"Lethal Mutations" a Misnomer or the Start of a Scientific Revolution?

The aim of this paper is to review the history surrounding the discovery of lethal mutations, later described as delayed reproductive death. Lethal mutations were suggested very early on, to be due to a generalised instability in a cell population and are considered now to be one of the first demonstrations of "radiation-induced genomic instability" which led later to the establishment of the field of "non-targeted effects." The phenomenon was first described by Seymour et al. in 1986 and was confirmed by Trott's group in Europe and by Little and colleagues in the United States before being extended by Mendonca et al. in 1989, who showed conclusively that the distinguishing feature of lethal mutation occurrence was that it happened suddenly after about 9-10 population doublings in progeny which had survived the original dose of ionizing radiation. However, many authors then suggested that in fact, lethal mutations were implicit in the original experiments by Puck and Marcus in 1956 and were described in the extensive work by Sinclair in 1964, who followed clonal progeny for up to a year after irradiation and described "small colony formation" as a persistent consequence of ionizing radiation exposure. In this paper, we examine the history from 1956 to the present using the period from 1986-1989 as an anchor point to reach into the past and to go forward through the evolution of the field of low dose radiobiology where non-targeted effects predominate.

Investigation of presence and impact of radiation-induced bystander effect in Acheta domesticus.

PURPOSE: 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. RESULTS: 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. CONCLUSION: 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.

X-ray-induced bio-acoustic emissions from cultured cells.

PURPOSE: We characterize for the first time the emission of acoustic waves from cultured cells irradiated with X-ray photon radiation. METHODS AND MATERIALS: 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. RESULTS: 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. CONCLUSIONS: 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.

From tangled banks to toxic bunnies; a reflection on the issues involved in developing an ecosystem approach for environmental radiation protection.

The objective of this paper is to present the results of discussions at a workshop held as part of the International Congress of Radiation Research (Environmental Health stream) in Manchester UK, 2019. The main objective of the workshop was to provide a platform for radioecologists to engage with radiobiologists to address major questions around developing an Ecosystem approach in radioecology and radiation protection of the environment. The aim was to establish a critical framework to guide research that would permit integration of a pan-ecosystem approach into radiation protection guidelines and regulation for the environment. The conclusions were that the interaction between radioecologists and radiobiologists is useful in particular in addressing field versus laboratory issues where there are issues and challenges in designing good field experiments and a need to cross validate field data against laboratory data and vice versa. Other main conclusions were that there is a need to appreciate wider issues in ecology to design good approaches for an ecosystems approach in radioecology and that with the capture of 'Big Data', novel tools such as machine learning can now be applied to help with the complex issues involved in developing an ecosystem approach.

Isolation of the effects of alpha-related components from total effects of radium at low doses.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

A pilot study of radiation-induced bystander effect in radio-adapting frogs at a radiologically contaminated site located on the chalk river laboratories property.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Relative Biological Effectiveness and Non-Poissonian Distribution of Dicentric Chromosome Aberrations following Californium-252 Neutron Exposures of Human Peripheral Blood Lymphocytes.

Cells exposed to fast neutrons often exhibit a non-Poisson distribution of chromosome aberrations due to the high ionization density of the secondary reaction products. However, it is unknown whether lymphocytes exposed to californium-252 (252Cf) spectrum neutrons, of mean energy 2.1 MeV, demonstrate this same dispersion effect at low doses. Furthermore, there is no consensus regarding the relative biological effectiveness (RBE) of 252Cf neutrons. Dicentric and ring chromosome formations were assessed in human peripheral blood lymphocytes irradiated at doses of 12-135 mGy. The number of aberrations observed were tested for adherence to a Poisson distribution and the maximum low-dose relative biological effectiveness (RBEM) was also assessed. When 252Cf-irradiated lymphocytes were examined along with previously published cesium-137 (137Cs) data, RBEM values of 15.0 ± 2.2 and 25.7 ± 3.8 were found for the neutron-plus-photon and neutron-only dose components, respectively. Four of the five dose points were found to exhibit the expected, or close to the expected non-Poisson over-dispersion of aberrations. Thus, even at low doses of 252Cf fast neutrons, when sufficient lymphocyte nuclei are scored, chromosome aberration clustering can be observed.

Relative Biological Effectiveness and Non-Poissonian Distribution of Dicentric Chromosome Aberrations after Californium-252 Neutron Exposures of Human Peripheral Blood Lymphocytes.

Cells exposed to fast neutrons often exhibit a non-Poisson distribution of chromosome aberrations due to the high ionization density of the secondary reaction products. However, it is unknown whether lymphocytes exposed to californium-252 (252Cf) spectrum neutrons, of mean energy 2.1 MeV, demonstrate this same dispersion effect at low doses. Furthermore, there is no consensus regarding the relative biological effectiveness (RBE) of 252Cf neutrons. Dicentric and ring chromosome formation was assessed in human peripheral blood lymphocytes irradiated at doses of 12-135 mGy. The number of aberrations observed were tested for adherence to a Poisson distribution and the maximum low-dose relative biological effectiveness (RBEM) was also assessed. When 252Cf-irradiated lymphocytes were examined along with previously published cesium-137 (137Cs) data, RBEM values of 15.0 ± 2.2 and 25.7 ± 3.8 were found for the neutron-plus-photon and neutron-only dose components, respectively. Four of the five dose points were found to exhibit the expected, or close to the expected non-Poisson over-dispersion of aberrations. Thus, even at low doses of 252Cf fast neutrons, when enough lymphocyte nuclei are scored, chromosome aberration clustering can be observed.

Quantifying Biophoton Emissions From Human Cells Directly Exposed to Low-Dose Gamma Radiation.

Biophoton emission leading to bystander effects (BEs) was shown in beta-irradiated cells; however, technical challenges precluded the analysis of the biophoton role in gamma-induced BEs. The present work was to design an experimental approach to determine if, what type, and how many biophotons could be produced in gamma-irradiated cells. Photon emission was measured in HCT116 p53+/+ cells irradiated with a total dose of 22 mGy from a cesium-137 source at a dose rate of 45 mGy/min. A single-photon detection unit was used and shielded with lead to reduce counts from stray gammas reaching the detector. Higher quantities of photon emissions were observed when the cells in a tissue culture vessel were present and being irradiated compared to a cell-free vessel. Photon emissions were captured at either 340 nm (in the ultraviolet A [UVA] range) or 610 nm. At the same cell density, radiation exposure time, and radiation dose, HCT116 p53+/+ cells emitted 2.5 times more UVA biophotons than 610-nm biophotons. For the first time, gamma radiation was shown to induce biophoton emissions from biological cells. As cellular emissions of UVA biophotons following beta radiation lead to BEs, the involvement of cellular emissions of the same type of UVA biophotons in gamma radiation-induced BEs is highly likely.

5-HT2A and 5-HT3 receptors contribute to the exacerbation of targeted and non-targeted effects of ionizing radiation-induced cell death in human colon carcinoma cells.

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.

One-Decade-Spanning transgenerational effects of historic radiation dose in wild populations of bank voles exposed to radioactive contamination following the chernobyl nuclear disaster.

The concept of historic radiation doses associated with accidental radioactive releases and their role in leading to radiation-induced non-targeted effects on affected wild animals are currently being evaluated. Previous research studying Fukushima butterfly, Chernobyl bird and fruit fly populations shows that the effects are transgenerational, underlined by the principles of genomic instability, and varied from one species to another. To further expand on the responses of and their sensitivity in different taxonomically distinct groups, the present study sought to reconstruct historic radiation doses and delineate their effects on bank voles (Clethrionomys glareolus) found within a 400-km radius of the Chernobyl Nuclear Power Plant meltdown site. Historic dose reconstruction from the whole-body dose rates for the bank vole samples for their parental generation at the time of radioactive release was performed. Relationships between the historic doses and cytogenetic aberrations and embryonic lethality were examined via graphical presentations. Results suggest that genomic instability develops at the historic dose range of 20-51 mGy while a radioadaptive response develops at the historic dose range of 51-356 mGy. The Linear No-Threshold (LNT) relationship was absent at historic doses of lower than 356 mGy at all generations. However, LNT was apparent when the very high historic dose of 10.28 Gy in one sampling year was factored into the dose response curve for the bank vole generation 21-22. It is worth being reminded that natural mutation accumulation and other environmental stressors outside the realm of dose effects could contribute to the observed effects in a multiple-stressor environment. Nevertheless, the consistent development of genomic instability and radio-adaptive response across generations and sampling sites unearths the utmost fundamental radiobiological principle of transgenerational non-targeted effects. As a result, it calls for better attention and regulation from global governing bodies of environmental health protection.

Serotonin and 5-HT3 receptors sensitize human skin cells to direct irradiation cell death but not to soluble radiation-induced bystander signals.

Ionizing radiation (IR) is an environmental carcinogen and the biological damages it elicits are mechanistically distinct between high and low doses. Non-targeted effects occurring in nonirradiated cells such as the radiation-induced bystander effect predominate at low doses of IR. However, the role of non-targeted effects in environmental radiation protection is often overlooked because the governing mechanisms are complex and multifactorial. An improved understanding of the signaling molecules and their capacity to sensitize specific cell types are essential in establishing environmental IR risks. In particular, serotonin (5-HT) has been identified to exacerbate both direct irradiation and bystander-induced cell death (CD) in certain cell types, although not all cell types are responsive to 5-HT in this respect. In this study, we further characterize the role of 5-HT and 5-HT receptors (5-HTR) in the amplification of CD following IR exposure in human keratinocytes. We examined the survival of HaCaT cells treated with 5-HT and the 5-HTR antagonists ketanserin (5-HT2A) and ondansetron (5-HT3) following exposure to direct IR and irradiated cell condition medium (ICCM). Nonirradiated cell survival was consistent with the vehicle control among 5-HT concentrations ranging from 0.001 to 100 µM. Significant 5-HT concentration-dependent increases in CD occurred following direct IR exposure. Nonirradiated ICCM-recipient CD was not altered by 5-HT (0.001-100 µM) when present during donor cell irradiation among all IR doses. Increases in direct irradiation CD evoked by 5-HT were significantly attenuated by ondansetron, blocking the effect of 5-HT, whereas ketanserin did not alter CD. Western blotting of these target 5-HTRs revealed protein expression of the 5-HT3 receptor, while the 5-HT2A receptor was not detected. We have demonstrated a definitive role for 5-HT in the exacerbation of CD following direct IR exposure and identified the 5-HT3 receptor as a potential target for ameliorating radiation damage in keratinocytes.

Effects of Dose Rate on the Reproductive Cell Death and Early Mitochondrial Membrane Potential in Different Human Epithelium-Derived Cells Exposed to Gamma Rays.

Dose rate is one of the most varied experimental parameters in radiation biology research. In this study, effects of dose rates on the radiation responses of 2 different types of human epithelium-derived cells, immortalized keratinocytes (HaCaT), and colorectal cancer cells (HCT116 p53+/+ and HCT116 p53-/-) were systematically studied. Cells were γ-irradiated at one of the 4 dose rates (24.6, 109, 564, and 1168 mGy/min) to a total dose of 0.5 to 2 Gy. Clonogenic survival and mitochondrial membrane potential (MMP) were measured to assess the levels of reproductive cell death and damage to mitochondrial physiology, respectively. It was found that clonogenic survival was similar at all 4 tested dose rates in the 3 cell lines. The loss of MMP occurred at all tested dose rates in all 3 cell lines except for one case where the MMP increased in HCT116 p53+/+cells after exposure to 0.5 Gy at 24.6 mGy/min. In HCT116 cells, the loss of MMP was the most severe at high dose/dose rate combination exposure and when p53 was expressed. In contrast, no effect in dose rate was observed with HaCaT cells as the reduction level of MMP was similar at the tested dose rates.

Effects of historic radiation dose on the frequency of sex-linked recessive lethals in Drosophila populations following the Chernobyl nuclear accident.

Contrary to the effects of high doses of radiation, the effects of low doses of radiation are still being investigated. Low doses and their non-targeted effects in particular are of special interest for researchers. The accident that occurred at the Chernobyl Nuclear Power Plant (NPP) gives researchers the opportunity to view these effects outside of a laboratory environment. For this paper, the relationship between low historic radiation doses and the persistent genetic damage observed in populations of fruit flies (Drosophila melanogaster) around the Chernobyl NPP over 3 years will be investigated. Data from Zainullin et al. (1992) on the frequency of sex-linked recessive lethals (SLRLs) in D. melanogaster around the Chernobyl NPP. To calculate the absorbed historic external dose, a method based on the Gaussian plume model was used to find the external dose from both plume shine and ground shine. The dose attributed to the ground shine dose made a greater contribution to the overall absorbed external historic radiation dose than the plume shine dose. For earlier generations of Drosophila living in the radioactive contaminated sites, the SLRL frequencies appeared to correlate with the dose in a linear no-threshold relationship. The later descendent generations appeared to have developed a radio-adaptive-like response. This work contributes to the understanding of historic dose effects on wildlife health following the accidental release of high mount of radioactive materials into the environment.

The common field lampricide 3-trifluoromethyl-4-nitrophenol is a potential radiosensitizer in fish cells.

PURPOSE: To evaluate if the common field lampricide 3-trifluoromethyl-4-nitrophenol (TFM) that is intended to eradicate the invasive species sea lampreys in the Great Lakes has the potential to sensitize radiation responses in cells from non-targeted native fish MATERIALS AND METHODS: The TFM toxicity was assessed acutely and chronically with the clonogenic fish cell line eelB. The acute toxicity (24-h exposure) was determined by the fluorescent cell viability probe Alamar Blue. The chronic toxicity was determined either by Alamar Blue (7-d exposure) or the clonogenic survival assay (14-d exposure). Pre- and post-exposure of fish cells to environmentally relevant TFM concentrations following gamma irradiation were performed. Clonogenic survival was determined to assess the damage level of radiation-induced reproductive cell death. RESULTS: The chronic toxicity tests were more sensitive than the acute toxicity tests. The 14-d EC50 using the clonogenic survival endpoint was 2.09 ±â€¯0.28 µg/mL and was statistically similar to the 7-d EC50 (1.85 ±â€¯0.07 µg/mL) based on the Alamar Blue-based cytotoxicity endpoint. Post-exposure of cells to environmentally relevant TFM concentrations following irradiation did not have any effect as compared to the irradiation alone group. In contrast, pre-exposure of cells to TFM following irradiation had a negative additive effect when the total radiation dose was 2 Gy, but not 0.1 or 0.5 Gy. CONCLUSION: Our results suggest that the common field lampricide TFM is a potential radiation sensitizer in cells from non-targeted native fish. This could be a health problem of concern for non-targeted native fish if a large accidental radioactive release occurs.

Transgenerational effects of historic radiation dose in pale grass blue butterflies around Fukushima following the Fukushima Dai-ichi Nuclear Power Plant meltdown accident.

Low dose radiation effects have been investigated in Chernobyl for many years but there is uncertainty about initial doses received by many animal species. However, the Fukushima Dai-ichi Nuclear Power Plant accident opens an opportunity to study the effects of the initial low historic dose on directly exposed species and their progeny during a time where the contaminating radionuclides are decaying. In this paper, it is proposed that historic acute exposure and its resulting non-targeted effects (NTEs) may be partially involved in the high mortality/abnormality rates seen across generations of pale grass blue butterflies (Zizeeria maha) around Fukushima. Data from Hiyama et al. (2012) on the morphological abnormality frequencies in Z. maha collected around Fukushima and their progeny were used in this paper. Two dose reconstruction methods based on the Gaussian plume model were used to determine the external absorbed dose to the first exposed generation from both ground shine and plume shine. One method involved the use of the dose rate recorded at the time of collection and only took Cs-137 into account. The other involved using release rates and atmospheric conditions to determine the doses and considered Cs-137 and Cs-134. The reconstructed doses were plotted against the mortality rates and abnormality frequencies across generations. The mortality rates of the progeny from irradiated progenitors increased linearly with the increasing historic radiation doses reconstructed using both Cs-137 and Cs-134 sources. Additionally, a higher level of morphological abnormalities was observed in progeny than in the progenitors. The mean abnormality frequencies also increased throughout generations. As these results are a sign of NTEs being involved, it can be suggested that increasing mutation levels across generations may result, in part, from NTEs induced by the initial low dose received by the first exposed generation. However, continual accumulation of mutations over generations in their natural contaminated habitats remains a likely contributor into the observed outcome.

Parallel comparison of pre-conditioning and post-conditioning effects in human cancers and keratinocytes upon acute gamma irradiation.

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.

Radiobiological characteristics of descendant progeny of fish and amphibian cells that survive the initial ionizing radiation dose.

PURPOSE: To evaluate the development of delayed lethal mutations, the production of medium borne lethal bystander signals, and the acquirement of radiosensitive or radioresistant traits in distant descendant progeny of fish and amphibian cells surviving ionizing radiation MATERIALS AND METHODS: American eel brain endothelial cells (eelB) and African clawed frog epithelial cells (A6) were initially irradiated with gamma rays at 0.5 Gy or 2 Gy. Ionizing radiation (IR)-surviving cells were grown for 27 population doublings (PDs) for eelB and 43 PDs for A6. Reproductive cell death as quantified by clonogenic survival assays was used to determine the development of delayed lethal mutations, the production of medium borne lethal bystander signals, and the acquirement of radiosensitive or radioresistant traits in the progeny survivors. RESULTS: Only medium borne bystander signals produced by 2-Gy-irradiated eelB progeny survivors at 12 PDs could reduce the clonogenic survival of the bystander reporter cells. IR-induced delayed lethal mutations occurred in irradiated eelB cells at 15-18 PDs; however, subsequently propagated progeny cells retained normal replicative abilities. No IR-induced delayed lethal mutations developed in progeny of irradiated A6 cells at up to 43 PDs. eelB progeny survivors did not develop new radiosensitive or radioresistant traits while A6 progeny survivors acquired a new radiosensitive characteristic. CONCLUSION: This study enriches the current literature on the radiobiological characteristics of distant surviving progeny of irradiated fish and amphibian cells and highlights cell-type/species-dependent differential responses to IR. This study is the first to examine the potential transgenerational effects of progenitor irradiation in amphibian cells.

Cell Line-Specific Direct Irradiation and Bystander Responses are Influenced by Fetal Bovine Serum Serotonin Concentrations.

The radiation-induced bystander effect is mechanistically complex, involving many different signaling components. Serotonin, present in fetal bovine serum (FBS), has been implicated in the modulation of cellular responses to radiation. However, the role of this ubiquitous signaling molecule has yet to be elucidated with regard to cell line-specific radiation responses. In this study, cell survival was measured in HCT116 p53 wild-type (HCT116+/+) and HaCaT cell cultures treated with media containing serotonin-depleted FBS and compared to our standard FBS-supplemented media, using clonogenic assays. We utilized an enzyme-linked immunosorbent assay to quantify the difference (4.3 ± 1.3 ng/ml) in serotonin concentrations among the media. Serotonin-depleted media significantly reduced survival in both nonirradiated cell lines. Furthermore, we sought to determine the effects to cells in this media exposed to direct irradiation as well as bystander media from irradiated cells. Cell survival was significantly increased when HCT116+/+ cells were directly irradiated in serotonin-depleted media, while HaCaT cells showed no significant difference in survival between the media. Bystander investigations demonstrated that HCT116+/+ cells were only able to generate a bystander effect when cultured in standard media conditions containing greater serotonin levels. Conversely, HaCaT cells were unaffected by the different media in terms of producing a bystander response, generating bystander effects irrespective of the media. Previous research linking serotonin receptors to the bystander effect, together with our results, indicate that receptor heterogeneity among cell types may underlie serotonin sensitivity in direct irradiation and bystander responses through serotonin receptor-mediated cell signaling cascades.

Irradiation of rainbow trout at early life stages results in a proteomic legacy in adult gills. Part B; the effect of a second radiation dose, after one year, on the proteomic responses in the irradiated and non-irradiated bystander fish.

This study extends the investigation of the legacy effects of exposure to a single radiation dose at one of four early life stages, in adult rainbow trout (Part A), by examining the effects of a second identical dose after one year; i.e. egg 48 h after fertilisation (48 h egg) + 1 year, eyed egg + 1 year, yolk sac larvae (YSL) + 1 year and first feeder + 1 year. This included the induction of a bystander effect in non-irradiated trout which had swam with the irradiated fish. The second radiation dose negated any beneficial proteomic responses following early life stage irradiation only, particularly irradiation of 48 h eggs and eyed eggs (Part A). Instead the responses after early life stage + 1 year irradiation are consistently associated with tumorigenesis, cancer progression, or are otherwise damaging: upregulation of alpha-globin 1 (YSL + 1 year and first feeders + 1 year) and downregulation of histone H1, type II keratin, malate dehydrogenase 2-2, Na/K ATPase alpha subunit isoform 1b, nucleoside diphosphate kinase (48 h egg + 1 year), electron transfer flavoprotein subunit alpha (eyed egg + 1 year), 60 S ribosomal protein L30 (YSL + 1 year) and haemoglobin subunit beta-4 (first feeder + 1 year). Most significantly the second radiation dose also negated the overwhelmingly beneficial bystander effect proteomic responses induced by trout irradiated at an early life stage only (Part A). Instead the bystander effect proteomic changes induced by trout irradiated at an early life stage and again at 1 year have been associated with uncertain, with respect to tumorigenesis, or detrimental effects; upregulation of alpha-globin 1 (YSL + 1 year and first feeder + 1 year) and downregulation of malate dehydrogenase 2-2, nucleoside diphosphate kinase (48 h egg + 1 year), transferrin precursor (eyed egg + 1 year), 60 S ribosomal protein L30 (YSL + 1 year) and serine / threonine-protein phosphatase 2 A 65 kDa (first feeder + 1 year). This difference between the bystander effect induced proteomic changes following early life stage irradiation only and early life stage + 1 year irradiation may indicate a fundamental change in the non-targeted effects of radiation following multiple exposure to radiation.