Skip to main content

Toxicity of Copper on Isolated Liver Mitochondria: Impairment at Complexes I, II, and IV Leads to Increased ROS Production

Cell Biochemistry and Biophysics volume 70, pages 367–381 (2014)Cite this article

Abstract

Oxidative damage has been implicated in disorders associated with abnormal copper metabolism and also Cu2+ overloading states. Besides, mitochondria are one of the most important targets for Cu2+, an essential redox transition metal, induced hepatotoxicity. In this study, we aimed to investigate the mitochondrial toxicity mechanisms on isolated rat liver mitochondria. Rat liver mitochondria in both in vivo and in vitro experiments were obtained by differential ultracentrifugation and the isolated liver mitochondria were then incubated with different concentrations of Cu2+. Our results showed that Cu2+ induced a concentration and time-dependent rise in mitochondrial ROS formation, lipid peroxidation, and mitochondrial membrane potential collapse before mitochondrial swelling ensued. Increased disturbance in oxidative phosphorylation was also shown by decreased ATP concentration and decreased ATP/ADP ratio in Cu2+-treated isolated mitochondria. In addition, collapse of mitochondrial membrane potential (MMP), mitochondrial swelling, and release of cytochrome c following of Cu2+ treatment were well inhibited by pretreatment of mitochondria with CsA and BHT. Our results showed that Cu2+ could interact with respiratory complexes (I, II, and IV). This suggests that Cu2+-induced liver toxicity is the result of metal’s disruptive effect on liver hepatocyte mitochondrial respiratory chain that is the obvious cause of Cu2+-induced ROS formation, lipid peroxidation, mitochondrial membrane potential decline, and cytochrome c expulsion which start cell death signaling.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Abbreviations

ROS:

Reactive oxygen species

GSH:

Reduced glutathione

DCFH-DA:

2′,7′-dichlorofluorescin diacetate

TBARs:

Thiobarbituric acid reactive substances

Cs A:

Cyclosporin A

MDA:

Malondialdehyde

Rh123:

Rhodamine 123

BSA:

Bovine serum albumin

MPT:

Mitochondrial permeability transition

MMP:

Mitochondrial membrane potential

BHT:

Butylated hydroxytoluene

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

TTFA:

Thenoyltrifuoroacetone

AA:

Antimycin A

DTNB:

Dithiobis-2-nitrobenzoic acid

Rot:

Rotenone

DMSO:

Dimethyl sulfoxide

TEP:

Tetramethoxypropane

References

  1. Gaetke, L. M., & Chow, C. K. (2003). Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicology, 189, 147–163.

    CAS  PubMed  Article  Google Scholar 

  2. Pourahmad, J., & O’Brien, P. J. (2000). Contrasting role of Na + ions in modulating Cu2 + or Cd2 + induced hepatocyte toxicity. Chemico-Biological Interactions, 126, 159–169.

    CAS  PubMed  Article  Google Scholar 

  3. Schilsky, M. L. (1996). Wilson’s disease: genetic basis of copper toxicity and natural history. Seminars in Liver Disease, 16(l), 83–95.

    CAS  PubMed  Article  Google Scholar 

  4. Britton, R. S. (1996). Metal-induced hepatotoxicity. Seminars in Liver Disease, 16, 3–12.

    CAS  PubMed  Article  Google Scholar 

  5. Pourahmad, J., & O’Brien, P. J. (2000). A comparison of hepatocyte cytotoxic mechanisms for Cu2+ and Cd2+. Toxicology, 143, 263–273.

    CAS  PubMed  Article  Google Scholar 

  6. Pourahmad, J., Mihajlovic, A., & O’Brien, P. J. (2001). Hepatocyte lysis induced by environmental metal toxins may involve apoptotic death signals initiated by mitochondrial injury. Advances in Experimental Medicine and Biology, 5002, 49–52.

    Google Scholar 

  7. Pourahmad, J., Ross, S., & O’Brien, P. J. (2001). Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but not Cd2+. Free Radical Biology and Medicine, 30, 89–97.

    CAS  PubMed  Article  Google Scholar 

  8. Winge, D. R., & Mehra, R. K. (1990). Host defenses against copper toxicity. International Review of Experimental Pathology, 31, 47–83.

    CAS  PubMed  Article  Google Scholar 

  9. Zhang, S. S. Z., Noordin, M. M., Rahman, S. O. A., & Haron, J. (2000). Effects of copper overload on hepatic lipid peroxidation and antioxidant defense in rats. Veterinary and Human Toxicology, 42, 261–264.

    CAS  PubMed  Google Scholar 

  10. Mehta, R., Templeton, D. M., & O’Brien, P. J. (2006). Mitochondrial involvement in genetically determined transition metal toxicity II. Copper toxicity. Chemico-Biological Interactions, 163, 77–85.

    CAS  PubMed  Article  Google Scholar 

  11. Brewer, G. J. (2005). Anti-copper therapy against cancer and diseases of inflammation and fibrosis. Drug Discovery Today, 10, 1103–1109.

    CAS  PubMed  Article  Google Scholar 

  12. Arciello, M., Rotilio, G., & Rossi, L. (2005). Copper-dependent toxicity in SH-SY5Y neuroblastoma cells involves mitochondrial damage. Biochem and Biophysical Research Communications, 327, 454–459.

    CAS  Article  Google Scholar 

  13. Kawakama, M., Inagawa, R., Hosokawa, T., Satio, T., & Kurasaki, M. (2008). Mechanism of apoptosis induced by copper in PC12 cells. Food and Chemical Toxicology, 46, 2157–2164.

    Article  Google Scholar 

  14. Valko, M., Morris, H., & Cronin, M. T. (2005). Metals, toxicity and oxidative stress. Current Medicinal Chemistry, 12, 1161–1208.

    CAS  PubMed  Article  Google Scholar 

  15. Boveris, A., Musacco-Sebio, R., Ferrarotti, N., Saporito-Magriñá, C., Torti, H., Massot, F., et al. (2012). The acute toxicity of iron and copper: biomolecule oxidation and oxidative damage in rat liver. Journal of Inorganic Biochemistry, 116, 63–69.

    CAS  PubMed  Article  Google Scholar 

  16. Ghazi-khansari, M., Mohammadi-Bardbori, A., & Hosseini, M.-J. (2006). Using Janus Green B to study paraquat toxicity in rat liver mitochondria role of ACE inhibitors (Thiol and Nonthiol ACEi). Annals of the New York Academy of Sciences, 1090, 98–107.

    CAS  PubMed  Article  Google Scholar 

  17. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    CAS  PubMed  Article  Google Scholar 

  18. Gao, X., Zheng, C. Y., Yang, L., Tang, X. C., & Zhang, H. Y. (2009). Huperzine A protects isolated rat brain mitochondria against β-amyloid peptide. Free Radical Biology and Medicine, 46, 1454–1462.

    CAS  PubMed  Article  Google Scholar 

  19. Barja, G. (2002). The Quantitative Measurement of H2O2 Generation in Isolated Mitochondria. Journal of Bioenergetics and Biomembranes, 34, 227–233.

    CAS  PubMed  Article  Google Scholar 

  20. Zhao, Y., Ye, L., Liu, H., Xia, Q., Zhang, Y., Yang, X., et al. (2010). Vanadium compounds induced mitochondria permeability transition pore (MPT) opening related to oxidative stress. Journal of Inorganic Biochemistry, 104, 371–378.

    CAS  PubMed  Article  Google Scholar 

  21. Zhang, F., Xu, Z., Gao, J., Xu, B., & Deng, Y. (2008). In vitro effect of manganese chloride exposure on energy metabolism and oxidative damage of mitochondria isolated from rat brain. Environmental Toxicology and Pharmacology, 26, 232–236.

    CAS  PubMed  Article  Google Scholar 

  22. Sadegh, C., & Schreck, R. P. (2003). The spectroscopic determination of aqueous sulfite using Ellman’s reagent. MIT Undergraduate Research Journal, 8, 39–43.

    Google Scholar 

  23. Baracca, A., Sgarbi, G., Solaini, G., & Lenaz, G. (2003). Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F(0) during ATP synthesis. Biochimica et Biophysica Acta, 1606, 137–146.

    CAS  PubMed  Article  Google Scholar 

  24. Tafreshi, N. K., Hosseinkhani, S., Sadeghizadeh, M., Sadeghi, M., Ranjbar, B., & Naderi-Manesh, H. (2007). The influence of insertion of a critical residue (Arg356) in structure and bioluminescence spectra of firefly luciferase. Journal of Biological Chemistry, 282, 8641–8647.

    CAS  PubMed  Article  Google Scholar 

  25. Drew, B., & Leeuwenburgh, C. (2003). Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology, 28, 1259–1268.

    Google Scholar 

  26. Belyaeva, E. A., Korotkov, S. M., & Saris, N. E. (2011). In vitro modulation of heavy metal-induced rat liver mitochondria dysfunction: a comparison of copper and mercury with cadmium. Journal of Trace Elements in Medicine and Biology, 25, 63–73.

    Article  Google Scholar 

  27. White, A. R., Huang, X., Jobling, M. F., Barrow, C. J., Beyreuther, K., Masters, C. L., et al. (2001). Homocysteine potentiates copper- and amyloid beta peptide peptide-mediated toxicity in primary neuronal cultures: possible risk factors in the Alzheimer’s-type neurodegenerative pathways. Journal of Neurochemistry, 76, 1509–1520.

    CAS  PubMed  Article  Google Scholar 

  28. Goldfrank, L. R., Flomenbaum, F. E., & Lewin, N. A. (1998). Goldfrank’s toxicologic emergencies,6 (th ed., pp. 1339–1340). Stamford: Appleton and Lange.

    Google Scholar 

  29. Pourahmad, J., Stohs, S. J., & Bagchi, D. (1995). Oxidative mechanisms in the toxicity of metal ions. Free Radical Biology and Medicine, 18, 321–3685.

    Article  Google Scholar 

  30. Pourahmad, J., Hosseini, M.-J., Bakan, S., & Ghazi-Khansari, M. (2011). Hepatoprotective activity of angiotensin-converting enzyme (ACE) inhibitors, captopril and enalapril, against paraquat toxicity. Pesticide Biochemistry and Physiology, 99, 105–110.

    CAS  Article  Google Scholar 

  31. Belyaeva, E. A., Dymkowska, D., Wieckowski, M. R., & Wojtczak, L. (2008). Mitochondria as an important target in heavy metal toxicity in rat hepatoma AS30D cells. Toxicology and Applied Pharmacology, 231, 34–42.

    CAS  PubMed  Article  Google Scholar 

  32. Fattoretti, P., Vecchiet, J., Felzani, G., Gracciotti, N., Solazzi, M., Caselli, U., et al. (2001). Succinic dehydrogenase activity in human muscle mitochondria during aging: a quantitative cytochemical investigation. Mechanisms of Ageing and Development, 122, 1841–1848.

    CAS  PubMed  Article  Google Scholar 

  33. Nadanaciva, S., Bernal, A., Aggeler, R., Capaldi, R., & Will, Y. (2007). Target identification of drug induced mitochondrial toxicity using immunocapture based OXPHOS activity assays. Toxicology in Vitro, 21, 902–911.

    CAS  PubMed  Article  Google Scholar 

  34. Jones, T. T., & Brewer, G. J. (2010). Age-related deficiencies in complex I endogenous substrate availability and reserve capacity of complex IV in cortical neuron electron transport. Biochimica et Biophysica Acta, 1797, 167–176.

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  35. Seth, R., Yang, S., Choi, S., Sabean, M., & Roberts, E. A. (2004). In vitro assessment of copper-induced toxicity in the human hepatoma line, Hep G2. Toxicology in Vitro, 18, 501–509.

    CAS  PubMed  Article  Google Scholar 

  36. Zoratti, M., & Szabo, I. (1995). The mitochondrial permeability transition. Biochimica et Biophysica Acta, 1241, 139–176.

    PubMed  Article  Google Scholar 

  37. Krumschnabel, G., Manzl, C., Berger, C., & Hofer, B. (2005). Oxidative stress, mitochondrial permeability transition, and cell death in Cu-exposed trout hepatocytes. Toxicology and Applied Pharmacology, 209, 62–73.

    CAS  PubMed  Article  Google Scholar 

  38. Eguchi, Y., Shimizu, S., & Tsujimoto, Y. (1997). Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Research, 57, 1835–1840.

    CAS  PubMed  Google Scholar 

  39. Simmons, T. D., Slater, K. J., & Crouch, S. P. M. (2001). Changes in intracellular ADP:ATP ratio as a marker of apoptosis. Miami Nature Biotechnology-Winter Symposium, 1, 58.

    Google Scholar 

  40. Suzuki, S., Higuchi, M., Proske, R. J., Oridate, N., Hong, W. K., & Lotan, R. (1999). Implication of mitochondria-derived reactive oxygen species, cytochrome C and caspase-3 in N-(4-hydroxyphenyl) retinamide-induced apoptosis in cervical carcinoma cells. Oncogene, 18, 6380–6387.

    CAS  PubMed  Article  Google Scholar 

  41. Fortunato, F., Deng, X., Gates, L. K., McClain, C. J., Bimmler, D., Graf, R., et al. (2006). Pancreatic response to endotoxin after chronic alcohol exposure: switch from apoptosis to necrosis? American Journal of Physiology. Gastrointestinal and Liver Physiology, 290, 232–241.

    Article  Google Scholar 

  42. Orrenius, S., Gogvadze, V., & Zhivotovsky, B. (2007). Mitochondrial oxidative stress: implications for cell death. Annual Review of Pharmacology and Toxicology, 47, 143–183.

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgments

The investigation described in this paper was extracted from Ph.D. thesis of Dr. Mir-Jamal Hosseini (graduated from Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences in 2012). The thesis conducted under supervision of Prof. Jalal Pourahmad at Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran. This study was supported by a grant from Shahid Beheshti University of Medical Sciences, Deputy of Research (88-01-94-6677, 2011).

Author information

Affiliations

  1. Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, P. O. Box: 14155-6153, Tehran, Iran

    Mir-Jamal Hosseini, Fatemeh Shaki & Jalal Pourahmad

  2. Department of Pharmacology and Toxicology, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran

    Mir-Jamal Hosseini

  3. Department of Pharmacology and Toxicology, School of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran

    Fatemeh Shaki

  4. Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Mahmoud Ghazi-Khansari

Authors
  1. Mir-Jamal Hosseini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Fatemeh Shaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mahmoud Ghazi-Khansari
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jalal Pourahmad
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jalal Pourahmad.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hosseini, MJ., Shaki, F., Ghazi-Khansari, M. et al. Toxicity of Copper on Isolated Liver Mitochondria: Impairment at Complexes I, II, and IV Leads to Increased ROS Production. Cell Biochem Biophys 70, 367–381 (2014). https://doi.org/10.1007/s12013-014-9922-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12013-014-9922-7

Keywords

  • Copper (Cu2+)
  • Isolated mitochondria
  • Toxicity
  • Respiratory complexes