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GAMMA RAY-INDUCED MUTATIONS I N DROSOPHZLA
MELANOGASTER OOCYTES: THE PHENOMENON OF DOSE RATE'
ELEANOR HIMOE MARKOWITZZ
Department of Zoology, Uniuersity of Wisconsin, Madison, Wisconsin 53706
Received November 3, 1969
RIOR to 1958, it had been generally accepted that radiation-induced mutation
frequencies are independent of dose rate. This was based mainly on findings
1931; TIMOFEEFF-RESSOVfor irradiated spermatozoa in Drosophila (PATTERSON
SKY and ZIMMER
1935j RAY-CHAUDHURI
1939,1944). In 1955, HERSKOWITZ
and
ABRAHAMSON
reported that the frequencies of sex-linked recessive lethals induced
in oocytes of Drosophila females might be intensity-dependent, since a higher
frequency of lethals was obtained after an acute dose of X rays than after a fractionated treatment. However, subsequent experiments on Drosophila females
with somewhat different doses and dose rates failed to show this difference in
frequency of sex-linked lethals ( HERSKOWITZ
1960).
et al. observed, for irradiated mouse spermatogonial cells,
In 1958, RUSSELL,
that the frequency, per roentgen, of induced specific-locus mutations was about
three times higher after an acute (80 to 9Or per minute) X irradiation than a
chronic (80 to 90r per week) cobalt-60 gamma-irradiation (RUSSELL
and KELLY
1958; RUSSELL,RUSSELL
and KELLY1958). They suggested that although the
mutation process in spermatozoa is apparently independent of dose rate, this
might not be the case in metabolically active cells such as spermatogonia. No doserate effect was observed in irradiated mouse spermatozoa, paralleling the results
for Drosophila spermatozoa. PHILLIPS
(1961 ) independently confirmed the doserate effect for spermatogonia in male mice. Experiments performed with female
mice again demonstrated an effect of radiation intensity, and the effect was
greater on oocytes than on spermatogonia (RUSSELL,RUSSELL,GOWERand
MADDUX
1958; RUSSELL,
RUSSELL
and CUPP1959).
Following this, other organisms were examined to see if corroborative evidence
for a dose-rate effect might be found. TAZIMA,
KONDO
and SADO(1961) measured
the frequencies of specific-locus mutations induced by gamma rays in spermatogonia and oogonia of the silkworm, Bombyx mori, and found two kinds of doserate effects, depending on the age of the treated larvae. BALDWIN(1962, 1965)
measured visible mutation frequencies recovered from treated oogonial cells of
the wasp, Dahlbominus, followed by acute X irradiation and chronic gamma
irradiation. The mutation frequency obtained from the acute dose was about 1.5
times greater than that from the chronic dose.
IP
From a thesis submitted to the Graduate School of the University of Wisconsin in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Supported by research grants from the National Science Foundation
(G-19394) and the Wisconsin Alumni Research Foundation.
Present address. Department of Anatomy, University of Wisconsin, Madison, Wisconsin 53706.
Genetics 64: 31S322 February 1970
314
E. H.
MARKOWITZ
With respect to dose-rate effects, it is important to distinguish between mutational events which are single-hit and those which are multiple-hit events resulting from more than one ionization track. I n metabolically active cells, the frequency of two-hit chromosome aberrations may depend on the dose rate due to
the fact that two chromosome breaks, resulting from two separate hits, must be
present at the same time in order to interact. If the breaks stay open for a certain
period of time, then protracting an exposure to irradiation beyond normal rejoining time reduces the probability that the two breaks will be present simultaneously and decreases the frequency of this kind of aberration. Although a similar
kind of explanation for an intensity effect on single-hit events is not as obvious,
there is evidence that dose-rate effects do exist for these events. I n experiments
with mouse spermatogonia and oocytes, the evidence suggests that the specificlocus mutations studied are single-hit (RUSSELL
1965). However, WOLFF(1967)
believes that these mutations are two-hit chromosome rearrangements. His view
is based primarily on the observed high relative biological effectiveness of neutrons to X rays, expected if the mutations are two-hit aberrations. The specificlocus mutations observed in the silkworm (TAZIMA,
KONDO
and SADO1961) and
in Dahlbominus (BALDWIN
1965) are presumed to be single-hit events, although
evidence on this point is not strongly convincing.
In 1942, NEWCOMBE
examined X-ray induced chromosome changes in Tradescantia and found, in contrast to others in his time, that both one-hit and two-hit
aberrations varied with radiation intensity (NEWCOMBE
1942). He suggested
that rejoining of broken chromosome ends is inhibited at both high doses and
dose rates. BREWEN(1963), in cytological studies on the corneal epithelium of
the Chinese hamster, found a significant decrease in the frequency of chromatid
aberrations at a dose rate of 2r per minute when compared to a dose rate of 600r
per minute. In the dose range used, 50 to lOOr, the frequency of two-hit aberrations is quite low, so the effect involves the frequency of one-hit aberrations. His
evidence, using an inhibitor of protein synthesis, supports the view that damage
to the chromosome-rejoining system is dose rate dependent.
In Drosophila, although there have been a number of studies on the effect of
radiation dose rate on induced recessive lethal frequencies, unequivocal evidence
for a dose rate dependent difference has not been obtained. PURDOM,
studying
recessive lethals in Drosophila spermatogonia, did find a significantly lower mutation rate at one low-intensity irradiation of 0.01 rads per minute of cobalt-60
gamma rays, but he did not consider this convincing in the light of his other
1963; PURDOM
and MCSHEEHY1961, 1963). Muller and coresults (PURDOM
workers have done an extensive series of dose-rate experiments on Drosophila
oogonia using cobalt-60 gamma rays (OSTER,ZIMMERING
and MULLER1959;
MULLER,OSTERand ZIMMERING
1963). Although their results were originally
suggestive, unresolved problems of repeatability under different conditions and
of accurate dose measurement rendered any conclusions doubtful. MULLERlater
came to favor the view that for this system there is no significant difference in the
mutagenic effectiveness of gamma rays over the range of dose rates from about
l r per hour to 3000r per hour (MULLER1965).
Y RAY-INDUCED M U T A T I O N
315
Preliminary experiments carried out in our laboratory using cesium-I37
gamma rays and examining sex-linked recessive lethals induced in Drosophila
oocytes indicated that there might be a difference in the frequency of lethals
recovered after each of two different dose rates. To further explore this possibility,
it was felt that the stages of irradiated oocytes sampled should be restricted in
order to avoid sampling from a heterogeneous population of cells, as was the case
previously. Observations were to be confined to those oocytes which were in
stage 7, according to the classification of KING,at the time of irradiation (KING,
RUBINSONand SMITH1956). It is known, from dose-fractionation experiments,
that at this stage the oocyte is capable of repairing chromosome breaks (PARKER
and HAMMOND
1958; PARKER
and MCCRONE
1958). Therefore, it is reasonable
to expect that if any kind of intracellular repair processes were acting, they might
be found here. It was further proposed that a sample of lethals recovered from the
low dose rate and from the high dose rate be examined cytologically in the
salivary gland chromosomes for visible abnormalities, to exclude obvious two-hit
aberrations.
MATERIALS A N D METHODS
The irradiated females were Canton-S wild-type virgins, which were generated in the following manner to avoid the presence of a large number of preexisting sex-linked lethals. Hemizygous, lethal-free Canton-S males were crossed to females with a multiply-inverted, dominantly
marked, X chromosome. The heterozygous F, females were again crossed to Canton-S males, and
the homozygous wild-type progeny were used for treatment. Using this procedure, spontaneous
sex-linked lethals could have accumulated for at most one generation prior to irradiation. Although a method exists which enables the identification and thus the elimination of preexisting
lethals, it does not allow the experiment to be carried out on females whose X chromosomes are
both structurally normal. Thus, it may have the undesirable side effect of permitting the induction of lethals and rearrangements at positions opposite the break points of the inversion(s) in
the homolog (THOMPSON
1961, 1962).
In order to insure that the oocytes sampled were in stage 7 at the time of irradiation, 0 to 4
hour-old virgins were collected. These females would be no more than twelve hours old a t the
completion of the treatment. Newly emerged females have about 19 stage-7 oocytes, and none
more mature than this stage (KING1957). Mature eggs are not produced in substantial quantities
for a period of about 48 hours. A maximum treatment of twelve hours duration was used to minimize heterogeneity of treated stages. Within 24 hours, the stage 7 oocytes present a t eclosion have
progressed to and beyond stage 8.
The females were divided into four equal groups for treatment and placed in plastic vials containing a half-inch of sugar-agar medium, stoppered with plastic foam plugs. Each vial contained
100 to 150 flies. A control g r m p received no irradiation, while the other three received appropriate gamma radiation. All treatments were completed before any females were over twelve
hours old.
The irradiations were performed with a cesium-I37 gamma ray source, made available
through the courtesy of the Department of Radiology, University of Wisconsin. During the irradiation, the vials were inverted so that the radiation passed through the agar, and the flies were
confined close to the agar surface.
The first irradiation. designated intense-I, was delivered a t a dose rate of 334r per minute
for approximately 12 minutes, for a total dose of 4000r. The dose was measured with a Victoreen
dosimeter and a high energy chamber. The distance from the radiation source was 13 cm in all
intense irradiations. The next irradiated group was given a dilute dose at a distance of 85 cm.
This irradiation was given a t the rate of 9.5r per min for about 7 hr, again delivering a total dose
316
E. H. MARKOWITZ
of 4000r. Upon completion of the dilute dose, the final group, intense-2, was irradiated in a
manner identical to intense-I.At the time of this irradiation, the flies were approximately 7 hr
older than they were at the time of the intense-1 irradiation. Two replicates of this experiment
were performed, and in a third experiment the dilute dose was delivered at a lower rate of 4.8r
per min, at a distance of 121.5 cm. To keep the total treatment time within the twelve hour
maximum, the dose delivered was now 2000r to both the dilute and intense groups.
Following treatment, the females were placed in quarter-pint milk bottles, containing sugaragar medium, for two days. This aging period, prior to mating, allowed the treated stage 7
oocytes to mature, while the sugar-agar medium inhibited egg laying. preventing the loss of
treated oocytes. The females were then mass-mated to FM6 males in bottles for a standard X
chromosome lethal test. The multiply-inverted F’M6 chromosome prevents crossing over between
the two homologous X chromosomes and allows the irradiated X chromosome to be followed
intact. It is marked with the dominant gene B (bar) and the two recessives yS1d (yellow) and
din (diminutive). One hour prior to these matings. a sample of control females was mated individually with at least two males per female in a plastic egg-laying unit placed over a Petri
dish with standard cornmeal-molasses agar medium seeded with live yeast. Counts of the number
of eggs laid per female were made until this number reached about 17 per female; a t this point
all parents were discarded.
In the F, generation, virgin females were collected and individually mated to FM6 males in
vials, each mating representing a test of one irradiated X chromosome. When offspring appeared
in these vials, they were scored for sex-linked lethals. The criterion chosen for lethality was the
appearance of at least 20 male offspring, of which no more than ten percent were wild type. In
the first two experiments, counts on all lethal cultures were continued until the eighteenth day
after mating; a confirmatory cross was made and counted for each lethal. Stocks of all lethalbearing cultures were kept for further analysis. In the third experiment, counts on suspected
lethals were made only until there were at least twenty FM6 males and no wild-type males, at
which time the culture was discarded and recorded as a lethal.
RESULTS
I n Experiments I and 11, where the total dose was 4000r, no significant differences were observed between the frequencies of sex-linked recessive lethals
induced in any of the treated groups (see Table 1). In Experiment I, the frequency of induced lethals, corrected for the control frequency, for the dilute
treatment was 2.92 +- 0.55% (estimate +- standard error), while the intense-I
and intense-2 treatments yielded 2.42 k 0.49% and 2.75 0.58% inducedlethals,
respectively. In Experiment 11,the induced lethal frequencies were 4.43 * 0.69%
for the dilute treatment, 3.65 * 0.62% for intense-I, and 4.49 k 0.77% for
intense-2. Chi-square values were calculated to test for differences among the
treated groups, according to the method of MARKOWITZ
(1966). The chi-square
values of Experiments I and I1 may be added to give a total chi-square of 1.68
with 4 df (0.7 < P < 0.8), showing that taking both experiments together, there
are no significant differences between the different treatments. It is clear from the
data that the lethal frequencies observed in Experiment I are different from those
in Experiment 11, and that the only valid comparisons are those made within
a single experiment. The reasons for between-experiment variation are unknown,
but it is not uncommon in experiments of this sort.
In Experiment 111, the total dose was reduced to 2000r, in order to reduce the
low intensity dose rate to 4.8r per min without extending the total irradiation
time. The results are given in Table 2. For the dilute treatment, the induced
*
Y RAY-INDUCED
31 7
MUTATION
TABLE 1
Sex-linked recessive lethals induced in stage 7 oocytes b y 4000r cesium-I37 gumma rays
Exposure time Total tests Lethals
EXPERIMENT I
Control
Dilute
(9.4 r/min)
Intense-l
Intense-2
(334 r/min)
EXPERIMENT I1
Control
Dilute
(9.6 r/min)
Intense-I
Intense-2
(312 r/min)
Rate (%) zk
SE
0
1271
4
0.31 k 0.16
7 hr 5 min
12 min
12 min
1116
1247
950
36
34
29
2.92* t 0.55
2.42* f 0.49
2.75* f 0.58
0
911
5
0.55 f 0.25
7hr5min
12.5 min
12.5 min
1150
1219
897
57
51
45
4.43* f 0.69
3.65' t 0.62
4.49* t 0.77
~
xz = 0.534,
.7
x2
2 df
< P < .8
= 1.143, 2df
.5
< P < .7
~
* Coi-rected for control rate.
lethal frequency was 1.98 f 0.29%. The frequency for intense-I was 1.73 2
0.29% and for intense-2 was 2.76 t 0.36%. Again, statistical analysis showed
no significant differences among the three treated groups. It may be concluded
that in irradiated stage 7 oocytes, for a range of dose rates from 4.8r per min to
over 300r p x min, there is no evidence that the recessive sex-linked lethal frequency is dependent on the dose rate at which an irradiation is given.
A corollary experiment was carried out by Dr. HELENU. MEYER(results
reproduced here with her permission), using dose rates similar to those of Experiments I and 11, but testing for recessive lethals induced on the second chromosome, which is roughly twice the length of the X chromosome. The total dose
delivered to each group was 3200r. Her results are given in Table 3. They show
no lessened effect from the low dose rate treatment, and in fact the mutation
rate is highest for this treatment. The conclusion that there is no dose-rate effect
for stage 7 oocytes is therefore confirmed for chromosome 2, and presumably is
true for the whole genome.
TABLE 2
Sex-linked recessive lethals induced in stage 7 oocytes b y 2000r cesium-I37 gumma ruys
~~
~
Exposure time Total tests Lethals
EXPERIMENT I11
Control
Dilute
(4.8 r/min)
Intense-1
Intense-2
(347.2 r/min)
~
~~
Rate (%) f SE
0
1809
0
0.0
7 hr
5.75min
5.75 min
2326
2019
2026
46
35
56
1.98 t 0.29
1.73 f 0.29
2.76 t 0.36
x2
= 5.075, 2 df
.07
< P < .08
318
E. H. MARKOWITZ
TABLE 3
Chromosome-2recessive lethals induced in stage 7 oocytes by 3200r cesium-137 gamma rayst
Exposure time Total tests Lethals
Control
Dilute
(6.6 r/min)
Intense-I
Intense-2
(466 r/min)
Rate (%) rfi
SE
0
864
8
0.93 C 0.33
8hr
6.87min
6.87 min
1087
952
949
94
58
74
7.79' f 0.91
5.21* 0.81.
6.94* f 0.93
*
x2
= 5.194, 2df
.05
< P < .IO
* Corrected for control rate.
t The author wishes to thank Dr. HELENMEYERfor permissionto use these data.
As originally proposed, a sample of lethals recovered from each of the two
dose-rate irradiations in Experiment I was subjected to further analysis, consisting of first genetically localizing the approximate position of each lethal on the
X chromosome, and then examining the salivary gland chromosomes of larvae
heterozygous for that lethal to see if it was associated with a visible aberration.
Of 24 lethals from the dilute group analyzed cytologically, one aberration was
found. This was a deficiency extending from approximately 9B to 10A, or a
maximum of 45 salivary bands in length. 28 lethals from the intense-1 group were
analyzed. Again, only one chromosome aberration was found, a translocation between the X chromosome and chromosome 2. The break point in the X chromosome was at about 11F, and in the second chromosome, near the tip of the left
arm, at about 22E-F. The observed frequencies of gross aberrations are, therefore, for the dilute group, 1/24, or 4.17 * 4.1% and for the intense group, 1/28,
or 3.57 * 3.5%. Since the size of the sample is so small, the errors are large, but
there is no indication of a great difference in aberration frequencies between the
two groups. The majority of lethals appear not to be associated with any gross
structural change.
There has been some previous work involving cytological analyses of mutations
induced in Drosophila females. GLASSand METTLER(1958) found no visible
chromosome aberrations in a sample of 31 sex-linked lethals recovered from
X irradiation of oogonial cells of 80 hour-old female larvae. VALENCIA
and
VALENCIA
(1961) analyzed 54 specific-locus mutations induced by 4000r X ray
treatment of adult females. A high proportion (80%) of those mutations recovered from mature oocytes were associated with visible chromosome aberrations, mostly small deficiencies, while those recovered from oogonia rarely were.
Since both of these experiments involved somewhat different conditions than
those in the present study, no attempt at comparisons will be made.
DISCUSSION
These experiments have provided no evidence for the existence of a dose-rate
effect in irradiated Drosophila oocytes. Earlier indications of such an effect in
Drosophila could be attributed to heterogeneity of treated oocyte stages. The
Y RAY-INDUCED MUTATION
319
lowest dose rate given to oocytes in the present study, 4.8r per min, is still
considerably higher than the low dose rates of 0.1 to 0.001r per min often employed in successful dose-rate experiments with other organisms, where the low
intensity irradiation can be given over a long period of time. I n restricing this
experiment to stage 7 oocytes, the shorter time allowable for irradiation made it
impossible to use a dose rate this low and still obtain meaningful mutation frequencies. Perhaps, if an effect is to be found for oocytes, it might only be apparent
at much lower dose rates or doses. However, it should be noted that X rays delivered at 9r per min did produce a significantly lower mutation frequency than
those at 90r per min in an experiment on mouse spermatogonia (RUSSELL
1963).
In Drosophila oogonia and spermatogonia, experiments have been performed with
low intensity irradiation as low as 0.01r per min, yet these experiments have
failed to provide convincing evidence for a dose-rate effect (PURDOM
1963;
MULLER,OSTERand ZIMMERING
1963). Although it is difficult to prove conclusively the absence of an effect, it does appear that, for Drosophila, in the range
of dose rates studied, there is no difference of the magnitude of that found for the
mouse spermatogonia and oocytes.
In reviewing dose-rate experiments performed on different organisms, a general set of interpretations is only beginning to emerge. Dose-rate-dependent differences which are due to the interaction of two separate hits, or due to selection
among a heterogeneous population of cells, reflect phenomena which, although
factors of biological interest, are not directly concerned with the mutation process
itself.
In the results from the mouse, the data on oocytes provide evidence against
the suggestion that the difference is attributable to selective cell killing or stage
heterogeneity. In the ovary of the adult female mouse, there are no oogonia, and
the majority of the oocytes are uniformly in the dictyate state, a modified resting
stage occurring after diplotene. The completion of the first meiotic division just
precedes ovulation. It has been argued that since the dictyate state is relatively
uniform and of long duration, the dose-rate effect is not the result of differential
stage-sensitivity in the female, and presumably this is true also for the male.
Furthermore, if the data are restricted to those oocyte stages which, on the basis
of fertility results and histological study, are reasonably free of cell killing, a significant difference between the mutation rates obtained from chronic and acute
irradiation still exists (RUSSELL,
RUSSELL
and KELLY1960). Thus, the interpretation favored by RUSSELL
is that there is a repair system for premutational
damage and that this system is either saturated at high dose rates or is damaged
by the radiation by a dose-rate-dependent process. The effects found in the silkworm may also involve an intracellular repair process, but the results are
sufficiently confounded with age and stage differences and cell selection, that
interpretation can only be very tentative (TAZIMA,
KONDO
and SADO1961).
In the experiments with Dahlbominus, not only was the magnitude of the
difference much smaller than that observed in the mouse, but the results from
radiation sources of different quality have been compared (BALDWIN
1962,1965).
It was argued that the high energy (2 MVp) X rays used were comparable to
320
E. H. MARKOWITZ
gamma rays. Also, the observation that there was no difference in the mutation
rates from acute 300 kV and 2 MV X rays suggests that radiation quality is not
an important factor. However, unless both the acute and the chronic doses are
from the same radiation source, any inferences about a dose-rate effect are open
et al. (1958) also
to some question. Although the original experiments of RUSSELL
employed radiations of different quality, later experiments were performed using
the same source for both acute and chronic doses, and a significant dose rate effect
was observed (RUSSELL,
RUSSELL
and KELLY1960; RUSSELL
1963).
The question of the one-hit nature of the mutations observed is a critical one.
That thzre is some doubt concerning the single-hit nature of specific-locusmutations in mice leaves open the possibility that the dose-rate effects observed are
simply two-hit aberration phenomena (WOLFF1967). This view, however, may
be an oversimplified interpretation of all the available data.
MULLER(1965) has argued that the difference between the response of mammals and that of flies to dose rate can be understood on evolutionary grounds. Due
to th-. longer life span of mammals, and their greater sensitivity to irradiation, a
selective advantage would be conferred on those lines which evolve a protective
mechanism against naturally occurring chronic irradiation. In the case of flies,
the influence of natural radiation would be so slight as not to exert much, if any,
selective pressure.
et al. (1968) provide possible
Observations made on Neurospora by DE SERRES
insight as to factors responsible for divergmt results obtained from different
organisms. He has demonstrated a dose-rate effect for lesions in the ad-3 region
of Neurospora, presumed to be chromosome breaks, which interact in pairs to
produce “deletions”, but not for those qualitatively different lesions which appear
to be ‘Lpoint”mutations. It was suggested that there are a variety of repair
mechanisms acting on different kinds of lesions. At the lowest dose used, there
is an indication that the “deletions” are produced by single hits, yet there is still
a dose-rate dependency in their frequency.
It is clear that more work needs to be done characterizing the nature of doserate effscts where they have been found. Until there is more information of a
descriptive nature, further attempts at interpretation are premature.
I wish to thank Dr. SEYMOUR
ABRAHAMSON
for his advice, criticism, and generous assistance
during the course of this work. I thank Miss LINDALowmv+”N for her assistance with the
localizations, and Mrs. JUDYVOIGTLANDER
and all the members of the Drosophila laboratory
who have helped me in carrying out these experiments.
SUMMARY
Drosophila melanogaster females were irradiated with cesium-137 gamma rays
at dose rates ranging from 4.8r per minute to over 300r per minute. The frequencies of sex-linked lethal mutations recovered from treated stage 7 oocytes
were measured, and indicated no difference due to the dose rate of the irradiation.
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Y RAY-INDUCED M U T A T I O N
321
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