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Quantification and in situ localisation of abcb1 and abcc9genes in toxicant-exposed sea urchin embryos

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Abstract

A multixenobiotic resistance (MXR) mechanism mediated by ABC binding cassette (ABC) transport proteins is an efficient chemical defence mechanism in sea urchin embryos. The aim of our work was to evidence whether exposure to sub-lethal doses of specific contaminants (oxybenzone (OXI), mercuric chloride (HgCl2) and trybutiltin (TBT)) would induce MXR transporter activity during embryonic development (from zygote to blastula stage) in purple sea urchin (Paracentrotus lividus) embryos. Further, we present data on molecular identification, transport function, expression levels and gene localisation of two ABC efflux transporters—P-glycoprotein (ABCB1/P-gp) and sulfonylurea-receptor-like protein (ABCC9/SUR-like). Partial cDNA sequences of abcb1 and abcc9 were identified and quantitative PCR (qPCR) evidenced an increase in mRNA transcript levels of both ABC transporters during the two-cell, as well as an overall decrease during the blastulae stage. Calcein-AM efflux activity assay indicated the activation of multidrug resistance-associated protein/ABCC-like transport in the presence of HgCl2 and TBT in exposed blastulae. The in situ hybridisation of the two-cell and blastula stages showed ubiquitous localisation of both transcripts within cells, supporting qPCR data. In conclusion, ABCB1 and ABCC9 are constitutive, as are HgCl2, TBT and OXI-inducible ABC membrane transporters, coexpressed in the zygote, two-cell and blastula stages of the P. lividus. Their ubiquitous cell localisation further fortifies their protective role in early embryonic development.

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Acknowledgements

The study was funded by the European Community—Research Infrastructure Action under the FP7 “Capacities” Specific Programme, referring to ASSEMBLE grant agreement (Project no. 227799) and the Croatian Ministry of Science, Education and Sport (Project No.001-0000000-3633). Special acknowledgements go to Dr. Maria Ina Arnone and Dr. Rossella Annunziata for help with technical advices and inspiring scientific talks at the Stazione Zoologica Anton Dohrn in Naples, Italy. For administrative and technical help during our stay, we thank Dr. Euan Brown. We thank Dr. Roko Žaja and Dr. Tvrtko Smital (Ruđer Bosković Institute, Zagreb, Croatia) and Dr. Lidija Šver (Faculty of Food Technology and Biotechnology, Zagreb, Croatia) for help with ABC transporter sequences. We also thank Dr. Tvtko Smital for help with reading the manuscript. And finally we thank Dr. Till Luckenbach of the Helmholtz Centre for Environmental Research (UFZ) (Leipzig, Germany) for the critical suggestions and reading of our manuscript.

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Authors and Affiliations

  1. Laboratory for Biology and Microbial Genetics, Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, Pierottijeva 6, Zagreb, Croatia

    Ivana Bošnjak

  2. Laboratory for Aquaculture, Institute of Oceanography and Fisheries, Setaliste Ivana Mestrovica 63, 21000, Split, Croatia

    Ivana Lepen Pleić & Ivona Mladineo

  3. Molecular Biology Service, Stazione Zoologica Anton Dohrn, Villa Comunale, 80121, Napoli, Italy

    Marco Borra

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  1. Ivana Bošnjak
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  2. Ivana Lepen Pleić
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  3. Marco Borra
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  4. Ivona Mladineo
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Correspondence to Ivona Mladineo.

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Responsible editor: Henner Hollert

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Bošnjak, I., Lepen Pleić, I., Borra, M. et al. Quantification and in situ localisation of abcb1 and abcc9genes in toxicant-exposed sea urchin embryos. Environ Sci Pollut Res 20, 8600–8611 (2013). https://doi.org/10.1007/s11356-013-1819-2

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