Cholinesterase activities in the scallop Pecten jacobaeus: Characterization and effects of exposure to aquatic contaminants

SC IE N CE OF T HE TOT AL E N V I RO N ME N T 3 9 2 ( 2 00 8 ) 9 9–1 09

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v

Cholinesterase activities in the scallop Pecten jacobaeus: Characterization and effects of exposure to aquatic contaminants Bonacci Stefano⁎, Corsi Ilaria, Focardi Silvano Department of Environmental Sciences “G. Sarfatti”, University of Siena, Via P.A. Mattioli 4, I-53100 Siena, Italy

AR TIC LE I N FO

ABS TR ACT

Article history:

Nearshore marine environments of industrialized countries are increasingly threatened by

Received 11 February 2007

anthropogenic pollution. It is therefore a priority task to investigate the sensitivity of new

Received in revised form

ecotoxicological warning signals of the occurrence and effects of aquatic pollutants. The

26 November 2007

main aims of the present study were: 1) to characterize the biochemical properties of ChEs in

Accepted 26 November 2007

tissues of the bivalve Pecten jacobaeus, using different specific substrates and selective

Available online 3 January 2008

inhibitors; 2) to measure sensitivity of ChE activities to in vitro exposure to the OPs azamethiphos and DFP and to the heavy metals cadmium and zinc. Our final aim was to

Keywords:

carry out a preliminary evaluation of the suitability of ChEs measurement in tissues of the

Pollution

scallop for monitoring marine environmental quality and neurotoxic compounds

Monitoring

contamination in the Mediterranean Sea. Responses to specific inhibitors have suggested

Biomarker

that ChEs in adductor muscle share many characteristics with vertebrate acetylcholinesterase.

Cholinesterase

Dose-dependent inhibition of ChE was observed in response to in vitro exposure to

Bivalve

environmental contaminants such as cadmium and azamethiphos. Sensitivity to zinc and DFP was lower. ChEs in P. jacobaeus might therefore have potential as a sensitive biomarker for monitoring marine pollution. Results of the present study will be useful to focus further experiment of exposure to pollutants under in vivo conditions. Capsule: Cholinesterase activities in scallop Pecten jacobaeus were observed to be sensitive to contaminants in vitro and may therefore have potential as biomarkers for monitoring water pollution. © 2007 Elsevier B.V. All rights reserved.

1.

Introduction

The aquatic environment is threatened by increasing levels of contaminants originating from human activities (Ali and Sreekrishnan, 2001; Chouksey et al., 2004). This situation endangers the health of organisms including humans (Dawe, 1990; Ternes et al., 2007). The highest concentrations of hazardous chemicals are often measured in nearshore and anthropized areas (Perin et al., 1988; Carbery et al., 2006). The Mediterranean Sea is surrounded by highly industrialized

countries. It is considered a threatened marine area because of the accumulation of anthropogenic compounds which have negative effects on ecosystems (Kuetting, 1994; Bertolotto et al., 2003; Vizzini and Mazzola, 2006). A wide range of biological indicators (biomarkers) has been developed to detect and assess exposure and effects of contaminants at sublethal levels. Biomarkers are now routinely used in biomonitoring programs (Prichard et al., 1997; Moore et al., 2004; Hyne and Maher, 2003; Wang et al., 2005; Bonacci et al., 2007; De la Torre et al., 2007; Kopecka and Pempkowiak, 2007).

⁎ Corresponding author. Tel.: +39 0577 23 28 77; fax: +39 0577 23 28 06. E-mail addresses: [email protected] (B. Stefano), [email protected] (C. Ilaria), [email protected] (F. Silvano). 0048-9697/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2007.11.029

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Among aquatic biota, bivalves have often been chosen as bioindicator species for pollution monitoring and biomarker measurements due to their sedentary lifestyle and accumulation of pollutants (Andral et al., 2005; Cheggour et al., 2005). Cholinesterases (ChEs) are a class of serine hydrolases ubiquitous in the animal kingdom (Silver, 1974; Massoulié et al., 1993). ChEs are classified as acetylcholinesterase (AChE, EC 3.1.1.7) and the less specific butyrylcholinesterase (BChE, EC 3.1.1.8) (Silver, 1974; Massoulié and Toutant, 1998). AChE is known to play a major role in cholinergic neurotransmission, hydrolyzing the neurotransmitter acetylcholine (ACh) at cholinergic synapses (Toutant et al., 1985; Talesa et al., 2002). BChE seems to have no specific natural substrates and has been proposed as a scavenging enzyme for certain classes of toxic compound (Massoulié et al., 1993). In vertebrates, ChEs may be distinguished quite easily as their functional characteristics of substrate specificity and susceptibility to diagnostic inhibitors are very different (Austin and Berry, 1953; Sturm et al., 1999; Radic et al., 1991; Arufe et al., 2007; Sturm et al., 2007). Among selective inhibitors currently used to classify vertebrate ChEs, phenylmethylsulphonyl fluoride (PMSF) is a general inhibitor of enzymes having serine residues in the active site, such as ChEs (Galloway et al., 2002), while tetra (monoisopropyl) pyrophosphor-tetramide (Iso-OMPA) and 1,5-bis (4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284c51) are considered selective for AChE and BChE, respectively, in vertebrates (Silver, 1974; Massoulié and Toutant, 1998; Sturm et al., 1999; Varó et al., 2007). However, the classification used for vertebrates may be inappropriate for invertebrates such as bivalves (Walker and Thompson, 1991). In marine bivalves, ChEs have been clearly observed and recent studies have attempted to classify them by substrate preference and sensitivity to specific inhibitors. This led to the identification of different ChE isoforms which have different degrees of similarity to true vertebrate ChE (Galloway et al., 2002; Talesa et al., 2002; Valbonesi et al., 2003; Brown et al., 2004; Corsi et al., 2004a; Bonacci et al., 2006). Great variability of ChE enzyme characteristics has also been found between different mollusc species and different tissues of the same organism (Escartin and Porte, 1997; Basack et al., 1998; Mora et al., 1999; Talesa et al., 2001; Bonacci et al., 2004; Cunha et al., 2007). ChEs and especially AChE forms are target enzymes of organophosphorus (OP) and carbamate (CB) pesticides and measurement of ChE inhibition in bivalve species is widely used as a biomarker of exposure to and effects of these neurotoxic chemicals (Escartin and Porte, 1997; Galloway et al., 2002; Owen et al., 2002; Binelli et al., 2006; Cooper and Bidwell, 2006; Bolton-Warberg et al., 2007; Donnini et al., 2007). Among OPs, the effects of exposure to azamethiphos and diisopropylphosphorofluoridate (DFP) on ChEs have been studied in bivalves used for biomonitoring, enabling exhaustive comparison with data in other references (Talesa et al., 2001; Talesa et al., 2002; Brown et al., 2004; Corsi et al., 2007; Valdez Domingos et al., 2007). An increasing number of studies provide evidence that ChE activities may be affected by a wide range of contaminants other than OPs and CBs, including heavy metals, polycyclic aromatic hydrocarbons (PAHs) and components of complex mixtures of contaminants (Najimi et al., 1997; Kang and Fang, 1997; Cunha et al., 2005; Gaitonde et al., 2006; Oropesa et al., 2007; Raftopoulou et al.,

2006; Roméo et al., 2006; Vioque-Fernández et al., 2007; Elumalai et al., 2007; Frasco et al., 2007). A more general use of this biomarker for the assessment of environmental quality is therefore under way (Matozzo et al., 2005; Zaccaron da Silva et al., 2005; Magni et al., 2006; Hannam et al., in press; Humphrey et al., 2007; Ozmen et al., in press; Tsangaris et al., 2007). Among heavy metals, cadmium and zinc, which are often of environmental concern in polluted marine areas (Nendza et al., 1997; Vandecasteele et al., 2002; Adamo et al., 2005; Smaoui-Damaka et al., 2006), were observed to elicit dose-dependent responses in ChEs of marine mollusc species exposed either in vitro or in vivo (Najimi et al., 1997; Bonacci et al., 2006; Roméo et al., 2006). To sum up, there is the evidence in the literature that ChE measurements in marine bivalve are often sensitive biomarkers for pollution monitoring and to extend researches to uninvestigated species it is suggested, so that the above ecotoxicological tool may be implemented to the most suited species for each monitored ecosystem. Nevertheless, a gap does exist in actual scientific knowledge. In fact, ChE measurements have been extensively investigated in mussels and applied in biomonitoring studies but much less effort has been dedicated to other mollusc species. More knowledge is needed, even underlining the large differences in ChE sensitivity to pollutants of different bivalve species, as well as the occurrence of ChE isoforms resistant to insecticides (Park et al., 2004; Bonacci et al., 2006). This gap is also pertinent to species such as scallops. Our hypothesis is that scallop ChEs may be more sensitive than those of mussels, at least to exposure to certain classes of contaminants. It is based on promising preliminary results showing similar or higher sensitivity of scallops ChEs with respect to those of other bivalve species (Owen et al., 2002; Bonacci et al., 2004; Corsi et al., 2004b; Bonacci et al., 2006). Moreover, scallops have very different ecological and physiological features to other marine bivalves, especially Mytilidae (which have been the subject of most studies in this field). Because scallops occupy a different ecological niche with respect to mussels (for instance, scallops have a benthic lifestyle while Mytilidae are mainly sessile), scallops may be exposed to contaminants of different quality and quantity with respect to mussels in a given area. ChEs may therefore have different features in the above families (such as different activity levels and different sensitivity to contaminants). Thus, scallops may be useful as bioindicators in biomonitoring studies, together with mussels. Examples include S. Chandrinou et al. (2007), where Pecten jacobaeus was successfully used together with other bivalves as a bioindicator for monitoring organotin contamination in the Aegean Sea. Further investigation of this issue is therefore worthwhile and may help to optimize this widely used biomarker for pollution monitoring and aquatic environmental quality assessment. To accomplish this, it is necessary to design experiments to characterize of ChEs. Assays would involve exposure to specific inhibitors and dangerous aquatic pollutants. P. jacobaeus (Linnaeus, 1758) [class: Bivalvia, order: Ostreoida (Pteroida), family: Pectinidae] is an ecologically significant temperate scallop found on rocky and sandy bottoms from depths of 15 to over 100 m. Recent studies (Katsanevakis, 2005) also report juvenile specimens in

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shallow water (4–8 m). P. jacobaeus is a filter-feeder like other bivalve species used in biomonitoring programs, e.g. Mytilus galloprovincialis (Baumard et al., 1998; Pisoni et al., 2004) and Mytilus edulis (Bocquené et al., 1990; Zauke et al., 2003). Exposure to contaminants has been reported to affect physiological functions in scallops (Le Pennec and Le Pennec, 2001; Camus et al., 2002). Finally, P. jacobaeus is widely distributed in the Mediterranean Sea and occurs in exploitable quantities in the northern Adriatic (Pagotto and Zatta, 1985; Mojetta and Ghisotti, 1994). This area is subject to heavy anthropogenic pressure, as documented by several biomonitoring studies (Binelli and Provini, 2003; Petrović et al., 2004; Moret et al., 2005) and implementation of sensitive biochemical methods applicable to monitoring of environmental health is needed. Studies reporting on ChE activities in scallops are quite scarce. For instance, Owen et al. (2002) reported AChE and BChE activities in haemolymph of the tropical scallop Euvola (Pecten) ziczac. Inhibition of enzyme activity following in vivo exposure to the OP pesticide chlorpyrifos was observed and the authors suggested that assay of haemolymph ChE activities in E. ziczac may be a sensitive tool for assessment of exposure to OPs and CBs. ChE activities versus various substrates, such as acetylthiocholine iodide (ASCh), butyrylthiocholine iodide (BSCh) and propionylthiocholine iodide (PrSCh) were measured in tissues of the Antarctic species Adamussium colbecki, as well as its sensitivity to in vitro exposure to OPs (Bonacci et al., 2004; Corsi et al., 2004a; Bonacci et al., 2006). It therefore appeared interesting to investigate responses of ChEs in P. jacobaeus to well-known inhibitors such as OPs and dangerous aquatic pollutants such as heavy metals. In this context, in vitro exposure to determine the inhibitory potential of contaminants seems particularly useful to focus further expensive and time-consuming in vivo exposure studies. The general objective of the present work was to carry out a preliminary evaluation of the suitability of ChE measurements in tissues of the scallop P. jacobaeus for monitoring marine environmental quality and contamination due to neurotoxic compounds. To better address future studies of exposure to contaminants, we also characterized the biochemical properties of ChEs in tissues of the bivalve, using different specific substrates and selective inhibitors. The second step was to measure sensitivity of ChE activities to in vitro exposure to the OPs azamethiphos and DFP and to the heavy metals cadmium and zinc. Gills, adductor muscle and digestive gland were chosen as sources of enzymes as their ChE activities have been extensively investigated in other marine bivalves (Escartin and Porte, 1997; Mora et al., 1999; Brown et al., 2004). The final aim of the present and of future studies will be to include this new ecotoxicological tool in biomonitoring in the Mediterranean Sea alongside, being placed side by side with well-known sentinel species, such as mussels. Differences in ecology, physiology and response to contaminants between species of the two genera (scallop and mussel) are welldocumented (Peña-Llopis et al., 2002; Chandrinou et al., 2007; Leverone et al., 2007) and suggest that their simultaneous use may increase the effectiveness and flexibility of environmental quality assessment. The few existing studies in this direction are quite promising (Chandrinou et al., 2007).

2.

Materials and methods

2.1.

Sample collection

101

Ten specimens of P. jacobaeus were obtained by SCUBA diving off Chioggia in the northern Adriatic (north-eastern Mediterranean Sea) in September 2004. The scallops were immediately dissected and tissues (gills, digestive gland and adductor muscle) frozen in liquid nitrogen to prevent enzyme deterioration. They were then shipped to the laboratory and stored at −80 °C until analysis.

2.2.

Sample preparation

The following reagents were obtained from Sigma-Aldrich S.r.l.: ASCh, BSCh, PrSCh, DTNB, Tris–HCl, Triton X-100, Iso-OMPA, BW284c51 and PMSF. BIORAD protein assay reagent was purchased from Bio-Rad Laboratories Gmbh. Tissues from each specimens were pooled, homogenized 1:16 (w/v) with homogenization buffer (0.1 M Tris–HCl, pH 7.2, 0.25 M sucrose, 0.1% Triton X-100) and centrifuged at 2100 rpm for 10 min. The pellet containing cell debris was discarded. The supernatant was collected and used for determination of ChE activities preferably fresh or after storage at −80 °C. All procedures were carried out keeping tissues at 4 °C in all phases of sample preparation or on ice.

2.3.

Biochemical determinations

2.3.1.

Initial screening with different substrates

Total protein was measured according to Bradford (1976) and values were expressed in mg protein per ml supernatant. ChE activities were estimated by the method of Ellman et al. (1961). Various thiocholines (ASCh, BSCh, PrSCh) were used as substrates for preliminary screening of ChE types in tissues. Basal conditions in the reaction mixture (final volume 300 μl) of the ChE assay were as follows: 25 mM Tris–HCl buffer (pH 7.6, 1 mM CaCl2), 40 μl DTNB (0.333 mM, final concentration), 25 °C temperature and 40 μl supernatant. The reaction was started by adding 40 μl substrate (2 mM final concentration). Reaction rates were quantified using a BIO-RAD max tunable microplate reader (Model 550), measuring the rate of change in absorbance at 405 nm for 4.5 min after addition of substrate at 30 °C. Each sample was determined at least in quadruplicate and spontaneous hydrolysis was measured in the absence of supernatant. ChE activities were expressed as nanomoles of substrate hydrolyzed per minute per milligram of total homogenate protein (nmol min− 1 mg prot− 1).

2.3.2.

Effects of substrate concentration

ASCh was the only substrate assayed to determine optimum substrate concentration and inhibition tests, as it is the preferred substrate for true-AChE activity and the most commonly used to measure ChE activity. Substrate concentrations ranged from 0.05 to 10 mM.

2.3.3. In vitro effects of selective inhibitors and environmental contaminants Sensitivity of ChE versus ASCh activities to potential contaminants and laboratory chemicals was tested in vitro in

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(OPs azamethiphos and DFP and heavy metals cadmium and zinc) for 15 min after which residual activities, determined as previously described, were compared with those of samples incubated with carrier only. Effects of exposure were measured at seven/eight concentrations scaled by one order of magnitude, ranging from 10− 9 to 10− 2 M, a suitable range for discriminating IC50 values (if any). PMSF was tested at 10− 3 M only as this concentration is considered sufficient to discern the action of serine-dependent enzymes from that of spontaneous substrate hydrolysis (Galloway et al., 2002).

2.4.

Fig. 1 – Substrate specificity of cholinesterase activities in gills, adductor muscle and digestive gland of P. jacobaeus. For each substrate, values that do not share at least one letter (a, b, c) are significantly different. For each tissue, values that do not share at least one symbol (†, #) are significantly different.

P. jacobaeus. Inhibition studies were performed, incubating the reaction mixture with various concentrations of specific inhibitors (PMSF, BW284c51 and Iso-OMPA) or pollutants

Statistical analysis

The results were expressed as mean ± standard deviation. Statistical comparison among means was performed by oneway analysis of variance (ANOVA) or Kruskal–Wallis test. Differences with p b 0.05 were considered significant. Statistica 6.0 software (StatSoft Inc., 2001, USA) was used for data analysis.

3.

Results

3.1.

Initial screening with different substrates

Preliminary screening to determine ChE substrate preferences in the three tissues was carried out at the fixed substrate

Fig. 2 – Effects of substrate concentration on ChE versus ASCh activities and Lineweaver–Burke plots in tissues of P. jacobaeus.

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Table 1 – Effects of 15 min in vitro exposure to selective inhibitors of ASCh-cleaving ChE activity in different tissues from P. jacobaeus Tissue

PMSF (M)

BW284C51 (M)

Iso-OMPA (M)

10− 3

10− 10

10− 9

10− 8

10− 7

10− 6

10− 5

10− 4

10− 3

10− 9

10− 8

10− 7

10− 6

10− 5

10− 4

10− 3

10− 2

Gills

nd#

Adductor muscle Digestive gland

nd#

99 ± 4.8a # 95.8 ± 4.7a # 93.5 ± 4.2a ⁎ #

40.4 ± 3.7b ⁎ # 50.6 ± 6.7b ⁎ † 43.7 ± 2.8b #

13.7 ± 2.9c # 22.7 ± 2.9c d † 18.6 ± 2.8c $

8.4 ± 1.6d # 19.2 ± 1.8c d † 8.1 ± 3.1d #

11.1 ± 2.2c d # 16 ± 2.8c † 4.8 ± 2.2d $

15.8 ± 4.3e # 25.2 ± 5.5d † 7.9 ± 2.3d #

6.5 ± 1.9f # 20.8 ± 2.6c d † 2.9 ± 2.9d $

6.4 ± 1.3d f # 20.6 ± 2.9c d † NDe $

81.6 ± 10a ⁎ # 110.3 ± 6.6a † 100 ± 10.9a †

82.6 ± 6.5a # 93.3 ± 2.1a b # 74.9 ± 24.7a b ⁎ #

84 ± 5.2a # 97.4 ± 3.3a b † 69.4 ± 4.2a b $

76.1 ± 8.4a # 102.1 ± 3.6a b † 59.5 ± 2.5b $

83 ± 9.9a # 91.4 ± 5.9a b † 65.6 ± 23.6b $

81.8 ± 5.3a # 98.4 ± 9a b # 44.1 ± 7.5b †

87.6 ± 8.2a # 101.6 ± 4a b # 30.1 ± 11c †

50.4 ± 5.4b # 84.1 ± 13.6b † 21.9 ± 3.8c $

nd#

Values are means ± standard deviation of at least four determinations expressed as % residual activity with respect to samples exposed to carrier only. For each tissue, values that do not share at least one letter (a, b, c) are significantly different. For each inhibitor concentration, values that do not share at least one symbol (†, $, #) are significantly different. ⁎Exposure dose at which inhibition of activity starts to be significant ( p b 0.05) with respect to controls. ND not detected.

concentration of 2 mM. The results are shown in Fig. 1. The highest ChE activities were found in gills, followed by adductor muscle (for ASCh- and PrSCh-cleaving ChE activities) and digestive gland (for ChE versus BSCh activity). The highest ChE activities were observed when ASCh, PrSCh and either BSCh or PrSCh were used as substrates, in adductor muscle, digestive gland and gills, respectively.

3.2.

Effects of substrate concentration

The effects of ASCh concentrations ranging from 0.05 to 10 mM on ChE activities in gills, adductor muscle and digestive gland of P. jacobaeus are reported in Fig. 2. In all tissues, variations in substrate concentration strongly affected ChE activities and the patterns of response observed were quite similar. In gills, ChE versus ASCh activity increased sharply with increasing substrate concentrations in the range 0.05–0.5 mM. Increases in activity were less marked in the range 0.5–2 mM. A decrease or plateau in enzyme activity was observed at 3.0 mM ASCh, while activity increased again to substrate concentrations of 10 mM. In parallel, variations in activities of ChE versus ASCh in adductor muscle were similar to those in gills. Activity increased sharply with increasing substrate concentrations in the range 0.05–1.0 mM. Differences were much smaller for ASCh concentrations in the range 1.0–10.0,

and a plateau can be inferred. Similar results were obtained in digestive gland: dose-dependent increases in ChE versus ASCh activity were observed for ASCh concentrations in the range 0.05–1.0 mM, while a plateau occurred for higher substrate concentrations. Lineweaver–Burk plots for ChE versus ASCh activities in tissues of P. jacobaeus are also shown in Fig. 2. For the substrate concentrations tested, linear plots, without substrate inhibition, were displayed by ChE versus ASCh activity in gills and adductor muscle. Kinetic parameters KM and Vmax for ChE versus ASCh enzyme activity were 274.8 μM and 15.26 nmol min− 1 mg prot− 1, respectively, in gills and 233.9 and 12.85, respectively, in adductor muscle. Their ratios (Vmax/KM, ml min− 1 mg prot− 1), indicating the catalytic efficiency of the enzyme, were 5.55 × 10− 2 and 5.49 × 10− 2, in gills and adductor muscle, respectively. The plot of enzyme activity in digestive gland was not linear but inhibition by substrate did not occur.

3.3.

In vitro effects of selective inhibitors

Results are summarized in Table 1. Exposure to 10− 3 M PMSF resulted in total inhibition of ASCh-dependent ChE activities in all three tissues. Effects of exposure to BW284c51 in the concentration range 10− 10–10− 3 M on ChE activity resulted in IC50 = 3.08 × 10− 9, 4.56 × 10− 9 and 4.78 × 10− 9 M in gills, adductor muscle and digestive gland, respectively. Residual activities

Table 2 – Effects of 15 min in vitro exposure to OP pesticides azamethiphos and DFP on ASCh-cleaving ChE activity in different tissues from P. jacobaeus Tissue

Gills Adductor muscle Digestive gland

Azamethiphos concentration (M)

DFP concentration (M)

10− 9

10− 8

10− 7

10− 6

10− 5

10− 4

10− 3

10− 9

10− 8

10− 7

103.8 ± 4.8a # 98.0 ± 6.7a # 94.4 ± 16.2a #

100 ± 5.0a # 98 ± 8.1a # 98.2 ± 8a #

93.7 ± 6.1b ⁎ # 103.8 ± 2.4a † 90.8 ± 12.1a #

66.2 ± 7.4c # 99.4 ± 8.9a † 95.9 ± 11.6a †

32.8 ± 3.3d # 92.6 ± 4a † 74 ± 6.6b ⁎ $

26.2 ± 4.5e # 53.4 ± 7.5b ⁎ † 40.4 ± 15.6b # †

17.1 ± 3.1f # 17.9 ± 8.6c # 37.1 ± 3.9b †

111.1 ± 5.1a # 93 ± 3.6a ⁎ † 87.2 ± 2.4a ⁎ $

117.9 ± 12.7a # 92.7 ± 2.6a † 90.7 ± 8.6a †

83 ± 11.5b ⁎ # 92.5 ± 4.5a # 78.3 ± 6b †

†

10− 6

10− 5

10− 4

10− 3

90.2 ± 11.2b # 84.7 ± 5b # 75.5 ± 5.7c †

36.1 ± 7.6c # 84.1 ± 4.6b † 66 ± 7.1d $

28.3 ± 5.3c # 79.2 ± 2.4c † 52.2 ± 8.3e $

16.9 ± 3.7d # 66.7 ± 3.9d † 56 ± 2.4e $

Values are means ± standard deviation of at least four determinations expressed as % residual activity with respect to samples exposed to carrier only. For each tissue, values that do not share at least one letter (a, b, c) are significantly different. For each pesticide concentration, values that do not share at least one symbol (†, $, #) are significantly different. ⁎Exposure dose at which inhibition of activity starts to be significant ( p b 0.05) with respect to controls.

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Table 3 – Effects of 15 min in vitro exposure to heavy metals cadmium or zinc on ASCh-cleaving ChE activity in different tissues from P. jacobaeus Tissue

Cd concentration (M) −9

10 Gills Adductor muscle Digestive gland

87.8 ± 8a # 83.6 ± 13a # 87.1 ± 7.8a #

−8

10

88.1 ± 7.3a # 104.3 ± 10.8a b † 101.7 ± 10.9b †

−7

10

90.5 ± 7.8a # 114.5 ± 12.4b † 91.5 ± 10.9a #

−6

10

87.4 ± 5.1a # 102.4 ± 8.5a b † 89.5 ± 6.5a #

Zn concentration (M)

−5

−4

10

77.7 ± 7b ⁎ # 100 ± 11.9a b 89.3 ± 6.9a $

10

†

55.6 ± 8.8c # 78 ± 5.4a ⁎ † 57.9 ± 7c ⁎ #

−3

10

36.3 ± 5.2d # 45.3 ± 8.9c # 14.1 ± 3.3d †

−9

10

89.4 ± 6.3a # 104.2 ± 7.6a † 95.6 ± 9.7a # †

−8

10− 7

10− 6

10− 5

10− 4

10− 3

94 ± 7.6a # 106.3 ± 6.4a † 99.1 ± 5a # †

95.0 ± 10.5a # 103.2 ± 10.7a # 101.7 ± 5.2a #

97.9 ± 5a # 102.8 ± 6a # 96.7 ± 7.7a #

68.2 ± 8.3b⁎ # 109.8 ± 4.6a † 82.8 ± 7.1b ⁎ $

46 ± 6.8b # 89.5 ± 8.3a ⁎ † 21.1 ± 5.7c $

10

101.8 ± 8.1a # 100 ± 8.5a # 104.1 ± 6.2a #

Values are means ± standard deviation of at least four determinations expressed as % residual activity with respect to samples exposed to carrier only. For each tissue, values that do not share at least one letter (a, b, c) are significantly different. For each metal concentration, values that do not share at least one symbol (†, $, #) are significantly different. ⁎Exposure dose at which inhibition of activity starts to be significant ( p b 0.05) with respect to controls.

were measured in gills and adductor muscle (6.4% and 20.6% respectively) while doses of 10− 4 M BW2845c51 or over completely depleted enzyme activity in digestive gland. In the latter tissue, significant inhibition (− 6.5% with respect to controls) was observed even at the lowest exposure dose. Effects of exposure to Iso-OMPA in the concentration range 10− 9–10− 2 resulted in IC50 ≈ 10− 2, not detectable and 4.59 × 10− 6 M in gills, adductor muscle and digestive gland, respectively. In gills, ChE showed low sensitivity to Iso-OMPA doses below 10− 2 M, which elicited the highest inhibition (50.4% of residual activity). No significant inhibition was detected in adductor muscle for the tested concentration range of Iso-OMPA and IC50 could not be determined. In digestive gland a dose-dependent pattern of inhibition was found, with 21.9% of residual activity at the highest dose of inhibitor.

3.4.

In vitro effects of environmental contaminants

ChE versus ASCh activities in tissues of P. jacobaeus after 15 min in vitro exposure to OP insecticides azamethiphos and DFP are summarized in Table 2. Those after exposure to heavy metals (Cd and Zn) are summarized in Table 3. The effects of exposure to azamethiphos in the concentration range 10− 9– 10− 3 M resulted in IC50 = 6.53 × 10− 6, 1.76 × 10− 4 and 8.54 × 10− 5 in gills, adductor muscle and digestive gland, respectively. ASCh-cleaving ChE activity was reduced by exposure to azamethiphos in all tissues, but significant differences were observed. In gills the enzyme activity was significantly different from control for 10− 7 M of pesticide and decreased with a dose-dependent pattern for increasing azamethiphos concentrations. Residual activity with respect to control was 17.1% at the highest concentration tested. In adductor muscle, ASCh-cleaving ChE activity was almost unaffected by azamethiphos in the range 10− 9–10− 5 M. Significant inhibition was elicited by a dose of 10− 4 M azamethiphos with 17.9% residual activity with respect to control at the highest concentration tested. In digestive gland, a dose–response pattern of inhibition of ChE versus ASCh activity was observed. Significant inhibition with respect to control organisms only occurred above 10− 5 M. Exposure to 10− 3 M azamethiphos reduced enzyme activity to 37.1% of controls. The effects of exposure to DFP in the concentration range 10− 9–10− 3 M resulted in IC50 = 5.75 × 10− 6 in gills whereas 50% reduction in enzyme activity was never reached in the other tissues. Gills were the

only tissue in which IC50 could be estimated and also the tissue with the most irregular pattern of response to increasing doses of DFP. A residual activity of 16.9% (the strongest inhibition detected) with respect to control was measured at the highest concentration tested, with decreases becoming significant for doses of 10− 7 M and over. In adductor muscle, ASCh-cleaving ChE activity was significantly affected by DFP even at the lowest concentration, and a clearer dose-dependent response was observed. It was also the tissue in which the highest percentage of residual activity (66.7% with respect to control) was measured at the highest DFP dose. Similar results were obtained in digestive gland. As for heavy metal effects on ASCh-cleaving ChE activity (Table 3), exposure to cadmium in the concentration range 10− 9–10− 3 M resulted in IC50 = 9.66 × 10− 5, 5.58 × 10− 4 and 1.15 × 10− 4 in gills, adductor muscle and digestive gland, respectively. Residual ChE versus ASCh activities at the highest cadmium concentration tested were 36.3%, 45.3% and 14.1% of control, in gills, adductor muscle and digestive gland, respectively. Exposure to zinc in the concentration range 10− 9–10− 3 M resulted in IC50 = 9.50 × 10− 4 and 7.79 × 10− 4, in gills and digestive gland, respectively, whereas 50% reduction in enzyme activity was never reached in the adductor muscle. ASCh-cleaving ChE activity had similar patterns of response in gills and digestive gland, but less sensitivity in adductor muscle. In gills a residual activity of 46% (the strongest inhibition detected) with respect to control was measured at the highest concentration tested, decreases becoming significant above 10− 4 M. Similar results were obtained in digestive gland with a residual activity of 21.1% of control (the strongest inhibition) recorded at the highest concentration tested with decreases becoming significant at the two highest concentrations tested. In adductor muscle, ASCh-cleaving ChE activity was only significantly affected by the highest concentration of zinc, showing the lowest residual activity (89.5% of control).

4.

Discussion

4.1. Characterization of ChE activities: tissue distribution, substrate specificity and sensitivity to specific inhibitors To our knowledge this is the first time that ChE activities have been demonstrated in tissues of the Mediterranean scallop

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P. jacobaeus. Close parallels as regards basal levels of enzyme activities, substrate specificity and tissue distribution were found with scallop species such as the Antarctic A. colbecki (Bonacci et al., 2004; Corsi et al., 2004a), and other aquatic bivalves (Bocquené et al., 1990; Escartin and Porte, 1997; Najimi et al., 1997; Mora et al., 1999; Galloway et al., 2002; Valbonesi et al., 2003; Corsi et al., 2007). Our results with P. jacobaeus are also consistent with those recorded in other bivalve species as regards ChE dosedependence on substrate concentration (M. galloprovincialis, Mora et al., 1999; M. edulis, Galloway et al., 2002; A. colbecki, Bonacci et al., 2004; Dreissena polymorpha, Binelli et al., 2006) and levelling off and/or lack of clear substrate inhibition of ChE activity by ASCh excess (Corbicula fluminea, Basack et al., 1998; M. galloprovincialis and Ostrea edulis, Valbonesi et al., 2003). Our results suggest that different suites of ChE enzymes may occur in different tissues of P. jacobaeus, and that the same or similar ChE enzymes may be shared. These conclusions are sustained by differences in enzyme activities and characteristics of the three tissues. Linear and non-linear Lineweaver–Burke plots (Cornish-Bowden, 1995) indicate that ASCh-cleaving activities may be related to single enzymes in gills and adductor muscle and to multiple enzyme activities in digestive gland. Of course, as effects of variations in substrate concentration were only tested for ASCh, our results do not rule out that: 1) these ChEs might be characterized by lowsubstrate specificity and therefore also metabolise substrates other than ASCh; 2) other ChEs with high specificity for different substrates may be present. As regards sensitivity to specific inhibitors, it was observed that 10− 3 M PMSF led to total inhibition of ChE versus ASCh activities in all three tissues, demonstrating that hydrolysis of the substrates was due exclusively to the action of serinedependent enzymes and that spontaneous hydrolysis was negligible (Galloway et al., 2002). Sensitivity to BW284c51 was high, dose-dependent and with similar patterns in all tissues, while sensitivity to Iso-OMPA was much lower, especially in gills and adductor muscle, indicating that these particular ChEs share this characteristics with vertebrate AChE (Silver, 1974). Our results seem to strengthen the hypothesis of different ASCh-cleaving ChEs, characterized by different degrees of similarity to vertebrate AChE and BChE, in P. jacobaeus. ChEs in gills seem to have the characteristics of true AChE (sensitivity to BW284c51) and BChE (preference for substrates other than ASCh, partial sensitivity to Iso-OMPA). We therefore infer a variant of vertebrate AChE or an intermediate form between vertebrate AChE and vertebrate BChE. We suggest that ChE from adductor muscle of the Mediterranean scallop has most characteristics of true AChE, such as substrate preference for ASCh, high sensitivity to BW284c51 and resistance to IsoOMPA. We therefore infer an AChE very similar to vertebrate AChE. Our results in digestive gland suggest that at least two enzymes catalyse hydrolysis of ASCh, having characteristics of classic AChE (sensitivity to BW284c51) and BChE (preference for substrates other than ASCh, partial sensitivity to IsoOMPA). However exposure to the highest doses of BW284c51 resulted in total inhibition, whereas 21% of the activity was Iso-OMPA resistant. This suggests two distinct ChE isoforms: one typical AChE, sensitive to BW284c51 but not to Iso-OMPA,

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and an atypical form of ChE sensitive to both inhibitors. ChEs with non-classic properties, such as atypical response to selective inhibitors and/or overlapping substrate preference, have been reported in various studies in vertebrate (reviewed by Frasco et al., 2006; Jung et al., 2007; Varó et al., 2007) and invertebrate marine species (Bonacci et al., 2006; Cunha et al., 2007). As regards inhibition by substrate excess, which was not observed for the present enzymes, it is worth considering that in vertebrate AChE it is due to a secondary substrate-binding site, called the peripheral anionic site. It is located at the entrance of the active site of the enzyme, regulating catalyses in an allosteric fashion by binding a substrate molecule and resulting in conformational changes in the AChE active centre (reviewed by Marcel et al., 1998). It has been demonstrated that these effects are determined by the nature of the residue in position 330 of the signal transmission chain between the peripheral anionic site and the active site. Substrate inhibition occurs only when residue 330 is aromatic and the phenomenon is eliminated by a single mutation of the residue (reviewed by Moralev and Rozengart, 2001). We suggest that this may be the case of ChEs in P. jacobaeus. Further studies are programmed to obtain more information about this issue. It is difficult to speculate about the different physiological roles of the various ChEs. Even in vertebrates, where ChEs have been much more widely investigated, the role of BChE is still unclear. They are inferred to have protective functions, either by sequestering circulating anticholinergic compounds and thus decreasing their effect on classic AChE or by scavenging certain classes of toxic compounds (Russell and Overstreet, 1987). It has been suggested that non-classic AChE may at least partially fulfil the role of the deficient AChE (Massoulié et al., 1993; Xie et al., 2000). Similar inferences about a role of ChEs with non-classic AChE functions in synapses have also been formulated for the worm C. elegans (reviewed by Villatte and Bachman, 2002). We suggest that when only one ChE isoform occurs in a tissue, as seems to be the case in gills and adductor muscle of P. jacobaeus, its only or major role is presumably classic hydrolysis of the neurotransmitter ACh at cholinergic synapses. When two or more ChEs are present, such as in digestive gland of the Mediterranean scallop, those with non-classic AChE features presumably have different roles, such as scavenging neurotoxic compounds, since this is the tissue mainly dedicated to chemical bioaccumulation and detoxifying activities in invertebrates (Livingstone, 1991; den Besten, 1998). Further studies are needed to elucidate this point.

4.2.

Sensitivity to contaminants: in vitro exposure

The main aim of this study was a preliminary evaluation of the sensitivity of ChE activities in the scallop P. jacobaeus for monitoring exposure to contaminants. The present results show that ChE activities in tissues of the Mediterranean mollusc P. jacobaeus are affected by in vitro exposure to OP pesticides and heavy metals, in line with observations in other scallop species (Owen et al., 2002; Bonacci et al., 2004; Corsi et al., 2004a). Comparison of tissues showed that gills are probably more sensitive to OPs than the other two tissues, even if enzymes in digestive gland seem the most susceptible

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to DFP doses in the range 10− 9–10− 6 M. Digestive gland seems to be the most susceptible to heavy metals. Adductor muscle was much less sensitive than the other tissues to Zn exposure. Differences in ChE sensitivity between tissues and when different chemicals were tested have been repeatedly reported for marine bivalves (Escartin and Porte, 1997; Najimi et al., 1997; Talesa et al., 2001; Galloway et al., 2002; Valbonesi et al., 2003). Comparison with data for bivalve species in the literature suggests that ChEs more and less sensitive to OPs occur in other aquatic bivalves (Escartin and Porte, 1997; Mora et al., 1999; Galloway et al., 2002; Valbonesi et al., 2003; Canty et al., 2007; Corsi et al., 2007). In a previous study in the Antarctic scallop A. colbecki, we observed dose-dependent inhibition of ChE versus ASCh activity in gills after 5 min incubation with 0.1–100 μM chlorpyrifos, significant inhibition occurring even at the lowest concentration (Bonacci et al., 2004). On the other hand, low sensitivity to DFP was observed in the same species for doses up to 10− 3 M, which resulted in only 25.3% inhibition of ChE versus ASCh activity (Bonacci et al., 2006). As regards sensitivity to heavy metals, the IC50 calculated for ChE versus ASCh activity in adductor muscle of A. colbecki after in vitro exposure to CdCl2 was very similar to that found by us in P. jacobaeus. The same is true for residual ChE activity after exposure to 10− 3 M Cd (Bonacci et al., 2006). Najimi et al. (1997) found dose-dependent inhibition of ChE versus ASCh activity in whole body extract of two species of mussels (M. galloprovincialis and the African mussel P. perna) after 30 min in vitro exposure to increasing doses of heavy metals such as Cd and Zn. As significant inhibitory effects were observed for metal concentrations of 10− 3 M or higher and the IC50 was much higher in the two mussels (P. perna 9.3 × 10− 3 M and 1.0 × 10− 2 M for Cd and Zn exposure respectively; M. galloprovincialis 1.0 × 10− 2 M for Cd and Zn) than in P. jacobaeus, the Mediterranean scallop is presumably more sensitive to heavy metals. In that study the results of in vivo exposure were much more difficult to interpret, as significant decreases and enhancements of ASCh-cleaving ChE activity were detected. This may parallel the increase in ChE activity observed by us in adductor muscle of P. jacobaeus after exposure to Cd (significant for 10− 7 M Cd). Bocquené et al. (1990) reported 17% inhibition of ASCh-cleaving ChE activity in whole body of M. galloprovincialis after 40 min incubation with 10− 3 M cadmium, suggesting lower sensitivity than observed by us in P. jacobaeus. In the same study, a reduction was also observed after exposure to zinc, but the results are more difficult to interpret and a clear comparison with the scallop cannot be made. Relatively low sensitivity of ChE activity of bivalve species to exposure to Zn and DFP has also been observed in vivo (A. colbecki, ZnCl2, Corsi et al., 2004b) and in vitro (benthic clam Scapharca inaequivalvis, DFP, Talesa et al., 2002). To sum up, our results clearly show that ChE versus ASCh activity tends to have dose-dependent inhibition patterns in response to in vitro exposure to environmental contaminants such as azamethiphos and CdCl2 (at least in the tested concentration range), suggesting that ChE measurements would be used in P. jacobaeus for monitoring exposure to and effects of the above contaminants. Enzyme susceptibility may

somehow also be inferred with regard to exposure to ZnCl2 and DFP but sensitivity seems to be much lower and significant inhibition sometimes occurs only at the highest concentrations. The present study is only a first approach to the question. The results will be useful to set up experiments focused on enzyme sensitivity to exposure in vivo, with the final aim of validating ChEs in tissues of P. jacobaeus as a useful tool for monitoring environmental quality and exposure to pollutants in marine environments. Further studies should also investigate enzyme sensitivity to dangerous water pollutants which are not known as classic ChE-inhibitors, such as PAHs and PCBs, and discover which combinations of tissues and ChE substrates could be the most suitable biomarkers of exposure and effects for biomonitoring projects using P. jacobaeus. Specifically, molecular studies of the tissue distribution and characteristics of enzyme isoforms would be useful to identify the ChEs most sensitive to chemical exposure in the scallop and thus most appropriate for biomonitoring purposes. Finally, similarities with results obtained by Valbonesi et al. (2003) in the oyster O. edulis suggest that the two species may be combined as bioindicator species for assessing marine pollution in the northern Adriatic Sea. Regarding the implications for neurotoxicity in the Mediterranean scallop, the present results clearly show that ChEs in this species may be sensitive to exposure to contaminants. This may be of particular concern for gills, the primary organs of respiratory and ionic exchange in most aquatic animals, where the highest susceptibility was observed (Lyndon and Houlihan, 1998). A certain sensitivity was also found for digestive gland, but detoxification by resident enzymes should provide at least partial protection, as previously described. Adductor muscle is the tissue where the most resistant ChEs seem to occur and where the risk of neurotoxicity is therefore lower. To sum up, although it is arduous to correlate pollutant concentrations used for in vitro exposure and those effectively occurring in the environment, the present results show that increasing impact of neurotoxic compounds may be a serious threat for P. jacobaeus populations in the northern Adriatic. This observation is significant because the species occurs in areas subject to heavy pollution, and enhancement of neurotoxicity and inhibition of ChEs has been reported as a consequence of additive or more than additive interaction of different chemicals (Walker et al., 1993; Johnston, 1995; Tahara et al., 2005; Binelli et al., 2006).

5.

Conclusions

The present study assessed and characterized ChE activities in tissues of the Mediterranean scallop P. jacobaeus. Molecular polymorphism for ASCh-cleaving ChEs may reasonably be hypothesized in the digestive gland. Enzymes from different tissues seem to display different degrees of similarity with vertebrate AChE or BChE. ChEs which catalyse ASCh hydrolysis in adductor muscle seem to be those with most characteristics of true AChE. Further molecular characterization of single isoforms would be useful. Strong parallels were observed with results reported in other bivalves and any discrepancies were in line with the accepted view that large

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variations exist between species. Even if different degrees of sensitivity to disruption caused by in vitro exposure to pollutants were observed, the overall results suggest that ChE activities in tissues of P. jacobaeus are potentially sensitive biomarkers for monitoring exposure to (and probably effects of) pollutants in marine organisms and therefore merit further in vitro and in vivo exposure studies.

Acknowledgements The present study is part of S. Bonacci's PhD. thesis. We thank Helen Ampt for revising the English language. We also acknowledge an anonymous referee, whose insightful comments helped us to improve the paper.

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