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Veterans from the 1991 Gulf War (GW) have suffered from Gulf War illness (GWI) for nearly 30 years. This illness encompasses multiple body systems, including the central nervous system (CNS). Diagnosis and treatment of GWI is difficult because there has not been an objective diagnostic biomarker. Recently, we reported on a newly developed blood biomarker that discriminates GWI from GW healthy controls, and symptomatic controls with irritable bowel syndrome (IBS) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). The present study was designed to compare levels of these biomarkers between men and women with GWI, as well as sex-specific effects in comparison to healthy GW veterans and symptomatic controls (IBS, ME/CFS). The results showed that men and women with GWI differ in 2 of 10 plasma autoantibodies, with men showing significantly elevated levels. Men and women with GWI showed significantly different levels of autoantibodies in 8 of 10 biomarkers to neuronal and glial proteins in plasma relative to controls. In summary, the present study addressed the utility of the use of plasma autoantibodies for CNS proteins to distinguish among both men and women veterans with GWI and other healthy and symptomatic control groups.
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For the past 30 years, there has been a lack of objective tools for diagnosing Gulf War Illness (GWI), which is largely characterized by central nervous system (CNS) symptoms emerging from 1991 Gulf War (GW) veterans. In a recent preliminary study, we reported the presence of autoantibodies against CNS proteins in the blood of veterans with GWI, suggesting a potential objective biomarker for the disorder. Now, we report the results of a larger, confirmatory study of these objective biomarkers in 171 veterans with GWI compared to 60 healthy GW veteran controls and 85 symptomatic civilian controls (n = 50 myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and n = 35 irritable bowel syndrome (IBS)). Specifically, we compared plasma markers of CNS autoantibodies for diagnostic characteristics of the four groups (GWI, GW controls, ME/CFS, IBS). For veterans with GWI, the results showed statistically increased levels of nine of the ten autoantibodies against neuronal "tubulin, neurofilament protein (NFP), Microtubule Associated Protein-2 (MAP-2), Microtubule Associated Protein-Tau (Tau), alpha synuclein (α-syn), calcium calmodulin kinase II (CaMKII)" and glial proteins "Glial Fibrillary Acidic Protein (GFAP), Myelin Associated Glycoprotein (MAG), Myelin Basic Protein (MBP), S100B" compared to healthy GW controls as well as civilians with ME/CFS and IBS. Next, we summed all of the means of the CNS autoantibodies for each group into a new index score called the Neurodegeneration Index (NDI). The NDI was calculated for each tested group and showed veterans with GWI had statistically significantly higher NDI values than all three control groups. The present study confirmed the utility of the use of plasma autoantibodies for CNS proteins to distinguish among veterans with GWI and other healthy and symptomatic control groups.
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INTRODUCTION: Military and civil aviation have documented physiological episodes among aircrews. Therefore, continued efforts are being made to improve the internal environment. Studies have shown that exposures to many organic compounds present in emissions are known to cause a variety of physiological symptoms. We hypothesize that these compounds may reversibly inhibit acetylcholinesterase, which may disrupt synaptic signaling. As a result, neural proteins leak through the damaged blood-brain barrier into the blood and in some, elicit an autoimmune response. MATERIALS AND METHODS: Neural-specific autoantibodies of immunoglobulin-G (IgG) class were estimated by the Western blotting technique in the sera of 26 aircrew members and compared with the sera of 19 normal healthy nonaircrew members, used as controls. RESULTS: We found significantly elevated levels of circulating IgG-class autoantibodies to neurofilament triplet proteins, tubulin, microtubule-associated tau proteins (Tau), microtubule-associated protein-2, myelin basic protein, and glial fibrillary acidic protein, but not S100 calcium-binding protein B compared to healthy controls. CONCLUSION: Repetitive physiological episodes may initiate cellular injury, leading to neuronal degeneration in selected individuals. Diagnosis and intervention should occur at early postinjury periods. Use of blood-based biomarkers to assess subclinical brain injury would help in both diagnosis and treatment.
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Personal Militar/estadística & datos numéricos , Fenómenos Fisiológicos/fisiología , Medicina Aeroespacial/métodos , Medicina Aeroespacial/estadística & datos numéricos , Aeronaves , Autoanticuerpos/análisis , Autoanticuerpos/sangre , Biomarcadores/análisis , Biomarcadores/sangre , Western Blotting/métodos , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/sangre , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina G/sangre , Proteínas Asociadas a Microtúbulos/análisis , Proteínas Asociadas a Microtúbulos/sangre , Proteína Básica de Mielina/análisis , Proteína Básica de Mielina/sangre , Proteínas de Neurofilamentos/análisis , Proteínas de Neurofilamentos/sangre , Proteínas S100/análisis , Proteínas S100/sangre , Tubulina (Proteína)/análisis , Tubulina (Proteína)/sangreRESUMEN
INTRODUCTION: Posttraumatic stress disorder (PTSD) can develop during the aftermath of traumatic events. Although many are impacted by several stressors, nearly 3.6% suffer from PTSD in the United States with higher incidence reported in military service personnel. Any injury to the blood-brain barrier can ignite an array of biological signaling molecules in the immune-privileged brain parenchyma, which can disrupt the synaptic neural network, resulting in altered behavior. MATERIALS AND METHODS: In this preliminary study, we compared 20 PTSD veterans with age-matched healthy veterans to identify plasma levels of brain-specific protein markers using enzyme-linked immunosorbent assay/immunofluorometric sandwich assay for neurotrophic factors and neuropoietic cytokines, and catalytic activity of matrix metalloproteinase (MMP) by zymography. RESULTS: We observed an increased level of glial fibrillary acidic protein, tumor necrosis factor-alpha, interleukin 6, and MMP2 and MMP9 but decreased level of brain-derived neurotrophic factor, nerve growth factor-beta, and negligible difference in astroglial marker S100 calcium-binding protein B compared to controls. CONCLUSION: Identification of neural biomarkers is essential to understand the subclinical symptoms for the diagnosis PTSD, which may not be visible by magnetic resonance imaging (MRI/fMRI) and may take years to clinically manifest.
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Trastornos por Estrés Postraumático/etiología , Adulto , Síntomas Conductuales/etiología , Síntomas Conductuales/psicología , Factor Neurotrófico Derivado del Encéfalo/análisis , Factor Neurotrófico Derivado del Encéfalo/sangre , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/sangre , Humanos , Interleucina-6/análisis , Interleucina-6/sangre , Imagen por Resonancia Magnética/métodos , Masculino , Persona de Mediana Edad , Factor de Crecimiento Nervioso/análisis , Factor de Crecimiento Nervioso/sangre , Subunidad beta de la Proteína de Unión al Calcio S100/análisis , Subunidad beta de la Proteína de Unión al Calcio S100/sangre , Trastornos por Estrés Postraumático/diagnóstico , Trastornos por Estrés Postraumático/psicología , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/sangre , Estados UnidosRESUMEN
OBJECTIVES: Serum repositories are foundations for seroepidemiological data, revealing targeted information about morbidities and existing heterogeneity in human populations. With the recent technological advances, we can perform high-throughput screening at an affordable cost using minimal plasma. Monitoring brain health after an injury is critical since mild Traumatic Brain Injury (mTBI) and other neurological symptoms are under-diagnosed. Our objective in this study is to present our preliminary serological data from one of our ongoing studies on mTBI. METHODS: In this retrospective study, we used stored plasma samples to understand biomarkers of mTBI. We compared plasma samples from five patients with mTBI following their first concussive episode to five gender and age-matched healthy controls. We assessed multiple biomarkers to show the importance of biorepositories. RESULTS: Most of the estimated plasma factors in mTBI subjects at baseline were comparable to normal healthy individuals except for the astroglial markers S100B and glial fibrillary acidic protein. Fluctuations of these biomarkers can affect the homeostasis of brain parenchyma by altering the neural network signaling, which in turn may result in intermittent behavioral symptoms. CONCLUSION: Biorepositories are powerful resources for understanding the spectrum of morbidity. Biomarkers serve as a valuable diagnostic and therapeutic tool.
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Biomarcadores/análisis , Conmoción Encefálica/sangre , Guerra , Adulto , Biomarcadores/sangre , Conmoción Encefálica/fisiopatología , Factor Neurotrófico Derivado del Encéfalo/análisis , Factor Neurotrófico Derivado del Encéfalo/sangre , Estudios de Cohortes , Complemento C3/análisis , Proteína 1 de la Respuesta de Crecimiento Precoz/análisis , Proteína 1 de la Respuesta de Crecimiento Precoz/sangre , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/sangre , Humanos , Interleucina-6/análisis , Interleucina-6/sangre , Estudios Longitudinales , Masculino , Factor de Activación Plaquetaria/análisis , Estudios Retrospectivos , Subunidad beta de la Proteína de Unión al Calcio S100/análisis , Subunidad beta de la Proteína de Unión al Calcio S100/sangre , Factor de Necrosis Tumoral alfa/análisis , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Autism spectrum disorders (ASDs) are the most common neurodevelopmental disorders with unidentified etiology. The behavioral manifestations of ASD may be a consequence of genetic and/or environmental pathology in neurodevelopmental processes. In this limited study, we assayed autoantibodies to a panel of vital neuronal and glial proteins in the sera of 40 subjects (10 children with ASD and their mothers along with 10 healthy controls, age-matched children and their mothers). Serum samples were screened using Western Blot analysis to measure immunoglobulin (IgG) reactivity against a panel of 9 neuronal proteins commonly associated with neuronal degeneration: neurofilament triplet proteins (NFP), tubulin, microtubule-associated proteins (tau), microtubule-associated protein-2 (MAP-2), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), α-synuclein (SNCA) and astrocytes proteins such as glial fibrillary acidic protein (GFAP) and S100B protein. Our data show that the levels of circulating IgG class autoantibodies against the nine proteins were significantly elevated in ASD children. Mothers of ASD children exhibited increased levels of autoantibodies against all panel of tested proteins except for S100B and tubulin compared to age-matched healthy control children and their mothers. Control children and their mothers showed low and insignificant levels of autoantibodies to neuronal and glial proteins. These results strongly support the importance of anti-neuronal and glial protein autoantibodies biomarker in screening for ASD children and further confirm the importance of the involvement of the maternal immune system as an index that should be considered in fetal in utero environmental exposures. More studies are needed using larger cohort to verify these results and understand the importance of the presence of such autoantibodies in children with autism and their mothers, both as biomarkers and their role in the mechanism of action of autism and perhaps in its treatment.
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Neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by progressive neuronal loss and pathological accumulation of some proteins. Developing new biomarkers for both diseases is highly important for the early diagnosis and possible development of neuro-protective strategies. Serum antibodies (AIAs) against neuronal proteins are potential biomarkers for AD and PD that may be formed in response to their release into systemic circulation after brain damage. In the present study, two AIAs (tubulin and tau) were measured in sera of patients of PD and AD, compared to healthy controls. Results showed that both antibodies were elevated in patients with PD and AD compared to match controls. Curiously, the profile of elevation of antibodies was different in both diseases. In PD cases, tubulin and tau AIAs levels were similar. On the other hand, AD patients showed more elevation of tau AIAs compared to tubulin. Our current results suggested that AIAs panel could be able to identify cases with neuro-degeneration when compared with healthy subjects. More interestingly, it is possible to differentiate between PD and AD cases through identifying specific AIAs profile for each neurodegenerative states.
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Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/metabolismo , Enfermedad de Parkinson/diagnóstico por imagen , Enfermedad de Parkinson/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
In the present study, we screened the sera of subjects chronically exposed to mixtures of pesticides (composed mainly of organophosphorus compounds (OPs) and others) and developed neurological symptoms for the presence of autoantibodies against cytoskeletal neural proteins. OPs have a well-characterized clinical profile resulting from acute cholinergic crisis. However, some of these compounds cause neuronal degeneration and demyelination known as organophosphorus compound-induced delayed neurotoxicity (OPIDN) and/or organophosphorus compound-induced chronic neurotoxicity (OPICN). Studies from our group have demonstrated the presence of autoantibodies to essential neuronal and glial proteins against cytoskeletal neural proteins in patients with chemical-induced brain injury. In this study, we screened the serum of 50 pesticide-exposed subjects and 25 non-exposed controls, using Western blot analysis against the following proteins: neurofilament triplet proteins (NFPs), tubulin, microtubule-associated tau proteins (Tau), microtubule-associated protein-2 (MAP-2), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), glial fibrillary acidic protein (GFAP), calcium-calmodulin kinase II (CaMKII), glial S100-B protein, and alpha-synuclein (SNCA). Serum reactivity was measured as arbitrary chemiluminescence units. As a group, exposed subjects had significantly higher levels of autoantibody reactivity in all cases examined. The folds of increase in of autoantibodies against neural proteins of the subjects compared to healthy humans in descending order were as follows: MBP, 7.67, MAG 5.89, CaMKII 5.50, GFAP 5.1, TAU 4.96, MAP2 4.83, SNCA 4.55, NFP 4.55, S-100B 2.43, and tubulin 1.78. This study has demonstrated the presence of serum autoantibodies to central nervous system-specific proteins in a group of farmers chronically exposed to pesticides who developed neurological signs and symptoms of neural injury. These autoantibodies can be used as future diagnostic/therapeutic target for OP-induced neurotoxicity.
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Autoanticuerpos/inmunología , Biomarcadores/sangre , Proteínas de Neurofilamentos/sangre , Plaguicidas/toxicidad , Adulto , Anciano , Autoanticuerpos/sangre , Proteínas del Citoesqueleto/metabolismo , Femenino , Proteína Ácida Fibrilar de la Glía/sangre , Humanos , Masculino , Persona de Mediana Edad , Proteína Básica de Mielina/metabolismo , Proteínas de Neurofilamentos/efectos de los fármacos , Síndromes de Neurotoxicidad/sangre , Síndromes de Neurotoxicidad/diagnóstico , Compuestos Organofosforados/farmacología , Enfermedades del Sistema Nervioso Periférico/inducido químicamenteRESUMEN
Gulf War illness (GWI) is primarily diagnosed by symptom report; objective biomarkers are needed that distinguish those with GWI. Prior chemical exposures during deployment have been associated in epidemiologic studies with altered central nervous system functioning in veterans with GWI. Previous studies from our group have demonstrated the presence of autoantibodies to essential neuronal and glial proteins in patients with brain injury and autoantibodies have been identified as candidate objective markers that may distinguish GWI. Here, we screened the serum of 20 veterans with GWI and 10 non-veteran symptomatic (low back pain) controls for the presence of such autoantibodies using Western blot analysis against the following proteins: neurofilament triplet proteins (NFP), tubulin, microtubule associated tau proteins (Tau), microtubule associated protein-2 (MAP-2), myelin basic protein (MBP), myelin associated glycoprotein (MAG), glial fibrillary acidic protein (GFAP), calcium-calmodulin kinase II (CaMKII) and glial S-100B protein. Serum reactivity was measured as arbitrary chemiluminescence units. As a group, veterans with GWI had statistically significantly higher levels of autoantibody reactivity in all proteins examined except S-100B. Fold increase of the cases relative to controls in descending order were: CaMKII 9.27, GFAP 6.60, Tau 4.83, Tubulin 4.41, MAG 3.60, MBP 2.50, NFP 2.45, MAP-2 2.30, S-100B 1.03. These results confirm the continuing presence of neuronal injury/gliosis in these veterans and are in agreement with the recent reports indicating that 25years after the war, the health of veterans with GWI is not improving and may be getting worse. Such serum autoantibodies may prove useful as biomarkers of GWI, upon validation of the findings using larger cohorts.
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Autoanticuerpos/sangre , Proteínas del Tejido Nervioso/inmunología , Síndrome del Golfo Pérsico/sangre , Veteranos , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Sarin (GB, O-isopropyl methylphosphonoï¬uoridate) is a potent organophosphorus (OP) nerve agent that inhibits acetylcholinesterase (AChE) irreversibly. The subsequent build-up of acetylcholine (ACh) in the central nervous system (CNS) provokes seizures and, at sufficient doses, centrally-mediated respiratory arrest. Accumulation of ACh at peripheral autonomic synapses leads to peripheral signs of intoxication and overstimulation of the muscarinic and nicotinic receptors, which is described as "cholinergic crisis" (i.e. diarrhea, sweating, salivation, miosis, bronchoconstriction). Exposure to high doses of sarin can result in tremors, seizures, and hypothermia. More seriously, build-up of ACh at neuromuscular junctions also can cause paralysis and ultimately peripherally-mediated respiratory arrest which can lead to death via respiratory failure. In addition to its primary action on the cholinergic system, sarin possesses other indirect effects. These involve the activation of several neurotransmitters including gamma-amino-butyric acid (GABA) and the alteration of other signaling systems such as ion channels, cell adhesion molecules, and inflammatory regulators. Sarin exposure is associated with symptoms of organophosphate-induced delayed neurotoxicity (OPIDN) and organophosphate-induced chronic neurotoxicity (OPICN). Moreover, sarin has been involved in toxic and immunotoxic effects as well as organophosphate-induced endocrine disruption (OPIED). The standard treatment for sarin-like nerve agent exposure is post-exposure injection of atropine, a muscarinic receptor antagonist, accompanied by an oxime, an AChE reactivator, and diazepam.
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Sustancias para la Guerra Química/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Sistema Nervioso/efectos de los fármacos , Sarín/toxicidad , Acetilcolinesterasa/metabolismoRESUMEN
This descriptive study reports the results of assays performed to detect circulating autoantibodies in a panel of 7 proteins associated with the nervous system (NS) in sera of 12 healthy controls and a group of 34 flight crew members including both pilots and attendants who experienced adverse effects after exposure to air emissions sourced to the ventilation system in their aircrafts and subsequently sought medical attention. The proteins selected represent various types of proteins present in nerve cells that are affected by neuronal degeneration. In the sera samples from flight crew members and healthy controls, immunoglobin (IgG) was measured using Western blotting against neurofilament triplet proteins (NFP), tubulin, microtubule-associated tau proteins (tau), microtubule-associated protein-2 (MAP-2), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and glial S100B protein. Significant elevation in levels of circulating IgG-class autoantibodies in flight crew members was found. A symptom-free pilot was sampled before symptoms and then again afterward. This pilot developed clinical problems after flying for 45 h in 10 d. Significant increases in autoantibodies were noted to most of the tested proteins in the serum of this pilot after exposure to air emissions. The levels of autoantibodies rose with worsening of his condition compared to the serum sample collected prior to exposure. After cessation of flying for a year, this pilot's clinical condition improved, and eventually he recovered and his serum autoantibodies against nervous system proteins decreased. The case study with this pilot demonstrates a temporal relationship between exposure to air emissions, clinical condition, and level of serum autoantibodies to nervous system-specific proteins. Overall, these results suggest the possible development of neuronal injury and gliosis in flight crew members anecdotally exposed to cabin air emissions containing organophosphates. Thus, increased circulating serum autoantibodies resulting from neuronal damage may be used as biomarkers for chemical-induced CNS injury.
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Contaminantes Ocupacionales del Aire/efectos adversos , Autoanticuerpos/sangre , Aviación , Proteínas del Tejido Nervioso/inmunología , Síndromes de Neurotoxicidad/etiología , Medicina Aeroespacial , Biomarcadores/sangre , Espacios Confinados , Proteína Ácida Fibrilar de la Glía/inmunología , Humanos , Inmunoglobulina G/sangre , Exposición por Inhalación , Masculino , Proteínas Asociadas a Microtúbulos/inmunología , Persona de Mediana Edad , Proteína Básica de Mielina/inmunología , Factores de Crecimiento Nervioso/inmunología , Proteínas de Neurofilamentos/inmunología , Síndromes de Neurotoxicidad/sangre , Síndromes de Neurotoxicidad/inmunología , Enfermedades Profesionales , Exposición Profesional/efectos adversos , Subunidad beta de la Proteína de Unión al Calcio S100 , Proteínas S100/inmunología , Tubulina (Proteína)/inmunología , Proteínas tau/inmunologíaRESUMEN
Organophosphorus ester-induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by ataxia progressing to paralysis with a concomitant central and peripheral distal axonapathy. Diisopropylphosphorofluoridate (DFP) produces OPIDN in the chicken, which results in mild ataxia in 7-14 days and severe paralysis as the disease progresses with a single dose. White leghorn layer hens were treated with DFP (1.7 mg/kg, sc) after prophylactic treatment with atropine (1 mg/kg, sc) in normal saline and eserine (1 mg/kg, sc) in dimethyl sulfoxide. Control groups were treated with vehicle propylene glycol (0.1 mL/kg, sc), atropine in normal saline and eserine in dimethyl sulfoxide. The hens were sacrificed at different time points such as 2, 4, and 8 h, as well as 1, 2, 5, 10 and 20 days, and the tissues from cerebrum, midbrain, cerebellum brainstem and spinal cord were quickly dissected and frozen for protein (western) and mRNA (northern) studies. Subcellular fractionation, SDS-PAGE and immunoblotting of the nuclear and supernatant fractions using standard protocols from spinal cord and cerebrum showed differential expression of protein levels of PKA, CREB and phosphorylated CREB (p-CREB). There was an increase in PKA level in spinal cord nuclear fraction after 4 h (130+/-5%) and 8 h (133+/-6 %), while cerebrum nuclear fraction showed decrease (77+/-5%) at 4 h and remained at the same level at 8 h. No change was seen in either spinal cord or cerebrum soluble fraction at any time points. There was an increase in CREB level in the spinal cord supernatant (133+/-3%) after 5 days, while nuclear and supernatant fraction of the cerebrum did not show any alterations at any time point. p-CREB was induced in the spinal cord nuclear fraction at 1 day (150+/-3%) and 5 days (173+/-7%) of treatment, in contrast to the decreased levels p-CREB (72+/-4%) at 10 days in cerebrum nuclear fraction. Supernatant fraction of spinal cord and cerebrum did not show any changes in pCREB at time points studied. Similarly another set of animals were treated with DFP and perfused using standard protocols and immunohistochemistry for p-CREB in the brain and spinal cord confirmed the overall protein expression pattern identified by western analysis. Expression of beta-tubulin subtypes (1, 2, 3, and 4), studied by Northern blotting showed complex and differential pattern, while immunohistochemistry of the anti-beta-tubulin for the entire period of OPIDN developmental stages showed early induction and persistence even in the disintegrating axonal and non-neuronal structures of the CNS. These data thus strongly suggest that early cytoskeletal damage at molecular level mediated by PKA/p-CREB pathways leads to the culmination of gross (microscopically observable) level cytoskeletal changes in various components of central nervous system (CNS), consistent with our earlier findings. Thus, the differential protein expression of PKA, CREB, p-CREB and beta-tubulin subtypes appear to contribute to the initiation, progression and development of OPIDN, probably by recruiting other molecular pathways specific to various components of nervous system.
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Sistema Nervioso Central/efectos de los fármacos , Inhibidores de la Colinesterasa/toxicidad , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Isoflurofato/toxicidad , Enfermedades Neurodegenerativas/inducido químicamente , Síndromes de Neurotoxicidad/etiología , Tubulina (Proteína)/metabolismo , Animales , Antídotos/farmacología , Atropina/farmacología , Northern Blotting , Western Blotting , Sistema Nervioso Central/enzimología , Sistema Nervioso Central/patología , Cerebro/efectos de los fármacos , Cerebro/enzimología , Pollos , Progresión de la Enfermedad , Femenino , Inmunohistoquímica , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/prevención & control , Síndromes de Neurotoxicidad/enzimología , Síndromes de Neurotoxicidad/patología , Síndromes de Neurotoxicidad/prevención & control , Fosforilación , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/enzimología , Factores de Tiempo , Tubulina (Proteína)/genéticaRESUMEN
Splenda is comprised of the high-potency artificial sweetener sucralose (1.1%) and the fillers maltodextrin and glucose. Splenda was administered by oral gavage at 100, 300, 500, or 1000 mg/kg to male Sprague-Dawley rats for 12-wk, during which fecal samples were collected weekly for bacterial analysis and measurement of fecal pH. After 12-wk, half of the animals from each treatment group were sacrificed to determine the intestinal expression of the membrane efflux transporter P-glycoprotein (P-gp) and the cytochrome P-450 (CYP) metabolism system by Western blot. The remaining animals were allowed to recover for an additional 12-wk, and further assessments of fecal microflora, fecal pH, and expression of P-gp and CYP were determined. At the end of the 12-wk treatment period, the numbers of total anaerobes, bifidobacteria, lactobacilli, Bacteroides, clostridia, and total aerobic bacteria were significantly decreased; however, there was no significant treatment effect on enterobacteria. Splenda also increased fecal pH and enhanced the expression of P-gp by 2.43-fold, CYP3A4 by 2.51-fold, and CYP2D1 by 3.49-fold. Following the 12-wk recovery period, only the total anaerobes and bifidobacteria remained significantly depressed, whereas pH values, P-gp, and CYP3A4 and CYP2D1 remained elevated. These changes occurred at Splenda dosages that contained sucralose at 1.1-11 mg/kg (the US FDA Acceptable Daily Intake for sucralose is 5 mg/kg). Evidence indicates that a 12-wk administration of Splenda exerted numerous adverse effects, including (1) reduction in beneficial fecal microflora, (2) increased fecal pH, and (3) enhanced expression levels of P-gp, CYP3A4, and CYP2D1, which are known to limit the bioavailability of orally administered drugs.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Heces/microbiología , Intestinos/efectos de los fármacos , Sacarosa/análogos & derivados , Edulcorantes/farmacología , Animales , Mucosa Intestinal/metabolismo , Intestinos/enzimología , Masculino , Ratas , Ratas Sprague-Dawley , Sacarosa/farmacologíaRESUMEN
Imidacloprid, a neonicotinoid, is one of the fastest growing insecticides in use worldwide because of its selectivity for insects. The potential for neurotoxicity following in utero exposure to imidacloprid is not known. Timed pregnant Sprague-Dawley rats (300-350 g) on d 9 of gestation were treated with a single intraperitoneal injection (i.p.) of imidacloprid (337 mg/kg, 0.75 x LD50, in corn oil). Control rats were treated with corn oil. On postnatal day (PND) 30, all male and female offspring were evaluated for (a) acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity, (b) ligand binding for nicotinic acetylcholine receptors (nAChR) and muscarinic acetylcholine receptors (m2 mAChR), (c) sensorimotor performance (inclined plane, beam-walking, and forepaw grip), and (d) pathological alterations in the brain (using cresyl violet and glial fibrillary acidic protein [GFAP] immunostaining). The offspring of treated mothers exhibited significant sensorimotor impairments at PND 30 during behavioral assessments. These changes were associated with increased AChE activity in the midbrain, cortex and brainstem (125-145% increase) and in plasma (125% increase). Ligand binding densities for [3H]cytosine for alpha4beta2 type nAchR did not show any significant change, whereas [3H]AFDX 384, a ligand for m2mAChR, was significantly increased in the cortex of offspring (120-155% increase) of imidacloprid-treated mothers. Histopathological evaluation using cresyl violet staining did not show any alteration in surviving neurons in various brain regions. On the other hand, there was a rise in GFAP immunostaining in motor cortex layer III, CA1, CA3, and the dentate gyrus subfield of the hippocampus of offspring of imidacloprid-treated mothers. The results indicate that gestational exposure to a single large, nonlethal, dose of imidacloprid produces significant neurobehavioral deficits and an increased expression of GFAP in several brain regions of the offspring on PND 30, corresponding to a human early adolescent age. These changes may have long-term adverse health effects in the offspring.
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Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Imidazoles/toxicidad , Insecticidas/toxicidad , Nitrocompuestos/toxicidad , Desempeño Psicomotor/efectos de los fármacos , Acetilcolinesterasa/metabolismo , Animales , Encéfalo/metabolismo , Butirilcolinesterasa/sangre , Femenino , Masculino , Intercambio Materno-Fetal , Neonicotinoides , Embarazo , Ratas , Ratas Sprague-Dawley , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismoRESUMEN
P-glycoprotein (P-gp), the most extensively studied ATP-binding transporter, functions as a biological barrier by extruding toxic substances and xenobiotics out of the cell. This study was carried out to determine the effect of N,N-diethyl-m-toluamide (DEET) and pyridostigmine bromide (PB), alone and in combination, on P-gp expression using Escherichia coli leaky mutant transformed with Mdr1 gene (pT5-7/mdr1), which codes for P-gp or lactose permease (pT5-7/lacY) as negative control. Also, daunomycin (a known P-gp sustrate) was used as a positive control and reserpine (a known P-gp inhibitor) served as a negative control. An in vitro cell-resistant assay was used to monitor the potential of test compounds to interact with P-gp. Following exposure of the cells to pyridostigmine bromide or daunomycin, P-gp conferred significant resistance against both compounds, while reserpine and DEET significantly inhibited the glycoprotein. Cells were grown in the presence of noncytotoxic concentrations of daunomycin, pyridostigmine bromide, reserpine, or DEET, and membrane fractions were examined by Western immunoblotting for expression of P-gp. Daunomycin induced P-gp expression quantitatively more than pyridostigmine bromide, while reserpine and DEET significantly inhibited P-gp expression in cells harboring mdr1. Photoaffinity labeling experiment performed with the P-gp ligand [125I]iodoarylazidoprazosin demonstrated that compounds that induced or inhibited P-gp transport activity also bound to P-gp. DEET was also found to be a potent inhibitor of P-gp-mediated ATPase activity, whereas pyridostigmine bromide increased P-gp ATPase activity. Cells expressing P-gp or lac permease were exposed to pyridostigmine bromide and DEET, alone and in combination. Noncytotoxic concentrations of DEET significantly inhibited P-gp-mediated resistance against pyridostigmine bromide, resulting in a reduction of the number of effective drug interactions with biological targets. An explanation of these results might be that DEET is a third-generation inhibitor of P-gp; it has high potency and specificity for P-gp, it inhibits hydrolysis of ATP, it exerts no appreciable impact on cytochrome P-450 3A4, and it prevents transport of xenobiotics, such as pyridostigmine bromide, out of the cell. This conclusion explains, at least in part, the increased toxicity and bioavailability of pyridostigmine bromide following combined administration with DEET. This study improves our understanding of the basis of chemical interactions with DEET by defining the ability of drugs to interact with P-gp either as inhibitors or substrates, which may in turn lead to altered efficacy or toxicity.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Inhibidores de la Colinesterasa/farmacología , DEET/farmacología , Escherichia coli/metabolismo , Repelentes de Insectos/farmacología , Bromuro de Piridostigmina/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Interacciones Farmacológicas , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Proteínas de Escherichia coli , Genes MDR , Técnicas In Vitro , Proteínas de Transporte de Monosacáridos , Plásmidos , SimportadoresRESUMEN
We have studied sarin-induced global gene expression patterns at an early time point (2 h: 0.5 x LD50) using Affymetrix Rat Neurobiology U34 chips and male Sprague-Dawley rats. A total of 46 genes showed statistically significant alterations from control levels. Three gene categories contained more of the altered genes than any other groups: ion channel (8 genes) and calcium channel and binding proteins (6 genes). Alterations were also found in the following gene groups: ATPases and ATP-based transporters (4), growth factors (4), G-protein-coupled receptor pathway-related molecules (3), neurotransmission and neurotransmitter transporters (3), cytoskeletal and cell adhesion molecules (2), hormones (2), mitochondria-associated proteins (2), myelin proteins (2), stress-activated molecules (2), cytokine (1), caspase (1), GABAnergic (1), glutamergic (1), immediate early gene (1), prostaglandin (1), transcription factor (1), and tyrosine phosphorylation molecule (1). Persistent alteration of the following genes also were noted: Arrb1, CaMKIIa, CaMKIId, Clcn5, IL-10, c-Kit, and Plp1, suggesting altered GPCR, kinase, channel, and cytokine pathways. Selected genes from the microarray data were further validated using relative RT-PCR. Some of those genes (GFAP, NF-H, CaMKIIa, Calm, and MBP) have been shown by other laboratories and ours, to be involved in the pathogenesis of sarin-induced pathology and organophosphate-induced delayed neurotoxicity (OPIDN). Induction of both proapoptotic (Bcl2l11, Casp6) and antiapoptotic (Bcl-X) genes, besides suppression of p21, suggest complex cell death/protection-related mechanisms operating early on. Principal component analysis (PCA) of the expression data confirmed that the changes in gene expression are a function of sarin exposure, since the control and treatment groups separated clearly. Our model (based on current and previous studies) indicates that both degenerative and regenerative pathways are activated early and contribute to the level of neurodegeneration at a later time, leading to neuro-pathological alterations.
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Encéfalo/efectos de los fármacos , Sustancias para la Guerra Química/toxicidad , Perfilación de la Expresión Génica , Sarín/toxicidad , Animales , Encéfalo/metabolismo , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Componente Principal , Ratas , Ratas Sprague-Dawley , Pruebas de Toxicidad AgudaRESUMEN
This study was carried out to investigate the effect of in utero exposure to the cholinotoxicants, nicotine and chlorpyrifos, alone or in combination on neurobehavioral alterations and neuronal morphology latter in adult age. In the present study, 90 days old (corresponding to a human adult age) male and female offspring rats were evaluated for neurobehavioral, and neuropathological alterations following maternal, gestational exposure to nicotine and chlorpyrifos (O,O-diethyl-O-3,5,6-trichloro-2-pyridinyl phosphorothioate), alone and in combination. Female Sprague-Dawley rats (300-350 g) with timed-pregnancy were treated with nicotine (3.3 mg/kg/day, in bacteriostatic water via s.c. implantation of mini osmotic pump), chlorpyrifos (1.0 mg/kg, daily, dermal, in 75% ethanol, 1.0 ml/kg) or a combination of both chemicals, on gestational days (GD) 4-20. Control animals received bacteriostatic water via s.c. implantation of mini osmotic pump and dermal application of 70% ethanol. The offspring at postnatal day (PND) 90 were evaluated for neurobehavioral performance, changes in the activity of plasma butyrylcholinesterase (BChE) and acetylcholinesterase (AChE), and neuropathological alterations in the brain. Neurobehavioral evaluations included beam-walk score, beam-walk time, incline plane performance and forepaw grip time. Male and female offspring from mothers treated with nicotine and CPF, alone or in combination showed impairments in the performance of neurobehavioral tests, indicating sensorimotor deficits. Female offspring from mothers treated with a combination of nicotine and chlorpyrifos showed significant increase in plasma BChE activity. Brain regional AChE activity showed differential increases in male and female offspring. Brainstem and cerebellum of female offspring from mothers treated with nicotine or chlorpyrifos, alone or in combination showed increased AChE activity, whereas brainstem of male offspring from mothers treated with nicotine alone or a combination of nicotine and chlorpyrifos showed increase in AChE activity. Also, male offspring exposed in utero to nicotine exhibited increased AChE activity. Histopathological evaluations using cresyl violet staining showed a decrease in surviving Purkinje neurons in the cerebellum in offspring of all treatments groups. An increase in glial fibrillary acidic protein (GFAP) immuno-staining was observed in cerebellum white matter as well as granular cell layer (GCL) of cerebellum following all exposures. These results indicate that in utero exposure to nicotine and chlorpyrifos, alone and in combination produced significant sensorimotor deficits in male and female offspring, differential increase in brain AChE activity, a decrease in the surviving neurons and an increased expression of GFAP in cerebellum in adult offspring rats at a corresponding human adult age. Collectively, this study demonstrates that maternal exposure to environmental neurotoxic chemicals, i.e., nicotine and chlorpyrifos leads to developmental abnormalities in the offspring that persist latter into adulthood.
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Conducta Animal/efectos de los fármacos , Cloropirifos/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Exposición Materna/efectos adversos , Nicotina/toxicidad , Efectos Tardíos de la Exposición Prenatal , Desempeño Psicomotor/efectos de los fármacos , Células de Purkinje/efectos de los fármacos , Administración Tópica , Animales , Muerte Celular/efectos de los fármacos , Colinesterasas/sangre , Quimioterapia Combinada , Femenino , Bombas de Infusión Implantables , Inyecciones Subcutáneas , Masculino , Embarazo , Células de Purkinje/patología , RatasRESUMEN
We have studied sarin-induced global gene expression patterns at an early time point (15 min; 0.5xLD50) and a later time point (3 months; 1xLD50) using Affymetrix: Rat Neurobiology U34 chips in male, Sprague-Dawley rats and have identified a total of 65 (early) and 38 (late) genes showing statistically significant alterations from control levels at 15 min and 3 months, respectively. At the early time point, those that are classified as ion channel, cytoskeletal and cell adhesion molecules, in addition to neuropeptides and their receptors predominated over all other groups. The other groups included: cholinergic signaling, calcium channel and binding proteins, transporters, chemokines, GABAnergic, glutamatergic, aspartate, catecholaminergic, nitric oxide synthase, purinergic, and serotonergic signaling molecules. At the late time point, genes that are classified as calcium channel and binding proteins, cytoskeletal and cell adhesion molecules and GABAnergic signaling molecules were most prominent. Seven molecules (Ania-9, Arrb-1, CX-3C, Gabab-1d, Nos-2a, Nrxn-1b, PDE2) were identified that showed altered persistent expression in both time points. Selected genes from each of these time points were further validated using semi quantitative RT-PCR approaches. Some of the genes that were identified in the present study have been shown to be involved in organophosphate-induced neurotoxicity by both other groups as well as ours. Principal component analysis (PCA) of the expression data from both time points was used for comparative analysis of the gene expression, which indicated that the changes in gene expression were a function of dose and time of euthanasia after the treatment. Our model also predicts that besides dose and duration of post-treatment period, age and possibly other factors may be playing important roles in the regulation of pathways, leading to the neurotoxicity.
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Encéfalo/efectos de los fármacos , Perfilación de la Expresión Génica/métodos , Sarín/toxicidad , Animales , Encéfalo/metabolismo , Canales de Calcio/genética , Sustancias para la Guerra Química/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Análisis por Conglomerados , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Inyecciones Intramusculares , Masculino , Óxido Nítrico Sintasa/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Ratas , Ratas Sprague-Dawley , Receptores de GABA/genética , Receptores de N-Metil-D-Aspartato/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de TiempoRESUMEN
Maternal smoking during pregnancy is known to be a significant contributor to developmental neurological health problems in the offspring. In animal studies, nicotine treatment via injection during gestation has been shown to produce episodic hypoxia in the developing fetus. Nicotine delivery via mini osmotic pump, while avoiding effects due to hypoxia-ischemia, it also provides a steady level of nicotine in the plasma. In the present study timed-pregnant Sprague-Dawley rats (300-350 g) were treated with nicotine (3.3 mg/kg, in bacteriostatic water via s.c. implantation of mini osmotic pump) from gestational days (GD) 4-20. Control animals were treated with bacteriostatic water via s.c. implantation of mini osmotic pump. Offspring on postnatal day (PND) 30 and 60, were evaluated for changes in the ligand binding for various types of nicotinic acetylcholine receptors and neuropathological alterations. Neurobehavioral evaluations for sensorimotor functions, beam-walk score, beam-walk time, incline plane and grip time response were carried out on PND 60 offspring. Beam-walk time and forepaw grip time showed significant impairments in both male and female offspring. Ligand binding densities for [3H]epibatidine, [3H]cytisine and [3H]alpha-bungarotoxin did not show any significant changes in nicotinic acetylcholine receptors subtypes in the cortex at PND 30 and 60. Histopathological evaluation using cresyl violet staining showed significant decrease in surviving Purkinje neurons in the cerebellum and a decrease in surviving neurons in the CA1 subfield of hippocampus on PND 30 and 60. An increase in glial fibrillary acidic protein (GFAP) immuno-staining was observed in cerebellum white matter as well as granular cell layer of cerebellum and the CA1 subfield of hippocampus on PND 30 and 60 of both male and female offspring. These results indicate that maternal exposure to nicotine produces significant neurobehavioral deficits, a decrease in the surviving neurons and an increased expression of GFAP in cerebellum and CA1 subfield of hippocampus of the offspring on PND 30 and 60. The results show that although 60-day-old male and female rat offspring of mothers exposed to nicotine during gestation did not differ from control in body weight gain or nicotinic acetylcholine receptors ligand binding, they exhibited significant sensorimotor deficits that were consistent with the neuropathological alterations seen in the brain. These neurobehavioral and pathological deficits indicate that maternal nicotine exposure may produce long-term adverse health effects in the offspring.