Biodosimetry for High-Dose Exposures Based on Dicentric Analysis in Lymphocytes Released from the G2-Block by Caffeine

Abstract

High-dose assessments using the conventional dicentric assay are essentially restricted to doses up to 5 Gy and only to lymphocytes that succeed to proceed to first post-exposure mitosis. Since G2-checkpoint activation facilitates DNA damage recognition and arrest of damaged cells, caffeine is used to release G2-blocked lymphocytes overcoming the mitotic index and dicentric yield saturation problems, enabling thus dicentric analysis even at high-dose exposures. Using the fluorescence in situ hybridization technique with telomere and centromere peptide nucleic acid probes, the released lymphocytes, identified as metaphases with decondensed chromosomes following 1.5 h caffeine treatment, show increased yield of dicentrics compared to that obtained in lymphocytes that reach metaphase without G2-checkpoint abrogation by caffeine. Here, a 3-h caffeine/colcemid co-treatment before harvesting at 55 h post-exposure is used so that the dicentric analysis using Giemsa staining is based predominantly on lymphocytes released from the G2-block, increasing thus dicentric yield and enabling construction of a dose-response calibration curve with improved precision of high-dose estimates.