Inhibition of poly(ADP-ribose) polymerase causes increased DNA strand breaks without decreasing strand rejoining in alkylated HeLa cells

Treatment of alkylated HeLa cells with 3-aminobenzamide, an inhibitor of poly(ADP-ribose) polymerase, increased the number of DNA strand breaks but did not affect the rate of strand rejoining. This suggests that an increase in DNA incision, not a decrease in ligation, results from the inhibition ofpoly(ADP-ribose) polymerase in cells recovering from DNA damaged by alkylating agents. Poly(ADP-ribose) DNA strand break DNA repair     

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.     

DNA strand breaks in mammalian tissues induced by methylarsenics

DNA damage induced by administration of dimethylarsinic acid (DMAA) to rats and mice was investigated. At 12 h after administration of DMAA, DNA single-strand breaks were induced markedly in lung. The majority of dimethylarsine, one of the main metabolites, in the expired air was excreted within 6–18 h after administration of DMAA to rats. In vitro experiments using nuclei isolated from lung of mice indicated that DNA strand breaks were caused by dimethylarsine. Furthermore, the strand breaks after exposure to dimethylarsine were reduced in the presence of catalase and/or superoxide dismutase. These results strongly suggest that the strand breaks are induced not by dimethylarsine itself but by active oxygen, e.g., O ₂ ^? and ·OH, produced both by dimethylarsine and molecular oxygen. When DNA was exposed to dimethylarsine, thiobarbituric acid (TBA)-reactive intermediates andcis-thymine glycol were produced. Dimethylarsine appears to induce DNA damage by the mechanism similar to the damage produced by ionizing radiation.     

Chromosomal aberrations induced by double strand DNA breaks

It has been suggested that introduction of double strand DNA breaks (DSBs) into mammalian chromosomes can lead to gross chromosomal rearrangements through improper DNA repair. To study this phenomenon, we employed a model system in which a double strand DNA break can be produced in human cells in vivo at a predetermined location. The ensuing chromosomal changes flanking the breakage site can then be cloned and characterized. In this system, the recognition site for the I-SceI endonuclease, whose 18 bp recognition sequence is not normally found in the human genome, is placed between a strong constitutive promoter and the Herpes simplex virus thymidine kinase (HSV-tk) gene, which serves as a negative selectable marker. We found that the most common mutation following aberrant DSB repair was an interstitial deletion; these deletions typically showed features of non-homologous end joining (NHEJ), such as microhomologies and insertions of direct or inverted repeat sequences. We also detected more complex rearrangements, including large insertions from adjacent or distant genomic regions. The insertion events that involved distant genomic regions typically represented transcribed sequences, and included both L1 LINE elements and sequences known to be involved in genomic rearrangements. This type of aberrant repair could potentially lead to gene inactivation via deletion of coding or regulatory sequences, or production of oncogenic fusion genes via insertion of coding sequences.     

DNA strand breaks induced by low-energy heavy ions

The DNA unwinding method was used to estimate DNA breakage in Chinese hamster cells exposed to heavy ions with LET in the range of 750-5000 keV/micron. Comparison of the primary induced unwinding rate per dose unit for ions with various track diameters but similar LET showed a pronounced influence on the track diameter. Low-energy ions, producing thin tracks with diameters (penumbra) in the submicrometer region, were almost two orders of magnitude less efficient than more energetic ions producing tracks with diameters of several micrometers and about three orders of magnitude less efficient than X-rays. For the thin tracks, clustering of breaks was indicated by comparison of the DNA unwinding rates in two different alkaline solutions. The results indicate that the unwinding rate cannot be used as a good measurement for DNA breaks in this case. The residual unwinding remaining after 4 h of repair at 37 degrees C correlated well with the ability of the various ions to produce cell-killing.     

Metal-mediated DNA damage induced by curcumin in the presence of human cytochrome P450 isozymes

Although curcumin is known to exhibit antitumor activity, carcinogenic properties have also been reported. To clarify the potentiality of carcinogenesis by curcumin, we have examined whether curcumin can induce DNA damage in the presence of cytochrome P450 (CYP) using [32P]-5(')-end-labeled DNA fragments obtained from genes relevant to human cancer. Curcumin treated with CYP 2D6, CYP1A1, or CYP1A2 induced DNA damage in the presence of Cu(II). CYP2D6-treated curcumin caused base damage, especially at 5(')-TG-3('), 5(')-GC-3('), and GG sequences. The DNA damage was inhibited by both catalase and bathocuproine, suggesting that reactive species derived from the reaction of H(2)O(2) with Cu(I) participate in DNA damage. Formation of 8-oxo-7,8-dihydro-2(')-deoxyguanosine was significantly increased by CYP2D6-treated curcumin in the presence of Cu(II). Time-of- flight mass spectrometry demonstrated that CYP2D6 catalyzed the conversion of curcumin to O-demethyl curcumin. Therefore, it is concluded that curcumin may exhibit carcinogenic potential through oxidative DNA damage by its metabolite.     

Protection of DNA from gamma-radiation induced strand breaks by Epicatechin

Epicatechin (EC), a polyphenolic antioxidant compound found in tea, apples and chocolate offered protection to DNA against ionizing radiation induced damages. Under in vitro conditions of radiation exposure, plasmid pBR322 DNA was protected by EC in a concentration dependent manner. The dose modifying factor for 0.2 mM EC for 50% protection of the plasmid DNA was found to be 6.0. EC when administered to mice 1 h prior to exposure to 4 Gy gamma-radiation protected cellular DNA against radiation-induced strand breaks in peripheral blood leukocytes, as revealed in alkaline comet assay studies. Thus, EC was found to protect DNA from gamma-radiation indiced strand breaks under in vitro as well as in vivo conditions of radiation exposure.     

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.     

Deciphering the chromatin landscape induced around DNA double strand breaks

DNA double strand breaks (DSBs) are among the most deleterious forms of lesions and deciphering the details of the chromatin landscape induced around DSBs represents a great challenge for molecular biologists.

Chromatin Immunoprecipitation, followed by microarray hybridisation (ChIP-chip) or high-throughput sequencing (ChIP-seq), are powerful techniques that provide high-resolution maps of protein-genome interactions. However, applying these techniques to study chromatin changes induced around DSBs was previously hindered due to a lack of suitable DSB induction techniques.

We have recently developed an experimental system utilizing a restriction enzyme fused to a modified oestrogen receptor ligand binding domain (AsiSI-ER), which generates multiple, sequence-specific and unambiguously positioned DSBs across the genome upon induction with 4-hydroxytamoxifen (4OHT) ¹. Cell lines expressing this construct represent a powerful tool to study specific chromatin changes during DSB repair, enabling high-resolution profiling of DNA repair complexes and chromatin modifications induced around DSBs. Using this system, we have recently produced the first map of gH2AX, a DSB-induced chromatin modification, on two human chromosomes and have investigated its spreading properties ¹. Here we provide additional data characterizing the cell lines, present a genome-wide profile of gH2AX obtained by ChIP-seq, and discuss the potential of our system towards investigations of previously uncharacterized aspects of DSB repair.     

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.     

Site-specific strand breaks in RNA produced by (125)I radiodecay

Decay of ¹²⁵I produces a shower of low energy electrons (Auger electrons) that cause strand breaks in DNA in a distance-dependent manner with 90% of the breaks located within 10 bp from the decay site. We studied strand breaks in RNA molecules produced by decay of ¹²⁵I incorporated into complementary DNA oligonucleotides forming RNA/DNA duplexes with the target RNA. The frequencies and distribution of the breaks were unaffected by the presence of the free radical scavenger dimethyl sulfoxide (DMSO) or by freezing of the samples. Therefore, as was the case with DNA, most of the breaks in RNA were direct rather than caused by diffusible free radicals produced in water. The distribution of break frequencies at individual bases in RNA molecules is narrower, with a maximum shifted to the 3′-end with respect to the distribution of breaks in DNA molecules of the same sequence. This correlates with the distances from the radioiodine to the sugars of the corresponding bases in A-form (RNA/DNA duplex) and B-form (DNA/DNA duplex) DNA. Interestingly, when ¹²⁵I was located close to the end of the antisense DNA oligonucleotide, we observed breaks in RNA beyond the RNA/DNA duplex region. This was not the case for a control DNA/DNA hybrid of the same sequence. We assume that for the RNA there is an interaction between the RNA/DNA duplex region and the single-stranded RNA tail, and we propose a model for such an interaction. This report demonstrates that ¹²⁵I radioprobing of RNA could be a powerful method to study both local conformation and global folding of RNA molecules.     

DNA strand breaks induced by the anti-topoisomerase II bis-dioxopiperazine ICRF-193

The bis-dioxopiperazine ICRF-193 has long time been considered as a pure topoisomerase II catalytic inhibitor able to exert its inhibitory effect on the enzyme without stabilization of the so-called cleavable complex formed by DNA covalently bound to topoisomerase II. In recent years, however, this concept has been challenged, as a number of reports have shown that ICRF-193 really "poisons" the enzyme, most likely through a different mechanism from that shown by the classical topoisomerase II poisons used in cancer chemotherapy. In the present investigation, we have carried out a study of the capacity of ICRF-193 to induce DNA strand breaks, as classical poisons do, in cultured V79 and irs-2 Chinese hamster lung fibroblasts using the comet assay and pulsed-field gel electrophoresis (PFGE). Our results clearly show that ICRF-193 readily induces breakage in DNA through a mechanism as yet poorly understood.     

Differences in the repair of DNA strand breaks induced by 9-hydroxy-benzo(a)pyrene and trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene in cultured human fibroblasts

Two benzo(a)pyrene metabolites were found to induce DNA strand breaks in cultured human fibroblasts. DNA strand breaks induced by the non- or weakly carcinogenic 9-hydroxy-benzo(a)pyrene were repaired within two hours, while those induced by the strongly carcinogenic trans-7,8-dihydro-7,8-dihydroxy-benzo(a)pyrene were repaired at a much slower rate.     

Three classes of DNA strand breaks induced by X-irradiation and internal beta-rays

Repair kinetics of DNA strand breaks were investigated after exposing exponentially growing CHO cells to X-radiation or to internal beta-rays from incorporated tritium, respectively. DNA strand breaks were analysed by the alkaline unwinding technique followed by chromatography on hydroxyapatite. For either type of radiation, the repair kinetics are statistically best described by a sum of three exponential components. The half-times determined are tau I approximately 2 min, tau II approximately 20 min and tau III approximately 170 min; they are identical for both types of radiation. But the initial fractions of the components are different for X- and internal beta-rays; X-rays; fI = 0.70, fII = 0.25, fIII = 0.05; internal beta-rays: fI = 0.40, fII = 0.40, fIII = 0.20. Components I and II are considered to represent the repair of two different classes of single-strand breaks and component III the repair of double-strand breaks. Two alternative interpretations for the occurrence of the two classes of single-strand breaks are discussed.     

Inhibition of repair of DNA single strand breaks in mouse leukemia cells by actinomycin D

The effects of Actinomycin D, cytosine arabinoside and temperature shifts on the repair of single strand breaks produced in murine leukemia cell DNA by ionizing radiation have been studied. A recently introduced modification of the alkaline sucrose sedimentation methods was used, allowing breaks to be demonstrated following clinical range irradiation doses. The results contrast to previous data using standard gradient procedures and indicate that low concentrations of Actinomycin D can inhibit single strand break repair, while cytosine arabinoside is ineffective. Inhibition can also be demonstrated by temperature shifts to 3° but not 24°, paralleling previous results from cellular repair studies (Elkind-Sutton repair). The results are consistent with the hypothesis that the accumulation of sublethal radiation damage in mammalian cells may be based on residual non-repaired single strand breaks.     

Induction of DNA double strand breaks by arsenite: comparative studies with DNA breaks induced by X-rays

Chinese hamster ovary (CHO-K1) cell line and two of its DNA double strand break (DSB) repair deficient mutant cell lines, xrs-5 (Ku80 mutant) and irs-20 (DNA-PKcs mutant), were treated with various concentrations of sodium arsenite for 2.5h, and the colony forming abilities were studied. The wild type cells showed the highest cell survival, while xrs-5 cells showed the lowest survival, and irs-20 cells had an intermediate survival. These results are very similar to the cell survival curves induced by X-rays in these three cell lines. Our data also show the dose dependent induction of DNA-DSBs in these cell lines exposed to arsenite. However, in order to obtain a similar cell survival in wild type cells, twice as many DNA-DSBs are necessary with arsenite exposure when compared with X-rays, suggesting that the types of DNA lesions leading to DSB induced by arsenite are different from those by X-rays. Based on these data, further mechanistic investigations including the involvement of DNA-DSB repair proteins are warranted in the recovery process from arsenic (As) exposure.     

Apoptosis of unstimulated human lymphocytes and DNA strand breaks induced by the topoisomerase II inhibitor etoposide (VP-16)

Etoposide (VP-16)-induced DNA strand breaks and repair and apoptosis of unstimulated human lymphocytes have been studied using DNA comet assay, electrophoresis of low-molecular-weight DNA extracts, and fluorescence microscopy. Incubation of unstimulated human lymphocytes with VP-16 (50-200 microg/ml) for 3 or 24 h induced apoptosis. This conclusion is supported by results of morphological studies, evaluation of the proportion of hypodiploidy and internucleosomal degradation of DNA in lymphocytes. Etoposide-induced formation of DNA strand breaks preceded the appearance of these conventional apoptotic manifestations. The number of single-strand breaks depended on VP-16 concentration, and 2-3 h after its removal from the incubation medium they were repaired. The hydroxyl group at the C-4; position of the etoposide dimethoxyphenol ring may be responsible for the formation of single-strand breaks. Double-strand breaks were unrepaired 20 h after the change of the incubation medium. The number of double-strand breaks and a proportion of apoptotic cells did not exhibit any dependence on VP-16 concentration and/or duration of cell exposure to this agent. We suggest that the cytotoxic effect of VP-16 on unstimulated lymphocytes is mediated by a topoisomerase II isoform, topoisomerase II-beta, which is localized in the nucleolus and is not related to the cell cycle.     

Interspecies differences in DNA single strand breaks caused by benzo(a)pyrene and marine environment

The presence of DNA single strand breaks in untreated specimens of selected species, mosquito fish Gambusia affinis, painted comber Serranus scriba, blue mussel Mytilus galloprovincialis, spiny crab Maja crispata and sea cucumber Holothuria tubulosa as well as in 10 microg/g benzo(a)pyrene (BaP) treated mosquito fish, blue mussel and spiny crab was measured, using alkaline filter elution. Interspecies differences in alkaline elution profiles were observed and attributed to different lengths of DNA from different sources and to differences in the number of strand breaks present during normal cellular events in different phyla. Spiny crab hemocytes are more sensitive to action of BaP then blue mussel hemocytes and mosquito fish hepatocytes that could be explained by differences in the rates of distinct metabolic reactions and DNA repair among the investigated species. In field study, DNA single strand breaks were measured in hepatocytes of painted comber and in hemocytes of blue mussel and spiny crab from natural population specimens collected at eight sampling sites along Istrian coast, Croatia. Spatial variations in DNA integrity for each species were detected and revealed for the first time that spiny crab is responsive to different environmental conditions. Interspecies variations in the DNA integrity due to environmental conditions, confirmed species specific susceptibility to genotoxicity of certain environment that in long-term may modify the structure of marine communities. The multi-species approach in designing biomonitoring studies was suggested.