Differential killing and radio-modifying effects of iodoacetate in mammalian normal and cancer cells.

To explore possible applications of iodoacetate (IA), a glycolytic inhibitor, in cancer treatment, we screened its cytotoxicity and radioprotective/sensitizing efficacy in three different mammalian cell lines; A549 (human lung carcinoma), MCF7 (human mammary cancer), a non-cancerous CHO (Chinese hamster ovary) cells and human lymphocytes. Experiments were carried out using IA concentrations ranging from 0.01 to 2.5 µg/ml, with or without 60Coγ-radiation. In the outcomes, IA was found to exhibit higher toxicity in the cancer cells, whereas it was non-toxic/marginally toxic to the non-cancerous cells. Considerably higher glucose uptake in both cancer cells lines was observed indicating higher rates of glycolysis. IA significantly inhibited glycolysis as reflected by GAPDH activity inhibition. Radiomodifying effects of IA were found to be concentration dependent in both cancerous and non-cancerous cells. The response in non-cancerous was found to be biphasic: at lower concentrations, it offered significant radioprotection; however, the protection decreased with increasing concentration. Moreover, at the highest tested concentration, marginal radiosensitization was also observed (as indicated by clonogenic assay). In both cancer cells, IA offered significant amount of radiosensitization which was considerably high at higher concentrations. Further experiments were carried out to estimate the Dose Modification Factor (DMF) to quantify and compare relative radiosensitization by IA in cancer and normal cell lines. The DMF was calculated for three different concentrations of IA, 0.5, 1, and 1.5 µg/ml, and corresponding values were found to be 1.26, 1.43, and 1.89 for A549 cancer cells, whereas for normal CHO cells, it was 1.13, 1.13, and 1.24. In conclusion, differential killing and radiosensitizing effects of IA suggest that it may have potential use as a anticancer agent and radiosensitizer in cancer therapy.

G0-PCC-FISH derived multi-parametric biodosimetry methodology for accidental high dose and partial body exposures.

High dose radiation exposures are rare. However, medical management of such incidents is crucial due to mortality and tissue injury risks. Rapid radiation biodosimetry of high dose accidental exposures is highly challenging, considering that they usually involve non uniform fields leading to partial body exposures. The gold standard, dicentric assay and other conventional methods have limited application in such scenarios. As an alternative, we propose Premature Chromosome Condensation combined with Fluorescent In-situ Hybridization (G0-PCC-FISH) as a promising tool for partial body exposure biodosimetry. In the present study, partial body exposures were simulated ex-vivo by mixing of uniformly exposed blood with unexposed blood in varying proportions. After G0-PCC-FISH, Dolphin's approach with background correction was used to provide partial body exposure dose estimates and these were compared with those obtained from conventional dicentric assay and G0-PCC-Fragment assay (conventional G0-PCC). Dispersion analysis of aberrations from partial body exposures was carried out and compared with that of whole-body exposures. The latter was inferred from a multi-donor, wide dose range calibration curve, a-priori established for whole-body exposures. With the dispersion analysis, novel multi-parametric methodology for discerning the partial body exposure from whole body exposure and accurate dose estimation has been formulated and elucidated with the help of an example. Dose and proportion dependent reduction in sensitivity and dose estimation accuracy was observed for Dicentric assay, but not in the two PCC methods. G0-PCC-FISH was found to be most accurate for the dose estimation. G0-PCC-FISH has potential to overcome the shortcomings of current available methods and can provide rapid, accurate dose estimation of partial body and high dose accidental exposures. Biological dose estimation can be useful to predict progression of disease manifestation and can help in pre-planning of appropriate & timely medical intervention.

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.

Thorium promotes lung, liver and kidney damage in BALB/c mouse via alterations in antioxidant systems.

Thorium (232Th), long lived (14.05 billion years) most stable thorium isotope, is thrice naturally abundant than uranium. 232Th occurs as rocky deposits and black monazite sands on the earth's crust geographically distributed in coastal South India and other places globally. Monazite sand comprises of cerium and large quantities of radioactive thorium. The environmental hazard lies in monazite rich area being termed as High Background Radiation Area (HBRA). In this study, we mimicked the HBRA under controlled chamber conditions using thorium oxalate as a thorium source for BALB/c mice exposure. Furthermore, sequential radio-disintegration of 232 Th leads to thoron (220Rn), the noble gas and other daughter products/progeny predominantly via alpha decay/emissions. Such progeny tend to attach to aerosol and dust particles having potential inhalation hazard followed by alpha emissions and damages that we evaluated in mouse lung tissues post thoron inhalation. Secondly, along with the radio disintegration and alpha emission, high energy gamma is also generated that can travel to various distant organs through the systemic circulation, as significant findings of our study as damages to the liver and kidney. The mechanistic findings include the damages to the hematological, immunological and cellular antioxidant systems along with activation of canonical NF-κß pathway via double stranded DNA damage.

Refined premature chromosome condensation (G0-PCC) with cryo-preserved mitotic cells for rapid radiation biodosimetry.

Mitotic cell fusion induced Premature Chromosome Condensation (G0-PCC) assay in human lymphocytes allows rapid detection of cytogenetic damage in interphase stage, within few hours after blood collection. Hence, it is the most suitable method for rapid and high dose biodosimetry. Mitotic cells, used for G0-PCC could be either freshly isolated or previously cryo-preserved. However, under emergency scenarios, only cryo-preserved cells can be relied upon, fresh isolation will only delay the process by 18-24 h. Impact of cryopreservation on mitotic cells and their efficacy to induce PCC are not reported. In the present study, we investigated effect of cryopreservation on mitotic cells and refined the parameters for G0-PCC. More than 95% of the cells were recoverable after 4 months of cryopreservation, within 20 min recovery at 37 °C, without significant change in the mitotic index or viability. Recovered mitotic cells have shown mitotic index of 89 ± 4% and viability of 90 ± 4%, similar to that of freshly isolated cells. Decrease in metaphases was observed within 40 min after recovery as the mitotic cells progressed through cell cycle and reduced to 21% at 1.5 h. Nevertheless, in presence of Colcemid, the cells progressed slowly and considerably high metaphase index (60%) persisted up to ~ 2 h. The recovered cells efficiently fused with lymphocytes and induced PCC. Average PCC index varied from 10 to 20%, which did not change with cryopreservation duration. Post fusion incubation duration of 2 h was found to be optimum for proper chromosome condensation. In conclusion, use of cryo-preserved mitotic cells is the most practical approach for rapid biodosimetry. The cells can be recovered quickly and efficiently without alteration in viability or mitotic index. Recovered cells are fully competent to induce G0-PCC.

Low energy proton beam induces efficient cell killing in A549 lung adenocarcinoma cells.

The aim of the current study was to determine the signaling differences between gamma- and proton beam-irradiations. A549 lung adenocarcinoma cells were irradiated with 2 Gy proton beam or gamma-radiation. Proton beam was found to be more cytotoxic than gamma-radiation. Proton beam-irradiated cells showed phosphorylation of H2AX, ATM, Chk2, and p53. The mechanism of excessive cell killing in proton beam-irradiated cells was found to be upregulation of Bax and downregulation of Bcl-2. The noteworthy finding of this study is the biphasic activation of the sensor proteins, ATM, and DNA-PK and no activation of ATR by proton irradiation.

Lung damage by thoron progenies versus possible damage redemption by lung stem cells: a perspective.

PURPOSE: Natural radiation is the major source of human exposure to ionizing radiation. About 52% of the total dose received from the high natural background radiations (HNBR) areas are due to inhalation dose from radon (222Rn)/thoron (220Rn) and their progenies. Hence, we reviewed the biological effects of 222Rn/220Rn and their progenies on lung tissue, and the possible role of lung stem cells in salvaging the damage caused by 222Rn/220Rn and their progenies. MATERIALS AND METHOD: We have extensively reviewed articles among several hits obtained in PubMed, Scopus, and Elsevier databases with keywords 'Radon/Thoron' OR Thoron progeny/Radon progeny OR 'Thoron/Radon inhalation and lungs', and proceed for further analysis. Also, databases related to oxidative damage to lung stem cells by radiation and the repair mechanisms involved by the lung stem cells were also included. RESULTS: Based on the existing epidemiological data on radon in residential buildings, we found that evidence exists on the association of radon induced lung carcinogenesis, but the data regarding the role of thoron induced lung damage is very limited and inconclusive. We also found that limited information has been provided based on ecological designs, leading to poor documentation of health statistics, in particular, organ-specific cancer rates. Finally, we tried to elucidate the possible mechanisms of lung injury induced by thoron inhalation and the probable role of lung stem cell toward the redemption of such oxidative damages. CONCLUSION: Existing epidemiological data on thoron inhalation and associated health outcomes are limited and inconclusive. Further, in vivo experiments, with respect to radon/thoron inhalation dose rate ranges corresponding to the HNBR areas will be helpful in understanding the cellular and molecular effects.

DOSE-RESPONSE STUDY USING MICRONUCLEUS CYTOME ASSAY: A TOOL FOR BIODOSIMETRY APPLICATION.

The present study is aimed at obtaining in vitro dose-response data for the induction of micronucleus (MN) and nucleoplasmic bridges (NPBs) in human lymphocytes using 60Co-gamma rays and 8 MeV pulsed electron beam. An attempt was made to validate the possibility of applying NPBs as new biodosimetry endpoint in the dose range of 0-6 Gy. A total of 1000 binucleated cells (BNCs) per dose point were evaluated for the frequency of MN and NPBs. From the study, it is clear that the dose-response increase of MN and NPBs is linear quadratic in nature. The study provides linear and quadratic parameter for biodosimetry application. The relative biological effectiveness value of the 8 MeV electron beam was estimated using slope values and is found to be 1.18 ± 0.01 for MN/BNCs, 1.27 ± 0.02 for the fraction of BNCs with MN, 1.16 ± 0.13 for MN/(BNCs with MN) and 1.09±0.01 for NPBs.

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.

Dose rate effect of pulsed electron beam on micronucleus frequency in human peripheral blood lymphocytes.

The micronucleus assay in human peripheral blood lymphocytes is a sensitive indicator of radiation damage and could serve as a biological dosimeter in evaluating suspected overexposure to ionising radiation. Micronucleus (MN) frequency as a measure of chromosomal damage has also extensively been employed to quantify the effects of radiation dose rate on biological systems. Here we studied the effects of 8 MeV pulsed electron beam emitted by Microtron electron accelerator on MN induction at dose rates between 35 Gy min-1 and 352.5 Gy min-1. These dose rates were achieved by varying the pulse repetition rate (PRR). Fricke dosimeter was employed to measure the absorbed dose at different PRR and to ensure uniform dose distribution of the electron beam. To study the dose rate effect, blood samples were irradiated to an absorbed dose of (4.7+/-0.2) Gy at different rates and cytogenetic damage was quantified using the micronucleus assay. The obtained MN frequency showed no dose rate dependence within the studied dose rate range. Our earlier dose effect study using 8 MeV electrons revealed that the response of MN was linear-quadratic. Therefore, in the event of an accident, dose estimation can be made using linear-quadratic dose response parameters, without adding dose rate as a correction factor.