Brain Cholinesterase Research Articles

Optimization of bioactivities of solvent extracts of bark and heartwood of Pterocarpus marsupium and exploration of neuroprotective effect linking to the major Phytoconstituents

The study aims to evaluate the neuroprotective properties of Pterocarpus marsupium Roxb. The bioactive optimized hydroalcohol-extracted fractions of bark (PMBHA) and heartwood (PMHHA) at 300 and 400 mg/kg were examined in rats with scopolamine-induced neurotoxicity to study oxidative, neurochemical, and neuroinflammation biomarkers. Phenol and flavonoid-rich radical scavengers in contributing total antioxidant capacities of PMBHA and PMHHA quenched peroxyl radicals and protected the cellular membrane and lipoproteins from ROS in DPPH, ORAC, and CAP-e tests. In anticholinesterase activities, PMBHA inhibited AChE (IC50: 54.40 ± 0.98 µg/mL) and BChE (IC50: 50.95 ± 0.98 µg/mL) mixed-competitively, while PMHHA inhibited cholinesterase activities (AChE: 42.23 ± 0.54, and BChE: 47.19 ± 0.65 µg/mL) competitively. Dosage at 300 and 400 mg/kg of PMBHA and PMHHA improved serum and brain oxidative indicators (MDA, SOD, CAT, and GSH), and restored proinflammatory cytokines (TNF-α, and IL-6) leading to enhanced brain cholinesterase and β-secretase levels in neurotoxic rats. GC-MS reported antioxidant, anti-inflammatory, and neuroprotective compounds of PMBHA catechol, thiamet-G, d-Gala-l-ido-octonic amide, falcarinol, D-mannose, ribitol, 1,2,4-cyclopentanetrione, 3-(2-pentenyl)-, hexadecanoic acid methyl ester, n-hexadecanoic acid, oleic acid, and 3,5-Dimethoxybenzyl alcohol, α-epi-7-epi-5-Eudesmol, 1 R,4aR,7 R,8aR)-7-(2-Hydroxypropan-2-yl)-1,4a-dimethyldecahydro naphthalen-1-ol, arctiol, 1,1,4,7-tetramethyldecahydro-1 H-cyclopropa[e]azulene-4,7-diol, 2-Propen-1-ol, 3-(2,6,6-trimethyl-1-cyclohexen-1-yl), 4-t-Butyl-2-[4-nitrophenyl]phenol in PMHHA were responsible in neuroprotection in neurotoxic rats. However, heartwood demonstrated better neuroprotective activities than the bark of P. marsupium due to some of the key compounds (1 R,4aR,7 R,8aR)-7-(2-Hydroxypropan-2-yl)-1,4a-dimethyldecahydronaphthalen-1-ol, 2-Propen-1-ol, 3-(2,6,6-trimethyl-1-cyclohexen-1-yl)-, 4-t-Butyl-2-[4-nitrophenyl]phenol, 1,1,4,7-Tetramethyldecahydro-1 H-cyclopropa[e]azulene-4,7-diol.

Alterations of Neurobehavioral Performance, Blood and Brain Cholinesterase Activities and Cholesterol Levels by Repeated Statin Treatments in Mice

Alterations of Neurobehavioral Performance, Blood and Brain Cholinesterase Activities and Cholesterol Levels by Repeated Statin Treatments in Mice

Formulation and characterization of polymeric nanoparticle of Rivastigmine for effective management of Alzheimer’s disease

Memory loss or dementia is a progressive disorder, and one of its common forms is Alzheimer’s disease (AD), effecting mostly middle aged and older adults. In the present study, we developed Rivastigmine (RIV) nanoparticles using poly(lactic-co-glycolic acid) (RIV-loaded PLGA NPs) and polyvinyl alcohol (PVA). The prepared RIV-PLGA nanoparticles was evaluated for the management of Alzheimer's disease (AD). The nanoparticles were prepared by the slightly modified nano-precipitation technique. The developed formulations were evaluated for particle size, zeta potential (ZP), polydispersibility index (PDI) and surface morphology and drug content. The experimental result revealed that prepared RIV-loaded PLGA NPs (F1) was optimized having particle size (61.2 ± 4.6 nm), PDI (0.292), ZP (−11.2 ± 1.2). SEM study confirms the prepared nanoparticles depicted non-aggregated as well smooth surface particles without any fracture. This formulation (F1) was further assessed for in vivo studies on animal model. A pharmacological screening on an animal model of Alzheimer's disease revealed that RIV-loaded PLGA NPs formulations treat CNS disorders like Alzheimer's effectively. In addition to that, an in-vivo brain cholinesterase estimation study found that, animals treated with optimized formulation significantly (p < 0.01) reduced brain cholinesterase activity when compared to scopolamine-treated animals. According to the above results, it can be concluded that RIV-loaded PLGA NPs are ideal carriers for delivering the drug at a specific target site in the brain, thus may treat Alzheimer's disease efficiently and improve patient compliance.

Adverse neurobehavioral changes with reduced blood and brain cholinesterase activities in mice treated with statins.

Pleiotropic effects of hypolipidemic statins with behavioral outcomes have been suggested in humans and laboratory animals. There is limited information on the neurobehavioral effects of statins in mice. The aim of the present study was to examine changes in neurobehavioral performance and cholinesterase (ChE) activity in mice after high doses of three commonly used statins (atorvastatin, simvastatin, and rosuvastatin). Two hours after vehicle (control) or statin dosing at 250, 500, 750, or 1000 mg/kg orally, each mouse was subjected to 5 min open-field activity, negative geotaxis at an angle of 45°/60 s, 5 min head pocking, and forced swimming endurance. Plasma, erythrocyte, and brain ChE activities were determined spectrophotometrically 2 and 24 h after oral dosing of statins at 500 and 1000 mg/kg. The statins variably, but dose-dependently and significantly (p < 0.05) delayed the latency to move in the open-field arena, decreased locomotion and rearing, reduced head pocking, and delayed negative geotaxis performance. However, statins significantly increased the duration of forced swimming and decreased the duration of immobility in the swimming tank. Statins significantly and dose-dependently decreased plasma, erythrocyte, and brain ChE activity 2 and 24 h after dosing. Plasma and brain ChE activities recovered by 5%-32.9% and 5.7%-14.4% 24 h later from the 2 h ChE values, respectively. High doses of statins differentially modulate neurobehavioral outcomes in mice in association with reduced plasma, erythrocyte, and brain ChE activity. Plasma or erythrocyte ChE may be used for biomonitoring of the adverse/therapeutic effects of statins.

Effects of fenobucarb based-Excel Basa 50EC on brain cholinesterase of juvenile snakehead fish (Channa striata) in the Vietnamese Mekong Delta's rice fields.

Fenobucarb is one of most common insecticides applied to rice crops in the Vietnamese Mekong Delta. Paddy fields are preferred habitats for snakehead fish (Channa striata). Therefore, the probability of exposure risks and growth effects is highly. This paper aimed to examine the effects of using fenobucarb based - Excel Basa 50EC on the brain cholinesterase (ChE) of snakehead fish. Two rice fields, in which a single dose of Excel Basa 50EC was applied to one field, whilst the other acted as a control. Each field was subdivided into three plots by earthen dikes. In each plot, one fish cage (1.2 m x 1.2 m x 1.2 m) was installed that was stocked with 20 snakehead fish. The Excel Basa 50EC was applied once at the indication dose. The results highlighted that the concentration of fenobucarb in water at 1 hr after application was 116.72 ppb ± 12.64, which decreased to 23.96 ± 6.61 ppb after d and then to below detection limits (0.02 ppb). For fish living in this field, no mortality was seen, but ChE was significantly inhibited for 31 % on the first day and recovery following 7 days of exposure. Residues of fenobucarb in soil and fish should also be investigated furthermore.

Differential effects of statins on plasma and brain cholinesterase activities in chicks

Statins used to treat dyslipidemia may differentially modulate cholinesterase (ChE) activity impacting neuronal function. This study examines the effects of three statins (atorvastatin, fluvastatin, and simvastatin) on plasma and brain ChE activities and cholesterol levels in a chick model of 7-14 days old. Chicks were dosed orally with single doses of each statin at 50, 100, and 200mg/kg or repeated doses at 100mg/kg/day for 14 consecutive days. Plasma and whole brain ChE activities were measured electrometrically, whereas cholesterol levels were measured using a commercial colourimetric kit. In vitro ChE inhibition by the statins was initiated at 37°C for 10 mins. Data were statistically analysed using analysis of variance followed by the least significant difference test. Atorvastatin and fluvastatin did not significantly affect plasma ChE activities 2 hours after the oral administration, whereas simvastatin at 100 and 200mg/kg significantly increased (28% and 16%, respectively) plasma ChE activity. Repeated oral doses of the statins did not significantly affect plasma ChE activity. However, only simvastatin significantly decreased whole brain ChE activity by 33%. Repeated treatments with the three statins significantly reduced cholesterol levels in the plasma but not in the whole brain. The three statins inhibited in vitro plasma and whole brain ChE activities by 10-33% and 8-43%, respectively. The results suggested that the statins differentially modulated ChE activity in vivo and in vitro in chicks. Additional in vivo studies are warranted on statin effects on ChE activity in different brain regions of animal models.

First meta-analysis study of cholinesterase inhibition in experimental animals by organophosphate or carbamate insecticides under the influence of diphenhydramine.

Diphenhydramine is an H1-antihistamine that counteracts the toxic effects of organophosphate and carbamate insecticides that inhibit cholinesterase (ChE) activity. This meta-analysis aimed to investigate the effects of diphenhydramine on ChE inhibition induced by these insecticides in the plasma, erythrocytes, or whole brain of experimental animals. A data search was performed on erythrocyte, plasma, and brain ChE inhibition caused by organophosphate and carbamate insecticides in experimental animals (mice, rats, and chicks) treated with the antihistamine diphenhydramine in accordance with preferred reporting items for systematic reviews and meta-analysis, which was done by the two-group random-effects model meta-analysis. The meta-analysis included 18 records extracted from six studies that, appeared from 1996 to 2022. Using the random-effects model, a two-group meta-analysis revealed that the combined effect size (ChE inhibition) was significantly more favorable in the control group than in the diphenhydramine intervention, as shown by a forest plot. The combined effect size (standardized mean difference) was 0.67, with a standard error of 0.3, a lower limit of 0.04, and an upper limit of 1.29 (p = 0.025). The heterogeneity was moderate, as I2 of the combined effect size was 74%, with a significant Cochrane Q-test result (Q = 65, p < 0.0001). Subgroup analysis indicated that, with brain ChE inhibition, the heterogeneity (I2) became 5%, which was lower than ChE inhibition in plasma (84%) and erythrocytes (78%). No publication bias was identified using the funnel plot and Egger's test. This meta-analysis suggests that, in addition to its documented antidotal action against ChE-inhibiting insecticides, diphenhydramine can also reduce the extent of ChE inhibition, especially in the brain, which is the main site of toxicity of these insecticides. There is a need for additional studies to assess such enzyme inhibition in different parts of the brain.

The efficacy of 2-formyl benzoic acid in reactivating diazinon inhibited murine cholinesterase.

Oximes, as classical acetylcholinesterase (AChE) reactivators, have some pharmacokinetics/pharmacodynamics disadvantages. During the synthesis of non-oxime compounds, we encountered the compound 2-formylbenzoic acid (2-FBA) with promising in vitro and in vivo cholinesterase (ChE) reactivating properties in the acute exposure to diazinon (DZN). For in vitro experiments, the healthy mice serum and brain homogenate were freshly prepared and exposed to DZN (160 µg/mL). After 10 minutes, 2-FBA was added to the poisoned samples, and ChE activity was measured afterward. For the in vivo assay, the mice were poisoned with DZN subcutaneous (SC) injection (50 mg/kg), and after 1 hour, either 2-FBA or Pralidoxime (2-PAM) was injected intravenously (IV). After 3 h, ChE activity was measured in the serum and brain homogenate samples. The LD50 (IV) for 2-FBA in mice was measured as well. 2-FBA effectively reactivated the inhibited ChE in serum and brain homogenate samples in vitro. In the in vivo experiments, while 2-FBA could significantly reactivate the brain ChE even better than 2-PAM, they failed to reactivate the serum ChE by single IV injection. LD50 of 2-FBA was calculated to be 963 mg/kg. There were no general toxicity signs in any treatment groups. The in silico results support the potential ability of 2-FBA efficacy via possibly Witting reaction mechanism. Our findings indicate that 2-FBA seems to be a suitable non-oxime candidate for AChE reactivation with minimal side effects. Further toxicokinetic studies on this compound are strongly recommended to be performed before conducting the clinical trial in humans.

Assessment of Cholinesterase inhibition activity of birds inhabiting pesticide exposed croplands and protected area in hot semi-arid region of Pakistan.

Acetylcholinesterase (AChE) activity levels can be used as an indicator for AChE inhibition due to pesticide poisoning in bird species. We assessed the comparative brain cholinesterase (AChE) activity level of five bird species inhabiting pesticide exposed croplands and Protected Area i.e. Deva Vatala National Park (DVNP), Bhimber by using a spectrophotometric method. AChE activity levels ranged from 56.3 to 85.9 µmol/min/g of brain tissue of birds representing DVNP. However, AChE activity levels ranged from 27.6 to 79.9 µmol/min/g of brain tissue of birds representing croplands. AChE activity levels observed in Jungle babbler, Common babbler, and Red-vented bulbul showed significant differences (P < 0.05) at two sites. However, White wagtail and Black drongo demonstrated non-significant differences (P > 0.05). Maximum inhibition was recorded in Jungle babbler (53%) followed by Common babbler (35%), Red-vented bulbul (18%), White wagtail (15%), and Black drongo (7%). The brain cholinesterase inhibition levels under-protected ecosystems (DVNP, Bhimber) and agricultural landscape suggest insecticidal contamination and its impact on avifauna diversity. The study also emphasizes on the importance of pesticide-free zones to protect the biodiversity of birds.

Impacts of Dietary Chrysophyllum albidum Fruit Pulp on Brain Cholinesterase Function in High-Fat Diet/Streptozotocin-Induced Diabetic Rats

Epidemiologic studies have shown strong correlations between Alzheimer’s disease and diabetes mellitus. The exact mechanism through which this happens remains unclear. However, the dependence on glucose for brain function has been proposed as one possible mechanism. Hence, this study investigated the neuroprotective potential of Chrysophyllum albidum fruit pulp (CAPP) with hypoglycaemic properties in diabetic rats induced with high-fat diet/streptozotocin (STZ). The animals were grouped into seven units as follows: control, STZ-induced, STZ + metformin (positive control), STZ + 5% CAPP, STZ + 10% CAPP, control + 5% CAPP and control + 10% CAPP and each group was made up of six rats. The animals were first placed on normal diet (non-diabetic groups) and high fat diet (diabetic groups) for a fortnight, respectively before induction with STZ and were treated with diets containing 5 and 10% CAPP for 14 days. After the experiment, the rat brain cholinesterase and antioxidant activities were determined. The results revealed that acetylcholinesterase (AChE), butylcholinesterase (BuChE), arginase, adenosine deaminase (ADA) and antioxidant activities were altered in STZ-diabetic group in comparison to the control. However, a significant decrease at p &lt; 0.05 was found in the activities of AChE, BuChE, arginase and ADA. In addition, there was a concomitant rise in the levels of antioxidant in all the groups administered supplemented diets and the group treated with metformin in comparison to the STZ-diabetic group. Conclusively, we can suggest that the fruit pulp prevents neurological damage in diabetic rats via anticholinesterase activity and improvement of brain antioxidant status.

Recognition and Assessment of Antidotal Effects of Diphenhydramine against Acute Carbaryl Insecticide Poisoning in a Chick Model

Diphenhydramine antagonizes poisoning produced by cholinesterase (ChE) inhibiting insecticides. This study examines the effects of diphenhydramine against acute poisoning induced by the carbamate insecticide carbaryl in a chick model. The effects of diphenhydramine on the 24 h median Lethal Dose (LD50), and acute toxicity of carbaryl were assessed in chicks (7-15 days old). The plasma and whole brain ChE activities were measured electrometrically in vitro and in vivo. Diphenhydramine at 10mg/Kg Body wt. administered intramuscularly 15 min before carbaryl dosing increased the oral LD50 value of carbaryl (207 mg/Kg Body wt.) by 62%. Carbaryl at 250 mg/Kg Body wt. has orally produced toxidrome of cholinergic poisoning with 100% lethality in 24 h. Diphenhydramine (10mg/ Kg Body wt.) used 15 min before carbaryl (250mg/Kg Body wt., orally) was the most effective dose (vs 5 and 20mg/Kg Body wt.) in delaying carbaryl-toxicity and increasing survivals in chicks. The intramuscular median effective dose (ED50) of diphenhydramine which prevented 24 h carbaryl-death in chicks was 8.6mg/ Kg Body wt. The antidotal response to diphenhydramine was similar to that of the standard antidote atropine sulfate. Diphenhydramine at 10mg/Kg Body wt., given immediately after carbaryl (200mg/Kg Body wt.), reduced the percentages of plasma and whole brain ChE inhibitions in vivo by 12- and 13%, respectively. Carbaryl (10μmol/L) in vitro inhibited ChE activities in the plasma and brain by 53 and 77%, respectively; these inhibitions were reduced by 13- and 14%, respectively, when diphenhydramine (10μmol/L) was added to in vitro reactions. Diphenhydramine exerted antidotal action against a model of acute and lethal carbaryl intoxication in chicks.

Moringa oleifera Ameliorates Chlorpyrifos Induced Oxidative Stress in Male Wistar Rats

Background: Chlorpyrifos is a widely used organophosphate pesticide. It exerts its toxic effect by inhibiting of cholinesterases and induction of oxidative stress. Methods: In the present study,Soya oil, Chlopyrifos,Moringa oleifera, Chlopyrifos + Moringa oleifera, were given to male rats through gavage for 2 weeks.The influence of chlorpyrifos on brain cholinesterase activity and indicators of oxidative stress malondialdehydecatalase, glutathione, superoxide dismutaseand also histopathological changes in the brain of Wistar rats was investigated compared to control group. Results: There was significant increase in the brain malondialdehyde of the chlopyrifos group compared to the control and chlopyrifos+Moringa oleifera group. Also there were significant decreases in the superoxide dismutase, catalase, glutathione and acetylcholinesterase concentrations in the chlopyrifosgroup compared to the control and chlopyrifos + Moringa oleifera group. The rats showed significant changes in body, liver, kidneys and heart weights No significant haematological changes were seen following treatment. While some histopathological changes were detected in brain tissues in the chlopyrifos treated group, less histopathological changes were observed in chlopyrifos +Moringa oleifera treated groups at the end of the 2 weeks’ study. Conclusion: Overall, these results support the hypothesis that chlopyrifos causes oxidative stress and Moringa oleiferaextract can help reduce the changes induced by chlopyrifos.

Effects of Sargassum wightii Methanolic Extract on L-Methionine Induced Experimental Vascular Dementia like Syndrome in Albino Wistar Rats

Sargassum wightii is brown seaweeds with high nutritive value with natural bioactive compounds having diverse therapeutic activity. In recent years, research on Sargassum wightii has gain much interest in neuropharmacological field. Basing on neuroprotective effect of Sargassum wightii against Parkinson’s disease animal models, the possible protective effect on dementia is hypothesised. Addressing this question the present study was designed to investigate the effect of S. wightii on L-methionine induced vascular dementia like syndrome in male wistar albino rats. Vascular dementia was induced by oral administration of L-methionine (1.7 g/kg) per body weight for 4weeks S. wightii was given from day1 to day32. The neurobehavioral assessment was performed by Morris water maze test for learning and memory ability and blood sample was collected for serum nitrite and homocysteine analysis. The whole brain content was subjected to a series of biochemical analysis that included brain cholinesterase, malondialdehyde, reduced glutathione and histopathological examination. L-methionine impaired learning and memory, increased serum homocysteine and decreased serum nitrite level significantly (p&lt;0.001). It also increased brain thiobarbituric acid reactive substances, reduced glutathione and increased acetylcholinesterase activity significantly (p&lt;0.001). Sargassum wightii at 200mg/kg and 400mg/kg body weight reversed the L-methionine induced learning and memory deficits (p&lt;0.01) and (p&lt;0.001) respectively. The L-methionine induced biochemical alterations in serum and brain as well as histopathological aberration in cortex and hippocampus were restored by Sargassum wightii (p&lt;0.01). This study concluded that Sargassum wightii ameliorated L-methionine induced vascular dementia like syndrome and provided a new concept in anti-dementia therapy as a neuroprotectants.

Effects of Chlorpyrifos Ethyl on Cholinesterase and Growth of Silver Barb (Barbonymus gonionotus)

Chlorpyrifos ethyl is a popular insecticide widely used in agriculture within the Vietnamese Mekong delta, including for rice farming. Here, local farmers often apply pesticides at very high rates which leads to contamination of the surrounding environment. Silver barb (Barbonymus gonionotus) is a fish species indigenous to the delta, which resides in a variety of water bodies and is also commonly cultivated in rice–fish systems. As a result, this species is at high risk of exposure to chlorpyrifos ethyl. This study aims to determine the lethal concentration (LC50) of chlorpyrifos ethyl, as well as the effects of sub-lethal concentrations on the activity of cholinesterase and growth of Silver barb. Lethal concentration testing was conducted in a static non-renewed system. Three concentrations of chlorpyrifos ethyl (1%, 10% and 20% LC50-96 h) were conducted in triplicate to assess the effects of chlorpyrifos ethyl on the brain cholinesterase (ChE) of fingerling fish for 15 days, and on their growth for 60 days. Results showed that chlorpyrifos ethyl was highly toxic to fingerling Silver barb with a LC50-96 h of 0.119 ppm. The lowest observed effect concentration (LOEC) was 1%LC50-96 h for ChE and 10%LC50-96 h for growth. No observed effect concentration (NOEC) of chlorpyrifos ethyl for growth was 1%LC50-96 h. The result from this study suggests that ChE activity is significantly inhibited at environmentally realistic concentrations in the Vietnamese Mekong delta and can be used as a biomarker of pesticide exposure. Further study in the rice fields as well as in the canals or rivers is required.

Micro/nano-plastics cause neurobehavioral toxicity in discus fish (Symphysodon aequifasciatus): Insight from brain-gut-microbiota axis

Numerous studies have investigated neurobehavioral toxicity of microplastics, but no studies have illustrated mechanism via brain-gut axis. Here, juvenile discus fish (Symphysodon aequifasciatus) were exposed for 96 h to microfibers (900 µm, fiber, MFs) or nanoplastics (~88 nm, bead, NPs) with three concentrations (0, 20 and 200 µg/L). Accumulation in fish gut was independent of plastics type and concentration. MFs reduced growth performance while NPs weakened swimming and predatory performance of post-exposed discus. For brain cholinesterase activity, acetylcholinesterase was activated by NPs while NPs/MFs exposure inhibited butyrylcholinesterase. Concentrations of neurotransmitters (acetylcholine, dopamine and γ-aminobutyric acid) increased in brain but decreased in gut after NPs or MFs exposure. For gut microbiota, increased richness under MFs exposure was observed. At phylum level, Proteobacteria proportion was lower in NPs but higher in MFs. Abundance of Clostridia and Fusobacteriia (Bacillus), potentially secreting neurotransmitters, increased in NPs but decreased in MFs. Brain transcriptomics revealed seven upregulated and four downregulated genes concerning neural-activities. Pathways of neuroactive ligand-receptor interaction and serotonergic synapse were enriched in both MFs and NPs, but dopaminergic synapse pathway was enriched only in MFs. These results established a novel mechanism by which microplastics might cause behavioral toxicities via brain-gut-microbiota axis.

Persistent proteomic changes in glutamatergic and GABAergic signaling in the amygdala of adolescent rats exposed to chlorpyrifos as juveniles

Chlorpyrifos (CPF) remains one of the most widely used organophosphorus insecticides (OPs) despite the concerns about its developmental neurotoxicity. Developmental exposure to CPF has long-lasting negative impacts, including abnormal emotional behaviors. These negative impacts are observed at exposure levels do not cause inhibition of acetylcholinesterase, the canonical target of OPs. Exposure to CPF at these levels inhibits the endocannabinoid metabolizing enzyme fatty acid amide hydrolase (FAAH) but it is not clear what the persistent effects of this inhibition are. To investigate this, male rat pups were exposed orally to either corn oil, 0.75 mg/kg CPF, or 0.02 mg/kg PF-04457845 (PF; a specific inhibitor of FAAH) daily from postnatal day 10 (PND10) - PND16. This dosage of CPF does not inhibit brain cholinesterase activity but inhibits FAAH activity. On PND38 (adolescence), the protein expression in the amygdala was determined using a label-free shotgun proteomic approach. The analysis of control vs CPF and control vs PF led to the identification of 44 and 142 differentially regulated proteins, respectively. Gene ontology enrichment analysis revealed that most of the proteins with altered expression in both CPF and PF treatment groups were localized in the synapse-related regions, such as presynaptic membrane, postsynaptic density, and synaptic vesicle. The different biological processes affected by both treatment groups included persistent synaptic potentiation, glutamate receptor signaling, protein phosphorylation, and chemical synaptic transmission. These results also indicated disturbances in the balance between glutamatergic (↓ Glutamate AMPA receptor 2, ↓ Excitatory amino acid transporter 2, and ↑ vesicular glutamate transporter 2) and GABAergic signaling (↑ GABA transporter 3 and ↑ glutamate decarboxylase 2). This imbalance could play a role in the abnormal emotional behavior that we have previously reported. These results suggest that there is a similar pattern of expression between CPF and PF, and both these chemicals can persistently alter emotional behavior as a consequence of inhibition of FAAH.

Synthetisation of an affinity matrix (Procainamide Sepharose Cl-6b) for brain cholinesterase purification and separation source from Monopterus albus

Affinity chromatography for acetylcholinesterase; AChE namely Procainamide Sepharose CL-6B was synthesised through the coupling method between soluble procainamide hydrochloride immobilised by a cross-linked agarose size exclusion, sepharose CL-6B. 1, 4-butanediol diglycidyl ether plays a role in building up a productive and rigid connecting of a biospecific ligand (Procainamide hydrochloride) to an insoluble matrix (Sephacryl CL-6B). Local freshwater eel brain was extracted and centrifuge at high speed. The supernatant was collected, and seven different volumes were separately loaded on to the column for isocratic purification where 12 fractions were collected at the end of elution stage. This study shows 1000 μL of extractant; considered as the maximum volume to load onto the column. Lastly, a stepwise elution was performed with five different concentrations of NaCl, and each of 1 mL fraction was collected then assay for determination of enzyme activity and protein content. The data shows AChE was successfully purified with percentage recovery of 38 % after 21 purification fold. Kinetic study strengthens the data where the efficient coefficient ratio of ATCi was much higher compared to PTCi and BTCi at 3.03, 2.67 and 1.52 Vmax⋅Km −1, respectively, prove that the collected fraction predominantly contained with AChE, which is a targeted enzyme to be used as a sensitive biosensor to detect the presence of carbamate and organophosphate contamination in the environment.

Neurobehavioral anomalies in zebrafish after sequential exposures to DDT and chlorpyrifos in adulthood: Do multiple exposures interact?

A sequence of different classes of synthetic insecticides have been used over the past 70 years. Over this period, the widely-used organochlorines were eventually replaced by organophosphates, with dichlorodiphenyltrichloroethane (DDT) and chlorpyrifos (CPF) as the principal prototypes. Considerable research has characterized the risks of DDT and CPF individually, but little is known about the toxicology of transitioning from one class of insecticides to another, as has been commonplace for agricultural and pest control workers. This study used adult zebrafish to investigate neurobehavioral toxicity following 5-week chronic exposure to either DDT or CPF, to or their sequential exposure (DDT for 5 weeks followed by CPF for 5 weeks). At the end of the exposure period, a subset of fish were analyzed for brain cholinesterase activity. Behavioral effects were initially assessed one week following the end of the CPF exposure and again at 14 months of age using a behavioral test battery covering sensorimotor responses, anxiety-like functions, predator avoidance and social attraction. Adult insecticide exposures, individually or sequentially, were found to modulate multiple behavioral features, including startle responsivity, social approach, predator avoidance, locomotor activity and novel location recognition and avoidance. Locomotor activity and startle responsivity were each impacted to a greater degree by the sequential exposures than by individual compounds, with the latter being pronounced at the early (1-week post exposure) time point, but not 3–4 months later in aging. Social approach responses were similarly impaired by the sequential exposure as by CPF-alone at the aging time point. Fleeing responses in the predator test showed flee-enhancing effects of both compounds individually versus controls, and no additive impact of the two following sequential exposure. Each compound was also associated with changes in recognition or avoidance patterns in a novel place recognition task in late adulthood, but sequential exposures did not enhance these phenotypes. The potential for chemical x chemical interactions did not appear related to changes in CPF metabolism to the active oxon, as prior DDT exposure did not affect the cholinesterase inhibition resulting from CPF. This study shows that the effects of chronic adult insecticide exposures may be relevant to behavioral health initially and much later in life, and that the effects of sequential exposures may be unpredictable based on their constituent exposures.

Non-linear model analysis of the relationship between cholinesterase activity in rats exposed to 2, 2-dichlorovinyl dimethylphosphate (dichlorvos) and its metabolite concentrations in urine

Urinary dialkylphosphates (DAPs) are measured to assess exposure to organophosphorus pesticides (OPs), but they are common metabolites of OPs and not specific indices for individual agents. Biomonitoring (BM) of urinary DAPs has been widely adopted as an assessment of individual exposure in general environments, however, guidance values for DAPs based on health effects have yet to be established. The present study aimed to clarify the relationship between the amount of urinary dimethylphosphate (DMP), a metabolite of dichlorvos (DDVP), and the inhibition of cholinesterase (ChE) activity in rats exposed to DDVP. The relationship was analyzed using a nonlinear model analysis, and the excretion level of urinary DMP equivalent to ChE 20 % inhibition (EL20) and the lower limit of the 95 % confidence interval of EL20 (ELL20) were estimated. EL20 and ELL20 (mg/24 h urine) of brain, erythrocyte, and plasma ChE activities after 10-day administration of DDVP were 0.21 and 0.15, 0.11 and 0.06, and 0.23 and 0.09, respectively. Extrapolating ELL20 of the brain ChE to humans, the range of 24 h urinary DMP concentration according to the 20 % inhibition of cholinesterase activity was estimated to be 20.5–30.8 mg/l. In conclusion, the amount of urinary DMP as ELL20 for DDVP exposure was identified and could probably be used as a novel index for the assessment of risk from OP exposure. Further studies are needed to clarify the ELL20 s derived from OPs other than DDVP, for informing efforts to establish guidance values of urinary OP metabolites that should prevent neurotoxicity.

In vivo cholinesterase sensitivity of gilthead seabream (Sparus aurata) exposed to organophosphate compounds: Influence of biological factors

Two cholinesterases have been found in vertebrates, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). These enzymes are present in the gilthead seabream, AChE in the brain and muscle and BChE in the muscle. Cholinesterases have been used as biomarker of effect in environmental monitoring studies. However, there are few studies about the influence of biometric parameters on ChE. This paper studies the possible influence of biological factors on brain and muscle cholinesterase (ChE) in Sparus aurata. Our results show that ChE activity in brain and muscle tissues changes depending on several biological variables. ChE activity in these tissues decreased when the age (48–152 week), body length (14.15–28.95 cm) and body weight (42.73–380.74 g) of the fishes studied increased. The relationships between brain and muscle ChE activity and several biometric factors were curvilinear.On the other hand, in vivo sensitivity of cholinesterase in the gilthead seabream exposed to organophosphorus pesticides (azinphosmethyl, dimethoate and dichlorvos) was studied in order to learn about recovery from cholinesterase present (brain and muscle) after exposure to a sublethal dose. The recovery of muscle AChE was similar to cerebral AChE, while muscular BChE showed a slower recovery.