Abstract
Abstract Nonfluorescent drug DCFHA was digested to DCFH by intracellular esterase enzyme. Consequently, DCFH reacts with intracellular reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) to form DCF fluorescence. Applied DCF fluorescence assay, a dose-dependent increase of DCF fluorescence intensity was found in CHO-K1 cells after 0-400 mM SbCl3 treatment for 4 h. In addition, a dose-dependent increase of nitrite (NO2-) level, a stable product of nitric oxide (NO) was also detected with DAN fluorescent dye in phenol red-free medium of same cell cultures after same treatment protocol. In cultures without cells, neither DCFHDA nor DAN reacted with 500 mM SbCl3 to increase DCF or DAN fluorescence intensity. DCF fluorescence intensity or nitrite level was decreased in SbCl3-treated cells after co-treatment with NOS inhibitor, 0.8-3.2 mM NAME or 25 mM MTC. However, the antimony accumulation was no difference in treated samples with or without NOS inhibitors. Furthermore, either NAME or MTC reduced SbCl3-induced micronuclei formation (MN) in cells. Catalase inhibitor, 40 mM 3AT (increase H2O2 level) did increase DCF fluorescence in cells after 160 mM H2O2 treatment for 30 min. However, it did not change DCF fluorescence in cells after 400 mM SbCl3 treatment for 4 h. SbCl3-induced nitrite level was suppressed in cells co-treated with 0.5 mM mannitol (to scavenge hydroxyl radical-OH.) or with 200 mM trolox (to scavenge OH. and peroxynitrite ONOO-). Recent studies from our laboratory indicated that SbCl3 treatment in CHO-K1 cells increased Fluo-3/AM probed intracellular calcium accumulation. In addition, either 1.6 mM NAME or 25 mM MTC did not affect SbCl3-treated intracellular calcium accumulation. In the present study, a suppressed SbCl3-increased DCF fluorescence was found in cells co-treated with 4-8 mM EGTA (calcium chelator). The intensity was decreased from 4.5-fold to 1.2-fold. And an increase of the intensity was shown in cells co-treated with 100-200 mM verapamil (vp, calcium channel blocker). SbCl3-enhanced nitrite level was significantly reduced in cells co-treated with 10-40 mM W7 (calmodulin inhibitor), and potentiated in cells co-treated with 1-5 mg/ml A23187. SbCl3-induced MN formation was suppressed from cell samples with 4-8 mM EGTA, or 80 mM BAPTA cotreatment, and potentiated from cell samples with 1 mg/ml A23187 or 100 mM Vp cotreatment. These results suggested that SbCl3-increased intracellular calcium accumulation may directly affect the intracellular ROS/RNS level, and induce MN formation. Calcium-dependent NO generation is involved in SbCl3-induced MN formation. In addition, nitrite depletion without decreasing intracellular calcium accumulation did reduced MN formation in cells.