- Email: [email protected]
genotoxic effects induced by β-oestradiol in vitro
Pergamon
Toxicology in Vitro 10 (1996) 637442
Genotoxic Effects Induced by fl-Oestradiol In Vitro I. ECKERT
and H. STOPPER*
Department of Toxicology, University of Wiirzburg, Versbacher Str. 9, 97078, Wiirzburg, Germany (Accepted 23 April 1996) Abstract-The carcinogenic endogenous hormone P-oestradiol induces genotoxic effects in short-term mammalian tests. In this study we investigated micronucleus induction in V79 cells. The early induction of primarily kinetochore positive micronuclei indicated a mechanism involving mitotic disturbances. Metaphase arrangements that showed displaced chromosomes suggested that these chromosomes may be enclosed in micronuclei. However, observation of live cells revealed that most of the displaced chromosomes reintegrated. In some of these cells chromatid separation at anaphase was impaired and a small chromatin fragment was separated from the remaining chromatin. Copyright 0 1996 Elsevier Science Ltd.
Introduction The endogenous
hormone
hormone-dependent et
al.,
1982).
anti-human antibody were purchased from Sigma Chemie GmbH (Deisenhofen, Germany). CREST-serum (anti-kinetochore antibody) was purchased from Biermann GmbH (Bad Nauheim, Germany). goat
j?-oestradiol
types Since
of cancer
P-oestradiol
has a role in (Henderson also
induces
genotoxic effects in short-term tests (Degen and Metzler, 1987), not all /3-oestradiol-related changes in cells can be explained by hormone receptor-dependent mechanisms. Examples are the induction of aneuploidy (Barrett et a/., 1985; Wheeler ef al., 1986), cell transformation (Purdy et al., 1983) and micronucleus formation (Wheeler et al., 1987) in vitro. In this study we investigated fi-oestradiolinduced micronucleus formation in vitro in the Chinese hamster cell line V79. Wheeler et al. (1987)
described the occurrence of displaced metaphase chromosomes after treatment with /I-oestradiol. It could be hypothesized that such displaced chromosomes are enclosed in micronuclei. To investigate this hypothesis we first quantified micronucleus induction and the occurrence of such displaced metaphase chromosomes after treatment with /?-oestradiol. Next, we followed live mitotic cells that contained such displaced chromosomes through the further course of mitosis with the method of supravital UV-microscopy (Schiffmann and DeBoni, 1991). Materials and Methods Chemicals
Acridine orange, bisbenzimide 33258 and 33342, cytochalasin B, P-oestradiol and FlTC-conjugated
*Author for correspondence. Abbre2Gations: DMSO = dimethyl sulfoxide; MEM = modified Eagle’s medium; PBS = phosphate buffered saline.
Ceil culture V79 Chinese hamster cells were cultured in modified Eagle’s medium (MEM) medium (Sigma) supplemented with antibiotics and 10% foetal calf serum (Gibco, Germany). Cell cultures were grown in humidified atmosphere with 5% CO? in air at 37°C. Micronucleus assay
Exponentially growing V79 cells were treated with /I-oestradiol for 4 hr. The solvent control contained 1% dimethyl sulfoxide (DMSO) (the maximal amount of DMSO being present from dissolving test substances). After removing P-oestradiol by medium replacement, the cells were incubated for O-25 hr for time course and 5 hr for dose-response experiments. After trypsinization the cells were placed onto glass slides by cytospin centrifugation. Fixation was performed with methanol (-2O”C, 1 hr). For staining, the slides were washed with distilled water incubated with bisbenzimide 33258 (5 pg/ml, 3 min), washed three times with distilled water and mounted for microscopy. Using a magnification of x 1000, the number of nuclei and micronuclei were scored. For the cytochalasin B method, 6 pg cytochalasin B/ml were added together with 75 pM /Loestradiol. After 4 hr the medium was replaced by medium containing 6 pg cytochalasin B/ml and 5 hr later the cells were prepared and fixed as described above. For staining of the cells, the slides were incubated for 4 min in acridine orange (62.5 &ml in Saerensen buffer: 67 mM Na2HP0,/KH$0,, pH 6.8). Slides
0887-2333/96/!§15.00+ 0.00 Copyright 0 1996 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII SO887-2333(96)00035-5
638
1. Eckert and H. Stopper
were washed twice in Scerensen buffer (5 min) and mounted in buffer for microscopy. Micronuclei were scored in binucleate cells using a x 1000 magnification. Each data point represents the mean of three slides with 2000 nuclei (or 1000 binucleate cells) evaluated per slide. All experiments were repeated twice with consistent results. Kinetochore staining
Kinetochore staining was achieved by incubating the fixed cell preparations (after washing for 5 min in phosphate buffered saline (PBS)/O.l% Tween 20) with CREST serum for 75 min in a humidified chamber at 37’C. After washing twice for 5 min in PBS/O.S% Tween 20 again, the cells were incubated as before for 30 min with FlTC-conjugated goat-antihuman antibody (diluted 1:lOO in PBS/O.S% Tween 20, pH 7.4), washed again twice in PBS/O. 1% Tween 20 and counterstained with bisbenzimide 33258 (5 pg/ml, 3 min). Using a magnification of x 1000 nuclei and micronuclei were evaluated for the presence of kinetochore signals. Metaphase ring arrangements V79 cells were treated for 4 hr with B-oestradiol (75 PM) or DMSO (1%) as a solvent control. Then the culture medium was replaced with fresh medium and after 5 min cells were trypsinized and placed onto glass slides with cytospin centrifugation. From withdrawal of P-oestradiol to the start of cytospin preparation a total time of 10 min passed. Cells were fixed in cold ( - 20°C) methanol-acidic acid (3: 1, v/v) and stained as described for micronuclei previously. Because of the cytospin preparation, many metaphases show a ring arrangement. Only those metaphases were evaluated for chromosomes dislocated from the ring.
after p-oestradiol treatment contained kinetochore signals. Thus, /I-oestradiol induced predominantly kinetochore containing micronuclei in V79 cells shortly after its removal. Wheeler et al. (1987) also found an induction of micronuclei after fl-oestradiol treatment at similar concentrations. As these authors did not allow for recovery between substance removal and cell preparation their experiments for time dependence of micronucleus formation are not comparable with ours. Early induction of micronuclei that are kinetochore-positive is typical for substances that act by way of mitotic disturbance (Stopper et al., 1992 and 1994). One method for examining disturbance of mitosis is to score metaphase ring arrangements for displaced chromosomes in fixed cells. After cytospin preparation, metaphases appear as rings and displaced chromosomes can readily be identified (Plate la,b). Of 300 metaphase ring arrangements in the control, 6 (2%) showed a displaced chromosome, whereas after treatment with p-oestradiol 25 of 300 (8.3%) ring arrangements showed such a displaced chromosome. Wheeler et al. (1987) also described the occurrence of misaligned metaphase chromosomes after P-oestradiol treatment in Chinese hamster (Don) cells. We next investigated the course of mitosis in live cells using supravital UV microscopy. With this method every cell has to be followed through mitosis individually and therefore only limited numbers of observations can be performed. We
(4 __ 125-
;r;
E
Supraoital WV microscopy
Supravital UV microscopy was performed according to the method of Schiffmann and DeBoni (1991), using V79 cells. This method allows the observation of the spatial arrangement of chromatin elements throughout mitosis in live cells. Chromatin was stained with 0.1 pg bisbenzimide 33342/ml and cells were treated with P-oestradiol (75 pM) for 3 to 4 hr. Then the medium was replaced (now containing only bisbenzimide) and observation began. As every single mitosis has to be followed over time, only limited numbers of observations can be achieved with this method.
0.
’
0
I 20
I 40
I 60
I 80
I 100
Concentration[w]
(b)
Results and Discussion /?-Oestradiol induced micronuclei in V79 cells in a dose-dependent manner (Fig. la). The time course of micronucleus induction showed a maximum at 5 hr after substance withdrawal (Fig. lb). Detection of kinetochores revealed that 20.7 + 0.5% of the spontaneous and 65.5 If 2.3% of the micronuclei
Fig. 1. Micronucleus induction by P-oestradiol in V79 cells. Dose response (a) and time course after the removal of B-oestradiol (75 PM) (b). BG: spontaneous micronucleus frequency.
Plate
1. Metaphase ring arrangements of V79 cells after cytospin preparation. (a) Control, (b) after treatment with 75 pM /I-oestradiol. The displaced chromosome is indicated by an arrow.
639
Genotoxicity of oestradiol began observation as soon as possible (less than 5 min) after substance withdrawal. First, we found an enriched population of prometaphase cells. After about 10 min these had progressed into metaphase and in some cases we detected a dislocated chromosome (Plate 2). As the cells were alive metaphases did not appear as rings with this method (Plate 2). We followed 22 such metaphases that contained dislocated chromosomes. Of these, only one proceeded further during mitosis with the chromosome staying dislocated and forming a micronucleus. The likely cause for this chromosome to be dislocated and become enclosed in a micronucleus may be explained as follows: 65.5% of the micronuclei after B-oestradiol treatment contained kinetochores. After subtraction of the spontaneous micronuclei (and their kinetochores) 72.9% of the /I-oestradiol-induced micronuclei would contain a kinetochore. As this compound is not known to break chromosomes, all of the induced micronuclei should contain at least one chromatid and therefore a kinetochore. We previously demonstrated that some displaced metaphase chromosomes can be without detectable kinetochore (Kirchner et al., 1993) and discussed that the missing spindle attachment of these might conceivably render them prone to enclosure into micronuclei. If the binding of kinetochore proteins to DNA is impaired after g-oestradiol treatment the relatively infrequent formation of micronuclei by enclosure of a displaced chromosome may be due to this mechanism. Centromere probes that would demonstrate the possible presence of centromeric DNA even in the absence of kinetochor proteins are not yet available for Chinese hamster cells. During the supravital UV microscopy observations in all of the remaining 21 cells the dislocated chromosome was realigned with the other metaphase chromosomes. 11 of these cells were unable to perform a normal anaphase chromatid separation. Four were not able to perform chromatid separation at all resulting in a presumptive endomitosis. Cells were followed until the chromatin showed an interphase-like appearance and in seven cells small fragments of chromatin were eventually separated from the remaining chromatin (Plate 2). From their experiments with fixed cells Wheeler et al. (1987) concluded that most of the displaced chromosomes realigned in metaphase. This is in agreement with our supravital UV video observations and with a study by Nicklas and Kubai (1985) in which it was demonstrated that chromosomes in grasshopper spermatocytes that had been displaced by micromanipulation showed microtubuli-assisted reorientation. However, our study was the first that used supravital UV microscopy to follow live cells through this process of realignment. With supravital UV microscopy it is not possible to distinguish whether cell division had taken place in cells that showed separation of small chromatin fragments. To address this question cytochalasin B
641
was applied. This compound prevents cells from dividing, resulting in binucleate cells. Usually only the binucleate cells are evaluated for the presence of micronuclei assuming that only those are actively cycling cells able to form micronuclei and that the mononucleate cells are cytostatic due to test substance treatment and are unable to form micronuclei. We treated cells with 75 PM /I-oestradiol and we also examined mononucleate cells for the presence of micronucleus-like structures in addition to binucleate cells. As a result of three independent experiments we found that 68 + 7.5% of the micronuclei or micronucleus-like structures (chromatin structures that are smaller than l/3 of the area of a nucleus) after /I-oestradiol treatment were in mononucleate cells. A possible explanation is that cytochalasin B did not prevent cytokinesis, especially if combined with /I-oestradiol. This view is supported by the investigation of Antoccia et al. (1993) in which colchicine induced micronuclei in human fibroblasts without the treatment with cytochalasin B. However, after a combined treatment with colchicine and cytochalasin B micronucleus induction in binucleate cells was impaired. Thus, the inclusion of cytochalasin B in the micronucleus assay may sometimes be misleading in investigations of micronucleus induction by aneugens. As another explanation, we cannot exclude that some of these micronucleus-like structures in mononucleate cells were the result of mitotic disturbances of the kind that we had observed in seven of 22 mitoses (example shown in Plate 2). In the supravital UV-microscopy observations it seemed that the separation of the small fragment occurred rather late in mitosis and it might have been impossible for the cells to divide after that. In some cases (where the separated fragment is small enough) the observed structures may-if fixed and evaluated at that time-be interpreted as one nucleus and one micronucleus, although it is debatable whether such structures can be defined as micronuclei. In fixed cell preparations structures are generally counted as micronuclei only if they are smaller than about l/3 of the area of a nucleus. However, the size difference between the fragment and the remaining chromatin in Plate 2 would increase after fixation because cells would be flattened. In cytospin preparations in particular the size change of nuclei due to flattening of cells is considerable. In conclusion, /I-oestradiol induced different types of changes in the genomic arrangement of cells and is thus clearly genotoxic in vitro. Acknowledgemenr-This study was supported by the SFB
172 of the Deutsche Forschungsgemeinschaft. REFERENCES
Antcccia A., Tanzarella C., Modesti D. and Degrassi F. (1993) Cytokinesis-block micronucleus assay with
642
I. Eckert and H. Stopper
kinetochore detection in colchicine-treated human fibroblasts. Mutation Research 287, 93-99. Barrett J. C., Hesterberg T. W., Oshimura M. and Tsutsui T. (1985) Role of chemically induced mutagenic events in neoplastic transformation of Syrian hamster embryo cells. In Mechanisms of Carcinogenesis and Assays for Carcinogens. Edited by J. A. Barrett and R. W. Tennant. pp. 123-137. Raven Press, New York. Degen G. H. and Metzler M. (1987) Sex hormones and neoplasia: genotoxic effects in short term assays; mouse liver tumors. Archives of Toxicology 10, 264278. Henderson B. E., Ross R. R., Pike M. C. and Casagrande J. T. (1982) Endogenous hormones as a major factor in human cancer. Cancer Research 42, 3232-3239. Kirchner S., Stopper H., Papp T., Eckert I., Yoo H. J., Vig B. K. and Schiffmann D. (1993) Cytogenetic changes in primary, immortalized and malignant mammalian cells. Toxicology Letters 67, 283-295.
Nicklas R. B. and Kubai D. F. (1985) Microtubules, chromosome movement, and reorientation after chromosomes are detached from the spindle by micromanipulation. Chromosoma 92, 3 13-324. Purdy R. H., Goldzieher J. W., LeQuesne P. W., Abdel Baky S., Durocher C. K., Moore P. H. and Rhim J. S.
(1983) Active intermediates in carcinogenesis. In Catechol Estrogens. Edited by G. L. Merriam and M. B. Lipsett. pp. 123-140. Raven Press, New York. Schiffmann D. and DeBoni U. (1991) Dislocation of chromatin elements in prophase induced by diethylstilbestrol: a novel mechanism by which micronuclei can arise. Mutation Research 246, 113-122. Stopper H., Eckert I., Schiffmann D., Spencer D. L. and Caspary W. J. (1994) Is micronucleus induction by aneugens an early event leading to mutagenesis?. Mutagenesis 9, 411416. Stopper H., Pechan R. and Schiffmann D. (1992) 5Azacytidine induces micronuclei in and morphological transformation of Syrian hamster embryo fibroblasts in the absence of unscheduled DNA svnthesis. Mutation Research 283, 2 l-28.
Wheeler W. J., Cherry L. M., Downs T. and Hsu T. C. (1986) Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in Chinese hamster cells in vitro. Mutation Research 171, 3141. Wheeler W. J., Hsu T. C., Tousson A. and Brinkley B. R. (1987) Mitotic inhibition and chromosome displacement induced by estradiol in Chinese hamster cells. Cell Motility and the Cytoskeleton 7, 235-247.
Toxicology in Vitro 10 (1996) 637442
Genotoxic Effects Induced by fl-Oestradiol In Vitro I. ECKERT
and H. STOPPER*
Department of Toxicology, University of Wiirzburg, Versbacher Str. 9, 97078, Wiirzburg, Germany (Accepted 23 April 1996) Abstract-The carcinogenic endogenous hormone P-oestradiol induces genotoxic effects in short-term mammalian tests. In this study we investigated micronucleus induction in V79 cells. The early induction of primarily kinetochore positive micronuclei indicated a mechanism involving mitotic disturbances. Metaphase arrangements that showed displaced chromosomes suggested that these chromosomes may be enclosed in micronuclei. However, observation of live cells revealed that most of the displaced chromosomes reintegrated. In some of these cells chromatid separation at anaphase was impaired and a small chromatin fragment was separated from the remaining chromatin. Copyright 0 1996 Elsevier Science Ltd.
Introduction The endogenous
hormone
hormone-dependent et
al.,
1982).
anti-human antibody were purchased from Sigma Chemie GmbH (Deisenhofen, Germany). CREST-serum (anti-kinetochore antibody) was purchased from Biermann GmbH (Bad Nauheim, Germany). goat
j?-oestradiol
types Since
of cancer
P-oestradiol
has a role in (Henderson also
induces
genotoxic effects in short-term tests (Degen and Metzler, 1987), not all /3-oestradiol-related changes in cells can be explained by hormone receptor-dependent mechanisms. Examples are the induction of aneuploidy (Barrett et a/., 1985; Wheeler ef al., 1986), cell transformation (Purdy et al., 1983) and micronucleus formation (Wheeler et al., 1987) in vitro. In this study we investigated fi-oestradiolinduced micronucleus formation in vitro in the Chinese hamster cell line V79. Wheeler et al. (1987)
described the occurrence of displaced metaphase chromosomes after treatment with /I-oestradiol. It could be hypothesized that such displaced chromosomes are enclosed in micronuclei. To investigate this hypothesis we first quantified micronucleus induction and the occurrence of such displaced metaphase chromosomes after treatment with /?-oestradiol. Next, we followed live mitotic cells that contained such displaced chromosomes through the further course of mitosis with the method of supravital UV-microscopy (Schiffmann and DeBoni, 1991). Materials and Methods Chemicals
Acridine orange, bisbenzimide 33258 and 33342, cytochalasin B, P-oestradiol and FlTC-conjugated
*Author for correspondence. Abbre2Gations: DMSO = dimethyl sulfoxide; MEM = modified Eagle’s medium; PBS = phosphate buffered saline.
Ceil culture V79 Chinese hamster cells were cultured in modified Eagle’s medium (MEM) medium (Sigma) supplemented with antibiotics and 10% foetal calf serum (Gibco, Germany). Cell cultures were grown in humidified atmosphere with 5% CO? in air at 37°C. Micronucleus assay
Exponentially growing V79 cells were treated with /I-oestradiol for 4 hr. The solvent control contained 1% dimethyl sulfoxide (DMSO) (the maximal amount of DMSO being present from dissolving test substances). After removing P-oestradiol by medium replacement, the cells were incubated for O-25 hr for time course and 5 hr for dose-response experiments. After trypsinization the cells were placed onto glass slides by cytospin centrifugation. Fixation was performed with methanol (-2O”C, 1 hr). For staining, the slides were washed with distilled water incubated with bisbenzimide 33258 (5 pg/ml, 3 min), washed three times with distilled water and mounted for microscopy. Using a magnification of x 1000, the number of nuclei and micronuclei were scored. For the cytochalasin B method, 6 pg cytochalasin B/ml were added together with 75 pM /Loestradiol. After 4 hr the medium was replaced by medium containing 6 pg cytochalasin B/ml and 5 hr later the cells were prepared and fixed as described above. For staining of the cells, the slides were incubated for 4 min in acridine orange (62.5 &ml in Saerensen buffer: 67 mM Na2HP0,/KH$0,, pH 6.8). Slides
0887-2333/96/!§15.00+ 0.00 Copyright 0 1996 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII SO887-2333(96)00035-5
638
1. Eckert and H. Stopper
were washed twice in Scerensen buffer (5 min) and mounted in buffer for microscopy. Micronuclei were scored in binucleate cells using a x 1000 magnification. Each data point represents the mean of three slides with 2000 nuclei (or 1000 binucleate cells) evaluated per slide. All experiments were repeated twice with consistent results. Kinetochore staining
Kinetochore staining was achieved by incubating the fixed cell preparations (after washing for 5 min in phosphate buffered saline (PBS)/O.l% Tween 20) with CREST serum for 75 min in a humidified chamber at 37’C. After washing twice for 5 min in PBS/O.S% Tween 20 again, the cells were incubated as before for 30 min with FlTC-conjugated goat-antihuman antibody (diluted 1:lOO in PBS/O.S% Tween 20, pH 7.4), washed again twice in PBS/O. 1% Tween 20 and counterstained with bisbenzimide 33258 (5 pg/ml, 3 min). Using a magnification of x 1000 nuclei and micronuclei were evaluated for the presence of kinetochore signals. Metaphase ring arrangements V79 cells were treated for 4 hr with B-oestradiol (75 PM) or DMSO (1%) as a solvent control. Then the culture medium was replaced with fresh medium and after 5 min cells were trypsinized and placed onto glass slides with cytospin centrifugation. From withdrawal of P-oestradiol to the start of cytospin preparation a total time of 10 min passed. Cells were fixed in cold ( - 20°C) methanol-acidic acid (3: 1, v/v) and stained as described for micronuclei previously. Because of the cytospin preparation, many metaphases show a ring arrangement. Only those metaphases were evaluated for chromosomes dislocated from the ring.
after p-oestradiol treatment contained kinetochore signals. Thus, /I-oestradiol induced predominantly kinetochore containing micronuclei in V79 cells shortly after its removal. Wheeler et al. (1987) also found an induction of micronuclei after fl-oestradiol treatment at similar concentrations. As these authors did not allow for recovery between substance removal and cell preparation their experiments for time dependence of micronucleus formation are not comparable with ours. Early induction of micronuclei that are kinetochore-positive is typical for substances that act by way of mitotic disturbance (Stopper et al., 1992 and 1994). One method for examining disturbance of mitosis is to score metaphase ring arrangements for displaced chromosomes in fixed cells. After cytospin preparation, metaphases appear as rings and displaced chromosomes can readily be identified (Plate la,b). Of 300 metaphase ring arrangements in the control, 6 (2%) showed a displaced chromosome, whereas after treatment with p-oestradiol 25 of 300 (8.3%) ring arrangements showed such a displaced chromosome. Wheeler et al. (1987) also described the occurrence of misaligned metaphase chromosomes after P-oestradiol treatment in Chinese hamster (Don) cells. We next investigated the course of mitosis in live cells using supravital UV microscopy. With this method every cell has to be followed through mitosis individually and therefore only limited numbers of observations can be performed. We
(4 __ 125-
;r;
E
Supraoital WV microscopy
Supravital UV microscopy was performed according to the method of Schiffmann and DeBoni (1991), using V79 cells. This method allows the observation of the spatial arrangement of chromatin elements throughout mitosis in live cells. Chromatin was stained with 0.1 pg bisbenzimide 33342/ml and cells were treated with P-oestradiol (75 pM) for 3 to 4 hr. Then the medium was replaced (now containing only bisbenzimide) and observation began. As every single mitosis has to be followed over time, only limited numbers of observations can be achieved with this method.
0.
’
0
I 20
I 40
I 60
I 80
I 100
Concentration[w]
(b)
Results and Discussion /?-Oestradiol induced micronuclei in V79 cells in a dose-dependent manner (Fig. la). The time course of micronucleus induction showed a maximum at 5 hr after substance withdrawal (Fig. lb). Detection of kinetochores revealed that 20.7 + 0.5% of the spontaneous and 65.5 If 2.3% of the micronuclei
Fig. 1. Micronucleus induction by P-oestradiol in V79 cells. Dose response (a) and time course after the removal of B-oestradiol (75 PM) (b). BG: spontaneous micronucleus frequency.
Plate
1. Metaphase ring arrangements of V79 cells after cytospin preparation. (a) Control, (b) after treatment with 75 pM /I-oestradiol. The displaced chromosome is indicated by an arrow.
639
Genotoxicity of oestradiol began observation as soon as possible (less than 5 min) after substance withdrawal. First, we found an enriched population of prometaphase cells. After about 10 min these had progressed into metaphase and in some cases we detected a dislocated chromosome (Plate 2). As the cells were alive metaphases did not appear as rings with this method (Plate 2). We followed 22 such metaphases that contained dislocated chromosomes. Of these, only one proceeded further during mitosis with the chromosome staying dislocated and forming a micronucleus. The likely cause for this chromosome to be dislocated and become enclosed in a micronucleus may be explained as follows: 65.5% of the micronuclei after B-oestradiol treatment contained kinetochores. After subtraction of the spontaneous micronuclei (and their kinetochores) 72.9% of the /I-oestradiol-induced micronuclei would contain a kinetochore. As this compound is not known to break chromosomes, all of the induced micronuclei should contain at least one chromatid and therefore a kinetochore. We previously demonstrated that some displaced metaphase chromosomes can be without detectable kinetochore (Kirchner et al., 1993) and discussed that the missing spindle attachment of these might conceivably render them prone to enclosure into micronuclei. If the binding of kinetochore proteins to DNA is impaired after g-oestradiol treatment the relatively infrequent formation of micronuclei by enclosure of a displaced chromosome may be due to this mechanism. Centromere probes that would demonstrate the possible presence of centromeric DNA even in the absence of kinetochor proteins are not yet available for Chinese hamster cells. During the supravital UV microscopy observations in all of the remaining 21 cells the dislocated chromosome was realigned with the other metaphase chromosomes. 11 of these cells were unable to perform a normal anaphase chromatid separation. Four were not able to perform chromatid separation at all resulting in a presumptive endomitosis. Cells were followed until the chromatin showed an interphase-like appearance and in seven cells small fragments of chromatin were eventually separated from the remaining chromatin (Plate 2). From their experiments with fixed cells Wheeler et al. (1987) concluded that most of the displaced chromosomes realigned in metaphase. This is in agreement with our supravital UV video observations and with a study by Nicklas and Kubai (1985) in which it was demonstrated that chromosomes in grasshopper spermatocytes that had been displaced by micromanipulation showed microtubuli-assisted reorientation. However, our study was the first that used supravital UV microscopy to follow live cells through this process of realignment. With supravital UV microscopy it is not possible to distinguish whether cell division had taken place in cells that showed separation of small chromatin fragments. To address this question cytochalasin B
641
was applied. This compound prevents cells from dividing, resulting in binucleate cells. Usually only the binucleate cells are evaluated for the presence of micronuclei assuming that only those are actively cycling cells able to form micronuclei and that the mononucleate cells are cytostatic due to test substance treatment and are unable to form micronuclei. We treated cells with 75 PM /I-oestradiol and we also examined mononucleate cells for the presence of micronucleus-like structures in addition to binucleate cells. As a result of three independent experiments we found that 68 + 7.5% of the micronuclei or micronucleus-like structures (chromatin structures that are smaller than l/3 of the area of a nucleus) after /I-oestradiol treatment were in mononucleate cells. A possible explanation is that cytochalasin B did not prevent cytokinesis, especially if combined with /I-oestradiol. This view is supported by the investigation of Antoccia et al. (1993) in which colchicine induced micronuclei in human fibroblasts without the treatment with cytochalasin B. However, after a combined treatment with colchicine and cytochalasin B micronucleus induction in binucleate cells was impaired. Thus, the inclusion of cytochalasin B in the micronucleus assay may sometimes be misleading in investigations of micronucleus induction by aneugens. As another explanation, we cannot exclude that some of these micronucleus-like structures in mononucleate cells were the result of mitotic disturbances of the kind that we had observed in seven of 22 mitoses (example shown in Plate 2). In the supravital UV-microscopy observations it seemed that the separation of the small fragment occurred rather late in mitosis and it might have been impossible for the cells to divide after that. In some cases (where the separated fragment is small enough) the observed structures may-if fixed and evaluated at that time-be interpreted as one nucleus and one micronucleus, although it is debatable whether such structures can be defined as micronuclei. In fixed cell preparations structures are generally counted as micronuclei only if they are smaller than about l/3 of the area of a nucleus. However, the size difference between the fragment and the remaining chromatin in Plate 2 would increase after fixation because cells would be flattened. In cytospin preparations in particular the size change of nuclei due to flattening of cells is considerable. In conclusion, /I-oestradiol induced different types of changes in the genomic arrangement of cells and is thus clearly genotoxic in vitro. Acknowledgemenr-This study was supported by the SFB
172 of the Deutsche Forschungsgemeinschaft. REFERENCES
Antcccia A., Tanzarella C., Modesti D. and Degrassi F. (1993) Cytokinesis-block micronucleus assay with
642
I. Eckert and H. Stopper
kinetochore detection in colchicine-treated human fibroblasts. Mutation Research 287, 93-99. Barrett J. C., Hesterberg T. W., Oshimura M. and Tsutsui T. (1985) Role of chemically induced mutagenic events in neoplastic transformation of Syrian hamster embryo cells. In Mechanisms of Carcinogenesis and Assays for Carcinogens. Edited by J. A. Barrett and R. W. Tennant. pp. 123-137. Raven Press, New York. Degen G. H. and Metzler M. (1987) Sex hormones and neoplasia: genotoxic effects in short term assays; mouse liver tumors. Archives of Toxicology 10, 264278. Henderson B. E., Ross R. R., Pike M. C. and Casagrande J. T. (1982) Endogenous hormones as a major factor in human cancer. Cancer Research 42, 3232-3239. Kirchner S., Stopper H., Papp T., Eckert I., Yoo H. J., Vig B. K. and Schiffmann D. (1993) Cytogenetic changes in primary, immortalized and malignant mammalian cells. Toxicology Letters 67, 283-295.
Nicklas R. B. and Kubai D. F. (1985) Microtubules, chromosome movement, and reorientation after chromosomes are detached from the spindle by micromanipulation. Chromosoma 92, 3 13-324. Purdy R. H., Goldzieher J. W., LeQuesne P. W., Abdel Baky S., Durocher C. K., Moore P. H. and Rhim J. S.
(1983) Active intermediates in carcinogenesis. In Catechol Estrogens. Edited by G. L. Merriam and M. B. Lipsett. pp. 123-140. Raven Press, New York. Schiffmann D. and DeBoni U. (1991) Dislocation of chromatin elements in prophase induced by diethylstilbestrol: a novel mechanism by which micronuclei can arise. Mutation Research 246, 113-122. Stopper H., Eckert I., Schiffmann D., Spencer D. L. and Caspary W. J. (1994) Is micronucleus induction by aneugens an early event leading to mutagenesis?. Mutagenesis 9, 411416. Stopper H., Pechan R. and Schiffmann D. (1992) 5Azacytidine induces micronuclei in and morphological transformation of Syrian hamster embryo fibroblasts in the absence of unscheduled DNA svnthesis. Mutation Research 283, 2 l-28.
Wheeler W. J., Cherry L. M., Downs T. and Hsu T. C. (1986) Mitotic inhibition and aneuploidy induction by naturally occurring and synthetic estrogens in Chinese hamster cells in vitro. Mutation Research 171, 3141. Wheeler W. J., Hsu T. C., Tousson A. and Brinkley B. R. (1987) Mitotic inhibition and chromosome displacement induced by estradiol in Chinese hamster cells. Cell Motility and the Cytoskeleton 7, 235-247.