M. Bhat et al., FLUOROMICROSCOPIC STUDIES OF BLEOMYCIN-INDUCED INTRACELLULAR OXIDATION IN ALVEOLAR MACROPHAGES AND ITS INHIBITION BY TAURINE, Environmental health perspectives, 102, 1994, pp. 91-96
The mechanism of bleomycin-induced pulmonary fibrosis is not yet clear
. Recent studies have shown that alveolar macrophages (AM) can be stim
ulated by bleomycin in vitro releasing inflammatory cytokines, suggest
ing 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 bleomyci
n, we have developed a fluoromicroscopic method using 2',7'-dichlorofl
uorescin diacetate (DCFHDA) as an oxidative fluorescence probe to stud
y the bleomycin-induced intracellular oxidation in rat AM and the inhi
bition of the oxidation by taurine, a compound known to inhibit the bl
eomycin-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, inhib
its cell response to zymosan-induced secretion of superoxide anion. At
4 mu g/ml, bleomycin has no effect on cell membrane integrity or morp
hology but results in a significant increase in intracellular oxidatio
n. This oxidative process is Fe2+-dependent and is accompanied by an i
ncrease in intracellular calcium (35 nM). Both the intracellular oxida
tion and calcium rise induced by internalized bleomycin ale inhibited
by pretreatment of cells with varying concentrations of taurine (25, 1
25, and 187.5 mu M). The inhibitory effect on intracellular oxidation
was found to be 36, 57, and 60%, respectively. These results demonstra
te a stimulation-inhibition relationship between bleomycin and taurine
on the cellular oxidation at a subcytotoxic dose of bleomycin, sugges
ting that the oxidative effect of the intracellular bleomycin-Fe2+ com
plex is important in the initiation of the fibrotic process.