The antioxidant response of the liver of male Swiss mice raised on a AIN 93 or commercial diet.

0.21 MB·English

by  Caetano, Aline C

Caetanoetal.BMCPhysiology2013,13:3
http://www.biomedcentral.com/1472-6793/13/3
RESEARCH ARTICLE Open Access
The antioxidant response of the liver of male
Swiss mice raised on a AIN 93 or commercial diet
Aline C Caetano1, Lucimara F da Veiga2, Flávia R Capaldi3, Severino M de Alencar2, Ricardo A Azevedo3
and Rosangela MN Bezerra4*
Abstract
Background: Reactive oxygenspecies (ROS) are formedunder natural physiological conditions and are thought
to play an importantrolein many humandiseases.A wide range ofantioxidants are involved in cellular defense
mechanisms against ROS, which can be generatedin excess during stressfulconditions, these include enzymes and
non-enzymatic antioxidants. The aim of this study was to evaluate the antioxidant responses of mice to two diets
control, commercialand thepurified AIN 93 diet, commonly used inexperimentswith rodents.
Results: Malondialdehyde (MDA) and hydrogenperoxide(H O ) concentrations and superoxide dismutase (SOD)
2 2
and glutathionereductase (GR) activities determined intheliver were lower inthe group of mice fed with theAIN
93 diet,whilecatalase (CAT) activity was higher inthesame group, when comparedto the group fed on the
commercial diet.Liver glutathioneperoxidase (GSH-Px) activity was similar inthe groups fed oneither AIN 93 or
thecommercialdiets. Two SOD isoforms, Mn-SODII and a Cu/Zn-SODV, were specifically reduced intheliver ofthe
AIN 93 diet fed animals.
Conclusions: The clear differencesin antioxidant responses observed inthe livers of mice fed onthe two diets
suggest thatthe macro- and micro-nutrient components with antioxidantproperties, including vitamin E, can
promote changes in theactivityof enzymes involved in theremovalof the ROS generated by cell metabolism.
Keywords: AIN 93 diet,Antioxidant enzymes, Commercialdiet, Lipid peroxidation, Liver, Mice, Oxidative stress,
Reactive oxygen species
Background accumulationofintracellularfreeradicals[4]andeventual
Differences inthe compositionof antioxidantcompounds cellular damage. The presence of vitamins and other nu-
in diets and nutritional supplements are known to cause trients with antioxidant activity, acting in conjunction
changes in the responses of enzymes involved in cellu- withantioxidantenzymeshasbeenshowntohavebenefi-
lar defense mechanisms against freeradicals[1,2]. Experi- cial effects against free radicals produced under normal
mental studies using a commercial diet for rodents as a physiologicalandpathophysiologicalconditions[5].
control diet, revealed a smaller antioxidant response in Although an aerobic existence provides many advan-
the control animals when compared to obese animals tages, the use of oxygen by cells results in the produc-
grown on a high fat diet. It was suggested that such a tion of free radicals, which can be defined as molecules
differencemay be due tothe amount of vitamins, suchas or molecular fragments containing one or more unpai-
vitamin E in the diet [3]. Under normal metabolic condi- red electron in atomic or molecular orbitals [6]. Reactive
tions, components of the defense mechanism of the liver, oxygen species (ROS) are defined as oxygen-containing
such as the enzymes superoxide dismutase, catalase and molecules, which may or may not have unpaired elec-
glutathione peroxidase, and non-enzymatic antioxidants trons, but are highly reactive in biological tissues. In re-
such as glutathione, vitamin A, C and E, may prevent the centyearsithasbecomeapparentthatlowconcentrations
of hydrogen peroxide (H O ) may be required for normal
2 2
*Correspondence:[email protected] cellular function and intracellular signaling. Physiological
4SchoolofAppliedSciences,UniversityofCampinas(UNICAMP),1300,Pedro
ROS are generated at the plasma membrane and endo-
ZaccariaSt,JdStaLuiza,13484-350Limeira,SãoPaulo,Brazil
Fulllistofauthorinformationisavailableattheendofthearticle membranesbyNADPHoxidase[7].
©2013Caetanoetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Caetanoetal.BMCPhysiology2013,13:3 Page2of6
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Studies have shown that the presence of antioxidants followingthemethodofHeath&Packer[10].Theconcen-
inthedietincreasesthecellulardefensemechanisms,re- trationofmalondialdehyde(MDA)equivalentswascalcula-
ducing the levels of ROS generated during cell meta- tedusinganextinctioncoefficientof1.55×10-5.mol-1.cm-1.
bolism to normal cell conditions [8]. In this study, the
effect of a purified AIN 93 diet and a commercial diet Hydrogenperoxideconcentration
on the antioxidant responses of the liver of male Swiss H O was measured spectrophotometrically (Lambda 40,
2 2
strain mice,were compared. Perkin Elmer) after reaction with potassium iodide (KI)
[11]. The reaction mixture consisted of 0.2 mL 0.1% tri-
Methods chloroaceticacid(TCA)containingtheliverextractsuper-
Animalsanddiets natant,0.2mLof100mMK-phosphatebufferand0.8mL
Three-week-old male Swiss strain mice (Sw/Uni) free of reagent(1MKI(w/v)infreshdouble-distilledwater).The
specific pathogens were obtained from the State Univer- blankconsistedof1%TCAintheabsenceofliverextract.
sity of Campinas Breeding Center (CEMIB-UNICAMP, The reaction was developed for 1h in darkness at room
Brazil); the animals were housed in individual cages at temperature and the absorbance measured at 390 nm.
20°C with a 12 h light/ 12h dark cycle and were given The amount of H O was calculated using a standard
2 2
free access to the diet and water. Six mice were fed with curve prepared with known concentrations of H O .
2 2
a commercial diet for rodents (CD) and six were fed
with the AIN 93 purified diet for 56 days. The commer- Extraction,determinationofproteinconcentrationand
cial diet contained 25.6% kcal protein, 62.6% kcal car- analysisofantioxidantenzymes
bohydrate, 11.8% kcal lipid and 0.006% diet vitamin E Thefollowing stepswere carried out at 4°C unless stated
(NUVILAB- CR1, Nuvital Nutrientes S/A), whereas the otherwise. The liver tissue was homogenized (5:1 buffer
AIN93 purified diet contained 19.9% kcal protein,64.4% volume:fresh weight liver) inamortarwith apestlewith
kcal carbohydrate, 15.7% kcallipid and 0.015% vitaminE 100 mM potassium phosphate buffer (pH 7.5) contain-
[9] (Table 1). The animals were anesthetized, and after ing 1 mM ethylenediaminetetraacetic acid (EDTA) and 3
loss of corneal and paw reflexes, the liver tissue was mM DL-dithiothreitol [12]. The homogenate was cen-
collected. All mice experiments were approved by the trifuged at 12,100 × g for 30 min and the supernatant
BioethicsCommitteeofOdontologyCollegeofPiracicaba was kept stored in separate aliquots at −80°C, prior to
(FOP/UNICAMP),underprotocoln°CEEA888–1. the determination of protein concentration, superoxide
dismutase (SOD),catalase (CAT), glutathioneperoxidase
Lipidperoxidation (GSH-Px) and glutathione reductase (GR) activity. The
Lipidperoxidationwasdeterminedbyestimatingthecon- protein concentration of all the samples was determined
tent of thiobarbituric acid reactive substances (TBARS) by the method of Bradford [13] using bovine serum
albumin as a standard. SOD activity was determined
as described by Giannopolitis & Reis [14] and the SOD
isoform determination was carried out as described by
Table1CommercialandAIN93dietscompoundsand
chemicalcomposition Azevedo et al. [12], following native polyacrylamide gel
electrophoresis (PAGE). CAT and GR activities were
NUVILAB-CR1-Nuvital AIN93G/M
assayedasdescribedbyCiaetal.[15].GSH-Pxwasdeter-
wholecornground casein
minedasdescribedbyFlohé&Günzler[16].
soybran dextrinizedcornstarch
wheatbran cornstarch Statisticalanalysis
calciumcarbonate cellulose The data are reported as means±standard error of
dicalciumphosphate mineralmix the mean (SEM). Statistical analysis was performed by
an unpaired two-tailed t test, Mann Whitney test using
sodiumchloride vitaminmix
GraphPadPrism6software.P<0.05wasconsideredstatis-
vitaminandmineralmix L-cystine
ticallysignificant.
aminoacids cholinebitartrate
soyoil Results
sucrose The concentration of MDA was used as a biomarker of
Proteins(min) 25.6%kcal Protein 19,9%/14.5%kcal lipid peroxidation (Figure 1A). The results revealed that
the concentration of MDA was higher in the livers of
Lipids(min) 11.8%kcal Lipids 15.7%/9.3%kcal
thecommercialdietfedmice(P=0.0022)thanintheAIN
Carbohydrate 62.6%kcal Carbohydrate 64.4%/76.2%kcal
93fed group. The concentration ofH O (Figure1B) was
2 2
VitaminE 0.006g% VitaminE 0.015g% alsohigherinthesamecommercialgroup(P=0.0152).
Caetanoetal.BMCPhysiology2013,13:3 Page3of6
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compared to those fed on the commercial diet, as shown
A
inTable2.
ht) 4 Toassessthenext stageoftheantioxidantdefensesys-
g
h wei3.5 * tem, which is the degradation of H2O2 produced by the
es 3 SOD reaction, or any other metabolic reactions that may
mol/g fr2.25 epnrozdymuceeseixncethsseHliv2Oer2s,othfethaectmiviitcieessuobfjtehcrteeedatnotitohxeidtawnot
n
nt (1.5 diets were determined and are shown in Table 1. CAT
e
ont 1 activity was higher in the livers of the AIN 93 fed ani-
MDA c0.5 0.92 3.16 minaalsstihmainlarinmthanenceormtomeCrAciTa,ledxiehtibfietdedananimianlcsr.eGasSeHd-Pacx-,
0
AIN93 Commercial tivity trend in the livers of AIN-93 fed animals when
compared to the commercial fed animals, but such an
B
increase was not statistically significant. In contrast, GR
weight) 11..68 * awchtievnitycowmapsalroewdetroitnhethceomlivmeresrcoifalthdeietAgINrou9p3.fed group
h 1.4
s
nmol/g fre 01..182 DAislacrugsesniounmber of studies have shown that ROS can act
nt ( 0.6 as intracellular signaling molecules in many normal phy-
e
nt 0.4 siological processes; however, an increase in such ROS
o
Oc2 0.2 1.04 1.43 can lead to an imbalance causing oxidative stress [6].
H2 0 Several factors such as the diet composition, increased
AIN93 Commercial energy intake, intense physical exercise, and hypercat-
Figure1Lipidperoxidation(MDAcontent)andhydrogen abolic conditions are associated with increased cellular
peroxideconcentrationintheliversofmaleSwissmicefedon
levels of ROS and /or decrease in antioxidant defense
twodifferentdiets.ThemicewerefedwithAIN93purifieddiet
systems triggering a state of oxidative stress in different
(AIN93)orcommercialdietforrodents(CT)for56days.Theanimals
wereanesthetized,andafterlossofcornealandpawreflexes,the tissues/organs [17,18]. The oxidative process such as
liverwascollected.Lipidperoxidationwasdeterminedbyestimating lipid peroxidation of biomembranes, produces several
thecontentofthiobarbituricacidreactivesubstances(TBARS)and compounds that are used as molecular markers, among
HO wasmeasuredspectrophotometricallyafterreactionwith
2 2 them MDA is one of the most widely used indicators of
potassiumiodideinlivertissue.MDA(A)andhydrogenperoxide
the cellularredox state[19].Inthisstudy,the concentra-
content(B)oftheliversofanimalfedwithAIN93andcommercial
dietfor56days.P<0.05,unpairedtwo-tailedttest,MannWhitney tion of the lipid peroxidation marker MDA in the liver
test.Eachvalueisexpressedasmean±SEM. was higher in the group fed with the commercial diet.
AccordingtoSohetetal.[3],commercialdietscontained
Up to five distinct SOD isoforms were identified fol-
lowing native PAGE of liver extracts of the mice fed on
the two different diets (Figure 2). These were character- Standard AIN93 Control
ized as: Mn-SOD (Bands I and II) that were resistant to
Mn-SOD I
KCN and H2O2, a class of SOD isoforms that has been Mn-SOD II
shown to be present in mitochondria and three Cu/
Zn-SOD isoforms (III, IV and V), that were inactivated
Cu/Zn-SOD III
in the presence of KCN and H O , which are normally Cu/Zn-SOD IV
2 2
located in the cytoplasm. The SOD activity band inten- Cu/Zn-SOD V
sity of Mn-SOD II, Cu/Zn-SOD Vand to a lesser extent
Mn-SOD1 were considerably reduced if not absent, fol- Figure2SODisoformsofmaleSwissmicefedontwodifferent
lowing PAGE of the liver extracts of AIN 93, when com- diets.ElectrophoresiswasperformedasdescribedinMaterialsand
Methods.Thegelwasplacedinadarkboxwithdyesolutionfor
pared to the commercial diet fed animals. Cu/Zn-SOD
30min.Afterincubation,thegelwasilluminatedandkeptinwater
III and IV were present in the livers of mice fed on both
untilthedevelopmentofcolorlessbandsofSODactivityinapurple-
diets and did not exhibit any apparent differences in stainedgelwasvisible.Thepictureillustratestheidentificationof
band intensity followingPAGE. Thereduced bandinten- SODisoformsintheliversofmicefedwithaAIN93orcommercial
sity ofthe threeSOD isoformsdetected following PAGE, diet.FivepredominantSODisoformswereidentifiedintheliverof
commercialdietfedanimals,whereasonlySODisoformsI,IIIandIV
could readily explains the lower total SOD activity de-
wereclearlyvisibleinAIN93dietfedanimals.
tected in the liver extracts of the AIN 93 fed mice, when
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Table2Superoxidedismutase(SOD),catalase(CAT),glutathione-peroxidase(GSH-Px)andglutathionereductase(GR)
enzymeactivitiesintheliversofmaleSwissmicefedontwodifferentdiets
Diet SOD(units/mgprotein) CAT(μmol/min/mgprotein) GSH-Px(μmol/min/mgprotein) GR(μmol/min/mgprotein)
AIN93 654.9±5.3 203.1±5.8 7.2±0.3 0.4±0.0
Commercial 106.1±0.7 154.9±3.1 6.3±0.5 0.7±0.0
Pvalues 0.0022 0.0022 0.3052 0.0022
Resultsareexpressedasmean±standarderrorofthemean(SEM).n=6forbothdiets.
lower levels of antioxidant vitamins such as vitamin E, diet group, suggesting that vitamin E might play an im-
resulting in higher values of MDA in hepatic tissue of portant role in lipid peroxidation and, indirectly, in regu-
animals. In another report using mice treated with 0.004, lating SOD activity by maintaining a reduced level of
0.008 and 0.032% vitamin E, a progressive decrease in superoxideinthecellsystem.TheSODisoformsIIandV
MDA levels was detected [20]. In a study where male werehardlydetectablefollowingPAGEofliverextractsof
Wistar rats were submitted to exhaustive stress and trea- micefedontheAIN93diet,whichcouldaccountforthe
ted with gavage administration of vitamin E, decreased reduction in total SOD activity observed in Table 2. This
production of MDA in the kidney tissue was observed is an important result, since the increased concentration
whencomparedwiththecontrolgroup[2]. ofvitaminEandpossiblyothercompoundsoftheAIN93
As well as MDA, the concentration of H O has also diet clearly affected specific SOD isoforms, one located in
2 2
been used as an indicator of oxidative stress. High con- themitochondria(SODII)andmorestronglyonelocated
centrations of H O are closely related with lipid peroxi- in the cytosol (SOD V), since the latter accounts for a
2 2
dation, where the H O in the cell can be converted by higher SOD activity when compared to SOD II. Within
2 2
theFentonreactiontothehydroxylradical,ahighlyreact- the cell,vitaminEpartitionsinto thehydrophobiccoreof
ive compound involved in the initiation of lipid peroxida- thevariouscellmembranes,includingtheinnerandouter
tion [21]. H O can be formed from the degradation of membrane ofthemitochondria [25],although the relative
2 2
superoxide produced during aerobic respiration, and by concentrationof vitaminEdiffersfrom onemembraneto
the exposure of cells to physical, chemical and biological another [26]. Furthermore, α-tocopherol supplementation
agents[7]. in human subjects and animal models has been shown to
Venditti et al. [22] showed that vitamin E decreased decrease lipid peroxidation and superoxide production by
H O release during basal respiration. This effect led to impairing the assembly of NADPH oxidase, as well as by
2 2
reduced ROS flow from the mitochondria to the cytosol, decreasing the expression of scavenger receptors (SR-A
limiting oxidative damage to the liver. In this current and CD36) [27], to which our results appear to match
study, the vitamin E content of the AIN 93 diet was 2.5- suchapossibility.ThereductionobservedinSODactivity
fold higher (0.015%) than that of the commercial diet inthe livers ofanimals subjected totheincreasedvitamin
(0.006%), which might suggest participationofVitamin E, E diet alsosuggests thatthe productionof the superoxide
along with other nutrients in the diet, in the responses radicalislikelytobediminishedmorelikelyinthecytosol
observed. For instance, in the livers of the animals fed and the mitochondria, which agrees and can be clearly
with AIN 93 there was less accumulation of H O , when correlatedtothespecificdepletionofSODIIandSODV.
2 2
compared tothe animalson the commercialdiet, indicat- Moreover, vitamin E has also been shown to prevent the
ingthatvitaminEmaybeinterferinginROSlevelsduring inductionofmetallothioneinsynthesisaswellaslipidper-
normalcellmetabolism. oxidationinthe liverofmice administered the mitochon-
Vitamin E is an important fat-soluble antioxidant in drial inhibitor 2,4-dinitrophenol [28], which agrees with
the body and operates with some of the antioxidant the findingsobserved hereof the depletion of the specific
enzymes tested in this study, such as superoxide dis- SOD isoforms and reduction in lipid peroxidation. More-
mutase (SOD), catalase (CAT) and glutathione peroxid- over,Fe-SODisoforms,whichcanbefoundinlivingcells,
ase (GSH-Px), to protect cells from attack by ROS [23]. but not necessarily in all living organisms, were not
SOD provides the first line of defense against oxygen detectedfollowingPAGEinthiswork.
derived free radicals [6]. Under stress conditions, high The enzymes CAT and GSH-Px are part of the next
SOD activity reflects a compensatory mechanism to re- step of the antioxidant defense mechanism, converting
duce the superoxide radical. Male rats Wistar fed with H O to water [29]. Alper et al. [30] reported a decrease
2 2
the control diet supplemented with 0.01% of vitamin E in CAT activity in rats fed with a diet deficient in vita-
showed a reduction in SOD activity [24]. In the results min E. The authors suggested that the decrease in CAT
presented here, there was lower SOD activity in the li- activitymightbedue tothesuppression of heme biosyn-
versoftheAIN93fedgroupcomparedtothecommercial thesis. Heme is a prosthetic group that consists of an
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iron atom contained in the center of a heterocyclic or- tested so that the effects on the antioxidant responses are
ganic ring termed porphyrin, which is present in the moreclearlyunderstoodandtheputativekeyroleof vita-
molecule of CAT, and is synthesized in the liver and min E is confirmed. Based on the composition of both
erythroid tissues [31]. Studies with animals deficient in diets, it cannot be ruled out that other components may
vitamin E showed a decrease in hepatic activity of heme have had an indirect effect on mouse metabolism, which
proteins such as CATand microsomal cytochrome P450 might have resulted in a response by the antioxidant sys-
andb [30].Inthisstudy,therewasasignificantincrease tem. Nevertheless, we must state that the fact that a high
5
in CATactivity in the livers of mice grown on the AIN vitamin E concentration was used and that among the
93 diet, which contained 2.5 fold higher more vitamin E componentsofthedietvitaminEisclearlytheonlydirect
than the commercial diet, supporting a role for this vita- and the major non-enzymatic antioxidant included, we
min in improving the rate of removal of H O in meta- mayassumethatVitaminE appearstohaveanimportant
2 2
bolically normal animals. Thus, the higher CATactivity participationintheresponsesobserved.
observed in animals fed with the AIN 93 diet could
explain the lower concentration of H2O2 observed Conclusion
(Figure 1B). Similar results were described by Ryan
Inconclusion,ourresultssuggestthatthedifferentcom-
et al. [32] in a study on the muscles of rats fed with
position of the two diets in particular the one with vita-
supplemented vitamin E or normal non-supplemented
min E included, with or without the co-participation of
rat chow. GSH-Px can act directly on H O , however,
2 2 other diet components, appears to have a considerable
this enzyme also acts in the inactivation of organic
effectonthe defense mechanism againstfree radicals ge-
hydroperoxides [33]. There was little difference in the
nerated during normal metabolism in male Swiss strain
activity of GSH-Px in the livers of the mice fed with
mice. One key aspect of such an effect is likely to be the
the two different diets. Similar results were reported
generation of the superoxide radical in the mitochondria
in a study with rats fed with diets supplemented with
and cytosol, based on reduction in activity of specific
vitamin E and a control diet [25]. GR is an enzyme
isoforms of SOD present in the liver of animals fed on
that is used for the regeneration of reduced glutathione
the AIN 93 diet. Therefore, the results further suggest
from oxidized glutathione, especially when the cell is ex-
that a diet rich in vitamin E may naturally give extra
posedtofreeradicals.Shireenetal.[34]workingwithrats
protection to the liver against any oxidative stress condi-
fed with an AIN 76 diet and AIN 76 supplemented with
tion that may occur, leading to excess ROS production,
vitaminEandC,demonstratedthattherewasasignificant
as a result of metabolic changes produced if the animal
increase in the activity of both GSH-Px and GR, in the
issubjected toa stressfulcondition.
animals fed with the vitamin supplemented diet. In this
study, the activity of GR in the livers of animals fed with Abbreviations
the AIN 93 diet was significantly lower than in animals AIN:Americaninstituteofnutrition;CAT:Catalase;GR:Glutathionereductase;
GSH-Px:Glutathioneperoxidase;MDA:Malondialdehyde;ROS:Reactive
fed with the commercial diet, suggesting that the AIN 93
oxygenspecies;SOD:Superoxidedismutase.
dietgrouphasalowerdemandforreducedglutathionein
thecellulardefensemechanisms,orthatGRmaynothave
Competinginterests
a major role as a defense enzyme under the conditions Theauthorsdeclarethatthereisnoconflictofinterestinthedissemination
tested.Awiderangeofantioxidantenzymesexistinorder ofthisdata.
to keep the redox state of the cell, so they are important
Authors’contributions
not only under normal metabolic conditions, but also
ACCparticipatedintheacquisitionofdata,performedthestatisticalanalysis,
whendealing withstressful conditions.Althoughinterest- interpretedthedataanddraftedthemanuscript.LFVparticipatedin
ingresponsesinenzymeactivitiesweredetectedinthisre- conductingtheanimalexperimentsandtheacquisitionofdata.FRC
participatedintheacquisitionofdata,interpretedthedataandrevisedthe
search, the role of other peroxidases or antioxidant
manuscript.SMAandRAAparticipatedintherevisionofthemanuscriptand
enzymescannotberuledoutandshouldbe consideredin RMNBconceivedthestudy,determinedthedesign,interpretedthedataand
futureresearch. draftedthemanuscript.Allauthorsreadandgavefinalapprovalforthe
versionsubmittedforpublication.
Before any major conclusions can be drawn, it is im-
portant to state that this study did not investigate varying
Acknowledgements
concentrations of vitamin E or of any other individual WewouldliketothankProf.Dr.PedroLuizRosalenbygrantingtheroom
components of the diets, which limits the impact of the forexperimentationanimalsattheCollegeofOdontologyofPiracicaba
(FOP/UNICAMP)andFernandoPiottoforguidanceandsupervisiononthe
results and how they are correlated with the major
statisticalanalysisofallresults.ThisworkwassupportedbyFundaçãode
changes observed. Even with such a limitation, the data AmparoàPesquisadoEstadodeSãoPaulo(FAPESP—Grantn°04/1381-0
obtained are not invalidated. However, future studies and08/52721-5).F.R.C.andR.A.A.wouldliketothankFAPESPforpost-
doctoralfellowship(Grantn°06/60473-6).R.A.A.alsothankstheConselho
should be performed in a way that varying concentra-
NacionaldeDesenvolvimentoCientíficoeTecnológico(CNPq,Brazil),forthe
tionsofvitaminEandothercomponentsofbothdietsare fellowshipgranted.
Caetanoetal.BMCPhysiology2013,13:3 Page6of6
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