Effects of dissolved gases and an echo contrast agent on apoptosis induced by ultrasound and its mechanism via the mitochondria-caspase pathway

Human histiocytic lymphoma U937 cells were exposed to continuous 1-MHz ultrasound (US) for therapeutic use, (0∼6.5 W/cm² (I_SPTA). Apoptosis and its related end points were examined by flow cytometry. Fraction of cells with low mitochondria membrane potential were observed after sonication and significant superoxide and peroxide formation, increased activity of caspase-3, and DNA fragmentation revealed biochemically, were also found. The fraction of early apoptosis and secondary necrosis increased with the incubation time after sonication. Early apoptosis observed at 6 h after sonication reached its maximum at 2 min of sonication and gradually decreased. On the other hand, secondary necrosis increased with the duration of sonication. When the effects of dissolved gases, Ar, N₂, O₂, air, N₂O and CO₂, on free radical formation due to inertial cavitation were investigated by electron spin resonance (ESR) spin trapping, formation of hydroxyl radicals and hydrogen atoms was found in solutions saturated with Ar, N₂, O₂ and air, but not with N₂O and CO₂. Apoptosis induced by US was also dependent on the dissolved gases in the order Ar = N₂ = O₂ = air ≫ N₂O = CO₂ ∼ 0. These results suggest that US-induced apoptosis, which is mitochondria-caspase dependent, was linked to inertial cavitation. However, quantities of free radicals did not influence the fraction of early apoptosis and secondary necrosis. When the cells were sonicated in the presence of an echo contrast agent, Levovist™; synergistic enhancement of secondary necrosis induced by US was observed at concentrations of more than 20 mg/mL. In contrast, an additive increase of early apoptosis was observed in the combined treatments. These results suggest that Levovist™; acting as cavitation nuclei, enhances secondary necrosis induced by US due to an increase in the membrane damage.