Fluoromicroscopic studies of bleomycin-induced intracellular oxidation in alveolar macrophages and its inhibition by taurine.

Abstract

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 property 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 micrograms/ml has a moderate stimulatory effect (1.87- to 2.66-fold) on the secretion of superoxide anion. A high concentration of bleomycin (20 micrograms/ml), however, inhibits cell response to zymosan-induced secretion of superoxide anion. At 4 micrograms/ml, bleomycin has no effect on cell membrane integrity or morphology but results in a significant increase in intracellular oxidation. This oxidative process is Fe(2+)-dependent and is accompanied by an increase in intracellular calcium (35 nM). Both the intracellular oxidation and calcium rise induced by internalized bleomycin are inhibited by pretreatment of cells with varying concentrations of taurine (25, 125, and 187.5 microM). The inhibitory effect on intracellular oxidation was found to be 36, 57, and 60%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Taurine blunts LPS-induced increases in intracellular calcium and TNF-α production by Kupffer cells

“…Thus, it is shown here for the first time in isolated Kupffer cells that taurine, at physiologically attainable concentrations, significantly blunted LPS-induced elevations in [Ca 2ϩ ] i and that this process is most likely linked to the observed reduction in TNF-␣ production. Taurine, at concentrations used here, also blocked the bleomycin-induced increase in [Ca 2ϩ ] i in isolated alveolar macrophages [42]. However, it did not prevent TNF-␣ production completely, most likely because Kupffer cells contain receptor-mediated pathways (i.e., scavenger receptor) that, although dependent on calcium [7], may not be affected by taurine.…”

Time-Dependent Apoptosis of Alveolar Macrophages From Rats Exposed to Bleomycin: Involvement of TNF Receptor 2

“…Another in vitro study showed that BLM induces a substantial rise in intracellular calcium and a significant generation of intracellular reactive oxygen species in rat AM (Bhat et al, 1994). These studies suggest that intracellular oxidative events in AM may play an important role in BLM-mediated inflammatory lung injury.…”

“…This BLM-induced apoptosis, lung inflammation, and fibrosis can be significantly attenuated by treatment with a caspase inhibitor (Kuwano et al, 2001). In a previous study, it was demonstrated that BLM induced intracellular oxidation in AM, and this action was accompanied by a rise in intracellular calcium levels (Bhat et al, 1994). Both reactive oxygen species and intracellular calcium are known to alter mitochondrial functions (Green & Reed, 1998), suggesting that BLM may produce apoptosis through a mitochondrial pathway.…”