Advertisement

Analytical and Bioanalytical Chemistry

, Volume 410, Issue 9, pp 2385–2394 | Cite as

Cu isotope fractionation response to oxidative stress in a hepatic cell line studied using multi-collector ICP-mass spectrometry

  • María R. Flórez
  • Marta Costas-Rodríguez
  • Charlotte Grootaert
  • John Van Camp
  • Frank Vanhaecke
Research Paper

Abstract

Reactive oxygen species (ROS) are generated in biological processes involving electron transfer reactions and can act in a beneficial or deleterious way. When intracellular ROS levels exceed the cell’s anti-oxidant capacity, oxidative stress occurs. In this work, Cu isotope fractionation was evaluated in HepG2 cells under oxidative stress conditions attained in various ways. HepG2 is a well-characterised human hepatoblastoma cell line adapted to grow under high oxidative stress conditions. During a pre-incubation stage, cells were exposed to a non-toxic concentration of Cu for 24 h. Subsequently, the medium was replaced and cells were exposed to one of three different external stressors: H2O2, tumour necrosis factor α (TNFα) or UV radiation. The isotopic composition of the intracellular Cu was determined by multi-collector ICP-mass spectrometry to evaluate the isotope fractionation accompanying Cu fluxes between cells and culture medium. For half of these setups, the pre-incubation solution also contained N-acetyl-cysteine (NAC) as an anti-oxidant to evaluate its protective effect against oxidative stress via its influence on the extent of Cu isotope fractionation. Oxidative stress caused the intracellular Cu isotopic composition to be heavier compared to that in untreated control cells. The H2O2 and TNFα exposures rendered similar results, comparable to those obtained after mild UV exposure. The heaviest Cu isotopic composition was observed under the strongest oxidative conditions tested, i.e., when the cell surfaces were directly exposed to UV radiation without apical medium and in absence of NAC. NAC mitigated the extent of isotope fractionation in all cases.

Keywords

Multi-collector ICP-mass spectrometry MC-ICP-MS Isotope fractionation Oxidative stress Hep G2 cell line Copper isotope ratio Liver 

Notes

Acknowledgements

The Flemish Research Foundation FWO-Vlaanderen (research project “G023014N”) is acknowledged for financial support. María R. Flórez thanks the Special Research Fund of Ghent University (BOF-UGent) for her postdoctoral grant and Marta Costas-Rodriguez thanks FWO-Vlaanderen for her postdoctoral grant.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    Thannickal VJ, Fanburg BL. Reactive oxygen species in cell signalling. Am J Physiol Lung Cell Mol Physiol. 2000;279:L1005–28.CrossRefGoogle Scholar
  2. 2.
    Nose K. Role of reactive oxygen species in the regulation of physiological functions. Biol Pharm Bull. 2000;23:897–903.CrossRefGoogle Scholar
  3. 3.
    Sauer H, Wartenberg M, Hescheler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Physiol Biochem. 2001;11:173–86.CrossRefGoogle Scholar
  4. 4.
    Davies KJA. The broad spectrum of responses to oxidants in proliferating cells: a new paradigm for oxidative stress. IUBMB Life. 1999;48:41–7.CrossRefGoogle Scholar
  5. 5.
    Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007;39:44–84.CrossRefGoogle Scholar
  6. 6.
    Valko M, Izakovic M, Mazur M, Rhodes CJ, Telser J. Role of oxygen radicals in DNA damage and cancer incidence. Mol Cell Biochem. 2004;266:37–56.CrossRefGoogle Scholar
  7. 7.
    de Andrade KQ, Moura FA, dos Santos JM, de Araújo ORP, de Farias Santos JC, Goulart MOF. Oxidative stress and inflammation in hepatic diseases: therapeutic possibilities of N-acetylcysteine. Int J Mol Sci. 2015;16:30269–308.CrossRefGoogle Scholar
  8. 8.
    Sun SY. N-acetylcysteine, reactive oxygen species and beyond. Cancer Biol Ther. 2010;9:109–10.CrossRefGoogle Scholar
  9. 9.
    Bleackley MR, MacGillivray RT. Transition metal homeostasis: from yeast to human disease. Biometals. 2011;24:785–809.CrossRefGoogle Scholar
  10. 10.
    Prousek J. Fenton chemistry in biology and medicine. Pure Appl Chem. 2007;79:2325–38.CrossRefGoogle Scholar
  11. 11.
    Vanhaecke F, Degryse P. Isotopic analysis—fundamentals and applications using ICP-MS. Wiley-VCH: Weinheim; 2012.CrossRefGoogle Scholar
  12. 12.
    Vanhaecke F, Balcaen L, Malinovsky D. Use of single-collector and multi-collector ICP-mass spectrometry for isotopic analysis. J Anal At Spectrom. 2009;24:863–86.CrossRefGoogle Scholar
  13. 13.
    Walczyk T, von Blanckenburg F. Natural iron isotope variations in human blood. Science. 2002;295:2065–6.CrossRefGoogle Scholar
  14. 14.
    Balter V, da Costa AN, Bondanese VP, Jaouen K, Lamboux A, Sangrajrang S, et al. Natural variations of copper and sulfur stable isotopes in blood of hepatocellular carcinoma patients. Proc Natl Acad Sci U S A. 2015;112:982–5.CrossRefGoogle Scholar
  15. 15.
    Gordon GW, Monge J, Channon MB, Wu Q, Skulan JL, Anbar AD, et al. Predicting multiple myeloma disease activity by analyzing natural calcium isotopic composition. Leukemia. 2014;28:2112–6.CrossRefGoogle Scholar
  16. 16.
    Larner F, Woodley LN, Shousha S, Moyes A, Humphreys-Williams E, Strekopytov S, et al. Zinc isotopic compositions of breast cancer tissue. Metallomics. 2015;7:112–7.CrossRefGoogle Scholar
  17. 17.
    Costas-Rodríguez M, Delanghe J, Vanhaecke F. High-precision isotopic analysis of essential mineral elements in biomedicine: natural isotope ratio variations as potential diagnostic and/or prognostic markers. TrAC Trends Anal Chem. 2016;76:182–93.CrossRefGoogle Scholar
  18. 18.
    Lauwens S, Costas-Rodríguez M, Van Vlierberghe H, Vanhaecke F. Cu isotopic signature in blood serum of liver transplant patients: a follow-up study. Sci Rep UK. 2016;6:30683.CrossRefGoogle Scholar
  19. 19.
    Costas-Rodríguez M, Anoshkina Y, Lauwens S, Van Vlierberghe H, Delanghe J, Vanhaecke F. Isotopic analysis of Cu in blood serum by multicollector ICP-mass spectrometry: a new approach for the diagnosis and prognosis of liver cirrhosis? Metallomics. 2015;7:491–8.CrossRefGoogle Scholar
  20. 20.
    Bondanese VP, Lamboux A, Simon M, Lafont JE, Albalat E, Pichat S, et al. Hypoxia induces copper stable isotope fractionation in hepatocellular carcinoma, in a HIF-independent manner. Metallomics. 2016;8:1177–84.CrossRefGoogle Scholar
  21. 21.
    Paredes E, Avazeri E, Malard V, Vidaud C, Reiller PE, Ortega R, et al. Evidence of isotopic fractionation of natural uranium in cultured human cells. Proc Natl Acad Sci U S A. 2016;113:14007–12.CrossRefGoogle Scholar
  22. 22.
    Cadiou J-L, Pichat S, Bondanese VP, Soulard A, Fujii T, Albarede F, et al. Copper transporters are responsible for copper isotopic fractionation in eukaryotic cells. Sci Rep. 2017;7:44533.CrossRefGoogle Scholar
  23. 23.
    Flórez MR, Anoshkina Y, Costas-Rodríguez M, Grootaert C, Van Camp J, Delanghe J, et al. Natural Fe isotope fractionation in an intestinal Caco-2 cell line model. J Anal At Spectrom. 2017;32:1713–20.CrossRefGoogle Scholar
  24. 24.
    Navarrete JU, Borrok DM, Viveros M, Ellzey JT. Copper isotope fractionation during surface adsorption and intracellular incorporation by bacteria. Geochim Cosmochim Acta. 2011;75:784–99.CrossRefGoogle Scholar
  25. 25.
    Kafantaris FCA, Borrock DM. Zinc isotope fractionation during surface adsorption and intracellular incorporation by bacteria. Chem Geol. 2014;366:42–51.CrossRefGoogle Scholar
  26. 26.
    Amor M, Busigny V, Louvat P, Gélabert A, Cartigny P, Durand-Dubief M, et al. Mass-dependent and-independent signature of Fe isotopes in magnetotactic bacteria. Science. 2016;352:705–8.CrossRefGoogle Scholar
  27. 27.
    Knowles BB, Howe CC, Aden DP. Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen. Science. 1980;209:497–9.CrossRefGoogle Scholar
  28. 28.
    Alía M, Ramos S, Mateos R, Bravo L, Goya L. Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2). J Biochem Mol Toxicol. 2005;19:119–28.CrossRefGoogle Scholar
  29. 29.
    Van Meerloo J, Kaspers GJL, Cloos J. Cell sensitivity assays: the MTT assay. In: Cree IA, editor. Cancer cell culture. Methods in molecular biology (methods and protocols). New York: Humana Press; 2011. p. 237–45.Google Scholar
  30. 30.
    Hissin PJ, Hilf R. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal Biochem. 1976;74:214–26.CrossRefGoogle Scholar
  31. 31.
    Baxter DC, Rodushkin I, Engström E, Malinovsky D. Revised exponential model for mass bias correction using an internal standard for isotope abundance ratio measurements by multi-collector inductively coupled plasma mass spectrometry. J Anal At Spectrom. 2006;21:427–30.CrossRefGoogle Scholar
  32. 32.
    Yu BP. Cellular defenses against damage from reactive oxygen species. Physiol Rev. 1994;74:139–62.CrossRefGoogle Scholar
  33. 33.
    Jiménez I, Speisky H. Effects of copper ions on the free radical-scavenging properties of reduced gluthathione: implications of a complex formation. J Trace Elem Med Biol. 2000;14:161–7.CrossRefGoogle Scholar
  34. 34.
    Turnlund JR. Human whole-body copper metabolism. Am J Clin Nutr. 1998;67:960S–4S.CrossRefGoogle Scholar
  35. 35.
    Peña MMO, Lee J, Thiele DJ. A delicate balance: homeostatic control of copper uptake and distribution. J Nutr. 1999;129:1251–60.CrossRefGoogle Scholar
  36. 36.
    Jiménez I, Aracena P, Letelier ME, Navarro P, Speisky H. Chronic exposure of HepG2 cells to excess copper results in depletion of glutathione and induction of metallothionein. Toxicol in Vitro. 2002;16:167–75.CrossRefGoogle Scholar
  37. 37.
    Song MO, Li J, Freedman JH. Physiological and toxicological transcriptome changes in HepG2 cells exposed to copper. Physiol Genomics. 2009;38:386–401.CrossRefGoogle Scholar
  38. 38.
    Aston NS, Watt N, Morton IE, Tanner MS, Evans GS. Copper toxicity affects proliferation and viability of human hepatoma cells (HepG2 line). Hum Exp Toxicol. 2000;19:367–76.CrossRefGoogle Scholar
  39. 39.
    Pham AN, Xing G, Miller CJ, Waite TD. Fenton-like copper redox chemistry revisited: Hydrogen peroxide and superoxide mediation of copper-catalyzed oxidant production. J Catal. 2013;301:54–64.CrossRefGoogle Scholar
  40. 40.
    Wajant H, Pfizenmaier K, Scheurich P. Tumor necrosis factor signaling. Cell Death Differ. 2003;10:45–65.CrossRefGoogle Scholar
  41. 41.
    Han D, Ybanez MD, Ahmadi S, Yeh K, Kaplowitz N. Redox regulation of tumor necrosis factor signaling. Antioxid Redox Signal. 2009;11:2245–63.CrossRefGoogle Scholar
  42. 42.
    Schrader M, Wodopia R, Fahimi HD. Induction of tubular peroxisomes by UV irradiation and reactive oxygen species in HepG2 cells. J Histochem Cytochem. 1999;47:1141–8.CrossRefGoogle Scholar
  43. 43.
    Vile GF, Tyrrell RM. UVA radiation-induced oxidative damage to lipids and proteins in vitro and in human skin fibroblasts is dependent on iron and singlet oxygen. Free Radic Biol Med. 1995;18:721–30.CrossRefGoogle Scholar
  44. 44.
    Zafarullah M, Li WQ, Sylvester J, Ahmad M. Molecular mechanisms of N-acetylcysteine actions. Cell Mol Life Sci. 2003;60:6–20.CrossRefGoogle Scholar
  45. 45.
    De Flora S, Izzotti A, D'agostini F, Balansky RM. Mechanisms of N-acetylcysteine in the prevention of DNA damage and cancer, with special reference to smoking-related end-points. Carcinogenesis. 2001;22:999–1013.CrossRefGoogle Scholar
  46. 46.
    Lu SC. Regulation of glutathione synthesis. Mol Asp Med. 2009;30:42–59.CrossRefGoogle Scholar
  47. 47.
    Balter V, Lamboux A, Zazzo A, Télouk P, Leverrier Y, Marvel J, et al. Contrasting cu, Fe, and Zn isotopic patterns in organs and body fluids of mice and sheep, with emphasis on cellular fractionation. Metallomics. 2013;5:1470–82.CrossRefGoogle Scholar
  48. 48.
    Mandal S, Das G, Askari H. Interactions of N-acetyl-l-cysteine with metals (Ni2+, Cu2+ and Zn2+): An experimental and theoretical study. Struct Chem. 2014;25:43–51.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • María R. Flórez
    • 1
  • Marta Costas-Rodríguez
    • 1
  • Charlotte Grootaert
    • 2
  • John Van Camp
    • 2
  • Frank Vanhaecke
    • 1
  1. 1.Department of Chemistry, Atomic & Mass Spectrometry – A&MS Research UnitGhent UniversityGhentBelgium
  2. 2.Department of Food Safety and Food Quality, Food Chemistry and Human Nutrition – nutriFOODchem Research UnitGhent UniversityGhentBelgium

Personalised recommendations