Methylation Status of IGF-Axis Genes in the Placenta of South Indian Neonates with Appropriate and Small for Gestational Age.

OBJECTIVE: Altered methylation patterns of insulin-like growth factor (IGF)-axis genes in small for gestational age (SGA) have been reported in different populations. In the present study, we analyzed the methylation status of IGF-axis genes in the placenta of appropriate for gestational age (AGA) and SGA neonates of South Indian women. METHODS: Placental samples were collected from AGA (n = 40) and SAG (n = 40) neonates. The methylation of IGF-axis genes promoter was analyzed using MS-PCR. RESULTS: IGF2, H19, IGF1, and IGFR1 genes promoter methylation was 2.5, 1.5, 5, and 7.5% lower in SGA compared to AGA, respectively. Co-methylation of IGF-axis genes promoter was 40% and 20% in AGA and SGA, respectively. IGF-axis gene promoter methylation significantly (p < 0.05) influenced the levels of IGFBP3 protein, birth weight, mitotic index, gestational weeks, and IGFR1 and IGFR2 gene expression. CONCLUSION: IGF-axis genes methylation was lower in SGA than in AGA, and the methylation significantly influenced the IGF-axis components.

Insights on the radiation-induced adaptive response at the cellular level and its implications in cancer therapy.

BACKGROUND: Development of resistance upon exposure to small doses of ionizing radiation followed by higher doses is known as radiation-induced adaptive response (RIAR). Traditionally, the induction of the RIAR phenomenon at the cellular level has been examined in cell lines, animal models, and epidemiological studies where people live in high natural background radiation. SUMMARY: The primary intention of the earlier studies was to corroborate the existence of RIAR and the mechanism involved in mediating the response surveyed by exposure to a low dose of radiation (<500 mGy) as priming dose towards the radiation protection point of view. However, the investigation has shifted the focus to understand the relevance of this phenomenon at clinically relevant set-ups (high doses in the order of Gy) and can be exploited during radiotherapy as RIAR is considered a mechanism for the development of radioresistance. Although the knowledge of molecular mechanisms at the cellular level has evolved significantly in multi-fractionated radiotherapy regimes, its relevance in developing radioresistance at low doses remains elusive. The authors recapitulate the existing knowledge on RIAR at cellular levels, specifically after low-dose exposure as an adaptive dose, and discussed its potential implications in clinical radiotherapy outcomes. KEY MESSAGES: Recent studies contributed to understand the signaling molecules, pathways, and inhibitors to mitigate RIAR-mediated radiation resistance and persistent radio-tolerance at the cellular level. Monitoring the disease progression in tumor samples or liquid biopsies before, during, and after therapy with suitable biomarkers has been proposed as a strategy to translate the phenomena into clinical scenario.

Candidate Gene Expression in Regional Population and Its Relevance for Radiation Triage.

Quantification of gene expression signatures has been substantiated as a potential and rapid marker for radiation triage and biodosimetry during nuclear emergencies. Similar to the established biodosimetry assays, the gene expression assay has drawbacks such as being highly dynamic and transient, not specific to ionizing radiation, and also influenced by confounding factors such as gender, health status, lifestyle, and inflammation. In view of that, prior knowledge of baseline expression of certain candidate genes in a population could complement the discrimination of the unexposed from the exposed individuals without the need for individual pre-exposure controls. We intended to establish a baseline expression of reported radiation-responsive genes such as CDKN1A, DDB2, FDXR, and PCNA in the blood samples of healthy human participants and then compare it with diabetic/hypertension participants (as a chronic inflammatory condition) drawn from south Indian population. Further, we have examined the appropriateness of the assay for radiation triage-like situations; i.e., the expression profiles of those genes were examined in the participants who underwent X-ray-based medical imaging. Acute inflammation induced by lipopolysaccharide exposure in the blood significantly increased the fold expression of those genes (p < 0.0001) compared to the control. Whereas the basal expression level of those genes among the participants with the inflammatory condition is marginally higher than those observed in the healthy participants; despite the excess, the fold increase in those genes between the groups did not differ significantly. Consistent with the inflammatory participants, the basal expression level of those genes in the blood sample of participants who received X-radiation during neuro-interventional and computed tomography imaging is marginally higher than those observed in the pre-exposure of respective groups. Nevertheless, the fold increase in those genes did not differ significantly as the fold change fell within the two folds. Thus, overall results suggest that the utility of CDKN1A, DDB2, FDXR, and PCNA gene expression for radiation triage specific after very low-dose radiation exposure needs to be interpreted with caution for a much more reliable triage.

The Role of Insulin-like Growth Factor-Axis and Mitotic Index in South Indian Neonates with Small for Gestational Age.

OBJECTIVE: IGF-axis and mitotic capacity of cells play a vital role in fetal growth. We compared IGF1, IGF2, and IGFBP3 protein levels, mitotic indices, IGFR1 and IGFR2 mRNA expression in appropriate for gestational age (AGA) and small for gestational age (SGA) neonates of Indian women. METHODS: Cord blood (n = 80) and placental samples (n = 40) were collected from AGA and SGA neonates. Plasma IGF1, IGF2, and IGFBP3 proteins were measured by ELISA. IGFR1 and IGFR2 mRNA expression in the placenta were analyzed by qRT-PCR. Cord blood was cultured in vitro and mitotic index was obtained. RESULTS: IGF1 (p = 1), and IGF2 (p = 0.69) protein levels did not differ, whereas IGFBP3 (p = 0.02) was significantly less in SGA compared to AGA neonates. Down-regulation of IGFR1 (3.9-folds) and IGFR2 (2.8-folds) mRNA and reduced mitotic index of lymphocytes was observed in SGA (p = 0.001) compared to AGA neonates. CONCLUSION: Our results showed that, SGA neonates displayed down-regulated IGFR1 and IGFR2 mRNA, decreased IGFBP3 protein and mitotic index.

Effect of chronic sleep deprivation and sleep recovery on hippocampal CA3 neurons, spatial memory and anxiety-like behavior in rats.

Sleep deprivation-induced degenerative changes in the brain lead to the impairment of memory, anxiety, and quality of life. Several studies have reported the effects of sleep deprivation on CA1 and dentate gyrus regions of the hippocampus; in contrast, there is less known about the impact of chronic sleep deprivation (CSD) and sleep recovery on CA3 neurons and behavior. Hence, the present study aimed to understand the effect of CSD and sleep recovery on hippocampal CA3 neurons and spatial memory, and anxiety-like behavior in rats. Sixty male rats (Sprague Dawley) were grouped as control, environmental control (EC), CSD, 5 days sleep recovery (CSD + 5D SR), and 21 days sleep recovery (CSD + 21D SR). CSD, CSD + 5D SR and, CSD + 21D SR group rats were sleep deprived for 21 days (18 h/day). After CSD, the CSD + 5D SR and CSD + 21D SR rats were sleep recovered for 5- and 21-days respectively. Oxidative stress, dendritic arborization of CA3 neurons, spatial memory, and anxiety-like behavior was assessed. Spatial memory, basal, and apical dendritic branching points/intersections in hippocampal CA3 neurons were reduced, and anxiety-like behavior and oxidative stress increased significantly in the CSD group compared to control (p < 0.001). The CSD + 21D SR showed a significant improvement in spatial memory, reduction in anxiety-like behavior, and oxidative stress when compared to the CSD group (p < 0.05). The basal and apical dendritic branching points/intersections in hippocampal CA3 neurons were increased after CSD + 21D SR, however, it was not significant (p > 0.05). Even though the CSD + 21D SR showed a significant improvement in all the parameters, it did not reach the control level. There was an improvement in all the parameters after CSD + 5D SR but this was not significant compared to the CSD group (p > 0.05). Overall results indicate that the CSD-induced impairment of spatial memory and anxiety-like behavior was associated with oxidative stress and reduced dendritic arborization of hippocampal CA3 neurons. The CSD + 21D SR significantly reduced the damage caused by CSD, but it was not sufficient to reach the control level.

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.

Measurement of γ-H2AX foci, miRNA-101, and gene expression as a means to quantify radiation-absorbed dose in cancer patients who had undergone radiotherapy.

Radiological accidents and nuclear terrorism pose an increased threat to members of the public who, following such an event, would need to be assessed for medical care by fast triage. Assay methods such as chromosome aberrations (CA), cytokinesis-block micronucleus (CBMN) and fluorescence in situ hybridization (FISH) techniques have been well established for dose estimation and their potential for handling more samples has also been proved with automation. However, culturing of lymphocytes is an inevitable step, which limits the potential of these markers for triage. In vitro analysis of gamma-H2AX (γ-H2AX), gene and microRNA (miRNA) markers do not require culturing of lymphocytes, and as such have been suggested as attractive tools for triage. Despite studies reporting in vitro dose-response curves, limited evidence is available evaluating the suitability of these assays in real situations. In this study, we have measured the absorbed dose using γ-H2AX, gene (GADD45A, FDXR, and CDKN1A) and miRNA-101 expression in blood samples of cancer patients (n = 20) who had undergone partial-body radiotherapy and compared with the derived equivalent whole-body doses (EWBD). The obtained results from all patients showed a significant (p < 0.05) increase of γ-H2AX foci in post-irradiated as compared to pre-irradiated samples. Moreover, estimated doses using γ-H2AX foci showed a correlation with the derived EWBD (r2 = 0.60, p = 0.0003) and was also shown to be dependent on the irradiated body volume. Consistent with γ-H2AX foci frequency, an increase in fold change expression of genes and miRNA-101 was observed. However, the estimated dose significantly varied among the subjects and showed poor correlation (r2 = 0.09, 0.04, 0.01 and 0.03 for GADD45A, FDXR, CDKN1A and miRNA-101, respectively) with EWBD. The overall results suggest that the established in vitro γ-H2AX assay is suitable for the detection of radiation exposure and can also provide an estimate of the dose in in vivo irradiated samples. The genes and miRNA-101 markers showed increased expression; nevertheless, there is a need for further improvements to measure doses accurately using these markers.

Frequency of gamma H2AX foci in healthy volunteers and health workers occupationally exposed to X-irradiation and its relevance in biological dosimetry.

Gamma-H2AX (γ-H2AX) assay is a marker to measure double-strand breaks in the deoxyribonucleic acid. Variables such as age, oxidative stress, temperature, genetic factors and inter-individual variation have been reported to influence the baseline γ-H2AX focus levels. Therefore, knowledge on baseline frequency of γ-H2AX foci in a targeted population would facilitate reliable radiation triage and dose estimation. The objective of the present study was to establish the baseline data using blood samples from healthy volunteers (n = 130) differing in age, occupation and lifestyle as well as from occupationally exposed health workers (n = 20). The γ-H2AX focus assay was performed using epifluorescence microscopy. In vitro dose-response curve for γ-H2AX foci was constructed in blood samples (n = 3) exposed to X-rays (30 min post-exposure). The mean γ-H2AX focus frequency obtained in healthy volunteers was 0.042 ± 0.001 and showed an age-related increase (p < 0.001). Significantly higher (p < 0.005) focus frequencies were observed in health workers (0.066 ± 0.005) than in healthy volunteers. A sub-group analysis did not show a significant (p > 0.1) difference in γ-H2AX focus frequency among sexes. Blood exposed in vitro to X-rays showed dose-dependent increase in γ-H2AX foci frequency (Y = 0.1902 ± 0.1363 + 2.9020 ± 0.3240 * D). Baseline frequency of γ-H2AX foci obtained from different age groups showed a significant (p < 0.01) influence on the dose-response coefficients. The overall results demonstrated that the γ-H2AX assay can be used as a reliable biomarker for radiation triage and estimating the radiation absorbed dose by considering variables such as age, occupation and lifestyle factors.

Impact of chronic sleep deprivation and sleep recovery on hippocampal oligodendrocytes, anxiety-like behavior, spatial learning and memory of rats.

Sleep and its quality play an important role in memory, cognition, and quality of life. Sleep deprivation-induced changes in hippocampal neurons and behavior have been studied widely, in contrast, the extent of damage to oligodendrocytes have not been fully understood. The present study aims to investigate chronic sleep deprivation (CSD) and sleep recovery-induced changes in oligodendrocytes of the hippocampus, cognition, and behavior of rats. Male Sprague-Dawley rats (n = 48) were grouped as control, sham control (SC), CSD, and CSD+sleep recovery (CSD+SR) (n = 12/group). CSD and CSD+SR group rats were sleep deprived for 21-days. After CSD, the CSD+SR group rats sleep recovered for 21-days. Oxidative markers, CNPase+ve oligodendrocytes, CNPase intensity, and CNPase gene expression were measured in the hippocampus, and the anxiety-like behavior, spatial learning, and memory were assessed. The 21-days of CSD significantly (p < 0.001) increased oxidative stress and significantly (p < 0.001) reduced the number of CNPase+ve oligodendrocytes, CNPase intensity, and CNPase gene expression when compared to controls. The increased oxidative stress was correlated with reduced CNPase+ve oligodendrocytes, CNPase intensity, and CNPase gene expression (r = -0.9). In-line with cellular changes, an increased (p < 0.01) anxiety-like behavior and impaired spatial memory were observed in the CSD group compared to controls. The 21-days of sleep recovery significantly (p < 0.01) reduced oxidative stress and anxiety-like behavior, improved spatial memory, increased CNPase intensity and CNPase gene expression, and non-significant (p > 0.05) increase in CNPase+ve oligodendrocytes compared to CSD. Overall, the 21-days of CSD reduced the number of CNPase+ve oligodendrocytes in the hippocampus, increased anxiety, and impaired spatial memory in rats. Though the 21-day sleep recovery showed an improvement in all parameters, it was not sufficient to completely reverse the CSD-induced changes to the control level.

Impact of X-radiation in the management of COVID-19 disease.

Coronaviruses are a diverse group of viruses that infect both animals and humans. Even though the existence of coronavirus and its infection to humans is not new, the 2019-novel coronavirus (nCoV) caused a major burden to individuals and society i.e., anxiety, fear of infection, extreme competition for hospitalization, and more importantly financial liability. The nCoV infection/disease diagnosis was based on non-specific signs and symptoms, biochemical parameters, detection of the virus using reverse-transcription polymerase chain reaction (RT-PCR), and X-ray-based imaging. This review focuses on the consolidation of potentials of X-ray-based imaging modality [chest-X radiography (CXR) and chest computed tomography (CT)] and low-dose radiation therapy (LDRT) for screening, severity, and management of COVID-19 disease. Reported studies suggest that CXR contributed significantly toward initial rapid screening/diagnosis and CT- imaging to monitor the disease severity. The chest CT has high sensitivity up to 98% and low specificity for diagnosis and severity of COVID-19 disease compared to RT-PCR. Similarly, LDRT compliments drug therapy in the early recovery/Less hospital stays by maintaining the physiological parameters better than the drug therapy alone. All the results undoubtedly demonstrated the evidence that X-ray-based technology continues to evolve and play a significant role in human health care even during the pandemic.

Potential application of γ-H2AX as a biodosimetry tool for radiation triage.

Radiation triage and biological dosimetry are two initial steps in the medical management of exposed individuals following radiological accidents. Well established biodosimetry methods such as the dicentric (DC) assay, micronucleus (MN) assay, and fluorescence in-situ hybridization (FISH) translocation assay (for residual damage) have been used for this purpose for several decades. Recent advances in scoring methodology and networking among established laboratories have increased triage capacity; however, these methods still have limitations in analysing large sample numbers, particularly because of the ∼ 48 h minimum culture time required prior to analysis. Hence, there is a need for simple, and high throughput markers to identify exposed individuals in case of radiological/nuclear emergencies. In recent years, a few markers were identified, one being phosphorylated histone 2AX (γ-H2AX), which measured a nuclear foci or nuclear staining intensity that was found to be suitable for triage. Measurement of γ-H2AX foci formed at and around the sites of DNA double-strand breaks is a rapid and sensitive biodosimetry method which does not require culturing and is thus promising for the analysis of a large number of samples. In this review, we have summarized the recent developments of γ-H2AX assay in radiation triage and biodosimetry, focusing chiefly on: i) the importance of baseline frequency and reported values among different laboratories, ii) the influence of known and unknown variables on dose estimation, iii) quality assurance such as inter-laboratory comparison between scorers and scoring methods, and iv) current limitations and potential for future development.

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.

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.

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.