Transcriptomic Analysis of Skin Biopsies: A New Approach to Understand the Effects of Emerging Contaminants on Bottlenose Dolphin (Tursiops truncatus)
Marine pollution and the decline of the ecosystem health (as well as the health of the organisms living in it) have raised concern in regard to posed environmental threats. Changes in the health status of the marine ecosystem can be mostly related to anthropogenic impacts such as overfishing, coastal habitat destruction, deep sea mining, oil and gas exploration, tourism, water sport, ocean acidification. Anthropogenic impacts are also strictly correlated to industrial application and the release of chemical contaminants and pollutants including a large amount of marine debris, which mainly consists of synthetic polymers or plastics.1 Plastics are dispersed by winds and currents over long distances across the globe, becoming ubiquitous in the marine environment. Large plastic debris degrade into smaller fragments, creating microsized plastics (microplastics) potentially available to an increasing number of marine species, posing toxicological hazard due to plastic additives. An example of plastic additive is bisphenol A (BPA), a monomer used in epoxy resins and polycarbonate plastics, present in many hard plastic bottles and metal-based food and beverage. BPA is among the most worldwide distributed compounds and is considered a dangerous factor because of its agonist or antagonist effects on endocrine receptors.2,3 We applied an ex vivo assay using skin biopsy slices from common bottlenose dolphin (Tursiops truncatus), combined with global gene expression analysis in response to BPA chemical exposure.4,5 The skin was collected from a stranded dolphin on the coast of Tuscany. Small slices of skin biopsy were excised, cultured and independently treated with different concentrations of BPA (0.1 mg/ml, 1 mg/ml, 100 mg/ml). RNA from the different treatments was extracted and hybridized to a species-specific custom-made microarray representing almost 25,000 dolphin unigenes. Genes involved with the activation of an immune response (e.g., B-cell receptor-associated protein - BCAP, nuclear factor of activated T-cells - NFAT, interferon regulatory factor 2 - IRF-2, immunoglobulins, lymphotoxin beta - LT-beta), endocrine pathways (e.g., progestin receptor alpha-like - NR3C3, estrogen response element binding protein - ERE-BP, thyroid hormone receptor beta - TR-beta), lipid homeostasis and adipogenesis (e.g., adipogenesis regulatory factor - ADIRF and the nuclear receptor subfamily 1 - NR1H3) were among the genes found to be differentially expressed. We also used real time-PCR (qPCR) to correctly quantify the expression of a suite of genes to validate the microarray results and to select potential biomarkers of BPA exposure. Preliminary results for genes such as ADIRF and NR1H3 indicated consistency between the two methods and more genes are currently being tested with qPCR. The expression of genes representing the final panel of potential biomarkers will also be investigated in samples from other animals. If successful, this could be informative not only of the impact of microplastics and endocrine disruptors on dolphins and marine mammals in general, but also on the threats posed to the marine ecosystem.
The authors wish to thank the Osservatorio Toscano Cetacei, the University of Siena and the Accademia dei Fisiocritici for the dolphin necropsy under the supervision of Dr. Letizia Marsili. The authors also wish to thank Marcella Birtele, former Biology student at the University of Ferrara, who contributed for a short time to the technical progress of the work.
* Presenting author
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