Elucidating the importance of mussel carboxylesterase activity as exposure biomarker of environmental contaminants of current concern: An in vitro study

Carboxylesterases (CEs) are alpha,beta-hydrolase fold proteins that catalyse the hydrolysis of a wide range of endogenous and exogenous compounds. In mammals, these enzymes are involved in the detoxification of certain pesticides and drugs. However, this toxicological role of CEs has received little attention in marine organisms such as the mussel Mytilus galloprovincialis. Therefore, the purpose of this study was to examine whether mussel CE activity is sensitive to inhibition by environmental chemicals of current concern (human pharmaceuticals and personal care products or PPCPs; Le fluoxetine, loperamide, simvastatin, fenofibrate, nonylphenol and triclosan), so this chemical interaction may be considered as a detoxification mechanism comparable to that of organophosphorus pesticides. First, we examined the basal levels of CE activity in multiple tissues of M. galloprovincialis, using a wide range of colorimetric substrates, i.e., p-nitrophenyl acetate (pNPA), p-nitrophenyl butyrate (pNPB), 1-naphthyl acetate (1-NA), 1-naphthyl butyrate (1-NB) and 2-naphtyl acetate (2-NA). Second, we tested for a substrate-dependence of detecting CE inhibition using the model organophosphorus dichlorvos. Finally, some PPCPs were tested for in vitro CE inhibition using multiple substrates. Results showed that long-chain esters (pNPB and 1-NB) provided the highest CE-mediated hydrolysis rates compared with pNPA or 1-NA. Moreover, the digestive gland and gills displayed the highest CE activities compared with haemolymph. As expected, the esterase enzyme was very sensitive to dichlorvos (IC50 s in the nM range), but dose-inhibition relationships were markedly dependent on the type of substrate used for enzyme assay. The in vitro inhibition kinetics identified triclosan as a potential CE inhibitor (IC50 = 7.07-27.2 mu M for digestive gland, IC50 = 10.8-104 mu M for gill CE activity), although other PPCPs such as simvastatin, fenofibrate and nonylphenol also decreased the enzyme activity to a lesser extent. In-gel esterase staining after non-denaturing polyacrylamide gel electrophoresis confirmed the inhibitory effect of triclosan upon CE activity, which was more pronounced with the substrate 1 NB. These findings suggest that CE inhibition may be a suitable biomarker of PPCP exposure to be incorporated into the battery of sub-individual indicators of PPCP exposure and toxicity. However, the selection of appropriate substrates is a key issue. Our results indicated that pNPB and 1-NB are the most suitable reporters for detecting inhibition of CE by both organophosphorus and PPCPs in mussels.