Inter-individual variation in DNA double-strand break repair in human fibroblasts before and after exposure to low doses of ionizing radiation

DNA double-strand breaks (DSB) are generally considered the most critical lesion induced by ionizing radiation (IR) and may initiate carcinogenesis and other disease. Using an immunofluorescence assay to simultaneously detect nuclear foci of the phosphorylated forms of histone H2AX and ATM kinase at sites of DSBs, we examined the response of 25 apparently normal and 10 DNA repair-deficient (ATM, ATR, NBN, LIG1, LIG4, and FANCG) primary fibroblast strains irradiated with low doses of ¹³⁷Cs γ-rays. Quiescent G₀/G₁-phase cultures were exposed to 5, 10, and 25 cGy and allowed to repair for 24 h. The maximum level of IR-induced foci (0.15 foci per cGy, at 10 or 30 min) in the normal strains showed much less inter-individual variation (CV ≈ 0.2) than the level of spontaneous foci, which ranged from 0.2–2.6 foci/cell (CV ≈ 0.6; mean ± SD of 1.00 ± 0.57). Significantly slower focus formation post-irradiation was observed in seven normal strains, similar to most mutant strains examined. There was variation in repair efficiency measured by the fraction of IR-induced foci remaining 24 h post-irradiation, curiously with the strains having slower focus formation showing more efficient repair after 25 cGy. Interestingly, the ranges of spontaneous and residual induced foci levels at 24 h in the normal strains were as least as large as those observed for the repair-defective mutant strains. The inter-individual variation in DSB foci parameters observed in cells exposed to low doses of ionizing radiation in this small survey of apparently normal people suggests that hypomorphic genetic variants in genomic maintenance and/or DNA damage signaling and repair genes may contribute to differential susceptibility to cancer induced by environmental mutagens.     

ER stress suppresses DNA double-strand break repair and sensitizes tumor cells to ionizing radiation by stimulating proteasomal degradation of Rad51

In this study, we provide evidence that endoplasmic reticulum (ER) stress suppresses DNA double-strand break (DSB) repair and increases radiosensitivity of tumor cells by altering Rad51 levels. We show that the ER stress inducer tunicamycin stimulates selective degradation of Rad51 via the 26S proteasome, impairing DSB repair and enhancing radiosensitivity in human lung cancer A549 cells. We also found that glucose deprivation, which is a physiological inducer of ER stress, triggered similar events. These findings suggest that ER stress caused by the intratumoral environment influences tumor radiosensitivity, and that it has potential as a novel target to improve cancer radiotherapy.     

G2 repair and chromosomal damage in lymphocytes from workers occupationally exposed to low-level ionizing radiation

The effect of the G2 repair of chromosomal damage in lymphocytes from workers exposed to low levels of X- or gamma-rays was evaluated. Samples of peripheral blood were collected from 15 radiation workers, 20 subjects working in radiodiagnostics, and 30 healthy control donors. Chromosomal aberrations (CA) were evaluated by scoring the presence of chromatid and isochromatid breaks, dicentric and ring chromosomes in lymphocytes with/without 5 mM caffeine plus 3 mM-aminobenzamide (3-AB) treatment during G2. Our results showed that the mean value of basal aberrations in lymphocytes from exposed workers was higher than in control cells (p < 0.001). The chromosomal damage in G2, detected with caffeine plus 3-AB treatment was higher than the basal damage (untreated conditions), both in control and exposed populations (p < 0.05). In the exposed workers group, the mean value of chromosomal abnormalities in G2 was higher than in the control (p < 0.0001). No correlation was found between the frequency of chromosome type of aberrations (basal or in G2), and the absorbed dose. Nevertheless, significant correlation coefficients (p < 0.05) between absorbed dose and basal aberrations yield (r = 0.430) or in G2 (r = 0.448) were detected when chromatid breaks were included in the total aberrations yield. Under this latter condition no significant effect of age, years of employment or smoking habit on the chromosomal aberrations yield was detected. However, analysis of the relationship between basal aberrations yield and the efficiency of G2 repair mechanisms, defined as the percentage of chromosomal lesions repaired in G2, showed a significant correlation coefficient (r = -0.802; p < 0.001). These results suggest that in addition to the absorbed dose, the individual G2 repair efficiency may be another important factor affecting the chromosomal aberrations yield detected in workers exposed to low-level ionizing radiation.     

TP53 is not required for the constitutive or induced repair of DNA damage produced by ionizing radiation at the G1/S-phase border

We have previously described a novel DNA repair response that is induced in cells irradiated with ionizing radiation at the G1/S-phase border and is characterized by the formation of very long repair patches (VLRP) containing at least 150 nucleotides. In the current study, we examined whether there is a requirement for TP53 in this induced repair process. We find that in normal cells, the endogenous levels of TP53 are elevated at the G1/S-phase border, and that these levels are not further increased after irradiation with 5 Gy. In cells expressing the E6 oncoprotein of human papillomavirus, which inactivates TP53 function, there is a greatly accentuated induction of the VLRP that nearly masks the constitutive repair response. Incubation of cells in the presence of cycloheximide, which inhibits the induced repair, reveals the presence of the constitutive repair patches. All cells examined continue to replicate their DNA after exposure to ionizing radiation. In contrast, cells irradiated with UV radiation at the G1/S-phase border show an induction of TP53 protein and halt DNA synthesis, but do not induce the VLRP. Our results show that TP53 is not required for the constitutive or induced repair of DNA damage induced by ionizing radiation. In addition, these results suggest that TP53 may suppress the formation of VLRP and that the progression of cells through S phase after exposure to ionizing radiation signals the induced repair response.     

Micronuclei in humans induced by exposure to low level of ionizing radiation: influence of polymorphisms in DNA repair genes

Understanding the risks deriving from protracted exposure to low doses of ionizing radiation has remarkable societal importance in view of the large number of work settings in which sources of IR are encountered. To address this question, we studied the frequency of micronuclei (MN), which is an indicator of DNA damage, in a population exposed to low levels of ionizing radiation and in matched controls. In both exposed population and controls, the possible influence of single nucleotide polymorphisms in XRCC1, XRCC3 and XPD genes on the frequency of micronuclei was also evaluated. We also considered the effects of confounding factors, like smoking status, age and gender. The results indicated that MN frequency was significantly higher in the exposed workers than in the controls [8.62+/-2.80 versus 6.86+/-2.65; P=0.019]. Radiological workers with variant alleles for XRCC1 or XRCC3 polymorphisms or wild-type alleles for XPD exon 23 or 10 polymorphisms showed a significantly higher MN frequency than controls with the same genotypes. Smoking status did not affect micronuclei frequency either in exposed workers or controls, while age was associated with increased MN frequency in the exposed only. In the combined population, gender but not age exerted an influence on the yield of MN, being higher in females than in males. Even though there is a limitation in this study due to the small number of subjects, these results suggest that even exposures to low level of ionizing radiation could have genotoxic effects and that XRCC3, XRCC1 and XPD polymorphisms might contribute to the increased genetic damage in susceptible individuals occupationally exposed to chronic low levels of ionizing radiation. For a clear conclusion on the induction of DNA damage caused by protracted exposure to low doses of ionizing radiation and the possible influence of genetic polymorphism in DNA repair genes larger studies are needed.     

Induction of chromosome aberrations in G0 human lymphocytes by low doses of ionizing radiations of different quality

Human peripheral blood lymphocytes from two donors were exposed to low doses (0.05 to 2.0 Gy) of gamma rays, X rays, or fast neutrons of different energies. Chromosome aberrations were analyzed in metaphase of first-division cells after a culture time of 45-46 hr. At this time, less than 5% of the cells were found in second division. Different dose-response relationships were fitted to the data by using a maximum likelihood method; best fits for radiation-induced dicentric aberrations were obtained with the linear-quadratic law for all radiations. The linear component of this equation predominated, however, for neutrons in the range of doses studied, and the frequency of dicentrics induced by d(16)+Be neutrons up to 1.0 Gy could also be described by a linear relationship. The relative biological efficiency (RBE) of X rays and d(16)+Be, d(33)+Be, and d(50)+Be neutrons compared to 60Co gamma rays in the low dose range was calculated from the dose-effect relationships for the dicentrics produced. The RBE increased with decreasing neutron dose and with decreasing neutron energy from d(50)+Be to d(16)-+Be neutrons. The limiting RBE at low doses (RBEo) was calculated to be about 1.5 for X rays and 14.0, 6.2, and 4.7 for the d(16)+Be, d(33)+Be, and d(50)+Be neutrons, respectively.     

Phototriggered formation and repair of DNA containing a site-specific single strand break of the type produced by ionizing radiation or AP lyase activity

DNA strand breaks are produced by a variety of agents and processes such as ionizing radiation, xenobiotics, oxidative metabolism, and enzymatic processing of DNA base damage. One of the major types of strand breaks produced by these processes is a single nucleotide gap terminating in 5'- and 3'-phosphates. Previously, we had developed a method for sequence-specifically producing such phosphate-terminated strand breaks in an oligodeoxynucleotide by way of two photochemically activated (caged) building blocks placed in tandem. We now report the design and synthesis of a single caged building block consisting of 1,3-(2-nitrophenyl)-1,3-propanediol, for producing phosphate-terminated strand breaks, and its use producing such a break at a specific site in a double-stranded circular DNA vector. To produce the site-specific break in a duplex vector, a primer containing the caged single strand break was extended opposite the single strand form of a circular DNA vector followed by enzymatic ligation and purification. The single strand break could then be formed in quantitative yield by irradiation of the vector with 365 nm light. In contrast to a previous study, it was found that the strand break can be repaired by Escherichia coli DNA polymerase I and E. coli DNA ligase alone, though less efficiently than in the presence of the 3'-phosphate processing enzyme E. coli endonuclease IV. Repair in the absence of endonuclease IV could be attributed to hydrolysis of the 3'-phosphate in the presence of dNTP and to a lesser extent to exonucleolytic removal of the 3'-phosphate-bearing terminal nucleotide by way of the 3' --> 5' exonuclease activity of polymerase I. This work demonstrates that specialized 3'-end processing enzymes such as endonuclease IV or exonuclease III are not absolutely required for repair of phosphate-terminated gaps. In addition to preparing single strand breaks, the caged building block described should also be useful for preparing double strand breaks and multiply damaged sites that might otherwise be difficult to prepare by other methods due to their lability.     

Detection of partial-body exposure to ionizing radiation by the automatic detection of dicentrics

In accidental exposure to ionizing radiation, it is essential to estimate the dose received by the victims. Currently dicentric scoring is the best biological indicator of exposure. The standard biological dosimetry procedure (500 metaphases scored manually) is suitable for a few dose estimations, but the time needed for analysis can be problematic in the case of a large-scale accident. Recently, a new methodology using automatic detection of dicentrics has greatly decreased the time needed for dose estimation and preserves the accuracy of the estimation. However, the capability to detect nonhomogeneous partial-body exposures is an important advantage of dicentric scoring-based biodosimetry, and this remains to be tested with automatic scoring. Thus we analyzed the results obtained with in vitro blood dilutions and in real cases of accidental exposure (partial- or whole-body exposure) using manual scoring and automatic detection of dicentrics. We confirmed that automatic detection allows threefold quicker dicentric scoring than the manual procedure with similar dose estimations and uncertainty intervals. The results concerning partial-body exposures were particularly promising, and homogeneously exposed samples were correctly distinguished from heterogeneously exposed samples containing 5% to 75% of blood irradiated with 2 Gy. In addition, the results obtained for real accident cases were similar whatever the methodology used. This study demonstrates that automatic detection of dicentrics is a credible alternative for recent and acute cases of whole- and partial-body accidental exposures to ionizing radiation.     

Frequency of micronuclei in peripheral blood lymphocytes from subjects occupationally exposed to low levels of ionizing radiation

The aim of this study was to evaluate the genotoxic effect of low dose of occupational radiation exposure in Nuclear Medicine Department employees, by using cytokinesis-blocked micronucleus assay in peripheral blood lymphocytes. The study included 46 exposed individuals together with 27 from the same area without occupational exposure to radiation which served as controls. The results obtained were evaluated with respect to age, gender, smoking habits, pathological condition and the occupational exposure to radiation of the individuals. The frequency of micronuclei increased significantly with the age of the subjects (P = 0.007). However there were no significant differences in micronucleus frequency with gender, smoking habits and occupational exposure. The frequency of micronuclei was significantly higher in individuals with presence of pathological condition (P < 0.0001) in comparison to healthy population irrespective of their exposure status.     

Redox status in workers occupationally exposed to long-term low levels of ionizing radiation: A pilot study

Objectives: Reactive oxygen species (ROS), including superoxide (O₂^??), play an important role in the biological effects of ionizing radiation. The human body has developed different antioxidant systems to defend against excessive levels of ROS. The aim of the present study is to investigate the redox status changes in the blood of radiologic technologists and compare these changes to control individuals.

Methods: We enrolled 60 medical workers: 20 occupationally exposed to ionizing radiation (all radiologic technologists), divided in three subgroups: conventional radiography (CR), computerized tomography (CT), and interventional radiography (IR) and 40 age- and gender-matched unexposed controls. Levels of O₂^?? and malondialdehyde (MDA) in blood were measured as an index of redox status, as were the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase. Redox status was also assessed by measuring levels of reduced and oxidized glutathione (GSH, GSSG, respectively).

Results: Levels of O₂^?? and MDA, and SOD activity in the blood of IR and CT-exposed subjects were significantly higher than both the CR-exposed subjects and control individuals. However, there were no statistically significant differences in the levels of catalase, GSH and ratio of GSH/GSSG between exposed workers and control individuals.

Discussion: This study suggests that healthcare workers in CT and IR occupationally exposed to radiation have an elevated circulating redox status as compared to unexposed individuals.     

Base excision repair of ionizing radiation-induced DNA damage in G1 and G2 cell cycle phases

Background

Major genomic surveillance mechanisms regulated in response to DNA damage exist at the G₁/S and G₂/M checkpoints. It is presumed that these delays provide time for the repair of damaged DNA. Cells have developed multiple DNA repair pathways to protect themselves from different types of DNA damage. Oxidative DNA damage is processed by the base excision repair (BER) pathway. Little is known about the BER of ionizing radiation-induced DNA damage and putative heterogeneity of BER in the cell cycle context. We measured the activities of three BER enzymes throughout the cell cycle to investigate the cell cycle-specific repair of ionizing radiation-induced DNA damage. We further examined BER activities in G2 arrested human cells after exposure to ionizing radiation.

Results

Using an in vitro incision assay involving radiolabeled oligonucleotides with specific DNA lesions, we examined the activities of several BER enzymes in the whole cell extracts prepared from synchronized human HeLa cells irradiated in G1 and G2 phase of the cell cycle. The activities of human endonuclease III (hNTH1), a glycosylase/lyase that removes several damaged bases from DNA including dihydrouracil (DHU), 8-oxoguanine-DNA glycosylase (hOGG1) that recognizes 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) lesion and apurinic/apyrimidinic endonuclease (hAPE1) that acts on abasic sites including synthetic analog furan were examined.

Conclusion

Overall the repair activities of hNTH1 and hAPE1 were higher in the G1 compared to G2 phase of the cell cycle. The percent cleavages of oligonucleotide substrate with furan were greater than substrate with DHU in both G1 and G2 phases. The irradiation of cells enhanced the cleavage of substrates with furan and DHU only in G1 phase. The activity of hOGG1 was much lower and did not vary within the cell cycle. These results demonstrate the cell cycle phase dependence on the BER of ionizing radiation-induced DNA damage. Interestingly no evidence of enhanced BER activities was found in irradiated cells arrested in G2 phase.     

Cytogenetic analyses by fluorescence in situ hybridization (FISH) in hospital workers occupationally exposed to low levels of ionizing radiation

An occupationally exposed population has been studied to evaluate the suitability of FISH painting techniques to detect chronic exposures to very low doses of ionizing radiation by the analysis of translocations. Whole-chromosome painting probes for chromosomes 1, 4 and 11 in combination with a pancentromeric probe have been employed. For comparison, a matched control population has also been studied. The mean genomic frequencies per 100 cells of total translocations in the control and exposed populations were 0.90 +/- 0.12 and 1.04 +/- 0.11, respectively. In the occupationally exposed population, no correlation between the frequencies of translocations and the doses received was found. When the two populations were compared, no significant differences were observed for the frequencies of the different chromosomal abnormalities examined. The absence of differences between control and exposed populations could be attributed to the very low-dose exposures recorded in the occupationally exposed population and to the wide range of individual frequencies of translocations observed.     

A new assay capturing chromosome fusions shows a protection trade-off at telomeres and NHEJ vulnerability to low-density ionizing radiation

Chromosome fusions threaten genome integrity and promote cancer by engaging catastrophic mutational processes, namely chromosome breakage–fusion–bridge cycles and chromothripsis. Chromosome fusions are frequent in cells incurring telomere dysfunctions or those exposed to DNA breakage. Their occurrence and therefore their contribution to genome instability in unchallenged cells is unknown. To address this issue, we constructed a genetic assay able to capture and quantify rare chromosome fusions in budding yeast. This chromosome fusion capture (CFC) assay relies on the controlled inactivation of one centromere to rescue unstable dicentric chromosome fusions. It is sensitive enough to quantify the basal rate of end-to-end chromosome fusions occurring in wild-type cells. These fusions depend on canonical nonhomologous end joining (NHEJ). Our results show that chromosome end protection results from a trade-off at telomeres between positive effectors (Rif2, Sir4, telomerase) and a negative effector partially antagonizing them (Rif1). The CFC assay also captures NHEJ-dependent chromosome fusions induced by ionizing radiation. It provides evidence for chromosomal rearrangements stemming from a single photon–matter interaction.     

Modeling radioprotective mechanisms in the dose effect relation at low doses and low dose rates of ionizing radiation

A new model (Random Coincidence Model--Radiation Adapted (RCM-RA)) is proposed which explains a possible pseudo threshold for stochastic radiation effects. It describes the formation of cancer in the case of multistep fixation of lesions in the critical regions of tumor associated genes such as proto-oncogenes or tumor-suppressor genes. The RCM-RA contains two different possibilities of DNA damage to complementary nucleotides. The damage may be caused either by radiation or by natural processes such as cellular radicals or thermal damage or by chemical cytotoxins. The model is based on the premise that radiation initially is bionegative, damaging organisms at their different levels of organization. The radiation, however, also induces various cellular radioprotective mechanisms which decrease the damage by natural processes. Considering both effects together, the theory explains apparent thresholds in the dose-response relation for radiation carcinogenesis without contradiction to the classical assumption that radiation is predominantly bionegative at doses typically found in occupational exposures.