1、Oralexposuretodiisodecylphthalateaggravatesallergicdermatitisbyoxidative stress and enhancement of thymic stromal lymphopoietinShiping Shena,1,Jinquan Lia,1,Huihui Youa,Zhuo Wua,Yang Wub,Yun Zhaoa,YuqingZhua,QingGuoa,XiaoxiaoLia,RuiLia,PingMab,XuYanga,*,MingqingChena,*aHubeiKeyLaboratoryofGeneticReg
2、ulationandIntegrativeBiology,SchoolofLifeSciences,CentralChinaNormalUniversity,Wuhan430079,Hubei,ChinabCollege of Basic Medical Sciences,Hubei University of Science and Technology,Xianning 437100,ChinaarticleinfoArticle history:Received 14 June 2016Received in revised form27 September 2016Accepted 1
3、7 November 2016Available online 18 November 2016Keywords:Allergic contact dermatitisDiisodecyl phthalate(DIDP)NF-kBOxidative stressThymic stromal lymphopoietin(TSLP)abstractDiisodecylphthalate(DIDP)isextensivelyusedasanenvironmentallyfriendlyplasticizer.However,littleis known about the adverse effec
4、ts and the underlying mechanisms of DIDP exposure on immunologicaldiseases.We aimed to determine the role and mechanisms of DIDP exposure in allergic contactdermatitis-like skin lesions.We show that oral DIDP exposure can aggravate allergic dermatitis in mice.Moreover,an increase of ROS,total serum
5、IgE and IL-4 levels were concomitant with this deterioration.We detectedtheexpressionof Thymicstromallymphopoietin(TSLP)andtheactivationofSTATsandNF-kBsignalpathways.ThedataindicatedthatDIDPincombinationwithFITCtriggersTSLP production.Ourresults also suggested that DIDP exacerbated the activation of
6、 NF-kB signal pathways,with anenhancement in TSLP expression,which potentiated the activation of STATs and the degranulation ofmastcellsintheskin,and nallyexacerbatedallergicdermatitis.ThestudyalsosuggestedthatmelatoninenhancedtheexpressionofNrf2,up-regulatedtheantioxidantgenesHO-1andNQO1,reducedthe
7、levelsof oxidative stress and TSLP,and alleviated allergic dermatitis.The results demonstrated that DIDPexacerbated allergic dermatitis through oxidative stress and enhanced TSLP production.2016 Elsevier Ltd.All rights reserved.1.IntroductionDiisodecyl phthalate(DIDP)is widely used in food packaging
8、,home furnishings,medical equipment and other consumer prod-ucts(Gimeno et al.,2014;Kasper-Sonnenberg et al.,2012).Diet isone of the major sources of human exposure to DIDP(Sakhi et al.,2014).Some metabolites of DIDP have been detected in humanurine(Kochetal.,2013;SaravanabhavanandMurray,2012).Young
9、children mouthing PVC toys and ingesting house dust may be at ahigher risk of exposure to DIDP than adults who are exposed fromfood,dust,andindoorair(Stringeretal.,2000).Untilrecently,DIDPwas considered to be a low toxic phthalate.It showed no effect onreproduction or development(Chen et al.,2014;Pa
10、tyna et al.,2006),nor did it show carcinogenic potential in a two-year carci-nogenicity study using F344 rats(Cho et al.,2008).However,studies did show that oral exposure to DIDP can cause liverenlargement in rats.Of particular interest is that there is evidencethat phthalates,including DIDP,have an
11、 impact on immune andallergic responses(Kimber and Dearman,2010).Surveys haveshown that current asthma was modestly associated with urinaryconcentrations of DIDP metabolites in Norwegian children(Bertelsen et al.,2013).Positive patch test reaction to DIDP wasfound in some workers(Lensen et al.,2012)
12、.However the associ-ation between allergic dermatitis and exposure to DIDP has notbeen fully elucidated and the mechanism behind DIDP relatedallergic dermatitis is still unclear.Among the various types of allergic dermatitis,one of the mostcommon skin allergies in western countries is allergic conta
13、ctdermatitis(ACD),alsoreferredtoascontacthypersensitivity(CHS).ACD is induced by an allergic response to a multitude of chemicalsubstances which are the result of environmental contamination(Vocanson et al.,2009).Epidemiological studies suggest thatphthalateexposuremayberelatedtotheriskofallergyinch
14、ildrenand workers in the plastics industries(Bornehag and Nanberg,2010;Kimber and Dearman,2010).The prevalence of ACD hasincreased with the increase in industrial pollution,suggesting thatcertainenvironmentaltoxins,suchasDIDP,mayplayanimportant*Corresponding author.*Corresponding author.E-mail addre
15、sses:(X.Yang),(M.Chen).1These authors contributed equally to this work.Contents lists available at ScienceDirectFood and Chemical Toxicologyjournal homepage:Elsevier Ltd.All rights reserved.Food and Chemical Toxicology 99(2017)60e69role in the enhancement of allergic diseases by increasing the po-te
16、ncy of allergens.Contact hypersensitivity(CHS)is induced by epicutaneouscontactwithanallergen(sensitization)leadingtothedevelopmentof immunologic memory and enhanced in ammatory skin re-sponses at sites of re-exposure to the allergen(Watanabe et al.,2002).It is believed that CHS is accompanied by a
17、switch in thelocal cytokine pro le,from Th1 to Th2 with enhanced serumimmunoglobulin E(IgE)levels(Shigeno et al.,2009).Studies sug-gest that Th2 conditions can induce pruritus and barrier dysfunc-tions(Kabashima,2013).Reactive oxygen species(ROS)-inducedoxidativestressisconsideredtobeoneoftheimporta
18、ntpathogenicmechanisms involved in various in ammatory skin diseases,including ACD(Bowler and Crapo,2002;Fuchs et al.,2001;Luset al.,2014;Tsukahara et al.,2003).It has been suggested that theskin sensitizer 1-uoro-2,4-dinitrobenzene(DNFB)leads to a ROS-dependentactivationofERstressindendriticcells(D
19、C)(Lusetal.,2014).ROS is also found to be involved in mast cell activation.Inacquired immunity,the cross-linking of receptor-bound IgE canactivate mast cells,after which some mediators involved in theallergic in ammatory response are released(Otsuka andKabashima,2015;Swindle and Metcalfe,2007).The t
20、ranscription factorNrf2,which is a powerful redox sensor,can be activated in response to oxidative stress(Hu et al.,2010).Various antioxidant genes,such as heme oxygenase-1(HO-1)andNADPHquinoneoxidoreductase1(NQO1),canberegulatedbyNrf2(Kobayashi and Yamamoto,2005).HO-1 is considered to be themain an
21、tioxidant gene among the Nrf2 target genes(Rushworthand MacEwan,2008).Melatonin(MT)is produced by the pineal gland,especially atnight.Itisakindofhormone(N-acetyl-5-methoxytryptamine)thatplays an important role in the human body.Melatonin is an effec-tive antioxidant with anti-in ammatory functions t
22、hat canprotectorganisms from oxidative stress(Reiter et al.,2003).Both physio-logical and pharmacological concentrations of melatonin are ableto protect against free radical destruction,which has been docu-mented by in vitro and in vivo studies(Rao and Chhunchha,2010;Tan et al.,2002).In addition,Mel
23、atonin,as a natural constituentof certain foods,is widely used as a nutritional food additive(Hardeland et al.,2006).The cytokine thymic stromal lymphopoietin(TSLP)is mainlyproduced by epithelial cells,and plays an essential role in thepathogenesisofallergicdermatitisandasthma(Larsonetal.,2010;Leyva
24、-Castillo and Li,2014;Nakajima et al.,2012).Signaling be-tween epithelial cells and innate immune cells via TSLP is consid-ered to drive AD as well as the atopic march.It was reported thatepithelialcellscanpromoteitchingbydirectlycommunicatingwithcutaneous sensory neurons via TSLP(Wilson et al.,2013
25、).Studieshave shown that the expression of TSLP is induced by variousenvironmental stimuli,and is also related to numerous gene mu-tations.TSLP has been implicated in the development and pro-gressionofallergicin ammationinbothhumansandmice,andhasbeen shown to promote a Th2-type response(Larson et al
26、.,2010;Moniaga et al.,2013;Siracusa et al.,2011).It was reported thatTSLPRexpressionwas elevated onTh2 instead of Th1 or Th17 cellsin rodents(Kitajima et al.,2011).In another study it was reportedthat the transcription factors NF-kB regulate human TSLP geneexpression(Takai,2012).TSLP production can
27、also be induced orenhanced by proin ammatory cytokines,Th2-related cytokinesand IgE(Takai,2012).The signal transducers and activators oftranscription STAT5 is critical in dendritic cells(DC)for develop-ment of Th2-dependent immunity and was required for TSLP-dependent DC activation(Bell et al.,2013)
28、.In the human body,studieshaveshownthat,inadditiontoSTAT5,TSLPstimulationalsoactivated STAT 1,3,4,5,and 6(Arima et al.,2010).Fluorescein isothiocyanate(FITC)is used as a hapten in studies,tobuildacontacthypersensitivity(CHS)model.Interestingly,FITC-induced CHS is an acute model which is mainly regul
29、ated by Th2rather than by Th1 cells(Shigeno et al.,2009).Inthisstudy,thedeleteriouseffectofDIDPonallergicdermatitisin an FITC-induced allergic dermatitis model is presented,and thelevels of in ammatory factors,oxidative stress,TSLP expression,STATs and the NF-kB signal pathway in skin lesions of mod
30、el miceare also investigated.We then evaluate the protective effect ofmelatoninonACDandinvestigateitsmechanismasanantioxidant.2.Materials and methods2.1.Ethics statementAll protocols used in these studies were approved by the Of ceof Scienti c Research Management of Central China Normal Uni-versity,
31、with the certi cation on the Application for the Use ofAnimals dated 5 December 2013(approval ID:CCNU-SKY-2011-008).All experiments in this study were performed in accordancewith the approved guidelines and regulations.2.2.Experimental animalsSpeci c Pathogen Free Balb/c mice(Male;about 6e7weeks;22e
32、23g)wereboughtfromtheHubeiExperimentalAnimalCenter(Wuhan,China),and all mice were housed under SPF raising con-ditions,and were provided a commercial diet and ltered wateradlibitum.Allmicewerefedfor7daysbeforethestudy.Sevenmicewere used in each group in order to minimize the number ofexperimental an
33、imals needed and still ensure statistical validity.2.3.Main reagents and kitsTween-80 was obtained from Amresco(Solon,OH,USA).DIDP(99%),DBP(99%),FITC and pentobarbital sodium were obtainedfromSigmaeAldrich(St.Louis,MO,USA).TheROSandGSHtestkitswere obtained from Nanjing Jiancheng Bioengineering Insti
34、tute(Nanjing,Jiangsu,China).The protein test kit was provided byBeijing Dingguo Changsheng Biotechnology Co.LTD(Beijing,China).Mouse enzyme-linked immunosorbent assay(ELISA)kitsfortotalIgEwereobtainedfromBiolegend(SanDiego,CA,USA).AnELISA kits for IL-4 and interferon(IFN)-g were obtained fromeBiosci
35、ence(San Diego,CA,USA).All other chemicals were ofanalytical grade.All the operations were performed according tomanufacturers instructions.2.4.Exposure and immunizationThe mice were divided randomly into seven groups of sevenmice each.The groups were treated as follows:(1)Saline oralexposure combin
36、ed with a saline-treated group(Saline);(2)200mg/(kg.d)DIDP oral exposure combined with a saline-treatedgroup(DIDP200);(3)Saline oral exposure combined with a 0.5%FITC sensitized group(FITC);(4)2 mg/(kg.d)DIDP oral exposurecombined with a 0.5%FITC sensitized group(FITC DIDP2);(5)20 mg/(kg.d)DIDP oral
37、 exposure combined with a 0.5%FITCsensitized group(FITC DIDP20);(6)200 mg/(kg.d)DIDP oralexposure combined with a 0.5%FITC sensitized group(FITCDIDP200);(7)200mg/(kg.d)DIDPand10mg/(kg.d)MToralexposure combined with a 0.5%FITC sensitized group(FITCDIDP200MT).TheparticularprotocolsareshowninFig.1.Groups(1)e(7)were given a daily gavage of saline or one of thethree different concentrations of DIDP,while group(7)was alsogiven a gavage of MT once a day for a continuous 21-day period.S.Shen et al./Food and Chemical Toxicology 99(2017)60e69 61
