Mutagenesis

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Mutagenesis, Vol. 18, No. 2, 187-194, March 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press

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 ¹ National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan

	Abstract

Top Abstract Introduction Materials and methods Results Discussion References

 
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 -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

Top Abstract Introduction Materials and methods Results Discussion References

 
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 non-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 centromere-positive (CEN⁺) MNi are scored separately because a large proportion of spontaneous MNi are CEN⁺ and radiation-induced 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 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 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.

View larger version (29K): [in this window] [in a new window]   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.

 
In this paper we provide evidence for the proposed mechanisms 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 -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

Top Abstract Introduction Materials and methods Results Discussion References

 
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 L-glutamine (Sigma, Australia). Samples were irradiated using a CIS BIO International IBL 437 blood cell irradiator comprising three stationary ¹³⁷Cs doubly encapsulated radiation sources emitting -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.

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.

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 2x 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 0.3x10⁶ 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 0.5x10⁶ 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 non-parametric 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

Top Abstract Introduction Materials and methods Results Discussion References

 
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 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.

View larger version (24K): [in this window] [in a new window]   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).

 
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 -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).

View this table: [in this window] [in a new window]   Table I. . Dose–response characteristics of chromosomal damage measurements in lymphocytes exposed to -rays

 

View larger version (20K): [in this window] [in a new window]   Fig. 3. . -Ray dose–response characteristics for MNi and acentric chromosome fragments. Data represent the mean ± SEM (n = 3).

 

View larger version (25K): [in this window] [in a new window]   Fig. 4. . -Ray dose–response characteristics for NPB, dicentric chromosomes and ring chromosomes. Data represent the mean ± SEM (n = 3).

 

View this table: [in this window] [in a new window]   Table II. . Correlation matrix of chromosome damage biomarkers in the classical metaphase and CBMN assays

 
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 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.

View this table: [in this window] [in a new window]   Table III. . Ratios of corresponding chromosome damage biomarker frequency in the classical metaphase analysis and CBMN assays

 
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.

View this table: [in this window] [in a new window]   Table IV. . Centromere-positive (CEN+) and centromere-negative (CEN-) chromosome rings in irradiated cultures

 
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 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.

View larger version (48K): [in this window] [in a new window]   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.

 
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).

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
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 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 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 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 chromosome-specific 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 WIL2-NS 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 chromatid-type 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.

	Acknowledgments

 
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.

	Notes

 
² To whom correspondence should be addressed. Tel: +618 8303 8880: Fax: +618 8303 8899: Email: michael.fenech{at}csiro.au

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Received on September 5, 2002; revised on November 14, 2002; accepted on November 14, 2002.

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