U/frasound in Med. & Biol. Vol. 12, No. 9, pp. 685-688, Printed in the U.S.A.
$3.00 + .oO Journals Ltd.
A. Department Bioeffect
in viva are often of parameters
sured with the required unduly
thetic media and contained within artificial exposure chambers are more liable to damage by a cavitational mechanism than similar cells in their normal environment in vivo. Despite these limitations, in vitro bioeffect observations play a key role in our understanding of the interaction of ultrasound with living matter and in the identification of effects which may subsequently be in vivo. One criticism of our current demonstrated bioeffect data base is that much of it was obtained using acoustic exposure parameters similar to those employed in ultrasonic therapy. There is therefore a need for well-designed studies employing pulsed ultrasound where the pulses are similar to those being emitted by the diagnostic equipment in current use.
can be meaand without
you are trying
sure. Consequently, the majority of the investigative studies which are reported tend to utilize suspensions of living cells or isolated organelles or even macromolecules as the biological target. The use of these in vitro systems enables the investigators not only to measure a wider range of experimental variables, but also to measure these parameters with a greater degree of precision than would be possible in viva An additional advantage of using in vitro exposure systems is that the homeostatic mechanisms which normally operate in vivo to maintain a constant cellular environment are not present and so one would expect that an in vitro biological system would be perturbed more easily and therefore should enable us to detect even the most subtle interactions with the acoustic field. However, in vitro bioeffect studies have several inherent disadvantages. The major one is that it is virtually impossible to extrapolate an in vitro bioeffect observation directly so as to predict a potential hazard in viva. Nevertheless, the identification of an effect in vitro should enable a suitable in vivo experimental system to be designed to determine whether or not that same effect could occur within the mammalian body. One of the greatest strengths on in vitro experimentation is the ease with which the physical and biological exposure conditions can be varied or manipulated so that once a bioeffect has been identified it is usually possible to obtain a reasonable indication as to the interaction mechanism which was responsible for that effect. Unfortunately in virro and in vivo exposure situations are usually so different that it is highly probable that different interaction mechanisms will dominate. For example, cells within an intact tissue are more liable to damage by a thermal mechanism than the same cells in dilute suspension in a nonabsorbing medium even though they are being exposed to the same ultrasonic beam. Conversely, cells suspended in syn-
REFERENCES Barnett S. B. and Kossoff G. (1984) Temporal peak intensity as a critical parameter in ultrasound dosimetry. J. Ultrasound in Med. 3,385-389. Bause G. S.. Niebyl J. R. and Sanders R. C. (1983) Doppler ultrasound and maternal erythrocyte fragility. Obstet. Gynecol. 62, 7. Chapman I. V. (1974) The effect of ultrasound on the potassium content of rat thymocytes in vitro. Br. J. Radio/. 47,41 1. Child S. Z.. Carstensen E. L. and Lam S. K. (198 1) Effects of ultrasound on Drosophila III. Exposure of larvae to low temperatureaverage-intensity, pulsed irradiation. Ultrasound in Med. & Biol. 7, 167. Child S. Z. and Carstensen
E. L. (1982)
Effects of ultrasound
Drosophila IV. Pulsed exposure of eggs. Ultrasound in Med. & Biol. 8,31 I. Ciaravino V.. Flynn H. G. and Miller M. W. (1981) Pulsed enhancement of acoustic cavitation: A postulated mode. Ultrasound in Med. & Biol. 7, 159. Flynn G. H. (1982) Generation of transient cavities in liquids by microsecond pulses of ultrasound. J. Acoust. Sot. .4m. 72, 1926. Love L. A. and Kremkau F. W. (1980) Intracellular temperature distribution produced by ultrasound. J. .4coust. Sot. Am. 67, 1045. Miller D. L., Nyborg W. L. and Whitcomb C. C. (1979) Platelet aggregation induced by ultrasound under specialised conditions
in vitro. Science 205, 505. Nyborg W. L. (1982) Ultrasonic microstreaming and related phenomena. Br. J. Cancer 45, Suppl. V, 156. Sanada M.. Hattori A.. Watanabe T., et al. (1977) The in vivo effect of ultrasound upon human blood platelets. Nikon Choompa Igakukai, Keon rombunshu Nov: 149- 150. Williams A. R. and Miller D. L. (1980) Photometric detection of ATP release from human erythrocytes exposed to ultrasonically activated gas-filled pores. l_Qrasound in Med. & Biol. 6,25 l-256. 685
September 1986, Volume 12. Number 9
Ultrasound in Medicine and Biology
SUMMARY Whilst heating is an important mechanism for irz viw irradiation it can be eliminated in the nonabsorbing medium of in vitro cell suspension. (Love & Kremkau, 1980). Also the use of short pulses and the absence of plane strong reflectors minimise standing wave effects. Even when present their damaging effects are apparently mediated by cavitational activity. In vifrv mechanisms of interaction which have been clearly identified involve gas bubble activation, where radiation pressure forces attract cells to oscillating bubbles (Nyborg. 1982) and acoustic microstreaming fields disrupt cells. The only in vitro studies showing repeatable effects from diagnostic pulsed conditions employed a cavitation phenomenon as in the formation of platelet aggregation (Miller et al., 1979; Barnett and Kossoff, 1984) and killing of Drosophilu larvae (Child and Carstensen, 198 1) and eggs (Child and Carstensen. 1982). Unconfirmed effects at diagnostic intensities have been reported on the cell surface (reduced attachment, abnormal surface activity, immunological changes. electrophonetic activity). on the nucleus (sister chromatid exchanges) or on cell locomotion. Examples of implausible reports of bioeffects from diagnostic ultrasound were given as: platelet damage from in rlivo insonation (Sanada. 1977); and erythrocyte osmolarity changes, from ultrasound during labour (Bause et al., 1983). In vitro exposures tend to enhance cavitation mechanisms (Ciaravino et al.. 198 1). I!I vitro cavitation
may result in the effect of released chemicals from lysed cells (e.g. Williams and Miller, 1980). Investigators should be encouraged to include an additional control incubated with a small amount of homogenised cells. Other proposed mechanisms include the biological effect of free radicals in the medium which may be liberated as a result of cavitational activity during pulses of diagnostic ultrasound (Riesz, 1985).
DISCUSSION Itr vitro experiments are useful: to determine the toxicity of an agent in increasingly complex steps (molecular, cellular, tissue, organ, whole body); to study mechanisms of action of the agent in simple biological systems; to restrict the complexity of the experimental system thereby reducing the possibility of subtle effects being masked by gross or dominant effects; and to serve as a basis for the design of in vivo studies. The International Radiation Protection Association (IRPA) holds that: “A health risk analysis cannot be made solely on the basis of in vitro studies ofcellular systems,” and such studies serve as a useful means of identifying effects that may occur in vivo. The existing bioeffect data base is heavily biased towards continuous wave exposures at therapeutic intensity levels. Well-designed experimental studies are therefore needed which investigate both existing and novel endpoints using series of repeated short pulses of high intensity ultrasound similar to those emitted by common diagnostic devices.
DEVELOPMENTAL EFFECTS OF ULTRASOUND EXPOSURE IN MAMMALS M.
Batelle Pacific North West Laboratories,
The extensive literature on the developmental effects of ultrasound in laboratory animals and relevant physical and experimental considerations have been reviewed (NCRP Report, 1983: AIUM Bioeffects Committee. 1984: Sikov and Hildebrand, 1979; Stewart and Stratmeyer, 1982). We will use these data to examine response patterns since sensitivities and responses to insult by physical and chemical agents change throughout prenatal development. In the absence of accepted measures of “dose,” we will try to relate pat-
terns of effect to various descriptors of exposure and to the conditions involved in clinical exposures. ItI vitro studies have shown that high CW exposures can affect rodent morulae/blastocysts viu thermal mechanisms, but pulsed exposures were ineffective (Akamatsu. 198 1). The available data suggests that in ilii~) exposures of pregnant rodents before implantation do not produce direct ell’ects on the conceptus at CW intensities up to 1 W/cm’ (Sikov and Collins, 1982). However, whole-abdomen exposures at these intensities