Effect of 3-aminobenzamide on DNA strand-break rejoining and cytotoxicity in CHO cells treated with hydrogen peroxide

The influence of the nuclear ADP-ribosyltransferase inhibitor 3-aminobenzamide on the DNA strand-break rejoining kinetics and cytotoxicity in Chinese hamster ovary cells following H2O2 treatment was investigated. For the DNA damage studies, cells were treated on ice with H2O2 (0-20 microM) for 1 h in serum-free medium, after which the H2O2 was removed and the cells were allowed to repair their damage in complete medium at 37 degrees C in the presence or absence of 3-aminobenzamide (5 mM) for periods up to 2 h. The DNA strand breaks remaining as a function of time were then estimated by alkaline elution. A linear relationship between the H2O2 concentration and the initial level of DNA single-strand breaks (zero time allowed for repair) was observed. No double-strand breaks or DNA-protein cross-links were detected at these doses. The rejoining of single-strand breaks after H2O2 (20 microM) alone was characterized by a single exponential process with a t1/2 of approx. 5 min. However, in the presence of 3-aminobenzamide, rejoining was much slower and biphasic, with t1/2 of approx. 10 and 36 min. The inhibitory action of 3-aminobenzamide was concentration-dependent and completely reversible in that, when the 3-aminobenzamide was removed from the treated cultures, the strand-break rejoining kinetics rapidly returned to the t1/2 of 5 min typical of H2O2 alone. Considerably higher concentrations of H2O2 (up to 600 microM) were required for cell killing compared to the DNA damage studies. Cell killing by H2O2 alone was characterized by a shoulderless, exponential survival curve (D0 = 880 microM). The cytotoxicity was potentiated when the cells were treated with 3-aminobenzamide (5 mM) for 1 h after the H2O2 treatment; the survival curve with 3-aminobenzamide also assumed a biphasic character (D0 of 212 microM and 520 microM). These results are consistent with the theory that OH.-induced single-strand breaks do not normally represent lethal lesions to the cell because of their rapid, efficient repair. However, interference with these repair processes (in this case by 3-aminobenzamide) can alter this relationship, possibly allowing lesion fixation.     

Pretreatment with oxygen species increases the resistance of mammalian cells to hydrogen peroxide and gamma-rays

Pretreatment of Chinese hamster ovary (CHO) or H4 (rat hepatoma) cells with low non-toxic doses of H2O2 or xanthine-xanthine oxidase renders the cells more resistant to the toxic effect of H2O2 and gamma-rays. This increased resistance is observed both in exponentially growing and in plateau-phase cells. Cells pretreated with xanthine-xanthine oxidase are less mutated than control cultures when challenged with ionizing radiation. The number of DNA single-strand breaks (measured by nucleoid sedimentation) induced by a high dose of gamma-rays or H2O2 is lower in cells pretreated with xanthine-xanthine oxidase compared to control cultures. However, the pretreatment does not modify the rate of DNA single-strand breaks rejoining in cells challenged with H2O2 or gamma-rays. The catalase activity is not modified in pretreated cells, but the superoxide dismutase activity is increased about 2-fold.     

Normal response of Fanconi's anemia cells to high concentrations of O2 as determined by alkaline elution

In this investigation, normal and Fanconi's anemia fibroblasts were exposed to high concentrations of oxygen and the effects of this treatment on DNA were analyzed by alkaline elution. No DNA single-strand breaks were detected in either cell type with up to 20 h incubation in high (50–95%) concentrations of O₂. No evidence of DNA damage by O₂ could be detected with an endonuclease preparation from Micrococcus luteus. Cells which have been treated with various DNA-damaging agents in the presence of the polymerase inhibitor cytosine arabinoside have been shown to accumulate DNA single-strand breaks during DNA excision repair. When cells were treated with the polymerase inhibitor in 50 or 95% O₂, a low level of DNA single-strand breaks accumulated in both cell types. However, no significant differences in the frequency of DNA single-strand breaks were detected between normal and Fanconi's anemia cells after exposure to high O₂.     

DNA damage and repair in embryonic mouse limbs exposed to teratogenic doses of methylnitrosourea

The alkaline elution assay was used to monitor DNA single-strand breaks in embryonic tissue following exposure to the DNA-damaging teratogen N-methyl-N-nitrosourea (MNU, CAS No. 694-93-5). An animal model was developed in which nearly every fetus exposed to the highest dose of MNU had malformations of the hindlimbs while the fetuses exposed to the lowest dose of MNU had none. Hindlimbs pooled within litters were analyzed for DNA single-strand breaks by alkaline elution conducted at rapid (0.35 ml/min) and slow (0.35 ml/min) speeds. Breaks in the DNA of hindlimbs exposed to teratogenic doses of MNU were readily detected by alkaline elution only if slower speeds were used in the assay. Using the more sensitive procedure, DNA breakage was monitored over a 24-h period. DNA breakage peaked in the MNU-exposed hindlimbs in a dose-dependent manner 4 h after injection. While the elution profiles of hindlimbs exposed to the lower doses of MNU returned to control levels 8 h after injection, single-strand breaks persisted in the hindlimbs exposed to the highest dose of MNU for at least 20 h. These latter data suggest that the highly teratogenic dose of MNU induced DNA damage that was more slowly repaired than that produced at lower doses, possibly by saturation of DNA repair systems. Although some necrosis did occur in hindlimbs exposed at teratogenic dose levels, it was not severe and it did not appear to influence the alkaline elution results. These experiments show that alkaline elution is a sensitive assay for the detection of DNA damage in embryonic tissues.     

Growth inhibition and DNA damage induced by benzo[a]pyrene and formaldehyde in primary cultures of rat tracheal epithelial cells

The effects of benzo[a]pyrene (BAP) and formaldehyde (HCHO), alone and combined, on cell growth and DNA damage were determined in primary cultures of rat tracheal epithelial cells dissociated from rat tracheas. Cell cultures treated with 25 microM BAP for 24 h or 200 microM HCHO for 90 min did not have a marked reduction in cell growth. However, their combined treatment reduced cell growth by 60% of control when cultures were exposed to BAP followed by HCHO as well as the reverse order. None of these treatments significantly decreased cell viability as judged by dye exclusion, nor did they enhance cell terminal differentiation as measured by cornified envelope formation. Alkaline elution analysis of DNA damage detected both DNA-protein crosslinks (DPC) and DNA single-strand breaks (SSB) as a result of HCHO treatment, whereas BAP treatment caused only SSB. While HCHO-induced SSB were repaired within 2 h, BAP-induced SSB were detected 3 days after treatment. Combined treatment of cell cultures with BAP followed by HCHO resulted in more SSB than was obtained from either agent alone, but less DPC than was detected from HCHO alone. The increased number of SSB obtained from this combined treatment may be related to the marked enhancement of carcinogenesis observed in earlier in vivo-in vitro studies.     

Hypochlorous acid potentiates hydrogen peroxide-mediated DNA-strand breaks in human mononuclear leucocytes.

In this study the formation of DNA single-strand breaks in MNL in close proximity to activated phagocytes, or in contact with added H2O2 and/or HOCl, were evaluated. Neutrophils activated by phorbol myristate acetate (PMA), induced DNA-strand breaks in neighboring lymphocytes which increased after 1-2 h incubation in a repair medium. These DNA-strand breaks could be prevented by the addition of catalase or substitution of the neutrophils with cells from a patient with chronic granulomatous disease. Inclusion of the myeloperoxidase (MPO) inhibitor, sodium azide (NaN3), to the system was associated with less damage after 1-2 h incubation and a faster repair rate. Exposure of MNL to added reagent H2O2 (12-100 microM) was also accompanied by DNA damage. Addition of reagent HOCl (3-25 microM) did not induce any DNA-strand breaks. However, when combined with H2O2 (12.5 microM), HOCl increased H2O2-mediated DNA damage and compromised the repair process. Interactions between the phagocyte-derived reactive oxidants H2O2 and HOCl are probably involved in the etiology of inflammation-related cancer.     

DNA damage induced by nitropyrenes in primary mouse hepatocytes and in rat H4-II-E hepatoma cells

The capacity of nitropyrenes to cause DNA damage in primary mouse hepatocytes (C57BL/6N mice) and rat H4-II-E hepatoma cells was studied by estimating single-strand breaks using the alkaline elution technique. 1-Nitropyrene (10-200 microM) caused clear dose-dependent increases in DNA strand breaks in both cell types, whereas no increase in DNA strand breaks was observed in hepatocytes treated with 1.3-, 1,6-, 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene under standard assay conditions (5-20 microM 30-min incubation). However, 1,8-dinitropyrene (1,8-DNP) caused dose-dependent increases in DNA strand breaks when incubated with the H4-II-E cells for 48 h, while no single-strand breaks were observed following treatment with 1,6-dinitropyrene (1,6-DNP) under the same conditions. Neither 1,6-DNP nor 1,8-DNAP induced DNA crosslinks in the H4-II-E cells. These data indicate that substrate specificity exists in the metabolic activation of nitropyrenes in murine liver.     

Clusters of S1 nuclease-hypersensitive sites induced in vivo by DNA damage.

DNA end-labeling procedures were used to analyze both the frequency and distribution of DNA strand breaks in mammalian cells exposed or not to different types of DNA-damaging agents. The 3' ends were labeled by T4 DNA polymerase-catalyzed nucleotide exchange carried out in the absence or presence of Escherichia coli endonuclease IV to cleave abasic sites and remove 3' blocking groups. Using this sensitive assay, we show that DNA isolated from human cells or mouse tissues contains variable basal levels of DNA strand interruptions which are associated with normal bioprocesses, including DNA replication and repair. On the other hand, distinct dose-dependent patterns of DNA damage were assessed quantitatively in cultured human cells exposed briefly to menadione, methylmethane sulfonate, topoisomerase II inhibitors, or gamma rays. In vivo induction of single-strand breaks and abasic sites by methylmethane sulfonate was also measured in several mouse tissues. The genomic distribution of these lesions was investigated by DNA cleavage with the single-strand-specific S1 nuclease. Strikingly similar cleavage patterns were obtained with all DNA-damaging agents tested, indicating that the majority of S1-hypersensitive sites detected were not randomly distributed over the genome but apparently were clustered in damage-sensitive regions. The parallel disappearance of 3' ends and loss of S1-hypersensitive sites during post-gamma-irradiation repair periods indicates that these sites were rapidly repaired single-strand breaks or gaps (2- to 3-min half-life). Comparison of S1 cleavage patterns obtained with gamma-irradiated DNA and gamma-irradiated cells shows that chromatin structure was the primary determinant of the distribution of the DNA damage detected.     

Iron-overload induces oxidative DNA damage in the human colon carcinoma cell line HT29 clone 19A

Dietary iron may contribute to colon cancer risk via production of reactive oxygen species (ROS). The aim of the study was to determine whether physiological ferric/ferrous iron induces oxidative DNA damage in human colon cells. Therefore, differentiated human colon tumour cells (HT29 clone 19A) were incubated with ferric-nitrilotriacetate (Fe-NTA) or with haemoglobin and DNA breaks and oxidised bases were determined by microgelelectrophoresis. The effects of Fe-NTA were measured with additional H(2)O(2) (75microM) and quercetin (25-100microM) treatment. Analytic detection of iron in cell cultures, treated with 250microM Fe-NTA for 15 min to 24h, showed that 48.02+/-5.14 to 68.31+/-2.11% were rapidly absorbed and then detectable in the cellular fraction. Fe-NTA (250-1000microM) induced DNA breaks and oxidised bases, which were enhanced by subsequent H(2)O(2) exposure. Simultaneous incubation of HT29 clone 19A cells with Fe-NTA and H(2)O(2) for 15 min, 37 degrees C did not change the effect of H(2)O(2) alone. The impact of Fe-NTA and H(2)O(2)-induced oxidative damage is reduced by the antioxidant quercetin (75-67% of H(2)O(2)-control). Haemoglobin was as effective as Fe-NTA in inducing DNA damage. From these results we can conclude that iron is taken up by human colon cells and participates in the induction of oxidative DNA damage. Thus, iron or its capacity to catalyse ROS-formation, is an important colon cancer risk factor. Inhibition of damage by quercetin reflects the potential of antioxidative compounds to influence this risk factor. Quantitative data on the genotoxic impact of ferrous iron (e.g. from red meat) relative to the concentrations of antioxidants (from plant foods) in the gut are now needed to determine the optimal balance of food intake that will reduce exposure to this type of colon cancer risk factor.     

DNA damage induced by the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in mammalian cells in vitro and in mice

3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) formed during chlorination of water containing natural organic substances, is a very potent bacterial mutagen. Recently, tumours at multiple sites were reported in rats given MX-containing drinking water. We have investigated the genotoxicity of MX in mammalian cells exposed in vitro and in vivo using alkaline filter elution to detect DNA single-strand breaks and/or alkali-labile sites (SSBs). Concentrations as high as 100 and 300 microM MX were required to induce detectable levels of SSBs in the HL-60 cells. If MX treatment was carried out in the presence of DNA repair inhibitors (AraC plus hydroxyurea), the sensitivity of the assay to detect MX-induced SSBs was increased by a factor of 100. The presence of serum proteins during exposure resulted in a minor reduction of the MX-induced DNA damage in HL-60 cells at the lowest MX concentrations. In primary cultures of testicular cells as well as in resting human peripheral blood mononuclear cells (PBMC), a slightly increased level of SSBs was observed at MX-concentrations above 30 microM, this effect was not further increased by repair inhibitors. In LLC-PK1 renal proximal tubular epithelial cells and in growth stimulated human peripheral PBMC, increased SSBs were detected at MX concentrations as low as low as 3-10 microM and higher using repair inhibitors, and at 10 times higher concentrations without repair inhibitors. No dose dependent DNA damage was detected in the liver, kidney, spleen and colon of male B6C3F1 mice administrated high doses of MX (40 and 80 mg kg-1). Moderately increased and dose dependent SSBs were detected in the liver and kidney in the presence of DNA repair inhibitors during MX treatment, but no such increase was observed in the spleen and colon.     

DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells: bolus addition versus continuous generation of H(2)O(2)

Aspects of the molecular mechanism(s) of hydrogen peroxide-induced DNA damage and cell death were studied in the present investigation. Jurkat T-cells in culture were exposed either to low rates of continuously generated H(2)O(2) by the action of glucose oxidase or to a bolus addition of the same agent. In the first case, steady state conditions were prevailing, while in the latter, H(2)O(2) was removed by the cellular defense systems following first order kinetics. By using single-cell gel electrophoresis (also called comet assay), an initial increase in the formation of DNA single-strand breaks was observed in cells exposed to a bolus of 150 microM H(2)O(2). As the H(2)O(2) was exhausted, a gradual decrease in DNA damage was apparent, indicating the existence of an effective repair of single-strand breaks. Addition of 10 ng glucose oxidase in 100 microl growth medium (containing 1.5 x 10(5) cells) generated 2.0 +/- 0.2 microM H(2)O(2) per min. This treatment induced an increase in the level of single-strand breaks reaching the upper limit of detection by the methodology used and continued to be high for the following 6 h. However, when a variety of markers for apoptotic cell death (DNA cell content, DNA laddering, activation of caspases, PARP cleavage) were examined, only bolus additions of H(2)O(2) were able to induce apoptosis, while the continuous presence of this agent inhibited the execution of the apoptotic process no matter whether the inducer was H(2)O(2) itself or an anti-Fas antibody. These observations stress that, apart from the apparent genotoxic and proapoptotic effects of H(2)O(2), it can also exert antiapoptotic actions when present, even at low concentrations, during the execution of apoptosis.     

DNA damage induced by phorbol ester-stimulated neutrophils is augmented by extracellular cofactors. Role of histidine and metals

Activated neutrophils cause extensive DNA damage in neighboring nonphagocytic cells. To determine whether compounds in the extracellular milieu participate in the DNA damage process, murine neutrophils were cocultivated with target tumor cells in media of varying composition. Using the alkaline elution assay, it was found that the level of strand breaks induced was significantly higher (2.8-fold) in complex cell culture media than in minimal phosphate-buffered saline. Addition of amino acids in general and of histidine in particular increased the level of damage nearly to that observed in complete media (2.7- and 2.1-fold, respectively). The histidine stimulation was concentration-dependent and reached a maximum at 100-400 microM. The mechanism whereby this occurred is not proven but probably derived from chelation of metals and participation in a site-specific Fenton reaction. Addition of the cell-impermeable chelator EDTA dramatically inhibited induction of strand breaks by neutrophils in complete media and prevented the enhancement of damage induced by histidine in phosphate-buffered saline. None of the effects on neutrophil-induced damage could be attributed to modulation of the oxidative burst activity of the cells (O2- and H2O2 production). Histidine also enhanced induction of strand breaks by reagent H2O2. However, EDTA had no effect or actually increased the level of damage induced by both a bolus of H2O2 and a flux of H2O2 generated by glucose oxidase. The cell-permeable chelator o-phenanthroline inhibited both neutrophil- and H2O2-induced damage. The results indicate that secondary reactions involving extracellular amino acids and metals contribute significantly to neutrophil-induced DNA damage to neighboring cells. Moreover, the data show that the mechanism whereby neutrophils induce this damage cannot be attributed solely to secretion of H2O2.     

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.     

Susceptibility of DNA to oxidative stressors in young and aging mice

The changes that accompany aging may be a result of oxidative damage to DNA that accumulates as a result of aging and age-related illnesses. Furthermore, a higher susceptibility is thought to be more common among elderly than young individuals. In the present study, we examined the severity of DNA damage caused by carbon tetrachloride (CCl4) and H2O2 in cells from young (2 month old) and older (14 month old) mice using both in vivo and in vitro exposures. CCl(4) is known to generate radical oxidative species (ROS) throughout its biotransformation in the liver. Therefore, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxdGuo) was quantified in liver DNA obtained from young and older mice treated with CCl4. In addition, DNA single-strand breaks were measured by the Comet assay in primary lung fibroblasts cultured from young and older mice and treated in vitro with H2O2. Intracellular ROS production and mitochondrial enzyme activity were determined in parallel. 8-oxodGuo levels were significantly higher in older mouse liver DNA than younger, and increased significantly with CCl4 treatment. When the basal DNA damage was subtracted, the net damage was almost equal for both. In addition, untreated cells cultured from older mice had significantly greater levels of strand breaks than cells derived from young mice. H2O2 increased the level of damage in both cell cultures. Our findings indicate that the DNA damage observed in older animals probably results from the accumulation of endogenous damage with age, perhaps due to insufficient repair, which enhances the injury caused by exposure to the toxic agents.     

Photolysis of N-hydroxpyridinethiones: a new source of hydroxyl radicals for the direct damage of cell-free and cellular DNA.

N-Hydroxypyridine-2-thione (2-HPT), known to release hydroxyl radicals on irradiation with visible light, and two related compounds, viz. N-hydroxypyridine-4-thione (4-HPT) and N-hydroxyacridine-9-thione (HAT), were tested for their potency to induce DNA damage in L1210 mouse leukemia cells and in isolated DNA from bacteriophage PM2. DNA single-strand breaks and modifications sensitive to various repair endonucleases (Fpg protein, endonuclease III, exonuclease III, T4 endonuclease V) were quantified. Illumination of cell-free DNA in the presence of 2-HPT and 4-HPT gave rise to damage profiles characteristic for hydroxyl radicals, i.e. single-strand breaks and the various endonuclease-sensitive modifications were formed in the same ratios as after exposure to established hydroxyl radical sources. In contrast, HAT plus light gave rise to a completely different DNA damage profile, namely that characteristic for singlet oxygen. Experiments with various scavengers (t-butanol, catalase, superoxide dismutase) and in D2O as solvent confirmed that hydroxyl radicals are directly responsible for the DNA damage caused by photoexcited 2-HPT and 4-HPT, while the damage by HAT plus light is mediated by singlet oxygen and type I reactions. The type of DNA damage characteristic of hydroxyl radicals was also observed in L1210 mouse leukemia cells when treated with 2-HPT plus light or with H2O2 at 0 degrees C. t-Butanol (2%) inhibited the cellular DNA damage by approximately 50%. A dose of 2-HPT plus light that generated single-strand breaks at a frequency of 5 x 10(-7)/bp was associated with 50% cell survival. No DNA damage and cytotoxicity was observed after treatment with 2-HPT in the dark. We propose that 2-HTP and 4-HTP may serve as new agents to study the consequences of DNA damage induced by hydroxyl radicals in cells. In addition, the data provide direct evidence that hydroxyl radicals are ultimately responsible for the genotoxic effects caused by H2O2 in the dark.     

Effect of age on endogenous DNA single-strand breakage, strand break induction and repair in the adult housefly, Musca domestica

It has been suggested that genomic alterations involving DNA damage and the ability to repair such damage play an important role in cellular senescence. In this study, endogenous DNA single-strand breaks, the susceptibility of DNA to induced strand breakage and the capacity to repair these breaks were compared in postmitotic cells from young (3-day-old) and old (23-day-old) houseflies. DNA single-strand breaks did not accumulate during normal aging in the housefly. However, cells of the old flies exhibited a greater sensitivity to single-strand breakage induced by gamma-radiation and UV light. The capacity to repair these exogenously induced single-strand breaks declined with age. Results do not support the view that DNA single-strand breaks are a causal factor in aging in the housefly. An age-related increase in the susceptibility to undergo single-strand breakage suggests alterations in chromatin during the aging process.     

2- and 4-Aminobiphenyls induce oxidative DNA damage in human hepatoma (Hep G2) cells via different mechanisms

4-Aminobiphenyl (4-ABP) and its analogue, 2-aminobiphenyl (2-ABP), were examined for their ability to induce oxidative DNA damage in Hep G2 cells. Using the alkaline comet assay, we showed that 2-ABP and 4-ABP (25-200 microM) were able to induce the DNA damage in Hep G2 cells. With both compounds, formation of intracellular reactive oxygen species (ROS) was detected using flow cytometry analysis. Post-treatment of 2-ABP and 4-ABP-treated cells by endonuclease III (Endo III) or formamidopyrimidine-DNA glycosylase (Fpg) to determine the formation of oxidized pyrimidines or oxidized purines showed a significant increase of the extent of DNA migration. This indicated that oxidative DNA damage occurs in Hep G2 cells after exposure to 2-ABP and 4-ABP. This assumption was further substantiated by the fact that the spin traps, 5,5-dimethyl-pyrroline-N-oxide (DMPO) and N-tert-butyl-alpha-phenylnitrone (PBN), decreased DNA damage significantly. Furthermore, addition of the catalase (100 U/ml) caused a decrease in the DNA damage induced by 2-ABP or 4-ABP, indicating that H(2)O(2) is involved in ABP-induced DNA damage. Pre-incubation of the cells with the iron chelator desferrioxamine (DFO) (1mM) and with the copper chelator neocupronine (NC) (100 microM) also decreased DNA damage in cells treated with 200 microM 2-ABP or 200 microM 4-ABP, while the calcium chelator {1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester}(BAPTA/AM) (10 microM) decreased only DNA strand breaks in cells exposed to 4-ABP. This suggested that ions are involved in the formation of DNA strand breaks. Using RT-PCR and Western blotting, lower inhibition of the expression of the OGG1 gene and of the OGG1 protein was observed in cells treated with 4-ABP, and 2-ABP-treated cells showed a marked reduction in the expression of OGG1 gene and OGG1 protein. Taken together, our finding indicated the mechanisms of induced oxidative DNA damage in Hep G2 cell by 2-ABP and 4-ABP are different, although both tested compounds are isomers.     

Soluble metals as well as the insoluble particle fraction are involved in cellular DNA damage induced by particulate matter

Exposure to ambient particulate matter has been reported to be associated with increased rates of lung cancer. Previously we showed that total suspended particulate matter (PM) induces oxidative DNA damage in epithelial lung cells. The aim of the present study was to further investigate the mechanism of PM-induced DNA damage, in which soluble iron-mediated hydroxyl radical (.OH) formation is thought to play a crucial role. Using electron spin resonance (ESR) we showed that PM suspensions as well as their particle-free, water-soluble fractions can generate .OH in the presence of hydrogen peroxide (H2O2), an effect which was abrogated by both deferoxamine and catalase. In addition, PM was also found to induce the .OH-specific DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in the presence of H2O2 as assessed by dot-blot analysis of calf thymus DNA using an 8-OHdG antibody. In human alveolar epithelial cells (A549), both PM suspensions and the particle-free soluble fraction elicited formation of DNA strand breaks (comet-assay). Unlike the acellular DNA assay, in epithelial cells the DNA-damaging capacity of the particle suspensions appeared to be stronger than that of their corresponding particle-free filtrates. In conclusion, our findings demonstrate that the water-soluble fraction of PM elicits DNA damage via transition metal-dependent .OH formation, implicating an important role of H2O2. Moreover, our data indicate that direct 'particle' effects contribute to the genotoxic hazard of ambient particulate matter in lung target cells.     

Daunorubicin-induced DNA lesions in isolated rat hepatocytes and mammary epithelial cells

Daunorubicin, an anticancer drug, induces primarily mammary adenocarcinoma in Sprague-Dawley rats. We investigated daunorubicin-induced DNA lesions in enzymatically isolated mammary epithelial cells and hepatocytes from 7-8-week-old female Sprague-Dawley rats. Differences were observed in the type and quantity of DNA lesions in mammary epithelial cells and hepatocytes as determined by alkaline elution analysis. DNA single-strand breaks and proteinase-K-sensitive cross-linking lesions were observed in mammary epithelial cells. Hepatocytes appeared to have significantly lower relative frequencies of single-strand breaks than mammary epithelial cells when treated with daunorubicin (1.5-10.0 micrograms/10(6) cells). Hepatocytes displayed two types of cross-link. One form was sensitive to proteinase-K digestion, whereas the other form was insensitive. The metabolism of daunorubicin to the aglycone metabolites was substantially lower in mammary cells than in hepatocytes. However, the total uptake of the drug was similar in these two cell types. A metabolite, 7-deoxydaunorubicinol aglycone, was unable to induce single-strand breaks or cross-linking lesions in mammary epithelial cells. Both cell types exhibited a similar ability to repair radiation-induced single-strand breaks of DNA. However, the mammary cells may be less able to repair daunorubicin-mediated DNA damage. These results revealed that mammary epithelial cells are less able to metabolize the active mutagen/carcinogen, daunorubicin, than are hepatocytes. This, coupled with the observations of greater apparent DNA damage in mammary cells, may be of primary importance in the drug-induced carcinogenicity in the rat mammary tissue.     

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.