Nuclear Innovation Institute 2021 Annual Public Health and Environment Research Report by Ontario NII

Nuclear Innovation Institute 2021 Annual Public Health and Environment Research Report April 2022

Prepared by the Nuclear Innovation Institute

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Table of Contents Introduction ....................................................................................................................... 4 Public Health Research Programs ..................................................................................... 5 NII-CHER (2016-2021) ..................................................................................................... 5 Research Activities and Results ................................................................................. 5 Outcomes ................................................................................................................... 6 Ultra-Low Dose (2016-2022) .......................................................................................... 6 Facilities, Equipment and Methodology ................................................................... 7 Research Activities and Results ............................................................................... 11 Outcomes ................................................................................................................. 15 2022 Research Plan .................................................................................................. 16 Key Researchers ....................................................................................................... 17 Fetal Programming (2015-2021) .................................................................................. 18 Research Activities and Results ............................................................................... 18 Outcomes ................................................................................................................. 21 2022 Research Plan .................................................................................................. 22 Key Researchers ....................................................................................................... 22 Lens of the Eye (2017-2021) ......................................................................................... 23 Research Activities and Results ............................................................................... 23 Outcomes ................................................................................................................. 25 2022 Research Plan .................................................................................................. 25 Key Researchers ....................................................................................................... 26 Low-dose Radiation Immunology (2020-2021) ........................................................... 26 Research Activities and Results ............................................................................... 27 2022 Research Plan .................................................................................................. 28 Key Researchers ....................................................................................................... 28 Harnessing Genetically Modified Microbes to Combat the Effects of Ionizing Radiation (2020-2021) ................................................................................................................... 28 Research Activities and Results ............................................................................... 29 2022 Research Plan .................................................................................................. 30 Key Researchers ....................................................................................................... 31 NEUDOSE (2019-2022).................................................................................................. 32 Research Activities and Results ............................................................................... 32 nii.ca/environment-at-nii

Outcomes ................................................................................................................. 35 2022 Research Plan .................................................................................................. 35 Key Researchers ....................................................................................................... 35 Environment Research Programs .................................................................................... 36 Aquatic Biota (2018-2022) ............................................................................................ 36 Research Activities and Results ............................................................................... 36 Outcomes ................................................................................................................. 40 2022 Research Plan .................................................................................................. 42 Key Researchers ....................................................................................................... 42 Environmental DNA (eDNA) (2021-2022) ..................................................................... 43 Research Activities and Results ............................................................................... 43 2022 Research Plan .................................................................................................. 47 Key Researchers ....................................................................................................... 47 Fairy Lake (2021-2022) ................................................................................................. 48 Research Activities and Results ............................................................................... 49 2022 Research Goals ................................................................................................ 49 Key Researchers ....................................................................................................... 50 Conclusion ........................................................................................................................ 51

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Introduction Environment@NII is home to the Nuclear Innovation Institute’s (NII) projects assessing the impact of energy generation on human health and the environment. Delivering actionable intelligence from leading-edge researchers focused on fostering a clean and healthy environment, NII’s focus is three-fold: • •

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On the future of energy – advancing knowledge and practices in the nuclear industry to help the world transition to a clean energy future On the future of health – accelerating research and advocacy for medical isotopes, from improved cancer diagnoses and treatments to expanded use in food production and industrial safety On the future environment – researching the impact of the nuclear fuel lifecycle on our water, land and air while also supporting efforts to fight climate change.

Located in Saugeen Shores, Ontario, NII is focused on new thinking for a new energy era. NII is supported by an engaged group of founding members including Bruce Power, BWXT Canada, Cameco, E.S. Fox Ltd., Kinectrics, SNC-Lavalin and Bruce County. The Annual Public Health and Environment Research Report The purpose of this report is to briefly describe the public health and environment research programs supported by Bruce Power through NII. NII supports public health and environment research with over $1 million annually from Bruce Power in direct research funding. NII support allows researchers to succeed in applying for competitive, peer-reviewed funding from federal and provincial agencies. The receipt of these matching funding grants demonstrates the scientific rigor of the academic research that Bruce Power is supporting. This report presents the research progress for the NII’s diverse public health and environment programs and provides an update on research plans for 2022. We expect that researchers will continue to make excellent progress in 2022 and NII looks forward to seeing the results of their research. Research results will be collected and summarized by NII, who will also assist in incorporating the results into relevant regulatory and business initiatives at Bruce Power. It should be noted that due to the Covid-19 pandemic, several research projects suffered significant delays. These have been omitted from the body of the report for brevity.

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Public Health Research Programs NII-CHER (2016-2021) Based at the Northern Ontario School of Medicine in Sudbury, Ontario, NII-CHER (Nuclear Innovation Institute – Research Centre for Health, Environment and Radiation, formerly the Bruce Power – Research Centre for Health, Environment and Radiation) was established in late 2016. The goals of the Centre are: 1. To sponsor and promote research on the effects of radiation at low doses and dose rates 2. To support education and understanding of the effects of radiation at low doses and dose rates 3. To contribute to guideline development for the use of radiation in industry and science In November 2016, funding was announced for the Northern Ontario School of Medicine/NII-CHER. The $1 million in annual funding for five years is used to continue research that has taken place during the first four years of the existence of the Bruce Power Chair in Radiation and Health, including: • Impacts of low-dose radiation on health • Environmental impacts of radiation and how they impact health • Effects of radiation and diagnostic imaging on fetal programming • Effect of radiation on specific species of fish • Impact of radiation on Indigenous communities NII-CHER allows the NII to consolidate and better coordinate research collaborations. Research Activities and Results Several additional sources of research funding were secured, allowing the expansion of research programs. This includes a Northern Cancer Foundation grant to explore the immune modulating effects of ionizing radiation and a New Frontiers in Research Fund grant to study the radiation response in genetically engineered microbes. Research has also continued on the lens of the eye project, which was initiated in 2019, and the NII funded SNOLAB (Sudbury Neutrino Observatory Laboratory) project. The additional grants received include a MITACS (Mathematics of Information Technology and Complex Systems) Research Training award, a New Frontiers in Research grant and an NSERC (Natural Sciences and Engineering Research Council) Undergraduate Student Research award, allowing the NII-research team to grow to nii.ca/environment-at-nii

include one lab technologist, two post-doctoral fellows, one Ph.D. student, six MSc students and one undergraduate student. One senior research scientist (Dr. Anthony G. Brooks), along with two NII funded PostDoctoral Fellows (now assistant professors), reviewed over 400 scientific papers (20122016) on risk estimation and the epidemiology of the Linear No Threshold (LNT) concept for biological responses to low dose radiation exposure. These contributions were included in a special toxicology journal issue dedicated to the subject matter (see publications below). Research activities and results are listed in the respective programs, except where they are not directly related to a specific program. Outcomes Table 1: Scientific reports produced as part of the NII-CHER collaboration Source Zarnke, A. M., Tharmalingam, S., Boreham, D. R., and Brooks, A. L. (2019) BEIR VI radon: The rest of the story. Chemico-biological interactions 301, 81-87 Tharmalingam, S., Sreetharan, S., Brooks, A. L., and Boreham, D. R. (2019) Re-evaluation of the linear no-threshold (LNT) model using new paradigms and modern molecular studies. Chemico-biological interactions 301, 54-67 McDonald A., Smith N., 2017. Foreword. Radiation Research 188(4.2):469-469

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Ultra-Low Dose (2016-2022) The laboratory established in the SNOLAB allows researchers to examine the biological effects of prolonged exposure to a sub-natural background radiation environment. The 2 km of overhead rock effectively shields out cosmic radiation. Because living organisms have evolved in the continual presence of natural background ionizing radiation, researchers believe it may be essential for life and helps to maintain genomic stability. Prolonged exposure to less than normal-background radiation environments may be detrimental to biological systems. The ultra-low dose research program has explored the effect of ultra-low levels of background radiation on Lake Whitefish embryo survival and development. Lake Whitefish embryos were chosen due to availability, to be used as a multi-cellular organism for comparison with cell studies. Single-cell models grown in typical ambient nii.ca/environment-at-nii

radiation and under ultra-low dose SNOLAB conditions were evaluated for markers of cancer development including mutation frequency, chromosomal aberrations and differentiation. This research will help us understand the impact that natural background radiation has on organisms, providing evidence and mechanistic understanding of low-dose radiation effects on organisms. The goal is to more accurately describe dose effects at natural and slightly elevated levels, testing the linear-no-threshold model of risk. In 2018, this project successfully received a 5-year NSERC grant to match the Bruce Power funding and will now be extended until 2022. Additional matching funding from MITACS for a SNOLAB postdoctoral fellow was obtained in 2019 for three years. In 2020, matching funding from MITACS for three years was obtained for an additional postdoctoral fellow for this project. Facilities, Equipment and Methodology The following sections outline the experimental tools used to perform the ultra-lowdose research. The experiments performed using this equipment are outlined below in the following section. Low-Radon Specialized Tissue Culture Incubator Due to radiological decay in the surrounding rock, underground radon levels are higher than on the surface (approximately 130 Bq/m3 underground compared to 5 Bq/m3 on surface). A low-radon specialized tissue culture incubator was designed in 2017 to reduce radon levels below natural background and allow researchers to achieve much lower radiation levels underground compared to the surface. SNOLAB management reviewed and approved the technical design of the underground laboratory for the Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR) project. Researchers worked with a design team at SNOLAB to build a low-radon specialized tissue culture incubator that will be used for culturing human cells. Construction and installation were completed for both the low-radon glovebox and all equipment required for the underground laboratory (figure 1). The low-radon specialized tissue culture incubator will enable researchers to culture cells in an ultralow background radiation environment to better understand fundamental mechanisms of low-dose radiation exposure. In 2019, training and testing of the specialized tissue culture incubator monitoring systems was completed. This included work to quantify and calculate the levels of natural background radiation (NBR) components (i.e., radon, gamma, neutrons) in the SNOLAB, both in the underground REPAIR laboratory and within the custom low-radon

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glovebox, and at the above ground laboratory. This work demonstrated that the lowradon specialized tissue culture incubator successfully reduced the major component gamma, neutron and radon radiological contaminants to a sub-NBR environment. Methodology for the transportation and growth of cell lines in the underground laboratory was tested in preparation for longer-term experiments.

Figure 1. SNOLAB project cell culture systems. A) Cell culture systems underground in SNOLAB. B) Schematic of low-radon specialized tissue culture incubator underground in the SNOLAB C) Low-radon specialized tissue culture incubator in-situ in the SNOLAB.

Characterization of the radiation environment within the glovebox has been conducted and commissioning reveals excellent reduction of natural background radiation components. This represents a significant achievement in the field of low-dose radiation research and has novel implications for our future sub-natural background radiation experiments. A manuscript detailing the engineering, construction, and commissioning of the STCI (Specialized Tissue Culture Incubator) (Kennedy 2021). A Monte Carlo simulation was conducted on the REPAIR project framework underground and above ground at NOSM, which includes the STCI geometry. Work since has been focused on getting the resulting manuscript from this work ready for publication. There have been several changes that have gone into refining and improving the simulation. One such change was improving the efficiency of the GPS (General Particle Source) within the simulation. As a result, the simulation has become much more accurate and representative of what is occurring within the different radiation environments. This simulation and measurement of the radiation environment both underground and above ground is one of the most comprehensive and detailed of any of the other labs who are actively conducting sub-natural background radiation research. The resultant dose rates from the simulation show substantial reductions in cosmic and terrestrial radiation from the STCI configuration (as well as the result of having the chemical

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sciences laboratory deep underground in SNOLAB). This specialized facility enables the research team to explore the effects of radiation, or lack thereof, on living organisms. The following sections outline that research using the facility. Cell Culture System Work has been performed in the NOSM laboratory with the cell culture systems and endpoints to be used underground. This included the FADU (Fluorescence Analysis DNA Unwinding), comet and transformation genetic assays. Transformation assays have been run using the CGL-1 and WI-38 cell lines to study cancer induction. The CGL-1 cell line is a hybridization between the tumorigenic human HeLa cell and a normal human cell and its extensive use for radiation exposure research has established specific cellular and genetic endpoints. These endpoints include the ability to identify specific genetic changes that occur after radiation exposure and that differ between tumorigenic (i.e., tumor causing) and non-tumorigenic cells. The WI-38 cell line consists of normal human cells and can serve as a control when compared to other cell lines. A modern molecular biology endpoint has been optimized that will allow the selective removal of genes of interest from the cell culture model systems. The removal of specific genes will allow researchers to explore the mechanisms driving any radiation-induced effects observed in the SNOLAB. Irradiator At the NOSM West Campus, installation of the X-RAD 320 x-ray cabinet irradiator was completed in 2020. This is the same model of irradiator that is currently being used at the East campus. The installation of the irradiator has allowed for parallel research to be performed at the West Campus and serve as a second surface lab site to perform radiation studies, which is a critical control for confirming results. Prior to installation of the irradiator, the clonogenic survival assay was performed using a chemical stressor, tert-butyl hydroperoxide. The clonogenic assay has been performed using the CGL-1 cell line for several radiation studies at NOSM East and is an appropriate endpoint for detecting the effects of low doses of radiation. Preliminary studies with the chemical showed a dose response relationship in the decrease of colony formation with increasing doses of chemical. Next Generation Sequencing using miRNA for Low-Dose Radiation Experiments The aim of this project is to identify novel biomarkers for low dose and sub-background radiation exposures, with particular focus on identifying small non-coding RNA molecules known as microRNA (miRNA) as radiation biomarkers.

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The methodology to sensitively detect specific miRNA using qPCR (quantitative polymerase chain reaction) technology was developed. This assay will be critical for detecting miRNA levels in-house. The process for extracting total miRNA from cell samples was streamlined, making it possible to collect miRNA on-site and accurately detect miRNA at the expected levels. Multiple sample sets of the two cell’s lines were irradiated with multiple doses and durations. The extracted RNA was verified to be of great quality and was subsequently used to generate a miRNA library. The generated miRNA library was sent off for next generation sequencing (NGS) to get miRNA of interest after radiation exposure was compared to the SNOLAB samples. This NGS assay allowed for unbiased detection and quantitation of all miRNA molecules in the samples using massively parallel sequencing technology. Large-scale miRNA profiling has been challenging since miRNA molecules are very short in length (18–22 base-pairs). In 2021, the research team overcame this challenge by developing a next-generation sequencing technology that can identify and profile millions of miRNA molecules in biological fluids. The researchers have utilized this technology to identify miRNA profiles in CGL-1 cells exposed to various doses of radiation. Furthermore, the team has verified the next-generation sequencing methodology for miRNA profiling using RT-qPCR technology. They have established a list of miRNAs that are significantly dysregulated upon radiation exposure and have evaluated the mRNA targets of the miRNA molecules and successfully showed reciprocal expression patterns. Overall, the result corroborates the hypothesis that the presence of miRNA molecules may be used to infer mRNA expression. This provides a novel pathway to perform and analyze the effects of low dose radiation on cells.

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Research Activities and Results The following sections outline a series of experiments performed on the CGL-1 human hybrid cell line and the WI-38 fibroblasts under various low-dose radiation conditions. The overall goal of this work is to understand the effects of low dose radiation on wellstudied cell lines commonly used for radiobiology experiments. These experiments expanded to include MDA-MB-231 cells in 2021, to study the molecular mechanisms for breast cancer induction. Lastly, there is a new line of research started in 2021 in collaboration with NASA to use a yeast as a model organism for radiation research. The Effects of Sub-Background Radiation and Tumour-Suppressor Genes on Neoplastic Transformation Frequency using the CGL-1 Tumorigenesis Model CRISPR-based genetic tools have been developed and optimized to knock-out and activate specific genes in the CGL-1 cell-line. This technological achievement will allow researchers to generate new CGL-1 cell-lines with changes to candidate tumour suppressor genes. These cell-lines will then be grown long-term at the SNOLAB subbackground radiation research facility to identify how changes to tumor-suppressor genes alter the effects of sub-background radiation on neoplastic transformation frequency. To test the capabilities of the newly developed tools, Cas9 and dCas9 lentivirus particles and CGL-1 cells that either express the Cas9 or the dCas9 protein have been successfully produced (see figures 2 and 3). These experiments confirm that the CRISPR-based tools work as expected, and can be used to activate or knock-out specific genes in cell lines.

Figure 2: The image to the left shows CGL-1 cells expressing the Cas9 protein in their cytoplasm. The image to the right shows CGL-1 cells stained with DAPI (blue) that show the nucleus.

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Figure 3: The image to the left shows CGL-1 cells expressing the dCas9 protein in their cytoplasm. The image to the right shows CGL-1 cells stained with DAPI (blue) that shows the nucleus.

Molecular Mechanisms Involved in Radiation Resistant Breast Cancer This research program has been developed to understand the molecular mechanisms that contribute to the development of radiation resistant breast cancers. The goal of this research program is to identify master gene regulators that are fundamental to the formation of radiation resistant phenotypes, and design novel therapeutic approaches to reverse these gene targets using CRISPR technology. This will thereby sensitize radioresistant cancer variants to radiation therapy. The short-term objective of this research program is to perform a genome-wide CRISPR based functional genomics screen (with gene activation) in MDA-MB-231 breast cancer cells using repeated radiation challenge to identify gene perturbations that promote radiation resistant survival. Radiation challenge experiments were completed to determine the percentage of cells resistant to 2 Gy and 4 Gy doses of radiation. Radiation experiments to induce a radiation resistant phenotype successfully produced radiation resistant MDA-MB-231 cells, however, only a small population of cells survived. These cells will continue to be maintained to allow for growth to occur so that RNA and/or DNA can be extracted from them. In 2021 three significant milestones were achieved. First, three biological replicates of the CRISPR activation screen were completed in a staggered fashion in MDA-MB-231 cells. These cell libraries were exposed to 12 weeks of weekly radiation exposure (57 Gy total) to identify radiation resistant cells. Cells were collected after every radiation dose to be processed in preparation for deep next-generation sequencing. nii.ca/environment-at-nii

Second, optimizations were performed on the gRNA PCR reaction for next-generation sequencing library prep. The appropriate annealing temperature, cycle number and input amount of DNA was determined. A method for library preparation of the samples for gRNA sequencing was established. Lastly, qPCR experiments were performed on the radiation resistant cell lines using a gene panel created to assess genes involved in the epithelial-mesenchymal transition (EMT). Results showed six genes involved in EMT (VIM, CK19, CDH2, CTNNB1, SNAI2 and FN1) were dysregulated in radiation resistant cell lines compared to their control cell line. DNA Damage response was also assessed in these cells using a gene array created in-house. After a 6 Gy radiation challenge, genes involved in cell cycle regulation showed dysregulation in a time-course experiment.

Elucidating the role of FOSL1 in radiation induced tumorigenesis. In 2021, a CRISPR-based methodology was established for upregulating and knockingdown gene expression in CGL-1 cells using Cas nucleases and gRNA molecules delivered in a lentivirus vehicle system. Using this approach, the researchers have successfully generated CGL-1 cell derivatives with (1) robust upregulation of FOSL1 and (2) robust down-regulation of FOSL1. Furthermore, radiation induced colonogenic survival assays for the CGL-1 cell types were completed. Cells were exposed to various radiation doses and survival was assessed nine days post-irradiation. Preliminary results demonstrate that there were no differences in cell survival with FOSL1 manipulation in CGL-1 cells. However, there is a trend towards reduced survival with FOSL1 knockout compared to wild type CGL-1 cells (see the figure below). And finally, Alkaline phosphatase activity is a marker of tumorigenesis in the CGL-1 model. The alkaline phosphatase (ALP) assay was performed for the CGL-1 cell types generated above. Although not statistically significant, activation of FOSL1 seems to demonstrate reduced ALP expression levels. This suggests that FOSL1 expression reduced basal levels of tumorigenesis.

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Figure 4: A survival fraction curve, showing the number of surviving cells as a fraction of the original culture at various total doses. CLG-1 FOSL1 knockout culture (orange diamond) had a significantly worse survival at higher doses than other CGL-1 cultures.

Adaptive Response in Yeast The adaptive response is a process where cells previously exposed to low doses of a stressor obtain a level of resistance as a form of protection against subsequent higher doses of the same or different environmental stressor. Several different stressors have been shown to induce the adaptive response, particularly ionizing radiation, and thermal stress. To better understand the adaptive response and the molecular mechanisms underlying the biological effects of low dose stresses, three different strains of yeast (Saccharomyces cerevisiae) were used as test organisms and subjected to thermal stress. The three strains of yeast include the BY4743 wild type strain which is the parental strain used to create collections of gene deletion mutants and two genetically engineered strains provided by the BioSentinel Group at NASA Ames Research Center. The BioSentinel strains consist of a wild type strain and a radiosensitive rad51 deletion mutant strain. A collaboration between NOSM/NII and NASA will study how these yeast strains respond to high dose space radiation (while travelling in deep space) compared to ultra-low shielded cosmic radiation environment in the SNOLAB. The experiments will begin in 2022, and work has already begun on culturing both the wild-type and genetically engineering yeast strains. The NASA work will examine the effects of the increased radiation dose in low Earth orbit on the yeast strains, to better understand the impacts of the additional stressors of space on cell survival, and to test our understanding of the molecular models involved. The SNOLAB will provide additional knowledge to the project by culturing the yeast in an ultra-low-dose

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environment and then provide the radiation stress. The goal is to better understand the effects of our natural radiation environment on overall cell survival. Heat shock response in the three yeast strains was assessed to determine a) the degree of cell killing and b) the optimal timing for the delivery of a priming heat shock to initiate an adaptive response prior to a higher heat shock. After determining the optimal dose of thermal stress to induce an adaptive response, whole transcriptomic analysis was performed to identify all the molecular pathways involved in initiating and maintaining this adaptive response. Evaluation of the transcriptomic data revealed that all three strains of Saccharomyces cerevisiae showed a distinct molecular response to the thermal stress when compared to the untreated controls. The data suggests that genes associated with protein folding, nutrient metabolism, and reproduction are significantly altered during the thermal adaptive response in this study.

Outcomes Table 2: Scientific reports for the Ultra-low dose (SNOLAB) research Source Kennedy K., LeBlanc A., Pirkkanen J., Thome C., Tai T.C., LeClair R., Boreham D.R., Dosimetric characterization of a sub-natural background radiation environment for radiobiology investigations Radiation Protection Dosimetry, Volume 195, Issue 2, June 2021, Pages 114–123 Thome, C., Tharmalingam, S., Pirkkanen, J., Zarnke, A., Laframboise, T., Boreham, D., 2017. The REPAIR project: Examining the biological impacts of sub-background radiation exposure within SNOLAB, a deep underground laboratory. Radiation Research 188(4.2):470-474 Kennedy K. 2020. Cellular and dosimetric characterization for biological studies of sub-natural background radiation. MSc Thesis, Laurentian. https://zone.biblio.laurentian.ca/handle/10219/3577 Jake Pirkkanen presented a virtual oral talk at Northern Health Research Conference titled: Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR): A Biological Investigation Deep Underground 2020. Jake Pirkkanen presented research at the Radiation Research Society’s annual meeting titled: Design and commissioning of a novel deep underground radiobiology project: Researching the Effects of the Presence and Absence of Ionizing Radiation (REPAIR) 2020 Wuroud Al-Kayyat presented a virtual oral talk at the Northern

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Health Research Conference in October 2020. Jake Pirkkanen, Andrew M. Zarnke, et al. 2020. A Research Environment 2 km Deep-Underground Impacts Embryonic Development in Lake Whitefish (Coregonus clupeaformis) www.frontiersin.org/articles/10.3389/feart.2020.00327/full Jake Pirkkanen, Taylor Laframboise, et al. 2020 A Novel Specialized Tissue Culture Incubator Designed and Engineered for Radiobiology Experiments in a Sub-Natural Background Radiation Research Environment. www.sciencedirect.com/science/article/pii/S0265931X2030758X?dg cid=author Pirkkanen, J., Boreham, D., Mendonca, M., 2017. The CGL1 (HeLa × normal skin fibroblast) human hybrid cell line: A history of ionizing radiation induced effects on neoplastic transformation and novel future directions in SNOLab. Radiation Research 188(4.2):512-524 Pirkkanen J, Tharmalingam S, Morais IH, Lam-Sidun D, Thome C, Zarnke AM, Benjamin LV, Losch AC, Borgmann AJ, Sinex HC, Mendonca MS, Boreham DR, 2019. Transcriptomic profiling of gamma ray induced mutants from the CGL1 human hybrid cell system reveals novel insights into the mechanisms of radiationinduced carcinogenesis. Free Radical Biology and Medicine, 145:300311. doi: 10.1016/j.freeradbiomed.2019.09.037.

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2022 Research Plan Molecular Mechanisms Involved in Radiation Resistant Breast Cancer • Perform next-generation sequencing analysis for all three CRISPR activation experiments to identify which gene manipulations promoted radiation resistant survival in MDA-MB-231 breast cancer cells. • Complete a research manuscript on the findings completed to date. Elucidating the role of FOSL1 in radiation induced tumorigenesis. • Perform transformation assays using the unique CGL-1 derivatives to identify the role of FOSL1 in radiation induced tumorigenesis. • Determine the role of FOSL1 in radiation induced DNA damage response and cell signaling. The researchers will utilize RT-qPCR based gene arrays encompassing gene families that include DNA damage response, apoptosis, cell cycle regulation, and the AP-1 complex. • Conduct transcriptomics analysis using FOSL1 manipulated CGL-1 cells with and without radiation challenge to identify the role of FOSL1 in radiobiology.

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Next Generation Sequencing using miRNA for Low-Dose Radiation Experiments • Perform an RNA-Seq transcriptomics experiment on three colonogenic radiation resistant cell lines compared to the parental control lines with sham and 6 Gy radiation exposures collected at 4 and 48 hours post challenge. • Perform γ-H2AX assay for DNA damage on radiation resistant cell lines after a radiation challenge. • Assess metabolic rate in radiation resistant cell lines using the MTT assay. • Summarize the novel miRNA profiling results in a peer-reviewed publication. • Assess whether the identified miRNA are dysregulated in the absence of background radiation using the SNOLAB research environment. • Complete a pilot experiment using radiation exposed rodent models to identify dysregulated miRNA profiles that can be detected in the blood or plasma. Key Researchers Dr. Simon J. Lees, Northern Ontario School of Medicine Dr. Sujeenthar Tharmalingam, NII Research Chair at the Northern Ontario School of Medicine Dr. Chris Thome, NII Research Chair at the Northern Ontario School of Medicine

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Fetal Programming (2015-2021) The fetal programming research program explores the effects of radiation exposure during pregnancy (fetal programming) in mice on cardiovascular and metabolic disease endpoints in offspring. The effect of low-dose radiation in attenuating induced fetal programming was explored. This research is important for the medical community to help characterize risks associated with radiation exposure during pregnancy. Sources of radiation exposure include both diagnostic imaging exposure and occupational exposure. The research is relevant to the CNSC (Canadian Nuclear Safety Commission) in adding to the knowledge base used to set dose limits and determine regulations surrounding occupational radiation exposure in pregnancy. NII funding for this research is matched by a peer reviewed NSERC grant. The findings of this work build off years of research to better understand the effects of radiation in natural and slightly elevated dose ranges. This work supports the hypothesis that significant acute doses are required to produce radiation induced effects in pregnant animals. This may provide some relief to pregnant workers, as there were no findings in mice receiving doses similar to a nuclear energy worker. Research Activities and Results In 2016, female C57BI/6J strain mice were irradiated on Day 15 of pregnancy with 0, 5, 10, 50, 100, 300 and 1000 mGy of 137Cs gamma radiation at 9.1 mGy/min. The mice pups had weekly blood pressures and weights completed until they reached 16 weeks. C57BI/6J mice pups irradiated in utero with doses of 1000mGy had significant growth restriction. The experiment was repeated with more radiosensitive BALB/c mice with similar results of growth restriction in both genders at the highest dose of 1000 mGy. No effects on blood pressure or heart rate were found in the BALB/c mice at doses between 5-300 mGy. Behavioral testing of C57BI/6J and BALB/c mice irradiated in utero showed no significant effect of radiation doses between 5 and 300 mGy. At 1000 mGy, there were no significant behavioural effects for the C57BI/6J mice. The male BALB/c mice in the 1000 mGy group displayed changes in behaviour related to dysregulation in the prefrontal cortex region of the brain (i.e., prosocial, anti-depressant behaviour consistent with potential phenotypical change). C57BI/6J female mice irradiated at 1000 mGy also had an increase in liver weight and a decrease in interscapular brown adipose tissue weight (iBAT) with increased glucose uptake by iBAT. iBAT presence and increased weight has been associated with longevity,

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improved glucose metabolism and better body weight control in animals in prior studies. In 2017, a new round of C57BI/6J mothers was irradiated at doses of 0, 50, 300 or 1000 mGy following the same procedures as in 2016, but with only a single transport to the irradiation facility. Gene expression involved in the liver functions of 1) lipid metabolism, 2) glucose regulation, 3) mitochondrial function and 4) DNA damage repair was measured in the prenatally irradiated C57BI/6J mice pups. Expression of proteins involved with insulin resistance and glucose production in both male and female mice were significant higher in the 1000 mGy group. Liver function was unaffected by prenatal radiation exposures up to 300 mGy. Liver weight was increased in females and liver triglyceride levels were increased in males at 1000 mGy. In 2018, two additional cohorts of mice were irradiated in utero at 10, 100 and 1000 mGy to assess changes in lung development. One of the cohorts received an induced acute lung injury to assess radiation-induced pulmonary immune response. A cohort of C57BI/6J mice received no treatment, a sham saline injection, or a dexamethasone steroid injection to examine the effects of glucocorticoid-induced fetal programming. In 2019, behavioural testing and gene expression data analysis was completed to correlate changes in offspring behaviour with cellular changes. From the mice that underwent behavioral testing, tissue analysis of gene expression was completed to look at various genes involved in several cellular pathways and to look for reactive oxidative species that would indicate potential stress that did not show a behavioral response. Gene expression changes were only detected for one gene at 1000 mGy. There were no significant changes in protein expression. There was a significant increase in glycogen content in the heart at higher doses, indicating increased glucose storage in heart tissue in response to adverse conditions. There were significant changes in enzyme activity related to the cellular response to oxidative stress caused by radiation. The assessment of the outcomes of glucocorticoid-induced fetal programming including PET imaging, glucose tolerance tests and tissue collection was completed in 2019. PET imaging showed some significant differences in glucose uptake in the brown adipose (fat) tissue related to dexamethasone treatments but no differences in the body weight of offspring or in the weight or size of the adipose (i.e., fat) tissue. In 2020, the goal was to determine how stress hormones affect the beiging process (the transition from white adipose tissue to brown adipose tissue) and metabolic dysfunction of adipose tissue. To study the activation of adipose tissue, the ß3adrenergic receptor (the receptor responsible for the production of heat through nonshivering thermogenesis in brown adipose tissue) was stimulated by the pharmaceutical compound mirabegron, a selective ß3-adrenergic receptor agonist administered in

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drinking water at two different concentrations. The stress hormone in mice, corticosterone, was administered via drinking water to observe its effects on adipose tissue metabolism. The individual effects of corticosterone and mirabegron were determined for adipose tissue metabolism and activation, the combination of the two treatments can provide answers to how the stress and non-shivering thermogenesis pathways interact, and how this interaction may be beneficial for combating metabolic diseases (Murray, 2021). In 2021, in vivo studies were completed on male C57BL/6J mice who were randomly divided into the five treatment groups and administered treatments via drinking water for 4 weeks. During the course of treatment, corticosterone treated mice drank significantly more water than all other treatment groups. After treatment was concluded, plasma and various tissues including the heart, soleus muscle, gastrocnemius and plantaris muscles, white (inguinal) adipose tissue (WAT) and brown adipose tissue (BAT) were collected. Analysis revealed corticosterone treated mice had significantly greater fasting body weights when compared to all other treatment groups and that when normalized to body weight, the corticosterone treatment resulted in both WAT and BAT having greater mass than other treatments. The corticosterone treated mice also had significantly lower heart, kidney, and gastrocnemius & plantaris muscle ratios than all other treatments. Fasting glucose and insulin concentrations were assessed across all mice groups. The treatments did not yield a significant effect on glucose levels, however insulin concentrations were significantly elevated in the corticosterone treatment. Use of a homeostatic model of assessment for insulin resistance (HOMA-IR) showed that corticosterone treatment induced insulin resistance in these mice. Corticosterone treated mice had significantly larger adipocytes in both white and brown adipose tissue samples. Mirabegron treated mice showed adipocyte sizes similar to both the control and vehicle mice. In order to study the changes occurring at the protein level, western blots for beiging and inflammation markers are in the process of being optimized.

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Outcomes Table 3: Scientific reports produced for Fetal Programming research Source Murray A., Tharmalingam S., Nguyen P., Tai T.C.. Untargeted metabolomics reveals sex-specific differences in lipid metabolism of adult rats exposed to dexamethasone in utero. Sci Rep. 2021 Oct 13;11(1) Sreetharan, S., 2017. Prenatal ionizing radiation exposure effects on cardiovascular health and disease in C57B1 mice. MSc thesis, McMaster University, September 2017 Nemec-Bakk et al. (2020). Long term effects of low dose radiation during late gestation on cardiac metabolism and oxidative stress. Experimental Biology 2020 Meeting. Sreetharan et al. (2020). 137Cs gamma radiation exposure during pregnancy in C57Bl mice: effects on offspring growth, cardiovascular physiology and gene expression following birth. 66th Annual Radiation Research Society Meeting. Bel et al. (2020). The effects of chronic stress on brown adipose tissue remodeling & metabolism. Northern Health Research Conference. Sreetharan et al. (2020). Ionizing radiation during pregnancy in C57Bl mice: effects on offspring following birth. Northern Health Research Conference. Bel J, Tai TC, Khaper N and Lees SJ (2020). Mirabegron: the most promising adipose tissue beiging agent. Physiol Rep. 2021 Mar;9(5) Davidson, C., Phenix, C.P., Tai, T.C., Khaper, N.K., Lees, S.J., 2018. Search for a novel PET probe for cardiac inflammation detection. Am. J. Nucl. Med. Mol. Imaging. In Press. Davidson CQ, Phenix CP, Tai TC, Khaper N and Lees SJ. (2018) Searching for novel PET radiotracers: imaging cardiac perfusion, metabolism and inflammation. American Journal of Nuclear Medicine and Molecular Imaging. 8(3): 200. Davidson CQ, Tharmalingam S, et al. (2020). Dose threshold for radiation induced fetal programming in a mouse model at 4 months of age: Hepatic expression of genes and proteins involved in glucose metabolism and glucose uptake in brown adipose tissue. PLoS ONE. 15(4): e0231650. Lalonde C, Sreetharan S, et al. Absence of Depressive and Anxious Behavior in Adult C57Bl/6J Mice After Prenatal Exposure to Ionizing Radiation. Radiation Research – In Press

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Type Publication

MSc thesis

Poster

Presentation

Publication Publication

Publication

McEvoy-May JH, Jones DE, et al. (2020). Unchanged cardiovascular and respiratory outcomes in healthy C57Bl/6 mice after in utero exposure to ionizing radiation. Int J Radiat Bio 2020 Dec 3;1-12. Sreetharan S, Stoa L, Cybulski ME, Jones DR, Lee AH, Tharmalingam S, Kulesza AV, Boreham DR, Tai TC and Wilson JY. (2019). Cardiovascular and growth outcomes of C57Bl mice offspring exposed to maternal stress and ionizing radiation during pregnancy. International Journal of Radiation Biology. 95(8): 10851093. Sreetharan S, Thome C, Tharmalingam S, Jones DE, Kulesza AV, Khaper N, Lees SJ, Wilson JY, Boreham DR and Tai TC. Ionizing radiation exposure during pregnancy: effects on postnatal development and life. Radiation Research 187(6):647-658 Tharmalingam S, Sreetharan S, Kulesza AV, Boreham DR and Tai TC. Low dose ionizing radiation exposure, oxidative stress and epigenetic programming of health and disease. Radiation Research 188(4.2):525-538

Publication

2022 Research Plan Although funding through the NII has ended for this project in 2021, the researchers noted there is some final work related to the development of pulmonary testing endpoints and the development of an Acute Lung Injury model to include prenatal radiation exposure. This work will continue in collaboration with the Genetic Models of Risk project, which will explore the risks of radiation exposure to the lungs during pregnancy.

Key Researchers Dr. T.C. Tai, Northern Ontario School of Medicine Dr. Simon Lees, Northern Ontario School of Medicine

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Lens of the Eye (2017-2021) Research Activities and Results The lens of the eye program aims to investigate the development of cataracts following exposure to ionizing radiation. Ionizing radiation exposure to the lens of the eye is a known cause of cataracts. Historically, it was believed that the threshold dose for cataract formation was 5 Sv and annual equivalent dose limits to the lens were set at 150 mSv. The International Commission on Radiological Protection (ICRP) and International Atomic Energy Agency (IAEA) have now reduced their threshold dose estimate for deterministic effects to 0.5 Gy and are now recommending an occupational limit of 20 mSv per year, averaged over 5 years, up to a maximum of 50 mSv in a single year. A comprehensive review of current evidence for the proposed new 20mSv per year dose limit to the lens of the eye was published in 2017 (see table 6). In 2018 and 2019, an epidemiological study was completed to examine the association between patient exposure to multiple head CT scans and subsequent cataract formation. A research proposal was submitted to the Institute for Clinical Evaluative Sciences (ICES) outlining a project to identify if there is a correlation between CT scans and cataract surgery in Ontario. The data set contained all individuals residing in Ontario between 1994 and 2015 (22 years), resulting in a sample size of over 16 million. Several groups were excluded from the analysis: individuals with a history of congenital/trauma induced cataracts or anyone receiving radiotherapy for head and neck cancers. Data were extracted on the presence/timing of cataract extraction surgery as well as the number and timing of head CT scans received. Subjects were grouped based on the number of head CT scans they received ranging from 0 to 10+ in the 5-year lag group or 0 to 6+ in the 10-year lag group. Data were also extracted for covariates previously linked to cataract formation. This included age, sex, diabetes, hypertension, and previous intraocular surgery. The full data set was analyzed using a multivariate cox proportional hazards survival model with 5-year and 10-year lags. There is a known latency period between radiation exposure and cataract formation, so it is unlikely that any cataract occurring within several years of a CT scan is due to radiation. The 5-year and 10-year lag omitted any head CT scan from the analysis that occurred within 5 or 10 years of a cataract surgical procedure respectively. Overall, the multivariable cox model did not show any significant correlation between head CT scans and cataract extraction surgery. Individuals receiving 1 to 3 head CT scans did have a small increased risk of cataract surgery. However, individuals receiving

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4 or more head CT scans did not have an increased risk. In general, an inverse dose response was observed, where the cataract risk decreased as the number of CT scans increased. Therefore, the conclusion of this study is that ionizing radiation exposure associated with head CT scans does not increase the risk of cataract formation. The paper was published in January 2020. During 2019, researchers also began in-vitro studies to investigate the mechanism behind radiation induced cataracts. Despite the large number of epidemiological studies on radiation cataracts, the exact mechanism by which radiation exposure in the lens can progress to opacifications remains unknown. It is hypothesized that radiation primarily interacts with lens epithelial cells (LEC), one of the two cell types found within the human lens. DNA damage and oxidative stress in LEC may result in impaired differentiation, proliferation, and migration. This could lead to a buildup of cells thereby reducing the normal transparency of the lens resulting in a cataract. Researchers are testing this hypothesis using a LEC cell line cultured in the laboratory. In 2020, cell culture experiments were conducted using a human lens epithelial cell line to examine the impacts of various doses of ionizing radiation on the rate of cell migration. Radiation-induced changes in cell migration is hypothesized to be one of the factors leading to lens opacities. Preliminary data suggests that higher doses of radiation (> 1 Gy) can increase the amount of cell migration in lens cells. The same effect was note seen at low doses of radiation (10 – 100 mGy). This suggests that there is a threshold level to radiation-induced changes in cell migration in the lens. Recent ICRP (International Commission on Radiological Protection) recommendations have stated that there is no dose rate effect within the lens, and that it is equally sensitive to acute and protracted exposures. To test this, cell survival curves across different dose rates (single acute exposure, 24-hour fractionated exposure, 48-hour fractionated exposure) were examined. It was found that cell survival is increased when radiation is delivered over multiple fractions, which suggests that dose rate effects do exist within cells of the lens. Therefore, these findings contradict the recommendations of the ICRP and warrants further investigation. Cell culture experiments were continued in 2021 using a human lens epithelial cell line (HLE-B3). The impacts of various doses of radiation were studied on cell migration, proliferation, and adhesion to begin to elucidate the mechanisms of radiation induced cataracts. This research produced some interesting results. In general, a non-linear radiation response was observed with the greatest effects occurring at a dose of 0.25 Gy. At this dose, a short-term reduction in proliferation occurred (82% of controls), which was followed by a long-term increase in proliferation (116% of controls). Cell migration

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was increased at both 12 hours and 7 days post-irradiation at a dose of 0.25 Gy, with migration occurring at respective rates 122% and 233% times greater than controls. Cell adhesion was consistently reduced above doses of 0.25 Gy on three different extracellular matrices. Transcriptional alterations were identified at these same doses in multiple genes related to proliferation, adhesion, and migration (Vigneux, 2022). In 2021, in-vivo animal studies were initiated. The goal of these studies is to examine changes in cell structure, apoptosis, and proliferation in the lens of mice which were exposed to x-rays. Irradiations were performed on the first set of mice in the spring of 2021. However, shortly after starting this project Laurentian University shut down their animal facility. As a result, this research project has been on hold since then. Outcomes Table 4: Scientific reports produced as part of the Lens of the Eye collaboration Source Type Vigneux G., Pirkkanen J., Laframboise T., Prescott H., Tharmalingam Publication S., Thome C. Radiation-Induced Alterations in Proliferation, Migration, and Adhesion in Lens Epithelial Cells and Implications for Cataract Development. Bioengineering (Basel). 2022 Jan 12;9(1):29 Prescott, H, Vigneux, G, Laframboise, T, Tharmalingam, S, Thome, C. Presentation 2020. Investigating Dose Rate Effects in Lens Epithelial Cells to Understand the Risk of Radiation-Induced Cataracts. Vigneux, G, Prescott, H, Thome, C. 2020. Radiation Induced Presentation Functional Changes in Lens Epithelial Cells and Implications for Cataract Formation. Gaudreau K*, Thome C*, Weaver B, Boreham DR. Cataract formation Publication and low dose radiation exposure from head computed tomography (CT) scans in Ontario, Canada, 1994 – 2015. Radiat Res. 2020. *Cofirst author. Thome, C., D.B. Chambers, A.M. Hooker, J.W. Thompson, and D.R. Publication Boreham. 2017. Deterministic effects to the lens of the eye following ionizing radiation exposure: Is there evidence to support a reduction in threshold dose? Health Physics. 114(3): 328-343 2022 Research Plan The research team will continue to investigate the mechanisms of radiation induced cataracts using the HLE-B3 cell line. Previous data suggests that a dose of 0.25 Gy has a significant impact on lens cell function and this radiation dose will be further investigated.

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The research to date has focused on the effects following a single acute radiation dose. Future work will examine the effects of different dose rates, to determine if the same changes in adhesion, proliferation, and migration will occur when radiation is delivered over fractionated doses. The researchers plan to run transcriptomic analysis on these irradiated cells. They have identified some of the genes involved in these responses, however, a full transcriptomic analysis will provide a more thorough picture of the regulatory mechanisms that are at play. Animal experiments are planned to restart in 2022. The animal facility in Thunder Bay has the same x-ray irradiator as in Sudbury. The research team is currently working out logistics of performing animal irradiations in Thunder Bay and then shipping the preserved tissue samples to Sudbury for analysis. Theu will also have to obtain a new animal ethics approval. This work will commence in early 2022. Key Researchers Dr. Chris Thome, NII Research Chair at the Northern Ontario School of Medicine

Low-dose Radiation Immunology (2020-2021)

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The aim of this project is to investigate the effects of low dose radiation exposure on the immune system. High dose ionizing radiation (HDR) is an established treatment modality for cancers. However, these high dose therapies often produce side effects of acute normal tissue toxicity. Unlike HDR induced cancer killing, there is growing evidence that low dose radiation (LDR) promotes tumour reduction via stimulation of the immune system. Using a small animal model this research project is investigating the effectiveness of LDR in treating tumours in mice, along with identifying which cells and molecules of the immune systems are involved in the LDR response. Research Activities and Results This research project commenced in 2020. Since then, animal ethics approval was obtained from Laurentian University. Wild type mice were ordered, and researchers used these animals to fully optimize our irradiation protocols. A major component of this project is to investigate the changes in immune cell populations in response to ionizing radiation exposure. To detect the various immune cells, a large number of antibodies are required. During the past four months, research has focused on optimizing the specific antibody panels that are required for immune cell identification. In early 2021, the research team completed the optimization of the antibody panels for flow cytometry analysis of immune cell types in mice. Mice were exposed to a single acute dose of 100, 250, 500 and 3,000 mGy. The 3,000 mGy dose was used as a high dose control, where immune suppression is expected. On the other hand, the three lower doses are in the range where immune stimulation will occur is expected. Two days post exposure, mice were sacrificed following which blood, spleen, and lymph node samples were collected. Flow cytometry analysis was performed to determine which immune cell types were impacted by radiation. Overall, the high dose exposure (3,000 mGy) caused a reduction in cell numbers for most cell types (T cells, B cell, NK cells, neutrophils), which was predicted. The low doses did not have a significant impact on most cell types, with the exception of natural killer (NK) cells. Mice receiving a 100 mGy exposure had an increase in the number of NK cells in the spleen 48 hours post exposure. NK cells are known to be involved in the immune systems defence against cancer. The initial plan was to complete additional animal experiments, where immune effects were examined at different time points post exposure (14 days) to look at fractionated vs acute exposure. However, the animal facility at Laurentian was shut down shortly after the first cohort was completed. Therefore, the animal work has been temporarily put on hold. While waiting for animal work to restart, cell culture experiments were started. The animal work suggested that low dose radiation can impact NK cells, so an NK cell line (NK-92) is being used. In the fall of 2021, protocols for culturing NK-92 cells in-vitro and exposing them to x-rays were optimized. Using this cell line, the ability of NK cells to kill

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tumour cells (cytotoxicity) can be measured. By co-culturing the NK-92 cells with a tumour cell line (k-562), how many of the tumour cells are killed by the NK cells is being quantified. Current experiments consist of irradiating NK cells, prior to co-culturing them, to investigate whether LDR can increase their cytotoxicity. Preliminary data suggests that a dose of 250 mGy can increase NK cell cytotoxicity 48 hours post irradiation. 2022 Research Plan The goal for 2022 is to continue with the NK cell in-vitro studies. The cytotoxicity experiments will be completed to confirm if LDR can increase the ability of NK cells to target and kill cancer cells. These experiments will help identify the optimal dose, timing, and fractionation regimen for radiation induced immune stimulation. Once these experiments are complete, the mechanisms of how LDR increases NK cytotoxicity, by quantifying changes in gene and protein expression will be examined. In 2022, the animal experiments will restart. This will most likely occur in Thunder Bay. Key Researchers Dr. Sujeenthar Tharmalingam, NII Research Chair at the Northern Ontario School of Medicine Dr. Chris Thome, NII Research Chair at the Northern Ontario School of Medicine

Harnessing Genetically Modified Microbes to Combat the Effects of Ionizing Radiation (2020-2021)

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The overall goal of this research project is to utilize state-of-the-art gene editing technology to generate genetically engineered microorganisms (GEMs) that are resistant to ionizing radiation, and to test the application of these GEMs as a revolutionary tool to substantially alter two long-standing problems: (1) Nuclear Waste Management: Develop GEMs that can survive toxic doses of radiation and demonstrate enhanced potential to concentrate and transform large amounts of radioactive waste to less toxic products. (2) Radiation Therapy Induced Bowel Injury: Develop genetically engineered variants of gut microbes that are resistant to high doses of radiation. Following that, the plan is to inoculate these GEMs in the gut of a rodent model and determine whether the radioresistant GI bacteria can help prevent radiationinduced bowel injury or accelerate recovery. Research Activities and Results Nuclear Waste Management As the project began in 2020, the research team established a microbiology laboratory at NOSM, including a hypoxic chamber for culturing anaerobic bacteria. The team is working with two bacterial strains: Lactobacillus reuteri and Bifidobactrium Bifidum by characterizing the growth of these strains in liquid culture and on agar plates. In 2021, research focused on the goal to develop genetically engineered bacteria that can survive toxic doses of radiation and demonstrate enhanced potential to concentrate and transform large amounts of radioactive waste to less toxic products. The research team established infrastructure and methodology for culturing anaerobic microbes to maintain Lactobacillus reuteri and Bifidobacterium bifidum cultures. Survival assays post radiation were used to demonstrate 50Gy radioresistance in L. reuteri and B. Bifidum. A compatible plasmid system for genetic engineering of L. reuteri was identified. Researchers have identified a methodology to stably integrate a foreign DNA plasmid system capable of delivering novel genes into the bacterial systems. Radiation Therapy Induced Bowel Injury Protocols for in-vivo analysis of the gastrointestinal tract and microbiome in mice have been established. The procedures have been optimized for animal dissections post irradiation, enabling the successful isolation of microbiome constituents and histological analysis of intestinal tissue. The following was also completed towards the research into the effects of radiation on the gut microbiome. Cohort 1 irradiations and sample collection have been completed for 40 male mice which were subjected to various radiation doses (0, 100, 250, 500 and nii.ca/environment-at-nii

3000 mGy) and euthanized 2 days post radiation. Various GI tissues (Duodenum, Jejunum, Ileum and Colon) and their respective luminal contents were collected for histological and microbiome composition analysis. A pilot 16S metagenomic analysis has been completed for the various GI tissue and luminal samples. This pilot experiment revealed that further optimizations were needed during the metagenomic library preparation in order to account for GI contaminants that interfere with the PCR steps. A second pilot experiment was conducted with optimized metagenomic library preparation procedures. This second experiment was a success. The researcher team has now completed the DNA extraction and library preparation for all GI and luminal samples for Cohort 1. GI tissues samples were collected to study changes in tissue morphology post- radiation exposure. Tissue embedding, sectioning and H&E staining have been completed for all GI tissues pertaining to Cohort 1. Moreover, basic histological analysis on GI crypt and villi lengths was completed in order to assess the effects of radiation on GI biology. 2022 Research Plan Nuclear Waste Management Supplies for next generation sequencing are been backordered due to COVID-19 which caused delays with completing the DNA extraction and library prep for the microbiome characterization analysis. Radiation Therapy Induced Bowel Injury Animal work at Laurentian is halted and efforts are being made to find an alternative location to continue work as soon as possible. Cohort 1 experiments will be repeated using female mice. A study on the effects of a fractionated radiation dosing regimen on GI biology is also planned. The plan in 2022 is to perform transcriptomic profiling of 50 Gy and sham exposed anaerobic cultures to identify genes that are dysregulated with radiation exposures. The identified genes will be examined for DNA repair capacity and ability to reduce heavy metals. Alongside the previous goal, the team also aims to Develop a CRISPR-based molecular biology methodology to manipulate the genome of the microbes in order to genetically alter the expression of genes that contribute to enhanced DNA repair and ability to reduce heavy metals. Further goals are to:

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• • •

Complete 16S metagenomics of the various rodent GI tissue collected post radiation. Complete the GI gross histology post-radiation analysis. Profile cell apoptosis markers to identify the cellular effects of radiation on the different GI cell types. Publish the histology and microbiome characterization results in a peerreviewed journal.

Key Researchers Dr. Sujeenthar Tharmalingam, NII Research Chair at the Northern Ontario School of Medicine Dr. Chris Thome, NII Research Chair at the Northern Ontario School of Medicine

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NEUDOSE (2019-2022) Started in 2015, NEUtron DOSimetry & Exploration (NEUDOSE, pronounced “new dose”) is a satellite mission that is being designed and built by researchers at McMaster University to measure the properties of radiation to which astronauts are exposed while performing spacewalks in low-Earth orbit. The official website for the NEUDOSE satellite project is updated weekly and can be accessed at https://mcmasterneudose.ca/. The scientific objective of NEUDOSE CubeSat is to increase our understanding of the risks associated with prolonged exposure to space radiation by investigating the contribution of charged particles and neutrons to the total ambient dose equivalent in low-Earth orbit (LEO). The free space radiation environment in LEO consists predominantly of charged particles trapped in the Van Allen belts, galactic cosmic rays (GCR) originating from deep space, solar energetic particles (SEPs), and neutrons produced by the interaction of GCRs with the Earth’s atmosphere. From a radiobiological perspective, neutrons present the greatest risk because they are difficult to measure and can deeply penetrate the body and affect blood-forming marrow in bones. However, in comparison with the number of measurements made of other types of ionizing radiation relatively few measurements have been made of neutrons and their contribution to total dose equivalent in LEO. If we are to commit astronauts to long term exposures in the near-Earth or deep space environment, we must provide a resolution for the ambiguous contribution of neutrons to the total ambient dose equivalent. To address the dosimetry challenges of current mission to the ISS, and future mission into deep space, the objective of our scientific instrument is to demonstrate the application of new techniques and on-board data processing to measure the neutron contribution of total ambient dose equivalent in a space environment. The NEUDOSE CubeSat represents an important platform for increasing the technological readiness of the instrument, making unique and new measurements of the neutron dose in LEO, and for furthering our understanding of risks associated with prolonged exposure to space radiation. Research Activities and Results In 2019, the NEUDOSE project successfully completed the following Canadian Space Agency milestones: • Mission Concept Review (MCR) – hosted by McMaster University on January 15th and 16th, 2019 • Preliminary Design Review (PDR) – hosted by Western University on October 10th and 11th, 2019

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The project also implemented software for configuration and data management and obtained licenses for basic and advanced radio licensing for team members to allow for communication and commanding of the radio transceiver on the satellite. Initial modelling work was completed on the electrical and power subsystem. A custom mechanical structure was designed. A prototype flight computer was designed and was fabricated. Initial ground-station design and testing is underway for the tracking telemetry and command station. The final engineering model for the data acquisition instruments to measure the properties of radiation in space was redesigned and tested successfully. In 2020, the NEUDOSE project successfully completed the following Canadian Space Agency milestones: • Preliminary Critical Design Review (Pre-CDR) – hosted virtually on Sept. 25, 2020 The NEUDOSE satellite will use amateur radio frequencies for communication, and this enables us to participate in the group licensing that will be managed by the Canadian Space Agency. However, operation of the ground station at McMaster satellite and commanding of the satellite requires certain members on the team to receive and maintain their basic and advanced amateur radio license. To date, six members of the team have received their basic license and two have received their advance licenses. In 2020, the team has successfully worked with the Spectrum Management Operations Branch of Innovation, Science and Economic Development (ISED) office to transfer of the callsign to McMaster NEUDOSE. The team has decided on a consumer-off-the-shelf (COTS) electrical power subsystem (EPS) that will be purchased for the satellite. The team has performed substantial design work on the placement and wiring of the physical deployment switches as well as the solar cell mounting procedure. Lastly, the team has built two solar panel simulators that will be used, in conjunction with AGI’s System Tool Kit, as a hardware-in-the-loop simulator for the EPS subsystem. The mechanical team made significant progress towards the final mechanical structure design for the NEUDOSE satellite. During this time, they have (1) redesigned the mechanical structure to make manufacturing easier and attain a better tolerance, (2) selected a new limit switch for deployment and decided on mounting them through the rails, (3) introduced a new harnessing method that simplifies the assembly process, and (4) completed all manufacturing drawings. The mechanical team has also worked with attitude control team to design mounts for the permanent magnets and hysteresis rods that make up the Passive Magnetic Attitude Control (PMAC) system. Lastly, the team has also completed various FEA simulations to verify the primary resonant modes of the structure as well as developed the vibration test plan.

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The thermal team continued their work on developing and verifying thermal modelling techniques to develop a thermal management solution for the satellite while in different operational modes. Additionally, the thermal team also developed a thermal cycling as well as thermal vacuum cycling plan for the entire satellite. The team is currently developing a Passive Magnetic Attitude Control (PMAC) system to be included on the NEUDOSE satellite. In 2020, the team has specified the strength and dimensions of the permanent magnet as well as hysteresis rods and worked with the mechanical team to ensure they can be mounted such that null vector in the dipole radiation pattern of the antenna does not point towards the ground station in an access window, which would hinder communication. The command & data handling subsystem has (1) purchased the flight computer as the primary flight computer for NEUDOSE, (2) completed the initial design for the secondary flight computer that is built by McMaster University students, and (3) made significant progress on the development of the flight software which is based on NASA’s Core Flight Software (cFS). The space segment of the communications subsystem has (1) completely defined the communications network topology for the entire satellite communication buses, (2) defined the communication protocols for telecommanding, (3) proposed a telecommand encryption scheme, and (4) fabricated/assembled two Rev 3.0 units of our custom-built communications module. Rev 3.0 is the newest, and most flight-like, version of our communications module and will be used as a platform for firmware/software development. The ground segment of the communications subsystem has (1) setup the ground station server, (2) developed and tested a processing pipeline for encoding and decoding packets, (3) finalized the design for the stationary antenna mount, and (4) completed preliminary layout for the McMaster NEUDOSE Satellite Operations Center. The science instrumentation team completed major design, fabrication, and testing on the instrument. At the mechanical assembly level, the students redesigned the structure to satisfy the NanoRacks venting requirements for air that may become trapped. Additionally, the team completed various FEA simulations to demonstrate the 1st resonant mode for all components is above 2 kHz and the pressure vessel can withstand the rapid change in outside pressure when placed in the NanoRacks deployer. With respect to the electronics, the team successfully completed the design work on the Data Acquisition Module (DAM) (Rev B) and has completed fabrication. The team received the fabricated DAM in late-December 2020 and has since verified the components work as expected.

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In 2021, the NEUDOSE project successfully completed the following Canadian Space Agency milestones: • Critical Design Review (CDR) and Test Readiness Review (TRR). The research team is constructing and testing the engineering and flight models of the satellite according to plans made in 2020 and presented to the CSA for comments. Final delivery of the satellite to the CSA for launch will occur Q4 2021. In 2021, the team successfully commissioned and completed the licensing and construction of the McMaster University ground station and cleanroom for satellite assembly and testing. Outcomes Table 5: Scientific reports produced as part of the NII-CHER collaboration Source Type Hanu A.R.,et al., 2017. NEUDOSE: A CubeSat Mission for Dosimetry Publication of Charged Particles and Neutrons in Low-Earth Orbit Radiation Research 187(1):42-49 https://doi.org/10.1667/RR14491.1 2022 Research Plan The team will finish the assembly of all qualification and flight components. The team will also be continuing development on the flight software and work alongside the communications and science instrument teams to perform integration testing at the FlatSat level. Once complete, the qualification satellite will undergo vibration and thermal tests to ensure survivability. Any final changes will be made to flight models and delivered to the CSA for launch. The major objectives for the science instrument are to (1) completely assemble and test 3 science instruments (all components are available on-hand but access to the lab is limited) and (2) perform a preliminary instrument calibration campaign at TRIUMF and/or on a high-altitude balloon. Key Researchers Dr. Andrei Hanu, Bruce Power Dr. Soo Hyun Byun, McMaster University Dr. Fiona McNeill, McMaster University Dr. Eric Johnston, Nuclear Innovation Institute, formerly Bubble Technology Industries

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Environment Research Programs Aquatic Biota (2018-2022) This research program investigates the research gaps identified in the course of the previous Whitefish research program. A Mitacs grant application was successfully submitted in 2017 seeking support for four post-doctoral fellows for an additional five years of research. These post-doctoral fellows are completing research on the following areas: 1. Determine the genetic population structure of Lake Whitefish, Round Whitefish, and Yellow Perch using advanced DNA analysis techniques. 2. Examine potential mechanisms causing mortality during embryogenesis and reduced fitness later in life by comparing transcriptomes of fish using advanced DNA analysis techniques. 3. Determine the survival and fitness of Lake and Round Whitefish subjected to thermal discharges during embryonic development. 4. Assess the survival of embryos from a model species (Yellow Perch) reared in varying temperatures reflective of thermal discharges. An NSERC application was received in 2018 in support of research addressing three remaining key questions: 1. Thermal effects in Lake and Round Whitefish hatchlings and juveniles: What are the effects of variable and increased incubation temperatures on Lake and Round Whitefish hatchlings and juveniles? 2. Thermal effects in spring spawning fish: Using Yellow Perch as a model species, what are the effects of variable and increased incubation temperatures on the embryonic, hatchling and juvenile stages of spring spawning fish? 3. Population structure of Lake and Round Whitefish and Yellow Perch: What are the underlying population structures and habitat use of Lake and Round Whitefish and Yellow Perch in Lake Huron and near Bruce Power? What are the boundaries of the populations near Bruce Power? Research Activities and Results Developmental effects of elevated rearing temperatures on Lake and Round Whitefish larval and juvenile stages This research is investigating whether thermal stress experienced during embryonic development of Lake Whitefish impacts fitness of fish during the larval and juvenile life stages. The objectives are to compare response of the following between species (Lake Whitefish and Round Whitefish) and among different thermal groups. Unfortunately, nii.ca/environment-at-nii

researchers were unable to obtain Round Whitefish during the fall of 2017, so the 2018 research was only conducted with Lake Whitefish examining the following endpoints: 1. 2. 3. 4.

Mortality rate and time of hatch during embryonic development. Mortality rate through the larval period (hatching to juvenile stage). Jaw development and transition to exogenous feeding during the larval period. Aggression and predator avoidance of juvenile whitefish.

An R-based analysis of morphometrics measurements has been developed. In the fall of 2018, Lake and Round Whitefish embryos from Lake Huron, Ontario, Superior and Simcoe were collected. Additional Lake Whitefish embryos were collected from Lake Diefenbaker in Saskatchewan. In 2018-2019, the experimental set-up was designed and the first studies of the thermal preferences of juvenile Lake Whitefish were completed. The 2019 studies examined the post-hatch survival, feeding, growth and behaviour effects of seasonal and elevated seasonal thermal regimes on post-hatch development. In 2021, Lake Whitefish embryos were incubated at either 0, 2, or 5oC until hatching, acclimated to 14-16oC, and reared until ~19 months before receiving a 6oC thermal stress event for two hours (followed 0-48 hours of recovery). Whole transcriptome sequencing was completed on liver and bioinformatics are ongoing. The team continues to analyze the liver transcriptome from Lake Whitefish reared at 0, 2 or 5 °C in embryogenesis which experienced heat shocks at 19-20 months of age. The heat shocks were +6°C for 2 h, with 0-48 h recovery time. This data will provide information on the changes in gene expression with incubation temperature, heat shock and recovery time. Overall, there was little separation in mRNA transcript levels between the 3 embryonic temperature groups. However, transcript levels did vary significantly over the course of recovery with genes related to the stress response, cell signaling, and protein processing in the endoplasmic reticulum being affected most. The Lake Whitefish genome has recently been published and may help with the identification and annotation of transcripts that are differentially expressed.

Developmental effects of elevated rearing temperatures on Lake and Round Whitefish embryos In 2018-2019, Round and Lake Whitefish embryos collected in the fall of 2018 were incubated under seasonal regimes starting at 8°C or 6°C and dropping by 1°C/week until 2°C, then staying at 2°C or 5°C per week for 6 weeks, followed by a rise of 1°C per week

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until hatch. An elevated seasonal regime was tested at +4°C above the seasonal regime. Data analysis is ongoing for these experiments but has been complicated by high overall mortality. Testing of acute heat shocks early in the seasonal decline for Lake Whitefish was also completed in 2018-2019. A threshold temperature of mortality was observed in one of the early incubation acute heat shock experiments, where exposure to 8 or 10 °C during early life had no effect while exposure to 12 °C induced and increase in embryonic mortality. (Harman, 2020) In 2020, the team completed their study on the temperature preference of Lake and Round Whitefish reared at different temperatures. Twelve-month-old Round Whitefish reared at 2°C and 6°C had significantly lower temperature preferences compared to those reared at 0.5°C. Interestingly, this relationship was not observed with Lake Whitefish at either 8 or 12 months of age, suggesting that Round Whitefish are more thermally sensitive than Lake Whitefish and that differences in embryonic incubation temperature can influence the preferred temperature of fish up to a year post-hatching. Round Whitefish preferred a colder temperature than Lake Whitefish. A difference in growth between treatment groups was not observed, as there were no difference in length or weight of either species at 8 or 12 months of age. This suggests that there may be reasonable “catch up” of growth for early hatched embryos, provide they are reared with plenty of food in optimal temperature post-hatching. There was a difference in growth across species; Lake Whitefish were significantly larger in both length and mass. (Harman, 2020) Performance effects of elevated rearing temperatures on Lake and Round Whitefish larval and juvenile stages The performance effects examined in this research look at the long-term effects of suboptimal rearing temperatures on 1) swim performance and metabolism and b) thermal stress responsiveness using transcriptomics. Experiments continued to examine the effects of sub-optimal incubation temperatures and daily heat shocks on thermal tolerance in Lake Whitefish. Swim tunnels were attempted unsuccessfully with 18-month-old Lake Whitefish but may be tried again with 2.5-year-old Lake Whitefish in the future. Lake Whitefish embryos were reared at seasonal, elevated seasonal or seasonal plus daily heat shock groups. Larvae from the elevated seasonal group in this experiment were found to hatch earlier and have a higher critical thermal maximum than those in the seasonal or seasonal plus daily heat shock group. No morphometric differences were found between groups. Heat shock experiments were completed on these larvae

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to examining the threshold for heat shock protein induction with heat shocks from 0 to 12℃. Work examining stress genes in larvae from these groups is ongoing. In 2020, the team completed morphometric measurements of remaining samples, and statistical analysis of data on hatching, post-hatching feeding, survival, and growth. In 2021, Swim performance was tested in those Lake Whitefish reared embryonically at 2 or 5°C with post-hatch rearing at 12°C for 12 months, followed by acclimation to 15 or 19°C and testing at 14-18 months post hatch (Harman, 2021). Minimum and maximum oxygen consumption rates, aerobic scope and critical swimming speed were determined. Aerobic scope is the capacity to increase the aerobic metabolic rate above those needed for basic maintenance. Critical swimming speed is defined as the fastest pace that can be maintained without exhaustion, an important measure of swim performance. Both aerobic scope and critical swimming speed were higher for fish reared at 2°C, with 15°C acclimation in the juvenile stage; suggesting an increase in swim performance for fish reared at colder temperatures. Thermal effects in spring spawning fish After several unsuccessful attempts, Yellow Perch embryos were successfully obtained in spring 2021. Population structure of Lake and Round Whitefish and Yellow Perch The research continues to expand upon the population structure work from prior Whitefish research. The recent focus has been on developing the use of single nucleotide polymorphisms (SNPs) as a tool for investigating Whitefish population structure. The data analysis has explored the trade-off between the cost of DNA sequencing and the resolution power provided by the resulting SNP data. Specifically, Search Bay shows relatively strong differentiation from the other sites within Lake Huron, while North Point and Hammond Bay showed slight differentiation in the ordination and maximum likelihood approaches. The area around Bruce Power shows no genetic differentiation in all three analyses with Scougall Bank, Douglas Point, McRae Point and Fishing Islands having very little differentiation in all three analyses. This indicates that there is not likely a distinct Lake Whitefish population in the Bruce Power area. On a larger scale, preliminary results suggest that the genetic diversity of Lake Whitefish may be higher outside of the Great Lakes region. The SNP Whitefish genetic population structure manuscript has been accepted for publication (Morgan, 2018). Researchers are continuing to develop a network to facilitate the collection of Round Whitefish specimens from additional sites in Lake Huron. This species is not commercially harvested and is therefore only available via targeted netting, such as that

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done by government and tribal biologists for population monitoring. These specimens will be used to delineate the population structure of Round Whitefish in Lake Huron using SNPs. Researchers are also developing a network to collect Yellow Perch specimens from Lake Huron. Two specimens are currently available and will be used to pilot basic genomic characterization for Yellow Perch. Lab evaluation is in progress for the completion of bulk- and compound-specific stable isotopes analyses of Lake and Round Whitefish and Yellow Perch from a variety of sites in Lake Huron. In 2020, analyses were ongoing of Lake Whitefish SNP variation geographically and across environmental gradients in a large area spanning the Great Lakes region and central Canada. Environmental parameters account for much less genetic variation than spatial connectivity of populations. Outcomes Table 6: Scientific reports for Aquatic Biota Source Eme, J., Mueller, C.A., Lee, A.H., Melendez,C., Manzon, R.G., Somers, C.M., Boreham, D.R., and Wilson, J.Y. 2018. Daily, repeating fluctuations in embryonic incubation temperature alter metabolism and growth of Lake whitefish (Coregonus clupeaformis). Journal of Comparative Physiology B, 226:49-56. Harman, A.A., Fuzzen, M., Stoa, L., Boreham, D.R., Manzon, R.G., Somers, C.M., Wilson, J.Y. (2021). Evaluating tank acclimation and trial length for dynamic shuttle box temperature preference assays in aquatic animals. Journal of Experimental Biology 224 Sessions, K.J., Whitehouse, L.M., Manzon, L.A., Boreham, D.R., Somers, C.M., Wilson, J.Y., Manzon, R.. (2021). The heat shock response shows plasticity in embryonic lake whitefish (Coregonus clupeaformis) exposed to repeated thermal stress. Journal of Thermal Biology 100, 103036. Mitz, C., Thome, T., Thompson, J., Cybulski, M.E., Somers, C.M., Manzon, R.G., Wilson, J.Y. Boreham, D.R. (2021). A model to predict embryonic development and hatching in lake whitefish (Coregonus clupeaformis) under variable incubation temperatures,Journal of Great Lakes Research 47: 494-503.

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Type Publication

Publication

Harman, A. Effect of elevated embryonic incubation temperature on the temperature preference of juvenile lake (Coregonus clupeaformis) and round whitefish (Prosopium cylindraceum). MSc Thesis, McMaster University 2020 Graham CF, Boreham DR, Manzon RG, Stott W, Wilson JY, et al. (2020) How “simple” methodological decisions affect interpretation of population structure based on reduced representation library DNA sequencing: A case study using the lake whitefish. PLOS ONE 15(1): e0226608. https://doi.org/10.1371/journal.pone.0226608 Harman, AA, Fuzzen, M, Stoa, L, Boreham, D, Manzon R, Somers CM, Wilson, JY (2020). Evaluating tank acclimation and trial length for shuttle box temperature preference assays. Submitted to Journal of Experimental Biology and BioRxiv – In Press Hulley, E.N., Taylor, N.D.J., Zarnke, A.M., Somers, C.M., Mazon, R,G., Wilson, J.Y., and Boreham, D.R., 2018. DNA barcoding vs. morphological identification of larval fish and eggs in Lake Huron: advantages to a molecular approach. Journal of Great Lakes Research 44: 1110-1116. Lim, M., Manzon, R.G., Somers, C.M., Boreham, D.R., and Wilson, J.Y., 2018. Impacts of temperature, morpholine, and chronic radiation on the embryonic development of round whitefish (Prosopium cylindraceum). Environmental Toxicology and Chemistry, 37:2593-2608. Mitz, C., Thome, C., Cybulski, M.E., Somers, C.M., Manzon, R.G., Wilson, J.Y., and Boreham, D.R., 2019. Thermal dependence of sizeat-hatch in the lake whitefish (Coregonus clupeaformis). Canadian Journal of Fisheries and Aquatic Sciences 76:2069-2079. Mitz. C, Thome C, Thompson J, Cybulski ME, Somers CM, Manzon RG, Wilson JY, Boreham DR. 2020. A heterograde model for embryonic development and hatching in lake whitefish (Coregonus clupeaformis) under variable incubation temperatures. J Great Lakes Res - In Press. Morgan, T.D.; Graham, C.F.; McArthur, A.G.; Raphenya, A.R.; Boreham, D.R.; Manzon, R.G.; Wilson, J.Y., Lance, S.L.; Howland, K.L.; Patrick, P.H.; Somers, C.M., 2018. Genetic population structure of the round whitefish (Prosopium cylindraceum) in North America: multiple markers reveal glacial refugia and regional subdivision. Canadian Journal of Fisheries and Aquatic Sciences, 75:836-849. Sreetharan, S., Thome, C., Tsang, K.K.; Somers, C.M.; Manzon, R.G.; Boreham, D.R.; Wilson, J.Y., 2018. Micronuclei formation in rainbow trout cells exposed to multiple stressors: morpholine, heat shock,

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Thesis

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and ionizing radiation. Toxicology In Vitro 47:38-47. Thome C, Laframboise T, Mitz C, Clancy E, Bates J, Somers CM, Manzon RG, Wilson JY, Gunn JM, Boreham DR. 2020. Modifying effects of a cobble substrate on thermal environments and implications for embryonic development in lake whitefish (Coregonus clupeaformis). J Fish Biol. 97 (1) 113-120.

Publication

2022 Research Plan The first goal in 2022 is to complete the analysis from the 2021 Yellow Perch samples. Next the team will extend experiments examining the long-term effects of embryonic rearing temperature on metabolic rate and swim performance studies in Yellow Perch. The final task is to perform detailed analysis on amino-acid specific stale isotopes data. The swim tunnels were attempted unsuccessfully with 18-month-old Lake Whitefish but will be tried again with 2.5-year-old Lake Whitefish in 2022.

Key Researchers Dr. Joanna Wilson, McMaster University Dr. Chris Somers, University of Regina Dr. Richard Manzon, University of Regina

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Environmental DNA (eDNA) (2021-2022) Water use in power generation, both directly through dams and indirectly in oncethrough cooling systems used by nuclear power plants, results in the entrainment of early-life stage fishes, collectively referred to as ichthyoplankton. Identifying species in ichthyoplankton samples is challenging due to the lack of unique morphological characters differentiating early-life stage fish. Unlike morphological classification, DNA sequence-based identification is not hampered in early-life stage fish, making it particularly useful for ichthyoplankton monitoring. Next-generation sequencing approaches, such as DNA metabarcoding, offer a promising high-throughput alternative to conventional DNA barcoding and make it possible to sequence samples containing DNA from many species and thousands of individuals. For ichthyoplankton, DNA metabarcoding increases the number of individuals that can be processed without significantly increasing costs and effort associated with sorting, extracting, and sequencing each individual specimen separately. DNA metabarcoding also makes It possible to identify species from environmental DNA samples to detect entrained species from DNA shed into the water-cooling systems of power generation facilities. Research Activities and Results DNA Metabarcoding Primers on Genomic DNA Numerous approaches for DNA metabarcoding have been developed for fish. These range from protocols designed to maximize detection and resolution for local fish diversity or for the global diversity of bony fish and have been implemented in largescale monitoring programs. These approaches, while often highly effective, need to be evaluated for the taxa inhabiting the systems that they are being used to monitor. The effectiveness of DNA-metabarcoding in the field will be further investigated using samples collected from the entrainment program beginning in spring 2022 for both larval fishes, and for a comparison of detections using eDNA and larval fish collected simultaneously. To evaluate the sensitivity and efficiency of DNA metabarcoding for the detection and quantification of entrained fishes, two primer sets targeting mitochondrial genes COI and 12S were tested, and one set targeting the nuclear gene rhodopsin (RH1). Rhodopsin primers were developed specifically for salmonid and cottid fish and targeted regions of the gene where adaptations to deepwater habitats have been observed (Van Nynatten et al. in prep). While species belonging to these two clades do not represent the entirety of fish entrained, they do account for fish important to local fisheries (Lake Whitefish, Ciscoes) and rare species of special concern or interest (Deepwater Sculpin, Round Whitefish).

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It is shown that the COI and 12S primer sets can amplify and differentiate species frequently entrained at the Bruce Power. Rhodopsin primers were only evaluated on the Deepwater Sculpin and Ciscoes, but were found to be highly effective, and the intraspecific variation targeted by these primers improves the resolution of cisco morphotypes (Lake Herring vs. Chub). All three primer sets were also evaluated using mock communities containing DNA from multiple species in known proportions. For the two mitochondrial primers, species were consistently detected when present in a sample at more than 2% of the total DNA. It also shows that all three primers recovered the complete species complement of bulk DNA extractions of larval fishes, of known species composition, collected from Lake Huron coastal wetlands. Developing and testing of metabarcoding primers on genomic DNA is now complete. All three metabarcoding approaches were tested and shown to be highly effective for species identification when the species was present at concentrations more than 2% of the total DNA in the sample. Determining the effectiveness of DNA metabarcoding at estimating species abundance Next-generation sequencing results produce hundreds of thousands of sequencing reads, which are generally representative of the proportions of barcode sequences generated during the PCR steps in DNA metabarcoding protocols. As a result, the proportions of these reads have been used to establish quantitative measures of species abundance, however, the accuracy of these estimates are debated. Differences in the expected proportion of sequencing reads to the relative proportion of species-specific biomass in samples are primarily attributed to primer bias, where barcode sequences are amplified preferentially in some species over others due to increased sequence similarity in binding regions. This relationship is being explored for the barcodes developed. Generation of species-specific primers In addition to the concerns associated with abundance using DNA-metabarcoding, it can also only recover relative abundance. While total species abundance can be inferred from dividing relative abundance by the number of individuals in ichthyoplankton surveys it cannot be estimated directly from eDNA samples. Studies using allele counting approaches based on forensic studies provide one possible alternative to conventional eDNA-metabarcoding for estimating abundance. This approach has been successfully applied to fish in diet studies and invasive species eDNA, but not in entrainment monitoring. A forensic-based approach for two commercially important species in Lake Huron, Lake Whitefish and Ciscoes, is outlined below for future work.

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Determining ecological tendencies of Lake Huron Larval Fish The species observed during the entrainment monitoring program could also vary due to changes in the community structures of fishes inhabiting the waters surrounding Bruce Power. This has been observed previously in entrainment monitoring programs, and can result from overall shifts in species abundance, or yearly fluctuations because of major weather events and climate change. Preliminary results from the first year of coastal monitoring of larval fishes from the Eastern Shore of Lake Huron are described. Also described is the use of an automated eDNA sampler which will be deployed in the source water of the power plant to compare eDNA concentrations outside and within the plant. To test how effectively DNA metabarcoding can determine abundance of species in bulk samples, abundance estimates recovered using DNA metabarcoding were compared to the true abundance of samples containing DNA from multiple species. Effectiveness was further compared of 12S primers on samples where the abundance of seven species was staggered so that each species was represented by 0% to 50% of the total DNA in the sample. The slope for the comparison of the true abundance to the abundance estimated by the sequencing reads ranged from 0.72 to 1.82, respectively, where a slope of one indicates consistency in the estimated and true abundance for each species included in the sample. All slopes measured were found to be strongly correlated with the input data. These in vitro tests of metabarcoding using controlled samples show that the 12S primers can provide approximate estimates of abundance. However, these results may need to be further refined for the entrainment project. Additional primers, balancing the potential for primer bias, are being actively developed in collaboration with Genfish (see https://gen-fish.ca/ for more information). In addition, an updated set of primers targeting the rhodopsin gene for wider breadth of species are being tested. Further work may also be required to ensure the results are consistent between eDNA samples and larval fishes. To generate species-specific forensic-based primers capable of estimating absolute abundance from bulk samples of DNA without a priori knowledge of the number of individuals contributing to the sample the genetic variation was surveyed in Lake Whitefish. Using previously published reduced-representation genomic libraries, the number of primers was simulated required to fully differentiate Lake Whitefish individuals by single nucleotide polymorphisms (SNPs), and by the expected number of unique alleles in a sample given the number of DNA contributors. By aligning these libraries to the Lake Whitefish genome, suitable genomic regions (loci) were identified for the development of primers for this approach.

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Reduced representation genomic datasets containing information regarding many thousands of SNPs differentiating individuals Lake Whitefish were used to determine the minimum number of SNPs required to differentiate each individual from a specific collection location in Lake Huron. A bioinformatics pipeline was also developed to locate SNPs ideal for primer development. This pipeline prioritizes loci where allelic variants are high-resolution (more than one SNP difference) and unlinked across the genome (different chromosomes or distant on chromosomes) for maximal diversity with fewest primers. A subset of primers based on this pipeline have been ordered for testing on larval fish samples. This analysis provides the groundwork to address evaluation of forensic approaches to determine the number of individuals in bulk tissue and eDNA samples. The bioinformatics pipeline for generating primers and number of primer sequences needed to count individuals will require further refinement as results are generated for larval Lake Whitefish, as these may differ from adult populations in terms of genetic differentiation among individuals. Similar data are being generated for ciscoes from Lake Huron in collaboration with the Great Lakes Fisheries Commission and will be incorporated into the design of this study when complete. To determine the ecological tendencies of Lake Huron larval fishes for comparison to the fish entrained at Bruce Power, larval fish were collected from multiple locations along the eastern shore of Lake Huron in collaboration with the Saugeen Ojibway Nation Coastal Waters Monitoring Program. These larval fish samples have been split into posterior and anterior halves for barcoding individually and as bulk samples where halved larval fishes are combined by day of capture. Measurements and comparisons of species identifications to other ecological factors will be used to determine ecological trends associated with each species and for comparison to trends observed in entrainment monitoring. These samples will also be used to further test and compare abundance estimates using a conventional metabarcoding approach and forensicbased approach. In total, 482 larval fishes were collected from 66 locations near the fishing islands of Lake Huron. To date, 225 of these 482 fishes have been extracted and 78 of these individuals have been barcoded for species identification individually. These samples are primarily comprised of ciscoes, Lake Whitefish, and Yellow Perch. Some preliminary spatial and temporal comparisons have been made. For example, Lake Whitefish found contemporaneously with ciscoes were generally larger, consistent with their earlier emergence times and larval Yellow Perch were found later in the season and nearshore, consistent with their spawning behaviours.

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These initial results for long-term monitoring of changes in species entrained have revealed interesting ecological trends in larval fish distributions, which can be compared to the fishes entrained at the nuclear plant in the future. They also provide many samples that can be used to test methods for quantifying abundance. 2022 Research Plan The DNA extractions and sequencing of larval fish collected from the fishing islands will be completed. These fish have been separated into posterior and anterior halves; the latter being used in a conventional barcoding project to determine the species identity of each fish. The posterior halves will be combined into bulk samples to compare different methodologies for estimating abundance. These larval samples and the genomic data available for Lake Whitefish and Ciscoes will be further analyzed using simulations to determine the best set of primers for the forensic-based approach. This will involve the utilization of population genetics methods to identify optimal alleles for the allele counting method. The results of the comparison of the two methods for estimating abundance, and the pipeline used to develop the primers for the forensicbased approach, will be prepared and submitted as a primary research article. The methodology may also be expanded to other species entrained at Bruce Power where sufficient genomic data is available. The assembly and set-up of the eDNA autosampler will be conducted. The autosampler will be deployed after ice-out (April). Planning of the eDNA sampling protocol for within the plant sampling will be continuously discussed during this time as the protocols for the deployment of the larval fish sampler develops. eDNA extractions as part of another study will also be worked on to determine the most appropriate methodology and protocols for amplifying eDNA using metabarcoding for seamless application of these methods when eDNA samples are collected this year. Additional rhodopsin primers targeting more species will be developed as an alternative to mitochondrial based metabarcoding primers and possibly include in the primer sets for the forensic-based approach for ciscoes. This is being undertaken by an undergraduate student as part of an undergraduate research project and will result in a peer-reviewed paper describing a more automated approach to developing primers for metabarcoding analyses. Key Researchers Alexander Van Nynatten, University of Toronto Dr. Nicholas Mandrak NE, University of Toronto

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Fairy Lake (2021-2022) This project commenced in May 2021. The researchers have recruited an assistant, Adrienne Mason, to carry out field work and archival work at the Bruce County Museum and Cultural Centre who has experience with that facility and prior experience sampling Fairy Lake. The 2.074 km2 drainage area for Fairy Lake was mapped, and the planned water, sediment, amphibian, and vegetation sampling was completed. That data has been collected and collated for analysis this fall. The necessary permits to sample the fish community were obtained and fish surveys began in 2021. Several components for the project website were drafted and the team developed a search strategy to carry out a systematic literature search for published scientific articles on the effects of cyprinid fish on lake water quality. Overall project progress is on track with minimal delays due to COVID-19.

Figure 5: Drainage area of Fairy Lake, measuring 2.074 km2, containing 0.030 km2 of wetland area and 0.022 km2 of lake area. The mean elevation of the drainage area is 203.895 m above sea level and the mean slope of the drainage area is 1.623%. The mean annual temperature is 7.2 deg C and the total annual precipitation measures 1145 mm.

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Research Activities and Results The research team succeeded in recruiting an excellent field assistant, Adrienne Mason, and obtaining the necessary field and COVID safety training for her to proceed, in large part thanks to project partners the Invasive Phragmites Control Centre. In 2021, the research goals were to survey water quality for the following analytes: • • • • • • • • • • • •

E. coli or total coliforms pH Conductivity Temperature (monitoring continuously at 5 locations around the lake) Secchi depth Nutrients Dissolved organic carbon Major and minor ions Chlorophyll-a Hardness/alkalinity Total suspended solids Metals

The researchers sought to sample the sediment to measure metals including mercury from three to five sites across the lake. They surveyed amphibians using Ontario’s Marsh Monitoring Protocol for dusk breeding choruses and fish with a combination of minnow traps and fyke nets. Incidental observations of fish and reptiles were recorded, with particular attention to turtles. Regular vegetation surveys were conducted to document the expansion and die back of invasive curly leaf pondweed (Potamogeton crispus), which peaked on June 8th at 75% coverage of the Lake’s surface area before proceeding to die back by mid-July. All field work was completed by mid-September, with the water and sediment sampling, amphibian, and vegetation surveys completed. The researchers were unable to complete archival research at the Bruce County Museum and Cultural Center in summer 2021 because it was closed under COVID-19 restrictions. However, the search strategy to systematically collect literature from peer reviewed scientific journals was revised, touching on invasive carp and water quality, as well as invasive curly leaf pondweed and fish.

2022 Research Goals 1) Outreach: Share a draft of the project website with project partners for review. 2) Field work: Complete fish community sampling.

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3) Data: Analyse fish, vegetation and water quality data collected during the summer of 2022 and load these analyses into the website draft. 4) Research: Search the Bruce County Museum and Cultural Centre’s archive for documents on the history of Fairy Lake. 5) Research: Implement the systematic search strategy to collect literature on carp, water quality, and curly leaf pondweed. 6) Reporting: Prepare an interim report on project progress for 2022 and complete fish sampling reporting requirements (permits and ethics). Key Researchers Dr. Rebecca Rooney, University of Waterloo Dr. Heidi Swanson, University of Waterloo Adrienne Mason, University of Waterloo

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Conclusion The outcomes of these studies will have ripple effects both in the academic community and in the nuclear industry for years past the publication of this report. Environment@NII is pleased to play a role in facilitating this research as well as in sharing stories and outcomes of these projects with audiences in nuclear and far beyond. From researcher profiles to the stories behind the research, Environment@NII will continue exploring this work in accessible, engaging ways. We expect that researchers will continue to make excellent progress in 2022, and NII looks forward to seeing the results of their research. Research results will be shared with NII members, who will be able to incorporate the findings into relevant regulatory and business initiatives. Specific research goals are outlined in each project section. The NII looks forward to continuing on-going research projects and developing new avenues of research in 2022. The use of cutting-edge techniques, like using environmental DNA (eDNA) and omics will be explored to further understand and characterize the environmental impacts to aquatic life. Similarly, NII will continue to support research into better detecting and understanding the effects that everyday radiation has on our lives. Our continued thanks to Bruce Power for funding these leading-edge projects and supporting scientific rigor and independent academic research in these critical areas.

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