Relative biological effectiveness of X-rays and fast neutrons in inducing translocations in mouse spermatogonia

Mutation Research, 19 (1973) 343-347

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

343

R E L A T I V E BIOLOGICAL E F F E C T I V E N E S S OF X-RAYS AND FAST N E U T R O N S IN I N D U C I N G TRANSLOCATIONS IN MOUSE SPERMATOGONIA

S. MURAMATSU, W. NAKAMURA AND H. ETO Division of Radiation Hazards, National Institute of Radiological Sciences, Chiba-~8o (Japan)

{Received April 9th, 1973)

SUMMARY"

The dose-response relationships for inducing translocations in the spermatogonia of mice were studied, and they were compared for 2oo kVp X-rays and 2 MeV fast neutrons. The dose response for fast neutrons was markedly convex; more precisely, the response obtained was linear in the dose range from 24 to 94 rad with a regression coefficient of II.36. IO-L but decreased for a further increase in dose up to 267 rad. On the other hand, that for X-rays showed a linear dose-response relationship from 48 to 672 rad with a regression coefficient of 2.69 • lO-4. The relative biological effectiveness for inducing translocations in the spermatogonia of mice was compared for the linear parts of the dose response in both types of radiation, and the relative biological effectiveness (RBE) value was 4.22.

INTRODUCTION As a rule, genetic effects of fast neutron irradiation on male mice are greater than those resulting from low-LET radiations such as X-rays or 7-rays as has been shown for various types of somatic damagel*, 16. The dose-response relationship for the induction of translocations in spermatogonia of mice b y X-rays is linear for exposures below 6oo or 8oo R*, 5-8. However, there is relatively little information on the effects of fast neutron irradiationn, 14. I t seems important, then, to accumulate information on the effectiveness of high-LET radiations on these criteria in order to assess the genetic effects on mice. The present study was carried out (z) to determine the dose-response relationships for the induction of translocations in the spermatogonia of mice after irradiation b y 200 kVp X-rays and 2 MeV fast neutrons, and (2) to compare the relative biological effectiveness of the two types of radiation. Some preliminary results have been reported elsewhereS, 9. Abbreviations: LET, linear-energy transfer; RBE, relative biological effectiveness.

344

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MATERIALS AND METHODS

io-week-old male mice of the ddY/SLC strain were used for this experiment. They were subjected to whole-body irradiation at various doses with either 200 kVp X-rays or 2 MeV fast neutrons. Fast neutrons were produced from the 9Be(d,n)X°B reaction b y bombarding a thick beryllium target with 2.8 MeV neutrons obtained from a Van de Graaff accelerator. The moderate neutron flux at this biological facilities and the dosimetry have been reported by INADA et al.3, 4. The y-ray contamination was estimated not to exceed lO% of the total dose, and the dose rate was about 3 rad/min. For X-ray exposure, a conventional therapy machine was operated at 200 kVp and 20 mA, with o.5-mm Cu and o.5-mm A1 filters, with a dose rate of about 85 R/min. Exposure doses were converted into absorbed doses in rad b y applying by a factor of 0.96. IO weeks later, preparations of meiotic spermatocytes were made by the airdrying method of EVANS et al. 1, and 200 to 400 meiotic diakinesis-metaphase figures were analyzed for each mouse. RESULT~

Non-irradiated control group. No chromosomal abnormality such as translocation or aneuploidy was observed in IO ooo cells analyzed from 30 control mice (Table I). In a few instances, cells having unattached X and Y univalents were recognized, and were counted as normal cells.

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Fig. i. Dose-response neutron doses. --O--,

r e l a t i o n s h i p s f o r t r a n s l o c a t i o n s p e r cell w i t h r e s p e c t t o t h e X - r a y a n d f a s t X - r a y s ; - - - © - - -, f a s t n e u t r o n s .

Irradiated groups. Results obtained for the frequencies of different dose levels with both types of radiation are given in Table I, and are plotted against exposure dose in Fig. I. A total of 56200 primary spermatocytes, 26200 from 9° X-irradiated mice and 30 ooo from 75 neutron-irradiated mice, were analyzed. Of 56 200 cells obtained from both groups of mice, 3845 (2257 from the X - r a y group and 1588 from the

X-RAY- AND NEUTRON-INDUCEDTRANSLOCATION8IN SPERMATOGONIA

345

TABLE I F R E Q U E N C Y OF P R E S U M P T I V E T R A N S L O C A T I O N S IN P R I M A R Y S P E R M A T O C Y T E S A F T E R V A R I O U S X-RAY

Dose (tad)

AND FAST NEUTRON DOSES

Number Total Number of translocations of mice number of o I 2 3 used cells analyzed

Cells with translocations (%)

Number of translocations per cell

Control

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3°

IOOOO

IOOOO

O

O

O

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O.OOO

300o 3000 4o0o 44oo 2600 3ooo 3°00 3200

2924 2889 3684 4o49 2372 2677 2595 2753

76 lO8 311 34° 214 294 367 430

o 3 5 I1 14 28 38 14

o o o o o I o 3

2.53 ±o.29 3.7o~:o.34 7.9o±0.43 7.98~o.4I 8.77~o.55 lO.77±o.57 13.5o±o.62 I3.97±o.61

o.o25 0.038 o.o8o o.o82 o.o93 o.131 o.148 o.212

3880 3829 3707 3605 3683 3846 I936 3926

113 I55 273 354 289 I32 55 73

6 I6 18 39 27 22 8 I

I o 2 2 I o I

3.00±0.27 4.28 ± 0.32 7.33 ± o . 4 I 9.88 ± 0.47 7.93 ±0.43 3.85 ± 0.3o 3.20±0.39 1.85 ~O.2I

0.032 0.047 o.079 o.IIO 0.087 0.044 0.037 O.O19

X-irradiation

48 96 192 288 384 48o 576 672

io IO 15 15 io io io 1o

Fast neutron irradiation

24 47 71 94 141 I78 I98 267

io lO IO io io IO 5 IO

4000 4000 4000 4°00 4000 4o0o 2000 4000

O

n e u t r o n group) were classified as a b n o r m a l spermatocytes. Most of t h e m were f o u n d with one t r a n s l o c a t i o n i n v o l v i n g four chromosomes in a ring ( 1 8 1 1 + R I V , 52.8%) or a chain ( I 8 I I + C h I V , 29.9%), a n d the rest were recorded as h a v i n g two or more translocations or small fragments. No difference was observed in the t y p e a n d freq u e n c y of these abnormalities for the two types of radiation. F o r the X - i r r a d i a t e d group (Table I) there was a linear relationship between the m e a n n u m b e r of translocations per cell a n d the exposure dose, which increased from 0.025 after 48 rad to o.212 after 672 rad. The linearity was o b t a i n e d at least over the dose range from 48 to 672 rad (Fig. I). This relationship between exposure dose a n d t r a n s l o c a t i o n frequency per cell can be expressed b y the following e q u a t i o n for a straight line: Y = (5.63 4- 4-34)"IO-S+(2-69 4- o.19)"IO-~ X, where Y is the m e a n yield of translocations per p r i m a r y spermatocyte, a n d X the X - r a y exposure dose in tad. A similar dose-response relationship was o b t a i n e d for the affected cells with a regression coefficient of 1.99. IO-*. The dose response for the fast n e u t r o n group was m a r k e d l y different from t h a t o b t a i n e d for the X-rays. The m e a n n u m b e r of translocations per s p e r m a t o c y t e increased linearly with the dose up to 94 rad, a n d reached a m a x i m u m of o.11o. Thereafter, it decreased for a f u r t h e r increase in dose u p to 267 rad. F u r t h e r m o r e , a n irradiation of 267 rad was less effective in i n d u c i n g translocation t h a n was t h a t of 24 rad. To compare these results with those o b t a i n e d from X-rays, the regression coefficient was calculated from the linear p a r t of the dose-response relationship o b t a i n e d in the

346

s. MURAMATSUet al.

neutron dose range from 24 to 94 rad. The slope calculated was (I1.36 ~- 0.69)-io -4, which is greater than that obtained with X-rays. Similar results were obtained for the affected spermatocytes. The results indicate that irradiation with fast neutrons in the dose range below 94 rad was more effective in inducing translocations than was that with the same dose of X-rays. DISCUSSION

The results presented here demonstrate the dose-response relationships of chromosome translocations induced in the spermatogonia of mice with X- and fastneutron radiations, and compare the relative biological effectiveness of the two types of radiation. No chromosome translocations were found in IOOOO primary spermatocytes obtained from 30 normal ddY/SLC males aged I4O days. The present results of the controls confirmed the previous findings of L]~ONARD AND DEKNUDT~ for BALB/c males and of SEARLE et al. 14 for (C3H/HeH × IoI/H)F1 males. It is inferred from these findings that the frequency of spontaneous translocation in spermatogonia is very low at younger ages (about 30o days and less) even for those that m a y occur spontaneously in non-irradiated males. After X-irradiation, translocation induction in spermatogonia increased linearly with exposure dose (Fig. I). The linearity of the translocation yield increase was obtained over the dose range from 48 to 672 rad. The present results are in general agreement with the findings of LI~ONARDAND DEKNUDTS,7 and EVANS et al. ~ in their linearity, except for a slight difference in slope. The regression coefficient of the present results (2.69.1o-4; t = 14.o 5, P < o.oooi) is larger than that (1.7.1o -4) obtained by LI~ONARDAND D E K N U D T 7, but smaller than that (2. 9 • 10 -4) obtained by EVANS et al. 2. The difference of slope m a y indeed be due to the radiosensitivity of the different strains as considered by EVANS et al. 2. The dose-response relationship for fast neutrons, on the other hand, was markedly convex with a m a x i m u m of o . i i o at 94 rad which is different from that for X-irradiation. SEARLE et al. 14 have reported results on the induction of reciprocal translocations in the spermatogonia of mice receiving 0. 7 MeV fast neutrons. The shape of the dose-response relationship for these results is also markedly convex, with a peak around IOO tad. Our results are rather similar to those of SEARLE et al. A humped dose-response curve has also been observed for the induction of translocations in spermatogonia of mice at higher X-ray doses than the dose range studied b y us, i.e., 800 R or moreT, TM. OFTEDAL has proposed a theoretical model for these humped dose-response curves that would result from the heterogeneous radiosensitivities of the spermatogonial populations 1°. If the radiosensitivities of spermatogonia for both cell destruction and translocation induction coincide, the present results m a y be interpreted using OFTEDAL'Smodel. However, as discussed by Lt~ONARD (ref. 5), further attempts to carry out a detailed analysis seem necessary in order to resolve the kinetics of these translocation inductions in the spermatogonia of mice. A comparison of the biological effectiveness for 2 MeV fast neutrons was made on the linear part of the dose-response curve. The R B E value was found to be 4.22 from the following regression coefficients: 2.2. IO-* for X-rays and 11.36. IO-* for fast neutrons. The present value of about 4 is not very different from the findings of

X-RAY- AND NEUTRON-INDUCED TRANSLOCATIONS IN SPERMATOGONIA

347

SEARLE et al. (3.7 for 0. 7 MeV fast neutrons) x* and of POMERANTSEVAet a/. (4.4 for 1.5 MeV fast neutrons)n. Hence, it may be concluded that the neutron irradiation was more effective than that of X-rays for inducing reciprocal translocations in the spermatogonia of mice, and that this feature is similar to that obtained with specific loci mutations, dominant lethal mutations, and various types of somatic injury in the samel~,l~. ACKNOWLEDGEMENTS

The authors thank to Dr. T. INADA, Division of Physics, National Institute of Radiological Sciences, Chiba, for the irradiation and dosimetry of fast neutrons. REFERENCES I EVANS, E. P., G. BRECKTON AND C. E. FORD, An air-drying method for meiotic preparation

from mammalian testis, Cytogenetics, 3 (I964) 289-294. 2 EVANS, E. P., C. E. FORD, A. G. SEARLE AND B. J. WEST, Studies on the induction of translocations in mouse spermatogonia, III. Effects of X-irradiation, Mutation Res., 9 (I97 o) 5Ol528. 3 INADA, T., T. HIRAOKA, Z. HABU AND H. MATSUZAWA,Dose distribution of fast neutrons from thick target *Be(d,n)X°B reaction (Japanese), Nippon Acta Radiol., 29 (I969) 52o-528. 4 INADA, T., T. HIRAOKA, T. HABU, H. MATSUZAWAAND K. OHMACHI, LET distribution and RBE of Be(d,n) neutrons (Japanese), Nippon A a a Radiol., 29 (1969) 513-519. 5 L~ONARD, A., Radiation-induced translocations in spermatogonia of mice, Mutation Res., i i (I97 I) 71-88. 6 L~ONARD,A., AND GH, DEENUDT, Relation between the X-ray dose and the rateof chromosome rearrangements in spermatogonia of mice, Radiation Res., 32 (i967) 35-4I. 7 L~ONAED, A., AND GH. DEKNUDT. Dose-response relationship for translocations induced by X-irradiation in spermatogonia of mice, Radiation Res., 4 ° (I969) 276-284. 8 MURAMATSU, S., W. NAXAMURA AND H. Ero, Radiation-induced translocations in mouse spermatogonia, Japan. J. Genet., 46 (I971) 281-283. 9 MUEA~ATSU, S., W. NAKAMURAAND H. ETO, Chromosome aberrations in spermatogonia of mice after irradiation with fast neutrons, Japan. J. Gena., 46 (I97 I) 429 (Abstr.). io OFT~DAL, P., A theoretical study of mutant yield and cell killing after treatment of heterogeneous cell populations, Hereclitas, 6o (I968} I77-2io. 1i POMERANTSEVA, M. D., L. K. RA~AJA AND M. G. DOMSHLACK, Mutagenic effects of fast neutrons and X-rays on mouse spermatogonia (Russian), Radiobiologiya, io (I97o) 864-873. 12 R B E COMMITTEE, Report of International Commissions of Radiobiological Protection and on Radiological Units and Measurements,Health Physics, 9 (I963) 357-386. I3 SAVKOVlC,N. V., AND M. F. LYON, Dose-response curve for X-ray-induced translocations in mouse spermatogonia, I. Single doses, Mutation Res., 9 (I97 o) 4o7-4o9. I4 SEARLE, A. G., E. P. EVANS AnD B. J. WEST, Studies on the induction of translocations in mouse spermatogonia, II. Effects of fast neutron irradiation, Mutation Res., 7 (x969) 235-24o. 15 SEARLE,A. G., AND R. J. S. PmLLn, S, Genetic effects of high-LET radiations in mice, Radiation Res., Suppl. 7 (I967) 294-3o3 •