Models of hepatoprotective activity assessment

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www.elsevier.es/rmuanl

REVIEW

ARTICLE

Models

of

hepatoprotective

activity

assessment

C. Delgado-Montemayor

a

,

P. Cordero-Pérez

b

,

R. Salazar-Aranda

a

,

N. Waksman-Minsky

a,∗

a_Department_of_Analytical_Chemistry,_Medical_School,_Autonomous_University_of_Nuevo_Leon,_Monterrey,_Mexico

b_Liver_Unit_at_the_‘‘Dr._José_E._{González’’}_University_Hospital_of_the_Autonomous_University_of_Nuevo_Leon,

Monterrey,Mexico

Received16January2015;accepted30October2015 Availableonline23April2016

KEYWORDS

Hepatoprotection; Invitromodels; Invivomodels; Exvivomodels; Liver;

Hepatocuration

Abstract Liverdiseasesareamajorhealthproblemworldwide,makingitnecessarytodevelop newmoleculesthathelpcounteractorpreventsuchdiseases.Onaccountofthisfact, inves-tigations aimingto obtainnatural and/or syntheticcompounds possessing hepatoprotective activityhavebeenundertaken.Thedevelopmentofnewdrugsconsistsofavarietyofsteps, rangingfromthediscoveryofthepharmacologicaleffectsincellularandanimalmodels,to finallydemonstratetheir efficacyandsafetyinhumans.Differentmodelsfor assessmentof thehepatoprotectiveactivityinvitro,exvivoandinvivocanbefoundinmedicalliterature. Thepurposeofthisreviewistoshowthefeatures,mainadvantagesanddisadvantagesofeach ofthemodels,thehepatotoxicagentsmostcommonlyused(CCl4,acetaminophen,ethanol,d

-galactosamine,t-BuOOH,thioacetamide)aswellasthebiochemicalparametersusefultoassess liverdamageinthedifferentmodels.

Abbreviations:DNA,deoxyribonucleicacid;ALT,alanine amino-transferase; RNA, ribonucleic acid; AST, aspartate aminotrans-ferase;CCl3, trichloromethylradical;CCl4,carbontetrachloride;

ALP,alkalinephosphatase;GGT,gamma-glutamyl-transpeptidase; GSH, reduced glutathione; GSSG, glutathione disulfide; NF-␬B, nuclearfactorkappabeta;LDH,lactatedeshydrogenase;NADPH, nicotinamideadenine dinucleotidephosphate;NAPQUI, n-acetyl-benzoquinoneimine; WHO, World Health Organization; PCTS, precision-cuttissueslices;t-BuOOH,tert-Butylhydroperoxide.

∗_{Corresponding}_author_at:_Departamento_Química_Analítica,

Fac-ultaddeMedicinaUANL,Av.MaderoyAguirrePeque˜noS/N,Col. MitrasCentro,CP.64460Monterrey,NuevoLeón,Mexico.

E-mailaddress:nwaksman@gmail.com(N.Waksman-Minsky).

Introduction

The liver is a key organ. It regulates different functions in the body, such as metabolism, secretion, storage and detoxifying.Liverdamageisusuallyassociatedwiththe dis-tortionofsomeofthesefunctions.Theliveriscontinuously exposed to an elevated amount of toxic agents, because theportalveinsuppliesbloodtothisorganafterintestinal absorption.1,2

TheWorld HealthOrganization (WHO)determined that around 2.4 million deaths yearlyare linkedto some liver disease,andthataround800thousandofthesedeathsare attributabletocirrhosis.3_On_the_other_hand,

epidemiologi-calstudiesconductedbytheNationalInstituteofStatistics

http://dx.doi.org/10.1016/j.rmu.2015.10.002

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Oxidative stress

Oxidative damage in DNA and proteins

↓ _ATP

ROS

H2O2

HO• •

O2 –

↑ Lipid peroxidation

Figure1 Mechanismofliverdamageduetooxidativestress.

and Geography (INEGI by its Spanish acronym) indicate that in 2013 in Mexico, over 600 thousand deaths were recorded.Themaincauseswerediabetesmellitus(14.25%), followedbyischemicheartdiseases(12.63%), cerebrovascu-lardiseases(5.29%)andliverdiseases(4.79%).Despitethe advancesinmodernmedicineandthedevelopmentofnew hepatoprotectivedrugs,4---6_the_incidence_of_hepatic_diseases

has not decreased or stopped; onthe contrary, statistics suggestthatthesecontinuetoincrease.7,8

Metabolism or biotransformation of hepatotoxicagents is a detoxifying process where molecules are surgically modifiedintolesstoxicshapesbydifferentenzymatic sys-tems.Thesemodificationscangeneratemetabolicproducts withvaryingdegreesofpharmacologicalactivityorinactive metabolites. Therearedifferent typesof metabolic reac-tions:phase1reactionsareusuallyoxidations,reductionsor hydrolysis(modifyingthestructureofthereactive group); phase2 reactions arethose inwhich the drugconjugates withglucuronicacid,sulfates,acetates,methylgroups, glu-tathioneoraminoacids,generallytoincreaseitssolubility andbeexcreted.Theliver’sabilitytobeabletocarryout thedifferentoxidativemetabolisms isassociatedwiththe highcytochromeP450cellcontent.9

Duetothehighmetabolitebiotransformationrate,free radicals can be generated continuously. Most hepatotoxic substances, mainly damage the liverbecause of the gen-eratedoxidativestress;oxygenreactive speciesinduced a riseinlipidperoxidation,areductionofATPandoxidative damageintheDNAandproteins(Fig.1).10---13

Protecting the liver from the harmful effects of hepatotoxins-whichmaybeingested-orcounteractingthe alterationsintheantirradicaldefensemechanisms,isvery important; the agents capable of doing this are called hepatoprotective.14

For this reason, researches have been developed in the search of natural and/or synthetic compounds withhepatoprotective activity.8 _The _development _of _new

pharmaceuticalsconsistsof avariety ofsteps, goingfrom thediscoveryofpharmacologicalsideeffectsincellularand

animal models, to finally prove its efficacy and safety in humanbeings.1

In vivo and well as ex vivo test models are used to evaluate hepatoprotective activity. These systems mea-sure the ability of the drug to prevent or cure hepatic toxicity(inducedbydifferenthepatotoxins)incellular cul-tures,organsorinexperimentalanimals(rats,mice,etc.) respectively.1

Evaluation

models

Nearlyeveryacute andchronic liverinjury can be exper-imentally induced; necrosis, steatosis, hepatic injuries, cirrhosisandcholestasis.Thesecanallbegeneratedin dif-ferentmodelsofliverdamage.

Theobjectiveofhepatoprotectivemodelsisforthe com-pounds, fractions or extracts being tested to counteract oravoidthedamagegeneratedbyhepatotoxins.The mag-nitude of the hepatoprotective effect can be measured throughbiochemical makers, survival rate or histology of theliver.

Testmethodsmaybeinvitro,exvivoorinvivo(Table1); andeach oneofthemcan beevaluatedtoseeifthe sub-stanceishepatoprotectiveorhepatocurative,dependingon ifthehepatoprotectiveagentisadministeredbeforeorafter thehepatotoxin.

Invitromodels

Freshhepatocytes,primaryhepatocyteculturesand immor-talizedcelllinesareusedtomeasurethehepatoprotective effect.Itispossibletoestablishactionmechanismsinthese models. These models represent the best option for the screeningandselectionofpotentialhepatoprotective com-poundsanditispossibletoestablishactionmechanismsat acellularandmolecularlevel.15,16

Primary hepatocytecultureshave thecharacteristic of maintainingnormalmetabolicliverproperties,butitisnot possibletomaintainthemforalongtime.Ontheotherhand, celllines maintaintheir properties stablefor a long time andcanbecryopreserved,butimmortalizedorcarcinogenic linesmaydifferinbiochemicalandmetabolicaspectsfrom normalcells.1

In order to evaluate protection, parameters like transaminase liberation, cell multiplication, morphology, macromolecular synthesis, oxygen consumption, etc., are measured.17,18

Advantagesofinvitromodelsare:Theyarequicktests (between2---3testingdays),theyrequiresmallamountsof thetest substances (milligramrange)and the experimen-talconditionsmaybestrictlycontrolled;differentsamples maybeanalyzedinthesametest,theyarecheaptestsand thereis littlevariability; therefore theyareconsidered a reproducibletest. Inthecase ofprimaryculturesor fresh hepatocytes,theyrequirefewexperimentalanimalsin com-parisontoinvivomodels.

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Table1 Modelsofevaluationsofhepatoprotectiveactivity.

Model Examples Advantages Disadvatages

Invitro *Freshhepatocytes *Primaryhepatocyte culture

*Immortalizedcelllines (HepG2,HUH7,HepRG)

*Quickandcheaptests *Requiresfewsamples *Highcontrolof variables;reproducible *Cananalysesvarious samplesinthesametest

*Duetoalackofcomplexitypresent intheorganofbiologicalsystem, resultsshouldbeinterpretedwith caution.

*Samplesdonotundergoany biotransformationprocess

Exvivo *Preciselivercuts

*Isolatedperfusedliver

*Resembletheinvivo environment

*Decreasethenumber ofanimalsexperimented on

*Ahumantissuemodel canbedeveloped

*Lowoxygenationrateinthe internalcells

*Lowcutviability(1---10días) *Therearesignificantdifferencesin sizeandfuctionbetweenhumanand murinetissue.

Invivo *Murinemodel *Widelyused

*Thereisagreater correlationwithwhat happensinhumans *Allbiochemicaland histologicalparameters canbemeasured

*Requiresalargenumberofanimals toexperimenton

*Interindividualvariationexists *Alargersimplesizeisrequired *Largeandexpensiveexperiments

beverified within vivosystems. Isolatedcells as well as celllineshavean elevatedcelldifferentiationratedueto thelossofnaturalenvironment.Thesubstancestested do notgo throughthe absorption anddistribution processes, whichoccursintheorganism.Thereislittletono cell-to-cellinteraction and thereis no complexity proper of the organ.19---21

Exvivomodels

Precisioncutliverslices(PCLS)areanexvivotissueculture which imitates multicellular characteristics of in vivo

organs.Cellularinteractionand spatialdisposition remain intactinthismodel,withthepossibilityofperforming mor-phologicalstudies. Liver slices have the characteristic of functionallymaintainingmetabolizingenzymesandbiliary canaliculus21_; _they_have _proven _to_be _a_valid _ex _vivo

sys-temtostudymetabolismandliverdamageandfunctionas abridgebetweeninvivosystemsandcellcultures.22

Isolated perfused livers represent a model combining

in vitro characteristics under in vivo circumstances. The

firstmodelwasdevelopedinporcineliversandlaterthe liv-ersofsmalleranimals(rats,miceandrabbits).Thismodel preservesthetridimensionalstructureaswellasthe cell-to-cellinteractionswiththepossibilityofcollectingbileinreal time.Ifbloodisusedasaperfusorliquid,thenhemodynamic parametersmaybestudied.23

Advantages of ex vivo models: they resemble in vivo

atmospheres,arelowcost,reproduciblemodels.InPCLSthe numberofexperimentalanimalsisreduced,alsothemodel canbedevelopedwithhumanorgans.

Disadvantagesof exvivomodels:in PCLSthe bileflow andfunctionalparameters,suchasportalflow,cannotbe analyzed.1 _There _is _poor _diffusion _of _oxygen _nutrients _to

themoreinternalcells,andevenwiththedevelopmentof new means of culture, the viabilityof the slicesremains short (8---10 Days).22 _In _small _labs, _because _of _space _and

budget,thebestoptionisthedevelopmentofperfusedrat liver;however,therearesignificantdifferencesinthesize, functionandgeometryofthemurinelivercomparedtothe human.1

Invivo_model

This model hasbeen widely used;through thismodel we areabletodeterminetheprotectionmechanism.The dam-ageproducedinexperimentalanimalsduetoknowndosage administration of different hepatotoxins and the magni-tude of the damage and/or protection is determined by the differentbiochemicaland metabolic markers,as well ashistopathologicaldeterminations.

Advantagesofinvivomodels:isthemodelwiththe high-estdegreeofcorrelationwithwhatoccursinhumansandall biochemicalandhistopathologicalparameterscanbe mea-sured.Theyletustakeintoaccountthepossibleeffectsof theimmuneandcentralnervoussystemsinthedevelopment ofhepaticdiseases.24

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Hepatotoxic

agents

and

their

action

mechanisms

Themoleculesresponsibleforliverdamagearecalled hep-atotoxins; nowadays it is possible to imitate any formof natural-originhepaticdiseasewithdifferentchemical sub-stancesandpharmaceuticals.

Hepatotoxinsmaybeclassifiedasintrinsiciftheagent’s behavior is predictable; there is a period of constant latencybetweenexposureandliverdamagedevelopment, or theinjury is dose-dependent(i.e. carbontetrachloride

{CCl4},thioacetamide, acetaminophen, ethanol). Another

classification is idiosyncratic, if the agents are not pre-dictable,butgenerateliverdamageinjustasmallportion of exposed individuals, the injury is not related to the dosage, it occurs after a variable latent period and it is notreproducible in experimentalanimals (i.e. halothane, sulfonamides,isoniazid).26,27

Carbontetrachloride(CCl₄)

CCl4toxicitydependsondosageandthedurationof

expo-sure.Inlowdose,effectslikelossofCa2+_homeostasis,_lipid

peroxidation, andrelease of cytokinesare produced, and apoptotic events may be generated, followed by cellular regeneration.Inhighdoses,orifthereisalongerexposure, theeffectsaremoresevereandthedamageoccursduring alongerperiodof time,the patientmay developfibrosis, cirrhosis,orevencancer.5,28,29

CCl4ismetabolizedbythecytochromeP450dependentof

monooxygenases,mainlythroughtheCYP2E1isoforminthe endoplasmicreticulumandmitochondria.16 _{Hepatotoxicity}

isproducedbytheformationofthetrichloromethylradical (CCl3), which is highly reactive. These radicals may

satu-ratetheorganism’santioxidantdefensesystem,reactwith proteins, attack unsaturated fatty acids, generating lipid peroxidation,reducetheamountofcytochromeP450,which leadstoafunctionalfailurewiththeconsequentloweringof proteinandaccumulationoftriglycerides(fattyliver),and alterwaterandelectrolyteequilibriumwithanincreaseof hepaticenzymesinplasma.30

Lipidperoxidationleadstoacascadeofreactions,such asthe destruction of membrane lipids, the generationof endogenoustoxicsubstances,whichoriginatemorehepatic complications and functional anomalies. For this reason, lipidperoxidationisconsideredacriticalfactorinthe patho-genesisofliverinjuriesinducedbyCCl4.15Theinhibitionof

the radical CCl3 generation is a key point in the

protec-tion against the damage generated. Because of this, this modeliswidelyusedfortheevaluationofpharmaceuticals andnaturalproductswithhepatoprotectiveandantioxidant activity.31,32

Acetaminophen

It is an analgesic antipyretic analgesic. In high doses, it producesacuteliverdamage,causingnecrosisofthe hepa-tocytes.Itisawidelyusedexperimentalmodelofclinical importanceasanexampleofdrug-inducedliverdamage.16

At therapeutic doses, it is mainly metabolized to glu-curonic or sulfated and excreted derivatives, the rest metabolizes to intermediate reactives, which are elimi-natedby conjugation withglutathione. At overdoses, the excess is oxidized by the cytochrome P450 (mainly the CYP2E1 isoform)33 _at _{N-acetyl-p-benzoquinone} _(NAPQI),

whichquicklyattachestoglutathione.Underexcessive con-ditionsofNAPQIandglutathionedepletion,acovalentbond ofmetabolitetoproteins,adductformation,mitochondrial dysfunctionandoxidativestressoccurs.Theresultis necro-sisorhepatocellulardeath.19,34

Ethanol

Theliveristhemostsusceptibleorgantothetoxiceffectsof ethanol.Thedamagemechanismisduetothemetabolism of ethanol by the CYP2E1 isoform of the cytochrome P450 producing oxidative stress with the generation of reactive species of oxygen and the increase of lipid per-oxidation, leading to the alteration of the compositions ofphospholipidsof thecellularmembrane.35,36 _Membrane

lipid peroxidation results in the loss of its structure and integrity,elevatingserumlevelsofglutamyl-transpeptidase, amembrane-bondingenzyme.Ethanol inhibitsglutathione peroxidase;itreducestheactivityofcatalaseanddismutase superoxide.16

Thedecreaseintheactivityofantioxidantenzymes, dis-mutasesuperoxideandperoxidaseglutathioneisbelievedto comeasaresultoftheharmfuleffectsoffreeradicals pro-ducedafterexposuretoethanol,oralternatively,theycould bea directeffect of acetaldehyde, a product of ethanol oxidation.

D-Galactosamine

Thishepatotoxingeneratesasimilardamagetoviral hepati-tisregardingmorphologicandfunctionalcharacteristics.A singledosecancausehepatocellularnecrosisandfattyliver. Itinducestheexhaustionoftheuracilnucleotide, result-ing in the inhibition of RNA synthesis and consequently of proteins.37 _The _toxicity _mechanism _causes _loss _of _the

activity of ion pumps and an increase in cellular mem-brane permeability, leading to enzyme liberation and an increasein intracellular Ca2+ _{concentration,} _which_is

con-sideredresponsibleforcellulardeath.16,36,38

Tert-Butylhydroperoxide(t-BuOOH)

Metabolized to free radicals by cytochrome P450 in hepatocytes generating lipid peroxidation, a decrease of glutathione,itreduces thepotentialof themitochondrial membraneandcellulardamage;generateddamageis simi-lartooxidativestress,whichoccursincellsandtissues.36,39

Alternatively,t-BuOOHcanbeconvertedbyglutathione peroxidaseintotetr-butylalcoholandglutathionedisulfide (GSSG).GSSGisconvertedintoreducedglutathione(GSH) bytheGSSGreductase,generatingtheoxidationofpyridine nucleotides(NAPD).Alltheseeventsalterthehomeostasis ofCa+2_which_is_considered_a_critical_event_to_provide

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Thioacetamide

Anorganiccompoundcontainingsulfur,originallyusedasa fungicideandcurrentlyusedforthetreatmentofleather,in labsandinthetextileandpaperindustries.29_It_can_induce

acuteandchronichepaticinjuriesandactsoverthe synthe-sisofprotein,DNA,RNAandover␥-glutamyltranspeptidase

(GGT)activity.

Thioacetamideisbio-activatedbytheCYP450and/orby themonooxigenasesystem,whichcontainsflavin, convert-ingthecompoundintosulfine(asulfoxide-typecompound) andlaterintosulfone-typecompounds.Sulfineisresponsible for generating an increase in the nucleus volume, nucle-olienlargement,anincreaseinintracellularconcentration of Ca+2_, _generating _changes _in _cellular _permeability _and

mitochondrialdysfunction.Ontheotherhand,Sulfone-type compoundsareresponsiblefortheliberationofnitricoxide synthaseand the nuclearfactor kappa B(NF-␬B), protein

denaturalizationandlipidperoxidation.41---43

Liver

function

markers

A decisive step when biological activity models are per-formedistheanalysisoftheactivityofthetestedanalyte. Depending on the selected model and its characteristics, thesurvivalrateandthedamagedbiochemicalmarkerscan bedetermined.Due tothe widevarietyof functions per-formedbytheliver,thereisawiderangeofmarkersthrough whichweareabletodeterminethefunctionalityordamage generatedbythisorganoritscells.28 _Although_there_is_no

biochemicalmarkerspecifictoliverdamage,the combina-tionof severalofthese,andknowingthecorrelation they havewiththeliver,willhelptobetterinterprettheresults ofthehepatoprotectivemodels.Markerscanbedividedinto tests related to the liver’s excretory function (bilirubin), testsrelatedtosyntheticfunction(albuminand prothrom-bintime)andtestsrelatedtotheintegrityofhepatocytes (transaminases,alkalinephosphatase,GGT).

Transaminasesoraminotransferases

Transaminasesoraminotransferasesareenzymesthat trans-fer a group of amino from an amino acid to an acid

␣-acetate. This process is an important step in the

metabolism of amino acids. The aspartate aminotrans-ferase (AST) and the alanine aminotransferase (ALT) are widely used enzymes; the increase in the liberation of thesetransaminasesislinkedtoliverdysfunction.ALT cat-alyzes the amino group transference of the L-alanine to

␣-ketoglutaratetoproducepyruvateandL-glutamate;itis

elevatedin hepaticand renaldiseases, i.e.hepatitis, cir-rhosisand mononucleosis. AST catalyzes the transference oftheaminogroupoftheL-aspartateto␣-ketoglutarateto

produceoxaloacetateandL-glutamate;theheart,liverand skeletalmuscle,areorgansrichwiththisenzymeandthe ASTliberationisproportionaltothedamagegenerated.Ina myocardialinfarctionitstartstoincreasebetween3and9h aftertheevent,reachingitspeakonthesecond day;the levelsnormalizebetweenthe fourthand thesixthday. In hepatitiscases,observedelevationsarebetween7and12

timesitsnormalconcentrations,withincreasesofupto100 times.28

Phosphatases

These enzymes belong to the hydrolases family and are known for their ability to hydrolyze a wide variety of organophosphate compounds with the formation of phos-phateionsandalcohols.Clinicallyrelevantphosphatasesare acidphosphataseandalkaline phosphatase.Alkaline phos-phatase(ALP)isproducedmainlyintheliverandbone;when there arenoosteogenic diseases, ALP elevation is gener-allylinkedtohepatobiliarydiseases. Itis morespecific in obstructivehepaticprocesses.28,44

Transpeptidase␥-glutamine(GGT)

This enzyme is boundto the plasmatic membrane, which catalyzes the transference of the ␥-glutamine group of a

peptide toitselforother peptides.Itis locatedmainly in hepatocytes;howeveritcanalsobefoundintheproximal renal tubules, intestinalepithelial cells and theprostate. High GGTP levels usually indicate infection in the liver, pancreaticand biliaryzones.The specificityof thetest is relativelylow,butsinceitisnotlinkedtobonediseases,it isusedtolinkhighALPlevelstoliverdamage.44

Bilirubin

Bilirubin is the most important metabolite of the heme group, found in hemoglobin, myoglobinand cytochromes. Itishighlyinsolubleinwaterinitsmostcommonisomeric form,and most ofit is transportedby albumin. The liver is responsible for eliminating bilirubin by turning it to a morehydrosolubecompound,thusallowingitselimination ofplasmaforitseventualexcretion.Itisthemostimportant test ofthehepaticmetabolicfunction;however,itis only possibletodetermineitininvivomodels.44

Totalproteins

Theliversynthetizesmostplasmaticproteins,andinmost hepaticdiseasesthelevelsarereduced.Albumin,␣-1

antit-rypsin,ceruloplasmin,and␣-fetoproteinareproteinslinked

toacuteliverdamage.

Lactatodeshydrogenase(LDH)

Lactatodeshydrogenaseisanenzymelocatedinthecellular cytoplasm. Itcatalyzes the interconversion ofthe lactate and pyruvate; LDH liberation may be interpreted as the opening of the cellularmembrane or cellular death. This enzyme is not specific to the liver and it is widely used

inin vitromodelsbecauseitis expressedinmostcellular

lines.45

AST, ALT and ALP are most commonly analyzed in all hepatoprotectivemodels,whilethequantificationoftotal proteinsandLDHaregenerallyusedasparametersofinvitro

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Conclusions

Liver diseases are a major health problem, domestically and around the world; thus, it is necessary to develop newmoleculeswhichhelpcounteractorpreventthem.The discovery and development of new drugs begins with the demonstrationofthepharmacologicaleffects,tolater con-ductsafetyand efficacystudies in humanbeings. Invitro

modelsarewidelyused;theyarefast,cheap,reproducible techniques andrequirealowersample. Nevertheless, the results ought tobe reevaluated by other models.Ex vivo

models are an intermediate point between in vivo and

invitromodels,butarelessutilized.Unliketheothertwo,

in vivomodelsprovidea widerangeof information.They arewidely usedtoverify theactivity of newcompounds, although they are more expensive and go through many experimentalanimals.Hence,theyaregenerallyusedafter

aninvitroorexvivoevaluation,asastepprevioustoclinical

trials.

Conflict

of

interest

Theauthorshavenoconflictsofinteresttodeclare.

Funding

Nofinancialsupportwasprovided.

Acknowledgements

SpecialthanksgototheMexicanNationalCouncilofScience andTechnology(abbreviatedCONACYT)fortheirsupportfor thenationalscholarshipno.359832andprojectno.180997 oftheBasicScientificResearchGrant2012.

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