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Biological effects of mixed-ion beams. Part 2: The relative biological effectiveness of CHO-K1 cells irradiated by mixed- and single-ion beams
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Biological effects of mixed-ion beams. Part 2: The relative biological effectiveness of CHO-K1 cells irradiated by mixed- and single-ion beams Joanna Czuba, , Dariusz Banaśa,b, Janusz Braziewicza,b, Iwona Buraczewskac, Marian Jaskóład, Urszula Kaźmierczake, Andrzej Kormand, Anna Lankoffg,c, Halina Lisowskag, Zygmunt Szeflińskie, Maria Wojewódzkac, Andrzej Wójcikf,g ⁎
a
Jan Kochanowski University, Institute of Physics, ul. Świętokrzyska 15, 25-406 Kielce, Poland Holy Cross Cancer Center, ul. Arwińskiego 3, 25-734 Kielce, Poland c Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195 Warsaw, Poland d National Centre for Nuclear Research, ul. Andrzeja Sołtana 7, 05-400 Otwock-Świerk, Poland e Heavy Ion Laboratory at the University of Warsaw, ul. Pasteura 5a, 02-093 Warsaw, Poland f Department of Molecular Bioscience, Centre for Radiation Protection Research, The Wenner-Gren Institute, Stockholm University, Universitetsvagen 10, 114 18 Stockholm, Sweden g Jan Kochanowski University, Institute of Biology, ul. Świętokrzyska 15, 25-406 Kielce, Poland b
HIGHLIGHTS
relative biological effectiveness (RBE) of low-energy ions was estimated. • The for the individual and mixed C and O beams was calculated. • RBE fractions of the CHO-K1 cells after such irradiations are presented. • Survival specific LET values, carbon and oxygen ions have equal RBEs. • AtSurvival fractions do not depend on the mixed beam composition. • 12
16
ARTICLE INFO
ABSTRACT
Keywords: Relative biological effectiveness Ions beams CHO-K1 cells
The relative biological effectiveness (RBE) values were determined for single- and mixed-ion beams containing carbon and oxygen ions. The CHO-K1 cells were irradiated with beams with the linear energy transfer (LET) values of 236–300 and 461–470 keV/μm for 12C and 16O ions, respectively. The RBE was estimated as a function of dose, survival fraction (SF) and LET. The SF was not affected by varying contributions of the constituent ions to the total mixed dose. The RBE has the same value for single-ion exposures with ions with LET 300 (12C) and 470 keV/μm (16O).
1. Introduction The relative biological effectiveness (RBE) is a parameter widely used in the ion therapy related to the biologically effective dose (BED) (Krämer et al., 2000). BED is used in the cancer therapy to take into account the physical dose absorbed by a live material as well as the behavior of cells after irradiation by ion beams (Krämer et al., 2000). The RBE is a component of the BED value that provides information on the biological response of irradiated cells (Krämer et al., 2000). Therefore, knowledge of RBE and its dependence on other important parameters, such as linear energy transfer (LET) and absorbed dose, is
⁎
important for improvement of the radiation therapy. Sørensen et al. (2011) reviewed available data on RBE for various cell, types and energies of single ions. For the Chinese hamster ovary (CHO-K1) cells, RBE values for a single exposure to carbon ions have been reported, albeit for a limited range of the linear energy transfer values; RBE values for other types of ions, such as neon, iron, argon ions was also reported (Sørensen et al., 2011). Also, Scholz et al. (1997) reported a survival fraction of the CHO-K1 cells after irradiations with the oxygen ions, but the data are only for a high energy of ions and simulation purposes, without information necessary to calculate RBE. There are other papers complimenting experimental data for the CHO-K1 cells in
Corresponding author. E-mail address: [email protected] (J. Czub).
https://doi.org/10.1016/j.apradiso.2018.12.001 Received 15 May 2017; Received in revised form 2 December 2018; Accepted 2 December 2018 0969-8043/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Czub, J., Applied Radiation and Isotopes, https://doi.org/10.1016/j.apradiso.2018.12.001
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previous publications (Weyrather et al., 1999; Mehnati et al., 2005; Czub et al., 2008, and review in Sørensen et al., 2011); in these studies, RBE was calculated for a single exposure to carbon ions with LETs of 236, 300 and 257 keV/μm, as well as oxygen ions with a LET of 470 keV/μm. Additionally, RBE was estimated as a function of dose, survival fraction and LET. The data for single exposures of the CHO-K1 cells to carbon and oxygen ions with specific LET values presented in this paper have, to our knowledge, not previously been reported. However, single-ion beams are rare in the nature, and mixed beams containing two or more ions and particles are most common. Mixed beams are always present, for example, in the outer space (Reitz et al., 1995) and, therefore, their effect on human cells is important for astronauts (Siranart et al., 2016). In a spacecraft, mixed beams appear when a flux of space ions and particles interact with parts of the craft (Obe et al., 1997; Edwards, 2001; Miller, 2003). Mixed beams also occur on Earth. One of such examples is the hadron therapy, in particular, the neon therapy, which is used against cancer cells to treat tumors in human bodies (Stelzer, 1998). In this therapy, neon ions undergo a process of fragmentation creating ions of other elements, such as carbon and oxygen (Stelzer, 1998). Therefore, it is important to study the impact of mixed ion beams, especially those containing carbon and oxygen ions, on live cells. In the past, only Kanai et al. (1997) studied the effect of two simultaneously accelerated ions (carbon and helium) on live cells. Furthermore, mixed beams containing photons and various types of ions or particles were used sequentially or simultaneously to irradiate cells of various types (a review in Staaf, 2012). Here, we examine the impact of two simultaneously accelerated heavy ions, namely, carbon and oxygen, on cell survival. The CHO-K1 cells were irradiated with three mixed-ion beams with the LET values of 236, 246 and 256 keV/ μm for the carbon ions, and 469, 465 and 461 keV/μm for the oxygen ions. RBE values were calculated for these mixed beams, which constitutes new scientific information.
2.2. Beam transport At this point, mixed- or single-ion beams from the cyclotron were scattered by a gold foil (20 mg/cm2 thick). After the scattering, a fraction of the ions were registered by a detector oriented at an angle of 20° to the direction of the primary ion beam (further on referred to as “20° Detector”), and a part of the ions passed through the gold foil. The dosimetry was based on readings of the 20° Detector (see the “Dosimetry” Section). The ions that had passed through the golden disc were collimated at a distance of 233 cm from the golden disc to a size of 1 × 1 cm2 and then passed through a Havar foil (2.3 mg/cm2 thick) at the end of the ion pipe and a specific air layer. Finally, the ions reached the cells inside a specially adapted Petri dish; the beam was oriented perpendicularly to the dish bottom. To change the energy of the incident ions, the Petri dishes were positioned in the air at various distances from the end of the ion pipe. 2.3. Procedure for irradiation of cells in the specially adapted Petri dish In this experimental system, we created a procedure to slide the Petri dish in a fixed beam with a step of 1 cm. This process was activated by an electrical pulse triggered by a certain number of counts in the 20° Detector. The values detector counts were necessary to calculate the dose absorbed by the cells (see the “Dosimetry” Section). The Petri dish moving process is controlled by a custom-written computer program. 2.4. Beam profile The values characterizing the beam profile were defined as ratios of the numbers of counts from the 0° and 20° Detectors. In this measurement, the position of the detector oriented at 0° to the primary beam (the 0° Detector) was changed at a specified step. The results of a measure of the mixed-beam profile were reported by Czub et al. (2018). The single-ion beam findings were reported in a paper by Czub et al. (2008). Based on that information, the estimated homogeneity of the mixed- and single-ion beams was 95%.
2. Materials and methods The experimental system and its the individual components including a special Petri dish, irradiation procedure, and results of the beam profile measurements were described previously (Czub et al., 2006, 2008, 2009). The production of mixed-ion beams was described by Czub et al. (2018).
2.5. Cell culture condition CHO-K1 cells were grown in the McCoy's 5A medium (No. BE12-ml 688F, Lonza, Switzerland) enriched with 10% Fetal Bovine Serum (No. ECS0180L, Euroclone, Italy) and 1% antibiotics (penicillin-streptomycin, No. 03-031-1B, Biological Industries, USA). The cells were passaged every two days with a 0.25% Trypsin-EDTA solution (No. 25200056, Gibco, England) to separate cells from the bottom of Petri dish. After four passages, the cells were used for the experiments.
2.1. Production of ion beam The mixed-ion beam containing carbon and oxygen ions was produced by a cyclotron at the Heavy Ion Laboratory of the University of Warsaw, Poland. The ions were mixed in an Electron Cyclotron Resonance category ion source. Two separate bottles with individual gases were connected to the ion source, one containing methane for the carbon ions and the other containing oxygen oxide for the oxygen ions. The mixed-ion beam from the ion source was injected into the cyclotron, where the mixture of the ions was accelerated. Two ions could be accelerated by a cyclotron simultaneously when their ratios q/m (q – charge, m - mass) have the same value (Kanai et al., 1997). In this case, q= +3 and + 4 for the carbon ions with m = 12 amu and oxygen ions with m = 16 amu, respectively. When the energy of the mixed-ion beam had reached a specified value, the beam was directed to a scattering chamber. This chamber was the first part of the experimental system intended for radiobiological studies. The single-ion beams containing only carbon or oxygen ions were also produced with an ECR-type ion source. In that case, the ion source was connected to a single gas cylinder with a suitable gas for a singleion beam. After acceleration, the beam was directed to the experimental hall of the cyclotron hosting a radiobiological setup used to irradiate live cells.
2.6. Survival test The CHO-K1 cells were seeded and cultured on a Mylar-foil (6 µm thick) bottom of a specially designed Petri dish. This Petri dish represented a plastic rim 1 cm high and 4.8 cm in diameter. The cells were plated at 150, 200, 300 and 500 cells per Petri dish, 10 h before the radiation exposure. The number of cells used in a single Petri dish depended on the absorbed dose and was selected experimentally in preliminary tests. These preliminary tests had shown that there was no risk that cell colonies would be connected when this number of cells per Petri dish were used. The cells were counted with a Countess Automated Cell Counter (No. C10227, Invitrogen, USA) with Countess Cell Counting Chamber Slides (No. C10312, Invitrogen, USA). In this counting procedure, cells in a single commercially available Petri dish (60 mm in diameter, No. 353004, Falcon, USA) were detached from the bottom with a trypsin solution (Biological Industries, No. 03–046-1A) administered for 3 min at 37 °C and 5% CO2. Next, a medium with additives (see the Cell culture condition Section) was added to the cells. 2
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Afterwards, 30 μL of the cell suspension was mixed with a trypan blue solution (No. T10282, Invitrogen, USA). Ten microliters of this mixture were then used for an automated cell counting. After the number of cells per milliliter was determined, the cell suspension was diluted with a phosphate buffered saline (PBS) solution and transferred into the specially designed Petri dish. Next, the specially designed Petri dish was fully filled with the McCoy's 5A medium containing additives (see the Cell culture condition Section). The Petri dish was then closed with a bespoke 3D-printed lid. That was done at the Institute of the Nuclear Chemistry and Technology in Warsaw, Poland. After that, the specially designed Petri dish was transported to the Heavy Ion Laboratory (HIL) of the University of Warsaw, Poland on iced water. After irradiation at HIL, the cells were taken back to the Institute of the Nuclear Chemistry and Technology on iced water to replace the medium. After that, the specially designed Petri dish was incubated at 37 °C in an atmosphere with 5% CO2 for 7 days. After the incubation, the cell colonies were fixed with methanol (Warchem, Poland) and stained with a 10% Giemsa solution (Pol-aura, Poland). Finally, the colonies were counted with an automated counter Scan500 (Interscience, France) or visually. The survival fraction was calculated as
SF =
F 12C =
12C N20 N0 12C , 12C N20
F 16O =
16O N20 N0 16O , 16O N20
where N is the number of counts of ions with superscripts specifying the type of the ions and the subscripts specifying the type of the detector used for the measurement; the apostrophe in the superscript indicates that the measurement was made before the cells were exposed (in other cases, the measurement was made during the cells irradiation). The sums over the i and k values are present in the absorbed dose equations because the beam spectra are not monoenergetic and the LET value changes when the beams passes through the cell layer, respectively. 2.8. Irradiation of cells with gamma rays from
60
Co
The procedure used to irradiate the cells with gamma rays from a Co source was described in detail in the paper by Czub et al. (2008); so, only a short description is provided here. The CHO-K1 cells were irradiated with gamma rays from 60Co to determine the RBE value. Gamma irradiations were performed at the Holy Cross Cancer Center (HCC) in Kielce, Poland. The cells were seeded in a Petri dish at the Jan Kochanowski University (JKU) in Kielce, Poland and transported to HCC. After the exposure, the cells were convoyed back to JKU, where the nutrient solution was refreshed.
60
A , B
where A is the number of colonies/number of cells in irradiated dishes and B is the number of colonies/number of cells in control dishes (Munshi et al., 2005) 2.7. Dosimetry The dosimetry for the single-ion and mixed-ion beams was described in detail in the papers by Czub et al. (2008) and Czub et al. (2018), respectively. Here, only a short description is provided. The measurement of the dose to be absorbed by the cells was performed before the exposure of the cells, and the dose delivery was controlled during irradiation of the live matter. For this purpose, we used the data collected with two silicon detectors (the 20° and 0° Detectors) with collimators of the diameters of 4 and 0.54 mm. Before irradiation of the cells, beam spectra obtained with these detectors were recorded, and the numbers of ions and the fluencies were calculated from these spectra. With the relationship between the counts by the two detectors known, it was possible to control the dose delivered to the cells using the counts registered only by the 20° Detector. The absorbed doses for the single- and mixed-ion beams were calculated using the following equations (Czub et al., 2008):
3. Results
a) for the single-ion beams
Fig. 1 shows the results of the survival test (i.e., survival curves for four single- and three mixed-ion irradiations, as well as the gamma exposure). The survival curves for the single- and the mixed-ion beam containing carbon ions with LET = 236 keV/μm and oxygen ions with LET = 470 keV/μm are also presented in our previous paper (Czub et al., 2018). The survival curve for the gamma irradiation was first published in the paper by Czub et al. (2008). A linear-quadratic model was fitted to the data in Fig. 1 using the equation (Weyrather et al., 1999)
9
D12C (Gy ) = 1.6 10
LETi12C
9 k=1
i
9
D16O (Gy ) = 1.6 10
LETi16O
9 k= 1
i
keV µm
Fi12C
keV µm
Fi16O
1 cm2
1 cm2
k
1
C
( )
C
( )
k
g cm3
1
g cm3
3.1. Linear energy transfers (LET) and the energies of the carbon and oxygen ions in the mixed-ion beams Table 1 lists the energies and the corresponding linear energy transfer values for the carbon and oxygen ions in the mixed ion beams. Both the values were determined at the cell entrance. The LET values presented in Table 1 differ from those calculated with the SRIM program (Ziegler et al., 2010) by 0.2–3%. This is due to the uncertainty of the fitting procedure used for the function between the energy and the LET of the ions. 3.2. Survival fractions in the absorbed dose function
,
;
aD bD2,
b) for the mixed-ion beams:
SF = e
D12C + 16O = D12C + D16O ,
where SF is the survival fraction, D is the absorbed dose, while a and b are coefficients from the fitting procedure listed in Table 2. The fitting was performed with the curve fitting toolbox in Matlab R2010b.
where , are the total absorbed doses from the mixedion beam, carbon ions and oxygen ions, respectively, ρ = 1 is the water density, i is the channel of the spectrum at the angle of 0° with respect to the incident beam, k is the cell thickness (Fisher et al., 1985), C is a constant corresponding to the ratio of the beam area (1 × 1 cm2) to the area of the collimator with the 0.54 mm diameter. F12C and F16O are the numbers of the carbon-ion (12C) and oxygen-ion (16O) irradiated cells calculated using the equations:
D12C + 16O
D12C ,
D16O
3.3. RBE values as functions of the dose, survival fraction (SF) and linear energy transfer (LET) Figs. 2–4 show the RBE values as functions of the dose, survival fraction and LET, respectively. 3
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Table 1 Energies and the linear energy transfer values at the entrances of the cell for the components of the mixed-ion beams. Mixed-ion beam
Ions in the mixed-ion beam Carbon
12
C(LET=236 keV/μm)+16O(LET=469 keV/μm) C(LET=246 keV/μm)+16O(LET=465 keV/μm) 12 C(LET=256 keV/μm)+16O(LET=461 keV/μm) 12
Oxygen
Energy (MeV)
LET (keV/μm)
Energy (MeV)
LET (keV/μm)
78 75 71
236 246 256
86 87 88
469 465 461
Fig. 1. Survival fractions as functions of the dose for single- and mixed-ion beams with different LET values. The error bars represent standard deviations.
contributions to the doses from the carbon and oxygen ions. Table 3 contains data for the mixed-ion beam 12C (LET=256 keV/μm)+16O (LET=461 keV/μm) corresponding to the survival curve shown in Fig. 5. Table 4 presents data for the mixed ion beam 12C (LET=236 keV/μm)+16O (LET=469 keV/μm) corresponding to the survival curve presented in Fig. 6. Contributions from the constituents of the 12C (LET=246 keV/μm)+16O (LET=465 keV/μm) beam are not presented because each survival fraction shown in Fig. 1 was measured only once. The sequence of data in Tables 3 and 4 corresponds to the order of the data in Figs. 5 and 6.
Table 2 Parameters of the linear-quadratic fits to the experimental data shown in Fig. 1. Mixed-ion beam 12
16
C (LET=236 keV/μm)+ O (LET=469 keV/μm) C (LET=246 keV/μm)+16O (LET=465 keV/μm) 12 C (LET=256 keV/μm)+16O (LET=461 keV/μm) Single-ion beam 12 C (LET=236 keV/μm) 12 C (LET=257 keV/μm) 12 C (LET=300 keV/μm) 16 O (LET=470 keV/μm) 60 Co 12
a
b
1.280 1.105 0.625 a 1.098 0.909 0.773 0.772 0.173
− 0.030 − 0.048 − 0.008 b 2.47•10−10
0.018
3.5. The RBEm values for single-ion beams as compared with data by others authors
3.4. Survival fractions after irradiations with the mixed-ion beams with varying ratios of the carbon- and oxygen-ion dose
Fig. 7 and Table 5 present the RBEm data calculated for a single-ion beam along with data from other papers, namely, Weyrather et al., 1999, Mehnati et al. (2005), and Czub et al. (2008). RBEm is the
Tables 3 and 4 list total mixed-ion doses with information on the
Fig. 2. RBE values as functions of the dose for the single- and mixed-ion beams. Error bars represent standard deviations. 4
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Fig. 3. RBE values as functions of the survival fractions for the single- and mixed-ion beams. Error bars represent standard deviations.
Fig. 4. RBE values as functions of the LET values for the single-ion beams and the arithmetical average LET values for the mixed-ion beams. Table 3 Total mixed-ion dose from the beams 12C (LET=256 keV/μm)+16O (LET=461 keV/μm) with various contributions from the two ions. Min. and Max. indicate the minimal and maximal contributions from the corresponding ions to the total dose.
Table 4 Total mixed-ion dose from the beams 12C (LET=236 keV/µm)+16O (LET=469 keV/µm) with various contributions from the two ions. Min. and Max. indicate the minimal and maximal contributions from the corresponding ions to the total dose.
Total mixed- ion dose
Carbon ion contribution to the dose (%)
Oxygen ion contribution to the dose (%)
Total mixed ion dose
Carbon ion contribution to the dose (%)
Oxygen ion contribution to the dose (%)
0.3 0.3 0.3 0.4 0.4 0.4 0.7 0.7 0.7 0.7 1 1 1 1 3 3 4 4 7 7 7 Min. Max. Average
66 64 67 35 63 63 44 33 44 37 61 48 53 35 43 25 37 43 45 36 39 25 67 47
34 36 33 65 37 37 56 67 56 63 39 52 47 65 57 75 63 57 55 64 61 33 75 53
0.2 0.2 0.2 0.4 0.4 1.5 1.5 3.0 3.0 3.8 3.8 Min. Max Mean
44 51 49 42 45 44 43 37 47 35 32 32 51 43
56 49 51 58 55 56 57 63 53 65 68 49 68 57
maximal value of RBE calculated by using only the parameter a of the fit (Weyrather et al., 1999). The RBEm values reported the literature (Fig. 7) were calculated by taking into account the gamma radiation values used in those studies. 4. Conclusions and discussion The RBE parameter is an important factor for the ion therapy and radiological protection, and its knowledge helps to better understand 5
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Fig. 5. Survival fraction as a function of the mixed-ion beam dose for
Fig. 6. Survival fraction as a function of the mixed-ion beam dose for
12
12
C (LET=256 keV/μm)+16O (LET=461 keV/μm). Error bars are equal to ± 10%.
C (LET=236 keV/μm)+16O (LET=469 keV/μm) beam. Error bars are equal to ± 10%.
effects of radiation on living cells. RBE values were obtained for cells of many types irradiated primarily with single-ion beams. However, few studies reported RBE values together with survival fractions for CHOK1 (Weyrather et al., 1999; Mehnati et al., 2005; Czub et al., 2008). The data generated in this study have supplemented this literature. However, the effects that mixed-ion beams exert on cell were studied for many years by many research groups who used various types of radiation, ways of irradiating cells (simultaneous or separate), cell types and biological tests (review in Staaf, 2012). To our knowledge, only Kanai et al. (1997) irradiated living cells with ions of two types accelerated simultaneously. However, the authors of that publication used
cells of a different type, namely, CHO-V79. Kanai et al. (1997) did not provide the survival curve fitting parameters for the mixed beams, making it impossible to determine the RBE parameters from their data. Data on simultaneous exposure to carbon and oxygen ions are not available in the literature. In this study, an experimental setup implemented in the Heavy Ion Laboratory at the University of Warsaw, Poland was used to irradiate live biological material. The CHO-K1 cells were exposed to four single- and three mixed-ion beams with different LET values. After the irradiations, survival tests were performed to determine the RBE values. These values were calculated and presented as functions of the absorbed dose, survival fraction (SF) and linear 6
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is new information for these cells. Additionally, survival fractions were calculated after irradiations with the mixed-ion beams with varying relative contributions to the dose from the constituent ions. These results have shown that variations of the relative contributions from the 12 C and 16O ions in the mixed beam to the dose have no effect on the survival fractions of the cells. These findings suggest that the mixed-ion beams exert additive effects on cells. Acknowledgements This work was supported by the statutory research No. 612 424 at the Jan Kochanowski University, Kielce, Poland. References Czub, J., Banaś, D., Błaszczyk, A., Braziewicz, J., Buraczewska, I., Choiński, J., Górak, U., Jaskóła, M., Korman, A., Lankoff, A., Lisowska, H., Łukaszek, A., Szefliński, Z., Wójcik, A., 2009. Cell survival and chromosomal aberrations in CHO-K1 cells irradiated by carbon ions. Appl. Radiat. Isot. 67, 447–453. https://doi.org/10.1016/j. apradiso.2008.06.016. Czub, J., Banaś, D., Braziewicz, J., Choiński, J., Jaskóła, M., Korman, A., Szefliński, Z., Wójcik, A., 2006. An irradiation facility with a horizontal beam for radiobiological studies. Radiat. Prot. Dosim. 122, 207–209. https://doi.org/10.1093/rpd/ncl518. Czub, J., Banaś, D., Błaszczyk, A., Braziewicz, J., Buraczewska, I., Choiński, J., Górak, U., Jaskóła, M., Korman, A., Lankoff, A., Lisowska, H., Łukaszek, A., Szefliński, Z., Wójcik, A., 2008. Biological effectiveness of 12C and 20Ne ions with very high LET. Int. J. Radiat. Biol. 84. https://doi.org/10.1080/09553000802389652. Czub, J., Banaś, D., Braziewicz, J., Buraczewska, I., Jaskóła, M., Kaźmierczak, U., Korman, A., Lankoff, A., Lisowska, H., Szefliński, Z., Wojewódzka, M., Wójcik, A., 2018. Biological effects of mixed-ion beams. Part 1: effect of irradiation of the CHOK1 cells with a mixed-ion beam containing the carbon and oxygen ions. Appl. Radiat. Isot. 139. https://doi.org/10.1016/j.apradiso.2018.05.028. Edwards, A.A., 2001. RBE of radiations in space and the implications for space travel. Phys. Med. 17 (Suppl 1), 147–152. Fisher, D.R., Frazier, M.E., Andrews Jr., T.K., 1985. Energy distribution and the relative biological effects of internal alpha emitters. Radiat. Prot. Dosim. 13, 223–227. https://doi.org/10.1093/rpd/13.1-4.223. Kanai, T., Furusawa, Y., Fukutsu, K., Itsukaichi, H., Eguchi-Kasai, K., Ohara, H., 1997. Irradiation of mixed beam and design of spread-out Bragg peak for heavy-ion radiotherapy. Radiat. Res. 147, 78–85. https://doi.org/10.2307/3579446. Krämer, M., Jäkel, O., Haberer, T., Kraft, G., Schardt, D., Weber, U., 2000. Treatment planning for heavy-ion radiotherapy: physical beam model and dose optimization. Phys. Med. Biol. 45, 3299–3317. https://doi.org/10.1088/0031-9155/45/11/313. Mehnati, P., Morimoto, S., Yatagai, F., Furusawa, Y., Kobayashi, Y., Wada, S., Kanai, T., Hanaoka, F., Sasaki, H., 2005. Exploration of “over kill effect” of high-LET Ar- and Feions by evaluating the fraction of non-hit cell and interphase death. J. Radiat. Res. 46, 343–350. https://doi.org/10.1269/jrr.46.343. Miller, J., 2003. Proton and heavy ion acceleration facilities for space radiation research. Gravit. Space Biol. Bull. 16, 19–28. Munshi, A., Hobbs, M., Meyn, R.E., 2005. Clonogenic cell survival assay. Methods Mol. Med. 110, 21–28. https://doi.org/10.1385/1-59259-869-2:021. Obe, G., Johannes, I., Johannes, C., Hallman, K., Reitz, G., Facius, R., 1997. Chromosomal aberrations in blood lymphocytes of astronauts after long-term space flights. Int. J. Radiat. Biol. 72, 727–734. https://doi.org/10.1080/095530097142889. Reitz, G., Facius, R., Sandler, H., 1995. Radiation protection in space. Acta Astronaut. 35 (4–5), 313–338. https://doi.org/10.1016/0094-5765(95)98735-R. Siranart, N., Blakely, E.A., Cheng, A., Handa, N., Sachs, R.K., 2016. Mixed beam murine harderian gland tumorigenesis: predicted dose-effect relationships if neither synergism nor antagonism occurs. Radiat. Res. 186, 577–591. https://doi.org/10.1667/ RR14411.1. Staaf, E., 2012. Cellular effects after exposure to mixed beams of ionizing radiation. (PhD work) 〈https://www.diva-portal.org/smash/get/diva2:557692/FULLTEXT01.pdf〉. Stelzer, H., 1998. Tumor therapy with heavy ions at GSI. Nucl. Phys. B (Proc. Suppl.) 61B, 650–657. https://doi.org/10.1016/S0920-5632(97)00633-6. Scholz, M., Kellerer, A.M., Kraft-Weyrather, W., Kraft, G., 1997. Computation of cell survival in heavy ion beams for therapy. Radiat. Environ. Biophys. 36, 59–66. https://doi.org/10.1007/s004110050055. Sørensen, B.S., Overgaard, J., Bassler, N., 2011. In vitro RBE-LET dependence for multiple particle types. Acta Oncol. 50, 757–762. https://doi.org/10.3109/0284186X.2011. 582518. Weyrather, W.K., Ritter, S., Scholz, M., Kraft, G., 1999. RBE for carbon track-segment irradiation in cell lines of differing repair capacity. Int. J. Radiat. Biol. 75, 1357–1364. https://doi.org/10.1080/095530099139232. Ziegler, J.F., Ziegler, M.D., Biersack, J.P., 2010. SRIM - The stopping and range of ions in matter (2010). Nucl. Instrum. Methods Phys. Res. Sect. B Beam Interact. Mater. At. 268, 1818–1823. https://doi.org/10.1016/j.nimb.2010.02.091.
Fig. 7. The RBEm values calculated for the single-ion beams presented alongside with data from others publications (Table 5). The numerical values indicate data from those studies. Table 5 A compilation of the LET values collected in this study and announced in the literature (also presented in Fig. 7). Paper
Ion
LET (keV/μm)
Weyrather et al. (1999) Weyrather et al. (1999) Mehnati et al. (2005) Weyrather et al. (1999) Mehnati et al. (2005) Mehnati et al. (2005) Mehnati et al. (2005) Weyrather et al. (1999) Weyrather et al. (1999) This work This work Weyrather et al. (1999) This work Weyrather et al. (1999) Czub et al. (2008) This work Weyrather et al. (1999) Czub et al. (2008) Czub et al. (2008) Mehnati et al. (2005) Mehnati et al. (2005) Mehnati et al. (2005) Mehnati et al. (2005) Mehnati et al. (2005) Czub et al. (2008) Czub et al. (2008) Czub et al. (2008) Mehnati et al. (2005) Mehnati et al. (2005) Mehnati et al. (2005) Mehnati et al. (2005)
12
13.7 16.8 20 32.4 60 82 99 103 153.5 232 257 275.1 300 339.1 438 470 482.7 576 832 102 120 171 228 341 933 1245 1616 1640 780 1200 2000
C C C 12 C 12 C 12 C 12 C 12 C 12 C 12 C 12 C 12 C 12 C 12 C 12 C 16 O 12 C 12 C 12 C 20 Ne 20 Ne 20 Ne 20 Ne 20 Ne 20 Ne 20 Ne 20 Ne 40 Ar 56 Fe 56 Fe 56 Fe 12 12
energy transfer (LET). Based on our data, we have concluded that RBE values decrease with the increasing dose and LET, and increase with the decreasing SF values. These findings are in line with the overkill effect and agree with the findings by Weyrather et al. (1999) for single-ion irradiations. The RBEm values calculated for the single-ion beams for the CHO-K1 cells and presented as functions of LET compliment the literature data for the cells of the other types reported in the papers by Weyrather et al. (1999), Mehnati et al. (2005), Czub et al. (2008). In this way, we have shown that the carbon and oxygen ions return the same RBEm parameter for the LET values of 300 and 470 keV/μm. This
7
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Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Biological effects of mixed-ion beams. Part 2: The relative biological effectiveness of CHO-K1 cells irradiated by mixed- and single-ion beams Joanna Czuba, , Dariusz Banaśa,b, Janusz Braziewicza,b, Iwona Buraczewskac, Marian Jaskóład, Urszula Kaźmierczake, Andrzej Kormand, Anna Lankoffg,c, Halina Lisowskag, Zygmunt Szeflińskie, Maria Wojewódzkac, Andrzej Wójcikf,g ⁎
a
Jan Kochanowski University, Institute of Physics, ul. Świętokrzyska 15, 25-406 Kielce, Poland Holy Cross Cancer Center, ul. Arwińskiego 3, 25-734 Kielce, Poland c Institute of Nuclear Chemistry and Technology, ul. Dorodna 16, 03-195 Warsaw, Poland d National Centre for Nuclear Research, ul. Andrzeja Sołtana 7, 05-400 Otwock-Świerk, Poland e Heavy Ion Laboratory at the University of Warsaw, ul. Pasteura 5a, 02-093 Warsaw, Poland f Department of Molecular Bioscience, Centre for Radiation Protection Research, The Wenner-Gren Institute, Stockholm University, Universitetsvagen 10, 114 18 Stockholm, Sweden g Jan Kochanowski University, Institute of Biology, ul. Świętokrzyska 15, 25-406 Kielce, Poland b
HIGHLIGHTS
relative biological effectiveness (RBE) of low-energy ions was estimated. • The for the individual and mixed C and O beams was calculated. • RBE fractions of the CHO-K1 cells after such irradiations are presented. • Survival specific LET values, carbon and oxygen ions have equal RBEs. • AtSurvival fractions do not depend on the mixed beam composition. • 12
16
ARTICLE INFO
ABSTRACT
Keywords: Relative biological effectiveness Ions beams CHO-K1 cells
The relative biological effectiveness (RBE) values were determined for single- and mixed-ion beams containing carbon and oxygen ions. The CHO-K1 cells were irradiated with beams with the linear energy transfer (LET) values of 236–300 and 461–470 keV/μm for 12C and 16O ions, respectively. The RBE was estimated as a function of dose, survival fraction (SF) and LET. The SF was not affected by varying contributions of the constituent ions to the total mixed dose. The RBE has the same value for single-ion exposures with ions with LET 300 (12C) and 470 keV/μm (16O).
1. Introduction The relative biological effectiveness (RBE) is a parameter widely used in the ion therapy related to the biologically effective dose (BED) (Krämer et al., 2000). BED is used in the cancer therapy to take into account the physical dose absorbed by a live material as well as the behavior of cells after irradiation by ion beams (Krämer et al., 2000). The RBE is a component of the BED value that provides information on the biological response of irradiated cells (Krämer et al., 2000). Therefore, knowledge of RBE and its dependence on other important parameters, such as linear energy transfer (LET) and absorbed dose, is
⁎
important for improvement of the radiation therapy. Sørensen et al. (2011) reviewed available data on RBE for various cell, types and energies of single ions. For the Chinese hamster ovary (CHO-K1) cells, RBE values for a single exposure to carbon ions have been reported, albeit for a limited range of the linear energy transfer values; RBE values for other types of ions, such as neon, iron, argon ions was also reported (Sørensen et al., 2011). Also, Scholz et al. (1997) reported a survival fraction of the CHO-K1 cells after irradiations with the oxygen ions, but the data are only for a high energy of ions and simulation purposes, without information necessary to calculate RBE. There are other papers complimenting experimental data for the CHO-K1 cells in
Corresponding author. E-mail address: [email protected] (J. Czub).
https://doi.org/10.1016/j.apradiso.2018.12.001 Received 15 May 2017; Received in revised form 2 December 2018; Accepted 2 December 2018 0969-8043/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Czub, J., Applied Radiation and Isotopes, https://doi.org/10.1016/j.apradiso.2018.12.001
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previous publications (Weyrather et al., 1999; Mehnati et al., 2005; Czub et al., 2008, and review in Sørensen et al., 2011); in these studies, RBE was calculated for a single exposure to carbon ions with LETs of 236, 300 and 257 keV/μm, as well as oxygen ions with a LET of 470 keV/μm. Additionally, RBE was estimated as a function of dose, survival fraction and LET. The data for single exposures of the CHO-K1 cells to carbon and oxygen ions with specific LET values presented in this paper have, to our knowledge, not previously been reported. However, single-ion beams are rare in the nature, and mixed beams containing two or more ions and particles are most common. Mixed beams are always present, for example, in the outer space (Reitz et al., 1995) and, therefore, their effect on human cells is important for astronauts (Siranart et al., 2016). In a spacecraft, mixed beams appear when a flux of space ions and particles interact with parts of the craft (Obe et al., 1997; Edwards, 2001; Miller, 2003). Mixed beams also occur on Earth. One of such examples is the hadron therapy, in particular, the neon therapy, which is used against cancer cells to treat tumors in human bodies (Stelzer, 1998). In this therapy, neon ions undergo a process of fragmentation creating ions of other elements, such as carbon and oxygen (Stelzer, 1998). Therefore, it is important to study the impact of mixed ion beams, especially those containing carbon and oxygen ions, on live cells. In the past, only Kanai et al. (1997) studied the effect of two simultaneously accelerated ions (carbon and helium) on live cells. Furthermore, mixed beams containing photons and various types of ions or particles were used sequentially or simultaneously to irradiate cells of various types (a review in Staaf, 2012). Here, we examine the impact of two simultaneously accelerated heavy ions, namely, carbon and oxygen, on cell survival. The CHO-K1 cells were irradiated with three mixed-ion beams with the LET values of 236, 246 and 256 keV/ μm for the carbon ions, and 469, 465 and 461 keV/μm for the oxygen ions. RBE values were calculated for these mixed beams, which constitutes new scientific information.
2.2. Beam transport At this point, mixed- or single-ion beams from the cyclotron were scattered by a gold foil (20 mg/cm2 thick). After the scattering, a fraction of the ions were registered by a detector oriented at an angle of 20° to the direction of the primary ion beam (further on referred to as “20° Detector”), and a part of the ions passed through the gold foil. The dosimetry was based on readings of the 20° Detector (see the “Dosimetry” Section). The ions that had passed through the golden disc were collimated at a distance of 233 cm from the golden disc to a size of 1 × 1 cm2 and then passed through a Havar foil (2.3 mg/cm2 thick) at the end of the ion pipe and a specific air layer. Finally, the ions reached the cells inside a specially adapted Petri dish; the beam was oriented perpendicularly to the dish bottom. To change the energy of the incident ions, the Petri dishes were positioned in the air at various distances from the end of the ion pipe. 2.3. Procedure for irradiation of cells in the specially adapted Petri dish In this experimental system, we created a procedure to slide the Petri dish in a fixed beam with a step of 1 cm. This process was activated by an electrical pulse triggered by a certain number of counts in the 20° Detector. The values detector counts were necessary to calculate the dose absorbed by the cells (see the “Dosimetry” Section). The Petri dish moving process is controlled by a custom-written computer program. 2.4. Beam profile The values characterizing the beam profile were defined as ratios of the numbers of counts from the 0° and 20° Detectors. In this measurement, the position of the detector oriented at 0° to the primary beam (the 0° Detector) was changed at a specified step. The results of a measure of the mixed-beam profile were reported by Czub et al. (2018). The single-ion beam findings were reported in a paper by Czub et al. (2008). Based on that information, the estimated homogeneity of the mixed- and single-ion beams was 95%.
2. Materials and methods The experimental system and its the individual components including a special Petri dish, irradiation procedure, and results of the beam profile measurements were described previously (Czub et al., 2006, 2008, 2009). The production of mixed-ion beams was described by Czub et al. (2018).
2.5. Cell culture condition CHO-K1 cells were grown in the McCoy's 5A medium (No. BE12-ml 688F, Lonza, Switzerland) enriched with 10% Fetal Bovine Serum (No. ECS0180L, Euroclone, Italy) and 1% antibiotics (penicillin-streptomycin, No. 03-031-1B, Biological Industries, USA). The cells were passaged every two days with a 0.25% Trypsin-EDTA solution (No. 25200056, Gibco, England) to separate cells from the bottom of Petri dish. After four passages, the cells were used for the experiments.
2.1. Production of ion beam The mixed-ion beam containing carbon and oxygen ions was produced by a cyclotron at the Heavy Ion Laboratory of the University of Warsaw, Poland. The ions were mixed in an Electron Cyclotron Resonance category ion source. Two separate bottles with individual gases were connected to the ion source, one containing methane for the carbon ions and the other containing oxygen oxide for the oxygen ions. The mixed-ion beam from the ion source was injected into the cyclotron, where the mixture of the ions was accelerated. Two ions could be accelerated by a cyclotron simultaneously when their ratios q/m (q – charge, m - mass) have the same value (Kanai et al., 1997). In this case, q= +3 and + 4 for the carbon ions with m = 12 amu and oxygen ions with m = 16 amu, respectively. When the energy of the mixed-ion beam had reached a specified value, the beam was directed to a scattering chamber. This chamber was the first part of the experimental system intended for radiobiological studies. The single-ion beams containing only carbon or oxygen ions were also produced with an ECR-type ion source. In that case, the ion source was connected to a single gas cylinder with a suitable gas for a singleion beam. After acceleration, the beam was directed to the experimental hall of the cyclotron hosting a radiobiological setup used to irradiate live cells.
2.6. Survival test The CHO-K1 cells were seeded and cultured on a Mylar-foil (6 µm thick) bottom of a specially designed Petri dish. This Petri dish represented a plastic rim 1 cm high and 4.8 cm in diameter. The cells were plated at 150, 200, 300 and 500 cells per Petri dish, 10 h before the radiation exposure. The number of cells used in a single Petri dish depended on the absorbed dose and was selected experimentally in preliminary tests. These preliminary tests had shown that there was no risk that cell colonies would be connected when this number of cells per Petri dish were used. The cells were counted with a Countess Automated Cell Counter (No. C10227, Invitrogen, USA) with Countess Cell Counting Chamber Slides (No. C10312, Invitrogen, USA). In this counting procedure, cells in a single commercially available Petri dish (60 mm in diameter, No. 353004, Falcon, USA) were detached from the bottom with a trypsin solution (Biological Industries, No. 03–046-1A) administered for 3 min at 37 °C and 5% CO2. Next, a medium with additives (see the Cell culture condition Section) was added to the cells. 2
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Afterwards, 30 μL of the cell suspension was mixed with a trypan blue solution (No. T10282, Invitrogen, USA). Ten microliters of this mixture were then used for an automated cell counting. After the number of cells per milliliter was determined, the cell suspension was diluted with a phosphate buffered saline (PBS) solution and transferred into the specially designed Petri dish. Next, the specially designed Petri dish was fully filled with the McCoy's 5A medium containing additives (see the Cell culture condition Section). The Petri dish was then closed with a bespoke 3D-printed lid. That was done at the Institute of the Nuclear Chemistry and Technology in Warsaw, Poland. After that, the specially designed Petri dish was transported to the Heavy Ion Laboratory (HIL) of the University of Warsaw, Poland on iced water. After irradiation at HIL, the cells were taken back to the Institute of the Nuclear Chemistry and Technology on iced water to replace the medium. After that, the specially designed Petri dish was incubated at 37 °C in an atmosphere with 5% CO2 for 7 days. After the incubation, the cell colonies were fixed with methanol (Warchem, Poland) and stained with a 10% Giemsa solution (Pol-aura, Poland). Finally, the colonies were counted with an automated counter Scan500 (Interscience, France) or visually. The survival fraction was calculated as
SF =
F 12C =
12C N20 N0 12C , 12C N20
F 16O =
16O N20 N0 16O , 16O N20
where N is the number of counts of ions with superscripts specifying the type of the ions and the subscripts specifying the type of the detector used for the measurement; the apostrophe in the superscript indicates that the measurement was made before the cells were exposed (in other cases, the measurement was made during the cells irradiation). The sums over the i and k values are present in the absorbed dose equations because the beam spectra are not monoenergetic and the LET value changes when the beams passes through the cell layer, respectively. 2.8. Irradiation of cells with gamma rays from
60
Co
The procedure used to irradiate the cells with gamma rays from a Co source was described in detail in the paper by Czub et al. (2008); so, only a short description is provided here. The CHO-K1 cells were irradiated with gamma rays from 60Co to determine the RBE value. Gamma irradiations were performed at the Holy Cross Cancer Center (HCC) in Kielce, Poland. The cells were seeded in a Petri dish at the Jan Kochanowski University (JKU) in Kielce, Poland and transported to HCC. After the exposure, the cells were convoyed back to JKU, where the nutrient solution was refreshed.
60
A , B
where A is the number of colonies/number of cells in irradiated dishes and B is the number of colonies/number of cells in control dishes (Munshi et al., 2005) 2.7. Dosimetry The dosimetry for the single-ion and mixed-ion beams was described in detail in the papers by Czub et al. (2008) and Czub et al. (2018), respectively. Here, only a short description is provided. The measurement of the dose to be absorbed by the cells was performed before the exposure of the cells, and the dose delivery was controlled during irradiation of the live matter. For this purpose, we used the data collected with two silicon detectors (the 20° and 0° Detectors) with collimators of the diameters of 4 and 0.54 mm. Before irradiation of the cells, beam spectra obtained with these detectors were recorded, and the numbers of ions and the fluencies were calculated from these spectra. With the relationship between the counts by the two detectors known, it was possible to control the dose delivered to the cells using the counts registered only by the 20° Detector. The absorbed doses for the single- and mixed-ion beams were calculated using the following equations (Czub et al., 2008):
3. Results
a) for the single-ion beams
Fig. 1 shows the results of the survival test (i.e., survival curves for four single- and three mixed-ion irradiations, as well as the gamma exposure). The survival curves for the single- and the mixed-ion beam containing carbon ions with LET = 236 keV/μm and oxygen ions with LET = 470 keV/μm are also presented in our previous paper (Czub et al., 2018). The survival curve for the gamma irradiation was first published in the paper by Czub et al. (2008). A linear-quadratic model was fitted to the data in Fig. 1 using the equation (Weyrather et al., 1999)
9
D12C (Gy ) = 1.6 10
LETi12C
9 k=1
i
9
D16O (Gy ) = 1.6 10
LETi16O
9 k= 1
i
keV µm
Fi12C
keV µm
Fi16O
1 cm2
1 cm2
k
1
C
( )
C
( )
k
g cm3
1
g cm3
3.1. Linear energy transfers (LET) and the energies of the carbon and oxygen ions in the mixed-ion beams Table 1 lists the energies and the corresponding linear energy transfer values for the carbon and oxygen ions in the mixed ion beams. Both the values were determined at the cell entrance. The LET values presented in Table 1 differ from those calculated with the SRIM program (Ziegler et al., 2010) by 0.2–3%. This is due to the uncertainty of the fitting procedure used for the function between the energy and the LET of the ions. 3.2. Survival fractions in the absorbed dose function
,
;
aD bD2,
b) for the mixed-ion beams:
SF = e
D12C + 16O = D12C + D16O ,
where SF is the survival fraction, D is the absorbed dose, while a and b are coefficients from the fitting procedure listed in Table 2. The fitting was performed with the curve fitting toolbox in Matlab R2010b.
where , are the total absorbed doses from the mixedion beam, carbon ions and oxygen ions, respectively, ρ = 1 is the water density, i is the channel of the spectrum at the angle of 0° with respect to the incident beam, k is the cell thickness (Fisher et al., 1985), C is a constant corresponding to the ratio of the beam area (1 × 1 cm2) to the area of the collimator with the 0.54 mm diameter. F12C and F16O are the numbers of the carbon-ion (12C) and oxygen-ion (16O) irradiated cells calculated using the equations:
D12C + 16O
D12C ,
D16O
3.3. RBE values as functions of the dose, survival fraction (SF) and linear energy transfer (LET) Figs. 2–4 show the RBE values as functions of the dose, survival fraction and LET, respectively. 3
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Table 1 Energies and the linear energy transfer values at the entrances of the cell for the components of the mixed-ion beams. Mixed-ion beam
Ions in the mixed-ion beam Carbon
12
C(LET=236 keV/μm)+16O(LET=469 keV/μm) C(LET=246 keV/μm)+16O(LET=465 keV/μm) 12 C(LET=256 keV/μm)+16O(LET=461 keV/μm) 12
Oxygen
Energy (MeV)
LET (keV/μm)
Energy (MeV)
LET (keV/μm)
78 75 71
236 246 256
86 87 88
469 465 461
Fig. 1. Survival fractions as functions of the dose for single- and mixed-ion beams with different LET values. The error bars represent standard deviations.
contributions to the doses from the carbon and oxygen ions. Table 3 contains data for the mixed-ion beam 12C (LET=256 keV/μm)+16O (LET=461 keV/μm) corresponding to the survival curve shown in Fig. 5. Table 4 presents data for the mixed ion beam 12C (LET=236 keV/μm)+16O (LET=469 keV/μm) corresponding to the survival curve presented in Fig. 6. Contributions from the constituents of the 12C (LET=246 keV/μm)+16O (LET=465 keV/μm) beam are not presented because each survival fraction shown in Fig. 1 was measured only once. The sequence of data in Tables 3 and 4 corresponds to the order of the data in Figs. 5 and 6.
Table 2 Parameters of the linear-quadratic fits to the experimental data shown in Fig. 1. Mixed-ion beam 12
16
C (LET=236 keV/μm)+ O (LET=469 keV/μm) C (LET=246 keV/μm)+16O (LET=465 keV/μm) 12 C (LET=256 keV/μm)+16O (LET=461 keV/μm) Single-ion beam 12 C (LET=236 keV/μm) 12 C (LET=257 keV/μm) 12 C (LET=300 keV/μm) 16 O (LET=470 keV/μm) 60 Co 12
a
b
1.280 1.105 0.625 a 1.098 0.909 0.773 0.772 0.173
− 0.030 − 0.048 − 0.008 b 2.47•10−10
0.018
3.5. The RBEm values for single-ion beams as compared with data by others authors
3.4. Survival fractions after irradiations with the mixed-ion beams with varying ratios of the carbon- and oxygen-ion dose
Fig. 7 and Table 5 present the RBEm data calculated for a single-ion beam along with data from other papers, namely, Weyrather et al., 1999, Mehnati et al. (2005), and Czub et al. (2008). RBEm is the
Tables 3 and 4 list total mixed-ion doses with information on the
Fig. 2. RBE values as functions of the dose for the single- and mixed-ion beams. Error bars represent standard deviations. 4
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Fig. 3. RBE values as functions of the survival fractions for the single- and mixed-ion beams. Error bars represent standard deviations.
Fig. 4. RBE values as functions of the LET values for the single-ion beams and the arithmetical average LET values for the mixed-ion beams. Table 3 Total mixed-ion dose from the beams 12C (LET=256 keV/μm)+16O (LET=461 keV/μm) with various contributions from the two ions. Min. and Max. indicate the minimal and maximal contributions from the corresponding ions to the total dose.
Table 4 Total mixed-ion dose from the beams 12C (LET=236 keV/µm)+16O (LET=469 keV/µm) with various contributions from the two ions. Min. and Max. indicate the minimal and maximal contributions from the corresponding ions to the total dose.
Total mixed- ion dose
Carbon ion contribution to the dose (%)
Oxygen ion contribution to the dose (%)
Total mixed ion dose
Carbon ion contribution to the dose (%)
Oxygen ion contribution to the dose (%)
0.3 0.3 0.3 0.4 0.4 0.4 0.7 0.7 0.7 0.7 1 1 1 1 3 3 4 4 7 7 7 Min. Max. Average
66 64 67 35 63 63 44 33 44 37 61 48 53 35 43 25 37 43 45 36 39 25 67 47
34 36 33 65 37 37 56 67 56 63 39 52 47 65 57 75 63 57 55 64 61 33 75 53
0.2 0.2 0.2 0.4 0.4 1.5 1.5 3.0 3.0 3.8 3.8 Min. Max Mean
44 51 49 42 45 44 43 37 47 35 32 32 51 43
56 49 51 58 55 56 57 63 53 65 68 49 68 57
maximal value of RBE calculated by using only the parameter a of the fit (Weyrather et al., 1999). The RBEm values reported the literature (Fig. 7) were calculated by taking into account the gamma radiation values used in those studies. 4. Conclusions and discussion The RBE parameter is an important factor for the ion therapy and radiological protection, and its knowledge helps to better understand 5
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Fig. 5. Survival fraction as a function of the mixed-ion beam dose for
Fig. 6. Survival fraction as a function of the mixed-ion beam dose for
12
12
C (LET=256 keV/μm)+16O (LET=461 keV/μm). Error bars are equal to ± 10%.
C (LET=236 keV/μm)+16O (LET=469 keV/μm) beam. Error bars are equal to ± 10%.
effects of radiation on living cells. RBE values were obtained for cells of many types irradiated primarily with single-ion beams. However, few studies reported RBE values together with survival fractions for CHOK1 (Weyrather et al., 1999; Mehnati et al., 2005; Czub et al., 2008). The data generated in this study have supplemented this literature. However, the effects that mixed-ion beams exert on cell were studied for many years by many research groups who used various types of radiation, ways of irradiating cells (simultaneous or separate), cell types and biological tests (review in Staaf, 2012). To our knowledge, only Kanai et al. (1997) irradiated living cells with ions of two types accelerated simultaneously. However, the authors of that publication used
cells of a different type, namely, CHO-V79. Kanai et al. (1997) did not provide the survival curve fitting parameters for the mixed beams, making it impossible to determine the RBE parameters from their data. Data on simultaneous exposure to carbon and oxygen ions are not available in the literature. In this study, an experimental setup implemented in the Heavy Ion Laboratory at the University of Warsaw, Poland was used to irradiate live biological material. The CHO-K1 cells were exposed to four single- and three mixed-ion beams with different LET values. After the irradiations, survival tests were performed to determine the RBE values. These values were calculated and presented as functions of the absorbed dose, survival fraction (SF) and linear 6
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is new information for these cells. Additionally, survival fractions were calculated after irradiations with the mixed-ion beams with varying relative contributions to the dose from the constituent ions. These results have shown that variations of the relative contributions from the 12 C and 16O ions in the mixed beam to the dose have no effect on the survival fractions of the cells. These findings suggest that the mixed-ion beams exert additive effects on cells. Acknowledgements This work was supported by the statutory research No. 612 424 at the Jan Kochanowski University, Kielce, Poland. References Czub, J., Banaś, D., Błaszczyk, A., Braziewicz, J., Buraczewska, I., Choiński, J., Górak, U., Jaskóła, M., Korman, A., Lankoff, A., Lisowska, H., Łukaszek, A., Szefliński, Z., Wójcik, A., 2009. Cell survival and chromosomal aberrations in CHO-K1 cells irradiated by carbon ions. Appl. Radiat. Isot. 67, 447–453. https://doi.org/10.1016/j. apradiso.2008.06.016. Czub, J., Banaś, D., Braziewicz, J., Choiński, J., Jaskóła, M., Korman, A., Szefliński, Z., Wójcik, A., 2006. An irradiation facility with a horizontal beam for radiobiological studies. Radiat. Prot. Dosim. 122, 207–209. https://doi.org/10.1093/rpd/ncl518. Czub, J., Banaś, D., Błaszczyk, A., Braziewicz, J., Buraczewska, I., Choiński, J., Górak, U., Jaskóła, M., Korman, A., Lankoff, A., Lisowska, H., Łukaszek, A., Szefliński, Z., Wójcik, A., 2008. Biological effectiveness of 12C and 20Ne ions with very high LET. Int. J. Radiat. Biol. 84. https://doi.org/10.1080/09553000802389652. Czub, J., Banaś, D., Braziewicz, J., Buraczewska, I., Jaskóła, M., Kaźmierczak, U., Korman, A., Lankoff, A., Lisowska, H., Szefliński, Z., Wojewódzka, M., Wójcik, A., 2018. Biological effects of mixed-ion beams. Part 1: effect of irradiation of the CHOK1 cells with a mixed-ion beam containing the carbon and oxygen ions. Appl. Radiat. Isot. 139. https://doi.org/10.1016/j.apradiso.2018.05.028. Edwards, A.A., 2001. RBE of radiations in space and the implications for space travel. Phys. Med. 17 (Suppl 1), 147–152. Fisher, D.R., Frazier, M.E., Andrews Jr., T.K., 1985. Energy distribution and the relative biological effects of internal alpha emitters. Radiat. Prot. Dosim. 13, 223–227. https://doi.org/10.1093/rpd/13.1-4.223. Kanai, T., Furusawa, Y., Fukutsu, K., Itsukaichi, H., Eguchi-Kasai, K., Ohara, H., 1997. Irradiation of mixed beam and design of spread-out Bragg peak for heavy-ion radiotherapy. Radiat. Res. 147, 78–85. https://doi.org/10.2307/3579446. Krämer, M., Jäkel, O., Haberer, T., Kraft, G., Schardt, D., Weber, U., 2000. Treatment planning for heavy-ion radiotherapy: physical beam model and dose optimization. Phys. Med. Biol. 45, 3299–3317. https://doi.org/10.1088/0031-9155/45/11/313. Mehnati, P., Morimoto, S., Yatagai, F., Furusawa, Y., Kobayashi, Y., Wada, S., Kanai, T., Hanaoka, F., Sasaki, H., 2005. Exploration of “over kill effect” of high-LET Ar- and Feions by evaluating the fraction of non-hit cell and interphase death. J. Radiat. Res. 46, 343–350. https://doi.org/10.1269/jrr.46.343. Miller, J., 2003. Proton and heavy ion acceleration facilities for space radiation research. Gravit. Space Biol. Bull. 16, 19–28. Munshi, A., Hobbs, M., Meyn, R.E., 2005. Clonogenic cell survival assay. Methods Mol. Med. 110, 21–28. https://doi.org/10.1385/1-59259-869-2:021. Obe, G., Johannes, I., Johannes, C., Hallman, K., Reitz, G., Facius, R., 1997. Chromosomal aberrations in blood lymphocytes of astronauts after long-term space flights. Int. J. Radiat. Biol. 72, 727–734. https://doi.org/10.1080/095530097142889. Reitz, G., Facius, R., Sandler, H., 1995. Radiation protection in space. Acta Astronaut. 35 (4–5), 313–338. https://doi.org/10.1016/0094-5765(95)98735-R. Siranart, N., Blakely, E.A., Cheng, A., Handa, N., Sachs, R.K., 2016. Mixed beam murine harderian gland tumorigenesis: predicted dose-effect relationships if neither synergism nor antagonism occurs. Radiat. Res. 186, 577–591. https://doi.org/10.1667/ RR14411.1. Staaf, E., 2012. Cellular effects after exposure to mixed beams of ionizing radiation. (PhD work) 〈https://www.diva-portal.org/smash/get/diva2:557692/FULLTEXT01.pdf〉. Stelzer, H., 1998. Tumor therapy with heavy ions at GSI. Nucl. Phys. B (Proc. Suppl.) 61B, 650–657. https://doi.org/10.1016/S0920-5632(97)00633-6. Scholz, M., Kellerer, A.M., Kraft-Weyrather, W., Kraft, G., 1997. Computation of cell survival in heavy ion beams for therapy. Radiat. Environ. Biophys. 36, 59–66. https://doi.org/10.1007/s004110050055. Sørensen, B.S., Overgaard, J., Bassler, N., 2011. In vitro RBE-LET dependence for multiple particle types. Acta Oncol. 50, 757–762. https://doi.org/10.3109/0284186X.2011. 582518. Weyrather, W.K., Ritter, S., Scholz, M., Kraft, G., 1999. RBE for carbon track-segment irradiation in cell lines of differing repair capacity. Int. J. Radiat. Biol. 75, 1357–1364. https://doi.org/10.1080/095530099139232. Ziegler, J.F., Ziegler, M.D., Biersack, J.P., 2010. SRIM - The stopping and range of ions in matter (2010). Nucl. Instrum. Methods Phys. Res. Sect. B Beam Interact. Mater. At. 268, 1818–1823. https://doi.org/10.1016/j.nimb.2010.02.091.
Fig. 7. The RBEm values calculated for the single-ion beams presented alongside with data from others publications (Table 5). The numerical values indicate data from those studies. Table 5 A compilation of the LET values collected in this study and announced in the literature (also presented in Fig. 7). Paper
Ion
LET (keV/μm)
Weyrather et al. (1999) Weyrather et al. (1999) Mehnati et al. (2005) Weyrather et al. (1999) Mehnati et al. (2005) Mehnati et al. (2005) Mehnati et al. (2005) Weyrather et al. (1999) Weyrather et al. (1999) This work This work Weyrather et al. (1999) This work Weyrather et al. (1999) Czub et al. (2008) This work Weyrather et al. (1999) Czub et al. (2008) Czub et al. (2008) Mehnati et al. (2005) Mehnati et al. (2005) Mehnati et al. (2005) Mehnati et al. (2005) Mehnati et al. (2005) Czub et al. (2008) Czub et al. (2008) Czub et al. (2008) Mehnati et al. (2005) Mehnati et al. (2005) Mehnati et al. (2005) Mehnati et al. (2005)
12
13.7 16.8 20 32.4 60 82 99 103 153.5 232 257 275.1 300 339.1 438 470 482.7 576 832 102 120 171 228 341 933 1245 1616 1640 780 1200 2000
C C C 12 C 12 C 12 C 12 C 12 C 12 C 12 C 12 C 12 C 12 C 12 C 12 C 16 O 12 C 12 C 12 C 20 Ne 20 Ne 20 Ne 20 Ne 20 Ne 20 Ne 20 Ne 20 Ne 40 Ar 56 Fe 56 Fe 56 Fe 12 12
energy transfer (LET). Based on our data, we have concluded that RBE values decrease with the increasing dose and LET, and increase with the decreasing SF values. These findings are in line with the overkill effect and agree with the findings by Weyrather et al. (1999) for single-ion irradiations. The RBEm values calculated for the single-ion beams for the CHO-K1 cells and presented as functions of LET compliment the literature data for the cells of the other types reported in the papers by Weyrather et al. (1999), Mehnati et al. (2005), Czub et al. (2008). In this way, we have shown that the carbon and oxygen ions return the same RBEm parameter for the LET values of 300 and 470 keV/μm. This
7