Effects of pyriproxyfen on zebrafish brain mitochondria and acetylcholinesterase.

Pyriproxyfen is an insecticide used worldwide that acts as a biomimetic of juvenile hormone. This study investigated metabolic and synaptic impairments triggered by pyriproxyfen using zebrafish acetylcholinesterase (zbAChE) and mitochondria as markers. A brain zbAChE assay was performed in vitro and in vivo covering a range of pyriproxyfen concentrations (0.001-10 µmol/L) to assess inhibition kinetics. Docking simulations were performed to characterize inhibitory interactions. Zebrafish male adults were acutely exposed to 0.001, 0.01 and 0.1 µg/mL pyriproxyfen for 16 h. Mitochondrial respiration of brain tissues was assessed. ROS generation was estimated using H2DCF-DA and MitoSOX. Calcium transport was monitored by Calcium Green™ 5 N. NO synthesis activity was estimated using DAF-FM-DA. Brain acetylcholinesterase showed an in vivo IC20 of 0.30 µmol/L pyriproxyfen, and an IC50 of 92.5 µmol/L. The inhibitory effect on zbAChE activity was competitive-like. Respiratory control of Complex I/II decreased significantly after insecticide exposure. The MitoSOX test showed that O2- generation had a pyriproxyfen dose-dependent effect. Brain tissue lost 50% of Ca2+ uptake capacity at 0.1 µg/mL pyriproxyfen. Ca2+ release showed a clear mitochondrial impairment at lower pyriproxyfen exposures. Thus, Ca2+ transport imbalance caused by pyriproxyfen may be a novel deleterious mechanism of action. Overall, the results showed that pyriproxyfen can compromise multiple and interconnected pathways: (1) zbAChE impairment and (2) the functioning of the electron transport chain, ROS generation and calcium homeostasis in zebrafish brain mitochondria. Considering the many similarities between zebrafish and human, more caution is needed when pyriproxyfen is used in both urban and agricultural pest control.

Impact on cholinesterase-inhibition and in silico investigations of sesquiterpenoids from Amazonian Siparuna guianensis Aubl.

The plant popularly known as "negramina" (Siparuna guianensis Aubl.), member of the family Siparunaceae produces an essential oil that presents several biological activities reported in literature. Here, the essential oil was obtained by hydrodistillation from fresh leaves collected in the state of Roraima, far north of the Amazon. Chemical composition of the essential oil was characterized by gas chromatography coupled to mass spectrometry (GC-MS) and flame ionization detector (GC-FID). The sesquiterpenoid shyobunone and its derivatives were identified as major compounds in the oil (>40%). The effect of S. guianensis essential oil on the acetylcholinesterase (AChE) activity from Crassostrea rhizophorae, Litopenaeus vannamei and Electrophorus electricus was tested by spectrophotometric assays. The essential oil has been identified as an AChE inhibitor. The mechanism of inhibition was investigated as well as spectrofluorimetric interactions between the essential oil and the enzyme. 1H NMR titration and molecular docking were also investigated. The spectrophotometric results revealed that shyobunone and its derivatives strongly interact with AChE with a kind of non-competitive inhibition. Interaction studies support the results of enzyme inhibition. Molecular coupling predicted that iso-shyobunone is the strongest ligand, corroborated by fluorescence suppression and 1H NMR titration results. In conclusion, Siparuna guianensis essential oil can be a new source of shyobunone and derivatives capable to reversibly inhibit AChE showing potential neuroprotective properties to be applied in the treatment of Alzheimer's disease.

Erythrocyte acetylcholinesterase as biomarker of pesticide exposure: new and forgotten insights.

Acetylcholinesterase (AChE) acts on the hydrolysis of acetylcholine, rapidly removing this neurotransmitter at cholinergic synapses and neuromuscular junctions as well as in neuronal growth and differentiation, modulation of cell adhesion ("electrotactins") and aryl-acylamidase activity (AAA). This enzyme is also found in erythrocyte, as 160 kDa dimer that anchors to the plasma membrane via glycophosphatidylinositol. The function of this enzyme in erythrocytes has not yet been elucidated; however, it is suspected to participate in cell-to-cell interactions. Here, a review on erythrocyte AChE characteristics and use as biomarker for organophosphorus and carbamate insecticides is presented since it is the first specific target/barrier of the action of these pesticides, besides plasma butyrylcholinesterase (BChE). However, some past and current methods have disadvantages: (a) not discriminating the activities of AChE and BChE; (b) low accuracy due to interference of hemoglobin in whole blood samples. On the other hand, extraction methods of hemoglobin-free erythrocyte AChE allows: (a) the freezing and transporting of samples; (b) samples free of colorimetric interference; (c) data from only erythrocyte AChE activity; (d) erythrocyte AChE specific activity presents higher correlation with the central nervous system AChE than other peripheral ChEs; (e) slow spontaneous regeneration against anti-ChEs agents of AChE in comparison to BChE, thus increasing the chances of detecting such compounds following longer interval after exposure. As monitoring perspectives, hemoglobin-free methodologies may be promising alternatives to assess the degree of exposure since they are not influenced by this interfering agent.

High sucrolytic activity by invertase immobilized onto magnetic diatomaceous earth nanoparticles.

Invertase immobilized on magnetic diatomaceous earth nanoparticles (mDE-APTES-invertase) with high sucrolytic activity was obtained by an easy and low-cost method. An experimental design was carried out to investigate the best immobilization conditions and it allowed obtaining an immobilized derivative with a residual specific activity equal to 92.5%. Then, a second experimental design selected the mDE-APTES-invertase with higher specific activity in relation to other derivatives reported in the literature (2.42-fold). Thermal and storage stability for immobilized invertase were found to be 35 °C for 60 min (85% retained activity) and 120 days storage period (80% retained activity), respectively. Besides, a residual activity higher than 60% and 50% were observed for mDE-APTES-invertase after reuse in short and long term, respectively. Given the simple and efficient method to obtain an immobilized derivative with high activity, the mDE nanoparticles appear to be a promising matrix for invertase immobilization as well as for other biomolecules.