Perinatal diesel exhaust exposure causes persistent changes in the brains of aged mice: An assessment of behavioral and biochemical endpoints related to neurodegenerative disease.

Epidemiological studies support an association between air pollution exposure, specifically particulate matter (PM), and neurodegenerative disease. Diesel exhaust (DE) is a principal component of ambient air pollution and a major contributor of PM. Our study aimed to examine whether early-life perinatal DE exposure is sufficient to affect behavioral and biochemical endpoints related to Alzheimer's disease later in life. To achieve this, mice were perinatally exposed (embryonic day 0-postnatal day 21) to DE (250-300 µg/m3 ) or filtered air (FA), and allowed to reach aged status (>18 months). Mice underwent behavioral assessment at 6 and 20 months of age, with tissue collected at 22 months for biochemical endpoints. At 6 months, minimal changes were noted in home-cage behavior of DE treated animals. At 20 months, an alternation deficit was noted with the T-maze, although no difference was seen in the object location task or any home-cage metrics. DE exposure did not alter the expression of Aß42, phosphorylated tau S199, or total tau. However, IBA-1 protein, a microglial activation marker, was significantly higher in DE exposed animals. Further, lipid peroxidation levels were significantly higher in the DE exposed animals compared to FA controls. Cytokine levels were largely unchanged with DE exposure, suggesting a lack of inflammation despite persistent lipid peroxidation. Taken together, the findings of this study support that perinatal exposure alone is sufficient to cause lasting changes in the brain, although the effects appear to be less striking than those previously reported in younger animals, suggesting some effects do not persist over time. These findings are encouraging from a public health standpoint and support the aggressive reduction of DE emissions to reduce lifetime exposure and potentially reduce disease outcome.

Rigor and reproducibility in rodent behavioral research.

Behavioral neuroscience research incorporates the identical high level of meticulous methodologies and exacting attention to detail as all other scientific disciplines. To achieve maximal rigor and reproducibility of findings, well-trained investigators employ a variety of established best practices. Here we explicate some of the requirements for rigorous experimental design and accurate data analysis in conducting mouse and rat behavioral tests. Novel object recognition is used as an example of a cognitive assay which has been conducted successfully with a range of methods, all based on common principles of appropriate procedures, controls, and statistics. Directors of Rodent Core facilities within Intellectual and Developmental Disabilities Research Centers contribute key aspects of their own novel object recognition protocols, offering insights into essential similarities and less-critical differences. Literature cited in this review article will lead the interested reader to source papers that provide step-by-step protocols which illustrate optimized methods for many standard rodent behavioral assays. Adhering to best practices in behavioral neuroscience will enhance the value of animal models for the multiple goals of understanding biological mechanisms, evaluating consequences of genetic mutations, and discovering efficacious therapeutics.

Prenatal and early life diesel exhaust exposure disrupts cortical lamina organization: Evidence for a reelin-related pathogenic pathway induced by interleukin-6.

Several epidemiological studies have shown associations between developmental exposure to traffic-related air pollution and increased risk for autism spectrum disorders (ASD), a spectrum of neurodevelopmental disorders with increasing prevalence rate in the United States. Though animal studies have provided support for these associations, little is known regarding possible underlying mechanisms. In a previous study we found that exposure of C57BL/6J mice of both sexes to environmentally relevant levels (250-300 µg/m3) of diesel exhaust (DE) from embryonic day 0 to postnatal day 21 (E0 to PND21) caused significant changes in all three characteristic behavioral domains of ASD in the offspring. In the present study we investigated a potential mechanistic pathway that may be of relevance for ASD-like changes associated with developmental DE exposure. Using the same DE exposure protocol (250-300 µg/m3 DE from E0 to PND21) several molecular markers were examined in the brains of male and female mice at PND3, 21, and 60. Exposure to DE as above increased levels of interleukin-6 (IL-6) in placenta and in neonatal brain. The JAK2/STAT3 pathway, a target for IL-6, was activated by STAT3 phosphorylation, and the expression of DNA methyltransferase 1 (DNMT1), a STAT3 target gene, was increased in DE-exposed neonatal brain. DNMT1 has been reported to down-regulate expression of reelin (RELN), an extracellular matrix glycoprotein important in regulating the processes of neuronal migration. RELN is considered an important modulator for ASD, since there are several polymorphisms in this gene linked to the disease, and since lower levels of RELN have been reported in brains of ASD patients. We observed decreased RELN expression in brains of the DE-exposed mice at PND3. Since disorganized patches in the prefrontal cortex have been reported in ASD patients and disrupted cortical organization has been found in RELN-deficient mice, we also assessed cortical organization, by labeling cells expressing the lamina-specific-markers RELN and calretinin. In DE-exposed mice we found increased cell density in deeper cortex (lamina layers VI-IV) for cells expressing either RELN or calretinin. These findings demonstrate that developmental DE exposure is associated with subtle disorganization of the cerebral cortex at PND60, and suggest a pathway involving IL-6, STAT3, and DNMT1 leading to downregulation of RELN expression that could be contributing to this long-lasting disruption in cortical laminar organization.

Prenatal and early-life diesel exhaust exposure causes autism-like behavioral changes in mice.

BACKGROUND: Escalating prevalence of autism spectrum disorders (ASD) in recent decades has triggered increasing efforts in understanding roles played by environmental risk factors as a way to address this widespread public health concern. Several epidemiological studies show associations between developmental exposure to traffic-related air pollution and increased ASD risk. In rodent models, a limited number of studies have shown that developmental exposure to ambient ultrafine particulates or diesel exhaust (DE) can result in behavioral phenotypes consistent with mild ASD. We performed a series of experiments to determine whether developmental DE exposure induces ASD-related behaviors in mice. RESULTS: C57Bl/6J mice were exposed from embryonic day 0 to postnatal day 21 to 250-300 µg/m3 DE or filtered air (FA) as control. Mice exposed developmentally to DE exhibited deficits in all three of the hallmark categories of ASD behavior: reduced social interaction in the reciprocal interaction and social preference tests, increased repetitive behavior in the T-maze and marble-burying test, and reduced or altered communication as assessed by measuring isolation-induced ultrasonic vocalizations and responses to social odors. CONCLUSIONS: These findings demonstrate that exposure to traffic-related air pollution, in particular that associated with diesel-fuel combustion, can cause ASD-related behavioral changes in mice, and raise concern about air pollution as a contributor to the onset of ASD in humans.

Acute exposure to diesel exhaust impairs adult neurogenesis in mice: prominence in males and protective effect of pioglitazone.

Adult neurogenesis is the process by which neural stem cells give rise to new functional neurons in specific regions of the adult brain, a process that occurs throughout life. Significantly, neurodegenerative and psychiatric disorders present suppressed neurogenesis, activated microglia, and neuroinflammation. Traffic-related air pollution has been shown to adversely affect the central nervous system. As the cardinal effects of air pollution exposure are microglial activation, and ensuing oxidative stress and neuroinflammation, we investigated whether acute exposures to diesel exhaust (DE) would inhibit adult neurogenesis in mice. Mice were exposed for 6 h to DE at a PM2.5 concentration of 250-300 µg/m3, followed by assessment of adult neurogenesis in the hippocampal subgranular zone (SGZ), the subventricular zone (SVZ), and olfactory bulb (OB). DE impaired cellular proliferation in the SGZ and SVZ in males, but not females. DE reduced adult neurogenesis, with male mice showing fewer new neurons in the SGZ, SVZ, and OB, and females showing fewer new neurons only in the OB. To assess whether blocking microglial activation protected against DE-induced suppression of adult hippocampal neurogenesis, male mice were pre-treated with pioglitazone (PGZ) prior to DE exposure. The effects of DE exposure on microglia, as well as neuroinflammation and oxidative stress, were reduced by PGZ. PGZ also antagonized DE-induced suppression of neurogenesis in the SGZ. These results suggest that DE exposure impairs adult neurogenesis in a sex-dependent manner, by a mechanism likely to involve microglia activation and neuroinflammation.

Modulation of paraoxonase 2 (PON2) in mouse brain by the polyphenol quercetin: a mechanism of neuroprotection?

Quercetin is a common flavonoid polyphenol which has been shown to exert neuroprotective actions in vitro and in vivo. Though quercetin has antioxidant properties, it has been suggested that neuroprotection may be ascribed to its ability of inducing the cell's own defense mechanisms. The present study investigated whether quercetin could increase the levels of paraoxonase 2 (PON2), a mitochondrial enzyme expressed in brain cells, which has been shown to have potent antioxidant properties. PON2 protein, mRNA, and lactonase activity were highest in mouse striatal astrocytes. Quercetin increased PON2 levels, possibly by activating the JNK/AP-1 pathway. The increased PON2 levels induced by quercetin resulted in decreased oxidative stress and ensuing toxicity induced by two oxidants. The neuroprotective effect of quercetin was significantly diminished in cells from PON2 knockout mice. These findings suggest that induction of PON2 by quercetin represents an important mechanism by which this polyphenol may exert its neuroprotective action.

Paraoxonase 2 (PON2) in the mouse central nervous system: a neuroprotective role?

The aims of this study were to characterize the expression of paraoxonase 2 (PON2) in mouse brain and to assess its antioxidant properties. PON2 levels were highest in the lung, intestine, heart and liver, and lower in the brain; in all tissues, PON2 expression was higher in female than in male mice. PON2 knockout [PON2(-/-)] mice did not express any PON2, as expected. In the brain, the highest levels of PON2 were found in the substantia nigra, the nucleus accumbens and the striatum, with lower levels in the cerebral cortex, hippocampus, cerebellum and brainstem. A similar regional distribution of PON2 activity (measured by dihydrocoumarin hydrolysis) was also found. PON3 was not detected in any brain area, while PON1 was expressed at very low levels, and did not show any regional difference. PON2 levels were higher in astrocytes than in neurons isolated from all brain regions, and were highest in cells from the striatum. PON2 activity and mRNA levels followed a similar pattern. Brain PON2 levels were highest around birth, and gradually declined. Subcellular distribution experiments indicated that PON2 is primarily expressed in microsomes and in mitochondria. The toxicity in neurons and astrocytes of agents known to cause oxidative stress (DMNQ and H(2)O(2)) was higher in cells from PON2(-/-) mice than in the same cells from wild-type mice, despite similar glutathione levels. These results indicate that PON2 is expressed in the brain, and that higher levels are found in dopaminergic regions such as the striatum, suggesting that this enzyme may provide protection against oxidative stress-mediated neurotoxicity.

Engineered recombinant human paraoxonase 1 (rHuPON1) purified from Escherichia coli protects against organophosphate poisoning.

The high-density lipoprotein-associated enzyme paraoxonase 1 (PON1) hydrolyzes lactones, aromatic esters, and neurotoxic organophosphorus (OP) compounds, including insecticide metabolites and nerve agents. Experiments with mice lacking PON1 (PON1(-/-) mice) have established that plasma PON1 protects against chlorpyrifos/chlorpyrifos-oxon and diazinon/diazoxon (DZO) exposure but does not protect against parathion/paraoxon or nerve agents. The catalytic efficiency of PON1 determines whether or not it will protect against a given OP exposure. Expression of active recombinant human PON1 (rHuPON1) in Escherichia coli provides a system in which PON1 can be engineered to achieve a catalytic efficiency sufficient to protect against or treat specific OP exposures. Here, we describe the generation of highly purified engineered rHuPON1(K192) that protects against DZO exposure when injected into PON1(-/-) mice. The injected rHuPON1 is nontoxic, persists in serum for at least 2 days after injection, and provides protection against DZO exposures of at least three times the median lethal dose value.

Evaluating Gait and Locomotion in Rodents with the CatWalk.

Motor deficits can significantly affect the completion of daily life activities and have a negative impact on quality of life. Consequently, motor function is an important behavioral endpoint to measure for in vivo pathophysiologic studies in a variety of research areas, such as toxicant exposure, drug development, disease characterization, and transgenic phenotyping. Evaluation of motor function is also critical to the interpretation of cognitive behavioral assays, as many rely on intact motor abilities to derive meaningful data. As such, gait analysis is an important component of behavioral research and can be achieved by manual or video-assisted methods. Manual gait analysis methods, however, are prone to observer bias and are unable to capture many critical parameters. In contrast, automated video-assisted gait analysis can quickly and reliably assess gait and locomotor abnormalities that were previously difficult to collect manually. Here, we describe the evaluation of gait and locomotion in rodents using the automated Noldus CatWalk XT system. We include a step-by-step guide for running an experiment using the CatWalk XT system and discuss theory and considerations when evaluating rodent gait. The protocol and discussion provided here act as a supplemental resource to the manual for this commercially available system and can assist CatWalk users in their experimental design and implementation. © 2021 Wiley Periodicals LLC.

Paraoxonase 2 deficiency in mice alters motor behavior and causes region-specific transcript changes in the brain.

Paraoxonase 2 (PON2) is an intracellular antioxidant enzyme shown to play an important role in mitigating oxidative stress in the brain. Oxidative stress is a common mechanism of toxicity for neurotoxicants and is increasingly implicated in the etiology of multiple neurological diseases. While PON2 deficiency increases oxidative stress in the brain in-vitro, little is known about its effects on behavior in-vivo and what global transcript changes occur from PON2 deficiency. We sought to characterize the effects of PON2 deficiency on behavior in mice, with an emphasis on locomotion, and evaluate transcriptional changes with RNA-Seq. Behavioral endpoints included home-cage behavior (Noldus PhenoTyper), motor coordination (Rotarod) and various gait metrics (Noldus CatWalk). Home-cage behavior analysis showed PON2 deficient mice had increased activity at night compared to wildtype controls and spent more time in the center of the cage, displaying a possible anxiolytic phenotype. PON2 deficient mice had significantly shorter latency to fall when tested on the rotarod, suggesting impaired motor coordination. Minimal gait alterations were observed, with decreased girdle support posture noted as the only significant change in gait with PON2 deficiency. Beyond one home-cage metric, no significant sex-based behavioral differences were found in this study. Finally, A subset of samples were utilized for RNA-Seq analysis, looking at three discrete brain regions: cerebral cortex, striatum, and cerebellum. Highly regional- and sex-specific changes in RNA expression were found when comparing PON2 deficient and wildtype mice, suggesting PON2 may play distinct regional roles in the brain in a sex-specific manner. Taken together, these findings demonstrates that PON2 deficiency significantly alters the brain on both a biochemical and phenotypic level, with a specific impact on motor function. These data have implications for future gene-environment toxicological studies and warrants further investigation of the role of PON2 in the brain.

The toxicity of mixtures of specific organophosphate compounds is modulated by paraoxonase 1 status.

Most chemical exposures involve complex mixtures. The role of paraoxonase 1 (PON1) and the Q192R polymorphism in the detoxication of individual organophosphorous (OP) compounds has been well-established. The extent to which PON1 protects against a given OP is determined by its catalytic efficiency. We used a humanized transgenic mouse model of the Q192R polymorphism to demonstrate that PON1 modulates the toxicity of OP mixtures by altering the activity of another detoxication enzyme, carboxylesterase (CaE). Chlorpyrifos oxon (CPO), diazoxon (DZO), and paraoxon (PO) are potent inhibitors of CaE, both in vitro and in vivo. We hypothesized that exposure of mice to these OPs would increase their sensitivity to the CaE substrate, malaoxon (MO), and that the degree of effect would vary among PON1 genotypes if the OP was a physiologically relevant PON1 substrate. When wild-type mice were exposed dermally to CPO, DZO, or PO and then, after 4 h, to different doses of MO, the toxicity of MO was increased compared to mice that received MO alone. The potentiation of MO toxicity by CPO and DZO was higher in PON1 knockout mice, which are less able to detoxify CPO or DZO. Potentiation by CPO was higher in Q192 mice than in R192 mice due to the decreased ability of PON1(Q192) to detoxify CPO. Potentiation by DZO was similar in the Q192 and R192 mice, due to their equivalent effectiveness at detoxifying DZO. PO exposure resulted in equivalent potentiation of MO toxicity among all four genotypes. These results indicate that PON1 status modulates the ability of CaE to detoxicate OP compounds from specific mixed insecticide exposures. PON1 status can also impact the capacity to metabolize drugs or other CaE substrates following insecticide exposure.

Engineering human PON1 in an E. coli expression system.

Expression and purification of recombinant human paraoxonase-1 (rHuPON1) from bacterial systems have proven elusive. Most systems for successful production of recombinant PON1 have relied on either eukaryotic expression in baculovirus or prokaryotic expression of synthetic, gene-shuffled rabbit-mouse-human PON1 hybrid molecules. We review here methods and protocols for the production of pure, native rHuPON1 using an E. coli expression system followed by conventional column chromatographic purification. The resulting rHuPON1 is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the organophosphorus (OP) compound diazoxon. Bacterially-derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that produces immunogenic complications when used as a therapeutic. The rHuPON1 should be useful for treating insecticide OP exposures and reducing risks of other diseases resulting from low PON1 status. The ease of mutagenesis in bacterial systems will also allow for the generation and screening of rHuPON1 variants with enhanced catalytic efficiencies against nerve agents and other OP compounds.

Developmental exposure to diesel exhaust upregulates transcription factor expression, decreases hippocampal neurogenesis, and alters cortical lamina organization: relevance to neurodevelopmental disorders.

BACKGROUND: Exposure to traffic-related air pollution (TRAP) during development and/or in adulthood has been associated in many human studies with both neurodevelopmental and neurodegenerative diseases, such as autism spectrum disorder (ASD) and Alzheimer's disease (AD) or Parkinson's disease (PD). METHODS: In the present study, C57BL/6 J mice were exposed to environmentally relevant levels (250+/-50 µg/m3) of diesel exhaust (DE) or filtered air (FA) during development (E0 to PND21). The expression of several transcription factors relevant for CNS development was assessed on PND3. To address possible mechanistic underpinnings of previously observed behavioral effects of DE exposure, adult neurogenesis in the hippocampus and laminar organization of neurons in the somatosensory cortex were analyzed on PND60. Results were analyzed separately for male and female mice. RESULTS: Developmental DE exposure caused a male-specific upregulation of Pax6, Tbr1, Tbr2, Sp1, and Creb1 on PND3. In contrast, in both males and females, Tbr2+ intermediate progenitor cells in the PND60 hippocampal dentate gyrus were decreased, as an indication of reduced adult neurogenesis. In the somatosensory region of the cerebral cortex, laminar distribution of Trb1, calbindin, and parvalbumin (but not of Ctip2 or Cux1) was altered by developmental DE exposure. CONCLUSIONS: These results provide additional evidence to previous findings indicating the ability of developmental DE exposure to cause biochemical/molecular and behavioral alterations that may be involved in neurodevelopmental disorders such as ASD.

Effects of air pollution on the nervous system and its possible role in neurodevelopmental and neurodegenerative disorders.

Recent extensive evidence indicates that air pollution, in addition to causing respiratory and cardiovascular diseases, may also negatively affect the brain and contribute to central nervous system diseases. Air pollution is comprised of ambient particulate matter (PM) of different sizes, gases, organic compounds, and metals. An important contributor to PM is represented by traffic-related air pollution, mostly ascribed to diesel exhaust (DE). Epidemiological and animal studies have shown that exposure to air pollution may be associated with multiple adverse effects on the central nervous system. In addition to a variety of behavioral abnormalities, the most prominent effects caused by air pollution are oxidative stress and neuro-inflammation, which are seen in both humans and animals, and are supported by in vitro studies. Among factors which can affect neurotoxic outcomes, age is considered most relevant. Human and animal studies suggest that air pollution may cause developmental neurotoxicity, and may contribute to the etiology of neurodevelopmental disorders, including autism spectrum disorder. In addition, air pollution exposure has been associated with increased expression of markers of neurodegenerative disease pathologies, such as alpha-synuclein or beta-amyloid, and may thus contribute to the etiopathogenesis of neurodegenerative diseases, particularly Alzheimer's disease and Parkinson's disease.

Paraoxonase 1 (PON1) modulates the toxicity of mixed organophosphorus compounds.

A transgenic mouse model of the human hPON1(Q192R) polymorphism was used to address the role of paraoxonase (PON1) in modulating toxicity associated with exposure to mixtures of organophosphorus (OP) compounds. Chlorpyrifos oxon (CPO), diazoxon (DZO), and paraoxon (PO) are potent inhibitors of carboxylesterases (CaE). We hypothesized that a prior exposure to these OPs would increase sensitivity to malaoxon (MO), a CaE substrate, and the degree of the effect would vary among PON1 genotypes if the OP was a physiologically significant PON1 substrate in vivo. CPO and DZO are detoxified by PON1. For CPO hydrolysis, hPON1(R192) has a higher catalytic efficiency than hPON1(Q192). For DZO hydrolysis, the two alloforms have nearly equal catalytic efficiencies. For PO hydrolysis, the catalytic efficiency of PON1 is too low to be physiologically relevant. When wild-type mice were exposed dermally to CPO, DZO, or PO followed 4-h later by increasing doses of MO, toxicity was increased compared to mice receiving MO alone, presumably due to CaE inhibition. Potentiation of MO toxicity by CPO and DZO was greater in PON1(-/-) mice, which have greatly reduced capacity to detoxify CPO or DZO. Potentiation by CPO was more pronounced in hPON1(Q192) mice than in hPON1(R192) mice due to the decreased efficiency of hPON1(Q192) for detoxifying CPO. Potentiation by DZO was similar in hPON1(Q192) and hPON1(R192) mice, which are equally efficient at hydrolyzing DZO. Potentiation by PO was equivalent among all four genotypes. These results indicate that PON1 status can have a major influence on CaE-mediated detoxication of OP compounds.

Developmental impact of air pollution on brain function.

Air pollution is an important contributor to the global burden of disease, particularly to respiratory and cardiovascular diseases. In recent years, evidence is accumulating that air pollution may adversely affect the nervous system as shown by human epidemiological studies and by animal models. Age appears to play a relevant role in air pollution-induced neurotoxicity, with growing evidence suggesting that air pollution may contribute to neurodevelopmental and neurodegenerative diseases. Traffic-related air pollution (e.g. diesel exhaust) is an important contributor to urban air pollution, and fine and ultrafine particulate matter (PM) may possibly be its more relevant component. Air pollution is associated with increased oxidative stress and inflammation both in the periphery and in the nervous system, and fine and ultrafine PM can directly access the central nervous system. This short review focuses on the adverse effects of air pollution on the developing brain; it discusses some characteristics that make the developing brain more susceptible to toxic effects, and summarizes the animal and human evidence suggesting that exposure to elevated air pollution is associated with a number of behavioral and biochemical adverse effects. It also discusses more in detail the emerging evidence of an association between perinatal exposure to air pollution and increased risk of autism spectrum disorder. Some of the common mechanisms that may underlie the neurotoxicity and developmental neurotoxicity of air pollution are also discussed. Considering the evidence presented in this review, any policy and legislative effort aimed at reducing air pollution would be protective of children's well-being.

Behavioral Phenotyping for Autism Spectrum Disorders in Mice.

Autism spectrum disorder (ASD) represents a heterogeneous group of disorders characterized by alterations in three behavioral symptom domains: Social interactions, verbal and nonverbal communication, and repetitive behaviors. Increasing prevalence of ASD in recent years suggests that exposure to environmental toxicants may be critical in modulating etiology of this disease. As clinical diagnosis of autism still relies on behavioral evaluation, it is important to be able to assess similar behavioral traits in animal models, to provide biological plausibility of associations between environmental exposures and ASD. Rodents naturally exhibit a large number of behaviors that can be linked to similar behaviors in human. In this unit, behavioral tests are described that are relevant to the domains affected in ASD. For the repetitive domain, the T-maze spontaneous alternation test and marble burying test are described. For the communication domain, neonatal ultrasonic vocalization and olfactory habituation test toward social and non-social odor are described. Finally, for the sociability domain, the three-chambered social preference test and the reciprocal interaction test are presented. © 2017 by John Wiley & Sons, Inc.

Developmental Neurotoxicity of Traffic-Related Air Pollution: Focus on Autism.

PURPOSE OF REVIEW: Epidemiological and animal studies suggest that air pollution may negatively affect the central nervous system (CNS) and contribute to CNS diseases. Traffic-related air pollution is a major contributor to global air pollution, and diesel exhaust (DE) is its most important component. RECENT FINDINGS: Several studies suggest that young individuals may be particularly susceptible to air pollution-induced neurotoxicity and that perinatal exposure may cause or contribute to developmental disabilities and behavioral abnormalities. In particular, a number of recent studies have found associations between exposures to traffic-related air pollution and autism spectrum disorders (ASD), which are characterized by impairment in socialization and in communication and by the presence of repetitive and unusual behaviors. The cause(s) of ASD are unknown, and while it may have a hereditary component, environmental factors are increasingly suspected as playing a pivotal role in its etiology, particularly in genetically susceptible individuals. Autistic children present higher levels of neuroinflammation and systemic inflammation, which are also hallmarks of exposure to traffic-related air pollution. Gene-environment interactions may play a relevant role in determining individual susceptibility to air pollution developmental neurotoxicity. Given the worldwide presence of elevated air pollution, studies on its effects and mechanisms on the developing brain, genetic susceptibility, role in neurodevelopmental disorders, and possible therapeutic interventions are certainly warranted.

Metals and Paraoxonases.

The paraoxonases (PONs) are a three-gene family which includes PON1, PON2, and PON3. PON1 and PON3 are synthesized primarily in the liver and a portion is secreted in the plasma, where they are associated with high-density lipoproteins (HDLs), while PON2 is an intracellular enzyme, expressed in most tissues and organs, including the brain. PON1 received its name from its ability to hydrolyze paraoxon, the active metabolite of the organophosphorus (OP) insecticide parathion, and also more efficiently hydrolyzes the active metabolites of several other OPs. PON2 and PON3 do not have OP-esterase activity, but all PONs are lactonases and are capable of hydrolyzing a variety of lactones, including certain drugs, endogenous compounds, and quorum-sensing signals of pathogenic bacteria. In addition, all PONs exert potent antioxidant effects. PONs play important roles in cardiovascular diseases and other oxidative stress-related diseases, modulate susceptibility to infection, and may provide neuroprotection (PON2). Hence, significant attention has been devoted to their modulation by a variety of dietary, pharmacological, lifestyle, or environmental factors. A number of metals have been shown in in vitro, animal, and human studies to mostly negatively modulate expression of PONs, particularly PON1, the most studied in this regard. In addition, different levels of expression of PONs may affect susceptibility to toxicity and neurotoxicity of metals due to their aforementioned antioxidant properties.

Neurotoxicity of traffic-related air pollution.

The central nervous system is emerging as an important target for adverse health effects of air pollution, where it may contribute to neurodevelopmental and neurodegenerative disorders. Air pollution comprises several components, including particulate matter (PM) and ultrafine particulate matter (UFPM), gases, organic compounds, and metals. An important source of ambient PM and UFPM is represented by traffic-related air pollution, primarily diesel exhaust (DE). Human epidemiological studies and controlled animal studies have shown that exposure to air pollution, and to traffic-related air pollution or DE in particular, may lead to neurotoxicity. In particular, air pollution is emerging as a possible etiological factor in neurodevelopmental (e.g. autism spectrum disorders) and neurodegenerative (e.g. Alzheimer's disease) disorders. The most prominent effects caused by air pollution in both humans and animals are oxidative stress and neuro-inflammation. Studies in mice acutely exposed to DE (250-300µg/m3 for 6h) have shown microglia activation, increased lipid peroxidation, and neuro-inflammation in various brain regions, particularly the hippocampus and the olfactory bulb. An impairment of adult neurogenesis was also found. In most cases, the effects of DE were more pronounced in male mice, possibly because of lower antioxidant abilities due to lower expression of paraoxonase 2.