C. Loss of heterozygosity (LOH) (see theallele difference in this figure)
4. Hybridization time should be for M- FISH at least 60 h, while for MCB/ mBAND probes 16 h is normally suffi cient
5. Reliable pseudocolor depictions like those shown in Fig. 1 can- not always be achieved in mFISH . In case of poor hybridiza- tion quality several candidate chromosomes may by suggested to be involved in a certain rearrangement after molecular karyotyping . This can be helpful to continue with only two or three specifi c whole chromosome painting probes to verify the M-FISH results. Also features provided by the software should be used to establish experiment-specifi c pseudocolors. For MCB/mBAND also the possibility of creating fl uorochrome profi les along the analyzed chromosomes (Fig. 1A -2) should be used besides the multicolor- banding feature.
Acknowledgments
Supported in parts by the Wilhelm Sander Stiftung (2013.032.1) and German academic exchange service (DAAD).
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Thomas S.K. Wan (ed.), Cancer Cytogenetics: Methods and Protocols, Methods in Molecular Biology, vol. 1541, DOI 10.1007/978-1-4939-6703-2_17, © Springer Science+Business Media LLC 2017
Chapter 17
Cytogenetics for Biological Dosimetry
Michelle Ricoul , Tamizh Gnana-Sekaran , Laure Piqueret-Stephan , and Laure Sabatier
Abstract
Cytogenetics is the gold-standard in biological dosimetry for assessing a received dose of ionizing radiation.
More modern techniques have recently emerged, but none are as specifi c as cytogenetic approaches, par- ticularly the dicentric assay. Here, we will focus on the principal cytogenetic techniques used for biological dosimetry: the dicentric assay in metaphase cells, the micronuclei assay in binucleated cells, and the prema- ture condensed chromosome (PCC) assay in interphase cells. New fl uorescence in situ hybridization (FISH) techniques (such as telomere–centromere hybridization) have facilitated the analysis of the dicen- tric assay and have permitted to assess the dose a long time after irradiation by translocation analysis (such as by Tri-color FISH or Multiplex-FISH). Telomere centromere staining of PCCs will make it possible to perform dose assessment within 24 h of exposure in the near future.
Key words Chromosomal aberrations , Radiation effects , Biological dosimetry , Telomere , Centromere
1 Introduction
Biological dosimetry is routinely performed for the estimation of the absorbed dose in the individuals exposed to radiation. Radiation is a form of energy that comes from naturally occurring radionu- clides or man-made sources. One of the earliest and most direct methods of dose determination following radiation exposure is the recording of daily counts of various cell types circulating in the peripheral blood ; the extent and duration of the decline and subse- quent recovery of specifi c cell-types correlate well with the received dose [ 1 ] despite poor sensitivity. Biological samples are used to quantify radiation damage, hence the term biological dosimetry.
There are many biological indicators of radiation exposure such as mutations [ 2 – 4 ], gene expression [ 5 ], cytogenetics [ 6 ], proteins such as γ -H2AX [ 7 ], metabolic intermediates [ 8 ], and proteomics [ 9 ]. Cytogenetic biomarkers are considered to be the most sensi- tive and reliable of the various biological indicators reported to quantify the absorbed dose of radiation. The radiation absorbed by
exposed cells can induce DNA strand breaks on the chromosomes, which are subsequently repaired by the DNA repair machinery of the cell. Misrepaired breaks can result in abnormal chromosome structures. Various types of abnormal chromosomes can be identi- fi ed and counted, of which the number is related to the dose, pro- viding a reliable dose-effect relationship.
The most common aberration is the dicentric chromosome (DC). It is an aberrant chromosome with two centromeres which is formed when two chromosome segments, each with a centro- mere, fuse end to end, with rejoining or not of their acentric frag- ments. DCs are unstable, highly specifi c to ionizing radiation, and can be used to estimate the unknown absorbed dose during a radi- ation emergency by counting their frequency [ 6 ]. Biodosimetry based on DC counts can also discriminate between whole and par- tial body exposures as DC formation is not infl uenced by any other factors. The background frequency is very low (0.001/cell) and the sensitivity of the DC assay is 0.1 Gray (Gy), hence it is the
“gold standard” for biodosimetry applications [ 6 ]. The assay has been used in many accidental incidents in Chernobyl [ 10 ], Istanbul [ 6 ], Goiania [ 11 ], and Bangkok [ 12 ]. However, it is time consum- ing, laborious, and requires skilled and highly trained personnel to score the chromosomal aberrations. The other principal cytoge- netic marker is micronuclei (MN). These are formed from lagging chromosomal fragments or whole chromosomes at anaphase which are not included in the nuclei of daughter cells. They are seen as distinct, separate, and small spherical objects in the cytoplasm of the daughter cells with the same morphology and staining proper- ties as nuclei [ 13 ]. MN refl ect chromosomal damage and are a useful index for monitoring environmental effects on genetic mate- rial in human cells [ 14 ]. This assay has been shown to be a promis- ing and potential tool for triage in the medical management of a nuclear emergency due to its simplicity and the rapidity of scoring.
However, its sensitivity is only 0.25 Gy due to a spontaneous MN frequency of 0.002–0.036/cell [ 6 ]. This assay was used in the Chernobyl [ 14 ] and Istanbul [ 15 ] radiation accidents. The above- mentioned techniques require an incompressible culture time and the report can only be generated after a minimum of 72 h. During a mass radiation exposure event, the potentially exposed individu- als cannot wait for 72 hr to start treatment. Thus, a technique was introduced by Johnson and Rao [ 16 ] in which the mitotic cells of Chinese hamster ovary (CHO) cells induce the condensation of chromosomes in interphase cells of lymphocytes following fusion using polyethylene glycol or Sendai virus [ 16 ]. This technique allows the study of radiation-induced damage without stimulating the cells and the aberrations can be assessed within 2 h of exposure [ 17 ]; thus, the chances of losing information due to interphase cell death are reduced. Another advantage of this assay is that it can be used for high dose (>5 Gy) estimations because conventional
cytogenetic dosimetry based on the frequency of chromosomal aberrations becomes diffi cult due to mitotic delay and the disap- pearance of lymphocytes in peripheral blood circulation [ 18 , 19 ].
The minimum dose detection limit of this technique is 0.05 Gy [ 6 ]. The premature condensed chromosome (PCC) assay using peripheral blood lymphocytes (PBLs) is recommended as a rapid method for biodosimetry [ 20 ]. This technique was also performed on three seriously exposed victims of the Tokaimura criticality acci- dent in Japan [ 21 ].
Potential scenarios of radiation exposure resulting in mass casualties require individual, early, and defi nitive radiation dose assessment to provide medical aid within days of the occurrence of a disaster. The preliminary dose estimation and segregation of exposed and nonexposed individuals are the main steps in triage medical management. Biological dosimetry in “triage” mode must provide an answer as quickly as possible. A rough estimate of the dose is suffi cient as long as it permits the classifi cation of the vic- tims into three categories that will guide medical follow up (<1 Gy, 1–2 Gy, and >2 Gy). Alternative strategies are being developed to meet the demands of triage. These include the use of automated scoring, as manual scoring of classical cytogenetic methods (DC, MN, and PCC) is time consuming.
The quantifi cation of DC is not reliable for retrospective dosimetry (>1 year after exposure), as they decrease by 50 % with each cell division. Stable chromosome rearrangements, such as translocations, are mostly scored using Tri-color FISH . Multiplex- FISH (M-FISH) can permit the detection of translocations involv- ing any chromosome but it is time consuming and expensive. Some approaches are focused on the detection of radiation-induced inversions, the most stable chromosomal aberrations, using cross- species FISH (RxFISH) or directional genomic hybridization [ 22 ].
Sharing of the workload among expert groups (i.e., the European RENEB (Realizing the European Network of Biodosimetry) network [ 23 ], the IAEA RANET (International Atomic Energy Agency Response and Assistance Network), REMPAN (Radiation Emergency Medical Preparedness and Assistance Network), and WHO BioDoseNet (World Health Organization biodosimetry network) is necessary, especially for tri- age. It also permits expert training, protocol harmonization, and dissemination of up-to-date developments, such as the automation of analytical methods and the use of early markers of ionizing radi- ation that are among the most recent advances in biodosimetry . In this chapter, we will focus on the principal techniques used for biological dosimetry in cytogenetics consisting of the dicentric assay and translocation assays in metaphase cells, the micronuclei assay in bi-nucleate cells, and the premature condensed chromo- some ( PCC ) assay in interphase cells.
2 Materials