RECENT TECHNIQUES IN CYTOGENETIC BIODOSIMETRY

1. RECENT TECHNIQUES IN CYTOGENETIC BIODOSIMETRY Güçlü İ., Dörter G., Kırbaşoğlu Ç., Dalcı D. deniz.dalci@taek.gov.tr Çekmece Nuclear Research and Training Center

2. CONTENT • CHROMOSOME ABERRATION ANALYSIS • MICRONUCLEI ASSAY • FLOURESCENCE IN SITU HYBRIDISATION (FISH) • APPLICATION OF CYTOGENETIC BIODOSIMETRY IN TURKEY

3. Most of the mutagenic and carcinogenic agents induce chromosomal aberrations in-vivo and iv-vitro • Spontaneous and induced chromosome aberrations have been studied over more than a century.

4. The resolution of detection of aberrations is depended on the improvement of available techniques. Chromosome aberration (CA) analysis has been used as Biological dosimeter for actual or suspected radiation exposures since mid 1960s.

5. In the mid 1980s a major technical innovation was introduced. • This is a method for blocking cytokinesis in cultured lymphocytes by adding cytochalasin-B to the medium. • This method without inhibiting nuclear division creates so called cytokinesis blocked (CB or MN) cells.

6. The stable translocations persist many years after exposure to radiation. • The technique of highlighting translocations is called fluorescence in-situ hybridization (FISH). • Scientific articles describing the production and use of FISH were first published in 1986. • FISH has opened up a re-examination of possibility that translocation yield can be used as • a biological dosimeter when blood samples are taken many years after the exposure.

7. The wide use of radioactive sources and X-rays, for medical, industrial, agricultural, research and military purposes • increases the risk of overexposure of radiation workers and individuals of general population. • Biological dosimetry based on the study chromosome aberration frequencies in human peripheral blood lymphocytes are often used for assessing exposure to clastogenic agents and ionizing radiation. • The analysis of unstable chromosome exchanges such as dicentrics or centricrings which are lost during cell division are useful for quantifying recent and acute exposures to different radiation qualities.

8. In the investigation of radiation accidents it is important to estimate the dose to exposed person for several reasons. • In case of high exposures (>1 Gy acute) information on doses • assists in the planning of therapy and alerting physician to likely deterministic health consequences that could arise in following/subsequent weeks and months.

9. For exposures below the levelwhere treatment is needed, • dosimetric information is important for the physician in counseling irradiated persons on the risk their developing late stochastic diseases i.e. cancer.

10. For persons whose exposures is very low (<50 mGy), the knowledge that no significant elevation in chromosomal damage could be found is frequently very reassuring. • This is particularly the case where details of events are poorly known and no physical dose measurements or calculations are available.

11. CHROMOSOME ABERRATION ANALYSIS • Chromosome aberration (CA) analysis of peripheral blood lymphocytes has been the only method routinely used in the biological dosimetry of exposure to ionizing radiation. • The dicentrics and centric rings are the aberration of choice because of easily scored, control level is low and caused of radiation. • The lymphocytes are chosen because it is easily obtained from a blood sample and it remains in the body for a relatively long time without dividing.

12. Calibration curves relating dicentric yields to absorbed dose exist for many radiation qualities. • The effect of dose rate on the shape of the calibration curves is also well known. • In addition it is possible, following a brief exposure, to detect serious non-uniformity in distribution of dose over body. • Overall an average absorbed to the body of about 0.1 Gy of γ rays can be detected.

13. The CA analysis works well, when the irradiation is more or less uniform over the whole body and • the blood sample is taken just after exposure. • The disadvantage of the dicentrics is that it does not persist in circulating peripheral blood lymphocytes because it is unstable aberration. • Therefore cells with dicentrics disappear with a half-life thought to be in three years.

14. Figure A: Picture of normal metaphase cell • Figure B: Picture of irradiated metaphase cell carrying dicentric Figure A: Picture of normal metaphase cell Figure B: Picture of irradiated metaphase cell carrying dicentric

16. MICRONUCLEI ASSAY • Micronuclei (MN) are small round bodies found in cytoplasm outside the main nucleus which they • resemble in shape, structure and staining properties so called micronuclei in binucleate cytokinesis blocked lymphocytes. • MN arises from acentric fragments or be formed by entire chromosomes that lag behind during mitosis due to a failure of mitotic spindle, or by • complex chromosomal configurations that pose problems during anaphase. • Thus, formation of MN can be induced by both clastogenic agents and mitotic inhibitors.

17. Numerous studies have been performed on the chemical induction of MN in-vitro human lymphocytes. • Because of its simplicity, low cost and speed of evaluation. • The MN test attractive for Biodosimetry • when screening large populations happen to exposed to radiation, • hypersensitivities to radiation or clastogenic agents or rapid estimation of acute radiation of overexposures when serious health are expected. • This assay is however comparatively less sensitive than dicentric measurement. 

19. FLOURESCENCE IN SITU HYBRIDISATION (FISH) • A recognized drawback of the dicentrics and MN assay is that the damage is unstable and • therefore is eliminated from the peripheral blood lymphocyte pool at the rate that cell renewal occurs. • Unstable chromosome exchanges such as dicentrics and rings can be easily detected and quantified by employing solid staining such as Giemsa.

20. However, symmetrical exchanges such as translocations, which are more persistent types of damage, cannot be detected accurately by solid staining. • Chromosome banding techniques, which became available in late 1960s, • could be employed for detection translocations of but they are time consuming, labor intensive and not practical, when large number of cells is to be scored.

21. The technique employs specific sequences of DNA which can be used as probes to particular part of the genome and then attachment of various fluorochromes to highlight the regions in different colours. • Translocations are seen as coloured rearrangements in a fluorescence microscope.

22. Picture A and B: FISH stained metaphase cells with and without translocation.

23. Application of FISH techniques has revealed the complexity of the types of chromosomal aberrations that are formed following irradiation. • Combination of probes specific to different regions of a chromosome has enabled us to detect and quantify the different types of aberrations. • The combined detection of translocations and dicentrics will increase the sensitivity of biological dosimetry. • In view of an expected correlation between the frequencies of translocations and past exposure, it is clear that FISH based translocation measurements can provide to extent useful information for a retrospective biodosimetric interpretation.

24. APPLICATION OF CYTOGENETIC BIODOSIMETRY IN TURKEY • ÇNAEM Biodosimetry laboratory was established in 1988, • provides routinely CA analysis to evaluate irradiated people or suspected of being over exposed to ionizing radiation and • person who applied to get licenses for importing medical or industrial radiological instruments. • It is well established dicentric and MN analysis techniques since 1988 and for FISH analysis since 1998. • Standard procedures and methods are used according to IAEA technical Report No: 405. • The laboratory was established own dose-response curves for gamma rays according to own laboratory working conditions in 1990’s.

25. The dose is estimated by using the observed yields of unstable chromosomal aberrations with standard dose response curve established by our laboratory following in-vitro irradiation of human lymphocytes for Co-60 γ rays. • ÇNAEM Biodosimetry laboratory serves to all of Turkey. • In Turkey radiation sources and radioactive materials have been used mainly in industry and research. • Until now more than 500 people’s CA analyses have been made.