Assessment of Micronuclei Frequency in the Peripheral Blood of Adult and Pediatric Patients Receiving Fractionated Total Body Irradiation.

The cytokinesis-block micronucleus (CBMN) assay is an established method for assessing chromosome damage in human peripheral blood lymphocytes resulting from exposure to genotoxic agents such as ionizing radiation. The objective of this study was to measure cytogenetic DNA damage and hematology parameters in vivo based on MN frequency in peripheral blood lymphocytes (PBLs) from adult and pediatric leukemia patients undergoing hematopoietic stem cell transplantation preceded by total body irradiation (TBI) as part of the conditioning regimen. CBMN assay cultures were prepared from fresh blood samples collected before and at 4 and 24 h after the start of TBI, corresponding to doses of 1.25 Gy and 3.75 Gy, respectively. For both age groups, there was a significant increase in MN yields with increasing dose (p < 0.05) and dose-dependent decrease in the nuclear division index (NDI; p < 0.0001). In the pre-radiotherapy samples, there was a significantly higher NDI measured in the pediatric cohort compared to the adult due to an increase in the percentage of tri- and quadri-nucleated cells scored. Complete blood counts with differential recorded before and after TBI at the 24-h time point showed a rapid increase in neutrophil (p = 0.0001) and decrease in lymphocyte (p = 0.0006) counts, resulting in a highly elevated neutrophil-to-lymphocyte ratio (NLR) of 14.45 ± 1.85 after 3.75 Gy TBI (pre-exposure = 4.62 ± 0.49), indicating a strong systemic inflammatory response. Correlation of the hematological cell subset counts with cytogenetic damage, indicated that only the lymphocyte subset survival fraction (after TBI compared with before TBI) showed a negative correlation with increasing MN frequency from 0 to 1.25 Gy (r = -0.931; p = 0.007). Further, the data presented here indicate that the combination of CBMN assay endpoints (MN frequency and NDI values) and hematology parameters could be used to assess cytogenetic damage and early hematopoietic injury in the peripheral blood of leukemia patients, 24 h after TBI exposure.

Comparative analysis of physical doses and biomarker changes in subjects underwent Computed Tomography, Positron Emission Tomography-Computed Tomography, and interventional procedures.

Even though the medical uses of ionizing radiation are well-acknowledged globally as vital tools for the improvement of human health, they also symbolize the major man-made sources of radiation exposure to the population. Estimation of absorbed dose and biological changes after radiation-based imaging might help to better understand the effects of low dose radiation. Because of this, we measured the Entrance Surface Dose (ESD) at different anatomical locations using Lithium tetraborate doped with manganese (Li2B4O7: Mn), recorded Dose Length Product (DLP) and Dose Area Product (DAP), analyzed Chromosomal Aberration (CA), Micronucleus (MN), gamma-H2AX (γ-H2AX), and p53ser15 proteins in the blood lymphocytes of patients (n = 267) underwent Computed Tomography (CT), Positron Emission Tomography-CT (PET/CT), and interventional procedures and healthy volunteers (n = 19). The DLP and effective doses obtained from PET/CT procedures were significantly higher (p < 0.05) when compared to CT. Fluoroscopic time and DAP were significantly higher (p < 0.05) in therapeutic compared to diagnostic interventional procedures. All the anatomical locations registered a significant amount of ESD, the ESD obtained from CT and interventional procedures were significantly (p < 0.05) higher when compared to PET/CT. Fluoroscopic time did not correlate with the ESD (eye, head, thyroid, and shoulder; R2 = 0.03). CA frequency after PET/CT was significantly higher (p < 0.001) when compared to CT and interventional procedures. MN frequency was significantly higher in 24-hs (p < 0.001) post-interventional procedure compared to 2-hs. The mean ± SD of mean fluorescence intensity of γ-H2AX and p53ser15 obtained from all subjects underwent PET/CT and interventional procedures did not show a significant difference (p > 0.05) between pre- and post-procedure. However, the relative fluorescence intensity of γ-H2AX and p53ser15 was >1 in 58.5 % and 65.8 % of subjects respectively. Large inter-individual variation and lack of correlation between physical dose and biomarkers suggest the need for robust dosimetry with a large sample size to understand the health effects of low dose radiation.

The relative biological effectiveness of high-energy clinical 3 and 6 MV X-rays for micronucleus induction in human lymphocytes.

PURPOSE: In the modern era of radiotherapy, use of conventional radiation modalities (based on γ-rays) is being replaced by high-energy linear accelerator-based X-rays. As a result of mishandling of equipment or mechanical errors, health workers can be exposed to these high-energy X-rays. Especially in the absence of personnel monitoring devices, biodosimetry with a lower energy X-ray calibration curve may not provide an acceptable dose estimate. Moreover, the relative biological effectiveness (RBE) value assigned for X-rays is the same (ONE) regardless of beam energy (V), employed in diagnosis, interventional medicine, and radiotherapy. Therefore, the purpose of the study is to examine the induced biological effects, measured through micronucleus (MN) formation, of X-rays of different energies (3 and 6 MV X-rays), and to investigate the RBE relative to 225 kVp X-rays. MATERIALS AND METHODS: Peripheral blood lymphocytes (PBLs) from healthy donors (n = 6), were irradiated with 225 kVp, 3 MV, and 6 MV energy X-rays and induced biological damage was quantified as MN formation using the cytokinesis blocked MN (CBMN) assay. RESULTS: The MN per cell in the X-irradiated samples for the three different X-ray energies showed a significant (p<.0001) dose-dependent increase, when compared to unexposed samples. Aberration frequencies obtained at the same dose for the three different energies showed significant (p<.05) difference for the MN per cell among the energy levels; however, the in vitro dose-response curve parameters (slope, intercept, and coefficient) did not show any significant differences. The estimated dose in the blinded sample was within the 95% confidence intervals of each of the calibration curves. However, overall, the 6 MV dose-response curve coefficients yielded the closest dose estimate to that of the true dose. The calculated RBE values at 5% induced MN for 3 and 6 MV LINAC X-rays were 2.0 ± 0.04 and 0.70 ± 0.01, respectively, and the average RBE for the complete dose-response curves were 1.13 ± 0.04 and 0.80 ± 0.02 relative to 225 kVp X-rays as standard radiation. CONCLUSION: The established dose-response curves obtained for PBL exposed to different energy levels of X-rays of 225 kVp, 3 MV, and 6 MV are ready to use for biological dosimetry purposes. The calculated RBE values for the higher energies of X-rays relative to 225 kVp X-rays in this study suggest that RBE of X-rays may not be equal to one, with the true value dependent on the beam energy, the dose and dose rate, and the endpoint investigated.

18F-FDG PET/CT scanning: Biological effects on patients: Entrance surface dose, DNA damage, and chromosome aberrations in lymphocytes.

Positron Emission Tomography/Computed Tomography (PET/CT), a combination of PET and CT, is used in tumor staging, therapy planning, and treatment response monitoring. During PET imaging, patients receive low doses of radiation, which can induce an adaptive response and necessitate higher doses for therapeutic efficacy. Higher doses may augment toxicity to normal cells. We are examining the effects of short-term, low-dose exposures to ionizing radiation. Entrance Surface Dose (ESD) to head, shoulders, and pelvis regions were measured using Li2B4O7: Mn thermoluminescent dosimeters. Induced DNA damage in lymphocytes was measured using γ-H2AX, p53Ser-15, chromosome aberrations, and micronucleus formation in subjects (n = 25) who underwent 18F-FDG PET/CT. The mean ESD ± SD value obtained were 32.40 ± 16.86, 32.58 ± 14.22, 32.02 ± 15.42, 43.55 ± 18.25 and 42.80 ± 24.67 mGy for the head, right shoulder, left shoulder, right pelvic, and left pelvic regions, respectively. The effective doses of PET and CT ranged from 4.01 to 6.61 and 16.40-72.18 mSv, respectively, and the obtained Dose Length Product (DLP) varied from 1093 to 4812 mGy*cm. There was no correlation between DLP and ESD (r2 = 0.1). The chromosome aberration assay showed a significant increase (p < 0.05), post-scanning vs. pre-scanning; the γ-H2AX, p53Ser-15, and micronucleus assays did not show significant increases. Induced DNA damage showed inter-individual variation among the study subjects. Our results imply that the patients received a biologically significant dose during 18F-PET/CT scanning and precautions may be needed to reduce any long-term risk of exposure.

Association between occupational heat stress and DNA damage in lymphocytes of workers exposed to hot working environments in a steel industry in Southern India.

Occupational heat stress apart from adverse heat-related health consequences also induces DNA damage in workers exposed to high working temperatures. We investigated the association between chronic heat exposures and Micronuclei (MN) frequency in lymphocytes of 120 workers employed in the steel industry. There was a significant increase in the MN-frequency in exposed workers compared to the unexposed workers (X2 = 47.1; p < 0.0001). While exposed workers had higher risk of DNA damage (Adj. OR = 23.3, 95% CI 8.0-70.8) compared to the unexposed workers, among the exposed workers, the odds of DNA damage was much higher for the workers exposed to high-heat levels (Adj. OR = 81.4; 95% CI 21.3-310.1) even after adjusting for confounders. For exposed workers, years of exposure to heat also had a significant association with higher induction of MN (Adj. OR = 29.7; 95% CI 2.8-315.5). Exposures to chronic heat stress is a significant occupational health risk including damages in sub-cellular level, for workers. Developing protective interventions to reduce heat exposures is imperative in the rising temperature scenario to protect millions of workers across the globe.

Does proliferation capacity of lymphocytes depend on human blood types?

In vitro human lymphocyte culture methodology is well established yet certain confounding factors such as age, medical history as well as individual's blood type may potentially modulate in vitro proliferation response. These factors have to be carefully evaluated to release reliable test report in routine cytogenetic evaluation for various genetic conditions, radiation biodosimetry, etc. With this objective, the current study was focused on analyzing the proliferation response of lymphocytes drawn from 90 individuals (21-29 years) with different blood types. The proliferation response was assessed in the cultured lymphocytes by cell cycle, mitotic index (MI), and nuclear division index (NDI) after stimulation with phytohaemagglutinin (PHA). To investigate the toxic effect on proliferation, MI was calculated in representative samples of each blood type were X-irradiated. The results showed that there was no significant difference among the cell cycle phases of lymphocytes in different blood types (P > 0.05). Similarly, both MI and NDI of lymphocytes derived from different blood types also did not show significant difference ( P > 0.05). The extensive interindividual variation within and among the blood types is likely responsible for the lack of significant difference in lymphocyte proliferation. Although spontaneous proliferation efficiency of lymphocytes of different blood types after PHA stimulation was grossly similar, the MI observed after radiation exposure showed a significant difference ( P < 0.05) indicating a differential proliferation response among the blood types. Our results suggest that the blood types did not have any impact on PHA-induced proliferation; however, a specific differential lymphocyte proliferation observed after radiation exposure needs to be considered.

Entrance surface dose and induced DNA damage in blood lymphocytes of patients exposed to low-dose and low-dose-rate X-irradiation during diagnostic and therapeutic interventional radiology procedures.

The ionizing radiation received by patients and health workers due to radiological imaging may increase the risks of radiation effects, such as cancer and cataracts. We have investigated the dose received by specific areas around the head and related this to DNA damage in the blood lymphocytes of subjects exposed to interventional imaging. The entrance surface doses (ESD) to the forehead, neck, and shoulder were measured with a thermoluminescence dosimeter (CaSO4 disc or polycrystalline powder of lithium tetraborate doped with Mn) and compared with that of dose area product (DAP). DNA damage was measured by γ-H2AX, p53ser15, chromosomal aberration (CA), and micronucleus (MN) assays in lymphocytes of patients (n=75), before and 2 and 24h after exposure. The measured ESD values were 230.5±4.9, 189.5±3.55 and 90.7±3.4mGy for the forehead, neck, and shoulder, respectively. The DAP varied from 1.8 to 2047 Gy*cm2, showing a correlation with fluoroscopy time (r=0.417). Received doses did not increase early markers of DNA damage (γ-H2AX and p53ser15 assays), but residual damage (CA and MN frequencies) showed a significant (p<0.001) increase at 2 and 24h post-exposure compared to pre-imaging, despite poor correlation with DAP (r=0.1). Our results show that interventional imaging procedures deliver significant radiation doses and induce measurable DNA damage in lymphocytes of subjects, highlighting the need for rigorous patient safety protocols.

Radiation signature on exposed cells: Relevance in dose estimation.

The radiation is considered as a double edged sword, as its beneficial and detrimental effects have been demonstrated. The potential benefits are being exploited to its maximum by adopting safe handling of radionuclide stipulated by the regulatory agencies. While the occupational workers are monitored by personnel monitoring devices, for general publics, it is not a regular practice. However, it can be achieved by using biomarkers with a potential for the radiation triage and medical management. An ideal biomarker to adopt in those situations should be rapid, specific, sensitive, reproducible, and able to categorize the nature of exposure and could provide a reliable dose estimation irrespective of the time of the exposures. Since cytogenetic markers shown to have many advantages relatively than other markers, the origins of various chromosomal abnormalities induced by ionizing radiations along with dose-response curves generated in the laboratory are presented. Current status of the gold standard dicentric chromosome assay, micronucleus assay, translocation measurement by fluorescence in-situ hybridization and an emerging protein marker the γ-H2AX assay are discussed with our laboratory data. With the wide choice of methods, an appropriate assay can be employed based on the net.

Assessment of dose and DNA damages in individuals exposed to low dose and low dose rate ionizing radiations during computed tomography imaging.

PURPOSE: Computed tomography (CT) is a frequently used imaging modality that contributes to a tenfold increase in radiation exposure to the public when compared to other medical imaging modalities. The use of radiation for therapeutic need is always rationalized on the basis of risk versus benefit thereby increasing concerns on the dose received by patients undergoing CT imaging. Therefore, it was of interest to us to investigate the effects of low dose and low dose-rate X-irradiation in patients who underwent CT imaging by recording the doses received by the eye, forehead and thyroid, and to study the levels of damages in the lymphocytes in vivo. MATERIALS AND METHODS: Lithium manganese borate doped with terbium (LMB:Tb) thermo luminescence dosimeters (TLD) were used to record the doses in the patient's (n = 27) eye, forehead, and thyroid and compared with the dose length product (DLP) values. The in vivo DNA damages measured were compared before and after CT imaging using chromosomal aberration (CA) and micronucleus (MN) assays. RESULTS: The overall measured organ dose ranged between 2 ± 0.29 and 520 ± 41.63 mGy for the eye, 0.84 ± 0.29 and 210 ± 20.50 mGy for the forehead, and 1.79 ± 0.43 and 185 ± 0.70 mGy for the thyroid. The in vivo damages measured from the blood lymphocytes of the subjects showed an extremely significant (p < 0.0001) increase in CA frequency and significant (p < 0.001) increase in MN frequency after exposure, compared to before exposure. CONCLUSION: The results suggest that CT imaging delivers a considerable amount of radiation dose to the eye, forehead, and thyroid, and the observed increase in the CA and MN frequencies show low dose radiation effects calling for protective regulatory measures to increase patient's safety. This study is the first attempt to indicate the trend of doses received by the patient's eye, forehead and thyroid and measured directly in contrast to earlier values obtained by extrapolation from phantoms, and to assess the in vivo low dose effects in an Indian patient population undergoing CT procedures.