, Volume 17, Issue 5, pp 516–527 | Cite as

Hepatitis B vaccine induces apoptotic death in Hepa1–6 cells

  • Heyam Hamza
  • Jianhua Cao
  • Xinyun Li
  • Changchun LiEmail author
  • Mengjin Zhu
  • Shuhong Zhao
Original Paper


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.


Hepatitis B vaccine Hepa1–6 cells Apoptosis Caspase-dependent pathway TUNEL Flow cytometry 



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.


  1. 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 124:1821–1831PubMedCrossRefGoogle Scholar
  2. 2.
    Goto N, Akama K (1982) Histopathological studies of reactions in mice injected with aluminum-adsorbed tetanus toxoid. Microbiol Immunol 26(12):1121–1132PubMedGoogle Scholar
  3. 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):286–292PubMedCrossRefGoogle Scholar
  4. 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 15:1314–1318CrossRefGoogle Scholar
  5. 5.
    Rana SV (2008) Metals and apoptosis: recent developments. J Trace Elem Med Biol 22(4):262–284PubMedCrossRefGoogle Scholar
  6. 6.
    Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516PubMedCrossRefGoogle Scholar
  7. 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):1895–1898CrossRefGoogle Scholar
  8. 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):329–339PubMedCrossRefGoogle Scholar
  9. 9.
    Shaw CA, Petrik MS (2009) Aluminum hydroxide injections lead to motor deficits and motor neuron degeneration. J Inorg Biochem 103(11):1555–1562PubMedCrossRefGoogle Scholar
  10. 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):83–100CrossRefGoogle Scholar
  11. 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):1831–1833PubMedCrossRefGoogle Scholar
  12. 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):4029–4039PubMedGoogle Scholar
  13. 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. 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):796–808PubMedCrossRefGoogle Scholar
  15. 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):257–267PubMedCrossRefGoogle Scholar
  16. 16.
    Joshi JG (1990) Aluminum, a neurotoxin which affects diverse metabolic reactions. Biofactors 2(3):163–169PubMedGoogle Scholar
  17. 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):221–230PubMedCrossRefGoogle Scholar
  18. 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):284–296PubMedCrossRefGoogle Scholar
  19. 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):1285–1289PubMedCrossRefGoogle Scholar
  20. 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):3755–3759PubMedGoogle Scholar
  21. 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):1122–1126PubMedCrossRefGoogle Scholar
  22. 22.
    Sutterwala FS, Ogura Y, Flavell RA (2007) The inflammasome in pathogen recognition and inflammation. J Leukoc Biol 82(2):259–264PubMedCrossRefGoogle Scholar
  23. 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):317–327PubMedCrossRefGoogle Scholar
  24. 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):869–882PubMedCrossRefGoogle Scholar
  25. 25.
    Osinska E, Kanoniuk D, Kusiak A (2004) Aluminum hemotoxicity mechanisms. Ann Univ Mariae Curie Sklodowska Med 59(1):411–416PubMedGoogle Scholar
  26. 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):329–348PubMedCrossRefGoogle Scholar
  27. 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 991:111–119PubMedCrossRefGoogle Scholar
  28. 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):565–574PubMedCrossRefGoogle Scholar
  29. 29.
    Kaufmann SH, Hengartner MO (2001) Programmed cell death: alive and well in the new millennium. Trends Cell Biol 11(12):526–534PubMedCrossRefGoogle Scholar
  30. 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):11–23PubMedCrossRefGoogle Scholar
  31. 31.
    Huang X, Hazlett LD (2003) Analysis of Pseudomonas aeruginosa corneal infection using an oligonucleotide microarray. Invest Ophthalmol Vis Sci 44(8):3409–3416PubMedCrossRefGoogle Scholar
  32. 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):103–110PubMedCrossRefGoogle Scholar
  33. 33.
    Nagata S (2000) Apoptotic DNA fragmentation. Exp Cell Res 256(1):12–18PubMedCrossRefGoogle Scholar
  34. 34.
    Sakahira H, Enari M, Nagata S (1998) Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391:196–199Google Scholar
  35. 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 12:621–632PubMedCrossRefGoogle Scholar
  36. 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):537–540PubMedCrossRefGoogle Scholar
  37. 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):339–345PubMedCrossRefGoogle Scholar
  38. 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):405–413Google Scholar
  39. 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):549–554PubMedCrossRefGoogle Scholar
  40. 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):479–489PubMedCrossRefGoogle Scholar
  41. 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):423–432PubMedCrossRefGoogle Scholar
  42. 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):281–292PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Heyam Hamza
    • 1
  • Jianhua Cao
    • 1
  • Xinyun Li
    • 1
  • Changchun Li
    • 1
    Email author
  • Mengjin Zhu
    • 1
  • Shuhong Zhao
    • 1
  1. 1.Key Lab of Agricultural Animal Genetics, Breeding, and Reproduction of Ministry of Education, College of Animal Science and TechnologyHuazhong Agricultural UniversityWuhanPeople’s Republic of China

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