Deciphering the molecular landscape of ionising radiation-induced eye damage with the help of genomic data mining

The Comparative Toxicogenomics Database (CTD; https://ctdbase.org), more and more often referred to as the “Golden Set Database”, is a publicly available database that evaluates and summarises the data on associations between chemicals, genes, and diseases, and also provides information about the gene ontology (biological processes, molecular functions), molecular pathways, and phenotype (21, 22). It is updated regularly to ensure that all the information it contains is reliable, consistent, and easily accessible. In our study, we relied on CTD to assess cellular response to ionising radiation and identify a set of genes linked to the eye injury based on the listed diseases and interacting genes. Using the key word “radiation” to search through gene ontology (GO) annotations, we obtained 31 matches. Among these, “cellular response to radiation” was the broadest term encompassing all radiation types in the CTD database. From the available options we selected “response to ionising radiation”, as this gene ontology term covers all types of ionising radiation, including cellular response to gamma radiation and X-rays. Then we filtered diseases of the eye associated with “response to ionising radiation” (“eye diseases”) and identified six genes associated with the listed eye diseases for further analysis. Our decision to consider all listed eye diseases was driven by a wish to gain a deeper and more comprehensive insight into the effects of ionising radiation on eye health across a range of clinical conditions.

The GeneMANIA Cytoscape plug-in (https://apps.cytoscape.org/apps/genemania) generates a list of the genes most similar to the ones entered into the query and explores the links between them (23). We used this plug-in to explore gene interactions, but also to expand the original set with 20 related genes [test organism: H. sapiens (human)]. The obtained gene set for further analysis now consisted of 26 genes. There are several possible interactions identified by the GeneMANIA server: protein-protein interactions, co-expressions (where gene expression levels show similarity across conditions in a gene expression study), gene interactions (functional associations observed when one gene disruption affects the other gene), shared protein domains, co-localisation (genes expressed in the same tissue or proteins found in the same location), pathway sharing (gene products participate in the same reaction within a pathway), and finally, predicted functional relationships, most often protein interactions, derived from known functional relationships from another organism based on orthology (two proteins are predicted to interact if their orthologues are known to interact in that organism) (24).

ToppGene Suite (https://toppgene.cchmc.org) is an online tool that uses functional descriptors and a protein association network to prioritise candidate genes. The ToppGene ToppFun feature (https://toppgene.cchmc.org/enrichment.jsp) allows exploration of ontologies (gene ontology, pathway), phenotype, pharmacome, miRNA, and other parameters (25). In this study, the ToppGeneSuite portal (ToppFun function) was used to identify probable molecular mechanisms involved in radiation-associated eye injury based on the above mentioned set of 26 genes to better understand their role in the context of a larger biological system. Biological processes, molecular functions, cellular components, molecular pathways, and miRNA were selected as the main functions of interest [p-value: 0.05, corrected for false discovery rate (FDR)]. The obtained miRNA were ranked by the mirSVR, a new machine learning method for ranking microRNA target sites by a down-regulation score (26).

Metascape (https://metascape.org) is a web-based portal and a comprehensive resource for gene list annotation and analysis (27) which uses the Molecular Complex Detection (MCODE) algorithm to locate dense gene clusters in a network based on their topology (27, 28). We used this algorithm to identify highly interconnected subnetworks of the identified genes and related biological processes or diseases of interest. An MCODE network contains a subset of proteins that form physical interactions with at least one other member in the list. If the network contains between three and 500 proteins, the algorithm is applied to identify densely connected network components (27).

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 (https://apps.cytoscape.org/apps/mcode)].

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 ATM, CRYAB, SIRT1, TGFB1, TREX1, and YAP1.

The ATM gene is part of the cellular response to DNA damage and plays a critical role in response pathway. ATM protein kinase regulates many signalling pathways by phosphorylating and controlling its substrates, a process whose failure results in genome-wide instability (29).

The CRYAB gene encodes a protein called alpha-crystallin B. CRYAB binds to crystallins and prevents them from aggregating, while alpha-crystallin B acts as an intracellular chaperone that counteracts oxidative stress-induced damage and apoptosis (30). Crystallins are abundant in the lens and to a smaller degree in other cells, including the retina. They are involved in cytoplasmic organisation and complex molecular mechanisms that regulate cell architecture and function (31). For the lens to be transparent, crystallins must remain densely packed (32), or else mutations increase the risk of developing cataracts (33).

The SIRT1 gene encodes a protein called Sirtuin 1, which is an epigenetic regulator involved in DNA repair (34) but also in a variety of biological functions such as metabolic regulation, cell maintenance, optimal ageing, and tumorigenesis. It is also active in apoptosis and cell proliferation in reaction to various stressors and metabolic imbalances (35, 36).

The TGFB1 gene encodes a protein called transforming growth factor beta 1 (TGF-β1), involved in the regulation of cell growth and often associated with anti-proliferative effects (37).

The TREX1 gene encodes a protein called three prime repair exonuclease 1, whose primary function is to degrade cytoplasmic single-stranded DNA or mispaired 3′ termini of DNA duplexes (38). Mutations in this gene have been associated with the development of age-related cataracts (39).

Finally, the YAP1 gene encodes a protein called yes-associated protein 1, important for the regulation of cell proliferation (40).

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 SIRT1 gene, silences rDNA and shields mammalian cells from energy-linked apoptosis (49). Others, such as the ATRATRIP complex, are key players in DNA damage response, triggering repair mechanisms upon radiation exposure (47). Additionally, components like PML bodies and the MutS complexes are involved in DNA repair processes crucial for maintaining genomic integrity (50, 51), while growth factor signalling pathways may improve tissue repair mechanisms post-exposure (53).

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 BARD1 signalling pathway involves the BARD1 protein in checkpoint activities and DNA damage response and repair (54), while molecules associated with elastic fibres are involved in the formation and maintenance of connective tissue.

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.