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Transient permeabilization of cell membranes by ultrasound-exposed microbubbles is related to formation of hydrogen peroxide

¹Institute for Cardiovascular Research, VU University Medical Center, Amsterdam; and ²Interuniversity Cardiology Institute of the Netherlands, Utrecht, The Netherlands

Submitted 24 October 2005 ; accepted in final form 12 April 2006

In the present study, we addressed the interactions among ultrasound, microbubbles, and living cells as well as consequent arising bioeffects. We specifically investigated whether hydrogen peroxide (H₂O₂) is involved in transient permeabilization of cell membranes in vitro after ultrasound exposure at low diagnostic power, in the presence of stable oscillating microbubbles, by measuring the generation of H₂O₂ and Ca²⁺ influx. Ultrasound, in the absence or presence of SonoVue microbubbles, was applied to H9c2 cells at 1.8 MHz with a mechanical index (MI) of 0.1 or 0.5 during 10 s. This was repeated every minute, for a total of five times. The production of H₂O₂ was measured intracellularly with CM-H₂DCFDA. Cell membrane permeability was assessed by measuring real-time changes in intracellular Ca²⁺ concentration with fluo-4 using live-cell fluorescence microscopy. Ultrasound, in the presence of microbubbles, caused a significant increase in intracellular H₂O₂ at MI 0.1 of 50% and MI 0.5 of 110% compared with control (P < 0.001). Furthermore, we found increases in intracellular Ca²⁺ levels at both MI 0.1 and MI 0.5 in the presence of microbubbles, which was not detected in the absence of extracellular Ca²⁺. In addition, in the presence of catalase, Ca²⁺ influx immediately following ultrasound exposure was completely blocked at MI 0.1 (P < 0.01) and reduced by 50% at MI 0.5 (P < 0.001). Finally, cell viability was not significantly affected, not even 24 h later. These results implicate a role for H₂O₂ in transient permeabilization of cell membranes induced by ultrasound-exposed microbubbles.

membrane permeability; calcium; ultrasound contrast agents

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