Cavitational bio-effects at 1.5 MHz

Cavitational bio-effects at 1.5 MHz E. GRAHAM,

M. HEDGES,

S. LEEMAN

and P. VAUGHAN

The effects of continuous wave ultrasound on three different classes of biosystems have been investigated at a frequency of 1.5 MHz. The criteria for cavitation are given, and these are applied to experimentally observed growth retardation of plant roots, cell death and DNA degradation in bacteria and pyknosis of human lymphocytes. An attempt is being made to find common physical mechanisms for all these biological responses, and cavitation prccesses in particular are examined here. A description is given of the techniques used to monitor the presence of cavitation, and indirect evidence, drawn from pulsed field and elevated pressure experiments, is presented to show that other non-linear processes are also operative.

Introduction Ultrasound in the megahertz frequency range is being used increasingly for medical purposes. It is essential, therefore, to investigate its action on biological systems, with a view to discovering its basic biomechanisms, and to establish safety levels for medical applications. A number of mechanisms of ultrasound action could produce biological responses, but much interest has recently been focused on thermal and cavitational effects. Three systems of different biological complexity, that is, plant, bacterial and mammalian, have been investigated by us to evaluate the diverse consequences of cavitational events. Cavitation

monitoring

We have adopted the following working definition of cavitation: it is the non-linear oscillations of bubbles, induced by an acoustic field in a liquid’. By this definition it is immaterial whether the bubbles are gas or vapour filled, or whether they are preexisting or created by the field, but the restriction to non-linearity of motion excludes gentle ‘degassing’ processes from the class of cavitational phenomena, and includes both stable and transient cavitation as special cases of non-linear motion. A practical consequence of the above definition is that non-linear bubble motion may be detected by the associated emission of higher and fractional harmonics. In particular, the emission of the first subharmonic has been considered an indicator of cavitation’ and this is entirely in accord with our concepts. To establish suitable methods for monitoring cavitation and its associated effects, preliminary experiments were carried out with aerated water, under controlled ambient conditions (pressure and temperature). Subharmonic emission, sonoE. Graham, S. Leeman and P. Vaughan are at the Department of Medical Physics, Hammersmith Hospital, Du Cane Road, London W12 OHS, UK; M. Hedges is at the MRC Cyclotron Unit, at the same address. Paper received 13 December 1979. Revised 27 March 1980.

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luminescence, and oxidation of ionic to molecular iodine have been measured. Of these, subharmonic emission is potentially the most useful indication, since it accords directly with the concept of cavitation adopted here, and may be monitored even through optically opaque soft tissues. The experimental arrangement is shown schematically in Fig. 1. A cylindrical 45 ml sample holder with acoustically transparent windows is suspended in the path of the beam, which can be either focused or plane wave in travelling or standing-wave mode. The generator can supply either continuous or pulsed ultrasound over a wide range of frequencies, pulse lengths and duty ratios, and can produce intensities well in excess of those encountered in present medical applications. The work reported here is mainly with continuous, plane travelling wave ultrasound. The coupling medium is well degassed and autoclaved circulating water maintained at constant temperature (sample holder contents maintained to f 1°C) throughout the experiments. It is essential to monitor whether or not subharmonic signals are generated within the coupling medium itself, and this is checked during each experiment over the range of intensities used. On occasions, subharmonic signals of short duration are picked up from the coupling medium, and these are attributed to stray bubbles in the system, as has been demonstrated by deliberately introducing these into the field. These transient emissions are eliminated by the addition of surfactant to the water and this precaution is adopted throughout. The subharmonic signals are detected by a 2.5 cm diameter PZT4 transducer set in the side of the chamber, with its axis aligned orthogonally to the driving beam. This transducer was undamped, with its thickness-mode resonance frequency chosen at 750 kHz, in order to enhance its sensitivity for subharmonic detection. The probe output is amplified (up to 70 dB) filtered (50 kHz band-width), and displayed on an oscilloscope screen. The position of the drive transducer can be altered to provide either near-field or far-field conditions at the sample. The beam profile was investigated in

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both field modes, and it was concluded that most consistent results were obtained with the sample in the far-field. Fig. 2 shows the acoustic beam profile as measured with a small piezoelectric probe at the sample-point, which is the position of the last axial maximum of the field. Field intensities are quoted as space-averaged values, as determined by radiation pressure balance measurements over a reflection area equal to the transducer face. The measured ratio of the spatial peak intensity in the beam, at the centre of the sample holder, to the quoted average value is % 5. Sonoluminescence was detected by a photomultiplier tube placed over the sample holder, the whole apparatus being placed inside a light-tight box, and the amplified signal displayed on a chart recorder. Subharmonic output can be monitored concurrently with the sonoluminescence. Our experiments do not reveal any sonoluminescence without subharmonic activity. The sonochemical experiments were performed with dilute aqueous (0.1 M) potassium iodide solution which had been previously shaken with carbon tetrachloride, and separated. The liberated iodine forms a coloured complex with the added starch and the concentration is assayed using a spectrophotometer, measuring absorbence at 575 nm. Fig. 3 shows the correlation between sonoluminescence and iodine release, with the subharmonic threshold indicated. In gassy water, the threshold for subharmonication is s 1 .I W and sonochemical threcm-‘, and the sonoluminescence sholds can be seen to be coincident and slightly higher, at s 1.2 W cm-*. Another phenomenon has been observed which we think has been noticed by other workers and interpreted differently3. If the sample-holder was rotated through 5 - 10” about its vertical axis, subharmonic activity often started where none had occurred at that particular input power. This ‘tweaking’ movement need only be very slight, and there is no need to continuously rotate the cell. The same effect resulted from adjusting the frequency control of the signal generator slightly. Indeed, the effect seems related to perturbation of the field, rather than to rotation. In most cases, with a stationary sample, subharmonic activity commenced at the threshold value indicated in Fig. 3. Occasionally, however, this intensity threshold could be exceeded without any, or with only a relatively weak, subharmonic signal - it is under these circumstances that ‘tweaking’ could excite the subharmonic emissions, with a strength appropriate to that expected for the applied ultrasound intensity. Rotation of the sample vessel, as preferred by Hill and his

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Fig. 2 Acoustic beam profile at three positions in sample volume. Measured in open tank

co-workers3, might well reduce the spread of data points in Fig. 3, since we do not find iodine release or sonoluminescence without subharmonic activity. However, since such rotation is not possible with our experimental arrangement during the monitoring of sonoluminescence, it was considered more consistent to perform all experiments with stationary samples. Effect of insonation

of plant roots (Vicia fabal

The sonically-induced reduction of the (linear) growth rate of the main tap root of Vicia Faba (broad bean) has been investigated, and some experimental details reported4. A 1.0

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convenient experimental end-point is the growth rate reduction in the first 24 h post-sonication relative to unsonicated controls. A possible mechanism for the ultrasound damage has been postulated to be the forced pulsation of air pockets between the cells of the root5 with the resultant cytoplasmic streaming in the adjacent cells causing the biological damage. Some of the broad aspects of the observed sonic damage may be summarized; the magnitude of the effect is frequency dependent and drops off rapidly in the range 0.75 to 3 MHz4. This is evidence of a non-thermal effect, since ultrasound absorption would be expected to rise with increasing frequency. Moreover, a sharp intensity threshold for the effect exists, a circumstance also compatible with a ‘mechanical’ origin of the damage. However, this threshold has been found to occur at % 0.4 W cmm2(linear average over the first 12 mm of root tip), which is less than half the subharmonic threshold in gassy water. This is clear evidence that the origin of the damaging effect, if cavitational, is not in the coupling water.

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The threshold intensity for damage is found to be frequencyindependent, within the range of experimental error. This may be ascribed to the existence of a range of size air cavities within the root. It does not, however, explain why the magnitude of the effect is so markedly frequency-dependent above the threshold. The effect of increasing ambient pressure to 0 .15 MPa (1.5 bar) was also investigated, and little or no change was found (Fig. 4). We note that Hill3 finds that an increased cavitation threshold intensity results from increasing ambient pressure, although this result is at variance with the theoretical prediction of Neppiras and Noltingk6 and the experimental finding of Plotskii (see Weissler’) that there is a cavitation effect maximum at ‘L 0.2 MPa. However, the observed absence of a dependence of the ultrasound growth-reduction effect on the ambient pressure indicates again that its origin does not lie with cavitation in the coupling medium (degassed water). Moreover, if cavitation occurs within the bean root itself (as seems to be supported by direct observations13) then the pressure findings suggest that it is not a ‘conventional’ cavitation process - viz: acoustic generation, growth and forced pulsation of gas bubbles, where none existed before. Our experimental findings are not contraindicatory of a cavitation mechanism, but the results cannot be attributed solely to non-linear (cavitation) bubble motion; indeed, the 1.0

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growth reduction may well be due to linear (that is, precavitational) pulsations of preexisting intercellular air pockets in the bean root. Effect of insonation B/rJ

of bacteria (Escberichia

Coli

The effect of insonation of cultures of E.coli (substrain B/r) has been investigated, the two experimental end-points being the monitoring of cell survival (as indicated by the ability to replicate and form colonies) and the direct assessment of damage to cellular DNA. In this system we have the interesting circumstance that, in the intact cell, the DNA is present in a long circular chain, attached to the cell membrane only at a relatively small number of attachment sites, the integrity of which are crucial for cellular replication.

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The survival curve, Fig. 5, shows an exponential behaviour. The bacteria are seen to be fairly sensitive to ultrasound, but the existence of an intensity threshold at a magnitude close to that for subharmonic generation in the suspension medium indicates that the primary mechanism is cavitation there and not within the cells.

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A technique has recently been developed* to establish whether the DNA is damaged, by assessment of strand breaks. In this method, the amount of double-stranded DNA measured is directly proportional to the extent of DNA breakage. Results show clearly (Fig. 6) that, in contrast to ionizing radiation, ultrasound does not damage intracellular DNA at this intensity, unless exposure times are greater than s 15 min. This finding confirms the view held by many that experiments with DNA in solution, which can be degraded at very low intensities3, have little relevance to the inter-

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enzyme staining appeared rapidly (that is, within the 25 min incubation period) in a large number of cells following incubation with the substrate (Fig. 7b), and appears to be concentrated around the nucleus in the majority, as was also observed by Reynolds and Wills after ionizing radiation. This effect was also seen very clearly in the polymorphs which are always present in our lymphocyte cultures. Insonated cells precultured with cortisone showed no indication of enzyme staining until times similar to those of the control cultures. Cortisone itself had no effect on our lymphocyte cultures at the concentrations used. Thus lysosomal enzyme leakage occurs very rapidly after, or possibly even during, sonication, and inclines us to agree with the view that lysosome membrane may be damaged by the ultrasound action itself.

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pretation of ultrasound bioeffects in vivo. Also, at the intensities used, the bacteria were observed to remain intact.

Such early lysosomal enzyme leakage is not usually associated with response to other forms of cell injury, but is seen as a consequence of hyperthermia, which, in itself, damages membranes16. However, the existence of an intensity threshold for the ultrasound effect argues against a dominantly thermal mechanism, and it would be reasonable to assume, on the basis of the magnitude of the intensity threshold, that it is

We tentatively conclude from these results that shear forces set up by pulsating bubbles within the suspension medium act upon the bacteria with insufficient force to rupture the cells, but are sufficient to sever the more delicate attachment sites of the DNA to the membrane, the DNA chain itself remaining intact. Prolonged sonication of the detached DNA chains, however, leads to strand breaks (seen here as a ‘fatigue’ effect). Effects of insonation lymphocytes)

of mammalian cells (human

Human lymphocytes, as one of the cellular constituents of blood, will inevitably undergo insonation in any application of diagnostic or therapeutic ultrasound. It is therefore desirable to study the effect of ultrasound on these cells. Cultures of lymphocytes have been exposed to ultrasound and the numbers of pyknotic cells present up to 30 h postinsonation monitored. Pyknosis is a non-specific indicator of cell death, and is seen as degeneration of the nucleus followed by the eventual disruption of the entire cell”. It has been reported” that an intensity threshold for the effect exists, and is very similar to that for cavitation in gassy water. Preculturing with a lysosomal membrane stabilizer, hydrocortisone’4, for 24 h, almost completely nullifies the effect of ultrasound I1 . This may be interpreted as giving credence to the hypothesis that insonation may damage lysosomal membraneIs , with the consequent leakage of the contained enzymes and the attendant cell death visualized by pyknosis. However, it still remains unclear whether the lysosomal enzyme leakage is triggered directly by the ultrasound, or whether it occurs merely in response to some other, more generalized, damage to the cell. Further experiments using the apparatus described” were set up to investigate this, the technique used being the modified Gomori staining method as used by Reynolds and Wills12 on gammairradiated HeLa cells. In this method, insoluble lead phosphate is precipitated at the site of enzyme activity. Lysosomal enzyme staining in control cultures is not observed until at least 55 min incubation with substrate (Fig. 7a), when a small percentage of the cells will start to undergo pyknosis. However, in samples of cells taken immediately after insonation (1.5 MHz continuous wave at 1.6 W cmm2for 5 min)

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Fig. 7 a - Photomicrograph of cell from control culture. No evidence of staining is visible; b - lnsonated cells show rapid development of stain, particularly around lymphocyte nuclei (arrows), and in cytoplasm (cy)

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cavitation within the suspension medium which creates the damaging events. However, the cell membrane appears to remain unharmed since cell fragments are rarely seen after sonic&ion at these intensities. Moreover, sonication with pulsed fields (70 /AS,1: 1 pulses, 1 h exposure) at an intensity (1.7 W cm-’ space-time average) well above the subharmonic threshold for continuous wave ultrasound, results in an apparently unscathed population, with the first signs of damage developing, inexplicably, only 15 h later; this effect is also nullified by cortisone”. Such delayed effects are not generally associated with the violence of cavitational effects. Cavitation, as indicated by subharmonic emission, was not present in gassy water for pulses as short as those used in these experiments, and it is tempting to infer that these results are evidence of ‘direct’ (that is, not mediated by bubble activity in the medium) ultrasound action on the cells. These responses would not be seen in the continuous wave case, as they would be expected to be masked by the immediate pyknosis sequence generated concurrently with such irradiations. However, if such ‘direct action’ exists for continuous waves, it must have an intensity threshold above that for cavitation in aerated water, otherwise it would have been evident in experiments conducted below that level. This important system clearly merits further investigation, but it may be concluded that cavitation plays some role in the damage of the lysosomal membranes. Whether a component of the damage arises from direct ultrasound action within the cell, and the reason for the early involvement of

the lysosomal membranes, are questions that must, unfortunately, remain unanswered at present.

References 1

Hedges, M.J., Leeman, S., Vaughan, P., Acoustic Cavitation,

Proc. Underwater Acoustics Group, Institute of Acoustics, No. 5 (1977) 19 Flynn, H.G., Physical Acoustics, ed. W.P. Mason (1964), vol 16, chapter 9, 57 Hill, C.R., J. Acoust. Sot. Am., 52 (1972), 667 Leeman, S., Khokhar, M.T., Oliver, R., Brit. J. Radio., 48, (1975) 954 Nyborg, W.L., Miller, D.L., Gershoy, A., Proc. 7th Rochester

Int. Conf. on Environmental Toxicity (1975) (Plenum Press) 271 Neppiras, E.A., Noltingk, B.E.,Proc Phys. Sot., B64 (1951)

9 10 11 12 13 14

1032 Weissler, A.,J. Acoust. Sot. Am. 25 (1953) 651 Ahnstrom, G., Edvardson, K.A., ht. J. Radiat. Biol., 26 (1974) 493 Hedges, M J., Lewis, M., Lunec, J., Cramp, WA., ht. J. Radial Biol. 37 (1980) 103 Trowel& O&J. Path. Bact. 64 (1952) 687 Hedges,MJ., Leeman, S.,Int. J. Radiat. Biol., 35 (1979) 301 Reynolds, C., Wills, CD.,lnt. J. Radiat, Biol., 25 (1974) 113 Lehmann, J.F., Herrick, J.F., Krusen, F.H., Arch. Phys. Med. & Rekab., 35 (1954) 141 Slater, T.F., Experimental Study of the Effects of Drugs on

the Liver. I.C.S. No. 115 (1966).__30. (Amsterdam : Excerpta . Medica) ’ 15 16

Dyson, M., Pond, J.B., Woodward, B., Broadbent, J., Ultrasound&fed. Biol., l(l974) 133 Hume, S.P., Rogers, M.A., Field, SB.,Znt. J. Radiat. Biol., 34 (1978) 401

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