This selective review of the biological effects of ultrasound presents a synopsis of our current understanding of how cells insonated in vitro are affected by inertial cavitation from the standpoint of physical and chemical mechanisms. The focus of this review is on the physical and chemical mechanisms of action of inertial cavitation which appear to be effective in causing biological effects. There are several fundamental conditions which must be satisfied before cavitation-related bioeffects may arise. First, bubbles must be created and then brought into proximity to cells. Exposure methods are critical in this regard, and simple procedures such as rotation of a vessel containing the cells during exposure can drastically alter the results. Second, once association is achieved between bubbles and cells, the former must interact with the latter to produce a bioeffect. It is not certain that the inertial event is the prime mechanism by which cells are lysed; there is evidence that the turbulence associated with bubble translation may cause lysis. Additionally, there appear to be chemical and other physical mechanisms by which inertial cavitation may affect cells; these include the generation of biologically effective sonochemicals and the apparent emission of ultraviolet (UV) and soft X-rays. The evidence for inertial cavitation occurring within cells is critically reviewed.

A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective

The statement is often made that free radicals will be produced during the violent collapse of transient cavities in liquids, that may result from the passage of ultrasonic waves in the low megahertz frequency range. Evidence for such free radicals is mostly indirect. We have insonated some argon-saturated aqueous solutions of biological molecules (trypsin, Dulbecco's Minimum Essential Medium, fetal bovine serum, and human blood plasma) and whole human blood, all containing a spin trapping compound, at 1 MHz, continuous wave, 6.6 W/cm2 (SPTA) for 15 min at 4-8 degrees C. Electron spin resonance spectra of the samples were recorded 10 and 30 min. after cessation of insonation. The spectra included those of spin adducts formed with .OH and .H radicals, which were absent in the cases of the sham treated controls. Thus, we have demonstrated the formation of the highly reactive .OH and .H radicals in the insonated media, even in the presence of natural radical scavengers in the mammalian derived products.

Evidence for free radical production by ultrasonic cavitation in biological media

L1210 cells were exposed in suspension to shock waves generated with a Dornier XL1 lithotripter. After 1000 discharges at 25 kV, the number of nondisrupted cells was 15% and the number of trypan blue excluding cells was 7% as compared to 100% in sham treated controls; the shock-wave effect was more prominent at higher voltages and less prominent at higher discharge numbers when compared at similar electrical input energies. Overall proliferation of cells which were trypan blue negative after exposure exceeded 70% of the proliferation of sham treated controls, except after 1000 shocks at 25 kV, where proliferation was reduced to 42%. The latter reduction in proliferation was found to be due to a reduced growth for 24 h after exposure, with a return to normal proliferation during the following days. Limiting dilution analysis revealed that the reduced growth was mainly due to a transitory increase of the doubling time and not to a reduction of the number of proliferating cells. Cell disruption by shock waves was completely inhibited by exposing the cells at an elevated pressure of 101 atmospheres, pointing to the possible involvement of cavitation in the shock wave effect.

Biological effects of shock waves: cell disruption, viability, and proliferation of L1210 cells exposed to shock waves in vitro.

Ultrasonically induced alterations of cultured tumour cells

5 – Effects and Mechanisms

Chinese hamster V-79 cells were exposed in suspensions for 1–15 min to 1.1 MHz continuous wave (CW) ultrasound at axial intensifies from 0.25–30 W/cm2. Cell lysis was evidenced by a decrease in the number of intact cells, and loss of reproductive integrity was evidenced by a decrease in the plating efficiency of the remaining intact cells. The magnitude of these effects was a function of both intensity and exposure duration. Mitotically synchronized cells displayed a differential sensitivity depending upon cell cycle position. The M and S phases of the cell cycle were more resistant with respect to cell lysis and loss of reproductive integrity than were the G1 and G2 phases.

Ultrasound lethality to synchronous and asynchronous chinese hamster V-79 cells

Monolayer cultures of JU56 wallaby cells were exposed to germicidal U.V. and/or photoreactivating (PR) light. The U.V. exposures induced dose-dependent cell-death. The survival data are consistent with a common extrapolation number (n) of 6 x 17 +/- 0 x 98 with a D(0) of 123 x 0 +/- 6 x 8 erg/mm2 for photo-reactivated cells and a D0 of 87 x 3 +/- 4 x 9 erg/mm2 for non-photoreactivated cells; the photoreactivation protected the cells with a dose-modification factor of 1 x 41 +/- 0 x 02. Therefore PR is not a shoulder phenomenon and so has no relationship to the repair of sub-lethal damage.

Ying-Kai Fu

Papers

Ultrasound lethality to synchronous and asynchronous chinese hamster V-79 cells

The absence of an effect of photoreactivation on sub-lethal damage accumulation in a photoreactive wallaby cell-line.