The effects of low doses of ionizing radiation on living things is a continually evolving area of research. Importantly, low dose effects were historically overlooked and not properly accounted for the assessment of risk to human health, as is the case with the contentious linear no-threshold model. These low dose effects are now known to be relevant to human health in both accidental and intentional exposures, including doses relevant to medical diagnostics and therapeutics. Furthermore, there is a relative dearth of information on low dose effects in non-human species, which necessitates further investigation and evaluation of radiosensitivity. Radiation-induced bystander effects occur in organisms due to the receipt of signals from directly irradiated cells, which act to communicate radiation damage to surrounding cells. Recent research has identified one type of bystander signal which is carried by photons of biological origin, however the effects produced in bystander cells receiving these photons has not been extensively investigated. It was suspected, based on previous research, that reactive oxygen species participate in the manifestation of this bystander effect. Three mammalian cell lines were assessed for their ability to produce bystander photons upon direct irradiation; subsequently, radiologically unexposed cells were exposed to the resulting photons and assayed for biological effects. The human cell lines used exhibited significant photon emissions and oxidative stress, clonogenic cell death, reduced cellular metabolism, and compromised mitochondrial oxidative phosphorylation following exposure to these photons. The use of a melanocyte cell line indicated that these effects are attenuated by melanin, and this is suspected to occur through photoabsorption or antioxidant mechanisms. Additionally, the same assays were conducted following cell exposure to hydrogen peroxide at low concentrations to assess responses to oxidative stress relevant to bystander responses, indicating less overall sensitivity in the examined melanocytes. These findings are significant because they contribute to our understanding of the mechanisms behind low dose biological effects, because they further challenge the linear no-threshold model and other models based on target theory, because they provide evidence for differential responses to the physical bystander signal in non-human species, and because secondary photon emissions are likely relevant to the medical radiation sciences. / Thesis / Master of Science (MSc) / Low doses of ionizing radiation interact with living things differently than high doses. Low dose effects are now known to be relevant to human health and protection of the environment. Radiation-induced bystander effects occur in cells due to the receipt of signals from irradiated cells which act to communicate radiation damage to surrounding cells. One type of bystander signal is carried by photons emitted from directly irradiated cells, however the effects produced in bystander cells receiving these photons has not been extensively investigated. This thesis investigates the cellular effects of these “biophotons”, including cell survival, oxidative stress, and metabolism.

The communication of information between irradiated and non-irradiated bystander cell populations and the subsequent expression of radiation-like responses in the non-irradiated population, formally referred to as the radiation-induced bystander effect, is a very well established phenomenon in the study of radiobiology. Intercellular communication of bystander signals is known to occur via the exchange of soluble factors through biological fluids and via the transfer of molecules between adjacent cells via gap-junctions. Both of these communication methods require some degree of physical contact between biological entities. However, observations made in the literature demonstrating the induction of radiation effects in optically-coupled, yet chemically-separated organisms raises the hypothesis that alternative radiation bystander communication mechanisms may exist that have not yet been explored. Following the detection of significant photon emission from human keratinocyte cells exposed to ionizing beta-radiation by Ahmad in 2013, the involvement of an electromagnetic bystander signal was proposed. While not yet established in the field of radiobiology, intercellular communication via electromagnetic signalling is widely studied in the field of biophotonics. The emission of electromagnetic radiation from biological material, called biophoton emission, and the subsequent communication of effects using those signals has been characterized both spontaneously and as a result of perturbation by various stressors. This thesis therefore aimed to investigate intercellular communication via electromagnetic signalling stimulated by low-dose ionizing radiation to identify a possible convergence between the fields of biophoton communication and radiation-induced bystander effects. The characterization of biophoton emission from human cell cultures was accomplished using a single photon counting photomultiplier tube. The results revealed that biophoton emission is exacerbated by external stimulation (beta-radiation), it possesses a dependence upon the activity of radiation delivered, the density of the irradiated cell culture, and cell viability. These results suggest that biophoton emission is governed by physical transitions between excited and ground states and may further be modulated by metabolic processes. An effect of beta-radiation-induced biophoton emission upon non-irradiated bystander cells was identified and manifested as a reduction in cell survival. The modulatory effects observed following the application of photomodulating agents to the bystander cultures support ultraviolet electromagnetic radiation as a responsible factor in the communication of bystander signals. Observation of photon emission across the entire ultraviolet, visible and infrared spectra lead to the suggestion that ultraviolet wavelengths are only a portion of the signal responsible for eliciting bystander responses and that coherent interaction of multiple wavelengths is probable in the intercellular exchange of information. The possibility of a link between biophoton bystander signalling and soluble factor mediated bystander effects was investigated next by isolating exosomes from biophoton-exposed bystander cultures. Positive bystander responses were exhibited by secondary reporter cells incubated with the exosomes isolated from the biophoton-exposed bystander cultures, thereby suggesting that biophoton signalling is a possible form of biological redundancy where it acts as an intermediary to trigger soluble factor release and further reinforce intercellular communication. Finally, the effect of beta-radiation-induced biophoton signals upon mitochondrial activity was assessed and revealed the capacity for biophotons to downregulate Complex I and ATP synthase activity. The demonstrated effect of biophotons upon mitochondria elucidates a candidate mechanism worthy of further exploration to determine how biophotons may trigger responses in bystander cells. Overall, this thesis elucidates an additional mechanism for intercellular communication between biological systems perturbed by low doses of ionizing radiation, in the form of an electromagnetic signal. This work contributes to the current perspective on biophoton bystander signalling as a potential source of biological redundancy, facilitating a means of intercellular communication when optical coupling but not chemical contact is available in a given system. / Thesis / Doctor of Philosophy (PhD)

The radiation-induced bystander effect is an important phenomenon in the field of radiation biology. It has been shown that cells, after exposure to radiation, can communicate with surrounding cells and affect their physiology. Otherwise-healthy recipient cells can be influenced to undergo cellular senescence or apoptosis through this process. This has potential utilizations for radiation oncology and as well as our understanding of radiation safety. The radiation-induced bystander effect has been extensively investigated since the 1990s, but the scientific community struggles to come to a unanimous decision on how strongly these signals impact the survival of bystander cells. Results show various degrees of impact on cell survival whereas certain studies refute the existence of a radiation-induced bystander effect. This may be due to the fact that there is a great deal of study heterogeneity within the radiation-induced bystander effect community. Most experiments follow a similar general bystander protocol but often use different donor and reporter cell lines that vary in sex, organ of origin, and p53 status. The type of radiation and dose rate also typically differ between experimental designs. In this analysis, 67 in vitro, medium-transfer, radiation-induced, bystander effect studies were retrospectively graphed and analyzed to determine which intrinsic and external factors contributed significantly to the overall survival percentage change observed in reporter cells. A Two-Way ANOVA was conducted on each variable and showed that the reporter cell line, p53 status, and radiation type had a statistically significant effect on survival percentage change. These findings may explain the variation in results seen in past experiments and may help standardize future research allowing for more direct comparisons. / Thesis / Master of Science (MSc)