Environ Sci Pollut Res DOI 10.1007/s11356-014-4023-0
MOLECULAR AND CELLULAR EFFECTS OF CONTAMINATION IN AQUATIC ECOSYSTEMS
Differential gene transcription, biochemical responses, and cytotoxicity assessment in Pacific oyster Crassostrea gigas exposed to ibuprofen Miguel A. S. Serrano & Maria Gonzalez-Rey & Jacó J. Mattos & Fabrício Flores-Nunes & Álvaro C. P. Mello & Flávia L. Zacchi & Clei E. Piazza & Marília N. Siebert & Rômi S. Piazza & Diana Alvarez-Muñoz & Sara Rodriguez-Mozaz & Damià Barceló & Maria João Bebianno & Carlos H. A. M. Gomes & Cláudio M. R. Melo & Afonso C. D. Bainy
Received: 15 August 2014 / Accepted: 17 December 2014 # Springer-Verlag Berlin Heidelberg 2015
D. Barceló Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain
aim of this study was to evaluate the effects on antioxidant status caused by ibuprofen (IBU) in oysters Crassostrea gigas exposed for 1, 4, and 7 days at concentrations 1 and 100 μg L−1. Levels of IBU in tissues of oysters, as well as cell viability of hemocytes, were measured. The transcription of cytochrome P450 genes (CYP2AU2, CYP356A1, CYP3071A1, CYP30C1), glutathione S-transferase isoforms (GST-ω-like and GST-πlike), cyclooxygenase-like (COX-like), fatty acid binding protein-like (FABP-like), caspase-like, heat shock proteinlike (HSP70-like), catalase-like (CAT-like), and the activity of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST) were also evaluated in the gills of oysters. The highest levels of IBU were observed in animals exposed to 100 μg L−1. A significant upregulation of CYP2AU1, CYP356A1, CYP3071A1, GST-ω-like, GST-π-like, COXlike, and FABP-like was observed in oysters exposed to IBU under different experimental conditions. Oysters exposed to 1 μg L−1 for 7 days showed a significantly higher transcription of CYP2AU2, CYP356A1, CYP3071A1, GST-ω-like, and GST-π-like but lower GR activity. In conclusion, C. gigas exposed to environmentally relevant concentrations of IBU (1 μg L−1) exhibited increased transcription of certain genes and alterations on antioxidant and auxiliary enzymes, which could, in the the long term, cause damages to exposed organisms.
C. H. A. M. Gomes : C. M. R. Melo Laboratory of Marine Mollusk, Department of Aquaculture, Center of Agricultural Science, Federal University of Santa Catarina, Florianopolis, SC 88040900, Brazil
Keywords Crassostrea gigas . Pharmaceuticals . Ibuprofen . Oxidative stress . Gene transcription
Abstract Pharmaceuticals, such as anti-inflammatory nonsteroidal drugs, are frequently detected in aquatic ecosystems. Studies about the effects of these substances in nontarget organisms, such as bivalves, are relevant. The Responsible editor: Markus Hecker Electronic supplementary material The online version of this article (doi:10.1007/s11356-014-4023-0) contains supplementary material, which is available to authorized users. M. A. S. Serrano : F. Flores-Nunes : Á. C. P. Mello : F. L. Zacchi : C. E. Piazza : M. N. Siebert : R. S. Piazza : A. C. D. Bainy (*) Laboratory of Biomarkers of Aquatic Contamination, Department of Biochemistry, Federal University of Santa Catarina, Florianopolis, SC 88040900, Brazil e-mail: [email protected] M. Gonzalez-Rey : M. J. Bebianno CIMA, Centre for Marine and Environmental Research, University of Algarve, Campus de Gambelas, 8000135 Faro, Portugal J. J. Mattos Aquaculture Pathology Research Center—NEPAQ, Federal University of Santa Catarina, Florianópolis, Brazil D. Alvarez-Muñoz : S. Rodriguez-Mozaz : D. Barceló Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, H2O Building, Emili Grahit 101, 17003 Girona, Spain
Environ Sci Pollut Res
Introduction The presence of pharmaceuticals and personal care products (PPCPs) in the aquatic environment and their effects on biota are still poorly known (Daughton and Ternes 1999; Madden et al. 2009). The growing demand for these compounds (including medicines, perfumes, and cosmetics) applied in both human and veterinary health practice results in an increased production and development of new and more efficient PPCPs every year (Fent et al. 2006; IMS 2011; Ternes et al. 2004;). In the USA, the use of PPCPs increased 2 to 3.9 billion prescriptions annually between 1999 and 2009 (Tong et al. 2011). PPCPs are released into aquatic systems from diffuse sources (e.g., agriculture or aquaculture) or point sources (e.g., hospital effluents and untreated sewage effluents) (Daughton and Ternes 1999) and have been detected at varying concentrations in the environment (Fent et al. 2006; Kümmerer 2009; Santos et al. 2010). The most commonly detected PPCPs in surface waters are steroids and nonprescription medicines. Among the detected therapeutic drugs are antibiotics, analgesics, anti-inflammatories, lipid regulators, beta blockers, antiepileptics, and synthetic steroids (Hernando et al. 2006; Santos et al. 2010). Ibuprofen (IBU) is the most used nonsteroidal antiinflammatory drug (NSAID) worldwide (Fent et al. 2006; Rao and Knaus 2008). As a consequence, IBU was detected in wastewater and surface waters at concentrations in the nanogram per liter to microgram per liter range (Ashton et al. 2004; Farré et al. 2001; Weigel et al. 2004). Moreover, IBU concentrations reported in the literature vary among countries. In European countries, IBU concentrations range between 0.042 and 7.1 μg L−1 (Ashton et al. 2004; Bendz et al. 2005; Santos et al. 2010) while in major rivers of Korea, it reached 0.03 μg L−1 (Kim et al. 2007). In surface water levels in Canada, they reached 6.7 μg L−1 (Verenitch et al. 2006). Relatively high concentrations of IBU were detected in wastewater treatment plant (STP) influents (up to 84 μg L−1) and effluents (up to 85 μg L−1) (Zuccato et al. 2005; Brun et al. 2006; Gómez et al. 2007; Farré et al. 2001). IBU is a NSAID that promotes the nonselective inhibition of cyclooxygenase isoforms (COX-1 and COX-2) involved in the biosynthesis of pro-inflammatory prostaglandins (PGs) from phospholipid arachidonic acid (AA) (Fent et al. 2006; Gierse et al. 1999; Rao and Knaus 2008). AA is oxidized by the enzyme cyclooxygenase and converted to prostaglandin G2 (PGG2) and prostaglandin H2 (PGH2). The inhibition of COX by IBU may cause an accumulation of AA, generating reactive oxygen species and consequently oxidative stress in cells (Hermes-Lima 2004). IBU is biotransformed by cytochrome P450 (CYP) mostly by isoforms belonging to the CYP2C subfamily (Hamman et al. 1997). IBU metabolites are conjugated by phase II enzymes, such as glutathione S-transferases (GSTs) and
sulfotransferases (ST), among others, to enhance their polarity for excretion (Regoli et al. 2011). Information concerning IBU toxicity to nontarget organisms is limited to a few freshwater (Parolini et al. 2009) and marine species (Ericson et al. 2010; Gonzalez-Rey and Bebianno 2011; 2012). Lysosomal membrane destabilization was described in hemocytes of clam Dreissena polymorpha incubated with IBU at concentrations from 45 to 909 μg L−1 (Parolini et al. 2009). The same species exposed to IBU 0.2, 2, and 8 μg L−1 showed changes in antioxidant activity (Parolini et al. 2011) and increased oxidative stress in the digestive gland (Contardo-Jara et al. 2011). In mussel Mytilus galloprovincialis, IBU was characterized by an increase of alkali-labile phosphate (ALP) levels, particularly in males which leads to mussels’ reproductive fitness impairment highlighting an impact of IBU as an endocrine disrupter (Gonzalez-Rey and Bebianno 2011; 2012). Mytilus edulis trossulus exposed to IBU showed a significant reduction in growth and byssus strength (Ericson et al. 2010). Changes in immune responses were observed in clam Ruditapes philippinarum exposed to sublethal concentrations of IBU (100, 500, and 1000 μg L−1) for 7 days (Matozzo et al. 2012). The Pacific oyster Crassostrea gigas is a very important species in estuarine environments and constitutes an important food source for marine organisms, as well as for human consumption (Markert et al. 2010). C. gigas is responsible for 32 % of the total value of global aquaculture production, which is worth in excess of US$3 billion annually (Wright et al. 2014). Any impact caused by chemical contaminants, such as IBU, may cause a significant impact on ecosystem function and the production of protein across the globe. Therefore, the objective of this study was to evaluate some biochemical, molecular, and cytotoxic biomarkers in oyster C. gigas exposed to two concentrations of IBU (1 and 100 μg L−1) over different periods of exposure (1, 4, and 7 days) under laboratory conditions, in order to get more information about the potential risk of exposure to this pharmaceutical.
Materials and methods Oyster acclimation and maintenance conditions Oysters C. gigas (6.5±0.5 cm) were obtained from an oyster farming area at the Marine Mollusk Laboratory (LMM, UFSC, Brazil). The specimens were selected, cleaned of fouling, and immediately transported to the laboratory for an acclimation period of 12 days. Under controlled conditions, the oysters were held in aerated filtered seawater (0.45 μm), temperature (22 °C), and salinity (30 ppt) and fed with a maintenance diet (mix: 50 %—105 cells mL−1 Isochrysis galbana, 50 %—105 cells mL−1 Chaetoceros muelleri).
Environ Sci Pollut Res
IBU exposure conditions and tissue collection
Analysis of IBU in tissues of C. gigas
Ibuprofen (2-(4-isobutylphenyl) propanoic acid) (I4883, ≥98 % GC, CAS: 15687-27-1) was acquired from SigmaAldrich (Steinheim, Germany). The oysters were placed in three glass aquaria of 60 L (n=50 oysters/aquarium) comprising the control (0) and two IBU treatments (1 and 100 μg L−1). The selected nominal concentrations used were based on the data presented on the toxicity of IBU in bivalve mollusks (Contardo-Jara et al. 2011; Ericson et al. 2010; Matozzo et al. 2012; Milan et al. 2013; Parolini et al. 2009; Pounds et al. 2008). During the exposure period, the oysters were fed once a day with the same acclimation diet 2 h before exchanging water. The following sequence was used: withdrawal of 100 % of the aquarium water and the cleaning of the inner walls of tanks and the valves of oysters with seawater. The remaining water was removed, and clean water containing microalgae was added to the aquarium to feed the oysters. The water used to feed the oysters was removed, and the tank was re-filled with water containing IBU (1 and 100 μg L−1) or ethanol (ethanol 0.02 %), for the treatments and control, respectively. Specimen collection was performed after 1, 4, and the end of 7 days for each treatment (1 and 100 μg L−1) and control (CT=without IBU). The oysters were weighed and measured, and the hemolymph was collected directly from the adductor muscle (n=10), stored on ice, and covered with foil until analysis. For the biochemical analysis, gills were dissected (n=10), immediately frozen in liquid nitrogen, and stored at −80 °C. For the molecular analysis, gills were immediately placed in RNAlater® and stored at −20 °C. After each exposure period, the water level in each aquarium was reduced by 10 L to maintain a ratio of one animal to 1 L of water, keeping the ratio of food and IBU added.
Samples of 0.5 g of lyophilized tissues from each experimental group were extracted in triplicate from each sample using a unit of accelerated solvent extraction (ASE 350, Dionex®). After extraction, the sample was dried to approximately 30 % of its original volume and prepared for HPLC according to Ferrando-Climent et al. (2012). The detection limit was 0.01 μg kg−1. To check for any signal suppression caused by the analyte in the sample matrix, extracts from solid-phase extraction (SPE) were purified, concentrated, and mixed with different concentrations of IBU (1, 5, 10, 25, 50, and 100 mg kg−1).
Tissue preparation for biochemical analysis Gills (n=10) were individually weighed and homogenized in 1:5 (w:v) chilled buffer (50 mM Tris-HCl, pH 7.6, containing 0.5 M sucrose, 1 mM DTT, 1 mM EDTA, 0.15 M KCl, 0.1 mM PMSF) using the tissue homogenizer TissueTearorTM. The homogenates were centrifuged at 9000×g for 30 min at 4 °C, followed by a second centrifugation of the supernatant at 100,000×g for 1 h at 4 °C to obtain the cytosolic fraction. The resulting supernatant was used for biochemical analysis. Total protein levels were quantified in the supernatant, according to Bradford (1976), using bovine serum albumin as standard. After 7 days of exposure, two sets of oysters (n=~15–19 oysters/per treatment; ~80 g of whole wet tissue in duplicate) were collected from each treatment, homogenized, and stored at −80 °C. The homogenate was lyophilized and stored according to Huerta et al. (2012) for subsequent IBU quantification.
Neutral red assay Neutral red assay (NRA) was performed in duplicate using 150 μL of hemolymph collected from oysters (n=10). The stock neutral red solution (NRS) was diluted in marine phosphate buffer solution (PBS) (50 μL VN+10 mL PBS). Immediately after collection, the hemolymph samples were centrifuged (1500×g; 10 min at 4 °C), the supernatant discarded, and 150 μL of NRS was added to the hemocytes followed by a 3-h incubation period in the dark at 25 °C. After the incubation period, the samples were re-centrifuged (1500×g for 10 min at 4 °C), the supernatant discarded, and the pellet dissolved in marine PBS. This washing step was repeated twice. The washed pellet from each sample was dissolved in 200 μL acidified alcohol (50 % ethanol+1 % acetic acid) and stored at −80 °C. After 24 h, the homogenized samples were centrifuged (1000×g for 10 min at 4 °C). The supernatants were analyzed in a spectrophotometer using 96 microplate wells at 550 nm. Readings were performed in duplicate along with two blank samples (marine and acidified alcohol PBS) for each reading.
Enzyme assays Catalase (CAT) activity was measured by the decrease in absorbance at 240 nm by H2O2 decomposition, according to Aebi (1984). Glutathione peroxidase (GPx) activity was measured indirectly by monitoring the NADPH oxidation rate at 340 nm according to Wendel (1981) using cumene hydroperoxide (CuOOH) as substrate. Glutathione reductase (GR) activity was quantified by the NADPH oxidation rate at 340 nm (Carlberg and Mannervik 1985). Glutathione S-transferase (GST) activity was assayed by increasing absorbance at 340 nm, using 1-chloro-2,4 dinitrobenzene (CDNB) as substrate (Keen et al. 1976). All enzyme assays using visible wavelengths were carried out using the 96-well plate reader Spectramax 250 (Molecular Devices, Sunnyvale, CA).
Environ Sci Pollut Res
Gene transcription: RNA extraction and cDNA synthesis
Table 1 Selected primer sequence (forward and reverse) data for oyster species Crassostrea gigas—IBU exposure treatment
Total RNA was extracted according to QIAzol Lysis Reagent from gill tissues (100 mg) of all experimental groups (n=10 each), according to the manufacturer’s protocol (Qiagen 2009a), and the concentration in each sample was determined using a NanoDrop ND-1000 spectrophotometer at 260 nm. The reverse transcription was performed with QuantiTect® Reverse Trancription Kit (Qiagen 2009b), using 1 μg of total RNA. The complementary DNA (cDNA) quantification from each sample was measured using NanoDrop at 260 nm.
Gene
Genes of interest COX-like F COX-like R CASPASE-like F CASPASE-like R
Quantitative real-time PCR analysis
CYP2AU2F
Primers were designed with OligoAnalyzer® and PrimerQuest® (IDT, http://www.idtdna.com) software based on the complete and/or partial messenger RNA (mRNA) sequences of each gene obtained from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm. nih.gov/) and Mytibase (http://mussel.cribi.unipd.it/) databases. The selected genes of interest were COX-like, Caspase, Catalase (CAT), CYP30C1, CYP2AU2, CYP3071A1, CYP356A1, FABP-like, HSP70, GST-ω-like, and GST-π-like. The transcription of these genes was normalized against reference genes: glyceraldehyde 3phosphate dehydrogenase-like (GAPDH-like) and β-tubulinlike (β-TUB-like). The primer sequences and GenBank access numbers are described in Table 1. Real-time reactions were performed with 100 ng of template cDNA per reaction following QuantiFast® SYBR® Green PCR kit methodology (Qiagen 2011) using real-time cycler Rotor Gene Q Qiagen® and Rotor Gene 6000 Series software. The cycling conditions were set as 1) PCR initial heat activation (95 °C for 5 min), 2) denaturation (95 °C for 10 s), and 3) combined annealing/ extension (60 °C for 30 s). For all reactions, 40 cycles of denaturation and annealing/extension were performed.
CYP2AU2 R CYP30C1-like F CYP30C1-like R CYP3071A1-like F CYP3071A1-like R CYP356A1 F CYP356A1 R FABP-like F FABP-like R GST Ω-like F GST Ω-like R GST π-like F GST π-like R CAT-like F
Analysis of gene transcription The levels of gene transcription were analyzed using a thermal cycler Rotor-Ge ne 6000 TM (Corbett Life Science). In order to grant a robust normalization of each gene of interest, the geometric mean of two normalizing genes was calculated (βTubulin-like and GAPDH-like). Finally, the data was calibrated to the respective controls.
CAT-like R HSP70-like F HSP70-like R Normalizing genes β-TUB-like F β-TUB-like R
Statistical analysis All results were calculated as mean (±standard deviation (SD)). Data was subjected to outlier verification (Grubbs test); the normality and homoscedasticity were verified using Shapiro-Wilk and Levene’s tests, respectively. In some cases,
GAPDH-like F GAPDH-like R
Primer
Access number
CCGTATTCTGTGAAG GGTCTGATGGCAA GAACCCAACCTCTCC ACCAAACGTAGAA AAGCGATGAGCCCAG AGTGTGTTTCT CGCTGTCTGTATTGTA GTGGCAACTGGT GCCACTTCTAGTCCA TCTTCACCTGC GCTCTTCGATTACTT CATTTGCGAACCC TCTAAAGCCGGACTC TTGAAGCCAGA TTTCGCCTTTCAGGT GCGTGGTTC ACCGTGTGTGTTT AGGCTCG CTGTTTGTTAGTC CTGCGTTCGG ATGAAACCCGCGA AACCAGA TAAATTCGGCTTC ACGCCCT
FJ375303.2
TCCGACGGAAAG ATGATGACGCTTT ACGCCATTGCATGTT GCTGT TGATGAGTTCACCAC CGCAA TTCAAACCATGGCCA CAGCA CACCATTCACGACTTT GTGGCAGA TCAGCCATTTCGGTAG CCTCTCTT AGCTAATCGTTTGTCT GCCGAGGA ACACTTTGGTCACATC GAACGGGT
EU069496
HQ425703.1
EKC26764.1
EKC28276.1
EKC42568.1
EF645271
AJ557141
AJ557140
EF687775
ACCCGTTCCAGAGTT CTTGGGTTT ATCGGACGAGGGCCA CAGTTATT
ABJ55915
CCAGCAGATGTTCGA CGCCAAGAA AACRGGCAGCAACGG TGAGGTAG AAGCAACAAGGATTG GCGTGGT AACTGGTACGCGGAA AGCCATT
AB296534
CAD67717
Environ Sci Pollut Res
the data was normalized using the logarithmic transformation (Y=log (Y)) (Zar 1999). Two-way ANOVA was performed for cell viability and enzymatic activity parameters to investigate possible time and dose-effect relationships, using time of exposure and IBU concentrations as factors. One-way ANOVA was used in gene transcription data analysis because the RNA extraction and cDNA synthesis from the groups at different periods were carried out in different days. Likewise, the quantitative realtime PCR analysis (qPCR) run to quantify the different genes was grouped by period of exposure since it was decided to compare treatments with their respective control group in each exposure time. Both one- and two-way ANOVA were followed by Tukey’s post hoc test to evaluate any significant differences (p
MOLECULAR AND CELLULAR EFFECTS OF CONTAMINATION IN AQUATIC ECOSYSTEMS
Differential gene transcription, biochemical responses, and cytotoxicity assessment in Pacific oyster Crassostrea gigas exposed to ibuprofen Miguel A. S. Serrano & Maria Gonzalez-Rey & Jacó J. Mattos & Fabrício Flores-Nunes & Álvaro C. P. Mello & Flávia L. Zacchi & Clei E. Piazza & Marília N. Siebert & Rômi S. Piazza & Diana Alvarez-Muñoz & Sara Rodriguez-Mozaz & Damià Barceló & Maria João Bebianno & Carlos H. A. M. Gomes & Cláudio M. R. Melo & Afonso C. D. Bainy
Received: 15 August 2014 / Accepted: 17 December 2014 # Springer-Verlag Berlin Heidelberg 2015
D. Barceló Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Jordi Girona 18-26, 08034 Barcelona, Spain
aim of this study was to evaluate the effects on antioxidant status caused by ibuprofen (IBU) in oysters Crassostrea gigas exposed for 1, 4, and 7 days at concentrations 1 and 100 μg L−1. Levels of IBU in tissues of oysters, as well as cell viability of hemocytes, were measured. The transcription of cytochrome P450 genes (CYP2AU2, CYP356A1, CYP3071A1, CYP30C1), glutathione S-transferase isoforms (GST-ω-like and GST-πlike), cyclooxygenase-like (COX-like), fatty acid binding protein-like (FABP-like), caspase-like, heat shock proteinlike (HSP70-like), catalase-like (CAT-like), and the activity of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST) were also evaluated in the gills of oysters. The highest levels of IBU were observed in animals exposed to 100 μg L−1. A significant upregulation of CYP2AU1, CYP356A1, CYP3071A1, GST-ω-like, GST-π-like, COXlike, and FABP-like was observed in oysters exposed to IBU under different experimental conditions. Oysters exposed to 1 μg L−1 for 7 days showed a significantly higher transcription of CYP2AU2, CYP356A1, CYP3071A1, GST-ω-like, and GST-π-like but lower GR activity. In conclusion, C. gigas exposed to environmentally relevant concentrations of IBU (1 μg L−1) exhibited increased transcription of certain genes and alterations on antioxidant and auxiliary enzymes, which could, in the the long term, cause damages to exposed organisms.
C. H. A. M. Gomes : C. M. R. Melo Laboratory of Marine Mollusk, Department of Aquaculture, Center of Agricultural Science, Federal University of Santa Catarina, Florianopolis, SC 88040900, Brazil
Keywords Crassostrea gigas . Pharmaceuticals . Ibuprofen . Oxidative stress . Gene transcription
Abstract Pharmaceuticals, such as anti-inflammatory nonsteroidal drugs, are frequently detected in aquatic ecosystems. Studies about the effects of these substances in nontarget organisms, such as bivalves, are relevant. The Responsible editor: Markus Hecker Electronic supplementary material The online version of this article (doi:10.1007/s11356-014-4023-0) contains supplementary material, which is available to authorized users. M. A. S. Serrano : F. Flores-Nunes : Á. C. P. Mello : F. L. Zacchi : C. E. Piazza : M. N. Siebert : R. S. Piazza : A. C. D. Bainy (*) Laboratory of Biomarkers of Aquatic Contamination, Department of Biochemistry, Federal University of Santa Catarina, Florianopolis, SC 88040900, Brazil e-mail: [email protected] M. Gonzalez-Rey : M. J. Bebianno CIMA, Centre for Marine and Environmental Research, University of Algarve, Campus de Gambelas, 8000135 Faro, Portugal J. J. Mattos Aquaculture Pathology Research Center—NEPAQ, Federal University of Santa Catarina, Florianópolis, Brazil D. Alvarez-Muñoz : S. Rodriguez-Mozaz : D. Barceló Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, H2O Building, Emili Grahit 101, 17003 Girona, Spain
Environ Sci Pollut Res
Introduction The presence of pharmaceuticals and personal care products (PPCPs) in the aquatic environment and their effects on biota are still poorly known (Daughton and Ternes 1999; Madden et al. 2009). The growing demand for these compounds (including medicines, perfumes, and cosmetics) applied in both human and veterinary health practice results in an increased production and development of new and more efficient PPCPs every year (Fent et al. 2006; IMS 2011; Ternes et al. 2004;). In the USA, the use of PPCPs increased 2 to 3.9 billion prescriptions annually between 1999 and 2009 (Tong et al. 2011). PPCPs are released into aquatic systems from diffuse sources (e.g., agriculture or aquaculture) or point sources (e.g., hospital effluents and untreated sewage effluents) (Daughton and Ternes 1999) and have been detected at varying concentrations in the environment (Fent et al. 2006; Kümmerer 2009; Santos et al. 2010). The most commonly detected PPCPs in surface waters are steroids and nonprescription medicines. Among the detected therapeutic drugs are antibiotics, analgesics, anti-inflammatories, lipid regulators, beta blockers, antiepileptics, and synthetic steroids (Hernando et al. 2006; Santos et al. 2010). Ibuprofen (IBU) is the most used nonsteroidal antiinflammatory drug (NSAID) worldwide (Fent et al. 2006; Rao and Knaus 2008). As a consequence, IBU was detected in wastewater and surface waters at concentrations in the nanogram per liter to microgram per liter range (Ashton et al. 2004; Farré et al. 2001; Weigel et al. 2004). Moreover, IBU concentrations reported in the literature vary among countries. In European countries, IBU concentrations range between 0.042 and 7.1 μg L−1 (Ashton et al. 2004; Bendz et al. 2005; Santos et al. 2010) while in major rivers of Korea, it reached 0.03 μg L−1 (Kim et al. 2007). In surface water levels in Canada, they reached 6.7 μg L−1 (Verenitch et al. 2006). Relatively high concentrations of IBU were detected in wastewater treatment plant (STP) influents (up to 84 μg L−1) and effluents (up to 85 μg L−1) (Zuccato et al. 2005; Brun et al. 2006; Gómez et al. 2007; Farré et al. 2001). IBU is a NSAID that promotes the nonselective inhibition of cyclooxygenase isoforms (COX-1 and COX-2) involved in the biosynthesis of pro-inflammatory prostaglandins (PGs) from phospholipid arachidonic acid (AA) (Fent et al. 2006; Gierse et al. 1999; Rao and Knaus 2008). AA is oxidized by the enzyme cyclooxygenase and converted to prostaglandin G2 (PGG2) and prostaglandin H2 (PGH2). The inhibition of COX by IBU may cause an accumulation of AA, generating reactive oxygen species and consequently oxidative stress in cells (Hermes-Lima 2004). IBU is biotransformed by cytochrome P450 (CYP) mostly by isoforms belonging to the CYP2C subfamily (Hamman et al. 1997). IBU metabolites are conjugated by phase II enzymes, such as glutathione S-transferases (GSTs) and
sulfotransferases (ST), among others, to enhance their polarity for excretion (Regoli et al. 2011). Information concerning IBU toxicity to nontarget organisms is limited to a few freshwater (Parolini et al. 2009) and marine species (Ericson et al. 2010; Gonzalez-Rey and Bebianno 2011; 2012). Lysosomal membrane destabilization was described in hemocytes of clam Dreissena polymorpha incubated with IBU at concentrations from 45 to 909 μg L−1 (Parolini et al. 2009). The same species exposed to IBU 0.2, 2, and 8 μg L−1 showed changes in antioxidant activity (Parolini et al. 2011) and increased oxidative stress in the digestive gland (Contardo-Jara et al. 2011). In mussel Mytilus galloprovincialis, IBU was characterized by an increase of alkali-labile phosphate (ALP) levels, particularly in males which leads to mussels’ reproductive fitness impairment highlighting an impact of IBU as an endocrine disrupter (Gonzalez-Rey and Bebianno 2011; 2012). Mytilus edulis trossulus exposed to IBU showed a significant reduction in growth and byssus strength (Ericson et al. 2010). Changes in immune responses were observed in clam Ruditapes philippinarum exposed to sublethal concentrations of IBU (100, 500, and 1000 μg L−1) for 7 days (Matozzo et al. 2012). The Pacific oyster Crassostrea gigas is a very important species in estuarine environments and constitutes an important food source for marine organisms, as well as for human consumption (Markert et al. 2010). C. gigas is responsible for 32 % of the total value of global aquaculture production, which is worth in excess of US$3 billion annually (Wright et al. 2014). Any impact caused by chemical contaminants, such as IBU, may cause a significant impact on ecosystem function and the production of protein across the globe. Therefore, the objective of this study was to evaluate some biochemical, molecular, and cytotoxic biomarkers in oyster C. gigas exposed to two concentrations of IBU (1 and 100 μg L−1) over different periods of exposure (1, 4, and 7 days) under laboratory conditions, in order to get more information about the potential risk of exposure to this pharmaceutical.
Materials and methods Oyster acclimation and maintenance conditions Oysters C. gigas (6.5±0.5 cm) were obtained from an oyster farming area at the Marine Mollusk Laboratory (LMM, UFSC, Brazil). The specimens were selected, cleaned of fouling, and immediately transported to the laboratory for an acclimation period of 12 days. Under controlled conditions, the oysters were held in aerated filtered seawater (0.45 μm), temperature (22 °C), and salinity (30 ppt) and fed with a maintenance diet (mix: 50 %—105 cells mL−1 Isochrysis galbana, 50 %—105 cells mL−1 Chaetoceros muelleri).
Environ Sci Pollut Res
IBU exposure conditions and tissue collection
Analysis of IBU in tissues of C. gigas
Ibuprofen (2-(4-isobutylphenyl) propanoic acid) (I4883, ≥98 % GC, CAS: 15687-27-1) was acquired from SigmaAldrich (Steinheim, Germany). The oysters were placed in three glass aquaria of 60 L (n=50 oysters/aquarium) comprising the control (0) and two IBU treatments (1 and 100 μg L−1). The selected nominal concentrations used were based on the data presented on the toxicity of IBU in bivalve mollusks (Contardo-Jara et al. 2011; Ericson et al. 2010; Matozzo et al. 2012; Milan et al. 2013; Parolini et al. 2009; Pounds et al. 2008). During the exposure period, the oysters were fed once a day with the same acclimation diet 2 h before exchanging water. The following sequence was used: withdrawal of 100 % of the aquarium water and the cleaning of the inner walls of tanks and the valves of oysters with seawater. The remaining water was removed, and clean water containing microalgae was added to the aquarium to feed the oysters. The water used to feed the oysters was removed, and the tank was re-filled with water containing IBU (1 and 100 μg L−1) or ethanol (ethanol 0.02 %), for the treatments and control, respectively. Specimen collection was performed after 1, 4, and the end of 7 days for each treatment (1 and 100 μg L−1) and control (CT=without IBU). The oysters were weighed and measured, and the hemolymph was collected directly from the adductor muscle (n=10), stored on ice, and covered with foil until analysis. For the biochemical analysis, gills were dissected (n=10), immediately frozen in liquid nitrogen, and stored at −80 °C. For the molecular analysis, gills were immediately placed in RNAlater® and stored at −20 °C. After each exposure period, the water level in each aquarium was reduced by 10 L to maintain a ratio of one animal to 1 L of water, keeping the ratio of food and IBU added.
Samples of 0.5 g of lyophilized tissues from each experimental group were extracted in triplicate from each sample using a unit of accelerated solvent extraction (ASE 350, Dionex®). After extraction, the sample was dried to approximately 30 % of its original volume and prepared for HPLC according to Ferrando-Climent et al. (2012). The detection limit was 0.01 μg kg−1. To check for any signal suppression caused by the analyte in the sample matrix, extracts from solid-phase extraction (SPE) were purified, concentrated, and mixed with different concentrations of IBU (1, 5, 10, 25, 50, and 100 mg kg−1).
Tissue preparation for biochemical analysis Gills (n=10) were individually weighed and homogenized in 1:5 (w:v) chilled buffer (50 mM Tris-HCl, pH 7.6, containing 0.5 M sucrose, 1 mM DTT, 1 mM EDTA, 0.15 M KCl, 0.1 mM PMSF) using the tissue homogenizer TissueTearorTM. The homogenates were centrifuged at 9000×g for 30 min at 4 °C, followed by a second centrifugation of the supernatant at 100,000×g for 1 h at 4 °C to obtain the cytosolic fraction. The resulting supernatant was used for biochemical analysis. Total protein levels were quantified in the supernatant, according to Bradford (1976), using bovine serum albumin as standard. After 7 days of exposure, two sets of oysters (n=~15–19 oysters/per treatment; ~80 g of whole wet tissue in duplicate) were collected from each treatment, homogenized, and stored at −80 °C. The homogenate was lyophilized and stored according to Huerta et al. (2012) for subsequent IBU quantification.
Neutral red assay Neutral red assay (NRA) was performed in duplicate using 150 μL of hemolymph collected from oysters (n=10). The stock neutral red solution (NRS) was diluted in marine phosphate buffer solution (PBS) (50 μL VN+10 mL PBS). Immediately after collection, the hemolymph samples were centrifuged (1500×g; 10 min at 4 °C), the supernatant discarded, and 150 μL of NRS was added to the hemocytes followed by a 3-h incubation period in the dark at 25 °C. After the incubation period, the samples were re-centrifuged (1500×g for 10 min at 4 °C), the supernatant discarded, and the pellet dissolved in marine PBS. This washing step was repeated twice. The washed pellet from each sample was dissolved in 200 μL acidified alcohol (50 % ethanol+1 % acetic acid) and stored at −80 °C. After 24 h, the homogenized samples were centrifuged (1000×g for 10 min at 4 °C). The supernatants were analyzed in a spectrophotometer using 96 microplate wells at 550 nm. Readings were performed in duplicate along with two blank samples (marine and acidified alcohol PBS) for each reading.
Enzyme assays Catalase (CAT) activity was measured by the decrease in absorbance at 240 nm by H2O2 decomposition, according to Aebi (1984). Glutathione peroxidase (GPx) activity was measured indirectly by monitoring the NADPH oxidation rate at 340 nm according to Wendel (1981) using cumene hydroperoxide (CuOOH) as substrate. Glutathione reductase (GR) activity was quantified by the NADPH oxidation rate at 340 nm (Carlberg and Mannervik 1985). Glutathione S-transferase (GST) activity was assayed by increasing absorbance at 340 nm, using 1-chloro-2,4 dinitrobenzene (CDNB) as substrate (Keen et al. 1976). All enzyme assays using visible wavelengths were carried out using the 96-well plate reader Spectramax 250 (Molecular Devices, Sunnyvale, CA).
Environ Sci Pollut Res
Gene transcription: RNA extraction and cDNA synthesis
Table 1 Selected primer sequence (forward and reverse) data for oyster species Crassostrea gigas—IBU exposure treatment
Total RNA was extracted according to QIAzol Lysis Reagent from gill tissues (100 mg) of all experimental groups (n=10 each), according to the manufacturer’s protocol (Qiagen 2009a), and the concentration in each sample was determined using a NanoDrop ND-1000 spectrophotometer at 260 nm. The reverse transcription was performed with QuantiTect® Reverse Trancription Kit (Qiagen 2009b), using 1 μg of total RNA. The complementary DNA (cDNA) quantification from each sample was measured using NanoDrop at 260 nm.
Gene
Genes of interest COX-like F COX-like R CASPASE-like F CASPASE-like R
Quantitative real-time PCR analysis
CYP2AU2F
Primers were designed with OligoAnalyzer® and PrimerQuest® (IDT, http://www.idtdna.com) software based on the complete and/or partial messenger RNA (mRNA) sequences of each gene obtained from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm. nih.gov/) and Mytibase (http://mussel.cribi.unipd.it/) databases. The selected genes of interest were COX-like, Caspase, Catalase (CAT), CYP30C1, CYP2AU2, CYP3071A1, CYP356A1, FABP-like, HSP70, GST-ω-like, and GST-π-like. The transcription of these genes was normalized against reference genes: glyceraldehyde 3phosphate dehydrogenase-like (GAPDH-like) and β-tubulinlike (β-TUB-like). The primer sequences and GenBank access numbers are described in Table 1. Real-time reactions were performed with 100 ng of template cDNA per reaction following QuantiFast® SYBR® Green PCR kit methodology (Qiagen 2011) using real-time cycler Rotor Gene Q Qiagen® and Rotor Gene 6000 Series software. The cycling conditions were set as 1) PCR initial heat activation (95 °C for 5 min), 2) denaturation (95 °C for 10 s), and 3) combined annealing/ extension (60 °C for 30 s). For all reactions, 40 cycles of denaturation and annealing/extension were performed.
CYP2AU2 R CYP30C1-like F CYP30C1-like R CYP3071A1-like F CYP3071A1-like R CYP356A1 F CYP356A1 R FABP-like F FABP-like R GST Ω-like F GST Ω-like R GST π-like F GST π-like R CAT-like F
Analysis of gene transcription The levels of gene transcription were analyzed using a thermal cycler Rotor-Ge ne 6000 TM (Corbett Life Science). In order to grant a robust normalization of each gene of interest, the geometric mean of two normalizing genes was calculated (βTubulin-like and GAPDH-like). Finally, the data was calibrated to the respective controls.
CAT-like R HSP70-like F HSP70-like R Normalizing genes β-TUB-like F β-TUB-like R
Statistical analysis All results were calculated as mean (±standard deviation (SD)). Data was subjected to outlier verification (Grubbs test); the normality and homoscedasticity were verified using Shapiro-Wilk and Levene’s tests, respectively. In some cases,
GAPDH-like F GAPDH-like R
Primer
Access number
CCGTATTCTGTGAAG GGTCTGATGGCAA GAACCCAACCTCTCC ACCAAACGTAGAA AAGCGATGAGCCCAG AGTGTGTTTCT CGCTGTCTGTATTGTA GTGGCAACTGGT GCCACTTCTAGTCCA TCTTCACCTGC GCTCTTCGATTACTT CATTTGCGAACCC TCTAAAGCCGGACTC TTGAAGCCAGA TTTCGCCTTTCAGGT GCGTGGTTC ACCGTGTGTGTTT AGGCTCG CTGTTTGTTAGTC CTGCGTTCGG ATGAAACCCGCGA AACCAGA TAAATTCGGCTTC ACGCCCT
FJ375303.2
TCCGACGGAAAG ATGATGACGCTTT ACGCCATTGCATGTT GCTGT TGATGAGTTCACCAC CGCAA TTCAAACCATGGCCA CAGCA CACCATTCACGACTTT GTGGCAGA TCAGCCATTTCGGTAG CCTCTCTT AGCTAATCGTTTGTCT GCCGAGGA ACACTTTGGTCACATC GAACGGGT
EU069496
HQ425703.1
EKC26764.1
EKC28276.1
EKC42568.1
EF645271
AJ557141
AJ557140
EF687775
ACCCGTTCCAGAGTT CTTGGGTTT ATCGGACGAGGGCCA CAGTTATT
ABJ55915
CCAGCAGATGTTCGA CGCCAAGAA AACRGGCAGCAACGG TGAGGTAG AAGCAACAAGGATTG GCGTGGT AACTGGTACGCGGAA AGCCATT
AB296534
CAD67717
Environ Sci Pollut Res
the data was normalized using the logarithmic transformation (Y=log (Y)) (Zar 1999). Two-way ANOVA was performed for cell viability and enzymatic activity parameters to investigate possible time and dose-effect relationships, using time of exposure and IBU concentrations as factors. One-way ANOVA was used in gene transcription data analysis because the RNA extraction and cDNA synthesis from the groups at different periods were carried out in different days. Likewise, the quantitative realtime PCR analysis (qPCR) run to quantify the different genes was grouped by period of exposure since it was decided to compare treatments with their respective control group in each exposure time. Both one- and two-way ANOVA were followed by Tukey’s post hoc test to evaluate any significant differences (p
