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Radiation-induced dominant lethal mutations in oocytes of Musca domestica
153
Mutation Research, 63 ( 1 9 7 9 ) 1 5 3 - - 1 6 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
RADIATION-INDUCED DOMINANT L E T H A L MUTATIONS IN OOCYTES OF Musca domestica
H A M I L T O N JO.~O T A R G A
Departamento de Biologia, lnstituto de Bioci~ncias, Universidade de S~do Paulo, Caixa Postal 11 461, 05421 S~doPaulo (Brazil) and C L O V I S A. P E R E S *
Departamento de Estat{stica, Instituto de Matemdtica e Estatistica, Universidade de S~do Paulo, Caixa Postal 20570, 01451 S~o Paulo (Brazil) (Received 15 A u g u s t 1977) (Revision received 14 J u n e 1979) ( a c c e p t e d 18 J u n e 1979)
Summary Dominant lethal mutations induced by ?-radiation were measured in stage-7 and stage-14 oocytes o f Musca domestica. At both stages the data are consistent with the multi-hit theory on radiation induction of dominant lethals. This conclusion is supported b y fractionation experiments which indicate that both $7 and S14 oocytes are capable of repairing, in different periods o f time, a similar a m o u n t of dominant lethal damage. Interest in the cell's capacity to repair the damage induced by ionizing radiation has increased in the past several years. Differential radiosensitivity of female germ cells has been related to the meiotic or growth stage reached by the o o c y t e and surrounding cells as well as to physical factors on exposure. Studies on the effects of ionizing radiation in female germ cells of insects have been carried o u t mainly in Drosophila melanogaster [14], Habrobracum ]uglandis [20] and B o m b y x mori [19]. The oogenesis o f Musca domestica is similar to that of D. melanogaster, both species showing the same pattern in the development of the egg chmnber [1,5]. However, the synchronous development of the egg chamber in all ovarioles o f the polytrophic meroistic ovaries of Musca domestica makes these insects a useful tool for studies on mutagenesis induced by radiation or drugs. * Present address: Nahonal Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 (U.S.A.).
154 This is reinforced by the fact that by feeding the females a protein-free diet, the development of the egg chambers can be maintained for several days at a pre-vitelogenic stage. Soon after the adults are supplied with a normal proteinrich diet, egg-chamber development is resumed and mature eggs are obtained 3--4 days later [3,5]. In this paper we report our observations on the induction of dominant lethals, when stage-7 and stage-14 oocytes of Musca domestica, following Goodman's terminology [5], were exposed to several doses of acute and fractionated irradiation. Materials and methods Females of a Dallas DDT-sensitive strain of Musca domestica were used as biological material. Two culture media were used in the experiments. The larval medium consisted of a mixture of fermented pig chow and hulled rice (2 : 1, v/v). Adults were reared, as described by Adams and Nelson [2], in a mixture of powdered non-fat milk, sugar and powdered egg (6 : 6 : 1, v/v). Eggs were collected from the population cages and then spread on larval medium in plastic cups. The cups were maintained in screened plastic boxes with sawdust in the b o t t o m . At pupation, when the larvae had migrated into the sawdust, the cups with the old food were removed from the boxes. Two days before eclosion, pupae were collected and distributed into several other screened plastic boxes containing adult diet and water. Samples of stage-7 o o c y t e s ($7) were obtained from virgin females, hatched in a 4-h period and aged for 72 h in an aqueous commercial 0.1 M sugar solution [ 5]. For stage-14 oocytes ($14) we used 5-day-old virgin females, hatched in the same interval as above, and supplied continuously with adult diet. In each experimental set, t w o groups o f 30 females were exposed to each dose of radiation. Simultaneously, t w o non-treated groups with the same number of flies were run as controls. In the experiments with $7 oocytes, the irradiated females were immediately caged with non-irradiated males (30 females : 30 m a l e s ) i n screened plastic boxes supplied with adult diet and water. On the third and fourth days after irradiation, the females were allowed to oviposit during 6 h in plastic containers filled with the larval medium. Eggs were collected and spread in a petri dish having a thin layer of 3% plain agar base. The relative survival for each dose was normalized to the survival of the control group which was considered as 1.00. When $14 oocytes were irradiated, the females were mated overnight to nonirradiated males (30 females : 30 males). In the 24 to 48 h post-irradiation period, the females were allowed to oviposit for a 6-h period, as described above. Hatchability was measured according to the procedure described for the experiments with $7 oocytes. Radiation treatment was given by a caesium-137 source at an exposure rate of 33.3 rad/min. Results
(1) Dose--survival relationship of stage-7 and stage-14 oocytes The relative survivals obtained in all treatments of $7 and $14 oocytes with
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different doses of 7-radiation are presented, respectively, in Figs. 1 and 2, and the pooled data at each exposure level are given in Table 1. The data from o o c y t e s of each stage, were fitted, by the least-square method, to the model logey = ax + bx 2. , where y is the corrected survival, x stands for doses in krad, a and b correspond to the one-target and two-target components, respectively. We can give to the model the same interpretation as given b y Sankaranarayanan [12] to the classical dose--response relationship y = e -Ax ( 1 - (1--e-BX)2), since the former is derived from the latter by the Taylor expansion up to second order terms and the parameters of the t w o models are related in the following way: a = --A and b = --B 2. The least-square estimates with their respective standard errors, and information on goodness of fit to the model, are given in Table 2. Some interesting observations can be made from the results in Table 2. (1) The R-square values and the F statistics for lack of fitness show that the model was well fitted to the data in the two stages. (2) Although the contribution of the two-target c o m p o n e n t in stage 7 is statistically significant, it is small in relation to the one-target component. This result is consistent with those obtained in fractionation experiments, as will be discussed later. (3) In stage 14 the two c o m p o n e n t s can be considered important to explain the relationship between survival and radiation. This result is also consistent with those obtained in the fractionation experiments. (2) D o s e f r a c t i o n a t i o n f o r o o c y t e s at stages 7 and 14
The pooled results obtained from experiments in which $7 oocytes exposed * I f w e w a n t t o e x p r e s s t h e s u r v i v a l as a p e r c e n t a g e , t h e n the m o d e l 1 0 0 + a x + b x 2. T h e p a r a m e t e r s are s a m e .
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TABLE 1 DOSE DEPENDENCE OF SURVIVAL AFTER OOCYTES OF Musca domestlca (POOLED DATA) Exposure in R units
7-IRRADIATION
Stage 7
OF
STAGE-7
AND
STAGE-14
S t a g e 14
Number of eggs laid
Number hatched
Relative surv. (%)
Number of e g g s laid
Number hatched
Control
23 8 3 6
21 9 9 9
92.3
14 4 8 8
13 1 5 2
90.8
500 1000 1500
7 801 13 6 1 5 11 7 0 9
6 378 10 6"/8 7 616
88.6 85.0 70.5
4 016 13 7 0 4 4 600
3 324 9 867 2 717
91.2 79.3 65.0
2000 2500 3000
20 2 7 2 10 0 8 8 16 4 0 2
12 1 2 8 4 787 6 231
64.8 51.4 41.2
14 0 8 2 3 574 12 8 8 3
5 515 1 121 2 465
43.1 34.5 21.1
3500 4000 4500 5000
10 14 5 5
3 250 3 599 1 256 867
32.9 27.6 26.8 18.5
3 984 10 4 5 2 ---
693 113 069 078
468 904 ---
Relative surv. (%)
12.9 9.5 ---
157 TABLE 2 LEAST-SQUARE
Oocyte
ESTIMATES OF ONE-TARGET AND TWO-TARGET COMPONENTS
a (on.target)
b (two-target)
R 2 (%)
F statistics for lack of fitness a
--O.1908 ± 0.0358 ---O.1'764 ± 0 . 0 5 4 5
--0.0295 ± 0.0097 --0.1059 ± 0.0166
94.4 97.5
0 . 2 9 7 (P ~ 0 . 9 5 ) 0 . 3 5 7 (P > 0 . 9 0 )
stage 7 14
a We w e r e able to u s e t h i s t e s t b e c a u s e t h e m o d e l is h n e a r in t h e p a r a m e t e r s a n d w e h a d m o r e t h a n o n e o b s e r v a t i o n f o r e a c h d o s e [ 17 ].
to 2000 rad delivered in two halves with different time intervals are summarized in Table 3. As can be seen, the d o m i n a n t lethal frequencies obtained in all fractionated treatments were somewhat lower than in the unfractionated control group. These differences increased significantly with enlargement of the time interval, reaching an almost stable value at the time of 120 min. In this interval the frequency of d o m i n a n t lethals in the fractionated group was 27.5% lower than that obtained in the control group. Stage-14 oocytes were also exposed to 2000 rad, delivered in two half doses separated by different time intervals. The pooled results of two experimental sets are shown in Table 3. As shown by these data, S14 oocytes of M.domestica TABLE 3 DOMINANT LETHAL STAGE-14 OOCYTES
FREQUENCIES
INDUCED
BY D O S E F R A C T I O N A T I O N
IN STAGE-7 AND
Doses dehvered in two equal fractions separated by variable time intervals Fractionatlon interval (mm)
Number of rephcates
Number of eggs
Number unhatched
Dominant lethals (%)
Repaired dominant lethals (%)
Control 2000 tad unffac~ona~d
2 2
4142 4202
192 1868
-41.8
---
30 60 90 120 150 360
2 2 2 2 2 2
4794 4145 4626 3992 3501 4199
1975 1660 1713 1337 1160 1464
38.4 37.2 35.8 30.3 29.9 31.7
8.1 11.0 14.4 27.5 28.5 24.2
Control 2000 rad unffacgona~d
4 4
7751 8750
707 5727
-62.0
---
30 45 60 90 120 150 180 210 270
4 2 4 4 4 4 4 4 2
8661 5056 7944 8114 6898 7503 7861 7972 5234
5503 3172 5070 5160 4041 4512 4618 4097 2779
59.9 59.1 60.2 59.9 59.4 56.1 54.6 46.5 48.4
3.4 4.7 2.9 3.4 12.3 9.5 11.9 25.0 21 9
S~ge 7
S ~ g e 14
158 TABLE 4 LEAST-SQUARE ESTIMATES OF REGRESSION LETHALS OVER TIME INTERVALS
COEFFICIENTS
OF
REPAIRED
DOMINANT
O o c y t e s stage
Coefficient
S.E.
tO
P (t ~ to)
7 14
0.1910 0.0720
0.0345 0.0322
5.5402 2.2373
0.005 < P < 0.010 0.05 ~ P ~ 0.10
were also capable of repairing dominant lethals induced by ionizing radiation. When doses were separated by a 120-min intervals, 12.3% of the induced dominant lethals were found to be repaired. A significant increase in the frequency of repaired dominant lethals was observed when the two half doses were separated by longer intervals, i.e. 210 and 270 min. The percentages obtained at these intervals, 25 and 21.9, respectively, were similar to those found for dominant lethals repaired in $7 oocytes. Although both $7 and S14 oocytes were capable of repair and the amount of repaired dominant lethal increased with the time interval, there were two main differences that can explain the sigmoid dose--response relationship for stage 14 and the almost exponential dose--response relationship (very small twotarget component) for stage 7. (1) In each time interval from 30 to 150 min the amount of dominant lethal damage repaired in stage 7 was higher than in stage 14 (see last column in Table 3). (2) When we fit by regression a straight line using repaired dominant lethal (%) as dependent variable and fractionation intervals (30, 60, 90, 120 and 150 min) as independent variable, the slope for stage 7 is significantly higher than for stage 14 (see Table 4). Discussion
Radiation-induced dominant lethals are related to chromosome breaks produced by the treatment, followed by bridge formation and loss of genetic material [6,9]. Radiation sensitivity differences, measured by the frequency of this kind of mutation, have been found during the different stages of the pocyte development. In D. rnelanogaster [7,11,12,18] and at comparable stages of oogenesis in Habrobracum juglandi8 [20,21] and Cochliomyia hominivorax [8,10], a sigmoid dose--effect curve was obtained when survival of oocytes irradiated at prophase I (stage 7) was analysed. An exponential relationship was obtained when similar studies were done with metaphase I (stage 14) oocytes of the above species [ 7,8,10,11,13,18,20,21 ]. In M. domestica we found slightly different results when $7 and S14 oocytes were treated by 7-radiation. In relation to stage 7, we observed a dose--survival curve following a sigmoid model, but with a very small two-target component. In respect to stage 14, a sigmoid model fits our results very well, the two components (one-target and two-target) having the same importance to the model. According to these findings the two-or-more-hit theory would account for the origin of radiation-induced dominant lethal mutations in both $7 and S14 oocytes of M. domestica. This conclusion is also supported by our results
159 on fractionation treatments, since statistically significant reductions in the frequencies of dominant lethals were observed in all cases when $7 and $14 o o c y t e s were treated, respectively, by doses separated in time by more than 120 and 210 min. According to the multi-hit theory, such reduction should be expected if all breaks were n o t available for reunion all the time, because, for interaction, all breaks must be open simultaneously. Although the sigmoid model can be accepted for b o t h stages, an important point remains: that is, the very small contribution of the two-target component in $7 when compared with that observed in the S14 oocytes. This divergence can be explained if we consider the influence of dose rate in the induction of chromosome breaks as shown first by Sax [15,16] and later confirmed b y several other authors [4]. According to these authors, the frequency of multi-hit chromosomal aberrations is greatly influenced by the dose rate used in the experiments. With a low dose rate, the frequency of exchanges will be lower than expected, because the first break can be repaired before the second occurs. In all treatments we used a low dose rate (33.3 rad/min) and the repair of dominant lethals was fast in $7 oocytes, probably starting even before the 30min interval we have analysed. This will permit the repair of many breaks that will n o t be available for interaction with those produced later in the treatment. In this way, many multi-hit dominant lethal mutations will be prevented from occurring and as the one-hit effect remains, the mutation frequencies will exhibit an almost exponential relationship (two-target c o m p o n e n t very small). Two important points arise from the discussion above. The first is the possibility of only one t y p e of mechanism, the multi-hit system, operating as the primary step in the radiation induction of dominant lethal mutations in b o t h $7 and $14 oocytes of M. domestica. The second is the existence of a repair mechanism acting in mature oocytes (S14) before fertilization, a situation that appears not to occur in oocytes of other species of insect irradiated at this stage of oogenesis [7,8,11,13,20]. Also important is the observation that, besides the action of this repair mechanism(s) being slower than that of the S7 oocytes, both stages are capable of repairing a similar a m o u n t of dominant lethals induced b y radiation. It cannot be ascertained by these experiments h o w this mechanism operates, nor whether it is unique for all steps of oogenesis. According to Wolff [ 22,23], b o t h energy and protein synthesis are required for rejoining of radiation-induced chromosome breaks. As dominant lethal mutations are related to chromosome breaks it is possible that both factors are responsible for the repair of such kinds of mutation. If this be so, the repair would occur fast in those stages of oogenesis showing a more intense metabolism and would decrease as the oocytes reach maturity. Our data seem to support this idea because the repair of S14 is slower than that observed for $7 oocytes. Further studies in this field would help to clarify this idea. Acknowledgements We thank T.E.M. Murto, Telma P. Stiinkel for technical assistance and Dr. A.L.P. Perondini for critical reading of the manuscript. This work was partly supported by the Conselho Nacional de Desenvolvimento Cientffico e TecnolSgico, Contract No. SIP/04-042.
160
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Mutation Research, 63 ( 1 9 7 9 ) 1 5 3 - - 1 6 0 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
RADIATION-INDUCED DOMINANT L E T H A L MUTATIONS IN OOCYTES OF Musca domestica
H A M I L T O N JO.~O T A R G A
Departamento de Biologia, lnstituto de Bioci~ncias, Universidade de S~do Paulo, Caixa Postal 11 461, 05421 S~doPaulo (Brazil) and C L O V I S A. P E R E S *
Departamento de Estat{stica, Instituto de Matemdtica e Estatistica, Universidade de S~do Paulo, Caixa Postal 20570, 01451 S~o Paulo (Brazil) (Received 15 A u g u s t 1977) (Revision received 14 J u n e 1979) ( a c c e p t e d 18 J u n e 1979)
Summary Dominant lethal mutations induced by ?-radiation were measured in stage-7 and stage-14 oocytes o f Musca domestica. At both stages the data are consistent with the multi-hit theory on radiation induction of dominant lethals. This conclusion is supported b y fractionation experiments which indicate that both $7 and S14 oocytes are capable of repairing, in different periods o f time, a similar a m o u n t of dominant lethal damage. Interest in the cell's capacity to repair the damage induced by ionizing radiation has increased in the past several years. Differential radiosensitivity of female germ cells has been related to the meiotic or growth stage reached by the o o c y t e and surrounding cells as well as to physical factors on exposure. Studies on the effects of ionizing radiation in female germ cells of insects have been carried o u t mainly in Drosophila melanogaster [14], Habrobracum ]uglandis [20] and B o m b y x mori [19]. The oogenesis o f Musca domestica is similar to that of D. melanogaster, both species showing the same pattern in the development of the egg chmnber [1,5]. However, the synchronous development of the egg chamber in all ovarioles o f the polytrophic meroistic ovaries of Musca domestica makes these insects a useful tool for studies on mutagenesis induced by radiation or drugs. * Present address: Nahonal Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709 (U.S.A.).
154 This is reinforced by the fact that by feeding the females a protein-free diet, the development of the egg chambers can be maintained for several days at a pre-vitelogenic stage. Soon after the adults are supplied with a normal proteinrich diet, egg-chamber development is resumed and mature eggs are obtained 3--4 days later [3,5]. In this paper we report our observations on the induction of dominant lethals, when stage-7 and stage-14 oocytes of Musca domestica, following Goodman's terminology [5], were exposed to several doses of acute and fractionated irradiation. Materials and methods Females of a Dallas DDT-sensitive strain of Musca domestica were used as biological material. Two culture media were used in the experiments. The larval medium consisted of a mixture of fermented pig chow and hulled rice (2 : 1, v/v). Adults were reared, as described by Adams and Nelson [2], in a mixture of powdered non-fat milk, sugar and powdered egg (6 : 6 : 1, v/v). Eggs were collected from the population cages and then spread on larval medium in plastic cups. The cups were maintained in screened plastic boxes with sawdust in the b o t t o m . At pupation, when the larvae had migrated into the sawdust, the cups with the old food were removed from the boxes. Two days before eclosion, pupae were collected and distributed into several other screened plastic boxes containing adult diet and water. Samples of stage-7 o o c y t e s ($7) were obtained from virgin females, hatched in a 4-h period and aged for 72 h in an aqueous commercial 0.1 M sugar solution [ 5]. For stage-14 oocytes ($14) we used 5-day-old virgin females, hatched in the same interval as above, and supplied continuously with adult diet. In each experimental set, t w o groups o f 30 females were exposed to each dose of radiation. Simultaneously, t w o non-treated groups with the same number of flies were run as controls. In the experiments with $7 oocytes, the irradiated females were immediately caged with non-irradiated males (30 females : 30 m a l e s ) i n screened plastic boxes supplied with adult diet and water. On the third and fourth days after irradiation, the females were allowed to oviposit during 6 h in plastic containers filled with the larval medium. Eggs were collected and spread in a petri dish having a thin layer of 3% plain agar base. The relative survival for each dose was normalized to the survival of the control group which was considered as 1.00. When $14 oocytes were irradiated, the females were mated overnight to nonirradiated males (30 females : 30 males). In the 24 to 48 h post-irradiation period, the females were allowed to oviposit for a 6-h period, as described above. Hatchability was measured according to the procedure described for the experiments with $7 oocytes. Radiation treatment was given by a caesium-137 source at an exposure rate of 33.3 rad/min. Results
(1) Dose--survival relationship of stage-7 and stage-14 oocytes The relative survivals obtained in all treatments of $7 and $14 oocytes with
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different doses of 7-radiation are presented, respectively, in Figs. 1 and 2, and the pooled data at each exposure level are given in Table 1. The data from o o c y t e s of each stage, were fitted, by the least-square method, to the model logey = ax + bx 2. , where y is the corrected survival, x stands for doses in krad, a and b correspond to the one-target and two-target components, respectively. We can give to the model the same interpretation as given b y Sankaranarayanan [12] to the classical dose--response relationship y = e -Ax ( 1 - (1--e-BX)2), since the former is derived from the latter by the Taylor expansion up to second order terms and the parameters of the t w o models are related in the following way: a = --A and b = --B 2. The least-square estimates with their respective standard errors, and information on goodness of fit to the model, are given in Table 2. Some interesting observations can be made from the results in Table 2. (1) The R-square values and the F statistics for lack of fitness show that the model was well fitted to the data in the two stages. (2) Although the contribution of the two-target c o m p o n e n t in stage 7 is statistically significant, it is small in relation to the one-target component. This result is consistent with those obtained in fractionation experiments, as will be discussed later. (3) In stage 14 the two c o m p o n e n t s can be considered important to explain the relationship between survival and radiation. This result is also consistent with those obtained in the fractionation experiments. (2) D o s e f r a c t i o n a t i o n f o r o o c y t e s at stages 7 and 14
The pooled results obtained from experiments in which $7 oocytes exposed * I f w e w a n t t o e x p r e s s t h e s u r v i v a l as a p e r c e n t a g e , t h e n the m o d e l 1 0 0 + a x + b x 2. T h e p a r a m e t e r s are s a m e .
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TABLE 1 DOSE DEPENDENCE OF SURVIVAL AFTER OOCYTES OF Musca domestlca (POOLED DATA) Exposure in R units
7-IRRADIATION
Stage 7
OF
STAGE-7
AND
STAGE-14
S t a g e 14
Number of eggs laid
Number hatched
Relative surv. (%)
Number of e g g s laid
Number hatched
Control
23 8 3 6
21 9 9 9
92.3
14 4 8 8
13 1 5 2
90.8
500 1000 1500
7 801 13 6 1 5 11 7 0 9
6 378 10 6"/8 7 616
88.6 85.0 70.5
4 016 13 7 0 4 4 600
3 324 9 867 2 717
91.2 79.3 65.0
2000 2500 3000
20 2 7 2 10 0 8 8 16 4 0 2
12 1 2 8 4 787 6 231
64.8 51.4 41.2
14 0 8 2 3 574 12 8 8 3
5 515 1 121 2 465
43.1 34.5 21.1
3500 4000 4500 5000
10 14 5 5
3 250 3 599 1 256 867
32.9 27.6 26.8 18.5
3 984 10 4 5 2 ---
693 113 069 078
468 904 ---
Relative surv. (%)
12.9 9.5 ---
157 TABLE 2 LEAST-SQUARE
Oocyte
ESTIMATES OF ONE-TARGET AND TWO-TARGET COMPONENTS
a (on.target)
b (two-target)
R 2 (%)
F statistics for lack of fitness a
--O.1908 ± 0.0358 ---O.1'764 ± 0 . 0 5 4 5
--0.0295 ± 0.0097 --0.1059 ± 0.0166
94.4 97.5
0 . 2 9 7 (P ~ 0 . 9 5 ) 0 . 3 5 7 (P > 0 . 9 0 )
stage 7 14
a We w e r e able to u s e t h i s t e s t b e c a u s e t h e m o d e l is h n e a r in t h e p a r a m e t e r s a n d w e h a d m o r e t h a n o n e o b s e r v a t i o n f o r e a c h d o s e [ 17 ].
to 2000 rad delivered in two halves with different time intervals are summarized in Table 3. As can be seen, the d o m i n a n t lethal frequencies obtained in all fractionated treatments were somewhat lower than in the unfractionated control group. These differences increased significantly with enlargement of the time interval, reaching an almost stable value at the time of 120 min. In this interval the frequency of d o m i n a n t lethals in the fractionated group was 27.5% lower than that obtained in the control group. Stage-14 oocytes were also exposed to 2000 rad, delivered in two half doses separated by different time intervals. The pooled results of two experimental sets are shown in Table 3. As shown by these data, S14 oocytes of M.domestica TABLE 3 DOMINANT LETHAL STAGE-14 OOCYTES
FREQUENCIES
INDUCED
BY D O S E F R A C T I O N A T I O N
IN STAGE-7 AND
Doses dehvered in two equal fractions separated by variable time intervals Fractionatlon interval (mm)
Number of rephcates
Number of eggs
Number unhatched
Dominant lethals (%)
Repaired dominant lethals (%)
Control 2000 tad unffac~ona~d
2 2
4142 4202
192 1868
-41.8
---
30 60 90 120 150 360
2 2 2 2 2 2
4794 4145 4626 3992 3501 4199
1975 1660 1713 1337 1160 1464
38.4 37.2 35.8 30.3 29.9 31.7
8.1 11.0 14.4 27.5 28.5 24.2
Control 2000 rad unffacgona~d
4 4
7751 8750
707 5727
-62.0
---
30 45 60 90 120 150 180 210 270
4 2 4 4 4 4 4 4 2
8661 5056 7944 8114 6898 7503 7861 7972 5234
5503 3172 5070 5160 4041 4512 4618 4097 2779
59.9 59.1 60.2 59.9 59.4 56.1 54.6 46.5 48.4
3.4 4.7 2.9 3.4 12.3 9.5 11.9 25.0 21 9
S~ge 7
S ~ g e 14
158 TABLE 4 LEAST-SQUARE ESTIMATES OF REGRESSION LETHALS OVER TIME INTERVALS
COEFFICIENTS
OF
REPAIRED
DOMINANT
O o c y t e s stage
Coefficient
S.E.
tO
P (t ~ to)
7 14
0.1910 0.0720
0.0345 0.0322
5.5402 2.2373
0.005 < P < 0.010 0.05 ~ P ~ 0.10
were also capable of repairing dominant lethals induced by ionizing radiation. When doses were separated by a 120-min intervals, 12.3% of the induced dominant lethals were found to be repaired. A significant increase in the frequency of repaired dominant lethals was observed when the two half doses were separated by longer intervals, i.e. 210 and 270 min. The percentages obtained at these intervals, 25 and 21.9, respectively, were similar to those found for dominant lethals repaired in $7 oocytes. Although both $7 and S14 oocytes were capable of repair and the amount of repaired dominant lethal increased with the time interval, there were two main differences that can explain the sigmoid dose--response relationship for stage 14 and the almost exponential dose--response relationship (very small twotarget component) for stage 7. (1) In each time interval from 30 to 150 min the amount of dominant lethal damage repaired in stage 7 was higher than in stage 14 (see last column in Table 3). (2) When we fit by regression a straight line using repaired dominant lethal (%) as dependent variable and fractionation intervals (30, 60, 90, 120 and 150 min) as independent variable, the slope for stage 7 is significantly higher than for stage 14 (see Table 4). Discussion
Radiation-induced dominant lethals are related to chromosome breaks produced by the treatment, followed by bridge formation and loss of genetic material [6,9]. Radiation sensitivity differences, measured by the frequency of this kind of mutation, have been found during the different stages of the pocyte development. In D. rnelanogaster [7,11,12,18] and at comparable stages of oogenesis in Habrobracum juglandi8 [20,21] and Cochliomyia hominivorax [8,10], a sigmoid dose--effect curve was obtained when survival of oocytes irradiated at prophase I (stage 7) was analysed. An exponential relationship was obtained when similar studies were done with metaphase I (stage 14) oocytes of the above species [ 7,8,10,11,13,18,20,21 ]. In M. domestica we found slightly different results when $7 and S14 oocytes were treated by 7-radiation. In relation to stage 7, we observed a dose--survival curve following a sigmoid model, but with a very small two-target component. In respect to stage 14, a sigmoid model fits our results very well, the two components (one-target and two-target) having the same importance to the model. According to these findings the two-or-more-hit theory would account for the origin of radiation-induced dominant lethal mutations in both $7 and S14 oocytes of M. domestica. This conclusion is also supported by our results
159 on fractionation treatments, since statistically significant reductions in the frequencies of dominant lethals were observed in all cases when $7 and $14 o o c y t e s were treated, respectively, by doses separated in time by more than 120 and 210 min. According to the multi-hit theory, such reduction should be expected if all breaks were n o t available for reunion all the time, because, for interaction, all breaks must be open simultaneously. Although the sigmoid model can be accepted for b o t h stages, an important point remains: that is, the very small contribution of the two-target component in $7 when compared with that observed in the S14 oocytes. This divergence can be explained if we consider the influence of dose rate in the induction of chromosome breaks as shown first by Sax [15,16] and later confirmed b y several other authors [4]. According to these authors, the frequency of multi-hit chromosomal aberrations is greatly influenced by the dose rate used in the experiments. With a low dose rate, the frequency of exchanges will be lower than expected, because the first break can be repaired before the second occurs. In all treatments we used a low dose rate (33.3 rad/min) and the repair of dominant lethals was fast in $7 oocytes, probably starting even before the 30min interval we have analysed. This will permit the repair of many breaks that will n o t be available for interaction with those produced later in the treatment. In this way, many multi-hit dominant lethal mutations will be prevented from occurring and as the one-hit effect remains, the mutation frequencies will exhibit an almost exponential relationship (two-target c o m p o n e n t very small). Two important points arise from the discussion above. The first is the possibility of only one t y p e of mechanism, the multi-hit system, operating as the primary step in the radiation induction of dominant lethal mutations in b o t h $7 and $14 oocytes of M. domestica. The second is the existence of a repair mechanism acting in mature oocytes (S14) before fertilization, a situation that appears not to occur in oocytes of other species of insect irradiated at this stage of oogenesis [7,8,11,13,20]. Also important is the observation that, besides the action of this repair mechanism(s) being slower than that of the S7 oocytes, both stages are capable of repairing a similar a m o u n t of dominant lethals induced b y radiation. It cannot be ascertained by these experiments h o w this mechanism operates, nor whether it is unique for all steps of oogenesis. According to Wolff [ 22,23], b o t h energy and protein synthesis are required for rejoining of radiation-induced chromosome breaks. As dominant lethal mutations are related to chromosome breaks it is possible that both factors are responsible for the repair of such kinds of mutation. If this be so, the repair would occur fast in those stages of oogenesis showing a more intense metabolism and would decrease as the oocytes reach maturity. Our data seem to support this idea because the repair of S14 is slower than that observed for $7 oocytes. Further studies in this field would help to clarify this idea. Acknowledgements We thank T.E.M. Murto, Telma P. Stiinkel for technical assistance and Dr. A.L.P. Perondini for critical reading of the manuscript. This work was partly supported by the Conselho Nacional de Desenvolvimento Cientffico e TecnolSgico, Contract No. SIP/04-042.
160
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