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1、Contents lists available at ScienceDirectFood and Chemical Toxicologyjournal homepage: phthalate aggravates the formaldehyde-exposure-inducedlearning and memory impairment in miceShuzhen Gea,1,Biao Yana,1,Jiawei Huangb,Yingying Chena,Mingqing Chenb,Xu Yangb,Yang Wua,Dingwen Shena,Ping Maa,aLaborator
2、y of Environment-immunological and neurological Diseases,Research Center of Basic Medical Sciences,Hubei University of Science and Technology,Xianning,437100,ChinabLaboratory of Environment Biomedicine,Central China Normal University,Wuhan,430079,ChinaA R T I C L E I N F OKeywords:Diisodecyl phthala
3、teFormaldehydeLearning and memoryOxidative stress17-estradiolVitamin EA B S T R A C TDiisodecyl phthalate(DIDP)is a new type of phthalate used in the coating of pharmaceutical pills and in plasticfood wrappers.This research was conducted to investigate whether DIDP could cause learning and memoryimp
4、airment in mice,using formaldehyde(FA)to construct a positive control.Behavioral analysis showed thatoral administration of 15 mgkg1d1DIDP combined with inhalation of 1mgm3FA led to learning andmemory impairment in mice.Histopathological observations of the brain showed that the pathological altera-
5、tions in the hippocampi.Detection of testosterone(T)and estradiol(E2)levels in the brain and serum showedthat E2 levels were associated with learning and memory disorders.Reactive oxygen species(ROS),reducedglutathione(GSH),malondialdehyde(MDA),and 8-hydroxy-2-deoxyguanosine(8-OH-dG)revealed the in-
6、creased oxidative stress levels.Detection of caspase-3,NF-B,the phosphorylated cAMP response-elementbinding protein(p-CREB)and the brain derived neurotrophic factor(BDNF)showed that the protective effectmediated by BDNF,is reduced.However,some of these effects were blocked by the administration of V
7、itmin E(VitE,100 mgkg1d1)or 17-estradiol(17-E2,100 gkg1).These data suggest that DIDP may aggravatethe FA-exposure-induced learning and memory impairment in mice,and that 17-E2 could be utilized to avoidthese adverse effects.1.IntroductionPhthalates(PAEs)are the most widely used artificial plasticiz
8、ers,while diisodecyl phthalate(DIDP)is a new type of PAE used in theproduction of plastic to increase flexibility,and is even used in thecoatings of pharmaceutical pills and in plastic food wrappers(Ejaredaret al.,2015).There has been recent concern in both the USA and theEuropean Union regarding DI
9、DP toxicity and bioaccumulation.TheEuropean Union has set a maximum specific migration limit for the sumof DIDP and diisononyl phthalate(DINP)from food contact materials of9mgkg1food(Cao,2010).Plasticizers,including DIDP,were de-tected over the limit for the first time in food products in Taiwan in2
10、011(Yang et al.,2013).Subsequently,the Department of Health of theHKSARGovernmentfoundthattheplasticizercomponentinGlaxoSmithKlines antibiotic“Antibacterial”(Augmentin)made inFrance,is twice the specified upper limit for plasticizers in food contactmaterials in Europe.The toxicity of DIDP in humans
11、is not comprehensively understoodnor entirely clear.Like other phthalates,DIDP is not covalently boundto plastics,and can therefore be easily emitted into the environmentand subsequently make its way into the human body(Halden,2010).Evidence from animal studies suggest that DIDP at dietary levels ra
12、ngedfrom 0.02 to 0.8%(or approximately 15600mgkg1d1)for 10weeks increased the liver weights and histopathologic changes(Hushkaet al.,2001;European Food Safety Authority(EFSA),2008).Severalstudies have suggested that there is a possible link between exposure tophthalates and negative behavioral effec
13、ts(Min et al.,2014;Ma et al.,2015;Tang et al.,2015).Studies have confirmed that exposure to di(2-https:/doi.org/10.1016/j.fct.2019.02.024Received 24 November 2018;Received in revised form 10 February 2019;Accepted 11 February 2019Corresponding author.Hubei University of Science and Technology,No.88
14、Xianning Avenue,Xianning,437100,China.Corresponding author.Hubei University of Science and Technology,No.88 Xianning Avenue,Xianning,437100,China.E-mail addresses:(S.Ge),(B.Yan),(J.Huang),(Y.Chen),(M.Chen),(X.Yang),(Y.Wu),(D.Shen),(P.Ma).1These authors contributed equally to this work.Food and Chemi
15、cal Toxicology 126(2019)152161Available online 19 February 20190278-6915/2019 Elsevier Ltd.All rights reserved.Tethylhexyl)phthalate(DEHP)and benzyl butyl phthalate(BBP)canimpair the learning and memory abilities of mice(Min et al.,2014;Tang et al.,2015),and that DINP can induce cognitive deficits a
16、ndanxiety in mice(Ma et al.,2015).It is not known whether DIDP canhave these same effects.Formaldehyde(FA)is the simplest of the aldehydes found in nature,and has become the main pollutant in indoor air.It is harmful to humanhealth and is known to be carcinogenic.In view of its widespread use,toxici
17、ty,and volatility,FA has been implicated as being an importantagent responsible for the development of neurocognitive disorders.Increasing evidence shows that prolonged exposure to FA is sig-nificantly associated with symptoms such as depression,memory de-cline and emotional instability(Songur et al
18、.,2010).The results fromanimal experiments have shown that FA exposure can induce learningand memory disabilities(Lu et al.,2010;Tong et al.,2013).DIDP and FA are widely present in the general environment.It ispossible for humans to ingest DIDP(the main route is oral)and also tobe exposed to FA(the
19、primary route is inhalation).Children are usuallythe most likely to be exposed to DIDP and FA(Le et al.,2011),and areat greater risk from exposure to these chemicals than adults,since theirbrains are still in the development stage,and their central nervoussystems are more vulnerable to toxic chemica
20、ls(Weiss,2000).Although the estrogenic effect of DIDP itself is not as strong as thatof DEHP and DINP,it is still possibly enter the brain through the blood-brain barrier because of its fat-solubility,and affect the estrogen levels(Joensen et al.,2012).Estradiol(E2)is the primary and the most active
21、estrogen in the brain(Toran-Allerand,2005).An increasing amount ofdata suggest a neuroprotective role for 17-Estradiol(17-E2)(Kimet al.,2016;Zhu et al.,2017),whereby it rapidly increases the dendriticspine density of pyramidal cells in the hippocampus and consolidateshippocampal memory(Frick,2015).I
22、t is unclear whether DIDP ex-posure interferes with the E2-mediated neuroprotective effects onlearning and memory.This is a scientific question worthy of our study.In addition,the brain is another important target of oxidative stressbecause it produces reactive oxygen species(ROS)in the metabolicpro
23、cess.Indeed,both oral exposure to DIDP and inhalation of FA resultin oxidative stress responses(Ferguson et al.,2011;Shen et al.,2016;Wei et al.,2017).Tang et al.(2015)have shown that the antioxidantvitamin E(VitE)plays a protective role by down regulating oxidativestress induced by DEHP.Based on th
24、e above evidence,we hypothesized that:(1)A decreasedE2 level induced by DIDP in the brain may affect the learning andmemory abilities of mice;(2)DIDP may also cause learning andmemory impairment through oxidative stress in FA-exposed mice.Thus,DIDP may aggravate the FA-induced learning and memory di
25、sabilitiesin mice by both oxidative stress and abnormal E2 levels in the brain(Graphical abstract).In this study 3-week-old Kunming(KM)mice were exposed to gas-eous FA to build a model of learning and memory impairment.We usedthe model mice as the positive control,and these were also orally ex-posed
26、 to DIDP at the same time for 3 weeks with pathway blocker 17-E2 or VitE as a protective agent,to explore the oxidative damage to thebrain caused by combined exposure to FA and DIDP,and whether thecombined exposure has a synergistic effect to induce more severelearning and memory impairment.2.Materi
27、als and methods2.1.Animal careSpecificpathogen-freemaleKMmice(3-week-old,15 2gweight)were purchased from the Experimental Animal Center of HubeiProvince(Wuhan,China)and housed under standard laboratory con-ditions(temperature,2025C;humidity,5070%;and 12h day/nightcycles).The mice were given a standa
28、rd chow diet and water ad li-bitum,except during exposure periods.All the mice spent one week inthis environment before initiation of the study.All animal experimentswere conducted in accordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals,and were approvedb
29、y the Office of Scientific Research Management of Hubei University ofScience and Technology(Xianning,China)with a Certificate approvalID:HBUST-IACUC-2018-001.2.2.Reagents and kitsDIDP(99%,CAS:26761-40-0),FA(10%,CAS:50-00-0),17-E2(98%,CAS:79037-37-9),Vit E(99%,CAS:59-02-9),2,7-di-chlorodihydrofluores
30、cein(DCFH-DA),and 2-thiobarbituric acid(TBA)were purchased from Sigma-Aldrich(St.Louis,MO,USA).Tween 80(CAS:9005-65-6)was obtained from Amresco(Solon,OH,USA).Themouse kit for reduced glutathione(GSH)was obtained from NanjingJiancheng Bioengineering Institute(Nanjing,China).Mouse ELISA kitsfor 8-hydr
31、oxy deoxy guanosine(8-OH-dG),cysteine-aspartic acid pro-tease 3(caspase-3),nuclear factor B(NF-B),phosphorylated cAMPresponse element binding protein(p-CREB),brain derived neurotrophicfactor(BDNF)were obtained from eBioscience(San Diego,CA,USA).MousekitsforTandE2wereobtainedfromBeijingNorthBiotechno
32、logy Institute(Beijing,China).All other chemicals were of thehighest grade available commercially,or as indicated.2.3.Experimental protocolNinety KM mice were randomly divided into nine groups:(A)ne-gative control(saline)group;(B)0.15 mgkg1d1DIDP group;(C)1.5 mgkg1d1DIDP group;(D)15mgkg1d1DIDP group
33、;(E)150mgkg1d1DIDP group;(F)1mgm3FA group;(G)1mgm3FA+15 mg kg1d1DIDP group;(H)1mgm3FA+15 mg kg1d1DIDP+100mgkg1d1VitEgroup,(I)1mgm3FA+15 mg kg1d1DIDP+100 g kg1d117-E2 group.The micereceived repeated exposure over a period of 3 weeks.The water mazeexperiment started on the 13th day of exposure as show
34、n in Fig.1A.The brain tissue of each mouse was taken for tissue sectioning and thepreparation of an homogenate.The homogenate was then used to de-termine the levels of ROS,GSH,malondialdehyde(MDA),8-OH-dG,caspase-3,NF-B,p-CREB,and BDNF.2.4.Chemical exposureAccording to the official recommendation of
35、 EFSA(2008),the dailyintake limit of DIDP for normal adults is 0.15 mgkg1d1.Based onthis,we chose DIDP concentrations of 0.15,1.5,15,150mgkg1d1for our experiment.DIDP and Tween 80 were mixed according to thevolume ratio of 1:1.The corresponding groups were given a daily ga-vage according to body wei
36、ght of 0.01 mLg1for 21 days.The con-centration of VitE administered was chosen to be 100 mgkg1ac-cording to Yousef et al.(2006).VitE and Tween 80 were mixed to a1:1 vol ratio.The solution was prepared with sterilized normal salineand administered by daily gavage at the rate of 0.01 mLg1.Theconcentra
37、tion of 17-E2 was set to 100 g kg1according to the ex-periment of Zhang et al.(2012),in which Zhang et al.(2012)providedthe evidence that 17-E2 protects against neuronal apoptosis.17-E2was dissolved in DMSO with a concentration of 272g/100 L.0.01 mLg1body weight was administered three times a week.T
38、heconcentration of Tween 80 used in our experiments has been shown inprevious pharmacological experiments in vivo to be inert(Wu et al.,2017)and has no toxic side effects on the organism,therefore the ne-gative control group was given only saline.According to previous studies by Wei et al.(2017),1mg
39、 m3FA cancause a decline in the learning and memory abilities in mice.On thisbasis,the concentration of FA was set to be 1mgm3.The mice wereplaced in a glass tank connected to a small intelligent environmentalclimate chamber(transformed from an 8.4L aerated dryer)thatS.Ge,et al.Food and Chemical Tox
40、icology 126(2019)152161153continuously and steadily produced gaseous FA with a concentration of1mgm3.The humidity in the tank was 45%5%and the air flowrate was 2L min1.According to occupational work times(work for 5days,8h a day and rest for 2 days per week),mice in model groupswere exposed to gaseo
41、us FA for 5 days per week for 8h per day,andlasted 3 weeks.Neither food nor water was provided during the periodof FA inhalation.DIDP and VitE were administered via gavage at a regular time everyday,after which all the mice in the(F),(G),(H)and(I)groups wereexposed to FA.The(A)(E)groups were subject
42、ed to the water mazetest after gavage,while the(F),(G),(H)and(I)groups were subjectedto the water maze test after FA exposure.The experimental schedule isshown in Fig.1B.2.5.Morris water maze(MWM)MWM is mainly used in behavioral neuroscience to study spatiallearning and memory abilities of test rode
43、nts.The experimental deviceis shown in Fig.2A.The circular pool is divided into four quadrants I,II,III and IV.A black circular escape platform is placed at a fixed position1cm below the water surface.The water was kept at a temperature of24 2C.The training included releasing each mouse facing the p
44、ool wallfrom a fixed position in the I,II,and III quadrants,and allow them tofind the fixed escape platform.A camera connected to a computer,tracks the swimming mouse from the time it enters the water for 60s.If,within the 60s tracking period,the mouse locates the platform,thetime to find the platfo
45、rm is called the escape latency.If the platformcould not be found within 60s,the escape latency is recorded as 60s,the experimenter then guides the mouse to the escape platform where itremains for 15 s.This training is given daily over a 7-day trainingperiod,and is known as the navigation test,and i
46、s used to evaluatemouse learning abilities.On the 8th day of the MWM experiment,the mice were kept awayfrom the water maze,giving them time to forget the location of theplatform.On the 9th day,the platform was removed from the pool forthe spatial probe test.The mice were put into the pool in same ma
47、nneras before,to test their memory.The camera device recorded theswimming trajectories of the mice,and the amount of swimming timespent in the target quadrant.The experimental data were analyzedusing AnyMaze software.2.6.Histological preparation of brain tissueOn the 22nd day,the mice were sacrifice
48、d by cervical dislocation.The brains were removed and fixed in 4%paraformaldehyde for 24h.After routinely embedded in paraffin,serial sections of samples weremade vertically from central region by using rotary microtome(RM2245,Leica,Germany)at 10m thickness.Sections were attachedonto the glass slide
49、 on its exact position and dried in the 42C slidewarmer for 6h,and then deparaffinized by xylene.And they werestained by hematoxylin-eosin and Nissl separately.The stained hippo-campal regions were examined under a light microscope(BX51,Olympus,Japan).2.7.Tissue sample preparationThe collected brains were weighed in a fully automatic electronicbalance.The tissue was placed in 10mLg1ice-cold 1phosphate-buffered saline(PBS,pH=7.5)and homogenized using a glass homo-genizer.Homogenate was centrifuged at 9800 g for 10min at 4C.Supernatants were collected and frozen at 70C until needed.Theprotein