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Radiation-induced bystander effect (RIBE), a non-targeted effect of ionizing radiation in which non-irradiated individuals behave as if they have been irradiated after interactions with irradiated individuals, has been well documented in vertebrates. However, little research has been done investigating RIBE in terrestrial insects, this paucity of invertebrate RIBE leads to lack of knowledge on invertebrates living in fallout and exclusion zones. This paper aims to better understand the impacts of RIBE on terrestrial insects. Methods and materials: House crickets who have interacted with irradiated crickets were examined to investigate population effects of ionizing radiation exposure to better understand RIBE in insects. The results demonstrated RIBE in crickets and found that cohabitated males had higher growth rate (mg/day) when compared to non-cohabitated males. Further, cohabitated males and females matured significantly faster with no significant difference in maturation weight than non-cohabitated populations. Experiment with adult irradiated crickets found saturability of bystander signals and similar shifts in maturation parameters. These results highlight that bystander signals can impacted development and maturation in crickets. Given long-term impacts of RIBE in insects, these results may have significant implications for interactions between insects inhabiting fringe nuclear exclusion zones and those outside of it. [ABSTRACT FROM AUTHOR]

We characterize for the first time the emission of acoustic waves from cultured cells irradiated with X-ray photon radiation. Human cancer cell lines (MCF-7, HL-60) and control cell-free media were exposed to 1 Gy X-ray photons while recording the sound generated before, during and after irradiation using custom large-bandwidth ultrasound transducer. The effects of dose rate and cell viability were investigated. We report the first recorded acoustic signals captured from a collective pressure wave response to ionizing irradiation in cell culture. The acoustic signal was co-terminous with the radiation pulse, its magnitude was dependent on radiation dose rate, and live and dead cells showed qualitatively and quantitatively different acoustic signal characteristics. The signature of the collective acoustic peaks was temporally wider and with higher acoustic power for irradiated HL-60 than for irradiated MCF-7. We show that X-ray irradiation induces two cultured cancer cell types to emit a characteristic acoustic signal for the duration of the radiation pulse. The rapid decay of the signal excludes acoustic emissions themselves from contributing to the inter-organism bystander signal previously reported in intact animals, but they remain a potential component of the bystander process in tissues and cell cultures. This preliminary study suggests that further work on the potential role of radiation-induced acoustic emission (RIAE) in the inter-cellular bystander effect is merited. [ABSTRACT FROM AUTHOR]

To measure medium borne bystander effects, to study the influence of radioadaptive response (RAR) on bystander response, and to discover reliable radioresponsive biomarkers in radio-adapting frogs from Duke Swamp contaminated with an above-background radiation level and in naïve frogs from Twin Lake as the background control site. Frogs were captured at Duke Swamp and Twin Lake and brought to the lab at the Canadian Nuclear Laboratories facility. Half of the frogs from each site were irradiated with 4 Gy while the other half of the frogs were left with no further radiation treatment. Frog bladders were removed and placed in sterile culture media. Upon arrival at McMaster University, the bladders were processed for tissue cultures. After 48 h, the culture media conditioned by the bladder explants were harvested for clonogenic reporter survival assay and calcium flux measurements for assessing bystander effects. HPV-G cells were used as bystander reporter cells in all radiation-induced bystander effect (RIBE) assays. The frog bladder cultures were incubated for another 10–12 days followed by immunochemical staining for bcl-2 and c-myc expressions to analyze cellular anti-apoptotic (pro-survival) and pro-apoptotic (pro-death) responses, respectively. Only culture media conditioned by bladders from 4-Gy-irradiated naïve frogs from Twin Lake induced bystander effects (reduction of HPV-G reporter cells' clonogenic survival and presence of strong calcium flux activities). The 4 Gy irradiation dose increased pro-apoptotic c-myc expression in naïve frogs' bladder explants. Culture media conditioned by bladders from radio-adapting frogs from Duke Swamp enhanced HPV-G's clonogenic survival and a 4 Gy irradiation challenge did not change the enhanced clonogenic survival nature nor induce calcium flux. In bladder explants from both control and 4-Gy-irradiated radio-adapting frogs, anti-apoptotic bcl-2 expression for pro-survival responses was ubiquitous while c-myc expression for pro-death responses was limited to a small fraction of cells. The clonogenic RIBE reporter assay using HPV-G and calcium flux measurements are useful diagnostic tools for RIBE assessment of field biological samples, specifically those from frogs. RAR induced by environmentally relevant low-dose radiation induces protective bystander response. Bcl-2 and c-myc are reliable biomarkers for evaluating low dose radiation responses in wild populations of amphibians. Overall, this pilot study emphasizes the importance of looking at non-targeted effects (NTEs) in natural populations of non-human biota that could be vulnerable to chronic low-dose radiation exposures. [ABSTRACT FROM AUTHOR]

Radium is the most common source of alpha radiation exposure to humans and non-human species in the environment but the dosimetry is complicated by the decay chain which involves gamma exposure due to radon daughters. This paper seeks to determine the separate contributions of alpha and gamma doses to the total dose and total direct and non-targeted effect in a fish and a human cell line. This study aimed to isolate the effect of alpha particles following exposure to low doses of radium in cells, and their progeny which received no further exposure. This was initially done by comparing the survival values of a human keratinocyte cell line (HaCaT) and an embryonic Chinook salmon cell line (CHSE-214) exposed to gamma radiation, from survival of the same cell lines exposed to mixed alpha and gamma radiation through exposure to Ra-226 and its decay products. A Monte Carlo simulation was later performed to determine the contributions of radium decay products including radon daughters. The human cell line showed increased radioresistance when exposed to low doses of alpha particles. In contrast the fish cell line, which demonstrated radioresistance to low dose gamma radiation, showed increased lethality when exposed to low doses of alpha particles. Significant and complex levels of non-targeted effects were induced in progeny of irradiated cells. The simulation showed that gamma and beta decay products did not contribute significant dose and the highest beta dose was below the threshold for inducing non-targeted effects. The results confirm the need to consider the dose-response relationship when developing radiation weighting factors for low dose exposures, as well as the need to be aware of possible cell line and species differences. [ABSTRACT FROM AUTHOR]

Purpose: Serotonin (5-HT) is implicated in the underlying mechanisms which mediate cell death following ionizing radiation exposure, however, effects appear to be cell type-dependent. We sought to further characterize the role of 5-HT and 5-HT receptors (5-HTRs) in the exacerbation of cell death following ionizing radiation exposure in human colon carcinoma cells. Materials and methods: We examined the clonogenic survival of colon carcinoma HCT116 cells treated with 5-HT and the selective 5-HTR antagonists ketanserin (5-HT2A) and ondansetron (5-HT3), following exposure to direct ionizing radiation and irradiated cell-conditioned medium (ICCM). The relative expression of these target receptors was measured using western blotting. Results: Western blotting results revealed that relative protein levels of the 5-HT2A and 5-HT3 receptors were similar. 5-HT concentration-dependent increases in cell death that occurred following direct ionizing radiation exposure were abolished by both 5-HTR antagonists. Death of nonirradiated cells recipient of ICCM was increased in a concentration-dependent manner by 5-HT when present during donor cell irradiation. Both 5-HTR antagonists completely abolished the increases in bystander-induced cell death generated by 5-HT. Finally, we show that exposure of cells to 5-HT prior to receipt of ICCM can also dictate the degree of bystander-induced cell death. Conclusions: Our findings demonstrate a definitive role for 5-HT in the exacerbation of cell death following ionizing radiation exposure in colon carcinoma cells and highlight 5-HTRs as potential markers for predicting cellular radiosensitivity. [ABSTRACT FROM AUTHOR]

Purpose: To determine and compare the effects of pre-conditioning and post-conditioning towards gamma radiation responses in human cancer cells and keratinocytes. Material and methods: The clonogenic survival of glioblastoma cells (T98G), keratinocytes (HaCaT), and colorectal carcinoma cells (HCT116 p53+/+ and p53−/−) was assessed following gamma ray exposure from a Cs-137 source. The priming dose preceded the challenge dose in pre-conditioning whereas the priming dose followed the challenge dose in post-conditioning. The priming dose was either 5 mGy or 0.1 Gy. The challenge dose was 0.5-5 Gy. Results: In both pre- and post-conditioning where the priming dose was 0.1 Gy and the challenge dose was 4 Gy, RAR developed in T98G but not in HaCaT cells. In HCT116 p53+/+, pre-conditioning had either no effect or a radiosensitizing effect and whereas post-conditioning induced either radiosensitizing or radioadaptive effect. The different observed outcomes were dependent on dose, the time interval between the priming and challenge dose, and the time before the first irradiation. Post-conditioning effects could occur with a priming dose as low as 5 mGy in HCT116 p53+/+ cells. When HCT116 cells had no p53 protein expression, the radiosensitizing or radioadaptive response by the conditioning effect was abolished. Conclusions: The results suggest that radiation conditioning responses are complex and depend on at least the following factors: the magnitude of priming/challenge dose, the time interval between priming and challenge dose, p53 status, cell seeding time prior to the first radiation treatment. This work is the first parallel comparison demonstrating the potential outcomes of pre- and post-conditioning in different human cell types using environmentally and medically relevant radiation doses. [ABSTRACT FROM AUTHOR]

Purpose: To determine if the legacy of early life stage irradiation seen in rainbow trout is modified in adult fish by acute exposure to waterborne aluminium (Al). Methods: Two-year-old trout which had been irradiated as either eggs 48 h after fertilization, eyed eggs, yolk sac larvae or as first feeders, were exposed to 100 or 200 μg l−1Al. These fish were then paired with untreated fish. Gill and skin samples from both the irradiated and Al-exposed, and the untreated bystander fish, were then assayed for the production of toxic or pro-death signals, using the HPV-G reporter cell line. Results: Depending on the life stage, irradiated and the tissue assayed acute Al exposure modified both the radiation legacy, and the radiation-induced bystander effect in untreated fish, either by reducing reporter cell survival or increasing reporter cell growth. Conclusions: The effects of early irradiation and adult Al exposure were complex but clearly demonstrated that both the direct radiation effects and the communication of a radiation-induced bystander effect can be modified by a second environmental stressor. [ABSTRACT FROM AUTHOR]

Purpose: To investigate if aluminium (Al) modifies the rainbow trout response to radiation exposure and/or the induction of a radiation-induced bystander effect. Methods: Rainbow trout were exposed to 100 or 200 μg l−1Al (for 3 h), a 0.5 Gy X-ray dose or Al followed immediately by irradiation. The exposed fish were then swum with completely untreated bystander fish. A human reporter cell clonogenic assay was used to determine whether Al exposure modified the effects of irradiation on the skin and gills from directly exposed fish and also the radiation-induced bystander effect in untreated fish. Results: Al exposure did not modify the response to direct irradiation by the skin, or the gill. Al did not modify the bystander effect in the skin. However Al did modify the bystander effect in the gill. Gills of bystander fish swum with fish exposed to 200 μg l−1Al, followed by irradiation, caused a greater reduction in HPV-G cell survival than was caused by irradiation only. Interestingly Al exposure only also caused a bystander effect (reduced HPV-G survival) in the gill. Conclusion: This study shows that, in a multiple stressor scenario, the communication of radiation-induced stress signals is modified on a tissue-specific basis by acute Al exposure. Aside from the implications this has for radiological protection this response may also have potential for environmental monitoring where detection of the bystander effect could act as an indicator of radiation exposure when direct exposure responses are not evident. [ABSTRACT FROM PUBLISHER]

Purpose: To investigate changes in the gill proteome of fathead minnows continually fed an environmentally relevant dietary dose of 226Ra for 2 years. Methods: The fish were fed a commercial diet containing 10 mBq-10 Bq 226Ra g−1. After 6 months and 2 years the gill proteome was analyzed by two-dimensional electrophoresis (2-DE). Protein spots which exhibited a significant change were identified using mass spectrometry. Results: Six proteins were found to be increased: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase, protein disulphide isomerase precursor, ATP synthase, glial cell fibrillary protein (GFAP), apolipoprotein A1 (ApoA1). One protein was found to be decreased; malate dehydrogenase. The majority of these changes occurred predominantly at the lowest 226Ra doses, within 6 months and were maintained for 2 years. Conclusions: These proteomic changes suggested an adaptive or protective response to radiation induced reactive oxygen species (ROS). Increased GFAP indicated the induction of oxidative stress. Increased GAPDH and enolase indicated enhanced ROS scavenging from glycolytic metabolites. Increased protein disulphide isomerase precursor indicated an enhanced source of radioprotective thiols. Decreased malate dehydrogenase indicated enhanced ROS scavenging within the mitochondria. Increased ATP synthase indicated enhanced protection of healthy cells and increased ApoA1 indicated enhanced protection of the gill lamellae. [ABSTRACT FROM AUTHOR]

Purpose: To assess the dosimetry of Ra-226 in a two-year chronic ingestion study in laboratory maintained fathead minnow fed environmentally relevant levels of the isotope known to occur in gut contents of fish from a uranium mining area. Methods: Fish were established on reconstituted commercial fish food containing 10 mBq-10 Bq Ra-226.g− 1 dry food. The fish were sampled at 1, 6, 18 and 24 months and the Ra-226 levels in the whole fish were directly determined using measurement performed using inorganic mass spectrometry. Pilot experiments using higher doses were also done during development of a liquid scintillation detection system which support some data. Results: The data show that after 1 month the levels of accumulation in the fish were below detection. At 6 months there was an activity dependent accumulation which was relatively higher in the low activity groups. By 18 and 24 months the radium was very low in all groups - well below 6 month levels suggesting considerable loss of radium from the fish. These data were confirmed in a small and shorter study using higher dietary activities. The highest dose calculated for any measurement point was 16 μGy.h− 1 in the 6-month-old fish fed the diet containing 10 Bq.g− 1 Conclusions: We conclude that environmentally relevant levels of Ra-226 have a maximum impact at early time-points when the fish are still growing. After that they appear to depurate accumulated radium. In terms of environmental impact, the maximum accumulation peaks at the age where fish could be spawning but is extremely low leading to μGy.year− 1 doses even after exposure to the high activity diets. [ABSTRACT FROM AUTHOR]

Purpose: To extend the investigations of bystander effect induction in fish of the same species as the irradiated fish, to bystander effect induction between fish species and between trophic levels. Materials and methods: To investigate interspecies bystander effect induction, zebrafish and medaka were irradiated with a 0.5 Gy X-ray dose and then swum with non-irradiated fish of the same and opposite species. To investigate trophic level bystander effect induction, California blackworms were irradiated with the same X-ray dose and then fed to non-irradiated rainbow trout. Results: Reductions in clonogenic survival of the HPV-G (non-transformed human keratinocytes, immortalized with the human papilloma virus) reporter cell line, treated with tissue explant media, revealed that zebrafish and medaka induced a pro-apoptotic bystander effect in the other species and that, in trout, the normally anti-apoptotic effect caused by the consumption of non-irradiated blackworms was significantly reduced or lost if the blackworms had been irradiated. Conclusions: These results are the first to show that a radiation- induced bystander effect can transcend taxonomic group and trophic level in fish. This provides further evidence that bystander signals are widespread and conserved and may be transmitted through an ecosystem, as well as between individuals of the same species. [ABSTRACT FROM AUTHOR]

Purpose: To further evaluate the use of microbeam irradiation (MBI) as a potential means of non-invasive brain tumor treatment by investigating the induction of a bystander effect in non-irradiated tissue. Methods: Adult rats were irradiated with 35 or 350 Gy at the European Synchotron Research Facility (ESRF), using homogenous (broad beam) irradiation (HI) or a high energy microbeam delivered to the right brain hemisphere only. The proteome of the frontal lobes were then analyzed using two-dimensional electrophoresis (2-DE) and mass spectrometry. Results: HI resulted in proteomic responses indicative of tumourigenesis; increased albumin, aconitase and triosphosphate isomerase (TPI), and decreased dihydrolipoyldehydrogenase (DLD). The MBI bystander effect proteomic changes were indicative of reactive oxygen species mediated apoptosis; reduced TPI, prohibitin and tubulin and increased glial fibrillary acidic protein (GFAP). These potentially anti-tumourigenic apoptotic proteomic changes are also associated with neurodegeneration. However the bystander effect also increased heat shock protein (HSP) 71 turnover. HSP 71 is known to protect against all of the neurological disorders characterized by the bystander effect proteome changes. Conclusions: These results indicate that the collective interaction of these MBI-induced bystander effect proteins and their mediation by HSP 71, may confer a protective effect which now warrants additional experimental attention. [ABSTRACT FROM AUTHOR]

Purpose: The nature of the transferrable factor which goes from irradiated objects to bystander objects remains undefined. Most agree that a chemical entity is the likely 'factor' although some authors have produced in vitro evidence for the involvement of a physical component or a very potent volatile capable of traveling through air distances. In this paper we test the hypothesis that the communicated signal may be physical at least in part. Methods: The in vivo fish model was used to allow signal production and response to occur in organisms in vivo without any shared blood or central nervous system (CNS) connections. A reporter assay and calcium flux measurements were used to detect signal production when irradiated fish were separated from unirradiated fish by (a) a plastic container, and (b) a foil-wrapped plastic container. Results: The unirradiated fish showed bystander effects in both cases. The use of foil excludes the possibility of a light signal and although a highly active volatile could travel from one tank to another, the arrangement of sham and irradiated tanks makes it highly unlikely that this could explain our result. Conclusion: We conclude that there must be a physical component in the mechanism such as a weak acoustic or electromagnetic signal. [ABSTRACT FROM AUTHOR]

Purpose: A number of contradictory studies have reported a role or not for p53 (protein 53) in the production of radiation-induced bystander effects. Most of these studies looked at a range of cell lines with normal or compromised p53 function. Methods: In this study, Human Colon Tumour line 116 (HCT 116) cells with confirmed wild type p53 function and a corresponding p53 null HCT 116 line were used to test for bystander signal production and response to bystander signals in a mix/match protocol using the medium transfer technique. Results: The results showed that both the null cells and the wild type cells produced bystander signals. However, only the p53 wild type cells responded to signals from either cell line. The Human Papilloma Virus transfected keratinocyte line G (HPV-G) reporter cell line used routinely in our laboratory was used to confirm that the null cells were producing signals. Conclusions: We conclude that in this system the p53 pathway is involved in response of cells to bystander signals but that signals can be produced by cells which do not have functional p53. If these results apply in vivo, they could be important in radiotherapy where tumours may have compromised p53 function but surrounding (and distant) normal tissue may have wild type functional p53. [ABSTRACT FROM AUTHOR]

Purpose: To determine the effect of acute high dose X-rays on the direct and bystander response of chronically exposed medaka in vivo using the fish communication model. Methods: Medaka were obtained from the Low Dose Rate Irradiation Facility (LoDIF) located at the Savannah River Ecology Laboratory (SREL), University of Georgia, Aiken, South Carolina, USA where they had been exposed over 264 days to cumulative total doses of 0, 0.03, 0.66 and 5.88 Gy. They were exposed to the acute dose at McMaster University and then allowed to swim with unexposed medaka. All groups were sacrificed and fins were cultured as explants and assayed using an established technique and reporter assay. Results: Directly irradiated medaka with no chronic exposure showed a classic in vivo bystander response. Chronic pre-exposure resulted in a chronic dose-dependent increase in reporter cell survival in directly exposed fish. A 'pro-survival' response was also seen in the bystander fish. The proteins bcl-2 (b cell lymphoma 2) and c-Myc (myelocytomatosis oncogene cellular) in tissue explants were good predictors of pro-life or pro-death signals. Conclusions: Environmentally relevant chronic exposure to medaka in vivo results in adaptive responses in fish subsequently irradiated with high acute doses and in communication of protective signals to fish swimming with exposed fish. The data have implications for interpretation of radiation effects in biota. [ABSTRACT FROM AUTHOR]

Purpose: Neutron irradiations at the McMaster Tandetron Accelerator were performed to study direct and bystander effects of neutrons in a live organism. Methods: The neutrons were produced through 7Li(p,n)7Be reaction. Although the gamma contamination of the neutron beam cannot be completely eliminated, it was designed to be as low as possible and remain below a threshold already established for bystander effects. Microdosimetric methods using a tissue-equivalent proportional counter have been used to measure the neutron and gamma doses for the cell irradiation. Previous data for a cell line exposed in vitro suggested that neutrons did not produce bystander effects at doses below 300 mGy. The current experiments sought to confirm this using a live whole organism (zebrafish) where tissue samples harvested 2 h after exposure were examined for direct evidence of apoptosis and tested for secretion of bystander factors using an established bioassay. Fish were either exposed directly to the beam or were allowed to swim with or in water previously occupied by irradiated fish. Results: Using the zebrafish model it was found that there was significant direct cell death seen both by apoptosis scores and clonogenic assay when the neutron dose was approximately 100 mGy. An equivalent dose of gamma rays produced a more toxic effect. It was further found that neutrons did not induce a bystander effect in fish receiving signals from irradiated fish. Conclusion: The results confirm in vitro experiments which suggest neutrons do not induce bystander signaling. In fact they may suppress gamma induced signaling suggesting a possible intriguing new and as yet unclear mechanism. [ABSTRACT FROM AUTHOR]

Purpose: Communication of signals from irradiated to non-irradiated fish has been demonstrated by our group for adults. Major questions are however, whether the effects persist for significant lengths of time (meaning there are memories or legacies of the exposure) and whether they are induced in young animals or very early stages in the life cycle. Methods: To address these questions we used a reporter cell clonogenic bioassay to detect the effects of radiation exposure and of 'bystander' signals, emitted from irradiated fish, on non-irradiated fish. The legacy of radiation exposure or receipt of bystander signals was investigated in rainbow trout irradiated as eggs at 48 h, eyed eggs at one month, yolk sac larvae (YSL) at two months and juveniles at three months after fertilisation. The irradiated and bystander fish together with shams and unhandled husbandry controls were grown on in a hatchery and examined as they reached each of the remaining life stages. They were also re-examined as one-year-olds with and without further irradiation and finally examined as sexually mature two-year-olds. Results: The data indicate a clear legacy effect of irradiation at any early life stage in the adult fish. Conclusion: The data suggest that bystander signals can be transmitted in vivo and once induced are persistent during the animals' lifespan. [ABSTRACT FROM AUTHOR]

Purpose: A previous study comparing the bystander effect and low-dose hypersensitivity found that they were inversely correlated. In the current study seven cell lines with established bystander effect and hyper-radiosensitivity/increased radioresistance (HRS/IRR) were further screened for the presence of an adaptive response. Materials and methods: Cell survival after exposure to direct radiation with or without a 0.1 Gy priming dose, was determined using the colony forming assay for seven human cell lines (HaCAT, HPV-G, SW48, T98G, U373, HGL21 and HT29). Furthermore, the impact of the bystander effect on cell survival after exposure to irradiated cell culture medium was measured concurrently. Results: An adaptive response was induced in four cell lines (U373, T98G, HGL21 and HT29) causing increased cell survival. In agreement with previous publications, a bystander effect was induced in three cell lines (HPV-G, HaCAT and SW48); while no bystander effect was seen in U373, T98G, HGL21 and HT29. An adaptive response was detected in cell lines known to produce hypersensitive response, and was inversely correlated with the bystander effect. Conclusions: These results suggest that for the cell lines tested the ability to induce an adaptive response may be mutually exclusive to the bystander effect. [ABSTRACT FROM AUTHOR]