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

Awadhesh N. Jha,James W. Readman,Mohamed Banni,Audrey Barranger,Volker M. Arlt,Caterina Millino,Beniamina Pacchioni,Michael N. Moore,Aldo Viarengo,Lorna J. Dallas,Gerolamo Lanfranchi,Yann Aminot,Caterina Oliveri,Susanna Sforzini

doi:10.1371/journal.pone.0178460

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 μg/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 32P-postlabeling method also showed the presence of bulky DNA adducts (i.e. dG-N2-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.

Highlights

In the last two decades, genomics-based approaches have provided insights into the mechanisms underlying the biological responses in natural biota following exposures to environmental pollutants
We have presented the development of a new low density oligonucleotide microarray to study alterations in mussel gene expression profiles after exposure to anubiquitous environmental stressor
Even though the number of differentially expressed genes (DEGs) involved in the oxidative stress response present in the new platform (i.e., 10) was less than other marked processes, the results indicate clear effects as 80% of the genes involved in the oxidative stress response were affected in the gills and digestive gland (DG) of mussels exposed to higher B[a]P concentrations

Summary

Introduction

In the last two decades, genomics-based approaches have provided insights into the mechanisms underlying the biological responses in natural biota following exposures to environmental pollutants. Recent developments in ‘omics’ technologies have raised our expectations to elucidate crucial biological processes following exposure to pollutants to which biota are exposed chronically at low dose/ concentrations. The accessibility of new genomic resources, high-throughput molecular technologies such as microarray, pyrosequencing and generation sequencing have provided information and helped to identify molecular targets of pollutants (e.g. genes) that contribute to stress in natural populations [6,7]. Several cDNA microarrays were developed recently in Mytilus galloprovincialis, Mytilus edulis, and Mytilus trossulus, putatively identified partial transcripts limit their use in generating reliable gene expression profiles in terms of pathway identification[11]

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Conclusion