•Set up a cost-effective automated piezoelectric spraying for biological and enzymatic assays.•Bioprofiling of Peganum harmala seed extract via piezoelectric spaying versus immersion.•Selective ...cholinesterase assays (AChE/BChE) to find the active ingredients.•Cholinesterase inhibitory equivalency referred to physostigmine via HPTLC-AChE/BChE.•In situ HRMS measurement of the active zones in the P. harmala seed extract.
Bioanalytical questions are more and more solved by bioassays directly in situ the planar separation. If compared to chemical derivatization in situ, several reagent applications on the same chromatogram make the workflow for enzymatic and biological assays more complex. Hence, if compared to piezoelectric spraying of chemical derivatization reagents, an assay transfer to the piezoelectric spraying technique was much more challenging. Important aspects were investigated, i.e., plate pre-wetting, spraying nozzle type and applied volumes for microorganism suspension as well as enzyme and substrate-chromogenic solutions. Finally, with the newly developed piezoelectric spraying procedures for the application of biological (Aliivibrio fischeri) and enzymatic (acetyl- and butyrylcholinesterase) assays, several obstacles of the state-of-the-art automated immersion were avoided such as the (1) required high volumes of solutions, (2) tailing of highly water-soluble zones upon slow plate withdrawal, (3) zone distortion or shift observed after previous buffer salt applications or long/slow immersion times/speeds, (4) gradual inactivation of the enzyme solution along with its ongoing re-use, and (5) lack of covering the whole plate surface. The benchmarking of both techniques also showed that simplicity remains the key argument for immersion. As proof of concept, piezoelectrically sprayed autograms were compared with those of immersion, by taking the example of Peganum harmala (P. h.) seed extract. The plate background and thus homogeneity of the applied solutions were found to be almost comparable. Three bands among the pronounced fluorescent bands were responsible for the most antibacterial activity of P. h. seed extract in the A. fischeri bioassay and were also inhibiting the AChE. These AChE and three further BChE inhibitors were detected, whereby the AChE inhibition was twice as strong as the BChE inhibition. By their in situ HRMS spectra, the active zones in the P. h. seed extract were assigned to be the AChE-inhibiting β-carboline alkaloids, harmine, harmaline and ruine, as well as the BChE-inhibiting quinazoline alkaloids, vasicine and deoxyvasicine, and the β-carboline alkaloid harmol. For the first time, the found inhibitors were calculated equivalently to the well-known ChE-inhibitor physostigmine, and thus, piezoelectric spraying was proven to be suited for quantifications.
Breast cancer is the leading cause of cancer-related death in women worldwide. Several studies have addressed the association between cancer in humans and agricultural pesticide exposure. Evidence ...indicates that exposure to organophosphorous pesticides such as parathion and malathion occurs as a result of occupational factors since they are extensively used to control insects. On the other hand, estrogens have been considered beneficial to the organism; however, epidemiological studies have pointed out an increased breast cancer risk in both humans and animals. Experimental female rat mammary gland cancer models were developed after exposure to parathion, malathion, eserine, an acetylcholinesterase inhibitor, and estrogen allowing the analysis of the signs of carcinogenicity as alteration of cell proliferation, receptor expression, genomic instability, and cell metabolism in vivo and in vitro. Thus, pesticides increased proliferative ducts followed by ductal carcinoma; and 17β-estradiol increased proliferative lobules followed by lobular carcinomas. The combination of both pesticides and either eserine or estrogen induced tumors with both types of structures followed by mammary gland tumors and metastasis to the lung and kidneys after 240 days of a 5-day treatment. Studies also showed that these pesticides and eserine decreased three to five times the acetylcholinesterase activity in the serum compared to controls whereas terminal end buds increased in number, being inhibited by atropine. Genomic instability was analyzed in such tissues (mp53, CYP1A2, c-myc, c-fos, ERα, M2R) and pesticides increased protein expression that was stimulated by estrogens but inhibited by atropine. Eserine also transformed the epithelium of the rat mammary gland in the presence of estrogen and increased the number of terminal end buds after treatment inducing mammary carcinomas. Then, enzymatic digestion of such structures gave rise to cells with increased DNA synthesis and induced anchorage independence. Thus, there were changes in the epithelium of the mammary gland influencing breast carcinogenesis. Furthermore, these substances and acetylcholine also showed the signs of carcinogenicity in vitro as cell proliferation, receptor expression (ERα, ErbB2, M2R), genomic instability (c-myc, mp53, ERα, M2R), and cell metabolism. A unique cellular model is also presented here based on the use of MCF-10 F, a non-tumorigenic cell line that represents a valuable clinically translatable experimental approach that identifies mechanistic links for pesticides and estrogen as suspect human carcinogenic agents.
In this work, 4,4-difluoro-8-(4-hydroxyphenyl)− 2,6-diethynly-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) having double terminal ethynyl groups was synthesized. Three dimensional ...single walled carbon nanotube (SWCNT)-BODIPY hybrid material (3D SWCNT-BODIPY) was synthesized by the reaction of BODIPY bearing double terminal ethynyl groups with azido containing SWCNTs via “Click” reaction. The structural properties and electrochemical detection of eserine (a pesticide) on BODIPY functionalized SWCNTs as a three dimensional (3D) material were investigated. A glassy carbon electrode (GCE) was modified by 3D SWCNT-BODIPY hybrid material for the determination of eserine in the range of 0.25–2.25 μM. In the study by the square wave voltammetry (SWV), the bare GCE showed no response, while the new peak at − 0.6 V appeared in the case of the modified electrode. The detection limit and quantification were determined as 160 nM and 528 nM for eserine on the 3D SWCNT-BODIPY modified electrode, respectively. Eserine was also determined with a standard addition method in different brands of orange juices, and the recovery of eserine was obtained to be in the range of 102.09% and 103.22%. This study clearly indicates that the 3D SWCNT-BODIPY modified electrode tested as an electrochemical sensor was found to be highly selective and sensitive to eserine.
•The 3D SWCNT-BODIPY nanomaterial was synthesized via “Click” reaction.•The electrochemical detection of eserine (a pesticide) on 3D SWCNT-BODIPY was investigated.•Selectivity was studied towards paration, fenitrothion, methomly, chlorptrifos methyl and diazinon.•Eserine was also determined with a standard addition method in different brands of orange juices.
5-Arylidene-N,N-diethylthiobarbiturates 1-25 were evaluated for their potential against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes and displayed varying degree of ...inhibition. Based on the IC50 values, compound 6 (IC50 = 92.51 and#177; 0.01 and#181;M) was the only active compound against acetylcholinesterase, however this compound was also weakly active against butyrylcholinesterase with an IC50 value 124.76 and#177; 0.02 and#181;M. Six compounds 1, 6, 7, 16, 17, and 18 showed weak inhibition against BChE, among them compound 18 (IC50 = 82.25 and#177; 0.07and#181;M) showed good inhibition against BChE. Nevertheless, compounds 1, 7, 16, 17, and 18 were selectively active against BChE enzyme.
1. Esterases may play a major role in the clearance of drugs with functional groups amenable to hydrolysis, particularly in the case of ester prodrugs. To understand the processes involved in the ...elimination of such drugs, it is necessary to determine the esterases involved. However, the tools currently available for this enzyme phenotyping are relatively scarce.
2. The work was aimed at summarizing the selectivity of esterase inhibitors for carboxylesterases 1 and 2 (CES1 and CES2) in the human liver to clarify their suitability for esterase phenotyping. Eserine, at around 10 μM, was found to be a highly specific CES2 inhibitor, whereas other esterase inhibitors turned out less selective. When used together with tacrine, which inhibits cholinesterases but not CES, and ethylenediaminetetraacetic acid (inhibitor of paraoxonases), the involvement of the hydrolyzing esterases in the hepatic clearance of a drug can be elucidated.
3. The second approach to esterase phenotyping is based on data from recombinant or isolated esterases, together with relative activity factors, which relate their activities to those of the same enzymes in subcellular fractions.
4. These two approaches will help to characterize the hydrolytic metabolism of drug candidates in a similar manner as practiced routinely for the oxidative metabolism by cytochrome P450 enzymes.
Breast cancer is a major health threat to women worldwide and the leading cause of cancer-related death. The use of organophosphorous pesticides has increased in agricultural environments and urban ...settings, and there is evidence that estrogen may increase breast cancer risk in women. The mammary gland is an excellent model for examining its susceptibility to different carcinogenic agents due to its high cell proliferation capabilities associated with the topography of the mammary parenchyma and specific stages of gland development. Several experimental cellular models are presented here, in which the animals were exposed to chemical compounds such as pesticides, and endogenous substances such as estrogens that exert a significant effect on normal breast cell processes at different levels. Such models were developed by the effect of malathion, parathion, and eserine, influenced by estrogen demonstrating features of cancer initiation in vivo as tumor formation in rodents; and in vitro in the immortalized normal breast cell line MCF-10F, that when transformed showed signs of carcinogenesis such as increased cell proliferation, anchorage independence, invasive capabilities, modulation of receptors and genomic instability. The role of acetylcholine was also demonstrated in the MCF-10F, suggesting a role not only as a neurotransmitter but also with other functions, such as induction of cell proliferation, playing an important role in cancer. Of note, this is a unique experimental approach that identifies mechanistic signs that link organophosphorous pesticides with breast carcinogenesis.
A new electrode modification technique, “click-electrochemistry” (CEC), was introduced for the electrochemical applications of a functional cobalt phthalocyanine. In this newly constructed modified ...electrode (GCE/PANI-N3/TA-CoPc), a terminal-alkynyl (TA) substituted cobalt phthalocyanine (CoPc-TA) was electrochemically bonded to the azido end of polymerized 4-azidoaniline (PANI-N3), which was electropolymerized on the GCE electrochemically previously.
•A new electrode modification technique via “click-electrochemistry”.•Terminal-alkynyl of phthalocyanines was electrochemically bonded to the azido of azidoaniline.•Modified electrode was electrochemically and spectroscopically characterized.•Modified electrode gave three redox couples assigned to cobalt phthalocyanine and azidoaniline.
A new electrochemical sensor electrode was constructed with a new electrode modification technique, “click electrochemistry” (CEC) based on a functional cobalt phthalocyanine. In this newly constructed modified electrode (GCE/PANI-N3/TA-CoPc (CEC)), a terminal-alkynyl (TA) substituted cobalt phthalocyanine (CoPc-TA) was electrochemically bonded to the azido end of electro-polymerized 4-azidoaniline (PANI-N3), which was electropolymerized on the GCE electrochemically previously. GCE/PANI-N3/TA-CoPc (CEC) electrode was characterized with voltammetric, electrochemical impedance spectroscopy (EIS), UV–vis spectroscopy, and scanning electron microscopy (SEM) techniques. GCE/PANI-N3/TA-CoPc electrode gave three redox couples assigned to the CoPc and PANI in nonaqueous and aqueous electrolyte systems, which is a desired factor for the application of a modified electrode in various electrochemical applications such as electrocatalytic and electrosensing fields. Pesticide sensing responses of GCE/PANI-N3/TA-CoPc (CEC) electrode were tested with SWV method for usability as an electrochemical pesticide sensor for eserine. Basic sensor parameters, calibration lines, and detection limits (LOD) of GCE/PANI-N3/TA-CoPc (CEC) electrode were derived from SWV responses of the electrode. Amperometric sensing analyses of SVW responses of the electrode give a linear range between 1.56×10−7 and 2.45×10−6moldm−3 eserine with a sensitivity of 1.15Adm3mol−3 and LOD of 1.75×10−7moldm−3.
The anterolateral cell group of the bed nucleus of the stria terminalis (BNSTALG) serves as an important relay station in stress circuitry. Limbic inputs to the BNSTALG are primarily glutamatergic ...and activity-dependent changes in this input have been implicated in abnormal behaviors associated with chronic stress and addiction. Significantly, local infusion of acetylcholine (ACh) receptor agonists into the BNST trigger stress-like cardiovascular responses, however, little is known about the effects of these agents on glutamatergic transmission in the BNSTALG. Here, we show that glutamate- and ACh-containing fibers are found in close association in the BNSTALG. Moreover, in the presence of the acetylcholinesterase inhibitor, eserine, endogenous ACh release evoked a long-lasting reduction of the amplitude of stimulus-evoked EPSCs. This effect was mimicked by exogenous application of the ACh analog, carbachol, which caused a reversible, dose-dependent, reduction of the evoked EPSC amplitude, and an increase in both the paired-pulse ratio and coefficient of variation, suggesting a presynaptic site of action. Uncoupling of postsynaptic G-proteins with intracellular GDP-β-S, or application of the nicotinic receptor antagonist, tubocurarine, failed to block the carbachol effect. In contrast, the carbachol effect was blocked by prior application of atropine or M2 receptor-preferring antagonists, and was absent in M2/M4 receptor knockout mice, suggesting that presynaptic M2 receptors mediate the effect of ACh. Immunoelectron microscopy studies further revealed the presence of M2 receptors on axon terminals that formed asymmetric synapses with BNST neurons. Our findings suggest that presynaptic M2 receptors might be an important modulator of the stress circuit and hence a novel target for drug development.
► Endogenous ACh suppresses glutamate release in the BNSTALG. ► Effects are mimicked by muscarinic receptor agonists. ► Effects are mediated by presynaptic M2 receptors. ► EM studies confirm presynaptic M2 receptor expression in the BNSTALG.
A highly sensitive acetylcholinesterase biosensor was developed for detection of carbamate drugs based on TTF–TCNQ–ionic liquid gel thiocholine sensor. The TTF–TCNQ–ionic/ionic liquid gel was ...characterized by FT-IR and scanning electron microscopy. The electrocatalytic behavior of TTF–TCNQ–ionic liquid gels toward oxidation of thiocholine was thoroughly investigated. 1-Ethyl-3-methylimidazolium tetracyanoborate gel based sensor allowed amperometric detection of thiocholine at +400mV vs. Ag/AgCl with a high sensitivity of 55.9±1.2μAmM−1cm−2 and a low detection limit equal to 7.6μM. The catalytic rate constant and diffusion constant of thiocholine were estimated from chronoamperometric data. The proposed biosensor based on AChE immobilized in sol–gel matrix was used for the detection of two carbamate therapeutic drugs. Very low detection limits of 26pM eserine and 0.3nM neostigmine were achieved. The analysis of spiked tap water proved the biosensor capability to be used as a screening method for detection of carbamate drugs in wastewaters.
► The first biosensing application of TTF–TCNQ–ionic liquid gel. ► Highly sensitive AChE biosensor. ► Very low detection limits for eserine and neostigimine drugs.
PDF