Reduction in free-radical-induced DNA strand breaks and base damage through fast chemical repair by flavonoids

This paper provides evidence that dietary flavonoids can repair a range of oxidative radical damages on DNA, and thus give protection against radical-induced strand breaks and base alterations. We have irradiated dilute aqueous solutions of plasmid DNA in the absence and presence of flavonoids (F) in a "constant *OH radical scavenging environment", k of 1.5 x 10(7) s(-1) by decreasing the concentration of TRIS buffer in relation to the concentration of added flavonoids. We have shown that the flavonoids can reduce the incidence of single-strand breaks in double-stranded DNA as well as residual base damage (assayed as additional single-strand breaks upon post-irradiation incubation with endonucleases) with dose modification factors of up to 2.0+/-0.2 at [F] < 100 microM by a mechanism other than through direct scavenging of *OH radicals. Pulse radiolysis measurements support the mechanism of electron transfer or H* atom transfer from the flavonoids to free radical sites on DNA which result in the fast chemical repair of some of the oxidative damage on DNA resulting from *OH radical attack. These in vitro assays point to a possible additional role for antioxidants in reducing DNA damage.     

Reduction in free-radical-induced DNA strand breaks and base damage through fast chemical repair by flavonoids

This paper provides evidence that dietary flavonoids can repair a range of oxidative radical damages on DNA, and thus give protection against radical-induced strand breaks and base alterations. We have irradiated dilute aqueous solutions of plasmid DNA in the absence and presence of flavonoids (F) in a “constant ^·OH radical scavenging environment”, k of 1.5 × 10⁷ s^-1 by decreasing the concentration of TRIS buffer in relation to the concentration of added flavonoids. We have shown that the flavonoids can reduce the incidence of single-strand breaks in double-stranded DNA as well as residual base damage (assayed as additional single-strand breaks upon post-irradiation incubation with endonucleases) with dose modification factors of up to 2.0 ± 0.2 at [F] < 100 μM by a mechanism other than through direct scavenging of ^·OH radicals. Pulse radiolysis measurements support the mechanism of electron transfer or H^· atom transfer from the flavonoids to free radical sites on DNA which result in the fast chemical repair of some of the oxidative damage on DNA resulting from ^·OH radical attack. These in vitro assays point to a possible additional role for antioxidants in reducing DNA damage.     

Yield of DNA strand breaks after base oxidation of plasmid DNA

We have irradiated aerobic aqueous solutions of plasmid DNA with 137Cs gamma rays in the presence of inorganic radical scavengers including nitrite, iodide, azide, thiocyanate and bromide. These scavengers react with the strongly oxidizing hydroxyl radical (*OH) to produce less powerful oxidants. Of these scavengers, only thiocyanate and bromide result in the formation of oxidizing species [(SCN)2*- and Br2*-, respectively] which are capable of reacting with the bases in DNA. The oxidized bases were detected after incubation of the irradiated plasmid with the two E. coli DNA base excision repair endonucleases, formamidopyrimidine-DNA N-glycosylase and endonuclease III. Depending on the experimental conditions, the intermediate base radicals may ultimately form stable oxidized bases in very high yields (within an order of magnitude of the *OH yield), and possibly also single-strand breaks (SSBs) in much lower yield (between 0.1 and 1% of the total yield of base damage). By competing for (SCN)2*- with an additional species (nitrite), it was possible to estimate the second-order rate constant for the reaction of (SCN)2*- with DNA as 1.6 x 10(4) dm3 mol(-1) s(-1), and also to demonstrate a correlation between the large yield of damaged bases and the much smaller increase in the yield of SSBs over background levels due to *OH. The efficiency of transfer of damage from oxidized base to sugar is estimated as about 0.5% or 5%, depending on whether purine or pyrimidine base radicals are responsible for the base to sugar damage transfer.     

A plant 3'-phosphoesterase involved in the repair of DNA strand breaks generated by oxidative damage

Two novel, structurally and functionally distinct phosphatases have been identified through the functional complementation, by maize cDNAs, of an Escherichia coli diphosphonucleoside phosphatase mutant strain. The first, ZmDP1, is a classical Mg(2+)-dependent and Li(+)-sensitive diphosphonucleoside phosphatase that dephosphorylates both 3'-phosphoadenosine 5'-phosphate (3'-PAP) and 2'-PAP without any discrimination between the 3'- and 2'-positions. The other, ZmDP2, is a distinct phosphatase that also catalyzes diphosphonucleoside dephosphorylation, but with a 12-fold lower Li(+) sensitivity, a strong preference for 3'-PAP, and the unique ability to utilize double-stranded DNA molecules with 3'-phosphate- or 3'-phosphoglycolate-blocking groups as substrates. Importantly, ZmDP2, but not ZmDP1, conferred resistance to a DNA repairdeficient E. coli strain against oxidative DNA-damaging agents generating 3'-phosphate- or 3'-phosphoglycolate-blocked single strand breaks. ZmDP2 shares a partial amino acid sequence similarity with a recently identified human polynucleotide kinase 3'-phosphatase that is thought to be involved in DNA repair, but is devoid of 5'-kinase activity. ZmDP2 is the first DNA 3'-phosphoesterase thus far identified in plants capable of converting 3'-blocked termini into priming sites for reparative DNA polymerization.     

Cell cycle effect on the induction of DNA double-strand breaks by X rays

Filter elution was used to compare X-ray-induced DNA single- and double-strand breaks in proliferating (P) and quiescent (Q) cells of the 66 and 67 mouse mammary tumor lines. There was no difference either between cell type or between growth states in the amount of single-strand breaks as defined by elution at pH 12.2. In contrast, Q cells appeared to sustain a much larger amount of double-strand break damage per Gray than P cells, when the damage was measured by elution at either pH 7.2 or pH 9.6. Experiments which combined centrifugal elutriation with pH 7.2 elution demonstrated that G1-P cells were similar to Q (greater than or equal to 95% G1) cells in the induction of elution-detectable double-strand breaks, while the S-phase enriched fractions sustained less damage than G1-P, Q, or asynchronous P populations. Studies in which P populations were pulse labeled with [14C]thymidine confirmed this finding. Mathematical analysis of the elution kinetics of irradiated P, Q, and S-phase cells supports a model in which the complex elution profiles observed for P cells could be explained as the sum of the one-component exponential elution profiles of G1- and S-phase subpopulations. Also, the correlation between damage measured by pH 7.2 elution and cell survival was tested by examining the dose response for stimulated 66 cells (St4), which like Q cells are greater than or equal to 95% in G1 but are more resistant to X-ray-induced cytotoxicity than are the 66 Q cells. However, the induction of double-strand breaks in St4 cells was identical to that in Q cells. Thus we conclude that there is not necessarily a correlation between the amount of elution-detectable X-ray-induced double-strand breaks and cell survival.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Abstract

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) after the incubation of 2′-deoxyguanosine (dGuo) with ChOOH and Cu²⁺. In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after the incubation of 2'-deoxyguanosine (dGuo) with ChOOH and Cu(2+). In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

Quantitative assessment of the contribution of clustered damage to DNA double-strand breaks induced by 60Co gamma rays and fission neutrons

The induction of DNA strand breaks by fission neutrons was studied in aqueous plasmid (pBR322) DNA under aerobic conditions for a wide range of hydroxyl radical (*OH) scavenger concentrations and was compared to the induction of strand breaks by 6OCo gamma rays. Strand breaks were measured using agarose gel electrophoresis coupled with sensitive 32P-based phosphor imaging. Yields are reported for DNA single-strand breaks (SSBs) and double-strand breaks formed linearly with dose (alphaDSBs). The fraction of alphaDSBs that were dependent on the multiply damaged site (MDS) or clustered damage mechanism was also calculated using a model. G values for SSBs and alphaDSBs declined with increasing *OH scavenging capacity. However, with increasing *OH scavenging capacities, the decrease in yields of strand breaks for fission neutrons was not as pronounced as for gamma rays. The percentage of alphaDSBs for gamma rays was dependent on *OH scavenging capacity, appearing negligible at low scavenging capacities but increasing at higher scavenging capacities. In contrast, fission neutrons induced high percentages of alphaDSBs that were approximately independent of *OH scavenging capacity. The levels of alphaDSBs formed by the MDS mechanism after exposure to fission neutrons are consistent with the expected distinctive features of high-LET energy deposition events and track structure. The results also confirm observations made by others that even for low-LET radiation, the MDS mechanism contributes significantly to DNA damage at cell-like scavenging conditions.     

The effect of temperature shock on the yield of gamma-induced single strand breaks in bacterial DNA

The existence of the super fast repairing system for single-strand breaks in DNA, induced in cells exposed to gamma-irradiation at 0°C in a phosphate buffer under aerobic conditions, has been shown in three strains of Escherichia coli K-12 (AB 1157 $\text{rec}{}^{\mathrm{+}}$, AB 2463 $\text{rec A}\text{13}$ and JC 4457 $\text{rec B}$). Super fast repairing system rejoins up to 90–95 % of initial single-strand breaks during irradiation. An estimation of the lower limit of the rate of repairing the breaks by this system has been made.     

The effect of superhelical density on the yield of single-strand breaks in gamma-irradiated plasmid DNA.

Using agarose gel electrophoresis, the formation of DNA single-strand breaks (SSBs) by 137Cs gamma irradiation was quantified in negatively supercoiled topological isomers of plasmid pUC18. The G value for SSB formation falls slightly from 1 x 10(8) to 8 x 10(-9) SSB Gy-1 Da-1 as the superhelical density varies from 0.00 to -0.08. This result is not in agreement with recent observations by others which suggest that increasing the negative superhelical density of plasmid DNA increases its sensitivity to X irradiation.     

Ferrous ion-induced strand breaks in the DNA plasmid pBR322 are not mediated by hydrogen peroxide

Ferrous ion-induced generation of single and multiple strand breaks in the DNA plasmid pBR322 induces the formation of two new plasmid forms with altered electrophoretic mobility. The yield of these plasmid forms, the circular relaxed and the linear forms, depended on the applied Fe²⁺ concentration. This property was independent of the presence of hydrogen peroxide in the incubation mixture indicating the lack of Fenton chemistry to explain the DNA degradation. The removal of dioxygen or the presence of superoxide dismutase diminished partially the yield of ferrous ion-induced DNA plasmid degradation, while catalase was without any effect. Autoxidation of divalent iron as followed by the formation of a coloured iron-phenanthroline complex was enhanced in a concentration-dependent manner by phosphate and bicarbonate and very efficiently using a mixture of 0.15 M NaCl, 1.2 mM phosphate, 23.8 mM bicarbonate, pH 7.4, that concentrations correspond closely to the intracellular values of buffer components. Thus, the formation of a yet unknown reactive species from Fe²⁺, and dioxygen, that is complexed to buffer components especially phosphate and its contribution in DNA plasmid degradation is more likely than the often cited formation of hydroxyl radicals in result of the Fenton reaction from Fe²⁺ and hydrogen peroxide. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Measurement of the initial levels of DNA damage in human lymphocytes induced by 29 kV X rays (mammography X rays) relative to 220 kV X rays and gamma rays

Experiments using the alkaline comet assay, which measures all single-strand breaks regardless of their origin, were performed to evaluate the biological effectiveness of photons with different energies in causing these breaks. The aim was to measure human lymphocytes directly for DNA damage and subsequent repair kinetics induced by mammography 29 kV X rays relative to 220 kV X rays, 137Cs gamma rays and 60Co gamma rays. The level of DNA damage, predominantly due to single-strand breaks, was computed as the Olive tail moment or percentage DNA in the tail for different air kerma doses (0.5, 0.75, 1, 1.5, 2 and 3 Gy). Fifty cells were analyzed per slide with a semiautomatic imaging system. Data from five independent experiments were transformed to natural logarithms and fitted using a multiple linear regression analysis. Irradiations with the different photon energies were performed simultaneously for each experiment to minimize interexperimental variation. Blood from only one male and one female was used. The interexperimental variation and the influence of donor gender were negligible. In addition, repair kinetics and residual DNA damage after exposure to a dose of 3 Gy were evaluated in three independent experiments for different repair times (10, 20, 30 and 60 min). Data for the fraction of remaining damage were fitted to the simple function F(d) = A/(t + A), where F(d) is the fraction of remaining damage, t is the time allowed for repair, and A (the only fit parameter) is the repair half-time. It was found that the comet assay data did not indicate any difference in the initial radiation damage produced by 29 kV X rays relative to the reference radiation types, 220 kV X rays and the gamma rays of 137Cs and 60Co, either for the total dose range or in the low-dose range. These results are, with some restrictions, consistent with physical examinations and predictions concerning, for example, the assessment of the possible difference in effectiveness in causing strand breaks between mammography X rays and conventional (150-250 kV) X rays, indicating that differences in biological effects must arise through downstream processing of the damage.     

On the nature and origin of DNA strand breaks in elongating spermatids

Transient DNA strand breaks are generated in the whole population of elongating spermatids and are perfectly coincident with histone H4 hyperacetylation at chromatin-remodeling steps. Given the limited DNA repair capacity of elongating spermatids, chromatin remodeling may present a threat to genetic integrity of the male gamete. The nature of the DNA strand breakage, the enzymes involved, and the role of H4 hyperacetylation in the process must be determined to further investigate the potential mutagenic consequences of this important transition. We used the metachromatic dye acridine orange in combination with fluorescence-activated cell sorting to achieve separation of spermatids according to their condensation state. Using single-cell electrophoresis (comet assay), in both alkaline and neutral conditions, we demonstrated that double-stranded breaks account for most of the DNA fragmentation observed in purified elongating spermatids. DNA strand breaks were generated in round spermatids as a result of de novo histone hyperacetylation induced by trichostatin A, whereas an increase in endogenous DNA strand breaks was observed in elongating spermatids. Using a short-term culture of testicular cells, we demonstrated that DNA strand breaks in spermatids were abolished on incubation with two functionally different topoisomerase II inhibitors. Hence, topoisomerase II appears as the unique enzyme responsible for the transient double-stranded breaks in elongating spermatids but depends on histone hyperacetylation for its activity.     

Processing of clustered DNA damage generates additional double-strand breaks in mammalian cells post-irradiation

Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cells. Mutant hamster cells (xrs-5), deficient in non-homologous end joining (NHEJ), were irradiated at 37 degrees C to determine whether any additional double-strand breaks (DSBs) are formed during processing of gamma-radiation-induced DNA clustered damage sites. A class of non-DSB clustered DNA damage, corresponding to approximately 30% of the initial yield of DSBs, is converted into DSBs reflecting an artefact of preparation of genomic DNA for pulsed field gel electrophoresis. These clusters are removed within 4 min in both NHEJ-deficient and wild-type CHO cells. In xrs-5 cells, a proportion of non-DSB clustered DNA damage, representing approximately 10% of the total yield of non-DSB clustered DNA damage sites, are also converted into DSBs within approximately 30 min post-gamma but not post-alpha irradiation through cellular processing at 37 degrees C. That the majority of radiation-induced non-DSB clustered DNA damage sites are resistant to conversion into DSBs may be biologically significant at environmental levels of radiation exposure, as a non-DSB clustered damage site rather than a DSB, which only constitutes a minor proportion, is more likely to be induced in irradiated cells.     

Measurement of oxidatively generated base damage in cellular DNA

This survey focuses on the critical evaluation of the main methods that are currently available for monitoring single and complex oxidatively generated damage to cellular DNA. Among chromatographic methods, HPLC-ESI-MS/MS and to a lesser extent HPLC-ECD which is restricted to a few electroactive nucleobases and nucleosides are appropriate for measuring the formation of single and clustered DNA lesions. Such methods that require optimized protocols for DNA extraction and digestion are sensitive enough for measuring base lesions formed under conditions of severe oxidative stress including exposure to ionizing radiation, UVA light and high intensity UVC laser pulses. In contrast application of GC-MS and HPLC-MS methods that are subject to major drawbacks have been shown to lead to overestimated values of DNA damage. Enzymatic methods that are based on the use of DNA repair glycosylases in order to convert oxidized bases into strand breaks are suitable, even if they are far less specific than HPLC methods, to deal with low levels of single modifications. Several other methods including immunoassays and (32)P-postlabeling methods that are still used suffer from drawbacks and therefore are not recommended. Another difficult topic is the measurement of oxidatively generated clustered DNA lesions that is currently achieved using enzymatic approaches and that would necessitate further investigations.     

Lethal and mutagenic lesions induced by ionizing radiations inE. coli and DNA strand breaks

Summary The inactivation doses forE. coli exposed to alpha particles and protons of different LETs and to gamma rays have been measured. Strains derived fromE. coli B/r showed a maximum sensitivity at LETs of 30 keV/ whilst B_s–1 and other strains known to be deficient in repair capacity had sensitivities which decreased monotonically with increasing LET. These results can be interpreted in terms of two types of lethal damage to the bacterial genome. Damage of type 1 affects only one strand of the DNA macromolecule and is partially reparablein vivo whilst damage of type 2, inflicted by one track intersection of the DNA, is irreparable. The identity of both types of damage is uncertain but type 1 probably gives rise to a lesion recognizablein vitro as a single strand break. Type 2 damage probably corresponds to double strand scission of DNA as observedin vitro.Mutations to prototrophy of three auxotrophic strains ofE. coli are induced with an effectiveness which decreases steadily with increasing LET. This form of LET dependence implies that these mutations involve damage to one target only, probably one strand of the DNA duplex.Paper read at the 6th Annual Meeting of the European Society for Radiobiology, Interlaken, 5.–8. June, 1968. Round Table: Radiation Effectsin vitro andin vivo. Correlations and Discrepancies.     

Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells

We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O6-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase beta were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.