SpringerLink
Log in

Arsenic trioxide and lead acetate induce apoptosis in adult rat hepatic stem cells

  • Published:
Cell Biology and Toxicology Aims and scope Submit manuscript

Abstract

In the present study, the toxicity of arsenic trioxide and lead acetate was assessed in adult hepatic stem cells induced in the 2-acetyl-aminofluorene/partial hepatectomy rat model. Isolated oval cells were incubated separately for 6 h with 40 μM each of arsenic trioxide and lead acetate. 3-(4,5-Dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay denoted significant time-dependent cell death in arsenic and lead treated oval cells. The degree of stress imposed by these metals was evidenced by induction of heat shock protein (HSP) 70 and HSP 90. Arsenic and lead were found to trigger apoptosis as revealed by DNA ladder formation, Western blots of apoptotic factors, and reverse transcriptase polymerase chain reaction analyses of bax and bcl-2. Results clearly indicate that both arsenic and lead induced apoptosis is caspase-mediated and accompanied by extracellular signal-regulated kinase (ERK) dephosphorylation. Full-length BH3-interacting-domain death agonist expression in presence of caspase 3 inhibitor unravels a direct involvement of caspase in As and Pb induced apoptosis. Expression patterns of apoptosis inducing factor, B cell lymphoma-2 (Bcl-2) antagonist of cell death, Bcl-2-associated X protein, and Bcl2 also signify mitochondrial regulation of apoptosis effected by lead and arsenic. It is concluded that stimulation of caspase cascade and simultaneous ERK dephosphorylation are the most significant operative pathways directly associated with apoptotic signals triggered by arsenic and lead in the oval cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

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
Fig. 7

Similar content being viewed by others

Abbreviations

AAF:

2-acetylaminofluorene

AIF:

Apoptosis-inducing factor

AP-1:

Activator protein-1

Apaf-1:

Apoptotic protease activating factor-1

Bcl-2:

B cell lymphoma-2

Bax:

Bcl-2-associated X protein

Bad:

Bcl-2 antagonist of cell death

Bid:

BH3-interacting-domain death agonist

CK-19:

cytokeratin 19

DMA:

Dimethylarsinic acid

DMEM:

Dulbecco’s minimum essential medium

ERK:

Extracellular signal-regulated kinase

GAPDH:

Glyceraldehyde phosphate dehydrogenase

HBSS:

Hanks balanced salt solution

HSP:

Heat shock protein

MMA:

Monomethylarsonic acid

MTT:

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

NFkB:

Nuclear factor kB

OV1:

Oval cell marker 1

OV6:

Oval cell marker 6

PH:

Partial hepatectomy

RT PCR:

Reverse transcriptase polymerase chain reaction

SCF:

Stem cell factor

ZFL:

Zebra fish liver cell line

References

  • Adams JM. Ways of dying: multiple pathways to apoptosis. Genes Dev. 2003;17:2481–95.

    Article  PubMed  CAS  Google Scholar 

  • Alison MR, Poulsom R, Forbes SJ. Update on hepatic stem cells. Liver 2001;21:367–73.

    Article  PubMed  CAS  Google Scholar 

  • Ayala-Fierro F, Barber DS, Rael LT, Carter DE. In vitro tissue specificity for arsine and arsenite toxicity in the rat. Toxicol Sci. 1999;52:122–29.

    Article  PubMed  CAS  Google Scholar 

  • Best DH, Coleman WB. Treatment with 2-AAF blocks the small hepatocyte-like progenitor cell response in retrorsine-exposed rats. J Hepatol. 2007;46:1055–63.

    Article  PubMed  CAS  Google Scholar 

  • Bidère N, Lorenzo HK, Carmona S, Laforge M, Harper F, Dumont C, et al. Cathepsin D triggers Bax activation, resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis. J Biol Chem. 2003;278:31401–11.

    Article  PubMed  CAS  Google Scholar 

  • Bode AM, Dong Z. The paradox of arsenic: molecular mechanisms of cell transformation and chemotherapeutic effects. Crit Rev Oncol Hematol. 2002;42:5–24.

    Article  PubMed  Google Scholar 

  • Boudreau RTM, Conrad DM, Hoskin DW. Apoptosis induced by protein phosphatase 2A (PP2A) inhibition in T leukemia cell is negatively regulated by PP2A—associated p38 mitogen-activated protein kinase. Cell Signal. 2007;19:139–51.

    Article  PubMed  CAS  Google Scholar 

  • Brambilla G, Martelli A. Cytotoxicity, DNA fragmentation and DNA repair synthesis in primary human hepatocytes. In: O’Hare S, Atterwill CK, editors. In vitro toxicity testing protocols. Totowa, New Jersey: Humana; 1995. p. 59–66.

    Google Scholar 

  • Chen N-Y, Ma W-Y, Yang CS, Dong Z. Inhibition of arsenite-induced apoptosis and AP-1 activity by epigallocatechin-3-gallate and theoflavins. J Environ Pathol Toxicol Oncol. 2000;19:287–95.

    PubMed  CAS  Google Scholar 

  • Columbano A, Ledda-Columbano GM, Coni PP, Faa G, Liguori C, Santa Cruz G, et al. Occurrence of cell death (apoptosis) during the involution of liver hyperplasia. Lab Invest. 1985;52:670–5.

    PubMed  CAS  Google Scholar 

  • Datta S, Saha DR, Ghosh D, Majumdar T, Bhattacharya S, Mazumder S. Sub-lethal concentration of arsenic interferes with the proliferation of hepatocytes and induces in vivo apoptosis in Clarias batrachus L. Comp Biochem Physiol Part C. 2007;145:339–49.

    Google Scholar 

  • Davila JC, Cezar GG, Thiede M, Strom S, Miki T, Trosko J. Use and application of stem cells in toxicology. Toxicol Sci. 2004;79:214–23.

    Article  PubMed  CAS  Google Scholar 

  • Dunsford HC, Sell S. Production of monoclonal antibodies to preneoplastic liver cell populations induced by chemical carcinogen in rats and transplantable Morris hepatomas. Cancer Res. 1989;49:4887–93.

    PubMed  CAS  Google Scholar 

  • Earnshaw WC, Martins LM, Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem. 1999;68:383–424.

    Article  PubMed  CAS  Google Scholar 

  • Fausto N. Liver regeneration: from laboratory to clinic. Liver Transplant. 2001;276:835–44.

    Article  Google Scholar 

  • Green DR, Kroemer G. The pathophysiology of mitochondrial cell death. Science 2004;305:626–9.

    Article  PubMed  CAS  Google Scholar 

  • Gross A, Yin XM, Wang K, Wei MC, Jockel J, Milliman C, et al. Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-R1/Fas death. J Biol Chem. 1999;274:1156–63.

    Article  PubMed  CAS  Google Scholar 

  • He ZP, Tan WQ, Tang YF, Zhang HJ, Feng MF. Activation, isolation, identification and in vitro proliferation of oval cells from adult rat livers. Cell Prolif. 2004;37:177–87.

    Article  PubMed  CAS  Google Scholar 

  • Ikeyama S, Kusumoto K, Miyake H, Rokutan K, Tashiro S. A non-toxic heat shock protein 70 inducer, geranylgeranylacetone, suppresses apoptosis of cultured rat hepatocytes caused by hydrogen peroxide and ethanol. J Hepatol. 2001;55:53–61.

    Article  Google Scholar 

  • Inglis JK. Introduction to laboratory animal science and technology. 1st ed. Oxford: Pergamon; 1980. p. 37–126.

    Google Scholar 

  • Kann S, Estes C, Reichard JF, Huang M, Sartor MA, Schwemberger S, et al. Butylhydroquinone protects cells genetically deficient in glutathione biosynthesis from arsenite-induced apoptosis without significantly changing their prooxidant status. Toxicol Sci. 2005;87:365–84.

    Article  PubMed  CAS  Google Scholar 

  • Kapur R, Chandra S, Cooper R, McCarthy J, Williams DA. Role of p38 and ERK MAP kinase in proliferation of erythroid progenitors in response to stimulation by soluble and membrane isoforms of stem cell factor. Blood 2002;100:1287–93.

    PubMed  CAS  Google Scholar 

  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5.

    Article  PubMed  CAS  Google Scholar 

  • Lanza R. Essentials of stem cell biology. USA: Elsevier; 2006.

    Google Scholar 

  • Li GC, Werb Z. Correlation between the synthesis of heat shock proteins and the development of thermotolerance in Chinese hamster fibroblasts. Proc Natl Acad Sci USA. 1982;79:3218–22.

    Article  PubMed  CAS  Google Scholar 

  • Li H, Zhu H, Xu CJ, Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 1998;94:491–501.

    Article  PubMed  CAS  Google Scholar 

  • Li L, Luo X, Wang X. Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 2001;412:95–9.

    Article  PubMed  CAS  Google Scholar 

  • Liedtke C, Trautwein C. The role of JNK2 in toxic liver injury. J Hepatol. 2006;45:762–4.

    Article  PubMed  CAS  Google Scholar 

  • Lin YW, Chuang SM, Yang JL. Persistent activation of ERK1/2 by lead acetate increases nucleotide excision repair synthesis and confers anti-cytotoxicity and anti-mutagenicity. Carcinogenesis 2003;24:53–61.

    Article  PubMed  Google Scholar 

  • Lindquist S, Craig EA. The heat shock proteins. Annu Rev Genet. 1998;22:631–37.

    Article  Google Scholar 

  • Liu ZM, Huang HS. As2O3-induced c-Src/EGFR/ERK signaling is via Sp1 binding sites to stimulate p21WAF1/CIP1 expression in human epidermoid carcinoma A431 cells. Cell Signal. 2005;18:244–55.

    Article  PubMed  CAS  Google Scholar 

  • Look AT. Arsenic and apoptosis in the treatment of acute promyelocytic leukemia. J Natl Cancer Inst. 1998;90:86–8.

    Article  PubMed  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Measurement of protein with the Folin phenol reagent. J Biol Chem. 1951;193:265–75.

    PubMed  CAS  Google Scholar 

  • Ludwig S, Hoffmeyer A, Goebeler M, Kilian K, Hafner H, Neufeld B, et al. The stress inducer arsenite activates mitogen-activated protein kinases extracellular signal-regulated kinases 1 and 2 via a MAPK kinase 6/p38-dependent pathway. J Biol Chem. 1998;273:1917–22.

    Article  PubMed  CAS  Google Scholar 

  • Luo X, Budiharjo H, Zou H, Slauhter C, Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 1998;94:481–90.

    Article  PubMed  CAS  Google Scholar 

  • Marceau N. Biology of disease. Cell lineage and differentiation programs in epidermal; urothelial and hepatic tissues and their neoplasms. Lab Invest. 1990;63:4–20.

    PubMed  CAS  Google Scholar 

  • Milton AG, Zalewski PD, Ratnaike RN. Zinc protects against arsenic-induced apoptosis in a neuronal cell line, measured by DEVD-caspase activity. Biometals 2004;17:707–13.

    Article  PubMed  CAS  Google Scholar 

  • Mosser DD, Caron AW, Bourget L, Larose CD, Massie B. Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. Mol Cell Bio. 1997;17:5317–27.

    CAS  Google Scholar 

  • Mosser DD, Caron AW, Bourget L, Meriin AB, Sherman AY, Morimoto RI, et al. The chaperone function of hsp70 is required for protection against stress-induced apoptosis. Mol Cell Biol. 2000;20:7146–59.

    Article  PubMed  CAS  Google Scholar 

  • Nollen EA, Morimoto RI. Chaperoning signaling pathways: molecular chaperones as stress-sensing ‘heat shock’ proteins. J Cell Sci. 2002;115:2809–16.

    PubMed  CAS  Google Scholar 

  • Parsell DA, Lindquist S. The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu Rev Genet. 1993;27:437–96.

    Article  PubMed  CAS  Google Scholar 

  • Petit PX, Susin SA, Zamzami N, Mignotte B, Kroemer G. Mitochondria and programmed cell death: back to the future. FEBS Lett. 1996;396:7–13.

    Article  PubMed  CAS  Google Scholar 

  • Pulido MD, Parrish AR. Metal-induced apoptosis: mechanisms. Mutat Res. 2003;533:227–41.

    PubMed  CAS  Google Scholar 

  • Qin AL, Zhou XQ, Zhang W, Yu H, **e Q. Characterization and enrichment of hepatic progenitor cells in adult rat liver. World J Gastroenterol. 2004;10:1480–6.

    PubMed  CAS  Google Scholar 

  • Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol. 1997;9:180–86.

    Article  PubMed  CAS  Google Scholar 

  • Roy S, Bhattacharya S. Arsenic-induced histopathology and synthesis of stress proteins in liver and kidney of Channa punctatus. Ecotox Environ Saf. 2006;65:218–29.

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T. Molecular cloning—a laboratory manual. 2nd ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory; 1989.

    Google Scholar 

  • Seok SH, Baek MW, Lee HY, Kim DJ, Na YR, Noh YR, et al. Arsenite-induced apoptosis is prevented by antioxidants in zebra fish liver cell line. Toxicol in Vitro. 2007;21:870–7.

    PubMed  CAS  Google Scholar 

  • Shimano K, Satake M, Okaya A, Kitanaka J, Kitanaka N, Takemura M, et al. Hepatic oval cell have the side population phenotype defined by expression of ATP binding cassette transporter ABCG2/BCRP1. American J Pathol. 2003;163(1):3–9.

    CAS  Google Scholar 

  • Snedecor GW, Cochran WG. Statistical methods. 6th ed. Iowa: The Iowa State University Press; 1967.

    Google Scholar 

  • Subjeck JR, Sciandra JJ, Johnson RJ. Heat shock proteins and thermotolerance; a comparison of induction kinetics. Br J Radiol. 1982;55:579–84.

    Article  PubMed  CAS  Google Scholar 

  • Sun X-M, MacFarlane M, Zhuang J, Wolf BB, Green DR, Cohen GM. Distinct caspase cascade are initiated in receptor-mediated and chemical-induced apoptosis. J Biol Chem. 1999;274:5053–60.

    Article  PubMed  CAS  Google Scholar 

  • Supino R. MTT assays. In: O’Hare S, Atterwill CK, editors. In vitro toxicity testing protocols. Totowa, New Jersey: Humana; 1995. p. 137–49.

    Google Scholar 

  • Trosko JE. Human stem cells as targets for the aging and diseases of aging processes. Med Hypotheses. 2003;60:439–47.

    Article  PubMed  Google Scholar 

  • Tsujimoto Y, Shimizu S. Bcl-2 family: life-or-death switch. FEBS Lett. 2001;466:6–10.

    Article  Google Scholar 

  • Wang X. The expanding role of mitochondria in apoptosis. Genes Dev. 2002;15:2922–33.

    Google Scholar 

  • Wang K, Yin XM, Chao DT, Milliman CL, Korsmeyer SJ. BID: a novel BH3 domain-only death agonist. Genes Dev. 1996;10:2859–69.

    Article  PubMed  CAS  Google Scholar 

  • Waskiewicz AJ, Cooper JA. Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals and yeast. Curr Opin Cell Biol. 1995;7:798–805.

    Article  PubMed  CAS  Google Scholar 

  • Yin XM, Wang K, Gross A, Zhao Y, Zinkel S, Klocke B, et al. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 1999;400:886–91.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

SB, SM, and SR gratefully acknowledge the Council of Scientific and Industrial Research for project grant (Project No. 37(1179)/04/EMR-II) and the CAS (UGC) grants to the department which enabled the present study. AR and SA are thankful for the UGC Research Fellowships. Authors are expressing their sincere gratitude to Professor Kaoru Kubokawa, Ocean Research Institute, University of Tokyo, Japan for providing Actinase-E for the present study. Authors also express their sincere gratitude to Mr Hemanta Yadav for his technical assistance.

Author information

Author notes

  1. Sonali Roy

    Present address: Ocean Research Institute, University of Tokyo, Tokyo, Japan

Authors and Affiliations

  1. Environmental Toxicology Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan, 731235, India

    Soumik Agarwal, Atish Ray & Shelley Bhattacharya

  2. Immunobiology Laboratory, Department of Zoology, Visva-Bharati University, Santiniketan, 731235, India

    Shibnath Mazumder

Authors
  1. Soumik Agarwal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sonali Roy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Atish Ray
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shibnath Mazumder
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shelley Bhattacharya
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shelley Bhattacharya.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Agarwal, S., Roy, S., Ray, A. et al. Arsenic trioxide and lead acetate induce apoptosis in adult rat hepatic stem cells. Cell Biol Toxicol 25, 403–413 (2009). https://doi.org/10.1007/s10565-008-9094-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10565-008-9094-6

Keywords

Search

Navigation