Global transcriptome profile reveals abundance of DNA damage response and repair genes in individuals from high level natural radiation areas of Kerala coast

PLOS ONE, Nov 2017

The high level natural radiation areas (HLNRA) of Kerala coast in south west India is unique for its wide variation in the background radiation dose (<1.0mGy to 45mGy/year) and vast population size. Several biological studies conducted in this area did not reveal any adverse effects of chronic low dose and low dose rate radiation on human population. In the present study, global transcriptome analysis was carried out in peripheral blood mono-nuclear cells of 36 individuals belonging to different background dose groups [NLNRA, (Group I, ≤1.50 mGy/year) and three groups of HLNRA; Group II, 1.51–5.0 mGy/year), Group III, 5.01-15mGy/year and Group IV, >15.0 mGy/year] to find out differentially expressed genes and their biological significance in response to chronic low dose radiation exposure. Our results revealed a dose dependent increase in the number of differentially expressed genes with respect to different background dose levels. Gene ontology analysis revealed majority of these differentially expressed genes are involved in DNA damage response (DDR) signaling, DNA repair, cell cycle arrest, apoptosis, histone/chromatin modification and immune response. In the present study, 64 background dose responsive genes have been identified as possible chronic low dose radiation signatures. Validation of 30 differentially expressed genes was carried out using fluorescent based universal probe library. Abundance of DDR and DNA repair genes along with pathways such as MAPK, p53 and JNK in higher background dose groups (> 5.0mGy/year) indicated a possible threshold dose for DDR signaling and are plausible reason of observing in vivo radio-adaptive response and non-carcinogenesis in HLNRA population. To our knowledge, this is the first study on molecular effect of chronic low dose radiation exposure on human population from high background radiation areas at transcriptome level using high throughput approach. These findings have tremendous implications in understanding low dose radiation biology especially, the effect of low dose radiation exposure in humans.

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Global transcriptome profile reveals abundance of DNA damage response and repair genes in individuals from high level natural radiation areas of Kerala coast

November Global transcriptome profile reveals abundance of DNA damage response and repair genes in individuals from high level natural radiation areas of Kerala coast Vinay Jain 0 1 Birajalaxmi Das 0 1 0 Low Level Radiation Research Section, Radiation Biology and Health Sciences Division, Bio-Science Group, Bhabha Atomic Research Centre , Trombay, Mumbai , India , 2 Homi Bhabha National Institute , Anushakti Nagar, Mumbai , India 1 Editor: Kamaleshwar P Singh, Texas Tech University , UNITED STATES The high level natural radiation areas (HLNRA) of Kerala coast in south west India is unique for its wide variation in the background radiation dose (<1.0mGy to 45mGy/year) and vast population size. Several biological studies conducted in this area did not reveal any adverse effects of chronic low dose and low dose rate radiation on human population. In the present study, global transcriptome analysis was carried out in peripheral blood mono-nuclear cells of 36 individuals belonging to different background dose groups [NLNRA, (Group I, 1.50 mGy/year) and three groups of HLNRA; Group II, 1.51±5.0 mGy/year), Group III, 5.0115mGy/year and Group IV, >15.0 mGy/year] to find out differentially expressed genes and their biological significance in response to chronic low dose radiation exposure. Our results revealed a dose dependent increase in the number of differentially expressed genes with respect to different background dose levels. Gene ontology analysis revealed majority of these differentially expressed genes are involved in DNA damage response (DDR) signaling, DNA repair, cell cycle arrest, apoptosis, histone/chromatin modification and immune response. In the present study, 64 background dose responsive genes have been identified as possible chronic low dose radiation signatures. Validation of 30 differentially expressed genes was carried out using fluorescent based universal probe library. Abundance of DDR and DNA repair genes along with pathways such as MAPK, p53 and JNK in higher background dose groups (> 5.0mGy/year) indicated a possible threshold dose for DDR signaling and are plausible reason of observing in vivo radio-adaptive response and non-carcinogenesis in HLNRA population. To our knowledge, this is the first study on molecular effect of chronic low dose radiation exposure on human population from high background radiation areas at transcriptome level using high throughput approach. These findings have tremendous implications in understanding low dose radiation biology especially, the effect of low dose radiation exposure in humans. - Data Availability Statement: The database has been submitted to GEO repository under the accession number GSE95279. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Introduction Studying the biological effects of low dose and low dose-rate ionizing radiation (IR) in humans has important implications to human health and radiation protection science. Increasing evidences suggest that the effect of IR at low and high doses of exposures is qualitatively and quantitatively different [ 1,2 ]. At high acute dose exposures, IR may lead to deleterious effects at cellular and molecular level thus leading to adverse consequences like cell death, accumulation of mutations, chromosomal aberrations and carcinogenesis. However, there is still uncertainty regarding the risk/effect due to low dose and low dose rate exposures to humans, below 100 mSv. At such low dose exposures, biological mechanisms such as adaptive response, bystander effects, hypersensitivity and genomic instability may play important role which is not clearly understood yet. Hence, efforts have been made to undertake studies to strengthen future areas of research on biological effects of IR at cellular, molecular and /or single cell level [3]. The linear no threshold (LNT) hypothesis is based on the fact that even the smallest doses of radiation exposure have the potential to cause an increase in health risk to humans. However, the extrapolation of data from high dose exposures to low dose region of the dose response curve is one of the criticisms about LNT. Also, defence mechanisms such as DNA repair processes and elimination of damaged cells have not been taken into consideration for LNT hypothesis [ 4 ]. DNA repair process helps the cells to recover from various types of DNA lesions produced by a spectrum of genotoxic agents including IR. However, the threshold dose at which DNA damage response signal functions at chronic low dose exposures is not yet known. In daily life, humans are exposed to low dose and low dose-rate ionizing radiation from various sources such as natural background, occupational, accidental, medical applications (diagnostic and therapeutic) etc. Natural chronic low dose exposure prevailing in high level natural radiation areas (HLNRA) in the world offers unique opportunity to study the biological effects of low dose radiation directly on humans. The HLNRA of Kerala is a narrow coastal line extending from Neendakara panchayat (Kollam district) in the south to Purakkad panchayat (Allapuzzha district) in the north. It has patchy distribution of monazite in the beach sand containing 8±10% of thorium-232 and therefore the human population (approximately 4,00, 000) is exposed continuously to natural chronic low dose radiation. Because of varied level of background radiation dose (<1.0 to 45.0 mGy/year), this area is suitable for dose response studies. Studying the biological effect of radiation exposure in humans at low dose and low dose-rates especially below 100mSv is highly challenging, but extremely essential for risk estimation. Human population residing in high background radiation areas provide the most appropriate opportunities to study the effect of IR at such low doses as they have been exposed continuously at all stages of development/organogenesis from birth to death. Several investigations have been carried out in this area, which includes studies in wild rats [ 5 ], plants [ 6,7 ], demographic characterization of human population[8]. In addition, epidemiological studies are carried out to find out cancer incidence in adults, congenital anomalies in newborns and a case control study of mental retardation and cleft lip/palate in this population [ 9,10,11,12,13 ]. So far, no significant changes are observed at phenotype level. This population has also been investigated for several biological end points such as chromosome aberrations, micronuclei, telomere length measurement and quantitation of DNA damage [ 14,15,16,17,18,19,20,21,22 ].None of the above DNA damage end points have shown significant difference between the population from HLNRA and the adjacent normal level natural radiation areas (NLNRA). The spontaneous level of DNA double strand breaks (DSBs) has not shown any increase in DSBs, rather showed marginal reduction in HLNRA individuals 2 / 28 belonging to background dose group >5mGy/year [ 18 ]. Interestingly, DNA damage and repair study in peripheral blood mono-nuclear cells (PBMC) using gamma H2AX marker and comet assay revealed that individuals from HLNRA groups (>5.0mGy/year) have better repair efficiency as compared to NLNRA group [ 21,23 ]. Our group has also shown in vivo radioadaptive response among HLNRA group of individuals after a challenge dose of 1.0 and 2.0Gy of gamma radiation[24]. IR induces a spectrum of DNA damages in human cells that gets repaired through efficient DNA repair pathways depending upon the type of lesions. DSBs are highly deleterious. Other damages such as oxidized purines or pyrimidines, abasic sites, single strand breaks and clustered DNA lesions cannot be underestimated because un-repaired or mis-repaired DNA damages may lead to accumulation of mutations, chromosomal aberrations and carcinogenesis. DNA damage response (DDR) may signal a number of cellular and molecular pathways which alter the expression profile of many genes, proteins and regulating miRNAs, that may help in maintaining genome integrity. Studies have shown that human PBMC exposed to moderate doses of acute gamma radiation leads to alteration of expression profile of DDR and DNA repair genes and proteins [ 25,26,27,28,29 ]. There is a growing interest in the emerging areas of low dose radiation biology and development of biomarkers of radiation exposures. Development of new biomarkers may be useful for identifying radiation signatures in population exposed to radiation, accidental exposure situation like Chernobyl and Fukushima Daiichi nuclear disasters, biological dosimetry and population monitoring purposes [ 30,31,32,33,34,35,36 ]. There are established biological end points, which are used as radiation induced markers in the genome such as dicentrics, translocations and inversions. The sensitivity of these assays is low as compared to newly developed high throughput molecular biology techniques. In recent years, sensitive and specific assays such as gamma-H2AX foci, gene expression profile, miRNA and protein profile have emerged as biomarkers for radiation exposure [ 18,37,38,39,40 ]. Global gene expression profile in circulating lymphocytes provides rapid, non-invasive method to identify differential expression of genes in response to a variety of genotoxic agents including IR. There are very few reports available on acute and chronic low dose exposures to human population, where gene expression changes at specific genes are used as radiation signatures [30,32±33,35,36,41±45]. Global transcriptional profiling has been used to gain insights into the molecular mechanisms induced by low dose exposures in a variety of cell types and cell cultures such as human myeloid cells [ 1,46 ], human skin fibroblasts and keratinocytes [ 2,47,48 ], PBMC [ 41,44,49,50 ], umbilical vein endothelial cells [ 51 ], lymphoblastoid cells [ 52,53 ] and human embryonic stem cells [54]. There are reports, which have shown the induction of transcriptional changes after ex-vivo acute exposure to doses as low as 1 cGy [ 55 ]. Studies have also shown changes in transcriptional profile after in vivo low-dose exposures. For instance, Yin et al. (2003) identified several genes with modulated transcript levels after exposures of 10cGy in brain tissue from irradiated mice. However, limited information is available, where in vivo gene expression profile is carried out in human PBMC exposed to chronic low dose radiation such as occupational and accidental exposures [ 41,49,50 ]. Most importantly, till date, no report is available on gene expression profile in PBMC of human population living in high level natural radiation areas. It is also important to find out the threshold dose at which differential gene expression at low doses can be seen in humans. In the present study, attempt has been made to study global gene expression profile in PBMC of individuals from normal and high level natural radiation areas to find out differentially expressed genes and their role in various cellular and molecular pathways in response to chronic low dose and/or low dose rate radiation exposure. 3 / 28 Materials and methods Ethics statement, study design and sample collection Blood samples were collected from 36 healthy male individuals (volunteers) with written informed consent, which was approved by Medical Ethics Committee, Bhabha Atomic Research Centre, Trombay, Mumbai, India. All the methods were carried out in "accordance" with the approved guidelines by the above committee. Approximately, 10 ml of blood samples were collected in sterile EDTA containing vaccutainers (BD vaccutainers systems, U.S.A) from each individual (age range: 28±52 year). Individuals were stratified into four different dose groups based on the annual background dose received by them i.e., Group I (NLNRA, control group 1.5 mGy/year, N = 9), Group II (HLNRA, 1.51±5.0 mGy/year, N = 9), Group III (HLNRA, 5.01±15.0 mGy/year, N = 11) and Group IV (HLNRA, > 15.0 mGy/year, N = 7). All these individuals studied from different dose groups were having similar life style and dietary habits without any chronic illness. The information on age, gender, personal habits such as smoking, chewing, occupation, previous radiation exposure and medical history was collected in a detailed questionnaire. The overall mean age of 36 individuals was 40.4 ±5.7 years (age range: 28±52 years), which was similar to mean age of individuals in different background dose groups. The mean age among the individuals from Group I, Group II, Group III and Group IV was 39.7±4.7, 41.9±6.8, 40.5±6.9 and 39.3±4.3 years, respectively. Individual dosimetry The external gamma-radiation levels were measured using a halogen quenched Geiger Muller (GM) tube-based survey meter (Type ER-709, Nucleonix Systems, India) in each individual's house. The survey meter readings measured absorbed doses in air (μR h-1) due to gamma rays and were converted to annual dose (mGy year-1) using a conversion factor of 0.0765 (= 0.873 ×24 h × 365 days ×10−5). The individual dose contributed by the gamma rays was derived as sum of 0.5 (occupancy factor) × the annual indoor dose and 0.5 (occupancy factor) × the annual outdoor dose. The occupancy factor taken for the calculation of individual dose was based on the sex and age specific occupancy factors estimated in a previous study conducted by Nair et al, 2005 [ 56 ]. Isolation of peripheral blood mononuclear cells (PBMC) PBMC were isolated by density gradient centrifugation using Histopaque-1077 solution (Sigma-aldrich, St. Louis, MO, USA). Equal volume of blood was layered over histopaque solution and centrifuged at 400g for 30 minutes at room temperature. After centrifugation, opaque interface layer containing mono-nuclear cells was carefully separated and washed with chilled isotonic phosphate buffered saline (PBS) and centrifuged at 250g for 10 minutes. The cell pellet was washed twice with PBS. The cells were divided into small aliquots, suspended in RNA later (Sigma-aldrich) and stored atÐ20ÊC until further use. Extraction of total RNA Total RNA was isolated from PBMC using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) as per manufacturer's instructions. Quality and quantity of isolated total RNA was checked using Nano Drop1 ND-1000 spectrophotometer (Nano Drop Technologies, USA). The integrity of isolated RNA was evaluated with microfluidic capillary electrophoresis using the Agilant 2100 Bioanalyzer (Agilant technologies, U.S.A). RNA integrity number (RIN) was calculated for all the extracted RNA samples. RIN values 8.0 were taken for microarray experiment as well as validation of genes in real time quantitative PCR (RT q-PCR). 4 / 28 Preparation of cRNA and hybridization using Affymetrix Gene Chip Biotin labelled cRNA was prepared using Gene Chip130 IVT Express Labeling Kit (Affymetrix, Santa Clara, CA, USA). All the procedures followed were as per the manufacturer's protocol (Affymetrix). Briefly, 100 ng of total RNA was used to synthesize double stranded cDNA using an oligo(dT) primer containing the T7 RNA polymerase promoter site provided with GeneChip130 IVT Express Labeling Kit (Affymetrix, Santa Clara, CA). Purification of cRNA was done to remove unincorporated nucleotide triphosphates, salts, enzymes, and inorganic phosphates prior to fragmentation and hybridization onto Human Genome U133 Plus 2.0 Gene Chip expression arrays. Genome arrays were hybridized for 16 hours overnight at 45ÊC as per the affymetrix protocol. Following washing and staining, the Gene Chips were scanned using the Affymetrix Gene Chip Scanner 3000. The raw data file formats were generated using Gene Chip operating software (GCOS). Differential gene expression and statistical analysis All the samples were normalized, filtered and analysed using R software. Robust Multichip Analysis (RMA) normalization method was used for background correction, normalization and calculation of expression values. COMBAT software developed by Johnson et al (2007) [ 57 ] was used to adjust the expression values of samples processed in different batches. The normalized expression data was used to identify probe sets/genes that are differentially regulated across the comparison of interest. Identification of differentially expressed genes was performed with the Linear Models for Microarray Data (LIMMA) package available on BioConductor. To obtain differentially expressed genes, moderated t-statistic was applied. Multiple testing corrections were performed using Benjamin & Hochberg (BH) correction adjusting for the false discovery rate (FDR). A threshold adjusted p-value was set < 0.05, and the foldchange threshold was set to 1.3. These settings were retained throughout the analysis to select gene list across different group comparisons. Gene ontology and pathway analysis The list of genes obtained through various comparisons was studied for their overabundance in different GO terms as well as Pathways. Fisher's exact test was used to determine the significance of the GO analysis and significance level p < 0.05 was set for enriched genes and biological relevance of the associated genes was explored. Bioinformatic analysis was carried out using Genowiz™ ver. 4.0 (Ocimum Biosolutions, Hyderabad, India) software. In the present study, for GO analysis, the data from Gene Ontology consortium was used, while for pathways, human KEGG pathways were referred. Validation of microarray data using real time quantitative PCR (RTqPCR) The differentially expressed genes from microarray results were selected for validation using fluorescent based hydrolysis probes in Light Cycler 480 (Roche Diagnostics Pvt. Ltd, GmbH, Germany). Two independent groups of samples were taken for validating the microarray data. The first group consisted of 30 individuals which were included in microarray study. Validation was not possible for six individuals due to the limitation of samples in that particular aliquot (cDNA prepared for these six samples were insufficient to carry out validation in real time q-PCR). The second group consisted of freshly collected independent set of 24 individuals from Kerala coast. For each sample, total RNA was isolated, checked for purity and integrity. cDNA was synthesized using High fidelity cDNA synthesis Kit (Roche Diagnostics). For all the 5 / 28 genes, intron spanning PCR primers were designed using Probe finder software version 1.1 and specific hydrolysis probes from Universal Probe library set for Humans (Roche Diagnostics) were used. Primer sequences used in the study are given in S1 Table. RT-q PCR reactions were performed on 96 multi-well plates in triplicates and û-actin (ACTB) was used as a housekeeping gene for normalization. Fold change was calculated by dividing average normalized value in HLNRA individuals with average normalized value in NLNRA individuals. Results Transcriptome analysis was carried out in PBMC of 36 random, healthy individuals belonging to different background dose groups [Group I (NLNRA; 1.5 mGy/year, N = 9), Group II (HLNRA; 1.51±5.0mGy/year, N = 9), Group III (HLNRA; 5.01±15.0 mGy/year, N = 11), and Group IV (HLNRA; > 15.0 mGy/year, N = 7)] from Kerala coast using affymetrix Human Genome U133 Plus 2.0 Gene Chip. The objective was to find out differentially expressed genes in different HLNRA dose groups (Group II, Group III and Group IV) with respect to NLNRA (control group) and their biological significance in response to chronic low dose radiation. The NLNRA (group I) group of individuals were considered as control population and the expression profile observed in this group was considered as background level changes in gene expression at a particular gene. Analysis was carried out to identify differentially expressed genes at a threshold fold change value of 1.3, 1.5 and 2.0 fold with adjusted p-value < 0.05. The differentially expressed genes (up and down-regulated) in each HLNRA dose group (Group II, Group III and Group IV) as compared to NLNRA (Group I) is given in Table 1. At a fold change threshold value of 2.0, only 6 genes (3 Up and 3 down) were differentially expressed in Group I vs. II, 24 (15 up and 9 down) genes in Group I vs. III and 97 genes (72 up and 25 down) in Group I vs. IV, respectively.At a fold change threshold value of 1.5, we observed 27 (10 up and 17 down) genes were differentially expressed in Group I vs. II, 332 genes (167 up and 165 down) in Group I vs. III and 769 genes (347 up and 422 down) in Group I vs. IV, respectively (Fig 1). In order to get a large number of biologically significant genes from various molecular and cellular pathways that are differentially expressed in different background dose groups, analysis was carried out at a fold change threshold of 1.3 and adjusted p-values < 0.05. Our data revealed a total of 138 (39 up and 99 down), 1361 (611 up and 750 down) and 2427 (889 up and 1538 down) genes to be differentially expressed between Group I vs. Group II, Group I vs. Group III and Group I vs. Group IV, respectively (Figs 1 and 2). Interestingly, we observed a background dose related increase in the number of differentially expressed genes in different HLNRA groups as compared to NLNRA group. Up 39 Fold change 1.3 Down 99 611 889 750 1538 P-value set at < 0.05 Fold change 1.5 Up Down 10 17 167 347 165 422 Up 3 Fold change 2.0 Down 3 15 72 9 25 6 / 28 Fig 1. Histograms showing the number of differentially expressed genes in different HLNRA groups Grp II, Grp III and Grp IV) with respect to NLNRA group (Grp I). (a) Upregulated genes with adjusted p value < 0.05 (b) Downregulated genes with adjusted p value < 0.05. 7 / 28 Fig 2. Volcano plots representing the differentially expressed genes (up and down-regulated) at 1.3 fold in different HLNRA groups (Group II, III and IV) as compared to NLNRA (Group I). A) Group I vs. II, B) Group I vs. III, C) Group I vs. IV. Blue dots represent significant (p<0.05) upregulated genes and red dots represent significant down- regulated genes. Identification of common genes among different background radiation dose groups As shown in the Venn diagram (Fig 3), a total of 82 common genes (13 up-regulated and 69 down-regulated) were found to be differentially expressed in all the three HLNRA groups. Further analysis showed a total of 92 (19 up and 73 down), 109 (17 up and 92 down) and 937 (376 and 561) genes were common between Group II and III, Group II and IV and Group III and IV, respectively. Similarly, a total of 24 (16 up and 8 down), 558 (299 up and 259 down) and 1793 (668 up and 1125 down) genes were uniquely expressed in Groups II, III and IV, respectively. Interestingly, higher number of common genes was observed in high dose groups (Group III and Group IV). Detailed characteristics and function of the common genes are given in Tables 2 and 3. We have observed some of the important up-regulated (EIF1, PDE4B, USP36 and SNRPA1) and down-regulated (NT5E, NUPL2, GTF2E1, GRB10, IL16, SLC4A7, METTL13, TMEM184C, TRIM36, PPIL1, KDM5A, NDUFAF4, TUBD1, DHFRL1) common genes among the three groups. These common genes are involved in RNA processing, cell cycle regulation, apoptosis, microtubule formation, nucleotide metabolism, transcription initiation and regulation, cell growth, signal transduction, protein folding, cytokine activity and ion transport etc. Gene ontology analysis of differentially expressed genes in HLNRA groups as compared to NLNRA Gene ontology analysis was carried out in order to find out the biological significance and molecular function of the differentially expressed genes. Analysis was done to find out overrepresented biological processes, cellular components, molecular functions and critical 8 / 28 Fig 3. Venn diagram showing common as well as unique genes expressed in different group comparisons. Genes showing 1.3 fold change was considered for analysis. pathways with a threshold fold change of 1.3 and a statistical significance of p <0.05. Overall analysis has revealed that biological processes such as regulation of transcription (JUN, NR4A2), apoptosis (SIAH1, PMAIP1), regulation of cell cycle (CDKN1A, BTG1), response to DNA damage (GADD45B, DDIT3), metabolic processes (NAMPT, NT5E), RNA processing (PAPD4, SRRM1), Immune response (IL8, KIR3DS1), signal transduction (MAPK6, AKT2), Fold change Grp I vs Grp II 1.38 1.55 1.85 1.50 1.42 1.64 1.75 1.39 1.44 2.01 2.67 1.39 1.47 Fold change Grp I vs Grp III 1.39 1.59 1.66 1.72 1.39 1.61 1.54 1.38 1.32 3.30 4.19 1.44 1.67 (Continued) 10 / 28 Fold change Grp I vs Grp II -1.40 -1.55 -1.34 -1.38 -1.46 -1.42 -1.37 -1.47 -1.41 -1.43 -1.35 -1.37 -1.51 -1.59 -1.40 -1.57 -1.31 -1.38 -1.33 -1.33 -1.39 -1.38 DNA repair (POLB, LIG4), protein transport (SEC31A, NUPL1), histone/chromatin modification (SETDB2, SIRT2), response to oxidative stress (SOD2, OXR1), protein ubiquitination (USP36, UBE2B.) are over-represented in higher dose groups (Group III and Group IV) as compared to Group I (Fig 4). In terms of cellular location, 23±29% genes coded for nuclear proteins, 15±22% coded for cytoplasmic proteins, 4±8% coded for mitochondrial genes and 15±17% were involved in membrane bound activities. Some of the significantly overrepresented molecular function in higher dose groups (Group III and IV) included transcription factor activity, protein kinase activity, DNA/RNA and protein binding, signal transducer activity, histone binding activity, metal ion binding, ligase activity, helicase activity, methyltransferase activity, ubiquitin-protein ligase activity and oxidoreductase activity. Pathway analysis of differentially expressed genes in HLNRA groups as compared to NLNRA Pathway analysis was done in all the groups (Group II, Group III and Group IV) as compared to NLNRA to find out relevant cellular and molecular pathways activated in response to chronic low dose exposure in HLNRA individuals. Our analysis showed that in higher dose groups (Group III and IV), various important pathways such as MAPK signaling, Jak-STAT signaling, p53 signaling, T cell receptor signaling, B cell receptor signaling, insulin signaling, purine metabolism, apoptosis, cell cycle, DNA repair, ubiquitin mediated proteolysis, focal adhesion, gap junction etc. were found to be over-represented (Table 4). Representative heat maps showing the genes from each pathway in higher dose groups especially Group IV (HLNRA) as compared to Group I (NLNRA) is shown in Fig 5A and 5B. 11 / 28 Fig 4. Representative biological processes (GO analysis) over-represented in differentially expressed genes of high dose groups A) Group III (5.01±15.0 mGy/y) and B) Group IV (>15.0 mGy/y) of HLNRA as compared to Group I (NLNRA). DNA damage response and repair genes in HLNRA individuals Interestingly, our data revealed that differentially expressed genes in Group II are involved in biological processes such as immune response (IL1A, IGHG1), apoptosis (WDR92, APH1B), No. of genes / database count (24 / 272) (14 / 108) (9 / 65) (14 / 263) (10 / 155) (12 / 139) (6 / 69) (10 / 203) (7 / 96) Fig 5. Representative heat maps for the respective pathways of up-regulated and down regulated genes in Group IV (high dose group, HLNRA) as compared to NLNRA (Group I). (a) Representative heat maps for up-regulated genes in Group IV vs Group I (b) Representative heap maps for down regulated genes in Group IV vs Group I. Color panel shows expression intensity levels of genes involved in each pathway. 13 / 28 Fig 6. Gene ontology analysis showing over-representation of DDR related biological processes to be highly represented in high dose groups (Group III and Group IV) than in Group II. * shows statistical significance at p < 0.05. RNA splicing (SNRPA1, HNRNPM) and translational initiation (EIF1, EIF4G3) etc. However, high dose groups (>5.0 mGy/year) have higher representation/abundance of genes involved in DNA damage response that include DNA repair, response to oxidative stress, immune response, chromatin modification (methylation and histone modification), mRNA processing, cell cycle, and apoptosis (Fig 6). Several genes involved in different DNA repair pathways were present in higher dose groups (Group III and Group IV: > 5.0 mGy/year). These includes genes involved in nonhomologous end joining repair (XRCC4, LIG4, DCLRE1C, DCLRE1B, DCLRE1A), base excision repair (APEX2, UNG, POLB), nucleotide excision repair (RAD23B, GTF2H1, GTF2H3, GTF2H5, ERCC4), homologous recombination repair (RAD54B, RAD51B), mismatch repair (PMS1) and other repair genes (RAD21, RAD 18, RMI1, POLH, UBE2B, UBE2T, FANCE, FANCF, PALB2, REV3L, HLTF). DDR regulated pathways such as MAPK signaling, p53 signaling, JAK-STAT signaling were also activated in higher dose groups (Group III and IV). Although expression levels of p53 gene did not show any change, but p53 regulated genes such as CDKN1A, TRAF4, ATF3, TNFRSF10B, APAF1, DUSP1, PMAIP1, GADD45B, BCL6, PLK3 etc were differentially expressed in our data. Some of the genes involved in MAPK pathway were DUSP1, DUSP10, AKT2, ATF2, BCL10, CD38, CDC42, CSNK1A1, DDIT3, DUSP5, GFRAL, GNAS, HIPK3, IL8 etc. Several transcription factors and signaling molecules, which are involved in regulating DNA damage response and repair such as cJUN, FOSB, JUND, CREBZF, FOXO3, ATF2, HEY1, NR4A2 etc were found to be activated in higher dose groups. 14 / 28 A representative list of important genes involved in DDR processes is given in Table 5. Representative heat maps (up and down regulated genes) showing over-represented DDR and repair genes in higher dose group (Group IV) with respect to NLNRA (Group 1) is shown in Fig 7. Dose responsive genes in HLNRA individuals We have also identified 64 genes (36 up-regulated and 28 down-regulated) which were highly expressed ( 2.0 fold) in Group IV and showed background radiation dose dependent increase or decrease (up and down regulation) in their expression profile. Fig 8 represents the fold changes in the expression of these genes in different background dose groups (Group I vs Group II, Group I vs Group III and Group I vs Group IV). Gene ontology analysis have revealed that most of these genes are involved in Cell cycle regulation (GADD45B, BTG1, GNAS, GIMAP8, GIMAP4), immune system (NFKB2, CXCR1, TNFSF10, CCR2), stimulus to DNA damage (DDIT3, DUSP1, JUN, GADD45B, JUND, BTG1), apoptosis (PMAIP1, PPIF, TSC22D2, SIK3, NLRC4), mRNA processing (CRNKL1, SFRS3, SFRS5, FUSIP1, PHAX), transcriptional regulation (JUN, JUND, KLF6, ATXN1, MED13, MED26, ZNF302, NFKB2 ZNF207, ZNF658) and signal transduction (DUSP1, DUSP10, LPXN, MYLIP, LGALS3, PAQR8) and ubiquitin dependent proteolysis (SQSTM1, UBQLN2) etc. A representative scatter plot showing the microarray intensity values of selected genes in individuals of different background dose groups is given in Fig 9. Validation of differentially expressed genes from microarray data using RT q-PCR Thirty differentially expressed genes (22 up-regulated and 8 down-regulated) were validated using RT q-PCR. Validation of these genes was carried out in 54 individuals, which include 30 individuals used for microarray analysis and another independent set of 24 individuals from the same dose groups of Kerala population. These genes were selected from different dose group comparisons involved in various biological processes, having different expression levels in order to get a good representation from microarray data. These include PMAIP1, PAPD4, DDIT3, GIMAP8, PPIF, CSRNP1, BTG1, CDKN1A, DUSP10, GADD45B, TSC22D2, METTL13, cJUN, DUSP1, KIR3DS1, JUND, EIF1, ATXN1, SNRPA1, DHFRL1, NAMPT, ZNF167, THAP2, KLF6, BBS10, PLK3, PDK4, SETDB2, KDM6B, CCR2. As shown in Fig 10, the average fold change values obtained from microarray experiments from 36 individuals. The fold change values obtained from RT q-PCR experiments in 30 individuals (microarray samples) and 24 individuals (new set of individuals) showed similar trend and good correlation for all the 30 genes studied. Our results showed consistent gene expression profile of the genes in microarray experiment as well as RT q-PCR experiments. We also observed similar expression values of the genes in new random set of individuals suggesting the results obtained are consistent in the population. Discussion The present study is focused on the cellular responses to chronic low dose and low dose-rate radiation in humans. The baseline gene expression is very important to understand the exvivo/ in-vivo effect of low dose and dose-rate exposure 55. Human population living in high level natural radiation areas (HLNRA) is exposed to chronic low dose background radiation since generations and provides unique opportunity to understand the in-vivo effects of low dose radiation exposure directly on humans. Most importantly, the unique feature of HLNRA of Kerala coast is the non-uniform distribution of monazite in the beach sand which leads to varying level of background radiation (<1.0 to 45mGy/year) along the 55 km long stretch 15 / 28 allowing us to investigate in-vivo dose response, if any at various biological end points. Several epidemiological (congenital malformation, cancer incidence etc) and biological studies carried out in this population have not revealed any significant difference between NLNRA and HLNRA population [ 9,11,16,17,18,20,22 ]. However, recent studies have shown a lower induction and efficient repair of DNA damage in HLNRA individuals [ 21,23 ]. In the present study, global gene expression profile was carried out to understand biological response of human cells exposed to chronic low dose and low dose-rate radiation. The individuals included in the study are classified into four different background dose groups based on the individual dose received annually by them. The first group receives a dose 1.5mGy/year and are considered as control population (NLNRA) and the other three groups are from HLNRA receiving a dose >1.5mGy/year. Our results have shown that a large number of genes are differentially expressed in HLNRA individuals as compared to NLNRA. Interestingly, a dose dependent increase in the number of differentially expressed genes with respect to background radiation levels was observed in HLNRA groups. The plausible explanation could be that individuals belonging to higher background dose groups (> 5.0mGy/year) have accumulated larger doses and thus stimulating many genes from different cellular and molecular pathways to maintain genome integrity. Further, detailed gene ontology analysis revealed an overrepresentation of genes involved in DNA damage response (DDR), DNA repair, cell cycle 19 / 28 Fig 7. Representative heat maps showing intensity values of up and down regulated DNA damage response and repair genes in high dose group (Group IV) of HLNRA as compared to Group I. regulation, mRNA processing, protein transport, stress response, histone/chromatin modification, methylation, apoptosis, transcriptional regulation, signal transduction, and immune response in individuals belonging to higher dose groups (Group III and Group IV) of HLNRA. The above findings are supportive of the results obtained from DNA damage and repair study in HLNRA, where significantly lower induction of DNA damage and efficient repair of DNA strand breaks observed in individuals belonging to higher dose groups (>5.0 mGy/year) of HLNRA [ 18,21,23 ]. Our results have also shown that, Group II ( 5.0mGy/year) has very few significantly differentially expressed genes as compared to Group III and Group IV of HLNRA (>5.0mGy/ year). Most of these genes are involved in transcriptional regulation, protein transport and RNA processing. Interestingly, an abundance or over-representation of DDR and DNA repair genes was observed in higher dose groups (>5.0 mGy/year). These included DNA repair genes such as UNG and APEX2 involved in base excision repair, RAD23B and ERCC4 genes which play important role in nucleotide excision repair, PMS1 from mismatch repair pathway and DCLRE1C (Artemis), XRCC4, LIG4 genes involved in NHEJ pathway. The differentially expressed genes clearly indicate the active involvement of all the molecular pathways of DNA repair in HLNRA individuals (>5.0 mGy/year). For instance, APEX2 is the regulatory gene of 20 / 28 Fig 8. Dose responsive genes showing background dose dependent increase or decrease in expression among different dose groups. (A) Dose responsive genes showing background dose dependent increase in expression among different dose groups. (B) Dose responsive genes showing background dose dependent decrease in expression among different dose groups. Genes were divided in to three groups A, B and C on the basis of dose response trend shown by them. NLNRA (Group I), HLNRA (Group II, Group III and Group IV). BER which controls both short patch and long patch pathways. Uracil DNA glycosylase (UNG) is the major glycosylase for the removal of uracil from DNA and initiates base excision repair [ 58 ] indicating that BER is active in HLNRA population. Similarly, presence of DCLRE1C (Artemis), XRCC4, LIG4 indicates that DSB repair is occurring in HLNRA and perhaps mediated through artemis-dependent pathway. Additionally, differential expression of ERCC4 and RAD23B suggests possible involvement of nucleotide excision repair pathway. Activation of genes involved in DDR response (DDIT3, SOD2), cell cycle check points (GADD45, CDKN1A, BTG1, BTG3, CCNG2), apoptosis (PMAIP1, PPP1R15A, PPP2R5C, SIAH1, APAF1) and signal transduction pathways such as mitogen activated protein kinase (MAPK) pathway, Jun amino-terminal kinases (JNK) pathway, p53 pathway, T cell receptor signaling, B-cell receptor signaling, Jak-stat signaling, purine metabolism, ubiquitin mediated proteolysis, and gap junction etc are suggestive of DDR to be active in this population exposed to chronic low dose radiation. Our observation that more number of DNA damage or stress response genes in higher background dose groups suggests that perhaps a certain level of minimum damage is required 21 / 28 Fig 9. Scatter plot showing the expression level of different genes in the individuals belonging to different background dose groups (NLNRA, Group I and HLNRA. Group II, III and IV) in microarray analysis. Each dot represents normalized value in one individual. Different colored dots represent individuals belonging to different dose groups. Group I (N = 9), Group II (N = 9), Group III (N = 11), Group IV (N = 7). for the cells to sense and activate DDR signaling [ 4,59 ]. Our results indicate a threshold dose of ~ 5.0mGy/year for chronic low dose exposure in HLNRA population for activation of molecular pathways of DNA damage response and better cell survival. The signal transduction pathways such as mitogen activated protein kinase (MAPK) pathway, Jun amino-terminal kinases (JNK) pathway and p53 pathway have been shown to play an important role in low dose radiation induced adaptive response [ 60,61,62 ]. Our results have shown over-representation of genes involved in MAPK pathway (DUSP1, DUSP10, AKT2, ATF2, DDIT3, DUSP5, GNAS, HIPK3, IL8 etc.) and p53 pathway (CDKN1A, MDM2, TRAF4, TNFRSF10B, APAF1, PMAIP1, GADD45B etc.) in higher dose groups of HLNRA (>5.0 mGy/ year). Several known radiation responsive genes such as CDKN1A and GADD45B etc involved in cell cycle check point activation were upregulated in HLNRA individuals. Immune response, cell to cell communication and gap junction genes and proteins are known to play important role in radio-adaptive response [ 63,64,65 ]. However, such information on PBMC at G0/G1 exposed to chronic low dose exposure is limited. In the present study, representation of genes involved in modulation of immune response (TNFRSF10B, IL16, IL8, CCR2, KIR3DS1, IFNG etc.), cell-cell communication and gap junction (TUBB3, GNAS, TUBB2C, LPAR1 etc) in HLNRA population suggest their possible role in radio-adaptive response. Another interesting finding is the transcriptional induction of a number of transcription factors such as c-JUN, JUND, FOS, ATF2, NR4A2, Sp1, FOXO3, CREBZF, which are known to play important role in DDR signaling and repair. Genes such as cJUN, JUND, FOS, ATF2, ATF3 and CREBZF belong to activating protein 1 (AP1) family of transcription factors (TFs). AP1 family TFs are stress responsive transcription activators and has been implicated in DNA repair by its ability to regulate a large set of genes functioning in DNA repair [ 66 ]. Recently, it 22 / 28 Fig 10. Validation of differentially expressed genes using RT q-PCR experiment. Comparison of fold change values obtained in two different sets of individuals along with microarray results are shown. Blue bar represents microarray results, Red bar shows RT q-PCR results of microarray samples, Green bar shows RT q-PCR results of new set of samples. Fold change values are shown above each bar in all the gene. has been shown that TFs such as ATF2 and NR4A2 not only regulates the transcription of genes involved in DNA repair, but also translocate to the site of DNA lesion and play a direct role in DNA repair [ 66,67 ]. NR4A2 has been shown to get translocated to DNA repair foci, where it gets phosphorylated by DNA-PKcs and co-localizes with other repair proteins such as gamma-H2AX, DDB2 and XPC [ 68,69 ] Increased expression of these transcription factors in higher dose groups of HLNRA individuals are suggestive of active involvement of DDR signaling and repair in high dose groups of HLNRA individuals (> 5.0 mGy/year). Studies on alteration of gene expression profile in human cells in response to low dose ionizing radiation are limited. Few studies have been carried out where transcriptional response in occupational workers exposed to very low dose ionizing radiation have been reported [ 41,50 ]. Morandi et al (2009) reported over-representation of histone modification genes involved in DNA packaging, chromatin architecture and DNA metabolism in medical workers exposed to low doses of radiations[50]. The role of histone modification/chromatin changes in DNA repair after acute exposure to IR has been reported [ 70,71,72,73 ]. In our study, we also observed large number of genes (HIST1H1E, H3F3B, HIST1H2BC, HP1BP3, KDM6B, ING3, MYST4, SETDB2, MECP2, MLL, SUV420H1 etc) involved in epigenetic processes such as histone/chromatin modification, DNA packaging, methylation, and DNA metabolism to be overrepresented. It indicates the alteration of chromatin structure is one of the important cellular response to chronic low dose exposure. Further, Fachin et al. (2009) observed several biological processes such as ubiquitin cycle (UHRF2 and PIAS1), DNA repair (LIG3, XPA, ERCC5, 23 / 28 RAD52, DCLRE1C), cell cycle regulation/proliferation (RHOA, CABLES2, TGFB2, IL16), and stress response (GSTP1, PPP2R5A, DUSP22) to be active chronic low level radiation exposures [41]. Interestingly, such pathways are over-represented in our data suggesting their response to chronic low dose radiation exposure. We have also identified a set of dose responsive genes DDIT3, GADD45B, JUN, PMAIP1, DUSP1, PAPD4, DUSP10, BTG1, PPIF, TSC22D2, TMEM132C, GIMAP8, METTL13, CSRNP1, KIR3DS1, TNFSF10 etc. which have potential to be used as possible low dose radiation signatures in humans. In summary, our findings clearly indicated that individuals exposed to background doses of >5mGy/year have shown alteration in expression of many genes involved in important functions or pathways. These included DDR signaling, DNA repair, RNA metabolism, epigenetic changes (histone/chromatin modifications), cell cycle regulation, immune response, apoptosis etc. These may be the primary reason of not getting any detectable change at phenotype or DNA damage levels in HLNRA individuals exposed to chronic low level background dose exposures. Sixty four dose responsive genes were identified, which are the possible radiation signatures for chronic low dose radiation exposure. In conclusion, global gene expression profile in response to natural chronic low dose radiation revealed active DDR and repair processes and their interaction with various cellular and molecular pathways in HLNRA individuals belonging to higher dose groups >5mGy/year. These findings indicated a possible threshold dose of 5mGy/year for signaling DDR and a plausible reason of observing in vivo radio-adaptive response and non-carcinogenesis in HLNRA population. These findings have tremendous implications in understanding the molecular effect of low dose radiation biology, especially the effect of low dose radiation in humans at low dose region. Supporting information S1 Table. Details of primer sequences and UPL probe numbers of the genes studied using real time PCR. (DOCX) Acknowledgments We profusely thank the volunteers who are part of the study. We also thank all the staff members of LLRRL and LLRRS for the continued support and help through-out the work. We thank Dr. Sandhya P. Kiran (Ocimum Biosolutions, Hyderabad) for her help in data analysis. We thank Dr. M. Seshadri, Former Head, Radiation Biology and Health Sciences Division and Mr. V. D. Cheriyan for their constant help and support. We profusely thank Dr. S. Chattopadhyay, Director, Bio-Science Group for constant support and guidance. Formal analysis: Vinay Jain, Birajalaxmi Das. Investigation: Vinay Jain, Birajalaxmi Das. Methodology: Vinay Jain, Birajalaxmi Das. Project administration: Birajalaxmi Das. Resources: Birajalaxmi Das. 24 / 28 Supervision: Birajalaxmi Das. Validation: Vinay Jain, Birajalaxmi Das. Visualization: Birajalaxmi Das. Writing ± original draft: Vinay Jain, Birajalaxmi Das. Writing ± review & editing: Birajalaxmi Das. 25 / 28 26 / 28 27 / 28 1. Amundson SA , Lee RA , Koch-Paiz CA , Bittner ML , Meltzer P , Trent JM , et al. ( 2003 ) Differential responses of stress genes to low dose-rate gamma irradiation . Mol Cancer Res 1 : 445 ± 452 . PMID: 12692264 2. Ding LH , Shingyoji M , Chen F , Hwang JJ , Burma S , Lee C , et al. ( 2005 ) Gene expression profiles of normal human fibroblasts after exposure to ionizing radiation: a comparative study of low and high doses . Radiat Res 164 : 17 ± 26 . PMID: 15966761 3. Morgan WF , Bair WJ ( 2013 ) Issues in low dose radiation biology: the controversy continues. A perspective . Radiat Res 179 : 501 ± 510 . https://doi.org/10.1667/RR3306.1 PMID: 23560636 4. Tubiana M , Feinendegen LE , Yang C , Kaminski JM ( 2009 ) The linear no-threshold relationship is inconsistent with radiation biologic and experimental data . Radiology 251 : 13 ± 22 . https://doi.org/10.1148/ radiol.2511080671 PMID: 19332842 5. Gruneberg H ( 1964 ) Genetical research in an area of high natural radioactivity in South India . Nature 204 : 222 ± 224 . PMID: 14225503 6. Gopal-Ayengar AR , Nayar GG , George KP , Mistry KB ( 1970 ) Biological effects of high background radioactivity: studies on plants growing on the monazite bearing areas of Kerala coast and adjoining regions . Indian J Exp Biol 8 : 313 ± 318 . PMID: 5502335 7. Nayar GG , George KP , Gopal-Ayengar AR ( 1970 ) On the biological effects of high background radioactivity: studies on Tradescancia grown in radioactive monazite sand . Radiat Bot 10 : 287 ± 292 . 8. Gopal-Ayengar AR , Sundaram K , Mistry KB , Suntha CM , Nambi KSV , Kathuria SP , et al. ( 1971 ) Evaluation of the long term effects of high background radiation on selected population groups of Kerala coast . In Proceedings of the IV International Conference on Peaceful uses of Atomic Energy , United Nations,New York. 2 : 31 ± 61 . 9. Jaikrishan G , Andrews VJ , Thampi MV , Koya PK , Rajan VK , Chauhan PS ( 1999 ) Genetic monitoring of the human population from high-level natural radiation areas of Kerala on the southwest coast of India. I. Prevalence of congenital malformations in newborns . Radiat Res 152 : S149 ± 153 . PMID: 10564958 10. Jaikrishan G , Sudheer KR , Andrews VJ , Koya PK , Madhusoodhanan M , Jagadeesan CK , et al. ( 2013 ) Study of stillbirth and major congenital anomaly among newborns in the high-level natural radiation areas of Kerala, India . J Community Genet 4 : 21 ± 31 . https://doi.org/10.1007/s12687-012 -0113-1 PMID: 22875746 11. Nair RR , Rajan B , Akiba S , Jayalekshmi P , Nair MK , Gangadharan P , et al. ( 2009 ) Background radiation and cancer incidence in Kerala, India-Karanagappally cohort study . Health Phys 96 : 55 ± 66 . https://doi. org/10.1097/01.HP. 0000327646 .54923.11 PMID: 19066487 12. Koya PK , Chougaonkar MP , Predeep P , Jojo PJ , Cheriyan VD , Mayya YS , et al. ( 2012 ) Effect of low and chronic radiation exposure: a case-control study of mental retardation and cleft lip/palate in the monazite-bearing coastal areas of southern Kerala . Radiat Res 177 : 109 ± 116 . PMID: 21980927 13. Koya PK , Jaikrishan G , Sudheer KR , Andrews VJ , Madhusoodhanan M , Jagadeesan CK , et al. ( 2015 ) Sex ratio at birth: scenario from normal- and high-level natural radiation areas of Kerala coast in southwest India . Radiat Environ Biophys 54 : 453 ± 463 . https://doi.org/10.1007/s00411-015 -0615-4 PMID: 26343038 14. Cheriyan VD , Kurien CJ , Das B , Ramachandran EN , Karuppasamy CV , Thampi MV , et al. ( 1999 ) Genetic monitoring of the human population from high-level natural radiation areas of Kerala on the southwest coast of India. II. Incidence of numerical and structural chromosomal aberrations in the lymphocytes of newborns . Radiat Res 152 : S154 ± 158 . PMID: 10564959 15. Das B , Karuppasamy CV ( 2009 ) Spontaneous frequency of micronuclei among the newborns from high level natural radiation areas of Kerala in the southwest coast of India . Int J Radiat Biol 85 : 272 ± 280 . https://doi.org/10.1080/09553000902751462 PMID: 19296343 16. Das B , Saini D , Seshadri M ( 2009 ) Telomere length in human adults and high level natural background radiation . PLoS One 4 : e8440 . https://doi.org/10.1371/journal.pone. 0008440 PMID: 20037654 17. Das B , Saini D , Seshadri M ( 2012 ) No evidence of telomere length attrition in newborns from high level natural background radiation areas in Kerala coast, south west India . Int J Radiat Biol 88 : 642 ± 647 . https://doi.org/10.3109/09553002. 2012 .699135 PMID: 22668000 18. Jain V , Kumar PR , Koya PK , Jaikrishan G , Das B ( 2016 ) Lack of increased DNA double-strand breaks in peripheral blood mononuclear cells of individuals from high level natural radiation areas of Kerala coast in India . Mutat Res 788 : 50 ± 57 . https://doi.org/10.1016/j.mrfmmm. 2016 . 03 .002 PMID: 27063255 19. Karuppasamy CV , Ramachandran EN , Kumar VA , Kumar PR , Koya PK , Jaikrishan G , et al. ( 2016 ) Peripheral blood lymphocyte micronucleus frequencies in men from areas of Kerala, India, with high vs normal levels of natural background ionizing radiation . Mutat Res Genet Toxicol Environ Mutagen 800 ± 801 : 40 ± 45 . 20. Kumar PR , Cheriyan VD , Seshadri M ( 2012 ) Evaluation of spontaneous DNA damage in lymphocytes of healthy adult individuals from high-level natural radiation areas of Kerala in India . Radiat Res 177 : 643 ± 650 . PMID: 22175299 21. Kumar PR , Seshadri M , Jaikrishan G , Das B ( 2015 ) Effect of chronic low dose natural radiation in human peripheral blood mononuclear cells: Evaluation of DNA damage and repair using the alkaline comet assay . Mutat Res 775 : 59 ± 65 . https://doi.org/10.1016/j.mrfmmm. 2015 . 03 .011 PMID: 25879710 22. Ramachandran EN , Karuppasamy CV , Cheriyan VD , Soren DC , Das B , Anil Kumar V et al. ( 2013 ) Cytogenetic studies on newborns from high and normal level natural radiation areas of Kerala in southwest coast of India . Int J Radiat Biol 89 : 259 ± 267 . https://doi.org/10.3109/09553002. 2013 .747014 PMID: 23134065 23. Jain V , Saini D , Vivek kumar PR , Jaikrishan G , Das B ( 2017 ) Efficient repair of DNA double strand breaks in individuals from high level natural radiation areas of Kerala coast, south-west India . Mutation Research, Fundamental and Molecular mechanisms of mutagenesis 806: 39 ± 50 . https://doi.org/10. 1016/j.mrfmmm. 2017 . 09 .003 PMID: 28963924 24. Ramachandran EN , Karuppasamy CV , Anilkumar V , Soren DC , Vivekkumar PR , Koya PKM , et al. ( 2017 ) Radio-adaptive response in peripheral blood lymphocytes of individuals residing in high level natural radiation areas of Kerala in the southwest coast of India . Mutagenesis 32 : 267 ± 273 . https://doi.org/ 10.1093/mutage/gew057 PMID: 27831478 25. Jain V , Das B , Seshadri M ( 2011 ) Transcriptional expression of H2B, CTP synthase and PLK3 genes in whole blood exposed to 60Co gamma radiation . International Journal of Low Radiation 8 : 55 ± 65 . 26. Saini D , Shelke S , Mani Vannan A , Toprani S , Jain V , Das B , et al. ( 2012 ) Transcription profile of DNA damage response genes at G(0) lymphocytes exposed to gamma radiation . Mol Cell Biochem 364 : 271 ± 281 . https://doi.org/10.1007/s11010-012 -1227-9 PMID: 22258824 27. Shelke S , Das B ( 2015 ) Dose response and adaptive response of non-homologous end joining repair genes and proteins in resting human peripheral blood mononuclear cells exposed to gamma radiation . Mutagenesis 30 : 365 ± 379 . https://doi.org/10.1093/mutage/geu081 PMID: 25473122 28. Toprani SM , Das B ( 2015 ) Role of base excision repair genes and proteins in gamma-irradiated resting human peripheral blood mononuclear cells . Mutagenesis 30 : 247 ± 261 . https://doi.org/10.1093/mutage/ geu065 PMID: 25381310 29. Toprani SM , Das B ( 2015 ) Radio-adaptive response of base excision repair genes and proteins in human peripheral blood mononuclear cells exposed to gamma radiation . Mutagenesis. 30. Amundson SA , Fornace AJ Jr. ( 2001 ) Gene expression profiles for monitoring radiation exposure . Radiat Prot Dosimetry 97 : 11 ± 16 . PMID: 11763352 31. Amundson SA , Fornace AJ Jr. ( 2003 ) Monitoring human radiation exposure by gene expression profiling: possibilities and pitfalls . Health Phys 85 : 36 ± 42 . PMID: 12852469 32. Amundson SA , Grace MB , McLeland CB , Epperly MW , Yeager A , Zhan Q , et al. ( 2004 ) Human in vivo radiation-induced biomarkers: gene expression changes in radiotherapy patients . Cancer Res 64 : 6368 ± 6371 . https://doi.org/10.1158/ 0008 - 5472 .CAN-04-1883 PMID: 15374940 33. Ghandhi SA , Smilenov LB , Elliston CD , Chowdhury M , Amundson SA ( 2015 ) Radiation dose-rate effects on gene expression for human biodosimetry . BMC Med Genomics 8 : 22 . https://doi.org/10. 1186/s12920-015-0097 -x PMID : 25963628 34. Pernot E , Hall J , Baatout S , Benotmane MA , Blanchardon E , Bouffler S , et al. ( 2012 ) Ionizing radiation biomarkers for potential use in epidemiological studies . Mutat Res 751 : 258 ± 286 . https://doi.org/10. 1016/j.mrrev. 2012 . 05 .003 PMID: 22677531 35. Paul S , Amundson SA ( 2008 ) Development of gene expression signatures for practical radiation biodosimetry . Int J Radiat Oncol Biol Phys 71 : 1236 ± 1244 . https://doi.org/10.1016/j.ijrobp. 2008 . 03 .043 PMID: 18572087 36. Paul S , Amundson SA ( 2011 ) Gene expression signatures of radiation exposure in peripheral white blood cells of smokers and non-smokers . Int J Radiat Biol 87 : 791 ± 801 . https://doi.org/10.3109/ 09553002. 2011 .568574 PMID: 21801107 37. Chaudhry MA , Omaruddin RA , Kreger B , de Toledo SM , Azzam EI ( 2012 ) Micro RNA responses to chronic or acute exposures to low dose ionizing radiation . Mol Biol Rep 39 : 7549 ± 7558 . https://doi.org/ 10.1007/s11033-012 -1589-9 PMID: 22367372 38. Nylund R , Kuster N , Leszczynski D ( 2010 ) Analysis of proteome response to the mobile phone radiation in two types of human primary endothelial cells . Proteome Sci 8 : 52 . https://doi.org/10.1186/ 1477 - 5956-8-52 PMID: 20955554 39. Sokolov M , Neumann R ( 2016 ) Global Gene Expression Alterations as a Crucial Constituent of Human Cell Response to Low Doses of Ionizing Radiation Exposure . Int J Mol Sci 17. 40. Rothkamm K , Lobrich M ( 2003 ) Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses . Proc Natl Acad Sci U S A 100 : 5057 ± 5062 . https://doi.org/10.1073/ pnas.0830918100 PMID: 12679524 41. Fachin AL , Mello SS , Sandrin-Garcia P , Junta CM , Ghilardi-Netto T , Donadi EA , et al. ( 2009 ) Gene expression profiles in radiation workers occupationally exposed to ionizing radiation . J Radiat Res 50 : 61 ± 71 . PMID: 19218781 42. Kabacik S , Mackay A , Tamber N , Manning G , Finnon P , Paillier F et al. ( 2011 ) Gene expression following ionising radiation: identification of biomarkers for dose estimation and prediction of individual response . Int J Radiat Biol 87 : 115 ± 129 . https://doi.org/10.3109/09553002. 2010 .519424 PMID: 21067298 43. Amundson SA , Fornace AJ Jr. ( 2003 ) Microarray approaches for analysis of tumor suppressor gene function . Methods Mol Biol 223 : 141 ± 154 . https://doi.org/10.1385/1-59259-329-1:141 PMID: 12777727 44. Fachin AL , Mello SS , Sandrin-Garcia P , Junta CM , Donadi EA , Passos GAS , et al. ( 2007 ) Gene expression profiles in human lymphocytes irradiated in vitro with low doses of gamma rays . Radiat Res 168 : 650 ± 665 . https://doi.org/10.1667/RR0487.1 PMID: 18088177 45. Paul S , Barker CA , Turner HC , McLane A , Wolden SL , Amundson SA ( 2011 ) Prediction of in vivo radiation dose status in radiotherapy patients using ex vivo and in vivo gene expression signatures . Radiat Res 175 : 257 ± 265 . https://doi.org/10.1667/RR2420.1 PMID: 21388269 46. Fornace AJ Jr., Amundson SA , Do KT , Meltzer P , Trent J , Bittner M ( 2002 ) Stress-gene induction by low-dose gamma irradiation . Mil Med 167 : 13 ± 15 . PMID: 11873496 47. Franco N , Lamartine J , Frouin V , Le Minter P , Petat C , Leplat JJ et al. ( 2005 ) Low-dose exposure to gamma rays induces specific gene regulations in normal human keratinocytes . Radiat Res 163 : 623 ± 635 . PMID: 15913394 48. Warters RL , Packard AT , Kramer GF , Gaffney DK , Moos PJ ( 2009 ) Differential gene expression in primary human skin keratinocytes and fibroblasts in response to ionizing radiation . Radiat Res 172 : 82 ± 95 . https://doi.org/10.1667/RR1677.1 PMID: 19580510 49. Albanese J , Martens K , Karanitsa LV , Schreyer SK , Dainiak N ( 2007 ) Multivariate analysis of low-dose radiation-associated changes in cytokine gene expression profiles using microarray technology . Exp Hematol 35 : 47 ± 54 . https://doi.org/10.1016/j.exphem. 2007 . 01 .012 PMID: 17379087 50. Morandi E , Severini C , Quercioli D , Perdichizzi S , Mascolo MG , Horn W et al. ( 2009 ) Gene expression changes in medical workers exposed to radiation . Radiation Research 172 : 500 ± 508 . https://doi.org/ 10.1667/RR1545.1 PMID: 19772471 51. Lanza V , Pretazzoli V , Olivieri G , Pascarella G , Panconesi A , Negri R ( 2005 ) Transcriptional response of human umbilical vein endothelial cells to low doses of ionizing radiation . J Radiat Res 46 : 265 ± 276 . PMID: 15988146 52. Long XH , Zhao ZQ , He XP , Wang HP , Xu QZ , An J , et al. ( 2007 ) Dose-dependent expression changes of early response genes to ionizing radiation in human lymphoblastoid cells . Int J Mol Med 19 : 607 ± 615 . PMID: 17334636 53. Wyrobek AJ , Manohar CF , Krishnan VV , Nelson DO , Furtado MR , Bhattacharya MS , et al. ( 2011 ) Low dose radiation response curves, networks and pathways in human lymphoblastoid cells exposed from 1 to 10cGy of acute gamma radiation . Mutat Res 722 : 119 ± 130 . https://doi.org/10.1016/j.mrgentox. 2011 . 03 .002 PMID: 21497671 54. Sokolov M , Neumann R ( 2014 ) Effects of low doses of ionizing radiation exposures on stress-responsive gene expression in human embryonic stem cells . Int J Mol Sci 15 : 588 ± 604 . https://doi.org/10. 3390/ijms15010588 PMID: 24398983 55. Fujimori A , Okayasu R , Ishihara H , Yoshida S , Eguchi-Kasai K , Nojima K , et al. ( 2005 ) Extremely low dose ionizing radiation up-regulates CXC chemokines in normal human fibroblasts . Cancer Res 65 : 10159 ± 10163 . https://doi.org/10.1158/ 0008 - 5472 .CAN-05-2015 PMID: 16287999 56. Nair RRK , Akiba S , Binu VS , Jayalekshmi P , Gangadharan P , Nair MK , et al. ( 2005 ) Individual dose estimationÐour experience with the Karunagapally study in Kerala, India . International Congress Series. 1276 : 41 ± 45 . 57. Johnson WE , Rabinovic A , Li C ( 2007 ) Adjusting batch effects in microarray expression data using Empirical Bayes methods . Biostatistics 8 ( 1 ): 118 ± 127 . Biostatistics 8: 118 ± 127 . https://doi.org/10.1093/ biostatistics/kxj037 PMID: 16632515 58. Visnes T , Akbari M , Hagen L , Slupphaug G , Krokan HE ( 2008 ) The rate of base excission repair of uracil is controlled by the initiating glycosylase . DNA repair 7: 1869 ± 1881 . https://doi.org/10.1016/j. dnarep. 2008 . 07 .012 PMID: 18721906 59. Grudzenski S , Raths A , Conrad S , Rube CE , Lobrich M ( 2010 ) Inducible response required for repair of low-dose radiation damage in human fibroblasts . Proc Natl Acad Sci U S A 107 : 14205 ± 14210 . https:// doi.org/10.1073/pnas.1002213107 PMID: 20660770 60. Shimizu T , Kato T Jr., Tachibana A , Sasaki MS ( 1999 ) Coordinated regulation of radioadaptive response by protein kinase C and p38 mitogen-activated protein kinase . Exp Cell Res 251 : 424 ± 432 . https://doi.org/10.1006/excr. 1999 .4582 PMID: 10471327 61. Takahashi A , Su X , Suzuki H , Omori K , Seki M , Hashizume T , et al. ( 2010 ) p53-dependent adaptive responses in human cells exposed to space radiations . Int J Radiat Oncol Biol Phys 78 : 1171 ± 1176 . https://doi.org/10.1016/j.ijrobp. 2010 . 04 .062 PMID: 20729005 62. Tang FR , Loke WK ( 2015 ) Molecular mechanisms of low dose ionizing radiation-induced hormesis, adaptive responses, radioresistance, bystander effects, and genomic instability . Int J Radiat Biol 91 : 13 ± 27 . https://doi.org/10.3109/09553002. 2014 .937510 PMID: 24975555 63. Feinendegen LE ( 2005 ) Evidence for beneficial low level radiation effects and radiation hormesis . Br J Radiol 78 : 3±7 . https://doi.org/10.1259/bjr/63353075 PMID: 15673519 64. Nenoi M , Wang B , Vares G ( 2015 ) In vivo radioadaptive response: a review of studies relevant to radiation-induced cancer risk . Hum Exp Toxicol 34 : 272 ± 283 . https://doi.org/10.1177/0960327114537537 PMID: 24925363 65. Vaiserman AM ( 2010 ) Radiation hormesis: historical perspective and implications for low-dose cancer risk assessment . Dose Response 8 : 172 ± 191 . https://doi.org/10.2203/dose-response. 09 - 037 . Vaiserman PMID: 20585444 66. Bhoumik A , Takahashi S , Breitweiser W , Shiloh Y , Jones N , Ronai Z ( 2005 ) ATM-dependent phosphorylation of ATF2 is required for the DNA damage response . Mol Cell 18 : 577 ± 587 . https://doi.org/10. 1016/j.molcel. 2005 . 04 .015 PMID: 15916964 67. Hayakawa J , Mittal S , Wang Y , Korkmaz KS , Adamson E , English C , et al. ( 2004 ) Identification of promoters bound by c-Jun/ATF2 during rapid large-scale gene activation following genotoxic stress . Mol Cell 16 : 521 ± 535 . https://doi.org/10.1016/j.molcel. 2004 . 10 .024 PMID: 15546613 68. Jagirdar K , Yin K , Harrison M , Lim W , Muscat GE , Sturm RA et al. ( 2013 ) The NR4A2 nuclear receptor is recruited to novel nuclear foci in response to UV irradiation and participates in nucleotide excision repair . PLoS One 8 : e78075 . https://doi.org/10.1371/journal.pone. 0078075 PMID: 24223135 69. Malewicz M , Kadkhodaei B , Kee N , Volakakis N , Hellman U , Kristina V , et al. ( 2011 ) Essential role for DNA-PK-mediated phosphorylation of NR4A nuclear orphan receptors in DNA double-strand break repair . Genes Dev 25 : 2031 ± 2040 . https://doi.org/10.1101/gad.16872411 PMID: 21979916 70. Falk M , Lukasova E , Gabrielova B , Ondrej V , Kozubek S ( 2007 ) Chromatin dynamics during DSB repair . Biochim Biophys Acta 1773 : 1534 ± 1545 . https://doi.org/10.1016/j.bbamcr. 2007 . 07 .002 PMID: 17850903 71. Hunt CR , Ramnarain D , Horikoshi N , Iyengar P , Pandita RK , Shay JW , et al. ( 2013 ) Histone modifications and DNA double strand break repair after exposure to ionizing radiations . Radiation Research 179 : 383 ± 392 . https://doi.org/10.1667/RR3308.2 PMID: 23373901 72. Pandita TK , Richardson C ( 2009 ) Chromatin remodeling finds its place in the DNA double-strand break response . Nucleic Acids Res 37 : 1363 ± 1377 . https://doi.org/10.1093/nar/gkn1071 PMID: 19139074 73. Jain V , Hassan PA , Das B ( 2014 ) Radiation-induced conformational changes in chromatin structure in resting human peripheral blood mononuclear cells . Int J Radiat Biol 90 : 1143 ± 1151 . https://doi.org/10. 3109/09553002. 2014 .925605 PMID: 24844371


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Vinay Jain, Birajalaxmi Das. Global transcriptome profile reveals abundance of DNA damage response and repair genes in individuals from high level natural radiation areas of Kerala coast, PLOS ONE, 2017, DOI: 10.1371/journal.pone.0187274