Relative biological effectiveness and microdosimetry of a mixed energy field of protons up to 200 MeV

Adv. Space Res. Vol. 14, No. 10, pp. (10)271-(10)275, 1994 Copyright © 1994 COSPAR Printed in Great Britain. All rights reserved. 0273-1177/94 $7.00 + 0.00

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RELATIVE BIOLOGICAL EFFECTIVENESS AND MICRODOSIMETRY OF A MIXED ENERGY FIELD OF PROTONS UP TO 200 MeV J. B. Robertson,* J. M. Eaddy,* J. O. Archambeau,** G. B. Coutrakon,** D. W. Miller,** M. F. Moyers,** J. V. Siebers,** J. M. Slater** and J. F. Dicello*** * East Carolina University, Greenville, NC 27858, U.S.A. ** Loma Linda University Medical Center, Loma Linda, CA 92354, U.S.A. *** Clarkson University, Potsdam, NY 13699, U.S.A.

ABSTRACT We have studied radiation effects utilizing the new 250 MeV Synchrotron at Loma Linda University Medical Center. In this paper we present the data collected for the survival of Chinese hamster lung (V79) cells, that were irradiated with a beam of mixed energy protons up to 200 MeV. The RBE for protons, when compared to 6°Co gamma rays, ranged from a low of 1.2 at the high energy portion of the field to 1.3+ at the low energy portion of the field. These results are consistent with the measured lineal energy (microdosimetric) spectra. INTRODUCTION As we have consistently pointed out,/1-3/, the radiation exposure to individuals beyond the protection of the earth's magnetic fields consists of the relatively constant galactic cosmic radiation spectrum which is about 85 percent protons and the transient radiation from solar particle events, where 90 percent or more of the particles are protons/4,5/. Clearly, protons are the most frequently encountered particles in the free space radiation environment beyond the earth's radiation belts. Protons from solar particle events pose the only natural acute radiation hazard that will be encountered during interplanetary space flight or during normal planetary and lunar base operations. This will also be the case for individuals living on a space station if its orbit includes regions outside the area protected by the earth's magnetosphere. All designs for human habitation must include some form of radiation protection from solar flare protons. Since these protons are of mixed energies, it is important to study and understand the radiation biology of mixed energy fields of protons. As we have stated earlier, underestimation of the risk could lead to serious harm to the exposed individuals while overestimation of the risk will cause unacceptable inefficiency, and neither of these alternatives is tolerable/1/. Mixed energy proton fields with energies up to 250 MeV are available for such studies at the Loma Linda University Proton Treatment Facility. In this paper we report on the results obtained when cells are exposed to mixed fields of protons with energies ranging from 200 MeV to stopping protons. METHODOLOGY Cell Culture The cells used in these experiments were the V79 Chinese hamster lung fibroblast cells. They. were maintained in Eagle's Minimum Essential Medium (EMEM) supplemented with non-essential armno acids; 50 U/ml penicillin, 0.05 mg/ml streptomycin sulfate (Sigma Chemical Co., cat # M 0643) and 10% Fetal Bovine Serum (FBS) (Hyclone Laboratories, cat # A-1111-L, Lot # 1111946). Stock cultures were maintained in 25 cm 2 Corning tissue culture flasks (Fisher Scientific Co., Corning# 25100) at 37°C in 5% CO2, 95% air at maximum humidity. Two days prior to irradiation, the cells were placed into 75 cm 2 Corning tissue culture flasks (Fisher 5 Scientific Co., Coming # 25110) at a density of 4x10/flask to attain a population of 5-6x10 6 cells/flask in exponential growth. The cells were then harvested in exponential growth approximately eight hours prior to irradiation using 0.5 g/L trypsin-EDTA solution (Sigma Chemical Co., cat # T9395) and Hanks' Balanced Salt Solution (HBSS) (Sigma Chemical Co., cat #H2387) and seeded into 25 cm 2 Corning tissue culture flasks at cell concentrations calculated to produce 100-200 survivors per flask and allowed to attach for a minimum of 4 hours. Approximately one hour before irradiations began, the flasks were ( 10)271

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filled with EMEM supplemented with 1% FBS. After filling, the flasks were transported by hand carrying to the irradiation sites in an insulated box to maintain temperature. After irradiations were completed, the flasks were again hand carried to the lab where the medium was decanted and replaced with 5 ml of complete EMEM. The flasks were allowed to incubate undisturbed for seven days, then the colonies were stained and scored; colonies containing 50 or more cells were scored as survivors. Irradiation Conditions Protons at the Loma Linda University Proton Therapy Facility are first accelerated in a synchrotron with a 2.2 second cycle time. After a 300 ms extraction time, they are transported to one of several beam lines in various rooms. Irradiations for these experiments were performed on the horizontal beam line and gantry I beamline. The protons enter either of the exposure nozzles 3 meters upstream from isocenter. They are then spread by a double scattering system to uniformly cover a 15 cm by 15 cm field at isocenter. The range of the 200 MeV protons after traversing the scattering system and monitoring devices is 21.1 cm of water as defined by the depth of the distal 90% of maximum dose. The beam range was additionally modulated in depth by a rotating stepped polycarhonate wheel giving a mesa with a proximal 90% dose at 13.0 cm depth. The polystyrene flasks containing the cells for proton beam irradiation were placed in a row in a tank filled with 37°C water with the bottom of the flask covered with cells in a vertical position. The proton beam was incident on the tank horizontally and onto the bottom of the flasks. Alignment of the tank containing the cell flasks was facilitated by positioning lasers. The upstream side of the tank was placed at isocenter for the horizontal beam line and 32 cm upstream of isocenter for the gantry beamline. The proton field was defined by a square Lipowitz metal aperture placed in the nozzle giving a size at the tank of approximately 15 cm by 15 cm thus completely irradiating the tank. Measurement of the dose in the proton beam was accomplished by irradiating a 0.05 cm 3 tissue-equivalent thimble ionization chamber in polystyrene at the center of the mesa region. The chamber had a 6°Co exposure calibration factor traceable to the NIST. Dose to muscle was calculated from the ionization m e a s u r e m e n t s using an N type f o r m a l i s m similar to the A m e r i c a n Association of Physicists in Medicine (AAPM) Task d ~ u p 21 p r o t o c o l / 6 / . This method gave results identical to those from calculations based on the AAPM Heavy Charged Particle protocol/7/. The dose rate delivered to the cells was approximately 1.0 Gy/min. Cobalt irradiations were performed using an AECL Eldorado Model G cobalt irradiator. For these irradiations, the beam was incident on the front surface of the flasks from above with an SSD of 64.4 cm. The surface of the flasks covered with cells was on the bottom, thereby placing the cells 2.25 cm from the front. Four flasks, placed in each of four quadrants of the field, were irradiated simultaneously. The field size at the level of the cells was approximately 18 cm, which completely surrounded the flasks. No additional side scattering material was provided. Measurement of the dose in the cobalt beam was accomplished by placing a 0.5 cm 3 air-equivalent thimble ionization chamber at the same SSD and depth in polystyrene as the cells. The chamber had a 6°Co exposure calibration factor traceable to the NIST. Dose to muscle was calculated from the ionization measurements using the AAPM Task Group 21 protocol/6/. The dose rate delivered to the cells was approximately 0.45 Gy/min. An additional check of the cobalt calibration procedures was provided by the University of Texas M.D. Anderson Cancer Center mail thermoluminescent dosimetry service. Microdosimetry Microdosimetry spectra were measured concurrently as a function of position for the beams used for the biological studies. The general procedure used was that established by Task Group #20 of the AAPM (Lyman et al. and Dicello et al.)/7,9/. The data were obtained with a custom designed EG&G/Far-West spherical proportional counter. The detector has an inside diameter of 0.127-cm and is constructed of A150 tissue equivalent plastic. It is embedded in a Lucite block and measurements were taken as a function of the thickness of absorbing plastic in the same phantom routinely used for dosimetric calibrations. The macroscopic dose as a function of depth is first obtained for each beam with the same proportional counter used for the microdosimetric measurement in order to know its precise location and to compensate for slight differences in the displacement factor in comparison with the dosimeters used for the biology measurements. Because the spectra extend over almost five decades in energy deposited and ten or more decades in probabilities, the measurements are taken in several overlapping sections. The method is described in more detail in the referenced publications. In order to relate the microdosimetric measurements to mammalian cell responses, we used a model developed by Zaider and Dicello /10/ which compares changes in radiation quality with changes in

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survival. The model uses the measured microdosimetric spectra as input to the theory of dual radiation action to calculate an RBE. The model is capable of compensating for sublethal damage-type repair by taking into account the dose rate and dose modifying factors such as the oxygen effect and it can estimate the effects of changes in the fractionation schedule. A recently modified version also takes into account repopulation according to the configuration model of Shymko, Hanser, and Archambeau/11/, although repopulation was not considered for the present analysis. RESULTS AND DISCUSSION There was no significant difference in the calculated RBE values for survival levels of 0.1 and 0.01. The average RBE was 1.21 in both cases as presented in Figure 1. The average values ranged from 1.24 at the entrance (2.08 cm) to a low of 1.18 (10.74 cm) and then increased to a value of 1.32 near the distant Bragg peak. 3.0

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Distance (cm) Fig. 1. The calculated RBE values for protons when compared to 6°Co photons, as a function of position (energy) in the proton field. Open circles represent calculated RBE values for surviving fractions of 0.1. Closed circles represent RBE values for surviving fractions of 0.01.

Microdosimetric spectra as a function of depth for the 200 MeV horizontal proton beam at Loma Linda University Medical Center are presented in Figure 2/12/. The probability of a given dose per logarithmic interval of lineal energy, yd(y), is plotted as a function of lineal energy, so the area under the curve between two values of lineal energy is proportional to the fraction of the dose in that interval; the curve is normalized so the integral of yd(y)dy is equal to one. The microdosimetric spectra show several distinctive regions. In the region with lineal energies between about 1 and 100 keV/~tm, there is a peak in the distribution produced by the primary protons as they cross the sensitive volume. The peak occurs at a relatively low lineal energy in the entrance region and shifts to higher lineal energies with depth as the stopping power of the primary protons increases. In the region between from about 30 keV/Ixm down to a fraction of a keV/Ixm, there is a broad distribution of events produced by the secondary electrons and photons. Above 100 keV/ixm, the secondary heavier particles from the primary protons and from the secondary neutrons dominate, with events primarily from secondary protons, then alpha particles, and then heavier ions as the lineal energy increases with cut-offs determined by their maximum stopping powers. Recoils from oxygen, for example, can have lineal energies which exceed 1000 keV/~m. Most of the events have lineal energies comparable to those produced by photons. For this reason, highenergy proton beams are generally assumed to be low LET (linear energy transfer). In the entrance region of this beam, however, there is a small fraction of the dose which has higher lineal energies. Such events have the potential of producing significantly more biological damage per event for the same macroscopic dose, i.e., they have an RBE which is greater than one in comparison with photons. Correspondingly, as the primary protons near the end of their range, their lineal energy approaches 100 keV/txm,

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a region which frequently can be more biologically effective in comparison with events from photons or electrons.

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y (keV/um) Fig. 2. Microdosimetric spectra at specified depths in a plastic phantom for a 200 MeV mixed energy proton beam at the Loma Linda University Medical Center. The distribution in dose per logarithmic interval of lineal energy, yd(y), is plotted as a function of linear energy, y. The area under the curve has been normalized so the integral of yd(y)dy is one. The area under the curve, then, between two values of y is proportional to the fractional contribution of that region to the total dose. Open circles, 9.4 cm penetration (surface); X's, 17.8 cm penetration; solid circles, 20.4 cm penetration; and squares, 21.5 cm penetration (stopping protons). Because of these two different factors, the nuclear secondaries in the region of high-energy protons and the high stopping power of the protons nearing the end of their range at depth, protons can produce an RBE significantly greater than one in comparison with irradiations with cobalt-60. For the system under consideration in this beam, the predictions of the biological model are consistent with the measured values. Moreover, the model suggests that, for larger volumes, that is higher initial proton energies, more penetration, and larger areal cross sections, the events of larger lineal energies, especially those produced by secondary neutrons, would be more uniformly distributed throughout the volume. This effect has been observed and explained earlier for the case of beams of negative pions by Dicello and Brenner/13/. REFERENCES 1. J.B. Robertson, W.C. Glisson, J.O. Axchambeau, G.B. Coutrakon, D.W. Miller, M.E Moyers, J.E Siebers, J.M. Slater, and J.F. Dicello. The relative biological effectiveness of attenuated protons, in: Biological Effects and Physics of Solar and Galactic Cosmic Radiation, ed. C.E. Swenberg, G. Horneck, and E.G. Stassinopoulos, Plenum Press: New York, in press (1992).

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2. J.B. Robertson, A.M. Koehler, P.A. Weideman, and P.J. McNulty. High energy proton induced mutations in cultured Chinese hamster cells, in: Terrestrial Space Radiation and its Biological Effects, ed. P.D. McCormack, C.W. Swenberg, and H. Bucker, Plenum Press: New York, 1988, p. 329. 3. J.F. Dieello. HZE cosmic rays in space. Is it possible that they are not the major radiation hazard? Submitted for publication in Radiation Proton Dosimetry (1992). 4. E.G. Stassinopoulos. Earth's trapped and transient space radiation environment, in: Terrestrial Space Radiation and its Biological Effects, ed. P.D. McCormack, C.E. Swenberg, and H. Bucker, Plenum Press: New York, 1988, p. 5. 5. NCRP Report No. 98. Guidance on radiation received in space activities: National Council on Radiation Protection and Measurements, Bethesda, Maryland (1989). 6. R. Nath, L. Anderson, D. Jones, C. Ling, R. Loevinger, J. Williamson, and W. Hanson. Specification of Brachytherapy source strength. AAPM Report No. 21, American Institute of Physics. New York (1987). 7. J.T. Lyman, M. Awchalom, P. Berardo, H. Bichsel, G.T.Y. Chen, J. Dicello, P. Fessenden, M. Goitein, G. Lam, J.C. McDonald, A.R. Smith, R. Ten Haken, L. Verhey, and S. Zink. Protocol for heavy charged-particle therapy beam dosimetry. AAPM Report No. 16, American Institute of Physics, New York (1986). 8. J.E Dicello, M. Wasoiolek, and M. Zaider. Measured microdosimetric spectra of energetic ion beams of Fe, Ar, Ne, and C: Limitations of LET distributions and quality factor in space research and radiation effects. IEEE Trans. Nucl. Science NS-38, in press, (1991). 9. J.F. Dicello, J.T. Lyman, J.C. McDonald, and L.J. Verhey. A portable system for microdosimetric intercomparisons by Task Group #20 of the American Association of Physicists in Medicine (AAPM). Nucl. Instr. Meth. 844, 724 (1990). 10. M. Zaider and J.F. Dicello. RBEOER. Los Alamos Report LA-7196-MS, Los Alamos National Laboratory, New Mexico (1978). 11. R.M. Shymko, D.L. Hauser, and J.O. Archambeau. Field size dependence of radiation sensitivity and dose fractionation response in skin. Int. J. Radiat. Oncology Biol. Phys. 11:1143 (1985). 12. J.E Dicello, J.O. Archambeau, J.M. Slater, G.B. Coutrakon, J. Johanning, D.W. Miller, M.E Moyers, J.V. Siebers, A. Kim, M. Zaider, and J.B. Robertson. Biological and clinical implications of microdosimetric distributions of high-energy protons. Presented at the Eleventh Symposium on Microdosimetry, Gatlinburg, TN (1992). In preparation for publication in Radiat. Prot. Dosimetry (1993). 13. J.E Dicello and D.J. Brenner. Radiation quality of beams of negative pions, in: Pion and Heavy Ion Radiotherapy: Pre-Clinical Studies, ed. L.D. Skarsgard, Elsevier Science Publishing Co., Inc.: New York, 1983, p. 63.