- Email: [email protected]
Biophysical effects of high-energy pulsed ultrasound on human cells
Ultrasound
in Med.
& Biol.,
Vol.
22, No. 9, pp. 1267-1275,
1996
Copyright 0 1996 World Federationfor Ultrasound in Medicine & Biology Printed
ELSEVIER
in the USA. All 0301.5629/96
rights reserved $15.00 + .OO
PII: SO301-5629( 96)00151-2
l Original Contribution BIOPHYSICAL
EFFECTS
OF HIGH-ENERGY ON HUMAN CELLS
PULSED
ULTRASOUND
T. FEIGL, B. VOLKLEIN, H. IRO, C. ELL and T. SCHNEIDER Departments of ENT and Medicine I, University of Erlangen-Nuremberg, Erlangen, Germany (Received
28 March
1996; injnal
form
23 July
1996)
Abstract-Human benign and malignant cellsof diierent human origin (pancreas, liver, kidney, pharynx, tongue, lip) were exposed to high-energy pulsed ultrasound (HEPUS) in vitro to evaluate the effects of various physical parameters and sonicationconditions on cell viability. This included the number of pulses, focal pressure, pulse repetition rate, pulse shape,cell suspensionvolume, water level of the basin and cell density. Cell viability was found to depend significantly on the number of pulses(exponential), the focal pressure(Linear) and the pulse repetition rate (minimum at 1 Hz). Other parameters showedno marked influence. Furthermore, electron microscopy revealed intracellular damage,and proliferation rates of cells surviving sonication were normal after HEPUS exposure. The experimental piezoelectric ultrasound transducer usedin the experiments generatedoscillating bipolar pulseswith high negative pressureamplitudes. Measurements were made of the pulse shape and ultrasonic field of the experimental device and of a conventional lithotripter for comparison. Copyright 0 1996 World Federation for Ultrasound in Medicine &
Biology. Key Words: High-energy pulsedultrasound, Biological effects, Cell viability, Cell destruction, Cell proliferation, Human carcinoma cells, Ultrasound treatment, Cancer therapy, Cavitation, Focus, Focal pressure, Pulseshape,Lithotripsy.
undesired tissue damage (Coleman and Saunders 1987; Riedlinger et al. 1989). The present study is based on the principles of piezoelectric ultrasound pulse generation. It was devised to investigate the influence of high-energy pulsed ultrasound (HEPUS) sonication parameters on six different human cell lines in vitro. Two of the cell lines used were benign and four were malignant. The dependencies of cell viability rates on various parameters and experimental set-up conditions of HEPUS exposure were investigated to ascertain the mechanisms of cell destruction. To evaluate intracellular damage after HEPUS, electron microscopy was carried out and cell proliferation rates were analysed. The ultrasonic fields of a HEPUS generator and a lithotripter were measured to determine the influence of pulse shape and maximum pressure on cell viability rates.
INTRODUCTION When implementing extracorporeal shock wave lithotripsy to treat kidney, gall bladder and salivary disorders noninvasively, the acoustic shock waves used can cause hemorrhage or other trauma to surrounding tissue, especially if shock wave energy is misapplied (Fischer et al. 1988; Lingeman et al. 1988). During recent years, numerous investigations with conventional and experimental lithotripters have shown that the growth and viability of tumor cells in vitro and in vivo can be affected by sonication with high-energy ultrasound shock waves (Briimrner et al. 1989, 1992; Debus et al. 1991; Feigl et al. 1992; Riedlinger et al. 1989; Russo et al. 1986; Smits et al. 1991). It has already been determined by clinically established lithotripsy that it is the negative pressure component of a shock wave that is the primary factor causing
MATERIALS Address correspondence to: Dr. rer. nat. Thomas Feigl, Klinik mit Poliklinik ftir Hals-Nasen-Ohrenkranke der Friedrich-AlexanderUniversitit Erlangen-Ntimberg. Waldstrape 1, D-91054 Erlangen, Germany.
AND METHODS
Pulse generation and HEPUS sonication An experimental piezoceramic (3300 elements, PZT-4) burst signal transducer was used for the ultra1267
1268 Focal zone (-6dB -line) b
Ultrasound in Medicine and Biology Water bath
z-axis I
/
A Laser II 1
Volume 22, Number 9, 1996
Germany), a polyvinylidene difluoride (PVDF) coaxial hydrophone (Dept. of Acoustics IHE, University of Karlsruhe, Germany) and a Marconi membrane hydrophone (Marconi Coplanar IPO35, GEC-Marconi, Chehnsford, UK). Individual high-energy pulsesdemonstrateda high degreeof reproducibility. Further pressure measurementswere carried out using small plastic tubes made of polyethylene, polypropylene and polystyrene (all obtained from NUNC, Germany) placed in the focal zone of the transducer. The polyethylene vesselswere best suited for the HEPUS experiments becausethey caused the least reduction in pressure (approximately 7%) and did not alter the shapeof the ultrasound pulses
;a b 2 3
Fig. 1. Diagramof the GMW2 piezoelectrictest generator. The sampletubescontainingthe cell suspension were positioned into the acoustic focus by meansof the two laser beamsand then exposedto HEPUS.
sound pulse generation. The transducer is bowl shaped and self-focusing ( 0 = 362 mm, f = 200 mm, C = 230 nF; GMW2, Fig. 1). A conventional shock wave lithotripter (Piezolith 2300: 4000 elements,bowl shaped and self-focusing ( @ = 400 mm, f = 400 mm; R. Wolf, Germany) wasusedto comparethe pulseform and evaluate the role of the maximum positive pressure. The shape of the pressurevs. time profile of the GMW2 is characterized by multiple exponentially decreasing bipolar oscillations and high negative pressure amplitudes to produce transient cavitation in the focal area‘. The major differences of HEPUS and conventional shock waves in the pulse shapeare shown in Fig. 2. The sonication of cell suspensiontubes was performed using partially degassedwater (<2.3 mg 02/ L, T = 37 2 1°C) as the coupling medium and by positioning the specimen at the acoustic focus of the transducer by means of two lasers (GMW2) and two diagnostic ultrasound-B-scanners integrated in the transducer (PL2300). The base of the tubes was located 14 mm below the focus and the surface of the coupling medium 30 mm above the focus. Before the cell experiments were carried out, extensive in vitro pressuremeasurementswere conductedwith a needleprobe hydrophone (hnotec 300/005/04; hnotec, ‘The term “transient cavitation” refers to the formation and violent collapse of small gas- and vapour-containing bubbles in ultrasonic fields, inducing extreme physical conditions (Suslick 1988), including secondary shock waves, temperatures of up to several thousand degrees and pressures of up to several thousand bar.
e n
3c
a
lime &s]
z E, f! a 8 t
150
100
50
0
-50
0
1
2
3
4
5
Time [ps]
Fig. 2. (Top) Pressurevs. time profile (MPa vs. /IS) of an ultrasound
pulse generated by the GMW2
at maximum
volt-
ageappliedfor the cell experiments.P+,,,.~= 30 MPa. (Bottom) Pressure vs. time profile (MPa vs. ps) of a shockwave generated
by the PL2300
at maximum 151 MPa.
intensity. p+,,,= =
Biophysical effects of high-energy 0 T. FEEL et al.
asthey passedthrough the material. The size of the polyethylene sampletubeswasreducedto more closely match the geometry of the focal zone within the spatial limits imposed by our experimental set-up. Several pressure vs. time profiles (produced by different electical controls) were tested to assesstheir efficiency in producing transient cavitation. Cell material and preparation Table 1 lists the cell lines (all except FIBRO obtained from the American Type Culture Collection, Rockville, MD, USA), their names, ATCC numbers and media used. The media of each cell line were enriched with fetal calf serum (FCS) , 1% penicillin-streptomycin, 1% L-glutamine and 1% sodium pyruvate (all obtained from Gibco/BRL, Germany). Cells were cultivated in tissue culture flasks (Greiner, Germany), grown as a monolayer in an incubator under standard conditions (relative air humidity, 97%; atmospheric CO7 content, 8%; pH 7.4; 37°C). The cells were rinsed twice with physiological saline and suspendedin phosphate-buffered saline solution containing low-dose trypsin (0.25%) and 0.1% EDTA. They were subsequently transferred to polyethylene tubes, which were shortened to 44 X 11 mm lengths (2-mL volume, l-mm wall thickness) and sealed with lids. Evaluation parameters The fraction of living cells present was determined before and after exposure of each tumour/norma1cell suspensionto ultrasound by trypan-blue dye exclusion (Lindl and Bauer 1989). At least three samples were examined per fixed parameter setting. Intact cells were counted in a haemocytometer in quadruplicate. Identically prepared control cell samples were positioned at the edge of the water bath outside the ultrasonic field and subsequently handled by the same procedure as the treated cells. Depending on the cell line, assayable enzymes were found in the supernatant of the fluid after centri-
1269
fuging suspendedcells. The assayableenzymes found were lactate dehydrogenase (LDH) , aspartate aminotransferase ( AST) , alanine aminotransferase ( ALT) , y-glutamyltransferase ( y-GT) , cr-amylase, lipase and total serum protein. A routine check was made for all enzymes. The enzyme levels measured in individual sampleswere normalized to enzyme values of corresponding cell suspensionssubjected to sonication by an ultrasonic oscillator (Branson, Danbury, CT, USA), ensuring complete cell destruction. Transmission electron microscopy was performed on representative samples of the cell lines to detect ultrastructural changes in the sonicated cells. Standard parameter settings (SP) Varying one single parameter of sonication, all other parameters were fixed as follows: Number of pulsesapplied n = 400 pulses; focal pressurep = pmax; pulse repetition frequency f = 1 Hz: cell density pcell = l-3 lo6 mL PI; suspensionvolume vSusp= V,,, = 2 mL. Variable parameters of sonication and variable set-up conditions On the basis of the standard values (SP) given above, individual parameters of ultrasound application varied within the following ranges for all cell lines: Number of pulsesapplied n = 50-2000; focal pressure ( 1. positive peak) p+ (MPa) = 10-30; pulse repetition frequency f(Hz) = 0.6-S; cell density p celllo6 rnI-’ = 0.5-6. Parameters were tested only for the PANC cell line as follows: Water level (mm above focus) = 2050; cell suspensionvolume VsUsp (mL) in the test tube to exclude artificial reflection effects at the air-water interface = 0.5-3.5. Cell viabilty rates were measuredwith and without residual air in the test tube to determine the level of artificially induced cavitation. The dependence of cell viability on pulse repetition rate acrossthe entire range was also evaluated for a filling volume of 1 mL instead of 2 mL (completely
Table 1. Cell lines. Human cell line
Name
Pancreatic carcinoma Liver carcinoma Normal liver cells Kidney carcinoma Pharynx carcinoma Tongue carcinoma Normal lip fibroblasts
PANC- 1 SK-HEP- 1 Chang Liver Caki-1 FaDu XC-4 FIBRO
ATCC # CRL HTB CCL HTB HTB CRL
1469 52 13 46 43 1624
(Biopsy)
Medium
FCS
RPM1 DMEM Eagle’s BM McCoy’s 5A EMEM Ham’s F12/DMEM DMEM
10% 10% 10% 10% 10% 20% 10%
1270
Ultrasound in Medicine and Biology
a40 b er 3 bt 30
I
I
1
20
10
0
0
L
/’
l-
-I
1
2 Voltage
3
4
Wl
Fig. 3. Linear (r = 0.998) pressure vs. voltage dependence in the focus of the GMW2. The maximum pressure applied in the experiments was 30 MPa at 3.2 kV transducer voltage.
filled) and a focal pressure of 22.5 MPa instead of 30 MPa (75% of pm=). This was done to rule out the possibility that induced streaming in the test tube contributed artificially to cell damage. The total electric input energy was increased from 200-560 J via the number of pulses (a factor of 2.8) and via the applied voltage (a factor of 1.7) to prove the impact of changing these parameters. A comparison of cell viability rates after sonication under standard conditions was carried out with the GMW2 and the PL2300. Cell proliferation rates after HEPUS sonication In order to investigate any potential antiproliferative effect of HEPUS, the cell proliferation rates after HEPUS sonication were determined for all cells lines under two conditions ( = 1% and = 30% surviving cells, respectively). The sonication was performed either applying 100 pulses, reducing the cell population to z 30%, and applying 1000/1500 pulses, leaving about 1% viable cells. These cells were recultivated in tissue culture flasks as described above. The fraction of vital cells was evaluated by trypan-blue dye exclusion test every 24 h over a total period of 240 h after HEPUS treatment.
Volume 22, Number 9, 1996
Cavitation effects stop the bipolar oscillations immediately after the first positive pressure peak. The maximum positive pressure ( 1. peak) used for the sonication of cells was 30 MPa. The maximum pressure of the PL2300 at highest intensity setting was 15 1 MPa (Fig. 2b). The dependence of the pressure in the focus of the GMW2 on the voltage was shown to be exactly linear (r = 0.998). This is important for later discussion of cell destruction mechanisms (Fig. 3). The spatial pressure profile yielded a focus area of 11 X 3 X 3 mm (z,y,x; 50% isobar = -6 dB line; Fig. 4) when the maximum transducer voltage was applied. The -6 dB line for the PL2300 was 17 X 3 x3mm. Cell viability rates The dependency of the cell viability rates on the following parameters and set-up conditions is referred to the sham-treated control samples at settings for standard parameters (SP) . With increasing number of pulses, the cell survival rates were observed to decrease exponentially, reaching < 1% (or even < 1%0) after 800 pulses (PANC), 1000 pulses (Caki), 600 pulses (SK-HEP), 1600 pulses (Chang) and 2000 pulses (FIBRO and FaDu) (Fig. 5). With increasing focal peak pressure, the decrease in the number of cells surviving HEPUS application was found to be linear (due to the pressure vs. voltage dependence) (Fig. 6). T a !P 1
100
8 : E g! 3 t P
75
50
25
0 -15
RESULTS Measurements of the ultrasonic field Figure 2a shows the pressure vs. time profile in the focus of the GMW2 at the maximum voltage.
-10
-5 Die
0 from focus
5
10
15
[mm]
Fig. 4. Spatial pressure profile of the focal area of the GMW2. Pressure (%p,=) vs. focal distance (mm; minus = toward transducer) : -6 dB line (z,y,x): 11 X 3 X 3 mm.
Biophysical effects of high-energy0 T. FEEL et a/.
-A--b
SKHEP Chang
r = 0.99 r = 0.97
-+
FIBRO
r = 0.99
1271
20
0 0
500
1000 Number
1500
2000 0
of pulses
0
Fig. 5. Cell viability (% of control) dependent on the number of pulses applied (mean; n = 3). Correlation coefficients (exponential fit): r = 0.95-0.99. Standard settings: f= 1 Hz;p =P,,,~; pee,, = 2 X lo6 cells II-II-‘.
By varying the pulse repetition frequency in repeated mesurement cycles, maximum cell destruction was shown to occur at a frequency around 1 Hz (Fig. 7). Changing the repetition rate to higher or lower frequencies resulted in an increase in viability for all cell lines.
-A- SK-HEP -A.-Chang
2
6 Pulse
repetitltn
frequency
Fiir]
Fig. 7. Cell viability (% of control) dependenton the pulse repetition rate (mean; IZ = 3) . Standardsettings:p = prnax ; no. of pulses= 400; pcell= 2 X IO6 cellsml.-‘.
No correlation was detected between cell destruction rates and the cell density of cells in the suspension in the range examined (Fig. 8, exemplary PANC cells, mean t SD: 6.9% -+ 4.6%, r = 0.1). The water level above the focus/tubes did not influence the cell count results.
r = 0.01 r=O.99
0 15
25
20 Focal
pressure
30
[Ml%]
Fig. 6. Cell viability (% of control) dependenton the focal peakpressure(MPa; mean;n = 3). Correlationcoefficients (linear fit): r = 0.87-0.99. Standardsettings:f = 1 Hz; no. of pulses= 400; pcell= 2 X lo6 cells mL- ’
0
1
2 Cell density
4
3
5
6
[miolml]
Fig. 8. Cell viability (% of control) dependenton cell density pcell(millions per millilitre; mean;n = 3, exemplaryPANC cells). Correlation coefficient: r = 0.1. Standardsettings: .f= 1 Hz;p = ~max;no. of pulses= 400.
Ultrasound in Medicine and Biology
Volume 22, Number 9, 1996
60
60
: 0
1
2
3
Pulse
repetition
4
5 frequency
6
7
6
[Hz]
Fig. 9. Cell viability (% of control) dependent on the pulse repetition rate for a suspension volume of 1 mL and focal pressure of 22.5 MPa (exemplary PANC cells, mean; n = 3). Standard settings: p = p,,; no. of pulses = 400; pcell= 2 X lo6 cells mI-‘.
The cell suspension volume in the test tube had no significant influence on cell destruction rates, except with a completely filled tube, which reached maximum cell destruction. No difference in viability rates was observed for tubes with and without residual air. A reduction of the suspension volume or of the focal peak pressure showed qualitatively the same dependence on frequency as the result indicated above, i.e., cell viability was observed to be at a minimum at a frequency of 1 Hz (Fig. 9). Increasing the total electric input energy from 200 J (43% surviving cells after HEPUS) to 560 J via the number of pulses and the applied voltage, respectively, led to the same percentage of surviving PANC cells (3.4% and 2.1%), i.e., a rise in the number of pulses (a factor of 2.8) is less efficient than a rise in the voltage (a factor of 1.7). A comparison between cell sonication under standard conditions by the GMW2 and the conventional lithotripter PL2300 revealed 2.2% and 8.1% surviving PANC cells, respectively. Although the viability rates are of the same order, nearly four times more cells managed to survive the treatment using the PL2300. The SCC-4 cells could not be counted because of clotting, but the enzyme levels obtained agreed with the enzyme levels and the cell count results of all other cell lines.
0
1
2
3
Pulse repetition
4
5
6
frequency
[Hz]
7
6
Fig. 10. Lactate dehydrogenase (% of max. LDH) levels in the supematant of cell suspensions after HEPUS dependent on the pulse repetition rate (exemplary PANC cells, mean; n = 3). Standard settings: p = pm=; no. of pulses = 400; pcell r 2 X lo6 cells mL-‘.
Enzyme levels Measurements of the different enzymes in the supernatant fluid confirmed both qualitatively and quantitatively all data obtained by cell counting (Fig. 10, exemplary PANC cells, LDH enzyme dependent on pulse repetition frequency: maximum LDH level at 1 Hz). Transmission electron microscopy Transmission electron microscopy of the sonicated cells revealed changes in cellular structure, such
Fig. 11. Transmission electron micrograph of sonicated cells (exemplary FaDu cells after 400 HEPUS pulses) revealed changes in the cellular structure, such as vacuoles (V.), localized ruptures and complete cell fragmentation (arrow).
Biophysical
effects
of high-energy
0 T. FEIGL et al.
FIBRO
Hep after HEPUS
I
PANC
1273
n
control 100 pulses 1000 pulses
Fig. 12. Cell proliferation rates after HEPUS exposure. Percentage of the initial number of cells surviving sonication vs. time (h) for each cell line (control samples, and after 100 and 1000/1500 pulses).
as vacuoles, localized ruptures and complete cell fragmentation, depending on the number of pulses applied (exemplary FaDu cells after 400 pulses, Fig. 11). Cell proliferation rates after HEPUS exposure When determining the cell proliferation rates after sonication, a reduction in growth was found during the first 48 h after HEPUS expoure, but the rates returned to normal during the following days. Over the total time period, a normal, i.e., exponential cell regrowth was observed with all cell lines under both conditions ( 1% and 30% surviving cells after HEPUS treatment) except for the FaDu cells, in which no proliferation was detected over the total time period (Fig. 12).
DISCUSSION By increasing the number of pulses, a near-total destruction of human normal and carcinoma cells in
vitro was achieved. The exponential decrease in viability can be explained by the fact that the focus volume is smaller than the volume of the exposure vessel. As a result, only the cells that are currently at the focus are destroyed with each single ultrasound pulse. In contrast to the benign cells, the malignant cells proved to be slightly more sensitive (but not significantly) to HEPUS exposure. The FaDu cells were an exception, which might be explained by the fact that they are squamous cell carcinoma cells, i.e., the cell membrane is more resistant. Cell death seems to depend directly on the absolute-positive and/or negative-pressure (at a constant pulse shape; the pressure output was proportional to the voltage applied; Fig. 3) as opposed to stone disintegration with conventional shock waves, where the total energy is the important parameter and fragmentation efficiency does not correlate with pressure (Granz and Kijhler 1992). Because the pressure output
1274
Ultrasound in Medicine and Biology
of the PL2300 is five times higher than that of the GMW2 (and hereby the energy 25 times higher), the absolute positive peak pressure is not the primary cause of cell death, as shown in the comparison of the two generators GMW2 and PL2300. Cavitation effects increase the viability rates at pulse repetition frequencies > 1 Hz. This can be explained by the fact that the energy transported by a subsequent pulse is shielded and dissipated by the cavitation cloud produced outside the tube by the preceding pulse. At frequencies < 1 Hz, the lifetime of the bubbles is shorter than the interval before the next pulse. This forces the implosion of the bubbles, which is necessary for cell destruction inside the test tube. Previous measurements of the lifetime of cavitation bubbles and the size of cavitation clouds generated by the GMW2 have shown that such effects must be taken into account (Riedlinger et al. 1989). This phenomenon together with the explanation could also be observed in animal experiments in the in viva situation. Pulse frequencies around 1 Hz were best suited for tissue damage on the surface or directly under the skin as opposed to deeper tissue layers (proved up to 13 cm), where pulse repetition frequencies up to 100 Hz showed the best results with respect to destruction efficiency (unpublished data). We can exclude the possibility of cell death being caused by collisions between cells or collisions between cells and the tube wall. Although the number of collisions increases with increasing cell concentration, the cell survival rate was shown to be independent of cell density. When counting the cells, no morphologically intact but dead (i.e., trypan-positive) cells could be detected. Therefore, we conclude that the main reason for cell death is rupture of the cell membrane. This assumption is strengthened by the finding of high levels of intracellular enzymes (e.g., LDH) in the supernatant fluid after HEPUS. Exclusion of other artijicial injuences other parameters and sonication conditions, such as reflections at the water-au interface, residual air in the test tube or streaming in the test tube, can be excluded as artificial contributions to cell destruction (see Results). None of these parameters influenced the results. Transmission electron microscopy of the sonicated cells revealed that intracellular damage and ultrastructural changes had occurred. However, the proliferation of surviving cells showed that the intracellular damage induced by HEPUS (and detected by transmission electron microscopy) might be reversible, and 100% cell destruction is necessary to permanently stop
Volume 22, Number 9, 1996
cell growth. HEPUS does not provide a long-term inhibiting effect on tumour cell proliferation as long as some cells survive sonication. Other authors have reported similar results when applying shock waves to cells in vitro, leading initially to reduced proliferation followed by normal growth (Gambihler et al. 1990; Jones et al. 1992; Nicolai et al. 1994). SUMMARY The survival rates of malignant and nonmalignant human cells showed a significant dependence on sonication parameters of HEPUS (number of pulses, repetition frequency, focal pressure, pulse shape). The positive peak pressure as such does not provide efficiency for cell destruction in vitro as opposed to pulse shapes with negative phases and much lower positive pressures. A cavitation-induced rupture of the cell membrane has been identified as the most likely reason for cell death. Acknowledgement-The authors would like to thank Dr.-Ing. R. Riedlinger, Department of Acoustics IHE, University of Karlsruhe, Germany, for providing generous accessto the GMW2 for the experiments.
REFERENCES Brtlmmer F, Bremrer J, Brauner T, Htilser DF. Effect of shock waves on suspended and immobilized L1210 cells. Ultrasound Med Biol 1989; 15:229-239. Briimmer F, Suhr D, Htilser D. Sensitivity of normal and malignant cells to shock waves. J Stone Dis 1992;4:243-248. Coleman AJ, Saunders JE. Comparison of extracorporeal shockwave lithotripters. In: Coptcoat MJ, Miller RA, Wickham JEA, eds. Lithotripsy II. London: BDI Publishing, 1987: 121- 13 1. Debus J, Peschke P, Hahn EW, Lorenz WJ, Lorenz A. Treatment of the dunning prostate rat tumor R3327-AT1 with pulsed high energy shock waves (PHEUS): Growth delay and histomorphologicchanges. J Urol 1991; 146:1143-1146.Feial T. Schneider HT. Riedlinner R. Lohr M. Hahn EG. Beschalrung von humaneh Pankr~astumorzelleh mit hochenergetischem gepulstem Ultraschall. Min Inv Med MedTech 1992; 3:139-143. Fischer N, Muller HM, Gulban A, Sohn M, Deutz F-J. Cavitation effects: Possible cause of tissue injury during extracorporeal shock wave lithotripsy. J Endourol 1988;2:215-220. Gambihler S, Delius M, Brendel W. Biological effects of shock waves: Cell disruption, viability, and proliferation of L1210 cells exposed to shock waves in vitro. Ultrasound Med Biol 1990; 16587-594. Gram B, Kohler G. What makes a shock wave efficient in lithotriODSV?J Stone Dis 1992;4:123-128. Jones BJ, McHale AP, Butler MR. Effect of high-energy shock wave frequency on viability of malignant cell lines in vitro. Eur Urol 1992;22:70-73. Lindl T, Bauer J. Zell- und Gewebekultur. Stuttgart/New York: Gustav Fischer Verlag, [2. Auflage], 1989:169. Lingeman JE, McAteer JA, Kempson SA, Evan AP. Bioeffects of extracorporeal shock wave lithotripsy. Urol Clin North Am 1988; 15:507-514.
Biophysical effects of high-energy 0 T. FEIGL et al. Nicolai H, Steinbach P, Knuechel Clarke R, Grimm D, Roessler W. Proliferation of tumor spheroids after shock-wave treatment. J Cancer Res Clin Oncol 1994; 120~438-441. Riedlinger RE, Brttmmer F, Htllser DF. Pulsed high-power-sonication of concrements, cancer cells and rodent tumors in vivo. Ultrasound International, 89, Conference Proceedings 1989:305312. Russo P, Stephenson RA, Mies C, Huryk R, Heston WDW. High-
1275
energy shock waves suppress tumor growth in vitro and in viva. J Urol 1986; 135626-628. Smits GAHJ, Oosterhof GON, de Ruyter AE, Schalken JA, Debru yne FMJ. Cytotoxic effects of high-energy shock waves in different in vitro models: Influence of the experimental set-up. J Urol 1991;145:171-175. Suslick KS. Ultrasound. 1stchemical, physical and biological effects. New York: VCH Publishers, Inc., 1988:130.
in Med.
& Biol.,
Vol.
22, No. 9, pp. 1267-1275,
1996
Copyright 0 1996 World Federationfor Ultrasound in Medicine & Biology Printed
ELSEVIER
in the USA. All 0301.5629/96
rights reserved $15.00 + .OO
PII: SO301-5629( 96)00151-2
l Original Contribution BIOPHYSICAL
EFFECTS
OF HIGH-ENERGY ON HUMAN CELLS
PULSED
ULTRASOUND
T. FEIGL, B. VOLKLEIN, H. IRO, C. ELL and T. SCHNEIDER Departments of ENT and Medicine I, University of Erlangen-Nuremberg, Erlangen, Germany (Received
28 March
1996; injnal
form
23 July
1996)
Abstract-Human benign and malignant cellsof diierent human origin (pancreas, liver, kidney, pharynx, tongue, lip) were exposed to high-energy pulsed ultrasound (HEPUS) in vitro to evaluate the effects of various physical parameters and sonicationconditions on cell viability. This included the number of pulses, focal pressure, pulse repetition rate, pulse shape,cell suspensionvolume, water level of the basin and cell density. Cell viability was found to depend significantly on the number of pulses(exponential), the focal pressure(Linear) and the pulse repetition rate (minimum at 1 Hz). Other parameters showedno marked influence. Furthermore, electron microscopy revealed intracellular damage,and proliferation rates of cells surviving sonication were normal after HEPUS exposure. The experimental piezoelectric ultrasound transducer usedin the experiments generatedoscillating bipolar pulseswith high negative pressureamplitudes. Measurements were made of the pulse shape and ultrasonic field of the experimental device and of a conventional lithotripter for comparison. Copyright 0 1996 World Federation for Ultrasound in Medicine &
Biology. Key Words: High-energy pulsedultrasound, Biological effects, Cell viability, Cell destruction, Cell proliferation, Human carcinoma cells, Ultrasound treatment, Cancer therapy, Cavitation, Focus, Focal pressure, Pulseshape,Lithotripsy.
undesired tissue damage (Coleman and Saunders 1987; Riedlinger et al. 1989). The present study is based on the principles of piezoelectric ultrasound pulse generation. It was devised to investigate the influence of high-energy pulsed ultrasound (HEPUS) sonication parameters on six different human cell lines in vitro. Two of the cell lines used were benign and four were malignant. The dependencies of cell viability rates on various parameters and experimental set-up conditions of HEPUS exposure were investigated to ascertain the mechanisms of cell destruction. To evaluate intracellular damage after HEPUS, electron microscopy was carried out and cell proliferation rates were analysed. The ultrasonic fields of a HEPUS generator and a lithotripter were measured to determine the influence of pulse shape and maximum pressure on cell viability rates.
INTRODUCTION When implementing extracorporeal shock wave lithotripsy to treat kidney, gall bladder and salivary disorders noninvasively, the acoustic shock waves used can cause hemorrhage or other trauma to surrounding tissue, especially if shock wave energy is misapplied (Fischer et al. 1988; Lingeman et al. 1988). During recent years, numerous investigations with conventional and experimental lithotripters have shown that the growth and viability of tumor cells in vitro and in vivo can be affected by sonication with high-energy ultrasound shock waves (Briimrner et al. 1989, 1992; Debus et al. 1991; Feigl et al. 1992; Riedlinger et al. 1989; Russo et al. 1986; Smits et al. 1991). It has already been determined by clinically established lithotripsy that it is the negative pressure component of a shock wave that is the primary factor causing
MATERIALS Address correspondence to: Dr. rer. nat. Thomas Feigl, Klinik mit Poliklinik ftir Hals-Nasen-Ohrenkranke der Friedrich-AlexanderUniversitit Erlangen-Ntimberg. Waldstrape 1, D-91054 Erlangen, Germany.
AND METHODS
Pulse generation and HEPUS sonication An experimental piezoceramic (3300 elements, PZT-4) burst signal transducer was used for the ultra1267
1268 Focal zone (-6dB -line) b
Ultrasound in Medicine and Biology Water bath
z-axis I
/
A Laser II 1
Volume 22, Number 9, 1996
Germany), a polyvinylidene difluoride (PVDF) coaxial hydrophone (Dept. of Acoustics IHE, University of Karlsruhe, Germany) and a Marconi membrane hydrophone (Marconi Coplanar IPO35, GEC-Marconi, Chehnsford, UK). Individual high-energy pulsesdemonstrateda high degreeof reproducibility. Further pressure measurementswere carried out using small plastic tubes made of polyethylene, polypropylene and polystyrene (all obtained from NUNC, Germany) placed in the focal zone of the transducer. The polyethylene vesselswere best suited for the HEPUS experiments becausethey caused the least reduction in pressure (approximately 7%) and did not alter the shapeof the ultrasound pulses
;a b 2 3
Fig. 1. Diagramof the GMW2 piezoelectrictest generator. The sampletubescontainingthe cell suspension were positioned into the acoustic focus by meansof the two laser beamsand then exposedto HEPUS.
sound pulse generation. The transducer is bowl shaped and self-focusing ( 0 = 362 mm, f = 200 mm, C = 230 nF; GMW2, Fig. 1). A conventional shock wave lithotripter (Piezolith 2300: 4000 elements,bowl shaped and self-focusing ( @ = 400 mm, f = 400 mm; R. Wolf, Germany) wasusedto comparethe pulseform and evaluate the role of the maximum positive pressure. The shape of the pressurevs. time profile of the GMW2 is characterized by multiple exponentially decreasing bipolar oscillations and high negative pressure amplitudes to produce transient cavitation in the focal area‘. The major differences of HEPUS and conventional shock waves in the pulse shapeare shown in Fig. 2. The sonication of cell suspensiontubes was performed using partially degassedwater (<2.3 mg 02/ L, T = 37 2 1°C) as the coupling medium and by positioning the specimen at the acoustic focus of the transducer by means of two lasers (GMW2) and two diagnostic ultrasound-B-scanners integrated in the transducer (PL2300). The base of the tubes was located 14 mm below the focus and the surface of the coupling medium 30 mm above the focus. Before the cell experiments were carried out, extensive in vitro pressuremeasurementswere conductedwith a needleprobe hydrophone (hnotec 300/005/04; hnotec, ‘The term “transient cavitation” refers to the formation and violent collapse of small gas- and vapour-containing bubbles in ultrasonic fields, inducing extreme physical conditions (Suslick 1988), including secondary shock waves, temperatures of up to several thousand degrees and pressures of up to several thousand bar.
e n
3c
a
lime &s]
z E, f! a 8 t
150
100
50
0
-50
0
1
2
3
4
5
Time [ps]
Fig. 2. (Top) Pressurevs. time profile (MPa vs. /IS) of an ultrasound
pulse generated by the GMW2
at maximum
volt-
ageappliedfor the cell experiments.P+,,,.~= 30 MPa. (Bottom) Pressure vs. time profile (MPa vs. ps) of a shockwave generated
by the PL2300
at maximum 151 MPa.
intensity. p+,,,= =
Biophysical effects of high-energy 0 T. FEEL et al.
asthey passedthrough the material. The size of the polyethylene sampletubeswasreducedto more closely match the geometry of the focal zone within the spatial limits imposed by our experimental set-up. Several pressure vs. time profiles (produced by different electical controls) were tested to assesstheir efficiency in producing transient cavitation. Cell material and preparation Table 1 lists the cell lines (all except FIBRO obtained from the American Type Culture Collection, Rockville, MD, USA), their names, ATCC numbers and media used. The media of each cell line were enriched with fetal calf serum (FCS) , 1% penicillin-streptomycin, 1% L-glutamine and 1% sodium pyruvate (all obtained from Gibco/BRL, Germany). Cells were cultivated in tissue culture flasks (Greiner, Germany), grown as a monolayer in an incubator under standard conditions (relative air humidity, 97%; atmospheric CO7 content, 8%; pH 7.4; 37°C). The cells were rinsed twice with physiological saline and suspendedin phosphate-buffered saline solution containing low-dose trypsin (0.25%) and 0.1% EDTA. They were subsequently transferred to polyethylene tubes, which were shortened to 44 X 11 mm lengths (2-mL volume, l-mm wall thickness) and sealed with lids. Evaluation parameters The fraction of living cells present was determined before and after exposure of each tumour/norma1cell suspensionto ultrasound by trypan-blue dye exclusion (Lindl and Bauer 1989). At least three samples were examined per fixed parameter setting. Intact cells were counted in a haemocytometer in quadruplicate. Identically prepared control cell samples were positioned at the edge of the water bath outside the ultrasonic field and subsequently handled by the same procedure as the treated cells. Depending on the cell line, assayable enzymes were found in the supernatant of the fluid after centri-
1269
fuging suspendedcells. The assayableenzymes found were lactate dehydrogenase (LDH) , aspartate aminotransferase ( AST) , alanine aminotransferase ( ALT) , y-glutamyltransferase ( y-GT) , cr-amylase, lipase and total serum protein. A routine check was made for all enzymes. The enzyme levels measured in individual sampleswere normalized to enzyme values of corresponding cell suspensionssubjected to sonication by an ultrasonic oscillator (Branson, Danbury, CT, USA), ensuring complete cell destruction. Transmission electron microscopy was performed on representative samples of the cell lines to detect ultrastructural changes in the sonicated cells. Standard parameter settings (SP) Varying one single parameter of sonication, all other parameters were fixed as follows: Number of pulsesapplied n = 400 pulses; focal pressurep = pmax; pulse repetition frequency f = 1 Hz: cell density pcell = l-3 lo6 mL PI; suspensionvolume vSusp= V,,, = 2 mL. Variable parameters of sonication and variable set-up conditions On the basis of the standard values (SP) given above, individual parameters of ultrasound application varied within the following ranges for all cell lines: Number of pulsesapplied n = 50-2000; focal pressure ( 1. positive peak) p+ (MPa) = 10-30; pulse repetition frequency f(Hz) = 0.6-S; cell density p celllo6 rnI-’ = 0.5-6. Parameters were tested only for the PANC cell line as follows: Water level (mm above focus) = 2050; cell suspensionvolume VsUsp (mL) in the test tube to exclude artificial reflection effects at the air-water interface = 0.5-3.5. Cell viabilty rates were measuredwith and without residual air in the test tube to determine the level of artificially induced cavitation. The dependence of cell viability on pulse repetition rate acrossthe entire range was also evaluated for a filling volume of 1 mL instead of 2 mL (completely
Table 1. Cell lines. Human cell line
Name
Pancreatic carcinoma Liver carcinoma Normal liver cells Kidney carcinoma Pharynx carcinoma Tongue carcinoma Normal lip fibroblasts
PANC- 1 SK-HEP- 1 Chang Liver Caki-1 FaDu XC-4 FIBRO
ATCC # CRL HTB CCL HTB HTB CRL
1469 52 13 46 43 1624
(Biopsy)
Medium
FCS
RPM1 DMEM Eagle’s BM McCoy’s 5A EMEM Ham’s F12/DMEM DMEM
10% 10% 10% 10% 10% 20% 10%
1270
Ultrasound in Medicine and Biology
a40 b er 3 bt 30
I
I
1
20
10
0
0
L
/’
l-
-I
1
2 Voltage
3
4
Wl
Fig. 3. Linear (r = 0.998) pressure vs. voltage dependence in the focus of the GMW2. The maximum pressure applied in the experiments was 30 MPa at 3.2 kV transducer voltage.
filled) and a focal pressure of 22.5 MPa instead of 30 MPa (75% of pm=). This was done to rule out the possibility that induced streaming in the test tube contributed artificially to cell damage. The total electric input energy was increased from 200-560 J via the number of pulses (a factor of 2.8) and via the applied voltage (a factor of 1.7) to prove the impact of changing these parameters. A comparison of cell viability rates after sonication under standard conditions was carried out with the GMW2 and the PL2300. Cell proliferation rates after HEPUS sonication In order to investigate any potential antiproliferative effect of HEPUS, the cell proliferation rates after HEPUS sonication were determined for all cells lines under two conditions ( = 1% and = 30% surviving cells, respectively). The sonication was performed either applying 100 pulses, reducing the cell population to z 30%, and applying 1000/1500 pulses, leaving about 1% viable cells. These cells were recultivated in tissue culture flasks as described above. The fraction of vital cells was evaluated by trypan-blue dye exclusion test every 24 h over a total period of 240 h after HEPUS treatment.
Volume 22, Number 9, 1996
Cavitation effects stop the bipolar oscillations immediately after the first positive pressure peak. The maximum positive pressure ( 1. peak) used for the sonication of cells was 30 MPa. The maximum pressure of the PL2300 at highest intensity setting was 15 1 MPa (Fig. 2b). The dependence of the pressure in the focus of the GMW2 on the voltage was shown to be exactly linear (r = 0.998). This is important for later discussion of cell destruction mechanisms (Fig. 3). The spatial pressure profile yielded a focus area of 11 X 3 X 3 mm (z,y,x; 50% isobar = -6 dB line; Fig. 4) when the maximum transducer voltage was applied. The -6 dB line for the PL2300 was 17 X 3 x3mm. Cell viability rates The dependency of the cell viability rates on the following parameters and set-up conditions is referred to the sham-treated control samples at settings for standard parameters (SP) . With increasing number of pulses, the cell survival rates were observed to decrease exponentially, reaching < 1% (or even < 1%0) after 800 pulses (PANC), 1000 pulses (Caki), 600 pulses (SK-HEP), 1600 pulses (Chang) and 2000 pulses (FIBRO and FaDu) (Fig. 5). With increasing focal peak pressure, the decrease in the number of cells surviving HEPUS application was found to be linear (due to the pressure vs. voltage dependence) (Fig. 6). T a !P 1
100
8 : E g! 3 t P
75
50
25
0 -15
RESULTS Measurements of the ultrasonic field Figure 2a shows the pressure vs. time profile in the focus of the GMW2 at the maximum voltage.
-10
-5 Die
0 from focus
5
10
15
[mm]
Fig. 4. Spatial pressure profile of the focal area of the GMW2. Pressure (%p,=) vs. focal distance (mm; minus = toward transducer) : -6 dB line (z,y,x): 11 X 3 X 3 mm.
Biophysical effects of high-energy0 T. FEEL et a/.
-A--b
SKHEP Chang
r = 0.99 r = 0.97
-+
FIBRO
r = 0.99
1271
20
0 0
500
1000 Number
1500
2000 0
of pulses
0
Fig. 5. Cell viability (% of control) dependent on the number of pulses applied (mean; n = 3). Correlation coefficients (exponential fit): r = 0.95-0.99. Standard settings: f= 1 Hz;p =P,,,~; pee,, = 2 X lo6 cells II-II-‘.
By varying the pulse repetition frequency in repeated mesurement cycles, maximum cell destruction was shown to occur at a frequency around 1 Hz (Fig. 7). Changing the repetition rate to higher or lower frequencies resulted in an increase in viability for all cell lines.
-A- SK-HEP -A.-Chang
2
6 Pulse
repetitltn
frequency
Fiir]
Fig. 7. Cell viability (% of control) dependenton the pulse repetition rate (mean; IZ = 3) . Standardsettings:p = prnax ; no. of pulses= 400; pcell= 2 X IO6 cellsml.-‘.
No correlation was detected between cell destruction rates and the cell density of cells in the suspension in the range examined (Fig. 8, exemplary PANC cells, mean t SD: 6.9% -+ 4.6%, r = 0.1). The water level above the focus/tubes did not influence the cell count results.
r = 0.01 r=O.99
0 15
25
20 Focal
pressure
30
[Ml%]
Fig. 6. Cell viability (% of control) dependenton the focal peakpressure(MPa; mean;n = 3). Correlationcoefficients (linear fit): r = 0.87-0.99. Standardsettings:f = 1 Hz; no. of pulses= 400; pcell= 2 X lo6 cells mL- ’
0
1
2 Cell density
4
3
5
6
[miolml]
Fig. 8. Cell viability (% of control) dependenton cell density pcell(millions per millilitre; mean;n = 3, exemplaryPANC cells). Correlation coefficient: r = 0.1. Standardsettings: .f= 1 Hz;p = ~max;no. of pulses= 400.
Ultrasound in Medicine and Biology
Volume 22, Number 9, 1996
60
60
: 0
1
2
3
Pulse
repetition
4
5 frequency
6
7
6
[Hz]
Fig. 9. Cell viability (% of control) dependent on the pulse repetition rate for a suspension volume of 1 mL and focal pressure of 22.5 MPa (exemplary PANC cells, mean; n = 3). Standard settings: p = p,,; no. of pulses = 400; pcell= 2 X lo6 cells mI-‘.
The cell suspension volume in the test tube had no significant influence on cell destruction rates, except with a completely filled tube, which reached maximum cell destruction. No difference in viability rates was observed for tubes with and without residual air. A reduction of the suspension volume or of the focal peak pressure showed qualitatively the same dependence on frequency as the result indicated above, i.e., cell viability was observed to be at a minimum at a frequency of 1 Hz (Fig. 9). Increasing the total electric input energy from 200 J (43% surviving cells after HEPUS) to 560 J via the number of pulses and the applied voltage, respectively, led to the same percentage of surviving PANC cells (3.4% and 2.1%), i.e., a rise in the number of pulses (a factor of 2.8) is less efficient than a rise in the voltage (a factor of 1.7). A comparison between cell sonication under standard conditions by the GMW2 and the conventional lithotripter PL2300 revealed 2.2% and 8.1% surviving PANC cells, respectively. Although the viability rates are of the same order, nearly four times more cells managed to survive the treatment using the PL2300. The SCC-4 cells could not be counted because of clotting, but the enzyme levels obtained agreed with the enzyme levels and the cell count results of all other cell lines.
0
1
2
3
Pulse repetition
4
5
6
frequency
[Hz]
7
6
Fig. 10. Lactate dehydrogenase (% of max. LDH) levels in the supematant of cell suspensions after HEPUS dependent on the pulse repetition rate (exemplary PANC cells, mean; n = 3). Standard settings: p = pm=; no. of pulses = 400; pcell r 2 X lo6 cells mL-‘.
Enzyme levels Measurements of the different enzymes in the supernatant fluid confirmed both qualitatively and quantitatively all data obtained by cell counting (Fig. 10, exemplary PANC cells, LDH enzyme dependent on pulse repetition frequency: maximum LDH level at 1 Hz). Transmission electron microscopy Transmission electron microscopy of the sonicated cells revealed changes in cellular structure, such
Fig. 11. Transmission electron micrograph of sonicated cells (exemplary FaDu cells after 400 HEPUS pulses) revealed changes in the cellular structure, such as vacuoles (V.), localized ruptures and complete cell fragmentation (arrow).
Biophysical
effects
of high-energy
0 T. FEIGL et al.
FIBRO
Hep after HEPUS
I
PANC
1273
n
control 100 pulses 1000 pulses
Fig. 12. Cell proliferation rates after HEPUS exposure. Percentage of the initial number of cells surviving sonication vs. time (h) for each cell line (control samples, and after 100 and 1000/1500 pulses).
as vacuoles, localized ruptures and complete cell fragmentation, depending on the number of pulses applied (exemplary FaDu cells after 400 pulses, Fig. 11). Cell proliferation rates after HEPUS exposure When determining the cell proliferation rates after sonication, a reduction in growth was found during the first 48 h after HEPUS expoure, but the rates returned to normal during the following days. Over the total time period, a normal, i.e., exponential cell regrowth was observed with all cell lines under both conditions ( 1% and 30% surviving cells after HEPUS treatment) except for the FaDu cells, in which no proliferation was detected over the total time period (Fig. 12).
DISCUSSION By increasing the number of pulses, a near-total destruction of human normal and carcinoma cells in
vitro was achieved. The exponential decrease in viability can be explained by the fact that the focus volume is smaller than the volume of the exposure vessel. As a result, only the cells that are currently at the focus are destroyed with each single ultrasound pulse. In contrast to the benign cells, the malignant cells proved to be slightly more sensitive (but not significantly) to HEPUS exposure. The FaDu cells were an exception, which might be explained by the fact that they are squamous cell carcinoma cells, i.e., the cell membrane is more resistant. Cell death seems to depend directly on the absolute-positive and/or negative-pressure (at a constant pulse shape; the pressure output was proportional to the voltage applied; Fig. 3) as opposed to stone disintegration with conventional shock waves, where the total energy is the important parameter and fragmentation efficiency does not correlate with pressure (Granz and Kijhler 1992). Because the pressure output
1274
Ultrasound in Medicine and Biology
of the PL2300 is five times higher than that of the GMW2 (and hereby the energy 25 times higher), the absolute positive peak pressure is not the primary cause of cell death, as shown in the comparison of the two generators GMW2 and PL2300. Cavitation effects increase the viability rates at pulse repetition frequencies > 1 Hz. This can be explained by the fact that the energy transported by a subsequent pulse is shielded and dissipated by the cavitation cloud produced outside the tube by the preceding pulse. At frequencies < 1 Hz, the lifetime of the bubbles is shorter than the interval before the next pulse. This forces the implosion of the bubbles, which is necessary for cell destruction inside the test tube. Previous measurements of the lifetime of cavitation bubbles and the size of cavitation clouds generated by the GMW2 have shown that such effects must be taken into account (Riedlinger et al. 1989). This phenomenon together with the explanation could also be observed in animal experiments in the in viva situation. Pulse frequencies around 1 Hz were best suited for tissue damage on the surface or directly under the skin as opposed to deeper tissue layers (proved up to 13 cm), where pulse repetition frequencies up to 100 Hz showed the best results with respect to destruction efficiency (unpublished data). We can exclude the possibility of cell death being caused by collisions between cells or collisions between cells and the tube wall. Although the number of collisions increases with increasing cell concentration, the cell survival rate was shown to be independent of cell density. When counting the cells, no morphologically intact but dead (i.e., trypan-positive) cells could be detected. Therefore, we conclude that the main reason for cell death is rupture of the cell membrane. This assumption is strengthened by the finding of high levels of intracellular enzymes (e.g., LDH) in the supernatant fluid after HEPUS. Exclusion of other artijicial injuences other parameters and sonication conditions, such as reflections at the water-au interface, residual air in the test tube or streaming in the test tube, can be excluded as artificial contributions to cell destruction (see Results). None of these parameters influenced the results. Transmission electron microscopy of the sonicated cells revealed that intracellular damage and ultrastructural changes had occurred. However, the proliferation of surviving cells showed that the intracellular damage induced by HEPUS (and detected by transmission electron microscopy) might be reversible, and 100% cell destruction is necessary to permanently stop
Volume 22, Number 9, 1996
cell growth. HEPUS does not provide a long-term inhibiting effect on tumour cell proliferation as long as some cells survive sonication. Other authors have reported similar results when applying shock waves to cells in vitro, leading initially to reduced proliferation followed by normal growth (Gambihler et al. 1990; Jones et al. 1992; Nicolai et al. 1994). SUMMARY The survival rates of malignant and nonmalignant human cells showed a significant dependence on sonication parameters of HEPUS (number of pulses, repetition frequency, focal pressure, pulse shape). The positive peak pressure as such does not provide efficiency for cell destruction in vitro as opposed to pulse shapes with negative phases and much lower positive pressures. A cavitation-induced rupture of the cell membrane has been identified as the most likely reason for cell death. Acknowledgement-The authors would like to thank Dr.-Ing. R. Riedlinger, Department of Acoustics IHE, University of Karlsruhe, Germany, for providing generous accessto the GMW2 for the experiments.
REFERENCES Brtlmmer F, Bremrer J, Brauner T, Htilser DF. Effect of shock waves on suspended and immobilized L1210 cells. Ultrasound Med Biol 1989; 15:229-239. Briimmer F, Suhr D, Htilser D. Sensitivity of normal and malignant cells to shock waves. J Stone Dis 1992;4:243-248. Coleman AJ, Saunders JE. Comparison of extracorporeal shockwave lithotripters. In: Coptcoat MJ, Miller RA, Wickham JEA, eds. Lithotripsy II. London: BDI Publishing, 1987: 121- 13 1. Debus J, Peschke P, Hahn EW, Lorenz WJ, Lorenz A. Treatment of the dunning prostate rat tumor R3327-AT1 with pulsed high energy shock waves (PHEUS): Growth delay and histomorphologicchanges. J Urol 1991; 146:1143-1146.Feial T. Schneider HT. Riedlinner R. Lohr M. Hahn EG. Beschalrung von humaneh Pankr~astumorzelleh mit hochenergetischem gepulstem Ultraschall. Min Inv Med MedTech 1992; 3:139-143. Fischer N, Muller HM, Gulban A, Sohn M, Deutz F-J. Cavitation effects: Possible cause of tissue injury during extracorporeal shock wave lithotripsy. J Endourol 1988;2:215-220. Gambihler S, Delius M, Brendel W. Biological effects of shock waves: Cell disruption, viability, and proliferation of L1210 cells exposed to shock waves in vitro. Ultrasound Med Biol 1990; 16587-594. Gram B, Kohler G. What makes a shock wave efficient in lithotriODSV?J Stone Dis 1992;4:123-128. Jones BJ, McHale AP, Butler MR. Effect of high-energy shock wave frequency on viability of malignant cell lines in vitro. Eur Urol 1992;22:70-73. Lindl T, Bauer J. Zell- und Gewebekultur. Stuttgart/New York: Gustav Fischer Verlag, [2. Auflage], 1989:169. Lingeman JE, McAteer JA, Kempson SA, Evan AP. Bioeffects of extracorporeal shock wave lithotripsy. Urol Clin North Am 1988; 15:507-514.
Biophysical effects of high-energy 0 T. FEIGL et al. Nicolai H, Steinbach P, Knuechel Clarke R, Grimm D, Roessler W. Proliferation of tumor spheroids after shock-wave treatment. J Cancer Res Clin Oncol 1994; 120~438-441. Riedlinger RE, Brttmmer F, Htllser DF. Pulsed high-power-sonication of concrements, cancer cells and rodent tumors in vivo. Ultrasound International, 89, Conference Proceedings 1989:305312. Russo P, Stephenson RA, Mies C, Huryk R, Heston WDW. High-
1275
energy shock waves suppress tumor growth in vitro and in viva. J Urol 1986; 135626-628. Smits GAHJ, Oosterhof GON, de Ruyter AE, Schalken JA, Debru yne FMJ. Cytotoxic effects of high-energy shock waves in different in vitro models: Influence of the experimental set-up. J Urol 1991;145:171-175. Suslick KS. Ultrasound. 1stchemical, physical and biological effects. New York: VCH Publishers, Inc., 1988:130.