Inhibition of Gap Junctional Intercellular Communication in Rat Liver Epithelial Cells Induced by BHT and Propyl Gallate

간상피세포에서 BHT와 propyl gallate에 의한 gap junctional intercellular communication 억제 효과

  • Published : 2007.10.31


This study was conducted to analyze the cytotoxic effects of butylated hydroxytoluene (BHT) and propyl gallate (PG) in WB-F344 rat liver epithelial cells. Here we measured the inhibition level of gap junctional intercellular communication (GJIC) and elucidated the relationships between GJIC and mitogen-activated protein kinases (MAPKs) such as ERK, JNK, and p38. The cytotoxicities of BHT and PG appeared at concentrations of 1.0mM and 0.25mM, respectively, in the WB-F344 cells; and GJIC inhibition, which was analyzed by a scrape-loading/dye transfer assay and Western blotting analysis, appeared at 0.6mM for BHT and 0.1mM for PG, respectively. Also, the phosphorylations of Cx43, ERK, JNK, and p38 increased in dose-dependent manners. This suggests that BHT and PG treatments inhibited GJIC by the phosphorylation of MAPKs prior to cell damage.


  1. Branen AL, Davidson PM, Salminen S. Food Additives. Marcel Dekker Inc., New York, NY, USA. pp. 143-145 (1990)
  2. Branen AL. Toxicology and biochemistry of butylated hydroxyanisole and butylated hydroxytoluene. J. Am. Oil Chem. Soc. 52: 59-63 (1975)
  3. WHO Guideline for the study of dietary intakes of chemical contaminants. Report prepared by the Joint UNEP/FAO/WHO Food Contamination Monitoring Programme. WHO Offset Publication No. 87. Geneva, Switzerland (1985)
  4. Loewenstein WR. Junctional intercellular communication: The cell-to-cell membrane channel. Physiol. Rev. 61: 829-913 (1981)
  5. Kang KS, Wilson MR, Hayashi T, Chang CC, Trosko JE. Inhibition of gap junctional intercellular communication in normal human breast epithelial cells after treatment with pesticides, PCBs and PBBs alone or in mixtures. Environ. Health Persp. 104: 192- 200 (1996)
  6. Marshall CJ. Specificity of receptor tyrosine kinase signalling: Transient versus sustained extracellular signal-regulated kinase activation. Cell 80: 175-185 (1995)
  7. El-Fouly MH, Trosko JE, Chang CC. Scrape-loading and dye transfer: A rapid and simple technique to study gap junctional intercellular communication. Exp. Cell Res. 168: 422-430 (1987)
  8. Kim MR. Food Safety. Shinjeong, Seoul, Korea. pp. 119-124 (2004)
  9. Eghbali B, Kessler JA, Reid LM, Roy C, Spray DC. Involvement of gap junctions in tumorigenesis: transfection of tumor cells with connexin32 cDNA retards growth in vivo. P. Natl. Acad. Sci. USA 88: 10701-10705 (1991)
  10. Cho JH, Cho SD, Hu H, Kim SH, Lee SK, Lee YS, Kang KS. The roles of ERK1/2 and p38 MAP kinases in the preventive mechanisms of mushroom Phellinus linteus against the inhibition of gap junctional intercellular communication by hydrogen peroxide. Carcinogenesis 23: 1163-1169 (2002)
  11. Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of the mitogen-activated protein kinase cascade. P. Natl. Acad. Sci. USA 92: 7686-7689 (1995)
  12. Roulston A, Reinhard C, Amiri P, Williams LT. Early activation of c-Jun N-terminal kinase and p38 kinase regulate cell survival in response to tumor necrosis factor $\alpha$. J. Biol. Chem. 273: 10232-10239 (1998)
  13. Kang KS, Kang BC, Lee BJ, Che JH, Li GX, Trosko JE, Lee YS. Preventive effect of epicatechin and ginsenoside Rb(2) on the inhibition of gap junctional intercellular communication by TPA and $H_2O_2$. Cancer Lett. 152: 97-106 (2000)
  14. Saez JC, Connor JA, Spray DC, Bennett MV. Hepatocyte gap junctions are permeable to the second messenger, inositol 1,4,5- trisphosphate and to calcium ions. P. Natl. Acad. Sci. USA 86: 2708-2712 (1989)
  15. Hwang JW, Park JS, Jo EH, Kim SJ, Yoon BS, Kim SH, Lee YS, Kang KS. Chinese cabbage extracts and sulforaphane can protect $H_2O_2$-induced inhibition of gap junctional intercellular communication through the inactivation of ERK1/2 and p38 MAP kinases. J. Agr. Food Chem. 53: 8205-8210 (2005)
  16. Rivedal E, Opsahl H. Role of PKC and MAP kinase in EGFand TPA-induced connexin43 phosphorylation and inhibition of gap junction intercellular communication in rat liver epithelial cells. Carcinogenesis 22: 1543-1550 (2001)
  17. Peers KE, Coxon DT, Chan HW. Auto-oxidation of methyl linoleate: The effect of antioxidants on product distribution. J. Sci. Food Agr. 35: 813-817 (1984)
  18. Kumar NM, Gilula NB. The gap junction communication channel. Cell 84: 381-388 (1996)
  19. Bruzzone R, White TW, Paul DL. Connections with connexins: The molecular basis of direct intercellular signaling. Eur. J. Biochem. 15: 1-27 (1996)
  20. Oh SY, Grupen CG, Murray AW. Phorbol ester induces phosphorylation and down-regulation of connexin 43 in WB cells. Biochim. Biophys. Acta 1094: 243-245 (1991)
  21. Yang SR, Cho SD, Ahn NS, Jung JW, Park JS, Park JS, Jo EH, Hwang JW, Jung JY, Kim TY, Yoon BS, Lee BH, Kang KS, Lee YS. Role of gap junctional intercellular communication (GJIC) through p38 and ERK1/2 pathway in the differentiation of rat neuronal stem cells. J. Vet. Med. Sci. 67: 291-294 (2005)
  22. Kim BS, Lee CW, Lee YJ, Hong KH, Lee CH, Park GG, Kim YK, Lee MH. Study on determination method of TBHQ in foods. The Report of National Institute of Health 28: 366-371 (1991)
  23. Carystinos GD, Bier A, Batist G. The role of connexin-mediated cell-cell communiucation in breast cancer metastasis. J. Mammary Gland Biol. 6: 431-461 (2001)
  24. Choe SY, Yang KH. Toxicological studies of antioxidants, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). Korean J. Food Sci. Technol. 14: 283-288 (1982)
  25. Rivedal E, Yamasaki H, Sanner T. Inhibition of gap junctional intercellular communication in Syrian hamster embryo cells by TPA, retinoic acid, and DDT. Carcinogenesis 15: 689-694 (1994)
  26. Kamibayashi Y, Oyamada Y, Mori M, Oyamada M. Aberrant expression of gap junction proteins (connexins) is associated with tumor progression during multistage mouse skin carcinogenesis in vivo. Carcinogenesis 16: 1287-1297 (1995)
  27. Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of tetrazolium-based semiautomated colorimetric assay: Assesment of chemosensitivity testing. Cancer Res. 47: 936-942 (1987)
  28. Kang KS, Yun JW, Yoon B, Lim YK, Lee YS. Preventive effect of germanium dioxide on the inhibition of gap junctional intercellular communication by TPA. Cancer Lett. 152: 97-106 (2000)
  29. SAS/STATTM User's Guide Release. 6.03 Edition. SAS Institute, Cary, NC, USA (1988)