Air Pollution-Related Brain Metal Dyshomeostasis as a Potential Risk Factor for Neurodevelopmental Disorders and Neurodegenerative Diseases

Increasing evidence links air pollution (AP) exposure to effects on the central nervous system structure and function. Particulate matter AP, especially the ultrafine (nanoparticle) components, can carry numerous metal and trace element contaminants that can reach the brain in utero and after birth. Excess brain exposure to either essential or non-essential elements can result in brain dyshomeostasis, which has been implicated in both neurodevelopmental disorders (NDDs; autism spectrum disorder, schizophrenia, and attention deficit hyperactivity disorder) and neurodegenerative diseases (NDGDs; Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and amyotrophic lateral sclerosis). This review summarizes the current understanding of the extent to which the inhalational or intranasal instillation of metals reproduces in vivo the shared features of NDDs and NDGDs, including enlarged lateral ventricles, alterations in myelination, glutamatergic dysfunction, neuronal cell death, inflammation, microglial activation, oxidative stress, mitochondrial dysfunction, altered social behaviors, cognitive dysfunction, and impulsivity. Although evidence is limited to date, neuronal cell death, oxidative stress, and mitochondrial dysfunction are reproduced by numerous metals. Understanding the specific contribution of metals/trace elements to this neurotoxicity can guide the development of more realistic animal exposure models of human AP exposure and consequently lead to a more meaningful approach to mechanistic studies, potential intervention strategies, and regulatory requirements.


Epidemiological Studies Increasingly Associate Air Pollution with Neurodevelopmental Disorders and Neurodegenerative Diseases
Over the past 10 years, it has become increasingly clear that the brain is a major target of air pollution (AP) [1,2]. For example, numerous epidemiological studies over the past several years have now linked the metrics of AP exposures with autism spectrum disorder (ASD) [3][4][5][6][7][8][9][10][11][12][13][14][15][16], a highly heterogeneously expressed neurodevelopmental disorder (NDD) [17][18][19] diagnosed on the basis of social communication/interaction deficits and restricted and repetitive behaviors, but that can also include hyperactivity, aggression, behavioral inflexibility, sensory-motor disturbances [20], impaired response inhibition, and impulsive responding [21,22]. AP has also now been associated with multiple reports of attention deficits or diagnoses of attention deficit hyperactivity disorder (ADHD) [23][24][25][26][27][28][29], another highly heterogeneous disorder [30], the features of which can be inattention, distractibility, impulsivity, and hyperactivity [31,32], but that can also include aggression, oppositional and externalizing behaviors, and alterations in social behaviors [33,34]}. Schizophrenia (SCZ), which     Studies from our laboratory in mice also demonstrate the potential for inhaled ambient AP to alter in vivo both the profile and level of metals in the brain [92]. Laser ablation ICP-MS analyses of a randomly chosen section of a male CAPs vs. air-exposed brains following postnatal CAPs exposures ( Figure 3) confirmed a marked elevation of metal levels in the brain, including Fe, S, Cu, Ca, and Al, while concurrently suggesting reductions in Mn and Zn.

Evidence for the Involvement of Metals in NDDs and NDGDs
A role for trace elements/metal alterations in the etiology of NDDs and NDGDs has been suggested in numerous studies over the years. Metal/elemental disturbances have been associated with NDDs [98], suggesting a role of metal dyshomeostasis in their etiology, albeit with much of the focus of studies on NDDs on peripheral markers rather than the brain. For example, alterations in the levels of metals and/or trace metals in serum or in hair or nails are reported and sometimes correlate with the features or severity of ASD [99][100][101][102][103][104][105][106][107]; the observed metal profiles can differ by sex [108] and geographic location. Alterations in the brain metal levels in ASD have been reported [109,110], as have correlations of trace metal levels with neuroinflammatory markers [111]. Similar findings are reported in SCZ, particularly for Zn and Cu [112][113][114][115][116][117][118][119], as well as in ADHD [120][121][122][123][124][125][126]. During development, the brain acquires mechanisms to tightly regulate the levels of essential metals. However, increases in the brain in the levels of either essential or non-essential metals, particularly redox-active but required metals-e.g., Fe and Cu-results in imbalance (i.e., brain metal dyshomeostasis) that can then progress to oxidative stress and neuroinflammation, disrupting brain development and subsequent function [127].
A strong case for brain trace metal disturbances has emerged for NDGDs. For example, numerous NDGDs are associated with excess brain Fe accumulation. Fe accumulates in the human brain with age; however, excesses above aging-related increases appear in multiple NDGDs and are thought to underlie associated oxidative stress, protein aggregation, demyelination, and neuronal loss [73,[128][129][130]. Both Fe and Cu have been reported to promote the formation and or accumulation of β-amyloid plaques, as well as the hyperphosphorylation of tau protein associated with AD [131], thereby contributing to the formation of intracellular plaques and tangles [73]. The accumulation of such metals can displace other essential metals such as Zn, mitigating its anti-inflammatory and anti-redox properties and its ability to precipitate aggregation intermediates [73]. Fe deposition has been shown to correlate with cognitive decline in AD [132]. Another metal that has repeatedly been implicated in AD is aluminum (Al) [110,[133][134][135], which can likewise contribute to β-amyloid formation and tau protein aggregation.
PD, which includes the loss of brain dopamine and the presence of Lewy bodies with inclusions, most notably of α-synuclein, likewise includes excess Fe accumulation, particularly in the substantia nigra, the site of nigrostriatal dopamine system cell bodies. Fe has been reported to directly bind α-synuclein, with a resulting pathophysiological structural reorganization and post-translational modifications of the protein (e.g., nitration, phosphorylation) [136]. Cu has also been associated with the oligomerization of α-synuclein, as well as oxidative stress [73]. While Mn appears to have a poor binding affinity to α-synuclein, studies show that it can trigger the misfolding and accumulation of this protein [137,138]. Mn also influences numerous neurotransmitter systems, including dopamine systems where striatal reductions in dopamine release and in the levels of the D2 dopamine receptor have been reported [139,140].
Excess Fe has also been considered a key driver in MS, with effects hypothesized to occur through mechanisms including oxidative stress, such as could occur due to the release of Fe from Fe-rich oligodendrocytes, the precursor cells for myelination. Excess Fe is also likely, via oxidative stress mechanisms, to increase proinflammatory cytokines and thereby promote demyelination [73,141]. Low levels of Zn may result from the excess accumulation of other metals in the brain that then suppress its anti-inflammatory protective capacity.
Several metals have been implicated in ALS. As with other NDGDs, this includes Fe, considered a biomarker for ALS [142], likely through Fenton reaction-generated oxidative stress [143]. Albeit considered less important, Cu may be involved through the formation of aggregates of superoxide dismutase 1 and Cu transport proteins [127]. In epidemiological studies, ALS has been associated with reduced serum Zn levels and increased Cu levels [144], as well as with blood and bone levels of Pb [145].
Calcium is another required element that is found in AP. No studies to date appear to have examined the impact of Ca overload in brain via instillation or inhalation. It is notable, however, that voltage-gated calcium channel gene variants have been linked to the pathogenesis of ASD [146], and proteomic analyses of ASD brains have identified the upregulation of proteins involved in Ca signaling [147]. Ca-binding protein dysregulation is also seen in the brains of individuals with SCZ [148]. Alterations in mitochondrial Ca homeostasis are considered to contribute to neuronal loss in PD [149], and has also been postulated to serve as a key mechanism in the etiology of AD [150] and ALS [151].

Could AP-Related Brain Metals Contribute to Brain Metal Dyshomeostasis?
As the above suggests, metals may play pathophysiological roles in NDDs and NDGDs. Fe (chemical species unknown) has been reported to increase in the human brain with normal aging [152][153][154][155][156], whereas the increases in Cu appear to be less pronounced, at least as measured in the human substantia nigra [157]; however, the findings have been equivocal, with reports of no increase [158] as well as increases in some cases [159]. Similarly, the levels of Zn in the human brain have been reported to show age-related increases [158], whereas in a study focused on the frontal cortex and hippocampus the Zn levels significantly declined with age [159]. Such discrepancies likely reflect differences in various parametric features of individual studies, such as the chemical formulation of the metal/trace element, dose, exposure durations, ages, and sample sizes, etc., but all findings are consistent with brain metal dyshomeostasis. The mechanism(s) of this aging-related brain metal dyshomeostasis remain unclear, but the possibilities proposed have been age-related loss of the homeostatic control of metal levels, including in the periphery and the brain. Another is aging-related pathophysiological changes in barriers such as the blood brain barrier, or the gastrointestinal tract [160][161][162].
However, what appears never to have been considered is the possible life-long exposures to metals via air pollution. Inhaled metal or trace element contaminants in AP can reach the brain at any stage of life. During gestation, metals can move across the placenta, even against a concentration gradient [163,164], into the fetal blood stream and then be distributed to developing organs. For example, non-heme (i.e., unbound) Fe is detectable in rat brain choroid plexus epithelial cells by gestational day (GD)14 and in the developing axonal tracts of fetal corpus callosum by GD19 [165]. S moves across the placenta in humans as sulfate via sulfate transporters [166]; the fetus relies directly on maternal sources [166]. Numerous other nanoparticle-adsorbed or incorporated metal and trace elements also show placental transfer and accumulation in fetal tissue-e.g., As, Pb, Cd, Hg, Cu, Ni, Mn, Al, and V [167][168][169][170][171][172][173][174][175][176]. As the Fe regulatory proteins 1 and 2 and Fe transporters such as the transferrin receptor and divalent metal transporter-1, critical to the tight brain regulation of Fe levels, for example, are not fully expressed until PND 15-20 in rodents [177,178], an extended window is open during which excess Fe can reach and directly influence brain development. Similarly, the homeostatic regulation of Fe absorption appears in human infants at about 6-9 months of age [179].
At least two routes allow AP-related metals and trace elements to reach the brain by inhalation following birth. Particle passage, particularly of UFP, proceeds from deposits in the alveolar region of the lung into the bloodstream [180], followed by distribution to secondary organs [181]. In addition, uptake via sensory nerves in the upper respiratory tract (olfactory and trigeminal [182]) is known to occur. For example, proof of principle nasal olfactory uptake of nanosized elemental AP particles has been demonstrated in both humans and rodents [183], which for the latter has included the uptake of Fe, Mn, Cd, Ni, Hg, Al, Co, Zn, and Cu [183][184][185][186][187][188][189][190] via inhalation or intransal instillation, moving across olfactory epithelial barriers in the nasal passages, likely via uptake by dendrites of neuronal olfactory cells reaching out with cilia directly into the nasal lumen, followed by transport to the olfactory bulb and then to other brain regions [191]. With olfactory or trigeminal nerve uptake, trace elements and metals actually bypass the tight blood brain barrier, such that increased levels of trace elements in the brain may not be reflected in peripheral markers. For example, the intranasal instillation of Fe 2 O 3 nanoparticles to rats over 7 days increased the Fe brain levels while significantly reducing the serum Fe levels [192]. Such discrepancies between the brain and serum levels are also seen in neurodegenerative diseases that include elevated brain Fe-e.g., PD [193][194][195]. In addition, sensory nerves in the upper and lower respiratory tract can translocate particles [196] that reach, e.g., the trigeminal ganglion, possibly via the hypoglossal nerve and tongue [182,191,197].
It is also interesting to consider AP as a source of neurotoxicity risk in the context of the dual-hit hypothesis proposed for the origins of PD [198]. According to this hypothesis, brain PD pathology actually originates in the olfactory bulb and brain stem via the vagal nerve ( Figure 4). Either such route could potentially result from AP exposure-i.e., the olfactory/trigeminal uptake of metal-containing UFPs-or potentially from vagal transfer into the brain stem [199][200][201], as has been demonstrated for metals [202] but is as yet unknown for NPs.
Collectively, such findings raise the question of whether AP-derived metals contribute a risk for NDDs or NDGDs. One way to begin to address this question is to examine the consequences of inhalational exposures to AP-related metals and trace elements in relation to the features of NDDs and NDGDs. This paper examines that premise based upon inhalation exposure studies; some studies using intranasal instillation are also included, but it is important to note that non-physiological bolus-type instillation exposures are significantly different in terms of the exorbitant dose and dose rate, as well as, importantly, deposition throughout the respiratory tract. Furthermore, particles in suspension will have altered surface chemistry and agglomeration/aggregation properties compared to those in an aerosol [203]. Nevertheless, intranasal instillation studies can be confirmatory of the trigeminal pathway of uptake, despite their poor real-world relevance. This chapter also did not include studies utilizing engineered NPs, given that such reformulations could change activity and outcome.
Atmosphere 2020, 11, x FOR PEER REVIEW 8 of 40 Figure 4. Schematic of brain with arrows depicting origins of the brain pathology-i.e., brain stem and olfactory bulb in Parkinson's disease based on extensive autopsies. Corresponding routes allow ultrafine particle air pollution to enter the brain while bypassing the blood brain barrier.
To facilitate this assessment, the current review focuses on features that are common across NDDs and NDGDs that should also assist in determining the breadth of metal-related AP exposures as a risk factor. To date, the number of studies based on inhalation or intranasal instillation is relatively limited. Further, the studies also differ in relation to the particular trace element or metal under study; comparisons can be hindered by the fact that exposure concentrations and specific metal chemistry and other exposure parameters can vary widely across such studies, rendering direct comparisons difficult. Nevertheless, the assessment of the ability of AP-related inhaled trace element and metal NP exposures to reproduce specific known features of NDDs and NGDGs also provides the basis for hypothesis-forming studies for more precisely defining future research directions.

Shared Features of Neurodevelopmental Disorders and Neurodegenerative Diseases
While NDDs and NDGDs both have unique features, they also share multiple neuropathological and behavioral characteristics and hypothesized mechanisms [19,24,34,, suggesting that if AP-related trace element exposures produce these characteristics, they would be a significant risk factor for a wide range of disorders with high economic and societal costs. For example, as shown in Table 1, ventriculomegaly-i.e., enlarged lateral ventricles-is found in ASD, ADHD, and SCZ. This has been shown to persist across life in individuals with ASD [292][293][294][295]; ventriculomegaly is considered a hallmark characteristic of SCZ [207][208][209][210][211][212]. Ventriculomegaly has also been seen in studies of ADHD [23, 204,205]. White matter reductions (i.e., loss of myelination) are seen not only in ASD [213][214][215][216][217], but are also a prominent feature of SCZ [219][220][221][222] and are reported in ADHD as well [214,218,296,297]. Ventriculomegaly is seen in AD [257,[298][299][300][301], as well as in PD [302,303], ALS [304], and MS [305,306]. Table 1. Shared characterristics of neurodevelopment disorders and neurodegenerative diseases and current understanding of the impact of inhaled nanoparticulate metals in reproducing these characteristics. Figure 4. Schematic of brain with arrows depicting origins of the brain pathology-i.e., brain stem and olfactory bulb in Parkinson's disease based on extensive autopsies. Corresponding routes allow ultrafine particle air pollution to enter the brain while bypassing the blood brain barrier.
To facilitate this assessment, the current review focuses on features that are common across NDDs and NDGDs that should also assist in determining the breadth of metal-related AP exposures as a risk factor. To date, the number of studies based on inhalation or intranasal instillation is relatively limited. Further, the studies also differ in relation to the particular trace element or metal under study; comparisons can be hindered by the fact that exposure concentrations and specific metal chemistry and other exposure parameters can vary widely across such studies, rendering direct comparisons difficult. Nevertheless, the assessment of the ability of AP-related inhaled trace element and metal NP exposures to reproduce specific known features of NDDs and NGDGs also provides the basis for hypothesis-forming studies for more precisely defining future research directions.

Current Evidence That AP-Related Metals and Trace Elements Can Reproduce Shared Characteristics of NDDs and NDGDs
Fe is the most abundant metal in the atmosphere [424] and is often present in the form of hematite (Fe 2 O 3 ) or magnetite (Fe 3 O 4 ), but can also be found as sulfates or carbonates. That atmospheric Fe associated with nanosized PM can reach the brain, and can do so via olfactory and trigeminal nerve uptake, bypassing the blood brain barrier, has been demonstrated. Mice of 4 weeks of age were exposed via intranasal instillation of the right nasal cavity to 40 mg/kg body weight of fine Fe 2 O 3 particles (mean diameter 280 nm), with outcomes assessed 14 days later [184]; these exposures produced increases in olfactory bulb and brain stem Fe concentrations, accompanied by neuronal degeneration in the hippocampus. A subsequent study comparing the effects of particle sizes of 40 vs. 280 nm Fe 2 O 3 in response to intranasal instillation in male mice found that smaller-sized Fe 2 O 3 particles were present in the axons of the olfactory neurons, as well as in the mitochondria and lysosomes in the hippocampus [425]. In another nose-only inhalation study, exposures of rats to 1.0 to 1.14 mg/m 3 of Fe 2 O 3 nanoparticles (mean diameter 14 nm) for 4 h/day, 5 days/week, for 3, 6, or 10 months demonstrated the presence of Fe NPs within damaged axonal sheaths of the olfactory bulb [426]. Similarly, a study of the whole-body inhalation of Fe (40 ug/m 3 ) soot (200 ug/m 3 ) for 6 h/day for 5 days/week for 5 weeks in female mice showed that Fe reached brain via the olfactory bulb, where it produced neuroinflammation, including increased numbers of activated microglial cells as well as levels of the pro-inflammatory cytokine IL-1β [427]. Studies of the human frontal cortex have found the presence of magnetite NPs in the human brain with a composition, according to the authors, consistent with external rather than internal formation [428]. Levels of the neurotransmitters dopamine and norepinephrine were markedly increased in male rats seven days after the intranasal instillation of 10 mg/kg of 30 nm-diameter Fe 2 O 3 for 7 days [192]. That such exposures can induce oxidative damage was demonstrated in a study in which intranasal instillation of 20 ug Fe 3 O 4 (462-570 nm in diameter) in rats for 7 days increased striatal and hippocampal H 2 O 2 , the latter measured via colorometric assay, and also reduced oxidized glutathione in the striatum [429]. Assessment of behavioral function appears to be limited to a single study that reported that airborne Fe levels were negatively associated with fine motor function across four cohorts of children from 1 to 9 years of age [430] (Summarized in Supplemental Table S1).
Collectively, while limited in scope, studies using environmentally relevant inhaled or less relevant intranasally instilled Fe indicate that nanoparticles (NPs) can reach the brain, bypass the blood brain barrier, and produce features consistent with the hypothesized mechanisms of both NDDs and NDGDs, which, based on current studies, include effects on myelination, neuronal cell death, inflammation, and microglial activation, as well as oxidative stress and mitochondrial dysfunction (Table 1 and  Supplemental Table S1). As noted above, such exposures would also be expected to be lifelong, as they can begin in utero and continue after birth. Further exacerbating the potential toxicity of elevated brain Fe in particular, studies have demonstrated its extremely slow turnover in rodent brains (half-time of ca. 9 mos), with estimates in the order of decades when extrapolated to humans, although this depends upon the underlying mechanisms of clearance and potential dissolution [431][432][433].
The Cu levels in AP can also be quite high depending upon geography [434]. Like any airborne nanosized particle, including Fe, Cu nanoparticles also reach brain via routes that include the olfactory bulb, where they produce effects consistent with those seen in NDDs and NDGDs. For example, female mice exposed via intranasal instillation to one of three doses (1, 10, and 40 mg/kg body weight, respectively) of Cu NPs (CuCl 2 ·2H 2 O) for a total of either 7 or 15 days showed significant increases in Cu concentrations in the olfactory bulb, as well as numerous neurochemical changes in multiple brain regions [435]. These included reductions in dopamine (DA) and homovanillic acid (HVA) with increases 3,4-dihydoxyphenylacetic acid (DOPAC), as well as increased intracellular DA turnover in the olfactory bulb, whereas DA, DOPAC, and HVA were all increased in the hippocampus and cerebellum, as was DA turnover in the cerebellum. Increases in DA were observed in the striatum and cortex at the highest concentrations. The levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were also markedly elevated in the olfactory bulb [435]. A subsequent study by this group using the same exposure concentrations administered every other day for 19 days found dose-related increases in Cu concentrations in the hippocampus, cortex, cerebellum, and striatum, accompanied by a modified elemental distribution in the brain of Fe, Zn, and Ca, consistent with brain metal dyshomeostasis [436]. Damaged neurons were particularly evident in the cortex following exposures, with similar trends in the hippocampus, while astrocytic activation was present throughout the hippocampus. Reductions in cortical and cerebellar dopamine were accompanied by marked elevations in its metabolites, as well as in both intracellular and extracellular DA turnover, whereas the levels of DA and metabolites were increased in the cortex, accompanied by reductions in turnover. Some increases in 5-HT and reductions in 5-HT turnover were also found in the cerebellum. In another study, female mice exposed via intranasal instillation to either 1 mg/kg body weight or 40 mg/kg of 25 nanometer Cu particles (CuCl 2 ·2H 2 O) or a comparable low dose of micro Cu nanoparticles (17 um), each delivered a total of three times across successive days, showed increased astrocytic activation and altered endoplasmic reticulum structure and dissociation in the hippocampus, as well as chromatin condensation and the aggregation of mitochondria in the olfactory bulb when examined 48 h after the termination of exposure [437], although 17 um particles are not likely to be inhalable by humans.
Interestingly, a study of 2897 children from 7 to 11 years of age from the BREATHE project revealed a positive trend between outdoor ambient Cu exposure and inattentiveness (p = 0.076), as measured by a computerized version of the Attentional Network test; more pronounced associations were found in children with polymorphisms of the ATPase copper-transporting beta gene (ATP7B) [438]. In another report incorporating 2836 children of 8-12 years of age, airborne Cu exposure was found to be significantly associated with poorer motor performance; in addition, higher Cu levels were significantly associated with higher fractional anisotropy, as measured via diffusion tensor imaging in white matter close to and within the caudate nucleus-i.e., areas surrounding the lateral ventricles [72]. In summary, to date, Cu nanoparticle inhalation reproduces features of NDDs and NDGDs that include alterations in myelination, neuronal cell death, inflammation, and microglial activation, as well as mitochondrial dysfunction, and have likewise been associated with inattention.
Studies of Zn NP exposure are limited and show that, like other metals, it too is transported via olfactory neurons directly into the brain [439,440]. In a recent study in 6-week-old male Wistar rats, daily intranasal instillation of ZnO NPs (10-30 nm in size; 20 ug/g body weight) was carried out for 15 or 30 days. Transmission electron micrographs were presented to show multiple ultrastructural changes in the olfactory bulb, hippocampus, striatum, and cortex that included mitochondrial changes, as well as changes in the endoplasmic reticulum, although these are difficult to visualize in the paper. Also reported was the presence of NPs in the cytoplasm and nerve fibers, along with histological changes in the hippocampus and striatum comprised of disordered cell arrangement, cell degeneration, and inflammatory cell infiltration. Changes in oxidative stress markers were quantified and revealed increased levels of malondialdehyde, TNF-α, and IL-1β in the olfactory bulb, striatum, hippocampus, and cortex at both 15 and 30 days, in conjunction with a corresponding reduction in glutathione [441]. Thus, inhaled Zn particles have been found to reproduce the neuronal cell death, inflammation, oxidative stress, and mitochondrial dysfunction features of NDDs and NDGDs.
Numerous studies have examined the impact of inhaled Mn, which has also been shown to be taken up via olfactory transport [442], as well as via the trigeminal nerve in Fischer 344/N rats and FVB/N mice [443], with the latter showing a clearance rate half-time of 9-10% per day that did not differ by sex. A MnCl 2 inhalation study in CD-1 male mice to 0.02M for 1 h twice/week for a 12-week period confirmed mitochondrial and Golgi damage in olfactory neurons, vacuolization beginning at week 4, and a significant increase in the percentage of apoptotic cells from 8 weeks on [444]. Assessments in mice in which exocytosis results in green fluorescent protein fluorescence in the olfactory bulb nerve terminals following the intranasal instillation of 2-200 µg of MnCl 2 demonstrated dose-dependent reductions in odorant-evoked neurotransmitter release even at the 2 µg levels, confirming a central rather than peripheral basis for these effects [445].
The subchronic inhalation exposure of male Sprague Dawley rats (34-38 days of age) to MnPO 4 at 3000, 300, 30, or 0.3 µg/m 3 of coarse and fine particulates for 6 h/day for 5 days/week for 13 consecutive weeks resulted in significant dose-dependent increases in the Mn concentrations in the olfactory bulb, frontal cortex, globus pallidus, caudate putamen, and cerebellum, but was not found to alter the neuronal cell counts in either the caudate or globus pallidus, nor to alter locomotor activity [446]. A subsequent study by this group using the same exposure paradigm but Mn SO 4 rather than MnPO 4 resulted in increases in the brain Mn in all regions at the two higher exposure concentrations, with reductions in neuronal counts in the globus pallidus and in ambulatory activity in both the diurnal and nocturnal periods [447]. MnSO 4 exposure concentrations of 0.03, 0.3, or 3.0 mg Mn/m3 for 6 h/day for 7 days/week for 14 consecutive days in 6-week-old male Crl;CD (SD)BR rats increased the brain Mn levels in the order of olfactory bulb > striatum > cerebellum at medium and high doses; glutamine synthetase protein levels were increased in the olfactory bulb, and, to a lesser extent, in the hypothalamus. Total glutathione levels were significantly reduced and metallothionein levels increased only in the hypothalamus [448]. A subsequent study using young male and female and aged male CD rats with exposures to MnSO 4 of 0.01, 0.1, and 0.5 mg Mn/m 3 or MnPO 4 at 0.1 mg Mn/m 3 showed effects of Mn that were sex-, age-, and brain region-dependent [449] in relation to alterations of glutamine synthetase protein level mRNA, metallothionein levels, and total glutathione [450], as well as in corresponding pharmacokinetics [451]. Exposures of CD rats to MnSO 4 (0.05, 0.5, or 1 mg/Mn/m 3 ; mean particle diameter 1.0 um) from 28 days prior to breeding until postnatal day 18 resulted in sex-dependent differences in offspring at postnatal day 19, consisting of reductions in TH mRNA and glutathione in the olfactory bulb and striatum, while reductions in the ratio of glutathione to oxidized glutathione were present in the hippocampus, olfactory bulb, and striatum [452]. Three weeks post-termination of exposure, reductions in glutamine synthetase and metallothionein mRNA levels in females in the striatum were still present at all exposure concentrations, while reductions in glutathione levels were present in males in the cerebellum and in females in the olfactory bulb [453].
Intranasal instillation of MnCl 2 solute (40 µL of aqueous 200mM) in rats resulted in olfactory bulb uptake as well as the impairment of olfactory discrimination [454]. Short term exposure of male Sprague Dawley rats (nose only to 1.2 µm, 39 mg/m 3 for 5 days/week for 3 weeks) produced a Mn concentration gradient decreasing from olfactory bulb to striatum; while this exposure did not produce changes in rotarod performance, reductions in dopamine transporter levels were present in striatum [455]. Of direct relevance to PD, over the course of exposure of adult male Wistar rats to 0.04 M MnCl 2 and 0.02 M Mn(OAc) 3 for 1 h 3× weekly for 6 months (72 inhalations), beam walk test time increased significantly, and numbers of successful reaches in a single-pellet reaching task declined. Furthermore, the numbers of tyrosine hydroxylase positive neurons declined dramatically in the substantia nigra, but were, as in PD, not affected in the ventral tegmental area [456]. A study in male Fischer 344 rats of inhalational exposures to~500 MnO ug/m 3 NPs (30 nm diameter) with either both nostrils patent or the right nostril occluded demonstrated that inhaled Mn is translocated via the olfactory neuronal pathway as a solid UFP to the central nervous system, where it produced inflammatory changes [457].
A series of studies in male rhesus monkeys of Mn solutes likewise confirmed olfactory uptake following inhalational exposure to MnSO 4 solute at ≥0.6 mg Mn/m 3 and increases in the brain regional Mn concentrations in a rostral-caudal plane [458]. As in studies with rodents, inhalational exposures of juvenile male rhesus monkey (0.06, 0.3 or 1.5 mg Mn/m 3 for 6 h/day for 5 days/week for 65 days, or 1.5 mg Mn/m 3 for 6 h/day for 5 days/week for 15, 33 or 65 days followed by a 45 or 90 day post exposure recovery period) led to brain region-specific changes in markers of oxidative stress and glutamate function, as well as evidence of reversibility post-exposure [459,460]. Some indirect findings in humans are indicated in a study of 70 children of 7-12 years of age living near a ferro-manganese alloy plant; in these studies, Mn in hair, used as a surrogate biomarker, was found to be inversely related to IQ and measures of verbal working memory. However, it must be recognized that the relationship between hair Mn and brain Mn is not clear, and the potential for other metal contaminants, e.g., excess Fe, is also possible [461]. As these studies indicate, Mn solutes and nanoparticles can likewise influence measures common to both NDDs and NDGDs, including to date, producing glutamatergic dysfunction and neuronal cell death as well as oxidative stress and mitochondrial impairments.
Sulfur is a trace element within AP as well as a requisite to human health [462], with sulfur dioxide (SO 2 ) levels regulated by the U.S. Environmental Protection Agency (EPA). When inhaled, SO 2 can be hydrated to sulfurous acid that then dissociates to produce sulfite/bisulfite and hydrogen ions; bisulfite can produce sulfur trioxide radical anions that react with oxygen leading to peroxyl radicals. When administered as SO 2 to young (3 mos), middle-aged (12 mos) or old (24 mos) Swiss albino rats (sex not specified) at 10 ppm for 1 h/day for 7 days/week for 6 weeks, increases in the antioxidant enzyme Cu, Zn-SOD and decreases in glutathione peroxidase were seen in all three age groups, as were increases in the lipid peroxidation marker, thiobarbituric acid reactive substances (TBARS) [463,464]. In contrast, reductions in these measures were seen in a study that exposed Kunming albino mice of both sexes to 22, 56 or 112 mg/m 3 of SO 2 for 6 h day for 7 days, with no systematic evidence of sex differences [465][466][467]. Further analyses revealed increased levels of sulfite in brains as measured 18 hr following the exposures [468]; exposures to 28 or 56 mg/m 3 were associated with brain ultrastructural changes that included glial cell damage, comprised of swelling and disrupted mitochondria, mitochondrial swelling within neurons, and the detachment of myelin along with mitochondrial vacuolization and outer membrane rupture [469].
Exposures of male Wistar rats to lower, but still relatively high levels of SO 2 (3.5, 7 or 14 mg/m 3 for 4 h/day for 30 days) resulted in increased mitochondrial biogenesis in the cortex, which the authors interpreted as an adaptive response to mitochondrial depletion following oxidative damage [470]. Assessment of synaptic plasticity at the two highest concentrations revealed ultrastructural changes in hippocampus (widening of the synaptic cleft, thinning of the postsynaptic density, a shortening of the synaptic active zone and reductions of synaptic vesicles), concentration-related reductions in synaptophysin expression, a marker of presynaptic terminals, as well as in protein phosphorylation markers related to synaptic plasticity (ERK1/2 and p-CREB) and the scaffolding marker PSD-95 [471]. Importantly, the role of exposure duration was examined by these authors in comparing the effects of 3.5 or 7 mg/m 3 for 6 h/day for 4 weeks to an equal mass concentration of 14 or 28 mg/m 3 for 6 h day for 1 week. In this comparison, measures such as glutamate receptor subunits mRNA in the hippocampus were inhibited in both exposure conditions, while synaptic plasticity markers such as protein kinase A and protein kinase C were changed in opposite directions. Importantly, this suggests that trying to equate the effects of acute exposures at higher concentrations to the effects of a lower concentration over a longer period of time, as is sometimes done to suggest that the more acute high level exposures are actually relevant to lower exposure concentrations over a more protracted period, is likely invalid [472,473], as previously reported for inhalation [474]. In another study by this group [475], exposures to SO 2 of 3.5 or 7.0 mg/m 3 (4-8 times the U.S. EPA standard) for 90 days was reported to impair memory in the probe trial of a water maze paradigm at the high concentration, while both concentrations reduced glutamate receptor subunit mRNA levels and increased levels of the pro-inflammatory cytokines TNFα, IL-1β, and IL-6 [475]. With respect to NDDs and NDGDs, S exposures have been shown to reproduce features including altered myelination, both glutamatergic and mitochondrial dysfunction, and oxidative stress.
Calcium is another required element that is found in AP. However, to date, no studies appear to have examined the impact of Ca overload in brain via instillation or inhalation, despite its potential involvement in both NDDs and NDGDs as cited above.
Studies have also examined the impact of such exposures to non-essential metals. Al has repeatedly been implicated in AD [133,476,477] and reported in high concentrations in brain in ASD [109]; it is considered to be a consistent component of ambient particulate matter. Like other metals, studies have demonstrated that exposure of rabbits to Al dust (otherwise not specified) at a dose of 0.56 mg Al/m 3 for 8 h/day for 5 days/week for 160 days resulted in a 247% increase in brain Al concentrations which were not seen in kidney, heart or bone [478]; notably, Al in serum was only slightly raised, indicating that serum Al was not a good marker for brain Al levels, findings similar to those reported since for other metals [193]. Intranasal instillation in 7-week-old male Sprague Dawley rats with Al NPs (5-100 nm diameter) at a dose of 1, 20 or 40 mg/kg body weight a total of 3 times within one week resulted in dose-related increases in olfactory bulb and whole brain levels of Al as determined 48 h post termination of exposure. It also resulted in significant changes in expression of genes primarily related to signal transduction, cell differentiation, transcription, transport, response to stress and apoptosis and in protein levels of MAPK signaling proteins p38 and ERK1, critical to signal transduction [479]. In a study of male and female mice exposed via inhalation to 0.5 mg/m 3 Al 2 O 3 NPs (average 40 nm diameter) for 6 h/day for 28 consecutive days, increases in cortex but not midbrain Al levels were seen in both sexes, but only females showed behavioral alterations that included increased time spent in the periphery of an open field, as well as increased immobility in a forced swim test, which the authors interpreted as a depressive-like behavioral phenotype [480]. H&E staining did not reveal any brain pathology, but numerous sex by Al exposure-related interactions were found in the altered expression of genes involved in neurotransmission, phosphatidic acid synthesis, and voltage-gated ion channel function. While a significant literature has accumulated demonstrating Al neurotoxicity, it has largely been via oral exposure routes, hence the ability to assess its impact on brain specifically from airborne pollution is as yet unknown.
Similarly, despite high emissions of Si, limited information is currently available on its in vivo toxicity via inhalation [481], although it is known to be translocated to brain via routes that include olfactory neurons [191]. Intranasal exposure of male Wistar rats to either 10 nm or 80 nm Si NPs (150 µg/50 µL) daily for 30 days [482] resulted in marked accumulation of Si in frontal cortex, hippocampus and striatum where it markedly increased levels of malondialdehyde, a marker of lipid peroxidation, as well as H 2 O 2 levels, while decreasing levels of antioxidant function, including Mn SOD, glutathione reductase, catalase, and reduced glutathione. Additionally, increases in the protein levels of NF-κB, TNF-α, and IL-1β were also observed, consistent with oxidative stress-based effects.
Some recent studies have examined the impact of PbO NPs. In one such study, mice were exposed in whole-body inhalation chambers to PbO for 4-72 h (acute exposure: 4.05 × 10 6 PbO NPs/cm 3 ) or for 1-11 weeks (subchronic) to a lower (3.83 × 10 5 particles/cm 3 ) or higher (1.93 × 10 6 particles/cm 3 ) concentration. Pb accumulation in brain increased over time in response to subchronic exposures, and histopathological analyses revealed changes in the hippocampus after 11 weeks consisting of spongiform changes consistent with degeneration, neuronal vacuolization and necrotic neurons [483]. In addition to producing an increase in brain Pb concentrations over time, a study of continuous inhalation exposure of mice to 1.93 × 106 nanoparticles/cm 3 for 2, 5, or 13 weeks reported increases in brain in the oxidative stress-related markers TBARS, 8-isoPGF 2α , and 8-isoPGE 2 [484]. A 5-day exposure for 4 h/day via nose only of female rats to 1.3 mg/m 3 PbO nanoparticles (mean diameter of 36 nm) revealed nanoparticle inclusion in neurons and vacuolized cytoplasm, as well as membrane demyelination [485]. Thus, inhaled Pb nanoparticles have been related to altered myelination, neuronal cell death, and oxidative stress.

Conclusions and Future Research Needs
To date, the number of studies examining in vivo inhalation exposures to the profile of trace elements and metals within AP in relation to central nervous system deficits is relatively limited, and comparisons can be hindered by the fact that the exposure concentrations and specific metal chemistry as well as exposure parameters do vary across studies, rendering direct comparisons difficult. Furthermore, not all studies have examined all of the shared characteristics (Table 1). Nevertheless, three generally consistent effects have begun to emerge across metals and trace elements in these studies to date-namely, neuronal death, oxidative stress, and mitochondrial dysfunction, thus including two features considered mechanistic drivers of NDDs and NDGDs. Similar trends for metals and trace elements reproducing characteristics of NDDs and NDGDs are also seen for alterations in myelination, as well as inflammation and microglial activation.
Unfortunately, however, there remain more questions in understanding than answers. For example, is one element more potent than others in producing these characteristics-i.e., which element most faithfully reproduces these characteristics? Alternatively, are combinations of metals responsible? It could be that single elements or even combinations of elements are primarily involved in specific NDDs or NDGDs, or that particular elements relate to specific features of these diseases and disorders. The determination of these relationships will require additional assessments of such shared features in response to exposures to individual metals and trace elements, followed by combinations of elements. In addition to the missing information, as seen in Table 1, very little information on the behavioral consequences of such exposures is available. An additional method that may help identify the role of an element in a specific NDD or NDGD is to examine its impact on behavioral functions known to be unique to specific NDDs or NDGDs, particularly the behavioral aberrations that are most characteristic of the disorder. For example, deficits in communications and social behaviors are distinctive in ASD, while alterations in pre-pulse inhibition are well documented in SCZ.
Most NDDs and NDGDs are highly heterogeneous in phenotypic expression. It is very likely in the case of NDDs in particular that this could reflect differences in the timing during the period of brain development when exposures to risk factors occur. In the case of metal and trace element contaminants from AP, there will be differences across populations of pregnant women based on geography in the profile of trace element metal exposures from AP, as well as in the timing of those exposures. Such differences in exposure timing mean that these metals and trace elements are impacting the brain at different points in development, and thus could contribute to the heterogeneity of NDDs; studies examining different periods of exposure during brain development will be critical to assess this possibility. In the case of NDGDs, in addition to differences in the profiles of trace elements within AP, the duration of exposures may be an important consideration, particularly as it relates to the cumulative level of increase in the brain metal concentrations or reductions. Understanding the role played by these factors will likewise be important.
Notably, with only a few exceptions, studies to date have utilized only males in assessments. While this might enhance sensitivity to the role of AP-related trace elements in NDDs, most of which are male-biased, it eliminates any understanding of the differences between the sexes that may serve as an underlying point of vulnerability and thus aid the development of therapeutic interventions. In addition, while NDDs certainly exist in females, the associated symptoms often differ by sex. Moreover, some NDGDs, particularly AD and MS, have a female bias. Consequently, the inclusion of both sexes in these studies is imperative for a full understanding.
Inhalation as the route of exposure for studies of the questions posed here is the only directly relevant method. Many studies have utilized non-physiological intranasal or intratracheal instillation routes as a substitute, but deposition directly onto tissue via an aqueous medium differs from that in an airborne state resulting from inhalation; the physico-chemical processes underlying target cell-toxicant interactions are different. As just one example, although entering the blood stream after deposition in the respiratory tract, nanoparticles are unlikely to enter the CNS via the very tight blood brain barrier; however, they can directly access the brain through olfactory and trigeminal neural pathways, bypassing the blood brain barrier. Such limitations are also relevant to studies reported to assess air pollution using in vitro approaches, as the significant particle chemistry that would have occurred in air and other biological sites before it reaches isolated cells of interest is lacking.
Once uptake occurs, NPs adsorb biomolecules, forming a protein corona; some of these biomolecules adsorb with greater affinity and some with lesser affinity, such that the protein corona remains in a state of flux with the composition varying with time, resulting in both nanoparticle-protein and protein-protein interactions [486]. The composition of the protein corona has been shown to be size-dependent [487] and is influenced by composition of the media in in vitro studies [488]. The composition of the protein corona influences cellular uptake [489] as well. Collectively, these factors influence the biological and physiological responses of NPs, including toxicity. Of particular relevance to the impact on the brain, a recent study of engineered NPs found that the composition of the protein corona of gold NPs underwent marked molecular modifications, both quantitative and qualitative, during passage from the "blood" to the "brain" side of an in vitro transwell model of the blood brain barrier [490]. Clearly, a far better understanding of the protein corona composition and its impact on effects and associated dynamic changes in vivo for different NPs are needed, particularly for the brain.
Finally, many studies to date have utilized high or extremely high levels of exposure, even recognizing the differences in the routes of uptake (nasal breathing vs. oro-nasal breathing) and other kinetic differences between humans and rodents. Clearly, exposure-dose-response information will be important for furthering public health protection in determining the need for regulations.