The Role of Natural Background Radiation in Maintaining Genomic Stability in the CGL1 Human Hybrid Model System.

Natural background ionizing radiation is present on the earth's surface; however, the biological role of this chronic low-dose-rate exposure remains unknown. The Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR) project is examining the impacts of sub-natural background radiation exposure through experiments conducted 2 km underground in SNOLAB. The rock overburden combined with experiment-specific shielding provides a background radiation dose rate 30 times lower than on the surface. We hypothesize that natural background radiation is essential for life and maintains genomic stability and that prolonged exposure to sub-background environments will be detrimental to biological systems. To evaluate this, human hybrid CGL1 cells were continuously cultured in SNOLAB and our surface control laboratory for 16 weeks. Cells were assayed every 4 weeks for growth rate, alkaline phosphatase (ALP) activity (a marker of cellular transformation in the CGL1 system), and the expression of genes related to DNA damage and cell cycle regulation. A subset of cells was also exposed to a challenge radiation dose (0.1 to 8 Gy of X rays) and assayed for clonogenic survival and DNA double-strand break induction to examine if prolonged sub-background exposure alters the cellular response to high-dose irradiation. At each 4-week time point, sub-background radiation exposure did not significantly alter cell growth rates, survival, DNA damage, or gene expression. However, cells cultured in SNOLAB showed significantly higher ALP activity, a marker of carcinogenesis in these cells, which increased with longer exposure to the sub-background environment, indicative of neoplastic progression. Overall, these data suggest that sub-background radiation exposure does not impact growth, survival, or DNA damage in CGL1 cells but may lead to increased rates of neoplastic transformation, highlighting a potentially important role for natural background radiation in maintaining normal cellular function and genomic stability.

Protracted Exposure to a Sub-background Radiation Environment Negatively Impacts the Anhydrobiotic Recovery of Desiccated Yeast Sentinels.

ABSTRACT: Experiments that examine the impacts of subnatural background radiation exposure provide a unique approach to studying the biological effects of low-dose radiation. These experiments often need to be conducted in deep underground laboratories in order to filter surface-level cosmic radiation. This presents some logistical challenges in experimental design and necessitates a model organism with minimal maintenance. As such, desiccated yeast ( Saccharomyces cerevisiae ) is an ideal model system for these investigations. This study aimed to determine the impact of prolonged sub-background radiation exposure in anhydrobiotic (desiccated) yeast at SNOLAB in Sudbury, Ontario, Canada. Two yeast strains were used: a normal wild type and an isogenic recombinational repair-deficient rad51 knockout strain ( rad51 Δ). Desiccated yeast samples were stored in the normal background surface control laboratory (68.0 nGy h -1 ) and in the sub-background environment within SNOLAB (10.1 nGy h -1 ) for up to 48 wk. Post-rehydration survival, growth rate, and metabolic activity were assessed at multiple time points. Survival in the sub-background environment was significantly reduced by a factor of 1.39 and 2.67 in the wild type and rad51 ∆ strains, respectively. Post-rehydration metabolic activity measured via alamarBlue reduction remained unchanged in the wild type strain but was 26% lower in the sub-background rad51 ∆ strain. These results demonstrate that removing natural background radiation negatively impacts the survival and metabolism of desiccated yeast, highlighting the potential importance of natural radiation exposure in maintaining homeostasis of living organisms.

Phenotypic and transcriptional changes in lens epithelial cells following acute and fractionated ionizing radiation exposure.

PURPOSE: Exposure to ionizing radiation is one of the known risk factors for the development of lens opacities. It is believed that radiation interactions with lens epithelial cells (LEC) are the underlying cause of cataract development, however, the exact mechanisms have yet to be identified. The aim of this study was to investigate how different radiation dose and fractionation impact normal LEC function. MATERIALS AND METHODS: A human derived LEC cell line (HLE-B3) was exposed to a single acute x-ray dose (0.25 Gy) and 6 fractionated doses (total dose of 0.05, 0.1, 0.25, 0.5, 1, and 2 Gy divided over 5 equal fractions). LEC were examined for proliferation using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and migration using a Boyden chamber assay at various time points (0.25, 0.5, 1, 2, 4, 7, 9, 11, and 14 d) post-irradiation. Transcriptomic analysis through RNA sequencing was also performed to identify differentially expressed genes and regulatory networks in cells following 4 different acute exposures and 1 fractionated exposure. RESULTS: Exposure to an acute dose of 0.25 Gy significantly increased proliferation and migration rates, peaking at 7 d post irradiation (20% and 240% greater than controls, respectively), before returning to baseline levels by day 14. Fractionated exposures had minimal effects up to a dose of 0.5 Gy, but significantly reduced proliferation and migration after 1 and 2 Gy by up to 50%. The largest transcriptional response occurred 12 h after an acute 0.25 Gy dose, with 362 genes up-regulated and 288 genes down-regulated. A unique panel of differentially expressed genes was observed between moderate versus high dose exposures, suggesting a dose-dependent transcriptional response in LEC that is more pronounced at lower doses. Gene ontology and upstream regulator analysis identified multiple biological processes and molecular functions implicated in the radiation response, in particular differentiation, motility, receptor/ligand binding, cell signaling and epithelial-mesenchymal cell transition. CONCLUSIONS: Overall, this research provides novel insights into the dose and fractionation effects on functional changes and transcriptional regulatory networks in LEC, furthering our understanding of the mechanisms behind radiation induced cataracts.

Overexpression of FRA1 (FOSL1) Leads to Global Transcriptional Perturbations, Reduced Cellular Adhesion and Altered Cell Cycle Progression.

FRA1 (FOSL1) is a transcription factor and a member of the activator protein-1 superfamily. FRA1 is expressed in most tissues at low levels, and its expression is robustly induced in response to extracellular signals, leading to downstream cellular processes. However, abnormal FRA1 overexpression has been reported in various pathological states, including tumor progression and inflammation. To date, the molecular effects of FRA1 overexpression are still not understood. Therefore, the aim of this study was to investigate the transcriptional and functional effects of FRA1 overexpression using the CGL1 human hybrid cell line. FRA1-overexpressing CGL1 cells were generated using stably integrated CRISPR-mediated transcriptional activation, resulting in a 2-3 fold increase in FRA1 mRNA and protein levels. RNA-sequencing identified 298 differentially expressed genes with FRA1 overexpression. Gene ontology analysis showed numerous molecular networks enriched with FRA1 overexpression, including transcription-factor binding, regulation of the extracellular matrix and adhesion, and a variety of signaling processes, including protein kinase activity and chemokine signaling. In addition, cell functional assays demonstrated reduced cell adherence to fibronectin and collagen with FRA1 overexpression and altered cell cycle progression. Taken together, this study unravels the transcriptional response mediated by FRA1 overexpression and establishes the role of FRA1 in adhesion and cell cycle progression.

The REPAIR Project, a Deep-Underground Radiobiology Experiment Investigating the Biological Effects of Natural Background Radiation: The First 6 Years.

In 2017, a special edition of Radiation Research was published [Oct; Vol. 188 4.2 (https://bioone.org/journals/radiation-research/volume-188/issue-4.2)] which focused on a recently established radiobiology project within SNOLAB, a unique deep-underground research facility. This special edition included original articles, reviews and commentaries relevant to the research goals of this new project which was titled Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR). These research goals were founded in understanding the biological effects of terrestrial and cosmic natural background radiation (NBR). Since 2017, REPAIR has evolved into a sub-NBR radiobiology research program which investigates these effects using multiple model systems and various biological endpoints. This paper summarizes the evolution of the REPAIR project over the first 6-years including its experimental scope and capabilities as well as research accomplishments.

Radiation-Induced Alterations in Proliferation, Migration, and Adhesion in Lens Epithelial Cells and Implications for Cataract Development.

The lens of the eye is one of the most radiosensitive tissues. Although the exact mechanism of radiation-induced cataract development remains unknown, altered proliferation, migration, and adhesion have been proposed as factors. Lens epithelial cells were exposed to X-rays (0.1-2 Gy) and radiation effects were examined after 12 h and 7 day. Proliferation was quantified using an MTT assay, migration was measured using a Boyden chamber and wound-healing assay, and adhesion was assessed on three extracellular matrices. Transcriptional changes were also examined using RT-qPCR for a panel of genes related to these processes. In general, a nonlinear radiation response was observed, with the greatest effects occurring at a dose of 0.25 Gy. At this dose, a reduction in proliferation occurred 12 h post irradiation (82.06 ± 2.66%), followed by an increase at 7 day (116.16 ± 3.64%). Cell migration was increased at 0.25 Gy, with rates 121.66 ± 6.49% and 232.78 ± 22.22% greater than controls at 12 h and 7 day respectively. Cell adhesion was consistently reduced above doses of 0.25 Gy. Transcriptional alterations were identified at these same doses in multiple genes related to proliferation, migration, and adhesion. Overall, this research began to elucidate the functional changes that occur in lens cells following radiation exposure, thereby providing a better mechanistic understanding of radiation-induced cataract development.

A novel specialized tissue culture incubator designed and engineered for radiobiology experiments in a sub-natural background radiation research environment.

Extensive research has been conducted investigating the effects of ionizing radiation on biological systems, including specific focus at low doses. However, at the surface of the planet, there is the ubiquitous presence of ionizing natural background radiation (NBR) from sources both terrestrial and cosmic. We are currently conducting radiobiological experiments examining the impacts of sub-NBR exposure within SNOLAB. SNOLAB is a deep underground research laboratory in Sudbury, Ontario, Canada located 2 km beneath the surface of the planet. At this depth, significant shielding of NBR components is provided by the rock overburden. Here, we describe a Specialized Tissue Culture Incubator (STCI) that was engineered to significantly reduce background ionizing radiation levels. The STCI was installed 2 km deep underground within SNOLAB. It was designed to allow precise control of experimental variables such as temperature, atmospheric gas composition and humidity. More importantly, the STCI was designed to reduce radiological contaminants present within the underground laboratory. Quantitative measurements validated the STCI is capable of maintaining an appropriate experimental environment for sub-NBR experiments. This included reduction of sub-surface radiological contaminants, most notably radon gas. The STCI presents a truly novel piece of infrastructure enabling future research into the effects of sub-NBR exposure in a highly unique laboratory setting.

The REPAIR Project: Examining the Biological Impacts of Sub-Background Radiation Exposure within SNOLAB, a Deep Underground Laboratory.

Considerable attention has been given to understanding the biological effects of low-dose ionizing radiation exposure at levels slightly above background. However, relatively few studies have been performed to examine the inverse, where natural background radiation is removed. The limited available data suggest that organisms exposed to sub-background radiation environments undergo reduced growth and an impaired capacity to repair genetic damage. Shielding from background radiation is inherently difficult due to high-energy cosmic radiation. SNOLAB, located in Sudbury, Ontario, Canada, is a unique facility for examining the effects of sub-background radiation exposure. Originally constructed for astroparticle physics research, the laboratory is located within an active nickel mine at a depth of over 2,000 m. The rock overburden provides shielding equivalent to 6,000 m of water, thereby almost completely eliminating cosmic radiation. Additional features of the facility help to reduce radiological contamination from the surrounding rock. We are currently establishing a biological research program within SNOLAB: Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR project). We hypothesize that natural background radiation is essential for life and maintains genomic stability, and that prolonged exposure to sub-background radiation environments will be detrimental to biological systems. Using a combination of whole organism and cell culture model systems, the effects of exposure to a sub-background environment will be examined on growth and development, as well as markers of genomic damage, DNA repair capacity and oxidative stress. The results of this research will provide further insight into the biological effects of low-dose radiation exposure as well as elucidate some of the processes that may drive evolution and selection in living systems. This Radiation Research focus issue contains reviews and original articles, which relate to the presence or absence of low-dose ionizing radiation exposure.