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.

본 연구에서는 정상 세포인 간상피세포를 이용하여 BHT와 PG가 GJIC에 미치는 효과 및 그 작용 기전을 살펴보았다. BHT와 PG를 WB-F344세포에 각각 1.0mM, 0.75mM 이상 처리한 결과, 세포 생육이 80%이하로 저하되어 세포 독성을 보였고, BHT와 PG의 GJIC에 대한 억제효과는 이보다 낮은 농도인 0.6mM, 0.1mM에서 나타났으며 처리에 따른 Cx43 단백질의 발현 및 인산화를 측정한 결과, BHT와 PG 모두 농도의존적으로 Cx43의 인산화가 증가하였고, Cx43의 구조적 변화와 관련된 MAP kinase는 주요 biomarker인 ERK, p38, JNK의 발현 및 인산화를 측정한 결과, 농도의존적으로 ERK와 p38의 인산화가 증가하는 것으로 나타났다. 이는 WB-F344세포에 BHT와 PG의 처리가 세포독성을 나타내기 전 농도에서 GJIC의 억제하였음을 의미하며 이는 식품첨가물의 안전성 평가에도 활용될 수 있을 것으로 기대된다.



  1. Kim MR. Food Safety. Shinjeong, Seoul, Korea. pp. 119-124 (2004)
  2. Branen AL, Davidson PM, Salminen S. Food Additives. Marcel Dekker Inc., New York, NY, USA. pp. 143-145 (1990)
  3. 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)
  4. 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)
  5. Branen AL. Toxicology and biochemistry of butylated hydroxyanisole and butylated hydroxytoluene. J. Am. Oil Chem. Soc. 52: 59-63 (1975)
  6. Choe SY, Yang KH. Toxicological studies of antioxidants, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA). Korean J. Food Sci. Technol. 14: 283-288 (1982)
  7. 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)
  8. Loewenstein WR. Junctional intercellular communication: The cell-to-cell membrane channel. Physiol. Rev. 61: 829-913 (1981)
  9. 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)
  10. Kumar NM, Gilula NB. The gap junction communication channel. Cell 84: 381-388 (1996)
  11. 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)
  12. Bruzzone R, White TW, Paul DL. Connections with connexins: The molecular basis of direct intercellular signaling. Eur. J. Biochem. 15: 1-27 (1996)
  13. 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)
  14. 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)
  15. 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)
  16. 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)
  17. 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)
  18. 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)
  19. 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)
  20. 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)
  21. 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)
  22. Marshall CJ. Specificity of receptor tyrosine kinase signalling: Transient versus sustained extracellular signal-regulated kinase activation. Cell 80: 175-185 (1995)
  23. 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)
  24. SAS/STATTM User's Guide Release. 6.03 Edition. SAS Institute, Cary, NC, USA (1988)
  25. 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)
  26. 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)
  27. 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)
  28. 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)
  29. 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)