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Ozone (O 3 ) is a widespread air pollutant that produces cardiovascular and pulmonary dysfunction possibly mediated by activation of central stress centers. Epidemiological data suggest that sedentary lifestyles may exacerbate responses to air pollutants such as O 3 . We sought to assess neurological changes in response to O 3 exposure and an active lifestyle. We developed an animal model in which female Long-Evans rats were either sedentary or active with continuous access to running wheels starting at postnatal day (PND) 22 until the age of PND 100 and then exposed to O 3 (0, 0.25, 0.5 or 1.0 ppm) 5 h/day for two consecutive days. We found significantly more reactive microglia within the hippocampus (HIP) in animals exposed to O 3 in both sedentary and active rats. No changes were detected in astrocytic coverage. We next analyzed mitochondrial bioenergetic parameters (complex I, complex II and complex IV). Complex I activity was significantly affected by exercise in hypothalamus (HYP). Complex II activity was significantly affected by both exercise and O 3 exposure in the HIP. Concomitant with the changes in enzymatic activity, there were also effects on expression of genes related to mitochondrial bioenergetics and antioxidant production. These results demonstrate that O 3 induces microglia reactivity within stress centers of the brain and that mitochondrial bioenergetics are altered. Some of these effects may be augmented by exercise, suggesting a role for lifestyle in O 3 effects on brain mitochondrial bioenergetics parameters in agreement with our previous reports on other endpoints., (Published by Elsevier Inc.)

The Hard-Soft Acid and Base hypothesis can be used to predict the potential bio-reactivity (electrophilicity) of a chemical with intracellular proteins, resulting in neurotoxicity. Twelve chemicals predicted to be neurotoxic were evaluated in vitro in rat dorsal root ganglia (DRG) for effects on cytotoxicity (%LDH), neuronal structure (total neurite length/neuron, NLPN), and neurophysiology (mean firing rate, MFR). DRGs were treated acutely on days in vitro (DIV) 7 (1-100 μM) with test chemical; %LDH and NLPN were measured after 48 h. 4-cyclohexylhexanone (4-C) increased %LDH release at 50 (29%) and 100 μM (56%), citronellal (Cit) and 1-bromopropane increased %LDH at 100 μM (22% and 26%). 4-C, Cit, 2,5 Hexanedione (2,5Hex), phenylacetylaldehyde (PAA) and 2-ethylhexanal decreased mean NLPN at 48 h; 50 and 100 μM for 4-C (28% and 60%), 100 μM Cit (52%), 100 μM 2,5- Hex (37%) 100 μM PAA (41%) and 100 μM for 2-ethylhexanal (23%). Separate DRG cultures were treated on DIV 14 and changes in MFR measured. Four compounds decreased MFR at 50 or 100 μM: Acrylamide (-83%), 3,4-dichloro-1-butene (-93%), 4-C (-89%) and hexane (-79%, 50 μM). Changes in MFR and NLPN occurred in absence of cytotoxicity. While the current study showed little cytotoxicity, it gave insight to initial changes in MFR. Results provide insight for future chronic exposure experiments to evaluate neurotoxicity., Competing Interests: Declaration of Competing Interest None., (Published by Elsevier Ltd.)

Dietary supplementation with omega-3 and omega-6 fatty acids offer cardioprotection against air pollution, but these protections have not been established in the brain. We tested whether diets rich in omega-3 or -6 fatty acids offered neuroprotective benefits, by measuring mitochondrial complex enzyme I, II and IV activities and oxidative stress measures in the frontal cortex, cerebellum, hypothalamus, and hippocampus of male rats that were fed either a normal diet, or a diet enriched with fish oil olive oil, or coconut oil followed by exposure to either filtered air or ozone (0.8 ppm) for 4 h/day for 2 days. Results show that mitochondrial complex I enzyme activity was significantly decreased in the cerebellum, hypothalamus and hippocampus by diets. Complex II enzyme activity was significantly lower in frontal cortex and cerebellum of rats maintained on all test diets. Complex IV enzyme activity was significantly lower in the frontal cortex, hypothalamus and hippocampus of animals maintained on fish oil. Ozone exposure decreased complex I and II activity in the cerebellum of rats maintained on the normal diet, an effect blocked by diet treatments. While diet and ozone have no apparent influence on endogenous reactive oxygen species production, they do affect antioxidant levels in the brain. Fish oil was the only diet that ozone exposure did not alter. Microglial morphology and GFAP immunoreactivity were assessed across diet groups; results indicated that fish oil consistently decreased reactive microglia in the hypothalamus and hippocampus. These results indicate that acute ozone exposure alters mitochondrial bioenergetics in brain and co-treatment with omega-6 and omega-3 fatty acids alleviate some adverse effects within the brain.

There is growing interest in understanding how maternal diet might affect the sensitivity of offspring to environmental exposures. Previous studies demonstrated that adult rat offspring (approximately 6-months-old) from dams given a high-fat diet (HFD) prior to, during, and after pregnancy displayed elevated pulmonary responses to an acute ozone (O 3 ) exposure. The aim of this study was to examine the influence of maternal and perinatal HFD on pulmonary and metabolic responses to O 3 in male and female young-adult offspring (approximately 3-month old). One-month-old F0 female Long-Evans rats commenced HFD (60% kcal from fat) or control diet (CD; 10.5% kcal from fat) and were bred on PND 72. Offspring were maintained on respective HFD or CD until PND 29 when all groups were switched to CD. The 3-months-old female and male offspring (n = 10/group) were exposed to air or 0.8 ppm O 3 for 5hr/day for 2 consecutive days. Maternal and perinatal HFD significantly increased body weight and body fat % in offspring regardless of gender. Ozone exposure, but not maternal and perinatal diet, induced hyperglycemia and glucose intolerance in the offspring. Ozone-induced alterations in pulmonary function were exacerbated by maternal and perinatal HFD in both offspring genders. Pulmonary injury/inflammation markers in response to O 3 exposure such as bronchoalveolar lavage fluid total protein, lactate dehydrogenase, total cells, and neutrophils were further augmented in offspring (males>females) from dams fed the HFD. Data suggest that maternal and perinatal HFD may enhance the susceptibility of offspring to O 3 -induced pulmonary injury and that these effects may be sex-specific.

Oxidative stress (OS) contributes to the neurological and cardio/pulmonary effects caused by adverse metabolic states and air pollutants such as ozone (O₃). This study explores the interactive effects of O₃ and diet (high-fructose (FRUC) or high⁻fat (FAT)) on OS in different rat brain regions. In acute exposure, there was a decrease in markers of reactive oxygen species (ROS) production in some brain regions by diet and not by O₃. Total antioxidant substances (TAS) were increased in the cerebellum (CER) and frontal cortex (FC) and decreased in the striatum (STR) by both diets irrespective of O₃ exposure. Protein carbonyls (PC) and total aconitase decreased in some brain regions irrespective of exposure. Following subacute exposure, an increase in markers of ROS was observed in both diet groups. TAS was increased in the FC (FAT only) and there was a clear O₃ effect where TAS was increased in the FC and STR. Diet increased PC formation within the CER in the FAT group, while the hippocampus showed a decrease in PC after O₃ exposure in controls. In general, these results indicate that diet/O₃ did not have a global effect on brain OS parameters, but showed some brain region- and OS parameter-specific effects by diets.

There is a need for methods to screen and prioritize chemicals for potential hazard, including neurotoxicity. Microelectrode array (MEA) systems enable simultaneous extracellular recordings from multiple sites in neural networks in real time and thereby provide a robust measure of network activity. In this study, spontaneous activity measurements from primary neuronal cultures treated with three neurotoxic or three non-neurotoxic compounds was evaluated across four different laboratories. All four individual laboratories correctly identifed the neurotoxic compounds chlorpyrifos oxon (an organophosphate insecticide), deltamethrin (a pyrethroid insecticide) and domoic acid (an excitotoxicant). By contrast, the other three compounds (glyphosate, dimethyl phthalate and acetaminophen) considered to be non-neurotoxic ("negative controls"), produced only sporadic changes of the measured parameters. The results were consistent across the different laboratories, as all three neurotoxic compounds caused concentration-dependent inhibition of mean firing rate (MFR). Further, MFR appeared to be the most sensitive parameter for effects of neurotoxic compounds, as changes in electrical activity measured by mean frequency intra burst (MFIB), and mean burst duration (MBD) did not result in concentration-response relationships for some of the positive compounds, or required higher concentrations for an effect to be observed. However, greater numbers of compounds need to be tested to confirm this. The results obtained indicate that measurement of spontaneous electrical activity using MEAs provides a robust assessment of compound effects on neural network function., (Published by Elsevier B.V.)

Epidemiological and experimental data suggest that obesity exacerbates the health effects of air pollutants such as ozone (O 3 ). Maternal inactivity and calorically rich diets lead to offspring that show signs of obesity. Exacerbated O 3 susceptibility of offspring could thus be manifested by maternal obesity. Thirty-day-old female Long-Evans rats were fed a control (CD) or high-fat (HF) (60% calories) diet for 6 wks and then bred. GD1 rats were then housed with a running wheel (RW) or without a wheel (SED) until parturition, creating four groups of offspring: CD-SED, CD-RW, HF-SED and HF-RW. HF diet was terminated at PND 35 and all offspring were placed on CD. Body weight and %fat of dams were greatest in order; HF-SED > HF-RW > CD-SED > CD-RW. Adult offspring were exposed to O 3 for two consecutive days (0.8 ppm, 4 h/day). Glucose tolerance tests (GTT), ventilatory parameters (plethysmography), and bronchoalveolar fluid (BALF) cell counts and protein biomarkers were performed to assess response to O 3 . Exercise and diet altered body weight and %fat of young offspring. GTT, ventilation and BALF cell counts were exacerbated by O 3 with responses markedly exacerbated in males. HF diet and O 3 led to significant exacerbation of several BALF parameters: total cell count, neutrophils and lymphocytes were increased in male HF-SED versus CD-SED. Males were hyperglycemic after O 3 exposure and exhibited exacerbated GTT responses. Ventilatory dysfunction was also exacerbated in males. Maternal exercise had minimal effects on O 3 response. The results of this exploratory study suggest a link between maternal obesity and susceptibility to O 3 in their adult offspring in a sex-specific manner.

Pyrethroid insecticides exert their insecticidal and toxicological effects primarily by disrupting voltage-gated sodium channel (VGSC) function, resulting in altered neuronal excitability. Numerous studies of individual pyrethroids have characterized effects on mammalian VGSC function and neuronal excitability, yet studies examining effects of complex pyrethroid mixtures in mammalian neurons, especially in environmentally relevant mixture ratios, are limited. In the present study, concentration-response functions were characterized for five pyrethroids (permethrin, deltamethrin, cypermethrin, β-cyfluthrin and esfenvalerate) in an in vitro preparation containing cortical neurons and glia. As a metric of neuronal network activity, spontaneous mean network firing rates (MFR) were measured using microelectorde arrays (MEAs). In addition, the effect of a complex and exposure relevant mixture of the five pyrethroids (containing 52% permethrin, 28.8% cypermethrin, 12.9% β-cyfluthrin, 3.4% deltamethrin and 2.7% esfenvalerate) was also measured. Data were modeled to determine whether effects of the pyrethroid mixture were predicted by dose-addition. At concentrations up to 10μM, all compounds except permethrin reduced MFR. Deltamethrin and β-cyfluthrin were the most potent and reduced MFR by as much as 60 and 50%, respectively, while cypermethrin and esfenvalerate were of approximately equal potency and reduced MFR by only ∼20% at the highest concentration. Permethrin caused small (∼24% maximum), concentration-dependent increases in MFR. Effects of the environmentally relevant mixture did not depart from the prediction of dose-addition. These data demonstrate that an environmentally relevant mixture caused dose-additive effects on spontaneous neuronal network activity in vitro, and is consistent with other in vitro and in vivo assessments of pyrethroid mixtures., (Published by Elsevier B.V.)

The hippocampus maintains a capacity for neurogenesis throughout life, a capacity that is reduced in models of adult onset hypothyroidism. The effects of developmental thyroid hormone (TH) insufficiency on neurogenesis in the adult hippocampus, however, has not been examined. Graded degrees of TH insufficiency were induced in pregnant rat dams by administration of 0, 3 or 10ppm of 6-propylthiouracil (PTU) in drinking water from gestational day (GD) 6 until weaning. Body, brain, and hippocampal weight were reduced on postnatal day (PN) 14, 21, 78 and hippocampal volume was smaller at the 10 but not 3ppm dose level. A second experiment examined adult hippocampal neurogenesis following developmental or adult onset hypothyroidism. Two male offspring from 0 and 3ppm exposed dams were either maintained on control water or exposed to 3ppm PTU to create 4 distinct treatment conditions (Control-Control; Control-PTU, PTU-Control, PTU-PTU) based on developmental and adult exposures. Beginning on the 28th day of adult exposure to 0 or 3ppm PTU, bromodeoxyuridine (BrdU, 50mg/kg, ip) was administered twice daily for 5days, and one male from each treatment was sacrificed 24h and 28days after the last BrdU dose and brains processed for immunohistochemistry. Although no volume changes were seen in the hippocampus of the neonate at 3ppm, thinning of the granule cell layer emerged in adulthood. Developmental TH insufficiency produced a reduction in newly born cells, reducing BrdU+ve cells at 1 with no further reduction at 28-days post-BrdU. Similar findings were obtained using the proliferative cell marker Ki67. Neuronal differentiations was also altered with fewer doublecortin (Dcx) expressing cells and a higher proportion of immature Dcx phenotypes seen after developmental but not adult TH insufficiency. An impaired capacity for neurogenesis may contribute to impairments in synaptic plasticity and cognitive deficits previously reported by our laboratory and others following moderate degrees of developmental TH insufficiency induced by this PTU model., (Published by Elsevier B.V.)