Copper-induced non-monotonic dose response in Caco-2 cells

In Vitro Cellular & Developmental Biology - Animal volume 55, pages221–225(2019)Cite this article

Abstract

Copper is an essential dietary micronutrient in humans for proper cell function; however, in excess, it is toxic. The human cell line Caco-2 is popular as an in vitro model for intestinal absorption and toxicology. This study investigated the response of exponentially growing Caco-2 cells to prolonged copper exposure (120 h). An unexpected non-monotonic dose-response profile was observed in Caco-2 cells. Exposure to media supplemented with 3.125 μM CuSO4 resulted in decreased cell yield vs. untreated. However, toxicity was progressively reduced from 90% at 3.125 μM to 60% at 25 μM. This effect was documented between 48 and 120 h continuous exposure (p < 0.05). This triphasic toxicity curve was observed to be specific to copper in Caco-2 cells, as iron, manganese and zinc displayed monotonic dose-response profiles. Two inorganic copper forms, copper sulphate and copper chloride, were shown to conserve the non-monotonic dose-response curve. The triphasic effect was shown to be specific to Caco-2 cells. These results have implications for research investigating the effect of copper and other micronutrients using Caco-2 cells.

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References

  1. Arredondo M, Martínez R, Núñez MT, Ruz M, Olivares M (2006) Inhibition of iron and copper uptake by iron, copper and zinc. Biol Res 39(1):95–102

    Article  CAS  PubMed  Google Scholar 

  2. Barceloux DG, Barceloux D (1999) Copper. J Toxicol Clin Toxicol 37(2):217–230 Available at: http://www.tandfonline.com/doi/full/10.1081/CLT-100102421. Accessed 11 Aug 2016

    Article  CAS  PubMed  Google Scholar 

  3. Bartłomiejczyk T, Lankoff A, Kruszewski M, Szumiel I (2013) Silver nanoparticles – allies or adversaries? Ann Agric Environ Med 20(1):48–54

    PubMed  Google Scholar 

  4. Calabrese EJ (2013) Hormetic mechanisms. Crit Rev Toxicol 43(7):580–606

    Article  CAS  PubMed  Google Scholar 

  5. Calabrese EJ (2014) Hormesis: from mainstream to therapy. J Cell Commun Signal 8(4):289–291

    Article  PubMed  PubMed Central  Google Scholar 

  6. Calabrese EJ et al (2001) Hormesis: a generalizable and unifying hypothesis. Crit Rev Toxicol 8444(31):353–424

    Article  Google Scholar 

  7. Chaube R, Mishra S, Singh RK (2010) In vitro effects of lead nitrate on steroid profiles in the post-vitellogenic ovary of the catfish Heteropneustes fossilis. Toxicol in Vitro 24(7):1899–1904. Available at: https://doi.org/10.1016/j.tiv.2010.07.021

  8. Chen A, Donovan S (2004) Genistein at a concentration present in soy infant formula inhibits Caco-2BBe cell proliferation by causing G2/M cell cycle arrest. J Nutr 134(6):1303–1308 Available at: http://www.ncbi.nlm.nih.gov/pubmed/15173388%5Cn; http://jn.nutrition.org/content/134/6/1303.short. Accessed 6 June 2018

    Article  CAS  PubMed  Google Scholar 

  9. Collins JF, Knutson MD (2010) Metabolic crossroads of iron and copper. Nutr Rev 68(3):133–147

    Article  PubMed  PubMed Central  Google Scholar 

  10. Conolly RB, Lutz WK (2004) Nonmonotonic dose-response relationships: mechanistic basis, kinetic modeling, and implications for risk assessment. Toxicol Sci 77(1):151–157

    Article  CAS  PubMed  Google Scholar 

  11. Fogh J, Trempe G (1975) New human tumor cell lines. In: Fogh J (ed) Human tumor cells in vitro. Springer US, Boston, pp 115–159. Available at: https://doi.org/10.1007/978-1-4757-1647-4_5

  12. Gaetke LM, Chow-Johnson HS, Chow CK (2014) Copper: toxicological relevance and mechanisms. Arch Toxicol 88(11):1929–1938 Available at: http://link.springer.com/10.1007/s00204-014-1355-y. Accessed 29 July 2015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gao C, Zhu L, Zhu F, Sun J, Zhu Z (2014) Effects of different sources of copper on Ctr1, ATP7A, ATP7B, MT and DMT1 protein and gene expression in Caco-2 cells. J Trace Elem Med Biol 28(3):344–350. Available at: https://doi.org/10.1016/j.jtemb.2014.04.004

  14. Goddard WP et al (1997) Iron uptake by isolated human enterocyte suspensions in vitro is dependent on body iron stores and inhibited by other metal cations. J Nutr 127(September 1996):177–183

    Article  CAS  PubMed  Google Scholar 

  15. He W, Feng Y, Li XL, Wei YY, Yang XE (2008) Availability and toxicity of Fe ( II ) and Fe ( III ) in Caco-2 cells. J Zhejiang Univ Sci B 9(9):707–712

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Helmestam M, Stavreus-Evers A, Olovsson M (2010) Cadmium chloride alters mRNA levels of angiogenesis related genes in primary human endometrial endothelial cells grown in vitro. Reprod Toxicol 30(3):370–376. Available at: https://doi.org/10.1016/j.reprotox.2010.05.003

  17. Hidalgo IJ, Raub TJ, Borchardt RT (1989) Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 96(3):736–749

    Article  CAS  PubMed  Google Scholar 

  18. Keenan J, Horgan K, Clynes M, Sinkunaite I, Ward P, Murphy R, O’Sullivan F (2018) Unexpected fluctuations of trace element levels in cell culture medium in vitro: caveat emptor. In Vitro Cell Dev Biol Anim Available at: 54:555–558. https://doi.org/10.1007/s11626-018-0285-z

    Article  CAS  PubMed  Google Scholar 

  19. Kim HJ, Huh D, Hamilton G, Ingber DE (2012) Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow. Lab Chip 12(12):2165–2174 Available at: http://www.ncbi.nlm.nih.gov/pubmed/22434367. Accessed 14 July 2014]

    Article  CAS  Google Scholar 

  20. Kozlowski H et al (2009) Copper, iron, and zinc ions homeostasis and their role in neurodegenerative disorders (metal uptake, transport, distribution and regulation). Coord Chem Rev 253(21–22):2665–2685 http://linkinghub.elsevier.com/retrieve/pii/S0010854509001258. Accessed 19 Oct 2014

    Article  CAS  Google Scholar 

  21. Lagarde F, Beausoleil C, Belcher SM, Belzunces LP, Emond C, Guerbet M, Rousselle C (2015) Non-monotonic dose-response relationships and endocrine disruptors: a qualitative method of assessment. Environ Health 14(1):13 Available at: http://www.ehjournal.net/content/14/1/13. Accessed 6 June 2018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Linder MC (2012) The relationship of copper to DNA damage and damage prevention in humans. Mutat Res Fundam Mol Mech Mutagen 733(1–2):83–91 Available at: http://www.sciencedirect.com/science/article/pii/S0027510712000796. Accessed 28 Jan 2016

    Article  CAS  Google Scholar 

  23. Mantha M, Jumarie C (2010) Cadmium-induced hormetic effect in differentiated caco-2 cells: ERK and p38 activation without cell proliferation stimulation. J Cell Physiol 224(1):250–261

    CAS  PubMed  Google Scholar 

  24. Michael Davis J, Svendsgaard DJ (1990) U-shaped dose-response curves: their occurrence and implications for risk assessment. J Toxicol Environ Health 30(2):71–83

    Article  Google Scholar 

  25. Natoli M et al (2011) Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer. J Cell Physiol 226(6):1531–1543 Available at: http://www.ncbi.nlm.nih.gov/pubmed/20945374. Accessed 3 Nov 2014

    Article  CAS  PubMed  Google Scholar 

  26. Nishito Y, Kambe T (2018) Absorption mechanisms of iron, copper, and zinc: an overview. J Nutr Sci Vitaminol 64:1–7 Available at: https://www.jstage.jst.go.jp/article/jnsv/64/1/64_1/_pdf/-char/en. Accessed 20 Nov 2018

    Article  CAS  PubMed  Google Scholar 

  27. Schulz H (1888) Ueber Hefegifte. Pflügers Arch 42(1):517–541

    Article  Google Scholar 

  28. Shah P, Jogani V, Bagchi T, Misra A (2006) Role of Caco-2 cell monolayers in prediction of intestinal drug absorption. Biotechnol Prog 22(1):186–198

    Article  CAS  PubMed  Google Scholar 

  29. Shao Y, Wolf PG, Guo S, Guo Y, Gaskins HR, Zhang B (2017) Zinc enhances intestinal epithelial barrier function through the PI3K/AKT/mTOR signaling pathway in Caco-2 cells. J Nutr Biochem 43:18–26 Available at: http://linkinghub.elsevier.com/retrieve/pii/S0955286316305459. Accessed 5 Nov 2017

    Article  CAS  PubMed  Google Scholar 

  30. Spence JR, Mayhew CN, Rankin SA, Kuhar MF, Vallance JE, Tolle K, Hoskins EE, Kalinichenko VV, Wells SI, Zorn AM, Shroyer NF, Wells JM (2011) Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470(7332):105–109 Available at: http://www.ncbi.nlm.nih.gov/pubmed/21151107%5Cn; http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3033971&tool=pmcentrez&rendertype=abstract. Accessed 23 May 2016

    Article  CAS  PubMed  Google Scholar 

  31. Tennant J, Stansfield M, Yamaji S, Srai SK, Sharp P (2002) Effects of copper on the expression of metal transporters in human intestinal Caco-2 cells. FEBS Lett 527:239–244

    Article  CAS  PubMed  Google Scholar 

  32. Titma T, Shimmo R, Siigur J, Kahru A (2016) Toxicity of antimony, copper, cobalt, manganese, titanium and zinc oxide nanoparticles for the alveolar and intestinal epithelial barrier cells in vitro. Cytotechnology 68(6):2363–2377

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Vandenberg LN, Colborn T, Hayes TB, Heindel JJ, Jacobs DR Jr, Lee DH, Shioda T, Soto AM, vom Saal FS, Welshons WV, Zoeller RT, Myers JP (2012) Hormones and endocrine-disrupting chemicals: low-dose effects and nonmonotonic dose responses. Endocr Rev 33(3):378–455

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zödl B, Zeiner M, Sargazi M, Roberts NB, Marktl W, Steffan I, Ekmekcioglu C (2003) Toxic and biochemical effects of zinc in Caco-2 cells. J Inorg Biochem 97(4):324–330

    Article  CAS  PubMed  Google Scholar 

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Funding

This work was funded by a joint Enterprise Ireland Innovative Partnership programme (IP/2015/0375).

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Author notes

  1. O’Doherty C and Keenan J were involved at all stages of the research, co-wrote the manuscript and are joint-first authors. O’Sullivan F and Clynes M contributed equally to this work.

Affiliations

  1. National Institute for Cellular Biotechnology, Dublin City University, Glasnevin, Dublin, D09 W6Y4, Ireland

    Charles O’Doherty, Joanne Keenan, Finbarr O’Sullivan & Martin Clynes

  2. Alltech, Dunboyne, Meath, Ireland

    Karina Horgan & Richard Murphy

Authors
  1. Charles O’Doherty
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  2. Joanne Keenan
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  3. Karina Horgan
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  4. Richard Murphy
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  5. Finbarr O’Sullivan
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  6. Martin Clynes
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Corresponding author

Correspondence to Charles O’Doherty.

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Editor: Tetsuji Okamoto

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O’Doherty, C., Keenan, J., Horgan, K. et al. Copper-induced non-monotonic dose response in Caco-2 cells. In Vitro Cell.Dev.Biol.-Animal 55, 221–225 (2019). https://doi.org/10.1007/s11626-019-00333-8

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Keywords

  • Dose response
  • Copper
  • Caco-2
  • Triphasic
  • Trace elements
  • Toxicity