Skip to main content
SpringerLink
Log in

Biochemical effects of copper nanomaterials in human hepatocellular carcinoma (HepG2) cells

  • Original Article
  • Published:
Cell Biology and Toxicology Aims and scope Submit manuscript

Abstract

In dose-response and structure-activity studies, human hepatic HepG2 cells were exposed for 3 days to nano Cu, nano CuO or CuCl2 (ions) at doses between 0.1 and 30 ug/ml (approximately the no observable adverse effect level to a high degree of cytotoxicity). Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function, and oxidative stress. With nano Cu and nano CuO, few indications of cytotoxicity were observed between 0.1 and 3 ug/ml. In respect to dose, lactate dehydrogenase and aspartate transaminase were the most sensitive cytotoxicity parameters. The next most responsive parameters were alanine aminotransferase, glutathione reductase, glucose 6-phosphate dehydrogenase, and protein concentration. The medium responsive parameters were superoxide dismutase, gamma glutamyltranspeptidase, total bilirubin, and microalbumin. The parameters glutathione peroxidase, glutathione reductase, and protein were all altered by nano Cu and nano CuO but not by CuCl2 exposures. Our chief observations were (1) significant decreases in glucose 6-phosphate dehydrogenase and glutathione reductase was observed at doses below the doses that show high cytotoxicity, (2) even high cytotoxicity did not induce large changes in some study parameters (e.g., alkaline phosphatase, catalase, microalbumin, total bilirubin, thioredoxin reductase, and triglycerides), (3) even though many significant biochemical effects happen only at doses showing varying degrees of cytotoxicity, it was not clear that cytotoxicity alone caused all of the observed significant biochemical effects, and (4) the decreased glucose 6-phosphate dehydrogenase and glutathione reductase support the view that oxidative stress is a main toxicity pathway of CuCl2 and Cu–containing nanomaterials.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

ALP :

alkaline phosphatase

ALT :

alanine aminotransferase

AOP :

adverse outcome pathway

AST :

aspartate transaminase

BET :

specific surface area/porosity as determined by the Brunauer, Emmett, Teller test

CAT :

catalase

DLVO :

Derjaguin, Landau, Verwey, and Overbeek theory

DPBS :

Dulbecco’s phosphate-buffered saline

EDX :

energy-dispersive x-ray analysis

FTIR :

Fourier transform infrared spectroscopy

GGT :

gamma glutamyltranspeptidase

G6PDH :

glucose 6-phosphate dehydrogenase

GPx :

glutathione peroxidase

GRD :

glutathione reductase

GSH :

reduced glutathione concentration

HepG2 :

human hepatocellular carcinoma cells, ATCC catalog number HB-8065

LDH :

lactate dehydrogenase

MIA :

microalbumin

MTS :

4-[5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-1,3-thiazol-2-yl)tetrazol-3-ium-2-yl]benzenesulfonate

MTT :

3-[4,5-dimethyl-2-thiazol]-2,5-diphenyl-2H-tetrazolium bromide

PBS :

phosphate buffered saline

ROS :

reactive oxygen species

SEM :

scanning electron microscopy

SOD :

superoxide dismutase

TBARS :

thiobarbituric acid reactive substances

T BIL :

total bilirubin

TEM :

transmission electron microscopy

THRR :

thioredoxin reductase

TRIG :

triglycerides

XRD :

X-ray diffraction

References

  • Arnal N, de Alaniz MJ, Marra CA. Effect of copper overload on the survival of HepG2 and A-549 human-derived cells. Hum Exp Toxicol. 2013;32(3):299–315.

    Article  CAS  PubMed  Google Scholar 

  • Blasco J, Puppo J. Effect of heavy metals (Cu, Cd and Pb) on aspartate and alanine aminotransferase in Ruditapes philippinarum (Mollusca: Bivalvia). Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1999;122(2):253–63.

    Article  CAS  PubMed  Google Scholar 

  • Boulard M, Blume KG, Beutler E. The effect of copper on red cell enzyme activities. J Clin Invest. 1972;51(2):459–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chusuei CC, Wu CH, Mallavarapu S, Hou FY, Hsu CM, Winiarz JG, et al. Cytotoxicity in the age of nano: the role of fourth period transition metal oxide nanoparticle physicochemical properties. Chem Biol Interact. 2013;206(2):319–26.

    Article  CAS  PubMed  Google Scholar 

  • Cuillel M, Chevallet M, Charbonnier P, Fauquant C, Pignot-Paintrand I, Arnaud J, et al. Interference of CuO nanoparticles with metal homeostasis in hepatocytes under sub-toxic conditions. Nanoscale. 2014;6(3):1707–15.

    Article  CAS  PubMed  Google Scholar 

  • Dastjerdi R, Montazer M. A review on the application of inorganic nano-structured materials in the modification of textiles: focus on anti-microbial properties. Colloids Surf B: Biointerfaces. 2010;79(1):5–18.

    Article  CAS  PubMed  Google Scholar 

  • Deiss A, Lee GR, Cartwright GE. Hemolytic anemia in Wilson's disease. Ann Intern Med. 1970;73(3):413–8.

    Article  CAS  PubMed  Google Scholar 

  • Dobryszycka W, Owczarek H. Effects of lead, copper, and zinc on the rat’s lactate dehydrogenase in vivo and in vitro. Arch Toxicol. 1981;48(1):21–7.

    Article  CAS  PubMed  Google Scholar 

  • Fairbanks VF. Copper sulfate-induced hemolytic anemia. Inhibition of glucose-6-phosphate dehydrogenase and other possible etiologic mechanisms. Arch Intern Med. 1967;120(4):428–32.

    Article  CAS  PubMed  Google Scholar 

  • Faixová Z, Faix S, Makova Z, Prosbová M. Effect of divalent ions on ruminal enzyme activities in sheep. Acta Vet Brno. 2006;56(1):17–23.

    Article  Google Scholar 

  • Flikweert JP, Hoorn R, Stall G. The effect of copper on human erythrocyte glutathione reductase. Int J BioChemiPhysics. 1974;5:649–53.

    Article  CAS  Google Scholar 

  • Griffitt RJ, Hyndman K, Denslow ND, Barber DS. Comparison of molecular and histological changes in zebrafish gills exposed to metallic nanoparticles. Toxicol Sci. 2009;107(2):404–15.

    Article  CAS  PubMed  Google Scholar 

  • Hendren CO, Lowry GV, Unrine JM, Wiesner MR. A functional assay-based strategy for nanomaterial risk forecasting. Sci Total Environ. 2015;536:1029–37.

    Article  CAS  PubMed  Google Scholar 

  • Holsapple MP, Farland WH, Landry TD, Monteiro-Riviere NA, Carter JM, Walker NJ, et al. Research strategies for safety evaluation of nanomaterials, part II: toxicological and safety evaluation of nanomaterials, current challenges and data needs. Toxicol Sci. 2005;88(1):12–7.

    Article  CAS  PubMed  Google Scholar 

  • Hu W, Zhi L, Zhuo MQ, Zhu QL, Zheng JL, Chen QL, et al. Purification and characterization of glucose 6-phosphate dehydrogenase (G6PD) from grass carp (Ctenopharyngodon idella) and inhibition effects of several metal ions on G6PD activity in vitro. Fish Physiol Biochem. 2013;39(3):637–47.

    Article  CAS  PubMed  Google Scholar 

  • Hu HL, Ni XS, Duff-Canning S, Wang XP. Oxidative damage of copper chloride overload to the cultured rat astrocytes. Am J Transl Res. 2016;8(2):1273–80.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kitchin KT, Robinette BL, Richards J, Coates NH, Castellon BT. Biochemical effects in HepG2 cells exposed to six TiO2 and four CeO2 nanomaterials. J Nanosci Nanotechnol. 2016;16(9):9505–34.

    Article  CAS  Google Scholar 

  • Kitchin KT, Stirdivant S, Robinette BL, Castellon BT, Liang X. Metabolomic effects of CeO2, SiO2 and CuO metal oxide nanomaterials on HepG2 cells. Particle Fibre Toxicol. 2017;14:50 Published online 2017 Nov 29. https://doi.org/10.1186/s12989-017-0230-4.

    Article  CAS  Google Scholar 

  • Kitchin KT, Richards JA, Robinette BL, Wallace KA, Coates NH, Castellon BT, Grulke EA. Biochemical effects of some CeO2, SiO2 and TiO2 nanomaterials in HepG2 cells. Cell Bio and Toxicol. 2019;35(2):129–49. https://doi.org/10.1007/s10565-018-9445x

  • Lai JC, Blass JP. Neurotoxic effects of copper: inhibition of glycolysis and glycolytic enzymes. Neurochem Res. 1984;9(12):1699–710.

    Article  CAS  PubMed  Google Scholar 

  • Liu J, Hurt RH. Ion release kinetics and particle persistence in aqueous nano-silver colloids. Environ Sci Technol. 2010;44(6):2169–75.

    Article  CAS  PubMed  Google Scholar 

  • Mackevica A, Revilla P, Brinch A, Hansen S. Current uses of nanomaterials in biocidal products and treated articles in the EU. Environ Sci: Nano. 2016;3:1195–205.

    CAS  Google Scholar 

  • Manna P, Ghosh M, Ghosh J, Das J, Sil PC. Contribution of nano-copper particles to in vivo liver dysfunction and cellular damage: role of IkappaBalpha/NF-kappaB, MAPKs and mitochondrial signal. Nanotoxicology. 2012;6(1):1–21.

    Article  CAS  PubMed  Google Scholar 

  • Marambio-Jones C, Hoek EMV. A review of the antibacterial effects of silver nanomaterials and potential implications for human health and the environment. J Nanopart Res. 2010;12(5):1531–51.

    Article  CAS  Google Scholar 

  • Merrifield RC, Wang ZW, Palmer RE, Lead JR. Synthesis and characterization of polyvinylpyrrolidone coated cerium oxide nanoparticles. Environ Sci Technol. 2013;47(21):12426–33.

    Article  CAS  PubMed  Google Scholar 

  • Mishchuk NA. The model of hydrophobic attraction in the framework of classical DLVO forces. Adv Colloid Interf Sci. 2011;168(1-2):149–66.

    Article  CAS  Google Scholar 

  • Mittal M, Siddiqui MR, Tran K, Reddy SP, Malik AB. Reactive oxygen species in inflammation and tissue injury. Antioxid Redox Signal. 2014;20(7):1126–67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mize CE, Langdon RG. Hepatic glutathione reductase. I. Purification and general kinetic properties. J Biol Chem. 1962;237:1589–95.

    Article  CAS  PubMed  Google Scholar 

  • Monteiro-Riviere NA, Inman AO, Zhang LW. Limitations and relative utility of screening assays to assess engineered nanoparticle toxicity in a human cell line. Toxicol Appl Pharmacol. 2009;234(2):222–35.

    Article  CAS  PubMed  Google Scholar 

  • Nel A, Xia T, Madler L, Li N. Toxic potential of materials at the nanolevel. Science. 2006;311(5761):622–7.

    Article  CAS  PubMed  Google Scholar 

  • Park EJ, Park K. Oxidative stress and pro-inflammatory responses induced by silica nanoparticles in vivo and in vitro. Toxicol Lett. 2009;184(1):18–25.

    Article  CAS  PubMed  Google Scholar 

  • Porter D, Sriram K, Wolfarth M, Jefferson A, Schwegler-Berry D, Andrew M, et al. A biocompatible medium for nanomparticle dispersion. Nanotoxicology. 2009;2:144–54.

    Article  Google Scholar 

  • Price C, Alberti K. Biochemical assessment of liver function. In: Wright RM, Alberti K, Karran S, Millward-Sadler G, editors. Liver and biliary disease-pathophysiology, diagnosis, management. London: W. B. Saunders; 1979. p. 381–416.

    Google Scholar 

  • Rafter GW. Copper inhibition of glutathione reductase and its reversal with gold thiolates, thiol, and disulfide compounds. Biochem Med. 1982a;27(3):381–91.

    Article  CAS  PubMed  Google Scholar 

  • Rafter GW. The effect of copper on glutathione metabolism in human leukocytes. Biol Trace Elem Res. 1982b;4(2-3):191–7.

    Article  CAS  PubMed  Google Scholar 

  • Sarkar A, Das J, Manna P, Sil PC. Nano-copper induces oxidative stress and apoptosis in kidney via both extrinsic and intrinsic pathways. Toxicology. 2011;290(2-3):208–17.

    Article  PubMed  Google Scholar 

  • Serafini MT, Romeu A, Arola L. Zn(II), Cd(II) and Cu(II) interactions on glutathione reductase and glucose-6-phosphate dehydrogenase. Biochem Int. 1989;18(4):793–802.

    CAS  PubMed  Google Scholar 

  • Shi M, De Mesy Bently KL, Palui G, Mattoussi H, Elder A, Yang H. The roles of surface chemistry, dissolution rate, and delivered dose in the cytotoxicity of copper nanoparticles. Nanoscale. 2017;9:4739–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Song MF, Li YS, Kasai H, Kawai K. Metal nanoparticle-induced micronuclei and oxidative DNA damage in mice. J Clin Biochem Nutr. 2012;50(3):211–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sotiriou GA, Pratsinis SE. Antibacterial activity of nanosilver ions and particles. Environ Sci Technol. 2010;44(14):5649–54.

    Article  CAS  PubMed  Google Scholar 

  • Thai SF, Wallace KA, Jones CP, Ren H, Castellon BT, Crooks J, et al. differential genomic effects on signaling pathways by two different CeO2 nanoparticles in HepG2 Cells. J Nanosci Nanotechnol. 2015;15(12):9925–37. https://doi.org/10.1096/fj.09-135731.

    Article  CAS  PubMed  Google Scholar 

  • Thompson TL, Yates JT Jr. Surface science studies of the photoactivation of TiO2--new photochemical processes. Chem Rev. 2006;106(10):4428–53.

    Article  CAS  PubMed  Google Scholar 

  • Thounaojam MC, Jadeja RN, Valodkar M, Nagar PS, Devkar RV, Thakore S. Oxidative stress induced apoptosis of human lung carcinoma (A549) cells by a novel copper nanorod formulation. Food Chem Toxicol. 2011;49(11):2990–6.

    Article  CAS  PubMed  Google Scholar 

  • Walker NJ, Bucher JR. A 21st century paradigm for evaluating the health hazards of nanoscale materials? Toxicol Sci. 2009;110(2):251–4.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang Z, Von Dem Bussche A, Kabadi PK, Kane AB, Hurt RH. Biological and environmental transformations of copper-based nanomaterials. ACS Nano. 2013;7:8715–27.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Warheit DB, Borm PJ, Hennes C, Lademann J. Testing strategies to establish the safety of nanomaterials: conclusions of an ECETOC workshop. Inhal Toxicol. 2007;19(8):631–43.

    Article  CAS  PubMed  Google Scholar 

  • Yang CC, Wu ML, Deng JF. Prolonged hemolysis and methemoglobinemia following organic copper fungicide ingestion. Vet Hum Toxicol. 2004;46(6):321–3.

    PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful for the participation of many individuals in this study. Particularly we thank Will Boyes and Maribel Bruno for reviewing this manuscript as part of EPA clearance procedures.

Author information

Authors and Affiliations

  1. Integrated Systems Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, 109 Alexander Drive, Mail Drop B105-03, Research Triangle Park, NC, 27711, USA

    Kirk T. Kitchin, Brian L. Robinette & Kathleen A. Wallace

  2. Environmental Public Health Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC, 27711, USA

    Judy A. Richards & Najwa H. Coates

  3. Institute of Biomedical Studies and Department of Environmental Science, Baylor University, Waco, TX, 76798, USA

    Benjamin T. Castellon

  4. Chemical & Materials Engineering, University of Kentucky, Lexington, KY, 20506-0046, USA

    Eric A. Grulke

Authors
  1. Kirk T. Kitchin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Judy A. Richards
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Brian L. Robinette
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kathleen A. Wallace
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Najwa H. Coates
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Benjamin T. Castellon
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Eric A. Grulke
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kirk T. Kitchin.

Ethics declarations

Competing interests

The authors declare no competing interests.

Disclaimer

The information in this document has been funded wholly by the US Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents necessarily reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Below is the link to the electronic supplementary material.

10565_2022_9720_MOESM1_ESM.pptx

Supplementary file1 (PPTX 92 kb) Supplementary Figure 1 Media, cellular and total LDH enzyme activity following Cu treatment of HepG2 cells

Supplementary file2 (PPTX 83 kb) Supplementary Figure 2 % LDH released

10565_2022_9720_MOESM3_ESM.pptx

Supplementary file3 (PPTX 103 kb) Supplementary Figure 3 Media, cellular and total AST enzyme activity following Cu treatment of HepG2 cells

Supplementary file4 (PPTX 86 kb) Supplementary Figure 4 % AST released

10565_2022_9720_MOESM5_ESM.pptx

Supplementary file5 (PPTX 95 kb) Supplementary Figure 5 Media, cellular and total ALT enzyme activity following Cu treatment of HepG2 cells

Supplementary file6 (PPTX 105 kb) Supplementary Figure 6 % ALT released

10565_2022_9720_MOESM7_ESM.pptx

Supplementary file7 (PPTX 79 kb) Supplementary Figure 7 Effects of 3 Cu containing materials on HepG2 cellular protein content

Supplementary file8 (PPTX 75 kb) Supplementary Figure 8 Effects of 3 Cu containing materials on G6PDH activity

Supplementary file9 (PPTX 73 kb) Supplementary Figure 9 Effects of 3 Cu containing materials on GRD activity

10565_2022_9720_MOESM10_ESM.doc

Supplementary file10 (DOC 659 kb) Supplementary Table 1. Cytotoxic and Biochemical effects from 3 different Cu containing materials: experimental values useful for modelers

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kitchin, K.T., Richards, J.A., Robinette, B.L. et al. Biochemical effects of copper nanomaterials in human hepatocellular carcinoma (HepG2) cells. Cell Biol Toxicol 39, 2311–2329 (2023). https://doi.org/10.1007/s10565-022-09720-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10565-022-09720-6

Keywords

Search

Navigation