Apigenin promotes antibacterial activity via regulation of nitric oxide and superoxide anion production

Apigenin is a naturally occurring flavone isolated from the medicinal herb, Aster yomena. The present study was designed to elucidate the apoptosis-like antibacterial mechanism of apigenin in Escherichia coli. Administration of apigenin resulted in a rapid increase in intracellular calcium accompanied by an increase in reactive nitrogen species (RNS) and nitric oxide (NO) levels. Furthermore, apigenin increases reactive oxygen species (ROS), superoxide anion (O₂^–) which left E. coli with no ability to activate superoxide dismutase. Finally, we found that perturbance of the membrane lipid bilayer leading to glutathione oxidation and formation 8-hydroxy-2′-deoxyguanosine occurred during the process and apoptosis-like death hallmarks were further observed. Furthermore, we applied the NO synthase inhibitor ( l -NAME) and the O₂^– scavenger (Tiron) and observed attenuation in apoptotic markers under their presence. Taken together, these results suggest that apigenin induces bacterial apoptosis via activation of cellular oxidative pathways dependent on the production and accumulation of RNS/ROS.     

Helicobacter pylori: a ROS-inducing bacterial species in the stomach

Background

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been reported to impact gastric inflammation and carcinogenesis. However, the precise mechanism by which Helicobacter pylori induces gastric carcinogenesis is presently unclear.

Aim

This review focuses on H. pylori-induced ROS/RNS production in the host stomach, and its relationship with gastric carcinogenesis.

Results

Activated neutrophils are the main source of ROS/RNS production in the H. pylori-infected stomach, but H. pylori itself also produces ROS. In addition, extensive recent studies have revealed that H. pylori-induced ROS production in gastric epithelial cells might affect gastric epithelial cell signal transduction, resulting in gastric carcinogenesis. Excessive ROS/RNS production in the stomach can damage DNA in gastric epithelial cells, implying its involvement in gastric carcinogenesis.

Conclusion

Understanding the molecular mechanism behind H. pylori-induced ROS, and its involvement in gastric carcinogenesis, is important for developing new strategies for gastric cancer chemoprevention.     

Cell cycle-dependent repair of double-strand DNA breaks in a γ-ray-sensitive Chinese hamster cell

A Chinese hamster cell mutant has been isolated which is extremely sensitive to killing by -irradiation in the G₁, and early S phases of the cell cycle (LD₅₀ of 20 vs. 250 rads for parent), but which has nearly normal resistance in late S. The mutant cell is able to repair single-stranded DNA breaks introduced by -radiation. However, in comparison to its parental cell, the mutant is deficient in the repair of double-stranded DNA breaks produced by -irradiation during the sensitive G₁-early S period, while in the resistant late S period, the repair is nearly the same for both cell types. This correlation between -ray sensitivity and repair strongly suggests that an inability to repair double-strand DNA breaks in G₁ is the basis for the hypersensitivity of the mutant to killing by -rays in this phase of the cell cycle. It also provides direct evidence in mammalian cells that the ability to repair double-strand DNA breaks induced by ionizing radiation is an important biochemical function in cell survival and supports the hypothesis that unrepaired double-strand breaks are a major lethal lesion in mammalian cells. A plausible explanation for the appearance of the cell cycle phenotype of the mutant is that in normal cells there are at least two pathways for the repair of double-strand breaks, one of which functions primarily in late S phase, and the other, either throughout the cell cycle or only in the G₁ and early S phases.     

Redox sensing and signaling by malaria parasite in vertebrate host

Plasmodium parasites, which is responsible to cause malaria, are also exceedingly receptive to oxidative stress during their intraerythrocytic life stage as they devour haemoglobin inside their food vacuoles and engender toxic haem moieties and reactive oxygen species (ROS). Other than, several studies suggest that the generation of reactive oxygen and nitrogen species (ROS and RNS) associated with oxidative stress, plays a decisive role in the ripeness of systemic complications caused by malaria. Malaria infection provokes the generation of hydroxyl radicals (OH^?), which most probably is the main reason for the induction of oxidative stress and apoptosis. In this study, it has been described to understand how redox molecules and NO carry out their diverse functions in both parasites and host. It is very important to understand the chemical reactions that produce those outcomes and how its regulation carried out by parasite during erythrocytic phase.

     

Auto-protective redox buffering systems in stimulated macrophages

Background

Macrophages, upon encounter with micro-organisms or stimulated by cytokines, produce various effector molecules aimed at destroying the foreign agents and protecting the organism. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are front line molecules exerting strong cytotoxic activities against micro-organisms and many cells, including macrophages themselves. Using cells of the murine macrophage cell line (RAW 264.7) stimulated in vitro with lipopolysaccharide (LPS) and/or interferon (IFN-γ), which induce strong endogenous NO production, we examined by which mechanisms a fraction of activated macrophages protect themselves from nitrosative stress and manage to escape destruction?

Results

We observed that survivors (10–50% depending on the experiments) had acquired a resistant phenotype being capable to survive when further exposed in vitro to an apoptosis inducing dose of the NO donor compound DETA-NO. These cells expressed an increased steady-state levels of Mn SOD, CuZn SOD and catalase mRNA (130–200%), together with an increased activity of the corresponding enzymes. Intracellular concentration of glutathione was also increased (× 3.5 fold at 6 hours, still maintained × 5.2 fold at 48 hours). Neither mRNA for glutathione peroxydase, γ-glutamylcysteine synthase and glutathione reductase, nor thioredoxine and thioredoxine reductase, were significantly modified. Additional experiments in which RAW 264.7 cells were stimulated with LPS and/or IFN-γ in the presence of relatively specific inhibitors of both Mn and Cu/Zn SOD, aminotriazol (ATZ) catalase inhibitor and buthionine sulfoximine (BSO) glutathione inhibitor, showed that inhibiting LPS-induced up-regulation of intracellular redox buffering systems also prevented acquisition of the resistant phenotype.

Conclusions

Our data suggest a direct causal relationship between survival of a fraction of macrophages and a up-regulation of key sets of auto-protective intracellular redox buffering systems, occurring simultaneously with modulation of expression of apoptotic molecules of the Bcl₂-Bcl-_XL/Bax-Bad family.     

Areca nut‐induced micronuclei and cytokinesis failure in Chinese hamster ovary cells is related to reactive oxygen species production and actin filament deregulation

Epidemiological studies have shown a strong association between environmental exposure to betel quid (BQ) and oral cancer. Areca nut (AN), an ingredient of BQ, contains genotoxic and mutagenic compounds. In this study, we found that AN extract (ANE) inhibited the growth of Chinese hamster ovary cells (CHO‐K1) in a dose‐ and time‐dependent manner. Intracellular reactive oxygen species (ROS) levels and micronuclei (MN) frequency were significantly increased following ANE treatment in CHO‐K1 cells. Addition of catalase markedly inhibited ANE‐induced MN formation, indicating that ANE‐induced genotoxicity was correlated with intracellular H₂O₂. Incubation of CHO‐K1 cells with ANE (400–800 μg/ml) for 24 hr caused G2/M arrest, and prolonged exposure to ANE (800 μg/ml) significantly induced cell death. Surprisingly, ANE itself caused cytokinesis failure and subsequent increase in binucleated cell formation. Coexposure to catalase (2,000 U/ml) and ANE (800 μg/ml) reduced the generation of binucleated cells, indicating that ANE‐induced cytokinesis failure was associated with oxidative stress. Following prolonged exposure to ANE, an accumulation of hyperploid/aneuploid cells concomitant with bi‐, micro‐ or multinucleated cells was found. In summary, our results demonstrate that ANE exposure to CHO‐K1 cells caused increased MN frequency, G2/M arrest, cytokinesis failure, and an accumulation of hyperploid/aneuploid cells. These events are associated with an increase in intracellular H₂O₂ level and actin filament disorganization. Environ. Mol. Mutagen., 2009. © 2009 Wiley‐Liss, Inc.     

Biochemical and cellular toxicology of peroxynitrite: implications in cell death and autoimmune phenomenon

Reactive nitrogen species include nitric oxide (·NO), peroxynitrite (ONOO^?) and nitrogen dioxide radical (NO₂·). Peroxynitrite is a reactive oxidant, produced from nitric oxide (·NO) and superoxide anion (O₂^·?–), that reacts with a variety of biological macromolecules. It is produced in the body in response to physiological stress and environmental toxins. It is a potent trigger of oxidative protein and DNA damage-including DNA strand breakage and base modification. It activates the nuclear enzyme poly-ADP ribose polymerase (PARP) resulting in energy depletion and apoptosis/necrosis of cells. Peroxynitrite generation is a crucial pathological mechanism in stroke, diabetes, inflammation, neurodegeneration, cancer, etc. Peroxynitrite modified DNA may also lead to the generation of autoantibodies in various autoimmune disorders such as systemic lupus erythematosus (SLE). In chronic inflammatory diseases, peroxynitrite formed by phagocytic cells may cause damage to DNA, generating neoepitopes leading to the production of autoantibodies. Hence, understanding the pathophysiology of peroxynitrite could lead to important therapeutic interventions.     

Hypersensitivity to mutation and sister-chromatid-exchange induction in CHO cell mutants defective in incising DNA containing UV lesions

Five UV-sensitive mutant strains of CHO cells representing different genetic complementation groups were analyzed for their ability to perform the incision step of nucleotide excision repair after UV exposure. The assay utilized inhibitors of DNA synthesis to accumulate the short-lived strand breaks resulting from repair incisions. After 6 J/m², each of the mutants showed <10% of the incision rate of the parental AA8 cells. After 50 J/m², the rate in AA8 was similar to that at 6 J/m², but the rates in the mutants were significantly higher (20% of the rate of AA8). Thus by this incision assay the mutants were phenotypically indistinguishable. Each of the mutants were hypersensitive to mutation induction at both thehprt andaprt loci by a factor of 10, and in the one strain tested ouabain resistance was induced sevenfold more efficiently than in AA8 cells. Sister chromatid exchange was also induced with sevenfold increased efficiency in the two mutant strains examined. Thus, these CHO mutants resemble xeroderma pigmentosum cells in terms of their incision defects and their hypersensitivity to DNA damage by UV.     

Chromosome aberration induction in Chinese hamster ovary cells by restriction enzymes with different methylation sensitivity

The isoschizomer pair MspI and HpaII were used to investigate whether the putative specificity of restriction endonucleases would be maintained when they were introduced into mammalian cells. Although both enzymes recognize the sequence CCGG, HpaII will cut only if the internal cytosine is unmethylated, whereas MspI will cut regardless of the methylation status. Cleavage results in a cohesive-end DNA double-strand break, which can lead to the formation of chromosome aberrations. Since mammalian DNA is heavily methylated, one would expect MspI to be much more effective than HpaII at inducing chromosome aberrations in Chinese hamster ovary cells. In fact, during G₁, MspI induced a >90-fold higher number of aberrations than did HpaII. Cell cycle studies indicated that during early S there was a 30-fold increase in HpaII-induced aberrations. This increase may be due to increased accessibility of replicating hypomethylated DNA. Cells that were treated with the demethylating agent 5-aza-2-deoxycytidine (AzdC) displayed only a moderate increase in HpaII-induced aberrations during G₁. This observation, together with the results of restriction enzyme analysis of genomic DNA, indicated that demethylation was incomplete. The effects of AzdC on the induction of aberrations by MspI suggested that AzdC increases chromatin accessibility. Our results were consistent with the expected specificity of MspI and HpaII. Thus, it appears that restriction endonucleases can play a useful role in determining the biological consequences of DNA double-strand breaks.     

Le stress oxydant,composante physiopathologique de l'athérosclérose

Reactive oxygen and nitrogen species (ROS–RNS) are constitutionally generated by the various cells of the arterial wall. Several intracellular enzymes are implicated in this ROS/RNS formation: NAD(P)H oxidases, NO synthases, mitochondrial respiratory chain pathway… Both enzymatic and nonenzymatic antioxidants allow the degradation of the ROS/RNS, thus maintaining a physiological equilibrium between pro-oxidants and antioxidants. In pathophysiological circumstances, a alteration of redox status occurs, that generates vascular cell dysfunction. Risk factors for the atherosclerotic process such as hypertension, hypercholesterolemia, diabetes…generate an excess of ROS production by stimulation of either NAD(P)H oxidases or the mitochondrial respiratory chain, or both. Overproduction of superoxide anion results in its combination with nitric oxide to form peroxinitrite, thereby decreasing the biodisponibility of nitric oxide. Such pro-oxidant conditions favour LDL oxidation, the release of pro-inflammatory biofactors and/or growth factors. They also provoke both the apoptotic process and necrosis, that are important features responsible for atherosclerotic lesion progression and rupture, leading to the clinical events of atherosclerosis.     

Synergistic genotoxicity caused by low concentration of titanium dioxide nanoparticles and p,p′‐DDT in human hepatocytes

The use of titanium dioxide nanoparticles (nano‐TiO₂) for the degradation of dichlorodiphenyltrichloroethane (p,p′‐DDT) increases the risk of exposure to trace nano‐TiO₂ and p,p′‐DDT mixtures. The interaction of p,p′‐DDT and nano‐TiO₂ at low concentrations may alter toxic response relative to nano‐TiO₂ or p,p′‐DDT alone. In this work, the combined genotoxicity of trace nano‐TiO₂ and p,p′‐DDT on human embryo L‐02 hepatocytes without photoactivation was studied. Nano‐TiO₂ (0.1 g/L) was mixed with 0.01–1 mmol/L p,p′‐DDT to determine adsorption isotherms. L‐02 cells were exposed to different levels of p,p′‐DDT (0, 0.001, 0.01, and 0.1 μmol/L) and nano‐TiO₂ (0, 0.01, 0.1, and 1 μg/mL) respectively. The adsorption of p,p′‐DDT by nano‐TiO₂ was approximately 0.3 mmol/g. Cell viability, apoptosis, and DNA double strand breaks were similar among all test groups. Nano‐TiO₂ alone (0.01–1 μg/mL) increased the levels of oxidative stress and oxidative DNA adducts (8‐OHdG), but it did not induce DNA breaks or chromosome damage. Addition of trace nano‐TiO₂ with trace p,p′‐DDT synergistically enhanced genotoxicity via increasing oxidative stress, oxidative DNA adducts, DNA breaks, and chromosome damage in L‐02 cells. Low concentrations of nano‐TiO₂ and p,p′‐DDT increased oxidativestress by reactive oxygen species (ROS) formation and lipid oxidation. Oxidative stress is a major pathway for DNA and chromosome damage. Dose‐dependent synergistic genotoxicity induced by combined exposure of trace p,p′‐DDT and nano‐TiO₂ suggests a potential environmental risk of nano‐TiO₂ assisted photocatalysis. Environ. Mol. Mutagen., 2010. © 2009 Wiley‐Liss, Inc.     

Interaction of the antiepileptic drug lamotrigine with recombinant rat brain type IIA Na+ channels and with native Na+ channels in rat hippocampal neurones

Actions of the new antiepileptic drug lamotrigine (LTG, Lamictal) were characterised using recombinant rat brain type IIA Na⁺ channels expressed in Chinese hamster ovary (CHO) cells and native Na⁺ channels in rat hippocampal pyramidal neurones, using whole-cell recording and intracellular recording techniques. In CHO cells, LTG caused a tonic inhibition of Na⁺ currents in a concentration-dependent and voltage-dependent manner. The half-maximal inhibitory concentration (IC₅₀) of approximately 500 M was obtained at a holding potential (V _h) of –90 mV compared with an IC₅₀ of 100 M at a V _h of –60 mV. LTG (50 M) caused a 10–mV negative shift in the slow, steady-state inactivation curve and delayed considerably the recovery from inactivation, but had no significant effects on the voltage dependence of activation or fast inactivation, suggesting that LTG acts mainly on the slow inactivated state. The affinity for the inactivated channels was estimated at 12 M. The tonic inhibition was augmented by a use-dependent action in which a further inhibition by the drug developed during rapid repetitive stimulation using a train of 20-ms duration pulses (11 Hz). These results were consistent with the drug action being on firing properties of pyramidal neurones. Only in those epileptiform bursts which caused cumulative inactivation of Na⁺ spikes did LTG produce a potent inhibition. Our data suggest that the inactivated channel is a primary target for LTG action at therapeutic concentrations.     

Biomarkers of inflammation and oxidative stress in ophthalmic disorders

ABSTRACT

The review article focuses on free radicals and oxidative stress involved in ophthalmological diseases such as retinopathy, cataract, glaucoma, etc. Oxidative stress is considered as a key factor involved in the pathology of many chronic diseases including ophthalmic complication and inflammatory process. Oxidative stress and inflammation are closely related pathophysiological processes and are simultaneously found in many pathological conditions. The free radicals produced oxidize cellular components such as lipids and phospholipids leading to lipid peroxidation and trigger the onset of retinopathy. Cataract is a significant cause of visual disability and it is proposed that the high incidence is related to oxidative stress induced by continued intraocular penetration of light and consequent photochemical generation of free radical oxidants. Glaucoma is the leading cause of irreversible blindness and comprises a group of diseases characterized by progressive optic nerve degeneration. Oxidative injury and altered antioxidant defense mechanisms in glaucoma appear to play a role in the pathophysiology of glaucomatous neurodegeneration that is characterized by death of retinal ganglion cells. The UVB radiations through this way may cause a number of diseases like photo-keratitis, pterygium, damage to epithelium, edema, and corneal cell apoptosis.

Abbreviations: ROS: reactive oxygen species; RNS: reactive nitrogen species; O_2.: superoxide anion; H₂O₂: hydrogen peroxide;. OH: hydroxyl radicals; ONOO^?, ONO₂^?: peroxynitrite; NO: nitric oxide; IOP: intraocular pressure; RGC: retinal ganglion cells. WHO: World Health Organization; IAPB: International Agency for the Prevention of Blindness.     

Inhibition of inflammatory mediators and reactive oxygen and nitrogen species by some depsidones and diaryl ether derivatives isolated from Corynespora cassiicola,an endophytic fungus of Gongronema latifolium leaves

Abstract

In this study, some depsidones and diaryl ether derivatives isolated from Corynespora cassicola, a fungi endophyte of Gongronema latifolium, were assessed for their anti-inflammatory potentials. The isolated metabolites corynesidone A (1), corynesidone C (2), corynesidone D (3) and corynether A (4) were screened for their effects on tumour necrosis factor-α (TNF-α), inducible nitric oxide (iNO), and reactive oxygen species (ROS) and reactive nitrogen species (RNS) production by stimulated RAW264.7 macrophages. Concentration of 1, 2, 3 and 4 up to 100?μM did not remarkably affect the viability of treated macrophages. The compounds were found to cause a concentration-dependent decrease in lipopolysaccharide-induced TNF-α and iNO in RAW264.7 cells. Pre-treatment with 100?μM of 1, 2, 3 and 4 suppressed iNO by as much as 96.28%, 95.71%, 78.14% and 73.28%; with IC₅₀ of 8.16, 9.49, 15.29 and 26.52?μM, respectively. Similarly, pre-treatment with 100?μM of 1, 2, 3 and 4 caused an inhibition of 99.17%, 99.59%, 95.02% and 74.07% in the formation of iNO production, respectively, with IC₅₀ of 1.88, 3.99, 7.48 and 37.22?μM. Treatment of with compounds 1–4 (10, 30 and 100?µM) followed by stimulation with phorbol 12-myristate 13-acetate (1?µM) caused significant (p?<?0.05) suppression of ROS/RNS-evoked chemiluminescence of luminol by as much as 100.96?±?1.88%, 98.59?±?1.38%, 87.35?±?1.41% and 79.22?±?0.30%, respectively at 100?µM. The depsidone derivatives (1–4) showed more potent inhibition of TNF-α and NO production and better scavenging ROS/RNS than the diaryl ether derivative (4). These chemical scaffolds can serve as suitable lead molecules for further development into novel anti-inflammatory and/or anti-cancer agents.     

Nitric Oxide, Superoxide, and Peroxynitrite Effects on the Insulin Secretion and Viability of βTC3 Cells

The onset of insulin-dependent diabetes mellitus (IDDM) is often associated with the infiltration of pancreatic cells by macrophages. Upon activation, macrophages release nitric oxide (NO) and superoxide (O ₂ ^- ). These species or their reactive intermediates can be cytoxic, mutagenic, or carcinogenic. Previous studies have reported both positive and negative effects of extracellularly generated NO on insulin secretion and viability of pancreatic cells. Inherent problems of several previous studies assessing the effects of NO on insulin secretion include unsteady state NO concentration exposures and the generation of other potentially damaging species. In this study, these problems were eliminated by using a modified experimental system in which NO delivery was achieved via diffusion across a gas-permeable tube and O ₂ ^- delivery was maintained using an enzymatic reaction. The delivery rates were constant, leading to steady state concentrations of NO and O ₂ ^- in the experimental system. Based on reaction kinetics, a model was developed to predict NO, O ₂ ^- and peroxynitrite ONOO^- concentrations during the experiment. This study showed that NO, O ₂ ^- and ONOO^- at predicted concentrations as high as 2.8 M, 0.25 M, and 0.1 nM, respectively, do not affect the insulin secretion rates of TC3 pancreatic cells over short times. © 2000 Biomedical Engineering Society. PAC00: 8717-d     

Prostaglandin E2 suppresses phytohemagglutin-induced immune responses of normal human monoculear cells by decreasing intracellular glutathione generation,but not due to increased DNA strand breaks or apoptosis

Prostaglandin E₂ (PGE₂) at concentrations more than 1×10^–8 M markedly suppressed the cell proliferation and release of soluble molecules of interleukin-2 receptor (sIL-2R), CD4 (sCD4) and CD8 (sCD8) from phytohemagglutinin (PHA)-stimulated normal human mononuclear cells (MNC) in a dose-related manner. To further elucidate the subcellular mechanism of the inhibitory effect of PGE₂ on PHA-stimulated MNC, intracellular concentration of glutathione (GSH) in PHA-stimulated MNC was sequentially measured from day 1 to day 3 by enzymic method. Furthermore, the effect of PGE₂ on nuclear DNA including DNA strand breaks in alkali treatment and DNA fragmentation (apoptosis) of PHA-stimulated MNC were also measured. We found intracellular GSH levels were significantly decreased in the early stage of lymphocyte activation (day 1), but no evidence of increased DNA stand breaks or apoptotic process appeared in 3-day culture. In addition, butathione sulfoximine (a specific GSH inhibitor) and dibutyryl cyclic AMP also exhibited both proliferation inhibition and GSH-decreasing effect on PHA-stimulated MNC as well as PGE₂. These results suggest that the immunosupressive effect of PGE₂ is mediated by the decreased generation of intracellular GSH, but not by the increased DNA strand breaks or apoptotic mechanism in the cells.     

Decreased production of nitric oxide by human neutrophils during septic multiple organ dysfunction syndrome

The objective of this study was to determine nitric oxide (NO) and superoxide anion release (O ₂ ^– ) by neutrophils (PMNs) in the septic multiple organ dysfunction syndrome (MODS) and to compare them with the response of normal cells to lipopolysaccharide (LPS) and cytokines. NO production was measured by the release of nitrites in the medium, its maximal production rate by a modified oxyhemoglobin assay and O ₂ ^– by standard methods. Normal cells were incubated with LPS, gamma interferon (IFN-), or tumor necrosis factor (TNF-) alone or in combination. Results showed that PMN release of both NO and O ₂ ^– was reduced in septic samples; in contrast, an association of LPS, IFN-, and TNF- promoted maximal NO release by normal cells (40–50%). We conclude that while interaction of normal PMNs with cytokines increases NO and O ₂ ^– release, progression of sepsis to a multiple organ dysfunction impairs these responses in both functions.