Ultrasound in Med. & Biol., Vol. 31, No. 1, pp.

129 134, 2005 Copyright 2005 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/05/$see front matter

doi:10.1016/j.ultrasmedbio.2004.09.012

Original Contribution
EFFECT OF ULTRASOUND ON NUCLEATED ERYTHROCYTES KATARZYNA MILOWSKA,* TERESA GABRYELAK,* GRAZYNA LYPACEWICZ, RYSZARD TYMKIEWICZ, and ANDRZEJ NOWICKI
*Department of General Biophysics, University of Lodz, Lodz, Poland; and Department of Ultrasound, Institute of Fundamental Technological Research, Polish Academy of Sciences, Warsaw, Poland
(Received 5 April 2004; revised 23 September 2004; accepted 28 September 2004)

AbstractIn this experimental study, carp erythrocytes were used as nucleated cell models to test the hypothesis that ultrasound (US) exposure can cause the change in plasma membranes and hemoglobin. To identify target cell damage, we studied hemolysis, osmotic fragility, lipid peroxidation of red blood cells and oxidation of hemoglobin in the erythrocytes. The erythrocytes were exposed to 1 MHz continuous-wave US at the intensities of 0.61 to 2.44 W/cm2 for 5 min. These results showed that US sonication at the intensities of 1.90 and 2.44 W/cm2 led to an increase in the degree of hemolysis and, at the highest used intensity (2.44 W/cm2), an increase in osmotic fragility. Ultrasound in all the used intensities induced an increase in lipid peroxidation. The results obtained showed that the level of methemoglobin has a tendency to increase after US exposure. Our results suggest that the changes in biomembranes can be due to inertial cavitation induced by US, but we cannot say which stage of the inertial event causes the damage. (E-mail: tgabryl@biol.uni.lodz.pl) 2005 World Federation for Ultrasound in Medicine & Biology. Key Words: Ultrasound, Lipid peroxidation, Hemolysis, Methemoglobin, Osmotic fragility.

INTRODUCTION The mechanisms of ultrasound (US) wave action on biologic material can be divided into thermal and nonthermal. Thermal effects, due to the absorption and dissipation of US energy, are used in physical therapy. Nonthermal mechanisms can be classied as cavitational and noncavitational. Nonthermal, noncavitational mechanisms include effects due to radiation pressure, radiation force, radiation torque and acoustic streaming (Riesz and Kondo 1992). Cavitational process may be divided into related categories by which US interaction with microbubbles may cause biologic effects (Riesz and Kondo 1992). These are: 1. gas body activation (formerly identied as stable cavitation) that is brought about by shear stress due to microstreaming caused by oscillating resonant bubbles, and 2. inertial cavitation (formerly identied as transient cavitation) that induces shock waves and free radical formation as a result of the implosive collapse of cavitation bubbles. There is similarity between the two phenomena: both cause a wide variety of biologic effects (Miller et al. 1996).
Address correspondence to: Dr. Teresa Gabryelak, Department of General Biophysics, University of Lodz, Banacha 12/16, Lodz 90-237 Poland. E-mail: tgabryl@biol.uni.lodz.pl 129

Riesz and Kondo (1992) have shown that sonochemical reactions take place in three different regions. The rst one is the interior of collapsing gas bubbles, in which high temperature and pressure exist. In this region, hydrogen atoms and hydroxyl radicals (H and OH) are formed by thermal dissociation of water. The second region is the interface between the collapsing gas bubbles and bulk of the liquid, where high temperature and pressure gradients exist. The third one is the bulk of solution at ambient temperature. In this region, the H atoms and OH radicals combine to form H2 and H2O2 and react with solutes to form products similar to those observed in aqueous radiation chemistry (Riesz and Kondo 1992). As a result of those reactions, free radicals are highly reactive and may cause cell and tissue damage by interacting with cell membranes and organelles (ztrk et al. 2003). The biologic effect of US can be measured in many components of cells. It has been noticed that cavitation-related bioeffects include hemolysis (Daniels et al. 1995; Everbach et al. 1997; Williams et al. 1991), blood vessel damage (Penney et al. 1993) and DNA damage (Fuciarelli et al. 1995; Kondo et al. 1993). The ability of continuous-wave (CW) US to lyse cells is well established and it is widely assumed that the basic mechanism is related to the formation of gas bub-

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bles. Cell lysis occurs rapidly from gas body activation and inertial cavitation. Hemolysis in dilute suspensions is useful as a cavitation indicator in studies of nucleation and evolution of cavitation (Miller and Williams 1989). Also, lipid peroxidation and osmotic fragility are useful coefcients of changes in plasma membrane. Extensive lipid peroxidation in biologic membranes causes loss of uidity, decrease in membrane potential, increased permeability to ions and eventual rupture leading to release of cell and organelle contents (Gutteridge 1995). Abnormal susceptibility of red blood cell (RBC) lipids to peroxidation is known to reect similar abnormalities in other organs and tissues (ztrk et al. 2003). Another very important indicator of damage in cells is the level of methemoglobin. The oxidation of hemoglobin to methemoglobin can take place in RBCs as a result of oxidation of Fe2 to Fe3, which can be caused by free radical and reactive oxygen species as well as metals and other chemical compounds. This project tested the hypothesis that US exposure causes changes in membrane and hemoglobin of carp erythrocytes in vitro. Fish erythrocytes are a useful model to investigate the aspect of toxicology in vitro because their membranes are rich in long-chain polyunsaturated fatty acids that could be oxidised under oxidative stress conditions induced by chemical or physical factors. These erythrocytes are nucleated, attened and ellipsoidal and they possess, in addition to hemoglobin (Hb), mitochondria, endoplasmatic reticulum and other organelles typical of somatic cells. Their cytoskeletal system consists principally of a membrane skeleton, containing actin and spectrin-family proteins, and intermediate laments of the desmin class (Cohen et al. 1996). In our examinations, we used this cellular model just on account of the presence of Hb and all organelles that red blood cells of humans and other mammals do not possess. Furthermore, carp blood is easily collected and many cellular functions and experimental approaches have been well-described. Our study is the rst stage in the investigation of US exposure to biologic material; therefore, at the beginning of our work, we prepared the experimental setup and we measured physical parameters of the apparatus. Next, we measured hemolysis, osmotic fragility, lipid peroxidation and level of methemoglobin in carp erythrocytes after US exposure. MATERIALS AND METHODS Chemicals Thiobarbituric acid (TBA) was purchased from Sigma (St. Louis, MO). All other chemicals came from Polish Chemical Reagents (Gliwice, Poland) and were of analytical grade.

All solutions were made with double-distilled water or water puried by the Mili-Q system. Cell preparations The cells used in this study were obtained from Cyprinus carpio L. The sh of both genders, weighing 1 to 2 kg, were collected from the local sh farm and were acclimated for a few days in aquarium water (temperature 14 to 16C). Blood was withdrawn by caudal puncture with heparinised syringes and centrifuged for 5 min with 1500 g. After the removal of the plasma, the erythrocytes were washed 3 times with the isotonic buffer (0.6 %) NaCl solution. After washing, red blood cells were diluted in the incubation medium (90.5 mM NaCl, 3 mM KCl, 1.3 mM CaCl2, 0.5 mM MgSO4, 6 mM glucose, 1 mM pyruvate, 1 mM Tris-HCl, pH 7.4) to 5% hematocrit. The erythrocytes suspended in incubation medium (0.2 mL) were put into small foil bags 1 cm 1 cm (width and height) for each trial. The bag was made out of thin polyethylene foil, 0.05-mm thickness. We always used the same volume of erythrocytes and they were evenly distributed inside the bag. During the exposure, erythrocytes did not fall to the bottom and the whole sample was sonicated to the same degree, so the cells did not demand rotation. Each sample of erythrocytes (except the control) was exposed to 1-MHz continuous-wave US at the intensities of 0.61 to 2.44 W/cm2 for 5 min. The controls were the erythrocytes that were not exposed to US and were kept in these same conditions. The procedures of sh treatment were approved by the Local Ethics Committee in Lodz in compliance with the Polish law. Ultrasound and exposure system Ultrasound was produced by an apparatus for the supersonic therapy, BTL-07p produced by Medical Technologies s.r.o. (Prague, Czech Republic). The pressure eld measurements were carried out in the setup shown in Fig. 1. The 1-MHz, 12-mm diameter transducer was immersed in a water container in front of the PVDF bilaminar shielded membrane hydrophone (Sonic Technologies, serial no. 804043; Hatboro, PA). The hydrophone was mounted on the xyz micromanipulator arm. To diminish the inuence of the standing wave on the pressure eld measurements and insonication of blood samples, the wedge made of Plexiglas was positioned behind the hydrophone. The reection coefcient R is a function of the angle of the incident ultrasonic wave according to the formula (Filipczyn ski and Lypacewicz 1974; Krautkrmer 1961):

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in each sample was expressed as percentage of the absorbance in distilled water. Methemoglobin formation (the oxidation of the Fe2 in the hemoglobin molecule to Fe3) was determined spectrophotometrically in the visible region after hypotonic hemolysis. Reference values (complete oxidation) were estimated by addition of ferricyanide. Osmotic fragility of erythrocytes After US exposure, red blood cells were suspended in media containing NaCl at concentrations ranging from 0.6 to 0.15%. After 30 min incubation at 15C, the samples were centrifuged. The degree of hemolysis was estimated from the absorbance at 540 nm of the hemoglobin in the supernatant. These absorbances were calculated as a lysis percentage in relation to control samples that had been 100% hemolysed by adding water (Dacie 1960). Lipid peroxidation Lipid peroxidation was followed by measuring the thiobarbituric acid-reactive substances (TBARS) expressed as MDA equivalents of lipid peroxidation products that react with thiobarbituric acid (TBA) to form a colored complex that has maximum absorbance at 532 nm (Placer et al. 1966). All reactions were conducted in the presence of traces of butylated hydroxytoluene (BHT). TBARS concentrations are presented in nmol/g Hb from six independent measurements. The amount of hemoglobin was determined by the method described by Drabkin (1946). Statistical analysis The results are presented as mean SD from six individual experiments. Each experiment was repeated 3 times. Statistical evaluation of the difference between control and treated groups was performed using Students ttest; p 0.05 was accepted as statistically signicant. RESULTS Carp erythrocyte hemolysis was determined by measuring the hemoglobin content after US exposure at
100 refflection coefficient [%] 80 60 40 20 0

Fig. 1. Experimental setup. The distance from the hydrophone to the transducer is 5.5 cm. The blood samples under examination were positioned at exactly the same distance.

cT 2 sin(2L) sin(2T) cos2(2T) cL

pr , pi cT 2 sin(2L) sin(2T) cos2(2T) cL

c cos(L) pcL cos()

(1)

c cos(L) pcL cos()

where pr and pi are reected and incident acoustical pressure, and p are densities of water and Plexiglas, respectively, c is the velocity of the wave in water, cL, cT are longitudinal and transverse waves velocities in Plexiglas, is an angle of incident wave in water and L and T are angles of diffraction of the longitudinal and transverse waves in Plexiglas. The wedge angle was carefully chosen, assuring the minimum reection of the probing wave back to the transducer. The plot of the reection coefcient vs. angle is shown in Fig. 2. As we can see, the smallest wave reection occurs for the incident angle from 40 to 58. For the 45 wedge, the reection coefcient does not exceed 5 %, even for a slightly divergent incident wave, and the standing wave can be neglected. The lateral ultrasonic pressure distribution measured at 5.5 cm from the transducer face is shown in Fig. 3. The blood samples under examination were positioned at exactly the same distance. The erythrocytes (0.2 mL) were put into small foil bags 1 cm 1 cm (width and height). The temperature of the coupling water was maintained at 15C. The characteristic output values for the ultrasonic medical unfocused transducers are given in Table 1. Hemolysis and methemoglobin Hemolysis was determined by measuring the hemoglobin content in the supernatant at 540 nm. Hemolysis

5%
0 10 20 30 40 50 angle [degrees] 60 70 80 90

Fig. 2. Reection coefcient vs. incident angle.

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250 200
pressure [kPa]

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-3 dB -6 dB

150 100 50 0

10

4 2 0 2 4 distance from axis [mm]

10

Fig. 3. Lateral eld distribution for axial distance equal to 5.5 cm. ( ) 3 dB; (-) 6 dB pressure drop.

intensities in the range of 0.61 to 2.44 W/cm2. The results are shown in Fig. 4. We observed that US induced hemolysis at the intensities of 1.90 and 2.44 W/cm2, as well as that there was a signicant trend for the increase in hemolysis from control to the highest intensity. Mean values of hemolysis percentages were plotted against saline concentration and the osmotic fragility curves of control and ultrasonication of erythrocytes was shown in Fig. 5. We observed that the mean corpuscular fragility of erythrocytes was signicantly higher after sonication of cells at 2.44 W/cm2 as compared with the control, but the other intensities used had no effect on erythrocyte fragility. Therefore, the control cells were less fragile and able to withstand the hypotonicity, whereas sonication erythrocytes were more fragile and less resistant to hypotonicity. Lipid peroxidation was measured in carp erythrocytes after US exposure at different intensities. To determine peroxidation properties, the spectrophotometry method was used to measure the level of TBARS. As seen in Fig. 6, all intensities of US signicantly increased lipid peroxidation compared with the untreated control. We found that there is an apparent trend for the increase in TBARS and the values grow with the increase in US intensity. Thus, these physical agents can induce the lipid peroxidation in carp erythrocytes. The percentages of methemoglobin in the carp red blood cells after US exposure and control are shown in Fig. 7. There was no signicant difference in the levels of hemoglobin between the control and sonication erythrocytes. However, we observed a tendency for an increase in the level of methemoglobin in red blood cells after sonication. DISCUSSION The response of biologic tissue to US exposure can be variable and depends on the acoustic and biologic

properties as well as on the location and function of the exposed tissue. The consequences of interactions with ultrasonic energy depend on the susceptibility of the cells to damage and on their differentiation or functional status (Barnett et al.1997). A wide spectrum of US-induced bioeffects has been studied in numerous cellular and animal models (Mik and Riesz 1999). The erythrocytes may prove to be a good model for elucidating mechanisms of action of US on cells. Erythrocytes from mice (Miller and Battaglia 2003), dogs (Gross et al. 1985), humans (Daniels et al. 1995; Miller et al. 2003c; Pohl et al. 1995), rabbits (Abramowicz et al. 2003) and cows (Miller et al. 2000) have been used for experiments aimed at providing insights into important physical factors pertinent to US-induced biologic effect (Miller and Brayman 1997). However, information obtained by studying this process in mammalian erythrocytes is limited by the fact that these cells are structurally simple, devoid of nucleus and mitochondria (Tiano et al. 2001). More information can be obtained by studying these processes in erythrocytes having these organelles (for instance, sh erythrocytes). In this study, US activities on carp erythrocytes have been demonstrated. We examined the level of hemolysis, osmotic fragility, lipid peroxidation and formation of methemoglobin after the treatment of the cells with 1 MHz continuous-wave US at the intensities of 0.61 to 2.44 W/cm2 for 5 min. Our results showed that there is a signicant trend for the increase in hemolysis from 3.5% (control value) to about 5.1% (highest intensity). The values grow along with the increase in the intensities used. It is known that sonolysis depends on many factors: there include cell concentration (Miller and Williams 1989), cell size (Abramowicz et al. 2003, Miller and Battaglia 2003), rotation of the sample vessel (Miller and Williams 1989) and physical parameters of US wave. There is considerable uncertainty as to the mechanism of action causing the cell lysis. It has been reported that the extent of US-induced hemolysis correlates with indications of the extent of inertial cavitation (Miller et al. 2003b, 2003c; Everbach

Table 1. The characteristic output values for the ultrasonic medical transducer (BTL-07p, produced by Medical Technologies s.r.o.).
Transducer output of the apparatus (W/cm2) Parameter Positive pressure amplitude p (MPa) Negative pressure amplitude p (MPa) Maximum intensity Im (W/cm2) Ispta (W/cm2) Isata (W/cm2) Index MI 0.5 0.17 0.18 0.61 0.15 0.06 0.18 1 0.23 0.24 1.21 0.30 0.12 0.23 1.5 0.30 0.29 1.90 0.47 0.17 0.29 2 0.34 0.33 2.44 0.61 0.24 0.33

Ultrasound and erythrocytes K. MILOWSKA et al.

6 5 hemolysis [%] 4 3 2 1 0 control 0.61 1.21 [W/cm2] 1.90 2.44 * ***

133
***

500 ** TBARS [nmol/g Hb] 400 300 200 100 0 control 0.61 1.21 [W/cm2] 1.90 ** ***

2.44

Fig. 4. The percentage hemolysis of carp erythrocytes exposed to 1 MHz US (continuous-wave) for 5 min. The data were obtained from six individual experiments. Errors bars denote SD * p 0.05; *** p 0.001 compared with the untreated control cells.

Fig. 6. TBARS concentration in carp erythrocytes induced by 1 MHz US after 5 min of sonication. The data were obtained from six individual experiments. Errors bars denote SD ** p 0.01, *** p 0.001 compared with the untreated control cells.

et al. 1997). However, it is not certain which stage of the inertial event causes the damage. That may be shock wave/shear forces of inertial cavitation or free radicals induced by US. Free radicals and reactive oxygen species are toxic to biomembranes and may lead to the peroxidation of lipids. There are some data about lipid peroxidation induced by US in model systems (Das et al. 1985; Jana et al. 1990; Yumita et al. 1996). Hristov et al. (1997) observed an increase in the lipid peroxidation products from Ehrlich tumor cells after sonication. We examined lipid peroxidation in carp erythrocytes after sonication. In our study, we found that there is also an apparent trend for an increase in TBARS above substantial control levels and this trend is compared with that of hemolysis. The increase in the concentration of TBARS suggested a lipid peroxidation process. However, TBARS is a small part of lipid peroxidation products (Hristov et al. 1997) and it only partially characterises the level of lipid peroxidation. The end products of lipid peroxidation, such as malondialdehyde, could induce modication in struc-

ture, uidity and permeability of erythrocyte membrane leading to membrane damage, decreased stability and increased sensitivity of the cells toward hypotonic saline (Devasena et al. 2001). On the basis of the abovementioned ndings, we assumed that, if there is an increase in lipid peroxidation in sonication of erythrocytes, the osmotic fragility should be increased as well. The osmotic fragility test of red blood cells is often performed to evaluate the sensitivity of erythrocyte toward hypotonic saline (i.e., this test reects the ability of RBCs to take up a certain amount of water before lysing). The ability of a normal red cell to withstand its hypotonicity allows the cell to increase its volume before the surface membrane is ruptured. When the limit is reached, lysis occurs (Devasena et al. 2001). Therefore, measurement of osmotic fragility provides a useful indicator as to whether or not the sonicated RBCs are normal. We got similar results when studying osmotic fragility as in the case of hemolysis. US causes an increase in osmotic fragility after an exposure of 2.44 W/cm2, which indicates lower resistance to hypotonicity. These results show that the control erythrocytes and sonication

120 100 hemolysis [%] 80 60 40 20 0 0,1 0,2 0,3 0,4 0,5 0,6 NaCl [%]
control
0.61 W/cm2
1.21 W/cm2
1.90 W/cm2
2.44 W/cm2

8 7 methemoglobin [%] 6 5 4 3 2 1 0 control 0.61 1.21 [W/cm2] 1.90 2.44

Fig. 5. Osmotic fragility curves of control and ultrasonication carp RBCs. The erythrocytes were exposed to 1 MHz US for 5 min. The values were obtained from six individual experiments as mean SD; signicant effect (p 0.05) relative to control were obtained after US exposure at intensity 2.44 W/cm2.

Fig. 7. The percentage of methemoglobin of carp erythrocytes after 5 min exposure to 1 MHz US. The data were obtained from six individual experiments.

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Volume 31, Number 1, 2005 Fuciarelli AF, Sisk EC, Thomas RM, Miller DL. Induction of base damage in DNA solutions by ultrasonic cavitation. Free Radic Biol Med 1995;18:231238. Gross DR, Miller DL, Williams AR. A search for ultrasonic cavitation within the canine cardiovascular system. Ultrasound Med Biol 1985;11:8597. Gutteridge JMC. Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin Chem 1995;41:1819 1828. Hristov PK, Petrov LA, Russanov EM. Lipid peroxidation induced by ultrasonication in Ehrlich ascetic tumor cells. Cancer Lett 1997; 121:710. Jana AK, Agarwal S, Chatterjee SN. The induction of lipid peroxidation in liposomal membrane by ultrasound and the role of hydroxyl radicals. Radiat Res 1990;142:714. Kondo T, Kodaira T, Kano E. Free radical formation induced by ultrasound and its effects on strand breakage in DNA cultured FM3A cells. Free Radic Res Commun 1993;19:S193S200. Krautkrmer JH. Werkstoffprfung mit Ultraschall. Berlin, Gttingen/ Heidelberg: Springer-Verlag, 1961. Miller DL, Williams AR. Bubble recycling as an explanation of the promotion of ultrasonic cavitation in a rotating tube exposure system. Ultrasound Med Biol 1989;15:641 648. Miller MW, Battaglia LF. The relevance of cell size on ultrasoundinduced hemolysis in mouse and human blood in vitro. Ultrasound Med Biol 2003;29:1479 1485. Miller MW, Brayman AA. Comparative sensitivity of human erythrocytes and lymphocytes to sonolysis by 1-MHz ultrasound. Ultrasound Med Biol 1997;23:635 638. Miller MW, Battaglia LF, Mazza S. Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: Medium tonicity. Ultrasound Med Biol 2003a;29:713724. Miller MW, Everbach EC, Miller WM, Battaglia LF. Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: 2. Medium dissolved gas p(O2) content. Ultrasound Med Biol 2003b;29:93102. Miller MW, Miller DL, Brayman AA. A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective. Ultrasound Med Biol 1996;22:11311154. Miller MW, Miller WM, Battaglia LF. Biological and environmental factors affecting ultrasound-induced hemolysis in vitro: 3. antioxidant (Trolox) inclusion. Ultrasound Med Biol 2003c;29:103112. Miller MW, Sherman TA, Brayman AA. Comparative sensitivity of human and bovine erythrocytes to sonolysis by 1-MHz ultrasound. Ultrasound Med Biol 2000;26:13171326. Mik V, Riesz P. EPR characterization of free radical intermediates formed during ultrasound exposure of cell culture media. Free Radic Biol Med 1999;26:936 943. ztrk L, Mansour B, Yksel M, et al. Lipid peroxidation and osmotic fragility of red blood cells in sleep-apnea patients. Clin Chim Acta 2003;332:83 88. Penney DP, Schenk EA, Maltby K, et al. Morphological effects of pulsed ultrasound in the lung. Ultrasound Med Biol 1993;19:127135. Placer ZA, Cushman L, Johnson BC. Estimation of products of lipid peroxidation malonyl dialdehyde in biochemical systems. Anal Biochem 1966;16:359 364. Pohl EE, Rosenfeld EH, Pohl P, Millner R. Effects of ultrasound on agglutination and aggregation of human erythrocytes in vitro. Ultrasound Med Biol 1995;21:711719. Riesz P, Kondo T. Free radical formation induced by ultrasound and its biological implications. Free Radic Biol Med 1992;13:247270. Tiano L, Fedeli D, Ballarini P, et al. Mitochondrial membrane potential in density-separated trout erythrocytes exposed to oxidative stress in vitro. Biochim Biophys Acta 2001;1505:226 237. Williams AR, Kubowicz G, Cramer E, et al. The effects of the microbubble suspension SH U454 (Echovist) on ultrasound-induced cell lysis in a rotating tube exposure system. Echocardiography 1991;8:423 433. Yumita N, Umemura S, Magario N, Umemura K, Nishigaki R. Membrane lipid peroxidation as a mechanism of sonodynamically induced erythrocyte lysis. Int J Radiat Biol 1996;69:397 404.

erythrocytes at lower intensities were less fragile and able to withstand the hypotonicity. We also determined the oxidation of hemoglobin in the erythrocytes. The results obtained showed that there is a tendency to increase the level of methemoglobin. However, the values are not signicant compared with control. High SD can result from differences among individuals from whom blood was taken. In summary, there is evidence that US-induced inertial cavitation activity is associated with US-induced change in plasma membrane. We can suggest that this process leads to the formation of radicals and subsequent membrane damage, but we are not sure whether or not the shock wave induces this damage. Our results conrm the hypothesis that US induced plasma membrane change in carp red blood cells in the range of applied intensities. But, on the basis of the obtained results, we cannot conrm or exclude the hypothesis about the changes in hemoglobin. This problem demands additional investigation. In the future, it will be very important to include in the studies the scavenger of free radicals, the check of whether or not US induces formation of free radicals in our model system and examination of the parameters of sonication. In addition, we are going to examine the inuence of US on different parameters of lipid bilayer as well as on DNA. REFERENCES
Abramowicz JS, Miller MW, Battaglia LF, Mazza S. Comparative haemolytic effectiveness of 1 MHz ultrasound on human and rabbit blood in vitro. Ultrasound Med Biol 2003;29:867 873. Barnett SB, Rott HD, Haar GR, Ziskin MC, Maeda K. The sensitivity of biological tissue to ultrasound. Ultrasound Med Biol 1997;23: 805 812. Cohen WD, Sanchez I, Rayos N, Dadacay A-V. Utility of smooth dogsh erythrocytes for studies of cellular morphogenesis and the cytoskeleton. Mar Mod Elec Rec 1996 http://www.mbl.edu/ biologicalbulletin/mmer/coh/cohtit.html. Dacie JV. Hemolytic anemia: Part I. The congenital anemias. 2nd ed. New York: Grune and Stratton, 1960:35 42. Daniels S, Kodoma T, Price DJ. Damage to red blood cells induced by acoustic cavitation. Ultrasound Med Biol 1995;21:105111. Das M, Mukhtar H, Greenspan ER, Bickers D. Photoenhancement of lipid peroxidation associated with the generation of reactive oxygen species in hepatic microsomes of hematoporphyrin-treated rats. Cancer Res 1985;45:6328 6330. Devasena T, Lalitha S, Padma K. Lipid peroxidation, osmotic fragility and antioxidant status in children with acute post-streptococcal glomerulonephritis. Clin Chim Acta 2001;308:155161. Drabkin DL. Spectrophotometric studiem. XIV the crystallographic and optical properties of the hemoglobin of man in comparison with those of other species. J Biol Chem 1946;164:703723. Everbach EC, Makin IRS, Azadniv M, Meltzer RS. Correlation of ultrasound-induced hemolysis with cavitation detector output in vitro. Ultrasound Med Biol 1997;23:619 624. Filipczyn ski L, Lypacewicz G. Overall sensitivity of ultrasonic pulse devices and its signicance in the diagnostics diseases of eyes (in Polish). Archiwum Akustyki 1974;9:67 64.