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    • This study was funded and

    2024-03-27

    This study was funded and supported by Tehran University of Medical Sciences and Health Services; and Iran National Science Foundation.
    Introduction The enzyme acetylcholinesterase (AChE) plays a central role in the signal transduction in the nervous system, hydrolyzing the neurotransmitter acetylcholine at cholinergic synapses and neuromuscular junctions, thus terminating the transmission of individual action potentials from pre- to postsynaptic neurons (Hrabovska and Krejci, 2014; Tripathi and Srivastava, 2008). AChE is located as a protein complex on the post-synaptic membranes and constituted of an AChE-homotetramer associated to the proline-rich membrane anchor (PRiMA) protein (Chen et al., 2011; Perrier et al., 2002). Apart from its role in the nervous system, non-neuronal AChE has been associated with a number of additional (“non-canonical”) biological processes such as apoptosis (Xie et al., 2011), cell cycle regulation (Pérez-Aguilar et al., 2015) or reproduction (Lu et al., 2012). The most commonly applied approach for assessment of AChE activity is the Ellman method (Ellman et al., 1961; Worek and Thiermann, 2012), which utilizes the substrate derivative acetylthiocholine, which is cleaved by the enzyme. In a second step, the thiol group reacts with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent), thus giving rise to products with a strong nefiracetam at 405 nm, which can readily be quantified photometrically. Since the amount of product is usually normalized to protein contents to yield the specific activity, an additional step for protein quantification is required for each enzyme preparation. AChE activity can be inhibited by several classes of chemical substances, most prominently by organophosphorus compounds and carbamate derivatives (Colovic et al., 2013; Thapa et al., 2017), which are applied in large quantities primarily as insecticides in agriculture (Lionetto et al., 2013). Homologous AChE enzymes originating from different organisms display varying susceptibilities towards certain inhibitors (Ehrich et al., 1995; Johnson and Wallace, 1987), a property which may even be harnessed for the development of more insect- or even species-specific inhibitors (Lang et al., 2012). The effects of many compounds are modulated by hepatic biotransformation processes, either generating their final AChE-inhibitory potential (Glickman et al., 1984) or allowing their detoxification (Moser and Padilla, 2011). Inhibition of AChE as a result from acute intoxication is associated with various symptoms in the respective organisms, ranging from disruption of motoric control by paralysis towards seizures to perturbation of other nerve-associated functions such as cardiac and respiratory depression, potentially resulting in death (King and Aaron, 2015; Pearson and Patel, 2016). Sub-lethal exposures have been associated with teratogenicity (Sotomayor et al., 2015), neurotoxicity (Branch and Jacqz, 1986), immunotoxicity (Xing et al., 2015) and behavioral alternations (Khalil et al., 2013), all of which are likely to compromise the ability of affected individuals to cope with daily environmental challenges. Inhibition of AChE activity is a parameter frequently assessed in environmental studies, both for water (Neale et al., 2017; Sumith et al., 2012) and sediment samples (Kais et al., 2015). AChE inhibition may be assessed either directly in individuals sampled in situ, in organisms exposed to the samples under laboratory conditions (both limited for example by the influence of compensation mechanisms, Falisse et al., 2017), or alternatively analyzing only the interaction of the sample with the enzyme in vitro. In the latter case, the enzyme is usually extracted from the tissue of animals (e.g.Sato et al., 2007; De Lima et al., 2013), similar to earlier studies of AChE from the electroplaxes of Torpedo californica (Reed et al., 1975), representing an frequently used, albeit time-consuming method for the generation of the enzyme. Alternatives for recombinant generation of AChE from other organisms have been developed (e.g.Gibney and Taylor, 1990; Kronman et al., 1992; Zhao et al., 2010); yet, tissue extraction has remained the main source for most direct AChE inhibition studies, requiring the processing of experimental organisms, associated with both ethical implications and significant methodological effort. More elaborate approaches are based on the in vivo exposure of whole organisms or embryos (Kais et al., 2015), which cover more complex biological aspects such as bioavailability, biotransformation and endogenous compensation mechanisms. In contrast, in vitro assays based on the simple reaction of enzyme preparations with test compounds are largely independent of interference with cellular biotransformation and other defense mechanisms, as they do not suffer from those complex organismic interactions.