Baarli, Johan

Bianchi, M

Quintiliani, M

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EUROPEAN ORGANIZATION FOR NUCLEAR RESEARCH DI/HP/144 Heal th Physics 5 July 197l A DISCUSSION OF THE :BIOLOGICAL SYSTEM USED FOR THE STUDY OF THE RJ3E IN THE PION :BEAMS AT CERN M. Bianchi and J. J3aarli CERN, Geneva, Switzerland M. Quintiliani Istituto Superiore di Sanita Rome, Italy The testis is known to be one of the most radio-sensitive organs in mammals and is for this reason very suitable for studies of biological effects due to small radiation doses. The depletion of the seminiferous epithelium appears after a small exposure and is due to spermatogonia death and gives rise to a testis weight loss and a reduction of fertility. The radiation effect on spermatogonia of mice has been exten-sively studied by Oakberg1 ), using the acrosome of the developing spermatids for identifying the stages in the cycle of the epithe-lium2). He also studied the duration of the spermatogenesis in mice and the timing of the cycle of the seminiferous epi theliurr_ and the development of the sperrratogonia3 ). The spermatogonia represent the mitotic stage of the spermato-genesis and by the multiplication of these cells a large population is produced. During the multiplication the spermatogonia change morphologically and three types of spermatogonia can be distinguished: type A, type Intermediate, and type J3 spermatogonia; it is also possible to identify four generations of the type A spermatogonia. Oakberg found for the type A spermatogonia a heterogeneous sensitivity toy rays with an effect comparable to Intermediate and type J3 for doses below 25 R but with much less efficiency for doses above 50 R. The ~O (50% effective dose) of type A spermatogonia was found to be 22.5 R for y rays. The Intermediate and type B spermato-gonia show a comparable sensi ti vi ty with an ED 50 eqval to 21. 4 R1 ). Presented at Meeting on :Biomedical Application of Pion :Beam from Isochronous Cyclotron Zurich, 14-15 May, 1971 ... 2 -Monesi suggests that the d:ifference in r.adiosensi ti vi ty could be correlated to the length of the post-DNA synthesis time which is different for the different types of spermatogonia4) and proves that after the DNA synthesis (during the prophase) the type J3 spermatogonia are much less sensitive to radiation than during or before the DNA synthesis (interphase). The primary factor for the radiation depletion of spermatogonia is the death of the cells5 ) which occurs when the cells come to a critical stage of the mitotic cycle, late interphase or very early prophase for the type A and I spermatogonia, anaphase or teleophase 4\ for the type B spermatogonia ). It has been shown that the spermatogonia survival after y · 1 1 · d d t f th dose-rate6 ' 7 ). F t A exposure is arge y in epen en o e or ype spermatogonia, Oakberg et al. found no significant difference in survival of mice irradiated at a dose-rate between 86 R/min and 0.8 R/min: a marginal effect of dose-rate was observed at the highest doses when mice were irradiated at a dose-rate of 0.009 R/min, and also a significant variation of the survival has been observed when mice were irradiated at a dose-rate of 0.0001 R/min. It has also been found, in our experiments, that the survival of type B spermatogonia was largely independent of dose-rate between 1 I 7 ,8) 4.5 and O. rad min of y rays, as shown in Fig. 1 The spermatogonia survival was not observed to be affected by ' 6) the stress procedures, as also reported by Oakberg et al. as well as by our group7). The lethal effect on spermatogonia appears to depend only on the dose absorbed by the testes, and not on the irra-diation of the rest of the animal. In addition, it was found that the spermatogonia survival was a very reproducible test system for radiation effects at low doses and also offered the possibility to use few animals and obtain a large number of information. In view of what has been mentioned, the spermatogonia survival has been considered as one of the most suitable systems for the study of the RBE of present pion beams which is limited by the very low dose-rates and small areas of uniform dose distribution (dose-rate of about 4 rad/hover a sphere of 2 cm in diameter). ( - 3 -MATERIALS AND METHODS Mice CrH/RL/Cas, 9 to 12 weeks old, were used. The mice were exposed singly in plastic tubes in the pion beam absorption peak, as shown in Fig. 2. The animals were sacrificed 46 hours after the start of irra-diation and the testes removed and fixed in Orth fluid and embedded in paraffin. Sections of 5 µ thick were made and stained, using the periodic fuchsin sulphurous acid technique and counter-stained with acid hematoxylin. Seminiferous tubules were classified according to Oakberg2) on the basis of the acrosoma development of spermatids as shown in Fig. 3. The type B spermatogonia cells when irradiated in the late stage IV and at the beginning of stage V are Resting Primary Spermatocytes in stage VII 46 hours later in accordance with the duration of the stages of the seminiferous cycle of the mouse shown by Oakberg3) (Fig. 4). Tubules in stage VII having a circular cross-section were selected and the number of RPS and the number of Sertoli cells in each tubule were scored. The number of Sertoli cells were scored to check the normality of the tubule. Figures 5 and 6 show an example of the difference between an irradiated and a not irradiated tubule. A minimum number of three animals irradiated at each dose and a minimum of 50 tubules for each animal were used for scoring. RESULTS AND DISCUSSION The average number of RPS and SC per tubule scored at each dose is shown in Table 1. The per cent of survival is represented in Fig. 7, in which the y-ray dose-response is also reported. The error bars represent the 95% confidence limits. The results obtained show for the negative pion beam a decrea-sing RBE with the increase of the dose. For the positive pion beams a straight line seems to be the best choice. Tbe RBE for the ED 50 was 2.3 for the positive pion beam, and 3.7 for the negative pion beam. - 4 -The difference in the effectiveness between the negative and the positive pion beam is most likely due to the difference in inter-action processes which occur at the end of the range of the particles. One of the possible explanations for this dose-response relation-ship could be the heterogeneity of population under irradiation. In that case it will be necessary to assume that the population shows the same sensitivity to X and y radiations but different sensitivity to pions. It appears from the results which we have obtained with the low-intensity pion beam that many other radiobiological experiments would be necessary to provide a better estimate of the biological effectiveness of stopped negative pion beams. Many factors influence these results, such as depth-dose characteristics, radiation composi-tions, OER ratios, etc., and it is of importance to establish these experimentally before any use is made of negative pion beams for cancer treatment. - 5 -REFERENCES 1. E.F. Oakberg, Exptl. J. Zool., l:l.i' 343 (1957). ( 2. E.F. Oakberg, Am. J. Anat., 99, 391 (1956). 3. E.F. Oakberg, Am. J. Anat., _22, 507 (1956). 4. V. Monesi, Rad. Res., 17, 809 (1969). 5. E.F. Oakberg, J. Morphol., 97, 39 (1955). 6. E.F. Oakberg, Japan J. Genetics, Suppl., 40, 119 (1964). 7. M. Bianchi and M. Quintiliani, Ann. Ist. Super. Sanita, _2., 39 (1969). Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. - 6 -FIGURE CAPTIONS Survival fraction (per cent) of the RPS (Resting Primary Spermatocytes stage VII) as a function of dose for various dose-rates of y radiation. Experimental arrangement used for exposing the mice in the pion beams. The various stages of the development of spermatids during the spermiogenesis (as described by Oakberg) after staining with APS and haematoxylin. The acro-some of spermatids appear black in the picture but is red on the slides. This is used to identify the stage of the tubules. Kinetics of spermatogenesis in mice with the duration of each stage of seminiferous epithelium as estab-lished by Oakberg. Cross-section of tubules in stage VII after irradiation. Cross-section of tubule in stage VII from an unirradiated animal. Survival fraction (per cent) of the RPS (Resting Primary Spermatocytes stage VII) as a function of dose for captured negative and positive pion beams. Dose - 7 -Type B spermatogonia surviving after positive and negative pion irradiations Total RPS* per Mean RPS (rad) tubules tubule surviving scored (:!: 0-) fraction 1. Positive £ions 0 2101 > 30.8 + 4.5 1.00 1.1 151 26.5 + 4,1 0.86 2.3 188 22.1 + 3.7 0.72 5.7 234 19.8 + 3.8 o.64 8.9 147 13.4 + 4,5 0.43 11.4 204 11.4 + 4.4 0.37 12.6 183 9.5 + 4,4 0.31 2. Negative £ions 0 2501 ) 30.8 + 4,5 1.00 1.2 156 23.2 + 3.3 0.75 2.3 201 20. 0 + 3.5 0.65 4.4 213 15.9 + 4.2 o. 51 4.6 148 15.0 + 3.4 0.49 9,3 207 10.2 + 4.6 0.33 11.1 203 7.3 + 3.9 0.24 *) RPS = Resting Primary Spermatocytes **) SC = Sertoli Cells 1) The control refers to 11 animals, 5 for the negative pion beam and 6 for the positive pion beam. SC** per tubule 8.7 8.3 8.1 8.6 8.5 8.6 8.9 8.4 8.4 8.7 8.7 8.6 8.4 8.7 cc cc c ~lil~ "O "O "O -0 "O EE EE E r--.. LO ('I) "7 LO N...:too~ eo x<JO '? ~ CX> !:':-<'? -0 0 0 0 <l• M 0 N 0 LO M LO -0 -. 0\ ·-LL ( N SIS/R/16871 V) ::>~ .... ----_, w .... w 15 ~~ c...; 5 ~~ --t;;o --A limits between stages of a the seminiferous epitheliLm I mitosis STAGE VIII (20.sh9 SPERMATOZOA 13 Dia diakinesis I M-1 first meiotic division Fig. 4 S-11 secondary spermatocytes M-11 second meiotic division Fig. 5 Fig. 6 Ul ~ ~ ~ "O c "' )( 0 . -L.() 0 I / / / / I I "O t!! / I / I Ln / -I I 0 -ClJ r:---. Ul 0 0\ "O ·-LL -0 

A discussion of the biological system used for the study of the RBE in the pion beams at CERN

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