Deposition of energy leading to occurrence of cataracts

Emma Carrothers¹, Meghan Appleby¹, Vita Lai¹, Tatiana Kozbenko¹, Dalya Alomar¹, Benjamin Smith¹, Robyn Hocking³, Carole Yauk², Ruth Wilkins¹, Vinita Chauhan¹ 

(1) Consumer and Clinical Radiation Protection Bureau, Environmental and Radiation Health Sciences Directorate, Health Canada, Ottawa, Ontario  

(2) Department of Biology, University of Ottawa, Ottawa, Ontario  

(3) Learning and Knowledge and Library Services, Health Canada, Ottawa, Ontario, Canada 

Consultants

Nobuyuki Hamada¹, Patricia Hinton², Elizabeth A. Ainsbury³

(1) Biology and Environmental Chemistry Division, Sustainable System Research Laboratory, Central Research Institute of Electric Power Industry (CRIEPI), Komae, Tokyo, Japan

(2) Canadian Forces Environmental Medicine Establishment, Toronto, Ontario, Canada  

(3) Radiation, Chemical and Environmental Hazards Division, UK Health Security Agency, United Kingdom.

Environmental Research Group within the School of Public Health, Faculty of Medicine at Imperial College of Science, Technology and Medicine, London, UK.

An AOP was developed describing a simplified path from “deposition of energy” (MIE; KE#1686) to cataracts (AO; KE#2083). The AOP is initiated by deposition of energy resulting in oxidative stress (KE#1392) within cells from increased free radical generation. If this exceeds antioxidant defence mechanisms, the oxidative stress, in turn, can damage molecules, the most well-studied include DNA and proteins.  Within the lens of the eye modified proteins (KE#2081) can aggregate, such as crystalline, and if not eliminated, can accumulate resulting in lens opacity. Concurrently, unmanaged oxidative stress can increase oxidative DNA damage (KE#1634) leading to DNA strand breaks (KE#1635). If these lesions are inadequately repaired (KE#155), an increase mutation frequency (KE#185) in critical genes and chromosomal aberrations (KE#1636) can occur. Mutations in genes associated with cell cycling can lead to uncontrolled cell proliferation (KE#870) of lens epithelial cells and the eventual AO, cataracts. The overall assessment of this AOP indicates high biological plausibility of the KERs as they are well established and understood; moderate levels of evidence support the essentiality and the Bradford-Hill empirical evidence criteria; low weight of evidence was identified for quantitative understanding across adjacent relationships, with some uncertainties and inconsistencies in mechanisms. Broadly, the information presented in this AOP can be used to support the review of radiation effects classification and broadly the system of radiological protection. 

Cataracts, one of the leading causes of blindness, are a progressive condition in which the lens of the eye develops opacities and becomes cloudy, resulting in blurred vision as well as glare and haloes around lights (National Eye Institute, 2022). For this AOP, a cataract is defined when over 5% of the lens is opacified. Cataracts typically occur after the age of 50 in humans, as an age-related disease (Liu et al., 2017); however, progression of this disease can be initiated or accelerated after exposure to a variety of agents, one of which is radiation.  

For radiation induced cataracts, most research shows that the anatomical location is within the posterior sub capsular region of the eye with limited occurrence in the cortical and nuclear region. Available epidemiological evidence confirms a positive statistically significant association between radiation exposure and cataracts (Nakashima et al., 2006; Worgul et al., 2007; Chylack et al., 2012; Little et al., 2018). The data comes from Chernobyl workers, radiologic technologists, and patients exposed to radiation through medical procedures, with the most compelling evidence derived from atomic bomb survivors. Although there is concern for the role of long duration space flight missions in cataract formation, there is limited data from astronauts.  

In 2012, the International Commission on Radiological Protection (ICRP) recommended lowering the occupational eye lens dose limit from 150 mSv per year to an average of 20 mSv, with no single year exceeding 50 mSv. This revision was based on new evidence from both radiobiological studies and relevant epidemiological data. Assessment of the literature indicated a threshold dose for radiation induced cataracts of about 0.5 Gy (ICRP, 2012). This change in exposure limit has led to a need to further understand radiation-induced effects at lower doses and dose-rates. It is believed the progression of cataracts at high  doses and s higher dose-rates generally induce more damage than lower dose-rates (Brooks et al., 2016).

This AOP provides a summary of the relevant studies and endpoints that can inform future research designed to understand the role of radiation in causing cataracts. Assays and study designs spanning biological levels of organization across relevant models were identified, with the end goal to improve testing strategies and understanding in risk from low dose low dose-rate exposures. 

The present AOP is a component of a broader network that links the deposition of energy with three AOs of vascular remodeling, bone loss, and learning and memory impairment. The network is intended to demonstrate the interconnectivity in mechanisms of diseases. The strategy for developing this network began with the creation of a hypothesized set of KEs and KERs identified through narrative review of key literature as well as extensive subject matter expert consultation. 

Many KEs were proposed for the preliminary network; however, attention was focused on those deemed most established for disease progression, routinely and robustly measurable and which had greatest biological plausibility for connectivity to the rest of the pathway. The preliminary network served as the basis for the next stage of development in which a scoping review methodology was used to collect a weight of evidence (WOE). Details of the methodology are described by Kozbenko et al. and are summarized below (Kozbenko et al., 2022).  

In short, literature collection consisted of structured database searches, followed by prioritization and screening stages. Focused searches were completed for each of the KERs in the pathway using combinations of key words related to involved KEs, as well as an overarching search, collecting all references broadly related to the MIE and AO. Following the literature searches, the results were processed in two stages to determine their inclusion in the pathway’s WOE. Both stages determined reference relevance according to inclusion and exclusion criteria that had been outlined prior to screening in a PEOE (Population, Exposure, Outcome, and Endpoint) statement.  

In the first phase, search results were prioritized using the SWIFT-Review software (Sciome, Durham NC, https://www.sciome.com/swift-review/). This software was used to identify the references most closely related to the PEOE statement as determined by the software created tags. Relevant references identified by SWIFT were then screened in Distiller SR (Evidence Partners) using the defined PEOE criteria. Human screeners additionally evaluated references for their suitability to meet the Bradford-Hill criteria. At the end of the process, screened-in studies were used for the WOE evaluation.  

The present cataract AOP was continually refined throughout the screening process based on evidence extracted from the literature. The final pathway includes several KEs previously existing in the AOP Wiki: deposition of energy (KE #1686), increased oxidative stress (KE #1392), increased DNA strand breaks (KE #1635), increased oxidative damage to DNA (KE #1634), inadequate repair (KE #155), increased mutations (KE #185), increased chromosomal aberrations (KE #1636), and increased cell proliferation (KE #870). Newly created KEs for this pathway include modified proteins (KE #2081) and cataracts (KE #2083). It should be noted that the WOE supporting this AOP is predominantly from space exposures, unless there was no available evidence, in which case other types of radiation stressors supported the response-response relationships. Human and animal studies were prioritized unless data was limited, and then in vitro studies were used to support the KERs. Due to the regulatory application, studies using relevant eye lens models were prioritized. 

Summary of evidence (KE & KER relationships and evidence) 

This assessment provides an overview of the pathway. Further details and references can be found in the individual KEs and KERs and within the AOP report. 

Biological Plausibility 

This AOP begins with an MIE (deposition of energy) and then branches to cataract formation either from modified proteins or through DNA damage processes. Ionization events from deposition of energy interact directly or indirectly with the DNA. Indirect damage can also occur when water molecules dissociate producing radicals such as reactive oxygen species (ROS) that induce DNA breaks (Ahmadi et al., 2022). Moreover, a cascade of ionization events can cause the formation of clustered damage (Joiner and van der Kogel, 2009). Many studies use ultraviolet radiation as a stressor, and it is important to note that ionizing and non-ionizing radiation interact through  different mechanisms when inducing cataracts. Ionizing radiation can remove tightly bound electrons from atoms to create charged particles,  but also excite molecules without ionization. The absorption of non-ionizing radiation results in heat generation from molecular vibrations (Alcócer et al., 2020).

Deposition of energy can also lead to high levels of ROS and reactive nitrogen species (RNS) (collectively RONS) (Tangvarasittichai & Tangvarasittichai, 2019). There are several pathways leading to ROS, but radiolysis is the most prominent. Free radicals can combine to produce hydrogen peroxide, hydroxide, superoxide, and hydroxyl (Tian et al., 2017; Venkatesulu et al., 2018). Interactions with NO can also lead to RNS (Wang et al., 2019). Additionally, deposited energy can directly upregulate enzymes involved in reactive RONS production (de Jager et al., 2017). Activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) within mitochondria can generate more ROS (Soloviev & Kizub, 2019). Energy absorption by an unstable molecule, such as the chromophore NADPH (Jurja et al., 2014), is another route for radical production. Overwhelming amounts of free radicals can decrease antioxidant levels, causing oxidative stress (Wang et al., 2019). 

Protein damage leading to cataracts 

Alongside DNA as a target to energy deposition, other macromolecules can be damaged. In terms of cataracts, there is much evidence to show that protein modifications such as phosphorylation, deamidation, oxidation, disulfide bonds (Hanson et al., 2000), increased cross-linking, altered water-solubility, and increased protein aggregation are critical to disease progression (Fochler & Durchschlag, 1997; Reisz et al., 2014; Wang et al. 2020; Chandrasekher et al., 2004 ). ROS can also cause many alterations including conformational changes, protein cross-link formation, oxidation of amino acid side chains (Uwineza et al., 2019), and protein aggregation (Moreau et al., 2012). For example, alpha crystallin aggregation can be induced by free radicals oxidizing the thiol groups (Cabrera & Chihuailaf, 2011; Moreau & King 2012; Stohs, 1995). Finally, these modified proteins can lead directly to cataracts, the AO. Aggregated proteins cause improper lens epithelial cell (LEC) organization and increases light scattering, therefore resulting in lens opacity (Hamada et al., 2014). Additionally, modifications to connexin protein can lead to improper LEC layering, which has been linked to cataracts in humans (NCRP, 2016).  

DNA damage leading to cataracts 

Oxidative stress is also directly connected to increased DNA strand breaks. Cells under oxidative stress have excessive levels of ROS, molecules that can oxidize and remove nitrogenous bases, producing nicks in the DNA strand known as single strand breaks (SSB). Under circumstances when multiple SSBs are in close proximity, they may combine to form double strand breaks (DSB). Furthermore, these strand breaks and a combination of various DNA abnormalities occurring in close proximity can create complex lesions that are more difficult to repair (Nickoloff et al., 2020). The formation of SSBs induces base excision repair (BER), a DNA repair mechanism; however, cells are often unable to support multiple sites of repair in one area, leading to residual unrepaired SSBs that will increase the number of DSBs. It has been shown that radiation-generated ROS are more likely to produce clustered damage (Cannan & Pederson, 2016). Increased oxidative stress can also lead to increased oxidative DNA damage. In this case, ROS can induce DNA lesions, such as oxidized nucleotides or DNA breaks (Collins, 2014). 

DNA strand breaks can lead to inadequate DNA repair. DSBs, the most detrimental form of this damage (Iliakis et al., 2015), are often formed in the G1 phase of the cell cycle. Cells utilize various systems to repair DNA damage, the most error-prone pathway being non-homologous end-joining (NHEJ). Since NHEJ is an active pathway for DNA DSB repair in the G1 phase of the cell cycle, DSBs are repaired using this pathway, leading to decreased repair accuracy (Jeggo et al., 1998). Although NHEJ is predominantly the preferred repair mechanism throughout the cell cycle, homologous recombination (HR) and single-stranded annealing (SSA) are favored during the S and G2 phases in scenarios where the NHEJ repair pathway is inhibited. The absence of HR leading to an increase in SSA activity is still a matter to debate (Ceccaldi et al., 2016). Furthermore, clustered damage generated by high linear energy transfer radiation (Nikitaki et al., 2016), overwhelms the repair systems, leading to increased probability of inadequate repair (Tsao, 2007). 

Similarly, increased oxidative damage to DNA can also lead to inadequate repair. Repair systems are unable to deal with increased levels of lesions within a small area, resulting in decreased repair ability and therefore, inadequate repair (Georgakilas et al., 2013). Moreover, unrepaired oxidative lesions may be incorrectly bypassed during DNA replication, leading to the insertion of incorrect bases opposite unrepaired lesions (Shah et al., 2018). Imbalances between the level of oxidative DNA lesions and cellular repair capacity can also lead to inadequate repair (Brenerman et al., 2014). Non-DSB oxidative DNA damage can alter nuclease or glycosylase activity, resulting in decreased local DNA repair ability (Georgakilas et al., 2013). 

One of the possible outcomes of inadequate repair is increased mutations. DNA repair mechanisms, such as NHEJ (Sishc & Davis, 2017), break-induced replication (BIR), and microhomology-mediated break-induced replication (MMBIR) can be error-prone, leading to increased mutagenesis and genomic instability (Kramara et al., 2018). 

Inadequate repair can also lead to increased chromosomal aberrations (CA). The best-known model for this KER holds that unrepaired DSBs eventually lead to CAs (Schipler & Iliakis, 2013). Alternate models suggests that CAs occur when the enzymes responsible for binding DNA strands during the repair of enzyme-induced DNA breaks dysfunctions. Failure of different binding enzymes would lead to different forms of CAs (Bignold, 2009).  

Increased mutations and increased CAs are both linked to increased cell proliferation; however, as no lens-specific data was found, the existing relationships in the Wiki (KER: 1978 and 1979) have not been altered. This presents a possible focus for future research. 

Finally, increased cell proliferation of the metabolically active LECs can lead to cataracts. The lens is composed of several zones, with the germinative zone (GZ) being the only one that is mitotically active. In healthy lenses, cells in the GZ replicate and differentiate into lens fiber cells (LFCs). The LFCs are organelle-free, allowing light to pass through the lens. However, in cases of increased proliferation, cells are forced out of the GZ before forming fully differentiated LFCs. These improperly differentiated cells have not lost all of their organelles, resulting in reduced lens transparency (Ainsbury et al., 2016; Hamada, 2017; McCarron et al., 2022). As the lens is a closed system with little turnover, these cells are not removed, and their accumulation contributes to the cataractogenic load, a gradual lens opacification throughout life, which can eventually lead to cataracts (Ainsbury et al., 2016; Uwineza et al., 2019).  

Time- and dose- and incidence-concordance 

Overall evidence of time- and dose-concordance is moderate to low throughout the AOP. Certain relationships, particularly those directly connected with deposition of energy, are well supported with measurable changes in expression in a temporal and dose concordant manner. However, KEs at the cellular and organ level are generally supported by a weaker WOE with inconsistencies. The use of different models, time-points and radiation types across studies may be the reason for inconsistencies.  

Evidence of time concordance is demonstrated by the occurrence of upstream KEs at earlier timepoints than the downstream KEs. Time concordance involving deposition of energy is well supported by studies showing the deposition of energy followed by downstream changes later in a time course. Studies using in vitro and in vivo models have found downstream effects occurring within minutes to years of the MIE. For example, oxidative stress in human LECs can occur within an hour following 0.25 Gy γ-rays exposure (Ahmadi et al., 2022), while it may take months to years for cataract development under radiotherapy or space radiation exposure (Gragoudas et al., 1995; Cucinotta et al., 2001). However, cellular and organ level events are not well-studied to demonstrate consistent time concordance.  

Studies in the AOP provide evidence of upstream KEs observed at the same doses or lower doses as the downstream KEs. The KERs directly involving the deposition of energy contain  the most evidence for dose concordance. Downstream KERs have limited support for dose concordance in a lens model but are supported with evidence from other cell types.  

A few studies demonstrate greater changes produced by the upstream KE than the downstream KE following a stressor (incidence concordance). One KER showing incidence concordance is oxidative stress to DNA strand breaks. For example, there was a 5x increase of DNA strand breaks, while only a ~1.4-fold increase in oxidative stress marker in human LECs (Liu et al., 2013a).   

As the International Commission on Radiological Protection works to review literature on health effects from radiation exposure, the collected knowledge presented in this AOP will provide a structured approach to guide future recommendations. With better designed experiments that cross biological levels of organization, more informative quantitative data will be generated that can then inform risk assessment strategies. A stronger evidence base can provide better justification to support guidelines and standards for future space missions and settings related to occupational, environmental, and medical exposures, where cataracts are of concern.

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