characterisation of choline esterases and their tissue and subcellular distribution in mussel...

Characterisation of choline esterases and theirtissue and subcellular distribution in

mussel (Mytilus edulis)

Margaret Browna,*, Ian M. Daviesb, Colin F. Moffatb,John Redshawc, John A. Crafta

aBiological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Rd, Glasgow G4 0BA, UKbFisheries Research Services Marine Laboratory, PO Box 101, 375 Victoria Road, Aberdeen, AB11 9DB, UK

cScottish Environment Protection Agency, 5 Redwood Crescent, Peel Park, East Kilbride G74 5PP, UK

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 organo-

phosphates (OP) in the marine environment. This study looked at cholinesterase activityin subcellular fractions of various tissues from the common mussel, Mytilus edulis. Measure-ment 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 thesecond highest specific activity with a useful level of recovery and therefore was the mostsuitable tissue fraction for biomarker applications. Comparative studies of alternative alkyl-thiocholine 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 gavean 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

Marine Environmental Research 57 (2004) 155–169

www.elsevier.com/locate/marenvrev

0141-1136/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/S0141-1136(03)00067-9

* Corresponding author. Tel.: +44-1413313222; fax: +44-1413313208.

E-mail address: [email protected] (M. Brown).

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1. Introduction

The expansion of salmon farming in Scotland and Ireland has greatly benefitedlocal economies but concerns have been raised about the effects aquaculture may behaving on marine ecosystems (Ernst et al. 2001; Gillibrand & Turreil, 1997; Wu1995). Intensive farming methods employed to enhance production in fish farmshave been associated with an increase in the incidence of sea-lice infestations infarmed stocks (Naylor et al., 2000). The medicines currently authorised for thecontrol of sea lice include the organophosphorous (OP) compound azamethiphosbut potential impacts of this compound on marine ecosystems remain uncertain. Astudy by Ernst et al. (2001), looking at toxicity to non-target aquatic organismsconcluded that ‘use of azamethiphos for sea lice control presents a low to moderateenvironmental risk’. However, sublethal effects may occur that could impact onprocesses such as differentiation, growth or reproduction. There is a pressing need tomonitor use and effects of OPs at a biochemical level in the marine environment andthus suitable sentinel species and relevant tests need to be identified. It has beensuggested (Galloway et al., in press; Rodger, Galgani & Truquet, 1999) that the bluemusselMytilus edulis, a semi-sessile filter feeder, is most suitable as a sentinel speciesto 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 crudehomogenate (Bocquene, Galgani & Truquet, 1990; McHenery, Linley-Adams,Moore, Rodger, & Davies, 1997; Robertson, Madden, Moore, & Davies, 1992), orthe soluble fraction recovered after treatment of gill tissue homogenate with deter-gent and/or phosphatidyl inositol phospholipase C (PIPLC) (Mora, Michel, &Narbonne, 1999) to release membrane-bound enzyme forms. However, cholinesteraseactivity 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) assubstrate. For instance it has been claimed that the enzyme in this species is notinhibited by exposure to OPs (Galgani & Bocquere, 1990). However, detectionof inhibition (reduced enzyme activity) is clearly compromised by assays whichhave low background, control activity and which may measure in parallel severalenzyme activities some of which may not be subject to inhibition. In vertebrates atleast 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 inparticular (Moreira, Coimbra, & Guilhermino, 2001; Talesa, Romani, Antognelli,Giovannini, & Rosi, 2001; Von Wachtendonk & Neef, 1979) has not been estab-lished. Results from a study by Mora, Fournier, & Narbonne, (1999), investigatingcholinesterase (ChE) forms in Mytilus edulis and Mytilus galloprovincialis, althoughnot conclusive appear to be consistent with the presence of only one pharma-cological 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

156 M. Brown et al. /Marine Environmental Research 57 (2004) 155–169

review by Fulton and Key (2001) raises the issue of whether low inhibition of musselcholinesterases to OPs by comparison to other species is due wholly to species-specificdifferences or signifies a requirement to measure activity in specific tissue frommussel and/or to account for cholinesterase types present which are not inhibited.Recently in marine vertebrates classification of cholinesterase types by substratepreferences and selectivity of inhibitors relevant to characteristics in mammals hasbeen carried out (Chuiko, 2000; Sturm, da Silva de Assis, & Hansen, 1999). Thismethodology can be applied to investigate characteristics of invertebrate cholin-esterases (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 throughimproved selection of target tissue and substrate. Tissue and subcellular distributionof cholinesterase activity in mussel are explored, allowing the differential assay ofmembrane-bound and soluble forms of cholinesterases. In addition, we have useddiagnostic 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 otherwisestated.

2.2. Animals

Farmed common mussels, Mytilus edulis (4–7 cm long), were obtained from acommercial shellfish farm in Loch Etive, Scotland. Animals were either transportedto holding tanks in an environmental room (9 �C) or were dissected on site. Trans-ported animals were held for at least 48 h prior to use. Tissues (gill, posterioradductor muscle, foot, digestive gland) were removed, snap-frozen in liquid nitrogenand 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
Substrate Inhibitor
Selective
Non-selective Selective Non-selective
Acetylcholinesterase

AChE (EC 3.1.1.7)

Acetyl-b-methylthiocholine

A-b-MSCh

Acetylthiocholine

ASCh

BW284C51
eserine
Butyrylcholinesterase

BChE (EC 3.1.1.8)

Butyrylthiocholine BuSCh
iso-OMPA
Propionylcholinesterase

PrChE

Propionylthiocholine PrSCh

M. Brown et al. /Marine Environmental Research 57 (2004) 155–169 157

2.3. Subcellular fractionation

Gill, posterior adductor muscle, foot and digestive gland tissues from each of 15mussels were pooled according to type in ice-cold isotonic sucrose (Solution A) (0.25M sucrose, containing 50 mM sodium phosphate buffer pH 7.6, 10 mM MgSO4 and 1mM EDTA). The tissues were then homogenised in ice-cold Solution A (3 ml g�1

wet weight tissue) using an Omni 2000 (Omni International, Virginia, USA) at speedsetting five for two 30 s bursts with a 30 s interval. Tissue from whole animalwas homogenised identically but in 2 ml g�1 wet weight tissue. Homogenates werecentrifuged (Beckman Avanti high speed centrifuge) at 700 g max for 10 min afterwhich the supernatants were transferred to clean tubes. These were then centrifugedat 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 Aand were centrifuged at 24 Kg max for 10 min in parallel with the 7000 gsupernatants. The resulting supernatants were ultracentrifuged (Centrikon T-2050)at 105 Kg max for 100 min. These procedures generated successively fractions whichare 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 priorto resuspension in the same medium using a glass-Teflon homogeniser. Theprotein content of each fraction was determined in triplicate by the method ofBradford (1976) (BIORAD) with bovine organophosphorous serine albumin (BSA)as standard. Absorbance at 595 nm was measured in a GeneQuantProspectrophotometer.

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 dissolu-tion in 0.1 M Tris–HCl (pH 7.6 at 20 �C) held on ice and used within 1 h ofpreparation. Enzyme activity was assayed with the various alkylthiocholine sub-strates in 0.09 M Tris–HCl (pH 7.6 at 20 �C) containing 0.7 mM 5,50-dithio-bis-(2-nitrobenzoic acid) (DTNB) as chromagen reagent. Reaction rates were quantifiedusing a Beckman DU650 measuring the rate of change of absorbance at 405 nmfrom 1 to 4 min after addition of enzyme at room temperature. The rate of spontan-eous substrate hydrolysis was found to be negligible, as was the rate of reaction ofDTNB with other thiols, and was disregarded. Various alkylthiocholine substratesacetylthiocholine (ASCh), acetyl-b-methylthiocholine (AbMSCh), butyrylthiocholine(BuSCh) and propionylthiocholine (PrSCh) selective for ChEs, acetylcholinesterase(AChE), butyrylcholinesterase (BuChE) and propionylcholinesterase (PrChE) wereused 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 tetra-isopropyl pyrophosphoramide, (iso-OMPA) are selective for ChE, AChE andBuChE respectively. Inhibition studies were conducted by incubation of enzyme


preparation with 10 or 100 mM inhibitor for 30 mins, after which residual activitieswere 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 aza-methiphos (Qmx Laboratories Ltd) was carried out in ‘microsomal’ fraction of gill.Enzyme preparations were incubated with the OP as described above for varioustimes between 0 and 60 min before addition of 1 mM ASCh as substrate andresidual activities measured.

3. Results

3.1. Subcellular fractionation

Cholinesterase activity of subcellular fractions from various tissues of mussel wasmeasured with acetyl thiocholine as substrate. Specific activities and recoveredactivities are shown in Fig. 1A and B respectively. Measurable activities were foundin all fractions of all tissues including the ‘soluble’ fractions. The specific activities ofeach of the fractions of digestive gland were low and were not significantly differentbetween fractions. Higher activities were found in the fractions of adductor muscleand foot with highest activity in the ‘mitochondrial’ fraction (P<0.001 compared togill ‘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 tes-ted. Higher activities than in digestive gland were also found in gill, but in gill thehighest activity was observed in the ‘microsomal’ fraction and the magnitude of thiswas second only to that of foot ‘mitochondrial’ fraction. The pattern of specificactivity 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 littleactivity was recovered in ‘mitochondrial’ or ‘microsomal’ fractions. The exception tothis was in the gill where significant activity was recovered in the ‘microsomal’fraction (equivalent to that of the ‘soluble’ fraction). For practical reasons the rela-tively high specific activity of ‘microsomal’ fraction of gill combined with therecovery of activity in this fraction determined its use in further studies. Acetyl-thiocholine esterase activity was measured in ‘microsomal’ fraction of gill with sub-strate concentration varied between 0.01 and 5 mM. The resulting kinetic plotsindicate a Michaelis Menten mechanism with saturation at substrate concentrationshigher than 1 mM and a linear Lineweaver–Burke plot (Fig. 2A). An investigationof activity in ‘soluble’ fraction of gill was also carried out. Activity was very low bycomparison to that in ‘microsomal’ fraction with saturation at substrate concentra-tions 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 presentwould not be useful for environmental monitoring purposes.


3.2. Kinetic studies with alternate substrates

Relative enzyme activities in subcellular fractions of gill were then determinedwith a series of alternate alkyl-substituted thiocholines (acetylthiocholine, acetyl-b-methylthiocholine, butyrylthiocholine and propionylthiocholine) (Fig. 3). Thedistribution of activity in the subcellular fractions with acetylthiocholine was simi-lar to that observed in the previous experiments (Fig. 1). In each of the fractionsactivity was highest with acetylthiocholine as substrate although activity withpropionylthiocholine was equivalent to that with acetylthiocholine in the ‘soluble’fraction. In ‘nuclear’, ‘mitochondrial’ and ‘microsomal’ fractions propionyl-, acetyl-b-methyl- and butyryl-thiocholines had approximately 60, 50 and 10% of the rates

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 protein�S.D., n=3) and (B) the activity recovered (nmol/min�S.D., n=3) in each frac-

tion. Each data point is the pooled data of triplicate determinations for two separate tissue fractionations.


with acetylthiocholine respectively, while in ‘soluble’ fraction this was 100, 50 and60%. To further investigate the relationship between esterase activities with thealternative substrates an inhibition study was conducted in which hydrolysis of thevarious alkylthiocholines was competed by the presence of acetylcholine (ACh) inthe gill ‘microsomal’ fraction. The relevant alkylthiocholine substrate was present ata fixed concentration of 1mM while the concentration of the acetylcholine comp-etitor was varied (0.0, 0.4, 1.0 mM) and the results are shown in Fig. 4. In the absenceof 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 protein�SEM, n=3) and corresponding

Lineweaver–Burk graphs. Pooled gill from 15 mussels were homogenised and subjected to subcellular

fractionation as described in section 2.


the original experiment (Fig. 3). The presence of acetylcholine decreased rates ofhydrolysis with all the alkylthiocholine substrates proportionally and in a concen-tration dependent manner with the exception of butyrylthiocholine. The experimentwas 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 canthus differentiate these activities from other esterases. The esterase activity of gill‘microsomal’ fraction with acetylthiocholine (1 mM) was measured after exposureto eserine (5 mM) for 30 min. Enzyme activity was inhibited by more than 90% (datanot shown). Cholinesterases of vertebrates can be further distinguished withenzyme-selective inhibitors and BW284C51 is acetylcholine esterase-selective whileiso-OMPA affects butyrylcholinesterase. The effect of these compounds at two con-centrations (10 and 100 mM) on esterase activity was determined with the acetyl- andacetyl-b-methylthiocholine substrates (2 mM). The selectivity and effectiveness ofthe inhibitors in our hands was initially demonstrated using a purified preparationof acetylcholinesterase from bovine erythrocytes and a rat brain ‘microsomal’ fraction(Fig. 5A). The inhibitors were effective and selective as expected with BW284C51inhibiting AChE, with little cross inhibition by iso-OMPA, and these effectswere observed at the lower inhibitor concentration. Activity in ‘microsomal’ frac-tion of gill was not affected by iso-OMPA but was sensitive to BW284C51inhibition 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 protein�S.D.) was determined in the resulting fractions

using alternate alkylthiocholine substrates (1 mM) as indicated.


‘microsomal’ preparation (P<0.001). To further establish selectivity a preparationcontaining only butyryl cholinesterase (horse serum) was used. The results showediso-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 theeffects of azamethiphos, a biocide used in the salmon farming industry, was investi-gated in vitro. Microsomes from gill of mussel were incubated up to 60 min with thecompound at 3 and 10 mM prior to determination of enzyme activity. The com-pound 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 exposurewhile 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 anIC50 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 frac-tion for assay of cholinesterase activity in mussel with a view to using this as a bio-marker of OP inhibition. It was expected that cholinesterase activity would behighest in tissue innervated for movement and this guided the selection of foot andposterior adductor muscle but gill and digestive gland were also investigated. Wholeanimal was included for comparative purposes. Subcellular fractionation achievedpartial purification of cholinesterase activity into various operationally described

Fig. 4. Effect of acetylcholine (ACh) concentration on esterase activity (nmol/min/mg protein�S.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).


fractions (‘nuclear’, ‘mitochondrial’, ‘microsomal’, ‘soluble’). While measurableactivity was obtained in all tissues and fractions (Fig. 1A) ‘mitochondrial’ fractionof foot and ‘microsomal’ fraction of gill provided significantly greater specific cho-linesterase activity than was measured in whole mussel fractions. It was expectedthat the highly innervated foot would have significant activity but its presence in‘mitochondrial’ fraction is surprising. In vertebrate species AChE is found in post-synaptic membranes and might be expected to be recovered in ‘microsomal’ frac-tions. However, mussel foot was consistently resistant to mechanical homogenisation

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 protein�S.D.). Gill from 15 mussels were homogenised and subjected to subcellular frac-

tionation as in Fig. 1 (n=3).


and it is likely that disruption was incomplete with the result that activity wasrecovered in the ‘mitochondrial’ fraction. The total activity recovered from this tis-sue fraction (Fig. 1B) was very low and coupled with its resistance to homogenisation,the tissue would be impractical for use in biomarker applications. Therefore althoughthe data of Fig. 1A suggests that foot ‘mitochondrial’ fraction (98 nmol/min/mgprotein) is the preparation of choice for biomarker measurements an analysis of thedata in terms of enzyme units recovered suggests otherwise. In contrast a significantlevel of activity was recovered in the ‘microsomal’ fraction of gill, which also had thesecond highest specific activity (35 nmol/min/mg). ‘microsomal’ fraction of gilltherefore appears to be the most suitable tissue/fraction for measurement of inhib-ition of cholinesterase activity using acetylthiocholine as substrate. This specificactivity reflected at least a three-fold increase on rates found under similar assayconditions 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 fractionof gill (M. galloprovincialis) of�24 nmol/min/mg protein. Several other studies reporthighest 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 protein�SEM) (B) effect of inhibitor concentration on hydrolysis of ASCh (nmol/min/mg

protein�SEM). Pooled gill from 15 mussels were homogenised and subjected to subcellular fractionation

as in Fig. 1 (n=3).


et al., 1997) however, direct comparison is often confounded by differences betweenboth sample preparation methods and use of varying arbitrary units to describedata.

A number of strategies were adopted to establish the possible multiplicity ofesterases in mussel. In the first of these the rate of acetylthiocholine hydrolysis wasstudied as a function of substrate concentration. Multiple enzymes using the samesubstrate but with different kinetic parameters generate non-linear Lineweaver–Burke plots (Cornish-Bowden, 1995). The Lineweaver-Burke plot for rates of activ-ity measured in ‘soluble’ fraction of gill was non-linear suggesting more than oneenzyme may be present (Fig. 2B). However, activity in this fraction was below alevel suitable for biomarker use so this was not pursued further. In ‘microsomal’fraction of gill linear Lineweaver–Burke plots showed no evidence for the presenceof more than one enzyme (Fig. 2A). The apparent value of Km (40 mM) for thisenzyme is similar to values reported previously (�12 mM in S9 fraction of gill fromMytilus edulis (Mora, Fournier, & Narbonne, 1999b); �30 mM in S9 fraction of gillof M. galloprovincialis (Mora, Michel, & Narbonne, 1999a); �73 mM in whole ani-mal for M. galloprovincialis (Najimi et al., 1997).

In a second approach to detection of multiple enzymes in mussel, activity wasmeasured in gill with alternate alkyl-substituted thiocholines. The ranked order ofrates of hydrolysis with substituted alkylthiocholines was ASCh>PrSCh>A-b-MSCh>BuSCh in all fractions (Fig. 3) and this was in agreement with studies usingalternative enzyme preparations (Mora, Fournier, & Narbonne, 1999b; Talesa et al.,2001). A slightly different result was found in soluble fraction in which activity withBuSCh was marginally greater than with A-b-MSCh. Although a non-linear Line-weaver–Burke plot was generated for this fraction (Fig. 2B), these data do not pro-vide sufficient evidence for two separate cholinesterase enzymes (a membrane-boundAChE and a separate soluble BuChE). In a variant of this experiment the ability ofacetylcholine to compete for hydrolysis of the alkylthiocholine substrates in ‘micro-somal’ fraction of gill was tested. Acetylcholine was equally effective as an inhibitorwith each of the alkylthiocholines in a concentration dependent manner (Fig. 4). Itmay therefore be that a single enzyme type is present that is capable of binding andhydrolysing all of the alkylthiocholine substrates but with different rates.

The effectiveness of the inhibitors BW284C51 and iso-OMPA (selective for AChEand BuChE) on gill ‘microsomal’ fraction was determined (Fig. 5A and B) and thisprovided a further test for the possibility of separate AChE and BuChE (Sturm etal., 1999). The absence of significant inhibition of cholinesterase activity in gill‘microsomal’ fraction with iso-OMPA suggests detectable BuChE activity is notpresent in these preparations. A control experiment with purified BuChE from horseserum demonstrated the efficacy of iso-OMPA as a selective inhibitor of this enzyme(results not shown). Comparison of inhibition of gill ‘microsomal’ cholinesteraseactivity to that in purified AChE and rat brain ‘microsomal’ preparation byBW284C51, an inhibitor known to be AChE-specific in vertebrates indicated sig-nificantly less inhibition in gill at both concentrations (Fig. 5A). This difference insensitivity of gill to BW284C51 suggests that cholinesterase activity present in thistissue fraction may have characteristics atypical of classical AChE. A further


experiment with a supposedly more AChE-specific substrate, A-b-MSCh, producedvery similar results reinforcing the view of a single AChE enzyme with atypicalproperties (Fig. 5B). A definitive view of the multiplicity of cholinesterase enzymesin mussel awaits genomic characterization.

Investigation of the effects of azamethiphos showed that the dissolved OP inhib-ited gill ‘microsomal’ cholinesterases in a time- and concentration-dependent man-ner with an IC50 of approximately 100 mM in vitro (Fig. 6A and B). This valuecompares to toxicity results of LC50 0.6 mM in stickleback (96 h exposure), Gaster-osteus 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 theeffects of azamethiphos is difficult because of the use of different biological end-points. Speculating on the basis of the results in context of aquaculture, the disper-sion of azamethiphos (administered therapeutic dosage 150 mM) by marine dilutionwould result in benign concentrations being available any further than one or twocage width distance from source. Repeated exposures however, may have moredeleterious effects. Whilst inhibition of AChE in mussel gill has been demonstratedthe effectiveness of the inhibitor in vivo still needs to be established.

AChE is a classical endpoint for environmental toxicology studies and was origin-ally introduced to reflect adverse effects of exposure to toxicants affecting the ner-vous system. The use of this enzyme in mussel is complicated by a number of factorsincluding low activity in crude tissue homogenates. Recent work (Galloway, Mill-ward, Browne, & Depledge, 2002; Moreira et al., 2001; Talesa et al., 2001) has sug-gested that haemolymph contains a more active AChE enzyme and that this mayprovide the necessary simplicity and sensitivity for monitoring purposes. Whilehaemolymph provides a simple assay medium the origin and function of the enzymeactivities in this preparation are unclear and may not be related to nervous systemfunction. Thus the enzyme(s) of haemolymph may provide a biomarker of exposurebut not necessarily of effect. Here we show that subcellular fractionation of gill willprovide a toxicologically relevant membrane-bound preparation enriched for AChEactivity relative to crude homogenates and which is inhibited by the OP compoundazamethiphos. Since gill is a ‘first-pass’ target the approach described here providesa significant advance in providing a monitoring tool. It would be highly desirable toextend this study to investigate the effects of in vivo azamethiphos exposures onAChE in mussel to broaden our understanding of the environmental impact of thiscompound.

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characterisation of choline esterases and their tissue and subcellular distribution in mussel...

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