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.     

Autophagy acts as a safeguard mechanism against G-quadruplex ligand-mediated DNA damage

G-quadruplex ligands have attracted considerable interest as novel anticancer therapeutics due to their capability to interfere with guanosine-rich DNA/RNA sequences, such as telomeres. Elucidation of the structures of telomeric G-quadruplexes has led, in the past few years, to the rational development of effective G-quadruplex-stabilizing small molecules. In the present study, we showed that short-term exposure of melanoma cells to Ant1,5—an anthracene-based ligand able to stabilize telomeric G-quadruplexes—impaired cell growth without inducing cell senescence or apoptosis. Conversely, drug-treated cells were characterized by the occurrence of typical biochemical and morphological features associated with autophagy, such as an increase in the lipidated form of the autophagic marker LC3B and the accumulation of autophagosomes. Such drug-induced autophagy occurred as a consequence of DNA damage induction, at least in part dependent on drug-mediated telomere uncapping, through a pathway converging on the cyclin-dependent kinase inhibitor 1A (CDKN1A/p21). Indeed, melanoma cells depleted for CDKN1A did not show evidence of autophagic markers upon exposure to Ant1,5. The inhibition of autophagy by a pharmacologic inhibitor or through RNAi-mediated depletion of the ATG5 gene enhanced the cytotoxic activity of Ant1,5, as revealed by the marked increase in drug-induced apoptosis. Our data outline a molecular scenario in which G-quadruplex ligand-induced telomeric dysfunctions and DNA damage are translated into an autophagic response and provide the first evidence of autophagy as a safeguard mechanism activated by melanoma cells to counteract G-quadruplex ligand-mediated cellular stress.     

Superoxide dismutases enhance H2O2-induced DNA damage and alter its site specificity

Superoxide dismutases (SODs) are involved in the protection of cells from oxygen toxicity. However, several papers have reported that the overexpression of CuZn-SOD causes oxidative damage to cells. We investigated a mechanism by which an excess of SODs accelerates oxidative stress. The presence of CuZn-SOD, Mn-SOD or Mn(II) enhanced the frequency of DNA damage induced by hydrogen peroxide (H2O2) and Cu(II), and altered the site specificity of the latter: H2O2 induced Cu(II)-dependent DNA damage with high frequency at the 5'-guanine of poly G sequences; when SODs were added, the frequency of cleavages at thymine and cytosine residues increased. SODs also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by H2O2 and Cu(II). We conclude that SODs may increase carcinogenic risks, e.g. of tumors in Down syndrome.     

L-DOPA oxidation products prevent H2O2-induced oxidative damage to cellular DNA

Using the single-cell gel electrophoresis ("Comet") assay, we show that tyrosinase-generated L-DOPA oxidation products prevent H2O2-induced oxidative DNA damage in cultured tissue cells. We propose that these oxidation products trigger cellular processes that up-regulate the overall antioxidant status of the cell, and could be incorporated into treatments of pathological conditions associated with elevated oxidative DNA damage and other manifestations of increased oxidative stress.     

The involvement of singlet oxygen in copper-phenanthroline/H2O2-induced DNA base damage: a chemiluminescent study

Copper in the presence of excess 1,10-phenanthroline, a reducing agent, and H2O2 causes DNA base damage as well as strand breakage. We have reported in previous work that a strong chemiluminescence was followed by DNA base damage in this system, which is characteristic of guanine. In the present work, the mechanism of the chemiluminescence was studied. Results show that the luminescence was inhibited by all three classes of reactive oxygen species (*OH, O2-, (1)O2) scavengers to different degrees. Singlet oxygen scavengers showed the most powerful inhibition while the other two classes of scavengers were relatively weaker. The emission intensity in D2O was 3-fold that in H2O. Comparing the effect of scavengers on the luminescence of DNA with that of dGMP, the ratio of inhibition was similar. On the other hand, DNA breakage analysis showed that inhibition by the singlet oxygen scavenger NaN3 of strand breakage was strong and comparable to that of the scavengers of the two oxygen radicals. The results suggest that singlet oxygen may be a major factor for the chemiluminescence of guanine, while DNA strand breakage may be caused by many active species.     

Acetaminophen is cardioprotective against H2O2-induced injury in vivo

Here we report our ongoing investigation of the cardiovascular effects of acetaminophen, with emphasis on oxidation-induced canine myocardial dysfunction. The objective of the current study was to investigate whether acetaminophen could attenuate exogenous H(2)O(2)-mediated myocardial dysfunction in vivo. Respiratory, metabolic, and hemodynamic indices such as left ventricular function (LVDP and +/-dP/dt(max)), and percent ectopy were measured in anesthetized, open-chest dogs during intravenous administration of 0.88 mM, 2.2 mM, 6.6 mM H(2)O(2). Following 6.6 mM H(2)O(2), tissue from the left ventricle was harvested for electron microscopy. Left ventricular function did not vary significantly between vehicle and acetaminophen groups under baseline conditions. Acetaminophen-treated dogs regained a significantly greater fraction of baseline function after high concentrations of H(2)O(2) than vehicle-treated dogs. Moreover, the incidence of H(2)O(2)-induced ventricular arrhythmias was significantly reduced in the acetaminophen-treated group. Percent ectopy following 6.6 mM concentrations of H(2)O(2) was 1 +/- 0.3 vs. 0.3 +/- 0.1 (P < 0.05) for vehicle- and acetaminophen-treated dogs, respectively. Additionally, electron micrograph images of left ventricular tissue confirmed preservation of tissue ultrastructure in acetaminophen-treated hearts when compared to vehicle. We conclude that, in the canine myocardium, acetaminophen is both functionally cardioprotective and antiarrhythmic against H(2)O(2)-induced oxidative injury.     

Cellular sites of H2O2-induced damage and their protection by nitroxides

While the exact mechanism of H2O2-induced cytotoxicity is unknown, there is considerable evidence implicating DNA as a primary target. A recent study showed that a cell-impermeable nitroxide protected mammalian cells from H2O2-induced cell killing and suggested that the protection was mediated through cell membrane-bound or extracellular factors. To further define the protective properties of nitroxides, Chinese hamster V79 cells were exposed to H2O2 with or without cell-permeable and impermeable nitroxides and selected metal chelators. EPR spectroscopy and paramagnetic line broadening agents were used to distinguish between intra- and extracellular nitroxide distribution. To study the effectiveness of nitroxide protection, in the absence of a cell membrane, H2O2-mediated damage to supercoiled plasmid DNA was evaluated. Both deferrioxamine and Tempol cross the cell membrane, and inhibited H2O2-mediated cell killing, whereas the cell-impermeable DTPA and nitroxide, CAT-1, failed to protect. Similar protective effects of the chelators and nitroxides were observed when L-histidine, which enhances intracellular injury, was added to H2O2. In contrast, when damage to plasmid DNA was induced (in the absence of a cell membrane), both nitroxides were protective. Collectively, these results do not support a role for membrane-bound or extracellular factors in mediating H2O2 cytotoxicity in mammalian cells.     

Protective effect of Nelumbo nucifera (Gaertn.) against H2O2-induced oxidative stress on H9c2 cardiomyocytes

Molecular Biology Reports - Ischemic heart disease (IHD), a severe condition of myocardium facing impediment in the supply of basic needs for cellular metabolism is caused by atherosclerosis....     

Histone peptide AKRHRK enhances H(2)O(2)-induced DNA damage and alters its site specificity

Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxicity. However, several studies have demonstrated that the metal-binding histone reacts with H(2)O(2), leading to oxidative damage to a nucleobase. We investigated whether histone can accelerate oxidative DNA damage, using a minimal model for the N-terminal tail of histone H4, CH(3)CO-AKRHRK-CONH(2), which has a metal-binding site. This histone peptide enhanced DNA damage induced by H(2)O(2) and Cu(II), especially at cytosine residues, and induced additional DNA cleavage at the 5'-guanine of GGG sequences. The peptide also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and ESR spin-trapping signal from H(2)O(2) and Cu(II). Cyclic redox reactions involving histone-bound Cu(II) and H(2)O(2), may give rise to multiple production of radicals leading to multiple hits in DNA. It is noteworthy that the histone H4 peptide with specific sequence AKRHRK can cause DNA damage rather than protection under metal-overloaded condition.     

Influence of salicylic acid on H2O2 production, oxidative stress, and H2O2-metabolizing enzymes. Salicylic acid-mediated oxidative damage requires H2O2.

We investigated how salicylic acid (SA) enhances H2O2 and the relative significance of SA-enhanced H2O2 in Arabidopsis thaliana. SA treatments enhanced H2O2 production, lipid peroxidation, and oxidative damage to proteins, and resulted in the formation of chlorophyll and carotene isomers. SA-enhanced H2O2 levels were related to increased activities of Cu,Zn-superoxide dismutase and were independent of changes in catalase and ascorbate peroxidase activities. Prolonging SA treatments inactivated catalase and ascorbate peroxidase and resulted in phytotoxic symptoms, suggesting that inactivation of H2O2-degrading enzymes serves as an indicator of hypersensitive cell death. Treatment of leaves with H2O2 alone failed to invoke SA-mediated events. Although leaves treated with H2O2 accumulated in vivo H2O2 by 2-fold compared with leaves treated with SA, the damage to membranes and proteins was significantly less, indicating that SA can cause greater damage than H2O2. However, pretreatment of leaves with dimethylthiourea, a trap for H2O2, reduced SA-induced lipid peroxidation, indicating that SA requires H2O2 to initiate oxidative damage. The relative significance of the interaction among SA, H2O2, and H2O2-metabolizing enzymes with oxidative damage and cell death is discussed.     

Protective effects of a wheat germ peptide (RVF) against H2O2-induced oxidative stress in human neuroblastoma cells

RVF (Arg-Val-Phe), a peptide derived from wheat germ, shows antioxidant properties. Here, the neuroprotective efficacies of RVF were investigated in human neuroblastoma cells (SH-SY5Y) that were pretreated with RVF (150–250 μM, 4 h) and exposed to H₂O₂ (200 μM). RVF increased viable cell numbers by 37 % and reduced the release of lactate dehydrogenase. Pretreatment with RVF also inhibited H₂O₂-induced accumulation of reactive oxygen species and maintained the mitochondrial transmembrane potential as well as preventing intracellular Ca²⁺ dysregulation during H₂O₂ exposure. Furthermore, pretreatment with RVF increased the Bcl-2/Bax ratio and blocked cleavage poly(ADP-ribose) polymerase by inhibiting caspase-3 activation, thus decreasing apoptosis.     

Vitamin C modulation of H2O2-induced damage and iron homeostasis in human cells

Vitamin C (ascorbic acid, AA) is an important antioxidant in human plasma. It is clear, however, that AA has other important, nonantioxidant roles in cells. Of particular interest is its involvement in iron metabolism, since AA enhances dietary iron absorption, increases the activity of Fe(2+)-dependent cellular enzymes, promotes Fenton reactions in vitro, and was reported to have deleterious effects in individuals with iron overload. Nevertheless, the ability of AA to modulate iron metabolism and enhance iron-dependent damage in cells, tissues, and organisms has not been fully elucidated. Here we investigated the effect of AA on iron-mediated oxidative stress in normal human fibroblasts. Incubation with physiologically relevant concentrations of AA was not harmful but sensitised cells toward H(2)O(2)-induced, iron-dependent DNA strand breakage and cell death. We also report that AA increased the levels of intracellular catalytic iron and concomitantly modulated the expression of two well-established iron-regulated genes, ferritin and transferrin receptor. In summary, we present evidence of a novel, nonantioxidant role of AA in human cells, where it increases iron availability and enhances ROS-mediated, iron-dependent damage. We suggest that AA may exacerbate the deleterious effects of metals in vivo and promote normal tissue injury in situations associated with elevated ROS production.     

Effect of H2O2-induced oxidative stress on some yeast membrane functions

This work was supported by the NATO Linkage Grant H TECH.LG 930 686 and by grant no. 204/93/2224 of the Grant Agency of the Czech Republic.     

Ascorbate and H2O2 induced oxidative DNA damage in Jurkat cells

The effect of vitamin C (ascorbate) on oxidative DNA damage was examined by first incubating cells with dehydroascorbate, which boosts the intracellular concentration of ascorbate, and then exposing cells to H₂O₂. Oxidative DNA damage was estimated by the analysis of 5-hydroxy-2′-deoxycytidine (oh⁵dCyd) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo⁸dGuo). The presence of a high concentration of ascorbate (30 mM), compared to the absence of ascorbate in cells, when exposed to H₂O₂ (200 μM), resulted in a remarkable sensitization of oh⁵dCyd from 2.7 ± 0.6 to 40.8 ± 6.1 lesions /10⁶ dCyd (15-fold). In contrast, the level of oxo⁸dGuo increased from 8.4 ± 0.4 to 12.1 ± 0.5 lesions/10⁶ dGuo (50%). The formation of oh⁵dCyd was also observed at lower concentrations of intracellular ascorbate and exogenous H₂O₂. Additional studies showed that replacement of H₂O₂ with tert-butyl hydroperoxide completely abolished damage, and that preincubation with iron and desferroxamine increased and decreased this damage, respectively. The latter studies suggest that a Fenton reaction is involved in the mechanism of damage. In conclusion, we report a novel model system in which ascorbate sensitizes H₂O₂-induced oxidative DNA damage in cells, leading to elevated levels of oh⁵dCyd and oxo⁸dGuo, with a strong bias toward the formation of oh⁵dCyd.     

Natural products inspired synthesis of neuroprotective agents against H2O2-induced cell death

Stroke is a debilitating disease and the third leading cause of death in the USA, where over 2000 new stroke cases are diagnosed every day. Treatment options for stroke-related brain damage are very limited and there is an urgent need for effective neuroprotective agents to treat these conditions. Comparison of the structures of several classes of neuroprotective natural products such as limonoids and cardiac glycosides revealed the presence of a common structural motif which may account for their observed neuroprotective activity. Several natural product mimics that incorporate this shared structural motif were synthesized and were found to possess significant neuroprotective activity. These compounds enhanced cell viability against H₂O₂ induced oxidative stress or cell death in PC12 neuronal cells. The compounds were also found to enhance and modulate Na⁺/K⁺-ATPase activity of PC12 cells, which may suggest that the observed neuroprotective activity is mediated, at least partly, through interaction with Na⁺/K⁺-ATPase.     

Differential protective effects of O-phenanthroline and catalase on H2O2-induced DNA damage and inhibition of protein synthesis in endothelial cells.

The respective roles of H2O2 and .OH radicals was assessed from the protective effects of catalase and the iron chelator o-phenanthroline on 1) the inhibition of protein synthesis, and 2) DNA damage and the related events (activation of the DNA repairing enzyme poly(ADP)ribose polymerase with the associated depletion of NAD and ATP stores) in cultured endothelial cells exposed to the enzyme reaction hypoxanthine-xanthine oxidase (HX-XO) or pure H2O2. Catalase added in the extracellular phase completely prevented all of these oxidant-induced changes. O-phenanthroline afforded a complete protective effect against DNA strand breakage and the associated activation of the enzyme poly(ADP)ribose polymerase. By contrast, iron chelation was only partially effective in maintaining the cellular NAD and ATP contents, as well as the protein synthetic activity. In addition, the ATP depletion following oxidant injury was much more profound than NAD depletion. These results indicate that: 1) .OH radical was most likely the ultimate O2 species responsible for DNA damage and activation of poly(ADP)ribose polymerase; 2) both H2O2 and .OH radicals were involved in the other cytotoxic effects (inhibition of protein synthesis and reduction of NAD and ATP stores); and 3) NAD and ATP depletion did not result solely from activation of poly(ADP)ribose polymerase, but other mechanisms are likely to be involved. These observations are also compatible with the existence of a compartmentalized intracellular iron pool.     

Peroxiredoxin 2, glutathione peroxidase,and catalase in the cytosol and membrane of erythrocytes under H2O2-induced oxidative stress

Abstract

Erythrocytes are continuously exposed to risk of oxidative injury due to oxidant oxygen species. To prevent damage, they have antioxidant agents namely, catalase (Cat), glutathione peroxidase (GPx), and peroxiredoxin 2 (Prx2). Our aim was to contribute to a better understanding of the interplay between Prx2, Cat, and GPx under H₂O₂-induced oxidative stress, by studying their changes in the red blood cell cytosol and membrane, in different conditions. These three enzymes were quantified by immunoblotting. Malondialdehyde, that is, lipoperoxidation (LPO) in the erythrocyte membrane, and membrane-bound hemoglobin (MBH) were evaluated, as markers of oxidative stress. We also studied the erythrocyte membrane protein profile, to estimate how oxidative stress affects the membrane protein structure. We showed that under increasing H₂O₂ concentrations, inhibition of the three enzymes with or without metHb formation lead to the binding of Prx2 and GPx (but not Cat) to the erythrocyte membrane. Prx2 was detected mainly in its oxidized form and the linkage of metHb to the membrane seems to compete with the binding of Prx2. Catalase played a major role in protecting erythrocytes from high exogenous flux of H₂O₂, since whenever Cat was active there were no significant changes in any of the studied parameters. When only Cat was inhibited, Prx2 and GPx were unable to prevent H₂O₂-induced oxidative stress resulting in increasing MBH and membrane LPO. Additionally, the inhibition of one or more of these enzymes induced changes in the anchor/linker proteins of the junctional complexes of the membrane cytoskeleton–lipid bilayer, which might lead to membrane destabilization.     

Lycium barbarum polysaccharides: Protective effects against heat-induced damage of rat testes and H2O2-induced DNA damage in mouse testicular cells and beneficial effect on sexual behavior and reproductive function of hemicastrated rats

Lycium barbarum, a famous Chinese medicinal herb, has a long history of use as a traditional remedy for male infertility. Polysaccharides are the most important functional constituent in L. barbarum fruits. We systematically investigated the effect of L. barbarum polysaccharides (LBP) on rat testis damage induced by a physical factor (43 degrees C heat exposure), on DNA damage of mouse testicular cells induced by a chemical factor (H2O2), and on sexual behavior and reproductive function of hemicastrated male rats. The results showed that LBP provided a protective effect against the testicular tissue damage induced by heat exposure. When compared with negative control, a suitable concentration of LBP significantly increased testis and epididymis weights, improved superoxide dismutase (SOD) activity, and raised sexual hormone levels in the damaged rat testes. LBP had a dose-dependent protective effect against DNA oxidative damage of mouse testicular cells induced by H2O2. LBP improved the copulatory performance and reproductive function of hemicastrated male rats, such as shortened penis erection latency and mount latency, regulated secretion of sexual hormones and increased hormone levels, raised accessory sexual organ weights, and improved sperm quantity and quality. The present findings support the folk reputation of L. barbarum fruits as an aphrodisiac and fertility-facilitating agent, and provide scientific evidence for a basis for the extensive use of L. barbarum fruits as a traditional remedy for male infertility in China.