Abstract
Tamoxifen (TAM) is a non-steroidal anti-estrogen used widely in the treatment and chemoprevention of breast cancer. TAM treatment can lead to DNA damage, but the mechanism of this process is not fully understood and the experimental data are often inconclusive. We compared the DNA-damaging potential of TAM in normal human peripheral blood lymphocytes and MCF-7 breast cancer cells by using the comet assay. In order to assess whether oxidative DNA damage may contribute to TAM-induced lesions, we employed two DNA repair enzymes: endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg). The kinetics of repair of DNA damage was also measured. In order to evaluate the involvement of free radicals in the genotoxicity of TAM we pre-treated the cells with nitrone spin traps: DMPO and POBN. The use of common antioxidants: vitamin C, amifostine and genistein, helped to assess the contribution of free radicals. TAM damaged DNA in both normal and cancer cells, inducing mainly DNA strand breaks but not alkali-labile sites. The drug at 5 and 10 μM induced DNA double strand breaks (DSBs) in lymphocytes and at 10 μM in MCF-7 cells. We observed complete repair of DSBs in cancer cells by contrast with incomplete repair of these lesions in lymphocytes. In both types of cells TAM induced oxidized purines and pyrimidines. Incubation of the cells with nitrone spin traps and antioxidants decreased, with exception of amifostine in MCF-7 cells, the extents of DNA damage in both kinds of cells, but the results were more distinct in cancer cells. Our results indicate that TAM can be genotoxic for normal and cancer cells by free radicals generation. It seems to have a higher genotoxic potential for normal cells, which can be the result of incomplete repair of DNA DSBs. Free radicals scavengers can modulate TAM-induced DNA damage interfering with its antitumour activity in cancer cells.
Similar content being viewed by others
References
Ashby JA, Tinwell H, Lefevre PA, Browne MA (1995) The single cell gel electrophoresis assay for induced DNA damage (comet assay): measurement of tail length and moment. Mutagenesis 10:85–90
Belzile JP, Choudhury SA, Cournoyer D, Chow TY (2006) Targeting DNA-repair proteins a promising avenue for cancer gene therapy. Curr Gene Ther 6:111–123
Blasiak J, Kowalik J (2000) A comparison of the in vitro genotoxicity of tri- and hexavalent chromium. Mutat Res 469:135–145
Blasiak J, Gloc E, Mlynarski W, Drzewoski J, Skorski T (2002) Amifostine differentially modulates DNA damage evoked by idarubicin in normal and leukemic cells. Leukemia Res 26:1093–1096
Blasiak J, Gloc E, Drzewoski J, Wozniak K, Zadrozny M, Skorski T, Pertynski T (2003) Free radical scavengers can differentially modulate the genotoxicity of amsacrine in normal and cancer cells. Mutat Res 535:25–34
Clemons M, Danson S, Howell A (2002) Tamoxifen (Nolvadex): a review. Cancer Treat Rev 28:165–180
Collins AR, Duthie SJ, Dobson VL (1993) Direct enzymatic detection of endogenous base damage in human lymphocyte DNA. Carcinogenesis 14:1733–1735
Ding J, Miao ZH, Meng LH, Geng MY (2006) Emerging cancer therapeutic opportunities target DNA-repair systems. Trends Pharmacol Sci 27:338–344
Evans MD, Podmore ID, Daly GJ, Perrett D, Lunec J, Herbert KE (1995) Detection of purine lesions in cellular DNA using single cell gel electrophoresis with Fpg protein. Biochem Soc Trans 23:434S
Fisher B, Costantino JP, Wickerham DL, Redmond CK, Kavanah M, Cronin WM, Vogel V, Robidoux A, Dimitrov N, Atkins J, Daly M, Wieand S, Tan-Chiu E, Ford L, Wolmark N (1998) Tamoxifen for prevention of breast cancer: report of the national surgical adjuvant breast and bowel project P-1 study. J Natl Cancer Inst 90:1371–1388
Giatromanolaki A, Sivridis E, Maltezos E, Koukourakis MI (2002) Down-regulation of intestinal-type alkaline phosphatase in the tumor vasculature and stroma provides a strong basis for explaining amifostine selectivity. Semin Oncol 29:14–21
Grdina DJ, Kataoka Y, Murley JS (2000) Amifostine: mechanism of action underlying cytoprotection and chemoprevention. Drug Metab Drug Interact 16:237–279
Greaves P, Goonetilleke R, Nunn G, Topham J, Orton T (1993) Two-year carcinogenicity study of tamoxifen in Alderley Park Wistar-derived rats. Cancer Res 53:3919–3924
Han XL, Liehr JG (1992) Induction of covalent DNA adducts in rodents by tamoxifen. Cancer Res 52:1360–1363
IARC (1996) Tamoxifen. In IARC Monographs on the evolution of carcinogenic risks to humans, No. 66, Some Pharmaceutical Drugs. IARC, Lyon, pp 253–365
Klaude M, Eriksson S, Nygren J, Ahnstrom G (1996) The comet assay: mechanisms and technical considerations. Mutat Res 363:89–96
Kotamraju S, Konorev EA, Joseph J, Kalyanaraman B (2000) Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. J Biol Chem 275:33585–33592
Krokan HE, Standal R, Slupphaug G (1997) DNA glycosylases in the base excision repair. Biochem J 325:1–16
Lim CK, Yuan Z-X, Lamb JH, White IN, De Matteis F, Smith LL (1994) A comparative study of tamoxifen metabolism in female rat, mouse and human liver microsomes. Carcinogenesis 15:589–593
Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362:709–715
Loft S, Poulsen HE (1996) Cancer risk and oxidative DNA damage in man. J Mol Med 74:297–312
Martin EA, Brown K, Gaskell M, Al-Azzawi F, Garner RC, Boocock DJ, Mattock E, Pring DW, Dingley K, Turteltaub KW, Smith LL, White INH (2003) Tamoxifen DNA damage detected in human endometrium using accelerator mass spectrometry. Cancer Res 63:8461–8465
McCall MR, Frei B (1999) Can antioxidant vitamins materially reduce oxidative damage in humans? Free Radic Biol Med 26:1034–1053
Miura T, Muraoka S, Fujimoto Y, Zhao K (2000) DNA strand break and 8-hydroxyguanine formation induced by 2-hydroxyestradiol dispersed in liposomes. J Steroid Biochem Mol Biol 74:93–98
Mizutani A, Okada T, Shibutani S, Sonoda E, Hochegger H, Nishigori Ch, Miyachi Y, Takeda S, Yamazoe M (2004) Extensive chromosomal breaks are induced by tamoxifen and estrogen in DNA repair-deficient cells. Cancer Res 64:3144–3147
Obrero M, Yu DV, Shapiro DJ (2002) Estrogen receptor-dependent and estrogen receptor-independent pathways for tamoxifen and 4-hydroxytamoxifen-induced programmed cell death. J Biol Chem 277:45695–45703
Pagano G, de Biase A, Deeva IB, Degan P, Doronin YK, Iaccarino M, Oral R, Trieff NM, Warnau M, Korkina LG (2001) The role of oxidative stress in developmental and reproductive toxicity of tamoxifen. Life Sci 68:1735–1749
Phillips DH (2001) Understanding the genotoxicity of tamoxifen? Carcinogenesis 22:839–849
Poirier MC, Schild LJ (2003) The genotoxicity of tamoxifen: extent and consequences, Kona Hawaii, January 23, 2003. Mutagenesis 18:395–399
Poon GK, Chui YC, McCague R, Llnning PE, Feng R, Rowlands MG, Jarman M (1993) Analysis of phase I and phase II metabolites of tamoxifen in breast cancer patients. Drug Metab Dispos 21:1119–1124
Salami S, Karami-Tehrani F (2003) Biochemical studies of apoptosis induced by tamoxifen in estrogen receptor positive and negative breast cancer cell lines. Clin Biochem 36:247–253
Shang Y, Brown M (2002) Molecular determinants for the tissue specificity of SERMs. Science 295:2465–2468
Sharma M, Shubert DE, Sharma M, Rodabaugh KJ, McGarrigle BP, Vezina ChM, Bofinger DP, Olson JR (2003) Antioxidant inhibits tamoxifen-DNA adducts in endometrial explant culture. Biochem Biophys Res Commun 307:157–164
Singh NP, McCoy T, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–192
Singh NP, Stephens RE (1997) Microgel electrophoresis: sensitivity, mechanisms and DNA electrostretching. Mutat Res 383:671–675
Tanos V, Brzezinski A, Drize O, Strauss N, Peretz T (2002) Synergistic inhibitory effects of genistein and tamoxifen on human dysplastic and malignant epithelial breast cells in vitro. Obstet Gynecol 102:188–194
Acknowledgments
We would like to thank Ms. Milena Iwaszko for her technical assistance. This work was supported by the University of Lodz, grant number 505/363 and “Spoleczny Komitet Walki z Rakiem” Foundation, Lodz, Poland.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Wozniak, K., Kolacinska, A., Blasinska-Morawiec, M. et al. The DNA-damaging potential of tamoxifen in breast cancer and normal cells. Arch Toxicol 81, 519–527 (2007). https://doi.org/10.1007/s00204-007-0188-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00204-007-0188-3