FLUOROMICROSCOPIC STUDIES OF BLEOMYCIN-INDUCED INTRACELLULAR OXIDATION IN ALVEOLAR MACROPHAGES AND ITS INHIBITION BY TAURINE

The mechanism of bleomycin-induced pulmonary fibrosis is not yet clear. Recent studies have shown that alveolar macrophages (AM) can be stimulated by bleomycin in vitro releasing inflammatory cytokines, suggesting that the interaction of bleomycin with AM is an important step in the drug-induced fibrotic process. Bleomycin is known to bind DNA and generate oxygen radicals through complexation with Fe2+ and oxygen. To provide more insight into the cellular oxidative properly of bleomycin, we have developed a fluoromicroscopic method using 2',7'-dichlorofluorescin diacetate (DCFHDA) as an oxidative fluorescence probe to study the bleomycin-induced intracellular oxidation in rat AM and the inhibition of the oxidation by taurine, a compound known to inhibit the bleomycin-induced fibrosis. Bleomycin at 5 to 20 mu g/ml has a moderate stimulatory effect (1.87- to 2.66-fold) on the secretion of superoxide anion. A high concentration of bleomycin (20 mu g/ml), however, inhibits cell response to zymosan-induced secretion of superoxide anion. At 4 mu g/ml, bleomycin has no effect on cell membrane integrity or morphology but results in a significant increase in intracellular oxidation. This oxidative process is Fe2+-dependent and is accompanied by an increase in intracellular calcium (35 nM). Both the intracellular oxidation and calcium rise induced by internalized bleomycin ale inhibited by pretreatment of cells with varying concentrations of taurine (25, 125, and 187.5 mu M). The inhibitory effect on intracellular oxidation was found to be 36, 57, and 60%, respectively. These results demonstrate a stimulation-inhibition relationship between bleomycin and taurine on the cellular oxidation at a subcytotoxic dose of bleomycin, suggesting that the oxidative effect of the intracellular bleomycin-Fe2+ complex is important in the initiation of the fibrotic process.