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Previous work in our laboratories has shown that, amongst other effects, irradiation of frog skin with low intensity ultrasound causes reductions in the chemical driving force of the short-circuit current. This indicated that either the Na/K dependent ATPase or ATP availability were being reduced. We measured the effect of ultrasound irradiation on ATP and NA/K-dependent ATPase from inverted erythrocyte ghosts and on firefly luciferin and luciferase activity. Our findings demonstrate that ultrasonic cavitation-induced sonochemical reactions were responsible for irreversible inactivation of luciferase and ATPase but had little or no effect on ATP and luciferin. We measured the levels of hydrogen peroxide generated by ultrasound under the conditions of our experiments and found that it could account for only part of the enzyme inactivation observed. Free radical scavengers/antioxidants were capable of fully protecting the enzymes from ultrasound-induced inactivation. These findings demonstrate that, in addition to hydrogen peroxide, free radicals generated by ultrasound are responsible for the effects.

Ultrasound has been used in physical therapy for > 4 decades. Recent studies indicate that non-thermal mechanisms such as cavitation are involved in the observed effects. Free radicals and other highly reactive compounds are known to form during sonochemical reactions associated with acoustic cavitation. Using frog skin as a biological model, the possibility that the increase in ionic conductance (Gt) upon exposure to therapeutic ultrasound is due to the effect of free radicals generated by sonochemical reactions, was investigated. It was found that the presence of cystamine, cysteamine and sodium ascorbate significantly reduced the increase in conductance caused by the exposure to 300 mW/cm2 (1 MHz CW) therapeutic ultrasound. The attenuation in the effects was dependent on the concentration of the radical scavengers/antioxidants used, the incubation time, and the intensity of ultrasound. The effects were also dependent on the lipid solubility of free radical scavengers/antioxidants. The time constant for the recovery process of Gt in the presence of free radical scavengers and antioxidants after exposure to ultrasound was found to be not significantly different from control. These results suggest that the increase in Gt due to ultrasound is induced by free radicals and other reactive species generated from acoustic cavitation. This study provides an indirect evidence to the contingent that free radicals are generated and act inside the cells. Furthermore, the radical scavengers and antioxidants used provide protection from oxidative damage without being involved in the recovery of Gt towards steady state values after sonication.