Sodium fluoride induces apoptosis in the kidney of rats through caspase-mediated pathways and DNA damage

Long-term excessive sodium fluoride (NaF) intake can cause many bone diseases and nonskeletal fluorosis. The kidneys are the primary organs involved in the excretion and retention of NaF. The objective of the present study was to determine the effects of NaF treatment on renal cell apoptosis, DNA damage, and the protein expression levels of cytosolic cytochrome C (Cyt C) and cleaved caspases 9, 8, and 3 in vivo. Male Sprague-Dawley rats were divided randomly into four groups (control, low fluoride, medium fluoride, and high fluoride) and administered 0, 50, 100, and 200 mg/L of NaF, respectively, via drinking water for 120 days. Histopathological changes in the kidneys were visualized using hematoxylin and eosin staining. Renal cell apoptosis was examined using flow cytometry, and renal cell DNA damage was detected using the comet assay. Cytosolic Cyt C and cleaved caspases 9, 8, and 3 protein expression levels were visualized using immunohistochemistry and Western blotting. The results showed that NaF treatment increased apoptosis and DNA damage. In addition, NaF treatment increased the protein expression levels of cytosolic Cyt C and cleaved caspases 9, 8, and 3. These results indicated that NaF induces apoptosis in the kidney of rats through caspase-mediated pathway, and DNA damage may be involved in this process

Caspase-3-dependent apoptosis in vascular smooth muscle cell by proteasome inhibition.

The effects of a number of substances on neointima formation following angioplasty have been investigated in animal models. It was suggested that delivering of proteasome inhibitor to the site of vascular injury would be a potential therapeutic approach in prevention of vascular restenosis. But the mechanisms underlying biologic activities of proteasome inhibition in vascular smooth muscle cells (VSMCs) are largely unknown. We have investigated effects of proteasome inhibition on VSMCs using proteasome inhibitor MG115. MG115 induced apoptotic death in VSMCs as determined by viability, morphology, and DNA fragmentation. Proteasome inhibition was accompanied by up-regulation of p53, p21, and p27. In contrast, there were no appreciable alterations in the levels of Bcl-2 and Bax. Proteasome inhibition was followed by activation of caspase-3 but not of -8. The induction of apoptosis was suppressed by treatment with a selective inhibitor of the caspase-3 family, z-DEVD-fmk but not by NG-monomethyl-L-arginine. These results indicate that proteasome inhibition induces apoptosis in VSMCs by activation of caspase-3.

A method for functional evaluation of caspase activation pathways in intact lymphoid cells using electroporation-mediated protein delivery and flow cytometric analysis.

The purpose of the study was to develop a rapid technique for determining the functional status of caspase activation pathways in intact lymphocytes. Proteins known to activate caspase-family cell death proteases (cytochrome c; granzyme-B; caspase-8) were introduced into human leukemia and lymphoma cell lines, as well as freshly isolated lymphocytes and leukemia cells, by electroporation. Fluorochrome-labeled proteins with a wide range of molecular weights (from 15 to 150 kDa) were used to evaluate electroporation efficiency by flow cytometry and to compare the efficiency of protein delivery using various electroporation conditions. Caspase activity was monitored using a cleavable, cell-permeable fluorogenic substrate. Conditions were identified for efficient delivery of proteins of +150 kDa into lymphoid cells. Caspase activation induced by various proteins was compared in normal and leukemic lymphocytic cells, revealing impaired caspase activation pathways in some malignant cells. We conclude that electroporation of apoptotic proteins into intact lymphoid cells can be used to contrast the status of various caspase activation pathways, thereby providing insights into the pathological defects in apoptosis regulation that exist in individual patient specimens.