Comparative protection against oxyradicals by three flavonoids on cultured endothelial cells

Oxygen-derived free radicals are known to injure the endothelium of aorta in diverse disorders. In this study we compared the cytoprotective effects of three flavonoids against oxyradical damage to porcine aortic endothelial cells in vitro. Cultured porcine aortic endothelial cells were exposed to oxyradicals generated by xanthine oxidase--hypoxanthine (XO-HP). The cytoprotective activities of morin, quercetin, and catechin on these systems were compared using established morphologic criteria. The results in the XO-HP system showed that morin at 0.125, 0.25, and 0.5 mM delayed cell necrosis to 27.4 +/- 1.3, 46.8 +/- 1.8, and longer than 70 min, respectively, compared with 12.0 +/- 1.3 min in the control group. These degrees of protection were significantly stronger than those provided by quercetin and catechin at corresponding concentrations (p < 0.01). Morin and quercetin were moderate inhibitors of xanthine oxidase on the basis of the oxygen consumption rate, whereas catechin at the same concentrations had little inhibitory effect. The data from uric acid formation and cytochrome c reduction were consistent with the oxygen consumption measurement for the three flavonoids.     

The role of iron in neurotoxicity: a study of novel antimalarial drugs

The antimalarial drug artemisinin and its derivatives display neurotoxicity in animal studies in vivo and in neuronal cells in vitro. Their toxicity may be due to an interaction of iron with the endoperoxide bridge of the derivative to produce toxic free radicals and/or other toxic metabolites. In this study, 0.3 microM artemether (AEM) in the presence of 2 microM haemin significantly inhibited the outgrowth of neurites from differentiating NB2a neuroblastoma cells by up to 76%. The antioxidants ascorbic acid and glutathione completely protected against this toxicity at a concentration of 100 microM. AEM was found to be partially converted to two isomeric products, which were identified as the tetrahydrofuran acetate isomer of AEM and 3alpha-hydroxydesoxyartemether.     

Effects of silibinin and antioxidants on high glucose-induced alterations of fibronectin turnover in human mesangial cell cultures

To elucidate the primary mechanism of high glucose cytotoxicity, the cytoprotective properties of antioxidants against metabolical disorders were assessed in human mesangial cell (HMC) cultures. An 8-day incubation of HMC with high glucose concentration (30 mM) resulted in an extracellular accumulation of the matrixprotein fibronectin (FN), owing to both an expansion of the matrix-associated pericellular FN and a 60% increase of the soluble molecule in the culture medium. The high glucose-induced FN alterations were not due to osmotical effects, as assessed by an iso-osmotic mannitol control. Rather, they are mediated by oxygen-free radicals because the combined treatment of HMC with high glucose and either the antioxidative flavonoid silibinin (given as the water soluble derivative silibinin-C-2,3-dihydrogensuccinate disodium salt) or a radical scavenger cocktail totally prevented the extracellular FN accumulation. This is corroborated further by the determination of malondialdehyde, a product of lipid peroxidation. Incubation of HMC with high glucose resulted in an increase of malondialdehyde in cell homogenates which was completely counteracted by either silibinin or a radical scavenger cocktail. Silibinin alone had no effects on protein synthesis and culture growth. The data presented are compatible with oxidative stress induced by high glucose concentration in HMC cultures. The study further substantiates the proposed role of silibinin in the amelioration of glucose cytotoxicity in renal cells.     

A new trinuclear complex of platinum and iron efficiently promotes cleavage of plasmid DNA

The compound [[Pt(trpy)]2Arg-EDTA]+ is synthesized in five steps, purified, and characterized by 1H, 13C, and 195Pt NMR spectroscopy, mass spectrometry, UV-vis spectrophotometry, and elemental analysis. The binuclear [[(Pt(trpy)]2Arg]3+ moiety binds to double-stranded DNA, and the chelating EDTA moiety holds metal cations. In the presence of ferrous ions and the reductant dithiothreitol, the new compound cleaves DNA. It cleaves a single strand in the pBR322 plasmid nearly as efficiently as methidiumrpropyl-EDTA (MPE), and it cleaves a restriction fragment of the XP10 plasmid nonselectively and more efficiently than [Fe(EDTA)]2-. The mechanism of cleavage was studied in control experiments involving different transition-metal ions, superoxide dismutase, catalase, glucose oxidase with glucose, metal-sequestering agents, and deaeration. These experiments indicate that adventitious iron and copper ions, superoxide anion, and hydrogen peroxide are not involved and that dioxygen is required. The cleavage apparently is done by hydroxyl radicals generated in the vicinity of the DNA molecule. The reagent [[Pt(trypy)]2Arg-EDTA]+ differs from methidiumpropyl-EDTA in not containing an intercalator. This difference in binding modes between the binuclear platinum(II) complex and the planar heterocycle may cause useful differences between the two reagents in cleavage of nucleic acids.     

Free radicals and antioxidants

The data obtained from the author's laboratory were used to make this review. The author's classification of free radicals, approaches, the origin and metabolism of primary radicals, the contribution of iron ions to the production of secondary radicals and the mechanisms of antioxidative protection of cells and tissues from damage are considered. According to the classification proposed, the radicals may be divided into primary (superoxide, semiquinones and nitric oxide), secondary (hydroxyl and lipid radicals) and tertiary (radicals of antioxidants). The primary radicals are formed by enzymatic systems and perform biologically important functions. The secondary radicals are formed from hydroperoxides in the reactions of divalent iron ions and damage to cell structures. In the cells and blood plasma, there is a complicated system of antioxidants that prevent the production of secondary radicals. All antioxidants may be arbitrarily divided into water-soluble and hydrophobic. The first group involves the enzymes catalase and glutathione peroxidase, iron ion chelators (such as ceruloplasmin and transferrin in the blood and carnosine in other tissues), and, probably, hydroxyl radical traps, such as uric acid and ascorbate. The hydrophobic antioxidants include primarily the free radical traps alpha-tocopherol, flavonoids, and carotenes. Studies of lipid peroxidation kinetics in the membranous structures, carried out by chemiluminescence and mathematical modeling of the reactions have shown that the radicals of antioxidants (such as alpha-tocopherol) enter the further reactions in the lipid phase, including those with lipid hydroperoxides.     

Toxicity of fotemustine in rat hepatocytes and mechanism-based protection against it

Fotemustine is a relatively novel DNA-alkylating 2-chloroethyl-substituted N-nitrosourea (CENU) drug, clinically used for the treatment of disseminated malignant melanoma in different visceral and non-visceral tissues. Thrombocytopenia has been observed in patients treated with fotemustine and liver and renal toxicities as well. In this study, firstly the metabolism of fotemustine was investigated in vitro and secondly the undesired cytotoxicity of fotemustine as well as different ways of protection against it. In rat hepatocytes, chosen as a model system, fotemustine was shown to cause lactate dehydrogenase (LDH) leakage, glutathione (GSH) depletion, GSSG-formation and lipid peroxidation (LPO). A reactive metabolite, DEP-isocyanate, is most likely responsible for these undesired cytotoxic effects. Based on the observed cytotoxicity mechanisms, chemoprotection with several sulfhydryl-containing nucleophiles and antioxidants was investigated. The sulfhydryl nucleophiles; GSH, N-acetyl-L-cysteine (NAC) and glutathione isopropylester (GSH-IP) protected almost completely against fotemustine-induced LDH-leakage and LPO. NAC and GSH protected partly against fotemustine-induced GSH-depletion. The antioxidant, vitamin E protected completely against fotemustine-induced LPO, but only partly against fotemustine-induced LDH-leakage and not against GSH-depletion. Ebselen, a peroxidase-mimetic organoselenium compound, did not show protective effects against the cytotoxicity of fotemustine, possibly because GSH is required for the bioactivation of ebselen. It is concluded that co-administration of sulfhydryl nucleophiles, in particular NAC and GSH-IP, possibly in combination with antioxidants, such as vitamin E, are effective against the toxicity of fotemustine in vitro. It might, therefore, be worthwhile to investigate the cytoprotective potency of these agents against undesired toxicities of fotemustine in vivo as well.     

Mechanism of brain protection by nitroxide radicals in experimental model of closed-head injury

Reactive oxygen-derived species were previously implicated in mediation of post-traumatic brain damage; however, the efficacy of traditional antioxidants in preventing/reversing the damage is sometimes limited. The present work focused on the mechanisms underlying the neuroprotective activity of cell permeable, nontoxic, antioxidants, namely stable nitroxide radicals in an experimental model of rat closed-head injury. Brain damage was induced by the weight-drop method and the clinical status was evaluated according to a neurological severity score at 1 h and 24 h, where the difference between these scores reflects the extent of recovery. The metal chelator deferoxamine as well as three nitroxide derivatives, differing in hydrophilicity and charge, and one hydroxylamine (a reduced nitroxide) facilitated the clinical recovery and decreased the brain edema. The nitroxides, but neither the hydroxylamine nor deferoxamine, protected the integrity of the blood-brain barrier. Superoxide dismutase also improved the clinical recovery but did not affect brain edema or the blood-brain barrier. The results suggest that by switching back and forth between themselves, the nitroxide and hydroxylamine act catalytically as self-replenishing antioxidants, and protect brain tissue by terminating radical-chain reactions, oxidizing deleterious metal ions, and by removal of intracellular superoxide.     

Action of phenolic antioxidants on various active oxygen species

The action of phenolic antioxidants, such as probucol, on various active oxygen species was investigated using luminol chemiluminescence and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The various active oxygen species, including hydroxyl radicals (Fenton reaction), superoxide anions, singlet oxygen and hypochlorite ions were examined with phenolic antioxidants under aqueous and nonaqueous conditions. Probucol showed a quenching effect on both superoxide anions and hypochlorite ions in nonaqueous solution. However, it had no effect on hydroxyl radicals. alpha-Tocopherol, a natural phenolic antioxidant, showed a stronger quenching effect on superoxide anions and hypochlorite ions than probucol, and quenched hydroxyl radicals in nonaqueous solution. Furthermore, Trolox showed a quenching effect on all active oxygen species in both aqueous and nonaqueous solution. The antioxidants were studied under comparable conditions in a series of test systems and the reactivity profiles depicted as 'radar charts' which are helpful for characterizing antioxidant action.     

The nitric oxide donor SIN-1 protects endothelial cells from tumor necrosis factor-alpha-mediated cytotoxicity: possible role for cyclic GMP and heme oxygenase

In cultured endothelial cells, incubation with TNF-alpha (50 ng/ml) for 72 h markedly reduced viability of endothelial cells. A 6-h pre-incubation with the nitric oxide (NO) donor linsidomine (SIN-1, 10-150 microM) protected endothelial cells in a concentration-dependent manner and increased viability by up to 59% of control. The unmetabolized parent compound molsidomine and the NO-free metabolite of SIN-1 3-morpholinoiminoacetonitrile (SIN-1C) were without cytoprotective effect. Cytoprotection by SIN-1 was completely abolished by the NO scavenger 2-phenyl-4,4,5,5, -tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 30 microM). A cytoprotective effect comparable to SIN-1 was observed when preincubating the cells with dibutyryl cyclic GMP (10-100 microM). Moreover, no protection by SIN-1 occurred in the presence of cycloheximide (1 microM) or 1H--1,2,4-oxadiazole-4, 3-a-quinoxalin-1-one (ODQ, 0.1 microM), a selective inhibitor of soluble guanylyl cyclase. Tin protoporphyrin-IX (SnPP, 25 microM), an inhibitor of heme oxygenase, was found to attenuate SIN-1-induced cytoprotection. Our results demonstrate that SIN-1 produces a long-term endothelial protection against cellular injury by TNF-alpha, presumably via a cyclic GMP-dependent pathway leading to up-regulation of protective proteins such as heme oxygenase.     

Sporicidal action of peracetic acid and protective effects of transition metal ions

Although peracetic acid (PAA) is used widely for cold sterilization and disinfection, its mechanisms of sporicidal action are poorly understood. PAA at high concentrations (5-10%) can cause major loss of optical absorbance and microscopically-visible damage to bacterial spores. Spores killed by lower levels of PAA (0.02-0.05%) showed no visible damage and remained refractile. Treatment of spores of Bacillus megaterium ATCC 19213 with PAA at concentrations close to the lethal level sensitized the cells to subsequent heat killing. In addition, PAA was found to act in concert with hypochlorite and iodine to kill spores. Antioxidant sulfhydryl compounds or ascorbate protected spores against PAA killing. Trolox, a water-soluble form of alpha-tocopherol, was somewhat protective, while other antioxidants, including alpha-tocopherol, urate, bilirubin, ampicillin and ethanol were not protective. Chelators, including dipicolinate, were not protective, but transition metal ions, especially the reduced forms (Co2+, Cu+ and Fe2+) were highly protective. The net conclusions are that organic radicals formed from PAA are sporicidal and that they may act as reducing agents for spores that are normally in a highly oxidized state, in addition to their well known actions as oxidizing agents in causing damage to vegetative cells.     

Neuroprotection by nitric oxide against hydroxyl radical-induced nigral neurotoxicity

We investigated the effects of nitric oxide on an in vitro and in vivo generation of hydroxyl radicals, and in vivo neurotoxicity caused by intranigral infusion of ferrous citrate in rats. The formation of hydroxyl radicals in vitro, without exogenous hydrogen peroxide, was dose-dependent. Some nitric oxide donors (e.g. sodium nitroprusside) stimulated, while others (nitroglycerin, diethylamine/nitric oxide, nitric oxide in Ringer's solution) suppressed hydroxyl radical generation in vitro. A significant increase in extra-cellular hydroxyl radicals was detected in a brain microdialysis study. Intranigral infusion of ferrous citrate caused long-lasting lipid peroxidation and dopamine depletion in the ipsilateral nigral region and striatum, respectively. Sub-acute dopamine depletion in the striatum was positively correlated with acute lipid peroxidation in substantia nigra. Intranigral administration of nitric oxide did not affect striatal dopamine. Interestingly, nitric oxide in Ringer's protected nigral neurones against the oxidative injury. The results demonstrate that a regional increase in the levels of iron can result in hydroxyl radical generation and lipid peroxidation leading to neurotoxicity. It also demonstrates that exogenous nitric oxide can act as hydroxyl radical scavenger and protect neurones from oxidative injury.     

Transport of GM1 and GM1 inner ester across an in vitro model of the blood-brain barrier

Gangliosides, especially GM1, attenuate the in vivo damage caused by various neurotoxins. The chemically neutral inner ester of GM1 may be a better cytoprotective agent against some neurotoxins than the parent GM1 compound, because it may cross the blood-brain barrier (BBB) more easily than the anionic GM1. Using an in vitro bovine brain endothelial cell model of the BBB, we show the inner ester more readily transverse the tight junction barrier of this model than does GM1. Further, it is demonstrated that the GM1 inner ester is stable for several hours at pH values between 7.0 and 8.2 at 37 degrees C. Finally, the results illustrate that the BBB model may be useful for testing other gangliosides and their various derivatives for increased ability to cross the BBB.     

Relationships between the anthelmintic activity of eight derivatives of benzimidazole carbamates against Trichinella spiralis and their chemical structures

Efficacy of eight recently developed and used anthelmintics of the benzimidazole carbamates; mebendazole, flubendazole, oxfendazole, albendazole, oxibendazole, 790163 proflubendazole, 780118 "cyanide" benzimidazole and 780120 "selenium" benzimidazole was tested orally against the enteral immature larval and adult stages of Trichinella spiralis in mice. Six of these derivatives of methyl benzimidazole-2-carbamates have an aryl and two have an alkyl substituent at the 5'-position of the parent benzimidazole ring. The nature of these substituents was found to be related to the antitrichinellous activity of the compounds. Compounds with the 5'-substituent linked to the parent benzimidazole ring by either a carbon, sulfur or an oxygen atom are more potent than those bridged by selenium or by the carbon with an attached-CN group. The result clearly indicates that the benzimidazoles are invariably more potent against immature enteral phase than the adult worms. This finding would be of importance in a targeted synthesis of new, effective derivatives of benzimidazole, e.g., in the screening for more important tissue-dwelling nematodes like filarial worms.     

Leiomyomatosis peritonealis disseminata. Two case reports

AIM: To study the action of quercetin (Que) on inhibiting platelet aggregation. METHODS: Active oxygen free radicals produced by xanthine/xanthine oxidase (Xan/XO) reaction was used, platelet aggregation was determined by the turbidimetric method, and the Xan/XO oxyradicals generating reaction by luminol-dependent chemiluminescence (Che) method. RESULTS: Active oxygen free radicals enhanced the platelet aggregation induced by ADP 1.6 mumol.L-1. The rate of maximal aggregation increased from 29%-38% for ADP to 59%-70% for ADP + Xan/XO. The enhancement was abolished by the treatment of platelet-rich plasma (PRP) with Que 650 mumol.L-1 or hydrocortisone (Hyd) 900 mg.L-1. Both Que and Hyd scavenged the active oxyradicals in vitro. The Che was decreased by 75.7% (Que 4 mumol.L-1) and 79.0% (Hyd 900 mg.L-1) as compared with control. CONCLUSION: Active oxygen free radicals participated in the platelet aggregation, and scavenging oxyradicals by Que was one of mechanisms of inhibiting platelet aggregation.     

Peroxynitrite-induced apoptosis in epithelial (T84) and macrophage (RAW 264.7) cell lines: effect of legume-derived polyphenols (phytolens)

Peroxynitrite (ONOO-) has been proposed as a mediator of gut inflammation and as an inducer of cell death by apoptosis. Phytolens (PHY), a water-soluble extract of polyphenolic antioxidants from nonsoy legumes (Biotics Research Corp, patent pending), was evaluated as a cytoprotective agent in human colonic (T84) and murine macrophage (RAW 264.7) cell lines. In the antioxidant testing, PHY showed a significant free radical scavenging ability against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and superoxide (O2.) radicals with an IC50 of 4.44 and 5.87 microg/ml against DPPH and O2., respectively. Apoptosis (DNA fragmentation) was measured by an ELISA technique. Cells were exposed to oxidative stress by treating them with peroxynitrite (100-300 microM) for 4 h in the presence and absence of PHY. Peroxynitrite elicited a dose-dependent increase in DNA fragmentation in both cell lines compared to the control group receiving decomposed ONOO-. PHY (10, 30, or 50 microg/ml) significantly attenuated the degree of apoptosis in T84 cells induced by ONOO- (P < 0.05). PHY (10-100 microg/ml) did not directly affect T84 cell viability or induce apoptosis after 4 h or overnight exposure. RAW 264.7 cells exposed to PHY alone (>30 microg/ml) for 4 h displayed decreased cell viability (P < 0.05) and increased apoptosis (P < 0.05). Phytolens may have beneficial effects on inflammation by attenuating peroxynitrite-induced apoptosis. The sparing of epithelial cells while compromising the viability of macrophages suggests that PHY may be beneficial in autoimmune disorders.     

Toxic action of ethylene oxide on pulmonary cells (L132) cultured under aerobic conditions

Various derivatives of oligoribonucleotides were synthesized by the H-phosphonate method. Different modifications of the ribophosphate backbone were designed in order to protect the derivatives against nucleolytic enzymes present in the biological media. These modifications include coupling of fluorescein moiety to 3'-terminal ribose, 2'-O-methylation of ribose, introduction of phosphorothioate internucleotide bonds throughout the molecule, replacement of the two last 3'-terminal phosphodiester bonds by phosphoroamidates and coupling of the last 3'-terminal nucleotide via the 3'-3'-phosphodiester bond. All modifications were tested for their effect on the stability of the derivatives against phosphodiesterase from snake venom and nucleases of the cell culture media. 2'-O-methylated oligoribonucleotides containing either terminal 3'-3'-linkage or two 3'-terminal phosphoroamidate internucleotide bonds appeared to be the most stable under the most severe conditions used. The results demonstrate a possibility to use protected oligoribonucleotide derivatives for experiments in vivo when the use of deoxy-analogues might be ineffective. The uptake of 2'-O-methylated derivatives and their 5'-cholesterol conjugates (coupled via a disulfide bond) by human carcinoma cells did not differ from that of the corresponding oligodeoxyribonucleotides. 85% of the bound derivatives were found in the membrane-cytosolic fraction, while only 15% were found in the nuclear fraction. The oligonucleotide moiety of 2'-O-methyloligoribonucleotide-cholesterol conjugate was not translocated through the cellular membrane. After cleavage of the linkage between cholesterol and oligonucleotide by dithiothreitol the major portion of the oligonucleotide moiety was released into the media. The derivatives, as well as their 5'-cholesterol conjugates, which entered the cells, were stable and protected from action of dithiothreitol dissolved in culture media. These results demonstrate an endocytosis mechanism of penetration as observed in similar experiments using oligodeoxyribonucleotides.     

Structural aspects of the inhibitory effect of glabridin on LDL oxidation

The inhibitory effects of glabridin, an isoflavan isolated from licorice (Glycyrrhiza glabra) root, and its derivatives on the oxidation of LDL induced by copper ions or mediated by macrophages were studied, in order to evaluate the contribution of the different parts of the isoflavan molecule to its antioxidant activity. The peak potential (E1/2) of the isoflavan derivatives, their radical scavenging capacity toward 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical and their ability to chelate heavy metals were also analyzed and compared to their inhibitory activity on LDL oxidation. In copper ion-induced LDL oxidation, glabridin (1), 4'-O-methylglabridin (2), hispaglabridin A (3), and hispaglabridin B (4), which have two hydroxyl groups at positions 2' and 4' or one hydroxyl at position 2' on ring B, successfully inhibited the formation of conjugated dienes, thiobarbituric acid reactive substances (TBARS) and lipid peroxides, and inhibited the electrophoretic mobility of LDL under oxidation. Compounds 1-3 exhibited similar activities, whereas compound 4 was less active. In macrophage-mediated LDL oxidation, the TBARS formation was also inhibited by these isoflavans (1-4) at a similar order of activity to that obtained in copper ion-induced LDL oxidation. On the other hand, 2'-O-methylglabridin (5), a synthesized compound, whose hydroxyl at 2'-position is protected and the hydroxyl at 4'-position is free, showed only minor inhibitory activity in both LDL oxidation systems. 2',4'-O-Dimethylglabridin (6), whose hydroxyls at 2'- and 4'-positions are both protected, was inactive. Resorcinol (7), which is identical to the phenolic B ring in glabridin, presented low activity in these oxidation systems. The isoflavene glabrene (8), which contains an additional double bond in the heterocyclic C ring, was the most active compound of the flavonoid derivatives tested in both oxidation systems. The peak potential of compounds 1-5 (300 microM), tested at pH 7.4, was similar (425-530 mV), and that for compound 6 and 8 was 1078 and 80 mV, respectively. Within 30 min of incubation, compounds 1, 2, 3, 4, 8 scavenged 31%, 16%, 74%, 51%, 86%, respectively, of DPPH radical, whereas compounds 5 and 6, which almost did not inhibit LDL oxidation, also failed to scavenge DPPH. None of the isoflavan derivatives nor the isoflavene compound were able to chelate iron, or copper ions. These results suggest that the antioxidant effect of glabridin on LDL oxidation appears to reside mainly in the 2' hydroxyl, and that the hydrophobic moiety of the isoflavan is essential to obtain this effect. It was also shown that the position of the hydroxyl group at B ring significantly affected the inhibitory efficiency of the isoflavan derivatives on LDL oxidation, but did not influence their ability to donate an electron to DPPH or their peak potential values.     

NAT2, GSTM-1, cigarette smoking, and risk of colon cancer

There is overwhelming evidence to indicate that free radicals cause oxidative damage to lipids, proteins and nucleic acids and are involved in the pathogenesis of several degenerative diseases. Therefore, antioxidants, which can neutralize free radicals, may be of central importance in the prevention of these disease states. The protection that fruits and vegetables provide against disease has been attributed to the various antioxidants contained in them. Recently, an anti-inflammatory and analgesic activity of a water-soluble fraction from shark cartilage has been described. Using electrophoretical assays, bacteria survival and transformation and the Salmonella/mammalian-microsome assay, we investigated the putative role of shark cartilage-containing preparation in protecting cells against reactive oxygen species induced DNA damage and mutagenesis. If antimutagens are to have any impact on human disease, it is essential that they are specifically directed against the most common mutagens in daily life. Our data suggest that shark cartilage-containing preparation can play a scavenger role for reactive oxygen species and protects cells against inactivation and mutagenesis.     

Lipid hydroperoxide generation, turnover, and effector action in biological systems

Lipid peroxidation is a well known example of oxidative damage in cell membranes, lipoproteins, and other lipid-containing structures. Peroxidative modification of unsaturated phospholipids, glycolipids, and cholesterol can occur in reactions triggered by i) free radical species such as oxyl radicals, peroxyl radicals, and hydroxyl radicals derived from iron-mediated reduction of hydrogen peroxide or ii) non-radical species such as singlet oxygen, ozone, and peroxynitrite generated by the reaction of superoxide with nitric oxide. Lipid hydroperoxides (LOOHs) are prominent non-radical intermediates of lipid peroxidation whose identification can often provide valuable mechanistic information, e.g., whether a primary reaction is mediated by singlet oxygen or oxyradicals. Certain cholesterol-derived hydroperoxides (ChOOHs) have been used very effectively in this regard, both in model systems and cells. Being more polar than parent lipids, LOOHs perturb membrane structure/function and can be deleterious to cells on this basis alone. However, LOOHs can also participate in redox reactions, the nature and magnitude of which often determines whether peroxidative injury is exacerbated or prevented. Exacerbation may reflect iron-catalyzed one-electron reduction of LOOHs, resulting in free radical-mediated chain peroxidation, whereas prevention may reflect selenoperoxidase-catalyzed two-electron reduction of LOOHs to relatively non-toxic alcohols. LOOH partitioning between these two pathways in an oxidatively stressed cell is still poorly understood, but recent cell studies involving various ChOOHs have begun to shed light on this important question. An aspect of related interest that is under intensive investigation is lipid peroxidation/LOOH-mediated stress signaling, which may evoke a variety of cellular responses, ranging from induction of antioxidant enzymes to apoptotic death. Ongoing exploration of these processes will have important bearing on our understanding of disease states associated with peroxidative stress.     

Immune response associated with nonmelanoma skin cancer

The effect of antioxidants and reducing agents on glutamate-induced cytotoxicity was examined using PC12 cells. The antioxidants vitamin E, idebenone, and selegiline protected cells against the cytotoxicity observed 24 h after exposure to 0.5 or 10 mM glutamate, as determined by lactate dehydrogenase leakage, even when added 3 h after glutamate. The reducing agents, glutathione (GSH) and dithiothreitol (DTT), also provided protection against the cytotoxicity of glutamate. Preincubation of PC12 cells with the antioxidants mentioned above, or the incubation with those antioxidants after exposure to glutamate for 3 h, prevented the reduction of viability caused by glutamate. Cystine uptake was inhibited by exposure of cells to glutamate, as determined by L-[35S]-cystine uptake. Incubation of cells with 0.5 or 10 mM glutamate caused a marked decrease in cellular GSH levels, not prevented by antioxidants. The activity of GSSG reductase was decreased by glutamate and this inhibition was reverted in the presence of the reducing agents GSH and DTT. These results indicate that glutamate toxicity on PC12 cells results from the inhibition of cystine uptake with consequent GSH depletion and oxidative stress, suggesting that antioxidants may reduce the cellular damage in pathologic conditions associated with excessive glutamate release.