Low Fluences Of Alpha Particles Research Articles

Influence of Elevated Temperature on Color Centers in LiF Crystals and Their Photoluminescence

The radiation-induced photoluminescence (PL) of LiF has found its way into many applications for the detection and imaging of ionizing radiation. In this work, the influence of thermal treatment at temperatures up to 400 °C on absorption and PL emission spectra as well as fluorescent nuclear tracks in irradiated LiF crystals was investigated. It was found that carrying out PL measurements with the crystals kept at the temperature of about 80 °C leads to a considerable increase in luminescence emission of F3+ color centers at 525 nm. This enhancement of PL intensity allows for the microscopic imaging of the fluorescent nuclear tracks using only F3+ emission, which is not possible at room temperature. It was also found that heating the irradiated crystals before measurement at temperatures from 100 °C to 200 °C increases the concentration of F3+ centers. However, the related enhancement of PL emission is insufficient in terms of enabling the observation of the fluorescent tracks in this part of the spectrum. In the case of the main PL emission at 670 nm related to F2 centers, the thermal treatment at around 290 °C substantially increases the intensity of fluorescent tracks. This effect, however, was found to occur only at low fluences of alpha particles (up to about 109 cm-2); therefore, it is barely visible in the emission spectrum and not noticeable in the absorption spectrum.

Differential radiosensitivity phenotypes of DNA-PKcs mutations affecting NHEJ and HRR systems following irradiation with gamma-rays or very low fluences of alpha particles.

We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.

Studying effects of Magnolol on alpha-particle induced bystander effects using PADC-film based dishes

Radiation-induced bystander effect refers to the biological response found in cells (called bystander cells) which are not irradiated directly by ionizing radiation but are next to cells irradiated directly by ionizing radiation. In the present paper, the effects of Magnolol, an extract from the bark of Magnolia officinalis which is used as a traditional Chinese medicine, were studied on alpha-particle induced bystander effects. In our experiments, Chinese hamster ovary (CHO) cells were cultured in PADC-film based dishes and were irradiated with low fluences of alpha particles passing through the PADC films. The precise number of cells traversed or missed by alpha particles could be determined by studying the alpha-particle tracks developed on the PADC films upon subsequent chemical etching. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was employed to analyze the biological response of bystander cells in terms of DNA strand breaks. With the pretreatment of Magnolol, the DNA strand breaks in bystander cells were reduced, which showed that the alpha-particle induced bystander effects were suppressed with the presence of Magnolol. Since Magnolol is an antioxidant which can scavenge reactive oxygen species (ROS), our results give support to that ROS play a role in the bystander signal transmission in our experiments.

Distance distribution of bystander effects in alpha-particle irradiated cell populations using a CR-39-based culture dish

Propagation of induced biological effects from irradiated to non-irradiated cells is known to occur in cell cultures exposed to low fluences of charged particles. These bystander effects are currently investigated using microbeam or non-microbeam (broad beams) irradiation techniques. Identification of the targeted and non-targeted bystander cells is critical to our understanding of mechanisms underlying such effects. We developed a novel cell culture dish where the base consists of a thin CR-39 sheet grafted on a thin polyethylene terephthalate (PET) foil. The validity of this device in identifying not only irradiated cells, but also the cellular compartment traversed by the particle track is described. We have optimized track etch parameters that do not interfere with measurement of induced biological endpoints under normal incident irradiation. Thus the culture dishes can be used to determine distance distributions for the propagation of induced biological effects from a hit cell to bystander cells. We describe the computer code developed to determine the distance distributions of propagated biological stress responses in normal human fibroblast cells exposed to very low fluences of alpha particles.

Production and Validation of CR-39-Based Dishes for α-Particle Radiobiological Experiments

The study of radiobiological effects induced in vitro by low fluences of alpha particles would be significantly enhanced if the precise localization of each particle track in the cell monolayer was known. From this perspective, we developed a new method based on tailor-made UV-radiation-cured CR-39, the production of which is described. Its validation both as a petri dish and as solid-state nuclear track detectors is demonstrated. With respect to the demands on solid-state nuclear track detectors in such experiments, these biologically compatible detectors have a controlled micrometric thickness that allows them to be crossed by the alpha particles. In this study, we present a method for obtaining 10-mum-thick CR-39, its chemical characterization, and its properties as a solid-state nuclear track detector under the environmental conditions of radiobiological experiments. The experimental studies performed with 3.5 MeV alpha particles show that their transmitted energy is sufficient enough to cross the entire cellular volume. Under optimal conditions, etched tracks are clearly defined 2 h after etching. Moreover, the UV-radiation-cured CR-39 represents an essentially zero background that is due to the short time between the production and use of the polymer. Under a confocal microscope, this thin solid-state nuclear track detector allows the precise localization of the impact parameter at the subcellular level.

Characteristics and mechanisms of the bystander response in monolayer cell cultures exposed to very low fluences of alpha particles

When confluent cultures of mammalian cells are irradiated with very low fluences of alpha particles whereby only occasional cells receive any radiation exposure, genetic changes are observed in the non-irradiated (“bystander”) cells. Upregulation of the p53 damage–response pathway as well as activation of proteins in the MAPK family occurred in bystander cells; p53 was phosphorylated on the serine 15 residue suggesting that the upregulation of p53 was a consequence of DNA damage. Damage signals were transmitted to bystander cells through gap junctions, as confirmed by the use of genetically manipulated cells including connexin43 knockouts. Expression of connexin43 was markedly enhanced by irradiation. A moderate bystander effect was observed for specific gene mutations and chromosomal aberrations. This effect was markedly enhanced in cells defective in the non-homologous end joining DNA repair pathway. Finally, an upregulation of oxidative metabolism occurred in bystander cells; the increased levels of reactive oxygen species appeared to be derived from flavine-containing oxidase enzymes. We hypothesize that genetic effects observed in non-irradiated bystander cells are a consequence of oxidative base damage; >90% of mutations in bystander cells were point mutations. When bystander cells cannot repair DNA double strand breaks, they become much more sensitive to the induction of chromosomal aberrations and mutations, the latter consisting primarily of deletion mutants. While we propose that the genetic effects occurring in bystander cells are a consequence of oxidative stress, the nature of the signal that initiates this process remains to be determined.

Involvement of the nonhomologous end joining DNA repair pathway in the bystander effect for chromosomal aberrations.

Cells of mouse knockout cell lines for Ku80 (now known as Xrcc5), Ku70 (now known as G22p1), DNA-PKcs (now known as Prkdc) and PARP (now known as Adprt) were synchronized in G1 phase and exposed to very low fluences of alpha particles. The frequency of gross chromosomal aberrations was scored at the first postirradiation metaphase. At the two lowest doses examined, aberrations were induced in 4-9% of wild-type cells and 36-55% of Xrcc5-/- cells, whereas only 2-3% of the nuclei were traversed by an alpha particle and thus received any radiation exposure. G22p1-/- cells responded similarly to Xrcc5-/- cells, whereas Prkdc-/- and Adprt-/- cells showed an intermediate effect. The frequency of aberrations per nuclear traversal increased approximately 30-fold for Xrcc5-/- and G22p1-/- cells at the lowest mean dose examined (0.17 cGy), compared with 10-fold in Prkdc-/- cells and 3-fold in wild-type cells. Based on these and other findings, we hypothesize that the marked sensitization of repair-deficient bystander cells to the induction of chromosomal aberrations is a consequence of unrejoined DNA double-strand breaks occurring as a result of clustered damage arising from opposed oxidative lesions and single-strand breaks.

Increased bystander mutagenic effect in DNA double-strand break repair-deficient mammalian cells

Purpose : We have shown previously that when monolayer cultures of Chinese hamster ovary (CHO) cells are exposed to very low fluences of alpha-particles, HPRT mutations are induced in non-irradiated 'bystander' cells in the population. The present investigation was designed to examine the role of DNA repair in this process. Materials and methods : The DNA double-strand repair-deficient mutant cell line xrs-5 was exposed to mean doses of alpha-particles as low as 0.04 cGy whereby less than 1% of the nuclei were traversed by an alpha track and thus received any radiation exposure. Results : With this very low alpha-particle fluence, most of the cells in the xrs-5 population appeared to be at risk for the induction of mutations, indicating a much larger bystander effect than observed with wild-type CHO cells. Molecular structural analyses showed that xrs-5 mutants primarily involved partial and total gene deletions as opposed to wild-type cells where point mutations predominated in bystander cells. Conclusions : These results indicate a very large bystander effect in xrs-5 cells. They support the hypothesis that unrepaired or misrepaired double-strand breaks (DSB), arising from opposed DNA lesions, enhance the sensitivity of bystander cells in xrs-5 cultures to the induction of mutations.

Bystander effect for chromosomal aberrations induced in wild-type and repair deficient CHO cells by low fluences of alpha particles

We have previously shown that when confluent cultures of mammalian cells are exposed to very low fluences of alpha particles, fluences whereby as few as 1% of the cell nuclei are traversed by a single particle, genetic effects including specific gene mutations and sister chromatid exchanges (SCE) are induced in neighboring, non-irradiated “bystander” cells. The present investigation was designed to examine the induction of chromosomal aberrations in wild-type CHO cells and its DNA double strand break repair deficient mutant xrs-5 by a broad range of alpha particle fluences yielding mean doses of 0.17–200 cGy. The dose–response curve for the induction of aberrations was curvilinear for both cell lines, with a greater effect occurring at very low fluences owing to aberrations arising in bystander cells. These aberrations were predominately of the chromatid-type. With such fluences, the number of cells with induced aberrations per nucleus irradiated increased up to 4-fold in CHO cells and 15-fold in xrs-5 cells over that expected if aberrations occurred only in irradiated cells. These results are discussed in terms of the hypothesis that the primary DNA damage in bystander CHO cells is oxidative base damage leading to a relatively small bystander effect for gross chromosomal aberrations as compared with mutations or SCE; the larger bystander effect in xrs-5 cells is the result of oxidative damage and non-repaired DNA strand breaks which may result from opposed oxidative lesions.

Transmission of damage signals from irradiated to nonirradiated cells

Confluent monolayer cultures of human and rodent cells were exposed to very low fluences of alpha particles, fluences whereby as few as 1% of the cell nuclei were traversed by an alpha track and received any radiation exposure. An elevated frequency of micronuclei, a surrogate measure of DNA damage, and of HPRT mutations were observed in the nonirradiated “bystander” cells. This was associated with an ATM-dependent upregulation of the p53 damage-response pathway. Damage signals were transmitted from irradiated to nonirradiated cells in the population by gap junction mediated intercellular communication. The biological effects in bystander cells appeared to be associated with oxidative stress, consistent with the observation that most of the mutants induced by very low fluences of alpha particles were a result of point mutations rather than deletions. Preliminary evidence suggests that activation of oxidative stress-responsive signal pathways may also occur in bystander cells. These findings, particularly the evidence for the induction of mutations in nonirradiated bystander cells after irradiation with very low fluences of alpha particles, could be of significance in estimations of the risk of residential radon exposure in human populations.

Radiation risk to low fluences of alpha particles may be greater than we thought.

Based principally on the cancer incidence found in survivors of the atomic bombs dropped in Hiroshima and Nagasaki, the International Commission on Radiation Protection (ICRP) and the United States National Council on Radiation Protection and Measurements (NCRP) have recommended that estimates of cancer risk for low dose exposure be extrapolated from higher doses by using a linear, no-threshold model. This recommendation is based on the dogma that the DNA of the nucleus is the main target for radiation-induced genotoxicity and, as fewer cells are directly damaged, the deleterious effects of radiation proportionally decline. In this paper, we used a precision microbeam to target an exact fraction (either 100% or < or =20%) of the cells in a confluent population and irradiated their nuclei with exactly one alpha particle each. We found that the frequencies of induced mutations and chromosomal changes in populations where some known fractions of nuclei were hit are consistent with non-hit cells contributing significantly to the response. In fact, irradiation of 10% of a confluent mammalian cell population with a single alpha particle per cell results in a mutant yield similar to that observed when all of the cells in the population are irradiated. This effect was significantly eliminated in cells pretreated with a 1 mM dose of octanol, which inhibits gap junction-mediated intercellular communication, or in cells carrying a dominant negative connexin 43 vector. The data imply that the relevant target for radiation mutagenesis is larger than an individual cell and suggest a need to reconsider the validity of the linear extrapolation in making risk estimates for low dose, high linear-energy-transfer (LET) radiation exposure.

HPRT mutants induced in bystander cells by very low fluences of alpha particles result primarily from point mutations.

We have shown previously that damage signals may be transmitted from irradiated cells to nonirradiated cells in monolayer cultures, leading to changes in gene expression and an enhanced frequency of mutations in these "bystander" cells. The present study was designed to test the hypothesis that mutations occurring in bystander cells result from a different mechanism than those occurring in irradiated cells, and thus show differences in molecular structure. Structural changes in the HPRT gene of Chinese hamster ovary (CHO) cells were determined by multiplex PCR analysis. A total of 790 mutant clones derived from monolayer cultures exposed to mean doses of 0, 0.5 or 10 cGy of alpha-particle radiation (0, 3% or 44%, respectively, of nuclei traversed by one or more alpha particles) were examined. Whereas mutations induced by 10 cGy included a high frequency of deletions, nearly all mutations occurring in bystander cells in cultures irradiated with 0.5 cGy involved point mutations, confirming our hypothesis that they are induced by a different mechanism.

Direct evidence for the participation of gap junction-mediated intercellular communication in the transmission of damage signals from alpha -particle irradiated to nonirradiated cells.

It has generally been considered that important biological effects of ionizing radiation arise as a direct consequence of DNA damage occurring in irradiated cells. We have examined this hypothesis by exposing cells to very low fluences of alpha-particles, similar to those emitted by radon gas, such that as few as 1% of the cells in a population are traversed by a particle and thus receive any radiation exposure. By using the endpoints of changes in gene expression and induction of DNA damage, we show that nonirradiated "bystander" cells participate in the overall response of confluent density-inhibited populations of cultured fibroblast and epithelial cells. By in situ immunofluorescence techniques and the use of cells genetically compromised in their ability to perform gap junction intercellular communication, we present direct evidence for the involvement of connexin43-mediated intercellular communication in the transmission of damage signals to nonirradiated cells. Induction of the stress-inducible p21(Waf1) protein in aggregates of neighboring cells far exceeding the fraction of cells whose nucleus has been traversed occurred in gap junction-competent cells only. These changes in p21(Waf1) expression correlated with both the induction of DNA damage (as measured by micronucleus formation) as well as increased Ser-15 phosphorylation of p53.

Direct evidence for the participation of gap junction-mediated intercellular communication in the transmission of damage signals from alpha -particle irradiated to nonirradiated cells

It has generally been considered that important biological effects of ionizing radiation arise as a direct consequence of DNA damage occurring in irradiated cells. We have examined this hypothesis by exposing cells to very low fluences of alpha-particles, similar to those emitted by radon gas, such that as few as 1% of the cells in a population are traversed by a particle and thus receive any radiation exposure. By using the endpoints of changes in gene expression and induction of DNA damage, we show that nonirradiated "bystander" cells participate in the overall response of confluent density-inhibited populations of cultured fibroblast and epithelial cells. By in situ immunofluorescence techniques and the use of cells genetically compromised in their ability to perform gap junction intercellular communication, we present direct evidence for the involvement of connexin43-mediated intercellular communication in the transmission of damage signals to nonirradiated cells. Induction of the stress-inducible p21(Waf1) protein in aggregates of neighboring cells far exceeding the fraction of cells whose nucleus has been traversed occurred in gap junction-competent cells only. These changes in p21(Waf1) expression correlated with both the induction of DNA damage (as measured by micronucleus formation) as well as increased Ser-15 phosphorylation of p53.

Intercellular Communication Is Involved in the Bystander Regulation of Gene Expression in Human Cells Exposed to Very Low Fluences of Alpha Particles

We demonstrate by western analysis that the expression levels of TP53 (formerly known as p53), CDKN1A (formerly known as p21Waf1), CDC2 (formerly known as p34cdc2), CCNB1 (cyclin B1) and RAD51 are significantly modulated in confluent, density-inhibited human diploid cell populations exposed to doses where only a small fraction of the nuclei are actually traversed by an alpha-particle track. The extent of modulation of TP53 and CDKN1A is significantly reduced in the presence of the gap junction inhibitor lindane and in irradiated low-density cell populations. In situ immunofluorescence studies show that at doses where about 2% of the nuclei would be traversed by an alpha particle, induction of CDKN1A occurs in more cells than predicted. Furthermore, the induced cells are present in isolated aggregates of neighboring cells. Therefore, our studies at the gene expression level indicate that similar signaling pathways are induced in bystander cells that are not traversed by an alpha particle as in traversed cells, and that biological effects in cell populations are not restricted to the response of individual cells to the DNA damage they receive.