Ethanol-induced Injury Research Articles

A Bicarbonate-Alkaline Mineral Water Protects from Ethanol-Induced Hemorrhagic Gastric Lesions in Mice

Ingestion of elevated amounts of ethanol in humans and rodents induces hemorrhagic gastric lesions, at least in part by increasing oxidative stress. The present study was undertaken in order to evaluate the influence of a bicarbonate-alkaline mineral water (Uliveto on ethanol-induced hemorrhagic gastric lesions in mice. Lesions were evaluated by both macroscopic and microscopic analysis. In a first set of experiments, mice were allowed to drink Uliveto or reference water ad libitum until 3 h prior to intragastric (i.g.) ethanol (23 ml/kg) administration. Neither Uliveto nor reference water did afford any protection. In a second set of experiments, acute exposure to reference water (35 ml/kg, i.g.), given 30 min before ethanol, did not inhibit gastric lesions. However, administration of the same amount of Uliveto caused a remarkable reduction in ethanol-evoked gastric lesions. Ethanol administration increased 4-hydroxy-2-nonenal levels, a byproduct of oxidative stress, in the luminal part of the gastric mucosa. This response was substantially reduced by about 70% by Uliveto, but not by reference water. Reference water, added with the bicarbonate content, present in the Uliveto water, protected against ethanol-induced lesions. Thus, acute pre-exposure to bicarbonate-alkaline mineral water (Uliveto) protects from both oxidative stress and hemorrhagic gastric lesions caused by ethanol. The elevated bicarbonate content of Uliveto likely accounts for the protection against ethanol-induced gastric injury.

Inhibition of endogenous hydrogen sulfide synthesis by PAG protects against ethanol-induced gastric damage in the rat

Hydrogen sulfide (H(2)S) is a gaseous mediator involved in a multitude of physiological functions; however the role of H(2)S in the gut is far from being understood completely. The aim of this study was to determine the effect of d-l-propargylglycine (PAG), an inhibitor of H(2)S synthesis, on ethanol-induced gastric injury in rat and to examine the role of l-cysteine, exogenous H(2)S, prostaglandins, non-protein sulphydryls groups, nitric oxide and K(ATP) channels in the gastroprotective effect of PAG. Administration of PAG (3.12 to 75mg/kg i.p.) or l-cysteine (0.3 to 300mg/kg, p.o.) exhibited a dose-dependent protective effect after intragastric administration of 1ml of ethanol to induce gastric injury. The gastroprotective effect of PAG (25mg/kg i.p.) was maintained after post-treatment with l-cysteine (10mg/kg p.o.), while NaHS (8.4mg/kg p.o.) inhibited this effect. The levels of gastric hydrogen sulfide were increased after ethanol-induced gastric damage and they were reverted by PAG while prostaglandin E(2) levels in gastric tissue were decreased by ethanol and PAG did not revert to this effect. Pretreatment with indomethacin (10mg/kg i.p.) and N-ethylmaleimide (NEM, 10mg/kg s.c.) resulted in a reversion of the gastroprotective effect of PAG while N(G)-nitro-l-arginine methyl ester (L-NAME, 70mg/kg s.c.), glibenclamide (1mg/kg i.p.) or diazoxide (3mg/kg i.p.) did not induce any changes. These results suggest that ethanol-induced gastric injury is related with an increment of endogenous H(2)S levels, and therefore a decrement of H(2)S levels by PAG is a benefit to protect gastric injury caused by ethanol.

Gastroprotective effect of barbatusin and 3-beta-hydroxy-3-deoxibarbatusin, quinonoid diterpenes isolated from Plectranthus grandis, in ethanol-induced gastric lesions in mice

Ethnopharmacological relevance Validate the popular use of Plectranthus grandis in gastric disorders through the active components. Aims Isolation of barbatusin (BB) and 3β-hydroxy-3-deoxibarbatusin (BBOH), diterpenes from Plectranthus grandis, and evaluation of their gastroprotective effect and possible mechanisms of action. Materials and methods Isolation and chemical characterization of diterpenes from Plectranthus grandis by chromatographic and spectroscopic methods and evaluation of gastroprotective action of the diterpenes through ethanol-induced gastric injury in mice model. It was evaluated the effect of capsazepine, indomethacin and the role of nitric oxide and K ATP− channels on the gastroprotective effect of BBOH and BB. Additionally it was measured the concentrations of gastric mucus, non-proteic-sulfhydryl groups and total thiobarbituric acid-reactive substances. Results Orally administered BBOH and BB at doses of 5 and 10 mg/kg, markedly reduced the gastric lesions by 59 and 96%, and 32 and 76%, respectively, with superior results as compared to N-acetylcysteine (150 mg/kg, i.p.), reference compound that caused 85% lesion suppression. Although BBOH presented a higher gastroprotection than BB they act by similar mechanisms in relation to N-acetylcysteine, and prevent the depletion of gastric mucus, gastric mucosal non-proteic-sulfhydryl groups as well as the increase in thiobarbituric acid-reactive species. Moreover, the gastroprotective effect of BB was effectively blocked in mice pretreated with TRPV1 antagonist capsazepine, by the non-selective cyclooxygenase inhibitor indomethacin, or by the nitric oxide synthase inhibitor l-NAME but not by K + ATP channel inhibitor glibenclamide. In contrast, the gastroprotective effect of BBOH was blocked only by indomethacin and glibenclamide pretreatments. Conclusion The protective role for BBOH and BB affording gastroprotection against gastric damage induced by ethanol indicates that these compounds contribute for the activity of Plectranthus species. The different modes of action are probably related to differences in their chemical structure.

Isolation and Identification of Urinary Metabolites of Kakkalide in Rats

Kakkalide is a major isoflavonoid from the flowers of Pueraria lobata (Willd.) Ohwi, possessing the protective effect against ethanol-induced intoxication and hepatic injury. The metabolism of kakkalide was investigated in rats. Thirteen metabolites were isolated by using solvent extraction and repeated chromatographic methods and identified by using spectroscopic methods including UV, IR, mass spectrometry, NMR, and circular dichroism experiments. Four new compounds were identified as irisolidone-7-O-glucuronide (M-1), tectorigenin-7-O-sulfate (M-2), tectorigenin-4'-O-sulfate (M-3), and biochanin A-6-O-sulfate (M-4) together with nine known compounds identified as irisolidone (M-5), tectorigenin (M-6), tectoridin (M-7), 5,7-dihydroxy-8,4'-dimethoxyisoflavone (M-8), isotectorigenin (M-9), biochanin A (M-10), genistein (M-11), daidzein (M-12), and equol (M-13). The metabolic pathway of kakkalide was proposed, which is important to understand its metabolic fate and disposition in humans.

Protective effect of a polysaccharide from Hizikia fusiformis against ethanol-induced cytotoxicity in IEC-6 cells

In the present study, we examined the signaling pathways related to the ethanol-protective effect of Hf-PS-1 in IEC-6 cells. Ethanol induced the death of IEC-6 cells in a dose-dependent manner, and pretreatment with Hf-PS-1 abrogated the ethanol toxicity. When we examined whether the effect of Hf-PS-1 on ethanol cytotoxicity was associated with insulin growth factor-I receptor signaling pathways, involving mitogen-activated protein kinase (MAPK), we found that ethanol treatment decreased the phosphorylation of Shc and the binding of Grb2 to Shc, and Hf-PS-1 pretreatment increased them. Ethanol treatment also induced the phosphorylation of JNK and ERK, whereas Hf-PS-1 pretreatment decreased JNK activation but not ERK activation. Using a JNK inhibitor (SP600125), we examined GSH levels to determine whether Hf-PS-1 pretreatment mi20 ght protect against ethanol-induced gastric intestinal damage by down-regulating JNK. Co-treatment with SP600125 and ethanol decreased GSH levels, indicating that JNK phosphorylation is a critical factor during ethanol-induced injury and that the effect of Hf-PS-1 occurs via JNK down-regulation. We have thus demonstrated the protective effect of Hf-PS-1 against ethanol-induced cellular damage. Therefore, Hf-PS-1 may be useful as a bio-functional food source to protect against ethanol-induced gastrointestinal injury.

Formation of γ-ketoaldehyde–protein adducts during ethanol-induced liver injury in mice

Ethanol metabolism promotes the formation of a variety of reactive aldehydes in the liver. These aldehydes can rapidly form covalent protein adducts. Accumulating evidence indicates that these protein adducts may contribute to ethanol-mediated liver injury. Overproduction of γ-ketoaldehydes, levuglandins (LGs) and isolevuglandins, is implicated in the pathogenesis of several chronic inflammatory diseases. γ-Ketoaldehydes can form protein adducts orders of magnitude more quickly than 4-hydroxynonenal (4-HNE) or malondialdehyde. We hypothesized that ethanol-induced oxidative stress in vivo results in overproduction of LGE 2– and iso[4]LGE 2–protein adducts in mouse liver. Female C57BL/6 mice were allowed free access to an ethanol-containing diet for up to 39 days or pair-fed control diets. Pathological markers of ethanol-induced hepatic injury including serum alanine aminotransferase, hepatic triglyceride, and CYP2E1 were elevated in response to ethanol feeding. Ethanol-induced formation of iso[4]LGE 2–, LGE 2–, and 4-HNE–protein adducts in mouse liver was dependent on both dose and duration of ethanol feeding. Deficiency of cyclooxygenase 1 or 2 did not prevent ethanol-induced iso[4]LGE 2 or LGE 2 adducts in the liver, but adduct formation was reduced in both TNFR1- and CYP2E1-deficient mice. In summary, ethanol feeding enhanced γ-ketoaldehyde–protein adduct production via a TNFR1/CYP2E1-dependent, but cyclooxygenase-independent, mechanism in mouse liver.

The phytotoxic effect of exogenous ethanol on Euphorbia heterophylla L.

This study investigated the effects of exogenously applied ethanol on Euphorbia heterophylla L., a troublesome weed in field and plantation crops. Ethanol at concentrations ranging from 0.25 to 1.5% caused a dose-dependent inhibition of germination and growth of E. heterophylla. Measurements of respiratory activity and alcohol dehydrogenase (E.C. 1.1.1.1) activity during seed imbibition and initial seedling growth revealed that ethanol induces a prolongation of hypoxic conditions in the growing tissues. In isolated mitochondria, ethanol inhibited the respiration coupled to ADP phosphorylation, an action that probably contributed to modifications observed in the respiratory activity of embryos. A comparison of the effects of methanol, ethanol, propanol and acetaldehyde on germination and growth of E. heterophylla indicates that alcohol dehydrogenase activity is required for the observed effects, with the conversion of ethanol to acetaldehyde playing a role in the ethanol-induced injuries.

Effects of Zinc on Intestinal Injury and Some Serum Parameters in Ethanol-Administered Rats

This study was designed to determine the morphological and biochemical effects of zinc sulfate and the role of metallothionein in ethanol-induced intestinal injury. Rats received zinc sulfate (100 mg/kg/d) for 3 consecutive d, 2 h prior to the administration of ethanol by gavage. Ethanol administration caused intestinal injury as determined by increased serum lactate dehydrogenase activity, urea, creatinine, uric acid, and sialic acid levels, intestinal lipid peroxidation level, decreased serum catalase activity, intestinal glutathione level, and metallothionein expression. Zinc sulfate pretreatment of the ethanol group caused a decrease in histological damage, serum lactate dehydrogenase activity, urea, creatinine, uric acid, sialic acid levels, and intestinal lipid peroxidation level, but increases in serum catalase activity, intestinal glutathione level, and metallothionein expression. The present study indicates that zinc sulfate has a protective effect against ethanol-induced intestinal injury. In addition, the protective effect of zinc on ethanol-induced intestinal injury might be mediated by metallothionein, as well as having antioxidative potential.

Potential Role of the Placenta in Fetal Alcohol Spectrum Disorder

Mediators of susceptibility to alcohol-related toxicity in the prenatal environment are relatively unknown. The placenta has been proposed as a potential source of variability in the fetal environment, primarily through its significant metabolic capabilities. Meconium is a toxicological matrix unique to the developing fetus that offers an opportunity to quantify fetal exposure to alcohol through the analysis of fatty acid ethyl esters (FAEE) [nonoxidative ethanol metabolites]. To explore the role of the placenta as a potential source of variability in mediating fetal alcohol exposure via meconium analysis of FAEE, by combining a clinical case report involving human dizygotic twins and a series of guinea pig littermates. Meconium was collected from a pair of dizygotic twins with clinical suspicion of prenatal alcohol exposure and analyzed for FAEE to confirm prenatal alcohol exposure. Meconium was also collected from a series of guinea pig pups prenatally exposed to alcohol at a daily dose of 4 g/kg/day administered to the pregnant dam. FAEE levels were analyzed by gas chromatography with flame ionization detection following liquid-liquid extraction from meconium. The dizygotic twin pair yielded positive meconium FAEE results (>2.00 nmoL/g) in the female twin (2.21 nmoL/g) and no detectable FAEE in the male twin. A total of 15 meconium samples were collected from 15 pups of five pregnant guinea pig litters. With the exception of one pair of littermates, meconium FAEE concentrations differed substantially within each litter (FAEE expressed in units of nmoL/g): litter 1 (0.996, 4.43, 1.36); litter 2 (5.17, 4.15, 0.00); litter 3 (5.16, 5.27); litter 4 (18.57, 8.26, 7.46); litter 5 (0.00, 4.32, 0.00, 1.27). Identical maternal ethanol exposure levels produced differing levels of fetal exposure in a dizygotic human twin pair and a series of guinea pig littermates as evidenced through FAEE meconium analysis. These data indicate that the placenta may have a previously unappreciated role in mediating ethanol-induced fetal injury.

Taurine supplementation prevents ethanol-induced decrease in serum adiponectin and reduces hepatic steatosis in rats #

Chronic ethanol feeding decreases expression of adiponectin by adipocytes and circulating adiponectin. Adiponectin treatment during chronic ethanol feeding prevents liver injury in mice. Chronic ethanol feeding also increases oxidative and endoplasmic reticulum (ER) stress in adipose tissue. Here we tested the hypothesis that supplemental taurine, an amino acid that functions as a chemical chaperone/osmolyte and enhances cellular antioxidant activity, would prevent ethanol-induced decreases in adiponectin expression and attenuate liver injury. Serum adiponectin concentrations decreased as early as 4 to 7 days after feeding rats a 36% ethanol diet. This rapid decrease was associated with increased oxidative, but not ER, stress in subcutaneous adipose tissue. Taurine prevented ethanol-induced oxidative stress and increased inflammatory cytokine expression in adipose tissue. Ethanol feeding also rapidly decreased expression of transcription factors regulating adiponectin expression (CCAAT/enhancer binding protein alpha; peroxisome proliferator-activated receptor alpha/gamma) in subcutaneous adipose tissue. Taurine prevented the ethanol-induced decrease in CCAAT/enhancer binding protein alpha and peroxisome proliferator-activated receptor alpha, normalizing adiponectin messenger (m)RNA and serum adiponectin concentrations. In the liver, taurine prevented ethanol-induced oxidative stress and attenuated tumor necrosis factor alpha expression and steatosis, at least in part, by increasing expression of genes involved in fatty acid oxidation. In subcutaneous adipose tissue, taurine decreased ethanol-induced oxidative stress and cytokine expression, as well as normalized expression of adiponectin mRNA. Taurine prevented ethanol-induced decreases in serum adiponectin; normalized adiponectin was associated with a reduction in hepatic oxidative stress, tumor necrosis factor alpha expression, and steatosis. Taken together, these data demonstrate that taurine has important protective effects against ethanol-induced tissue injury in both adipose and liver tissue.

A study on Cordyceps militaris polysaccharide purification, composition and activity analysis

In this study, raw Cordyceps militaris polysaccharide (CPS) was extracted with ultrasonic- assisted extraction method. Proteins in the raw CPS were removed with the enzymatic and Sevag methods. After dialysis, an extraction rate of 0.82% was achieved. Two components, namely CPS-1 and CPS-2, were obtained from DEAE-52 ionic exchange chromatography eluted with NaCl solution at a gradient from 0 to 0.6 mol•L-1. The ultraviolet scanning demonstrated that CPS-1 did not couple with protein and nucleic acid, but CPS-2 was coupled with a few proteins and nucleic acids. After product derivatization using acid hydrolysis, gas chromatography analysis revealed that these two CPSs were both composed of mannose and galactose, with a mole ratio of 0.96:1 and 1.04:1, respectively. The two CPSs can reverse ethanol-induced injury in primary cultured rat hepatocytes. Its effect appeared dose-dependent, having better effect when higher dose was applied. Its effect is probably due to its anti-oxidation function.

Protective effects of a polysaccharide from Hizikia fusiformis against ethanol toxicity in rats

Hizikia fusiformis is an edible brown alga that is widely consumed in Korea, Japan, and China and possesses a number of potentially beneficial compounds, including antioxidants and anticoagulants. No reports have investigated potential H. fusiformis protectants against ethanol-induced peptic injury. We extracted a polysaccharide from H. fusiformis ( Hf–PS-1) that exhibited protective effects against ethanol-induced peptic injury and related mechanisms in rats. Experimental animals were divided into three groups: control, ethanol-only, and ethanol + Hf–PS-1. The ethanol-only group exhibited decreased levels of total glutathione (GSH) and increased levels of jun N-terminal kinase (JNK) phosphorylation relative to the control group, whereas levels were significantly increased and decreased, respectively, in the ethanol + Hf–PS-1 group. The ethanol-only group also exhibited increased levels of extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation relative to the control group; these levels were not significantly different in the ethanol + Hf–PS-1 group. Hf–PS-1 appeared to reduce ethanol-induced gastric injury. Therefore, we suggest that Hf–PS-1 could protect against ethanol-induced peptic ulcers primarily through a mechanism associated with the inhibition of JNK activation.

Acid-sensitive channel inhibition prevents fetal alcohol spectrum disorders cerebellar Purkinje cell loss

Ethanol is now considered the most common human teratogen. Educational campaigns have not reduced the incidence of ethanol-mediated teratogenesis, leading to a growing interest in the development of therapeutic prevention or mitigation strategies. On the basis of the observation that maternal ethanol consumption reduces maternal and fetal pH, we hypothesized that a pH-sensitive pathway involving the TWIK-related acid-sensitive potassium channels (TASKs) is implicated in ethanol-induced injury to the fetal cerebellum, one of the most sensitive targets of prenatal ethanol exposure. Pregnant ewes were intravenously infused with ethanol (258+/-10 mg/dl peak blood ethanol concentration) or saline in a "3 days/wk binge" pattern throughout the third trimester. Quantitative stereological analysis demonstrated that ethanol resulted in a 45% reduction in the total number of fetal cerebellar Purkinje cells, the cell type most sensitive to developmental ethanol exposure. Extracellular pH manipulation to create the same degree and pattern of pH fall caused by ethanol (manipulations large enough to inhibit TASK 1 channels), resulted in a 24% decrease in Purkinje cell number. We determined immunohistochemically that TASK 1 channels are expressed in Purkinje cells and that the TASK 3 isoform is expressed in granule cells of the ovine fetal cerebellum. Pharmacological blockade of both TASK 1 and TASK 3 channels simultaneous with ethanol effectively prevented any reduction in fetal cerebellar Purkinje cell number. These results demonstrate for the first time functional significance of fetal cerebellar two-pore domain pH-sensitive channels and establishes them as a potential therapeutic target for prevention of ethanol teratogenesis.

Ablation of primary afferent neurons by neonatal capsaicin treatment reduces the susceptibility of the portal hypertensive gastric mucosa to ethanol-induced injury in cirrhotic rats

Primary sensory afferent neurons modulate the hyperdynamic circulation in cirrhotic rats with portal hypertension. The stomach of cirrhotic rats is prone to damage induced by ethanol, a phenomenon associated with reduced gastric hyperemic response to acid-back diffusion. The aim of this study was to examine the impact of ablation of capsaicin-sensitive neurons and the tachykinin NK1 receptor antagonist A5330 on the susceptibility of the portal hypertensive gastric mucosa to ethanol-induced injury and its effects on gastric cyclooxygenase (COX) and nitric oxide synthase (NOS) mRNA expression. Capsaicin was administered to neonatal, male, Wistar rats and the animals were allowed to grow. Cirrhosis was then induced by bile duct ligation in adult rats while controls had sham operation. Ethanol-induced gastric damage was assessed using ex vivo gastric chamber experiments. Gastric blood flow was measured as well as COX/NOS mRNA expression. Topical application of ethanol produced significant gastric damage in cirrhotic rats compared to controls, which was reversed in capsaicin- and A5330-treated animals. Mean arterial and portal pressure was normalized in capsaicin-treated cirrhotic rats. Capsaicin and A5330 administration restored gastric blood flow responses to topical application of ethanol followed by acid in cirrhotic rats. Differential COX and NOS mRNA expression was noted in bile duct ligated rats relative to controls. Capsaicin treatment significantly modified gastric eNOS/iNOS/COX-2 mRNA expression in cirrhotic rats. Capsaicin-sensitive neurons modulate the susceptibility of the portal hypertensive gastric mucosa to injury induced by ethanol via tachykinin NK1 receptors and signalling of prostaglandin and NO production/release.

Alleviation of acute ethanol-induced liver injury and impaired metabolomics of S-containing substances by betaine supplementation

Oxidative stress is suggested to play a key role in the development of alcoholic liver injury. We investigated the induction of oxidative damage in association with changes in hepatic concentrations of sulfur-containing substances in mice challenged with binge-like ethanol administration. Also the protective effect of dietary betaine against ethanol-induced liver injury was determined. Serum alanine aminotransferase activity, TNFα level, and hepatic malondialdehyde level were increased significantly by ethanol administration. Hepatic Cyp2e1 was induced to 250% of control. Ethanol administration decreased hepatic S-adenosylmethionine, cysteine, and glutathione, but elevated hypotaurine and taurine levels. Betaine supplied in drinking water for 2 weeks attenuated the induction of alcoholic liver injury and Cyp2e1 significantly. Reduction of hepatic S-adenosylmethionine and glutathione was alleviated, and elevation of hypotaurine and taurine was depressed. The results suggest that betaine may protect the liver against ethanol-induced oxidative injury most probably via its effects on the sulfur–amino acid metabolism.

NF-κB-activated tissue transglutaminase is involved in ethanol-induced hepatic injury and the possible role of propolis in preventing fibrogenesis

The increased expression and cross-linking activity of tissue transglutaminase (tTG) have been demonstrated in acute liver injury and fibrosis. We focused on the molecular mechanisms that contribute to ethanol-induced tTG expression and investigated the efficacy of propolis components in preventing both the tTG expression in vitro and fibrogenesis in vivo. We demonstrate herein that both ERK1/2 and PI3K/Akt pathways can regulate the effects of ethanol on NF-κB-dependent transcription and these signaling pathways may be involved in activation of ethanol-mediated tTG expression. We also found that administration of pinocembrin (PIN), one of the major components of propolis, inhibited tTG activation and significantly prevented the development of thioacetamide (TAA)-induced liver cirrhosis. The present study suggests that tTG may be an important member of the cascade of factors necessary for ethanol-induced liver fibrogenesis and PIN could serve as an anti-fibrogenic agent.

Oxidative stress disturbs energy metabolism of mitochondria in ethanol-induced gastric mucosa injury

To study the role of mitochondrial energy disorder in the pathogenesis of ethanol-induced gastric mucosa injury. Wistar rats were used in this study. A gastric mucosal injury model was established by giving the rats alcohol. Gross and microscopic appearance of gastric mucosa and ultrastructure of mitochondria were evaluated. Malondiadehyde (MDA) in gastric mucosa was measured with thiobarbituric acid. Expression of ATP synthase (ATPase) subunits 6 and 8 in mitochondrial DNA (mtDNA) was determined by reverse transcription polymerase chain reaction (RT-PCR). The gastric mucosal lesion index was correlated with the MDA content in gastric mucosa. As the concentration of ethanol was elevated and the exposure time to ethanol was extended, the content of MDA in gastric mucosa increased and the extent of damage aggravated. The ultrastructure of mitochondria was positively related to the ethanol concentration and exposure time. The expression of mtDNA ATPase subunits 6 and 8 mRNA declined with the increasing MDA content in gastric mucosa after gavage with ethanol. Ethanol-induced gastric mucosa injury is related to oxidative stress, which disturbs energy metabolism of mitochondria and plays a critical role in the pathogenesis of ethanol-induced gastric mucosa injury.

Combined Effects of Vitamin C, Vitamin E, and Sodium Selenate Supplementation on Absolute Ethanol-Induced Injury in Various Organs of Rats

In this study, the effect of combination of vitamin C (ascorbic acid), vitamin E (alpha -tocopherol), and selenium (sodium selenate) on ethanol-induced liver and intestine injury in rats was investigated. The ethanol-induced injury was produced by the administration of 1 ml of absolute ethanol to each rats. Animals received vitamin C (250 mg/kg), vitamin E (250 mg/kg), and sodium selenate (Se) (0.5 mg/kg) for 3 days; 1 h after the final antioxidant administration, they were sacrificed. Lipid peroxidation and glutathione levels, catalase (CAT), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and glutathione peroxidase (GP(x)) activities were determined in liver and intestine tissues. Myeloperoxidase (MPO), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT) were determined in liver tissue. Also, CAT activity, urea, creatinine, uric acid, and total lipid levels were determined in serum samples. In the ethanol group, serum urea, creatinine, uric acid, and total lipid levels; liver and intestine LDH; liver MPO, AST, ALP, ALT, and GGT activities; and liver and intestine LPO levels increased, whereas serum CAT activity, liver and intestine GSH levels, and CAT, SOD, and GP(x) activities decreased. On the other hand, treatment with vitamin C, vitamin E, and Se reversed these effects. As a result of these findings, we can say that the combination of vitamin C, vitamin E, and selenium has a protective effect on ethanol-induced changes in lipid peroxidation, glutathione levels, and antioxidant enzyme activities in liver and intestine tissues, and in some serum parameters of rats.

Gastric mucosal protection against ethanol by EP2and EP4signaling through the inhibition of leukotriene C4production

Prostaglandin (PG)E derivatives are widely used for treating gastric mucosal injury. PGE receptors are classified into four subtypes, EP(1), EP(2), EP(3), and EP(4). We have tested which EP receptor subtypes participate in gastric mucosal protection against ethanol-induced gastric mucosal injury and clarified the mechanisms of such protection. The gastric mucosa of anesthetized rats was perfused at 2 ml/min with physiological saline, agonists for EP(1), EP(2), EP(3), and EP(4), or 50% ethanol, using a constant-rate pump connected to a cannula placed in the esophagus. The gastric microcirculation of the mucosal base of anesthetized rats was observed by transillumination through a window made by removal of the adventitia and muscularis externa. PGE(2) and subtype-specific EP agonists were applied to the muscularis mucosae at the window. Application of 50% ethanol dilated the mucosal arterioles and constricted the collecting venules. Collecting venule constriction by ethanol was completely inhibited by PGE(2) and by EP(2) and EP(4) agonists (100 nM) but not by an EP(1) or an EP(3) agonist. Ethanol-induced mucosal injury was also inhibited by EP(2) and EP(4) agonists. When leukotriene (LT)C(4) levels in the perfusate of the gastric mucosa were determined by ELISA, intragastric ethanol administration elevated the LTC(4) levels sixfold from the basal levels. These elevated levels were significantly (60%) reduced by both EP(2) and EP(4) agonists but not by other EP agonists. Since LTC(4) application at the window constricted collecting venules strongly, and an LTC antagonist reduced ethanol-induced mucosal injury, reductions in LTC(4) generation in response to EP(2) and EP(4) receptor signaling may be relevant to the protective action of PGE(2). The present results indicate that EP(2) and EP(4) receptor signaling inhibits ethanol-induced gastric mucosal injury through cancellation of collecting venule constriction by reducing LTC(4) production.

Ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells: Role of fatty acid ethyl esters

To understand the mechanism(s) of alcoholic pancreatitis and role of fatty acid ethyl esters (FAEEs, non-oxidative metabolites of ethanol) in ethanol-induced pancreatic injury. A time- and concentration-dependent synthesis of FAEEs and the cytotoxicity of ethanol and its predominant fatty acid esters were studied in rat pancreatic tumour (AR42J) cells in cultures. Role of FAEEs in ethanol-induced cytotoxicity was investigated by measuring the synthesis of FAEEs, injury markers and apoptosis in cells incubated simultaneously with ethanol and FAEE synthase inhibitor, 3-benzyl-6-chloro-2-pyrone. The cells were pre-incubated with caspase-3 inhibitor (N-acetyl-DEVD-CHO) to measure the effect of caspase-3 inhibition on ethanol-induced apoptosis. The levels of FAEEs synthesized in cell cultures incubated with 800 mg% ethanol for 6 h were approximately 10-fold higher (60 nmol/25 x 10(6) cells) than those in cells incubated with 100 mg% ethanol (5.4 nmol/25 x 10(6) cells). Ethanol exposure resulted in a concentration-dependent apoptosis (10, 12 and 13% at 200, 400 and 800 mg% ethanol, respectively, vs 5% in controls). A similar concentration-dependent apoptosis was also found in the cells incubated with ethyl oleate (one of the predominant FAEEs reported in alcoholic patients). Inhibition of FAEE synthesis and resultant apoptosis was found in the cells incubated simultaneously with pancreatic FAEE synthase inhibitor and ethanol. Ethanol-induced apoptosis was significantly inhibited in cells pre-incubated with caspase-3 inhibitor. These results support our hypothesis that ethanol-induced cytotoxicity in AR42J cells is mediated by the non-oxidative metabolite(s) of ethanol, and caspase-3 mediated apoptosis could be one of the mechanisms involved in ethanol-induced pancreatic injury.