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Abstracts from Fourteenth International Symposium on Pollutant Responses in Marine Organisms (PRIMO 14) - Environmental Genomics
Marine Environmental Research 66 (2008) 137–141
Contents lists available at ScienceDirect
Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev
Abstracts from Fourteenth International Symposium on Pollutant Responses in Marine Organisms (PRIMO 14) – Environmental Genomics A genomic analysis of embryo-larval development in plaice (Pleuronectes platessa) with emphasis on pollutant metabolism and detoxification systems Amer Diab a, Paul Hodgson a, Kevin Chipman b, Stephen George Institute of Aquaculture, University of Stirling, UK b University of Birmingham, Birmingham, UK
a
a
Abstract Mortalities during early larval development of fish are naturally very high and these are exacerbated by exposure to pollutants, therefore it has been proposed that the intracellular detoxification systems may not be fully developed during this period. Plaice were artificially reared and total RNA was extracted from replicate samples (five batches over 3 years) of 75–100 eggs, embryos and larvae every 3–5 days up to metamorphosis (ca. 30 days post hatch). Gene transcription patterns were determined with the GENIPOL flounder liver 13,824 element cDNA microarray and results validated by northern blot analysis with probes for specific genes. Hierarchical clustering, principle component and analyses of annotated GO and KEGG terms all revealed development-stage specific distributions in gene expression. For the pollutant detoxification systems, significant amounts of Vitamin E, Rho class GST and MT mRNA’s were present in eggs indicating good antioxidant protection; there were fivefold higher levels of UGT1B1 mRNA in gastrula stage embryos than those post somitogenesis; there was a steady increase in plasma protein precursor, catalase, CYP1A, Se-GPX, GST, superoxide dismutase and ST expression from the heart beat stage (approx. 2 days pre hatch) until the onset of exogenous feeding. Enzyme activity measurements also showed functionality of the systems from hatching. Supported by EU GENIPOL Grant ENV-2001-0057 and NE/C507688/1.
Differentiation of transcriptional responses to BFR’S, PCB’S, HCH and PAH’S in flounder with the Genipol microarray 0141-1136/$ - see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.marenvres.2008.02.045
Amer Diab a, Vicky Sabine a, Matt Gubbins b, Dick Vethaak c, Tim Williams d, Kevin Chipman d, Stephen George a a Institute of Aquaculture, University of Stirling, UK b FRS Marine Laboratory, Aberdeen, UK c RIKZ, The Hague, The Netherlands d University of Birmingham, Birmingham, UK
Abstract Polybrominated diphenyl ethers (PBDEs) have been used extensively as flame retardants in polymeric materials, however, due to their structural similarity to chlorinated biphenyl’s, high bioconcentration factors (over 25,000) and environmental persistence (sediment t1/2 of 2 years), their use has now been phased out in many countries. Toxicological information is poor, however, hepato- and neuro-toxicity, disruption of the thyroid hormone system and activation of the Ah receptor have been reported in mammals. In fish effects are equivocable. In this study we used the GENIPOL liver 13,824 element cDNA microarray to compare hepatic transcriptomic responses in male flounders after 3 months exposure to decadally increasing concentrations of a penta-PBDE mixture added to the sediments (0.7–700 mg/kg TOC) and to the diet(1.4 ìg to 14 mg/ kg lipid) with those elicited 16 days after i.p. injection with a pcb mixture (Arochlor 1254), lindane (gHCH) or 3-methylcholanthrene. Arrays were analysed by hierarchical clustering and principal component analysis using GeneSpring and diagnostic gene sets capable of distinguishing between each of the treatments were derived, thus enabling the arrays to be used diagnostically. Our results showed that only at the highest level of BFR exposure was there a detectable significant estrogenic response (unscheduled VTG and CHG mRNA synthesis) or induction of CYP1A expression. This work was supported by EU grants ENV-2001-005 and QLK4-CT-200200596 and SOAFD.
Gene expression profiling in four Atlantic cod populations Kai K. Lie; Pål A. Olsvik National Institute of Nutrition and Seafood Research, Pb. 2029 Nordnes, N-5817 Bergen, Norway
Abstract In order to investigate toxicological stress responses in cod (Gadus morhua), we have designed a small-scale custom-made cDNA microarray with a total of 746 clones containing genes encoding stress and immune-relevant proteins. Four hundred and seventy one clones were amplified from extracted plasmids while 278 clones were amplified directly from lysed bacterial colonies. The microarray is partitioned into four sub-arrays making it possible to hybridise four differentially labelled cDNA samples on each slide. Optimization of hybridisation was done using different hybridisation temperature regimes and different amounts of labelled cDNA
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in the hybridisation mixture. Eighty to hundred nanograms of labelled cDNA was needed to obtain high intensity spots with low background. The 4 3600 spots design, in addition to the low amounts of labelled cDNA needed for hybridisation, makes the hybridization procedure less time consuming. The custom-made array was used to examine gene expression patterns in liver sampled from four cod populations; two populations from polluted harbour areas, one population from fish caught in an unpolluted area and compared to one farmed fish population. The results showed few differentially expressed genes between the three natural populations compared to the farmed fish population. However, the preliminary results indicate a significant down-regulation of phospholipid hydroperoxide glutathione peroxidase, and a significant up-regulation of complement C4, complement C8 and CYP24A in the wild caught fish compared to the farmed fish (two-fold change). Further QPCR analysis will be done to validate these results. The research was funded by The Norwegian Research Council, project number: 159197/I20.
Are there biological justifications for the use of housekeeping genes in toxicological gene response biomarker studies? Augustine Arukwe Department of Biology, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, 7491 Trondheim, Norway
Abstract The use of housekeeping genes in toxicological gene biomarkers studies implies that their expression patterns are constant regardless of experimental conditions. Housekeeping gene is defined as a gene needed for the sustenance of basic cellular functions. Thus, housekeeping genes are defined by specific gene promoter elements and are expressed constitutively in every cell and used in a normal cell to maintain basic cellular functions. The function of these genes does not necessarily imply that their expressions are not regulated in cells. Indeed, this basic concept has become a misconception of reasonable concern in toxicological research, as these so-called housekeeping genes have been shown to vary considerably across different experimental conditions and thereby lead to an erroneous interpretation of the expression profile of a target gene. This study was performed to evaluate some of the most commonly used housekeeping genes in toxicology (b-actin, b-tubulin, 18S ribosomal RNA (18S rRNA) and elongation factor-1a (EF-1a)) for their stability as reference genes using in vivo (TBT and EE2; DDE and T4) and in vitro (PCB-77 and nonylphenol (NP)) systems. All experiments were performed in a time and concentration exposure conditions either singly or in combination and gene expressions were analyzed using validated qPCR method. Our data showed that these toxicological housekeeping genes were modulated based on random exposure condition and time, in both in vivo and in vitro test systems. Since the choice of reference gene will definitely influence statistical interpretation of toxicological data, it is essential to validate potential reference genes prior to their use, in order to establish their suitability for a specific experimental purpose. Based on the data presented herein and elsewhere, there are very few biological justifications for a generalized use of anything as housekeeping gene in real-time PCR assays for toxicological research. Therefore, this generalized used of the housekeeping gene concept should be avoided. Nevertheless, the absolute need for normalization genes to correct
for sample-to-sample variations should be emphasized. Therefore, the choice of internal control gene should be determined empirically based on the individual exposure condition and by individual researcher. Supported by the Norwegian research Council (NFR). We thank Anne S. Mortensen for technical help during sampling and analysis.
The transcriptional response to oxidative stress in fish embryos and cells exposed to tert-butyl hydroquinone (tBHQ) or 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) Mark E. Hahn a, Sibel I. Karchner a, Diana G. Franks a, Bruce R. Woodin a, Katie L. Barott a, M.J. Cipriano b, Andrew G. McArthur c a Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA b Lawrence Berkeley National Laboratory, One Cyclotron Road, Berkeley, CA, USA c 11 Roanoke Road, Hamilton, Ontario, Canada L8S 3P6
Abstract Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis as well as to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. The developmental expression and inducibility of anti-oxidant defenses through activation of NF-E2-related factor 2 (NRF2) affect susceptibility to oxidants, but the embryonic response to oxidants is not well understood. To assess the response to chemicals at different stages of development, zebrafish (Danio rerio) embryos were exposed to DMSO (0.1%), TCDD (2 nM), or tBHQ (10 mM) for 6 h. Transcript abundance was assessed by real-time qRT-PCR and microarray (Agilent 22k). qRT-PCR showed strong (4- to 5-fold) induction of gstp1 by tBHQ as early as 1 dpf. tBHQ also induced gclc (2 dpf), but not sod1, nqo1, or cyp1a. TCDD induced cyp1a but none of the other genes. By microarray, 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated embryos at 4 dpf. Of these, 220 were =2-fold up-regulated and 108 were =2-fold downregulated by tBHQ. For TCDD, 17 probes were =2-fold up-regulated and 6 were =2-fold down-regulated. Genes induced by tBHQ, included genes involved in glutathione synthesis and utilization (gclc, gclm, ggt, gst), signal transduction (fos, jun, C/EBP eta, cox-2), and DNA damage/stress response (hsp70, gadd45b, atf3). These data show that zebrafish embryos are responsive to oxidative stress as early as 1 dpf, and that microarrays are capable of detecting altered expression of known and novel oxidant-responsive genes in whole embryos. Supported by R01ES006272 and Walter A. and Hope Noyes Smith.
Cross species application of DNA microarrays in fish. . .How many arrays do we need? Stephen George a, Michael Leaver a, Kevin Chipman b, Andrew Cossins c, Moshe Tom d Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, UK b University of Birmingham, Birmingham, UK c University of Liverpool, UK d IOLR, Haifa, Israel
a
Abstracts / Marine Environmental Research 66 (2008) 137–141
Abstract Currently there is much interest in the application of genomic technologies, particularly DNA microarrays, to study pollutant effects in fish from a large geographic area and from a wide variety of habitats. Many of these studies are focussed upon elucidating mechanisms of toxicity which can be extrapolated across species, whilst others may be focused on toxic effects in particular ecological niches e.g. extremes of salinity, temperature, oxia, pressure, etc. or at appraisal of organism health or of the biological availability of contaminants. Fish are the largest vertebrate class with nearly 30,000 different living species, most of which are ray-finned bony fish and they have evolved to populate this wide range of habitats. This diversity is not only seen in their morphology and physiology but also at the level of their gene structures and fish of different taxonomic orders can show quite low sequence homologies. Since construction of DNA microarrays is both expensive and labour intensive it would be beneficial if they could be used across species wherever possible. DNA microarrays from more than a dozen fish species across a wide range of Orders and Families have thus far been constructed. We can make theoretical predictions on their cross species utility from DNA sequence data and in this study we report experimental validation of cross hybridisation utility between different fish species of varying phllyic variance.
Genomic responses to chemicals in the European flounder and identification of gene – sets predictive of origin of fish from the environment Tim Williams a, Fernando Ortega a, Amer Diab b, Francesco Falciani a, Steven George b, James K. Chipman a a School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK b Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK
Abstract Expression of stress responsive genes has been the basis of various biomarkers of pollutant exposure in the environment. Genomic technologies offer opportunities to gain a more global assessment of the health status of an organism through an understanding of the functional pathways that are responding to pollutant exposure. Flounder taken from different sites in Northern Europe (and of different pollution status) can be distinguished according to their hepatic gene expression profile using bioinformatic approaches. To determine which gene expression differences may relate to pollutant impact, we have completed complementary laboratory exposures of flounder to selected toxicants and determined the associated gene expression profiles. Flounders were treated by intraperitoneal injection with 3-methylcholanthrene (25 mg/kg in olive oil), Aroclor 1254 (50 mg/kg in olive oil), perfluoro-octanoic acid (100 mg/kg in olive oil), cadmium chloride (50mg/kg in 0.9% saline), tert-butylhydroperoxide (5 mg/kg in 0.9% saline), lindane (25 mg/kg in olive oil) or olive oil or saline carriers and sampled at days 1, 2, 4, 8 and 16 after exposure. Gene expression in liver tissue was determined using a 13K cDNA microarray and treated groups were compared to the relevant time-matched control groups to generate lists of genes whose expression was altered. As well as providing potential novel individual biomarkers, gene ontology (GO) analyses using Blast2GO allowed the identification of pathways modulated by toxicant stress; for example chaperones, protein synthesis and degradation, cytoskeleton, apoptosis and cell cycle with cadmium. Using a multivariate variable selection cou-
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pled with a statistical modeling procedure (GALGO). We demonstrate that signatures associated with exposure to individual chemicals have some predictive power for geographical site. However, by combining the signatures derived from laboratory exposure to individual chemicals, more accurate models for classification of all the different environmental sites was achieved. This work was funded by the NERC, CEFAS and EU and has involved additional GENIPOL collaborations.
Toxicogenomic tools for Atlantic cod (Gadus morhua) Pål A. Olsvik; Kai K. Lie National Institute of Nutrition and Seafood Research, Pb. 2029 Nordnes, N-5817 Bergen, Norway
Abstract The aim of this project was to develop toxicogenomic tools for the Atlantic cod Gadus morhua. We have designed a small-scale microarray containing 746 clones to be used for gene expression analysis. The cDNA array contains app. Four hundred and fifty genes encoding stress-responsive proteins, 90 immuno genes, 80 metabolism genes and 70 reference (housekeeping) genes. The gene list can be found at the project home page. To be able to measure gene expression with methods like real-time PCR and microarray in an animal, the nucleotide sequences of the studied genes need to be known. So far we have sequenced about 25,000 expressed sequenced tags (EST’s) from seven cDNA libraries in the Atlantic cod. The cDNA libraries were made from liver, gill, head kidney and intestinal tissues, in addition to stem cells. Because the expression of many stress-responsive genes can be low during non-stress situations, we exposed juvenile cod to a mixture of toxicants to induce the mRNA levels of these genes. The toxicant mixture contained PAH’s (1,5 demethylnaphthalene, phenanthrene, fluoanthene and benzo(a)pyren), PCB (aroclor 1254), Cu and Cd. We have also sequenced EST’s from fish caught in polluted harbor waters in Norwegian towns. With the applied shotgun sequencing technique, we were able to find EST’s encoding many of the stress-responsive proteins we were searching for, including proteins involved in the cellular stress response, oxidative stress, apoptosis, acute phase proteins, heat shock proteins and detoxification.
Molecular effects of Ni and clorpyrifos in the digestive gland tissue of Mytilus galloprovincialis: A systems biology approach Francesco Dondero a, Oliver A.H. Jones b, Mohamed Banni a, Lara Boatti a, Pamela Minicozzi a, Alessandro Negri a, Julian L. Griffin b, Aldo Viarengo a a Environmental and Life Sciences Department (DISAV), Università A. Avogadro, Alessandria, Italy b Department of Biochemistry, University of Cambridge, Cambridge, UK
Abstract A systems biology approach based on transcriptomics, proteomics, metabolomics, and biomarkers was applied to study the effects of two prioritized environmental pollutants, i.e. nickel and
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the organophosphate clorpyrifos. Mussels were exposed for 4 days to equitoxic amounts of each substance, i.e. the EC50 for digestive gland lysosomal membrane destabilization. Effects of a mixture of the two chemicals were also assessed (EC25 plus EC25). Microarray analysis (digestive gland) was performed using a 2K MytArray DNA chip [Venier et al., 2006. Mutat. Res. (602), 121–34), providing distinct gene expression patterns between the two treatments. In animals exposed to Ni, about 120 genes were found to be differentially expressed, and these were involved in carbohydrate metabolism (down-regulation of glycan pathways); proteolysis (e.g. induction of catheptsin L), ribosome biogenesis and assembly, signal transduction, immune functions (lectins and galectins). A metallothionein 10IV gene was also induced. Chlorpyrifos exposure involved a lower number of genes, which were mostly up-regulated and mainly involved in aminosugar metabolism (chitinase, chitriosidase, etc.). Ferritin and an hsp70 gene were also up-regulated. Proteomic analysis based on 2D electrophoresis and de novo peptide sequencing tandem (ESI Q TOF) mass spectrometry clearly revealed a different molecular fingerprinting. Differentially expressed proteins (about a dozen in all cases) were mostly down-regulated because of the physiological conditions of mussels, i.e. a dramatic lysosomal membrane destabilization. Polypeptides were partially sequenced but only a few were identified because of the lack of similarity with known orthologues. Interestingly, in CP-exposed mussels we recognized a putative iron responsive element (a modulator of ferritin expression), suggesting a disruption of iron homeostasis. Finally metabolomic based analysis using nuclear magnetic resonance (NMR) spectroscopy and gas chromatography–mass spectrometry (GC–MS) coupled with computer assisted multivariate statistics and pattern recognition techniques gave results that complemented the transcriptomic and proteomic results. Nickel appeared to more toxic than chlorpyrifos and produced changes in amino acid and carbohydrate metabolism. Chlorpyrifos was found to inhibit acetyl cholinesterase activity and also produced changes in amino acid and sugar metabolism. In conclusion, this work demonstrated the applicability of a systems toxicology approach in mussels, a well-established ecotoxicological model species, notwithstanding its available genomic information being still limited.
A microarray meta-analysis approach to find sensitive and robust estrogen induced responses in fish Lina Gunnarsson a, Erik Kristiansson b, Lars Förlin c, Olle Nerman b, Joakim D.G. Larsson a a Department of Neuroscience and Physiology, The Sahlgrenska Academy at Göteborg University, Göteborg, Sweden b Department of Mathematical Statistics, Chalmers University of Technology, Göteborg, Sweden c Department of Zoology/Zoophysiology, Göteborg University, Göteborg, Sweden
Abstract Vitellogenin (VTG) is an established marker for estrogenic exposure in fish. Effects on gonadal differentiation at concentrations not sufficient to induce VTG suggest that more sensitive markers would be useful. The aim of our study was to find sensitive and robust markers of estrogenic exposure. Liver mRNA expression profiles were characterized in estrogen exposed juvenile rainbow trout using microarray. The higher concentration was used to guide the subsequent identification of generally more subtle responses at the low concentration not sufficient to induce vitellogenin. A meta-
analysis was performed with data from the present study and similar microarray studies using different fish species and platforms. Within the generated list of presumably robust responses, several well-known estrogen-regulated genes were identified. Two genes, confirmed by quantitative RT-PCR (qPCR), fulfilled both the criteria of high sensitivity and robustness; the induction of the genes encoding zona pellucida protein 3 and a nucleoside diphosphate kinase (nm23). The cross-species, cross-platform meta-analysis correctly identified several robust responses. This adds confidence to our approach used for identifying candidate biomarkers. Specifically, we propose that analyses of the nm23 together with zona pellucida genes may increase the possibilities to detect an exposure to low levels of estrogenic compounds in fish.
Development of a stickleback (Gasterosteus aculeatus) cDNA microarray and gene expression responses to dibenzanthracene, ethinyl-estradiol and copper Tim D. Williams a, Margaret Brown b, James K. Chipman a, Francesco Falciani a, Fernando Ortega a, Fleur Geoghegan a, John A. Craft b, Ioanna Katsiadaki c, John Ball d, Charles R. Tyler d, Eduarda Santos d a School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK b School of Life Sciences, Glasgow Caledonian University, Glasgow G4 0BA, UK c Centre for Environment, Fisheries and Aquaculture Science, Weymouth DT4 8UB, UK d School of Biosciences, The University of Exeter, Exeter, Devon EX4 4QJ, UK
Abstract The Three-spined stickleback (Gasterosteus aculeatus) is a fish species that inhabits European estuarine and freshwater environments and is a useful model for ecotoxicology and, as a genomesequenced species, ecotoxicogenomics. We have constructed a cDNA array for the stickleback using SSH and normalised liver cDNA libraries (Brown et al.) and a whole body cDNA library (Prof. Kingsley, Stanford, USA). Bioinformatic analyses of the sequenced clones have been carried out with a view to identification and functional annotation by Gene Ontology category (GO). This has used the Blast2GO program (A. Conesa, S. Goetz). Additional annotation has been derived from the stickleback genome project. Our initial microarray experiments with the stickleback have characterized their acute hepatic responses to dibenzanthracene (DbA), ethinyl-estradiol (EE2) and copper (Cu) treatment. Male stickleback were exposed to waterborne DbA at various concentrations between 10 and 50,000 ng/l, EE2 between 0.1 and 100 ng/l or Cu between 3.2 and 128 ìg/l for 4 days. The microarray was used to compare gene expression of exposed fish to those of relevant controls. Microarrays were carried out using both pooled samples and samples from individual fish. Concentration/dose responses were found for known biomarker genes CYP1A (r2 = 0.90), vitellogenin (r2 = 0.97) and metallothionein (r2 = 0.88) to DbA, EE2 and Cu, respectively. Additional responsive genes and Gene Ontology categories will be described, that provide insights into the mechanistic modes of action of these three model pollutants and our findings will highlight additional biomarkers for exposure to these chemicals. Funding was provided by UK Natural Environment Research Council Post Genomics and Proteomics grant NE/ C507661/1.
Abstracts / Marine Environmental Research 66 (2008) 137–141
Gene expression in oyster Crassostrea gigas exposed to sewage contaminated sites Igor D. Medeiros a,b, Juliano Zanette a, Marília N. Sieber a, Guilherme Toledo-Silva a, Thiago B. Rodrigues a, Fabrício F. Nunes a,c, Maria R.F. Marques a, Afonso C.D. Bainy a a Laboratório de Biomarcadores de Contaminação Aquática e Imunoquímica, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil b Laboratório de Ciências Marinhas, Universidade do Sul de Santa Catarina, Palhoça, Santa Catarina, Brazil c Centro Municipal de Educação Ambiental Escola do Mar, São José, Santa Catarina, Brazil
Abstract Exposure to sewage effluents may affect the biotransformation systems of aquatic organisms. The pattern of these responses could be used complementarily as an early warning tool for environmental monitoring. The aim of this work was to analyze the expression of genes in gill of oysters exposed to sewage contaminated sites. Six-month-old-Crassostrea gigas were transplanted from a farming area to four stations distant from a source of sewage 0.01, 0.4, 1.5 and 4.5 km (sites 1, 2, 3 and 4, respectively). After 2 weeks, the oysters were collected and the gills were excised for total RNA extraction and cDNA synthesis. Gene expression analysis was performed using semi-quantitative RT-PCR reaction, with specific primers for the genes cytochrome P450 (CYP356A1), fatty acid binding protein (FABP), glutathione S-transferase (GST) and multidrug resistance protein (MDR). Actin (ACT) gene was used as a housekeeping gene to normalize the data. Oysters exposed to the sewage-contaminated site (Site 1) showed an induction in the expression of FABP, CYP356A1 and GST. The MDR expression was higher in the oysters kept at the Site 3. The induction of these genes in gills of oysters from the contaminated site indicates that discharges of domestic sewage into the coastal water affect the biotransformation systems of this organism, suggesting that after a long-term exposure, high mortality rate might be observed. We suggest the use of these biological markers to evaluate exposure and susceptibility in future biomonitoring programs of coastal waters. Supported by CNPq-Universal to ACDB.
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Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies Giacomo Spinsanti a, Cristina Panti a, Letizia Marsili b, Silvia Casini Maria Cristina Fossi b a Department of Evolutionary Biology, University of Siena, Siena, Italy b Department of Environmental Sciences, University of Siena, Siena, Italy
b
,
Abstract Quantitative real-time PCR is a sensitive molecular technique for the detection of variations in gene expression using minimal amounts of biological materials. The application of qRT-PCR to cetacean skin biopsies, obtained with non lethal techniques from freeranging animals, has the potential of representing a powerful tool for the quantification of gene induction caused by the exposure to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. The expression of 10 potential control genes was examined in 30 striped dolphin (Stenella coeruleoalba) skin biopsy samples, obtained from specimens inhabiting the north-western Mediterranean Sea. The stability of selected control genes was determined using three different softwares: geNorm, NormFinder and BestKeeper. Glyceraldehyde-3P-dehydrogenase (GAPDH) and tyrosine-3-monooxygenase (YWHAZ) always rank as the two most stably expressed HKGs and ribosomal protein L4 (RPL4) and S18 (RPS18) also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC), phosphoglycerate kinase1 (PGK1), hypoxanthine ribosyltransferase (HPRT1) and b-2-microglobin (B2M) show variable expression among the studied samples and appear as less suitable reference genes. Our results suggest that the genes encoding for YWHAZ and GAPDH have the most stable expression patterns and appear as highly reliable controls. Potentially useful reference genes are also those encoding for the ribosomal proteins L4 and S18. This work provides background essential information for studying expression patterns of several potential genes of interest as biomarkers of exposure to contaminants of free-ranging marine mammals.
Contents lists available at ScienceDirect
Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev
Abstracts from Fourteenth International Symposium on Pollutant Responses in Marine Organisms (PRIMO 14) – Environmental Genomics A genomic analysis of embryo-larval development in plaice (Pleuronectes platessa) with emphasis on pollutant metabolism and detoxification systems Amer Diab a, Paul Hodgson a, Kevin Chipman b, Stephen George Institute of Aquaculture, University of Stirling, UK b University of Birmingham, Birmingham, UK
a
a
Abstract Mortalities during early larval development of fish are naturally very high and these are exacerbated by exposure to pollutants, therefore it has been proposed that the intracellular detoxification systems may not be fully developed during this period. Plaice were artificially reared and total RNA was extracted from replicate samples (five batches over 3 years) of 75–100 eggs, embryos and larvae every 3–5 days up to metamorphosis (ca. 30 days post hatch). Gene transcription patterns were determined with the GENIPOL flounder liver 13,824 element cDNA microarray and results validated by northern blot analysis with probes for specific genes. Hierarchical clustering, principle component and analyses of annotated GO and KEGG terms all revealed development-stage specific distributions in gene expression. For the pollutant detoxification systems, significant amounts of Vitamin E, Rho class GST and MT mRNA’s were present in eggs indicating good antioxidant protection; there were fivefold higher levels of UGT1B1 mRNA in gastrula stage embryos than those post somitogenesis; there was a steady increase in plasma protein precursor, catalase, CYP1A, Se-GPX, GST, superoxide dismutase and ST expression from the heart beat stage (approx. 2 days pre hatch) until the onset of exogenous feeding. Enzyme activity measurements also showed functionality of the systems from hatching. Supported by EU GENIPOL Grant ENV-2001-0057 and NE/C507688/1.
Differentiation of transcriptional responses to BFR’S, PCB’S, HCH and PAH’S in flounder with the Genipol microarray 0141-1136/$ - see front matter Ó 2008 Published by Elsevier Ltd. doi:10.1016/j.marenvres.2008.02.045
Amer Diab a, Vicky Sabine a, Matt Gubbins b, Dick Vethaak c, Tim Williams d, Kevin Chipman d, Stephen George a a Institute of Aquaculture, University of Stirling, UK b FRS Marine Laboratory, Aberdeen, UK c RIKZ, The Hague, The Netherlands d University of Birmingham, Birmingham, UK
Abstract Polybrominated diphenyl ethers (PBDEs) have been used extensively as flame retardants in polymeric materials, however, due to their structural similarity to chlorinated biphenyl’s, high bioconcentration factors (over 25,000) and environmental persistence (sediment t1/2 of 2 years), their use has now been phased out in many countries. Toxicological information is poor, however, hepato- and neuro-toxicity, disruption of the thyroid hormone system and activation of the Ah receptor have been reported in mammals. In fish effects are equivocable. In this study we used the GENIPOL liver 13,824 element cDNA microarray to compare hepatic transcriptomic responses in male flounders after 3 months exposure to decadally increasing concentrations of a penta-PBDE mixture added to the sediments (0.7–700 mg/kg TOC) and to the diet(1.4 ìg to 14 mg/ kg lipid) with those elicited 16 days after i.p. injection with a pcb mixture (Arochlor 1254), lindane (gHCH) or 3-methylcholanthrene. Arrays were analysed by hierarchical clustering and principal component analysis using GeneSpring and diagnostic gene sets capable of distinguishing between each of the treatments were derived, thus enabling the arrays to be used diagnostically. Our results showed that only at the highest level of BFR exposure was there a detectable significant estrogenic response (unscheduled VTG and CHG mRNA synthesis) or induction of CYP1A expression. This work was supported by EU grants ENV-2001-005 and QLK4-CT-200200596 and SOAFD.
Gene expression profiling in four Atlantic cod populations Kai K. Lie; Pål A. Olsvik National Institute of Nutrition and Seafood Research, Pb. 2029 Nordnes, N-5817 Bergen, Norway
Abstract In order to investigate toxicological stress responses in cod (Gadus morhua), we have designed a small-scale custom-made cDNA microarray with a total of 746 clones containing genes encoding stress and immune-relevant proteins. Four hundred and seventy one clones were amplified from extracted plasmids while 278 clones were amplified directly from lysed bacterial colonies. The microarray is partitioned into four sub-arrays making it possible to hybridise four differentially labelled cDNA samples on each slide. Optimization of hybridisation was done using different hybridisation temperature regimes and different amounts of labelled cDNA
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Abstracts / Marine Environmental Research 66 (2008) 137–141
in the hybridisation mixture. Eighty to hundred nanograms of labelled cDNA was needed to obtain high intensity spots with low background. The 4 3600 spots design, in addition to the low amounts of labelled cDNA needed for hybridisation, makes the hybridization procedure less time consuming. The custom-made array was used to examine gene expression patterns in liver sampled from four cod populations; two populations from polluted harbour areas, one population from fish caught in an unpolluted area and compared to one farmed fish population. The results showed few differentially expressed genes between the three natural populations compared to the farmed fish population. However, the preliminary results indicate a significant down-regulation of phospholipid hydroperoxide glutathione peroxidase, and a significant up-regulation of complement C4, complement C8 and CYP24A in the wild caught fish compared to the farmed fish (two-fold change). Further QPCR analysis will be done to validate these results. The research was funded by The Norwegian Research Council, project number: 159197/I20.
Are there biological justifications for the use of housekeeping genes in toxicological gene response biomarker studies? Augustine Arukwe Department of Biology, Norwegian University of Science and Technology (NTNU), Høgskoleringen 5, 7491 Trondheim, Norway
Abstract The use of housekeeping genes in toxicological gene biomarkers studies implies that their expression patterns are constant regardless of experimental conditions. Housekeeping gene is defined as a gene needed for the sustenance of basic cellular functions. Thus, housekeeping genes are defined by specific gene promoter elements and are expressed constitutively in every cell and used in a normal cell to maintain basic cellular functions. The function of these genes does not necessarily imply that their expressions are not regulated in cells. Indeed, this basic concept has become a misconception of reasonable concern in toxicological research, as these so-called housekeeping genes have been shown to vary considerably across different experimental conditions and thereby lead to an erroneous interpretation of the expression profile of a target gene. This study was performed to evaluate some of the most commonly used housekeeping genes in toxicology (b-actin, b-tubulin, 18S ribosomal RNA (18S rRNA) and elongation factor-1a (EF-1a)) for their stability as reference genes using in vivo (TBT and EE2; DDE and T4) and in vitro (PCB-77 and nonylphenol (NP)) systems. All experiments were performed in a time and concentration exposure conditions either singly or in combination and gene expressions were analyzed using validated qPCR method. Our data showed that these toxicological housekeeping genes were modulated based on random exposure condition and time, in both in vivo and in vitro test systems. Since the choice of reference gene will definitely influence statistical interpretation of toxicological data, it is essential to validate potential reference genes prior to their use, in order to establish their suitability for a specific experimental purpose. Based on the data presented herein and elsewhere, there are very few biological justifications for a generalized use of anything as housekeeping gene in real-time PCR assays for toxicological research. Therefore, this generalized used of the housekeeping gene concept should be avoided. Nevertheless, the absolute need for normalization genes to correct
for sample-to-sample variations should be emphasized. Therefore, the choice of internal control gene should be determined empirically based on the individual exposure condition and by individual researcher. Supported by the Norwegian research Council (NFR). We thank Anne S. Mortensen for technical help during sampling and analysis.
The transcriptional response to oxidative stress in fish embryos and cells exposed to tert-butyl hydroquinone (tBHQ) or 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) Mark E. Hahn a, Sibel I. Karchner a, Diana G. Franks a, Bruce R. Woodin a, Katie L. Barott a, M.J. Cipriano b, Andrew G. McArthur c a Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA, USA b Lawrence Berkeley National Laboratory, One Cyclotron Road, Berkeley, CA, USA c 11 Roanoke Road, Hamilton, Ontario, Canada L8S 3P6
Abstract Oxidative stress is an important mechanism of chemical toxicity, contributing to teratogenesis as well as to cardiovascular and neurodegenerative diseases. Developing animals may be especially sensitive to chemicals causing oxidative stress. The developmental expression and inducibility of anti-oxidant defenses through activation of NF-E2-related factor 2 (NRF2) affect susceptibility to oxidants, but the embryonic response to oxidants is not well understood. To assess the response to chemicals at different stages of development, zebrafish (Danio rerio) embryos were exposed to DMSO (0.1%), TCDD (2 nM), or tBHQ (10 mM) for 6 h. Transcript abundance was assessed by real-time qRT-PCR and microarray (Agilent 22k). qRT-PCR showed strong (4- to 5-fold) induction of gstp1 by tBHQ as early as 1 dpf. tBHQ also induced gclc (2 dpf), but not sod1, nqo1, or cyp1a. TCDD induced cyp1a but none of the other genes. By microarray, 1477 probes were significantly different among the DMSO-, tBHQ-, and TCDD-treated embryos at 4 dpf. Of these, 220 were =2-fold up-regulated and 108 were =2-fold downregulated by tBHQ. For TCDD, 17 probes were =2-fold up-regulated and 6 were =2-fold down-regulated. Genes induced by tBHQ, included genes involved in glutathione synthesis and utilization (gclc, gclm, ggt, gst), signal transduction (fos, jun, C/EBP eta, cox-2), and DNA damage/stress response (hsp70, gadd45b, atf3). These data show that zebrafish embryos are responsive to oxidative stress as early as 1 dpf, and that microarrays are capable of detecting altered expression of known and novel oxidant-responsive genes in whole embryos. Supported by R01ES006272 and Walter A. and Hope Noyes Smith.
Cross species application of DNA microarrays in fish. . .How many arrays do we need? Stephen George a, Michael Leaver a, Kevin Chipman b, Andrew Cossins c, Moshe Tom d Institute of Aquaculture, University of Stirling, Stirling, FK9 4LA, UK b University of Birmingham, Birmingham, UK c University of Liverpool, UK d IOLR, Haifa, Israel
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Abstract Currently there is much interest in the application of genomic technologies, particularly DNA microarrays, to study pollutant effects in fish from a large geographic area and from a wide variety of habitats. Many of these studies are focussed upon elucidating mechanisms of toxicity which can be extrapolated across species, whilst others may be focused on toxic effects in particular ecological niches e.g. extremes of salinity, temperature, oxia, pressure, etc. or at appraisal of organism health or of the biological availability of contaminants. Fish are the largest vertebrate class with nearly 30,000 different living species, most of which are ray-finned bony fish and they have evolved to populate this wide range of habitats. This diversity is not only seen in their morphology and physiology but also at the level of their gene structures and fish of different taxonomic orders can show quite low sequence homologies. Since construction of DNA microarrays is both expensive and labour intensive it would be beneficial if they could be used across species wherever possible. DNA microarrays from more than a dozen fish species across a wide range of Orders and Families have thus far been constructed. We can make theoretical predictions on their cross species utility from DNA sequence data and in this study we report experimental validation of cross hybridisation utility between different fish species of varying phllyic variance.
Genomic responses to chemicals in the European flounder and identification of gene – sets predictive of origin of fish from the environment Tim Williams a, Fernando Ortega a, Amer Diab b, Francesco Falciani a, Steven George b, James K. Chipman a a School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK b Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK
Abstract Expression of stress responsive genes has been the basis of various biomarkers of pollutant exposure in the environment. Genomic technologies offer opportunities to gain a more global assessment of the health status of an organism through an understanding of the functional pathways that are responding to pollutant exposure. Flounder taken from different sites in Northern Europe (and of different pollution status) can be distinguished according to their hepatic gene expression profile using bioinformatic approaches. To determine which gene expression differences may relate to pollutant impact, we have completed complementary laboratory exposures of flounder to selected toxicants and determined the associated gene expression profiles. Flounders were treated by intraperitoneal injection with 3-methylcholanthrene (25 mg/kg in olive oil), Aroclor 1254 (50 mg/kg in olive oil), perfluoro-octanoic acid (100 mg/kg in olive oil), cadmium chloride (50mg/kg in 0.9% saline), tert-butylhydroperoxide (5 mg/kg in 0.9% saline), lindane (25 mg/kg in olive oil) or olive oil or saline carriers and sampled at days 1, 2, 4, 8 and 16 after exposure. Gene expression in liver tissue was determined using a 13K cDNA microarray and treated groups were compared to the relevant time-matched control groups to generate lists of genes whose expression was altered. As well as providing potential novel individual biomarkers, gene ontology (GO) analyses using Blast2GO allowed the identification of pathways modulated by toxicant stress; for example chaperones, protein synthesis and degradation, cytoskeleton, apoptosis and cell cycle with cadmium. Using a multivariate variable selection cou-
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pled with a statistical modeling procedure (GALGO). We demonstrate that signatures associated with exposure to individual chemicals have some predictive power for geographical site. However, by combining the signatures derived from laboratory exposure to individual chemicals, more accurate models for classification of all the different environmental sites was achieved. This work was funded by the NERC, CEFAS and EU and has involved additional GENIPOL collaborations.
Toxicogenomic tools for Atlantic cod (Gadus morhua) Pål A. Olsvik; Kai K. Lie National Institute of Nutrition and Seafood Research, Pb. 2029 Nordnes, N-5817 Bergen, Norway
Abstract The aim of this project was to develop toxicogenomic tools for the Atlantic cod Gadus morhua. We have designed a small-scale microarray containing 746 clones to be used for gene expression analysis. The cDNA array contains app. Four hundred and fifty genes encoding stress-responsive proteins, 90 immuno genes, 80 metabolism genes and 70 reference (housekeeping) genes. The gene list can be found at the project home page. To be able to measure gene expression with methods like real-time PCR and microarray in an animal, the nucleotide sequences of the studied genes need to be known. So far we have sequenced about 25,000 expressed sequenced tags (EST’s) from seven cDNA libraries in the Atlantic cod. The cDNA libraries were made from liver, gill, head kidney and intestinal tissues, in addition to stem cells. Because the expression of many stress-responsive genes can be low during non-stress situations, we exposed juvenile cod to a mixture of toxicants to induce the mRNA levels of these genes. The toxicant mixture contained PAH’s (1,5 demethylnaphthalene, phenanthrene, fluoanthene and benzo(a)pyren), PCB (aroclor 1254), Cu and Cd. We have also sequenced EST’s from fish caught in polluted harbor waters in Norwegian towns. With the applied shotgun sequencing technique, we were able to find EST’s encoding many of the stress-responsive proteins we were searching for, including proteins involved in the cellular stress response, oxidative stress, apoptosis, acute phase proteins, heat shock proteins and detoxification.
Molecular effects of Ni and clorpyrifos in the digestive gland tissue of Mytilus galloprovincialis: A systems biology approach Francesco Dondero a, Oliver A.H. Jones b, Mohamed Banni a, Lara Boatti a, Pamela Minicozzi a, Alessandro Negri a, Julian L. Griffin b, Aldo Viarengo a a Environmental and Life Sciences Department (DISAV), Università A. Avogadro, Alessandria, Italy b Department of Biochemistry, University of Cambridge, Cambridge, UK
Abstract A systems biology approach based on transcriptomics, proteomics, metabolomics, and biomarkers was applied to study the effects of two prioritized environmental pollutants, i.e. nickel and
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the organophosphate clorpyrifos. Mussels were exposed for 4 days to equitoxic amounts of each substance, i.e. the EC50 for digestive gland lysosomal membrane destabilization. Effects of a mixture of the two chemicals were also assessed (EC25 plus EC25). Microarray analysis (digestive gland) was performed using a 2K MytArray DNA chip [Venier et al., 2006. Mutat. Res. (602), 121–34), providing distinct gene expression patterns between the two treatments. In animals exposed to Ni, about 120 genes were found to be differentially expressed, and these were involved in carbohydrate metabolism (down-regulation of glycan pathways); proteolysis (e.g. induction of catheptsin L), ribosome biogenesis and assembly, signal transduction, immune functions (lectins and galectins). A metallothionein 10IV gene was also induced. Chlorpyrifos exposure involved a lower number of genes, which were mostly up-regulated and mainly involved in aminosugar metabolism (chitinase, chitriosidase, etc.). Ferritin and an hsp70 gene were also up-regulated. Proteomic analysis based on 2D electrophoresis and de novo peptide sequencing tandem (ESI Q TOF) mass spectrometry clearly revealed a different molecular fingerprinting. Differentially expressed proteins (about a dozen in all cases) were mostly down-regulated because of the physiological conditions of mussels, i.e. a dramatic lysosomal membrane destabilization. Polypeptides were partially sequenced but only a few were identified because of the lack of similarity with known orthologues. Interestingly, in CP-exposed mussels we recognized a putative iron responsive element (a modulator of ferritin expression), suggesting a disruption of iron homeostasis. Finally metabolomic based analysis using nuclear magnetic resonance (NMR) spectroscopy and gas chromatography–mass spectrometry (GC–MS) coupled with computer assisted multivariate statistics and pattern recognition techniques gave results that complemented the transcriptomic and proteomic results. Nickel appeared to more toxic than chlorpyrifos and produced changes in amino acid and carbohydrate metabolism. Chlorpyrifos was found to inhibit acetyl cholinesterase activity and also produced changes in amino acid and sugar metabolism. In conclusion, this work demonstrated the applicability of a systems toxicology approach in mussels, a well-established ecotoxicological model species, notwithstanding its available genomic information being still limited.
A microarray meta-analysis approach to find sensitive and robust estrogen induced responses in fish Lina Gunnarsson a, Erik Kristiansson b, Lars Förlin c, Olle Nerman b, Joakim D.G. Larsson a a Department of Neuroscience and Physiology, The Sahlgrenska Academy at Göteborg University, Göteborg, Sweden b Department of Mathematical Statistics, Chalmers University of Technology, Göteborg, Sweden c Department of Zoology/Zoophysiology, Göteborg University, Göteborg, Sweden
Abstract Vitellogenin (VTG) is an established marker for estrogenic exposure in fish. Effects on gonadal differentiation at concentrations not sufficient to induce VTG suggest that more sensitive markers would be useful. The aim of our study was to find sensitive and robust markers of estrogenic exposure. Liver mRNA expression profiles were characterized in estrogen exposed juvenile rainbow trout using microarray. The higher concentration was used to guide the subsequent identification of generally more subtle responses at the low concentration not sufficient to induce vitellogenin. A meta-
analysis was performed with data from the present study and similar microarray studies using different fish species and platforms. Within the generated list of presumably robust responses, several well-known estrogen-regulated genes were identified. Two genes, confirmed by quantitative RT-PCR (qPCR), fulfilled both the criteria of high sensitivity and robustness; the induction of the genes encoding zona pellucida protein 3 and a nucleoside diphosphate kinase (nm23). The cross-species, cross-platform meta-analysis correctly identified several robust responses. This adds confidence to our approach used for identifying candidate biomarkers. Specifically, we propose that analyses of the nm23 together with zona pellucida genes may increase the possibilities to detect an exposure to low levels of estrogenic compounds in fish.
Development of a stickleback (Gasterosteus aculeatus) cDNA microarray and gene expression responses to dibenzanthracene, ethinyl-estradiol and copper Tim D. Williams a, Margaret Brown b, James K. Chipman a, Francesco Falciani a, Fernando Ortega a, Fleur Geoghegan a, John A. Craft b, Ioanna Katsiadaki c, John Ball d, Charles R. Tyler d, Eduarda Santos d a School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK b School of Life Sciences, Glasgow Caledonian University, Glasgow G4 0BA, UK c Centre for Environment, Fisheries and Aquaculture Science, Weymouth DT4 8UB, UK d School of Biosciences, The University of Exeter, Exeter, Devon EX4 4QJ, UK
Abstract The Three-spined stickleback (Gasterosteus aculeatus) is a fish species that inhabits European estuarine and freshwater environments and is a useful model for ecotoxicology and, as a genomesequenced species, ecotoxicogenomics. We have constructed a cDNA array for the stickleback using SSH and normalised liver cDNA libraries (Brown et al.) and a whole body cDNA library (Prof. Kingsley, Stanford, USA). Bioinformatic analyses of the sequenced clones have been carried out with a view to identification and functional annotation by Gene Ontology category (GO). This has used the Blast2GO program (A. Conesa, S. Goetz). Additional annotation has been derived from the stickleback genome project. Our initial microarray experiments with the stickleback have characterized their acute hepatic responses to dibenzanthracene (DbA), ethinyl-estradiol (EE2) and copper (Cu) treatment. Male stickleback were exposed to waterborne DbA at various concentrations between 10 and 50,000 ng/l, EE2 between 0.1 and 100 ng/l or Cu between 3.2 and 128 ìg/l for 4 days. The microarray was used to compare gene expression of exposed fish to those of relevant controls. Microarrays were carried out using both pooled samples and samples from individual fish. Concentration/dose responses were found for known biomarker genes CYP1A (r2 = 0.90), vitellogenin (r2 = 0.97) and metallothionein (r2 = 0.88) to DbA, EE2 and Cu, respectively. Additional responsive genes and Gene Ontology categories will be described, that provide insights into the mechanistic modes of action of these three model pollutants and our findings will highlight additional biomarkers for exposure to these chemicals. Funding was provided by UK Natural Environment Research Council Post Genomics and Proteomics grant NE/ C507661/1.
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Gene expression in oyster Crassostrea gigas exposed to sewage contaminated sites Igor D. Medeiros a,b, Juliano Zanette a, Marília N. Sieber a, Guilherme Toledo-Silva a, Thiago B. Rodrigues a, Fabrício F. Nunes a,c, Maria R.F. Marques a, Afonso C.D. Bainy a a Laboratório de Biomarcadores de Contaminação Aquática e Imunoquímica, Universidade Federal de Santa Catarina, Florianópolis, Santa Catarina, Brazil b Laboratório de Ciências Marinhas, Universidade do Sul de Santa Catarina, Palhoça, Santa Catarina, Brazil c Centro Municipal de Educação Ambiental Escola do Mar, São José, Santa Catarina, Brazil
Abstract Exposure to sewage effluents may affect the biotransformation systems of aquatic organisms. The pattern of these responses could be used complementarily as an early warning tool for environmental monitoring. The aim of this work was to analyze the expression of genes in gill of oysters exposed to sewage contaminated sites. Six-month-old-Crassostrea gigas were transplanted from a farming area to four stations distant from a source of sewage 0.01, 0.4, 1.5 and 4.5 km (sites 1, 2, 3 and 4, respectively). After 2 weeks, the oysters were collected and the gills were excised for total RNA extraction and cDNA synthesis. Gene expression analysis was performed using semi-quantitative RT-PCR reaction, with specific primers for the genes cytochrome P450 (CYP356A1), fatty acid binding protein (FABP), glutathione S-transferase (GST) and multidrug resistance protein (MDR). Actin (ACT) gene was used as a housekeeping gene to normalize the data. Oysters exposed to the sewage-contaminated site (Site 1) showed an induction in the expression of FABP, CYP356A1 and GST. The MDR expression was higher in the oysters kept at the Site 3. The induction of these genes in gills of oysters from the contaminated site indicates that discharges of domestic sewage into the coastal water affect the biotransformation systems of this organism, suggesting that after a long-term exposure, high mortality rate might be observed. We suggest the use of these biological markers to evaluate exposure and susceptibility in future biomonitoring programs of coastal waters. Supported by CNPq-Universal to ACDB.
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Selection of reference genes for quantitative RT-PCR studies in striped dolphin (Stenella coeruleoalba) skin biopsies Giacomo Spinsanti a, Cristina Panti a, Letizia Marsili b, Silvia Casini Maria Cristina Fossi b a Department of Evolutionary Biology, University of Siena, Siena, Italy b Department of Environmental Sciences, University of Siena, Siena, Italy
b
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Abstract Quantitative real-time PCR is a sensitive molecular technique for the detection of variations in gene expression using minimal amounts of biological materials. The application of qRT-PCR to cetacean skin biopsies, obtained with non lethal techniques from freeranging animals, has the potential of representing a powerful tool for the quantification of gene induction caused by the exposure to contaminants. A limitation for the application of qRT-PCR is the need for appropriate reference genes which allow the correct quantification of gene expression. The expression of 10 potential control genes was examined in 30 striped dolphin (Stenella coeruleoalba) skin biopsy samples, obtained from specimens inhabiting the north-western Mediterranean Sea. The stability of selected control genes was determined using three different softwares: geNorm, NormFinder and BestKeeper. Glyceraldehyde-3P-dehydrogenase (GAPDH) and tyrosine-3-monooxygenase (YWHAZ) always rank as the two most stably expressed HKGs and ribosomal protein L4 (RPL4) and S18 (RPS18) also exhibit a remarkable stability of their expression levels. On the other hand, transferrin receptor (TFRC), phosphoglycerate kinase1 (PGK1), hypoxanthine ribosyltransferase (HPRT1) and b-2-microglobin (B2M) show variable expression among the studied samples and appear as less suitable reference genes. Our results suggest that the genes encoding for YWHAZ and GAPDH have the most stable expression patterns and appear as highly reliable controls. Potentially useful reference genes are also those encoding for the ribosomal proteins L4 and S18. This work provides background essential information for studying expression patterns of several potential genes of interest as biomarkers of exposure to contaminants of free-ranging marine mammals.