Impact of 1.8-GHz radiofrequency radiation (RFR) on DNA damage and repair induced by doxorubicin in human B-cell lymphoblastoid cells

In the present in vitro study, a comet assay was used to determine whether 1.8-GHz radiofrequency radiation (RFR, SAR of 2 W/kg) can influence DNA repair in human B-cell lymphoblastoid cells exposed to doxorubicin (DOX) at the doses of 0 μg/ml, 0.05 μg/ml, 0.075 μg/ml, 0.10 μg/ml, 0.15 μg/ml and 0.20 μg/ml. The combinative exposures to RFR with DOX were divided into five categories. DNA damage was detected at 0 h, 6 h, 12 h, 18 h and 24 h after exposure to DOX via the comet assay, and the percent of DNA in the tail (% tail DNA) served as the indicator of DNA damage. The results demonstrated that (1) RFR could not directly induce DNA damage of human B-cell lymphoblastoid cells; (2) DOX could significantly induce DNA damage of human B-cell lymphoblastoid cells with the dose–effect relationship, and there were special repair characteristics of DNA damage induced by DOX; (3) E–E–E type (exposure to RFR for 2 h, then simultaneous exposure to RFR and DOX, and exposure to RFR for 6 h, 12 h, 18 h and 24 h after exposure to DOX) combinative exposure could obviously influence DNA repair at 6 h and 12 h after exposure to DOX for four DOX doses (0.075 μg/ml, 0.10 μg/ml, 0.15 μg/ml and 0.20 μg/ml) in human B-cell lymphoblastoid cells.     

XRCC1 deficiency influences the cytotoxicity and the genomic instability induced by Me-lex,a specific inducer of N3-methyladenine

Me-lex is a sequence-specific alkylating agent synthesized to preferentially (>90%) generate N3-methyladenine (3-mA) in the minor groove of double-strand DNA, in A-T rich regions. In this paper we investigated the effect of XRCC1 deficiency in the processing of 3-mA adducts generated by Me-lex, through the molecular analysis of the Hprt mutations and the evaluation of cytogenetic end points such as sister chromatid exchanges (SCEs), micronuclei (MN) and nucleus fragmentation. EM-C11 cells, deficient in XRCC1 activity, showed a 2.5-fold higher sensitivity to the toxicity of Me-lex compared to the DNA repair proficient parental CHO-9 cells, but were not hyper mutable. The spontaneous mutation spectrum at the Hprt locus generated in EM-C11 cells revealed a high percentage of genomic deletions. After Me-lex treatment, the percentage of genomic deletions did not increase, but a class of mutations which appeared to target regulatory regions of the gene significantly increased (p = 0.0277), suggesting that non-coding Hprt genomic sequences represent a strong target for the rare mutations induced by Me-lex. The number of SCEs per chromosome increased 3-fold above background in 50 μМ Me-lex treated CHO-9 cells, while at higher Me-lex concentrations a sharp increase in the percentage of MN and fragmented nuclei was observed. In EM-C11 cells the background level of SCEs (0.939 ± 0.182) was approximately 10-fold higher than in CHO-9 (0.129 ± 0.027) and higher levels of multinucleated cells and MN were also found. In EM-C11, even low doses of Me-lex (25 μM) led to a significant increase in genomic damage. These results indicate that XRCC1 deficiency can lead to genomic instability even in the absence of an exogenous genotoxic insult and low levels of Me-lex-induced lesions, i.e., 3-mA and/or a BER intermediate, can exacerbate this instability.     

Effect of dithiocarbamate pesticide zineb and its commercial formulation,azzurro: IV. DNA damage and repair kinetics assessed by single cell gel electrophoresis (SCGE) assay on Chinese hamster ovary (CHO) cells

Single cell gel electrophoresis (SCGE) was used to analyse dithiocarbamate zineb- and the zineb-containing technical formulation azzurro-induced DNA damage and repair in CHO cells. Cells were treated with zineb (50.0 μg/ml) or azzurro (100.0 μg/ml) for 80 min, washed and reincubated in pesticide-free medium for 0–12 h until SCGE. Viability of treated cells (0 h) did not differ from control remaining unchanged up to 6 h of incubation. After 12 h, viability decreased up to 70 and 54% in zineb- and azzurro-treated cultures, respectively. SCGE revealed at 0 h the absence of undamaged cells and an increase of slightly damaged and damaged cells in zineb-treated cultures or by an increase in damaged cells in azzurro-treated cultures. For both chemicals, a time-dependent repair of pesticide-induced DNA damage within a 0–12 h post-treatment incubation period was observed. Overall, damaged cells decreased as a function of the repair time for both pesticides while the slightly damaged cells decreased as a function of the repair time of zineb-induced DNA damage. Concomitantly, a time-dependent increase of undamaged cells was observed within the 0.5–12 h repair time for both pesticides. At 12 h after treatment, no differences in the frequencies of undamaged, slightly damaged and damaged cells were found between both zineb- or azzurro-treated cultures and control values as well as between zineb- and azzurro-treated cells. Immediately after exposure, nuclear DNA from zineb and azzurro-treated cells were larger and wider than nuclear DNA from untreated cells. When damaged cells were allowed to repair, a time-dependent decrease of the amount of free DNA migrating fragments was observed committed only to damaged cells but not in slightly or undamaged cells. On the other hand, no time-dependent alteration on nuclear DNA width within the 0–12 h repair period was observed.     

Both genetic and dietary factors underlie individual differences in DNA damage levels and DNA repair capacity

The interplay between dietary habits and individual genetic make-up is assumed to influence risk of cancer, via modulation of DNA integrity. Our aim was to characterize internal and external factors that underlie inter-individual variability in DNA damage and repair and to identify dietary habits beneficial for maintaining DNA integrity.Habitual diet was estimated in 340 healthy individuals using a food frequency questionnaire and biomarkers of antioxidant status were quantified in fasting blood samples. Markers of DNA integrity were represented by DNA strand breaks, oxidized purines, oxidized pyrimidines and a sum of all three as total DNA damage. DNA repair was characterized by genetic variants and functional activities of base and nucleotide excision repair pathways.Sex, fruit-based food consumption and XPG genotype were factors significantly associated with the level of DNA damage. DNA damage was higher in women (p = 0.035). Fruit consumption was negatively associated with the number of all measured DNA lesions, and this effect was mediated mostly by β-cryptoxanthin and β-tocopherol (p < 0.05). XPG 1104His homozygotes appeared more vulnerable to DNA damage accumulation (p = 0.001). Sex and individual antioxidants were also associated with DNA repair capacity; both the base and nucleotide excision repairs were lower in women and the latter increased with higher plasma levels of ascorbic acid and α-carotene (p < 0.05).We have determined genetic and dietary factors that modulate DNA integrity. We propose that the positive health effect of fruit intake is partially mediated via DNA damage suppression and a simultaneous increase in DNA repair capacity.     

Oxidative stress induces an ATM-independent senescence pathway through p38 MAPK-mediated lamin B1 accumulation

We report crosstalk between three senescence-inducing conditions, DNA damage response (DDR) defects, oxidative stress (OS) and nuclear shape alterations. The recessive autosomal genetic disorder Ataxia telangiectasia (A-T) is associated with DDR defects, endogenous OS and premature ageing. Here, we find frequent nuclear shape alterations in A-T cells, as well as accumulation of the key nuclear architecture component lamin B1. Lamin B1 overexpression is sufficient to induce nuclear shape alterations and senescence in wild-type cells, and normalizing lamin B1 levels in A-T cells reciprocally reduces both nuclear shape alterations and senescence. We further show that OS increases lamin B1 levels through p38 Mitogen Activated Protein kinase activation. Lamin B1 accumulation and nuclear shape alterations also occur during stress-induced senescence and oncogene-induced senescence (OIS), two canonical senescence situations. These data reveal lamin B1 as a general molecular mediator that controls OS-induced senescence, independent of established Ataxia Telangiectasia Mutated (ATM) roles in OIS.     

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.     

Differential binding kinetics of replication protein A during replication and the pre- and post-incision steps of nucleotide excision repair

The ability of replication protein A (RPA) to bind single-stranded DNA (ssDNA) underlines its crucial roles during DNA replication and repair. A combination of immunofluorescence and live cell imaging of GFP-tagged RPA70 revealed that RPA, in contrast to other replication factors, does not cluster into replication foci, which is explained by its short residence time at ssDNA. In addition to replication, RPA also plays a crucial role in both the pre- and post-incision steps of nucleotide excision repair (NER). Pre-incision factors like XPC and TFIIH accumulate rapidly at locally induced UV-damage and remain visible up to 4 h. However, RPA did not reach its maximum accumulation level until 3 h after DNA damage infliction and a chromatin-bound pool remained detectable up to 8 h, probably reflecting its role during the post-incision step of NER. During the pre-incision steps of NER, RPA could only be visualized at DNA lesions in incision deficient XP-F cells, however without a substantial increase in residence time at DNA damage. Together our data show that RPA is an intrinsically highly dynamic ssDNA-binding complex during both replication and distinct steps of NER.     

Genetic factors in individual radiation sensitivity

Cancer risk and radiation sensitivity are often associated with alterations in DNA repair, cell cycle, or apoptotic pathways. Interindividual variability in mutagen or radiation sensitivity and in cancer susceptibility may also be traced back to polymorphisms of genes affecting e.g. DNA repair capacity. We studied possible associations between 70 polymorphisms of 12 DNA repair genes with basal and initial DNA damage and with repair thereof. We investigated DNA damage induced by ionizing radiation in lymphocytes isolated from 177 young lung cancer patients and 169 cancer-free controls. We also sought replication of our findings in an independent sample of 175 families (in total 798 individuals). DNA damage was assessed by the Olive tail moment (OTM) of the comet assay. DNA repair capacity (DRC) was determined for 10, 30 and, 60 min of repair.Genes involved in the single-strand-repair pathway (SSR; like XRCC1 and MSH2) as well as genes involved in the double-strand-repair pathway (DSR; like RAD50, XRCC4, MRE11 and ATM) were found to be associated with DNA damage. The most significant association was observed for marker rs3213334 (p = 0.005) of XRCC1 with basal DNA damage (B), in both cases and controls. A clear additive effect on the logarithm of OTM was identified for the marker rs1001581 of the same LD-block (p = 0.039): B_CC = −1.06 (95%-CI: −1.16 to −0.96), B_CT = −1.02 (95%-CI: −1.11 to −0.93) and B_TT = −0.85 (95%-CI: −1.01 to −0.68). In both cases and controls, we observed significantly higher DNA basal damage (p = 0.007) for carriers of the genotype AA of marker rs2237060 of RAD50 (involved in DSR). However, this could not be replicated in the sample of families (p = 0.781). An alteration to DRC after 30 min of repair with respect to cases was observed as borderline significant for marker rs611646 of ATM (involved in DSR; p = 0.055), but was the most significant finding in the sample of families (p = 0.009).Our data indicate that gene variation impacts measurably on DNA damage and repair, suggesting at least a partial contribution to radiation sensitivity and lung cancer susceptibility.     

Sodium orthovanadate associated with pharmacological doses of ascorbate causes an increased generation of ROS in tumor cells that inhibits proliferation and triggers apoptosis

Pharmacological doses of ascorbate were evaluated for its ability to potentiate the toxicity of sodium orthovanadate (Na₃VO₄) in tumor cells. Cytotoxicity, inhibition of cell proliferation, generation of ROS and DNA fragmentation were assessed in T24 cells. Na₃VO₄ was cytotoxic against T24 cells (EC₅₀ = 5.8 μM at 24 h), but in the presence of ascorbate (100 μM) the EC₅₀ fell to 3.3 μM. Na₃VO₄ plus ascorbate caused a strong inhibition of cell proliferation (up to 20%) and increased the generation of ROS (4-fold). Na₃VO₄ did not directly cleave plasmid DNA, at this aspect no synergism was found occurring between Na₃VO₄ and ascorbate once the resulting action of the combination was no greater than that of both substances administered separately. Cells from Ehrlich ascites carcinoma-bearing mice were used to determine the activity of antioxidant enzymes, the extent of the oxidative damage and the type of cell death. Na₃VO₄ alone, or combined with ascorbate, increased catalase activity, but only Na₃VO₄ plus ascorbate increased superoxide dismutase activity (up to 4-fold). Oxidative damage on proteins and lipids was higher due to the treatment done with Na₃VO₄ plus ascorbate (2–3-fold). Ascorbate potentiated apoptosis in tumor cells from mice treated with Na₃VO₄. The results indicate that pharmacological doses of ascorbate enhance the generation of ROS induced by Na₃VO₄ in tumor cells causing inhibition of proliferation and apoptosis. Apoptosis induced by orthovanadate and ascorbate is closer related to inhibition on Bcl-xL and activation of Bax. Our data apparently rule out a mechanism of cell demise p53-dependent or related to Cdk2 impairment.     

Juglans regia L. protects against UVB induced apoptosis in human epidermal keratinocytes

The present study was aimed to investigate the photoprotective effect of the male flower of J. regia L. (MEJR) against ultraviolet-B induced apoptosis in human skin cells. Human skin epidermal keratinocytes were pretreated with the MEJR (80 µg/ml, has been selected after MTT assay), prior to 30 min UVB-irradiation at a dose of 20 mJ/cm². Mitochondrial membrane potential was evaluated using Rhodamine-123 staining; the % apoptosis by Hoechst staining and acridine orange staining; DNA damage was measured by comet assay. The levels of p53, Bax, Bcl-xL, Bcl-2, Cytochrome c, Caspase-9 and Caspase-3 expression in HaCaT cells were analyzed by western blotting and RT-PCR. Pretreatment with MEJR 80 µg/ml prior to UVB-irradiation significantly prevents apoptotic characteristics, DNA damage and loss of mitochondrial membrane potential. Thus, MEJR protects UVB-mediated human skin cells, by modulating the expression of apoptotic markers and UVB-induced DNA damage in HaCaT cells.     

Assessment of alterations in X-ray irradiation-induced DNA damage of glioma cells by using proton nuclear magnetic resonance spectroscopy

Glioma is one of the most common types of brain tumors. DNA damage is closely associated with glioma cell apoptosis induced by X-ray irradiation. Alterations of metabolites in glioma can be detected noninvasively by proton nuclear magnetic resonance (1H NMR) spectroscopy. To noninvasively explore the micro mechanism in X-ray irradiation-induced apoptosis, the relationship between metabolites and DNA damage in glioma cells was investigated. Three glioma cell lines (C6, U87 and U251) were randomly designated as control (0 Gy) and treatment groups (1, 5, 10, 15 Gy). After X-ray exposure, each group was separated into four parts: (i) to detect metabolites by 1H NMR spectroscopy; (ii) to make cell colonies; (iii) to detect cell cycle distribution and apoptosis rate by flow cytometry; and (iv) to measure DNA damage by comet assay. The metabolite ratios of lactate/creatine and succinate/creatine decreased (lactate/creatine: C6, 22.17–66.27%; U87, 15.93–44.56%; U251, 26.27–74.48%. succinate/creatine: C6, 14.41–48.35%; U87, 22.03–70.62%; U251, 17.33–60.06%) and choline/creatine increased (C6, 52.22–389.68%; U87, 56.15–82.36%; U251, 31.87–278.62%) in the treatment groups compared with the control group (each P < 0.05), which linearly depended on DNA damage. An increasing dose of X-ray irradiation increased numbers of apoptotic cells (P < 0.01), and the DNA damage parameters were dose-dependent (P < 0.05). The colony-forming rate declined (P < 0.01) and the percentage of cells at G1 stage increased when exposed to 1 Gy X-ray (three cell lines, P < 0.05). Metabolite alterations detected by 1H NMR spectroscopy can be used to determine DNA damage induced by X-ray irradiation. 1H NMR spectroscopy is a noninvasive method to predict DNA damage of glioma cell at the micro level.     

Consequences of ionizing radiation-induced damage in human neural stem cells

Cranial irradiation remains a frontline treatment for brain cancer, but also leads to normal tissue damage. Although low-dose irradiation (≤ 10 Gy) causes minimal histopathologic change, it can elicit variable degrees of cognitive dysfunction that are associated with the depletion of neural stem cells. To decipher the mechanisms underlying radiation-induced stem cell dysfunction, human neural stem cells (hNSCs) subjected to clinically relevant irradiation (0–5 Gy) were analyzed for survival parameters, cell-cycle alterations, DNA damage and repair, and oxidative stress. hNSCs showed a marked sensitivity to low-dose irradiation that was in part due to elevated apoptosis and the inhibition of cell-cycle progression that manifested as a G2/M checkpoint delay. Efficient removal of DNA double-strand breaks was indicated by the disappearance of γ-H2AX nuclear foci. A dose-responsive and persistent increase in oxidative and nitrosative stress was found in irradiated hNSCs, possibly the result of a higher metabolic activity in the fraction of surviving cells. These data highlight the marked sensitivity of hNSCs to low-dose irradiation and suggest that long-lasting perturbations in the CNS microenvironment due to radiation-induced oxidative stress can compromise the functionality of neural stem cells.     

DNA fragmentation in human fibroblasts under extremely low frequency electromagnetic field exposure

Extremely low frequency electromagnetic fields (ELF-EMFs) were reported to affect DNA integrity in human cells with evidence based on the Comet assay. These findings were heavily debated for two main reasons; the lack of reproducibility, and the absence of a plausible scientific rationale for how EMFs could damage DNA. Starting out from a replication of the relevant experiments, we performed this study to clarify the existence and explore origin and nature of ELF-EMF induced DNA effects. Our data confirm that intermittent (but not continuous) exposure of human primary fibroblasts to a 50 Hz EMF at a flux density of 1 mT induces a slight but significant increase of DNA fragmentation in the Comet assay, and we provide first evidence for this to be caused by the magnetic rather than the electric field. Moreover, we show that EMF-induced responses in the Comet assay are dependent on cell proliferation, suggesting that processes of DNA replication rather than the DNA itself may be affected. Consistently, the Comet effects correlated with a reduction of actively replicating cells and a concomitant increase of apoptotic cells in exposed cultures, whereas a combined Fpg-Comet test failed to produce evidence for a notable contribution of oxidative DNA base damage. Hence, ELF-EMF induced effects in the Comet assay are reproducible under specific conditions and can be explained by minor disturbances in S-phase processes and occasional triggering of apoptosis rather than by the generation of DNA damage.     

Induced Pluripotent Stem Cells Are Sensitive to DNA Damage

Induced pluripotent stem cells(iPSCs)resemble embryonic stem cells(ESCs)in morphology,gene expression and in vitro differentiation,raising new hope for personalized clinical therapy.While many efforts have been made to improve reprogramming effciency,signifcant problems such as genomic instability of iPSCs need to be addressed before clinical therapy.In this study,we try to fgure out the real genomic state of iPSCs and their DNA damage response to ionizing radiation(IR).We found that iPSC line 3FB4-1 had lower DNA damage repair ability than mouse embryonic fbroblast(MEF)cells,from which 3FB4-1line was derived.After the introduction of DNA damage by IR,the number of c-H2AX foci in 3FB4-1 increased modestly compared to a large increase seen in MEF,albeit both signifcantly(P<0.01).In addition,whole-genome sequencing analysis showed that after IR,3FB4-1 possessed more point mutations than MEF and the point mutations spread all over chromosomes.These observations provide evidence that iPSCs are more sensitive to ionizing radiation and their relatively low DNA damage repair capacity may account for their high radiosensitivity.The compromised DNA damage repair capacity of iPSCs should be considered when used in clinical therapy.     

Evaluation of the genotoxic and antigenotoxic effects after acute and subacute treatments with açai pulp (Euterpe oleracea Mart.) on mice using the erythrocytes micronucleus test and the comet assay

Açai, the fruit of a palm native to the Amazonian basin, is widely distributed in northern South America, where it has considerable economic importance. Whereas individual polyphenolics compounds in açai have been extensively evaluated, studies of the intact fruit and its biological properties are lacking. Therefore, the present study was undertaken to investigate the in vivo genotoxicity of açai and its possible antigenotoxicity on doxorubicin (DXR)-induced DNA damage. The açai pulp doses selected were 3.33, 10.0 and 16.67 g/kg b.w. administered by gavage alone or prior to DXR (16 mg/kg b.w.) administered by intraperitoneal injection. Swiss albino mice were distributed in eight groups for acute treatment with açai pulp (24 h) and eight groups for subacute treatment (daily for 14 consecutive days) before euthanasia. The negative control groups were treated in a similar way. The results of chemical analysis suggested the presence of carotenoids, anthocyanins, phenolic, and flavonoids in açai pulp. The endpoints analyzed were micronucleus induction in bone marrow and peripheral blood cells polychromatic erythrocytes, and DNA damage in peripheral blood, liver and kidney cells assessed using the alkaline (pH >13) comet assay. There were no statistically significant differences (p > 0.05) between the negative control and the groups treated with the three doses of açai pulp alone in all endpoints analyzed, demonstrating the absence of genotoxic effects. The protective effects of açai pulp were observed in both acute and subacute treatments, when administered prior to DXR. In general, subacute treatment provided greater efficiency in protecting against DXR-induced DNA damage in liver and kidney cells. These protective effects can be explained as the result of the phytochemicals present in açai pulp. These results will be applied to the developmental of food with functional characteristics, as well as to explore the characteristics of açai as a health promoter.     

The canonical NF-κB pathway differentially protects normal and human tumor cells from ROS-induced DNA damage

DNA damage responses (DDR) invoke senescence or apoptosis depending on stimulus intensity and the degree of activation of the p53-p21(Cip1/Waf1) axis; but the functional impact of NF-κB signaling on these different outcomes in normal vs. human cancer cells remains poorly understood. We investigated the NF-κB-dependent effects and mechanism underlying reactive oxygen species (ROS)-mediated DDR outcomes of normal human lung fibroblasts (HDFs) and A549 human lung cancer epithelial cells. To activate DDR, ROS accumulation was induced by different doses of H(2)O(2). The effect of ROS induction caused a G2 or G2-M phase cell cycle arrest of both human cell types. However, ROS-mediated DDR eventually culminated in different end points with HDFs undergoing premature senescence and A549 cancer cells succumbing to apoptosis. NF-κB p65/RelA nuclear translocation and Ser536 phosphorylation were induced in response to H(2)O(2)-mediated ROS accumulation. Importantly, blocking the activities of canonical NF-κB subunits with an IκBα super-repressor or suppressing canonical NF-κB signaling by IKKβ knock-down accelerated HDF premature senescence by up-regulating the p53-p21(Cip1/Waf1) axis; but inhibiting the canonical NF-κB pathway exacerbated H(2)O(2)-induced A549 cell apoptosis. HDF premature aging occurred in conjunction with γ-H2AX chromatin deposition, senescence-associated heterochromatic foci and beta-galactosidase staining. p53 knock-down abrogated H(2)O(2)-induced premature senescence of vector control- and IκBαSR-expressing HDFs functionally linking canonical NF-κB-dependent control of p53 levels to ROS-induced HDF senescence. We conclude that IKKβ-driven canonical NF-κB signaling has different functional roles for the outcome of ROS responses in the contexts of normal vs. human tumor cells by respectively protecting them against DDR-dependent premature senescence and apoptosis.     

Prompt repair of hydrogen peroxide-induced DNA lesions prevents catastrophic chromosomal fragmentation

Iron-dependent oxidative DNA damage in vivo by hydrogen peroxide (H₂O₂, HP) induces copious single-strand(ss)-breaks and base modifications. HP also causes infrequent double-strand DNA breaks, whose relationship to the cell killing is unclear. Since hydrogen peroxide only fragments chromosomes in growing cells, these double-strand breaks were thought to represent replication forks collapsed at direct or excision ss-breaks and to be fully reparable. We have recently reported that hydrogen peroxide kills Escherichia coli by inducing catastrophic chromosome fragmentation, while cyanide (CN) potentiates both the killing and fragmentation. Remarkably, the extreme density of CN + HP-induced chromosomal double-strand breaks makes involvement of replication forks unlikely. Here we show that this massive fragmentation is further amplified by inactivation of ss-break repair or base-excision repair, suggesting that unrepaired primary DNA lesions are directly converted into double-strand breaks. Indeed, blocking DNA replication lowers CN + HP-induced fragmentation only ∼2-fold, without affecting the survival. Once cyanide is removed, recombinational repair in E. coli can mend several double-strand breaks, but cannot mend ∼100 breaks spread over the entire chromosome. Therefore, double-strand breaks induced by oxidative damage happen at the sites of unrepaired primary one-strand DNA lesions, are independent of replication and are highly lethal, supporting the model of clustered ss-breaks at the sites of stable DNA-iron complexes.