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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, an activator of phospholipase A 2 , 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 ug/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 A 2 can induce necrosis but not apoptosis and thus is not a necessary step in the signaling cascade that initiates apoptosis in thymocytes.

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.

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 A P and lipoxygenase inhibitors stimulated the proliferation of P-388 cells. At higher concentrations, the inhibitors suppressed cell proliferation by blocking the G I -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 W WM. z 1998 Federation of European Biochemical Societies.

The effects of avermectins on radiation-and dexamethasone-induced apoptosis of rat thymocytes have been studied. In particular, the properties of abamectin (avermectin B 1 ), ivermectin (22,23-dihydroavermectin B 1 , H 2 B 1 ) and aversectin C were examined. Aversectin C is a mixture of eight naturally occurring avermectins of the following composition: B 1 (42%), B 2 (22%), A 1 (13%) and A 2 (23%). This mixture is currently widely used as active ingredient in veterinary antiparasitic formulations produced by Pharmbiomed Co. Apoptosis was estimated by membrane damage, nuclear pycnosis (or chromatin condensation), DNA fragmentation and percentage of cells with subdiploid DNA. It was shown that aversectin C inhibits thymocyte apoptosis induced by both agents. The IC 50 dose was in the range of 0.1-0.3 µg/ml for aversectin C, whereas abamectin and ivermectin produced no effect up to 1 µg/ml. It is suggested that avermectins of the A series are responsible for the high anti-apoptotic activity exhibited by aversectin C. Thus, aversectin C is not only an effective antiparasitic drug but is also capable of exhibiting some other useful activities, e.g. a cytoprotective effect exerted on the mammalian immune cells. Avermectins, antiparasitic drugs, apoptosis, thymocytes, dexamethasone, gamma raysThe avermectins are a family of closely related 16-membered macrocyclic lactones produced by the fungus Streptomyces avermitilis. Nearly all the avermectins exhibit a broad spectrum of activity against nematode and arthropode parasites, with the B 1a compound being the most potent (Campbell 1989). Accordingly, it was used as active ingredient in commercial antiparasitic formulations. Until recently, there were basically two types of such avermectin-based active ingredients, i.e. ivermectin (consisting mainly of avermectin H 2 B 1a ) and abamectin (predominantly containing avermectin B 1a ). However, several years ago the Pharmbiomed Co. started manufacturing the veterinary and plant protection formulations based on a new active ingredient -aversectin C which is an intrinsic mixture of eight naturally occurring avermectins. Preliminary experiments demonstrated that aversectin C (presumably due to the presence of avermectins of both the A-and B-series) possesses some new useful features compared to ivermectin and abamectin, e.g. with the same high efficacy, the spectrum of activity is broader, level of residues is lower and, hence, the shorter withdrawal periods could be recommended (Simetsky et al. 1994;Berezkina et al. 1996). Furthermore, degradation of aversectin C in soil and water was found to be more effective compared to abamectin and ivermectin ( M o sin et al. 1998) and, hence, ecologically aversectin C is safer.

The effect of inhibitors and activators of protein kinase C and phospholipase A, 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 I-(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 Az prevents apoptosis induced by all the factors. The results obtained indicate that both protein kinase C and phospholipase A, are involved in the thymocyte apoptosis.