Antioxidant and pro-oxidant actions of flavonoids: effects on DNA damage induced by nitric oxide, peroxynitrite and nitroxyl anion

Antioxidant and pro-oxidant activities of flavonoids have been reported. We have studied the effects of 18 flavonoids and related phenolic compounds on DNA damage induced by nitric oxide (NO), peroxynitrite, and nitroxyl anion (NO⁻). Similarly to our previous findings with catecholamines and catechol-estrogens, DNA single-strand breakage was induced synergistically when pBR322 plasmid was incubated in the presence of an NO-releasing compound (diethylamine NONOate) and a flavonoid having an ortho-trihydroxyl group in either the B ring (e.g., epigallocatechin gallate) or the A ring (e.g., quercetagetin). Either NO or any of the above flavonoids alone did not induce strand breakage significantly. However, most of the tested flavonoids inhibited the peroxynitrite-mediated formation of 8-nitroguanine in calf-thymus DNA, measured by a new HPLC-electrochemical detection method, as well as the peroxynitrite-induced strand breakage. NO⁻ generated from Angeli’s salt caused DNA strand breakage, which was also inhibited by flavonoids but at only high concentrations. On the basis of these findings, we propose that NO⁻ and/or peroxynitrite could be responsible for DNA strand breakage induced by NO and a flavonoid having an ortho-trihydroxyl group. Our results indicate that flavonoids have antioxidant properties, but some act as pro-oxidants in the presence of NO.     

Anthocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth muscle and hepatoma cells

Anthocyanins are flavonoids present in a variety of pigmented food and, like other flavonoids, seem to play a role in preventing human pathologies related to oxidative stress. In fact, anthocyanins have been shown to exert antiproliferative effects in cell cultures and exhibit antiinflammatory and vasoprotective activities in animal models. Although these biological activities have been related to their antioxidant properties, little is known on the molecular mechanism of action of anthocyanins. The effects of pretreatment with the anthocyanins delphinidin, cyanidin, and their glycoside and rutinoside derivatives against induction of DNA damage induced by tert-butyl-hydroperoxide (TBHP) were evaluated in rat smooth muscle and in rat hepatoma cell lines using alkaline single cell gel electrophoresis (Comet test). In addition, a possible protection exerted by anthocyanins on cell killing, lipid peroxidation, and redox state alterations induced by TBHP was also investigated. It was found that the treatment with TBHP induces the formation of DNA single strand breaks (SSB) and oxidised bases, along with cell killing, lipid peroxidation and redox state alteration. Our data demonstrate that anthocyanins are effective against cytotoxicity, DNA SSB formation and lipid peroxidation induced by TBHP, but they do not have any detectable effect against impairment by TBHP of cellular redox state and on protection against DNA bases oxidation. The presence of a sugar moiety in anthocyanin derivatives reduced this protective effect, mainly in rat hepatoma cells. The different activity of anthocyanins and their derivatives may be explained taking into account a structure/function relationship that could also influence anthocyanin intracellular localisation.     

Nitroxide radicals protect cultured rat embryos and yolk sacs from diabetic-induced damage

BACKGROUND: Diabetic teratogenicity relates, partly, to embryonic oxidative stress and the extent of the embryonic damage can apparently be reduced by antioxidants. We investigated the effects of superoxide dismutase-mimics nitroxides, 2,2,6,6-tetramethyl piperidine-N-oxyl (TPL) as an effective antioxidant, on diabetes-induced embryopathy. METHODS: Embryos (10.5 day old) and their yolk sacs from Sabra female rats were cultured for 28 h in the absence or in the presence of nitroxides at 0.05-0.4 mM in control, diabetic subteratogenic, or diabetic teratogenic media, and monitored for growth retardation and congenital anomalies. The oxidant/antioxidant status was examined by oxygen radical absorbance capacity and lipid peroxidation assays, whereas the yolk sac function was evaluated by endocytosis assay. RESULTS: Diabetic culture medium inhibited embryonic and yolk sac growth, induced a high rate of NTDs, reduced yolk sac endocytosis and embryonic antioxidant capacity, and increased lipid peroxidation. These effects were more prominent in the embryos with NTD compared to those without NTD. TPL added to diabetic teratogenic medium improved embryonic and yolk sac growth, reduced the rate of NTDs, and improved yolk sac function. The oxidant/antioxidant status of embryos was also improved. TPL at 1 mM did not damage the embryos cultured in control medium. CONCLUSIONS: In diabetic culture medium, oxidative damage is higher in the malformed rat embryos compared to those without anomalies; the nitroxide provides protection against diabetes-induced teratogenicity in a dose-dependent manner. The yolk sac damage, apparently caused by the same mechanism, might be an additional contributor to the embryonic damage observed in diabetes.     

The chemistry of DNA damage from nitric oxide and peroxynitrite

Nitric oxide is a key participant in many physiological pathways; however, its reactivity gives it the potential to cause considerable damage to cells and tissues in its vicinity. Nitric oxide can react with DNA via multiple pathways. Once produced, subsequent conversion of nitric oxide to nitrous anhydride and/or peroxynitrite can lead to the nitrosative deamination of DNA bases such as guanine and cytosine. Complex oxidation chemistry can also occur causing DNA base and sugar oxidative modifications. This review describes the different mechanisms by which nitric oxide can damage DNA. First, the physiological significance of nitric oxide is discussed. Details of nitric oxide and peroxynitrite chemistry are then given. The final two sections outline the mechanisms underlying DNA damage induced by nitric oxide and peroxynitrite.     

Relationships between nitric oxide, nitroxyl ion, nitrosonium cation and peroxynitrite.

This review is concerned mainly with the three redox-related, but chemically distinct, species NO-, NO. and NO+, with greatest emphasis being placed on the chemistry and biology of the nitroxyl ion. Biochemical routes for the formation of nitroxyl ion and methods for showing the intermediacy of this species are discussed, together with chemical methods for generating nitroxyl ion in solution. Reactions of nitroxyl ion with NO., thiols, iron centres in haem and with dioxygen are reviewed The significance of the reaction between NO- and dioxygen as a source of peroxynitrite is assessed, and attention drawn to the possible significance of the spin state of the nitroxyl ion in this context. The biological significance of nitrosation and the importance of S-nitrosothiols and certain metal nitrosyl complexes as carriers of NO+ at physiological pH is stressed. Some features in the chemistry of peroxynitrite are noted.     

Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient

Indolinonic nitroxide radicals efficiently scavenge oxygen- and carbon-centered radicals. They protect lipid and protein systems against oxidative stress, but little is known about their capacity to protect DNA against radical-mediated damage. We compare indolinonic nitroxides and the piperidines TEMPO and TEMPOL for their ability to inhibit strand breaks inflicted on DNA when it is illuminated in vitro in the presence of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a UVA-absorbing sunscreen. We used spin-trapping EPR to examine the formation of radicals and plasmid nicking assays to evaluate DNA strand breakage. The results have a two-fold interest. First, they show that all the nitroxides tested efficiently prevent DNA damage in a dose-dependent fashion. Vitamin E had no effect under the conditions used. Second, they show that carbon-centered radicals are produced on illumination of DBM and its relative and that their formation is probably responsible for the direct strand breaks found when naked DNA is illuminated in vitro in their presence. Additional work on the ability of sunscreens to enter human cells and their response to the light that penetrates sunscreen-protected skin would be necessary before any conclusion could be drawn as to whether the results reported here are relevant to human use of sunscreens.     

Exogenous nitric oxide protect cucumber roots against oxidative stress induced by salt stress.

Mitochondria are subcellular organelles with an essentially oxidative type of metabolism. The production of reactive oxygen species (ROS) in it increases under stress conditions and causes oxidative damage. In the present study, effects of exogenous sodium nitroprusside (SNP), a nitric oxide (NO) donor, on both the ROS metabolism in mitochondria and functions of plasma membrane (PM) and tonoplast were studied in cucumber seedlings treated with 100mM NaCl. NaCl treatment induced significant accumulation of H(2)O(2) and led to serious lipid peroxidation in cucumber mitochondria, and the application of 50muM SNP stimulated ROS-scavenging enzymes and reduced accumulation of H(2)O(2) in mitochondria of cucumber roots induced by NaCl. As a result, lipid peroxidation of mitochondria decreased. Further investigation showed that application of SNP alleviated the inhibition of H(+)-ATPase and H(+)-PPase in PM and/or tonoplast by NaCl. While application of sodium ferrocyanide (an analog of SNP that does not release NO) did not show the effect of SNP, furthermore, the effects of SNP were reverted by addition of hemoglobin (a NO scavenger).     

Estrogens protect against hydrogen peroxide and arachidonic acid induced DNA damage

The ability of estrogens to protect against DNA damage induced by either hydrogen peroxide or arachidonic acid alone or in combination with Cu²⁺ was investigated. DNA strand breaks were determined by conversion of double stranded supercoiled ØX-174 RFI DNA to double stranded open circular DNA and linear single stranded DNA. Estradiol-17β significantly decreased the formation of single and double strand breaks in DNA induced by H₂O₂ alone or with Cu²⁺. Equilin (an equine estrogen) was more effective than estradiol-17β at the doses tested. Arachidonic acid in the presence of Cu²⁺ caused the formation of high levels of linear DNA which was protected by estrogen with equilen being more effective. These studies suggest that estrogens through this protective effect on DNA damage might contribute to cardioprotection.     

Linoleic acid and antioxidants protect against DNA damage and apoptosis induced by palmitic acid

Polyunsaturated fats are the main target for lipid peroxidation and subsequent formation of mutagenic metabolites, but diets high in saturated fats are more strongly associated with adverse health effects. We show that the common saturated fatty acid, palmitic acid, is a potent inducer of DNA damage in an insulin-secreting cell line, and in primary human fibroblasts. Damage is not associated with upregulation of inducible nitric oxide synthase, but is prevented by two different antioxidants, alpha-lipoic acid and 3,3'-methoxysalenMn(III) (EUK134), which also partly prevent palmitic acid-induced apoptosis and growth inhibition. Since mutagenic metabolites can be formed from peroxidation of polyunsaturated fatty acids, co-administration of palmitic and a polyunsaturated fatty acid might be particularly harmful. Palmitic acid-induced DNA damage is instead prevented by linoleic acid, which is acting here as a protective agent against oxidative stress, rather than as a source of mutagenic metabolites. These results illustrate the complexity of the relationship of dietary fat intake to genotoxicity.     

Sodium deoxycholate causes nitric oxide mediated DNA damage in oesophageal cells

Patients with chronic gastro-oesophageal reflux disease experience the reflux of acid and bile into the distal oesophagus. The secondary bile salt sodium deoxycholate (NDC) is implicated in the induction of mucosal injury during reflux episodes. This study hypothesized that NDC damages DNA in oesophageal cells by an oxidative mechanism. In the oesophageal cell line HET1-A, increased production of nitric oxide (NO) was measured in NDC-treated cells. Protection from DNA strand breaks induced by NDC (10 µm) was observed in cells coincubated with the nitric oxide scavenger C-PTIO (p<0.012) or pre-incubated with the NO synthase inhibitor L-NAME (p<0.009) or the NFκB inhibitor, TPCK (p<0.036). Collectively these data implicate the involvement of NFκB and nitric oxide synthase in the DNA damage induced by NDC in oesophageal cells. In conclusion, NDC-driven NO production may play an important role in inducing DNA damage during episodes of gastro-oesophageal reflux and thereby contribute to reflux-related carcinogenesis.     

Time- and concentration-dependent production of superoxide anion, nitric oxide, DNA damage and cellular death by ricin in the J774A.1 macrophage cells

The time- and concentration-dependent effects of ricin on some biomarkers of cellular toxicity, including production of superoxide anion (O2-), nitric oxide (NO), and DNA single strand breaks (SSB), as well as cellular death, have been examined in the J774A.1 macrophage cell cultures. Various concentrations of ricin have been added to various cell cultures, and the cells were incubated for 12, 24, 36, and 48 hours. Following 12 hour incubation, ricin did not cause significant increases in any of those biomarkers. However, time- and concentration-dependent increases were observed in the induction of all the biomarkers after incubation for 24-48 hours. Approximately twofold increases in the production of O2- were observed after incubation with 1 and 10 ng/mL of ricin for 24 and 36-48 hours, respectively. The concentrations of ricin that caused approximately twofold increases in the rate of DNA-SSB are 10 and 1-10 ng/mL after 24 and 36-48 hours incubation, respectively. Approximately twofold increases in NO production were only observed after incubation of the cultures with 1-10 ng/mL of ricin for 36-48 hours. Fifty percent reductions in cellular viability were also observed with ricin concentrations of 10-100, 10, and 1-10 ng/mL, after incubation for 24, 36, and 48 hours, respectively.     

NO-mesalamine protects colonic epithelial cells against apoptotic damage induced by proinflammatory cytokines

The activation of a self-amplifying cascade of caspases, of which caspase-8 is the apical protease, mediates Fas-, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-, and TNF-alpha-induced apoptosis in colon cell lines. Nitric oxide (NO) protects from apoptosis induced by Fas and TNF-alpha. We examined whether NCX-456, an NO-releasing derivative of mesalamine, protects colon epithelial cells from cytokine-induced apoptosis. Caco-2 and HT-29 cell lines express death factor receptors and are driven to apoptosis in response to incubation with Fas-agonistic antibody, TNF-alpha/interferon-gamma, and TRAIL. The two novel observations reported here are that 1) cotreatment of cells with NCX-456, but not mesalamine, resulted in concentration-dependent protection against death factor-induced apoptosis and inhibition of caspase activity, and 2) exposure to dithiothreitol, an agent that effectively removes NO from thiol groups, resulted in a 70% recovery of caspase activity, which is consistent with S-nitrosation as a major mechanism for caspase inactivation. These data suggest that caspase S-nitrosation represents a mechanism for protection of colonic mucosal epithelial cells from death factor-induced death.     

Selenium and GPx-1 overexpression protect mammalian cells against UV-induced DNA damage

Supplementation of the culture media of human MCF-7 breast carcinoma cells or mouse fibroblasts with low levels of selenium (30 nM) provided as sodium selenite was shown to protect these cells from ultraviolet (UV)-induced chromosome damage, as quantified by micronucleus assay. Selenium supplementation was also effective in reducing UV-induced gene mutations as measured in the lacI shuttle vector model. Protection was dependent on functional BRCA1 activity, a protein implicated in breast cancer risk and DNA damage repair. In addition, overexpression of GPx-1, a selenoprotein with antioxidant activity, also attenuated UV induced micronuclei formation in the absence of selenium supplementation. Combining selenium supplementation with GPx-1 overexpression further reduced UV-induced micronucleus frequency. These data provide evidence that the benefits of selenium supplementation might be either through the prevention or repair of DNA damage, and they implicate at least one selenoprotein (GPx-1) in the process.     

Methylglyoxal-induced nitric oxide and peroxynitrite production in vascular smooth muscle cells

Methylglyoxal (MG) is a metabolite of glucose. Our previous study demonstrated an elevated MG level with an increased oxidative stress in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. Whether MG causes the generation of nitric oxide (NO) and superoxide anion (O2*-), leading to peroxynitrite (ONOO-) formation in VSMCs, was investigated in the present study. Cultured rat thoracic aortic SMCs (A-10) were treated with MG or other different agents. Oxidized DCF, reflecting H2O2 and ONOO- production, was significantly increased in a concentration- and time-dependent manner after the treatment of SMCs with MG (3-300 microM) for 45 min-18 h (n = 12). MG-increased oxidized DCF was effectively blocked by reduced glutathione or N-acetyl-l-cysteine, as well as L-NAME (p < 0.05, n = 12). Both O2*- scavenger SOD and NAD(P)H oxidase inhibitor DPI significantly decreased MG-induced oxidized DCF formation. MG significantly and concentration-dependently increased NO and O2*- generation in A-10 cells, which was significantly inhibited by L-NAME and SOD or DPI, respectively. In conclusion, MG induces significant generation of NO and O2*- in rat VSMCs, which in turn causes ONOO- formation. An elevated MG level and the consequential ROS/RNS generation would alter cellular signaling pathways, contributing to the development of different insulin resistance states such as diabetes or hypertension.     

Regulation of xanthine oxidase by nitric oxide and peroxynitrite

Xanthine oxidase (XO) is a central mechanism of oxidative injury as occurs following ischemia. During the early period of reperfusion, both nitric oxide (NO(*)) and superoxide (O-*(2)) generation are increased leading to the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the presence and nature of the interactions of NO(*) or ONOO(-) with XO and the role of this process in regulating oxidant generation. Therefore, we determined the dose-dependent effects of NO(*) and ONOO(-) on the O-*(2) generation and enzyme activity of XO, respectively, by EPR spin trapping of O-*(2) using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and spectrophotometric assay. ONOO(-) markedly inhibited both O-*(2) generation and XO activity in dose-dependent manner, while NO(*) from NO(*) gas in concentrations up to 200 microM had no effect. Furthermore, we observed that NO(*) donors such as NOR-1 also inhibited O-*(2) generation and XO activity; however, these effects were O-*(2)-dependent and blocked by superoxide dismutase or ONOO(-) scavengers. Finally, we found that ONOO(-) totally abolished the Mo(V) EPR spectrum. These changes were irreversible, suggesting oxidative disruption of the critical molybdenum center of the catalytic site. Thus, ONOO(-) formed in biological systems can feedback and down-regulate XO activity and O-*(2) generation, which in turn may serve to limit further ONOO(-) formation.     

Prion protein protects against DNA damage induced by paraquat in cultured cells

Exposure of cells to paraquat leads to production of superoxide anion (O2*-). This reacts with hydrogen peroxide to give the hydroxyl radical (*OH), leading to lipid peroxidation and cell death. In this study, we investigated the effects of cellular prion protein (PrPC) overexpression on paraquat-induced toxicity by using an established model system, rabbit kidney epithelial A74 cells, which express a doxycycline-inducible murine PrPC gene. PrPC overexpression was found to significantly reduce paraquat-induced cell toxicity, DNA damage, and malondialdehyde acid levels. Superoxide dismutase (total SOD and CuZn-SOD) and glutathione peroxidase activities were higher in doxycycline-stimulated cells. Our findings clearly show that PrPC overexpression plays a protective role against paraquat toxicity, probably by virtue of its superoxide dismutase-like activity.     

Exogenous nitric oxide improves seed germination in wheat against mitochondrial oxidative damage induced by high salinity

Effects of exogenous nitric oxide (NO) on starch degradation, oxidation in mitochondria and K⁺/Na⁺ accumulation during seed germination of wheat were investigated under a high salinity level. Seeds of winter wheat (Triticum aestivum L., cv. Huaimai 17) were pre-soaked with 0 mM or 0.1 mM of sodium nitroprusside (SNP, as nitric oxide donor) for 20 h just before germination under 300 mM NaCl. At 300 mM NaCl, exogenous NO increased germination rate and weights of coleoptile and radicle, but decreased seed weight. Exogenous NO also enhanced seed respiration rate and ATP synthesis. In addition, seed starch content decreased while soluble sugar content increased by exogenous NO pre-treatment, which was in accordance with the improved amylase activities in the germinating seeds. Exogenous NO increased the activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6); whereas decreased the contents of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), and superoxide anions (O₂^??) release rate in the mitochondria. Exogenous NO also decreased Na⁺ concentration while increased K⁺ concentration in the seeds thereby maintained a balance between K⁺ and Na⁺ during germination under salt stress. It is concluded that exogenous NO treatment on wheat seeds may be a good option to improve seed germination and crop establishment under saline conditions.     

Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H(2)O(2) induced oxidative damage

Hydroxytyrosol (2-(3′,4′-dihydroxyphenyl)ethanol; HT), the most active ortho-diphenolic compound, present either in free or esterified form in extravirgin olive oil, is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites. We investigated the capacity of three glucuronide metabolites of HT, 3′-O-β-d-glucuronide and 4′-O-β-d-glucuronide derivatives and 2-(3′,4′-dihydroxyphenyl)ethanol-1-O-β-d-glucuronide, in comparison with the parent compound, to inhibit H₂O₂ induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line. H₂O₂ treatment exerted a toxic effect inducing cell death, interacting selectively within the pro-death extracellular-signal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways. It also produced direct oxidative damage initiating the membrane lipid peroxidation process. None of the tested glucuronides exhibited any protection against the loss in renal cell viability. They also failed to prevent the changes in the phosphorylation states of ERK and Akt, probably reflecting their inability to enter the cells, while HT was highly effective. Notably, pretreatment with glucuronides exerted a protective effect at the highest concentration tested against membrane oxidative damage, comparable to that of HT: the formation of malondialdehyde, fatty acid hydroperoxides and 7-ketocholesterol was significantly inhibited.