한국식품과학회지 (Korean Journal of Food Science and Technology)

한국식품과학회 (Korean Society of Food Science and Technology)
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MTT formazan의 발색에 미치는 zinc protoporphyrin의 영향

Changes in Color Response of MTT formazan by Zinc Protoporphyrin

  • 박경아 (서울여자대학교 자연과학대학 식품공학과) ;
  • 최현아 (서울여자대학교 자연과학대학 식품공학과) ;
  • 김미리 (서울여자대학교 자연과학대학 식품공학과) ;
  • 최유미 (서울여자대학교 자연과학대학 식품공학과) ;
  • 김현정 (서울여자대학교 자연과학대학 식품공학과) ;
  • 홍정일 (서울여자대학교 자연과학대학 식품공학과)
  • Park, Kyung-A (Department of Food Science and Technology, College of Natural Science, Seoul Women's University) ;
  • Choi, Hyun-A (Department of Food Science and Technology, College of Natural Science, Seoul Women's University) ;
  • Kim, Mi-Ri (Department of Food Science and Technology, College of Natural Science, Seoul Women's University) ;
  • Choi, Yoo-Mi (Department of Food Science and Technology, College of Natural Science, Seoul Women's University) ;
  • Kim, Hyun-Jung (Department of Food Science and Technology, College of Natural Science, Seoul Women's University) ;
  • Hong, Jung-Il (Department of Food Science and Technology, College of Natural Science, Seoul Women's University)
  • 투고 : 2011.08.31
  • 심사 : 2011.10.31
  • 발행 : 2011.12.31
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초록

본 연구는 세포의 사멸 및 성장변화 등의 평가에 널리 이용되는 MTT assay에서 생성된 formazan dye에 미치는 ZnPP의 영향을 조사하였다. ZnPP는 생체 내에 자연적으로 생성되거나 다양한 관련 실험에 인위적으로 첨가해주는 물질로서, MTT formazan dye의 빛에 의한 탈색을 가속화시키는 것으로 밝혀졌다. Formazan dye의 분해는 5와 $10{\mu}M$ ZnPP 존재 시 반감기를 기준하여 각각 10 및 20배 가량 가속화되었으며, 빛이 차단된 조건에서는 영향을 미치지 않았다. ZnPP 구조 중 Zn는 formazan dye의 탈색에 영향을 미치지 않았으나, porphyrin 구조와 공통적인 감광체 성질을 나타내는 MB에 의해서 ZnPP 존재 시와 유사하게 dye의 탈색을 가속화 시켰다. 이러한 ZnPP와 MB에 의한 formazan dye의 탈색반응은 NAC와 ${\beta}$-carotene에 의해 지연되었으나, BHT에 의한 저해효과는 나타나지 않았다. 본 결과는 세포 중에 존재하는 ZnPP 등의 감광체류가 MTT tetrazolium으로부터 환원된 formazan dye의 빛에 의한 신속한 분해를 유도하며, 관련 실험에서 빛의 차단 등의 조치와 함께 각별히 유의해야함을 시사한다.

Zinc protoporphyrin (ZnPP) is produced endogenously during heme metabolism and treated in cells as a heme oxygenase inhibitor. In the present study, the effects of ZnPP on the color response of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a commonly-used method for analyzing cell viability, were investigated. ZnPP induced rapid decolorizaion of MTT formazan under light; the degradation rates were 10- and 20- folds faster in the presence of 5 and $10{\mu}M$ ZnPP, respectively. Methylene blue (MB), another type of photosensitizer, also accelerated degradation of formazan under light. Butylated hyroxytoluene did not inhibit ZnPP- or MB-induced formazan degradation. The color degradation of formazan dye was signficantly delayed in the presence of N-acetylcysteine or ${\beta}$-carotene. The present results suggest that certain photosensitizing compounds may affect the color and stability of MTT formazan, which should be carefully considered when conducting the MTT assay.

키워드

참고문헌

  1. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55-63 (1983) https://doi.org/10.1016/0022-1759(83)90303-4
  2. Twentyman PR, Luscombe M. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Brit. J. Cancer 56: 279-285 (1987) https://doi.org/10.1038/bjc.1987.190
  3. Gerlier D, Thomasset N. Use of MTT colorimetric assay to measure cell activation. J. Immunol. Methods 94: 57-63 (1986) https://doi.org/10.1016/0022-1759(86)90215-2
  4. Green LM, Reade JL, Ware CF. Rapid colormetric assay for cell viability: Application to the quantitation of cytotoxic and growth inhibitory lymphokines. J. Immunol. Methods 70: 257-268 (1984) https://doi.org/10.1016/0022-1759(84)90190-X
  5. Edmondson JM, Armstrong LS, Martinez AO. A rapid and simple MTT-based spectrophotometric assay for determining drug sensitivity in monolayer cultures. J. Tissue Cult. Meth. 11: 15-17 (1988) https://doi.org/10.1007/BF01404408
  6. Labbe RF, Vreman HJ, Stevenson DK. Zinc protoporphyrin: A metabolite with a mission. Clin. Chem. 45: 2060-2072 (1999)
  7. Hastka J, Lasserre J, Schwarzbeck A, Strauch M, Hehlmann R. Zinc protoporphyrin in anemia of chronic disorders. Blood 81: 1200-1204 (1993)
  8. Lamola AA, Joselow M, Yamane T. Zinc protoporphyrin (ZPP): A simple, sensitive, fluorometric screening test for lead poisioning. Clin. Chem. 21: 93-97 (1975)
  9. Fang J, Sawa T, Akaike T, Greish K, Maeda H. Enhancement of chemotherapeutic response of tumor cells by a heme oxygenase inhibitor, pegylated zinc protoporphyrin. Int. J. Cancer 109: 1-8 (2004) https://doi.org/10.1002/ijc.11644
  10. Brouard S, Otterbein LE, Anrather J, Tobiasch E, Bach FH, Choi AMK, Soares MP. Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J. Exp. Med. 192: 1015-1026 (2000) https://doi.org/10.1084/jem.192.7.1015
  11. Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, Toma T, Ohta K, Kasahara Y, Koizumi S. Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J. Clin. Invest. 103: 129-130 (1999) https://doi.org/10.1172/JCI4165
  12. Deshane J, Wright M, Agarwal A. Heme oxygenase-1 expression in disease states. Acta Biochim. Pol. 52: 273-284 (2005)
  13. Fang J, Akaike T, Maeda H. Antiapoptotic role of heme oxygenase (HO) and the potential of HO as a target in anticancer treatment. Apoptosis 9: 27-35 (2004) https://doi.org/10.1023/B:APPT.0000012119.83734.4e
  14. Tanaka S, Akaike T, Fang J, Beppu T, Ogawa M, Tamura F, Miyamoto Y, Maeda H. Antiapoptotic effect of heme oxygenase-1 induced by nitric oxide in experimental solid tumour. Brit. J. Cancer 88: 902-909 (2003) https://doi.org/10.1038/sj.bjc.6600830
  15. Lee TS, Chau LY. Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice. Nat. Med. 8: 240-246 (2002) https://doi.org/10.1038/nm0302-240
  16. Bruggisser R, von Daeniken K, Jundt G, Schaffner W, Tullberg-Reinert H. Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay. Planta Med. 68: 445-448 (2002) https://doi.org/10.1055/s-2002-32073
  17. Talorete TP, Bouaziz M, Sayadi S, Isoda H. Influence of medium type and serum on MTT reduction by flavonoids in the absence of cells. Cytotechnology 52: 189-198 (2006)
  18. Plumb JA. Cell sensitivity assays: The MTT assay. pp. 165-169. In: Cancer Cell Culture: Methods and Protocols. Langdon SP (ed). Humana Press Inc., Totowa, NJ, USA (2004)
  19. Maines MD. Zinc. protoporphyrin is a selective inhibitor of heme oxygenase activity in the neonatal rat. BBA-Gen Subjects 673: 339-350 (1981) https://doi.org/10.1016/0304-4165(81)90465-7
  20. Kennedy JC, Pottier RH, Pross DC. Photodynamic therapy with endogenous protoporphyrin: IX: Basic principles and present clinical experience. J. Photoch. Photobio. B 6: 143-148 (1990) https://doi.org/10.1016/1011-1344(90)85083-9
  21. Moan J. Porphyrin photosensitization and phototherapy. Photochem. Photobiol. 43: 681-690 (1986) https://doi.org/10.1111/j.1751-1097.1986.tb05647.x
  22. Tardivo JP, Del Giglio A, Oliveira CS, Gabrielli DS, Junqueira HC, Tada DB, Severino D, Turchiello RF, Baptista MS. Methylene blue in photodynamic therapy: From basic mechanisms to clinical applications. Photodiagn. Photodyn. 2: 175-191 (2005) https://doi.org/10.1016/S1572-1000(05)00097-9
  23. Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci. Technol. 28: 25-30 (1995) https://doi.org/10.1016/S0023-6438(95)80008-5
  24. Fukumoto LR, Mazza G. Assessing antioxidant and prooxidant activities of phenolic compounds. J. Agr. Food Chem. 48: 3597-3604 (2000) https://doi.org/10.1021/jf000220w
  25. Hirayama O, Nakamura K, Hamada S, Kobayasi Y. Singlet oxygen quenching ability of naturally occurring carotenoids. Lipids 29: 149-150 (1994) https://doi.org/10.1007/BF02537155
  26. Foote CS, Denny RW. Chemistry of singlet oxygen. VII. Quenching by $\beta$-carotene. J. Am. Chem. Soc. 90: 6233-6235 (1968) https://doi.org/10.1021/ja01024a061