V. V. Shaposhnikova

Role of arachidonic acid metabolism in thymocyte apoptosis after irradiation

The present study demonstrates that DNA fragmentation, nuclear pycnosis and trypan blue staining of irradiated thymocytes is prevented by inhibition of the lipoxygenase pathway of arachidonic acid metabolism and is not affected by cyclooxygenase inhibition. Exposed to irradiation [3H]arachidonic acid‐labeled thymocytes release radioactive products to the external medium. The process is blocked by the lipoxygenase inhibitor, nordihydroguaiaretic acid. Thus, it can be concluded that irradiation activates arachidonic acid metabolism and that lipoxygenase metabolites play an important role in thymocyte apoptosis.

Detection of Reactive Oxygen Species Induced by Radiation in Cells Using the Dichlorofluorescein Assay

Abstract Korystov, Y. N., Shaposhnikova, V. V., Korystova, A. F. and Emelyanov, M. O. Detection of Reactive Oxygen Species Induced by Radiation in Cells Using the Dichlorofluorescein Assay. Radiat. Res. 168, 226–232 (2007). The goal of this study was to determine the amount of reactive oxygen species (ROS) that arises inside cells irradiated in medium containing blood serum using the 2′7′-dichlorofluorescein (DCF) assay. DCF fluorescence in cells and medium was recorded on an MF44 Perkin Elmer fluorimeter, and fluorescence in cells only was recorded on a Partec flow-through cytometer. Human larynx tumor HEp-2 cells and lympholeukosis P388 cells were irradiated with X rays at a dose rate of 1.12 Gy/min. The factors (temperature, pH, serum concentration) affecting the oxidation of 2′7′-dichlorofluorescin (DCFH) to DCF were studied, and errors in the dichlorofluorescein assay of ROS were minimized. The amount of ROS registered by the DCF assay in cells was found to depend on the concentration of serum in the medium during irradiation. In the presence of 10% serum, radiation had no effect on the amount of detectable ROS. The effect of radiation on the formation of intracellular ROS was almost completely abolished if the irradiated medium was removed immediately after radiation exposure. The increase in the formation of ROS in cells irradiated in medium with a low serum content is due mainly to the radiolytic products of water that arise in medium and oxidize DCFH located in cells.

The effect of melittin on proliferation and death of thymocytes

The effect of melittin, an activator of phospholipase A2, on proliferation and death of rat thymocytes in a broad concentration range was studied. Cell proliferation was estimated by the accumulation of colchicin metaphases, necrotic death was determined from lysis and staining of cells with trypan blue, and apoptosis was assessed from the type of DNA fragmentation, the amount of fragmented DNA, and the percentage of cells with subdiploid DNA. It was shown that low melittin concentrations (below 5 μg/ml) stimulate thymocyte proliferation. At high melittin concentrations, thymocytes die by the primary necrosis type. Throughout the concentration range studied, melittin does not produce apoptosis in thymocytes. Conversely, high melittin concentrations even inhibit thymocyte apoptosis in the control and after irradiation. An inhibitor of RNA synthesis actinomycin D does not affect thymocyte death in the presence of melittin. It is concluded that the activation of phospholipase A2 can induce necrosis but not apoptosis and thus is not a necessary step in the signaling cascade that initiates apoptosis in thymocytes.

Dependence of thymocyte apoptosis on protein kinase C and phospholipase A2

The effect of inhibitors and activators of protein kinase C and phospholipase A2 on radiation‐induced apoptosis of rat and mouse thymocytes has been studied. It is shown that the apoptosis is prevented by the protein kinase C inhibitor 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperasine dihydrochloride and is potentiated by protein kinase C activator phorbol 12‐myristate 13‐acetate, calcium ionophore A23187 and concanavalin A. The protein kinase C activators initiate apoptosis in mouse but not in rat thymocytes. The inhibitor of phospholipase A2 prevents apoptosis induced by all the factors. The results obtained indicate that both protein kinase C and phospholipase A2 are involved in the thymocyte apoptosis.

The effect of inhibitors of arachidonic acid metabolism on proliferation and death of tumor cells

The effect of inhibitors of arachidonic acid metabolism on proliferation and death of tumor P‐388 cells in a broad concentration range was studied. Cell proliferation was estimated by the metaphase frequency and the proportion of cells in S phase; cell death was determined from lysis, staining of cells with trypan blue, nuclear damage, percentage of cells with subdiploid DNA and the type of DNA fragmentation. It was shown that low concentrations of phospholipase A2 and lipoxygenase inhibitors stimulated the proliferation of P‐388 cells. At higher concentrations, the inhibitors suppressed cell proliferation by blocking the G1‐S transition and induced cell death of the apoptosis type. Indomethacin, an inhibitor of cyclooxygenase, did not initiate cell death, nor did it affect the proliferation of P‐388 cells at concentrations of up to 10 μM.

Intercellular Interactions in the Interphase Death of Irradiated Thymocytes

The effect of the interaction of different types of cells on the interphase death and pycnosis of thymocytes irradiated in vitro was studied. When removed from the thymus suspension of cells with natural killer activity, medullary thymocytes and macrophages did not change the radiation-induced death of cortical thymocytes. On the other hand, postirradiation incubation of cortical thymocytes together with unirradiated thymocytes or with cells of certain other cell lines diminished thymocyte death. Mixing the cell suspensions and changing the incubation medium decreased thymocyte death. All of these results indicate that these cells produce soluble mediators that are toxic to the cells that secrete them. The possible nature of these autotoxic mediators has been studied using inhibitors of arachidonic acid metabolism. Inhibitors of phospholipase A2 or lipoxygenase reduced interphase death markedly, while an inhibitor of cyclooxygenase did not. These data suggest that some lipoxygenase products may serve as autotoxic mediators in the interphase death of thymocytes.

Protection of microcirculation in rat brain against late radiation injury by gammaphos

The ability of a radioprotector-gammaphos to modify the development of late vascular changes in the rat brain after local irradiation with 40 and 60 Gy was investigated by means of an angiographic method. The relative number of animals with gross vascular abnormalities, the size of foci with vascular lesions, and the changes in size and number of vessels were less in the drug-treated groups. Thus, it may be possible to increase the tolerance of host tissues to curative radiation therapy by chemical radioprotectors such as gammaphos.

The mechanism of radiation-induced interphase death of lymphoid cells: A new hypothesis

The interphase death of irradiated rat thymocytes depends on their concentration during postirradiation incubation. The kinetics of pycnosis and cell death determined with the trypan blue exclusion test in the samples with the highest cell concentration (1-2 x 10(7) cells/ml) is consistent with the data available in the literature, whereas the samples with the lowest concentration (2 x 10(5) cells/ml) undergo almost no pycnosis and death after irradiation with doses up to 50 Gy. On the basis of these results, we suggest a new mechanism of interphase death involving an interaction between irradiated thymocytes and the fraction of thymus cells possessing cytocidal activity. The observed correlation between the cytocidal activity and interphase death of thymocytes from animals of different ages favors our mechanism. It was found that the inhibitors which prevent the conjugation of killer cells and their targets do not influence interphase death, while the substances which block the secretion of cytotoxic factors or their action on the target membrane do protect from interphase death. Thus we suggest that the irradiation activates the killer cells to secrete some cytotoxic factors which induce pycnosis and interphase death of thymocytes.

Synthesis of polyarylenephthalides prospective as smart polymers

The data on the synthesis of polyarylenephthalides, their analogs, and derivatives are surveyed. The main attention is given to the synthesis of polyarylenes through the polycondensation of pseudoacid chlorides according to several variants, predominantly via the self-condensation of pseudoacid chlorides, which may be depicted by the general scheme nOpen image in new window n(−RH is the radical of aromatic or heterocyclic polynuclear hydrocarbon.). The reaction affords polyarylenephthalides (I) (X = CO, Y = O), polyarylenephthalimidines (II) (X = CO, Y = NR1, where R1 is an aromatic hydrocarbon), and polyarylenesulfophthalides (III) (X = SO2, Y = O). Polymers I and II hold promise for designing thermostable, heat-(T g ≥ 420–470°C) and chemoresistant, and functional materials. One should distinguish the valuable functional properties of polymers I and III that make them prospective as smart polymers, namely, the switching effect induced by temperature, pressure, and electric and magnetic fields along with changes in color, electro-and photoluminescence. Owing to the presence of sulfophthalide groups, polymers III are candidates for use as latent polyelectrolytes. With consideration for the foregoing reasoning, phthalide-containing polyarylenes of several other classes, namely, polyaryleneketones, poly(arylene ether ketones), polyarylates, oligomer resols, and crosslinked systems on their basis, which are prospective as smart polymers, have been synthesized.

Effects of taxifolin on the activity of angiotensin-converting enzyme and reactive oxygen and nitrogen species in the aorta of aging rats and rats treated with the nitric oxide synthase inhibitor and dexamethasone

The action of taxifolin on the angiotensin-converting enzyme (ACE) and the formation of reactive oxygen and nitrogen species (ROS/RNS) in the aorta of aging rats and rats treated with nitric oxide synthase inhibitor (Nω-nitro-l-arginine methyl ester (L-NAME)) or dexamethasone have been studied. The ACE activity in aorta sections was determined by measuring the hydrolysis of hippuryl-l-histidyl-l-leucine, and the ROS/RNS production was measured by oxidation of dichlorodihydrofluorescein. It was shown that taxifolin at a dose of 30–100xa0μg/kg/day decreases the ACE activity in the aorta of aging rats and of rats treated with L-NAME or dexamethasone to the level of the ACE activity in young control rats. Taxifolin (100xa0μg/kg/day) was found to also reduce the amount of ROS/RNS in the aorta that increased as a result of L-NAME intake. L-NAME treatment increases the contribution of 5-lipoxygenase and NADPH oxidase to ROS/RNS production in the aorta, while taxifolin (100xa0μg/kg/day) decreases the contribution of these enzymes to the normal level.