Journal of Radiation Protection and Research

The Korean Association for Radiation Protection (대한방사선방어학회)
권호 표지

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
Download PDF
⟨ Previous Next ⟩

Abstract

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.

방사선에 의해 유도되는 사람 말초혈액 림프구 미소핵 관찰빈도를 높이면서 생물학적 선량평가법으로서 활용 가능성을 확인하고자 본 연구를 수행하였다. 우선 5명의 건강한 사람으로부터 혈액을 제공 받아 선량영역을 0에서 800cGy로 하여 감마선$(^{137}Cs)$을 조사 한 후 김사와 아크리딘 오렌지 형광 염색하고 미소핵 출현빈도를 비교하였다. 아크리딘 오렌지 염색법을 이용하여 미소핵을 관찰하였을 때, 김사 염색법에 비교하여 적갈색의 비특이성 과립과 녹황색의 DNA가 붉은색의 세포질을 배경으로 명확히 구별되었을 뿐만 아니라 선량이 증가하면서 검출율도 높았다. 아울러, 말초혈액 T와 B-림프구에 대하여 선량영역을 0에서 50cGy로 하여 방사선을 조사한 후 미소핵 출현빈도를 아크리딘 오렌지 염색하여 김사염색과 비교한 바, B-림프구에서 선량이 증가하면서 적어도 2배 이상 높게 관찰되었다. 본 실험 결과, 사람 말초 혈액 B-림프구를 대상으로 한 아크리딘 오렌지 형광염색 미소핵 분석법은 저선량 방사선 인체영향 평가나 과피폭 선량추정시 활용이 가능 할 것으로 생각된다.

Keywords

References

  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) https://doi.org/10.1016/0165-7992(83)90096-9
  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) https://doi.org/10.1016/0165-1218(92)90218-O
  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) https://doi.org/10.1002/eji.1830241150
  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) https://doi.org/10.1002/(SICI)1097-0320(19971101)29:3<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) https://doi.org/10.1111/j.1601-5223.1993.00099.x
  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) https://doi.org/10.1080/095530098142103
  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 L.de 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) https://doi.org/10.1093/mutage/9.5.439
  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) https://doi.org/10.1002/1098-2280(2001)37:1<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) https://doi.org/10.1016/0165-7992(83)90100-8
  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) https://doi.org/10.1080/0955300001004237
  24. A.A. Freitas and B.B Rocha, "Lymphocyte lifespans: homeostasis, selection and competiton', Immunology, 14, 25-29(1993)