No description available.

Our laboratory has invented a series of oxime acetylcholinesterase (AChE) reactivators (US Patent 9,227,937) that enter the brain, reduce time to cessation of seizure-like activities, and prevent organophosphorus compound (OP) neuropathology, not seen with the current U.S. approved AChE reactivator, pralidoxime (2-PAM). Thus, 2-PAM fails to protect the brain against damage and long-term cognitive and behavioral deficits seen in humans after OP exposure. However, the mechanisms by which these novel oximes provide central neuroprotection through preservation of neuronal cell structures from damage in a rat model are not fully understood by AChE reactivation alone. This dissertation investigated neurotoxic mechanisms of NIMP as potential targets for additional direct and indirect neuroprotection by our lead in vivo AChE reactivator, Oxime 20. Male Sprague Dawley rats exposed to NIMP experienced neurotoxic effects in areas critical to OP-induced seizure generation (e.g., hippocampus and piriform cortex) such as the inhibition of multiple serine hydrolases (i.e., fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MAGL)), necrotic cell death evident by increased necrotic receptor-interacting serine/threonine-protein kinase 1 (RIPK1) levels and no apoptotic caspase-3 activity, and increased levels of neuroinflammation via elevated levels of pro-inflammatory oxylipins 4 days post lethal exposure. However, due to the lack of statistical significance, NIMP exposure did not definitively affect the subcellular location of either phosphorylated excitatory N-methyl-D-aspartate (NMDA) receptor or inhibitory γ-aminobutyric acid (GABA) receptor subunits. Results suggested that Oxime 20 therapy provided neuroprotection after NIMP exposure, such as limited reactivation of other serine hydrolase targets, significantly decreased RIPK1 levels (i.e., necrotic environment) in the hippocampus, and significantly decreased inflammatory oxylipins 4 days post-NIMP exposure. Thus, reducing OP-induced neuroinflammation might be the main contributor to the neuroprotection (i.e., neuronal cell structure preservation) previously observed in our laboratory.

High doses of 3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") are known to reduce levels of various serotonergic markers outside of the raphe nuclei. To test the hypothesis that these deficits reflect a degeneration of distal axons/terminals, we investigated the effects of an MDMA binge (10mg/kg x 4) on the relative protein and genetic expression of several serotonergic markers in rats, as well as the effects of this compound on the quantity of serotonergic terminals in these animals. In experiment I, we examined whether MDMA alters serotonin transporter (SERT) levels as determined by lysate binding and immunoblotting analyses. Both methods of analysis revealed MDMA-induced reductions in regional SERT content. Experiment II investigated MDMA-induced changes in terminal-specific levels of SERT and the vesicular monoamine transporter 2 (VMAT-2) in the hippocampus, a region with sparse dopaminergic innervation, after lesioning noradrenergic input with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). Animals were administered 100 mg/kg DSP-4 or saline 1 week prior to MDMA (or saline). As determined by immunoblotting of synaptosomal tissue, the DSP-4/MDMA group showed little change in hippocampal VMAT-2 protein expression compared to DSP-4/Saline controls, despite large reductions in SERT levels in all regions examined in the MDMA-treated animals. Experiment III examined whether MDMA alters genetic expression of SERT and VMAT-2. When compared to saline-treated controls, animals given MDMA showed a striking decrease in SERT gene expression (and a lesser effect on VMAT-2) in dorsal/median raphe as assessed by quantitative RT-PCR. Experiment IV(a) investigated the effects of MDMA on gene and protein expression of tryptophan hydroxylase (TPH) in the hippocampus. Levels of TPH protein were unchanged between treatment groups, while transcript levels were decreased 15-fold in the dorsal/median raphe. In experiment IV(b), flow cytometry was used to measure whether MDMA alters the quantity of serotonergic terminals in the hippocampus. MDMA-treated animals showed an increase in the number of serotonergic synaptosomes identified by co-labeling for synaptosome-associated protein of 25 kDa (SNAP-25) and TPH. These results demonstrate that MDMA causes substantial regulatory changes in the expression of serotonergic markers with no evidence for synaptic loss, questioning the need to invoke distal axotomy as an explanation of MDMA-related serotonergic deficits.

No description available.

No description available.

No description available.

No / The chemical and biological nonproliferation regime stands at a watershed moment, when failure seems a real possibility. After the unsuccessful outcome of the 2016 Eighth Review Conference, the future of the Biological and Toxin Weapons Convention is uncertain. As the Chemical Weapons Convention (CWC) approaches its Fourth Review Conference in 2018, it has almost completed removing the huge stocks of chemical weapons, but it now faces the difficult organizational task of moving its focus to preventing the reemergence of chemical weapons at a time when the international security situation appears to be increasingly more difficult and dangerous. In this article, we assess the current and near-term state (5–10 years) and impact of three related areas of science and technology that could be of dual-use concern: targeted delivery of agents to the central nervous system (CNS), particularly by means of nanotechnology; direct impact of nanomaterials on synaptic functions in the CNS; and neuronal circuits in the brain that might be targeted by those with hostile intent. We attempt to assess the implications of our findings, particularly for the consideration of the problem of state-level interest in so-called nonlethal incapacitating chemical agents for law enforcement at the CWC Review Conference in 2018, but also more generally for the longer-term future of the chemical and biological nonproliferation regime.

Some organophosphorus (OP) esters induce a central-peripheral distal axonopathy called organophosphorus ester-induced delayed neurotoxicity (OPIDN). In the chicken model neurological deficits and microscopic lesions develop 7-21 days after exposure. Neurotoxic esterase (NTE) is thought to be the initial target in OPIDN. Evidence indicates that neuropathic OP esters have to bind NTE and chemically ?age? for OPIDN induction. It was hypothesized that phenyl saligenin phosphate (PSP), a neuropathic OP ester that essentially irreversibly inhibits NTE as it undergoes the chemical aging process, results in changes in spinal cord gene expression that do not occur with phenylmethylsulfonyl fluoride (PMSF), a non-neuropathic compound that inhibits NTE without aging. This hypothesis was tested in Gallus gallus in experiments designed to detect differences in spinal cord gene expression between PSP, PMSF and vehicle-treated birds 24 hours after exposure. Two approaches were used. Targeted display was developed and used to screen approximately 15000 gel bands. Three candidate genes were identified by targeted display. One, designated P1 has 100% homology with expressed sequence tag pgp1n.pk010.m23, another, P2, is homologous to human KIAA1307, and a third, P3, is unidentified. Northern blotting was used to measure spinal cord expression of a-tubulin and other genes previously reported to be differentially expressed following exposure to di-isopropryl phosphorofluoridate, another agent causing OPIDN. Only expression of a-tubulin was altered in PSP-treated hens. Time course experiments were undertaken to determine spinal cord expression changes of P1, P2, P3 and a-tubulin transcripts at 12, 24, 36 and 48 hours post-exposure. Findings indicated decreases and increases, respectively, of P1 (22%, p=0.0011) and P2 (26%, p=0.0055) transcripts at 12 hours in PSP treated hen spinal cord compared to DMSO controls. An ~2.5 kb a-tubulin transcript was decreased across most time points with maximum change at 48 hours (33%, p=0.0479); an ~4.5 kb a-tubulin transcript was upregulated at 12 hours (38%, p=0.0125) and down regulated at 48 hours (28%, p=0.0576). Responses to PMSF were different than responses to PSP. Spinal cord in-situ hybridization experiments revealed, 1.) mainly neuronal expression of P1, P2 and a-tubulin transcripts, and, 2.) decreased expression of neuronal P1 and a-tubulin transcripts at 12 and 48 hours, respectively. Results indicate that PSP can induce changes in gene expression distinct from those induced with the non-neuropathic NTE inhibitor, PMSF. However, expression changes were low in frequency and magnitude, and their mechanistic importance remains to be fully established. / Ph. D.

Several epidemiological studies have suggested a role for environmental chemicals in the etiology of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Endosulfan (an organochlorine) and zineb (zinc-ethylene-bis-dithiocarbamate) are used as pesticides on a variety of crops worldwide and pose potential health risks to humans and animals. Both endosulfan and zineb are known to affect nervous system. Because the dopaminergic system continues to develop postnatally, we hypothesized that developmental exposure to endosulfan or zineb alone or in combination would result in alteration of nigrostrial neurotransmitters and would render the nigrostrial dopamine system more susceptible to chemical challenge later in life. The objectives of this study were (1) to determine the effects of endosulfan and zineb individually and in combination on dopaminergic or cholinergic pathways in vivo, (2) to investigate the effects of exposure to endosulfan, zineb and their mixtures administered in early life (during brain development) on subsequent exposure to these pesticides on the dopaminergic and cholinergic systems, in vivo, (3) to investigate the mechanism(s) of induction of neuronal cell death caused by these pesticides using human neuroblastoma SH-SY5Y cells in culture, (4) to define the role of oxidative stress in pesticide-induced neuronal cell death in vitro. Male C57Bl/6 mice of 7-9 months old exposed to zineb (50 and 100 mg/kg), endosulfan (1.55, 3.1 and 6.2 mg/kg) and their mixtures every other day over a 2-week period exhibited higher levels of dopamine accumulation in the striatum. Both pesticide-treated groups displayed significantly lower norepinephrine levels in the striatum (Ï ≤ 0.05) than the controls. The developmental exposure to zineb, endosulfan and their combination enhanced the vulnerability to subsequent neurotoxic challenges occurring later in life. Thus, C57BL/6 mice exposed to zineb, endosulfan and their mixtures as juveniles (postnatal days 5 to 19) and re-exposed at 8 months of age showed a significant depletion of striatal dopamine, to 22%, 16%, and 35% of control, respectively. Acetylcholinesterase activity in the cerebral cortex was found to be significantly increased in all pesticide treated groups. Mice given mixtures of pesticides also showed significantly increased levels of normal and aggregated alpha-synuclein, a hallmark of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. The results of these studies indicate that exposure to these pesticides as neonates and re-exposure as adults could result in neurochemical changes that did not reveal at adulthood when the exposure was at juvenile age only. We further investigated the mechanism(s) of activation of pesticide-induced neuronal cell death in vitro. The characteristic of cell death in SH-SY5Y human neuroblastoma cells was examined. These cells are known to retain catecholaminergic phenotype. Cells were exposed to endosulfan, zineb and mixtures of two pesticides, in concentrations ranging from 50 μM to 400 μM. These exposures caused both apoptotic and necrotic cell death in SH-SY5Y cells as evaluated by lactate dehydrogenase release, 7-aminoactinomycin-D and Annexin-V/PI assays. Exposure to mixtures of the pesticides enhanced both the early apoptosis and late apoptosis/necrosis compared to either chemical alone. Visual evaluation using DNA ladder assay and fluorescence Annexin V/PI assay confirmed the contribution of both apoptotic and necrotic events. Furthermore, endosulfan and zineb alone and in combination altered the caspase-3 activity indicating that both pesticides exposure exert their apoptotic effect via the caspase-3 pathway. Because there has been increasing evidence of the role of reactive oxygen species (ROS) and oxidative stress in pesticide-induced neuronal cell death (apoptosis and necrosis), the levels of ROS and antioxidant enzymes were examined. Cells treated with pesticides were found to enhance the generation of superoxide anion and hydrogen peroxide both in a dose- and time-dependent manner. Mixture of pesticides significantly enhanced the production of these reactive oxygen species compared to cells exposed to individual pesticide. Cells treated with pesticides showed a decrease in superoxide dismutase, glutathione peroxidase, and catalase levels. These pesticides also induced lipid peroxides (thiobarbituric acid reactive products) formation in SH-SY5Y cells. Furthermore, cells exposed to these pesticides were found to have increased in the expression of NFkappaB activity in the nucleus. These data support the hypothesis that oxidative stress was induced in neuronal cells by exposing to these pesticides in vitro. Taken together, the results of this study support the above hypothesis and suggest that the cytotoxicity of endosulfan and zineb and their combinations may, at least in part, be associated with the generation of ROS. Furthermore, mice exposed at early age and re-exposed at adulthood become more susceptible to alteration of neurotransmitter levels compared to mice exposed to these pesticides only as juveniles. These findings could add to the growing body of knowledge on the mechanism of pesticide-induced dopaminergic neuronal cell death and could hold tremendous implication for the future understanding of the possible involvement of environmental risk factors in the pathogenesis of Parkinson's disease. / Ph. D.

Organophosphates (OP) such as malathion are organic derivatives of phosphoric acid with broad use in everyday life throughout the world, especially as insecticides. Lead particles can accumulate in soil and from there leach into our water supplies. Interaction with the environment offers opportunities for multiple exposures to combinations of different toxicants (such as lead and malathion). Thus, it is important to assess effects that these compounds exert not only on the nervous system, but also on the blood-brain barrier (BBB). The BBB consists of specialized endothelial cells that form the vasculature of the brain; it regulates passage of nutrients, while preventing potentially damaging substances from entering the brain. The main feature of the BBB is the presence of tight junctions between cells, which provide the BBB with its low permeability. The work presented in this dissertation tests the hypothesis that lead and malathion disrupt BBB integrity by affecting tight junctions of the BBB. The hypothesis suggests that disruptions involve changes in protein levels and gene expression as well as activation of transient receptor potential canonical channels (TRPC) that in turn increase intracellular calcium levels affecting tight junction structure. The hypothesis was tested by assessing lead-malathion interactions in an in vitro BBB model. This model was constructed with rat astrocytes and rat brain endothelial cells (RBE4). Assessments of cell toxicity in response to different concentrations of the neurotoxicants tested showed that concentrations between 10-5 µM and 10-6 µM were ideal to assess combinations of neurotoxicants. In general, protein levels of occludin, claudin 5, ZO1, and ZO2 decreased at all times, however, qPCR analysis of gene expression for all the proteins did not correlate with the assessments on protein levels. TRPC channel protein levels increased in response to neurotoxicant insult, which correlated with results for gene expression. This study suggests that at least one of the mechanisms that neurotoxicants lead and malathion utilize to disrupt permeability of the BBB is by affecting tight junction structure. This effect could be regulated by increases in gene expression of TRPC1 and TRPC4 that are associated with increases in the number of TRPC channels on the membrane of endothelial cells of the cerebral microvasculature. / Ph. D.