Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, Sankt-Petersburg, Russia.
Toxicol In Vitro. 2011 Dec;25(8):1609-18. doi: 10.1016/j.tiv.2011.06.006. Epub 2011 Jun 16.
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 0.5-16 mM NaF for 1, 5 and 24 h. 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 24 h. The cells underwent dramatic morphological alterations resulted in appearance of shrunken echinocytes after 1h and swollen spherocytes within 24 h. 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 16 mM NaF within 24 h 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 0.5 mM 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 5 h and transition to necrosis within 24 h. An increase in intracellular [Ca2+] appears to be crucial mechanism implicated in development of NaF-induced apoptosis in rat erythrocytes.
虽然低浓度的氟（F）对牙齿和骨骼的发育是必不可少的，但它的过量摄入会导致细胞代谢和生理功能的许多有害异常，常常导致细胞死亡。本研究旨在建立体外诱导大鼠红细胞死亡的有毒 F 效应。将细胞在 0.5-16 mM NaF 存在下培养 1、5 和 24 h。通过细胞活力（钙黄绿素测定法）、细胞膜完整性（溶血测定法）、细胞形态学改变（光镜）和大小（流式细胞术前向散射）、质膜混乱（ annexin V 结合）监测红细胞死亡的进展。为了阐明氟诱导细胞死亡的分子机制，测定了胞质 Ca2+活性（Fluo-3 荧光）和神经酰胺形成（FITC 标记抗体结合）。暴露于 NaF 的大鼠红细胞的活力明显降低，并在 24 h 内导致部分细胞溶血。细胞发生剧烈的形态改变，1 h 后出现皱缩的棘红细胞，24 h 内出现肿胀的球红细胞。NaF 诱导的红细胞死亡伴随着质膜外 PS 的逐渐向外转位，在 24 h 内，用 16 mM NaF 处理，约 45%的细胞对 annexin V 呈阳性，一小部分细胞表现出坏死特征。细胞死亡前伴随着大量游离胞质 Ca2+的积累，早在 1 h 孵育 0.5 mM NaF 时，观察到 Fluo-3 阳性红细胞的数量就有统计学意义的增加。NaF 还诱导中等程度的神经酰胺形成。总之，大鼠红细胞暴露于 NaF 以剂量和时间依赖的方式引发其快速死亡，在 1 和 5 h 后出现凋亡细胞，24 h 内过渡为坏死。细胞内[Ca2+]的增加似乎是氟诱导大鼠红细胞凋亡发展的关键机制。
