Hydrogen peroxide is critical for UV-induced apoptosis inhibition

Abstract

Apoptosis is an important cell death system that deletes damaged and mutated cells, preventing the induction of cancer. We previously have reported that UV irradiation inhibited the apoptosis induced by serum starvation and cell detachment. This phenomenon is suitable for clarifying the relationship between cancer and the dysregulation of apoptosis by UV irradiation. Here, we have studied the factors responsible for this inhibition of apoptosis, focusing on reactive oxygen species (ROS) and DNA damage. Treatment with xanthine oxidase in the presence of hypoxanthine, which is known to produce superoxide anion (O₂^??) and hydrogen peroxide (H₂O₂), inhibited the induction of apoptosis. The xanthine oxidase-induced anti-apoptotic effect was suppressed in the presence of an H₂O₂-eliminating enzyme, catalase, but not in the presence of an O₂^??-eliminating enzyme, superoxide dismutase. Treatment with H₂O₂ itself significantly inhibited the induction of apoptosis. Furthermore, the effect of the inhibition of cell death by UVB irradiation and by H₂O₂ treatment decreased in H₂O₂-resistant cells. Although both UVB and H₂O₂ are known to induce DNA damage, other DNA damaging agents, like γ-irradiation and treatment with cisplatin and bleomycin, showed no inhibition of apoptosis. These findings suggested that H₂O₂ was essential to the inhibition of apoptosis, in which DNA damage had no role.     

Effects of Different Sources and Levels of Zinc on H2O2-Induced Apoptosis in IEC-6 Cells

Zinc has been shown to be an inhibitor of apoptosis for many years. The present study was designed to investigate effects of three zinc chemical forms on H₂O₂-induced cell apoptosis in IEC-6 cells via analysis of cell vitality, LDH activity, apoptosis percentage, caspase-3 activity, and Bcl-2, Bax, and caspase-3, -8, and -9 gene expression. Cells were divided into H₂O₂ and zinc sources+H₂O₂ groups, and there are three different zinc sources [zinc oxide nanoparticle (nano-ZnO), zinc oxide (ZnO), and zinc sulfate (ZnSO₄)] and three concentrations (normal = 25 μM, medium = 50 μM, and high = 100 μM) used in this article. In the present study, we found the striking cytotoxicity of H₂O₂ higher than 200 μM on cell vitality, LDH activity, and apoptosis percentage in the cells using five different concentrations (50, 100, 200, 400, and 800 μM) of H₂O₂ for 4 h. Moreover, we observed that cell vitality was increased, LDH activity and apoptotic percentage were decreased, and gene expression level of Bax and caspase-3 and -9 was markedly reduced, while gene expression level of Bcl-2 and ratio of Bcl-2/Bax were increased in normal concentration groups of nano-ZnO and ZnSO₄ compared with H₂O₂ group, but no significant difference was observed in caspase-8 gene expression. Furthermore, medium or, more intensely, high concentrations of nano-ZnO and ZnSO₄ enhanced H₂O₂-induced cell apoptosis. Compared with nano-ZnO and ZnSO₄, ZnO showed weakest protective effect on H₂O₂-induced apoptosis at normal concentration and was less toxic to cells at high level. Taken together, we proposed that preventive and protective effects of zinc on H₂O₂-induced cell apoptosis varied in IEC-6 cells with its chemical forms and concentrations, and maybe for the first time, we suggested that nano-ZnO have a protective effect on H₂O₂-induced cell apoptosis in IEC-6 cells.     

Experimental approaches to free radicals and ageing

Abstract

Probucol, a clinically used cholesterol lowering and antioxidant drug, was investigated for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) plus hydrogen peroxide (H₂O₂). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H₂O₂-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Fe-NTA is associated with a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation and DNA damage are the principal manifestation of Fe-NTA induced toxicity, which could be mitigated by probucol. Incubation of renal microsomal membrane and/or calf thymus DNA with H₂O₂ (40 mM) in the presence of Fe-NTA (0.1 mM) induces renal microsomal lipid peroxidation and DNA damage to about 2.4-fold and 5.9-fold, respectively, as compared to control (P < 0.05). Induction of renal microsomal lipid peroxidation and DNA damage was inhibited by probucol in a concentration-dependent manner. In lipid peroxidation protection studies, probucol treatment showed a concentration-dependent inhibition (10–34% inhibition; P <0.05) of Fe-NTA plus H₂O₂-induced lipid peroxidation as measured by thiobarbituric acid reacting species' (TBARS) formation in renal microsomes. Similarly, in DNA damage protection studies, probucol treatment also showed a concentration-dependent strong inhibition (36–71% inhibition; P < 0.05) of DNA damage. From these studies, it was concluded that probucol inhibits peroxidation of microsomal membrane lipids and DNA damage induced by Fe-NTA plus H₂O₂. However, because the lipid peroxidation and DNA damage studied here are regarded as early markers of carcinogenesis, we suggest that probucol may be developed as a cancer chemopreventive agent against renal carcinogenesis and other adverse effects of Fe-NTA exposure in experimental animals, in addition to being a cholesterol-lowering drug, useful for the control of hypercholestrolemia.     

The ginsenoside protopanaxatriol protects endothelial cells from hydrogen peroxide-induced cell injury and cell death by modulating intracellular redox status

Ginsenosides, the active components of the famous Chinese herb ginseng, have been suggested to possess cardiovascular-protective effects. The mechanism of ginsenosides is believed to be associated with their ability to prevent cellular oxidative stress. The purpose of this study was to explore the cytoprotective effects of the ginsenoside protopanaxatriol (PPT) on hydrogen peroxide (H₂O₂)-induced endothelial cell injury and cell death. Pretreatment of human umbilical vein endothelial cells (HUVECs) with PPT for 24 h was able to protect the cells against H₂O₂-induced injury. In addition to cell death, pretreatment with PPT could also reduce H₂O₂-induced DNA damage, overactivation of the DNA repair enzyme PARP-1, and concomitant depletion of the intracellular substrate NAD⁺. Furthermore, PPT could reverse the decrease in ATP/ADP ratio caused by H₂O₂. The metabolism of glutathione was also changed. H₂O₂ could induce a significant decrease in GSH level resulting in a decrease in the GSH/GSSG ratio. This could be prevented by pretreatment with PPT. The action was associated with increasing activities of the GSH-metabolizing enzymes glutathione reductase and glutathione peroxidase. These findings suggest that the ginsenoside PPT could protect HUVECs against H₂O₂-induced cell death via its action against oxidative stress, which may be responsible for the cardiovascular-protective action of ginseng.     

LINE‐1 hypomethylation induced by reactive oxygen species is mediated via depletion of S‐adenosylmethionine

Whether long interspersed nuclear element‐1 (LINE‐1) hypomethylation induced by reactive oxygen species (ROS) was mediated through the depletion of S‐adenosylmethionine (SAM) was investigated. Bladder cancer (UM‐UC‐3 and TCCSUP) and human kidney (HK‐2) cell lines were exposed to 20 μM H₂O₂ for 72 h to induce oxidative stress. Level of LINE‐1 methylation, SAM and homocysteine (Hcy) was measured in the H₂O₂‐exposed cells. Effects of α‐tocopheryl acetate (TA), N‐acetylcysteine (NAC), methionine, SAM and folic acid on oxidative stress and LINE‐1 methylation in the H₂O₂‐treated cells were explored. Viabilities of cells treated with H₂O₂ were not significantly changed. Intracellular ROS production and protein carbonyl content were significantly increased, but LINE‐1 methylation was significantly decreased in the H₂O₂‐treated cells. LINE‐1 methylation was restored by TA, NAC, methionine, SAM and folic acid. SAM level in H₂O₂‐treated cells was significantly decreased, while total glutathione was significantly increased. SAM level in H₂O₂‐treated cells was restored by NAC, methionine, SAM and folic acid; while, total glutathione level was normalized by TA and NAC. Hcy was significantly decreased in the H₂O₂‐treated cells and subsequently restored by NAC. In conclusion, in bladder cancer and normal kidney cells exposed to H₂O₂, SAM and Hcy were decreased, but total glutathione was increased. Treatments with antioxidants (TA and NAC) and one‐carbon metabolites (SAM, methionine and folic acid) restored these changes. This pioneer finding suggests that exposure of cells to ROS activates glutathione synthesis via the transsulfuration pathway leading to deficiency of Hcy, which consequently causes SAM depletion and eventual hypomethylation of LINE‐1. Copyright © 2015 John Wiley & Sons, Ltd.     

Honokiol ameliorates oxidative stress-induced DNA damage and apoptosis of c2c12 myoblasts by ROS generation and mitochondrial pathway

ABSTRACT

Honokiol is one of the main active components of Magnolia officinalis, and has been demonstrated to have multiple pharmacological activities against a variety of diseases. Recently, this phenolic compound is known to have antioxidant activity, but its mechanism of action remains unclear. The purpose of the current study was to evaluate the preventive effects of honokiol against oxidative stress-induced DNA damage and apoptosis in C2C12 myoblasts. The present study found that honokiol inhibited hydrogen peroxide (H₂O₂)-induced DNA damage and mitochondrial dysfunction, while reducing reactive oxygen species (ROS) formation. The inhibitory effect of honokiol on H₂O₂-induced apoptosis was associated with the up-regulation of Bcl-2 and down-regulation of Bax, thus reducing the Bax/Bcl-2 ratio that in turn protected the activation of caspase-9 and -3, and inhibition of poly (ADP-ribose) polymerase cleavage, which was associated with the blocking of cytochrome c release to the cytoplasm. Collectively, these results demonstrate that honokiol defends C2C12 myoblasts against H₂O₂-induced DNA damage and apoptosis, at least in part, by preventing mitochondrial-dependent pathway through scavenging excessive ROS.     

Study of the Mechanism of Action of p-Chloromercuribenzoate on Endonuclease from the Bacterium Serratia marcescens

The mechanism of action of p-chloromercuribenzoate (PCMB) on Serratia marcescens nuclease was investigated. The analysis showed that PCMB forms complexes with DNA. Binding of C₇H₅O₂Hg⁺ to DNA changes the secondary structure of the DNA. These changes alter the enzymatic activity of S. marcescens nuclease, which was previously found to be sensitive to the secondary structure of the substrates. The nuclease activity was either suppressed or stimulated in the presence of PCMB depending on the C₇H₅O₂Hg⁺ to nucleotide equivalent ratio. Binding of C₇H₅O₂Hg⁺ to DNA did not form an abortive enzyme–substrate complex. Binding of Mg²⁺ to the C₇H₅O₂Hg–DNA complex caused appropriate changes in secondary structure of the substrate. Since Mg²⁺ and C₇H₅O₂Hg⁺, though differing in the type of metal cation, are similar in their mechanisms of influence on enzymatic activity of S. marcescens nuclease, the identity of other metal-containing effectors in their mechanism of action on Serratia marcescens nuclease is assumed.     

Mechanism of DNA Damage and Apoptosis Induced by Tetrahydropapaveroline, a Metabolite of Dopamine

Tetrahydropapaveroline (THP), a metabolite of dopamine, has been suspected to be associated with dopaminergic neurotoxicity of L-DOPA. THP induced apoptosis in human leukemia cell line HL-60 cells, but did not in its hydrogen peroxide (H₂O₂)-resistant clone HP100. THP-induced DNA ladder formation in HL-60 cells was inhibited by a metal chelator. THP induced damage to ³²P-labeled DNA fragments in the presence of metals. In the presence of Fe(III)EDTA, THP caused DNA damage at every nucleotide. The DNA damage was inhibited by free hydroxy radical (^·OH) scavengers and catalase, suggesting that the Fe(III)EDTA-mediated DNA damage is mainly due to ^·OH generation. In the presence of Cu(II), THP caused DNA damage mainly at T and G of 5′-TG-3′ sequence. The inhibitive effect of catalase and bathocuproine on Cu(II)-mediated DNA damage suggested that H₂O₂ and Cu(I) participate in the DNA damage. This study demonstrated that THP-induced apoptosis via reactive oxygen species generated from reaction of H₂O₂ and metals plays an important role in cytotoxicity of L-DOPA.     

Micromolar concentrations of hydrogen peroxide induce oxidative DNA lesions more efficiently than millimolar concentrations in mammalian cells

Reactive oxygen species produce oxidized bases, deoxyribose lesions and DNA strand breaks in mammalian cells. Previously, we demonstrated that aldehydic DNA lesions (ADLs) were induced in mammalian cells by 10 mM hydrogen peroxide (H₂O₂). Interestingly, a bimodal H₂O₂ dose–response relationship in cell toxicity has been reported for Escherichia coli deficient in DNA repair as well as Chinese hamster ovary (CHO) cells. Furthermore, it has been demonstrated that H₂O₂ causes single-strand breaks in purified DNA in the presence of iron and induces mitochondrial DNA damage in CHO cells with a biphasic dose–response curve. Here we show that H₂O₂ produces ADLs at concentrations as low as 0.06 mM in HeLa cells and that lower concentrations of H₂O₂ were much more efficient at inducing ADLs than higher concentrations. This dose–response curve is strikingly similar to that for cell killing effects in E.coli deficient in DNA repair exposed to H₂O₂. Interestingly, serial treatment of submillimolar levels of H₂O₂ induced a massive accumulation of ADLs. The toxicity arising from H₂O₂ determined by intracellular NAD(P)H in cells correlated well with the formation of ADLs. The addition of dipyridyl, an iron (II)-specific chelator, significantly protected against DNA damage and cell toxicity from submillimolar, but not millimolar, amounts of H₂O₂. These results suggest that ADLs induced by submillimolar levels of H₂O₂ may be due to a Fenton-type reaction between H₂O₂ and intracellular iron ions in mammalian cells.     

Interindividual differences in initial DNA repair capacity when evaluating H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced DNA damage in extended-term cultures of human lymphocytes using the comet assay

It has been suggested that extended-term cultures of human lymphocytes could be used as a complement to cell lines based on transformed cells when testing the genotoxicity of chemicals. To investigate whether the pattern of induced DNA damage and its subsequent repair differs significantly between cultures based on different blood donors, hydrogen peroxide (H₂O₂)-induced DNA damage was measured in cultures from four different subjects using the comet assay. The DNA damage was significantly increased in all cultures after 10 min exposure to 0.25 mmol/L H₂O₂, and there was a significant decrease in the H₂O₂-induced DNA damage in all cultures after 30 min of DNA repair. The level of damage varied between the different donors, especially after the repair. Using PCR and DNA sequencing, exon 5 of the p53 gene was sequenced in the lymphocytes from the donors with the lowest and highest residual damage. No such mutation was found. Mouse lymphoma L5178Y cells carrying the p53 mutation in exon 5 were included as a reference. These cells were found to be less sensitive toward the H₂O₂-induced DNA damage, and they were also found to have a rather low DNA repair capacity. The demonstrated variation in H₂O₂-induced DNA damage and DNA repair capacity between the cultures from the different subjects may be important from a risk assessment perspective, but is obviously not of decisive importance when it comes to the development of a routine assay for genotoxicity.     

The free radical scavenging and anti-inflammatory activities of gallate-chitooligosaccharides in human lung epithelial A549 cells

In this study, gallic acid-conjugated chitooligosaccharides (gallate-COS) was evaluated for its enhancing capabilities in free radical scavenging and anti-inflammatory activities in human lung epithelial A549 cells. It was found that gallate-COS possesses significantly high DPPH radical scavenging activity and protective effect against H₂O₂-induced DNA damage. Likewise, gallate-COS decreased the production of intracellular reactive oxygen species in H₂O₂-stimulated A549 cells. Moreover, the suppressive effects of gallate-COS on COX-2 expression and PGE₂ production from LPS-stimulated A549 cells were evidenced. Finally, the productions of cytokines including IL-8 and TNF-α were also inhibited by gallate-COS in a dose-dependent manner. Collectively, these results suggest that gallate-COS could be used as a functional ingredient with potent antioxidant and anti-inflammatory benefits.     

Mammalian MutY homolog (MYH or MUTYH) protects cells from oxidative DNA damage

MutY DNA glycosylase homologs (MYH or MUTYH) reduce G:C to T:A mutations by removing misincorporated adenines or 2-hydroxyadenines paired with guanine or 8-oxo-7,8-dihydroguanine (8-oxo-G). Mutations in the human MYH (hMYH) gene are associated with the colorectal cancer predisposition syndrome MYH-associated polyposis. To examine the function of MYH in human cells, we regulated MYH gene expression by knockdown or overproduction. MYH knockdown human HeLa cells are more sensitive to the killing effects of H₂O₂ than the control cells. In addition, hMYH knockdown cells have altered cell morphology, display enhanced susceptibility to apoptosis, and have altered DNA signaling activation in response to oxidative stress. The cell cycle progression of hMYH knockdown cells is also different from that of the control cells following oxidative stress. Moreover, hMYH knockdown cells contain higher levels of 8-oxo-G lesions than the control cells following H₂O₂ treatment. Although MYH does not directly remove 8-oxo-G, MYH may generate favorable substrates for other repair enzymes. Overexpression of mouse Myh (mMyh) in human mismatch repair defective HCT15 cells makes the cells more resistant to killing and refractory to apoptosis by oxidative stress than the cells transfected with vector. In conclusion, MYH is a vital DNA repair enzyme that protects cells from oxidative DNA damage and is critical for a proper cellular response to DNA damage.     

Genoprotection and genotoxicity of green tea (Camellia sinensis): Are they two sides of the same redox coin?

Abstract

Objectives

Regular intake of green tea associates with lower DNA damage and increased resistance of DNA to oxidant challenge. However, in vitro pro-oxidant effects of green tea have been reported. Both effects could be mediated by hydrogen peroxide (H₂O₂) which is generated by autoxidation of tea catechins. In large amounts, H₂O₂ is genotoxic, but low concentrations could activate the redox-sensitive antioxidant response element (ARE) via the Keap-1/Nrf2 redox switch, inducing genoprotective adaptations. Our objective was to test this hypothesis.

Methods

Peripheral lymphocytes from healthy volunteers were incubated for 30 minutes at 37°C in freshly prepared tea solutions (0.005, 0.01, 0.05%w/v (7, 14, 71 µmol/l total catechins) in phosphate buffered saline (PBS), with PBS as control) in the presence and absence of catalase (CAT). H₂O₂ in tea was measured colorimetrically. Oxidation-induced DNA lesions were measured by the Fpg-assisted comet assay.

Results

H₂O₂ concentrations in 0.005, 0.01, and 0.05% green tea after 30 minutes at 37°C were, respectively, ～3, ～7, and ～52 µmol/l. Cells incubated in 0.005 and 0.01% tea showed less (P < 0.001) DNA damage compared to control cells. Cells treated with 0.05% green tea showed ～50% (P < 0.001) more DNA damage. The presence of CAT prevented this damage, but did not remove the genoprotective effects of low-dose tea. No significant changes in expression of ARE-associated genes (HMOX1, NRF2, KEAP1, BACH1, and hOGG1) were seen in cells treated with tea or tea + CAT.

Conclusion

Genoprotection by low-dose green tea could be due to direct antioxidant protection by green tea polyphenols, or to H₂O₂-independent signalling pathways.     

Divalent-metal-dependent nucleolytic activity of Cu, Zn superoxide dismutase

The known action of Cu, Zn superoxide dismutase (holo SOD) that converts O₂ ⁻ to O₂ and H₂O₂ plays a crucial role in protecting cells from toxicity of oxidative stress. However, the overproduction of holo SOD does not result in increased protection but rather creates a variety of unfavorable effects, suggesting that too much holo SOD may be injurious to the cells. In the in vitro study, we report a finding that the holo SOD from bovine erythrocytes and its apo form possess a divalent-metal-dependent nucleolytic activity, which was confirmed by UV–vis absorption titration of calf thymus DNA (ctDNA) with the holo SOD, quenching of holo SOD intrinsic fluorescence by ctDNA, and by gel electrophoresis monitoring conversion of DNA from the supercoiled DNA to nicked and linear forms, and fragmentation of a linear λDNA. Moreover, the DNA cleavage activity was examined in detail under certain reaction conditions. The steady-state study indicates that DNA cleavage supported by both forms of SOD obeys Michaelis–Menten kinetics. On the other hand, the assays with some other proteins indicate that this new function is specific to some proteins including the holo SOD. Therefore, this study reveals that the divalent-metal-dependent DNA cleavage activity is an intrinsic property of the holo SOD, which is independent of its natural metal (copper and zinc) sites, and may provide an alternative insight into the link between SOD enzymes and neurodegenerative disorders.     

Human cancer cell (Jurkat) killing by the cyanobacterial metabolite calothrixin A

Calothrixin A is a bioactive metabolite of the cyanobacterium Calothrix and has been shown to be active at nanomolar concentrations against human HeLa cancer cells. It induces apoptotic killing of human Jurkat cancer cells in a time- and concentration-dependent manner, as revealed by flow cytometry, morphological evidence from electron microscopy and DNA fragmentation. It also causes G2/M cell cycle arrest, which is indicative of intracellular DNA damage. It was shown to be redox-active, as measured by oxygen uptake in the presence of dithiothreitol, and caused cleavage of plasmid DNA, as revealed by electrophoresis. This cleavage was blocked by the H₂O₂ scavenger catalase, but not by other scavengers of reactive oxygen species (ROS). Calothrixin A appeared to induce intracellular formation of ROS as measured by fluorescence of dichlorodihydrofluorescein diacetate. Conditions were not found, however, under which the calothrixin A-induced apoptosis could be reversed by the scavengers, or by the metal chelator desferal. Menadione was compared with calothrixin A. The IC₅₀ at which this compound caused apoptosis was significantly higher. It also induced cell cycle arrest at higher concentrations, although caused plasmid DNA cleavage at lower concentrations. It is postulated that the greater effectiveness of calothrixin A in killing cells is related to its ring structure, which has the characteristics of a DNA intercalator, thereby localizing its quinone-dependent effects.     

Chloroethylnitrosourea-induced cell death and genotoxicity: Cell cycle dependence and the role of DNA double-strand breaks,HR and NHEJ

Chloroethylnitrosureas (CNUs) are powerful DNA-reactive alkylating agents used in cancer therapy. Here, we analyzed cyto- and genotoxicity of nimustine (ACNU), a representative of CNUs, in synchronized cells and in cells deficient in repair proteins involved in homologous recombination (HR) or nonhomologous end-joining (NHEJ). We show that HR mutants are extremely sensitive to ACNU, as measured by colony formation, induction of apoptosis and chromosomal aberrations. The NHEJ mutants differed in their sensitivity, with Ku80 mutants being moderately sensitive and DNA-PKcs mutated cells being resistant. HR mutated cells displayed a sustained high level of γH2AX foci and displayed co-staining with Rad51 and 53BP1, indicating DNA double-strand breaks (DSB) to be formed. Using synchronized cells, we analyzed whether DSB formation after ACNU treatment was replication-dependent. We show that γH2AX foci were not induced in G₁ but increased significantly in S phase and remained at a high level in G₂, where a fraction of cells became arrested and underwent, with a delay of > 12 h, cell death by apoptosis and necrosis. Rad51, ATM, MDC-1 and RPA-2 foci were also formed and shown to co-localize with γH2AX foci induced in S phase, indicating that the DNA damage response was activated. All effects observed were abrogated by MGMT, which repairs O⁶-chloroethylguanine that is converted into DNA cross-links. We deduce that the major genotoxic and killing lesion induced by CNUs are O⁶-chloroethylguanine-triggered cross-links, which give rise to DSBs in the treatment cell cycle, and that HR, but not NHEJ, is the major route of protection against this group of anticancer drugs. Base excision repair had no significant impact on ACNU-induced cytotoxicity.     

Phosphorothioate DNA as an antioxidant in bacteria

Diverse bacteria contain DNA with sulfur incorporated stereo-specifically into their DNA backbone at specific sequences (phosphorothioation). We found that in vitro oxidation of phosphorothioate (PT) DNA by hydrogen peroxide (H₂O₂) or peracetic acid has two possible outcomes: DNA backbone cleavage or sulfur removal resulting in restoration of normal DNA backbone. The physiological relevance of this redox reaction was investigated by challenging PT DNA hosting Salmonella enterica cells using H₂O₂. DNA phosphorothioation was found to correlate with increasing resistance to the growth inhibition by H₂O₂. Resistance to H₂O₂ was abolished when each of the three dnd genes, required for phosphorothioation, was inactivated. In vivo, PT DNA is more resistant to the double-strand break damage caused by H₂O₂ than PT-free DNA. Furthermore, sulfur on the modified DNA was consumed and the DNA was converted to PT-free state when the bacteria were incubated with H₂O₂. These findings are consistent with a hypothesis that phosphorothioation modification endows DNA with reducing chemical property, which protects the hosting bacteria against peroxide, explaining why this modification is maintained by diverse bacteria.     

Protective effects of cyclosativene on H2O2-induced injury in cultured rat primary cerebral cortex cells

Sesquiterpenes have attracted much interest with respect to their protective effect against oxidative damage that may be the cause of many diseases including several neurodegenerative disorders and cancer. Our previous unpublished work suggested that cyclosativene (CSV), a tetracyclic sesquiterpene, has antioxidant and anticarcinogenic features. However, little is known about the effects of CSV on oxidative stress induced neurotoxicity. We used hydrogen peroxide (H₂O₂) exposure for 6 h to model oxidative stress. Therefore, this experimental design allowed us to explore the neuroprotective potential of CSV in H₂O₂-induced toxicity in new-born rat cerebral cortex cell cultures for the first time. For this aim, MTT and lactate dehydrogenase release assays were carried out to evaluate cytotoxicity. Total antioxidant capacity (TAC) and total oxidative stress (TOS) parameters were used to evaluate oxidative changes. In addition to determining of 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels, the single cell gel electrophoresis (or Comet assay) was also performed for measuring the resistance of neuronal DNA to H₂O₂-induced challenge. Our results showed that survival and TAC levels of the cells decreased, while TOS, 8-OH-dG levels and the mean values of the total scores of cells showing DNA damage (Comet assay) increased in the H₂O₂ alone treated cultures. But pre-treatment of CSV suppressed the cytotoxicity, genotoxicity and oxidative stress which were increased by H₂O₂. On the basis of these observations, it is suggested that CSV as a natural product with an antioxidant capacity in mitigating oxidative injuries in the field of neurodegenerative disorders.     

The mechanism of bilirubin-photosensitized DNA strand breakage in human cells exposed to phototherapy light

Exposure of normal human fibroblasts to visible light (420–490 nm) in the presence of exogenously added 1–100 μg/ml bilirubin enhanced the level of DNA strand breakage compared with cells irradiated in the absence of added bilirubin. Treatment of cells in the dark with an irradiated bilirubin solution also induced DNA strand breaks. However, strand breakage was not detected in cells treated with an irradiated bilirubin solution that had been incubated with catalase (H₂O₂: H₂O₂ oxidoreductase EC 1.11.1.6). Examination of irradiated bilirubin solutions demonstrated the presence of hydrogen peroxide although, apparently, not at concentrations sufficient to account for the level of DNA strand breakage detected. Hence, irradiation of bilirubin results in the generation of hydrogen peroxide and possibly other peroxides that can cause DNA damage.     

3-Bromopyruvate antagonizes effects of lactate and pyruvate, synergizes with citrate and exerts novel anti-glioma effects

Oxidative stress-energy depletion therapy using oxidative stress induced by D-amino acid oxidase (DAO) and energy depletion induced by 3-bromopyruvate (3BP) was reported recently (El Sayed et al., Cancer Gene Ther., 19, 1–18, 2012). Even in the presence of oxygen, cancer cells oxidize glucose preferentially to produce lactate (Warburg effect) which seems vital for cancer microenvironment and progression. 3BP is a closely related structure to lactate and pyruvate and may antagonize their effects as a novel mechanism of its action. Pyruvate exerted a potent H₂O₂ scavenging effect to exogenous H₂O₂, while lactate had no scavenging effect. 3BP induced H₂O₂ production. Pyruvate protected against H₂O₂-induced C6 glioma cell death, 3BP-induced C6 glioma cell death but not against DAO/D-serine-induced cell death, while lactate had no protecting effect. Lactate and pyruvate protected against 3BP-induced C6 glioma cell death and energy depletion which were overcome with higher doses of 3BP. Lactate and pyruvate enhanced migratory power of C6 glioma which was blocked by 3BP. Pyruvate and lactate did not protect against C6 glioma cell death induced by other glycolytic inhibitors e.g. citrate (inhibitor of phosphofructokinase) and sodium fluoride (inhibitor of enolase). Serial doses of 3BP were synergistic with citrate in decreasing viability of C6 glioma cells and spheroids. Glycolysis subjected to double inhibition using 3BP with citrate depleted ATP, clonogenic power and migratory power of C6 glioma cells. 3BP induced a caspase-dependent cell death in C6 glioma. 3BP was powerful in decreasing viability of human glioblastoma multiforme cells (U373MG) and C6 glioma in a dose- and time-dependent manner.