LYMHOCYTE CHROMOSOMAL ABERRATION ASSAY IN RADIATION BIODOSIMETRY

Transcription

1 LYMHOCYTE CHROMOSOMAL ABERRATION ASSAY IN RADIATION BIODOSIMETRY Dr. Birutė Gricienė 1,2 1 Radiation Protection Centre 2 Vilnius University

2 Introduction Ionising radiation is a well-known mutagenic and carcinogenic agent. The intensive use of ionizing radiation sources and development of radiation technology is related with increased exposure risk. In the case of nuclear or radiological event it is important to identify exposed individuals in order to determine whether the medical intervention is needed In many emergency cases the dose assessments can only be conducted using biological assays. Chromosomal aberrations assay in peripheral blood lymphocyte is a most reliable, sensitive and mostly used biological dosimetry method for assessing radiation dose.

3 Biological indicators commonly used for biodosimetry Rate of decline in lymphocytes Absorbed dose Future health risk Immediate Medical Management Long-term effects Cancer risk Hereditary effects J. Albanese, PhD Building Connecticut s clinical biodosimetry laboratory surge capacity to mitigate the health consequences of radiological and nuclear disasters 17th Nuclear Medical Defence Conference, 2007

4 DNA Chromosomes The target of induction of aberrations is DNA

5 Ionising radiation damage in DNA IR can induce various types of damage in DNA: single strand breaks, double strand breaks base damage, DNA-DNA cross links DNA-protein cross links The most important - double strand breaks, they directly forms chromosome aberations

6 1) Mutation repaired No effects 2) Cell dies Deterministic Effects (>~1Sv) 3)Cell survives but mutated Stochastic effects

7 Radiation Effects on lymphocytes Undamaged cells contain 46 chromosomes, each with 1 centromere Damaged cells may display a number of types of aberrations including dicentrics, centric rings, acentric fragments and translocations, all of which may be related to radiation dose Abnormal chromosomes showing fragments, rings and dicentrics due to exposure to radiation

8 Clases of chromosome aberrations Chromosome type aberrations -dicentrics -rings -translocations -acentric fragments result if cell is irradiated early in interphase (G1), before chromosome material has been duplicated Chromatide type aberrations -exchanges -breaks -result if cell is irradiated in S and G2 stages

9 Cytogenetic biomarkers of ionising radiation exposure Citogenetic biomarkers: Unstable chromosome aberrations Reciprocal translocations Micronuclei Sister chromatid exchanges Chromosome aberrations Micronuclei The most sensitive and widely used method unstable chromosome aberation analysis in peripheral blood lymphocytes Sister chromatid exchanges Translocations

10 Chromosome aberration analysis. Phytohaemagglutinin stimulated cultures incubated at 37 C for hours in RPMI 1640 medium Peripheral blood samples collected by venipuncture into blood collection tubes. The harvested lymphocytes treated with hypotonic KCl (0.075 M) and then fixed in methanol-glacial acetic acid (3:1) Slides containing methaphase spreads stained with Giemsa or FISH probes Chromosomes analysed by optical microscope

11 No. of dicentrics/cell Determination of absorbed dose using dicentric analysis The frequency of dicentric chromosomes in lymphocytes gives a good approximation of radiation dose Neutrons x-rays -rays By comparing the number of dicentric chromosomes in peripheral blood lymphocytes to that of lymphocytes irradiated in vitro, radiation dose estimates can be derived. Radiation Dose

12 Production of gamma radiation (Co-60) dose-response curves In 2009 was produced gamma radiation dose-response curve at Vilnius University, faculty of Nature sciences. Experiment was designed as follows: blood samples from three donors were irradiated with Co-60 (0,256 Gy/min) to various doses (0.5-4 Gy). Samples were cultured according to the standard procedures first cycle metaphases were analysed for each radiation dose per donor after the preparation and staining of the slides. For fitting a linear-quadratic dose-response relationship the CABAS software was used. Gricienė B, Slapšytė G, Mierauskienė J. Gama spinduliuote sukeltų chromosomų aberacijų dozės ir atsako kreivės sudarymas bei panaudojimas biologinei dozimetrijai. Visuomenės sveikata. 2009;1:19-23.

13 No of dicentric chromossome/cell Production of gamma radiation (Co-60) doseresponse curves Dose-response curve showed a linearquadratic response Y= D+ D 2 +c, where Y is the frequency of dicentrics, D is the dose, c is the background frequency. the linear coefficient 0.056±0.005, the dose squared coefficient β 0.075± There is a good agreement between our dose-response curve with similar published studies. Curve has been used for biological dose estimation in two cases of suspected overexposure of radiation workers Absorbed dose, Gy Dose-response curve for induction of dicentrics Y 0, D 0.076D 2 Gricienė B, Slapšytė G, Mierauskienė J. Gama spinduliuote sukeltų chromosomų aberacijų dozės ir atsako kreivės sudarymas bei panaudojimas biologinei dozimetrijai. Visuomenės sveikata. 2009;1:19-23.

14 Application of lymhocyte chromosomal aberration assay in radiation biodosimetry Dose-response curves are used: For accidental exposure dose assessment (Chernobyl NPP, 1986; Brazil, 1987; Estonia, 1994; Japan, 1999 ir kt.) For epidemiological studies to investigate absorbed doses long time after exposure (Japan A bomb survivors, exposed persons after Chernobyl NPP accident) For determination of individual radiosensitivity

15 Strengths and weakness of the dicentric assay Dicentric assay considered to be the gold standard for estimating radiation dose specific to ionizing radiation low level of occurrence in unirradiated persons (about 1/1000 cells) sensitive to 0.1 Gy exhibits dose-response relationship number of dicentrics chromosomes increase with radiation dose Weakness: dicentric analysis is time consuming Voisin et al Why can t we find a better biological indicator of Dose. Radiation Protection Dosimetry, 112:

16 Limitations of scoring dicentrics Major limitation of using dicentrics for dosimetry is loss of lymphocytes from the blood Yield of measured dicentrics after an irradiation would decrease with time (1/2 life is about 3 yrs), although this is uncertain When replaced by stem cell division, new lymphocytes will tend not to contain dicentrics due to elimination at anaphase unstable aberration since it is lethal to the cell

17 FISH method and translocations Cells which contain balanced translocations tend not to be eliminated at cell division Translocations can persist in the body for a long time Difficult to see in a conventional preparation, but easy to observe with Flourescence in situ hibridisation (FISH) Since this is a stable aberration, the advantage of this method is its application to persons exposed to radiation at some time in the past Reciprocal translocation ucker.pdf

18 National Biological Dosimetry laboratory establishment at Radiation Protection Centre It is important that every country would have the laboratory, which could perform biological dosimetry (IAEA, 2001) Currently the Radiation Protection Centre prepared the national IAEA project for establishment of biological dosimetry laboratory in It is planed to be used for public and occupational accidental exposure dose assessment, epidemiological studies and radiosensitivity studies of patients.

19 Summary Ionising radiation induces chromosome aberrations at all stages of the cell cycle. Since in vitro and in vivo irradiation of lymphocytes induces similar yields of chromosome damage per unit dose, the absorbed dose to individual can be estimated by comparing the aberration yield to calibration curve generated in vitro. Biological dosimetry based on chromosome aberration analysis can be used for accidental exposure dose assessment, radiosensitivity studies of radiotherapy patients. Methods are available to perform biodosimetry using dicentric frequencies following the ionising radiation exposure. Dicentrics are unstable aberrations and eliminated with time. FISH technique using chromosome specific libraries has proven to be very efficient to detect translocations, which are stable and persist in the body for many years and allows estimating absorbed dose from past accidents.