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A dose-rate effect on translocation induction by X-irradiation of mouse spermatogonia
SHORT COMMUNICATIONS
89
14 SOBELS, F. H., Rates of forward and reverse mutation in Drosophila after exposure to mustard gas and X-rays, Genetica, 33 (1962) 31-44 . 15 STEVENS, W. L., Accuracy of mutation rates, J. Genetics, 43 (1942) 3°1-3°7. 16 WOODRUFF, 1R. C., J. T. BOWMANAND J. R. SIMMONS, Some parameters of the reversion of the mutant forked-3n in Drosophila melanogaster, Genetics (Abstract), 64 (197 o) s66-s67.
Received October 7th, 1971 Revision received J a n u a r y 3Ist, 1972 Mutation Res., 15 (1972) 86-89 Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands
A dose-rate effect on translocation induction by X-irradiation of mouse spermatogonia Introduction
The frequency of reciprocal translocations in mouse spermatocytes, after acute X-irradiation of the spermatogonia from which they arise, shows an approximately linear relationship to gonadal dose up to about 700 rad~-L but then declinesS, °. Thus a humped type of curve results. This suggests that the primary dose-response curve has been distorted by events intervening between initiation of the structural changes in spermatogonia and their detection several cell generations later~, ~. If so, this primary dose-response relationship m a y well have both a linear and a dose-squared (interaction) component, as would be expected on theoretical grounds. To throw further light on this possibility, SEARLE et al. 1° studied the effect of X - r a y and y-ray dose-rate on the frequency of these translocations. They found a marked effect of T-ray dose-rate: the translocation frequency after 600 rad increased steadily from 1.4% at 0.o2 rad/min to 12.1% at 86 rad/min. With 60o rad X-irradiation, however, there was no significant change in the frequency of spermatocytes carrying translocations over a dose-rate range of 0.8 to 91o rad/min, although the lowest frequency was found at the o.8-rad/min point. Although various possible explanations for the apparent lack of a dose-rate effect with X-rays were put forward, it was felt that further experiments would be required before the problem was solved. It was suggested that these should include X-irradiation at an even lower dose rate than 0.8 rad/min to fill part of the gap between the X- and 7-ray dose-rate ranges. Methods
A new dose-rate experiment of the type envisaged has now been carried out. The total X - r a y dose was 300 rad (250 kVp, HVT 1.2 m m Cu), or half that in the previous experiment, in order to remove the possibility of any saturation effect. IO Fz ( C 3 H / H e H ~ × I o I / H ~ ) male mice aged 8-11 weeks were given continuous wholebody irradiation at intensities of 93, 0.87 and 0.09 rad/min. They were killed lO-14 weeks later and meiotic preparations were made by an air-drying technique which ensures randomization of cells 1. 50 spermatocytes at diakinesis-metaphase I were scored from each testis by C.V.B. The experiment was coded. Results
Translocation frequencies at 0.87 and 0.09 rad/min were less than half those at Mutation Res., 15 (1972) 89-91
90 TABLE
SHORT COMMUNICATIONS I
DISTRIBUTION OF PRESUMPTIVE TRANSLOCATION$ IN PRIMARY SPERMATOCYTES AFTER A 300-RAD SPERMATOGONIAL X - R A Y DOSE AT THREE DIFFERENT INTENSITIES
Intensity (rad/min)
Number of mice
Translocations per cell o • 2 3
Total in sample
Translocations per cell ( % )
Cells with translocations ( % )
93 0.87 o.09
io io io
882 97 ° 97 °
95 oa IOOO IOOO
7.7 4- 1.8 3.1 4- o.8 3.4 4- 0.8
7.2 ± 1.6 3.0 ± 0.8 3.0 ± 0.5
a Preparations
64 29 26
3 I 4
i o o
f r o m o n e t e s t i s of o n e m o u s e w e r e r e j e c t e d f o r t e c h n i c a l r e a s o n s .
93 rad/min (Table I). The differences are highly significant, both with respect to translocations per cell [F(2,54), 5-7; P, o.oo61 and to affected cells EF(2,54), 6.0; P, o.oo41, but results for the two lower dose-rates do not differ significantly from each other. An analysis of variance showed overall significant heterogeneity between testes for both measures of mutation (Z227, 48.5 ; P, 0.007 for translocations per cell; Z227, 43.6; P , 0.o23 for affected cells) but no overall significant heterogeneity between mice. However, it is interesting to note that at the 93-rad/min dose rate, where there was no significant heterogeneitybetween testes, there was significant heterogeneity between mice,while at the two lower dose rates the situation was reversed. The complete spectrum of translocations diagnosed in the spermatocytes was as follows (R, ring; C, chain): R I V (76), CIV (32), C I I I + I (ii), R I V + R I V (4), RVI (4), R V I I I (I). Discussion
This new experiment extends the range of X-ray dose-rates studied with respect to translocation induction by a factor of nine, since the previous lowest rate was 0.8 rad/min. In the previous experiment involving 600 rad X-irradiation n the translocation frequency at 0.8 rad/min was lower than at any other dose-rate but not significantly so; in the present experiment significantly lower frequencies were found at both 0.87 and 0.o 9 rad/min. The more pronounced effect in the present experimenL m a y be connected with the lower total dose and resultant reducl ion in spermatogonial killing, which in turn might be expected to cause less distortion of the primary response. The frequency of 7,2 % cells with translocations at the highest dose-rate is in good agreement with that found after whole-body doses of 300 rad acute X-irradiation in a previous experiment 2. The dose-rate effect seems less pronounced than after y-irradiation, which in the previous experiment n led to a fall from 12.o8% to 2.94% in the frequency of translocation-carrying cells as the dose-rate fell from 83 tad/rain to 0.09 rad/min after a 6oo-rad exposure. These results suggest that both the X-ray and y-ray dose-responses have one-track and two-track components, but that the one-track component is larger with X- than with y-irradiation. If so, one would expect that there would also be more effect of dose reduction after y- than after X-irradiation. SEARLE et al. 1° failed to find any significant departure from linearity in the y-ray dose-response curve for translocation induction in spermatogonia, although there was more concavity over the 0-400 R range than with X-irradiation. However, LYON et al. 7 found a marked effect of reducing the size of individual doses on the incidence of spermatocy~es carrying translocations after a total dose of 300 rad spermatogonial X-irradiation at 17 rad/min. While the incidence after a single 3oo-rad dose was 6.3%, frequencies Mutation Res., 15 (1972) 8 9 - 9 1
91
SHORT COMMUNICATIONS
after successive daily doses of 6o, IO or 5 tad (totalling 3oo rad in all) were 3.4 %, 1.3 % and 1.7% respectively, the last two not being significantly different. In a later experiment% 30 daily doses of lO. 4 tad acute v-irradiation (17-18 rad/min) gave a frequency of spermatocytes carrying translocations of 1.6°/o . Thus this comparison provides no evidence for a greater effect of dose reduction after v-irradiation. However, LYON et al. ~ pointed out that other factors besides the existence of a secondorder term in the dose-response relationship might be responsible for these effects of dose fractionation. In their v-radiation experiment, in fact, the authors concluded that a change in susceptibility of the spermatogonial population after repeated irradiation was largely responsible for the reduced frequencies. From their own results, SEARLE et al. lo considered that this phenomenon might also occur with protracted continuous irradiation and thus help to explain the much reduced translocation frequency after chronic v-irradiationn. Effects on repair mechanisms, like those postulated by RUSSELL8 to explain the dose-rate phenomena with the induction of specific locus mutations, may also be involved. LYON et al. 6 did not rule out the possibility that yields from single as well as from repeated small doses would be less than the linear expectation on the basis of large single doses. The reduced effects of both X-ray and v-ray doses when they are administered over a few hours rather than a few minutes make this possibility increasingly likely. M . R . C . Radiobiology Unit, Harwell, Didcot, Berkshire (Great Britain)
A. G. SEARLE C. V. BEECHEY
M. J.
CORP
D. G. PAPWORTH I EVANS, E. P., G. BRECKON AND C. E. FORD, An air-drying m e t h o d for meiotic p r e p a r a t i o n s from m a m m a l i a n testes, Cytogenetics, 3 (1964) 289-294. 2 EVANS, E. P., C. E. FORD, A. G. SEARLE AND B. J. WEST, Studies on the induction of t r a n s locations in mouse spermatogonia, I I I . Effects of X-irradiation, Mutation Res., 9 (197 o) 5Ol-5O6. 3 L~ONARD, A., AND G. DEKNUDT, Relation between the X - r a y dose and the rate of c h r o m o s o m e r e a r r a n g e m e n t s in s p e r m a t o g o n i a of mice, Radiation Res., 32 (1967) 35-41. 4 LI~ONARD, A., AND G. DEKNUDT, Chromosome r e a r r a n g e m e n t s after low X - r a y doses given to s p e r m a t o g o n i a of mice, Can. J. Genet. Cytol., IO (1968) 119-124. 5 LYON, M. F., AND T. MORRIS, G e n t and c h r o m o s o m e m u t a t i o n s after large fractionated or u n f r a c t i o n a t e d radiation doses to mouse spermatogonia, Mutation Res., 8 (1969) 191-198. 6 LYON, M. F., R. J. S. PHILLIPS AND P. GLENISTER, D o s e - r e s p o n s e curve for the yield of translocations in mouse s p e r m a t o g o n i a after repeated small radiation doses, Mutation Res., io (197 ° ) 497-5Ol. 7 LYON, M. F., T. MORRIS, P. GLENISTER AND S. E. O'GRADY, I n d u c t i o n of translocations in mouse s p e r m a t o g o n i a b y X - r a y doses divided into m a n y small fractions, Mutation Res., 9 (197 o) 219-223. 8 RUSSELL, W. L., Repair m e c h a n i s m s in radiation m u t a t i o n induction in the mouse, Broohhaven Syrup. Biol., 20 (1967) 179-189. 9 SAVKOVIC, ~ . V., AND i . F. LYON, D o s e - r e s p o n s e curve for X-ray-induced translocations in mouse spermatogonia, Mutation Res., 9 (197 o) 407-409. IO SEARLE, A. G., C. V. BEECHEY, E. P. EVANS, C. E. FORD AND D. G. PAPWORTH, Studies on the induction of translocations in mouse spermatogonia, IV. Effects of acute gamma-irradiation, Mutation Res., 12 (1971) 4 I I - 4 1 6 . I I SEARLE, A. G., E. R. EVANS, C. E. FORD, B. J. WEST AND D. G. PAPWORTH, Studies on the induction of translocations in mouse spermatogonia, 1. The effect of dose rate, Mutation Res., 6 (1968) 427-436.
Received January 27th , 1972 Mutation Res., 15 (1972) 8 9 - 9 I
89
14 SOBELS, F. H., Rates of forward and reverse mutation in Drosophila after exposure to mustard gas and X-rays, Genetica, 33 (1962) 31-44 . 15 STEVENS, W. L., Accuracy of mutation rates, J. Genetics, 43 (1942) 3°1-3°7. 16 WOODRUFF, 1R. C., J. T. BOWMANAND J. R. SIMMONS, Some parameters of the reversion of the mutant forked-3n in Drosophila melanogaster, Genetics (Abstract), 64 (197 o) s66-s67.
Received October 7th, 1971 Revision received J a n u a r y 3Ist, 1972 Mutation Res., 15 (1972) 86-89 Mutation Research Elsevier Publishing Company, Amsterdam Printed in The Netherlands
A dose-rate effect on translocation induction by X-irradiation of mouse spermatogonia Introduction
The frequency of reciprocal translocations in mouse spermatocytes, after acute X-irradiation of the spermatogonia from which they arise, shows an approximately linear relationship to gonadal dose up to about 700 rad~-L but then declinesS, °. Thus a humped type of curve results. This suggests that the primary dose-response curve has been distorted by events intervening between initiation of the structural changes in spermatogonia and their detection several cell generations later~, ~. If so, this primary dose-response relationship m a y well have both a linear and a dose-squared (interaction) component, as would be expected on theoretical grounds. To throw further light on this possibility, SEARLE et al. 1° studied the effect of X - r a y and y-ray dose-rate on the frequency of these translocations. They found a marked effect of T-ray dose-rate: the translocation frequency after 600 rad increased steadily from 1.4% at 0.o2 rad/min to 12.1% at 86 rad/min. With 60o rad X-irradiation, however, there was no significant change in the frequency of spermatocytes carrying translocations over a dose-rate range of 0.8 to 91o rad/min, although the lowest frequency was found at the o.8-rad/min point. Although various possible explanations for the apparent lack of a dose-rate effect with X-rays were put forward, it was felt that further experiments would be required before the problem was solved. It was suggested that these should include X-irradiation at an even lower dose rate than 0.8 rad/min to fill part of the gap between the X- and 7-ray dose-rate ranges. Methods
A new dose-rate experiment of the type envisaged has now been carried out. The total X - r a y dose was 300 rad (250 kVp, HVT 1.2 m m Cu), or half that in the previous experiment, in order to remove the possibility of any saturation effect. IO Fz ( C 3 H / H e H ~ × I o I / H ~ ) male mice aged 8-11 weeks were given continuous wholebody irradiation at intensities of 93, 0.87 and 0.09 rad/min. They were killed lO-14 weeks later and meiotic preparations were made by an air-drying technique which ensures randomization of cells 1. 50 spermatocytes at diakinesis-metaphase I were scored from each testis by C.V.B. The experiment was coded. Results
Translocation frequencies at 0.87 and 0.09 rad/min were less than half those at Mutation Res., 15 (1972) 89-91
90 TABLE
SHORT COMMUNICATIONS I
DISTRIBUTION OF PRESUMPTIVE TRANSLOCATION$ IN PRIMARY SPERMATOCYTES AFTER A 300-RAD SPERMATOGONIAL X - R A Y DOSE AT THREE DIFFERENT INTENSITIES
Intensity (rad/min)
Number of mice
Translocations per cell o • 2 3
Total in sample
Translocations per cell ( % )
Cells with translocations ( % )
93 0.87 o.09
io io io
882 97 ° 97 °
95 oa IOOO IOOO
7.7 4- 1.8 3.1 4- o.8 3.4 4- 0.8
7.2 ± 1.6 3.0 ± 0.8 3.0 ± 0.5
a Preparations
64 29 26
3 I 4
i o o
f r o m o n e t e s t i s of o n e m o u s e w e r e r e j e c t e d f o r t e c h n i c a l r e a s o n s .
93 rad/min (Table I). The differences are highly significant, both with respect to translocations per cell [F(2,54), 5-7; P, o.oo61 and to affected cells EF(2,54), 6.0; P, o.oo41, but results for the two lower dose-rates do not differ significantly from each other. An analysis of variance showed overall significant heterogeneity between testes for both measures of mutation (Z227, 48.5 ; P, 0.007 for translocations per cell; Z227, 43.6; P , 0.o23 for affected cells) but no overall significant heterogeneity between mice. However, it is interesting to note that at the 93-rad/min dose rate, where there was no significant heterogeneitybetween testes, there was significant heterogeneity between mice,while at the two lower dose rates the situation was reversed. The complete spectrum of translocations diagnosed in the spermatocytes was as follows (R, ring; C, chain): R I V (76), CIV (32), C I I I + I (ii), R I V + R I V (4), RVI (4), R V I I I (I). Discussion
This new experiment extends the range of X-ray dose-rates studied with respect to translocation induction by a factor of nine, since the previous lowest rate was 0.8 rad/min. In the previous experiment involving 600 rad X-irradiation n the translocation frequency at 0.8 rad/min was lower than at any other dose-rate but not significantly so; in the present experiment significantly lower frequencies were found at both 0.87 and 0.o 9 rad/min. The more pronounced effect in the present experimenL m a y be connected with the lower total dose and resultant reducl ion in spermatogonial killing, which in turn might be expected to cause less distortion of the primary response. The frequency of 7,2 % cells with translocations at the highest dose-rate is in good agreement with that found after whole-body doses of 300 rad acute X-irradiation in a previous experiment 2. The dose-rate effect seems less pronounced than after y-irradiation, which in the previous experiment n led to a fall from 12.o8% to 2.94% in the frequency of translocation-carrying cells as the dose-rate fell from 83 tad/rain to 0.09 rad/min after a 6oo-rad exposure. These results suggest that both the X-ray and y-ray dose-responses have one-track and two-track components, but that the one-track component is larger with X- than with y-irradiation. If so, one would expect that there would also be more effect of dose reduction after y- than after X-irradiation. SEARLE et al. 1° failed to find any significant departure from linearity in the y-ray dose-response curve for translocation induction in spermatogonia, although there was more concavity over the 0-400 R range than with X-irradiation. However, LYON et al. 7 found a marked effect of reducing the size of individual doses on the incidence of spermatocy~es carrying translocations after a total dose of 300 rad spermatogonial X-irradiation at 17 rad/min. While the incidence after a single 3oo-rad dose was 6.3%, frequencies Mutation Res., 15 (1972) 8 9 - 9 1
91
SHORT COMMUNICATIONS
after successive daily doses of 6o, IO or 5 tad (totalling 3oo rad in all) were 3.4 %, 1.3 % and 1.7% respectively, the last two not being significantly different. In a later experiment% 30 daily doses of lO. 4 tad acute v-irradiation (17-18 rad/min) gave a frequency of spermatocytes carrying translocations of 1.6°/o . Thus this comparison provides no evidence for a greater effect of dose reduction after v-irradiation. However, LYON et al. ~ pointed out that other factors besides the existence of a secondorder term in the dose-response relationship might be responsible for these effects of dose fractionation. In their v-radiation experiment, in fact, the authors concluded that a change in susceptibility of the spermatogonial population after repeated irradiation was largely responsible for the reduced frequencies. From their own results, SEARLE et al. lo considered that this phenomenon might also occur with protracted continuous irradiation and thus help to explain the much reduced translocation frequency after chronic v-irradiationn. Effects on repair mechanisms, like those postulated by RUSSELL8 to explain the dose-rate phenomena with the induction of specific locus mutations, may also be involved. LYON et al. 6 did not rule out the possibility that yields from single as well as from repeated small doses would be less than the linear expectation on the basis of large single doses. The reduced effects of both X-ray and v-ray doses when they are administered over a few hours rather than a few minutes make this possibility increasingly likely. M . R . C . Radiobiology Unit, Harwell, Didcot, Berkshire (Great Britain)
A. G. SEARLE C. V. BEECHEY
M. J.
CORP
D. G. PAPWORTH I EVANS, E. P., G. BRECKON AND C. E. FORD, An air-drying m e t h o d for meiotic p r e p a r a t i o n s from m a m m a l i a n testes, Cytogenetics, 3 (1964) 289-294. 2 EVANS, E. P., C. E. FORD, A. G. SEARLE AND B. J. WEST, Studies on the induction of t r a n s locations in mouse spermatogonia, I I I . Effects of X-irradiation, Mutation Res., 9 (197 o) 5Ol-5O6. 3 L~ONARD, A., AND G. DEKNUDT, Relation between the X - r a y dose and the rate of c h r o m o s o m e r e a r r a n g e m e n t s in s p e r m a t o g o n i a of mice, Radiation Res., 32 (1967) 35-41. 4 LI~ONARD, A., AND G. DEKNUDT, Chromosome r e a r r a n g e m e n t s after low X - r a y doses given to s p e r m a t o g o n i a of mice, Can. J. Genet. Cytol., IO (1968) 119-124. 5 LYON, M. F., AND T. MORRIS, G e n t and c h r o m o s o m e m u t a t i o n s after large fractionated or u n f r a c t i o n a t e d radiation doses to mouse spermatogonia, Mutation Res., 8 (1969) 191-198. 6 LYON, M. F., R. J. S. PHILLIPS AND P. GLENISTER, D o s e - r e s p o n s e curve for the yield of translocations in mouse s p e r m a t o g o n i a after repeated small radiation doses, Mutation Res., io (197 ° ) 497-5Ol. 7 LYON, M. F., T. MORRIS, P. GLENISTER AND S. E. O'GRADY, I n d u c t i o n of translocations in mouse s p e r m a t o g o n i a b y X - r a y doses divided into m a n y small fractions, Mutation Res., 9 (197 o) 219-223. 8 RUSSELL, W. L., Repair m e c h a n i s m s in radiation m u t a t i o n induction in the mouse, Broohhaven Syrup. Biol., 20 (1967) 179-189. 9 SAVKOVIC, ~ . V., AND i . F. LYON, D o s e - r e s p o n s e curve for X-ray-induced translocations in mouse spermatogonia, Mutation Res., 9 (197 o) 407-409. IO SEARLE, A. G., C. V. BEECHEY, E. P. EVANS, C. E. FORD AND D. G. PAPWORTH, Studies on the induction of translocations in mouse spermatogonia, IV. Effects of acute gamma-irradiation, Mutation Res., 12 (1971) 4 I I - 4 1 6 . I I SEARLE, A. G., E. R. EVANS, C. E. FORD, B. J. WEST AND D. G. PAPWORTH, Studies on the induction of translocations in mouse spermatogonia, 1. The effect of dose rate, Mutation Res., 6 (1968) 427-436.
Received January 27th , 1972 Mutation Res., 15 (1972) 8 9 - 9 I