Mutation Research, 244 (1990) 147-151 Elsevier
147
MUTLET 0352
Induction of micronuclei by mitomycin C and colchicine in the marine mussel
Mytilus galloprovincialis Franca Majone, Riccardo Brunetti, Ornella Fumagalli, Marco Gabriele and A.G. Levis Department of Biology, Universityof Padua, 1-35131 Padua (Italy)
(Accepted29 December 1989)
Keywords: Mytilus galloprovincialis; MitomycinC; Colchicine;Micronuclei;Antikinetochoreantibodies
Summary The frequencies of micronuclei (MNi) in the gill cells of the mussel Mytilus galloprovincialis were determined over a long period (up to 28 days) following a 48-h treatment with colchicine. The frequency of MNi at the end of treatment was significantly higher than in controls and 24 h later it had increased even more. After this period, the frequency of MNi rapidly declined until a plateau level was reached on day 2-3, which was significantly higher than the control baseline level, and persisted until the end of the experiment (28th day). In the same cell system we previously reported a persistence of an increased frequency of MNi after treatment with mitomycin C (MMC) (Majone et al., 1987). In order to establish the origin of MNi, the difference between their size distribution in MMC- and colchicine-treated animals was determined at the end o f treatment as well as during the plateau phase. The difference was statistically significant (P < 0.001) at the end o f treatment, the MNi induced by MMC being smaller than those induced by colchicine. However, the difference during the plateau phase was not statistically significant. H u m a n CREST antikinetochore fluorescent antibodies reacted with chromosome centromeres of Mytilus and were applied to gill cells at the end of a 48-h treatment with MMC or colchicine. About 60% of the MNi induced by colchicine but only 30% of those produced by MMC reacted positively with the fluorescent antibodies. This result indicates that the majority of MNi observed at the end of a 48-h treatment with MMC or colchicine originate, respectively, from acentric chromosome fragments and from whole lagging chromosomes.
We previously studied the induction and the persistence of micronuclei (MNi) in the gill cells of Mytilus after treatment with mitomycin C (MMC) (Majone et al., 1987). The frequency o f MNi was Correspondence: Dr. F. Majone, Department of Biology, University of Padua, Via Loredan 10, 1-35131, Padua (Italy).
significantly increased by MMC and declined after treatment until it reached a plateau level on the 8th day, significantly higher (about twice) than the control value, which persisted even after 40-52 days. The continuous production o f MNi for a considerable time after treatment suggested that the frequency o f MNi in the gills of mussels from
0165-7992/90/$ 03.50 © 1990Elsevier SciencePublishers B.V. (BiomedicalDivision)
148
natural populations could be used as a parameter for evaluating the genotoxicity of water pollutants even if acting long before sampling (Brunetti et al., 1988). In the present paper we determined the frequency of MNi in the gills of Mytilus at different times after treatment with colchicine. MMC and colchicine are known to produce, in other cell systems, MNi deriving from chromosome fragments or whole chromosomes, respectively (Frackowiak et al., 1986; Degrassi and Tanzarella, 1988; Thomson and Perry, 1988). Therefore we determined the origin of MNi produced in mussels by the above substances by analyzing the size distribution of the induced MNi, which depends on the kind of chromosomal alteration induced, the MNi derived from lagging chromosomes being, as a rule, significantly larger than those produced from chromosome fragments (Yamamoto and Kikuchi, 1980; H6gstedt and Karlsson, 1985). Moreover, we applied the technique based on the use of human CREST antikinetochore antibodies and fluorescence staining (Moroy et al., 1980), which, through identifying kinetochore-containing micronuclei, is much more precise in discriminating the micronuclei containing whole chromosomes (CREST + ) from those containing acentric chromosome fragments (CREST - ).
of Dispase I (Boehringer, Mannheim, F.R.G.) in 50 ml of phosphate-buffered saline (PBS), and shaking vigorously 2-3 times for some seconds. Then 8 ml of Carnoy fixative was added to each tube, the cell suspension was filtered and centrifuged and the pellet was resuspended in fixative. The cell suspension was finally spread on slides, airdried and stained with 5% Giemsa (Majone et al., 1988). 15 animals and 2000 cells per animal were considered per experimental point.
Indirect immunofluorescence Human serum containing antikinetochore autoantibodies was a generous gift of Dr. Amelia Ruffatti (Institute of Internal Medicine, University of Padua). The serum obtained from a patient with progressive systemic sclerosis, known as the CREST syndrome, was used in an indirect immunofluorescence technique. The target of the antibodies was first checked on in vitro established
in
• o
U
3
o
Material and methods
Cell preparation The freshly excised gills were placed for l0 min, at room temperature, in 10-ml test tubes containing 1 ml Dispase solution prepared by dissolving 5 mg
12_
i
~
Test animals and treatment Adult specimens of Mytilus galloprovincialis Lmk. (major axis about 5 cm) were collected from a mussel park in the lagoon of Venice and acclimatized for a week in our laboratory in running sea water (~35%0 salinity) at 18___I°C. Then the animals were treated for 48 h with 0.5 mg/1 colchicine (Merck, Darmstadt, F.R.G.) or 0.018 mg/l mitomycin C (Kyowa Hakko Kogyo Co., Tokyo, Japan) and sampled at different times for a month.
14
U Idt
10_ _
a_
E
-Q
E 3 r
GO
o
~
4_ _
2_
o o3
10 , DaWS
18 , after
2~8
treatment
Fig. 1. Pattern of the frequency of micronuclei induced in the gill tissue of Mytilus by a 48-h treatment with colchicine (0.5 mg/l), e : treated; • : control. Vertical bars represent the 95o/o confidence interval of the mean. Statistical comparison (Wilcoxon test): day 0 vs. day 1: P < 0 . 0 0 1 ; day 1 vs. day 2: P < 0 . 0 0 1 ; day 2 vs. days 3, 10, 18 and 28: NS; day 1 vs. corresponding control: P < 0.001; day 10 vs. corresponding control: P < 0 . 0 1 ; day 28 vs. corresponding control: P < 0 . 0 0 1 ; a m o n g controls: NS.
149
HEp2 cells. In metaphase HEp2 cells the fluorescence appeared associated with each chromosome, producing a positive line in the middle of the mitotic spindle, which is the typical distribution of the anticentromere antibodies (Moroy et al., 1980). In order to prepare the cells for the immunofluorescence staining, the gills from 10 animals were rinsed in 1% NaC1 and treated for 15 min in a hypotonic solution consisting of 19 parts NaCI 1%0 and 1 part KCI 6%o. Then the gills were dissolved in Dispase for 5 min and fixed overnight with methanol at -20°C. Then the slides were prepared as above and dried for 20 min. After rinsing 3 times in ice-cold PBS solution for 10 min, the cell preparations were treated for 30 min with antikinetochore serum diluted 1:40 with PBS. The cell preparations were washed 3 times in PBS for 10 min and then incubated for 30 min with fluorescein-labeled sheep anti-human immunoglobulin G (IgG; Wellcome, U.K.) diluted 1:20 with PBS. After extensive washing in PBS, the slides were stained for not more than 1 min with 0.01 mg/ml ethidium bromide (Sigma Chemical Co., St. Louis, MO) and, after 2 further washes with PBS, mounted with a PBS-glycerol (1:1) mixture. The cell preparations were finally examined with an Olympus BHS microscope with BH2-RFL fluorescent equipment under blue illumination. The size of the induced MNi was expressed as the ratio between the area of the micronucleus and that of the main cell nucleus (H6gstedt and Karlsson, 1985). 50 micronucleated cells from 10 animals per experimental point were examined. Results Fig. 1 shows the frequencies of MNi induced by a 48-h treatment with 0.5 mg/l colchicine in cells of the gill tissue of Mytilus. The frequency of MNi at the end of treatment was significantly higher (P
147
MUTLET 0352
Induction of micronuclei by mitomycin C and colchicine in the marine mussel
Mytilus galloprovincialis Franca Majone, Riccardo Brunetti, Ornella Fumagalli, Marco Gabriele and A.G. Levis Department of Biology, Universityof Padua, 1-35131 Padua (Italy)
(Accepted29 December 1989)
Keywords: Mytilus galloprovincialis; MitomycinC; Colchicine;Micronuclei;Antikinetochoreantibodies
Summary The frequencies of micronuclei (MNi) in the gill cells of the mussel Mytilus galloprovincialis were determined over a long period (up to 28 days) following a 48-h treatment with colchicine. The frequency of MNi at the end of treatment was significantly higher than in controls and 24 h later it had increased even more. After this period, the frequency of MNi rapidly declined until a plateau level was reached on day 2-3, which was significantly higher than the control baseline level, and persisted until the end of the experiment (28th day). In the same cell system we previously reported a persistence of an increased frequency of MNi after treatment with mitomycin C (MMC) (Majone et al., 1987). In order to establish the origin of MNi, the difference between their size distribution in MMC- and colchicine-treated animals was determined at the end o f treatment as well as during the plateau phase. The difference was statistically significant (P < 0.001) at the end o f treatment, the MNi induced by MMC being smaller than those induced by colchicine. However, the difference during the plateau phase was not statistically significant. H u m a n CREST antikinetochore fluorescent antibodies reacted with chromosome centromeres of Mytilus and were applied to gill cells at the end of a 48-h treatment with MMC or colchicine. About 60% of the MNi induced by colchicine but only 30% of those produced by MMC reacted positively with the fluorescent antibodies. This result indicates that the majority of MNi observed at the end of a 48-h treatment with MMC or colchicine originate, respectively, from acentric chromosome fragments and from whole lagging chromosomes.
We previously studied the induction and the persistence of micronuclei (MNi) in the gill cells of Mytilus after treatment with mitomycin C (MMC) (Majone et al., 1987). The frequency o f MNi was Correspondence: Dr. F. Majone, Department of Biology, University of Padua, Via Loredan 10, 1-35131, Padua (Italy).
significantly increased by MMC and declined after treatment until it reached a plateau level on the 8th day, significantly higher (about twice) than the control value, which persisted even after 40-52 days. The continuous production o f MNi for a considerable time after treatment suggested that the frequency o f MNi in the gills of mussels from
0165-7992/90/$ 03.50 © 1990Elsevier SciencePublishers B.V. (BiomedicalDivision)
148
natural populations could be used as a parameter for evaluating the genotoxicity of water pollutants even if acting long before sampling (Brunetti et al., 1988). In the present paper we determined the frequency of MNi in the gills of Mytilus at different times after treatment with colchicine. MMC and colchicine are known to produce, in other cell systems, MNi deriving from chromosome fragments or whole chromosomes, respectively (Frackowiak et al., 1986; Degrassi and Tanzarella, 1988; Thomson and Perry, 1988). Therefore we determined the origin of MNi produced in mussels by the above substances by analyzing the size distribution of the induced MNi, which depends on the kind of chromosomal alteration induced, the MNi derived from lagging chromosomes being, as a rule, significantly larger than those produced from chromosome fragments (Yamamoto and Kikuchi, 1980; H6gstedt and Karlsson, 1985). Moreover, we applied the technique based on the use of human CREST antikinetochore antibodies and fluorescence staining (Moroy et al., 1980), which, through identifying kinetochore-containing micronuclei, is much more precise in discriminating the micronuclei containing whole chromosomes (CREST + ) from those containing acentric chromosome fragments (CREST - ).
of Dispase I (Boehringer, Mannheim, F.R.G.) in 50 ml of phosphate-buffered saline (PBS), and shaking vigorously 2-3 times for some seconds. Then 8 ml of Carnoy fixative was added to each tube, the cell suspension was filtered and centrifuged and the pellet was resuspended in fixative. The cell suspension was finally spread on slides, airdried and stained with 5% Giemsa (Majone et al., 1988). 15 animals and 2000 cells per animal were considered per experimental point.
Indirect immunofluorescence Human serum containing antikinetochore autoantibodies was a generous gift of Dr. Amelia Ruffatti (Institute of Internal Medicine, University of Padua). The serum obtained from a patient with progressive systemic sclerosis, known as the CREST syndrome, was used in an indirect immunofluorescence technique. The target of the antibodies was first checked on in vitro established
in
• o
U
3
o
Material and methods
Cell preparation The freshly excised gills were placed for l0 min, at room temperature, in 10-ml test tubes containing 1 ml Dispase solution prepared by dissolving 5 mg
12_
i
~
Test animals and treatment Adult specimens of Mytilus galloprovincialis Lmk. (major axis about 5 cm) were collected from a mussel park in the lagoon of Venice and acclimatized for a week in our laboratory in running sea water (~35%0 salinity) at 18___I°C. Then the animals were treated for 48 h with 0.5 mg/1 colchicine (Merck, Darmstadt, F.R.G.) or 0.018 mg/l mitomycin C (Kyowa Hakko Kogyo Co., Tokyo, Japan) and sampled at different times for a month.
14
U Idt
10_ _
a_
E
-Q
E 3 r
GO
o
~
4_ _
2_
o o3
10 , DaWS
18 , after
2~8
treatment
Fig. 1. Pattern of the frequency of micronuclei induced in the gill tissue of Mytilus by a 48-h treatment with colchicine (0.5 mg/l), e : treated; • : control. Vertical bars represent the 95o/o confidence interval of the mean. Statistical comparison (Wilcoxon test): day 0 vs. day 1: P < 0 . 0 0 1 ; day 1 vs. day 2: P < 0 . 0 0 1 ; day 2 vs. days 3, 10, 18 and 28: NS; day 1 vs. corresponding control: P < 0.001; day 10 vs. corresponding control: P < 0 . 0 1 ; day 28 vs. corresponding control: P < 0 . 0 0 1 ; a m o n g controls: NS.
149
HEp2 cells. In metaphase HEp2 cells the fluorescence appeared associated with each chromosome, producing a positive line in the middle of the mitotic spindle, which is the typical distribution of the anticentromere antibodies (Moroy et al., 1980). In order to prepare the cells for the immunofluorescence staining, the gills from 10 animals were rinsed in 1% NaC1 and treated for 15 min in a hypotonic solution consisting of 19 parts NaCI 1%0 and 1 part KCI 6%o. Then the gills were dissolved in Dispase for 5 min and fixed overnight with methanol at -20°C. Then the slides were prepared as above and dried for 20 min. After rinsing 3 times in ice-cold PBS solution for 10 min, the cell preparations were treated for 30 min with antikinetochore serum diluted 1:40 with PBS. The cell preparations were washed 3 times in PBS for 10 min and then incubated for 30 min with fluorescein-labeled sheep anti-human immunoglobulin G (IgG; Wellcome, U.K.) diluted 1:20 with PBS. After extensive washing in PBS, the slides were stained for not more than 1 min with 0.01 mg/ml ethidium bromide (Sigma Chemical Co., St. Louis, MO) and, after 2 further washes with PBS, mounted with a PBS-glycerol (1:1) mixture. The cell preparations were finally examined with an Olympus BHS microscope with BH2-RFL fluorescent equipment under blue illumination. The size of the induced MNi was expressed as the ratio between the area of the micronucleus and that of the main cell nucleus (H6gstedt and Karlsson, 1985). 50 micronucleated cells from 10 animals per experimental point were examined. Results Fig. 1 shows the frequencies of MNi induced by a 48-h treatment with 0.5 mg/l colchicine in cells of the gill tissue of Mytilus. The frequency of MNi at the end of treatment was significantly higher (P
