Application of FISH based G2-PCC assay for the cytogenetic assessment of high radiation dose exposures: Potential implications for rapid triage biodosimetry

Abstract

The main goal of this study is to test the utility of calyculin A induced G2-PCC assay as a biodosimetry triage tool for assessing a wide range of low and acute high radiation dose exposures of photons. Towards this initiative, chromosome aberrations induced by low and high doses of x-rays were evaluated and characterized in G2-prematurely condensed chromosomes (G2-PCCs) by fluorescence in situ hybridization (FISH) using human centromere and telomere specific PNA (peptide nucleic acid) probes. A dose dependent increase in the frequency of dicentric chromosomes was observed in the G2-PCCs up to 20 Gy of x-rays. The combined yields of dicentrics and rings in the G2-PCCs showed a clear dose dependency up to 20 Gy from 0.02/cell for 0.1 Gy to 14.98/cell for 20 Gy. Centric rings were observed more frequently than acentric ring chromosomes in the G2-PCCs at all the radiation doses from 1 Gy to 20 Gy. A head-to-head comparison was also performed by FISH on the yields of chromosome aberrations induced by different doses of x-rays (0 Gy -7.5 Gy) in colcemid arrested metaphase chromosomes and calyculin A induced G2-PCCs. In general, the frequencies of dicentrics, rings and acentric fragments were slightly higher in G2-PCCs than in colcemid arrested metaphase chromosomes at all the radiation doses, but the differences were not statistically significant. To reduce the turnaround time for absorbed radiation dose estimation, attempt was made to obtain G2-PCCs by reducing the culture time to 36 hrs. The absorbed doses estimated in x-rays irradiated (0,1,2 and 4 Gy) G2-PCCs after 36 hrs of culture were grossly like that of G2-PCCs and colcemid arrested metaphase chromosomes prepared after 48 hrs of culture. Our study indicates that the shortened version of calyculin A induced G2-PCC assay coupled with the FISH staining technique can serve as an effective triage biodosimetry tool for large-scale radiological/nuclear incidents.

Fig 1. Representative pictures of calyculin A induced G2-PCCs prepared from mock and irradiated lymphocytes with different doses of x-rays.

Dicentric and multicentric chromosomes are indicated by arrows. CF-Compound fragment, TF- Terminal fragment, IF-Interstitial fragment, MC-Multicentric chromosomes, AcR-Acentric ring and CR-Centric ring.

Fig 2. Frequencies of dicentric (A) and ring chromosomes (B) induced by different doses of x-rays in calyculin A induced G2-PCCs.

Yields of dicentric, and ring chromosomes induced by 0.1 to 2 Gy of x-rays are shown in the inserts. Error bars indicate SD of the mean.

Fig 3. Combined yields of dicentric chromosomes and ring chromosomes in calyculin A induced G2-PCCs after different doses of x-rays are shown.

Error bars indicate SD of the mean.

Fig 4. Frequencies x-rays induced centromere positive and negative ring chromosomes observed in G2-PCCs are shown.

Error bars indicate SD of the mean.

Fig 5. Comparative analysis on the yields of x-rays induced unstable chromosome aberrations (dicentrics, rings and fragments) in colcemid arrested metaphase chromosomes (Col) and calyculin A (Cal A) induced G2-PCCs performed in the blood samples of two donors are shown.

Error bars indicate SEM.

Fig 6

(A). Percentages of G2 cells detected in the lymphocyte cultures harvested at different times following treatment for 30 min with calyculin A. Cultures harvested at 36 hrs and 40 hrs showed the enrichment of cells with G2-PCCs. (B) Frequencies of dicentric chromosomes detected in calyculin A induced G2-PCCs after fixation of lymphocytes at 36 hrs and 48 hrs relative to conventional colcemid arrested metaphase chromosomes prepared from lymphocytes cultured for 48 hrs. Error bars indicate SEM.