Production of Thymine Base Damage in Ultrasound-Exposed EMT6 Mouse Mammary Sarcoma Cells

Abstract

Mouse mammary sarcoma cells, line EMT6/Ro, were exposed for 1 min to 1 MHz continuous wave ultrasound over a range of intensities from 0.5 to 30 W/cm2 (spatial peak). The presence of thymine base damage (TBD) products of the 5,6-dihydroxydihydrothymine type was determined by an alkali degradation assay. Production of damage was found to be greatest (approximately 2.7 X 10(-3%) t'/T) at an intensity of 10 W/cm2 and fell off rapidly above and below this intensity. The amount of base damage produced at 10 W/cm2 ultrasound was approximately equivalent to the damage produced by a gamma-ray absorbed dose of 12 krad. Assay of cells immediately after sonication at 10 W/cm2 showed that approximately 14% of the cells had been lysed. Tests showed that it was the DNA of the intact cells, however, which sustained all of the TBD. Survival data demonstrated that of the remaining unlysed cell population approximately 5% were viable, whereas cells exposed to 12 krad showed no survival. Additionally, cells were exposed for up to 5 min at 5 W/cm2. An increase in TBD was demonstrated with increasing time of exposure such that the rate of production at 5 min was approximately three times greater than that of a 1-min exposure. TBD was found to be completely suppressed when cells were sonicated at 10 W/cm2 for 2 min under 4 bar of hydrostatic pressure. Addition of the radical scavengers beta-MEA and cystamine eliminated TBD but had minimal effect on survival. The pressure and scavenger experiments demonstrate that TBD results from cavitation-induced free radicals. Based on the values for both the half-life and diffusion distance of such radicals, our results indicate that at least part of the bubble collapse occurs intracellularly.