Acridine Orange Stained Micronucleus Assay in Human B and T-lymphocytes after Low Dose ${\gamma}-irradiation$

아크리딘 오렌지 형광염색법을 이용한 저선량 감마선 유도 말초혈액 B와 T-림프구 미소핵 분석

  • Choi, Jeong-Mi (Radiation Health Research Institute, Korea Hydro and Nuclear Power Corporation) ;
  • Kim, Hee-Sun (Radiation Health Research Institute, Korea Hydro and Nuclear Power Corporation) ;
  • Yang, Kwang-Hee (Radiation Health Research Institute, Korea Hydro and Nuclear Power Corporation) ;
  • Kim, Cha-Soon (Radiation Health Research Institute, Korea Hydro and Nuclear Power Corporation) ;
  • Lim, Yong-Khi (Radiation Health Research Institute, Korea Hydro and Nuclear Power Corporation) ;
  • Kim, Chong-Soon (Radiation Health Research Institute, Korea Hydro and Nuclear Power Corporation) ;
  • Woon, Jae-Ho (National Veterinary Research and Quarantine Service)
  • 최정미 (한국수력원자력(주) 방사선보건연구원) ;
  • 김희선 (한국수력원자력(주) 방사선보건연구원) ;
  • 양광희 (한국수력원자력(주) 방사선보건연구원) ;
  • 김차순 (한국수력원자력(주) 방사선보건연구원) ;
  • 임영기 (한국수력원자력(주) 방사선보건연구원) ;
  • 김종순 (한국수력원자력(주) 방사선보건연구원) ;
  • 운재호 (국립수의과학검역원)
  • Published : 2004.03.30


Firstly, we compared the two staining techniques, Giemsa and Acridine orange, to determine micronuclei on samples of cultures of five healthy human peripheral blood lymphocytes after ${\gamma}-irradiation\;(^{137}Cs)$ in dose ranges of 0 to 800cGy. It was found that the Acridine orange staining method gives more reliable results than the usual Giemsa staining method in micronucleus tests. Moreover, the frequency of micronuclei in cytokinesis-blocked human B-lymphocytes was studied after in vitro irradiation in dose ranges of 0 to 50cGy. After setting and separating the B-lymphocytes, the frequency of radiation-induced micronuclei were observed as the end-point markers for the low-dose radiation dosimetry after staining with Giemsa and Acridine orange dyes. The micronuclei frequency in B-lymphocytes was significantly elevated from 10 to 30cGy ${\gamma}-irradiation$. The determination of micronuclei in B-lymphocytes after staining with Acridine orange was higher than that of Giemsa. The frequency of micronuclei in B-lymphocytes was observed to be at least two times higher than those of T-lymphocytes Giemsa in dose increasing. Therefore, the determination of low-dose radiation-induced micronuclei in B-lymphocytes after staining with Acridine orange is likely to have the greatest potential in the estimation of low dose radiation exposure.


  1. W, Schmid, 'The micronucleus test', Mutat Res., 31, 9-15(1975)
  2. D.E. Roony and B.H. Czepulkowski, 'Human chromosome preparation essential techniques', Wiley, New York, pp. 50-59(1997)
  3. J.S. Prosser, J.E. Moquet, D.C. Lloyd and A.Edwards, 'Radiation induction of micronuclei in human lymphocytes', Mutat Res., 199, 37-45(1998)
  4. M. Fenech, 'Important variables that influence base-line micronuclus frequency in cytokinesis-blocked lymphocytes-a bio-maker for DNA damage in human populations', Mutat Res., 404, 155-165(1998)
  5. M. Fenech, 'The cytokinesis-block micro-nucleus technique and its application to genetoxicity studies in human populations', Environ Health Perspect., 101, 101-107(1993)
  6. M. Kirsch-Volders, A. Elhajouji, E. Cundari and P. Van Hummelen, 'The in vitro micronucleus test: a multi-endpoint assay to detect simultaneously mitotic delay, apoptosis, chromosome breakage, chromosome loss and non-disjunction', Mutat Res., 392, 19-30(1997)
  7. H. Thierens, A. Vral and de Ridder, 'Biological dosimetry using the micro-nucleus assay for lymphocytes: inter individaul differences in dose response', Health Physics, 61, 623-630(1991)
  8. B. Gollapudi and O.P. Kamra, 'Application of a simple Giemsa-staining methods in the micronucleus test', Mutat. Res., 64, 45-46(1976)
  9. M. Fenech, 'The advantages and disadvantages of the cytokinesis-block micro-nucleus method', Mutat Res., 392, 11-18(1997)
  10. M. Hayashi, T. Sofuni and M. Ishidate, 'An application of acridine orange fluorescent staining to the micronucleus test', Mutat Res., 120, 241-247(1983)
  11. K. Kondo and S. Ozawa, 'Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides', Mutat Res., 278, 109-111(1992)
  12. H. Seki, H. Kanegane, K. Iwai, A. Konno, K.Ohta, A. Yachie, N. Taniguchi and T.Miyawaki, 'Ionizing radiation induces apoptotic cell death in human TcRgamma/delta + T and natural killer cells without detectable P53 protein', Eur J Immunol., 24, 2914-2917(1994)
  13. J. Philippe, H. Louagie, H. Thierens, A. Vral, M. Cornelissen and L. de Ridder, 'Quantification of apoptosis in lymphocyte subsets and effect of apoptosis on apparent expression of membrane antigens', Cytometry, 29, 242-249(1997)<242::AID-CYTO7>3.0.CO;2-D
  14. J.L. Schwartz, J.C. Darr and M.E. Gaulden, 'Survival and PHA stimulation of gamma-irradiated human peripheral blood T-lymphocyte subpopulations', Mutat Res., 107, 413-425(1983)
  15. B. Hogsted, A. Karlsson, I. Bratt and A.H. Page, 'Micronucleus induction in human B and T lymphocytes separated by an immunomagnetic techique', Hereditas., 119, 99-103(1993)
  16. A. Holemen, A. Karlsson, I. Bratt and B. Hogstedt, 'Micronulei and mitotic index in B, T4 and T8 cells treated by mitomycin C and r-irradiation', Mutat Res., 309, 93-99(1994)
  17. A. Vral, H. Thierens, J. Philippe, M. Cornelissen and L. de Ridder, 'Micronucleus frequencies in cytokinesis-blocked human B lymphocytes after low dose gamma-irradiation', Int J Radial Biol., 73, 549-555(1998)
  18. M.S. Sasaki, 'Use of lymphocyte chromosome aberrations in biological dosimetry, Radiation-induced chromosomal damage in man'. pp. 585-604, YN, Liss(1983)
  19. A. Vral, F. Verhaegen, H. Thierens and Ridder, 'The in vitro cytokinesis-block micronucleus assay: a detailed description of an improved slide preparation of micronuclei in human lymphocytes', Mutagenesis, 9, 439-443(1994)
  20. B. Stefano and F. Michael, "Human micronucleus project: International database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei', Environ Mol Mutagen., 37, 31-45(2001)<31::AID-EM1004>3.0.CO;2-P
  21. J.T. MacGregor, C.M. Wehr and R.G. Langlos, 'A simple fluorescent staining procedure for micronuclei and RNA in erythrocytes using Hoechst 33258 and Pyronin Y', Mutat. Res., 120, 269-275(1983)
  22. C. muller, C. Dusseau, P. Calsou and B.Salles, 'Human normal periphral blood B-lymphocytes are deficient in DNA-dependent protein kinase activity due to the expression of a variant form of the Ku86 protein', Oncogene, 26, 1553-1560(1998)
  23. A. Vral, H. Thierens, P. Bryant and L.Ridder, 'A higher micronucleus yield in B-versus T-cells after low-dose gamma-irradiation is not linked with defective Ku86 protein', Int J Radiat. Biol., 77, 329-339(2001)
  24. A.A. Freitas and B.B Rocha, "Lymphocyte lifespans: homeostasis, selection and competiton', Immunology, 14, 25-29(1993)