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Effect of X-irradiation on the DNA content of individual nuclei of rabbit bone marrow
Experimental
Cell
Research
17, 405-413
405
(1959)
EFFECT OF X-IRRADIATION ON THE DNA CONTENT OF INDIVIDUAL NUCLEI OF RABBIT BONE MARROW1 E. M. UYEKI, Atomic
CECILIE
LEUCHTENBERGER
and P. R. SALERNO
Energy Medical Research Project and The Institute of Pathology, Western Reserve Universily, Cleveland, Ohio, U.S.A. Received
October
27, 1958
OUR earlier studies dealing with phosphatase activity [lo] and the incorporation of labeled leucine into protein [ 111, in comparable populations of bone marrow cells, revealed that changes observed after X-irradiation were concomitant with changes in cell types. With the disappearance of the younger forms of marrow cells, there was a decrease in the leucine incorporation into protein and an increase in the phosphatase activity. These observations were interpreted as indicating that younger cells have a greater capacity to incorporate leucine into proteins, while the phosphatase activity is more marked in the remaining cell population which consists of the more mature elements and the primitive stem cells. Although such a mixed cell assay gives some biochemical information as to the predominant cell types which contribute the activity, it does not assess the individual cells and the possible modified response of individual cells to injury. In view of the heterogeneity of marrow tissue, variations in sensitivity towards X-irradiation are not unexpected. Therefore, studies on individual cells which can be directly correlated with the morphology, degree of maturation and the mitotic stage would be desirable. The influence of ionizing radiation as studied biochemically on whole tissues by other workers [7, 91 indicates variable effects on desoxyribonucleic acid (DNA) synthesis. The variations have been shown to depend on the particular tissue, radiation dose, time interval after radiation, and the physiological state of the animal. Generally, it can be stated that a depression of DNA synthesis is usually observed by means of isotopic tracer studies. Howard [a] has critically analyzed the factors which bring about this inhibition of DNA synthesis. Concerning the effects of radiation on the bone marrow, similar decreases in DNA synthesis were reported by Smellie et al. [7] and Thomson et nl. [Y]. However, Thomson et al. noted that if the DNA content was expressed in 1 This work was performed under Atomic Energy Commission Contract with Western Reserve University, and supported in part by Research Grant Institutes of Health, U.S. Public Health Service. Experimental
No. W31-109~eng-78 No. C-1814, National
Cell Research
17
406
E. M. Uyeki,
Cecilie Leuchtenberger
and P. R. Salerno
terms of cell population instead of tissue weight [7], the mean DNA content per cell did not show a significant change after irradiation. Although the Thomson study on cell population facilitates the interpretation of the DNA data, a computed mean value on a heterogeneous cell population such as the hone marrow does not permit an interpretation in regard to the individual cells. Consequently, from the studies of the workers noted above on bone marro\\ tissue, one cannot ascertain whether or not the DNA synthesis in the individual cells is changed after irradiation. A direct assessment of the DNA content of single cells is obviously needed. Feulgen microspectrophotometry, which permits the analysis of DNA in the cells preserving their morphology and location within the bone marrow tissue, was utilized in our studies. The validity of the quantititarc study of DNA on single cells as studied by the microspectrophotometric technique has been established [6]; to the best of our knowledge, the effect of X-irradiation on individual bone marrow cells by this technique has not been studied.
MATERIALS
AND
METHODS
Male albino rabbits weighing from 1.5 to 2.5 kg were employed for these studies and were maintained in air conditioned cages and fed ad libitum. The animals were anesthetized with Nembutal prior to X-irradiation. A single dose of 800 r of X-rays was administered to one-half of the body on the longitudinal axis, the other half being protected from X-irradiation by lead shielding. Radiation was delivered at a dose rate of 17.9 r per minute; other radiation factors were: 220 KVP, 12 ma., 0.5 mm Cu and 1.0 mm Al filters. The animals were killed and the marrows were rapidly removed from the distal epiphyseal portion of the femurs and were fixed immediately in Lavdowsky’s fixative. For studies on the control bone marrow, the marrows were taken from non-irradiated rabbits and also from the non-irradiated marrows of the half-body S-rayed rabbits. The same animal served both as control and X-rayed and thus minimized cellular composition differences of individual animals. The specimens were fixed, dehydrated, cleared and embedded in paraffin under uniform conditions. Sections were cut at 7 micra in thickness. For the determination of DNA in these sections, microspectrophotometry was carried out as previously described [6]. The nuclei for DNA measurement were selected under the following considerations: (1) the selection of nuclei that were neither cut nor overlapped; (2) in the Feulgen stained sections, the nuclei from control femoral marrows were selected and measured on the basis of nuclear size, presence of nucleoli, clumping of chromatin, and were numerically compared with similar cell types from the irradiated femurs. The cells of the Feulgen stained sections were compared with hematoxylin-eosin-azure sections. In view of the variable makeup of normal bone marrow it was necessary to ascertain the basic values for the normal controls. Therefore, we have utilized femurs from non-irradiated rabbits and non-irradiated femurs from 800 r hemi-body S-rayed rabbits.
X-rays
on bone marrow DNA
407
In our series of experiments the most pronounced effect occurred at 24 and 48 hours post-irradiation. At 6 and 12 hours after X-irradiation a lesser degree of cell loss was observed. Paralleling the time interval after irradiation that the animal was sacrificed, there was a corresponding decrease in the number of cells that could be measured, due in part to the damage to the nuclei and also to the selective depletion of these cells from the bone marrow. Since the tissue sections showed less destruction of the cells than the smear technique, it permitted a more accurate percentage composition of the cell types. Thus, it was used in preference to smears although histograms of DNA values were comparable. RESULTS
Fig. 1 compares the distribution of the DNA in the individual cells of rabbit bone marrow of the different controls and also shows a histogram of the distribution of DNA of the individual nuclei of normal liver cells. Comparison of the distribution of DNA content of the femoral marrows of non-irradiated rabbits and non-irradiated femoral marrows of the 800 r hemi-body X-rayed rabbit indicates that there is no significant difference in the DNA distribution. The histograms of the marrow cells reveal that the greater number of cells lies in the DNA values between 6 and 12 x 10-s mg per nucleus. On the other hand, the DNA content of individual nuclei from the liver of rabbits indicates MARROW N’=l42
30
20
G 2 f ”
IO
RABBIT
NON-IRRADIATED MARROW OF HEMI-BODY X-RAYED RABBIT 124 HRS AFTER 8OOrl N’= 140
E 30 d = 5
OF NON-IRRADIATED
n
0
E
LIVER OF NON-IRRADIATED N-=40
40
RABBIT
30 L
Fig. l.--DNA nuclei from the livers of rabbits. measured.
content
of individual femoral marrows and No, number of nuclei
--E
20 IO 0
a-
E
I 2
4 AMOUNT
27 - 593706
I 8
I
I I IO I2 OF DNA IN 10-eMGM
Experimental
I4
Cell Research
17
E. M.
Uyeki,
Cecilie Leuchtenberger
and P. R. Salerno
that the normal distribution of cells lies essentially below mg. Prior investigations by Leuchtenberger [a] as well cated that liver cells of most adult mammals fall into Therefore, in our studies, nuclei below values of 6 Fig. 1 as the shaded area, were classified as diploid. both control marrows, over 85 per cent of the measured values between diploid and tetraploid values. These
Fig. 2.-Bone marrow of nuclei measured.
the values of 6 X 10-S as Swift [S] have indidistinct DNA classes. X 10-S mg, shown on It can be seen that in nuclei are in the DNA cells may be looked
nuclear sizes in cubic micra of 800 r hemi-body X-rayed rabbits. No, number C, non-irradiated femur; X, irradiated femur; NI, non-irradiated rabbit.
upon as cells that are synthesizing DNA or cells that have entered the DNA synthetic period. A study was made of the changes in DNA content of individual nuclei of the bone marrow brought about by 800 r of hemi-body X-irradiation. Animals were sacrificed at 1, 6, 12, 18, 24 and 48 hours after exposure. As a routine procedure, groups of 20 nuclei from the irradiated femoral marrow were morphologically compared with the non-irradiated femoral marrow from the same animal for DNA content in individual nuclei. Fig. 2 shows the nuclear size relationships for the nuclei which were compared. Since size represented an important parameter in comparing cell types between the X-rayed and its control, the nuclear size, in terms of cubic micra, was computed and compared with each other. The nuclear sizes were arbitrarily classified into the following four categories: (1) lo-20 cubic micra-cell types which were generally contained in this range were polychromatic and acidophilic erythroblasts; (2) 20-30 cubic micra-cells which were included in this range were myelocytes and the basophilic erythroblasts; (3) 30-40 cubic micrapromyelocytes and blast forms were included in this range; (4) 40 + cubic micra-blast forms as well as hemocytoblasts were included in this range. Experimental
Cell Research
17
X-rays
on bone marrow DNA
400
Megakaryocytes, granulocytes, and cells below 10 cubic micra were not measured. Overlapping of other cell types into the different nuclear size categories is not unusual because of some difficulty in cell identification. However, categorizing cells in this manner does visually facilitate the comparisons of the cell types between the X-rayed and its control femur. In most instances, in terms of nuclear size relationships, Fig. 2 does indicate that equivalent nuclei were compared.
TIME AFTER IRRADIATION
Fig. 3.--Per cent distribution of DNA content body X-rayed rabbits. No, number of nuclei femur; NI, non-irradiated rabbit.
of individual measured.
bone marrow C, non-irradiated
nuclei from 800 r hemifemur; X, irradiated
Fig. 3 compares DNA content of these equivalent nuclei. On groups of 38 nuclei each, comparing the X-rayed and control femoral marrow from a rabbit sacrificed one hour after 800 r of hemi-body radiation, the DNA values indicate there is no appreciable difference in the control and X-rayed marrows. The G-hour post-irradiation values indicate somewhat variable results betvveen individual rabbits. In view of the varying susceptibilities of individual animals to ionizing radiations during the first few days after X-ray, these results are not too surprising. Similarly, the 12-hour and l&hour postirradiation data indicate varying sensitivities in individual rabbits. Only when one measures the nuclei from the 24-hour post-irradiated femurs does one find a consistent decrease in the number of cells in the diploid to tetraploid range (synthesizing cells). The magnitude of this decrease, however, varies with individual rabbits. The 4%hour post-irradiation values indicate a further decrease in the synthesizing cells. Fig. 4 shows a composite histogram of the DNA content of individual nuclei of the bone marrow by 800 r hcmi-body X-irradiation 6, 12, and 24 hours after exposure. The data indicate that at 6 and 12 hours after X-ray there E.zperimenlul
Cell
Research
17
E. M. Uyeki,
410
Cecilie Leuchfenberger
and P. R. Salerno
is a decrease in the number of synthesizing cells. The significance of this change awaits the analysis of larger samples of nuclei. Fig. 4 shows the individual nuclei DNA values from control femurs and femurs taken from rabbits 24 hours after HO0 r of hemi-body X-irradiation. A comparison of the histograms indicates that whereas 85 per cent of the nuclei from non-irradiated femurs are above the diploid value, as shown by the non-shaded area, the irradiated femoral marrow from the same rabbit
6 HNS K-60
MEN
x-RA”
24 ms BFTEN Km 100
x-F&w
30
d-l 2
4
AMOUNT
Fig. 4.-Comparison femoral marrows
of DNA of hemi-body
content X-rayed
NUCLEAR
Nm.24
3.B
8 DNA
IN
10
12
10
p MG,,,
- 3.3
T
14
of individual nuclei rabbits. No, number
DIAMETERS
=I= 3. 0 l
6 OF
2
K.17
2.9-3
I
2.n-
6
8
from irradiated and of nuclei measured.
(ARBITRARY
311 - 3.3
4
N’- I0
1 231-
2.7
.
24661012
P-27
Al3 246610
246610 AMOUNT
Cdl
Research
17
es
A0
1.
Experimental
non-irradiated
I 0ELow
P.26
Fig. 5.-Distribution of DNA size (24 hour 800 r hemi-body
14
“NITS,
2.6
AL 24660
2466OQ
12
Y-*26
J
NP. 24
10
content X-rayed
OF ONA
2 4 6 IN lo-*
0 0
2 4 6
6 IO
246
A 2466012
MGM
of individual bone marrow nuclei on the basis rabbits). No, number of nuclei measured.
of nuclear
X-rays
on bone marrow DNA
indicates a decrease of the synthesizing cells to 59 per cent. Conversely, whereas the normal bone marrow cell distribution indicated only 14 per cent of the cells are in the diploid range, the 24-hour post-irradiated marrow cells increased to 41 per cent in the diploid range, indicating a threefold increase in the non-synthesizing cells. On the basis of this comparison the 800 r hemibody X-irradiation results in a marked decrease in the synthesizing cells. In an attempt to focus the X-ray changes on the cell types involved, histograms of the non-irradiated and irradiated femoral marrow nuclei of the 24-hour post-irradiation experiments were categorized according to nuclear diameters. Fig. 5 represents the composite histogram of the 24-hour postirradiation samples. Although aware of the small sample population per nuclear diameter group, the histograms reveal: (1) a consistent decrease in the synthesizing cells of the X-irradiated femoral marrow nuclei compared vvith their control in the nuclear size ranges 2.71-2.9, 2.91-3.1, 3.11-3.3, 3.3-3.5 and 3.5 + (arbitrary units). In the nuclear diameters below 2.7, there is no significant difference between the irradiated cells and however, their non-irradiated controls. It can be seen that these cells lie in the DNA content range designated as diploid. In the case of the erythrohlasts between nuclear diameters of 2.0-2.7 (the cell identification made by its characteristic clumping of chromatin) there is no significant effect of X-irradiation. It is seen in the histograms, however, that these cells are predominantly in the DNA content range between the diploid to tetraploid range. DISCUSSION
Investigations by Lajtha et crl. on the effect of ionizing radiations on individual cells of bone marrow tissue [3], using the radioautographic technique, revealed a significant inhibition of DNA synthesis. They indicated that there is a period in the interphase which is particularly susceptible to inhibition and designated this period as the “DNA synthesizing period” or the “S” period. Recently Lajtha et al. [4] confirmed their prior studies on DNA synthesis inhibition in bone marrow cultures and also pointed out that there is a system which is connected with but not identical to DNA synthesis vvhich appears to be more sensitive than DNA synthesis itself. Our investigations, using Feulgen microspectrophotometry and performed on histologic sections of non-irradiated and irradiated femoral marrow of rabbits, generally coincide with the work of previous investigators. Limiting our studies to the cells capable of undergoing mitosis, the major portion of Exprrimental
Cell
Research
17
412
E. M.
Uyeki,
Cecilie Leuchfenberger
and P. R. Salerno
non-irradiated marrow cells (greater than 85 per cent) contain amounts of DNA in the range of 6 and 12 x 10-9 mg. This may be contrasted with liver nuclei in which the major portion of the measured cells is grouped around 6 x 10-S mg of DNA. Consequently, the major portion of the measured marrow nuclei from non-irradiated femurs falls between the diploid and tetraploid values and the designation synthesizing cells has been used to categorize these cells. The main consequence of S-irradiation to exposed femurs has been to reduce the number of the synthesizing cells. Comparison of the X-rayed marrow cells with the shielded marrow cells from the same animal tends to minimize cell composition variations from animal to animal. Studies conducted at 24 hours post-irradiation from individual rabbit studies (Fig. 3) and coinposite histograms (Fig. 4) clearly reveal a consistent decrease in the number of these synthesizing cells. From the standpoint of comparing DNA content of nuclei of comparable size (Fig. 5), the reduction of the synthesizing cells is clearly revealed at the larger nuclear sizes. Generally the larger nuclear sizes are indicative of the more immature cells of the bone marrow. At nuclear diameters below 2.7 (arbitrary units), comparisons of histograms from irradiated and nonirradiated femurs reveal no significant differences. Since the DIVA content of most of these smaller nuclei are grouped around the diploid value, it is possible that they have matured beyond the stage in which they are capable of undergoing further mitosis and DNA synthesis. In the case of the erythroblasts, between the nuclear diameter size range of 2.0-2.7 (arbitrary units), there are not significant differences between Xrayed and control. Since the major portion of these cells are synthesizing according to our criteria (cells bet\veen 6 and 12 x 10-S mg), these results are opposed to the view that the erythroblastic series are quite sensitive to the influence of ionizing radiation [l]. These cells are darkly stained,however, and it may be that the photometric method is less sensitive at these high extinctions. The analysis and interpretation of the results are speculative; in view of the number of parameters involved in our system, it is not possible to attribute to one series of biochemical events the lethal effects of X-irradiation to the cell. Since the intervening biochemical events between the end of prophase and the eventual resynthesis of DN.4 of the next cycle remain fragmentary, the exact inhibition scheme is open to a number of possibilities. It would be important to demonstrate the temporal sequence of biochemical events of the long interphase period leading to eventual mitosis. Esperimentul
Cell
Research
17
X-rays
on bone marrow DNA
413
SUMMARY
1. Feulgen microspectrophotometric studies of DNA were conducted on individual nuclei of non-irradiated and irradiated femoral marrows of rabbits exposed to 800 r hemi-body X-irradiation. 2. The major portion of the measured marrow nuclei from the non-irradiated marrow cells are in the range between 6 and 12 x 10-S mg of DNA per nucleus. These cells have been designated as synthesizing cells. S. The main consequence of X-irradiation to the exposed femurs has been to reduce the number of synthesizing c,ells. Studies conducted at 24 and 48 hours post-irradiation reveal a consistent decrease in the number of synthesizing cells. At intervals of X-irradiation less than 24 hours, the results were variable although indicative of a decrease in synthesizing cells. 4. Studies on comparable nuclear sizes of X-rayed and non-X-rayed femoral marrovv reveal a reduction of synthesizing cells in the larger nuclei (2.773.5 + arbitrary units). In the case of nuclear size ranges below 2.7 and erythroblasts (nuclear size range 2.62.7 arbitrary units) there are no significant effects of X-irradiation. REFERENCES 1. BLOOM, 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
JI. A., Histopathology of Irradiation, p. 162, Mc-Gnaw-Hill Book Co., Inc., New York, 1948. HOWARD, A., Ciba Foundation Symposium on Ionizing Radiations and Cell Metabolism, p. 196, Little, Brown and Co., 1956. LAJTHA, L. G., OLIVER, R. and ELLIS, F., Brit. J. Cancer 8, 367 (1954). LAJTHA, L. G., OLIVER, R., KUMATORI, T. and ELLIS, F., Radiation Research 8, 1 (1958). LEUCHTENBERGER, C., Science 120, 1022 (1954). ~ General Cytochemical Methods. Vol. I, p. 219. Academic Press, Inc., New York, 1958. SMELLIE, R. M. S., HUMPHREY, 6. F., KAY, E. R. M. and DAVIDSON, J. N., Biochem. J. 60, 177 (1955). SWIFT, H., Physiol. Zool. 23, 169 (1950). THOMSOS, J. F., TOURTELLOTTE, W. E., CARTTAR, M. S. and STORER, J. B., Arch. Biochem. Biophys. 42, 185 (1953). UYEI(I, E. hI. and SALERNO, P. R., NYO-4933(1957). -NYO-2057 (1957).
Experimental
Cell Research
17
Cell
Research
17, 405-413
405
(1959)
EFFECT OF X-IRRADIATION ON THE DNA CONTENT OF INDIVIDUAL NUCLEI OF RABBIT BONE MARROW1 E. M. UYEKI, Atomic
CECILIE
LEUCHTENBERGER
and P. R. SALERNO
Energy Medical Research Project and The Institute of Pathology, Western Reserve Universily, Cleveland, Ohio, U.S.A. Received
October
27, 1958
OUR earlier studies dealing with phosphatase activity [lo] and the incorporation of labeled leucine into protein [ 111, in comparable populations of bone marrow cells, revealed that changes observed after X-irradiation were concomitant with changes in cell types. With the disappearance of the younger forms of marrow cells, there was a decrease in the leucine incorporation into protein and an increase in the phosphatase activity. These observations were interpreted as indicating that younger cells have a greater capacity to incorporate leucine into proteins, while the phosphatase activity is more marked in the remaining cell population which consists of the more mature elements and the primitive stem cells. Although such a mixed cell assay gives some biochemical information as to the predominant cell types which contribute the activity, it does not assess the individual cells and the possible modified response of individual cells to injury. In view of the heterogeneity of marrow tissue, variations in sensitivity towards X-irradiation are not unexpected. Therefore, studies on individual cells which can be directly correlated with the morphology, degree of maturation and the mitotic stage would be desirable. The influence of ionizing radiation as studied biochemically on whole tissues by other workers [7, 91 indicates variable effects on desoxyribonucleic acid (DNA) synthesis. The variations have been shown to depend on the particular tissue, radiation dose, time interval after radiation, and the physiological state of the animal. Generally, it can be stated that a depression of DNA synthesis is usually observed by means of isotopic tracer studies. Howard [a] has critically analyzed the factors which bring about this inhibition of DNA synthesis. Concerning the effects of radiation on the bone marrow, similar decreases in DNA synthesis were reported by Smellie et al. [7] and Thomson et nl. [Y]. However, Thomson et al. noted that if the DNA content was expressed in 1 This work was performed under Atomic Energy Commission Contract with Western Reserve University, and supported in part by Research Grant Institutes of Health, U.S. Public Health Service. Experimental
No. W31-109~eng-78 No. C-1814, National
Cell Research
17
406
E. M. Uyeki,
Cecilie Leuchtenberger
and P. R. Salerno
terms of cell population instead of tissue weight [7], the mean DNA content per cell did not show a significant change after irradiation. Although the Thomson study on cell population facilitates the interpretation of the DNA data, a computed mean value on a heterogeneous cell population such as the hone marrow does not permit an interpretation in regard to the individual cells. Consequently, from the studies of the workers noted above on bone marro\\ tissue, one cannot ascertain whether or not the DNA synthesis in the individual cells is changed after irradiation. A direct assessment of the DNA content of single cells is obviously needed. Feulgen microspectrophotometry, which permits the analysis of DNA in the cells preserving their morphology and location within the bone marrow tissue, was utilized in our studies. The validity of the quantititarc study of DNA on single cells as studied by the microspectrophotometric technique has been established [6]; to the best of our knowledge, the effect of X-irradiation on individual bone marrow cells by this technique has not been studied.
MATERIALS
AND
METHODS
Male albino rabbits weighing from 1.5 to 2.5 kg were employed for these studies and were maintained in air conditioned cages and fed ad libitum. The animals were anesthetized with Nembutal prior to X-irradiation. A single dose of 800 r of X-rays was administered to one-half of the body on the longitudinal axis, the other half being protected from X-irradiation by lead shielding. Radiation was delivered at a dose rate of 17.9 r per minute; other radiation factors were: 220 KVP, 12 ma., 0.5 mm Cu and 1.0 mm Al filters. The animals were killed and the marrows were rapidly removed from the distal epiphyseal portion of the femurs and were fixed immediately in Lavdowsky’s fixative. For studies on the control bone marrow, the marrows were taken from non-irradiated rabbits and also from the non-irradiated marrows of the half-body S-rayed rabbits. The same animal served both as control and X-rayed and thus minimized cellular composition differences of individual animals. The specimens were fixed, dehydrated, cleared and embedded in paraffin under uniform conditions. Sections were cut at 7 micra in thickness. For the determination of DNA in these sections, microspectrophotometry was carried out as previously described [6]. The nuclei for DNA measurement were selected under the following considerations: (1) the selection of nuclei that were neither cut nor overlapped; (2) in the Feulgen stained sections, the nuclei from control femoral marrows were selected and measured on the basis of nuclear size, presence of nucleoli, clumping of chromatin, and were numerically compared with similar cell types from the irradiated femurs. The cells of the Feulgen stained sections were compared with hematoxylin-eosin-azure sections. In view of the variable makeup of normal bone marrow it was necessary to ascertain the basic values for the normal controls. Therefore, we have utilized femurs from non-irradiated rabbits and non-irradiated femurs from 800 r hemi-body S-rayed rabbits.
X-rays
on bone marrow DNA
407
In our series of experiments the most pronounced effect occurred at 24 and 48 hours post-irradiation. At 6 and 12 hours after X-irradiation a lesser degree of cell loss was observed. Paralleling the time interval after irradiation that the animal was sacrificed, there was a corresponding decrease in the number of cells that could be measured, due in part to the damage to the nuclei and also to the selective depletion of these cells from the bone marrow. Since the tissue sections showed less destruction of the cells than the smear technique, it permitted a more accurate percentage composition of the cell types. Thus, it was used in preference to smears although histograms of DNA values were comparable. RESULTS
Fig. 1 compares the distribution of the DNA in the individual cells of rabbit bone marrow of the different controls and also shows a histogram of the distribution of DNA of the individual nuclei of normal liver cells. Comparison of the distribution of DNA content of the femoral marrows of non-irradiated rabbits and non-irradiated femoral marrows of the 800 r hemi-body X-rayed rabbit indicates that there is no significant difference in the DNA distribution. The histograms of the marrow cells reveal that the greater number of cells lies in the DNA values between 6 and 12 x 10-s mg per nucleus. On the other hand, the DNA content of individual nuclei from the liver of rabbits indicates MARROW N’=l42
30
20
G 2 f ”
IO
RABBIT
NON-IRRADIATED MARROW OF HEMI-BODY X-RAYED RABBIT 124 HRS AFTER 8OOrl N’= 140
E 30 d = 5
OF NON-IRRADIATED
n
0
E
LIVER OF NON-IRRADIATED N-=40
40
RABBIT
30 L
Fig. l.--DNA nuclei from the livers of rabbits. measured.
content
of individual femoral marrows and No, number of nuclei
--E
20 IO 0
a-
E
I 2
4 AMOUNT
27 - 593706
I 8
I
I I IO I2 OF DNA IN 10-eMGM
Experimental
I4
Cell Research
17
E. M.
Uyeki,
Cecilie Leuchtenberger
and P. R. Salerno
that the normal distribution of cells lies essentially below mg. Prior investigations by Leuchtenberger [a] as well cated that liver cells of most adult mammals fall into Therefore, in our studies, nuclei below values of 6 Fig. 1 as the shaded area, were classified as diploid. both control marrows, over 85 per cent of the measured values between diploid and tetraploid values. These
Fig. 2.-Bone marrow of nuclei measured.
the values of 6 X 10-S as Swift [S] have indidistinct DNA classes. X 10-S mg, shown on It can be seen that in nuclei are in the DNA cells may be looked
nuclear sizes in cubic micra of 800 r hemi-body X-rayed rabbits. No, number C, non-irradiated femur; X, irradiated femur; NI, non-irradiated rabbit.
upon as cells that are synthesizing DNA or cells that have entered the DNA synthetic period. A study was made of the changes in DNA content of individual nuclei of the bone marrow brought about by 800 r of hemi-body X-irradiation. Animals were sacrificed at 1, 6, 12, 18, 24 and 48 hours after exposure. As a routine procedure, groups of 20 nuclei from the irradiated femoral marrow were morphologically compared with the non-irradiated femoral marrow from the same animal for DNA content in individual nuclei. Fig. 2 shows the nuclear size relationships for the nuclei which were compared. Since size represented an important parameter in comparing cell types between the X-rayed and its control, the nuclear size, in terms of cubic micra, was computed and compared with each other. The nuclear sizes were arbitrarily classified into the following four categories: (1) lo-20 cubic micra-cell types which were generally contained in this range were polychromatic and acidophilic erythroblasts; (2) 20-30 cubic micra-cells which were included in this range were myelocytes and the basophilic erythroblasts; (3) 30-40 cubic micrapromyelocytes and blast forms were included in this range; (4) 40 + cubic micra-blast forms as well as hemocytoblasts were included in this range. Experimental
Cell Research
17
X-rays
on bone marrow DNA
400
Megakaryocytes, granulocytes, and cells below 10 cubic micra were not measured. Overlapping of other cell types into the different nuclear size categories is not unusual because of some difficulty in cell identification. However, categorizing cells in this manner does visually facilitate the comparisons of the cell types between the X-rayed and its control femur. In most instances, in terms of nuclear size relationships, Fig. 2 does indicate that equivalent nuclei were compared.
TIME AFTER IRRADIATION
Fig. 3.--Per cent distribution of DNA content body X-rayed rabbits. No, number of nuclei femur; NI, non-irradiated rabbit.
of individual measured.
bone marrow C, non-irradiated
nuclei from 800 r hemifemur; X, irradiated
Fig. 3 compares DNA content of these equivalent nuclei. On groups of 38 nuclei each, comparing the X-rayed and control femoral marrow from a rabbit sacrificed one hour after 800 r of hemi-body radiation, the DNA values indicate there is no appreciable difference in the control and X-rayed marrows. The G-hour post-irradiation values indicate somewhat variable results betvveen individual rabbits. In view of the varying susceptibilities of individual animals to ionizing radiations during the first few days after X-ray, these results are not too surprising. Similarly, the 12-hour and l&hour postirradiation data indicate varying sensitivities in individual rabbits. Only when one measures the nuclei from the 24-hour post-irradiated femurs does one find a consistent decrease in the number of cells in the diploid to tetraploid range (synthesizing cells). The magnitude of this decrease, however, varies with individual rabbits. The 4%hour post-irradiation values indicate a further decrease in the synthesizing cells. Fig. 4 shows a composite histogram of the DNA content of individual nuclei of the bone marrow by 800 r hcmi-body X-irradiation 6, 12, and 24 hours after exposure. The data indicate that at 6 and 12 hours after X-ray there E.zperimenlul
Cell
Research
17
E. M. Uyeki,
410
Cecilie Leuchfenberger
and P. R. Salerno
is a decrease in the number of synthesizing cells. The significance of this change awaits the analysis of larger samples of nuclei. Fig. 4 shows the individual nuclei DNA values from control femurs and femurs taken from rabbits 24 hours after HO0 r of hemi-body X-irradiation. A comparison of the histograms indicates that whereas 85 per cent of the nuclei from non-irradiated femurs are above the diploid value, as shown by the non-shaded area, the irradiated femoral marrow from the same rabbit
6 HNS K-60
MEN
x-RA”
24 ms BFTEN Km 100
x-F&w
30
d-l 2
4
AMOUNT
Fig. 4.-Comparison femoral marrows
of DNA of hemi-body
content X-rayed
NUCLEAR
Nm.24
3.B
8 DNA
IN
10
12
10
p MG,,,
- 3.3
T
14
of individual nuclei rabbits. No, number
DIAMETERS
=I= 3. 0 l
6 OF
2
K.17
2.9-3
I
2.n-
6
8
from irradiated and of nuclei measured.
(ARBITRARY
311 - 3.3
4
N’- I0
1 231-
2.7
.
24661012
P-27
Al3 246610
246610 AMOUNT
Cdl
Research
17
es
A0
1.
Experimental
non-irradiated
I 0ELow
P.26
Fig. 5.-Distribution of DNA size (24 hour 800 r hemi-body
14
“NITS,
2.6
AL 24660
2466OQ
12
Y-*26
J
NP. 24
10
content X-rayed
OF ONA
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0 0
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6 IO
246
A 2466012
MGM
of individual bone marrow nuclei on the basis rabbits). No, number of nuclei measured.
of nuclear
X-rays
on bone marrow DNA
indicates a decrease of the synthesizing cells to 59 per cent. Conversely, whereas the normal bone marrow cell distribution indicated only 14 per cent of the cells are in the diploid range, the 24-hour post-irradiated marrow cells increased to 41 per cent in the diploid range, indicating a threefold increase in the non-synthesizing cells. On the basis of this comparison the 800 r hemibody X-irradiation results in a marked decrease in the synthesizing cells. In an attempt to focus the X-ray changes on the cell types involved, histograms of the non-irradiated and irradiated femoral marrow nuclei of the 24-hour post-irradiation experiments were categorized according to nuclear diameters. Fig. 5 represents the composite histogram of the 24-hour postirradiation samples. Although aware of the small sample population per nuclear diameter group, the histograms reveal: (1) a consistent decrease in the synthesizing cells of the X-irradiated femoral marrow nuclei compared vvith their control in the nuclear size ranges 2.71-2.9, 2.91-3.1, 3.11-3.3, 3.3-3.5 and 3.5 + (arbitrary units). In the nuclear diameters below 2.7, there is no significant difference between the irradiated cells and however, their non-irradiated controls. It can be seen that these cells lie in the DNA content range designated as diploid. In the case of the erythrohlasts between nuclear diameters of 2.0-2.7 (the cell identification made by its characteristic clumping of chromatin) there is no significant effect of X-irradiation. It is seen in the histograms, however, that these cells are predominantly in the DNA content range between the diploid to tetraploid range. DISCUSSION
Investigations by Lajtha et crl. on the effect of ionizing radiations on individual cells of bone marrow tissue [3], using the radioautographic technique, revealed a significant inhibition of DNA synthesis. They indicated that there is a period in the interphase which is particularly susceptible to inhibition and designated this period as the “DNA synthesizing period” or the “S” period. Recently Lajtha et al. [4] confirmed their prior studies on DNA synthesis inhibition in bone marrow cultures and also pointed out that there is a system which is connected with but not identical to DNA synthesis vvhich appears to be more sensitive than DNA synthesis itself. Our investigations, using Feulgen microspectrophotometry and performed on histologic sections of non-irradiated and irradiated femoral marrow of rabbits, generally coincide with the work of previous investigators. Limiting our studies to the cells capable of undergoing mitosis, the major portion of Exprrimental
Cell
Research
17
412
E. M.
Uyeki,
Cecilie Leuchfenberger
and P. R. Salerno
non-irradiated marrow cells (greater than 85 per cent) contain amounts of DNA in the range of 6 and 12 x 10-9 mg. This may be contrasted with liver nuclei in which the major portion of the measured cells is grouped around 6 x 10-S mg of DNA. Consequently, the major portion of the measured marrow nuclei from non-irradiated femurs falls between the diploid and tetraploid values and the designation synthesizing cells has been used to categorize these cells. The main consequence of S-irradiation to exposed femurs has been to reduce the number of the synthesizing cells. Comparison of the X-rayed marrow cells with the shielded marrow cells from the same animal tends to minimize cell composition variations from animal to animal. Studies conducted at 24 hours post-irradiation from individual rabbit studies (Fig. 3) and coinposite histograms (Fig. 4) clearly reveal a consistent decrease in the number of these synthesizing cells. From the standpoint of comparing DNA content of nuclei of comparable size (Fig. 5), the reduction of the synthesizing cells is clearly revealed at the larger nuclear sizes. Generally the larger nuclear sizes are indicative of the more immature cells of the bone marrow. At nuclear diameters below 2.7 (arbitrary units), comparisons of histograms from irradiated and nonirradiated femurs reveal no significant differences. Since the DIVA content of most of these smaller nuclei are grouped around the diploid value, it is possible that they have matured beyond the stage in which they are capable of undergoing further mitosis and DNA synthesis. In the case of the erythroblasts, between the nuclear diameter size range of 2.0-2.7 (arbitrary units), there are not significant differences between Xrayed and control. Since the major portion of these cells are synthesizing according to our criteria (cells bet\veen 6 and 12 x 10-S mg), these results are opposed to the view that the erythroblastic series are quite sensitive to the influence of ionizing radiation [l]. These cells are darkly stained,however, and it may be that the photometric method is less sensitive at these high extinctions. The analysis and interpretation of the results are speculative; in view of the number of parameters involved in our system, it is not possible to attribute to one series of biochemical events the lethal effects of X-irradiation to the cell. Since the intervening biochemical events between the end of prophase and the eventual resynthesis of DN.4 of the next cycle remain fragmentary, the exact inhibition scheme is open to a number of possibilities. It would be important to demonstrate the temporal sequence of biochemical events of the long interphase period leading to eventual mitosis. Esperimentul
Cell
Research
17
X-rays
on bone marrow DNA
413
SUMMARY
1. Feulgen microspectrophotometric studies of DNA were conducted on individual nuclei of non-irradiated and irradiated femoral marrows of rabbits exposed to 800 r hemi-body X-irradiation. 2. The major portion of the measured marrow nuclei from the non-irradiated marrow cells are in the range between 6 and 12 x 10-S mg of DNA per nucleus. These cells have been designated as synthesizing cells. S. The main consequence of X-irradiation to the exposed femurs has been to reduce the number of synthesizing c,ells. Studies conducted at 24 and 48 hours post-irradiation reveal a consistent decrease in the number of synthesizing cells. At intervals of X-irradiation less than 24 hours, the results were variable although indicative of a decrease in synthesizing cells. 4. Studies on comparable nuclear sizes of X-rayed and non-X-rayed femoral marrovv reveal a reduction of synthesizing cells in the larger nuclei (2.773.5 + arbitrary units). In the case of nuclear size ranges below 2.7 and erythroblasts (nuclear size range 2.62.7 arbitrary units) there are no significant effects of X-irradiation. REFERENCES 1. BLOOM, 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
JI. A., Histopathology of Irradiation, p. 162, Mc-Gnaw-Hill Book Co., Inc., New York, 1948. HOWARD, A., Ciba Foundation Symposium on Ionizing Radiations and Cell Metabolism, p. 196, Little, Brown and Co., 1956. LAJTHA, L. G., OLIVER, R. and ELLIS, F., Brit. J. Cancer 8, 367 (1954). LAJTHA, L. G., OLIVER, R., KUMATORI, T. and ELLIS, F., Radiation Research 8, 1 (1958). LEUCHTENBERGER, C., Science 120, 1022 (1954). ~ General Cytochemical Methods. Vol. I, p. 219. Academic Press, Inc., New York, 1958. SMELLIE, R. M. S., HUMPHREY, 6. F., KAY, E. R. M. and DAVIDSON, J. N., Biochem. J. 60, 177 (1955). SWIFT, H., Physiol. Zool. 23, 169 (1950). THOMSOS, J. F., TOURTELLOTTE, W. E., CARTTAR, M. S. and STORER, J. B., Arch. Biochem. Biophys. 42, 185 (1953). UYEI(I, E. hI. and SALERNO, P. R., NYO-4933(1957). -NYO-2057 (1957).
Experimental
Cell Research
17