(7.18)--2020 Dibutyl phthalate-mediated环境与健康环境与健康.pdf

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1、Contents lists available at ScienceDirectFood and Chemical Toxicologyjournal homepage: phthalate-mediated oxidative stress induces splenic injury in miceand the attenuating effects of vitamin E and curcuminXianliang Wanga,Xu Yana,Yuyan Yanga,Wenjing Yanga,Yujing Zhanga,Jiao Wanga,Dan Yea,Yang Wub,Pi

2、ng Mab,Biao Yanb,aNational Institute of Environmental Health,Chinese Center for Disease Control and Prevention,Beijing,100021,ChinabLaboratory of Environment-immunological and Neurological Diseases,Research Center of Basic Medical Sciences,Hubei University of Science and Technology,Xianning,437100,C

3、hinaA R T I C L E I N F OKeywords:Dibutyl phthalateVitamin ECurcuminSplenic injuryImmunotoxicityAntagonistic effectsA B S T R A C TDibutyl phthalate(DBP)is a ubiquitous environmental contaminant that at certain levels can be harmful tohuman health.Although DBP has been widely linked to immunotoxicit

4、y,any association between DBP exposureand splenic injury remains unknown.The purpose of this study was to investigate whether DBP exposure caninduce splenic injury and the antagonistic effects of two antioxidants,vitamin E(VitE)and curcumin(Cur),onDBP-induced splenic injury.The levels of ROS,GSH,T-A

5、OC,IL-1,TNF-,cytochrome C,caspase-8,caspase-9and caspase-3 in the spleen homogenate of mice were measured.Any histopathological changes in the spleenwere observed using H&E and toluidine blue staining.And the morphology of mitochondria was observed usingJanus Green B staining.The results indicate th

6、at exposure to 50 mg/kg DBP could cause histopathologicalchanges of the spleen and result in inflammation and apoptosis associated with oxidative stress,which may leadto splenic injury in mice.Moreover,both VitE and Cur could antagonize the oxidative stress induced by DBP toreduce splenic injury.The

7、se findings help to expand our understanding of DBP-mediated immunotoxicity,and toshow that VitE and Cur can alleviate DBP-induced splenic injury and the possible DBP-associated decline inimmune function.1.IntroductionPhthalates,frequently used as plasticizers in products such aspolyvinyl chloride(P

8、VC),are ubiquitous environmental contaminantsthat can be deleterious to the immune system at certain exposureconcentrations(Kimber and Dearman,2010;Xu et al.,2013).Sincephthalates are not covalently bound in PVC products,they can leach outof the PVC into the atmosphere where humans are exposed via f

9、ood,drink,personal care products or directly via inhalation or dermalsorption(Wormuth et al.,2006;Pei et al.,2014).Dibutyl phthalate(DBP)is one of the most common phthalates.Based on the urinaryconcentrations of phthalate metabolites,Guo et al.(2011)estimatedthat the daily intake of DBP in China was

10、 12.2 g/kgbw/day,exceededthe tolerable daily intake of 10g/kgbw/day,proposed for DBP,by theEuropean Food Safety Authority.Data from meta-analyses in childrenprovide some evidence showed that an association between high levelsof phthalates from PVC products and the risk of asthma fixed-effectsmodel:s

11、ummary odds ratio(OR),1.55;95%confidence interval(CI),1.182.05 and allergies(OR,1.32;95%CI,1.091.60)(Jaakkola andKnight,2008).A number of studies have been performed using mice toexamine the influence of DBP,via various exposure routes,on the im-mune responses(Kimber and Dearman,2010).In addition,on

12、e tox-icological study has demonstrated the immunotoxicity of DBP onmacrophages in mice(Li et al.,2013).Although DBP has been widelylinked to immunotoxicity,the current findings are not sufficient forclarifying the toxic effects of DBP on the immune system(Robinson andhttps:/doi.org/10.1016/j.fct.20

13、19.110955Received 18 July 2019;Received in revised form 2 November 2019;Accepted 6 November 2019Abbreviations:Dibutyl phthalate,DBP;Vitamin E,VitE;Curcumin,Cur;Reactive oxygen species,ROS;Glutathione,GSH;Total antioxidant capacity,T-AOC;Interleukin-1,IL-1;Necrosis factor-,TNF-;nuclear factor-B,NF-B;

14、Cytochrome C,Cyt C;Caspase-8,Casp-8;Caspase-9,Casp-9;Caspase-3,Casp-3;White pulp,WP;Red pulp,RP;Marginal zone,MZCorresponding author.Hubei University of Science and Technology,No.88 Xianning Avenue,Xianning,437100,China.Corresponding author.Hubei University of Science and Technology,No.88 Xianning A

15、venue,Xianning,437100,China.E-mail addresses:(X.Wang),(X.Yan),(Y.Yang),(W.Yang),(Y.Zhang),(J.Wang),(D.Ye),(Y.Wu),(P.Ma),(B.Yan).Food and Chemical Toxicology 136(2020)110955Available online 08 November 20190278-6915/2019 Elsevier Ltd.All rights reserved.TMiller,2015).New research to elucidate these m

16、echanisms is necessary.In particular,any association between DBP exposure and splenic injuryremains unknown since the process by which DBP could affect thespleen is very complex:DBP enters the body and is metabolized by avariety of enzymatic conversions in the blood before reaching thespleen.The spl

17、een is a peripheral immune organ and the largest lymphoidorgan in the human body.As the center of cellular and humoral im-munity,the spleen plays an important role in regard to the immunesystem.It can remove old red blood cells and preserve blood reserves,and it also monitors and removes aging,mecha

18、nically damaged orabnormal cells,as well as protecting the body from pathogens andexogenous substances(Mebius and Kraal,2005).Xu et al.(2013)de-monstrated that DBP exposure could induce antioxidant and immuneresponses in zebrafish embryos.The questions of whether exposure toDBP at toxic levels will

19、affect the spleen,and if so,whether this willalter its immune function,are problems worth exploring.Natural antioxidants protect cells against oxidative damage by di-rectly scavenging excess reactive oxygen species(ROS)in vivo,bypromoting the expression of the antioxidant enzyme defense system,and b

20、y inhibiting the production of ROS(Xu et al.,2017).Both VitE andCur are well-known natural antioxidants.VitE plays a protective role byacting as a radical scavenger,delivering a hydrogen atom to the DBPinduced free radicals(Yan et al.,2019).Other studies have shown thatVitE has potent pro-inflammato

21、ry regulatory functions in different ex-perimental models(Galli et al.,2017).Cur is designated as“GenerallyRecognized As Safe”(GRAS)by the US Food and Drug Administration(FDA)(Gupta et al.,2013;Susan and Douglas,2017),and is the mainpharmacologically active phytochemical component of turmeric.Themai

22、n proven biological activities of Cur are its antioxidant and anti-inflammatory effects at the cellular level due to its ability to target thespleen(Abdollahi et al.,2018).Increasing evidence also suggests thatCur can be involved in immune response modulation(Yadav et al.,2008;Momtazi-Borojeni et al

23、.,2018).This contributes to extensiveinvestigations being undertaken of the underlying mechanisms of ac-tion activated by curcumin.Interestingly,the attenuating effects of VitEand Cur on DBP exposure in the spleen should be taken into account.This study was designed to explore whether continuous DBP

24、 ex-posure can adversely affect the spleen.The effects of DBP on splenicoxidative stress(ROS,reduced glutathione(GSH)and total antioxidantcapacity(T-AOC),inflammation factor interleukin-1(IL-1),necrosisfactor-(TNF-),the phosphorylation of nuclear factor-B(NF-B),apoptosis related factors(cytochrome C

25、(Cyt C),caspase-8(Casp-8),caspase-9(Casp-9)and caspase-3(Casp-3)and the expression ofsplenic lymphocyte subset markers(CD3+and CD8+)in BALB/c micewere examined.We looked for histopathological changes to the spleenafter 21 days exposure to DBP using Haematoxylin&Eosin(H&E)andtoluidine blue staining m

26、ethods.The morphology of mitochondria insplenic tissue was examined by Janus Green B staining.On the basis ofthe above experiments,further blocking experiments were conducted tohighlight the possible roles of VitE and Cur in reducing DBP-inducedsplenic injury in mice.2.Materials and methods2.1.Anima

27、l careSpecific pathogen-free male BALB/c mice(5-week old,about 16.1g)were obtained from the Experimental Animal Center of Hubei Province(Wuhan,China)and housed under standard laboratory conditions(temperature:2025C,humidity:5070%,and 12h day/night cycles).All the mice were quarantined for 3 days pri

28、or to the study.The micewere given a standard chow diet and water ad libitum,except duringexposure periods.All experimental procedures were conducted in ac-cordance with the National Institutes of Health Guide for the Care andUse of Laboratory Animals,and were approved by the Office ofScientific Res

29、earch Management of Hubei University of Science andTechnology(Xianning,China),with a certificate of Application for theUse of Animals(approval ID:HBUST-IACUC-2018-001).2.2.Reagents and kitsDBP(99%,CAS:84-74-2),VitE(CAS:59-02-9)and Cur(CAS:458-37-7)were purchased from Sigma-Aldrich(St.Louis,MO,USA).M

30、ouse colorimetric kit for GSH and T-AOC were purchased fromNanjing Jiancheng Bioengineering Institute(Nanjing,China).MouseELISA kits for IL-1,TNF-,Cyt C,Casp-9 and Casp-3 were purchasedfrom eBioscience(San Diego,CA,USA).The rabbit anti-mouse CD3+(ab16669),CD8+(ab217344),NF-B p65(ab16502)and goat ant

31、i-rabbit IgG antibodies were purchased from Abcam(Cambridge,UK).The rabbit anti-mouse Casp-8 p43/p18(bs-0052 R)were purchasedfrom Bioss(Beijing,China).All other reagents were of the highest gradeavailable commercially,or as indicated.2.3.Experimental protocolThe BALB/c mice were divided randomly int

32、o six groups of ninemice each.The groups were treated as follows:(A)control(saline,oralexposure),(B)50 mg/kg/d DBP(oral exposure),(C)50 mg/kg/d VitE(oral exposure),(D)DBP+VitE(50 mg/kg/d VitE was given at thesame time as the intragastric administration of 50 mg/kg/d DBP),(E)2.5 mg/kg/d Cur(intraperi

33、toneal injection),(F)DBP+Cur(2.5 mg/kg/d Cur was given at the same time as the intragastric administrationof 50 mg/kg/d DBP).Based on previous studies carried out in our laboratory,the ex-posure dosage of DBP(50 mg/kg/d)was selected as the optimized do-sage according to Wu et al.(2017).The dosage of

34、 VitE(50 mg/kg/d)was selected according to Yan et al.(2019).Dose selection for Cur(2.5mg/kg/d)is based on a pharmacological report by Bhutani et al.(2009).Experimental mice received daily treatment for a period of 3weeks.After the 21 days,the mice were sacrificed,the spleens of themice were collecte

35、d for tissue sectioning and for the preparation of ahomogenate.The homogenate was then used to determine the levels ofROS,GSH,IL-1,TNF-,Cyt C,Casp-9 and Casp-3.2.4.Sampling and testingThe collected spleens were weighed in a fully automatic electronicbalance.The tissue was placed in 10 mL/g ice-cold

36、1phosphate-buffered saline(PBS,pH=7.5)and homogenized using a glass homo-genizer.The homogenate was centrifuged at 9391g for 10min at 4C.Supernatants were collected and kept frozen at 70C until needed.The protein concentration of the supernatant was determined using aFolin assay.ROS levels(fluoresce

37、nce intensity)in the spleen super-natant were determined by DCFH-DA fluorescent assay,at an excitationwavelength of 488 nm and an emission wavelength of 525nm using afluorescence reader(Hide Chameleon V,Hidex,Finland).The GSHconcentration in the spleen supernatant was measured using a colori-metric

38、kit.GSH levels were calculated as follows:GSH(molg1prot)=(measure OD405-blank OD405)/(standard OD405-blankOD405)standardconcentration sampledilutionfactor/homo-genate protein concentration.IL-1,TNF-,Cyt C,Casp-9 and Casp-3were measured using ELISA kits according to the manufacturers in-structions.Th

39、e sensitivities of the ELISA kits were 1.0 pg/mL for IL-1,1.0 pg/mL for TNF-,1.0 nmol/L for cyt C,1.0pmol/L for Casp-9,and1.0 pmol/L for Casp-3.2.5.Fractionation and specific staining of mitochondria in the spleenAfter the 21 days of exposure,one mouse from each treatment groupwas randomly selected

40、and sacrificed by cervical dislocation.About 1gof spleen tissue was collected quickly,then washed and placed in aX.Wang,et al.Food and Chemical Toxicology 136(2020)1109552small plate,and 1mL precooled homogenate(0.25 mol/L sucrose,0.003 mol/L calcium chloride)was added.The homogenate was fil-tered,c

41、entrifuged at 376 g for 7min,then the collected supernatant wascentrifuged at 9391g for 5min.After that,the supernatant was dis-carded,the precipitate was resuspended,and centrifuged at 9391 g for5min again,and the obtained precipitate containing mitochondria wasmade into a smear,which was observed

42、under a BX53 microscope(Olympus,Tokyo,Japan)using Janus green B staining.2.6.Histological evaluationThe spleen was removed and immediately put into 4%paraf-ormaldehyde solution for fixation.After 24h,the specimens were de-hydrated in gradient alcohol,cleared in xylene,and embedded in par-affin wax.S

43、ections(5 m)were cut vertically from the central regionusing a Shandon Finesse 325 microtome(Thermo Electron Cooperation,Waltham,MA,USA),then separately stained with H&E and toluidineblue.AllsampleswerescannedusingNDP.view2software(Hamamatsu,Japan)and examined.2.7.Immunohistochemical analysis of NF-

44、B p65,Casp-8 p43/p18,CD3+and D8+The spleen tissue of mice was collected and fixed with 4%paraf-ormaldehyde.After 24h,the sections(5 m thickness)were dewaxedand mounted on glass slides.After antigen repair and inactivation ofendogenous enzymes,immunohistochemical analyses were performedusing the foll

45、owing antibodies.The first antibodies were NF-B p65(1:200),Casp-8 p43/p18(1:200),CD3+(1:100)and CD8+(1:500).Thesecond antibodies were goat anti-rabbit IgG antibodies.The expressionsof NF-B p65,Casp-8 p43/p18,CD3+and CD8+were observed under amicroscope respectively.Three different visual fields were

46、quantifiedfrom each section and the average optical density was statisticallyanalyzedusingImage-ProPlussoftware(version6.0;MediaCybernetics,Baltimore,MD,USA).2.8.Statistical analysisThe data obtained from the experiment were analyzed and statis-tical graphs were generated using GraphPad Prism 5.02(G

47、raphPadSoftware Inc.,CA,USA).Values were presented as mean standarderror of the mean(SEM).The differences between groups were ana-lyzed for significance using a one-way analysis of variance(ANOVA)followed by Tukeys test.A probability 0.05 was regarded as statis-tically significant.3.Results3.1.VitE

48、and Cur alleviate spleen tissue damage of mice exposed to DBPIn the control group,the junction of spleen cortex and pulp wasclear,the red pulp(RP)was more obviously red,and the white pulp(WP)was more prominently blue.There is a dense distribution oflymphocytes in RP and WP,as shown in Fig.1A(a).In t

49、he DBP ex-posure group,the interface between WP and RP was blurred,the ar-rangement was sparse,the number of lymphocytes was relatively re-duced,and focal multinucleated giant cell infiltration or granulomatouslesions could be seen in the RP,as shown in Fig.1A(b).Compared withthe DBP exposure group,the junctions of WP and RP in the DBP+VitEgroup and the DBP+Cur group were more obvious,and there weremore lymphocytes.This can be seen in Fig.1A(d,f).There are a large number of mast cells(MC)in the spleen,and thesecells contain heterochromic substances such as heparin and histamine,which can be