Biomedical Microdevices

, Volume 14, Issue 4, pp 709–720 | Cite as

Concentration-dependent cytotoxicity of copper ions on mouse fibroblasts in vitro: effects of copper ion release from TCu380A vs TCu220C intra-uterine devices

  • Bianmei Cao
  • Yudong Zheng
  • Tingfei Xi
  • Chuanchuan Zhang
  • Wenhui Song
  • Krishna Burugapalli
  • Huai Yang
  • Yanxuan Ma


Sustained release of copper (Cu) ions from Cu-containing intrauterine devices (CuIUD) is quite efficient for contraception. However, the tissue surrounding the CuIUD is exposed to toxic Cu ion levels. The objective for this study was to quantify the concentration dependent cytotoxic effects of Cu ions and correlate the toxicity due to Cu ion burst release for two popular T-shaped IUDs - TCu380A and TCu220C on L929 mouse fibroblasts. Fibroblasts were cultured in 98 well tissue culture plates and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphehyltetrazolium bromide (MTT) assay was used to determine their viability and proliferation as a function of time. For cell seeding numbers ranging from 10,000 to 100,000, a maximum culture time of 48 h was identified for fibroblasts without significant reduction in cell proliferation due to contact inhibition. Thus, for Cu cytotoxicity assays, a cell seeding density of 50,000 and a maximum culture time of 48 h in 96 well plates were used. 24 h after cell seeding, culture media were replaced with Cu ion containing media solutions of different concentrations, including 24 and 72 h extracts from TCuIUDs and incubated for a further 24 h. Cell viability decreased with increasing Cu ion concentration, with 30 % and 100 % reduction for 40 μg/ml and 100 μg/ml respectively at 24 h. The cytotoxic effects were further evaluated using light microscopy, apoptosis and cell cycle analysis assays. Fibroblasts became rounded and eventually detached from TCP surface due to Cu ion toxicity. A linear increase in apoptotic cell population with increasing Cu ion concentration was observed in the tested range of 0 to 50 μg/ml. Cell cycle analysis indicated the arrest of cell division for the tested 25 to 50 μg/ml Cu ion treatments. Among the TCuIUDs, TCu220C having 265 mm2 Cu surface area released 9.08 ± 0.16 and 26.02 ± 0.25 μg/ml, while TCu380A having 400 mm2 released 96.7 ± 0.11 and 159.3 ± 0.15 μg/ml respectively following 24 and 72 h extractions. The effects of TCuIUD extracts on viability, morphology, apoptosis and cell cycle assay on L929 mouse fibroblasts cells, were appropriate for their respective Cu ion concentrations. Thus, a concentration of about 46 μg/ml (~29 μM) was identified as the LD50 dose for L929 mouse fibroblasts when exposed for 24 h based on our MTT cell viability assay. The burst release of lethal concentration of Cu ions from TCu380A, especially at the implant site, is a cause of concern, and it is advisable to use TCuIUD designs that release Cu ions within cytotoxic limits yet therapeutic, similar to TCu220C.


Intrauterine devices Copper Cell behavior Material-cell interactions 



The authors acknowledge the financial support from National Science and Technology Support Project of China (Grant No.2006BAI15B08), National Natural Science Foundation of China Project (Grant No. 51073024) and the Royal Society-NSFC international joint project grant (No. 5111130207).


  1. V. Arancibia, C. Pena, H.E. Allen et al., Characterization of copper in uterine fluids of patients who use the copper T-380A intrauterine device. Clin. Chim. Acta 332, 69–78 (2003)CrossRefGoogle Scholar
  2. R. Araya, H. Gomez-Mora, R. Vera et al., Human spermatozoa motility analysis in a Ringer’s solution containing cupric ions. Contraception 67, 161–163 (2003)CrossRefGoogle Scholar
  3. N. Arnal, M.J. de Alaniz, C.A. Marra, Alterations in copper homeostasis and oxidative stress biomarkers in women using the intrauterine device TCu380A. Toxicol. Lett. 192, 373–378 (2010)CrossRefGoogle Scholar
  4. N.S. Aston, N. Watt, I.E. Morton et al., Copper toxicity affects proliferation and viability of human hepatoma cells (HepG2 line). Hum. Exp. Toxicol. 19, 367–376 (2000)CrossRefGoogle Scholar
  5. M.J. Beltran-Garcia, A. Espinosa, N. Herrera et al., Formation of copper oxychloride and reactive oxygen species as causes of uterine injury during copper oxidation of Cu-IUD. Contraception 61, 99–103 (2000)CrossRefGoogle Scholar
  6. M.J. Burkitt, Copper–DNA adducts. Methods Enzymol. 234, 66–79 (1994)CrossRefGoogle Scholar
  7. S. Cai, X. Xia, C. Xie, Corrosion behavior of copper/LDPE nanocomposites in simulated uterine solution. Biomaterials 26, 2671–2676 (2005)CrossRefGoogle Scholar
  8. B. Cao, T. Xi, Y. Zheng, Release behavior of cupric ions for TCu380A and TCu220C IUDs. Biomed. Mater. 3, 044114 (2008)CrossRefGoogle Scholar
  9. C.C. Chang, H.J. Tatum, F.A. Kincl, The effect of intrauterine copper and other metals on implantation in rats and hamsters. Fertil. Steril. 21, 274–278 (1970)Google Scholar
  10. M.C. Cortizo, M.A. De Mele, A.M. Cortizo, Metallic dental material biocompatibility in osteoblastlike cells correlation with metal ion release. Biol. Trace Elem. Res. 100, 151–168 (2004)CrossRefGoogle Scholar
  11. D. De la Cruz, A. Cruz, M. Arteaga et al., Blood copper levels in Mexican users of the T380A IUD. Contraception 72, 122–125 (2005)CrossRefGoogle Scholar
  12. M. DiDonato, B. Sarkar, Copper transport and its alterations in Menkes and Wilson diseases. Biochim. Biophys. Acta 1360, 3–16 (1997)Google Scholar
  13. C.A. Grillo, M.A. Reigosa, M.A. de Mele, Does over-exposure to copper ions released from metallic copper induce cytotoxic and genotoxic effects on mammalian cells? Contraception 81, 343–349 (2010)CrossRefGoogle Scholar
  14. C.A. Grillo, M.A. Reigosa, M.F. Lorenzo de Mele, Effects of copper ions released from metallic copper on CHO-K1 cells. Mutat. Res. 672, 45–50 (2009)Google Scholar
  15. K. Hagenfeldt, Intrauterine contraception with the copper-T device. 4. Influence on protein and copper concentrations and enzyme activities in uterine washings. Contraception 6, 219–230 (1972)CrossRefGoogle Scholar
  16. M. Hayashi, S. Fuse, D. Endoh et al., Accumulation of copper induces DNA strand breaks in brain cells of Long-Evans Cinnamon (LEC) rats, an animal model for human Wilson Disease. Exp. Anim. 55, 419–426 (2006)CrossRefGoogle Scholar
  17. F. Hefnawi, O. Kandil, H. Askalani et al., Copper levels in women using intrauterine devices or oral contraceptives. Fertil. Steril. 25, 556–561 (1974)Google Scholar
  18. G.G. Hov, F.E. Skjeldestad, T. Hilstad, Use of IUD and subsequent fertility–follow-up after participation in a randomized clinical trial. Contraception 75, 88–92 (2007)CrossRefGoogle Scholar
  19. D. Hubacher, P.L. Chen, S. Park, Side effects from the copper IUD: do they decrease over time? Contraception 79, 356–362 (2009)CrossRefGoogle Scholar
  20. D. Hubacher, R. Lara-Ricalde, D.J. Taylor et al., Use of copper intrauterine devices and the risk of tubal infertility among nulligravid women. N. Engl. J. Med. 345, 561–567 (2001)CrossRefGoogle Scholar
  21. D. Hubacher, R. Vilchez, R. Gmach et al., The impact of clinician education on IUD uptake, knowledge and attitudes: results of a randomized trial. Contraception 73, 628–633 (2006)CrossRefGoogle Scholar
  22. ISO10993-5, Biological evaluation of medical devices, Part 5: Tests for in vitro cytotoxicity. 9 (2009).Google Scholar
  23. J. Kang, C. Lin, J. Chen et al., Copper induces histone hypoacetylation through directly inhibiting histone acetyltransferase activity. Chem. Biol. Interact. 148, 115–123 (2004)CrossRefGoogle Scholar
  24. B. Kaplan, R. Orvieto, M. Hirsch et al., The impact of intrauterine contraceptive devices on cytological findings from routine Pap smear testing. Eur. J. Contracept. Reprod. Health Care 3, 75–77 (1998)CrossRefGoogle Scholar
  25. T. Kishimoto, Y. Fukuzawa, M. Abe et al., Injury to cultured human vascular endothelial cells by copper (CuSO4). Nihon Eiseigaku Zasshi 47, 965–970 (1992)CrossRefGoogle Scholar
  26. A. Kjaer, K. Laursen, L. Thormann et al., Copper release from copper intrauterine devices removed after up to 8 years of use. Contraception 47, 349–358 (1993)CrossRefGoogle Scholar
  27. R. Kulier, P.A. O’Brien, F.M. Helmerhorst, et al., Copper containing, framed intra-uterine devices for contraception, Cochrane Database Syst Rev, CD005347 (2007).Google Scholar
  28. M.E. Letelier, A.M. Lepe, M. Faundez et al., Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity. Chem. Biol. Interact. 151, 71–82 (2005)CrossRefGoogle Scholar
  29. M.C. Linder, Biochemistry of Copper, in Biochemistry of the elements, ed. by E. Freiden (Plenum Publishing Co., New York, 1991), pp. 163–239Google Scholar
  30. Y.-H. Lu, H.-B. Xu, J. Wang et al., Size effect on the corrosion behavior of Copper wires in NaCl solution. Acta Phys. -Chim. Sin. 24, 1907–1911 (2008)CrossRefGoogle Scholar
  31. A.I. Meisler, Studies on contact inhibition of growth in the mouse fibroblast, 3T3. II. Effects of amino acid deprivation and serum on growth rate. J. Cell. Sci. 12, 861–873 (1973a)Google Scholar
  32. A.I. Meisler, Studies on contract inhibition of growth in the mouse fibroblast, 3 T3. I. Changes in cell size and composition during ‘unrestricted’ growth. J. Cell. Sci. 12, 847–859 (1973b)Google Scholar
  33. H. Obata, N. Sawada, H. Isomura et al., Abnormal accumulation of copper in LEC rat liver induces expression of p53 and nuclear matrix-bound p21(waf 1/cip 1). Carcinogenesis 17, 2157–2161 (1996)CrossRefGoogle Scholar
  34. T. Okereke, I. Sternlieb, A.G. Morell et al., Systemic absorption of intrauterine copper. Science 177, 358–360 (1972)CrossRefGoogle Scholar
  35. M.E. Ortiz, H.B. Croxatto, C.W. Bardin, Mechanisms of action of intrauterine devices. Obstet. Gynecol. Surv. 51, S42–51 (1996)CrossRefGoogle Scholar
  36. M.D. Pereda, M. Reigosa, M. Fernandez Lorenzo de Mele, Relationship between radial diffusion of copper ions released from a metal disk and cytotoxic effects. Comparison with results obtained using extracts. Bioelectrochemistry 72, 94–101 (2008)CrossRefGoogle Scholar
  37. R. Prasad, G. Kaur, R. Nath et al., Molecular basis of pathophysiology of Indian childhood cirrhosis: role of nuclear copper accumulation in liver. Mol. Cell. Biochem. 156, 25–30 (1996)CrossRefGoogle Scholar
  38. L. Roblero, A. Guadarrama, T. Lopez et al., Effect of copper ion on the motility, viability, acrosome reaction and fertilizing capacity of human spermatozoa in vitro. Reprod. Fertil. Dev. 8, 871–874 (1996)CrossRefGoogle Scholar
  39. K. Shimizu, T. Nishikawa, M. Nozaki et al., Effects of an intrauterine copper device on serum copper, endometrial histology, and ovarian, hepatic, and renal functions in the Japanese monkey (Macaca fuscata fuscata). J. Med. Primatol. 20, 277–283 (1991)Google Scholar
  40. E. Shubber, N.S. Amin, B.H. El-Adhami, Cytogenetic effects of copper-containing intrauterine contraceptive device (IUCD) on blood lymphocytes. Mutat. Res. 417, 57–63 (1998)Google Scholar
  41. R.P. Singh, S. Kumar, R. Nada et al., Evaluation of copper toxicity in isolated human peripheral blood mononuclear cells and it’s attenuation by zinc: ex vivo. Mol. Cell. Biochem. 282, 13–21 (2006)CrossRefGoogle Scholar
  42. I. Sivin, Utility and drawbacks of continuous use of a copper T IUD for 20 years. Contraception 75, S70–75 (2007)CrossRefGoogle Scholar
  43. J. Stanback, D. Grimes, Can intrauterine device removals for bleeding or pain be predicted at a one-month follow-up visit? A multivariate analysis. Contraception 58, 357–360 (1998)CrossRefGoogle Scholar
  44. M. Thiery, H. Van der Pas, H. Van Kets, A decade of experience with the TCu220C. Adv. Contracept. 1, 313–318 (1985)CrossRefGoogle Scholar
  45. V. Toder, A. Madanes, N. Gleicher, Immunologic aspects of IUD action. Contraception 37, 391–403 (1988)CrossRefGoogle Scholar
  46. J.C. Wahata, P.E. Lockwood, A. Schedle et al., Ag, Cu, Hg and Ni ions alter the metabolism of human monocytes during extended low-dose exposures. J. Oral. Rehabil. 29, 133–139 (2002)CrossRefGoogle Scholar
  47. WHO, The intrauterine device (IUD)—worth singing about, Progress in reproductive health research, 60, (2002).Google Scholar
  48. M. Yin, P. Zhu, H. Luo et al., The presence of mast cells in the human endometrium pre- and post-insertion of intrauterine devices. Contraception 48, 245–254 (1993)CrossRefGoogle Scholar
  49. J.A. Zipper, H.J. Tatum, L. Pastene et al., Metallic copper as an intrauterine contraceptice adjunct to the “T” device. Am. J. Obstet. Gynecol. 105, 1274–1278 (1969)Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Bianmei Cao
    • 1
    • 5
  • Yudong Zheng
    • 1
  • Tingfei Xi
    • 1
    • 2
  • Chuanchuan Zhang
    • 2
  • Wenhui Song
    • 3
  • Krishna Burugapalli
    • 4
  • Huai Yang
    • 1
  • Yanxuan Ma
    • 1
  1. 1.School of Materials Science and EngineeringUniversity of Science and Technology BeijingBeijingPeople’s Republic of China
  2. 2.National Institute for the Control of Pharmaceutical & Biological ProductsBeijingPeople’s Republic of China
  3. 3.Wolfson Center for Materials ProcessingSchool of Engineering and Design, Brunel UniversityWest LondonUK
  4. 4.Brunel Institute for BioengineeringBrunel UniversityUxbridgeUK
  5. 5.Beijing Institute of Medical Device TestingBeijingPeople’s Republic of China

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