Human beings are exposed to various ionizing radiations throughout their lives from different sources, including natural and artificial sources. Since the discovery of X-rays by Rontgen in 1895, ionizing radiations have become very important tools in diagnostic and therapeutic medical applications. Under the linear no-threshold (LNT) hypothesis, ionizing radiations cause detrimental health effects even at low doses, and act as mutagen and/or carcinogen. However, recent research findings revealed the presence of non-traditional radiobiological effects, including the radiation-induced bystander effect (RIBE), radiation-induced rescue effect (RIRE) and hormesis, which challenged the linearity between radiation dose and response assumed in the LNT hypothesis. However, the molecular mechanisms underlining these non-traditional radiobiological effects are still not fully understood. Better understanding on these non-traditional radiobiological effects would be required for more accurate assessment on the health risks from medical, environmental as well as occupational radiation exposures.

The present thesis was devoted to investigating some factors that affected the non-traditional effects induced by ionizing radiation. The thesis consisted of six chapters.

Chapter 1 gave the introduction and literature review.

Chapter 2 described the work on biphasic and triphasic dose responses in zebrafish embryos to low-dose 150 kV X-rays with different hardness. The in vivo low-dose responses of zebrafish embryos to 150 kV X-rays with different levels of hardness were examined through the number of apoptotic events revealed at 24 h post fertilization (hpf) by vital dye acridine orange (AO) staining. Our results suggested that a triphasic dose response was likely a common phenomenon in living organisms irradiated by X-rays, which comprised an ultra-low-dose inhibition, low-dose stimulation and high-dose inhibition. Our results also suggested that the hormetic zone (or the stimulation zone) was shifted towards lower doses with application of filters. The non-detection of a triphasic dose response in previous experiments could likely be attributed to the use of hard X-rays, which shifted the hormetic zone into an unmonitored ultra-low-dose region. In such cases where the subhormetic zone was missed, a biphasic dose response would be reported instead.

Chapter 3 described the work on some properties of the signals involved in unirradiated zebrafish embryos rescuing α-particle irradiated zebrafish embryos. The in vivo radiation-induced bystander effect (RIBE) and radiation-induced rescue effect (RIRE) induced between embryos of the zebrafish by α-particle irradiation were studied through the number of apoptotic signals revealed at 24 h hpf through AO staining. The medium transfer experiment where irradiated zebrafish embryos were rescued through immersion in the medium previously conditioned by a larger number of irradiated zebrafish embryos showed (a) the involvement of a released stress signal in the induction of RIRE, and (b) RIBE and RIRE signals had the same function. With the help of 500 μM of the specific nitric oxide (NO) scavenger cPTIO (2-(4carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1- oxyl-3-oxide), NO was confirmed as an essential signaling molecule for inducing both the RIBE and RIRE. On the other hand, the treatment with 20 μM of the carbon monoxide (CO) releasing chemical CORM-3 (tricarbonylchloro(glycinato)ruthenium (II)) suppressed the manifestations of RIBE but did not suppress RIRE. These suggested that unirradiated zebrafish embryos need NO but not NO-induced damages to rescue α-particle irradiated zebrafish embryos.

Chapter 4 described the work on the X-ray-induced targeted effect in irradiated zebrafish embryos, as well as a non-targeted effect in bystander naïve embryos partnered with irradiated embryos, and examined the influence of exogenous NO on these targeted and non-targeted effects. The exogenous NO was generated using an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). The targeted and non-targeted effects, as well as the toxicity of the SNAP, were assessed using the number of apoptotic events in the zebrafish embryos at 24 hpf revealed through AO staining. SNAP with concentrations of 20 and 100 µM were first confirmed to have no significant toxicity on zebrafish embryos. The targeted effect was mitigated in zebrafish embryos if they were pretreated with 100 µM SNAP prior to irradiation with an X-ray dose of 75 mGy but was not alleviated in zebrafish embryos if they were pretreated with 20 µM SNAP. On the other hand, the non-targeted effect was eliminated in the bystander naïve zebrafish embryos if they were pretreated with 20 or 100 µM SNAP prior to partnering with zebrafish embryos having been subjected to irradiation with an X-ray dose of 75 mGy. These findings revealed the importance of NO in the protection against damages induced by ionizing radiations or by radiation-induced bystander signals, and could have important impacts on development of advanced cancer treatment strategies.

Chapter 5 described the work on metabolic cooperation for RIRE through induction of autophagy and interleukin 6 (IL-6) secretion in bystander cells. We proposed that RIRE shared similar mechanisms with “metabolic cooperation” where nutrient-deprived cancer cells prompted normal cells to provide nutrients. Under the proposed unified scheme, the nutrient-deprived cancer cells and the irradiated cells (IRCs) were generalized as “stressed cells”, while the normal cells metabolically cooperating with the cancer cells and the bystander unirradiated cells (UICs) partnering with the IRCs were generalized as “bystander cells”. Our data demonstrated that X-ray irradiation induced autophagy in HeLa cells, which could last at least 18 h, and proved that the IRCs resorted to breakdown their own intracellular components to supply molecules required for cell-repair enhancement (e.g., to activate the NF-κB pathway) in the absence of support from bystander UICs. Furthermore, autophagy accumulation in IRCs was significantly reduced when they were partnered with UICs, and more so with UICs with pre-induced autophagy before partnering (through starvation using Earle’s Balanced Salt Solution), which showed that autophagy induced in UICs supported the IRCs. Our results also showed that IL-6 was secreted by bystander UICs, particularly the UICs with pre-induced autophagy, when they were cultured in the medium having previously conditioned irradiated HeLa cells. It was established that autophagy could activate the signal transducer and activator of transcription 3 (STAT3) which was required for the IL-6 production in the autophagy process. Taken together, the metabolic cooperation of RIRE was likely initiated by the bystander factors released from IRCs, which induced autophagy and activated STAT3 to produce IL-6 in bystander UICs, and was finally manifested by the activation of the NF-κB pathway in IRCs by the IL-6 secreted by the UICs. Some insights were gained from the proposed unified scheme, including (a) inactivation of autophagy pathways in UICs might potentially lower the resistance of cancer cells to radiotherapies, (b) identification of the bystander factors participating in cancers involving metabolic cooperation and inhibition of the pathways for secreting these bystander factors might be alternative therapy methods, e.g., for pancreatic cancers, and (c) potential explanation of some unexpected results from previous studies on RIRE such as exacerbated detrimental effect in IRCs, upon recognizing that both IRCs and UICs could be “stressed” cells, and that the cells destined to provide more support than the support they received would show exacerbated effects from its initial sustained stress.

Chapter 6 gave the conclusion and potential future work.