Metabolical effects of Folidol 600 on the neotropical freshwater fish matrinxã, Brycon cephalus

The neotropical freshwater fish matrinx?, Brycon cephalus (Günther, 1869), was exposed to 1/3 of 96 h-LC50 of Folidol 600 (methyl parathion) for 96 h and allowed to recover for 24, 48, 96, and 192 h. Acetylcholinesterase (AChE), alanine aminotransferase (ALAT), aspartate aminotransferase (AAT), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and glutamate dehydrogenase (GDH) were assayed in brain, liver, muscle, and plasma. Plasma and brain AChE activities were strongly inhibited (64% and 87%, respectively), remaining low in the brain during recovery. Liver ALAT decreased 59.4% and plasma ALAT increased 94.2%. This response, associated with GDH reduction during the recovery period, was attributed to impairment of amino acid metabolism and to liver damage. The increase of heart and plasma AAT suggested tissue injury. Liver and plasma LDH and MDH did not change during the exposure to Folidol, but liver MDH decreased 34% during recovery, indicating a failure of oxidative metabolism in such tissue. Hepatic glycogen and glucose levels decreased 80.4 and 55%, respectively, followed by glucose mobilization to the plasma. The hepatic and muscular profile of lactate after recovery suggested gluconeogenesis without tissue hypoxia. These results revealed that methyl parathion affects the intermediary metabolism of matrinx? and that the assayed enzymes can work as good biomarkers of organophosphorus pesticide contamination.     

Maternal exposure to nicotine and chlorpyrifos,alone and in combination,leads to persistently elevated expression of glial fibrillary acidic protein in the cerebellum of the offspring in late puberty

We previously showed that maternal exposure to nicotine, alone or in combination with chlorpyrifos, caused an increase in glial fibrillary acidic protein (GFAP) immunostaining in the CA1 subfield of hippocampus and cerebellum in postnatal day (PND) 30 offspring. In the present study, PND 60 offspring were evaluated for histopathological and cholinergic effects following maternal exposure to nicotine and chlorpyrifos, alone and in combination. Timed-pregnant Sprague-Dawley rats (300–350 g) were treated daily with nicotine (1 mg/kg, s.c., in normal saline) or chlorpyrifos (0.1 mg/kg, dermal, in ethanol) or a combination of nicotine and chlorpyrifos from gestational days (GD) 4 to 20. Control animals were treated with saline and ethanol. On PND 60, the offspring were evaluated for cholinergic changes and pathological effects. Plasma butyrylcholinesterase (BChE) activity in the female offspring from chlorpyrifos treated mothers showed a significant increase (~183% of control). Male offspring from mothers treated with either chlorpyrifos or nicotine alone showed a significant increase in the acetylcholinesterase (AChE) activity in the brainstem while female offspring from mothers treated with either nicotine or a combination of nicotine and chlorpyrifos showed a significant increase (~134 and 126% of control, respectively) in AChE activity in the brainstem. No significant changes were observed in the ligand binding densities for 42 and 7 nicotinic acetylcholine receptors in the cortex. Histopathological evaluation using cresyl violet staining showed a significant decrease in surviving Purkinje neurons in the cerebellum of the offspring from nicotine treated mothers. An increase in GFAP immunostaining in cerebellar white matter was observed in the offspring from the mothers treated with nicotine. These results suggest that maternal exposure to real-life levels of nicotine and/or chlorpyrifos causes differential regulation of brainstem AChE activity. Also, nicotine caused a decrease in the surviving neurons and an increased expression of GFAP in cerebellar white matter of the offspring on PND 60. These changes can lead to long-term neurological adverse health effects later in life.     

Design,synthesis and biological evaluation of ambenonium derivatives as AChE inhibitors

Ambenonium (1), an old AChE inhibitor, is endowed with an outstanding affinity and a peculiar mechanism of action that, taken together, make it a very promising pharmacological tool for the treatment of Alzheimer's disease (AD). Unfortunately, the bisquaternary structure of 1 prevents its passage through the blood brain barrier. In a search of centrally active ambenonium derivatives, we planned to synthesize tertiary amines of 1, such as 2 and 3. In addition, to add new insights into the binding mechanism of the inhibitor, we designed constrained analogues of ambenonium by incorporating the diamine functions into cyclic moieties (4-12). The biological evaluation of the new compounds has been assessed in vitro against human AChE and BChE. All tertiary amine derivatives resulted more than 1000-fold less potent than 1 and, unlike prototype, did not show any selectivity between the two enzymes. This result, because of recent findings concerning the role of BChE in AD, makes our compounds, endowed with a well-balanced profile of AChE/BChE inhibition, valuable candidates for further development. To better clarify the interactions that account for the high affinity of 1, docking simulations and molecular dynamics studies on the AChE-1 complex were also carried out.     

Developmental exposure to chlorpyrifos alters reactivity to environmental and social cues in adolescent mice

Neonatal mice were treated daily on postnatal days (pnds) 1 through 4 or 11 through 14 with the organophosphate pesticide chlorpyrifos (CPF), at doses (1 or 3 mg/kg) that do not evoke systemic toxicity. Brain acetylcholinesterase (AChE) activity was evaluated within 24 h from termination of treatments. Pups treated on pnds 1-4 underwent ultrasonic vocalization tests (pnds 5, 8, and 11) and a homing test (orientation to home nest material, pnd 10). Pups in both treatment schedules were then assessed for locomotor activity (pnd 25), novelty-seeking response (pnd 35), social interactions with an unfamiliar conspecific (pnd 45), and passive avoidance learning (pnd 60). AChE activity was reduced by 25% after CPF 1-4 but not after CPF 11-14 treatment. CPF selectively affected only the G(4) (tetramer) molecular isoform of AChE. Behavioral analysis showed that early CPF treatment failed to affect neonatal behaviors. Locomotor activity on pnd 25 was increased in 11-14 CPF-treated mice at both doses, and CPF-treated animals in both treatment schedules were more active when exposed to environmental novelty in the novelty-seeking test. All CPF-treated mice displayed more agonistic responses, and such effect was more marked in male mice exposed to the low CPF dose on pnds 11-14. Passive avoidance learning was not affected by CPF. These data indicate that developmental exposure to CPF induces long-term behavioral alterations in the mouse species and support the involvement of neural systems in addition to the cholinergic system in the delayed behavioral toxicity of CPF.     

Acetylcholinesterase induces the expression of the beta-amyloid precursor protein in glia and activates glial cells in culture

Acetylcholinesterase (AChE) activities in CNS physiopathology are increasingly diverse and range from neuritogenesis, through synaptogenesis, to enhancement of amyloid fiber assembly. In Alzheimer's disease, senile plaques and neurodegeneration specially affect regions enriched for cholinergic synapses. In this study we show an effect of AChE that could contribute to the increased deposition of Abeta in certain regions. Affinity-purified AChE induced the expression of amyloid-beta-precursor protein (beta-APP) in glial cells in a concentration-dependent manner up to 5 nM. In glia, AChE also increased inducible nitric oxide synthase (iNOS) assessed by immunocytochemistry and decreased reductive metabolism as evidence of cell activation. AChE could increase the expression of beta-APP in astrocytes and microglia as result of the activation of glial cells. As a whole, we found that AChE has additional effects that could result in an increased synthesis of Abeta, both by increasing beta-APP expression of astrocytes and by further activating glial cells.     

Distribution of choline acetyltransferase and acetylcholinesterase in the vocal motor system of zebra finches

The distribution of choline acetyltransferase immunoreactivity (ChAT-IR) was surveyed in the vocal motor system of adult male and female zebra finches and was compared with the pattern of histochemical acetylcholinesterase (AChE-His). In the vocal motor system the most prominent accumulation of ChAT-IR somata was found in lobus parolfactorius (LPO) including Area X. Immunoreactive neuropil was found to be concentrated in pericellular networks of fibers in male's Area X while the corresponding area in females could not be demarcated within the LPO. The density of ChAT-IR fiber networks was much higher in LPO, paleostriatum augmentatum and in a shelf region around nucleus robustus archistriatalis (RA) than in neostriatal and hyperstriatal parts of the telencephalon. AChE positive neurons and neuropil were observed in all ChAT-IR regions and, in addition, in the vocal motor nuclei nucleus hyperstriatum ventrale pars caudalis (HVc), nucleus magnocellularis in the anterior neostriatum (MAN), nucleus interfacialis (NIF) and RA. However, none of the latter nuclei contained ChAT-IR cell bodies. They were characterized by rare ChAT-IR neuropil. MAN and RA exhibited shelf regions with a higher degree of stained fibers. The discrepancy between the localization of AChE-His and ChAT-IR can hardly be explained by different classes of ChAT isoenzymes in neurons within the basal forebrain and the neostriatal, hyperstriatal and archistriatal vocal control nuclei not detected by our antibody. On the other hand, vocal control centers while receiving cholinergic inputs, might - except for Area X - not possess cholinergic efferent projections within the telencephalon.     

Inhibition of acetylcholinesterase by two genistein derivatives: kinetic analysis,molecular docking and molecular dynamics simulation

In this study two genistein derivatives (G1 and G2) are reported as inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and differences in the inhibition of AChE are described. Although they differ in structure by a single methyl group, the inhibitory effect of G1 (IC₅₀=264 nmol/L) on AChE was 80 times stronger than that of G2 (IC₅₀=21,210 nmol/L). Enzyme-kinetic analysis, molecular docking and molecular dynamics (MD) simulations were conducted to better understand the molecular basis for this difference. The results obtained by kinetic analysis demonstrated that G1 can interact with both the catalytic active site and peripheral anionic site of AChE. The predicted binding free energies of two complexes calculated by the molecular mechanics/generalized born surface area (MM/GBSA) method were consistent with the experimental data. The analysis of the individual energy terms suggested that a difference between the net electrostatic contributions (ΔE_ele+ΔG_GB) was responsible for the binding affinities of these two inhibitors. Additionally, analysis of the molecular mechanics and MM/GBSA free energy decomposition revealed that the difference between G1 and G2 originated from interactions with Tyr124, Glu292, Val294 and Phe338 of AChE. In conclusion, the results reveal significant differences at the molecular level in the mechanism of inhibition of AChE by these structurally related compounds.KEY WORDS: Genistein derivatives, Acetylcholinesterase (AChE), Kinetics analysis, Molecular docking, Molecular dynamics simulation, MM/GBSAAbbreviations: ACh, acetylcholine; AChEIs, acetylcholinesterase inhibitors; AChE, acetylcholinesterase; AD, Alzheimer׳s disease; BuChE, butyrylcholinesterase; BuSCh, S-butyrylthiocholine chloride; CAS, catalytic active site; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); GAFF, generalized AMBER force field; G1, 3-(4-methoxyphenyl)-7-(2-(piperidin-1-yl)ethoxy)-4H-chromen-4-one; G2, (S)-3-(4-methoxyphenyl)-7-(2-(2-methylpiperidin-1-yl)ethoxy)-4H-chromen-4-one; iso-OMPA, tetraisopropyl pyrophosphoramide; MD, molecular dynamics; MM/GBSA, molecular mechanics/generalized born surface area; PAS, peripheral anionic site; PDB, protein data bank; PME, particle mesh Ewald; RMSD, root-mean-square deviation; S-ACh, acetylthiocholine iodide; ΔE_ele, electrostatic energy contribution; ΔE_MM, gas-phase interaction energy between receptor and ligand; ΔE_vdw, van der Waals energy contribution; SASA, solvent accessible surface area; ΔG_exp, experimental binding free energy; ΔG_GB, polar desolvation energy term; ΔG_pred, total binding free energy; ΔG_SA, nonpolar desolvation energy term; ΔS, conformational entropy contribution     

Ameliorating effects of constituents from Cortex Acanthopanacis Radicis on memory impairment in mice induced by scopolamine

OBJECTIVE：To investigate the effects of active con- stituents extracted from Cortex Acanthopanacis Radicison improving the impaired memory in mice models. METHODS： The mice models of memory impair- ment were established using scopolamine. Amelio- rating effects of the fractions and constituents on scopolamine-induced memory impairment in vivo were investigated using passive avoidance and Morris water-maze task tests, and their anti-acetyl- cholinesterase（AChE） and antioxidant activities in vitro examined. The isolation of constituents was performed by chromatographic methods and their structures were identified on the basis of instru- mental analysis. RESULTS： Among the fractions tested, ethylacetate fraction exhibited the anti-AChE activity（25.83%± 0.23%） properly and excellent 2,2-diphenyl-1-picryl- hydrazyl（DPPH） radical and superoxide anion scav- enging capacity（87.50% ± 0.83% and 60.22% ± 0.43%, respectively）. However, the methylene chlo- ride fraction was much more active than the ethyl- acetate fraction in the passive avoidance task test（167.5% increase of step-through latency time） and Morris water-maze task test（33.3% decrease of es-cape latency time）. Four constituents, β-sitosterol, stigmasterol, sesamin, and hyperin were isolated from the methylene chloride fraction, among them, hyperin showed anti-acetylcholinesterase and anti-oxidant activities remarkably. Moreover, hyperin exerted a potent effect（146±38） s on mem- ory improvement in terms of passive avoidance task test compared with the reference compound tacrine（162±43） s at a dose of 2.5 mg/kg. CONCLUSION： Hyperin, a flavonoid glucoside iso- lated from Cortex Acanthopanacis Radicis, inhibited AChE activity and potently ameliorated scopol- amine-induced memory impairment, and its action may be partially mediated by the acetylcholine-en- hancing cholinergic nervous system.     

Hesperidin,a citrus flavonoid,protects against l-methionine-induced hyperhomocysteinemia by abrogation of oxidative stress,endothelial dysfunction and neurotoxicity in Wistar rats

Context: Hesperidin (HSP), a flavanoglycone found in citrus fruits, has antioxidant, anti-inflammatory and neuroprotective properties.

Objective: This study evaluates the protective effect of HSP on l-methionine-induced hyperhomocysteinemia (HHcy) in rats.

Materials and methods: Male Wistar rats were randomly divided into seven groups as DMSO, l-methionine, HSP (25, 50 and 100?mg/kg), HSP-per se (100?mg/kg) and donepezil (0.1?mg/kg). HHcy was induced by oral administration of l-methionine (1.7?g/kg) for 32 days. From the 14^th day of study HSP (25, 50 and 100?mg/kg) and donepezil was administered orally to l-methionine-treated rats. Cognitive impairment induced by HHcy was determined using the Morris water maze (MWM) and Y-maze on video tracking system (28^th–32^nd day). Different biomarkers of HHcy in serum and brain and vascular reactivity were evaluated and histopathology (thoracic aorta and brain) was done.

Results: HSP (100?mg/kg) treatment in l-methionine-treated rats exhibited significant (p?p?l-methionine on acetylcholine-induced endothelial-dependent relaxation and increased serum nitrite and vascular nitric oxide bioavailability along with the restoration of histological aberrations.

Conclusion: HSP exerts a protective effect on HHcy by abrogating oxidative stress, ED and neurotoxicity.     

巴豆醛致雄性大鼠神经毒性作用的研究

目的探讨巴豆醛暴露致雄性大鼠神经毒性作用,分析其可能的作用机制。方法于2019年7至10月,将24只SPF级雄性Wistar大鼠随机分为对照组和2.5、4.5、8.5 mg/kg染毒组,每组6只,分别经口给予0.0、2.5、4.5、8.5 mg/kg体重巴豆醛溶液,每周5次,连续染毒90 d。染毒结束后,测量大鼠体重,麻醉解剖取大鼠大脑组织和肝组织。测定大脑组织乙酰胆碱酯酶(AChE)活力及肝组织乙酰胆碱(ACh)水平;检测大脑组织中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活力以及丙二醛(MDA)和还原型谷胱甘肽(GSH)水平;酶联免疫吸附测定(ELISA)法检测大脑组织中白细胞介素6(IL-6)、白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)水平。结果与对照组比较,2.5、4.5、8.5 mg/kg染毒组大鼠大脑组织中AChE活力明显降低,8.5 mg/kg染毒组大鼠肝组织中ACh水平明显升高,差异均有统计学意义(P<0.05);与对照组比较,4.5、8.5 mg/kg染毒组大鼠大脑组织中MDA水平明显升高,GSH水平和SOD、GSH-Px活力明显降低,差异均有统计学意义(P<0.05);与对照组比较,2.5、4.5、8.5 mg/kg染毒组大鼠大脑组织中TNF-α、IL-6水平明显升高,4.5、8.5 mg/kg染毒组IL-1β水平明显升高,差异均有统计学意义(P<0.05)。结论巴豆醛暴露可致大鼠神经系统损伤,可能与氧化平衡状态改变及上调大脑组织炎性因子表达等作用有关。