Oxidative Properties of 12-O-Tetradeca noyIphorbol-13-acetate-stimulated Human Blood Monomorphonuclear Leukocytes and Their Toxicity against a Human Lung Carcinoma Cell Line1

Human monomorphonuclear leukocytes (MMNs) stimulated with 12-O-tetradecanoylphortx)l-13-acetate (TPA) were found to be toxic towards human A549 lung carcinoma cells which have been desensitized against the direct growth-inhibitory effect of TPA. This toxicity was dependent on the TPA concentration and the ratio of MMNs to A549 cells. Using a TPA concentration of 10~7 M and an effectortarget cell ratio of 30:1, experiments were performed to give clues as to the mechanisms by which TPAstimulated MMNs cause toxicity. Levels of the endogenous thiol glutathione were reduced by 37% in MMNs exposed to TPA for 24 h, but the glutathione levels in the A549 target cells were not markedly affected by TPA-stimulated MMNs. The supernatant of incubations of MMNs with TPA contained a species which was capable of oxidizing the thiol agent 5-thio-2-nitrobenzoic acid. Within 2 h, 9 nmol of this oxidant were produced by 107 MMNs. The oxidant exhibited a half-life of 20 h, and its formation was abolished by adding catatase (150 units/ml), azide (1 HIM), or cyanide (1 mw) to the incubations of MMNs with TPA. The addition of Superoxide dismutase (100 units/ml) enhanced oxi dant formation. These results indicate that its generation was dependent on the myeloperoxidase:H202:halide system. Large amounts of an oxidizing species with properties identical to those described here have been characterized recently in polymorphonuclear leukocytes [S. J. Weiss, M. B. Lampert, and S. T. Test. Science (Wash. DC), 222: 625-627,1983]. The toxicity exerted by TPA-stimulated MMNs was partially inhibited by Superoxide dismutase and by retinoic acid (30 UM)but not at all by catatase, azide, or cyanide. Therefore, the 5-thio-2-nitrobenzoic acid oxi dant does not appear to be involved in the process which led to cytotoxicity by TPA-stimulated MMNs in A549 cells.