Efficient mutation induction by 125I and 131I decays in DNA of human cells

To examine the role of radiation energy deposition in DNA on cellular effects, we investigated the ability of 125IdUrd and 131IdUrd to kill cells and induce mutations at the hprt locus. We employed human lymphoblastoid cells proficient (TK6) or deficient (SE30) in the ability to incorporate a thymidine analog into DNA by way of the thymidine kinase (TK) scavenger pathway. Iodine-125 releases a shower of low-energy Auger electrons upon decay which deposit most of their energy within 20 nm of the decay site, whereas 131I is a high-energy beta/gamma emitter that is generally considered to emit sparsely ionizing radiation. Although 125IdUrd incorporated into cellular DNA was very effective at producing toxic and mutagenic effects in TK6 cells, virtually no effect was seen in TK-deficient cells incubated with similar levels of 125IdUrd in the extracellular medium. In response to 131IdUrd treatment, 0.45 X 10(-6) mutants were induced per centigray dose deposited within the nucleus in TK-proficient cells, whereas few mutations were induced in TK-deficient cells at doses up to 38 cGy from 131I decays occurring in the medium. The differences in biological response between TK6 and SE30 cells cannot be explained by differential radiosensitivity or IdUrd sensitization of the cell lines involved. We conclude that both 125I and 131I decays occurring while incorporated into DNA are more effective at inducing cell killing and mutations in human cells than either nonincorporated decays or low-LET radiations. These results suggest that localized energy deposition is an important factor in producing biologically important damage by both of these isotopes, and that residual lesions following the decay of DNA-incorporated radioisotopes may contribute to the toxic and mutagenic effects observed in TK-proficient cells. Furthermore, they emphasize that certain beta/gamma-emitting isotopes such as 131I may be particularly hazardous when incorporated into DNA.     

Nativity of the macromolecule of isologous DNA as a condition for its reparative effect on radiation damage

The effect of isologous DNA on the course of postirradiation reparation of meristematic cells ofVicia faba primary roots was studied in detail. A considerable interest was devoted to determinations of fundamental qualitative and quantitative conditions of the above effect of isologous DNA. Main criteria of the effect were both mitotic activity of irradiated cellular population and dynamics of chromosome aberrations induced by radiation. One set of experiments compared the course of reparation as occurred in regard to applied dose of ionizing radiation in native isologous DNA, DNA denaturated by heat and degraded by DNAase, and post-irradiation reparation of induced damages was favorably affected by native isologous DNA only. Another set of results evaluated the dependence of positive reparative effect of native isologous DNA on the length of the molecule demonstrating that in the process of reparation the presence of a complete DNA macromolecule was not essential. The last experimental group was focused on observations on the dependence of the rate of native isologous DNA effect on concentration of applied solution of the macromolecule.     

Modeling radiation damage to DNA using radionuclides incorporated into polynucleotides

Because of a great variety and different reparability of radiation-induced DNA lesions it is difficult to evaluate the radiobiological significance of certain individual alterations. It is suggested that the radionuclides incorporated into DNA can be used to imitate different types of radiation damages to DNA. Both qualitative and quantitative aspects of the problem are discussed.     

DNA conformation of Chinese hamster V79 cells and sensitivity to ionizing radiation

Chinese hamster V79 cells grown for 20 h in suspension culture form small clusters of cells (spheroids) which are more resistant to killing by ionizing radiation than V79 cells grown as monolayers. This resistance appears to be due to the greater capacity of cells grown in contact to repair radiation damage. Attempts to relate this "contact effect" to differences in DNA susceptibility or DNA repair capacity have provided conflicting results. Two techniques, alkaline sucrose gradient sedimentation and alkaline elution, show no difference in the amounts of radiation-induced DNA single-strand breakage or its repair between suspension or monolayer cells. However, using the alkali-unwinding assay, the rate of DNA unwinding is much slower for suspension cells than for monolayer cells. Interestingly, a decrease in salt concentration or in pH of the unwinding solution eliminates these differences in DNA unwinding kinetics. A fourth assay, sedimentation of nucleoids on neutral sucrose gradients, also shows a significant decrease in radiation damage produced in suspension compared to monolayer cultures. It is believed that this assay measures differences in DNA conformation (supercoiling) as well as differences in DNA strand breakage. We conclude from these four assays that the same number of DNA strand breaks/Gy is produced in monolayer and spheroid cells. However, changes in DNA conformation or packaging occur when cells are grown as spheroids, and these changes are responsible for reducing DNA damage by ionizing radiation.     

Action of secondary radiation from 70-Gev protons on the cellular DNA of mammals

A study was made of the effect of secondary radiation of 70 GeV protons on DNA of Chinese hamster cells. With a reference to fibroblast DNA, lymphoid cell DNA, and the lethal effect of radiation on the survival of Chinese hamster cells the RBE was 1.6-7.6, 1.1-3.8 and 1.14-1.7, respectively. DNA breaks were repaired to an equal level after exposure to secondary radiation from the accelerator and gamma-radiation from 60Co in equally effective doses.     

Interaction of a natural modifier of lethal effect of radiation with HeLa cells

Polypeptide 14C-aspartyl glutamate, a chemical analogue of a natural modified of a lethal effect of radiation, has been synthesized. The modifier was shown to react readily with nucleic acids and proteins of non-irradiated cells: the reaction was considerably enhanced when the modifier was administered simultaneously with radiation of cells. As is known from the literature, a molar concentration of the incorporated polypeptide is considerably lower than the intracellular molar concentration of an active radiosensitizer, BUdr, which produces an effect on mammalian cells comparable with that of polypeptide.     

Cancer as an emergent phenomenon in systems radiation biology

Radiation-induced DNA damage elicits dramatic cell signaling transitions, some of which are directed towards deciding the fate of that particular cell, while others lead to signaling to other cells. Each irradiated cell type and tissue has a characteristic pattern of radiation-induced gene expression, distinct from that of the unirradiated tissue and different from that of other irradiated tissues. It is the sum of such events, highly modulated by genotype that sometimes leads to cancer. The challenge is to determine as to which of these phenomena have persistent effect that should be incorporated into models of how radiation increases the risk of developing cancer. The application of systems biology to radiation effects may help to identify which biological responses are significant players in radiation carcinogenesis. In contrast to the radiation biology paradigm that focuses on genomic changes, systems biology seeks to integrate responses at multiple scales (e.g. molecular, cellular, organ, and organism). A key property of a system is that some phenomenon emerges as a property of the system rather than of the parts. Here, the idea that cancer in an organism can be considered as an emergent phenomenon of a perturbed system is discussed. Given the current research goal to determine the consequences of high and low radiation exposures, broadening the scope of radiation studies to include systems biology concepts should benefit risk modeling of radiation carcinogenesis. Presented at the First International Workshop on Systems Radiation Biology, February 14–16 2007, GSF-Research Centre, Neuherberg, Germany.     

RecBC enzyme overproduction affects UV and gamma radiation survival of Deinococcus radiodurans

Deinococcus radiodurans recovering from the effect of acute dose of gamma (gamma) radiation shows a biphasic mechanism of DNA double strands breaks repair that involves an efficient homologous recombination. However, it shows higher sensitivity to near-UV (NUV) than Escherichia coli and lacks RecBC, a DNA strand break (DSB) repair enzyme in some bacteria. Recombinant Deinococcus expressing the recBC genes of E. coli showed nearly three-fold improvements in near-UV tolerance and nearly 2 log cycle reductions in wild type gamma radiation resistance. RecBC over expression effect on radiation response of D. radiodurans was independent of indigenous RecD. Loss of gamma radiation tolerance was attributed to the enhanced rate of in vivo degradation of radiation damaged DNA and delayed kinetics of DSB repair during post-irradiation recovery. RecBC expressing cells of Deinococcus showed wild type response to Far-UV. These results suggest that the overproduction of RecBC competes with the indigenous mechanism of gamma radiation damaged DNA repair while it supports near-UV tolerance in D. radiodurans.     

The effect of gamma radiation on DNA methylation

The effect of 60Co gamma radiation on DNA methylation was studied in four cultured cell lines. In all cases a dose-dependent decrease in 5-methylcytosine was observed at 24, 48, and 72 h postexposure to 0.5-10 Gy. Nuclear DNA methyltransferase activity decreased while cytoplasmic activity increased in irradiated (10 Gy) V79A03 cells as compared to controls. No DNA demethylase activity was detected in the nuclei of control or irradiated V79A03 cells. Additionally, gamma radiation resulted in the differentiation of C-1300 N1E-115 cells, a mouse neuroblastoma line, in a dose- and time-dependent manner. These results are consistent with the hypothesis that (1) genes may be turned on following radiation via a mechanism involving hypomethylation of cytosine and (2) radiation-induced hypomethylation results from decreased intranuclear levels of DNA methyltransferase.     

A sense of danger from radiation

Tissue damage caused by exposure to pathogens, chemicals and physical agents such as ionizing radiation triggers production of generic "danger" signals that mobilize the innate and acquired immune system to deal with the intrusion and effect tissue repair with the goal of maintaining the integrity of the tissue and the body. Ionizing radiation appears to do the same, but less is known about the role of "danger" signals in tissue responses to this agent. This review deals with the nature of putative "danger" signals that may be generated by exposure to ionizing radiation and their significance. There are a number of potential consequences of "danger" signaling in response to radiation exposure. "Danger" signals could mediate the pathogenesis of, or recovery from, radiation damage. They could alter intrinsic cellular radiosensitivity or initiate radioadaptive responses to subsequent exposure. They may spread outside the locally damaged site and mediate bystander or "out-of-field" radiation effects. Finally, an important aspect of classical "danger" signals is that they link initial nonspecific immune responses in a pathological site to the development of specific adaptive immunity. Interestingly, in the case of radiation, there is little evidence that "danger" signals efficiently translate radiation-induced tumor cell death into the generation of tumor-specific immunity or normal tissue damage into autoimmunity. The suggestion is that radiation-induced "danger" signals may be inadequate in this respect or that radiation interferes with the generation of specific immunity. There are many issues that need to be resolved regarding "danger" signaling after exposure to ionizing radiation. Evidence of their importance is, in some areas, scant, but the issues are worthy of consideration, if for no other reason than that manipulation of these pathways has the potential to improve the therapeutic benefit of radiation therapy. This article focuses on how normal tissues and tumors sense and respond to danger from ionizing radiation, on the nature of the signals that are sent, and on the impact on the eventual consequences of exposure.     

3H]-adenine metabolism and radiation damage during in vitro development of the kidney.

We followed the early post-induction changes in nucleic acid synthesis of the metanephric kidney anlage in vitro. Enhanced incorporation of [3H]-thymidine and [3H]-adenine was detected, but several factors were shown to influence the interpretation of such in vitro experiments. The incorporation is dependent not only on the stage of development of the target organ but also on its transfer to organ culture, as early rudiments require an "adaptive" pre-cultivation to stabilize their metabolism; at more advanced stages growth and DNA synthesis proceed without delay. Another potential artifact is radiation damage which is caused by the incorporated radioisotope and can be detected in prolonged cultures. A [3H]-adenine pulse of more than 1 microCi/ml for 2 hr leads to definite growth retardation, and a 10-microCi/ml pulse causes extensive cell death and atrophy on a 4- to 6-day subculture. The radiation damage is dose-dependent and of variable severity in the different cell lineages within the organ. Since the radioisotope doses were in the range of those commonly used for monitoring cell proliferation and metabolism, we stress the risk of obtaining artifactual results, especially in prolonged cultures after pulse-labeling.     

The indirect effect of radiation reduces the repair fidelity of NHEJ as verified in repair deficient CHO cell lines exposed to different radiation qualities and potassium bromate

The complexity of DNA lesions induced by ionizing radiation is mainly dependent on radiation quality, where the indirect action of radiation may contribute to different extent depending on the type of radiation under study. The effect of indirect action of radiation can be investigated by using agents that induce oxidative DNA damage or by applying free radical scavengers. The aim of this study was to investigate the role of the indirect effect of radiation for the repair fidelity of non-homologous end-joining (NHEJ), homologous recombination repair (HRR) and base excision repair (BER) when DNA damage of different complexity was induced by gamma radiation, alpha particles or from base damages (8-oxo-dG) induced by potassium bromate (KBrO(3)). CHO cells lines deficient in XRCC3 (HRR) irs1SF, XRCC7 (NHEJ) V3-3 and XRCC1 (BER) EM9 were irradiated in the absence or presence of the free radical scavenger dimethyl sulfoxide (DMSO). The endpoints investigated included rate of cell proliferation by the DRAG assay, clonogenic cell survival and the level of primary DNA damage by the comet assay. The results revealed that the indirect effect of low-LET radiation significantly reduced the repair fidelity of both NHEJ and HRR pathways. For high-LET radiation the indirect effect of radiation also significantly reduced the repair fidelity for the repair deficient cell lines. The results suggest further that the repair fidelity of the error prone NHEJ repair pathway is more impaired by the indirect effect of high-LET radiation relative to the other repair pathways studied. The response to bromate observed for the two DSB repair deficient cell lines strongly support earlier studies that bromate induces complex DNA damages. The significantly reduced repair fidelity of irs1SF and V3-3 suggests that NHEJ as well as HRR are needed for the repair, and that complex DSBs are formed after bromate exposure.     

Radiofrequency (microwave) radiation exposure of mammalian cells during UV-induced DNA repair synthesis

The effect of continuous-wave (CW) and pulsed-wave (PW) radiofrequency radiation (RFR) in the microwave range on UV-induced DNA repair has been investigated in MRC-5 normal human diploid fibroblasts. RFR exposure at power densities of 1 (or 5) and 10 mW/cm2 gave a maximum specific absorption rate (SAR) (at 10 mW/cm2) of 0.39 +/- 0.15 W/kg for 350 MHz RFR, 4.5 +/- 3.0 W/kg for 850 MHz RFR, and 2.7 +/- 1.6 W/kg for 1.2 GHz RFR. RFR exposures for 1 to 3 h at 37 degrees C, in either continuous-wave or pulsed-wave modes, had no effect on the rate of repair replication label incorporated into preexisting UV-damaged DNA. RFR exposures (PW), with a constant medium temperature of 39 degrees C at 350 and 850 MHz during the repair period after UV damage, also had no effect. Assay for induction of repair synthesis by RFR exposure alone in non-UV irradiated cells was negative for the 350-, 850-, and 1200-MHz CW and PW RFR at 37 degrees C and the 350- and 850-MHz PW RFR at 39 degrees C. RFR does not induce DNA repair under these exposure conditions. In preliminary experiments--with the tissue culture medium maintained at 39 degrees C and RFR exposures (PW) at the frequencies of 350, 850, and 1200 MHz--no effect on incorporation of [3H]thymidine into DNA undergoing semiconservative synthesis was observed.     

Molecular radiation biology: Future aspects

Summary Future aspects of molecular radiation biology may be envisaged by looking for unsolved problems and ways to analyse them. Considering the endpoints of cellular radiation effects as cell inactivation, chromosome aberrations, mutation and transformation, the type of DNA damage in the irradiated cell and the mechanisms of DNA repair as excision repair, recombination repair and mutagenic repair are essential topics. At present, great efforts are made to identify, to clone and to sequence genes involved in the control of repair of DNA damage and to study their regulation. There are close relationships between DNA repair genes isolated from various organisms, which promises fast progress for the molecular analysis of repair processes in mammalian cells. More knowledge is necessary regarding the function of the gene products, i.e. enzymes and proteins involved in DNA repair. Effort should be made to analyse the enzymatic reactions, leading to an altered nucleotide sequence, encountered as a point mutation. Mislead mismatch repair and modulation of DNA polymerase might be possible mechanisms.Paper given at the workshop Molecular Radiation Biology. German Section of the DNA Repair Network, München-Neuherberg, 21.–23.3.90     

Mammalian cell killing by ultrasoft X rays and high-energy radiation: an extension of the MK model

An alternate formulation of the microdosimetric-kinetic (MK) model is presented that applies to irradiation of mammalian cells with ultrasoft X rays as well as high-energy radiations of variable linear energy transfer (LET). Survival and DNA double-strand break measurements for V79 cells from the literature are examined to illustrate application of the model. It is demonstrated that the linear component of the linear-quadratic survival relationship (alpha) is enhanced because repairable potentially lethal lesions formed from a single ultrasoft X-ray energy deposition event, when closer on average than for a single high-energy radiation event, are more likely to combine to form a lethal lesion. The quadratic component (beta) of the linear-quadratic survival relationship is increased because the potentially lethal lesions formed by ultrasoft X rays are created with greater efficiency than those of high-energy radiation. In addition, potentially lethal lesions from very low-energy carbon K-shell X rays may be enriched in structural forms that favor combination to form lethal lesions instead of repair. These features account for the increased effectiveness of killing of V79 cells by ultrasoft X rays compared to cobalt-60 gamma radiation. The importance of pairwise combination of potentially lethal lesions to form exchange chromosome aberrations that become lethal lesions is discussed. The extended MK model explains and reconciles differences between the MK model and the theory of dual radiation action on the one hand, and on the other, the view that variation in the RBE with radiation quality is explained by differences in energy deposition in nanometer- rather than micrometer-size volumes.     

Oxazole derivatives as compounds modifying radiation effects in the body of mice

A new class of substances exhibiting radioprotective and radiosensitizing effects depending on the concentration of the substance has been found. The radioprotective effect is probably due to the resonant absorption of radiation energy and its transformation into low-energy forms, as well as reactions with water radiolysis products. We studied the effects of 2,5-difeniloxazole and di[2-(5-feniloxazolil)]benzene in various concentrations in conjunction with irradiation on the growth of melanoma B-16 in mice and the average time of their lives. When using individual doses of irradiation and doses of preparations, we observed an increase in the average lifetime of mice and a reduced tumor size. These data allow us to conclude about the possibility of using these substances in the radiotherapy of tumors.     

Protective effects of melatonin against oxidation of guanine bases in DNA and decreased microsomal membrane fluidity in rat liver induced by whole body ionizing radiation

The aim of the study was to examine the potential protective effect of melatonin against whole body ionizing radiation (800 cGy). Changes in 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels, an index of DNA damage, and alterations in membrane fluidity (the inverse of membrane rigidity) and lipid peroxidation in microsomal membranes, as indices of damage to lipid and protein molecules in membranes, were estimated. Measurements were made in rat liver, 12 h after their exposure to radiation. To test the potential protective effects of melatonin, the indole was injected (i.p. 50 mg/kg b.w.) at 120, 90, 60 and 30 min prior to radiation exposure. Both 8-OH-dG levels and microsomal membrane rigidity increased significantly 12 h after radiation exposure. Melatonin completely counteracted the effects of ionizing radiation. Changes in 8-OH-dG levels and membrane fluidity are early sensitive parameters of DNA and microsomal membrane damage, respectively, induced by ionizing radiation and our findings document the protective effects of melatonin against ionizing radiation.     

Cellular responses to ionizing radiation: effects of interrupting DNA repair with chemical agents

This review is concerned with the influence of different classes of chemical agents on cellular repair of DNA damage induced by ionizing radiation. Single-strand break rejoining is little affected by inhibitors of DNA synthesis; however, such inhibitors do lead to a persistence of double-strand breaks in the DNA, and this correlates with an enhancement of chromosome aberrations and cell killing. Experiments with antagonists of topoisomerase II suggest an intriguing role for this DNA unwinding enzyme in double-strand break repair. Interference with poly(ADP-ribose) synthesis, by means of the inhibitor 3-aminobenzamide, does not have a clear-cut effect on recovery from ionizing radiation damage. Various substances (for example, caffeine and trypsin) affect DNA repair via a modulation of the cell cycle, altering the time available to the cell for repairing potentially lethal DNA damage before such damage is 'fixed' by the process of DNA replication. Finally, disturbing cellular energy metabolism, and depressing the level of ATP, can inhibit the repair of radiation damage.     

Nontargeted effects of ionizing radiation: implications for low-dose exposures

The traditional thinking has been that the biological effects of ionizing radiation occur in irradiated cells as a consequence of the DNA damage they incur. This implies that: 1) biological effects occur only in irratiated cells, 2) radiation traversal through the nucleus of the cell is a prerequisite to produce a biological response, and 3) DNA is the target molecule in the cell. Evidence has been emerging, however, for non-DNA targeted effects of radiation; that is, effects including mutations, chromosomal aberrations, and changes in gene expression which occur in cells that in themselves receive no radiation exposure. Two of these phenomena will be described in this paper. The first is radiation-induced genomic instability whereby biological effects, including elevated frequencies of mutations and chromosomal aberrations, arise in the distant descendants of irradiated cells. The second phenomenon has been termed the "bystander effect", whereby in a mixed population of irradiated and nonirradiated cells, biological effects arise in those cells that receive no radiation exposure. The damage signals are transmitted from cell to cell through gap junction channels, and the genetic effects observed in bystander cells appear to result from an upregulation of oxidative stress. The possible influence of these non-targeted effects of radiation of the respounse to low-dose exposures is discussed.