Major factors involved in the inhibition of ultrasound-induced free radical production and cell killing by pre-sonication incubation or by high cell density

To identify the factors involved in the inhibition of ultrasound (US)-induced free radical production and cell killing by pre-sonication incubation or by high cell density, we used different densities of U937 cells, and with (up to 2 h) or without pre-sonication incubations, the cell suspensions were exposed to 1 MHz US (10% duty factor at 100 Hz pulse rate; intensities 0.1-0.5 W/cm2 for 1 min). The intensity 0.3 W/cm2 was used for cell killing experiments and 0.5 W/cm2 for free radical experiments. Free radical production was determined by electron paramagnetic resonance (EPR)-spin trapping with DMPO while cell killing was determined by assays for lysis, loss of cell viability, apoptosis and necrosis. The results show that at higher cell densities, CO2 in the medium rapidly increased, with shorter pre-sonication incubation required to attain complete inhibition of both free radical production and cell killing. Cell killing at 0.3 W/cm2 and free radical production at 0.5 W/cm2 were both inhibited at 10 million cells/ml without incubation, and at 2 million cells/ml incubated for 2 h before sonication. Level of CO2 alone could not account for the inhibition; consumption of gases in the medium is also considered in the inhibitory effect of pre-sonication, while suppression of cavitational activities due to the “viscosity effect” is considered a more important factor in the inhibition by high cell density.