Copper, aluminum, iron and calcium inhibit human acetylcholinesterase in vitro

doi:10.1016/j.etap.2014.01.001

Environmental Toxicology and Pharmacology

Volume 37, Issue 1, January 2014, Pages 455-459

https://doi.org/10.1016/j.etap.2014.01.001 Get rights and content

Highlights

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Copper, aluminum, iron and calcium have ability to inhibit acetylcholinesterase.
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No effect was caused by sodium, magnesium and potassium.
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Noncompetitive mechanism of inhibition was proved.
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Copper is the most potent inhibitor from tested metals.
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Inhibition constants K_i were calculated using Dixon plot.

Abstract

Acetylcholinesterase (AChE) is an important part of cholinergic nerves where it participates in termination of neurotransmission. AChE can be inhibited by e.g. some Alzheimer disease drugs, nerve agents, and secondary metabolites. In this work, metal salts aluminum chloride, calcium chloride, cupric chloride, ferric chloride, potassium chloride, magnesium chloride and sodium chloride were tested for their ability to inhibit AChE. Standard Ellman assay based on human recombinant AChE was done and inhibition was measured using Dixon plot. No inhibition was proved for sodium, potassium and magnesium ions. However, aluminum, cupric, ferric and calcium ions were able to inhibit AChE via noncompetitive mechanism of inhibition. Though the inhibition is much weaker when compared to e.g. drugs with noncompetitive mechanism of action, biological relevance of the findings can be anticipated.

Introduction

Currently, two enzymes hydrolyzing esters of choline are known. Acetylcholinesterase (AChE; EC 3.1.1.7.) is an enzyme hydrolyzing neurotransmitter acetylcholine and it can be mentioned as the first. Butyrylcholinesterase (BChE; EC 3.1.1.8.) is the second enzyme able to hydrolyze choline esters (Pohanka, 2011, Pohanka, 2012b, Pohanka, 2012c). AChE plays an important role in cholinergic neurotransmission where it terminates stimulation in neurosynaptic cleft by hydrolyzing of acetylcholine (Wessler and Kirkpatrick, 2008). BChE is abundantly presented in plasma and tissues; however, its biological role is not well understood (Masson et al., 2009). The most significant effect of BChE in the body is detoxification of some drugs including procaine and muscle relaxant succinylcholine (Yuan et al., 2007).

The both cholinesterases have large structural similarities. They are a type α/β hydrolase folded and the both contains Ser-His-Glu in its active site (Cygler et al., 1993). The active site is localized in a cavity with peripheral (or β) anionic site in its entrance followed by aromatic gorge. As the last part, α anionic site responsible for proper orientation of substrate toward the catalytic triade can be mentioned (Macdonald et al., 2012).

Cholinesterases can be inhibited by many compounds including drugs, natural toxins, military used nerve agents and others. Inhibitors binding into active site of cholinesterases have typically equal affinity toward AChE and BChE. Nerve agents sarin, VX and some pesticides such as carbofuran can be exampled (Knaack et al., 2012, Pohanka, 2011). Different situation can be learned for inhibitors binding into aromatic gorge or peripheral anionic site which are more extensively developed in AChE than in BChE (Macdonald et al., 2012).

Cholinergic system and activity of AChE can be influenced by heavy metals as proved using some in vivo models (Gioda et al., 2013, Saidi and Shojaie, 2012). The effect is not; however, well understood. In this work, an attempt to identify one of the possible molecular mechanisms where heavy metals are involved in cholinergic system was made. In order to identify molecular mechanism, affinities of three biologically and toxicologically relevant metals: calcium, copper, iron, sodium, magnesium and aluminum to AChE were measured. The chosen metals are quite common and they are occurring as either biogenic elements or elements with high occurrence in environment. Salts of all these elements are soluble in water so the assay is well reproducible and is not sensitive to a methodical error. Beside this, the good solubility in water is an important attribute that the compound will be easily distributed in the body which is necessary for a biological impact related to cholinesterases. Possibility that the metals act as inhibitors is hypothesized prior to the experiment.

Section snippets

Reagents

In the experiment, human recombinant AChE (expressed in HEK 293 cells, lyophilized powder ≥1500 U/mg of protein) was purchased from Sigma-Aldrich (Saint Louis, Missouri, USA). 5,5′-Dithiobis-(2-nitrobenzoic) acid and acetylthiocholine chloride were received from Sigma-Aldrich as a chromogen and substrate in purity at least 98%. The enzymes, thiocholine esters and 5,5′-dithiobis-(2-nitrobenzoic) acid were dissolved in phosphate buffered saline pH 7.4 with composition 137 mmol/l NaCl, 2.7 mmol/l

Results and discussion

Sodium chloride, magnesium chloride and potassium chloride caused no inhibition of AChE. The activity measured for control assay was not differing (tested by ANOVA on the both probability levels) to activity of AChE in presence of the upper concentration of sodium, potassium or magnesium. The finding is not surprising. In the body, the metals are dissolved and presented in bulky concentration in free, solved ionic form (Aramli et al., 2013, Asadi et al., 2010, McCallum et al., 2013). For the

Conclusions

In the present paper, inhibition of human AChE was proved. Though the found inhibition is weaker than typical for e.g. Alzheimer disease drugs, the inhibiting metals are potent enough to play a role in regulation cholinergic nerves. The inhibition caused by metals probably will not have any significance for pharmacological application. However, biological significance deserves further attention. The findings can be a reason of processes related to disparate neurodegenerations. Interaction of

Conflict of interest statement

None.

Acknowledgments

The Ministry of Education, Youth and Sports of the Czech Republic is gratefully acknowledged for project LH11023. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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View full text

Copper, aluminum, iron and calcium inhibit human acetylcholinesterase in vitro

Highlights

Abstract

Introduction

Section snippets

Reagents

Results and discussion

Conclusions

Conflict of interest statement

Acknowledgments

Am. J. Clin. Nutr.

Anal. Biochem.

Neurotoxicology

Pharmacol. Ther.

J Chromatogr. A

Study of sperm concentration, seminal plasma composition and their physiological correlation in the Persian sturgeon, Acipenser persicus

Reprod. Domest. Anim.

Serum electrolyte and nonelectrolyte status in freshwater juvenile Persian sturgeon Acipenser persicus

J. Aquat. Anim. Health

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Curr. Med. Chem.

A simple graphical method for determinating the inhibition constants of mixed, uncompetitive and non-competitive inhibitors

Biochem. J.

Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins

Protein Sci.

The determination of enzyme inhibitor constants

Biochem. J.

High doses of zinc and copper alter neither cerebral metal levels nor acetylcholinesterase activity of suckling rats

Exp. Clin. J.

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Sublethal zinc and copper exposure affect acetylcholinesterase activity and accumulation in different tissues of Leporinus obtusidens

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