Alcohol toxicity is one of the major health problems worldwide . Ethanol is a fat-soluble nonelectrolyte, which is readily absorbed from the gastrointestinal tract . After ethanol is absorbed, it is distributed to all tissues and body fluids in direct proportion to the blood levels . Ethanol affects most vital functions of almost all organs including the liver, kidney, brain, pancreas, and intestine . Ethanol causes extensive damage to the intestinal tract from the oropharynx to the rectum . However, alcohol liver disease is a medical complication of alcohol abuse worldwide . Acute exposure of the small intestine to alcohol may induce a variety of structural and functional abnormalities, ranging from mucosal hemorrhages, erosions, and induction of blebs, to upset active transport mechanisms [6,7]. Also, ethanol metabolites may directly or indirectly induce oxidative stress as a result of a disturbed balance between pro-oxidative and antioxidative processes . Oxidative stress refers to an enhanced generation of reactive oxygen species (ROS) and/or depletion in the antioxidant defense system .
Ethanol administration is accompanied by morphological liver changes that include the increased production of apoptotic cells [10,11]. Some characteristics of the molecular control of apoptosis have emerged recently. Perhaps one of the most significant among these is the expression of the gene Bcl-2, which can delay, inhibit, or regulate apoptosis. Bcl-2 can confer a survival advantage to rapidly proliferating cells . Bcl-2 plays a critical role in the development of the immune system, as well as attenuation of apoptosis; therefore, when Bcl-2 is in excess, almost all cells are protected . Inhibition of apoptosis by Bcl-2 is considered to occur through its interactions with a proapoptotic homologue, bax .
Pathogenesis of ethanol-induced injury in the small intestine involves the generation of ROS, resulting in enhanced lipidperoxidation . The free radicals released initiate lipid peroxidation by attacking polyunsaturated fatty acids in the cell membrane . Malondialdehyde (MDA) is one of the biomarkers of lipid peroxidation [17,18].
ROS generated in the tissues are efficiently scavenged by the enzymatic antioxidant system such as catalase (CAT) and superoxide dismutase (SOD) and by nonenzymatic antioxidants such as vitamins A, C, and E . SOD and CAT counteract free radicals and thereby protect the cell membrane against free radical-mediated peroxidation. SOD also catalyzes the destruction of superoxide anion free radicals .
Aim of the work
The aim of the present study was to determine the possible protective effect of vitamins C and E on ethanol-induced histopathological changes in rat liver and jejunum, lipid peroxidation, and antioxidant enzyme activities in these tissues and some serum parameters.
Materials and methods
Sixty adult male albino rats of an average weight (150–200 g) were obtained from the breeding animal house in Menofia governorate. They were kept under good hygienic conditions and maintained at normal room temperature. The rat diet included standard animal food and tap water ad libitum.
Drugs and chemicals
Ethyl alcohol (99%) was obtained from the El-Gomhoureya Pharmaceutical Company (Cairo, Egypt).
Vitamin C: 1 g effervescent tablets were obtained from Cid Pharmaceutical Company (Cairo, Egypt). The calculated dose was dissolved in 25 ml distilled water.
Vitamin E: 400 mg oily capsules were obtained from Pharco Pharmaceutical Company (Cairo, Egypt). Then, the estimated dose was dissolved in 8 ml corn oil.
Experimental design and treatment of animals
Animal experiments were carried out ethically following the guidelines set by Ethical Committee of Menofia University. The animals were divided randomly into four equal groups of 15 animals each.
Group I: The control group was administered the same amount of vehicles (distilled water and corn oil) orally during the entire experiment (4 days).
Group II: The control group received vitamins C and E (250 mg/kg/day) for 4 days.
Group III: Rats were administered 5 ml/kg 10% ethanol solution for 4 days.
Group IV: Animals received vitamins C and E (250 mg/kg/day) 1 h before the administration of a 10% ethanol solution. The ethanol solution and antioxidants were administered orally to each animal using a curved needle-like tube to be introduced directly into the stomach (a gavage process) [21,22].
At the end of the experiment, blood samples were taken 1 h after the administration of ethanol solution from the retro-orbital plexus using a heparinized capillary tube and were allowed to percolate along the wall of the centrifuge tube to prevent the risk of hemolysis . The animals were then sacrificed by cervical dislocation. Specimens of the right lobe of liver and the first part of jejunum were excised then immersed in isotonic saline. The tissues were divided and subjected to the following studies.
Specimen of the right lobe of the liver and the first part of the jejunum were fixed in 10% formal-saline and then processed to obtain paraffin blocks, from which 5-μm-thick sections were cut and stained with H&E and PAS .
For this study, 4-μm-thick paraffin sections were mounted on glass slides coated on pol-l-lysine, deparaffinized, and rehydrated. Sections were incubated in hydrogen peroxide (3% H2O2 in absolute methyl alcohol) for 10–15 min in a humidity chamber. For antigen retrieval, the heat-induced epitope retrieval (HIER) procedure was used (New Marker immune histopathology catalogue, 2000). Ultra V Block was applied for 5 min. Sections were incubated overnight with the monoclonal primary antibody raised against Bcl-2 (diluted at a 1:50), Dako, code M 0887 (Clone 124), (Dakocytomation, Copenhagen, Denmark). After washing with PBS, biotinylated secondary anti-immunoglobulin (LSAB 2 system-HRP; Dakocytomation, Copenhagen, Denmark) was applied for 40 min at room temperature. The specimens were washed in PBS and incubated with streptavidin peroxidase for 10 min. While the slides were in PBS, the DAB chromogen substrate was prepared and then applied on slides for 3 min. Finally, the specimens were counterstained with Mayer's hematoxylin. Normal lymphoid tissue was used as a positive control. Negative control was obtained by omitting the primary antibody step; consequently, no immunostaining occurred .
In this study, biochemical investigations were carried out on serum, liver, and intestinal tissues. In the serum, the following were assayed: CAT, alanine transaminase (ALT), aspartate transaminase (AST), and total serum lipid. One milliliter of blood was withdrawn, and transferred slowly into a plain tube. The sample was centrifuged for 5 min at 3000 rpm. The clear supernatant was separated and kept frozen at 20°C until analyses of the previous parameters were carried out. Colorimetric determinations of AST and ALT activity were carried out using the Biomereiux and colorimetric determination of total lipids with a sulfophosphovanillin mixture (Biomerieux Vitek Inc., Biomerieux Vitek Inc. Dourham, France) [26,27].
Specimens of the right lobe of the liver and the first part of the jejunum were kept frozen until the day of the experiment. These tissue samples were homogenized in cold 0.9% NaCl with a glass homogenizer to make up 10% (w/v) tissue homogenate. The homogenates were centrifuged and the supernatant fraction was removed for the determination of CAT, SOD, and MDA .
Kinetic colorimetric determination of CAT in the serum, liver, and jejunal tissue homogenates was carried out with hydrogen peroxide as a substrate. The decomposition of H2O2 was followed directly by a decrease in extinction at 240 nm at 25°C . The protein content in the supernatant was estimated using BSA as a standard . Colorimetric determination of MDA in the liver and jejunal homogenate was carried out, in which tissue homogenate was boiled with thiobarbituric acid; the colored product obtained was measured at 532 nm . Kinetic colorimetric determination of SOD activity was measured at 460 nm, at 25°C, and absorbance readings were taken at 0 and 8 min of illumination .
Biochemical data were expressed as arithmetic mean ± SD. The Student test was used to test the significant change in each biochemical parameter in the experimental animals of groups II, III, and IV in comparison with the corresponding control animals of group I, as well as in group III and IV rats in comparison with group II. The statistical analysis of data was carried out using Excel and Statistical Package for the Social Science Software, version 11 (SPSS, Inc., Chicago, USA.) on an IBM compatible computer . Results were considered significant when the P-value was less than 0.05.
Histopathology of the liver
Light microscopic examination of the liver sections of the control group I stained by H&E showed that the hepatocytes were arranged in the form of branching and anatomizing cords radiating from the central vein and extending to the periphery of the hepatic lobule. The hepatocytes were polygonal in shape and had an acidophilic granular cytoplasm and vesicular nuclei. The cords of cells were separated by blood sinusoids, which were lined by flat endothelial cells (Fig. 1). Normal glycogen distribution was detected by PAS stain in the cytoplasm of hepatocytes (Fig. 2). Nuclei and cytoplasm of almost all hepatocytes showed negative Bcl-2 immunoreactivity; thus, they appeared blue in color (Fig. 3). The histological profile of control group II was more or less comparable to that of control group I.
In ethanol-treated rats in group III, different forms of liver injury were found. The liver section showed loss of normal hepatic architecture with vacuolated hepatocytes, congested blood vessels, lymphocytic infiltration, and an empty space inside hepatocyte cells with a signet ring nucleus, most probably microvesicular steatosis (Fig. 4). Hepatic sections stained by PAS showed variable degrees of glycogen depletion in some hepatocytes (Fig. 5). The nuclei and cytoplasm of degenerated hepatocytes showed moderate immunoreactivity for Bcl-2. They appeared brown in color (Fig. 6).
In group IV, almost normal liver architecture was observed, except in small areas, so that most hepatocytes were normal and some cells were degenerated (Fig. 7). Glycogen distribution returned normal in most hepatocytes, whereas some cells still showed glycogen depletion (Fig. 8). Mild Bcl-2 immunoreactivity was observed in the nuclei and the cytoplasm of some hepatocytes. They appeared brown in color, whereas other hepatocytes showed negative Bcl-2 immunoreactivity in their nuclei and cytoplasm, which appeared blue in color (Fig. 9).
Biochemical data of the liver and serum
Table 1 presents the activity of serum CAT, ALT, AST, and total serum lipids of the experimental animals (groups I, II, III, and IV). It also shows liver CAT, SOD, and MDA. The serum CAT level in the ethanol group III was significantly reduced compared with the control group (P2<0.001 and P4<0.001). In group IV (given ethanol, vitamin C, and E), CAT activity was elevated compared with group III (P<0.001). Serum ALT and AST were found to be markedly increased in group III compared with control groups I and II (P2<0.001 and P4<0.001). Both were significantly reduced in group IV (P<0.001). The total serum lipid level was significantly elevated in the ethanol group (P2<0.001 and P4<0.001). The administration of antioxidants led to a significant decrease in their levels compared to group III (P<0.001).
Liver CAT and SOD activities were significantly decreased in the ethanol group compared with the control group (P2<0.001 and P4<0.001). Vitamins C and E led to a marked increase in liver CAT and SOD activities of group IV (P<0.001). In terms of liver MDA, it was significantly increased as compared with the control groups (P2<0.001 and P4<0.001). After administration of vitamins C and E, a significant decrease in liver MDA was observed when compared with the ethanol group (P<0.001).
Histopathology of the jejunum
Light microscopic study of H&E-stained jejunal sections of control group I showed that the jejunum consisted of three layers: the mucosa, submucosa, and musculosa (Fig. 10). The mucosa showed villi and crypts. The villi appeared as tapering finger-like projections from the surface covered by simple columnar epithelium (enterocytes) with goblet cells (Fig. 11). Using PAS with the hematoxylin counterstain, the brush border of columnar cells covering the villi and goblet cells showed a PAS-positive magenta red color in the jejunal villi and crypts (Fig. 12). Bcl-2 in the nuclei and cytoplasm of columnar cells covering the villi showed the same moderate immunoreactivity, except at the tip of the villi for all groups. The nuclei and cytoplasm of cells in the jejunal crypts showed negative immunoreactivity; thus, they appeared bluish in color (Fig. 13). The histological profile of control group II was more or less comparable to that of control group I.
In ethanol-treated rats in group III, different forms of tissue injury were found. Study of H&E-stained sections showed shortening broadening as well as fusion of the villi; inflammatory infiltrate was observed in lamina propria extending to the submucosa, with separation and denudation of the surface epithelium from the underlying connective tissue at the tip of the villi because of the presence of subepithelial space. In addition, shedding of the epithelial covering and desquamation of some cells in the intestinal lumen were observed (Figs 14–16). With the PAS stain, no goblet cells were found in the epithelial covering of the apparently damaged intestinal villi, but many goblet cells were observed in the intestinal crypts (Fig. 17) as compared with the control group (Fig. 12). The nuclei and cytoplasm of cells of jejunal crypts showed moderate Bcl-2 immunoreactivity (Fig. 18).
In contrast, in animals of group IV, pretreated with vitamins C and E and then challenged with ethanol, the histological changes were less as compared with group III. H&E-stained sections showed that some villi were tapering and covered with simple columnar epithelium and goblet cells. The inflammatory infiltrate in the core of villi was less observed, with no subepithelial space (Fig. 19), compared with the ethanol group (Fig. 16). There was a mild PAS positive reaction of goblet cells in intestinal villi. They appeared magenta red in color (Fig. 20). Immunoreactivity was mild positive in the nuclei and cytoplasm of cells of jejunal crypts (Fig. 21).
Biochemical data of the intestine
Intestinal CAT and SOD in the ethanol group was significantly decreased compared with the control groups (P2<0.001 and P4<0.001), which increased after the administration of antioxidants (group IV) in comparison with group III (P<0.001). The rats in the ethanol group showed an increased level of intestinal MDA as compared with the control groups (P2<0.001 and P4<0.001). In group IV, the administration of antioxidants before alcohol led to a significant reduction in intestinal MDA in relation to group III (P<0.001) as reported in Table 2.
Ethanol is a direct systemic toxin that induces injury to all tissues depending on the dose, duration of exposure, and the affected organ. Ethanol consumption and the increase in oxidative stress that is associated with its metabolism lead to the development of a number of illnesses in both hepatic and extrahepatic tissues . Supplementation of antioxidant inhibited acute alcohol-induced fatty liver and lipid peroxidation . Also, it has been applied widely to prevent alcoholic liver injury in both animal models and human clinical trials .
This study shows the possible protective effect of vitamins C and E, as vitamin C is associated with the regeneration of α-tocopherol. It supplies electrons necessary for hydroperoxide reductase that directly reduces α-tocopheroxy radical to α-tocopherol, thus contributing toward the protection of cellular membranes . Vitamin E (α-tocopherol) is the primary membrane-bound, lipid-soluble, chain-breaking antioxidant that has been reported to protect against lipid peroxidation-induced tissue damage . It has two important functions in the membrane: as a liposoluble antioxidant that prevents ROS damage in polyunsaturated fatty acids and also as a membrane stabilizer agent acting against the damage caused by phospholipids .
Vitamins C and E significantly attenuate histopathological changes in the liver and jejunum as well as the biochemical parameters induced by ethanol in rats. Furthermore, it provides data that may explain the potential mechanisms of vitamin C-induced and vitamin E-induced protection in the liver and jejunum. Our results are in agreement with the previous reports .
An intact intestinal mucosa is critical for barrier function. Acute ethanol ingestion at a high concentration may lead to alterations in small intestinal barrier function, which facilitates the absorption of endotoxin from rat small intestine, which may play a critical role in alcohol-induced liver injury [38,39].
One of the most common histopathological changes in the rat liver after acute ethanol administration is fatty liver (microvesicular steatosis), which was suggested in this study. Fatty liver is potentially reversible . Defects in fat metabolism are responsible for the pathogenesis of fatty liver disease, which may be because of an imbalance in energy consumption and its combustion. Energy imbalance may result in lipid storage or may be a consequence of peripheral resistance to insulin. The transport of fatty acids from adipose tissue to the liver is increased, resulting in lipolysis, and an increase in triglyceride synthesis, resulting in hyperlipidemia . Ethanol oxidation by the alcohol dehyrogenase pathway results in the production of NADH, which might contribute toward enhanced lipid synthesis . In the present study, it was reflected by a significant elevation in the total serum lipid level in ethanol-treated rats and a significant decrease in their levels after the administration of vitamins E and C, with the absence of hepatic steatosis in the antioxidant group. It appears that an antioxidant supplement could be used to improve the lipid metabolism.
In ethanol-treated rats, different forms of liver injury were found. The liver sections showed cytoplasmic vacuolation, nuclear dissolution, lymphocytic infiltration, and dilated congested central veins. The same changes have been observed by pervious workers [39,43]. They reported that the liver is the major target of ethanol toxicity and the role of oxidative stress in the pathogenesis of liver injury has been confirmed repeatedly. Oxidative stress plays an important role in the pathogenesis of ethanol toxicity. The close relation between ethanol and liver is because of the fact that more than 80% of ingested ethanol is metabolized in the liver without a feedback mechanism. In addition, acetaldehyde, which is a metabolite of ethanol, when increased in level, markedly altered the intracellular redox status, induced fat deposits, and triggered the inflammatory and immune responses . In this study, supplementation of antioxidants was shown to prevent ethanol induced injury in the liver. The same result has been reported by others .
In the present study, some depletion of the glycogen content of the liver was found in the ethanol-treated group of rats. This finding was in agreement with previous researchers  who concluded that excessive ethanol intake impairs carbohydrate metabolism. This impairment is believed to involve the inhibition of the intestinal absorption of nutrients. Also, ethanol-induced perturbation in the redox state is considered to be an important factor in the inhibition of hepatic gluconeogenesis. However, the administration of Vitamins C and E before ethanol intake led to improved glycogen storage in the liver as compared with ethanol-intoxicated rats.
Different histological changes were found in the jejunal sections of the rats treated with ethanol including the formation of short broad villi, subepithelial space, lymphocytic infiltration, hemorrhage, and denudation of surface epithelium. These changes can be considered as injuries to the intestinal villi and are in accordance with the reports of others [47,48]. However, massive degeneration of duodenal villi, hemorrhage, partial villous atrophy, increase in lamina propria infiltrate, and intraepithelial lymphocytes have been reported by others as the main findings in the alcoholic group [39,49]. It may be possible that the intestinal histological changes and dysfunction in acute ethanol toxicity may be because of decreased intestinal barrier function against small and large antigens, with the latter implicating an increase in transcellular permeability through epithelial layer disruption. Others  have suggested that reduced intestinal blood flow may be a causative factor for villi injury. In the present study, these histopathological findings mostly normalized with the uptake of vitamins C and E before acute ethanol toxicity.
In the present study, decreased number of goblet cells was found in rats of the ethanol-treated group. This result was in agreement with the results obtained by other workers  who reported inhibited cell production in the jejunum and distal aspect of the ileum and goblet cells after the administration of ethanol. The increase in the number of goblet cells after the administration of vitamins C and E in rats represents a protective mechanism by producing excess mucous. The increase in goblet cells may be attributed to an increase in the DNA and protein content (mucoprotein) in the jejunal mucosa .
Bcl-2 was located at the mitochondrial membrane and cristae, nuclear envelope, plasma membrane, and endoplasmic reticulum . It is abundantly expressed in the thymus, spleen, and cerebrum, but not in the liver . Bcl-2 is not expressed in hepatocytes under normal physiological conditions . In the present study, the control liver showed no immunoreactivity for Bcl-2. This can be attributed to the fact that apoptosis is rare in normal liver tissues. This was in agreement with other workers  who have reported that there are only two to four apoptotic cells per 10 000 hepatic or biliary cells. In this study, liver sections of the ethanol-treated group showed moderate immunoreactivity to Bcl-2. This was related to ethanol-induced inflammation as well as degeneration of hepatocytes and continuous proliferation and regeneration in response to ethanol stress. Also, it represents an adaptive response to limit liver injury. Other workers  have reported that increased numbers of apoptotic cells were observed in rats developing ethanol-induced pathological liver injury. They reported that increased Bcl-2 protein concentrations are associated with the presence of inflammatory cells and lipid peroxidation and found a significant correlation between Bcl-2 protein and the number of inflammatory cells/mm2. In our study, liver sections of the protective group showed mild Bcl-2 immunoreactivity in some hepatocytes; others were nonimmunoreactive, which could be explained by the fewer number of inflammatory cells and more normal hepatocytes because of the protective effect of vitamins C and E on ethanol-induced liver injury.
In the gastrointestinal tract, Bcl-2 protein expression is not uniform in the intestinal epithelium . In the present study, in the control group, the jejunal crypt cells showed negative immunoreactivity to Bcl-2, whereas the epithelial covering of the villi was immunoreactive to Bcl-2, except near the tip of the villus because of apoptosis of mature cells. This was in agreement with other workers  who observed that Bcl-2 protein was not expressed in the base of crypts of normal small intestine, but may be expressed in the surface epithelial cells of the villus. Other workers  have postulated that, under normal conditions, the absorptive columnar enterocytes, mucus-producing goblet cells, and enteroendocrine cells emerge from the crypt and complete their differentiation as they migrate up to adjacent villi in vertical coherent columns. When mature cells approach the apical extrusion zone of the villus, they enter programmed cell death. In the ethanol group, the jejunal crypt cells showed immunoreactivity to Bcl-2 with increased apoptosis because of the stress of ethanol toxicity but they showed less immunoeactivity after the administration of vitamins C and E before ethanol uptake. This was in accordance with other researchers  who found that Bcl-2 expression plays a significant role in attenuating both spontaneous and induced apoptosis in the crypts of the small intestine that correspond to the stem cell position. The apoptotic deletion of cells in the small intestine, spontaneously or after carcinogen treatment, was greatest at this region, followed by the villi.
In this research, changes in serum CAT were studied in different animal groups. It was observed that the administration of ethanol significantly reduced serum CAT as compared with the control group. The reduced activity of CAT is especially important, because it may lead to the breakdown of antioxidant barrier. This finding is in agreement with that of other workers . Ethanol metabolism is accompanied by the generation of very reactive metabolites, that is acetaldehyde and free radicals, especially superoxide anions, which can inactivate CAT. Moreover, CAT may be inactivated by an l-hydroxyethyl radical, which is another ethanol metabolite . The reduced activity of CAT results in the accumulation of these highly reactive free radicals, leading to deleterious effects such as loss of cell membrane integrity and membrane function. In our study, the administration of antioxidant, that is, vitamins C and E before the administration of ethanol in group IV rats led to significantly elevated CAT activity as compared with those on ethanol treatment alone, which is in agreement with the result reported by another researcher .
Hepatic cells contain several enzymes. AST and ALT are the most sensitive for the diagnosis of hepatic diseases . In the present study, it was observed that rats treated with ethanol showed an increase in the serum levels of ALT and AST. The enzymatic elevation can probably be attributed to the damaged structural integrity of the hepatic cells, because these enzymes are located in the cytoplasm and are released into circulation after cellular damage. If injury involves other organelles, such as the mitochondria, the soluble enzymes such as AST compartmentalized may also be similarly released, indicating that alcohol consumption causes both plasma membrane and organelle membrane damage. In rats administered vitamins C and E before ethanol, a decrease in the levels of serum AST and ALT was observed, indicating the capacity of these vitamins to preserve normal liver structure . In this research, on measuring liver and intestine CAT and SOD, a significant decrease in their activities was observed in group III rats as compared with the control group. Also, a decrease in the level of liver CAT enzyme was found to be correlated to hepatocyte degeneration. In addition, a significant correlation was found between intestinal histopathological changes and the decrease in the level of intestinal CAT enzyme in ethanol-treated animals. This may result in the hampered dismutation of superoxide anions and inefficient detoxification of H2O2, which results in the formation of OH− ions enhancing the peroxidation of membrane lipids, thereby leading to oxidative damage in many tissues . The observed restoration of the SOD and CAT activities on vitamin supplementation may be because of a direct stimulatory effect of vitamins C and E on SOD and CAT.
In this study, liver and intestine MDA was increased in the ethanol group as compared with the control group. The attendant elevation in MDA indicates that the damage is related to increased lipid peroxidation . This is consistent with the findings of a previous study . Again, after the administration of vitamins C and E, in group IV before ethanol gavage, a significant decreased level of liver and intestine MDA was observed. The inhibitory activity of vitamins C and E could result from the scavenging of ROS, which initiate lipid peroxidation .
From the present results, it can be concluded that, ethanol could induce hepatotoxicity as it affected liver function tests, histological structure and immunoreactivity of the liver. Also, ethanol led to histopathological changes on the jejunum and affected the intestinal biochemical parameters. Preadministration of vitamins C and E could protect and reduce the ethanol induced histopathological changes, lipid peroxidation, and antioxidant enzyme activities in liver and jejunal tissues.
We are indebted to Prof. Dr. Magda Dr. Hanan Sleem, lecturer, Department of Histology, Faculty of Medicine, Monoufyia University for providing us with help, assistance, support and experience.
Conflicts of interest
There is no conflict of interest to declare.
Singha PK, Roy S, Dey S. Protective activity of andrographolide and arabinogalactan proteins from Andrographis paniculata
Nees. against ethanol
-induced toxicity in mice. J Ethnopharmacol. 2007;111:13–21
Cho M-H, Shim S-M, Lee S-R, Mar W, Kim G-H. Effect of Evodiae fructus
extracts on gene expressions related with alcohol metabolism and antioxidation in ethanol
-loaded mice. Food Chem Toxicol. 2005;43:1365–1371
Ramírez-Farías C, Madrigal-Santillán E, Gutiérrez-Salinas J, Rodríguez-Sánchez N, Martínez-Cruz M, Valle-Jones I, et al. Protective effect of some vitamins against the toxic action of ethanol
regeneration induced by partial hepatectomy in rats. World J Gastroenterol. 2008;14:899–907
Rajagopal SK, Manickam P, Periyasamy V, Namasivayam N. Activity of Cassia auriculata
leaf extract in rats with alcoholic liver
injury. J Nutr Biochem. 2003;14:452–458
Nakagawa K, Adachi J, Wong MCY, Ueno Y. Protective effect of daidzein against acute ethanol
-induced lipid peroxidation in rat jejunum
. Kobe J Med Scis. 2006;52:141–149
Bode C, Bode JC. Effect of alcohol consumption on the gut. Best Pract Res Clin Gastroenterol. 2003;17:575–592
Bujanda L. The effect of alcohol consumption upon the gastrointestinal tract. Am J Gastroenterol. 2000;95:3374–3382
Stańczyk M, Gromadzińska J, Wąsowicz W. The effect of vitamin C
and glutathione on ethanol
cytotoxicity and selected parameters of pro- and antioxidative processes in mouse fibroblasts 3T3-L1. Pol J Environ Stud. 2006;15:131–137
Devipriya N, Srinivasan M, Sudheer AR, Menon VP. Effect of ellagic acid, a natural polyphenol, on alcohol-induced prooxidant and antioxidant imbalance: a drug dose dependent study. Singapore Med J. 2007;48:311–318
Valente M, Calabrese F. Liver
and apoptosis. Ital J Gastroenterol Hepatol. 1999;31:73–77
Deaciuc IV, Fortunato F, D'Souza NB, Hill DB, Schmidt J, Lee EY, McClain CJ. Modulation of caspase-3 activity and fas ligand mRNA expression in rat liver
cells in vivo
by alcohol and lipopolysaccharide. Alcohol Clin Exp Res. 1999;23:349–356
Athanassios CT, Konstantinos T, Charalambos G, Konstantinos T, Theodore K, Panagiota R, et al. Expresssion of Bcl-2 oncoprotein in cases of acute and chronic viral hepatitis type B and type C: a clinicopathologic study Dig Dis Sci. 2004;47:1618–1624
Guo L-L, Xiao S, Guo Y. Detection of bcl-2 and bax expression and bcl-2/JH fusion gene in intrahepatic cholangiocarcinoma. World J Gastroenterol. 2004;10:3251–3254
Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo
with a conserved homolog, Bax, that accelerates programed cell death. Cell. 1993;74:609–619
Stewart S, Jones D, Day CP. Alcoholic liver
disease: new insights into mechanisms and preventative strategies. Trends Mol Med. 2001;7:408–413
Aydin S, Benian A, Madazli R, Uludag S, Uzun H, Kaya S. Plasma malondialdehyde, superoxide dismutase, sE-selectin, fibronectin, endothelin-1 and nitric oxide levels in women with preeclampsia. Eur J Obstet Gynecol Reprod Biol. 2004;113:21–25
Aksoy H, Taysi S, Altinkaynak K, Bakan E, Bakan N, Kumtepe Y. Antioxidant potential and transferrin, ceruloplasmin, and lipid peroxidation levels in women with preeclampsia. J Investig Med. 2003;51:284–287
Murray RKRobert KM, David AB, Kathleen MB, Peter AM, Victor WR, Anthony PW. Red and white blood cells (chapter 51) Harper Illustrated Biochemistry. 2009 McGraw-Hill:617 In: , editors. p.
Yanardag R, Ozsoy-Sacan O, Ozdil S, Bolkent S. Combined effects of vitamin C
, vitamin E
, and sodium selenate supplementation on absolute ethanol
-induced injury in various organs of rats. Int J Toxicol. 2007;26:513–523
Poranen A-K, Ekblad U, Uotila P, Ahotupa M. Lipid peroxidation and antioxidants in normal and pre-eclamptic pregnancies. Placenta. 1996;17:401–405
MacDonald J, French JE, Gerson RJ, Goodman J, Inoue T, Jacobs A, et al. The utility of genetically modified mouse assays for identifying human carcinogens: a basic understanding and path forward. Toxicol Sci. 2004;77:188–194
Stine KE, Brown TM. Neurotoxicology (chapters 1,9,10, 11, and 12 Principles of toxicology. 20062nd ed. London Taylor and Francis
Halpern RS, Ceaune S COC Bil 2000; 145–146. International federation of clinical chemistry (IFCC).
Stevens A, Wilson IGBancroft JD, Stevens A. The haematoxylin and eosin Theory and practice of histological techniques. 19964th ed. New York Churchill Livingstone:99–112 In: editors. pp.
Sun K, Liu ZS, Sun Q. Role of mitochondria in cell apoptosis during hepatic ischemia-reperfusion injury and protective effect of ischemic postconditioning. World J Gastroenterol. 2004;10:1934–1938
Keiding R, Horder M, Gerhardt W. Recommended methods for the determination of four enzymes in blood. Scand J Clin Lab Investig. 1974;33:291–306
Christopher S, Frings CS, Fendley TW, Dunn RT, Queen CA. Improved determination of total serum lipids by the sulfophosphovanillin reaction. Clin Chem. 1972;18:673–674
Aebi H. Catalase in vitro
. Method Enzymol. 1984;105:121–126
Lowry OH, Rosebrough A, Randal RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951;193:265–275
Ledwozyw A, Michalak J, Stepien A, Kadziolka A. The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clin Chim Acta. 1986;155:275–284
Mylroie AA, Collins H, Umbles C, Kyle J. Erythrocyte superoxide dismutase activity and other parameters of copper status in rats ingesting lead acetate. Toxicol Appl Pharmacol. 1986;82:512–520
Peat J, Barton B. Medical Statistics A Guide to data analysis and critical appraisal. 2005First Edition. Wiley-Blackwell
Di Luzio NR. Prevention of the acute ethanol
-induced fatty liver
by the simultaneous administration of anti-oxidants. Life Sci. 1964;3:113–119
Mullen KD, Dasarathy S. Potential new therapies for alcoholic liver
disease. Clin Liver
Chepda T, Cadau M, Lassabliere F, Reynaud E, Perier C, Frey J, Chamson A. Synergy between ascorbate and α-tocopherol on fibroblasts in culture. Life Sci. 2001;69:1587–1596
Sushma B, Kanwaljit C, Praveen R. Vitamin E
supplementation modulates endotoxin-induced liver
damage in a rat model. Am J Biomed Sci. 2010;2:51–62
Parra-Vizuet J, Camacho-Luis A, Madrigal-Santillan E, Bautista M, Esquivel-Soto J, Esquivel-Chirino C, et al. Hepatoprotective effects of glycine and vitamin E
, during the early phase of liver
regeneration in the rat. Afr J Pharm Pharmacol. 2009;3:384–390
Mathurin P, Deng Q-G, Keshavarzian A, Choudhary S, Holmes EW, Tsukamoto H. Exacerbation of alcoholic liver
injury by enteral endotoxin in rats. Hepatology. 2000;32:1008–1017
Lambert JC, Zhou Z, Wang L, Song Z, McClain CJ, Kang YJ. Preservation of intestinal structural integrity by zinc is independent of metallothionein in alcohol-intoxicated mice. Am J Pathol. 2004;164:1959–1966
Adams LA, Lymp JF, St Sauver J, Sanderson SO, Lindor KD, Feldstein A, Angulo P. The natural history of nonalcoholic fatty liver
disease: a population-based cohort study. Gastroenterology. 2005;129:113–121
Reddy JK, Rao MS. Lipid metabolism and liver
inflammation. II. Fatty liver
disease and fatty acid oxidation. Am J Physiol Gastrointest Liver
Park KJ, Lee MJ, Kang H, Kim KS, Lee S-H, Cho I, Lee HH. Saeng-Maek-San, a medicinal herb complex, protects liver
cell damage induced by alcohol. Biol Pharm Bull. 2002;25:1451–1455
Zhou Z, Sun X, Lambert JC, Saari JT, Kang YJ. Metallothionein-independent zinc protection from alcoholic liver
injury. Am J Pathol. 2002;160:2267–2274
Uzun H, Simsek G, Aydin S, Unal E, Karter Y, Yelmen NK, et al. Potential effects of L-NAME on alcohol-induced oxidative stress. World J Gastroenterol. 2005;11:600–604
Mansouri A, Demeilliers C, Amsellem S, Pessayre D, Fromenty B. Acute ethanol
administration oxidatively damages and depletes mitochondrial DNA in mouse liver
, brain, heart, and skeletal muscles: protective effects of antioxidants. J Pharmacol Exp Ther. 2001;298:737–743
Gopumadhavan S, Mohammed R, Azeemuddin M, Mitra SK. Ameliorative effect of Partysmart in rat model of alcoholic liver
disease. Indian J Exp Biol. 2008;46:132–137
Kaur M, Kaur J, Ojha S, Mahmood A. Ethanol
effects on lipid peroxidation and glutathione-mediated defense in rat small intestine: role of dietary fats. Alcohol. 1998;15:65–69
Peres WAF, Carmo MGT, Zucoloto S, Iglesias AC, Braulio VB. Ethanol
intake inhibits growth of the epithelium in the intestine of pregnant rats. Alcohol. 2004;33:83–89
Bhonchal S, Nain CK, Prasad KK, Nada R, Sharma AK, Sinha SK, Singh K. Functional and morphological alterations in small intestine mucosa of chronic alcoholics. J Gastroenterol Hepatol. 2008;23(Pt 2):e43–e48
Horie Y, Ishii H. Effect of alcohol on organ microcirculation: its relation to hepatic, pancreatic and gastrointestinal diseases due to alcohol. Nihon Arukoru Yakubutsu Igakkai Zasshi. 2001;36:471–485
Javed TM, Saeed MK, Irfan M, Siddique M, Cagiola M. Effect of ethanol
on different organs and on FCR in quails. Pak Vet J. 2008;28:119–124
Soontornchai S, Glinsukon T, Toskulkao C, Tontisirin T. Effect of capsaicin and ethanol
feeding on growth and disaccharidase activity in rats. J Natl Res Council Thailand. 1989;21:18–36
Akao Y, Otsuki Y, Kataoka S, Ito Y, Tsujimoto Y. Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes. Cancer Res. 1994;54:2468–2471
Miyashita T, Krajewski S, Krajewska M, Wang HG, Lin HK, Liebermann DA, et al. Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro
and in vivo
. Oncogene. 1994;9:1799–1805
Souto EO, Miyoshi H, Raymond N, Dubois RN, Gores GJ. Kupffer cell-derived cyclooxygenase-2 regulates hepatocyte Bcl-2 expression in choledocho-venous fistula rats. Am J Physiol Gastrointest Liver
Peng LM, Wang ZL Foundation and clinic of apoptosis. 20001st ed. Beijing Renmin Weisheng Chubanshe:394 p.
Yacoub LK, Fogt F, Griniuviene B, Nanji AA. Apoptosis and bcl-2 protein expression in experimental alcoholic liver
disease in the rat. AlcoholClin Exp Res. 1995;19:854–859
Merritt AJ, Potten CS, Watson AJ, Loh DY, Nakayama K, Nakayama K, Hickman JA. Differential expression of bcl-2 in intestinal epithelia. Correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia. J Cell Sci. 1995;108(Pt 6):2261–2271
Gao C, Wang AY. Significance of increased apoptosis and Bax expression in human small intestinal adenocarcinoma. J Histochem Cytochem. 2009;57:1139–1148
Hall PA, Coates PJ, Ansari B, Hopwood D. Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci. 1994;107(Pt 12):3569–3577
Nakayama K, Negishi I, Kuida K, Sawa H, Loh DY. Targeted disruption of Bcl-2 alpha beta in mice: occurrence of gray hair, polycystic kidney disease, and lymphocytopenia. Proc Natl Acad Sci USA. 1994;91:3700–3704
Polavarapu R, Spitz DR, Sim JE, Follansbee MH, Oberley LW, Rahemtulla A, Nanji AA. Increased lipid peroxidation and impaired antioxidant enzyme function is associated with pathological liver
injury in experimental alcoholic liver
disease in rats fed diets high in corn oil and fish oil. Hepatology. 1998;27:1317–1323
Łuczaj W, Skrzydlewska E. Antioxidant properties of black tea in alcohol intoxication. Food Chem Toxicol. 2004;42:2045–2051
Lee J-S. Supplementation of Pueraria radix
water extract on changes of antioxidant enzymes and lipid profile in ethanol
-treated rats. Clin Chim Acta. 2004;347:121–128
Ae L, Bnrl J, Nf N. Protective effect of Abrus precatorius
seed extract following alcohol induced renal damage. Eur J Sci Res. 2009;25:428–436
Senthilkumar R, Viswanathan P, Nalini N. Effect of glycine on oxidative stress in rats with alcohol induced liver
injury. Pharmazie. 2004;59:55–60