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Effect of X-Ray and UV irradiation of the C60 fullerene aqueous solution on biological samples
Carbon 42 (2004) 1199–1201 www.elsevier.com/locate/carbon
Effect of X-Ray and UV irradiation of the C60 fullerene aqueous solution on biological samples P. Scharff a, L. Carta-Abelmann a, C. Siegmund a, O.P. Matyshevska b, S.V. Prylutska b, T.V. Koval b, A.A. Golub c, V.M. Yashchuk d, K.M. Kushnir d, Yu.I. Prylutskyy b,* b
a Institute of Physics, TU Ilmenau, Ilmenau D-98684, Germany Department of Biophysics, Faculty of Biology, Kyiv National Shevchenko University, Volodymyrska Str., 64,Kyiv 01033, Ukraine c Faculty of Chemistry, Kyiv National Shevchenko University, Volodymyrska Str., 64, Kyiv 01033, Ukraine d Faculty of Physics, Kyiv National Shevchenko University, Volodymyrska Str., 64, Kyiv 01033, Ukraine
Available online 6 February 2004
Abstract The dependence of the optical absorption density on the UV irradiation time of a biological sample (the suspension of thymocytes, isolated from Wistar rat thymus) and the DNA structural state in cells under X-ray irradiation in the presence of the C60 fullerene aqueous solution were investigated and analyzed in detail. The significant pro-oxidant effect of this material at the low concentration of fullerene C60 (105 M) was revealed. 2004 Elsevier Ltd. All rights reserved. Keywords: A. Fullerene; C. Adsorption; D. Bioactivity
1. Introduction
2. Experiment
C60 fullerenes have some potential biological activity in vitro and in vivo [1], e.g. inhibition of HIV protease and lipid peroxidation, antiviral and antioxidant effects, specific cleavage of DNA, which has attracted considerable attention and has become a challenging research field at present. However, the desire to study the biological effects of C60 fullerenes is hindered by the strong hydrophobicity of these merely carbon molecules for they have difficulties in reacting with biological systems. In that case, for example, it is the key to prepare watersoluble fullerene derivatives to be suitable for biological study [2]. But the recently obtained C60 fullerene aqueous solutions (C60 FAS) with different C60 concentrations [3] opens the prospect for their intensive use in biological studies. The purpose of this work was to investigate the bioactivity of C60 FAS in the biological medium in dependence on the UV and X-ray irradiation.
The samples of C60 FAS were prepared as follows. We have used a saturated solution of pure C60 (a purity >99.5%) in toluene and the same amount of distilled water in an open beaker. Two phases are formed. Then we applied an ultrasonic bath as long as the toluene needs to evaporate completely. Meanwhile the water phase became yellow colored, indicating that a water– fullerene solution has been formed. Thereafter we filtered the water solution from undissolved C60 . As a result we prepared the C60 FAS samples with different concentration of C60 fullerene in water up to 1.4 mg/ml (for comparison, the solubilities of C60 are 1.7 and 2.9 mg/ml in benzene and toluene, respectively). Thymus (100 mg) of decapitated Wister rats was filtered through the nylon sieve into the buffer (3 mM Na2 HPO4 Æ 12H2 O, 5 mM KCl, 120 mM NaCl, 10 mM glucose, 10 mM HEPES, pH 7.4, 4 mM NaHCO3 , 1 mM CaCl2 , 1 mM MgCl2 ). The total volume of cell suspension was 10 ml. The cells were washed twice by centrifugation (1500g, 10 min). The sediment was resuspended in the same buffer to the concentration 107 cells/ml. The DNA fragmentation was estimated by the polydesoxyribonucleotides (the low-molecular fragments of
*
Corresponding author. Tel.: +380-44-266-5408; fax: +380-44-2520827. E-mail address: [email protected] (Yu.I. Prylutskyy). 0008-6223/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbon.2003.12.055
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P. Scharff et al. / Carbon 42 (2004) 1199–1201
DNA) accumulation in the cells. Cells were lysed in the buffer (5 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.5% Triton X-100). The samples were centrifuged (1500g, 20 min) for the separation of intact chromatin (in the pellet) from DNA fragments in supernatant. DNA content was determined by Burton reaction. The UV absorption spectra of the investigated samples at room temperature are presented in Fig. 1. Two wide absorption bands in range of k1 ¼ 225 nm and k2 ¼ 270 nm based on the p–p transition of nucleic acid bases are observed for the suspension of thymocytes (curve 1). Addition of C60 FAS to the cell suspension (curve 2) leads to the increase in the value of optical absorption density, to the small shift of the absorption spectrum (k1 ¼ 220 nm and k2 ¼ 275 nm) in comparison with the cell suspension absorption (curve 1), that is the result of the light scattering effect, and also to the appearance of pure C60 FAS absorption band at k3 ¼ 350 nm [4]. We have investigated the influence of 90 min UV irradiation (k ¼ 365 nm) with the light power 1016 photon/(cm2 s) on the optical absorption spectra of samples. The dependence of the optical absorption density at k2 ¼ 275 nm on the time of the investigated sample irradiation is presented in Fig. 2. The observed curves of absorption 1 and 2 were normalized for the initial irradiation times. After 90 min UV irradiation of sample 1 (the thymocytes suspension) without the C60 FAS (curve 1) the increase of the optical absorption density, probably caused by cell DNA double-strand structure injury, is observed. On the contrary, a strong effect is observed for
Fig. 1. UV absorption spectra of the investigated samples at room temperature (relative to buffer): 1––the thymocyte suspension; 2––the thymocyte suspension + C60 FAS.
Fig. 2. Dependence of the optical absorption density on the UV irradiation time by the light (k ¼ 365 nm) for the investigated samples: 1––the thymocyte suspension; 2––the thymocyte suspension + C60 FAS.
sample 2 (the C60 FAS in the thymocytes suspension) in comparison with sample 1: the sharp decrease in the optical absorption density takes place at 30 min after irradiation of the C60 FAS, presented in the thymocytes suspension (curve 2). In this case the tangent value of the angle slope is increased, which testifies to the faster passing of biochemical processes connected with the change of the biological molecules oxidation level and structural conformation. The thymocytes suspension was irradiated by the use of an X-ray apparatus with the exposure dose of 2.58 · 102 C/kg (the absorbed dose was 4.5 Gy). The addition of C60 FAS to the non-irradiated thymocytes suspension does not influence the structural state of DNA cells during 1 h incubation at the temperature of 310 K (see Fig. 3A). However, the significant increase of the DNA fragments accumulation is observed after the irradiation of thymocytes in the presence of 105 M C60 FAS (see Fig. 3B): the content of
Fig. 3. The content of low-molecular DNA fragments (in % to the total DNA content) in the thymocyte suspension during the incubation without additions (1) and in the presence of 105 M C60 FAS (2) in a control (A) and after X-ray irradiation (B); p < 0:05.
P. Scharff et al. / Carbon 42 (2004) 1199–1201
polydesoxyribonucleotides, accumulated for a period of 1 h incubation of the thymocytes suspension, irradiated in the presence of the C60 FAS reaches 195% in comparison with the irradiated suspension without C60 FAS presence in it. It is important to note that this to 42% is greater than in the case with the incubation of the irradiated thymocytes suspension in the presence of the fullerene-containing composite, synthesized on the basis of aminopropylaerosil [2]. Thus, the irradiation of the cellular suspension in the presence of C60 FAS is followed by the intensification of DNA cleavage and accumulation of low-molecular DNA fragments. This can occur, for example, as a result of the excited singlet oxygen interaction with oligonucleotides [5] within the framework of the following mechanism of C60 fullerene interaction with the biomedium under irradiation: p-electron system of the irradiated biomolecules excites C60 fullerene, transfering it as a result of the intercombination conversion from the basic singlet state to the low excited triplet state with energy 2.06 eV. The value of this energy for C60 fullerene is sufficient in order to transfer oxygen from the basic triplet state (3 O2 ) with energy 0.98 eV to the excited singlet state (1 O2 ) due to the non-radiating transmission energy. Excited oxygen is a high reactive molecule, which promotes the formation of the oxygen-containing active radicals and intensification of the oxidation reactions in the biological object. Actually, the comparative analysis of the number of living cells before and after irradiation showed that the quantity of living cells was about 45% after 10 min of irradiation, 20% after 20 min and only 7% after 30 min. Thus, the investigated irradiation effect can be used, for example, for cancer cell killing.
1201
3. Summary We have found that low-energy UV irradiation is sufficient to induce the pro-oxidant activity of C60 FAS and the DNA cleavage is caused by singlet oxygen generated by interaction of the photoexcited C60 group with molecular oxygen. Among the numerous implications of the present revealing, the most exciting prospect includes the use of C60 FAS for photodynamic therapy of transformed cells [1]. Acknowledgements This work was supported by BMBF grant Ukr 02007. S.V.P. is grateful to the DAAD, Germany for providing the Leonard-Euler Scholarship to carry out this research work. References [1] Wilson SR. Biological aspects of fullerenes. In: Fullerenes: chemistry, physics and technology. New York: John Wiley & Sons; 2000. p. 437–65. [2] Prylutskyy YI, Yashchuk VM, Kushnir KM, Golub AA, Kudrenko VA, Prylutska SV, et al. Biophysical studies of fullerenebased composite for bio-nanotechnology. Mater Sci Eng C 2003;23:109–11. [3] Andrievsky GV, Klochkov VK, Karyakina EL, Mchedlov-Petrosyan NO. Studies of aqueous colloidal solutions of fullerene C60 by electron microscopy. Chem Phys Lett 1999;300:392–6. [4] Bulavin L, Adamenko I, Prylutskyy Y, Durov S, Graja A, Bogucki A, et al. Structure of fullerene C60 in aqueous solution. Phys Chem Chem Phys 2000;2:1627–9. [5] Boutorine AS, Tokuyama H, Takasugi M, Isobe H, Nakamura E, Helene C. Singlet oxygen production from fullerene derivatives. Angew Chem Int Ed Engl 1994;33:2462–5.
Effect of X-Ray and UV irradiation of the C60 fullerene aqueous solution on biological samples P. Scharff a, L. Carta-Abelmann a, C. Siegmund a, O.P. Matyshevska b, S.V. Prylutska b, T.V. Koval b, A.A. Golub c, V.M. Yashchuk d, K.M. Kushnir d, Yu.I. Prylutskyy b,* b
a Institute of Physics, TU Ilmenau, Ilmenau D-98684, Germany Department of Biophysics, Faculty of Biology, Kyiv National Shevchenko University, Volodymyrska Str., 64,Kyiv 01033, Ukraine c Faculty of Chemistry, Kyiv National Shevchenko University, Volodymyrska Str., 64, Kyiv 01033, Ukraine d Faculty of Physics, Kyiv National Shevchenko University, Volodymyrska Str., 64, Kyiv 01033, Ukraine
Available online 6 February 2004
Abstract The dependence of the optical absorption density on the UV irradiation time of a biological sample (the suspension of thymocytes, isolated from Wistar rat thymus) and the DNA structural state in cells under X-ray irradiation in the presence of the C60 fullerene aqueous solution were investigated and analyzed in detail. The significant pro-oxidant effect of this material at the low concentration of fullerene C60 (105 M) was revealed. 2004 Elsevier Ltd. All rights reserved. Keywords: A. Fullerene; C. Adsorption; D. Bioactivity
1. Introduction
2. Experiment
C60 fullerenes have some potential biological activity in vitro and in vivo [1], e.g. inhibition of HIV protease and lipid peroxidation, antiviral and antioxidant effects, specific cleavage of DNA, which has attracted considerable attention and has become a challenging research field at present. However, the desire to study the biological effects of C60 fullerenes is hindered by the strong hydrophobicity of these merely carbon molecules for they have difficulties in reacting with biological systems. In that case, for example, it is the key to prepare watersoluble fullerene derivatives to be suitable for biological study [2]. But the recently obtained C60 fullerene aqueous solutions (C60 FAS) with different C60 concentrations [3] opens the prospect for their intensive use in biological studies. The purpose of this work was to investigate the bioactivity of C60 FAS in the biological medium in dependence on the UV and X-ray irradiation.
The samples of C60 FAS were prepared as follows. We have used a saturated solution of pure C60 (a purity >99.5%) in toluene and the same amount of distilled water in an open beaker. Two phases are formed. Then we applied an ultrasonic bath as long as the toluene needs to evaporate completely. Meanwhile the water phase became yellow colored, indicating that a water– fullerene solution has been formed. Thereafter we filtered the water solution from undissolved C60 . As a result we prepared the C60 FAS samples with different concentration of C60 fullerene in water up to 1.4 mg/ml (for comparison, the solubilities of C60 are 1.7 and 2.9 mg/ml in benzene and toluene, respectively). Thymus (100 mg) of decapitated Wister rats was filtered through the nylon sieve into the buffer (3 mM Na2 HPO4 Æ 12H2 O, 5 mM KCl, 120 mM NaCl, 10 mM glucose, 10 mM HEPES, pH 7.4, 4 mM NaHCO3 , 1 mM CaCl2 , 1 mM MgCl2 ). The total volume of cell suspension was 10 ml. The cells were washed twice by centrifugation (1500g, 10 min). The sediment was resuspended in the same buffer to the concentration 107 cells/ml. The DNA fragmentation was estimated by the polydesoxyribonucleotides (the low-molecular fragments of
*
Corresponding author. Tel.: +380-44-266-5408; fax: +380-44-2520827. E-mail address: [email protected] (Yu.I. Prylutskyy). 0008-6223/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbon.2003.12.055
1200
P. Scharff et al. / Carbon 42 (2004) 1199–1201
DNA) accumulation in the cells. Cells were lysed in the buffer (5 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.5% Triton X-100). The samples were centrifuged (1500g, 20 min) for the separation of intact chromatin (in the pellet) from DNA fragments in supernatant. DNA content was determined by Burton reaction. The UV absorption spectra of the investigated samples at room temperature are presented in Fig. 1. Two wide absorption bands in range of k1 ¼ 225 nm and k2 ¼ 270 nm based on the p–p transition of nucleic acid bases are observed for the suspension of thymocytes (curve 1). Addition of C60 FAS to the cell suspension (curve 2) leads to the increase in the value of optical absorption density, to the small shift of the absorption spectrum (k1 ¼ 220 nm and k2 ¼ 275 nm) in comparison with the cell suspension absorption (curve 1), that is the result of the light scattering effect, and also to the appearance of pure C60 FAS absorption band at k3 ¼ 350 nm [4]. We have investigated the influence of 90 min UV irradiation (k ¼ 365 nm) with the light power 1016 photon/(cm2 s) on the optical absorption spectra of samples. The dependence of the optical absorption density at k2 ¼ 275 nm on the time of the investigated sample irradiation is presented in Fig. 2. The observed curves of absorption 1 and 2 were normalized for the initial irradiation times. After 90 min UV irradiation of sample 1 (the thymocytes suspension) without the C60 FAS (curve 1) the increase of the optical absorption density, probably caused by cell DNA double-strand structure injury, is observed. On the contrary, a strong effect is observed for
Fig. 1. UV absorption spectra of the investigated samples at room temperature (relative to buffer): 1––the thymocyte suspension; 2––the thymocyte suspension + C60 FAS.
Fig. 2. Dependence of the optical absorption density on the UV irradiation time by the light (k ¼ 365 nm) for the investigated samples: 1––the thymocyte suspension; 2––the thymocyte suspension + C60 FAS.
sample 2 (the C60 FAS in the thymocytes suspension) in comparison with sample 1: the sharp decrease in the optical absorption density takes place at 30 min after irradiation of the C60 FAS, presented in the thymocytes suspension (curve 2). In this case the tangent value of the angle slope is increased, which testifies to the faster passing of biochemical processes connected with the change of the biological molecules oxidation level and structural conformation. The thymocytes suspension was irradiated by the use of an X-ray apparatus with the exposure dose of 2.58 · 102 C/kg (the absorbed dose was 4.5 Gy). The addition of C60 FAS to the non-irradiated thymocytes suspension does not influence the structural state of DNA cells during 1 h incubation at the temperature of 310 K (see Fig. 3A). However, the significant increase of the DNA fragments accumulation is observed after the irradiation of thymocytes in the presence of 105 M C60 FAS (see Fig. 3B): the content of
Fig. 3. The content of low-molecular DNA fragments (in % to the total DNA content) in the thymocyte suspension during the incubation without additions (1) and in the presence of 105 M C60 FAS (2) in a control (A) and after X-ray irradiation (B); p < 0:05.
P. Scharff et al. / Carbon 42 (2004) 1199–1201
polydesoxyribonucleotides, accumulated for a period of 1 h incubation of the thymocytes suspension, irradiated in the presence of the C60 FAS reaches 195% in comparison with the irradiated suspension without C60 FAS presence in it. It is important to note that this to 42% is greater than in the case with the incubation of the irradiated thymocytes suspension in the presence of the fullerene-containing composite, synthesized on the basis of aminopropylaerosil [2]. Thus, the irradiation of the cellular suspension in the presence of C60 FAS is followed by the intensification of DNA cleavage and accumulation of low-molecular DNA fragments. This can occur, for example, as a result of the excited singlet oxygen interaction with oligonucleotides [5] within the framework of the following mechanism of C60 fullerene interaction with the biomedium under irradiation: p-electron system of the irradiated biomolecules excites C60 fullerene, transfering it as a result of the intercombination conversion from the basic singlet state to the low excited triplet state with energy 2.06 eV. The value of this energy for C60 fullerene is sufficient in order to transfer oxygen from the basic triplet state (3 O2 ) with energy 0.98 eV to the excited singlet state (1 O2 ) due to the non-radiating transmission energy. Excited oxygen is a high reactive molecule, which promotes the formation of the oxygen-containing active radicals and intensification of the oxidation reactions in the biological object. Actually, the comparative analysis of the number of living cells before and after irradiation showed that the quantity of living cells was about 45% after 10 min of irradiation, 20% after 20 min and only 7% after 30 min. Thus, the investigated irradiation effect can be used, for example, for cancer cell killing.
1201
3. Summary We have found that low-energy UV irradiation is sufficient to induce the pro-oxidant activity of C60 FAS and the DNA cleavage is caused by singlet oxygen generated by interaction of the photoexcited C60 group with molecular oxygen. Among the numerous implications of the present revealing, the most exciting prospect includes the use of C60 FAS for photodynamic therapy of transformed cells [1]. Acknowledgements This work was supported by BMBF grant Ukr 02007. S.V.P. is grateful to the DAAD, Germany for providing the Leonard-Euler Scholarship to carry out this research work. References [1] Wilson SR. Biological aspects of fullerenes. In: Fullerenes: chemistry, physics and technology. New York: John Wiley & Sons; 2000. p. 437–65. [2] Prylutskyy YI, Yashchuk VM, Kushnir KM, Golub AA, Kudrenko VA, Prylutska SV, et al. Biophysical studies of fullerenebased composite for bio-nanotechnology. Mater Sci Eng C 2003;23:109–11. [3] Andrievsky GV, Klochkov VK, Karyakina EL, Mchedlov-Petrosyan NO. Studies of aqueous colloidal solutions of fullerene C60 by electron microscopy. Chem Phys Lett 1999;300:392–6. [4] Bulavin L, Adamenko I, Prylutskyy Y, Durov S, Graja A, Bogucki A, et al. Structure of fullerene C60 in aqueous solution. Phys Chem Chem Phys 2000;2:1627–9. [5] Boutorine AS, Tokuyama H, Takasugi M, Isobe H, Nakamura E, Helene C. Singlet oxygen production from fullerene derivatives. Angew Chem Int Ed Engl 1994;33:2462–5.