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  • Our results for imidacloprid were unexpected considering

    2019-07-17

    Our results for imidacloprid were unexpected: considering that imidacloprid (Im) is a neonicotinoid insecticide with a different mode of action (agonist of the nicotinic CPTH2 hydrochloride to receptor) than OPs, we expected Es-ChE and T-ChE activity to be largely insensitive to changes in Im concentrations. However, significant reductions of both T-ChE and Es-ChE activity were observed in gills at the highest Im concentration (100mg/L), whereas, even more surprisingly, T-ChE and Es-ChE activities in digestive gland increased significantly at the highest Im concentration. Radwan and Mohamed (2013) also reported inhibition of AChE activity in ganglia tissue of the gastropod Helix aspersa, exposed to Im at concentrations ranging between 2.5–30mg/mL. In this context, it is interesting that exposure of Saccostrea sp. to Im (100mg/L) resulted in a reduction of not only Es-ChE activity, but of Er-ChE activity as well (corresponding to the light grey bar in Fig. 4), explaining the marked 3-fold decrease of T-ChE activity with respect to controls and low Im concentrations. Whereas measurement artefacts cannot be discounted, the low variation of absolute Es-ChE and T-ChE activity in the three replicate kinetic measurements suggests that the observed enzyme responses for Im are robust. This could indicate an indirect or non-specific impact by Im on protein expression in Saccostrea sp: given that Im does not interfere directly with AChE catalytic functionality (Radwan and Mohamed, 2013), reductions in T-ChE, Es-ChE and Er-ChE activity could, perhaps, be due to a reduction of bulk protein expression or protein residence time (i.e. protein turnover), in line with Mukadam and Kulkarni (2014) who observed an overall reduction of tissue protein concentrations. However, in our study, this effect was only observed at the highest exposure concentration (100mg/L). Accordingly, Malev et al. (2012) were unable to observe significant changes of AChE activity in the amphipod Gammarus fossarum at Im exposure concentrations ranging from 6.3 and 511.3µ/L, which are much lower than those used in the present study (10–100,000µg/L). On the other hand, Dondero et al. (2010) reported significant reduction of AChE activity in the mussel Mytilus galloprovincialis at Im concentrations of only 0.1 and 1mg/L, even though it is unclear whether total ChE was similarly reduced. Our observation that Es-ChE activity CPTH2 hydrochloride to in Saccostrea sp. decreased upon Im exposure, yet in synchrony with T-ChE activity (resulting in little change of relative Es-ChE activity), indicates that reductions of T-ChE and Es-ChE activity are not sufficiently specific biomarkers of exposure to “classic” AChE inhibitors (e.g. organophosphates), since reductions of Es-ChE could—in principle—also result from altered protein concentrations or protein turnover rates caused by other agents. Our suspicion of potential changes in Es-ChE protein cycling is further fueled by the finding that Im exposure (at 100mg/L) resulted in an increase of T-ChE and Es-ChE activity in the digestive gland. This would be consistent with Saccostrea re-partitioning protein resources from gills towards digestive gland tissue upon high acute Im exposure, perhaps to aid in de-toxification, even though this explanation remains purely speculative at this point. However, to be able to unequivocally discriminate reductions of cholinesterase enzyme functionality (i.e. catalytic activity) from reductions in enzyme concentrations (i.e. protein expression), simultaneous measurement of cholinesterase protein concentration (for each of the various enzyme types) as well as cholinesterase enzyme activity are necessary, perhaps combined with a proteomic approach, such as recently described by Meng et al. (2017), to detect and quantify differentially expressed proteins. The metal exposures with Cd and Cu revealed sensitivity of T-ChE and Es-ChE activity in Saccostrea at concentrations > 100µg/L Cd, most notably so in digestive gland, where activities of both enzyme fractions decreased nearly 3-fold relative to controls at the highest exposure concentration (1000µg/L, corresponding to whole oyster soft tissue Cd concentration of 297µg/g dw). Inhibition of ChE and AChE activity by Cd has also been reported for Adamussium colbecki and for Ruditapes decussatus (Cravo et al., 2012). For example, for Adamussium colbecki, Bonacci et al. (2006) reported a Cd concentration-dependent inhibition of ChE activity in adductor muscle, compared to controls, with an IC50 of 4.57 × 10−4M, which corresponds to a Cd2+ concentration of 11.2mg/L (or 11,200µg/L, nearly 10 times the highest Cd concentration used in the present study). While the mechanism by which Cd interferes with ChE and AChE activity remains obscure, enzyme inactivation by the metal cation has been proposed (Bonacci et al., 2006, and references therein). Non-specific, allosteric interference would be consistent with the observation that the proportion of Es-ChE activity to T-ChE activity remained similar (fluctuating around 60%) over the Cd concentration range tested, and Meng et al. (2017) have demonstrated significant alteration of general protein metabolism by Cd at the proteome as well as at the transcriptome level in the Pacific oyster Crassostrea gigas. Other authors, such as Magni et al. (2006), Senger et al. (2006) and Choi et al. (2011), have also reported altered activity of ChE in the presence of heavy metals, as well as PAHs, hydrocarbons, detergents and phytotoxins, concluding that cholinesterases are sensitive not only to OP and carbamate pesticides but also to many other toxicants.