The Korean Journal of Physiology and Pharmacology

The Korean Society of Pharmacology (대한약리학회)
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Development of Luciferase Reporter Gene-based Cell Bioassay for the Aromatic Hydrocarbon Receptor Agonists

  • Kim, Sun-Young (Department of Pharmacology, School of Medicine, Catholic University of Daegu) ;
  • Choi, Eun-Jung (Department of Pharmacology, School of Medicine, Catholic University of Daegu) ;
  • Yang, Jae-Ho (Department of Pharmacology, School of Medicine, Catholic University of Daegu)
  • Published : 2006.12.31
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Abstract

The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. The application of recombinant reporter plasmid such as the firefly luciferase gene has proven to be a very effective method to detect these chemicals. The bioassay system, CALUX, is sensitive in directly detecting AhR-agonists from a variety of environmental and biologic materials. However, responses of the AhR-dependent bioassays are dependent on the cell types used. Thus, we developed a sensitive bioassay using the recombinant mouse hepatoma cell (Hepa1c1c7) for the determination of dioxins. The recombinant cell line was stably transfected with firefly luciferase reporter gene (pGudLuc1.1). The transfected cells showed the highest induction of luciferase activity at 4.5 hr and a decrease beyond this time point. The system showed the highest sensitivity of detection ever reported. Upon TCDD exposure cells showed 2 fold increase at 10 pM and 7 fold increase at 100 pM, respectively. The passage number after the transfection played an important role in the sensitivity. The increase of passage number tended to increase the sensitivity of the cells up to 15. The media without phenol red showed a higher induction rate than with phenol red, suggesting the preferable use of phenol red-free media for the bioassay. Since each of the assays has unique characteristics that make them suitable for some screening applications and not others, development of sensitive bioanalytical methods based on a variety of cellular systems in a key to the successful determination of dioxins. The bioassay system developed in this study will contribute to further development of successful screening the AhR agonists among the environmental mixture. In addition, the rapid and sensitive nature of this cellular system can be applied as a valuable tool to screen the dioxin-like moieties among the prodrugs at the initial stage, thereby expediting the new drug discovery.

Keywords

References

  1. Denison MS, Fisher JM, Whitlock JP. The DNA recognition site for the dioxin-Ah receptor complex: Nucleotide sequence and functional analysis. J Biol Chem 263: 17221-1722, 1988
  2. Denison MS, Phelan D, Elferink CJ. The Ah receptor signal-transduction pathway. Toxicant-Receptor Interactions 3-33, 1998a
  3. Denison MS, Phelan D, Winter GM, Ziccardi MH. Carbaryl, a carbamate insecticide, is a ligand for the hepatic Ah (dioxin) receptor. Toxicol Appl Pharmacol 152: 406-414, 1998b https://doi.org/10.1006/taap.1998.9999
  4. El-Fouly MH, Richter C, Giesy JP, Denison MS. Production of a novel recombinant cell line for use as a bioassay for detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Environ Toxicol Chem 13: 1581-1588, 1994
  5. Fernandez-Salguero PM, Hilbert DM, Rudikoff S, Ward JM, Gonzalez FJ. Aryl-hydrocarbon receptor-deficient mice are resistant to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxicity. Toxicol Appl Pharmacol 140: 173-179, 1996 https://doi.org/10.1006/taap.1996.0210
  6. Garrison PM, Tullis K, Aarts JM, Brouwer A, Giesy JP, Denison MS. Species-specific recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-dioxin-like chemicals. Fundam Appl Toxicol 30: 194-203, 1996, 2006 https://doi.org/10.1006/faat.1996.0056
  7. Gizzi G, Hoogenboom LA, Von Holst C, Rose M, Anklam E. Determination of dioxins (PCDDs/PCDFs) and PCBs in food and feed using the DR CALUX bioassay: results of an international validation study. Food Addit Contam 22: 472-481, 2005 https://doi.org/10.1080/02652030500129196
  8. Han D, Nagy SR, Denison MS. Comparison of recombinant cell bioassays for the detection of Ah-receptor agonists. Biofactors 20: 11-22, 2004 https://doi.org/10.1002/biof.5520200102
  9. Heath-Pagliuso S, Rogers WJ, Tullis K, Seidel SD, Cenijn PH, Brouwer A, Denison MS. Activation of the Ah receptor by tryptophan and tryptophan metabolites. Biochemistry 37: 115083-11515, 1998
  10. Helferich WG, Denison MS. Ultraviolet photoproducts of tryptophan can act as dioxin agonists. Mol Pharmacol 40: 674-678, 1991
  11. Holmes JL, Pollenz RS. Determination of aryl-hydrocarbon receptor, nuclear translocator-protein concentration and subcellular localization in hepatic and non-hepatic cell culture lines: Development of quantitative Western blotting protocols for calculation of aryl-hydrocarbon receptor and aryl-hydrocarbon receptor nuclear-translocator protein in total cell lysates. Mol Pharmacol 52: 202-211, 1997 https://doi.org/10.1124/mol.52.2.202
  12. Jeong SH, Cho JH, Park JM, Denison MS. Rapid bioassay for the determination of dioxins and dioxin-like PCDFs and PCBs in meat and animal feeds. J Anal Toxicol 29: 156-162, 2005 https://doi.org/10.1093/jat/29.3.156
  13. Khan EM, Denison MS. Synergistic Activation of Ah receptordependent gene expression by activators of protein kinase C. Organohalogen Compounds 49: 143-146, 2000
  14. Nagy SR, Sanborn JR, Hammock BD, Denison MS. Development of a green fluorescent protein-based cell bioassay for the rapid and inexpensive detection and characterization of ah receptor agonists. Toxicol Sci 65: 200-210, 2002 https://doi.org/10.1093/toxsci/65.2.200
  15. Poland A, Knutson JC. 2,3,7,8,-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: examination of the mechanism of toxicity. Annu Rev Pharmacol Toxicol 22: 517- 554, 1982 https://doi.org/10.1146/annurev.pa.22.040182.002505
  16. Probst MR, Reisz-Porszasz S, Agbunag RV, Ong MS, Hankinson O. Role of the aryl hydrocarbon receptor nuclear translocator protein in aryl hydrocarbon (dioxin) receptor action. Mol Pharmacol 44: 511-518, 1993
  17. Safe S. Polychlorinated biphenyls (PCBs), dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and related compounds: environmental equivalency factors (TEFs). Crit Rev Toxicol 21: 51-88, 1990 https://doi.org/10.3109/10408449009089873
  18. Safe S, Davis D, Romkes M, Yao C, Keyes B, Piskorska-Pliszczynska J, Farrell K, Mason G, Denomme MA, Safe L, Zmudzka B, Holcomb M. Development and validation of in vitro bioassays for 2,3,7,8-TCDD equivalents. Chemosphere 19: 853-860, 1989 https://doi.org/10.1016/0045-6535(89)90421-9
  19. Schmidt JV, Bradfield CA. Ah receptor signaling pathways. Annu Rev Cell Dev Biol 12: 55-89, 1996 https://doi.org/10.1146/annurev.cellbio.12.1.55
  20. Seidel SD, Winters GM, Rogers WJ, Ziccardi MH, Li V, Keser B, Denison MS. Activation of the Ah receptor signaling pathway by prostaglandins. J Biochem Molec Toxicol 15: 187-196, 2001 https://doi.org/10.1002/jbt.16
  21. Yang JH, Rhim JS. 2,3,7,8-tetrachlorodibenzo-p-dioxin; molecular mechanism of carcinogenesis and implication of human in vitro model. Crit Rev Oncol Hematol 18: 111-127, 1995 https://doi.org/10.1016/1040-8428(94)00125-D
  22. Ziccardi MH, Gardner IA, Denison MS. Development and modification of a recombinant cell bioassay to directly detect halogenated and polycyclic aromatic hydrocarbons in serum. Toxicol Sci 54: 183-193, 2000 https://doi.org/10.1093/toxsci/54.1.183