Catechins induce oxidative damage to cellular and isolated DNA through the generation of reactive oxygen species

Green tea catechins have antimutagenic and anticarcinogenic activities. On the other hand, several epidemiological studies have indicated significant positive relationship between green tea consumption and cancer. Catechins enhance colon carcinogenesis in rats initiated with chemical carcinogen. To clarify the mechanism underlying the potential carcinogenicity, we investigated the DNA-damaging ability of catechins in human cultured cells. Catechin increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a characteristic oxidative DNA lesion, in human leukemia cell line HL-60 but not in HP100, a hydrogen peroxide (H

2

O

2

)-resistant cell line derived from HL-60. The catechin-induced formation of 8-oxodG in HL-60 cells significantly decreased by bathocuproine. Furthermore, we investigated DNA damage and its site-specificity induced by catechins, using

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P-labeled DNA fragments. Catechin and epicatechin induced extensive DNA damage in the presence of Cu(II). Catechin caused piperidine-labile sites at thymine and cytosine residues in the presence of Cu(II). Catalase and bathocuproine inhibited the DNA damage, indicating the involvement of H

2

O

2

and Cu(I). NADH enhanced catechins plus Cu(II)-induced 8-oxodG formation in calf thymus DNA, suggesting the redox cycle between catechins and their corresponding quinones, the oxidized forms of catechins. The DNA-damaging ability of epicatechin is stronger than that of catechin, possibly due to the greater turnover frequency of the redox cycle. The difference in their redox properties could be explained by their redox potentials estimated form an ab initio molecular orbital calculation. The present study demonstrated that catechins could induce metal-dependent H

2

O

2

generation during the redox reactions and subsequently damage to cellular and isolated DNA. Therefore, it is reasonably considered that green tea catechins may have the dual function of anticarcinogenic and carcinogenic potentials.     

Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation

Oncogene-induced reactive oxygen species (ROS) have been proposed to be signaling molecules that mediate proliferative cues. However, ROS may also cause DNA damage and proliferative arrest. How these apparently opposite roles can be reconciled, especially in the context of oncogene-induced cellular senescence, which is associated both with aberrant mitogenic signaling and DNA damage response (DDR)-mediated arrest, is unclear. Here, we show that ROS are indeed mitogenic signaling molecules that fuel oncogene-driven aberrant cell proliferation. However, by their very same ability to mediate cell hyperproliferation, ROS eventually cause DDR activation. We also show that oncogenic Ras-induced ROS are produced in a Rac1 and NADPH oxidase (Nox4)-dependent manner. In addition, we show that Ras-induced ROS can be detected and modulated in a living transparent animal: the zebrafish. Finally, in cancer we show that Nox4 is increased in both human tumors and a mouse model of pancreatic cancer and specific Nox4 small-molecule inhibitors act synergistically with existing chemotherapic agents.     

Hypoxic damage generates reactive oxygen species in isolated perfused rat liver

The aim of the present study was to investigate the possible role of reactive oxygen species in the pathogenesis of hypoxic damage in isolated perfused rat liver. One hour of hypoxia caused severe cell damage (lactate dehydrogenase release of greater than 12,000 mU/min/g liver wt) and total irreversible cholestasis which was accompanied by a loss of cellular ATP and a marked decrease in lactate efflux. Tissue glutathione disulfide (GSSG) content and GSSG efflux as a measure of hepatic reactive oxygen formation was less than 1% of total glutathione before and during hypoxia. Upon reoxygenation, however, hepatic GSSG content increased sharply to about twice the control values and GSSG efflux increased several-fold to around 3-4 nmol GSH-equivalents/min/g. The release of lactate dehydrogenase decreased upon reoxygenation and tissue ATP content recovered partially. When livers were reoxygenated at an earlier time interval than 1 hr of hypoxia, i.e., before the onset of damage, no enhanced GSSG formation was observed. The results demonstrate that hypoxic damage is a prerequisite to reactive oxygen formation during the subsequent reoxygenation period. Thus, reactive oxygen species appear unlikely to play a crucial role in the pathogenesis of hypoxic liver damage in the hemoglobin-free, isolated perfused liver model.     

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.     

Role of reactive oxygen species (ROS) in apoptosis induction

Reactive oxygen species (ROS) and mitochondria play an important role in apoptosis induction under both physiologic and pathologic conditions. Interestingly, mitochondria are both source and target of ROS. Cytochrome c release from mitochondria, that triggers caspase activation, appears to be largely mediated by direct or indirect ROS action. On the other hand, ROS have also anti-apoptotic effects. This review focuses on the role of ROS in the regulation of apoptosis, especially in inflammatory cells.     

Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA

Heat-induced formation of 8-oxoguanine was demonstrated in DNA solutions in 10^–3 M phosphate buffer, pH 6.8, by enzyme-linked immunosorbent assays using monoclonal antibodies against 8-oxoguanine. A radiation-chemical yield of 3.7 × 10^–2 µmol J^–1 for 8-oxoguanine production in DNA upon γ-irradiation was used as an adequate standard for quantitation of 8-oxoguanine in whole DNA. The initial yield of heat-induced 8-oxoguanine exhibits first order kinetics. The rate constants for 8-oxoguanine formation were determined at elevated temperatures; the activation energy was found to be 27 ± 2 kcal/mol. Extrapolation to 37°C gave a value of k₃₇ = 4.7 × 10^–10 s^–1. Heat-induced 8-oxoguanine formation and depurination of guanine and adenine show similarities of the processes, which implies that heat-mediated generation of reactive oxygen species (ROS) should occur. Heat-induced production of H₂O₂ in phosphate buffer was shown. The sequence of reactions of thermally mediated ROS formation have been established: activation of dissolved oxygen to the singlet state, generation of superoxide radicals and their dismutation to H₂O₂. Gas saturation (O₂, N₂ and Ar), D₂O, scavengers of ¹O₂, O₂^–• and OH^• radicals and metal chelators influenced heat-induced 8-oxoguanine formation as they affected thermal ROS generation. These findings imply that heat acts via ROS attack leading to oxidative damage to DNA.     

P2X7 receptor activation induces reactive oxygen species formation in erythroid cells

The presence of P2X7 on erythroid cells is well established, but its physiological role remains unclear. The current study aimed to determine if P2X7 activation induces reactive oxygen species (ROS) formation in murine erythroleukaemia (MEL) cells, a commonly used erythroid cell line. ATP induced ROS formation in a time- and concentration-dependent fashion. The most potent P2X7 agonist, 2′(3′)-O-(4-benzoylbenzoyl)ATP, but not UTP or ADP, also induced ROS formation. The P2X7 antagonist, A-438079, impaired ATP-induced ROS formation. The ROS scavenger, N-acetyl-l-cysteine, and the ROS inhibitor, diphenyleneiodonium, also impaired P2X7-induced ROS formation, but use of enzyme-specific ROS inhibitors failed to identify the intracellular source of P2X7-induced ROS formation. P2X7-induced ROS formation was impaired partly by physiological concentrations of Ca²⁺ and Mg²⁺ and almost completely in cells in N-methyl-d-glucamine chloride medium. The p38 MAPK inhibitors SB202190 and SB203580, and the caspase inhibitor Z-VAD-FMK, but not N-acetyl-l-cysteine, impaired P2X7-induced MEL cell apoptosis. ATP also stimulated p38 MAPK and caspase activation, both of which could be impaired by A-438079. In conclusion, these findings indicate that P2X7 activation induces ROS formation in MEL cells and that this process may be involved in events downstream of P2X7 activation, other than apoptosis, in erythroid cells.     

Mitochondrial permeability transition in rat hepatocytes after anoxia/reoxygenation: role of Ca2+-dependent mitochondrial formation of reactive oxygen species

Onset of the mitochondrial permeability transition (MPT) is the penultimate event leading to lethal cellular ischemia-reperfusion injury, but the mechanisms precipitating the MPT after reperfusion remain unclear. Here, we investigated the role of mitochondrial free Ca(2+) and reactive oxygen species (ROS) in pH- and MPT-dependent reperfusion injury to hepatocytes. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 h and then reoxygenated at pH 7.4 to simulate ischemia-reperfusion. Some cells were loaded with the Ca(2+) chelators, BAPTA/AM and 2-[(2-bis-[carboxymethyl]aono-5-methoxyphenyl)-methyl-6-methoxy-8-bis[carboxymethyl]aminoquinoline, either by a cold loading protocol for intramitochondrial loading or by warm incubation for cytosolic loading. Cell death was assessed by propidium iodide fluorometry and immunoblotting. Mitochondrial Ca(2+), inner membrane permeability, membrane potential, and ROS formation were monitored with Rhod-2, calcein, tetramethylrhodamine methylester, and dihydrodichlorofluorescein, respectively. Necrotic cell death increased after reoxygenation. Necrosis was blocked by 1 μM cyclosporin A, an MPT inhibitor, and by reoxygenation at pH 6.2. Confocal imaging of Rhod-2, calcein, and dichlorofluorescein revealed that an increase of mitochondrial Ca(2+) and ROS preceded onset of the MPT after reoxygenation. Intramitochondrial Ca(2+) chelation, but not cytosolic Ca(2+) chelation, prevented ROS formation and subsequent necrotic and apoptotic cell death. Reoxygenation with the antioxidants, desferal or diphenylphenylenediamine, also suppressed MPT-mediated cell death. However, inhibition of cytosolic ROS by apocynin or diphenyleneiodonium chloride failed to prevent reoxygenation-induced cell death. In conclusion, Ca(2+)-dependent mitochondrial ROS formation is the molecular signal culminating in onset of the MPT after reoxygenation of anoxic hepatocytes, leading to cell death.     

Chemiluminescence assay for reactive oxygen species scavenging activities and inhibition on oxidative damage of DNA in Deinococcus radiodurans.

Free radical scavenging effects of the cellular protein extracts from two strains of Deinococcus radiodurans and Escherichia coli against O2-, H2O2 and *OH were investigated by chemiluminescence (CL) methods. The cellular protein extracts of D. radiodurans R1 and KD8301 showed higher scavenging effects on O2- than that of E. coli. D. radiodurans R1 and KD8301 also strongly scavenged H2O2 with an EC50 (50% effective concentration) of 0.12 and 0.2 mg/mL, respectively, compared to that of E. coli (EC50 = 3.56 mg/mL). The two strains of D. radiodurans were effective in scavenging *OH generated by the Fenton reaction, with EC50 of 0.059 and 0.1 mg/mL, respectively, compared to that of E. coli (EC50 > 1 mg/mL). Results from the chemiluminescence assay of *OH-induced DNA damage and the plasmid pUC18 DNA double-strand break (DSB) model in vitro showed that D. radiodurans had remarkably inhibitory effect on the *OH-induced oxidative damage of DNA. The scavenging effects of D. radiodurans on reactive oxygen species (ROS) played an important role in the response to oxidation stress and preventing against DNA oxidative damage, and may be attributed to intracellular scavenging proteins, including superoxide dismutase (SOD) and catalase.     

Loss of oxidized and chlorinated bases in DNA treated with reactive oxygen species: implications for assessment of oxidative damage in vivo

Oxidative damage to DNA has been reported to occur in a wide variety of disease states. The most widely used "marker" for oxidative DNA damage is 8-hydroxyguanine. However, the use of only one marker has limitations. Exposure of calf thymus DNA to an .OH-generating system (CuCl(2), ascorbate, H(2)O(2)) or to hypochlorous acid (HOCl), led to the extensive production of multiple oxidized or chlorinated DNA base products, as measured by gas chromatography-mass spectrometry. The addition of peroxynitrite (ONOO(-)) (<200 microM) or SIN-1 (1mM) to oxidized DNA led to the extensive loss of 8-hydroxyguanine, 5-hydroxycytosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2-hydroxyadenine, 8-hydroxyadenine, and 4,6-diamino-5-formamidopyrimidine were lost at higher ONOO(-) concentrations (>200 microM). Exposure of DNA to HOCl led to the generation of 5-Cl uracil and 8-Cl adenine and addition of ONOO(-) (<200 microM) or SIN-1 (1mM) led to an extensive loss of 8-Cl adenine and a small loss of 5-Cl uracil at higher concentrations (>500 microM). An .OH-generating system (CuCl(2)/ascorbate/H(2)O(2)) could also destroy these chlorinated species. Treatment of oxidized or chlorinated DNA with acidified nitrite (NO(2)(-), pH 3) led to substantial loss of various base lesions, in particular 8-OH guanine, 5-OH cytosine, thymine glycol, and 8-Cl adenine. Our data indicate the possibility that when ONOO(-), nitrite in regions of low pH or .OH are produced at sites of inflammation, levels of certain damaged DNA bases could represent an underestimate of ongoing DNA damage. This study emphasizes the need to examine more than one modified DNA base when assessing the role of reactive species in human disease.     

Vorinostat induces reactive oxygen species and DNA damage in acute myeloid leukemia cells

Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents.     

The role of mitochondrial DNA damage in the citotoxicity of reactive oxygen species

Mitochondria contain their own genome, a small circular molecule of around 16.5 kbases. The mitochondrial DNA (mtDNA) encodes for only 13 polypeptides, but its integrity is essential for mitochondrial function, as all 13 proteins are regulatory subunits of the oxidative phosphorylation complexes. Nonetheless, the mtDNA is physically associated with the inner mitochondrial membrane, where the majority of the cellular reactive oxygen species are generated. In fact, the mitochondrial DNA accumulates high levels of oxidized lesions, which have been associated with several pathological and degenerative processes. The cellular responses to nuclear DNA damage have been extensively studied, but so far little is known about the functional outcome and cellular responses to mtDNA damage. In this review we will discuss the mechanisms that lead to damage accumulation and the in vitro models we are establishing to dissect the cellular responses to oxidative damage in the mtDNA and to sort out the differential cellular consequences of accumulation of damage in each cellular genome, the nuclear and the mitochondrial genome.     

Protective effect of metallothionein-III on DNA damage in response to reactive oxygen species

Metallothionein (MT)-III is a member of a brain-specific MT family, in contrast to MT-I and MT-II that are found in most tissues and are implicated in metal ion homeostasis and as an antioxidant. To investigate the defensive role of MT-III in terms of hydroxyl radical-induced DNA damage, we used purified human MT-III. DNA damage was detected by single-strand breaks of plasmid DNA and deoxyribose degradation. In this study, we show that MT-III is able to protect against the DNA damage induced by ferric ion-nitrilotriacetic acid and H(2)O(2), and that this protective effect is inhibited by the alkylation of the sulfhydryl groups of MT-III by treatment with EDTA and N-ethylmaleimide. MT-III was also able to efficiently remove the superoxide anion, which was generated from the xanthine/xanthine oxidase system. These results strongly suggest that MT-III is involved in the protection of reactive oxygen species-induced DNA damage, probably via direct interaction with reactive oxygen species, and that MT-III acts as a neuroprotective agent.     

DNA damage by reactive oxygen species in cryopreservation and the antioxidant properties of cryoprotectors

Several disorders in the DNA structure in the cells of vertebrate and invertebrate animals, plant and algae are reviewed. Some causes of the DNA disorders, the reactive oxygen species especially, are discussed. Some data are shown that the common used cryoprotectors such as dimethylsulfoxide, glycerol, methanol, sucrose and albumin, are OH^· scavengers. Some seldom used cryoprotectors, which scavenge several forms of active oxygen, are described. It is supposed that the antioxidant properties of the cryoprotectors are essential for their mechanism of action.     

Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis

Oxidative stress may be an important factor in the development of diabetic complications. Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes. We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin. Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro. DNA was exposed to either reactive oxygen species or advanced glycation end-products. Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects. Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains. Both pravastatin and simvastatin still lacked protective activity. Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.     

Sperm activation: role of reactive oxygen species and kinases

Reactive oxygen species (ROS), such as the superoxide anion (O(2)(-*)), hydrogen peroxide (H(2)O(2)) and nitric oxide (NO*), when generated at low and controlled levels, act as second messengers. ROS regulate sperm capacitation, which is the complex series of changes allowing spermatozoa to bind to the zona pellucida surrounding the oocyte, induce the acrosome reaction (exocytotic event by which proteolytic enzymes are released) and fertilize the oocyte. Capacitating spermatozoa produce controlled amounts of ROS that regulate downstream events: first, the increase in cAMP, protein kinase A (PKA) activation and phosphorylation of PKA substrates (arginine-X-X-serine/threonine motif; 15-30 min); second, the phosphorylation of MEK (extracellular signal regulated kinase [ERK] kinase)-like proteins (30-60 min) and then that of the threonine-glutamate-tyrosine motif (>1 h); finally, the late tyrosine phosphorylation of fibrous sheath proteins (>2 h). Although all these events are ROS-dependent, the regulation by various kinases, protein kinase C, PKA, protein tyrosine kinases, the ERK pathway, etc. is different. ROS also regulate the acquisition of hyperactivated motility and the acrosome reaction by spermatozoa. ROS action is probably mediated via the sulfhydryl/disulfide pair on sperm proteins. Redundancy, cross talk, and multiple systems acting in parallel point to an array of safeguards assuring the timely function of spermatozoa.     

Hypochlorous acid-induced DNA base modification: potentiation by nitrite: biomarkers of DNA damage by reactive oxygen species

Chronic inflammation results in increased nitric oxide formation and nitrite (NO-2) accumulation. Activated phagocytes release myeloperoxidase generating the cytotoxic agent hypochlorous acid (HOCl). Reaction of HOCl with NO-2 results in the formation of nitryl chloride (NO2Cl), a potent oxidising, nitrating and chlorinating species. Exposure of DNA to NO-2 alone (up to 250 microM) at pH 7.4 did not induce oxidative DNA base damage. However, incubation of DNA with NO-2 in the presence of HOCl led to increases in thymine glycol, 5-hydroxyhydantoin, 8-hydroxyadenine and 5-chlorouracil to levels higher than those achieved by HOCl alone. No significant increases in 8-hydroxyguanine, xanthine, hypoxanthine, 2-hydroxyadenine, FAPy guanine, FAPy adenine and 8-chloroadenine were observed. HOCl-induced depletion of FAPy guanine and 8-hydroxyguanine was reduced in the presence of NO-2. Modification of DNA by HOCl/NO-2 (presumably generating NO2Cl) produces a pattern of DNA base damage products in isolated DNA that is similar to the pattern produced by HOCl but not other reactive species.     

DNA damage-induced reactive oxygen species (ROS) stress response in Saccharomyces cerevisiae

Cells are exposed to both endogenous and exogenous sources of reactive oxygen species (ROS). At high levels, ROS can lead to impaired physiological function through cellular damage of DNA, proteins, lipids, and other macromolecules, which can lead to certain human pathologies including cancers, neurodegenerative disorders, and cardiovascular disease, as well as aging. We have employed Saccharomyces cerevisiae as a model system to examine the levels and types of ROS that are produced in response to DNA damage in isogenic strains with different DNA repair capacities. We find that when DNA damage is introduced into cells from exogenous or endogenous sources there is an increase in the amount of intracellular ROS which is not directly related to cell death. We have examined the spectrum of ROS in order to elucidate its role in the cellular response to DNA damage. As an independent verification of the DNA damage-induced ROS response, we show that a major activator of the oxidative stress response, Yap1, relocalizes to the nucleus following exposure to the DNA-alkylating agent methyl methanesulfonate. Our results indicate that the DNA damage-induced increase in intracellular ROS levels is a generalized stress response that is likely to function in various signaling pathways.     

External acidification leads to formation of reactive oxygen species in rat brain synaptosomes

Formation of reactive oxygen species in rat brain synaptosomes upon acidification of the incubation medium has been studied using a fluorescent probe DCFDA. It is shown that lowering the pH from 7.4 to 7.0 and 6.0 leads to a progressive increase in fluorescence that is indicative of oxidative stress. The effect is observed regardless of the presence of calcium ions in the incubation medium. In experiments without synaptosomes, acidification of the medium causes quenching of the fluorescence of DCF (pre-oxidized dye). This testifies that the fluorescence rise in synaptosomes is indeed associated with enhanced production of reactive oxygen species. Thus, even a small reduction of external pH to 7.0 is sufficient to cause oxidative stress in brain synaptosomes.