Bijoy K. Bhattacharya

Immobilization of acetylcholineesterase-choline oxidase on a gold-platinum bimetallic nanoparticles modified glassy carbon electrode for the sensitive detection of organophosphate pesticides, carbamates and nerve agents.

A novel, highly sensitive amperometric biosensor, based on electrodeposition of gold-platinum bimetallic nanoparticles onto 3-aminopropyltriethoxy silane modified glassy carbon electrode for the detection of paraoxon ethyl, aldicarb, and sarin has been developed. The biosensor consists of acetylcholineesterase (AChE)/choline oxidase (ChOx) immobilized by cross-linking with glutaraldehyde on a modified electrode. The properties of nanoparticles modified electrodes are characterized by scanning electron microscopy (SEM), energy dispersive X-ray (EDX), cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS). The synergistic action of Au and Pt nanoparticles showed excellent electrocatalytic activity with low applied potential for the detection of hydrogen peroxide (H(2)O(2)). The IC(50) and inhibition rate constant (K(i)) values were determined for the inhibitors using immobilized enzymes on modified electrode and the data were compared by spectrophotometric determination of these kinetic parameters using free enzymes in solution. Paraoxon ethyl, sarin, and aldicarb could be detected up to 150-200nM, 40-50nM, and 40-60 microM respectively at 30-40% inhibition level of AChE enzyme and followed linearity in wide range concentration.

In vivo binding of [1-14C]methylisocyanate to various tissue proteins

Liaison covalente de methyl-isocyanate MIC a differentes proteines tissulaires apres administration intraperitoneale et par inhalation. Les proteines sanguines et la globine subissent une carbamoylation apres injection IP. Ces resultats suggerent que le passage de la barriere «sang-tissu» se fait sous la forme active de MIC

Differential mRNA expression of acetylcholinesterase in the central nervous system of rats with acute and chronic exposure of sarin & physostigmine.

A time‐course study was carried out to measure the acetylcholinesterase (AChE) gene expression in the brain of female rats exposed to different doses of sarin and physostigmine. Short‐term effects were studied with an acute single subcutaneous dose (s.c.) of 80 µg kg−1 (0.5 × LD50) sarin. Cortex and cerebellum showed a significant decline in AChE mRNA expression at 2.5, 24 and 72 h. Biochemical studies showed that plasma butrylcholinesterase (BChE) and brain AChE activities were significantly decreased at 2.5 h, which came back to near control values by 24 h in both cases. For long‐term chronic studies, three groups of female rats received daily doses of physostigmine (0.1 mg kg−1 day−1) intramuscularly (i.m.), sarin (15 µg kg−1 day−1) s.c. independently and a combined dose of physostigmine (i.m.) (0.1 mg kg−1 day−1) followed by sarin (s.c.) (15 µg kg−1 day−1) continuously for 30 days. Differential AChE mRNA levels in cortex and cerebellum of rat brain were observed after 30 days and after a lag period of another 30 days with no further administration. Plasma (BChE) and brain (AChE) showed irregular inhibition profile in biochemical studies at 30 days and returned to control levels after 60 days. The acute single subcutaneous administration of sarin for short‐term as well as chronic long‐term studies showed that AChE inhibition alone does not lead to observed changes in mRNA expression of AChE gene. These observations further suggest that route of administration as well as dose exposure regimen also contributes to the regulation of AChE mRNA expression. Copyright

An electrochemical genosensor for Salmonella typhi on gold nanoparticles-mercaptosilane modified screen printed electrode.

In this work, we fabricated a system of integrated self-assembled layer of organosilane 3-mercaptopropyltrimethoxy silane (MPTS) on the screen printed electrode (SPE) and electrochemically deposited gold nanoparticle for Salmonella typhi detection employing Vi gene as a molecular marker. Thiolated DNA probe was immobilized on a gold nanoparticle (AuNP) modified SPE for DNA hybridization assay using methylene blue as redox (electroactive) hybridization indicator, and signal was monitored by differential pulse voltammetry (DPV) method. The modified SPE was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) method. The DNA biosensor showed excellent performances with high sensitivity and good selectivity. The current response was linear with the target sequence concentrations ranging from 1.0 × 10(-11) to 0.5 × 10(-8)M and the detection limit was found to be 50 (± 2.1)pM. The DNA biosensor showed good discrimination ability to the one-base, two-base and three-base mismatched sequences. The fabricated genosensor could also be regenerated easily and reused for three to four times for further hybridization studies.

Multiple signal transduction pathways alterations during nerve agent toxicity.

Nerve agent toxicity is primarily due to the synaptic build up of toxic levels of acetylcholine. The acute lethal effects of the nerve agents are generally attributed to respiratory failure caused by a combination of effects at both central and peripheral levels and are further complicated by copious secretions, muscle fasciculations, and convulsions. In addition to this, a range of non cholinergic effects have been observed. The development of effective treatment to block multiple effects resulting from nerve agent exposure is hampered by a limited understanding of the molecular changes responsible for their persistent effects. Excessive accumulation of acetylcholine leads to activation nicotinic and muscarinic acetylcholine receptors, these receptors activate diverse kind of cellular responses by distinct signaling pathways. Metabolism of cyclic nucleotides, membrane phospholipids, activation of a multitude of protein kinases and the induction of transcription factors are the key biochemical steps and pathways that have been investigated. This review will focus on the effects of nerve agents on signal transduction pathways; particularly, MAP kinases, protein kinase C isozymes, calcium calmodulin dependent protein kinase II (CaMKII) and on cytoskeletal proteins, calpain, and certain transcription factors and discusses how such changes may be involved in nerve agent induced neurotoxicity. Alterations in these key brain proteins could explain the neurological impairments following nerve agent exposure. A better understanding of the whole picture may lead to new pharmacological interventions aimed to improve or modulate those signal transduction pathways affected during nerve agent poisoning or associated pathologies that are responsible for neuronal disturbances.

Changes of protein oxidation, calpain and cytoskeletal proteins (alpha tubulin and pNF-H) levels in rat brain after nerve agent poisoning

Highly toxic organophosphorus (OP) nerve agents, sarin and soman act by inhibiting acetylcholinesterase (AChE) function at neuronal synapses and cause many toxic effects including death within minutes. The effect of nerve agents on protein oxidation, calpain, and cytoskeletal protein levels was not well known. In the present study we investigated these parameters after subcutaneous injection of sarin (120 μg/kg) and soman (80 μg/kg) in the rat brain. Results indicate that several rat brain proteins were intensely oxidized after nerve agent poisoning. Immunoreactivity levels of μ-calpain were significantly elevated in cerebral cortex and cerebellum regions of rat brain from 2.5 h to 30 days. Alpha tubulin levels reduced from 1 to 7 days in the supernatant and 1 to 3 days in the pellet fractions of cerebellum and cerebral cortex, where as phosphorylation of high molecular weight neurofilament (pNF-H) was increased significantly in nerve agent intoxicated rat brains as compared to control rats. AChE activity was inhibited up to 3 days after nerve agent exposure in plasma and brain. Results suggest that altered protein oxidation, calpain and cytoskeletal protein levels are due to multiple mechanisms of nerve agents actions and these changes might be involved in nerve agent induced complex neurotoxicity.

Application of bimetallic nanoparticles modified screen printed electrode for the detection of organophosphate compounds using an enzyme inhibition approach

A novel, unique gold–platinum bimetallic nanoparticles modified screen printed electrode (SPE) has been developed for the sensitive detection of paraoxon ethyl, carbofuran and simulant of nerve agent diisopropyl fluorophosphate (DFP), based on the enzyme inhibition approach. The gold–platinum nanoparticles were electrochemically deposited on a 3-aminopropyl triethoxy silane modified electrode. Following this acetylcholineesterase (AChE)/choline oxidase (ChOx) bienzymes were immobilized through cross-linking with glutaraldehyde onto a modified electrode. Electrodes were characterized by scanning electron microscopy (SEM), EDX (energy dispersive X-ray analysis) and cyclic voltammetry (CV). The bimetallic alloy nanoparticles have an excellent high surface area and the unique effect of electrocatalytic activity for the oxidation of hydrogen peroxide (H2O2). The IC50 values were determined for the inhibitors using immobilized enzymes on the modified electrode. Paraoxon ethyl, carbofuran, and DFP could be detected up to 0.20 μM, 0.20–0.25 μM, and 0.20–0.25 μM respectively at a 25–30% inhibition level of AChE enzyme (residual enzyme activity) with an incubation time of 10 min.

Regional alterations of JNK3 and CaMKIIα subunit expression in the rat brain after soman poisoning.

Calcium/calmodulin-dependent protein kinase II (CaMKII) and c-Jun N-terminal kinases (JNKs) exert numerous and diverse functions in the brain. However, their role in nerve agent poisoning is poorly understood. In the present study, rats were exposed to soman (80 µg/kg) subcutaneously to study the changes in the protein levels of calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) and JNK3 and activities of acetylcholinestarase (AChE) and CaMKII in the rat brain. Western blot analysis revealed that significant changes were found in both the protein kinases expression. Immunoreactivity levels of neural specific JNK3 isoform increased from 2.5 hours to 30 days after soman exposure in cerebral cortex, hippocampus, striatum and thalamus regions and decreased in the case of cerebellum. CaMKIIα expression levels were also increased from 2.5 hours to 30 days after soman exposure in cerebral cortex, hippocampus, thalamus and down regulated in cerebellum. AChE activity remained inhibited in plasma and brain up to 3 days post exposure. CaMKII activity was increased in cerebrum and decreased in cerebellum. Results suggest that altered expression of both the protein kinases play a role in nerve agent-induced long-term neurotoxic effects.

Attenuation of sulfur mustard toxicity by S-2(2-aminoethylamino)ethyl phenyl sulfide (DRDE-07) in mouse liver.

Sulfur mustard (SM) (bis-(2-chloroethyl) sulfide) is a chemical warfare agent. Evaluation of toxicity and protective effect of DRDE-07 (S-2(2-aminoethylamino)ethyl phenyl sulfide) was studied in mouse liver after SM challenging. Female mice were given orally 0.2 LD50 of DRDE-07 (249 mg/kg body weight) and exposed percutaneously with 1.0 LD50 of SM (8.1 mg/kg body weight). Gene expression profiles were determined using global genome microarray analysis at 3 days post-exposure. DRDE-07 alone treated animal showed significant upregulation to metabolism of xenobiotics by cytochrome P450 pathways. Genes related to cell adhesion molecules (CAMs), were downregulated. DRDE-07 pretreated SM exposed animals showed upregulation of xenobiotic cytochrome P450 pathway genes. Antigen presenting, cell adhesion molecules, cytokine, cytokine receptor metabolism, fatty acid metabolism, glutathione metabolism, cell cycle signaling pathway genes showed downregulation. The present study showed that SM-induced toxicity in mouse liver was attenuated by the pretreatment with DRDE-07.

Soman-induced alterations of protein kinase C isozymes expression in five discrete areas of the rat brain.

Soman is a highly neurotoxic chemical warfare agent and inhibits the neural enzyme, acetylcholinesterase (AChE). Protein kinase C (PKC) isozymes regulate a wide range of cellular functions to a variety of extracellular stimuli. However, their exact role in nerve-agent poisoning is not well understood. In the present study, we investigated the effect of soman (80 μg/kg−1, administered subcutaneously) on two PKC isozymes’ immunoreactivity levels and activities of PKC and AChE in different rat-brain areas. Results showed a significant induction in PKC βII and ζ isoenzyme expression levels from 2.5 hours to 14 days post-soman exposure periods in the hippocampus, cerebellum, thalamus and cerebral cortex. The striatum showed reduced expression levels of both the isozymes from 1 to 3 days after soman exposure. PKC activity was increased in the cerebrum and cerebellum up to 7 days post-soman exposure. The toxicity target enzyme, AChE activity remained inhibited in plasma and brain up to 3 days post exposure and thereafter recovered to control levels. The results suggest a possible role of PKC isozymes in nerve-agent–induced neurotoxicity.