Assessing the impact of Benzo[a]pyrene on Marine Mussels: Application of a novel targeted low density microarray complementing classical biomarker responses

Type Article
Date 2017-06
Language English
Author(s) Banni MohamedORCID1, 2, Sforzini Susanna1, Arlt Volker M.3, 4, Barranger AudreyORCID5, Dallas Lorna J.5, Oliveri Caterina1, Aminot YannORCID6, Pacchioni Beniamina7, Millino Caterina7, Lanfranchi Gerolamo7, Readman James W.6, 8, Moore Michael N.5, 8, 9, Viarengo Aldo1, Jha Awadhesh N.5
Affiliation(s) 1 : Univ Piemonte Orientale, Dept Sci & Technol Innovat DiSIT, Alessandria, Italy.
2 : Sousse Univ, ISA Chott Mariem, Lab Biochem & Environm Toxicol, Sousse, Tunisia.
3 : Kings Coll London, MRC PHE Ctr Environm & Hlth, Analyt & Environm Sci Div, London, England.
4 : Kings Coll London, Publ Hlth England, NIHR Hlth Protect Res Unit Hlth Impact Environm H, London, England.
5 : Univ Plymouth, Sch Biol & Marine Sci, Plymouth, Devon, England.
6 : Univ Plymouth, Ctr Chem Sci, Plymouth, Devon, England.
7 : Univ Padua, Padua, Italy.
8 : Plymouth Marine Lab, Prospect Pl, Plymouth, Devon, England.
9 : Univ Exeter, Med Sch, Knowledge Spa, ECEHH,Royal Cornwall Hosp, Truro, Cornwall, England.
Source Plos One (1932-6203) (Public Library Science), 2017-06 , Vol. 12 , N. 6 , P. e0178460 (26p.)
DOI 10.1371/journal.pone.0178460
WOS© Times Cited 41
Abstract

Despite the increasing use of mussels in environmental monitoring and ecotoxicological studies, their genomes and gene functions have not been thoroughly explored. Several cDNA microarrays were recently proposed for Mytilus spp., but putatively identified partial transcripts have rendered the generation of robust transcriptional responses difficult in terms of pathway identification. We developed a new low density oligonucleotide microarray with 465 probes covering the same number of genes. Target genes were selected to cover most of the well-known biological processes in the stress response documented over the last decade in bivalve species at the cellular and tissue levels. Our new 'STressREsponse Microarray' (STREM) platform consists of eight sub-arrays with three replicates for each target in each sub-array. To assess the potential use of the new array, we tested the effect of the ubiquitous environmental pollutant benzo[a] pyrene (B[a] P) at 5, 50, and 100 mu g/L on two target tissues, the gills and digestive gland, of Mytilus galloprovincialis exposed invivo for three days. Bioaccumulation of B[a] P was also determined demonstrating exposure in both tissues. In addition to the well-known effects of B[a] P on DNA metabolism and oxidative stress, the new array data provided clues about the implication of other biological processes, such as cytoskeleton, immune response, adhesion to substrate, and mitochondrial activities. Transcriptional data were confirmed using qRT-PCR. We further investigated cellular functions and possible alterations related to biological processes highlighted by the microarray data using oxidative stress biomarkers ( Lipofuscin content) and the assessment of genotoxicity. DNA damage, as measured by the alkaline comet assay, increased as a function of dose. DNA adducts measurements using 32 P-postlabeling method also showed the presence of bulky DNA adducts (i.e. dG-N-2-BPDE). Lipofiscin content increased significantly in B[a] P exposed mussels. Immunohistochemical analysis of tubulin and actin showed changes in cytoskeleton organisation. Our results adopting an integrated approach confirmed that the combination of newly developed transcriptomic approcah, classical biomarkers along with chemical analysis of water and tissue samples should be considered for environmental bioimonitoring and ecotoxicological studies to obtain holistic information to assess the impact of contaminants on the biota.

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Publisher's official version 26 6 MB Open access
S1 Fig. Concentration of benzo[a]pyrene (B[a]P) in seawater over the first 24 h of 3 d exposure at 0–100 μg L-1. 1 176 KB Open access
S2 Fig. A typical dual color hybridization analysis of Cy3/Cy5-labelled cDNAs from B[a]P-treated vs control mussels, obtained by means of a dual laser source microchip scanner. 1 240 KB Open access
S3 Fig. qRT-PCR confirmation of microarray data. 1 183 KB Open access
S1 Table. qRT-PCR primers and Taqman probes. 1 115 KB Open access
S2 Table. The total array sequence names. 27 KB Open access
S3 Table. M-Values of the 205 DEGs in gills tissues in at least one condition during the exposure to B[a]P concentrations (without B[a]P supply is considered as the reference). 24 KB Open access
4 Table. M-Values of the 109 DEGs indigestive gland tissues in at least one condition during the exposure to B[a]P concentrations (without B[a]P supply is considered as the reference). 17 KB Open access
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Banni Mohamed, Sforzini Susanna, Arlt Volker M., Barranger Audrey, Dallas Lorna J., Oliveri Caterina, Aminot Yann, Pacchioni Beniamina, Millino Caterina, Lanfranchi Gerolamo, Readman James W., Moore Michael N., Viarengo Aldo, Jha Awadhesh N. (2017). Assessing the impact of Benzo[a]pyrene on Marine Mussels: Application of a novel targeted low density microarray complementing classical biomarker responses. Plos One, 12(6), e0178460 (26p.). Publisher's official version : https://doi.org/10.1371/journal.pone.0178460 , Open Access version : https://archimer.ifremer.fr/doc/00476/58751/