Combined effects of n-TiO₂ and 2,3,7,8-TCDD in Mytilus galloprovincialis digestive gland: A transcriptomic and immunohistochemical study

Despite the growing concern over the potential biological impact of nanoparticles (NPs) in the aquatic environment, little is known about their interactions with other pollutants. In the marine mussel Mytilus galloprovincialis, exposure to nanosized titanium dioxide (n-TiO₂), one of the most widespread type of NPs in use, in combination with and 2,3,7,8-tetrachlorodibenzo-p-dioxins (TCDD), chosen as model organic xenobiotic, was shown to induce significant changes in different biomarkers in hemocytes, gills and digestive gland, with distinct effects depending on cell/tissue and type of response measured. In this work, the interactive effects of n-TiO₂ and TCDD at the tissue level were further investigated in mussel digestive gland using an integrated approach transcriptomics/immunohistochemistry. Mussels were exposed to n-TiO₂ (100μgL^-1) and TCDD (0.25μgL^-1), alone and in combination, for 96h. Transcriptomic analysis identified 48-, 49- and 62 Differentially Expressed Genes (DEGs) in response to n-TiO₂, TCDD and n-TiO₂/TCDD, respectively. Gene Ontology (GO) term analysis revealed distinct biological processes affected in different experimental conditions. n-TiO₂ mainly up-regulated cytoskeletal genes, while TCDD up-regulated endocrine and signal transduction related processes. Co-exposure induced transcriptional changes common to individual treatments, and identified a newly generated process, response to chemical stimulus. Transcription of selected genes was verified by qPCR. Moreover, expression of tubulin, as an example of target protein of interest identified by gene transcription data, was evaluated in tissue sections by immunolabelling. Tissue TCDD accumulation was evaluated by immunofluorescence with an anti-dioxins antibody.The results demonstrate both distinct and interactive effects of n-TiO₂ and TCDD in mussel digestive gland at the molecular and tissue level, identify the main molecular targets involved, and underline how exposure to the n-TiO₂/TCDD mixture does not result in increased TCDD accumulation and overall stressful conditions in the tissue. These represent the first data on transcriptional responses of marine invertebrates to exposure not only to n-TiO₂ as a model of NP, but also to a legacy contaminant like TCDD.