Skip to main content
SpringerLink
Log in

Development of stable HSPA1A promoter-driven luciferase reporter HepG2 cells for assessing the toxicity of organic pollutants present in air

  • Original Paper
  • Published:
Cell Stress and Chaperones Aims and scope

Abstract

HSPA1A (HSP70-1) is a highly inducible heat shock gene up-regulated in response to environmental stresses and pollutants. The aim of our study was to evaluate the sensitivity of the stable metabolically competent HepG2 cells containing a human HSPA1A promoter-driven luciferase reporter (HepG2-luciferase cells) for assessing the toxicity of organic pollutants present in air. The HepG2-luciferase cells were validated by heat shock treatment and testing three organic compounds (pyrene, benzo[a]pyrene, and formaldehyde) that are ubiquitous in the air. The maximal level of HSPA1A (HSP70-1) and relative luciferase activity induced by heat shock were over three and nine times the control level, respectively. Pyrene, benzo[a]pyrene, and formaldehyde all induced significantly elevated levels of relative luciferase activity in a dose-dependent manner. Extractable organic matter (EOM) from urban traffic and coke oven emissions in ambient air were tested on the HepG2-luciferase cells. The traffic EOM induced significant increase in relative luciferase activity at concentrations of picogram per liter. The coke oven EOM produced a strong dose-dependent induction of relative luciferase activity up to six times the control value. Significant increases in relative luciferase activity were observed at concentrations that were as low, or lower than the concentrations that the tested organic pollutants decreased cell viability, and increased malondialdehyde concentration, Olive tail moment, and micronuclei frequency. Therefore, we conclude that the HepG2-luciferase cells are a valuable tool for rapid screening of the overall toxicity of organic pollutants present in air.

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

Similar content being viewed by others

Abbreviations

EOM:

Extractable organic matter

MDA:

Malondialdehyde

HSPs:

Heat shock proteins

PAHs:

Polycyclic aromatic hydrocarbons

DMSO:

Dimethylsulfoxide

References

  • Aït-Aïssa S, Porcher J, Arrigo A, Lambré C (2000) Activation of the hsp70 promoter by environmental inorganic and organic chemicals: relationships with cytotoxicity and lipophilicity. Toxicology 145:147–157

    Article  PubMed  Google Scholar 

  • Aït-Aïssa S, Pandard P, Magaud H, Arrigo AP, Thybaud E, Porcher JM (2003) Evaluation of an in vitro hsp70 induction test for toxicity assessment of complex mixtures: comparison with chemical analyses and ecotoxicity tests. Ecotoxicol Environ Saf 54:92–104

    Article  PubMed  Google Scholar 

  • de Pomerai D (1996) Heat-shock proteins as biomarkers of pollution. Hum Exp Toxicol 15:279–285

    Article  PubMed  Google Scholar 

  • Deane EE, Woo NY (2006) Impact of heavy metals and organochlorines on hsp70 and hsc70 gene expression in black sea bream fibroblasts. Aquat Toxicol 79:9–15

    Article  PubMed  CAS  Google Scholar 

  • Doostdar H, Grant MH, Melvin WT, Wolf CR, Burke MD (1993) The effects of inducing agents on cytochrome P450 and UDP-glucuronyltransferase activities in human HEPG2 hepatoma cells. Biochem Pharmacol 46:629–635

    Article  PubMed  CAS  Google Scholar 

  • Fenech M (2007) Cytokinesis-block micronucleus cytome assay. Nat Protoc 2:1084–1104

    Article  PubMed  CAS  Google Scholar 

  • Gupta SC, Sharma A, Mishra M, Mishra RK, Chowdhuri DK (2010) Heat shock proteins in toxicology: how close and how far? Life Sci 86:377–384

    Article  PubMed  CAS  Google Scholar 

  • Hahn GM, Shiu EC, West B, Goldstein L, Li GC (1985) Mechanistic implications of the induction of thermotolerance in Chinese hamster cells by organic solvents. Cancer Res 45:4138–4143

    PubMed  CAS  Google Scholar 

  • He M, Guo H, Yang X, Zhang X, Zhou L, Cheng L, Zeng H, Hu FB, Tanguay RM, Wu T (2009) Functional SNPs in HSPA1A gene predict risk of coronary heart disease. PLoS One 4:e4851

    Article  PubMed  Google Scholar 

  • Hightower LE (1991) Heat shock, stress proteins, chaperones, and proteotoxicity. Cell 66:191–197

    Article  PubMed  CAS  Google Scholar 

  • IARC (1983) Polynuclear aromatic compounds, Part 1, Chemical, environmental and experimental data. IARC Monogr Eval Carcinog Risk Chem Hum 32:1–453. http://monographs.iarc.fr/ENG/Monographs/vol32/volume32.pdf. Accessed 17 April 1998

  • IARC (1985) Polynuclear aromatic compounds, Part 4, Bitumens, coal-tars and derived products, shale-oils and soots. IARC Monogr Eval Carcinog Risk Chem Hum 35:1–247. http://monographs.iarc.fr/ENG/Monographs/vol35/volume35.pdf. Accessed 20 April 1998

  • Kotoglou P, Kalaitzakis A, Vezyraki P, Tzavaras T, Michalis LK, Dantzer F, Jung JU, Angelidis C (2009) Hsp70 translocates to the nuclei and nucleoli, binds to XRCC1 and PARP-1, and protects HeLa cells from single-strand DNA breaks. Cell Stress Chaperones 14:391–406

    Article  PubMed  CAS  Google Scholar 

  • Luparello C, Sirchia R, Longo A (2011) Cadmium as a transcriptional modulator in human cells. Crit Rev Toxicol 41:75–82

    Article  PubMed  Google Scholar 

  • Mendez F, Kozin E, Bases R (2003) Heat shock protein 70 stimulation of the deoxyribonucleic acid base excision repair enzyme polymerase beta. Cell Stress Chaperones 8:153–161

    Article  PubMed  CAS  Google Scholar 

  • Morimoto RI (1993) Cells in stress: transcriptional activation of heat shock genes. Science 259:1409–1410

    Article  PubMed  CAS  Google Scholar 

  • NIOSH (1998) National institute for occupational safety and health. Polynuclear aromatic hydrocarbons by HPLC: method 5506. In: NIOSH manual of analytical methods, 4th edn. http://www.cdc.gov/niosh/docs/2003-154/pdfs/5506.pdf. Accessed 15 January 1998

  • NTP (2010) Final report on carcinogens background document for formaldehyde. Rep Carcinog Backgr Doc (10–5981):i-512. http://ntp.niehs.nih.gov/ntp/roc/twelfth/2009/November/Formaldehyde_BD_Final.pdf. Accessed 22 January 2010

  • Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–358

    Article  PubMed  CAS  Google Scholar 

  • Olive PL, Banáth JP (2006) The comet assay: a method to measure DNA damage in individual cells. Nat Protoc 1:23–29

    Article  PubMed  CAS  Google Scholar 

  • Parsell DA, Lindquist S (1993) The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu Rev Genet 27:437–496

    Google Scholar 

  • Simmons SO, Fan C, Ramabhadran R (2009) Cellular stress response pathway system as a sentinel ensemble in toxicological screening. Toxicol Sci 111:202–225

    Article  PubMed  CAS  Google Scholar 

  • Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354

    Article  PubMed  CAS  Google Scholar 

  • Truettner JS, Hu K, Liu CL, Dietrich WD, Hu B (2009) Subcellular stress response and induction of molecular chaperones and folding proteins after transient global ischemia in rats. Brain Res 1249:9–18

    Article  PubMed  CAS  Google Scholar 

  • Voellmy R (1996) Sensing stress and responding to stress. In: Feige U, Morimoto RI, Yahara I, Polla BS (eds) Stress-inducible cellular responses. Birkhäuser Verlag, Basel, pp 121–137

    Google Scholar 

  • Westerink WM, Schoonen WG (2007) Phase II enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Toxicol in Vitro 21:1592–1602

    Article  PubMed  CAS  Google Scholar 

  • Wu X, Lu W, Mersch-Sundermann V (2003) Benzo(a)pyrene induced micronucleus formation was modulated by persistent organic pollutants (POPs) in metabolically competent human HepG2 cells. Toxicol Lett 144:143–150

    Article  PubMed  CAS  Google Scholar 

  • Yang J, Liu X, Niu P, Zou Y, Gong Z, Yuan J, Wu T (2007a) Dynamic changes of XPA, XPC, XPF, XPG and ERCC1 protein expression and their correlations with levels of DNA damage in human bronchial epithelia cells exposed to benzo[a]pyrene. Toxicol Lett 174:10–17

    Article  PubMed  CAS  Google Scholar 

  • Yang X, Zheng J, Bai Y, Tian F, Yuan J, Sun J, Liang H, Guo L, Tan H, Chen W, Tanguay RM, Wu T (2007b) Using lymphocyte and plasma Hsp70 as biomarkers for assessing coke oven exposure among steel workers. Environ Health Perspect 115:1573–1577

    Article  PubMed  CAS  Google Scholar 

  • Zheng Y, Ritzenthaler JD, Sun X, Roman J, Han S (2009) Prostaglandin E2 stimulates human lung carcinoma cell growth through induction of integrin-linked kinase: the involvement of EP4 and Sp1. Cancer Res 69:896–904

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Special Program for Scientific Public Welfare Research of Public Health grant (200902006) and the National Basic Research Program grant (2011CB503800) to T.W. and the National Natural Scientific Foundation of China grant (30600491) to F.W. The research of R.W.C. is funded by Heart and Stroke Foundation of New Brunswick.

Author information

Authors and Affiliations

  1. Department of Occupational and Environmental Health, School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, 13 Hangkong Rd, Wuhan, 430030, Hubei, China

    Lili Xin, Xiaohai Li, Huaxin Deng, Dan Kuang, Xiayun Dai, Suli Huang, Meian He & Tangchun Wu

  2. School of Public Health and Primary Care, 4/F School of Public Health and Primary Care, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, NT, Hong Kong, China

    Feng Wang

  3. Department of Anatomy and Neurobiology, Faculty of Medicine, Dalhousie University, 5850 College Street, PO Box 15000, Halifax, NS, B3H 4R2, Canada

    R. William Currie

Authors
  1. Lili Xin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaohai Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huaxin Deng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dan Kuang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiayun Dai
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Suli Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Feng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Meian He
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. R. William Currie
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Tangchun Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tangchun Wu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Xin, L., Li, X., Deng, H. et al. Development of stable HSPA1A promoter-driven luciferase reporter HepG2 cells for assessing the toxicity of organic pollutants present in air. Cell Stress and Chaperones 17, 567–576 (2012). https://doi.org/10.1007/s12192-012-0332-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12192-012-0332-8

Keywords

Search

Navigation