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1、Contents lists available at ScienceDirectEcotoxicology and Environmental Safetyjournal homepage: E reduces the extent of mouse brain damage induced by combinedexposure to formaldehyde and PM2.5Xudong Liua,1,Yuchao Zhanga,1,Xu Yanga,b,aDepartment of Food Science and Engineering,Moutai Institute,Renhu
2、ai 564507,ChinabLaboratory of Environmental Biomedicine,Hubei Key Laboratory of Genetic Regulation and Integrative Biology,College of Life Science,Central China Normal University,Wuhan 430079,ChinaA R T I C L E I N F OKeywords:Vitamin EProtective effectsBrain damageCo-exposureA B S T R A C TExposure
3、 to specific air pollutants has been demonstrated to induce damage in the brain.However,these studiesignore the effects of a combination of contaminants,and there is a high likelihood that people will be exposed toa mixture of contaminants in daily life.Our previous study showed that co-exposure to
4、formaldehyde(FA)andPM2.5 induced damage in the mouse brain at the safe exposure level for FA or PM2.5 exposure alone,and thatoxidative stress and inflammation may be involved in the toxicity mechanisms.A universal strategy to protectpeople exposed to FA and PM2.5 is urgently needed.To explore whethe
5、r an exogenous substance could coun-teract the negative effects of exposure to these pollutants,we administered vitamin E(Vit E)to the experimentalanimals.The results showed that administration of Vit E in tandem with the FA and PM2.5 co-exposure,reducedthe extent of damage to the mouse brain.Down-r
6、egulation of oxidative stress and inflammation were proposedto explain the protective effects of Vit E.This research provides a universal strategy to effectively protect peoplewho are exposed to FA and PM2.5 simultaneously.1.IntroductionEnvironmental pollutants are substances that break down thenorm
7、al composition of the environment and cause damage directly orindirectly to people after entering the environment(Brannan andDiane,2012).They are mainly chemical substances produced as a resultof human activity,but also include materials released naturally(Wjtowicz,2015).Exposure to environmental to
8、xins can be thought ofas a multi-pollutant co-exposure system.In the case of air pollution,people are frequently exposed to PM2.5 and FA simultaneously.Air pollution is a multifaceted environmental pollutant,comprisingparticulate matter,gases,organic compounds and metals,and is presentin indoor and
9、outdoor air(Block and Caldern-Garcidueas,2009).It iswell-established that some health problems,such as asthma,heartdisease,and lung cancer are induced by air pollutants(Thomson et al.,2015;Brook and Rajagopalan,2007;Brunekreef and Holgate,2002).There is increasing evidence suggesting that air pollut
10、ion not onlyharms the heart and lungs,but may also result in brain damage(Underwood,2017).In our previous study,we showed that co-exposureto formaldehyde(FA)and PM2.5 induced AD-like changes in the mousebrain(Liu et al.,2017).According to the“Hygienic standard for formaldehyde in the indoorair of a
11、house(GB/T 16127-1995)”in China,the upper limit for anacceptable concentration of FA in indoor air is 0.08mg/m3.And ac-cording to the“Report on the State of the Environment in China”published by the Ministry of Environmental Protection(China),theactual PM2.5 daily exposure level is higher than the n
12、ational hygienicstandard,and can reach 100g/m3both indoors and outdoors.Peopletend to assume that when we are exposed to FA and PM2.5 at levels ator below the standard,that there will be zero or only minor adverseeffects.However,we ignore the fact that exposure to environmentaltoxins is a multi-poll
13、utant co-exposure system.In our previous study,we converted the standard exposure levels forhumans mentioned above to mice,which can result in equivalent effectson humans for FA and PM2.5 co-exposure.The results from that studysuggested that exposure to FA or PM2.5 alone at those concentrationshttps
14、:/doi.org/10.1016/j.ecoenv.2019.01.048Received 24 November 2018;Received in revised form 8 January 2019;Accepted 10 January 2019Abbreviations:FA,formaldehyde;AD,Alzheimers disease;Vit E,vitamin E;MWM,Morris water maze;EL,escape latency;H&E,hematoxylin and eosin;OD,averageoptical density;A142,beta-am
15、yloid plaques 142;Tau-P,hyper-phosphorylated tau;HIS,Immunohistochemistry;IF,Immunofluorescence;ROS,reactive oxygenspecies;GSH,glutathione;SOD,superoxide dismutase;COX-2,Cyclooxygenase 2Corresponding author at:Department of Food Science and Engineering,Moutai Institute,Renhuai 564507,China.E-mail ad
16、dress:(X.Yang).1These authors have contributed equally to this work.Ecotoxicology and Environmental Safety 172(2019)33390147-6513/2019 The Author(s).Published by Elsevier Inc.This is an open access article under the CC BY-NC-ND license(http:/creativecommons.org/licenses/BY-NC-ND/4.0/).Tresulted in l
17、ittle or no damage to the mouse brain.But when we ex-posed the mice to a combination of contaminants(FA+PM2.5)at thesame levels,some damage such as cognitive deficits,pathological brainalterations,A142and Tau-P accumulation,glia activation,oxidativestress and inflammation became evident(Liu et al.,2
18、017).These safelevels for single pollutant exposure were no longer safe when animalswere co-exposed to FA and PM2.5.Since it is very likely that people willbe exposed to a mixture of contaminants in the course of a normal day,especially indoors(Liu et al.,2017),a universal strategy is needed toeffec
19、tively protect people who are simultaneously exposed to FA andPM2.5.This study is an extension of our previous work.To address theproblem highlighted above,we chose Vit E as the agent to protect themouse brain against damage induced by co-exposure to pollutants.After a week of daily exposure accordi
20、ng to the experimental protocol,we investigated cognitive ability and brain damage,and looked at someupstream biological events.2.Materials and methodsAll experimental procedures were approved by the Office ofScientific Research Management of Central China Normal University(Wuhan,China),and animal e
21、xperiments were conducted under theNational Institutes of Health Guide for the Care and Use of LaboratoryAnimals,with a certificate of Application for the Use of Animals dated24 January 2016(approval ID:CCNU-IACUC-2016-003).2.1.AnimalsSPF male C57BL/6 mice(6 weeks old,22g)were supplied by theHubei P
22、rovince Experimental Animal Center(Wuhan,China).All micewere kept in pathogen-free cages at 2025 with 5070%humidityand a 12h light/dark cycle.They were provided with a commercial dietand filtered water ad libitum.2.2.Collection and component analysis of PM2.5A high traffic,total suspended particulat
23、es sampler(KC-1000,Qingdao,China)was used to collect ambient air composed ofPM 2.5m in Wuhan,China.The collected PM2.5 glass filter mem-branes were cut into small pieces,processed ultrasonically in ultrapurewater for 40min.The extracted liquid was vacuum freeze dried andcryogenically preserved.The e
24、lements in the samples were analyzedusing inductively coupled plasma atomic emission spectroscopy(ICP-AES,61E Trace and ICP-750,Thermo Jarrell Ash,MA)after acid di-gestion with a mixture of acids(68%nitric,38%hydrofluoric,and 70%perchloric=5:1:1).The analysis was performed at 180C for 3h.Thewater-so
25、luble components in the samples were analyzed using an ionchromatograph(DX-100,Dionex,Sunnyvale,CA)and ICP-AES(61ETrace,Thermo Jarrell-Ash).Polycyclic Aromatic Hydrocarbons(PAHs)were analyzed according to a previously reported method(He et al.,2016).The data are provided in Supplementary data.2.3.Ex
26、perimental protocolMice were randomly assigned to 3 groups of 9 animals:(1)control;(2)exposed to 0.155 mg/kg/day FA+0.193 mg/kg/day PM2.5(Liuet al.,2017);(3)exposed to 0.155 mg/kg/day FA+0.193 mg/kg/dayPM2.5+50 mg/kg/day VitE(Li et al.,2014).The mice were exposed tothe mixture of contaminants by int
27、ranasal instillation and to Vit E byintragastric administration(Li et al.,2014)daily,for 7 days.Followingthe exposure period,we investigated some pathological hallmarks ofbrain damage.To explore the possible mechanisms behind the resultingdamage,we examined some key upstream events.2.4.Morris water
28、maze(MWM)The MWM test was performed to measure the cognitive ability ofmice as described in our previous study(Liu et al.,2017).2.5.Histological examinationBrain samples were collected and sectioned into 10m slices forhematoxylin and eosin(H&E).These were examined,and cell numberscounted according t
29、o procedures described in our previous study(Liuet al.,2017).2.6.Immunohistochemistry(HIS)and immunofluorescence(IF)for A142,Tau-P,Iba1,GFAPBrain sections were incubated with diluted primary antibodies,andthen examined through HIS and IF as in our previous study(Liu et al.,2017).2.7.Assessment of re
30、active oxygen species(ROS),glutathione(GSH),superoxide dismutase(SOD)activity and Cyclooxygenase 2(COX-2)contentMouse brains were collected to make into sample homogenate,andthis was used to measure the ROS level,GSH content,SOD activity andCOX-2 content as described in our previous study(Liu et al.
31、,2017).2.8.Statistical analysesRepeated ANOVAs combined with a post-hoc Tukey test were usedto analyze the MWM data.All other data collected were analyzed by aone-way ANOVA followed by a Tukey test.Values of p 0.05 wereconsidered statistically significant.Data analyses were carried out usingSPSS 13.
32、0(Chicago,IL,USA)and statistical graphs were generatedusing GraphPad Prism 5.0(San Diego,CA,USA).3.Results3.1.Cognitive deficits in mice after exposureAfter the MWM swimming training,the average EL over5 days forthe co-exposure groups was higher than for the control group(Figs.1A,1B).On the seventh
33、day,the time that the mice stayed in the platformquadrant(SE quadrant)was remarkably short for the mice that wereexposed to FA and PM2.5 only(Fig.1C).The mice that had Vit E ad-ministered spent less time finding the escape platform,and spent moretime in the SE quadrant than did the pollutant-only gr
34、oup.3.2.Brain histological changes in mice after exposureCompared with the control group,after co-exposure the pyramidalcells in the CA1region of the hippocampus became loose and dis-ordered,and the cell shapes became swollen and deformed(Fig.2A).Inaddition,the circular structure of the synaptic glo
35、merulus in the ol-factory bulb was destroyed(Fig.2B).The Vit E group also sustained celldamage,however this damage was less than that seen in the co-ex-posure group,with most pyramidal cells remaining intact.Fig.2C andFig.2D show that although co-exposure did not induce structural da-mage in the cer
36、ebral and prefrontal cortex,the cell numbers were re-duced.The group receiving Vit E protection had a significantly highercell number in the cerebral and prefrontal cortex(Table 1)than the co-exposure group.3.3.Protein aggregates in the mouse brain after exposureFig.3 shows the protein aggregates pr
37、esent in the cerebral cortex ofX.Liu et al.Ecotoxicology and Environmental Safety 172(2019)333934mice,as well as the neuronal accumulation of A142and Tau-P afterco-exposure.The number of cells positive with A142and Tau-P in-creased,and increases in average optical density(OD)were detected inthe co-e
38、xposure mice(Table 1).When mice were treated with Vit E,theexpression of A142and Tau-P in brain tissues was reduced in com-parison with the co-exposure group.Fig.1.MWM test after exposure.(A)The EL for the first 5 days of training.(B)The average EL for the first 5 days of training.(C)The time spent
39、swimming in the SEquadrant on the seventh day.*p 0.05,*p 0.01,compared with the control group.#p 0.05,#p 0.01,compared with the co-exposure group.Fig.2.Histological observation of the mouse brain after exposure.(A)H&E staining for the hippocampus.(B)H&E staining for the olfactory bulb.(C)H&E stainin
40、gfor the cerebral cortex.(D)H&E staining for the prefrontal cortex.X.Liu et al.Ecotoxicology and Environmental Safety 172(2019)333935Table 1Cell numbers in the cerebral cortex,prefrontal cortex and the OD of HIS,IF.Group(mg/kg/day)Cerebral cortexPrefrontal cortexCell numberCell numberControl163.17 3
41、6.65162.33 26.34FA+PM2.5119 19.9*118.33 17.48*FA+PM2.5+Vit E152.8 17*142.83 17.03*Group(mg/kg/day)A142Tau-POD for HISOD for IFOD for HISOD for IFControl0.0018 1.520E40.0018 1.812E40.0060 0.00120.0034 7.541E4FA+PM2.50.0190 0.0021*0.0307 0.004*0.0080 9.368E4*0.0046 7.470E4*FA+PM2.5+Vit E0.0159 0.0019*
42、,*0.0156 0.0017*,*0.0069 2.157*0.0037 4.720 E4*Group(mg/kg/day)Iba1GFAPOD for HISOD for IFOD for HISOD for IFControl0.0010 4.001E40.0043 8.380E40.0502 0.01070.0090 0.0037FA+PM2.50.0108 0.0048*0.0317 0.0059*0.0633 0.0054*0.0219 0.0062*FA+PM2.5+Vit E0.0056 5.631 E4*,*0.0198 0.0070*,*0.0474 0.0049*0.01
43、50 0.0012*,*p 0.05,compared with the control group.*p 0.01,compared with the control group.*p 0.05,compared with the FA+PM2.5 group.*p 0.01,compared with the FA+PM2.5 group.Fig.3.Representative images of the expression of A142(A)and Tau-P(B).X.Liu et al.Ecotoxicology and Environmental Safety 172(201
44、9)3339363.4.Glia activation in the mouse brain after exposureAs Fig.4A shows,after co-exposure,Iba1-positive cell size in-creased,neurites retracted and coarsened,and some spines were foundon the neurites.In Fig.4B,astrocyte activation can also be observed.This activation is characterized by increas
45、ed GFAP-positive cell bodyvolume,and branches becoming more ramified.Significant changes inIba1 and GFAP levels were observed in the mice after co-exposure(Table 1).However,administration of Vit E resulted in a decrease in theexpression of Iba1 and GFAP.3.5.Effect of exposure on oxidative stress and
46、 inflammation levels in themouse brainThe protective effects of Vit E on oxidative stress and inflammationlevels are shown in Fig.5.According to the results,a significant in-crease in ROS and COX-2 levels,and a decrease in CSH content and SODactivity were observed in mice after co-exposure.For the m
47、ice receivingVit E,the GSH levels and SOD activity were significantly higher than forthe mice in the co-exposure group,and the ROS and COX-2 levels weresignificantly lower than the co-exposure group.4.DiscussionEpidemiological studies have shown that there is a link betweenexposure to air pollutants
48、 and brain damage,however,toxicologicalstudies have not fully confirmed this link,particularly the effects re-sulting from exposure to a mixture of contaminants.FA and PM2.5 arecommon indoor and outdoor pollutants to which people are frequentlysimultaneously exposed.FA is generally found in higher c
49、oncentrationsindoors,being widely used in furnishings,while PM2.5 generally entersbuildings by infiltration and ventilation.Indoor sources of PM2.5 willincrease the concentration of indoor PM2.5 such that it will tend to bethe same as outdoor levels,and in some cases,may be even higher.Since people
50、typically spend more than 90%of their time indoors,co-exposure to PM2.5 and FA is highly likely in the indoor environment(Marques and Pitarma,2016).Our previous study suggested that one week of co-exposure to FAand PM2.5 resulted in mouse brain damage through oxidative stressand inflammation.Accordi