Mutagenesis vol. 18 no. 6 pp. 487-490,
November 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Aneugenic and clastogenic effects of doxorubicin in human lymphocytes
Industrial Toxicology Research Centre, PO Box-80, M.G.Marg, Lucknow 226 001, Uttar Pradesh, India, 1Centre for Molecular Genetics and Toxicology, University of Wales, Swansea SA2 8PP, UK and 2Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, UK
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Abstract |
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Doxorubicin, a benzanthroquinone anticancer agent, was examined for its effect on micronucleus induction in cultured human lymphocytes. A statistically significant dose-dependent increase in micronucleus frequency (P < 0.001) in binucleated cells was seen and an increase in the kinetochore-positive (P < 0.001) and kinetochore-negative micronuclei (P < 0.001) was observed. An increase was also observed in the number of necrotic cells, but the frequency of apoptotic cells remained almost constant. This confirms that doxorubicin is both clastogenic and aneugenic.
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Introduction |
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Doxorubicin (adriamycin, CAS no. 25316-40-9) is a benzanthroquinone drug, which is useful in the treatment of several types of human malignancies (Fisherman et al., 1996
; Misset et al., 1996). It is cytotoxic and mutagenic in both bacterial and mammalian systems. One type of interaction with the DNA is associated with the production of reactive free radicals (Akman et al., 1992), but its cytotoxic activity has been related to its interaction with nuclear topoisomerase II (Wassermann, 1996). Doxorubicin is known to be a cell cycle-specific agent. Doxorubicin was shown to produce an increase in DNA strand breakage and in the percentage of abnormal frequencies of chromosomal damage in the FISH and conventional chromosomal aberration assays (Anderson et al., 1997). In vivo and in vitro studies in mouse and in different cell lines have shown that doxorubicin increases the frequency of micronuclei (Boucher et al., 1993; Amara-Mokrane et al., 1996; Jagetia and Vijayashree, 1996; Duffaud et al., 1998). The present single study has investigated the effect of low doses of doxorubicin in human lymphocytes using the micronucleus assay using kinetochore staining. This is a test procedure recommended in the Committee on Mutagenicity Guidelines of the Department of Health, UK, for determining the mutagenicity of chemicals (Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment, 2000). |
Materials and methods |
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Chemicals
Micronucleus assay Doxorubicin hydrochloride (adriamycin, CAS no. 25316-40-9) and dimethyl sulphoxide were purchased from Sigma Chemical Co. (Poole, UK). Phytohaemagglutinin (PHA) was obtained from Abbott Laboratories, Maidenhead, UK. RPMI 1640 growth medium and foetal calf serum were purchased from Gibco BRL (Paisley, UK).
Kinetochore labelling Primulin stain (ICN, UK) was used at a concentration of 0.5 µg/ml. Commercial anti-kinetochore antibody (Quadratech, Epsom, UK) and fluorescein isothiocyanate (FITC)-conjugated anti-human antibody (Sigma Chemical Co.) were diluted 1:1 and 1:13, respectively, in phosphate-buffered saline (PBS). Bovine serum albumin (BSA) (Sigma Chemical Co.) was used to remove the unbound antibody. Vectoshield Antifade (Vector, Peterborough, UK) was used to dilute DAPI (Cambio, Cambridge, UK) for staining the nuclei.
Preparation of media
Growth medium (GR10) consisted of final concentrations of 20% heat-inactivated foetal calf serum (HIFCS) and a 1:1:1 mix of 10 000 IU/ml (10 000 µg/ml) penicillin/streptomycin, 100 mM sodium pyruvate and 200 mM L-glutamine, and 1250 IU/ml recombinant interleukin-2 (Chiron, Emeryville, CA) in RPMI 1640 medium.
Stimulating medium (SR10) consisted of a final concentration of 0.4 µg/ml phytohaemagglutinin in GR10 medium.
Culture of human lymphocytes
Peripheral blood was obtained from a healthy non-smoking male donor (30 years of age). Lymphocytes were separated using standard procedures as described previously (Dey et al., 2001) and were then frozen in liquid nitrogen until the experiments were performed. After removal from the liquid nitrogen freezer, cell stocks were thawed rapidly in a water bath at 37°C for 5 min. The thawed cell suspension was transferred to a sterile Universal tube containing 10 ml of wash medium (RPMI 1640 + 10% HIFCS). After gently mixing, the cells were centrifuged at 500 r.p.m. for 10 min, the supernatant was removed and the pellet was resuspended. These cells (at a concentration of 
1 x 105) were then transferred to a small tissue culture flask containing 10 ml of stimulating medium. The flask was gassed with CO2, sealed and placed at an angle of 45° in an incubator at 37°C. On day 5, cells were disaggregated and counted to check for growth and prepared for the in vitro micronucleus assay.
Micronucleus assay
The micronucleus assay was performed using the method of Fenech and Morley (1985) as modified by Ellard and Parry (1993). On day 5 after thawing the cells, two flasks were set up for each dose and incubated for another 24 h. Thereafter the cells were treated with doxorubicin (1% v/v, 100 µl/10 ml culture) until harvest. At 44 h after setting up the flasks, cytochalasin B was added to the cultures at a final concentration of 6 µg/ml. Harvesting was performed at 72 h. Each culture was transferred to a correspondingly labelled Universal tube and centrifuged at 1500 r.p.m. for 10 min. The supernatant was discarded and the pellets resuspended in 1–2 ml of RPMI.
Slide preparation
Aliquots of 100–300 µl of lymphocyte suspensions were cytocentrifuged (Shandon Cytospin, Runcorn, UK) onto microscope slides at 500 r.p.m. for 10 min. The slides were air dried prior to fixation with 90% methanol at –20°C for 8 min and the slides were kept at –20°C until they were stained for kinetochores or with Giemsa.
Slides were stained with 5% Giemsa for 3 min and washed with PBS. Coverslips were mounted using Depex mounting cement.
Kinetochore labelling
Immunofluorescent staining of kinetochores was done using CREST serum containing anti-kinetochore antibodies as described by Ellard et al. (1991) with minor modifications.
Slides were removed from the –20°C freezer and rehydrated in PBS at room temperature. Slides were immersed in a 0.5 µg/ml (in PBS) solution of primulin for 1 min and then placed in a fresh PBS solution. Slides were removed and excess PBS around the cytodot (a dot of cells obtained after centrifugation) was carefully removed with absorbent paper. An aliquot of 50 µl of commercial anti-kinetochore antibody, diluted 1:1 with PBS, was applied to the cytodot. A plastic coverslip was lowered gently, to avoid air bubbles, onto the slide. The slides were placed in a pre-warmed humidified chamber, which was placed in an incubator at 37°C for 45 min. Following incubation, any unbound antibody was removed by a series of washes: 3 x 5 min in PBS + 1% BSA, followed by 3 x 5 min in PBS. After a series of washings, excess PBS around the cytodot was removed with absorbent paper. An aliquot of 50 µl of the second antibody, FITC-conjugated goat anti-human IgG, diluted 1:13 with PBS, was added to the slides and the slides were washed as before.
After the second wash, the slides were rinsed with distilled water and allowed to dry in the dark. Dry slides were stored at –20°C. Slides were mounted with 25 µl of DAPI/antifade solution (1 µl DAPI + 9 µl dH2O + 990 µl antifade) and visualized using an Olympus BH2-RFL fluorescent microscope with a triple bandpass filter to allow the simultaneous observation of both the kinetochore signals and the DAPI counterstain and single bandpass filters (Olympus) for green and blue spectra, to allow the signals and DAPI to be observed separately.
Slide analysis
The slides were coded prior to analysis. The criteria for MN were as described by Fenech (1993, 1996). Scoring of necrotic and apoptotic cells was done according to Fenech et al. (1999). Giemsa stained slides were analysed using an Olympus BH2 light microscope at 1000x magnification and mono-, bi- and multi-nucleated cells were recorded. Micronuclei were scored in a total of 1000 binucleate cells per culture (2000 cells/dose).
The kinetochore labelled slides were viewed under an Olympus BH2-RFL fluorescence microscope. A total of 100 (where possible) micronuclei were observed for the presence or absence of a kinetochore signal.
Statistics
The 
2 test was used to compare the treated cultures with controls.
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Results |
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A pilot experiment was conducted to assess the dose–response relationship of doxorubicin. Doses above a concentration of 0.1 µM showed no binucleate cells (data not shown), hence the maximum dose taken in this study was 0.1 µM.
As shown in Table I, a dose-dependent increase in the mean frequency of binucleated cells containing micronuclei was observed. This increase was 12-, 22- and 27-fold higher in comparison with the negative control and was highly statistically significant. There was a corresponding decrease in the mean percentage of binucleated cells, which was greatly reduced at doses of 0.05 and 0.10 µM.
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Kinetochore studies revealed that among the micronuclei counted, there was a significant decrease (P < 0.001) in the proportion of kinetochore-negative (K–) micronuclei after treatment with doxorubicin, with a concomitant significant increase (P < 0.001) in the kinetochore-positive (K+) micronuclei (Table II). However, the frequency of binucleated cells containing either K– or K+ micronuclei showed a significant dose-dependent increase (P < 0.001). At the lowest dose (0.01 µM), an almost 5-fold increase was observed in the K– micronucleated cells, although a 59-fold increase was observed in the K+ micronuclei. At a dose of 0.10 µM, no micronuclei could be scored for kinetochore staining (Table II).
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An increase was also observed in the frequency of necrotic cells in a dose-dependent manner and there was a slight decrease in the percentage of apoptotic cells (Figure 1).
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Dicussion |
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The results have shown that doxorubicin produces a dose-dependent increase in micronucleus formation in human lymphocytes in vitro and an increase in kinetochore-positive and kinetochore-negative stained micronuclei, indicating the induction of both aneugenicity and clastogenicity. An increase in micronucleus formation in human lymphocytes, over a similar dose range, was also observed by Migliore et al. (1987
). The results of clastogenicity confirm the findings of previous studies (Migliore et al., 1987; Jagetia and Vijayashree, 1996). This study also confirms the aneugenic effect of doxorubicin in vitro reported by Aly et al. (1999), where in lymphocytes from healthy individuals and cancer patients, an increase in the trisomy of chromosomes 7 and 17, using FISH DNA-specific probes, was observed.
The micronucleus index reduction at the 0.10 µM dose compared with the lower doses could be due to the fact that higher necrosis was observed in this dose range. Doxorubicin binds to DNA by intercalation and its antitumour activity has been associated with the production of protein-concealed DNA strand breaks as a result of DNA topoisomerase II poisoning. It is thought that interference of the DNA–topoisomerase complex with replication or transcription may induce the lethal event (Wassermann, 1996). It may also be due to the response of the cells to the genotoxin causing necrosis or mitotic slippage, as discussed by Kirsch-Volders and Fenech (2001). The necrosis observed at higher doses of doxorubicin suggests that while apoptosis has an important role in the elimination of cells with DNA damage, the majority of cells were eliminated by necrosis in lymphocyte cultures. This has also been reported in an earlier study where hydrogen peroxide was used as the DNA-damaging agent (Fenech et al., 1999). This is an important parameter in understanding the toxicity of a compound and has been extensively reviewed (Fenech, 2000; Kirch-Volders and Fenech, 2001).
In an earlier study by Anderson et al. (1997), dose-dependent clastogenicity was observed over a similar dose range to the present study, when measured using FISH and conventional chromosomal analysis. This previous observation corresponds to the findings of the present study and probably explains the observed dose-dependent increase in micronucleus formation. This single study, using kinetochore staining, has additionally shown that along with clastogenicity, doxorubicin also exerts aneugenic effects in human lymphocytes under in vitro conditions.
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Acknowledgements |
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The technical assistance of Ms Margaret Clatworthy is gratefully acknowledged. The authors wish to thank the Department of Science and Technology, Government of India, for a BOYCAST Fellowship (no. HRU/BYS/L-11/99) to A.D.
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Notes |
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3To whom correspondence should be addressed. Tel: +44 1274 233569; Fax: +44 1274 309742; Email: d.anderson1{at}bradford.ac.uk
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Received on April 2, 2002; accepted on July 11, 2003.
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