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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 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 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.