Archives of Toxicology

, Volume 83, Issue 5, pp 417–427 | Cite as

Oxidative stress and apoptotic changes in primary cultures of rat proximal tubular cells exposed to lead

  • Lin Wang
  • Heng Wang
  • Maozhi Hu
  • Jin Cao
  • Dawei Chen
  • Zongping Liu
Inorganic Compounds

Abstract

Lead is a known nephrotoxic element. In this study, primary cultures of rat proximal tubular (rPT) cells were treated with different concentrations of lead acetate (0.25, 0.5 and 1 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability together with a significant increase in the number of apoptotic and necrotic cells and lactate dehydrogenase release were seen in the experiment. Simultaneously, elevation of reactive oxygen species levels and intracellular [Ca2+]i, depletion of mitochondrial membrane potential and intracellular glutathione were revealed during the lead exposure. In addition, apoptotic morphological changes induced by lead exposure in rPT cells were demonstrated by Hoechst 33258 staining. The apoptosis was markedly prevented by N-acetyl-l-cysteine, while the necrosis was not affected. Moreover, catalase and superoxide dismutase activities in the living cells rose significantly. In conclusion, exposure of rPT cells to low-concentration lead led to cell death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death induced by oxidative stress was the chief mechanism. Meanwhile, a group of cells survived lead action, mediated by their ability to activate antioxidant defense systems.

Keywords

Lead Oxidative stress Apoptosis Proximal tubular cells Primary cell culture 

References

  1. Aleo MD, Wyatt RD, Schnellmann RG (1991) Mitochondrial dysfunction is an early event in ochratoxin a but not osoporein toxicity to rat renal proximal tubules. Toxicol Appl Pharmacol 107:73–80PubMedCrossRefGoogle Scholar
  2. Alvarez-Barrientos A, O’Connor JE, Nieto Castillo R, Moreno Moreno AB, Prieto P (2001) Use of flow cytometry and confocal microscopy techniques to investigate early CdCl2-induced nephrotoxicity in vitro. Toxicol Vitro 15:407–412CrossRefGoogle Scholar
  3. Chakraborti T, Das S, Mondal M, Roychoudhury S, Chakraborti S (1999) Oxidant, mitochondria and calcium: an overview. Cell Signal 11:77–85PubMedCrossRefGoogle Scholar
  4. Chen F, Vallyathan V, Castranova V, Shi X (2001) Cell apoptosis induced by carcinogenic metals. Mol Cell Biochem 222:183–188PubMedCrossRefGoogle Scholar
  5. Cheng YJ, Yang BC, Hsieh WC, Huang BM, Liu MY (2002) Enhancement of TNF-α expression does not trigger apoptosis upon exposure of glial cells to lead and lipopolysaccharide. Toxicology 78:183–191CrossRefGoogle Scholar
  6. Chikahisa L, Oyama Y, Okazaki E, Noda K (1996) Fluorescent estimation of H2O2-induced changes in cell viability and cellular nonprotein thiol level of dissociated rat thymocytes. Jpn J Pharmacol 71:299–305PubMedCrossRefGoogle Scholar
  7. Crompton M (1999) The mitochondrial permeability transition pore and its role in cell death. Biochem J 341:233–249PubMedCrossRefGoogle Scholar
  8. Dalton TP, Shertzer HG, Puga A (1999) Regulation of gene expression by reactive oxygen. Annu Rev Pharmacol Toxicol 39:67–101PubMedCrossRefGoogle Scholar
  9. Dyatlov VA, Dyatlova OM, Parsons PJ, Lawrence DA, Carpenter DO (1998) Lipopolysaccharide and interleukin-6 enhance lead entry into cerebellar neurons: application of a new and sensitive flow cytometric technique to measure intracellular lead and calcium concentrations. Neurotoxicology 19:293–302PubMedGoogle Scholar
  10. Ercal N, Treeratphan P, Hammond TC, Mattews RH, Grannemann N, Spitz D (1996) In vivo indices of oxidative stress in lead exposed C57BL/6 mice are reduced by treatment with meso-2, 3-dimercaptosuccinic acid or N-acetyl cysteine. Free Rad Biol Med 21:157–161PubMedCrossRefGoogle Scholar
  11. Fleury C, Mignotte B, Vayssière JL (2002) Mitochondrial reactive oxygen species in cell death signaling. Biochimie 84:131–141PubMedCrossRefGoogle Scholar
  12. Foster KA, Galeffi F, Gerich FJ, Turner DA, Müller M (2006) Optical and pharmacological tools to investigate the role of mitochondria during oxidative stress and neurodegeneration. Prog Neurobiol 79:136–171PubMedCrossRefGoogle Scholar
  13. Fox DA, He L, Poblenz AT, Medrano CJ, Blocker YS, Srivastava D (1998) Lead-induced alterations in retinal cGMP phosphodiesterase trigger calcium overload, mitochondrial dysfunction and rod photoreceptor apoptosis. Toxicol Lett 102–103:359–361PubMedCrossRefGoogle Scholar
  14. Goyer RA (1989) Mechanisms of lead and cadmium nephrotoxicity. Toxicol lett 46:153–162PubMedCrossRefGoogle Scholar
  15. Grammatopoulos TN, Johnson V, Moore SA, Andres R, Weyhenmeyer JA (2004) Angiotensin type 2 receptor neuroprotection against chemical hypoxia is dependent on the delayed rectifier K+ channel, Na+/Ca2+ exchanger and Na+/K+ ATPase in primary cortical cultures. Neurosci Res 50:299–306PubMedCrossRefGoogle Scholar
  16. Hervouet E, Simonnet H, Godinot C (2007) Mitochondria and reactive oxygen species in renal cancer. Biochimie 89:1080–1088PubMedCrossRefGoogle Scholar
  17. Kadir MM, Janjua NZ, Kristensen S, Fatmi Z, Sathiakumar N (2008) Status of children’s blood lead levels in Pakistan: implications for research and policy. Public health 122:708–715PubMedCrossRefGoogle Scholar
  18. Koh JY, Choi DW (1987) Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J Neurosci Methods 20:83–90PubMedCrossRefGoogle Scholar
  19. Lavrentiadou SN, Chan C, Kawcak T, Ravid T, Tsaba A, Der Vliet AV, Rasooly R, Goldkorn T (2001) Ceramide-mediated apoptosis in lung epithelial cells is regulated by glutathione. Am J Respir Cell Mol Biol 25:676–684PubMedGoogle Scholar
  20. Loghman-Adham M (1997) Renal effects of environmental and occupational lead exposure. Environ Health Perspect 105:928–939PubMedCrossRefGoogle Scholar
  21. López E, Arce C, Oset-Gasque MJ, Cañadas S, González MP (2006) Cadmium induces reactive oxygen species generation and lipid peroxidation in cortical neurons in culture. Free Radic Biol Med 40:940–951PubMedCrossRefGoogle Scholar
  22. Lühe A, Hildebrand H, Bach U, Dingermann T, Ahr HJ (2003) A new approach to studying ochratoxin A (OTA)-induced nephrotoxicity: expression profiling in vivo and in vitro employing cDNA microarrays. Toxicol Sci 73:315–328PubMedCrossRefGoogle Scholar
  23. Marklund S, Marklund G (1974) Involvement of superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47:469–474PubMedCrossRefGoogle Scholar
  24. Masella R, Benedetto R, Varì R, Filesi C, Giovannini C (2005) Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione-related enzymes. J Nutr Biochem 16:577–586PubMedCrossRefGoogle Scholar
  25. Matés JM, Pérez-Gómez C, Núñez de Castro I (1999) Antioxidant enzymes and human diseases. Clin Biochem 32:595–603PubMedCrossRefGoogle Scholar
  26. Moran LK, Guteridge JM, Quinlan GJ (2001) Thiols in cellular redox signaling and control. Curr Med Chem 8:763–772PubMedGoogle Scholar
  27. Muntner P, Menke A, Batuman V, Rabito FA, He J, Todd AC (2007) Association of tibia lead and blood lead with end-stage renal disease: a pilot study of African-Americans. Environ Res 104:396–401PubMedCrossRefGoogle Scholar
  28. Nicholls DG (1985) A role for the mitochondrion in the protection of cells against calcium overload? Prog Brain Res 63:97–106PubMedCrossRefGoogle Scholar
  29. Nouwen EJ, Dauwe S, Van der Biest I, De Broe ME (1993) Stage- and segment-specific expression of cell-adhesion molecules N-CAM, A-CAM, and L-CAM in the kidney. Kidney Int 44:147–158PubMedCrossRefGoogle Scholar
  30. Okada Y, Oyama Y, Chikahisa L, Satoh M, Kanemaru K, Sakai H, Noda K (2000) Tri-n-butyltin-induced change in cellular level of glutathione in rat thymocytes: a flow cytometric study. Toxicol Lett 117:123–128PubMedCrossRefGoogle Scholar
  31. Orrenius S, Mccabe MJ, Nicotera P (1992) Ca2+-dependent mechanisms of cytotoxicity and programmed cell death. Toxicol Lett 64:357–364PubMedCrossRefGoogle Scholar
  32. Patra RC, Swarup D, Dwidedi SK (2001) Antioxidant effects of α tocopherol, ascorbic acid and l-methionine on lead-induced oxidative stress of the liver, kidney and brain in rats. Toxicology 162:81–88PubMedCrossRefGoogle Scholar
  33. Pulido MD, Parrish AR (2003) Metal-induced apoptosis: mechanisms. Mutat Res 533:227–241PubMedGoogle Scholar
  34. Rekasi Z, Czompoly T, Schally AV, Boldizsar F, Varga JL, Zarandi M, Berki T, Horvath RA, Nemeth P (2005) Antagonist of growth hormone releasing hormone induces apoptosis in LNCaP human prostate cancer cells through a Ca2+-dependent pathway. Proc Natl Acad Sci 102:3435–3440PubMedCrossRefGoogle Scholar
  35. Sandhir R, Gill KD (1995) Effect of lead on lipid peroxidation in livers of rats. Biol Trace Elem Res 48:91–97PubMedCrossRefGoogle Scholar
  36. Sandhir R, Julka D, Gill KD (1994) Lipoperoxidative damage on lead exposure in rat brain and its implications on membrane bound enzymes. Pharmacol Toxicol 74:66–71PubMedCrossRefGoogle Scholar
  37. Schafer FQ, Buettner GR (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Rad Biol Med 30:1191–1212PubMedCrossRefGoogle Scholar
  38. Sivaprasad R, Nagaraj M, Varalakshmi P (2003) Combined efficacies of lipoic acid and meso-2, 3-dimercaptosuccinic acid on lead-induced erythrocyte membrane lipid peroxidation and antioxidant status. Hum Exp Toxicol 22:183–192PubMedCrossRefGoogle Scholar
  39. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995) A novel assay for apoptosis, flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein-labeled Annexin V. J Immunol Meth 184:39–51CrossRefGoogle Scholar
  40. Voehringer DW (1999) Bcl-2 and glutathione: alterations in cellular redox state that regulate apoptosis sensitivity. Free Rad Biol Med 27:945–950PubMedCrossRefGoogle Scholar
  41. Wallace DC (2005) A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 39:359–407PubMedCrossRefGoogle Scholar
  42. Xu J, Ji LD, Xu LH (2006) Lead-induced apoptosis in PC 12 cells: involvement of p53, bcl-2 family and caspase-3. Toxicol Lett 166:160–167PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Lin Wang
    • 1
  • Heng Wang
    • 1
  • Maozhi Hu
    • 2
  • Jin Cao
    • 1
  • Dawei Chen
    • 1
  • Zongping Liu
    • 1
  1. 1.College of Veterinary MedicineYangzhou UniversityYangzhouPeople’s Republic of China
  2. 2.Testing Center of Yangzhou UniversityYangzhouPeople’s Republic of China

Personalised recommendations