Mutagenesis vol.18 no.2 pp.187–194, 2003
Nucleoplasmic bridges are a sensitive measure of chromosome
rearrangement in the cytokinesis-block micronucleus assay
Philip Thomas, Keizo Umegaki1 and Michael Fenech2
CSIRO Health Sciences and Nutrition, PO Box 10041 Adelaide BC,
Adelaide, SA 5000, Australia and 1National Institute of Health and
Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan
We have performed experiments using the WIL2-NS
human B lymphoblastoid cell line and primary human
lymphocytes to: (i) determine the importance of including
measurements of nucleoplasmic bridges (NPB) in the cytokinesis-block micronucleus (CBMN) assay; (ii) provide
evidence that NPB originate from dicentric chromosomes
and centric ring chromosomes. In addition, we describe
theoretical models that explain how dicentric chromosomes
and centric ring chromosomes may result in the formation
of NPB at anaphase. The results with WIL2-NS showed
that it was possible to distinguish genotoxic effects induced
by different oxidizing agents in terms of the NPB/micronucleus frequency ratio. The results with lymphocytes
indicated a strong correlation: (i) between NPB, centric ring
chromosomes and dicentric chromosomes in metaphases
(r > 0.93, P < 0.0001); (ii) between micronuclei (MNi),
acentric chromosome fragments and acentric ring chromosomes (r > 0.93, P < 0.0001). The dose–response curves
with γ-rays were very similar for NPB, ring chromosomes
and dicentric chromosomes, as were the dose–response
curves for MNi, acentric rings and fragments. However,
not all acentric chromosomes and dicentric chromosomes/
centric rings were converted to MNi and NPB respectively,
depending on the dose of radiation. Preliminary data, using
FISH, suggest that NPB often represent DNA from a
structural rearrangement involving only one or two homologous chromosomes. The results from this study validate
the inclusion of NPB in the CBMN assay which provides a
valuable measure of chromosome breakage/rearrangement
that was otherwise not available in the micronucleus assay.
The CBMN assay allows NPB measurement to be achieved
reliably because inhibition of cytokinesis prevents the loss
of NPB that would otherwise occur if cells were allowed
to divide.
Introduction
The cytokinesis-block micronucleus (CBMN) assay is an
established cytogenetic method for the measurement of chromosome breakage and loss in nucleated cells (Fenech, 2000).
By blocking cytokinesis using cytochalasin-B (cyt-B) it is
possible to specifically score micronuclei (MNi) in once
divided cells, which are recognized by their appearance as
binucleated cells (BNCs). It is practical to use molecular
probes to identify centromeres or kinetochores within MNi and
thus distinguish between MNi containing acentric chromosome
fragments and MNi containing whole chromosomes. The
use of chromosome-specific centromeric probes allows non2To
disjunction events to be readily scored in BNCs even when
no MNi are induced. The above measures together with
cell division kinetic data obtained by scoring the ratios of
mononucleated cells, BNCs, multinucleated cells, necrotic
cells and apoptotic cells makes the assay useful to obtain
comprehensive toxicological data. More recently, nuclear buds
have also been validated in this system as a potential marker
of gene amplification which correlates positively with micronucleus (MN) frequency (Crott et al., 2001; Fenech and
Crott, 2002).
The CBMN assay is particularly effective in the study of
chromosome damage induced by ionizing radiation both for
the purpose of biological dosimetry after accidental exposure
(Fenech et al., 1997; Thierens et al., 1999; Touil et al., 2002)
and in determining the intrinsic sensitivity of cells to this
genotoxin (Scott et al., 1999; Rothfuss et al., 2000). The
dicentric chromosome index is probably the best established
biomarker of exposure to and effect of ionizing radiation and
has the added advantage that background frequencies are
usually very low (IAEA, 2001). In contrast, baseline MN
frequencies are usually relatively high, they increase with age
and are increased in females relative to males (Bonassi et al.,
2001), making the estimation of exposure somewhat more
difficult when baseline frequencies are not known beforehand.
However, this problem can be minimized and sensitivity
increased if centromere-negative (CEN–) and centromerepositive (CEN⫹) MNi are scored separately because a large
proportion of spontaneous MNi are CEN⫹ and radiationinduced MNi following acute exposures are mainly due to
lagging of acentric fragments and therefore CEN– (Kryscio
et al., 2001; Touil et al., 2002).
The CBMN assay in its current format does not provide
information on chromosomal structural rearrangement, an
event considered important in carcinogenesis and in biological
dosimetry of radiation exposure (IAEA, 2001). Therefore, we
have examined whether it is possible to use the CBMN assay
to obtain a measure of dicentric chromosome and chromosome
ring formation because, theoretically, these abnormal chromosomes could produce a nucleoplasmic bridge (NPB) between
the nuclei of BNCs during anaphase. Figure 1A–D illustrates
the formation of dicentric chromatids, dicentric chromosomes
and dicentric ring chromosomes and the resulting NPB and
MNi between the nuclei of a BNC. Dicentric ring chromosomes
may occur due to sister chromatid exchange (SCE) between
sister ring chromatids producing either one large dicentric ring
following one SCE or two concatenated ring chromosomes
following two SCEs (Figure 1C) (Therman and Susman, 1993;
Gardner and Sutherland, 1996). There are a number of ways
that the dicentric chromatids, chromosomes or rings can
distribute themselves at anaphase depending on whether both
centromeres travel to the same pole of the cell or whether they
travel to opposite poles of the cells (Figure 1B–D). It is in the
latter case that one may expect a NPB to occur, which in
whom correspondence should be addressed. Tel: ⫹618 8303 8880: Fax: ⫹618 8303 8899: Email: [email protected]
© UK Environmental Mutagen Society/Oxford University Press 2003 all rights reserved
187
P.Thomas, K.Umegaki and M.Fenech
Fig. 1. (A) Initiating events in the formation of NPB. (Top) Double-strand DNA break (DSB) is induced in one chromosome but remains unrepaired until
after replication. Misjoining of the broken ends after replication leads to the formation of a dicentric chromatid and an acentric fragment. (Middle) Two DSBs
on either side of the centromere of one chromosome are induced. The broken ends are misrepaired producing a ring chromatid and two acentric fragments
which are subsequently replicated. (Bottom) DSBs are induced in two homologous or non-homologous chromosomes. Misjoining of the broken ends leads to
the formation of a dicentric chromatid and acentric fragment which are subsequently replicated. (B) (Top) Centromeres of a dicentric chromatid are pulled to
opposite poles of the cell at anaphase to form a NPB and the acentric chromatid fragment lags behind to form a MN. (Bottom) Centromeres of a dicentric
chromatid are both pulled to the same pole of the cell and no NPB is formed, however, the lagging acentric chromatid fragment results in the formation of a
MN. (C) (Top) The ring chromatids are segregated normally but the acentric fragments lag at anaphase to form two MNi. (Middle) The two ring chromatids,
after completing one SCE, are transformed into one large dicentric ring chromatid which leads to the formation of a NPB when the centromeres are pulled to
the opposite poles of the cell at anaphase. The accompanying acentric fragments lag at anaphase to form two micronuclei. (Bottom) The two ring chromatids,
after completing two SCEs, become concatenated, which leads to the formation of a NPB when the centromeres are pulled to the opposite poles of the cell at
anaphase. The accompanying acentric fragments lag at anaphase to form two MNi. (D) (Top) Centromeres of dicentric chromatids travel to the same pole of
the cell and no NPB are formed. (Middle) Centromeres of one of the dicentric chromatids are pulled to opposite poles of the cell leading to the formation of
one NPB. (Bottom) Centromeres of both dicentric chromatids are pulled to opposite poles of the cell leading to the formation of two NPBs. In each of the
above cases one MN is produced from the lagging acentric chromosome fragment accompanying the dicentric chromosome.
theory should be accompanied by a lagging acentric fragment
which may become a MN. The formation of NPBs between
daughter nuclei has been noted previously in studies on
oxidative stress and folate deficiency (Umegaki and Fenech,
2000; Crott et al., 2001). These studies showed strong positive
correlations of NPB with MN and nuclear buds. However, the
origin of NPB was not properly validated by comparison
188
with dicentric and ring chromosome formation nor were the
mechanisms of formation carefully deduced. The CBMN assay
is ideal to study the NPB phenomenon because inhibition of
cytokinesis with cyt-B should prevent the breakage of the
NPB, which occurs when the cell divides, allowing NPB
events to be accumulated in BNCs for scoring.
In this paper we provide evidence for the proposed mechan-
Nucleoplasmic bridges measure chromosome rearrangement
isms of NPB formation. Because the measurement of NPBs
has the potential to improve the chromosome damage profile
measured in the CBMN assay, we have studied the relationship
between NPB and MNi in WIL2-NS cells exposed to hydrogen
peroxide, superoxide and activated neutrophils and the dose–
response relationships between MNi, NPB and acentric, dicentric and ring chromosomes in human lymphocytes exposed to
γ-rays. The results obtained suggest that the NPB measure is
easily incorporated in the CBMN assay, providing a sensitive
biomarker with a lower background level than the MN index.
In addition, NPBs indicate chromosome breakage/translocation
events without the need for kinetochore/centromere staining
of MNi and allow a more refined picture of chromosome
damage to be derived from the CBMN assay.
Materials and methods
Blood collection, lymphocyte isolation, cell irradiation and cell culture
The collection of blood for the purpose of this study was approved by the
Human Ethics Committee of CSIRO Health Sciences and Nutrition. The
experiments were performed using lymphocytes from three donors (two
females aged 41 and 42 years and one male aged 38 years). An aliquot of
40 ml of whole blood was collected per subject into a lithium–heparin
Vacutainer tube. Lymphocytes were isolated from the heparinized blood
samples by diluting 1:2 with Hanks balanced salt solution (HBSS) (Sigma,
Australia) and using Ficoll Hypaque gradients (Pharmacia Biotech, Uppsala,
Sweden). The isolated lymphocytes were then washed twice in HBSS before
estimating cell concentration using a Zb model Coulter counter. Cells were
added at a cell density of 106 cells/ml to RPMI 1640 (Trace, Australia)
containing 10% foetal calf serum (FCS) (Trace, Australia) and 2 mM Lglutamine (Sigma, Australia). Samples were irradiated using a CIS BIO
International IBL 437 blood cell irradiator comprising three stationary 137Cs
doubly encapsulated radiation sources emitting γ-radiation at 5.91 Gy/min.
Duplicate cultures were set up for each radiation dose of 0, 1, 2 and 4 Gy,
for both the CBMN assay and for 48 and 72 h chromosome metaphase
analysis. Post-irradiation, 10 µl/ml phytohemagglutinin (PHA) (Murex Biotech,
UK) was added to each culture and then incubated at 37°C. The unirradiated
control culture was treated in the same way as the irradiated samples.
Fig. 2. Correlation between NPB and MNi in WIL2-NS cultures exposed to
increasing doses of hydrogen peroxide or superoxide or activated
neutrophils (data derived from experiments described in Umegaki and
Fenech, 2000).
CBMN assay in lymphocytes
Forty-four hours after PHA stimulation, 4.5 µg/ml cyt-B (Sigma, Australia)
was added to the cultures to accumulate cells that had completed one nuclear
division at the binucleate stage. Cells were harvested between 68 and 72 h
after PHA stimulation. Dimethyl sulphoxide (5%) (Sigma, Australia) was
added to the cell suspension to minimize cellular clumping and to better
define cellular boundaries. Slides were prepared using a cytocentrifuge
(Shandon Southern Products, UK). Slides were air dried for 10 min before
being fixed and stained using Diff Quik (Lab Aids, Australia). Each slide was
scored for the following: number of MNi, micronucleated cells and NPBs
found in 1000 BNCs and number of BNCs containing both NPBs and MNi
in 1000 BNCs. Scoring criteria for selection of BNCs, MN and NPB were as
described previously (Fenech, 2000). However, in the case of NPB scoring
we were careful to include only those BNCs in which the main nuclei were
clearly separated because it is difficult to determine the presence of a NPB
when nuclei are touching. Slides were coded and scored by the same,
single person.
Chromosome metaphase analysis
For the purpose of comparison with the CBMN assay, in which cells are
blocked between 44 and 72 h of culture, we decided to examine metaphases
at both 48 and 72 h post-PHA stimulation. At 48 or 72 h, 10 µl of colchicine
(10 mg/ml) (Sigma, Australia) was added to each of the cultures for 60 min.
Cultures were spun down and treated with 0.075 M KCl (Sigma, Australia)
and allowed to stand for 20 min at 37°C. Cells were fixed using cold 3:1
ethanol/acetic acid (Univar, Australia/Ajax Chemicals, Australia). Cells and
slides were chilled prior to slide preparation to aid in chromosomal spread.
Cells were dropped onto dry slides and allowed to air dry prior to being
stained with Giemsa (Sigma, Australia). Each slide was scored for the
following: total number of dicentric (Dic), acentric (Ace) and ring chromosomes and the number of cells containing both dicentric and acentric fragments
in 100 good quality metaphases. Scoring criteria were as described previously
(IAEA, 2001). Slides were coded and scored by the same, single person who
scored the lymphocyte MN slides.
Fig. 3. γ-Ray dose–response characteristics for MNi and acentric
chromosome fragments. Data represent the mean ⫾ SEM (n ⫽ 3).
C-banding
C-banding was performed to demonstrate the constitutive heterochromatin of
the chromosome set, which is useful in determining the presence or position
of pericentromeric regions in rearranged chromosomes (Sumner, 1972). This
methodology was used to determine whether structural rings were CEN⫹ or
CEN–. Slides were placed in 0.2 N HCl (Sigma, Australia) for 1 h and then
rinsed in distilled water. Slides were then rinsed in freshly prepared saturated
barium hydroxide (Sigma, Australia) solution for 2 min. The slides were
further rinsed in distilled water and in 2⫻ SSC (0.3 M sodium chloride,
0.03 M sodium citrate) at 62°C for 1 h prior to staining with 5% Giemsa for
5 min. Slides were coded and scored by the same, single person who scored
the lymphocyte MN slides.
Fluorescence in situ hybridization
Whole chromosome paint for chromosome 4 was used to paint NPBs in the
4 Gy CBMN culture slides. Paints were from Cambio (UK) and used as per
the manufacturer’s instructions. Throughout the study all images were taken
using a Spot Jr cooled CCD camera (Diagnostic Instruments Inc.) and Image
Pro software linked to an E600 Nikon fluorescence microscope and triple
band filter.
CBMN assay of WIL2-NS cultures
The methods for culture, CBMN assay and treatment with hydrogen peroxide,
superoxide and activated neutrophils were as described previously (Umegaki
and Fenech, 2000). Briefly, cells were cultured in RPMI 1640 medium
(containing 5% FCS) at 0.3⫻106 cells/ml. Twenty-four hours later cells were
exposed to hydrogen peroxide, superoxide or activated neutrophils in HBSS.
The cells were then washed in HBSS and cultured at a cell density of 0.5⫻106
189
P.Thomas, K.Umegaki and M.Fenech
Table I. Dose–response characteristics of chromosomal damage measurements in lymphocytes exposed to γ-rays
γ-Irradiation
Biomarker
MNi per 1000 BNCs
NPB per 1000 BNCs
Ace per 1000 mets (48 h)
Dic per 1000 mets (48 h)
Rings per 1000 mets (48 h)
Ace per 1000 mets (72 h)
Dic per 1000 mets (72 h)
Rings per 1000 mets (72 h)
P (ANOVA)
0 Gy
1 Gy
11.7 ⫾ 5.7
5.3 ⫾ 0.9
6.7 ⫾ 3.2
0.0 ⫾ 0.0
0.0 ⫾ 0.0
9.9 ⫾ 4.9
0.0 ⫾ 0.0
0.0 ⫾ 0.0
55.0
47.0
50.0
16.6
20.0
56.6
36.7
53.3
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
6.0
11.5
5.4
4.1
4.9
6.2
8.9
13.1
2 Gy
4 Gy
131.7 ⫾ 22.2
127.0 ⫾ 12.7
289.8 ⫾ 48.9
103.4 ⫾ 10.3
159.9 ⫾ 15.9
166.6 ⫾ 28.1
99.9 ⫾ 9.9
73.3 ⫾ 7.3
680.0
331.3
1027.0
516.5
356.5
913.2
399.9
403.2
⫾
⫾
⫾
⫾
⫾
⫾
⫾
⫾
15.5
22.7
23.4
35.4
24.4
20.8
27.4
27.6
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
⬍0.0001
Results represent the mean and SEM for cultures from three different subjects.
Ace, acentric fragments; BNCs, binucleated cells; Dic, dicentric chromosomes; mets, metaphases; MNi, micronuclei; NPB, nucleoplasmic bridges.
Table II. Correlation matrix of chromosome damage biomarkers in the classical metaphase and CBMN assays
Biomarker
NPB per 1000
BNCs
MNi per 1000 BNCs
0.96
NPB per 1000 BNCs
Ace per 1000 mets (48 h)
Dic per 1000 mets (48 h)
Rings per 1000 mets (48 h)
Ace per 1000 mets (72 h)
Dic per 1000 mets (72 h)
Ace per 1000
mets (48 h)
Dic per 1000
mets (48 h)
Rings per 1000
mets (48 h)
Ace per 1000
mets (72 h)
Dic per 1000
mets (72 h)
Rings per 1000
mets (72 h)
0.99
0.98
0.99
0.98
0.98
0.94
0.99
0.97
0.96
0.99
0.97
0.99
0.99
0.94
0.99
0.99
0.98
0.99
0.97
0.98
0.99
0.97
0.97
0.99
0.94
0.98
0.99
Values shown are the correlation coefficient r. All P values were ⬍0.0001.
Ace, acentric fragments; BNCs, binucleated cells; Dic, dicentric chromosomes; mets, metaphases; MNi, micronuclei; NPB, nucleoplasmic bridges.
and hydrogen peroxide at physiologically relevant doses
(Umegaki and Fenech, 2000). In a further analysis of these
data we now show that: (i) there is a strong positive correlation
between NPB frequency and MN frequency (r2 ⫽ 0.43–0.86,
P ⬍ 0.0001); (ii) the NPB frequency/MN frequency ratio is
5.4 times greater after exposure to activated neutrophils relative
to superoxide or hydrogen peroxide, as deduced from the slope
of the relationship between NPB and MN frequency (Figure
2). Superoxide and hydrogen peroxide were indistinguishable
from each other with regard to this ratio.
Fig. 4. γ-Ray dose–response characteristics for NPB, dicentric chromosomes
and ring chromosomes. Data represent the mean ⫾ SEM (n ⫽ 3).
cells/ml in RPMI 1640 medium containing 10% FCS and 4.5 µg/ml cyt-B.
Cells were harvested 42 h later using a cytocentrifuge and slides scored for
the frequency of MNi and NPB in BNCs as described previously (Fenech,
2000). Slides were coded and scored by a single person.
Statistical methods
The significance of dose–response effects was evaluated using the nonparametric Kruksall–Wallis ANOVA. The correlation matrix values were
obtained using the Spearman rank order correlation test. These tests were
performed using Prism 3.0 (GraphPad Inc., USA) and CSS Statistica (Statsoft,
USA) software.
Results
WIL2-NS
In previous experiments with WIL2-NS cells using the CBMN
assay we had shown that both NPB and MN increased
significantly in a dose-related manner after exposure to reactive
oxygen species generated by activated neutrophils, superoxide
190
Lymphocytes
We compared MN and NPB in BNCs with chromosome
aberrations at 48 and 72 h because BNCs are accumulated in
the 44–72 h period but metaphases are only accumulated for
1 h. In this manner we could make comparisons between the
CBMN assay data (which includes both late and early dividing
cells) and chromosome aberrations from both early and late
dividing metaphases.
There was a highly significant dose-dependent increase in
MNi, NPB, Ace, Dic and ring chromosome frequency as a
result of exposure to γ-rays (P trend ⬍ 0.0001 for all dose–
response effects) (Table I and Figures 3 and 4). These indices
of chromosome damage were tightly positively correlated with
each other (r ⬎ 0.93, P ⬍ 0.0001) (Table II).
To obtain an estimate of the conversion rate of Ace to MNi
and Dic to NPB we calculated the ratios of their frequencies
in the irradiated cultures. These measurements are listed in
Table III. An analysis of the Ace/MNi ratio in irradiated
cultures indicates that the Ace can be almost completely
accounted for by the MNi frequency in cultures exposed to
1 Gy because the ratio is close to 1.0 (0.90 and 1.03 for
48 and 72 h metaphases, respectively). However, this ratio
Nucleoplasmic bridges measure chromosome rearrangement
Table III. Ratios of corresponding chromosome damage biomarker
frequency in the classical metaphase analysis and CBMN assays
γ-Ray dose Metaphase Ace/MNi Ace ⫹ CEN– Dic/NPB Dic ⫹ CEN⫹
harvest time
ring/MNi
ring/NPB
1 Gy
2 Gy
4 Gy
1 Gy
2 Gy
4 Gy
Meana
48
48
48
72
72
72
aArithmetic
h
h
h
h
h
h
0.90
2.20
1.51
1.03
1.26
1.34
1.38
1.07
2.75
1.75
1.42
1.49
1.58
1.68
0.35
0.81
1.56
0.78
0.79
1.21
0.92
0.59
1.51
2.16
1.46
1.14
1.94
1.47
mean of all values in the column.
Table IV. Centromere-positive (CEN⫹) and centromere-negative (CEN–)
chromosome rings in irradiated cultures
Harvest
time
48 h
Total
72 h
Total
γ-Radiation
dose
No. of
metaphases
examined
1 Gy
2 Gy
4 Gy
300
300
300
1 Gy
2 Gy
4 Gy
300
300
300
Total
no. of rings
7
39
99
145
18
26
89
133
CEN⫹
rings
CEN–
rings
n
%
n
5
20
54
79
12
14
54
80
2
19
45
55.5 66
6
12
35
60.2 53
%
III). The combined average for all doses and harvest times for
the Dic ⫹ CEN⫹ ring/NPB ratio was 1.47, which indicates
that 32% of dicentric and centric ring chromosomes are not
coverted to NPB.
Whole chromosome painting of chromosome 4 verified that
NPB may occasionally contain DNA from more than one
(non-homologous) chromosome (Figure 5a) but often contain
DNA from a single chromosome (Figure 5b and c). The latter
supports the possibility that a significant proportion of the
NPB are the result of rearrangements involving homologous
chromosomes. In fact, most of the chromosome 4 bridges
observed (eight of nine) were completely painted by the
chromosome 4 probe cocktail and did not appear to involve
other chromosomal material. Figure 5d–f shows examples of
BNCs with one or more NPB.
Theoretically every bridge should be accompanied by a MN
originating from an acentric fragment that resulted from the
asymmetrical exchange that produced the ring or dicentric
chromosome. However, in this study only 15, 28 and 59% of
BNCs with NPB included one or more MN in cultures exposed
to 1, 2 and 4 Gy, respectively. There was a significant positive
correlation between the frequency of metaphases exhibiting
both dicentric and acentric fragments and BNCs with both
NPB and MN (r ⫽ 0.99, P ⫽ 0.0017).
45.5
Discussion
39.8
increases at the higher radiation doses, indicating that fewer
acentric fragments are observed as MNi at doses ⬎1 Gy. The
combined average for all doses and harvest times for the Ace/
MNi ratio was 1.38.
The Dic/NPB ratio in cultures exposed to 1 or 2 Gy was
⬍1.0, suggesting that more NPB were produced than could
be accounted for by the frequency of Dic. However, at 4 Gy
the ratio was 1.56 and 1.21 for the 48 and 72 h metaphases,
respectively. The combined average for all doses and harvest
times for the Dic/NPB ratio was 0.92. This suggests that there
may be other mechanisms contributing to NPB formation.
As illustrated in Figure 1, it is possible that NPBs could
also arise from centric ring chromosomes that have undergone
one or more SCEs. Furthermore, acentric ring chromosomes
could also result in MN formation because they would be
expected to lag at anaphase in the same way that acentric
fragments would.
Given the high frequency of ring chromosomes induced in
the irradiated cultures we decided to determine the proportion
of ring chromosomes that were centric and those that were
acentric. The results (shown in Table IV) indicate that 45.5
and 39.8% were CEN– and 55.5 and 60.2% of ring chromosomes were CEN⫹ in the 48 and 72 h metaphases, respectively.
Because both acentric fragments and ring chromosomes can
give rise to MNi we determined the ratio of the combined
frequency of acentric fragments plus acentric rings and MNi
frequency (Table III). The combined average for all doses and
harvest times for the Ace ⫹ CEN– ring/MNi ratio was 1.68,
which indicates that 40% of acentric chromosomes are not
observed as MNi. Similarly, because both dicentric chromosomes and centric ring chromosomes can give rise to NPB we
determined the ratio of the combined frequency of dicentric
chromosomes plus centric rings and MNi frequency (Table
The results we have obtained validate the use of NPB frequency
in BNCs as a biomarker of DNA damage and chromosome
rearrangement. In addition, they show that the inclusion of
NPB measurement in the CBMN assay increases its versatility
as a genotoxicity test because not only does it allow a
measurement of chromosome rearrangement that was previously unavailable in this test, but our results also show that
one can more reliably distinguish between similar genotoxic
agents by their differences in the NPB/MN ratio. The 5.4-fold
difference in the NPB/MN ratio observed for exposure to
activated neutrophils as compared with hydrogen peroxide or
superoxide suggests that oxidative damage induced by activated
neutrophils in WIL2-NS cells cannot be explained solely by
generation of hydrogen peroxide and/or superoxide but must
include other agents, such as the highly toxic hypochlorous
acid (which the neutrophil synthesizes from hydrogen peroxide)
(Roos and Winterbourn, 2002), which may be more effective
in inducing dicentric chromosomes and/or rings. Therefore,
the NPB/MN ratio provides an important fingerprint for distinguishing the toxic effects of different agents. For example, the
induction of MNi in the absence of NPB could be indicative
of an aneugenic agent, such as a spindle poison, because MNi
would not be the result of chromosome breaks, which could
generate NPB, but the result of chromosome loss events due
to spindle failure. Therefore, at one extreme an aneugenic
agent such as a spindle poison would be expected to exhibit
a NPB/MN ratio that is 0 or close to 0, while at the other
extreme a strong clastogenic agent such as the ionizing
radiation used in this experiment is observed to have a NPB/
MN ratio of 0.77.
The full accountability of acentric frequency by MN frequency in the 1 Gy lymphocyte cultures is in accordance with
the work of Littlefield et al. (1989), who came to the same
conclusion in studies on human peripheral blood lymphocyte
cultures when exposed to low doses of ionizing radiation.
However, at higher radiation doses, and when including the
191
P.Thomas, K.Umegaki and M.Fenech
Fig. 5. (a) Binucleated lymphocyte with a NPB that contains DNA from chromosome 4 (green fluorescence) partially across the width and almost entirely
along the length of the NPB. There are also four other chromosome 4 domains (two in each nucleus), suggesting other complex rearrangements of
chromosome 4 or aneuploidy. The incomplete staining across the width of the bridge suggests the involvement a second dicentric or ring chromosome within
the NPB involving another chromosome(s). (b and c) Binucleated lymphocytes with a NPB that appears to consist entirely of chromosome 4 DNA along the
width and length of the NPB. These NPBs represent either an asymmetrical rearrangement between the two chromosome 4 homologues, or a dicentric
chromosome 4 ring chromosome or, alternatively, the NPBs may have originated from an asymmetrical rearrangement between non-homolgous chromosomes
in which chromosome 4 happens to contribute the great majority of the DNA in the bridge. (d) Binucleated lymphocyte with one NPB and one MN. (e)
Binucleated lymphocyte with two NPB and one MN. (f) Binucleated lymphocyte with one NPB but no MN.
contribution of acentric rings, it was evident that acentric
chromosomes are not always efficiently observed as or converted to MNi. This may be because (i) some MNi are hidden
behind the main nuclei in the slide preparation and/or (ii)
because some of the acentric rings and fragments become
incorporated into the nuclei at anaphase due to their proximity
to the rest of the chromosomes at the poles of the cell and/or
(iii) inclusion of two or more chromosome fragments into one
MN at the higher radiation doses. Minissi et al. (1999) provided
192
evidence suggesting that reduced polar distance at anaphase
may favour the re-incorporation of lagging chromosomes or
fragments into the daughter nuclei. It is plausible that NPB
formation may restrict polar distance depending on the size of
the rearranged chromosome causing the bridge.
The fact that not all dicentrics and rings can be accounted
for by the frequency of NPB is not entirely surprising because,
as explained in Figure 1A–D, it is possible that some dicentric
chromosomes may travel to one pole only and thus not form
Nucleoplasmic bridges measure chromosome rearrangement
a NPB. Only those ring chromosomes that undergo one or
more SCE prior to mitosis to form either a large dicentric ring
chromosome or a concatenated double ring have the potential
to form a NPB, unless the centromeres travel to the same pole.
The relative propensity of dicentric or ring chromosomes to
form NPB is yet to be determined. This could be achieved
using whole chromosome painting and same chromosomespecific painting with sub-telomeric sequences and a different
chromophore tag. The ring chromosomes, unlike the dicentric
chromosomes, are expected to be negative for the sub-telomeric
sequences which should be observed within the nuclei in close
proximity to the ends of the NPB.
Our preliminary observation, on a very small sample, that
the great majority of chromosome 4 painted bridges were
completely painted suggests that NPBs may originate mainly
from asymmetrical rearrangements within the same chromosome or between homologous chromosomes. Clearly, our data
are scanty preliminary observations and this hypothesis has to
be tested more rigorously by scoring a statistically meaningful
number of NPBs and including all-chromosome paints using
spectral karyotyping methods. The relatively frequent absence
of MNi in BNCs with NPBs could be due to inefficient
conversion of acentric fragments to MNi, as explained above,
or because the dicentric or ring chromosomes were caused
by breaks in the telomeric or sub-telomeric regions of the
chromosome with only very small losses of chromatin that
may not be visible by microscopy as a MN.
To further verify that NPB do in fact originate from dicentric
rings and chromosomes it would be necessary to use whole
chromosome painting and centromeric probes which are ideally
specific to the chromosomes involved in the NPB. We have
not achieved this goal, although Saunders et al. (2000) in their
study on genomic instability in oral cancer cells used this
methodology to show NPB in BN cells involving chromosome
11 only with centromeric sequences at either end of the NPB
(see figure 4 Saunders et al., 2000). It would be of interest to
know the proportion of NPB that involve only one chromosome
or two or more, as this would help to ascertain which of the
models illustrated in Figure 1 is the most significant contributor
to NPB formation.
It is important to note that the comparative data for ring
and dicentric chromosomes in relation to NPB frequency
observed in binucleated lymphocytes do not necessarily apply
to WIL2-NS cells or other cell lines. Exposure of WIL2NS cells to hydrogen peroxide, superoxide and activated
neutrophils occurred at a variety of stages of the cell cycle
because the cultures were not synchronized, while, on the
other hand, lymphocytes were exposed to ionizing radiation
in G0 phase. Strand breaks and base lesions induced in S phase
may lead to the generation of complex assymetrical chromatidtype exchanges and the formation of multiradial chromosomes
with a dicentric chromatid (Natarajan and Obe, 1982). The
efficiency with which the latter are expressed as NPBs in
BNCs has yet to be determined and will be a focus for
future research.
Measurement of NPB has the advantage that, unlike MN,
the background frequency is low and should not be influenced
by gender, although there is insufficient data at this point to
verify the absence of a gender effect. The use of the NPB
biomarker should be useful in biological dosimetry following
radiation accidents or chronic exposure to cosmic radiation,
which induces dicentric chromosomes as well as MNi
(Hiemers, 2000). Measurement of NPB in the CBMN assay
adds important information on asymmetrical chromosome
rearrangement that is otherwise only achievable using metaphase analysis of dicentric and ring chromosomes. Measurement of NPBs in the CBMN assay should provide a useful
complement to the use of dicentric and rings chromosomes in
biological dosimetry of radiation exposure (IAEA, 2001). The
inclusion of NPB may also enhance the capacity of the CBMN
assay to identify those individuals with reduced DNA repair
capacity, such as those with a BRCA1 mutation (Rothfuss
et al., 2000), because it may provide a measure of both reduced
repair capacity as well as misrepair leading to chromosome
rearrangement.
Finally, these data, together with those published previously
(Fenech et al., 1999; Umegaki and Fenech, 2000; Crott et al.,
2001; Fenech and Crott, 2002), indicate that the CBMN assay
has matured into a comprehensive method for assessing both
genotoxicity and cytotoxicity so that it now includes the
measurement of chromosome breakage and loss (MN with
or without centromere staining), chromosome rearrangement
(NPB), gene amplification and/or DNA repair complexes
(nuclear buds), non-disjunction (by centromere-specific staining), necrosis, apoptosis and cytostasis. These measures allow
a much more thorough assessment of the type of toxicity
experienced by a cell than was achievable previously and
should be implemented in future studies.
Acknowledgements
We acknowledge the contribution of the volunteers who donated the blood
samples as well as earlier unpublished preliminary studies by Will Greenrod
that helped in establishing the NPB measurements in lymphocytes.
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Received on September 5, 2002; revised on November 14, 2002; accepted on
November 14, 2002
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