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  • A more refined picture of cholinesterase activity patterns m


    A more refined picture of cholinesterase activity patterns may be obtained by measuring ChE activity of the same sample before and after addition of certain cholinesterase inhibitors, with differential specificity for different components of total ChE activity, such as BW284c51 (BW1,5-bis-(4-allyldimethylammoniumphenyl) pentan-3-one dibromide = a specific AChE inhibitor), isotetramonoisopropyl pyrophosphortetramide (iso-OMPA = a specific BChE inhibitor in vertebrates) and eserine (physostigmine, or (1’methylpyrrolidino (2’:3’:2:3)1,3-dimethylindolin-5-yl N-methylcarbamate = a total cholinesterase inhibitor). Using this approach, a sub-fraction of cholinesterase activity may be identified that responds with greater sensitivity to a given contaminant, and several laboratory and field studies have described distinct ChE fractions with differential sensitivity to organophosphates and carbamates this way (Bocquené et al., 1997, Galloway et al., 2002, Monserrat et al., 2002, Sandahl and Jenkins, 2002, Nunes and Resende, 2017). Among these, Bocquené et al. (1997) electrophoretically and chromatographically separated two ChE fractions in Crassostrea gigas, one fraction (“A cholinesterase”) whose activity was strongly inhibited by general ChE inhibitors such as paraoxon and eserine, and a second (“B cholinesterase”) whose activity was insensitive to these compounds. Both enzyme forms showed highest activity when acetylthiocholine was presented as substrate (with Km ranging between of 10–100µM) and somewhat lower activity (81–93%) when presented with propionylthiocholine, whereas virtually no activity was detected for butyrylthiocholine as substrate (0–9%), consistent with no observable inhibition by the specific butyrylcholinesterase inhibitor iso-OMPA. Nevertheless, despite the temptation to define the two enzyme forms of C. gigas as functionally resembling AChEs, the authors chose to refer to them generically as ChEs, for lack of a detailed molecular characterization. Despite being sensitive to organophosphate and carbamates, the specificity of ChE or AChE activity inhibition as biomarkers of exposure to this group of Halopemide mg has become increasingly questionable, since cholinesterase activity may also be altered by exposure to other environmental toxicants, including metals or other types of biocides. For example, several studies have found that certain metals, including Cd and Cu, may cause changes in AChE and/or ChE activity in mollusks (Magni et al., 2006, Bonacci et al., 2006, Gold-Bouchot et al., 2007, Tsangaris et al., 2010, Choi et al., 2011, Cravo et al., 2012). Cd competes with essential metals in cellular metabolism, altering general patterns of protein expression (Meng et al., 2017) and cell function (Jarup and Akesson, 2009; Thévenod and Lee, 2013). Correspondingly, Bonacci et al. (2006) observed inhibition of ChE activity in the scallop Adamussium colbecki exposed to Cd concentrations greater than 10-6M (approx. 112µg/L), as did Najimi et al. (1997), who reported reductions of ChE activity in M. galloprovincialis and Perna perna exposed to CdCl2 at 10-2M. Similarly, Bocquené et al. (1997), found a reduction of ChE activity in crude extract of M. edulis exposed to Cd concentrations of 10-3M for 40min, and Cravo et al. (2012) reported low AChE activity in field-collected clams Ruditapes decussatus having elevated concentrations of tissue Cd (0.2–0.9µg/g dw) and Cu (4.6–37.2µg/g dw). Chronic exposure to Cu at concentrations as low as 6µg/L can be toxic (Brown et al., 2004; Gaetke and Chow, 2003; Al-Subiai et al., 2011), causing a variety of adverse effects, ranging from inhibition of feeding and burrowing behavior to reduced growth and reproduction and histopathological as well as biochemical anomalies (Bonnard et al., 2009). Aside from oxidative stress (Nicholson, 2003, Gaetke and Chow, 2003, Zorita et al., 2006; Fitzpatrick et al., 2008), reduction, increase as well as no effect on AChE activity have been reported in mollusks and crustaceans upon Cu exposure: Brown et al. (2004) observed reduction of AChE activity in the green shore crab Carcinus maenas upon Cu exposure at 68µg/L, in contrast to a significant increase of AChE activity in the limpet mollusk Patella vulgaris at Cu concentrations of 6µg/L and no effect on AChE activity in Mytilus edulis at Cu concentrations of 68µg/L. Other authors have suggested that Cu affects AChE activity via mechanisms other than those involving the organophosphate-sensitive active site (Viarengo, 1989, Najimi et al., 1997).