IN VITRO TOXI - AND ECOTOXICOLOGICAL ASSESSMENT OF PORPHYRINE NANOMATERIALS BY FLOW CYTOMETRY USING NUCLEATED ERYTHROCYTES

The use of nanoparticles for biological and medical applications has rapidly increased and the potential for human and ecological toxicity is a growing area of investigation. For assessing cytotoxicity of nanoparticles we developed a new experimental cell system based on the use of nucleated erythrocytes (RBCs) from fish and batrachians, which are directly exposed to pollutants or nanoparticles absorbed by different ways and we have evaluated the toxic effects by flow cytometric analysis. The two modes of cell death (apoptosis and necrosis) differ fundamentally in their morphology, biochemistry and biological relevance. We and others have recently shown that programmed cell death of nucleated erythrocytes is related to an apoptotic mechanism. The toxicological analysis were performed comparatively on porphyrin base or metalloporphyrin and for each porphyrin bare derivative on two correspondent porphyrin-silica-hybrid nanomaterials obtained by one step acid catalysis and by two steps acid-base catalysis. To evaluate cell-nanoparticles interactions, nucleated RBCs were exposed to different concentrations of nanocomposites and analyzed by flow cytometry, after 24h incubation endpoints for morphological changes (FSC/SSC), apoptosis/necrosis analysis (FITC-annexin-V labeling/PI) and viability (using calcein-AM method). The investigation showed that the type of cellular death of nucleated erythrocytes is related to an apoptotic mechanism and that flow cytometric analysis of nucleated RBCs viability and cell death discrimination could provide a rapid and accurate analytical tool for evaluating in vitro the biological responses towards of nanoparticles for environmental protection. Nucleated erythrocytes can be a new experimental cellular model easy to use, with no costs for culture and for maintaining in the culture. The results reported in the present study indicate that our new flow cytometric protocols can be used to create dose-response curves which allow us to determine EC50 for toxicity or ecotoxicity tests. It is also generally applicable for identifying harmful effects associated with general antropic impact for the aquatic environment and for its biomonitoring.