Abstract
In this study, the effect of combination of vitamin C (ascorbic acid), vitamin E (α-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 (GPx) activities were determined in liver and intestine tissues. Myeloperoxidase (MPO), aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), γ-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 GPx 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.
Keywords
Alcohol toxicity is one of the world’s major health problems, as significant numbers of peoples are affected due to several fatal diseases caused by alcohol (Singha et al. 2007). For this, we have chosen ethanol-induced toxicity in rats model to study the effects of ethanol on liver and intestine.
Ethanol is a fat-soluble nonelectrolyte, which is readily absorbed from the gastrointestinal tract, diffuses rapidly into circulation, and is distributed uniformly throughout the body (Cho et al. 2005). Ethanol affects most vital functions of virtually all organs, including liver, kidney, brain, pancreas, and intestine (Rajagopal et al. 2003). Ethanol manifests its harmful effects either through free radical species that react with most of the cell components, changing their structures and functions, or by contributing to other mechanisms that finally promote enhanced oxidative damage (Ostrowska et al. 2004). The liver is the major target of ethanol toxicity, and the role of oxidative stress in the pathogenesis of alcohol-related disease, particularly in the liver, has been repeatedly confirmed (Lieber 1997).
Oxidative stress, in the cells or tissues, refers to an enhanced generation of reactive oxygen species (ROS) and/or a depletion in antioxidant defense system, causing an imbalance between prooxidants and antioxidants (Devipriya et al. 2007; Schlorff, Husain, and Somani 1999). Peroxidative damage occurs in alcoholic patients and enhanced lipid peroxidation products can be correlated with increased ethanol consumption animals and humans (Husain and Somani 1997). ROS generated in the tissues are efficiently scavenged by enzymatic antioxidant system such as catalase, glutathione peroxidase, glutathione reductase, and superoxide dismutase and nonenzymatic antioxidants such as reduced gluthathione (GSH) and vitamins A, C, and E (Schlorff, Husain, and Somani 1999).
Antioxidants are essential in preventing the cellular damage caused by free radical–modified lipid peroxidation. In normal metabolism, there is a balance between the generation of free radicals and antioxidant defense mechanism. Excessive ethanol use commonly leads to vitamin deficiency (Ozdil et al. 2004a, 2004b). Recent studies have shown that both vitamins C and E are reduced in alcoholics (Suresh et al. 1999). The antioxidant pretreatment data clearly indicated that both vitamins C and E can prevent the generation of 1-hydroxyethyl radicals following acute ethanol treatment (Navasumrit et al. 2000). These antioxidants probably exert their effects through their ability to scavenge reactive oxidants. Vitamins C and E or vitamin E and Se exert synergistic effects in the prevention of biological membranes from oxidants (Navasumrit et al. 2000).
The aim of the present study was to determine whether a combination of vitamin C, vitamin E, and selenium had a protective effect on ethanol-induced changes in lipid peroxidation and glutathione levels and antioxidant enzymes activities in the liver and intestine tissues and some serum parameters of rats.
MATERIALS AND METHODS
Animals
Forty, 4- to 5-month-old female Sprague-Dawley rats (Istanbul University Institute of Experimental Medicine [DETAE]) were used in metal cages maintained at normal room temparature. The experiments were reviewed and approved by the Local Institute’s Animal Care and Use Committees. Their diet consisted of standart animal pellet food and tap water ad libitum. All rats were clinically healthy.
Experimental Design and Treatment of Animals
The animals were randomly divided into four groups, as follows. Group I: Intact animals (control). Group II: Control animals receiving vitamin C (250 mg/kg/day), vitamin E (250 mg/kg/day), and sodium selenate (0.5 mg/kg/day) for 3 days, by gavage technique. Group III: Rats receiving 1 ml absolute ethanol. Group IV: Animals receiving vitamin C (250 mg/kg/day), vitamin E (250 mg/kg/day), and sodium selenate (0.5 mg/kg/day) for 3 days, 1 h before the administration of absolute ethanol. The animals were fasted overnight (18 h) prior to the experiment, but they were allowed free access to water. The rats were sacrificed at 1 h after ethanol, with exposure to ether. The absolute ethanol and antioxidants were given to rats by gavage.
Animal Model for Liver and Duodenal Mucosal Lesions
Liver and duodenal damage were induced by oral administration at a constant volume by 1 ml absolute ethanol per rat. The animals were sacrificed by ether 1 h after treatment with absolute ethanol (Kanter, Coskun, and Uysal 2006) and the blood samples were taken, the liver and intestine tissues were removed.
Biochemical Assays
In this study, biochemical investigations were made in serum, liver, and intestine tissue from all groups. For biochemical analyses, the tissue samples of intestine and liver were washed with saline and kept frozen until the day of the experiments. These tissue samples were homogenized in cold 0.9% NaCl with a glass homogenizer to make up 10% (w/v) homogenate. The homogenates were centrifuged. The supernatant fraction was removed for the determination of GSH, lipid peroxidation (LPO), and protein levels and enzyme activities.
Lipid peroxidation in liver and intestine homogenates were assayed by the method of Ledwozyw et al. (1986). In brief, the adducts formed following boiled tissue homogenate with thiobarbutiric acid is extracted with n-butanol. The difference in optical density at 532 nm is measured in terms of the liver malondialdehyde (MDA) content, also of thiobarbituric acid-reactive substances (TBARS), which is undertaken as an index of lipid peroxidation. Results were expressed as nmol MDA/mg protein.
Reduced GSH was determined according to Beutler’s method using Ellman’s reagent (Beutler 1975). The procedure is based on the reduction of Ellman’s reagent by –SH groups to form 5,5′-dithiobis (2-nitrobenzoic acid) with an intense yellow color, measured spectrophotometrically at 412 nm using a Shimadzu Spectrophotometer. Results were expressed as nmol GSH/mg protein.
The protein content in the supernatants were estimated by the method of Lowry using Bovine serum albumin as standart (Lowry et al. 1951).
Catalase (CAT) activity was assayed in serum, liver and intestine tissue homogenates by the method of Aebi (1984), with hydrogen peroxide as substrate. The decomposition of H2O2was followed directly by the decrease in extinction at 240 nm and 25°C .
Serum uric acid levels were determined by Caraway method (Caraway 1955). In alkaline solution, uric acid reduces a complex phosphotungstate with the production of a blue color, which is photometrically measured at 760 nm.
Serum urea levels were determined by the method of acetylmonooxime (Barker 1944). In the oxime method, diacetyl monoxime in the presence of hydrolyzes to produce the unstable compound diacetyl. The diacetyl reacts with urea to produce a yellow diazine derivative. The color of this product is intensified by addition of thiosemicarbazide. Serum creatinine, levels were determined by the method of Jaffe’s reaction (Bonsnes and Taussky 1945). The serum is first deproteinised by the addition of picric acid. After alkalinization, the picric acid reacts with creatinine with formation of an orange-red dyestuff which is measured spectrophotometrically measured at 520 nm. Serum total lipid levels were determined by the method and sulphophosphovanilin (Frings et al. 1972). The serum is heated with concentrated sulfuric acid without prior deproteinization, and is then mixed with phosphoric acid/vanillin reagent. In this sulfo-phospho-vanillin reaction, the serum lipids form a pink dyestuff, which is measured spectrophotometrically measured at 530 nm. The supernatant of the tissue homogenate was used for the assay of lactate dehydrogenase (LDH) activity (Wroblewski 1957). This method is based on the reverse (pyruvate-lactate) reaction and uses a pH of 7.4 and a temperature of 25°C. The reaction is follewed by measuring the decrease in absorbance at 340 nm as NADH is oxidized to NAD.
Superoxide dismutase (SOD) activity (Mylroie et al. 1986), was measured in cuvettes containing 2.7 ml 50 mM potassium phosphate (pH 7.8) with 0.1 mM EDTA, 0.1 ml 0.39 mM riboflavin in 10 mM potassium phosphate (pH 7.5), 0.1 ml of 6 mM o-dianisidine·2HCl in deionized water, and tissue samples 0.1 ml. Cuvettes containing all components were illuminated with 20-W fluorescent tubes placed 5 cm above and to one side of the cuvettes and maintained at 25°C. Absorbances were measured at 460 nm with a spectrophotometer. Absorbance readings were taken at 0 and 8 min of illumination, and the net absorbance was calculated. A standart curve was prepared routinely with bovine SOD as a reference.
Glutathione peroxidase (GPx) activity was determined by the method described by Paglia and Valentine (1967) and modified by Wendel (1981). The enzymatic reaction in the tube containing NADPH, reduced GSH, sodium azide, and glutathione reductase was initiated by addition of H2O2, and the change in absorbance at 340 nm was monitored by a spectrophotometer. Activity was given in units per gram of protein (U/g protein).
Myeloperoxidase (MPO) activity was measured in liver tissue in a procedure similar to that of Hillegass et al. (1990). Liver samples were homogenized in 50 mM Potassium phosphate buffer (PB; pH 6.0) and centrifuged at 41,400 × g (10 min); pellets were suspended in 50 mM PB containing 0.5 % hexadecyltrimethylammonium bromide (HETAB). After three freeze and thaw cycles with sonication between cycles, the samples were centrifuged at 41,400 × g for 10 min. Aliquots (0.3 ml were added to 2.3 ml of reaction mixture containing 50 mM PB, o-dianisidine, and 20 mM H2O2 solution. One unit of enzyme activity was defined as the amount of the MPO that caused a change in absorbance measured at 460 nm for 3 min. Results are expressed as U/g tissue.
Liver aspartate transaminase (AST) and alanine transaminase (ALT) activities were determined by Reitman-Frankel methods (Reitman and Frankel 1957). The tissue homogenized is allowed to act in a buffered solution on ketoglutarate and aspartate, and on ketoglutarate and alanine, respectively, and the quantity of oxalacetate and of pyruvate, respectively, formed is measured. The reaction products can be measured photometrically as the corresponding 2,4-dinitrophenyl-hydrazones in alkaline medium at 490 nm.
Liver γ-glutamyltransferase (GGT) activities were estimated by Szasz (1969). The enzyme GGT catalyzes the transfer of a γ-glutamyl group from a γ-glutamyl peptide to an amino acid or other peptide. The reaction is followed by measuring the absorbance of the liberated nitroaniline at 405 nm.
Liver alkaline phosphatase (ALP) activity were estimated by two points (Walter and Schütt 1974) methods. ALP catalyzes the hydrolysis of p-nitrophenyl phosphate, forming phosphate and free p-nitrophenol, which in dilute acid solutions are colorless. Under alkaline conditions, this is coverted to the nitrophenolateion, which assumes a quinoid structure with a very intense yellow color at 405 nm.
Statistical Analysis
All data are expressed as mean ± SD. The results were evaluated using an unpaired t test and analysis of variance (ANOVA) variance analysis using the NCSS statistical computer package.
RESULTS
Table 1 shows the effects of vitamin C, vitamin E, and selenium on liver GSH and lipid peroxidation. Liver LPO levels in ethanol group were significantly increased as compared to the control group ( a P t -test = 0.006).Vitamin C, vitamin E, and selenium administration caused remarkable decreases in LPO levels in ethanol group ( b P t -test = 0.010). The concentration of GSH levels was decreased in the liver tissue given ethanol compared to control groups alone (P t -test = 0.009). The concentration of GSH significantly increased in liver tissues of rat given vitamin C, vitamin E, and Se (P t -test = 0.026).
The mean intestine GSH and LPO levels of the four groups are given in Table 2. Ethanol administration markedly stimulated LPO levels in intestine tissues and MDA content was elevated by about 53.19% compared to control animals ( a P t -test = 0.066). Treatment with vitamin C, vitamin E, and Se reduced the MDA content in rat intestine by 25% compared to animals given alcohol alone. GSH levels was insignificantly decreased by ethanol administration in the intestine ( b P t -test = 0.355). However, after the administration of antioxidants, a considerable increase was observed in the GSH levels of ethanol group (P t -test = 0.003).
Tables 3 gives the activity of CAT and the levels of urea, creatinine, uric acid, and total lipid in serum of experimental animals. Serum CAT activity in ethanol group were significantly lowered compared to control group ( a P t -test =0.0001). In ethanol, vitamin C, vitamin E, and Se group, serum CAT activity was high compared to the ethanol group (P t -test = 0.0001). The serum urea, creatinine, uric acid, and total lipid levels in the ethanol group were found to markedly increased compared to the control group ( b P t -test = 0.0001; c P t -test =0.004; d P t -test = 0.0001; and e P t -test = 0.049, respectively). The administration of antioxidant decreased urea, creatinine, uric acid, and total lipid levels considerably.
The liver CAT, LDH, SOD, GPx, and MPO activities are given in Table 4. Liver CAT, SOD, and GPx activities were considerably decreased in ethanol groups as compared to control groups ( a P t -test = 0.0001; c P t -test = 0.0001; and d P t -test = 0.001, respectively). Vitamin C, vitamin E, and Se had caused a remarkable increase in the liver CAT, SOD, and GPx activities in ethanol group (P t -test = 0.0001). This situation was opposite for the liver LDH and MPO activities. An increase in the ethanol group ( b P t -test = 0.0001; and e P t -test = 0.001, respectively) and a decrease in the vitamin C, vitamin E, and Se groups were observed (P t -test = 0.0001; and P t -test = 0.009, respectively).
The liver AST, ALT, ALP, and GGT activities are given in Table 5. Liver AST, ALT, ALP, and GGT activities were considerably increased in ethanol groups as compared to control groups ( a,b,c,d all P t -test = 0.0001). Vitamin C, vitamin E, and Se had caused a remarkable decrase in the liver AST, ALT, ALP, and GGT activities in the ethanol group (P t -test = 0.0001).
The intestine CAT, LDH, SOD, and GPx activities are given in Table 6. Intestine CAT, SOD, and GPx activities in ethanol group were significantly lowered when compared to control group ( a P t -test = 0.0001; c P t -test = 0.0001; and d P t -test = 0.009, respectively). Again, on the contrary, the administration of vitamin C, vitamin E, and Se to rats had caused an increase in the intestine CAT, SOD, and GPx activities in the ethanol group (P t -test = 0.0001, P t -test = 0.002 and P t -test = 0.001) (Table 6). In ethanol group, tissue LDH activity were found to increase remarkably than in those in the control group ( b P t -test = 0.0001). Administration of vitamin C, vitamin E, and Se caused a significant decrease in LDH activity in ethanol group (P t -test = 0.0001).
DISCUSSION
Ethanol is a direct systemic toxin that produces injury to all tissues, depending on dose and duration of exposure (Kanbak, Inal, and Baycu 2001). The degree of injury varies among organ systems. Ethanol is capable of generating oxygen radicals, inhibiting glutathione synthesis, loss of glutathione in the tissue, increasing malondialdehyde levels and imparing antioxidant defense systems in humans and animals (Ozaras et al. 2003).
Free radicals and associated oxidative stress have been implicated in eliciting pathological changes, including atherosclerosis, diabetes mellitus, chronic alcohol toxicity, muscular dystrophy, aging, and other diseases (Senthilkumar, Viswanathan, and Nalini 2004). We showed the protective effects of antioxidants on ethanol-induced gastric mucosal, intestinal, and liver injuries in our other studies (Ozdil et al. 2004a, 2004b; Koyuturk et al. 2004; Bolkent et al. 2006; Arda-Pirincci, Bolkent, and Yanardag 2006). Ethanol is also known to induce hyperlipidemia, leading to enhanced lipid peroxidation. The increased peroxidation is due to the increased oxidative stress in hepatic and extrahepatic tissues induced by ethanol and its oxidation. In the present study, the increased lipid peroxidation products (TBARS) in liver and intestine were observed in rats given with ethanol.
Ascorbate, the major water soluble antioxidant, is beneficial in reducing oxidative stress, but is harmful depending on the sensitive balance of its concentration. Ascorbate has been shown to efficiently scavenge superoxide, hydrogen peroxide, hypochloride, hydroxyl radicals, and peroxyl radicals, and to restore the antioxidant properties of fat-soluble α-tocopherol (Skrzdlewska and Farbiszewski 1999). Vitamin E prevents damage generated by toxic substances and the development of various disease such as cancer, diabetes, and ulcer by neutralizing or reducing free radicals (Chow 1991; Yanardag, Bolkent, and Kizir 2001). Selenium is an essential component of glutathione peroxidase, whose main role is to decompose safely mainly hydrogen peroxide and organic peroxides with the help of reduced glutathione (Zima et al. 2001; Yanardag and Orak 2001). Antioxidants are essential in preventing the cellular damage caused by free radicals and free radical–modified lipid peroxidation. In normal metabolism, there is a balance between the generation of free radicals and antioxidant defense mechanism. Excessive ethanol use commonly leads to vitamin deficiency (Ozdil et al. 2004a, 2004b). A number of investigations have revealed that vitamin C, vitamin E, and Se levels decreased by ethanol exposure. The antioxidants considered could act by reducing free radical production, trapping free radicals themselves, interrupting the peroxidation process, or reinforcing the natural antioxidant defence (Nordmann 1994). Combination of vitamin E, vitamin C, and Se play a complementary role in the prevention of oxidative damage in cellular and subcellular membranes (Ozdil et al. 2004a, 2004b). In our study, there were significantly elevated levels of TBARS in the liver and intestine of rats on ethanol treatment. These results are in agreement with the observations of previous studies (Senthilkumar, Viswanathan, and Nalini 2004). On supplementing antioxidants to ethanol-treated rats, we observed significantly decreased levels of TBARS in liver and intestine tissues. The ethanol-induced LPO in liver and intestine was inhibited by vitamin E, vitamin C, and Se. The inhibitor activity of antioxidants (vitamin E, vitamin C, and Se) could result from the scavenging of reactive oxygen species, which initate LPO. Ineffective scavenging of free radicals, due to depletion of antioxidants, plays a crucial role in cell injury. The decrease in those increasing LPO levels shows that vitamin E, vitamin C, and Se prevented damage in the liver and intestine.
GSH and glutathione-related enzymes are substances with an important role in cell detoxification and protection from hazardous compounds (Nechay 1985). Reduced GSH is one of the nonenzymic antioxidants. Glutathione is the most important nonprotein sulfhydryl compound in living organisms, playing a critical role in detoxification reactions (Kaplowitz, Aw, and Ookhtens 1985). Tissue GSH concentration reflects the potential for detoxification. It is well known that GSH is involved in the protection of normal cell structure and function by maintaining the redox homeostasis, by quenching free radicals, and by participating in detoxification reactions. Insufficiency in nonenzymatic antioxidant status in tissues of ethanol-intoxicated rats could be the consequence of increased utilization for trapping free radicals. Acetaldehyde promotes peroxidation reaction by binding to cysteine and/or glutathione, which causes depletion of GSH (Pushpakiran, Mahalakshmi, and Anuradha 2004). Previous studies show that on ethanol administration GSH levels were markedly decreased (Rajakrishnan, Viswanathan, and Menon 1996). Our studies also showed significantly decreased levels of GSH in liver and intestine of ethanol-treated rats as compared to control. The decrease in GSH content may be due to increased utilization of GSH to scavenge the toxic intermediates, which are formed on feeding ethanol. Administration of vitamin E, vitamin C, and Se significantly increased in liver and intestine GSH levels in the group given ethanol. This effect may be due to the activating role of selenium on GPx activity and indicates that these three substances effectively protect membrane integrity. The histopathologic results are given in our previous studies (Ozdil et al. 2004a; Koyuturk et al. 2004). These studies contains the protective effects of vitamin E, vitamin C, and Se combination therapy on ethanol-induced liver and duodenal injury, morphologically and biochemically (Ozdil et al. 2004a; Koyuturk et al. 2004). It has been revealed that, during ethanol metabolism, free radicals are generated (Luczaj and Skrzydlewska 2004). They play a major role in ethanol-induced oxidative stress, which may be additionally enhanced by depletion in antioxidant defense system and as a consequence by an imbalance between oxidants and antioxidants. Results of this study have confirmed that ethanol intoxication causes significant change in antioxidant parameters in blood. The decrease in catalase activity is especially important, because it may lead to the breakdown of antioxidant barrier. After ethanol consumption, this effect was also observed by other authors (Polavarapu, Spitz, and Sim 1998). Moreover, ethanol metabolism is accompanied by generation of very reactive metabolites, i.e., acetaldehyde and free radicals that can readily react with amino and sulphydryl groups of protein molecules; especially superoxide anions can inactivate catalase, whereas hydrogen peroxide inhibits superoxide dismutase. Moreover, catalase may be inactivated by another ethanol metabolite 1-hydroxyethyl radical (Luczaj and Skrzydlewska 2004). In our study, activity of CAT were significantly lower in alcohol-treated rats as compared with those of control rats. Lowered 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. Administration of antioxidant to ethanol-treated rats significantly elevated CAT activity as compared with those on ethanol treatment alone, which corresponds to the result reported by another researcher (Lee 2004).
Serum levels of urea and creatinine were used as indicators of renal protection. Elevated blood urea is known to be correlated with an increased protein catabolism in mammals and/or the conversion of ammonia to urea as a result of increased synthesis of arginase enzyme involved in urea production. In agreement with this fact, the results of another study made by us show that serum total protein concentrations were decreased in animals treated with ethanol. The administration of absolute ethanol to rats leads to a marked elevation of serum urea and creatinine levels in agreement with the results obtained by other researchers (El-Demerdash et al. 2004). This elevation of urea and creatinine levels reflects the degree of renal injury. In our study, serum urea and creatinine levels were increased by application of ethanol, but not vitamin E, vitamin C, and Se, and it shows that antioxidants prevent the damage caused by ethanol. It can be concluded from these results that antioxidants possess a remarkable protective effect on absolute ethanol-induced renal injuries.
The ethanol-fed rats in our study showed significant rise in serum uric acid concentrations. Hyperuricemia has been reported to be an independent predictor of cardiovascular risk (Ward 1998). In another study, it was stated that hyperuricemia is a renal prognostic factor (Sanchez-Lozada et al. 2005). One of the mechanisms by which hyperuricemia may lead to renal prognosis is the linkage between hyperuricemia and inflammatory response. Another mechanism is the relationship between hyperuricemia and atherosclerosis. Increased oxidant stress and oxidation of low-density lipoprotein (LDL) in arterial wall by peroxynitrite play an important role in the development of atherosclerosis. In this sense, the finding of elevated serum uric acid concentrations may reflect the bodily response to an increased production of endogenous oxygen species, because uric acid is a potent scavenger of peroxynitrite (Hooper et al. 1998). Suarna et al. (1995) reported a more direct role uric acid in the progression of the atherosclerotic process, because urate crystals trigger an inflammatory response in the vascular plaque. In our study, serum uric acid levels were significantly higher in the ethanol groups than in control groups. Administration of vitamin C, vitamin E, and Se significantly decreases uric acid in the serum of the ethanol group.
Exposure of intestinal mucosa to ethanol causes morphological injuries, impairs the absorption of solutes, and alters lipid metabolism and enzyme activities (Kaur et al. 1998). Many studies indicate that alcohol intake significantly increases both serum and hepatic lipid levels resulting in hyperlipidemia and fatty liver (Park et al. 2002). Ethanol oxidation by the alcohol dehyrogenase pathway results in the production of NADH, which might contribute to enhanced lipid synthesis. Ethanol itself is converted into acetate, which is the building block of fatty acid synthesis. Ethanol oxidation has been shown to increase α-glycerophosphate levels, which can lead to enhance triglyceride synthesis and lipid levels. In our study, serum total lipid levels were increased by application of ethanol, but vitamin E, vitamin C, and Se show that antioxidants prevent the ethanol-caused damage. The antioxidants in ethanolic diet were beneficial in lowering the lipid content. It appears that antioxidant supplement could be used to improve the lipid metabolism.
Activities of SOD and CAT the two enzymes that help to scavenge superoxide ions and hydroxyl ions, respectively, were significantly lower in alcohol administered rats as compared with those of control rats. Lowered activities of SOD and CAT will result in the accumulation of these highly reactive free radicals leading to deleterious effects such as loss of cell membrane integrity and membrane function (Reedy and Lokesh 1992). Superoxide ion and hydroxyl radical are known to cause marked injuries to the surrounding tissues and organs. Some natural or synthetic compounds with antioxidant properties may help to alleviate the liver damage totally or partially. Therefore removing superoxide ion and hydroxyl radical is probably one of the most effective defense mechanisms againts a variety of diseases (Rajagopal et al. 2003). The observed restoration of the SOD and CAT activities on vitamin E, vitamin C, and Se supplementation may be due to a direct stimulatory effect of vitamin E, vitamin C, and Se on SOD and CAT. The decrease in the tissue lipid peroxidation on vitamin E, vitamin C, and Se treatment can also be correlated with the elevated SOD and CAT activities.
LDH is an enzyme that exists in many tissues and organs such as heart, muscle, kidney, liver, etc. When those tissues or organs are damaged, LDH is released in to the blood from the cells. Leakage of this enzyme due to the change in the permeability of plasma membrane resulted as a consequence of peroxidation of membrane by oxygen-and ethanol-derived free radicals (Saravanan and Pugalendi 2006). Coadministration of antioxidant gave protection againts ethanol by reversing the changes produced by ethanol. The protective ability of vitamin E, vitamin C, and Se may be due its cell membrane stabilizing effect and radical scavenging potency.
Glutathione peroxidase provides defense againts oxidative tissue damage to various tissues after administration of ethanol. GPx and antioxidant enzymes significantly inhibited by ethanol administration, which indicates that inhibition of GPx activity was responsible for oxidative tissue damage to liver and intestine after administration of ethanol. Vitamin E, vitamin C, and Se supplementation caused an increase in GPx activity in liver and intestine tissue of the ethanol group. GPx activity significantly decreased in liver and intestine tissue of the rats receiving ethanol. GPx runs nonspecifically to scavenge and decompose excess hydroperoxides, including H2O2, which may be prevalent under oxidative stress (Augustyniak, Waszkiewicz, and Skrzydlewska 2005)
Activated neutrophils are known to induce tissue injury through the production and release of reactive oxygen metabolites and cytotoxic proteins (MPO, lactoferrin) into the extracellular fluid. MPO is an essential enzyme for normal neutrophil function. When neutrophils are stimulated by various stimulants, MPO as well as other tissue-damaging substances are released from the cells (Kettle and Winterbourn 1997). An increase in MPO activity due to ethanol cause inflammation and damage in the organs. In our study, liver MPO activities were increased by application of ethanol. Vitamin E, vitamin C, and Se treatment significantly decreased the enzyme activity and prevented neutrophil infiltration into the damaged tissue. These results suggest that ethanol-induced oxidative damage involves the interaction of neutrophils and the protective effect of vitamin E, vitamin C, and Se is mediated in part by blocking neutrophil infiltration into the tissues.
Hepatic cells participate in a variety of metabolic activities and contain a lot of enzymes. Among these, AST, ALT, and ALP are commonly employed biological markers for hepatic injury and efficacy of hepatoprotective interventions (Rosser and Gores 1995). In the present study, it has been observed that the levels of AST, ALT, and ALP activities increased in rats liver tissue following administration of ethanol. Ethanol may responsible for the increment of the AST and ALT, important marker enzyme of the liver function test (Singha et al. 2007). Administration of vitamin E, vitamin C, and Se of alcoholic rats decreased AST and ALT activities. This shows that vitamin E, vitamin C, and Se can preserve the structural integrity of the liver from the adverse effects of ethanol. ALP is distributed in lysosomal fraction of cells and this is the indicators of toxicity in metabolic organs. After ethanol treatment, ALP was a significant increase in the activity of ALP was observed in liver by Singha et al. (2007). This might be due to some alteration in lysosomal enzyme activity in these metabolic organs after ethanol treatment. In the present study, the activity of GGT was elevated, indicating the toxicity induced by ethanol. This was brought down to almost normal levels by the combined action of vitamin E, vitamin C, and Se.
As a result, the biochemical evaluations reveal 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 as well as in some serum parameters. In addition, a decrease in serum urea, creatinine, and uric acid levels indicate an improving effect in renal injury.
In conclusion, the combination of vitamin C, vitamin E, and selenium significantly inhibits MDA production with a concamitant replenishment of tissue GSH content, indicating a reduction in lipid peroxidation and cellular damage, which protects the liver and intestine tissues against ethanol-induced oxidative damage. The results suggest that vitamin E, vitamin C, and Se, with their potent free radical–scavenging and antioxidant properties, may be promising agents againts ethanol toxicity by protecting tissues againts oxidative damage. We can conclude that combined treatment with vitamin C, vitamin E, and selenium can be used in the therapy of ethanol-induced injury.
Footnotes
Tables
This work was supported by Faculty of Engineering and Faculty of Science of The University of İstanbul.