Radiation and Environmental Biophysics

Rad. and Environm. Biophys. 16, 261--265 (1979)

© Springer-Verlag 1979

Effect of Negative Pions, Relative to 140 kV -- 29 MeV Photons and 20 MeV Electrons, on Proliferation of Ehrlich Ascites Carcinoma Cells K. R. Rao, H. Blattmann, I. Cordt, and H. Fritz-Niggli Strahlenbiologisches Institut der Universitgt Ziifich, August Forel-StraBe 7, CH-8029 Zfirich, Switzerland

Summary. The effect of negative pions (peak and plateau), photons (140 kV and 29 MeV), and 20 MeV electrons on the proliferative capacity of Ehrlich ascites carcinoma cells was investigated. Proliferative survival curves plotted for the modalities employed are presented. Under the experimental conditions used, the peak pions were more effective than plateau pions by a factor of about 1.4. For 50% survival, 140 kV X-rays had the same effect as peak pions but the latter was more effective (factor 1.2) at 10% survival level. When 140 kV X-rays were taken as the standard, following are the RBE values calculated at 50% survival level: plateau pions -0.73; peak pions --about 1.0; 29 MeV photons -0.73 and 20 MeV electrons --0.6. The results obtained are compared with those reported on other tumor systems and biological test objects. Introduction

Since the prediction of the potential advantages of applying negative pions in tumor radiotherapy (Fowler and Perkins, 1961) considerable efforts are being made at various research centers such as Los Alamos, Zurich, Vancouver etc. to evaluate the application of this modality in medicine and the initial radiobiological experiences gained are reviewed (Fritz-Niggli, 1974; Raju et al., 1977). Though considerable data is available on a variety of biological systems, there are relatively few reports on experimental tumors. The reason may be that solid tumors require high radiation doses and the pion dose-rate available for experiments was low. However in the following experiments with Ehrlich ascites tumor (EAT) we employed a vary sensitive assay system. Materials and Methods Modalities

The meson facility of the Swiss Institute of Nuclear Research was used. Pions were produced by bombarding a beryllium target with the 590 MeV proton beam. The

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tal composition) were filled into a cylinder-shaped tube of 12 cm length and 3.2 cm in diameter at 37 ° C. The gelatine turns fluid at 37°C whereas at the radiation temperature of 17° C, it remains solid. The tube was placed in a water phantom and irradiated with the tube aligned with the axis of the beam. After irradiation, the gel was sliced in intervals of 2 mm at 10° C. This allows to observe the biological effect of pion irradiation at various points of the depth dose profile. A central part of 2.5 cm was cut out of each slice and dissolved in 37 ° C medium. Samples of each slice were plated in 4 - 5 plastic Petri dishes. After 8 days of incubation, the cellcolonies were fixed and stained with toluidine blue. The proliferation capacity was estimated by counting the number of clones of more than 50 cells per cell-colony. Microclones (2--49 cells per clone) were taken into consideration as well. The gel/medium mixture has a density of 1.07, this means the stopping power is about 7% greater than that of water. All depth values presented here are corrected by this factor. The physical measurements of the pion beam are described in detail elsewhere (Fritz-Niggli and Blattmann, 1977a and b). The dose rate of the pion beam in the Bragg peak varied from 4-8.5 rad/min. Cells were put in identical tubes and exposed to the same experimental conditions as in the pion irradiation with 140 kV Xrays (HVL 0.93 mm Cu; 1 mm A1 + 0.25 mm Cu + 0.4 mm Sn) with a dose rate of 4.5 rad/min.

Results

Depending on the pion dose delivered, the resulting depth profiles for cell survival were different. In the lower frame of Fig. 1, the biological profiles are plotted against depth in water. In the upper part the relative physical dose of the pion beam is depicted. The resulting depth profiles for cell survival are so closely related to the physical depth dose profile, that we have really evidence of the suitability of this gel method and the cell line used. Along the depth dose profiles one can construct complete survival curves as many as are desirable and determine RBE as a function of dose for any depth. In Fig. 2 cell survivals are plotted from data taken from the plateau region at 14-15 cm in depth (50% relative dose), from the peak at 19 cm, the post peak I at 21 cm (80% relative dose) and from the post peak II at 23 cm (30% relative dose). The survival measurements of the peak and post peak I can be adequately accomodated by one single survival curve. This suggests, that the biological effect in this system of the beam does not change significantly throughout these two regions. In contrast the survival curve of the plateau region shows a large initial shoulder. The slope of the exponential part of the cell survival curves for plateau and peak pions are similar. The survival curve of the post peak II is characterized by almost the same shoulder as the X-ray response. The RBE values (140 kV X-rays = RBE 1) calculated at 0.5 and 0.1 surviving fractions are 1.3 for the peak and post peak region I and 1.1 for the post peak region II. The RBE for the plateau region are 0.8 and 1.0, respectively, at surviving fractions of 0.5 and 0.1. The plateau peak relation is 1.6 and 1.3, respectively, surviving fractions of 0.5 and 0.1.

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For investigation of further differences between effects of high LET and low LET radiations, we counted all small cell colonies which developed after irradiation. In Fig. 3, frequencies of the microclones are plotted against the dose. For higher doses the fraction of clones with cells with "non-lethal" damage increases. The microclones, not contained in the survival curves probably have a wide range of damage. We observed microclones with only giant cells, microclones with micronuclei and/or mitosis as well as pycnotic nuclei. The depicted frequency does not take into account these kinds of damage. We recorded the data of 140 kV Xrays and of four different depth in the pion profile as mentioned above. Compared to the X-ray response the number of microclones in peak, post peak I and plateau increases considerably more from approximately 700 rad onwards. The resulting RBE values for peak pions are comparable with those obtained from the survival curve. It seems that the RBE values for plateau, post peak and post peak II are somewhat higher than found in cell survival studies. The statistical evaluation, however, has not yet been carried out.

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Cell Survival over the Depth Profile after Irradiation

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Discussions The biological effectiveness depends strongly on the different local energy deposition spectra for negative pions in depth. Menzel et al. (1978) report, that the microdosimetric spectra in the plateau region of the pion beam was similar to those observed of other low LET radiation (e.g., gamma rays) except a small number of events above 10 keV/p~m, whereas for the peak and 3 cm beyond, a marked increase of events above 100 keV/p.m was observed. The wide shoulder in the survival curves obtained with plateau pions may indicate that sublethal damage, produced by low LET, is accumulated before cell killing actually results. In contrast to this the small shoulder of the survival curve in the peak and 2 cm beyond implies sublethal damage to a lesser degree. The final slope, similar for plateau and peak, is often interpreted as result of the double-strand break of the DNA which depends on the high LET particles of the radiation (Chadwick and Leenhouts, 1973). On the other hand, experiments by Fritz-Niggli and Blattmann (1969), studying the proliferation kinetics of the yeast cells, point out that several different damage-causing mechanisms must be considered in order to interpret dose effect curves. The authors conclude that cytoplasmatic damage gains importance along with increase of the dose. Also microclones must be seen from this point of view. The relative number of "non vital" clones grows with increasing dose. Our data show that not only low but also high LET-particles produce "non lethal" damage. The probability of survival as microcolony is greater than as macrocolony for X-rays and pions. This means that with increasing dose the number of undamaged colonies becomes small in relation to the number of surviving colonies. Observations of Westra and Barendsen (1969) put emphasis on the fact that after a certain time a fraction of microclones regain the capacity of unlimited proliferation and become macroclones. Looking at macro- and microclones, also some other aspects must be considered. In order to get sufficient cell survival we must for technical reasons raise the initially plated number of cells with increasing dose. The higher total number of cells may cause intercellular effects which can influence the colony-forming ability. In comparing the results of plateau and peak irradiations it must be taken into account, that the dose rates were different for different positions in the irradiated tube and in addition they varied from experiment to experiment. All experiments with low doses got a low dose rate of about 2 rad/min in the plateau region, whereas for high doses it was about 4.5. This different dose rate could influence the survival curves and the frequency of the microclones as well. The presented results demonstrate a marked difference in the shoulder of the survival curve between plateau and peak irradiation. The pions in the peak region are, compared to the plateau region more effective in cell inactivation at low doses, which is of clinical interest for fractionated treatment. Within certain restriction (due to different cell lines, irradiation techniques, reference radiation) the results are consistent with the most recent data from the pion facilities at Vancouver and Los Alamos. Skarsgard et al. (1977) in Vancouver got an RBE of 1.3 in the peak center whereas Todd et al. (1975) in Los Alamos observed an RBE of 1.4--1.5 with human kidney cells. Acknowledgements. The authors wish to thank Mrs. D. Bohrer, Mrs. B. Mustur, and Mr. P. Binz for skilfultechnicalassistance. Specialthanks also go to Prof. Skarsgardfor his sincereinterestin the subject matter and for sendingthe gelatinewhichwas apparantlynot availablein Europe.

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References 1. Chadwick, K. D., Leenhouts, H. P.: A molecular theory of cell survival. Phys. Med. Biol. 18, 78-87 (1973) 2. Fritz-Niggli, H., Blattmann, H.: Verschiedene Mechanismen der Strahleninaktivierung in Abh/ingigkeit vonder Dosis und physiologischen Parametern. Biophysik 6, 46--62 (1969) 3. Fritz-Niggli, H., Blattmann, H.: Different genetic and cellular responses to negative pi mesons in dependence of intrinsic properties of reaction systems: A new concept of RBE. Rad. and Environm. Biophys. 14, 95--108 (1977a) 4. Fritz-Niggli, H., Blattmann, H.: Preclinical experiments with the SIN negative pi meson beam. IAEA-SM-212/71 Radiobiological Research and Radiotherapy, Vol. II, pp. 31-47 (1977b) 5. Menzel, H. G., Schuhmacher, H., Blattmann, H.: Experimental microdosimetry at high-LET radiation therapy beams. Proceedings of the 6th Symposium on Microdosimetry (1978, in press) 6. Skarsgard, L. D., Palcic, B.: Pretherapeutic researchprogrammes at negative pion facilities. International Congress Series No. 339. Radiology 2, 447--454 (1974) 7. Skarsgard, L. D., Palcic, B., Henkelmann, R. M., Lain, G. K. Y.: RBE measurements on the negative pi meson beam at TRIUMF. IAEA, Advis. Group Meeting 19.-21. 12. 1977 8. Todd, P., Shonk, C. R.,West, G., Kligermann, M. M.,Dicello, J.: Spatialdistribution ofeffects ofnegative pions on cultured human cells. Radiology 116, 179--180 (1975) 9. Westra, A., Barendsen, G. W.: Proliferation characteristics ofcultured mammaliancells after irradiation with sparsely and densely ionizing radiations. Int. J. Radiat. Biol. 11, 447-485 (1966) Received February 15, 1979