- Email: [email protected]
Analysis of radiation dose-response curve obtained with cytokinesis block micronucleus assay
ISSN 0969-8051/97/$17.00 + 0.00 PII S0969-8051 (97)00004-8
Nuclear Medicine & Biology, Vol. 24, pp. 413-416, 1997 Copyright © 1997 Elsevier Science Inc. ELSEVIER
Analysis of Radiation Dose-Response Curve Obtained with Cytokinesis Block Micronucleus Assay Solomon F. D. Paul, P. Venkatachalam and R. K. Jeevanram* HEALTH A N D SAFETY DIVISION, SAFETY RESEARCH A N D HEALTH PHYSICS GROUP, IND1RA GANDHI CENTRE FOR ATOMIC RESEARCH, KALPAKKAM 603 102, 1NDIA
ABSTRACT. The frequency of micronuclei and acentrics obtained with different doses of 6°Co gamma radiation was examined. When compared to acentric frequency the micronuclei frequency was found to be higher at about 115% for doses below 1 Gy. However, it dropped to about 65% as the dose was increased to 4 Gy. This paper discusses the causes for the reduced frequency of micronuclei at higher doses by taking into account the possibility of their being masked from view by the daughter nuclei in the binucleated cell. NUCL MED BlOt. 24;5:413-416, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Micronuclei, Daughter nuclei, Cytokinesis, Acentric fragments, Radiation dose response
INTRODUCTION Measurement of micronuclei frequency in human lymphocytes is gaining importance as a technique for estimating radiation dose because it is simple and permits easy scoring of micronuclei even by an inexperienced scorer. Micronuclei are formed both from acentric fragments and a whole chromosome (4). Whole chromosomes that have become detached from the mitotic spindle and consequently excluded from incorporation into either daughter nucleus may also form some of the micronuclei. Though the frequency of micronuclei increases with dose, most of the studies have reported that the occurrence of micronuclei is less compared to that of dicentric chromosomes at higher doses--i.e., above about 1.5 Gy (11, 12). It has been suggested by Savage that, at higher doses, micronuclei get included into the main nucleus (13). We felt that a considerable number of micronuclei may be masked by daughter nuclei at higher doses and may not be counted. To study this, (i) dose-response curves for micronuclei and acentric fragments were constructed, (ii) the areas occupied by micronuclei, daughter nuclei, and cytoplasm were measured, and (iii) the sizes of the micronuclei obtained with both high and low dose were measured. The results obtained are analysed in this paper to explain the reduced frequency of micronuclei at high dose. MATERIALS A N D M E T H O D S
Peripheral blood samples (3.5 mL) obtained from normal volunteers were irradiated with different doses ranging between 0.25 and 4.0 Gy with 6°Co gamma radiation at a dose rate of 0.6 Gy/min. The irradiated samples were maintained at 37°C for 1 h to enable repair of chromosomal damages. Blood samples were cultured in F-10 HAM medium (Sigma, Catalogue No. N-6013), supplemented with 20% fetal calf serum and 0.2 mL phytohemagglutinin (PHA-M) (1) (Difco, No. 052856). For the micronucleus assay, Cytochalasin-B (Sigma, No. C-6762) at a final concentration of 3 Ixg/mL was added to each sample at 44 h and the cells harvested after a further incubation of *Author for correspondence. Received 28 October 1995. Accepted 4 February 1997.
28 h (3). The slides for scoring micronuclei were prepared by fixing the cells with 3:1 mixture of methanol and acetic acid after a brief hypotonic treatment. The cell suspensions were dropped onto clear, cold slides from a height of 1 cm and stained with 10% Giemsa solution (pH 6.8) and scored for micronuclei. Cells with two daughter nuclei, surrounded by cytoplasm and cell membrane, were scored for the presence of micronuclei according to the modified criteria of Countryman and Heddle (2)--i.e., material staining as nuclear material having less than half of the diameter of either daughter nucleus and not overlapped by either of the main nuclei. The distribution of micronuclei in binucleated cells was studied. In the case of control and 0.25-Gy dose, the number of cells scored were 2000, whereas for doses between 0.5 and 4.0 Gy, the number of cells scored totaled 1000. The method of examining the distribution is described by Papworth (10). This is done by the standard u test using the formula du-
(N-I)
~d)
Where N is the total number of cells scored, d is the coefficient of dispersion (N - 1 ) ~2/y, Where Y is the mean number of observed aberrations, o2/Y is the relative variance and Var d is the variance o f d given by 2(N - 1) (1 - 1/NY). The diameters of the cytoplasm, the daughter nuclei, and the micronuclei were measured using an ocular micrometer. Using these values the cellular and nuclear areas were calculated by assuming the cytoplasm and nuclei to have circular shapes. The chromosome aberration assay was set up by the modified procedure described by Moorhead et al. (8) and Hungefford (5). The procedure is the same as that reported for micronucleus assay up to the stage of addition of PHA. For metaphase chromosome preparation, colcemid at a final concentration of 0.1 Ixg/mL was added to each sample at 46 h and incubation continued for another 2 h. At the end of culture, cells were given hypotonic treatment with 0.45 % KC1, washed, and then fixed in 3:1 mixture of methanol and acetic acid. The cell suspensions were dropped onto the precooled slides from a height of 10 cm and stained in the same way mentioned for micronucleus assay. These slides were scored for chromosomal aberrations.
414
S . F . D . Paul et al.
TABLE 1. Distribution of Micronuclei After Various Doses of 6°Co G a m m a Radiation Dose
M N Distribution pattern
(Gy)
0
1
0.00 0.25 0.50
1986 1950 946
14 47 51
1.00 2.00 3.00 4.00
888 801 636 526
104 177 274 298
2
3
. 3 i
.
. -2
6 18 64 118
1 2 23 43
il
~rzl y
0.21 + 2.78 +4.05
0.993 1.087 i.I 79
+ 2.74 +2.04 +3.53 +5.68 Average
1.122 1.091 1.158 1.254 1.109
4 .
. ---
---
1 2 2 10
--1 5
u, Dispersionindex; ~2/y, relative variance; MN, micronuclei;Y, mean number of observed aberrations. RESULTS
The distribution of micronuclei obtained with different gamma doses is given in Table 1. The table shows that u, the distribution of micronuclei in binucleated cells, varies between -0.21 and +5.68. The average areas occupied by the micronucleus, both daughter nuclei and the cytoplasm unmasked by daughter nuclei, were 20.37 -+ 21.18 x 10 12 m 2, 1013.86 _+ 134.68 × 10 -i2 m 2, and 1580.83 -+ 432.27 × 10 -12 m 2, respectively. These average areas occupied by the macronuclei and micronuclei were calculated in those samples that were not given radiation dose. In relative terms, these areas were found to have average value of 0.78 -+ 0.81%, 38.77 -+ 5.55%, and 60.45 _+ 13.87%, respectively. Figure 1 compares the acentric and micronuclei frequency obtained at different doses of gamma radiation; it shows that at higher doses the micronuclei frequency is lower than that obtained for acentric frequency. Both curves in Figure 1 were fitted using the linear quadratic equation. The micronuclei frequency shown in Figure 1 accounts only for those micronuclei as observed under microscope without taking into account those buried under daughter nuclei. 1.5
1.5
1.0-
//
/
//
/
/
The uncorrected micronuclei frequency was plotted as a function of acentric frequency, which showed a correlation of 0.99 (Fig. 2, continuous line). However, the angle of the slope of the regression line was 58 °. Because both daughter nuclei occupy about 38% of the cytoplasmic area, a correction factor was introduced to account for the number of micronuclei masked by these daughter nuclei. Micronuclei frequency, corrected for the area masked by daughter nuclei, is given in Figure 3. The figure shows that both curves are overlapping one above the other. This can also be seen from the angle of the slope ( - 4 5 °) of the regression line (dotted) in Figure 2. Both the size and the distribution of micronuclei obtained at 0.5 and 4 Gy were also measured. The results show that at the higher dose, the percentage of micronuclei having larger size is greater than that obtained at low dose (Table 2). DISCUSSION The distribution of micronuclei as per u test indicates overdispersion as six out of seven values showed a value more than _+1.96--/. e., only 5% probability exists that the magnitude of u will be greater than this value when the distribution is a Poisson distribution. The present study clearly shows that, at higher doses, the observed micronuclei frequency is low compared to acentric
ij
,,'/t
1.2
,./"
1.0
/J
, , / " AFTER ,_1 ,_1
---I,
I,.i.I c.)
0.5
0.5.
,._1 ,.,J
"'
n,"
0 0.8 DC
o
o
u.I
uJ
///"I/
//
z
0.0
'
0
I
1
2 DOSE
/
0.0
4
(0/)
o.o 0.0
q .//
I
I
I
0.4
0.8
1.2
MN PER CELL
FIG. 1. Comparison o f acentric and micronuclei frequency as a function of dose. T h e vertical bar on the curves indicates SE obtained at each dose.
FIG. 2. MN/Cell obtained before and after correction plotted as a function of acentric/cell.
Radiation Dose Response Curve for Micronucleus Assay
1.5
415
1.5
//'/ 1.0
1.0
v
m
,,q o
-.I n,,
0.5
0.5
12. o
z
0.0 0
I
I
2
4
0.0
DOSE (~) FIG. 3. Comparison of acentric and corrected micronuclei frequency as a function of dose. The vertical bar on the curves indicates SE obtained at each dose. frequency (Fig. 1). However, micronuclei frequency was somewhat higher compared to acentric frequency at doses less than 1 Gy, and this could be due to interference of high background frequency of micronuclei (1). Similar results have been reported by others (11, 12). Balakrishnan and Bhatt (1) have shown that for a gamma dose of 2 Gy, when 432 acentric fragments were present, the micronucleus yield was only 242 in 1000 cells. The reason for the lower micronuclei frequency at higher dose is not very clear. Savage has suggested the possibility of two or more fragments fusing to form one micronucleus at higher dose (14). The flow cytometric studies carried out by Nusse et al. indicate that the DNA contents in the micronuclei obtained at higher doses have higher DNA content than the one obtained at low dose (9). Using fluorescence in situ hybridisation, Miller et al. (7) have shown micronuclei carrying two or more acentric fragments. Although fusion of acentric fragments may lead to a decrease in micronuclei frequency, a concomitant increase in the size of the micronuclei would be expected. According to Littlefield et al. the size difference is insufficient to explain TABLE 2. Size Distribution of Micronuclei (MN) at Low and High Doses
Dose (Gy)
Number of cells with M N scored
0.50
200
4.00
200
Area range (p.m z) 1-40 41-80 >81 1-40 41-80 >81
(148) (71) (6) (129) (83) (52)
Percentage (%) 65.7 31.6 2.7 48.9 31.4 19.7
The number of micronuclei analysed for different sizes are given in parentheses.
this phenomenon (6). The other hypothesis put forward by Savage is that with increasing radiation dose the micronuclei get included into the main nucleus (13). In the present study, it was found that both daughter nuclei on an average mask 38.8% of the cytoplasmic area. If we consider that the distribution of micronuclei within the cell is uniform, then a portion of about 38% of cytoplasmic area goes unscored. In other words, the number of micronuclei scored are only those that are seen in the cytoplasmic area, unmasked by daughter nuclei--i.e., in 60.5% of the total area. It was reasonable to assume that the number of micronuclei being masked or buried by daughter nuclei would be considerable at higher doses. This suggested that a correction should be applied to get the actual number of micronuclei that are formed. As the micronuclei scored were only in 60% of the area, a correction was applied to get the actual number in 100% of the area. Following the above corrections, the two curves became superimposable, which can be seen in Fig. 3. This was verified by measuring the angle of the slope of the regression line (i) micronuclei per cell as observed when plotted as a function of acentric per cell. (ii) micronuclei per cell as obtained after correction plotted as a function of acentric per cell. Following a nucleus-masked correction, the angle of the slope of the regression line came very close to 45 °, indicating that masking of micronuclei by daughter nuclei is the major factor for the reduced micronuclei frequency at higher doses compared to acentric frequency. Though it has been pointed out by Littlefield et al. (6) that the size difference in micronuclei obtained due to fusion of micronuclei is not adequate to explain the reason for lower micronuclei frequency at higher dose, we made an attempt to study the size of the micronucleus obtained at both low and high doses. Although the size and the distribution of such micronuclei show some variation with respect to dose, the present study indicates that the contribution of fusion of micronuclei in reducing the micronuclei frequency may be insignificant.
CONCLUSION The present study indicates that the observed low micronuclei frequency at higher doses of 6°CO gamma radiation compared to acentric frequency is mainly due to masking of micronuclei by daughter nuclei, and the role of other factors if any could be only of minor consequence.
We wish to express our sincere thanks to Mr. L. V. Krishnan, Director, Safety Research and Health Physics Group, and to Mr. A. R. Sundararajan, Head, Health and Safety Division, for the encouragement given during the course of the study. We also wish to acknowledge Dr. M. Jagadeesan, Director, Bernard Institute of Radiology, Government General Hospital, Madras, for permitting us to use the teletherapy unit to irradiate the blood sample. The authors wish to thank Mrs. Shyamala Devi for scoring acentric aberrations.
References 1. Balakrishnan S. and Bhatt B. (1989) Studies of radiation-induced micronuclei: A rapid method for screening suspected overexposed individuals.Bull. Radiat. Protect. 12, 86-90. 2. Countryman P. 1. and Heddle J. A. (1976) The production of micronuclei from chromosome aberrations in irradiated culture of human lymphocytes. Murat. Res. 41,321-332. 3. Fenech M. and Morley A. A. (1985) Measurement of micronuclei in lymphocytes. Murat. Res. 147, 29-36.
416
4. Fenech M. and Morley A.A. (1986) Cytokinesis block micronucleus method in human lymphocytes: Effect of in vivo ageing and low dose x-irradiation. Mutat. Res. 161, 193-198. 5. Hungerford D.A. (1965) Leukocytes cultured from small innocula of whole blood and the preparation of metaphase chromosome by treatment with hypotonic KCl. Stain. Tech. 40, 333. 6. Littlefield L. G., Sayer A. M. and Frome E. L. (1989) Comparison of dose-response parameters for radiation-induced acentric fragments and micronuclei observed in cytokinesis-arrested lymphocytes. Mutagenesis 4, 265-270. 7. Miller B. M., Werner T., Weier H. U. and Nusse M. (1992) Analysis of radiation-induced micronuclei by fluorescence in situ hybridization (FISH) simultaneously using telomeric and centromeric DNA probes. Radiat. Res. 131, 177-185. 8. Moorhead P.S., Nowell P.C., Mellman W.J., Battips D. M. and Hungerford D.A. (1960) Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell Res. 20, 613-616.
S . F . D . Paul et al.
9. Nusse M., Kramer J. and Miller B. M. (1992) Factors influencing the DNA content of radiation-induced micronuclei. Int. J. Radiat. Biol. 62, 587-602. 10. Papworth D. G. (1970) Appendix to paper by J. R. K. Savage (1970) Sites of radiation-induced chromosome exchanges. Curr. Top. Radiat. Res. 6, 129-194. 11. Prosser J.S., Moquet J.E., Lloyd D.C. and Edwards A.A. (1988) Radiation induction of micronuclei in human lymphocytes. Mutat. Res. 199, 37-45. 12. Prosser J. S., Lloyd D. C. and Edwards A. A. (1989) A comparison of chromosomal and micronuclear methods for radiation accident dosimetry. Proceedings of the Fourth International Symposium of the Society for Radiological Protection, pp. 133-138. Malvern, United Kingdom. 13. Savage J. R. K. (1988) A comment on the quantitative relationship between micronuclei and chromosomal aberrations. Murat. Res. 207, 33-36. 14. Savage J. R. K. (1989) Acentric chromosomal fragments and micronuclei: The time displacement factor. Mutat. Res. 225, 171-173.
Nuclear Medicine & Biology, Vol. 24, pp. 413-416, 1997 Copyright © 1997 Elsevier Science Inc. ELSEVIER
Analysis of Radiation Dose-Response Curve Obtained with Cytokinesis Block Micronucleus Assay Solomon F. D. Paul, P. Venkatachalam and R. K. Jeevanram* HEALTH A N D SAFETY DIVISION, SAFETY RESEARCH A N D HEALTH PHYSICS GROUP, IND1RA GANDHI CENTRE FOR ATOMIC RESEARCH, KALPAKKAM 603 102, 1NDIA
ABSTRACT. The frequency of micronuclei and acentrics obtained with different doses of 6°Co gamma radiation was examined. When compared to acentric frequency the micronuclei frequency was found to be higher at about 115% for doses below 1 Gy. However, it dropped to about 65% as the dose was increased to 4 Gy. This paper discusses the causes for the reduced frequency of micronuclei at higher doses by taking into account the possibility of their being masked from view by the daughter nuclei in the binucleated cell. NUCL MED BlOt. 24;5:413-416, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Micronuclei, Daughter nuclei, Cytokinesis, Acentric fragments, Radiation dose response
INTRODUCTION Measurement of micronuclei frequency in human lymphocytes is gaining importance as a technique for estimating radiation dose because it is simple and permits easy scoring of micronuclei even by an inexperienced scorer. Micronuclei are formed both from acentric fragments and a whole chromosome (4). Whole chromosomes that have become detached from the mitotic spindle and consequently excluded from incorporation into either daughter nucleus may also form some of the micronuclei. Though the frequency of micronuclei increases with dose, most of the studies have reported that the occurrence of micronuclei is less compared to that of dicentric chromosomes at higher doses--i.e., above about 1.5 Gy (11, 12). It has been suggested by Savage that, at higher doses, micronuclei get included into the main nucleus (13). We felt that a considerable number of micronuclei may be masked by daughter nuclei at higher doses and may not be counted. To study this, (i) dose-response curves for micronuclei and acentric fragments were constructed, (ii) the areas occupied by micronuclei, daughter nuclei, and cytoplasm were measured, and (iii) the sizes of the micronuclei obtained with both high and low dose were measured. The results obtained are analysed in this paper to explain the reduced frequency of micronuclei at high dose. MATERIALS A N D M E T H O D S
Peripheral blood samples (3.5 mL) obtained from normal volunteers were irradiated with different doses ranging between 0.25 and 4.0 Gy with 6°Co gamma radiation at a dose rate of 0.6 Gy/min. The irradiated samples were maintained at 37°C for 1 h to enable repair of chromosomal damages. Blood samples were cultured in F-10 HAM medium (Sigma, Catalogue No. N-6013), supplemented with 20% fetal calf serum and 0.2 mL phytohemagglutinin (PHA-M) (1) (Difco, No. 052856). For the micronucleus assay, Cytochalasin-B (Sigma, No. C-6762) at a final concentration of 3 Ixg/mL was added to each sample at 44 h and the cells harvested after a further incubation of *Author for correspondence. Received 28 October 1995. Accepted 4 February 1997.
28 h (3). The slides for scoring micronuclei were prepared by fixing the cells with 3:1 mixture of methanol and acetic acid after a brief hypotonic treatment. The cell suspensions were dropped onto clear, cold slides from a height of 1 cm and stained with 10% Giemsa solution (pH 6.8) and scored for micronuclei. Cells with two daughter nuclei, surrounded by cytoplasm and cell membrane, were scored for the presence of micronuclei according to the modified criteria of Countryman and Heddle (2)--i.e., material staining as nuclear material having less than half of the diameter of either daughter nucleus and not overlapped by either of the main nuclei. The distribution of micronuclei in binucleated cells was studied. In the case of control and 0.25-Gy dose, the number of cells scored were 2000, whereas for doses between 0.5 and 4.0 Gy, the number of cells scored totaled 1000. The method of examining the distribution is described by Papworth (10). This is done by the standard u test using the formula du-
(N-I)
~d)
Where N is the total number of cells scored, d is the coefficient of dispersion (N - 1 ) ~2/y, Where Y is the mean number of observed aberrations, o2/Y is the relative variance and Var d is the variance o f d given by 2(N - 1) (1 - 1/NY). The diameters of the cytoplasm, the daughter nuclei, and the micronuclei were measured using an ocular micrometer. Using these values the cellular and nuclear areas were calculated by assuming the cytoplasm and nuclei to have circular shapes. The chromosome aberration assay was set up by the modified procedure described by Moorhead et al. (8) and Hungefford (5). The procedure is the same as that reported for micronucleus assay up to the stage of addition of PHA. For metaphase chromosome preparation, colcemid at a final concentration of 0.1 Ixg/mL was added to each sample at 46 h and incubation continued for another 2 h. At the end of culture, cells were given hypotonic treatment with 0.45 % KC1, washed, and then fixed in 3:1 mixture of methanol and acetic acid. The cell suspensions were dropped onto the precooled slides from a height of 10 cm and stained in the same way mentioned for micronucleus assay. These slides were scored for chromosomal aberrations.
414
S . F . D . Paul et al.
TABLE 1. Distribution of Micronuclei After Various Doses of 6°Co G a m m a Radiation Dose
M N Distribution pattern
(Gy)
0
1
0.00 0.25 0.50
1986 1950 946
14 47 51
1.00 2.00 3.00 4.00
888 801 636 526
104 177 274 298
2
3
. 3 i
.
. -2
6 18 64 118
1 2 23 43
il
~rzl y
0.21 + 2.78 +4.05
0.993 1.087 i.I 79
+ 2.74 +2.04 +3.53 +5.68 Average
1.122 1.091 1.158 1.254 1.109
4 .
. ---
---
1 2 2 10
--1 5
u, Dispersionindex; ~2/y, relative variance; MN, micronuclei;Y, mean number of observed aberrations. RESULTS
The distribution of micronuclei obtained with different gamma doses is given in Table 1. The table shows that u, the distribution of micronuclei in binucleated cells, varies between -0.21 and +5.68. The average areas occupied by the micronucleus, both daughter nuclei and the cytoplasm unmasked by daughter nuclei, were 20.37 -+ 21.18 x 10 12 m 2, 1013.86 _+ 134.68 × 10 -i2 m 2, and 1580.83 -+ 432.27 × 10 -12 m 2, respectively. These average areas occupied by the macronuclei and micronuclei were calculated in those samples that were not given radiation dose. In relative terms, these areas were found to have average value of 0.78 -+ 0.81%, 38.77 -+ 5.55%, and 60.45 _+ 13.87%, respectively. Figure 1 compares the acentric and micronuclei frequency obtained at different doses of gamma radiation; it shows that at higher doses the micronuclei frequency is lower than that obtained for acentric frequency. Both curves in Figure 1 were fitted using the linear quadratic equation. The micronuclei frequency shown in Figure 1 accounts only for those micronuclei as observed under microscope without taking into account those buried under daughter nuclei. 1.5
1.5
1.0-
//
/
//
/
/
The uncorrected micronuclei frequency was plotted as a function of acentric frequency, which showed a correlation of 0.99 (Fig. 2, continuous line). However, the angle of the slope of the regression line was 58 °. Because both daughter nuclei occupy about 38% of the cytoplasmic area, a correction factor was introduced to account for the number of micronuclei masked by these daughter nuclei. Micronuclei frequency, corrected for the area masked by daughter nuclei, is given in Figure 3. The figure shows that both curves are overlapping one above the other. This can also be seen from the angle of the slope ( - 4 5 °) of the regression line (dotted) in Figure 2. Both the size and the distribution of micronuclei obtained at 0.5 and 4 Gy were also measured. The results show that at the higher dose, the percentage of micronuclei having larger size is greater than that obtained at low dose (Table 2). DISCUSSION The distribution of micronuclei as per u test indicates overdispersion as six out of seven values showed a value more than _+1.96--/. e., only 5% probability exists that the magnitude of u will be greater than this value when the distribution is a Poisson distribution. The present study clearly shows that, at higher doses, the observed micronuclei frequency is low compared to acentric
ij
,,'/t
1.2
,./"
1.0
/J
, , / " AFTER ,_1 ,_1
---I,
I,.i.I c.)
0.5
0.5.
,._1 ,.,J
"'
n,"
0 0.8 DC
o
o
u.I
uJ
///"I/
//
z
0.0
'
0
I
1
2 DOSE
/
0.0
4
(0/)
o.o 0.0
q .//
I
I
I
0.4
0.8
1.2
MN PER CELL
FIG. 1. Comparison o f acentric and micronuclei frequency as a function of dose. T h e vertical bar on the curves indicates SE obtained at each dose.
FIG. 2. MN/Cell obtained before and after correction plotted as a function of acentric/cell.
Radiation Dose Response Curve for Micronucleus Assay
1.5
415
1.5
//'/ 1.0
1.0
v
m
,,q o
-.I n,,
0.5
0.5
12. o
z
0.0 0
I
I
2
4
0.0
DOSE (~) FIG. 3. Comparison of acentric and corrected micronuclei frequency as a function of dose. The vertical bar on the curves indicates SE obtained at each dose. frequency (Fig. 1). However, micronuclei frequency was somewhat higher compared to acentric frequency at doses less than 1 Gy, and this could be due to interference of high background frequency of micronuclei (1). Similar results have been reported by others (11, 12). Balakrishnan and Bhatt (1) have shown that for a gamma dose of 2 Gy, when 432 acentric fragments were present, the micronucleus yield was only 242 in 1000 cells. The reason for the lower micronuclei frequency at higher dose is not very clear. Savage has suggested the possibility of two or more fragments fusing to form one micronucleus at higher dose (14). The flow cytometric studies carried out by Nusse et al. indicate that the DNA contents in the micronuclei obtained at higher doses have higher DNA content than the one obtained at low dose (9). Using fluorescence in situ hybridisation, Miller et al. (7) have shown micronuclei carrying two or more acentric fragments. Although fusion of acentric fragments may lead to a decrease in micronuclei frequency, a concomitant increase in the size of the micronuclei would be expected. According to Littlefield et al. the size difference is insufficient to explain TABLE 2. Size Distribution of Micronuclei (MN) at Low and High Doses
Dose (Gy)
Number of cells with M N scored
0.50
200
4.00
200
Area range (p.m z) 1-40 41-80 >81 1-40 41-80 >81
(148) (71) (6) (129) (83) (52)
Percentage (%) 65.7 31.6 2.7 48.9 31.4 19.7
The number of micronuclei analysed for different sizes are given in parentheses.
this phenomenon (6). The other hypothesis put forward by Savage is that with increasing radiation dose the micronuclei get included into the main nucleus (13). In the present study, it was found that both daughter nuclei on an average mask 38.8% of the cytoplasmic area. If we consider that the distribution of micronuclei within the cell is uniform, then a portion of about 38% of cytoplasmic area goes unscored. In other words, the number of micronuclei scored are only those that are seen in the cytoplasmic area, unmasked by daughter nuclei--i.e., in 60.5% of the total area. It was reasonable to assume that the number of micronuclei being masked or buried by daughter nuclei would be considerable at higher doses. This suggested that a correction should be applied to get the actual number of micronuclei that are formed. As the micronuclei scored were only in 60% of the area, a correction was applied to get the actual number in 100% of the area. Following the above corrections, the two curves became superimposable, which can be seen in Fig. 3. This was verified by measuring the angle of the slope of the regression line (i) micronuclei per cell as observed when plotted as a function of acentric per cell. (ii) micronuclei per cell as obtained after correction plotted as a function of acentric per cell. Following a nucleus-masked correction, the angle of the slope of the regression line came very close to 45 °, indicating that masking of micronuclei by daughter nuclei is the major factor for the reduced micronuclei frequency at higher doses compared to acentric frequency. Though it has been pointed out by Littlefield et al. (6) that the size difference in micronuclei obtained due to fusion of micronuclei is not adequate to explain the reason for lower micronuclei frequency at higher dose, we made an attempt to study the size of the micronucleus obtained at both low and high doses. Although the size and the distribution of such micronuclei show some variation with respect to dose, the present study indicates that the contribution of fusion of micronuclei in reducing the micronuclei frequency may be insignificant.
CONCLUSION The present study indicates that the observed low micronuclei frequency at higher doses of 6°CO gamma radiation compared to acentric frequency is mainly due to masking of micronuclei by daughter nuclei, and the role of other factors if any could be only of minor consequence.
We wish to express our sincere thanks to Mr. L. V. Krishnan, Director, Safety Research and Health Physics Group, and to Mr. A. R. Sundararajan, Head, Health and Safety Division, for the encouragement given during the course of the study. We also wish to acknowledge Dr. M. Jagadeesan, Director, Bernard Institute of Radiology, Government General Hospital, Madras, for permitting us to use the teletherapy unit to irradiate the blood sample. The authors wish to thank Mrs. Shyamala Devi for scoring acentric aberrations.
References 1. Balakrishnan S. and Bhatt B. (1989) Studies of radiation-induced micronuclei: A rapid method for screening suspected overexposed individuals.Bull. Radiat. Protect. 12, 86-90. 2. Countryman P. 1. and Heddle J. A. (1976) The production of micronuclei from chromosome aberrations in irradiated culture of human lymphocytes. Murat. Res. 41,321-332. 3. Fenech M. and Morley A. A. (1985) Measurement of micronuclei in lymphocytes. Murat. Res. 147, 29-36.
416
4. Fenech M. and Morley A.A. (1986) Cytokinesis block micronucleus method in human lymphocytes: Effect of in vivo ageing and low dose x-irradiation. Mutat. Res. 161, 193-198. 5. Hungerford D.A. (1965) Leukocytes cultured from small innocula of whole blood and the preparation of metaphase chromosome by treatment with hypotonic KCl. Stain. Tech. 40, 333. 6. Littlefield L. G., Sayer A. M. and Frome E. L. (1989) Comparison of dose-response parameters for radiation-induced acentric fragments and micronuclei observed in cytokinesis-arrested lymphocytes. Mutagenesis 4, 265-270. 7. Miller B. M., Werner T., Weier H. U. and Nusse M. (1992) Analysis of radiation-induced micronuclei by fluorescence in situ hybridization (FISH) simultaneously using telomeric and centromeric DNA probes. Radiat. Res. 131, 177-185. 8. Moorhead P.S., Nowell P.C., Mellman W.J., Battips D. M. and Hungerford D.A. (1960) Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell Res. 20, 613-616.
S . F . D . Paul et al.
9. Nusse M., Kramer J. and Miller B. M. (1992) Factors influencing the DNA content of radiation-induced micronuclei. Int. J. Radiat. Biol. 62, 587-602. 10. Papworth D. G. (1970) Appendix to paper by J. R. K. Savage (1970) Sites of radiation-induced chromosome exchanges. Curr. Top. Radiat. Res. 6, 129-194. 11. Prosser J.S., Moquet J.E., Lloyd D.C. and Edwards A.A. (1988) Radiation induction of micronuclei in human lymphocytes. Mutat. Res. 199, 37-45. 12. Prosser J. S., Lloyd D. C. and Edwards A. A. (1989) A comparison of chromosomal and micronuclear methods for radiation accident dosimetry. Proceedings of the Fourth International Symposium of the Society for Radiological Protection, pp. 133-138. Malvern, United Kingdom. 13. Savage J. R. K. (1988) A comment on the quantitative relationship between micronuclei and chromosomal aberrations. Murat. Res. 207, 33-36. 14. Savage J. R. K. (1989) Acentric chromosomal fragments and micronuclei: The time displacement factor. Mutat. Res. 225, 171-173.