Development of a biomarker tissue-array for marine pollution biomonitoring

Supervisor: Urtzi Izagirre, Larraitz Garmendia (PIE-UPV/EHU)
The application of biomarkers in marine pollution monitoring programmes is recognised by international intergovernmental institutions (ICES/OSPAR, UNEP…). Although detailed Standard Operating Procedures (SOPs) are available for many biomarkers, the development of improved protocols and novel procedures to speed up measurements and to reduce their cost are major challenges at present. For instance, cell and tissue level biomarkers require considerable time and effort, relatively expensive reagents and may be subjected to unpredictable variability between sample batches. Most of these restrains can be overcome with the application of currently available high-throughput technologies such as tissue arrays (TAs). The present investigation is aimed at developing TA approaches suitable for the application of cell and tissue-level biomarkers in sentinel species used in marine pollution monitoring programmes. TAs ensure good preservation (both molecular and structural) of the target tissues and can provide quick and cost-effective protocols (both for frozen and paraffin embedded samples) and homogeneous conditions (e.g. quantity and intensity of staining) for large numbers of samples. In addition, TAs permit easy access to different samples grouped together on one single slide. In order to design and construct TAs on sentinel species, samples from the Biscay Bay Environmental Biospecimen Bank were employed. In the present study, twenty different fish species collected in the last throw from two Oceanography campaigns of the Biscay Bay (2010-2011) and Mytilus galloprovincialis samples from a reference sampling site (Plentzia, Basque Coast) were used. The description of processing instructions to the case of marine species ensured good preservation of samples and confirmed the TA application on the aquatic organisms tested. Preparation of fish TA blocks containing different fish species helped in the screening of potential new sentinel species. A selection of cell and tissue-level biomarkers was included for the TA validation: lysosomal membrane stability, lysosomal structural changes, intracellular neutral lipid accumulation, cell type composition in the digestive gland epithelium and structural changes of digestive alveoli. Among TA advantages, good quality tissue preservation was observed as well as the applicability of biomarkers in the selected tissues and organisms. Those results confirmed the potential of TA to improve the protocols and procedures in marine pollution biomonitoring programmes. The TA technique also demonstrated good preservation of donor blocks from the Biospecimen Bank, which confirms that the applicability of TA could be towards Biospecimen Banking programmes in the close future.