption reduces endothelium-dependent relaxation induced by acetylcholine, but does not affect SNP or EFS-induced relaxation, suggesting that ethanol disrupts the endothelial function. Despite the overexpression of eNOS and iNOS in ethanol-treated rats, the impaired relaxation induced by acetylcholine may suggest that chronic ethanol consumption induces endothelial dysfunction.

Effects of chronic ethanol consumption in blood: A time dependent study on rat.

Alcohol consumption and health outcomes are complex and multidimensional. Ethanol (1.6g / kg body weight/ day) exposure initially affects liver function followed by renal function of 16-18 week-old male albino rats of Wistar strain weighing 200-220 g. Chronic ethanol ingestion increased in thiobarbituric acid reactive substances level and glutathione s-transferase activity; while decreased reduced gluatathione content and activities of catalase, glutathione peroxidase and glutathione reductase in a time dependent manner in the hemolysate. Though superoxide dismutase activity increased initially might be due to adaptive response, but decreased later. Elevation of serum nitrite level and transforming growth factor-b(1) activity indicated that long-term ethanol consumption may cause hepatic fibrosis and can elicit pro-angiogenic factors. However, no alteration in vascular endothelial growth factor-C activity indicated that ethanol consumption is not associated with lymphangiogenesis. Therefore, we conclude that long-term ethanol-induced toxicity is linked to an oxidative stress, which may aggravate to fibrosis and elevate pro-angiogenic factors, but not associated with lymphangiogenesis.

Overexpression of Peroxiredoxin 6 Does Not Prevent Ethanol-Mediated Oxidative Stress and May Play a Role in Hepatic Lipid Accumulation

Oxidative stress is implicated in the etiology of many diseases, including alcoholic liver disease (ALD). Peroxiredoxin 6 is a cytosolic peroxidase that has been demonstrated to protect various tissues, such as skin, lung, and cardiac muscle, against acute oxidative insults. Consequently, peroxiredoxin 6 was hypothesized to also protect the liver from oxidative stress generated during the process of chronic ethanol ingestion. To test this, wild-type peroxiredoxin 6 knockout mice (KO), and transgenic peroxiredoxin 6 overexpressing mice (TG) were fed an ethanol-containing diet. Various biomarkers of ALD were assessed, along with the effects of chronic ethanol consumption on the antioxidant defenses. After 9 weeks of ethanol consumption, all backgrounds exhibited elevations of plasma alanine aminotransferase activity, hepatosteatosis, CYP2E1 induction, and lipid peroxidation; however, hepatic triglyceride accumulation seemed to be exacerbated in ethanol-fed TG mice. Differences in antioxidant protein expression and activity in response to chronic ethanol consumption were also observed. Examples include significant inductions of catalase and glutathione transferase activity in ethanol-fed KO and TG mice, along with elevated levels of glutathione peroxidase activity. These alterations in antioxidant defenses could be attributed to either compensatory responses due to the genetic manipulations or ethanol-mediated responses. In conclusion, both ethanol-fed KO and ethanol-fed TG mice developed early stage ALD and peroxiredoxin 6 may play a role in ethanol-mediated hepatic lipid accumulation.

Betaine (Trimethylglycine) as a nutritional agent prevents oxidative stress after chronic ethanol consumption in pancreatic tissue of rats

In this study, we investigated the free radical-mediated cytotoxic effects of chronic ethanol consumption on the pancreatic tissue and a possible cytoprotective effect of betaine as a methyl donor and an important participant in the methionine cycle. Twenty-four male Wistar rats were divided into control, ethanol, and ethanol+betaine groups. Prior to sacrifice, all groups were fed 60 mL/diet per day for two months. Rats in the ethanol group were fed with ethanol 8 g/kg/day. The ethanol+betaine groups were fed ethanol plus betaine (0.5 % w/v). Malondialdehyde levels and adenosine deaminase, superoxide dismutase, and xanthine oxidase activities were determined in pancreatic tissues of rats. Compared to control group, MDA levels increased significantly in the ethanol group (p<0.05). MDA levels in the ethanol+betaine group were significantly decreased compared to the ethanol group (p<0.05). ADA activity in the ethanol+betaine group decreased significantly when compared to the ethanol group (p<0.05). XO activities in ethanol-fed rats were decreased significantly compared to the control group (p<0.05). XO activity in the betaine group was increased significantly (p<0.05) compared to the ethanol group. SOD activity in the ethanol group decreased significantly compared to control group (p<0.001). SOD activity in the ethanol+betaine group decreased significantly (p<0.05) compared to the control group. We think that betaine, as a nutritional methylating agent, may be effective against ethanol-mediated oxidative stress in pancreatic tissue.

Chronic ethanol exposure increases the association of hippocampal mu-opioid receptors with G-protein receptor kinase 2

Opioid receptors (ORs) have been shown to have a significant role in the central nervous system (CNS) effects of chronic ethanol consumption. The OR antagonist, naltrexone, is used clinically to reduce continued intake. We previously observed that chronic ethanol consumption, by adult male Sprague-Dawley rats, induced a reduction in functional coupling of mu- and delta-ORs to G-proteins in rat CNS regions, including the hippocampus. G-protein receptor kinase (GRK) 2 phosphorylates G-protein coupled receptors, including ORs, after agonist binding, as part of normal regulation and desensitization. We tested the hypothesis that chronic ethanol exposure affects the association of the GRK2 with the mu-OR. Co-immunoprecipitation methods were used to determine if mu-OR association with GRK2 is elevated in the hippocampus after chronic ethanol, when compared to controls. Hippocampal homogenates from chronic ethanol and pair-fed control rats were treated with affinity-purified rabbit polyclonal antibodies (ab) to mu-OR, and immune complexes were probed for GRK2 by immunoblotting techniques. Results demonstrate an association of GRK2 with mu-ORs in chronic ethanol-treated rats, but not in the controls. Possible changes in GRK2 association with ORs after chronic ethanol may be related to levels of phosphorylation and subsequent trafficking of the receptors.

Altered pattern of Na,K-ATPase activity and mRNA during chronic alcohol consumption by juvenile and adolescent rats.

The effect of chronic ethanol consumption on cerebral cortical activity of Na,K-ATPase was determined in Long-Evans (LE) rats fed an ethanol-containing diet beginning at different stages of development. Na,K-ATPase activity was operationally resolved into alpha1 and alpha2/3 isozyme activities. There was no significant difference in Na,K-ATPase activities before and after alcohol consumption in the preparations from adult rats. However, for rats beginning alcohol consumption as adolescents, the alpha2/3 activity was significantly elevated following chronic alcohol consumption. Both LE and Sprague-Dawley rats showed this same selective increase in cortical alpha2/3 activity when rats began alcohol consumption as juveniles. The shift in cortical alpha2/3 activity was not observed in cerebellum or subcortical forebrain and was reversible when rats were fed ethanol throughout the normal adolescent period and then withdrawn and tested 2 weeks later (during the adult period). Levels of isoform-specific mRNA were determined in preparations of cerebral cortices of rats showing elevated alpha2/3 isozyme activities. In these preparations, isoform specific alpha2 and alpha3 mRNA was significantly elevated. There was no effect of ethanol feeding on cortical alpha1 mRNA. These findings indicate that the longer term effects of ethanol on the developing brain include elevated Na,K-ATPase activity and a mechanism that is pre-translational and isoform specific.

Tissue-specific expression of ALA synthase-1 and heme oxygenase-1 and their expression in livers of rats chronically exposed to ethanol

5-Aminolevulinic acid synthase-1 (ALAS1) and heme oxygenase-1 (HO-1) are the rate-controlling enzymes for heme biosynthesis and degradation, respectively. Expression of these two genes showed tissue-specific expression pattern at both mRNA and protein levels in selected non-treated rat tissues. In the livers of rats receiving oral ethanol for 10 weeks, ALAS1 mRNA levels were increased by 65%, and the precursor and mature ALAS1 protein levels were increased by 1.8- and 2.3-fold, respectively, while no changes were observed in HO-1 mRNA and protein levels, compared with pair-fed controls. These results provide novel insights into the effects of chronic ethanol consumption on hepatic heme biosynthesis and porphyrias.

Effects of chronic ethanol consumption on memory and molecular changes in hippocampus of young adult wistar rats

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Abrogation of the Antifibrotic Effects of Natural Killer Cells/Interferon-γ Contributes to Alcohol Acceleration of Liver Fibrosis

Chronic alcohol drinking accelerates liver fibrosis in patients with viral hepatitis that cannot be fully explained by ethanol-enhanced liver damage. Here, we identified a novel mechanism by which alcohol accelerates liver fibrosis: inhibition of the antifibrotic effects of natural killer (NK) cells and interferon-gamma (IFN-gamma). Alcohol administration was achieved by feeding mice with a liquid diet containing 5% ethanol for 8 weeks. Liver fibrosis was induced by administration of carbon tetrachloride (CCl(4)) for 2 weeks. Hepatic stellate cells (HSCs) were also isolated and cultured for in vitro studies. CCl(4) treatment induced greater fibrosis and less apoptosis of HSCs in ethanol-fed mice compared with pair-fed mice. Polyinosinic-polycytidylic acid (Poly I:C) or IFN-gamma treatment inhibited liver fibrosis in pair-fed but not in ethanol-fed mice. Poly I:C activation of NK cell cytotoxicity against HSCs was attenuated in ethanol-fed mice compared with pair-fed mice, which was due to reduced natural killer group 2 member D (NKG2D), tumor necrosis factor-related apoptosis-inducing ligand, and IFN-gamma expression on NK cells from ethanol-fed mice. In vitro, HSCs from ethanol-fed mice were resistant to IFN-gamma-induced cell cycle arrest and apoptosis compared with pair-fed mice. Such resistance was due to diminished IFN-gamma activation of signal transducer and activator of transcription 1 (STAT1) in HSCs from ethanol-fed mice caused by the induction of suppressors of cytokine signaling proteins and the production of oxidative stress. Finally, HSCs from ethanol-fed mice were resistant to NK cell killing, which can be reversed by transforming growth factor-beta1 (TGF-beta1) neutralizing antibody. Chronic ethanol consumption attenuates the antifibrotic effects of NK/IFN-gamma/STAT1 in the liver, representing new and different therapeutic targets with which to treat alcoholic liver fibrosis.

Effects of Chronic Ethanol Consumption on Brain Synaptosomes and Protective Role of Betaine

To evaluate the cytotoxic effects of chronic ethanol consumption on brain cerebral synaptosomes and preventive role of betaine as a methyl donor and S-adenosylmethionine precursor, 24 male Wistar rats were divided into three groups: control, ethanol (8 g/kg/day) and ethanol plus betaine(0.5% w/v) group. Animals were fed 60 ml/diet per day for two months, then sacrificed. Malondialdehyde (MDA), protein carbonyl contents and adenosine deaminase (ADA) activities were determined in synaptosomal/mitochondrial enriched fraction isolated from rat cerebral cortexes. When compared to controls, ethanol containing diet significantly increased MDA levels (P < 0.05), also increased protein carbonyl levels and adenosine deaminase activities. But these were not statistically significant (P > 0.05). However, adding betaine to ethanol containing diet caused a significant decrease in MDA, protein carbonyl levels and adenosine deaminase activities (P < 0.05). These results indicate that betaine may appear as a protective nutritional agent against cytotoxic brain damage induced by chronic ethanol consumption.

Effects of 4-hydroxynonenal on mitochondrial 3-hydroxy-3-methylglutaryl (HMG-CoA) synthase

Chronic ethanol consumption causes increased production of reactive oxygen species in hepatic mitochondria accompanied by elevations in products of lipid peroxidation such as 4-hydroxynonenal (4-HNE). In the current study we investigated the effects of chronic ethanol consumption on a prominent protein–4-HNE adduct in liver mitochondria. Male Sprague-Dawley rats were fed a liquid diet for 31 days in which ethanol constituted 36% of total calories. Immunoblot analyses of liver mitochondria from ethanol-fed and control animals, using an antibody to a 4-HNE–protein adduct, demonstrated elevated 4-HNE binding (+ 50%) to a mitochondrial protein of ∼ 55 kDa due to chronic ethanol consumption. Analysis of this protein using AspN digestion and tandem mass spectrometry identified it as the mitochondrial form of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. Activity of the activated form of this enzyme was unchanged in livers from ethanol-fed animals, but the protein level was elevated by 36%, which suggests a compensatory mechanism to maintain constant levels of synthase activity in the mitochondrion in the face of continuous inactivation by 4-HNE. Treatment of isolated mitochondria with 4-HNE demonstrated that the enzyme activity decreased as a function of 4-HNE concentration and with time of exposure. This study demonstrates that ethanol consumption increases the formation of a 4-HNE adduct with mitochondrial HMG-CoA synthase, which has the potential to inactivate the enzyme in situ.

Effect of chronic ethanol consumption on the expression of complement components and acute-phase proteins in liver

The complement system can provoke but also participate in the repair of liver injury. Here we investigated by microarray analysis the effect of chronic ethanol consumption on hepatic mRNA expression of complement components and acute-phase proteins in complement C3-deficient ( C3 −/−) and wild-type ( C3 +/+) mice. Up-regulation by ethanol of factor B, C1qA-chain and clusterin but down-regulation of factor H, Masp-2, factor D and the terminal components C6, C8α and C9 was seen in both strains. Ethanol up-regulated C2 and down-regulated C4bp only in C3 +/+ mice, while in C3 − /− mice up-regulation of C1qB-chain and vitronectin was observed. The expression of factor B, C6, C1qB and factor I was lower but that of factor D higher in C3 − /− than in C3 +/+ mice. Ethanol induced mRNA synthesis of many acute-phase proteins including SPARC and lipocalin-2, but reduced the expression of SAP. The induction of early classical and alternative pathway components and suppression of terminal pathway components and soluble regulators may thus contribute to alcohol-induced liver injury. Lipocalin-2 and SPARC emerge as new candidate markers for early detection of liver damage.