Recent years have seen a significant increase in the use of radiation-emitting materials, devices, and ionising radiation technology, particularly in sectors such as industry, agriculture, and medicine (1). With the widespread adoption of interventional radiology procedures worldwide, workers involved in these operations may face substantial levels of radiation exposure due to the complexity and duration of each procedure (2). According to the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR), about 4.2 billion medical procedures were performed between 2009 and 2018, including 24 million interventional radiology procedures, involving around 11 million workers between 2010 and 2014 (3).
The International Commission on Radiological Protection (ICRP) Report 103 (4) recommended a review of non-cancerous effects of ionising radiation on normal tissues at low doses, including the sensitivity of the eye to radiation (5). The development of cataracts was previously considered a common tissue reaction, with effective dose thresholds established by the ICRP in 2007 at 5 Sv for chronic exposures and 2 Sv for acute exposures (4, 6). As for the absorbed dose, based on new epidemiological evidence, the ICRP has lowered the dose threshold for the eye lens to 0.5 Gy, having taken into account the latency period and the possibility of cataracts occurring at much lower doses, particularly with chronic exposure to relatively small doses. Consequently, the annual effective dose limit for the eye lens has been lowered from 150 mSv to 20 mSv (7).
Cardiologists are estimated to receive an average cumulative dose of 6 Sv without modifications to personal protective equipment, while support staff may receive around 1.5 Sv (8, 9). Moreover, a study tracking over 35,000 radiological technicians for 20 years revealed that even a relatively low cumulative dose of up to 60 mGy throughout their working lives could induce radiation injuries and elevate the risk of cataract development (10, 11), impaired vision and, ultimately, blindness as significant ocular adverse effects associated with exposure to ionising radiation (12, 13). Radiation retinopathy, on the other hand, presents as a progressive series of vascular changes, primarily affecting the macula. The onset, progression, and severity of retinopathy are mainly determined by the total radiation dose and treatment schedule, although factors such as concurrent chemotherapy and pre-existing diabetes may exacerbate vasculopathy by intensifying the attack of oxygen-derived free radicals on vascular cells (14).
However, the mechanisms underlying radiation-induced eye damage remain insufficiently understood. In Serbia, ethical concerns and considerations for animal welfare prohibit radiation testing on animals (15). Alternative methods, such as
Taking all of this into consideration, the primary aim of our study was to explore the mechanisms of radiation-induced eye injury using gene databases, software, and tools. Additionally, we aimed to demonstrate the utility of these resources in effectively identifying the effects and causes of damage resulting from radiation exposure.
Data presented in this article were obtained in July 2023. Figure 1 shows all the steps of the applied bioinformatics analysis explained in detail later in the text.
Detailed step-by-step diagram showing different phases of gene database analysis applied to investigate the relationship between the ionising radiation and eye injury
The Comparative Toxicogenomics Database (CTD;
The GeneMANIA Cytoscape plug-in (
ToppGene Suite (
Metascape (
To construct the figures, the obtained network was downloaded from Metascape and adjusted in the Cytoscape software [i.e. extracted from the pre-constructed GeneMANIA network by the Cytoscape MCODE plug-in (
Table 1 shows 13 eye diseases and six genes (
Diseases and interacting genes associated with eye injury caused by radiation (
Cellular response to gamma radiation | Cataract | |
Cellular response to ionising radiation | Diabetic retinopathy | |
Cellular response to ionising radiation | Retinal diseases | |
Cellular response to gamma radiation | Coloboma, ocular, with or without hearing impairment, cleft lip/palate, and/or impaired intellectual development | |
Cellular response to gamma radiation | Cataract 16, multiple types | |
Regulation of cellular response to gamma radiation | Cataract | |
Cellular response to gamma radiation | Myopathy, myofibrillar, fatal infantile hypertonic, alpha-B crystallin-related | |
Cellular response to X-ray | Cataract | |
Cellular response to gamma radiation | Alpha-B crystallinopathy | |
Cellular response to ionising radiation | Dry eye syndromes | |
Cellular response to ionising radiation | Cataract | |
Cellular response to gamma radiation | Vasculopathy, retinal, with cerebral leukodystrophy | |
Cellular response to ionising radiation | Graves disease |
Table 2 shows the expanded gene set, including the six genes from the original query and 20 related genes. The interactions for all 26 genes were physical (Figure 2), indicating that they might be involved in the same biological processes or pathways and that their products may interact to carry out specific functions.
GeneMANIA network of genes associated with eye injury caused by ionising radiation (black) together with the 20 related genes (grey). Interaction type: 100 % physical interactions (GeneMANIA;
Gene set linked to the eye injury caused by ionising radiation based on CTD and GeneMANIA analysis (
ATM serine/threonine kinase | 472 | |
Crystallin alpha B | 1410 | |
Sirtuin 1 | 23411 | |
Transforming growth factor beta 1 | 7040 | |
Three prime repair exonuclease 1 | 11277 | |
Yes1 associated transcriptional regulator | 10413 | |
TEA domain transcription factor 2 | 8463 | |
Ribosomal RNA processing 8 | 23378 | |
Citrate synthase | 1431 | |
TCDD inducible poly(ADP-ribose) polymerase | 25976 | |
Crystallin gamma C | 1420 | |
HIC ZBTB transcriptional repressor 1 | 3090 | |
Eukaryotic translation elongation factor 1 epsilon 1 | 9521 | |
Latent transforming growth factor beta binding protein 4 | 8425 | |
Nibrin | 4683 | |
MutS homolog 2 | 4436 | |
WW domain binding protein 1 | 23559 | |
Angiomotin like 1 | 154810 | |
Bone morphogenetic protein 3 | 651 | |
LBH regulator of WNT signaling pathway | 81606 | |
Crystallin gamma S | 1427 | |
Intraflagellar transport 20 | 90410 | |
Integrin subunit beta 8 | 3696 | |
Ficolin 1 | 2219 | |
Crystallin beta B2 | 1415 | |
ATR serine/threonine kinase | 545 |
Table 3 shows the top 15 gene ontology (molecular functions, biological processes, cellular components) and molecular pathways listed by statistical significance.
Top 15 gene ontology (molecular functions, biological processes) and molecular pathways associated with eye injury caused by ionising radiation (
Molecular functions | GO:0005212 | structural constituent of eye lens | 2.806E-8 | 4 | 25 |
GO:0032405 | MutLalpha complex binding | 6.840E-8 | 3 | 7 | |
GO:0032404 | mismatch repair complex binding | 3.214E-7 | 3 | 11 | |
GO:0032407 | MutSalpha complex binding | 3.412E-5 | 2 | 7 | |
GO:0044877 | protein-containing complex binding | 2.123E-4 | 9 | 1726 | |
GO:0003950 | NAD+ ADP-ribosyltransferase activity | 6.938E-4 | 2 | 30 | |
GO:0047485 | protein N-terminus binding | 7.323E-4 | 3 | 137 | |
GO:0050699 | WW domain binding | 8.919E-4 | 2 | 34 | |
GO:0004108 | citrate (Si)-synthase activity | 1.304E-3 | 1 | 1 | |
GO:0160011 | NAD-dependent protein decrotonylase activity | 1.304E-3 | 1 | 1 | |
GO:0160012 | NAD-dependent histone decrotonylase activity | 1.304E-3 | 1 | 1 | |
GO:0036440 | citrate synthase activity | 1.304E-3 | 1 | 1 | |
GO:0016763 | pentosyltransferase activity | 2.491E-3 | 2 | 57 | |
GO:0106231 | protein-propionyllysine depropionylase activity | 2.603E-3 | 1 | 2 | |
GO:0032129 | histone deacetylase activity (H3-K9 specific) | 2.603E-3 | 1 | 2 | |
Biological processes | GO:0071479 | cellular response to ionising radiation | 1.415E-11 | 7 | 90 |
GO:0042770 | signal transduction in response to DNA damage | 8.815E-11 | 8 | 200 | |
GO:0010212 | response to ionising radiation | 9.545E-11 | 8 | 202 | |
GO:0072331 | signal transduction by p53 class mediator | 9.545E-11 | 8 | 202 | |
GO:0071480 | cellular response to gamma radiation | 5.615E-10 | 5 | 34 | |
GO:0030330 | DNA damage response, signal transduction by p53 class mediator | 7.424E-10 | 6 | 83 | |
GO:0043516 | regulation of DNA damage response, signal transduction by p53 class mediator | 2.173E-9 | 5 | 44 | |
GO:0043517 | positive regulation of DNA damage response, signal transduction by p53 class mediator | 4.584E-9 | 4 | 17 | |
GO:0097190 | apoptotic signaling pathway | 7.241E-9 | 10 | 715 | |
GO:0071478 | cellular response to radiation | 8.951E-9 | 7 | 225 | |
GO:0010332 | response to gamma radiation | 4.104E-8 | 5 | 78 | |
GO:0009314 | response to radiation | 5.117E-8 | 9 | 646 | |
GO:0045786 | negative regulation of cell cycle | 8.888E-8 | 8 | 483 | |
GO:1901798 | positive regulation of signal transduction by p53 class mediator | 9.931E-8 | 4 | 35 | |
GO:0097193 | intrinsic apoptotic signalling pathway | 2.361E-7 | 7 | 363 | |
Cellular components | GO:0061773 | eNoSc complex | 4.112E-6 | 2 | 3 |
GO:0140552 | TEAD-YAP complex | 4.112E-6 | 2 | 3 | |
GO:0033553 | rDNA heterochromatin | 8.218E-6 | 2 | 4 | |
GO:0000781 | chromosome, telomeric region | 4.993E-5 | 4 | 173 | |
GO:0005677 | chromatin silencing complex | 1.237E-4 | 2 | 14 | |
GO:0140513 | nuclear protein-containing complex | 1.424E-4 | 8 | 1386 | |
GO:0016605 | PML body | 4.055E-4 | 3 | 122 | |
GO:0098687 | chromosomal region | 1.439E-3 | 4 | 419 | |
GO:0070310 | ATR-ATRIP complex | 2.389E-3 | 1 | 2 | |
GO:0032301 | MutSalpha complex | 2.389E-3 | 1 | 2 | |
GO:0034686 | integrin alphav-beta8 complex | 2.389E-3 | 1 | 2 | |
GO:0032302 | MutSbeta complex | 2.389E-3 | 1 | 2 | |
GO:0099126 | transforming growth factor beta complex | 3.582E-3 | 1 | 3 | |
GO:1902636 | kinociliary basal body | 3.582E-3 | 1 | 3 | |
GO:0005657 | replication fork | 3.703E-3 | 2 | 76 | |
Molecular pathways | M9703 | role of BRCA1, BRCA2 and ATR in Cancer Susceptibility | 3.010E-8 | 4 | 22 |
M39490 | DNA IR-damage and cellular response via ATR | 1.245E-7 | 5 | 81 | |
M39598 | DNA IR-double strand breaks and cellular response via ATM | 1.358E-6 | 4 | 55 | |
M648 | cell Cycle: G1/S Check Point | 1.012E-5 | 3 | 28 | |
137959 | BARD1 signalling events | 1.128E-5 | 3 | 29 | |
M258 | BARD1 signalling events | 1.128E-5 | 3 | 29 | |
1270252 | molecules associated with elastic fibres | 1.384E-5 | 3 | 31 | |
1309108 | HDR through Single Strand Annealing (SSA) | 2.580E-5 | 3 | 38 | |
1309104 | presynaptic phase of homologous DNA pairing and strand exchange | 3.016E-5 | 3 | 40 | |
M40049 | DNA repair pathways, full network | 3.166E-5 | 4 | 121 | |
1309097 | sensing of DNA Double Strand Breaks | 3.389E-5 | 2 | 6 | |
1270251 | elastic fibre formation | 3.497E-5 | 3 | 42 | |
1309103 | homologous DNA Pairing and Strand Exchange | 3.756E-5 | 3 | 43 | |
M39628 | integrated cancer pathway | 4.310E-5 | 3 | 45 | |
M39518 | ATM signalling in development and disease | 4.606E-5 | 3 | 46 |
Abbreviations: ATM – ataxia telangiectasia Mutated; ATR – ataxia telangiectasia and Rad3-related; ATRIP – ATR-interacting protein Complex; BARD1 – BRCA1-associated RING domain 1; BRCA1 – breast cancer type 1 susceptibility protein; BRCA2 – breast cancer type 2 susceptibility protein; DNA – deoxyribonucleic acid; HDR – homology directed repair; IR – ionising radiation; MutSalpha – mismatch repair protein MutS alpha; MutSbeta – mismatch repair protein MutS beta; NAD – nicotinamide adenine dinucleotide; PML – promyelocytic leukemia protein; SSA – single-strand annealing; TEAD-YAP – TEA domain transcription factor-Yes-associated protein; eNoSC – embryonic nuclear silencing complex; p53 – tumour protein 53
Figure 3A shows all input genes (n=26) forming a subnetwork associated with eye injury caused by ionising radiation, while Figure 3B shows highly interconnected genes within this subnetwork. The most important gene ontologies it identifies are DNA damage checkpoint signalling, DNA integrity checkpoint signalling, and signal transduction in response to DNA damage, which are all part of cellular response to ionising/gamma radiation (Table 4).
Subnetwork of interconnected genes associated with eye injury caused by ionising radiation (yellow) obtained with MCODE algorithm (
Gene ontology terms linked to the obtained MCODE network [Metascape software (
GO:0010212 | response to ionising radiation | −10.5 |
GO:0071479 | cellular response to ionising radiation | −10.4 |
GO:0071480 | cellular response to gamma radiation | −10.4 |
GO:0000077 | DNA damage checkpoint signalling | −12.3 |
GO:0031570 | DNA integrity checkpoint signalling | −12.2 |
GO:0042770 | signal transduction in response to DNA damage | −11.7 |
Table 5 shows that the identified miRNAs, namely hsa-miR-183 and hsa-miR-589, play a significant role in eye injury caused by ionising radiation, while hsa-miR-892b, hsa-miR-708, hsa-miR-3118, hsa-miR-3166, and hsa-miR-589 play a weaker role.
miRNAs linked to eye injury caused by ionising radiation (
hsa-miR-892b:mirSVR lowEffct | hsa-miR-892b:mirSVR non-conserved low effect-0.1-0.5 | 2.811E-7 | 8 | 1596 |
hsa-miR-183:mirSVR highEffct | hsa-miR-183:mirSVR conserved high effect-0.5 | 1.597E-6 | 6 | 853 |
hsa-miR-708:mirSVR lowEffct | hsa-miR-708:mirSVR non-conserved low effect-0.1-0.5 | 1.629E-6 | 7 | 1376 |
hsa-miR-3688-3p | 2.792E-6 | 6 | 940 | |
hsa-miR-3118:mirSVR lowEffct | hsa-miR-3118:mirSVR non-conserved low effect-0.1-0.5 | 4.640E-6 | 7 | 1613 |
hsa-miR-19a-3p | 6.859E-6 | 6 | 1100 | |
hsa-miR-19b-3p | 7.002E-6 | 6 | 1104 | |
hsa-miR-590-5p | 1.105E-5 | 4 | 317 | |
hsa-miR-3166:mirSVR lowEffct | hsa-miR-3166:mirSVR non-conserved low effect-0.1-0.5 | 1.146E-5 | 7 | 1853 |
hsa-miR-589:mirSVR highEffct | hsa-miR-589:mirSVR non-conserved high effect-0.5 | 1.182E-5 | 7 | 1862 |
hsa-miR-548ap-3p | 1.281E-5 | 6 | 1228 | |
hsa-miR-548t-3p | 1.281E-5 | 6 | 1228 | |
hsa-miR-548aa | 1.281E-5 | 6 | 1228 | |
hsa-miR-21-5p | 1.455E-5 | 4 | 340 | |
hsa-miR-19b:PITA | hsa-miR-19b:PITA TOP | 1.615E-5 | 5 | 741 |
Non-conserved miRNAs: miRNAs specific to particular species or closely related groups, contrasting with widely preserved conserved ones. Conserved miRNA: miRNAs highly preserved across diverse species, exhibiting similar sequences and functions, crucial for gene regulation
The CTD database listed several diseases/conditions associated with eye injury caused by ionising radiation, including cataract myopathy, alpha-b crystallinopathy, retinal vasculopathy, retinal diseases, dry eye syndromes, and ocular coloboma. These diseases/conditions are linked to six genes, namely
The
The
The
The
The
Finally, the
As expected, some of the identified molecular functions were related to DNA repair mechanisms (MutLalpha complex binding and mismatch repair complex binding), while others were related to protein binding and enzymatic activities (i.e., protein-containing complex binding). Other functions could be characterised as metabolic processes (citrate synthase activity and citrate (Si)-synthase activity) or post-translational modifications of proteins (protein-propionyllysine depropionylase activity).
On the other hand, the list of biological processes (including those obtained with the MCODE algorithm) was focused on DNA damage and more specifically on the role of the p53 signalling pathway in regulating DNA repair. Ionising radiation can penetrate tissues, disrupt the DNA helix, and cause breaks in one or both strands (41). The resulting DNA damage triggers a cascade of cellular responses, including DNA repair mechanisms and cell cycle checkpoints. If the damage is severe or remains unrepaired, it can lead to genome instability, mutations, cell death, or other adverse outcomes (42). In the context of eye injury caused by ionising radiation, such DNA damage can affect the integrity and function of ocular cells, and if a sufficient number of cells is affected, it can lead to the functional impairment of the lens. As the DNA damage accumulates in the secondary fibre cells for several months after the initial incident, new structures build up that scatter light (43). Wolf et al. (44) reported that 11 Gy of soft x-irradiation, specifically targeting the head region of mice, induced the development of cortical cataracts within the first month, which progressed to an advanced stage 5–11 months after exposure. Although the initial DNA strand breaks were repaired within 30 minutes, DNA damage was persistent over the first 72 h after irradiation, as indicated by the presence of the DNA adduct 8-hydroxyguanosine (8-OHG) and the DNA repair protein X-ray repair cross-complementing protein 1 (XRCC1). This persistence suggests that DNA repair mechanisms may be overwhelmed by radiation-induced DNA lesions and unable to prevent the development of advanced cortical cataracts.
When DNA is damaged, a cascade of signalling events is triggered, which results in the activation of several proteins involved in cell cycle arrest, DNA repair, and apoptosis (45). Lower radiation doses result in lower damage, which allows better repair and reduces the number of cells stuck in the G1/S phase (46). Namely, DNA damage triggers the checkpoint signalling system to prevent the cell from continuing its cycle until the damage has been repaired. Part of this process is the induction of the ATM, ATR, and Chk1/2 proteins, which start cell cycle arrest and DNA repair (47). DNA double strand breaks trigger the ATM/Chk2 pathway, whereas DNA single strand breaks or complex lesions generally start the ATR/Chk1 pathway (47). Markiewicz et al. (48) reported that double strand breaks got repaired more slowly in mouse lens epithelial cells after exposure to 20 than 100 mGy. As a consequence of changes in cell proliferation and density the lens aspect ratio in treated mice changed 10 months after irradiation (48), which suggests impaired DNA repair and checkpoint activation.
Cellular components associated with eye injury (Table 3) consist of regulator complexes such as eNoSC and chromatin silencing. The eNoSC (energy-dependent nucleolar silencing complex), which includes the
As for molecular pathways listed in Table 3, some include cancer susceptibility and some DNA damage and cellular response or double strand breaks and cellular response. After exposing retinal photoreceptor cells to ionising radiation doses of 2, 4, 6, 8, 10, and 20 Gy, Yao et al. (52) found increased phosphorylation levels of Chk1 and p53 downstream the ATM pathway (52), which suggests that these signalling events are part of the cellular response to DNA damage and are necessary for the cell to initiate repair mechanisms or apoptosis.
The
Homology-directed repair through single-strand annealing (SSA) and homologous DNA pairing and strand exchange are involved in the mechanisms of DNA repair (55, 56), while the presynaptic stage of homologous DNA pairing and strand exchange pathway is a specific stage in the process of homologous recombination (57).
The list of miRNAs obtained on the basis of genes involved in eye injury caused by radiation could provide valuable insights into the molecular mechanisms underlying this condition. miRNAs are small non-coding RNAs that play important roles in post-transcriptional regulation of gene expression (58) and pathways, and their dysregulation has been implicated in various diseases, including cancer and neurodegenerative disorders (59). Our study has identified several miRNAs with a high (hsa-miR-183 and hsamiR-589) or low effect (hsa-miR-892b, hsa-miR-708, hsa-miR-3118, and hsa-miR-3166) on the target genes. The (miR)183 cluster microRNAs, i.e., miRs-183, -96, and -182, have closely synchronised expression during development and are necessary for sensory organ maturation. They are particularly abundant in retinal photoreceptors and are light-responsive (60).
Our study demonstrates the potential utility of the proposed toxicogenomics data mining in exploring molecular mechanisms of ionising radiation. However, data mining relies on the reliability and completeness of interactions described in online sources such as the CTD database. Furthermore, the obtained data are based on statistical associations between stressor-gene-disease relationships and do not take into account important factors like the dose-response relationship, exposure route, exposure duration, and individual sensitivity.
This study has identified