Monochloramine produces reactive oxygen species in liver by converting xanthine dehydrogenase into xanthine oxidase

In the present study, we assessed the influence of monochloramine (NH₂Cl) on the conversion of xanthine dehydrogenase (XD) into xanthine oxidase (XO) in rat liver in vitro. When incubated with the partially purified cytosolic fraction from rat liver, NH₂Cl (2.5-20 μM) dose-dependently enhanced XO activity concomitant with a decrease in XD activity, implying that NH₂Cl can convert XD into the reactive oxygen species (ROS) producing form XO. The NH₂Cl (5 μM)-induced XD/XO interconversion in the rat liver cytosol was completely inhibited when added in combination with an inhibitor of NH₂Cl methionine (25 μM). A sulfhydryl reducing agent, dithiothreitol at concentrations of 0.1, 1 and 5 mM also dose-dependently reversed the NH₂Cl (5 μM)-induced XD/XO interconversion. These imply that NH₂Cl itself acts on the XD/XO interconversion, and that this conversion occurs at the cysteine residues in XD. Furthermore, using the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate, it was found that NH₂Cl could increase ROS generation in the cytoplasm of rat primary hepatocyte cultures, and that this increase might be reversed by an XO inhibitor, allopurinol. These results suggest that NH₂Cl has the potential to convert XD into XO in the liver, which in turn may induce the ROS generation in this region.     

Biological durability and oxidative potential of a stonewool mineral fibre compared to crocidolite asbestos fibres

Experiments are described concerning the differences in redox properties and biodurability of natural asbestos fibres and an experimental stonewool fibre incubated in Gamble solution and reconstructed surfactant fluid. Crocidolite exhibits a significantly higher oxidative potential compared to the tested stonewool fibres. The oxidative acitivity of both types of fibres is not constant during incubation over several weeks, but rather shows a sinoidal curve including reactivities much higher than those at the beginning of the incubation. A continuous loss of mass is concluded not to be definitively connected with a continuous loss of toxicity. Received: 19 November 1996 / Accepted: 27 March 1997     

Evaluation of the effects of felodipine,verapamil and hydralazine on the survival rate of rats subjected to lethal effects of oxygen free radicals

Summary Intravenous administration of xanthine (X: 0.225 mg/kg, i.v.) plus xanthine oxidase (XO: 3.0 units/ kg, i.v.) to anesthetized rats resulted in a rapid fall in the arterial pressure and a mortality rate of over 80% during 120 min observation period. Pretreatment of the rats with superoxide dismutase (SOD) or SOD plus catalase significantly enhanced survival rate to 60% confirming that the toxicity after [X + XO] administration is due to the generation of oxygen free radicals. Pretreatment of the rats with either felodipine, a dihydropyridine calcium antagonist or verapamil, a structurally different Ca²⁺-channel blocker was most effective in promoting survival rate to 90%; in contrast, hydralazine, an arteriolar dilator but not a calcium antagonist, was ineffective in significantly enhancing survival.In the vehicle treated groups, mortality of the rats after [X + XO] administration was associated with significant increases in serum creatine phosphokinase (CPK) levels; both the calcium antagonists as well as hydralazine prevented any significant changes in CPK levels. Since only the calcium antagonists but not hydralazine were effective in providing significant protection against mortality, the data suggests that CPK may not be a reliable indicator to predict prevention of lethal toxicity induced by free radicals. Hence, the observation that calcium antagonists can promote survival would suggest that calcium overload may be the ultimate mediator of tissue toxicity. These observations can account for the remarkable efficacy of various calcium antagonists in preventing ischemia-reperfusion induced damage to organs, such as heart and kidneys, in which a role for free radicals has been postulated. Correspondence to B. S. Jandhyala, at the above address     

Uncoupling of cytochrome P450 1A and stimulation of reactive oxygen species production by co-planar polychlorinated biphenyl congeners

The non-ortho-polychlorinated biphenyl (PCB) congener 3,3'4,4'-tetrachlorobiphenyl (PCB 77) can uncouple the catalytic cycle of fish (scup) cytochrome P4501A (CYP1A) and mammalian (rat, human) CYP1A1, stimulating release of reactive oxygen species (ROS). PCB 77 also inactivates CYP1A in an NADPH-, oxygen-, and time-dependent process, linked to uncoupling. We addressed a hypothesis that planar halogenated hydrocarbons generally will uncouple CYP1A. Thus, additional PCB congeners including non-ortho-3,3',4,4',5'-pentachlorobiphenyl (PCB 126) and 3,3',4,4',5,5'-hexachlorobiphenyl (PCB 169), mono-ortho-2,3,3',4,4'-pentachlorobiphenyl (PCB 105) and di-ortho-2,2',5,5'-tetrachlorobiphenyl (PCB 52), as well as the polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P), were examined for their ability to stimulate microsomal ROS production and to inactivate CYP1A. Incubated without NADPH, non-ortho-PCB 126 and -PCB 169 both inhibited microsomal CYP1A activity (ethoxyresorufin O-deethylase; EROD). When NADPH was included, these congeners caused a progressive inactivation of CYP1A, in addition to the inhibition. The determined K(Inact) values for inactivation were 0.14 and 0.08 microM, respectively, for PCB 126 and PCB 169, similar to the 0.05 microM for PCB 77 previously reported. The mono-ortho-PCB 105 weakly inhibited and weakly inactivated CYP1A. The di-ortho-PCB 52 neither inhibited nor inactivated CYP1A. Alone, B[a]P strongly inhibited CYP1A, but when NADPH was added that inhibition was reversed, apparently by metabolic depletion of the substrate, and there was no inactivation. PCB 126 and PCB 169 stimulated release of ROS from induced liver microsomes, while B[a]P, PCB 52 and PCB 105 did not. ROS release and CYP1A inactivation stimulated by the non-ortho-PCB 126 and PCB 169 indicate an uncoupling of CYP1A like that previously shown with PCB 77. The uncoupling and release of ROS further suggest a participation of CYP1A in the oxidative stress associated with some planar halogenated aryl hydrocarbon receptor agonists.     

Effect of chronic apocynin treatment on nitric oxide and reactive oxygen species production in borderline and spontaneous hypertension

The purpose of this study was to investigate the effect of NAD(P)H oxidase inhibitor – apocynin (4-hydroxy-3-methoxyacetophenone) on the increase of systolic blood pressure (SBP) in borderline (BHR) and spontaneously hypertensive rats (SHR). Young 6-week-old male BHR (offspring of SHR dams andWistar Kyoto sires) and SHR were treated with apocynin (30 mg/kg/day) for six weeks. SBP was measured by tail-cuff plethysmography. Nitric oxide synthase (NOS) activity was determined in the left ventricle and aorta. Protein expression of nuclear factor kappa B (NF-κB) and NAD(P)H oxidase subunits p67phox and p22phox as well as concentration of cGMPwere determined for the left ventricle. Apocynin significantly decreased SBP in all groups investigated. Administration of apocynin had no effect on NOS activity in either tissue studied. However, apocynin decreased protein expression of NF-κB (p65) and NAD(P)H oxidase subunit p22phox in both hypertensive groups and p67phox subunit in the SHR group. Moreover, apocynin was able to prevent a decrease in cGMP concentration in the left ventricle of both hypertensive groups. In conclusion, our study demonstrated that apocynin treatment partially prevented SBP rise in borderline and spontaneously hypertensive rats, yet without increasing activity of NOS in the left ventricle and aorta. However, apocynin was able to decrease production of reactive oxygen species in hypertensive rats; thus preventing the decrease in cGMP formation.     

Suppression of rat neutrophil reactive oxygen species production and adhesion by the diterpenoid lactone andrographolide

The present study was to examine whether andrographolide, a diterpenoid lactone isolated from the anti-inflammatory herbal medicine Andrographis paniculata (Burm. f.) Nees. (Acanthaceae), has the ability to prevent phorbol-12-myristate-13-acetate (PMA)-induced reactive oxygen species (ROS) production, as well as N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced adhesion by rat neutrophils. Results demonstrated that PMA (100 ng/ml) induced rapid accumulation of H2O2 and O2. in neutrophils within 30 minutes. Andrographolide (0.1 to 10 microM) pretreatment (10 min, 37 degrees C) significantly attenuated the accumulation of these two oxygen radical metabolites. Administration of andrographolide also significantly prevented fMLP-induced neutrophil adhesion. These data suggest that preventing ROS production and neutrophils adhesion may confer andrographolide the ability to be an anti-inflammatory drug.     

Induction of apoptosis and reactive oxygen species production by N-nitrosopiperidine and N-nitrosodibutylamine in human leukemia cells

N-nitrosopiperidine (NPIP) and N-nitrosodibutylamine (NDBA) belong to a group of N-nitrosamines that are widely distributed in foodstuffs and the occupational environment. In the present study, the human promyelocytic leukemia cell line HL-60, was used to characterize the apoptotic effects of N-nitrosamines, and to examine the production of reactive oxygen species (ROS). Apoptotic cells were identified by (i) chromatin condensation (ii) flow cytometry analysis and (iii) poly(ADP-ribose) polymerase (PARP) cleavage. NPIP and NDBA induced morphological changes consistent with apoptotic events in HL-60 cells. Flow cytometry analysis showed that both N-nitrosamines induced apoptotic cell death in a concentration and time dependent-manner. It was observed that NDBA was stronger than NPIP, since it induced a significant apoptotic cell death after 18 h starting from a concentration of 2 mm, whereas NPIP was effective at 10 mm. Furthermore, PARP was markedly cleaved with 0.5 mm of NDBA and 5 mm of NPIP after treatments for 3 and 18 h, respectively. Finally, the ROS level was found to be elevated after 0.5 h of treatment with both N-nitrosamines. Antioxidant N-acetylcysteine (NAC) completely inhibited the ROS production induced by NPIP and NDBA. However, this action seems not to be associated with the apoptosis because NAC did not block N-nitrosamines-induced apoptosis. The data demonstrate that NPIP and NDBA induce apoptosis and ROS production in HL-60 cells.     

Post-translational activation of human phenylalanine 4-monooxygenase from an endobiotic to a xenobiotic enzyme by reactive oxygen and reactive nitrogen species

1. An investigation into the post-translational activation of cDNA-expressed human phenylalanine 4-monooxygenase and human hepatic cytosolic fraction phenylalanine 4-monooxygenase activity with respect to both endobiotic metabolism and xenobiotic metabolism revealed that the reactive oxygen species (hydrogen peroxide and hydroxyl radical) and reactive nitrogen species (nitric oxide and peroxynitrite) could elicit the post-translational activation of the enzyme with respect to both of these biotransformation reactions.

2. In virtually all instances, the K_m values were decreased and the V_max values were increased; the only exceptions observed being with hydrogen peroxide and L-phenylalanine.

3. These effects were shown to occur at activator concentrations known to exist in physiological situations and, hence, suggest that reactive oxygen and reactive nitrogen species may cause, and may be involved with, the post-translational activation of phenylalanine 4-monooxygenase within the human body.

4. This mechanism, in response to free-radical bursts, may enable the enzyme to expand its substrate range and to process certain xenobiotics as and when required.

     

The production of reactive oxygen species in peripheral blood neutrophils is modulated by airway mucous

Neutrophils are a major source of reactive oxygen species (ROS). The role of airway mucous on ROS production is unknown. The aim of our study was to investigate the direct influence of bronchoalveolar lavage fluid (BALF) and induced sputum (IS) alone or in combination with chemical/biological stimulus on ROS production in peripheral blood neutrophils during chronic obstructive pulmonary disease (COPD). Neutrophils were isolated from peripheral blood of 47 patients with moderate COPD and 14 healthy individuals (HI). BALF/RPMI (1:1) or IS/RPMI (1:1) from COPD patients were used to stimulate neutrophils alone or in combination with phorbolmyristate- acetate (PMA) (0.1–30 nM) or Staphylococcus aureus bacteria (0.7–500 bact/neutrophil). Relative generation of ROS was measured flow cytometrically. BALF/RPMI and in combination with relatively low PMA or all bacteria concentrations stimulated ROS; while, combination with relatively high PMA concentrations suppressed ROS in of COPD patients and HI. IS/RPMI and its combination with PMA inhibited ROS generation in both groups; whereas, IS stimulated or had a tendency to stimulate ROS production with relatively high bacteria concentrations. In conclusion, BALF and IS directly or in combination with chemical/biological factors modulated ROS production. This effect was stronger in neutrophils from COPD patients and depended on chemical/biological stimulus intensity.     

Induction of apoptosis and modulation of production of reactive oxygen species in human endothelial cells by diphenyleneiodonium

Diphenyleneiodonium (DPI) inhibits activity of flavoenzymes like NADPH oxidase, the major source of superoxide anion in cardiovascular system, but affects also other oxidoreductases. Contradictory data have been published concerning the effect of diphenyleneiodonium on the production of reactive oxygen species in cells, both inhibitory and stimulatory action of DPI being reported. We have examined the effect of DPI on the cellular production of reactive oxygen and nitrogen species (ROS/RNS) and on the proliferation and apoptosis of human vascular endothelial cells. We found increased oxidation of ROS-sensitive probes (dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate) when DPI (20 microM-100 microM) was present in the treated cells. However, oxidation of the fluorogenic probes was inhibited if DPI (20 microM-100 microM) was removed from the reaction medium after cell preincubation. These results suggest an artifactual oxidation of the fluorogenic probes by DPI or its metabolites. A similar pattern of influence of DPI on the production of NO (measured with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) was observed. Modulation of generation of reactive oxygen and nitrogen species in DPI-treated cells influenced the nitration of tyrosine residues of cellular proteins, estimated by Western blotting. Decreased level of nitration generally paralleled the lowered production of ROS. A decreased 3-(4,5-dimethylthiazolyl)-3-3(4-sulphophenyl) tetrazolium (MTT) reducing activity of cells for was observed immediately after 1h treatment of human endothelial cells with DPI (1 microM-100 microM), in spite of lack of changes in cell viability estimated by other methods. These results point to a next limitation of MTT in estimation of viability of cells treated with oxidoreductase inhibitors. DPI inhibited the proliferation of HUVECs as well as immortalized cell line HUVEC-ST, as assessed by acid phosphatase activity test and measurement of total nucleic acid content. Proapoptotic action of DPI was observed 12 h after incubation with this compound.     

Mechanism of apoptosis induced by apigenin in hepg2 human hepatoma cells: involvement of reactive oxygen species generated by NADPH oxidase

Although plant-derived flavonoids have been reported to have anti-cancer activities, the exact mechanism of these actions is not completely understood. In this study we investigated the role for reactive oxygen species (ROS) as a mediator of the apoptosis induced by apigenin, a widespread flavonoid in plant, in HepG2 human hepatoma cells. Apigenin reduced cell viability, and induced apoptotic cell death in a dose-dependent manner. In addition, it evoked a dose-related elevation of intracellular ROS level. Treatment with various inhibitors of the NADPH oxidase (diphenylene iodonium, apocynin, neopterine) significantly blunted both the generation of ROS and induction of apoptosis induced by apigenin. These results suggest that ROS generated through the activation of the NADPH oxidase may play an essential role in the apoptosis induced by apigenin in HepG2 cells. These results further suggest that apigenin may be valuable for the therapeutic management of human hepatomas.     

Cell death and production of reactive oxygen species by murine macrophages after short term exposure to phthalates

The effects of four phthalates, i.e., di-2-ethylhexyl phthalate (DEHP), butyl benzyl phthalate (BBP), dibutyl phthalate (DBP) and diisobutyl phthalate (DIBP) on necrotic and apoptotic cell death, and production of reactive oxygen species (ROS) were studied on mouse macrophage cell line RAW 264.7. All the phthalates caused negligible and non-dose-dependent ROS production compared to control experiment. DEHP and BBP did not cause significant necrotic nor apoptotic cell death at any of the studied doses. Both DIBP and DBP caused dose-dependent necrotic cell death at the two highest concentrations (100 μM and 1 mM). Both doses (500 μM and 1 mM) of DIBP increased apoptosis by 31- and 60-fold, respectively, whereas the increase in apoptotic cell death caused by DBP was only two and fourfold, that however, was not statistically significant. In conclusion, DIBP caused a substantially different apoptotic cell death effect on murine macrophages from the three other phthalates, and this effect was not related to ROS production. Thus, toxicological and health risks of DIBP and DBP should be assessed separately in the future.     

Contributions of reactive oxygen species and mitogen-activated protein kinase signaling in arsenite-stimulated hemeoxygenase-1 production

Hemeoxygenase-1 (HO-1) is an oxidative stress responsive gene upregulated by various physiological and exogenous stimuli. HO-1 has cytoprotective activities and arsenite is a potent inducer of HO-1 in many cell types and tissues, including epidermal keratinocytes. We investigated the potential contributions of reactive oxygen species (ROS) generation and mitogen-activated protein kinase (MAPK) activation to arsenite-dependent regulation of HO-1 in HaCaT cells, an immortalized human keratinocyte line. Both epidermal growth factor (EGF) and arsenite stimulated ROS production was detected by dihydroethidium (DHE) staining and fluorescence microscopy. Arsenite induced HO-1 in a time- and concentration-dependent manner, while HO-1 expression in response to EGF was modest and evident at extended time points (48-72 h). Inhibition of EGF receptor, MEK I/II or Src decreased arsenite-stimulated HO-1 expression by 20-30%. In contrast, addition of a superoxide scavenger or inhibition of p38 activity decreased the arsenite-dependent response by 80-90% suggesting that ROS and p38 are required for HO-1 induction. However, ROS generation alone was insufficient for the observed arsenite-dependent response as use of a xanthine/xanthine oxidase system to generate ROS did not produce an equivalent upregulation of HO-1. Cooperation between ERK signaling and ROS generation was demonstrated by synergistic induction of HO-1 in cells co-treated with EGF and xanthine/xanthine oxidase resulting in a response nearly equivalent to that observed with arsenite. These findings suggest that the ERK/MAPK activation is necessary but not sufficient for optimal arsenite-stimulated HO-1 induction. The robust and persistent upregulation of HO-1 may have a role in cellular adaptation to chronic arsenic exposure.     

Nicotine- and tar-free cigarette smoke induces cell damage through reactive oxygen species newly generated by PKC-dependent activation of NADPH oxidase

We examined cytotoxic effects of nicotine/tar-free cigarette smoke extract (CSE) on C6 glioma cells. The CSE induced plasma membrane damage (determined by lactate dehydrogenase leakage and propidium iodide uptake) and cell apoptosis {determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction activity and DNA fragmentation}. The cytotoxic activity decayed with a half-life of approximately 2 h at 37°C, and it was abolished by N-acetyl-l-cysteine and reduced glutathione. The membrane damage was prevented by catalase and edaravone (a scavenger of ^?OH) but not by superoxide dismutase, indicating involvement of ^?OH. In contrast, the CSE-induced cell apoptosis was resistant to edaravone and induced by authentic H₂O₂ or O₂^? generated by the xanthine/xanthine oxidase system, indicating involvement of H₂O₂ or O₂^? in cell apoptosis. Diphenyleneiodonium [NADPH oxidase (NOX) inhibitor] and bisindolylmaleimide I [BIS I, protein kinase C (PKC) inhibitor] abolished membrane damage, whereas they partially inhibited apoptosis. These results demonstrate that 1) a stable component(s) in the CSE activates PKC, which stimulates NOX to generate reactive oxygen species (ROS), causing membrane damage and apoptosis; 2) different ROS are responsible for membrane damage and apoptosis; and 3) part of the apoptosis is caused by oxidants independently of PKC and NOX.[Supplementary methods and Figure: available only at http://dx.doi.org/10.1254/jphs.11166FP]     

A comparison of the production of reactive oxygen species by suspended particulate matter and diesel exhaust particles with macrophages

Oxidative stress has emerged as a pivotal mechanism that underlies the toxic pulmonary effects of suspended particulate matter (SPM). Experimental evidence shows that redox-active transition metals, redox-cycling quinoids, and polycyclic aromatic hydrocarbons (PAHs) contained in SPM act synergistically, producing reactive oxygen species (ROS). The direct production of superoxide anion and the damaging hydroxyl radical has been studied in aqueous and dimethyl sulfoxide (DMSO) suspensions of SPM both with and without H2O2; however, no study has reported on the release of ROS from ingesting macrophages with SPM. We investigated the time course of the ability to induce lucigenin-dependent chemiluminescence (CL) from human monocyte-derived macrophages exposed to SPM, carbon black particles, and diesel exhaust particles (DEP). We also examined hydroxyl radical generation from the same experimental system using the 2-deoxy-d-robse method. We found an increase of CL for SPM, but not for carbon black particles or for DEP. Hydroxyl radical generation was observed in both SPM and DEP, but the release from DEP was more frequent than that from SPM. These results suggest that certain components of SPM are important in the response of ROS from ingesting macrophages with SPM, and that those components are discharged from SPM into the atmosphere.     

Anti-aging effects of chlorpropamide depend on mitochondrial complex-II and the production of mitochondrial reactive oxygen species

Sulfonylureas are widely used oral anti-diabetic drugs. However, its long-term usage effects on patients’ lifespan remain controversial, with no reports of influence on animal longevity. Hence, the anti-aging effects of chlorpropamide along with glimepiride, glibenclamide, and tolbutamide were studied with special emphasis on the interaction of chlorpropamide with mitochondrial ATP-sensitive K⁺ (mitoK-ATP) channels and mitochondrial complex II. Chlorpropamide delayed aging in Caenorhabditis elegans, human lung fibroblast MRC-5 cells and reduced doxorubicin-induced senescence in both MRC-5 cells and mice. In addition, the mitochondrial membrane potential and ATP levels were significantly increased in chlorpropamide-treated worms, which is consistent with the function of its reported targets, mitoK-ATP channels. Increased levels of mitochondrial reactive oxygen species (mtROS) were observed in chlorpropamide-treated worms. Moreover, the lifespan extension by chlorpropamide required complex II and increased mtROS levels, indicating that chlorpropamide acts on complex II directly or indirectly via mitoK-ATP to increase the production of mtROS as a pro-longevity signal. This study provides mechanistic insight into the anti-aging effects of sulfonylureas in C. elegans.     

Silver nanoparticle induced toxicity to human sperm by increasing ROS(reactive oxygen species) production and DNA damage

Silver nanoparticles (AgNPs) have been used in medical products and industrial coatings, due to their antimicrobial properties. Excessive use of AgNPs can have adverse effects on the human body, however, their toxicity characteristics to human sperm and the potential mechanisms are not entirely clear. In this study, we exposed human sperm to different doses of AgNPs (0, 50 μg ml⁻¹, 100 μg ml⁻¹, 200 μg ml⁻¹ or 400 μg ml⁻¹) for various times (15 min, 30 min, or 60 min), followed by analyses of the sperm viability, motility and the ratio of abnormal to normal sperm.Then, transmission electron microscopy(TEM) was used to explore the sperm ultrastructural characteristics. Reactive oxygen species production and DNA fragmentation were tested using standard kits and the sperm chromatin dispersion method, respectively. The results showed a dose- and time-dependent decline in sperm viability and motility and an increased ratio of abnormal to normal sperm after 30 min and 60 min of exposure to AgNPs at 200 μg ml⁻¹ and 400 μg ml⁻¹. The most common abnormalities were sperm heads with disrupted chromatin or absent acrosomes, bent tails, and curved mid-pieces. The ultrastructural characteristics of AgNP-treated sperm included disrupted, swollen, granular and vacuolar defects of the chromatin. In addition, ROS(reactive oxygen species)production and DNA fragmentation were markedly increased after 60 min of exposure to AgNPs at 200 μg ml⁻¹ and 400 μg ml⁻¹. Our results indicated that AgNPs caused detrimental changes in human sperm characteristics, and the excessive use of AgNPs should be carried out with caution.     

Effects of methoxylation of apocynin and analogs on the inhibition of reactive oxygen species production by stimulated human neutrophils.

Owing to their multiple side effects, the use of steroidal drugs is becoming more and more controversial, resulting in an increasing need for new and safer anti-inflammatory agents. In the inflammatory process, reactive oxygen species produced by phagocytic cells are considered to play an important role. We showed that apocynin (4'-hydroxy-3'-methoxy-acetophenone or acetovanillone), a non-toxic compound isolated from the medicinal plant Picrorhiza kurroa, selectively inhibits reactive oxygen species production by activated human neutrophils. Apocynin proved to be effective in the experimental treatment of several inflammatory diseases such as arthritis, colitis and atherosclerosis. These features suggest that apocynin could be a prototype of a novel series of non-steroidal anti-inflammatory drugs (NSAIDs). So far, apocynin is mainly used in vitro to block NADPH oxidase-dependent reactive oxygen species generation by neutrophils. In order to get a better insight in what chemical features play a role in the anti-inflammatory effects of apocynin, a structure-activity relationship study with apocynin analogs was performed. We show here that especially substances with an additional methoxy group at position C-5 display enhanced anti-inflammatory activity in vitro. Our approach may lead to the development of more effective anti-inflammatory agents which are safe and which lack the side effects of steroids.     

Understanding the biology of reactive oxygen species and their link to cancer: NADPH oxidases as novel pharmacological targets

Reactive oxygen species (ROS), the cellular products of myriad physiological processes, have long been understood to lead to cellular damage if produced in excess and to be a causative factor in cancer through the oxidation and nitration of various macromolecules. Reactive oxygen species influence various hallmarks of cancer, such as cellular proliferation and angiogenesis, through the promotion of cell signalling pathways intrinsic to these processes and can also regulate the function of key immune cells, such as macrophages and regulatory T cells, which promote angiogenesis in the tumour environment. Herein we emphasize the family of NADPH oxidase enzymes as the most likely source of ROS, which promote angiogenesis and tumourigenesis through signalling pathways within endothelial, immune and tumour cells. In this review we focus on the pharmacological inhibitors of NADPH oxidases and suggest that, compared with traditional anti‐oxidants, they are likely to offer better alternatives for suppression of tumour angiogenesis. Despite the emerging enthusiasm towards the use of NADPH oxidase inhibitors for cancer therapy, this field is still in its infancy; in particular, there is a glaring lack of knowledge of the roles of NADPH oxidases in in vivo animal models and in human cancers. Certainly a clearer understanding of the relevant signalling pathways influenced by NADPH oxidases during angiogenesis in cancer is likely to yield novel therapeutic approaches.