Protective effects of endomorphins, endogenous opioid peptides in the brain, on human low density lipoprotein oxidation

Neurodegenerative disorders are associated with oxidative stress. Low density lipoprotein (LDL) exists in the brain and is especially sensitive to oxidative damage. Oxidative modification of LDL has been implicated in the pathogenesis of neurodegenerative diseases. Therefore, protecting LDL from oxidation may be essential in the brain. The antioxidative effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid peptides in the brain, on LDL oxidation has been investigated in vitro. The peroxidation was initiated by either copper ions or a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). Oxidation of the LDL lipid moiety was monitored by measuring conjugated dienes, thiobarbituric acid reactive substances, and the relative electrophoretic mobility. Low density lipoprotein oxidative modifications were assessed by evaluating apoB carbonylation and fragmentation. Endomorphins markedly and in a concentration-dependent manner inhibited Cu2+ and AAPH induced the oxidation of LDL, due to the free radical scavenging effects of endomorphins. In all assay systems, EM1 was more potent than EM2 and l-glutathione, a major intracellular water-soluble antioxidant. We propose that endomorphins provide protection against free radical-induced neurodegenerative disorders.     

Antioxidative and free radical scavenging effects of ecdysteroids from Serratula strangulata

The antioxidative and free radical scavenging effects of four ecdysteroids, 20-hydroxyecdysone (E1), 25-deoxy-11,20-dihydroxyecdysone (E2), 24-(2-hydroxyethyl)-20-hydroxyecdysone (E3), and 20-hydroxyecdysone-20,22-monoacetonide (E4), isolated from the Chinese herb Serratula strangulata have been investigated in vitro. These ecdysteroids could protect human erythrocytes against oxidative hemolysis induced by a water-soluble azo initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). They could also inhibit the peroxidation of rat liver microsomes induced by hydroxyl radicals, as monitored by the formation of thiobarbituric acid reactive substances (TBARS), and prevent radical-induced decrease of membrane fluidity as determined by fluorescence polarization. They reacted with galvinoxyl radicals in homogeneous solution, and the pseudo-first-order rate constants were determined spectrophotometrically by following the disappearance of galvinoxyl radicals. Compounds E1 and (or) E3 were the most active in both antioxidative and radical-scavenging reactions.     

Endomorphins and morphine limit anoxia-reoxygenation-induced brain mitochondrial dysfunction in the mouse

The protection of brain mitochondria from oxidative stress is an important therapeutic strategy against ischemia-reperfusion injury and neurodegenerative disorders. Isolated brain mitochondria subjected to a 5 min period of anoxia followed by 5 min reoxygenation mirrored the effect of oxidative stress in the brain. The present study attempts to evaluate the protective effects of endomorphin 1 (EM1), endomorphin 2 (EM2), and morphine (Mor) in an in vitro mouse brain mitochondria anoxia-reoxygenation model. Endomorphins (EM1/2) and Mor were added to mitochondria prior to anoxia or reoxygenation. EM1/2 and Mor markedly improved mitochondrial respiratory activity with a decrease in state 4 and increases in state 3, respiratory control ratio (RCR) and the oxidative phosphorylation efficiency (ADP/O ratio), suggesting that they may play a protective role in mitochondria. These drugs inhibited alterations in mitochondrial membrane fluidity, lipoperoxidation, and cardiolipin (CL) release, which indicates protection of the mitochondrial membranes from oxidative damage. The protective effects of these drugs were concentration-dependent. Furthermore, these drugs blocked the enhanced release of cytochrome c (Cyt c), and consequently inhibited the cell apoptosis induced by the release of Cyt c. Our results suggest that EM1/2 and Mor effectively protect brain mitochondria against oxidative stresses induced by in vitro anoxia-reoxygenation and may play an important role in the prevention of deleterious effects during brain ischemia-reperfusion and neurodegenerative diseases.     

The effects of nitroxide radicals on oxidative DNA damage

The indolinonic and quinolinic aromatic nitroxides synthesized by us are a novel class of biological antioxidants, which afford a good degree of protection against free radical-induced oxidation in different lipid and protein systems. To further our understanding of their antioxidant behavior, we thought it essential to have more information on their effects on DNA exposed to free radicals. Here, we report on the results obtained after exposure of plasmid DNA and calf thymus DNA to peroxyl radicals generated by the water-soluble radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the protective effects of the aromatic nitroxides and their hydroxylamines, using a simple in vitro assay for DNA damage. In addition, we also tested for the potential of these nitroxides to inhibit hydroxyl radical-mediated DNA damage inflicted by Fenton-type reactions using copper and iron ions. The commercial aliphatic nitroxides 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), and bis(2,2, 6,6-tetramethyl-1-oxyl-piperidin-4-yl)sebacate (TINUVIN 770) were included for comparison. The results show that the majority of compounds tested protect: (i) both plasmid DNA and calf thymus DNA against AAPH-mediated oxidative damage in a concentration-dependent fashion (1-0.1 mM), (ii) both Fe(II) and Cu(I) induced DNA oxidative damage. However, all compounds failed to protect DNA against damage inflicted by the presence of the transition metals in combination with H(2)O(2). The differences in protection between the compounds are discussed in relation to their molecular structure and chemical reactivity.     

Peroxyl radical mediated oxidative DNA base damage: implications for lipid peroxidation induced mutagenesis

Endogenous DNA damage induced by lipid peroxidation is believed to play a critical role in carcinogenesis. Lipid peroxidation generates free radical intermediates (primarily peroxyl radicals, ROO(*)) and electrophilic aldehydes as the principal genotoxicants. Although detailed information is available on the role of aldehyde base adducts in mutagenesis and carcinogenesis, the contribution of peroxyl radical mediated DNA base damage is less well understood. In the present study we have mapped oxidative base damage induced by peroxyl radicals in the supF tRNA gene and correlated this information with peroxidation-induced mutations in several human fibroblast cell lines. Nearly identical patterns of oxidative base damage were obtained from reaction of DNA with either peroxidizing arachidonic acid (20:4omega6) or peroxyl radicals generated by thermolysis of ABIP in the presence of oxygen. Oxidative base damage primarily occurred at G and C. Transversions at GC base pairs in the supF gene were the major base substitution detected in all cell lines. Peroxyl radical induced tandem mutations were also observed. Many mutation hot spots coincided with sites of mapped oxidative lesions, although in some cases hot spots occurred adjacent to the damaged base. Evidence is presented for the involvement of 8-oxodG in the oxidation of DNA by ROO(*). These results are used to interpret some key features of previously published mutation spectra induced by lipid peroxidation in human cells.     

Immunofluorescent detection of 8-oxoguanine DNA lesions in liver cells from aging OXYS rats, a strain prone to overproduction of free radicals

Production of free radicals in animals is accompanied with a number of pathologic conditions, some of which may be manifested through DNA damage. Studies of mechanisms of oxidative DNA damage by free radicals in vivo are hindered by the lack of good animal models with significant overgeneration of or increased sensitivity to free radicals. An inbred rat strain (OXYS) is characterized by inherited overgeneration of free radicals, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases. We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative age-dependent amounts and distribution of 8-oxoG in liver cells from OXYS and Wistar rats. 8-OxoG increased with age in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. Statistical analysis indicates that 8-oxoG does not uniformly accumulate in all cells with advancing age or increasing free radical load, but rather concentrates in a minor fraction of cells with a high damage level.     

Reactivity toward oxygen radicals and antioxidant action of thiol compounds

Thiol compounds exert diverse functions in the defense network against oxidative stress in vivo. Above all, the role of glutathione in the enzymatic removal of hydrogen peroxide and lipid hydroperoxides has been well established. The scavenging of reactive free radicals is one of the many functions. In this study, the reactivities of several thiol compounds toward oxygen- and nitrogen-centered radicals were measured from their reaction with galvinoxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and also from their sparing effects on the decay of fluorescein, pyrogallol red, and BODIPY induced by peroxyl radicals. Furthermore, the antioxidant capacity against lipid peroxidation was assessed in the oxidation of methyl linoleate induced by free radicals in micelle systems. Cysteine, homocysteine, and glutathione exhibited considerable reactivity toward galvinoxyl, DPPH, and peroxyl radicals in this order but methionine did not. Bovine serum albumin (BSA) was less reactive toward these radicals than cysteine on molar base. Cysteine, homocysteine, and glutathione suppressed the oxidation of methyl linoleate in micelle systems, but methionine did not. The reactivity toward free radicals and antioxidant capacity of these thiol compounds were less than that of ascorbic acid, but higher than that of uric acid.     

Manda,a Fermented Natural Food,Suppresses Lipid Peroxidation in the Senescent Rat Brain

The level of lipid peroxidation reflects the degree of free radical-induced oxidative damage in brain tissue of the elderly. We examined the effects of Manda, a product prepared by yeast fermentation of several fruits and black sugar, on lipid peroxidation in the senescent rat brain as model of aging. Senescent rats were provided with a diet containing 50 g/100 g Manda for 8 days, supplemented on day 8 with an intragastric administration of Manda (6.0 g/kg body wt.) twice daily. The hydroxyl radical scavenging activity was generated by the FeSO₄-H₂O₂ system and analyzed by electron spin resonance spectrometry. Using this method, the addition of Manda (2.88 mg/ml) to brain homogenates of adult rats (0.06 mg/ml) had an additive inhibitory effect on lipid peroxidation compared with control adult rats not treated with Manda. Incubation of brain homogenates with Manda for 2 h and 3 h, significantly inhibited the increase in lipid peroxides (malondialdehydes and 4-hydroxyalkenals) levels in aged rats due to auto-oxidation. In addition, oral administration of Manda significantly suppressed the age-related increase in lipid peroxidation in the hippocampus and striatum, although such change was not observed in the cerebral cortex. Although Manda contains trace level of -tocopherol, the level of -tocopherol in Manda did no correlate with its antioxidant effect. Our results suggest that Manda protects against age-dependent oxidative neuronal damage caused by oxidative stress and that this protective effect may be due, in part, to its scavenging activity against free radicals.     

Oxidative stress following traumatic brain injury in rats: quantitation of biomarkers and detection of free radical intermediates

Oxidative stress may contribute to many pathophysiologic changes that occur after traumatic brain injury. In the current study, contemporary methods of detecting oxidative stress were used in a rodent model of traumatic brain injury. The level of the stable product derived from peroxidation of arachidonyl residues in phospholipids, 8-epi-prostaglandin F(2alpha), was increased at 6 and 24 h after traumatic brain injury. Furthermore, relative amounts of fluorescent end products of lipid peroxidation in brain extracts were increased at 6 and 24 h after trauma compared with sham-operated controls. The total antioxidant reserves of brain homogenates and water-soluble antioxidant reserves as well as tissue concentrations of ascorbate, GSH, and protein sulfhydryls were reduced after traumatic brain injury. A selective inhibitor of cyclooxygenase-2, SC 58125, prevented depletion of ascorbate and thiols, the two major water-soluble antioxidants in traumatized brain. Electron paramagnetic resonance (EPR) spectroscopy of rat cortex homogenates failed to detect any radical adducts with a spin trap, 5,5-dimethyl-1-pyrroline N:-oxide, but did detect ascorbate radical signals. The ascorbate radical EPR signals increased in brain homogenates derived from traumatized brain samples compared with sham-operated controls. These results along with detailed model experiments in vitro indicate that ascorbate is a major antioxidant in brain and that the EPR assay of ascorbate radicals may be used to monitor production of free radicals in brain tissue after traumatic brain injury.     

Antioxidant activity of extract from Polygonum cuspidatum

Numerous diseases are induced by free radicals via lipid peroxidation, protein peroxidation and DNA damage. It has been known that a variety of plant extracts have antioxidant activities to scavenge free radicals. Whether Polygonum cuspidatum Sieb. et Zuce has antioxidant activity is unknown. In this study, dried roots of Polygonum cuspidatum were extracted by ethanol and the extract was lyophilized. Free radical scavenging assays, superoxide radical scavenging assays, lipid peroxidation assays and hydroxyl radical-induced DNA strand scission assays were employed to study antioxidant activities. The results indicate that the IC50 value oí Polygonum cuspidatum extract is 110 microg/ml in free radical scavenging assays, 3.2 microg/ml in superoxide radical scavenging assays, and 8 microg/ml in lipid peroxidation assays, respectively. Furthermore, Polygonum cuspidatum extract has DNA protective effect in hydroxyl radical-induced DNA strand scission assays. The total phenolics and flavonoid content of extract is 641.1 +/- 42.6 mg/g and 62.3 +/- 6.0 mg/g. The results indicate that Polygonum cuspidatum extract clearly has antioxidant effects.     

Antioxidant activity of extract from Polygonum aviculare L

Free radicals induce numerous diseases by lipid peroxidation, protein peroxidation, and DNA damage. It has been reported that numerous plant extracts have antioxidant activities to scavenge free radicals. Whether Polygonum aviculare L. (Polygonaceae) has antioxidant activity is unknown. In this study, dried Polygonum aviculare L. was extracted by ethanol, and the extract was lyophilized. The antioxidant activities of extract powder were examined by free radical scavenging assays, superoxide radical scavenging assays, lipid peroxidation assays and hydroxyl radical-induced DNA strand scission assays. The results show that the IC50 value of Polygonum aviculare L. extract is 50 microg/ml in free radical scavenging assays, 0.8 microg/ml in superoxide radical scavenging assays, and 15 microg/ml in lipid peroxidation assays, respectively. Furthermore, Polygonum aviculare L. extract has DNA protective effect in hydroxyl radical-induced DNA strand scission assays. The total phenolics and flavonoid content of extract is 677.4 +/- 62.7 microg/g and 112.7 +/- 13 microg/g. The results indicate that Polygonum aviculare L. extract clearly has antioxidant effects.     

Antioxidant properties of two gallotannins isolated from the leaves of Pistacia weinmannifolia

Pistacia weinmannifolia J. Poisson ex Franch (Anacardiaceae) is a shrub or arbor widely found in Yunnan province of China and its leaves are used as traditional Chinese medicine by herbalists. The leaves of P. weinmannifolia are rich in phenolic compounds, among which two novel gallotannins, Pistafolin A and Pistafolin B, are identified. In the present investigation, the antioxidant efficiency of Pistafolin A and Pistafolin B in preventing lipid, protein and DNA from reactive oxygen species-mediated damage was studied. Both Pistafolin A and Pistafolin B inhibited the peroxyl-radical induced lipid peroxidation of l-alpha-phosphatidylcholine liposomes dose-dependently and prevented the bovine serum albumin from peroxyl-induced oxidative damage. Pistafolin A and Pistafolin B also inhibited copper (II)-1,10-phenanthroline complex-induced DNA oxidative damage. Both Pistafolin A and Pistafolin B scavenged the hydrophilic 2,2'-azinobis(3-ethylbenzothiozoline-6-sulphonic acid) diammonium salt-free radicals and the hydrophobic 1,1-dipheny-2-picrylhydrazyl radicals effectively, suggesting they may act as hydrogen donating antioxidants. The protective effects of the two gallotannins against oxidative damage of biomacromolecules were due to their strong free radical scavenging ability. Pistafolin A with three galloyl moieties showed stronger antioxidant ability than Pistafolin B with two galloyl moieties.     

Free radical-mediated degradation of proteins: the protective and deleterious effects of membranes

Lipid membranes have been shown to scavenge free radicals generated by various means. However, under oxidative conditions, unsaturated lipids within membranes can produce damaging free radicals. We have determined the relative importance of these two conflicting properties of lipid membranes with the use of liposomal membrane studies. (1) Liposome membranes can protect extra-liposomal albumin from free radicals derived from sources other than peroxidizing lipid. When albumin or copper (an essential component of the free radical generating systems used) were encapsulated, protein damage was further reduced. (2) Using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) we demonstrate that the exposure of albumin to peroxidizing liposome membranes results in both cross-linking and degradation. Our results indicate that protein damage is substantially less than in the case of other biologically relevant free radical generating systems. We discuss our findings with respect to membrane function and the in vivo exposure of cells to free radicals.     

脂质过氧化引起的DNA损伤研究进展

脂质过氧化可以引起各种碱基损伤、DNA链断裂和各种荧光产物生成,并对DNA分子鸟嘌呤碱基具有选择性损伤.过渡金属离子可以明显加深脂质过氧化对DNA的损伤程度.多种抗氧化剂、活性氧自由基清除剂对脂质过氧化引起的DNA损伤有一定程度的保护作用.具有致突、致癌作用的8－羟基鸟嘌呤已经观察到．脂质过氧化的致突变、致癌变作用机制引起了人们的极大兴趣．     

Reduction of ultraviolet light-induced oxidative stress by amino acid-based iron chelators

The generation of free radicals by ultraviolet (UV) light accelerates skin aging, which is known as photoaging. Cutaneous iron catalyzes the generation of free radicals. We designed novel antioxidants that suppressed the iron-catalyzed free radical generation and the ensuing UV-induced damage by mimicking the binding site of iron sequestering proteins. These antioxidants, N-(2-hydroxybenzyl)amino acids, were prepared by condensation of amino acids such as glycine and L-serine with salicylaldehyde and followed by catalytic reduction. The compounds formed a 2:1 complex to iron ion. These amino acid derivatives inhibited the iron-induced hydroxyl radical generation (the Fenton reaction). The compounds also suppressed UV-induced lipid peroxidation in murine dermal fibroblast homogenates. In addition, N-(2-hydroxybenzyl)-L-serine showed protective activity against UV-induced cytotoxicity in murine dermal fibroblasts. Desferrioxamine, a strong iron sequestering compound, was effective in inhibiting the Fenton reaction and the lipid peroxidation, but it was ineffective in protecting against UV-induced cytotoxicity. The results suggest that UV-induced oxidative stress can be reduced by these amino acid derivatives.     

Phenolic chain-breaking antioxidants--their activity and mechanisms of action

This tutorial review is focused on some mechanistic aspects of peroxidation process and chemistry of phenolic chain-breaking antioxidants. Lipids are susceptible to oxidative degradation caused by radicals and during autoxidation (peroxidation) the chain reaction is mediated by peroxyl radicals leading to damage of integrity and the protective and organizational properties of biomembranes. Phenolic antioxidants provide active system of defence against lipid peroxidation, however, the effectiveness of their antioxidant action depends on several important parameters. Stoichiometry of the reaction with free radicals, fate of a phenoxyl radical, polarity of the microenvironment, localization of antioxidant molecules, their concentration and mobility, kinetic solvent effects, and interactions with other co-antioxidants are considered. Principal mechanisms of reaction between phenols and free radicals (Hydrogen Atom Transfer, Proton Coupled Electron Transfer and two mechanisms based on separate electron transfer and proton transfer steps) are described.     

Resveratrol, melatonin, vitamin E, and PBN protect against renal oxidative DNA damage induced by the kidney carcinogen KBrO3.

Free radical scavengers can protect against the genotoxicity induced by chemical carcinogens by decreasing oxidative damage. The protective effect of the antioxidants melatonin, resveratrol, vitamin E, butylated hydroxytoluene and 2-mercaptoethylamine, and the spin-trapping compound alpha-phenyl-N-tert-butyl nitrone (PBN) against oxidative DNA damage was studied in the kidney of rats treated with the kidney-specific carcinogen potassium bromate (KBrO3). KBrO3 was given to rats previously treated with melatonin, resveratrol, PBN, vitamin E, butylated hydroxytoluene, or 2-mercaptoethylamine. Oxidative damage to kidney DNA was estimated 6 hours afterwards by measuring 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo8dG) referred to deoxyguanosine (dG) by means of high performance liquid chromatography with electrochemical-coulometric and ultraviolet detection. Levels of oxo8dG in the renal genomic DNA significantly increased by more than 100% after the KBrO3 treatment. This increase was completely abolished by the treatment with resveratrol and was partially prevented by melatonin, PBN and vitamin E. Resveratrol and PBN also prevented the increase in relative kidney weight induced by KBrO3. These results show that various different antioxidants and a free radical trap, working in either the water-soluble or the lipid-soluble compartments, can prevent the oxidative DNA damage induced in the kidney by the carcinogen KBrO3.     

Ginsenoside Rd attenuates early oxidative damage and sequential inflammatory response after transient focal ischemia in rats

We previously found that ginsenoside Rd (Rd), one of the main active ingredients in Panax ginseng, attenuates neuronal oxidative damage in vitro induced by hydrogen peroxide and oxygen-glucose deprivation. In this study, we sought to investigate the potential protective effects and associated mechanisms of Rd in a rat model of focal cerebral ischemia. Rats administered with Rd (0.1-200mg/kg) or vehicle was subjected to transient middle cerebral artery occlusion. Rd at the dose of 10-50mg/kg significantly reduced the infarct volume and improved the long-term neurological outcome up to 6 weeks after ischemia. To evaluate the underlying mechanisms, in vivo free radical generation was monitored using microdialysis, oxidative DNA damage was identified by 8-hydroxy-deoxyguanosine immunostaining, oxidative protein damage was identified by the assessment of protein carbonyl and advanced glycosylation end products, and lipid peroxidation was estimated by determining the malondialdehyde and 4-hydroxynonenal formations. Microdialysis results displayed a prominent inhibitory effect of Rd on the hydroxy radical formation trapped as 2,3- and 2,5-DHBA. Early accumulations of DNA, protein and lipid peroxidation products were also suppressed by Rd treatment. Although Rd partly preserved endogenous antioxidant activities in the ischemic penumbra, in sham rats without stroke, endogenous antioxidant activities were not affected by Rd. Furthermore, we assayed sequential inflammatory response in a later phase after ischemia. Rd significantly eliminated inflammatory injury as indicated by the suppression of microglial activation, inducible nitric oxide synthase and cyclooxygenase-2 expression. Collectively, these findings demonstrated that Rd exerts neuroprotection in transient focal ischemia, which may involve early free radicals scavenging pathway and a late anti-inflammatory effect.