Mutagenesis

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (12) Request Permissions Google Scholar Articles by Fischer, W. H. Articles by Stopper, H. Search for Related Content PubMed PubMed Citation Articles by Fischer, W. H. Articles by Stopper, H. Social Bookmarking          What's this?

Mutagenesis, Vol. 16, No. 3, 209-212, May 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press

Increased formation of micronuclei after hormonal stimulation of cell proliferation in human breast cancer cells

Wolfgang H. Fischer, Adriana Keiwan, Elmar Schmitt and Helga Stopper^1,

Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany

	Abstract

Top Abstract Introduction Materials and methods Results and discussion References

 
The carcinogenicity of sex hormones is considered to be the result of a combination of genotoxic and epigenetic modes of action. For estrogens, genotoxic activities include DNA damage by reactive metabolites and indirect genotoxicity by redox cycling and production of reactive oxygen species. Here, we present data on the induction of micronuclei in estrogen receptor-positive (MCF-7) and -negative (MDA) human breast cancer cell lines treated with estradiol to support an additional mechanism of chromosomal damage. MCF-7 cells, but not MDA cells, treated with estradiol in the picomolar concentration range showed an increase in micronucleus formation which correlated with the estradiol-induced cell proliferation. Addition of the specific estradiol-receptor antagonist hydroxytamoxifen suppressed the estradiol-induced formation of micronuclei in MCF-7 cells. Increased frequencies were also seen after normalization of the data to the number of cell divisions by additional treatment of the cells with cytochalasin B. Thus, formation of micronuclei was not due to the chromosomal damaging activity of estradiol. The induced genomic damage may be explained by a hormone-specific forcing of responsive cells through the cell cycle, thereby overriding checkpoints operating under homeostatic control of the cell cycle.

	Introduction

Top Abstract Introduction Materials and methods Results and discussion References

 
Tumors in hormone-responsive tissues are among the most prevalent fatal cancer types. The natural hormone estradiol induces tumors in rodents and contributes to carcinogenesis in humans. Sex-hormone-induced carcinogenicity is considered to be the result of a combination of epigenetic and genotoxic mechanisms (for review see Roy and Liehr, 1999).

Initially, estrogens were considered to be epigenetic and nongenotoxic carcinogens largely based on their well-established hormone-receptor-mediated effects on cell proliferation. Later, genotoxic effects of estrogens were described. Types of estrogen-induced genomic damage include direct covalent binding of estrogen metabolites to DNA and indirect genotoxic damage by redox cycling and production of reactive oxygen species (Blackburn et al., 1974; Roy and Liehr, 1999). In general, estrogens do not induce gene mutation in classical bacterial and mammalian mutation assays (Roy and Liehr, 1999), though there have been some positive reports. At the chromosomal level of mutation, the majority of studies have reported that estrogens do not induce chromosomal breaks or aberrations (e.g. Banduhn and Obe, 1985; Schuler et al., 1998). On the other hand, alterations in chromosome number evidently occur. At concentrations of 10 µM, ploidy changes have been observed (Schuler et al., 1998; Wheeler et al., 1986). Micronucleus induction at 20 µM has been described and was explained by mitotic disturbances in accordance with ploidy changes reported in that concentration range (Eckert and Stopper, 1996).

High-fidelty maintenance of genomic integrity is ensured by DNA repair and cell cycle checkpoints, surveillance pathways that respond to DNA damage by inhibiting critical cell cycle events (Weinert, 1998). Impaired fidelity of checkpoint control may give cells the opportunity to proceed through mitosis without adequate DNA repair, resulting in increased genomic damage. This could be achieved by loss of function of genes involved in these pathways or by compounds interfering with cell cycle regulation. Mailand et al. (2000) have reported that overexpression of Cdc25A, a phosphatase required for progression from G₁ to S phase of the cell cycle, increased genomic damage (Mailand et al., 2000). Caffeine overrides the G₂–M cell cycle checkpoint, sensitizing cells to genomic damage (Kiefer and Wiebel, 1998; Zhou et al., 2000).

Overriding checkpoints operating under homeostatic control of the cell cycle could also be a result of forcing cells through the cycle by stimulation of cell proliferation. One consequence of this might be that there is insufficient time for adequate control and repair of DNA damage. Since hormones are known to increase the rate of cell proliferation in responsive cells, it seems possible that this mechanism could contribute to the process of hormone-induced cancer. In the present study we demonstrate that estrogen-induced increase in cell proliferation of estrogen-receptor-positive human breast cancer cells is correlated with increased genomic damage.

	Materials and methods

Top Abstract Introduction Materials and methods Results and discussion References

 
Cell culture and micronucleus test
MCF-7 and MDA-MB231 cells (ATTC) were cultured in RPMI 1640 medium supplemented with antibiotics, L-glutamine (0.25 mg/ml), sodium pyruvate, human insulin (0.2 ng/ml) and 5% fetal bovine serum (FBS) (all from Sigma Chemie GmbH, Deisenhofen, Germany). Cell cultures were grown in a humidified atmosphere with 5% CO₂ in air at 37°C. For experiments, cells (0.8–1.0 x 10⁵/ml) were seeded in culture flasks (25 ml). Twenty-four hours later, medium was exchanged. Fresh medium contained charcoal/dextran-treated FBS (4.5%), normal FBS (0.5%) and test compunds as indicated. At 96 h, medium was exchanged again. Cells were harvested at 144 h (MCF-7) or 120 h (MDA-MB231). Cell numbers were determined using a Coulter counter. In experiments with cytochalasin B, cells were incubated for 120 h in the presence of 2.5 µg/ml cytochalasin B.

After harvesting, cells were collected on glass slides by cytospin centrifugation and fixed with methanol (–20°C, 1 h). To stain nuclei and micronuclei, the slides were incubated with acridine orange [0.00625% (w/v) in Sorensen buffer, pH 6.8] for 4 min. Slides were washed twice with buffer and mounted for microscopy. Numbers of nuclei and micronuclei were scored at a magnification of 500x. In experiments with cytochalasin B, micronuclei were evaluated in binucleated cells only. Each data point represents the mean of two slides from one experiment with 1000 cells/binucleate cells analyzed for the number of micronuclei per slide. Objects were classified as micronuclei if they were clearly separated from the nuclei, were round or oval, had an area of less than a quarter of the area of a nucleus and showed staining characteristics similar to those of nuclei. Statistical analysis was performed using Student's t-test.

Gene expression assay
Cells were transiently transfected with the plasmids pSVGal and ptkERE2Luc using the Lipofectamine method according to the manufacturer's instructions (Boehringer Mannheim, Germany). Briefly, cells were seeded in 96-well plates at a density of 16 000 cells/well in Phenol-red-free medium containing 5% charcoal/dextran-treated FBS. The next day, cells were transfected in serum-free, Phenol-red-free medium using Lipofectamine and plasmids for 6.5 h. After transfection, medium was removed from the wells by aspiration and replaced with Phenol-red-free medium containing 5% charcoal/dextran-treated FBS and the indicated test compounds. Twenty hours later, medium was removed and cells were incubated in lysis buffer for 30 min at 25°C. Collected supernantants were divided into two portions. One was analysed for luciferase activity while the other was used for the determination of ß-galactosidase activity. Luciferase activity was determined in a Berthold LB96P luminometer using the manufacturer's reagents and instructions. ß-Galactosidase activity was measured after addition of 40 µg galactosidase substrate and subsequent incubation at 37°C for 1 h. Optical density was measured at 420 nm. Gene expression, measured as luciferase activity, was normalized for ß-galactosidase activity and protein concentration. The luciferase activity of samples treated with test compounds divided by the normalized luciferase activity of those treated with vehicle was used to determine fold induction. The data shown are representative of three independent experiments with three replicates.

	Results and discussion

Top Abstract Introduction Materials and methods Results and discussion References

 
The `E-screen' (Payne et al., 2000) is a commonly used system for evaluating the ability of chemicals to induce a hormonal response. This test is based on the cell proliferation of the human estrogen-receptor-positive breast cancer cell line, MCF-7. In addition to cell proliferation, we investigated the induction of chromosomal damage in this system. As a measure of chromosomal damage, we analyzed the induction of micronuclei and detected genotoxicity after treatment with high concentrations of several phytohormones (Boos and Stopper, 2000). In these experiments a low dose (1 nM) of estradiol was used as a positive control for hormone-induced cell proliferation. To our surprise, an increase in chromosomal damage after treatment with estradiol was detected (Figure 1). However, identical treatment of the estrogen-receptor-negative human breast cancer cell line, MDA, did not induce micronuclei in these cells, indicating that the applied estradiol concentration was not genotoxic in this test. The occurrence of micronuclei in estrogen-receptor-positive (MCF-7), but not in -negative (MDA) breast cancer cells indicated that the estrogen receptor had a role in the observed micronucleus formation. To verify that the estrogen receptor was expressed and biologically active in the MCF-7 cells used here, MCF-7 cells were transiently transfected with a plasmid carrying the estrogen-responsive element conjugated with the reporter enzyme luciferase (Klotz et al., 1996). Treatment of transfected MCF-7 cells with estradiol (1 nM) resulted in an increased luciferase activity, i.e. increased gene expression (Figure 2). The known estrogen-receptor antagonist, hydroxytamoxifen (50 nM), significantly reduced the activity induced by estradiol. In control experiments with transfected MDA cells, no induction of gene expression was observed (data not shown). Thus, induction of micronuclei under these experimental conditions was only found in cells expressing a biologically active estrogen receptor.

View larger version (17K): [in this window] [in a new window]   Fig. 1. . Formation of micronuclei after treatment with vehicle [0.1% dimethylsulfoxide (DMSO)], 1 nM estradiol or 4 µg/ml mitomycin C (MMC; positive control). Estrogen-receptor-positive MCF-7 cells (left) and estrogen-receptor-negative MDA cells (right) were incubated for 140 h (MCF-7) or 115 h (MDA) in the presence or absence of estradiol and MMC. After harvesting, cells were prepared by cytospin, stained with acridine orange and analyzed microscopically. *P < 0.05.

 

View larger version (19K): [in this window] [in a new window]   Fig. 2. . Induction of estrogen-specific gene expression after treatment with vehicle (0.1% ethanol), estradiol (1 nM), hydroxytamoxifen (OH-T; 50 nM), or estradiol and OHT. MCF-7 cells were transiently transfected with the plasmids pSV Gal and ptkERE2Luc using the Lipofectamine method according to the manufacturer's instructions (Boehringer Mannheim). The plasmid pSV Gal was used as an internal standard for normalizing luciferase activity. Luciferase activity was determined by measuring chemoluminescence in a Berthold LB96P Microlumat and represents the estrogen-dependent gene expression after binding of ligand–receptor complex to the estrogen-responsive elements located on ptkERE2Luc. *P < 0.05.

 
Next, we investigated whether micronucleus formation after stimulation with estradiol was concentration-dependent in MCF-7 cells (Figure 3A). Concentration dependency was observed between 5 and 1000 pM estradiol. To exclude the possibility that the observed effect was due to the described gentoxic potential of estradiol, MDA cells were treated with estradiol. Even with a five-fold higher concentration, we detected no induction of micronucleus formation in these receptor-negative cells (Figure 3D). This is in accordance with findings by Banduhn and Obe (1985) and Schuler et al. (1998), which showed that estradiol induces chromosomal damage only in the micromolar range. More recently, Ahmad et al. (2000) demonstrated the lack of chromosomal damage in human lymphocytes after treatment with 36 µM estradiol. Incubation with 92 µM estradiol induced chromosomal damage (Ahmad et al., 2000). In our experiments, the concurrently analyzed cell proliferation also showed a concentration-dependency in MCF-7 cells but not in MDA cells (Figure 3B and E). Thus, a correlation between the rate of cell proliferation and micronucleus formation was found in MCF-7 cells: the higher the rate of cell proliferation, the higher the number of micronuclei (Figure 3C). In order to assess whether the observed formation of micronuclei was mediated by the estrogen receptor, we applied the specific receptor antagonist hydroxytamoxifen. Simultaneous treatment with estradiol (1 nM) and hydroxytamoxifen (0.5 nM) resulted in reduced formation of micronuclei (Figure 4). Hydroxytamoxifen by itself had no effect.

View larger version (27K): [in this window] [in a new window]   Fig. 3. . Formation of micronuclei (A and D) or induction of cell proliferation (B and E) after treatment with vehicle (0.1% ethanol) or the indicated concentrations of estradiol. MCF-7 cells or MDA cells were incubated for 140 h in the presence or absence of estradiol. After harvesting, cells were counted to assess cell proliferation and prepared by cytospin, stained with acridine orange and analyzed microscopically for the presence of micronuclei. (C) Correlation between cell proliferation (B) and micronucleus formation (A) in MCF-7 cells. The significance of this correlation was analyzed by Spearman's rank test (P < 0.05).

 

View larger version (22K): [in this window] [in a new window]   Fig. 4. . Formation of micronuclei after treatment with vehicle (0.1% DMSO), 1 nM estradiol, 0.5 nM hydroxytamoxifen (OH-T) or estradiol in combination with OH-T. MCF-7 cells were incubated for 140 h. After harvesting, cells were prepared by cytospin, stained with acridine orange and analyzed microscopically. *P < 0.05.

 
In the next set of experiments, we normalized the induction of micronucleus formation to the number of cell divisions in untreated control cells and in estradiol-stimulated cells. Experimentally, this was achieved by adding cytochalasin B, which inhibits cell division but not mitosis, yielding binucleate cells (Fenech, 1993). If the analysis of micronucleus frequencies is limited to these binucleate cells, all analyzed cells have divided exactly once during the experiment. It can be assumed that the endogenous damage present at the beginning of the experiment is identical in controls and cells that will be treated with estradiol. Therefore, one cell division should lead to the same number of micronuclei based on the expression of endogenous DNA-damage in both groups. Thus, if the same number of binucleate cells is analyzed from controls and estradiol-treated cells, no differences in micronucleus numbers should be observed. However, in our experiments the frequency of micronuclei was 1.5-fold higher in estradiol stimulated cells than in controls in three independent experiments (Figure 5).

View larger version (18K): [in this window] [in a new window]   Fig. 5. . Increase in micronucleus formation in binucleate MCF-7 cells. Cells were treated for 120 h with vehicle (0.1% DMSO) or 1 nM estradiol in the presence of cytochalasin B (2.5 µg/ml). After harvesting, cells were prepared by cytospin and stained with acridine orange. Binucleate cells were analyzed for the presence of micronuclei.

 
These data indicate that cell division in estradiol-treated cells differs from that in controls. Stimulation of MCF-7 cells by estradiol leads to increased cell proliferation accompanied by shorter cell cycles. The resulting time limitations could impair the fidelity of control and/or repair of genomic integrity. This is comparable to a situation in which DNA repair is inhibited or in which chemicals impair cell-cycle checkpoints. It is well known that inhbitors of DNA repair induce micronuclei (Fenech and Neville, 1992). However, estradiol does not impair DNA repair (Coibion et al., 1989; Epstein and Smith, 1988). Kiefer and Wiebel (1998) demonstrated that caffeine potentiated the formation of micronuclei caused by environmental chemical carcinogens in V79 Chinese hamster cells. They suggested that caffeine might be able to override the delay caused by DNA-damaging agents in the growth cycle of mammalian cells. Zhou et al. (2000) demonstrated recently that caffeine abolishes the mammalian G₂–M DNA damage checkpoint. In accordance with this, it has been shown that dysregulated cell-cycle checkpoint control sensitizes cells to induction of DNA damage (Suganuma et al., 1999). In addition, Schwartz et al. (1996) described an inverse correlation between the length of G₂-phase delay after radiation exposure and the frequency of induced chromosomal terminal deletions in 10 human tumor cell lines. Therefore, it seems likely that our observations can be explained by a mechanism involving cell-cycle control.

In conclusion, our results demonstrate that hormonal stimulation of cells can lead to increased chromosomal damage. We suggest that this may be caused by disturbances in cell-cycle control or progression. Further work will shed light on these relationships and will elucidate their possible role in hormone-related cancers.

	Acknowledgments

 
We thank Martina Ruppert for her technical assistance. S.F.Arnold and J.A.McLachlan are gratefully acknowledged for providing the plasmids used in the gene expression assays. We thank S.O.Müller for assistance with transfection experiments. This study was supported by the Universitätsbund Würzburg.

	Notes

 
¹ To whom correspondence should be addressed. Tel: +49 931 201 3427; Fax: +49 931 201 3446; Email: stopper{at}toxi.uni-wuerzburg.de

	References

Top Abstract Introduction Materials and methods Results and discussion References

 

Ahmad,M.E., Shadab,G.G.H.A., Hoda,A. and Afzal,M. (2000) Genotoxic effects of estradiol-17ß on human lymphocyte chromosomes. Mutat. Res., 466, 109–115.[Medline]

Banduhn,N. and Obe,G. (1985) Mutagenicity of methyl-2-benzimidazolcarbamate, diethylstilbestrol and estradiol: structural chromosomal aberrations, sister-chromatid exchanges, C-mitoses, polyploidies, and micronuclei. Mutat. Res., 156, 199–218.[ISI][Medline]

Blackburn,G.M., Flavell,A.J. and Thompson,M.H. (1974) Oxidative and photochemical linkage of diethylstilbestrol to DNA in vitro. Cancer Res., 34, 2015–2019.[Abstract/Free Full Text]

Boos,G. and Stopper,H. (2000) Genotoxicity of several clinically used topoisomerase II inhibitors. Toxicol. Lett., 116, 7–16.[ISI][Medline]

Coibion,M., Kiss,R., Jossa,V., de Launoit,Y., Van Geloven,O., Malengreau,A., Mattheiem,W., Pasteels,J.L. and Paridaens,R. (1989) In vitro influence of estradiol or progesterone on the thymidine labeling indices of human benign breast tumors. Anticancer Res., 9, 475–482.[ISI][Medline]

Eckert,I. and Stopper,H. (1996) Genotoxic effects induced by ß-oestradiol in vitro. Toxicol. In Vitro, 10, 637–642.

Epstein,R.J. and Smith,P.J. (1988) Estrogen-induced potentiation of DNA damage and cytotoxicity in human breast cancer cells treated with topoisomerase II-interactive antitumor drugs. Cancer Res., 48, 297–303.[Abstract/Free Full Text]

Fenech,M. (1993) The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat. Res., 285, 35–44.[ISI][Medline]

Fenech,M. and Neville,S. (1992) Conversion of excision-repairable DNA lesions to micronuclei within one cell cycle in human lymphocytes. Environ. Mol. Mutagen., 19, 27–36.[ISI][Medline]

Kiefer,F. and Wiebel,F.J. (1998) Caffeine potentiates the formation of micronuclei caused by environmental chemical carcinogens in V79 Chinese hamster cells. Toxicol. Lett., 96/97, 131–136.[Medline]

Klotz,D.M., Beckman,B.S., Hill,S.M., McLachlan,J.A., Walters,M.R. and Arnold,S.F. (1996) Identification of environmental chemicals with estrogenic activity using a combination of in vitro assays. Environ. Health Perspect., 104, 1084–1089.[ISI][Medline]

Mailand,N., Falck,J., Lukas,C., Syljuasen,R.G., Welcker,M., Bartek,J. and Lukas, J. (2000) Rapid destruction of human Cdc25A in response to DNA damage. Science, 288, 1425–1429.[Abstract/Free Full Text]

Payne,J., Jones,C., Lakhani,S. and Kortenkamp,A. (2000) Improving the reproducibility of the MCF-7 cell proliferation assay for the detection of xenoestrogens. Sci. Total Environ., 248, 51–62.[Medline]

Roy,D. and Liehr,J.G. (1999) Estrogen, DNA damage and mutations. Mutat. Res., 424, 107–115.[ISI][Medline]

Schuler,M., Hasegawar,L., Parks,R., Metzler,M. and Eastmond,D.A. (1998) Dose–response studies of the induction of hyperdiploidy and polyploidy by diethylstilbestrol and 17ß-estradiol in cultured human lymphocytes using multicolor fluorescence in situ hybridization. Environ. Mol. Mutagen., 31, 263–273.[ISI][Medline]

Schwartz,J.L., Cowan,J., Grdina,D.J. and Weichselbaum,R.R. (1996) Attenuation of G₂-phase cell cycle checkpoint control is associated with increased frequencies of unrejoined chromosome breaks in human tumor cells. Radiat. Res., 146, 139–143.[ISI][Medline]

Suganuma,M., Kawabe,T., Hori,H., Funabiki,T. and Okamoto,T. (1999) Sensitization of cancer cells to DNA damage-induced cell death by specific cell cycle G₂ checkpoint abrogation. Cancer Res., 59, 5887–5891.[Abstract/Free Full Text]

Weinert,T. (1998) DNA damage and checkpoint pathways: molecular anatomy and interactions with repair. Cell, 94, 555–558.[ISI][Medline]

Wheeler,W.J., Cherry,L.M., Downs,T. and Hsu,T.C. (1986) Mitotic inhibition and aneuploidy induction by naturally occurring and sythetic estrogens in chinese hamster cells in vitro. Mutat. Res., 171, 31–41.[ISI][Medline]

Zhou,B.-B.S., Chaturvedi,P., Spring,K., Scott,S.P., Johanson,R.A., Mishra,R., Mattern,M.R., Winkler,J.D. and Khanna,K.K. (2000) Caffeine abolishes the mammalian G₂/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity. J. Biol. Chem., 275, 10342–10348.[Abstract/Free Full Text]

Received on October 27, 2000; accepted on November 24, 2000.

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				O Torres-Bugarin, R Covarrubias-Bugarin, A L Zamora-Perez, B M G Torres-Mendoza, M Garcia-Ulloa, and F G Martinez-Sandoval Anabolic androgenic steroids induce micronuclei in buccal mucosa cells of bodybuilders Br. J. Sports Med., September 1, 2007; 41(9): 592 - 596. [Abstract] [Full Text] [PDF]

				O. I. Kalantzi, R. Hewitt, K. J. Ford, L. Cooper, R. E. Alcock, G. O. Thomas, J. A. Morris, T. J. McMillan, K. C. Jones, and F. L. Martin Low dose induction of micronuclei by lindane Carcinogenesis, April 1, 2004; 25(4): 613 - 622. [Abstract] [Full Text] [PDF]

				H. Stopper, E. Schmitt, C. Gregor, S. O. Mueller, and W. H. Fischer Increased cell proliferation is associated with genomic instability: elevated micronuclei frequencies in estradiol-treated human ovarian cancer cells Mutagenesis, May 1, 2003; 18(3): 243 - 247. [Abstract] [Full Text] [PDF]

				C. Davis, S. Bhana, A. J. Shorrocks, and F. L. Martin Oestrogens induce G1 arrest in benzo[a]pyrene-treated MCF-7 breast cells whilst enhancing genotoxicity and clonogenic survival Mutagenesis, September 1, 2002; 17(5): 431 - 438. [Abstract] [Full Text] [PDF]

				E. Yared, T. J. McMillan, and F. L. Martin Genotoxic effects of oestrogens in breast cells detected by the micronucleus assay and the Comet assay Mutagenesis, July 1, 2002; 17(4): 345 - 352. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (12) Request Permissions Google Scholar Articles by Fischer, W. H. Articles by Stopper, H. Search for Related Content PubMed PubMed Citation Articles by Fischer, W. H. Articles by Stopper, H. Social Bookmarking          What's this?

Online ISSN 1464-3804 - Print ISSN 0267-8357

Copyright © 2008 United Kingdom Environmental Mutagen Society

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics