Modulation of cisplatin-induced reactive oxygen species production by fullerene C(60) in normal and transformed lymphoid cells

The early response of normal (Wistar rat thymocytes) and transformed (mice lymphoid leukemia L1210) cells to treatment with anticancer drug cisplatin or to combined treatment with cisplatin and carbon nanostructure fullerene C60 was studied. We demonstrated with fluorescent probes DCFH-DA and TMRE that cisplatin at concentration 1 µg/ml induced reactive oxygen species (ROS) production and decreased the value of mitochondrial membrane potential in both cell types. The combined treatment with cisplatin (1 µg/ml) and fullerene C60 (7.2 µg/ml) was shown to be followed by oppositely directed modulation of ROS production in thymocytes and L1210 cells. Cisplatin-induced ROS production was intensified in L1210 cells, while in thymocytes it was decreased. It is supposed that the different effects of combined treatment are associated with peculiarities of fullerene C60 accumulation and localization in normal and cancer cells.

[Generation of active oxygen forms in rat thymocytes under action of hydrogen peroxide and fullerene C60].

The dynamics of active oxygen forms (AOF) generation in rat thymocytes 50 min after treatment with 0.1 and 0.5 mM H2O2 was estimated with the use of fluorescent probe DCFDA. Both enhanced AOF generation, which was dependent on H2O2 concentration, and glutathione peroxidase and superoxide dismutase activation, followed by a decrease of thymocytes viability were demonstrated. Preincubation of cells with 10(-5) M fullerene C60 was shown not only to prevent H2O2--induced AOF generation but to increase viability of H2O2-treated thymocytes at more prolonged time period. The data obtained indicate to fullerene C60 ability to prevent oxidative stress in thymocytes.

[Change in concentration of cytosolic Ca2+ caused by extracellular ATP and ecto-ATP-ase activity in thymocytes and transformed MT-4 cells].

The comparative study of extracellular ATP (ATP0) effect on free cytosolic calcium concentration ([Ca2+]i) in normal (isolated rat thymocytes) and transformed (leukosis MT-4 line) T-cells was carried out. Addition of 1 mM ATP to Ca-free incubation medium of both types of cells, loaded with indo-1, had no effect on [Ca2+]i level. Upon subsequent addition of 1 mM CaCl2 to the incubation medium the rapid and significant increase of [Ca2+]i in MT-4 cells was registered. This effect was maintained within 10 min and was not inhibited by phospholipase C inhibitor 0.2 mM neomycin, that was induced by cation entry into the cells from the extracellular medium. Both types of cells were shown to demonstrate ecto-ATPase activity in the presence of 1 mM MgCl2 or CaC12 in the incubation medium. Estimation of kinetic parameters has indicated that the maximum rate of extracellular ATP hydrolysis by MT-4 cells is higher and Mg2+ and Ca2+ activation constants are lower as compared to respective parameters of ATP hydrolysis by thymocytes. The possible functional significance of the increased level of ecto-ATPase activity in malignantly transformed cells is discussed.

[Change in glutathione content in rat thymocytes under apoptosis induced by H2O2 or irradiation].

Glutathione (GSH) content as well as GSH-peroxidase and GSH-reductase activity in isolated rat thymocytes X-irradiated in a dose of 4.5 Gy or treated with 0.1 mM H2O2 were studied in a period preceding the appearance of apoptosis morphological symptoms. The early adaptive response of thymocytes to radiation - increase of both GSH content and glutathione peroxidase and glutathione reductase activity was revealed. On the contrary the rapid fall of GSH level in H2O2-treated thymocytes was observed simultaneousely with glutathione reductase inhibition and enhanced GSH consumption by glutathione peroxidase, this disbalance of GSH-dependent antioxidant system probably facilitates mitochondrial way of apoptosis.

[Oxidative properties of photoexcited fullerenes C60 and C60-containing composites in suspension of thymocytes and Ehrlich ascites carcinoma cells].

Biological effects of photoexcited in UV/VIS region water-soluble fullerenes C60 and synthesized C60-containing composites after its addition to cell incubation medium are studied. It is shown by EPR method, that in the presence of photoexcited C60 (10(-5) M) reactive oxygen species (ROS) are generated both in water solution and cell suspensions. ROS generation is accelerated if C60 is included into aminopropylaerosyl or antracenaliminopropylaerosyl. Photodynamic damage of Erlich ascites carcinoma cells by photoexcited C60-composites is confirmed by a decrease of cell viability, enhanced content of Schiff base fluorescent products of malonaldehyde as indicator of lipid peroxidation and the decrease in respiratory activity, estimated by MTT-test. No effects of photoexcited C60-composites were detected in thymocytes suspension. The data obtained testity to perspective utility of C60-composites for optimization of photodynamic therapy methods.

[Changes of chromatin structure state in thymocites at the early stage of apoptosis induced by hydrogen peroxide and radiation]. / Zminy strukturnoho stanu khromatynu v tymotsytakh na rann'omu etapi apoptozu za induktsiï peroksydom vodniu i radiatsiieiu.

The paper deals with changes in the structural state of chromatin in isolated thymocites at the early stage of apoptosis induced by hydrogen peroxide and radiation. Content of necrosis and apoptosis cells in the suspension of the isolated rat thymocites, during 3-hour incubation after X-ray irradiation in a dose of 4.5 Gy or with the presence of 0.1 microM of H2O2 by the method of double lifetime staining by fluorescent dye Hehst 33342 and propydium iodide has been estimated. Apoptogenic effect of the studied effects has been found out, the dynamics of condensation and internucleosomic chromatin fragmentation has been established. It has been shown that 100 microM alpha-tocopherol inhibited completely DNA fragmentation in the cells incubated with H2O2 and only partially in irradiated cells. Introduction of postmitochondrial supernatant, isolated from the incubated control or irradiated cells, into the cell-free system which included the ATP-regenerating system and nuclei of control thymocites did not affect the level of DNA fragmentation, while the increase of the level of fragmented DNA in nuclei was observed in the presence of the supernatant obtained by centrifugation of the cells treated by H2O2. Differences of mechanisms of thymocite apoptosis initiation, as affected by hydrogen peroxide and ionizing radiation, is discussed.

[Particulars on the regulation of Ca(2+) in the cell nucleus]. / Osoblyvosti rehuliatsiï kontsentratsiï Ca(2+) v iadri.

The data of literature on the structural and functional features of nuclear pore complexes, the role of receptor-activated Ca(2+)-channels and Ca(2+)-ATPase of nuclear membranes are presented. The possible ways of Ca(2+)-signal transmission into the nucleus and the role of nuclear Ca2+ in apoptosis are discussed.

[Formation of inositol triphosphate in lymphocytes of rat spleen in the early stages after x-irradiation]. / Utvorennia inozytoltryfosfatu v limfotsytakh selezinky shchuriv na rann'omu etapi pislia renthenivs'koho oprominennia.

Stimulation of 32P-phosphoinositides turnover and phosphatidylinositol-4,5-biphosphate hydrolysis is observed 6 h after animals X-ray irradiation in a dose 1 Gy. Both mitogenic lectins LPS and PHA (10 mg/ml), but not 0.5 microM A23187 induce inositol phosphates release from endogenous [3H]inositol--phospholipids in lymphocytes. The level of inositol triphosphate is increased within 1-6 h and decreased in 12 h after irradiation. 45Ca2+ influx through lymphocytes plasma membrane is not changed within 3 h, but increases greatly 12 h after exposure. Data obtained indicate that IP3 formation is an early signal for radiation-induced changes in calcium homeostasis.

[Endonucleolysis of chromatin in rat thymus and spleen lymphocytes after irradiation]. / Endonukleoliz khromatynu u limfotsytakh selezinky ta tymusa shchuriv pislia oprominennia.

The level of soluble DNA fragments (polydesoxynucleotides) in thymus and spleen lymphocytes is enhanced 12 h after the whole-body X-ray irradiation in dose of 0.5 and 1 Gy. The level of Ca2+, Mg(2+)-DNAase activity in lysed cells extracts, measured with calf spleen DNA, was enhanced in the region of 0.1-0.5 mM CaCl2 in incubation medium and decreased at 5 mM CaCl2. Monitoring of DNA fragmentation by electrophoresis in agarose gels showed the presence of fragments with approximately 200, 400, 800 bp length. X-ray induced DNA fragmentation is supposed to be associated with Ca2+, Mg(2+)-endonuclease activation.