Genotoxic damage in non-irradiated cells: contribution from the bystander effect

It has always been accepted dogma that the deleterious effects of ionising radiation such as mutagenesis and carcinogenesis are due mainly to direct damage to DNA. Using the Columbia University charged-particle microbeam and the highly sensitive AL cell mutagenic assay, it is shown here that non-irradiated cells acquire the mutagenic phenotype through direct contact with cells whose nuclei are traversed with 2 alpha particles each. Pre-treatment of cells with lindane, a gap junction inhibitor, significantly decreased the mutant yield. Furthermore, when irradiated cells were mixed with control cells in a similar ratio as the in situ studies, no enhancement in bystander mutagenesis was detected. Our studies provide clear evidence that genotoxic damage can be induced in non-irradiated cells, and that gap junction mediated cell-cell communication plays a critical role in the bystander phenomenon.     

Coupling of cell fate selection model enhances DNA damage response and may underlie BE phenomenon

Double‐strand break‐induced (DSB) cells send signal that induces DSBs in neighbour cells, resulting in the interaction among cells sharing the same medium. Since p53 network gives oscillatory response to DSBs, such interaction among cells could be modelled as an excitatory coupling of p53 network oscillators. This study proposes a plausible coupling model of three‐mode two‐dimensional oscillators, which models the p53‐mediated cell fate selection in globally coupled DSB‐induced cells. The coupled model consists of ATM and Wip1 proteins as variables. The coupling mechanism is realised through ATM variable via a mean‐field modelling the bystander signal in the intercellular medium. Investigation of the model reveals that the coupling generates more sensitive DNA damage response by affecting cell fate selection. Additionally, the authors search for the cause‐effect relationship between coupled p53 network oscillators and bystander effect (BE) endpoints. For this, they search for the possible values of uncertain parameters that may replicate BE experiments’ results. At certain parametric regions, there is a correlation between the outcomes of cell fate and endpoints of BE, suggesting that the intercellular coupling of p53 network may manifest itself as the form of observed BEs.Inspec keywords: biological effects of ionising particles, molecular biophysics, biochemistry, DNA, cellular biophysics, physiological models, biomolecular effects of radiation, cellular effects of radiation, biological effects of X‐rays, oscillations, proteinsOther keywords: three‐mode two‐dimensional oscillators, p53‐mediated cell fate selection, globally coupled DSB‐induced cells, coupled model consists, coupling mechanism, ATM variable, bystander signal, intercellular medium, sensitive DNA damage response, coupled p53 network oscillators, intercellular coupling, cell fate selection model, double‐strand break‐induced cells, DSBs, neighbour cells, oscillatory response, excitatory coupling, plausible coupling model     

A review of the bystander effect and its implications for low-dose exposure

Current models for the interaction between ionising radiation and living cells or tissues are based on direct genetic damage produced by energy deposition in cellular DNA. An important observation which has questioned this basic assumption is the radiation-induced bystander response, in which cells which have not been directly targeted respond if their neighbours have been exposed. This response predominates at low doses of relevance to radiation risk analysis (<0.2 Gy) and therefore needs to be fully characterised. The development of microbeams, which allow individual cells within populations to be targeted with precise doses of radiation, has provided a useful tool for quantifying this response. The authors' studies have targeted individual human and mouse cells with counted protons and helium ions and monitored neighbouring cells for the production of bystander responses. Bystander responses have been measured after exposures as low as a single proton or helium ion delivered to an individual cell. An important aspect is that these responses saturate with increasing dose to the single target cell, thus the relative roles of direct and indirect (non-targeted) responses change with dose. Studies with multicellular, tissue-based models are providing evidence that bystander responses may have a complex phenotype involving multiple pathways and the overall response may be a balance between multiple signalling processes and responses to radiation exposure. Current models for radiation risk assume a linear non-threshold response and have generally been extrapolated from high-dose exposures. The involvement of competing processes at low doses may have important consequences for understanding the effects of low-dose exposure.     

A microbeam study of DNA double-strand breaks in bystander primary human fibroblasts

Radiation-induced bystander effect has been well documented. However, the mechanisms are poorly understood. How we incorporate this effect into the classical models of risk assessment remains an open question. Here, the induction of bystander effect was studied by assessing DNA double-strand break (DSB) formation in situ with the rapid and sensitive gamma-H2AX focus formation assay. Utilising the Columbia University single-cell microbeam system to deliver 2 or 20 individual alpha particles to selected cell nuclei in a precisely known proportion of cells in a population, the induced DNA DSB incidences were quantified 30 min and 18 h post-IR. The increase in DNA DSB incidence in bystander cells lacked of a linear dose response indicating that neither the dose of irradiation nor proportion of irradiated cells in a population, is a critical parameter. This study confirms a binary all-or-nothing model of triggering the bystander response. The delay and persistence of the bystander response suggests a different mechanism of DSB induction in bystander cells than in directly irradiated cells.     

Track structure calculations on hypothetical subcellular targets for the release of cell-killing signals in bystander experiments with medium transfer

Track structure studies using PARTRAC have been performed with the aim to investigate the possibility of revealing information on initiating targets and mechanisms of bystander effects mediated by signals released into the culture medium. Dependences on radiation dose have been assessed for alternative signal emission scenarios, defined by required energy deposits in a number of subcellular targets, mimicking e.g. mitochondria as hypothetical targets for the release of signals. The simulation results agree with target theory, and elucidate the characteristic dose for signal emission as a function of target topology, size and activation energy. The observed dose dependence of bystander cell kill in medium transfer experiments is not as steep as predicted by the considered simple signal emission scenarios with a single or even multiple hits to the hypothetical targets. This has been resolved by accounting for variations in cellular characteristics among the irradiated cells.     

The sensitivity of the alkaline comet assay in detecting DNA lesions induced by X rays, gamma rays and alpha particles

Experiments were designed and performed in order to investigate whether or not the different cellular energy deposition patterns of photon radiation with different energies (29 kV, 220 kV X rays; Co-60, Cs-137-gamma-rays) and alpha-radiation from an Am-241 source differ in DNA damage induction capacity in human cells. For this purpose, the alkaline comet assay (single cell gel electrophoresis) was applied to measure the amount of DNA damage in relation to the dose received. The comet assay data for the parameters '% DNA in the tail' and 'tail moment' for human peripheral lymphocytes did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiations, 220 kV X rays and the gamma rays, whether for the total mean dose range of 0-3 Gy nor in the low-dose range. In contrast, when the 'tail length' data were analysed saturation of the fitted dose response curve appeared for X rays at about 1.5 Gy but was not apparent for gamma rays up to 3 Gy. Preliminary data for alpha exposures of HSC45-M2 cells showed a significant increase in DNA damage only at high doses (>2 Gy Am-241), but the damage at 2 Gy exceeded the damage induced at 2 Gy by Cs-137-gamma-rays by a factor of 2.5. In contrast, other experiments involving different cell systems and DNA damage indicators such as chromosomal aberrations have detected a significant increase in DNA damage at much lower doses, that is at 0.02 Gy for Am-241 and depicte a higher biological effectiveness. These results indicate that differences in biological effects arise through downstream processing of complex DNA damage.     

Humic acid induced genotoxicity in human peripheral blood lymphocytes using comet and sister chromatid exchange assay

Humic acid (HA) in well water used by the inhabitants for drinking is one of the possible etiological factors for blackfoot disease (BFD). Moreover, within BFD endemic areas cancers occur at significantly higher rates than in areas free of BFD. In this study, the genotoxic potential of HA is assessed using human peripheral blood lymphocytes. The cells were exposed to HA (0-200 microg/mL for 2 h), and the induction of DNA primary damage in cellular DNA was evaluated by single-cell gel electrophoresis (comet assay). HA-induced DNA damage was decreased by superoxide (O(2)(-)), hydrogen peroxide (H(2)O(2)), and reactive oxygen species (ROS) scavengers (superoxide dismutase, catalase, and Trolox), and nitric oxide (NO) synthase inhibitors (N(G)-nitro-l-arginine methyl ester and N(G)-methyl-l-arginine). Moreover, formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Endo III), known to catalyze the excision of oxidized bases, increase the amount of DNA migration in HA-treated cells. Pretreatment of the cells with both the Ca(2+)-chelator BAPTA and EGTA completely inhibited HA-induced DNA damage, indicating that HA-induced changes in Ca(2+)-homeostasis are the predominant pathways for the HA induction of genotoxicity. Furthermore, sister chromatid exchange was found in the HA-treated lymphocytes. Our findings suggest that HA can induce oxidative DNA damage and genotoxicity in human lymphocytes.     

H2AX phosphorylation in response to DNA double-strand break formation during bystander signalling: effect of microRNA knockdown

Upon DNA double-strand break (DSB) formation, hundreds of H2AX molecules in the chromatin flanking the break site are phosphorylated on serine residue 139, termed gamma-H2AX, so that virtually every DSB site in a nucleus can be visualised within 10 min of its formation using an antibody to gamma-H2AX. One application of this sensitive assay is to examine the induction of DNA double-strand damage in subtle non-targeted cellular effects such as the bystander effect. Here whether microRNA (miRNA) serve as a primary signalling mechanism for bystander effect propagation by comparing matched human colon carcinoma cell lines with wild-type or depleted levels of mature miRNAs was investigated. No major differences were found in the levels of induced gamma-H2AX foci in the tested cell lines, indicating that though miRNAs play a role in bystander effect manifestation, they appear not to be the primary bystander signalling molecules in the formation of bystander effect-induced DSBs.     

A model for the induction of chromosome aberrations through direct and bystander mechanisms

A state vector model (SVM) for chromosome aberrations and neoplastic transformation has been adapted to describe detrimental bystander effects. The model describes initiation (formation of translocations) and promotion (clonal expansion and loss of contact inhibition of initiated cells). Additional terms either in the initiation model or in the rate of clonal expansion of initiated cells, describe detrimental bystander effects for chromosome aberrations as reported in the scientific literature. In the present study, the SVM with bystander effects is tested on a suitable dataset. In addition to the simulation of non-linear effects, a classical dataset for neoplastic transformation in C3H 10T1/2 cells after alpha particle irradiation is used to show that the model without bystander features can also describe LNT-like dose responses. A published model for bystander induced neoplastic transformation was adapted for chromosome aberration induction and used to compare the results obtained with the different models.     

Non-targeted effects of radiation: bystander responses in cell and tissue models

The standard paradigm for radiation effects in cellular systems has involved direct damage to DNA and in particular, DNA double strand breaks as the triggering lesions leading to mutation, cell death and transformation. Recently, however, a growing body of evidence has reported non-targeted effects, which are not a direct consequence of the initial lesions produced in cellular DNA. These have included bystander responses, genomic instability, gene induction, adaptive responses and low dose hypersensitivity. A common observation of these responses is that they dominate at low doses and saturate with increasing dose. Non-targeted effects may therefore have consequences for extrapolation of risk estimates to low doses if these are important in vivo. A range of experimental techniques is being used to study non-targeted responses, including microbeam approaches. Microbeams have considerable advantages in that they allow individual cells and subcellular targets to be selected within populations with precise low doses and, if required, exact dose rates. Recent advances also allow targeting of 3-D cell systems. The mechanisms underlying non-targeted responses appear to involve production of reactive oxygen species and direct cell-to-cell signalling via gap junctional intercellular communication although significant differences exist in different cell types. The triggering lesions for these responses remain unclear however. Some non-targeted responses may be inter-related, for example in the case of bystander responses and instability and may be part of a general stress response system in irradiated populations. Some non-targeted effects may also act as protective mechanisms; if they lead to the removal of potentially damaged cells from the population.     

Simulation of light ion induced DNA damage patterns

The biophysical simulation code PARTRAC was extended by a module to handle ions heavier than alpha particles. Cross sections for ion-electron interactions were taken from He(++) ions of the same velocity and scaled by Z(eff(2))/4. Calculated linear energy transfer values, radial dose distributions and secondary electron spectra were found in agreement with experimental results. DNA damage due to irradiation of human fibroblast cells by several light ions from H to S was calculated for various energies complemented by 220 kV(p) X rays as reference radiation. With increasing linear energy transfer, the calculated total yield of double-strand breaks per dose showed saturation behaviour at about twice the value for reference radiation. When data analysis methods for experimental double-strand break yield determination were applied to the simulated DNA damage patterns, the two data sets were found in accord. The calculated patterns of DNA damage clusters were analysed on local and regional scale finding regional clusters in closer correlation to experimental cell inactivation data.     

Detection of low dose radiation induced DNA damage using temperature differential fluorescence assay.

A rapid and sensitive fluorescence assay for radiation-induced DNA damage is reported. Changes in temperature-induced strand separation in both calf thymus DNA and plasmid DNA (puc 19 plasmid from Escherichia coli) were measured after exposure to low doses of radiation. Exposures of between 0.004 and 1 Gy were measured with doses as low as 0.008 Gy yielding significant responses. The double-strand, sensitive dye PicoGreen was used as an indicator of DNA denaturation. Calibration plots indicate that fluorescence changes corresponding to amounts as low as 1 ng of double stranded DNA (10(6) copies for plasmid puc 19) are detected by this method.     

ATM-dependent cellular response to DNA double strand breaks plays a pivotal role in the maintenance of the integrity of the genome

ATM-dependent cellular response to DNA double strand breaks plays a pivotal role in the maintenance of the integrity of the genome. Upon irradiation, activated ataxia-telangiectasia mutated (ATM) proteins phosphorylate various downstream mediators and effectors, such as histone H2AX, MDC1, 53BP1 and NBS1. These proteins create discrete foci within the nuclei, which are detectable under fluorescence microscopes. Interestingly, the size of the foci is also increasing as increasing the time after irradiation. Particularly, the residual foci form large foci, the sizes of which reach approximately 2 μm in diameter. We confirmed that such 'foci growth' is a mechanism, by which DNA damage signal is amplified. Especially, a proper DNA damage response of cells to lower doses of ionising radiation required amplification of the ATM-dependent damage signal by recruiting the DNA damage checkpoint factors to the site of chromatin.     

Bystander-induced apoptosis and premature differentiation in primary urothelial explants after charged particle microbeam irradiation

The ureter primary explant technique was developed to study bystander effects under in vivo like conditions where stem and differentiated cells are present. Irradiation was performed with a 3He2+ charged particle microbeam available at the Gray Cancer Institute, with high (approximately 2 microns) precision. Tissue sections from porcine ureters were pre-irradiated with the microbeam at a single location with 10 3He2+ particles (5 MeV; LET 70 keV.micron-1). After irradiation, the tissue section was incubated for 7 days, thus allowing the explant outgrowth to form. Total cellular damage (total fraction of micronucleated and apoptotic cells) was measured according to morphological criteria. Apoptosis was also assessed using a 3'-OH DNA end-labelling technique. Premature differentiation was estimated using antibodies to uroplakin III, a specific marker of terminal urothelial differentiation. Results of our experiments demonstrated a significant bystander-induced differentiation and a less significant increase in apoptotic and micronucleated cells. A hypothesis based on the protective nature of the bystander effect is proposed.     

Online imaging of initial DNA damages at the PTB microbeam

In an inter-disciplinary collaboration of Physikalisch-Technische Bundesanstalt (PTB), German Collection of Microorganisms and Cell Cultures (DSMZ) and Heinrich-Heine University, live-cell imaging has been established at the charged-particle microbeam facility of PTB. Candidate genes participating in DNA strand-break repair pathways such as PARP-1, MRE11, MSH2, MDC1 and p53BP1 have been modified to generate fluorescent fusion proteins. Using multi-cistronic expression vectors, stable genomic integration was achieved in HT-1080 fibroblasts. The aim of this study is to characterise and use these highly reliable cell lines for studying initial steps of DNA damage responses and kinetics of repair after microbeam irradiation with high- and low-linear energy transfer (LET) particles in living cells at physiological conditions.     

Experimental techniques for studying bystander effects in vitro by high and low-LET ionising radiation

Ionising radiation can induce responses within non-exposed neighbouring (bystander) cells which potentially have important implications on the estimates of risk from low dose or low dose rate exposures of ionising radiations. A range of strategies have been developed for investigating bystander effects in vitro for both high-LET alpha particles or low-LET ultrasoft X rays using either partial shielding (grids, half-shields and slits) or by using a co-culture system where two physically separated populations of cells can be cultured together, allowing one population of cells to be irradiated while the second population remains unirradiated. The techniques described provide a useful tool to study bystander effects and complement microbeam studies. Studies using these systems show significant increases in the unirradiated bystander cells for various end points including the induction of chromosomal instability in haemopoetic stem cells and transformation in CGL1 cells.     

The effects of hemodialysis treatment on the level of DNA strand breaks and oxidative DNA lesions measured by the comet assay

Hemodialysis patients have a higher risk for oxidative stress‐related complications, such as cardiovascular disease and cancer. The increased level of oxidative stress is due to several factors, e.g., the hemodialysis treatment itself and the uremic state. In the present study, the effects of dialysis treatment on the level of DNA breaks and oxidative DNA lesions in mononuclear cells were measured with the comet assay. Factors possibly affecting DNA damage (reported as % DNA in tail) such as the duration of dialysis, time since last dialysis session, years of dialysis treatment, nutritional status (measured as protein catabolic rate), age, and diabetes were also investigated. The levels of DNA breaks (13.6 ± 4.7 before dialysis) and oxidative DNA lesions (7.9 ± 4.8 before dialysis) were significantly higher in dialysis patients (n = 31) compared to the levels of DNA breaks (5.8 ± 1.1) and oxidative DNA lesions (3.4 ± 1.7) in 10 healthy controls (P < 0.001). A decrease of DNA breaks was observed after dialysis (P = 0.038), and the level of oxidative DNA lesions was higher when the time between two treatment sessions were 68 hours compared to 44 hours (P < 0.001). Older subjects had a higher level of DNA breaks (P = 0.003), a good nutritional status predicted a lower level of DNA breaks (P < 0.001), and the duration of the dialysis session was inversely correlated with oxidative DNA lesions (P = 0.014). Diabetes or years of dialysis treatment did not affect DNA damage. The observations in the present study suggest that accumulation of uremic toxins induce DNA damage. The hemodialysis treatment seems to change the DNA damage.     

Gamma ray-induced bystander effect in tumour glioblastoma cells: a specific study on cell survival, cytokine release and cytokine receptors

Recent experimental evidence has challenged the paradigm according to which radiation traversal through the nucleus of a cell is a prerequisite for producing genetic changes or biological responses. Thus, unexposed cells in the vicinity of directly irradiated cells or recipient cells of medium from irradiated cultures can also be affected. The aim of the present study was to evaluate, by means of the medium transfer technique, whether interleukin-8 and its receptor (CXCR1) may play a role in the bystander effect after gamma irradiation of T98G cells in vitro. In fact the cell specificity in inducing the bystander effect and in receiving the secreted signals that has been described suggests that not only the ability to release the cytokines but also the receptor profiles are likely to modulate the cell responses and the final outcome. The dose and time dependence of the cytokine release into the medium, quantified using an enzyme linked immunosorbent assay, showed that radiation causes alteration in the release of interleukin-8 from exposed cells in a dose-independent but time-dependent manner. The relative receptor expression was also affected in exposed and bystander cells.