Marine Environmental Research 69 (2010) S31–S33 Contents lists available at ScienceDirect Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev Differential gene expression in Poecilia vivipara exposed to diesel oil water accommodated fraction Jaco J. Mattos a, Marília N. Siebert a, Karim H. Luchmann a, Ninna Granucci a, Tarquin Dorrington a, Patrícia H. Stoco b, Edmundo C. Grisard b, Afonso C.D. Bainy a,* a b LABICAI/UFSC, Florianópolis, SC, Brazil Laboratório de Protozoologia, CCB, UFSC, SC, Brazil a r t i c l e Keywords: Gene expression Diesel oil Poecilia vivipara SSH Biotransformation Fish i n f o a b s t r a c t Diesel fuel is a potential contaminant of estuarine and mangrove areas, particularly because it is the main fuel used in small boats and larger vessels. The aim of this work was to identify genes differentially expressed in the liver of Poecilia vivipara (Guppy) exposed to 10% diesel fuel water accommodated fraction (WAF), employing the subtractive suppressive hybridization (SSH) method. The results showed 27 differentially expressed gene fragments, 12 up-regulated and 15 down-regulated. Among the up-regulated genes were CYP1A, UDPGT1a, ABCC4, Methyltransferase and Apolipoprotein A1. Down-regulated genes included Vitellogenins, C1 Inhibitor and Complement Component 3c. The identiﬁed genes are associated with different metabolic functions like biotransformation, membrane transport and immune system, indicating the susceptibility and/or molecular responses of this organism to the toxic effects elicited by diesel fuel WSF. Ó 2009 Elsevier Ltd. All rights reserved. Of the different types of pollutants, petroleum products are amongst the most relevant to aquatic ecotoxicology. Oil exploration, production and transport pose considerable risks to the marine environment (Simonato et al., 2008). The evaluation and prediction of the effects of crude petroleum and its by-products on aquatic organisms has become an urgent and important issue for the implementation of risk assessment studies. Field monitoring studies usually involve water chemical analysis and the activity of various enzymes (biochemical biomarkers) in exposed organisms, but frequently the obtained results are insufﬁcient to evaluate cellular responses against petroleum products (Arukwe et al., 2008). In order to better understand the mechanisms involved in short-term cellular responses against oil-related compounds, the subtractive suppressive hybridization (SSH) method was employed to identify genes up- and down-regulated in the liver of the ﬁsh Poecilia vivipara (Guppy) after exposure to diesel oil water accommodated fraction (WAF), under controlled laboratory conditions. Diesel fuels are complex mixtures of alkanes, cycloalkanes and aromatic hydrocarbons with carbon numbers in the range of C9–C28 (WHO, 1996). The diesel oil WAF was prepared as described by Singer et al. (2000). Fish were caught at Conceição Lagoon, Florianópolis, SC, Brazil and split into two 50-L glass aquaria (15 ﬁsh * Corresponding author. Tel.: +55 48 37216391; fax: +55 48 37219672. E-mail address: [email protected] (A.C.D. Bainy). 0141-1136/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2009.11.002 each). The animals were submitted to an acclimation period of 2 months, with constant aeration, temperature (22 ± 2 °C), and salinity (25), with daily water changes. After that, one group of 15 females ﬁsh was statically exposed for 24 h to 10% diesel oil WAF (v/v), and a control group containing 15 females ﬁsh kept in non-contaminated aquaria. The 10% diesel oil WAF (v/v) was chosen based on the proﬁle of the activity of biotransformation enzyme changes elicited at this concentration observed in a previous laboratory study (data not shown). During the exposure period, both aquaria were maintained at 22 ± 2 °C, a salinity of 25, with minimal aeration. Following the exposure, all ﬁsh were killed and the liver from 15 ﬁsh from each group excised and immediately frozen in liquid nitrogen in pools until RNA extraction. Total RNA was extracted in TRIZOL (Invitrogen Life Technologies, Carlsbad, CA). Messenger RNA was isolated using the MicropolyA mRNA kit (Ambion). Sample purity, mRNA concentration and integrity were conﬁrmed at 260 and 280 nm using a Nanodrop spectrophotomer and by 2% agarose gel electrophoresis. SSH was performed according to the PCR Select cDNA subtraction kit instructions (Clontech, Palo Alto, CA). Subtracted libraries were obtained from 2 lg mRNA of liver from exposed and control groups, respectively. Secondary (nested) PCR-ampliﬁed subtracted cDNA populations were cloned into the pGEM-T easy vector system and transformed into JM-109 Escherichia coli cells (Promega, Madison, WI). Plasmids were extracted and puriﬁed using perfectprep plasmid mini kit (Eppendorf, Hamburg). One hundred and six clones were sequenced using the ABI prism big S32 J.J. Mattos et al. / Marine Environmental Research 69 (2010) S31–S33 Table 1 List of genes up-regulated in liver of Poecilia vivipara exposed to diesel fuel oil water accommodated fraction. Biological role Putative match Species match e-Value GenBank accession number Phase I biotransformation Cytochrome P450 1A (CYP1A) Cytochrome P450 2P2 (CYP2P2) Gluthathione S transferase (GST) UDP glycosyltransferase 1 family a (UDPGT1A) Predicted O-methyltransferase ATP binding cassete CC4 (ABCC4) Cytochrome c oxidase subunit I (COXI) Presenilin enhancer – like (PEN) Fundulus heteroclitus Fundulus heteroclitus Pleuronectes platessa Danio rerio Danio rerio Danio rerio Cubiceps paradoxus Oreochromis mossambicus Epinephelus coioides Oreochromis mossambicus Danio rerio Platichthys ﬂesus 1.00E 3.00E 2.00E 3.00E 6.00E 5.00E 9.00E 9.00E AF026800.1 AF026800 AF117342.1 X63761.1 PPGLUST BC 109404.1 BC090471.1 EU586042.1 AB205442.1 AY522633.1 Phase II biotransformation Phase III biotransformation Electron transfer chain Anti-apoptotic Pro-apoptotic Chaperone Immune system Cholesterol efﬂux and Immune system Integral membrane protein 2B (ITM2b) Nascent polypeptide-associated complex alpha polypeptide-like Alpha-1-Microglobulin/bikunin precursor, like Apolipoprotein AI precursor 76 04 46 40 118 39 154 36 1.00E 17 3.00E 20 FJ43 8523.1 AY522625.1 7.00E 29 4.00E 32 BC 150464.1 AJ844288.1 Table 2 List of genes down-regulated in liver of Poecilia vivipara exposed to diesel fuel oil water accommodated fraction. Biological role Putative match Species match e-Value GenBank accession number Reproduction Vitellogenin A (VgA) Vitellogenin B (VgB) Vitellogenin C (VgC) Complement component C3 C1 inhibitor Heat shock protein 90ab (HSP90ab) Solute carrier organic anion transporter family, member 1c1 (OATP2) Transferrin UTP glucose-1-phosphate uridyltransferase Hemoglobin Alfa hemoglobin A AcylCoA binding protein Integrin beta 1 protein-like Ribossomal protein L5 40S ribossomal S5 Poecilia reticulata Poecilia reticulata Gambusia a/ﬁnis Paralichthys olivaceus Oncorhynchus mykiss Solea senegalensis Mus musculus 9.00E-115 7.00E-25 4.00E-43 4.00E-65 1.00E-59 4.00E-61 5.00E-23 EF408838.1 EF456700.1 AB181837.1 AB021653.1 AJ519929.1 OMY519929 AB367527.1 BC078456.1 Acanthopagrus schlegelii Salmo salar Salmo salar Seriola quinqueradiata Oryzias latipes Danio rerio Salmo salar Pagrus major 4.00E-32 2.00E-27 8.00E-48 5.00E-48 1.00E-39 7.00E-34 2.00E-27 3.00E-85 AY365052.1 BT045585.1 BT060431.1 AB034639.1 FJ843607.1 DO149101.1 BT045585.1 AY190724.1 Immune system Chaperone Organic anion bidirectional transport Immune response and iron metabolism Glycogen metabolism Oxygen transport Fatty acid metabolism Cell signaling Translational machinery dye terminator cycle sequencing kit (PE applied biosystems). Deduced protein sequences were subsequently subjected to homology search through the BLAST program (http://www.ncbi. nlm.nih.gov/BLAST). Among the 106 cDNA sequences, 90 were identiﬁed, resulting in 27 differentially expressed genes (Tables 1 and 2). The 12 up-regulated sequences are genes associated with different metabolic functions such as the biotransformation Phase I Cytochrome P450 1A and 2P2 (CYP1A and CYP2P2), Phase II Gluthathione S transferase (GST), UDP glucuronosyltransferase 1 family a (UDPGT1A) and Methyltransferase, Phase III membrane transport (ABCC4) and immune system genes Apolipoprotein AI (ApoAI) and Alpha-1-Microglobulin/ bikunin precursor (AMBP). The down-regulated genes are associated with the immune system (Complement Component C3 and C1 inhibitor) and reproduction (Vitellogenin A, B and C). One interesting result was the up-regulation of CYP1A with concomitant down-regulation of different Vitellogenins. Other studies, using cultured ﬁsh hepatocytes, revealed that the AhR-ligands inhibited Vitellogenin genes or protein expression and induced the CYP1A gene (or protein expression thereof), suggesting a role for AhR in this response (Bemanian et al., 2004; Navas and Segner, 2000). The genes involved in innate immune response showed contradictory results with the concurrent down-regulation of C1 inhibitor, responsible for complement system inhibition and Complement Component C3, a component of the complement system cascade. However, the induction of ApoAI, can act as an inhibitor of the membrane attack complex C5b-9 and can interact with Complement Component C3 (Magnadóttir and Lange, 2004). Therefore, the complement system cascade may be inhibited in these organisms, enhancing their susceptibility to pathogens. Other up-regulated genes involved with the immune system included AMBP, which is responsible for coding two plasma glycoproteins: alpha-1-Microglobulin (A1M) and bikunin. A1M plays a role as a carrier for small hydrophobic molecules and may have a key role in the network of immune regulation (Tyiagi et al., 2002), while bikunin has been shown to prevent tumor cell invasion and metastasis in vitro (Kobayashi et al., 1994). The majority of up-regulated genes in this work show a shortterm molecular response against diesel oil WAF components. However, long-term laboratory studies are necessary to understand the effects of WAF exposure on ﬁsh reproduction, immunity and other biological effects before ﬁeld validation. Conﬂict of interest statement The authors state they have no conﬂict of interest. Acknowledgements This work was supported by CNPq-CTPetro (Grant #550706/ 2005-4) to ACDB. ACDB and ECG are recipients of Productivity Fellowship from CNPq. J.J. Mattos et al. / Marine Environmental Research 69 (2010) S31–S33 References Arukwe, A., Nordtug, T., Kortner, T.M., Mortensen, A.S., Brakstad, O.G., 2008. Environmental Research 107, 362–370. 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