Evaluation of a Brain Acetylcholinesterase Extraction Method and Kinetic Constants after Methyl-Paraoxon Inhibition in Three Brazilian Fish Species

Abstract

Acetylcholinesterase (AChE) is an important enzyme in the control of the neuronal action potential and sensitive to organophosphate inhibition. Brain fish AChE is less sensitive to organophosphate inhibition than AChE from terrestrial animals, although this sensitivity is variable among species and has not yet been fully evaluated in fish species. In this setting, inhibition kinetic constants for progressive irreversible inhibition of brain acetylcholinesterase due to methyl-paraoxon exposure were determined in three fish species (Mugil liza, Genidens genidens and Lagocephalus laevigatus) and hen (Gallus domesticus). Enzyme extraction using a detergent was shown to be adequate, and samples presented activity inhibition in high substrate concentrations and suppression of inhibition by methyl-paraoxon in the presence of the substrate, similar to kinetic patterns from purified enzyme preparations. Catfish (G. genidens) AChE presented the highest sensitivity among the evaluated fish species (IC50 = 1031.20 nM ± 63.17) in comparison to M. liza and L. laevigatus (IC50: 2878.83 ± 421.94 and 2842.5 ± 144.63 nM respectively). The lower dissociation constant (Kd = 20.3 ± 2.95 μM) of catfish AChE showed greater enzyme affinity for methyl-paraoxon, explaining this species higher sensitivity to organophosphates. Hen AChE presented higher ki (900.57 ± 65.3 mM-1min-1) and, consequently, greater sensitivity to methyl-paraoxon, explained by a lower Kd (0.6 ± 0.13 μM). Furthermore, hen AChE did not differentiate between the propionylthiocholine and acetylthiocholine substrates, indicating easier access of methyl-paraoxon to the hen enzyme activity site. The results obtained herein indicate a suitable extraction of AChE and, despite different inhibition kinetic constants, demonstrate that fish AChE is less sensitive to methyl-paraoxon, probably due to reduced access to the catalytic center which provides greater enzyme substrate selectivity.

Acetylcholinesterase Activities in Adult Houseflies Musca Domestica L. of the Chlorfenapyr-Resistant Strain

“…Published studies show an increase in activity and a change in quality indicators of AChE in insects, resistant not only to OPs and carbamate classes [28] but also to pyrethroids [34,35]. The possible involvement of this enzyme in the detoxification of insecticides through hydrolysis [13] and sequestration [14,15] has been previously reported. This study focused on the activity and kinetic parameters of AChE in adults of M. domestica of Lab (chlorfenapyr-unselected) and ChlA (chlorfenapyr-selected) strains.…”

“…The resistance development to these compounds is often realized through the mechanism of decreasing the enzyme's sensitivity to them [12]. AChE can also contribute to the formation of insecticide resistance through detoxification, as evidenced by the ability of the enzyme with a high affinity for choline ethers to hydrolyze other ethers, including OPs [13]. In addition, it is assumed that solubilized AChE isoforms in insects can be involved in the sequestration of xenobiotics, including insecticides [14,15].…”

Development of an antibody technique for acetylcholinesterase expression detection in the gill of Nile tilapia (Oreochromis niloticus) as a glyphosate-based herbicide biomarker

“…AChE from fish brains was less sensitive to organophosphate inhibition than that from terrestrial animals. Although this sensitivity is known to vary among species, it has not been evaluated in fish [19] . Many forms of AChE molecules can anchor to the membrane, ensuring the immobilisation necessary for the enzyme activity found in synapses and the motor end-plate.…”

“…Many forms of AChE molecules can anchor to the membrane, ensuring the immobilisation necessary for the enzyme activity found in synapses and the motor end-plate. AChE purification is an essential step to prepare carefully; therefore, the non-specific binding of esterase activity should affect false-positive result analysis [19] . Modesto and Martinez [20] reported a significant decrease in AChE enzyme activity at 96 h post-exposure to Round up® in the fish brain ( Prochilodus lineatus ) and at 32 h post-exposure in fish muscle.…”

Impact of Insecticides at Sublethal Concentrations on the Enzyme Activities in Adult Musca domestica L.

“…The resistance development to these compounds is often realized through the mechanism of decreasing the enzyme’s sensitivity to them [ 24 , 25 ]. An assumption was made that solubilized AChE isoforms in insects can be involved in the sequestration of xenobiotics, including insecticides [ 26 , 27 ]. In addition to esterases, cytochrome P450 monooxygenase (EC 1.14.14.1, P450) and glutathione-S-transferase (EC 2.5.1.18, GST) contribute to protecting insects against insecticides [ 16 ].…”