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Cytogenetic effects of 3,4-dichloroaniline in human lymphocytes and V79 Chinese hamster cells
Mutation Research, 226 (1989) 197-202
197
Elsevier MUTLET 0226
Cytogenetic effects of 3,4-dichloroaniline in human lymphocytes and V79 Chinese hamster cells M. Bauchinger, U. K u l k a a n d E. S c h m i d lnstitut fffr Strahlenbiologie, Gesellschaft fiir Strahlen- und Umweltforschung ( GSF), D-8042 Neuherberg/ Munich ( F.R.G.) (Accepted 21 March 1989)
Keywords: 3,4-Dichloroaniline; Clastogenicity; Sister-chromatid exchange; Spindle poison; Human lymphocytes; V79 cells
Summary 3,4-Dichloroaniline (3,4-DCA), an intermediate in various chemical syntheses, has been detected as an environmental contaminant in surface waters and in the effluents from dye-manufacturing plants. Tested for clastogenicity in human lymphocytes in vitro the compound was inactive in the chromosome aberration assay yet exhibited a positive sister-chromatid exchange response in the presence of a mammalian metabolic activation system. Exposure of V79 Chinese hamster cells to 3,4-DCA caused a concentration-dependent increase in the incidence of spindle disturbances, predominantly of the initial c-mitotic type. The results indicate that 3,4-DCA might induce aneuploidy in mammalian cells by interaction with the mitotic apparatus.
To define the hazards of environmental chemicals data on their genotoxic and carcinogenic properties are required. For 3,4-dichloroaniline (3,4-DCA), a compound selected in a priority list of existing chemicals (BUA, 1985), only insufficient data are available. It is an intermediate in the synthesis of dyes, pigments and several urea and acylanilide herbicides (Di Muccio et al., 1984). After degradation of these herbicides by microorganisms in soil or water (Bartha and Pramer, 1967; Deuel et al., 1977; E1-Dib and Aly, Correspondence: Dr. M. Bauchinger, Institut fiir Strahlenbiologie, Gesellschaft for Strahlen- und Umweltforschung (GSF), D-8042 Neuherberg/Munich (F.R.G.).
1976) 3,4-DCA has been detected in various surface waters in western Europe (Wegman and de Korte, 1981), in flooded rice field cultures (Deuel et al., 1977) and in the effluents from dyemanufacturing plants (Games and Hites, 1977). In a hepatocyte/DNA-repair test the compound failed to induce a DNA-repair response (Yoshimi et al., 1988). The present study examines whether chromosome aberrations (CA) and sisterchromatid exchanges (SCE) can be induced in human lymphocytes treated in vitro in the presence or absence of a metabolic activation system. V79 Chinese hamster cells are used to test whether spindle disturbances can be caused.
0165-7992/89/$ 03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
198
Material and methods
Chromosome and SCE analysis 3,4-DCA with a purity of 99% was purchased from Aldrich Chemie (Steinheim, F.R.G.). For the experiments duplicate lymphocyte cultures of a healthy female donor were set up with 0.5 ml whole blood, 4 ml RPMI 1640 medium, 0.5 ml fetal calf serum and 0.13 ml phytohemagglutinin; the cultures were incubated at 37°C. 24 h after culture initiation the test substance, dissolved in dimethyl sulfoxide (DMSO), was added for 1 h, either with or without $9 mix (protein content 5 mg/ml mix). The $9 fraction was prepared from Clophen A 50-induced male Wistar rats with minor modifications according to Ames et al. (1975). A detailed description of the metabolic activation system used has been published (G6ggelmann, 1984; Kugler et al., 1987). After the 3,4-DCA exposure cells were washed twice with RPMI medium and new cultures were set up supplemented with bromodeoxyuridine
in a final concentration of 7.75 #M and incubated for a further 29 or 47 h, with 0.1 ~g/ml Colcemid added for the final 3 h. The total incubation time was thus 54 or 72 h. Cultures exposed to 0.33% DMSO served as solvent control. 0.05 mM cyclophosphamide (CP, Serva) was used as a positive control for the metabolic activation system. Chromosome preparation and fluorescence plus Giemsa staining (FPG) were carried out according to our standardized laboratory procedures (Kolin-Gerresheim and Bauchinger, 1981; Apelt et al., 1981). Analysis of CA was performed exclusively in complete first-division metaphases (M1, 54 h culture time) identified by uniformly stained sister chromatids. SCEs were scored in harlequin-stained second-division metaphases
100-
"~ 8 0 o
~h
~5o-
100"~ ' ~ 4 0 -
"////" .....
20
8O
0
,111~ ~11//
/ i / /
"E/;<4 /i//~
100-
~ ~o
~ 80I ieoIE
:~ ~4ou,, 6 20-
O-
0
0
,
0.125 0.25 0.5 3,4 - Dichlor'oeniline ( m M )
1.0
Fig. 1. Effect of 3,4-DCA on the frequency of total mitoses in V79 cells. Cells were treated and processed as described in Material and methods. Results (means +- SD from 3 × 1000 cells in 2 slides from duplicate cultures) of 2 independent experiments (open and hatched columns) are shown.
0
0.125 0.25 0.5 1.0 3,4- Dichlo¢'ooniline ( r a m )
Fig. 2. Effects of 3,4-DCA on the frequency of metaphases in V79 cells. Cells were treated and processed as described in Material and methods. Results are pooled data of 2 independent experiments. (A) Frequency of total metaphases. (B) Frequency of c-metaphases (open columns, initial c-mitoses; hatched colu m n s , complete c-mitoses).
199
TABLE
1
FREQUENCY
OF
CHROMOSOMAL
METABOLICALLY
A C T I V E $9 M I X
CHANGES
INDUCED
BY
3,4-DCA
IN
THE
PRESENCE
AND
ABSENCE
OF
Experiment
Conc.
Gaps a
S-cells a
A b e r r a t i o n s p e r cell a
S C E s p e r cell b
(exposure conditions)
(mM)
p e r cell
(%)
(chromatid type)
+ SEM
Breaks 3,4-Dichloroaniline ( - $9 mix)
3,4-Dichloroaniline ( + $9 mix)
Exchanges
0
0.025
-
-
0.25
0.020
-
-
7.30 ± 0.55 8.26 ± 0.55
0.50
0.030
0.5
0.005
-
7.42 ± 0 . 5 0
1.0
0.050
0.5
0.005
-
9.24 ± 0.43
0
0.020
1.0
0.010
-
7.58 ± 0 . 5 2
0.125
0.045
1.0
0.010
-
15.18 ± 0.58
0.25
0.035
0.5
0.005
-
18.50 ± 0.57
0.50
0.045
2.0
0.020
-
23.30 ± 0.66
1.0
0.035
3.0
0.025
0.005
23.38 ± 0.74
0.05
0.410
44.0
0.625
0.110
Cyclophosphamide ( + $9 mix)
- 100
S-cells, cells w i t h s t r u c t u r a l c h r o m o s o m e a b e r r a t i o n s . Cells p e r s a m p l e : a 200, b 50.
(M2, 72 h culture time). Cells containing structural CA were classified as S-cells.
Analysis of spindle disturbances V79 Chinese hamster cells were used to study the induction of spindle disturbances by 3,4-DCA. The cells were cultured in Dulbecco's modified Eagle's minimum essential medium (Biochrom, Berlin, F.R.G.) supplemented with 10070 fetal calf serum (Flow, Meckenheim, F.R.G.), 100 U/ml penicillin and 100 /zg/ml streptomycin at 37°C in a humidified atmosphere containing 7°70 CO2. For the experiments 0.5-1 × 105 cells were seeded in 60-mm dishes containing 25 x 30 mm coverslips. After 24 h cells were washed with phosphatebuffered saline (PBS) and exposed to 3,4-DCA in serum-free medium for the last 3 h of culture time. Controls received DMSO only. After exposure cells were washed with Hanks' balanced salt solution and carefully fixed without hypotonic and Colcemid treatment directly on the coverslips (Bauchinger et al., 1988). For the analysis of spindle disturbances 500 cells were scored from duplicate cultures of 2 independent experiments.
The mitotic index was determined by scoring 3 × 1000 cells in 2 slides of duplicate cultures. The polyploidy index gives the number of polyploid mitoses as the percentage of total mitoses scored. Results and discussion
The highest dose of 3,4-DCA which did not impair cell proliferation of human lymphocytes was 1 mM. The results of the chromosome analysis are given in Table 1. The structural CA observed in M 1 were exclusively of the chromatid type with single or isolocus breaks and were accompanied by gaps. With and without metabolic activation there was no evidence of significant differences in the incidences of chromatid breaks, gaps and S-cells between 3,4-DCA-treated and untreated cultures ( p < 0 . 0 5 in a double-sided Wilcoxon-MannWhitney rank test; Lehmann, 1975). Compared to controls the incidence of SCEs was significantly increased in the absence of $9 mix at 1 mM 3,4-DCA only and in the presence of $9 mix at each concentration (Table 1). Metabolic activation induced SCE frequencies almost 3-fold.
200 TABLE 2 MEAN VALUES (°70 ± SEM) OF N O R M A L A N D D A M A G E D M I T O T I C FIGURES OBSERVED IN D U P L I C A T E CULTURES OF 2 EXPERIMENTS W I T H V79 CELLS AFTER 3 h TREATMENT W I T H 3,4-DCA 3,4-Dichloroaniline (mM) Cells analyzed Normalmitoticfigures Prophases Metaphases Ana-telophases Mitoses with spindle disturbances Initial c-mitoses Prophases Metaphases c-Mitoses Prophases Metaphases Multipolar ana-telophases Anaphases with lagging or non-disjunction
0
0.125
0.25
0.5
1.0
1000
1000
1000
1000
1000
90.4±0.9 16.1±1.2 55.8±1.6 18.5±1.2
90.5±0.9 15.3±1.1 56.1±1.6 19.1±1.2
87.9±1.0 16.4±1.2 54.7±1.6 16.8±1.2
71.8±1.4 10.8±1.0 51.8±1.6 9.2±0.9
24.5±1.4 7.1±0.8 16.8±1.2 0.6±0.2
23.2 17.8 1.2 16.6 4.9 0
69.5 62.3 1.4 60.9 7.0 0
5.5 3.8 0.6 3.2 1.1 0
± ± ± ± ±
0.7 0.6 0.2 0.6 0.3
6.5 4.6 0.8 3.8 1.1 0
± ± + + ±
0.8 0.7 0.3 0.6 0.3
8.5 7.0 0.5 6.5 1.3 0
± 0.9 ±_ 0.8 + 0.2 ± 0.8 ± 0.4
± ± ± ± ±
1.3 1.2 0.3 1.2 0.7
± ± ± ± ±
1.5 1.5 0.4 1.5 0.8
1.1 ± 0.3 0.3 ± 0.2
1.1 ± 0.3 0.5 + 0.2
1.3 ± 0.4 0.2 ± 0.1
4.9 ± 0.7 0.3 ± 0.2
7.0 ± 0.8 0
0.3 ± 0.2
0.3 ± 0.2
0
0.2 ± 0.1
0.2 ± 0.1
Ana-telophases with bridges a n d / o r fragments
0.3 ± 0.2
0.4 ± 0.2
0.4 ± 0.2
0.1±0.1
0
Mitoses with stickiness and pycnosis
1.2±0.3
0.8±0.4
0.9±0.3
2.4±0.5
2.7±0.5
Polyploidy index
2.6±0.5
1.8±0.4
2.3±0.5
2.5±0.5
3.3±0.6
The effect of a 3-h exposure of V79 cells on cell proliferation is demonstrated in Fig. 1. At 0.5 and 1.0 mM 3,4-DCA the mitotic index was significantly increased compared to controls. At 2.0 mM (not shown) only highly pycnotic mitotic figures were observed indicating cytotoxicity. The results on the induction of spindle disturbances by 3,4-DCA are shown in Table 2. The substance caused a concentration-dependent increase in the number of abnormal mitotic figures. At 1.0 mM 3,4-DCA their incidence was increased about 13-fold above that found in controls. A c-mitotic effect was reflected in the appearance of a metaphase block (Fig. 2A) and the concomitant disappearance of ana-telophases. As shown in Fig. 2B metaphases were predominantly of the initial c-mitotic type (Levan, 1954; Schmid and Bauchinger, 1976). About 60°70 had the appearance of so-called ball
metaphases with chromosomes concentrated in the cell centre, about 30°70 had their chromosomes arranged in several groups and about 10% showed non-congression, i.e., lack of correct arrangement of single chromosomes onto the metaphase plate. The picture of typical c-metaphases with contracted chromosomes scattered in the cytoplasm was only infrequently observed (Fig. 2B). Multipolar ana-telophases or non-disjunction were not found more frequently than in controls. The polyploidy index was not higher in treated cultures than in controls. The frequency of ana-telophases with bridges and/ or fragments never exceeded 0.4°70. At 0.5 and 1.0 mM 3,4-DCA the number of mitotic figures with stickiness and pycnosis was about twice that observed in controls. In conclusion, the data show that 3,4-DCA fails
201
to induce a significant clastogenic effect in human lymphocytes in the presence and absence of an in vitro metabolizing activation system. According to UKEMS guidelines on mutagenicity testing (Scott, 1983; Scott et al., 1983) where at least a doubling in SCE frequency should be accepted as a positive response, 3,4-DCA is active as an SCE-inducing agent in the presence of rat liver $9 mix. The induction of SCEs is generally referred to the induction o f DNA damage. However, one should bear in mind that phenomena other than DNA reactivity may contribute to the induction of SCEs. Evidence of this comes from a study of Montaldi et al. (1985) who found that interaction of the chelating agent nitrilotriacetic acid with heavy metals led to enhanced SCEs. Thus, an isolated positive SCE response in the absence of a clastogenic response does not allow a clear decision whether a chemical must be regarded as an in vitro genotoxin. Beyond this the significance of SCEs to mutagenicity and carcinogenicity in humans is still not known. 3,4-DCA is, however, a potent spindle poison in V79 cells. Although mitotic arrestants are not necessarily aneuploidy-inducing agents, due to its ability to cause spindle disturbances 3,4-DCA must be considered to have genotoxic activity.
Acknowledgements We thank R. Giesen and F. Zelles for technical assistance. This work has been supported by the Bayerisches Staatsministerium ftir Landesentwicklung und Umweltfragen.
References Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/ mammalian microsome mutagenicity test, Mutation Res., 31, 347-365. Apelt, F., I. Kolin-Gerresheim and M. Bauchinger (1981) Azathioprine, a clastogen in human somatic cells? Analysis of chromosome damage and SCE in lymphocytes after exposure in vivo and in vitro, Mutation Res., 88, 61-72. Bartha, R., and A. Pramer (1967) Pesticide transformation to
aniline and azo compounds in soil, Science, 156, 1617-1618. Bauchinger, M., E. Schmid, F.J. Wiebel and E. Roscher (1988) 1,6-Dinitropyrene causes spindle disturbances and chromosomal damage in V79 Chinese hamster cells, Mutation Res., 208, 213-218. BUA (1985) Beratergremium fiir umweltrelevante Altstoffe, Kriterien und Stoffliste, Gesellschaft Deutscher Chemiker. Deuel, L.E., K.W. Brown, F.C. Turner, D.G. Westfall and J.D. Price (1977) Persistence of propanil, DCA and TCAB in soil and water under flooded rice culture, J. Environ. Qual., 6, 127-132. Di Muccio, A., 1. Camoni and R. Dommarco (1984) 3,3',4,4'-Tetrachloroazobenzene and 3,3',4,4'-tetrachloroazooxybenzene in technical grade herbicides: propanil, diuron, linuron, and neburon, Ecotoxicol. Environ. Safety, 8, 511-515. EI-Dib, M.A., and O.A. Aly (1976) Persistence of some phenylamide pesticides in the aquatic environment. 1II. Biological degradation, Water Res., 10, 1055-1059. Games, L.M., and R.A. Hites (1977) Composition, treatment efficiency, and environmental significance of dye manufacturing plant effluents, Anal. Chem., 49, 1433-1440. G6ggelmann, W. (1984) Problems of microbial mutagenicity testing as exemplified by Salmonella/microsome test, in: R. Bass, V. Glocking, P. Grossdanoff, D. Henschler, B. Kilbey, D. Mtiller and D. Neubert (Eds.), Critical Evaluation of Mutagenicity Tests, MMV Medizin Verlag, Munich, pp. 243-258. Kolin-Gerresheim, I., and M. Bauchinger (1981) Dependence of the frequency of harlequin-stained cells on BrdU concentration in human lymphocyte cultures, Mutation Res., 91, 251-254. Kugler, U., M. Bauchinger, E. Schmid and W. G#ggelmann (1987) The effectiveness of $9 and microsomal mix on activation of cyclophosphamide to induce genotoxicity in human lymphocytes, Mutation Res., 187, 151-156. Lehmann, E.L., and H.J.M. D'Abrera (1975) Nonparametrics, Holden-Day, San Francisco/McGraw Hill, New York, p. 210. Levan, A. (1954) Colchicine-induced C-mitoses in two mouse ascites tumors, Hereditas, 40, 1-64. Montaldi, A., L. Zentilin, P. Venier, I. Goli, V. Bianchi, S. Paglialunga and A.G. Levis (1985) Interaction of NTA with heavy metals in the induction of SCE in cultured mammalian cells, Environ. Mutagen., 7, 381-390. Schmid, E., and M. Bauchinger (1976) The cytogenetic effect of an X-ray contrast medium in Chinese hamster cell cultures, Mutation Res., 43,291-298. Scott, D. (1983) An evaluation of cytogenetic tests, including SCE and micronuclei, used in genotoxicity studies, Colloque INSERM/EEMS, 119, 125-134. Scott, D., B. Danford, D. Dean, D.J. Kirkland and C. Richardson (1983) In vitro chromosome aberration assay, in: B.J. Dean (Ed.), UKEMS Guidelines for Mutagenicity Testing, UKEMS, pp. 41-46.
202 Wegman, R.C.C., and G.A.L. de Korte (1981) Aromatic amines in surface waters of The Netherlands, Water Res., 15, 391-394. Yoshimi, N., S. Sugie, H. Iwata, K. Niwa, H. Mori, C. Hashida and H. Shimizu (1988) The genotoxicity of a variety of aniline derivatives in a DNA repair test with primary cultured rat hepatocytes, Mutation Res., 206, 183-191. Communicated by H. Greim
197
Elsevier MUTLET 0226
Cytogenetic effects of 3,4-dichloroaniline in human lymphocytes and V79 Chinese hamster cells M. Bauchinger, U. K u l k a a n d E. S c h m i d lnstitut fffr Strahlenbiologie, Gesellschaft fiir Strahlen- und Umweltforschung ( GSF), D-8042 Neuherberg/ Munich ( F.R.G.) (Accepted 21 March 1989)
Keywords: 3,4-Dichloroaniline; Clastogenicity; Sister-chromatid exchange; Spindle poison; Human lymphocytes; V79 cells
Summary 3,4-Dichloroaniline (3,4-DCA), an intermediate in various chemical syntheses, has been detected as an environmental contaminant in surface waters and in the effluents from dye-manufacturing plants. Tested for clastogenicity in human lymphocytes in vitro the compound was inactive in the chromosome aberration assay yet exhibited a positive sister-chromatid exchange response in the presence of a mammalian metabolic activation system. Exposure of V79 Chinese hamster cells to 3,4-DCA caused a concentration-dependent increase in the incidence of spindle disturbances, predominantly of the initial c-mitotic type. The results indicate that 3,4-DCA might induce aneuploidy in mammalian cells by interaction with the mitotic apparatus.
To define the hazards of environmental chemicals data on their genotoxic and carcinogenic properties are required. For 3,4-dichloroaniline (3,4-DCA), a compound selected in a priority list of existing chemicals (BUA, 1985), only insufficient data are available. It is an intermediate in the synthesis of dyes, pigments and several urea and acylanilide herbicides (Di Muccio et al., 1984). After degradation of these herbicides by microorganisms in soil or water (Bartha and Pramer, 1967; Deuel et al., 1977; E1-Dib and Aly, Correspondence: Dr. M. Bauchinger, Institut fiir Strahlenbiologie, Gesellschaft for Strahlen- und Umweltforschung (GSF), D-8042 Neuherberg/Munich (F.R.G.).
1976) 3,4-DCA has been detected in various surface waters in western Europe (Wegman and de Korte, 1981), in flooded rice field cultures (Deuel et al., 1977) and in the effluents from dyemanufacturing plants (Games and Hites, 1977). In a hepatocyte/DNA-repair test the compound failed to induce a DNA-repair response (Yoshimi et al., 1988). The present study examines whether chromosome aberrations (CA) and sisterchromatid exchanges (SCE) can be induced in human lymphocytes treated in vitro in the presence or absence of a metabolic activation system. V79 Chinese hamster cells are used to test whether spindle disturbances can be caused.
0165-7992/89/$ 03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
198
Material and methods
Chromosome and SCE analysis 3,4-DCA with a purity of 99% was purchased from Aldrich Chemie (Steinheim, F.R.G.). For the experiments duplicate lymphocyte cultures of a healthy female donor were set up with 0.5 ml whole blood, 4 ml RPMI 1640 medium, 0.5 ml fetal calf serum and 0.13 ml phytohemagglutinin; the cultures were incubated at 37°C. 24 h after culture initiation the test substance, dissolved in dimethyl sulfoxide (DMSO), was added for 1 h, either with or without $9 mix (protein content 5 mg/ml mix). The $9 fraction was prepared from Clophen A 50-induced male Wistar rats with minor modifications according to Ames et al. (1975). A detailed description of the metabolic activation system used has been published (G6ggelmann, 1984; Kugler et al., 1987). After the 3,4-DCA exposure cells were washed twice with RPMI medium and new cultures were set up supplemented with bromodeoxyuridine
in a final concentration of 7.75 #M and incubated for a further 29 or 47 h, with 0.1 ~g/ml Colcemid added for the final 3 h. The total incubation time was thus 54 or 72 h. Cultures exposed to 0.33% DMSO served as solvent control. 0.05 mM cyclophosphamide (CP, Serva) was used as a positive control for the metabolic activation system. Chromosome preparation and fluorescence plus Giemsa staining (FPG) were carried out according to our standardized laboratory procedures (Kolin-Gerresheim and Bauchinger, 1981; Apelt et al., 1981). Analysis of CA was performed exclusively in complete first-division metaphases (M1, 54 h culture time) identified by uniformly stained sister chromatids. SCEs were scored in harlequin-stained second-division metaphases
100-
"~ 8 0 o
~h
~5o-
100"~ ' ~ 4 0 -
"////" .....
20
8O
0
,111~ ~11//
/ i / /
"E/;<4 /i//~
100-
~ ~o
~ 80I ieoIE
:~ ~4ou,, 6 20-
O-
0
0
,
0.125 0.25 0.5 3,4 - Dichlor'oeniline ( m M )
1.0
Fig. 1. Effect of 3,4-DCA on the frequency of total mitoses in V79 cells. Cells were treated and processed as described in Material and methods. Results (means +- SD from 3 × 1000 cells in 2 slides from duplicate cultures) of 2 independent experiments (open and hatched columns) are shown.
0
0.125 0.25 0.5 1.0 3,4- Dichlo¢'ooniline ( r a m )
Fig. 2. Effects of 3,4-DCA on the frequency of metaphases in V79 cells. Cells were treated and processed as described in Material and methods. Results are pooled data of 2 independent experiments. (A) Frequency of total metaphases. (B) Frequency of c-metaphases (open columns, initial c-mitoses; hatched colu m n s , complete c-mitoses).
199
TABLE
1
FREQUENCY
OF
CHROMOSOMAL
METABOLICALLY
A C T I V E $9 M I X
CHANGES
INDUCED
BY
3,4-DCA
IN
THE
PRESENCE
AND
ABSENCE
OF
Experiment
Conc.
Gaps a
S-cells a
A b e r r a t i o n s p e r cell a
S C E s p e r cell b
(exposure conditions)
(mM)
p e r cell
(%)
(chromatid type)
+ SEM
Breaks 3,4-Dichloroaniline ( - $9 mix)
3,4-Dichloroaniline ( + $9 mix)
Exchanges
0
0.025
-
-
0.25
0.020
-
-
7.30 ± 0.55 8.26 ± 0.55
0.50
0.030
0.5
0.005
-
7.42 ± 0 . 5 0
1.0
0.050
0.5
0.005
-
9.24 ± 0.43
0
0.020
1.0
0.010
-
7.58 ± 0 . 5 2
0.125
0.045
1.0
0.010
-
15.18 ± 0.58
0.25
0.035
0.5
0.005
-
18.50 ± 0.57
0.50
0.045
2.0
0.020
-
23.30 ± 0.66
1.0
0.035
3.0
0.025
0.005
23.38 ± 0.74
0.05
0.410
44.0
0.625
0.110
Cyclophosphamide ( + $9 mix)
- 100
S-cells, cells w i t h s t r u c t u r a l c h r o m o s o m e a b e r r a t i o n s . Cells p e r s a m p l e : a 200, b 50.
(M2, 72 h culture time). Cells containing structural CA were classified as S-cells.
Analysis of spindle disturbances V79 Chinese hamster cells were used to study the induction of spindle disturbances by 3,4-DCA. The cells were cultured in Dulbecco's modified Eagle's minimum essential medium (Biochrom, Berlin, F.R.G.) supplemented with 10070 fetal calf serum (Flow, Meckenheim, F.R.G.), 100 U/ml penicillin and 100 /zg/ml streptomycin at 37°C in a humidified atmosphere containing 7°70 CO2. For the experiments 0.5-1 × 105 cells were seeded in 60-mm dishes containing 25 x 30 mm coverslips. After 24 h cells were washed with phosphatebuffered saline (PBS) and exposed to 3,4-DCA in serum-free medium for the last 3 h of culture time. Controls received DMSO only. After exposure cells were washed with Hanks' balanced salt solution and carefully fixed without hypotonic and Colcemid treatment directly on the coverslips (Bauchinger et al., 1988). For the analysis of spindle disturbances 500 cells were scored from duplicate cultures of 2 independent experiments.
The mitotic index was determined by scoring 3 × 1000 cells in 2 slides of duplicate cultures. The polyploidy index gives the number of polyploid mitoses as the percentage of total mitoses scored. Results and discussion
The highest dose of 3,4-DCA which did not impair cell proliferation of human lymphocytes was 1 mM. The results of the chromosome analysis are given in Table 1. The structural CA observed in M 1 were exclusively of the chromatid type with single or isolocus breaks and were accompanied by gaps. With and without metabolic activation there was no evidence of significant differences in the incidences of chromatid breaks, gaps and S-cells between 3,4-DCA-treated and untreated cultures ( p < 0 . 0 5 in a double-sided Wilcoxon-MannWhitney rank test; Lehmann, 1975). Compared to controls the incidence of SCEs was significantly increased in the absence of $9 mix at 1 mM 3,4-DCA only and in the presence of $9 mix at each concentration (Table 1). Metabolic activation induced SCE frequencies almost 3-fold.
200 TABLE 2 MEAN VALUES (°70 ± SEM) OF N O R M A L A N D D A M A G E D M I T O T I C FIGURES OBSERVED IN D U P L I C A T E CULTURES OF 2 EXPERIMENTS W I T H V79 CELLS AFTER 3 h TREATMENT W I T H 3,4-DCA 3,4-Dichloroaniline (mM) Cells analyzed Normalmitoticfigures Prophases Metaphases Ana-telophases Mitoses with spindle disturbances Initial c-mitoses Prophases Metaphases c-Mitoses Prophases Metaphases Multipolar ana-telophases Anaphases with lagging or non-disjunction
0
0.125
0.25
0.5
1.0
1000
1000
1000
1000
1000
90.4±0.9 16.1±1.2 55.8±1.6 18.5±1.2
90.5±0.9 15.3±1.1 56.1±1.6 19.1±1.2
87.9±1.0 16.4±1.2 54.7±1.6 16.8±1.2
71.8±1.4 10.8±1.0 51.8±1.6 9.2±0.9
24.5±1.4 7.1±0.8 16.8±1.2 0.6±0.2
23.2 17.8 1.2 16.6 4.9 0
69.5 62.3 1.4 60.9 7.0 0
5.5 3.8 0.6 3.2 1.1 0
± ± ± ± ±
0.7 0.6 0.2 0.6 0.3
6.5 4.6 0.8 3.8 1.1 0
± ± + + ±
0.8 0.7 0.3 0.6 0.3
8.5 7.0 0.5 6.5 1.3 0
± 0.9 ±_ 0.8 + 0.2 ± 0.8 ± 0.4
± ± ± ± ±
1.3 1.2 0.3 1.2 0.7
± ± ± ± ±
1.5 1.5 0.4 1.5 0.8
1.1 ± 0.3 0.3 ± 0.2
1.1 ± 0.3 0.5 + 0.2
1.3 ± 0.4 0.2 ± 0.1
4.9 ± 0.7 0.3 ± 0.2
7.0 ± 0.8 0
0.3 ± 0.2
0.3 ± 0.2
0
0.2 ± 0.1
0.2 ± 0.1
Ana-telophases with bridges a n d / o r fragments
0.3 ± 0.2
0.4 ± 0.2
0.4 ± 0.2
0.1±0.1
0
Mitoses with stickiness and pycnosis
1.2±0.3
0.8±0.4
0.9±0.3
2.4±0.5
2.7±0.5
Polyploidy index
2.6±0.5
1.8±0.4
2.3±0.5
2.5±0.5
3.3±0.6
The effect of a 3-h exposure of V79 cells on cell proliferation is demonstrated in Fig. 1. At 0.5 and 1.0 mM 3,4-DCA the mitotic index was significantly increased compared to controls. At 2.0 mM (not shown) only highly pycnotic mitotic figures were observed indicating cytotoxicity. The results on the induction of spindle disturbances by 3,4-DCA are shown in Table 2. The substance caused a concentration-dependent increase in the number of abnormal mitotic figures. At 1.0 mM 3,4-DCA their incidence was increased about 13-fold above that found in controls. A c-mitotic effect was reflected in the appearance of a metaphase block (Fig. 2A) and the concomitant disappearance of ana-telophases. As shown in Fig. 2B metaphases were predominantly of the initial c-mitotic type (Levan, 1954; Schmid and Bauchinger, 1976). About 60°70 had the appearance of so-called ball
metaphases with chromosomes concentrated in the cell centre, about 30°70 had their chromosomes arranged in several groups and about 10% showed non-congression, i.e., lack of correct arrangement of single chromosomes onto the metaphase plate. The picture of typical c-metaphases with contracted chromosomes scattered in the cytoplasm was only infrequently observed (Fig. 2B). Multipolar ana-telophases or non-disjunction were not found more frequently than in controls. The polyploidy index was not higher in treated cultures than in controls. The frequency of ana-telophases with bridges and/ or fragments never exceeded 0.4°70. At 0.5 and 1.0 mM 3,4-DCA the number of mitotic figures with stickiness and pycnosis was about twice that observed in controls. In conclusion, the data show that 3,4-DCA fails
201
to induce a significant clastogenic effect in human lymphocytes in the presence and absence of an in vitro metabolizing activation system. According to UKEMS guidelines on mutagenicity testing (Scott, 1983; Scott et al., 1983) where at least a doubling in SCE frequency should be accepted as a positive response, 3,4-DCA is active as an SCE-inducing agent in the presence of rat liver $9 mix. The induction of SCEs is generally referred to the induction o f DNA damage. However, one should bear in mind that phenomena other than DNA reactivity may contribute to the induction of SCEs. Evidence of this comes from a study of Montaldi et al. (1985) who found that interaction of the chelating agent nitrilotriacetic acid with heavy metals led to enhanced SCEs. Thus, an isolated positive SCE response in the absence of a clastogenic response does not allow a clear decision whether a chemical must be regarded as an in vitro genotoxin. Beyond this the significance of SCEs to mutagenicity and carcinogenicity in humans is still not known. 3,4-DCA is, however, a potent spindle poison in V79 cells. Although mitotic arrestants are not necessarily aneuploidy-inducing agents, due to its ability to cause spindle disturbances 3,4-DCA must be considered to have genotoxic activity.
Acknowledgements We thank R. Giesen and F. Zelles for technical assistance. This work has been supported by the Bayerisches Staatsministerium ftir Landesentwicklung und Umweltfragen.
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