Characterization of cholinesterases present in brain and muscle tissues of juvenile blue shark (<i>Prionace glauca</i>)

• Main Authors: Luís Miguel Fonseca Alves, Sara Calçada Novais
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2014-06-01
• Series: Frontiers in Marine Science
• Subjects: Cholinesterases; Sharks; marine pollution; Prionace glauca; Chlorpyrifos-oxon
• Online Access: http://journal.frontiersin.org/Journal/10.3389/conf.fmars.2014.02.00007/full

<b>1. Introduction</b> Acetylcholinesterase (AChE) is a vital enzyme for the normal functioning of the neuromuscular system, where it is responsible for the hydrolysis of acetylcholine into choline and acetic acid, thus preventing continuous nervous impulses (Murphy, 1986; Walker and Thompson, 1991). The measurement of AChE and the activity of other cholinesterases is a widely applied method in pollution monitoring mainly due to their high sensitivity to anticholinergic chemicals, such as organophosphorus pesticides and carbamates (Payne et al, 1996; Fulton and Key, 2001; Chambers et al., 2002; Arufe et al., 2007). Additionally, ChEs are among the less variable biomarkers, which reinforces their use in pollution monitoring (Solé et al., 2008). Due to the lack of information on the forms of ChEs present in many organisms, studies that report screening for AChE may, in reality, be dealing with other ChE types (Magnotti et al., 1994; Oliveira et al., 2007; Silver, 1974). Currently, there are three known types of ChEs in fish: AChE, butyrylcholinesterase (BChE) and propionylcholinesterase (PChE) (Sturm et al., 1999, Sturm et al., 2000; Kirby et al., 2000; Solé et al., 2008). Being top predators, sharks help maintaining populations of other fish balanced in the marine ecosystem. Their predatory nature makes them particularly vulnerable to environmental contamination via bioaccumulation and biomagnification and, due to this, sharks tend to have higher levels of metals and other compounds in their bodies than most fish species (Porcella, 1994; Hueter et al., 1995). Blue sharks (<i>Prionace glauca</i>) are one of the most abundant and heavily caught fishes in the world, with an estimated 20 million individuals caught annually as target or by-catch, making them a very suitable organism for ecotoxicology studies (Bonfil, 1994; Stevens, 2009). To our knowledge, the only available study where ChEs of shark species have been characterized is the one from Solé and colleagues (2008) where muscle tissue of <i>Scyliorhinus canicula</i> and <i>Galeus melastomus</i> was used. The main goal of this research was to characterize the cholinesterases present in the brain and muscle of <i>P. glauca</i>, and to validate its use as a biomarker of effect with an in vitro exposure to an organophosphate insecticide, in order to optimize tissue selection and assay protocols for future biomonitoring studies in marine ecosystems. <b>2. Materials and methods</b> Muscle and brain samples from eight juvenile blue sharks (<i>P. glauca</i>) were collected aboard a commercial sword fishing boat, as soon as the animals were captured, and all samples were immediately kept on ice until they were stored in the lab at −80 °C. The eight organisms used in this study consisted in four males and four females ranging from 105 to 157 cm and from 113 to 167 cm, respectively. Samples were homogenized and cholinesterase characterization was performed in each tissue using substrates with higher affinity for specific enzymes: acetylthiocoline iodide (ATCh) for AChE, butyrylthiocholine iodide (BTCh) for BChE, and propionylthiocholine iodide (PTCh) for PChE. Substrate concentrations varied from 0.01 to 20.48mM. To calculate the catalytic efficiency of the enzyme with each substrate, experimental curves were fitted (monotonic increase part of the curve) using the Michaelis-Menten equation, in order to determine the ChE kinetic parameters: maximal velocity (Vmax) and Michaelis-Menten constant (Km). Eserine sulfate, BW-284C51 and iso-OMPA were used as selective inhibitors for total ChEs, AChE and BChE, respectively. An additional in vitro test was performed using the organophosphate insecticide chlorpyrifos-oxon, whose mode of action is to inhibit AChE, as a model compound to assess the potential of this biomarker for effect assessment in the blue shark. <b>3. Results and discussion</b> The results of ChE substrate preference in the brain are shown in figure 1. The substrate with higher hydrolysis rate was ATCh, followed by BTCh and PTCh. The enzymatic catalytic efficiency shown by the parameters of the Michaelis–Menten equation also shows the preference for the substrate ATCh. An inhibition of hydrolysis by excess of all three substrates was also verified. Eserine sulfate, a generic inhibitor of ChE, inhibited enzyme activity over 90% at concentrations higher than 50µM, showing that the enzymatic activity measured is mainly due to ChEs and not to other nonspecific esterases. Regarding the specific inhibitors for AChE and BChE, BW-284C51 caused over 90% inhibition but only at the highest concentration tested (800µM) while Iso-OMPA has also caused a dose-response inhibition with a maximum of 40% decrease in enzyme activity. With these results it is possible to conclude that the brain of <i>P. glauca</i> seems to contain atypical ChEs, displaying mixed properties of AChE and BChE. However, as the substrate with higher hydrolysis rates was ATCh this is the most suitable substrate to be used in future studies, with an optimal concentration 0.64 mM. In muscle tissue, the enzyme clearly preferred the substrate ATCh presenting much higher hydrolysis rates and catalytic efficiency than with the other substrates (Fig. 2). As it was observed for the brain tissue, also in muscle incubation with eserine caused an almost complete inhibition of enzymatic activity at concentrations higher than 50µM and therefore no other esterases are being assessed. The ChE present in the muscle showed to be highly sensitive to BW-284C51 and little affected by iso-OMPA, making it possible to conclude that the muscle of <i>P. glauca</i> seems to contain mainly AChE, and that the most suitable substrate to be used in future studies is ATCh, at an optimal concentration 0.64 mM. Regarding the in vitro exposure to chlorpyrifos-oxon, we have observed a dose-response pattern showing higher ChE inhibitions with increasing pesticide concentrations and with more than 90% inhibition in the highest concentrations, which reveals the potential of this biomarker for biomonitoring studies and contaminant effect assessment in the blue shark.