LDLensRad (the European CONCERT project starting in 2017): Towards a full mechanistic understanding of low dose radiation induced cataracts

Cataract is the leading cause of visual impairment which can result in blindness. Cataract formation has been associated with radiation exposure; however, the mechanistic understanding of this phenomenon is still lacking. The goal of this study was to investigate mechanisms of cataract induction in isolated lens epithelial cells (LEC) exposed to ionizing radiation. Human LECs from different genetic backgrounds (SV40 immortalized HLE-B3 and primary HLEC cells) were exposed to varying doses of 137Cs gamma rays (0, 0.1, 0.25 and 0.5 Gy), at low (0.065 Gy/min) and higher (0.3 Gy/min) dose rates. Different assays were used to measure LEC response for, e.g., viability, oxidative stress, DNA damage studies, senescence and changes to telomere length/telomerase activity at two time points (1 h and 24 h, or 24 h and 15 days, depending on the type of assay and expected response time). The viability of cells decreased in a dose-dependent manner within 24 h of irradiation. Measurement of reactive oxygen species showed an increase at 1 h postirradiation, which was alleviated within 24 h. This was consistent with DNA damage results showing high DNA damage after 1 h postirradiation which reduced significantly (but not completely) within 24 h. Induction of senescence was also observed 15 days postirradiation, but this was not attributed to telomere erosion or telomerase activity reduction. Overall, these findings provide a mechanistic understanding of low-dose radiation-induced cataractogenesis which will ultimately help to inform judgements on the magnitude of risk and improve existing radiation protection procedures.

Recent epidemiological and experimental animal data, as well as reanalyses of data previously accumulated, indicate that the lens of the eye is more radiosensitive than was previously thought. This has resulted in a reduction of the occupational lens dose limit within the European Union countries, Japan and elsewhere. This Commentary introduces the work done by the LDLensRad Consortium contained within this Focus Issue, towards advancement of understanding of the mechanisms of low dose radiation cataract.

Ionising radiation interacts with lenses and retinae differently. In human lenses, posterior subcapsular cataracts are the predominant observation, whereas retinae of adults are comparably resistant to even relatively high doses. In this study, we demonstrate the effects of 2 Gy of low linear energy transfer ionising radiation on eyes of B6C3F1 mice aged postnatal day 2. Optical coherence tomography and Scheimpflug imaging were utilised for the first time to monitor murine lenses and retinae in vivo. The visual acuity of the mice was determined and histological analysis was conducted. Our results demonstrated that visual acuity was reduced by as much as 50 % approximately 9 months after irradiation in irradiated mice. Vision impairment was caused by retinal atrophy and inner cortical cataracts. These results help to further our understanding of the risk of ionising radiation for human foeti (∼ 8 mo), which follow the same eye development stages as neonatal mice.

Ionizing radiation is widely known to induce various kinds of lens cataracts, of which posterior subcapsular cataracts (PSCs) have the highest prevalence. Despite some studies regarding the epidemiology and biology of radiation-induced PSCs, the mechanism underscoring the formation of this type of lesions and their dose dependency remain uncertain. Within the current study, our team investigated the in vivo characteristics of PSCs in B6C3F1 mice (F1-hybrids of BL6 × C3H) that received 0.5-2 Gy γ-ray irradiation after postnatal day 70. For purposes of assessing lenticular damages, spectral domain optical coherence tomography was utilized, and the visual acuity of the mice was measured to analyze their levels of visual impairment, and histological sections were then prepared in to characterize in vivo phenotypes. Three varying in vivo phenotype anterior and posterior lesions were thus revealed and correlated with the applied doses to understand their marginal influence on the visual acuity of the studied mice. Histological data indicated no significantly increased odds ratios for PSCs below a dose of 1 Gy at the end of the observation time. Furthermore, our team demonstrated that when the frequencies of the posterior and anterior lesions were calculated at early time points, their responses were in accordance with a deterministic model, whereas at later time points, their responses were better described via a stochastic model. The current study will aid in honing the current understanding of radiation-induced cataract formation and contributes greatly to addressing the fundamental questions of lens dose response within the field of radiation biology.

Recent epidemiological findings and reanalysis of historical data suggest lens opacities resulting from ionizing radiation exposures are likely induced at lower doses than previously thought. These observations have led to ICRP recommendations for a reduction in the occupational dose limits for the eye lens, as well as subsequent implementation in EU member states. The EU CONCERT LDLensRad project was initiated to further understand the effects of ionizing radiation on the lens and identify the mechanism(s) involved in radiation-induced cataract, as well as the impact of dose and dose-rate. Here, we present the results of a long-term study of changes to lens opacity in male and female adult mice from a variety of different genetic (radiosensitive or radioresistant) backgrounds, including mutant strains Ercc2 and Ptch1, which were assumed to be susceptible to radiation-induced lens opacities. Mice received 0.5, 1 and 2 Gy 60Co gamma-ray irradiation at dose rates of 0.063 and 0.3 Gy min–1. Scheimpflug imaging was used to quantify lens opacification as an early indicator of cataract, with monthly observations taken post-irradiation for an 18-month period in all strains apart from 129S2, which were observed for 12 months. Opacification of the lens was found to increase with time post-irradiation (with age) for most mouse models, with ionizing radiation exposure increasing opacities further. Sex, dose, dose rate and genetic background were all found to be significant contributors to opacification; however, significant interactions were identified, which meant that the impact of these factors was strain dependent. Mean lens density increased with higher dose and dose rate in the presence of Ercc2 and Ptch1 mutations. This project was the first to focus on low (<1 Gy) dose, multiple dose rate, sex and strain effects in lens opacification, and clearly demonstrates the importance of these experimental factors in radiobiological investigations on the lens. The results provide insight into the effects of ionizing radiation on the lens as well as the need for further work in this area to underpin appropriate radiation protection legislation and guidance.

Lens epithelial cell proliferation and differentiation are naturally well regulated and controlled, a characteristic essential for lens structure, symmetry and function. The effect of ionizing radiation on lens epithelial cell proliferation has been demonstrated in previous studies at high acute doses, but the effect of dose and dose rate on proliferation has not yet been considered. In this work, mice received single acute doses of 0.5, 1 and 2 Gy of radiation, at dose rates of 0.063 and 0.3 Gy/min. Eye lenses were isolated post-irradiation at 30 min up until 14 days and flat-mounted. Then, cell proliferation rates were determined using biomarker Ki67. As expected, radiation increased cell proliferation 2 and 24 h post-irradiation transiently (undetectable 14 days post-irradiation) and was dose dependent (changes were very significant at 2 Gy; P = 0.008). A dose-rate effect did not reach significance in this study (P = 0.054). However, dose rate and lens epithelial cell region showed significant interactions (P < 0.001). These observations further our mechanistic understanding of how the lens responds to radiation.

Epidemiological studies suggest an increased incidence and risk of cataract after low-dose (,2 Gy) ionizing radiation exposures. However, the biological mechanism(s) of this process are not fully understood. DNA damage and repair are thought to have a contributing role in radiation-induced cataractogenesis. Recently we have reported an inverse doserate effect, as well as the low-dose response, of DNA damage and repair in lens epithelial cells (LECs). Here, we present further initial findings from two mutated strains (Ercc2+/– and Ptch1+/–) of mice, both reportedly susceptible to radiationinduced cataract, and their DNA damage and repair response to low-dose and low-dose-rate gamma rays. Our results support the hypothesis that the lens epithelium responds differently to radiation than other tissues, with reported radiation susceptibility to DNA damage not necessarily translating to the LECs. Genetic predisposition and strain(s) of mice have a significant role in radiation-induced cataract susceptibility.

In 2011, the International Commission on Radiological Protection (ICRP) recommended reducing the occupational equivalent dose limit for the lens of the eye from 150 mSv/year to 20 mSv/year, averaged over five years, with no single year exceeding 50 mSv. With this recommendation, several important assumptions were made, such as lack of dose rate effect, classification of cataracts as a tissue reaction with a dose threshold at 0.5 Gy, and progression of minor opacities into vision-impairing cataracts. However, although new dose thresholds and occupational dose limits have been set for radiation-induced cataract, ICRP clearly states that the recommendations are chiefly based on epidemiological evidence because there are a very small number of studies that provide explicit biological and mechanistic evidence at doses under 2 Gy. Since the release of the 2011 ICRP statement, the Multidisciplinary European Low Dose Initiative (MELODI) supported in April 2019 a scientific workshop that aimed to review epidemiological, clinical and biological evidence for radiation-induced cataracts. The purpose of this article is to present and discuss recent related epidemiological and clinical studies, ophthalmic examination techniques, biological and mechanistic knowledge, and to identify research gaps, towards the implementation of a research strategy for future studies on radiation-induced lens opacities. The authors recommend particularly to study the effect of ionizing radiation on the lens in the context of the wider, systemic effects, including in the retina, brain and other organs, and as such cataract is recommended to be studied as part of larger scale programs focused on multiple radiation health effects.

The lens of the eye is thought to be one of the most radiosensitive tissues. Cataracts were one of the first observed biological effects following ionising radiation exposure. The recent change in regulations for eye lens dose limits has led to the urgent need to make sure our biological understanding is sufficient. The anterior of the lens is covered by lens epithelial cells (LEC), that are critical to maintaining normal lens function and producing fibre cells. Damage or disruption to LECs can have detrimental consequences to the lens. Low dose (<500 mGy) radiation-induced DNA damage and repair, cell proliferation and lens opacity were investigated post-exposure in or amongst four mouse strains (C57BL/6,129S2, BALB/c and CBA/Ca). Mice were sacrificed up to 24 hours post-exposure and lenses removed and epithelia isolated for analyses. Immunofluorescent staining for DNA double strand break (DSB) repair (53BP1) and cell proliferation (Ki67) were performed. Dose, dose-rates were varied during exposures to seek experimental evidence to support the epidemiological studies. Peripheral blood lymphocytes were collected for comparison with LEC. 120 female mice were irradiated and their lenses analysed for opacity at monthly intervals over 18 months. An inverse dose-rate effect was observed in the DSB repair response, as well as slower repair at low IR doses and a significant strain dependency. A nonlinear response to IR was observed for LEC proliferation that was bimodal; inhibition at low dose (<50 mGy), and a significant interaction effect between dose-rate and region. Lens opacity also increased over time. These results give the first biological evidence for an inverse dose-rate response in the lens. They highlight the importance of dose-rate in low-dose cataract formation represent the first evidence that LECs process radiation damage differently to blood lymphocytes. More work is needed to support lens dose limits.

Young organisms are known to be more sensible to ionizing radiation (IR). Previous irradiation studies with neonatal mice have shown early changes in the eye lens (cataracts) but no retinal changes. Nonetheless, it was shown that visible light might induce retinal atrophy already, because of enhanced ROS production. Here, we demonstrate the impact of ionizing radiation (2 Gy) on wild-type and heterozygous Ercc2 +/-mice (Ercc2 = excision repair cross-complementation group 2), both F1-hybrids of C3HeB/FeJ x C57BL/6J, in order to assess lens and retina damages. For the first time, visual acuity in such impaired mice will be quantified. A dose of 2 Gy causes in BL6/C3H hybrids photoreceptor cell hypoplasia in the retina and in almost every mouse lens cataracts with a latent period of maximal 10 weeks after irradiation. In the most extreme cases, retinae completely lacked photoreceptor cells and up to one eighth of the lenses were totally damaged with extrusions of fibre cell material into the vitreous. Surprisingly, despite those severe lens and retina phenotypes, visual acuity was reduced only be up to 50% and only some mice were totally hampered in their vision. Yet, in total, damages were more often and severe, then in neonatal inbred CD1 or BL6 mice in previous studies. This demonstrates the influence of genetic background on the effectiveness of IR damage on the eye. Methodological excurse: Mice were irradiated with 2 Gy of IR (dose rate = 0.3 Gy/min), 2 days after birth. In vivo measurements of eye lenses with optical coherence tomography (OCT) and subsequent experiments with the optokinetic drum were conducted. Eyes were collected 8.5 months after irradiation, cut and stained for histology or immunohistochemistry.

One noxious agent for cataract formation is ionizing radiation (IR). For doses till or equal 2 Gy it is still unclear, whether opacification occurs and if, how cataract type depends on the animals age at irradiation. Wild‐type and heterozygous Ercc2 +/‐ mice were whole‐body irradiated by 0.5 Gy, 1 Gy and 2 Gy of γ‐radiation (dose rate = 0.3 Gy/min), 10 weeks after birth (P70). Another cohort was exposed to 2 Gy, 2 days after birth (P2). All cohorts were investigated by optical coherence tomography (OCT) and eyes were collected for histology after death. OCT revealed frequent lens alteration in the mice irradiated at P2 and P70. These lesions were only subcapsular located in P70 mice and are dose‐dependent in size. Mice irradiated at P2 showed scattering structures within the posterior cortex, 8.5 mth after irradiation. At least 80% of the irradiated animals displayed at least mild posterior cortical cataracts. Histology of irradiated eyes (P70) unravelled the OCT‐detected lesions as cataractous accumulations of enlarged fibre cells, accompanied by subcapsular placed cells with nuclei. Lesions of P2 mice displayed no correlate to the in vivo data. Therefore, we conclude that IR leads to lens opacification in a dose‐dependent manner and its magnitude correlates strongly with age at irradiation. These results help to assess the sensibility of eye lenses to IR in case of exposure. The LDLensRad project received funding from Euratom in the framework of CONCERT, grant No 662287.

The eye lens displayes a variety of phenotypes in the wake of genetical modifications or environmental influences. Therefore, a high-resolution in vivo imaging method for the lens is desirable. Optical coherence tomography (OCT) has become a powerful imaging tool in ophthalmology, especially for retinal imaging in small animal models such as mice. Here, we demonstrate an optimized approach specifically for anterior eye segment imaging with spectral domain OCT (SD-OCT) on several known murine lens cataract mutants. Scheimpflug and histological section images on the same eye were used in parallel to assess the observed pathologies. With SD-OCT images, we obtained detailed information about the different alterations from the anterior to the posterior pole of the lens. This capability makes OCT a valuable high-resolution imaging modality for the anterior eye segment in mouse.

The influence of dose rate on radiation cataractogenesis has yet to be extensively studied. One recent epidemiological investigation suggested that protracted radiation exposure increases radiation-induced cataract risk: cumulative doses of radiation mostly <100 mGy received by US radiologic technologists over 5 years were associated with an increased excess hazard ratio for cataract development. However, there are few mechanistic studies to support and explain such observations. Low-dose radiation-induced DNA damage in the epithelial cells of the eye lens (LECs) has been proposed as a possible contributor to cataract formation and thus visual impairment. Here, 53BP1 foci was used as a marker of DNA damage. Unexpectedly, the number of 53BP1 foci that persisted in the mouse lens samples after γ-radiation exposure increased with decreasing dose-rate at 4 and 24 h. The C57BL/6 mice were exposed to 0.5, 1 and 2 Gy ƴ-radiation at 0.063 and 0.3 Gy/min and also 0.5 Gy at 0.014 Gy/min. This contrasts the data we obtained for peripheral blood lymphocytes collected from the same animal groups, which showed the expected reduction of residual 53BP1 foci with reducing dose-rate. These findings highlight the likely importance of dose-rate in low-dose cataract formation and, furthermore, represent the first evidence that LECs process radiation damage differently to blood lymphocytes.

Ionizing radiation (IR) damages DNA and other macromolecules, including proteins and lipids. Most cell types can repair DNA damage and cycle continuously their macromolecules as a mechanism to remove defective proteins and lipids. In those cells that lack nuclei and other organelles, such as lens fiber cells and mammalian erythrocytes, IR-induced damage to macromolecules is retained because they cannot be easily replenished. Whilst the life span for an erythrocyte is several months, the life span of a human lens is decades. There is very limited turnover in lens macromolecules, therefore the aging process greatly impacts lens structure and function over its lifetime. The lens is a tissue where biomolecular longevity, lifelong retention of its components and continued growth are integral to its homeostasis. These characteristics make the lens an excellent model to study the contribution of retained macromolecular damage over time. Epidemiological data have revealed a significant association between exposure to IR, the loss of lens optical function and the formation of cataracts (cataractogenesis) later in life. Lifestyle, genetic and environmental factors all contribute to cataractogenesis due to their effect on the aging process. Cataract is an iconic age-related disease in humans. IR is a recognised cause of cataract and the occupational lens dose limit is reduced from 150 to 20 mGy / year averaged over 5 years (ICRP Publication 118). Understanding the effects of low dose IR on the lens and its role in cataractogenesis is therefore very important. So we redefine “cataractogenic load” as a term to account for the combined lifestyle, genetic and environmental processes that increase biomolecular damage to lens macromolecules. These processes weaken metabolic defenses, increase post-translational protein modifications, and alter the lipid structure and content of the lens. IR exposure is a significant insult to the lens because of free radical generation and the ensuing oxidative stress. We support the concept that damage caused by IR compounds the aging process by increasing the cataractogenic load, hereby accelerating lens aging and its loss of function.

The radiosensitive nature of the lens has been increasingly reported, although the exact mechanistic details of the radiation response pathways for cataractogenesis are unclear. Radiation-induced DNA damage and the subsequent impairment of repair pathways within the lens epithelium are involved. Here, two distinct regions of the murine lens epithelium have been analysed for their differences in double strand break (DSB) repair responses to ionising radiation. The responses of epithelial cells located at the anterior pole (central region) have been compared to those in other locations, including the proliferative compartment, and including the very periphery of the monolayer (peripheral region). Described here are the responses between the two regions, across four strains, over a low dose (0–25 mGy) X-irradiation range up to 24 hours. Damage visualised through biomarker 53BP1 staining was present across the epithelium, repair kinetics appear non-uniform. Epithelial cells in the central region have significantly more 53BP1 positive foci. In this study, BALB/c were identified as the most suitable strain for low dose ionising radiation exposure investigation.

The lens of the eye has long been considered as a radiosensitive tissue, but recent research has suggested that the radiosensitivity is even greater than previously thought. The recent recommendations of the International Commission on Radiological Protection (ICRP) to substantially reduce the annual occupational equivalent dose limit for the ocular lens have now been adopted in the European Union and are under consideration around the rest of the world. However, ICRP clearly states that the recommendations are chiefly based on epidemiological evidence because there are a very small number of studies that provide explicit biological, mechanistic evidence at doses <2 Gy. This paper aims to present a review of recently published information on the biological and mechanistic aspects of cataracts induced by exposure to ionizing radiation (IR). The data were compiled by assessing the pertinent literature in several distinct areas which contribute to the understanding of IR induced cataracts, information regarding lens biology and general processes of cataractogenesis. Results from cellular and tissue level studies and animal models, and relevant human studies, were examined. The main focus was the biological effect of low linear energy transfer IR, but dosimetry issues and a number of other confounding factors were also considered. The results of this review clearly highlight a number of gaps in current knowledge. Overall, while there have been a number of recent advances in understanding, it remains unknown exactly how IR exposure contributes to opacification. A fuller understanding of how exposure to relatively low doses of IR promotes induction and/or progression of IR-induced cataracts will have important implications for prevention and treatment of this disease, as well as for the field of radiation protection.