Cadangan Vitamin E

© Copyright 2001 by Humana Press Inc.All rights of any nature, whatsoever, reserved.0163–4984/01/8102–0145 $13.00

A Comparative Study on Effectof Dietary Selenium and Vitamin E

on Some Antioxidant Enzyme Activities of Liver and Brain Tissues

BELMA TURAN,*,1 N. LEYLA ACAN,2 N. NURAY ULUSU,2

AND E. FERHAN TEZCAN2

1Department of Biophysics, Faculty of Medicine, Ankara University, Ankara, Turkey; and 2Department of Biochemistry,

Faculty of Medicine, Hacettepe University, Ankara, TurkeyReceived April 25, 2000; Revised September 6, 2000;

Accepted September 19, 2000

ABSTRACT

Since selenium and vitamin E have been increasingly recognized as an essential element in biology and medicine, current research activities in the field of human medicine and nutrition are devoted to the possibilities of using these antioxidants for the prevention or treatment of many diseases. The present study was aimed at investigating and comparing the effects of dietary antioxidants on glutathione reductase and glutathione peroxidase activities as well as free and protein-bound sulfhydryl contents of rat liver and brain tissues. For12–14 wk, both sex of weanling rats were fed a standardized selenium- deficient and vitamin E-deficient diet, a selenium-excess diet, or a control diet. It is observed that glutathione reductase and glutathione peroxidase activities of both tissues of the rats fed with a selenium- deficient or excess diet were significantly lower than the values of the control group. It is also shown that free and bound sulfhydryl concentrations of these tissues of both experimental groups were significantly lower than the control group. The percentage of glutathione reductase and glutathione peroxidase activities of the deficient group with respect to the control were 50% and 47% in liver and 66% and61% in the brain, respectively; while these values in excess group were 51% and 69% in liver and 55% and 80% in brain, respectively. Free sulfhydryl contents of the tissues in both experimental groups

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

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142 Turan et al.

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showed a parallel decrease. Furthermore, the decrease in protein- bound sulfhydryl values of brain tissues were more pronounced than the values found for liver. It seems that not only liver but also the brain is an important target organ to the alteration in antioxidant sys- tem through either a deficiency of both selenium and vitamin E or an excess of selenium alone in the diet.

Index Entries: Sodium selenite; selenium deficiency; vitamin E;selenium toxicity; glutathione reductase; glutathione peroxidase.

INTRODUCTION

The biochemistry and pharmacology of selenium (Se) are subjects of intense current interest (1–3). Se, long known to be an important dietary antioxidant, is now recognized as an essential component of the active sites of a number of known enzymes; additionally several new mammalian selenoproteins have recently been identified (4–6). Moreover, dietary Se deficiency has been linked to diseases related to abnormalities of several organs, including the liver, brain, heart, striated muscles, and pancreas (7–12). Combined deficiency of vitamin E (Vit.E) and Se leads to cardiovascular abnormalities in humans and laboratory animals (9,13). However, the onset of the symptoms and lesions produced show great variability from one species to another. There is an interest in supple- menting human subjects with Se for prevention of these abnormalities. However, there is still uncertainty concerning the optimum dosage and chemical form of the element. On the other hand, Se toxicity in livestock that consumed Se-accumulating plants can be traced back to Marco Polo (5). When dietary Se is in excess of 4 ppm in the mammalian diet (14), this toxic effect is more pronounced. Experimental chronic Se toxicity, particularly with sodium selenite, has been shown to cause cellular dysfunction in number of tissues, including liver, spleen, heart, kidneys, and pancreas (15–17). Just as the mechanism of its deficiency, the mechanism of Se toxicity is not clearly established yet. It might be related to its abil- ity to form covalent linkages with intracellular proteins and reduced glutathione (18).

Selenium has been identified biochemically in several tissues and is known to be an integral component of a few oxidoreductases and a vital cofactor in glutathione peroxidase (GPx), which controls intracellular peroxide levels in mitochondria and cytoplasm (6). The glutathione redox cycle plays an important role in the oxidant defense mechanism of the cell. The components of the cycle, glutathione (GSH), GPx, and glutathione reductase (GR), participate in the protection of the

Dietary Se and Enzyme Activities in Liver and Brain 143


cell from the toxic effects of endogenous and exogenous hydroperoxides.

Although various effects of Se excess or deficiency have been reported in humans and animals, most of these studies give little infor- mation on the status of relationship between Vit.E and Se in those cases.


144 Turan et al.

This point is of importance because Vit.E is known to decrease the threshold for dietary Se needed to elicit deficiency symptoms in several species (19). In previous studies on animal models, several antioxidants were either used separately (20,21) or in combination (22). However, there are also some reports that compare the combined effect of Vit.E and Se to the effects of each supplement separately (23). Although antioxidants are known to interact with one another, it is not yet known how Vit.E and Se might interact to affect the structure and function of biological systems. The present study was therefore designed to investigate the effects of either combined deficiency of dietary Se and Vit.E or sole Se excess on GR and GPx activities of rat liver and brain. The results are compared with the previous observations obtained for rabbit tissues to demonstrate whether these effects are species-specific as well as tissue-specific.

METHODS

Animals and HousingWistar rats were divided randomly into three equal groups

and housed in stainless-steel, wire-bottomed cages initially at a density of three per cage, and as they grew, they were caged individually. They were maintained at an ambient air temperature of 22 ± 1°C and a 12-h light/dark cycle. The Se and Vit.E status of the animals were verified by determination of their levels in blood.

Diets and FeedingThe deficient diets were obtained commercially (7,8). Se

and Vit.E were supplemented in adequate and excess diets with sodium selenite and -tocopherol acetate. Based on the analysis of random batches of diet, the Se concentration of the deficient diet was 9.8 g/kg diet and the adequate and excess diets contained 225 g Se/kg diet and 4.2 mg Se/kg diet, respectively. The Se content of deionized distilled water was negli- gible (<1 g/L). The animals were fed with either an adequate diet (control group), a deficient diet (Se- and Vit.E-deficient group), or an excess diet (Se-excess group). The animals were permitted free access to the diets and water for approx 12–14 wk.

Tissue Preparation and AnalysisRats were heparinized and anesthetized with sodium

pentobarbital (30 mg/kg). Blood samples for Se and Vit.E


Dietary Se and Enzyme Activities in Liver and Brain 145investigations were collected by cardiac puncture. Liver and brain tissues were removed for measurement of enzyme activities. The tissues were homogenized with three vol- umes of 50 mM potassium phosphate buffer, pH 7.4, in a glass–glass


146 Turan et al.

homogenizer. 14,000g supernatant of the homogenates were used for the measurements of the enzyme activities.

For histological studies, liver tissues were fixed in 10% neutral- buffered formalin. After dehydration, they were embedded in paraffin. Serial sections were cut at 5–6 m thickness. Sections were examined for structural detail with the use of hematoxylin and eosin and for metachro- mosy of most cells with the toluidine blue stains. Micrographs were taken with a Carl Zeiss photomicroscope.

Measurement of Glutathione Reductase ActivityThe activity of this enzyme was measured according to

modified Staal method (24). The incubation mixture contained 100 mM sodium phosphate buffer, pH 7.4, 1 mM oxidized glutathione (GSSG), 100 M reduced nicotinamide adenine dinucleotide phosphate (NADPH), and the tissue homogenates. A decrease in the absorbance of NADPH at 340 nm was monitored spectrophotometrically at 37°C. A unit of activity (U) was defined as the amount of enzyme that catalyzes the oxidation of 1 mol of NADPH in 1 min under these conditions.

Measurement of GlutathionePeroxidase ActivityThe GPx activity was measured by the modified Paglia

and Valen- tine method (25). The tissue homogenate samples were incubated with100 mM sodium phosphate buffer, pH 7.4, 1 mM GSH, 1 U/mL glutathione reductase, 4 mM sodium azide, and 200 M NADPH at 37°C, for 10 min. After the incubation period, 1 mM H2O2 was added into the incubation medium and the decrease in the absorbance at 340 nm was monitored. A similar mixture excluding GSH was used as a blank. The definition of the unit was the same as that of GR.

Determination of Sulfhydryl GroupsSulphydryl groups (SH) were measured according to the

Ellman method (26). For total SH measurement, 5 L of homogenate was added to a 1.095-mL of mixture containing 0.114 mM 5,5 -dithiobis- (2-nitrobenzoic acid) (DTNB), 82 mM Tris-HCl buffer, pH 7.8, and 2.45 mM ethylenediaminetetraacetic acid (EDTA). Absorbance at 412 nm was measured against a similar mixture that does not contain DTNB.

For free SH measurement, an equal volume of 8%


Dietary Se and Enzyme Activities in Liver and Brain 147HClO4 was added to the sample to remove proteins. The pH of the supernatant was adjusted to 7.8 by adding 0.7 M K3PO4. One hundred microliters of this solution was added to 1 mL of 0114 mM DTNB, 82 mM Tris-HCl buffer, pH 7.8, and 2.45 mM EDTA. Absorbance at 412 nm was measured against a similar mixture that does not contain DTNB. Concentration of


148 Turan et al.

the protein-bound SH was calculated from the difference of total and free SH concentrations.

Protein DeterminationProteins were measured by the Bradford method (27).

Bovine serum albumin was used as the standard.

Chemicals and InstrumentsAll the reagents were analytical grade. The Se content of

the diets was determined by using a graphite furnace atomic absorption spectrophotometer (Varian Spectrophotometer AA-30/40). The enzyme activities were measured by using LKB Ultraspec Plus spectrophotometer, whereas SH and protein contents of the tissues were measured by using a Shimadzu UV-120-02 spectrophotometer.

Statistical AnalysisAll data were presented as mean (±SD). Mann–Whitney U-

test was used for the statistical evaluation of the results.

RESULTS

In our previous publications, Se deficiency or Se excess was verified by measuring Se concentrations in plasma of all animals (7,8). Vit.E deficiency was also confirmed by measurement of Vit.E levels in plasma. Plasma Se and Vit.E levels in deficient rats were 50% of the control animals. Feeding the rats with a Se-excess diet caused a significant increase in plasma Se levels (280% of the control).

Effect of Dietary Selenium on TissueAntioxidant Enzyme ActivitiesThe GR and GPx activities, free and bound SH contents

of liver and brain tissues of all groups are summarized in Tables 1 and 2, respectively. Both liver and brain GR and GPx activities of the rats receiving either Se-deficient and Se-excess diets were significantly lower than the control groups (p < 0.05). Free and bound SH levels of the tissues obtained for both of the experimental groups were also significantly lower than that of the controls (p < 0.05). In other words, these changes are parallel to the changes in GR and GPx activities.

Effect of Dietary Selenium on LiverTissue Histopathology

Vol. 81, 2001Biological Trace Element Research

149Dietary Se and Enzyme Activities in Liver and BrainThe histopathological changes in liver tissues of both the

experimental groups implicated the deleterious degenerations within tissues by the


150 Turan et al.

Table 1

Effect of Selenium and Vitamin E Deficiency and Selenium Excess on Enzyme

Activities and Free SH, Protein-Bound SH Values in Rat Liver*

Control Se-deficient diet Se-excess diet

GR (U/mg) 0.372 + 0.172 0.182 + 0.038** (49.9) 0.188 + 0.042** (50.5)GPx (U/mg) 22.989 + 6.630 10.833 + 2.202** (47.1) 15.767 + 3.807** (68.6)Free -SH (*mol/mg) 0.467 + 0.123 0.125 + 0.034** (26.8) 0.151 + 0.045** (32.3)Bound -SH (*mol/mg) 0.422 + 0.365 0.233 + 0.365** (55.2) 0.198 + 0.105 **(46.9)

Note: All values are means (±SD). Se-adequate, Se-deficient, Vit.E-deficient, and Se- excess are used for the groups of animals fed with a selenium and vitamin E-adequate (control), a selenium and vitamin E-deficient, and a selenium-excess, and vitamin E- adequate diets. Each parameter was measured from each tissue taken from each animal.

*For each group, n = 8; figures in parentheses are the percentages of the values with respect to the control.

**p < 0.05.

Table 2

Effect of Selenium and Vitamin E Deficiency and Selenium Excess on Enzyme

Activities and Free SH, Protein-Bound SH Values in Rat Brain*

Control Se-deficient diet Se-excess diet

GR (U/mg) 0.055 + 0.019 0.036 + 0.007** (65.5) 0.030 + 0.007** (54.5)GPx (U/mg) 0.075 + 6.630 0.046 + 0.015** (61.3) 0.060 + 0.014** (80.0)Free -SH (*mol/mg) 0.096 + 0.016 0.027 + 0.006** (28.1) 0.032 + 0.003** (33.3)Bound -SH (*mol/mg) 0.617 + 0.465 0.146 + 0.076** (23.6) 0.170 + 0.109 **(27.6)

Note: All values are means (±SD). Se-adequate, Se-deficient, Vit.E-deficient, and Se- excess are used for the groups of animals fed with a selenium and vitamin E-adequate (control), a selenium and vitamin E-deficient, and a selenium-excess, and vitamin E-adequate diets. Each parameter was measured from each tissue taken from each animal.

*For each group, n = 8; figures in parentheses are the percentages of the values with respect to the control.

**p < 0.05.

selenium content of the diet. Histopathologically, the major alterations seen in livers from the deficient-group rats were hydropic swelling of hepatocytes in the portal region, a large nucleus, pronounced congestion showing early degeneration in


Dietary Se and Enzyme Activities in Liver and Brain 151hepatocytes sinusoids, and abnormal granular cytoplasm. When the preparations were stained with a meto- chromatic technique (toluidine blue), an increased mast cell number was observed (Fig. 1a). Dilatation in sinusoidal capillaries and small local necrotic regions were observed (Fig. 1b) in liver tissue of the animals fed with a selenium-excess diet.


152 Turan et al.

Fig. 1. Effect of dietary antioxidants on rat liver tissue. The liver tissue was investigated under light microscopy and sections were examined for the metachromasy of most cells with toluidine blue stains. Se and Vit.E deficiency affected the structure of liver and an increase in the number of mast cells in the portal region was observed (a). Se excess in diet caused some toxic effects in liver and local necrotic regions and periportal local infiltration were observed (b). Magnification 250.



DISCUSSION

The main purpose of this study was to investigate the effects of either combined deficiency of dietary Se and Vit.E or sole Se excess on GR and GPx activities of rat liver and brain. To compare the pathological changes in liver of rat with that of the rabbit tissue fed with a Se-deficient and Se-excess diets was also a goal of the study.

Previous data on plasma Se and Vit.E-levels of these animals revealed that Se and Vit.E deficiency and Se-excess status were attained by the described diet (7). Plasma Se levels for control, Se-deficient, and Se- excess groups were 4.21, 2.16, and 11.91 nmol/mL, respectively, and plasma Vit.E levels for these groups were 7.2, 3.9, and 8.0 g/mL, respectively. Liver Se values for the same groups were also measured and found to be 13.05, 3.08, and 41.18 nmol/mL for control, Se-deficient, and Se-excess groups, respectively.

In this study, we have found that both liver and brain GR and GPx activities of rats fed with a Se-deficient and Se-excess diets were significantly lower than the values of the Se-adequate group. We have also shown that free and bound SH concentrations of these tissues obtained from Se-deficient and Se-excess groups were significantly lower than the Se-adequate group. In other words, free and bound SH changes in both tissues of rats fed either with a Se-deficient or Se-excess diet are parallel to the changes in GR and GPx activities of these groups. This is expected because almost all of the free intracellular SH is in the form of GSH, which is a component of the glutathione redox cycle. It is thought that in the Se-excess group, excess Se in the form of selenite ion interacts with intracellular GSH and reduces its concentration (18,28) whereas Se deficiency appears to be related with increased free-radical and/or oxidant damage in tissues (29,30).

The percentage alterations of GR and GPx activities of the deficient group with respect to the control were 50% and 47% in the liver and 66% and 61% in the brain, respectively, whereas these values for the excess group were 51% and 69% in the liver and 55% and 80% in the brain, respectively. On the other hand, free SH contents of both tissues in both experimental groups were decreased in the same ranges with respect to the control (27–33%). Furthermore, the decrease in protein- bound SH values of brain tissues of both experimental groups with respect to the control group were more pronounced than the values in liver tissues. For deficient and excess groups, the values are 24% and 28% of control, respectively, for the brain, and 55% and 47% of control, respectively, for the liver (Tables 1 and 2). It was mentioned in most


154 Turan et al.studies that the liver is one of the main target tissues to the toxicity and/or oxidant stress (31–33). The present study implies that the brain is also an important target for the effects of Se. This result is supported by others who had shown that brain tissue is affected by Se supplementation in animal models (33,34).



In a previous study, GR and GPx activities of rabbit liver and brain at deficiency and excess of Se were investigated (35). In that study, it was found that GPx activity of the deficient group was not different from the control group, whereas the same parameter was significantly higher in the Se-excess group with respect to the control group. On the other hand, liver GR activity was lower in the deficient group and higher in the excess group with respect to the control, whereas brain GR activities of both tissues were the same in the experimental and the control groups.

Our histopathological investigations revealed similar pathological degenerations in rat and rabbit liver tissues (7,8) of both deficient and excess groups; this finding is in accordance with other reports (36). When we compare these data with the current data, we can conclude that deficiency and excess of Se cause several hazardous alterations in the living organisms through different mechanisms. The effects of its deficiency or excess on the glutathione redox cycle enzymes differ with respect to the species and the tissue. It is also observed that several protection and compensation mechanisms exist in living organisms. We must point out that in the study with rabbit tissues (35), Vit.E of the Se-deficient group was at adequate levels. This might partly account for the insignificant changes found in the activities of GPx at Se deficiency in rabbit tissues.

Selenium is located at the catalytic site of the enzyme GPx and its deficiency is associated mainly with decreased GPx activity. Severe dietary Se deficiency is known to produce a set of distinct pathological signs depending on the species and the age, as well as on the age of onset and duration of the deficiency (29). Most of the pathology associated with dietary Se deficiency appears to be attributable to the increased free-radical and/or oxidant damage in tissues (29,30). At sufficiently high concentrations however, Se may have an opposite effect, directly or indi- rectly related to the increased endogenous release of hydrogen peroxide in cell (37). In addition to the species and tissue specificities, the dosage and the mode of intake and the duration of the exposure influence the severity of these effects.

Ytrehus et al. (38) observed a reduced tolerance against enzymatically generated oxygen radicals in isolated Langendorff preparations and minor damages within tissues in hearts from Se-deficient but Vit.E- adequate rats. On the other hand, in two separate studies, it was shown that sole Se deficiency did not significantly affect the contractile force of the electrically stimulated left atria and ECG values (13) and left ventric- ular function of Langendorff preparations (39) in rat


156 Turan et al.hearts. The results of these studies were supported by a previous report in which the altered contractile force of Langendorff preparations and electrically stimulated papillary muscle strips could not be observed (7).

Our results suggest that although Se in combination with Vit.E is important in maintaining normal functions of the biological systems, Se compounds, when used in high concentration in diet, can give rise toxic effects on some organ structure and functions, and, therefore, the



estimation of the therapeutic and toxic doses seems to be critical for human beings. It is now well known that antioxidants are clearly important in cellular regulation, but the appropriate balance between oxidative and antioxidative processes in cells under various conditions still needs to be defined.

ACKNOWLEDGMENTS

This research was supported through the Ankara University Re- search Fund, Projects No. 98-09-00-05 and 99-09-00-10, and the Hacettepe University Research Fund, Project No. 9601 101 011, and State Planning Organization, Project No. 99K-120190 . Thanks are due to Dr. A. Sayal and Dr. Y. Saran for their technical contributions.

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Cadangan Vitamin E

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