Date Published: May 6, 2019
Publisher: Public Library of Science
Author(s): Terri L. Ryan, Antonio G. Pantelias, Georgia I. Terzoudi, Gabriel E. Pantelias, Adayabalam S. Balajee, Roberto Amendola.
A sensitive biodosimetry tool is required for rapid individualized dose estimation and risk assessment in the case of radiological or nuclear mass casualty scenarios to prioritize exposed humans for immediate medical countermeasures to reduce radiation related injuries or morbidity risks. Unlike the conventional Dicentric Chromosome Assay (DCA), which takes about 3–4 days for radiation dose estimation, cell fusion mediated Premature Chromosome Condensation (PCC) technique in G0 lymphocytes can be rapidly performed for radiation dose assessment within 6–8 hrs of sample receipt by alleviating the need for ex vivo lymphocyte proliferation for 48 hrs. Despite this advantage, the PCC technique has not yet been fully exploited for radiation biodosimetry. Realizing the advantage of G0 PCC technique that can be instantaneously applied to unstimulated lymphocytes, we evaluated the utility of G0 PCC technique in detecting ionizing radiation (IR) induced stable and unstable chromosomal aberrations for biodosimetry purposes. Our study demonstrates that PCC coupled with mFISH and mBAND techniques can efficiently detect both numerical and structural chromosome aberrations at the intra- and inter-chromosomal levels in unstimulated T- and B-lymphocytes. Collectively, we demonstrate that the G0 PCC technique has the potential for development as a biodosimetry tool for detecting unstable chromosome aberrations (chromosome fragments and dicentric chromosomes) for early radiation dose estimation and stable chromosome exchange events (translocations) for retrospective monitoring of individualized health risks in unstimulated lymphocytes.
Exposure to ionizing radiation (IR) can adversely affect human health including mortality from acute radiation syndrome with a LD50/30 value of 3.5 Gy-4.5 Gy without treatment and 6.5 Gy- 7.5 Gy with appropriate therapy. Therefore, it is imperative to determine the absorbed radiation dose to initiate appropriate medical countermeasures. Bender and Gooch (1962) reported for the first time that the detection of dicentric chromosomes (DCs) in peripheral blood lymphocytes can be reflective of the absorbed radiation dose in exposed humans. Since then, Dicentric Chromosome Assay (DCA) has been routinely used for radiation dose assessment of either occupationally or accidentally exposed humans. DCA was effectively utilized in the past for radiation dose assessment in the victims of many well-known accidents such as Chernobyl [1–3], Goiania [4–6] and Fukushima-Daiichi [7–9]. The conventional DCA requires the stimulation of T-lymphocytes in vitro by Phytohaemagglutinin-M (PHA-M) for at least 48 hrs with an additional time of 24–48 hrs for cell fixation, DC analysis and radiation dose estimation. Although DCA is considered as the “gold standard” for radiation dose assessment, its labor intensive and time-consuming nature make DCA largely unsuitable for mass casualty incidents. Rapid individualized dose assessment is an absolute requirement for segregating people with moderate or high radiation exposure from non-exposed but “worried- well” population so that individuals who need urgent care can be prioritized for treatment.
The G0 PCC-FISH technique is distinctly advantageous because the turnaround time for radiation dose assessment is dramatically reduced (6–8 hrs after the receipt of blood samples relative to 72–96 hrs by the conventional DCA). Previous studies on G0 PCC have focused mainly on dicentric chromosomes for radiation dose assessment [34, 35, 41]. The current study was undertaken to determine the applicability of PCC-FISH technique for detecting both inter- and intra-chromosomal aberrations using a wide variety of multicolor DNA probes.
DCA is considered to be the gold standard for radiation dose assessment but it requires proliferation of lymphocytes in vitro for 48 hrs to obtain metaphase chromosomes for analysis. Therefore, a turnaround time of 3–4 days is required at minimum for DCA to estimate an individual’s absorbed radiation dose. Further, it is somewhat difficult to perform conventional DCA for radiation doses higher than 5 Gy because lymphocytes, owing to their radiation sensitivity, may fail to proliferate or undergo apoptotic death at high radiation doses. Realizing the time consuming and laborious nature of DCA, an alternative method was developed by preparing prematurely condensed chromosomes in unstimulated human G0 lymphocytes to detect IR induced chromosomal aberrations. As the PCC assay bypasses the need for lymphocyte proliferation, chromosomal aberrations induced by a wide range of radiation exposures up to 20 Gy can be easily measured within 6–8 hrs of blood collection. PCC assay has been successfully utilized in earlier studies for analyzing IR induced dicentric chromosomes, rings and excess chromosome fragments [31, 32, 34, 43–45]. Recent studies have assessed the utility of G0 PCCs for triage biodosimetry by using the FISH technique with telomere and centromere specific PNA probes [35, 37, 41]. Based on the ex vivo data obtained, Lamadrid Boada et al.,  suggested the use of PCC-rings for dose estimation in severely exposed humans. Interestingly, the frequency of rings remained essentially the same in ex vivo irradiated lymphocytes irrespective of the post-recovery times used for PCC preparation (8 hrs and 24 hrs). Recently, Pantelias and Terzoudi  developed an automatable micro-PCC assay for rapid individualized dose estimation during large-scale radiological emergencies. This technique will enhance the practical applicability of G0 PCC as an effective triage tool.