Formation Of Reactive Oxygen Intermediates Research Articles

KIDNEY INJURY IN CANCER THERAPY

Кidney injury is a frequent and significant complication of cancer and cancer therapy. The kidneys are susceptible to injury from malignant infiltration, damage by metabolites of malignant cells, glomerular injury, nephrotoxic drugs including chemotherapeutic agents. Also bone marrow transplantation complications, infections with immune suppression (including septicemia), tumor lysis syndrome should be taken into account. Chemotherapeutic agents are a common cause of acute kidney injury but can potentially lead to chronic kidney disease development in cancer patients. This article summarizes risk factors of acute kidney injury in cancer patients. Risk factors are divided into two groups. The systemic are decrease of total circulating blood volume, infiltration of kidney tissue by tumor cells, dysproteinemia, electrolyte disturbances. The local (renal) risk factors are microcirculation disturbances, drugs biotransformation with formation of reactive oxygen intermediates, high concentration of nephrotoxic agents in proximal tubules and its sensitivity to ischemia. Drug-related risk factors include: drugs combination with cytotoxic effect high doses long term use necessity, direct cytotoxic effect of not only chemotherapeutic agents but also its metabolites, mean solubility forming intratubular precipitates. Early diagnosis, timely prevention and treatment of these complications provide significantly improve nononcologic results of treatment.

Ozone uptake on glassy, semi-solid and liquid organic matter and the role of reactive oxygen intermediates in atmospheric aerosol chemistry.

Heterogeneous and multiphase reactions of ozone are important pathways for chemical ageing of atmospheric organic aerosols. To demonstrate and quantify how moisture-induced phase changes can affect the gas uptake and chemical transformation of organic matter, we apply a kinetic multi-layer model to a comprehensive experimental data set of ozone uptake by shikimic acid. The bulk diffusion coefficients were determined to be 10(-12) cm(2) s(-1) for ozone and 10(-20) cm(2) s(-1) for shikimic acid under dry conditions, increasing by several orders of magnitude with increasing relative humidity (RH) due to phase changes from amorphous solid over semisolid to liquid. Consequently, the reactive uptake of ozone progresses through different kinetic regimes characterised by specific limiting processes and parameters. At high RH, ozone uptake is driven by reaction throughout the particle bulk; at low RH it is restricted to reaction near the particle surface and kinetically limited by slow diffusion and replenishment of unreacted organic molecules. Our results suggest that the chemical reaction mechanism involves long-lived reactive oxygen intermediates, likely primary ozonides or O atoms, which may provide a pathway for self-reaction and catalytic destruction of ozone at the surface. Slow diffusion and ozone destruction can effectively shield reactive organic molecules in the particle bulk from degradation. We discuss the potential non-orthogonality of kinetic parameters, and show how this problem can be solved by using comprehensive experimental data sets to constrain the kinetic model, providing mechanistic insights into the coupling of transport, phase changes, and chemical reactions of multiple species in complex systems.

Circadian redox signaling in plant immunity and abiotic stress.

Plant crops are critically important to provide quality food and bio-energy to sustain a growing human population. Circadian clocks have been shown to deliver an adaptive advantage to plants, vastly increasing biomass production by efficient anticipation to the solar cycle. Plant stress, on the other hand, whether biotic or abiotic, prevents crops from reaching maximum productivity. Stress is associated with fluctuations in cellular redox and increased phytohormone signaling. Recently, direct links between circadian timekeeping, redox fluctuations, and hormone signaling have been identified. A direct implication is that circadian control of cellular redox homeostasis influences how plants negate stress to ensure growth and reproduction. Complex cellular biochemistry leads from perception of stress via hormone signals and formation of reactive oxygen intermediates to a physiological response. Circadian clocks and metabolic pathways intertwine to form a confusing biochemical labyrinth. Here, we aim to find order in this complex matter by reviewing current advances in our understanding of the interface between these networks. Although the link is now clearly defined, at present a key question remains as to what extent the circadian clock modulates redox, and vice versa. Furthermore, the mechanistic basis by which the circadian clock gates redox- and hormone-mediated stress responses remains largely elusive.

Hepatotropic growth factors protect hepatocytes during inflammation by upregulation of antioxidative systems

To investigate effects of hepatotropic growth factors on radical production in rat hepatocytes during sepsis. Rat hepatocytes, isolated by collagenase perfusion, were incubated with a lipopolysaccharide (LPS)-containing cytokine mixture of interleukin-1β, tumor necrosis factor-α and interferon-γ to simulate sepsis and either co-incubated or pre-incubated with hepatotropic growth factors, e.g. hepatocyte growth factor, epidermal growth factor and/or transforming growth factor-α. Cells were analyzed for glutathione levels. Culture supernatants were assayed for production of reactive oxygen intermediates (ROIs) as well as NO(2) (-), NO(3) (-) and S-nitrosothiols. To determine cellular damage, release of aspartate aminotransferase (AST) into the culture medium was analyzed. Activation of nuclear factor (NF)-κB was measured by electrophoretic mobility shift assay. Rat hepatocytes treated with the LPS-containing cytokine mixture showed a significant increase in ROI and nitrogen oxide intermediate formation. AST leakage was not significantly increased in cells treated with the LPS-containing cytokine mixture, independent of growth-factor co-stimulation. However, pretreatment with growth factors significantly reduced AST leakage and ROI formation while increasing cellular glutathione. Application of growth factors did not result in increased NF-κB activation. Pretreatment with growth factors further increased formation of NO(2) (-), NO(3) (-) and S-nitrosothiols in hepatocytes stimulated with LPS-containing cytokine mixture. Thus, we propose that, together with an increase in glutathione increased NO(2) (-), NO(3) (-) formation might shift their metabolism towards non-toxic products. Our data suggest that hepatotropic growth factors positively influence sepsis-induced hepatocellular injury by reducing cytotoxic ROI formation via induction of the cellular protective antioxidative systems.

The Glycosylphosphatidylinositol-Anchored Protease Sap9 Modulates the Interaction ofCandida albicanswith Human Neutrophils

Human polymorphonuclear neutrophils (PMNs) play a major role in the immune defense against invasive Candida albicans infection. This fungal pathogen produces a set of aspartic proteases that directly contributes to virulence properties such as adhesion, tissue invasion, and immune evasion. We show here that, in contrast to other secreted proteases, the cell surface-associated isoform Sap9 has a major impact on the recognition of C. albicans by PMNs. SAP9 is required for the induction of PMN chemotaxis toward C. albicans filaments, an essential prerequisite of effective PMN activation. Furthermore, deletion of SAP9 leads to a mitigated release of reactive oxygen intermediates (ROI) in human PMNs and decreases C. albicans-induced apoptosis triggered by ROI formation. In confrontation assays, killing of a SAP9 deletion mutant is reduced in comparison to wild-type C. albicans. These data clearly implicate Sap9 protease activity in the initiation of protective innate immunity and suggest novel molecular mechanisms in C. albicans-host interaction leading to neutrophil activation.

NCF1 gene and pseudogene pattern: association with parasitic infection and autoimmunity

BackgroundNeutrophil cytosolic factor 1, p47phox (NCF1) is a component of the leukocyte NADPH oxidase complex mediating formation of reactive oxygen intermediates (ROI) which play an important role in host defense and autoimmunity. An individual genomic pattern of ncf1 and its two types of pseudogenes (reflected by the ΔGT/GTGT ratio) may influence the individual capacity to produce ROI.MethodsNCF1ΔGT/GTGT ratios were correlated with clinical parameters and ROI production during Plasmodium falciparum malaria and with susceptibility to the autoimmune disease multiple sclerosis (MS).ResultsAmong Gabonese children with severe malaria, ROI production from peripheral blood tended to be higher in individuals with a ΔGT/GTGT ratio ≤ 1:1. ΔGT/GTGT ratios were not associated with susceptibility to MS, but to age-of-onset among MS patients.ConclusionThe genomic pattern of NCF1 and its pseudogenes might influence ROI production but only marginally influence susceptibility to and outcome of malaria and MS.

The environmental carcinogen 3-nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells

The environmental contaminant 3-nitrobenzanthrone (3-NBA) is highly mutagenic and a suspected human carcinogen. We aimed to evaluate whether 3-NBA is able to deregulate critical steps in cell cycle control and apoptosis in human lung epithelial A549 cells. Increased intracellular Ca 2+ and caspase activities were detected upon 3-NBA exposure. As shown by cell cycle analysis, an increased number of S-phase cells was observed after 24 h of treatment with 3-NBA. Furthermore, 3-NBA was shown to inhibit cell proliferation when added to subconfluent cell cultures. The main metabolite of 3-NBA, 3-ABA, induced statistically significant increases in tail moment as judged by alkaline comet assay. The potential of 3-NBA and 3-ABA to enhance the production of reactive oxygen species (ROS) was demonstrated by flow cytometry using 2′,7′-dichlorofluorescein-diacetate (DCFH-DA). The enzyme inhibitors allopurinol, dicumarol, resveratrol and SKF525A were used to assess the impact of metabolic conversion on 3-NBA-mediated ROS production. Resveratrol decreased dichlorofluorescein (DCF) fluorescence by 50%, suggesting a role for CYP1A1 in 3-NBA-mediated ROS production. Mitochondrial ROS production was significantly attenuated (20% reduction) by addition of rotenone (complex I inhibition) and thenoyltrifluoroacetone (TTFA, complex II inhibition). Taken together, the results of the present study provide evidence for a genotoxic potential of 3-ABA in human epithelial lung cells. Moreover, both compounds lead to increased intracellular ROS and create an environment favorable to DNA damage and the promotion of cancer.

Cytoprotective Effect of ICL670 (Deferasirox) on At20 Endocrine Cells Susceptible to Iron-Evoked Oxidative Damage.

A major cause of biological damage associated with tissue iron accumulation is the cellular acquisition of circulating labile plasma iron (LPI) by endocytic mechanisms and ensuing metal-catalyzed oxidations. Endocrine cells are presumed to have endocytic abilities and/or high susceptibility to the formation of reactive oxygen intermediates (ROIs), particularly those generated in the presence of labile iron. As these properties might be implicated in the early onset of endocrine dysfunctions observed in transfusional hemosiderosis, a major goal of iron chelation therapy is to prevent iron ingress into endocrine cells and ensuing toxicity and/or removal of cell-accumulated iron. Such properties of chelators have been afforded in cardiac cells (Glickstein et al. 2006, Blood In press) by treating LPI-carrying plasma with pharmacologically attainable concentrations of orally active iron chelators in clinical practice. The latter also evoked neutralization of metal-catalyzed ROS formation, by iron chelation and extrusion from cells and thereby supported cardiac functional restoration. We explored here whether addition of ICL670 (deferasirox) could rescue acutely iron-overloaded At20 pituitary cells from iron-toxicity, using fluorescence-plate reader and microscopy-imaging for live monitoring of cell functions and assessing toxicity in terms of:cell integrity (calcein cell retention assay);metabolic activities (mitochondrial Alamar-Blue assay); andintracellular (ACTH) hormone levels. Our studies indicate that in cell culture conditions and at pharmacological chelator concentrations, ICL670 can preserve both At20 endocrine cell viability and functional properties that were compromised by iron-catalyzed formation of ROIs.

Postprandial hyperglycemia alters inflammatory and hemostatic parameters

Glucose or glucose derived products are increased in blood during the postprandial phase and are, to a certain extent, related to meal composition. Glucose and glucose derived products such as advanced glycation end products (AGEs) can be formed in the intracellular compartment but can also be absorbed as AGEs or AGE precursors present in food. Glucose, glucose metabolites and AGEs alter endothelial cell functions, induce adhesion molecule overexpression (ICAM-1, VCAM), cytokine release (IL-6, MCP-1) and tissue factor production. Tumor necrosis factor alpha systemic level is increased during the postprandial phase as are augmented C reactive protein and fibrinogen level. Hyperglycemia induced an increase in plasminogen activator inhibitor, and shortened fibrinogen half life. Hyperglycemia and AGEs provoked an oxidant stress. The formation of reactive oxygen intermediates perturbates NO (Nitric oxide) formation and are deleterious for cell functions. All the modifications observed in the postprandial phase are not too deleterious but their iterative characteristics may lead to vascular dysfunction.

Regulation of adenosine receptor subtypes during cultivation of human monocytes: role of receptors in preventing lipopolysaccharide-triggered respiratory burst.

Adenosine is a potent anti-inflammatory agent that modulates the function of cells involved in the inflammatory response. Here we show that it inhibits lipopolysaccharide (LPS)-induced formation of reactive oxygen intermediates (ROI) in both freshly isolated and cultured human monocytes. Blocking of adenosine uptake and inactivation of the adenosine-degrading enzyme adenosine deaminase enhanced the inhibitory action of adenosine, indicating that both pathways regulate the extracellular adenosine concentration. Adenosine-mediated inhibition could be reversed by XAC (xanthine amine congener), an antagonist of the adenosine receptor A(2A), and MRS 1220 [N-9-chloro-2-(2-furanyl)[1, 2, 4]-triazolo[1,5-c]quinazolin-5-benzeneacetamide], an A(3) receptor antagonist, in both cell populations, while DPCPX (1,3-dipropyl-8-cyclopentylxanthine), an A(1) receptor antagonist, had no effect. Similar to what was seen with adenosine, CGS 21680, an A(2A) and A(3) receptor agonist, and IB-MECA, a nonselective A(1) and A(3) receptor agonist, dose dependently prevented ROI formation, indicating the involvement of A(3) and probably also A(2A) in the suppressive effect of adenosine. Pretreatment of monocytes with adenosine did not lead to changes in the LPS-induced increase in intracellular calcium levels ([Ca(2+)](i)). Thus, participation of [Ca(2+)](i) in the action of adenosine seems unlikely. The adenosine-mediated suppression of ROI production was found to be more pronounced when monocytes were cultured for 18 h, a time point at which changes in the mRNA expression of adenosine receptors were observed. Most prominent was the increase in the A(2A) receptor mRNA. These data demonstrate that cultivation of monocytes is accompanied by changes in the inhibitory action of adenosine mediated by A(3) and probably also the A(2A) receptor and that regulation of adenosine receptors is an integral part of the monocyte differentiation program.

The metastasis suppressor gene C33/CD82/KAI1 induces apoptosis through reactive oxygen intermediates.

Here we describe the isolation of C33/CD82/KAI1 in a screen for apoptosis-inducing genes. C33 is a gene that is downregulated in many metastatic tumor cells and the expression of which can attenuate the process of metastases formation in a variety of tumors. In accordance, we observed cell death induction by C33 in many different cell types. C33 seems to promote cell death by the generation of reactive oxygen intermediates (ROIs). These ROIs, however, are not derived from the mitochondrial respiratory chain as in most other scenarios leading to apoptosis. We observed that C33 renders cells sensitive to ROIs by causing the specific release of the intracellular antioxidant glutathione (GSH) from cells. Moreover, C33 activates the GTPase Cdc42, which mediates GSH release and apoptosis induction and allows to detect the formation of ROIs.

Proteoglycan isolated from Phellinus linteus activates murine B lymphocytes via protein kinase C and protein tyrosine kinase

Medicinal mushrooms are increasingly used to treat a wide variety of disease processes. Aqueous extract from the fruiting body or mycelia of Phellinus linteus has been reported to produce antitumor and immunomodulatory activities in vivo and in vitro. However, the therapeutic mechanism has not been known. In the present study, we investigated whether proteoglycan (PL) isolated from P. linteus has an effect on the immunomodulatory activities of the murine splenic lymphocytes (MSLs). Treatment with PL caused a four-fold augmentation in [ 3H]thymidine incorporation compared to untreated control group in MSLs. Flow cytometric analysis indicated that the affected cell population was mainly CD19 + cells, but not CD3 + cells. These data suggested that the main target of PL was the B cells, but not T cells. PL also enhanced the expression of co-stimulatory molecules, CD80 and CD86, in murine B cells in a time-dependent manner. Accordingly, we investigated if intracellular [Ca 2+] and reactive oxygen intermediates (ROI) were the principal downstream components that contributed to PL-induced activation, with respect to both increases of proliferation and induction of co-stimulatory molecules. However, PL has no influence on the [Ca 2+] concentration and the ROI formation in murine B cells, whereas the genistein, protein tyrosine kinase (PTK) inhibitor or staurosporine, protein kinase C (PKC), blocked the proliferation and the induction of co-stimulatory molecules, CD80 and CD86, in B cells stimulated with PL. Taken together, these data suggest that PL is a biological response modifier that stimulates proliferation and expression of co-stimulatory molecules in B cells, probably by regulating PTK and PKC signaling pathways.

Induction of hepatic heme oxygenase-1 by diclofenac in rodents: role of oxidative stress and cytochrome P-450 activity

Background/Aims: The role of oxidative stress in diclofenac hepatotoxicity is still not clear. This study examined whether the drug induced heme oxygenase-1 (HO-1), a stress protein. Methods: HO-1 mRNA and HO activity were measured in mouse liver and in rat hepatocytes after treatment with diclofenac parallel to release of serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) as a marker of hepatic damage. Results: HO-1 was transcriptionally and dose-dependently induced by diclofenac in mouse liver and rat hepatocytes. HO-1 mRNA, ALT and SDH peaked at the same time. Mechanistic studies revealed that the drug synergized with buthionine sulfoximine (BSO) in lowering hepatic glutathione, increased the formation of reactive oxygen intermediates and activated NF-κB and AP-1 in rat hepatocytes. HO-1 induction and hepatic damage were increased by BSO and only HO-1 induction was attenuated by the antioxidant N-acetylcysteine. HO-1 induction was also reduced by the cytochrome P-450 inhibitors ketoconazole and tranylcypromine, concomitantly with a significant decrease in the formation of diclofenac oxidative metabolites, which may give rise to reactive compounds. Conclusions: Acute treatment with diclofenac induces HO-1 in rodent hepatocytes. Induction is influenced by changes in the cellular redox states and by cytochrome P-450 activity and gives a new insight into the response of the liver to diclofenac.

Glucose increases endothelial-dependent superoxide formation in coronary arteries by NAD(P)H oxidase activation: attenuation by the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor atorvastatin.

Increased vascular superoxide anion (O(2)(-)) formation is essentially involved in the pathophysiology of atherosclerosis. Chronic hyperglycemia induces endothelial dysfunction, probably due to increased formation of reactive oxygen intermediates. However, little is known about the localization, modulators, and molecular mechanisms of vascular O(2)(-) formation during hyperglycemia. In porcine coronary segments, high glucose significantly increased O(2)(-) formation (1,703.5 +/- 394.9 vs. 834.1 +/- 91.7 units/mg for control, n = 64, P < 0.05; measured by lucigenin-enhanced chemiluminescence). This effect was completely blocked after removal of the endothelium. Coincubation with 10 micromol/l atorvastatin, a lipophilic inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, attenuated basal and glucose-induced O(2)(-) formation (328.1 +/- 46.5 and 332.8 +/- 50.3 units/mg, P < 0.05 vs. without atorvastatin). Incubation with mevalonic acid reversed this effect. High glucose increased mRNA expression of the oxidase subunit p22(phox), which was blocked by 10 micromol/l atorvastatin, whereas expression of gp91(phox) was unchanged. In conclusion, glucose-induced increase of vascular O(2)(-) formation is endothelium dependent and is probably mediated by increased p22(phox) subunit expression. Beneficial effects of statins in diabetic patients may be explained in part by attenuation of vascular O(2)(-) formation independent of lipid lowering.

Reactive oxygen intermediates and glutathione regulate the expression of cytosolic ascorbate peroxidase during iron-mediated oxidative stress in bean.

Excess of free iron is thought to harm plant cells by enhancing the intracellular production of reactive oxygen intermediates (ROI). Cytosolic ascorbate peroxidase (cAPX) is an iron-containing, ROI-detoxifying enzyme induced in response to iron overload or oxidative stress. We studied the expression of cAPX in leaves of de-rooted bean plants in response to iron overload. cAPX expression, i.e., mRNA and protein, was rapidly induced in response to iron overload. This induction correlated with the increase in iron content in leaves and occurred in the light as well as in the dark. Reduced glutathione (GSH), which plays an important role in activating the ROI signal transduction pathway as well as in ROI detoxification, was found to enhance the induction of APX mRNA by iron. To determine whether cAPX induction during iron overload was due to an increase in the amount of free iron, which serves as a co-factor for cAPX synthesis, or due to iron-mediated increase in ROI production, we tested the expression of APX in leaves under low oxygen pressure. This treatment, which suppresses the formation of ROI, completely abolished the induction of cAPX mRNA during iron overload, without affecting the rate of iron uptake by plants. Taken together, our results suggest that high intracellular levels of free iron in plants lead to the enhanced production of ROI, which in turn induces the expression of cAPX, possibly using GSH as an intermediate signal. We further show, using cAPX-antisense transgenic plants, that cAPX expression is essential to prevent iron-mediated tissue damage in tobacco.

C60-Fullerene monomalonate adducts selectively inactivate neuronal nitric oxide synthase by uncoupling the formation of reactive oxygen intermediates from nitric oxide production.

C(60)-Fullerene monomalonate adducts inactivate selectively the neuronal nitric oxide synthase isoform in a manner completely preventable by the concurrent presence of superoxide dismutase and catalase. This inactivation is time-, fullerene concentration-, and turnover-dependent and is not reversible by dilution. The di(carboxypropan-3-ol)methano-[60]-fullerene (diol adduct) has no effect on NADPH consumption by nNOS as measured in the absence of arginine substrate, but dramatically increases NADPH consumption in the presence of arginine. This fullerene-enhanced NADPH consumption is linked to oxygen as electron acceptor and is accompanied by the increased production of hydrogen peroxide. These effects of fullerene monomalonate adducts are unique to the nNOS isoform and are not observed using either the iNOS or the eNOS isoform. The inhibitory effects of fullerene monomalonate adducts are unaltered and insurmountable by increased concentrations of arginine, tetrahydrobiopterin, or calmodulin. These observations indicate that fullerene monomalonate adducts uncouple in the presence of arginine the formation of reactive oxygen intermediates from NO production by nNOS. These reactive oxygen intermediates dissociate from the enzyme and, acting from solution, inactivate NOS NO forming activity.

Influence of diabetes on cardiac nitric oxide synthase expression and activity

There is some evidence that the endothelium dependent vasodilatation of coronary arteries is impaired in both types of diabetes. The underlying mechanisms are not yet clear, in particular whether this defect is caused by a direct effect of diabetes on the activity and the expression of nitric oxide synthases (NOS) or indirectly by an enhanced inactivation of nitric oxide. Methods: To study this question we determined the activity (conversion of L-arginine to citrulline) and the mRNAs encoding the isoforms of NOS (using polymerase chain reaction after reverse transcription of the mRNAs into cDNAs by reverse transcriptase) in hearts of streptozotocin diabetic rats and in rat heart endothelial cells (RHEC). The formation of reactive oxygen intermediates (ROI) was measured by the dichloro-dihydro-fluorescein method. Results: The activity of total NOS and the amounts of mRNAs encoding ecNOS and iNOS were dependent on the duration of diabetes. After a diabetes duration of 4 to 6 weeks both the total activity as well as the mRNAs encoding ecNOS and iNOS were elevated. A reduction of NOS activity and the amounts of mRNAs of ecNOS and iNOS was only seen after a diabetes duration longer than 20 weeks, a time at which a loss of endothelium has been described. In RHEC, high glucose (22 mM) and H 2O 2 (100 μM) were able to increase the mRNA encoding ecNOS, but not iNOS. This increase in ecNOS mRNA was inhibited by lipoic acid (1 μM). In addition, high glucose (22 and 30 mM) led to an enhanced formation of ROI and to activation of the transcription NFκB. Conclusion: These observations suggest that diabetes causes a temporary increase in NOS activity and ecNOS mRNA in the rat heart which is presumably the consequence of an enhanced oxidative stress exerted by hyperglycaemia. Together with previously published observations, our data suggest that the impairment of endothelium dependent vasodilatation in rat heart is not the consequence of a reduced activity and expression of NOS, but is caused by an enhanced inactivation of nitric oxide by ROI.

Effects of hepatocellular iron imbalance on nitric oxide and reactive oxygen intermediates production in a model of sepsis

Background/Aims: In mammals iron homeostasis is most important, as imbalance of iron such as iron overload may lead to severe diseases. Recently, it has been shown that the iron regulatory protein-1 is partially controlled by nitric oxide and reactive oxygen intermediates, molecules frequently seen in inflammatory events. The aim of the present study was to investigate the effects of impaired iron homeostasis on the interaction of nitric oxide, and reactive oxygen intermediate formation in hepatocytes in a model of acute inflammation. Methods: Hepatocytes isolated from Corynebacterium parvum (C. parvum)-injected rats were used to examine the formation of nitrogen and oxygen intermediates by iron deprivation and iron overload in the presence of lipopolysaccharide. In addition, we investigated the RNA binding and aconitase activity of iron regulatory protein-1. Results: In the present study we show that iron overload in lipopolysaccharide-treated C. parvum-primed hepatocytes downregulated the RNA binding of iron regulatory protein-1 and aconitase activity. Subsequently, we observed a reduced formation of nitrite/ nitrate and S-nitrosothiols but an increased production of reactive oxygen species, and hepatocellular damage. Moreover, the addition of iron to cell cultures caused a further increase in cellular damage, a drop in the cellular glutathione pool, and an increase in peroxynitrite and hydroxyl-like radicals. In contrast, addition of deferoxamine (an iron chelator) to lipopolysaccharide-treated C. parvum-primed hepatocytes protected cells by stabilizing the GSH content, maintaining the nitric oxide formation, and by reducing Fenton oxidants. Conclusions: Our results show that the antioxidative effects of iron chelators prevent the formation of toxic Fenton oxidants in severe inflammatory events, which should be considered in the treatment of disorders characterized by an iron imbalance.

Suppression of the reactive oxygen intermediates production of human macrophages by colorectal adenocarcinoma cell lines.

Although some in vitro studies indicate that macrophages exert cytotoxic responses against tumour cells by production of reactive oxygen intermediates (ROI), no obvious impairment of tumour cell growth is visible in various human malignant tumours, which contain a large number of tumour-associated macrophages (TAM). We made use of an in vivo-like co-culture model of multicellular tumour spheroids of three colon carcinoma cell lines (HRT-18, HT-29, CX-2) and three functionally different phenotypes of human macrophages (27E10, RM3/1, 25F9) to investigate if tumour cells deactivate macrophage cytotoxicity. The production of ROI was measured by a lucigenin-amplified chemiluminescence assay in a 96-well-microplate luminometer. Different capabilities to produce ROI by different macrophage phenotypes were observed. However, independent of the macrophage phenotype and the tumour cell type a significant inhibition of ROI formation was found in co-cultures after 1 hr, 1 and 2 days. Macrophages were also suppressed by tumour cell supernatants, which contained anti-inflammatory cytokines transforming growth factor-beta1 (TGF-beta1) and negligible levels of interleukin-4 (IL-4) and IL-10 as shown by enzyme-linked immunosorbent assay (ELISA). Although recombinant human cytokines TGF-beta1, IL-10 and IL-4 inhibited the production of ROI in freshly isolated monocytes, these cytokines had no effect on differentiated macrophage phenotypes, indicating that these cytokines are not involved in mediating tumour-induced suppression of ROI production by human macrophages.

Tumor necrosis factor-alpha plus actinomycin D-induced apoptosis of L929 cells is prevented by nitric oxide.

Treatment with the nitric oxide-(NO)-generating compound S-nitroso-N-acetylpenicillamine protected cul-tured L929 cells from apoptosis induced by tumor necrosis factor-alpha (TNF-alpha) plus actinomycin D, as determined by the detection of DNA fragmentation and morphological changes. NO also prevented an enhancement of the production of reactive oxygen intermediates by TNF-alpha plus actinomycin D, as assessed by the oxidation of dihydrorhodamine 123 and hydroethidine. Because the inhibition of mitochondrial respiration by rotenone or antimycin A suppressed the increased oxidation of both dihydrorhodamine 123 and hydroethidine, it was suggested that TNF-alpha accelerated the leakage of reactive oxygen intermediates from the mitochondrial electron transport system. Polarography showed that NO reversibly inhibited mitochondrial respiration at either complexes I-III, II-III, or IV, thus suggesting the inhibition of cytochrome oxidase. Taken together, these findings indicate that the decreased mitochondrial formation of reactive oxygen intermediates in the presence of NO might have a protective effect against TNF-alpha plus actinomycin D-induced apoptosis.