苯胺及其衍生物聯(lián)合毒性的研究畢業(yè)論文

苯胺及其衍生物聯(lián)合毒性的研究畢業(yè)論文目錄1緒論 ）的冪值分別計(jì)算（表1）。Logistic方程提供了一個(gè)良好的適合馬拉硫磷（r2=0.987），對(duì)硫磷（r2=0.987），和胡椒基?。╮2=0.998）濃度-反應(yīng)數(shù)據(jù)。這展出的兩個(gè)有機(jī)磷農(nóng)藥相似的毒性特點(diǎn)。相對(duì)于有機(jī)磷物質(zhì)，胡椒基丁的毒性想到較弱，而且冪值大約是有機(jī)磷物質(zhì)的一半。儲(chǔ)存器（盒子）的分配根據(jù)IAI的分析圖，將不同濃度的有機(jī)磷酸脂配置到同一暗盒中，在暗盒中可近似地計(jì)算出某一附加濃度值來(lái)確定其毒性。用附加濃度來(lái)建立有機(jī)磷酸酯的聯(lián)合毒性分析圖，準(zhǔn)確度是由兩種毒性相當(dāng)?shù)挠袡C(jī)磷酸酯多次聯(lián)合實(shí)驗(yàn)后來(lái)確定的。用濃縮反映來(lái)評(píng)價(jià)二元混合物在數(shù)學(xué)統(tǒng)計(jì)上是難以分辨的。因此，馬拉硫磷和對(duì)硫磷對(duì)最終混合物的毒性作用結(jié)果需用單一的有機(jī)磷酸酯來(lái)建分析圖。有機(jī)磷酸脂抑制乙酰膽堿酯酶活性的普通作用模式可根據(jù)實(shí)驗(yàn)數(shù)據(jù)來(lái)確定。對(duì)比之下，胡椒基丁并沒(méi)有抑制乙酰膽堿酯酶的活性。因此胡椒基丁可配置到它對(duì)應(yīng)的暗盒中，在暗盒里，混有這種成分的混合物的毒性與使用了反應(yīng)添加劑的混合物的毒性完全復(fù)合?；瘜W(xué)相互作用我們假設(shè)胡椒基丁可以以一種形式和有機(jī)磷酸酯的成分完全混合，并可以減小它的毒性。圖2直接表明了胡椒基丁撤銷(xiāo)有機(jī)磷酸脂對(duì)乙酰膽堿酯酶的抑制能力。胡椒基丁經(jīng)過(guò)長(zhǎng)期對(duì)有機(jī)磷酸脂的毒性作用后可證明為拮抗作用，然后做出馬拉硫磷的濃度反應(yīng)曲線(xiàn)（圖3A）和對(duì)硫磷（圖3b）的濃縮反應(yīng)曲線(xiàn)。胡椒基丁的抵制作用是用來(lái)表K-函數(shù)的濃度（如圖4）在混合溶液中，這些K-函數(shù)常被用于最終IAI模型中，他可以減小被胡椒基丁濃度控制的馬拉硫磷和對(duì)硫磷的有效濃度?；旌衔锒拘缘墓浪愀鶕?jù)實(shí)驗(yàn)確定的30種成分的三元混合物與被指定的毒物進(jìn)行了實(shí)驗(yàn)測(cè)定和比較，繪出濃度加法模型圖（等式2），反應(yīng)加法模型圖（等式3），綜合加法模型圖（等式4），IAI模型圖（等式6）。并不是濃度加法，反應(yīng)加法和綜合加法模型圖能描述混合物的毒性（r2<0.10）。而是所有模型都遠(yuǎn)遠(yuǎn)超出其混合毒性。因此，IAI模型圖對(duì)各種毒性混合物可提供準(zhǔn)確的數(shù)據(jù)。83%的混合元素在表2中能精確估算出其毒性。討論這項(xiàng)研究的結(jié)果表明，毒物動(dòng)力學(xué)相互作用可以運(yùn)用到綜合加法模型圖中來(lái)估算混合毒性。最近的研究表明，濃縮及反應(yīng)加法模型都能用于創(chuàng)造簡(jiǎn)單加法模型，并用此模型來(lái)計(jì)算無(wú)相互化學(xué)作用的混合物的毒性（Altenburgeretal.,2005年；OlmsteadandLeBlanc，2005年；Teuschler等，2004年）。我們可以根據(jù)活性毒藥和混合成分之間的聯(lián)合作用建立其結(jié)構(gòu)模型。根據(jù)定義，建立混合毒性模型時(shí)，因?yàn)楹?jiǎn)單的加和作用使化學(xué)相互作用表現(xiàn)的很不正常。量化這些相互作??用，預(yù)期要增大混合物毒性，必須先確定選擇適當(dāng)?shù)哪Ｐ凸浪闾砑恿?，并?zhǔn)備解釋其相互作用的結(jié)果。美國(guó)環(huán)保局評(píng)估混合毒性建議使用濃度模型（2000）。濃度加法模型與反應(yīng)加法模型相比是一種更趨向保守估計(jì)混合物毒性濃度反的方法（Drescher和Boedecker，1995）。不過(guò)，不加區(qū)別的應(yīng)用濃度加法模型會(huì)導(dǎo)致缺乏正確的機(jī)械基礎(chǔ)，這就增加了混合物毒性的的不確定因素。綜合加法描述了在近期研究中（Altenburger，2005年；OlmsteadandLeBlanc，2005年）提供了一種基礎(chǔ)的方法來(lái)評(píng)估混合物的毒性。起初，類(lèi)似于這種機(jī)制的化學(xué)反應(yīng)都被置于群體或暗匣中。每個(gè)暗盒的毒性可以參考濃度加法模型，不同暗盒的總毒性可以參考反應(yīng)加法模型（圖6）。綜合加法模式由Altenburger等人創(chuàng)建（2005年），奧姆斯特德和勒布朗（2005年）定理在概念上是等同，在計(jì)算方法上有點(diǎn)輕微的差別。綜合加法模型在評(píng)估非相互作用的化學(xué)混合物毒性上市一個(gè)重大的進(jìn)步。但是，這種模式并不是具備管理之間的相互作用，化學(xué)物質(zhì)影響該混合物的毒性。在環(huán)境中兩個(gè)或更多的化學(xué)物質(zhì)之間發(fā)生協(xié)調(diào)作用的可能性很大，也許只有研究混合毒性才能解釋這種現(xiàn)象。明確界定的互動(dòng)關(guān)系的例子包括：預(yù)暴露在kepone的四氯化碳對(duì)肝的毒性（Klingensmith和Mehendale，1982年）和涉及激素受體拮抗劑和激素合成抑制劑的相互作用（木和勒布朗，2004年）。相互作用時(shí)可利用機(jī)制作用推出的化學(xué)成分。例如，用于本研究中的細(xì)胞色素P450酶抑制劑增效醚是假設(shè)對(duì)抗通過(guò)降低其代謝活化抵消馬拉硫磷和對(duì)硫磷的毒性。然而，一些互動(dòng)顯然對(duì)某些機(jī)制成分無(wú)反應(yīng)，綜合加法模式有可能找出些意想不到的相互作用。實(shí)際上，實(shí)驗(yàn)結(jié)果預(yù)測(cè)模型的重大偏差就意味著互動(dòng)。只有通過(guò)遵循模式推理或?qū)嶒?yàn)量化與團(tuán)之間的相互作用來(lái)確定源的相互作用。毒物動(dòng)力學(xué)的相互作用可以通過(guò)一個(gè)質(zhì)“重證據(jù)”的方法或定量的方法評(píng)估混合物性質(zhì)。這兩種方法在概念上非常相似，兩者都改變了化學(xué)物質(zhì)的有效濃度。不過(guò)，不同的辦法的運(yùn)用其表現(xiàn)也不同，“重證據(jù)”的辦法（穆塔茲和德金，1992年;赫茲伯格等人修改，1999年）是目前推薦的EPA的混合物毒性準(zhǔn)則（2000年）。簡(jiǎn)言之，就互動(dòng)而言，界定一種化學(xué)品在另一種上所產(chǎn)生效應(yīng)根據(jù)預(yù)測(cè)所含的化學(xué)物質(zhì)相互作用幅度（實(shí)驗(yàn)確定或默認(rèn)值）為2293nm。危害藥品（暴露量除以參考劑量或參考濃度）在混合物中各化學(xué)品乘以相互作用項(xiàng)。減小危害后總結(jié)得出混合物的風(fēng)險(xiǎn)指數(shù)（赫茲伯格和麥克唐內(nèi)爾，2002年）。危害指數(shù)為量綱，只是提供了一個(gè)與相關(guān)的危險(xiǎn)混合物一般估計(jì)。它有助于查明有潛在危險(xiǎn)的混合物，但它不提供一個(gè)精確混合毒性的計(jì)算。另外，Mu和LeBlanc于2004年嚴(yán)格描述了該定量方法，它是基于對(duì)K-值，或K-函數(shù)的概念最早由芬尼于1942年提出。根據(jù)濃度-反應(yīng)曲線(xiàn)，量化隨著增加一種化學(xué)品的誘導(dǎo)濃度逐步轉(zhuǎn)變。這項(xiàng)研究的主要目標(biāo)是確定減小函數(shù)（即K-函數(shù)）是否可以用綜合加法模式來(lái)增強(qiáng)化學(xué)互動(dòng)對(duì)混合物成分的毒性影響。這項(xiàng)工作的第二個(gè)目的是加強(qiáng)我們對(duì)如何建立化學(xué)品類(lèi)機(jī)理或暗匣及混合物的函數(shù)的理解。例如，有證據(jù)表明，某些化學(xué)品表現(xiàn)為穩(wěn)定的互動(dòng)模式（Durkinetal.1995年）。根據(jù)一個(gè)暗匣對(duì)另一個(gè)的化學(xué)作用我們可以看出這種穩(wěn)定性增加了產(chǎn)生K-函數(shù)的可能。然而，同類(lèi)型的互動(dòng)并不意味著化學(xué)品可以表現(xiàn)出同樣規(guī)模的互動(dòng)。在目前的研究中，胡椒基丁對(duì)馬拉硫磷和對(duì)硫磷都表現(xiàn)為抵抗作用，不過(guò)，兩有機(jī)磷酸酯之間對(duì)立程度明顯不同，生成的K-函數(shù)具體到每一個(gè)不同的有機(jī)磷酸脂。運(yùn)用K-函數(shù)，在馬拉硫磷、胡椒基丁的相互作用下，整個(gè)有機(jī)磷酸脂暗匣遠(yuǎn)遠(yuǎn)低于混合物毒性。另外，一些有機(jī)磷酸酯不需代謝活化，p450s都能去除其毒性。這些化合物可適當(dāng)予以分配到有機(jī)磷酸酯暗盒與胡椒基丁混合來(lái)計(jì)算聯(lián)合后有機(jī)磷酸酯的毒性，但他們需要用K-函數(shù)來(lái)表示他們的協(xié)調(diào)作用，而不是對(duì)立的。早在60年前布利斯（1939年）用IAI模式積分得到精確數(shù)據(jù)后就確定了這三個(gè)混合物概念。IAI模型為三元混合物毒性提供了合理的預(yù)測(cè)方法。該模型與加法基本模式相比是一個(gè)重大的改進(jìn)。在觀(guān)察和模擬結(jié)果期間出現(xiàn)變異可能是由于以下幾個(gè)因素。不同的有機(jī)生物反應(yīng)導(dǎo)致了固有生物變異，試驗(yàn)期間也可能觀(guān)察到若干變異。假設(shè)K-函數(shù)運(yùn)用到二元高階化學(xué)混合物時(shí)是不完全正確的。進(jìn)一步用復(fù)雜的混合物測(cè)試的IAI模型將有助于闡明基本原則和限制K-函數(shù)的相關(guān)應(yīng)用。這種模式是比較易于應(yīng)用，并通?？梢詮臉?biāo)準(zhǔn)濃度-效應(yīng)分析輸入?yún)?shù)。不過(guò)，化學(xué)品之間的相互量化作用需要嚴(yán)格的實(shí)驗(yàn)要求。未來(lái)的研究可能揭示不管是否有限或?qū)嶒?yàn)是否定指標(biāo)都能提供互動(dòng)量化所需的資料。這種毒性互動(dòng)是不太常見(jiàn)的（赫茲伯格和麥克多內(nèi)爾，2002年），但仍然是混合毒性重要貢獻(xiàn)者。IAI模型可以提高化學(xué)混合物危害和風(fēng)險(xiǎn)評(píng)估的準(zhǔn)確率，減少在估算混合物毒性的不確定性。致謝這項(xiàng)研究是由環(huán)保局科學(xué)院AchieveResults提供R829358和NIEHS的培訓(xùn)中心提供ES07046的資金。作者衷心感謝AllenOlmstead博士和王桂榮女士為他們提供援助和咨詢(xún)。附錄B外文原文TOXICOLOGICALSCIENCES87(2),520-528(2005)doi:10.1093/toxsci/kfi247AdvanceAccesspublicationJuly7,2005AnIntegratedAdditionandInteractionModelforAssessingToxicityofChemicalMixturesCynthiaV.RiderandGeraldA.LeBlance1DepartmentofEnvironmentalandMolecularToxicology,NorthCarolinaStateReceivedMay2,2005;acceptedJune24,2005ABSTRACT

Thehighpropensityforsimultaneousexposuretomultipleenvironmentalchemicalsnecessitatesthedevelopmentanduseofmodelsthatprovideinsightintothetoxicityofchemicalmixtures.Inthisstudy,wedevelopedamathematicalmodelthatcombinesconceptsofconcentrationaddition,responseaddition,andtoxicokineticchemicalinteractiontoassesstoxicityofchemicalmixtures.Aternarymixtureofacetylcholinesteraseinhibitingorganophosphates(malathionandparathion)andtheP450inhibitorpiperonylbutoxidewasusedtomodeltoxicity.Concentration-responsecurvesweregeneratedforindividualchemicalsaswellasformixturesofthechemicalsusingacutetoxicitytestswithDaphniamagna.Thetoxicityofbinarycombinationsofmalathionandparathionadheredtotheprinciplesofconcentrationaddition.Thecontributionofpiperonylbutoxidetomixturetoxicitywasintegratedusingamodelforresponseaddition.Piperonylbutoxidealsomodifiedthetoxicityoftheorganophosphatesbyinhibitingtheirmetabolicactivation.Theantagonisticeffectsofpiperonylbutoxidetowardstheorganophosphateswerequantifiedascoefficientsofinteractions(K-functions)andincorporatedintothemixturemodel.Finally,toxicityoftheternarymixturewasmodeledat30differentmixtureformulationsusingthreeadditivemodelsthatassumednointeraction(concentrationaddition,responseaddition,andintegratedaddition)andusingtheintegratedadditionandinteraction(IAI)model.Toxicityofthe30mixtureswasthenexperimentallydeterminedandcomparedtomodelresults.OnlytheIAImodelaccuratelypredictedthetoxicityofthemixtures.TheIAImodelholdspromiseasameansforassessinghazardofcomplexchemicalmixtures.

KeyWords:synergy;cumulativetoxicity;predictivemodel;toxicodynamic;hazardassessment;riskassessment.

INTRODUCTION

Surveysofagriculturalandurbanstreamsandgroundwaterhavebroughtpublicattentiontowidespreadchemicalmixturecontamination(Battaglinetal.,2003;Kolpinetal.,2002).Theinfinitenumberofpotentialchemicalcombinations(intermsofbothconstituentsandconcentrationsofconstituents)limitstheutilityofstandardtoxicitytestingmethodsforestablishinghazardassociatedwithchemicalmixtures.Modelingapproachescouldaugmentthestandardtoxicitytestingparadigmwhenevaluatinghazardsassociatedwithexposuretochemicalmixtures.Chemicalconstituentsofamixturecanelicitsimilaraction,dissimilaraction,orinteraction(Bliss,1939;Casseeetal.,1998).Modelsofmixturetoxicityhavefocusedprimarilyonquantifyingthe"no-interaction"scenarios,whilecasesofinteractionoftenappearasqualitativeobservations(HertzbergandMacDonell,2002).Concentrationaddition(Loeweadditivity)andresponseaddition(Blissindependence)(Grecoetal.,1992)arecommonlyusedtomodelthetoxicityofnon-interactingchemicalswithinamixture.

Concentrationadditionmodelsrelyupontheassumptionthatmixturecomponentscontributetotoxicitythroughacommonmechanismofaction.Calculatingmixturetoxicitybaseduponconcentrationadditionrequiresassessingtherelativecontributionofeachconstituenttothetotaltoxicantpool.Thetoxicityofthispoolisthenmodeledasasingletoxicant.Concentrationadditionisthebasisofthe"toxicequivalency"approachcommonlyusedtoassesstoxicityofchemicalsofthesameclasssuchasdioxins(Safe,1990).Ampleevidencesupportstheuseoftheconcentrationadditionmodelforassessingmixturestoxicityoflike-actingchemicals(Altenburgeretal.,2000;Deneeretal.,1988;Knemann,1981).Theresponseadditionmodel,alsoreferredtoastheindependentjointactionmodel,hasbeenusedtocomputetoxicityofmixtureswhenchemicalconstituentshavedifferentmechanismsofaction(Backhausetal.,2000;Walteretal.,2002).Intheresponseadditionmodel,combinedeffectsofthechemicalsarebasedupontheprobabilitythatindividualconstituentsofthemixturewillaffecttheexposedorganisms.

Theconcentrationadditionandresponseadditionmodelsarelimitedintheirapplicationtocomplexmixturesinthattheydonotaddresschemicalinteractions.Toxicokineticinteractionscanoccurbetweenchemicalsinwhichonechemicalalterstheeffectiveconcentrationofanother(AndersenandDennison,2004).Alternatively,toxicodynamicinteractionscanoccurbetweenchemicalsinwhichonechemicalinfluencestheresponseoftheorganismtoanotherchemical(AndersenandDennison,2004).Bothtoxicokineticandtoxicodynamicinteractionscansignificantlyimpactthetoxicityofchemicalmixtures.TheimportanceofaddressingchemicalinteractionswashighlightedbytheUSEPAintheirrecommendationsforevaluatingriskassociatedwithchemicalmixtures(USEPA,2000).

Recently,Altenburgeretal.(2005)andOlmsteadandLeBlanc(2005)demonstratedthatconcentrationadditionandresponseadditionmodelscouldbeintegratedintoacomprehensivemodelforuseinevaluatingtoxicityofnon-interactingchemicalmixtures.Theintentofthepresentstudywastoexpandthisapproachtoincorporateinteractionsamongchemicalconstituentswhentheyarepredictedtooccur.Importantissuesaddressedinthisworkinclude:(1)evaluatingwhethersingleinteractionmodifierscanbeappliedtoclassesofchemicalsand(2)establishingwhetherclearlydefinedbinaryinteractionspersistinhigherordercombinations.Thestrengthoftheintegratedadditionandinteraction(IAI)modelwasassessedbycomparingmodelresultstoexperimentallydeterminedtoxicityof30differentderivationsofaternarymixture.

MATERIALSANDMETHODS

Daphnidculture.

AlltoxicologicalexperimentswereperformedwiththedaphnidDaphniamagna.Daphnidswereacquiredfromlong-standingculturesinourlaboratorythatwereoriginallyobtainedfromtheUSEnvironmentalProtectionAgency,Mid-ContinentEcologyDivision–Duluth,MN.Daphnidsweremaintainedinreconstituteddeionizedwater(192mg/lCaSO4·H2O,192mg/lNaHCO3,120mg/lMgSO4,8.0mg/lKCl,1.0μg/lseleniumand1.0μg/lvitaminB12).Culturesweremaintainedin1-literbeakersatadensityof50daphnids/lmediumandculturemediumwaschangedthreetimesperweek.Adultdaphnidswerediscardedafterthreeweeksandreplacedwithneonates.Culturebeakersandallexperimentsweremaintainedinincubatorswitha16/8-hlight/darkcycleataconstanttemperatureof20°C.Culturedaphnidswerefed2.0ml(1.4x108cells)oftheunicellulargreenalgaeSelenastrumcapricornutumand1.0ml(4mgdryweight)ofTetrafinfishfoodsuspension(PetInternational,Chesterfill,NewSouthWales,Australia).TheSelenastrumwasculturedinthelaboratoryusingBold'sbasalmedium.

Acutetoxicityassays.

Chemicalsusedinmixtureanalyses(malathion,parathion,andpiperonylbutoxide)wereacquiredfromChemServices(WestChester,PA).Absoluteethanolwasusedasthecarrierforallofthechemicals.Alltoxicityassessmentswereinitiatedwithneonatal(24hold)daphnids.Eachtreatmentconsistedoftwo50mlbeakerscontaining40mlofexposuremediumand10neonates.Selanastrum(7x106cells)andfishfoodhomogenate(0.2mgdryweight)wereprovidedtoeachbeakerasfoodatthestartofeachexposure.Allbeakers,includingcontrols,contained0.01%carrier(ethanol).Beakerswerelabeledonthebottomandrandomlyrearranged,sothattheexposureconcentrationineachbeakerwasnotknowntotheinvestigatorwhenassessingresponseoforganisms.At48h,neonateswereevaluatedforresponse.Theresponseendpoint,immobilization,wasjudgedbytheinabilityoftheneonatetooccupythewatercolumnduring10sofobservation.

Acetylcholinesteraseanalyses.

AcetylcholinesteraseactivitywasmeasuredaccordingtoEllmanetal.(1961)asmodifiedforusewithmicrotiterplates(Fisheretal.,2000)withminoradditionalmodifications.Exposuregroupsconsistedofthree250mlbeakerscontaining200mlsolutionand40neonates(24hold).Algae(1.4x107cells)andfishfood(0.4mgdryweight)wereaddedtoeachbeakeronceperday.Solutionswererenewedat24h.Followingthe48-hexposureperiod,neonatesweretransferredto1.5mlmicrofugetubes.Mediawasremovedfromtubes;neonateswererinsed,andhomogenizedin35μlicecold0.02Mphosphatebuffer,pH8.0with1%Triton-X-100usingaTeflonpestle.Anadditional315μlphosphatebuffer,pH8.0withoutTriton-X-100wasthenaddedandsamplesweremixed.Sampleswerecentrifugedat14,000xgfor4minat4°Candsupernatantwastransferredtoacleanpre-cooledmicrofugetube.Approximately100μlofthesupernatantwasstoredat–20°Cforproteinanalysis.Thefollowingsolutionswereaddedtoeachwellina96-wellplate:100μlof8mM5,5'-dithio-bis(2-nitrobenzoate)(D-1830Sigma),50μlsupernatant(phosphatebufferwith0.1%Triton-X-100wasusedforsupernatantblanks),50μlof16mMacetylthiocholineiodide(A-5751Sigma).Absorbancewasmeasuredkineticallyfor15minat420nmusingaFusionUniversalMicroplateAnalyzer(PerkinElmer,Boston,MA).ProteinwasmeasuredaccordingtoBradford(1976)usingBio-RadProteinAssaydyeconcentrate(Hercules,CA)andastandardcurvegeneratedwithbovineserumalbumin.Themolarextinctioncoefficient(13,300M–1·cm–1)(Massonetal.,2004)wasusedtocalculatetheamountofyellowanion,5-thio-2-nitrobenzoate,formedover15minandthisratewasnormalizedtotheamountofproteinaddedtotheassay(nmol/min/mg).AnalysesofvarianceandTukey-KramerHSDwereusedtodetermineifsignificant(p0.05)differencesexistedbetweentreatments.

(1)

whereRistheresponse(%immobilization),Cisthechemicalconcentration,isthepowerorslopeofthecurve,andEC50istheexposureconcentrationelicitingimmobilizationin50%ofexposedanimals.Theseindividualconcentration-responsecurvesweresubsequentlyusedinmixturemodelingasdescribedbelow.

MixtureModeling

Concentrationaddition.

AccordingtoOlmsteadandLeBlanc's(2005)integratedadditionmodel,likeactingchemicalsareassignedtoacommoncassette(i.e.,grouping).Toxicityassociatedwiththecassetteisthencalculatedusingaconcentrationadditionapproach.Accordingly,malathionandparathionwereassignedtoacommoncassette,theorganophosphate(OP)cassette.ToestablishwhetherthetoxicityofthechemicalswithintheOPcassetteconformedtoaconcentrationadditionmodel,fiveratios(Table2)ofthechemicals(malathion:parathion)wereeachtestedatsixdifferentconcentrations.Parathionconcentrationswereexpressedintermsofmalathionequivalents.AllfiveratioswereequitoxicbaseduponcharacterizationofthetoxicityoftheindividualOPs.Thesixconcentrationsofeachbinarymixtureusedintheexperimentswereselectedtodefinetheconcentration-responsecurveforthemixture.Thejointtoxicityofthesebinarymixturesoflike-actingchemicalswascomputedusingthefollowingequation(OlmsteadandLeBlanc,2005):

(2)whereRistheresponsetothemixture,Ciistheconcentrationofchemicaliinthemixture,EC50iistheconcentrationofchemicalithatcausesa50%response,and'istheaveragepowerassociatedwiththechemicalsinthecassette.Theaveragepowerwasusedbecausechemicalswithinacassetteshouldhavesimilarslopes,aswasthecasewithmalationandparathion.Concentration-responseresultsfromeachbinarymixturewerethenusedtocalculateEC50valuesasdescribedforindividualchemicals.Analysesofvariancewereperformedtodetectsignificant(p0.05)differencesamongthefiveratiosusingSAS8.2software(SASInstitute,Cary,NC).

Responseaddition.

TheconceptofresponseadditionwasusedbyOlmsteadandLeBlanc(2005)tocomputethejointtoxicityassociatedwiththedifferentchemicalcassetteswithinamixture.Theresponseadditionmodelwasusedbecauseeachcassetteisassumedtoelicitaresponsethroughdifferentmechanisms.Theresponseadditionmodelcanbedepictedas:

(3)hereRrepresentstheresponsetothemixtureandRiistheresponsetochemicalsincassettei.

Equations2and3wereintegratedtoestablishtheresponseassociatedwithindividualcassetteswithinamixtureandtosumtheresponsesassociatedwiththecassettes(OlmsteadandLeBlanc,2005).Theresultingequationisacombinationofconcentrationandresponseadditionequations:

(4)

Chemicalinteractions.

TheabilityofonechemicalinthemixturetomodifytheeffectiveconcentrationofanotherwasdefinedbycoefficientsofinteractionsorK-functions(Finney,1942;MuandLeBlanc,2004).Specifically,K-functions,definedthedegreetowhichtheconcentrationofPBOinthemixturealteredtheeffectiveconcentration(i.e.,oxonmetabolite)ofeitherorganophosphateinthemixture.K-functionsweredescribedbyexperimentallyderivingtheeffectofconcentrationsofPBOontheEC50valuesderivedforeachorganophosphate.K-functionswerecalculatedforeachofthePBOconcentrationswiththefollowingequation:

(5)whereEC50OPistheconcentrationoforganophosphatethatimmobilized50%oftheexposedanimalsandEC50OP+PBOxistheEC50oftheorganophosphatewhenexposureoccurredinthepresenceofxconcentrationofPBO.TheseK-functionswerethenplottedagainsttheconcentrationofPBOfromwhichtheywerederived.ThelogisticequationthatdefinedthisrelationshipwasusedtocalculateK-functionswhenmodelingmixturetoxicity.K-functionswereintegratedintothismodeltodescribetoxicokineticinteractionsbetweenPBOandtheorganophosphates:

(6)whereka,irepresentsafunctiondescribingtheextenttowhichchemicala(PBO)presentinthemixtureatconcentrationCaalterstheeffectiveconcentrationofchemicali(malathionorparathion).

Theresponsetothirtycombinationsofthethreechemicalswascomputedusingtheconcentrationadditionmodel(Equation2),theresponseadditionmodel(Equation3),theintegratedadditionmodel(Equation4),andtheIAImodel(Equation6).Inaddition,theactualtoxicityofthe30mixtureswasmeasuredandresultswerecomparedtothefourmodelresults.The30mixtureformulationsweredesignedsothattheratioofthethreechemicalsvariedamongthemixtureformulations.Modelpredictionswerecomparedtoexperimentaldatausingcoefficientsofdetermination(r2;Zar,1996).Anr2valueof0.70orgreaterwasconsideredagoodfitoftheobserveddatatothemodel(QualityAmerica,2004).

RESULTS

IndividualChemicalToxicityAnalyses

TheIAImodelrequirestoxicitydescriptionfortheindividualchemicalswithinamixture.Concentration-responsecurvesweregeneratedformalathion,parathion,andpiperonylbutoxide(Fig.1)fromwhichEC50valuesandcorresponding95%confidenceintervals,andpowerofthecurves()werederived(Table1).Thelogisticequationprovidedagoodfittothemalathion(r2=0.987),parathion(r2=0.987),andpiperonylbutoxide(r2=0.998)concentration-responsedata.Thetwoorganophosphatesexhibitedsimilartoxicitycharacteristics.Piperonylbutoxidewasconsiderablylesstoxicascomparedtotheorganophosphatesandhadapowerapproximatelyone-halfthatoftheorganophosphates.

CassetteAssignment

AccordingtotheIAImodel,theorganophosphateswouldbeassignedtothesamecassetteandtoxicityassociatedwiththecassettewouldbeassessedusingaconcentrationadditionapproach.Thevalidityofusingconcentrationadditiontomodelthetoxicityassociatedwiththeorganophosphatecassettewasdeterminedusingseveralcombinationsofthetwoorganophosphatesdeemedtobeequitoxicbaseduponconcentrationadditivity.Indeed,theconcentration-responseassessmentsofthesebinarymixtureswerestatisticallyindistinguishable(Table2).Therefore,thecontributionsofmalathionandparathiontothetoxicityofthefinalmixturesweremodeledasasingleorganophosphatecassette.

Thecommonmodeofactionoftheorganophosphates—theinhibitionofacetylcholinesteraseactivity—wasconfirmedexperimentally(Fig.2).Incontrast,piperonylbutoxidedidnotinhibitacetylcholinesteraseactivity.Piperonylbutoxidewas,therefore,assignedtoitsowncassettewherethetoxicityofthismixturecomponentwasintegratedintothetoxicityofthemixtureusingtheresponseadditionmodel.

ChemicalInteraction

Wehypothesizedthatpiperonylbutoxidewouldinteractwiththeconstituentsoftheorganophosphatecassetteinamannerthatwouldmodifythetoxicityassociatedwiththiscassette.Theabilityofpiperonylbutoxidetoabrogatetheacetylcholinesterase-inhibitingpotentialofeachorganophosphatewasdemonstrateddirectly(Fig.2).Theantagonisticeffectofpiperonylbutoxideonthetoxicityoftheorganophosphateswasfurtherdemonstratedbytheprogressiveshiftingoftheconcentration-responsecurvesformalathion(Fig.3A)andparathion(Fig.3B).Thismodifyingeffectofpiperonylbutoxidewasquantifiedasconcentration-dependentK-functions(Fig.4).TheseK-functionswereusedinthefinalIAImodeltomodifytheeffectiveconcentrationsofmalathionandparathionasdictatedbytheconcentrationofpiperonylbutoxideinthemixture.

MixturesToxicityAssessment

Thetoxicityof30combinationsoftheternarymixture(Table3)wasexperimentallydeterminedandcomparedtopredictedtoxicityusingtheconcentrationadditionmodel(Equation2),theresponseadditionmodel(Equation3),theintegratedadditionmodel(Equation4),andtheIAImodel(Equation6).Neithertheconcentrationaddition,responseadditionnorintegratedadditionmodelsaccuratelydescribedthetoxicityofthemixtures(r2<0.10).Rather,allmodelsgrosslyoverestimatedmixturetoxicity(Figs.5A–5C).However,theIAImodelprovidedagood(r2=0.716)assessmentofthetoxicityofthevariousmixtureformulations(Table3,Fig.5D).Toxicitywasaccuratelyestimatedwithinafactorof2for83%ofthemixtureformulations.

DISCUSSION

Theresultsofthisstudydemonstratethattoxicokineticinteractionscanbeincorporatedintoanintegratedadditionmodeltoassessmixturetoxicity.Recentstudieshaveshownthatconcentrationandresponseadditionmodelscanbeusedincombinationtocreateacomprehensiveadditivemodeltocalculatethetoxicityofnon-interactingchemicalmixtures(Altenburgeretal.,2005;OlmsteadandLeBlanc,2005;Teuschleretal.,2004).Here,webuilduponthatmodelingframeworkbyincorporatingtoxicokineticinteractionsbetweenmixtureconstituents.

Bydefinition,chemicalinteractionsrepresentadeviationfromsimpleadditivitywhenmodelingmixturetoxicity.Toquantifytheseinteractions,theexpectedadditivetoxicityofthemixturemustfirstbedetermined.Choosingtheappropriatemodeltoassessadditivityisessentialforaccurateinterpretationofinteractionresults.USEPAguidelinesforassessingmixturetoxicitysuggestadefaultmodelofconcentrationaddition(2000).Thisrecommendationisbasedonatendencytowardsmoreconservativeestimatesofmixturetoxicitywithconcentrationadditionthanwithresponseadditionmodeling(DrescherandBoedecker,1995).However,indiscriminateapplicationofconcentrationadditionlacksasoundmechanisticbasisandthereforeincreasestheuncertaintyassociatedwithpredictingmixturetoxicity.Theintegratedadditionmodeldescribedinrecentworks(Altenburgeretal.,2005;OlmsteadandLeBlanc,2005)providesamechanism-basedalternativetoassessingmixturetoxicity.Initially,chemicalswithsimilarmechanismsofactionareplacedintogroups,orcassettes.Thetoxicitywithineachcassetteismodeledwithconcentrationadditionandoveralltoxicityofthedifferentcassettesisthenmodeledwithresponseaddition(Fig.6).TheintegratedadditionmodelspresentedbyAltenburgeretal.(2005)andOlmsteadandLeBlanc(2005)areconceptuallyequivalentanddifferonlyslightlyintheirmethodsofcalculation.Theintegratedadditionmodelrepresentsasignificantadvanceinassessingtoxicityofnon-interactingchemicalmixtures.Thismodel,however,isnotequippedtomanageinteractionsamongchemicalsthatimpacttoxicityofthemixture.

Thepossibilityofsignificantsynergisticinteractionsoccurringbetweentwoormorechemicalsintheenvironmentisperhapsthemostcompellingreasontostudymixturetoxicity.Well-definedexamplesofsynergyincludeenhancedhepatotoxicityofcarbontetrachloridewithpre-exposuretokepone(KlingensmithandMehendale,1982)andinteractionsinvolvinghormonereceptorantagonistsandhormonesynthesisinhibitors(MuandLeBlanc,2004).Interactionsoftencanbepredictedbasedonmechanismsofactionofconstituentchemicals.Forexample,theP450inhibitorpiperonylbutoxideusedinthepresentstudywashypothesizedtoantagonizethetoxicityofmalathionandparathionbydecreasingtheirmetabolicactivation.However,someinteractionswillnotbeapparentfromconstituentmechanismsofaction.Theintegratedadditionmodelhasthepotentialtoidentifytheseunexpectedinteractions.Ineffect,significantdeviationofexperimentalresultsfrommodelpredictionsimpliesinteraction.Oncethesourceoftheinteractionisidentified,eitherthroughinferenceorexperimentation,quantificationandincorporationoftheinteractionintothemodelfollow.

Toxicokineticinteractionscanbeincorporatedintomixtureassessmentsviaaqualitative"weightofevidence"approachoraquantitativeapproach.Thetwoapproachesareconceptuallyquitesimilarinthatbothmodifytheeffectiveconcentrationsofchemicalsinaneffectorconcentration-dependentmanner.However,theapproachesdiffersignificantlyintheirapplication.The"weightofevidence"approach(MumtazandDurkin,1992;modifiedbyHertzbergetal.,1999)iscurrentlyrecommendedintheEPAmixturetoxicityguidelines(2000).Briefly,interactiontermsthatdefinetheeffectofonechemicaluponanotheraregeneratedbaseduponthepredictedmagnitudeofinteraction(experimentallydeterminedordefaultvalue)asafunctionoftheconcentrationsoftheinteractingchemicals.Hazardquotients(exposureleveldividedbyreferencedoseorreferenceconcentration)ofindividualchemicalsinthemixturearemultipliedbytheinteractionterm.Themodifiedhazardquotientsarethensummedtoarriveatthehazardindexofthemixture(HertzbergandMacDonell,2002).Thehazardindexisdimensionlessandsimplyprovidesageneralestimateofthehazardassociatedwiththemixture.Itisusefulforidentifyingpotentiallyhazardousmixtures,butitdoesnotprovideanaccuratecalculationofmixturetoxicity.Alternatively,astrictlyquantitativeapproachwasdescribe