MARINE ENVIRONMENTAL RESEARCH Marine Environmental Research 62 (2006) S109–S112 www.elsevier.com/locate/marenvrev
Short communication
Cloning and expression pattern of peroxisomal enzymes in the mussel Mytilus galloprovincialis and in the thicklip grey mullet Chelon labrosus: Generation of new tools to study peroxisome proliferation Eider Bilbao, Oihane Dı´az de Cerio, Miren P. Cajaraville, Ibon Cancio * Laboratory of Cell Biology and Histology, Department of Zoology and Animal Cell Biology, School of Science and Technology, University of the Basque Country, E-48080 Bilbo, Basque Country, Spain
Abstract Aquatic organisms living in coastal/estuarine areas show peroxisome proliferation after exposure to different environmentally relevant pollutants. In order to generate new tools to assess peroxisome proliferation in aquatic animals, peroxisomal enzymes were cloned using degenerate primers in the mussel Mytilus galloprovincialis and in the thicklip grey mullet Chelon labrosus. Fragments of catalase (CAT), thiolase (THIO), polyamine oxidase (POX) and xanthine oxidoreductase (XOR) were cloned and their expression pattern studied in different tissues by semi-quantitative RT-PCR. In mussels, CAT, THIO, POX and XOR were expressed in digestive gland, mantle and gills while in mullets CAT, THIO and POX were expressed in liver, spleen, brain, heart, muscle and gills. XOR was mainly expressed in liver and heart. Mature mullets showed the highest expression of peroxisomal enzymes in liver, spleen and brain, while in juveniles expression was mainly found in muscle tissues, liver and gills. Laboratory experiments of exposure to organic pollutants are being performed to study the usefulness of these tools to study peroxisome proliferation in pollution biomonitoring programmes. Ó 2006 Elsevier Ltd. All rights reserved.
*
Corresponding author. Tel.: +34946012734. E-mail address:
[email protected] (I. Cancio).
0141-1136/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.marenvres.2006.04.004
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Keywords: Peroxisome proliferation; Gene expression; Catalase; Thiolase; Polyamine oxidase; Xanthine oxidoreductase; Chelon labrosus; Mytilus galloprovincialis
During the last decades, a considerable research effort has been devoted to developing sensitive early warning biomarkers of pollutant exposure and effects. Peroxisome proliferation (PP) is one of these emerging biomarkers of exposure to organic pollutants. Peroxisomes of marine organisms including bivalve molluscs and fish have shown capacity to proliferate under experimental and field exposure to organic xenobiotics (Cancio and Cajaraville, 2000; Cajaraville et al., 2003). PP is accompanied by induction of peroxisomal enzyme activities, especially those involved in lipid homeostasis. This induction occurs at the transcriptional level. In this sense, the aim of this work was to generate new tools to assess PP in aquatic animals by cloning genes that may allow to study changes in expression patterns in animals exposed to peroxisome proliferators (PPs). Mussels (Mytilus galloprovincialis) and mullets (Chelon labrosus) were selected for this study. Mussels are used worldwide as sentinels of pollution in marine environments and mullets are abundant in Eastern-Atlantic estuaries where they are able to endure highly polluted environments. Cloning of these genes coding for peroxisomal enzymes may be relevant for future applications of PP as biomarker of exposure to organic pollutants in pollution biomonitoring programmes. Mussels, M. galloprovincialis and thicklip grey mullets C. labrosus were collected from Arriluze, Biscay Bay (43°20 0 N, 003°01 0 W) in winter. Mussels were immediately processed after capture, and mullets were dissected after classification as mature male or female and juveniles (2 individuals per group). Total RNA was isolated using Trizol (Invitrogen) and cDNA synthesis was carried out using random hexamers. Degenerate primers generated aligning known sequences from different species were employed to amplify polyamine oxidase (POX), catalase (CAT), thiolase (THIO) and xanthine oxidoreductase (XOR) fragments in mussels and mullets. For mussel THIO an EST sequence without assigned homology was obtained from the Genbank (AJ624743). PCR amplificates were purified and cloned using a TOPO-TA cloning vector. Sequencing of plasmids was performed by Sanger’s method using the Fw-M13 primer. For expression studies specific primers that amplify fragments around 200 bp were designed according to the new sequences obtained. Monoplex-PCR conditions for the amplification of target cDNAs were optimised using Taq polymerase (Invitrogen). PCR products were visualized by gel electrophoresis and analysed using a computer-aided gel analyser (Gel-Doc-2000, Bio-Rad). Results were expressed in arbitrary semi-quantitative units, using b-actin and 18S rRNA as housekeeping genes for normalisation. Peroxisomal genes were partially cloned in mussels and mullets (Table 1). The mullet XOR fragment showed 85% amino acid identity with Poecilia reticulata XOR (e-value for BlastN algorithm: N 1e54) while mussel PCR product showed highest homology with Strongylocentrotus purpuratus XOR. XOR is involved in purine metabolism acting either as oxidase or dehydrogenase. Both forms produce superoxide anions but xanthine dehydrogenase has also been implicated in the metabolism of xenobiotics (Kooij, 1994). The dehydrogenase form has been previously found in mussel tissues using enzyme histochemistry and immunohistochemistry (Cancio and Cajaraville, 1999). Cloned POX fragments showed 90% amino acid identity with zebrafish POX (N 4e70). POX is a flavin oxidase
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Table 1 Putative identity of PCR amplification products belonging to genes coding for peroxisomal enzymes in Chelon labrosus and Mytilus galloprovincialis Gene
Size of RT-PCR product (bp)
Amino acid identity with known sequences
Accession number
Chelon labrosus Xanthine oxidoreductase Polyamine oxidase Catalase Thiolase
146 346 273 303
85% 90% 94% 78%
AY876387 AY876389 AY743715 DQ021958
Mytilus galloprovincialis Xanthine oxidoreductase Polyamine oxidase Catalase
290 326 489
55% Strongylocentrotus purpuratus 87% Danio rerio 94% Mytilus californianus
Poecilia reticulata Danio rerio Oplegnathus fasciatus Danio rerio
AY876388 AY876390 AY743716
induced after exposure to PPs (Hayashi and Miwa, 1989) that, as other oxidases within peroxisomes, generates H2O2 (Seiler, 1995) contributing to tissue damage. H2O2 is eliminated in peroxisomes by CAT. Mullet CAT was cloned with a 94% amino acid identity
Fig. 1. Expression pattern of polyamine oxidase (POX), catalase (CAT), xanthine oxidoreductase (XOR) and peroxisomal thiolase (THIO) in different tissues of juvenile and mature male and female mullets Chelon labrosus, collected in December (2004) in the Bay of Biscay. b-actin (data not shown) and 18S rRNA were included as housekeeping genes.
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with fish Oplegnathus fasciatus CAT (N 0.0), while mussel sequence was identical to other Mytilidae CATs. Mullet THIO was cloned showing 78% amino acid identity with Danio rerio 3-ketoacyl-Coa thiolase (e-value for BlastX algorithm: X 6e34). THIO catalyses the last step of peroxisomal b-oxidation of long-chain fatty acids. In rat two distinct 3ketoacyl-CoA thiolases with identical substrate specificity have been identified, THIO-A and B (Antonenkov et al., 1999). While THIO-A is constitutively expressed, THIO-B expression is induced by PPs (Hijikata et al., 1990). The mullet and mussel fragments studied here belong to a highly conserved domain within mammalian THIOs, thus only cloning of the whole ORFs will clarify the existence of differentially regulated THIOs in aquatic species. The tissue expression pattern of these genes was studied in juvenile and adult mullets (Fig. 1). CAT, THIO and POX were expressed in all tissues studied. Expression was highest in brain, liver and spleen of mature individuals, while in juveniles expression was mainly observed in muscle, heart, brain, liver and gills. THIO expression pattern demonstrates that piscine peroxisomal b-oxidation takes place mainly in liver, as it occurs in mammals (Nemali et al., 1988). Future studies of exposure to PPs will focus on liver as main detoxification tissue and gills as tissue highly exposed to dissolved chemicals. Comparing both tissues, expression differences along development were detected, with highest POX, THIO and CAT expression in juvenile gills. On the contrary, hepatic CAT expression was highest in mature individuals. THIO expression, was highest in female livers. Regarding XOR, expression was detected in all tissues in mature individuals except in gills while in juveniles expression was exclusively hepatic. Although no semi-quantitative studies were performed in mussels, expression of CAT, THIO, POX and XOR was found in digestive gland, mantle and gills. Laboratory experiments of exposure to pollutants are being conducted in mullets and mussels in order to determine the usefulness of these new tools to study PP as biomarker of exposure to organic pollutants in biomonitoring programmes. Acknowledgements Funded by Spanish-MEC (BIOMTOOLS, REN:2002-02982/MAR; PRESTEPSE, VEM2003-20082-C06), Basque Government (ETORTEK-IMPRES) and University of the Basque-Country (grant to consolidated research groups and predoctoral fellowship to Eider Bilbao). References Antonenkov, V.D., Van Veldhoven, P.P., Waelkens, E., Mannaerts, G.P., 1999. Biochimica et Biophysica Acta 1437, 136–141. Cajaraville, M.P., Cancio, I., Ibabe, A., Orbea, A., 2003. Microscopy Research and Technique 61, 191–202. Cancio, I., Cajaraville, M.P., 1999. Biology of the Cell 199, 201–293. Cancio, I., Cajaraville, M.P., 2000. International Review of Cytology 199, 201–293. Hayashi, H., Miwa, A., 1989. Biochimica et Biophysica Acta 99 (2), 310–316. Hijikata, M., Wen, J.K., Osumi, T., Hashimoto, T., 1990. Journal of Biological Chemistry 265, 4600–4606. Kooij, A., 1994. Histochemical Journal 26, 889–915. Nemali, M.R., Usuda, N., Reddy, M.K., Oyasu, K., Hashimoto, T., Osumi, T., Rao, M.S., Reddy, J.K., 1988. Cancer Research 48, 5316–5324. Seiler, N., 1995. Progress in Brain Research 106, 333–344.
