Global transcriptome profile reveals abundance of DNA damage response and repair genes in individuals from high level natural radiation areas of Kerala coast
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.
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 [
]. 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
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 [
]. 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
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
], plants [
], demographic characterization of human population. 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
]. 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
].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
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belonging to background dose group >5mGy/year [
]. 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 [
]. 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.
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 [
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 [
]. 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 [
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
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 [
], human skin fibroblasts and keratinocytes [
], PBMC [
umbilical vein endothelial cells [
], lymphoblastoid cells [
] and human embryonic stem
cells . There are reports, which have shown the induction of transcriptional changes after
ex-vivo acute exposure to doses as low as 1 cGy [
]. 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 [
]. 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.
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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.
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 [
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).
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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)
] 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
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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.
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.
Fold change 1.3
P-value set at < 0.05
Fold change 1.5
Fold change 2.0
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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.
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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
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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),
Grp I vs Grp II
Grp I vs Grp III
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Grp I vs Grp II
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.
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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.
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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.
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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.
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
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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 [
]. However, recent studies have shown a lower
induction and efficient repair of DNA damage in HLNRA individuals [
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
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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 [
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
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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
] 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
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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 [
]. 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 [
]. 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 [
]. 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
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 [
]. Recently, it
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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 [
]. 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 [
] 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
]. 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. The role of histone modification/chromatin changes in
DNA repair after acute exposure to IR has been reported [
]. 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,
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RAD52, DCLRE1C), cell cycle regulation/proliferation (RHOA, CABLES2, TGFB2, IL16), and
stress response (GSTP1, PPP2R5A, DUSP22) to be active chronic low level radiation exposures
. 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
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.
S1 Table. Details of primer sequences and UPL probe numbers of the genes studied using
real time PCR.
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.
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Supervision: Birajalaxmi Das.
Validation: Vinay Jain, Birajalaxmi Das.
Visualization: Birajalaxmi Das.
Writing ± original draft: Vinay Jain, Birajalaxmi Das.
Writing ± review & editing: Birajalaxmi Das.
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26 / 28
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