. -- - I OFT ORNL P 3085 4. EEEFEFFE 1.25 1..4 LG MICROCOPY RESOLUTION TEST CHART NATIONAL BUREAU OF STANDARDS - 1963 - -. -. . ."' ... 'TGALILI GLALTER EGET MATT FT- H .n . n * Wri , d. r** * ORNO -3081 Conf. 67039stups . JÚN 2 2 1962 MASTES en veckas CISTI RUCES Relative Biological Effectiveness of Gamma Rays, X-rays, Protons, and Neutrons for Spermatogonial Killing* E. F. Oakberg INTRODUCTION High sensitivity to radiation-induced cell death and ease of quantitation of surviving cells make spermatogonia of the mouse a good radiobiological test system (Oakberg, 1957). With appropriate techniques, the effect of doses in the range of 2-1000 rads can be studied. These attributes have led to the present experiments on the comparison of the biological effectiveness of different types of radiation. Three types of spermatogonia can be recognized in the mouse (Oakberg, 1956a). Type A spermatogonia are the stem cells or the seminiferous epithelium, and through the process of stem cell renewal (Leblond and Clermont, 1952) maintain their own numbers while giving rise to an unlimited number of differentiated cells. Intermediate spermatogonia are derived from type A, and divide once to form spermatogonia of type B. B spermatogonia divide once to forni primary spermatocytes (Oakberg, 1956a). Intermediate and B spermatogonia. are of homogeneous sensitivity, and when log- survival is plotted against dose, give survival curves with a shoulder at doses of 10 R or l.ess, and a steeper, exponential decrease in survival at higher doses. LD50's in the range of 21-25 R of Co60 gamma rays have been reported for these cells (Oakberg, 1957). Survival at 100 R is essentially zero. Type A spermatogonia are of heterogeneous sensitivity, with survival comparable to intermediate and B types at doses below 25 R. The survival curve then flattens markedly at doses of 50-1000 R presumably because of a resistant com- ponent in the population. Thus at doses of 25 R or less, data on survival of type A spermatogonia can be combined with that of late type A and intermediate spermatogonia to obtain larger numbers of cells and greater statistical :: reliability of estimates of radiation effects. The resistant component of the A population can be used for doses in the range of 100 to 1000 R. . MATERIALS AND METHODS Male F, hybrid mice from the cross of inbred 1.01 strain females with inbred C3H males have been used exclusively. All animals were 12 weeks old at the time of irradiation. Mice were killed 72 hours after irradiation when doses were 200 R or less and five days after irradiation for doses of over 200 R. Testes were fixed in. * Research sponsored by the V. S. Atomic Energy Commission under contract with the Union Carbide Corporation. DISTRIBUTION OF THIS DOCUMENT IS UNLIMITED, . vi . --- der in winter senere . ' R . Zenker-formol, embedded in paraffin, sectioned at 5u, and stained by the PAS technique. One hundred tubule cross sections, distributed among the stages of the cycle of the seminiferous epithelium on the basis of a previously determined frequency distribution for control mice (Oakberg, 1956b) were scored for each mouse. Apparently normal spermatogonia, in all tubules, and pre-leptotene . spermatocytes in stage VII were counted. Data were expressed as experimental control ratios. RESULTS No significant difference was observed between 250 and 280 kv x-rays, and the data were pooled to provide a more reliable base of comparisons for the pre- liminary estimates of RBE given in Table 1. Slopes were fitted to the data and the ratio of the slopes compared to obtain the RBE. At present, data are restricted to late A and late A+ intermediate spermatogonia. Although the actual values vary from 0.76 for 130 and 730 Mev, protons for late type A to 1.38 for Cobo ganima rays for late A+ intermediate spermatogonia, RBE'S of 1 should be acceptable for these radiations. In an earlier study (Oakberg and Clark, 1961), the relative effectiveness of C060 gamma rays, 2.5 Mev and 14.1 Mev neutrons was compared. These data are given in Table 2. Two sets of RBE'S were computed, one for each cell type over the entire dose range, and a second set for the combined survival of all spermatogonial types at low doses. The RBE for both levels of neutron energies is greater than 1, and probably about 2 when the comparison is based on all the data. Limitation of the comparison to low doses raised the point estimate to 8 for 24.1 Mer and to 3 for 2.5 Mev neutrons. Since the confidence limits overlap, the RBE's for 2.5 and 14.1 Mev energies are not significantly different. Recently, cell survival after irradiation with fission neutrons with an average energy of 1-2 Mev has been measured (Figure 1). These data are for cells irradiated and scored as type A spermatogonia, i.e., a different cell type from those represented by the data of tables I and 2. C06 ganma rays, 250 kv x-rays, and 730 Mev protons gave equal survival. The neutron RBE, however, was based only on the x-ray data. A value of 6.5 was obtained for neutron doses of 18-101 rads, and of 4.7 for doses of 172-258 rads. DISCUSSION AND SUMMARY Statistical analyses of the data are not complete, especially for cells irradiated and scored as type A spermatogonia. On the basis of preliminary results presented here, it is reasonable to assume an RBE of 1 for 250 kv x-rays, 280 kv x-rays, 130 Mev protons, 730 Mev protons, and C060 gamma rays. Previously, we had attributed the lack of a difference in RBE between 14.1 and 2.5 Mev neutrons to a "wasting" of energy with the more densely ionized track of th: 2.5 Mev neutrons (Oakberg and Clark, 1961). This explanation no longer appears tenable in view of the RBE'S of 6.5 and 4.7 for fission neutrons, with an average energy of 1-2 Mev. Obviously, there is no simple relationship be- tween her and biological effect, as already pointed out by Smith and Rossi (1966). The phenomenon of higher neutron RBE's at low doses, as given in Table 2 and Figure 1, have been observed previously. The logical explanation of this . . . phenomenon is that there is an area of less slope (shoulder) on the x- arid ganuma ray survival curves; whereas the neutron dose curves are linear. 'Accordingly, divergence of x or grama ray and neutron curves will be greatest at low doses. This presentation is obviously over-simplified, for a continuous scale of RBE piicbably exists for each energy comparison. The RBE of neutron v8. ionizing radiation therefore is quite arbitrary owing to the comparison of a dose-effect curve which is curvilinear ionizing radiation) with a linear dose- effect relationship (neutrons). Use of systems which give linear effects with Lonizing radiation is not the answer either, since quals biological effect usually occur. In summary, X-rays, gamma rays, and 130 as well as 730 Mev protons all appear to yield RBE's of 1. Neutron RBE's are a function of LET and dose, being greater with both low doses and higher LET. No simple relationship exists, however, between HPE and either dose or LET. ACKNOWLEDGEMENTS The author wishes to express his appreciation to Dr. A. H. Koehler and other staff members of the Harvard Cyclotron Laboratory for the 1.30 Mev proton irradiation; to the staff of the Lawrence Radiation Laboratory at Berkeley, California, for the 730 Mev proton irradiations; to Dr. M. L. Randolph for the 2.5 and 14.1 Mev neutron irradiations; to the Health Physics Division of ORNL for the fission neutron exposures; and to Dr. D. G. Gosslee for statistical analysis of the data. REFERENCES 1. Leblond, C. P., and Y. Clermont, Spermiogenesis of rat, mouse, hamster, and guinea pig as revealed by the "periodic acid-fuchsin sulfurour acid" technique. Am. J. Anat., 90: 167 (1952). 2. Oakberg, E. F., A description of spermiogenesis in the mouse and its use in' analysis of the cycle of the seminiferous epithelium and germ cell. renewal. Am. J. Anat. 99: 391 (1956a). Dakberg, E. F., Duration of spermatogenesis in the mouse and timing of stages of the cycle of the seminiferous epithelium. Am. J. Anat. 99: 507 (1956b). 4. Dakberg, E. F., Gamma-ray sensitivityof spermatogonia of the mouse. J. Exp. Zool. 13lt: 343, 1957. 5. Dakberg, E. F., The effects of dose, dose rate and quality of radiation on the dynamics and survival of the spermatogonial population of the 6. Oakberg, E. F., and E. Clark, Effect of dose and dose rate on radiation damage to mouse spermatogonia and oocytes as measured by cell survival. J. Cell Comp. Physiol. Suppl. 1, v. 58: 173 (2961). ... 7. Smith, H. H., and H. H. Rossi, Energy requirements and relative biological: effectiveness for producing cytogenic phenomena in maize by irradiating seeds with x-rays and monoenergetic neutrons. Rad. Res. 28: 302 (1966). . ......... .. . . . . . . 1 ** * * W Buit. .. . I [. .. ... ALI . t Ver. . .. . LE . . : Table 1 Estimates of RBE'S of Cobo gamma rays, 730 Mev protons, and 130 Mev protons to 250 and 280 kv x-reys based on spermatogonial killing : . I. Late A spermatogonia Source: RBE 730 Mev Protons .765 130 Mev Protons .755 60 Gamma .799 250-280 Kv X 1.000 II. Late A+ Intermediate spermatogonia Source RBE 730 Mev Protons .973 130 Mev Protons 1.013 60 Gamma 1.379 250-280 Kv X 1.000 LEGAL NOTICE This report was prepared as an account of Govorament sponsored work. Noithor the United States, nor the Commission, nor any person icung on behalf of the Commission: A, Makes any warranty or representation, expressed or implied, with resject to the accu. : racy, completeness, or usefulness of the information coatuined in this report, or that the we of any information, appart tus, method, or procesu disclosed in the report may not Infringe : privately owned righto; or B. Assumos any liabiliuos with respect to the use of, or for damagos resulting from the use of any information, apparatus, method, or procede disclosed in this report. As used in the above, "period acting on behalf of the Commission" includes way om- ployee or contractor of the Commission, or etapioy of such contractor, to the extent that such employee or contractor of the Commission, or employee of much contractor properes, disseminates, or provides accos to, any information pursuant to his employment or contract with the Cosmission, or his omployment with such contractor. - - - - - - + DAR Table 2 RBE of neutrons to Coºo y rays for spermatogonial killing RBE Neutron energ Cell type scored Lower 95 % confidence limit Point estimate Upper 95 % confidence Limit : А B RPS pooled 14.1 Mev 1.41 2.19 2.11 4.39 1.76 2.52 2.38 2.76 2.89 2.69 16.42 8.18 , А B 2.5 Mev 1.26 1.22 0.73 0.68 1.89 1.85 1.10 3.03 3.32 2.53 1.49 6.80 RPS pooled* *All three cell types combined for low dose ranges only: (1-23 rad for Cocº y rays, 0.5-5.0 rad for 14.2 Mev neutrons, 0.25-7.42 rad for 2.5 mev neutrons). (From Oakberg, E. F., and E. Clark, J. Cell Comp. Physiol. Suppl. to Vol. 58: 173-182, 1961.) ' - 1. FIGURE LEGEND Figure 1 Survival of type A spermatogonia of the mouse after gamma-ray, X-ray, 730 Mev proton, and neutron irradiation. (. = gamma rays, o * X-rays, A = 730 Mev protons, A = neutrons). (From Oakberg, E. F., Jap. J. Genetics 40: 219-127, 1964). 6. 7 'Er'17 .. T- - . UN Tii .... - nu.1.1 t .. HPV irtu:56.-...... - - - - - +-- - - - - - "}: - + - M, L, - -- t + - rs. iury w Wa 13,648 .-y RAY 0-X RAY A-NEUTRONS 4-730 Mev PROTONS o ******************************* SURVIVING FRACTION 0.001- 100 200 300 400 500 600 700 800 900 1000 DOSE (rads) F112 - . . END DATE FILMEI 8 / 23/07