Establishment of in vitro Calibration Curve for 60Co-γ-rays Induced Phospho-53BP1 Foci, Rapid Biodosimetry and Initial Triage, and Comparative Evaluations With γH2AX and Cytogenetic Assays.

A rapid and reliable method for biodosimetry of populations exposed to ionizing radiation in the event of an incident or accident is crucial for initial triage and medical attention. DNA-double strand breaks (DSBs) are indicative of radiation exposure, and DSB-repair proteins (53BP1, γH2AX, ATM, etc.) are considered sensitive markers of DSB quantification. Phospho-53BP1 and γH2AX immunofluorescence technique serves as a sensitive, reliable, and reproducible tool for the detection and quantification of DSB-repair proteins, which can be used for biological dose estimations. In this study, dose-response curves were generated for 60Co-γ-rays induced phospho-53 Binding Protein 1 (phospho-53BP1) foci at 1, 2, 4, 8, 16, and 24 h, post-irradiation for a dose range of 0.05-4 Gy using fluorescence microscopy. Following ISO recommendations, minimum detection limits (MDLs) were estimated to be 16, 18, 25, 40, 50, and 75 mGy for dose-response curves generated at 1, 2, 4, 8, 16, and 24 h post-irradiation. Colocalization and correlation of phospho-53BP1 and γH2AX were also measured in irradiated peripheral blood lymphocytes (PBLs) to gain dual confirmation. Comparative evaluation of the established curve was made by γH2AX-immunofluorescence, dicentric chromosome assay (DCA), and reciprocal translocation (RT) assays by reconstructing the dose of 6 dose-blinded samples. Coefficients of respective in-house established dose-response curves were employed to reconstruct the blind doses. Estimated doses were within the variation of 4.124%. For lower doses (0.052 Gy), phospho-53BP1 and γH2AX assays gave closer estimates with the variation of -4.1 to + 9% in comparison to cytogenetic assays, where variations were -8.5 to 24%. For higher doses (3 and 4 Gy), both the cytogenetic and immunofluorescence (phospho-53BP1 and γH2AX), assays gave comparable close estimates, with -11.3 to + 14.3% and -10.3 to -13.7%, variations, respectively.

Protocol for one-step selective lysis of red blood cells and platelets with long-term preservation of white blood cells (human) at ambient temperature.

Current protocols for storage of white blood cells (WBCs) rely on constant refrigeration. The protocol described below explains the preparation of a fixative combination saline (FCS) formulation, which allows fixation of human WBCs and lysis of red blood cells and platelets (at ambient temperature, 4-35oC) in whole blood samples in one step. FCS can be used for storing and transporting blood at ambient temperatures for up to 4 months, without altering the nuclear morphology and genomic integrity of WBCs.

Biodosimetry for high dose accidental exposures by drug induced premature chromosome condensation (PCC) assay.

The conventional dicentric assay does not provide an accurate dose estimate in the case of accidental exposure to ionizing radiation above 6 Gy due to mitotic delay and poor mitotic index. The present study aims to establish a simple and rapid dose assessment technique based on scoring of rings and fragments in PCC spreads of stimulated lymphocytes. Human peripheral blood lymphocytes were gamma irradiated to different doses (6.2-24.5 Gy), cultured for two days with PHA and were forced to condense prematurely using 500 nM Okadaic acid (OA). The chromosome spreads were prepared, stained with Giemsa and observed under a microscope. The PCC index, PCC rings, and PCC fragments were scored for each dose point to arrive at the dose effect curve for various end points such as induction of rings and fragments and dicentrics. The PCC index varied from 12-18% up to 18 Gy and thereafter dropped to 6-8% at higher doses. The dose dependent increase in rings and fragments was found to be linear with a slope of 0.054+/-0.001 Gy(-1) for rings and 0.45+/-0.03 Gy(-1) for PCC fragments. An experiment was carried out to simulate partial-body exposure by mixing 10 Gy in vitro irradiated blood with un-irradiated blood in different proportions. The ratio of frequency of damaged cells among the total number of cells analyzed was found to be a good index of partial-body exposure. The culture duration was extended to 72 h to overcome the cell cycle delay induced by high doses of radiation. The conventional dicentrics rings and fragments also showed a dose response at high doses. The response can be best fitted to a linear model with a slope of 0.28+/-0.0007 Gy(-1) for the induction of dicentrics. However, long culture duration, technical skill and time required to analyse multi-aberrant cells makes the dicentric assay less suitable for high dose exposures requiring a rapid dose estimate. The PCC assay can be performed in 50 h with biodosimetric information about the irradiated fraction in cases of acute radiation exposures. The automated finding of PCC spreads significantly increased the speed of scoring PCC fragments.

Multifaceted applications of pre-mature chromosome condensation in radiation biodosimetry.

BACKGROUND: Biodosimetry with persistent cytogenetic indicators in peripheral blood lymphocytes (PBLs) plays crucial role in regulatory/medical management of individuals overexposed to radiation. Conventional methods require ∼48 h culture and have limited dose range (0.1-5Gy) applications due to checkpoint arrest/poor stimulation. G0-Phase Premature chromosome condensation (G0-PCC) allows chromosome aberration analysis within hours after blood collection. Due to high skill demand, applications of G0-PCC were not very well explored and being re-visited worldwide. Among all aberrations, analysis of excess chromosomal fragments is quickest. Radiation dose response curve for the fragments has been reported. PURPOSE: In present study, excess fragment analysis has been addressed in detail, in addition to validation of radiation dose response curve, gender variation in the response, dose dependent repair kinetics, minimum detection limit (MDL), duration and accuracy of final dose estimation with 5blindfolded, ex vivo irradiated samples have been studied. In extension, feasibility of multiparametric dosimetry with Fluorescent in situ hybridization (FISH) based endpoints were qualitatively explored. MATERIAL AND METHODS: PBLs were exposed to Gamma-Radiation and G0-PCC was performed at different time points. Decay kinetics and dose response curve were established. Gender Variation of the frequency of the fragments was assessed at 0, 2 and 4 Gy. FISH was performed with G0-PCC applying centromere probe, whole chromosome paints, multi-color FISH and multi-color banding probes. RESULTS: Radiation response curve for fragments was found to be linear (Slope 1.09 ± 0.031 Gy-1). Background frequency as well as dose response did not show significant gender bias. Based on variation in background frequency of fragments MDL was calculated to be ∼0.3 Gy. Kinetics of fragment tested at 0, 4, 8, 16 and 24 h showed exponential decay pattern from 0 to 8 h and without further decay. Final dose estimation of five samples was completed within 13 man-hours. Dicentric chromosomes, translocations, insertions and breaks were identifiable in combination with centromere FISH and WCP. Advanced methods employing multicolor FISH and multi-color banding were also demonstrated with PCC spreads. CONCLUSION: G0-PCC, can be useful tool for high dose biodosimetry with quick assessment of fragment frequency. Further, it holds potential for multi-parametric dosimetry in combination with FISH.

Standardization of CalyculinA induced PCC assay and its advantages over Okadaic acid PCC assay in Biodosimetry applications.

OBJECTIVE: In the present study an attempt was made to estimate coefficients of dose response curves for PCC aberrations induced by CalyculinA and Okadaic acid, using 60Co gamma radiation and 8 MeV pulsed electron beam for biodosimetry application. MATERIALS AND METHODS: The modified method outlined by Puig et al. 2013 was used to conduct Calyculin A and Okadaic acid induced PCC assay in human blood lymphocytes.Chemical treatment was given for the last 1 h of a 48 h culture. The study was carried out in the dose range 2.5 to 20 Gy using 60Co gamma rays and 8 MeV pulsed electron beam. RESULTS AND CONCLUSIONS: Results show a linear dose dependent increase with a slope of 0.047 ± 0.001 from Calycalin A PCC and 0.048 ± 0.002 form Okadaic acid PCC. The slope of the fragments curve was 0.327 ± 0.006 from Calyculin A and 0.328 ± 0.006 from Okadaic acid PCC. Further, dose calibration studies were carried out for 8 MeV electron using Calyculin A PCC assay and the obtained slope from ring yield was 0.054 ± 0.002 and 0.427 ± 0.009 from fragment yield.

Differential modifying effects of food additive butylated hydroxytoluene toward radiation and 4-nitro-quinoline 1-oxide-induced genotoxicity in yeast.

The modifying effect of butylated hydroxytoluene (BHT) on 60Co gamma radiation and 4-nitro-quinoline 1-oxide-induced gene conversion and back mutation frequencies was investigated using diploid yeast Saccharomyces cerevisiae D7. Cells were exposed to 100 or 400 Gy in the presence of 0.025-0.25 mM BHT. BHT exhibited radioprotection and significantly reduced radiation-induced gene conversion and back mutation frequencies as well as cell killing. In another set of experiments, cells were simultaneously treated with 0.025-0.1 mM BHT and 0.5 µM 4-NQO. BHT significantly enhanced 4-NQO-induced gene conversion and back mutation frequencies. BHT post-treatment did not modify radiation-induced genetic events but enhanced 4-NQO-induced back mutation frequencies, indicating its potential to act as a tumor-promoting agent with 4-NQO.