, Volume 20, Issue 1, pp 1–11 | Cite as

Copper-1,10-Phenanthroline-Induced Apoptosis in Liver Carcinoma Bel-7402 Cells Associates with Copper Overload, Reactive Oxygen Species Production, Glutathione Depletion and Oxidative DNA Damage



The mechanism of cytotoxicity on liver carcinoma Bel-7402 cells induced by copper-1,10-phenanthroline, Cu(OP)2, has been studied. Cell viability and apoptotic rate were examined in cells treated with Cu(OP)2 or Cu2+ alone. It was found that the apoptosis induced by Cu(OP)2 could not be induced by Cu2+ or OP alone in our experimental conditions. Total copper content in cells was measured by atomic absorption spectrophotometry, and the abnormal elevation of intracellular copper transported by lipophilic OP ligand may play the role of initial factor in the apoptosis, which caused subsequent redox state changes in cells. Intracellular levels of reactive oxygen species (ROS) were detected by fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Reduced (GSH) and total glutathione (GSSG + GSH) were determined by High-performance liquid chromatography (HPLC) after derivatization, and the ratios of GSH/GSSG were subsequently calculated. The overproduction of ROS and the decreased GSH/GSSG ratio were observed in cells which represented the occurrence of oxidative stress in the apoptosis. Oxidative DNA damage was also found in cells treated with Cu(OP)2 in the early stage of the apoptosis, and it suggests that the activation of DNA repair system may be involved in the pathway of the apoptosis induced by Cu(OP)2.


apoptosis copper-1 10-phenanthroline DNA damage glutathione reactive oxygen species 


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This work was supported by the grant from National Natural Science Foundation of China (Grant No. 30370366, 30470111) and The Research Fund for the Doctoral Program of Higher Education (RFDP).


  1. Barbara SB, Stadtman ER (1997) Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 272:20313–20316CrossRefGoogle Scholar
  2. Bradford S (1976) Protein determination. Anal Biochem 72:248–252PubMedCrossRefGoogle Scholar
  3. Burkitt MJ, Milne L, Nicotera P, Orrenius S (1996) 1,10-Phenanthroline stimulates internucleosomal DNA fragmentation in isolated rat-liver nuclei by promoting the redox activity of endogenous copper ions. Biochem J 313:163–170PubMedGoogle Scholar
  4. Buttke TM, Sandstrom PA (1994) Oxidative stress as a mediator of apoptosis. Immunol Today 15:7–10PubMedCrossRefGoogle Scholar
  5. Cereser C, Guichard J, Drai J (2001) Quantitation of reduced and total glutathione at the femtomole level by high-performance liquid chromatography with fluorescence detection: application to red blood cells and cultured fibroblasts. J Chromatogr B 752:123–132CrossRefGoogle Scholar
  6. Chen F, Shi X (2002) Intracellular signal transduction of cells in response to carcinogenic metals. Crit Rev Oncol Hematol 42:105–121PubMedCrossRefGoogle Scholar
  7. Collins AR (2004) The comet assay for DNA damage and repair. Mol Biotechnol 26:249–261PubMedCrossRefGoogle Scholar
  8. Cotgreave IA, Gerdes RG (1998) Recent trends in glutathione biochemistry—glutathione–protein interactions: a molecular link between oxidative stress and cell proliferation? Biochem Biophys Res Commun 242:1–9PubMedCrossRefGoogle Scholar
  9. Curtin JF, Donovan M, Cotter TG (2002) Regulation and measurement of oxidative stress in apoptosis. J Immunol Methods 265:49–72PubMedCrossRefGoogle Scholar
  10. Danks DM (1988) Copper deficiency in humans. Annu Rev Nutr 8:235–257PubMedCrossRefGoogle Scholar
  11. Dean RT, Fu S, Stocker R, Davies MJ (1997) Biochemistry and pathology of radical-mediated protein oxidation. Biochem J 324:1–18PubMedGoogle Scholar
  12. Dizdaroglu M, Aruoma OI, Halliwell B (1990) Modification of bases in DNA by copper ion-1,10-phenanthroline complexes. Biochemistry 29:8447–8451PubMedCrossRefGoogle Scholar
  13. Gaetke LM, Chow CK (2003) Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicology 189:147–163PubMedCrossRefGoogle Scholar
  14. Galaris D, Evangelou A (2002) The role of oxidative stress in mechanisms of metal-induced carcinogenesis. Crit Rev Oncol Hematol 42:93–103PubMedCrossRefGoogle Scholar
  15. Gerschenson LE, Rotello RL (1992) Apoptosis: A different type of cell death. FASEB J 6:2450–2455PubMedGoogle Scholar
  16. Halliwell B, Aruoma OI (1991) DNA damage by oxygen-derived species. Its mechanism and measurement in mammalian system. FEBS Lett 281:9–19PubMedCrossRefGoogle Scholar
  17. Jabs T (1999) Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochem Pharmacol 57:231–245PubMedCrossRefGoogle Scholar
  18. Klatt P, Lamas S (2000) Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur J Biochem 267:4928–4944PubMedCrossRefGoogle Scholar
  19. Krzysztof K, Anna L, Anna B, et al. (2003) A cross-platform public domain PC image-analysis program for the comet assay. Mut Res 534:15–20Google Scholar
  20. Linder MC (2001) Copper and genomic stability in mammals. Mut Res 475:141–152Google Scholar
  21. Ma Y, Cao L, Kawabata T, Yoshino T, Yang BB, Okada S (1998) Cupric nitrilotriacetate induces oxidative DNA damage and apoptosis in human leukemia HL-60 cells. Free Radic Biol Med 25:568–575PubMedCrossRefGoogle Scholar
  22. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63PubMedCrossRefGoogle Scholar
  23. Norbury CJ, Zhivotovsky B (2004) DNA damage-induced apoptosis. Oncogene 23:2797–2808PubMedCrossRefGoogle Scholar
  24. Pourahmad J, Ross S, O‘Brien PJ (2001) Lysosomal involvement in hepatocyte cytotoxicity induced by Cu2+ but not Cd2+. Free Radic Biol Med 30:89–97PubMedCrossRefGoogle Scholar
  25. Pulg S, Thiele DJ (2002) Molecular mechanism of copper uptake and distribution. Curr Opin Chem Biol 6:171–180CrossRefGoogle Scholar
  26. Ravia JJ, Stephen RM, Ghishan FK, Collins JF (2005) Menkes copper ATPase (Atp7a) is a novel metal-responsive gene in rat duodenum, and immunoreactive protein is present on brush-border and basolateral membrane domains. J Biol Chem 280:36221–36227PubMedCrossRefGoogle Scholar
  27. Schafer FQ, Buettner GR (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 30:1191–1212PubMedCrossRefGoogle Scholar
  28. Schulz JB, Lindenau J, Seyfried J, Dichgans J (2000) Glutathione, oxidative stress and neurodegeneration. Eur J Biochem 267:4904–4911PubMedCrossRefGoogle Scholar
  29. Sigman DS, Graham DR, D’Aurora V, Stern AM (1979) Oxygen-dependent cleavage of DNA by the 1,10-phenanthroline cuprous complex. Inhibition of Escherichia coli DNA polymerase I. J Biol Chem 254:12269–12271PubMedGoogle Scholar
  30. Singh RP, Kumar S, Nada R, Prasad R (2006) Evaluation of copper toxicity in isolated human peripheral blood mononuclear cells and it‘s attenuation by zinc: ex vivo. Mol Cell Biochem 282:13–21PubMedCrossRefGoogle Scholar
  31. Sun Y, Oberley LW (1996) Redox regulation of transcriptional activators. Free Radic Biol Med 21:335–348PubMedCrossRefGoogle Scholar
  32. Theophanides T, Anastassopoulou J (2002) Copper and carcinogenesis. Crit Rev Oncol Hematol 42:57–64PubMedCrossRefGoogle Scholar
  33. Toyokuni S, Sagripanti JL (1996) Association between 8-hydroxy-2′-deoxyguanosine formation and DNA strand breaks mediated by copper and iron. Free Radic Biol Med 20:859–864PubMedCrossRefGoogle Scholar
  34. Young MC, Yun SB, Soo YL (2003) Molecular ordering of ROS production, mitochondrial changes, and caspase activation during sodium salicylate-induced apoptosis. Free Radic Biol Med 34:434–442CrossRefGoogle Scholar
  35. Zhou H, Zheng C, Zou G, Tao D, Gong J (2002) G1-phase specific apoptosis in liver carcinoma cell line induced by copper-1,10-phenanthroline. Int J Biochem Cell Biol 34:678–684PubMedCrossRefGoogle Scholar
  36. Zwart LL, Meerman JHN, Commandeur JNM, Vermeulen NPE (1999) Biomarkers of free radical damage. Applications in experimental animals and in humans. Free Radic Biol Med 26:202–226PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  1. 1.State Key Laboratory of Virology, Department of Biotechnology, College of Life SciencesWuhan UniversityWuhanChina

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