Increased lymphocyte DNA strand breaks in rubber workers

Objective: To study the effect of occupational exposure to rubber processing, smoking, and alcohol drinking on lymphocyte DNA damage. Subjects and Methods: Of 371 employees (197 men and 174 women) from a rubber factory in Guangzhou, 281 were rubber processing workers from five production sections and 90 were managerial workers. Information on occupational exposure, smoking, and drinking was collected by interviews. Blood samples were taken in the morning by venipuncture. DNA damages were measured by the Comet assay. Possible DNA-protein crosslinks were broken down by proteinase K. Tail moment, measured by Komet 4.0 image analysis software, was the measure of DNA damage. Results: The rubber processing workers had larger tail moment than the managerial workers (Geometric mean, 95%CI) [1.77 μm (1.64–1.90) versus 1.52 μm (1.36–1.71), P=0.04]. Both smoking [1.93 μm (1.74–2.13) versus 1.59 μm (1.47–1.71), P=0.003] and alcohol drinking [2.21 μm (1.87–2.62) versus 1.63 μm (1.53–1.74), P<0.001] increased tail moment. Tail moment differed significantly among job categories (F=3.21, P=0.008), the largest was observed in mixers. In the non-smoking and non-drinking workers, rubber processing workers had larger tail moment than managerial workers after adjusting for age (P=0.033). General linear model analysis showed that after adjusting for each other, occupational exposure (P=0.027), smoking (P=0.012), and alcohol drinking (P=0.013) was associated with larger tail moment, whereas age and gender had no effect. Conclusions: Occupational exposure to rubber processing, smoking, and alcohol drinking can cause DNA damage.     

Genome-wide mapping of DNA strand breaks

Determination of cellular DNA damage has so far been limited to global assessment of genome integrity whereas nucleotide-level mapping has been restricted to specific loci by the use of specific primers. Therefore, only limited DNA sequences can be studied and novel regions of genomic instability can hardly be discovered. Using a well-characterized yeast model, we describe a straightforward strategy to map genome-wide DNA strand breaks without compromising nucleotide-level resolution. This technique, termed "damaged DNA immunoprecipitation" (dDIP), uses immunoprecipitation and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (TUNEL) to capture DNA at break sites. When used in combination with microarray or next-generation sequencing technologies, dDIP will allow researchers to map genome-wide DNA strand breaks as well as other types of DNA damage and to establish a clear profiling of altered genes and/or intergenic sequences in various experimental conditions. This mapping technique could find several applications for instance in the study of aging, genotoxic drug screening, cancer, meiosis, radiation and oxidative DNA damage.     

Coping with DNA double strand breaks

The repair of DNA double strand breaks (dsb) is important for maintaining the physical and genetic integrity of the genome. Moreover, in humans it is associated with the prevention of diseases such as immune deficiencies and cancer. This review briefly explores the fundamental strategies for repairing dsb, examines how cells maximize the fidelity of dsb repair in the cell cycle and discusses the requirements for dsb repair in the context of chromatin.     

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.     

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.     

Eumelanin causes DNA strand breaks and kills cells

Synthetic eumelanin prepared by autooxidation of D,L-DOPA causes DNA strand breaks, as determined by alkaline elution after cell lysis with detergent and proteolysis, in B16CL4 mouse melanoma cells. The melanin is toxic to the cells in the range of doses that causes strand breaks. When the melanin was incubated with the cells at 37 degrees C in tissue culture medium, it was maximally effective after 15 to 20 min at causing strand breaks in the DNA. The extent of damage is concentration dependent, but the effect plateaus at 1 mg/ml. The nature of the interaction of the cellular DNA with melanin is consistent with strand breaks, not DNA-DNA crosslinks. The strand break damage is repaired, even in the continued presence of melanin, but repair is more rapid if the cells are washed and the melanin is removed. The form of the melanin is important for obtaining the effect. Sonication for 3 min abrogates the effect to a considerable extent, and repeated cycles of sonication can completely destroy the activity. Lost activity returns slowly with storage at 4 degrees C. Melanin is more effective at damaging DNA in a protein-free medium. It is also DNA-damaging at 4 degrees C, but less so than at 37 degrees C. Preliminary studies indicate that the strand breaks caused by melanin are additive with those caused by ionizing radiation. The extent of DNA strand breaks and alkali-labile sites caused by several other melanins was also determined. Some melanins did not cause frank strand breaks, but were active in causing alkali-labile sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased frequency of oxidant-mediated DNA strand breaks in mononuclear leucocytes exposed to activated neutrophils from cigarette smokers

Following activation with the synthetic chemotactic tripeptide FMLP, potentiated by cytochalasin B (CB), blood neutrophils from cigarette smokers generated greater amounts of both extracellular and intracellular reactive oxidants than cells from non-smoking control subjects. FMLP/CB-activated neutrophils from cigarette smokers also inflicted increased oxidant-mediated damage to the DNA of cocultured mononuclear leucocytes, which was prevented by the inclusion of superoxide dismutase and catalase individually and in combination. These observations demonstrate that cigarette smoking primes phagocytes to generate increased amounts of potentially carcinogenic reactive oxidants.     

Joining of nonhomologous DNA double strand breaks in vitro.

Extracts of Xenopus laevis eggs can efficiently join ends of duplex DNA that differ in structure and sequence. This was analysed by recircularisation of linear plasmid DNA molecules with dissimilar termini, generated by successive cuts with two different restriction enzymes within the pSP65 polylinker. Use of various enzymes provided blunt ended or 4 nucleotides long 3' and 5' protruding single strand (PSS) termini which were successfully joined in vitro in any tested combination. Sequence analysis of numerous junctions from cloned reaction products of 7 terminus combinations reveal: apart from very rare base exchanges and single nucleotide insertions less than 10% deletions (1 to 18 nucleotides long) were detected. Blunt/PSS or 3'PSS/5'PSS terminus pairs undergo simple "blunt end" joining which preserves PSS ends by fill-in. In contrast, equally polar 3'PSS/3'PSS or 5'PSS/5'PSS terminus pairs are joined by a complex mode: PSS ends overlap by a defined number of nucleotides, set by matching basepairs. Even one basematch suffices to define the setting. This then determines the final mismatch repair and fill-in pattern. We propose that yet unknown terminal DNA-binding proteins stabilize the energetically highly unfavorable configuration of single matching basepairs and help to support defined overlap structures.     

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 nervous predisposition to unrepaired DNA double strand breaks

Ataxia-telangiectasia (A–T) has for a long time stood apart from most other human neurodegenerative syndromes by the characteristic failure of cells derived from these patients to properly repair DNA damage-induced by ionizing radiation. The discovery of mutations in the ATM gene as being the underlying cause for A–T and the demonstration that the ATM protein functions as a DNA damage-responsive kinase has defined current research focusing on decoding how the cell responds to genotoxic stress. Yet, despite significant advances in delineating the cellular DNA damage response pathways coordinated by ATM, very little headway has been made toward understanding how loss of ATM leads to progressive cerebellar ataxia and whether this can be attributed to an underlying defect in DNA double strand break repair (DSBR). Since its identification, A–T has been used as the archetypal model for how a deficiency in DNA repair affects both the development and maintenance of the nervous and immune systems in humans as well as contributing to the process of tumourigenesis. However, following the growing availability and cost effectiveness of next generation sequencing technologies, the increasing recognition of novel human disorders associated with abnormal DNA repair has demonstrated that the neuropathology typified by A–T is an ‘exception’ rather than the ‘rule’. As a consequence, this throws into doubt the longstanding hypothesis that the neurodegeneration seen in A–T is due to the progressive loss of damaged neurons that have acquired toxic levels of unrepaired DNA lesions over time. Therefore, this review aims to address the question: Is defective DNA double strand break repair an underlying cause of neurodegeneration?     

Hyperthermic potentiation of unrejoined DNA strand breaks following irradiation

Previous reports have suggested that the potentiation of cellular radiation sensitivity by hyperthermia may be due to its inhibition of the repair of single-strand breaks in DNA. Such inhibition could result in increased numbers of unrejoined breaks at long times following irradiation, lesions that are presumed to be lethal to the cell. As a test of this hypothesis, the amounts of residual strand-break damage in cells following combined hyperthermia and ionizing radiation were measured. The results show that hyperthermia does significantly enhance the relative number of unrejoined strand breaks as measured by the technique of alkaline elution and that the degree of enhancement is dependent on both the temperature and duration of the hyperthermia treatment. For example, compared to unheated cells, the proportion of unrejoined breaks measured 8 hr after irradiation was increased by a factor of 1.5 in cells that were treated for 30 min at 43 degrees C, by a factor of 6 for cells treated for 30 min at 45 degrees C, and by a factor of 4 for cells treated at 43 degrees C for 2 hr. In experiments in which the sequence of heat and irradiation were varied, a high degree of correlation was observed between the resulting level of cell killing and the relative numbers of unrejoined strand breaks. The greatest effects on both of these parameters were observed in those protocols in which the irradiation was delivered either during, just before, or just after the heat treatment.     

RecA protein-facilitated DNA strand breaks. A mechanism for bypassing DNA structural barriers during strand exchange

RecA protein promotes an unexpectedly efficient DNA strand exchange between circular single-stranded DNA and duplex DNAs containing short (50-400-base pair) heterologous sequences at the 5' (initiating) end. The major mechanism by which this topological barrier is bypassed involves DNA strand breakage. Breakage is both strand and position specific, occurring almost exclusively in the displaced (+) strand of the duplex within a 15-base pair region of the heterology/homology junction. Breakage also requires recA protein, ATP hydrolysis, and homologous sequences 3' to the heterology. Although the location of the breaks and the observed requirements clearly indicate a major role for recA protein in this phenomenon, the molecular mechanism is not yet clear. The breakage may reflect a DNA structure and/or some form of structural stress within the DNA during recA protein-mediated DNA pairing which either exposes the DNA at this precise position to the action of a contaminating nuclease or induces a direct mechanical break. We also find that when heterology is located at the 3' end of the linear duplex, strand exchange is halted (without DNA breakage) about 500 base pairs from the homology/heterology junction.     

Induction and repair of DNA strand breaks in Bacteroides fragilis

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair.     

Checkpoint mediators: relaying signals from DNA strand breaks

The recently identified checkpoint mediator MDC1 facilitates recruitment of DNA repair proteins to damaged sites and establishment of the intra-S-phase cell-cycle checkpoint. Increasing evidence suggests that proteins like MDC1 provide the framework necessary for transducing signals from DNA double-strand breaks.