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The persistence of aberrations in mice induced by gamma radiation as measured by chromosome painting
Fundamental and Molecular Mechanisms of Mutagenesis Mutation Research 356 (1996) 135-145
The persistence of aberrations in mice induced by gamma radiation as measured by chromosome painting Michelle D. Spruill a,‘, Marilyn J. Ramsey b, Roy R. Swiger b, Joginder Nath a, James D. Tucker b2* a Genetics and Developmental b Biology and Biotechnology
College of Agriculture and Forestry. P.O. Box 6108, West Virginia Uniwrsit?;. Morgantoun. WV 26506.6108, USA
Biology Program, Research
Program,
L-452,
P.O. Box 808. Lawrence 94551-9900,
Received 21 August
Lkermore
Nutional Laboratory,
Lirlermore.
CA
USA
1995; revised 24 October
1995: accepted
16 November
1995
Abstract Fluorescence in situ hybridization, or chromosome painting, has become an invaluable tool in the cytogenetic evaluation of historical or chronic exposure because it can be used to detect stable genetic damage, such as translocations, which persist through cell division, quickly and easily. The recent development of chromosome-specific composite DNA probes for the mouse has allowed the use of chromosome painting in this commonly used animal model. In order to measure the persistence of radiation-induced translocations, C57BL/6 female mice were given a whole body acute dose of 0, 1, 2, 3 or 4 Gy ‘37Cs gamma rays at 8 weeks of age. Metaphase chromosomes from both peripheral blood and bone marrow cells were obtained from four mice in each dose group at 1, 8, 15 and 30 days post-irradiation. Chromosomes 2 and 8 were painted, while the remaining chromosomes were counterstained with propidium iodide. DAPI counterstain was used to differentiate between translocations and dicentrics because it brightly labels the centromeric heterochromatin. The equivalent of 100 cells from each tissue was scored from each mouse. The results show that the percentage of reciprocal translocations, at least at doses of 3 Gy or lower, did not decrease with time in either tissue. In contrast, the frequency of non-reciprocal translocations induced by doses of 3 Gy or lower, remained unchanged in the peripheral blood, but decreased after a week in the bone marrow, then remained constant. An increase in these two types of aberration was observed between 15 and 30 days in the bone marrow and may have been due to clonal expansion. Dicentrics decreased with time in both tissues, almost none remained in the bone marrow after 8 days. These data suggest that reciprocal translocations are persistent and will serve as an effective biodosimeter for radiation exposure. Keywords:
Chromosome
painting;
Persistent
translocation:
Dicentrics;
Mouse model; Gamma radiation
1. Introduction
* Corresponding author. Tel.: I (510) 423-8154: Fax: + 1 (510) 422-2282. ’ Tel.: 1 (304) 293-6256; Fax: + I (304) 293-3740. 0027-5107/96/$15.00 Published SSDI 0027-5107(95)00218-9
by Elsevier Science B.V
The
relationship
between
exposure
to ionizing
radiation and chromosome damage has been well studied with in vitro systems. However, with expo-
sure in vivo. at least in humans, it is frequently difficult to know the dose received because the subjects typically have been victims of events such as the atomic bombs in Japan or nuclear accidents such as the one at Chernobyl. Accurate dose estimates have been difficult because most cytogenetic analyses have focused upon dicentrics. which are inherently unstable with time (Buckton et al.. 1978). The instability of dicentrics has also been supported by studies with mice exposed to radiation (Kligerman et al.. 1990). When evaluating chronic or historical exposures. persistent rearrangements such as chromosome translocations must be measured. Dicentrics are unstable because the two centromeres may be pulled to opposite poles during cell division causing a chromosome break and the loss of essential genetic material, leading to cell death. Similarly. the accompanying acentric fragment may be lost during cell division due to its lack of a centromere. Conversely, reciprocal translocations represent stable genetic damage because each resulting chromosome has only one centromere and there is no apparent loss of genetic material. Due to their stability, translocations are not only important for biodosimetry, but are implicated in cancer and a variety of genetic diseases and thus represent a health risk (Jorde et al.. 1994). The analysis of stable aberrations has generally required chromosome banding. which is laborious and expensive to perform. Fluorescence in situ hybridization with chromosome-specific composite painting’. has been DNA probes, ‘chromosome shown to be an accurate method for detecting translocations quickly and easily (Lucas et al., 1989. 1992a; Natarajan et al., 1992; Bauchinger et al.. 1993; Tucker et al.. 1993, 1995a). The advent of chromosome painting has allowed the detection of stable genetic damage in the atomic bomb survivors (Lucas et al.. 1992a) and in radiation workers at Chernobyl (Tucker et al.. unpublished data). The mouse is an excellent model for estimating human risk from adverse environmental exposure. However. the mouse karyotype consists entirely of acrocentric chromosomes that are similarly sized, which previously made cytogenetic studies of stable damage difficult to perform. The recent development of chromosome-specific composite DNA probes for the mouse (Breneman et al., 1993. 1995: Rabbitts et
al., 1995) has greatly simplified translocation detection in this animal. Distinguishing between translocations and dicentrics is facilitated by counterstaining the slides with the DNA binding dye DAPI, which labels the centromeric heterochromatin brightly. Although translocations are generally stable, breaks at critical loci may lead to cell selection. Clonal expansion of cells bearing stable translocations is also possible as the stem cells carrying them replenish the cell population. This study is the first to assess the stability of radiation-induced aberrations by chromosome painting in a rodent model. Three main types of aberrations were examined: reciprocal translocations. non-reciprocal translocations and dicentrics. The persistence of each type of aberration was measured in both non-dividing (peripheral blood) and rapidly dividing (bone marrow) tissues following exposure to 0, I. 2, 3 or 4 Gy 17’Cs.
2. Materials
and methods
Eight-week-old C57BL/6 female mice were given a whole-body acute dose of 0, 1. 2, 3 or 4 Gy 13’Cs gamma rays at a dose rate of approximately 80 cGy/min. Peripheral blood and bone marrow were taken from four mice in each dose group at I, 8, IS and 30 days post-irradiation. Peripheral blood was obtained via cardiac puncture using a 2 1g 1” needle and a 3 ml syringe containing 0.1 ml heparin. The blood was placed in a wash medium [RPM1 1640 supplemented with 1% heparin and 1% penicillinstreptomycin (100 U and 100 kg/ml. respectively)]. Bone marrow was flushed from each femur with I.5 ml fetal bovine serum using a 3 ml syringe with a 2lg 1” needle. 2.2. Cell cultiwe and slide preparation Following two rinses in wash media. peripheral blood was cultured for 36 h at 37°C. 5%’ CO1 in a medium consisting of RPM1 1640 supplemented with 12.5% fetal bovine serum. 1% heparin, 3 mM I~glutamine. I % penicillin-streptomycin (100 U and 100 kg/ml, respectively) and 60 pg/ml lipopolysaccharide. Colcemid (0.1 kg/ml) was added to the
M.D. Spruill er al. / Mutation Research
peripheral blood cultures after 24 h. Colcemid (0.2 kg/ml) was added to the bone marrow/FBS solution for a short-term culture of 2 h at 37°C to obtain metaphase cells. Cells from each tissue were swollen in 75 mM KC1 for 1 h at 37°C. Cell pellets were fixed three times in methanol/acetic acid (3 : 1) and dropped onto warm slides that had been lightly sprayed with water to aid in spreading. After air drying for 24 h, the slides were stored at - 20°C in an atmosphere of N, and in the presence of CaSO, desiccant. 2.3. Chromosome
painting
Biotinylated composite DNA probes specific for mouse chromosomes 2 and 8, developed by the method of Breneman et al. (19931, were used for in situ hybridization. Probe DNA was added to the hybridization mix [50% formamide, 2 X SSC (1 X SSC is 0.15 M NaCl, 0.015 M sodium citrate), 10% dextran sulfate, 1.5 p,l herring sperm DNA (1.0 kg/l& and 2.0 kg mouse Cot 1 DNA (1.0 p,g/p,l) (Gibco-BRL)] for a final volume of 15 t~,l and denatured at 70°C for 5 min. Mouse metaphase chromosomes on slides were denatured at 70°C for 5 min in 70% formamide, 2 X SSC, pH 7.0 and then dehydrated through a series of 5 min ethanol washes (70%, 85%, 100%). The denatured probe solution was applied to the denatured metaphase chromosomes, covered with ethanol-cleaned glass coverslips (22 mm*) and sealed with rubber cement. After incubation for about 48 h at 37”C, the unbound probe was removed by three 5 min washes in 50% formamide, 2 X SSC, pH 7.0 (45”C), and 5 min washes in 2 X SSC (45”C), 2 X SSC with 1% Nonidet NP-40 (45”C), and in sodium phosphate (PN) buffer (0.1 M NaPO,, pH 8.0, 0.1% Nonidet NP-40) at room temperature. After a 5 min blocking step at room temperature with PNM (3% powdered non-fat milk in PN buffer), avidin-FITC (fluorescein isothiocyanate; 1 : 400 in PNM) was applied to the metaphase chromosomes for 30 min at room temperature. Excess avidin-FITC was removed with three 5 min washes in PN buffer at room temperature. A layer of biotinylated anti-avidin (1 : 100 in PNM, Vector Laboratories) was applied for 30 min at room temperature followed by three more 5 min PN washes also at room temperature. A final layer of avidin-FITC was
356 (19961 135- 145
137
added for 30 min and the excess fluorophore was again removed by three 5 min PN washes. Slides were then rinsed with water and dried with a gentle stream of N, gas. The metaphase chromosomes were then counterstained with propidium iodide (1.0 (DAPI) ug/ml) and 4’, 6-d iamidino-2-phenylindole (0.5 (*g/ml) and mounted in an antifade solution (Johnson and Nogueira Araujo, 1981). The slides were refrigerated until they could be scored. 2.4. Slide scoring A Zeiss Axioskop or Axiophot microscope was used to observe metaphase spreads. Photographs were taken of all abnormal cells using Kodak Ektachrome 400 Elite color slide film. The criteria used in scoring were: (1) the metaphase cell must appear complete and must contain the material derived from four painted chromosomes; and (2) only those aberrations involving painted chromosomes were counted in the results. Aberrations were categorized by the PAINT system (Tucker et al., 1995b). In this system, each abnormal chromosome is given one or more symbols to represent its particular rearrangement(s) (i.e., t = translocation, die = dicentric, or ace = acentric fragment). Following this symbol are letters inside parentheses that describe the painting pattern. The letters ‘A’ and ‘a’ represent an unpainted portion of a chromosome, while the letters ‘B’ and ‘b’ indicate a portion of a chromosome which is painted. A capital letter denotes a portion of the chromosome that contains a centromere, while a lowercase letter is indicative of a segment that does not contain a centromere. All slides were coded prior to scoring to minimize observer bias. Cell equivalents were calculated by multiplying the total number of metaphase cells scored by the fraction of exchanges detectable by painting. Since chromosome painting detects only those exchanges that result in bicolored chromosomes, the fraction of detectable exchanges is equal to 2pq; where p is the fraction of the genome that is painted and q is the unpainted fraction (Lucas et al., 1989). For mouse chromosomes 2 and 8, p = 0.115 (Disteche et al., 1981>, so 2pq = 0.205. Therefore, approximately 5 metaphase cells must be scored to obtain the same amount of information as in one G-banded cell. One hundred cell equivalents (ap-
proximately 500 actual metaphase cells) were scored from each tissue for each mouse where available. At the 4 Gy dose, no metaphase cells were present in either tissue at 1 day post-exposure. The bone marrow yielded the desired number of cell equivalents at all other doses and times. At least 85 cell equivalents were obtained from the blood of all mice at 8 days post-exposure and by 15 days. 100 cell equivalents were easily obtained from every mouse. 2.5. Statistical aim1ysi.s Since the variances were non-uniform, the data were square root-transformed. ANOVA was performed on data from each aberration type and tissue to test for a significant effect of dose, time and the interaction between these variables. Because dose was significant in every case, a one-way ANOVA was then performed on each independent dose from each tissue to test only the effect of time. When time since exposure was found to be significant ( p < 0.05), a regression analysis was performed to find the slope of that line. A t-test was used to evaluate the similarity of the mean frequency of translocations and dicentrics induced at day 1. An cy level of 0.05 was used in all analyses. Raw data will be made available by the authors upon request.
3. Results and discussion 3.1. Aberration
detection
We were able to detect and differentiate between different types of genetic damage by chromosome painting. Fig. 1 shows a mouse metaphase cell in which chromosomes 2 and 8 are clearly painted. A reciprocal translocation is evident by the two bicol-
ored chromosomes. The pericentric heterochromatic DNA is not painted, but is labeled brightly with DAPI (Fig. lb). Fig. 2 gives an example of a non-reciprocal translocation, t(Ab) (Tucker et al.. 1995b), in which part of a painted chromosome is translocated onto an unpainted chromosome, but a reciprocal color junction is not seen. Fig. 3 illustrates a dicentric with its corresponding acentric fragment. The two centromeres of the dicentric and the lack of a centromere in the fragment are apparent when the same cell is viewed by DAPI excitation and emission (Fig. 3b). 3.2. Induction and persistence
of aberrations
There was no significant difference between the mean number of translocations and dicentrics observed in the peripheral blood at day 1 after exposure. Translocations and dicentrics are expected to be induced at equal frequencies (Evans, 1962), but this has not always been observed in human lymphocytes (Lucas et al., 1989; Natarajan et al.. 1992: Bauchinger et al., 1993; Tucker et al., 1993, 1995a; Schmid et al., 1992) or in mouse lymphocytes (Breneman et al., 1993). Our data. and that of Boei et al. (1994). support the hypothesis of equal induction of translocations and dicentrics in mouse lymphocytes. The induction of these aberrations could not be compared accurately in the bone marrow, since at least one mitotic division had occurred by the first sampling time of 24 h after irradiation. The disparity between our data and that of Breneman et al. (1993) could be due to our longer incubation in the presence of Colcemid, which was timed to ensure that no second division cells would be present. The persistence of aberrations in peripheral blood lymphocytes is shown in Fig. 4. The frequency of reciprocal translocations, at least in mice exposed to doses of 3 Gy or lower, did not decrease after 30
Fig. 1. A mouse metaphase cell bearing a reciprocal translocation [t(Ab) and t(Ba)] as viewed (a) with fluorescein excitation and emission. and (b) with DAPI excitation and emission. The arrows indicate points of breakage. Fig. 2. A mouse metaphase cell bearing a non-reciprocal and emission. The arrows indicate points of breakage.
tranalocatmn
[t(Ab)]
and propidium
iodide
a\ I. viewed with fluorescein and propidium iodide excitation
Fig. 3. A mouse metaphse cell bearing a dicentric and corresponding acentrlc fragment [dic(AB) and acecab)] as viewed (a) with tluorescein and propidium iodide excitation and emission, and (b) with DAPI excitation and emission. The arrows indicate points of breakage.
M.D. Spruill et al. /Mutation
Research 356 (19961 135-145
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Time (Days)
0
i0
+
OGy
--+
1 Gy
+
7Gy
20 Ttme
so -,C
--c -0.-
0
-=F ---
(Days)
10
20
OGy ?Gy
30
Time (Days)
Fig. 4. The persistence of (a) reciprocal translocations, (b) non-rrciprocal translocation and Cc) dicentrics in peripheral blood lymphocytes. Vertical bars represent the standard errors. Some of the points are offset for clarity.
days post-exposure (Fig. 4a). There was a significant linear decrease in reciprocal translocations over time in mice exposed to 4 Gy gamma radiation ( p = 0.013, slope = -0.047 aberrations per 100 cells/ day). Time elapsed after exposure was also significant at the 3 Gy dose (p = 0.0361: however, a linear
model did not fit the data. Similar results were obtained for the persistence of non-reciprocal translocations (Fig. 4b). Mice exposed to the 4 Gy dose again showed a significant linear decrease in aberrations over time ( p = 0.047, slope = -0.080 aberrations per 100 cells/day). Dicentrics decreased almost 50% in the peripheral blood after 2 weeks (Fig. 4~). Mice exposed to the 3 and 4 Gy doses showed linear decreases in dicentrics over time with slopes of - 0.097 and - 0.220 aberrations per 100 cells/day, respectively. While the overall number of dicentrics in peripheral blood lymphocytes in our study is lower than that of Kligerman et al. (19901, the decay curve for dicentrics induced at 3 Gy is very similar to theirs through 30 days. Past 30 days. the decrease in dicentrics is expected to be non-linear, approaching baseline as seen in other studies (Buckton et al.. 1978; Kligerman et al., 19901. In their study, Kligerman et al. used phytohemagglutinin (PHA) to stimulate T lymphocytes in the peripheral blood, while we used lipopolysaccharide (LPS) as a mitogen which stimulates B cell division. Their higher frequency of dicentrics may suggest an increased sensitivity of T cells to radiation; however, no difference in the sensitivity of T cells and splenic B cells was observed in their study. The persistence of aberrations in bone marrow cells is shown in Fig. 5. Reciprocal translocations did not decrease in frequency after 30 days (Fig. 5a). regardless of the dose. Instead, there was an increase between I5 and 30 days post-irradiation, possibly due to clonal expansion. This increase is significant in mice exposed to the 3 Gy dose (p = 0.0081. However, the increase at 4 Gy was found to be non-significant. The mean number of aberrations at 30 days was elevated by one very high outlier. Non-reciprocal translocations (Fig. 5b) decreased significantly after a week and then remained stable out to 30 days in mice exposed to doses of 3 Gy or lower. Again, some clonal expansion may have occurred between 15 and 30 days at the 3 and 4 Gy doses. The increase in non-reciprocal translocations in mice exposed to 4 Gy was found to be non-significant due to one very high outlier, which was not the same mouse that produced the outlier in the reciprocal translocation data. As is expected of unstable events in rapidly dividing cells, the frequency
M.D. Spruill et al./Mutation
-+-
A
so
OGy
1
40
0
P
-O-
1Gy
t-
2Gy
+
3Gv
I
20
10
30
Time (Days)
B
30
--t
1
-1
“II
0
OGy
-+-
1Gy
-A-
2Gy
--(I
3Gy
-.-
4Gy
Y
30
20
10 Time (Days)
25,c
-f-
zo-
15-
OGy
+
1Gy
-A-
2Gy
--El--
3Gy
--.--
4Gy
10-
5-k \
si0: 0
20
10
30
Time (Days)
Fig. 5. The persistence of (A) reciprocal translocations, (B) nonreciprocal translocations, and (Cl dicentrics in bone marrow cells. Vertical bars represent the standard errors. Some of the points are offset for clarity.
of dicentrics in the bone marrow dropped quickly with almost none remaining after a week (Fig. 5~). A study of atomic bomb survivors (Awa et al., 1978) suggested that radiation-induced translocations were stable in vivo because they were detected decades after exposure. However, there was no initial cytogenetic measurement to confirm that their
Research 356 (1996) 135-145
141
frequency had remained unchanged since induction. In a study of one radiation worker accidentally exposed to tritium, it was found that the frequency of radiation-induced translocations in peripheral blood lymphocytes measured by chromosome painting 6 years post exposure was not significantly different from the dicentric frequency measured within 39 days of the accident (Lucas et al., 1992b). This result and the unpublished data from radiation workers at Chernobyl obtained by Tucker et al. suggest that translocations are persistent in human peripheral blood lymphocytes. However, these results do not allow us to make a quantitative determination of the fraction of translocations that may be lost with time. Our data show that translocations are persistent in both mouse peripheral blood and bone marrow cells exposed to doses of 3 Gy and below. The significant decrease in both reciprocal and non-reciprocal translocations in the peripheral blood of mice exposed to 4 Gy could be the result of a shortened life span of heavily damaged cells, thus giving the appearance of a decrease in aberrations. As the dose of radiation increases, the number of aberrations induced per cell also increases. At doses of 1 and 2 Gy the average number of aberrations per abnormal cell was close to 1, while the 3 and 4 Gy doses induced about 1.5 aberrations per abnormal cell. If a cell carrying a reciprocal translocation also carries an unstable event, then the life expectancy of that cell will obviously be shortened regardless of the stability of the translocation. At 4 Gy, the average number of aberrations per abnormal cell had dropped to 1.25 after 30 days, showing that heavily damaged cells were being eliminated. Additionally, some of the damage caused by exposures of 4 Gy will affect single genes and will not be observed at the chromosomal level. After significant amounts of cell death, the restoration of the lymphocyte population from undamaged stem cells will result in the dilution of abnormal cells (Buckton et al., 1978). The difference in the persistence of reciprocal and non-reciprocal translocations in the bone marrow suggests that non-reciprocal translocations have a reduced stability compared to reciprocal translocations. While classical cytogenetics theory states that all chromosome exchanges are reciprocal, Tucker and Senft (1994) showed that non-reciprocal translocations occurred too often to be explained by random
chromosome loss during slide preparation. Our data suggest that these non-reciprocal translocations are a biologically distinct class of aberrations. Bone marrow cells, unlike peripheral blood cells. are continuously dividing. Acute exposure, as with this study. will therefore produce chromatid as well as chromosome damage. Following mitosis, chromatid exchanges (‘quadriradials’) will give rise to non-reciprocal translocations, which are expected to be unstable due to the loss of material. In addition. some apparently non-reciprocal translocations may, in fact, be reciprocal events in which one of the color junctions is too subtle to be observed. This may account for at least a fraction of the stable aberrations. The increase in reciprocal and non-reciprocal translocations with time in the bone marrow cells of mice exposed to 3 and 4 Gy gamma radiation can be explained by the substantial cell killing at high doses of radiation (Till and McCulloch, 1961) and the subsequent ability of the hemopoietic stem cells to repopulate (Erslev and Weiss, 1983). It is by random chance that one of the stem cells carries an exchange involving either chromosome 2 or 8 and after clonal expansion, the cells carrying this event constitute a significant portion of the cell population. If banding techniques were used, the increase in variance shown by the large standard error bars on the mean number of translocations at the 4 Gy dose 30 days post-exposure (Fig. 5a and 5b) might not be seen. This is because banding theoretically detects all aberrations, not only those between certain chromosomes. If exchanges are distributed randomly throughout the genome. clonal expansion of cells containing stable aberrations involving any of the 18 other pairs of mouse chromosomes would be equally likely. The clonal expansion of normal stem cells should keep the mean frequency of translocations unchanged.
3.3. Relationship between centromeric mutirz crnd centromere actil,it>
heterochro-
While DAPI is a useful tool for differentiating translocations and dicentrics. two types of aberrations were encountered in which DAPI gave misleading results with regard to centromere location. The first. shown in Fig. 6, is thought to be a reciprocal translocation that resulted from a break in the long arm of a painted chromosome and a break within the centromeric heterochromatin of an unpainted chromosome. This cell, which appears to contain a bicolored dicentric and a painted acentric fragment (both of which would be unstable). was part of an eight cell clone observed in the bone marrow of a mouse exposed to 4 Gy. The clonal origin was verified by examining the photographs taken of the cells and discovering the presence of another aberration, similar to the one just described. involving an unpainted chromosome in all eight cells. Therefore, this clone appears to contain only stable aberrations, each chromosome possessing only one functional centromere. Additional evidence that the aberration is not a dicentric is that the chromatids are slightly separated below the primary constriction and there is not a second constriction within the lower brightly DAPIstained region. The second aberration in which DAPI can yield ambiguous results, shown in Fig. 7. is thought to be a dicentric that resulted from breaks within the centromeric heterochromatin of both a painted and an unpainted chromosome. The result appears to be a large metacentric chromosome and a circle of brightly staining heterochromatin. Based on the information gained from the previous cell (Fig. 6), we believe that the circle contains the centromeres of both chromosomes and that the large ‘metacentric chromosome’ is actually an acentric fragment. Similar to the
Fig. 6. A reciprocal translocation (arrows) that occunrd ah a result of a break within the centromeric heterochromatin as viewed (a) with fluorescein and propidium iodide excitation and emission. (b) with DAPI excitation and emission and Cc) schematically. where the circles represent the centromeric heterochromatin. The arrowhead indicates the aberration which confirmed clonal expansion. Fig. 7. A dicentric (arrow) and corresponding acentrlc fragment (arrowhead)
that occurred as a result of breaks within the centromeric
heterochromatin as viewed (a) with fluorescein and propidium iodide excitation and emission. (b) with DAPI excitation and emission and Cc) schematically. where the circles represent the centromeric heterochromatin.
M.D. Spruill et al./Mutation Research 356 (19961 135-145
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DNA Replication I
A -I
Fig. 6
a.
Fig. 7
b.
I44
M.D. Spruill et d.,‘Mut(~ttrwt Resrrtrch _{Sb f/996)
aberration in Fig. 6, the chromatids of this fragment are slightly separated and there is no constriction within the brightly DAPI-stained heterochromatin. These results show that DAPI may not always be a true marker for the functional centromere. There is no evidence that mouse minor satellite DNA plays a direct role in centromere function, although this is the area to which kinetochore proteins, specifically CENP-B, are thought to bind (Willard, 1990). Patterns of major and minor satellite DNA distribution are not conserved in the genus Mus (Wang et al.. 1990). This lack of conservation between species could explain our observation that this satellite DNA is not necessary for, or indicative of, kinetochore activity. The results of this study show that radiation-induced reciprocal translocations in mice, at least those induced at doses of 3 Gy and below, are in fact stable and will persist, not only in the peripheral blood but also in the rapidly dividing bone marrow cells. Fast, reliable and precise detection of these persistent rearrangements makes translocation analysis an efficient biodosimeter for radiation exposure.
Acknowledgements We thank Barry Brunckhorst, Todd Coble and Joe Serpa for their technical assistance. We also thank Dave Partsch for his help in statistical analyses. This work was performed in part under the auspices of the US DOE by the Lawrence Livermore National Laboratory under contract No. W-7405-ENG-48 and West Virginia University. Published with the approval of the Director of West Virginia Agriculture and Forestry Experiment Station as Scientific Paper Number 25 14.
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a pancentromeric DNA probe, Int. .J. Radiat. Biol., 64, 17% 184. Boei, J.J.W.A., AS. Balajee, P. DeBoer, W. Rem, J.A. Atrn. L.H.F. Mullenders and A.T. Natarajan (1994) Construction of mouse chromosome-specific DNA libraries and their use for the detection of X-ray-induced aberrations, Int. J. Radiat. Biol., 65, 583-590. Breneman, J.W., M.J. Ramsey, D.A. Lee, G.G. Eveleth. J.I.. Minkler and J.D. Tucker (1993) The development of chromosome-specific composite DNA probes for the mouse and their application to chromosome painting, Chromosoma. 102, SO I 598. Breneman, J.W.. R.R. Swiger, M.J. Ramsey, J.L. Minkler. G.G. Eveleth, R.A. Langlois and I.D. Tucker (1995) The development of painting probes for dual color and multiple chromesome analysis in the mouse, Cytogenet. Cell Genet.. 68. 197-202. Buckton. K.E.. G.E. Hamilton. L. Paton and A.O. Langlands ( 1978) Chromosome aberrations in irradiated ankylosing spondylitis patients. in: H.J. Evans and D.C. Lloyd (Eds.), Mutagen-Induced Chromosome Damage in Man, Edinburgh University Press, Edinburgh, pp. 142-150. Disteche. C.M.. A.V. Carrano. L.K. Ashworth. K. Burkhart-Shultr and S.A. Latt (1981) Flow sorting of the mouse Cattanach X chromosome, T(X;7) I Ct. in an active or inactive state. Cytogenet. Cell Genet.. 29, I89- 197. Erslev. A.J. and L. Weiss (1983) Structure and function of the marrow, in: W.J. Williams. E. Beutler. A.J. Erslev and M.A. Lichtman (Ed\.). Hematology. McGraw-Hill, New York. pp. 75-83. Evans, H.J. (1962) Chromosome aberrations induced by iomzing radiations, Int. Rev. Cytol., 13, 221-230. Johnson. G.D. and G.M. de C. Nogueira Araujo (1981) A himpie method of reducing the fading of immunofluorescence during microscopy, J. Immunol. Methods, 43, 349-3.50. Jorde, L.B., J.C. Carey, R.L. White (1994) Medical Genetic\. Mosby-Year Book, St. Louis, pp. 117-120, 126-127. 142 and 178. Kligerman. A.D., E.C. Halpetin, G.L. Erexson and G. Honore (1990) The persistence of lymphocytes with dicentric chromosomes following whole-body X irradiation of mice. Radiat. Res., 124, 22-27. Lucas. J.N., T. Tenjin, T. Straume, D. Pinkel, D. Moore II. M. Litt and J.W. Gray (1989) Rapid human chromosome aberration analysis using fluorescence in situ hybridization. Int. J. Radiat. Biol.. 56. 3%44 and 201 (Erratum). Lucas. J.N.. A. Awa. T. Straume, M. Poggensee, Y. Kodama, M. Nakano. K. Ohtaki, H.U. Weirr. D. Pinkel. J. Gray and G. Littlefield (1992a) Rapid translocation frequency analysis in humans decades after exposure to ionizing radiation. Int. J. Radiat. Biol., 62, 53-63. Lucas. J.N.. M. Poggensee and T. Straume (1992b) The persi\tence of chromosome translocations in a radiation worker accidentally exposed to tritium. Cytogenet. Cell Genet.. 60. 255-756. Natarajan. A.T., R.C. Vyas, F. Darroudi and S. Vermeulen ( IYYI) Frequencies of X-ray-induced chromosome translocations in
M.D. Spud1 et al./Mutation human peripheral lymphocytes as detected by in situ hybridization using chromosome-specific DNA libraries, Int. J. Radiat. Biol.. 61, 199-203. Rabbitts, P.. H. Impey, A. Heppell-Patton, C. Langford, C. Tease. N. Lowe, D. Bailey, M. Ferguson-Smith and N. Carter (1995) Chromosome specific paints from a high resolution flow karyotype of the mouse, Nature Genet., 9, 369-375. Schmid, E., H. Zitzelberger. H. Braselman, J.W. Gray and M. Bauchinger (19921 Radiation-induced chromosome aberrations analyzed by fluorescence in situ hybridization with triple combination of composite whole chromosome-specific DNA probes, bit. J. Radiat. Biol.. 62, 673-678. Till, J.E. and E.A. McCulloch (1961) A direct measurement of the radiation sensitivity of normal mouse bone marrow. Radiat. Res., 14. 213-222. Tucker, J.D. and J.R. Senft (1994) Analysis of naturally occurring and radiation-induced breakpoint locations in human chromosomes I. 2 and 4. Radiat. Res., 139, 31-36.
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Tucker, J.D., M.J. Ramsey. D.A. Lee and J.L. Minkler (1993) Validation of chromosome painting as a biodosimeter in human peripheral lymphocytes following acute exposure to ionizing radiation in vitro, Int. J. Radiat. Biol., 64, 27-37. Tucker, J.D., D.A. Lee and D.H. Moore II (1995a) Validation of chromosome painting. II. A detailed analysis of aberrations following high doses of ionizing radiation in vitro, Int. J. Radiat. Biol., 67, 19-28. Tucker, J.D., W.F. Morgan. A.A. Awa. M. Bauchinger. D. Blakey. M.N. Comforth, L.G. Littlefield, A.T. Natarajan and C. Shasserre ( 1995b) A proposed system for scoring structural aberrations detected by chromosome painting. Cytogenet. Cell Genet., 68, 211-221. Willard, H.F. (1990) Centromeres of mammalian chromosomes, Trends Genet., 6. 410-416. Wong, A.K.C., F.G. Biddle and J.B. Rattner (19901 The chromosomal distribution of the major and minor satellite is not conserved in the genus Mus, Chromosoma, 99, 190-195.
The persistence of aberrations in mice induced by gamma radiation as measured by chromosome painting Michelle D. Spruill a,‘, Marilyn J. Ramsey b, Roy R. Swiger b, Joginder Nath a, James D. Tucker b2* a Genetics and Developmental b Biology and Biotechnology
College of Agriculture and Forestry. P.O. Box 6108, West Virginia Uniwrsit?;. Morgantoun. WV 26506.6108, USA
Biology Program, Research
Program,
L-452,
P.O. Box 808. Lawrence 94551-9900,
Received 21 August
Lkermore
Nutional Laboratory,
Lirlermore.
CA
USA
1995; revised 24 October
1995: accepted
16 November
1995
Abstract Fluorescence in situ hybridization, or chromosome painting, has become an invaluable tool in the cytogenetic evaluation of historical or chronic exposure because it can be used to detect stable genetic damage, such as translocations, which persist through cell division, quickly and easily. The recent development of chromosome-specific composite DNA probes for the mouse has allowed the use of chromosome painting in this commonly used animal model. In order to measure the persistence of radiation-induced translocations, C57BL/6 female mice were given a whole body acute dose of 0, 1, 2, 3 or 4 Gy ‘37Cs gamma rays at 8 weeks of age. Metaphase chromosomes from both peripheral blood and bone marrow cells were obtained from four mice in each dose group at 1, 8, 15 and 30 days post-irradiation. Chromosomes 2 and 8 were painted, while the remaining chromosomes were counterstained with propidium iodide. DAPI counterstain was used to differentiate between translocations and dicentrics because it brightly labels the centromeric heterochromatin. The equivalent of 100 cells from each tissue was scored from each mouse. The results show that the percentage of reciprocal translocations, at least at doses of 3 Gy or lower, did not decrease with time in either tissue. In contrast, the frequency of non-reciprocal translocations induced by doses of 3 Gy or lower, remained unchanged in the peripheral blood, but decreased after a week in the bone marrow, then remained constant. An increase in these two types of aberration was observed between 15 and 30 days in the bone marrow and may have been due to clonal expansion. Dicentrics decreased with time in both tissues, almost none remained in the bone marrow after 8 days. These data suggest that reciprocal translocations are persistent and will serve as an effective biodosimeter for radiation exposure. Keywords:
Chromosome
painting;
Persistent
translocation:
Dicentrics;
Mouse model; Gamma radiation
1. Introduction
* Corresponding author. Tel.: I (510) 423-8154: Fax: + 1 (510) 422-2282. ’ Tel.: 1 (304) 293-6256; Fax: + I (304) 293-3740. 0027-5107/96/$15.00 Published SSDI 0027-5107(95)00218-9
by Elsevier Science B.V
The
relationship
between
exposure
to ionizing
radiation and chromosome damage has been well studied with in vitro systems. However, with expo-
sure in vivo. at least in humans, it is frequently difficult to know the dose received because the subjects typically have been victims of events such as the atomic bombs in Japan or nuclear accidents such as the one at Chernobyl. Accurate dose estimates have been difficult because most cytogenetic analyses have focused upon dicentrics. which are inherently unstable with time (Buckton et al.. 1978). The instability of dicentrics has also been supported by studies with mice exposed to radiation (Kligerman et al.. 1990). When evaluating chronic or historical exposures. persistent rearrangements such as chromosome translocations must be measured. Dicentrics are unstable because the two centromeres may be pulled to opposite poles during cell division causing a chromosome break and the loss of essential genetic material, leading to cell death. Similarly. the accompanying acentric fragment may be lost during cell division due to its lack of a centromere. Conversely, reciprocal translocations represent stable genetic damage because each resulting chromosome has only one centromere and there is no apparent loss of genetic material. Due to their stability, translocations are not only important for biodosimetry, but are implicated in cancer and a variety of genetic diseases and thus represent a health risk (Jorde et al.. 1994). The analysis of stable aberrations has generally required chromosome banding. which is laborious and expensive to perform. Fluorescence in situ hybridization with chromosome-specific composite painting’. has been DNA probes, ‘chromosome shown to be an accurate method for detecting translocations quickly and easily (Lucas et al., 1989. 1992a; Natarajan et al., 1992; Bauchinger et al.. 1993; Tucker et al.. 1993, 1995a). The advent of chromosome painting has allowed the detection of stable genetic damage in the atomic bomb survivors (Lucas et al.. 1992a) and in radiation workers at Chernobyl (Tucker et al.. unpublished data). The mouse is an excellent model for estimating human risk from adverse environmental exposure. However. the mouse karyotype consists entirely of acrocentric chromosomes that are similarly sized, which previously made cytogenetic studies of stable damage difficult to perform. The recent development of chromosome-specific composite DNA probes for the mouse (Breneman et al., 1993. 1995: Rabbitts et
al., 1995) has greatly simplified translocation detection in this animal. Distinguishing between translocations and dicentrics is facilitated by counterstaining the slides with the DNA binding dye DAPI, which labels the centromeric heterochromatin brightly. Although translocations are generally stable, breaks at critical loci may lead to cell selection. Clonal expansion of cells bearing stable translocations is also possible as the stem cells carrying them replenish the cell population. This study is the first to assess the stability of radiation-induced aberrations by chromosome painting in a rodent model. Three main types of aberrations were examined: reciprocal translocations. non-reciprocal translocations and dicentrics. The persistence of each type of aberration was measured in both non-dividing (peripheral blood) and rapidly dividing (bone marrow) tissues following exposure to 0, I. 2, 3 or 4 Gy 17’Cs.
2. Materials
and methods
Eight-week-old C57BL/6 female mice were given a whole-body acute dose of 0, 1. 2, 3 or 4 Gy 13’Cs gamma rays at a dose rate of approximately 80 cGy/min. Peripheral blood and bone marrow were taken from four mice in each dose group at I, 8, IS and 30 days post-irradiation. Peripheral blood was obtained via cardiac puncture using a 2 1g 1” needle and a 3 ml syringe containing 0.1 ml heparin. The blood was placed in a wash medium [RPM1 1640 supplemented with 1% heparin and 1% penicillinstreptomycin (100 U and 100 kg/ml. respectively)]. Bone marrow was flushed from each femur with I.5 ml fetal bovine serum using a 3 ml syringe with a 2lg 1” needle. 2.2. Cell cultiwe and slide preparation Following two rinses in wash media. peripheral blood was cultured for 36 h at 37°C. 5%’ CO1 in a medium consisting of RPM1 1640 supplemented with 12.5% fetal bovine serum. 1% heparin, 3 mM I~glutamine. I % penicillin-streptomycin (100 U and 100 kg/ml, respectively) and 60 pg/ml lipopolysaccharide. Colcemid (0.1 kg/ml) was added to the
M.D. Spruill er al. / Mutation Research
peripheral blood cultures after 24 h. Colcemid (0.2 kg/ml) was added to the bone marrow/FBS solution for a short-term culture of 2 h at 37°C to obtain metaphase cells. Cells from each tissue were swollen in 75 mM KC1 for 1 h at 37°C. Cell pellets were fixed three times in methanol/acetic acid (3 : 1) and dropped onto warm slides that had been lightly sprayed with water to aid in spreading. After air drying for 24 h, the slides were stored at - 20°C in an atmosphere of N, and in the presence of CaSO, desiccant. 2.3. Chromosome
painting
Biotinylated composite DNA probes specific for mouse chromosomes 2 and 8, developed by the method of Breneman et al. (19931, were used for in situ hybridization. Probe DNA was added to the hybridization mix [50% formamide, 2 X SSC (1 X SSC is 0.15 M NaCl, 0.015 M sodium citrate), 10% dextran sulfate, 1.5 p,l herring sperm DNA (1.0 kg/l& and 2.0 kg mouse Cot 1 DNA (1.0 p,g/p,l) (Gibco-BRL)] for a final volume of 15 t~,l and denatured at 70°C for 5 min. Mouse metaphase chromosomes on slides were denatured at 70°C for 5 min in 70% formamide, 2 X SSC, pH 7.0 and then dehydrated through a series of 5 min ethanol washes (70%, 85%, 100%). The denatured probe solution was applied to the denatured metaphase chromosomes, covered with ethanol-cleaned glass coverslips (22 mm*) and sealed with rubber cement. After incubation for about 48 h at 37”C, the unbound probe was removed by three 5 min washes in 50% formamide, 2 X SSC, pH 7.0 (45”C), and 5 min washes in 2 X SSC (45”C), 2 X SSC with 1% Nonidet NP-40 (45”C), and in sodium phosphate (PN) buffer (0.1 M NaPO,, pH 8.0, 0.1% Nonidet NP-40) at room temperature. After a 5 min blocking step at room temperature with PNM (3% powdered non-fat milk in PN buffer), avidin-FITC (fluorescein isothiocyanate; 1 : 400 in PNM) was applied to the metaphase chromosomes for 30 min at room temperature. Excess avidin-FITC was removed with three 5 min washes in PN buffer at room temperature. A layer of biotinylated anti-avidin (1 : 100 in PNM, Vector Laboratories) was applied for 30 min at room temperature followed by three more 5 min PN washes also at room temperature. A final layer of avidin-FITC was
356 (19961 135- 145
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added for 30 min and the excess fluorophore was again removed by three 5 min PN washes. Slides were then rinsed with water and dried with a gentle stream of N, gas. The metaphase chromosomes were then counterstained with propidium iodide (1.0 (DAPI) ug/ml) and 4’, 6-d iamidino-2-phenylindole (0.5 (*g/ml) and mounted in an antifade solution (Johnson and Nogueira Araujo, 1981). The slides were refrigerated until they could be scored. 2.4. Slide scoring A Zeiss Axioskop or Axiophot microscope was used to observe metaphase spreads. Photographs were taken of all abnormal cells using Kodak Ektachrome 400 Elite color slide film. The criteria used in scoring were: (1) the metaphase cell must appear complete and must contain the material derived from four painted chromosomes; and (2) only those aberrations involving painted chromosomes were counted in the results. Aberrations were categorized by the PAINT system (Tucker et al., 1995b). In this system, each abnormal chromosome is given one or more symbols to represent its particular rearrangement(s) (i.e., t = translocation, die = dicentric, or ace = acentric fragment). Following this symbol are letters inside parentheses that describe the painting pattern. The letters ‘A’ and ‘a’ represent an unpainted portion of a chromosome, while the letters ‘B’ and ‘b’ indicate a portion of a chromosome which is painted. A capital letter denotes a portion of the chromosome that contains a centromere, while a lowercase letter is indicative of a segment that does not contain a centromere. All slides were coded prior to scoring to minimize observer bias. Cell equivalents were calculated by multiplying the total number of metaphase cells scored by the fraction of exchanges detectable by painting. Since chromosome painting detects only those exchanges that result in bicolored chromosomes, the fraction of detectable exchanges is equal to 2pq; where p is the fraction of the genome that is painted and q is the unpainted fraction (Lucas et al., 1989). For mouse chromosomes 2 and 8, p = 0.115 (Disteche et al., 1981>, so 2pq = 0.205. Therefore, approximately 5 metaphase cells must be scored to obtain the same amount of information as in one G-banded cell. One hundred cell equivalents (ap-
proximately 500 actual metaphase cells) were scored from each tissue for each mouse where available. At the 4 Gy dose, no metaphase cells were present in either tissue at 1 day post-exposure. The bone marrow yielded the desired number of cell equivalents at all other doses and times. At least 85 cell equivalents were obtained from the blood of all mice at 8 days post-exposure and by 15 days. 100 cell equivalents were easily obtained from every mouse. 2.5. Statistical aim1ysi.s Since the variances were non-uniform, the data were square root-transformed. ANOVA was performed on data from each aberration type and tissue to test for a significant effect of dose, time and the interaction between these variables. Because dose was significant in every case, a one-way ANOVA was then performed on each independent dose from each tissue to test only the effect of time. When time since exposure was found to be significant ( p < 0.05), a regression analysis was performed to find the slope of that line. A t-test was used to evaluate the similarity of the mean frequency of translocations and dicentrics induced at day 1. An cy level of 0.05 was used in all analyses. Raw data will be made available by the authors upon request.
3. Results and discussion 3.1. Aberration
detection
We were able to detect and differentiate between different types of genetic damage by chromosome painting. Fig. 1 shows a mouse metaphase cell in which chromosomes 2 and 8 are clearly painted. A reciprocal translocation is evident by the two bicol-
ored chromosomes. The pericentric heterochromatic DNA is not painted, but is labeled brightly with DAPI (Fig. lb). Fig. 2 gives an example of a non-reciprocal translocation, t(Ab) (Tucker et al.. 1995b), in which part of a painted chromosome is translocated onto an unpainted chromosome, but a reciprocal color junction is not seen. Fig. 3 illustrates a dicentric with its corresponding acentric fragment. The two centromeres of the dicentric and the lack of a centromere in the fragment are apparent when the same cell is viewed by DAPI excitation and emission (Fig. 3b). 3.2. Induction and persistence
of aberrations
There was no significant difference between the mean number of translocations and dicentrics observed in the peripheral blood at day 1 after exposure. Translocations and dicentrics are expected to be induced at equal frequencies (Evans, 1962), but this has not always been observed in human lymphocytes (Lucas et al., 1989; Natarajan et al.. 1992: Bauchinger et al., 1993; Tucker et al., 1993, 1995a; Schmid et al., 1992) or in mouse lymphocytes (Breneman et al., 1993). Our data. and that of Boei et al. (1994). support the hypothesis of equal induction of translocations and dicentrics in mouse lymphocytes. The induction of these aberrations could not be compared accurately in the bone marrow, since at least one mitotic division had occurred by the first sampling time of 24 h after irradiation. The disparity between our data and that of Breneman et al. (1993) could be due to our longer incubation in the presence of Colcemid, which was timed to ensure that no second division cells would be present. The persistence of aberrations in peripheral blood lymphocytes is shown in Fig. 4. The frequency of reciprocal translocations, at least in mice exposed to doses of 3 Gy or lower, did not decrease after 30
Fig. 1. A mouse metaphase cell bearing a reciprocal translocation [t(Ab) and t(Ba)] as viewed (a) with fluorescein excitation and emission. and (b) with DAPI excitation and emission. The arrows indicate points of breakage. Fig. 2. A mouse metaphase cell bearing a non-reciprocal and emission. The arrows indicate points of breakage.
tranalocatmn
[t(Ab)]
and propidium
iodide
a\ I. viewed with fluorescein and propidium iodide excitation
Fig. 3. A mouse metaphse cell bearing a dicentric and corresponding acentrlc fragment [dic(AB) and acecab)] as viewed (a) with tluorescein and propidium iodide excitation and emission, and (b) with DAPI excitation and emission. The arrows indicate points of breakage.
M.D. Spruill et al. /Mutation
Research 356 (19961 135-145
139
Time (Days)
0
i0
+
OGy
--+
1 Gy
+
7Gy
20 Ttme
so -,C
--c -0.-
0
-=F ---
(Days)
10
20
OGy ?Gy
30
Time (Days)
Fig. 4. The persistence of (a) reciprocal translocations, (b) non-rrciprocal translocation and Cc) dicentrics in peripheral blood lymphocytes. Vertical bars represent the standard errors. Some of the points are offset for clarity.
days post-exposure (Fig. 4a). There was a significant linear decrease in reciprocal translocations over time in mice exposed to 4 Gy gamma radiation ( p = 0.013, slope = -0.047 aberrations per 100 cells/ day). Time elapsed after exposure was also significant at the 3 Gy dose (p = 0.0361: however, a linear
model did not fit the data. Similar results were obtained for the persistence of non-reciprocal translocations (Fig. 4b). Mice exposed to the 4 Gy dose again showed a significant linear decrease in aberrations over time ( p = 0.047, slope = -0.080 aberrations per 100 cells/day). Dicentrics decreased almost 50% in the peripheral blood after 2 weeks (Fig. 4~). Mice exposed to the 3 and 4 Gy doses showed linear decreases in dicentrics over time with slopes of - 0.097 and - 0.220 aberrations per 100 cells/day, respectively. While the overall number of dicentrics in peripheral blood lymphocytes in our study is lower than that of Kligerman et al. (19901, the decay curve for dicentrics induced at 3 Gy is very similar to theirs through 30 days. Past 30 days. the decrease in dicentrics is expected to be non-linear, approaching baseline as seen in other studies (Buckton et al.. 1978; Kligerman et al., 19901. In their study, Kligerman et al. used phytohemagglutinin (PHA) to stimulate T lymphocytes in the peripheral blood, while we used lipopolysaccharide (LPS) as a mitogen which stimulates B cell division. Their higher frequency of dicentrics may suggest an increased sensitivity of T cells to radiation; however, no difference in the sensitivity of T cells and splenic B cells was observed in their study. The persistence of aberrations in bone marrow cells is shown in Fig. 5. Reciprocal translocations did not decrease in frequency after 30 days (Fig. 5a). regardless of the dose. Instead, there was an increase between I5 and 30 days post-irradiation, possibly due to clonal expansion. This increase is significant in mice exposed to the 3 Gy dose (p = 0.0081. However, the increase at 4 Gy was found to be non-significant. The mean number of aberrations at 30 days was elevated by one very high outlier. Non-reciprocal translocations (Fig. 5b) decreased significantly after a week and then remained stable out to 30 days in mice exposed to doses of 3 Gy or lower. Again, some clonal expansion may have occurred between 15 and 30 days at the 3 and 4 Gy doses. The increase in non-reciprocal translocations in mice exposed to 4 Gy was found to be non-significant due to one very high outlier, which was not the same mouse that produced the outlier in the reciprocal translocation data. As is expected of unstable events in rapidly dividing cells, the frequency
M.D. Spruill et al./Mutation
-+-
A
so
OGy
1
40
0
P
-O-
1Gy
t-
2Gy
+
3Gv
I
20
10
30
Time (Days)
B
30
--t
1
-1
“II
0
OGy
-+-
1Gy
-A-
2Gy
--(I
3Gy
-.-
4Gy
Y
30
20
10 Time (Days)
25,c
-f-
zo-
15-
OGy
+
1Gy
-A-
2Gy
--El--
3Gy
--.--
4Gy
10-
5-k \
si0: 0
20
10
30
Time (Days)
Fig. 5. The persistence of (A) reciprocal translocations, (B) nonreciprocal translocations, and (Cl dicentrics in bone marrow cells. Vertical bars represent the standard errors. Some of the points are offset for clarity.
of dicentrics in the bone marrow dropped quickly with almost none remaining after a week (Fig. 5~). A study of atomic bomb survivors (Awa et al., 1978) suggested that radiation-induced translocations were stable in vivo because they were detected decades after exposure. However, there was no initial cytogenetic measurement to confirm that their
Research 356 (1996) 135-145
141
frequency had remained unchanged since induction. In a study of one radiation worker accidentally exposed to tritium, it was found that the frequency of radiation-induced translocations in peripheral blood lymphocytes measured by chromosome painting 6 years post exposure was not significantly different from the dicentric frequency measured within 39 days of the accident (Lucas et al., 1992b). This result and the unpublished data from radiation workers at Chernobyl obtained by Tucker et al. suggest that translocations are persistent in human peripheral blood lymphocytes. However, these results do not allow us to make a quantitative determination of the fraction of translocations that may be lost with time. Our data show that translocations are persistent in both mouse peripheral blood and bone marrow cells exposed to doses of 3 Gy and below. The significant decrease in both reciprocal and non-reciprocal translocations in the peripheral blood of mice exposed to 4 Gy could be the result of a shortened life span of heavily damaged cells, thus giving the appearance of a decrease in aberrations. As the dose of radiation increases, the number of aberrations induced per cell also increases. At doses of 1 and 2 Gy the average number of aberrations per abnormal cell was close to 1, while the 3 and 4 Gy doses induced about 1.5 aberrations per abnormal cell. If a cell carrying a reciprocal translocation also carries an unstable event, then the life expectancy of that cell will obviously be shortened regardless of the stability of the translocation. At 4 Gy, the average number of aberrations per abnormal cell had dropped to 1.25 after 30 days, showing that heavily damaged cells were being eliminated. Additionally, some of the damage caused by exposures of 4 Gy will affect single genes and will not be observed at the chromosomal level. After significant amounts of cell death, the restoration of the lymphocyte population from undamaged stem cells will result in the dilution of abnormal cells (Buckton et al., 1978). The difference in the persistence of reciprocal and non-reciprocal translocations in the bone marrow suggests that non-reciprocal translocations have a reduced stability compared to reciprocal translocations. While classical cytogenetics theory states that all chromosome exchanges are reciprocal, Tucker and Senft (1994) showed that non-reciprocal translocations occurred too often to be explained by random
chromosome loss during slide preparation. Our data suggest that these non-reciprocal translocations are a biologically distinct class of aberrations. Bone marrow cells, unlike peripheral blood cells. are continuously dividing. Acute exposure, as with this study. will therefore produce chromatid as well as chromosome damage. Following mitosis, chromatid exchanges (‘quadriradials’) will give rise to non-reciprocal translocations, which are expected to be unstable due to the loss of material. In addition. some apparently non-reciprocal translocations may, in fact, be reciprocal events in which one of the color junctions is too subtle to be observed. This may account for at least a fraction of the stable aberrations. The increase in reciprocal and non-reciprocal translocations with time in the bone marrow cells of mice exposed to 3 and 4 Gy gamma radiation can be explained by the substantial cell killing at high doses of radiation (Till and McCulloch, 1961) and the subsequent ability of the hemopoietic stem cells to repopulate (Erslev and Weiss, 1983). It is by random chance that one of the stem cells carries an exchange involving either chromosome 2 or 8 and after clonal expansion, the cells carrying this event constitute a significant portion of the cell population. If banding techniques were used, the increase in variance shown by the large standard error bars on the mean number of translocations at the 4 Gy dose 30 days post-exposure (Fig. 5a and 5b) might not be seen. This is because banding theoretically detects all aberrations, not only those between certain chromosomes. If exchanges are distributed randomly throughout the genome. clonal expansion of cells containing stable aberrations involving any of the 18 other pairs of mouse chromosomes would be equally likely. The clonal expansion of normal stem cells should keep the mean frequency of translocations unchanged.
3.3. Relationship between centromeric mutirz crnd centromere actil,it>
heterochro-
While DAPI is a useful tool for differentiating translocations and dicentrics. two types of aberrations were encountered in which DAPI gave misleading results with regard to centromere location. The first. shown in Fig. 6, is thought to be a reciprocal translocation that resulted from a break in the long arm of a painted chromosome and a break within the centromeric heterochromatin of an unpainted chromosome. This cell, which appears to contain a bicolored dicentric and a painted acentric fragment (both of which would be unstable). was part of an eight cell clone observed in the bone marrow of a mouse exposed to 4 Gy. The clonal origin was verified by examining the photographs taken of the cells and discovering the presence of another aberration, similar to the one just described. involving an unpainted chromosome in all eight cells. Therefore, this clone appears to contain only stable aberrations, each chromosome possessing only one functional centromere. Additional evidence that the aberration is not a dicentric is that the chromatids are slightly separated below the primary constriction and there is not a second constriction within the lower brightly DAPIstained region. The second aberration in which DAPI can yield ambiguous results, shown in Fig. 7. is thought to be a dicentric that resulted from breaks within the centromeric heterochromatin of both a painted and an unpainted chromosome. The result appears to be a large metacentric chromosome and a circle of brightly staining heterochromatin. Based on the information gained from the previous cell (Fig. 6), we believe that the circle contains the centromeres of both chromosomes and that the large ‘metacentric chromosome’ is actually an acentric fragment. Similar to the
Fig. 6. A reciprocal translocation (arrows) that occunrd ah a result of a break within the centromeric heterochromatin as viewed (a) with fluorescein and propidium iodide excitation and emission. (b) with DAPI excitation and emission and Cc) schematically. where the circles represent the centromeric heterochromatin. The arrowhead indicates the aberration which confirmed clonal expansion. Fig. 7. A dicentric (arrow) and corresponding acentrlc fragment (arrowhead)
that occurred as a result of breaks within the centromeric
heterochromatin as viewed (a) with fluorescein and propidium iodide excitation and emission. (b) with DAPI excitation and emission and Cc) schematically. where the circles represent the centromeric heterochromatin.
M.D. Spruill et al./Mutation Research 356 (19961 135-145
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DNA Replication I
A -I
Fig. 6
a.
Fig. 7
b.
I44
M.D. Spruill et d.,‘Mut(~ttrwt Resrrtrch _{Sb f/996)
aberration in Fig. 6, the chromatids of this fragment are slightly separated and there is no constriction within the brightly DAPI-stained heterochromatin. These results show that DAPI may not always be a true marker for the functional centromere. There is no evidence that mouse minor satellite DNA plays a direct role in centromere function, although this is the area to which kinetochore proteins, specifically CENP-B, are thought to bind (Willard, 1990). Patterns of major and minor satellite DNA distribution are not conserved in the genus Mus (Wang et al.. 1990). This lack of conservation between species could explain our observation that this satellite DNA is not necessary for, or indicative of, kinetochore activity. The results of this study show that radiation-induced reciprocal translocations in mice, at least those induced at doses of 3 Gy and below, are in fact stable and will persist, not only in the peripheral blood but also in the rapidly dividing bone marrow cells. Fast, reliable and precise detection of these persistent rearrangements makes translocation analysis an efficient biodosimeter for radiation exposure.
Acknowledgements We thank Barry Brunckhorst, Todd Coble and Joe Serpa for their technical assistance. We also thank Dave Partsch for his help in statistical analyses. This work was performed in part under the auspices of the US DOE by the Lawrence Livermore National Laboratory under contract No. W-7405-ENG-48 and West Virginia University. Published with the approval of the Director of West Virginia Agriculture and Forestry Experiment Station as Scientific Paper Number 25 14.
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