(7.13)--2019 Comparing the effects of di环境与健康环境与健康.pdf

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1、Contents lists available at ScienceDirectEcotoxicology and Environmental Safetyjournal homepage: the effects of diethylhexyl phthalate and dibutyl phthalateexposure on hypertension in miceXiaoman Xie1,Ting Deng1,Jiufei Duan,Shumao Ding,Junlin Yuan,Mingqing ChenHubei Key Laboratory of Genetic Regulat

2、ion and Integrative Biology,School of Life Sciences,Central China Normal University,Wuhan,Hubei 430079,ChinaA R T I C L E I N F OKeywords:DBPDEHPHypertensionEstradiolRAASA B S T R A C TEpidemiological studies have shown that high molecular weight phthalates(HMW)such as diethylhexylphthalate(DEHP),ar

3、e associated with hypertension in humans,while low molecular weight phthalates(LMW)such as dibutyl phthalate(DBP),have hardly any impact on the elevation of blood pressure.However,themolecular mechanisms responsible for this difference are not completely understood.In this experiment,micewere expose

4、d to 0.1/1/10mg/kg/day DEHP and 0.1/1/10 mg/kg/day DBP for 6 weeks,and their blood pressurewas monitored using the tail pressure method.The results showed that exposure to DEHP dosages of 1 or 10 mg/kg/day resulted in a sharp increase in blood pressure,while exposure to DBP did not induce any signif

5、icantchanges in blood pressure.Investigating the renin-angiotensin-aldosterone system(RAAS)and NO pathway inmice exposed to DEHP,we found that levels of angiotensin-converting enzyme(ACE)and angiotensin II(AngII)increased with increasing exposure to DEHP,and the expression of nitric oxide synthase(e

6、NOS)and the level ofNO decreased.Treatment with ACE inhibitor(ACEI)to block the ACE pathway inhibited the enhancement ofRAAS expression,inhibited the increase in blood pressure,and inhibited the decrease in NO levels induced byDEHP.However,the expression of ACE,AngII,AT1R,and eNOS in the DBP treatme

7、nt groups showed no sig-nificant changes.When examining estradiol in vivo,we found that exposure to DBP resulted in a significantincrease in the level of estradiol,while exposure to DEHP did not lead to a significant change.When ICI182780was used to block the estradiol receptors,any increase in the

8、level of NO induced by DBP exposure,was in-hibited.These results indicate that exposure to DEHP induces an increase in mouse blood pressure throughRAAS,and the different effects of DEHP and DBP on blood pressure are partly due to the different estradiol levelsinduced by DEHP and DBP.1.IntroductionPh

9、thalates are widely used in the production of pesticides,medicaldevices,cosmetics,childrens toys and some plastic packaging(Blountet al.,2000a,2000b;Latini,2005).In the past few decades,the use ofphthalates has increased dramatically,thus increasing the risk of hu-mans being exposed to their environ

10、mentally toxic components.Phthalates can be classified into two types,high molecular weight(HMW)phthalates and low molecular weight(LMW)phthalates.HMWphthalates,such as DEHP,dioctyl phthalate(DOP),butyl benzylphthalate(BBP)are commonly used in vinyl plastics for a variety ofapplications,including fl

11、ooring,transparent food packaging and in-travenous tubing(Hermann,2007).LMW phthalates,such as diethylphthalate(DEP),DBP and diisobutyl phthalate(DiBP)are often addedto shampoos,cosmetics,lotions and other personal care products topreserve the fragrance(Schettler,2006).Phthalates can enter thehuman

12、body via the respiratory tract,digestive tract,skin,and in-travenous infusion,causing toxicity in various tissues and organs.BothDEHP and DBP are rapidly absorbed and excreted following all uptakeroutes(Koch et al.,2006;Zeng et al.,2013).DEHP is cleaved into themonoester MEHP,which is extensively fu

13、rther metabolized by differentoxidation reactions(Koch et al.,2006).After 24h,about 67.0%of aDEHP dose is excreted in urine as its metabolites.Absorbed DBP isconverted to monobutyl phthalate(MBP)and accumulated in the liverin this form(Miura et al.,2019).MBP can be:excreted unchanged inurine;further

14、 metabolized;or conjugated to glucuronic acid.Studieshave shown that there are many links between phthalate exposure andhuman health,including reproductive system malformations,dysplasia,and cardiovascular diseases such as coronary artery disease,hyperten-sion,atherosclerosis,and myocardial infarcti

15、on(Ejaredar et al.,2015;https:/doi.org/10.1016/j.ecoenv.2019.02.067Received 28 November 2018;Received in revised form 19 February 2019;Accepted 20 February 2019Corresponding authors.E-mail addresses:(J.Yuan),(M.Chen).1Contributed equally to this paper.Ecotoxicology and Environmental Safety 174(2019)

16、75820147-6513/2019 Elsevier Inc.All rights reserved.TKhalil et al.,2014;Lind and Lind,2011;Mariana et al.,2018;Sanghyuk,2012;Trasande et al.,2013).Based on rodent experiments(rats)with the no-observable-adverse-effect level(NOAEL)of 5mg/kgbw/day DEHP and the lowest-observed-adverse-effect-level(LOAE

17、L)of2mg/kg bw/day DBP,the European Food Safety Authority(EFSA)re-commended tolerable daily intake(TDI)of 0.05mg/kg bw/day DEHPand 0.01mg/kg bw/day DBP for humans in 2005(Authority,2005;Duan et al.,2018a).Several studies have suggested a connection between exposure tophthalates and an increase in the

18、 risk of high blood pressure(Diamanti-Kandarakis et al.,2009;Mariana et al.,2016).According to Barry et al.,the DEHP metabolite,MEHP,resulted in a dose dependent,negativeinotropic effect on human atrial trabecula(Barry et al.,1990,1989),suggesting that high levels of MEHP in serum could have a cardi

19、otoxiceffect in humans.Werner et al.analyzed urine samples from 369pregnant women and found that there is a significant association be-tween BBzP metabolite levels and increased diastolic blood pressure(Werner et al.,2015).According to data from the National Health andNutrition Examination Survey,th

20、e concentration of certain HMWphthalates such as DEHP,diisononyl phthalate(DINP),and diisodecylphthalate(DIDP)in the urine of children and adolescents is positivelycorrelated with high systolic blood pressure.However,LMW phthalicacid ester metabolites are not related to blood pressure,according toth

21、e cross-sectional studies(Trasande et al.,2013).One study suggested that DEHP can change the metabolic char-acteristics of cardiomyocytes,making the heart susceptible to ischemiaand ventricular dysfunction(Schaedlich et al.,2015).The study by Leeet al.,showed that maternal exposure to DEHP can affec

22、t the bloodpressure of mice offspring(Lee et al.,2016).Moreover,these authorsbelieve that elevated blood pressure in the offspring of mice exposed toDEHP may be due to the dysregulation of eNOS activity,and an in-creased AngII-angiotensin II type 1 receptor(AT1R)signal.An increasein eNOS activity,as

23、 a result of an external stimulus,is closely related tothe development of cardiovascular disorders such as hypertension.In-creased eNOS activity can increase endogenous nitric oxide production,which plays an important role in regulating vascular tone(Davignonand Ganz,2004;Lee et al.,2016).Among the

24、various mechanisms re-sponsible for the development of hypertension,the RAAS plays a crucialrole in the initiation and maintenance of vascular inflammation,as wellas the vascular remodeling of hypertension.AngII,the main effector ofthe RAAS,mediates vasoconstriction,endothelial dysfunction,vascularr

25、emodeling,and inflammation through a variety of pathogenic me-chanisms(Montezano et al.,2014).AngII increases the expression ofchemokines and cytokines,resulting in the recruitment of leukocytesinto the vessel wall(Montezano et al.,2014;Suzuki et al.,2003).AngIIalso increases ROS production,resultin

26、g in decreased nitric oxidebioavailability followed by endothelial dysfunction(Pacurari et al.,2014;Suzuki et al.,2003).Estradiol has been shown to play an important role in regulatingblood pressure and preventing the development of hypertension(Smithand Ferguson,2016).The link between phthalates an

27、d estrogen may berelated to the cardiovascular system(Mariana et al.,2016;Smith andFerguson,2016).In a study exploring the effect of DEHP and DBP ex-posure on the reproductive system of female rats,it was shown that theHMW phthalate,DEHP,could reduce the level of estradiol in the rats,while DBP,one

28、of the LMW phthalates,was shown to increase the levelof estrogen(Rahmani et al.,2016).It can be speculated that the dif-ferent effects of DEHP and DBP on blood pressure might be related tothe different estradiol levels.Clinical studies have shown that the lowincidence of premenopausal cardiovascular

29、 system disorders is sig-nificantly different to the incidences of these disorders in men of thesame age.This advantage gradually disappears in women in the 6070year age group(Farquhar et al.,2005;Writing Group for the WomensHealth Initiative Investigators,2002).The possible reason for thisphenomeno

30、n is that female estrogen levels in postmenopausal womendeclines,leading to changes in estrogen concentration and function,suggesting that estrogen plays a significant role in cardiovascular dis-ease,and especially in hypertension(Burns and Korach,2012).It iscurrently believed that estrogen can prev

31、ent hypertension by itself,aswell as by activating both the NO and prostacyclin-mediated relaxationresponses,by relaxing the vasculature.In the peripheral vascularsystem,estrogen first binds to the receptor,and promotes the release ofNO by increasing the expression and activity of intracellular eNOS

32、,thereby promoting endothelium-resistant vasodilation(Santos et al.,2010).In this study,we compared the impact of DEHP and DBP on highblood pressure,and explored the underlying mechanisms.2.Materials and methods2.1.Animals117 male C57BL/6 strain mice(67 weeks old)were purchasedfrom the Experimental

33、Animal Center of Three Gorges University inHubei Province(Yichang,China).The animals were housed in a pa-thogen free cage at 2426C,5575%humidity with a 12-h light-darkcycle.The mice were given a standard diet(Hubei Experimental AnimalCenter)and water ad libitum.Nine mice were used per group to ensur

34、estatistical validity.All mouse care and experimental procedures wereapproved by the Central China Normal University Scientific ResearchManagement Office.The animal application certification was issued onMay 4,2018(approval ID:CCNU-SCXK-2017-0012)2.2.Reagents and kitsDEHP and DBP were purchased from

35、 Sigma-Aldrich(St.Louis,MO,USA),The specific antagonist of estradiol receptor inhibitor ICI182780,and Angiotensin converting enzyme inhibitor ACEI(Enalapril Maleate)were purchased from Shanghai Qianyuan Biomedical Technology Co.,Ltd.China.The mouse NO enzyme-linked immunosorbent assay kit waspurchas

36、ed from Nanjing Institute of Biotechnology,China and the en-zyme-linked immunosorbent assay kit for mouse AngII and estradiolwas purchased from Shanghai Yuanye Technology Co.,Ltd.,China(Fig.1).2.3.Experimental protocolThe 117 male C57BL/6 mice were randomly divided into 13 groups:(1)saline group(2)0

37、.5 mg/kg ICI182780 group(3)5mg/kg ACEIgroup(4)0.1mg/kg DEHP Group(5)1mg/kg DEHP group(6)10mg/kg DEHP group(7)10mg/kg DEHP+ICI182780 group(8)10mg/kgDEHP+ACEI group(9)0.1mg/kg DBP group(10)1mg/kg DBP group(11)10mg/kg DBP group(12)10mg/kg DBP+ICI182780 group(13)10mg/kg DBP+ACEI group.The mice were weig

38、hed every morning,and given,by intragastric(ig)administration,different concentrationsof DEHP and DBP according to the weight of the mice,and according totheir group protocol.The saline group received physiological saline viaig administration.In the afternoon,the mice in groups(2),(7),and(12)were in

39、traperitoneally(ip)injected with a dose of 0.5 mg/kg/d of theblocker ICI182780,while the mice in groups(3),(8),and(13)wereintraperitoneally injected with a dose of 5mg/kg/d of the blocker,Enalapril Maleate(ACEI).Throughout the experimental period,themice were able to eat and drink normally.Blood pre

40、ssure meters(BP-2010 Series,Softron)were used to measure diastolic blood pressure,systolic blood pressure and mean blood pressure,by tail-cuffplethys-mography on day 42.The recorded blood pressure was taken as theaverage of three,consecutive measurements.On day 46,the mice weresacrificed,and the blo

41、od,heart,kidney and aortic blood vessels of themice were collected to examine for various biological indicators.Thespecific experimental protocol is as follows:X.Xie,et al.Ecotoxicology and Environmental Safety 174(2019)7582762.4.Sampling and testingAfter the mice were anesthetized by intraperitonea

42、l injection of 1%pentobarbital,blood samples were collected by cardiac puncture,al-lowed to stand at room temperature for 30 min,and then centrifuged tocollect the upper serum,which was stored at 80C.The mice werethen sacrificed by cervical dislocation,and the blood vessels from thekidneys and aorta

43、s of the mice were collected.PBS buffer(pH 7.5)wasadded to these two tissues for thorough grinding,the resulting tissuewas then centrifuged under the corresponding conditions to collect thesupernatant.This was stored at 80C for later use.Serum NO,es-tradiol and AngII levels in the blood vessels were

44、 measured using NO,estradiol and AngII ELISA kits according to the manufacturers in-structions.2.5.Histopathological examinationOn day 46,the thoracic aortas and kidneys of the mice were col-lected from each group and fixed in 4%formalin at 25C for subsequenthistological evaluation.The collected blo

45、od vessel and kidney tissuesamples were sliced and then stained with hematoxylin and eosin(H&E).All sections were examined and photographed using a digital in-verted microscope(Leica DM 4000B,Germany).2.6.Immunohistochemical analysis of ACE,AT1R,eNOSThe thoracic aortic vessels of the mice were colle

46、cted and fixedovernight in 4%paraformaldehyde.Sections(3 m thickness)were cutand mounted on glass slides.Immunohistochemical analyses of ACE,AT1R and eNOS were performed by using the following monoclonalantibodies:anti-ACE(1:50,Bioss,Beijing,China);anti-AT1R(1:50,Proteintech Group,Inc.,Chicago,USA);

47、anti-eNOS(1:50,ProteintechGroup,Inc,Chicago,USA).These were performed as previously de-scribed(Duan et al.,2018b).The average optical density of images forACE,AT1R,and eNOS in the thoracic aortic vessel samples was ob-tained by using Image-Pro Plus software(Image-Pro Plus 6.0,MediaCybernetics).Avera

48、ge optical density values were used to statisticallyanalyze the expression score or activation score.2.7.Statistical analysisThe data obtained from the experiment were statistically analyzedwith SPSS 13.0 software,and plotted with Origin 6.0 and GraphPadPrism 5 software.A one-way ANOVA was used to a

49、nalyze the experi-mental data,following which an LSD test was used to make a pairwisecomparison.p 0.05,p 0.01 indicate a significant difference andan extremely significant difference,respectively.3.Results3.1.Effects of DEHP and DBP exposure on the blood pressure of miceAfter 42 days of exposure to

50、the various concentrations of DEHP orDBP,the blood pressure of each mouse was taken.The results areshown in Fig.2.Compared with the saline group,the diastolic bloodpressure,mean blood pressure and systolic blood pressure of the miceall increased with increasing DEHP concentrations.It can be seen tha