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Mutagenesis, Vol. 14, No. 4, 385-389, July 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press

A proposal for a simple way to distinguish aneugens from clastogens in the in vitro micronucleus test

Atsuko Matsuoka1, Katsuko Matsuura, Hiroko Sakamoto, Makoto Hayashi and Toshio Sofuni

Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
In our previous in vitro micronucleus (MN) study, we showed that aneugens, in addition to inducing micronuclei, induce a higher frequency of polynuclear (PN) and mitotic (M) cells than clastogens. We hypothesized that the frequency of PN and M cells induced can distinguish aneugens from clastogens. To test the hypothesis, we conducted the micronucleus tests with mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), vincristine (VINC) and diazepam in a Chinese hamster cell line (CHL) and VINC, benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) in a subclone of V79 cells (V79-MZ). All chemicals increased the frequency of M cells with statistical significance. All chemicals except diazepam increased the frequency of PN cells with statistical significance. Three of the aneugens (VINC, BP and DMBA) induced >=200 PN cells/1000 cells while the clastogens (MNNG and MMC) induced 100 PN cells at most. All the aneugens but no clastogens significantly increased the frequency of M cells. We propose that micronucleus test-positive chemicals that induce >=200 PN cells/1000 cells and significantly increase the frequency of M cells are aneugens and those that induce at most 100 PN cells/1000 cells and do not significantly increase the frequency of M cells in our MN test protocol are clastogens. Diazepam, however, did not induce PN cells, although it increased the frequency of M cells dose dependently. We explain this fact in relation to diazepam's mode of action. Our proposal suggests a quick, easy and practical way to distinguish aneugens from clastogens for screening purposes.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The micronucleus (MN) test detects both structural and numerical chromosome aberrations. It may be used as an alternative to the conventional in vitro chromosomal aberration test, but it does not distinguish aneugens from clastogens. That distinction can be made by analysis of MN size and morphology (Yamamoto and Kikuchi, 1980Go; Hogstedt and Karlsson, 1985Go; Tinwell and Ashby, 1991Go) or immunofluorescent staining with anti-kinetochore antibody in the in vivo (Gudi et al., 1990Go; Schriever-Schwemmer and Adler, 1994Go) and in vitro (Eastmond and Tucker, 1989Go) MN test. The fluorescence in situ hybridization (FISH) technique using centromeric probes has also been used for detecting aneuploidy in both the in vitro (Kirsch-Volders et al., 1997Go; Schuler et al., 1997Go) and in vivo (Hayashi et al., 1994Go; Schriever-Schwemmer and Adler, 1994Go; Mäki-Paakkanen et al., 1995Go) MN test. Chromosome-specific centromeric probes have been used as well to detect aneugens in human lymphocytes in interphase (Eastmond and Pinkel, 1990Go). These are sophisticated and time-consuming methods and we do not have centromeric probes for all species used in the MN test.

The conclusion that the in vitro MN test can be an alternative to the in vitro chromosomal aberration test was based on data from 14 chemicals (12 clastogens and two aneugens) studied in Chinese hamster lung (CHL) cells (Matsuoka et al., 1993Go). We found in that study that aneugens induce higher frequencies of polynuclear (PN) and mitotic (M) cells than clastogens do. As the number of PN and M cells appearing in the same microscopic field as 1000 interphase cells observed for MN cells was recorded as additional end-points, the number of cells to be observed for them was not defined. Therefore, a precise comparison of the number of PN and M cells could not be made in that study. In this study, we analyzed PN and M cells more carefully on 1000 cells to test whether the in vitro MN test can indeed be used to discriminate aneugens from clastogens.

We also found previously that benzo[a]pyrene (BP) and 7,12-dimethylbenz[a]anthracene (DMBA) induce aneuploidy and polyploidy, respectively, in V79-MZ cells (Matsuoka et al., 1997Go) in the absence of an exogenous metabolic activation system. We clarified that aneuploidy and polyploidy induction are due to the abnormality and inhibition of spindle formation after BP and DMBA treatment, respectively (Matsuoka et al., 1998Go). These are good model aneugens and we used them in the present study to test the above hypothesis.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cells
A Chinese hamster fibroblast cell line (CHL) (Ishidate and Odashima, 1977Go) was maintained in Eagle's minimum essential medium (MEM) (Gibco 61100-061) supplemented with 10% heat-inactivated calf serum. The doubling time was ~15 h and the modal chromosome number was 25.

Another Chinese hamster cell line, V79-MZ (Glatt et al., 1987Go), kindly supplied by Professor Glatt, was subcloned and maintained in Dulbecco's modified Eagle's medium (D-MEM) (Gibco 31600-034) supplemented with 5% fetal bovine serum (Flow Laboratories, Rockville, MD). The doubling time was ~12 h and the modal chromosome number was 22.

Chemicals
N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (CAS no. 70-25-7) purchased from K & K Laboratory (Plainview, NY), mitomycin C (MMC) (CAS no. 50-07-7) purchased from Kyowa Hakko Kogyo Co. (Tokyo, Japan) and vincristine sulfate (VINC) (CAS no. 2068-78-2) purchased from Wako Pure Chemical Industries (Osaka, Japan) were dissolved in physiological saline. Diazepam (CAS no. 439-14-5) was purchased from Wako Pure Chemical Industries and dissolved in dimethyl sulfoxide (DMSO). The aneugens BP (CAS no. 50-32-8) and DMBA (CAS no. 57-97-6) were purchased from Wako Pure Chemical Industries and dissolved in DMSO.

In vitro MN test
Cells were seeded at a density of 1.5x105/plate (60 mm in diameter). After 17 h incubation, they were treated with the test chemical for 24 (V79-MZ cells) or 30 h (CHL cells) and harvested immediately. MN preparations were made as reported (Matsuoka et al., 1993Go). Briefly, cells were treated with 0.05% trypsin in 0.02% EDTA and the cell suspension was incubated in hypotonic KCl solution for 5–10 min at room temperature. The cells were fixed twice with ice-cold fixative (glacial acetic acid:methanol 1:3) and then suspended in methanol containing 0.5–1% acetic acid. A drop of the suspension was placed on a clean glass slide and air dried. The cells were stained by mounting in 40 µg/ml acridine orange in phosphate-buffered saline and immediately observed at 400x magnification by fluorescence microscopy with a model Olympus BX50 and a U-MWBV filter.

All slides were coded and the number of micronucleated cells among 1000 intact interphase cells (those with cytoplasm around the main nucleus) was counted. Cells with a main nucleus and a single micronucleus (MN) were categorized into two groups, those with a MN smaller than one-third of the main nucleus in diameter and those with a large MN between one-third and half the size of the main nucleus. A MN attached to the main nucleus was not scored. A cell with two or more MN was recorded as a multi MN cell. In an additional 1000 total cells (those with cytoplasm around the main nucleus, polynuclei or chromosomes) we scored polynuclear (PN) cells (cells with fragmented nuclei), including karyorrhectic cells (cells with abnormally shaped nuclei) and mitotic (M) cells. PN and karyorrhectic cells were categorized morphologically. Those cells were recorded regardless of the presence of MN in the cell. Two scorers observed 500 cells each.

We analyzed the data using the {chi}2 test for treated versus control groups.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The number of MN cells/1000 intact interphase cells and the number of PN and M cells/1000 total cells after treatment are shown in Figures 1–3GoGoGo. The ratio of large MN and multi MN to total MN cells is shown in Table IGo. The historical solvent control values of MN, PN and M cells were 6.95 ± 2.36, 4.75 ± 2.41 and 21.75 ± 4.42, respectively, for CHL cells and 10.18 ± 5.91, 1.67 ± 1.76 and 24.67 ± 3.51, respectively, for V79-MZ cells.



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  		Fig. 1. The effect of MNNG and MMC (30 h treatments) in CHL cells. The black portion represents cells with a MN whose diameter is less than one-third the diameter of the main nucleus. The shadowed portion represents cells with a MN whose diameter is one-third to a half the diameter of the main nucleus. The white portion represents cells with multiple MN. The upper graphs indicate the number of MN cells/1000 intact interphase cells. The middle graphs indicate the number of PN cells/1000 total cells including karyorrhectic cells. The white portion shows karyorrhectic cells and the black portion shows PN cells. The bottom graphs indicate the number of M cells/1000 total cells. *P < 0.05, **P < 0.01.

 


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  		Fig. 2. The effect of VINC and diazepam (30 h treatments) in CHL cells and VINC (24 h treatment) in V79-MZ cells. Details as described in legend to Figure 1Go.

 


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  		Fig. 3. The effect of BP and DMBA (24 h treatments) in V79-MZ cells. Details as described in legend to Figure 1Go.

 

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  		Table I. Types of MN induced by chemicals after 24 (V79-MZ) or 30 h (CHL) treatment and the ratio of large MN and multi MN cells to total MN cells
 
The clastogens MNNG and MMC induced a dose-related increase in the frequency of MN cells (Figure 1Go) and in the ratio of multi MN to total MN cells (Table IGo). Both chemicals induced a slight (<100) but statistically significant number of PN cells/1000 cells but did not significantly increase the frequency of M cells (Figure 1Go).

The aneugens VINC and diazepam significantly induced MN in CHL and VINC in V79-MZ cells (Figure 2Go). The ratio of large MN to total MN cells induced by VINC peaked at 0.42 at the highest dose in CHL cells and was steady at ~0.2 in V79-MZ cells (Table IGo). The ratio of multi MN to total MN cells increased with dose up to 0.31 in CHL cells and was relatively constant at ~0.3 in V79-MZ cells. Diazepam induced a much lower increase in MN frequency than VINC and almost all the cells had MN (<1/3) (Table IGo). VINC induced PN and M cells dose dependently in both cell lines. Diazepam, in contrast, induced M cells but not PN cells. Diazepam had a much smaller effect than VINC and it caused severe cytotoxicity at 150 µg/ml.

The aneugens BP and DMBA induced all the end-points scored (Figure 3Go). Over the dose range 1–4 µg/ml, BP decreased the ratio of large MN to total MN cells from 0.29 to 0.21 and the ratio was ~0.2 at all doses of DMBA (Table IGo). BP and DMBA increased the ratio of multi MN to total MN cells (Table IGo). DMBA decreased the frequency of MN and PN cells, both dose dependently, but increased the frequency of M cells dose dependently.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Based on our previous study (Matsuoka et al., 1993Go), we predicted that the induction of large MN together with an elevated frequency of M cells distinguishes aneugens from clastogens.

In the present study, both clastogens and aneugens induced MN cells significantly. Clastogens induced at most 100 PN cells/1000 cells and did not cause a significant increase in M cell frequency. Aneugens, except diazepam, induced a high frequency of PN and M cells. Diazepam was unique in that it did not significantly induce PN cells; only the greater induction of M cells distinguished diazepam from clastogens. The induction of PN cells might identify aneugens that affect tubulin versus those with a different mechanism of action. VINC acts by inducing the formation of paracrystalline aggregates of tubulin, which leads to microtubule depolymerization, whereas diazepam mainly inhibits centriole separation at prophase.

Aneuploidy induction by diazepam is difficult to observe in the conventional chromosome aberration test (Ishidate, 1988Go; Ishidate et al., 1988Go). In a detailed in vitro investigation, Hsu et al. (1983) reported that diazepam induces aneuploidy and increases the number of anaphases with multipolar and lagging chromosomes. In the present study, diazepam increased the frequency of M cells but not PN cells, which may follow from multipolar anaphases. VINC increased the frequency of both PN and M cells. That difference between diazepam and VINC suggests that it may be possible to discriminate between a chemical that affects parts of the mitotic apparatus other than tubulin and a chemical that affects tubulin. On the other hand, Natarajan et al. (1993) reported that diazepam induces aneuploidy and decreases the mitotic index dose dependently. In the present study, diazepam caused a dose-dependent increase in the frequency of M cells and Warr et al. (1993) reported similar results. The reason for the discrepancy in the frequency of M cells is not known.

Glatt et al. (1990) reported negative results in the MN test for BP and DMBA at up to 1 µg/ml in V79 cells (the same cell line as V79-MZ in the present study). In the present study, BP was also negative at 1 µg/ml but was positive at higher doses, while DMBA was positive at 1 µg/ml but negative at 0.5 µg/ml. The negative result by Glatt et al. (1990) was due to low doses. Ellard et al. (1991) reported that BP tested in V79 cells at up to 25 µg/ml in the absence of S9 mix induces a small but statistically significant increase in MN frequency. That low increase may have been due to the shorter treatment time of 4 h. The effect of BP and DMBA in V79-MZ cells demonstrated that aneugens yield high frequencies of PN and M cells and that fits our proposal in another cell line in addition to CHL cells.

The induction of larger MN by spindle poisons than by clastogens has been reported in mouse bone marrow cells (Yamamoto and Kikuchi, 1980Go) and cultured human lymphocytes (Hogstedt and Karlsson, 1985Go). We obtained concordant results in that we observed a higher ratio of large MN to total MN cells with VINC, BP (only in V79-MZ cells) and DMBA (only in V79-MZ cells) than with MNNG and MMC. Tinwell and Ashby (1991) reported that in the in vivo mouse MN assay, crescent and large MN indicate aneugenic activity. They used the size and shape of MN to distinguish aneugens and clastogens. We found that the frequency of PN and M cells can also distinguish aneugens from clastogens.

In conclusion, the two clastogens tested in this study induced MN but not more than 200 PN or M cells. All the four aneugens increased the frequency of M and MN cells. In addition, three of the aneugens induced >200 PN cells/1000 cells. We propose here that in addition to the increase in MN cells, chemicals in our MN test protocol that induce >=200 PN cells/1000 cells and a statistically significant increase in M cells are aneugens and chemicals that induce <100 PN cells/1000 cells and do not increase M cell frequency are clastogens. Aneugens such as diazepam that act on parts of the mitotic apparatus other than tubulin might not induce a significant number of PN cells. Even so, the increase in M cell frequency can be an indicator for aneugens. A high ratio of large MN to total MN cells provides additional evidence that the tested chemical is an aneugen.


   Notes
 
1 To whom correspondence should be addressed. Tel: +81 3 3700 1141; Fax: +81 3 3700 2348; Email: matsuoka{at}nihs.go.jp Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

    Eastmond,D.A. and Pinkel,D. (1990) Detection of aneuploidy and aneuploidy-inducing agents in human lymphocytes using fluorescence in situ hybridization with chromosome-specific DNA probes. Mutat. Res., 234, 303–318.[Web of Science][Medline]

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    Glatt,H.-R., Gemperlein,I., Setiabudi,F., Platt,K.L. and Oesch,F. (1990) Expression of xenobiotic-metabolizing enzymes in propagatable cell cultures and induction of micronuclei by 13 compounds. Mutagenesis, 5, 241–249.[Abstract/Free Full Text]

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Received on November 24, 1998; accepted on March 18, 1999.


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