Main radiation pathways in the landscape of Armenia.

PURPOSE: To investigate sources, accumulation, and vertical migration of radionuclides in Armenia, and their impact on biota. CONCLUSIONS: This review describes the radiation status in the landscape of Armenia and features of the impact of natural and human-generated radiation on human and non-human biotas, according to studies of Armenian scientists carried out since the middle of the last century. The mountain landscape demonstrates the diversity, speciation, and radioresistance of the biota, which arise under radiation exposure in a variable environment. Although the effects of radiation have been described for a long time, some of them require further study. It is important to present the data collected in order to produce a base line for future studies of radiation effects and interactions with other stressors caused by climate change.

Towards an ecological modelling approach for assessing ionizing radiation impact on wildlife populations.

The emphasis of the international system of radiological protection of the environment is to protect populations of flora and fauna. Throughout the MODARIA programmes, the United Nations' International Atomic Energy Agency (IAEA) has facilitated knowledge sharing, data gathering and model development on the effect of radiation on wildlife. We present a summary of the achievements of MODARIA I and II on wildlife dose effect modelling, extending to a new sensitivity analysis and model development to incorporate other stressors. We reviewed evidence on historical doses and transgenerational effects on wildlife from radioactively contaminated areas. We also evaluated chemical population modelling approaches, discussing similarities and differences between chemical and radiological impact assessment in wildlife. We developed population modelling methodologies by sourcing life history and radiosensitivity data and evaluating the available models, leading to the formulation of an ecosystem-based mathematical approach. This resulted in an ecologically relevant conceptual population model, which we used to produce advice on the evaluation of risk criteria used in the radiological protection of the environment and a proposed modelling extension for chemicals. This work seeks to inform stakeholder dialogue on factors influencing wildlife population responses to radiation, including discussions on the ecological relevance of current environmental protection criteria. The area of assessment of radiation effects in wildlife is still developing with underlying data and models continuing to be improved. IAEA's ongoing support to facilitate the sharing of new knowledge, models and approaches to Member States is highlighted, and we give suggestions for future developments in this regard.

An Observed Effect of p53 Status on the Bystander Response to Radiation-Induced Cellular Photon Emission.

In this study, we investigated the potential influence of p53 on ultraviolet (UV) signal generation and response of bystander cells to the UV signals generated by beta-irradiated cells. Five cell lines of various p53 status (HaCaT, mutated; SW48, wild-type; HT29, mutated; HCT116+/+, wild-type; HCT116-/-, null) were irradiated with beta particles from tritium. Signal generation (photon emission at 340 ± 5 nm) was quantified from irradiated cells using a photomultiplier tube. Bystander response (clonogenic survival) was assessed by placing reporter cell flasks directly superior to irradiated signal-emitting cells. All cell lines emitted significant quantities of UV after tritium exposure. The magnitudes of HaCaT and HT29 photon emission at 340 nm were similar to each other while they were significantly different from the stronger signals emitted from SW48, HCT116+/+ and HCT116-/- cells. In regard to the bystander responses, HaCaT, HCT116+/+ and SW48 cells demonstrated significant reductions in survival as a result of exposure to emission signals. HCT116-/- and HT29 cells did not exhibit any changes in survival and thus were considered to be lacking the mechanisms or functions required to elicit a response. The survival response was found not to correlate with the observed signal strength for all experimental permutations; this may be attributed to varying emission spectra from cell line to cell line or differences in response sensitivity. Overall, these results suggest that the UV-mediated bystander response is influenced by the p53 status of the cell line. Wild-type p53 cells (HCT116+/+ and SW48) demonstrated significant responses to UV signals whereas the p53-null cell line (HCT116-/-) lacked any response. The two mutated p53 cell lines exhibited contrasting responses, which may be explained by unique modulation of functions by different point mutations. The reduced response (cell death) exhibited by p53-mutated cells compared to p53 wild-type cells suggests a possible role of the assessed p53 mutations in radiation-induced cancer susceptibility and reduced efficacy of radiation-directed therapy.

The influence of smoking on radiation-induced bystander signal production in esophageal cancer patients.

The relevance of radiation-induced bystander effects in humans is unclear. Much of the existing data relate to cell lines but the effect of bystander signals in complex human tissues is unclear. A phase II clinical study was untaken, where blood sera from 60 patients along with 15 cancer-free volunteers were used to detect whether measurable bystander factor(s) could be found in the blood following high dose rate (HDR) brachytherapy. Overall, there was no significant change in bystander signal production (measured in a human keratinocyte reporter system) before and after one treatment fraction of HDR brachytherapy (p>0.05). Further assessment of patient characteristics and environmental modifiable factors including smoking were also analyzed. Similar to previously published data, samples taken from smokers produced weaker signals compared to non-smokers (p<0.05). Although the number of non-smoking subjects was low, there was a clear decrease in cloning efficiency observed in keratinocyte cultures for these patients that requires further study. This study found that samples taken from smokers do not produce bystander signals, whereas samples taken from non-smokers can produce such signals following HDR brachytherapy. These findings highlight the importance of studying the interactions of multiple stressors including environmental modifiers with radiation, since some factors such as smoking may elicit protection in tumor cells which could counteract the effectiveness of radiation therapy.

Factors affecting ultraviolet-A photon emission from ß-irradiated human keratinocyte cells.

The luminescence intensity of 340±5 nm photons emitted from HaCaT (human keratinocyte) cells was investigated using a single-photon-counting system during cellular exposure to (90)Y ß-particles. Multiple factors were assessed to determine their influence upon the quantity and pattern of photon emission from ß-irradiated cells. Exposure of 1 x 10(4) cells/5 mL to 703 µCi resulted in maximum UVA photoemission at 44.8 x 10(3)±2.5 x 10(3) counts per second (cps) from live HaCaT cells (background: 1-5 cps); a 16-fold increase above cell-free controls. Significant biophoton emission was achieved only upon stimulation and was also dependent upon presence of cells. UVA luminescence was measured for (90)Y activities 14 to 703 µCi where a positive relationship between photoemission and (90)Y activity was observed. Irradiation of live HaCaT cells plated at various densities produced a distinct pattern of emission whereby luminescence increased up to a maximum at 1 x 10(4) cells/5 mL and thereafter decreased. However, this result was not observed in the dead cell population. Both live and dead HaCaT cells were irradiated and were found to demonstrate different rates of photon emission at low ß activities (⩽400 µCi). Dead cells exhibited greater photon emission rates than live cells which may be attributable to metabolic processes taking place to modulate the photoemissive effect. The results indicate that photon emission from HaCaT cells is perturbed by external stimulation, is dependent upon the activity of radiation delivered, the density of irradiated cells, and cell viability. It is postulated that biophoton emission may be modulated by a biological or metabolic process.

Dose calculations for [(131)i] meta-iodobenzylguanidine-induced bystander effects.

UNLABELLED: Targeted radiotherapy is a potentially useful treatment for some cancers and may be potentiated by bystander effects. However, without estimation of absorbed dose, it is difficult to compare the effects with conventional external radiation treatment. METHODS: Using the Vynckier - Wambersie dose point kernel, a model for dose rate evaluation was created allowing for calculation of absorbed dose values to two cell lines transfected with the noradrenaline transporter (NAT) gene and treated with [(131)I]MIBG. RESULTS: The mean doses required to decrease surviving fractions of UVW/NAT and EJ138/NAT cells, which received medium from [(131)I]MIBG-treated cells, to 25 - 30% were 1.6 and 1.7 Gy respectively. The maximum mean dose rates achieved during [(131)I]MIBG treatment were 0.09 - 0.75 Gy/h for UVW/NAT and 0.07 - 0.78 Gy/h for EJ138/NAT. These were significantly lower than the external beam gamma radiation dose rate of 15 Gy/h. In the case of control lines which were incapable of [(131)I]MIBG uptake the mean absorbed doses following radiopharmaceutical were 0.03 - 0.23 Gy for UVW and 0.03 - 0.32 Gy for EJ138. CONCLUSION: [(131)I]MIBG treatment for ICCM production elicited a bystander dose-response profile similar to that generated by external beam gamma irradiation but with significantly greater cell death.

[Genetic effects of bystander factors from the blood sera of people irradiated as the result of the Chernobyl accident].

The purpose of this work was the analysis of the effects of bystander factors from blood sera of people affected by the Chernobyl accident on human keratinocyte cell culture (HPV-G cells). A new method was developed for evaluation of the bystander factor presence in vivo in blood of the people irradiated by the Chernobyl accident. Affected population groups included liquidators of the Chernobyl accident and people living and working in areas of the Gomel region contaminated by radionuclides. The analysis has shown that bystander factors persist in Chernobyl liquidator blood samples for more than 20 years since irradiation. The data suggest that blood sera contain bystander factors, which are able to induce micronuclei and decrease the metabolic activity of HPV-G cells.

Induction of bystander response in human glioma cells using high-energy electrons: a role for TGF-beta1.

We examined bystander cell death produced in T98G cells by exposure to irradiated cell conditioned medium (ICCM) produced by high-energy 20 MeV electrons at a dose rate of 10 Gy min(-1) and doses up to 20 Gy. ICCM induced a bystander response in T98G glioma cells, reducing recipient cell survival by more than 25% below controls at 5 and 10 Gy. Higher doses increased survival to near control levels. ICCM was analyzed for the presence of transforming growth factor alpha (TGF-alpha) and transforming growth factor beta1 (TGF-beta1). Monoclonal antibodies for TGF-alpha (mAb TGF-alpha) and TGF-beta1 (mAb TGF-beta1) were added to the ICCM to neutralize any potential effect of the cytokines. The results indicate that TGF-alpha was not present in the ICCM and addition of mAb TGF-alpha to the ICCM had no effect on bystander cell survival. No active TGF-beta1 was present in the ICCM; however, addition of mAb TGF-beta1 completely abolished bystander death of reporter cells at all doses. These results indicate that bystander cell death can be induced in T98G glioma if a large enough radiation stress is applied and that TGF-beta1 plays a downstream role in this response.

Communication of radiation-induced signals in vivo between DNA repair deficient and proficient medaka (Oryzias latipes).

Radiation-induced bystander effects are established consequences of exposure to ionizing radiation. The operation of this mechanism has been seen in vitro and also between fish, mammals, and plants in vive where stress signals from treated organisms induce responses in neighbors. In vitro research shows that DNA repair deficient cells produce more toxic bystander responses. To test this in vivo two strains of Japanese medaka were tested. One is a mutant, repair deficient strain (ric2) and the other, the wildtype repair proficient strain (CAB). Irradiated fish swam with unirradiated partners in a strain mix and match protocol. The data suggest that medaka produce signals, when exposed to radiation, that induce unirradiated fish ofthe same strain swimming with them to produce an altered response to that seen in bystanders to sham irradiated fish. More apoptosis was seen in bystanders to repair deficient fish. When the strains are mixed, the bystanders of either strain respond like the donor strain. Measurements of Bcl-2 and cmyc proteins in the explants confirmed these observations. A possible role for p53 was also identified in that the use of reporters with mutant p53 demonstrated that CAB signals killed all the reporter cells by apoptosis. Use of a similar but p53 wildtype cell line had no such effect. The data add to the body of knowledge showing that bystander signals operate at hierarchical levels of organization greater than the individual and may therefore have relevance in radioecology and (eco)systems biology.

Bystander effect induced changes in apoptosis related proteins and terminal differentiation in in vitro murine bladder cultures.

PURPOSE: Radiation-induced bystander effects are now an established phenomenon seen in numerous cell and tissue culture models. The aim of this investigation was to examine the bystander signal and response in a multicellular primary tissue culture system in vitro. METHODS AND MATERIALS: Murine bladder samples were explanted and directly exposed to gamma radiation, or treated with irradiated tissue conditioned medium (ITCM) generated from the directly irradiated cultures. RESULTS: Results indicated that there was a strong bystander signal produced by the tissue that caused both dose-dependent and -independent changes in the ITCM treated tissue. Significantly increased B-cell lymphoma 2 (Bcl2) expression was noted after treatment with 0.5Gy and 5Gy ITCM (approximately 80%), while dose-dependent changes were observed in c-myelocytomatosis (cMyc) (39.48% at 0.5 Gy ITCM, 81.28% at 5 Gy ITCM) and the terminal differentiation marker uroplakin III (17.88% at 0.5 Gy). Nuclear fragmentation was also significantly increased at both doses of ITCM. CONCLUSION: These data suggest that the bystander signal produced in a multicellular environment induces complex changes in the ITCM-treated culture, and that these changes are reflective of a coordinated response to maintain integrity throughout the tissue.

Development of a zebrafish spleen cell line, ZSSJ, and its growth arrest by gamma irradiation and capacity to act as feeder cells.

A zebrafish spleen cell line, ZSSJ, was developed and its growth arrest by gamma radiation determined and its capacity to stimulate the proliferation of the zebrafish blastula cell line, ZEB2J, measured. ZSSJ was initiated by explant outgrowth, grew adherent with mainly an epithelial-like morphology, and stained strongly for alkaline phosphatase. ZSSJ was not only grown in L-15 with 15% fetal bovine serum at 26 degrees C to 28 degrees degrees C but also grew at room temperature. Cultures of ZSSJ have undergone approximately 40 population doublings, had few cells staining for b-galactosidase activity, which is commonly present in senescent cultures, and many cells with an aneuploid karyotype, which is frequently associated with immortalization. ZSSJ growth was arrested by 30 to 50 Gy of g-irradiation, whereas after 20 Gy, some slight growth was observed. By contrast, growth of the rainbow trout spleen stromal cell line, RTS34st, which has been used as a feeder for zebrafish ES cell cultures, was arrested completely by 20 Gy. In cocultures, nongrowth-arrested ZSSJ stimulated ZEB2J proliferation better than growth-arrested ZSSJ and better than RTS34st. ZSSJ should be useful as a feeder cell line for zebrafish ES cell cultures.

Effects of hTERT on genomic instability caused by either metal or radiation or combined exposure.

Genomic instability is considered to be an important component in carcinogenesis. It can be caused by low-dose exposure to agents, which appear to act through induction of stress-response pathways related to oxidative stress. These agents have been studied mostly in the radiation field but evidence is accumulating that chemicals, especially heavy metals such as Cr (VI), can also act in the same manner. Previous work showed that metal ions could initiate long-term genomic instability in human primary fibroblasts and this phenomenon was regulated by telomerase. The aim of this study was to examine the difference in clonogenic survival and cytogenetic damage after exposure to Cr (VI) and radiation both singly and in combination in normal human fibroblasts (hTERT- cells) and engineered human fibroblasts, infected with a retrovirus carrying a cDNA encoding hTERT, which rendered these cells telomerase positive and replicatively immortal (hTERT+ cells). Cr (VI) induced genomic instability in hTERT- cells but not in hTERT+ cells, whereas radiation induced genomic instability in hTERT+ cells and to a lesser extent in hTERT- cells. Combined exposure caused genomic instability in both types of cells. However, this genomic instability was more pronounced in hTERT- cells after radiation followed by Cr (VI) and more pronounced in hTERT+ cells after Cr (VI) followed by radiation. Moreover, the biological effects provoked by combined exposure of Cr (VI) and radiation also led to a synergistic action in both types of cells, compared to either Cr (VI) treatment only or radiation exposure only. This study suggests that telomerase can prevent genomic instability caused by Cr (VI), but not by radiation. Furthermore, genomic instability may be prevented by telomerase when cells are exposed to radiation and then Cr (VI) but not after exposure to Cr (VI) and then radiation.

Effects of contaminated sediment from Cork Harbour, Ireland on the cytochrome P450 system of turbot.

Hatchery-reared juvenile turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to inter-tidal sediments collected from polluted sites in Cork Harbour (Whitegate and Agahda) and a reference site at Ballymacoda Co., Cork, Ireland. The potential of the sediment exposure to induce cytochrome P450 activities and CYP1A1 in the fish was assessed. Chemical analysis revealed that the sediments originating from the reference and harbour sites were contaminated principally with PAHs-the harbour sites having double the levels of those at the reference site. Following 3 weeks exposure to the sediments western blotting demonstrated a strong immunogenic response for CYP1A1 in the liver, but not for gill or intestine. P450 activities were generally significantly higher than those exposed to reference site sediment. Liver was the most responsive tissue with significantly greater P450 activities compared with gill and intestinal tissues.

Environmentally relevant mixed exposures to radiation and heavy metals induce measurable stress responses in Atlantic salmon.

These experiments were designed to identify stress effects in 3 key organs in Atlantic Salmon (Salmo salar, L.) after exposure in vivo to very low doses of radiation, and subtoxic levels of aluminum (Al) and cadmium (Cd) alone or in combination. Six fish per group were sacrificed after exposure and the anterior kidney, fin, and gill were dissected and sentfor assay of bystander signal production as a stress response end point. Radiation doses as low as 4 mGy delivered over 5 h, alone or in combination with Cd and/or Al, caused bystander signals to be produced in tissues harvested from in vivo exposed salmon. The effects vary among different organs and are not consistently additive or synergistic for a given treatment although gill cells do show high degrees of synergism between radiation and metal exposure. Data for individual fish did not suggest any systemic sensitivity to the stressors. Interestingly, the data for Cd suggest that lower toxicity is found when the metal is used in combination with radiation exposure. Expression of two proteins associated with survival responses (Bcl-2) or death responses (cmyc) after radiation was measured in the tissue cultures and showed a highly significant correlation with response outcome. The results, although complex, indicate that these stress signal responses may aid in the mechanistic investigation of mixed contaminant effects in fish exposed to metals and radiation.

Effect of dose rate on the radiation-induced bystander response.

Radiation-induced biological bystander effects have become a well-established phenomenon associated with the interaction of radiation with cells. These so-called bystander effects have been seen across a variety of end points for both high and low linear energy transfer (LET) radiations, utilizing a variety of dose rates and radiation sources. In this study, the effect of dose rate and different low LET sources on the bystander cell survival fraction (SF) was examined. The cell line investigated was the human keratinocyte HPV-G. The bystander response was measured via clonogenic assay after medium transfer protocol. Cells were irradiated using (60)Co gamma-rays and 20 MeV electrons at doses of 0.5, 5 and 10 Gy with varying dose rates. Both gamma and electron irradiation decreased recipient SF at 0.5 Gy and 5 Gy, respectively. Subsequent recovery of the SF to control levels for 10 Gy was observed. There was no dose rate dependence for (60)Co irradiation. A significant difference in the survival fraction was observed for electron irradiation at 10 Gy and a high dose rate. Furthermore, survival fractions were compared between (60)Co and 20 MeV electron irradiations. This showed a significant increase in the survival fraction 'recovery' at 10 Gy for a (60)Co dose rate of 1.1 Gy min(-1) compared to 20 MeV electrons at 1.0 Gy min(-1). No such difference was observed when comparing at higher dose rates. Lastly, increases in survival fraction at 10 Gy were abolished and the SF decreased by the plating of increased numbers of recipient cells. Such evidence may help gain insight into the nature and mechanism(s) surrounding bystander signal production, how these phenomena are tested and their eventual application in a clinical setting.

Multiple stressor effects of radiation and metals in salmon (Salmo salar).

These experiments were designed to look at the cellular effects in key organs in Atlantic salmon (Salmo salar) after exposure in vivo to radiation and subtoxic levels of aluminum (Al) and cadmium (Cd), alone or in combination. Salmon (25g) were exposed to a single 0.5Gy dose of gamma-irradiation in water containing Cd, Al or Cd+Al. Three fish per group were sacrificed after 1h and the liver, pronephros, fin and gill of each was dissected. Small explants of each tissue were set up. After 2 days, the culture medium was harvested and filtered then placed on a reporter cell line for determination of stress signal activity (bystander effects). Radiation in combination with Cd and/or Al, caused bystander effects in tissues harvested from in vivo exposed salmon. The effects vary between different organs and are not consistently additive or synergistic for a given treatment. Tissue type appears to be critical. Liver cultures produce a toxic factor which is lethal to reporter cells, and therefore no liver data could be obtained. It is hoped that this stress signal response will prove to be a useful indicator of environmental stress in species inhabiting aquatic ecosystems.

A novel form of ataxia oculomotor apraxia characterized by oxidative stress and apoptosis resistance.

Several different autosomal recessive genetic disorders characterized by ataxia with oculomotor apraxia (AOA) have been identified with the unifying feature of defective DNA damage recognition and/or repair. We describe here the characterization of a novel form of AOA showing increased sensitivity to agents that cause single-strand breaks (SSBs) in DNA but having no gross defect in the repair of these breaks. Evidence for the presence of residual SSBs in DNA was provided by dramatically increased levels of poly (ADP-ribose)polymerase (PARP-1) auto-poly (ADP-ribosyl)ation, the detection of increased levels of reactive oxygen/nitrogen species (ROS/RNS) and oxidative damage to DNA in the patient cells. There was also evidence for oxidative damage to proteins and lipids. Although these cells were hypersensitive to DNA damaging agents, the mode of death was not by apoptosis. These cells were also resistant to TRAIL-induced death. Consistent with these observations, failure to observe a decrease in mitochondrial membrane potential, reduced cytochrome c release and defective apoptosis-inducing factor translocation to the nucleus was observed. Apoptosis resistance and PARP-1 hyperactivation were overcome by incubating the patient's cells with antioxidants. These results provide evidence for a novel form of AOA characterized by sensitivity to DNA damaging agents, oxidative stress, PARP-1 hyperactivation but resistance to apoptosis.

Bystander effects of ionizing radiation can be modulated by signaling amines.

Actual risk and risk management of exposure to ionizing radiation are among the most controversial areas in environmental health protection. Recent developments in radiobiology especially characterization of bystander effects have called into question established dogmas and are thought to cast doubt on the scientific basis of the risk assessment framework, leading to uncertainty for regulators and concern among affected populations. In this paper we test the hypothesis that small signaling molecules widely used throughout the animal kingdom for signaling stress or environmental change, such as 5-Hydroxytryptamine (5-HT, serotonin), l-DOPA, glycine or nicotine are involved in bystander signaling processes following ionizing radiation exposure. We report data which suggest that nano to micromolar concentrations of these agents can modulate bystander-induced cell death. Depletion of 5-HT present in tissue culture medium, occurred following irradiation of cells. This suggested that 5-HT might be bound by membrane receptors after irradiation. Expression of 5-HT type 3 receptors which are Ca(2+) ion channels was confirmed in the cells using immunocytochemistry and receptor expression could be increased using radiation or 5-HT exposure. Zofran and Kitryl, inhibitors of 5-HT type 3 receptors, and reserpine a generic serotonin antagonist block the bystander effect induced by radiation or by serotonin. The results may be important for the mechanistic understanding of how low doses of radiation interact with cells to produce biological effects.

A role for bioelectric effects in the induction of bystander signals by ionizing radiation?

The induction of "bystander effects" i.e. effects in cells which have not received an ionizing radiation track, is now accepted but the mechanisms are not completely clear. Bystander effects following high and low LET radiation exposure are accepted but mechanisms are still not understood. There is some evidence for a physical component to the signal. This paper tests the hypothesis that bioelectric or biomagnetic phenomena are involved. Human immortalized skin keratinocytes and primary explants of mouse bladder and fish skin, were exposed directly to ionizing radiation or treated in a variety of bystander protocols. Exposure of cells was conducted by shielding one group of flasks using lead, to reduce the dose below the threshold of 2mGy (60)Cobalt gamma rays established for the bystander effect. The endpoint for the bystander effect in the reporter system used was reduction in cloning efficiency (RCE). The magnitude of the RCE was similar in shielded and unshielded flasks. When cells were placed in a Faraday cage the magnitude of the RCE was less but not eliminated. The results suggest that liquid media or cell-cell contact transmission of bystander factors may be only part of the bystander mechanism. Bioelectric or bio magnetic fields may have a role to play. To test this further, cells were placed in a Magnetic Resonance Imaging (MRI) machine for 10 min using a typical head scan protocol. This treatment also induced a bystander response. Apart from the obvious clinical relevance, the MRI results further suggest that bystander effects may be produced by non-ionizing exposures. It is concluded that bioelectric or magnetic effects may be involved in producing bystander signaling cascades commonly seen following ionizing radiation exposure.