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2C - Biomarkers (TUP/94) Lysosomal enlargement and lysosomal membrane destabilisation in mussel digestive cells measured by an integrative index. Izagirre, Urtzi1, Zorita, Izaskun1, Cajaraville, Miren P.1, Marigómez, Ionan1, 1 University of Basque Country, Bilbo, Basque Country, Spain ABSTRACT- Lysosomal biomarkers in mussel digestive cells are used world-wide in monitoring biological effects of pollution. Such biomarkers are based on two different lysosomal responses, enlargement -measured through the LSC (Lysosomal Structural Changes) test after B-glucuronidase (Gus) histochemistry- and membrane destabilisation -measured through the LMS (Lysosomal Membrane Stability) test that uses hexosaminidase (Hex) as lysosomal marker-. Previous reports evidenced that the same degree of membrane destabilisation is not necessarily associated with the same level of enlargement when different mussel populations are compared. In addition, reactivity for lysosomal enzyme markers also varies between populations: a different incubation time is needed for (Gus) in Mediterranean and Atlantic mussels in order to avoid background that hinder image analysis. The aims of the present investigation were (i) to standardise a procedure suitable for interpopulation comparisons and (ii) to integrate both lysosomal responses in a single index. Intertidal and subtidal mussels were carried to the laboratory and kept under their respective tidal regimes at two temperatures (18°C, 30°C) for 24 h. According to LSC and LMS tests, lysosomal enlargement and membrane destabilisation occurred in mussels kept at 30°C. Further, both responses were measured on the same sections in an atempt to combine both techniques and use only one lysosomal enzyme marker (Hex). Once labilisation period (LP) was determined by LMS test, tissue sections corresponding to the time just before LP were selected to apply LSC, as an indication of maximum lysosomal enlargement occurring just before membrane destabilisation. An integrative index was calculated as the ratio between LSC parameters (i.e. Vv -Volume density-) and LP values. Mussels kept at 30°C presented significantly higher index values than those kept at 18°C. The same analytical approach was applied in a field study where mussels were collected from Portofino (reference) and inside the Genova harbour (polluted). Lysosomal enlargement and membrane destabilisation were recorded in mussels from Genova. In agreement the index was significantly higher in Genova than in Portofino. Funded by EC (BEEP Project, EVK3-CT2000-00025). U. I. is recipient of a fellowship from the Basque Government. Key words: digestive cells, biomarkers, lysosomal membrane stability, lysosomal structural changes |
