br Introduction To detect exposure

Introduction To detect exposure to and effects of neuroactive pesticides in aquatic organisms, one widely used biomarker is cholinesterase (ChE) activity, which, among other possible functions, catalyzes the hydrolysis of the neurotransmitter 98014 (ACh) in the cholinergic synapses of the central and peripheral nervous system of animals and occurs in many molecular forms (Talesa et al., 2002, Sturm et al., 2007). ChE activity has become a popular biomarker of exposure to organophosphates and carbamates, since these insecticides tend to inhibit the functionality of cholinesterase enzymes in neuronal synapses (Walker et al., 2012; Monserrat et al., 2002; Bernal-Hernández et al., 2010), leading to the accumulation of ACh in post-synaptic membrane receptors, causing hyperpolarization of the membrane due to the continued permeability of gated Na-channels and continued stimulation of the post-synaptic membrane, resulting in cholinergic toxicity and tetanized state (Bonacci et al., 2006). There is increasing evidence that ChE is actually an assortment of various enzymes with similar function. To date, three different types of cholinesterases have been described in mollusks: acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and propionylcholinesterase (PChE, Bocquené et al., 1997; Talesa et al., 2001; Nunes and Resende, 2017). The proportion of the different enzyme forms varies among species and tissues: for example, in the mangrove oyster Crassostrea rhizophorae, as well as in the Manila clam Ruditapes phillippinarum, AChE is the prevalent cholinesterase in gills (Alves et al., 2002) and whole tissue (De Marchi et al., 2017), respectively, whereas for the mussel Perna perna, it is BChE, and for Corbicula fluminea, Ostrea edulis and Solen marginatus, it is PChE (Mora et al., 1999, Valbonesi et al., 2003, Nunes and Resende, 2017). In practice, however, AChE, PChE and BChE are commonly measured together as “total cholinesterase activity” (ChE), by quantifying the hydrolysis of a model substrate, such as acetylthiocholine iodide, using the procedure of Ellman et al. (1961). Due to the low substrate specificity of cholinesterases, a decrease in the hydrolytic activity of acetylthiocholine iodide (as the result of exposure to organophosphates, for example) may, thus, indicate the reduction of either AChE, BChE or PChE activity, and not just simply AChE alone–even though it might be the component with the fastest kinetics–complicating interpretation of patterns. No published information currently exists on the expression levels of different cholinesterases in Saccostrea sp. in response to xenobiotics, nor on the dominant form of ChE. Over the last two decades, numerous studies have assessed the effect of carbamate and organophosphate pesticides by measuring inhibition of AChE activity, as measured by the Ellman et al. (1961) method or other components of ChE activity (Bocquené et al., 1997, Galloway et al., 2002, Rickwood and Galloway, 2004, Choi et al., 2011), using bivalve species such as Dreissena polymorpha (Binelli et al., 2006, Ricciardi et al., 2006), Perna perna (Alves et al., 2002), Mytilus galloprovincialis (Lionetto et al., 2003), Ruditapes phillippinarum (Choi et al., 2011), Adamussium colbecki (Bonacci et al., 2006) and Crassostrea rhizophorae (Alves et al., 2002, Valdez Domingos et al., 2007, Lee et al., 2002). Among these, Choi et al. (2011) observed inhibition of acetylthiocholine hydrolysis in the adductor muscle of Ruditapes phillippinarum by the organophosphates methidathion, chlorpyrifos and diazinon, reporting a significant reduction of ChE activity (35–67% compared to the control group). Doran et al. (2001) reported a significant reduction of “AChE activity” (as measured by the Ellman method) in the adductor muscle of the freshwater mussel Amblema plicata exposed to chlorpyrifos at concentrations as low as 0.1 and 2mg/L, and Matozzo et al. (2012) observed low AChE activities in gills of field-collected Ruditapes phillippinarum exposed to wastewater from agricultural activity containing organophosphate residues.