Model Of Gulf War Illness Research Articles

390 - Extracellular Superoxide Dismutase as a Therapeutic Target in a Mouse Model of Gulf War Illness

More than twenty five years after the end of the 1900-1991 Gulf War, a substantial contingent of veterans still suffer from a number of health problems such as chronic fatigue, muscle and joint pain, cognitive deficits, etc. – this constellation of symptoms is collectively termed Gulf War Illness. The etiology of Gulf War Illness remains elusive, yet likely involves exposure to various chemicals as well as physical and psychological stresses unique to the Gulf War theatre. We adapted a mouse model of Gulf War Illness by exposing mice to Gulf War-related chemicals in addition to immobilization stress and assessed their cognitive functions along with pain susceptibilities. The mouse model recapitulated several aspects of the human illness, namely deficits in working spatial memory and recognition memory, along with heightened pain sensitization—precursor to chronic pain, impaired descending inhibition of pain, and increased tonic pain. Neuropathological changes, including the presence of neuroinflammation and increased oxidative stress, were found in brain tissues long after the completion of Gulf War chemical exposure. Coinciding with these chronic conditions was a reduction in the perineuronal net encapsulating inhibitory neurons, suggesting an imbalance in the excitation and inhibition of neuronal signal transmission. To assess whether enhanced antioxidant capacity can suppress the aforementioned neuroinflammation and oxidative stress, and ultimately counter the symptoms of Gulf War Illness, transgenic mice overexpressing extracellular superoxide dismutase in neurons (EC-SOD mice) were utilized. These mice were also exposed to Gulf War-related chemicals and immobilization stress, after which cognitive functions along with pain susceptibilities were assessed. The negative impacts of Gulf War, including neuropathological changes, cognitive deficits, and pain susceptibilities were largely diminished in these EC-SOD mice, suggesting the benefit of antioxidants in improving symptoms of Gulf War Illness.

Translational potential of long-term decreases in mitochondrial lipids in a mouse model of Gulf War Illness

Gulf War Illness (GWI) affects 25% of veterans from the 1990-1991 Gulf War (GW) and is accompanied by damage to the brain regions involved in memory processing. After twenty-five years, the chronic pathobiology of GWI is still unexplained. To address this problem, we examined the long-term consequences of GW exposures in an established GWI mouse model to identify biological processes that are relevant to the chronic symptoms of GWI. Three-month old male C57BL6 mice were exposed for 10days to GW agents (pyridostigmine bromide and permethrin). Barnes Maze testing conducted at 15- and 16-months post-exposure revealed learning and memory impairment. Immunohistochemical analyses showed astroglia and microglia activation in the hippocampi of exposed mice. Proteomic studies identified perturbation of mitochondria function and metabolomics data showed decreases in the Krebs cycle compounds, lactate, β-hydroxybutyrate and glycerol-3 phosphate in the brains of exposed mice. Lipidomics data showed decreases in fatty acids, acylcarnitines and phospholipids, including cardiolipins in the brains of exposed mice. Pilot biomarker studies showed that plasma from exposed mice and veterans with GWI had increases in odd-chain, and decreases in long-chain, acylcarnitines compared to their respective controls. Very long-chain acylcarnitines were decreased in veterans with GWI compared to controls. These studies suggest that mitochondrial lipid disturbances might be associated with GWI and that further investigation is required to determine its role in the pathophysiology of this illness. Targeting mitochondrial function may provide effective therapies for GWI, and that lipid abnormalities could serve as biomarkers of GWI.

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.

Low‐intensity exercise training improves autonomic modulation and cardiac function in a potential model of Gulf War illness in mice with organophosphate (DFP) exposure: analyze by noninvasive method of cardiovascular measures

BackgroundSeveral conditions and exposures have been hypothesized to serve as instigating events that precipitated the recurring/ chronic symptoms associated with Gulf War illness (GWI). Our group was focused on the use of DFP as chemicals which can be used to produce models of GWI.PurposeThe objective in this new study was the determination of the influence of DFP on cardiac (Echo), autonomic (spectral analysis) parameters in association with long term exercise training (6 months).MethodsMale mice were divided into (n=8/group): Sedentary (S) and trained (T) groups. The exercise training was performed on an electronic wheel at low intensity for 1 h a day, 5 days/wk for 6 months. We used sc injection of DFP with dose of 1.5 mg/kg after 6 months of exercise program. Heart rate was recorded using the electronic platform provided by the echocardiography machine. The mice were kept under anesthesia (Isoflurane, 2 – 2.5% on 0.8l/min of O2) and positioned on the board with ECG sensors. The cardiac autonomic modulation was evaluated by spectral analysis. Cardiac function was assessed by echocardiography.ResultsT group showed better diastolic function with lower isovolumetric relaxation time (IVRT) (T: 14.5±0.3 vs S: 17.2±0.8 ms), E wave deceleration time (Desac. E) (T: 30.7±3 vs S: 15±0.91 ms), and cardiac global function by the myocardial performance index MPI (T: 0.29±0.007 vs S: 0.52±0.07). Regard to morphological parameters such as end diastolic diameter (EDD: T: 0.36±0.008 vs S: 0.32±0.008) end systolic diameter (ESD: T: 0.20±0.01 vs S: 0.15±0.01) and Left ventricular mass (LVM: T: 0.17±0.01 vs S: 0.13±0.007) were increased in T group compared to S group. Finally sympathetic modulation (LF) was lower in T groups (T: 24± 3 %) compared to S group (S: 40±4%). Vagal modulation was similar between both groups (T: 18±4 vs S: 30±7 %) as well as LF/HF ratio (T: 2.1±0.4 vs S: 1.5±0.3 beats/min).ConclusionOur findings demonstrated that Low‐intensity exercise training coupled with organophosphate exposure reduced sympathetic modulation and improved cardiac function which could result in an improved quality of life and longevity of this population.Support or Funding InformationAward Number: W81XWH‐13‐2‐0085

Challenges and new strategies for Gulf War illness research

Gulf War illness (GWI) research has generated an abundance of interesting but diverse data. While increased molecular mechanisms have been identified, the high levels of heterogeneity for initial trigger factors, cellular defects, and symptoms continuously challenge the efforts of clinical implications of the research, including the search for biomarkers and the common mechanism of GWI. In this analysis, I consider GWI as an adaptive illness condition where system stresses and genome instability-mediated cellular evolution play an important role. By further defining GWI as an environmental illness caused by extremely high levels of specific Gulf War (GW) stresses, the challenges for GWI research are briefly reviewed, with comparisons to other common and complex diseases such as cancer. Based on the new discovery that many GWI patients display elevated genome instability coupled with increased cellular stress, a general model of GWI is proposed to unify GW-specific stress, cellular damage, and genome-heterogeneity-mediated cellular adaptation and evolution, as well as diverse-related symptoms. Finally, some new strategies are suggested based on the general model of GWI.

Towards a Biopsychosocial Model of Gulf War Illness?

Towards a Biopsychosocial Model of Gulf War Illness?

Repeated low-dose organophosphate DFP exposure leads to the development of depression and cognitive impairment in a rat model of Gulf War Illness

Approximately 175,000–250,000 of the returning veterans from the 1991 Persian Gulf War exhibit chronic multi-symptom illnesses that includes neurologic co-morbidities such as depression, anxiety and cognitive impairments. Amongst a host of causative factors, exposure to low levels of the nerve agent Sarin has been strongly implicated for expression of Gulf War Illness (GWI). Nerve agents similar to pesticides are organophosphate (OP) compounds. There is evidence from civilian population that exposure to OPs such as in agricultural workers and nerve agents such as the survivors and first-responders of the Tokyo subway Sarin gas attack suffer from chronic neurological problems similar to GWI symptoms. Given this unique chemical profile, OPs are ideal to study the effects of nerve agents and develop models of GWI in civilian laboratories. In this study, we used repeated low-dose exposure to OP agent diisopropyl fluorophosphate (DFP) over a 5-day period to approximate the duration and level of Sarin exposure during the Persian Gulf War. We tested the rats at 3-months post DFP exposure. Using a battery of behavioral assays, we observed the presence of symptoms of chronic depression, anxiety and memory problems as characterized by increased immobility time in the Forced Swim Test, anhedonia in the Sucrose Preference Test, anxiety in the Elevated Plus Maze, and spatial memory impairments in the Object Location Test, respectively. Chronic low dose DFP exposure was also associated with hippocampal neuronal damage as characterized by the presence of Fluoro-Jade staining. Given that OP exposure is considered a leading cause of GWI related morbidities, this animal model will be ideally suited to study underlying molecular mechanisms for the expression of GWI neurological symptoms and identify drugs for the effective treatment of GWIs.

Alterations in Global DNA Methylation/Hydroxymethylation and MicroRNA Expression 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. Epidemiological studies have consistently identified neurotoxic chemical exposures during deployment as the strongest risk factors for the persistent symptoms affecting GW veterans, but after nearly 25 years, treatments are lacking and underlying cellular and molecular mechanisms involved in its pathobiology remain poorly understood. Epigenetic changes including DNA methylation and altered microRNA (miRNA) expression play an important role in in learning, memory, and emotion regulation and have been implicated in various neurological disorders. The aim of this study was to determine in a rat model of GWI whether chronic alterations in global DNA methylation/hydroxymethylation and miRNA expression are mechanisms involved in the pathobiology of GWI. One year after a 28-day exposure regimen of GW chemicals and stress, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) content and expression of 84 mature miRNAs were analyzed in the brains of GWI rats and vehicle controls by ELISA and PCR array, respectively. In GWI rats, hippocampal 5mC content increased 46%, while 5hmC content increased 29% in the cerebellum and decreased 45% in the cortex (p<0.01 for all). Ten miRNAs known to be involved in synaptic plasticity and memory function, DNA methylation, or adult neurogenesis were differentially expressed (>1.5 fold change, p<0.01) in the hippocampus. Results from this study implicate a role for epigenetic alterations in GWI.

Corticosterone primes the neuroinflammatory response to DFP in mice: potential animal model of Gulf War Illness

Gulf War Illness (GWI) is a multi‐symptom disorder with features characteristic of persistent sickness behavior. Among conditions encountered in the Gulf War (GW) theater were physiological stressors (e.g., heat/cold/physical activity/sleep deprivation), prophylactic treatment with the reversible AChE inhibitor, pyridostigmine bromide (PB), the insect repellent, N,N‐diethyl‐meta‐toluamide (DEET), and potentially the nerve agent, sarin. Prior exposure to the anti‐inflammatory glucocorticoid, corticosterone (CORT), at levels associated with high physiological stress, can paradoxically prime the CNS to produce a robust proinflammatory response to neurotoxicants and systemic inflammation; such neuroinflammatory effects can be associated with sickness behavior. Here, we examined whether CORT primed the CNS to mount neuroinflammatory responses to GW exposures as a potential model of GWI. Male C57BL/6 mice were treated with chronic (14 days) PB/ DEET, subchronic (7–14 days) CORT, and acute exposure (day 15) to diisopropyl fluorophosphate (DFP), a sarin surrogate and irreversible AChE inhibitor. DFP alone caused marked brain‐wide neuroinflammation assessed by qPCR of tumor necrosis factor‐α, IL6, chemokine (C‐C motif) ligand 2, IL‐1β, leukemia inhibitory factor, and oncostatin M. Pre‐treatment with high physiological levels of CORT greatly augmented (up to 300‐fold) the neuroinflammatory responses to DFP. Anti‐inflammatory pre‐treatment with minocycline suppressed many proinflammatory responses to CORT+DFP. Our findings are suggestive of a possible critical, yet unrecognized interaction between the stressor/environment of the GW theater and agent exposure(s) unique to this war. Such exposures may in fact prime the CNS to amplify future neuroinflammatory responses to pathogens, injury, or toxicity. Such occurrences could potentially result in the prolonged episodes of sickness behavior observed in GWI. Gulf War (GW) veterans were exposed to stressors, prophylactic medicines and, potentially, nerve agents in theater. Subsequent development of GW Illness, a persistent multi‐symptom disorder with features characteristic of sickness behavior, may be caused by priming of the CNS resulting in exaggerated neuroinflammatory responses to pathogens/insults. Nerve agent, diisopropyl fluorophosphate (DFP), produced a neuroinflammatory response that was exacerbated by pre‐treatment with levels of corticosterone simulating heightened stressor conditions. While prophylactic treatments reduced DFP‐induced neuroinflammation, this effect was negated when those treatments were combined with corticosterone.

Gulf War agent exposure causes impairment of long-term memory formation and neuropathological changes in a mouse model of Gulf War Illness.

Gulf War Illness (GWI) is a chronic multisymptom illness with a central nervous system component such as memory deficits, neurological, and musculoskeletal problems. There are ample data that demonstrate that exposure to Gulf War (GW) agents, such as pyridostigmine bromide (PB) and pesticides such as permethrin (PER), were key contributors to the etiology of GWI post deployment to the Persian GW. In the current study, we examined the consequences of acute (10 days) exposure to PB and PER in C57BL6 mice. Learning and memory tests were performed at 18 days and at 5 months post-exposure. We investigated the relationship between the cognitive phenotype and neuropathological changes at short and long-term time points post-exposure. No cognitive deficits were observed at the short-term time point, and only minor neuropathological changes were detected. However, cognitive deficits emerged at the later time point and were associated with increased astrogliosis and reduction of synaptophysin staining in the hippocampi and cerebral cortices of exposed mice, 5 months post exposure. In summary, our findings in this mouse model of GW agent exposure are consistent with some GWI symptom manifestations, including delayed onset of symptoms and CNS disturbances observed in GWI veterans.

Parvalbumin and neuropeptide Y expressing hippocampal GABA-ergic inhibitory interneuron numbers decline in a model of Gulf War illness.

Cognitive dysfunction is amongst the most conspicuous symptoms in Gulf War illness (GWI). Combined exposure to the nerve gas antidote pyridostigmine bromide (PB), pesticides and stress during the Persian Gulf War-1 (PGW-1) are presumed to be among the major causes of GWI. Indeed, our recent studies in rat models have shown that exposure to GWI-related (GWIR) chemicals and mild stress for 4 weeks engenders cognitive impairments accompanied with several detrimental changes in the hippocampus. In this study, we tested whether reduced numbers of hippocampal gamma-amino butyric acid (GABA)-ergic interneurons are among the pathological changes induced by GWIR-chemicals and stress. Animals were exposed to low doses of GWIR-chemicals and mild stress for 4 weeks. Three months after this exposure, subpopulations of GABA-ergic interneurons expressing the calcium binding protein parvalbumin (PV), the neuropeptide Y (NPY) and somatostatin (SS) in the hippocampus were stereologically quantified. Animals exposed to GWIR-chemicals and stress for 4 weeks displayed reduced numbers of PV-expressing GABA-ergic interneurons in the dentate gyrus and NPY-expressing interneurons in the CA1 and CA3 subfields. However, no changes in SS+ interneuron population were observed in the hippocampus. Furthermore, GABA-ergic interneuron deficiency in these animals was associated with greatly diminished hippocampus neurogenesis. Because PV+ and NPY+ interneurons play roles in maintaining normal cognitive function and neurogenesis, and controlling the activity of excitatory neurons in the hippocampus, reduced numbers of these interneurons may be one of the major causes of cognitive dysfunction and reduced neurogenesis observed in GWI. Hence, strategies that improve inhibitory neurotransmission in the hippocampus may prove beneficial for reversing cognitive dysfunction in GWI.

Object location and object recognition memory impairments, motivation deficits and depression in a model of Gulf War illness

Memory and mood deficits are the enduring brain-related symptoms in Gulf War illness (GWI). Both animal model and epidemiological investigations have indicated that these impairments in a majority of GW veterans are linked to exposures to chemicals such as pyridostigmine bromide (PB, an antinerve gas drug), permethrin (PM, an insecticide) and DEET (a mosquito repellant) encountered during the Persian Gulf War-1. Our previous study in a rat model has shown that combined exposures to low doses of GWI-related (GWIR) chemicals PB, PM, and DEET with or without 5-min of restraint stress (a mild stress paradigm) causes hippocampus-dependent spatial memory dysfunction in a water maze test (WMT) and increased depressive-like behavior in a forced swim test (FST). In this study, using a larger cohort of rats exposed to GWIR-chemicals and stress, we investigated whether the memory deficiency identified earlier in a WMT is reproducible with an alternative and stress free hippocampus-dependent memory test such as the object location test (OLT). We also ascertained the possible co-existence of hippocampus-independent memory dysfunction using a novel object recognition test (NORT), and alterations in mood function with additional tests for motivation and depression. Our results provide new evidence that exposure to low doses of GWIR-chemicals and mild stress for 4 weeks causes deficits in hippocampus-dependent object location memory and perirhinal cortex-dependent novel object recognition memory. An open field test performed prior to other behavioral analyses revealed that memory impairments were not associated with increased anxiety or deficits in general motor ability. However, behavioral tests for mood function such as a voluntary physical exercise paradigm and a novelty suppressed feeding test (NSFT) demonstrated decreased motivation levels and depression. Thus, exposure to GWIR-chemicals and stress causes both hippocampus-dependent and hippocampus-independent memory impairments as well as mood dysfunction in a rat model.

Mood and Memory Deficits in a Model of Gulf War Illness Are Linked with Reduced Neurogenesis, Partial Neuron Loss, and Mild Inflammation in the Hippocampus

Impairments in mood and cognitive function are the key brain abnormalities observed in Gulf war illness (GWI), a chronic multisymptom health problem afflicting ∼25% of veterans who served in the Persian Gulf War-1. Although the precise cause of GWI is still unknown, combined exposure to a nerve gas prophylaxis drug pyridostigmine bromide (PB) and pesticides DEET and permethrin during the war has been proposed as one of the foremost causes of GWI. We investigated the effect of 4 weeks of exposure to Gulf war illness-related (GWIR) chemicals in the absence or presence of mild stress on mood and cognitive function, dentate gyrus neurogenesis, and neurons, microglia, and astrocytes in the hippocampus. Combined exposure to low doses of GWIR chemicals PB, DEET, and permethrin induced depressive- and anxiety-like behavior and spatial learning and memory dysfunction. Application of mild stress in the period of exposure to chemicals exacerbated the extent of mood and cognitive dysfunction. Furthermore, these behavioral impairments were associated with reduced hippocampal volume and multiple cellular alterations such as chronic reductions in neural stem cell activity and neurogenesis, partial loss of principal neurons, and mild inflammation comprising sporadic occurrence of activated microglia and significant hypertrophy of astrocytes. The results show the first evidence of an association between mood and cognitive dysfunction and hippocampal pathology epitomized by decreased neurogenesis, partial loss of principal neurons, and mild inflammation in a model of GWI. Hence, treatment strategies that are efficacious for enhancing neurogenesis and suppressing inflammation may be helpful for alleviation of mood and cognitive dysfunction observed in GWI.

The Tokyo Attacks in Retrospect: Sarin Leads to Memory Loss

Science SelectionsOpen AccessThe Tokyo Attacks in Retrospect Sarin Leads to Memory Loss E Hood E Hood Search for more papers by this author Published:1 November 2001https://doi.org/10.1289/ehp.109-a542aCited by:10AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail "The Tokyo Attacks in Retrospect Sarin Leads to Memory Loss." Environmental Health Perspectives, 109(11), p. A542FiguresReferencesRelatedDetailsCited By Deshpande L, Blair R, Huang B, Phillips K and DeLorenzo R (2016) Pharmacological blockade of the calcium plateau provides neuroprotection following organophosphate paraoxon induced status epilepticus in rats, Neurotoxicology and Teratology, 10.1016/j.ntt.2016.05.002, 56, (81-86), Online publication date: 1-Jul-2016. Deshpande L, Blair R, Phillips K and DeLorenzo R (2016) Role of the calcium plateau in neuronal injury and behavioral morbidities following organophosphate intoxication, Annals of the New York Academy of Sciences, 10.1111/nyas.13122, 1374:1, (176-183), Online publication date: 1-Jun-2016. Phillips K and Deshpande L (2016) Repeated low-dose organophosphate DFP exposure leads to the development of depression and cognitive impairment in a rat model of Gulf War Illness, NeuroToxicology, 10.1016/j.neuro.2015.11.014, 52, (127-133), Online publication date: 1-Jan-2016. Kassa J, Bajgar J, Kuca K and Jun D (2015) Behavioral Toxicity of Nerve Agents Handbook of Toxicology of Chemical Warfare Agents, 10.1016/B978-0-12-800159-2.00035-X, (477-487), . Alexandrova E, Alkondon M, Aracava Y, Pereira E and Albuquerque E (2014) Galantamine prevents long-lasting suppression of excitatory synaptic transmission in CA1 pyramidal neurons of soman-challenged guinea pigs, NeuroToxicology, 10.1016/j.neuro.2014.07.005, 44, (270-278), Online publication date: 1-Sep-2014. Deshpande L, Phillips K, Huang B and DeLorenzo R (2014) Chronic behavioral and cognitive deficits in a rat survival model of paraoxon toxicity, NeuroToxicology, 10.1016/j.neuro.2014.08.008, 44, (352-357), Online publication date: 1-Sep-2014. Mamczarz J, Kulkarni G, Pereira E and Albuquerque E (2011) Galantamine counteracts development of learning impairment in guinea pigs exposed to the organophosphorus poison soman: Clinical significance, NeuroToxicology, 10.1016/j.neuro.2011.07.001, 32:6, (785-798), Online publication date: 1-Dec-2011. Kassa J, Bajgar J, Kuca K and Jun D (2009) Behavioral Toxicity of Nerve Agents Handbook of Toxicology of Chemical Warfare Agents, 10.1016/B978-012374484-5.00033-X, (481-492), . Genovese R, Benton B, Lee E, Shippee S and Jakubowski E (2007) Behavioral and biochemical evaluation of sub-lethal inhalation exposure to VX in rats, Toxicology, 10.1016/j.tox.2006.12.015, 232:1-2, (109-118), Online publication date: 1-Mar-2007. Genovese R, Benton B, Shippee S, Jakubowski E and Bonnell J (2007) Effects of Low-level Inhalation Exposure to Cyclosarin on Learned Behaviors in Sprague-Dawley Rats, Journal of Toxicology and Environmental Health, Part A, 10.1080/15287390600748153, 69:24, (2167-2180), Online publication date: 1-Jun-2006. Vol. 109, No. 11 November 2001Metrics About Article Metrics Publication History Originally published1 November 2001Published in print1 November 2001 Financial disclosures License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.