Abstract
Proton being the easiest light ion to accelerate and achieve desired beam profile, has been pursued as a popular particulate radiation for therapy applications. In the present study, Saccharomyces cerevisiae D7 strain was used to estimate the RBE values of the 3 MeV proton beam, and an attempt was made to derive mathematical formula for calculating RBE value with respect to the dose. Dosimetry studies were carried out using Fricke dosimetry and Semiconductor Surface Barrier detector to calibrate the absorbed doses of Gamma chamber-1200 and Folded Tandem Ion Accelerator respectively. Gold standard cell survival assay and gene conversion assay were used to compare gamma and proton radiation induced cell death and genetic endpoint. Multi target single hit model was used to derive mathematical formula for RBE estimation. The results show a linear survival-dose response after proton radiation and sigmoid survival-dose response after gamma radiation treatment. The calculated RBE value from the survival and gene conversion studies was 1.60 and 3.93, respectively. The derived mathematical formula is very useful in calculating RBE value, which varies from 3.61 to 1.80 with increasing dose. The estimated RBE value from the mathematical formula is comparable with the experimental values. With the help of the present mathematical formulation, RBE value at any dose can be calculated in the exponential and sigmoidal regions of the survival curve without actually extending the experiment in that dose region, which is not possible using conventional methods.
Keywords
- Saccharomyces cerevisiae
- relative biological effectiveness
- radiation dose
- cell survival and gene conversion
1. Introduction
Biological effects of heavy charged particles on humans play an important role in two different scientific fields; in radiation therapy using protons and heavier ions and in space research for understanding effects on space travelers from galactic cosmic radiation [1]. In addition, the low energy heavy ion accelerators have an important role in basic and applied sciences [2]. Proton being the easiest light ion to accelerate and achieve desired beam profile, has been pursued as a popular particulate radiation for therapeutic applications. Nonetheless, very less has been understood about biological effectiveness of these charged particles. Proton beams can provide highly localized, uniform doses of radiation to tumors, while sparing the surrounding normal tissues, compared with conventional modalities using photons or electrons [3]. In addition to therapeutic applications, energetic proton also finds its presence in space research, neutron dosimetry wherein due to elastic scattering of energetic neutrons lead to (n, p) reaction and creation of low energy protons in the tissues.
The radiobiological studies conforms that equal physical doses of different types of radiation do not produce equal biological effects, because of differences in their energy deposition patterns. This is taken into account by the concept of Relative Biological Effectiveness (RBE). RBE compares the severity of damage induced by a radiation under test, at a dose DT relative to the reference radiation dose DR for producing same biological effect. The reference radiation is commonly 60Co-gamma radiation. Generally, the RBE depends on many factors such as the radiation dose, linear energy transfer (LET) at a given tissue depth, dose rate, energy of the radiation, test system and studied biological endpoint. The RBE values of the radiation are very useful in risk estimation during accidental exposure of ionizing radiation (IR) [4]. Revisions in weighting factors for intermediate and very high energy neutrons as well as accelerated protons in the recent ICRP recommendation has drawn more attention to mechanistic approach of studies using radiobiological endpoints.
In the present study,
2. Materials and methods
Absorbed dose was calculated using the relation [Kraft et al. 1989]
Where fluence represents particles delivered per unit area and LET represents energy transferred per unit length. The LET of the present setup was estimated to be 13 KeV/μm. The fluence of the source detector was measured using
Where ‘rs’ represents, sample detector collimator radius. Number of particles on sample detector can be calculated by taking ratio between monitor detector and source detector counts
Substituting Eqs. (3) and (2) in (1) gives
Rearranging Eq. (4), gives
Eq. (5) was used to calculate required number of monitor detector counts for desired absorbed dose.
The single cell stationary-phase cultures were obtained by growing the cells Yeast extract: Peptone: Dextrose (YEPD) (1%:2%:2%) medium for several generations to a density of approximately 3 × 108 cells mL−1. Cells were washed thrice by centrifugation (2000 g for 5 min) and re-suspended to a cell concentration of 1 × 108 cells mL−1 (by counting in heamocytometer) in sterile double distilled water. For proton radiation, the cell suspension was mixed well and exactly 1× 108 cells were filtered using millipore filter assembly in aseptic condition. The filter paper having cells on the surface was placed inside sterile 3 cm diameter petri dish and irradiated for different doses. For gamma ray irradiation, polypropylene vials were used containing 1× 108 cells per ml. Cell suspensions were maintained at 0–4°C before and after irradiation till plating.
3. Results and discussion
The obtained experimental data were fit to multi-target single hit theory and the survival response of gamma and proton radiation were represented as SGamma = (1-(1-exp(0.00459 D))3.78) (with R2 = 0.99 and Chi2 = 0.00012) and SProton = (1-(1-exp(0.00736 D))1.50) (with R2 = 0.99 and Chi2 = 0.00056). The calculated D0 value, which is a reciprocal of the inactivation constant, is 218 and 136 Gy for gamma and proton radiation respectively. The RBE value in the exponential region can be calculated by taking ratio between inactivation constant of gamma and proton radiation and is found to be 1.60.
Presently RBE values are calculated on the basis of D0 doses, which give RBE value in the exponential region. In the present study, we formulated an equation, which can be used to calculate RBE value throughout the selected dose-region. Generally RBE is represented by taking the ratio between gamma radiation and test radiation doses, required to produce the same biological effectiveness.
Where, DG is gamma radiation dose and DT is test radiation (in this case proton radiation) dose. From multi-target single hit model, the survival can be represented as
Where S represents survival fraction, k is inactivation constant, D is dose and n gives number of targets. To calculate RBE value, we are considering same survival level with both the radiations, thus using Eq. (7), we can write
Simplifying (8), considering high radiation dose (D)
Eq. (9) gives the relation between RBE and dose. In Eq. (9), the DT, nT, kT represents dose, number of target and inactivation constant under test radiation condition respectively and nG, kG represents number of target, inactivation constant under gamma radiation condition respectively. The variance in the measurements was calculated using following equations, in Eq. (9) the kG, nG, kT and nT are variables
where y represents RBE value
Accordingly standard deviation was calculated. Figure 4 represents RBE value of 3 MeV proton beam at different doses, calculated using Eq. (9). The experimentally calculated RBE value and theoretically calculated RBE values were compared and presented in Figure 4. Very good correlation between experimental and theoretical data was observed.
Higher RBE values were observed at lower doses whereas remain constant at higher doses. RBE values varied in a range from 3.61 to 1.80; the maximum value at lower doses is mainly due to the absence of sub-lethal repair processes. In the case of gamma radiation, at lower doses induced damages are repaired but in the case of proton radiation a small dose also creates lethal damages, hence maximum RBE value was observed. At higher doses the damage due to peroxyl radicals and multi-ionizing events lead to lethal damage in gamma radiation, hence RBE value remains constant. Another reason for higher RBE value is energy deposition pattern of the 3 MeV proton radiation. The LET of the gamma radiation is 0.2–0.3 keV μm−1, whereas 3 MeV proton radiation is 13 keV μm−1. The higher RBE values for low energy protons were reported previously [16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30]. Belli et al. [16] has reported that the RBE depends on LET of the proton radiation. They studied SOBP region proton radiation using V79–753B cells. The RBE value for protons with LET 7.7 keV μm−1, 11 keV μm−1, 20 keV μm−1, 30.5 keV μm−1, 34.6 keV μm−1 and 37.8 keV μm−1 is 2.22 ± 0.27, 2.88 ± 0.37, 3.64 ± 0.41, 5.59 ± 0.54, 5.06 ± 0.51 and 4.50 ± 0.44, respectively [16]. Similar type observation was made by Folkard et al. [17] and reported an RBE value for protons with mean energies of 1.9, 1.15 and 0.76 MeV, using V79 chinese hamster cells. The RBE values for cell survival at 10% survival level are 1.6, 1.9 and 3.36 for protons with track-average LETs of 17, 24 and 32 keV μm−1, respectively.
In another report Mark Andrew [28] observed an RBE value of 2.6 ± 0.6 for 94 keV, 3.1 ± 0.4 for 250 keV, 3.9 ± 0.8 for 390 keV and 2.4 ± 0.5 for 1.2 MeV protons using V79 cell line. Belli et al. [18] studied four human cell lines, SCC25, SQ20B derived from human epithelium tumors of the tongue and larynx, respectively, the normal lines M/10, derived from human mammary epithelium, and HF19 derived from a lung fibroblast. The RBE of the proton beams with LET 30 keV μm−1 was 3.2, 1.8, 1.3 and 0.8 for SQ20B, M/10, SCC25, and HF19, respectively [18]. Similarly, Ristić-Fira et al. [29] reported RBE value for mid SOBP region proton particles using radio-resistant human HTB140 melanoma cells and is found to be 2.09 ± 0.36.
Recently, Wéra et al. [30] irradiated Human A549 alveolar adenocarcinoma cells with 4 MeV broad proton beam and calculated RBE value at 10% survival. They reported RBE value of the low energy proton radiation is independent of the dose rate and is equal to 1.9 ± 0.4 for 10 keV μm−1 and 2.9 ± 0.5 for 25 keV μm−1 [30]. In the same study they calculated RBE values at 77% survival level and were equal to 10.7 ± 3.3 and 3.6 ± 0.6 for 10 keV μm−1 and 25 keV μm−1 respectively [30]. These values suggest that RBE value depends on survival, which again depends on radiation dose. Britten et al. [22] studied human Hep2 laryngeal cancer cells and V79 cells at various positions along the SOBPs of beams with incident energies of 87 and 200 MeV. Using Hep2 cells, the RBE values were 1.46 at the middle of SOBP, 2.3 at the distal end of the SOBP [22]. For V79 cells, the RBE for the 87 MeV beams was 1.23 for the proximal end of the SOBP, 1.46 for the distal SOBP and 1.78 for the distal end of the SOBP [22]. Similar studies were conducted by Paganetti [23], Słonina et al. [24] and Aoki-Nakano et al. [26] to calculate SOBP region RBE value. They concluded that, the proton RBE value increases with increasing LET which ranges from 1.1 to 4.98. The RBE values for continuous and pulsed proton radiation also studied using human tumor cells [27]. No significant difference was observed between pulsed proton (RBE = 1.22 ± 0.19) and continuous proton (RBE = 1.10 ± 0.1) beam [27].
Previous studies reveal that there is a large variation in reported RBE values among laboratories with the same cell line and a similar LET. For example, Belli et al. [16] and Folkard et al. [17] measured an RBE value of 24 keV μm−1 protons as 1.9 and 2.4, respectively. On average, literature reported data concludes RBE value for low energy proton radiation varies from 0.9 to 6, which is comparable with the present findings.
4. Conclusion
The study confirms that, the 3 MeV proton beam is more lethal to biological system compare to gamma radiation and the dose response was found to be linear. Nearly 4 times higher gene conversion frequency was observed in proton radiation as compared to gamma radiation. The estimated RBE value estimated from the mathematical equation developed in the present study is comparable with the experimental values. The RBE value of the 3 MeV protons was found to decreases with the dose and varied from 3.61 to 1.80. With the help of the present mathematical formulation, RBE value at any dose can be calculated in the exponential region of the survival curve without actually extending the experiment in that dose region, which is not possible using conventional methods.
Acknowledgments
The authors from Mangalore University are thankful to Board of Research in Nuclear Sciences, Department of Atomic Energy, Government of India, for the financial support.
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