Fig. 3

Intracellular events underlying PS exposure in RBCs induced by TiO₂ NPs. Isolated human RBCs were treated with various concentrations (0, 5, 10 and 25 μg/mL) of TiO₂ NPs at 37 °C for 24 h. Next, phospholipid translocation was shown by (a) concentration-dependently increased scramblase activity and (b) no changes of flippase activity. (b) ROS generation was determined through preloading 5 μM CM-H₂DCF-DA for 30 min, and beta-lapachone was used as positive control. (c) Intracellular calcium, [Ca²⁺]_i, was examined by preloading 3 μM fluo-4 AM for 1 h. (d) Caspase 3 activity was increased as measured shown in Method. (e) Inhibition of PS exposure was performed by preloading various inhibitors, a calcium chelating agent (5 mM EGTA) or caspase inhibitors (Z-VAD-FMK and Q-VD-Oph) for 3 h prior to exposure to TiO2 NPs for 24 h. Values are mean ± S.E. of 3–5 independent experiments, * represents significant differences from the control group (p < 0.05)