Copyright 2001 by Humana Press Inc. All rights of any nature, whatsoe er, reser e!. 01"#$%&'%(01('102$01%) *1#.

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A Comparative Study on Effect of Dietary Selenium and Vitamin E on Some Antioxidant Enzyme Activities of Liver and Brain Tissues
+,-.A /01A2,3,1 2. -,4-A ACA2,2 2. 201A4 0-050,2 2 A26 ,. 7,1HA2 /,8CA2
Department of Biophysics, Faculty of Medicine, Ankara University, Ankara, Turkey; and 2Department of Biochemistry, Faculty of Medicine, acettepe University, Ankara, Turkey
1

1ecei e! April 2), 20009 1e ise! 5eptember ", 20009 Accepte! 5eptember 1&, 2000

ABSTRACT
5ince selenium an! itamin , ha e been increasingly recogni:e! as an essential element in biology an! me!icine, current research acti ities in the fiel! of human me!icine an! nutrition are !e ote! to the possibilities of using these antio;i!ants for the pre ention or treat< ment of many !iseases. /he present stu!y was aime! at in estigating an! comparing the effects of !ietary antio;i!ants on glutathione re!uctase an! glutathione pero;i!ase acti ities as well as free an! protein<boun! sulfhy!ryl contents of rat li er an! brain tissues. 7or 12$1% w=, both se; of weanling rats were fe! a stan!ar!i:e! selenium< !eficient an! itamin ,<!eficient !iet, a selenium<e;cess !iet, or a con< trol !iet. It is obser e! that glutathione re!uctase an! glutathione pero;i!ase acti ities of both tissues of the rats fe! with a selenium< !eficient or e;cess !iet were significantly lower than the alues of the control group. It is also shown that free an! boun! sulfhy!ryl con< centrations of these tissues of both e;perimental groups were signifi< cantly lower than the control group. /he percentage of glutathione re!uctase an! glutathione pero;i!ase acti ities of the !eficient group with respect to the control were )0> an! %?> in li er an! ""> an! "1> in the brain, respecti ely9 while these alues in e;cess group were )1> an! "&> in li er an! ))> an! '0> in brain, respecti ely. 7ree sulfhy!ryl contents of the tissues in both e;perimental groups
3Author to whom all correspon!ence an! reprint re@uests shoul! be a!!resse!.
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showe! a parallel !ecrease. 7urthermore, the !ecrease in protein< boun! sulfhy!ryl alues of brain tissues were more pronounce! than the alues foun! for li er. It seems that not only li er but also the brain is an important target organ to the alteration in antio;i!ant sys< tem through either a !eficiency of both selenium an! itamin , or an e;cess of selenium alone in the !iet. Index Entries 5o!ium selenite9 selenium !eficiency9 itamin ,9 selenium to;icity9 glutathione re!uctase9 glutathione pero;i!ase.

I!TR"D#CTI"!
/he biochemistry an! pharmacology of selenium A5eB are subCects of intense current interest (1)*+. 5e, long =nown to be an important !ietary antio;i!ant, is now recogni:e! as an essential component of the acti e sites of a number of =nown en:ymes9 a!!itionally se eral new mam< malian selenoproteins ha e recently been i!entifie! (,)-+. .oreo er, !ietary 5e !eficiency has been lin=e! to !iseases relate! to abnormalities of se eral organs, inclu!ing the li er, brain, heart, striate! muscles, an! pancreas (.)12+. Combine! !eficiency of itamin , ADit.,B an! 5e lea!s to car!io ascular abnormalities in humans an! laboratory animals (/,1*+. Howe er, the onset of the symptoms an! lesions pro!uce! show great ariability from one species to another. /here is an interest in supple< menting human subCects with 5e for pre ention of these abnormalities. Howe er, there is still uncertainty concerning the optimum !osage an! chemical form of the element. En the other han!, 5e to;icity in li estoc= that consume! 5e< accumulating plants can be trace! bac= to .arco Polo (0+. Fhen !ietary 5e is in e;cess of % ppm in the mammalian !iet (1,+, this to;ic effect is more pronounce!. ,;perimental chronic 5e to;icity, particularly with so!ium selenite, has been shown to cause cellular !ys< function in number of tissues, inclu!ing li er, spleen, heart, =i!neys, an! pancreas (10)1.+% Gust as the mechanism of its !eficiency, the mechanism of 5e to;icity is not clearly establishe! yet. It might be relate! to its abil< ity to form co alent lin=ages with intracellular proteins an! re!uce! glutathione (1&+. 5elenium has been i!entifie! biochemically in se eral tissues an! is =nown to be an integral component of a few o;i!ore!uctases an! a ital cofactor in glutathione pero;i!ase AHP;B, which controls intracellular pero;i!e le els in mitochon!ria an! cytoplasm (-+. /he glutathione re!o; cycle plays an important role in the o;i!ant !efense mechanism of the cell. /he components of the cycle, glutathione AH5HB, HP;, an! glu< tathione re!uctase AH1B, participate in the protection of the cell from the to;ic effects of en!ogenous an! e;ogenous hy!ropero;i!es. Although arious effects of 5e e;cess or !eficiency ha e been reporte! in humans an! animals, most of these stu!ies gi e little infor< mation on the status of relationship between Dit., an! 5e in those cases.

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/his point is of importance because Dit., is =nown to !ecrease the threshol! for !ietary 5e nee!e! to elicit !eficiency symptoms in se eral species (1/+. In pre ious stu!ies on animal mo!els, se eral antio;i!ants were either use! separately (2',21+ or in combination (22+. Howe er, there are also some reports that compare the combine! effect of Dit., an! 5e to the effects of each supplement separately (2*+. Although antio;i< !ants are =nown to interact with one another, it is not yet =nown how Dit., an! 5e might interact to affect the structure an! function of biolog< ical systems. /he present stu!y was therefore !esigne! to in estigate the effects of either combine! !eficiency of !ietary 5e an! Dit., or sole 5e e;cess on H1 an! HP; acti ities of rat li er an! brain. /he results are compare! with the pre ious obser ations obtaine! for rabbit tis< sues to !emonstrate whether these effects are species<specific as well as tissue<specific.

$ET%"DS
Animals and Housing
Fistar rats were !i i!e! ran!omly into three e@ual groups an! house! in stainless<steel, wire<bottome! cages initially at a !ensity of three per cage, an! as they grew, they were cage! in!i i!ually. /hey were maintaine! at an ambient air temperature of 22 I 1JC an! a 12<h light(!ar= cycle. /he 5e an! Dit., status of the animals were erifie! by !etermination of their le els in bloo!.

Diets and Feeding

/he !eficient !iets were obtaine! commercially (.,&+. 5e an! Dit., were supplemente! in a!e@uate an! e;cess !iets with so!ium selenite an! < tocopherol acetate. +ase! on the analysis of ran!om batches of !iet, the 5e concentration of the !eficient !iet was &.' g(=g !iet an! the a!e@uate an! e;cess !iets containe! 22) g 5e(=g !iet an! %.2 mg 5e(=g !iet, respecti ely. /he 5e content of !eioni:e! !istille! water was negli< gible AK1 g(-B. /he animals were fe! with either an a!e@uate !iet Acon< trol groupB, a !eficient !iet A5e< an! Dit.,< !eficient groupB, or an e;cess !iet A5e<e;cess groupB. /he animals were permitte! free access to the !iets an! water for appro; 12$1% w=.

Tissue Preparation and Analysis

1ats were heparini:e! an! anestheti:e! with so!ium pentobarbital A#0 mg(=gB. +loo! samples for 5e an! Dit., in estigations were collecte! by car!iac puncture. -i er an! brain tissues were remo e! for measure< ment of en:yme acti ities. /he tissues were homogeni:e! with three ol< umes of )0 mM potassium phosphate buffer, pH ?.%, in a glass$glass

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homogeni:er. 1%,000! supernatant of the homogenates were use! for the measurements of the en:yme acti ities. 7or histological stu!ies, li er tissues were fi;e! in 10> neutral< buffere! formalin. After !ehy!ration, they were embe!!e! in paraffin. 5erial sections were cut at )$" m thic=ness. 5ections were e;amine! for structural !etail with the use of hemato;ylin an! eosin an! for metachro< mosy of most cells with the tolui!ine blue stains. .icrographs were ta=en with a Carl 8eiss photomicroscope.

Measurement of Glutathione Reductase Activity

/he acti ity of this en:yme was measure! accor!ing to mo!ifie! 5taal metho! (2,+. /he incubation mi;ture containe! 100 mM so!ium phosphate buffer, pH ?.%, 1 mM o;i!i:e! glutathione AH55HB, 100 M re< !uce! nicotinami!e a!enine !inucleoti!e phosphate A2A6PHB, an! the tissue homogenates. A !ecrease in the absorbance of 2A6PH at #%0 nm was monitore! spectrophotometrically at #?JC. A unit of acti ity A0B was !efine! as the amount of en:yme that cataly:es the o;i!ation of 1 mol of 2A6PH in 1 min un!er these con!itions.

Measurement of Glutathione Peroxidase Activity

/he HP; acti ity was measure! by the mo!ifie! Paglia an! Dalen< tine metho! (20+. /he tissue homogenate samples were incubate! with 100 mM so!ium phosphate buffer, pH ?.%, 1 mM H5H, 1 0(m- glu< tathione re!uctase, % m. so!ium a:i!e, an! 200 M 2A6PH at #?JC, for 10 min. After the incubation perio!, 1 mM H2E2 was a!!e! into the incubation me!ium an! the !ecrease in the absorbance at #%0 nm was monitore!. A similar mi;ture e;clu!ing H5H was use! as a blan=. /he !efinition of the unit was the same as that of H1.

Determination of Sulfhydryl Groups

5ulphy!ryl groups A5HB were measure! accor!ing to the ,llman metho! (2-+. 7or total 5H measurement, ) - of homogenate was a!!e! to a 1.0&)<m- of mi;ture containing 0.11% mM ),) <!ithiobis< A2<nitroben:oic aci!B A6/2+B, '2 mM /ris<HCl buffer, pH ?.', an! 2.%) mM ethylene!iaminetetraacetic aci! A,6/AB. Absorbance at %12 nm was measure! against a similar mi;ture that !oes not contain 6/2+. 7or free 5H measurement, an e@ual olume of '> HClE% was a!!e! to the sample to remo e proteins. /he pH of the supernatant was a!Custe! to ?.' by a!!ing 0.? M L#PE%. Ene hun!re! microliters of this solution was a!!e! to 1 m- of 011% mM 6/2+, '2 mM /ris<HCl buffer, pH ?.', an! 2.%) mM ,6/A. Absorbance at %12 nm was measure! against a similar mi;ture that !oes not contain 6/2+. Concentration of
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the protein<boun! 5H was calculate! from the !ifference of total an! free 5H concentrations.

Protein Determination
Proteins were measure! by the +ra!for! metho! (2.+. +o ine serum albumin was use! as the stan!ar!.

hemicals and !nstruments

All the reagents were analytical gra!e. /he 5e content of the !iets was !etermine! by using a graphite furnace atomic absorption spec< trophotometer ADarian 5pectrophotometer AA<#0(%0B. /he en:yme acti < ities were measure! by using -L+ 0ltraspec Plus spectrophotometer, whereas 5H an! protein contents of the tissues were measure! by using a 5hima!:u 0D<120<02 spectrophotometer.

Statistical Analysis
All !ata were presente! as mean AI56B. .ann$Fhitney U<test was use! for the statistical e aluation of the results.

RES#LTS
In our pre ious publications, 5e !eficiency or 5e e;cess was erifie! by measuring 5e concentrations in plasma of all animals (.,&+. Dit., !efi< ciency was also confirme! by measurement of Dit., le els in plasma. Plasma 5e an! Dit., le els in !eficient rats were )0> of the control ani< mals. 7ee!ing the rats with a 5e<e;cess !iet cause! a significant increase in plasma 5e le els A2'0> of the controlB.

"ffect of Dietary Selenium on Tissue Antioxidant "n#yme Activities

/he H1 an! HP; acti ities, free an! boun! 5H contents of li er an! brain tissues of all groups are summari:e! in /ables 1 an! 2, respec< ti ely. +oth li er an! brain H1 an! HP; acti ities of the rats recei ing either 5e<!eficient an! 5e<e;cess !iets were significantly lower than the control groups Ap K 0.0)B. 7ree an! boun! 5H le els of the tissues obtaine! for both of the e;perimental groups were also significantly lower than that of the controls Ap K 0.0)B. In other wor!s, these changes are parallel to the changes in H1 an! HP; acti ities.

"ffect of Dietary Selenium on $iver Tissue Histopathology

/he histopathological changes in li er tissues of both the e;perimen< tal groups implicate! the !eleterious !egenerations within tissues by the
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/able 1 ,ffect of 5elenium an! Ditamin , 6eficiency an! 5elenium ,;cess on ,n:yme Acti ities an! 7ree 5H, Protein<+oun! 5H Dalues in 1at -i er3
Control H1 A0(mgB HP; A0(mgB 7ree <5H A3mol(mgB +oun! <5H A3mol(mgB 0.#?2 M 0.1?2 22.&'& M "."#0 0.%"? M 0.12# 0.%22 M 0.#") 5e<!eficient !iet 0.1'2 M 0.0#'33 A%&.&B 10.'## M 2.20233 A%?.1B 0.12) M 0.0#%33 A2".'B 0.2## M 0.#")33 A)).2B 5e<e;cess !iet 0.1'' M 0.0%233 A)0.)B 1).?"? M #.'0?33 A"'."B 0.1)1 M 0.0%)33 A#2.#B 0.1&' M 0.10) 33A%".&B

4ote5 All alues are means AI56B. 5e<a!e@uate, 5e<!eficient, Dit.,<!eficient, an! 5e< e;cess are use! for the groups of animals fe! with a selenium an! itamin ,<a!e@uate AcontrolB, a selenium an! itamin ,<!eficient, an! a selenium<e;cess, an! itamin ,< a!e@uate !iets. ,ach parameter was measure! from each tissue ta=en from each animal. 37or each group, n N '9 figures in parentheses are the percentages of the alues with respect to the control. 33p K 0.0).

/able 2 ,ffect of 5elenium an! Ditamin , 6eficiency an! 5elenium ,;cess on ,n:yme Acti ities an! 7ree 5H, Protein<+oun! 5H Dalues in 1at +rain3
Control H1 A0(mgB HP; A0(mgB 7ree <5H A3mol(mgB +oun! <5H A3mol(mgB 0.0)) M 0.01& 0.0?) M "."#0 0.0&" M 0.01" 0."1? M 0.%") 5e<!eficient !iet 0.0#" M 0.00?33 A").)B 0.0%" M 0.01)33 A"1.#B 0.02? M 0.00"33 A2'.1B 0.1%" M 0.0?"33 A2#."B 5e<e;cess !iet 0.0#0 M 0.00?33 A)%.)B 0.0"0 M 0.01%33 A'0.0B 0.0#2 M 0.00#33 A##.#B 0.1?0 M 0.10& 33A2?."B

4ote5 All alues are means AI56B. 5e<a!e@uate, 5e<!eficient, Dit.,<!eficient, an! 5e< e;cess are use! for the groups of animals fe! with a selenium an! itamin ,<a!e@uate AcontrolB, a selenium an! itamin ,<!eficient, an! a selenium<e;cess, an! itamin ,<a!e< @uate !iets. ,ach parameter was measure! from each tissue ta=en from each animal. 37or each group, n N '9 figures in parentheses are the percentages of the alues with respect to the control. 33p K 0.0).

selenium content of the !iet. Histopathologically, the maCor alterations seen in li ers from the !eficient<group rats were hy!ropic swelling of hepatocytes in the portal region, a large nucleus, pronounce! congestion showing early !egeneration in hepatocytes sinusoi!s, an! abnormal granular cytoplasm. Fhen the preparations were staine! with a meto< chromatic techni@ue Atolui!ine blueB, an increase! mast cell number was obser e! A7ig. 1aB. 6ilatation in sinusoi!al capillaries an! small local necrotic regions were obser e! A7ig. 1bB in li er tissue of the animals fe! with a selenium<e;cess !iet.

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7ig. 1. ,ffect of !ietary antio;i!ants on rat li er tissue. /he li er tissue was in estigate! un!er light microscopy an! sections were e;amine! for the metachromasy of most cells with tolui!ine blue stains. 5e an! Dit., !eficiency affecte! the structure of li er an! an increase in the number of mast cells in the portal region was obser e! &a'. 5e e;cess in !iet cause! some to;ic effects in li er an! local necrotic regions an! periportal local infiltration were obser e! &('. .agnification 2)0.

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DISC#SSI"!
/he main purpose of this stu!y was to in estigate the effects of either combine! !eficiency of !ietary 5e an! Dit., or sole 5e e;cess on H1 an! HP; acti ities of rat li er an! brain. /o compare the pathological changes in li er of rat with that of the rabbit tissue fe! with a 5e<!eficient an! 5e<e;cess !iets was also a goal of the stu!y. Pre ious !ata on plasma 5e an! Dit.,<le els of these animals re< eale! that 5e an! Dit., !eficiency an! 5e<e;cess status were attaine! by the !escribe! !iet (.+. Plasma 5e le els for control, 5e<!eficient, an! 5e< e;cess groups were %.21, 2.1", an! 11.&1 nmol(m-, respecti ely, an! plasma Dit., le els for these groups were ?.2, #.&, an! '.0 g(m-, respec< ti ely. -i er 5e alues for the same groups were also measure! an! foun! to be 1#.0), #.0', an! %1.1' nmol(m- for control, 5e<!eficient, an! 5e<e;cess groups, respecti ely. In this stu!y, we ha e foun! that both li er an! brain H1 an! HP; acti ities of rats fe! with a 5e<!eficient an! 5e<e;cess !iets were signifi< cantly lower than the alues of the 5e<a!e@uate group. Fe ha e also shown that free an! boun! 5H concentrations of these tissues obtaine! from 5e<!eficient an! 5e<e;cess groups were significantly lower than the 5e<a!e@uate group. In other wor!s, free an! boun! 5H changes in both tissues of rats fe! either with a 5e<!eficient or 5e<e;cess !iet are parallel to the changes in H1 an! HP; acti ities of these groups. /his is e;pecte! because almost all of the free intracellular 5H is in the form of H5H, which is a component of the glutathione re!o; cycle. It is thought that in the 5e<e;cess group, e;cess 5e in the form of selenite ion interacts with intracellular H5H an! re!uces its concentration (1&,2&+ whereas 5e !efi< ciency appears to be relate! with increase! free<ra!ical an!(or o;i!ant !amage in tissues (2/,*'+. /he percentage alterations of H1 an! HP; acti ities of the !eficient group with respect to the control were )0> an! %?> in the li er an! ""> an! "1> in the brain, respecti ely, whereas these alues for the e;cess group were )1> an! "&> in the li er an! ))> an! '0> in the brain, respecti ely. En the other han!, free 5H contents of both tissues in both e;perimental groups were !ecrease! in the same ranges with respect to the control A2?$##>B. 7urthermore, the !ecrease in protein< boun! 5H alues of brain tissues of both e;perimental groups with respect to the control group were more pronounce! than the alues in li er tissues. 7or !eficient an! e;cess groups, the alues are 2%> an! 2'> of control, respecti ely, for the brain, an! ))> an! %?> of control, respec< ti ely, for the li er A/ables 1 an! 2B. It was mentione! in most stu!ies that the li er is one of the main target tissues to the to;icity an!(or o;i!ant stress (*1)**+. /he present stu!y implies that the brain is also an impor< tant target for the effects of 5e. /his result is supporte! by others who ha! shown that brain tissue is affecte! by 5e supplementation in animal mo!els (**,*,+.

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In a pre ious stu!y, H1 an! HP; acti ities of rabbit li er an! brain at !eficiency an! e;cess of 5e were in estigate! (*0+. In that stu!y, it was foun! that HP; acti ity of the !eficient group was not !ifferent from the control group, whereas the same parameter was significantly higher in the 5e<e;cess group with respect to the control group. En the other han!, li er H1 acti ity was lower in the !eficient group an! higher in the e;cess group with respect to the control, whereas brain H1 acti ities of both tissues were the same in the e;perimental an! the control groups. Eur histopathological in estigations re eale! similar pathological !egenerations in rat an! rabbit li er tissues (.,&+ of both !eficient an! e;cess groups9 this fin!ing is in accor!ance with other reports (*-+. Fhen we compare these !ata with the current !ata, we can conclu!e that !efi< ciency an! e;cess of 5e cause se eral ha:ar!ous alterations in the li ing organisms through !ifferent mechanisms. /he effects of its !eficiency or e;cess on the glutathione re!o; cycle en:ymes !iffer with respect to the species an! the tissue. It is also obser e! that se eral protection an! compensation mechanisms e;ist in li ing organisms. Fe must point out that in the stu!y with rabbit tissues (*0+, Dit., of the 5e<!eficient group was at a!e@uate le els. /his might partly account for the insignificant changes foun! in the acti ities of HP; at 5e !eficiency in rabbit tissues. 5elenium is locate! at the catalytic site of the en:yme HP; an! its !eficiency is associate! mainly with !ecrease! HP; acti ity. 5e ere !ietary 5e !eficiency is =nown to pro!uce a set of !istinct pathological signs !epen!ing on the species an! the age, as well as on the age of onset an! !uration of the !eficiency (2/+. .ost of the pathology associ< ate! with !ietary 5e !eficiency appears to be attributable to the increase! free<ra!ical an!(or o;i!ant !amage in tissues (2/,*'+. At sufficiently high concentrations howe er, 5e may ha e an opposite effect, !irectly or in!i< rectly relate! to the increase! en!ogenous release of hy!rogen pero;i!e in cell (*.+% In a!!ition to the species an! tissue specificities, the !osage an! the mo!e of inta=e an! the !uration of the e;posure influence the se erity of these effects. 4trehus et al. (*&+ obser e! a re!uce! tolerance against en:ymati< cally generate! o;ygen ra!icals in isolate! -angen!orff preparations an! minor !amages within tissues in hearts from 5e<!eficient but Dit.,< a!e@uate rats. En the other han!, in two separate stu!ies, it was shown that sole 5e !eficiency !i! not significantly affect the contractile force of the electrically stimulate! left atria an! ,CH alues (1*+ an! left entric< ular function of -angen!orff preparations (*/+ in rat hearts. /he results of these stu!ies were supporte! by a pre ious report in which the altere! contractile force of -angen!orff preparations an! electrically stimulate! papillary muscle strips coul! not be obser e! (.+. Eur results suggest that although 5e in combination with Dit., is important in maintaining normal functions of the biological systems, 5e compoun!s, when use! in high concentration in !iet, can gi e rise to;ic effects on some organ structure an! functions, an!, therefore, the

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estimation of the therapeutic an! to;ic !oses seems to be critical for human beings. It is now well =nown that antio;i!ants are clearly impor< tant in cellular regulation, but the appropriate balance between o;i!ati e an! antio;i!ati e processes in cells un!er arious con!itions still nee!s to be !efine!.

AC)!"*LED+$E!TS
/his research was supporte! through the An=ara 0ni ersity 1e< search 7un!, ProCects 2o. &'<0&<00<0) an! &&<0&<00<10, an! the Hacettepe 0ni ersity 1esearch 7un!, ProCect 2o. &"01 101 011, an! 5tate Planning Ergani:ation, ProCect 2o. &&L<1201&0 . /han=s are !ue to 6r. A. 5ayal an! 6r. 4. 5aran for their technical contributions.

RE,ERE!CES
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