Radiation induced DNA DSBs: Contribution from stalled replication forks?

When cells are exposed to radiation serious lesions are introduced into the DNA including double strand breaks (DSBs), single strand breaks (SSBs), base modifications and clustered damage sites (a specific feature of ionizing radiation induced DNA damage). Radiation induced DNA damage has the potential to initiate events that can lead ultimately to mutations and the onset of cancer and therefore understanding the cellular responses to DNA lesions is of particular importance. Using γH2AX as a marker for DSB formation and RAD51 as a marker of homologous recombination (HR) which is recruited in the processing of frank DSBs or DSBs arising from stalled replication forks, we have investigated the contribution of SSBs and non-DSB DNA damage to the induction of DSBs in mammalian cells by ionizing radiation during the cell cycle. V79-4 cells and human HF19 fibroblast cells have been either irradiated with 0–20 Gy of γ radiation or, for comparison, treated with a low concentration of hydrogen peroxide, which is known to induce SSBs but not DSBs. Inhibition of the repair of oxidative DNA lesions by poly(ADP ribose) polymerase (PARP) inhibitor leads to an increase in radiation induced γH2AX and RAD51 foci which we propose is due to these lesions colliding with replication forks forming replication induced DSBs. It was confirmed that DSBs are not induced in G₁ phase cells by treatment with hydrogen peroxide but treatment does lead to DSB induction, specifically in S phase cells. We therefore suggest that radiation induced SSBs and non-DSB DNA damage contribute to the formation of replication induced DSBs, detected as RAD51 foci.     

Involvement of DNA polymerase beta in protection against the cytotoxicity of oxidative DNA damage

We had shown previously that DNA polymerase beta (beta-pol) null mouse fibroblasts, deficient in base excision repair (BER), are hypersensitive to monofunctional methylating agents but not to hydrogen peroxide (H2O2). This is surprising because beta-pol is thought to be involved in BER of oxidative as well as methylated DNA damage. We confirm these findings here in early-passage cells. However, with time in culture, beta-pol null cells become hypersensitive to H2O2 and other reactive oxygen species-generating agents. Analysis of in vitro BER reveals a strong deficiency in single-nucleotide BER of 8-oxoguanine (8-oxoG) by both early- and late-passage beta-pol null cell extracts. Therefore, in early-passage wild-type and beta-pol null cells, the capacity for single-nucleotide BER of 8-oxoG does not correlate with cellular sensitivity to H2O2. Expression of beta-pol protein in the late-passage null cells almost completely reverses the H2O2-hypersensitivity phenotype. Methoxyamine (MX) treatment sensitizes late-passage wild-type cells to H2O2 as expected for beta-pol-mediated single-nucleotide BER; however in beta-pol null cells, MX has no effect. The data indicate a role(s) of beta-pol-dependent repair in protection against the cytotoxicity of oxidative DNA damage in wild-type cells.     

Efficient repair of hydrogen peroxide-induced DNA damage by Escherichia coli requires SOS induction of RecA and RuvA proteins

The survival of Escherichia coli following treatment with a low dose (1-3 mM) of hydrogen peroxide (H(2)O(2)) that causes extensive mode-one killing of DNA repair mutants is stimulated by the induction of the SOS regulon. Results for various mutants indicate that induction of recA and RecA protein-mediated recombination are critical factors contributing to the repair of H(2)O(2)-induced oxidative DNA damage. However, because DNA damage activates RecA protein's coprotease activity essential to cleavage of LexA repressor protein and derepression of all SOS genes, it is unclear to what extent induction of RecA protein stimulates this repair. To make this determination, we examined mode-one killing of DeltarecA cells carrying plasmid-borne recA (P(tac)-recA(+)) and constitutively expressing a fully induced level of wild-type RecA protein when SOS genes other than recA are non-inducible in a lexA3 (Ind(-)) genetic background or inducible in a lexA(+) background. At a H(2)O(2) dose resulting in maximal killing, DeltarecA lexA3 (Ind(-)) cells with P(tac)-recA(+) show 40-fold greater survival than lexA3 (Ind(-)) cells with chromosomal recA having a low, non-induced level of RecA protein. However, they still show 10- to 15-fold lower survival than wild-type cells and DeltarecA lexA(+) cells with P(tac)-recA(+). To determine if the inducible RuvA protein stimulates survival, we examined a ruvA60 mutant that is defective for the repair of UV-induced DNA damage. This mutant also shows 10- to 15-fold lower survival than wild-type cells. We conclude that while induction of RecA protein has a pronounced stimulatory effect on the recombinational repair of H(2)O(2)-induced oxidative DNA damage, the induction of other SOS proteins such as RuvA is essential for wild-type repair.     

Evidence that hydrogen peroxide generated by 365-nm UVA radiation is not important in mammalian cell killing

We compared measurements of cell survival and DNA single-strand breaks (SSBs) caused by hydrogen peroxide (H2O2) and UVA radiation (365-nm) in both a parental and a H2O2-resistant variant of the Chinese hamster ovary HA1 line derived by culturing cells in progressively higher concentrations of H2O2. Both RNA slot blot analysis and enzyme analysis confirmed that the variant possesses high levels of both catalase activity and mRNA. The variant was completely resistant to the lethal effects of H2O2 over the concentration range tested (up to 480 microM), whereas the parental strain showed less than 1% survival at this concentration. Similarly, the H2O2-resistant strain exhibited far fewer SSBs after exposure to H2O2 than the parental strain. Addition of o-phenanthroline to the parental cells during H2O2 exposure almost completely inhibited SSB induction, evidence that these SSBs are produced via the Fenton pathway of Haber-Weiss reactions. Very little difference was found between the variant and the parent after exposure to 365-nm radiation: only a minor difference in survival kinetics and no difference is SSB induction were observed between the two cell lines. These results are consistent with a hypothesis that most lethal events caused in cells by UVA occur by pathways that do not involve the H2O2 that is produced by sensitized reactions within the cells.     

Calcium chelator Quin 2 prevents hydrogen-peroxide-induced DNA breakage and cytotoxicity

Exposure of cultured Chinese hamster ovary (CHO) cells to hydrogen peroxide results in the production of extensive DNA breakage which can be prevented by the intracellular calcium chelator Quin 2. This effect occurs at Quin 2 AM concentrations as low as 0.1 microM and is maximal at 1 microM. Addition of the extracellular calcium chelator, EGTA, does not affect the level of DNA breakage generated by H2O2. Quin 2 also significantly reduces cellular toxicity caused by the oxidant. Experiments with spin-trapping techniques demonstrate that Quin 2 does not affect the formation of hydroxyl radicals generated by the action of Fe2+ on H2O2. Quin 2 at high concentrations, similar to those reached within the cell, actually enhanced generation of hydroxyl radical in the absence of other iron chelators under our experimental conditions. These results suggest that H2O2 or H2O2-derived radicals do not directly induce DNA strand breakage in intact mammalian cells; rather, these radicals may disturb intracellular Ca2+ homeostasis which results in secondary reactions ultimately leading to DNA strand breakage. In addition to strand breakage, membrane and protein oxidation probably contribute to the cytotoxic effect of H2O2.     

Hydrogen peroxide is critical for UV-induced apoptosis inhibition

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O2*-) and hydrogen peroxide (H2O2), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H2O2-eliminating enzyme, catalase, but not in the presence of an O2*--eliminating enzyme, superoxide dismutase. Treatment with H2O2 itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H2O2 treatment decreased in H2O2-resistant cells. Although both UVB and H2O2 are known to induce DNA damage, other DNA damaging agents, like gamma-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H2O2 was essential to the inhibition of apoptosis, in which DNA damage had no role.     

Ionizing radiation induces a Yap1-dependent peroxide stress response in yeast

Repair of DNA damage is fundamental for cellular tolerance to ionizing radiation (IR) and many IR-induced DNA lesions are thought to occur as a result of oxidative stress. We investigated the physiological effects of IR in Saccharomyces cerevisiae by performing protein expression profiles in cells exposed to electron pulse irradiation. Transient induction of several antioxidant enzymes in wild-type cells, but not in cells lacking the oxidative stress regulator Yap1, indicated that IR exposure causes cellular oxidative stress. Yap1 activation involved oxidation to the intramolecular disulfide bond, a signature of activation by peroxide, and was dependent on the Yap1 peroxide sensor Orp1/Gpx3. H(2)O(2) was produced in the culture medium of irradiated cells and was both necessary and sufficient for IR-induced Yap1 activation. When IR was performed in the presence of N(2)O, obviating H(2)O(2) production and increasing hydroxyl radical ((*)OH) production, the Yap1 response was lost, indicating that Yap1 was unable to respond to (*)OH or (*)OH-induced damage. However, the Yap1 response to IR did not seem to be a primary determinant of cellular IR tolerance. Altogether, these data provide a molecular demonstration that cells experience in vivo peroxide stress during IR and indicate that the H(2)O(2) produced cannot account for IR toxicity.     

Hydrogen peroxide inhibits activation, not activity, of cellular caspase-3 in vivo

Oxidants such as H(2)O(2) can induce a low level of apoptosis at low concentrations but at higher concentrations cause necrosis. Higher concentrations of H(2)O(2) also inhibit the induction of apoptosis by chemotherapy drugs. One theory is that, at higher concentrations, H(2)O(2) causes direct oxidative inactivation of caspase-3 activity, thus preventing the apoptotic pathway from being used. We find that treatment of recombinant caspase-3 with H(2)O(2) can partially reduce its enzymatic activity: However, the following findings show that this does not occur in the cell. (1) The inhibition by H(2)O(2) of VP-16-induced apoptosis and cellular caspase-3 activity can be overcome by adding inhibitors of poly(ADP-ribose) polymerase (PARP) at sub-stoichiometric concentrations. (2) Delayed addition of H(2)O(2) to VP-16-treated cells prevents additional caspase induction but does not inhibit the caspase activity that has already been generated. (3) H(2)O(2) is a poor inhibitor of caspase-3 activity in cell lysates. (4) Addition of H(2)O(2) to cells inhibits activation of caspase-9, which is required for activation of caspase-3. We conclude that inhibition of caspase-3 activity in the cell occurs indirectly at a step located upstream of caspase-3 activation. H(2)O(2) acts in part by inducing DNA strand breaks and activating PARP, thus depleting the cells of ATP. When this pathway is blocked, even high concentrations of H(2)O(2) can induce caspase-9 and -3 activation and cause apoptosis.     

Mammalian cells are not killed by DNA single-strand breaks caused by hydroxyl radicals from hydrogen peroxide

Cell killing by ionizing radiation has been shown to be caused by hydroxyl free radicals formed by water radiolysis. We have previously suggested that the killing is not caused by individual OH free radicals but by the interaction of volumes of high radical density with DNA to cause locally multiply damaged sites (LMDS) (J. F. Ward, Radiat. Res. 86, 185-195, 1985). Here we test this hypothesis using hydrogen peroxide as an alternate source of OH radicals. The route to OH production from H2O2 is expected to cause singly damaged sites rather than LMDS. Chinese hamster V79-171 cells were treated with H2O2 at varying concentrations for varying times at 0 degree C. DNA damage produced intracellularly was measured by alkaline elution and quantitated in terms of Gray-equivalent damage by comparing the rate of its elution with that of DNA from gamma-irradiated cells. The yield of DNA damage produced increases with increasing concentration of H2O2 and with time of exposure. H2O2 is efficient in producing single-strand breaks; treatment with 50 microM for 30 min produces damage equivalent to that formed by 10 Gy of gamma irradiation. In the presence of a hydroxyl radical scavenger, dimethyl sulfoxide (DMSO), the yield of damage decreases with increasing DMSO concentration consistent with the scavenging of hydroxyl radicals traveling an average of 15 A prior to reacting with the DNA. In contrast to DNA damage production, cell killing by H2O2 treatment at 0 degree C is inefficient. Concentrations of 5 X 10(-2) M H2O2 for 10 min are required to produce significant cell killing; the DNA damage yield from this treatment can be calculated to be equivalent to 6000 Gy of gamma irradiation. The conclusion drawn is that individual DNA damage sites are ineffectual in killing cells. Mechanisms are suggested for killing at 0 degree C at high concentrations and for the efficient cell killing by H2O2 at 37 degrees C at much lower concentrations.     

Inducibility of the response of yeast cells to peroxide stress.

Exponential phase cells of the yeast, Saccharomyces cerevisiae when treated with a non-lethal concentration of hydrogen peroxide (H2O2; 0.2mM) for 60 min adapted to become resistant to the lethal effects of a higher dose of H2O2 (2mM). From studies using cycloheximide to inhibit protein synthesis it appears that protein synthesis is required for maximal induction of resistance but that some degree of protection from the lethal effects of peroxide can be acquired in the absence of protein synthesis. Treatment of cells with 50 micrograms cycloheximide ml-1 alone lead to them acquiring some protection from peroxide. Cells subjected to heat shock became more resistant to 2mM-H2O2; however, peroxide pretreatment did not confer thermotolerance. L-[35S]Methionine labelling of cells subjected to 0.2 mM-H2O2 stress showed that synthesis of at least ten polypeptides was induced by peroxide treatment. Some of these were also induced in cells subjected to heat shock (23 to 37 degrees C shift) but the synthesis of at least four polypeptides (45, 39.5, 38 and 24 kDa) was unique to peroxide-stressed cells. Resistance to peroxide was also inducible in an isogenic petite and an isogenic strain with a mutation in the HAP1 gene, indicating that the adaptive response does not require functional mitochondria.     

A common pathway for protection of bacteria against damage by solar UVA (334 nm, 365 nm) and an oxidising agent (H2O2)

Pre-exposure of growing bacterial populations to low concentrations of hydrogen peroxide (H2O2) protects a repair-proficient strain of Escherichia coli (AB1157) very strongly and a rec A strain (AB2463) to a lesser extent from the lethal action of subsequent exposure to 5 mM H2O2 in buffer. The conditioning procedure also protects AB1157 and AB2463 from the toxic effects of UVA (334 nm, 365 nm) radiation but not UVB (313 nm) or UVC (254 nm) radiations. Pretreatment of growing AB1157 with low fluences of UVA (365 nm) radiation leads to the induction of resistance to H2O2, an effect which apparently requires protein synthesis. As in a previous report, the treatment of growing populations with low concentrations of H2O2 enhanced the resistance of such populations to H2O2 challenge in the growth medium. However, when H2O2 (+ Cu2+)-treated bacteriophage were subsequently infected into AB1157 under optimal inducing conditions, their resistance was not enhanced relative to infection into untreated bacteria. We conclude that the primary mechanism for the inducible effects observed could be the induction of H2O2 scavenging activity by low concentrations of H2O2 either introduced into the growth medium directly or produced by low fluences of UVA irradiation.     

Induction by H2O2 of DNA and interphase chromosome damage in plateau-phase Chinese hamster ovary cells.

The induction by H2O2 of DNA breaks, DNA double-strand breaks (DSBs), and interphase chromatin damage and their relationship to cytotoxicity were studied in plateau-phase Chinese hamster ovary (CHO) cells. Damage in interphase chromatin was assayed by means of premature chromosome condensation (PCC); DNA DSBs were assayed by nondenaturing filter elution (pH 9.6), and DNA breaks by hydroxyapatite chromatography. Cells were treated with H2O2 in suspension at 0 degrees C for 30 min and treatment was terminated by the addition of catalase. Concentrations of H2O2 lower than 1 mM were not cytotoxic, whereas concentrations of 40 and 60 mM reduced cell survival to 0.1 and 0.004, respectively. An induction of DNA breaks that was dependent on H2O2 concentration was observed at low H2O2 concentrations that reached a maximum at approximately 1 mM; at higher H2O2 concentrations induction of DNA breaks either remained unchanged or decreased. Damage at the chromosome level was not evenly distributed among the cells, when compared to that expected based on a Poisson distribution. Three categories of cells were identified after exposure to H2O2: cells with intact, control-like chromosomes, cells showing chromosome fragmentation similar to that observed in cells exposed to ionizing radiation, and cells showing a loss in the ability of their chromatin to condense into chromosomes under the PCC reaction. The fraction of cells with fragmented chromosomes, as well as the number of excess chromosomes per cell, showed a dose response similar to that of DNA DSBs, reaching a maximum at 1 mM and decreasing at higher concentrations. The results indicate that induction of DNA and chromosome damage by H2O2 follows a complex dependence probably resulting from a depletion of reducing equivalents in the vicinity of the DNA. Reducing equivalents are required to recycle the transition metal ions that are needed to maintain a Fenton-type reaction. The absence of cell killing at H2O2 concentrations that yielded the maximum amount of DNA and chromosome damage suggests that this damage is nonlethal and repairable. It is suggested that lethal DNA and chromosome damage is induced at higher concentrations of H2O2 where cell killing is observed by an unidentified mechanism.     

Mechanisms of the bactericidal action of hydrogen peroxide

The comparative study of some aspects of the bactericidal action of H2O2 on Escherichia coli, Staphylococcus aureus and Bacterium subtilis wild-type cells and their mutants with lesions in the systems of the reparation of DNA has been carried out. Hydrogen peroxide has been shown to cause disturbances in the structure and permeability of the cell wall, the cytoplasmic membrane, as well as to induce ribosomal lesions and the ruptures of bacterial DNA. The activity of the systems responsible for the reparation of lesions in the cell genome plays an important role in the resistance of bacteria to H2O2.     

Apoptosis induced by dithiothreitol in HL-60 cells shows early activation of caspase 3 and is independent of mitochondria

Previous studies have shown that under certain conditions some thiol-containing compounds can cause apoptosis in a number of different cell lines. Herein, we investigated the apoptotic pathways in HL-60 cells triggered by dithiothreitol (DTT), used as a model thiol compound, and tested the hypothesis that thiols cause apoptosis via production of hydrogen peroxide (H2O2) during thiol oxidation. The results show that, unlike H2O2, DTT does not induce apoptosis via a mitochondrial pathway. This is demonstrated by the absence of early cytochrome c release from mitochondria into the cytosol, the lack of mitochondrial membrane depolarization at early times, and the minor role of caspase 9 in DTT-induced apoptosis. The first caspase activity detectable in DTT-treated cells is caspase 3, which is increased significantly 1 - 2 h after the start of DTT treatment. This was shown by following the cleavage of both a natural substrate, DFF-45/ICAD, and a synthetic fluorescent substrate, z-DEVD-AFC. Cleavage of substrates of caspases 2 and 8, known as initiator caspases, does not start until 3 - 4 h after DTT exposure, well after caspase 3 has become active and at a time when apoptosis is in late stages, as shown by the occurrence of DNA fragmentation to oligonucleosomal-sized pieces. Although oxidizing DTT can produce H2O2, data presented here indicate that DTT-induced apoptosis is not mediated by production of H2O2 and occurs via a novel pathway that involves activation of caspase 3 at early stages, prior to activation of the common 'initiator' caspases 2, 8 and 9.     

Changes of chromatin structure state in thymocites at the early stage of apoptosis induced by hydrogen peroxide and radiation

The paper deals with changes in the structural state of chromatin in isolated thymocites at the early stage of apoptosis induced by hydrogen peroxide and radiation. Content of necrosis and apoptosis cells in the suspension of the isolated rat thymocites, during 3-hour incubation after X-ray irradiation in a dose of 4.5 Gy or with the presence of 0.1 microM of H2O2 by the method of double lifetime staining by fluorescent dye Hehst 33342 and propydium iodide has been estimated. Apoptogenic effect of the studied effects has been found out, the dynamics of condensation and internucleosomic chromatin fragmentation has been established. It has been shown that 100 microM alpha-tocopherol inhibited completely DNA fragmentation in the cells incubated with H2O2 and only partially in irradiated cells. Introduction of postmitochondrial supernatant, isolated from the incubated control or irradiated cells, into the cell-free system which included the ATP-regenerating system and nuclei of control thymocites did not affect the level of DNA fragmentation, while the increase of the level of fragmented DNA in nuclei was observed in the presence of the supernatant obtained by centrifugation of the cells treated by H2O2. Differences of mechanisms of thymocite apoptosis initiation, as affected by hydrogen peroxide and ionizing radiation, is discussed.     

Glucose oxidase-produced H2O2 induces Ca2+-dependent DNA damage in human peripheral blood lymphocytes

DNA of lymphocytes from human peripheral blood was analyzed by using the single cell gel electrophoresis technique (comet assay). The cells were used either as received from the donors or after treatment with various concentrations of the H2O2-generating enzyme glucose oxidase, in order to achieve a continuous flow of H2O2. The formation of single strand breaks (SSB) was dose-related but the time course of the induction of SSB by relatively low concentrations of glucose oxidase was of a biphasic mode with a fast increase 2 to 5 min after the addition of glucose oxidase followed by a gradual decrease toward the original base level during the next 35 to 60 min. This response of the cells appears to be based on the activation of already existing defense system(s) because it was shown that H2O2 is continuously released during the reaction time and the inhibition of protein synthesis does not affect the observed pattern. Supplementation of the growth medium with various antioxidants resulted in substantial protection only when the agents were taken up by the cells. The presence of the intracellular calcium chelator BAPTA protected the cells from H2O2-induced DNA damage in a dose-dependent manner. Only at the higher rate of H2O2-generation considerable DNA damage was observed in the presence of BAPTA.These results suggest that H2O2, at low concentrations induces DNA damage through intracellular Ca2+ -mediated processes, which lead to DNA strand breaks possibly by endonuclease activation.     

Gamma-ray and hydrogen peroxide induction of gene amplification in hamster cells deficient in DNA double strand break repair

To investigate the role of DNA double strand breaks (DSBs) and of their repair in gene amplification, we analyzed this process in the V3 Chinese hamster cell line and in the parental line AA8, after exposure to gamma-rays and to hydrogen peroxide (H2O2). V3 is defective in DSB repair because of a mutation in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene, a gene involved in the non-homologous end-joining pathway. As a measure of gene amplification we used the frequency of colonies resistant to N-(phosphonacetyl)-L-aspartate (PALA), since in rodent cells PALA resistance is mainly achieved through the amplification of the CAD (carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase) gene. After treatment with different doses of gamma-rays and of H2O2, we found a dose related increase in the frequency of gene amplification and of chromosome aberrations. When the same doses of damaging agents were used, these increments were higher in V3 than in AA8. These results indicate that DSBs that are not efficiently repaired can be responsible for the induction of gene amplification. H2O2 stimulates gene amplification as well as gamma-rays, however, at similar levels of amplification induction, chromosome damage was about 50% lower. This suggests that gene amplification can be induced by H2O2 through pathways alternative to a direct DNA damage. Stimulation of gene amplification by H2O2, which is one of the products of the aerobic metabolism, supports the hypothesis that cellular metabolic products themselves can be a source of genome instability.