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DevelopmentalNeurotoxicityofInhaledAmbientUltrafineParticleAirPollution:ParallelswithNeuropathologicaland...

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Full length article

Developmental neurotoxicity of inhaled ambient ultrafine particle airpollution: Parallels with neuropathological and behavioral features ofautism and other neurodevelopmental disorders

J.L. Allena,*, G. Oberdorstera, K. Morris-Schaffera, C. Wonga, C. Klockea, M. Sobolewskia,K. Conrada, M. Mayer-Proschelb, D.A. Cory-Slechtaa

aDepartment of Environmental Medicine, University of Rochester School of Medicine, Rochester, NY 14642, United StatesbDepartment of Biomedical Genetics, University of Rochester School of Medicine, Rochester, NY 14642, United States

A R T I C L E I N F O

Article history:Received 12 November 2015Received in revised form 18 December 2015Accepted 18 December 2015Available online xxx

Keywords:Ultrafine particlesAir pollutionWhite matterCorpus callosumLateral ventricleMyelinationAutismSchizophreniaPeriventricular leukomalaciaExcitatory–inhibitory imbalance

A B S T R A C T

Accumulating evidence from both human and animal studies show that brain is a target of air pollution.Multiple epidemiological studies have now linked components of air pollution to diagnosis of autismspectrum disorder (ASD), a linkage with plausibility based on the shared mechanisms of inflammation.Additional plausibility appears to be provided by findings from our studies in mice of exposures frompostnatal day (PND) 4–7 and 10–13 (human 3rd trimester equivalent), to concentrated ambient ultrafine(UFP) particles, considered the most reactive component of air pollution, at levels consistent with hightraffic areas of major U.S. cities and thus highly relevant to human exposures. These exposures, occurringduring a period of marked neuro- and gliogenesis, unexpectedly produced a pattern of developmentalneurotoxicity notably similar to multiple hypothesized mechanistic underpinnings of ASD, including itsgreater impact in males. UFP exposures induced inflammation/microglial activation, reductions in size ofthe corpus callosum (CC) and associated hypomyelination, aberrant white matter development and/orstructural integrity with ventriculomegaly (VM), elevated glutamate and excitatory/inhibitoryimbalance, increased amygdala astrocytic activation, and repetitive and impulsive behaviors.Collectively, these findings suggest the human 3rd trimester equivalent as a period of potentialvulnerability to neurodevelopmental toxicity to UFP, particularly in males, and point to the possibilitythat UFP air pollution exposure during periods of rapid neuro- and gliogenesis may be a risk factor notonly for ASD, but also for other neurodevelopmental disorders that share features with ASD, such asschizophrenia, attention deficit disorder, and periventricular leukomalacia.

ã 2015 Elsevier Inc. All rights reserved.

Contents lists available at ScienceDirect

NeuroToxicology

1. Introduction

1.1. Air pollution exposure

Air pollution is a complex mixture of particles, gases, tracemetals and adsorbed organic contaminants. Particle sizes rangefrom coarse (2.5–10 mm) to fine (<2.5 mm) to ultrafine (UFP,<100 nm or 0.1 mm). Although not a significant component bymass, UFPs achieve orders of magnitude higher particle numberconcentrations and thus have more extensive surface areas thatcan adsorb toxic air pollutants (oxidant gases such as ozone, NOX,organic compounds, transition metals) per unit mass. AmbientUFPs arise primarily from combustion of fossil fuels (i.e., motor

* Corresponding author.E-mail address: joshua_allen@urmc.rochester.edu (J.L. Allen).

http://dx.doi.org/10.1016/j.neuro.2015.12.0140161-813X/ã 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: J.L. Allen, et al., Developmental neurotoxneuropathological and behavioral features of autism and other neurod10.1016/j.neuro.2015.12.014

vehicle traffic) (Lippmann et al., 2013). Levels of up to 50% ofinhaled UFP are deposited in pulmonary alveolar regions of lungfrom where they can traverse the alveolocapillary barrier to accesspulmonary interstitium and cross endothelial cells into bloodcirculation and subsequently impact other organs, including heartand brain, and thereby lead to more serious health consequences,which is why UFPs are considered among the most reactiveelements of air pollution (Oberdorster et al., 1994; Brown et al.,2001). Deposition can also occur in the nasal cavity allowingtranslocation to brain (Elder et al., 2006; Hunter and Dey, 1998;Lewis et al., 2005).

Air pollution is a worldwide environmental health problem, towhich exposures begin at gestation, and are cumulative over thelifespan. While air quality standards have clearly improved inmany countries, even as of 2012, the U.S. Census Bureau estimatedthat approximately 142 million U.S. residents live in countieswhere regulated levels of various air pollutants exceeded

icity of inhaled ambient ultrafine particle air pollution: Parallels withevelopmental disorders, Neurotoxicology (2016), http://dx.doi.org/

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standards set by the U.S. Environmental Protection Agency(EPA). Further, ongoing urbanization trends and expanding roadtraffic in many areas of the world are predicted to further increasepopulation exposures to UFP air pollution (Kumar et al., 2014).Currently, the U.S. EPA sets regulatory standards for levels ofPM10 and PM2.5. In addition to the fact that it represents asignificant technological challenge, regulation of UFPs is said toawait additional evidence of their toxicity. Considered in a globalcontext, new studies ascribe 3.3 million premature deaths peryear to outdoor air pollution (Lelieveld et al., 2015), and as of2013, air pollution was listed as the 12th leading global risk factorfor disability adjusted life year reductions (Forouzanfar et al.,2013).

1.2. Air pollution and brain

While the focus of air pollution research has long centered oncardiovascular and pulmonary systems, evidence now shows thatair pollution also adversely impacts brain, and does so bymechanisms similar to those operative in lung, i.e., via inflamma-tion and oxidative stress (Block and Calderon-Garciduenas., 2009).In children, various components of air pollution have beenassociated with impaired cognitive abilities (Harris et al., 2015;Suglia et al., 2008), deficits in attention-related behaviors (Chiuet al., 2013; Newman et al., 2013; Siddique et al., 2011), reducedmental development index and IQ scores (Perera et al., 2009),symptoms of anxiety/depression (Perera et al., 2006, 2012),decreased nonverbal reasoning ability (Edwards et al., 2010) anddelayed psychomotor development (Guxens et al., 2015). Animalmodels confirm behavioral and CNS toxicity in response togestational, postnatal and adult exposures to air pollution and/or its components (Block and Calderon-Garciduenas., 2009),including findings of depressive-like behaviors, impaired spatiallearning and memory, reduced apical dendritic spine density anddendritic branching in hippocampus (Fonken et al., 2011), reducednumbers of hippocampal neuronal GluA1glutamate receptorsubunits and altered neuronal differentiation of cortical neurons(Davis et al., 2013) and altered locomotor activity and levels ofbrain catecholamines (Suzuki et al., 2010).

Interestingly, the combined presentation of many of thesespecific impairments is consistent with the multifaceted nature ofAutism Spectrum disorders (ASD) that has now reached aworldwide prevalence as high as 1–2% (Lai et al., 2014; Elsabbaghet al., 2012), with a clear bias towards males being more affected(Elsabbagh et al., 2012; Frazier et al., 2014; Van Wijngaarden-Cremers et al., 2014; Werling and Geschwind, 2013). Diagnosis ofASD relies on the identification of impairments of core behavioralfeatures like social deficits, impaired communication, and repeti-tive/perseverative behaviors early in life. In fact, while the etiologyof this highly intractable and extremely heterogeneous neuro-developmental disorder remains unknown, numerous epidemio-logical studies of populations from different states within the U.S.and different countries now report associations of ASD diagnosiswith components of air pollution, including residential distancefrom the freeway (Volk et al., 2011), ambient levels of metals(Roberts et al., 2013; Windham et al., 2006), aromatic solvents(Roberts et al., 2013; Windham et al., 2006; Kalkbrenner et al.,2010), styrene and chromium (Talbott et al., 2015), gases (Becerraet al., 2013; Jung et al., 2013; Volk et al., 2013), diesel exhaust(Roberts et al., 2013; Windham et al., 2006) and particulate matter(PM2.5 and/or PM10) (Becerra et al., 2013; Jung et al., 2013; Volket al., 2013; Kalkbrenner et al., 2015; Raz et al., 2014). Some reportincreased odds ratios for ASD diagnoses in relation to levels ofPM2.5 (Jung et al., 2013; Volk et al., 2013; Raz et al., 2014), whichincludes UFPs, and/or NO2, a surrogate for UFP (Beckerman et al.,2008). The one exception to such findings was a recent study that

Please cite this article in press as: J.L. Allen, et al., Developmental neurotoxneuropathological and behavioral features of autism and other neurod10.1016/j.neuro.2015.12.014

combined data across 4 cohorts, but, unfortunately, each cohortused different measures of autistic traits, effectively limiting powerand comparability (Guxens et al., 2015).

Underlying reasons for an association of air pollution with ASDare not known, but several lines of evidence suggest that airpollution-induced inflammatory mechanisms during develop-ment could play a key role (Melillo and Leisman, 2009; Noriegaand Savelkoul, 2014; Meldrum et al., 2013; Depino, 2013; Onoreet al., 2012). For example, markers of inflammation are elevated inamniotic fluid (Abdallah et al., 2013), and through life (Gesund-heit et al., 2013; Emanuele et al., 2010; Vargas et al., 2005) in ASD,consistent with a chronic inflammatory state in both periphery(Depino , 2013; Emanuele et al., 2010; El-Ansary and Al-Ayadhi.,2012; Naik et al., 2011) and brain (Vargas et al., 2005; Rose et al.,2012; Wei et al., 2011; Li et al., 2009). Increased cytokine levels, aconsequence of inflammation, associate with severity of ASDdiagnostic features (Ashwood et al., 2011). Correspondingly,multiple components of air pollution including diesel exhaust,fine as well as UFP, and toxicants adsorbed to particles, such asendotoxin (LPS), also produce inflammatory changes in brain(Bolton et al., 2012; Campbell et al., 2009; Hartz et al., 2008;Levesque et al., 2011a,b; Morgan et al., 2011; van Berlo et al.,2010).

Mechanistically, it has been proposed that air pollution-associated CNS pathology reflects interactive pathways thatinclude systemic inflammation, as mediated e.g., by vagal nerveafferents (Oberdorster et al., 2004, 2009), in conjunction withdirect effects of particulate matter on brain tissue (Block andCalderon-Garciduenas., 2009). Perhaps not surprisingly, levels ofasthma, a disease associated with air pollution-induced inflam-mation (Wendt et al., 2014; Evans et al., 2014; MacIntyre et al.,2013; Gonzalez-Barcala et al., 2013), are substantially higher inchildren with ASD, consistent with common inflammatorymechanisms in lung and brain. In a nationally representativesample of 77,951 children, asthma was 35% more common inthose with ASD after controlling for age, gender, body mass index,race, brain injury, secondhand smoke and socioeconomic status(Kotey et al., 2014); further the prevalence of both ASD andasthma has been increasing (Becker, 2007; Becker and Schultz,2010).

Potential mechanistic links may also result from alterations inglutamate. Hyperglutamate function has been hypothesized tounderlie ASD (Jacob et al., 2011), and air pollution impacts brainglutamate function (Davis et al., 2013). Both developing and adultmicroglia have functional glutamate receptors and microglialactivation can engender glutamate release (Murugan et al., 2013;Takeuchi et al., 2005). Furthermore, glutamate release can alsoactivate microglia, leading to a vicious cycle (Biber et al., 2007), anda potential mechanism of chronic inflammation.

This paper summarizes the results of a series of studies,including previously published findings and new data describingthe impact of exposures of mice to concentrated ambientparticles (CAPS) in the ultrafine particle (UFP) range thatcollectively appear to provide biological plausibility for anassociation between air pollution and ASD. As presented below,exposures of mice to real time, road inlet ambient concentrated(10–20x) UFP from postnatal days (PND) 4–7 and 10–13(equivalent to human 3rd trimester (Clancy et al., 2007a,b; Riceand Barone, 2000), and a period of marked neuro- and gliogenesis(Bandeira et al., 2009), unexpectedly produced a pattern ofdevelopmental neurotoxicity notably similar to multiple hypoth-esized mechanistic underpinnings of ASD and other neuro-developmental disabilities. Our exposure paradigm is highlyrelevant to human exposures as UFPs, considered the mostreactive component of air pollution, occurred at levels consistentwith high traffic areas of major U.S. cities.

icity of inhaled ambient ultrafine particle air pollution: Parallels withevelopmental disorders, Neurotoxicology (2016), http://dx.doi.org/

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2. Methods

Methods presented herein are for results not previouslypublished; methods for findings presented in prior publicationsare in the corresponding cited references for those findings.

2.1. Animals, reagents, & exposures

Male and female C57BL6/J mice 8 weeks of age were purchasedfrom Jackson Laboratories (Bar Harbor, ME) and allowed toacclimate in the housing room for 1 week prior to breeding. Micewere co-housed for 3 days and bred monogamously, after whichmales were removed and pregnant dams were singly housed withlitters until weaning on PND21. Pups were exposed to filtered air(air) or concentrated ambient ultrafine particles (CAPS) using theHarvard University Concentrated Ambient Particle System(HUCAPS) fitted with a size-selective inlet and a high-volumeultrafine particle (UFPs, �100 nm) concentrator (10–20x) that takesin outdoor air at 5000 L per minute and concentrates ambient UFP,as previously described (Allen et al., 2013, 2014a,b,c). Exposureslasted for 4 h per day from 0700 to 1200 for 4 days per week fromPND4-7 and PND10-13, with exposure timing corresponding topeak vehicular traffic outside the intake valve of the HUCAPSinstrumentation. A condensation particle counter (TSI 3022A)provided particle counts. Mass concentration was calculated usingidealized particle density (1.5 g/cm3). A Scanning Mobility ParticleSizer (SMPS) was used to determine particle size distribution andmedian particle diameter + GSD. Due to the presence of the particleimpactor in the HUCAPS system, mice in the CAPS exposurechamber may be held at a slightly higher negative pressurecompared to the Air exposed mice in an adjacent chamber. Flow ofCAPS-enriched and filtered air were both maintained at 35–40%relative humidity and 77–79 �F.

To preclude any effect of anesthetics on measured endpoints,brains were extracted following decapitation without sedation toassess the immediate and persistent effects of CAPS on whitematter in the corpus callosum (CC), striatum (STR), and frontalcortex (FC). Brains were extracted and placed into 4% paraformal-dehyde for 24 h and then cryoprotected in 30% sucrose until theysank. Brains were stored at 4 �C until they were sectioned on afreezing microtome at 40 mm thickness. Sectioned tissue wasstored in sucrose- and ethylene glycol containing cryoprotectant

Fig. 1. CAPS exposure leads to male specific and time dependent increases in IBA-1. IBAmales and females for air and CAPS-treated groups at PND14 and PND55 in anterior comCAPS = concentrated ambient ultrafine particles; PND = postnatal day; air-14 = brains fromat postnatal day 55; CAPS-14 = brains collected at postnatal day 55 from offspring exposedday 14 from offspring exposed to concentrated ambient ultrafine particles; brains colleparticles; * = significantlydifferent from corresponding time point control. n = 4–5/grou

Please cite this article in press as: J.L. Allen, et al., Developmental neurotoxneuropathological and behavioral features of autism and other neurod10.1016/j.neuro.2015.12.014

and kept at �20 �C until immunostaining. To preclude litter-specific effects, only single pups/sex/litter were used in all of thesestudies. All mice used in this study were treated humanely andwith regard for alleviation of suffering and approved by theUniversity of Rochester Institutional Animal Care and UseCommittee.

2.2. Particle size distribution and number and mass concentration

Exposures in this study were to concentrated ambient UFPs(�100 nm). As previously reported (Allen et al., 2014a), meanparticle diameter for these exposures was consistently below100 nm; mean CAPS counts across the 8 days of exposures wasapproximately 200,000 particles/cm3, and the mean particle massconcentration was 96 mg/m3. These values are similar to valuesreported for expressway traffic in L. A. (Fruin et al., 2008;Westerdahl et al., 2005), and Minneapolis (Kittelson et al.,2004), and did not affect offspring body weights nor produceany overt toxicity.

2.3. Corpus callosum (CC) area determination

The size of the CC (approximate adult-equivalent Bregma range0.74–0.2 mm) was determined by tracing the area of interest in atleast 3 adjacent sections of slide-mounted brain using Neuroucida(MBF, Villiston, VT). Software enumerated the area of interest inmm2. Bregma for PND14 brains are approximate given that, to ourknowledge, no atlas at that specific point of early postnatal braindevelopment exists. Given difficulties in demarcation, all analysesof CC would also have included the cingulum, a primarilyipsilateral white matter tract lying above the CC that encirclesthe brain and provides communicative structure for components ofthe limbic system, critical to executive functions, decision-makingand emotion. Dysregulation of cingulate bundle white matterdevelopment has also been linked to developmental disordersincluding ASD (Billeci et al., 2012; Ikuta et al., 2014), schizophrenia(Samartzis et al., 2014) and ADHD (Chiang et al., 2015).

2.4. Myelin basic protein immunostaining & image analysis

To determine the extent of myelination, every 4th section(PND14) or every 6th section (PND270) was stained for myelin

-1 levels (group mean � S.E.) as percent of time point control plotted separately formissure and hippocampus (Panel A) and at PND 270 in corpus callosum (Panel B).

air exposed offspring at postnatal day 14; air-55 = brains from air exposed offspring to concentrated ambient ultrafine particles; CAPS-55 = brains collected at postnatalcted at postnatal day 55 from offspring exposed to concentrated ambient ultrafinep.

icity of inhaled ambient ultrafine particle air pollution: Parallels withevelopmental disorders, Neurotoxicology (2016), http://dx.doi.org/

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basic protein (MBP) to assess global development and develop-mental trajectory of myelination in the CC (separately examiningtruncus (immediately posterior to the genu), medial externalcapsule and lateral external capsule), striatum, and frontal cortex.Briefly, brain sections were washed of cryoprotectant and placedinto primary antibody for MBP (EMD Millipore; MAB386). Tissuewas then placed into biotinylated secondary antibody (Vector labs;BA-9401; 1:200 dilution) for 1 hour and the stain was visualizedusing 3-30-diaminobenzidine (DAB). Immunolabeled tissue wasmounted onto Superfrost Plus micro slides (VWR; Radnor, PA;48311-703) and cover-slipped using Cytoseal 60 (Fisher Scientific;Pittsburgh, PA; 23-244257).

Briefly, at least 3 images per region of interest were capturedusing an Olympus BX-41 brightfield microscope prior to imageanalysis. Expression of MBP was analyzed using Image Pro Plus 7.0(Mediacybernetics) using segmentation and thresholding toestimate a percent area of the region of interest that was positivefor MBP expression. For CC, percent of area myelinated wasdetermined, whereas total relative units of myelination weremeasured in frontal cortex and striatum.

2.5. Statistical analyses

Statistical analysis was carried out using JMP11 (Cary, NC) withinitial between groups ANOVAs that included all experimentalfactors (sex, group, PN) in full factorial designs. Main effects andinteractions were then pursued in post-hoc t-testing as appropri-ate. p values � 0.05 were considered statistically significant.

3. Results

3.1. Postnatal CAPS produces male-specific microglial (IBA-1)activation, reminiscent of inflammation and persistent microglialactivation seen in ASD (Zeidan-Chulia et al., 2014)

We previously reported persistent and male-specific IBA-1-labeling (a marker for microglia) in male but not female CAPS-treated mice, as found in anterior commissure at PND14 andhippocampus at PND55 (Fig. 1A) and in CC at PND 270 (Fig. 1B), atwhich point microglial numbers were approximately 350% of aircontrols, even though exposures had ended at PND13 (Allen et al.,2014a,b). Microglial activation has been frequently observed in thecontext of ASD. For example, adult males with ASD showed

Fig. 2. CAPS exposure leads to increase in lateral ventricle size; effects were graded with

to caudal section of air exposed males at PND 14 with lateral ventricle (LV) becoming visibCAPS-treated males at PND14. Note: LV and CC damage apparent rostrally; ArrowheadsMagnification 2�; CAPS = concentrated ambient ultrafine particles; PND = postnatal day

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increased binding of a microglial radiotracer in cerebellum,midbrain, pons, fusiform gyri and anterior cingulate and orbito-frontal cortices relative to age and IQ-matched healthy controls(Suzuki et al., 2013). Post-mortem brains from individuals withASD showed increased astrocyte- and microglial specific markersin frontal cortex (Edmonson et al., 2014; Young et al., 2011; Morganet al., 2010; Laurence and Fatemi, 2005). Inflammatory markershave also been detected in blood and urine in ASD (Vargas et al.,2005; Rossignol and Frye, 2014). Early microglial activation indeveloping CNS (Bilimoria and Stevens, 2014) could be the sourceof altered cytokine profiles where inflammatory signals have beenshown to predominate (Masi et al., 2014). Collectively, thesefindings are consistent with a persisting inflammatory state.

3.2. Postnatal CAPS and male-biased ventriculomegaly (VM) andwhite matter tract disruption mimic aberrant white matter tractdevelopment and VM noted in ASD (Aoki et al., 2013; Keller et al.,2007)

We found a number of white matter tract changes in CAPS-exposed mice that are consistent with changes seen in ASD:

3.2.1. Lateral ventricle sizeUnexpectedly, a male-specific VM of variable severity was seen

(Fig. 2) in CAPS males, but not in Air males or in any CAPS females(Allen et al., 2014a). Quantitation of VM size was determined at 3time points, and showed VM in males to be persistent, beingstatistically significantly lower than Air control values out to atleast PND 270 (Fig. 3) (Allen et al., 2014a).

3.2.2. CC size and developmental trajectoryBecause of the suggestion of a disrupted CC (Fig. 2E), CC area

was quantified. Prior studies have indicated rapid postnatal growthin CC with adult-like patterns of connections by PND15 (Wahlsten ,1984). In our studies, female CC size was slightly smaller thanmales at PND14 (Fig. 4A and C), but larger at PND 270 (Fig. 4B andD) as shown in the trajectory across time (Fig. 4E; sex by time point,F(1,41) = 57.9, p < 0.0001), findings consistent with previousreports of greater increases in CC size in female rats betweenadulthood and middle age (Yates and Juraska, 2007) that isconsidered to reflect the initially larger number and greater size ofmyelinated axons in males (Yates and Juraska, 2007) and more

the example demonstrating the most severe effects shown here. Panels A–C: Rostralle indicted by arrowhead in Panel C. Panels D–E matched rostral to caudal sections of

in Panel D shows bridge across ventricle (fusion) and coarctation in LV in Panel E.; FCtx = frontal cortex; Cpu = caudate putamen. n = 4–5/group.

icity of inhaled ambient ultrafine particle air pollution: Parallels withevelopmental disorders, Neurotoxicology (2016), http://dx.doi.org/

Fig. 3. CAPS exposure leads to male specific increased ventricular size. Lateral ventricle area (group mean � S.E) of males (left panel) and females (right panel) treated with airor postnatal CAPS were measured at three time points: PND 14 (defined 24 h post exposure), PND 55 and PND 270; n = 4–6/group, average of at least 3 sections/brain.CAPS = concentrated ambient ultrafine particles; PND = postnatal day. Male data modified from (Allen et al., 2014a) and female data unpublished.

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Fig. 4. CAPS treatment reduces size of CC in both sexes at PND14 with a trend towards greater magnitude effects in males. Panels A–D: CC size (mm2) of individual male andfemale mice following air of postnatal CAPS treatment as measured at PND14 and PND270. Each data point represents an individual value. Sample sizes of 3–5 for PND14 and7–11 for PND270. Panel E. Group mean � S.E. size of CC across time points for male and females in treatment groups as indicated. CAPS = concentrated ambient ultrafineparticles; PND = postnatal day; CC = corpus callosum.

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delayed maturation to the adult pattern in females (Kim andJuraska, 1997).

At PND14, both males and females exhibited statisticallysignificant CAPS-induced reductions in CC size (Fig. 4A and C),whereas no significant differences were found at PND270 (Fig. 4B

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and D). While CAPS-treated males showed a 30% size reductioncompared to 10% in CAPS-treated females at PND14 relative to Aircontrols (Fig. 4A and C), statistical analysis of PND14 dataconfirmed only an effect of treatment (F(1,14) = 8.1, p = 0.013),but not a treatment by sex interaction that would statistically

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confirm greater magnitude effects in males. However, as can beseen from the plotted individual data, differences among males atPND14 included a broader decrement with some overlap with Aircontrols, while evidence for female effects based on individual datawere less biologically compelling. As can be observed in Fig. 4E,there appears to be no disruption of the trajectory of CC size infemales, whereas an initial blunting is clear in males.

3.2.3. CC MyelinationThe potential for altered myelination patterns was examined

using MBP staining at three different sites along the CC, i.e., thetruncus, the medial external capsule (MEC) and the lateral externalcapsule (LEC). At PND14 (Fig. 5A and B), marked reductions ofapproximately 35% in MBP staining within the CC were seen inmales at the truncus that also extended to the medial externalcapsule (approximately 15%), whereas no significant differences inMBP staining area were seen in females at any point along the CC(overall ANOVA: treatment by sex by region, F(2,28) = 3.39,p = 0.048; post hoc p values for male PND14: site by treatment,p = 0.018; truncus, p = 0.005; medial external capsule, p = 0028). AtPND270 (Fig. 5C and D), no statistically significant treatmentrelated effects were found in either sex despite a visual trendtowards hypomyelination in CAPS-treated females.

Fig. 5E–G present time-course trajectory changes in myelina-tion for the truncus, medial external capsule and lateral externalcapsule, respectively. Consistent with the early sex-specifichypomyelination in males, significant treatment by sex � timepoint interactions were found for truncus and medial external

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Fig. 5. CAPS treatment induces male specific CC hypomyelination at PND14. Panels A–D. Farea at PND14 (Panels A,B) and PND270 (Panels C and D) for male and female air vs. postnexternal capsule and lateral external capsule for treatment groups as indicated. Shown imale and females in treatment groups as indicated for truncus, medial external capsule

indicates significant 3-way interaction in overall ANOVAs. Sample sizes of 3–5 for PNDultrafine particles; PND = postnatal day; CC = corpus callosum.

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capsule (F(1,42) = 12.73, p = 0.0009 and F(1,42) = 6.62, p = 0.0137,respectively). No CAPS-related changes in trajectory were found inthe lateral external capsule, but, consistent with observations intruncus and medial external capsule, increases in area myelinationoccurred between PND14 and PND270.

In contrast to the CC, striatal myelination levels of females,while significantly higher than those of males at both time points(Fig. 6A and B) and also when considered across time (Fig. 6C,overall ANOVA: sex by time point, F(1,40) = 4.19, p = 0.047), showedno effects of CAPS in either sex. Levels of MBP staining in fontalcortex were generally quite low (Fig. 7A and B), althoughincreasing between PND14 and PND270 (Fig. 7C), but did notdiffer by sex or CAPS treatment.

Some of these changes have also been reported to occur in ASD,including reductions in area and myelination of the corpuscallosum (CC), the largest white matter tract in the brain (Melilloand Leisman, 2009; Barnea-Goraly et al., 2004) and a tract that iscritical to inter-hemispheric communication/integration for sen-sory, motor and higher cognitive functions (Yates and Juraska,2007). The consequences of such hypomyelination and reduced CCinclude under-connectivity of the two hemispheres, and increasedlocalized connectivity. Both correlate with social and languagedeficits and altered hand preference/usage in ASD (Floris et al.,2013; Forrester et al., 2014; Geschwind and Levitt, 2007; Kanaet al., 2011; Wass, 2011), an inter-hemispherically-based behavior.The particular thinning of the genu of the CC in ASD is notable,given its importance to frontal cortical connectivity, and it has beenseen in conjunction with increased brain size and complexity of

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luorescent intensity of myelin basic protein labeling expressed as percent of total CCatal CAPS-treated mice as measured at 3 different sites along the CC: truncus, medials group mean � S.E. Panels E–G. Relative area of MBP labeling across time points for(MEC) and lateral external capsule (LEC), respectively. Treatment � sex � time point14 and 4–11 for PND270. Shown is group mean � S.E. CAPS = concentrated ambient

icity of inhaled ambient ultrafine particle air pollution: Parallels withevelopmental disorders, Neurotoxicology (2016), http://dx.doi.org/

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Fig. 6. CAPS exposure does not alter striatal myelination in either sex. Panels A–B: fluorescent intensity of myelin basic protein labeling expressed as arbitrary units instriatum at PND14 and PND270, respectively, for male and female air vs. postnatal CAPS-treated mice. Panel C: trajectory of density of myelin basic protein staining betweenPND14 and PND270 for males and females for indicated treatment groups. Sample sizes of 3–5 for PND14 and 4–11 for PND270. Shown is group mean � S.E.CAPS = concentrated ambient ultrafine particles; PND = postnatal day.

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Fig. 7. CAPS treatment does not alter frontal cortex myelination in either sex. Panels A–B: density of myelin basic protein staining (mm2) in frontal cortex at PND14 andPND270, respectively, for male and female air vs. postnatal CAPS-treated mice. Panel C: trajectory of density of myelin basic protein staining between PND14 and PND270 formales and females for indicated treatment groups. Sample sizes of 3–5 for PND14 and 4–11 for PND270. Data are group mean � S.E. CAPS = concentrated ambient ultrafineparticles; PND = postnatal day.

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gyrification (Vidal et al., 2006). Despite some inconsistencies inprofiles of white matter tract alterations in ASD (Koldewyn et al.,2014), diminished CC size has been a salient manifestation(Casanova and Pickett, 2013).

The observed VM we identified in CAPS exposure has also beenreported in ASD and while this is not a prototypical pathology inASD, it is a clinical indicator of white matter damage, and oftenaccompanies CC disruption in ASD, likely reflecting an expansion ofthe ventricles to fill available space (Leviton and Gilles, 1996) dueto delayed development and/or cell loss. In a low for birth weightcohort, ventricular enlargement in the first week of life wasassociated with a 7-fold increase in ASD diagnosis at 16 and 21years of age (Movsas et al., 2013), and children with VM are foundto be more likely to develop ASD (Piven et al., 1995).

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particles; PND = postnatal day; treatment = main effect of CAPS in statistical analysis;

modified from (Allen et al., 2014a) and (Allen et al., 2014b). Shown are group means �

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3.3. Postnatal CAPS persistently elevated glutamate levels and resultedin male-specific excitatory–inhibitory imbalance, a pathology that ismirrored in ASD (Page et al., 2006)

CAPS increased total levels of frontal cortex glutamate,glutamine and GABA levels in both sexes at various times followingpostnatal exposures, as we have previously reported (Allen et al.,2014a,b,c). In males, but not females, glutamate levels inhippocampus were also elevated, suggesting potential for greaterCAPS-associated excitotoxicity. Indeed, calculated as [Glu]/[GABA]ratios, only males showed significant hippocampal excitatory/inhibitory imbalance as shown for PND14 and PND60 (Fig. 8), asGABA increases were less pronounced than those of glutamate inmales. These findings are consistent with the male specificity of

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S.E.

icity of inhaled ambient ultrafine particle air pollution: Parallels withevelopmental disorders, Neurotoxicology (2016), http://dx.doi.org/

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microglial activation (Fig. 1) and its potential to produce glutamaterelease (Murugan et al., 2013; Takeuchi et al., 2005) seen in ASD.

It has been reported that children and adults with ASD showaltered glutamatergic balance, both in the periphery and CNS(Naushad et al., 2013; Kuwabara et al., 2013; Horder et al., 2013;Bejjani et al., 2012; Shimmura et al., 2011; Choudhury et al., 2012),which can correlate with altered social functioning (Horder et al.,2013). Single nucleotide polymorphisms in glutamate transportergenes (Jacob et al., 2011), and astrocyte specific dysfunctions thataffect glutamate metabolism (Kondapalli et al., 2013), have beenfound in genetic variants in ASD. These variants affect genes in theneurexin-neuroligin gene superfamily that are important toregulation of glutamatergic synapse formation (Chih et al.,2005). Collectively these finding contribute to the ‘hyperglutamatetheory’ of ASD (Jacob et al., 2011). As noted above, microglialactivation/inflammation could trigger glutamate release (Muruganet al., 2013; Takeuchi et al., 2005), and glutamate release furtheractivate microglia (Biber et al., 2007).

3.4. Postnatal CAPS produces astrocytic activation in amygdalaconsistent with the aberrant amygdala theory of ASD (Baron-Cohenet al., 2000; Cahill et al., 2004; Ruigrok et al., 2014)

We have identified significant increases in the astrocyte specificmarker GFAP in amygdala of CAPS-treated males at PND14 (Fig. 9).This adds yet another brain region impacted by UFPs that isconsidered key to aberrant behaviors involved in ASD (Baron-Cohen et al., 2000). Data from females and later time points are notyet examined, so the extent to which this effect is sex-differential isnot yet known.

ASD brains also show altered amygdala volume, marked byearly enlargement and normal or reduced volumes in adulthood,that can be linked to social abnormalities in ASD (Schumann et al.,2004, 2009). A recent report cited fewer amygdala neurons in somemales diagnosed with ASD (Morgan et al., 2010).

Fig. 9. CAPS exposure increased GFAP labeling in amygdala. Fluorescent intensity ofGFAP labeling in amygdala in male mice exposed to postnatal CAPS at PND14 andnormalized to air exposed controls. n = 5/gp. Shown is group mean � S.E. CAPS =concentrated ambient ultrafine particles; PND = postnatal day; GFAP = glialfibrillary acidic protein.

Please cite this article in press as: J.L. Allen, et al., Developmental neurotoxneuropathological and behavioral features of autism and other neurod10.1016/j.neuro.2015.12.014

3.5. Postnatal CAPS is associated with impulsive-like behavior that hasalso been reported in ASD

Where diagnosis is predicated on primary behavioral character-istics that include social deficits, impaired communication, andrepetition/perseveration (Lai et al., 2014). Executive functionsincluding set shifting can also be impaired in ASD and are frequentco-morbid features of the disorder (Vidal et al., 2006; Troyb et al.,2014; Ravizza et al., 2013; Rosenthal et al., 2013; Christ et al., 2011;Lemon et al., 2011; Scott-Van Zeeland et al., 2010).

The unexpected observations of disrupted CC white matterdevelopment, VM and excitatory/inhibitory imbalance inresponse to CAPS treatment were found only after ourcompletion of behavioral testing in our studies, and thereforeexplicit testing for these primary ASD behavioral domains hasnot yet been completed, nor were all behavioral assays thathave been implemented carried out in both sexes. Consequent-ly, the full extent to which postnatal CAPS reproduces ASDbehavioral features and its sex-dependence is not yet fullydelineated.

However, other behavioral dysfunctions associated with ASDhave been found in response to CAPS treatment, includingperseverative/impulsive behavior in CAPS males (females not yettested) on a Fixed Ratio waiting for reward paradigm thatrequired 25 lever press responses in an operant chamber toinitiate a waiting component during which free reward deliveriesoccurred. However, the time between each successive free rewarddelivery doubled during the wait component, thus requiringincreasingly longer wait times, and any lever press responseduring the waiting component re-set the 25 response require-ment to re-start the wait component. Postnatal CAPS markedlydecreased mean waiting time and increased numbers of fixedratio resets in males, despite the added cost in terms of numbersof responses required for each reward delivery (Allen et al., 2013).Fig. 10 presents cumulative records of an individual subject fromeach treatment group depicting this perseverative/impulsivebehavior, behaviors consistent with ASD-like repetition andimpulsivity.

Sex-dependent deficits in executive-type behavioral domainswere also found after CAPS, including alterations in learning,reversal learning, and memory even out to 14 mos (final timepoint of behavioral observations). Both sexes showed novelobject recognition deficits (considered to reflect short termmemory) (Allen et al., 2014b) and impairments in repeatedlearning (in preparation; data not shown), i.e., ability to learnnew 3-response chains for reward delivery in each successivebehavioral test session, but no deficits in performing a constantalready learned 3 response chains, consistent with a selectivelearning deficit.

4. Discussion

Postnatal exposures of mice to ambient UFP appear toreproduce many features consistent with characteristics ofASD, including inflammation/microglial activation (Fig. 1), re-duced CC size (Fig. 4), aberrant white matter development and/orstructural integrity (Figs. 2–5), hypomyelination (Fig. 5) and VM(Figs. 2–3), an altered ratio of glutamate and GABA, consistentwith excitatory/inhibitory imbalance (Fig. 8), and alterations inGFAP expression in the amygdala (Fig. 9), many of which wereeither male-specific or of greater magnitude in males. While weare still in the process of completing behavioral tests that defineASD-like behavior in animal models, we found deficits in learning,short-term memory (not shown) and, in males, repetitive andimpulsive-like behaviors (Fig. 10), which are part of the primarydiagnostic domain of ASD.

icity of inhaled ambient ultrafine particle air pollution: Parallels withevelopmental disorders, Neurotoxicology (2016), http://dx.doi.org/

Fig. 10. Cumulative records of individual male from indicated treatment groups on the fixed ratio (FR) wait schedule. Y axis response counter resets to baseline after each 30responses; pips (backslashes) = reward delivery, horizontal lines = periods of waiting, vertical lines = FR responding. Modified from (Allen et al., 2013).

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4.1. White matter damage

New analyses presented here reveal UFP-induced white matteralterations at PND14 selectively in commissural tracts (CC), witheffects that were of significantly greater magnitude in males,consistent with our previous observation of male-specific andpersistent microglial activation and VM (Allen et al., 2014a,b).These changes included both a significantly reduced CC size, aswell as male-specific hypomyelination of CC at PND 14, withmarked reductions particularly in the truncus and the medial, asopposed to the lateral external capsule. CC size ultimately reachedcontrol levels later in life following postnatal CAPS in males,whereas small but non-significant reductions in myelination weresuggestive in females at PND270, findings that could indicatedelayed neurotoxicity in this sex and perhaps repair in males. Incontrast, no changes were seen in white matter in either striatumor in frontal cortex in either sex.

The reduction of CC size and region-specific hypomyelinationcoupled with our prior observations of persistent microglialactivation and VM (Allen et al., 2014a) is markedly similar to theclinical conditions of diffuse periventricular leukomalacia or whitematter injury of prematurity/neonatal white matter damage(Leviton and Gilles, 1996; Rezaie and Dean, 2002) that can beproduced by fetal inflammatory or hypoxic-ischemic injury. VM isa clinical indicator of disrupted white matter development (Allenet al., 2014a,b) and can be accompanied by significant cognitivedeficits (Leviton and Gilles, 1996). Based on its well-knowninflammatory properties (Kelly and Fussell, 2015), UFP exposureswould be expected to activate brain microglia, resulting in therelease of proinflammatory cytokines (Baburamani et al., 2014;Harry and Kraft, 2012; Chew et al., 2013) that could lead tohypomyelination via toxicity to oligodendrocytes, the myelinatingcells of the brain (El Waly et al., 2014). Indeed, particularvulnerability of premyelinating olidgodendrocytes to such con-sequences of microglial activation has been described (Liu et al.,2013; Volpe et al., 2011). Such scenarios are considered theunderlying basis of disrupted white matter development associat-ed with prematurity (Rezaie and Dean, 2002).

Our collective findings raise several important questions. Forexample, what underlies the preferential male vulnerability to theearly reduction in CC size and hypomyelination? One hypothesisrelates to the greater number of colonizing activated microglia in

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male brain at the time of CAPS exposure (Schwarz et al., 2012).Neuro- and gliogenesis, which derive from germinal ventricularand subventricular zones next to the ventricle walls, occurs at peaklevels during the postnatal period when CAPS exposures occurred(Bandeira et al., 2009), and continues at a constant level into youngadulthood (Tabata et al., 2012; Tramontin et al., 2003), withactivated microglia actually required for neuro- and oligodendro-genesis through the release of cytokines (potentially in combina-tions), including IL-1b and IL-6 (Shigemoto-Mogami et al., 2014).However, the greater number of activated microglia, despite itsrequirement for neuro- and gliogenesis to proceed, could alsorender male brain microglia more vulnerable to dysfunction oreven cell death in response to an ongoing inflammatory insult.Females exhibit increased numbers and more activated microgliathan males only at a later postnatal age (PND30) (Schwarz et al.,2012), and thus CAPS exposures in these studies may havepreceded the period of female vulnerability.

Other questions raised by our data are what underlies thepreferential vulnerability of the truncus of the CC to CAPS-inducedhypomyelination, and, what accounts for the lack of vulnerabilityof striatal and frontal cortex (non-commissural) white mattermyelination? One potential contributor to these dichotomouseffects could be regional differences in timing of myelination inearly development relative to the CAPS exposure period. Interest-ingly, as in humans (Bird et al., 1989), myelination in mouse brainoccurs earliest in the caudal part of the CC (PND14) and only later inthe anterior segments, i.e., truncus and genu, e.g., between PND21-28 (Vincze et al., 2008). Myelination of regions such as striatumand cortex occurs even later, with studies showing myelinationlevels of only 1% in frontal cortex at PND21, up to about 15% atPND90, and up to approximately 70% at PND 180. At PND21,myelination of striatum is only about 5% of the area, and increasesto about 28% by PND90 (Mengler et al., 2014).

Thus, it may be that the absence of white matter myelinationchanges in striatum and frontal cortex reflects the fact that theseprocesses are occurring at a sufficiently later time relative to CAPSexposures and thus are spared, and/or that any ongoinginflammation levels have subsided in that period. With respectto the vulnerability of the truncus of the CC, its primarymyelination occurs 1–2 weeks post CAPS exposures, while morecaudal parts of CC are undergoing myelination during the period ofCAPS exposure itself. This also means that the entirety of CAPS

icity of inhaled ambient ultrafine particle air pollution: Parallels withevelopmental disorders, Neurotoxicology (2016), http://dx.doi.org/

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exposure (cumulative exposure) preceded the period associatedwith truncus (and genu) myelination, whereas a lesser ‘dose’ ofexposure occurred during myelination of more caudal regions.Thus, there may have been insufficient inflammation during theearlier periods of myelination, whereas a more protracted/higherlevel of inflammation period ended only days prior to myelinationof the truncus/genu. An additional consideration is that perinatalwhite matter injury can be accompanied by neuronal damage(Wahlsten, 1984; Leviton and Gressens, 2007), including corticalsubplate neurons that are critical to formation of axons for the CC,raising the possibility that these neurons were either dysfunctionalor dead the thus the infrastructure for myelination neverdeveloped, a hypothesis that has yet to be examined.

Hypomyelination at the truncus, i.e., just posterior to the genu,is of particular significance given that these fibers connect motorand prefrontal cortical areas, respectively, and preserve hemi-spheric connectivity (Pandya and Seltzer, 1986; Barbas and Pandya,1984; Paul et al., 2007). White matter alterations in these regionshave been associated in adolescents with a history of pretermbirth, e.g., with cognitive outcomes (Narberhaus et al., 2008) aswell as with deficits in social communication (Ewing-Cobbs et al.,2012). In the context of cognitive outcomes, it is notable that airpollution is also associated with prematurity and reductions inbirth weight (Shah and Balkhair, 2011; Stieb et al., 2012).

4.2. Air pollution as a broader risk factor: ASD shares features withother neurodevelopmental disorders

Different components of the observed profile of CAPS-inducedneuropathology are actually consistent with multiple neuro-developmental disorders, including ASD (Melillo and Leisman,2009; Aoki et al., 2013; Keller et al., 2007; Barnea-Goraly et al.,2004), schizophrenia (Lener et al., 2015), and attention deficitdisorder (Langevin et al., 2014; Chaim et al., 2014; Newbury andRosen, 2012; Paul, 2011; Gilliam et al., 2011; Qiu et al., 2011;Schnoebelen et al., 2010; Cao et al., 2010) among others, all ofwhich also show a higher prevalence in males, consistent with themale dominance of outcomes observed here. ASD and schizophre-nia, for example, particularly childhood onset schizophrenia, arefrequently considered components of a shared spectrum (Cheunget al., 2010; King and Lord, 2011; Meyer et al., 2011; Rapoport et al.,2009). Prior to 1971 these disorders were generally lumpedtogether as ‘childhood psychosis’, with ASD considered an earlymanifestation of schizophrenia. They can occur as co-morbidities,and behaviorally, both include deficits in social interactions,cognition, executive functions, emotional processing and sensori-motor gating. Both can involve auditory and visual hallucinations(Meyer et al., 2011; Barneveld et al., 2011), abnormalities ofserotonergic, dopaminergic and glutamatergic systems (Brownet al., 2013; Shimmura et al., 2013; Sodhi and Sanders-Bush., 2004)and shared genetic causes (de Lacy and King, 2013; Smoller et al.,2013). Schizophrenia and ASD also share neuropathologicalfeatures, including VM (Palmen et al., 2005; Sanfilipo et al.,2000; Shen et al., 2013), a clinical indicator of white matteralterations that are crucial to brain interhemispheric connectivity.

Adverse pregnancy outcomes, specifically preterm birth andreduced birth weight are often associated with brain white matteralterations (Elitt and Rosenberg, 2014; Volpe, 2003), as seen inconditions such as periventricular leukomalacia (Back, 2006), thathave been linked to inflammatory mechanisms (Melhem et al.,2000; Perlman, 1998; Wang et al., 2013), and can lead to cognitiveimpairments. In up to 50% of extremely and very preterm infants,alterations in brain white matter constitute the most importantbasis of axonal/neuronal damage associated with adverse neuro-cognitive and motor development (Back and Rosenberg, 2014). Asnoted above, links between prematurity and reduced birth weight

Please cite this article in press as: J.L. Allen, et al., Developmental neurotoxneuropathological and behavioral features of autism and other neurod10.1016/j.neuro.2015.12.014

with air pollution are increasingly reported (Shah and Balkhair,2011; Stieb et al., 2012). Alterations in brain white matter are alsoobserved in attention deficit hyperactivity disorder (Gilliam et al.,2011). Disconnectivity, i.e., functional and/or anatomical damageto white matter axons in communicating pathways found in ASDand schizophrenia (Geschwind and Levitt, 2007; Kana et al., 2011;Wass, 2011; Guo et al., 2013), is critical to language, social behaviorand some cognitive functions. VM associated with such deficitspersists after birth (Gilmore et al., 2001) and is more prevalent inmales (Gilmore et al., 1998). Prenatal VM is predictive of time tofirst-episode psychosis, and schizophrenia diagnosis (Sanfilipoet al., 2000; Gilmore et al., 1998, 2008; Fannon et al., 2000;Kyriakopoulou et al., 2013).

Animal models yielding a similar neuropathological profile tothat produced by CAPS treatment can result from exposures tohypoxia, ischemia, maternal-fetal infection and lipopolysaccharide(endotoxin) administration (Yates and Juraska, 2007). Frequentlyobserved in such pathology is an early necrosis in the subven-tricular zone adjacent to the lateral ventricle with depletion ofstem cells, followed by later proliferation, as also observed inconditions such as periventricular leukomalacia (Back, 2006).Numerous studies now demonstrate the role of reactive microglia,with a particular vulnerability of premyelinating oligodendroglia,to such insults (Leviton and Gilles, 1996; Back and Rosenberg,2014).

5. Conclusions

The findings reported here have two major implications: first,the data highlight the human 3rd trimester equivalent (Clancyet al., 2007a; Rice and Barone, 2000) as a period of potentialvulnerability to neurodevelopmental toxicity to ambient UFP,particularly in males, adding to a growing body of evidence thatbrain is a target of air pollution, and point to the possibility thatUFP exposure during periods of rapid neuro- and gliogenesis maybe a risk factor for neurodevelopmental disorders. The mostprominent period of myelination in the mouse begins atapproximately PND10 (Vincze et al., 2008; Bockhorst et al.,2008; Sturrock, 1980) and thus is encompassed by our CAPSexposures. One cited limitation of rodent models of inhaled airpollution has been that rodents, unlike humans, are obligate nose-breathers and thus dosing to mouse results in higher exposuresrelative to humans. However, this may be a minimal or negligentconcern according to a recent study based on computationalmodeling using an anatomically accurate model of the humannasal cavity that found that the olfactory dose per unit surface areais actually estimated to be higher in humans for 1–7 nm particlesbased on the larger inhalation rates of humans (Garcia et al., 2015).The latter is of particular note as emphasized in a recent editorial(Pedata et al., 2015), given the presence in fly ash and car exhaust ofa significant fraction of particles below 10 nm, often overlookedbecause of measurement difficulty (Ronkko et al., 2007).

Second, ASD phenotypic expression and severity shows broadvariability. If a risk factor for ASD, the variability of air pollutionlevels and composition across seasons/climate/geography couldassist in contributing to the heterogeneity of the phenotype, whichcould be further compounded by the developmental timing of theexposure. Current thinking posits that the multifactorial geneticand environmental risks for ASD superimposed on differentperiods of brain development, could ostensibly underlie thisvariability of the phenotype. Air pollution, as one such risk factor,could pose a greater threat in more highly polluted regions and/orin areas where it includes more toxic elements. Inconsistenciesacross ASD studies could in part reflect the convergence ofdifferent sets of risk factors occurring in geographically different

icity of inhaled ambient ultrafine particle air pollution: Parallels withevelopmental disorders, Neurotoxicology (2016), http://dx.doi.org/

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cohorts and significant differences in UFP exposures could alsooccur within even cohorts.

Conflict of interest

None.

Acknowledgements

Supported by T32 ES007026 (PI: B.P. Lawrence), P30 ES001247(PI: D.A. Cory-Slechta), K12 ES019852 (PI: D.A. Cory-Slechta), andR21 ES019105 (PI: D.A. Cory-Slechta).

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