Objective
To determine whether the width of the shoulder and the size of the bystander effect are correlated using clonal lineages derived from a cultured cell line.
Methods
HCT 116 (p53 wildtype) ...cells were grown at cloning density and individual viable colonies were picked off and grown to establish a series of cell lines from both unirradiated and irradiated progenitors. These cell lines were then irradiated to generate full survival curves. Highly variant clones were then tested to determine the level of the bystander effect using a medium transfer protocol.
Results
The multi-target model gave the best fit in these experiments and size of the shoulder n is assessed in terms of radiosensitivity. The parent cell line has an n value of 1.1 while the most variant clones have n values of 0.88 (Clone G) and 5.5 (Clone A). Clonal lines subject to irradiation prior to isolation differed in bystander signal strength in comparison to clonal lines which were not initially irradiated (P = .055).
Conclusions
Based on these experiments we suggest there may be a link between shoulder size of a mammalian cell line and the strength of a bystander effect produced in vitro. This may have implications for radiotherapy related to out-of-field effects.
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.
Mothersill, C. and Seymour, C. Radiation-Induced Bystander Effects: Past History and Future Directions. Radiat. Res. 155, 757–765 (2001). There has been a recent upsurge of interest in the phenomenon ...now known as radiation-induced bystander effects. This is largely due to the increased awareness of the contribution of indirect and delayed effects, such as genomic instability, to cellular outcomes after low-dose exposures. It is also due to the availability of tools such as the microbeam and advanced cell culture systems and to the ability to study end points such as gene or protein expression at low doses which were previously difficult to study. This review looks at the history of bystander effects in the earlier literature, in which the clastogenic effect of plasma from irradiated patients was well known. The effect was known to persist for several years and to cause transgenerational effects, making it similar to what we now call genomic instability. The review then examines the current data and controversies which are now beginning to resolve the questions concerning the mechanisms underlying the induction and transmission of both bystander effects and genomic instability. Finally, the possible impact of data concerning radiation-induced bystander effects on radiotherapy and radiation protection is discussed.
Radiation-induced biophotons are an electromagnetic form of bystander signalling. In human cells, biophoton signalling is capable of eliciting effects in non-irradiated bystander cells. However, the ...mechanisms by which the biophotons interact and act upon the bystander cells are not clearly understood. Mitochondrial energy production and ROS are known to be involved but the precise interactions are not known. To address this question, we have investigated the effect of biophoton emission upon the function of the complexes of oxidative phosphorylation (OXPHOS). The exposure of bystander HCT116 p53 +/+ cells to biophoton signals emitted from β-irradiated HCT116 p53 +/+ cells induced significant modifications in the activity of Complex I (NADH dehydrogenase or NADH:ubiquinone oxidoreductase) such that the activity was severely diminished compared to non-irradiated controls. The enzymatic assay showed that the efficiency of NADH oxidation to NAD+ was severely compromised. It is suspected that this impairment may be linked to the photoabsorption of biophotons in the blue wavelength range (492–455 nm). The photobiomodulation to Complex I was suspected to contribute greatly to the inefficiency of ATP synthase function since it resulted in a lower quantity of H+ ions to be available for use in the process of chemiosmosis. Other reactions of the ETC were not significantly impacted. Overall, these results provide evidence for a link between biophoton emission and biomodulation of the mitochondrial ATP synthesis process. However, there are many aspects of biological modulation by radiation-induced biophotons which will require further elucidation.
•This paper provides evidence that the biophoton signals known to be emitted by irradiated cells, interfere with the processes of oxidative phosphorylation (OXPHOS) in cells receiving the signals.•The paper demonstrates that Complex 1 activity is completely supressed by these biophotons resulting in downstream compromise of ATP production.•The mechanism may help explain fatigue and immune compromise effects of low dose exposure to ionising radiation.
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
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.
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.
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.
•Progeny of fish cells surviving 2 Gy produced lethal bystander signals.•Radiation-induced delayed lethal mutations occurred in fish cells at 15–18 PDs.•No radiation-induced delayed lethal mutations occurred in amphibian cells.•Surviving fish progeny cells did not develop new radio-sensitive/resistant traits.•Surviving amphibian progeny cells did acquire a radiosensitive trait.
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.
The practice of investigating pathological abnormalities in the breasts of females who are asymptomatic is primarily employed using X-ray mammography. The importance of breast screening is reflected ...in the mortality-based benefits observed among females who are found to possess invasive breast carcinoma prior to the manifestation of clinical symptoms. It is estimated that population-based screening constitutes a 17% reduction in the breast cancer mortality rate among females affected by invasive breast carcinoma. In spite of the significant utility that screening confers in those affected by invasive cancer, limitations associated with screening manifest as potential harms affecting individuals who are free of invasive disease. Disease-free and benign tumour-bearing individuals who are subjected to diagnostic work-up following a screening examination constitute a population of cases referred to as false positives (FPs). This article discusses factors contributing to the FP rate in mammography and extends the discussion to an assessment of the consequences associated with FP reporting. We conclude that the mammography FP rate in North America is in excess based upon the observation of overtreatment of in situ lesions and the disproportionate distribution of detriment and benefit among the population of individuals recalled for diagnostic work-up subsequent to screening. To address the excessive incidence of FPs in mammography, we investigate solutions that may be employed to remediate the current status of the FP rate. Subsequently, it can be suggested that improvements in the breast-screening protocol, medical litigation risk, image interpretation software and the implementation of image acquisition modalities that overcome superimposition effects are promising solutions.
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.
Radiation-induced communication of stress signals between rainbow trout (Oncorhynchus mykiss W) have recently been described by this group and linked to the bystander effect. This paper addresses the ...question of whether another totally unrelated fish species (Danio rerio L) can demonstrate the effect and also looks at attenuation of both the bystander signal, from irradiated fish, and the bystander effect, in naive fish. The data show that zebrafish produce bystander signals, and that, as with rainbow trout these can affect naïve (i.e., non-irradiated) fish placed in water with X-rayed fish or in water previously occupied by X-rayed fish. Skin explants from directly X-rayed fish still reduce HPV-G reporter cell growth 6 h after X-ray, but the bystander signal to naïve fish is lost. Twelve h after X-ray the signal is lost in X-rayed fish. The bystander effect is also attenuated if induction was by placing naïve fish in water which previously held the X-rayed fish. However, the effect is retained if induction was by placing X-rayed and naïve fish together. This suggests the signal is not retained by water for long periods of time. Individual fish data reveal unique responses by bystander fish which could indicate varying levels of sensitivity to signal strength among individuals.
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
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.
The chronic toxicity tests were more sensitive than the acute toxicity tests. The 14-d EC50 using the clonogenic survival endpoint was 2.09 ± 0.28 μg/mL and was statistically similar to the 7-d EC50 (1.85 ± 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.
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.
•Effects of the lampricide TFM on the fish cells’ reproductive death were studied.•Chronic TFM toxicity tests were more sensitive than acute toxicity tests.•Post-exposure of fish cells to TFM has no additive effect on the radiation responses.•Pre-exposure of fish cells to TFM sensitizes the radiation responses.
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