► After 96h of exposition, fish in the 2.4μgendosulfan/L group presented a significant decrease in brain AChE activity when compared to the control group. ► Exposure to 2.4μgendosulfan/L during 96h ...also impaired all exploratory parameters evaluated. ► Exposure to 2.4μgendosulfan/L during 96h did not compromise animals’ performance in the inhibitory avoidance apparatus. ► Our findings provide further evidence of the deleterious effects of endosulfan exposure in the nervous system.
Endosulfan is a broad spectrum organochlorine pesticide that is still widely in use in many developing countries. Following application, endosulfan can get to watercourses through surface runoff from agricultural fields and disturb the non-target aquatic animals including freshwater fish species. Given that the activity of the enzyme acetylcholinesterase (AChE) is one of the most recurrently used biomarkers of exposure to pesticides and there are controversial results concerning the effects of endosulfan exposure and AChE activity in fish, the aim of the present study was to evaluate the effects of endosulfan in brain AChE activity and its gene expression pattern using adult zebrafish (Danio rerio) as an animal model. Moreover, we have analyzed the effects of endosulfan exposure in different parameters of zebrafish swimming activity and in long-term memory formation. After 96h of exposition, fish in the 2.4μgendosulfan/L group presented a significant decrease in AChE activity (9.44±1.038μmolSChh−1mgprotein−1; p=0.0205) when compared to the control group (15.87±1.768μmolSChh−1mgprotein−1; p=0.0205) which corresponds to approximately 40%. The down-regulation of brain AChE activity is not directly related with the transcriptional control as demonstrated by the RT-qPCR analysis. Our results reinforce AChE activity inhibition as a pathway of endosulfan-induced toxicity in brain of fish species. In addition, exposure to 2.4μgendosulfan/L during 96h impaired all exploratory parameters evaluated: decreased line crossings (≈21%, 273.7±28.12 number of line crossings compared to the control group 344.6±21.30, p=0.0483), traveled distance (≈20%, 23.44±2.127m compared to the control group 29.39±1.585, p=0.0281), mean speed (≈25%, 0.03±0.003m/s compared to the control group 0.04±0.002, p=0.0275) and body turn angle (≈21%, 69,940±4871 absolute turn angle compared to the control group 88,010±4560, p=0.0114). These results suggest that endosulfan exposure significantly impairs animals’ exploratory performance, and potentially compromises their ecological and interspecific interaction. Our results also showed that the same endosulfan exposure did not compromise animals’ performance in the inhibitory avoidance apparatus. These findings provide further evidence of the deleterious effects of endosulfan exposure in the nervous system.
Alzheimer's disease (AD) is a widespread multifactorial aging-related pathology, which includes cholinergic deficit among its main causes. Following a multi-target design strategy, the structure of ...the approved drug donepezil was taken as the starting point for generating some new potential multi-functional compounds. Therefore, a series of twenty molecular hybrids were synthesized and assayed against three different enzymes, namely the well-established targets acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and the innovative one fatty acid amide hydrolase (FAAH). In silico studies confirmed the interaction of benzylpiperidine and the benzylpiperazine isostere with the catalytic anionic site (CAS) of AChE, while the aryloxycarbonyl portion appeared to be important for the interaction with the peripheral site (PAS). A QSAR study was carried out on AChE inhibition data, which revealed that the inhibition potency seems to depend upon the length of the spacer and the number of polar atoms. The docking poses of selected compounds within BChE and FAAH were also calculated. Furthermore, pharmacokinetics and drug-likeness properties were assessed by chemoinformatic tools. Several piperidine derivatives (in particular compound 10) showed interesting profiles as multi-target directed agents, while the lead piperazine derivative 12 (SON38) was found to be a more potent and selective AChE inhibitor (IC50 = 0.8 nM) than donepezil, besides being able to bind bivalent copper cations (pCu = 7.9 at physiological pH). Finally, the selected lead compounds (10 and 12, SON38) did not show significant cytotoxicity on SH-SY5Y and HepG2 cells at the highest tested concentration (100 μM) in a MTT assay.
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•Twenty compounds were designed and assayed for their multi-target activity.•Compound 10 showed an interesting profile as multi-target agent as ChEs and FAAH inhibitor.•Compound 12 (SON38) was found to be a potent and selective AChE inhibitor, as well as a chelator of Cu2+.
Eugenol was used as starting material to obtain some phenolic compounds. The synthesis of these phenolic compounds was performed in a two-step procedure. The structures of the formed products (novel ...eugenol derivatives 1–6) have been determined on the basis of NMR spectroscopy and other spectroscopic methods. The compounds were tested in terms of carbonic anhydrase (CA) inhibition potency. Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes, which catalyse the reaction between carbon dioxide (CO2) and water (H2O), to generate bicarbonate (HCO3−) and protons (H+). CO2, HCO3− and H+ are essential molecules and ions for many important physiologic processes occurring in all living organisms. Acetylcholinesterase (AChE, E.C.3.1.1.7) is found in high concentrations in the red blood cells and brain. Novel eugenol derivatives (1–6) were tested for the inhibition of two cytosolic CA isoforms I, and II (hCA I, and II) and AChE. These compounds demonstrated effective inhibitory profiles with Ki values in ranging of 113.48-738.69nM against hCA I, 92.35-530.81nM against hCA II, and 90.10-379.57nM against AChE, respectively. On the other hand, acetazolamide clinically used as CA inhibitor, shoed Ki value of 594.11nM against hCA I, and 120.68nM against hCA II, respectively. Also, AChE was inhibited by tacrine as an AChE inhibitor at the 71.18nM level.
This work introduced an efficient immobilization of acetylcholinesterase (AChE) onto amino functionalized carbon nanotubes (CNT-NH2), in order to fabricate high sensitive and practical ...organophosphorus pesticide (OPs) biosensors. Compared with the pristine, -COOH and -OH decorated CNTs, there were larger amount of enzymes adsorbed on the surface of CNT-NH2 with a favorable orientation and the best amperometric response was obtained on the AChE/CNT-NH2/GC electrode. Furthermore, the biosensor modified with CNT-NH2 showed a high affinity to acetylthiocholine chloride (ATCh) and could catalyze the hydrolysis of ATCh with an apparent Michaelis-Menten constant (Km) value of 67.4 µM. Using paraoxon as a model compound, wide linear ranges from 0.2 nM to 1 nM and 1 nM to 30 nM, and a low detection limit of 0.08 nM were obtained with satisfactory reproducibility and stability. Moreover, the biosensor had also been successfully employed for the determination of low concentrations of pesticides in real vegetable samples. This method could be extended to other functionalized nano-materials for their application in constructing biosensors.
The present study compares two approaches to evaluate the effects of inter-individual differences in the biotransformation of chlorpyrifos (CPF) on the sensitivity towards in vivo red blood cell ...(RBC) acetylcholinesterase (AChE) inhibition and to calculate a chemical-specific adjustment factor (CSAF) to account for inter-individual differences in kinetics (HK
AF
). These approaches included use of a Supersome
™
cytochromes P450 (CYP)-based and a human liver microsome (HLM)-based physiologically based kinetic (PBK) model, both combined with Monte Carlo simulations. The results revealed that bioactivation of CPF exhibits biphasic kinetics caused by distinct differences in the Km of CYPs involved, which was elucidated by Supersome
™
CYP rather than by HLM. Use of Supersome
™
CYP-derived kinetic data was influenced by the accuracy of the intersystem extrapolation factors (ISEFs) required to scale CYP isoform activity of Supersome
™
to HLMs. The predicted dose–response curves for average, 99th percentile and 1st percentile sensitive individuals were found to be similar in the two approaches when biphasic kinetics was included in the HLM-based approach, resulting in similar benchmark dose lower confidence limits for 10% inhibition (BMDL
10
) and HK
AF
values. The variation in metabolism-related kinetic parameters resulted in HK
AF
values at the 99th percentile that were slightly higher than the default uncertainty factor of 3.16. While HK
AF
values up to 6.9 were obtained when including also the variability in other influential PBK model parameters. It is concluded that the Supersome
™
CYP-based approach appeared most adequate for identifying inter-individual variation in biotransformation of CPF and its resulting RBC AChE inhibition.
The orphan drug dantrolene (DAN) is the only therapeutic treatment for malignant hyperthermia (MH), a pharmacogenetic pathology affecting 0.2 over 10,000 people in the EU. It acts by inhibiting ...ryanodine receptors, which are responsible for calcium recruitment in striatal muscles and brain. Because of its involvement in calcium homeostasis, DAN has been successfully investigated for its potential as neuroprotecting small molecule in several animal models of Alzheimer's disease (AD). Nevertheless, its effects at a molecular level, namely on putative targets involved in neurodegeneration, are still scarcely known. Herein, we present a prospective study on repurposing of DAN involving, besides the well-known calcium antagonism, inhibition of monoamine oxidase B and acetylcholinesterase, cytoprotection from oxidative insult, and activation of carnitine/acylcarnitine carrier, as concurring biological activities responsible for neuroprotection.
Protein engineering by resurfacing is an efficient approach to provide new molecular toolkits for biotechnology and bioanalytical chemistry. H39GFP is a new variant of green fluorescent protein (GFP) ...containing 39 histidine residues in the primary sequence that was developed by protein resurfacing. Herein, taking H39GFP as the signal reporter, a label-free fluorometric sensor for Cu2+ sensing was developed based on the unique multivalent metal ion-binding property of H39GFP and fluorescence quenching effect of Cu2+ by electron transfer. The high affinity of H39GFP with Cu2+ (K d, 16.2 nM) leads to rapid detection of Cu2+ in 5 min with a low detection limit (50 nM). Using acetylthiocholine (ATCh) as the substrate, this H39GFP/Cu2+ complex-based sensor was further applied for the turn-on fluorescence detection of acetylcholinesterase (AChE) activity. The assay was based on the reaction between Cu2+ and thiocholine, the hydrolysis product of ATCh by AChE. The proposed sensor is highly sensitive (limit of detection (LOD) = 0.015 mU mL–1) and is feasible for screening inhibitors of AChE. Furthermore, the practicability of this method was demonstrated by the detection of pesticide residue (carbaryl) in real food samples. Hence, the successful applications of H39GFP in the detection of metal ion and enzyme activity present the prospect of resurfaced proteins as versatile biosensing platforms.
2-oleyl-1,3-dipalmitoylglycerol (ODG) obtained from the bacuri seeds. In vitro tests showed inhibition of the enzyme acetylcholinesterase. However, ODG has low solubility in water. In order to ...increase its solubility, the inclusion complex between ODG and β-cyclodextrin (β-CD) was obtained by solubilization followed by lyophilization. The objective of this study was to prepare, characterize and evaluate the solubility of the complex that was characterized by infrared spectroscopy (IR), differential scanning calorimetry (DSC), thermogravimetry (TG), scanning electron microscopy (SEM), X-ray diffraction XRD), hydrogen magnetic resonance (1H NMR) and phase solubility. All results confirmed the formation of the inclusion complex between ODG and β-CD. By 1H NMR data, it was possible to predict that the ODG was encapsulated by the wider side of the β-CD cavity. The solubility isotherm allowed to determine the apparent stability constant (K = 339.38 L mol−1) and the inclusion efficiency (IE = 57.82%), as well as the 1:1 stoichiometry between ODG and β- CD. The rate of dissolution and solubility of the inclusion complex were significantly improved as compared to the pure drug. Therefore, the use of ODG-β-CD can effectively improve the solubility and dissolution rate of free ODG, being a promising approach to promote its clinical application.
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•The ODG substance was detected the inhibition of the enzyme acetylcholinesterase.•The inclusion complex showed faster dissolution rate than the free ODG.•All results confirmed the formation of the inclusion complex between ODG and β-CD.•The ODG was encapsulated by the wider side of the β-CD cavity.
The inhibition of acetylcholinesterase (AChE) has pharmaceutical applications as well as potential neurotoxic effects. The in vivo metabolites of some chemicals including organophosphorus pesticides ...can become more potent AChE inhibitors compared to their parental compounds. To account for the effects of biotransformation, we have developed and characterized a high-throughput screening method for identifying AChE inhibitors that become active or more potent following xenobiotic metabolism. In this study, an enzyme-based assay was developed in 1536-well plates using recombinant human AChE combined with human or rat liver microsomes. The AChE activity was measured by two methods with different readouts: colorimetric and fluorescent. The assay exhibited exceptional performance characteristics including large assay signal window, low well-to-well variability and high reproducibility. The performance of the assays with microsomes was characterized by testing a group of known AChE inhibitors including parent compounds and their metabolites. Large potency differences between the parent compounds and the metabolites were observed in the assay with microsome addition. Both assay readouts were required for maximal sensitivity. These results demonstrate that this platform is a promising method to profile large numbers of chemicals that require metabolic activation for inhibiting AChE activity.
•Many OPs require metabolic activation by P450 isoenzymes to inhibit AChE activity for their pesticidal activity•In vitro AChE assays with xenobiotic metabolism capability were developed and validated in a qHTS platform.•Potency differences between the parent compounds and their metabolites were observed in assays with metabolic activation.•The screening platform can be used to evaluate compounds for potential neurotoxicity from AChE inhibition.
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