Mami Nakata

Exposure of rat thymocytes to hydrogen peroxide increases annexin V binding to membranes: inhibitory actions of deferoxamine and quercetin

Effects of hydrogen peroxide (H(2)O(2)) on rat thymocytes were examined, using a flow cytometer and three fluorescent probes, annexin V-fluorescein isothiocyanate (annexin V-FITC) for detecting phosphatidylserine expressed on the membrane surface, ethidium bromide for estimating dead cells, and fluo-3-acetoxymethyl ester (fluo-3-AM) for monitoring changes in intracellular Ca(2+) concentration ([Ca(2+)](i)), to characterize H(2)O(2)-induced cytotoxicity. Exposure to H(2)O(2) (30 microM or more) increased the number of annexin V-positive live cells dose- and time-dependently while the number of dead cells increased at concentrations of 1 mM or more. H(2)O(2) (30 microM or more) increased [Ca(2+)](i) in a dose-dependent manner. Threshold concentration of H(2)O(2) to increase [Ca(2+)](i) was similar to that to increase annexin V binding to membranes. The H(2)O(2)-induced change in cell membranes was attenuated under Ca(2+)-free conditions. Therefore, it is likely that Ca(2+) is involved in the H(2)O(2)-induced cytotoxicity. Deferoxamine was effective to protect the cells suffering from H(2)O(2)-induced oxidative stress, suggesting a contribution of hydroxyl radicals generated by the Fenton reaction. Quercetin also exerted a potent protective action on cells suffering from H(2)O(2)-induced oxidative stress. The results indicate that the exposure of rat thymocytes to H(2)O(2) at micromolar concentrations increases annexin V binding to cell membranes in a Ca(2+)-dependent manner, suggesting the possibility that the oxidative stress caused by H(2)O(2) (and/or hydroxyl radicals) induces apoptosis via increasing [Ca(2+)](i).

Flow cytometric analysis on tri-n-butyltin-induced increase in annexin V binding to membranes of rat thymocytes

Effects of tri-n-butyltin chloride (TBT) on rat thymocytes were examined by using a flow cytometer and three fluorescent dyes (annexin V-FITC, ethidium bromide and fluo-3-AM) to further characterize its cytotoxic action. TBT at concentrations of 100 nM or greater, time- and dose-dependently increased the population of annexin V-positive live cells in the cell suspension. Most of cells became to be annexin V-positive within 60 min after the start of application of 300 nM TBT. Some of annexin V-positive live cells were further stained with ethidium, indicating that some of the cells were killed, in continued presence of TBT at 300 nM or greater. When the cells were exposed to 300 nM TBT only for 15 min, the population of annexin V-positive live cells increased after removal of TBT from incubation medium. TBT-induced increase in the population of annexin V-positive live cells was partly attenuated under Ca(2+)-free condition, although that was not the case for the dead cells. TBT at 30 nM or greater increased [Ca(2+)]i in a dose-dependent manner. Triethyltin and trimethyltin even at 1 μM did not increase the [Ca(2+)]i and the population of annexin V-positive live cells. The population of annexin V-positive live cells increased as the [Ca(2+)]i was increased by ionomycin, a calcium ionophore. Results suggest an involvement of Ca(2+) in some of TBT-induced cytotoxicity.

Methylmercury toxicity in dissociated rat brain neurons: modification by l-cysteine and trimethylbenzylmercaptan and comparison with dimethylmercury and N-ethylmaleimide

The effects of methylmercury (MeHg) on dissociated rat cerebellar neurons were compared with those of MeHg conjugated with l-cysteine (MeHg-Cys conjugate), dimethylmercury (DiMeHg), N-ethylmaleimide (NEM) and ionomycin using a flow cytometer and two fluorescent dyes, fluo-3-AM and ethidium bromide. The efficacies of MeHg to increase intracellular concentration of Ca(2+) ([Ca(2+)]i) and to decrease cell viability were greatly reduced by conjugating MeHg with l-cysteine. It was not due to a decreased lipophilic property of MeHg-Cys because the conjugation of MeHg with trimethylbenzylmercaptane, a lipophilic substance, also reduced the efficacies. It seems that the reactivity of MeHg to SH-groups is responsible for the MeHg-induced toxicity since NEM increased [Ca(2+)]i and decreased cell viability while DiMeHg did not significantly affect them. However, the toxicity of MeHg was not explained only by the reactivity of MeHg to SH-groups since NEM-induced changes in fluo-3 and ethidium fluorescence were different from MeHg-induced ones. Ionomycin-induced changes in those fluorescence were also different although ionomycin decreased cell viability after increasing [Ca(2+)]i. Therefore, it is suggested that the mechanism of MeHg toxicity is more complicated than those of NEM and ionomycin.