Comparative study of the formation of oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct from the nucleoside 2'-deoxyguanosine by transition metals and suspensions of particulate matter in relation to metal content and redox reactivity

An association between exposure to ambient particulate matter (PM) and increased incidence of mortality and morbidity due to lung cancer and cardiovascular diseases has been demonstrated by recent epidemiological studies. Reactive oxygen species (ROS), especially hydroxyl radicals, generated by PM, have been suggested by many studies as an important factor in the oxidative damage of DNA by PM. The purpose of this study was to characterize quantitatively hydroxyl radical generation by various transition metals in the presence of H₂O₂ in aqueous buffer solution (pH 7.4) and hydroxylation of 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG) under similar conditions. The order of metals' redox reactivity and hydroxyl radical production was Fe(II), V(IV), Cu(I), Cr(III), Ni(II), Co(II), Pb(II), Cd(II). Then, we investigated the generation of hydroxyl radicals in the presence of H₂O₂ by various airborne PM samples, such as total suspended particulate (TSP), PM₁₀, PM_2.5 (PM with aerodynamic diameter 10 and 2.5 μm), diesel exhaust particles (DEP), gasoline exhaust particles (GEP) and woodsmoke soot under the same conditions. When suspensions of PMs were incubated with H₂O₂ and dG at pH 7.4, all particles induced hydroxylation of dG and formation of 8-OHdG in a dose-dependent increase. Our findings demonstrated that PM's hydroxyl radical (HO√) generating ability and subsequent dG hydroxylation is associated with the concentration of water-soluble metals, especially Fe and V and other redox or ionizable transition metals and not their total metal content, or insoluble metal oxides, via a Fenton-driven reaction of H₂O₂ with metals. Additionally, we observed, by Electron paramagnetic resonance (EPR), that PM suspensions in the presence of H₂O₂ generated radical species with dG, which were spin-trapped by 2-methyl-2-nitroso-propane (MNP).     

Assessment of DNA damage in sperm after repeated non‐invasive sampling in zebrafish Danio rerio

Repeated non‐invasive sampling of zebrafish Danio rerio sperm was conducted, sperm counts were obtained and a method for measurement of DNA damage in sperm was developed and validated (single‐cell gel electrophoresis, comet, assay). DNA damage in sperm increased with concentration of hydrogen peroxide (H₂O₂, 0–200 µM), and in vitro exposure of sperm to 200 µM H₂O₂ produced 88·7 ± 3·9% tail DNA compared to unexposed controls [12 ± 0·7% tail DNA (mean ± s.e ., n = 3)]. Frequency of sperm sampling (sampled every 2, 4 or 7 days) did not affect DNA damage in sperm, but sperm counts decreased 57 and 22% for fish sampled every 2 or 4 days, respectively.     

DNA single stranded gaps formed during DNA repair synthesis induced by methyl methanesulfonate are filled by sequential action of aphidicolin- and dideoxythymidine sensitive DNA polymerases in HeLa cells

DNA repair synthesis induced by methyl methanesulfonate in preconditioned HeLa cells in which DNA replicative synthesis had been highly suppressed was inhibited by aphidicolin (an inhibitor of DNA polymerases and ) and dideoxythymidine (ddThR, an inhibitor of DNA polymerase ). Incomplete repair patches sensitive to exonuclease III were accumulated in the presence of aphidicolin while not in the presence of ddThR. These patches were comopleted by the combined action of Klenow fragment and T4 DNA ligase, indicating that the single-stranded gaps were formed during the repair synthesis. Moreover, ddThR had little effect on the repair synthesis in the presence of aphidicolin. Thus, the results suggest that the single-stranded gaps may be sealed first by aphidicolin-sensitive polymerase followed by ddThR-sensitive DNA polymerase on the same site of the repair patch.Abbreviations ddThR dideoxythymidine - MMS methyl methanesulfonate - dNTP deoxynucleoside triphosphate     

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

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

Copper increases the damage to DNA and proteins caused by reactive oxygen species

Copper [Cu(II)] is an ubiquitous transition and trace element in living organisms. It increases reactive oxygen species (ROS) and free-radical generation that might damage biomolecules like DNA, proteins, and lipids. Furthermore, ability of Cu(II) greatly increases in the presence of oxidants. ROS, like hydroxyl (·OH) and superoxide (·O₂) radicals, alter both the structure of the DNA double helix and the nitrogen bases, resulting in mutations like the AT→GC and GC→AT transitions. Proteins, on the other hand, suffer irreversible oxidations and loss in their biological role. Thus, the aim of this investigation is to characterize, in vitro, the structural effects caused by ROS and Cu(II) on bacteriophage λ DNA or proteins using either hydrogen peroxide (H₂O₂) or ascorbic acid with or without Cu(II). Exposure of DNA to ROS-generating mixtures results in electrophoretic (DNA breaks), spectrophotometric (band broadening, hypochromic, hyperchromic, and bathochromic effects), and calorimetric (denaturation temperature [T _d], denaturation enthalpy [ΔH], and heat capacity [C _p] values) changes. As for proteins, ROS increased their thermal stability. However, the extent of the observed changes in DNA and proteins were distinct, depending on the efficiency of the systems assayed to generate ROS. The resulting effects were most evident when Cu(II) was present. In summary, these results show that the ROS, ·O₂ and ·OH radicals, generated by the Cu(II) systems assayed deeply altered the chemical structure of both DNA and proteins. The physiological relevance of these structural effects should be further investigated.     

Protection by tropolones against H2O2-induced DNA damage and apoptosis in cultured Jurkat cells

Tropolones, the naturally occurring compounds responsible for the durability of heartwood of several cupressaceous trees, have been shown to possess both metal chelating and antioxidant properties. However, little is known about the ability of tropolone and its derivatives to protect cultured cells from oxidative stress-mediated damage. In this study, the effect of tropolones on hydrogen peroxide-induced DNA damage and apoptosis was investigated in cultured Jurkat cells. Tropolone, added to the cells 15 min before the addition of glucose oxidase, provided a dose dependent protection against hydrogen peroxide induced DNA damage. The IC₅₀ value observed was about 15 μM for tropolone. Similar dose dependent protection was also observed with three other tropolone derivatives such as trimethylcolchicinic acid, purpurogallin and β-thujaplicin (the IC₅₀ values were 34, 70 and 74 μM, respectively), but not with colchicine and tetramethyl purpurogallin ester. Hydrogen peroxide-induced apoptosis was also inhibited by tropolone. However, in the absence of exogenous H₂O₂ but in the presence of non-toxic concentrations of exogenous iron (100 μM Fe³⁺), tropolone dramatically increased the formation of single strand breaks at molar ratios of tropolone to iron lower than 3 to 1, while, when the ratio increased over 3, no toxicity was observed. In conclusion, the results presented in this study indicate that the protection offered by tropolone against hydrogen peroxide-induced DNA damage and apoptosis was due to formation of a redox-inactive iron complex, while its enhancement of iron-mediated DNA damage at ratios of [tropolone]/[Fe³⁺] lower than 3, was due to formation of a lipophilic iron complex which facilitates iron transport through cell membrane in a redox-active form.     

Distinct mechanisms of DNA damage in apoptosis induced by quercetin and luteolin

Quercetin has been reported to have carcinogenic effects. However, both quercetin and luteolin have anti-cancer activity. To clarify the mechanism underlying the carcinogenic effects of quercetin, we compared DNA damage occurring during apoptosis induced by quercetin with that occuring during apoptosis induced by luteolin. Both quercetin and luteolin similarly induced DNA cleavage with subsequent DNA ladder formation, characteristics of apoptosis, in HL-60 cells. In HP 100 cells, an H₂O₂-resistant clone of HL-60 cells, the extent of DNA cleavage and DNA ladder formation induced by quercetin was less than that in HL-60 cells, whereas differences between the two cell types were minimal after treatment with luteolin. In addition, quercetin increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in HL-60 cells but not in HP 100 cells. Luteolin did not increase 8-oxodG formation, but inhibited topoisomerase II (topo II) activity of nuclear extract more strongly than quercetin and cleaved DNA by forming a luteolin-topo II-DNA ternary complex. These results suggest that quercetin induces H₂O₂-mediated DNA damage, resulting in apoptosis or mutations, whereas luteolin induces apoptosis via topo II-mediated DNA cleavage. The H₂O₂-mediated DNA damage may be related to the carcinogenic effects of quercetin.     

Mechanisms of neutrophil-induced DNA damage in respiratory tract epithelial cells

Reactive oxygen species (ROS) released by neutrophils have been suggested to play an important role in cancer development. Since the mechanisms underlying this effect in the respiratory tract are still unclear, we evaluated DNA damage induced by neutrophils in respiratory tract epithelial cells in vitro and in vivo. For in vitro studies, rat lung epithelial cells (RLE) were co-incubated with activated neutrophils, neutrophil-conditioned medium, or hydrogen peroxide. For in vivo studies, we considered the human nose as a target organ, comparing neutrophilic inflammation in the nasal lavage fluid with the oxidative DNA lesion 8-hydroxydeoxyguanosine (8-OHdG) in epithelial cells obtained by nasal brush. Our in vitro data show that human neutrophils are able to induce both 8-OHdG and strand breaks in DNA from RLE cells. Our data also suggest that DNA damage induced by neutrophils is inhibited when neutrophil-derived H₂O₂ is consumed by myeloperoxidase. In contrast, in the nose no association between neutrophil numbers and 8-OHdG was found. Therefore, it remains unclear whether neutrophils pose a direct genotoxic risk for the respiratory tract epithelium during inflammation, and more in vivo studies are needed to elucidate the possible association between neutrophils and genotoxicity in the lung.     

Bulky adducts detected by P-postlabeling in DNA modified by oxidative damage in vitro. Comparison with rat lung I-compounds

Oxygen free radicals, such as the hydroxyl radical generated by interaction of Fe²⁺ and H₂O₂ (Fenton reaction), are produced in mammalian cells as a result of aerobic metabolism and under various pathological conditions and are known to elicit mutations and potentially other adverse effects by reacting with DNA bases. Several products thus formed have recently been characterized as hydroxylated derivatives of cytosine, thymine, adenine, and guanine and imidazole-ring-opened derivatives of adenine and guanine in DNA. As shown herein by ³²P-postlabeling, incubation of DNA under Fenton reaction conditions led to additional products which, by virtue of resistance to nuclease P1 catalyzed 3′-dephosphorylation and chromatographic behavior, appeared to be bulky adducts rather than small polar, hydroxylated or ring-opened nucleotide derivatives. Two major and five minor DNA derivatives were measured after ³²P-postlabeling and TLC mapping of DNA oxidized in vitro under conditions known to lead to formation of reactive oxygen species. Amounts of products formed depended on Fe²⁺ and H₂O₂ concentrations and increased in the presence of -ascorbic acid. One of the two major products was also detected in lung DNA of rats where its amount increased with animal age. Thus, at least one I-compound appeared to have its origin in the interaction of DNA with reactive oxygen species.     

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 against nuclear DNA damage offered by flavonoids in cells exposed to hydrogen peroxide: the role of iron chelation

The ability of a number of flavonoids belonging to the flavone, flavonol, flavanone, and flavan-3-ol subclasses to protect cellular DNA from H₂O₂-induced single-strand breaks and the underlying molecular mechanisms were investigated in this work. Formation of single-strand breaks on nuclear DNA, after exposure of Jurkat cells to continuously generated H₂O₂ in the presence or absence of the flavonoid compounds, was evaluated by the comet assay (single-cell gel electrophoresis). The results indicate the following structural requirements of flavonoids for effective DNA protection: (a) the ortho-dihydroxy structure in either ring A or ring B, (b) the hydroxyl moiety on position 3 in combination with the oxo group at position 4, and (c) the presence of a C₂, C₃ double bond in ring C. In contrast to free flavonoids, the ability of complexes of [Fe²⁺]/[flavonoid] to protect nuclear DNA was decreased as the ratio increased, and the complex was completely inactive when the ratio reached a certain value. Moreover, it was observed that several of the flavonoids tested were able to remove iron from calcein loaded into cells and that this property was in excellent correlation with their ability to protect DNA (Spearman's correlation coefficient, ρ = 0.9, p = 0.005). The antioxidant (electron donating) capacities of the same flavonoids were also evaluated by a conventional method, but no relation with their DNA-protective ability could be established even when their membrane-penetrating abilities were taken into account (p = 0.64). In conclusion, the results presented in this work strongly support the notion that intracellular binding of iron is responsible for the protection offered by flavonoids against H₂O₂-induced DNA damage.     

Enhancement of the repair of carcinogen-induced DNA damage in the hamster pancreas by dietary selenium

We measured single strand breaks (SSB) in pancreas DNA produced by N-nitrosobis(2-oxopropyl)amine (BOP) in hamsters fed purified diets containing added sodium selenite (Se) at 0.0, 0.1 and 5.0 ppm. There were fewer SSB in those given the 5.0 ppm Se diet throughout the experiment. One hour after dosing with BOP (20 mg/kg), there were 2.26 ± 0.47, 2.83 ± 0.43 and 1.74 ± 0.43 SSB per 10⁸ daltons (mean ± S.E.M.) respectively in the three groups. The SSB were repaired faster in the 5.0 ppm Se-fed group. The approximate half-lives of the SSB were 33, 30 and 8 days, respectively. In the hamsters fed 5.0 ppm Se there was a small, statistically significant increase in pancreatic DNA synthesis. Autoradiographic analysis indicated that this was repair synthesis. In a second experiment, hamsters were fed one of the three diets prior to and for 2 days after administration of a single dose of BOP (20 mg/kg). They were then fed the 5.0 ppm Se diet for 5 days. The number of SSB was compared with those in hamsters fed their original diet for 7 days after BOP dosing. There was a statistically significant difference in the number of SSB in the hamsters fed 0.1 ppm Se before and for 2 days after BOP. In these hamsters there were 1.21 ± 0.24 SSB per 10⁸ daltons compared with 3.19 ± 0.4 (mean ± S.E.M.). These results suggest high levels of dietary Se stimulate the repair of carcinogen-induced DNA damage.     

Hydrixyl radical generation by the tetracycline antibiotics with free radical damage to DNA, lipids and carbohydrate in the presence of iron and copper salts

Tetracycline antibiotics caused the degradation of carbohydrate in the presence of a ferric salt at pH 7.4. This degradation appeared to involve hydroxyl radicals since the damage was substantially reduced by the presence of catalase, superoxide dismutase, scavengers of the hydroxyl radical and metal chelators. Similarly, the tetracycline antibiotics in the presence of a ferric salt greatly stimulated the peroxidation of liposomal membranes. This damage, which did not implicate the hydroxyl radical, was significantly reduced by the addition of chain-breaking antioxidants and metal chelators. Only copper salts in the presence of tetracycline antibiotics, however, caused substantial damage to linear duplex DNA. Studies with inhibitors suggested that damage to DNA did involve hydroxyl radicals.     

Involvement of hydroxyl radical in NAD(P)H oxidation and associated oxygen reduction by the granule fraction of human blood polymorphonuclears.

The cyanide-insensitive NAD(P)H oxidase activities have been measured in particulate fractions isolated from resting or zymosan-stimulated polymorphonuclear leukocytes. The particulate fraction was primarily composed of granules. The activities were measured both in the presence and absence of Mn⁺⁺. It was found, in all experiments, that hydroxyl radical scavengers such as Tris, benzoate or mannitol, were powerful inhibitors of the NAD(P)H oxidase activities. This was taken as evidence for the involvement of hydroxyl radical as an intermediate in the aerobic oxidation of both NADH and NADPH. Possibles sources of hydroxyl radical are suggested, but none of them is demonstrated.     

Protein-mediated hydroxyl radical generation--the primary event in NADH oxidation and oxygen reduction by the granule rich fraction of human resting leukocytes.

The granule rich-fraction isolated from human resting polymorphonuclear leukocytes is capable of CN-insensitive NADH oxidation and O₂-uptake, accompanied by production of superoxide anion, hydroxyl radicals and H₂O₂. We showed that H₂O₂ initiates and maintains NADH oxidation and O₂-uptake but is also necessary for the formation of superoxide anion and hydroxyl radicals. It acts as a primary substrate for CN-insensitive protein-mediated formation of hydroxyl radicals, which in turn produce superoxide anions, probably through univalent oxidation of NADH as an intermediary.     

Single-strand breaks in DNA during repair of UV-induced damage in normal human and xeroderma pigmentosum cells as determined by alkaline DNA unwinding and hydroxylapatite chromatography: effects of hydroxyurea, 5-fluorodeoxyuridine and 1-beta-D-arabinofuranosylcytosine on the kinetics of repair.

A simple and sensitive technique for detection of strand breaks in DNA has been further developed. The method has been used to follow UV-induced excision-repair in human fibroblasts. It has been possible to study the kinetics of enzymic reactions in intact cells, in which strand breaks in DNA are produced and sealed again. Hydroxyurea, 5-fluorodeoxyuridine and 1-beta-D-arabinofuranosylcytosine, potent inhibitors of DNA synthesis, drastically increased the number of breaks observed during the repair process. This was probably due to a decreased polymerase activity, which will cause the strand breaks formed by endonuclease to remain open longer. The initial rate of strand-break formation did not seem to be influenced by hydroxyurea or araC, and was about 4000 breaks per minute in a diploid genome, at a dose of 20 J/m2. After 5--30 min, depending on the dose of UV, the number of breaks reached a maximum and started to decrease again. Hydroxyurea decreased the rate of polymerization in the sites under repair. However, there was no concomitant reduction of repair-induced incorporation of [3H]thymidine and no reduction of the excision of pyrimidine dimers. It therefore seems that the action of the polymerase was not a rate-limiting event, but rather an earlier step. It is likely that the endonucleolytic activity determined the rate of repair. As a consequence, the endonuclease and polymerase cannot be bound in a permanent complex. Under certain assumptions, the time for repair of a site, i.e. the time from incision to final ligase sealing, can be estimated as between 3 and 10 min. Essentially no breaks were produced in Xeroderma pigmentosum cells belonging to complementation group A, and there was no enhancement by hydroxyurea. Cells from the variant type of Xeroderma pigmentosum behaved like normal cells in this respect.