Oxidative stress and endoreduplication induced by blue light exposure to CHO cells.

Blue light is commonly used for the treatment of Neonatal Jaundice and as a photodynamic therapy for cancer. In comparison to ultraviolet light, blue light has a lower toxicity due to the differences in photon energies. However, blue light can still be mutagenic to cells. The proposed mechanism suggests blue light exposure induces reactive oxygen species inducing oxidative stress. In this study, we examined how blue light exposure caused genotoxic effects utilizing Chinese hamster ovary (CHO) cells and UV135 cells when exposed to fluorescent blue light. Cytotoxic effects of blue light exposure were quantified through cellular oxidative stress analysis, cell survival assay, and in cell cycle arrest experiments. Genotoxicity was studied in sister chromatid exchange (SCE) only, and endoreduplication formation. Following blue light exposure, an increase of cell cycle arrest, oxidative stress, and cytotoxicity was observed. Blue light treatment also produced an increased amount of SCE, and more importantly, induced endoreduplicated chromosomes. In conclusion, exposure to blue light resulted in significant genotoxicity of the treated cells.

PrimPol is required for the maintenance of efficient nuclear and mitochondrial DNA replication in human cells.

Eukaryotic Primase-Polymerase (PrimPol) is an enzyme that maintains efficient DNA duplication by repriming replication restart downstream of replicase stalling lesions and structures. To elucidate the cellular requirements for PrimPol in human cells, we generated PrimPol-deleted cell lines and show that it plays key roles in maintaining active replication in both the nucleus and mitochondrion, even in the absence of exogenous damage. Human cells lacking PrimPol exhibit delayed recovery after UV-C damage and increased mutation frequency, micronuclei and sister chromatin exchanges but are not sensitive to genotoxins. PrimPol is also required during mitochondrial replication, with PrimPol-deficient cells having increased mtDNA copy number but displaying a significant decrease in replication. Deletion of PrimPol in XPV cells, lacking functional polymerase Eta, causes an increase in DNA damage sensitivity and pronounced fork stalling after UV-C treatment. We show that, unlike canonical TLS polymerases, PrimPol is important for allowing active replication to proceed, even in the absence of exogenous damage, thus preventing the accumulation of excessive fork stalling and genetic mutations. Together, these findings highlight the importance of PrimPol for maintaining efficient DNA replication in unperturbed cells and its complementary roles, with Pol Eta, in damage tolerance in human cells.

Nucleoporin 54 contributes to homologous recombination repair and post-replicative DNA integrity.

The nuclear pore complex (NPC) machinery is emerging as an important determinant in the maintenance of genome integrity and sensitivity to DNA double-strand break (DSB)-inducing agents, such as ionising radiation (IR). In this study, using a high-throughput siRNA screen, we identified the central channel NPC protein Nup54, and concomitantly its molecular partners Nup62 and Nup58, as novel factors implicated in radiosensitivity. Nup54 depletion caused an increase in cell death by mitotic catastrophe after IR, and specifically enhanced both the duration of the G2 arrest and the radiosensitivity of cells that contained replicated DNA at the time of IR exposure. Nup54-depleted cells also exhibited increased formation of chromosome aberrations arisen from replicated DNA. Interestingly, we found that Nup54 is epistatic with the homologous recombination (HR) factor Rad51. Moreover, using specific DNA damage repair reporters, we observed a decreased HR repair activity upon Nup54 knockdown. In agreement with a role in HR repair, we also demonstrated a decreased formation of HR-linked DNA synthesis foci and sister chromatid exchanges after IR in cells depleted of Nup54. Our study reveals a novel role for Nup54 in the response to IR and the maintenance of HR-mediated genome integrity.

Cytogenetic monitoring of hospital staff exposed to ionizing radiation: optimize protocol considering DNA repair genes variability.

PURPOSE: Chronic occupational exposure to ionizing radiation (IR) induces a wide spectrum of DNA damages. The aim of this study was to assess the frequencies of micronucleus (MN), sister chromatid exchanges (SCE) and to evaluate their association with XRCC1 399 Arg/Gln and XRCC3 241 Thr/Met polymorphisms in Hospital staff occupationally exposed to IR. MATERIALS AND METHODS: A questionnaire followed by a cytogenetic analysis was concluded for each subject in our study. The exposed subjects were classified into two groups based on duration of employment (Group I < 15 years; Group II ≥15years). The genotypes of all individuals (subjects and controls) were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: DNA damage frequencies were significantly greater in IR workers compared with controls (p < .05). However, no association arised between XRCC1 399 Arg/Gln and XRCC3 241 Thr/Met polymorphisms, on one hand, and the severity of DNA damages in the studied cohort of Tunisian population, on the other hand. CONCLUSION: Our data provide evidence for an obvious genotoxic effect associated with IR exposure and reinforce the high sensitivity of cytogenetic assays for biomonitoring of occupationally exposed populations. These results indicate that workers exposed to IR should have periodic monitoring, along their exposure. The variants, rs25487 and rs861539, of XRCC1 and XRCC3 genes have obvious functional effects. Paradoxically, these variants are not associated with the severity of damages, according to used assays, in the studied cohort of Tunisian population, unlike other studies.

Influence of reduced glutathione on end-joining of DNA double-strand breaks: Cytogenetical and molecular approach.

Radiation induced DNA double-strand breaks (DSB) are the major initial lesions whose misrejoining may lead to exchange aberrations. However, the role of glutathione (GSH), a major cellular thiol, in regulating cell's sensitivity to DNA damaging agents is not well understood. Influence of endogenous GSH on the efficiency of X-rays and bleomycin (Blem) induced DNA DSBs end-joining has been tested here cytogenetically, in human lymphocytes and Hct116 cells. In another approach, oligomeric DNA (75bp) containing 5'-compatible and non-compatible overhangs mimicking the endogenous DSB were for rejoining in presence of cell-free extracts from cells having different endogenous GSH levels. Frequency of aberrations, particularly exchange aberrations, was significantly increased when Blem was combined with radiation. The exchange aberration frequency was further enhanced when combined treatment was given at 4°C since DNA lesions are poorly repaired at 4°C so that a higher number of DNA breaks persist and interact when shifted from 4°C to 37°C. The exchange aberrations increased further when the combined treatment was given to Glutathione-ester (GE) pre-treated cells, indicating more frequent rejoining of DNA lesions in presence of higher cellular GSH. This is further supported by the drastic reduction in frequency of exchange aberrations but significant increase in incidences of deletions when combined treatment was given to GSH-depleted cells. End-joining efficiency of DNA DSBs with compatible ends was better than for non-compatible ends. End-joining efficiency of testicular and MCF7 cell extracts was better than that of lungs and Hct116 cells. Cell extract made from GE-treated MCF-7 cells provided more efficient end-joining than from untreated and GSH-depleted cells. However, direct addition of GSH to the cell-free extracts showed considerable reduction in end-joining efficiency. Present data indicate that higher endogenous GSH favours rejoining of DNA DSBs (both restitution and illegitimate reunion) which in turn produce more exchange aberrations.

Solution Radioactivated by Hadron Radiation Can Increase Sister Chromatid Exchanges.

When energetic particles irradiate matter, it becomes activated by nuclear reactions. Radioactivation induced cellular effects are not clearly understood, but it could be a part of bystander effects. This investigation is aimed at understanding the biological effects from radioactivation in solution induced by hadron radiation. Water or phosphate buffered saline was activated by being exposed to hadron radiation including protons, carbon- and iron-ions. 1 mL of radioactivated solution was transferred to flasks with Chinese hamster ovary (CHO) cells cultured in 5 mL of complete media. The induction of sister chromatid exchanges (SCE) was used to observe any increase in DNA damage responses. The energy spectrum and the half-lives of the radioactivation were analyzed by NaI scintillation detector in order to identify generated radionuclides. In the radioactivated solution, 511 keV gamma-rays were observed, and their half-lives were approximately 2 min, 10 min, and 20 min. They respectively correspond to the beta+ decay of 15O, 13N, and 11C. The SCE frequencies in CHO cells increased depending on the amount of radioactivation in the solution. These were suppressed with a 2-hour delayed solution transfer or pretreatment with dimethyl sulfoxide (DMSO). Our results suggest that the SCE induction by radioactivated solution was mediated by free radicals produced by the annihilated gamma-rays. Since the SCE induction and DMSO modulation are also reported in radiation-induced bystander effects, our results imply that radioactivation of the solution may have some contribution to the bystander effects from hadron radiation. Further investigations are required to assess if radioactivation effects would attribute an additional level of cancer risk of the hadron radiation therapy itself.

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.

Increased γ-H2AX by exposure to a 60-Hz magnetic fields combined with ionizing radiation, but not hydrogen peroxide, in non-tumorigenic human cell lines.

PURPOSE: Genotoxic effects have been considered the gold standard to determine if an environmental factor is a carcinogen, but the currently available data for extremely low frequency time-varying magnetic fields (ELF-MF) remain controversial. As an environmental stimulus, the effect of ELF-MF on cellular DNA may be subtle. Therefore, a more sensitive method and systematic research strategy are warranted to evaluate genotoxicity. MATERIALS AND METHODS: We investigated the effect of ELF-MF in combination with ionizing radiation (IR) or H(2)O(2) on the DNA damage response of expression of phosphorylated H2AX (γ-H2AX) and production of γ-H2AX foci in non-tumorigenic human cell systems consisting of human lung fibroblast WI-38 cells and human lung epithelial L132 cells. RESULTS: Exposure to a 60-Hz, 2 mT ELF-MF for 6 h produced increased γ-H2AX expression, as well as γ-H2AX foci production, a common DNA double-strand break (DSB) marker. However, exposure to a 1 mT ELF-MF did not have the same effect. Moreover, 2 mT ELF-MF exposure potentiated the expression of γ-H2AX and γ-H2AX foci production when combined with IR, but not when combined with H(2)O(2). CONCLUSIONS: ELF-MF could affect the DNA damage response and, in combination with different stimuli, provide different effects on γ-H2AX.

The evaluation of long-term effects of ionizing radiation through measurement of current sister chromatid exchange (SCE) rates in radiology technologists, compared with previous SCE values.

Ionizing radiation is a strong physical mutagen, causing breakage of phosphodiester bonds in DNA at any stage of the mitotic cycle. Analysis of sister chromatid exchange (SCE) has come into use as a sensitive DNA-damage indicator. We investigated the SCE rates in radiology technologists who are occupationally and chronically exposed to ionizing radiation. The study included 39 radiology technologists and 35 sex- and age-matched healthy controls. There was a statistically significant difference in the SCE frequency between radiology technologists and controls (p<0.0001). Additionally, previous SCE data of 10 radiology technologists were compared with current results regarding radiation exposure time. There was statistically significant difference between previous and current SCE values (p=0.005). The significant increase in the frequency of SCE in radiology technologists emphasizes the importance of radiation-protection procedures in order to minimize radiation exposure and avoid possible genotoxic effects. Comparison of two studies that measured SCE values of radiology technologists after 8 years also suggests that the genotoxic effect is reversible. In conclusion, radiation is still an important mutagenic agent despite improvements in daily working hours and conditions.

Comparison of radiation-induced damage between CT angiography and conventional coronary angiography.

OBJECTIVE: Both computed tomography (CTA) and conventional angiography (CCA) can provide direct visualization of the coronary arteries. The aim of the present study was to compare the radiation exposure between CTA and CCA and to search whether this amount of radiation causes significant DNA damage. METHOD: Seventy-two patients who underwent CTA or CCA were enrolled prospectively. We recorded the radiation dosage that was used during the procedures and calculated the effective dose (ED). We determined the sister chromatid exchange (SCE) level from the blood samples which were drawn from the patients before and after the procedures. The change in SCE is used as the measure of DNA damage induced by the radiation. RESULTS: The patients in the CTA (n = 36) and CCA groups (n= 36) had similar baseline characteristics. The ED was higher in CTA examinations compared to CCA examinations (14.2 +/- 2.7 vs 6.4 +/- 3.1, P <0.001). The SCE level increased significantly after both angiography methods (P <0.001). When the change in SCE after angiography was compared, we did not find a significant difference among the groups (2.73 +/- 1.6 vs 2.54 +/- 1.22, P= NS). CONCLUSION: Although the patients who underwent CTA were exposed to a greater amount of radiation, the radiation-induced genetic damage was similar with both types of the procedures.

[The estimate of association between gene polymorphisms and the frequency and spectrum of cytogenetic abnormalities in the cohort of Siberian Group of Chemical Enterprises employees exposed to professional irradiation (microarray studies)].

The results from the research into the association between polymorphisms of genes-candidates for individual radiosensitivity and the frequency and spectrum of cytogenetic abnormalities are analyzed. The study was conducted among Siberian Group of Chemical Enterprises healthy employees exposed to professional irradiation in a dose range of 100-300 mSv. Genotyping of DNA samples from 96 employees was carried out by oligonucleotide microarray: "Cancer_SNP_Panel GT-17-211" ("Illumina") containing 1, 421 SNP-markers (Single Nucleotide Polymorphisms) of 406 genes. The standard cytogenetic analysis was performed in the entire examined group. We have also analyzed the association of these SNP with the frequencies of aberrant cells and following chromosomal aberrations: single chromatid fragments, chromatid exchanges, paired fragments, dicentric, ring and frequencies, translocations. We have found that 40 SNP (rs1800389, rs1051690, rs2392221, rs1041163, rs2114443, rs6083, rs1760904, rs4986894, rs488133, rs7462102, rs11249938, rs34206126, rs33945943, rs34324628, rs5742694, rs978458, rs5742667, rs2373721, rs2162679, rs889162, rs2233679, rs2010457, rs2873950, rs1574154, rs10934500, rs4688046, rs10934503, rs4624596, rs2288729, rs4227, rs1367696, rs751087, rs1269486, rs1149901, rs1800404, rs887477, rs696405, rs751087, rs81 92284, rs312016) are associated with the frequency of different types of chromosomal abnormalities (p-value with FDR of Benjamini-Hochberg--equal less than 0.05). 24 SNP (underlined) are associated with more than one type of chromosome abnormalities. In the future, we are going to confirm the results in further studies on the cohort of more than 600 persons.

SMC6 is an essential gene in mice, but a hypomorphic mutant in the ATPase domain has a mild phenotype with a range of subtle abnormalities.

Smc5-6 is a highly conserved protein complex related to cohesin and condensin involved in the structural maintenance of chromosomes. In yeasts the Smc5-6 complex is essential for proliferation and is involved in DNA repair and homologous recombination. siRNA depletion of genes involved in the Smc5-6 complex in cultured mammalian cells results in sensitivity to some DNA damaging agents. In order to gain further insight into its role in mammals we have generated mice mutated in the Smc6 gene. A complete knockout resulted in early embryonic lethality, demonstrating that this gene is essential in mammals. However, mutation of the highly conserved serine-994 to alanine in the ATP hydrolysis motif in the SMC6 C-terminal domain, resulted in mice with a surprisingly mild phenotype. With the neo gene selection marker in the intron following the mutation, resulting in reduced expression of the SMC6 gene, the mice were reduced in size, but fertile and had normal lifespans. When the neo gene was removed, the mice had normal size, but detailed phenotypic analysis revealed minor abnormalities in glucose tolerance, haematopoiesis, nociception and global gene expression patterns. Embryonic fibroblasts derived from the ser994 mutant mice were not sensitive to killing by a range of DNA damaging agents, but they were sensitive to the induction of sister chromatid exchanges induced by ultraviolet light or mitomycin C. They also accumulated more oxidative damage than wild-type cells.

Does formaldehyde induce aneuploidy?

Formaldehyde (FA) was tested for a potential aneugenic activity in mammalian cells. We employed tests to discriminate between aneugenic and clastogenic effects in accordance with international guidelines for genotoxicity testing. The cytokinesis-block micronucleus test (CBMNT) in combination with fluorescence in situ hybridisation (FISH) with a pan-centromeric probe was performed with cultured human lymphocytes and the human A549 lung cell line. FA induced micronuclei (MN) in binuclear cells of both cell types under standard in vitro test conditions following the OECD guideline 487. FISH analysis revealed that the vast majority of induced MN were centromere negative, thus indicating a clastogenic effect. A similar result was obtained for MN induced by γ-irradiation, whereas the typical aneugens colcemid (COL) and vincristine (VCR) predominantly induced centromere-positive MN. Furthermore, COL and VCR clearly enhanced the MN frequency in mononuclear lymphocytes in the CBMNT, whereas such an effect was not observed for γ-irradiation and FA. In experiments with the Chinese hamster V79 cell line, the aneugens COL and VCR clearly increased the frequency of tetraploid second division metaphases, whereas FA did not cause such an effect. Altogether, our results confirm the clastogenicity of FA in cultured mammalian cells but exclude a significant aneugenic activity.

DNA-PK-dependent RPA2 hyperphosphorylation facilitates DNA repair and suppresses sister chromatid exchange.

Hyperphosphorylation of RPA2 at serine 4 and serine 8 (S4, S8) has been used as a marker for activation of the DNA damage response. What types of DNA lesions cause RPA2 hyperphosphorylation, which kinase(s) are responsible for them, and what is the biological outcome of these phosphorylations, however, have not been fully investigated. In this study we demonstrate that RPA2 hyperphosphorylation occurs primarily in response to genotoxic stresses that cause high levels of DNA double-strand breaks (DSBs) and that the DNA-dependent protein kinase complex (DNA-PK) is responsible for the modifications in vivo. Alteration of S4, S8 of RPA2 to alanines, which prevent phosphorylations at these sites, caused increased mitotic entry with concomitant increases in RAD51 foci and homologous recombination. Taken together, our results demonstrate that RPA2 hyperphosphorylation by DNA-PK in response to DSBs blocks unscheduled homologous recombination and delays mitotic entry. This pathway thus permits cells to repair DNA damage properly and increase cell viability.

Roles of vertebrate Smc5 in sister chromatid cohesion and homologous recombinational repair.

The structural maintenance of chromosomes (Smc) family members Smc5 and Smc6 are both essential in budding and fission yeasts. Yeast smc5/6 mutants are hypersensitive to DNA damage, and Smc5/6 is recruited to HO-induced double-strand breaks (DSBs), facilitating intersister chromatid recombinational repair. To determine the role of the vertebrate Smc5/6 complex during the normal cell cycle, we generated an Smc5-deficient chicken DT40 cell line using gene targeting. Surprisingly, Smc5(-) cells were viable, although they proliferated more slowly than controls and showed mitotic abnormalities. Smc5-deficient cells were sensitive to methyl methanesulfonate and ionizing radiation (IR) and showed increased chromosome aberration levels upon irradiation. Formation and resolution of Rad51 and gamma-H2AX foci after irradiation were altered in Smc5 mutants, suggesting defects in homologous recombinational (HR) repair of DNA damage. Ku70(-/-) Smc5(-) cells were more sensitive to IR than either single mutant, with Rad54(-/-) Smc5(-) cells being no more sensitive than Rad54(-/-) cells, consistent with an HR function for the vertebrate Smc5/6 complex. Although gene targeting occurred at wild-type levels, recombinational repair of induced double-strand breaks was reduced in Smc5(-) cells. Smc5 loss increased sister chromatid exchanges and sister chromatid separation distances in mitotic chromosomes. We conclude that Smc5/6 regulates recombinational repair by ensuring appropriate sister chromatid cohesion.

Telomere sister chromatid exchange and the process of aging.

Telomeres are a hotspot for sister chromatid exchange (T-SCE). Any biological consequence of this form of instability remained obscure until quantitative modeling revealed a link between elevated T-SCE rates and accelerated cellular replicative senescence. This work strongly suggests that progressive telomere erosion is not the only determinant of replicative capacity; instead, T-SCE need to be considered as an independent factor controlling colony growth and senescence. Additionally high T-SCE rates have been observed in cells with deficiencies in WRN and BLM, the genes that are defective in Werner's and Bloom's syndromes, implying a connection to premature aging. In this Research Perspective we will explore some of the implications this recent work has for human health.

Transient activation of the ALT pathway in human primary fibroblasts exposed to high-LET radiation.

It is well established that high-LET radiations efficiently induce chromosome aberrations. However, data on the effect of protons on telomere maintenance, as involved in genomic stability, are scarce and contradictory. Here we demonstrate that high-LET protons induce telomere lengthening in human primary fibroblasts and that this elongation does not involve the telomerase enzyme, supporting the hypothesis that high-LET radiations are able to activate a telomerase-independent mechanism. In tumor cells that lack telomerase, one or more non-telomerase mechanisms for telomere maintenance are present, which are termed alternative lengthening of telomeres (ALT). Since ALT cells are characterized by recombinational events at telomeres, known as telomeric-sister chromatid exchanges (T-SCE), and colocalization of telomeres and premyelocytic leukemia protein (PML), we analyzed both T-SCE and PML. Our results show that high-LET protons induce a 2.5-fold increase of T-SCE and a colocalization of PML protein and telomeric DNA. Furthermore, our data show that the ALT pathway can be activated in human primary cells after induction of severe DNA damage. Thus, since telomeres are known to be involved in chromosome maintenance, the present work may contribute in the elucidation of the mechanism by which ionizing radiation induces genomic instability.

Rad21-cohesin haploinsufficiency impedes DNA repair and enhances gastrointestinal radiosensitivity in mice.

Approximately half of cancer-affected patients receive radiotherapy (RT). The doses delivered have been determined upon empirical experience based upon average radiation responses. Ideally higher curative radiation doses might be employed in patients with genuinely normal radiation responses and importantly radiation hypersensitive patients would be spared the consequences of excessive tissue damage if they were identified before treatment. Rad21 is an integral subunit of the cohesin complex, which regulates chromosome segregation and DNA damage responses in eukaryotes. We show here, by targeted inactivation of this key cohesin component in mice, that Rad21 is a DNA-damage response gene that markedly affects animal and cell survival. Biallelic deletion of Rad21 results in early embryonic death. Rad21 heterozygous mutant cells are defective in homologous recombination (HR)-mediated gene targeting and sister chromatid exchanges. Rad21+/- animals exhibited sensitivity considerably greater than control littermates when challenged with whole body irradiation (WBI). Importantly, Rad21+/- animals are significantly more sensitive to WBI than Atm heterozygous mutant mice. Since supralethal WBI of mammals most typically leads to death via damage to the gastrointestinal tract (GIT) or the haematopoietic system, we determined the functional status of these organs in the irradiated animals. We found evidence for GIT hypersensitivity of the Rad21 mutants and impaired bone marrow stem cell clonogenic regeneration. These data indicate that Rad21 gene dosage is critical for the ionising radiation (IR) response. Rad21 mutant mice thus represent a new mammalian model for understanding the molecular basis of irradiation effects on normal tissues and have important implications in the understanding of acute radiation toxicity in normal tissues.

Transient ATM kinase inhibition disrupts DNA damage-induced sister chromatid exchange.

Cells derived from ataxia telangiectasia (A-T) patients exhibit defective cell cycle checkpoints because of mutations in the gene encoding ATM (ataxia telangiectasia mutated). After exposure to ionizing radiation (IR), A-T cells exhibit sensitivity to IR-induced cellular damage that results in increased chromosome aberrations and cell death (radiosensitivity). ATM is a member of a family of kinases that become activated in response to DNA damage. We showed that even transient inhibition of ATM kinase for 1 hour, initiated 15 minutes after cellular irradiation, resulted in an accumulation of persistent chromosome aberrations and increased cell death. Using reversible inhibitors of DNA-PK (DNA-dependent protein kinase), another kinase involved in responding to DNA damage, and ATM, we showed that these two kinases acted through distinct DNA repair mechanisms: ATM resolved DNA damage through a mechanism involving sister chromatid exchange (SCE), whereas DNA-PK acted through nonhomologous end joining. Furthermore, because DNA damage-induced SCE occurred in A-T fibroblasts that lack functional ATM protein, and the inhibitors of ATM kinase had no effect on DNA damage-induced SCE in A-T fibroblasts, we showed that the consequences of short-term inhibition of the kinase activity of ATM and adaptation to ATM protein disruption were distinct. This suggests that A-T fibroblasts have adapted to the loss of ATM and have alternative mechanisms to initiate SCE.

Cytogenetic analysis of peripheral blood lymphocytes of hospital staff occupationally exposed to low doses of ionizing radiation.

Ionizing radiation is known to induce mutations and cell transformations, predominantly by causing single-strand and double-strand DNA breakage, thereby leading to chromosome instability and carcinogenesis. The aim of this study was to evaluate genotoxic effects in hospital staff exposed to low-dose ionizing radiation in comparison with a selected control group, by using the cytokinesis-blocked micronucleus (CBMN) and sister chromatid exchange (SCE) tests in peripheral blood lymphocytes. The study included 40 exposed radiology staff and 30 control subjects. The frequency of micronuclei (MN) was significantly increased in radiation-exposed groups compared with control persons (p < 0.05). The frequency of SCE did not show any significant difference in the exposed individuals in comparison to the controls. Our results showed that low-level chronic occupational exposure to ionizing radiation causes an increase of MN frequency in chromosomes, even though the absorbed doses were below the permissible limits. Our studies indicate that the CBMN assay is considered to be sensitive test in contrast to SCE analysis to evaluate chromosomal damage induced by ionizing radiation.