Mutagenesis, Vol. 15, No. 4, 329-336,
July 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
A mathematical model of the in vitro micronucleus assay predicts false negative results if micronuclei are not specifically scored in binucleated cells or in cells that have completed one nuclear division
CSIRO Health Sciences and Nutrition, PO Box 10041, Adelaide BC, SA 5000, Australia
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Abstract |
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A mathematical model is described that predicts the effect of altered nuclear/cell division kinetics and cytotoxicity on micronucleus expression in vitro when the micronucleus assay is performed without discriminating between cells that have divided once and cells that have not divided after genotoxic insult. The model is based on the probabilities of: (i) a viable cell completing nuclear division; (ii) micronucleus expression in a cell that completes nuclear division after genotoxic insult; (iii) a cell not dividing and surviving as a mononuclear cell; (iv) a cell dying by necrosis or apoptosis. The model predicts: (i) false negative results for relatively weak chromosome damaging agents that also inhibit nuclear division, if micronuclei are scored in mononucleated cells without discriminating between divided and non-divided cells; (ii) this tendency for a false negative result when scoring micronuclei without discriminating between non-divided and once-divided mononuclear cells increases with cell lines and culture conditions that do not result in optimal rates of nuclear division (i.e. >90% of dividing cells); (iii) the absolute increment in micronucleus frequency in binucleated cells is at least 2-fold greater than that observed in mononucleated cells when nuclear division is not inhibited and this difference increases with increasing nuclear inhibition. The number of dead cells does not influence the micronucleus frequency if only viable cells are considered when determining the micronucleus frequency ratio. The results from this model suggest that the micronucleus assay when performed by scoring mononucleated cells, without restricting the score to those cells that have divided once after genotoxic insult, is prone to produce false negative results and, therefore, cannot be considered reliable or conclusive. Scoring of micronuclei in cytokinesis-blocked binucleated cells is predicted by the model to provide consistent results under all culture conditions and based on these theoretical results should be considered the preferred choice.
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Introduction |
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The in vitro micronucleus (MN) assay is currently being considered as a suitable method for testing the genotoxicity of chemicals and pharmaceuticals. The reason for this stems from the relative ease of scoring MN and the versatility of the assay, which can detect chromosome breakage and chromosome loss (Evans, 1997; Fenech, 1997; reviewed by Kirsch-Volders, 1997). MN originate from lagging chromosome fragments (due to DNA strand breakage) or whole chromosomes (due to spindle, kinetochore or centromere damage) at anaphase and thus can only be expressed once a cell has completed nuclear division. An exception to this are tumour cells or drug-resistant cells with amplified genes which can extrude the amplified genes as nuclear blebs that may become MN independently of nuclear division (Miele et al., 1989
; Shimizu et al., 1996). The latter are not relevant to the current discussion, which relates to normal cells (e.g. lymphocytes or fibroblasts) without amplified genes that are commonly used for in vitro assays.
Currently two methods are used to perform the assay. In the original method proposed by Countryman and Heddle (1976) MN are scored in a dividing cell population without discriminating between cells that have completed nuclear division and those that have not following exposure to the genotoxic agent. It has been shown that this approach in human lymphocyte cultures tends to underestimate MN frequency when nuclear division is inhibited or when cells are allowed sufficient time to divide more than once (Fenech and Morley, 1985b, 1986; Fenech, 1997). In the latter case a reduced MN frequency may be due to loss of MN from micronucleated (MNed) cells and/or dilution of MNed cells that divided once by non-MNed cells that have divided more than once.
To improve the in vitro MN assay, Fenech and Morley (1985a, 1986) proposed that MN should only be scored in cells that had completed one nuclear division, both to obtain an accurate estimate of spontaneous MN frequency as well as a reliable estimate of MN induced by radiation or chemicals. This was particularly important for chemicals (and UV radiation) as genotoxic doses of several chemicals also tend to inhibit nuclear division (Fenech, 1985). Several methods were proposed and developed to score MN only in cells that complete nuclear division but of these only the cytokinesis-block micronucleus (CBMN) assay could efficiently and reliably identify such cells (Fenech and Morley, 1985a). The CBMN assay was subsequently adopted by numerous laboratories leading to an unabated interest in its use and application for human biomonitoring, radiosensitivity testing and in vitro genotoxicity studies (Scott et al., 1998; Fenech et al., 1999; reviewed by Kirsch-Volders, 1997).
Cytochalasin-B (cyt-B) does not induce MN expression in cytokinesis-blocked binucleated cells in non-neoplastic cell lines (e.g. human lymphocytes and Chinese hamster fibroblasts) over the concentration range that is normally used to block cytokinesis (i.e. 1–10 µg/ml) (Wakata and Sasaki, 1987; Prosser et al., 1988). A recent study suggests that MN expression induced by spindle poisons may be less than expected in the cytokinesis-blocked binucleated (BNed) cells because of pole-to-pole distance shortening which may increase the probability of re-inclusion of lagging chromosome fragments/whole chromosomes back into a nucleus (Minissi et al., 1999). Cyt-B concentration should be kept optimal throughout the culture period to prevent BNed cells emerging from cell division of multi-MNed multi-nucleated cells which could cause an artefactual increase in MN frequency in BNed cells (Shultz and Onfelt, 1994). Results from another study suggest that MN in BNed cells and MNed cells may contain a slightly different spectrum of chromosomes and chromosome aberrations or loss that may change with culture time (Falck et al., 1997). These potential, but resolvable, problems of the CBMN assay have also been reviewed elsewhere (Fenech, 1997).
There has been an increased interest in further exploring the possibility of performing the in vitro MN assay without the use of cyt-B to minimize the possibility of obtaining a false positive or false negative result due to interference of cyt-B with the toxic effects of the chemical being tested (Kalweit et al., 1999; Matsushima et al., 1999). However, this approach has the potential risk of obtaining a false negative result because of inadequate control of cell division kinetics, i.e. inhibition of nuclear division inhibits MN expression. Evidence of obtaining a false negative or false positive result with the CBMN assay is lacking. However, there is evidence that performing the MN assay in a manner that does not account for inhibition of nuclear division can lead to an underestimate of MN induction when using human lymphocytes (Fenech and Morley, 1985b, 1986; Fenech, 1997).
Currently there are numerous extensive studies underway comparing the results for the in vitro MN assay performed with and without cyt-B to identify which method is most likely to give reliable results and presumably also least likely to give a false negative result. To contribute to this body of work I have developed a mathematical model that predicts the dose–response curve and sensitivity of the two types of MN assays for genotoxins that are either strong or weak MN inducers, as well as being inhibitors of nuclear division and/or inducers of apoptosis or necrosis.
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Materials and methods |
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The mathematical equation used for the predictions is based on the following factors: pD, probability that a viable cell completes nuclear division; pMNed, probability that a divided cell expresses a micronucleus; pND, probability that a viable cell does not divide and survives; pDC, probability that a cell does not divide but becomes a dead cell via necrosis or apoptosis.
The following assumptions are made in this relatively simple model: the test is designed so that dividing cells only have sufficient time to divide once; the probability of MN expression in a BNed cell is the same as that for a cell that completes nuclear division and cytokinesis and produces two mononuclear (MO) cells; cells undergoing cytotoxicity do not induce DNA damage in bystander cells.
Because binucleate cells represent divided cells, the frequency of MNed cells amongst these cells (fMNed[BN]) is the same as the factor pMNed. This value is the theoretical frequency of MNed BNed cells that should be observed in the CBMN assay.
The frequency of MNed MO cells (fMNed[MO]) in a test system that does not discriminate between divided and non-divided cells is derived by the formula:
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The model was used to predict the MNed cell frequency in cultures exposed to the following type of chemicals: S1, a strong inducer of MNed cells that has negligible effects on nuclear division and cytotoxicity; S2, a strong inducer of MNed cells that inhibits nuclear division with negligible effects on cytotoxicity; S3, a strong inducer of MNed cells that inhibits nuclear division and is also an inducer of cytotoxicity; W1, a weak inducer of MNed cells that has negligible effects on nuclear division and cytotoxicity; W2, a weak inducer of MNed cells that inhibits nuclear division with negligible effects on cytotoxicity; W3, a weak inducer of MNed cells that inhibits nuclear division and is also an inducer of cytotoxicity.
The strong inducers of MNed cell frequency were based on a chemical that increases MN frequency 3-fold over baseline at the lowest dose with a doubling increase in effect with each doubling dose. The weak inducers of MNed cell frequency were based on a chemical that increases MNed cell frequency 0.5-fold over baseline at the lowest dose with a 33% increase in effect with each doubling dose. The chemicals that also inhibited nuclear division decreased nuclear division by 20% relative to control at the lowest dose with a doubling increase in effect with doubling dose. The chemicals that were also inducers of cytotoxicity, increased cytotoxicity 4-fold relative to baseline at the lowest dose with a doubling increase in effect with each doubling dose.
The level of cytotoxicity in control cultures was set at 4% so that only 96% of cells were potentially capable of dividing. The model was used to compare the predicted results for cultures in which 100, 66 and 33% of viable cells were able to divide under control conditions.
Necrosis and apoptosis were considered together for estimating the effect of cytotoxicity.
The selection of dose-related changes in MNed cell frequency, nuclear division and cytotoxicity are based on typical dose–response characteristics commonly observed in our studies with chemical agents and radiation, although they do not necessarily fit observations for a specific agent (Fenech, 1985; Fenech and Morley, 1986; Fenech et al., 1999).
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Results |
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Strong inducers of MN in dividing cells
Figure 2
and Tables I–III summarize the results for the S1, S2 and S3 types of chemical in cell culture conditions in which 100, 66 and 33% divide under control conditions. The results suggested that for S1 chemicals almost linear dose–response relationships are observed under all culture conditions, both with an assay that scores MN only in divided BNed cells and with an assay that does not discriminate between divided and non-divided MO cells. The slope of the dose–response curve did not change with different cell culture conditions when MN are scored in divided BNed cells but it became increasingly smaller with decreased nuclear division rate when MNed cells are scored in MO cells.
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The dose–response curve was not altered with S2 chemicals that also inhibit nuclear division if MN are scored specifically in divided BNed cells. However, inhibition of nuclear division had a marked negative effect on the dose–response curve when MNed cells are scored in all viable MO cells regardless of their nuclear division status: this effectively reduced the slope of the dose–response curve even further and produced a plateau effect in the dose–response relationship.
The results for S3 chemicals were effectively similar to those for S2 chemicals, suggesting that the extent of cytotoxicity has a minimal effect on the dose–response curve observed when scoring MNed cells in divided BNed cells or MO cells (divided and non-divided).
The fold increase and the absolute increase in fMNed was compared for S1, S2 and S3 chemicals at the highest test dose in cultures in which 100% of the cells completed nuclear division (Figure 4). There was no difference in the fold increase in fMNed when comparing results for S1 in BNed cells and MO cells, however, the fold increase in fMNed was 3 times greater for BNed cells than it was for MO cells when considering results for S2 and S3. With regard to the absolute increase in fMNed, there was no difference between S1, S2 and S3 chemicals if fMNed is scored in BNed cells. Furthermore, the absolute increase in fMNed was 2, 7.33 and 7.85 times greater in BNed cells relative to MO cells for the S1, S2 and S3 chemicals, respectively.
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Weak inducers of MN in dividing cells
Figure 3
and Tables I–III
summarize the results for the W1, W2 and W3 types of chemical in cell culture conditions in which 100, 66 and 33% divide under control conditions. The results suggested that for W1 chemicals clear dose–response relationships are observed under all culture conditions both with an assay that scores MN only in BNed cells and with an assay that does not discriminate between divided and non-divided MO cells. The slope of the dose–response curve did not change with different cell culture conditions when MNed cells are scored in BNed cells, but it became smaller with decreased nuclear division rate when MNed cells are scored in MO cells.
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The dose–response curve was not altered with W2 chemicals that also inhibit nuclear division if MNed cells are scored specifically in BNed cells. However, inhibition of nuclear division had a marked negative effect on the dose–response curve when MNed cells are scored in MO cells regardless of their nuclear division status: this effectively changed the slope of the dose–response curve from a positive to a negative slope.
The results for W3 chemicals were effectively similar to those for W2 chemicals, suggesting that the extent of cytotoxicity has a minimal effect on the dose–response curve observed when scoring MNed cells in viable BNed cells or viable MO cells (divided and non-divided).
The fold increase and the absolute increase in fMNed was compared for the W1, W2 and W3 chemicals at the highest test dose in cultures in which 100% of the cells complete nuclear division under control conditions (Figure 4). There was no difference in the fold increase in fMNed when comparing results for W1 in BNed cells and MO cells, however, the fold increase in fMNed was 3.0 and 3.1 times greater for BN cells than it was for MO cells when examining results for W2 and W3, respectively. With regard to the absolute increase in fMNed, there was no difference between W1, W2 and W3 chemicals if fMNed was scored in BNed cells. However, the absolute increase in fMNed was 2 times greater in BNed cells relative to MO cells for W1. With regard to W2 and W3, the absolute change in fMNed was actually negative in MO cells (–0.0005 and –0.0007, respectively) but positive for BNed cells (0.017 and 0.017, respectively), making the difference in results for BNed and MO cells even more pronounced.
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Discussion |
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The in vitro MN assay is being proposed as an alternative method for genotoxicity assessment of chemicals (Kirsch-Volders, 1997
). The main point of contention is whether it is necessary to restrict scoring of MN to cells that have completed one nuclear division after exposure to a presumed genotoxin or to ignore the division status of the cells scored when cell lines with `good' growth characteristics are used (Kirsch-Volders et al., 2000). The main questions relating to scoring MN only in BNed or MO cells (divided and/or non-divided) are the effects of this choice on: (i) false negative results; (ii) sensitivity of the test; (iii) shape of the dose–response curve; (iv) effects of cytotoxicity; (v) efficiency of MN expression in the cells scored.
The proposal that it is not necessary to restrict MN scoring to BNed cells in established cell lines with good growth characteristics is based, presumably, on the assumptions that: (i) virtually all the cells are dividing; (ii) the cell cycle time of all the dividing cells are the same; (iii) nuclear division rates, cell cycle time and culture conditions do not change from one experiment to the next within laboratories and between laboratories; (iv) nuclear division rates and cell cycle time are the same at all the doses of the chemical tested. To date there is only limited evidence to support some of these assumptions. Studies with V79 cells treated with strong MN-inducing agents such as mitomycin C and griseofulvin suggests similar results when the MN assay is performed with cyt-B (i.e. MN in BNed cells) or without cyt-B (i.e. MN in MO cells) (Kalweit et al., 1999). These results do not fit the proposed theoretical model and possible explanations include the following: (i) nuclear division was not significantly inhibited by the treatments; (ii) the culture conditions were optimal, allowing >90% of cells to complete nuclear division; (iii) MN in BNed cells were not as efficiently scored or expressed as MN in MO cells. A more recent collaborative validation study on 66 compounds (including clastogens of varying potency and polyploidy inducers) by Matsushima et al. (1999) showed an 88.7% concordance between results for the chromosome aberration test and the MN assay performed without cyt-B in a Chinese hamster lung cell line (CHL/IU). These data suggest that false negative results can be obtained in 10% of cases with some genotoxic chemicals when the MN assay is performed without cyt-B. Matsushima et al. (1999) also compared MN induction in tests performed with and without cyt-B in CHL/IU cells. Their results for mitomycin C, N-ethylnitrosourea, hydrogen peroxide and benzyl chloride (but not hydroquinone) showed that the absolute MN frequency in the BNed cells (in the test with cyt-B) was at least 2-fold greater than the MN frequency in MO cells (in the test without cyt-B). The latter result is in agreement with the predictions of the mathematical model presented in this paper. However, the fold increase in MN frequency relative to baseline in genotoxin-treated cultures in the same study tended to be greater in MO cells in cultures without cyt-B relative to BNed cells in cultures with cyt-B, which does not fit the model. Similar comparative experiments with weak MN-inducing agents that are strong inhibitors of nuclear division are warranted, for the reasons outlined below, because they may explain why false negative results have been obtained in the study of Matsushima et al. (1999) using the MN assay without cyt-B. The results from all of these comparative studies may also be used to refine the mathematical model proposed here.
The proposed model predicts: (i) that the most consistent dose–response curves are obtained when MN are scored specifically in BNed cells under all conditions and treatments considered; (ii) scoring MO cells is likely to produce false negative results when chemicals inhibit nuclear division because non-divided MO cells are included in the denominator of the fMNed ratio; (iii) a lower fold increase and an even lower absolute increase in fMNed in MO cells relative to BNed cells. Because the statistical power of the test is dependent on the absolute increment in fMNed (Motulsky, 1995), it is predictable that tests with MO cells are more likely to produce statistically non-significant results relative to tests based on BNed cells, unless the standard deviation of results with the MO cells is much lower than that for results with BNed cells. In addition, the dose–response curves observed for MO cells tend to have lower slopes than those for BNed cells and under conditions of reduced nuclear division the slopes may become negative for MO cells as opposed to positive in BNed cells. The latter suggests a greater propensity of tests with MO cells to yield false negative results. The requirement to test doses of chemicals that induce up to 50% toxicity is likely to produce false negative results if the CBMN assay is not used because nuclear division rate tends to decline with increased cytotoxicity. Our recent results in lymphocytes treated with hydrogen peroxide revealed a strong negative correlation between necrosis and the binucleate ratio (r = –0.75, P < 0.0001) (Fenech et al., 1999).
The results from this mathematical model suggest that the extent of cytotoxicity does not alter the outcome of fMNed measurements because these cells are essentially not included in the fMNed ratio. However, this may appear to be so because it was assumed that: (i) cytotoxicity does not specifically eliminate cells that had DNA damage; (ii) cytotoxicity itself does not induce DNA damage to `bystander' viable cells. In addition, agents that specifically inhibit apoptosis may enable heavily DNA-damaged cells to survive and thus increase the probability of MN expression. In view of the above, we have proposed that a better way of assessing the genotoxicity, cytotoxicity and cytostatic effects of a chemical is to integrate all of these measurements within the CBMN assay (Fenech et al., 1999).
One issue that has not been tackled in the model described above is the consequence of allowing cells to divide more than once. This is very likely to occur when scoring MO cells for fMNed in cell lines unless the culture time is minimized to less than one cell cycle time. Under acute exposure conditions one may expect that allowing cells to divide more than once will result in the dilution of MNed cells by daughter cells that do not contain MN. It is relatively simple to adapt the proposed model to take account of the effect of a proportion of cells dividing more than once and surviving. Figure 5 illustrates how this can be achieved using the example in Figure 1B and extending it to include a significant proportion of cells that divide twice instead of once. In the example it is assumed that the probability of a cell becoming MNed becomes increasingly diminished after a second division, with the result that fMNed in MO cells declines as cells are allowed to divide more than once. Restricting scoring to BNed cells ensures that only cells that divided once are included in the fMNed ratio and overcomes the confounding effect of cells that divide more than once. One other aspect to consider is the possibility that a particular genotoxin might induce genomic instability in daughter cells, as has been observed with 
-particle irradiation (Kadhim et al., 1992). In this case one might expect a sustained higher rate of MN expression over an extended number of cell cycles.
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The proposed model could also be modified to take into account other potential, events such as: (i) induction of mitotic arrest by spindle poisons followed by mitotic slippage leading to polyploid MNed cells with MN (Elhajouji et al., 1998
); (ii) the possibility that MNed MO and BNed cells may undergo apoptosis; (iii) the possibility that cyt-B may alter MN expression and sensitivity of cells to the genotoxic and cytotoxic effects of the tested chemical. In addition, there may be some concern that scoring MN in cytokinesis-blocked cells may interfere with the effect of the chemical being tested by restricting scoring to cells that have not experienced division delay because they incurred less DNA damage. This concern does not seem justifiable because cyt-B blocking usually occurs over a period of 24 h and can be extended to 48 h if division delay occurs. This extension of the cyt-B blocking time has resulted in an increased MN frequency when human lymphocytes are exposed to ionizing radiation (Scott et al., 1998) but no difference when human lymphocytes are exposed to hydrogen peroxide (Fenech et al., 1999), suggesting that division delay of DNA-damaged cells may be significant with some but not all genotoxic agents and is easily accounted for by altering the CBMN assay protocol. In conclusion, the proposed mathematical model of in vitro expressed MN predicts that: (i) scoring MN in BNed cells is the most reliable way of determining fMNed; (ii) scoring MN in MO cells (divided and non-divided) is likely to generate false negative results, particularly with chemicals that are relatively weak inducers of MN and relatively strong inhibitors of nuclear division at the doses tested. Consequently, negative results for fMNed obtained by scoring MO cells cannot be considered conclusive. It is evident that negative results with a MN assay without cyt-B should be confirmed using the CBMN assay.
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Acknowledgments |
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Dr Marilyn Aardema and Prof. Micheline Kirsch-Volders are thanked for their helpful and constructive comments.
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Notes |
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1 To whom correspondence should be addressed. Tel: +618 8303 8880; Fax: +618 8303 8899; Email: michael.fenech{at}hsn.csiro.au
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References |
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Received on December 21, 1999; accepted on March 23, 2000.
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