Genetic association between the APOE ε4 allele, toxicant exposures and Gulf war illness diagnosis.

INTRODUCTION: Exposure to nerve agents, pyridostigmine bromide (PB), pesticides, and oil-well fires during the 1991 Gulf War (GW) are major contributors to the etiology of Gulf War Illness (GWI). Since the apolipoprotein E (APOE) ε4 allele is associated with the risk of cognitive decline with age, particularly in the presence of environmental exposures, and cognitive impairment is one of the most common symptoms experienced by veterans with GWI, we examined whether the ε4 allele was associated with GWI. METHODS: Using a case-control design, we obtained data on APOE genotypes, demographics, and self-reported GW exposures and symptoms that were deposited in the Boston Biorepository and Integrative Network (BBRAIN) for veterans diagnosed with GWI (n = 220) and healthy GW control veterans (n = 131). Diagnosis of GWI was performed using the Kansas and/or Center for Disease Control (CDC) criteria. RESULTS: Age- and sex-adjusted analyses showed a significantly higher odds ratio for meeting the GWI case criteria in the presence of the ε4 allele (Odds ratio [OR] = 1.84, 95% confidence interval [CI = 1.07-3.15], p ≤ 0.05) and with two copies of the ε4 allele (OR = 1.99, 95% CI [1.23-3.21], p ≤ 0.01). Combined exposure to pesticides and PB pills (OR = 4.10 [2.12-7.91], p ≤ 0.05) as well as chemical alarms and PB pills (OR = 3.30 [1.56-6.97] p ≤ 0.05) during the war were also associated with a higher odds ratio for meeting GWI case criteria. There was also an interaction between the ε4 allele and exposure to oil well fires (OR = 2.46, 95% CI [1.07-5.62], p ≤ 0.05) among those who met the GWI case criteria. CONCLUSION: These findings suggest that the presence of the ε4 allele was associated with meeting the GWI case criteria. Gulf War veterans who reported exposure to oil well fires and have an ε4 allele were more likely to meet GWI case criteria. Long-term surveillance of veterans with GWI, particularly those with oil well fire exposure, is required to better assess the future risk of cognitive decline among this vulnerable population.

Impact of gulf war toxic exposures after mild traumatic brain injury.

Chemical and pharmaceutical exposures have been associated with the development of Gulf War Illness (GWI), but how these factors interact with the pathophysiology of traumatic brain injury (TBI) remains an area of study that has received little attention thus far. We studied the effects of pyridostigmine bromide (an anti-nerve agent) and permethrin (a pesticide) exposure in a mouse model of repetitive mild TBI (r-mTBI), with 5 impacts over a 9-day period, followed by Gulf War (GW) toxicant exposure for 10 days beginning 30 days after the last head injury. We then assessed the chronic behavioral and pathological sequelae 5 months after GW agent exposure. We observed that r-mTBI and GWI cumulatively affect the spatial memory of mice in the Barnes maze and result in a shift of search strategies employed by r-mTBI/GW exposed mice. GW exposure also produced anxiety-like behavior in sham animals, but r-mTBI produced disinhibition in both the vehicle and GW treated mice. Pathologically, GW exposure worsened r-mTBI dependent axonal degeneration and neuroinflammation, increased oligodendrocyte cell counts, and increased r-mTBI dependent phosphorylated tau, which was found to colocalize with oligodendrocytes in the corpus callosum. These results suggest that GW exposures may worsen TBI-related deficits. Veterans with a history of both GW chemical exposures as well as TBI may be at higher risk for worse symptoms and outcomes. Subsequent exposure to various toxic substances can influence the chronic nature of mTBI and should be considered as an etiological factor influencing mTBI recovery.

Neurogenesis and chronic neurobehavioral outcomes are partially improved by vagus nerve stimulation in a mouse model of Gulf War illness.

Gulf War illness (GWI) is a chronic, multi-symptom disorder that has impacted approximately one third of Gulf War veterans. GWI and its symptoms have been linked to the exposure to neurological chemicals, including the anti-nerve gas drug pyridostigmine bromide (PB) and the insecticide permethrin (PER), among others. Mouse models utilizing these chemicals have reported symptomology analogous to human GWI. These changes include behavioral and cognitive impairment, neuroinflammation and hippocampal pathogenesis. Disease modifying interventions that target these pathological components are desperately needed. Vagus nerve stimulation (VNS) is FDA approved for drug-resistant epilepsy and depression. VNS has also been used off-label to target a myriad of symptoms, some of which are encompassed within the Kansas and CDC definitions of clinical GWI symptomology. A GWI animal model in which mice are exposed to a daily injection of PB and PER for 10 consecutive days has been shown to exhibit cognitive impairment and hippocampal pathology. The purpose of this study was to determine if 2-4 weeks of continuous vagus nerve stimulation initiated at 32 weeks after exposure to PB and PER would improve cognitive performance and hippocampal pathology. The results of the study revealed that exposure to PB and PER produces long-term cognitive deficits and reduced hippocampal neurogenesis. The results also showed that the VNS treatment was anxiolytic, improved some aspects of pattern separation deficits, and mitigated the reduced hippocampal neurogenesis. Thus, VNS improves outcomes in a mouse model of GWI and should be examined as a potential therapeutic strategy for mitigating some symptomology associated with GWI.

Induction of distinct neuroinflammatory markers and gut dysbiosis by differential pyridostigmine bromide dosing in a chronic mouse model of GWI showing persistent exercise fatigue and cognitive impairment.

AIMS: To characterize neuroinflammatory and gut dysbiosis signatures that accompany exaggerated exercise fatigue and cognitive/mood deficits in a mouse model of Gulf War Illness (GWI). METHODS: Adult male C57Bl/6N mice were exposed for 28 d (5 d/wk) to pyridostigmine bromide (P.O.) at 6.5 mg/kg/d, b.i.d. (GW1) or 8.7 mg/kg/d, q.d. (GW2); topical permethrin (1.3 mg/kg), topical N,N-diethyl-meta-toluamide (33%) and restraint stress (5 min). Animals were phenotypically evaluated as described in an accompanying article [124] and sacrificed at 6.6 months post-treatment (PT) to allow measurement of brain neuroinflammation/neuropathic pain gene expression, hippocampal glial fibrillary acidic protein, brain Interleukin-6, gut dysbiosis and serum endotoxin. KEY FINDINGS: Compared to GW1, GW2 showed a more intense neuroinflammatory transcriptional signature relative to sham stress controls. Interleukin-6 was elevated in GW2 and astrogliosis in hippocampal CA1 was seen in both GW groups. Beta-diversity PCoA using weighted Unifrac revealed that gut microbial communities changed after exposure to GW2 at PT188. Both GW1 and GW2 displayed systemic endotoxemia, suggesting a gut-brain mechanism underlies the neuropathological signatures. Using germ-free mice, probiotic supplementation with Lactobacillus reuteri produced less gut permeability than microbiota transplantation using GW2 feces. SIGNIFICANCE: Our findings demonstrate that GW agents dose-dependently induce differential neuropathology and gut dysbiosis associated with cognitive, exercise fatigue and mood GWI phenotypes. Establishment of a comprehensive animal model that recapitulates multiple GWI symptom domains and neuroinflammation has significant implications for uncovering pathophysiology, improving diagnosis and treatment for GWI.

Pyridostigmine bromide, chlorpyrifos, and DEET combined Gulf War exposure insult depresses mitochondrial function in neuroblastoma cells.

Gulf War Illness (GWI) is defined by the Centers for Disease Control and Prevention (CDC) as a multi-symptom illness having at least one symptom from two of three factors, which include: fatigue, mood-cognition problems, and musculoskeletal disorders. The cluster of long-term symptoms is unique to military personnel from coalition countries including United States, Australia, and the United Kingdom that served in Operation Desert Storm from 1990 to 1991. Reporting of these symptoms is much lower among soldiers deployed in other parts of the world like Bosnia during the same time period. The exact cause of GWI is unknown, but combined exposure to N,N-diethyl-m-toluamide (DEET), organophosphates like chlorpyrifos (CPF), and pyridostigmine bromide (PB), has been hypothesized as a potential mechanism. Mitochondrial dysfunction is known to occur in most neurodegenerative diseases that share symptoms with GWI and has therefore been implicated in GWI. Although exposure to these and other toxicants continues to be investigated as potential causes of GWI, their combined impact on mitochondrial physiology remains unknown. In this study, the effects of combined GWI toxicant exposure on mitochondrial function were determined in a commonly used and readily available immortalized cell line (N2a), whose higher rate of oxygen consumption resembles that of highly metabolic neurons in vivo. We report that combined exposure containing pesticide CPF 71 µM, insect repellants DEET 78 µM, and antitoxins PB 19 µM, causes profound mitochondrial dysfunction after a 4-h incubation resulting in decreased mitochondrial respiratory states in the absence of proapoptotic signaling, proton leak, or significant increase in reactive oxygen species production.

Effects of Incubation of Human Brain Microvascular Endothelial Cells and Astrocytes with Pyridostigmine Bromide, DEET, or Permethrin in the Absence or Presence of Metal Salts.

Gulf War Illness (GWI) is a chronic, multi-symptom illness suffered by over one-third of American military veterans who served in the Persian Gulf War between 1990 and 1991. No current single-exposure scenario accounts for all the symptoms observed in GWI, and instead may be due to a multi-exposure scenario. As a larger effort to understand how one category of multi-exposure scenarios of organic compounds such as nerve gas prophylactic pyridostigmine bromide, or insecticides/pesticides such as N,N-diethyl-m-toluamide (DEET) and permethrin, plus heavy metals found in inhaled dust particles (Al, Fe, Ni, Sr, DU, Co, Cu, Mn, and Zn) might play a role in neural aspects of GWI, we begin this initial study to examine the toxicity and oxidative damage markers of human brain endothelial cell and human astrocyte cell cultures in response to these compounds. A battery of cytotoxicity assessments, including the MTT assay, Neutral Red uptake, and direct microscopic observation, was used to determine a non-toxic dose of the test compounds. After testing a wide range of doses of each compound, we chose a sub-toxic dose of 10 µM for the three organic compounds and 1 µM for the nine metals of interest for co-exposure experiments on cell cultures and examined an array of oxidative stress-response markers including nitric oxide production, formation of protein carbonyls, production of thiobarbituric acid-reactive substances, and expression of proteins involved in oxidative stress and cell damage. Many markers were not significantly altered, but we report a significant increase in nitric oxide after exposure to any of the three compounds in conjunction with depleted uranium.

Pyridostigmine bromide exposure creates chronic, underlying neuroimmune disruption in the gastrointestinal tract and brain that alters responses to palmitoylethanolamide in a mouse model of Gulf War Illness.

Gulf War Illness (GWI) is a chronic multisymptom illness that includes gastrointestinal disorders. Although the exact etiology of GWI is unknown, exposure to the drug pyridostigmine bromide (PB) is considered a major factor. Exposure to PB drives enteric neuroinflammation, promotes immunosuppression, and alters physiological functions of the colon in the short term but whether exposure to PB is sufficient to promote long term dysfunction is not known. Here, we tested whether exposure to PB is sufficient to drive long term changes that reflect GWI, and whether the endogenous anti-inflammatory mediator palmitoylethanolamide (PEA) is sufficient to reduce the detrimental effects of PB in the gut and brain of mice. Exposure to PB alone was not sufficient to cause major changes in neuromuscular transmission but did drive major changes by altering the effects of PEA. Calcium imaging data show that the mechanisms responsible include a shift in receptor signaling mediated by TRPV1, endocannabinoids, and peroxisome proliferator-activated receptors alpha (PPARα). Additional mechanisms include the development of glial reactivity and changes in enteric neurochemical coding and survival. PB and PEA caused major shifts in pro-inflammatory cytokines/chemokines in the brain and colon that persisted up to 5 months following exposure. Many of the effects of PB and PEA exhibit significant sex differences. Together, these results highlight novel mechanisms whereby PB promotes long-lasting changes in nervous system and immune function by inducing occult neuroplasticity that is revealed by subsequent exposure to unrelated drugs in a sex dependent manner.

Neurochemical and neuroinflammatory perturbations in two Gulf War Illness models: Modulation by the immunotherapeutic LNFPIII.

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.

Respiratory failure triggered by cholinesterase inhibitors may involve activation of a reflex sensory pathway by acetylcholine spillover.

Respiration failure during exposure by cholinesterase inhibitors has been widely assumed to be due to inhibition of cholinesterase in the brain. Using a double chamber plethysmograph to measure various respiratory parameters, we observed long "end inspiratory pauses" (EIP) during most exposure that depressed breathing. Surprisingly, Colq KO mice that have a normal level of acetylcholinesterase (AChE) in the brain but a severe deficit in muscles and other peripheral tissues do not pause the breathing by long EIP. In mice, long EIP can be triggered by a nasal irritant. Eucalyptol, an agonist of cold receptor (TRPM8) acting on afferent sensory neurons and known to reduce the EIP triggered by such irritants, strongly reduced the EIP induced by cholinesterase inhibitor. These results suggest that acetylcholine (ACh) spillover from the neuromuscular junction, which is unchanged in Colq KO mice, may activate afferent sensory systems and trigger sensory reflexes, as reversed by eucalyptol. Indeed, the role of AChE at the cholinergic synapses is not only to accurately control the synaptic transmission but also to prevent the spillover of ACh. In the peripheral tissues, the ACh flood induced by cholinesterase inhibition may be very toxic due to interaction with non-neuronal cells that use ACh at low levels to communicate with afferent sensory neurons.

The Val16Ala-SOD2 polymorphism affects cyto-genotoxicity of pyridostigmine bromide on human peripheral blood mononuclear cells.

Pyridostigmine bromide (PB), an acetylcholinesterase (AChE) enzyme inhibitor. Experimental evidence showed that when combined with other drugs or exercise, PB caused extensive neural and/or systemic oxidative stress. However, no studies have been conducted on the genetic influence associated with basal oxidative superoxide-hydrogen peroxide (S-HP) imbalance, such as that triggered by Val16Ala-SOD2 single nucleotide polymorphism (SNP, rs4880). This SNP, (homozygous genotypes) has been associated with several chronic degenerative disorders. Therefore, we evaluated whether the SOD-SNP could alter cyto-genotoxic effects triggered by different PB-concentrations in peripheral blood mononuclear cells (PBMCs). PBMCs were obtained from volunteers carrying different SOD2-genotypes and were cultured with various concentrations of PB. PB effects in quantity of enzyme AChE, mortality rate, oxidative stress markers, and DNA damage were assessed. Protein and gene expression of antioxidant enzymes, apoptotic markers and DNA repair enzyme, were evaluated in 24 h cultures. In general, PB up-regulated expression of antioxidant enzymes, and did not trigger apoptotic events. However, AA-PBMCs seemed more sensitive to PB exposure, in a protein decrease of the enzyme AChE by 10%, cell-mortality at concentrations of 20 and 40 ng/mL, protein carbonylation, and DNA damage, as analyzed by the Comet assay. Contrarily, PB demonstrated cyto-genoprotective effects on V-allele cells. These results indicated that genetic factors that increase HP-release may affect PB efficiency and safety.

Gastrointestinal neuroimmune disruption in a mouse model of Gulf War illness.

Gulf War illness (GWI) is a chronic multisymptom disorder that is prominent in Gulf War veterans. Major unexplained symptoms of GWI include functional gastrointestinal disorders and undiagnosed illnesses, including neurologic disorders. Exposure to the antinerve gas drug pyridostigmine bromide (PB) is linked to the development of GWI, but the exact mechanisms remain unclear. Here, we tested the hypothesis that PB alters gut function by disrupting the neural and immune systems of the intestine. We exposed male and female mice to physiologically comparable amounts of PB that match the dose, route, and time frame of exposure experienced by Gulf War veterans and assessed the acute and chronic impacts on gastrointestinal functions, the functional architecture of the enteric nervous system, and immune responses in the gut and brain. Exposure to PB drove acute alterations to colonic motility and structure in both male and female mice that transitioned to chronic changes in gut functions. PB drove acute alterations to enteric neural and glial activity, glial reactivity, and neuron survival with glial reactivity persisting into the chronic phase in male mice. Despite having no effect on colonic permeability, exposure to PB caused major shifts in the expression of proinflammatory cytokines and chemokines in the colon and brain that suggest immunosuppressive effects. Interestingly, immune disruption was still evident in the colon and brain in female animals at 1 mo following exposure to PB. Together, our results show that the paradigm of PB exposure experienced by veterans of the Persian Gulf War contributes to long-lasting pathophysiology by driving enteric neuroinflammation, promoting immunosuppression, and altering functional anatomy of the colon in a sex-dependent manner.-Hernandez, S., Fried, D. E., Grubisic, V., McClain, J. L., Gulbransen, B. D. Gastrointestinal neuroimmune disruption in a mouse model of Gulf War illness.

Corticosterone and pyridostigmine/DEET exposure attenuate peripheral cytokine expression: Supporting a dominant role for neuroinflammation in a mouse model of Gulf War Illness.

Gulf War Illness (GWI) is a chronic multi-symptom disorder experienced by as many as a third of the veterans of the 1991 Gulf War; the constellation of "sickness behavior" symptoms observed in ill veterans is suggestive of a neuroimmune involvement. Various chemical exposures and conditions in theater have been implicated in the etiology of the illness. Previously, we found that GW-related organophosphates (OPs), such as the sarin surrogate, DFP, and chlorpyrifos, cause neuroinflammation. The combination of these exposures with exogenous corticosterone (CORT), mimicking high physiological stress, exacerbates the observed neuroinflammation. The potential relationship between the effects of OPs and CORT on the brain versus inflammation in the periphery has not been explored. Here, using our established GWI mouse model, we investigated the effects of CORT and DFP exposure, with or without a chronic application of pyridostigmine bromide (PB) and N,N-diethyl-meta-toluamide (DEET), on cytokines in the liver and serum. While CORT primed DFP-induced neuroinflammation, this effect was largely absent in the periphery. Moreover, the changes found in the peripheral tissues do not correlate with the previously reported neuroinflammation. These results not only support GWI as a neuroimmune disorder, but also highlight the separation between central and peripheral effects of these exposures.

Gulf War agents pyridostigmine bromide and permethrin cause hypersensitive nociception that is restored after vagus nerve stimulation.

Gulf war illness (GWI) is a chronic multi-symptom disease that afflicts 25-33% of troops that were deployed in the 1990-1991 Gulf War. GWI symptoms include cognitive, behavioral and emotional deficits, as well as migraines and pain. It is possible that exposure to Gulf War agents and prophylactics contributed to the reported symptomology. Pyridostigmine bromide (PB) and permethrin (PER) were given to protect from nerve gas attacks and insect vector born disease, respectively. Previous studies have demonstrated that 10 days of exposure to these chemicals can cause symptoms analogous to those observed in GWI, including impairment of long-term memory in mice. Other studies using this model have shown chronic neuroinflammation, and chronic neuroinflammation can lead to altered nociceptive sensitivity. At 10-weeks after the 10-day PB and PER exposure paradigm, we observed lowered nociceptive threshold on the Von Frey test that was no longer evident at 28 weeks and 38 weeks post-exposure. We further determined that vagus nerve stimulation, initiated at 38 weeks after exposure, restores the lowered nociceptive sensitivity. Therefore, stimulating the vagus nerve appears to influence nociception. Future studies are need to elucidate possible mechanisms of this effect.

Gulf war illness-related chemicals increase CD11b/c+ monocyte infiltration into the liver and aggravate hepatic cholestasis in a rodent model.

Gulf War Illness (GWI) is a chronic multisymptom disorder affecting veterans of the 1990-91 Gulf war. GWI was linked with exposure to chemicals including the nerve gas prophylactic drug pyridostigmine-bromide (PB) and pesticides (DEET, permethrin). Veterans with GWI exhibit prolonged, low-level systemic inflammation, though whether this impacts the liver is unknown. While no evidence exists that GWI-related chemicals are hepatotoxic, the prolonged inflammation may alter the liver's response to insults such as cholestatic injury. We assessed the effects of GWI-related chemicals on macrophage infiltration and its subsequent influence on hepatic cholestasis. Sprague Dawley rats were treated daily with PB, DEET and permethrin followed by 15 minutes of restraint stress for 28 days. Ten weeks afterward, GWI rats or naïve age-matched controls underwent bile duct ligation (BDL) or sham surgeries. Exposure to GWI-related chemicals alone increased IL-6, and CD11b+F4/80- macrophages in the liver, with no effect on biliary mass or hepatic fibrosis. However, pre-exposure to GWI-related chemicals enhanced biliary hyperplasia and fibrogenesis caused by BDL, compared to naïve rats undergoing the same surgery. These data suggest that GWI patients could be predisposed to developing worse liver pathology due to sustained low-level inflammation of the liver when compared to patients without GWI.

Neuropsychological functioning in military pesticide applicators from the Gulf War: Effects on information processing speed, attention and visual memory.

1991 Gulf War (GW) veterans continue to experience debilitating cognitive and mood problems more than two decades following their return from deployment. Suspected causes for these cognitive complaints include additive and/or synergistic effects of the varying combinations of exposures to chemicals in theater, including pesticides and pyridostigmine bromide (PB) pills. This study was undertaken to address one of the key recommendations of the US Department of Defense Environmental Exposure Report on Pesticides, which was to conduct an epidemiological study to further evaluate the role of neurotoxicant exposures in the expression of central nervous system symptoms reported by GW veterans. This study evaluated the role of pesticides and/or PB in the development of chronic neuropsychological dysfunction in GW veterans. We examined the associations between self-reported measures of pesticide and PB exposures and performance on neuropsychological tests in a group of 159 GW-deployed preventative medicine personnel who had varying levels of pesticide exposures during their work as pesticide applicators or other preventative medicine roles. These veterans had a unique knowledge of pesticides and their usage during the war. It was hypothesized that pesticide applicator personnel with higher exposures would perform significantly worse on objective cognitive measures than lower-exposed personnel and that multiple chemical exposures (pesticide and PB) would further diminish cognitive functioning and increase mood complaints. Study results showed that the participants with both high pesticide and high PB exposure performed worse on specific measures than the groups with high single exposures or low exposures to both toxicants. High combined exposure was associated with significantly slower information processing reaction times, attentional errors, worse visual memory functioning, and increased mood complaints. In addition, stepwise regression analyses of individual pesticide exposures found that pest strip exposure was associated with slower reaction times and attentional errors, and that fly bait and delouser exposures predicted greater mood complaints.

Effects of Pyridostigmine bromide on SH-SY5Y cells: An in vitro neuroblastoma neurotoxicity model.

Pyridostigmine bromide (PB) is a reversible acetylcholinesterase (AChE) inhibitor and the first-choice for the treatment of symptoms associated with myasthenia gravis and other neuromuscular junction disorders. However, evidence suggested that PB could be associated with the Gulf War Illness characterised by the presence of fatigue, headaches, cognitive dysfunction, and musculoskeletal respiratory and gastrointestinal disturbances. Given that a potential neurotoxic effect of PB has not yet been completely elucidated, the present investigation used neural SH-SY5Y cells to evaluate the effect of PB on the cellular viability, cell apoptosis, modulation of the cell cycle, oxidative stress, and genotoxicity variables, which indicate neurodegeneration. As expected, a PB concentration curve based on the therapeutic dose of the drug showed an inhibition of the AChE activity. However, this effect was transient and did not involve differential AChE gene regulation by PB. These results confirmed that undifferentiated SH-SY5Y cells can be used as a cholinergic in vitro model. In general, PB did not trigger oxidative stress, and at a slightly higher PB concentration (80ng/mL), higher levels of protein carbonylation and DNA damage were detected, as determined by the marker 8-deoxyguanosine. The PB genotoxic effects at 80ng/mL were confirmed by the upregulation of the p53 and DNA methyltransferase 1 (DNMT1) genes, which are associated with cellular DNA repair. PB at 40ng/mL, which is the minimal therapeutic dose, led to higher cell proliferation and mitochondrial activity compared with the control group. The effects of PB were corroborated by the upregulation of the telomerase gene. In summary, despite the methodological constrains related to the in vitro protocols, our results suggested that exposure of neural cells to PB, without other chemical and physical stressors did not cause extensive toxicity or indicate any neurodegeneration patterns.

Novel potent pyridoxine-based inhibitors of AChE and BChE, structural analogs of pyridostigmine, with improved in vivo safety profile.

We report a novel class of carbamate-type ChE inhibitors, structural analogs of pyridostigmine. A small library of congeneric pyridoxine-based compounds was designed, synthesized and evaluated for AChE and BChE enzymes inhibition in vitro. The most active compounds have potent enzyme inhibiting activity with IC50 values in the range of 0.46-2.1µM (for AChE) and 0.59-8.1µM (for BChE), with moderate selectivity for AChE comparable with that of pyridostigmine and neostigmine. Acute toxicity studies using mice models demonstrated excellent safety profile of the obtained compounds with LD50 in the range of 22-326mg/kg, while pyridostigmine and neostigmine are much more toxic (LD50 3.3 and 0.51mg/kg, respectively). The obtained results pave the way to design of novel potent and safe cholinesterase inhibitors for symptomatic treatment of neuromuscular disorders.

Long-term epigenetic alterations in a rat model of Gulf War Illness.

Gulf War Illness (GWI) is a chronic, multisymptom illness that affects 25% of the 700,000 US veterans deployed to the Persian Gulf during the 1990-1991 Gulf War. Central nervous system impairments are among the most common symptoms reported, including memory dysfunction and depression. After 25 years, the diagnosis remains elusive, useful treatments are lacking, and the cause is poorly understood, although exposures to pyridostigmine bromide (PB) and pesticides are consistently identified to be among the strongest risk factors. Epigenetic changes including altered microRNA (miRNA) expression and DNA methylation play an important role in learning, memory, and emotion regulation and have been implicated in various neurological disorders. In this study, we used an established rat model of GWI to determine whether 1) chronic alterations in miRNA expression and global DNA methylation and DNA hydroxymethylation are mechanisms involved in the pathobiology of GWI, and 2) plasma exosome small RNAs may serve as potential noninvasive biomarkers of this debilitating disease. One year after a 28-day exposure regimen of PB, DEET (N,N-diethyl-3-methylbenzamide), permethrin, and mild stress, expression of 84 mature miRNAs and global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) content were analyzed in the brains of GWI rats and vehicle controls by PCR array and enzyme-linked immunosorbent assay, respectively. Plasma exosome RNA next-generation sequencing analysis was performed in pooled samples to discover potential noninvasive biomarkers. We found that combined exposure to low doses of GW-related chemicals and mild stress caused epigenetic modifications in the brain that persisted one year after exposure, including increased expression of miR-124-3p and miR-29b-3p in the hippocampus and regional alterations in global 5mC and 5hmC content. GW-relevant exposures also induced the differential expression of two piwi-interacting RNAs (piRNAs) in circulation (piR-007899 and piR-019162). Results from this study implicate a role for epigenetic alterations in GWI. Evaluation of the diagnostic potential of plasma exosome RNAs in veterans with GWI is warranted.

Gulf War illness: an overview of events, most prevalent health outcomes, exposures, and clues as to pathogenesis.

INTRODUCTION: During or very soon after the 1990-1991 Persian Gulf War, veterans of the conflict began to report symptoms of illness. Common complaints included combinations of cognitive difficulties, fatigue, myalgia, rashes, dyspnea, insomnia, gastrointestinal symptoms and sensitivity to odors. Gradually in the USA, and later in the UK, France, Canada, Denmark and Australia, governments implemented medical assessment programs and epidemiologic studies to determine the scope of what was popularly referred to as "the Gulf War syndrome". Attention was drawn to numerous potentially toxic deployment-related exposures that appeared to vary by country of deployment, by location within the theater, by unit, and by personal job types. Identifying a single toxicant cause was considered unlikely and it was recognized that outcomes were influenced by genetic variability in xenobiotic metabolism. METHODS: Derived from primary papers and key reports by the Research Advisory Committee on Gulf War Veterans' Illnesses and the Institute of Medicine, a brief overview is presented of war related events, symptoms and diagnostic criteria for Gulf War illness (GWV), some international differences, the various war-related exposures and key epidemiologic studies. Possible exposure interactions and pathophysiologic mechanisms are discussed. RESULTS: Exposures to pyridostigmine bromide, pesticides, sarin and mustard gas or combinations thereof were most associated with GWI, especially in some genotype subgroups. The resultant oxidant stress and background exposome must be assumed to have played a role. CONCLUSION: Gulf War (GW) exposures and their potential toxic effects should be considered in the context of the human genome, the human exposome and resultant oxidant stress to better characterize this unique environmentally-linked illness and, ultimately, provide a rationale for more effective interventions and future prevention efforts.

A delayed chronic pain like condition with decreased Kv channel activity in a rat model of Gulf War Illness pain syndrome.

Following their return from deployment, Gulf War (GW) veterans reported widespread joint and muscle pain at rates that far exceeded those of soldiers returning from other conflicts. It is widely believed that exposure to insecticides, repellants and nerve gas prophylactics contributed to the symptoms of Gulf War Illness (GWI), but an animal model of GW pain has been elusive. In our previous work, we observed that 4-8 weeks exposure to pyridostigmine bromide (PB), permethrin and chlorpyrifos could produce persistent alterations in the physiology of Nav1.9 and Kv7 expressed in deep tissue nociceptors of the dorsal root ganglion. However, behavioral assessments from these same rats were not consistent with a delayed pain syndrome similar to that of GWI pain. In the present studies, we intensified the exposure to anticholinesterases PB and chlorpyrifos while retaining the same dosages. Animals receiving the intensified protocol for 30 days exhibited significant increases in resting for about 8 weeks after exposure. Thereafter, all measures were comparable to controls. Animals treated with intensified anticholinesterases for 60 days exhibited increased resting and reduced movement 12 weeks post-exposure. In whole cell patch studies, muscle and vascular nociceptor KDR and Kv7 ion channels exhibited increased amplitude relative to controls (e.g., normalized current and/or peak conductance) at 8 weeks post-exposures; however, at 12 weeks post-exposure, the amplitude of these currents was significantly decreased in muscle nociceptors. In current clamp studies, muscle nociceptors also manifested increased action potential duration, afterhyperpolarization and increased discharge to muscarinic agonists 12 weeks post-exposure. The decline in activity of muscle nociceptor KDR and Kv7 channel proteins was consistent with increased nociceptor excitability and a delayed myalgia in rats exposed to GW chemicals.