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Objectives: Reports indicate that children of mothers who received docosahexaenoic acid (DHA) or egg yolk supplements during pregnancy have improved performance on cognitive tasks and brain growth; their combination has recently been demonstrated to modulate functional neuronal network connectivity in the human-relevant piglet brain. To expand upon this functional connectivity analysis, neurochemical evaluation to determine how dietary supplementation with one or both of these nutrients during the last trimester of pregnancy alters monoamine homeostasis in selected brain regions of piglets was done., Methods: Beginning gestation days 60-69 through weaning, pregnant sows were fed either control diet or diets supplemented with egg yolk powder, DHA, or both. Brains were then collected, and monoamine neurotransmitters and their metabolites were quantified from various brain regions with HPLC-ECD., Results: Relative to controls, egg yolk supplementation increased serotonin metabolite (5-HIAA) levels in the cerebellum, while DHA supplementation decreased serotonin (5-HT) levels in the prefrontal cortex; combined supplementation increased norepinephrine metabolite (MHPG) levels in the prefrontal cortex and cerebellum, but decreased 5-HT levels in the posterior hippocampus. Notably, all diets increased serotonin, dopamine, and their respective metabolite levels in the substantia nigra., Disscussion: This suggests both overlapping and specific effects of DHA and components of egg yolk in the context of maternal supplementation during pregnancy and lactation that might facilitate optimal neurodevelopment, with the nigrostriatal pathway being particularly sensitive. Such supplementations might impact brain function and facilitate development later in life through modulating monoamine homeostasis.

Manganese (Mn) is an essential and important metal; however, overexposures lead to adverse neurological outcomes. Nonoccupational Mn overexposure occurs primarily through consumption of Mn-contaminated drinking water (DW). Sex differences in terms of nervous and immune systems' responsiveness to excessive Mn in the DW are understudied. Thus, this study investigated behavioral and sex differences in response to Mn DW treatment (0.4 g Mn/L for up to 8 weeks) and a lipopolysaccharide (LPS) challenge of adult C57BL/6 mice with GFP-tagged monocytes/microglia. After 6 weeks, in motor function tests, Mn exposure resulted in decreased activity and gait deficits. In two different mood tests (open field test [OFT]/elevated zero maze), Mn-exposed mice exhibited decreased fear/anxiety-like behavior. Two weeks after behavioral assessment, when mice were challenged with LPS, circulating inflammatory cytokines, and acute phase proteins increased in both sexes. After 8 weeks of Mn exposure, liver and brain Mn levels were increased, but Mn alone did not affect circulating cytokines in either sex. Notably, Mn-exposed/LPS-challenged males had potentiated plasma cytokine output, whereas the reverse was seen in females. Males, but not females, continued to exhibit increased fearlessness (i.e., increased OFT center time), even when challenged with LPS. Overall, our results show that Mn DW exposure increases brain Mn levels and it leads to behavioral alterations in both sexes. However, males might be more susceptible to the effect of Mn on mood, and this effect is recalcitrant to an inflammagen challenge. Mn augmented post-LPS cytokine production only in males, further indicating that important Mn effects are sex-biased., (© 2024 The Author(s). Journal of Applied Toxicology published by John Wiley & Sons Ltd.)

γ-aminobutyric acid (GABA) is an abundant neurotransmitter that plays multiple roles in the vertebrate central nervous system (CNS). In the early developing CNS, GABAergic signaling acts to depolarize cells. It mediates several aspects of neural development, including cell proliferation, neuronal migration, neurite growth, and synapse formation, as well as the development of critical periods. Later in CNS development, GABAergic signaling acts in an inhibitory manner when it becomes the predominant inhibitory neurotransmitter in the brain. This behavior switch occurs due to changes in chloride/cation transporter expression. Abnormalities of GABAergic signaling appear to underlie several human neurological conditions, including seizure disorders. However, the impact of reduced GABAergic signaling on brain development has been challenging to study in mammals. Here we take advantage of zebrafish and light sheet imaging to assess the impact of reduced GABAergic signaling on the functional circuitry in the larval zebrafish optic tectum. Zebrafish have three gad genes: two gad1 paralogs known as gad1a and gad1b , and gad2. The gad1b and gad2 genes are expressed in the developing optic tectum. Null mutations in gad1b significantly reduce GABA levels in the brain and increase electrophysiological activity in the optic tectum. Fast light sheet imaging of genetically encoded calcium indicator (GCaMP)-expressing gab1b null larval zebrafish revealed patterns of neural activity that were different than either gad1b-normal larvae or gad1b -normal larvae acutely exposed to pentylenetetrazole (PTZ). These results demonstrate that reduced GABAergic signaling during development increases functional connectivity and concomitantly hyper-synchronization of neuronal networks.

Gulf War Illness (GWI) affects nearly 30% of veterans from the 1990-1991 Gulf War (GW) and is a multi-symptom illness with many neurological effects attributed to in-theater wartime chemical overexposures. Brain-focused studies have revealed persistent structural and functional alterations in veterans with GWI, including reduced volumes, connectivity, and signaling that correlate with poor cognitive and motor performance. GWI symptomology components have been recapitulated in rodent models as behavioral, neurochemical, and neuroinflammatory aberrations. However, preclinical structural imaging studies remain limited. This study aimed to characterize the progression of brain structural alterations over the course of 12 months in two established preclinical models of GWI. In the PB/PM model, male C57BL/6 J mice (8-9 weeks) received daily exposure to the nerve agent prophylactic pyridostigmine bromide (PB) and the pyrethroid insecticide permethrin (PM) for 10 days. In the PB/DEET/CORT/DFP model, mice received daily exposure to PB and the insect repellent DEET (days 1-14) and corticosterone (CORT; days 7-14). On day 15, mice received a single injection of the sarin surrogate diisopropylfluorophosphate (DFP). Using a Varian 7 T Bore MRI System, structural (sagittal T2-weighted) scans were performed at 6-, 9-, and 12-months post GWI exposures. Regions of interest, including total brain, ventricles, cortex, hippocampus, cerebellum, and brainstem were delineated in the open source Aedes Toolbox in MATLAB, followed by brain volumetric and cortical thickness analyses in ImageJ. Limited behavioral testing 1 month after the last MRI was also performed. The results of this study compare similarities and distinctions between these exposure paradigms and aid in the understanding of GWI pathogenesis. Major similarities among the models include relative ventricular enlargement and reductions in hippocampal volumes with age. Key differences in the PB/DEET/CORT/DFP model included reduced brainstem volumes and an early and persistent loss of total brain volume, while the PB/PM model produced reductions in cortical thickness with age. Behaviorally, at 13 months, motor function was largely preserved in both models. However, the GWI mice in the PB/DEET/CORT/DFP model exhibited an elevation in anxiety-like behavior., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Carpenter, Hughes and Filipov.)

Fescue toxicosis (FT) is produced by an ergot alkaloid (i.e., ergovaline [EV])-producing fungus residing in toxic fescue plants. Associations between EV, decreased weight gain and ruminal volatile fatty acids are unclear. Feces, rumen fluid, and blood were collected from 12 steers that grazed non-toxic (NT) or toxic (E +) fescue for 28 days. The E + group exhibited decreased propionate (P), increased acetate (A), and increased ruminal A:P ratio, with similar trends in feces. Plasma GASP-1 (G-Protein-Coupled-Receptor-Associated-Sorting-Protein), a myostatin inhibitor, decreased (day 14) only in E + steers. Ergovaline was present only in E + ruminal fluid and peaked on day 14. The lower ruminal propionate and higher A:P ratio might contribute to FT while reduced GASP-1 might be a new mechanism linked to E + -related weight gain reduction. Day 14 ergovaline zenith likely reflects ruminal adaptations favoring EV breakdown and its presence only in rumen points to local, rather than systemic effects., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fescue toxicosis is caused by grazing ergot alkaloid-producing endophyte ( Epichloë coenophiala )-infected tall fescue (E+). Summer grazing of E+ leads to decreased productivity, associated impaired thermoregulation, and altered behavior. The goal of this study was to determine the role of E+ grazing-climate interaction on animal behavior and thermoregulation during late fall. Eighteen Angus steers were placed on nontoxic (NT), toxic (E+) and endophyte-free (E-) fescue pastures for 28 days. Physiological parameters, such as rectal temperature (RT), respiration rate (RR), ear and ankle surface temperature (ET, AT), and body weights, were measured. Skin surface temperature (SST) and animal activity were recorded continuously with temperature and behavioral activity sensors, respectively. Environmental conditions were collected using paddocks-placed data loggers. Across the trial, steers on E+ gained about 60% less weight than the other two groups. E+ steers also had higher RT than E- and NT, and lower SST than NT post-pasture placement. Importantly, animals grazing E+ spent more time lying, less time standing, and took more steps. These data suggest that late fall E+ grazing impairs core and surface temperature regulation and increases non-productive lying time, which may be partly responsible for the observed decreased weight gains.

Chemical overexposures and war-related stress during the 1990-1991 Gulf War (GW) are implicated in the persisting pathological symptoms that many GW veterans continue to endure. These symptoms culminate into a disease known as Gulf War Illness (GWI) and affect about a third of the GW veteran population. Currently, comprehensive effective GWI treatment options are unavailable. Here, an established GWI mouse model was utilized to explore the (1) long-term behavioral and neuroinflammatory effects of deployment-related GWI chemicals exposure and (2) ability of the immunotherapeutic lacto-N-fucopentaose III (LNFPIII) to improve deficits when given months after the end of exposure. Male C57BL6/J mice (8-9 weeks old) were administered pyridostigmine bromide (PB) and DEET for 14 days along with corticosterone (CORT; latter 7 days) to emulate wartime stress. On day 15, a single injection of the nerve agent surrogate diisopropylfluorophosphate (DFP) was given. LNFPIII treatment began 7 months post GWI chemicals exposure and continued until study completion. A battery of behavioral tests for assessment of cognition/memory, mood, and motor function in rodents was performed beginning 8 months after exposure termination and was then followed by immunohistochemcal evaluation of neuroinflammation and neurogenesis. Within tests of motor function, prior GWI chemical exposure led to hyperactivity, impaired sensorimotor function, and altered gait. LNFPIII attenuated these motor-related deficits and improved overall grip strength. GWI mice also exhibited more anxiety-like behavior that was reduced by LNFPIII; this was test-specific. Short-term, but not long-term memory, was impaired by prior GWI exposure; LNFPIII improved this measure. In the brains of GWI mice, but not in mice treated with LNFPIII, glial activation was increased. Overall, it appears that months after exposure to GWI chemicals, behavioral deficits and neuroinflammation are present. Many of these deficits were attenuated by LNFPIII when treatment began long after GWI chemical exposure termination, highlighting its therapeutic potential for veterans with GWI., (© 2022 The Authors.)

Bovine fescue toxicosis (FT) is caused by grazing ergot alkaloid-producing endophyte (Epichloë coenophiala)-infected tall fescue. Endophyte's effects on the animal's microbiota and metabolism were investigated recently, but its effects in planta or on the plant-animal interactions have not been considered. We examined multi-compartment microbiota-metabolome perturbations using multi-'omics (16S and ITS2 sequencing, plus untargeted metabolomics) in Angus steers grazing non-toxic (Max-Q) or toxic (E+) tall fescue for 28 days and in E+ plants. E+ altered the plant/animal microbiota, decreasing most ruminal fungi, with mixed effects on rumen bacteria and fecal microbiota. Metabolic perturbations occurred in all matrices, with some plant-animal overlap (e.g., Vitamin B6 metabolism). Integrative interactomics revealed unique E+ network constituents. Only E+ had ruminal solids OTUs within the network and fecal fungal OTUs in E+ had unique taxa (e.g., Anaeromyces). Three E+-unique urinary metabolites that could be potential biomarkers of FT and targeted therapeutically were identified., (© 2022. The Author(s).)

Sex and age have distinct influences and roles in behavior and immune reactivity; yet, most studies use adult male rodents with little attention to middle age, a time associated with key physiological transitions in both sexes. Thus, this study investigated sex differences during middle age in behavior, immune response to lipopolysaccharide (LPS), and glucose regulation in C57BL/6 mice with GFP-tagged monocytes/microglia. Behaviorally, males performed better in tests of motor function (Open Field [OF], Grip Strength, Sticker Removal, Gait, and Pole tests) and displayed less depressive- and anxiety-like behaviors across multiple mood tests (OF, Elevated Zero Maze, Sucrose Preference, and Swim test). However, females performed better in tests of cognition (Barnes Maze and Novel Object Recognition). Following behavioral assessment, mice were given LPS to characterize sex-dependent inflammagen responses. Females displayed greater sickness behavior in the OF, higher levels of peripheral cytokines, and subtle neuroinflammation in the cortex, striatum, and hippocampus. A separate middle-aged cohort was used for glucose tolerance and insulin sensitivity testing. Both sexes had excessive blood glucose rebound after insulin challenge, but displayed differences following glucose administration, where males had higher baseline glucose and females remained hyperglycemic. This study suggests that during middle-age male mice have better emotional regulation and motor function, but not cognitive ability than females. Further, males are less sensitive than females to the acute effects of LPS peripherally and centrally, but both sexes showed sex-specific impairments in blood glucose regulation. Overall, it appears that middle age is an important transition point with multiple sex differences, some of which are unique to this stage of life., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Residual effects of the 1990-1991 Gulf War (GW) still plague veterans 30 years later as Gulf War Illness (GWI). Thought to stem mostly from deployment-related chemical overexposures, GWI is a disease with multiple neurological symptoms with likely immunological underpinnings. Currently, GWI remains untreatable, and the long-term neurological disease manifestation is not characterized fully. The present study sought to expand and evaluate the long-term implications of prior GW chemicals exposure on neurological function 6-8 months post GWI-like symptomatology induction. Additionally, the beneficial effects of delayed treatment with the glycan immunotherapeutic lacto-N-fucopentaose III (LNFPIII) were evaluated. Male C57BL/6J mice underwent a 10-day combinational exposure (i.p.) to GW chemicals, the nerve agent prophylactic pyridostigmine bromide (PB) and the insecticide permethrin (PM; 0.7 and 200 mg/kg, respectively). Beginning 4 months after PB/PM exposure, a subset of the mice were treated twice a week until study completion with LNFPIII. Evaluation of cognition/memory, motor function, and mood was performed beginning 1 month after LNFPIII treatment initiation. Prior exposure to PB/PM produced multiple locomotor, neuromuscular, and sensorimotor deficits across several motor tests. Subtle anxiety-like behavior was also present in PB/PM mice in mood tests. Further, PB/PM-exposed mice learned at a slower rate, mostly during early phases of the learning and memory tests employed. LNFPIII treatment restored or improved many of these behaviors, particularly in motor and cognition/memory domains. Electrophysiology data collected from hippocampal slices 8 months post PB/PM exposure revealed modest aberrations in basal synaptic transmission and long-term potentiation in the dorsal or ventral hippocampus that were improved by LNFPIII treatment. Immunohistochemical analysis of tyrosine hydroxylase (TH), a dopaminergic marker, did not detect major PB/PM effects along the nigrostriatal pathway, but LNFPIII increased striatal TH. Additionally, neuroinflammatory cells were increased in PB/PM mice, an effect reduced by LNFPIII. Collectively, long-term neurobehavioral and neurobiological dysfunction associated with prior PB/PM exposure was characterized; delayed LNFPIII treatment provided multiple behavioral and biological beneficial effects in the context of GWI, highlighting its potential as a GWI therapeutic., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Approximately one-third of Persian Gulf War veterans are afflicted by Gulf War Illness (GWI), a chronic multisymptom condition that fundamentally presents with cognitive deficits (i.e., learning and memory impairments) and neuroimmune dysfunction (i.e., inflammation). Factors associated with GWI include overexposures to neurotoxic pesticides and nerve agent prophylactics such as permethrin (PM) and pyridostigmine bromide (PB), respectively. GWI-related neurological impairments associated with PB-PM overexposures have been recapitulated in animal models; however, there is a paucity of studies assessing PB-PM-related aberrations in hippocampal synaptic plasticity and transmission that may underlie behavioral impairments. Importantly, FDA-approved neuroactive treatments are currently unavailable for GWI. In the present study, we assessed the efficacy of an immunomodulatory therapeutic, lacto-N-fucopentaose-III (LNFPIII), on ameliorating acute effects of in vivo PB-PM exposure on synaptic plasticity and transmission as well as trophic factor/cytokine expression along the hippocampal dorsoventral axis. PB-PM exposure resulted in hippocampal synaptic transmission deficits 48 h post-exposure, a response that was ameliorated by LNFPIII coadministration, particularly in the dorsal hippocampus (dH). LNFPIII coadministration also enhanced synaptic transmission in the dH and the ventral hippocampus (vH). Notably, LNFPIII coadministration elevated long-term potentiation in the dH. Further, PB-PM exposure and LNFPIII coadministration uniquely altered key inflammatory cytokine and trophic factor production in the dH and the vH. Collectively, these findings demonstrate that PB-PM exposure impaired hippocampal synaptic responses 48 h post-exposure, impairments that differentially manifested along the dorsoventral axis. Importantly, LNFPIII ameliorated GWI-related electrophysiological deficits, a beneficial effect indicating the potential efficacy of LNFPIII for treating GWI., (Copyright © 2021. Published by Elsevier B.V.)

Aims: The present study investigated if treatment with the immunotherapeutic, lacto-N-fucopentaose-III (LNFPIII), resulted in amelioration of acute and persisting deficits in synaptic plasticity and transmission as well as trophic factor expression along the hippocampal dorsoventral axis in a mouse model of Gulf War Illness (GWI)., Main Methods: Mice received either coadministered or delayed LNFPIII treatment throughout or following, respectively, exposure to a 15-day GWI induction paradigm. Subsets of animals were subsequently sacrificed 48 h, seven months, or 11 months post GWI-related (GWIR) exposure for hippocampal qPCR or in vitro electrophysiology experiments., Key Findings: Progressively worsened impairments in hippocampal synaptic plasticity, as well as a biphasic effect on hippocampal synaptic transmission, were detected in GWIR-exposed animals. Dorsoventral-specific impairments in hippocampal synaptic responses became more pronounced over time, particularly in the dorsal hippocampus. Notably, delayed LNFPIII treatment ameliorated GWI-related aberrations in hippocampal synaptic plasticity and transmission seven and 11 months post-exposure, an effect that was consistent with enhanced hippocampal trophic factor expression and absence of increased interleukin 6 (IL-6) in animals treated with LNFPIII., Significance: Approximately a third of Gulf War Veterans have GWI; however, GWI therapeutics are presently limited to targeted and symptomatic treatments. As increasing evidence underscores the substantial role of persisting neuroimmune dysfunction in GWI, efficacious neuroactive immunotherapeutics hold substantial promise in yielding GWI remission. The findings in the present report indicate that LNFPIII may be an efficacious candidate for ameliorating persisting neurological abnormalities presented in GWI., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Physiological homeostasis decline, immunosenescence, and increased risk for multiple diseases, including neurodegeneration, are all hallmarks of ageing. Importantly, it is known that the ageing process is sex-biased. For example, there are sex differences in predisposition for multiple age-related diseases, including neurodegenerative and autoimmune diseases. However, sex differences in age-associated immune phenotypes are not clearly understood., Results: Here, we examined the effects of age on immune cell phenotypes in both sexes of C57BL/6J mice with a particular focus on NK cells. We found female-specific spleen weight increases with age and concordant reduction in the number of splenocytes per gram of spleen weight compared to young females. To evaluate sex- and age-associated changes in splenic immune cell composition, we performed flow cytometry analysis. In male mice, we observed an age-associated reduction in the frequencies of monocytes and NK cells; female mice displayed a reduction in B cells, NK cells, and CD8 + T cells and increased frequency of monocytes and neutrophils with age. We then performed a whole blood stimulation assay and multiplex analyses of plasma cytokines and observed age- and sex-specific differences in immune cell reactivity and basal circulating cytokine concentrations. As we have previously illustrated a potential role of NK cells in Parkinson's disease, an age-related neurodegenerative disease, we further analyzed age-associated changes in NK cell phenotypes and function. There were distinct differences between the sexes in age-associated changes in the expression of NK cell receptors, IFN-γ production, and impairment of α-synuclein endocytosis., Conclusions: This study demonstrates sex- and age-specific alterations in splenic lymphocyte composition, circulating cytokine/chemokine profiles, and NK cell phenotype and effector functions. Our data provide evidence that age-related physiological perturbations differ between the sexes which may help elucidate sex differences in age-related diseases, including neurodegenerative diseases, particularly Parkinson's disease, where immune dysfunction is implicated in their etiology.

Rapid scientific advances are increasing our understanding of the way complex biological interactions integrate to maintain homeostatic balance and how seemingly small, localized perturbations can lead to systemic effects. The 'omics movement, alongside increased throughput resulting from statistical and computational advances, has transformed our understanding of disease mechanisms and the multi-dimensional interaction between environmental stressors and host physiology through data integration into multi-dimensional analyses, i.e., integrative interactomics. This review focuses on the use of high-throughput technologies in farm animal research, including health- and toxicology-related papers. Although limited, we highlight recent animal agriculture-centered reports from the integrative multi-'omics movement. We provide an example with fescue toxicosis, an economically costly disease affecting grazing livestock, and describe how integrative interactomics can be applied to a disease with a complex pathophysiology in the pursuit of novel treatment and management approaches. We outline how 'omics techniques have been used thus far to understand fescue toxicosis pathophysiology, lay out a framework for the fescue toxicosis integrome, identify some challenges we foresee, and offer possible means for addressing these challenges. Finally, we briefly discuss how the example with fescue toxicosis could be used for other agriculturally important animal health and welfare problems.

The microbiota's influence on host (patho) physiology has gained interest in the context of Gulf War Illness (GWI), a chronic disorder featuring dysregulation of the gut-brain-immune axis. This study examined short- and long-term effects of GWI-related chemicals on gut health and fecal microbiota and the potential benefits of Lacto-N-fucopentaose-III (LNFPIII) treatment in a GWI model. Male C57BL/6J mice were administered pyridostigmine bromide (PB; 0.7 mg/kg) and permethrin (PM; 200 mg/kg) for 10 days with concurrent LNFPIII treatment (35 μg/mouse) in a short-term study (12 days total) and delayed LNFPIII treatment (2×/week) beginning 4 months after 10 days of PB/PM exposure in a long-term study (9 months total). Fecal 16S rRNA sequencing was performed on all samples post-LNFPIII treatment to assess microbiota effects of GWI chemicals and acute/delayed LNFPIII administration. Although PB/PM did not affect species composition on a global scale, it affected specific taxa in both short- and long-term settings. PB/PM elicited more prominent long-term effects, notably, on the abundances of bacteria belonging to Lachnospiraceae and Ruminococcaceae families and the genus Allobaculum . LNFPIII improved a marker of gut health (i.e., decreased lipocalin-2) independent of GWI and, importantly, increased butyrate producers (e.g., Butyricoccus , Ruminococcous ) in PB/PM-treated mice, indicating a positive selection pressure for these bacteria. Multiple operational taxonomic units correlated with aberrant behavior and lipocalin-2 in PB/PM samples; LNFPIII was modulatory. Overall, significant and lasting GWI effects occurred on specific microbiota and LNFPIII treatment was beneficial.

Epidemiologic studies associate maternal docosahexaenoic acid (DHA)/DHA-containing seafood intake with enhanced cognitive development; although, it should be noted that interventional trials show inconsistent findings. We examined perinatal DHA supplementation on cognitive performance, brain anatomical and functional organization, and the brain monoamine neurotransmitter status of offspring using a piglet model. Sows were fed a control (CON) or a diet containing DHA (DHA) from late gestation throughout lactation. Piglets underwent an open field test (OFT), an object recognition test (ORT), and magnetic resonance imaging (MRI) to acquire anatomical, diffusion tensor imaging (DTI), and resting-state functional MRI (rs-fMRI) at weaning. Piglets from DHA-fed sows spent 95% more time sniffing the walls than CON in OFT and exhibited an elevated interest in the novel object in ORT, while CON piglets demonstrated no preference. Maternal DHA supplementation increased fiber length and tended to increase fractional anisotropy in the hippocampus of offspring than CON. DHA piglets exhibited increased functional connectivity in the cerebellar, visual, and default mode network and decreased activity in executive control and sensorimotor network compared to CON. The brain monoamine neurotransmitter levels did not differ in healthy offspring. Perinatal DHA supplementation may increase exploratory behaviors, improve recognition memory, enhance fiber tract integrity, and alter brain functional organization in offspring at weaning.

Although there has been an increasing appreciation for functional differences between the dorsal (dH) and ventral (vH) hippocampal sectors, there is a lack of information characterizing the cholinergic and noncholinergic mechanisms of acetylcholinesterase inhibitors on synaptic transmission along the hippocampal dorsoventral axis. Diisopropylfluorophosphate (DFP) is an organophosphate (OP) that is commonly employed as a nerve agent surrogate in vitro as well as in rodent models of disease states, such as Gulf War Illness. The present study investigated the cholinergic and noncholinergic mechanisms responsible for the effects of acute DFP exposure on dH and vH synaptic transmission in a hippocampal slice preparation. A paired-pulse extracellular recording protocol was used to monitor the population spike (PS) amplitude as well as the PS paired-pulse ratio (PS-PPR) in the CA1 subfield of the dH and the vH. We observed that DFP-induced PS1 inhibition was produced by a cholinergic mechanism in the dH, whereas a noncholinergic mechanism was indispensable in mediating the inhibitory effect of DFP on the PS1 in the vH. PS-PPR in both dH and vH sectors was increased by acute DFP exposure, an effect that was blocked by an N-methyl-D-aspartate receptor antagonist but not by cholinergic antagonists. Clinical reports have indicated dorsoventral-specific hippocampal abnormalities in cases of OP intoxications. Therefore, the observed dorsoventral-specific noncholinergic mechanisms underlying the effects of DFP on hippocampal synaptic transmission may have important implications for the treatment of OP overexposures. SIGNIFICANCE STATEMENT: It is unknown if acetylcholinesterase inhibitors differentially impact dorsal and ventral hippocampal synaptic transmission. The data in the present study show that an organophosphate, diisopropylfluorophosphate, impacts glutamatergic transmission along the dorsoventral axis in a hippocampal slice preparation via distinct cholinergic and noncholinergic mechanisms. These findings may provide insight into investigations of therapeutic agents that target noncholinergic mechanisms in cases of organophosphate overexposures., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Gulf War Illness (GWI) manifests a multitude of symptoms, including neurological and immunological, and approximately a third of the 1990-1991 Gulf War (GW) veterans suffer from it. This study sought to characterize the acute neurochemical (monoamine) and neuroinflammatory profiles of two established GWI animal models and examine the potential modulatory effects of the novel immunotherapeutic Lacto-N-fucopentaose III (LNFPIII). In Model 1, male C57BL/6 J mice were treated for 10 days with pyridostigmine bromide (PB) and permethrin (PM). In Model 2, a separate cohort of mice were treated for 14 days with PB and N,N-Diethyl-methylbenzamide (DEET), plus corticosterone (CORT) via drinking water on days 8-14 and diisopropylfluorophosphate (DFP) on day 15. LNFPIII was administered concurrently with GWI chemicals treatments. Brain and spleen monoamines and hippocampal inflammatory marker expression were examined by, respectively, HPLC-ECD and qPCR, 6 h post treatment cessation. Serotonergic (5-HT) and dopaminergic (DA) dyshomeostasis caused by GWI chemicals was apparent in multiple brain regions, primarily in the nucleus accumbens (5-HT) and hippocampus (5-HT, DA) for both models. Splenic levels of 5-HT (both models) and norepinephrine (Model 2) were also disrupted by GWI chemicals. LNFPIII treatment prevented many of the GWI chemicals induced monoamine alterations. Hippocampal inflammatory cytokines were increased in both models, but the magnitude and spread of inflammation was greater in Model 2; LNFPIII was anti-inflammatory, more so in the apparently milder Model 1. Overall, in both models, GWI chemicals led to monoamine disbalance and neuroinflammation. LNFPIII co-treatment prevented many of these disruptions in both models, which is indicative of its promise as a potential GWI therapeutic., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Impaired thermoregulation and lowered average daily gains (ADG) result when livestock graze toxic endophyte (Epichloë coenophialum)-infected tall fescue (E+) and are hallmark signs of fescue toxicosis (FT), a disease exacerbated by increased temperature and humidity (+temperature-humidity index; +THI). We previously reported FT is associated with metabolic and microbiota perturbations under thermoneutral conditions; here, we assessed the influence of E+ grazing and +THI on the microbiota:metabolome interactions. Using high-resolution metabolomics and 16S rRNA gene sequencing, plasma/urine metabolomes and the fecal microbiota of Angus steers grazing non-toxic or E+ tall fescue were evaluated in the context of +THI. E+ grazing affected the fecal microbiota profile; +THI conditions modulated the microbiota only in E+ steers. E+ also perturbed many metabolic pathways, namely amino acid and inflammation-related metabolism; +THI affected these pathways only in E+ steers. Integrative analyses revealed the E+ microbiota correlated and co-varied with the metabolomes in a THI-dependent manner. Operational taxonomic units in the families Peptococcaceae, Clostridiaceae, and Ruminococcaceae correlated with production parameters (e.g., ADG) and with multiple plasma/urine metabolic features, providing putative FT biomarkers and/or targets for the development of FT therapeutics. Overall, this study suggests that E+ grazing increases Angus steer susceptibility to +THI, and offers possible targets for FT interventions.

Tall fescue, the predominant southeastern United States cool-season forage grass, frequently becomes infected with an ergot alkaloid-producing toxic endophyte, Epichloë coenophialum Consumption of endophyte-infected fescue results in fescue toxicosis (FT), a condition that lowers beef cow productivity. Limited data on the influence of ergot alkaloids on rumen fermentation profiles or ruminal bacteria that could degrade the ergot alkaloids are available, but how FT influences the grazing bovine fecal microbiota or what role fecal microbiota might play in FT etiology and associated production losses has yet to be investigated. Here, we used 16S rRNA gene sequencing of fecal samples from weaned Angus steers grazing toxic endophyte-infected (E+; n  = 6) or nontoxic (Max-Q; n  = 6) tall fescue before and 1, 2, 14, and 28 days after pasture assignment. Bacteria in the Firmicutes and Bacteroidetes phyla comprised 90% of the Max-Q and E+ steer fecal microbiota throughout the trial. Early decreases in the Erysipelotrichaceae family and delayed increases of the Ruminococcaceae and Lachnospiraceae families were among the major effects of E+ grazing. E+ also increased abundances within the Planctomycetes , Chloroflexi , and Proteobacteria phyla and the Clostridiaceae family. Multiple operational taxonomic units classified as Ruminococcaceae and Lachnospiraceae were correlated negatively with weight gains (lower in E+) and positively with respiration rates (increased by E+). These data provide insights into how E+ grazing alters the Angus steer microbiota and the relationship of fecal microbiota dynamics with FT. IMPORTANCE Consumption of E+ tall fescue has an estimated annual $1 billion negative impact on the U.S. beef industry, with one driver of these costs being lowered weight gains. As global agricultural demand continues to grow, mitigating production losses resulting from grazing the predominant southeastern United States forage grass is of great value. Our investigation of the effects of E+ grazing on the fecal microbiota furthers our understanding of bovine fescue toxicosis in a real-world grazing production setting and provides a starting point for identifying easy-to-access fecal bacteria that could serve as potential biomarkers of animal productivity and/or FT severity for tall fescue-grazing livestock., (Copyright © 2019 American Society for Microbiology.)

Scope: Well-controlled glycation (generally limited to the early stages) has been proposed as a strategy to improve the physiochemical properties of dietary proteins, but the functional studies of glycation products are mostly on advanced glycation end-products (AGEs) rather than early glycation products (EGPs). Since cytokines are important modulators of various biological processes, this study aims to determine whether EGPs and AGEs affected immune homeostasis differentially and do so through modulating macrophage-derived factors., Methods and Results: Two systems (glycine-glucose and whey protein isolate (WPI)-glucose) are established to generate glycation products. They are applied to human macrophages (PMA-differentiated U937 cells), and cell viability and cytokine production are measured. Furthermore, EGPs, AGEs, and their conditioned medium (CM) from macrophages are applied to human prostate cancer (PCa) cells with different etiology (LNCaP and PC-3) and murine PCa cells (TRAMP-C2) to determine their direct and indirect effects on PCa cell proliferation. EGPs enhance the production of immunosuppressive cytokines, and this enhancement is associated with increased PCa cell proliferation. In contrast, AGEs inhibit macrophages to secret cytokines, but increase PCa cell proliferation directly., Conclusions: Our data suggest that EGPs promote the prostate tumor proliferation indirectly through modulating macrophages, while AGEs have a direct effect., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Alterations in lipid metabolism play a significant role in the pathogenesis of obesity-associated disorders, and dysregulation of the lipidome across multiple diseases has prompted research to identify novel lipids indicative of disease progression. To address the significant gap in knowledge regarding the effect of age and diet on the blood lipidome, we used shotgun lipidomics with electrospray ionization-mass spectrometry (ESI-MS). We analyzed blood lipid profiles of female C57BL/6 mice following high-fat diet (HFD) and low-fat diet (LFD) consumption for short (6weeks), long (22weeks), and prolonged (36weeks) periods. We examined endocannabinoid levels, plasma esterase activity, liver homeostasis, and indices of glucose tolerance and insulin sensitivity to compare lipid alterations with metabolic dysregulation. Multivariate analysis indicated differences in dietary blood lipid profiles with the most notable differences after 6weeks along with robust alterations due to age. HFD altered phospholipids, fatty acyls, and glycerolipids. Endocannabinoid levels were affected in an age-dependent manner, while HFD increased plasma esterase activity at all time points, with the most pronounced effect at 6weeks. HFD-consumption also altered liver mRNA levels of PPARα, PPARγ, and CD36. These findings indicate an interaction between dietary fat consumption and aging with widespread effects on the lipidome, which may provide a basis for identification of female-specific obesity- and age-related lipid biomarkers., (Copyright © 2017. Published by Elsevier B.V.)

Fescue toxicosis (FT) results from consumption of tall fescue (Lolium arundinaceum) infected with an endophyte (Epichloë coenophiala) that produces ergot alkaloids (EA), which are considered key etiological agents of FT. Decreased weight gains, hormonal imbalance, circulating cholesterol disruption, and decreased volatile fatty acid absorption suggest toxic (E+) fescue-induced metabolic perturbations. Employing untargeted high-resolution metabolomics (HRM) to analyze E+ grazing-induced plasma and urine metabolome changes, fescue-naïve Angus steers were placed on E+ or non-toxic (Max-Q) fescue pastures and plasma and urine were sampled before, 1, 2, 14, and 28 days after pasture assignment. Plasma and urine catecholamines and urinary EA concentrations were also measured. In E+ steers, urinary EA appeared early and peaked at 14 days. 13,090 urinary and 20,908 plasma HRM features were detected; the most significant effects were observed earlier (2 days) in the urine and later (≥14 days) in the plasma. Alongside EA metabolite detection, tryptophan and lipid metabolism disruption were among the main consequences of E+ consumption. The E+ grazing-associated metabolic pathways and signatures described herein may accelerate development of novel early FT detection and treatment strategies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

High-fat diet (HFD) induced obesity is associated not only with metabolic dysregulation, e.g., impaired glucose homeostasis and insulin sensitivity, but also with neurological dysfunction manifested with aberrant behavior and/or neurotransmitter imbalance. Most studies have examined HFD's effects predominantly in male subjects, either in the periphery or on the brain, in isolation and after a finite feeding period. In this study, we evaluated the time-course of selected metabolic, behavioral, and neurochemical effects of HFD intake in parallel and at multiple time points in female (C57BL/6) mice. Peripheral effects were evaluated at three feeding intervals (short: 5-6 weeks, long: 20-22 weeks, and prolonged: 33-36 weeks). Central effects were evaluated only after long and prolonged feeding durations; we have previously reported those effects after the short (5-6 weeks) feeding duration. Ongoing HFD feeding resulted in an obese phenotype characterized by increased visceral adiposity and, after prolonged HFD intake, an increase in liver and kidney weights. Peripherally, 5 weeks of HFD intake was sufficient to impair glucose tolerance significantly, with the deleterious effects of HFD being greater with prolonged intake. Similarly, 5 weeks of HFD consumption was sufficient to impair insulin sensitivity. However, sensitivity to insulin after prolonged HFD intake was not different between control, low-fat diet (LFD) and HFD-fed mice, most likely due to age-dependent decrease in insulin sensitivity in the LFD-fed mice. HFD intake also induced bi-phasic hepatic inflammation and it increased gut permeability. Behaviorally, prolonged intake of HFD caused mice to be hypoactive and bury fewer marbles in a marble burying task; the latter was associated with significantly impaired hippocampal serotonin homeostasis. Cognitive (short-term recognition memory) function of mice was unaffected by chronic HFD feeding. Considering our prior findings of short-term (5-6 weeks) HFD-induced central (hyperactivity/anxiety and altered ventral hippocampal neurochemistry) effects and our current results, it seems that in female mice some metabolic/inflammatory dysregulations caused by HFD, such as gut permeability, appear early and persist, whereas others, such as glucose intolerance, are exaggerated with continuous HFD feeding; behaviorally, prolonged HFD consumption mainly affects locomotor activity and anxiety-like responses, likely due to the advanced obesity phenotype; neurochemically, the serotonergic system appears to be most sensitive to continued HFD feeding., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Considering the limited information on the ability of chronic peripheral inflammation to induce behavioral alterations, including on their persistence after inflammatory stimuli termination and on associated neurochemical perturbations, this study assessed the effects of chronic (0.25 mg/kg; i.p.; twice weekly) lipopolysaccharide (LPS) treatment on selected behavioral, neurochemical and molecular measures at different time points in adult male C57BL/6 mice. Behaviorally, LPS-treated mice were hypoactive after 6 weeks, whereas significant hyperactivity was observed after 12 weeks of LPS and 11 weeks after 13 week LPS treatment termination. Similar biphasic responses, i.e., early decrease followed by a delayed increase were observed in the open field test center time, suggestive of, respectively, increased and decreased anxiety. In a forced swim test, mice exhibited increased immobility (depressive behavior) at all times they were tested. Chronic LPS also produced persistent increase in splenic serotonin (5-HT) and time-dependent, brain region-specific alterations in striatal and prefrontocortical dopamine and 5-HT homeostasis. Microglia, but not astrocytes, were activated by LPS early and late, but their activation did not persist after LPS treatment termination. Above findings demonstrate that chronic peripheral inflammation initially causes hypoactivity and increased anxiety, followed by persistent hyperactivity and decreased anxiety. Notably, chronic LPS-induced depressive behavior appears early, persists long after LPS termination, and is associated with increased splenic 5-HT. Collectively, our data highlight the need for a greater focus on the peripheral/central monoamine alterations and lasting behavioral deficits induced by chronic peripheral inflammation as there are many pathological conditions where inflammation of a chronic nature is a hallmark feature., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mounting experimental evidence, predominantly from male rodents, demonstrates that high-fat diet (HFD) consumption and ensuing obesity are detrimental to the brain. To shed additional light on the neurological consequences of HFD consumption in female rodents and to determine the relatively early impact of HFD in the likely continuum of neurological dysfunction in the context of chronic HFD intake, this study investigated effects of HFD feeding for up to 12weeks on selected behavioral, neurochemical, and electrophysiological parameters in adult female C57BL/6 mice; particular focus was placed on the ventral hippocampus (vHIP). Selected locomotor, emotional and cognitive functions were evaluated using behavioral tests after 5weeks on HFD or control (low-fat diet) diets. One week later, mice were sacrificed and brain regional neurochemical (monoamine) analysis was performed. Behaviorally naïve mice were maintained on their respective diets for an additional 5-6weeks at which time synaptic plasticity was determined in ex vivo slices from the vHIP. HFD-fed female mice exhibited increased: (i) locomotor activity in the open field testing, (ii) mean turn time on the pole test, (iii) swimming time in the forced swim test, and (iv) number of marbles buried in the marble burying test. In contrast, the novel object recognition memory was unaffected. Mice on HFD also had decreased norepinephrine and dopamine turnover, respectively, in the prefrontal cortex and the vHIP. HFD consumption for a total of 11-12weeks altered vHIP synaptic plasticity, evidenced by significant reductions in the paired-pulse ratio and long-term potentiation (LTP) magnitude. In summary, in female mice, HFD intake for several weeks induced multiple behavioral alterations of mainly anxiety-like nature and impaired monoamine pathways in a brain region-specific manner, suggesting that in the female, certain behavioral domains (anxiety) and associated brain regions, i.e., the vHIP, are preferentially targeted by HFD., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

Overexposure to the commonly used herbicide atrazine (ATR) affects several organ systems, including the brain. Previously, we demonstrated that short-term oral ATR exposure causes behavioral deficits and dopaminergic and serotonergic dysfunction in the brains of mice. Using adult male C57BL/6 mice, the present study aimed to investigate effects of a 10-day oral ATR exposure (0, 5, 25, 125, or 250mg/kg) on the mouse plasma metabolome and to determine metabolic pathways affected by ATR that may be reflective of ATR's effects on the brain and useful to identify peripheral biomarkers of neurotoxicity. Four hours after the last dosing on day 10, plasma was collected and analyzed with high-performance, dual chromatography-Fourier-transform mass spectrometry that was followed by biostatistical and bioinformatic analyses. ATR exposure (≥5mg/kg) significantly altered plasma metabolite profile and resulted in a dose-dependent increase in the number of metabolites with ion intensities significantly different from the control group. Pathway analyses revealed that ATR exposure strongly correlated with and disrupted multiple metabolic pathways. Tyrosine, tryptophan, linoleic acid and α-linolenic acid metabolic pathways were among the affected pathways, with α-linolenic acid metabolism being affected to the greatest extent. Observed effects of ATR on plasma tyrosine and tryptophan metabolism may be reflective of the previously reported perturbations of brain dopamine and serotonin homeostasis, respectively. ATR-caused alterations in the plasma profile of α-linolenic acid metabolism are a potential novel and sensitive plasma biomarker of ATR effect and plasma metabolomics could be used to better assess the risks, including to the brain, associated with ATR overexposure., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Atrazine (ATR) is one of the most frequently detected pesticides in the U.S. water supply. This study aimed to investigate neurobehavioral and neurochemical effects of ATR in C57BL/6 mouse offspring and dams exposed to a relatively low (3 mg/l, estimated intake 1.4 mg/kg/day) concentration of ATR via the drinking water (DW) from gestational day 6 to postnatal day (PND) 23. Behavioral tests included open field, pole, grip strength, novel object recognition (NOR), forced swim, and marble burying tests. Maternal weight gain and offspring (PND21, 35, and 70) body or brain weights were not affected by ATR. However, ATR-treated dams exhibited decreased NOR performance and a trend toward hyperactivity. Juvenile offspring (PND35) from ATR-exposed dams were hyperactive (both sexes), spent less time swimming (males), and buried more marbles (females). In adult offspring (PND70), the only behavioral change was a sex-specific (females) decreased NOR performance by ATR. Neurochemically, a trend toward increased striatal dopamine (DA) in dams and a significant increase in juvenile offspring (both sexes) was observed. Additionally, ATR exposure decreased perirhinal cortex serotonin in the adult female offspring. These results suggest that perinatal DW exposure to ATR targets the nigrostriatal DA pathway in dams and, especially, juvenile offspring, alters dams' cognitive performance, induces sex-selective changes involving motor and emotional functions in juvenile offspring, and decreases cognitive ability of adult female offspring, with the latter possibly associated with altered perirhinal cortex serotonin homeostasis. Overall, ATR exposure during gestation and lactation may cause adverse nervous system effects to both offspring and dams., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Natural leaching processes and/or anthropogenic contamination can result in ground water concentrations of the essential metal manganese (Mn) that far exceed the current regulatory standards. Neurological consequences of Mn drinking water (DW) overexposure to experimental animals, i.e., mice, including its brain deposition/distribution and behavioral effects are understudied. Adult male C57BL/6 mice were exposed to Mn via the DW for 8 weeks. After 5 weeks of Mn exposure, magnetic resonance imaging revealed significant Mn deposition in all examined brain regions; the degree of Mn deposition did not increase further a week later. Behaviorally, early hyperactivity and more time spent in the center of the arenas in an open field test, decreased forelimb grip strength and less time swimming in a forced swim test were observed after 6 weeks of Mn DW exposure. Eight-week Mn DW exposure did not alter striatal dopamine, its metabolites, or the expression of key dopamine homeostatic proteins, but it significantly increased striatal 5-hydroxyindoleacetic acid (a serotonin metabolite) levels, without affecting the levels of serotonin itself. Increased expression (mRNA) of glial fibrillary acidic protein (GFAP, an astrocyte activation marker), heme oxygenase-1 and inducible nitric oxide synthase (oxidative and nitrosative stress markers, respectively) were observed 8 weeks post-Mn DW exposure in the substantia nigra. Besides mRNA increases, GFAP protein expression was increased in the substantia nigra pars reticulata. In summary, the neurobehavioral deficits, characterized by locomotor and emotional perturbations, and nigral glial activation associated with significant brain Mn deposition are among the early signs of Mn neurotoxicity caused by DW overexposure.

Atrazine (ATR) is a widely used chlorotriazine herbicide, a ubiquitous environmental contaminant, and a potential developmental toxicant. To quantitatively evaluate placental/lactational transfer and fetal/neonatal tissue dosimetry of ATR and its major metabolites, physiologically based pharmacokinetic models were developed for rat dams, fetuses and neonates. These models were calibrated using pharmacokinetic data from rat dams repeatedly exposed (oral gavage; 5mg/kg) to ATR followed by model evaluation against other available rat data. Model simulations corresponded well to the majority of available experimental data and suggest that: (1) the fetus is exposed to both ATR and its major metabolite didealkylatrazine (DACT) at levels similar to maternal plasma levels, (2) the neonate is exposed mostly to DACT at levels two-thirds lower than maternal plasma or fetal levels, while lactational exposure to ATR is minimal, and (3) gestational carryover of DACT greatly affects its neonatal dosimetry up until mid-lactation. To test the model's cross-species extrapolation capability, a pharmacokinetic study was conducted with pregnant C57BL/6 mice exposed (oral gavage; 5mg/kg) to ATR from gestational day 12 to 18. By using mouse-specific parameters, the model predictions fitted well with the measured data, including placental ATR/DACT levels. However, fetal concentrations of DACT were overestimated by the model (10-fold). This overestimation suggests that only around 10% of the DACT that reaches the fetus is tissue-bound. These rodent models could be used in fetal/neonatal tissue dosimetry predictions to help design/interpret early life toxicity/pharmacokinetic studies with ATR and as a foundation for scaling to humans., (© 2013.)

Excessive exposure to the widely used herbicide atrazine (ATR) affects several organ systems, including the brain, where neurochemical alterations reflective of dopamine (DA) circuitry perturbation have been reported. The present study aimed to investigate effects of short-term oral exposure to a dose-range (0, 5, 25, 125, or 250 mg/kg) of ATR on behavioral, neurochemical, and molecular indices of toxicity in adult male C57BL/6 mice. The experimental paradigm included open field, pole and grip tests (day 4), novel object recognition (NOR) and forced swim test (FST; day 9), followed by tissue collection 4h post dosing on day 10. After 4 days of exposure, ATR decreased locomotor activity (≥125 mg/kg). On day 9, ATR-exposed mice exhibited dose-dependent decreased performance in the NOR test (≥25 mg/kg) and spent more time swimming and less time immobile during the FST (≥125 mg/kg). Neurochemically, short-term ATR exposure increased striatal DA and DA turnover (its metabolite homovanillic acid [HVA] and the HVA/DA ratio; ≥125 mg/kg). In addition, ATR exposure increased the levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the striatum (≥125 mg/kg) and it also increased DA turnover (≥125 mg/kg), 5-HIAA (125 mg/kg), and norepinephrine (≥125 mg/kg) levels in the prefrontal cortex. In the hippocampus, the only effect of ATR was to increase the norepinephrine metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG; 250 mg/kg). At the molecular level, the expression of key striatal (protein) or nigral (mRNA) markers associated with nigrostriatal DA function, such as tyrosine hydroxylase, DA transporter, vesicular monoamine transporter 2, and DA receptors, was not affected by ATR. These results indicate that short-term ATR exposure targets multiple monoamine pathways at the neurochemical level, including in the striatum, and induces behavioral abnormalities suggestive of impaired motor and cognitive functions and increased anxiety. Impaired performance in the NOR behavioral test was the most sensitive endpoint affected by ATR; this should be taken into consideration for future low-dose ATR studies and for the assessment of risk associated with overexposure to this herbicide., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aims: This study sought to determine the impact of in vitro exposure to the herbicide atrazine (ATR) or its major mammalian metabolite diaminochlorotriazine (DACT) on dopaminergic cell differentiation., Main Methods: N27 dopaminergic cells were exposed for 24 or 48 h to ATR or DACT (12-300 μM) and their effects on cell viability, ATP levels, ADP:ATP ratio and differentiation markers, such as soma size and neurite outgrowth, were assessed., Key Findings: Overall, intracellular ATP levels and soma size (decreased by ATR at ≥12 μM; 48 h) were the two parameters most sensitive to ATR exposure in undifferentiated and differentiating dopaminergic cells, respectively. At the morphological level, ATR, but not DACT, increased the percentage of morphologically abnormal undifferentiated N27 cells. On the other hand, exposure to DACT (300 μM; 48 h), but not ATR, increased the ADP:ATP ratio regardless of the differentiation state and it moderately disrupted thin neurite outgrowth. Only the highest concentration of ATR or DACT (300 μM) was cytotoxic after a longer exposure (48 h) and undifferentiated N27 cells were the least sensitive to the cytotoxic effects of ATR or DACT., Significance: Our results suggest that the energy perturbation and morphological disruption of dopaminergic neuronal differentiation induced by ATR and, to a lesser extent, DACT, may be associated with reported neurological deficits caused by developmental ATR exposure in rodents., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The objectives of this focused review are to (i) provide a systematic overview of recent advances pertaining to the role of glia, namely microglia and astrocytes, in the neuropathology associated with excessive exposure to manganese (Mn), (ii) highlight possible mechanisms and factors involved in Mn-modulated, glia-derived neuroinflammation, and (iii) discuss the implications of excessive neuroinflammation on neuronal injury within the context of Mn overexposure. As this is not meant to be a comprehensive review on the topic of Mn neurotoxicity, the reader may wish to refer to several broader and more comprehensive reviews. After a brief introduction to Mn neurotoxicity, we first discuss the role of glial cells in neurodegeneration. Next, we review existing in vitro and in vivo studies that implicate Mn as a modulator of glial activation and ensuing neuroinflammation. This is followed by an examination of recognized and potential mechanisms that are involved in the modulation of glial inflammatory output by Mn; here the common pathways activated by Mn in glial and neuronal cells, including outcomes of such activation, are also addressed. We finish with a discussion of the implications of Mn-modulated glial activation for neuronal survival and with a list of data gaps in the topic that need to be filled in the future., (Copyright © 2011 John Wiley & Sons, Ltd.)

Excessive manganese (Mn) exposure increases output of glial-derived inflammatory products, which may indirectly contribute to the neurotoxic effects of this essential metal. In microglia, Mn increases hydrogen peroxide (H(2)O(2)) release and potentiates lipopolysaccharide (LPS)-induced cytokines (TNF-α, IL-6) and nitric oxide (NO). Inducible heme-oxygenase (HO-1) plays a role in the regulation of inflammation and its expression is upregulated in response to oxidative stressors, including metals and LPS. Because Mn can oxidatively affect neurons both directly and indirectly, we investigated the effect of Mn exposure on the induction of HO-1 in resting and LPS-activated microglia (N9) and dopaminergic neurons (N27). In microglia, 24h exposure to Mn (up to 250 μM) had minimal effects on its own, but it markedly potentiated LPS (100 ng/ml)-induced HO-1 protein and mRNA. Inhibition of microglial HO-1 activity with two different inhibitors indicated that HO-1 is a positive regulator of the Mn-potentiated cytokine output and a negative regulator of the Mn-induced H(2)O(2) output. Mn enhancement of LPS-induced HO-1 does not appear to be dependent on H(2)O(2) or NO, as Mn+LPS-induced H(2)O(2) release was not greater than the increase induced by Mn alone and inhibition of iNOS did not change Mn potentiation of HO-1. However, because Mn exposure potentiated the LPS-induced nuclear expression of small Maf proteins, this may be one mechanism Mn uses to affect the expression of HO-1 in activated microglia. Finally, the potentiating effects of Mn on HO-1 appear to be glia-specific for Mn, LPS, or Mn+LPS did not induce HO-1 in N27 neuronal cells., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Atrazine (ATR) is a chlorotriazine herbicide that is widely used and relatively persistent in the environment. In laboratory rodents, excessive exposure to ATR is detrimental to the reproductive, immune, and nervous systems. To better understand the toxicokinetics of ATR and to fill the need for a mouse model, a physiologically based pharmacokinetic (PBPK) model for ATR and its main chlorotriazine metabolites (Cl-TRIs) desethyl atrazine (DE), desisopropyl atrazine (DIP), and didealkyl atrazine (DACT) was developed for the adult male C57BL/6 mouse. Taking advantage of all relevant and recently made available mouse-specific data, a flow-limited PBPK model was constructed. The ATR and DACT sub-models included blood, brain, liver, kidney, richly and slowly perfused tissue compartments, as well as plasma protein binding and red blood cell binding, whereas the DE and DIP sub-models were constructed as simple five-compartment models. The model adequately simulated plasma levels of ATR and Cl-TRIs and urinary dosimetry of Cl-TRIs at four single oral dose levels (250, 125, 25, and 5mg/kg). Additionally, the model adequately described the dose dependency of brain and liver ATR and DACT concentrations. Cumulative urinary DACT amounts were accurately predicted across a wide dose range, suggesting the model's potential use for extrapolation to human exposures by performing reverse dosimetry. The model was validated using previously reported data for plasma ATR and DACT in mice and rats. Overall, besides being the first mouse PBPK model for ATR and its Cl-TRIs, this model, by analogy, provides insights into tissue dosimetry for rats. The model could be used in tissue dosimetry prediction and as an aid in the exposure assessment to this widely used herbicide., (© 2010 Elsevier Inc. All rights reserved.)

Multiple studies demonstrate that manganese (Mn) exposure potentiates inflammatory mediator output from activated glia; this increased output is associated with enhanced mitogen activated protein kinase (MAPK: p38, ERK and JNK) activity. We hypothesized that Mn activates MAPK by activating the kinases upstream of MAPK, i.e. MKK-3/6, MKK-1/2 and MKK-4 (responsible for activation of p38, ERK, and JNK, respectively), and/or by inhibiting a major phosphatase responsible for MAPK inactivation, MKP-1. Exposure of N9 microglia to Mn (250 µm), LPS (100 ng ml⁻¹) or Mn + LPS increased MKK-3/6 and MKK-4 activity at 1 h; the effect of Mn + LPS on MKK-4 activation was greater than the rest. At 4 h, Mn, LPS, and Mn + LPS increased MKK-3/6 and MKK-1/2 phosphorylation, whereas MKK-4 was activated only by Mn and Mn + LPS. Besides activating MKK-4 via Ser257/Thr261 phosphorylation, Mn (4 h) prevented MKK-4's phosphorylation on Ser80, which negatively regulates MKK-4 activity. Exposure to Mn or Mn + LPS (1 h) decreased both mRNA and protein expression of MKP-1, the negative MAPK regulator. In addition, we observed that at 4 h, but not at 1 h, a time point coinciding with increased MAPK activity, Mn + LPS markedly increased TNF-α, IL-6 and Cox-2 mRNA, suggesting a delayed effect. The fact that all three major groups of MKKs, MKK-1/2, MKK-3/6 and MKK-4, are activated by Mn suggests that Mn-induced activation of MAPK occurs via traditional mechanisms, which perhaps involve the MAPKs furthest upstream, MKKKs (MAP3Ks). In addition, for all MKKs, Mn-induced activation was persistent at least for 4 h, indicating a long-term effect., (Copyright © 2010 John Wiley & Sons, Ltd.)

Administration of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to adult (2-month to 4-month-old) male C57BL/6 mice (MPTP-sensitive) is a valuable Parkinson's disease model. At comparable age, other strains, such as BALB/c, are minimally affected by MPTP (MPTP-resistant). However, the maintenance of resistance to MPTP throughout aging in MPTP-resistant strains has not been studied. Here, we show that, as previously reported, 1-month and 18-month-old C57BL/6 mice are least and most sensitive to MPTP, respectively. MPTP, as expected, did not affect the younger (1-month and 3-month-old) BALB/c mice, but it markedly decreased striatal dopamine in the older (10-month and 18-month-old) BALB/c mice. These data suggest that the sensitivity to MPTP is age dependent and that mice from an MPTP-resistant strain lose their resistance as they age.

2-Chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine, ATR) is a toxicologically important and widely used herbicide. Recent studies have shown that it can elicit neurological, immunological, developmental, and biochemical alterations in several model organisms, including in mice. Because disposition data in mice are lacking, we evaluated ATR's metabolism and tissue dosimetry after single oral exposures (5-250 mg/kg) in C57BL/6 mice using liquid chromatography/mass spectrometry (Ross and Filipov, 2006). ATR was metabolized and cleared rapidly; didealkyl ATR (DACT) was the major metabolite detected in urine, plasma, and tissues. Plasma ATR peaked at 1 h postdosing and rapidly declined, whereas DACT peaked at 2 h and slowly declined. Most ATR and metabolite residues were excreted within the first 24 h. However, substantial amounts of DACT were still present in 25- to 48-h and 49- to 72-h urine. ATR reached maximal brain levels (0.06-1.5 microM) at 4 h (5-125 mg/kg) and 1 h (250 mg/kg) after dosing, but levels quickly declined to <0.1 microM by 12 h in all the groups. In contrast, strikingly high concentrations of DACT (1.5-50 microM), which are comparable with liver DACT levels, were detectable in brain at 2 h. Brain DACT levels slowly declined, paralleling the kinetics of plasma DACT. Our findings suggest that in mice ATR is widely distributed and extensively metabolized and that DACT is a major metabolite detected in the brain at high levels and is ultimately excreted in urine. Our study provides a starting point for the establishment of models that link target tissue dose to biological effects caused by ATR and its in vivo metabolites.

Studies have shown that both in vivo and in vitro exposure to the herbicide atrazine (ATR) results in dopaminergic neurotoxicity manifested by decreased striatal dopamine (DA) levels. However, the mechanism behind this reduction is largely unknown. A decrease in striatal DA could be due to ATR exposure affecting vesicular and/or synaptosomal uptake resulting in disrupted vesicular storage and/or cellular uptake of DA. Hence, we investigated the effects of in vitro ATR exposure on DA uptake into isolated rat striatal synaptosomes and synaptic vesicles. In addition to ATR, effects of its major mammalian metabolites, didealkyl atrazine (DACT), desethyl atrazine (DE) and desiopropyl atrazine (DIP) were investigated. ATR (1-250 microM) inhibited DA uptake into synaptic vesicles in a dose-dependent manner. Of the three ATR metabolites tested, DACT did not affect vesicular DA uptake. DE and DIP, on the other hand, significantly decreased vesicular DA uptake with the effect of 100 microM DE/DIP being similar to the effect of the same concentration of ATR. Kinetic analysis of vesicular DA uptake indicated that ATR significantly decreased the V(max) while the K(m) value was not affected. Contrary to the inhibitory effects on vesicular DA uptake, synaptosomal DA uptake was marginally (6-13%) increased by ATR and DE, but not by DACT and DIP, at concentrations of

Background: Despite several reports on age-related phenotypic changes of the immune system's cells, studies that use a multipoint age comparison between the specific and innate immune cell populations of prototypical Th1- and Th2-type polarized mouse strains are still lacking., Results: Using a multipoint age comparison approach, cells from the two major immune system compartments, peripheral blood and spleen, and flow cytometry analysis, we found several principal differences in T cell and professional antigen presenting cell (APC) populations originating from a prototypical T helper (Th) 1 mouse strain, C57BL/6, and a prototypical Th2 strain, BALB/c. For example, regardless of age, there were strain differences in both peripheral blood mononuclear cells (PBMC) and spleens in the proportion of CD4+ (higher in the BALB/c strain), CD8+ T cells and CD11b+/CD11c+ APC (greater in C57BL/6 mice). Other differences were present only in PBMC (MHC class II + and CD19+ were greater in C57BL/6 mice) or differences were evident in the spleens but not in circulation (CD3+ T cells were greater in C57BL/6 mice). There were populations of cells that increased with age in PBMC and spleens of both strains (MHC class II+), decreased in the periphery and spleens of both strains (CD11b+) or did not change in the PBMC and spleens of both strains (CD8+). We also found strain and age differences in the distribution of naïve and memory/activated splenic T cells, e.g., BALB/c mice had more memory/activated and less naive CD8+ and CD4+ T cells and the C57BL/6 mice., Conclusion: Our data provide important information on the principal differences, within the context of age, in T cell and professional APC populations between the prototypical Th1 mouse strain C57BL/6 and the prototypical Th2 strain BALB/c. Although the age-related changes that occur may be rather subtle, they may be very relevant in conditions of disease and stress. Importantly, our data indicate that age and strain should be considered in concert in the selection of appropriate mouse models for immunological research.

Previous studies that investigated the role of inflammation in the neurotoxicity of manganese (Mn) found that Mn enhanced the production of inflammogen (lipopolysaccharide; LPS)-induced proinflammatory cytokines such as IL-6 and TNF-alpha. Although we have shown that the enhanced cytokine production occurs via a NF-kappaB-dependent mechanism, the role of upstream kinases in this Mn-induced enhancement has not been explored. As other studies have demonstrated that p38 mitogen activated protein kinase (p38) is necessary for LPS-induced, NF-kappaB-dependent expression of proinflammatory cytokines, we hypothesized that Mn enhancement of LPS-induced production of IL-6 and TNF-alpha may be associated with p38 activation and conducted a series of experiments to address our hypothesis. We found that pre-treatment of microglial cells with a p38-inhibitor (SB203580) prevented Mn+LPS-induced production of IL-6 and TNF-alpha. Moreover, potentiation of IL-6 and TNF-alpha production, which occurred in both concurrent and sequential (3h apart) exposures to Mn and LPS, was inhibited by inhibition of p38. Additionally, Mn exposure enhanced the phosphorylation and activity of p38 and this effect was persistent. Although p38 activity declined over time LPS-exposed cells, it persisted in cells exposed to Mn or Mn+LPS. Thus, the increased production of proinflammatory cytokines by LPS-activated microglia exposed to Mn is associated with increased and persistent activation of p38.

Exposure to either chlorpyrifos (CPS) or methyl parathion (MPS) results in the inhibition of acetylcholinesterase and leads to altered neuronal activity which normally regulates critical genes such as the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). The effects of postnatal exposure to CPS and MPS on the expression of messenger RNA (mRNA) and protein levels for NGF and BDNF were investigated in the frontal cerebral cortex (cortex) and hippocampus of rats. Oral administration of CPS (4.0 or 6.0 mg/kg), MPS (0.6 or 0.9 mg/kg), or the safflower oil vehicle was performed daily from postnatal day 10 (PND10) through PND20. Exposure induced significant effects on growth and cholinesterase activity. Increased NGF protein levels were observed in the hippocampus but not the cortex on PND20 with some reduction occurring on PND28 in both regions. These changes did not correlate with the changes in NGF mRNA. BDNF mRNA was increased in both regions on PND20 and PND28, whereas BDNF protein levels were increased on PND20. On PND12, c-fos mRNA, a marker of neuronal activation, was increased in both regions. Total BDNF protein was increased in the hippocampus but decreased in the cortex. No changes in NGF protein were observed. These results indicate that repeated developmental OP exposure during the postnatal period alters NGF and BDNF in the cortex and the hippocampus and the patterns of these alterations differ between regions.

Recent data suggest that some of the immunotoxic effects of the herbicide atrazine, a very widely used pesticide, may be due to perturbations in dendritic cell (DC) function. As consequences of atrazine exposure on the phenotypic and functional maturation of DC have not been studied, our objective was, using the murine DC line, JAWSII, to determine whether atrazine will interfere with DC maturation. First, we characterized the maturation of JAWSII cells in vitro by inducing them to mature in the presence of growth factors and selected maturational stimuli in vitro. Next, we exposed the DC cell line to a concentration range of atrazine and examined its effects on phenotypic and functional maturation of DC. Atrazine exposure interfered with the phenotypic and functional maturation of DC at non-cytotoxic concentrations. Among the phenotypic changes caused by atrazine exposure was a dose-dependent removal of surface MHC-I with a significant decrease being observed at 1 microM concentration. In addition, atrazine exposure decreased the expression of the costimulatory molecule CD86 and it downregulated the expression of the CD11b and CD11c accessory molecules and the myeloid developmental marker CD14. When, for comparative purposes, we exposed primary thymic DC to atrazine, MHC-I and CD11c expression was also decreased. Phenotypic changes in JAWSII DC maturation were associated with functional inhibition of maturation as, albeit at higher concentrations, receptor-mediated antigen uptake was increased by atrazine. Thus, our data suggest that atrazine directly targets DC maturation and that toxicants such as atrazine that efficiently remove MHC-I molecules from the DC surface are likely to contribute to immune evasion.

Following combustion of fuel containing the additive methylcyclopentadienyl-manganese-tricarbonyl (MMT), manganese phosphate (MnPO(4)) and manganese sulfate (MnSO(4)) are emitted in the atmosphere. Manganese chloride (MnCl(2)), another Mn(2+) species, is widely used experimentally. Using rat striatal slices, we found that MnPO(4) decreased tissue and media dopamine (DA) and media Dopac (a DA metabolite) levels substantially more than either MnCl(2) or MnSO(4); antioxidants were partially protective. Also, both MnCl(2) and MnPO(4) (more potently) oxidized DA and Dopac even in the absence of tissue in the media, suggesting a direct interaction between Mn and DA/Dopac. Because aminochrome is a major oxidation product of DA, we next determined whether MnPO(4) will be more potent in forming aminochrome than MnCl(2) or MnSO(4) which, indeed, was the case. Thus, a potential additional mechanism for the neurotoxic effects of environmentally-relevant forms of Mn, MnPO(4) in particular, is the generation of reactive DA intermediates.

A possible link between Parkinson's disease and pesticide exposure has been suggested, and recently it was shown that the herbicide atrazine (ATR) modulates catecholamine metabolism in PC12 cells and affects basal ganglia function in vivo. Hence, the objectives of this study were to: (i) determine if ATR is capable of modulating dopamine (DA) metabolism in striatal tissue slices in vitro and (ii) explore possible mechanisms of its effects. Striatal tissues from adult male Sprague-Dawley rats were incubated with up to 500 microM ATR in a metabolic shaker bath at 37 degrees C and an atmosphere of 95% O(2) and 5% CO(2) for 4h. At the end of incubation, samples were collected for both tissue and media levels of DA and its metabolites (3,4-dihydroxyphenylacetic acid, DOPAC and homovanillic acid, HVA), which were determined by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). To gain some mechanistic insight in to the way ATR affects DA metabolism, several pharmacological manipulations were performed. Striata exposed to ATR at concentrations of 100 microM and greater had a dose-dependent decrease of tissue levels of DA. At doses of ATR 50 microM and greater, the DOPAC+HVA/DA ratio was dose-dependently increased. Tyrosine hydroxylase (TH, the rate-limiting enzyme in DA synthesis) protein levels and activity were not affected by ATR treatment. However, high potassium-induced DA release into the medium was decreased, whereas the increase in media DA observed in the presence of the DA uptake inhibitor nomifensine was increased even further by ATR in a dose-dependent manner. All of these effects of ATR were observed at levels that were not toxic to the tissue, as LDH release into the medium (lactate dehydrogenase, an index of non-specific cytotoxicity) was not affected by ATR. Taken together, results from this study suggest that ATR decreases tissue DA levels not by affecting TH activity, but possibly by interfering with the vesicular storage and/or cellular uptake of DA.

The herbicide atrazine (ATR) is a very commonly used pesticide in the United States. and a major ground water contaminant. It has also been recently implicated as a potential basal ganglia toxicant. In the present study, our objective was to determine the effects of ATR exposure on striatal neurochemistry, on the number of dopaminergic neurons in the substantia nigra pars compacta (SNpc), and, as a reference, in the ventral tegmental area (VTA) of male juvenile C57BL/6 mice. Oral exposure to ATR for 14 days dose-dependently decreased the levels of dopamine (DA) and its metabolites in the striatum for up to a week post-treatment. ATR exposure also time- and dose-dependently decreased the number of tyrosine hydroxylase-positive (TH+) dopaminergic neurons in both SNpc and VTA (with effects being slightly more prominent in SNpc), such that the decreases were most evident at 7 weeks post-cessation of exposure to ATR. Together, these data indicate that, in the juvenile male C57BL/6 mouse, the neurotoxic effects of ATR appear to cause transient neurochemical alterations, whereas the loss of TH+ neurons appears to be persistent, possibly confined to basal ganglia dopaminergic neurons, but not exclusive to the SNpc.

Atrazine is a herbicide widely used on agricultural commodities. Existing analytical methods to analyze atrazine and its metabolites in biological matrices have various drawbacks. Thus, further development of such methods will be needed to correlate the growing number of toxicological effects associated with atrazine exposure with the concentrations of this compound and its metabolites in plasma, urine, and tissues. The purpose of this study was to develop a broad and sensitive LC-MS method for the analysis of atrazine and its metabolites in mouse urine and plasma. We were able to simultaneously measure atrazine and its major mammalian metabolites, which include didealkyl atrazine, desisopropyl atrazine, desethyl atrazine, atrazine-glutathione conjugate, and atrazine-mercapturate, using preparation procedures that used small sample volumes of plasma and urine (0.25 and 0.5 ml, respectively). Furthermore, derivatization of analytes prior to analysis was unnecessary. This method was used to analyze plasma and urine samples following single in vivo oral exposures of a limited number of mice to atrazine (doses, 5-250 mg/kg body weight) to demonstrate the utility of this LC-MS method. The data obtained from this study suggest that atrazine is rapidly metabolized in mice. Didealkyl atrazine was the most abundant metabolite detected in the urine and plasma samples (approximately 1000 microM in 24-h urine and approximately 100 microM in plasma following the highest dose of atrazine), with lesser quantities of mono N-dealkylated metabolites and thio conjugates of atrazine observed. We also used this methodology in a preliminary study of cytochrome P450-catalyzed metabolism of atrazine in vitro. The results obtained in this study suggest that this method will be a useful tool for the determination of atrazine and its metabolites in future pharmacokinetic studies and for the subsequent development and refinement of biologically based models of atrazine disposition.

The herbicide atrazine (ATR) is a very widely used pesticide; yet the immunotoxicological potential of ATR has not been studied extensively. Our objective was to examine the effect of ATR on selected immune parameters in juvenile mice. ATR (up to 250 mg/kg) was administered by oral gavage for 14 days to one-month-old male C57BL/6 mice. One day, one week, and seven weeks after the last ATR dose, mice were sacrificed, and blood, spleens, and thymuses were collected and processed for cell counting and flow cytometry. Thymus and spleen weights were decreased by ATR, with the thymus being more sensitive than the spleen; this effect was still present at seven days, but not at seven weeks after the last ATR dose. Similarly, organ cellularity was persistently decreased in the thymus and in the spleen, with the splenic, but not thymic cellularity still being depressed at seven weeks post ATR. Peripheral blood leukocyte counts were not affected by ATR. There were also alterations in the cell phenotypes in that ATR exposure decreased all phenotypes in the thymus, with the number of CD4(+)/CD8(+) being affected the least. At the higher doses, the decreases in the thymic T-cell populations were still present one week after the last ATR dose. In the spleen, the CD8(+) were increased and MHC-II(+) and CD19(+) cells were decreased one day after the last ATR dose. Also, ATR treatment decreased the number of splenic naïve T helper and T cytotoxic cells, whereas it increased the percentage of highly activated cytotoxic/memory T cells. Interestingly, the proportion of mature splenic dendritic cells (DC; CD11c(high)), was also decreased and it persisted for at least one week, suggesting that ATR inhibited DC maturation. In the circulation, ATR exposure decreased CD4(+) lymphocytes at one day, whereas at seven days after the last ATR dose, in addition to the decrease in CD4(+) lymphocytes, the MHC-II(+) cells were also decreased at the 250 mg/kg dose. Thus, ATR exposure appears to be detrimental to the immune system of juvenile mice by decreasing cellularity and affecting lymphocyte distribution, with certain effects persisting long after exposure has been terminated.