Journal of Hazardous Materials
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j h a z m a t
Automated cytochrome c oxidase bioassay developed for ionic liquids’
toxicity assessment
Susana P.F. Costa, Bárbara S.F. Martins, Paula C.A.G. Pinto
∗, M. Lúcia M.F.S. Saraiva
∗LAQV, Requimte, Departamento de Ciências Químicas, Laboratório de Química Aplicada, Faculdade de Farmácia, Universidade do Porto, Rua Jorge Viterbo Ferreira, N◦228, 4050-313 Porto, Portugal
h i g h l i g h t s
•Development of the first automated cytochrome c oxidase inhibition assay based on SIA.
•The developed methodology was val- idated through evaluation of 15 ILs toxicity.
•The influence of different structural elements on ILs toxicity was studied.
•A rigorous control of the analyti- cal parameters was obtained through computer control
g r a p h i c a l a b s t r a c t
a r t i c l e i n f o
Article history:
Received 24 September 2015
Received in revised form 1 January 2016 Accepted 1 February 2016
Available online 3 February 2016
Keywords: Cytochrome c oxidase SIA Ionic liquids Toxicity screening a b s t r a c t
A fully automated cytochrome c oxidase assay resorting to sequential injection analysis (SIA) was devel- oped for the first time and implemented to evaluate potential toxic compounds. The bioassay was validated by evaluation of 15 ionic liquids (ILs) with distinct cationic head groups, alkyl side chains and anions. The assay was based on cytochrome c oxidase activity reduction in presence of tested com- pounds and quantification of inhibitor concentration required to cause 50% of enzyme activity inhibition (EC50). The obtained results demonstrated that enzyme activity was considerably inhibited by BF4anion
and ILs incorporating non-aromatic pyrrolidinium and tetrabutylphosphonium cation cores. Emim [Ac] and chol [Ac], on contrary, presented the higher EC50values among the ILs tested.
The developed automated SIA methodology is a simple and robust high-throughput screening bioassay and exhibited good repeatability in all the tested conditions (rsd < 3.7%, n = 10). Therefore, it is expected that due to its simplicity and low cost, the developed approach can be used as alternative to tradi- tional screening assays for evaluation of ILs toxicity and identification of possible toxicophore structures. Additionally, the results presented in this study provide further information about ILs toxicity.
© 2016 Elsevier B.V. All rights reserved.
Abbreviations: ILs, ionic liquids; SIA, sequential injection analysis; CytCox, cytochrome c oxidase; FeC, ferrocytochrome c; emim [BF4], 1-ethyl-3-methylimidazolium tetrafluoroborate; emim [Tf2N], 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide; emim [Ms], 1-ethyl-3-methylimidazolium methanesulfonate; emim [TfMs], 1-ethyl-3-methylimidazolium trifluoromethanesulfonate; bmim [BF4], 1-butyl-3-methylimidazolium tetrafluoroborate; bmim [Cl], 1-butyl-3-methylimidazolium chloride; hmim [Cl], 1-hexyl-3-methylimidazolium chloride; bmpy [BF4], 1-butyl-4-methylpyridinium tetrafluoroborate; bmpyr [BF4], 1-butyl-1-methylpyrrolidinium tetrafluoroborate; bmpyr [Cl], 1-butyl-1-methylpyrrolidinium chloride; N4,4,4,4[BF4], tetrabutylammonium tetrafluoroborate; tbph [Ms], tetrabutylphosphonium methane- sulfonate; chol [Ac], choline acetate; bmim [Ac], 1-butyl-3-methylimidazolium acetate; emim [Ac], 1-ethyl-3-methylimidazolium acetate.
∗ Corresponding authors. Fax: +351 226093483.
Ionicliquids(ILs)raisedinterestinscientificcommunity,par- ticularly in the last two decades, as potential alternative to conventionalorganicsolvents.ILsaregenerallydefinedasorganic saltswithmeltingpointsbelow100◦C[1,2],andahugenumber ofcombinationbetweencationsandanionsarepossible[3].These compoundsexhibitlowvaporpressure,highthermalandchemical stability,non-flammability,highliquidrange,solvationabilityand relativelylowviscosity[2].Thesenoteworthycharacteristicsledto aremarkableincreaseinthenumberofpublicationsdescribingpos- sibleapplicationsinanalyticalproceduresinacademia[4–8]and industry[3].TheindiscriminateutilizationofILsatindustrialscale, infuture,raisestheissueofILseffectwhenreleasedtotheenviron- mentandpotentiallong-termconsequences.Itisthenrequiredthe studyoftheirpossible(eco)toxicitybeforethedischargeintothe environment.Somestudiesfocusedonthisthemeandtheresults demonstratedthatatleastsomeILspresentnegativeeffectonthe studiedorganisms[9–12].DespiteILsnegligiblevaporpressureand thereforelowairpollutionrisk,theirwatersolubilitypointoutthat theirreleaseintotheaquaticenvironmentshouldbeconsidered
[11,13].Inthelastyears,hugeeffortshavebeenmadeintermsof obtaininformationaboutILspotentialeffectintheenvironment byassessingtheirtoxicitythroughbiologicalelementsofdifferent complexity.Intheaquaticfield,fish(e.g.,Daniorerio)[14,15],bio- luminescentbacteria(e.g.,Vibriofischeri)[16,17],cladocerans(e.g., Daphniamagna)[18,19]andduckweed(e.g.,Lemnaminor)[20]have beencommonlyusedinthe(eco)toxicitystudies.
Ontheterrestrialenvironment,someauthorshavehypothe- sizedtheabilityofILstocontaminatesoilsthroughsorptiononto soiland/orbioaccumulationinlivingorganisms[21,22],whereby theassaysonthisfieldhasbeenfocusedontheirpossibleaccumu- lationinthesoils[11–13].Someinvestigatorsstudiedthesorption ofimidazoliumandpyridiniumbased-ILsontodifferenttypesof soilsandmarinesediments[23];others,evaluatedthephytotoxi- cityofILsinplants,suchaswheat(e.g.,Triticumaestivum)[24,25], andgardencress(e.g.,Lepidiumsativum)[26],bacterialstrains(e.g., Escherichiacoli,PichiapastorisandBacilluscereus)[27]andtheeffect intheorganismsCaenorhabditiselegansandEiseniafoetida[28,29]. In whatconcerns humantoxicity, it is ethically incorrectto useindividualsintoxicologicalassays,beingoftenusedanimals suchasmice,rats,monkeys,amongothers,sincetheypresentsim- ilaritieswithhumans.Fortunately, over theyearsthere wasan efforttodevelop newproceduresthatpreferablyavoid animals or,atleast,aremoresensibletotheirwelfare.Thesealternative methods present moreefficiency in terms of cost and time, as wellsimplicityofapplication.Theyhavebeenusedtopredictthe toxicityofnewproductsduringtheirdevelopment,aswell,the synthesizedintermediatesanddegradation products[30].Mod- elstopredictquantitativestructure-activityrelationships(QSAR), humancellslinesandenzymaticinhibitionassaysfillinthepro- posalsestablishedforthesealternativemethodologies.Inacellular level,promyeloticleukemiaratcelllineIPC-81[31,32],HeLa[33], HT29[34]andCaco-2[35]humancellslinesarethemostapplied in the study of ILscytotoxicity. The enzymatic inhibition data describedinliteraturefortheassessmentofILstoxicityincludesthe enzymes:acetylcholinesterase[32,36],carboxylesterase[37],AMP deaminase[38]andtheantioxidantenzymesystemofmouseliver
[39].Theseinvitrosystemsprovedtobebeneficialinthestudyof thexenobioticsatthemolecularlevel,includingitsmodeofaction andidentificationoftoxicophorestructures[30]andmightenable theextrapolationofinvitrodatawithregardtopossibleeffectson humans[40].Someenzymaticassayshavebeenappliedtopredict
membraneofbacteriaandmitochondriaofmammals.CytCoxcat- alyzestheelectrontransferfromcytochromectooxygenmolecule. Thiselectron-transferprocesscreatesaprotongradientacrossthe membrane,withproductionofATP,providingenergytothecell.It islocatedontheinnermitochondrialmembrane[41]andhasbeen usedforyearsasamarkerofmitochondrialmembraneintegrity
[42,43].RecentstudieshighlightedtheimportanceofCytCoxon toxicityevaluationofxenobioticsanditsintegrationinthestandard monitoringassays[44–46].
Inthiscontext,amethodologyfortheevaluationofILstoxic effectonCytCoxwasdevelopedandoptimizedbasedonsequen- tialinjectionanalysis(SIA)[47].Ina practicalperspective, flow techniques,likeSIA,enablestocircumventsomeoftheproblems associatedtobatchassays.Onitstraditionalmode,thebatchassays canbeaffected,inagreatextent,bytheoperatorabilitytosyn- chronizetheadditionofsamples,enzymeandsubstrateandread atpredefinedperiodsoftime.Thiscanbeaproblematicissuein assaysthatisnecessaryguaranteethesamecontacttimeforallthe samplesandcontrols.Amongflowtechniques,SIAalreadyproved tobearobustandaccuratesolutionfortheautomationofbioas- says,becauseitallowsaprecisecontrolofthereactionconditions intermsofspaceandtimethroughcomputercontrolofanalyti- calparameters.Indeed,itsimplementationinbio-procedureswith ILsprovedtobeaninterestingalternative[48–50],includingfor applicationin screeningtoxicologicalassays [17,37].Additional, thecomputercontrolenableslowoperatorinterferenceandrepro- ducibility of assays. The operating modeallows a reduction of sampleandreagentsconsumption,loweffluentsproduction,and highsampleanalysisrate.Itisalsoaversatiletechnique[51,52]
makingitaninterestingalternativemethodontheGreenChemistry perspective.
Inthiscontext,themainobjectiveofthisworkwasthedevelop- mentofanautomated,robust,simpleandeconomicmethodology suitableforhigh-throughputscreening,appliedintheevaluationof ILs’toxicitybasedontheinhibitionofCytCoxactivity.Itisexpected thattheresultsobtainedinthisstudyenablestheidentificationof sometoxicophoremoietiesandtheextrapolationofinvitrodata obtainedtopossibleeffectsonhumans.Thisinformationcanbe useful,consideringthatitisagoalofmanyresearcherstotunethe physico-chemicalpropertiesofILsbydesignofanionicandcationic componentssaferfortheenvironment.
2. Materialsandmethods
2.1. Reagents
All solutions were prepared using chemicals of analytical gradeandultrapurewater.Allthereagentswerepurchasedfrom Sigma–Aldrich. The carrier solution of the flow system was a Tris–HClassaybuffer,10mmolL−1pH7.0,withKCl120mmolL−1. CytCox from bovine heart (EC 1.9.3.1) contained 250g of enzyme,with5mgprotein/mL,correspondingto20U/mgprotein. The enzyme wasdissolved in Tris–HCl enzyme dilution buffer, 10mmolL−1 pH 7.0, containing sucrose 250mmolL−1 in final volumeof2mL.Thisstocksolutionwasdividedin12workingsolu- tionsandthenstoredat−20◦C.Daily,analiquotofworkingsolution
wasreconstitutedinenzymedilutionbufferatafinaldilutionfactor of20.
roborate; ≥97%), emim [Tf2N] (1-ethyl-3-methylimidazolium
bis(trifluoromethylsulfonyl)imide; ≥98%), emim [Ms] (1-ethyl- 3-methylimidazolium methanesulfonate; ≥95%), emim [TfMs] (1-ethyl-3-methylimidazolium trifluoromethanesulfonate;≥97%), bmim [BF4] (1-butyl-3-methylimidazolium tetrafluoroborate;
≥98%), bmim [Cl] (1-butyl-3-methylimidazolium chloride; ≥98%), hmim [Cl] (1-hexyl-3-methylimidazolium chloride;≥97%), bmpy [BF4] (1-butyl-4-methylpyridinium tetrafluoroborate; ≥97%),
bmpyr [BF4] (1-butyl-1-methylpyrrolidinium tetrafluoroborate;
≥97%), bmpyr [Cl] (1-butyl-1-methylpyrrolidinium chloride; ≥99%), N4,4,4,4 [BF4] (tetrabutylammoniumtetrafluoroborate;
≥99%), tbph [Ms] (tetrabutylphosphoniummethanesulfonate; ≥98%), chol [Ac] (choline acetate; ≥95%), bmim [Ac] (1- butyl-3-methylimidazolium acetate; ≥95%) and emim [Ac] (1-ethyl-3-methylimidazolium acetate; ≥97%). All the tested ILs were stored at room temperature in a carefully controlled anhydrous environment.
2.2. Apparatus
A 6300 Jenway spectrophotometer model set at 550 nm and equipped with an 18L flow cell (Helma 178.711QS, Mülheim, Balden, Germany) was used in the spectrophotometric measure- ments. The SIA system (Fig. 2) consisted of a syringe module Bu1S from Crison Instruments S.A. (Allela, Barcelona, Spain) and a 10- port multiposition CheminertTMselection valve. A glass syringe of
5 mL total dispense volume (Hamilton Bonaduz AG, Switzerland) was coupled to the syringe equipment and driven by a step- per motor. Solenoid head-valves allowed the commutation of the syringe either to the manifold or to the carrier. The communi- cation between the PC and the modules was done resorting to a RS232C serial protocol. The software used for the instrumental control was developed using visual basic and communication with instruments was accomplished by means of RS-232C asynchronous protocols, using embedded dynamic libraries. A sequential output of the commands and evaluation of the equipment status was per- formed through the implementation of the control algorithm based in the use of a set of interdependent timers. Analytical system con- trol enabled the control of flow rate, flow direction, valve position, stop flow duration, sample, enzyme and substrate volumes, as well as data acquisition and processing. Manifold components were con- nected by means of 0.8 mm i.d. PTFE tubing, which was also used for the holding and reaction coil (2 and 1 m, respectively). During the assays, the analytical signals were recorded on strip chart recorder (Kipp & Zonen BD 111) or acquired via computer.
2.3. Sequential injection procedure
The analytical cycle established for the implementation of the CytCox assay, in the flow system, is schematized inTable 1. In each cycle, were aspirated sequentially 5 aliquots of reagents to the hold- ing coil: 17.5L of FeC, 20 L of CytCox and after 12.5 L of sample, and again 20L of CytCox and 17.5 L of FeC. Then, the flow was reversed and the reaction zone was propelled by the carrier solu- tion to the reaction coil, during 20 s. The mixture remained in the reaction coil for 180 s. After the stop period, the reaction zone was propelled to the detector and a decrease in the signal proportional to the extent of FeC oxidation by CytCox was obtained.
Blank assays were performed for each concentration of IL deter- mined. The blank assays consisted in the replacement of enzyme aliquots for enzyme dilution buffer. Each condition was evaluated
Fig. 1. Chemical structures of the studied ILs. 1. emim [Ms]; 2. emim [BF4]; 3. bmim [BF4]; 4. bmim [Cl]; 5. bmpyr [Cl]; 6. tbph [Ms]; 7. bmpy [BF4]; 8. hmim [Cl]; 9. bmim [Ac]; 10. emim [Ac]; 11. chol [Ac]; 12. bmpyr [BF]; 13. emim [Tf2N]; 14.
W S HC SV C D S IL