Inhibition of rat liver microsomal lipid peroxidation by N-acyldehydroalanines: an in vitro comparative study

Captodative substituted olefins are radical scavengers which react with free radicals to form stabilized radical adducts. One of those compounds, N-(paramethoxyphenylacetyl)dehydroalanine (AD-5), may react and scavenge both superoxide anion (O-2) and alk-oxyl radicals (RO.), and in this way prevent the appearance of their mediated biological effects. Nitrofurantoin and tert-butyl hydroperoxide were used as model compounds to stimulate free radical production and their mediated lipid peroxidation in rat liver microsomes. In addition, lipid peroxidation was also initiated by exposure of rat liver microsomal suspensions to ionizing radiation (gamma rays). The microsomal lipid peroxidation induced by these chemicals and physical agents was inhibited by the addition of AD-5. These effects were dose-dependent in a millimolar range of concentration. In addition, AD-5 has no effect on microsomal electron transport, showing that NADPH-cytochrome P450 reductase activity was not modified. These data, together with the comparisons of the effects of AD-5 and some antioxidant molecules such as superoxide dismutase, uric acid, and mannitol, support the conclusion that inhibition of lipid peroxidation by AD-5 is the result of its free radical scavenger activity. In addition, the inhibitory effect of AD-5 on microsomal lipid peroxidation was dependent of the nature of the free radical species involved in the initiation of the process, suggesting that O-2 is scavenged more efficiently than RO.     

Cinnamophilin as a novel antiperoxidative cytoprotectant and free radical scavenger

The antioxidant properties of cinnamophilin were evaluated by studying its ability to react with relevant reactive oxygen species, and its protective effect on cultured cells and biomacromolecules under oxidative stress. Cinnamophilin concentration-dependently suppressed non-enzymatic iron-induced lipid peroxidation in rat brain homogenates with an IC50 value of 8.0+/-0.7 microM and iron ion/ADP/ascorbate-initiated rat liver mitochondrial lipid peroxidation with an IC50 value of 17.7+/-0.2 microM. It also exerted an inhibitory activity on NADPH-dependent microsomal lipid peroxidation with an IC50 value of 3.4+/-0.1 microM without affecting microsomal electron transport of NADPH-cytochrome P-450 reductase. Both 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azo-bis(2-amidinopropane) dihydrochloride-derived peroxyl radical tests demonstrated that cinnamophilin possessed marked free radical scavenging capacity. Cinnamophilin significantly protected cultured rat aortic smooth muscle cells (A7r5) against alloxan/iron ion/H2O2-induced damage resulting in cytoplasmic membranous disturbance and mitochondrial potential decay. By the way, cinnamophilin inhibited copper-catalyzed oxidation of human low-density lipoprotein, as measured by fluorescence intensity and thiobarbituric acid-reactive substance formation in a concentration-dependent manner. On the other hand, it was reactive toward superoxide anions generated by the xanthine/xanthine oxidase system and the aortic segment from aged spontaneously hypertensive rat. Furthermore, cinnamophilin exerted a divergent effect on the respiratory burst of human neutrophil by different stimulators. Our results show that cinnamophilin acts as a novel antioxidant and cytoprotectant against oxidative damage.     

Spin trapping and its application in the study of lipid peroxidation and free radical production with liver microsomes.

Lipid peroxidation in microsomes was studied using a spin-trapping technique. Free radical adducts of phenyltertiarybutylnitrone (PBN) were produced as detected by electron spin resonance during induced lipid peroxidation of microsomes with a system consisting of NADPH, Fe²⁺, and pyrophosphate. The adducts were identified as intermediates of the substrates added to the microsomal system and not OH · or HO₂ radicals. The production of the adduct parallels the NADPH-dependent formation of malondialdehyde (MDA). Analyses of the electron spin resonance hyperfine splitting constants allowed in some instances identification of the adducts. Purified preparations of cytochrome P-450 mimic the results of the microsomes. The carcinogens dimethyl and diethylnitrosoamine were metabolized in this system yelding reactive free radicals and free NO, suggesting an alternate mechanism for the activity of these compounds as ultimate carcinogens.     

Inhibition of liver microsomal lipid peroxidation by 13-cis-retinoic acid

The effects of 13-cis-retinoic acid on iron/ascorbate-dependent lipid peroxidation were investigated with rat liver microsomes. 13-cis-retinoic acid effectively inhibited malondialdehyde generation and molecular oxygen consumption associated with lipid peroxidation. Under the conditions employed, inhibition was complete at concentrations as low as 25 microM and the IC50 was 10 microM. Evidence for concomitant retinoid oxidation by microsomal unsaturated fatty acid-derived peroxyl radicals was demonstrated by detection of several retinoid-derived metabolites, including 5,8-oxy-13-cis-retinoic acid, generated during lipid peroxidation. The data indicate that 13-cis-retinoic acid inhibits lipid peroxidation by scavenging lipid peroxyl radicals with its conjugated polyene system. Its antioxidant properties may contribute to the pharmacological activities of this and related retinoids.     

Nitric oxide inhibition of free radical-mediated cholesterol peroxidation in liposomal membranes

The ability of nitric oxide ((*)NO) to inhibit propagative lipid peroxidation was investigated using unilamellar liposomes (LUVs) constituted with egg phosphatidylcholine (PC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), [(14)C]cholesterol (Ch), and a nonregenerable singlet oxygen-derived primer, 5alpha-hydroperoxycholesterol (5alpha-OOH). Exposing LUVs to ascorbate and a lipophilic iron chelate at 37 degrees C resulted in an exponential decay of 5alpha-OOH and accumulation of free radical-derived 7alpha- and 7beta-hydroperoxycholesterol (7alphabeta-OOH), as detected by high-performance liquid chromatography with electrochemical detection. Thiobarbituric acid-reactive species (TBARS) were generated concurrently in egg PC-containing LUVs. Including the (*)NO donor spermine NONOate (SPNO, 5-50 microM) or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 50-100 microM) in the reaction mixture had no effect on 5alpha-OOH decay (suggesting that iron was not redox-inhibited) but slowed TBARS and 7alphabeta-OOH accumulation in a strongly dose-dependent fashion. Decomposed SPNO or SNAP had no such effects, implying that (*)NO was the responsible agent. Accumulation of several [(14)C]Ch oxidation products, detected by high-performance thin-layer chromatography with phosphorimaging, was similarly diminished by active SPNO or SNAP. Concomitantly, a new band referred to as RCh.4 appeared, the radioactivity of which increased as a function of incubation time and (*)NO donor concentration. RCh.4 material was also generated via direct iron/ascorbate reduction of 7alpha-OOH in the presence of (*)NO, consistent with 7alpha-nitrite (7alpha-ONO) identity. However, various other lines of evidence suggest that RCh.4 is not 7alpha-ONO, but rather 5alpha-hydroxycholesterol (5alpha-OH) generated by reduction of 5alpha-ONO arising from 7alpha-ONO rearrangement. 5alpha-OH was only detected when (*)NO was present in the reaction system, thus providing indirect evidence for the existence of nitrosated Ch intermediates arising from (*)NO chain-breaking activity.     

The inhibition of radical-initiated peroxidation of microsomal lipids by both alpha-tocopherol and beta-carotene.

Rat liver microsomal lipids in hexane solution were exposed to the lipid-soluble radical initiator, azobis-isobutyronitrile (AIBN), and the antioxidant activities of alpha-tocopherol and beta-carotene have been compared. Lipid peroxidation was monitored both by conjugated diene formation at 233 nm, and by malondialdehyde (MDA) formation in the thiobarbituric acid assay at 535 nm. Diene formation was continuous for at least 120 min in the presence of 85 micrograms/ml lipid and 4 mM AIBN. Both alpha-tocopherol and beta-carotene acted as chain-breaking antioxidants, suppressing lipid peroxidation and producing an induction period at concentrations as low as 0.5 and 8 microM, respectively. When both of these lipid-soluble antioxidants were present together, the oxidation was strongly suppressed and the induction period was the sum of the individual antioxidants, alpha-Tocopherol and beta-carotene also inhibited MDA generation. In the presence of 170 micrograms/ml lipid and 8 mM AIBN, beta-carotene exhibited an IC50 of 1.1 microM and inhibited completely at 15 microM. Using beta-carotene, an induction period was observed, although much less pronounced than with alpha-tocopherol. Furthermore, beta-carotene inhibited MDA production in a concentration-dependent manner and exhibited an IC50 of 50 microM. In addition, added beta-carotene delayed the radical-initiated destruction of the endogenous alpha-tocopherol and gamma-tocopherol in this system.     

Free radical scavenging activity of a novel antioxidative peptide purified from hydrolysate of bullfrog skin, Rana catesbeiana Shaw

In the present study, a peptide having antioxidant properties was isolated from bullfrog skin protein, Rana catesbeiana Shaw. Bullfrog skin protein was hydrolyzed using alcalase, neutrase, pepsin, papain, alpha-chymotrypsin and trypsin. Antioxidant activities of respective hydrolysates were evaluated using lipid peroxidation inhibition assay and direct free radical scavenging activity by using electron spin resonance (ESR) spectrometer. Among hydrolysates, alcalase derived hydrolysate exhibited the highest antioxidant activities than those of other enzyme hydrolysates. In order to purity a peptide having potent antioxidant properties, alcalase hydrolysate was separated using consecutive chromatographic methods on a Hiprep 16/10 DEAE FF anion exchange column, Superdex Peptide 10/300 GL gel filtration column and highan octadecylsilane (ODS) C18 reversed phase column. Finally, a potent antioxidative peptide was isolated and its sequence was identified to be LEELEEELEGCE (1487 Da) by Q-TOF ESI mass spectroscopy. This antioxidant peptide from bullfrog skin protein (APBSP) inhibited lipid peroxidation higher than that of alpha-tocopherol as positive control and efficiently quenched different sources of free radicals: DPPH radical (IC(50)=16.1 microM), hydroxyl radical (IC(50)=12.8 microM), superoxide radical (IC(50)=34.0 microM) and peroxyl radical (IC(50)=32.6 microM). Moreover, MTT assay showed that this peptide does not exert any cytotoxicity on human embryonic lung fibroblasts cell line (MRC-5).     

Nitric oxide induces oxidative stress and apoptosis in neuronal cells

Within the central nervous system and under normal conditions, nitric oxide (NO) is an important physiological signaling molecule. When produced in large excess, NO also displays neurotoxicity. In our previous report, we have demonstrated that the exposure of neuronal cells to NO donors induced apoptotic cell death, while pretreatment with free radical scavengers L-ascorbic acid 2-[3, 4-dihydro-2,5,7,8-tetramethyl-2-(4,8, 12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1) or superoxide dismutase attenuated apoptosis effectively, suggesting that reactive oxygen species (ROS) may be involved in the cascade of events leading to apoptosis. In the present investigation, we directly studied the kinetic generation of ROS in NO-treated neuronal cells by flow cytometry using 2', 7'-dichloro-fluorescein diacetate and dihydrorhodamine 123 as redox-sensitive fluorescence probes. The results indicated that exposure of cerebellar granule cells to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced oxidative stress, which was characterized by the accumulation of cytosolic and mitochondrial ROS, the increase in the extracellular hydrogen peroxide level, and the formation of lipid peroxidation products. SNAP treatment also induced apoptotic cell death as confirmed by the formation of cytosolic mono- and oligonucleosomes. Pretreating cells with the novel antioxidant EPC-K1 effectively prevented oxidative stress induced by SNAP, and attenuated cells from apoptosis.     

Purification and characterization of antioxidant peptide from hoki (Johnius belengerii) frame protein by gastrointestinal digestion

To extract antioxidant peptide from hoki frame protein hydrolysate (APHPH), we employed six proteases (pepsin, trypsin, papain, alpha-chymotrypsin, Alcalase and Neutrase) for enzymatic hydrolysis, and the antioxidant activities of their hydrolysates were investigated using both lipid peroxidation inhibition assay and free radical scavenging assay by electron spin resonance spin-trapping technique. Among hydrolysates, peptic hydrolysate, having the highest antioxidant activity, further separated into four groups using ultrafiltration membranes and purified consecutive chromatographic methods. Finally, the purified peptide had a molecular mass of 1801 Da, and amino acid sequence was identified as Glu-Ser-Thr-Val-Pro-Glu-Arg-Thr-His-Pro-Ala-Cys-Pro-Asp-Phe-Asn. APHPH inhibited lipid peroxidation higher than that of alpha-tocopherol as positive control and efficiently quenched different sources of free radical: 1,1-diphenyl-2-pycryl-hydrazyl (IC(50)=41.37 microM), hydroxyl (IC(50)=17.77 microM), peroxyl (IC(50)=18.99 microM) and superoxide radicals (IC(50)=172.10 microM). Furthermore, APHPH decreased t-butylhydroperoxide-induced cytotoxicity on human embryonic lung fibroblasts and efficiently protected free-radical-induced DNA damage.     

The effect of chronic alcohol feeding on lipid peroxidation in microsomes: lack of relationship to hydroxyl radical generation

Chronic alcohol feeding causes microsomal induction including increased generation of hydroxyl radicals. Ethanol induced liver injury may be mediated by lipid peroxidation for which hydroxyl radicals have been proposed as major mediators. Ethanol promotes lipid peroxidation when given acutely but also may serve as a hydroxyl radical scavenger. Therefore, we studied the acute and chronic effects of alcohol on microsomal lipid peroxidation and hydroxyl radical generation. Chronic alcohol feeding in rats increased microsomal generation of hydroxyl radicals but lipid peroxidation of endogenous lipid was inversely related to hydroxyl radical generation. Ethanol (50mM) had a slight inhibitory effect on hydroxyl radical production in peroxidizing microsomes, no effect on endogenous lipid peroxidation and enhanced the lysis of RBCs added as targets of peroxidation. Enhanced microsomal generation of hydroxyl radicals following chronic alcohol feeding is not an important mediator of lipid peroxidation.     

Selective in vitro antioxidant properties of bisphosphonates

The aim of this study was to investigate the in vitro antioxidant profile of different bisphosphonates. Bisphosphonates were tested for their xanthine oxidase and microsomal lipid peroxidation inhibiting capacity. Furthermore, the effect of these different compounds on DPPH, a stable radical, was investigated. Clodronate, risedronate, and pyrophosphate were further tested for their hydroxyl radical scavenging activity. None of the tested compounds showed xanthine oxidase inhibiting activity or DPPH scavenging activity. All the tested bisphosphonates exhibited inhibiting capacities on the microsomal lipid peroxidation. The hydroxyl radical scavenging activity was dependent on the order of adding the different reagents and was highest for risedronate. Bisphosphonates possess an inhibiting activity on the microsomal lipid peroxidation and the Fenton reaction. In these reactions iron plays an important role suggesting that the selective in vitro antioxidant properties of the bisphosphonates are due to their iron chelating characteristics.     

Antioxidant enzyme inhibitors enhance nitric oxide-induced cell death in U937 cells

Nitric oxide (NO), a radical species produced by many types of cells, is known to play a critical role in many regulatory processes, yet it may also participate in collateral reactions at higher concentrations, leading to cellular oxidative damage. The protective role of antioxidant enzymes against NO-induced oxidative damage in U937 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole, and oxlalomalate, specific inhibitors of superoxide dismutase, catalase, and NADP(+)-dependent isocitrate dehydrogenase, respectively. Upon exposure to 1 mM S-nitroso-N-acetylpenicillamine (SNAP), the nitric oxide donor, to U937 cells, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage reflected by an increase in 8-hydroxy-2'-deoxyguanosine, were higher in inhibitor-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species, as measured by the oxidation of 2'7'-dichlorodihydrofluorescin as well as the significant decrease in the intracellular GSH level in inhibitor-treated U937 cells upon exposure to NO. Upon exposure to 0.2 mM SNAP, which induced apoptotic cell death, a clear inverse relationship was observed between the control and inhibitor-treated U937 cells in their susceptibility to apoptosis. These results suggest that antioxidant enzymes play an important role in cellular defense against NO-induced cell death including necrosis and apoptosis.     

Attenuation of daunorubicin-augmented microsomal lipid peroxidation and oxygen consumption by calcium channel antagonists.

Daunorubicin (20 microM) stimulated NADPH-dependent microsomal lipid peroxidation about 2-fold over control values and enhanced the rate of oxygen utilization by microsomes. The calcium channel blockers tested inhibited daunorubicin-augmented lipid peroxidation and O2 consumption to varying degrees. Inhibition of daunorubicin-stimulated lipid peroxidation was found to be dose dependent; the IC50 (drug concentration producing 50% inhibition of lipid peroxidation) values for verapamil, nifedipine and diltiazem were approximately 150 microM, 200 microM, and 600 microM respectively. Our in vitro studies suggest that calcium channel antagonists may modulate the free radical-mediated, cardiotoxic effects of daunorubicin.     

Nitric oxide donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, stimulate myoblast proliferation in vitro

Summary Nitric oxide (NO) is an inter- and intracellular messenger involved in a variety of physiologic and pathophysiologic conditions. The effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) and their effect on myoblast proliferation was examined. Both donors stimulated an increase in myoblast cell number over a range (1–10 μM) of donor concentrations. However, 50 μM SNAP inhibited myoblast proliferation. Cell numbers from cultures treated with degraded 10 μM SNAP were equivalent to the control. Therefore, it appears NO can stimulate as well as inhibit myoblast proliferation.     

Evaluation of antioxidant properties of root bark of Hemidesmus indicus R. Br. (Anantmul).

Hemidesmus indicus R. Br. (Asclepiadaceae) is a well known drug in Ayurveda system of medicine. In the present study, antioxidant activity of methanolic extract of H. indicus root bark was evaluated in several in vitro and ex vivo models. Further, preliminary phytochemical analysis and TLC fingerprint profile of the extract was established to characterize the extract which showed antioxidant properties. The in vitro and ex vivo antioxidant potential of root bark of H. indicus was evaluated in different systems viz. radical scavenging activity by DPPH reduction, superoxide radical scavenging activity in riboflavin/light/NBT system, nitric oxide (NO) radical scavenging activity in sodium nitroprusside/Greiss reagent system and inhibition of lipid peroxidation induced by iron-ADP-ascorbate in liver homogenate and phenylhydrazine induced haemolysis in erythrocyte membrane stabilization study. The extract was found to have different levels of antioxidant properties in the models tested. In scavenging DPPH and superoxide radicals, its activity was intense (EC50 = 18.87 and 19.9 microg/ml respectively) while in scavenging NO radical, it was moderate. It also inhibited lipid peroxidation of liver homogenate (EC50 = 43.8 microg/ml) and the haemolysis induced by phenylhydrazine (EC50 = 9.74 microg/ml) confirming the membrane stabilization activity. The free radical scavenging property may be one of the mechanisms by which this drug is effective in several free radical mediated disease conditions.     

Antioxidant mechanism of Mn(II) in phospholipid peroxidation.

Antioxidant action of Mn2+ on radical-mediated lipid peroxidation without added iron in microsomal lipid liposomes and on iron-supported lipid peroxidation in phospholipid liposomes or in microsomes was investigated. High concentrations of Mn2+ above 50 microM inhibited 2,2'-azobis (2-amidinopropane) (ABAP)-supported lipid peroxidation without added iron at the early stage, while upon prolonged incubation, malondialdehyde production was rather enhanced as compared with the control in the absence of Mn2+. However, in a lipid-soluble radical initiator, 2,2'-azobis (2,4-dimethyl-valeronitrile) (AMVN)-supported lipid peroxidation of methyl linoleate in methanol Mn2+ apparently did not scavenge lipid radicals and lipid peroxyl radicals, contrary to a previous report. At concentrations lower than 5 microM, Mn2+ competitively inhibited Fe(2+)-pyrophosphate-supported lipid peroxidation in liposomes consisting of phosphatidylcholine with arachidonic acid at the beta-position and phosphatidylserine dipalmitoyl, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in the presence of iron complex in microsomes. Iron reduction responsible for lipid peroxidation in microsomes was not influenced by Mn2+.     

C-phycocyanin: a potent peroxyl radical scavenger in vivo and in vitro

C-Phycocyanin (from Spirulina platensis) effectively inhibited CCl(4)-induced lipid peroxidation in rat liver in vivo. Both native and reduced phycocyanin significantly inhibited peroxyl radical-induced lipid peroxidation in rat liver microsomes and the inhibition was concentration dependent with an IC(50) of 11.35 and 12.7 microM, respectively. The radical scavenging property of phycocyanin was established by studying its reactivity with peroxyl and hydroxyl radicals and also by competition kinetics of crocin bleaching. These studies have demonstrated that phycocyanin is a potent peroxyl radical scavenger with an IC(50) of 5.0 microM and the rate constant ratios obtained for phycocyanin and uric acid (a known peroxyl radical scavenger) were 1.54 and 3.5, respectively. These studies clearly suggest that the covalently linked chromophore, phycocyanobilin, is involved in the antioxidant and radical scavenging activity of phycocyanin.     

Nitric oxide alleviates oxidative damage induced by high temperature stress in wheat

Effect of sodium nitroprusside (SNP), a donor of nitric oxide (NO) was examined in two wheat (Triticum aestivum L.) cultivars, C 306 (heat tolerant) and PBW 550 (comparatively heat susceptible) to study the extent of oxidative injury and activities of antioxidant enzyme in relation to high temperature (HT) stress. HT stress resulted in a marked decrease in membrane thermostability (MTS) and 2, 3, 5-triphenyl tetrazolium chloride (TTC) cell viability whereas content of lipid peroxide increased in both the cultivars. The tolerant cultivar C 306 registered less damage to cellular membranes compared to PBW 550 under HT stress. Activities of antioxidant enzymes viz, superoxide dismutase, catalase, ascorbate peroxidase, guaicol peroxidase and glutathione reductase increased with HT in both the cultivars. Following treatment with SNP, activities of all antioxidant enzymes further increased in correspondence with an increase in MTS and TTC. Apparently, lipid peroxide content was reduced by SNP more in shoots of heat tolerant cultivar C 306 indicating better protection over roots under HT stress. The up-regulation of the antioxidant system by NO possibly contributed to better tolerance against HT induced oxidative damage in wheat.     

Specificity of antioxidant enzyme inhibition in skeletal muscle to reactive nitrogen species donors

Nitric oxide (*NO) and its by-products modulate many physiological functions of skeletal muscle including blood flow, metabolism, glucose uptake, and contractile function. However, growing evidence suggests that an overproduction of nitric oxide contributes to muscle wasting in a number of pathologies including chronic heart failure, sepsis, COPD, muscular dystrophy, and extreme disuse. Limited data point to the potential of inhibition various enzymes by reactive nitrogen species (RNS), including (.)NO and its downstream products such as peroxynitrite, primarily in purified systems. We hypothesized that exposure of skeletal muscle to RNS donors would reduce or downregulate activities of the crucial antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). Diaphragm muscle fiber bundles were extracted from 4-month-old Fischer-344 rats and, in a series of experiments, exposed to either (a) 0 (control), 1, or 5 mM diethylamine NONOate (DEANO: *NO donor); (b) 0, 100, 500 microM, or 1 mM sodium nitroprusside (SNP: *NO donor); (c) 0 or 2 mM S-nitroso-acetylpenicillamine (SNAP: *NO donor); or (d) 0 or 500 microM SIN-1 (peroxynitrite donor) for 60 min. DEANO resulted in a 50% reduction in CAT, GPX, and a dose-dependent inhibition of Cu, Zn-SOD. SNP resulted in significantly lower activities for total SOD, Mn-SOD isoform, Cu, Zn-SOD isoform, CAT, and GPX in a dose-dependent fashion. Two millimolar SNAP and 500 microM SIN-1 also resulted in a large and significant inhibition of total SOD and CAT. These data indicate that reactive nitrogen species impair antioxidant enzyme function in an RNS donor-specific and dose-dependent manner and are consistent with the hypothesis that excess RNS production contributes to skeletal muscle oxidative stress and muscle dysfunction.