Mutagenesis, Vol. 14, No. 6, 569-580,
November 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Validation study of the in vitro micronucleus test in a Chinese hamster lung cell line (CHL/IU)
Japan Bioassay Research Center, 2445 Hirasawa, Hadano-shi, Kanagawa 257-0015, 1 Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, 2 Chromosome Research Center, Olympus Optical Co. Ltd, 2-3 Kuboyama-cho, Hachioji-shi, Tokyo 192-8512, 3 Department of Public Health and Environmental Medicine, The Jikei University School of Medicine, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105-8461, 4 Department of Hygiene and Preventive Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita-shi, Osaka 565-0871 and 5 National Institute of Industrial Health, 6-21-1, Nagao, Tama-ku, Kawasaki-shi, Kanagawa 214-8585, Japan
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We conducted a collaborative validation study, under the auspices of the Japanese Ministry of Labour, on the in vitro micronucleus test to see if it could be used as an alternative to the in vitro chromosome aberration test for evaluation of chemical safety. We used the Chinese hamster lung cell line (CHL/IU), which is the most widely used system for the latter test in Japan, and evaluated 66 chemicals, including clastogens and polyploidy inducers. The cytochalasin B cytokinesis blocking method, which is commonly used in human lymphocyte culture, was applied to the established cell line, but did not improve the detection of chemically-induced micronuclei in continuously growing cells. The highest micronucleus frequencies were obtained at 48 or 72 h continuous treatments. In short treatments (6 h), a 42 h recovery time yielded the best responses. Concordance between the results of the micronucleus test and the chromosomal aberration test was satisfactorily high (88.7%), and we concluded that the in vitro micronucleus test could be used in place of the chromosomal aberration test as a simple and rapid method for detecting clastogens and aneugens in vitro. We also propose a protocol for the test.
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Introduction |
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A chromosome aberration (CA) test in cultured mammalian cells is required by the genotoxicity guidelines for evaluation of chemical safety (OECD, 1983
; Japan/MHW, 1984; Japan/MOL, 1987; US/FDA, 1993; ICH3, 1996). For an in vivo cytogenetic assay, a rodent micronucleus (MN) test is a widely used alternative to a bone marrow CA test. It has been argued, therefore, that the in vitro MN test could, for regulatory purposes, be an acceptable alternative to the in vitro CA test.
Nowadays, the in vitro MN test with cultured human lymphocytes is used for monitoring occupational exposure. The method uses cytochalasin B (CB) to inhibit cytokinesis, thereby restricting the cell population scored to those that have undergone one cell division (Fenech and Morley, 1985). The cytokinesis blocking method has also been applied to the micronucleus test in a Chinese hamster established cell line (Wakata and Sasaki, 1987). It has been reported, however, that CB itself is cytotoxic (Lindholm et al., 1991) and that it can interact with test compounds, especially those that induce numerical CAs.
Although the MN test cannot give information about types of structural CAs, there have been many attempts to distinguish MNs caused by aneugens from those induced by clastogens (Yamamoto and Kikuchi, 1980; Högstedt and Karlsson, 1985; Vanderkerken et al., 1989; Tinwell and Ashby, 1991). For that purpose, centromere-specific staining methods are commonly used (Degrassi and Tanzarella, 1988; Miller et al., 1991; Schriever-Schwemmer and Adler, 1994; Mäki-Paakkanen et al., 1995).
A collaborative study to validate the in vitro MN test was begun in 1989 under the auspices of the Japanese Ministry of Labour, with the cooperation of the Japan Chemical Industry Ecology-Toxicology & Information Center (JETOC). Four institutes participated, and 66 compounds, including clastogens of varying potency and polyploidy inducers, were tested. The aim of the collaborative study was to validate the in vitro MN test for use as an alternative to the in vitro CA test, and to prepare guidelines for its use in Japan.
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Participants
The four institutes (and the investigators) involved in this project were the National Institute of Health Sciences (L1; T.Sofuni, M.Hayashi, A.Matsuoka, T.Suzuki, M.Honma, N.Yamazaki and H.Sakamoto), Olympus Optical Co. Ltd (L2; M.Ishidate,Jr, K.F.Miura, M.Hatanaka, C.O.Sasaoka and T.Satoh), the Jikei University School of Medicine (L3; H.Shimizu, Y.Suzuki, J.Lee, Y.Seki and H.Okonogi), and Osaka University School of Medicine (L4; K.Morimoto, H.Ogura and K.Mure).
Cells
CHL/IU cells obtained from The Japanese Collection of Research Bioresources (JCRB) were used throughout the collaborative study. They were maintained in Eagle's minimum essential medium supplemented with 10% heat inactivated (56°C for 30 min) calf serum. 1x104–1x105 cells were seeded in 60 mm plastic plates and treated with test chemicals on the second day.
Test chemicals and rat liver S9
The 66 test chemicals used in the collaborative study are listed in Table I. Initially we tested both direct-acting clastogens and those requiring metabolic activation to assess the basic performance of the in vitro MN test. Then we tested a variety of clastogens having different modes of action or with weak or marginal activity and polyploidy inducers (Ishidate, 1987; Ishidate et al., 1988). Occasionally we tested some structurally related chemicals even though CA data were not available for them in the literature. As a rule, one chemical was tested by one laboratory, but some were tested at two or more laboratories (Table I). Additional confirmation tests were carried out if necessary, though only one representative result is generally presented for each chemical (Table II and Figures 1–3).
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Chemicals were dissolved or suspended in appropriate solvent or vehicle (Table II
) immediately before treatment. To minimize interlaboratory variation, the same lot of rat liver S9 fraction prepared from phenobarbital- and 5,6-benzoflavone-pretreated male Sprague–Dawley rats (Kikkoman Corporation, Noda, Japan) was used for all experiments.
Test chemical treatment and slide preparation
As a rule, the cells were treated continuously for 24, 48 or 72 h in the absence of S9 mix (indicated as 24 h, 48 h, 72 h) and/or for 6 h with or without S9 mix followed by 18, 42 or 66 h recovery time (indicated as 6+18 h, 6+42 h, 6+66 h). The cells were then detached by trypsinization and treated with KCl hypotonic solution (75 mM) for 10 min at room temperature. The hypotonized cells were fixed by at least three changes of 1:3 acetic acid:ethanol. Finally, the cells were suspended in methanol containing 1–2% acetic acid and air dried on clean glass slides. After drying overnight, the cells were stained with either acridine orange (Matsuoka et al., 1992) or Giemsa. In several experiments, the cells were treated with test chemicals in the presence and in the absence of CB (3 µg/ml) until cell harvest.
Microscopic observation
All slides were coded and analyzed blind microscopically [with x20 objective lens for acridine orange stain, x40 (or more) for Giemsa stain]. We analyzed only well-outlined cells with a single main nucleus. With CB, only binucleated cells were scored for MNs. The candidate MNs were categorized into three groups: very small (pin point) inclusions stained homogeneously (type 1) (those were not included for result evaluation), typical, i.e., smaller in diameter than 
of the normal main nucleus (type 2), and large, i.e., between and 
the diameter of the normal main nucleus (type 3). The number of micronucleated cells per 1000 intact interphase cells was recorded. The number of mitoses and abnormal cells (e.g., multinucleated cells, cells with abnormal nucleus) that appeared in the same microscopic field were also recorded. We used in principle a single scorer/experiment in all laboratories, but scorers changed in each laboratory during the long-term (about 8 years) collaborative study. Meetings for standardization of identification and classification of micronuclei were held every other year using microscopy or microphotographs.
Statistical procedures
The frequencies of cells with type 2 and/or type 3 MN in the treatment groups were compared with those of the concurrent negative control by Fisher's exact test. The concentration–response relationship was evaluated by the Cochran–Armitage trend test (Cochran, 1954; Armitage, 1955). We called a result statistically significant when the P-value of the Fisher's exact test was smaller than 0.05 divided by the number of treatment groups and the P-value of the trend test was also smaller than 0.05.
Concurrent chromosome aberration tests
For some chemicals, chromosomal aberration tests were performed concurrently with the MN tests under the same experimental conditions. The cells were treated continuously for 24 or 48 h in the absence of S9 mix and/or for 6 h with or without S9 mix followed by 18 h recovery time (Ishidate, 1987).
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Results |
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Treatment and sampling time
In the continuous treatment experiments, longer treatments generally gave higher frequencies of micronucleated cells in the order of 72 h
48 h > 24 h (see acetaminophen, ethyl methanesulfonate, 5-fluorouracil, mitomycin C and potassium bromate in Figure 1). In the short (6 h) treatment experiments, the 42 h recovery period tended to give higher frequencies of micronucleated cells than the 18 and 66 h periods, regardless of the presence of an exogenous metabolic activation system [see benzo(a)pyrene, benzylchloride, cadmium chloride, cyclophosphamide, 7,12-dimethylbenz(a)anthracene and dimethylnitrosamine in Figure 1). When continuous and short treatments were compared, continuous treatments yielded higher frequencies of micronucleated cells (see MeA
C, ß-naphthoquinoline, m-phenylenediamine and vinblastine sulfate in Figure 1).
Combination with CB
The effect of CB on the detection of MN induced by five compounds (mitomycin C, N-ethyl-N-nitrosourea, hydroquinone, hydrogen peroxide and benzylchloride) is shown in Figure 4. The frequencies of micronucleated cells induced by all the compounds, except for hydroquinone, were significantly higher with CB than without CB (Figure 4, left). The frequencies of micronucleated cells in the negative control of the tests with CB were clearly higher than in the tests without CB. Therefore, fold increases of micronucleated cells over the negative control values were compared between the tests with and without CB (Figure 4, right). At all concentration levels of mitomycin C (top row, Figure 4), the fold increases without CB were higher than they were with CB. Other compounds showed a similar tendency, except for hydrogen peroxide, which showed the reverse owing to the relatively high frequency of micronucleated cells occasionally observed in the solvent control of the test without CB. At the highest concentrations of hydrogen peroxide and benzylchloride in the test with CB, strong cytotoxicity was seen and micronuclei were not observed, while without CB maximum responses were obtained at those concentrations. At high concentrations of hydroquinone and benzylchloride, the frequencies of micronucleated cells in the tests with CB reached a plateau, which seemed to be related to enhanced cytotoxicity, while the tests without CB showed dose-related increases of micronucleated cells.
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Correlation between micronuclei and chromosomal aberrations
The average frequencies of micronucleated cells in the solvent controls were 0.70% (n = 45), 0.83% (n = 74) and 0.78% (n = 65) in L1, L3 and L4, respectively, based on data accumulated between 1989 and 1994. In L2, the average frequency was 1.25% (n = 64) based on data obtained between 1992 and 1996. As the negative control values ranged from 0.1 to 2.0% (Table II
), chemicals yielding statistically significant MN frequencies with >4.0% were judged to be clear positives. When a compound induced a statistically significant MN frequency of <4.0%, it was judged a weak positive. When no statistical significance was obtained, it was judged negative. Exceptionally, some chemicals were judged negative if the biological significance of the data was doubtful, even though statistical significance was reached. These cases are explained in detail in the Discussion section.
Of 66 chemical compounds tested, 36 (54.5%) were clearly positive (+ in Table II). The dose–response curves of those compounds are illustrated in Figure 1. Seventeen compounds (25.8%) showed relatively weak responses with <4.0% of the maximum micronucleated cell frequency, although the increase was statistically significant (+w in Table II); the dose–response curves are shown in Figure 2. Thirteen compounds (19.7%) gave clear negative results (– in Table II) (Figure 3).
All 36 clear positive chemicals had been reported as positive in CA tests with cultured mammalian cells (Table III). Four compounds (colchicine, diethylstilbestrol, o-nitrotoluene and vinblastine sulfate) induced mainly polyploid cells. Of the 17 weak positive compounds, all except one (BCN) were positive in the CA tests. Two compounds [4,4'-methylenebis (2-chloroaniline) and m-nitrotoluene] induced mainly numerical aberrations, and for three (p-bromoaniline, p-fluoroaniline and mercuric acetate), we could find no CA data in the literature.
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Of the 13 negative compounds, four (3,5-diaminobenzoic acid, TPA, sodium chloride and urethane) yielded statistically significant increases in MN frequency. Detailed explanations for why these were judged to be negative are given in the Discussion section. Two compounds (mercuric chloride and tetrachloroethylene) were negative in the CA test, and six (p-chloroaniline, 2-chloro-4-nitroaniline, 2-methyl-4-nitroaniline, o-nitroaniline, p-nitrotoluene and phenacetin) were positive. Discrepancies between MN and CA test data for those compounds are dealt with in detail in the Discussion section. No data for the CA test were available for one MN-negative compound (p-iodoaniline).
When the four compounds without CA data are excluded, 49 (89.1%) of 55 CA-positive compounds were positive in the MN test, and six (85.7%) of seven CA-negative compounds were negative in the MN test (Table III). Thus concordance between the MN and CA tests was 88.7% (55/62).
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Four compounds yielded statistically significant increases in MN frequency, but were judged to be negative based on the following considerations.
3,5-Diaminobenzoic acid
The statistically significant increase in MN frequency was obtained by short treatment (6+66 h) with S9 mix, but it was not dose-dependent and the maximum frequency was only 1.7%, which was within the variations of the negative control value of the laboratory tested. In the CA test, positive results were obtained for structural aberrations with 24 and 48 h continuous treatments at 2500 and 3000 µg/ml (JETOC, 1996), but the MN test showed clear negative results with 24, 48 and 72 h continuous treatments at up to 3500 µg/ml. We cannot explain this discrepancy. In short treatments (6+18 h) with or without S9 mix, the CA test was negative (JETOC, 1996), and so the judgement for the short treatment MN test with S9 mix seems reasonable. As the positive finding in the CA test was obtained only at >10 mM (1520 µg/ml), it is biologically irrelevant as discussed for sodium chloride (see below). The MN and CA test results were identically negative.
TPA
In the first experiment, MN frequency increased with 72 h continuous treatment but no dose–response relationship was seen. In the second experiment, the increase in MN frequency was statistically significant for 72 h continuous treatment, but only at the highest concentration (50 µg/ml) (Figure 2). Because MN induction was not reproduced in the present test, our overall judgment was that TPA is negative, though additional tests may be needed to confirm this. The concurrent CA test showed no induction of chromosome aberrations. TPA did not induce any chromosome aberrations in CHL/IU cells (Ishidate, 1987), though it did in other cells (Ishidate et al., 1988). We judged TPA to be negative in both MN and CA tests in the CHL/IU system.
Sodium chloride
High concentrations of some chemical substances induce chromosome aberrations indirectly through non-physiologic osmolality or pH, or ion unbalance (Scott et al., 1991). In the present study, sodium chloride induced MNs at extremely high concentrations (2900–5800 µg/ml) and CA at 5000–7000 µg/ml (Ishidate, 1987). Such positive findings are irrelevant for the present study because sodium chloride was negative in both tests under the limit concentration of 10 mM.
Urethane
Urethane induced a statistically significant increase in MN frequency only in short treatments (6+42 h) with S9 mix. It induced the highest frequency at the middle concentration (1250 µg/ml), and frequencies decreased at the higher concentrations (2500 and 5000 µg/ml). This finding was considered biologically insignificant and urethane was judged negative. Urethane was also negative in the CA test with the short treatment (6+18 h) of up to 5000 µg/ml in the presence of S9 mix (unpublished data). It induced chromosome aberrations only at an extremely high concentration (8000 µg/ml) with 48 h continuous treatment (Ishidate, 1987), and this positive result was considered irrelevant.
Benzene, on the other hand, was judged positive in the present study based on 6 h treatment with 66 h recovery time in the presence of S9 mix; the maximum frequency was very low (1.6%), but response was statistically significant and the maximum frequency (1.6%) at 2340 µg/ml was just over the highest value for the negative controls (1.4%) in the tested laboratory. In the 6+18 h treatment at 2340 µg/ml, only 250 cells were available for analysis, and MN frequency was 6.2% (data not shown), but these data were not used for statistical analysis. Benzene at 1100–4400 µg/ml induced structural aberrations in short treatments (6+18 h) in the presence of S9 mix (Ishidate, 1987). The concentrations were extremely high, but smaller concentrations might have been effective because the chemical is volatile, though the tests were carried out in a closed system. Finally, this chemical was judged to be identically positive for both MN and CA tests.
The following six compounds were CA-positive but MN-negative, and some considerations on the discrepancies are indicated below.
p-Chloroaniline
The CA test was positive at concentrations of 200–400 µg/ml in the presence of S9 mix (Ishidate, 1987). However, no corresponding data are available for the MN test because the compound was toxic at >128 µg/ml. As the test chemical was diluted by factors of three, adequate concentrations might have been missed in the present study. Metabolic activation procedures were different for both tests: suspension treatment was used in the CA test while plate treatment was used in the MN test, and different metabolic enzyme-inducing agents were used as well. It is necessary to confirm the induction of chromosome aberrations under conditions similar to those used in the MN test.
2-Chloro-4-nitroaniline
Short treatment (6+18 h) with this compound induced structural aberrations with (26.5% at 200 µg/ml) and without (25% at 250 µg/ml) S9 mix (JETOC, 1996), though 24 and 48 h continuous treatments did not induce structural aberrations. On the other hand, short treatment MN tests with and without S9 mix up to 240 µg/ml showed negative results. Additional MN tests including relatively high concentrations (250 µg/ml or more) might need to confirm the present negative result. This chemical induced polyploid cells with 48 h continuous treatment (23% at 80 µg/ml), but there was no dose-dependency (2.0% and 8.5% at 40 and 120 µg/ml, respectively) (JETOC, 1996). Such pin-point responses might not be reproducible in any test system.
2-Methyl-4-nitroaniline
This compound induced polyploid cells with the 24 and 48 h continuous treatment: at 24 h a positive response (10.5%) was seen only at the middle concentration (100 µg/ml), and at 48 h a dose-dependent response was induced (14.0% and 35.6% at 100 and 200 µg/ml, respectively) (JETOC, 1997). Short treatment without S9 mix yielded marginal responses for structural and numerical aberrations, and with S9 mix it was negative for both types of aberrations. In the MN test with the 48 h continuous treatment at over 100 µg/ml, no data were available because of cytotoxicity. In the CA test carried out concomitantly, the compound induced polyploid cells at 100 µg/ml with 24 h (11.0%) and 48 h (10.3%) continuous treatment. Seventy-two-hour continuous treatment might be necessary for the MN test at concentrations of 100 µg/ml and more.
o-Nitroaniline
This compound, like 2-methyl-4-nitroaniline, induced polyploid cells with the 24 and 48 h continuous treatments. In the 24 h treatment, a marginal response (9.0%) was seen in CAs only at the lower concentration (130 µg/ml) of four concentrations (63–500 µg/ml), and in the 48 h treatment test, a dose-dependent response was seen (7.0% and 22% at 130 and 250 µg/ml, respectively) (JETOC, 1997). Short treatment without S9 mix induced polyploid cells without a dose–response relationship. The protocol also induced structural aberrations at the highest concentration (18.7% at 800 µg/ml) only. Short treatment in the presence of S9 mix induced structural aberrations with dose-dependency (10.0–35.4% at 200–800 µg/ml) (JETOC, 1997). Seventy-two-hour continuous treatment at the concentrations that induced polyploid cells in the CA test failed to induce MNs in two independent experiments. Additional MN tests using 48 h continuous treatments or narrow spacing between concentration levels might be needed. For the detection of structural aberrations, metabolic activation should be used, because clear structural aberration induction was observed in the test with S9 mix.
p-Nitrotoluene
This compound also induced polyploid cells but only at the highest concentration (250 µg/ml) with the 48 h continuous treatment (Ishidate, 1987). No structural chromosome aberrations were induced with the 24 and 48 h continuous treatments, though no data in the short treatment with and without S9 mix are available. In the present MN test, negative results were obtained in both short treatment with and without S9 mix and also in 24, 48 and 72 h continuous treatments. In the 48 h continuous treatment at 20 and 50 µg/ml, only 200 and 100 cells were available for analysis and MN frequencies were 1.0% and 4.0%, respectively (data not shown), but these data could not be used for results evaluation by our definition. Confirmation MN tests might be necessary especially using the 48 and 72 h continuous treatment, since o- and m-nitrotoluene were positive and weakly-positive, respectively, in the present MN test.
Phenacetin
In the presence of S9 mix, phenacetin induced structural aberrations at 400–1600 µg/ml (19–26%) (Ishidate, 1987). However, the CA test that we carried out concurrently with the MN test was negative with S9 mix; we obtained a weak positive response without S9 mix but saw no dose-related response. To explore this discrepancy among the CA tests, we conducted several experiments modifying different factors, such as the solvent (ethanol versus DMSO), the test chemical lot, cell lines (CHL versus V79), S9 mix pH, and concentrations up to 3200 µg/ml, but all eight independent tests yielded clear negative results (data not shown). One of the differences between the previous report and the present studies is the use of different compounds (Aroclor versus phenobarbital and 5,6-benzoflavone) to induce rat liver metabolizing enzymes that were in the S9 mix. Though identical negative results were obtained in both tests in this collaborative study, phenacetin was classified as a CA-positive, MN-negative chemical based on previous CA data. We still need to clarify why discrepant results were obtained between the previous and present CA tests.
Only one compound (BCN) was CA-negative and MN-positive. BCN induced MNs with short treatments (6+18 h) without S9 mix at 670–1330 µg/ml, while the frequency of CAs increased only slightly (7% and 8%) with 24 and 48 h continuous treatments at 500 µg/ml; at 1000 µg/ml, however, the aberration frequency decreased to 3% with continuous treatments (Ishidate, 1987). We found no short-treatment data for CAs. Therefore, the clastogenic potential should be confirmed using the same short treatment protocol and similar dose range as in the MN test.
Based on the above considerations, different test protocols might have caused the discordant findings. Therefore, it is likely that additional tests using the protocols suggested above will provide better concordance between the MN and CA tests.
Some cells with CAs, especially those with multiple aberrations, may die before mitosis is complete, but the micronucleated cells have survived through mitosis. These surviving aberrant cells are biologically more meaningful when interpreting genotoxicity. In addition, as interphase lasts considerably longer than metaphase, MNs in interphase may have a higher chance of being detected than CAs in metaphase. That higher frequencies of micronucleated cells were observed following the longer continuous treatments indicates that micronucleated cells accumulated during continuous treatment. In the case of CHL/IU cells, maximum sensitivity was generally observed after 48 or 72 h continuous treatment. After short treatments, on the other hand, the maximum sensitivity was observed at one sampling time (42 h after a 6 h treatment). Optimal treatment and sampling time, however, depends on the chemical, as discussed previously, and the conditions should be adjusted for each chemical.
The in vitro MN test using human lymphocyte cultures is performed by blocking cytokinesis with CB to restrict the cell population observed to those that have divided once following treatment (Fenech and Morley, 1985; Wakata and Sasaki, 1987). Such restriction may not be necessary for cells of established cell lines that grow continuously and homogeneously. According to the present study in a Chinese hamster cell line, the MN frequencies induced by clastogens and polyploid inducers were higher when CB was used than when it was not used. However, negative control values in tests with CB were higher too, while frequencies relative to the negative controls (fold increases) were higher in the tests without CB. We found cytotoxicity caused by CB as indicated in the published paper (Lindholm et al., 1991). When CB was used to test o-nitroaniline, which was negative without it, no significant increase of MN frequency was seen (data not shown). We could not find any reason to use CB in the present study in an established cell line. In addition, it is possible for a test chemical to interact with CB and enhance the cytotoxic effects. Considering these factors, at least when an established cell line is used, we believe that the CB cytokinesis block is not necessary and should not be used.
MNs are thought to be derived either from acentric chromosome fragments or whole chromosomes, as the result of clastogenic effects or disturbance of the mitotic apparatus, respectively. To distinguish those mechanisms, many attempts have been made with techniques such as kinetochore identification by fluorescent immunohistochemistry (Degrassi and Tanzarella, 1988), centromere detection by fluorescent in situ hybridization (Miller et al., 1991; Schriever-Schwemmer and Adler, 1994; Mäki-Paakkanen et al., 1995), sizing of MN (Yamamoto and Kikuchi, 1980; Högstedt and Karlsson, 1985) and C-banding (Vanderkerken et al., 1989). In the present study, we found that mitotic index and/or frequency of multinucleated cells could be used to identify numerical chromosome aberration inducers (Matsuoka et al., 1999), and this measurement could easily be made part of the routine analysis of micronuclei.
The present study revealed a high concordance between the in vitro MN and CA tests. The European collaborative study showed a similar concordance although their protocol was different from ours (Miller et al., 1997). The most important issue regarding the in vitro MN test is that an experimental protocol has not yet been internationally harmonized. In spite of the use of different experimental protocols, the in vitro MN test gave reliable outcomes, suggesting that it is as robust as the in vivo MN test. Thus, we believe that the in vitro MN test system could be used in place of the CA test as a simple and rapid method for detecting clastogens and aneugens in vitro.
Based on our experience in several laboratories, we propose the following basic protocol for the in vitro MN test: (i) cells should be treated in the presence and absence of an exogenous metabolic activation system; (ii) any proliferating mammalian cells can be used, e.g., established cell lines (CHL/IU, CHO, V79, L5178Y) or primary cell cultures (mitogenized human lymphocytes); (iii) a cytokinesis block is not required for continuously growing established cell lines; (iv) at least three concentration levels should be used to evaluate the concentration–response relationship, and concurrent negative (solvent/vehicle) and positive control groups should be included; (v) short treatments (e.g. 3–6 h) should be followed by recovery times that last as long as cells are growing exponentially, with and without an exogenous metabolic activation system, and when the result is negative cells should be treated continuously without an exogenous metabolic activation system as long as they are growing exponentially; (vi) the slide preparation method should preserve the cytoplasm; (vii) at least 1000 intact cells with a single main nucleus should be analyzed for the presence of MN; (viii) data should be evaluated statistically and biologically regarding the increase in the frequency of micronucleated cells, and concentration–response relationship or reproducibility; (ix) a positive response in the in vitro MN test indicates that the test chemical induces CAs (structural and/or numerical).
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Acknowledgments |
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This study was supported by a grant from Ministry of Labour, Japan and Japan Chemical Industry Ecology-Toxicology and Information Center (JETOC). We are indebted to Ms Matsuura for help in preparing this manuscript. We also thank Dr Noriho Tanaka, Dr Lutz Müller and Dr Miriam Bloom for their critical review of the manuscript and English editing.
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Notes |
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6 To whom correspondence should be addressed at present address: Chromosome Research Center, Olympus Optical Co. Ltd, 2-3 Kuboyama-cho, Hachioji-shi, Tokyo 192-8512, Japan. Tel: +81 426 91 7115; Fax: +81 426 91 7209; Email: t_sofuni{at}ot.olympus.co.jp
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Received on March 3, 1999; accepted on July 12, 1999.
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