Protection of peroxide-treated fish erythrocytes by coelenterazine and coelenteramine

European seabass ( Dicentrarchus labrax ) erythrocytes treated with tert -butyl hydroperoxide ( t -BHP) showed decreasing levels of reduced glutathione, increased lipid peroxidation and DNA damage, and ultimately underwent haemolysis. The addition of the marine luciferin coelenterazine (CLZn) markedly delayed the onset of the haemolytic process induced by t -BHP as well as lipid peroxidation and glutathione oxidation. CLZn also protected the red blood cells' DNA against t -BHP-triggered damage. CLZn's oxidation product coelenteramine (CLM) also delayed the lysis of the cells as well as the occurrence of oxidative stress indicators but it did not offer protection against DNA damage. Both compounds proved more efficient than the vitamin E analogue Trolox C ® at similar doses. These results demonstrate the ability of CLZn and CLM to protect fish cells against oxidative stress, providing further support to the evolutionary model suggesting that CLZn's first physiological role was that of an antioxidant in fish thriving in surface layers of the ocean, later evolving into its light-emitting function in deep-sea species.     

Green tea metabolite EGCG protects membranes against oxidative damage in vitro

Green tea polyphenols like epigallocatechin gallate (EGCG) have been proposed as a cancer chemopreventative. Several studies have shown that EGCG can act as an antioxidant by trapping proxyl radicals and inhibiting lipid peroxidation. The main propose of this study is to investigate the antioxidant capacity of EGCG using erythrocyte membrane-bound ATPases as a model. The effects of EGCG on t-butylhydroperoxide-induced lipid peroxidation and the activity of membrane-bound ATPases in human erythrocyte membranes were studied. The extent of oxidative damage in membranes was assessed by measuring lipid peroxidation, (TBARS, thiobarbituric acid reactive substances formation) and the activity of ATPases (Na(+)/K(+), Ca(2+), and CaM-activated Ca(2+) pump ATPases). EGCG blocked t-BHP induced lipid peroxidation in erythrocyte membranes, significantly (0.45 +/- 0.02 vs 0.20 +/- 0.01; t-BHP vs t-BHP + EGCG respectively, microm/L TBARS) (p < 0.05). EGCG also protected ATPases against t-BHP induced damage; for Na/K ATPase (2.4 +/- 0.2 vs 1.6 +/- 0.1 vs 2.44 +/- 0.2, nmol Pi/min/mg protein, control vs t-BHP vs t-BHP and EGCG respectively), for Ca ATPase (5.8 +/- 0.4 vs 3.9 +/- 0.3 vs 5.6 +/- 0.34, nmol Pi/min/mg protein, control vs t-BHP vs t-BHP and EGCG respectively) and for CaM-Ca ATPase (14.7 +/- 0.7 vs 7.3 +/- 0.4 vs 11.6 +/- 0.55, nmol Pi/min/mg protein, control vs t-BHP vs t-BHP and EGCG respectively) (p < 0.05). In conclusion our results indicate that EGCG is a powerful antioxidant that is capable protecting erythrocyte membrane-bound ATPases against oxidative stress.     

Schisandrin B protects against tert-butylhydroperoxide induced cerebral toxicity by enhancing glutathione antioxidant status in mouse brain

Schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from Fructus Schisandrae, has been shown to produce antioxidant effect on rodent liver and heart. A mouse model of tert-butylhydroperoxide (t-BHP) induced cerebral toxicity was adopted for examining the antioxidant potential of Sch B in the brain. Intracerebroventricular injection of t-BHP caused a time-dependent increase in mortality rate in mice. The t-BHP toxicity was associated with an increase in the extent of cerebral lipid peroxidation and an impairment in cerebral glutathione antioxidant status, as evidenced by the abrupt decrease in reduced glutathione (GSH) level and the inhibition of Se-glutathione peroxidase activity at 5 min following t-BHP challenge. Sch B pretreatment (1 or 2 mmol/kg/day × 3) produced a dose-dependent protection against t-BHP induced mortality. The protection was associated with a decrease in the extent of lipid peroxidation and an enhancement in glutathione antioxidant status in brain tissue detectable at 5 min post t-BHP challenge, with the assessed biochemical parameters being returned to normal values at 60 min in Sch B pretreated mice at a dose of 2 mmol/kg. The ensemble of results suggests the antioxidant potential of Sch B pretreatment in protecting against cerebral oxidative stress.     

Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels

In the present work, the potential hepatoprotective effects of five phenolic compounds against oxidative damages induced by tert-butyl hydroperoxide (t-BHP) were evaluated in HepG2 cells in order to relate in vitro antioxidant activity with cytoprotective effects. t-BHP induced considerable cell damage in HepG2 cells as shown by significant LDH leakage, increased lipid peroxidation, DNA damage as well as decreased levels of reduced glutathione (GSH). All tested phenolic compounds significantly decreased cell death induced by t-BHP (when in co-incubation). If the effects of quercetin are given the reference value 1, the compounds rank in the following order according to inhibition of cell death: luteolin (4.0) > quercetin (1.0) > rosmarinic acid (0.34) > luteolin-7-glucoside (0.30) > caffeic acid (0.21). The results underscore the importance of the compound's lipophilicity in addition to its antioxidant potential for its biological activity. All tested phenolic compounds were found to significantly decrease lipid peroxidation and prevent GSH depletion induced by t-BHP, but only luteolin and quercetin significantly decreased DNA damage. Therefore, the lipophilicity of the natural antioxidants tested appeared to be of even greater importance for DNA protection than for cell survival. The protective potential against cell death was probably achieved mainly by preventing intracellular GSH depletion. The phenolic compounds studied here showed protective potential against oxidative damage induced in HepG2 cells. This could be beneficial against liver diseases where it is known that oxidative stress plays a crucial role.     

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe2+ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than alpha-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than alpha-tocopherol; (e) to be a weaker antiradical than alpha-tocopherol in the reduction of the stable radical DPPH*. Resveratrol reduced Fe3+ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe3+, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like alpha-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.     

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe²⁺ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than α-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than α-tocopherol; (e) to be a weaker antiradical than α-tocopherol in the reduction of the stable radical DPPH^·. Resveratrol reduced Fe³⁺ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe³⁺, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like α-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.     

Membrane peroxidation: Inhibiting effects of water- soluble antioxidants on phospholipids of different charge types

Quantitative kinetic methods of autoxidation are used to determine the antioxidant activities of two water-soluble antioxidants of the chromanol type, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 6-hydroxy-2,5,7,8- tetramethyl-2-N,N,N-trimethylethanaminium methylbenzene-sulfonate (MDL 73404), during free radical peroxidation of phospholipid membranes of different charge types. The stoichiometric factor (n) for peroxyl radical trapping for both Trolox and MDL 73404 was found to be 2. Trolox was found to partition partially, approximately 20%, into the lipid phase of liposomes. The antioxidant activity of Trolox during peroxidation of membranes determined by measurements of the absolute rate constant for inhibition of oxygen uptake,k_inh, was found to vary with the membrane surface charge that is controlled by variation in pH. When peroxidation is initiated in the lipid phase by azo-bis-2,4-dimethylvaleronitrile (ADVN), using a typical zwitterionic liposome, dilinoleoylphosphatidyl choline (DLPC), the k_inh was found to be 2.98 × 10³ M⁻¹s⁻¹. The k_inh of Trolox increased approximately 2-fold for membranes that have positive surface, including DLPC at pH 4, DLPC containing stearylamine at pH 7, and for a membrane of dimyristoylphosphatidic acid containing linoleic acid (DMPA/LA). Conversely, Trolox does not inhibit peroxidation of negatively charged dilinoleoylphosphatidyl glycerol (DLPG) at pH 7–11. Studies made of the positively charged MDL 73404 show that its antioxidant activity using DLPC and DLPG is pH dependent. Trolox inhibits the peroxidations of DLPC initiated in the aqueous phase by azo-bis(2-amidinopropane·HCl)(ABAP) at pH 4 or 7. However, Trolox does not inhibit the peroxidation of DLPG at pH 7. The different antioxidant activities of Trolox and MDL 73404 are rationalized in terms of a peroxyl-radical diffusion model and specific charge interactions between antioxidants and membrane surface.     

Oxidative insult to human red blood cells induced by free radical initiator AAPH and its inhibition by a commercial antioxidant mixture.

This study was carried out to investigate sequel of oxidative insult to human erythrocytes induced by a water-soluble radical initiator, 2,2'-azobis-(amidinopropane) dihydrochloride (AAPH) and the effect of a commercially available mixed antioxidant (Blackmores, BioAce Excel), containing alpha-tocopherol, ascorbic acid, beta-carotene and some herbal extracts (containing grape seed catechins and milk thistle derived silybin), on lipid peroxidation, degradation of membrane proteins and haemolysis. We performed this study in order firstly to clarify aspects of the mechanism of AAPH induced free radical damage in human erythrocytes and secondly to establish in vitro conditions by which the efficacy of mixed antioxidant preparations may fairly and objectively be compared. In the process of oxidation initiated by peroxyl radical, a rapid loss of reduced glutathione occurred in the first 60 min. Formation of thiobarbitric acid-reactive substances indicative of lipid peroxidation increased subsequently and almost reached maximal levels at 180 min before significant apparent degradation of membrane proteins was detected. At this point, a significant haemolysis occurred. This sequence of events is consistent with the idea that haemolysis is a consequence of lipid peroxidation and the degradation of membrane proteins. The mixed commercial antioxidant, which suppressed lipid peroxidation and protected membrane proteins against degradation induced by peroxyl radicals, also effectively delayed AAPH induced haemolysis. The system we describe provides a sound objective basis for the in vitro comparison of the potential efficacy of the hundreds of antioxidant nutritional supplements currently available in the market place.     

Radiosensitive antioxidant membrane-bound factors in rat liver microsomes: I. The roles of glutathione and vitamin E

In vitro lipid peroxidation initiated by NADPH/ADP/Fe3+ reveals an alteration of rat liver microsomal antioxidant factors at day D+4 after whole-body gamma irradiation (8Gy). This alteration is partly reversed by GSH, and more efficiently by Trolox C, a water-soluble analog of vitamin E. This reversion by Trolox C, together with the observed 50% decrease in vitamin E content in microsomes of irradiated rats as compared to those of control animals, indicate that Trolox C acts as a free-radical scavenger like and in place of vitamin E. The antioxidant action of Trolox C is not improved in the presence of GSH, which suggests that the former acts earlier than the latter on the autoxidative free-radical chain reactions. Neither GSH, nor Trolox C, nor both antioxidants totally inhibit in vitro lipid peroxidation, which appeals attention on the possible role of extra-microsomal antioxidant factors, especially cytosolic ones.     

Incorporation of ascorbic acid and α-tocopherol to the extender media to enhance antioxidant system of cryopreserved sea bass sperm

Despite the overwhelming application of sperm cryopreservation in aquaculture and broodstock management, its detrimental effects on sperm quality must be taken into account. Imbalance of reactive oxygen species is considered one of the main triggers of cell damage after cryopreservation, because the spermatozoa antioxidant system is decimated during this process, mainly because the natural antioxidants present in seminal plasma diminish when sperm is diluted in extenders. It has been demonstrated that the addition of antioxidants to the extender improves the quality of thawed sperm. Thus, the aim of the present work was to evaluate the status of the antioxidant system in cryopreserved sea bass sperm, and the possibility of enhancing this system to reduce oxidation of the membrane compounds by extender supplementation with vitamins. To do this, sperm from European sea bass (Dicentrarchus labrax) was cryopreserved using an extender control (NAM), supplemented with 0.1 mm α-tocopherol or 0.1 mm ascorbic acid. Sperm motility (computer assisted sperm analysis (CASA) parameters), viability (SYBR Green/propidium iodide (PI)), lipid peroxidation (malondialdehyde (MDA) levels) and protein oxidation (DNPH levels) were analyzed, as well as the status of the sperm antioxidant system by determining glutathione peroxidase, glutathione reductase and superoxide dismutase (GPX, GSR and SOD) activity. The results demonstrated that extenders containing vitamins significantly increased sperm motility. Total motility, velocity and linearity increased from 31.2 ± 3.0 μm/sec, 18.3 ± 1.7 μm/sec and 46.9 ± 2.0% in extender containing 0.1 mm α-tocopherol or 30.6 ± 3.9 μm/sec, 19.5 ± 1.6 μm/sec and 47.9 ± 2.2% in extender containing 1 mm ascorbic acid respect to the extender control (20.7 ± 3.3 μm/sec, 13.8 ± 1.7 μm/sec and 37.3 ± 4.1%). However, viability and levels of lipid peroxidation and protein oxidation were not affected by the presence of these antioxidants, suggesting that membrane impairment could be more associated to osmotic shock or membrane destabilization than oxidative damage. The increased activity of both GPX and GSR after cryopreservation showed that the antioxidant system of sea bass sperm must play an important role in preventing oxidation of the membrane compounds. In conclusion, the addition of α-tocopherol and ascorbic acid to the extender media, together with the antioxidant system of the spermatozoa improved sea bass sperm motility, which is one of the impairment parameters most affected by cryopreservation.     

Hydroxy-urea protects erythrocytes against oxidative damage

Hydroxy-urea (OH-U) is used to treat sickle cell anemia by increasing hemoglobin fetal-fraction. It has been suggested that the sickle cell mutations lead to the formation of unstable HbS and release of iron, which can result in lipid peroxidation (LPO), and eventual cell damage. Since oxidative processes might be involved in pathogenesis of sickle cell disease, we investigated the antioxidant property of OH-U in a red blood cell (RBC) model. Intact RBCs or RBC membranes were exposed to t-butyl hydroperoxide (t-BHP, 0.75 mM) or iron (ferrous sulfate; 100 microM) at 37 degrees C for 60 min in the presence or absence of OH-U (1.25 mM). The extent of oxidative damage was measured by LPO (as thiobarbituric acid reactive substances, TBARS), hemoglobin oxidation (as percent of methemoglobin, metHb %), and decrease in the activities of membrane-bound Na+/K+-ATPase and Ca2+-ATPases. Our results show that OH-U inhibited t-BHP-induced LPO in fresh RBC membranes significantly (P <0.01). OH-U significantly inhibited t-BHP-mediated LPO (P <0.01) and metHb formation (P <0.01) in intact RBC. Also, OH-U inhibited iron-induced LPO and metHb formation in intact RBC (P <0.01). In addition, OH-U blocked t-BHP-mediated changes in membrane ATPase activities. Furthermore, OH-U blocked iron-mediated hydroxyl radical generation in a dose-dependent fashion. In conclusion, the observed antioxidant properties of OH-U might contribute to its therapeutic action in sickle cell disease.     

Celastrus paniculatus Willd. mitigates t-BHP induced oxidative and apoptotic damage in C2C12 murine muscle cells

Identification, exploration and scientific validation of antioxidant rich herbal extracts to mitigate the radical induced cell damage provide new insights in the field of ayurvedic research/therapies. In the present study, we evaluated the anti-oxidant and anti-apoptotic potential of Celastrus paniculatus seed extract (CPSE) against tertiary butyl hydroperoxide (t-BHP) induced mice muscle cell damage. The extract at a dose of 50 µg/ml protected the cells up to 70 % as evidenced by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell survival assay and also prevented LDH leakage against t-BHP induced cytotoxicity. CPSE showed potential antioxidant activity by restoring mitochondrial membrane potential and inhibited reactive oxygen species generation and lipid peroxidation. CPSE pretreatment also regulated the antioxidant markers such as superoxide dismutase and catalase enzymes content and proteins expression. Further CPSE showed anti-apoptotic effects by regulating cytochrome-C and heat shock protein-70 expression and also showed 43 % muscle cell DNA damage inhibitory activity against t-BHP challenge as observed by single cell gel electrophoresis assay. Overall the extract inhibits the muscle cell damage, thus explaining the possible anti-oxidant/anti-apoptotic defense status of the C. paniculatus seed extract.     

Evaluation of DNA damage in Dicentrarchus labrax sperm following cryopreservation

In this paper, DNA laddering analysis and single-cell gel electrophoresis (SCGE) or Comet assay, were used to detect DNA damage in response to a cryopreservation process in sea bass spermatozoa. The results obtained demonstrate that the cryopreservation protocol used to cryopreserve the sea bass sperm cause significantly damage at DNA level. In fact, the degree of DNA damage in frozen-thawed sperm (%DNAT=38.2+/-11.2, MT=498.9+/-166.4, n=3) was different (P<0.01) from that measured in fresh sperm (%DNAT=32.7+/-11.1, MT=375.2+/-190.7, n=3). Data here reported also demonstrated the fundamental role played by cryoprotectants (BSA and Me2SO) in reducing fish sperm DNA fragmentation. Finally, from our results, the ability of SCGE to reveal DNA fragmentation in fish sperm is also confirmed.     

Promoting effects of polyunsaturated fatty acids on chromosomal giant DNA fragmentation associated with cell death induced by glutathione depletion

Glutamate and buthionine sulfoximine (BSO) both reduce intracellular glutathione (GSH) concentration but by different mechanisms, and thereby induce cell death in C6 rat glioma cells. The effects of lipid peroxidation on chromosomal DNA damage during the GSH depletion-induced cell death were assessed. Polyunsaturated fatty acids (PUFA), such as arachidonic acid (AA), gamma-linolenic acid and linoleic acid enhanced lipid peroxidation, induced a loss of membrane integrity and consequently promoted 1-2 Mbp giant DNA fragmentation under both glutamate- and BSO-induced GSH-depletion. Treated C6 cells had 3'-OH termini in their DNA which were recognized by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) analysis. Antioxidants capable of scavenging reactive oxygen species and lipid radicals and iron or copper scavengers inhibited both lipid peroxidation and 1-2 Mbp giant DNA fragmentation, consequently protecting against cell death under GSH depletion. These results suggest that GSH depletion induces lipid peroxidation and leads to 1-2 Mbp giant DNA fragmentation; and that PUFAs can promote giant DNA fragmentation and 3'-OH termini in chromosomal DNA enhancing lipid peroxidation of C6 cells.     

Hyperglycemia can cause membrane lipid peroxidation and osmotic fragility in human red blood cells

The present study has examined the effect of elevated glucose levels on membrane lipid peroxidation and osmotic fragility in human red blood cells (RBC). Defibrinated whole blood or RBC were incubated with varying concentrations of glucose at 37 degrees C for 24 h. RBC incubated with elevated levels of glucose showed a significantly increased membrane lipid peroxidation when compared with control RBC. A significant positive correlation was observed between the extent of glucose-induced membrane lipid peroxidation and the osmotic fragility of treated RBC. Glucose-induced membrane lipid peroxidation and osmotic fragility were blocked when RBC were pretreated with fluoride, an inhibitor of glucose metabolism; with vitamin E, an antioxidant; with para-chloromercurobenzoate and metyrapone, inhibitors of the cytochrome P-450 system; or with dimethylfurane, diphenylamine, and thiourea, scavengers of oxygen radicals. RBC treated with elevated glucose concentrations also showed an increase in NADPH levels. Exogenous addition of NADPH to normal RBC lysate induced membrane lipid peroxidation similar to that observed in the glucose-treated RBC. These data suggest that elevated glucose levels can cause the peroxidation of membrane lipids in human RBC.     

Inhibition of Lipid Peroxidation by N-Acetylserotonin and Its Role in Retinal Physiology

N-Acetylserotonin (NAS) inhibits the peroxidation of linoleic acid induced by a water-soluble initiator, 2,2′-azobis(2-amidinopropane) (ABAP). Lipid peroxidation was detected by the formation of conjugated dienes, monitored spectrophotometrically at 236 nm. N-Acetylserotonin, at concentrations ranging from 200 nM to 20 μM, reduced the rate of ABAP-initiated lipid peroxidation in a concentration-dependent manner. The results of NAS-inhibited lipid peroxidation are compared to the antioxidant activities of melatonin, vitamin E, and a water-soluble vitamin E analog, Trolox. It is suggested that NAS acts as a physiological antioxidant in retinal photoreceptor cells.     

Effect of d-mannose on antioxidant defense and oxidative processes in etiolated wheat coleoptiles

Effect of d-mannose treatment on different antioxidants, phenolics, protease activity, lipid peroxidation, DNA damage and cell death was investigated in coleoptiles of etiolated wheat seedlings. Modulations in these biochemical parameters were monitored up to 96 h after treatment at 24 h intervals. With accelerating effect on initial signs of cell death, i.e., appearance of long DNA fragmentation and no effect on initiation of terminal stage, i.e., internucleosomal nDNA fragmentation, mannose treatment (1 % = 56 mM) diminished the antioxidant activities in wheat coleoptiles. Mannose treatment decreased the catalase activity at all intervals, while APX and POD activities decreased at 72 h. Peroxidation of lipids increased at 72 h after mannose treatment. Levels of most of antioxidants, i.e., SOD, peroxidases and phenolics were raised during initial time period (24–48 h) of mannose treatment probably as an attempt to counter the stress effect. Protease activity gradually increased and protein content decreased with time in both treated and non-treated coleoptiles. Sharp decrease in CAT, APX and peroxidase activities and increase in lipid peroxidation at 72 h overlaps with apoptotic internucleosomal nDNA fragmentation in this organ. This coincidence points towards the importance of compromised antioxidant defense and involvement of reactive oxygen species in initiation of terminal stage of programmed cell death in wheat coleoptile. In conclusion, accelerating effect on DNA fragmentation and lipid peroxidation along with diminished antioxidant activities at the time of internucleosomal nDNA fragmentation, provide evidence for pro-apoptotic effect of d-mannose in wheat coleoptile.     

Antioxidant Activity of Vitamin E and Trolox: Understanding of the Factors that Govern Lipid Peroxidation Studies In Vitro

Peroxidation of lipids is of significant interest owing to the evidence that peroxyl radicals and products of lipid peroxidation may be involved in the toxicity of compounds initiating a deteriorative reaction in the processing and storage of lipid-containing foods. In view of the significance of the antioxidant role of the dietary compound vitamin E and its water-soluble analogue Trolox in research of lipid-containing foods, it is desirable to determine more specifically how and where they operate its antioxidant activity in lipid membranes. In this study, unilamellar liposomes of phosphatidylcholine were used as membrane mimetic systems to estimate the antioxidant properties of vitamin E and Trolox and establish a relationship between their interactions with the membrane and their consequent antioxidant activity. Lipid peroxidation was initiated by the peroxyl radical (ROO^•) in lipid and aqueous media by the thermal decomposition of azocompounds and was assessed by the fluorescence intensity decay of the fluorescent probe diphenylhexatriene propionic acid. Results obtained showed that membrane lipoperoxidation is related not only to the scavenging characteristics of the compounds studied but also to their ability to interact with the lipid bilayers, and consequently liposomes provide additional information to that obtained currently from assays performed in aqueous buffer media.     

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.     

Macelignan protects HepG2 cells against tert-butylhydroperoxide-induced oxidative damage

In this study, we investigated the protective effect of macelignan, isolated from Myristica fragrans Houtt. (nutmeg) against tert-butylhydroperoxide (t-BHP)-induced cytotoxicity in a human hepatoma cell line, HepG2. The tetrazolium dye colorimetric test (MTT test) and lactate dehydrogenase (LDH) assay were used to monitor cell viability and necrosis, respectively. Lipid peroxidation [malondialdehyde (MDA) formation] was estimated by the fluorometric method. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA), and DNA damage was detected using single cell gel electrophoresis (comet assay). The results showed that macelignan significantly reduced the cell growth inhibition and necrosis caused by t-BHP. Furthermore, macelignan ameliorated lipid peroxidation as demonstrated by a reduction in MDA formation in a dose-dependent manner. It was also found that macelignan reduced intracellular ROS formation and DNA damaging effect caused by t-BHP. These results strongly suggest that macelignan has significant protective ability against oxidative damage caused by reactive intermediates.