Environmental Pollution 158 (2010) 401–408
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Environmental monitoring of Domingo Rubio stream (Huelva Estuary, SW Spain) by combining conventional biomarkers and proteomic analysis in Carcinus maenas Rafael Montes Nieto a, Tamara Garcı´a-Barrera b, Jose´-Luis Go´mez-Ariza b, Juan Lo´pez-Barea a, * a b
´ rdoba, Severo Ochoa Building, Rabanales Campus, Highway A4 Km 396a, 14071 Co ´ rdoba, Spain Department of Biochemistry and Molecular Biology, University of Co Department of Chemistry and Materials Sciences, University of Huelva, Faculty of Experimental Sciences, El Carmen Campus, 21007 Huelva, Spain
Pollution assessment at ‘‘Domingo Rubio’’ stream (Spain).
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 March 2009 Received in revised form 31 July 2009 Accepted 1 September 2009
Element load, conventional biomarkers and altered protein expression proﬁles were studied in Carcinus maenas crabs, to assess contamination of ‘‘Domingo Rubio’’ stream, an aquatic ecosystem that receives pyritic metals, industrial contaminants, and pesticides. Lower antioxidative activities – glucose-6phosphate and 6-phosphogluconate dehydrogenases, catalase – were found in parallel to higher levels of damaged biomolecules – malondialdehyde, oxidized glutathione –, due to oxidative lesions promoted by contaminants, as the increased levels of essential – Zn, Cu, Co – and nonessential – Cr, Ni, Cd – elements. Utility of Proteomics to assess environmental quality was conﬁrmed, especially after considering the six proteins identiﬁed by de novo sequencing through capLC-mESI-ITMS/MS and homology search on databases. They include tripartite motif-containing protein 11 and ATF7 transcription factor (upregulated), plus CBR-NHR-218 nuclear hormone receptor, two components of the ABC transporters and aldehyde dehydrogenase (downregulated). These proteins could be used as novel potential biomarkers of the deleterious effects of pollutants present in the area. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Element load Biochemical biomarkers Protein expression proﬁles Protein identiﬁcation by de novo sequencing Environmental quality
1. Introduction Ecosystem pollution can be assessed by using sentinel organisms as bioindicators. Several molecular parameters, known as biomarkers, respond early to environmental stressors and alert before severe and irreversible damages appear in the ecosystems. Some biochemical parameters are used as ‘‘conventional’’ biomarkers, based on a previous and deep knowledge of pollutant effects and organism’s defense systems. They include induction of biotransforming and antioxidative enzymes, increased oxidative damages to biomolecules or esterase inhibition (Bonilla-Valverde
Abbreviations: AChE, acetylcholinesterase; Cat, catalase; CbE, carboxylesterase; 2-DE, two-dimensional electrophoresis; DTT, dithiothreitol; DRS, ‘‘Domingo Rubio’’ stream; EDTA, ethylenediamine tetraacetic acid; EROD, ethoxyresoruﬁne-O-deethylase; G6PDH, glucose-6-phosphate dehydrogenase; GSH, GSSG, glutathione in its reduced, oxidized forms; GST, glutathione-S-transferase; MALDI-TOF, matrixassisted laser desorption-ionization and time-of-ﬂight; MDA, malondialdehyde; nESI-IT-MS/MS, microelectrospray ion trap and tandem mass spectrometry; 6PGDH, 6-phospho-gluconate dehydrogenase; SDS, sodium dodecyl sulphate; SeGSHPx, Se-glutathione peroxidase. * Corresponding author. Tel.: þ34 957218687; fax: þ34 957218688. E-mail address: [email protected]
(J. Lo´pez-Barea). 0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.09.005
et al., 2004; Vioque-Ferna´ndez et al., 2007a,b). Recently, we have shown the utility of Proteomics to assess environment quality, in order to ﬁnd potentially new and unbiased biomarkers, and to gain insight into toxicity mechanisms (Montes-Nieto et al., 2007; Vioque-Ferna´ndez et al., 2009), though this so-called ‘‘environmental proteomic approach’’ (Lo´pez-Barea and Gomez-Ariza, 2006; Dowling and Sheehan, 2006) is still at an early stage of development. Since popular bioindicators are non-model organisms that are absent or underrepresented in public gene or protein sequence databases, identiﬁcation of differentially expressed proteins by high-throughput MALDI-TOF-PMF is not always possible. The European green crab (Carcinus maenas), a decapod crustacean living on sediments where most contaminants accumulate (Elumalai et al., 2007), is widely distributed along worldwide coasts (Baeta et al., 2005), being a non-protected species readily sampled without restrictions, that is considered one of the ‘‘100 world’s worst invasive alien species’’. Since it accumulates heavy metals, among other contaminants, this crab is a suitable pollution bioindicator, useful to assess contamination of European estuaries (Martı´n-Dı´az et al., 2005, 2008). The Domingo Rubio stream (DRS, SW Spain, Fig. 1) is at a little ﬂoodable valley connected to Huelva Estuary by a swinging channel that ends in the left margin of Tinto River, before this watercourse
R. Montes Nieto et al. / Environmental Pollution 158 (2010) 401–408
Fig. 1. Localization of Domingo Rubio stream and Cadiz bay and DRS details.
joins with Odiel River to form the Huelva Estuary. A great variety of pollutant inﬂows reach DRS: 1) The ‘‘Juan Delgado’’ brook drains the ‘‘Nuevo Puerto’’ industrial area, where petrochemical and chemical activities are carried out. Although a hydrocarbonretention system was built years ago, pollutants from previous spills still enter DRS; several collectors also convey pluvial waters from this area. Accidental leaks are the most important alterations derived from the activities carried out in this heavily industrialized area. 2) The Tinto and Odiel river system carry acidic water and heavy metals from miner-metallurgical activity carried out in Huelva province (Sainz et al., 2004); other industrial areas located nearby add many pollutants to the system. Since the low-medium course of DRS is under tidal inﬂuence, both pollutant pools enter it at high tide. 3) Finally, strawberry crops are grown along the medium-high DRS course, using high amounts of biocides – as organochlorine pesticides – and nitrogen fertilizers. These two types of contaminants enter DRS through underground or surface waters that circulate in small brooks. The present work aimed to characterize the response of conventional biomarkers, and to assess changes in protein expression proﬁles in an aquatic organism exposed to a complex mixture of heavy metals, PAHs and pesticides. We have recently assessed the presence of some pollutants and the biochemical responses elicited by them in Mus spretus mice from the Domingo Rubio stream (Montes-Nieto et al., 2007), though its living habits and food web position renders this rodent less exposed to waterborne pollutants. Since C. maenas lives upon sediments, it is more directly exposed to the contaminants present in this area. To work with a model organism, such as the laboratory mouse, Mus musculus, or its free-living analog, M. spretus, has the advantage of the availability in protein/gene sequence databases that allows the identiﬁcation of proteins via a fast, cheap, and high-throughput MALDI-TOF-PMF approach. Although C. maenas is a non-model species, this limitation can be circumvented by de novo sequencing via tandem mass spectrometry. Thus, nESI-IT-MS/MS allowed us to unambiguously identify four cytoskeletal proteins and two enzymes as new potential pollutant-sensitive biomarkers in two clam species exposed to model contaminants or collected at
polluted sites of Guadalquivir Estuary (Rodrı´guez-Ortega et al., 2003; Romero-Ruiz et al., 2006). 2. Materials and methods 2.1. Sample collection and organ extraction Crabs were collected in July 2004 at 2 sites (Fig. 1): the mouth of Domingo Rubio stream (DR1) and a reference site at Ca´diz Bay (TOR), that is relatively free of contamination (Pe´rez et al., 2004; Hampel et al., 2007). Crabs were captured with ﬁshing baskets baited with cat feed; the animals were taken alive to a nearby laboratory, where their sex and weight were determined. Digestive glands, gills and neural tissues were extracted, frozen in liquid N2 and stored at 80 C. Biochemical assays were made in digestive glands and neural tissue, and gills were used for element analysis and differential protein expression. The study was approved by the Animal Ethics Committee of Co´rdoba University. 2.2. Chemicals, element analyses, cell-free extract preparation, and conventional biochemical biomarkers assays All chemicals were of analytical quality and purchased from Sigma (Alcobendas, Spain) or Merck (Mollet del Valle´s, Spain). Milli-Q water (MilliporeÒ, Bedford, MA, USA) was used throughout. For element analysis, gills (0.1 g fresh weight) from 3 crabs/site were mixed with 3.0 mL H2NO3 in closed Teﬂon vessels and were digested with microwave assistance. Metals were analyzed in a 4500 Inductively Coupled Plasma-Mass Spectroscopy instrument (Hewlett-Packard, Palo Alto, CA, USA) as previously reported (Romero-Ruiz et al., 2006). Analysis were made in 3 pools/site, representing a total of 9 individuals. The following biomarkers were assessed in soluble extracts of digestive glands: the activity of primary – Se-glutathione peroxidase (SeGSHPx), catalase (Cat) – and ancillary – glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) – antioxidant enzymes; the content of malondialdehyde (MDA), reduced and oxidized glutathione (GSH, GSSG), and the activity of inducible biotransforming enzymes, namely, microsomal ethoxyresoruﬁne-O-deethylase (EROD, phase I) and cytosolic glutathione transferase (GST, phase II). Neural tissue acetylcholinesterase (AChE) and digestive gland carboxylesterase (CbE) activities were analyzed as organophosphate and carbamate pesticide targets. Assays of digestive glands were made in individual samples, and AChE assays in neural tissue pooled from 6 animals per site. Published methods were used for the assays (Rodrı´guez-Ariza et al., 1992, 1993; Ruiz-Laguna et al., 2001; Bonilla-Valverde et al., 2004; Vioque-Ferna´ndez et al., 2007a,b) after optimizing the conditions for crab extracts. Tissues (w300 mg) were disrupted in an Ultra-Turrax homogenizer with 50 mM Tris/HCl buffer, pH 7.5, containing 0.1 mM EDTA and 1 mM phenylmethylsulphonylﬂuoride at 4 ml/g. MDA was determined ﬂuorimetrically in a part of
R. Montes Nieto et al. / Environmental Pollution 158 (2010) 401–408 each homogenate; the remainder of it was centrifuged (25.500 g, 15 min) and divided in two portions. Six cytosolic activities – 6PGDH, G6PDH, SeGSHPx, GST, Cat and CbE – were assayed spectrophotometrically in one (Rodrı´guez-Ariza et al., 1992, 1993). Microsomes were obtained from the other portion, by ultracentrifugation (105,000 g, 60 min) and pellet resuspension in 0.1 M potassium phosphate buffer, pH 7.5 containing 1 mM EDTA, to assay EROD activity (Rodrı´guez-Ariza et al., 1993). Digestive glands were also extracted with 1 M HClO3 containing 2 mM EDTA (15 ml/g) and centrifuged (25,500 g, 15 min), to assay GSH and GSSG by HPLC with electrochemical detection (Ruiz-Laguna et al., 2001). Neural tissue was homogenized with 0.1 M phosphate buffer, pH 8 (3.3 ml/g sample) and centrifuged (25.500 g, 2 min) to assay AChE (Vioque-Ferna´ndez et al., 2007a). Protein was measured by the Bradford (1976) method using bovine serum albumin as standard. Each biochemical analysis was determined in triplicate. Results are expressed as means SEM. Activities are expressed as units or mU/mg protein. Glutathione redox status was calculated as 2GSSG/GSH þ 2GSSG. Statistical comparisons were done by Student’s t-test. 2.3. 2-DE analysis Gills from 5 male crabs/site (w100 mg) were homogenized in 20 mM Tris–HCl buffer, pH 7.6, containing 0.5 M sucrose, 0.15 M KCl, 20 mM dithiothreitol (DTT), 1 mM phenylmethyl sulphonyl ﬂuoride and protease inhibitors (AEBSF, E-64, bestatin, leupeptin, aprotinin, EDTA) at 3 ml/g. The homogenates were centrifuged, treated with bezonase, and ultracentrifuged as previously described (Montes-Nieto et al., 2007). The extracted proteins (160 mg) were incubated for 30 min in 350 ml rehydration buffer and then loaded on 18 cm (pH 4–7) Amersham Immobiline DryStripsÒ. A ProteanÒ isoelectrofocusing cell (BioRad) was used for separation as previously described (Montes-Nieto et al., 2007). After freezing at 80 C, the strips were soaked 20 min in equilibration mix containing 65 mM DTT, drained and soaked again for 20 min in this mix but containing instead 25 mM iodoacetamide. SDSPAGE was carried out in 12,5% gels using the BioRad ProteanÒ Plus Dodeca cell (20 C) at 2.5 W/gel, 10 min, and 10 W/gel until separation was ﬁnished (w5 h) (Montes-Nieto et al., 2007). Gels were stained with SYPRO Ruby. A BioRad FX Multiimager was used to obtain 2-DE images of 4 replicates. Spot volumes, normalized by the total volume of all valid and matched spots in a set of gels, were quantiﬁed by the PDQuest software (V8.0, BioRad). Initially, spots with signiﬁcant differences (p < 0.001) between sampling sites in the Student’s t-test were considered. From all of them, only those over- or under-expressed at least 2.5-fold with respect to control animals were selected. 2.4. Trypsin digestion, MS analysis and protein identiﬁcation Differentially expressed spots were excised and in-gel digested with trypsin (Promega, Madison WI) using a Digest MSPro (Intavis, Koeln, Germany). Brieﬂy, gel slices were washed with water and 20 mM ammonium bicarbonate pH 7.8, reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide and digested with trypsin for 8 h at 37 C. Tryptic peptides were extracted with acetonitrile/water (1/1) containing 0.25% triﬂuoroacetic acid (TFA). Extracts were evaporated to dryness by vacuum and redissolved in 5 mL acetonitrile/water plus 0.1% TFA, and stored frozen (80 C) until MS analysis. Samples were initially analyzed by MALDI-TOF at the CSIC-UAB Proteomics Laboratory (Barcelona, Spain) in a Voyager DE PRO MALDI-TOF (Applied Biosystems, Foster City, CA, USA) working in positive reﬂectron mode. A 0.5 ml sample fraction was loaded in a 96 2 well plate, mixed with 0.5 ml of matrix (3 mg/ml a-ciano-4hidroxycinamic acid in acetonitrile/water 2/1 containing 0.1% TFA) and was allowed to dry. Spectra were externally calibrated using an standard mixture – des-Arg1-bradiquinine (Mr 904.46), Glu1-ﬁbrinopeptid B (Mr 1570.68), angiotensin-1, (Mr 1296.69), ACTH 1–17 (Mr 2093.09), ACTH 18–39 (Mr 2465.20), ACTH 7–38 (Mr 3657.93) – and, when possible, an internal calibration using the ions derived from the trypsin autodigestion was used. The proteins yielding intense peptides by MALDI-TOF were further analyzed by capLC-mESI-ITMS/MS at the CSIC-UAB Proteomics Laboratory using a linear LTQ ion trap equipped with a microESI ion source both from ThermoFisher (San Jose, CA).
Each peptide extract was diluted up to 40 mL with 1% formic acid. Samples were loaded in a chromatographic system consisting in a C18 preconcentration cartridge (Agilent Technologies, Barcelona, Spain) connected to a 10 cm long, 150 mm i.d. Vydac C18 column (Vydac, IL, USA). The separation was done at 1 mL/min in a 30 min acetonitrile gradient from 3 to 40% (solvent A: 0.1% formic acid, solvent B: acetonitrile in 0.1% formic acid). The HPLC system was composed by an Agilent 1200 capillary pump, binary pump, a thermostated microinjector and a micro switch valve. The LTQ instrument was operated in the positive ion mode with a spray voltage of 2 kV. The scan range of each full MS was m/z 400–2000. The spectrometric analysis was performed in an automatic dependent mode, acquiring for each sample a full scan and 8 MS/MS spectra of the most abundant signals. A dynamic exclusion was set to 1 to avoid the redundant selection of precursor ions. MS/MS fragmentation spectra were evaluated by ‘‘de novo’’ analysis using the PEAKS de novo search engine tool (PEAKS Studio 4.5, Bioinformatics Solutions Inc., Waterloo, ON, Canada). The search parameters were: mass tolerance 0.5 Da for precursor ions and 0.3 Da for fragment ions; the enzyme was set to trypsin and up to two missed cleavages were allowed; and static modiﬁcation involving carbamidomethylated cysteine (þ57 Da) was included. Sequences obtained by PEAKS were manually validated. De novo sequence tags with an identity score >80% were sent to the BLAST tool for protein identiﬁcation using the NCBI nonredundant (nr) protein sequences database (http://www.ncbi.nlm.nih.gov/).
3. Results and discussion 3.1. Element contents in gills The pollutant load sustained by crabs from problem DR1 and reference TOR sites was evaluated by taking the concentration of 11 elements as an indirect index, as in our previous study in M. spretus from DRS (Montes-Nieto et al., 2007). This would avoid the intricacy of measuring the multiple contaminants also present in the area. Given the small amount of digestive gland samples, elements and proteomic analysis were carried out in gills, a highly irrigated organ fully exposed to waterborne contaminants, thus sparing the digestive glands and the neural tissues for conventional biomarker assays. Table 1 shows that higher element contents, from 1.9- to 10.2-fold, were detected in crabs dwelling at DRS compared to those from the TOR reference site, with the only exception of Se. Signiﬁcant differences in essential – Zn, Cu, Co – and nonessential – Cr, Ni, Cd – elements were found. The lack of signiﬁcant differences in Fe, Pb, As, and Mn could be due to high interindividual variability, as suggested by their SEMs. Overall, the contents of essential and nonessential elements conﬁrmed the starting hypothesis, that animals living at DRS should be much more exposed to contaminants, as illustrated by elements of pyritic origin, than those from TOR reference site. 3.2. Conventional biochemical biomarkers Ten biochemical biomarkers were assayed in the digestive gland of crabs from the TOR and DR1 sites. Fig. 2 shows that three antioxidative enzymes, G6PDH, 6PGDH and Cat, had the same response pattern, lower in DR1 problem crabs than in those from the TOR reference site. The differences were extensive and
Table 1 Metal contents in Carcinus maenas gills from both sites studied. Site
Element concentration Fe
ES1a – TOR –
688b 144 357 59
118 21.8 32.5 6.15
66.4 10.0 33.4 3.48
17.2 6.06 1.68 0.53
10.3 4.31 2.92 0.22
9.28 3.16 4.29 0.91
2.11 0.38 0.63 0.22
1.96 0.33 2.17 0.30
1.15 0.18 0.36 0.02
0.53 0.33 0.24 0.01
0.67 0.12 0.11 0.11
n-fold p-value Signiﬁcance
1.9 0.100 ns
3.6 0.019 *
2.0 0.035 *
10.2 0.063 ns
3.5 0.161 ns
2.2 0.204 ns
3.3 0.029 *
0.9 0.655 ns
3.2 0.013 *
2.2 0.045 *
6.1 0.025 *
Crabs were sampled at two sites, ES1, at DRS mouth, and TOR, at Ca´diz Bay. Element concentrations were assessed in gills and are shown as ng/mg dry weight.
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Fig. 2. Antioxidant (G6PDH, Cat, 6PGDH, SeGSHPx) activities and oxidative damages (MDA, %GSSG). Signiﬁcant changes (DR1 vs TOR) are shown as: *(p < 0.05), **(p < 0.005).
statistically signiﬁcant, as shown by the decrease of 2.7-fold in G6PDH (p ¼ 0.034), of 3.8-fold in Cat (p ¼ 0.011), and of 7-fold in 6PGDH (p ¼ 0.004) in crabs from the DR1 site. Nevertheless, another antioxidative activity, SeGSHPx, failed to show signiﬁcant differences between reference and problem animals. In agreement to the low activity of 3 out of the 4 antioxidant enzymes assayed, crabs dwelling at DRS had higher lipid oxidative damages and a more oxidized glutathione redox status. Actually, the patterns of MDA and glutathione status were highly parallel, being near twofold higher in the DR1 problem crabs than in those from the reference site. Again, the differences in these oxidative stress biomarkers were signiﬁcant, both the 1.9-fold increase of MDA in DR1 crabs (p ¼ 0.002) and of 2.1-fold of glutathione redox status (p ¼ 0.012). Activities of the biotransforming enzymes had divergent patterns (Fig. 3). GST increased 1.8-fold in DR1 crabs (p ¼ 0.048), in parallel to higher lipid oxidative damages and the more oxidized glutathione status, while the P4501A-associated EROD activity decreased 1.3-fold in DR1 (p ¼ 0.025), as the three antioxidative enzymes referred above. Esterases used as speciﬁc pesticide targets showed also differential responses. While AChE increased 2.0-fold in the DR1 problem crabs (p < 0.001) compared to reference animals, only a small and non-signiﬁcant CbE increase (p ¼ 0.485) was observed in DR1 animals. DRS is a highly polluted area that receives contaminants from many sources, including petroleum derivatives, pesticides, and
elements of pyritic origin. Accumulation in C. maenas gills of seven elements, both essential and nonessential, indicates that water and sediments from DRS are polluted, conﬁrming the accumulation of toxic metals in kidneys of M. spretus from DRS banks compared to reference mice (Montes-Nieto et al., 2007). Metals and other prooxidant chemicals, probably present also in DRS, enter organisms and generate reactive oxygen species that promote oxidative stress, as shown in different animals (Ruiz-Laguna et al., 2001; Bonilla-Valverde et al., 2004; Romero-Ruiz et al., 2008), and in DRS mice (Montes-Nieto et al., 2007). As expected, DR1 crabs suffered a clear oxidative stress, conﬁrmed by the high oxidation in lipids and glutathione. Yet, in contrast to our previous studies showing higher antioxidative activities in polluted animals (Bonilla-Valverde et al., 2004; Romero-Ruiz et al., 2006, 2008) and the consensus in the biomarkers ﬁeld (Martı´n-Dı´az et al., 2008; Maria et al., 2009), most antioxidative enzymes, 6PGDH, G6PDH and Cat, were significantly lower in DRS crabs, and no differences were found in SeGSHPx, a biomarker induced in previous studies (Romero-Ruiz et al., 2006, 2008; Montes-Nieto et al., 2007). The differential response of these oxidative stress biomarkers could be attributed to the duration of exposure or to the high contaminant levels at the Doming Rubio stream. The decreases of glucose-6-phosphate and 6-phosphogluconate dehydrogenases, two enzymes pertaining to the pentose-phosphate pathway that provides NADPH for antioxidative processes, can be attributed to the inhibitory effects of metals, well established for
Fig. 3. Biotransforming and esterases activities. Signiﬁcant changes (DR1 vs TOR) are shown as: *(p < 0.05), ***(p < 0.001).
R. Montes Nieto et al. / Environmental Pollution 158 (2010) 401–408
Cu or Hg (Mu¨ller, 1986; Sarkar et al., 1995; Wolf and Baynes, 2007), or of prooxidant contaminants, probably via oxidation of –SH groups at their active sites (Winzer et al., 2002). Actually, these two antioxidative enzymes decreased in animals exposed to high metal loads, both in mice exposed to pyritic metals from Aznalcollar mining spill (Ruiz-Laguna et al., 2001) and in bivalves from the Guadalquivir Estuary living at sites of high metal levels (RomeroRuiz et al., 2008). Catalase is a primary antioxidative enzyme that decomposes the H2O2 formed by oxidative stress, that if not detoxiﬁed generates hydroxyl radicals. Although catalase should increase under oxidative stress, it has been shown to decrease in vitro when the enzyme is exposed to high H2O2 levels (Aebi, 1984), and in vivo, in metal-exposed mice (Ruiz-Laguna et al., 2001) and Cr-exposed daphnids (Jemec et al., 2008). The parallel decreases of catalase and EROD could have a common origin. Both enzymes are hemoproteins, where the Fe-protoporphyrin XI located at their active site is essential in catalysis. Heme oxygenase breaks free heme groups to biliverdin that is later reduced to bilirubin (Baranano et al., 2002). It is induced by heme and by stimuli that generate oxidative stress, such as UV radiations or metals (Maines and Gibbs, 2005; Ryter and Choi, 2005). Its overexpression in metalexposed animals would drastically diminish free heme levels and, thus, the activity of heme-containing enzymes, such as the catalase or P4501A-associated EROD activities. The higher GST activity of DR1 crabs probably reﬂects the presence at this area of PAHs or electrophilic compounds released by nearby industries or agricultural practices (Talalay, 2000). Actually, high GST activities were reported in mice (Ruiz-Laguna et al., 2001; Bonilla-Valverde et al., 2004) and bivalves (Funes et al., 2006) exposed to high metal concentrations. This agrees with the antioxidative capacity of some GSTs (Baez et al., 1997) and the presence of an antioxidant response element in some GST genes (Ikeda et al., 2002). A recent study with C. maenas from an eutrophic and metal contaminated coastal system shows higher GST activity and MDA levels and drastically lower EROD activity (Pereira et al., 2009). Contrary to our expectations, the esterases used as sensitive and speciﬁc targets to detect inhibition by organophosphate and carbamate pesticides remained unaltered (CbE) or were signiﬁcantly increased (AChE). It has been shown that several xenobiotics (Day and Scott, 1990; Printes and Callaghan, 2004) and metals (Calabrese and Baldwin, 2003; Brown et al., 2004) induce AChE activity. Thus, the 2-fold AChE increase observed in DR1 crabs could reﬂect a compromise between induction of this activity by some contaminants and the inhibitory effects of pesticides (Vioque-Ferna´ndez et al., 2007a,b, 2009).
2405, 3208, 4413) were over-expressed in DR1 gels from 5 to 57fold compared to TOR gels. These 15 spots were excised, trypsin digested and analyzed by MALDI-TOF. The proteins yielding intense peptides in this method were further analyzed by capLC-mESI-ITMS/MS, and the fragmentation spectra were evaluated by ‘‘de novo’’ analysis using the PEAKS search engine tool and manually validated. De novo sequences from six different spots successfully matched with annotated proteins after BLAST search on public databases (Table 2). Since the UniProtKB (http://www.uniprot.org/) database contains only 103 C. maenas proteins, far from the 63,888 M. musculus entries or the 155,754 Homo Sapiens proteins included, identiﬁcation of de novo sequenced peptides had to rely on search against the NCBI nonredundant (nr) protein sequence database. This, added to the small length of the sequences matched, would explain the high E-values obtained for the C. maenas proteins identiﬁed. Two of them, the tripartite motif-containing protein 11 and the ATF7 transcription factor, showed increased levels on DR1 problem crabs. Other four, the CBR-NHR-218 nuclear hormone receptor, the peptide ABC transporter permease protein, the ABC transporter G family protein, and the aldehyde dehydrogenase, were under-expressed in crabs dwelling at the DR1 site.
3.3. Protein expression proﬁles in gills
3.4. Biological relevance of the proteins over-expressed in DRS crabs
For proteomic analysis, gills of 5 male crabs (17–18 g) per site were randomly grouped. Cytosolic fractions were prepared in the TOR (reference) and DR1 (problem) groups and differences in protein expression proﬁles were analyzed by 2-DE. Over 400 spots were resolved in the 4–7 pH range corresponding to proteins of 14–45 kDa Mr. Image analysis of the 8 gels run showed that 24 spots had altered expression proﬁles (p < 0.001). Fig. 4 shows a 2-DE master gel generated from all gels run, with the locations of the 15 most intense spots (most likely to be identiﬁed by ESI-MS/MS) of the 24 initially found. Fig. 5 shows close-up views of these 15 spots comparing their intensity in gels from the reference and problem samples. The number in parenthesis indicates the fold-variation of each spot compared to its intensity in the TOR reference gel. Two spots (4520, 6422) were almost undetectable in DR1 problem crabs, and other seven (2317, 3522, 4514, 4620, 5428, 6414, 6424) diminished from 3.6 to 167-fold in DR1 animals compared to TOR crabs. Other six (1212, 2208, 2310,
The tripartite motif-containing protein 11 (TRIM11) regulates in mice the levels of a neuroprotective peptide through the proteasomal degradation pathway. It has a specialized type of Zn-ﬁnger (40–60 aas) involved in protein–protein interactions, with E3 ubiquitin-protein ligase activity (Niikura et al., 2003), and an SPRY domain whose distant homologues regulate Ca2þ-release by the ryanodine and/or the IP3 receptors (Ponting et al., 1997). Cd exposure increases neurodegenerative diseases in humans by collecting ubiquitinated and misfolded proteins in protein inclusion bodies, that contains among others TRIM25 (Song et al., 2008). Gene arrays show signiﬁcant increase of TRIM16 in human mammary epithelial cells exposed to benzo(a)pyrene-quinones, in a process mediated by xenobiotic and antioxidant response elements (Burchiel et al., 2007). The ATF family includes basic-region leucine zipper (bZIP) transcription factors with many physiological functions (Hai et al., 1989). Their basic region interacts with DNA, and their leucine
Fig. 4. Virtual master 2-DE gel of samples from TOR and DR1 crabs. Spots marked (O) are under-expressed (DR1 vs TOR); those marked (6) are over-expressed.
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Fig. 5. Close-up views of spots differentially expressed (DR1 vs TOR). Values under each spot are average intensities from 4 replicas/sample. A coefﬁcient of variation with respect control samples is also included.
zippers allows its dimerization (Hai and Curran, 1991). The ATF/ CREB (cAMP-response binding) members respond to environmental signals and maintain cellular homeostasis, by controlling cell proliferation and apoptosis (Persengiev and Green, 2003). Thus, ATF2, ATF3 and ATF6 mediate stress responses (Hai et al., 1999), as could be an oxidative stress (Fawcett et al., 1999). The most recently characterized component is ATF7, related in mammals to control of cellular proliferation and differentiation of some tissues, such as intestinal epithelium and hepatic tissue (Peters et al., 2001). Plasticity of the genome and its regulation support genomic reaction and adaptation to environmental stimuli, and provide evidence that chemicals placed in the environment can and do promote disease by altering gene expression. Thus, vanadium in the residual oil ﬂy ash of polluted air inhibit tyrosine phosphatases, phosphorylating NK-kB and other transcription factors, such as ATF2, leading to expression of inﬂammatory genes exacerbating respiratory distress (Edwards and Myers, 2007). 3.5. Biological relevance of the proteins under-expressed in DRS crabs Spot 4629, 5-fold less intense in DRS crabs, is homologous to the CBR-NHR-218 nuclear hormone receptor of Caenorhabditis briggsae. Such receptors are important transcriptional regulators with many
physiological functions. They act as dimers in nuclei to regulate the transcription of target genes. They have a highly conserved DNAbinding domain that recognises speciﬁc sequences via a Zinc ﬁnger, connected to a C-terminal ligand-binding domain, that after ligandbinding changes receptor conformation to induce a response, acting as a molecular switch to turn on transcriptional activity (Edwards, 2000). Thus, under-expression of this protein would have important effects in crabs living in a polluted site (Edwards and Myers, 2007). Two under-expressed spots were identiﬁed as ABC transporters, a wide group of proteins that translocate different molecules through cell membranes using energy from ATP hydrolysis (Higgins, 2001). While in prokaryotes they introduce essential nutrients, including sugars, metals or vitamins, in eukaryotes they eject to the surrounding lipids, steroids, peptides, ions, drugs or xenobiotics. One of the proteins is an integral transmembrane domain (TMD) probably responsible for substrate translocation across membrane (Saurin et al., 2006). TMD is fused in a variety of combinations to the ABC module (w200 amino acids), known to bind and hydrolyse ATP, thus coupling transport to ATP hydrolysis in many biological processes (Higgins, 2001). The ABCG subfamily expels cholesterol and some xenobiotics, including therapeutic drugs, and are responsible of acquired drug resistance (Kusuhara and Sugiyama, 2007). Their decrease in animals from polluted sites
Table 2 Differentially expressed Carcinus maenas proteins identiﬁed by de novo sequencing using capLC-mESI-ITMS/MS. Spot
DRS patterna MHþ
4413 3208 4620 3522 5428 6414
[ [ Y Y Y Y
a b c
1911 1811.4 1484.8 1174.1 1381.1 1993.6
De novo sequenceb
Matched protein organism [accession number]c
WWVRRLLVEEEQLR HRNGSDGMYSGSDGNTR HLEGGGEQFLNQR QNEEEEQLR SPEESNYENKGK SSVMDVDGVAR
Tripartite motif-containing protein 11 Bos taurus [NP_001071388.1; A0JN74] ATF7, cAMP-dependent transcription factor Caenorhabditis elegans [NP_497913.1; C07G2.2] CBR-NHR-218 nuclear hormone receptor Caenorhabditis briggsae [XP_001675312.1; A8XMZ8] Peptide ABC transporter, permease protein Croceibacter atlanticus [ZP_00951349.1; A3UB71] ABC transporter G family protein Dictyostelium discoideum [ZP_01811796.1; Q55DW4] Aldehyde dehydrogenase Lyngbya sp. [ZP_01620044.1; A0YLX3]
10/10 11/17 13/13 7/9 9/12 11/11
Protein spot that was signiﬁcantly up- ([) or downregulated (Y) in animals collected at the DRS. Underlined sequences correspond to those matched after BLAST search. Numbers at the NCBI and at the Uniprot databases, respectively.
0,69 4.0 137 772 331 466
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could be due to oxidation and loss of function, related to direct reaction with xenobiotics, due to their peripheral cellular location. Aldehyde dehydrogenase decreased also over 5-fold in DRS crabs. In Mytilus galloprovincialis, this enzymatic activity decreases in the presence of pollutants, in a process attributed to the antagonic effects of different chemicals (Nasci et al., 2002). In human saliva, it is inactivated by reversible oxidation promoted by some substances, such as caffeine, in a process proposed as a novel biomarker of oxidative stress (Wierzchowski et al., 2008). In addition to inactivation, oxidation of this protein labels it for proteasome degradation (Grune et al., 2003). Important changes of human aldehyde dehydrogenase 3 family, member A1, were detected by gene arrays and by qRT-PCR in human mammary epithelial cells exposed to B(a)P-quinones (Burchiel et al., 2007). 3.6. Concluding remarks The combined use of element analysis, biomarkers and proteomic approaches conﬁrms that crabs from the aquatic ecosystem of Domingo Rubio stream contain more contaminants than those from Ca´diz Bay, to which they respond with signiﬁcant changes of several conventional biomarkers. Decreased antioxidative enzymes – G6PDH, 6PGDH, Cat – paralleled increasingly damaged molecules – MDA, GSSG –, due to oxidative damages promoted, among others, by elements of pyritic origin. The simultaneous presence of electrophiles could explain the increase of detoxifying activities, such as GSTs, while mixed effects of pesticides and agrochemicals could explain the increased AChE activity, initially selected as speciﬁc and selective target of organophosphate and carbamate pesticides. Our study conﬁrms that DRS in addition to in its terrestrial ecosystem, as shown in a previous study with the aboriginal mouse, M. spretus (Montes-Nieto et al., 2007), is particularly polluted in its aquatic ecosystem, in agreement with the role of the Tinto–Odiel system in the transport of most pollutants present in the area, especially at high tide. Our work additionally conﬁrms the utility of Proteomics to assess environmental quality, not only throughout the alterations of protein expression signatures, as previously reported (Rodrı´guez-Ortega et al., 2003; Lo´pez-Barea and Gomez-Ariza, 2006; Vioque-Ferna´ndez et al., 2009), but especially in view of the proteins identiﬁed by the full proteomic approach carried out in this study, including de novo sequencing of proteins and their subsequent identiﬁcation by bioinformatics search against public databases. In fact, the two proteins induced in DR1 crabs that were identiﬁed, the tripartite motif-containing protein 11 and the ATF7 transcription factor, probably defend organisms living in polluted areas from the deleterious effects of pollutants. In the same way, the four proteins repressed in DR1 crabs that were identiﬁed, the CBR-NHR-218 nuclear hormone receptor, two components of the ABC family of transporters, and the aldehyde dehydrogenase, could be also considered as novel potential biomarkers of the biological effects of contaminants so abundant in the area studied. Acknowledgements This work was funded by grant CTM2006-08960-C02, from the Spanish Ministry of Education and Science, and by grant RNM-523, from the Innovation and Science Agency, Andalusian Government. References Aebi, H., 1984. Catalase in vitro. Methods Enzymol. 105, 121–126. Baeta, A., Cabral, H.N., Neto, J.M., Marques, J.C., Pardal, M.A., 2005. Biology, population dynamics and secondary production of the green crab Carcinus maenas (L.) in a temperate estuary. Estuar. Coast. Shelf Sci. 65, 43–52.
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