Skip to main content

Advertisement

SpringerLink
Log in

Hepatitis B vaccine induces apoptotic death in Hepa1–6 cells

  • Original Paper
  • Published:
Apoptosis Aims and scope Submit manuscript

Abstract

Vaccines can have adverse side-effects, and these are predominantly associated with the inclusion of chemical additives such as aluminum hydroxide adjuvant. The objective of this study was to establish an in vitro model system amenable to mechanistic investigations of cytotoxicity induced by hepatitis B vaccine, and to investigate the mechanisms of vaccine-induced cell death. The mouse liver hepatoma cell line Hepa1–6 was treated with two doses of adjuvanted (aluminium hydroxide) hepatitis B vaccine (0.5 and 1 μg protein per ml) and cell integrity was measured after 24, 48 and 72 h. Hepatitis B vaccine exposure increased cell apoptosis as detected by flow cytometry and TUNEL assay. Vaccine exposure was accompanied by significant increases in the levels of activated caspase 3, a key effector caspase in the apoptosis cascade. Early transcriptional events were detected by qRT-PCR. We report that hepatitis B vaccine exposure resulted in significant upregulation of the key genes encoding caspase 7, caspase 9, Inhibitor caspase-activated DNase (ICAD), Rho-associated coiled-coil containing protein kinase 1 (ROCK-1), and Apoptotic protease activating factor 1 (Apaf-1). Upregulation of cleaved caspase 3,7 were detected by western blot in addition to Apaf-1 and caspase 9 expressions argues that cell death takes place via the intrinsic apoptotic pathway in which release of cytochrome c from the mitochondria triggers the assembly of a caspase activation complex. We conclude that exposure of Hepa1–6 cells to a low dose of adjuvanted hepatitis B vaccine leads to loss of mitochondrial integrity, apoptosis induction, and cell death, apoptosis effect was observed also in C2C12 mouse myoblast cell line after treated with low dose of vaccine (0.3, 0.1, 0.05 μg/ml). In addition In vivo apoptotic effect of hepatitis B vaccine was observed in mouse liver.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Gherardi RK, Coquet M, Cherin P, Belec L, Moretto P, Dreyfus PA et al (2001) Macrophagic myofasciitis lesion assesses long-term persistence of vaccine-derived aluminum hydroxide in muscle. Brain 124:1821–1831

    Article  PubMed  CAS  Google Scholar 

  2. Goto N, Akama K (1982) Histopathological studies of reactions in mice injected with aluminum-adsorbed tetanus toxoid. Microbiol Immunol 26(12):1121–1132

    PubMed  CAS  Google Scholar 

  3. Good PF, Perl DP, Bierer LM, Schmeidler J (1992) Selective accumulation of aluminum and iron in the neurofibrilary tangles of Alzheimer’ disease. Ann Neurol 31(3):286–292

    Article  PubMed  CAS  Google Scholar 

  4. Richard EF, Stanley LH, Joe LW, David E, Mark AS, Anita C et al (1997) In vivo absorption of aluminum- containing vaccine adjuvants using 26Al. Vaccine 15:1314–1318

    Article  Google Scholar 

  5. Rana SV (2008) Metals and apoptosis: recent developments. J Trace Elem Med Biol 22(4):262–284

    Article  PubMed  CAS  Google Scholar 

  6. Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516

    Article  PubMed  CAS  Google Scholar 

  7. Walter JL, Maire EP, Theo PK (2005) Nanomolar aluminum induces pro-inflammatory and pro-apoptotic gene expression in human brain cells in Primary culture. J Inorg Biochem 99(9):1895–1898

    Article  Google Scholar 

  8. Johnson VJ, Kim SH, Sharma RP (2005) Aluminum-maltolate induces apoptosis and necrosis in neuro-2a cells: potential role for p53 signaling. Toxicol Sci 83(2):329–339

    Article  PubMed  CAS  Google Scholar 

  9. Shaw CA, Petrik MS (2009) Aluminum hydroxide injections lead to motor deficits and motor neuron degeneration. J Inorg Biochem 103(11):1555–1562

    Article  PubMed  CAS  Google Scholar 

  10. Petrik MS, Wong MC, Tabata RC, Garry RF, Shaw CA (2007) Aluminum adjuvant linked to Gulf War illness induces motor neuron death in mice. Neuromol Med 9(1):83–100

    Article  CAS  Google Scholar 

  11. Rook GA, Zumla A (1997) Gulf War syndrome: is it due to a systemic shift in cytokine balance towards a Th2 profile? Lancet 349(9068):1831–1833

    Article  PubMed  CAS  Google Scholar 

  12. Valensi Jp, Carlson JR, Van Nest GA (1994) Systemic cytokine profiles in BALB/c mice immunized with trivalent influenza vaccine containing MF59 oil emulsion and other advanced adjuvants. J Immunol 153(9):4029–4039

    PubMed  CAS  Google Scholar 

  13. Hamza H, Cao J, Li X, Zhao S (2011) In vivo study of hepatitis B vaccine effects on Inflammation and metabolism gene expression. Mol Biol Rep. doi:10.1007/s11033-011-1090-x. 21 Jun 2011

  14. Son YO, Jang YS, Heo JS, Chung WT, Choi KC, Lee JC (2009) Apoptosis-inducing factor plays a critical role in caspase independent, pyknotic cell death in hydrogen peroxide-exposed cells. Apoptosis 14(6):796–808

    Article  PubMed  CAS  Google Scholar 

  15. Platt B, Fiddler G, Riedel G, Henderson Z (2001) Aluminum toxicity in the rat brain: histochemical and immunocytochemical evidence. Brain Res Bull 55(2):257–267

    Article  PubMed  CAS  Google Scholar 

  16. Joshi JG (1990) Aluminum, a neurotoxin which affects diverse metabolic reactions. Biofactors 2(3):163–169

    PubMed  CAS  Google Scholar 

  17. Kaya M, Kalayci R, Arican N, Küçük M, Elmas I (2003) Effect of aluminum on the blood-brain barrier permeability during nitric oxide-blockade-induced chronic hypertension in rats. Biol Trace Elem Res 92(3):221–230

    Article  PubMed  CAS  Google Scholar 

  18. Aremu DA, Meshitsuka S (2005) Accumulation of aluminum by primary cultured astrocytes from aluminum amino acid complex and its apoptotic effect. Brain Res 1031(2):284–296

    Article  PubMed  CAS  Google Scholar 

  19. Nayak P, Chatterjee AK (2001) Effects of aluminum exposure on brain glutamate and GABA systems: an experimental study in rats. Food Chem Toxicol 39(12):1285–1289

    Article  PubMed  CAS  Google Scholar 

  20. Kool M, Pétrilli V, De Smedt T, Rolaz A, Hammad H, van Nimwegen M et al (2008) Cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome. J Immunol 181(6):3755–3759

    PubMed  CAS  Google Scholar 

  21. Eisenbarth SC, Colegio OR, O’Connor W, Sutterwala FS, Flavell RA (2008) Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 453(7198):1122–1126

    Article  PubMed  CAS  Google Scholar 

  22. Sutterwala FS, Ogura Y, Flavell RA (2007) The inflammasome in pathogen recognition and inflammation. J Leukoc Biol 82(2):259–264

    Article  PubMed  CAS  Google Scholar 

  23. Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichtenberger GS, Grant EP et al (2006) Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity 24(3):317–327

    Article  PubMed  CAS  Google Scholar 

  24. Kool M, Soullié T, van Nimwegen M, Willart MA, Muskens F, Jung S et al (2008) Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. J Exp Med 205(4):869–882

    Article  PubMed  CAS  Google Scholar 

  25. Osinska E, Kanoniuk D, Kusiak A (2004) Aluminum hemotoxicity mechanisms. Ann Univ Mariae Curie Sklodowska Med 59(1):411–416

    PubMed  Google Scholar 

  26. Gonzalez MA, Alvarez Mdel L, Pisani GB, Bernal CA, Roma MG, Carrillo MC (2007) Involvement of oxidative stress in the impairment in biliary secretory function induced by intraperitoneal administration of aluminum to rats. Biol Trace Elem Res 116(3):329–348

    Article  PubMed  CAS  Google Scholar 

  27. Fiskum G, Starkov A, Polster BM, Chinopoulos C (2003) Mitochondrial Mechanisms of neural cell death and neuroprotective interventions in Parkinson’s disease. Ann N Y Acad Sci 991:111–119

    Article  PubMed  CAS  Google Scholar 

  28. Toimela T, Tahti H (2004) Mitochondrial viability and apoptosis induced aluminum, mercuric mercury and methyl mercury in cell lines of neural origin. Arch Toxicol 78(10):565–574

    Article  PubMed  CAS  Google Scholar 

  29. Kaufmann SH, Hengartner MO (2001) Programmed cell death: alive and well in the new millennium. Trends Cell Biol 11(12):526–534

    Article  PubMed  CAS  Google Scholar 

  30. Fu HJ, Hu QS, Lin ZN, Ren TL, Song H, Cai CK et al (2003) Aluminum-induced apoptosis in cultured cortical neurons and its effect on SAPK/JNK signal transduction pathway. Brain Res 980(1):11–23

    Article  PubMed  CAS  Google Scholar 

  31. Huang X, Hazlett LD (2003) Analysis of Pseudomonas aeruginosa corneal infection using an oligonucleotide microarray. Invest Ophthalmol Vis Sci 44(8):3409–3416

    Article  PubMed  Google Scholar 

  32. Kijima K, Toyosawa K, Yasuba M, Matsuoka N, Adachi T, Komiyama M et al (2004) Gene expression analysis of the rat testis after treatment with di (2-ethylhexyl) phthalate using cDNA microarray and real-time RT-PCR. Toxicol Appl Pharmacol 200(2):103–110

    Article  PubMed  CAS  Google Scholar 

  33. Nagata S (2000) Apoptotic DNA fragmentation. Exp Cell Res 256(1):12–18

    Article  PubMed  CAS  Google Scholar 

  34. Sakahira H, Enari M, Nagata S (1998) Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391:196–199

    Google Scholar 

  35. Thomas DA, Du C, Xu M, Wang X, Ley TJ (2000) DFF45/ICAD can be directly process by granzyme B during the induction of apoptosis. Immunity 12:621–632

    Article  PubMed  CAS  Google Scholar 

  36. Halenbeck R, MacDonald H, Roulston A, Chen TT, Conroy L, Williams LT (1998) CPAN, a human nuclease regulated by the caspase-sensitive inhibitor DFF45. Curr Biol 8(9):537–540

    Article  PubMed  CAS  Google Scholar 

  37. Coleman ML, Sahai EA, Yeo M, Bosch M, Dewar A, Olson MF (2001) Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol 3(4):339–345

    Article  PubMed  CAS  Google Scholar 

  38. Zou H, Henzel WJ, Liu X, Lutschg A, Wang X (1997) Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90(3):405–413

    Google Scholar 

  39. Joza N, Susin SA, Daugas E, Stanford WL, Cho SK, Li CY et al (2001) Essential role of the mitochondrial apoptosis—inducing factor in programmed cell death. Nature 410(6828):549–554

    Article  PubMed  CAS  Google Scholar 

  40. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES et al (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91(4):479–489

    Article  PubMed  CAS  Google Scholar 

  41. Acehan D, Jiang X, Morgan DG, Heuser JE, Wang X, Akey CW (2002) Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding and activation. Mol Cell 9(2):423–432

    Article  PubMed  CAS  Google Scholar 

  42. Slee EA, Harte MT, Kluck RM, Wolf BB, Casiano CA, Newmeyer DD et al (1999) Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of Caspases-2, -3, -6, 7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 144(2):281–292

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This research was supported by National Natural Science Foundation of China and the creative team project of Education Ministry (IRT-0831) and by the Fundamental Research Funds for the Central Universities from Education Ministry of China (2009PY001). We thank Yang Feng, Niu Lili, Wei Wei and GU Ting, in our lab for suggestions on data analysis.

Author information

Authors and Affiliations

  1. Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, People’s Republic of China

    Heyam Hamza, Jianhua Cao, Xinyun Li, Changchun Li, Mengjin Zhu & Shuhong Zhao

Authors
  1. Heyam Hamza
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jianhua Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xinyun Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Changchun Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mengjin Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shuhong Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Changchun Li.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hamza, H., Cao, J., Li, X. et al. Hepatitis B vaccine induces apoptotic death in Hepa1–6 cells. Apoptosis 17, 516–527 (2012). https://doi.org/10.1007/s10495-011-0690-1

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10495-011-0690-1

Keywords

Search

Navigation