T H E EFFECT O F SINGLE AND DIVIDED DOSES O F HIGH
INTENSITY X-RAYS ON T H E EGGS
O F DROSOPHILA
CHARLES PACKARD
(From Colztmbia University, I n s t i t ~ ~ tofe Cancer Research, F. C . W o o d , Director)
The many studies on the relation between the intensity of radiation and the
magnitude of the biological reaction indicate that in a majority of cases high
intensities and brief exposures are more effective than low intensities and
correspondingly long exposures; and that single doses are more effective from
the biological point of view than divided doses. The opinion is sometimes
expressed or implied that experimental results which point to an opposite
conclusion are probably wrong. In the literature, reviewed by Griffith and
Zimmer ( 7 ) Pack and Quimby (16), and Schreiber (20), may be found many
instances of experiments in which the same test objects and technic were
used and yet the results were contradictory. Ascaris eggs, exposed in air,
are reported to be less affected by divided doses than by a single massive dose
(13, 81, and again to be more affected (23). Exposed in an atmosphere
deprived of its oxygen, these eggs show a simple cumulative effect of repeated
doses (8) ; that this treatment may produce a greater reaction than that which
follows a single dose is also claimed ( 1 1).
The behavior of tissue culture cells under different conditions of dosage
is also under dispute. Faber (6) states that the mitotic rate is equally retarded whether the dose is given at high or low intensities or in fractions,
while others (1, 22) believe that high intensities are the more effective and
that divided doses produce a greater reaction than single large doses. Drosophila eggs, according to Sievert and Forssberg (2 I ) , respond equally to all
intensities of x-rays within a very wide range; but Roesler and Henshaw (19)
find that high intensities are more effective than low.
Errors in technic may account for some of these conflicting results; but
without doubt many of the observed differences are real, and may be related
to differences in the physiological condition of the test objects at the time of
exposure. A review of some of the physical and biological conditions which
must be considered when carrying out experiments of this kind may make clear
why uniformity in results cannot be expected.
The terms " low " and " high " intensity are purely relative. What constitutes a low or subminimal intensity for any particular material cannot be
predicted; it must be determined by experiment. Radiations received at the
rate of 0.04 r/hour by the human skin are ineffective, no matter how long the
exposure may be (15). At the other extreme are some Protozoa which are
not injured by enormous doses repeated daily. As the intensity rises above
a certain minimum, whose value varies widely in different kinds of cells and
tissues, the effect produced often becomes proportional to the dose within
a wide range of intensities and periods of exposure. The epilation dose
130
for the human skin is 300 r whether it is delivered at the rate of 5 or 500 r/min.
(9). Pack and Quimby (16) report that the threshold skin dose produced
by the beta rays of radium is approximately the same whether given in forty
seconds or in four hours. Drosophila eggs respond equally to equal doses
when the intensities lie between 5 and 30 r/min. More frequently, however,
the effectiveness of radiations steadily increases with the intensity, as shown
by Holthusen (9) for the skin erythema dose. This is true also for the
fertilized egg of the chick ( 2 ) and some Protozoa (5). On the other hand,
in another section of this paper is presented evidence that under certain conditions the reverse is true; high intensities may be less effective than low intensities.
The terms " long " and " short " to designate the duration of exposure
are highly elastic. The same number of minutes of irradiation may constitute a long dose for one kind of cell and a short one for another kind. For
example, twenty minutes is a relatively long treatment for Drosophila eggs,
for in this brief interval all of the cells normally divide once, and many
divide a second time. The sensitivity also changes measurably in this space
of time. But for adult tissues this length of exposure is short; during twenty
minutes not a single cell may have entered mitosis, and the change in sensitivity is probably negligible. For Drosophila eggs exposed to low intensities
the length of the exposure, within definite limits, makes no difference in the
quantitative result. But this is not necessarily true for adult tissues. Pack
and Quimby (16) show that if the duration of exposure to radiation of low
intensity is comparable to the life span of the cell, that is, to the interval
between successive mitoses, the effect produced on the human skin is smaller
than that produced by a stronger source acting for a shorter time.
Uniformity of results should not be expected even when different kinds of
test objects are exposed to doses which are comparable in length and intensity.
The biological condition of the cells, especially in respect to their rate and
rhythm of mitosis, is a factor which in large measure controls the degree of
reaction. In some test objects, such as developing Drosophila eggs, cell division is continuous in the sense that there is practically no intermitotic period
of rest, the daughter cells at the conclusion of one division almost immediately preparing for the next. Under such conditions radiosensitivity remains
constant and high. But the cells of tissue culture preparations divide slowly,
perhaps once in two days. Actual mitosis requires about one hour and is
followed by a long resting stage. The sensitivity of the individual cells exhibits a wide fluctuation.
In other kinds of material, cell division is not constant but occurs in definite
rhythms during which radiosensitivity rises. Langendorff (12) reports that
in the mouse testis, spermatogonial divisions occur with greatest frequency
at 2 P.M. and a t 5 A.M. Among plants this phenomenon has long been
known. The cells of the onion root tip divide most actively at 10 A.M. and
11 P.M. Jiingling and Langendorff (10) find a maximum mitotic activity in
bean seedlings between 3 and 9 P.M. during which interval about 14 per cent
of the cells are in division. At other times the percentage falls considerably,
although mitosis is never completely absent. Radiations alter this rhythm.
Shortly after exposure there is an almost complete cessation of cell division.
132
CHARLES PACKARD
This period is followed by two waves of mitotic activity occurring about sixty
hours and one hundred and twenty hours after exposure. The amount of
effect produced by equal doses of radiation depends largely on the relation between the time of exposure and these rhythms. If exposure is made in the
morning, during the quiescent period, the effect is much smaller than if it is
made in the afternoon, when cell division is at a maximum. Alberti and
Politzer (1) also find that an initial irradiation of embryonic tissue from the
salamander is followed by a mitosis-free interval during which the sensitivity
is low. This is followed by a return of cell division and a rise in sensitivity.
I n view of these facts it is evident that an experiment to test the effect
of high or low intensities, or of divided doses, is of little value if only the
physical conditions of dosage are considered. T h e biological condition of
the test object a t the moment of exposure and the changes which it undergoes
during and immediately after irradiation are of prime importance. Unless
they are taken into account, the significance of the results may easily be misinterpreted.
I n the following section data are presented which show that under certain
conditions, high intensities are less effective on Drosophila eggs than low intensities, and that divided doses may produce an effect equal to or greater
than that of a single dose. I n the latter respect the response is similar to
that of bean rootlets and embryonic animal tissue.
EXPERIMENTAL
I n these experiments the term " low intensity " signifies an output of
10-30 r/min.; " high intensity" means 125-135 r/min. I n both series the
voltage was 120 kv. For the low intensity tests the filtration was 0.25 mm.
Cu and 1.0 mm. Al; for the high, 1.0 mm. Al. The half values are 0.32 mm.
and 0.13 mm. Cu respectively. The difference in the wavelength of the two
beams does not affect the biological results (17). Occasional tests with the
low intensity beam, using the 1.0 mm. A1 filter, gave results identical with
those obtained under the heavier filtration. Scattered radiation was practically absent; the eggs during exposure were supported on a strip of gauze
held in place by a wooden frame not in the direct beam. A newly calibrated
Victoreen dosimeter was used to determine dosage.
The eggs were prepared for irradiation in precisely the same manner that
has been followed in previous experiments (17). They are collected for two
hours, transferred to slips of black filter paper, and exposed one hour after
the end of the collection period. I n the first series of tests exposures were
made in two ways. Several samples were given an initial dose of one minute
or less; the beam was then interrupted while one or more slips of paper bearing
eggs were removed. T h e remaining samples were then given a second dose,
after which the beam was again interrupted and more samples taken. This
procedure continued until all of the slips had been removed. T h e interval
between successive exposures was not more than one half minute. In other
tests the samples received the entire dose without interruption. T h e response
of the eggs treated in these two ways is the same. The length of exposure
in the low intensity tests varied from four to twenty minutes; in the high intensity tests, from forty-five seconds to four minutes.
The criterion of effect is the percentage of eggs that hatch out as larvae
two days after exposure. These larvae develop into apparently normal flies;
the proportion that live and reproduce compares favorably with that observed
in the untreated controls. This is true even when the eggs receive a dose
sufficient to kill approximately 75 per cent of the sample. I t is evident,
therefore, that the survival rate of the treated eggs is a valid criterion of effect.
No attempt has been made to determine whether mutants have been produced
by the treatment.
The reaction of Drosophila eggs to beams of low intensity has been observed over a period of years (18). The total number of tests to determine
the relation between the dose, measured in roentgens, and the percentage of
survivors is now upwards of 250. The course of the survival curve is well
established (Fig. 1). In Table I are presented some of these data, selected
for comparison with the results of the high intensity experiments in which
the dosage in roentgens is the same. Each figure is an average of six to fifteen
separate determinations made during the past six years. The probable error
of the means indicates the amount of variation which is expected.
The number of tests to determine the quantitative effect of the high intensity beams now exceeds 200. The figures given in Table I under the
heading High Intensity are averages of ten to twenty tests. The degree of
134
CHARLES PACKARD
variation in this series is about the same as that found in the first. A comparison of the two series shows at once that, when equal doses are given, a
larger proportion of eggs survive under the high intensity beams than under
the low. The difference is very little for the smaller doses, but it becomes
obvious with large doses. Statistical analysis shows that the two series are
perfectly distinct. The difference between the average survival rates, in the
case of the larger doses, divided by the probable error of the difference, results in factors of 7 or more. In other words, the possibility that the two
series are the same is exceedingly remote. The high intensity beams are
definitely less effective on Drosophila eggs than low intensities.
TABLE
I : Comparison of the Effects of Low and IZigh Intensity Beams on Drosophila Eggs
High Intensity
Percentage of Survival
Low Intensity
Percentage of Survival
Dose
(roentgens)
-.
76.2
52.8
44.0
29.0
24.8
125
195
235
330
390
f 0.73
f 0.70
f 1.13
f 0.69
f 0.56
T,\r3~rr11: Effect of Two Doses of High Intensity X-rays Separated by Different Time Znterzlals
High Intensity
Percentage of Survival
Dose
(roentgens)
I
Single
Exposure
Five-minute
Interval
Ten-minute
Interval
Low Intensity
Percentage of Survival
1
1
I
Twenty-
~~~~~~~1
Single Exposure
This result is contrary to that obtained by Sievert and Forssberg (21)
and by Roesler and Henshaw (19), who also used these eggs. The former
investigators tested the effect of intensities ranging from 5.5 to 24,000 r/min.,
and report that throughout this enormous range the effect of equal doses is
about equal. There was some evidence that with the highest intensity fewer
eggs were killed. Their average dose was 165 roentgens. From the curves
shown in Fig. 1 it is evident that for this dose the difierence between t.he two
series is not large. If tests are made only at this point the conclusion might
be warranted that the two series are substantially the same. That they are
really different but converging cannot be demonstrated unless other points
along the curve are also determined. The results of Sievert and Forssberg,
therefore, do not prove that high and low intensity beams are equally effective.
Roesler and Henshaw (19) conclude that an intensity of 234 r/min. is more
effective than a much lower intensity. Some of their experimental conditions,
as they point out, were not the same as those used in the present investigation.
Their results are not strictly comparable with those here described.
The effect of divided doses of high intensity x-rays can be tested to only
a limited extent on these eggs because their sensitivity changes rapidly and
at a rate which is not constant (18). An experiment cannot safely be prolonged beyond twenty or thirty minutes. This brief interval, however, is
long enough to allow all of the cells in each egg to divide at least once, and
is biologically equivalent to the much longer periods required for a single
mitosis in less rapidly dividing material. Two exposures, separated by intervals of five to twenty minutes can be given. The slips of paper with eggs
are given an initial dose, then removed from the field. Subsequently some
are given a second dose five minutes after the first dose was begun, and others
are exposed ten or more minutes after the first exposure, the total dose for
each lot of eggs being the same.
The averaged results of a large number of such tests are given in Table 11.
The total dose is shown in the first column; next is the percentage of survivals
when this dose is given in one continuous exposure. Some of these figures
appear also in Table I. I n the succeeding columns are given the percentages
of survival of eggs exposed five, ten, and twenty minutes after the initial dose.
In the last column appear, for comparison, the percentage survivals of eggs
exposed to low intensity x-rays.
I t will be seen that the reaction of eggs exposed after an interval of five
minutes is not significantly different from that found in eggs given continuous
irradiation. After a ten minute rest the eggs are definitely more sensitive,
136
CHARLES PACKARD
as shown by the fact that fewer survive. After a lapse of twenty minutes
the sensitivity has increased still more. The difference between these series
becomes evident when the data are plotted as shown in Fig. 2, in which the
logarithms of the percentage survivals are plotted against the doses. The
evidence is clear that eggs can partially regain their sensitivity when the interval between two successive doses is sufficiently long. Thus the effect of
divided doses on Drosophila eggs may be equal to that of a single large dose,
or it may be greater, depending on the way in which the exposures are arranged.
The smaller effectiveness of high intensity x-rays as coinpared with low
intensity beams is clearly seen when the doses are comparatively large; 400
roentgens of the former result in a survival of 23 per cent of the eggs, while
the same dose of the latter is followed by a survival of only 10 per cent. But
small doses of both high and low intensities produce practically equal quantitative results. This indicates that under the former the eggs lose their
sensitiveness more rapidly than under the latter. This reaction is undoubtedly associated with a decline in the rate of mitosis during exposure.
A retard of cell division or its complete cessation is a common phenomenon
which occurs even after small doses of x-rays. Langendorff (12) shows that
the spermatogonial divisions in the mouse testis quickly cease following a
dose of only 100 roentgens. For tissue cultures, 50-75 roentgens suflice (4).
The retard begins almost as soon as irradiation commences and continues
until shortly after it stops. This reaction is not necessarily a sign of serious
injury; cell division may begin again at a rate which for a time exceeds the
normal. However, if the exposure has been severe, the subsequent mitosis
may be abnormal. I n this connection the fact is significant that high intensity rays are more effective in producing the retard in cell division than
low intensities ( 3 ) .
The sensitivity of cells declines during this period. Jiingling and Langendorff (10) report that if bean rootlets are exposed during a period of maximum
cell division, and subsequently given a second dose during the time when the
mitotic rate is declining, the effect of the two exposures is equal to that of a
single large dose. But if the second is applied when mitosis is at a minimum,
its effect is lost. Sensitivity is also at a minimum.
I t is reasonable to conclude that these same phenomena occur also in
Drosophila eggs, but a t a much quicker tempo. As soon as irradiation begins,
mitosis quickly ceases. Under high intensities this reaction is more marked
than under low intensities. As a result, the eggs under the former become
less sensitive than under the latter. The difference becomes more and more
obvious as the dose is prolonged.
When irradition stops, cell division begins again, and with its return is
associated a rise in sensitivity. If the second dose is administered during
this period, the effect of the two is greater than that of a single large dose
(10). Alberti and Politzer also show that embryonic tissue is more sensitive
during the period of returning mitoses, which in this case are abnormal, than
during the quiescent interval. These facts furnish an explanation for the
behavior of Drosophila eggs exposed under similar circumstances. A return
of cell division begins soon after the end of the first exposure, but sensitivity
does not rise appreciably within the first five minutes. I t becomes apparent
after ten minutes and is very marked after an interval of twenty minutes.
The chief difference, therefore, between the reaction of these eggs and that of
bean seedlings is the rate at which these changes take place. I t may be inferred that other test objects in which cell division is rapid may show the same
kind of response under analogous conditions of exposure, if the intervals between successive irradiations are properly adjusted.
These results are the opposite of those obtained in experiments on the
human skin, which have established the fact that fractional doses are less
effective in producing the erythema reaction than a single large dose. MacComb and Quimby ( 1 4 ) show that the threshold skin dose is 5 2 5 roentgens,
delivered at the rate of 6 0 r/min. But it is 9 2 5 roentgens if five fractional
doses are given in successive days. The response of the eggs and rootlets
to fractional doses differs from that of the skin because the two types of
material are in different physiological states at the time of exposure. I n
both there is partial recovery, that is, a return toward the condition obtaining
when the initial exposure was given. In the skin, the injured cell constituents
presumably are repaired or replaced; in rapidly dividing cells or tissues, there
is a more or less successful attempt to resume mitosis. Unless the initial dose
has been severe, cell division returns to normal, although its rhythm may be
altered. I n the skin, recovery is marked by a decrease in sensitivity, shown
by the fact that the total number of roentgens delivered in fractional doses
must be greater than that of a single dose to produce the same degree of reaction. On the other hand, the return of active mitosis in rootlets is accompanied by an increase in sensitivity. In these two types of material the
physiological state at the time of the initial exposure was not the same; recovery toward the original condition proceeds in different directions. I t is
to be expected, therefore, that the effect of radiations applied during this period
should not be the same.
High intensities (125-135 r/min.) are more effective than low intensities
(10-30 r/min.) on developing Drosophila eggs. The difference is most
clearly seen after large doses.
When the high intensity dose is divided into two portions, the effect of
the total dose may be equal to or greater than that of a single large dose, depending on the length of the interval between the two exposures.
The response of Drosophila eggs to divided doses differs from that observed in the skin because the biological condition of the two test objects is
not the same.
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138
CHARLES PACKARD
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