Fluoride-induced death of rat erythrocytes in vitro

Abstract

Although fluoride (F) in low concentrations is essential for teeth and bone development, its excessive consumption causes numerous deleterious abnormalities in cellular metabolism and physiology often leading to cell death. The present study was performed to establish the toxic F effects inducing the death of rat erythrocytes in vitro. The cells were cultured in the presence of 0.5–16mM NaF for 1, 5 and 24h. The progression of erythrocyte death was monitored by cell viability (calcein assay), membrane integrity (hemolysis assay), alterations in the cell morphology (light microscopy) and size (flow cytometry forward scatter), plasma membrane scrambling (annexin V binding). To elucidate the molecular mechanisms underlying F-induced cell death, the cytosolic Ca2+ activity (Fluo-3 fluorescence) and ceramide formation (binding of FITC-labeled antibodies) were determined. Exposure of the rat erythrocytes to NaF considerably suppressed their viability and caused partial cell hemolysis within 24h. The cells underwent dramatic morphological alterations resulted in appearance of shrunken echinocytes after 1h and swollen spherocytes within 24h. The development of NaF-induced erythrocyte death was accompanied by progressive PS externalization at the outer cell membrane, ∼45% of the cells were annexin V-positive in response to 16mM NaF within 24h with a small cell population exhibiting necrotic features. The cell death was preceded by considerable accumulation of the free cytosolic Ca2+, with statistically significant increase in the number of Fluo-3-positive erythrocytes observed as early as during 1-h incubation with 0.5mM NaF. NaF also induced moderate ceramide formation. Overall, exposure of the rat erythrocytes to NaF triggers rapid progression of their death in a dose- and time-dependent manner, with appearance of apoptotic cells after 1 and 5h and transition to necrosis within 24h. An increase in intracellular [Ca2+] appears to be crucial mechanism implicated in development of NaF-induced apoptosis in rat erythrocytes.