(7.1)--2013 Food and Chemical Toxicolog环境与健康环境与健康.pdf

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1、Authors personal copyApproach to distribution and accumulation of dibutyl phthalate in ratsby immunoassayQiang Zenga,Chenxi Weia,Yang Wua,Ke Lib,Shumao Dinga,Junlin Yuana,Xu Yanga,Mingqing Chena,aHubei Key Laboratory of Genetic Regulation and Integrative Biology,College of Life Science,Central China

2、 Normal University,Wuhan 430079,ChinabWuhan Institute for Food and Drug Control,Wuhan 430079,Chinaa r t i c l ei n f oArticle history:Received 15 May 2012Accepted 20 January 2013Available online 16 February 2013Keywords:Semi-volatile organic compoundsDibutyl phthalateBio-accumulationDistributionImmu

3、nofluorescence assayEnzyme-linked immunosorbent assaya b s t r a c tDibutyl phthalate(DBP)is mainly taken up by the general population from food intake.To estimateintake of phthalates,determining distribution and accumulation of DBP in biological materials was a crit-ical need.In this work,we set up

4、 two novel approaches with a monoclonal antibody specific to DBP todetermine the distribution and accumulation of DBP in vivo.The contents of DBP in liver,kidney,stomachand testes were detected by immunofluorescence assays and indirect competitive ELISA.This data givedirectly evidence that indicates

5、 the distribution and accumulation of DBP in vivo.Double-label immuno-fluorescence assay provides with a visual approach to determination of the distribution and accumulationof DBP.It indicated that DBP accumulated in subcutaneous tissue such as sweat gland,hair follicle.Bothof immunofluorescence as

6、say and ELISA can be used to detect the content of DBP in biological materials.Our assays showed that DBP accumulated in viscera being rich in fat,such as liver,kidney and could over-come physiological barriers to penetrate testes.The date suggested that the accumulations of DBPexposed through derma

7、l route were less than that of oral route and most of DBP was metabolized in 2or 3 days.?2013 Elsevier Ltd.All rights reserved.1.IntroductionIn recent years,plasticizer contamination triggers food securityfear in many countries and regions.Phthalates are a group of syn-thetic chemicals with widespre

8、ad use in the industrial productionof numerous consumer products.They are used to soften plasticsfound in numerous flexible plastics products and personal careproducts such as food packaging and food-contact materials,cos-metics,toys,bags,drugs and building materials.During the lastthirty years,nume

9、rous studies have been published concerningthe presence of phthalate compounds in food and packaging mate-rials(Cao,2010;Fierens et al.,2012).Phthalate esters are suspected to be involved in male reproduc-tive health problems and have also been associated with severalother health problems including

10、obesity,diabetes,allergy,andasthma(Borch et al.,2006;Bornehag and Nanberg,2010;Engelet al.,2010;Hauser et al.,2007;Matsuda et al.,2010;Ormondet al.,2009;Stahlhut et al.,2007;Stroheker et al.,2006).Amongall phthalate esters,dibutyl phthalate(DBP)is second only to di-(2-ethylhexyl)phthalate in terms o

11、f such effects(Huang et al.,1999;Yie,1993).DBP is a phthalate diester that is used mainly as a plasticizer fornitrocellulose,polyvinyl acetate,and polyvinyl chloride;a lubri-cant;an antifoaming agent;and a skin emollient(Koo and Lee,2004).DBP,the main kind of plasticizers used in China at present,is

12、 one of the main sources of indoor semi-volatile organic com-pounds(SVOCs)(Wang et al.,2010).DBP is a developmental toxi-cant in rats and mice(Fisher et al.,2003;Foster et al.,2000;LambIV et al.,1987).It is known to affect male fertility,cause testicularatrophy in young rats,and produce embryomortal

13、ity and teratoge-nicity,in rodents(Gray et al.,1999,2000;Kavlock et al.,2002;Moore et al.,2001;Mylchreest et al.,1998,1999,2000).Besidesits role as endocrine disruptor,DBP has been shown to alter theexpression of a number of genes.Expressions of 391 genes havebeen found significantly altered as a re

14、sult of the exposure to0278-6915/$-see front matter?2013 Elsevier Ltd.All rights reserved.http:/dx.doi.org/10.1016/j.fct.2013.01.045Abbreviations:BSA,albumin from bovine serum;CR,cross-reactivity;DBAP,dibutyl 4-aminophthalate;DBP,dibutyl phthalate;ELISA,enzyme-linked immuno-sorbent assay;HRP,Horsera

15、dish peroxidase;IC50,50%inhibiting concentration;IcELISA,indirect competitive ELISA;IFA,immunofluorescence assay;InELISA,indirect noncompetitive ELISA;IOD SUM,integrated option density;MAb,mono-clonal antibody;MBP,monobutyl phthalate;MBP-G,monobutyl phthalate glucuronide;NHANES,National Health and N

16、utrition Examination Survey;OCT,optimum cutting temperature;OPD,O-phenylenediamine;OVA,ovalbumin;PI,3,8-Diamino-5-3-(diethylmethylammonio)propyl-6-phenylphenanthridiniumdiio-dide;SVOCs,semi-volatile organic compounds.Corresponding author Address:Huazhong Normal University,College of LifeSciences,Hub

17、ei Key Laboratory of Genetic Regulation and Integrative Biology andLaboratory of Environmental Biomedicine,Building 5,No.152 Luo-Yu Road,WuhanCity 430079,China.Tel.:+86 27 87105935;fax:+86 27 67861936.E-mail address:(M.Chen).Food and Chemical Toxicology 56(2013)1827Contents lists available at SciVer

18、se ScienceDirectFood and Chemical Toxicologyjournal homepage: personal copyphthalates including DBP among approximately 30,000 genes que-ried(Euling et al.,2011;Liu et al.,2005).DBP is metabolized tomonobutyl phthalate(MBP)in the stomach and small intestineand enters the bloodstream.The pharmacokine

19、tics of DBP havebeen investigated in rats in experimental studies(Clewell et al.,2009).MBP and its primary metabolite,monobutyl phthalate glucuronide(MBP-G),were measured in maternal and fetal afterDBP doses where developmental effects have been found.MBPhas been reported to be the active species re

20、sponsible for adverseeffects from DBP exposure(Ema et al.,1996;Kremer et al.,2005).Ithas been reported that both DBP and MBP suppress steroidogene-sis by fetal-type Leydig cells in rats,but similar effects in vitro can-not be studied in primate(Hallmark et al.,2007).DBP enters the environment in sev

21、eral different ways.These in-clude:during production,distribution,and incorporation intopolyvinylchloride resin;disposal in industrial and municipal land-fills;waste incineration;and leaching from consumer productsduring use or after disposal.Because of its low vapor pressure,con-centrations of DBP

22、in outdoor air are low;they range from 100 to102 ng/m3(Luks-Betlej et al.,2001).Although outdoor concentra-tions of DBP are low,indoor concentrations of DBP are relativelyhigh(Weschler and Nazaroff,2008).It is among the most abundantmanmade SVOC commonly measured in indoor air and in settleddust.DBP

23、 is very stable and can enter the aquatic environment.There is a potential risk for ecotoxicological effects of DBP on aqua-tic species.Concentrations above 0.04 mg/L are shown to cause ad-verse effects on aquatic invertebrates and concentrations above0.2 mg/L can have lethal effects.Although DBP ca

24、n be biodegradedas a short chains phthalate,the degradation process is time-consuming in anaerobic environments.It has some potential tobioaccumulate in some organisms.Because of its large and widespread use DBP is taken up by thegeneral population from various sources(Clark et al.,2003;Wormuth et a

25、l.,2006).For humans,food intake is the most impor-tant exposure pathway for phthalates,followed by ingestion ofdust and inhalation of indoor(Dickson-Spillmann et al.,2009;Jan-jua et al.,2008;Petersen and Jensen,2010;Schlumpf et al.,2010;Weschler and Nazaroff,2012;Wormuth et al.,2006).Intake ofphthal

26、ates via food might cause different effects comparing withthe routes by ingestion of dust and inhalation of indoor air.Studiesto evaluate the potential adverse effects of exposure to dibutylphthalate through various routes were a critical need.Phthalate metabolite concentrations measured in urine sa

27、mpleswere used to estimate phthalate daily intake in previous re-searches(Ferguson et al.,2011).To evaluate the potential adverseeffects of exposure to DBP,the concentration of DBP and MBP,the major metabolite of DBP,in the blood and urine was oftendetermined by gas chromatography,liquid chromatogra

28、phy andmass spectroscopy(Jen and Liu,2006;Serodio and Nogueira,2006;Zhang et al.,2008).The mean concentrations of MBP in urinewere measured in the United States according to the NationalHealth and Nutrition Examination Survey(NHANES).While MBP,the principal metabolite of DBP,has been measured in blo

29、od andurine in numerous studies,there are no studies that have mea-sured DBP,itself,in blood or urine.And the environmental dataconsist primarily of estimates.DBP concentrations are often either at or below detection limits.It is expensive and difficult to determine the concentration of DBPin organi

30、sm.Although,the chromatographic techniques provide alow level of detection for phthalates,they are time consumingand have high instrumentation costs.To evaluate the potential ad-verse effects of exposure to dibutyl phthalate,methods for dibutylphthalate determination must be developed.In this work,w

31、e set up two novel approaches to determinationof distribution and accumulation of DBP in vivo with a monoclonalantibody specific to DBP(Wei et al.,2011).The described assay canprovide useful estimates of excretion,distribution and metabolismof DBP through different routes.2.Materials and methods2.1.

32、AnimalsSix-week-old male Wistar rats arrived from Hubei Center for Disease Controland were housed in the Animal Care Facility of Central China Normal University,which is accredited by the Association for Assessment and Accreditation of Labora-tory Animal Care National.The animals were housed 2 or 3/

33、cage on wood-chip bed-ding in an air-conditioned animal room at 22 3?C and 55%10%humidity with a12/12 h lightdark cycle and allowed free access to powdered diet and filtered tapwater.2.2.TreatmentDBP(SigmaAldrich)was dissolved in corn oil(99.5%pure,Shanghai SolventFactory,Shanghai,China).Wistar rats

34、(n=5/group)were administered either di-butyl-phthalate(DBP SigmaAldrich)or corn oil by epicutaneously exposure(cutaneous route)or gavage(oral route).For dermal exposure,rat abdomens wereshaved using small animal clippers one day before exposure.Rats were epicutane-ously smeared with DBP at 200,400 m

35、g/kg bw-d or corn oil.For oral route,Wistarrats(n=5/group)were administered either dibutyl-phthalate(DBP SigmaAl-drich)at dose of 200,400 mg/kg bw-d,by gavage(oral route),or corn oil(vehicle).Starting 24,48,72 h after the final treatment,the rats were euthanized.Skin,liver,kidney,stomach,testes from

36、 each rats were collected,snap frozen in liquid nitro-gen,and stored individually at?80?C.2.3.Preparation for monoclonal antibodyAnti-DBP monoclonal antibody(MAb)was prepared according to Wei et al.(2011).Dibutyl 4-aminophthalate(DBAP),an aminated hapten with structural fea-ture of DBP,was linked to

37、 BSA or OVA through diazotization to generate artificialantigen DBAP-BSA or DBAP-OVA.All the hapten-protein conjugates were character-ized by UVvis spectrometry.Then BALB/c mice were immunized with these artifi-cial antigens.Mice were tail-bled 710 days after the third and the fourth injection.Indir

38、ect noncompetitive ELISA(inELISA)and Indirect competitive ELISA(icELISA)with 1.0lg/mL heterologous coating protein conjugates were used to determinethe titer and sensitivity of antibody in the serum.The mouse that generated poly-clonal antibody with a combination of the best sensitivity and the high

39、est titer re-ceived a final intraperitoneal injection of 200lg of immunogen in 200lL of PBS,3 days before cell fusion.Cell fusion,hybridoma culture and selection were carriedout essentially as described by Wei et al.(2011).Then female BALB/c mice were in-jected intraperitoneally with 0.5 mL of paraf

40、fin oil 710 days before receiving anintraperitoneal injection of 2?106hybridoma cells suspended in PBS.Ascites fluiddeveloped 23 weeks after the injection of the cells and was collected every otherday for 6 days(James,1995).The ascites fluid was centrifuged at 450 g for 5 min toremove cell pellet.Th

41、e supernatant was diluted with equal volume of PBS andappropriate amount of silica dioxide(15 mg/mL)was added to the supernatantand incubated at room temperature for 30 min with rotation.The IgG from the pre-treated ascites fluid was purified by caprylic acid/ammonium sulfate precipitationtwice and

42、dialysis and then stored at?70?C.The concentration and purity of puri-fied MAb were determined by taking absorbance at 280 nm and SDSpolyacryl-amide gel electrophoresis,respectively.2.4.Determining for the titer of anti-DBP MAbCheckerboard titration,in which purified MAb was titrated against varying

43、amounts of the coating hapten-conjugate was used to measure the reactivity ofantibodies and to select an appropriate coating concentrations and antibody dilu-tions for icELISA(Crowther,2001).The checkerboard assays were performed withinELISA.Microtiter plates were coated with 100lL/well of hapten-pr

44、otein conju-gates(0,0.25,0.5,and 1lg/mL)in 0.05 mol/L carbonate buffer(pH 9.6)and incu-bated overnight at 4?C.The plates were washed three times with PBST(0.01 mol/L PBS containing 0.05%Tween-20,pH7.4,200lL/well)and were blocked with 3%(w/v)skim milk powder in PBS(250lL/well)for 1 h at 37?C.After an

45、other washingstep,100lL/well of purified anti-DBP MAb(1:200,1:400,1:800,1:1600,1:3200,1:6400)diluted in PBS containing 0.1%BSA was added.After incubation for 1.5 hat 25?C,the plates were washed Subsequently,the plates were incubated for 1 hat 37?C with 8000-fold diluted HRP anti-mIgG in PBS containi

46、ng 0.1%BSA(100lL/well).After another washing step,100lL/well of freshly prepared O-phen-ylenediamine(OPD)solution(0.4 mg/mL OPD and 0.012%H2O2in 0.05 mol/L cit-ratephosphate,pH 5.0)was added.The reaction was stopped with 2 mol/Lsulfuric acid(50lL/well)after an incubation of 15 min at room temperatur

47、e keep-ing away from light.The absorbance was recorded at a primary wavelength of492 nm with a reference wavelength of 630 nm.Q.Zeng et al./Food and Chemical Toxicology 56(2013)182719Authors personal copy2.5.Determining for the specificity of MAbTo determine the specificity of MAb,the ability of the

48、 MAb to recognize severalstructurally related compounds(analogues)was tested by performing haptencoated icELISA(Wei et al.,2011)and determining their respective IC50(the concen-tration of analyte giving 50%inhibition)values.The cross-reactivity(CR)valueswere calculated according to the following equ

49、ation CR(%)=IC50DBP/IC50ana-logue?100.The hapten coated icELISA was modified to determine whether MBPaffects the binding of DBP to the MAb.The increasing concentration of MBP(19.510,000lg/mL)was added to the 12.5lg/L of DBP,then twice as the titer ofthe anti-DBP Mab in 50lL of assay buffer was added

50、 to the wells in the competi-tion step of icELISA.The other procedures were the same as hapten coated icELISAunder optimal condition.2.6.Frozen sectionsEach excised sample was immediately frozen in liquid nitrogen.The specimenswere embedded in tissue freeze media optimum cutting temperature(OCT)com-