Haber-Weiss Reaction Research Articles

The utility of iron chelators in the management of inflammatory disorders.

Since iron can contribute to detrimental radical generating processes through the Fenton and Haber-Weiss reactions, it seems to be a reasonable approach to modulate iron-related pathways in inflammation. In the human organism a counterregulatory reduction in iron availability is observed during inflammatory diseases. Under pathological conditions with reduced or increased baseline iron levels different consequences regarding protection or susceptibility to inflammation have to be considered. Given the role of iron in development of inflammatory diseases, pharmaceutical agents targeting this pathway promise to improve the clinical outcome. The objective of this review is to highlight the mechanisms of iron regulation and iron chelation, and to demonstrate the potential impact of this strategy in the management of several acute and chronic inflammatory diseases, including cancer.

Melatonin and its metabolites as copper chelating agents and their role in inhibiting oxidative stress: a physicochemical analysis.

The copper sequestering ability of melatonin and its metabolites cyclic 3-hydroxymelatonin (3OHM), N(1) -acetyl-N(2) -formyl-5-methoxykynuramine (AFMK), and N(1) -acetyl-5-methoxykynuramine (AMK) was investigated within the frame of the Density Functional Theory. It was demonstrated that these compounds are capable of chelating copper ions, yielding stable complexes. The most likely chelation sites were identified. Two different mechanisms were modeled, the direct-chelation mechanism (DCM) and the coupled-deprotonation-chelation mechanism (CDCM). It is proposed that, under physiological conditions, CDCM would be the main chelation route for Cu(II). It was found that melatonin and its metabolites fully inhibited the oxidative stress induced by Cu(II)-ascorbate mixtures, via Cu(II) chelation. In the same way, melatonin, AFMK, and 3OHM also prevented the first step of the Haber-Weiss reaction, consequently turning off the ˙OH production via the Fenton reaction. Therefore, it is proposed that, in addition to the previously reported free radical scavenging cascade, melatonin is also involved in a concurrent 'chelating cascade', thereby contributing to a reduction in oxidative stress. 3OHM was identified as the most efficient of the studied compounds for that purpose, supporting the important role of this metabolite in the beneficial effects of melatonin against oxidative stress.

Oxidatively generated damage to cellular DNA by UVB and UVA radiation.

This review article focuses on a critical survey of the main available information on the UVB and UVA oxidative reactions to cellular DNA as the result of direct interactions of UV photons, photosensitized pathways and biochemical responses including inflammation and bystander effects. UVA radiation appears to be much more efficient than UVB in inducing oxidatively generated damage to the bases and 2-deoxyribose moieties of DNA in isolated cells and skin. The UVA-induced generation of 8-oxo-7,8-dihydroguanine is mostly rationalized in terms of selective guanine oxidation by singlet oxygen generated through type II photosensitization mechanism. In addition, hydroxyl radical whose formation may be accounted for by metal-catalyzed Haber-Weiss reactions subsequent to the initial generation of superoxide anion radical contributes in a minor way to the DNA degradation. This leads to the formation of both oxidized purine and pyrimidine bases together with DNA single-strand breaks at the exclusion, however, of direct double-strand breaks. No evidence has been provided so far for the implication of delayed oxidative degradation pathways of cellular DNA. In that respect putative characteristic UVA-induced DNA damage could include single and more complex lesions arising from one-electron oxidation of the guanine base together with aldehyde adducts to amino-substituted nucleobases.

English

A number of studies have demonstrated that geophagia can be beneficial and harmful. However, toxicological aspects of this earth, soil, clay eating habit are not fully investigated. In addition, low levels of antioxidants have been observed in pre-eclamptic women. Still, the molecular mechanism implicated oxidative stress in geophagia inducing pre-eclampsia has not been elucidated. Despite its absorbent properties in detoxifying phytotoxin, lowering the incidence of nausea, natural clay still remains toxic for human consumption in normal and long term conditions. Our previous studies have shown the implication of oxidative stress in pre-eclampsia characterized by maternal hypertension. Biomarkers of oxidative stress such as lipid peroxides (LPO), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPX) and 8-OHdG correlated with the extent of oxidative stress in pre-eclampsia. Fenton reaction induced by the presence of iron produces a more powerful reactive oxygen species (OH radical) which may contribute to the exacerbation of this status; while, clay eating is believed to protect pregnant women from pre-eclampsia by inhibiting iron uptake from gastrointestinal environment and so reducing the risk of production of OH radical during pregnancy where iron deficiency has been observed, it may be harmful however in iron overload conditions. We have demonstrated that cellular hepato-carcinoma can be indeed induced in rats being iron-loaded by ferrocene supplementation of their diet. Taking into account this possibility, we are postulating that during pre-eclamptic pregnancy, a number of factors including oxidative stress, activated macrophages and activation of xanthine oxidase (XO) can exacerbate this pregnancy complication. In addition, the availability of free iron from soil may as catalytic trace element involve in Harber-Weiss and Fenton reactions producing more ROS/RNS and especially OH radical. We are therefore suggesting that the subsequent activation of the nuclear factor NF-kB by ROS/RNS generated from activated macrophages stimulates the induction of COX-2. This enzymatic induction leading to the expression of inflammatory cytokines could be one of the molecular mechanisms underlying pre-eclamptic conditions. Supplementation of zinc or eating clay containing zinc could overcome such pregnancy complication.   Key words: Geophagia, pregnancy, molecular mechanism, oxidative stress, Haber-Weiss and Fenton reactions, superoxide dismutase (SOD), glutathione peroxidase (GPX), OH radical, 8-hydroxyl deoxyguanosine (8-OHdG).

Increasing Superoxide Production and the Labile Iron Pool in Tumor Cells may Sensitize Them to Extracellular Ascorbate.

Low millimolar concentrations of ascorbate are capable of inflicting lethal damage on a high proportion of cancer cells lines, yet leave non-transformed cell lines unscathed. Extracellular generation of hydrogen peroxide, reflecting reduction of molecular oxygen by ascorbate, has been shown to mediate this effect. Although some cancer cell lines express low catalase activity, this cannot fully explain the selective sensitivity of cancer cells to hydrogen peroxide. Ranzato and colleagues have presented evidence for a plausible new explanation of this sensitivity – a high proportion of cancers, via NADPH oxidase complexes or dysfunctional mitochondria, produce elevated amounts of superoxide. This superoxide, via a transition metal-catalyzed transfer of an electron to the hydrogen peroxide produced by ascorbate, can generate deadly hydroxyl radical (Haber–Weiss reaction). It thus can be predicted that concurrent measures which somewhat selectively boost superoxide production in cancers will enhance their sensitivity to i.v. ascorbate therapy. One way to achieve this is to increase the provision of substrate to cancer mitochondria. Measures which inhibit the constitutive hypoxia-inducible factor-1 (HIF-1) activity in cancers (such as salsalate and mTORC1 inhibitors, or an improvement of tumor oxygenation), or that inhibit the HIF-1-inducible pyruvate dehydrogenase kinase (such as dichloroacetate), can be expected to increase pyruvate oxidation. A ketogenic diet should provide more lipid substrate for tumor mitochondria. The cancer-killing activity of 42°C hyperthermia is to some degree contingent on an increase in oxidative stress, likely of mitochondrial origin; reports that hydrogen peroxide synergizes with hyperthermia in killing cancer cells suggest that hyperthermia and i.v. ascorbate could potentiate each other’s efficacy. A concurrent enhancement of tumor oxygenation might improve results by decreasing HIF-1 activity while increasing the interaction of ascorbic acid with oxygen. An increased pool of labile iron in cancer cells may contribute to the selective susceptibility of many cancers to i.v. ascorbate; antagonism of NF-kappaB activity with salicylate, and intravenous iron administration, could be employed to further elevate free iron in cancers.

Ceruloplasmin, hephaestin and zyklopen: the three multicopper oxidases important for human iron metabolism

Multi-copper oxidases are a group of proteins which demonstrate enzymatic activity and are capable of oxidizing their substrates with the concomitant reduction of dioxygen to two water molecules. For some multi-copper oxidases there has been demonstrated ferroxidase activity which is related to their specific structure characterized by the presence of copper centres and iron-binding sites. Three multi-copper oxidases have been included in this group: ceruloplasmin, hephaestin and zyklopen. Multi-copper oxidases which are expressed in different tissues are capable of oxidizing a wide spectrum of substrates. Multi-copper oxidases are capable of oxidizing a wide spectrum of substrates. Ceruloplasmin exhibits antioxidant activity as well as being involved in many other biological processes. The observations of phenotypic effects of absence or low expression of multi-copper ferroxidase-coding genes suggest that the main role of these proteins is taking part in iron metabolism. The main role of ceruloplasmin in iron turnover is oxidizing Fe2+ into Fe3+, a process which is essential for iron binding to transferrin (the main iron-transporting protein), as well as to ferritin (the main iron-storage protein). The function of hephaestin as ferroxidase is essential for iron binding to apotransferrin in the lamina propria of the intestinal mucosa, a process that is important for further transport of iron to the liver by the portal vein. Available data indicate that zyklopen is responsible for the placental iron transport. The presence of three multi-copper oxidases with ferroxidase activity emphasizes the significance of oxidation for iron metabolism. The distribution of multi-copper ferroxidases in many tissues ensures the proper iron turnover in the body as well as preventing toxic effects related to the presence of Fe2+ ions. These ions contribute to generation of free radicals, including the highly reactive hydroxyl radical, through the Fenton and Haber-Weiss reactions.

Oxidized Silicon Nanoparticles and Iron Oxide Nanoparticles for Radiation Therapy

Oxidized Silicon Nanoparticles and Iron Oxide Nanoparticles for Radiation Therapy Our research objective is to develop superparamagnetic iron oxide nanoparticles and silicon nanoparticles as radiosensitizers for cancer therapy. After internalization by breast tumor cells and irradiation with X-rays, the nanoparticles were observed to enhance the oxidative stress in tumor cells. While silicon nanoparticles increase the reactive oxygen species production under X-ray treatment due to their incompletely oxidized surface, positively charged amino-functionalized silicon nanoparticles enhance the formation of mitochondrial reactive oxygen species formation because of their direct interaction with the mitochondrial membrane. On the other hand, uncoated and citrate-coated superparamagnetic iron oxide nanoparticles were found to increase the reactive oxygen species formation in X-ray treated tumor cells via two particular surface features, being, first, the leakage of iron ions and second, the catalytic activity of nanoparticle surfaces. Both may initiate the Haber-Weiss and Fenton reaction.

The In Vitro and In Vivo Effects Of Fermented Papaya Preparation On Radiation Exposure

BackgroundVarious forms of radiation (radioactive, UV) cause cellular damage, which may lead to premature cell death or accumulation of somatic mutations which may lead to initiation and progression of malignancy. The damage is mediated in part by free radicals, particularly reactive oxygen species (ROS) (Reizenstein, Med Oncol Tumor Pharmacother. 1991;8:229). Fermented papaya preparation (FPP) is a product of yeast fermentation of Carica papaya Linn. FPP has been shown to have anti-oxidative potential by scavenging ROS, as well as by chelating excess iron (Prus & Fibach J Biol Regul Homeost Agents. 2012;26:203). Free iron species, such as the labile iron pool (LIP), participate in chemical reactions that generate ROS (Haber-Weiss and Fenton reactions). We studied the potential effect of FPP in preventing radiation-induced damage to various intracellular components in cultured human fibroblasts and myeloid leukemia (HL60) cells and in mice. MethodsCultured cells, human foreskin fibroblasts and myeloid leukemia (HL-60), were irradiated at various doses (0-18 Gy (Gammacell® 220, MDS Nordion, Excel, Canada). FPP (Osato Research Institute, Gifu, Japan) (10-100 µg/ml, was added either before or after irradiation. The cells were assayed after 1-3 days: Their survival was estimated by CellTiter 96® Aqueous Non-Radioactive Cell Proliferation assay. Apoptosis was determined by staining for phosphatidylserine exposure (by fluorescent Annexin V) and for intracellular uptake of propidium iodide. ROS was measured by staining with dichlorofluoresceine diacetate, and the LIP - with calcein-AM. The effects on the DNA were estimated by measuring 8-oxyguanine (a marker of DNA oxidation), using a fluorescent specific probe, and DNA instability was measured by the “comet assay”, a single-cell gel electrophoresis procedure. Apoptosis, ROS, LIP and 8-oxyguanine were quantified by flow cytometry. Experiments were also carried out on irradiated mice treated with FPP (by adding it to the drinking water) either before or after irradiation. The above mentioned parameters were assayed in their bone marrow cells in addition to determining their survival. ResultsThe results indicated that FPP has significant (P<0.05) ameliorating effects on radiation-induced increase of LIP and generation of ROS, as well as on the generation of 8-oxyguanine and DNA instability. In addition, apoptosis was decreased and consequently the survival of the cells was increased (Fig. 1A). Moreover, about 60% of 14 Gy-irradiated mice that received 100 mg/ml of FPP in their drinking water were still alive after 30 days (Fig. 1B). [Display omitted] ConclusionsFPP was able to protect the short-term effects of radiation on cultured cells by increasing their viability. A similar effect was found in mice. The improvement in DNA instability induced by FPP may influence the long-term effects of radiation, such as the development of secondary leukemia, in patients who receive radiotherapy for their primary tumors. Disclosures:Fibach:Osato Research Institute, Gifu, Japan: Consultancy, Research Funding. Rachmilewitz:Osato Research Institute, Gifu, Japan: Consultancy, Research Funding.

EGCG inhibit chemical reactivity of iron through forming an Ngal–EGCG–iron complex

Accumulated evidence indicates that the interconversion of iron between ferric (Fe(3+)) and ferrous (Fe(2+)) can be realized through interaction with reactive oxygen species in the Fenton and Haber-Weiss reactions and thereby physiologically effects redox cycling. The imbalance of iron and ROS may eventually cause tissue damage such as renal proximal tubule injury and necrosis. Many approaches were exploited to ameliorate the oxidative stress caused by the imbalance. (-)-Epigallocatechin-3-gallate, the most active and most abundant catechin in tea, was found to be involved in the protection of a spectrum of renal injuries caused by oxidative stress. Most of studies suggested that EGCG works as an antioxidant. In this paper, Multivariate analysis of the LC-MS data of tea extracts and binding assays showed that the tea polyphenol EGCG can form stable complex with iron through the protein Ngal, a biomarker of acute kidney injury. UV-Vis and Luminescence spectrum methods showed that Ngal can inhibit the chemical reactivity of iron and EGCG through forming an Ngal-EGCG-iron complex. In thinking of the interaction of iron and ROS, we proposed that EGCG may work as both antioxidant and Ngal binding siderphore in protection of kidney from injuries.

Deferasirox Reduces Oxidative Stress in Patients With Transfusion Dependency

BackgroundIron chelation therapy is useful against the over-accumulation of iron and is expected to reduce oxidative stress resulting from the Fenton reaction and Haber-Weiss reaction. We monitored oxidative status and serum ferritin levels after in vivo administration of deferasirox (DFS) and studied the in vitro effects of iron chelators on neutrophil function.MethodsNine patients suffering from transfusion dependency were recruited for this study, and derivatives of reactive oxygen metabolite (dROM) tests to detect serum hydroperoxide levels were evaluated in addition to serum ferritin levels. Human neutrophil reactive oxygen species (ROS) production was determined with flow cytometry.ResultsFerritin levels decreased after DFS treatment (P = 0.068), and a significant reduction in dROM levels was measured (P = 0.031). Fifty microM DFS significantly inhibited ROS production induced by fMLP in vitro (P < 0.0001), and tended to inhibit that induced by PMA. On the other hand, deferioxamine failed to inhibit ROS production even at high concentrations.ConclusionsIn vivo administration of DFS resulted in the reduction of oxidative stress, and this effect was considered to depend not only on a reduction in iron storage but also on the ability of DFS to inhibit neutrophil ROS production in vitro at clinically relevant plasma levels. Further studies are needed to examine the effects of iron chelators.

The effect of Fe-deficiency on antioxidant enzymes’ activity and lipid peroxidation in wheat leaves

Studying the physiological role of nutrient elements has an unraveling capacity in understanding plant’s behavioral pattern in order to acquire a high and stable yield. For this purpose, the durum wheat variety, P1252, was selected and planted in hydroponic way. To investigate the effects of Fe deficiency, the element was eliminated from media solutions. Results showed that the lack of Fe affected superoxide dismutase (SOD), guaicol peroxidase (GPX) and catalase (CAT) activities significantly. Meanwhile, ascorbate peroxidase (APX) was the only antioxidant enzyme not shows any significant with control. The SOD/(APX + GPX + CAT) ratio as an index of assessing the balance between hydrogen peroxide (H2O2)-producing and H2O2-scavenging enzymes increased leading to the accumulation of H2O2 in cell. The elevation of SOD/APX + GPX + CAT ratio and H2O2 accumulation indicates the occurrence of oxidative stress in the leave cells in the element-deletion conditions. Other oxidative stress indices, cell death as well as malondialdehyde (MDA) did not show any significant change in the absence of Fe. The reason is assumed to be the non-occurrence of Haber-Weiss reaction in Fe absence so that hydroxyl, a very dangerous radical, is produced, leading to increased damage to cell bio-molecules and apoptosis subsequently.

Non Transferrin Bound Iron: Nature, Manifestations and Analytical Approaches for Estimation

Iron is an essential trace element and plays a number of vital roles in biological system. It also leads the chains of pathological actions if present in excess and/or present in free form. Major portion of iron in circulation is associated with transferrin, a classical iron transporter, which prevent the existence of free iron. The fraction of iron which is free of transferrin is known as "non transferrin bound iron". Along with the incidence in iron over loaded patient non transferrin bound iron has been indicated in patients without iron overload. It has been suggested as cause as well as consequence in a number of pathological conditions. The major organs influenced by iron toxicity are heart, pancreas, kidney, organs involved in hematopoiesis etc. The most commonly suggested way for iron mediated pathogenesis is through increased oxidative stress and their secondary effects. Generation of free oxygen radicals by iron has been well documented in Fenton chemistry and Haber-Weiss reaction. Non transferrin bound iron has obvious chance to generate the free reactive radicals as it is not been shielded by the protective carrier protein apo transferrin. The nature of non transferrin bound iron is not clear at present time but it is definitely a group of heterogenous iron forms free from transferrin and ferritin. A variety of analytical approaches like colorimetry, chromatography, fluorimetry etc. have been experimented in different research laboratories for estimation of non transferrin bound iron. However the universally accepted gold standard method which can be operated in pathological laboratories is still to be developed.

Superparamagnetic iron oxide nanoparticles as radiosensitizer via enhanced reactive oxygen species formation

Internalization of citrate-coated and uncoated superparamagnetic iron oxide nanoparticles by human breast cancer (MCF-7) cells was verified by transmission electron microscopy imaging. Cytotoxicity studies employing metabolic and trypan blue assays manifested their excellent biocompatibility. The production of reactive oxygen species in iron oxide nanoparticle loaded MCF-7 cells was explained to originate from both, the release of iron ions and their catalytically active surfaces. Both initiate the Fenton and Haber–Weiss reaction. Additional oxidative stress caused by X-ray irradiation of MCF-7 cells was attributed to the increase of catalytically active iron oxide nanoparticle surfaces.

Exposure of Arabidopsis thaliana to Cd or Cu excess leads to oxidative stress mediated alterations in MAPKinase transcript levels

Metals, like cadmium (Cd) and copper (Cu), have the ability to induce the production of reactive oxygen species (ROS) at the cellular level. It is widely known that these ROS can cause irreversible damage to cellular components, like DNA, proteins and lipids. On the other hand, ROS can also act as signaling molecules and in this way they play an essential role in many normal physiological processes, but also in defense responses against stress. ROS signaling in plants uses mitogen-activated protein kinase (MAPK) pathways leading to the transcriptional control of target genes involved in the scavenging or production of ROS. Here, oxidative signaling induced by exposure to excess Cd or Cu was investigated in relation to anti-oxidative defense responses to these metals. Three-week-old Arabidopsis thaliana plants were exposed to environmentally realistic concentrations of Cu and Cd and immediate responses were measured at the level of hydrogen peroxide (H2O2) content, lipid peroxidation and transcript levels of genes involved in ROS homeostasis and signaling. Our findings show immediate (after 2h exposure) effects in the roots following Cu exposure, whereas effects in the leaves were generally more delayed. Effects of Cd exposure in leaves and roots were observed only after 24h exposure. On one hand, exposure of roots to Cu leads via activation of NADPH oxidases and Fenton reactions to H2O2 production that can induce MAPK and oxylipin signaling to control the cellular redox status. On the other hand, conversion of H2O2 to the more damaging hydroxyl radical by Fenton and Haber–Weiss reactions can initiate lipid peroxidation leading to membrane damage. In roots exposed to elevated Cd concentrations only oxidative signaling was initiated, possibly via NADPH oxidase-mediated ROS production. In leaves, time-dependent activation of MAPK and oxylipin signaling was seen after exposure to both metals, Cu or Cd, independent of changes in H2O2 content.

Hydroxyl Radical Generation Mechanism During the Redox Cycling Process of 1,4-Naphthoquinone

Airborne quinones contribute to adverse health effects of ambient particles probably because of their ability to generate hydroxyl radicals (·OH) via redox cycling, but the mechanisms remain unclear. We examined the chemical mechanisms through which 1,4-naphthoquinone (1,4-NQ) induced ·OH, and the redox interactions between 1,4-NQ and ascorbate acid (AscH(2)). First, ·OH formation by 1,4-NQ was observed in cellular and acellular systems, and was enhanced by AscH(2). AscH(2) also exacerbated the cytotoxicity of 1,4-NQ in Ana-1 macrophages, at least partially due to enhanced ·OH generation. The detailed mechanism was studied in an AscH(2)/H(2)O(2) physiological system. The existence of a cyclic 1,4-NQ process was shown by detecting the corresponding semiquinone radical (NSQ·-) and hydroquinone (NQH(2)). 1,4-NQ was reduced primarily to NSQ·- by O2·- (which was from AscH(2) reacting with H(2)O(2)), not by AscH(2) as normally thought. At lower doses, 1,4-NQ consumed O2·- to suppress ·OH; however, at higher doses, 1,4-NQ presented a positive association with ·OH. The reaction of NSQ·- with H(2)O(2) to release ·OH was another important channel for OH radical formation except for Haber-Weiss reaction. As a reaction precursor for O2·-, the enhanced ·OH response to 1,4-NQ by AscH(2) was indirect. Reducing substrates were necessary to sustain the redox cycling of 1,4-NQ, leading to more ·OH and a deleterious end point.

Genetic polymorphism of the iron-regulatory protein-1 and -2 genes in age-related macular degeneration

Iron can be involved in the pathogenesis of AMD through the oxidative stress because it may catalyze the Haber–Weiss and Fenton reactions converting hydrogen peroxide to free radicals, which can induce cellular damage. We hypothesized that genetic polymorphism in genes related to iron metabolism may predispose individuals to the development of AMD and therefore we checked for an association between the g.32373708 G>A polymorphism (rs867469) of the IRP1 gene and the g.49520870 G>A (rs17483548) polymorphism of the IRP2 gene and AMD risk as well as the modulation of this association by some environmental and life-style factors. Genotypes were determined in DNA from blood of 269 AMD patients and 116 controls by the allele-specific oligonucleotide-restriction fragment length polymorphism and the polymerase chain reaction-restriction fragment length polymorphism. An association between AMD, dry and wet forms of AMD and the G/G genotype of the g.32373708 G>A-IRP1 polymorphism was found (OR 3.40, 4.15, and 2.75). On the other hand, the G/A genotype reduced the risk of AMD as well as its dry or wet form (OR 0.23, 0.21, 0.26). Moreover, the G allele of the g.49520870 G>A-IRP2 polymorphism increased the risk of the dry form of the disease (OR 1.51) and the A/A genotype and the A allele decreased such risk (OR 0.43 and 0.66). Our data suggest that the g.32373708 G>A-IRP1 and g.49520870 G>A-IRP2 polymorphisms may be associated with increased risk for AMD.

Pro-oxidative effects of melanoidin–copper complexes on isolated and cellular DNA

High molecular weight Maillard reaction products (melanoidins) are described to possess metal-chelating properties. Whereas in food systems, this ability contributes to antioxidant properties, the consequences on biological systems are not quite clear. The study was aimed to evaluate the implication of metal complexation by melanoidins on DNA damage. Melanoidins prepared with d-glucose and different l-amino acids under water-free reaction conditions were charged with cupric ions. The effect on isolated DNA was investigated by the PM2 assay and on cellular systems in the human colon carcinoma cell line HCT-116 by alkaline unwinding. Independent of the amino acid composition, pure melanoidins (MW >14 kDa) did not cause significant DNA damage. By charging melanoidins with Cu2+ ions, a considerable DNA strand breaking activity was detectable, which was again amplified in an oxidative milieu (addition of hydrogen peroxide). Since Cu2+ normally does not provoke the formation of reactive oxygen species (ROS) via Fenton-type reaction, the results obtained have to be attributed to reducing properties of melanoidins. Thus, in melanoidin–copper complexes redox cycling may take place leading to Cu+ which subsequently undergoes Fenton-type and Haber–Weiss reactions. As a consequence, ROS are formed, which may explain the generation of DNA strand breaks.

Tryptamine-Gallic Acid Hybrid Prevents Non-steroidal Anti-inflammatory Drug-induced Gastropathy

We have investigated the gastroprotective effect of SEGA (3a), a newly synthesized tryptamine-gallic acid hybrid molecule against non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy with mechanistic details. SEGA (3a) prevents indomethacin (NSAID)-induced mitochondrial oxidative stress (MOS) and dysfunctions in gastric mucosal cells, which play a pathogenic role in inducing gastropathy. SEGA (3a) offers this mitoprotective effect by scavenging of mitochondrial superoxide anion (O(2)(·-)) and intramitochondrial free iron released as a result of MOS. SEGA (3a) in vivo blocks indomethacin-mediated MOS, as is evident from the inhibition of indomethacin-induced mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. SEGA (3a) corrects indomethacin-mediated mitochondrial dysfunction in vivo by restoring defective electron transport chain function, collapse of transmembrane potential, and loss of dehydrogenase activity. SEGA (3a) not only corrects mitochondrial dysfunction but also inhibits the activation of the mitochondrial pathway of apoptosis by indomethacin. SEGA (3a) inhibits indomethacin-induced down-regulation of bcl-2 and up-regulation of bax genes in gastric mucosa. SEGA (3a) also inhibits indometacin-induced activation of caspase-9 and caspase-3 in gastric mucosa. Besides the gastroprotective effect against NSAID, SEGA (3a) also expedites the healing of already damaged gastric mucosa. Radiolabeled ((99m)Tc-labeled SEGA (3a)) tracer studies confirm that SEGA (3a) enters into mitochondria of gastric mucosal cell in vivo, and it is quite stable in serum. Thus, SEGA (3a) bears an immense potential to be a novel gastroprotective agent against NSAID-induced gastropathy.

Involvement of four different intracellular sites in chloroacetaldehyde- induced oxidative stress cytotoxicity.

Chloroacetaldehyde (CAA) is a chlorination by-product in finished drinking water and a toxic metabolite of a wide variety of industrial chemicals (e.g. vinyl chloride) and chemotherapeutic agents (e.g. cyclophosphamide and ifosfamide). In this research, the cytotoxic mechanisms of CAA in freshly isolated rat hepatocytes were investigated.CAA cytotoxicity was associated with reactive oxygen species (ROS) formation and glutathione depletion suggesting that oxidative stress contributed to the CAA cytotoxic mechanism. CAA-induced oxidative stress cytotoxicity markers were significantly prevented by antioxidants, ROS scavengers, mitochondrial permeability transition (MPT) pore sealing agents, endocytosis inhibitors, ATP generators and xanthine oxidase inhibitor. In our study the hepatocyte mitochondrial membrane potential was rapidly decreased by CAA which was prevented by antioxidants and ROS scavenger indicating that mitochondrial membrane damage was a consequence of ROS formation. CAA cytotoxicity was also associated with lysosomal membrane rupture. OUR FINDINGS SHOWED THAT AT LEAST FOUR DIFFERENT INTRACELLULAR SOURCES INCLUDING: metabolic enzymes cytochrome P450 and xanthine oxidase, mitochondrial respiratory chain disruption and lysosomal Haber-weiss reaction, were involved in CAA induced ROS formation and other subsequent cytotoxic events. Our other interesting finding was that the lysosomotropic agents prevented CAA induced mitochondrial membrane potential collapse and mitochondrial MPT pore sealing agents inhibited lysosomal membrane damage caused by CAA. It can therefore be suggested that there is probably a toxic interaction (cross-talk) between mitochondrial and lysosomal oxidative stress generating systems, which potentiates each organelle damage and ROS formation in CAA- induced hepatotoxicity.

Comment on: Identification of antimicrobial activity among FDA-approved drugs for combating Mycobacterium abscessus and Mycobacterium chelonae

Sir, In the article titled ‘Identification of antimicrobial activity among FDA-approved drugs for combating Mycobacterium abscessus and Mycobacterium chelonae’ published in the July 2011 issue of JAC, it was reported that the FDA-approved drug metronidazole has potent activity (<0.015 mg/L; ∼87 nM) against the rapid-growing mycobacteria (RGM) Mycobacterium abscessus.1 This finding intrigued us and our clinical colleagues who treat M. abscessus-infected patients here at NIH. We therefore tried to repeat their experiment with M. abscessus ATCC 19977 and some recent patient isolates of M. abscessus to evaluate their susceptibility to metronidazole. We regret to report that we did not observe any MIC99, even at the highest concentration of the drug we tested (2 mM) after 48, 72 and 96 h. In accordance with the method published in the paper, we grew the M. abscessus ATCC 19977 (and four clinical isolates) from glycerol stock in 7H9 Middlebrook medium, passaged them once and let them grow to an OD650 of 0.2–0.4. The cell cultures were then diluted to an OD650 of 0.0002 in 7H9 medium and 50 μL of the cells was added to 50 μL of drug at various concentrations in a 96-well plate. They were incubated at 37°C for 48, 72 and 96 h and no killing of this RGM was observed (Figure 1a). Figure 1. (a) MICs of drugs for M. abscessus ATCC 19977 after 72 h. Each of the drugs was assayed in duplicate. MTZ, metronidazole. Each drug was serially diluted (50% each time) from well 1 to well 11, and well 12 does not have any drug. The concentration (in ... It is known that M. abscessus is susceptible to clarithromycin,2 and we therefore included this as a positive control in the experiment. PA-824, another nitroimidazole that is currently in Phase II clinical trials for the treatment of tuberculosis, has shown aerobic as well as anaerobic activity against the slow-growing mycobacteria Mycobacterium tuberculosis.3 It was also tested for activity against the RGM M. abscessus. The susceptibility of M. abscessus to clarithromycin clearly shows that there was no flaw in the experimental design, yet M. abscessus grew in the presence of 100 μM metronidazole and 50 μM PA-824 (Figure 1a). To investigate the susceptibility of M. abscessus ATCC 19977 and four clinical isolates to a higher concentration of metronidazole we determined the MIC99 starting with a 2 mM concentration of metronidazole, but we still did not observe any cidal effect of metronidazole even at such a high concentration (Figure 1b). In addition, no killing was observed with PA-824 at a concentration of 2 mM (data not shown). Strains tested have been identified as belonging to the M. abscessus group by partial sequencing of the secA gene4 and/or MALDI-TOF (matrix-assisted laser desorption ionization–time of flight) mass spectrometry.5 Metronidazole is known for its activity and has been used in the treatment of diseases caused by anaerobic microorganisms like Trichomonas vaginalis,6 Entamoeba histolytica7 and Clostridium difficile6 and microaerophilic bacteria like Helicobacter pylori.8 Though metronidazole is active against hypoxically adapted non-replicating M. tuberculosis, it is inactive against rapidly replicating M. tuberculosis under aerobic conditions.9 Metronidazole, which belongs to the class of 5-nitroimidazoles, has a single electron reduction potential of −0.4 to −0.5 V, which is out of the range normally accessible to the mammalian redox system. Under a limiting oxygen concentration in microbes the redox potential range is −0.42 V and lower and is suitable for the bioreductive activation of metronidazole. Upon single electron reduction of nitroimidazole, a nitro radical anion is produced,10 which could reduce oxygen in a microaerophilic organism to form superoxide.11 In the presence of transition elements like iron or copper, the hydrogen peroxide and superoxide react to form a hydroxyl radical by the Haber–Weiss reaction,10 causing inhibition of DNA synthesis and DNA strand breakage by this futile cycle. Metronidazole induces the expression of SOS response genes, which are responsible for DNA repair by the RecA pathway,10 as a result of which recA mutants in Escherichia coli and H. pylori are more susceptible to metronidazole treatment.12,13 Under normoxic conditions reduction of molecular oxygen by reduced metronidazole would be expected to render it harmless, as superoxide formed would be rapidly decomposed by the microbial superoxide dismutase.10 Our finding clearly demonstrates that metronidazole is inactive against M. abscessus under aerobic conditions, as has been reported. Metronidazole is potent against anaerobic organisms, but cannot be used in the treatment of infection with M. abscessus. Metronidazole does not kill RGM M. abscessus at a concentration of 2 mM (342.3 mg/L), which is more than 20 000-fold the concentration of what was reported by Chopra et al.1