Induction of oxidative cell damage by photo-treatment with zinc meta N-methylpyridylporphyrin

We have previously reported that isomeric Zn(II) N-methylpyridylporphyrins (ZnTM-2(3,4)-PyP^4 + ) can act as photosensitizers with efficacy comparable to that of hematoporphyrin derivative (HpD) in preventing cell proliferation and causing cell death in vitro. To better understand the biochemical basis of this activity, the effects of photo-activated ZnTM-3-PyP^4 +  on GSH/GSSG ratio, lipid peroxidation, membrane permeability, oxidative DNA damage, and the activities of SOD, catalase, glutathione reductase, and glutathione peroxidase were evaluated. Light exposure of ZnTM-3-PyP^4 + -treated colon adenocarcinoma cells caused a wide spectrum of oxidative damage including depletion of GSH, inactivation of glutathione reductase and glutathione peroxidase, oxidative DNA damage and peroxidation of membrane lipids. Cell staining with Hoechst-33342 showed morphological changes consistent with both necrotic and apoptotic death sequences, depending upon the presence of oxygen.     

Photosensitizing action of isomeric zinc N-methylpyridylporphyrins in human carcinoma cells

The success of photodynamic therapy (PDT), as a minimally invasive approach, in treating both neoplastic and non-neoplastic diseases has stimulated the search for new compounds with potential application in PDT. We have previously reported that Zn(II) N-alkylpyridylporphyrins (ZnTM-2(3,4)-PyP⁴⁺ and ZnTE-2-PyP⁴⁺) can act as photosensitizers and kill antibiotic-resistant bacteria. This study investigated the photosensitizing effects of the isomers of ZnTMPyP⁴⁺ (ZnTM-2(3,4)-PyP⁴⁺) and respective ligands on a human colon adenocarcinoma cell line. At 10 μM and 30 min of illumination the isomeric porphyrins completely inhibited cell growth, and at 20 μM killed approximately 50% of the cancer cells. All these effects were entirely light-dependent. The isomers of the ZnTMPyP⁴⁺ and the respective ligands show high photosensitizing efficiency and no toxicity in the dark. Their efficacy as photosensitizers is comparable to that of hematoporphyrin derivative (HpD).     

Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: implication in hemodialysis mediated atherogenic LDL modification

Objective: Al³⁺ stimulates Fe²⁺ induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al³⁺ and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al³⁺ on LDL oxidation.

Materials and methods: Using different LDL modifying systems (Fe²⁺, Cu²⁺, free radical generating compounds, human endothelial cells, hemin/H₂O₂ and HOCl), the influence of Al³⁺ on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation.

Results: Al³⁺ could stimulate the oxidizability of LDL by Fe²⁺, but not in the other systems tested. Al³⁺ and Fe²⁺ were found to bind to LDL and Al³⁺could compete with Fe²⁺ binding to the lipoprotein. Fluorescence polarization data indicated that Al³⁺ does not affect the phospholipid compartment of LDL.

Conclusions:The results indicate that increased LDL oxidation by Fe²⁺ in presence of Al³⁺ might be due to blockage of Fe²⁺ binding sites on LDL making more free Fe²⁺ available for lipid oxidation.     

Iron (II) Ions Induced Oxidation of Ascorbic Acid and Glucose

Lipid peroxidation (LPO) of polyunsaturated fatty acids (PUFAs) is suspected to be involved in the generation of chronic diseases. A model reaction for LPO is the air oxidation of PUFAs initiated by Fe²⁺ and ascorbic acid. In the course of such model reactions glycolaldehyde (GLA) was detected as main aldehydic product. Since it is difficult to explain the generation of GLA by oxidation of PUFAs, it was suspected that GLA might be derived by oxidation of ascorbic acid. This assumption was verified by treatment of ascorbic acid with Fe²⁺.

Produced aldehydic compounds were trapped by addition of pentafluorobenzylhydroxylamine hydrochloride (PFBHA-HCl), trimethylsilylated and finally identified by gas chromatography/mass spectrometry (GC/MS). Oxidation of ascorbic acid with O₂ in presence of iron ions produced not only glycolaldehyde (GLA), but also glyceraldehyde (GA), dihydroxyacetone (DA) and formaldehyde. Glyoxal (GO) and malondialdehyde (MDA) were detected as trace compounds.

The yield of the aldehydic compounds was increased by addition of lipid hydroperoxides (LOOH) or H₂O₂. The buffer influenced the reaction considerably: Iron ions react with Tris buffer by producing dihydroxyace-tone (DA). Since ascorbic acid is present in biological systems and Fe²⁺ ions are obviously generated by cell damaging processes, the production of GLA and other aldehydic components might add to the damaging effects of LPO.

Glucose suffers also oxidation to short-chain aldehydic compounds in aqueous solution, but this reaction requires addition of equimolar amounts of Fe²⁺ together with equimolar amounts of H₂O₂ or 13-hydroperoxy-9-cis-11-trans-octadecadienoic acid (13-HPODE). Therefore this reaction, also influenced by the buffer system, seems to be not of biological relevance.     

Increased levels of superoxide in brains from old female rats

Hypertension, aging and a range of neurodegenerative diseases are associated with increased oxidative damage. The present study examined whether superoxide (O₂^•-) levels in brain are increased during aging in female rats, and the role of superoxide dismutase (SOD) and oestrogen in regulating O₂^•- levels.

Young adult (3 month) and old (11 month) female spontaneously hypertensive stroke prone rats (SHRSP) and normotensive Wistar-Kyoto rats (WKY) were studied. O₂^•- levels were measured in brain homogenates by lucigenin chemiluminescence and SOD expression by Western blotting. Ageing significantly increased brain O₂^•- levels in WKY (cortex +216%, hippocampus +320%, striatum +225%) and to a greater extent in SHRSP (cortex +540%, hippocampus +580%, striatum +533%). Older SHRSP showed a decline in cortical Cu/Zn SOD expression compared to young adult SHRSP. Oestrogen did not attenuate O₂^•- levels.

The results show a significant age-dependent increase in brain O₂^•- levels which is exaggerated in SHRSP. The excess cortical O₂^•- levels in the SHRSP may be associated with a down-regulation of Cu/Zn SOD but are not related to a decrease in oestrogen.     

Copper-Ligand Interactions and Physiological Free Radical Processes. pH-Dependent Influence of Cu2+ Ions on Fe2+-Driven OH- Generation

Prior to comparative studies on the reactivity of various copper complexes with respect to OH radicals, the influence of free Cu²⁺ ions on the superoxide-independent generation of OH radicals through Fenton assays and water gamma radiolysis has been tested in the present work.

Cu²⁺ ions have been shown to behave in a distinct manner towards each of these two production systems. As was logically expected from the noninvolvement of copper in OH^- radical production through gamma radioiysis, no influence of Cu²⁺ ions has been observed on the amount of radicals detected in that case. In contrast, Cu²⁺ ions do influence OH^- radical generation through iron-driven Fenton reactions, but differently depending on copper concentration.

When present in high concentrations, Cu²⁺ ions significantly contribute to OH^- radical production, which confirms previous observations on the reactivity of these in the presence of hydrogen peroxide. At lower levels corresponding to copper/iron ratios below unity on the contrary, Cu²⁺ ions behave as inhibitors of the OH^- production in a pH-dependent manner over the 1-6 range investigated: the lower the pH, the greater the inhibition.

The possible origin of this previously unreported inhibitory effect is discussed.     

Application of High Enzyme Activities Present in Clostridium Thermoaceticum for the Efficient Regeneration of NADPH, NADP+, NADH AND NAD+

Crude extracts of Clostridium thermoaceticum DSM 521 contain various AMAPORs (artificial mediator accepting pyridine nucleotide oxidoreductases). The specific activities of this mixture of AMAPORs is about 8-9 U mg^-1 protein (µmoles mg^-1 min^-1) for NADPH and 3-4 U mg^-1 protein for NADH formation with reduced methylviologen (MV⁺⁺) as electron donor. These AMAPOR-activities are only slightly oxygen sensitive. The reoxidation of NADPH and NADH with carboxamido-methylviologen is catalysed by crude extracts with 2.0 and 1.6 U mg^-1 protein, respectively. The same crude extracts also catalyse the dehydrogenation of reduced pyridine nucleotides with suitable quinones such as anthraquinone-2,6-disulphonate. The reduced quinone can be reoxidised by dioxygen.

The K_m-values of these enzymes for the pyridine nucleotides and also for the artificial electron mediators are in a suitable range for preparative transformations.

Furthermore the crude extract of C. thermoaceticum contains about 2.5 U mg^-1 protein of an NADP⁺-dependent formate dehydrogenase (FDH), which is suitable for NADPH and/or MV⁺⁺ regeneration. The regeneration of MV⁺⁺ with FDH and formate as electron donor proceeds with a specific activity of about 5 U mg^-1 protein of the crude extract. The reduced viologen in turn reduces NAD(P)⁺ by AMAPOR. The formate dehydrogenase is sensitive to oxygen.

Examples of compounds which have been prepared by combination of AMAPORs or formate dehydrogenase with an oxidoreductase are: (S)-3-hydroxycarboxylates, esters of (S)-3-hydroxycarboxylates, (1R,2S)-1-hydroxypropane-tricarboxylate (D_s-(+)-isocitrate), L_s-(-)-isocitrate and 6-phosphogluconate.     

Oxidant Stress and Glomerular Prostanoid Production: Influence of Angiotensin Converting Enzyme Inhibition

1. The effect of H₂O₂ (4.7 × 10^-9 4.7 × 10^-3M) on prostanoid production by isolated glomeruli from normotensive (WKY) and, spontaneously hypertensive rats (SHR) has been studied.

2. Oxidant stress significantly increased synthesis of prostaglandin E₂(PGE₂), I₂(PGI₂)and thromboxane A₂ (TxA₂) by glomeruli from both strains whereas the ratio (PGE₂ + PGI₂)/TxA₂ increased in only SHR.

3. Pre-incubation of glomeruli with the angiotensin converting enzyme inhibitors captopril or lisinopril, had virtually no effect on H₂O₂-induced synthesis of individual prostanoids nor on the ratio (PGE₂ + PGI₂)/TA₂ by glomeruli from either WKY or SHR.

4. The findings suggest that H₂O₂-induced changes in glomerular function may be mediated, in part, by PGs but fail to support the suggestion that the ability of ACEI to protect glomeruli from H₂O₂-induced damage is determined by PGs.     

Decreased hepatic ischemia-reperfusion injury by manganese-porphyrin complexes

Reactive oxygen and nitrogen species have been implicated in ischemia-reperfusion (I/R) injury. Metalloporphyrins (MP) are stable catalytic antioxidants that can scavenge superoxide, hydrogen peroxide, peroxynitrite and lipid peroxyl radicals. Studies were conducted with three manganese-porphyrin (MnP) complexes with varying superoxide dimutase (SOD) and catalase catalytic activity to determine if the MnP attenuates I/R injury in isolated perfused rat livers. The release of the hepatocellular enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) was maximal at 1 min reperfusion, decreased rapidly and increased gradually by 90 min. Manganese tetrakis-(N-ethyl-2 pyridyl) porphyrin (MnTE-2-PyP) decreased ALT, AST, LDH at 1-90 min reperfusion, while manganese tetrakis-(N-methyl-2 pyridyl) porphyrin (MnTM-2-PyP) and manganese tetrakis-(ethoxycarbonyl) porphyrin (MnTECP) decreased ALT and LDH from 5 to 90 min reperfusion. The release of thiobarbituric acid-reacting substances (TBARS) was diminished by MnTE-2-PyP and MnTM-2-PyP at 90 min. The extent of protein nitration (nitrotyrosine, NT) was decreased in all three MnPs treated livers. These results demonstrate that MnP complexes can attenuate hepatic I/R injury and may have therapeutic implications in disease states involving oxidants.     

Ascorbic Acid Oxidation and DNA Scission Catalyzed by Iron and Copper Chelates

The asorbic acid (AH^-) auto-oxidation rates catalyzed by copper chelates of 1,10-phenanthroline (OP) or by iron chelates of bleomycin (BLM) are only slightly higher than the oxidation rates catalyzed by the metal ions. AH^- oxidation in the presence of DNA is accompanied by degradation of the DNA. The rates of DNA scission by the metal chelates are markedly higher than the rates induced by the free metal ions. AH^- oxidation is slowed down in the presence of DNA which forms ternary complexes with the chelates. The ternary complexes react slowly with AH^- but induce DNA double strand breaks more efficiently than the free metal chelates. With OP, DNA is degraded by the reaction of the ternary complex, DNA-(OP)₂Cu(I), withH₂O₂

AH^- oxidation in the presence of DNA was biphasic, showing a marked rate increase after DNA was cleaved. We suggest that this sigmoidal pattern of the oxidation curves reflects the low initial oxidative activity of the ternary complexes, accelerating as DNA is degraded.

Using O₂^-produced by pulse radiolysis as a reductant, we found that AH^- oxidation with (OP)₂Cu(II) induced more DNA double strand breaks per single strand break than bipyridine-copper.

The site specific DNA damaging reactions indicated by these results are relevant to the mechanism of cytotoxic activities of bleomycin and similar antibiotics or cytotoxic agents.     

Effects of hypochlorite on cultured respiratory epithelial cells

Neutrophils and eosinophils are involved in the pathogenesis of many respiratory diseases. The enzymes myeloperoxidase and eosinophil peroxidase catalyze the reaction of H₂O₂ with Cl to produce the reactive oxygen species HOCl.

Normal human bronchial epithelial (NHBE) cells were exposed to 0.18-0.90 mM HOCl for 48 h, and studied with immunohistochemical, metabolic and morphological studies.

The ability of the cells to attach to each other and/or to the matrix was altered. Immunohistochemical studies showed a decreased amount of desmosomes and focal adhesion sites, although the morphology of the cells was not affected. The ability of the mitochondria to oxidize glucose was reduced. HOCl-exposed cells had an increased production of NO, probably by an increased activity of cNOS, due to increased intracellular Ca²⁺. The antioxidant N-acetylcysteine inhibited both the NO production and the effects of HOCl on glucose oxidation. The cNOS-inhibitor N-propyl-L-arginine inhibited HOCl-induced NO production. X-ray microanalysis showed an increase in the intracellular Na⁺/K⁺ ratio, which indicates cell damage.

In conclusion, exposure to HOCl results in cell detachment and metabolic alterations in normal human bronchial epithelial cells. Oxygen radicals could in part mediate the effects. Oxygen radicals could hence contribute to the observed epithelial damage in respiratory diseases.     

Reduction of DNA fragmentation and hydroxyl radical production by hyaluronic acid and chondroitin-4-sulphate in iron plus ascorbate-induced oxidative stress in fibroblast cultures

Glycosaminoglycans (GAGs), components of extracellular matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. Oxidative stress is one of the most frequent causes of tissue and cell injury and the consequent lipid peroxidation is the main manifestation of free radical damage. It has been found to play an important role in the evolution of cell death. Since several reports have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) are able to inhibit lipid peroxidation during oxidative stress, We investigated the antioxidant capacity of these GAGs in reducing oxidative damage in fibroblast cultures.

Free radicals production was induced by the oxidizing system employing iron (Fe₂₊) plus ascorbate. We evaluated cell death, membrane lipid peroxidation, DNA damage, protein oxidation, hydroxyl radical (OH_•) generation and endogenous antioxidant depletion in human skin fibroblast cultures.

The exposition of fibroblasts to FeSO₄ and ascorbate caused inhibition of cell growth and cell death, increased OH_• production determined by the aromatic trap method; furthermore it caused DNA strand breaks and protein oxidation as shown by the DNA fragments analysis and protein carbonyl content, respectively. Moreover, it enhanced lipid peroxidation evaluated by the analysis of conjugated dienes (CD) and decreased antioxidant defenses assayed by means of measurement of superoxide dismutase (SOD) and catalase (CAT) activities.

When fibroblasts were treated with two different doses of HYA or C4S a protective effect, following oxidative stress induction, was shown. In fact these GAGs were able to limit cell death, reduced DNA fragmentation and protein oxidation, decreased OH_• generation, inhibited lipid peroxidation and improved antioxidant defenses.

Our results confirm the antioxidant activity of HYA and C4S and this could represent a useful step in the understanding of the exact role played by GAGs in living organisms.     

Enhanced Superoxide Production with on Change of the Antioxidant Activity in Gingival Fluid of Patients with Chronic Adult Periodontitis

In the gingival crevicular fluid (GCF) of control and chronic adult periodontitis (CAP) patients there is a spontaneous release of O₂^- radicals from polymorphonuclear leukocytes (PMN). The addition of the exogenous stimuli phorbol myrystate acetate (PMA) decreased the O₂^- formation in control GCF, while in CAP patients produced a marked enhancement of O₂^- generation.

The circulating PMN of control subjects did not show a spontaneous O₂^- formation, differently from CAP patients. On the contrary, a similar O₂^- production was measured when the circulating PMN were stimulated with PMA.

Moreover, the antioxidant activity measured in 10μl of cell free gingival supernatant (GS) of control and CAP patients had the same values by inhibiting 12.6% and 18.9% respectively of the O₂^- formation supported by a xanthine/xanthine oxidase system.

Probably, the protective or destructive effect of PMN in GCF of CAP patients depends on the variations of the rate of O₂^- formation in respect to the intrinsic antioxidant property of GS.     

低氧应激下甘肃鼢鼠与SD 大鼠心脏抗氧化酶和ATP 酶活性的比较

为探讨低氧应激下甘肃鼢鼠心脏对抗氧化损伤和电生理紊乱的可能机制,对甘肃鼢鼠和SD 大鼠在4. 5%氧浓度下分别进行2 h、4 h、6 h、8 h、10 h、16 h 低氧应激,比较常氧和各时程低氧下二者心脏超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽还原酶(GR)和Ca^{2 +} - ATP 酶、Ca^{2 +} - Mg^{2 +} - ATP 酶、Na ⁺ - K ⁺ - ATP酶活性,以丙二醛(MDA)含量作为机体氧化损伤指标。结果显示,常氧组甘肃鼢鼠GR 活性比SD 大鼠高,SOD、CAT、Ca^{2 +}-ATP 酶、Ca^{2 +} - Mg^{2 +} - ATP 酶和Na ⁺ - K ⁺ - ATP 酶活性及MDA 含量与SD 大鼠相比均无显著性差
异;低氧组甘肃鼢鼠SOD、CAT、GR、Ca^{2 +} - ATP 酶、Ca^{2 +} - Mg^{2 +} - ATP 酶和Na ⁺ - K ⁺ - ATP 酶活性迅速升高,显著高于SD 大鼠,MDA 含量则显著低于SD 大鼠。说明甘肃鼢鼠心脏通过提高抗氧化酶活性清除低氧诱导产生的多余自由基,并通过提高ATP 酶活性保证心电活动正常、心率稳定,应对低氧应激。     

Menadiol as an electron donor for reversed oxidative phosphorylation in submitochondrial particles

Dithiothreitol in the presence of menadione or N,N,N′,N′-tetramethyl-p-phenylenediamine provides the reducing equivalents for oxidative phosphorylation and the ATP-dependent reduction of NAD⁺ in submitochondrial particles. With menadione the reaction is nearly as fast as with succinate and it is insensitive to antimycin, indicating electron entry between the first and second sites of oxidative phosphorylation. The phenylenediamine-mediated reduction of NAD⁺ is nearly as fast as succinate-linked reduction and is antimycin sensitive.     

Copper-Ligand Interactions and Physiological free Radical Processes. Part 2. Influence of Cu2+ Ions on Cu+-Driven Oh Generation and Comparison with Their Effects on Fe2+-Driven Oh Production

In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu²⁺ ions on the Cu⁺/H₂O₂ reaction has been investigated.

This influence depends on the C_Cu²⁺/C_Cu⁺ ratio. At low Cu²⁺ concentrations, ·OH damage to various detector molecules decreases with increasing Cu²⁺ concentrations until C_Cu²⁺/C_Cu⁺ reaches unity. Above this value, ·OH damage increases sharply until C_Cu²⁺/C_Cu⁺ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu²⁺ cancels. Finally, at higher concentrations, Cu²⁺ ions logically add their own ·OH production to that normally expected from Cu⁺ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu⁺ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu²⁺ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand.     

Exogenous N-linoleoyl tyrosine marker as a tool for the characterization of cellular oxidative stress in macrophages

Oxidative stress and its resultant products continue to attract investigators. Numerous endogenous substances have been suggested as potential markers for the identification of oxidative stress in tissues and organisms. In this study, we present a novel concept whereby an exogenous marker is designed and synthesized for the characterization of oxidative stress. The designed marker is constructed from tyrosine (Tyr) and linoleic acid (LA), which are attached covalently to form N-linoleoyl tyrosine (N-LT). Each of the two components (Tyr and LA) is known to be easily oxidized upon exposure to different types of reactive species. Combining the two allows their distinction from the endogenous Tyr and LA in the tested biological samples. The ability of the N-LT marker to characterize oxidative stress in macrophage cell lines was first studied using different types of ROS/RNS. N-LT was found to interact with macrophages, binding to the cell membrane. Upon treatment of J-774 A.1 macrophages with N-LT (40 μM) and with various oxidants; HOCl (0.2, 0.4 mM), copper ions (20 μM), SIN-1 (0.1, 1.0 mM), specific oxidized N-LT (Ox-N-LT) products were formed, depending on the type of oxidant used. Exposing cells to HOCl (0.2 mM) resulted in exclusive attack of the LA residue of N-LT, preferentially forming an adduct of HOCl to the LA double bond (N-L(HOCl)T, 4.3%). In contrast, when SIN-1 (0.1 mM) was applied as the oxidant, the Tyr moiety of N-LT was most reactive, yielding a nitration product of the Tyr aromatic ring (N-LT(NO₂), 1.8%). Similar N-LT oxidation in cell-free systems yielded a significantly higher content of Ox-N-LT (10.8% N-L(HOCl)T, 7% N-LT(NO₂)). The designed marker was then tested with peritoneal macrophages taken from atherosclerotic apolipoprotein-deficient (E⁰) mice showing specific and selective oxidation of N-LT to yield N-LT-hydroperoxide (1.9% N-L(OOH)T), at significantly higher levels than resulted from similar experiments using peritoneal macrophages harvested from control BalbC mice (0.0% N-L(OOH)T). In contrast, the differences in N-L(epoxy)T level between BalbC and E⁰ mice were not significant using both types of peritoneal macrophages (E⁰ and BalbC), suggesting that N-L(OOH)T is characteristic of the atherosclerotic state. Thus, we show that the designed marker is sufficiently sensitive to detect oxidative stress imposed on cells and cell-free systems and to react selectively with the various ROS/RNS induced. Such a marker may be useful for characterizing oxidative stress in general, and possibly also in oxidative-stress-associated diseases.     

In vitro study of the antioxidant properties of non steroidal anti-inflammatory drugs by chemiluminescence and electron spin resonance (ESR)

Objectives. To determine the antioxidant activities of nonsteroidal anti-inflammatory drugs (NSAIDS), we examined by chemiluminescence (CL) and electron spin resonance (ESR) their scavenging properties towards lipid peroxides, hypochlorous acid and peroxynitrite.

Methods. The antioxidant properties of nimesulide (NIM), 4-hydroxynimesulide (4-HONIM), aceclofenac (ACLO), 4-hydroxyaceclofenac (4-HOA-CLO), diclofenac (DICLO) and indomethacin (INDO) were tested on four different reactive oxygen species (ROS) generating systems: (I) phorbol-myristate acetate (PMA)-activated neutrophils, (II) Fe²⁺/ascorbate-induced lipid peroxidation, (III) HOCl-induced light emission, (IV) the kinetics of ONOO^- decomposition followed by spectrophotometry. ROS production was monitored by luminol-enhanced CL or by ESR using two different spin traps.

Results. At 10 μM, ACLO, NIM, 4-HONIM, 4-HOA-CLO, and DICLO decreased luminol-enhanced CL generated by PMA-activated neutrophils. Inversely, INDO increased the luminol enhanced CL. Interestingly, hydroxylated metabolites were more potent antioxidants than the parent drugs. Furthermore, all drugs tested, excepted ACLO, lowered lipid peroxidation induced by Fe²⁺/ascorbate system. ACLO and DICLO, even at the highest concentration tested (100 μM), did not significantly lower HOCl induced CL, whereas the other drugs were potent scavengers. Finally, all the NSAIDS accelerated decomposition of ONOO^-, suggesting a potential capacity of the molecules to scavenge peroxynitrite.

Conclusion. The NSAIDs possess variable degrees of antioxidant activities, linked to their ability to react with HOCl, lipid peroxides or ONOO^-. These antioxidant activities could offer interesting targeted side-effects in the treatment of joint inflammatory diseases.     

Catalysis of catechol oxidation by metal-dithiocarbamate complexes in pesticides

Dithiocarbamate (DTC)-based pesticides have been implicated in Parkinson’s disease (PD) through epidemiological links to increased risk of PD, clinical reports of parkinsonism following occupational exposure to the DTC-based pesticide maneb, and experimental studies showing dopaminergic neurodegeneration with combined exposure of rats to maneb and paraquat. We hypothesize that the manganese-ethylene-bis-dithiocarbamate (MnEBDC) complex in maneb may produce oxidative stress by catalyzing catechol oxidation. We tested this hypothesis by performing a structure-function analysis of metal-EBDC and metal-diethyldithiocarbamate (DEDC) complexes of Mn²⁺, Zn²⁺, and Cu²⁺ to catalyze oxidation of N-acetyldopamine (NA-DA) and 3,4-dihydroxyphenylacetic acid (DP) in the presence and absence of N-acetylcysteine (NAC), a model of glutathione. Both Mn-DTCs retained the capacity of the parent ion to catalyze one-electron oxidation of NA-DA, but lost the ability to catalyze DP oxidation. Strikingly, while Zn²⁺ did not catalyze catechol oxidation, both Zn-DTCs catalyzed one-electron oxidation of NA-DA but not DP. While Cu²⁺ catalyzed oxidation of both catechols, Cu-DTCs were inert. Similar results were obtained with MnEBDC and dopamine or norepinephrine; however, zinc-ethylene-bis-dithiocarbamate was less efficient at catalyzing oxidation of these catechols. Our results point to the potential for manganese- and zinc-containing EBDC pesticides to promote oxidative stress in catecholaminergic regions of the brain.