Vitamin E and selenium administration as a modulator of antioxidant defense system

Vitamin E and SeleniumAdministration as a Modulator of Antioxidant Defense System

Biochemical Assessment and Modification

EMAN NOAMAN,*,1 AHMED M. ZAHRAN,1

AZZA M. KAMAL,2 AND MANAR F. OMRAN1

1Department of Biological Radiation, National Centre for Radiation Research and Technology, Atomic Energy Authority,Cairo, Egypt; and 2Department of Biochemistry. Animal HealthResearch Institute, Centre of Agriculture Research, Cairo, Egypt

Received June 29, 2001; Accepted August 30, 2001

ABSTRACT

Exposure of cells to ionizing radiation leads to the formation of reac-tive oxygen species (ROS) that are associated with radiation-induced cyto-toxicity. Because of the serious damaging potential of ROS, cells depend onthe elaboration of the antioxidant defense system (AODS), both enzymaticand nonenzymatic oxidant defense mechanisms. The deficiency in impor-tant components of the endogenous AODS leads to the accumulation ofoxidative stress inducing oxidative damage. The antioxidant enzymessuperoxide dismutase and glutathione peroxidase are key intracellularantioxidants in the metabolism of ROS.

In the current study, we investigated the potential role of theseantioxidant enzymes in radioresistance during the evaluation of the com-pensatory role of some exogenous micronutrients against oxidative stress

Animals were categorized into eight groups, receiving vitamin E (α-tocopherol) and/or selenium (Se) with or without whole-body γ-irradia-tion (6.5 Gy).

The results indicate that antioxidant pretreatments before irradiationmay have some beneficial effects against irradiation-induced injury. Theresults also indicate that selenium and vitamin E act alone and in an addi-tive fashion as radioprotecting agents. The results further suggest that sele-nium confers protection in part by inducing or activating cellularfree-radical scavenging systems and by enhancing peroxide breakdown,

Biological Trace Element Research 55 Vol. 86, 2002

© Copyright 2002 by Humana Press Inc.All rights of any nature, whatsoever, reserved.0163-4984/02/8601–0055 $12.50

*Author to whom all correspondence and reprint requests should be addressed.

whereas vitamin E appears to confer its protection by an alternate comple-mentary mechanism.

Index Entries: Vitamin E; selenium; γ-radiation; lipid peroxidation;glutathione; glutathione peroxidase; superoxide dismutase.

INTRODUCTION

Several studies postulated that reactive oxygen species (ROS) partici-pated in the etiology of many chronic health problems. Free radicals cancause tissue damage by reacting with polyunsaturated fatty acids (PUFA)in cellular membrane, nucleotides in DNA, and critical sulfhydryl bonds inproteins (1). These highly reactive species can originate endogenously fromnormal metabolic reactions or exogenously, through air pollutants,chemotherapeutics, and pesticides, as well as through exposure to ionizingradiation (2,3). In health, under normal conditions a delicate balance existsbetween the generation of ROS and the cellular antioxidant defense sys-tems (4). Defense against free-radical damage is initiated in some aspectthrough primary antioxidant metalloenzymes. These include glutathioneperoxidase (selenium), catalase (iron), and superoxide dismutase (copper,manganese). In another aspect, secondary antioxidants might trap free rad-icals, preventing chain reactions such as α-tocopherol (vitamin E), ascorbicacid (vitamin C), β-carotene, and reduced glutathione (5). Vitamin E is themost important lipid-soluble chain-breaking antioxidant in tissues, redcells, and plasma (6). The vitamin might protect cellular componentsagainst peroxidative damage via the free-radical scavenging mechanism oras a constituent of the membrane (7). Selenium has been shown to be essen-tial constituent of erythrocyte glutathione peroxidase in several species (8).Selenium was considered responsible for the balance and activity of cellu-lar glutathione peroxidase. In addition, the dietary selenium requirementsfor the expression of glutathione peroxidase and other newly discoveredselenoenzymes or proteins have been established (9). It has been assumedthat glutathione peroxidase functions as an antioxidant enzyme, by reduc-ing hydrogen peroxide and organic hydroperoxides. Its activity has beenused to assess body selenium status and nutritional requirements (10).

The present study was designed to evaluate the radioprotective roleof α-tocopherol (vitamin E) and/or selenium administration to acuteoxidative stress γ-irradiated rats. In order to evaluate the lipid peroxida-tion that might be induced as a result of exposure to ionizing radiation,thiobarbituric acid reactive substances (TBARS) content in plasma andliver tissues was estimated. The activity of glutathione peroxidase (GSH-Px) as the selenium-dependent enzyme was evaluated in blood andhepatic tissues to assess the selenium status as well as antioxidant enzyme.Moreover, the concentration of reduced glutathione (GSH) and the activi-ties of superoxide dismutase (SOD) were estimated in whole blood andliver tissues.

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MATERIAL AND METHODS

Experimental Animals

Eighty female albino rats (100–120 g) obtained from the EgyptianOrganization for Biological Product and Vaccines at Giza, Egypt were usedthroughout the present experiment. Animals were housed in cages undergood ventilation and illumination conditions and were allowed balancedstandard diet and water ad libitum.

Treatment

Vitamin E (α-tocopherol acetate) and selenium (sodium selenite) werepurchased from Sigma Co. Vitamin E was dissolved in corn oil before useand administered intraperitoneally to rats in a concentration of 200 mg/kgbody weight/dose for 10 successive days. Selenium was dissolved in salineand intraperitoneally administered (0.1 mg/kg body weight) for 10 succes-sive days. Radiation processing involved the following γ-Irradiation was per-formed by using cesium-137, a biological irradiator source (γ cell-40) locatedat the National Center for Radiation Research and Technology (NCRRT)Egypt. The dose rate was 0.7 Gy/min at the time of experiment. Animalswere exposed to whole-body γ-rays at a sublethal single dose of 6.5 Gy.

Experimental Design

The experimental animals were randomly divided into 8 groups eachof 10 rats. Vitamin E and selenium were administered to the animal as 10successive doses with or without radiation exposure; radiation processingwas performed 20 min after the last dose of treatment.

Group 1: animals were considered as negative controlGroup 2: animals administered with vitamin EGroup 3: animals injected with seleniumGroup 4: animals received a concomitant treatment of vitamin E

and seleniumGroup 5: animals were exposed to whole-body γ-irradiation at a

dose of 6.5 GyGroup 6: animals administered with vitamin E, then subjected to

whole-body γ-irradiationGroup 7: animals injected with selenium, then subjected to

whole-body γ-raysGroup 8: animals received concomitant treatment of vitamin E

and selenium, then subjected to whole body γ-irradiation

Experimental Parameters

Five animals of each group were sacrificed after 1 and 7 d after radia-tion exposure. Blood was collected in heparinized tubes by heart puncture,

Se and Vitamin E Protection Against Radiation 57


as quickly as possible; the liver was dissected out, homogenized in bidis-tilled water (10% homogenates), and kept in ice. The following parameterswere determined: Lipid peroxidation was indicated by measuring the thio-barbituric acid reactive substances (TBARS) content in plasma and liverhomogenates (11). Reduced glutathione (GSH) content was estimated inwhole blood and liver homogenate by measuring the optical density (OD)of the yellow color that developed when 5,5 dithiol-bis(2-nitrobenzoicacid) is added to sulfhydryl compounds (12). Estimation of glutathioneperoxidase (GSH-Px) in whole blood and liver homogenate was based onthe measurement of the amount of residual GSH left after exposure toenzyme activity for a fixed time (13). Superoxide dismutase activity wasestimated in whole blood and liver homogenate by the method thatdepends on detection superoxide anions by nitroblue tetrazoluim for-mazan color development (14). The results were presented as mean values± standard error and groups were compared using one-way analysis ofvariance (ANOVA) (15).

RESULTS

The data of the response of administration of α-tocopherol and/orselenium in different groups of animals with or without radiation expo-sure are represented in Tables 1–4.

The results for lipoperoxide contents, which were evaluated asTBARS concentration, are shown in Table 1. α-Tocopherol and seleniumadministration resulted in nonsignificant changes in the TBARS levels.There was a significant increase in lipoperoxidation in the group of ani-mals exposed to γ-radiation throughout the experimental time intervals.Administration of α-tocopherol and/or selenium preirradiation resultedin significant improvement in the levels of lipid peroxidation when com-pared to irradiated groups.

The levels of glutathione (reduced form) in both blood and hepatic tis-sues presented in Table 2 showed that rats exposed to whole-body γ-irra-diation revealed a significant decrease in glutathione content. Treatment ofanimals with α-tocopherol and/or selenium preradiation exposureimproved the glutathione profile compared with the irradiated group.

Results of glutathione peroxidase in the rats’ blood and liver tissuesare illustrated in Table 3. Glutathione peroxidase analysis revealed non-significant change when animals were administered with both α-toco-pherol and selenium. Rats exposed to whole-body γ-irradiation revealed adeleterious effect represented in a significant decrease in GHS-Px activities.When animals were administrated a course of α-tocopherol and/or sele-nium before radiation exposure, the inhibition recorded in the activities ofGSH-Px noticeably ameliorated when compared with irradiated groups.

Table 4 shows superoxide dismutase in whole blood and liver for dif-ferent groups. Irradiation with γ-rays caused some depression in the SOD

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Table 1Lipid Peroxidation (TBARS) Contents in Different Groups of Animals

Note: Each value represents the mean of five observations ± S.E.* Significant change between groups: Least significant difference (LSD) of blood

groups at 0.05 = 6.433; least significant difference (LSD) of tissues groups at 0.05 = 4.33.

Table 2Reduced Glutathione Contents in Different Groups of Animals

Note: Each value represents the mean of five observations ± S.E.* Significant change between groups and control: LSD of blood groups at 0.05 = 4.662;

LSD of liver tissue groups at 0.05 = 3.81.

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Table 3Glutathione Peroxidase Activities in Different Groups of Animals

Note: Each value represents the mean of five observations ± S.E.* Significant change between groups and control: LSD of blood groups at

0.05 = 4.722; LSD of liver tissue groups at 0.05 = 3.485.

Table 4Superoxide Dismutase Activities in Different Groups of Animals

Note: Each value represents the mean of five observations ± S.E.* Significant change between groups and control: LSD of blood groups at

0.05 = 4.573; LSD of tissues groups at 0.05 = 5.497.

activities, whereas administration of α-tocopherol or selenium before radi-ation rectified the changes that occurred in SOD activities.

DISCUSSION

Much of the current literature regarding the biological effects ofantioxidant nutrients has concentrated on their potential role in inhibitingor preventing tissue damage induced by free-radical species produced dur-ing metabolism (16). Vitamin E (α-tocopherol) has been known for manyyears as a one of the most important natural factors from the biologicalpoint of view. This lipid-soluble compound has an essential structuralcomponent of biological membranes, contributing to their stability (17). Itis needed for the mitochondria electron-transport function and preventsoxidation of various compounds (18). Several studies indicate that vitaminE diminishes the peroxidation of PUFA through scavenging free radicals(19,20). Selenium, which has been recognized as an essential trace elementfor mammals and humans, is part of the enzyme glutathione peroxidase(GSH-Px) and of other selenium-dependent enzymes. GSH-Px protects theorganism from oxidative damage (21). In the present study, whole-body γ-irradiation strongly initiates the process of lipid peroxide formation asshown in Table 1 and the hypothesis of lipid peroxidation formation, asindicated by the estimated lipid malonaldahyde (TBARS) contents in theplasma and liver tissues. When rats were submitted to ionizing radiation,plasma TBARS shows a marked significant elevation, which was observedalso in hepatic tissue homogenates. However, there was a pronounceddecrease in TBARS content in the groups of animals treated with vitamin Epreirradiation exposure when compared with the irradiated group,whereas the group of rats that received vitamin E and/or seleniumrevealed a normal range of TBARS. On the other hand, exposure to radia-tion decreased the antioxidants enzymes detected in the blood and liverhomogenate of rats, which recorded a significance decrease in glutathionecontent, glutathione peroxidase, and superoxide dismutase. This findingwas also observed in the previous study, which stated that a low dose ofradiation caused changes in the lipid peroxidation–antioxidant protectivesystem (22). The status of the antioxidant systems was variably affected bydietary vitamin E and selenium, which ameliorated the disturbances in thepro-oxidant–antioxidant balance (23). The amelioration has been verifiedby damage findings; by monitoring the activity of chosen scavengingenzymes in the blood and liver homogenate, we can accumulate informa-tion on ongoing biological oxidative stress. Because GSH contents aremaintained by the activities of glutathione reductase and GSH-PX, themoderate levels of GSH in rats receiving vitamin or selenium preirradia-tion may be the result of an adaptive response to increased oxidative stress(24). It is worth mentioning that ionizing radiation induced significantchanges in selenium content and distribution (25). The patients exposed to



ionizing radiation have diminished blood antioxidant defense associatedwith pronounced selenium deficiency (26). The most important metabolicrole of selenium in mammalian species depends on its function in theactive site of selenoenzyme glutathione peroxidase (GSH-PX) (27). More-over, erythrocyte GSH-Px activity is positively correlated to total bloodselenium concentration (28). In addition, selenium and vitamin E, whichpromote catabolism of homocysteine, have the potential to promote vascu-lar health by mitigating the adverse impact of superoxide-derived oxidantson endothelial function (29). This result is considered important becausemicrovascular networks, which control the delivery of oxygen and nutri-ents and the removal of metabolic waste, are the most sensitive part of thevascular system to ionizing radiation (30). As a biological tool to explorereaction mechanisms, SOD was a unique and valuable asset. Superoxidedismutase ability to inhibit radical reactions leading to oxidative damagein vitro often turned out to be the result of its ability to prevent reductionof iron ions by superoxide (31). This enzyme especially MnSOD, plays acentral role in protecting cells against ROS injury during ionizing radiationexposure among MnSOD, CuZnSOD, and GSH-Px (32). In the presentstudy, it was shown that after total-body γ-irradiation, there was a ten-dency toward a decrease in the activity of this enzyme. Administration ofvitamin E and/or selenium confers a significant protective effect againstradiation exposure. We postulate that this protective effect is mediated bya reduction in the oxidative load induced by irradiation. This explanationconfirmed in a previous study suggested that the preservation of SODmight be common mechanisms of vitamin E supplementation (33).

The results obtained from this study provide experimental evidenceon the effect of vitamin E and/or Se intake in vivo on the hydroperoxidesproduction that might be changed into the much less toxic hydroxymoeityproduct and permits regeneration of a membrane lipid molecule perox-idative. The findings also demonstrate that the type of trace elements (Se)and levels of vitamin E in the diet are important factors in determining cel-lular susceptibility to oxidative damage. Additionally, vitamin E workssynergistically with other antioxidant nutrients, including selenium, toquench free radicals, peroxides, and other potentially harmful substances.

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Vitamin E and selenium administration as a modulator of antioxidant defense system

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