Research Article Autism-Like Behavior and Epigenetic ...

Research ArticleAutism-Like Behavior and Epigenetic Changes Associated withAutism as Consequences of In Utero Exposure to EnvironmentalPollutants in a Mouse Model

Denise S Hill1 Robert Cabrera2 Deeann Wallis Schultz3 Huiping Zhu1 Wei Lu1

Richard H Finnell2 and Bogdan J Wlodarczyk2

1Center for Environmental and Genetic Medicine Institute of Biosciences and TechnologyTexas AampM University System Health Science Center Houston TX 77030 USA2Department of Nutritional Sciences The University of Texas at Austin Austin TX 78723 USA3Department of Biochemistry amp Biophysics Texas AampM University College Station TX 77843 USA

Correspondence should be addressed to Bogdan J Wlodarczyk bwlodarczykaustinutexasedu

Received 10 June 2015 Revised 1 October 2015 Accepted 1 October 2015

Academic Editor Joao Quevedo

Copyright copy 2015 Denise S Hill et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We tested the hypothesis that in utero exposure to heavy metals increases autism-like behavioral phenotypes in adult animals andinduces epigenetic changes in genes that have roles in the etiology of autismMouse damswere treatedwith cadmium lead arsenatemanganese and mercury via drinking water from gestational days (E) 1ndash10 Valproic acid (VPA) injected intraperitoneally once on(E) 85 served as a positive control Young male offspring were tested for behavioral deficits using four standardized behavioralassays In this study in utero exposure to heavy metals resulted in multiple behavioral abnormalities that persisted into adulthoodVPA and manganese induced changes in perseverativeimpulsive behavior and social dominance behavior arsenic caused changesonly in perseverativeimpulsive behavior and lead induced abnormalities in social interaction in comparison to the control animalsBrain samples fromMn Pb andVPA treated and control animalswere evaluated for changes inCpG islandmethylation in promoterregions and associated changes in gene expression The Chd7 gene essential for neural crest cell migration and patterning wasfound to be hypomethylated in each experimental animal tested compared to water-treated controls Furthermore distinct patternsof CpG island methylation yielded novel candidate genes for further investigation

1 Introduction

Autism spectrum disorder (ASD) commonly referred toas autism is diagnosed in 1 of every 68 children in theUnited States and is characterized by a range of com-plex neurodevelopment disorders Diagnostic and StatisticalManual of Mental Disorders (DSM-5) define ASD as aneurobehavioral disorder manifested by persistent deficits insocial and communication interaction deficits in developingunderstanding andmaintaining relationships and abnormaland fixed interests and repetitive behavior [1] Accumulatingevidence suggests that epigenetic factors play a strong role inthe etiology ofASD [2ndash6] Epigenetic disruption of regulatoryCpG islands has also been associated with autism For exam-ple a decrease in expression of the methyl binding protein

gene MECP2 was observed in the brain tissue of autisticpatients Downregulation of MeCP2 was also demonstratedin valproate animal model of autism [7] This gene functionsas a transcriptional repressor at specific gene promoterscrucial to brain development [8 9]

Valproic acid (VPA) is a commonly used antiepilepticdrug In utero exposure to VPA increases the risk for neuraltube defects (NTDs) in humans [10 11] and laboratoryrodents [12] VPA is also known to cause behaviors con-sistent with some aspects of autism and is employed asan established research model for inducing autism [13 14]Years of preliminary data suggest that there is a genetic basisunderlying VPA-sensitivity to the induction of neural deficitsin laboratory animals [15ndash17] VPA is also known to affect theepigenome as well as blood levels of folate and homocysteine

Hindawi Publishing CorporationBehavioural NeurologyVolume 2015 Article ID 426263 10 pageshttpdxdoiorg1011552015426263

2 Behavioural Neurology

both of which have the potential to dysregulate criticaldevelopmental genes that can result in adverse pregnancyoutcomes [18 19] A growing body of literature indicatesthat similar disruption is observed in some autistic patientsboth disrupted folate and homocysteine status [20ndash22] andepigenomic disruption of regulatory CpG islands have beenassociated with autism [23] Furthermore evidence suggeststhat exposure to environmental neurotoxins is a risk factorfor autism [24]

Industrial chemicals are being produced and released intothe environment at a staggering rate The average consumeris exposed to up to 10000 different chemicals every dayout of a world market that includes over 30000 industrialchemicals sold at quantities greater than 400 million tonsper year [25] These chemicals range in toxicity from beingbenign to being extremely hazardous and the environmentaland public health implications of this overwhelming andpervasive exposure have not been well characterized for thevast majority of chemicals Among the hazardous toxicantsheavy metals known neurotoxicants continue to be of agreat concern due to their increasing anthropogenic presencein the environment [26] Several epidemiological studiesreported a relationship between autism and heavy metalbiomarkers [27 28] Exposure to industrial chemicals hasbeen thought to play a role in the etiology of autism throughepigenetic mechanismsThis is based on evidence suggestingthe plausibility of a role for mercury in the etiology of autism[29 30] and mercury in maternal peripheral and cord bloodhas been shown to affect fetal epigenetic status [31] A handfulof reports also points out to other heavy metals neurotoxicityand epigenetic mechanism [32 33]

The experiments presented in this paper were designedto identify abnormal neurobehavioral phenotypes resultingfrom in utero exposure to several identified environmentalpollutants We hypothesized that in utero exposure to thesetoxicants results in epigenetic changes in key developmentalgenes affecting pathways that have direct roles in the etiologyof autism Collectively this work provides an assessment ofthe effect of in utero exposure to selected heavy metals onthe epigenome and associated autistic-like phenotypes in theoffspring that persist as adult behaviors

2 Materials and Methods

21 General Study Design C57BL6J mice were exposed inutero to selected toxicants and evaluated for their adultbehavioral phenotype VPA was used as a positive controlbecause it is a widely used mitochondriotoxic medicationthat induces a spectrum of teratogenic effects includingbehavioral disruption and is used as a rodent model forstudying autistic behavioral changes [34] Environmentaltoxicants including cadmium (Cd) lead (Pb) arsenic (As)manganese (Mn) and mercury (Hg) were investigated withnormal drinking water used as a negative control

22 Animals and Housing C57B6J mice were housed inthe Institute of Biosciences and Technology Vivarium whichis fully accredited by the Association for Assessment andAccreditation of Laboratory Animal Care The animals were

maintained in clear polycarbonate microisolator cages andwere allowed free access to food and water (Harlan TekladRodent Diet 8606 Ralston Purina St Louis MO) Themice were maintained on a 12 h lightdark cycle Nulligravidfemales 50ndash70 days of age were mated overnight with malesand examined for the presence of vaginal plugs the followingmorning and the onset of gestation was considered to be10 pm of the previous night the midpoint of the dark cycle

23 Treatment with Select Compounds Presumed graviddams C57BL6J were treated with one of seven agents drink-ingwater served as the negative control andVPA (CAS 1069-66-5 valproic acid sodium salt ge98 Sigma-Aldrich Chem-icals St Louis MO) administered at 600mgkg by intraperi-toneal injection on gestational day 85 only served as thepositive control Five groups were treated from the first dayof pregnancy through day 105 the end of neurulation whichgenerally occurs from E80ndash105 via treated drinking watercontaining 10 ppmCd as cadmium chloride (CAS 10108-64-2 9999 trace metals basis Sigma-Aldrich Chemicals StLouis MO) 300 ppm Pb as lead acetate (CAS 6080-56-4lead(II) acetate trihydrate 99999 tracemetals basis Sigma-Aldrich Chemicals St Louis MO) 05 ppm As as sodiumarsenate (CAS 10048-95-0 sodium arsenate heptahydrateACS Reagent ge98 Sigma-Aldrich Chemicals St LouisMO) 10 ppm Mn as manganese chloride (CAS 13446-34-9 manganese(II) chloride tetrahydrate 9999 trace metalsbasis Sigma-Aldrich Chemicals St Louis MO) or 20 ppmHg as mercury chloride (CAS 7487-94-7 mercury(II) chlo-ride ACS reagent ge999 Sigma-Aldrich Chemicals StLouis MO) Treated dams maintained in separate cages wereallowed to give birth Only animals from litters consisting of6ndash8 pups were admitted for further testing All experimentalmice were weaned at three weeks of age and maintained inseparate cages Because of the higher incidence of autismreported in male children as compared to female childrenmalemice were used exclusively in these studies Twelvemiceper group that is seventy two males in total were used inthis study Animals were assigned randomly to experimentalgroups and to reduce any litter effect only one male from agiven litter was assigned to the same treatment group (nolittermates in the same group)

24 Behavioral Assays and Statistical Analysis Thebehavioraltesting began when experimental animals reached twelveweeks of age and lasted until twenty weeks of age with atleast one-week break period between consecutive assays forany individual mouse All treatment groups were evaluatedwith 4 different neurobehavioral assays nest building tobegin to assess behavioral deficits marble burying for per-severativeimpulsive behaviors 3-chambered box for socialinteraction and tube test for social dominance Each assaywas performed sequentially such that the least stressful assaywas performed first and the most stressful performed lastnest building marble burying 3-chambered box for socialinteraction and tube test

241 Nest Building Upon reaching twelve weeks of agemice were tested for their ability to build nests C57 mice

Behavioural Neurology 3

are considered exceptionally robust nest builders a normalbehavior for both male and female mice Nesting has beenreported to be impaired by brain lesions including thoseto the hippocampus and medial prefrontal cortex [35]pharmaceutical intervention with selective serotonin reup-take inhibitors (SSRIs) [36] and disease (eg scrapie) [37]Nestlets (Ancare Bellmore NY) manufactured from pulpedvirgin cotton fiber and sterilized during the manufacturingprocess were routinely provided in each cage Three gramsof Nestlet was placed in each cleaned cage 1 hour prior tothe onset of the dark cycle and left undisturbed overnightNest building was assessed based on a 5-point scale 1 Nestletnot noticeably touched 2 Nestlet partially torn 3 Nestletmostly shredded but no identifiable nest 4 identifiable butflat nest and 5 a near perfect nest Data were analyzed usinga nonparametric ANOVA (Kruskal-Wallis test) with Dunnrsquosmultiple comparison test

242 Marble Burying Mice were tested for persevera-tiveimpulsive behavior using marble burying as an indexAs mice dig a normal behavior marbles are incidentallysubmerged in litter It is suggested that this is a geneticallyregulated behavior which is not related to anxiety or novelty[38 39] This repetitiveperseverative behavior has beenobserved to be disrupted by awide array of agents that includehippocampal lesions [40] NK1 antagonists [41] and manymore [42ndash44] Mice were presented with 16 marbles in a 4 times4 grid placed lightly on a gently tamped volume of aspen chipbedding 5 cm deep and allowed 30 minutes to bury marblesMarbles were considered buried if gt70 of the marblevolume was submerged The numbers of marbles buriedwere recorded and analyzed using a nonparametric ANOVA(Kruskal-Wallis test) with Dunnrsquos multiple comparisontest

243 Three-Chambered Social Testing Apparatus In thisassay we tested the normal preference of a mouse toinvestigate and spend time with a novel strange mouse asopposed to a novel objectThe three-chambered social testingapparatus was designed at NIH in order to measure variousaspects of sociability in mice [45] Three chambers are linkedby closeable gates transitions between chambers and timespent in each chamber may be recorded by a computerizedmonitoring program

The test mouse was placed in the closed central chamber(2) of the apparatus and allowed to acclimate for tenminutes Next a novel strange mouse was placed under aninverted wire cup in chambers 1 or 3 with a duplicatecup placed in the remaining chamber the gates between thechambers were raised and the behavior of the mouse wasrecorded for ten minutes Total time spent with the novelstrange mouse versus the novel object was analyzed with a2-tailed paired 119905-test for each treatment group Failure toachieve a statistically significant preference for spending timewith a novel stranger was considered indicative of abnormalbehavior A set of novel stranger mice had been previouslyacclimated to being restrained for 10-minute periods withinthe confines of the inverted cup The stranger mice werealternated from side one to side three to ensure that a

preference for a specific side by the test mice did notconfound the outcome Similarly the apparatus was housedin a room with an ambient noise level of 55 db providedby the ventilation fan and placed directly under a light Theapparatus was wiped down with 70 ethanol at the onset ofinvestigations and between mice thereafter

244 Tube Test for Social Dominance Mice were tested fora preference when challenged with a novel strange mouse atopposing ends of a 1210158401015840 long PVC pipe to yield to the novelstranger and back out of the tube (lose) or complete transit ofthe tube while the novel stranger yields (win) Disruption ofnormal behavior has been reported in mice that recapitulatemany characteristic features of autism-like behavior such asthosewith a truncatedMeCP2 that recapitulatemany featuresof Rett Syndrome [46] and chromosome deletions causingclassic features of Williams-Beuren syndrome such ashypersociability [47] Each test mouse was challenged threetimes with a novel strange mouse for a maximum allottedtime of four minutes (a match) Mice were placed headfirstat opposite ends of a tube and released simultaneously Thematch ended when one retreated from the tube and wasassigned a score of zero (lose) the remaining mouse wasassigned a score of one (win) Stranger mice were generallyused three times and nomore than four times Both test miceand stranger mice were separately acclimated to the testingapparatus in advance of testing Failure to achieve a winlossoutcome in four minutes or the mice crossing over eachother to exit opposite ends of the tube excluded the matchfrom scoring The outcome from the three matches for eachmouse was calculated as an average percentage of wins andthen the mean percentage of win matches was calculatedfor each treatment group These mean values were analyzedusing a nonparametric ANOVA (Kruskal-Wallis test) withDunnrsquos multiple comparison test

25 TissueHarvest andMolecular Assays Three animals fromnegative control group and from each of the groups thatexhibited changes in the greatest number of behavioral assays(water control VPA Mn and Pb) were evaluated for changesin frontal cortex CpG islandmethylation in promoter regionsand associated changes in gene expression that similarlypersisted into adulthood

After all behavioral assays were complete experimentalmice were euthanized via CO

2asphyxiation and brain tissue

immediately harvested The frontal cortex was dissectedout and divided for extraction of DNA and RNA used insubsequent molecular assays Agilentrsquos Mouse CpG IslandMicroarray and Genomic DNA Enzymatic Labeling Kit(Agilent Santa Clara CA) were used to identify changesin methylation of genes previously known to be importantfor early neurodevelopment as well as identifying novelcandidate genes The 2 times 105K format interrogates over16000 CpG islands using 97652 probes in or within 95 bp ofCpG islands and was sourced from UCSCmm8 (NCBI Build36)

Briefly genomic DNA was isolated and subsequent per-formance of the array was performed by MOgene (St LouisMO) Whole genomic DNA was sheared (reference DNA)


and labeled with Cy5 (blue) Methylated DNA was precipi-tated from each sample and labeled with Cy3 (red) The sam-ples were then hybridized to the slide for 40 hours followedby washing drying and imaging Data were extracted usingthe Feature Extraction software and methylation protocolIn order to view and search for high-level patterns in theresults for each experimental analysis hierarchical clusteringwas performed Data tables were prepared as tab-delimitedtext files and loaded intoHierarchical Clustering Explorer 30(University of Maryland College Park MD)

Upon completion of data analysis microarray resultswere validated by real-time quantitative PCR using TaqManGene Expression Assays on an ABI 3730 DNA 7900HTGenetic Analyzer (Life Technologies Carlsbad CA) for thosegenes identified as having altered histone methylation Gene-specific probes and primer sets were purchased from LifeTechnologies 50 ng cDNA of each sample was mixed with10 120583L TaqMan Gene Expression PCR Master Mix and 1 120583Lprobe and primer mixture in a total volume of 20 120583L in a384-well plate The assays were performed according to themanufacturerrsquos protocol on anABI PRISM7900HT SequenceDetection System The thermocycling started with a 10mindenaturing at 95∘C followed by 40 cycles of 95∘C for 15 secand 60∘C for 1min Data was analyzed using SDS softwarev21 Mouse Actb gene was used as internal control ΔΔCtmethodwas used to generate relative quantitative values (foldchange) Each reactionwas replicated two times and themeanvalue was used in final comparisons

3 Results

31 Behavioral Assays

311 Nest Building Assay All mice in this study made neststhat scored a 5 on a whole integer scale of 1ndash5 No statisticallysignificant differences between the nests of water-treatedcontrol mice and those of mice exposed to toxicants in uterowere observed

312 Marble-Burying Test C57 mice were vigorous diggerswith water-treated controls burying nearly 70 of the mar-bles placed on the surface of the lightly packed beddingAnimals from all treated groups buried fewer marbles butstatistically significant difference was confirmed only inthree groups VPA As and Mn exposed animals buriedsignificantly fewer marbles than water-treated control micewith the 119901 values less than 005 001 and 001 respectively(Figures 1 and 2)

313 Three-Chambered Testing Apparatus for Social Interac-tion Exposure to one toxicant Pb resulted in an abnormallack of preference for spending time with a novel strangermouse Statistical analysis revealed that mice treated with Pbdid not spend significantly more time with novel strangermouse than with the novel object (119901 = 0396) Water-treatedcontrol mice as well as all other treatment groups with theexception of Pb displayed a normal preference for spendingtime with a novel stranger mouse rather than with a novel

Table 1 Tube test for social dominance

Treatment wins 119901 valueCtl (119899 = 36) 495VPA (119899 = 36) 220 lt005Cd (119899 = 36) 303 gt005Pb (119899 = 36) 443a gt005As (119899 = 36) 525 gt005Mn (119899 = 36) 192 lt001Hg (119899 = 36) 289 gt005The mean values were analyzed using a nonparametric ANOVA (Kruskal-Wallis test) with Dunnrsquos multiple comparison testa25 of matches crossed over or ldquotimed outrdquo leaving the match unscored

object The observed differences (time with strange mouse minustime with a novel object) were statistically significant with a119901 values ranging from 0003 to 00001 (Figure 3)

314 Tube Test for Social Dominance Mice from water-treated control group on average won half (495) of theirmatches On the other hand the VPA-exposed mice experi-enced significantly more losses (only 22 wins) as comparedto water-treated controls (119901 lt 005) The Mn-exposed miceexperienced evenmore losses (only 192wins119901 lt 001)Thetube test identified also abnormal behavior of th Pb-exposedmice Even though the Pb-exposed group won 443 of theirmatches (not significantly different form control group 119901 gt005) these matches were very long and 25 of the matcheswere unscored when the animals crossed over one another toexit the tube or simply remained in the tube together for thefull 4 minutes of the match period This behaviour was notobserved in any other tested group (Table 1)

315 Overview of Behavioral Studies While none of thetreatment groups displayed abnormal behavior in all fourassays 3 treatment groups displayed abnormal behavior in 2of the assays when compared to water-treated controls VPA(marble burying and tube test)Mn (marble burying and tubetest) and Pb (three-chambered testing apparatus for socialinteraction and tube test) Molecular assays were performedusing tissue from the frontal cortex of representative animalsthree animals from each of the most affected groups (VPAMn and Pb) and water control were used (Table 2)

32 Molecular Assays

321MouseCpG IslandMicroarray forAlteredHistoneMethy-lation CpG array probe names were associated with geneIDs (UCSC Genome Browser) and analyzed for significantdifferences using Studentrsquos 119905-test (119901 lt 005) and a twofoldchange in average probe signal for treated versus controlgroups (Table 3) Clustering on the untransformed data usingAverage Linkage (UPGMA) with similarity represented byEuclideanDistance indicated that clusters fit expected groupsbased on treatment type (Figure 4) Further one region wasfound with lower regulation in all treatment groups Thisregion is 51015840 of the chromodomain-helicase-DNA-binding


(a) (b)

(c)

Figure 1 Marble-burying apparatus (a) Marble-burying apparatus ready for use (b) Marble-burying apparatus after use by representativeVPA-treated animal (c) Marble-burying apparatus after use by representative normal control animal

02468

101214

Control VPA Cd Pb As Mn Hg

Mean number of buried marbles

lowast

lowastlowast lowastlowast

Figure 2Marble-burying test for perseverativerepetitive behaviorVPA As and Mn exposed animals bury significantly fewer marblesthan controls Data were analyzed using the nonparametric ANOVA(Kruskal-Wallis test) with Dunnrsquos multiple comparison test lowastdenotes statistical significance (119901 lt 005) lowastlowast denotes statisticalsignificance (119901 lt 0001)

protein 7 (Chd7) gene which was found to be hypomethy-lated in each sample tested from the three experimentalgroups displaying the most altered behavior (Table 3)

322 Enrichment Analysis A web-based analysis toolkit(WEB-based Gene SeT AnaLysis Toolkit WebGestalt) [48]was utilized for enrichment analysis of genes adjacent toCpG enriched regions from the CpGmicroarray SpecificallyM musculus was queried using gene symbols Enrichment

0

50

100

150

200

250

300

350


(s)

Mean time spent in a chamber

With novel mouseWith novel object

lowast lowastlowast lowastlowastlowast

Figure 3Three-chambered testing apparatus for social interactionmice from all groups except the Pb exposed group exhibited anormal preference to spend significantly more time with a novelstranger mouse rather than with a novel object Data were analyzedwith a 2-tailed paired 119905-test for each treatment groupThe preferencewas statistically significant in all groups (0002 lt 119901 lt 004 range)but Pb treated group (119901 gt 005) lowast denotes statistical significance

analysis utilized genome query hypergeometric methodwith significance set at 119901 lt 005 with Bonferroni cor-rection Gene enrichment is reported for Gene Ontol-ogy For VPA anatomical structure formation involved inmorphogenesis (GO0048646) was the most significantly


MN-173MN-5-74MN-12-74VPA-8-71VPA-10-71VPA-5-70PB-2-72PB-8-73PB-3-72CTL1-69CTL10-70CTL-6-69

A68

P23471510

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P27865446

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P25616891

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P26954162

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P27910550

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P24589803

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P24834763

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P21319210

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P27571290

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P32118990

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P27903931

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P22880595

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P26347065

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P21339751

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P27897956

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P20961826

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P31925435

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P24627748

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P21934188

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P27560005

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P30374037

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P26813081

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P28261134

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P31948064

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P31929449

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P20058423

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P20723961

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P27850353

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P20218130

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P26872704

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P20598626

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P25693341

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P30472460

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P30926574

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P32099497

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P31918777

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P24776843

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P31601947

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P30484706

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P24674229

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P21928447

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P25677780

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P22716230

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P29222291

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P24293210

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P30392997

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P20720546

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P27996401

A68

P20137272

A68

P27710860

A68

P24593882

A_68

_P20597394

Figure 4 Mouse CpG Island Microarray for altered histone methylation Hierarchical clustering of the untransformed data was done usingAverage Linkage (UPGMA) with similarity represented by Euclidean Distance Data filtering the genomic results were filtered from theUCSC (httpgenomeucscedu) Mouse Genome Browser Gateway (NCBI37mm9) for being within a CpG island and 51015840 of a known geneThe samples clustered according to the treatment type

Table 2 Metatable for behavioral studies

Treatment Marble burying 3-chambered box Tube testVPA (119899 = 12) X XCd (119899 = 12)Pb (119899 = 12) X As (119899 = 12) XMn (119899 = 12) X XHg (119899 = 12)Groups that displayed behaviors significantly different from expected out-come are marked with X indicates that significant numbers of test animals had an unscored match

(119901 lt 0005) enriched biological process The genes in thisgroup (Rab3gap1 Ptprz1 Col2a1 Chd7 and Nf2) also popu-lated subsets specific to face development (GO0060324) andcamera-type eye development (GO0043010) The synapse(GO0045202) was the most significantly (119901 lt 0005)enriched cellular component in VPA treated animals Col-lectively altered facial and neurological development anddysregulated cellular signaling were indicated The mostsignificantly enrichedGOgroupswithMn exposure includedcellular biosynthetic process (119901 lt 001) under the biologicalprocess grouping (GO0044249) transferase (GO0016740)activity under molecular function (119901 lt 001) and intra-cellular membrane-bounded organelle (GO0043231) undercellular component (119901 lt 005)These data are consistent withan overall impact on biosynthesis with specific interactionof calmodulin and transferase activity In animals exposed tolead in utero the organelle (GO0043226) cellular componentgroup (119901 lt 005) and protein kinase inhibitor activity(GO0004860) molecular function group (119901 lt 0001) werethe most significantly enriched

Enrichment analysis was also conducted for animal phe-notypes Specifically these gene groups produce a commonphenotype when knocked out or mutated The most sig-nificantly enriched gene groups by phenotypes were abnor-mal CNS glial cell morphology (Ptprz1 Pi4k2a and AppMP0000952) and head bobbing (Col2a1 Chd7 MP0001410)for VPA treatment (119901 lt 005) decreased body weight(Ovol1 Trib2 Slc13a5 Lrp5 Hk2 Pi4k2a Chd7 and Tmeff2

MP0001262) for Mn (119901 lt 005) and impaired right-ing response (Trib2 Dst Chd7 and Bsn MP0001523) forPb (119901 lt 0001) Pb had an additional twelve signif-icantly enriched phenotype groups that included prema-ture death (MP0002083) abnormal involuntary movement(MP0003492) gliosis (MP0002183) and abnormal cochlearnerve compound action potential (MP0004415)

323 Real-Time Q-PCR Validation Chd7 was overexpressedin all three (Mn Pb and VPA) treatment groups as predictedby microarray data for altered histone methylation Whencompared to water control group the expression of Chd7was 24- 47- and 16-fold higher in Mn Pb and VPAtreated groups respectively While loss of function has beendemonstrated to be responsible for CHARGE syndrome inwhich affected children may have learning disabilities andcraniofacial malformations it is unknown at this time whateffects overexpression due to hypomethylation might confer

4 Discussion

C57 male mice were exposed in utero from the onset of ges-tation and during neural tube closure to toxicants suspectedof affecting neurodevelopment in humans VPA (the positivecontrol treatment) lead arsenic cadmium manganese andmercury Abnormal behavior was observed compared towater-treated controls in several treatment groups VPAmanganese and leadThe observed impacts of VPA treatmentwere consistent with those found in the literature [34 4950] These data also indicate that prenatal manganese andlead exposures are associated with adverse neurobehavioraldevelopment in the resultant offspring

In recent years growing evidence suggests that bothgenetic and environmental factors contribute to ASDsThereare indications that certain gene variants confer geneticsusceptibility [51] Additionally de novo mutations andadvanced parental age have also been identified as geneticallyrelated risk factors [52 53] The observed pathophysiologyof ASD in the central nervous system including oxidativestress neuroinflammation and mitochondrial dysfunctionis consistent with environmental exposures to air pollutionorganophosphates and heavy metals [54]


Table 3 Differentially methylated regions and associated genes

VPA Manganese LeadGene ID CpG VPA Ctl Gene ID CpG Mn Ctl Gene ID CpG Pb CtlCol2a1 chr15 minus125942 Atf1 chr15 minus240606 Lpo chr11 minus21686

Glt25d2 chr1 minus121282 Lrp5 chr19 minus153512 Alox5 chr6 minus194012

App chr16 minus115531 Ercc3 chr18 minus142284 Arhgef10 chr8 minus161243

Chd7 chr4 minus111644 Pnpla3 chr15 minus132399 Pkia chr3 minus129882

Ptprz1 chr6 minus10547 Gpr120 chr19 minus118607 Frmd4b chr6 minus128576

Nf2 chr11 1022569 Rasgrf2 chr13 minus113359 Chd7 chr4 minus114022Rab3gap1 chr1 1059968 Chd7 chr4 minus106245 Derl2 chr11 1021783

Nmnat2 chr1 1134621 Hivep2 chr10 1032248 Ctsf chr19 1023935

Eif5a chr11 1442196 Ctxn1 chr8 1063293 Bsn chr9 1028517

Pi4k2a chr19 1548454 Ovol1 chr19 1087982 Odz2 chr11 1066651

R3hdm1 chr1 2132963 Hk2 chr6 1132845 Rpp38 chr2 1205103

Slc13a5 chr11 1200217 Mtx2 chr2 1403068

Adcy1 chr11 1272176 Fads3 chr19 1511005

Scn5a chr9 1381722 Plxna1 chr6 1563187

Hs3st3a1 chr11 1454418 Zbtb5 chr4 1564412

Pi4k2a chr19 1532898 Dguok chr6 1565438

Dmtf1 chr5 1597027 Dnm2 chr9 1584182

Gdap1 chr1 1676317 Trib2 chr12 1654498

Csmd1 chr8 1730636 Prpsap2 chr11 1692807

Tmeff2 chr1 1771945 Dst chr1 2000012

Trib2 chr12 1790036

Hat1 chr2 1840714

Prmt8 chr6 1858983

Mrps28 chr3 1916268

Tnrc6b chr15 1969439

CpG array probe names were associated with gene IDs (UCSC Genome Browser) All array data was normalized to the internal sheered standard and controldata was compared to experimental data for differential methylation using log

2ratios Reported significant differences were defined by gt2-fold change 119905-test

(119901 lt 0005) The resulting changes in methylation for exposures were 11 for VPA (5 down 6 up) 25 for Mn (7 down 18 up) and 20 for Pb (6 down 14 up)

It has been reported that exposure to industrial chemicalsduring early fetal development can cause brain injury at dosesmuch lower than that affecting adult brain function [55] Theresearch on heavy metal exposure and ASD has largely beenfocusing on mercury however association between an ASDdiagnosis and other toxins including lead andmanganese hasbeen observed in epidemiology studies [56 57]

The knowledge on acute neurotoxic effect of lead can betraced back to the Roman times [58] During the 1970s widespread subclinical neurobehavioral deficits such as problemswith concentration memory and cognition were observedamong children with raised blood lead levels [59ndash64] Morerecent studies suggest that even low lead exposure couldhave greater than previously expected adverse effects onhuman brain development [65] Exposure to low doses ofmanganese in children who are particularly susceptible maylead to subclinical neurotoxicity [55] Recent studies havereported that manganese levels and psychomotor and mentaldevelopment in children are significantly associated and therelationship presents as an invertedU-shape [66ndash68] Despite

the profoundknowledge regarding neurotoxicity of industrialchemicals the relationship between prenatal exposure to lowdoses of neurotoxic metals and neurodevelopment in theoffspring remains unclear

One recent epidemiology study [69] in a Chinese pop-ulation revealed that high levels of manganese and leadin umbilical cord blood are associated with neurodevel-opmental deficit at two years of age Further there maybe an interactive effect between manganese and lead thataggravate this outcome We used rodent models to carefullyevaluate the spectrum of neurodevelopmental effects causedby common heavy metals Our results are consistent withLin and coworkers observation in humans and providedsupporting evidence to fill the data gap on in utero exposureto selected neurotoxins

When frontal cortex tissue from representative animalsfrom treatment groups and water-treated controls was eval-uated for changes in CpG island methylation data clusteringindicated that changes in methylation showed clear patternsclusters fit expected groups based on treatment type Further


one region was found with lower regulation in all treatmentgroups This region is 51015840 of the chromodomain-helicase-DNA-binding protein 7 (Chd7) gene Real-time qPCR con-firmed that Chd7 was overexpressed by several fold changesin all three treatment groups as predicted bymicroarray data

While loss of function of this gene has been demonstratedto be responsible for CHARGE syndrome in which affectedchildren often have learning disabilities and craniofacialmalformations it is unknown at this time what effectsoverexpression due to hypomethylation might confer

In this study in utero exposure to several toxicantsearly in development resulted in behavioral abnormalitiesand changes in methylation of CpG islands and associatedchanges in expression that were evident in adulthood Thestrength of the study is the combination of four behavioralassays as well as molecular assays on gene expression andmethylation status of genes responsive to exposure of toxi-cants Investigation of the role ofChd7 and other genes whosealtered methylation state is associated with disrupted socialbehavior is warranted Further epidemiology studies sug-gested potential convergent interactions among neurotoxinsand this hypothesis should be examined in rodent models asdescribed in this study

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was funded in part by Department of DefenseAward no W81XWH-09-1-0248 and NIH Grant noNS076465

References

[1] American Psychiatric Association Diagnostic and StatisticalManual of Mental Disorders American Psychiatric AssociationArlington Va USA 2013 httpdsmpsychiatryonlineorg

[2] L E Buxton and R N Murdoch ldquoLectins calcium ionophoreA23187 and peanut oil as deciduogenic agents in the uterus ofpseudopregnant mice effects of tranylcypromine indometh-acin iproniazid and propranololrdquo Australian Journal of Biologi-cal Sciences vol 35 no 1 pp 63ndash72 1982

[3] C Ladd-Acosta K D Hansen E Briem M D Fallin W EKaufmann and A P Feinberg ldquoCommon DNA methylationalterations in multiple brain regions in autismrdquo MolecularPsychiatry vol 19 no 8 pp 862ndash871 2014

[4] J M LaSalle ldquoA genomic point-of-view on environmentalfactors influencing the human brain methylomerdquo Epigeneticsvol 6 no 7 pp 862ndash869 2011

[5] J Lv Y Xin W Zhou and Z Qiu ldquoThe epigenetic switchesfor neural development and psychiatric disordersrdquo Journal ofGenetics and Genomics vol 40 no 7 pp 339ndash346 2013

[6] A M Persico and T Bourgeron ldquoSearching for ways out ofthe autism maze genetic epigenetic and environmental cluesrdquoTrends in Neurosciences vol 29 no 7 pp 349ndash358 2006

[7] K C Kim C S Choi J-W Kim et al ldquoMeCP2 modulates sexdifferences in the postsynaptic development of the valproateanimal model of autismrdquoMolecular Neurobiology 2014

[8] M Chahrour Y J Sung C Shaw et al ldquoMeCP2 a key contri-butor to neurological disease activates and represses transcrip-tionrdquo Science vol 320 no 5880 pp 1224ndash1229 2008

[9] R P Nagarajan A R Hogart Y Gwye M R Martin and JM LaSalle ldquoReduced MeCP2 expression is frequent in autismfrontal cortex and correlates with aberrant MECP2 promotermethylationrdquo Epigenetics vol 1 no 4 pp e1ndashe11 2006

[10] A Ornoy ldquoNeuroteratogens in man an overview with specialemphasis on the teratogenicity of antiepileptic drugs in preg-nancyrdquo Reproductive Toxicology vol 22 no 2 pp 214ndash2262006

[11] A Ornoy ldquoValproic acid in pregnancy how much are weendangering the embryo and fetusrdquo Reproductive Toxicologyvol 28 no 1 pp 1ndash10 2009

[12] R H Finnell G D Bennett S B Karras and V K MohlldquoCommon hierarchies of susceptibility to the induction ofneural tube defects in mouse embryos by valproic acid and its4-propyl-4-pentenoic acid metaboliterdquo Teratology vol 38 no4 pp 313ndash320 1988

[13] O S Cohen E I Varlinskaya C A Wilson S J Glatt and SMMooney ldquoAcute prenatal exposure to amoderate dose of val-proic acid increases social behavior and alters gene expressionin ratsrdquo International Journal of Developmental Neurosciencevol 31 no 8 pp 740ndash750 2013

[14] M R Favre T R Barkat D LaMendola G Khazen HMarkram and K Markram ldquoGeneral developmental health inthe VPA-rat model of autismrdquo Frontiers in Behavioral Neuro-science vol 7 article 88 2013

[15] R H Finnell B CWlodarczyk J C Craig J A Piedrahita andG D Bennett ldquoStrain-dependent alterations in the expressionof folate pathway genes following teratogenic exposure tovalproic acid in a mouse modelrdquo American Journal of MedicalGenetics vol 70 no 3 pp 303ndash311 1997

[16] D N Hovland Jr A F Machado W J Scott Jr and MD Collins ldquoDifferential sensitivity of the SWV and C57BL6mouse strains to the teratogenic action of single administrationsof cadmium given throughout the period of anterior neuroporeclosurerdquo Teratology vol 60 no 1 pp 13ndash21 1999

[17] B J Wlodarczyk A M Palacios T M George and R HFinnell ldquoAntiepileptic drugs and pregnancy outcomesrdquo Ameri-can Journal of Medical Genetics Part A vol 158 no 8 pp 2071ndash2090 2012

[18] A Duenas-Gonzalez M Candelaria C Perez-Plascencia EPerez-Cardenas E de la Cruz-Hernandez and L A HerreraldquoValproic acid as epigenetic cancer drug preclinical clinicaland transcriptional effects on solid tumorsrdquo Cancer TreatmentReviews vol 34 no 3 pp 206ndash222 2008

[19] B Monti E Polazzi and A Contestabile ldquoBiochemical molec-ular and epigenetic mechanisms of valproic acid neuroprotec-tionrdquo Current Molecular Pharmacology vol 2 no 1 pp 95ndash1092009

[20] R J Berry ldquoMaternal prenatal folic acid supplementation isassociated with a reduction in development of autistic disorderrdquoJournal of Pediatrics vol 163 no 1 pp 303ndash304 2013

[21] M A Faucher ldquoFolic acid supplementation before and in earlypregnancy may decrease risk for autismrdquo Journal of MidwiferyampWomenrsquos Health vol 58 no 4 pp 471ndash472 2013

[22] J Kałuzna-Czaplinska E Zurawicz M Michalska and JRynkowski ldquoA focus on homocysteine in autismrdquoActa Biochim-ica Polonica vol 60 no 2 pp 137ndash142 2013

[23] L R Schaevitz and J E Berger-Sweeney ldquoGene-environmentinteractions and epigenetic pathways in autism the importance


of one-carbon metabolismrdquo ILAR Journal vol 53 no 3-4 pp322ndash340 2012

[24] P E Goines and P Ashwood ldquoCytokine dysregulation in autismspectrum disorders (ASD) possible role of the environmentrdquoNeurotoxicology and Teratology vol 36 pp 67ndash81 2013

[25] F Dardenne S Van dongen I Nobels R Smolders W DeCoen and R Blust ldquoMode of action clustering of chemicalsand environmental samples on the bases of bacterial stress geneinductionsrdquo Toxicological Sciences vol 101 no 2 pp 206ndash2142008

[26] P B Tchounwou C G Yedjou A K Patlolla and D J SuttonldquoHeavy metal toxicity and the environmentrdquo in MolecularClinical and Environmental Toxicology vol 101 of ExperientiaSupplementum pp 133ndash164 Springer 2012

[27] D A Geier J K Kern P G King L K Sykes and M RGeier ldquoHair toxic metal concentrations and autism spectrumdisorder severity in young childrenrdquo International Journal ofEnvironmental Research and Public Health vol 9 no 12 pp4486ndash4497 2012

[28] D A Rossignol S J Genuis and R E Frye ldquoEnvironmentaltoxicants and autism spectrum disorders a systematic reviewrdquoTranslational Psychiatry vol 4 no 2 article e360 2014

[29] M Garrecht and D W Austin ldquoThe plausibility of a role formercury in the etiology of autism a cellular perspectiverdquo Toxi-cological and Environmental Chemistry vol 93 no 6 pp 1251ndash1273 2011

[30] J K Kern D A Geier T Audhya P G King L K Sykes andMR Geier ldquoEvidence of parallels between mercury intoxicationand the brain pathology in autismrdquo Acta Neurobiologiae Exper-imentalis vol 72 no 2 pp 113ndash153 2012

[31] Y Arai J Ohgane S Yagi et al ldquoEpigenetic assessment ofenvironmental chemicals detected in maternal peripheral andcord blood samplesrdquo Journal of Reproduction and Developmentvol 57 no 4 pp 507ndash517 2011

[32] D Lonsdale R J Shamberger and M E Obrenovich ldquoDysau-tonomia in autism spectrum disorder case reports of a familywith review of the literaturerdquo Autism Research and Treatmentvol 2011 Article ID 129795 7 pages 2011

[33] H YasudaM Kobayashi Y Yasuda and T Tsutsui ldquoEstimationof autistic children by metallomics analysisrdquo Scientific Reportsvol 3 no 1199 pp 1ndash7 2013

[34] T Schneider and R Przewłocki ldquoBehavioral alterations in ratsprenatally to valproic acid animal model of autismrdquo Neuropsy-chopharmacology vol 30 no 1 pp 80ndash89 2005

[35] S Macrı G Laviola M P Leussis and S L AndersenldquoAbnormal behavioral and neurotrophic development in theyounger sibling receiving less maternal care in a communalnursing paradigm in ratsrdquo Psychoneuroendocrinology vol 35no 3 pp 392ndash402 2010

[36] M Li R Budin A S Fleming and S Kapur ldquoEffects of chronictypical and atypical antipsychotic drug treatment on maternalbehavior in ratsrdquo Schizophrenia Research vol 75 no 2-3 pp325ndash336 2005

[37] M H Bassant M Picard D Olichon F Cathala and L CourtldquoChanges in the serotonergic noradrenergic and dopaminergiclevels in the brain of scrapie-infected ratsrdquo Brain Research vol367 no 1-2 pp 360ndash363 1986

[38] NAlbelda andD Joel ldquoAnimalmodels of obsessive-compulsivedisorder exploring pharmacology and neural substratesrdquo Neu-roscience and Biobehavioral Reviews vol 36 no 1 pp 47ndash632012

[39] M Angoa-Perez M J Kane D I Briggs D M Francescuttiand D M Kuhn ldquoMarble burying and nestlet shredding astests of repetitive compulsive-like behaviors in micerdquo Journalof Visualized Experiments vol 82 Article ID e50978 2013

[40] P Jedynak P Jaholkowski GWozniak C Sandi L Kaczmarekand R K Filipkowski ldquoLack of cyclin D2 impairing adult brainneurogenesis alters hippocampal-dependent behavioral taskswithout reducing learning abilityrdquo Behavioural Brain Researchvol 227 no 1 pp 159ndash166 2012

[41] M J Millan S Girardon J Mullot M Brocco and A DekeyneldquoStereospecific blockade of marble-burying behaviour in miceby selective non-peptidergic neurokinin1 (NK1) receptorantagonistsrdquo Neuropharmacology vol 42 no 5 pp 677ndash6842002

[42] S S Moy R J Nonneman G O Shafer et al ldquoDisruption ofsocial approach by MK-801 amphetamine and fluoxetine inadolescentC57BL6JmicerdquoNeurotoxicology andTeratology vol36 pp 36ndash46 2013

[43] T L Schaefer C V Vorhees and M T Williams ldquoMouse plas-macytoma-expressed transcript 1 knock out induced 5-HT dis-ruption results in a lack of cognitive deficits and an anxietyphenotype complicated by hypoactivity and defensivenessrdquoNeuroscience vol 164 no 4 pp 1431ndash1443 2009

[44] S N Umathe J M Vaghasiya N S Jain and P V Dixit ldquoNeu-rosteroids modulate compulsive and persistent behavior inrodents implications for obsessive-compulsive disorderrdquoProgress in Neuro-Psychopharmacology and Biological Psychia-try vol 33 no 7 pp 1161ndash1166 2009

[45] M Yang J L Silverman and J N Crawley ldquoUNIT 826Automated three-chambered social approach task for micerdquo inCurrent Protocols inNeuroscience chapter 8 JohnWileyamp Sons2011

[46] J L Neul ldquoThe relationship of Rett syndrome and MECP2 dis-orders to autismrdquoDialogues in Clinical Neuroscience vol 14 no3 pp 253ndash262 2012

[47] N M Williams ldquoMolecular mechanisms in 22q11 deletion syn-dromerdquo Schizophrenia Bulletin vol 37 no 5 pp 882ndash889 2011

[48] J Wang D Duncan Z Shi and B Zhang ldquoWEB-based geneset analysis toolkit (WebGestalt) update 2013rdquo Nucleic AcidsResearch vol 41 no 1 pp W77ndashW83 2013

[49] K Iwata H Matsuzaki N Takei T Manabe and N MorildquoAnimal models of autism an epigenetic and environmentalviewpointrdquo Journal of Central Nervous System Disease vol 2pp 37ndash44 2010

[50] K C Kim P Kim H S Go et al ldquoThe critical period of val-proate exposure to induce autistic symptoms in Sprague-Dawley ratsrdquo Toxicology Letters vol 201 no 2 pp 137ndash142 2011

[51] T Gaugler L Klei S J Sanders et al ldquoMost genetic risk forautism resides with common variationrdquo Nature Genetics vol46 no 8 pp 881ndash885 2014

[52] S J Sanders M T Murtha A R Gupta et al ldquoDe novomutations revealed by whole-exome sequencing are stronglyassociated with autismrdquo Nature vol 484 no 7397 pp 237ndash2412012

[53] S Idring C Magnusson M Lundberg et al ldquoParental age andthe risk of autism spectrum disorders findings from a Swedishpopulation-based cohortrdquo International Journal of Epidemio-logy vol 43 no 1 pp 107ndash115 2014

[54] M R Herbert ldquoContributions of the environment and environ-mentally vulnerable physiology to autism spectrum disordersrdquoCurrent Opinion in Neurology vol 23 no 2 pp 103ndash110 2010


[55] P Grandjean and P Landrigan ldquoDevelopmental neurotoxicityof industrial chemicalsrdquoTheLancet vol 368 no 9553 pp 2167ndash2178 2006

[56] E Blaurock-Busch O R Amin H H Dessoki and T RabahldquoToxic metals and essential elements in hair and severity ofsymptoms among children with autismrdquo Maedica vol 7 no 1pp 38ndash48 2012

[57] M Fujimoto K Sakai and T Ueda ldquoSignificance of amikacinadministered orally prior to operation for prevention of post-operative infections in colonic cancer surgeryrdquo The JapaneseJournal of Antibiotics vol 39 no 11 pp 2889ndash2896 1986

[58] S Hernberg ldquoLead poisoning in a historical perspectiverdquoAmerican Journal of Industrial Medicine vol 38 no 3 pp 244ndash254 2000

[59] P J Landrigan A S McKinney L C Hopkins W W RhodesJr W A Price and D H Cox ldquoChronic lead absorption Resultof poor ventilation in an indoor pistol rangerdquoThe Journal of theAmericanMedical Association vol 234 no 4 pp 394ndash397 1975

[60] P J Landrigan R H Baloh W F Barthel R H Whitworth NW Staehling and B F Rosenblum ldquoNeuropsychological dys-function in children with chronic low-level lead absorptionrdquoThe Lancet vol 305 no 7909 pp 708ndash712 1975

[61] H L Needleman ldquoLead levels and childrenrsquos psychologicperformancerdquo The New England Journal of Medicine vol 301no 3 p 163 1979

[62] H L Needleman C Gunnoe A Leviton et al ldquoDeficits in psy-chologic and classroom performance of children with elevateddentine lead levelsrdquo The New England Journal of Medicine vol300 no 13 pp 689ndash695 1979

[63] H Needleman and A Leviton ldquoLead and neurobehaviouraldeficit in childrenrdquoThe Lancet vol 314 no 8133 p 104 1979

[64] H L Needleman and A Leviton ldquoNeurologic effects of expo-sure to leadrdquo Journal of Pediatrics vol 94 no 3 pp 505ndash5061979

[65] B P Lanphear RHornung J Khoury et al ldquoLow-level environ-mental lead exposure and childrenrsquos intellectual function aninternational pooled analysisrdquo Environmental Health Perspec-tives vol 113 no 7 pp 894ndash899 2005

[66] B C Henn A S Ettinger J Schwartz et al ldquoEarly postnatalblood manganese levels and childrenrsquos neurodevelopmentrdquoEpidemiology vol 21 no 4 pp 433ndash439 2010

[67] Z Su M-F Chai P-L Lu R An J Chen and X-C WangldquoAtMTM1 a novel mitochondrial protein may be involved inactivation of the manganese-containing superoxide dismutasein Arabidopsisrdquo Planta vol 226 no 4 pp 1031ndash1039 2007

[68] L Takser D Mergler G Hellier J Sahuquillo and G HuelldquoManganese monoamine metabolite levels at birth and childpsychomotor developmentrdquo NeuroToxicology vol 24 no 4-5pp 667ndash674 2003

[69] C-C Lin Y-C Chen F-C Su et al ldquoIn utero exposure toenvironmental lead andmanganese and neurodevelopment at 2years of agerdquo Environmental Research vol 123 pp 52ndash57 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


both of which have the potential to dysregulate criticaldevelopmental genes that can result in adverse pregnancyoutcomes [18 19] A growing body of literature indicatesthat similar disruption is observed in some autistic patientsboth disrupted folate and homocysteine status [20ndash22] andepigenomic disruption of regulatory CpG islands have beenassociated with autism [23] Furthermore evidence suggeststhat exposure to environmental neurotoxins is a risk factorfor autism [24]

Industrial chemicals are being produced and released intothe environment at a staggering rate The average consumeris exposed to up to 10000 different chemicals every dayout of a world market that includes over 30000 industrialchemicals sold at quantities greater than 400 million tonsper year [25] These chemicals range in toxicity from beingbenign to being extremely hazardous and the environmentaland public health implications of this overwhelming andpervasive exposure have not been well characterized for thevast majority of chemicals Among the hazardous toxicantsheavy metals known neurotoxicants continue to be of agreat concern due to their increasing anthropogenic presencein the environment [26] Several epidemiological studiesreported a relationship between autism and heavy metalbiomarkers [27 28] Exposure to industrial chemicals hasbeen thought to play a role in the etiology of autism throughepigenetic mechanismsThis is based on evidence suggestingthe plausibility of a role for mercury in the etiology of autism[29 30] and mercury in maternal peripheral and cord bloodhas been shown to affect fetal epigenetic status [31] A handfulof reports also points out to other heavy metals neurotoxicityand epigenetic mechanism [32 33]

The experiments presented in this paper were designedto identify abnormal neurobehavioral phenotypes resultingfrom in utero exposure to several identified environmentalpollutants We hypothesized that in utero exposure to thesetoxicants results in epigenetic changes in key developmentalgenes affecting pathways that have direct roles in the etiologyof autism Collectively this work provides an assessment ofthe effect of in utero exposure to selected heavy metals onthe epigenome and associated autistic-like phenotypes in theoffspring that persist as adult behaviors

2 Materials and Methods

21 General Study Design C57BL6J mice were exposed inutero to selected toxicants and evaluated for their adultbehavioral phenotype VPA was used as a positive controlbecause it is a widely used mitochondriotoxic medicationthat induces a spectrum of teratogenic effects includingbehavioral disruption and is used as a rodent model forstudying autistic behavioral changes [34] Environmentaltoxicants including cadmium (Cd) lead (Pb) arsenic (As)manganese (Mn) and mercury (Hg) were investigated withnormal drinking water used as a negative control

22 Animals and Housing C57B6J mice were housed inthe Institute of Biosciences and Technology Vivarium whichis fully accredited by the Association for Assessment andAccreditation of Laboratory Animal Care The animals were

maintained in clear polycarbonate microisolator cages andwere allowed free access to food and water (Harlan TekladRodent Diet 8606 Ralston Purina St Louis MO) Themice were maintained on a 12 h lightdark cycle Nulligravidfemales 50ndash70 days of age were mated overnight with malesand examined for the presence of vaginal plugs the followingmorning and the onset of gestation was considered to be10 pm of the previous night the midpoint of the dark cycle

23 Treatment with Select Compounds Presumed graviddams C57BL6J were treated with one of seven agents drink-ingwater served as the negative control andVPA (CAS 1069-66-5 valproic acid sodium salt ge98 Sigma-Aldrich Chem-icals St Louis MO) administered at 600mgkg by intraperi-toneal injection on gestational day 85 only served as thepositive control Five groups were treated from the first dayof pregnancy through day 105 the end of neurulation whichgenerally occurs from E80ndash105 via treated drinking watercontaining 10 ppmCd as cadmium chloride (CAS 10108-64-2 9999 trace metals basis Sigma-Aldrich Chemicals StLouis MO) 300 ppm Pb as lead acetate (CAS 6080-56-4lead(II) acetate trihydrate 99999 tracemetals basis Sigma-Aldrich Chemicals St Louis MO) 05 ppm As as sodiumarsenate (CAS 10048-95-0 sodium arsenate heptahydrateACS Reagent ge98 Sigma-Aldrich Chemicals St LouisMO) 10 ppm Mn as manganese chloride (CAS 13446-34-9 manganese(II) chloride tetrahydrate 9999 trace metalsbasis Sigma-Aldrich Chemicals St Louis MO) or 20 ppmHg as mercury chloride (CAS 7487-94-7 mercury(II) chlo-ride ACS reagent ge999 Sigma-Aldrich Chemicals StLouis MO) Treated dams maintained in separate cages wereallowed to give birth Only animals from litters consisting of6ndash8 pups were admitted for further testing All experimentalmice were weaned at three weeks of age and maintained inseparate cages Because of the higher incidence of autismreported in male children as compared to female childrenmalemice were used exclusively in these studies Twelvemiceper group that is seventy two males in total were used inthis study Animals were assigned randomly to experimentalgroups and to reduce any litter effect only one male from agiven litter was assigned to the same treatment group (nolittermates in the same group)

24 Behavioral Assays and Statistical Analysis Thebehavioraltesting began when experimental animals reached twelveweeks of age and lasted until twenty weeks of age with atleast one-week break period between consecutive assays forany individual mouse All treatment groups were evaluatedwith 4 different neurobehavioral assays nest building tobegin to assess behavioral deficits marble burying for per-severativeimpulsive behaviors 3-chambered box for socialinteraction and tube test for social dominance Each assaywas performed sequentially such that the least stressful assaywas performed first and the most stressful performed lastnest building marble burying 3-chambered box for socialinteraction and tube test

241 Nest Building Upon reaching twelve weeks of agemice were tested for their ability to build nests C57 mice
















3 Results










32 Molecular Assays



(a) (b)

(c)


02468

101214



lowast





0

50

100

150

200

250

300

350


(s)








A68

P23471510

A68

P27865446

A68

P25616891

A68

P26954162

A68

P27910550

A68

P24589803

A68

P24834763

A68

P21319210

A68

P27571290

A68

P32118990

A68

P27903931

A68

P22880595

A68

P26347065

A68

P21339751

A68

P27897956

A68

P20961826

A68

P31925435

A68

P24627748

A68

P21934188

A68

P27560005

A68

P30374037

A68

P26813081

A68

P28261134

A68

P31948064

A68

P31929449

A68

P20058423

A68

P20723961

A68

P27850353

A68

P20218130

A68

P26872704

A68

P20598626

A68

P25693341

A68

P30472460

A68

P30926574

A68

P32099497

A68

P31918777

A68

P24776843

A68

P31601947

A68

P30484706

A68

P24674229

A68

P21928447

A68

P25677780

A68

P22716230

A68

P29222291

A68

P24293210

A68

P30392997

A68

P20720546

A68

P27996401

A68

P20137272

A68

P27710860

A68

P24593882

A_68

_P20597394








4 Discussion















Slc13a5 chr11 1200217 Mtx2 chr2 1403068

Adcy1 chr11 1272176 Fads3 chr19 1511005




Dmtf1 chr5 1597027 Dnm2 chr9 1584182

Gdap1 chr1 1676317 Trib2 chr12 1654498


Tmeff2 chr1 1771945 Dst chr1 2000012

Trib2 chr12 1790036

Hat1 chr2 1840714

Prmt8 chr6 1858983

Mrps28 chr3 1916268
















Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































3 Results










32 Molecular Assays



(a) (b)

(c)


02468

101214



lowast





0

50

100

150

200

250

300

350


(s)








A68

P23471510

A68

P27865446

A68

P25616891

A68

P26954162

A68

P27910550

A68

P24589803

A68

P24834763

A68

P21319210

A68

P27571290

A68

P32118990

A68

P27903931

A68

P22880595

A68

P26347065

A68

P21339751

A68

P27897956

A68

P20961826

A68

P31925435

A68

P24627748

A68

P21934188

A68

P27560005

A68

P30374037

A68

P26813081

A68

P28261134

A68

P31948064

A68

P31929449

A68

P20058423

A68

P20723961

A68

P27850353

A68

P20218130

A68

P26872704

A68

P20598626

A68

P25693341

A68

P30472460

A68

P30926574

A68

P32099497

A68

P31918777

A68

P24776843

A68

P31601947

A68

P30484706

A68

P24674229

A68

P21928447

A68

P25677780

A68

P22716230

A68

P29222291

A68

P24293210

A68

P30392997

A68

P20720546

A68

P27996401

A68

P20137272

A68

P27710860

A68

P24593882

A_68

_P20597394








4 Discussion















Slc13a5 chr11 1200217 Mtx2 chr2 1403068

Adcy1 chr11 1272176 Fads3 chr19 1511005




Dmtf1 chr5 1597027 Dnm2 chr9 1584182

Gdap1 chr1 1676317 Trib2 chr12 1654498


Tmeff2 chr1 1771945 Dst chr1 2000012

Trib2 chr12 1790036

Hat1 chr2 1840714

Prmt8 chr6 1858983

Mrps28 chr3 1916268
















Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c)


02468

101214



lowast





0

50

100

150

200

250

300

350


(s)








A68

P23471510

A68

P27865446

A68

P25616891

A68

P26954162

A68

P27910550

A68

P24589803

A68

P24834763

A68

P21319210

A68

P27571290

A68

P32118990

A68

P27903931

A68

P22880595

A68

P26347065

A68

P21339751

A68

P27897956

A68

P20961826

A68

P31925435

A68

P24627748

A68

P21934188

A68

P27560005

A68

P30374037

A68

P26813081

A68

P28261134

A68

P31948064

A68

P31929449

A68

P20058423

A68

P20723961

A68

P27850353

A68

P20218130

A68

P26872704

A68

P20598626

A68

P25693341

A68

P30472460

A68

P30926574

A68

P32099497

A68

P31918777

A68

P24776843

A68

P31601947

A68

P30484706

A68

P24674229

A68

P21928447

A68

P25677780

A68

P22716230

A68

P29222291

A68

P24293210

A68

P30392997

A68

P20720546

A68

P27996401

A68

P20137272

A68

P27710860

A68

P24593882

A_68

_P20597394








4 Discussion















Slc13a5 chr11 1200217 Mtx2 chr2 1403068

Adcy1 chr11 1272176 Fads3 chr19 1511005




Dmtf1 chr5 1597027 Dnm2 chr9 1584182

Gdap1 chr1 1676317 Trib2 chr12 1654498


Tmeff2 chr1 1771945 Dst chr1 2000012

Trib2 chr12 1790036

Hat1 chr2 1840714

Prmt8 chr6 1858983

Mrps28 chr3 1916268
















Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















A68

P23471510

A68

P27865446

A68

P25616891

A68

P26954162

A68

P27910550

A68

P24589803

A68

P24834763

A68

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_P20597394








4 Discussion















Slc13a5 chr11 1200217 Mtx2 chr2 1403068

Adcy1 chr11 1272176 Fads3 chr19 1511005




Dmtf1 chr5 1597027 Dnm2 chr9 1584182

Gdap1 chr1 1676317 Trib2 chr12 1654498


Tmeff2 chr1 1771945 Dst chr1 2000012

Trib2 chr12 1790036

Hat1 chr2 1840714

Prmt8 chr6 1858983

Mrps28 chr3 1916268
















Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























Slc13a5 chr11 1200217 Mtx2 chr2 1403068

Adcy1 chr11 1272176 Fads3 chr19 1511005




Dmtf1 chr5 1597027 Dnm2 chr9 1584182

Gdap1 chr1 1676317 Trib2 chr12 1654498


Tmeff2 chr1 1771945 Dst chr1 2000012

Trib2 chr12 1790036

Hat1 chr2 1840714

Prmt8 chr6 1858983

Mrps28 chr3 1916268
















Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References














































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Autism-Like Behavior and Epigenetic ...

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