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Reactive oxygen species and DNA damage after ultrasound exposure
Biomolecular Engineering 24 (2007) 263–267 www.elsevier.com/locate/geneanabioeng
Reactive oxygen species and DNA damage after ultrasound exposure Katarzyna Milowska, Teresa Gabryelak * Department of General Biophysics, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland Received 24 January 2006; accepted 1 February 2007
Abstract The aim of this work was to detect the formation of hydrogen peroxide and hydroxyl radicals after ultrasound (US) exposure and test the hypothesis that reactive oxygen species induced by ultrasound can contribute to DNA damage. Formation of reactive oxygen species was observed in incubated medium after sonication with 1 MHz continuous ultrasound at the intensities of 0.61–2.44 W/cm2. Free radicals and hydrogen peroxide produced by ultrasound exposure of cells can lead to DNA damage. Comet assay was used to assess the effect of ultrasound on the level of nuclear DNA damage. The nucleated erythrocytes from fish were exposed in vitro to ultrasound at the same intensities and frequency. It was noticed that ultrasound in all used intensities induced DNA damage. The effect was not eliminated by the addition of catalase, which indicates that DNA damage was not caused by hydrogen peroxide only. The results showed that the DNA damage can be repair and this mechanism was the most effective after 30 and 60 min after sonication. Furthermore, the ultrasound-induced DNA damage in the presence of sonosensitizer (Zn- and AlClphthalocyanine) was studied. It was noticed that phthalocyaniens (Pcs) alone or with ultrasound did not induce significant changes in the level of DNA damage. # 2007 Elsevier B.V. All rights reserved. Keywords: Ultrasound; Reactive oxygen species; DNA damage; Comet assay; Phthalocyanines
1. Introduction The expanding use of ultrasound (US) both in industries and in medicine has led to a wider-ranging of the research on the mechanism of ultrasound action and its influence on matter, especially on living cells and tissues (Feril and Kondo, 2004). Ultrasound can penetrate deeply into living tissues and is focused on a small region of interest in the tissue. It is employed for surgery, lithotripsy in urology, emulsification of cataracts in ophathamology, in physical therapy and cancer therapy. Biophysical modes of ultrasonic action can be divided into thermal and nonthermal mechanisms. Thermal effects are connected with absorption and dissipation of ultrasound energy. Nonthermal mechanisms can be classified as cavitational and noncavitational (effects due to radiation pressure, force and acoustic streaming). It is known that the collapse of cavitation bubbles induces local high temperatures and pressures accompanied by mechanical shear stress and free radical formation. Under these conditions, hydroxyl radicals (OH) and hydrogen atoms (H) are formed by the thermal dissociation of
* Corresponding author. Tel.: +48 42 635 44 74; fax: +48 42 635 44 74. E-mail address: [email protected] (T. Gabryelak). 1389-0344/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bioeng.2007.02.001
water. The H atoms and OH radicals either combine to form H2, H2O2 and water or attack cells or their components (Riesz and Kondo, 1992; Misˇ´ık et al., 1999; Naffrechoux et al., 2000; S¸ahin et al., 2004). Ultrasound induced physical and chemical bioeffects include direct and indirect cellular damage, DNA damage and radicals production. Therefore, factors determining the safety of diagnostic and therapeutic ultrasound have been studied for DNA in vitro and in vivo (Riesz and Kondo, 1992; S¸ahin et al., 2004; Fory´tkova` et al., 1995). DNA damage can be caused by the mechanical and sonochemical action of ultrasonic cavitation. Mechanical action occurs when cells and cavitation bubbles directly interact. Shear stress can lead to double and single strand breaks of DNA, which occur mainly between oxygen and carbon atoms, leading to DNA fragments with a phosphorylated 50 -terminus and a free alcohol at the 30 end (Fuciarelli et al., 1995). On the other hand, sonochemical action of ultrasound is a result of oxidative stress. Free radicals and hydrogen peroxide, formed during cavitation, also cause DNA damage. They can influence purine and pyrimidine bases leading to their modifications (Fuciarelli et al., 1995). The earlier reports demonstrate that hydrogen peroxide is an important DNA-damaging chemical in cultured Chinese hamster ovary (CHO) cells and Unio tumidus digestive cells
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(Miller et al., 1995; Miller and Thomas, 1996; Labieniec et al., 2003). Our present study is a continuation of the work on the effect of ultrasound on nucleated erythrocytes in vitro. The earlier results showed that ultrasound caused changes in carp erythrocytes membrane, which results in the increase in hemolysis and lipid peroxidation products. Red blood cells from carp are nucleated, flattened and ellipsoidal and they possess, beside hemoglobin (Hb), mitochondria, endoplasmatic reticulum and other organelles typical of somatic cells. In our examinations we used this cellular model because they possess two important properties: (a) they are nucleated, and (b) they contain hemoglobin (Milowska et al., 2005). The first aim of this work was to detect the formation of reactive oxygen species (hydroxyl radical and hydrogen peroxide) induced by ultrasound exposure in our model system and examine the parameters of sonication. Secondly, we tested whether the yield of reactive oxygen species induced by ultrasound correlated with DNA damage. Furthermore, two phthalocyanines, Pcs (zinc and chloroaluminum) were chosen for the testing of their DNA-damaging properties. The Pcs have been suggested as one of the most effective photosensitizers (Zavodnik et al., 1998; Ben-Hur et al., 1991). Our earlier experiments (Milowska and Gabryelak, 2005) present sonodynamic properties of these Pcs too. They show synergistic effect with ultrasound expressed by significantly increased hemolysis, osmotic fragility and lipid peroxidation in carp erythrocytes. 2. Materials and methods 2.1. Chemicals Low melting-point (LMP) and normal melting-point (NMP) agarose, 40 ,6diamidino-2-phenyl indole (DAPI), dimethyl sulfoxide (DMSO) and catalase were purchased from Sigma (St. Louis, MO, USA). ZnPc and AlClPc were obtained from Acros Organics (New Jersey, USA). All the other chemicals came from Polish Chemical Reagents (Gliwice, Poland) and were of analytical grade. All solutions were made with double-distilled water or water purified by the Mili-Q system.
2.2. Cell preparations
458 wedge, the reflection coefficient did not exceed 5%, even for a slightly divergent incident wave, and the standing wave could be neglected. Dosimetry was performed with the acoustic absorber in place, using the PVDF bilaminar shielded membrane hydrophone (Sonic Technologies, serial no. 804043; Hatboro, PA). The spatial peak intensities in these experiments were in the range of 0.61–2.44 W/cm2, corresponding to the measured peak positive and peak negative pressures of p+ = 0.17 MPa, p = 0.18 MPa and p+ = 0.34 MPa, p = 0.33 MPa, respectively. The experimental set-up, the plot of the reflection coefficient versus angle, lateral ultrasonic pressure distribution and characteristic output values for the ultrasonic unfocused transducer were described in detail and shown previously (Milowska et al., 2005).
2.4. Sonication The samples were put into small foil bags 1 cm 1 cm (width and height) for each trial. The bag was made of thin polyethylene foil, 0.05 mm thickness. We always used the same volume (0.2 ml) of erythrocytes diluted in medium to 5% hematocrit and they were evenly distributed inside the bag. The samples had not been degasified and rotated during the exposure. Suspension 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 ultrasound wave at the intensities of 0.61– 2.44 W/cm2 for 5 min. Control erythrocytes were simultaneously incubated without ultrasound exposure.
2.5. Chemical treatment of erythrocytes Catalase (1000 units/ml) was added to the erythtocytes before sonication to remove hydrogen peroxide formed during sonication. ZnPc and AlClPc were added to the suspension of erythrocytes to give a final concentration of 3 mM. The cells were incubated with the phthalocyanine for 20 min at 22 8C in the dark. After incubation the samples of erythrocytes (except the control) were exposed to 1 MHz continuous ultrasound wave at the intensity of 2.44 W/cm2 for 5 min. To remove the photosensitizer after sonication erythrocytes were washed before measuring the most cell parameters.
2.6. Hydrogen peroxide measurement H2O2 was determined by the iodometric method. To 0.2 ml of the sonicated sample (incubation medium without cells), 0.4 ml of a solution containing 0.4 M KI, 0.05 M NaOH, 1.6 104 M (NH4)6Mo7O244H2O (ammonium molybdate) and 0.4 ml of 0.1 M KHC8H4O4 (potassium hydroterephthalate) were added and the mixture was briefly vortexed. The concentration of H2O2 in the samples was assessed spectrophotometerically using the standard curve (350 nm) (Kondo et al., 1998).
2.7. Determination of OH
The fish (Cyprinus carpio L.) of both sexes, weighing 1–2 kg, were collected from the local fish farm. The whole blood was withdrawn by caudal puncture with heparinized syringes and centrifuged for 5 min with 1500 g at 4 8C. After the removal of the plasma, the erythrocytes were washed three 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 procedures of fish treatment were approved by the Local Ethics Committee in Lodz in compliance with the Polish law.
The presence of OH was determined by spectrofluorimetric method (Tang et al., 2005). Sodium terephthalate dissolved in incubation medium (without cells) was exposed to 1 MHz continuous-wave ultrasound at the intensities of 0.61–2.44 W/cm2. Immediately sodium terephthalate traps OH and sodium 2hydroxyterephthalate was produced, which had strong fluorescence. Fluorescence was excited at 315 nm and registered at 431 nm with a Perkin-Elmer LS50B spectrofluorimetr. The relative fluorescence intensity is proportional to the amount of OH.
2.3. Ultrasound and exposure system
2.8. Alkaline comet assay
Continuous-wave ultrasound was generated by an unfocused apparatus for ultrasonic therapy, BTL-07p, produced by Medical Technologies s.r.o. (Prague, Czech Republic). The 1 MHz, 12 mm diameter transducer was immersed in a distilled water container. The sample was centered in the beam at the distance of 5.5 cm from the transducer. To minimize reflected ultrasound, an acoustic reflector (Plexiglas wedge) was placed at the end of the tank opposite the transducer. Reflection coefficient was calculated for pressure amplitude. For the
The alkaline version of the comet assay was carried out according to the procedure of Singh et al. (1988) with slight modifications by Blasiak and Kowalik (2000). Briefly, 30 ml (300,000 cells) of the cell suspension was mixed with 50 ml 0.75% low melting-point agarose (LMP) at 37 8C, spread on a normal agarose (NMP) pre-coated microscope slide. The slides with cells were covered with a cover slip and subsequently placed on an ice-cold surface to solidify for about 10 min. The cover slips were removed and the slides were
K. Milowska, T. Gabryelak / Biomolecular Engineering 24 (2007) 263–267 placed in cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, 1% Triton X-100, 10% DMSO; the last two components were added freshly). Lysis was performed for 1 h at 4 8C in total darkness. The slides were then incubated in an electrophoretic buffer (300 mM NaOH, 1 mM EDTA pH > 13) for 20 min to allow the unwinding of DNA before electrophoresis. Electrophoresis was performed in the same buffer at 0.73 V/cm (280 mA) for 20 min to allow the damaged DNA or fragments to migrate towards the anode. The slides were then washed three times in water and dried by air. Finally, 30 ml DAPI (2 mg/ml) was added to stain DNA. The ability of fish erytrocytes to repair DNA damage was assessed using the same method (comet assay). To estimate DNA repair, the cells were placed in tubes with incubation medium, before lysis and incubated for 5–60 min at 4 8C. After the incubation the cells were placed on slides and then, the lysis, denaturation, electrophoresis and staining process were performed in the same manner as described above. The comets were analyzed by an Eclipse fluorescence microscope (Nikon, Tokyo, Japan) attached to COHU 4910 video camera (Cohu, San Diego, CA) equipped with UV-1 filter block (an excitation filter of 359 nm and a barrier filter of 461 nm) and connected to a personal computerbased image analysis system Lucia-Comet 4.51 (Laboratory Imaging, Praha, Czech Republic). The tail moment as a measure of DNA damage in the graphic presentation represents the mean S.E.M. of six individual experiments (100 cells from each slide).
2.9. Statistical analysis The results are presented as mean S.E.M. for the Comet assay and as mean S.D. for iodometric method and spectrofluorimetric method. Statistical evaluation of the difference between control and treated group was performed using Student’s t-test. P < 0.05 and below was accepted as statistically significant.
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Fig. 2. The level of OH in medium induced by 1 MHz ultrasound at different intensities after 5 min sonication. The data, obtained from 6 individual experiments, are shown as a ratio of the sample fluorescence after sonication (F) to the control sample fluorescence (Fo). Error bars denote S.D.; *P < 0.05; ***P < 0.001 compared with control.
determine OH, the spectrofluorimetric method was used to measure the level of sodium 2-hydroxyterephthalate, which is a product of the reaction of OH with sodium terephthalate. The data are shown as a ratio of the samples fluorescence after sonication (F) to the control sample fluorescence (Fo). As seen in Fig. 2, all intensities of ultrasound significantly increased the amount of sodium 2-hydroxyterephthalate, which indicates that OH are formed during ultrasound exposure. 3.3. Genotoxic effect of ultrasound
3. Results 3.1. Hydrogen peroxide measurement
The comet assay was performed on carp erythrocytes suspension exposed to ultrasound (1 MHz, 0.61–2.44 W/cm2)
Using the iodometric method, we measured the concentration of hydrogen peroxide in incubation medium (without cells) after ultrasound exposure. The obtained results are shown in Fig. 1. We observed that ultrasound induced the formation of hydrogen peroxide at all used intensities. The concentration of hydrogen peroxide increased from 4.2 mM (lowest intensity) to about 7.8 mM (highest intensity). 3.2. Determination of OH The formation of OH was measured in medium (without cells) after ultrasound exposure at different intensities. To
Fig. 1. The concentration of hydrogen peroxide in medium (presented as mean value with S.D.) induced by 1 MHz ultrasound at different intensities after 5 min sonication. Control sample was medium without ultrasound exposure (the concentration of H2O2 was 0 mM).
Fig. 3. Mean comet tail moment of carp erythrocytes exposed to ultrasound for 5 min. The number of cells in each of six individual treatments was 100. Error bars denote S.E.M.; ***P < 0.001 compared with untreated cells.
Fig. 4. Dependence of DNA damage on the concentration of hydrogen peroxide produced by ultrasound exposure.
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Fig. 5. Mean comet tail moment of carp erythrocytes exposed to ultrasound for 5 min without and with catalase. The number of cells in each of 6 individual treatments was 100. Error bars denote S.E.M.
as well as in a combination of ultrasound with catalase and ultrasound with phthalocyanines. The results, shown in Fig. 3, demonstrate that ultrasound at used intensities contributed to DNA strand breaks. There was a significant increase in DNA damage from control to the highest intensity. The dependence of DNA damage (tail moment) in carp erythrocytes against the estimated hydrogen peroxide generated in medium during the ultrasound exposure is shown in Fig. 4. Knowing that hydrogen peroxide is produced in medium after ultrasound exposure and that it can damage DNA cells, we tested whether or not H2O2 caused damage in carp erythrocytes. Catalase was added to the suspension of cells before ultrasound exposure. The cells were sonicated at the two highest intensities. As seen in Fig. 5, this effect was not eliminated
Fig. 6. Mean comet tail moment of carp erythrocytes exposed to ultrasound for 5 min (I = 2.44 W/cm2) and for different time of repair. The number of cells in each of 6 individual treatments was 100. Error bars denote S.E.M.; *P < 0.05; ***P < 0.001 compared with sonicated cells.
Fig. 7. Mean comet tail moment of carp erythrocytes exposed to: ultrasound, Pcs and Pcs with ultrasound. Ultrasound was used at the intensity of 2.44 W/cm2 and Pcs at the concentration of 3 mM. The number of cells in each of six individual treatments was 100. Error bars denote S.E.M.
by the addition of catalase. This result indicates that DNA damage was not due to the action of H2O2. We also checked the ability of DNA to repair the changes caused by ultrasound at the highest intensity. The cells were able to repair some of the strand breaks 15 min after sonication. The results are shown in Fig. 6. The repair mechanism is the most effective after 30 and 60 min after removing ultrasound. In our study we also investigated the influence of Pcs (Znand AlClPc) alone and in combination with ultrasound on carp red blood cells DNA. The data are shown in Fig. 7. We did not observe changes in DNA after the incubation with Pcs. The combined action of Pcs and ultrasound did not increase the tail moment in comparison with the data obtained for the cells sonicated without Pcs. DNA damage was on the same level after sonication with Pcs as after sonication without Pcs. 4. Discussion The aim of this in vitro study was to check whether ultrasound waves (1 MHz, 0.61–2.44 W/cm2), which are generated by apparatus for ultrasonic therapy, could produce free radicals. The presence of OH radicals was indicated by the formation of sodium 2-hydroxyterephthalate during the sonolysis of aqueous solution of sodium terephthalate. Hydroxyl radicals can combine to form hydrogen peroxide. Therefore, we investigated the hydroxyl radicals formation with the aim of the confirmation of the presence and amount of hydrogen peroxide after sonication. The results reported in this paper demonstrate that ultrasound at the used intensities and frequencies causes a formation of hydroxyl radicals and hydrogen peroxide, as a result of the reaction between hydroxyl radicals. Based on this information, we can claim, that ultrasound waves in these conditions induce cavitation. It is known that acoustic (inertial) cavitation induces shock waves and free radical formation as a result of the implosion of cavitation bubbles (Riesz and Kondo, 1992; Miller and Thomas, 1996). Previous study of Basta et al. (2003) on the effect of cardiac ultrasound which is currently used in clinical diagnostics showed that free radical production in the extracellular medium is a likely mediator of ultrasound effect. They noticed increase in intracellular oxidative stress on endothelial cells in vitro. Another point of our report was to test the working hypothesis that free radicals induced by ultrasound can contribute to DNA damage. We assessed DNA damage and DNA repair in carp erythrocytes as a model system. The reaction between DNA and oxygen radicals species leads to different alterations including damaged bases, mutations and DNA strand breaks (Labieniec and Gabryelak, 2004). Our results show that ultrasound at all used intensities induced DNA damage and this effect depended on the intensity used. Our results are in agreement with the observation of Miller et al. (Miller et al., 1995; Miller and Thomas, 1996) that ultrasound contributed to DNA lesions, which grow along with the increase in the doses of ultrasound. Kondo and Kano (1988) observed an increase in double strand breaks along with the growth of exposure time and a decrease in the survival of mouse
K. Milowska, T. Gabryelak / Biomolecular Engineering 24 (2007) 263–267
L cells. The obtained data (Fig. 4) could suggest that hydrogen peroxide formed during cavitation was responsible for the DNA damage. In order to check this suggestion, we added catalase before sonication to remove hydrogen peroxide from the medium. We noticed that the addition of catalase did not eliminate DNA lesions. The tail moment in the experiment with catalase did not change significantly in comparison with the results obtained for the cells sonicated without catalase (Fig. 5). This observation indicates that H2O2 is not the factor contributing to DNA damage in surviving cells. Probably, other factors of cavitation could be involved. One of these factors could be hydroxyl radicals generated during ultrasound action which have not been recombined with other radicals or chemicals and have not been scavenged. Hydroxyl radicals induced by sonolysis of water are believed to cause single strand breaks and/or double strand breaks of DNA which seem to be similar to those induced by radiolysis of water (Riesz and Kondo, 1992). Also, other radicals or mechanical action of acoustic cavitation could be responsible for DNA damage. The following research showed that some DNA damage caused by ultrasound can be repaired. It is suggested that some of these lesions are not permanent. The significant repair occurred 15, 30 and 60 min after stopping sonication. The tail moment after 30 and 60 min of repair was on a similar level. Another interesting aspect was to study the synergistic effect of Pcs and ultrasound on DNA. The synergistic effect of cytotoxic activity of chemicals (sonosensitizers) on tumor cells is a promising modality for cancer treatment (sonodynamic therapy). Pcs are a second generation sensitizers, which show photo- and sonosensitizing properties (Zavodnik et al., 2002). Yumita and Umemura (2004) noticed a significant antitumor effect of chloroaluminum phthalocyanine tetrasulfonate and ultrasound in vivo as evaluated by the decrease in the tumor size. Our earlier results show synergistic effect of Pcs with ultrasound in relation to erythrocytes membranes (Milowska and Gabryelak, 2005). In this work, we examined the influence of two Pcs (zinc and chloroaluminum) at the final concentration of 3 mM alone and with ultrasound at the intensity of 2.44 W/cm2 on DNA. The obtained results showed that ZnPc as well as AlClPc at the used concentration did not induce DNA damage. They did not enhance genotoxic properties of ultrasound, either. Although, Pcs showed sonosentizing properties in relation to plasma membrane, they did not influence the effect of ultrasound on DNA. They could have caused physical destabilization and increased sensitivity of erythrocytes membrane (Zavodnik et al., 2002). These effects can explain why Pcs bound with membrane, but did not penetrate to cellular nucleus. To summarize this work, the results presented here show that ultrasound waves generated by the apparatus for the ultrasonic
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therapy induce reactive oxygen species. Ultrasound can also cause DNA damage. This process was not due only to hydrogen peroxide formed during cavitation. The possible consequences of ultrasonically induced free radical and DNA damage are potential risks associated with the patient’s exposure to diagnostic and/or therapeutic ultrasound. Although the possibility of unwanted effects exists, current data indicate that the benefits to patients of the prudent use of diagnostic ultrasound outweigh the risks. Another consequence can be possible uses in cancer therapy in combination with anticancer drugs, but this aspect still demands many investigations.
Acknowledgement The authors would like to thank Prof. Janusz Blasiak for enabling us to analyze the comets.
References Basta, G., Venneri, L., Lazzerini, G., Pasanisi, E., Pianelli, M., Vesentini, N., Del Turco, S., Kusmic, C., Picano, E., 2003. Cardiovasc. Res. 58, 156–161. Ben-Hur, E., Freud, A., Canfi, A., Livne, A., 1991. Int. J. Radiat. Biol. 59, 797– 806. Blasiak, J., Kowalik, J., 2000. Mutat. Res. 469, 135–145. Feril Jr., L.B., Kondo, T., 2004. Int. Congr. Ser. 1274, 133–140. Fory´tkova`, L., Hrazdira, I., Mornstein, V., 1995. Ultrasound Med. Biol. 2, 585– 592. Fuciarelli, A.F., Sisk, E.C., Thomas, R.M., Miller, D.L., 1995. Free Radic. Biol. Med. 18, 231–238. Kondo, T., Kano, E., 1988. Int. J. Radiat. Biol. 54, 475–486. Kondo, T., Misˇ´ık, V., Riesz, P., 1998. Free Radic. Biol. Med. 25, 605–612. Labieniec, M., Gabryelak, T., 2004. Toxicol. In Vitro 18, 773–781. Labieniec, M., Gabryelak, T., Falcioni, G., 2003. Mutat. Res. 539, 19–28. Miller, D.L., Thomas, R.M., 1996. Ultrasound Med. Biol. 22, 681–687. Miller, D.L., Thomas, R.M., Buschbom, R.L., 1995. Ultrasound Med. Biol. 21, 841–848. Milowska, K., Gabryelak, T., 2005. Ultrasound Med. Biol. 31, 1707–1712. Milowska, K., Gabryelak, T., Lypacewicz, G., Tymkiewicz, R., Nowicki, A., 2005. Ultrasound Med. Biol. 31, 129–134. Misˇ´ık, V., Miyoshi, N., Riesz, P., 1999. Free Radic. Biol. Med. 26, 961–967. Naffrechoux, E., Chanoux, S., Petrier, C., Suptil, J., 2000. Ultrason. Sonochem. 7, 255–259. Riesz, P., Kondo, T., 1992. Free Radic. Biol. Med. 13, 247–270. ¨ ., Do¨nmez-Altuntas¸, H., Hizmelti, S., Hamurcu, Z., Imamog˘lu, N., S¸ahin, O 2004. Ultrasound Med. Biol. 30, 545–548. Singh, N.P., McCoy, M.T., Tice, R.R., Schneider, E.L., 1988. Exp. Cell Res. 175, 184–191. Tang, B., Zhang, L., Geng, Y., 2005. Talanta 65, 769–775. Yumita, N., Umemura, S., 2004. J. Med. Ultrason. 31, 35–40. Zavodnik, I.B., Zavodnik, L.B., Krajewska, E., Romanowicz-Makowska, H., Bryszewska, M., 1998. Curr. Top. Biophys. 22B, 230–237. Zavodnik, I.B., Zavodnik, L.B., Bryszewska, M.J., 2002. J. Photochem. Photobiol. B: Biol. 67, 1–10.
Reactive oxygen species and DNA damage after ultrasound exposure Katarzyna Milowska, Teresa Gabryelak * Department of General Biophysics, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland Received 24 January 2006; accepted 1 February 2007
Abstract The aim of this work was to detect the formation of hydrogen peroxide and hydroxyl radicals after ultrasound (US) exposure and test the hypothesis that reactive oxygen species induced by ultrasound can contribute to DNA damage. Formation of reactive oxygen species was observed in incubated medium after sonication with 1 MHz continuous ultrasound at the intensities of 0.61–2.44 W/cm2. Free radicals and hydrogen peroxide produced by ultrasound exposure of cells can lead to DNA damage. Comet assay was used to assess the effect of ultrasound on the level of nuclear DNA damage. The nucleated erythrocytes from fish were exposed in vitro to ultrasound at the same intensities and frequency. It was noticed that ultrasound in all used intensities induced DNA damage. The effect was not eliminated by the addition of catalase, which indicates that DNA damage was not caused by hydrogen peroxide only. The results showed that the DNA damage can be repair and this mechanism was the most effective after 30 and 60 min after sonication. Furthermore, the ultrasound-induced DNA damage in the presence of sonosensitizer (Zn- and AlClphthalocyanine) was studied. It was noticed that phthalocyaniens (Pcs) alone or with ultrasound did not induce significant changes in the level of DNA damage. # 2007 Elsevier B.V. All rights reserved. Keywords: Ultrasound; Reactive oxygen species; DNA damage; Comet assay; Phthalocyanines
1. Introduction The expanding use of ultrasound (US) both in industries and in medicine has led to a wider-ranging of the research on the mechanism of ultrasound action and its influence on matter, especially on living cells and tissues (Feril and Kondo, 2004). Ultrasound can penetrate deeply into living tissues and is focused on a small region of interest in the tissue. It is employed for surgery, lithotripsy in urology, emulsification of cataracts in ophathamology, in physical therapy and cancer therapy. Biophysical modes of ultrasonic action can be divided into thermal and nonthermal mechanisms. Thermal effects are connected with absorption and dissipation of ultrasound energy. Nonthermal mechanisms can be classified as cavitational and noncavitational (effects due to radiation pressure, force and acoustic streaming). It is known that the collapse of cavitation bubbles induces local high temperatures and pressures accompanied by mechanical shear stress and free radical formation. Under these conditions, hydroxyl radicals (OH) and hydrogen atoms (H) are formed by the thermal dissociation of
* Corresponding author. Tel.: +48 42 635 44 74; fax: +48 42 635 44 74. E-mail address: [email protected] (T. Gabryelak). 1389-0344/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bioeng.2007.02.001
water. The H atoms and OH radicals either combine to form H2, H2O2 and water or attack cells or their components (Riesz and Kondo, 1992; Misˇ´ık et al., 1999; Naffrechoux et al., 2000; S¸ahin et al., 2004). Ultrasound induced physical and chemical bioeffects include direct and indirect cellular damage, DNA damage and radicals production. Therefore, factors determining the safety of diagnostic and therapeutic ultrasound have been studied for DNA in vitro and in vivo (Riesz and Kondo, 1992; S¸ahin et al., 2004; Fory´tkova` et al., 1995). DNA damage can be caused by the mechanical and sonochemical action of ultrasonic cavitation. Mechanical action occurs when cells and cavitation bubbles directly interact. Shear stress can lead to double and single strand breaks of DNA, which occur mainly between oxygen and carbon atoms, leading to DNA fragments with a phosphorylated 50 -terminus and a free alcohol at the 30 end (Fuciarelli et al., 1995). On the other hand, sonochemical action of ultrasound is a result of oxidative stress. Free radicals and hydrogen peroxide, formed during cavitation, also cause DNA damage. They can influence purine and pyrimidine bases leading to their modifications (Fuciarelli et al., 1995). The earlier reports demonstrate that hydrogen peroxide is an important DNA-damaging chemical in cultured Chinese hamster ovary (CHO) cells and Unio tumidus digestive cells
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K. Milowska, T. Gabryelak / Biomolecular Engineering 24 (2007) 263–267
(Miller et al., 1995; Miller and Thomas, 1996; Labieniec et al., 2003). Our present study is a continuation of the work on the effect of ultrasound on nucleated erythrocytes in vitro. The earlier results showed that ultrasound caused changes in carp erythrocytes membrane, which results in the increase in hemolysis and lipid peroxidation products. Red blood cells from carp are nucleated, flattened and ellipsoidal and they possess, beside hemoglobin (Hb), mitochondria, endoplasmatic reticulum and other organelles typical of somatic cells. In our examinations we used this cellular model because they possess two important properties: (a) they are nucleated, and (b) they contain hemoglobin (Milowska et al., 2005). The first aim of this work was to detect the formation of reactive oxygen species (hydroxyl radical and hydrogen peroxide) induced by ultrasound exposure in our model system and examine the parameters of sonication. Secondly, we tested whether the yield of reactive oxygen species induced by ultrasound correlated with DNA damage. Furthermore, two phthalocyanines, Pcs (zinc and chloroaluminum) were chosen for the testing of their DNA-damaging properties. The Pcs have been suggested as one of the most effective photosensitizers (Zavodnik et al., 1998; Ben-Hur et al., 1991). Our earlier experiments (Milowska and Gabryelak, 2005) present sonodynamic properties of these Pcs too. They show synergistic effect with ultrasound expressed by significantly increased hemolysis, osmotic fragility and lipid peroxidation in carp erythrocytes. 2. Materials and methods 2.1. Chemicals Low melting-point (LMP) and normal melting-point (NMP) agarose, 40 ,6diamidino-2-phenyl indole (DAPI), dimethyl sulfoxide (DMSO) and catalase were purchased from Sigma (St. Louis, MO, USA). ZnPc and AlClPc were obtained from Acros Organics (New Jersey, USA). All the other chemicals came from Polish Chemical Reagents (Gliwice, Poland) and were of analytical grade. All solutions were made with double-distilled water or water purified by the Mili-Q system.
2.2. Cell preparations
458 wedge, the reflection coefficient did not exceed 5%, even for a slightly divergent incident wave, and the standing wave could be neglected. Dosimetry was performed with the acoustic absorber in place, using the PVDF bilaminar shielded membrane hydrophone (Sonic Technologies, serial no. 804043; Hatboro, PA). The spatial peak intensities in these experiments were in the range of 0.61–2.44 W/cm2, corresponding to the measured peak positive and peak negative pressures of p+ = 0.17 MPa, p = 0.18 MPa and p+ = 0.34 MPa, p = 0.33 MPa, respectively. The experimental set-up, the plot of the reflection coefficient versus angle, lateral ultrasonic pressure distribution and characteristic output values for the ultrasonic unfocused transducer were described in detail and shown previously (Milowska et al., 2005).
2.4. Sonication The samples were put into small foil bags 1 cm 1 cm (width and height) for each trial. The bag was made of thin polyethylene foil, 0.05 mm thickness. We always used the same volume (0.2 ml) of erythrocytes diluted in medium to 5% hematocrit and they were evenly distributed inside the bag. The samples had not been degasified and rotated during the exposure. Suspension 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 ultrasound wave at the intensities of 0.61– 2.44 W/cm2 for 5 min. Control erythrocytes were simultaneously incubated without ultrasound exposure.
2.5. Chemical treatment of erythrocytes Catalase (1000 units/ml) was added to the erythtocytes before sonication to remove hydrogen peroxide formed during sonication. ZnPc and AlClPc were added to the suspension of erythrocytes to give a final concentration of 3 mM. The cells were incubated with the phthalocyanine for 20 min at 22 8C in the dark. After incubation the samples of erythrocytes (except the control) were exposed to 1 MHz continuous ultrasound wave at the intensity of 2.44 W/cm2 for 5 min. To remove the photosensitizer after sonication erythrocytes were washed before measuring the most cell parameters.
2.6. Hydrogen peroxide measurement H2O2 was determined by the iodometric method. To 0.2 ml of the sonicated sample (incubation medium without cells), 0.4 ml of a solution containing 0.4 M KI, 0.05 M NaOH, 1.6 104 M (NH4)6Mo7O244H2O (ammonium molybdate) and 0.4 ml of 0.1 M KHC8H4O4 (potassium hydroterephthalate) were added and the mixture was briefly vortexed. The concentration of H2O2 in the samples was assessed spectrophotometerically using the standard curve (350 nm) (Kondo et al., 1998).
2.7. Determination of OH
The fish (Cyprinus carpio L.) of both sexes, weighing 1–2 kg, were collected from the local fish farm. The whole blood was withdrawn by caudal puncture with heparinized syringes and centrifuged for 5 min with 1500 g at 4 8C. After the removal of the plasma, the erythrocytes were washed three 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 procedures of fish treatment were approved by the Local Ethics Committee in Lodz in compliance with the Polish law.
The presence of OH was determined by spectrofluorimetric method (Tang et al., 2005). Sodium terephthalate dissolved in incubation medium (without cells) was exposed to 1 MHz continuous-wave ultrasound at the intensities of 0.61–2.44 W/cm2. Immediately sodium terephthalate traps OH and sodium 2hydroxyterephthalate was produced, which had strong fluorescence. Fluorescence was excited at 315 nm and registered at 431 nm with a Perkin-Elmer LS50B spectrofluorimetr. The relative fluorescence intensity is proportional to the amount of OH.
2.3. Ultrasound and exposure system
2.8. Alkaline comet assay
Continuous-wave ultrasound was generated by an unfocused apparatus for ultrasonic therapy, BTL-07p, produced by Medical Technologies s.r.o. (Prague, Czech Republic). The 1 MHz, 12 mm diameter transducer was immersed in a distilled water container. The sample was centered in the beam at the distance of 5.5 cm from the transducer. To minimize reflected ultrasound, an acoustic reflector (Plexiglas wedge) was placed at the end of the tank opposite the transducer. Reflection coefficient was calculated for pressure amplitude. For the
The alkaline version of the comet assay was carried out according to the procedure of Singh et al. (1988) with slight modifications by Blasiak and Kowalik (2000). Briefly, 30 ml (300,000 cells) of the cell suspension was mixed with 50 ml 0.75% low melting-point agarose (LMP) at 37 8C, spread on a normal agarose (NMP) pre-coated microscope slide. The slides with cells were covered with a cover slip and subsequently placed on an ice-cold surface to solidify for about 10 min. The cover slips were removed and the slides were
K. Milowska, T. Gabryelak / Biomolecular Engineering 24 (2007) 263–267 placed in cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, 1% Triton X-100, 10% DMSO; the last two components were added freshly). Lysis was performed for 1 h at 4 8C in total darkness. The slides were then incubated in an electrophoretic buffer (300 mM NaOH, 1 mM EDTA pH > 13) for 20 min to allow the unwinding of DNA before electrophoresis. Electrophoresis was performed in the same buffer at 0.73 V/cm (280 mA) for 20 min to allow the damaged DNA or fragments to migrate towards the anode. The slides were then washed three times in water and dried by air. Finally, 30 ml DAPI (2 mg/ml) was added to stain DNA. The ability of fish erytrocytes to repair DNA damage was assessed using the same method (comet assay). To estimate DNA repair, the cells were placed in tubes with incubation medium, before lysis and incubated for 5–60 min at 4 8C. After the incubation the cells were placed on slides and then, the lysis, denaturation, electrophoresis and staining process were performed in the same manner as described above. The comets were analyzed by an Eclipse fluorescence microscope (Nikon, Tokyo, Japan) attached to COHU 4910 video camera (Cohu, San Diego, CA) equipped with UV-1 filter block (an excitation filter of 359 nm and a barrier filter of 461 nm) and connected to a personal computerbased image analysis system Lucia-Comet 4.51 (Laboratory Imaging, Praha, Czech Republic). The tail moment as a measure of DNA damage in the graphic presentation represents the mean S.E.M. of six individual experiments (100 cells from each slide).
2.9. Statistical analysis The results are presented as mean S.E.M. for the Comet assay and as mean S.D. for iodometric method and spectrofluorimetric method. Statistical evaluation of the difference between control and treated group was performed using Student’s t-test. P < 0.05 and below was accepted as statistically significant.
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Fig. 2. The level of OH in medium induced by 1 MHz ultrasound at different intensities after 5 min sonication. The data, obtained from 6 individual experiments, are shown as a ratio of the sample fluorescence after sonication (F) to the control sample fluorescence (Fo). Error bars denote S.D.; *P < 0.05; ***P < 0.001 compared with control.
determine OH, the spectrofluorimetric method was used to measure the level of sodium 2-hydroxyterephthalate, which is a product of the reaction of OH with sodium terephthalate. The data are shown as a ratio of the samples fluorescence after sonication (F) to the control sample fluorescence (Fo). As seen in Fig. 2, all intensities of ultrasound significantly increased the amount of sodium 2-hydroxyterephthalate, which indicates that OH are formed during ultrasound exposure. 3.3. Genotoxic effect of ultrasound
3. Results 3.1. Hydrogen peroxide measurement
The comet assay was performed on carp erythrocytes suspension exposed to ultrasound (1 MHz, 0.61–2.44 W/cm2)
Using the iodometric method, we measured the concentration of hydrogen peroxide in incubation medium (without cells) after ultrasound exposure. The obtained results are shown in Fig. 1. We observed that ultrasound induced the formation of hydrogen peroxide at all used intensities. The concentration of hydrogen peroxide increased from 4.2 mM (lowest intensity) to about 7.8 mM (highest intensity). 3.2. Determination of OH The formation of OH was measured in medium (without cells) after ultrasound exposure at different intensities. To
Fig. 1. The concentration of hydrogen peroxide in medium (presented as mean value with S.D.) induced by 1 MHz ultrasound at different intensities after 5 min sonication. Control sample was medium without ultrasound exposure (the concentration of H2O2 was 0 mM).
Fig. 3. Mean comet tail moment of carp erythrocytes exposed to ultrasound for 5 min. The number of cells in each of six individual treatments was 100. Error bars denote S.E.M.; ***P < 0.001 compared with untreated cells.
Fig. 4. Dependence of DNA damage on the concentration of hydrogen peroxide produced by ultrasound exposure.
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Fig. 5. Mean comet tail moment of carp erythrocytes exposed to ultrasound for 5 min without and with catalase. The number of cells in each of 6 individual treatments was 100. Error bars denote S.E.M.
as well as in a combination of ultrasound with catalase and ultrasound with phthalocyanines. The results, shown in Fig. 3, demonstrate that ultrasound at used intensities contributed to DNA strand breaks. There was a significant increase in DNA damage from control to the highest intensity. The dependence of DNA damage (tail moment) in carp erythrocytes against the estimated hydrogen peroxide generated in medium during the ultrasound exposure is shown in Fig. 4. Knowing that hydrogen peroxide is produced in medium after ultrasound exposure and that it can damage DNA cells, we tested whether or not H2O2 caused damage in carp erythrocytes. Catalase was added to the suspension of cells before ultrasound exposure. The cells were sonicated at the two highest intensities. As seen in Fig. 5, this effect was not eliminated
Fig. 6. Mean comet tail moment of carp erythrocytes exposed to ultrasound for 5 min (I = 2.44 W/cm2) and for different time of repair. The number of cells in each of 6 individual treatments was 100. Error bars denote S.E.M.; *P < 0.05; ***P < 0.001 compared with sonicated cells.
Fig. 7. Mean comet tail moment of carp erythrocytes exposed to: ultrasound, Pcs and Pcs with ultrasound. Ultrasound was used at the intensity of 2.44 W/cm2 and Pcs at the concentration of 3 mM. The number of cells in each of six individual treatments was 100. Error bars denote S.E.M.
by the addition of catalase. This result indicates that DNA damage was not due to the action of H2O2. We also checked the ability of DNA to repair the changes caused by ultrasound at the highest intensity. The cells were able to repair some of the strand breaks 15 min after sonication. The results are shown in Fig. 6. The repair mechanism is the most effective after 30 and 60 min after removing ultrasound. In our study we also investigated the influence of Pcs (Znand AlClPc) alone and in combination with ultrasound on carp red blood cells DNA. The data are shown in Fig. 7. We did not observe changes in DNA after the incubation with Pcs. The combined action of Pcs and ultrasound did not increase the tail moment in comparison with the data obtained for the cells sonicated without Pcs. DNA damage was on the same level after sonication with Pcs as after sonication without Pcs. 4. Discussion The aim of this in vitro study was to check whether ultrasound waves (1 MHz, 0.61–2.44 W/cm2), which are generated by apparatus for ultrasonic therapy, could produce free radicals. The presence of OH radicals was indicated by the formation of sodium 2-hydroxyterephthalate during the sonolysis of aqueous solution of sodium terephthalate. Hydroxyl radicals can combine to form hydrogen peroxide. Therefore, we investigated the hydroxyl radicals formation with the aim of the confirmation of the presence and amount of hydrogen peroxide after sonication. The results reported in this paper demonstrate that ultrasound at the used intensities and frequencies causes a formation of hydroxyl radicals and hydrogen peroxide, as a result of the reaction between hydroxyl radicals. Based on this information, we can claim, that ultrasound waves in these conditions induce cavitation. It is known that acoustic (inertial) cavitation induces shock waves and free radical formation as a result of the implosion of cavitation bubbles (Riesz and Kondo, 1992; Miller and Thomas, 1996). Previous study of Basta et al. (2003) on the effect of cardiac ultrasound which is currently used in clinical diagnostics showed that free radical production in the extracellular medium is a likely mediator of ultrasound effect. They noticed increase in intracellular oxidative stress on endothelial cells in vitro. Another point of our report was to test the working hypothesis that free radicals induced by ultrasound can contribute to DNA damage. We assessed DNA damage and DNA repair in carp erythrocytes as a model system. The reaction between DNA and oxygen radicals species leads to different alterations including damaged bases, mutations and DNA strand breaks (Labieniec and Gabryelak, 2004). Our results show that ultrasound at all used intensities induced DNA damage and this effect depended on the intensity used. Our results are in agreement with the observation of Miller et al. (Miller et al., 1995; Miller and Thomas, 1996) that ultrasound contributed to DNA lesions, which grow along with the increase in the doses of ultrasound. Kondo and Kano (1988) observed an increase in double strand breaks along with the growth of exposure time and a decrease in the survival of mouse
K. Milowska, T. Gabryelak / Biomolecular Engineering 24 (2007) 263–267
L cells. The obtained data (Fig. 4) could suggest that hydrogen peroxide formed during cavitation was responsible for the DNA damage. In order to check this suggestion, we added catalase before sonication to remove hydrogen peroxide from the medium. We noticed that the addition of catalase did not eliminate DNA lesions. The tail moment in the experiment with catalase did not change significantly in comparison with the results obtained for the cells sonicated without catalase (Fig. 5). This observation indicates that H2O2 is not the factor contributing to DNA damage in surviving cells. Probably, other factors of cavitation could be involved. One of these factors could be hydroxyl radicals generated during ultrasound action which have not been recombined with other radicals or chemicals and have not been scavenged. Hydroxyl radicals induced by sonolysis of water are believed to cause single strand breaks and/or double strand breaks of DNA which seem to be similar to those induced by radiolysis of water (Riesz and Kondo, 1992). Also, other radicals or mechanical action of acoustic cavitation could be responsible for DNA damage. The following research showed that some DNA damage caused by ultrasound can be repaired. It is suggested that some of these lesions are not permanent. The significant repair occurred 15, 30 and 60 min after stopping sonication. The tail moment after 30 and 60 min of repair was on a similar level. Another interesting aspect was to study the synergistic effect of Pcs and ultrasound on DNA. The synergistic effect of cytotoxic activity of chemicals (sonosensitizers) on tumor cells is a promising modality for cancer treatment (sonodynamic therapy). Pcs are a second generation sensitizers, which show photo- and sonosensitizing properties (Zavodnik et al., 2002). Yumita and Umemura (2004) noticed a significant antitumor effect of chloroaluminum phthalocyanine tetrasulfonate and ultrasound in vivo as evaluated by the decrease in the tumor size. Our earlier results show synergistic effect of Pcs with ultrasound in relation to erythrocytes membranes (Milowska and Gabryelak, 2005). In this work, we examined the influence of two Pcs (zinc and chloroaluminum) at the final concentration of 3 mM alone and with ultrasound at the intensity of 2.44 W/cm2 on DNA. The obtained results showed that ZnPc as well as AlClPc at the used concentration did not induce DNA damage. They did not enhance genotoxic properties of ultrasound, either. Although, Pcs showed sonosentizing properties in relation to plasma membrane, they did not influence the effect of ultrasound on DNA. They could have caused physical destabilization and increased sensitivity of erythrocytes membrane (Zavodnik et al., 2002). These effects can explain why Pcs bound with membrane, but did not penetrate to cellular nucleus. To summarize this work, the results presented here show that ultrasound waves generated by the apparatus for the ultrasonic
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therapy induce reactive oxygen species. Ultrasound can also cause DNA damage. This process was not due only to hydrogen peroxide formed during cavitation. The possible consequences of ultrasonically induced free radical and DNA damage are potential risks associated with the patient’s exposure to diagnostic and/or therapeutic ultrasound. Although the possibility of unwanted effects exists, current data indicate that the benefits to patients of the prudent use of diagnostic ultrasound outweigh the risks. Another consequence can be possible uses in cancer therapy in combination with anticancer drugs, but this aspect still demands many investigations.
Acknowledgement The authors would like to thank Prof. Janusz Blasiak for enabling us to analyze the comets.
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