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Characterisation of choline esterases and their tissue and subcellular distribution in mussel (Mytilus edulis)
Marine Environmental Research 57 (2004) 155–169 www.elsevier.com/locate/marenvrev
Characterisation of choline esterases and their tissue and subcellular distribution in mussel (Mytilus edulis) Margaret Browna,*, Ian M. Daviesb, Colin F. Moffatb, John Redshawc, John A. Crafta a
Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Rd, Glasgow G4 0BA, UK Fisheries Research Services Marine Laboratory, PO Box 101, 375 Victoria Road, Aberdeen, AB11 9DB, UK c Scottish Environment Protection Agency, 5 Redwood Crescent, Peel Park, East Kilbride G74 5PP, UK
b
Received 14 November 2002; received in revised form 30 May 2003; accepted 18 June 2003
Abstract Acetylcholinesterase in mussel is potentially a useful biomarker of exposure to organophosphates (OP) in the marine environment. This study looked at cholinesterase activity in subcellular fractions of various tissues from the common mussel, Mytilus edulis. Measurement of enzyme rates demonstrated that although highest specific activity was found in foot ‘mitochondrial’ fraction, recovery of activity was very low. Gill ‘microsomal’ fraction had the second highest specific activity with a useful level of recovery and therefore was the most suitable tissue fraction for biomarker applications. Comparative studies of alternative alkylthiocholine substrates and competitive inhibitors suggest there is a single cholinesterase enzyme type present in this fraction. Inhibition of alkylcholine hydrolysis by BW284C51, specific to acetylcholinesterase in vertebrates, showed that cholinesterase activity in gill ‘microsomal’ fraction is inhibited by this compound but to a lesser extent than in vertebrate AChE. Inhibition of cholinesterase activity by azamethiphos in gill ‘microsomal’ fraction gave an IC50 of approximately 100 mM and showed both time and concentration dependence. However this indicates a lower potency compared to other animals and it is debatable whether mussel cholinesterase activity is useful as a biomarker of exposure in the field. # 2003 Elsevier Ltd. All rights reserved. Keywords: Biomarkers; Cholinesterase; Organophosphates; Azamethiphos; Mussel; Mytilus edulis
* Corresponding author. Tel.: +44-1413313222; fax: +44-1413313208. E-mail address: [email protected] (M. Brown). 0141-1136/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0141-1136(03)00067-9
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1. Introduction The expansion of salmon farming in Scotland and Ireland has greatly benefited local economies but concerns have been raised about the effects aquaculture may be having on marine ecosystems (Ernst et al. 2001; Gillibrand & Turreil, 1997; Wu 1995). Intensive farming methods employed to enhance production in fish farms have been associated with an increase in the incidence of sea-lice infestations in farmed stocks (Naylor et al., 2000). The medicines currently authorised for the control of sea lice include the organophosphorous (OP) compound azamethiphos but potential impacts of this compound on marine ecosystems remain uncertain. A study by Ernst et al. (2001), looking at toxicity to non-target aquatic organisms concluded that ‘use of azamethiphos for sea lice control presents a low to moderate environmental risk’. However, sublethal effects may occur that could impact on processes such as differentiation, growth or reproduction. There is a pressing need to monitor use and effects of OPs at a biochemical level in the marine environment and thus suitable sentinel species and relevant tests need to be identified. It has been suggested (Galloway et al., in press; Rodger, Galgani & Truquet, 1999) that the blue mussel Mytilus edulis, a semi-sessile filter feeder, is most suitable as a sentinel species to monitor for use of dissolved OPs in fish farming. A relevant biomarker for OP exposure is acetylcholinesterase (AChE) activity. Cholinesterase activity in mussel is usually measured in gill in either a crude homogenate (Bocquene, Galgani & Truquet, 1990; McHenery, Linley-Adams, Moore, Rodger, & Davies, 1997; Robertson, Madden, Moore, & Davies, 1992), or the soluble fraction recovered after treatment of gill tissue homogenate with detergent and/or phosphatidyl inositol phospholipase C (PIPLC) (Mora, Michel, & Narbonne, 1999) to release membrane-bound enzyme forms. However, cholinesterase activity is much lower in mussel than in mammals (Bocquene et al. 1990; Dauberschmidt, Dietrich, & Schlatter, 1997) when assayed by standard methods (Ellman, Courtney, Andres, & Featherstore, 1961) with acetylthiocholine (ASCh) as substrate. For instance it has been claimed that the enzyme in this species is not inhibited by exposure to OPs (Galgani & Bocquere, 1990). However, detection of inhibition (reduced enzyme activity) is clearly compromised by assays which have low background, control activity and which may measure in parallel several enzyme activities some of which may not be subject to inhibition. In vertebrates at least two families of enzyme exist, as classified by substrate preference (Massoulie, Pezzementi, Bon, Krejci, & Vallette, 1993) but the situation in molluscs (Basack, Oneto, Fuchs, Wood, & Kestern, 1998; Bocquene et al., 1997) and mussels in particular (Moreira, Coimbra, & Guilhermino, 2001; Talesa, Romani, Antognelli, Giovannini, & Rosi, 2001; Von Wachtendonk & Neef, 1979) has not been established. Results from a study by Mora, Fournier, & Narbonne, (1999), investigating cholinesterase (ChE) forms in Mytilus edulis and Mytilus galloprovincialis, although not conclusive appear to be consistent with the presence of only one pharmacological form common to both species. Results from a study by Talesa et al. (2001), meanwhile identified three possible ChE forms in Mytilus galloprovincialis. The consideration of these factors complicate interpretation of toxicity data and a
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review by Fulton and Key (2001) raises the issue of whether low inhibition of mussel cholinesterases to OPs by comparison to other species is due wholly to species-specific differences or signifies a requirement to measure activity in specific tissue from mussel and/or to account for cholinesterase types present which are not inhibited. Recently in marine vertebrates classification of cholinesterase types by substrate preferences and selectivity of inhibitors relevant to characteristics in mammals has been carried out (Chuiko, 2000; Sturm, da Silva de Assis, & Hansen, 1999). This methodology can be applied to investigate characteristics of invertebrate cholinesterases (Galloway et al., 2002; Moreira et al., 2001; Talesa et al., 2001). The aim of the current study is to improve the sensitivity of the assay through improved selection of target tissue and substrate. Tissue and subcellular distribution of cholinesterase activity in mussel are explored, allowing the differential assay of membrane-bound and soluble forms of cholinesterases. In addition, we have used diagnostic substrates and inhibitors relevant to vertebrate cholinesterase types (Table 1) to establish the number of distinct enzymes in Mytilus edulis.
2. Materials and methods 2.1. Chemicals Chemicals were supplied by Sigma Chemical Co., Poole, Dorset, unless otherwise stated. 2.2. Animals Farmed common mussels, Mytilus edulis (4–7 cm long), were obtained from a commercial shellfish farm in Loch Etive, Scotland. Animals were either transported to holding tanks in an environmental room (9 C) or were dissected on site. Transported animals were held for at least 48 h prior to use. Tissues (gill, posterior adductor muscle, foot, digestive gland) were removed, snap-frozen in liquid nitrogen and stored at 80 C. Table 1 Selectivity of various alkylthiocholine substrates and organophosphate and carbamate inhibitors of isozymes of cholinesterases, as characterised in mammalian species Enzyme
Acetylcholinesterase AChE (EC 3.1.1.7) Butyrylcholinesterase BChE (EC 3.1.1.8) Propionylcholinesterase PrChE
Substrate
Inhibitor
Selective
Non-selective
Selective
Non-selective
Acetyl-b-methylthiocholine A-b-MSCh Butyrylthiocholine BuSCh
Acetylthiocholine ASCh
BW284C51
eserine
Propionylthiocholine PrSCh
iso-OMPA
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2.3. Subcellular fractionation Gill, posterior adductor muscle, foot and digestive gland tissues from each of 15 mussels were pooled according to type in ice-cold isotonic sucrose (Solution A) (0.25 M sucrose, containing 50 mM sodium phosphate buffer pH 7.6, 10 mM MgSO4 and 1 mM EDTA). The tissues were then homogenised in ice-cold Solution A (3 ml g1 wet weight tissue) using an Omni 2000 (Omni International, Virginia, USA) at speed setting five for two 30 s bursts with a 30 s interval. Tissue from whole animal was homogenised identically but in 2 ml g1 wet weight tissue. Homogenates were centrifuged (Beckman Avanti high speed centrifuge) at 700 g max for 10 min after which the supernatants were transferred to clean tubes. These were then centrifuged at 7000 g max for 10 min and again supernatants were transferred to clean tubes. The pellets produced at 7000 g were washed by resuspension in 3 ml Solution A and were centrifuged at 24 Kg max for 10 min in parallel with the 7000 g supernatants. The resulting supernatants were ultracentrifuged (Centrikon T-2050) at 105 Kg max for 100 min. These procedures generated successively fractions which are operationally described here as: 700 g ‘nuclear’, 7000 g ‘mitochondrial’, 24 K g ‘microsomal’ and 105 Kg ‘microsomal’ pellets and a 105 Kg supernatant ‘soluble’ fraction. Each pellet was washed in situ with Solution A and then drained prior to resuspension in the same medium using a glass-Teflon homogeniser. The protein content of each fraction was determined in triplicate by the method of Bradford (1976) (BIORAD) with bovine organophosphorous serine albumin (BSA) as standard. Absorbance at 595 nm was measured in a GeneQuantPro spectrophotometer. 2.4. Cholinesterase activity Cholinesterase (ChE) activity was measured by the method of Ellman et al. (1961). All alkylthiol substrates were prepared immediately prior to the assays by dissolution in 0.1 M Tris–HCl (pH 7.6 at 20 C) held on ice and used within 1 h of preparation. Enzyme activity was assayed with the various alkylthiocholine substrates in 0.09 M Tris–HCl (pH 7.6 at 20 C) containing 0.7 mM 5,50 -dithio-bis-(2nitrobenzoic acid) (DTNB) as chromagen reagent. Reaction rates were quantified using a Beckman DU650 measuring the rate of change of absorbance at 405 nm from 1 to 4 min after addition of enzyme at room temperature. The rate of spontaneous substrate hydrolysis was found to be negligible, as was the rate of reaction of DTNB with other thiols, and was disregarded. Various alkylthiocholine substrates acetylthiocholine (ASCh), acetyl-b-methylthiocholine (AbMSCh), butyrylthiocholine (BuSCh) and propionylthiocholine (PrSCh) selective for ChEs, acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and propionylcholinesterase (PrChE) were used to define cholinesterase types present. Diagnostic inhibitors (Sturm et al., 1999) were used to further differentiate cholinesterase types. In vertebrates, eserine, 1,5-bis (4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51) and tetraisopropyl pyrophosphoramide, (iso-OMPA) are selective for ChE, AChE and BuChE respectively. Inhibition studies were conducted by incubation of enzyme
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preparation with 10 or 100 mM inhibitor for 30 mins, after which residual activities were determined as before with 2 mM alkylthiocholine substrate. 2.5. Azamethiphos inhibition of Cholinesterase activity in mussel in vitro A time course for in vitro inhibition of activity by 3 and 10 mM of the OP azamethiphos (Qmx Laboratories Ltd) was carried out in ‘microsomal’ fraction of gill. Enzyme preparations were incubated with the OP as described above for various times between 0 and 60 min before addition of 1 mM ASCh as substrate and residual activities measured.
3. Results 3.1. Subcellular fractionation Cholinesterase activity of subcellular fractions from various tissues of mussel was measured with acetyl thiocholine as substrate. Specific activities and recovered activities are shown in Fig. 1A and B respectively. Measurable activities were found in all fractions of all tissues including the ‘soluble’ fractions. The specific activities of each of the fractions of digestive gland were low and were not significantly different between fractions. Higher activities were found in the fractions of adductor muscle and foot with highest activity in the ‘mitochondrial’ fraction (P < 0.001 compared to gill ‘microsomal’ fraction). Indeed, the specific activity found in the ‘mitochondrial’ fraction of foot was at least three times higher than in any other fraction/tissue tested. Higher activities than in digestive gland were also found in gill, but in gill the highest activity was observed in the ‘microsomal’ fraction and the magnitude of this was second only to that of foot ‘mitochondrial’ fraction. The pattern of specific activity in the gill was similar to that observed in the whole organism. For each tissue the majority of the recovered activity was in the ‘nuclear’ and ‘soluble’ fractions with ‘nuclear’ always greater than ‘soluble’ (Fig. 1B). Very little activity was recovered in ‘mitochondrial’ or ‘microsomal’ fractions. The exception to this was in the gill where significant activity was recovered in the ‘microsomal’ fraction (equivalent to that of the ‘soluble’ fraction). For practical reasons the relatively high specific activity of ‘microsomal’ fraction of gill combined with the recovery of activity in this fraction determined its use in further studies. Acetylthiocholine esterase activity was measured in ‘microsomal’ fraction of gill with substrate concentration varied between 0.01 and 5 mM. The resulting kinetic plots indicate a Michaelis Menten mechanism with saturation at substrate concentrations higher than 1 mM and a linear Lineweaver–Burke plot (Fig. 2A). An investigation of activity in ‘soluble’ fraction of gill was also carried out. Activity was very low by comparison to that in ‘microsomal’ fraction with saturation at substrate concentrations higher than 1 mM and the results gave a non-linear Lineweaver–Burke plot (Fig. 2B). The fraction was not further analysed, as the level of activity present would not be useful for environmental monitoring purposes.
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Fig. 1. Choline esterase activity of subcellular fractions of various tissues of mussel. Tissues from 15 mussels were pooled and subjected to subcellular fractionation and choline esterase activity measured in each using acetyl thiocholine (ASCh) (3 mM) as substrate. (A) shows the specific activity of each fraction (nmol/min/mg proteinS.D., n=3) and (B) the activity recovered (nmol/minS.D., n=3) in each fraction. Each data point is the pooled data of triplicate determinations for two separate tissue fractionations.
3.2. Kinetic studies with alternate substrates Relative enzyme activities in subcellular fractions of gill were then determined with a series of alternate alkyl-substituted thiocholines (acetylthiocholine, acetyl-bmethylthiocholine, butyrylthiocholine and propionylthiocholine) (Fig. 3). The distribution of activity in the subcellular fractions with acetylthiocholine was similar to that observed in the previous experiments (Fig. 1). In each of the fractions activity was highest with acetylthiocholine as substrate although activity with propionylthiocholine was equivalent to that with acetylthiocholine in the ‘soluble’ fraction. In ‘nuclear’, ‘mitochondrial’ and ‘microsomal’ fractions propionyl-, acetylb-methyl- and butyryl-thiocholines had approximately 60, 50 and 10% of the rates
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with acetylthiocholine respectively, while in ‘soluble’ fraction this was 100, 50 and 60%. To further investigate the relationship between esterase activities with the alternative substrates an inhibition study was conducted in which hydrolysis of the various alkylthiocholines was competed by the presence of acetylcholine (ACh) in the gill ‘microsomal’ fraction. The relevant alkylthiocholine substrate was present at a fixed concentration of 1mM while the concentration of the acetylcholine competitor was varied (0.0, 0.4, 1.0 mM) and the results are shown in Fig. 4. In the absence of acetylcholine all alkylthiocholine substrates gave rates similar to those found in
Fig. 2. Effect of substrate concentration on esterase activity with acetylthiocholine as substrate in ‘microsomal’ (A) and ‘soluble’ (B) fraction of gill (nmol/min/mg proteinSEM, n=3) and corresponding Lineweaver–Burk graphs. Pooled gill from 15 mussels were homogenised and subjected to subcellular fractionation as described in section 2.
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the original experiment (Fig. 3). The presence of acetylcholine decreased rates of hydrolysis with all the alkylthiocholine substrates proportionally and in a concentration dependent manner with the exception of butyrylthiocholine. The experiment was repeated with ‘soluble’ fraction of gill and a similar pattern of results was found (results not shown). 3.3. Enzyme inhibition Vertebrate cholinesterases are selectively inhibited by eserine and this agent can thus differentiate these activities from other esterases. The esterase activity of gill ‘microsomal’ fraction with acetylthiocholine (1 mM) was measured after exposure to eserine (5 mM) for 30 min. Enzyme activity was inhibited by more than 90% (data not shown). Cholinesterases of vertebrates can be further distinguished with enzyme-selective inhibitors and BW284C51 is acetylcholine esterase-selective while iso-OMPA affects butyrylcholinesterase. The effect of these compounds at two concentrations (10 and 100 mM) on esterase activity was determined with the acetyl- and acetyl-b-methyl- thiocholine substrates (2 mM). The selectivity and effectiveness of the inhibitors in our hands was initially demonstrated using a purified preparation of acetylcholinesterase from bovine erythrocytes and a rat brain ‘microsomal’ fraction (Fig. 5A). The inhibitors were effective and selective as expected with BW284C51 inhibiting AChE, with little cross inhibition by iso-OMPA, and these effects were observed at the lower inhibitor concentration. Activity in ‘microsomal’ fraction of gill was not affected by iso-OMPA but was sensitive to BW284C51 inhibition although significantly less than in purified AChE (P < 0.001) or rat brain
Fig. 3. Comparison of esterase activity of mussel gill subcellular fractions with alternate alkylthiocholine substrates. Pooled gill from 15 mussels were homogenised and subjected to subcellular fractionation as in Fig. 1 (n=3). Esterase activity (nmol/min/mg proteinS.D.) was determined in the resulting fractions using alternate alkylthiocholine substrates (1 mM) as indicated.
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Fig. 4. Effect of acetylcholine (ACh) concentration on esterase activity (nmol/min/mg proteinS.D.) in ‘microsomal’ fraction of gill with alternative alkylthiocholines (2 mM) as substrate. Pooled gill from 15 mussels were homogenised and subjected to subcellular fractionation as in Fig. 1 (n=3).
‘microsomal’ preparation (P < 0.001). To further establish selectivity a preparation containing only butyryl cholinesterase (horse serum) was used. The results showed iso-OMPA inhibited hydrolysis of butyrylthiocholine while BW284C51 had no effect (not shown). To assess the potential of mussel AChE as a biomarker in the environment the effects of azamethiphos, a biocide used in the salmon farming industry, was investigated in vitro. Microsomes from gill of mussel were incubated up to 60 min with the compound at 3 and 10 mM prior to determination of enzyme activity. The compound inhibited the enzyme in both a time and concentration dependent manner (Fig. 6A). At 3 mM enzyme activity was inhibited by 80% after 60 min exposure while at 10 mM 80% inhibition was found after 5 min exposure and a limiting 95% inhibition was found by 25 min. Variation of inhibitor concentration indicated an IC50 of 100 mM azamethiphos in vitro (Fig. 6B).
4. Discussion The aim of this study was to identify a more sensitive target tissue or tissue fraction for assay of cholinesterase activity in mussel with a view to using this as a biomarker of OP inhibition. It was expected that cholinesterase activity would be highest in tissue innervated for movement and this guided the selection of foot and posterior adductor muscle but gill and digestive gland were also investigated. Whole animal was included for comparative purposes. Subcellular fractionation achieved partial purification of cholinesterase activity into various operationally described
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Fig. 5. Effects of selective inhibitors, (A) iso-OMPA (butyrylcholinesterase (BChE) inhibitor) and BW284C51 (acetylcholinesterase (AChE) inhibitor) on hydrolysis of acetylthiocholine (ASCh) 2 mM, in purified AChE (from bovine erythrocytes) and ‘microsomal’ fractions of rat brain and mussel gill. (B) Effect of BW284C51 on hydrolysis of acetyl-b-methylthiocholine (A-b-MTCh) in the same preparations (nmol/min/mg proteinS.D.). Gill from 15 mussels were homogenised and subjected to subcellular fractionation as in Fig. 1 (n=3).
fractions (‘nuclear’, ‘mitochondrial’, ‘microsomal’, ‘soluble’). While measurable activity was obtained in all tissues and fractions (Fig. 1A) ‘mitochondrial’ fraction of foot and ‘microsomal’ fraction of gill provided significantly greater specific cholinesterase activity than was measured in whole mussel fractions. It was expected that the highly innervated foot would have significant activity but its presence in ‘mitochondrial’ fraction is surprising. In vertebrate species AChE is found in postsynaptic membranes and might be expected to be recovered in ‘microsomal’ fractions. However, mussel foot was consistently resistant to mechanical homogenisation
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and it is likely that disruption was incomplete with the result that activity was recovered in the ‘mitochondrial’ fraction. The total activity recovered from this tissue fraction (Fig. 1B) was very low and coupled with its resistance to homogenisation, the tissue would be impractical for use in biomarker applications. Therefore although the data of Fig. 1A suggests that foot ‘mitochondrial’ fraction (98 nmol/min/mg protein) is the preparation of choice for biomarker measurements an analysis of the data in terms of enzyme units recovered suggests otherwise. In contrast a significant level of activity was recovered in the ‘microsomal’ fraction of gill, which also had the second highest specific activity (35 nmol/min/mg). ‘microsomal’ fraction of gill therefore appears to be the most suitable tissue/fraction for measurement of inhibition of cholinesterase activity using acetylthiocholine as substrate. This specific activity reflected at least a three-fold increase on rates found under similar assay conditions in gill S9 fraction by Mora, Fournier, & Marbonne, (1999) 9 nmol/min/ mg protein (M. edulis) and 7.5 nmol/min/mg protein (M. galloprovincialis). Escartin and Porte (1997) reported a specific cholinesterase activity in S12 fraction of gill (M. galloprovincialis) of 24 nmol/min/mg protein. Several other studies report highest specific activity in gill (Bocquene et al., 1990, Escartin & Porte, 1997, Najimi
Fig. 6. Effect of azamethiphos inhibition on esterase activity in ‘microsomal’ fraction of gill with acetylthiocholine (ASCh) 2 mM as substrate (A) time course with various concentrations of azamethiphos (nmol/min/mg proteinSEM) (B) effect of inhibitor concentration on hydrolysis of ASCh (nmol/min/mg proteinSEM). Pooled gill from 15 mussels were homogenised and subjected to subcellular fractionation as in Fig. 1 (n=3).
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et al., 1997) however, direct comparison is often confounded by differences between both sample preparation methods and use of varying arbitrary units to describe data. A number of strategies were adopted to establish the possible multiplicity of esterases in mussel. In the first of these the rate of acetylthiocholine hydrolysis was studied as a function of substrate concentration. Multiple enzymes using the same substrate but with different kinetic parameters generate non-linear Lineweaver– Burke plots (Cornish-Bowden, 1995). The Lineweaver-Burke plot for rates of activity measured in ‘soluble’ fraction of gill was non-linear suggesting more than one enzyme may be present (Fig. 2B). However, activity in this fraction was below a level suitable for biomarker use so this was not pursued further. In ‘microsomal’ fraction of gill linear Lineweaver–Burke plots showed no evidence for the presence of more than one enzyme (Fig. 2A). The apparent value of Km (40 mM) for this enzyme is similar to values reported previously (12 mM in S9 fraction of gill from Mytilus edulis (Mora, Fournier, & Narbonne, 1999b); 30 mM in S9 fraction of gill of M. galloprovincialis (Mora, Michel, & Narbonne, 1999a); 73 mM in whole animal for M. galloprovincialis (Najimi et al., 1997). In a second approach to detection of multiple enzymes in mussel, activity was measured in gill with alternate alkyl-substituted thiocholines. The ranked order of rates of hydrolysis with substituted alkylthiocholines was ASCh > PrSCh > A-bMSCh > BuSCh in all fractions (Fig. 3) and this was in agreement with studies using alternative enzyme preparations (Mora, Fournier, & Narbonne, 1999b; Talesa et al., 2001). A slightly different result was found in soluble fraction in which activity with BuSCh was marginally greater than with A-b-MSCh. Although a non-linear Lineweaver–Burke plot was generated for this fraction (Fig. 2B), these data do not provide sufficient evidence for two separate cholinesterase enzymes (a membrane-bound AChE and a separate soluble BuChE). In a variant of this experiment the ability of acetylcholine to compete for hydrolysis of the alkylthiocholine substrates in ‘microsomal’ fraction of gill was tested. Acetylcholine was equally effective as an inhibitor with each of the alkylthiocholines in a concentration dependent manner (Fig. 4). It may therefore be that a single enzyme type is present that is capable of binding and hydrolysing all of the alkylthiocholine substrates but with different rates. The effectiveness of the inhibitors BW284C51 and iso-OMPA (selective for AChE and BuChE) on gill ‘microsomal’ fraction was determined (Fig. 5A and B) and this provided a further test for the possibility of separate AChE and BuChE (Sturm et al., 1999). The absence of significant inhibition of cholinesterase activity in gill ‘microsomal’ fraction with iso-OMPA suggests detectable BuChE activity is not present in these preparations. A control experiment with purified BuChE from horse serum demonstrated the efficacy of iso-OMPA as a selective inhibitor of this enzyme (results not shown). Comparison of inhibition of gill ‘microsomal’ cholinesterase activity to that in purified AChE and rat brain ‘microsomal’ preparation by BW284C51, an inhibitor known to be AChE-specific in vertebrates indicated significantly less inhibition in gill at both concentrations (Fig. 5A). This difference in sensitivity of gill to BW284C51 suggests that cholinesterase activity present in this tissue fraction may have characteristics atypical of classical AChE. A further
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experiment with a supposedly more AChE-specific substrate, A-b-MSCh, produced very similar results reinforcing the view of a single AChE enzyme with atypical properties (Fig. 5B). A definitive view of the multiplicity of cholinesterase enzymes in mussel awaits genomic characterization. Investigation of the effects of azamethiphos showed that the dissolved OP inhibited gill ‘microsomal’ cholinesterases in a time- and concentration-dependent manner with an IC50 of approximately 100 mM in vitro (Fig. 6A and B). This value compares to toxicity results of LC50 0.6 mM in stickleback (96 h exposure), Gasterosteus aculeatus and LC50 of 30 mM in brine shrimp (24 h exposure), Artemia salina (Ernst et al., 2001). Direct comparison of our data with these other studies of the effects of azamethiphos is difficult because of the use of different biological endpoints. Speculating on the basis of the results in context of aquaculture, the dispersion of azamethiphos (administered therapeutic dosage 150 mM) by marine dilution would result in benign concentrations being available any further than one or two cage width distance from source. Repeated exposures however, may have more deleterious effects. Whilst inhibition of AChE in mussel gill has been demonstrated the effectiveness of the inhibitor in vivo still needs to be established. AChE is a classical endpoint for environmental toxicology studies and was originally introduced to reflect adverse effects of exposure to toxicants affecting the nervous system. The use of this enzyme in mussel is complicated by a number of factors including low activity in crude tissue homogenates. Recent work (Galloway, Millward, Browne, & Depledge, 2002; Moreira et al., 2001; Talesa et al., 2001) has suggested that haemolymph contains a more active AChE enzyme and that this may provide the necessary simplicity and sensitivity for monitoring purposes. While haemolymph provides a simple assay medium the origin and function of the enzyme activities in this preparation are unclear and may not be related to nervous system function. Thus the enzyme(s) of haemolymph may provide a biomarker of exposure but not necessarily of effect. Here we show that subcellular fractionation of gill will provide a toxicologically relevant membrane-bound preparation enriched for AChE activity relative to crude homogenates and which is inhibited by the OP compound azamethiphos. Since gill is a ‘first-pass’ target the approach described here provides a significant advance in providing a monitoring tool. It would be highly desirable to extend this study to investigate the effects of in vivo azamethiphos exposures on AChE in mussel to broaden our understanding of the environmental impact of this compound.
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Chuiko, G. M. (2000). Comparative study of acetylcholinesterase and butyrylcholinesterase in brain and serum of several freshwater fish: specific activities and in vitro inhibition by DDVP, an organophosphorus pesticide. Comparative Biochemistry and Physiology, 127, 233–242. Cornish-Bowden, A. (1995). Fundamentals of enzyme kinetics. London: Portland Press Ltd. Dauberschmidt, C., Dietrich, D. R., & Schlatter, C. (1997). Esterases in the zebra mussel Dreissena polymorpha: activities, inhibition, and binding to organophosphates. Aquatic Toxicology, 37, 295– 305. Ellman, G. L., Courtney, K. D., Andres Jr., V., & Featherstone, R. M. (1961). A new and rapid colometric determination of acetylcholinesterase activity. Biochemical Pharmacology, 7, 88–95. Ernst, W., Jackman, P., Doe, K., Page, F., Julien, G., Mackay, K., & Sutherland, T. (2001). Dispersion and toxicity to non-target aquatic organisms of pesticides used to treat sea lice on salmon in net pen enclosures. Marine Pollution Bulletin, 42, 433–444. Escartin, E., & Porte, C. (1997). The use of cholinesterase and carboxylesterase activities from Mytilus galloprovincialis in pollution monitoring. Environmental Toxicology and Chemistry, 16, 2090–2095. Fulton, M. H., & Key, P. B. (2001). Acetylcholinesterase inhibition in estuarine fish and invertebrates as an indicator of organophosphorous insecticide exposure and effects. Environmental Toxicology and Chemistry, 20, 37–45. Galgani, F., & Bocquene, G. (1990). In vitro inhibition of acetylcholinesterase from four marine species by organophosphates and carbamates. Bulletin of Environmental Contamination and Toxicology, 45, 243–249. Galloway, T. S., Millward, N., Browne, M. A., & Depledge, M. H. (2002). Rapid assessment of organophosphorous/carbamate exposure in the bivalve mollusc Mytilus edulis using combined esterase activities as biomarkers. Aquatic Toxicology, 61(3-4), 169–180. Gillibrand, P. A., & Turrell, W. R. (1997). The use of simple models in the regulation of the impact of fish farms on water quality in Scottish sea lochs. Aquaculture, 159, 33–46. Massoulie, J., Pezzementi, L., Bon, S., Krejci, E., & Vallette, F.-M. (1993). Molecular and cellular biology of cholinesterases. Progress in Neurobiology, 41, 31–91. McHenery, J. G., Linley-Adams, G. E., Moore, D. C., Rodger, G. K., & Davies, I. M. (1997). Experimental and field studies of effects of dichlorvos exposure on acetylcholinesterase activity in the gills of the mussel, Mytilus edulis L. Aquatic Toxicology, 38, 125–143. Mora, P., Fournier, D., & Narbonne, J.-F. (1999). Cholinesterases from the marine mussels Mytilus galloprovincialis Lmk. and M. edulis L. and from the freshwater bivalve Corbicula fluminea Muller. Comparative Biochemistry and Physiology Part C, 122, 353–361. Mora, P., Michel, X., & Narbonne, J.-F. (1999). Cholinesterase activity as potential biomarker in two bivalves. Environmental Toxicology and Pharmacology, 7, 253–260. Moreira, S. M., Coimbra, J., & Guilhermino, L. (2001). Acetylcholinesterase of Mytilus galloprovincialis LmK. haemolymph: a suitable biomarker. Bulletin of Environmental Contamination and Toxicology, 67, 470–475. Najimi, S., Bouhami, A., Daubeze, M., Zekhini, A., Pellerin, J., Narbonne, J-F., & Moukrim, A. (1997). Use of acetylcholinesterase in Perna perna and Mytilus galloprovincialis as a biomarker of pollution in Agadir Marine Bay (south of Morocco). Bulletin of Environmental Contamination and Toxicology, 58, 901–908. Naylor, R. L., Goldburg, R. J., Primavera, J. H., Kautsky, N., Beveridge, M. C. M., Clay, J., Folke, C., Lubchence, J., Mooney, H., & Troell, M. (2000). Effect of aquaculture on world fish supplies. Nature, 405, 1017–1024. Robertson, N. A., Madden, S., Moore, D. C., & Davies, I. M. (1992) Effects of short, repeated exposure of the mussel (Mytilus edulis L.) to CGA18809. Fisheries Research Services Report No. 8/92. Rodger, G.K., Davies, I.M. & Stagg, R.M. (1999) Development and application of biomarker/bioassay procedures for the environmental monitoring of sea lice treatment chemicals used in salmon farming— Phase 1 (Scoping Study), FRS Marine Laboratory. Sturm, A., da Silva de Assis, H. C., & Hansen, P.-D. (1999). Cholinesterases of marine teleost fish: enzymological characterisation and potential use in the monitoring of neurotoxic contamination. Marine Environmental Research, 47, 389–398.
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Talesa, V., Romani, R., Antognelli, C., Giovannini, E., & Rosi, G. (2001). Soluble and membrane-bound acetylcholinesterases in Mytilus galloprovincialis (Pelcypoda: Filibranchia) from the northern Adriatic sea. Chemico-Biological Interactions, 134, 151–166. Von Wachtendonk, D., & Neef, J. (1979). Isolation, purification and molecular properties of an acetylcholinesterase (E.C. 3.1.1.7) from the haemolymph of the sea mussel Mytilus edulis. Comparative Biochemistry and Physiology, 63, 279–286. Wu, R. S. S. (1995). The environmental impact of marine fish culture: toward a sustainable future. Marine Pollution Bulletin, 31, 159–166.
Characterisation of choline esterases and their tissue and subcellular distribution in mussel (Mytilus edulis) Margaret Browna,*, Ian M. Daviesb, Colin F. Moffatb, John Redshawc, John A. Crafta a
Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Rd, Glasgow G4 0BA, UK Fisheries Research Services Marine Laboratory, PO Box 101, 375 Victoria Road, Aberdeen, AB11 9DB, UK c Scottish Environment Protection Agency, 5 Redwood Crescent, Peel Park, East Kilbride G74 5PP, UK
b
Received 14 November 2002; received in revised form 30 May 2003; accepted 18 June 2003
Abstract Acetylcholinesterase in mussel is potentially a useful biomarker of exposure to organophosphates (OP) in the marine environment. This study looked at cholinesterase activity in subcellular fractions of various tissues from the common mussel, Mytilus edulis. Measurement of enzyme rates demonstrated that although highest specific activity was found in foot ‘mitochondrial’ fraction, recovery of activity was very low. Gill ‘microsomal’ fraction had the second highest specific activity with a useful level of recovery and therefore was the most suitable tissue fraction for biomarker applications. Comparative studies of alternative alkylthiocholine substrates and competitive inhibitors suggest there is a single cholinesterase enzyme type present in this fraction. Inhibition of alkylcholine hydrolysis by BW284C51, specific to acetylcholinesterase in vertebrates, showed that cholinesterase activity in gill ‘microsomal’ fraction is inhibited by this compound but to a lesser extent than in vertebrate AChE. Inhibition of cholinesterase activity by azamethiphos in gill ‘microsomal’ fraction gave an IC50 of approximately 100 mM and showed both time and concentration dependence. However this indicates a lower potency compared to other animals and it is debatable whether mussel cholinesterase activity is useful as a biomarker of exposure in the field. # 2003 Elsevier Ltd. All rights reserved. Keywords: Biomarkers; Cholinesterase; Organophosphates; Azamethiphos; Mussel; Mytilus edulis
* Corresponding author. Tel.: +44-1413313222; fax: +44-1413313208. E-mail address: [email protected] (M. Brown). 0141-1136/$ - see front matter # 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0141-1136(03)00067-9
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1. Introduction The expansion of salmon farming in Scotland and Ireland has greatly benefited local economies but concerns have been raised about the effects aquaculture may be having on marine ecosystems (Ernst et al. 2001; Gillibrand & Turreil, 1997; Wu 1995). Intensive farming methods employed to enhance production in fish farms have been associated with an increase in the incidence of sea-lice infestations in farmed stocks (Naylor et al., 2000). The medicines currently authorised for the control of sea lice include the organophosphorous (OP) compound azamethiphos but potential impacts of this compound on marine ecosystems remain uncertain. A study by Ernst et al. (2001), looking at toxicity to non-target aquatic organisms concluded that ‘use of azamethiphos for sea lice control presents a low to moderate environmental risk’. However, sublethal effects may occur that could impact on processes such as differentiation, growth or reproduction. There is a pressing need to monitor use and effects of OPs at a biochemical level in the marine environment and thus suitable sentinel species and relevant tests need to be identified. It has been suggested (Galloway et al., in press; Rodger, Galgani & Truquet, 1999) that the blue mussel Mytilus edulis, a semi-sessile filter feeder, is most suitable as a sentinel species to monitor for use of dissolved OPs in fish farming. A relevant biomarker for OP exposure is acetylcholinesterase (AChE) activity. Cholinesterase activity in mussel is usually measured in gill in either a crude homogenate (Bocquene, Galgani & Truquet, 1990; McHenery, Linley-Adams, Moore, Rodger, & Davies, 1997; Robertson, Madden, Moore, & Davies, 1992), or the soluble fraction recovered after treatment of gill tissue homogenate with detergent and/or phosphatidyl inositol phospholipase C (PIPLC) (Mora, Michel, & Narbonne, 1999) to release membrane-bound enzyme forms. However, cholinesterase activity is much lower in mussel than in mammals (Bocquene et al. 1990; Dauberschmidt, Dietrich, & Schlatter, 1997) when assayed by standard methods (Ellman, Courtney, Andres, & Featherstore, 1961) with acetylthiocholine (ASCh) as substrate. For instance it has been claimed that the enzyme in this species is not inhibited by exposure to OPs (Galgani & Bocquere, 1990). However, detection of inhibition (reduced enzyme activity) is clearly compromised by assays which have low background, control activity and which may measure in parallel several enzyme activities some of which may not be subject to inhibition. In vertebrates at least two families of enzyme exist, as classified by substrate preference (Massoulie, Pezzementi, Bon, Krejci, & Vallette, 1993) but the situation in molluscs (Basack, Oneto, Fuchs, Wood, & Kestern, 1998; Bocquene et al., 1997) and mussels in particular (Moreira, Coimbra, & Guilhermino, 2001; Talesa, Romani, Antognelli, Giovannini, & Rosi, 2001; Von Wachtendonk & Neef, 1979) has not been established. Results from a study by Mora, Fournier, & Narbonne, (1999), investigating cholinesterase (ChE) forms in Mytilus edulis and Mytilus galloprovincialis, although not conclusive appear to be consistent with the presence of only one pharmacological form common to both species. Results from a study by Talesa et al. (2001), meanwhile identified three possible ChE forms in Mytilus galloprovincialis. The consideration of these factors complicate interpretation of toxicity data and a
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review by Fulton and Key (2001) raises the issue of whether low inhibition of mussel cholinesterases to OPs by comparison to other species is due wholly to species-specific differences or signifies a requirement to measure activity in specific tissue from mussel and/or to account for cholinesterase types present which are not inhibited. Recently in marine vertebrates classification of cholinesterase types by substrate preferences and selectivity of inhibitors relevant to characteristics in mammals has been carried out (Chuiko, 2000; Sturm, da Silva de Assis, & Hansen, 1999). This methodology can be applied to investigate characteristics of invertebrate cholinesterases (Galloway et al., 2002; Moreira et al., 2001; Talesa et al., 2001). The aim of the current study is to improve the sensitivity of the assay through improved selection of target tissue and substrate. Tissue and subcellular distribution of cholinesterase activity in mussel are explored, allowing the differential assay of membrane-bound and soluble forms of cholinesterases. In addition, we have used diagnostic substrates and inhibitors relevant to vertebrate cholinesterase types (Table 1) to establish the number of distinct enzymes in Mytilus edulis.
2. Materials and methods 2.1. Chemicals Chemicals were supplied by Sigma Chemical Co., Poole, Dorset, unless otherwise stated. 2.2. Animals Farmed common mussels, Mytilus edulis (4–7 cm long), were obtained from a commercial shellfish farm in Loch Etive, Scotland. Animals were either transported to holding tanks in an environmental room (9 C) or were dissected on site. Transported animals were held for at least 48 h prior to use. Tissues (gill, posterior adductor muscle, foot, digestive gland) were removed, snap-frozen in liquid nitrogen and stored at 80 C. Table 1 Selectivity of various alkylthiocholine substrates and organophosphate and carbamate inhibitors of isozymes of cholinesterases, as characterised in mammalian species Enzyme
Acetylcholinesterase AChE (EC 3.1.1.7) Butyrylcholinesterase BChE (EC 3.1.1.8) Propionylcholinesterase PrChE
Substrate
Inhibitor
Selective
Non-selective
Selective
Non-selective
Acetyl-b-methylthiocholine A-b-MSCh Butyrylthiocholine BuSCh
Acetylthiocholine ASCh
BW284C51
eserine
Propionylthiocholine PrSCh
iso-OMPA
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2.3. Subcellular fractionation Gill, posterior adductor muscle, foot and digestive gland tissues from each of 15 mussels were pooled according to type in ice-cold isotonic sucrose (Solution A) (0.25 M sucrose, containing 50 mM sodium phosphate buffer pH 7.6, 10 mM MgSO4 and 1 mM EDTA). The tissues were then homogenised in ice-cold Solution A (3 ml g1 wet weight tissue) using an Omni 2000 (Omni International, Virginia, USA) at speed setting five for two 30 s bursts with a 30 s interval. Tissue from whole animal was homogenised identically but in 2 ml g1 wet weight tissue. Homogenates were centrifuged (Beckman Avanti high speed centrifuge) at 700 g max for 10 min after which the supernatants were transferred to clean tubes. These were then centrifuged at 7000 g max for 10 min and again supernatants were transferred to clean tubes. The pellets produced at 7000 g were washed by resuspension in 3 ml Solution A and were centrifuged at 24 Kg max for 10 min in parallel with the 7000 g supernatants. The resulting supernatants were ultracentrifuged (Centrikon T-2050) at 105 Kg max for 100 min. These procedures generated successively fractions which are operationally described here as: 700 g ‘nuclear’, 7000 g ‘mitochondrial’, 24 K g ‘microsomal’ and 105 Kg ‘microsomal’ pellets and a 105 Kg supernatant ‘soluble’ fraction. Each pellet was washed in situ with Solution A and then drained prior to resuspension in the same medium using a glass-Teflon homogeniser. The protein content of each fraction was determined in triplicate by the method of Bradford (1976) (BIORAD) with bovine organophosphorous serine albumin (BSA) as standard. Absorbance at 595 nm was measured in a GeneQuantPro spectrophotometer. 2.4. Cholinesterase activity Cholinesterase (ChE) activity was measured by the method of Ellman et al. (1961). All alkylthiol substrates were prepared immediately prior to the assays by dissolution in 0.1 M Tris–HCl (pH 7.6 at 20 C) held on ice and used within 1 h of preparation. Enzyme activity was assayed with the various alkylthiocholine substrates in 0.09 M Tris–HCl (pH 7.6 at 20 C) containing 0.7 mM 5,50 -dithio-bis-(2nitrobenzoic acid) (DTNB) as chromagen reagent. Reaction rates were quantified using a Beckman DU650 measuring the rate of change of absorbance at 405 nm from 1 to 4 min after addition of enzyme at room temperature. The rate of spontaneous substrate hydrolysis was found to be negligible, as was the rate of reaction of DTNB with other thiols, and was disregarded. Various alkylthiocholine substrates acetylthiocholine (ASCh), acetyl-b-methylthiocholine (AbMSCh), butyrylthiocholine (BuSCh) and propionylthiocholine (PrSCh) selective for ChEs, acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and propionylcholinesterase (PrChE) were used to define cholinesterase types present. Diagnostic inhibitors (Sturm et al., 1999) were used to further differentiate cholinesterase types. In vertebrates, eserine, 1,5-bis (4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51) and tetraisopropyl pyrophosphoramide, (iso-OMPA) are selective for ChE, AChE and BuChE respectively. Inhibition studies were conducted by incubation of enzyme
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preparation with 10 or 100 mM inhibitor for 30 mins, after which residual activities were determined as before with 2 mM alkylthiocholine substrate. 2.5. Azamethiphos inhibition of Cholinesterase activity in mussel in vitro A time course for in vitro inhibition of activity by 3 and 10 mM of the OP azamethiphos (Qmx Laboratories Ltd) was carried out in ‘microsomal’ fraction of gill. Enzyme preparations were incubated with the OP as described above for various times between 0 and 60 min before addition of 1 mM ASCh as substrate and residual activities measured.
3. Results 3.1. Subcellular fractionation Cholinesterase activity of subcellular fractions from various tissues of mussel was measured with acetyl thiocholine as substrate. Specific activities and recovered activities are shown in Fig. 1A and B respectively. Measurable activities were found in all fractions of all tissues including the ‘soluble’ fractions. The specific activities of each of the fractions of digestive gland were low and were not significantly different between fractions. Higher activities were found in the fractions of adductor muscle and foot with highest activity in the ‘mitochondrial’ fraction (P < 0.001 compared to gill ‘microsomal’ fraction). Indeed, the specific activity found in the ‘mitochondrial’ fraction of foot was at least three times higher than in any other fraction/tissue tested. Higher activities than in digestive gland were also found in gill, but in gill the highest activity was observed in the ‘microsomal’ fraction and the magnitude of this was second only to that of foot ‘mitochondrial’ fraction. The pattern of specific activity in the gill was similar to that observed in the whole organism. For each tissue the majority of the recovered activity was in the ‘nuclear’ and ‘soluble’ fractions with ‘nuclear’ always greater than ‘soluble’ (Fig. 1B). Very little activity was recovered in ‘mitochondrial’ or ‘microsomal’ fractions. The exception to this was in the gill where significant activity was recovered in the ‘microsomal’ fraction (equivalent to that of the ‘soluble’ fraction). For practical reasons the relatively high specific activity of ‘microsomal’ fraction of gill combined with the recovery of activity in this fraction determined its use in further studies. Acetylthiocholine esterase activity was measured in ‘microsomal’ fraction of gill with substrate concentration varied between 0.01 and 5 mM. The resulting kinetic plots indicate a Michaelis Menten mechanism with saturation at substrate concentrations higher than 1 mM and a linear Lineweaver–Burke plot (Fig. 2A). An investigation of activity in ‘soluble’ fraction of gill was also carried out. Activity was very low by comparison to that in ‘microsomal’ fraction with saturation at substrate concentrations higher than 1 mM and the results gave a non-linear Lineweaver–Burke plot (Fig. 2B). The fraction was not further analysed, as the level of activity present would not be useful for environmental monitoring purposes.
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Fig. 1. Choline esterase activity of subcellular fractions of various tissues of mussel. Tissues from 15 mussels were pooled and subjected to subcellular fractionation and choline esterase activity measured in each using acetyl thiocholine (ASCh) (3 mM) as substrate. (A) shows the specific activity of each fraction (nmol/min/mg proteinS.D., n=3) and (B) the activity recovered (nmol/minS.D., n=3) in each fraction. Each data point is the pooled data of triplicate determinations for two separate tissue fractionations.
3.2. Kinetic studies with alternate substrates Relative enzyme activities in subcellular fractions of gill were then determined with a series of alternate alkyl-substituted thiocholines (acetylthiocholine, acetyl-bmethylthiocholine, butyrylthiocholine and propionylthiocholine) (Fig. 3). The distribution of activity in the subcellular fractions with acetylthiocholine was similar to that observed in the previous experiments (Fig. 1). In each of the fractions activity was highest with acetylthiocholine as substrate although activity with propionylthiocholine was equivalent to that with acetylthiocholine in the ‘soluble’ fraction. In ‘nuclear’, ‘mitochondrial’ and ‘microsomal’ fractions propionyl-, acetylb-methyl- and butyryl-thiocholines had approximately 60, 50 and 10% of the rates
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with acetylthiocholine respectively, while in ‘soluble’ fraction this was 100, 50 and 60%. To further investigate the relationship between esterase activities with the alternative substrates an inhibition study was conducted in which hydrolysis of the various alkylthiocholines was competed by the presence of acetylcholine (ACh) in the gill ‘microsomal’ fraction. The relevant alkylthiocholine substrate was present at a fixed concentration of 1mM while the concentration of the acetylcholine competitor was varied (0.0, 0.4, 1.0 mM) and the results are shown in Fig. 4. In the absence of acetylcholine all alkylthiocholine substrates gave rates similar to those found in
Fig. 2. Effect of substrate concentration on esterase activity with acetylthiocholine as substrate in ‘microsomal’ (A) and ‘soluble’ (B) fraction of gill (nmol/min/mg proteinSEM, n=3) and corresponding Lineweaver–Burk graphs. Pooled gill from 15 mussels were homogenised and subjected to subcellular fractionation as described in section 2.
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the original experiment (Fig. 3). The presence of acetylcholine decreased rates of hydrolysis with all the alkylthiocholine substrates proportionally and in a concentration dependent manner with the exception of butyrylthiocholine. The experiment was repeated with ‘soluble’ fraction of gill and a similar pattern of results was found (results not shown). 3.3. Enzyme inhibition Vertebrate cholinesterases are selectively inhibited by eserine and this agent can thus differentiate these activities from other esterases. The esterase activity of gill ‘microsomal’ fraction with acetylthiocholine (1 mM) was measured after exposure to eserine (5 mM) for 30 min. Enzyme activity was inhibited by more than 90% (data not shown). Cholinesterases of vertebrates can be further distinguished with enzyme-selective inhibitors and BW284C51 is acetylcholine esterase-selective while iso-OMPA affects butyrylcholinesterase. The effect of these compounds at two concentrations (10 and 100 mM) on esterase activity was determined with the acetyl- and acetyl-b-methyl- thiocholine substrates (2 mM). The selectivity and effectiveness of the inhibitors in our hands was initially demonstrated using a purified preparation of acetylcholinesterase from bovine erythrocytes and a rat brain ‘microsomal’ fraction (Fig. 5A). The inhibitors were effective and selective as expected with BW284C51 inhibiting AChE, with little cross inhibition by iso-OMPA, and these effects were observed at the lower inhibitor concentration. Activity in ‘microsomal’ fraction of gill was not affected by iso-OMPA but was sensitive to BW284C51 inhibition although significantly less than in purified AChE (P < 0.001) or rat brain
Fig. 3. Comparison of esterase activity of mussel gill subcellular fractions with alternate alkylthiocholine substrates. Pooled gill from 15 mussels were homogenised and subjected to subcellular fractionation as in Fig. 1 (n=3). Esterase activity (nmol/min/mg proteinS.D.) was determined in the resulting fractions using alternate alkylthiocholine substrates (1 mM) as indicated.
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Fig. 4. Effect of acetylcholine (ACh) concentration on esterase activity (nmol/min/mg proteinS.D.) in ‘microsomal’ fraction of gill with alternative alkylthiocholines (2 mM) as substrate. Pooled gill from 15 mussels were homogenised and subjected to subcellular fractionation as in Fig. 1 (n=3).
‘microsomal’ preparation (P < 0.001). To further establish selectivity a preparation containing only butyryl cholinesterase (horse serum) was used. The results showed iso-OMPA inhibited hydrolysis of butyrylthiocholine while BW284C51 had no effect (not shown). To assess the potential of mussel AChE as a biomarker in the environment the effects of azamethiphos, a biocide used in the salmon farming industry, was investigated in vitro. Microsomes from gill of mussel were incubated up to 60 min with the compound at 3 and 10 mM prior to determination of enzyme activity. The compound inhibited the enzyme in both a time and concentration dependent manner (Fig. 6A). At 3 mM enzyme activity was inhibited by 80% after 60 min exposure while at 10 mM 80% inhibition was found after 5 min exposure and a limiting 95% inhibition was found by 25 min. Variation of inhibitor concentration indicated an IC50 of 100 mM azamethiphos in vitro (Fig. 6B).
4. Discussion The aim of this study was to identify a more sensitive target tissue or tissue fraction for assay of cholinesterase activity in mussel with a view to using this as a biomarker of OP inhibition. It was expected that cholinesterase activity would be highest in tissue innervated for movement and this guided the selection of foot and posterior adductor muscle but gill and digestive gland were also investigated. Whole animal was included for comparative purposes. Subcellular fractionation achieved partial purification of cholinesterase activity into various operationally described
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Fig. 5. Effects of selective inhibitors, (A) iso-OMPA (butyrylcholinesterase (BChE) inhibitor) and BW284C51 (acetylcholinesterase (AChE) inhibitor) on hydrolysis of acetylthiocholine (ASCh) 2 mM, in purified AChE (from bovine erythrocytes) and ‘microsomal’ fractions of rat brain and mussel gill. (B) Effect of BW284C51 on hydrolysis of acetyl-b-methylthiocholine (A-b-MTCh) in the same preparations (nmol/min/mg proteinS.D.). Gill from 15 mussels were homogenised and subjected to subcellular fractionation as in Fig. 1 (n=3).
fractions (‘nuclear’, ‘mitochondrial’, ‘microsomal’, ‘soluble’). While measurable activity was obtained in all tissues and fractions (Fig. 1A) ‘mitochondrial’ fraction of foot and ‘microsomal’ fraction of gill provided significantly greater specific cholinesterase activity than was measured in whole mussel fractions. It was expected that the highly innervated foot would have significant activity but its presence in ‘mitochondrial’ fraction is surprising. In vertebrate species AChE is found in postsynaptic membranes and might be expected to be recovered in ‘microsomal’ fractions. However, mussel foot was consistently resistant to mechanical homogenisation
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and it is likely that disruption was incomplete with the result that activity was recovered in the ‘mitochondrial’ fraction. The total activity recovered from this tissue fraction (Fig. 1B) was very low and coupled with its resistance to homogenisation, the tissue would be impractical for use in biomarker applications. Therefore although the data of Fig. 1A suggests that foot ‘mitochondrial’ fraction (98 nmol/min/mg protein) is the preparation of choice for biomarker measurements an analysis of the data in terms of enzyme units recovered suggests otherwise. In contrast a significant level of activity was recovered in the ‘microsomal’ fraction of gill, which also had the second highest specific activity (35 nmol/min/mg). ‘microsomal’ fraction of gill therefore appears to be the most suitable tissue/fraction for measurement of inhibition of cholinesterase activity using acetylthiocholine as substrate. This specific activity reflected at least a three-fold increase on rates found under similar assay conditions in gill S9 fraction by Mora, Fournier, & Marbonne, (1999) 9 nmol/min/ mg protein (M. edulis) and 7.5 nmol/min/mg protein (M. galloprovincialis). Escartin and Porte (1997) reported a specific cholinesterase activity in S12 fraction of gill (M. galloprovincialis) of 24 nmol/min/mg protein. Several other studies report highest specific activity in gill (Bocquene et al., 1990, Escartin & Porte, 1997, Najimi
Fig. 6. Effect of azamethiphos inhibition on esterase activity in ‘microsomal’ fraction of gill with acetylthiocholine (ASCh) 2 mM as substrate (A) time course with various concentrations of azamethiphos (nmol/min/mg proteinSEM) (B) effect of inhibitor concentration on hydrolysis of ASCh (nmol/min/mg proteinSEM). Pooled gill from 15 mussels were homogenised and subjected to subcellular fractionation as in Fig. 1 (n=3).
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et al., 1997) however, direct comparison is often confounded by differences between both sample preparation methods and use of varying arbitrary units to describe data. A number of strategies were adopted to establish the possible multiplicity of esterases in mussel. In the first of these the rate of acetylthiocholine hydrolysis was studied as a function of substrate concentration. Multiple enzymes using the same substrate but with different kinetic parameters generate non-linear Lineweaver– Burke plots (Cornish-Bowden, 1995). The Lineweaver-Burke plot for rates of activity measured in ‘soluble’ fraction of gill was non-linear suggesting more than one enzyme may be present (Fig. 2B). However, activity in this fraction was below a level suitable for biomarker use so this was not pursued further. In ‘microsomal’ fraction of gill linear Lineweaver–Burke plots showed no evidence for the presence of more than one enzyme (Fig. 2A). The apparent value of Km (40 mM) for this enzyme is similar to values reported previously (12 mM in S9 fraction of gill from Mytilus edulis (Mora, Fournier, & Narbonne, 1999b); 30 mM in S9 fraction of gill of M. galloprovincialis (Mora, Michel, & Narbonne, 1999a); 73 mM in whole animal for M. galloprovincialis (Najimi et al., 1997). In a second approach to detection of multiple enzymes in mussel, activity was measured in gill with alternate alkyl-substituted thiocholines. The ranked order of rates of hydrolysis with substituted alkylthiocholines was ASCh > PrSCh > A-bMSCh > BuSCh in all fractions (Fig. 3) and this was in agreement with studies using alternative enzyme preparations (Mora, Fournier, & Narbonne, 1999b; Talesa et al., 2001). A slightly different result was found in soluble fraction in which activity with BuSCh was marginally greater than with A-b-MSCh. Although a non-linear Lineweaver–Burke plot was generated for this fraction (Fig. 2B), these data do not provide sufficient evidence for two separate cholinesterase enzymes (a membrane-bound AChE and a separate soluble BuChE). In a variant of this experiment the ability of acetylcholine to compete for hydrolysis of the alkylthiocholine substrates in ‘microsomal’ fraction of gill was tested. Acetylcholine was equally effective as an inhibitor with each of the alkylthiocholines in a concentration dependent manner (Fig. 4). It may therefore be that a single enzyme type is present that is capable of binding and hydrolysing all of the alkylthiocholine substrates but with different rates. The effectiveness of the inhibitors BW284C51 and iso-OMPA (selective for AChE and BuChE) on gill ‘microsomal’ fraction was determined (Fig. 5A and B) and this provided a further test for the possibility of separate AChE and BuChE (Sturm et al., 1999). The absence of significant inhibition of cholinesterase activity in gill ‘microsomal’ fraction with iso-OMPA suggests detectable BuChE activity is not present in these preparations. A control experiment with purified BuChE from horse serum demonstrated the efficacy of iso-OMPA as a selective inhibitor of this enzyme (results not shown). Comparison of inhibition of gill ‘microsomal’ cholinesterase activity to that in purified AChE and rat brain ‘microsomal’ preparation by BW284C51, an inhibitor known to be AChE-specific in vertebrates indicated significantly less inhibition in gill at both concentrations (Fig. 5A). This difference in sensitivity of gill to BW284C51 suggests that cholinesterase activity present in this tissue fraction may have characteristics atypical of classical AChE. A further
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experiment with a supposedly more AChE-specific substrate, A-b-MSCh, produced very similar results reinforcing the view of a single AChE enzyme with atypical properties (Fig. 5B). A definitive view of the multiplicity of cholinesterase enzymes in mussel awaits genomic characterization. Investigation of the effects of azamethiphos showed that the dissolved OP inhibited gill ‘microsomal’ cholinesterases in a time- and concentration-dependent manner with an IC50 of approximately 100 mM in vitro (Fig. 6A and B). This value compares to toxicity results of LC50 0.6 mM in stickleback (96 h exposure), Gasterosteus aculeatus and LC50 of 30 mM in brine shrimp (24 h exposure), Artemia salina (Ernst et al., 2001). Direct comparison of our data with these other studies of the effects of azamethiphos is difficult because of the use of different biological endpoints. Speculating on the basis of the results in context of aquaculture, the dispersion of azamethiphos (administered therapeutic dosage 150 mM) by marine dilution would result in benign concentrations being available any further than one or two cage width distance from source. Repeated exposures however, may have more deleterious effects. Whilst inhibition of AChE in mussel gill has been demonstrated the effectiveness of the inhibitor in vivo still needs to be established. AChE is a classical endpoint for environmental toxicology studies and was originally introduced to reflect adverse effects of exposure to toxicants affecting the nervous system. The use of this enzyme in mussel is complicated by a number of factors including low activity in crude tissue homogenates. Recent work (Galloway, Millward, Browne, & Depledge, 2002; Moreira et al., 2001; Talesa et al., 2001) has suggested that haemolymph contains a more active AChE enzyme and that this may provide the necessary simplicity and sensitivity for monitoring purposes. While haemolymph provides a simple assay medium the origin and function of the enzyme activities in this preparation are unclear and may not be related to nervous system function. Thus the enzyme(s) of haemolymph may provide a biomarker of exposure but not necessarily of effect. Here we show that subcellular fractionation of gill will provide a toxicologically relevant membrane-bound preparation enriched for AChE activity relative to crude homogenates and which is inhibited by the OP compound azamethiphos. Since gill is a ‘first-pass’ target the approach described here provides a significant advance in providing a monitoring tool. It would be highly desirable to extend this study to investigate the effects of in vivo azamethiphos exposures on AChE in mussel to broaden our understanding of the environmental impact of this compound.
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