Upload
others
View
5
Download
0
Embed Size (px)
Citation preview
1Q2
2
3
4Q3
5
6
7891011
121314151617181920
3940
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
Comparative Biochemistry and Physiology, Part C xxx (2015) xxx–xxx
CBC-08084; No of Pages 9
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part C
j ourna l homepage: www.e lsev ie r .com/ locate /cbpc
Neurotoxicological effects on marine mussel Mytilus galloprovincialiscaged at petrochemical contaminated areas (eastern Sicily, Italy):1H NMR and immunohistochemical assays
OF
Tiziana Cappello, Maria Maisano ⁎, Alessia Giannetto, Vincenzo Parrino, Angela Mauceri, Salvatore FasuloDepartment of Biological and Environmental Sciences, University of Messina, Viale F. Stagno d'Alcontres 31, 98166 Messina, Italy
⁎ Corresponding author. Tel.: +39 090 391435; fax: +E-mail address: [email protected] (M. Maisano).
http://dx.doi.org/10.1016/j.cbpc.2014.12.0061532-0456/© 2014 Published by Elsevier Inc.
Please cite this article as: Cappello, T., et acontaminated areas (eastern Sicily, I..., Comp
Oa b s t r a c t
a r t i c l e i n f o21
22
23
24
25Q426
27
28Q529
30
31
32
33
Article history:Received 29 October 2014Received in revised form 17 December 2014Accepted 23 December 2014Available online xxxx
Keywords:1H NMR metabolomicsCaged musselsCholinergic systemDopaminergic systemEnvironmental pollutionGillsImmunohistochemistrySerotoninergic system
34
35
36
37
38
ECTED PRThe neurotoxicological potential of environmental pollution, mainly related to petrochemical activities, wasinvestigated in marine mussel Mytilus galloprovincialis. Bivalve mollusks, particularly mussels, are widely usedas sentinel organisms in biomonitoring studies for assessing the impact of anthropogenic contaminants. Thegills, mainly involved in nutrient uptake, digestion, gas exchange and neuronal signaling, are the first organ tobe affected by pollutants present in the external environment, and therefore were selected as the target organfor this study. Mussels from an aquaculture farm were caged at a highly polluted petrochemical area and areference site along the Augusta coastline (eastern Sicily, Italy) for onemonth. A battery of biomarkers indicativeof neuronal perturbationswas applied on gills in order to investigate on the serotonergic (i.e. serotonin, 5-HT, andits receptor, 5-HT3R), cholinergic (i.e. acetylcholine, acetylcholinesterase, AChE, and choline acetyltransferase,ChAT), and dopaminergic systems (i.e. tyrosine and tyrosine hydroxylase, TH). Overall, impairment in the normalciliary motility was found in mussels caged at the polluted site. Alterations in serotoninergic and cholinergicsystems were revealed, with enhancement of dopaminergic neurotransmission resulting in a cilio-inhibitoryeffect. However, the over-expression in 5-HT3R and ChAT at cellular level may indicate an adaptive responseof mussels to recover a regular physiological activity in gills. To our knowledge, this is the first study that uses1H NMR and immunohistochemical assays. Their concurrent use demonstrated to be sensitive and effective forassessing environmental influences on the health status of aquatic organisms, and thus suitable to be appliedin ecotoxicological studies.
© 2014 Published by Elsevier Inc.
R
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
UNCO
R1. Introduction
It is generally recognized that the quality of coastal marineenvironments is greatly affected by a variety of anthropogenic activities.These activities result in the release of a mixture of contaminantsinto the aquatic ecosystems, and pose a threat to both animal andhuman health. In order to assess the harmful effects of environmentalpollutants, field studies have been conducted on aquatic organisms(Cajaraville et al., 2000; Matozzo et al., 2005; Rank et al., 2007;Viarengo et al., 2007; Sureda et al., 2011; Cravo et al., 2012; Fasuloet al., 2012a, 2012b; Kwon et al., 2012), used as biological indicatorsof the health status of aquatic environments. Bivalve mollusks, particu-larly mussels, are widely used in biomonitoring studies because theyare sedentary and sessilefilter-feeders, havingwide geographical distri-bution, ability to accumulate toxic chemicals, and suitability for cagingexperiments at field sites (Viarengo et al., 2007; Ausili et al., 2008;Fasulo et al., 2012b; Cappello et al., 2013a, 2013b). The use of cagedmussels from a single population with uniform starting size/age, allows
78
79
8039 090 6765556.
l., Neurotoxicological effects. Biochem. Physiol., C (2015)
the assessment of the combined effects of environmental pollutantmixtures at locations where suitable natural populations are notavailable. Therefore the mussel caging, as an in situ approach, hasbecome a recognized tool in ecotoxicological studies (Rank et al., 2007;Ausili et al., 2008; Tsangaris et al., 2010; Cappello et al., 2013a, 2013b).
In our previous studies (Fasulo et al., 2012b; Cappello et al., 2013a,2013b), we presented evidence of the adverse and compromisingeffects of petrochemical environmental pollution on mussel Mytilusgalloprovincialis caged at the “Augusta–Melilli–Priolo” industrial site(eastern Sicily, Italy). It is one of the largest and most complexpetrochemical sites in Europe,with very high levels of Hg and polycyclicaromatic hydrocarbons (PAHs) in sediments (Di Leonardo et al., 2007,2008). After caging the mussels, we found high concentrations ofnaphthalene and fluoranthene, indicative of pyrolytic origin of thePAHs, and benzo(a)pyrene and dibenzo(a,b)anthracene, indicative ofurban and industrial contamination, in mussel digestive gland tissue(Fasulo et al., 2012b). As biological effects of petrochemical pollution,we found severe morphological damage in the mussel digestive gland(Fasulo et al., 2012b) and gills (Cappello et al., 2013b), as well asmetabolic disturbance investigated by proton nuclear magneticresonance (1H NMR) spectroscopic analysis. In detail, mussel digestive
on marine mussel Mytilus galloprovincialis caged at petrochemical, http://dx.doi.org/10.1016/j.cbpc.2014.12.006
TD P
RO
OF
81
82
83
84
85
86
87
88
89
90
91
92
93
94Q6
95
96
97
98
99
100
101
102
103
104Q7
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
t1:1Q1t1:2
t1:3
t1:4
t1:5
t1:6
t1:7
t1:8
t1:9
t1:10
t1:11
t1:12
Fig. 1.Map showing location of the mussel caging sites, Priolo (37°12′10″N; 15°13′44″E) and Vendicari (36°47′35″N; 15°08′52″E).
2 T. Cappello et al. / Comparative Biochemistry and Physiology, Part C xxx (2015) xxx–xxx
CO
RREC
glands showed changes in metabolites involved in energy and lipidmetabolism (Fasulo et al., 2012b), and activation of detoxificationand antioxidant defense mechanisms, namely cytochrome P4504Y1(CYP4Y1) and glutathione S-transferase (GST), indicative of xenobioticdetoxification, and catalase (CAT) as oxidative stress index (Cappelloet al., 2013a). The changes observed in the 1HNMRmetabolomic profileof gills suggested alteration in osmotic regulation, energy metabolismand neurotransmission (Cappello et al., 2013b).
The gills of marine mollusks, mainly involved in nutrient uptake,digestion, gas exchange and neuronal signaling, have been used inecotoxicological studies as indicators of aquatic pollution (Gregoryet al., 2002; Gagné et al., 2007; Fasulo et al., 2008; Zhang et al., 2011;Ciacci et al., 2012; Cappello et al., 2013b). Gill activity is regulated bysympathetic and parasympathetic innervations of the autonomicnervous system running through the branchial connective tissue(Catapane et al., 1974), with various neurotransmitters participat-ing in regulation of ciliary beating (Stefano, 1990). Serotonin, or5-hydroxytryptamine (5-HT), involved in the serotonergic system,plays a cilioexcitatory activity in the gills of lamellibranchiates(Gosselin, 1961; Carroll and Catapane, 2007). The molluskan gill
UN 121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
Table 1Details of primary antibodies.
Antigen Animalsource
Supplier Dilution
Serotonin (5-HT) Mouse Dako Cytomation,Milan, IT
1:50
Serotonin receptor(5-HT3R)
Rabbit Sigma-Aldrich,St. Louis, MO, USA
1:100
Acetylcholine esterase(AChE)
Mouse Chemicon International,Temecula, CA, USA
1:50
Choline Acetyltransferase(ChAT)
Rabbit Abcam, Cambridge, UK 1:250
Tyrosine hydroxylase(TH)
Mouse Sigma-Aldrich,St. Louis, MO, USA
1:100
Please cite this article as: Cappello, T., et al., Neurotoxicological effectscontaminated areas (eastern Sicily, I..., Comp. Biochem. Physiol., C (2015)
Econtains significant amounts of endogenous 5-HT, which producesprompt, sustained, and reversible stimulation of ciliary beat frequency,and the magnitude of the response is graded over a wide range of 5-HTlevels (Gosselin, 1961). Impairment in the serotoninergic system mayoccur after exposure to toxicant compounds, as observed in gills of themussel M. galloprovincialis exposed to chromium and copper, resultingin increase of the 5-HT-stimulated adenylate cyclase activity in vivoand over-expression of 5-HT receptors (Fabbri and Capuzzo, 2006).
Acetylcholine is a neural transmitter used in efferent nervoussystems, and it is synthesized in the cytoplasm of cholinergic neuronsby the enzyme choline acetyltransferase (ChAT) and split into cholineand acetate in cholinergic synapses and neuromuscular junctions byacetylcholinesterase (AChE) (Matozzo et al., 2005). AChE is essentialfor the normal functioning of the central and peripheral nervous system(Lionetto et al., 2013). The measurement of AChE activity is a validatedbiomarker of neurotoxic compounds in aquatic organisms (Cajaravilleet al., 2000; Matozzo et al., 2005; Ciacci et al., 2012; D'Agata et al.,2014). AChE activity is directly inhibited by pollutants like organophos-phate and carbamate pesticides (Lionetto et al., 2013), even thoughits responsiveness to other chemicals, such as heavy metals andhydrocarbons, has been demonstrated (Rank et al., 2007).
Tyrosine is a non-essential amino acid that serves as substrateprecursor for the synthesis of catecholamines, which includeadrenaline, noradrenaline, and dopamine. The conversion of tyrosineinto catecholamines is catalyzed by the enzyme tyrosine hydroxylase(TH), involved in the adrenergic–dopaminergic system that, inMytilus, has an inhibitory effect on lateral cilia beating (Zhu et al.,2005). TH is a mixed-function oxidase that uses molecular oxygenand tyrosine as its substrates, and biopterin as its cofactor (Shiman et al.,1971). TH activity may be affected by toxic compounds present in theenvironments, as reported by Ciacci et al. (2012) with a noticeabledose-dependent increase of TH in M. galloprovincialis exposed to Cr(VI),resulting in an enhancement of dopaminergic neurotransmission.
The aim of this study was to assess the neurotoxic potential ofenvironmental pollution, mainly related to Hg and PAHs, in marine
on marine mussel Mytilus galloprovincialis caged at petrochemical, http://dx.doi.org/10.1016/j.cbpc.2014.12.006
T
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159Q8
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184Q9
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
Fig. 3. 1H NMR measurements of serotonin (mM) from mussel gill tissue, expressed asmeans ± standard deviation (n = 12). Asterisks indicate significant differences relativeto control (**p b 0.005).
Fig. 2.Western blot (WB) of proteins from (A)whole cell lysates and (B)mussel gill tissueextracts. SKBR-3, HeLa and PC-12 whole cell lysates were used as WB positive control forAChE, ChAT and TH, respectively.
3T. Cappello et al. / Comparative Biochemistry and Physiology, Part C xxx (2015) xxx–xxx
UNCO
RREC
musselM. galloprovincialis caged in an eastern Sicily petrochemical area.In order to extend our previous 1H NMR findings (Cappello et al.,2013b), a battery of biomarkers indicative of neuro-endocrinal pertur-bations was applied on mussel gills in order to investigate on the(i) serotoninergic (i.e. 5-HT and its receptor, 5-HT3R), (ii) cholinergic(i.e. acetylcholine, AChE and ChAT), and (iii) dopaminergic systems(i.e. tyrosine and TH). The environmental metabolomics, based onNMR spectroscopy, is a qualitative and quantitative approach that canbe used to accurately determine metabolite concentration with a veryhigh sensitivity (Hines et al., 2007; Espina et al., 2009; Kwon et al.,2012). To our knowledge, this is the first study that has used 1H NMRmeasurements of neurotransmitter concentrations in combinationwith immunohistochemical assays.
2. Materials and methods
2.1. Study area
As reported previously (Ausili et al., 2008; De Domenico et al., 2011,2013; Fasulo et al., 2012b), the petrochemical area of “Augusta–Melilli–Priolo” was declared a “site of national interest” by the Italian Ministryof Environment (Law No. 426/98; Ministerial Decree of 10.01.2000)owing to the high level of pollution, mainly due to Hg and PAHs, andsubsequent risk for human health. Conversely, the natural reserveof Vendicari (southeastern Sicily) was considered as a reference sitenot known to be impacted by petrochemical contamination (Fig. 1).Physical and chemical parameters of water (i.e. temperature, salinity,pH, dissolved oxygen) were measured at both sampling sites at thecage deployment depth (8 m) three times, as reported in Cappelloet al. (2013a).
2.2. Experimental design
The experimental design was performed as detailed in our previouspapers (Fasulo et al., 2012b; Cappello et al., 2013a, 2013b). Briefly,mussels M. galloprovincialis (mean shell length: 6.1 ± 0.54 cm) werepurchased in October 2009 from an aquaculture farm in Goro (Ferrara,Italy), named Consortium of Fishermen. After maintaining in aeratedseawater in the laboratory for one week, mussels were transplanted tothe two selected sites at 8 m depth in stainless steel cages (about 200specimens at each site) for 30 days. The translocation period of onemonth allows the adaptation of the organisms to the new environmen-tal conditions, and it is used in a number of national and internationalMussel Watch programs (i.e. RAMOGE). From each area, twelve maleindividuals, sexed by microscopic observation of gonad tissue, of com-parable body length and mass (6.6 ± 0.46 cm shell length; 27.8 ±3.2 g wet weight) were selected randomly and sacrificed. Gill sampleswere rapidly excised and flash-frozen in liquid nitrogen, then trans-ferred to the laboratory and stored at−80 °C prior to the NMR analysis.Further, small pieces of each dissected tissuewere taken formorpholog-ical and immunohistochemical analyses.
2.3. 1H NMR-based metabolomic analysis
Polarmetabolites were extracted from gills (n=12 per site) using a“two-step” methanol/chloroform/water procedure (Wu et al., 2008),as previously described (Cappello et al., 2013b). Briefly, the gill tissue(ca. 100 mg) was homogenized in 4 ml/g of cold methanol and0.85 ml/g of cold water by an Ultraturrax homogenizer. The homog-enates were transferred to glass vials, and 4 ml/g chloroform and2 ml/g water were added. Samples were vortexed and centrifugedfor 5 min at 2000 g at 4 °C. The upper methanol layer with thepolar metabolites was transferred to glass vials, dried in a centrifugalvacuum concentrator (Eppendorf 5301) and stored at −80 °C. Prior toNMR analysis, the dried polar extracts were resolvated in 100 μl ofD2O (Armar AG, Döttingen, Switzerland) buffered in 240 mM sodium
Please cite this article as: Cappello, T., et al., Neurotoxicological effectscontaminated areas (eastern Sicily, I..., Comp. Biochem. Physiol., C (2015)
ED P
RO
OF
phosphate, pH 7.0, containing 1 mM 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) (Sigma-Aldrich Co) as chemical shift standard. Fiftymicroliters of each resuspended sample was then pipetted into4 mm-diameter zirconia rotors.
NMR analyses were performed using a Bruker Avance-700 NMRspectrometer operating at 6000 Hz at 298 K, and equipped with aHigh Resolution-Magic Angle Spinning (HR-MAS) probe used for ourconvenience. One-dimensional (1-D) 1H NMR spectra were obtainedas previously described (Cappello et al., 2013b), and manually phased,baseline-corrected, and calibrated (DSS at 0.0 ppm) using XWIN-NMRsoftware (version 3.5; Bruker). The peaks of interest (i.e. serotonin,acetylcholine and tyrosine) within the 1H NMR spectra were identifiedand quantified using Chenomx Profiler, a module of Chenomx NMRSuite software (version 5.1; Chenomx Inc., Edmonton, Canada), whichuses the concentration of a known DSS signal to determine theconcentrations of individual metabolites (Kwon et al., 2012).
2.4. Immunohistochemical analysis
Gill tissues of twelve mussels from each sampling site were fixed in4% paraformaldehyde (Immunofix, Bio-OpticaMilano, Italy) for 4 h, andthen rinsed in 0.1 M phosphate buffered saline (PBS, pH 7.4). After de-hydration in a graded series of ethanol and embedding in Paraplast(Bio-Optica Milano, Italy), 5 μm thick histological sections were cutwith a rotary automatic microtome (Leica Microsystems, Wetzlar,Germany), mounted on glass slides and processed for indirect immuno-fluorescence method (Mauceri et al., 1999) for localization of 5-HT andits receptor, 5-HT3R, AChE and ChAT, and TH. Briefly, non-specificbinding sites for immunoglobulins were blocked with normal goatserum (NGS) in PBS (1:5) by incubation for 1 h. The sections werethen incubated overnight at 4 °C in a humid chamber in the presenceof the primary antisera (dilutions and suppliers as indicated inTable 1). After a rinse in PBS for 10 min, binding sites of the primaryantibodies were visualized by corresponding fluorescein isothiocyanate
on marine mussel Mytilus galloprovincialis caged at petrochemical, http://dx.doi.org/10.1016/j.cbpc.2014.12.006
UNCO
RRECT
227
228
229
230
231
232
233
234
235
236
237
Fig. 4. Immunohistochemical investigation of the serotoninergic system inmussel gills. (A) Mus(B) individuals caged at Priolo displayed positive fibers. Scale bars, 20 μm. (E)Mean and standarcontrol (*p b 0.0001). (C) Control mussels displayed numerous 5-HT3R immunolabeled fibers apertrochemical site showed numerous positive cells and hemocytes, and few fibers. Scale bars,significant differences relative to control (*p b 0.0001).
t2:1 Table 2t2:2 1H chemical shift and percent changes in neurotransmitter concentrations betweent2:3 polluted and reference site caged mussel groups, presented together with theirt2:4 significance (Student's t-test). Letters in parentheses denote the peak multiplicities:t2:5 s, singlet; d, doublet; t, triplet; dd, doublet of doublet; m, multiplets.
t2:6 Metabolitest2:7 (Chemical formula)
Chemical shift and peakshape (ppm)
Changes (%) p-Value
t2:8 Serotonin (C10H12N2O) 3.10(t), 3.30(t), 6.87(dd),7.09(d), 7.28(s), 7.41(d)
42% ↓ b0.005
t2:9 Acetylcholine (C7H16NO2) 2.15(s), 3.20(s), 3.75(t) 64% ↓ b0.05t2:10 Tyrosine (C9H11NO3) 3.04(m), 3.18(m), 3.93(m),
6.89(d), 7.19(d)35% ↓ b0.05
4 T. Cappello et al. / Comparative Biochemistry and Physiology, Part C xxx (2015) xxx–xxx
Please cite this article as: Cappello, T., et al., Neurotoxicological effectscontaminated areas (eastern Sicily, I..., Comp. Biochem. Physiol., C (2015)
(FITC)-conjugated goat anti-rabbit IgG (Sigma), and tetramethylrodamineisothiocyanate (TRITC)-conjugated goat anti-mouse IgG (Sigma),diluted 1:50 for 2 h at room temperature. Positive controls for labelingspecificity of each peptide were performed by incubating sections withantiserum pre-absorbed with the respective antigen (10–100 g/ml).The pre-absorption procedures were carried out overnight at 4 °C. Inaddition, negative controls were also performed by substitution ofnon-immune sera (without antibodies) for the primary antisera.
All observations were made on five fields of one section per sampleusing a 40× oil-immersion objective with a motorized Zeiss Axio ImagerZ1 epifluorescence microscope (Carl Zeiss AG, Werk Göttingen, Germany),
ED P
RO
OF
sels caged at the reference site showed an intense 5-HT positivity in cells and fibers, whiled deviation (S.D.) of 5-HT positive cells. Asterisk indicates significant differences relative tolong the branchial epithelium and very few positive cells, while (D) mussels caged at the20 μm. (F) Mean and standard deviation (S.D.) of 5-HT3R positive cells. Asterisk indicates
on marine mussel Mytilus galloprovincialis caged at petrochemical, http://dx.doi.org/10.1016/j.cbpc.2014.12.006
T
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
Fig. 5. 1H NMRmeasurements of acetylcholine (mM) frommussel gill tissue, expressed asmeans± standard deviation (n= 12). Asterisk indicates significant differences relative tocontrol (*p b 0.05).
5T. Cappello et al. / Comparative Biochemistry and Physiology, Part C xxx (2015) xxx–xxx
UNCO
RREC
equipped with an AxioCam digital camera (Zeiss, Jena, Germany)for the acquisition of images. Sections were imaged using theappropriate filters for the excitation of FITC (480/525 nm) andTRITC (515/590 nm), and then processed by using AxioVisionRelease 4.5 software (Zeiss).
2.5. Western blotting
The specificity of the antibodies selected for this study was WesternBlot (WB) tested in marine mussels. According to the manufacturer'srecommendation, the antibodies suitable for use in WB analysis wereonly those against AChE, ChAT, and TH. SKBR-3, HeLa and PC-12whole cell lysates were used as WB positive control for anti-AChE,anti-ChAT and anti-TH antibodies, respectively, as suggested by theantibody suppliers.
Gill tissues were homogenized in a lysis buffer (20 mM Tris-HCl, 2%SDS, 5 M NaCl, 1 MMgCl2, 1 mM EDTA, 1 mM EGTA, pH 7.4) containingprotease inhibitormixture (Sigma-Aldrich) for 10min at 50 vibrations/susing a TissueLyser LT bead mill (Qiagen) with 3.2 mm stainless steelbeads, and centrifuged at 3000 g at 4 °C. The protein concentration ofthe supernatants was determined by using BCA Protein assay (Thermoscientific). Samples (40 μg total protein) were separated by SDS-PAGE(12%) and after electrophoresis, the proteins were transferred to apolyvinylidene difluoride (PVDF) membrane 0.45 μm (BioRad),successively stained with Ponceau red. After blocking the membranein 5% bovine serum albumin in TPBS (PBS pH 7.4, 0.1% Tween 20), theblot was incubated overnight at 4 °C with appropriate polyclonalantibodies against different proteins (1 μg/ml). The blot was thenwashed and incubated with goat anti-rabbit IgG or goat anti-mouseIgG conjugated to peroxidase (Sigma-Aldrich St. Louis, MO, USA).Antibody binding was detected by chemiluminescence stainingusing the ECL detection kit (BioRad).
2.6. Statistical analysis
The concentrations of the three neurotransmitters of interest,i.e. serotonin, acetylcholine and tyrosine, were expressed in mMas means ± standard deviation (S.D.), and Student's t-test wasapplied to determinate significant differences between the two cagedmussel groups induced by environmental pollutants, by using theGraphPad software (Prism 5.0, San Diego CA, USA). For 1H NMR-basedmetabolomic analysis, statistical significance was accepted at p b 0.05.
Quantification of immunoreactive cells was performed by countingthe number of positive cells on five fields per section using Axio VisionRelease 4.5 software (Zeiss, Göttingen, Germany). All the obtaineddata were statistically processed with one-way analysis of variance(ANOVA) using the GraphPad software (Instat, La Jolla, CA, USA),applying Student's two-tailed t-test for unpaired data. For immu-nohistochemical analysis, statistical significance was accepted atp b 0.0001.
3. Results
3.1. Serotoninergic system
A quantitative comparison of 1-D 700 MHz 1H NMR spectra of gilltissue extracts from themusselM. galloprovincialis revealed a significant42% depletion in serotonin level (p b 0.005) in mussels caged at thepetrochemical area in respect to mussels caged at Vendicari (Fig. 3).Proton chemical shift and percent change in serotonin concentrationare reported in Table 2.
In the gill tissue of specimens caged at Vendicari an intense 5-HTimmunoreactivity was seen in a number of ciliated cells and severalfibers (Fig. 4A). In contrast, the expression of 5-HT in the gills ofindividuals acclimatized in Priolo was found almost solely along the
Please cite this article as: Cappello, T., et al., Neurotoxicological effectscontaminated areas (eastern Sicily, I..., Comp. Biochem. Physiol., C (2015)
ED P
RO
OF
fibers (Fig. 4B). Statistical analysis of immunohistochemical results for5-HT (p b 0.0001) is shown in Fig. 4E.
The immunodetection of the serotonin receptor in mussels caged atthe reference site revealed a number of intensely immunolabeled fibersalong the branchial epithelium and very few positive cells (Fig. 4C).On the contrary, the gills of mussels caged at the petrochemical siteshowed numerous cells, hemocytes underlying the gill epithelium,and few fibers immunopositive to 5-HT3R (Fig. 4D). Statistical analysisof immunohistochemical results for 5-HT3R (p b 0.0001) is shown inFig. 4F.
3.2. Cholinergic system
Proton NMR-based metabolomic analysis showed a significantdecrease of 64% in acetylcholine concentration (p b 0.05) in gills ofmussels transplanted at Priolo relative tomussels caged at the referencesite (Fig. 5). Proton chemical shift and percent change in acetylcholinelevel are reported in Table 2.
The immunohistochemical investigation of AChE in mussels cagedat the reference site showed a high number of immunopositivecells, regularly distributed along the gill epithelium, and no AChEimmunopositive fibers (Fig. 6A). Conversely, in gills of mussels trans-ferred to the polluted area few cells limited to the area in which thestructural integrity was maintained were immunopositivite to AChE.However, a great number of AChE-immunopositive hemocytes distrib-uted along the epithelium was noticed (Fig. 6B). Statistical analysis ofimmunohistochemical results for AChE (p b 0.0001) is shown in Fig. 6E.
By using the antibody directed against ChAT, a low number ofcells and several fibers lining the branchial epithelium were foundto be immunopositive in mussels caged at the reference site (Fig. 6C).On the contrary, in mussels caged at the petrochemical site a veryhigh number of ChAT-immunopositive cells, mostly frontal cells situat-ed at the apex of the gill filament, were observed (Fig. 6D). Statisticalanalysis of immunohistochemical results for ChAT (p b 0.0001) isshown in Fig. 6F.
3.3. Dopaminergic system
Data from 1H NMR-based metabolomic analysis reported thattyrosine significantly declined by 35% (p b 0.05) in gills of musselscaged at the polluted site relative to mussels caged at the referencesite (Fig. 7). Proton chemical shift and percent change in tyrosine levelare reported in Table 2.
Inmussels caged at the reference site few TH-immunopositivite cellswere detected along the gill epithelium (Fig. 8A), while in the gills ofindividuals acclimatized in Priolo a number of TH-immunopositivecells and hemocyteswere observed, resulting in an intense TH immuno-reactivity (Fig. 8B). Statistical analysis of immunohistochemical resultsfor TH (p b 0.0001) is shown in Fig. 8C.
on marine mussel Mytilus galloprovincialis caged at petrochemical, http://dx.doi.org/10.1016/j.cbpc.2014.12.006
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
6 T. Cappello et al. / Comparative Biochemistry and Physiology, Part C xxx (2015) xxx–xxx
3.4. Western blotting
Immunoreaction with AChE antibody revealed the predicted bandof 68 kDa both in the SKBR-3 extract and mussel gills. Specific ChATimmunoreactive bands of molecular mass of 70 kDa, corresponding tothe expected size for ChAT, were observed in both HeLa and musselgill extracts. Finally, the antibody against TH in the PC-12 whole celllysate revealed the presence of the predicted band of approximately60 kDa. A similar band was visualized in mussel gill extract. The WBresults for AChE, ChAT and TH are reported in Fig. 2.
UNCO
RRECT
Fig. 6. Immunohistochemical investigation of the cholinergic system inmussel gills. (A)Musselsdistributed along the gill epithelium, while (B) inmussels caged at Priolo AChE-immunopositivistandard deviation (S.D.) of AChEpositive cells. Asterisk indicates significant differences relativeseveral fibers along the gill epithelium, while (D)mussels caged at Priolo showed numerous podeviation (S.D.) of ChAT positive cells. Asterisk indicates significant differences relative to cont
Please cite this article as: Cappello, T., et al., Neurotoxicological effectscontaminated areas (eastern Sicily, I..., Comp. Biochem. Physiol., C (2015)
4. Discussion
The neurotoxicological potential of environmental pollution, mainlyrelated to petrochemical activities, was assessed in the marine musselM. galloprovincialis caged in an eastern Sicily industrial area. The gills,mainly involved in nutrient uptake, digestion, gas exchange andneuronal signaling, were chosen as the target organ. Evidencesof metabolomic disturbances in neurotransmission were previouslyreported in Cappello et al. (2013b), therefore the neuronal pertur-bations on the serotoninergic, cholinergic, and dopaminergic
ED P
RO
OF
caged at the reference site showed a high number of AChE-immunopositive cells regularlytywas reduced, and numerous hemocytes were observed. Scale bars, 20 μm. (E)Mean andto control (*p b 0.0001). (C) Controlmussels displayed fewChAT-immunolabeled cells andsitive cells mostly at the apex of the gill filament. Scale bars, 20 μm. (F)Mean and standardrol (*p b 0.0001).
on marine mussel Mytilus galloprovincialis caged at petrochemical, http://dx.doi.org/10.1016/j.cbpc.2014.12.006
T
R
359
360
361
362
363
364
365
366
367Q10
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395Q11
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429Q12
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445Q13
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
Fig. 7. 1H NMR measurements of tyrosine (mM) from mussel gill tissue, expressed asmeans ± standard deviation (n = 12). Asterisks indicate significant differences relativeto control (*p b 0.05).
7T. Cappello et al. / Comparative Biochemistry and Physiology, Part C xxx (2015) xxx–xxx
UNCO
RREC
systems were examined more closely, by the use of 1H NMR andimmunohistochemical assays.
The environmental metabolomics is a well-established techniquebased on the identification of low molecular weight endogenousmetabolites within a biological system (i.e. cells, tissues, organs orwhole individuals), whose production and levels vary in response toenvironmental stressors, diseases or exposure to toxicants (Viantet al., 2003; Iacono et al., 2010; Liu et al., 2011; Zhang et al., 2011;Fasulo et al., 2012a, 2012b; Kwon et al., 2012; Cappello et al., 2013b).In this study, 1H NMR-based metabolomics measurements of themetabolites of interest frommussel gill extracts, allowed the successfulinvestigation on the changes in neurotransmitter levels betweenmussels caged in the reference site and those transplanted in thepolluted area in response to environmental stress.
The significant decline in serotonin in gills of mussels caged at thepetrochemical area relative to mussels caged at the reference site wasrevealed both by 1HNMRand immunohistochemical analyses. As amat-ter of fact, these results are consistent with the impairments in energymetabolism highlighted in our previous work (Cappello et al., 2013b),in which we supposed the occurrence of glycogenolysis that may beassociated with compensatory mechanisms elicited by mussels inresponse to compromised gill ciliary activity, likely due to interferencesof environmental pollutants with the serotoninergic system. In fact, it iswell-known that serotonin is involved in hormonal and neuronalpathways, and plays a key role in cilioexcitatory activity, reproductivesuccess and regulating food intake in invertebrates (Gosselin, 1961;Stefano, 1990). Noteworthy, a concomitant 5-HT3R over-expression atcellular level in mussels caged at the polluted area was detected,while in mussels caged at Vendicari solely nerve fibers were 5-HT3Rimmunopositive. The rise of 5-HT3R could be related to an adaptiveresponse mediated by paracrine signaling activities in order to recovera regular physiological activity in gills. Indeed, it has been reportedthat gill epithelial cells containing the lateral cilia present 5-HTreceptors to increasing the ciliary beating rate, if activated (Carroll andCatapane, 2007). Fabbri and Capuzzo (2006), after exposing the musselM. galloprovincialis to Cr(VI) and Cu(II), increased 5-HT-stimulatedadenylate cyclase activity in vivowas found, and suggested the possibil-ity that metal accumulation in mussel gills might induce the over-expression of 5-HT receptors. Further evidences of alterations in seroto-nin signaling pathwayswere provided by the significantly up-regulatedtranscription of 5-HT receptor in both sexes of M. galloprovincialisexposed to 0.1, 1, and 10 μg Cr(VI) L-1 animal−1 for 96 h, with malesshowing more sensitivity to metal treatment than females (Ciacciet al., 2012).
Quantitative 1H NMR analysis showed a 64% significant decreasein the concentration of acetylcholine, a cholinergic neurotransmitter,in gills of mussels caged at Priolo in respect to mussels caged atVendicari. In studies of ecotoxicology, measurements of AChE activ-ity have been used to test for neurotoxicity in aquatic organismssince it can be inhibited by the presence of contaminants in complexmixtures (Matozzo et al., 2005; Cajaraville et al., 2000; Tsangariset al., 2010; Cravo et al., 2012; D'Agata et al., 2014), by pollutantslike organophosphate and carbamate pesticides (Lionetto et al.,2013), or heavy metals and hydrocarbons (Rank et al., 2007; Ciacciet al., 2012). As expected, in mussels caged at the petrochemicalsite of Priolo AChE immunopositivity was reduced, and limited almostsolely to hemocytes. In marine mussels, hemocytes possess a complexcell signaling network with high homology with that of vertebrates,that allows them to modulate their own functions (Humphries andYoshino, 2003; Franzellitti and Fabbri, 2013). However, little it isknown on the physiological roles of these signaling pathways in musselhemocytes. Also, the presence of ChAT, which enzymatic action resultsin synthesis of acetylcholine, implicating a negative correlation withAChE, was found noticeably increased. As amatter of fact, the inhibitionof AChE and increment in ChAT are followed by accumulation ofacetylcholine. However, in the present study the opposite result of
Please cite this article as: Cappello, T., et al., Neurotoxicological effectscontaminated areas (eastern Sicily, I..., Comp. Biochem. Physiol., C (2015)
OO
F
reduced acetylcholine was found in gills of mussels transferred at thepetrochemical site for one month, likely to be ascribed to an ongoingacetylcholine synthesis process. Similar findings have been showed byZhang et al. (2011), which reported a decline in acetylcholine in gillsof adult Manila clams Ruditapes philippinarum after a 24 h exposure toboth low (10 μg L−1) and high (40 μg L−1) doses of Cu.
The concentration of tyrosine was found significantly reduced by35% in mussels caged at the petrochemical area in respect to musselscaged at the reference site. In the same individuals, the presence ofthe enzyme tyrosine hydroxylase (TH), which serves for conversion oftyrosine into catecholamines, was significantly induced in mussel gills.This data is consistent with the depletion of tyrosine, which is negative-ly correlated to the elevation of TH. Previous experimental studies havereported a noticeable dose-dependent increase of TH in the gills ofM. galloprovincialis exposed to 0.1, 1, 10 μg Cr(VI) L−1 animal−1 for96 h (Ciacci et al., 2012). The increase in THmay result in the enhance-ment of dopaminergic neurotransmission that has an inhibitory effecton lateral ciliary activity of mussel gills (Carroll and Catapane, 2007).Zhu et al. (2005) demonstrated that tyrosine and tyramine mayaugment endogenous ganglionic morphine and dopamine levelsin Mytilus edulis under in vitro and in vivo experiments, with theinvolvement of CYP2D6 and TH in the process, resulting in a cilio-inhibitory effect.
ED P5. Conclusions
This study provides evidence that environmental pollution, mainlyrelated to petrochemical activities, adversely affects the neurotrans-mission system in caged M. galloprovincialis individuals, resulting inimpairment in the normal ciliary motility, and thus in feeding effi-ciencies. 1H NMR-based environmental metabolomics offers an al-ternative method of measuring metabolite concentration. Indeed,the high sensitivity of 1H NMR allowed measurement of changesin neurotransmitter levels, namely serotonin, acetylcholine andtyrosine. The depletion in neurotransmitter concentrations wassupported by evidence of neuronal perturbations in mussel gills.In detail, alterations in serotoninergic and cholinergic systemswere revealed, together with enhancement of dopaminergic neuro-transmission, resulting in a cilio-inhibitory effect. However, theconcomitant over-expression in serotonin receptors and cholineacetyltransferase at cellular level may indicate an adaptive responsemediated by paracrine signaling activities in order to recover aregular physiological activity in gills. Therefore, the concurrent useof 1H NMR and immunohistochemical assays demonstrated to besensitive and effective for assessing environmental influences onthe health status of aquatic organisms, and thus suitable to beapplied in ecotoxicological studies.
on marine mussel Mytilus galloprovincialis caged at petrochemical, http://dx.doi.org/10.1016/j.cbpc.2014.12.006
TED P
RO
OF
470Q14
471
472
473
474
475
476
477478479480481482483484485486487488489490491492493494495496497498499500501502
503504505506507
Fig. 8. Immunohistochemical investigation of the dopaminergic system inmussel gills. (A) Controlmussels showed a slight TH-immunopositivity in cells of the gill epithelium,while (B) inmussels caged at Priolo an intense immunoreactivity of TH was revealed, with a number of positive cells and hemocytes. Scale bars, 20 μm. (C) Mean and standard deviation (S.D.) of THpositive cells. Asterisk indicates significant differences relative to control (*p b 0.0001).
8 T. Cappello et al. / Comparative Biochemistry and Physiology, Part C xxx (2015) xxx–xxx
C
6. Uncited references
Mauceri et al., 2005Wu and Wang, 2010
508509510511512513514515
RE
Acknowledgments
This research was supported by a National Interest Research Project(PRIN 2007-20079FELYB).
R 516517518519520521522523524525526527528529530531532533534535536537538539540541542543544545
UNCOReferences
Ausili, A., Gabellini, M., Cammarata, G., Fattorini, D., Benedetti, M., Pisanelli, B., Gorbi, S.,Regoli, F., 2008. Ecotoxicological and human health risk in a petrochemical districtof southern Italy. Mar. Environ. Res. 66, 217–219.
Cajaraville, M.P., Bebianno, M.J., Blasco, J., Porte, C., Sarasquete, C., Viarengo, A., 2000. Theuse of biomarkers to assess the impact of pollution in coastal environments of theIberian Peninsula: a practical approach. Sci. Total Environ. 247, 295–311.
Cappello, T., Maisano, M., D'Agata, A., Natalotto, A., Mauceri, A., Fasulo, S., 2013a. Effects ofenvironmental pollution in caged mussels (Mytilus galloprovincialis). Mar. Environ.Res. 91, 52–60.
Cappello, T., Mauceri, A., Corsaro, C., Maisano, M., Parrino, V., Lo Paro, G., Messina, G.,Fasulo, S., 2013b. Impact of environmental pollution on caged mussels Mytilusgalloprovincialis using NMR-based metabolomics. Mar. Pollut. Bull. 77, 132–139.
Carroll, M.A., Catapane, E.J., 2007. The nervous system control of lateral ciliary activity ofthe gill of the bivalve mollusc, Crassostrea virginica. Comp. Biochem. Physiol. A 148,445–450.
Catapane, E., Aiello, E., Stefano, G.B., 1974. Ganglionic mediationmechanism of lateral ciliain Mytilus edulis gill. Physiologist 17–372.
Ciacci, C., Barmo, C., Gallo, G., Maisano, M., Cappello, T., D'Agata, A., Leonzio, C., Mauceri, A.,Fasulo, S., Canesi, L., 2012. Effects of sublethal, environmentally relevant concentrationsof hexavalent chromium in the gills ofMytilus galloprovincialis. Aquat. Toxicol. 120–121,109–118.
Cravo, C., Pereira, C., Gomes, T., Cardoso, C., Serafim, A., Almeida, C., Rocha, T., Lopes, B.,Company, R., Medeiros, A., Norberto, R., Pereira, R., Araújo, O., Bebianno, M.J.,2012. A multibiomarker approach in the clam Ruditapes decussatus to assessthe impact of pollution in the Ria Formosa lagoon, South Coast of Portugal. Mar.Environ. Res. 75, 23–34.
Please cite this article as: Cappello, T., et al., Neurotoxicological effectscontaminated areas (eastern Sicily, I..., Comp. Biochem. Physiol., C (2015)
D'Agata, A., Cappello, T., Maisano, M., Parrino, V., Giannetto, A., Brundo, M.V., Ferrante, M.,Mauceri, A., 2014. Cellular biomarkers in the mussel Mytilus galloprovincialis(Bivalvia: Mytilidae) from Lake Faro (Sicily, Italy). Ital. J. Zool. 81 (1), 43–54.
De Domenico, E., Mauceri, A., Giordano, D., Maisano, M., Gioffrè, G., Natalotto, A.,D'Agata, A., Ferrante, M., Brundo, M.V., Fasulo, S., 2011. Effects of “in vivo” exposureto toxic sediments on juveniles of sea bass (Dicentrarchus labrax). Aquat. Toxicol.105, 688–697.
De Domenico, E., Mauceri, A., Giordano, D., Maisano, M., Giannetto, A., Parrino, V.,Natalotto, A., D'Agata, A., Cappello, T., Fasulo, S., 2013. Biological responses of juvenileEuropean sea bass (Dicentrarchus labrax) exposed to contaminated sediments.Ecotoxicol. Environ. Saf. 97, 114–123.
Di Leonardo, R., Bellanca, A., Capotondi, L., Cundy, A., Neri, R., 2007. Possible impacts of Hgand PAH contamination on benthic foraminiferal assemblages: an example from theSicilian coast, central Mediterranean. Sci. Total Environ. 388, 168–183.
Di Leonardo, R., Bellanca, A., Angelone, M., Leonardi, M., Neri, R., 2008. Impact of humanactivities on the central Mediterranean offshore: evidence from Hg distribution inbox-core sediments from the Ionian Sea. Appl. Geochem. 23, 3756–3766.
Espina, R., Yu, L., Wang, J., Tong, Z., Vashishtha, S., Talaat, R., Scatina, J., Mutlib, A., 2009.Nuclear magnetic resonance spectroscopy as a quantitative tool to determine theconcentrations of biologically produced metabolites: implications in metabolites insafety testing. Chem. Res. Toxicol. 22, 299–310.
Fabbri, E., Capuzzo, A., 2006. Adenylyl cyclase activity and its modulation in the gills ofMytilus galloprovincialis exposed to Cr6+ and Cu2+. Aquat. Toxicol. 76, 59–68.
Fasulo, S., Mauceri, A., Giannetto, A., Maisano, M., Bianchi, N., Parrino, V., 2008.Expression of metallothionein mRNAs by in situ hybridization in the gills ofMytilus galloprovincialis, from natural polluted environments. Aquat. Toxicol.88, 62–68.
Fasulo, S., Maisano, M., Sperone, E., Mauceri, A., Bernabo, I., Cappello, T., D'agata, A.,Tripepi, S., Brunelli, E., 2012a. Toxicity of Foroozan crude oil to ornate wrasse(Thalassoma pavo, Osteichthyes, Labridae): ultrastructure and cellular biomarkers.Ital. J. Zool. 79, 182–199.
Fasulo, S., Iacono, F., Cappello, T., Corsaro, C., Maisano, M., D'Agata, A., Giannetto, A., DeDomenico, E., Parrino, V., Lo Paro, G., Mauceri, A., 2012b. Metabolomic investigationof Mytilus galloprovincialis (Lamarck 1819) caged in aquatic environments.Ecotoxicol. Environ. Saf. 84, 139–146.
Franzellitti, S., Fabbri, E., 2013. Cyclic-AMPmediated regulation of ABCBmRNA expressionin mussel haemocytes. PLoS ONE 8 (4), e61634.
Gagné, F., Cejka, P., André, C., Hausler, R., Blaise, C., 2007. Neurotoxicological effectsof a primary and ozonated treated wastewater on freshwater mussels exposedto an experimental flow-through system. Comp. Biochem. Physiol. C Toxicol.Pharmacol. 146, 460–470.
Gosselin, R.E., 1961. The cilioexcitatory activity of serotonin. J. Cell. Comp. Physiol.58, 17–25.
on marine mussel Mytilus galloprovincialis caged at petrochemical, http://dx.doi.org/10.1016/j.cbpc.2014.12.006
546547548549550551552553554555Q15556557558559560561562563564565566567568569570571572573574575576577
578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607608609
9T. Cappello et al. / Comparative Biochemistry and Physiology, Part C xxx (2015) xxx–xxx
Gregory, M.A., Marshall, D.J., George, R.C., Anandraj, A., McClurg, T.P., 2002. Correlationsbetween metal uptake in the soft tissue of Perna perna and gill filament pathologyafter exposure to mercury. Mar. Pollut. Bull. 45, 114–125.
Hines, A., Yeung, W.H., Craft, J., Brown, M., Kennedy, J., Bignell, J., Stentiford, G.D., Viant,M.R., 2007. Comparison of histological, genetic, metabolomics, and lipid-basedmethods for sex determination in marine mussels. Anal. Biochem. 369, 175–186.
Humphries, J.E., Yoshino, T.P., 2003. Cellular receptors and signal transduction inmolluscan hemocytes: connections with the innate immune system of vertebrates.Integr. Comp. Biol. 43, 305–312.
Iacono, F., Cappello, T., Corsaro, C., Branca, C., Maisano, M., Gioffre, G., De Domenico, E.,Mauceri, A., Fasulo, S., 2010. Environmental metabolomics and multibiomarker ap-proaches on biomonitoring of aquatic habitats. Comp. Biochem. Physiol. A 157, S50-S50.
Kwon, Y.K., Jung, Y.S., Park, J.C., Seo, J., Choi, M.S., Hwang, G.S., 2012. Characterizing theeffect of heavy metal contamination on marine mussels using metabolomics. Mar.Pollut. Bull. 64, 1874–1879.
Law No. 426 of 9.12.1998, 14.12.1998. Nuovi interventi in campo ambientale. G.U. n. 291.Lionetto,M.G., Caricato, R., Calisi, A., Giordano,M.E., Schettino, T., 2013. Acetylcholinesterase
as a biomarker in environmental and occupational medicine: new insights and futureperspectives. Biomed Res. Int. 2013 (321213).
Liu, X., Zhang, L., You, L., Yu, J., Zhao, J., Li, L., Wang, Q., Li, F., Li, C., Liu, D., Wu, H., 2011. Differ-ential toxicological effects induced by mercury in gills from three pedigrees of manilaclam Ruditapes philippinarum by NMR-based metabolomics. Ecotoxicology 20, 177–186.
Matozzo, V., Tomei, A., Marin, M., 2005. Acetylcholinesterase as a biomarker of exposureto neurotoxic compounds in the clam Tapes philippinarum from the Lagoon of Venice.Mar. Pollut. Bull. 50, 1686–1693.
Mauceri, A., Fasulo, S., Ainis, L., Licata, A., Lauriano, E.R., Martinez, A., Mayer, B.,Zaccone, G., 1999. Neuronal nitric oxide synthase (nNOS) expression in theepithelial neuroendocrine cell system and nerve fibers in the gill of the catfish,Heteropneustes fossilis. Acta Histochem. 101, 437–448.
Mauceri, A., Fossi, M.C., Leonzio, C., Ancora, S., Minniti, F., Maisano, M., Lo Cascio, P.,Ferrando, S., Fasulo, S., 2005. Stress factors in the gills of Liza aurata (Perciformes,Mugilidae) living in polluted environments. Ital. J. Zool. 72, 285–292.
UNCO
RRECT
Please cite this article as: Cappello, T., et al., Neurotoxicological effectscontaminated areas (eastern Sicily, I..., Comp. Biochem. Physiol., C (2015)
RO
OF
Ministerial Decree of 10.01.2000, 23.02.2000. Perimetrazione del sito di interessenazionale di Gela e Priolo. G.U. n. 44.
Rank, J., Lehtonen, K.K., Strand, J., Laursen, M., 2007. DNA damage, acetylcholinesteraseactivity and lysosomal stability in native and transplanted mussels (Mytilusedulis) in areas close to coastal chemical dumping sites in Denmark. Aquat.Toxicol. 84, 50–61.
Shiman, R., Akino, M., Kaufman, S., 1971. Solubilization and partial purification of tyrosinehydroxylase from bovine adrenal medulla. J. Biol. Chem. 246, 1330–1340.
Stefano, G.B., 1990. Neurobiology of Mytilus edulis. Manchester University Press, ND(312 pp.).
Sureda, A., Box, A., Tejada, S., Blanco, A., Caixach, J., Deudero, S., 2011. Biochemicalresponses of Mytilus galloprovincialis as biomarkers of acute environmentalpollution caused by the Don Pedro oil spill (Eivissa Island, Spain). Aquat. Toxicol.101, 540–549.
Tsangaris, C., Kormas, K., Strogyloudi, E., Hatzianestis, I., Neofitou, C., Andral, B., Galgani, F.,2010. Multiple biomarkers of pollution effects in caged mussels on the Greekcoastline. Comp. Biochem. Physiol. C 151, 369–378.
Viant, M.R., Rosenblum, E.S., Tjeerdema, R.S., 2003. NMR-based metabolomics: a powerfulapproach for characterizing the effects of environmental stressors on organismhealth. Environ. Sci. Technol. 37, 4982–4989.
Viarengo, A., Lowe, D., Bolognesi, C., Fabbri, E., Koehler, A., 2007. The use of biomarkersin biomonitoring: a 2-tier approach assessing the level of pollutant-induced stresssyndrome in sentinel organisms. Comp. Biochem. Physiol. C 146, 281–300.
Wu, H., Wang,W.X., 2010. NMR-based metabolomic studies on the toxicological effects ofcadmium and copper on green mussels Perna viridis. Aquat. Toxicol. 100, 339–345.
Zhang, L.B., Liu, X.L., You, L.P., Zhou, D., Wu, H.F., Li, L.Z., Zhao, J.M., Feng, J.H., Yu, J.B., 2011.Metabolic responses in gills of Manila clam Ruditapes philippinarum exposed tocopper using NMR-based metabolomics. Mar. Environ. Res. 72, 33–39.
Zhu, W., Mantione, K.J., Shen, L.H., Cadet, P., Esch, T., Goumon, Y., Bianchi, E., Sonetti, D.,Stefano, G.B., 2005. Tyrosine and tyramine increase endogenous ganglionic morphineand dopamine levels in vitro and in vivo: cyp2d6 and tyrosine hydroxylasemodulation demonstrates a dopamine coupling. Med. Sci. Monit. 11, Br397–Br404.
ED P
on marine mussel Mytilus galloprovincialis caged at petrochemical, http://dx.doi.org/10.1016/j.cbpc.2014.12.006