Radiation-induced DNA base damage detected in individual aerobic and hypoxic cells with endonuclease III and formamidopyrimidine-glycosylase.

X-ray-induced DNA base damage can be detected using endonuclease III and formamidopyrimidine-glycosylase, which create DNA strand breaks at enzyme-sensitive sites. Strand breaks can then be measured with excellent sensitivity using the alkaline comet assay, a single-cell gel electrophoresis method that detects DNA damage in individual cells. In using this approach to measure the oxygen enhancement ratio (OER) for radiation-induced base damage, we observed that the number of enzyme-sensitive sites increased with dose up to 4 Gy in air and 12 Gy in hypoxic WIL2NS cells. After rejoining of radiation-induced strand breaks, base damage was detected more easily after higher doses. The number of radiation-induced enzyme-sensitive sites was similar under both air and nitrogen. Base damage produced by hydrogen peroxide and 4-nitroquinoline-N-oxide (4NQO) was also measured. Results with hydrogen peroxide (20 min at 4 degrees C) were similar to those observed for X rays, indicating that enzyme-sensitive sites could be detected most efficiently when few direct strand breaks were present. Removing DNA-associated proteins before irradiation did not affect the ability to detect base damage. Base damage produced by 4NQO (30 min at 37 degrees C) was readily apparent after treatment with low concentrations of the drug when few 4NQO-induced strand breaks were present, but the detection sensitivity decreased rapidly as direct strand breaks increased after treatment with higher concentrations. We conclude that: (1) the OER for base damage is approximately 1.0, and (2) the presence of direct DNA strand breaks (>2000-4000 per cell) prevents accurate detection of base damage measured as enzyme-sensitive sites with the alkaline comet method.     

Induction of DNA damage by dimethylarsine, a metabolite of inorganic arsenics, is for the major part likely due to its peroxyl radical

To reveal the mechanisms of previously reported lung-specific DNA strand scissions in murine after oral administration of dimethylarsinic acid (DMAA), a main metabolite of inorganic arsenics in mammals, the ultimate substance causing DNA lesion was investigated using dimethylarsine which was a further metabolite of DMAA. The alkaline elution assay using 3H-labeled DNA showed that a major portion of the strand breaks was not suppressed by SOD and catalase, suggesting an ultimate substance other than active oxygen participated in the DNA damage. By ESR analysis, a radical estimated to be (CH3)2AsOO. was detected as a reaction product of dimethylarsine and molecular oxygen. This peroxyl radical, rather than active oxygen, was assumed to play a major role in DNA damage.     

Photocleavage of DNA and photofootprinting of E. coli RNA polymerase bound to promoter DNA by azido-9-acridinylamines.

The long-wavelength ultraviolet (lambda approximately 420 nm) radiation induced reaction between 6-azido-2-methoxy-9-acridinylamines and supercoiled plasmid DNA results in single strand scissions and formation of covalent adducts (ratio approximately 1:10). By treating azidoacridine-photomodified DNA with piperidine at 90 degrees C, additional strand scissions are observed in a complex sequence dependent manner with an overall preference for T greater than or equal to G greater than C much greater than A. The resulting DNA fragments migrate as 5'-phosphates in polyacrylamide gels. Photofootprinting of the binding site of RNA-polymerase on promoter DNA is demonstrated with an azido-9-acridinylamino-octamethylene-9-aminoacridine. Similar experiments using 9-amino-6-azido-2-methoxyacridine indicate that this reagent recognizes changes in the DNA conformation induced by RNA polymerase binding, in relation to open complex formation.     

Alkaline elution of rat testicular DNA: detection of DNA cross-links after in vivo treatment with chemical mutagens

The alkaline elution technique was used to measure DNA damage in the rat testis after intraperitoneal injection of 3 chemicals known to cause heritable mutations in rodents. These 3 chemicals are triethylenemelamine (TEM), mitomycin C, and cyclophosphamide. All three of these chemicals produced DNA damage which was readily detectable by alkaline elution. Both TEM and mitomycin C produced DNA interstrand cross-links, although TEM was a more potent cross-linker on an equimolar basis than mitomycin C. Cyclophosphamide produced both DNA cross-links and DNA strand breaks. Alkaline elution in the absence of proteinase K indicated that some of the strand breaks appeared to be closely associated with protein. These studied indicate that the alkaline elution technique is capable of detecting DNA damage in mammalian germ cells produced by chemical mutagens. This technique may prove useful as a screening tool for identifying chemicals which cause heritable mutations in mammals.     

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)     

Elaboration of cellular DNA breaks by hydroperoxides.

Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.     

Characterization of intracellular DNA strand breaks induced by neocarzinostatin in Escherichia coli cells.

DNA strand breaks induced by Neocarzinostatin in Escherichia coli cells have been characterized. Radioactively labeled phage lambda DNA was introduced into lysogenic host bacteria allowing the phage DNA to circularize into superhelical molecules. After drug treatment DNA single- and double-strand breaks were measured independently after neutral sucrose gradient sedimentation. The presence of alkali-labile lesions was measured in parallel in alkaline sucrose gradients. The cell envelope provided an efficient protection towards the drug, since no strand breaks were detected unless the cells were made permeable with toluene or with hypotonic Tris buffer. In permeable cells, no double strand breaks could be detected, even at high NCS concentration (100 micrograms/ml). Induction of single-strand breaks leveled off after 15 min at 20 degrees C in the presence of 2 mM mercaptoethanol. Exposure to 0.3N NaOH doubled the number of strand breaks. No enzymatic repair of the breaks could be observed.     

Single-strand nicking of DNA in vitro by neocarzinostatin and its possible relationship to the mechanism of drug action.

Neocarzinostatin, a protein antibiotic with anti-tumor activity was found to place single-strand scissions in DNA in an in vitro reaction. The drug's cutting activity was strongly dependent on the presence of 2-mercaptoethanol or dithiothreitol but some cutting did take place in the absence of reducing agent at very high drug levels and prolonged incubation. The requirement for reducing agents could not be replaced with NAD+, FAD, NADH or H2O2 and the strand-scission reaction was not affected by Mg2+, EDTA or intercalating agents. Similar profiles of heat-inactivation of neocarzinostatin were found whether activity was measured by the scission of DNA strand either in vitro or in HeLa cells treated with the drug. Furthermore, both of these parameters corresponded closely with the ability of the modified drug to inhibit DNA synthesis and growth of HeLa cells. By column isoelectric focusing it was shown that all four activities are associated with the same protein band (pH 3.28). From these data we conclude that the cytotoxic activity of neocarzinostatin and the nicking of DNA strands in vitro appear to reside in the same protein.     

Degradation of HeLa S3 cell chromatin by auromomycin and its chromophore

The antitumor protein agent auromomycin was found to degrade chromatin structure primarily by inducing strand scissions in linker regions. The reaction was stimulated by dithiothreitol. The chromophore form of the drug caused similar effects on chromatin, but it appeared to function at a more rapid rate. There was no evidence that auromomycin could cause breakage in core regions of chromatin.     

Assessment of radiation-induced DNA strand breaks and their repair in unlabeled cells.

Radiation induced damage, i.e., the induction of DNA strand breaks, was studied on the level of single, unlabeled cells. DNA strand breaks were determined by direct partial alkaline unwinding in intact cell nuclei followed by staining with acridine orange, a development of a proposal first described by B. Rydberg (Int J Radiat Biol 46:521-527, 1984). The ratio of green fluorescence (double-stranded DNA) to red fluorescence (single-stranded DNA) in single cells was taken as a measure of DNA strand breaks. CHO-K1 and M3-1 cells irradiated with X-rays show a dose dependent induction of DNA strand breaks. Incubation at 37 degrees C after irradiation leads to repair of breaks. A repair halflife of about 10-11 min can be determined. Cell cycle specific differences in the induction of DNA strand breaks or repair behavior are not detectable at the resolution achieved so far. This new method offers two major advantages: the resolution of DNA damage and repair on the level of single cells and no need for labeling, thereby allowing for DNA damage and repair to be assessed in biopsy material from tumor patients.     

Effect of dose rate on cell killing and DNA strand break repair in CHO cells exposed to internal beta-rays from incorporated [3H]thymidine

Survival as well as repair of DNA strand breaks were studied in CHO cells after exposure to internal beta-rays from incorporated [3H]thymidine at 4 degrees C (equivalent to an exposure at 'infinitely high' dose rate) and at 37 degrees C (low dose rate). DNA strand breaks were determined by the alkaline unwinding technique. In cells exposed at 4 degrees C cell killing was five times higher (Do = 250 decays per cell) than in cells exposed at 37 degrees C (Do = 1280 decays per cell). Strand breaks induced by 3H decay at 37 degrees C were repaired with the same kinetics as those generated at 4 degrees C. Therefore the different degrees of cell killing at 4 degrees C and 37 degrees C cannot be attributed to a difference in the repair kinetics for DNA strand breaks.     

The induction and repair of DNA damage detected by the DNA precipitation assay in Chinese hamster ovary cells treated with acridine orange + visible light

The photodynamic effect of the dye acridine orange (AO) in combination with visible light (400-700 nm) was studied in Chinese hamster ovary (CHO) cells, the endpoints investigated being induction, as well as repair, of DNA strand breaks. Cells were treated for 20 min with AO (0.1-3.0 micrograms/ml), washed free of excess dye and subsequently exposed to low doses of visible light (2 x 40 W/8 W/m2) for 5-15 min. AO proved to be an efficient sensitizer for light-induced DNA strand breaks, detected with the DNA precipitation assay, and expressed as percentage of DNA precipitated. The induction of breaks was linear up to 0.5 micrograms/ml AO + 10 min of light, which corresponds to 55% precipitated DNA, and was dependent on the concentration of AO as well as on the dose of light delivered. As a comparison, 18 Gy of X-rays was required to yield an equivalent amount of induced DNA strand breaks. The rejoining of the light-induced DNA strand breaks was studied by incubating the AO-sensitized cells for 30-120 min at 37 degrees C directly after light exposure. A fast recover of 67-91% of the damage (compared to initial damage, recovery time = 0, and dependent on the concentration of AO) was observed during the first 30 min of incubation. However, a significant amount of DNA damage remained after 2 h of recovery. These remaining, long-lived lesions might be involved in the photoinduced and acridine-sensitized chromosomal aberrations and sister-chromatid exchanges (SCE). The significance of these observations is discussed in relation to AO-sensitized and photoinduced DNA damage and chromosomal alterations.     

Bleomycin-induced DNA-strand damage in isolated male germ cells

DNA strand damage in isolated male germ cells (MGC) was evaluated after in vitro exposure to bleomycin (BLM), a known genotoxin. The alkaline elution technique was used to determine DNA-strand breaks. Concentration-dependent strand damage was established following exposure to bleomycin for 1 h at 37 degrees C. Exposure at 0 degrees C resulted in an increase in the frequency of strand breaks as compared to those observed at 37 degrees C. Pretreatment of cells with deferoxamine (DM), an iron-selective chelating agent, abolished the DNA damage induced by bleomycin. Isolated male germ cells responded in a predictable and reproducible manner thus supporting their use in mechanistic studies of genotoxicity.     

Processive action of T4 endonuclease V on ultraviolet-irradiated DNA.

The action of the dimer-specific endonuclease V of bacteriophage T4 was studied on UV-irradiated, covalently-closed circular DNa. Form I ColE1 DNA preparations containing average dimer frequencies ranging from 2.5 to 35 pyrimidine dimers per molecule were treated with T4 endonuclease V and analysed by agarose gel electrophoresis. At all dimer frequencies examined, the production of form III DNA was linear with time and the double-strand scissions were made randomly on the ColE1 DNA genome. Since the observed fraction of form III DNA increased with increasing dimer frequency but the initial rate of loss of form I decreased with increasing dimer frequency, it was postulated that multiple single-strand scissions could be produced in a subset of the DNA population while some DNA molecules contained no scissions. When DNA containing an average of 25 dimers per circle was incubated with limiting enzyme concentrations, scissions appeared at most if not all dimmer sites in some molecules before additional strand scissions were produced in other DNA molecules. The results support a processive model for the interaction of T4 endonuclease V with UV-irradiated DNA.     

Role of active oxygen species in metal-induced DNA strand breakage in human diploid fibroblasts

The ability of 6 metal salts to induce DNA damage in human diploid fibroblasts was examined. Cadmium, magnesium, manganese, chromium(VI), zinc and selenite were all shown to induce DNA strand breaks as measured by two independent assays. DNA strand breaks were repaired within 2-4 h after removal of metal and this repair appeared not to be sensitive to "long-patch" repair inhibitors. With the exception of selenite, metal-induced DNA damage appeared to be mediated via the formation of active oxygen species since oxygen scavengers when administered simultaneously with the metal, antagonized strand break formation. Selenite-induced DNA damage (as previously reported) was dependent on the formation of a selenite-glutathione conjugant and was not affected by oxygen radical scavengers. Scavenger treatment did not enhance cloning ability of metal-treated cells suggesting that DNA strand breaks may not be important in metal-induced cytotoxicity.     

Catalytic properties of the HhaII restriction endonuclease

The catalytic properties of the HhaII restriction endonuclease were studied using plasmid pSK11 DNA containing a single 5'-G-A-N-T-C HhaII cleavage site as substrate. Reactions were followed by two methods: 1) gel electrophoretic analysis of nicked circular and linear DNA products, or 2) release of 32P-labeled inorganic phosphate from specifically labeled HhaII sites in a reaction coupled with bacterial alkaline phosphatase. The enzyme is optimally active at 37 degrees C in 10 mM Tris-HCl (pH 9.1) and 4-10 mM MgCl2 without added NaCl. Activity is stabilized by the presence of 2-mercaptoethanol and 0.2% Triton X-100 or 50 microgram/ml bovine serum albumin. At enzyme concentrations below 10 nM and using pSK11 as substrate, initial kinetic rates were dependent on the order of mixing of reactants. A lag of 3-4 min was observed if enzyme or substrate was added last. Preincubation of substrate and enzyme followed by initiation of the reaction with MgCl2 or preincubation of the enzyme with nonspecific DNA followed by initiation with substrate eliminated or reduced the lag, respectively, and speeded up the reactions. Under a wide range of reaction conditions, nicked pSK11 DNA accumulated early, while linear molecules appeared later, suggesting that HhaII cleaves one strand at a time in separate binding events. The apparent Km for covalently closed pSK11 DNA molecules was approximately 17 nM, and the turnover number for the conversion of covalent to nicked sites was 1.1 single strand scissions/min. Pre-steady state kinetic analysis indicated that cleavage of the first phosphodiester bond in a site is first order with a rate constant of about 0.8 min-1, while cleavage of the second phosphodiester bond is first order with a rate constant of about 0.2 min-1.     

Bleomycin fragmentation of duplex DNA occurs as staggered single-strand scissions.

Electron microscopy of purified full-length linear duplex molecules produced by bleomycin reaction with PM2 DNA revealed low frequencies of closed circular duplex molecules as well as linear duplex molecules with opposed ends (cyclized molecules which have dissociated to yield a gap between the termini). The occurrence of these latter forms indicates that double-strand scissions produced by bleomycin reaction consist of two single-strand scissions which are physically staggered on the complementary strands. Analysis of the temperature dependence for cyclization led to the estimate that an average of 1.7 +/- 0.44 base-pairs (2.6 +/- 0.5 base pairs without base-stacking energies) occur between the staggered breaks. The reassociated termini cannot be ligated with T4 ligase. When PM2 DNA was fragmented at several sites within each molecule, circular duplexes and linear duplexes with opposed ends with a range of sizes from 350 base pairs up to full-length PM2 DNA were observed. Analysis of the frequency distribution of lengths of these fragments indicates that most, if not all, of the specific sites for bleomycin-directed double-strand scissions in PM2 DNA contain representatives of the same two base single-stranded termini.     

Formation of endonuclease III-sensitive sites as a consequence of oxygen radical attack on DNA

Exposure of the plasmid pBR 322 to the aerobic xanthine oxidase reaction introduced single strand scissions and endonuclease III-sensitive sites. The latter may be residues of thymine glycol. Both forms of DNA damage were completely prevented by superoxide dismutase or catalase, whereas bovine serum albumin was much less effective. Mannitol and benzoate, added as scavengers of HO., and desferrioxamine or diethylene triamine pentaacetate, added to sequester Fe(III), also protected. These results indicate a metal-catalyzed interaction of O2- with H2O2, which produces HO. which, in turn, causes DNA strand scission and oxidation of thymine residues to thymine glycol. Plasmid isolated from aerobically-incubated cells contained more strand scissions and endonuclease III-sensitive sites than did plasmid from anaerobically-incubated cells, and a low molecular weight scavenger of O2- prevented the damage seen with the aerobic cells. Genetic defects in AP endonucleases rendered E. coli more susceptible to the dioxygen-dependent lethality of plumbagin, which mediates O2- production. Similarly, plasmid DNA, within the endonuclease-deficient cells, exhibited more strand scissions and endonuclease III-sensitive sites upon aerobic exposure to plumbagin than did endonuclease-sufficient cells, and a low molecular weight scavenger of O2- was protective. These results are consistent with the conclusions that strand scissions and formation of endonuclease III-sensitive sites are among the consequences of exposure of DNA to O2- plus H2O2, both in vitro and in vivo.     

Characterization of DNA strand breakage in vitro by the antitumor protein neocarzinostatin.

The antitumor protein antibiotic neocarzinostatin causes strand scission of DNA in vitro in the presence of a sulfhydryl compound. The breaks are single stranded in nature and bear 5'-phosphoryl termini. All four deoxymononucleotides are recoverable at the 5'-ends of the cleavage sites although a higher proportion of dGMP and TMP are consistently found. The lesions are not repairable with polynucleotide ligase from Escherichia coli. A quantitative assay was developed to determine the pH profile and time course of the reaction. Data from protection experiments with synthetic and natural DNAs indicate the requirement for thymidylic acid and deoxyadenylic acid in the DNA for cutting. In DNA-RNA hybrids, riboadenylic acid can substitute for deoxyadenylic acid, whereas ribouridylic acid cannot substitute for thymidylic acid. Release of thymine is detected, and the amount of release correlates well with the number of strand scissions.     

Quantitative measurement of DNA strand breaks and repair in gamma-irradiated human leukocytes from normal and ataxia telangiectasia donors

Fluorimetric analysis of DNA unwinding, which allows measurement of DNA strand breaks in human leukocytes, has been optimized by reducing the amount of cells required for the test and by modifying the DNA alkali unwinding conditions. This permitted measurement of DNA strand-break induction in cells irradiated with low (0.5-7 Gy) or high doses (5-20 Gy) of gamma rays. Linear dose-response curves were obtained for both dose ranges. Presence of cysteamine during irradiation caused a decrease in the extent of DNA strand breaks. The kinetics of the DNA strand-break rejoining process appeared to be biphasic over the dose range of 2-20 Gy when plotted on a linear vs linear axis (percentage of damage as a function of time). Since the rate of disappearance of damaged DNA was similar for any given dose and for all postirradiation incubation times tested, we have expressed the extent of repair after a given postirradiation incubation as the ratio of the slopes of the regression lines obtained from incubated and nonincubated cells. Leukocytes from 25 healthy donors were analyzed to determine an average value for controls. No difference in the level of DNA strand breaks and the rate of repair of these breaks was observed between leukocytes from three ataxia telangiectasia patients and those from normal donors.