Bioelectromagnetics 3:247-251 (1 982)

Temperature Dependence of Ultrasound= Induced Cell Killing: The Role of Membrane Fluidity

E. Ben-Hur and M. Green Department of Radiobiology, Nuclear Research Centre, Negev, Beer-Sheva, Israel

Chinese hamster cells in suspension were exposed to 20 lcHz ultrasound (US) at 54 Wicm' and various temperatures between 2 and 44 "C. Activation energies were 2.6 and 24 kcaVmole below and above 35 "C, respectively. Procaine, a local anaesthetic drug known to increase membrane fluidity, enhanced cellular inactivation by US above 41 "C, increasing the activation energy to 62 kcaUmole. The inactivation of the bacterium Salmonella typhimurium by US was also de- pendent on the exposure temperature, with an activation energy of 2.9 kcaUmole between 2 and 44 "C. These data are most simply explained by the hypothesis that membranes are a major target for cellular inactivation by US and that the fluidity of the membranes is important in this respect.

Key words: ultrasound, temperature-dependent cell killing, membrane fluidity

INTRODUCTION

Ultrasound (US) is widely used in medical diagnosis as well as for therapeutic purposes. Mammalian cells exposed to US suffer membrane damage reflected by changes in electrophoretic mobility [Repacholi et al, 19711 and permeability [Chapman et al, 19791. Above a certain US power threshold their colony forming ability is impaired [Martins et al, 19771. This effect of US is more pronounced when the treatment tem- perature is raised above 37 "C [Li et al, 19771.

Damage to insonated cells in vitro may result from at least three sources: heat production, cavitation phenomena, and direct mechanical forces. Proteins are the main target for thermal effects [Dewey et al, 19771. Cavitation phenomena most probably cause membrane damage due to the high tensile forces resulting from bubble oscillations [Flynn, 19641. Acoustic microstreaming results in mechanical shear forces on the cell membrane that are of a smaller magnitude than those observed to originate from cavitation but may be sufficient to damage the membrane or cause cell lysis [Williams et al, 19701.

Received for review September 8 , 198 1 ; revision received December 11, 198 1.

Address reprint requests to E. Ben-Hur, Department of Radiobiology, Nuclear Research Center, Negev, PO Box 9001, Beer-Sheva 84190, Israel.

0197-846218210302-0247$02.00 0 1982 Alan R. Liss, Inc.

248 Ben-Hur and Green

In the present work, the temperature dependence of US-induced cell killing was quantitated and the activation energies calculated. The role of membrane damage was probed with the aid of procaine, a membrane-fluidizing drug.

MATERIALS AND METHODS

Chinese hamster V79 fibroblasts were grown as a monolayer with Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf cerum. The cells doubled in number in about 9 h at 37 "C, in an atmosphere containing 5% C02 and a relative humidity near 100%.

Cell survival was determined using the technique of in vitro colony formation [Puck and Marcus, 19561.

Briefly, log-phase cells were suspended in DMEM (1 X lo5 cells/ml). After ex- posure to US, appropriate dilutions were made and the cells plated in Petri dishes to obtain - 200 colonies per plate. Colonies were stained with methylene blue after 8 days and counted. Results are expressed as the fraction of cells surviving a given treatment relative to the number of colony-forming cells in the starting suspension. Plating efficiency was over 80%.

Cells were exposed to US in 15-ml plastic tubes containing 3 ml of cell suspension in DMEM using 100 W Ultrasonic Disintegrator (Measuring and Scientific Equipment Ltd, London). The instrument consists of an electronic signal generator tunable over the range of 18 to 24 kHz, a power amplifier, a magneto-strictive transducer unit to convert the electrical energy into mechanical energy and a titanium probe (3.2 mm diameter) to translate mechanical energy into vibratory energy of an appropriate amplitude. The probe was immersed to a depth of 1 cm in the cell suspension. The instrument was operated at 20 kHz (frequency modulation). The average power density was 54 W/cm2. The tube containing the cell suspension was immersed in a temperature-regulated water bath. Temperature of the water bath was maintained to f 0.1 "C, and the temperature of the cell suspension did not rise more than 2 "C during exposure to US. The water used was not degassed. Temperature was monitored in the medium using a thermistor with a digital readout to 2 0.01 "C (Digitec, model 5810 thermometer, United System Corp, Dayton, Ohio).

Procaine hydrochloride (Sigma) was added to the cell suspension from 1 M stock solution 5 minutes prior to US exposure. Immediately after exposure, the cells were centrifuged and resuspended in 3 ml of fresh DMEM.

Salmonella typhymurium cells (strain TA100) were grown in nutrient broth plus 0.5% NaCl to a density of 1 X lo9 cells/ml and 3-ml samples were exposed to US as described above. Cell survival and mutagenicity were studied as previously described [Ben-Hur et al, 19801.

RESULTS

Figure 1 shows the survival curves of Chinese hamster cells exposed to US at various temperatures. Survival as a function of exposure time was strictly exponential and the only parameter required to describe the sensitivity of the cells is Do (the time of US exposure required to reduce survival by a factor of Ue). Evidently, the cells became progressively more sensitive to US as the exposure temperature was raised from 2 "C to 44 "C. In addition, it can be seen from Figure 1 that in the presence of 10-mM procaine, the sensitivity is enhanced at 42 "C and above.

Cell Killing by Ultrasound vs Temperature 249

1 I 0 10 20 3 0 0

SONICATION TIME (seconds)

Fig. 1 . Survival of Chinese hamster cells exposed to US for various periods A. Cell survival at various temperatures, as indicated. B . Cell survival at 41 "C, circles; 42 "C, triangles; 43 "C, squares. Filled symbols, 10-mM procaine present during sonication. Each datum point is the average of triplicate measures. Standard errors were less than 5% and are not shown. Lines were fitted to the points by eye.

To obtain an activation energy the sensitivity to US ( UD0) was plotted as a function of the reciprocal of the absolute temperature (Figure 2). There is a break point in the Arrhenius plot around 35 "C. From the slopes of the straight lines, the activation energy was calculated as 2.6 & 0.16 and 24 2 1.6 kcal/mole below and above 35 "C, respec- tively. In the presence of procaine, activation energy was increased to 62 & 5.9 kcal/ mole.

Also shown in Figure 2 is the Arrhenius plot for US inactivation of S. typhimurium. Although the bacteria were inactivated at a rate about 7-fold lower than mammalian cells (note the 10-fold difference in scales of the ordinates), activation energy was similar, 2.9 ? 0.12 kcal/mole. Unlike mammalian cells, activation energy for bacteria remained the same from 2 to 44 "C. The difference between the activation energies for bacteria and mammalian cells (2.9 and 2.6 kcaUmole, respectively) was not statistically significant.

Previous reports have shown that US can chemically modify nucleic acid bases in solution [McKee et al, 19771 and induce sister chromatid exchanges in human lymphocytes [Liebskind et al, 19791. Chromosome aberrations, however, were not observed [Rosebord et al, 19781. Therefore, we looked for possible mutagenic effects of US in S . typhimurium. The end point was reversion to histidine independence. Strain TA 100 and strain TA 98, which are sensitive to base-pair substitution mutagens and frameshift mutagens, respec- tively, have been used. Neither displayed a significant increase in the number of revertants above the spontaneous rate, as a result of US exposure (data not shown).

250 Ben-Hur and Green

Fig. 2. An Arrhenius plot of the sensitivity of Chinese hamster cells (circles) and S . typhimurium (triangles, slashed line) to US as a function of the reciprocal of the absolute temperature. Filled symbols denote cells treated with 10-mM procaine during exposure to US. Scale on left ordinate for Chinese hamster cells. Right ordinate for S typhimurium.

DISCUSSION

We have quantitated the temperature dependence of US-induced cell killing and calculated the activation energy for Chinese hamster cells and S. typhimurium over the temperature range 2-44 "C. While activation energy for bacteria was found to be 2.9 kcal/mole over this entire range, there was an abrupt increase to 24 kcal/mole for mam- malian cells above 35 "C. This could bc due to a phase transition in the membrane structure, which is known to occur at about 38 "C [Wallach, 19781. The further increase of the activation energy to 62 kcal/mole above 41 "C in the presence of procaine may be due to an additional phase transition brought about by the drug. Procaine, a local anaesthetic drug, increases membrane fluidity and at 10 mM was shown to enhance the response to hyperthermia of bacteria [Yatvin, 19771 and mammalian cells [Yau, 19791. We therefore suggest that enhanced membrane fluidity above 41 "C makes the cells more susceptible to US-induced inactivation.

With regard to the mechanism by which US inactivates the cells, macroscopic changes in temperature by US were ruled out under our experimental conditions. As discussed by Hahn et a1 119801, microscopic, localized heating of specific heat-sensitive targets, is also ruled out. By modifying the puhe duration of 1-MHz US, they showed that the temperature-dependent component of US-induced cell killing is not due to cav- itation. Therefore, by elimination, we are left with acoustic microstreaming as the mech- anism likely to be involved with the temperature-dependent US effects described in this

Cell Killing by Ultrasound vs Temperature 251

paper. The 7-fold lower sensitivity of bacteria compared with mammalian cells (Fig. 2) is similar to the ratio of the sizes of these cells. The effect of acoustic microstreaming is expected to be proportional to the size of the cell. Therefore, our results are consistant with the hypothesis that acoustic microstreaming is the major mechanism responsible for temperature-dependent US-induced cell killing under our experimental conditions.

REFERENCES Ben-Hur E, Prager A, Green M, Rosenthal I (1980): pH dependence of the phototoxic and photomutagenic

Chapman IV, MacNally NA, Tucker S (1979): Ultrasound-induced changes in rates of influx and efflux of

Dewey WC, Hopwood LE, Sapareto SA, Gerweck LE (1977): Cellular responses to combinations of hyper-

Flynn HG (1964): “Physics of Acoustic Cavitation in Liquids.” New York: Academic Press, vol IB. Hahn GM, h GC, Marmor JB, Pounds DW (1980): Thermal and nonthermal effects of ultrasound. In Meyn

RE, Withers HR (eds): “Radiation Biology in Cancer Research.” New York: Raven Press, pp 623-636. Li GC, Hahn GM, Tolmach LJ (1977): Cellular inactivation by ultrasound. Nature 267:163-165. Liebskind D, Bases R, Mendez F, Elequin E, Koenigsberg M (1979): Sister chromatid exchanges in human

Martins BI, Raju MR, Hayes TL, Tobias CA (1977): Survival of cultured mammalian cells exposed to ultrasound.

McKee JR, Christman CL, O’Brien WD, Wang SY (1977): Effects of ultrasound on nucleic acid bases.

Puck TT, Marcus PI (1956): Action of x-rays on mammalian cells. J Exp Med 103:653-666. Repacholi MH, Woodcock JP, Newman DL, Taylor KJW (1971): Interaction of low intensity ultrasound and

Rosebord JA, Buchanan P, Norman A, Stern R (1978): Effect of ultrasonic irradiation on mammalian cells and

Wallach DFM (1978): Action of hyperthermia and ionizing radiation on plasma membranes. In Strefer C (ed):

Williams AR, Hughes DL, Nyborg WL (1970): Hemolysis near a transversely oscillating wire. Science

Yatvin MB (1977): The influence of membrane lipid composition and procaine on hyperthermic death of cells.

Yau TM (1979): Procaine-mediated modification of membranes and of the response to x-irradiation and hy-

effects of chlorpromazine. Chem Biol Interact 29223-233.

potassium ions in rat thymocytes in vitro. Ultrasound Med Biol 6:47-58.

thermia and radiation. Radiology 123:463474.

lymphocytes after exposure to diagnostic ultrasound. Science 205: 1273-1 275.

Radiat Environ Biophys 14:243-252.

Biochemistry 16:46514654.

ionizing radiation with the tumour cell surface. Phys Med Biol 16:221-227.

chromosomes in vitro. Phys Med Biol 23:324-331.

“Cancer Therapy by Hyperthermia and Radiation.” Munich: Urban and Schwarzenberg, pp 19-28.

169~871-873.

Int J Radiat Biol 32:513-521.

perthermia in mammalian cells. Radiat Res 80523-541.