Free radical lipid oxidation and physical properties of lipid layer of biological membranes

The results obtained mainly by the author and coworkers are summarized. One efficient method to detect free radicals in biological samples is chemiluminescence (CL). In the absence of activators CL of membraneous systems is due to lipid peroxide free radicals, whereas in the presence of luminol it is initiated by oxygen radicals. Low levels of free radicals in the cells and blood plasma are maintained by antioxidants, enzymes included. Ferrous ions increase free radical concentrations in the cells and tissues. Deleterious action of hydroxyl radicals is the result of the breakage of DNA strains and of lipid peroxidation (LPO). The latter reaction brings about the damage of the membrane barriers due to a decrease of the electrical stability of the membrane lipid bilayer and "self-breakdown" of the membranes by potential differences produced in the living cells.     

Oxidation of indole-3-acetic acid by horseradish peroxidase induces apoptosis in G361 human melanoma cells

The combination of indole-3-acetic acid (IAA) and horseradish peroxidase (HRP) has recently been proposed as a novel cancer therapy. However, the mechanism underlying the cytotoxic effect involved is substantially unknown. Here, we show that IAA/HRP treatment induces apoptosis in G361 human melanoma cells, whereas IAA or HRP alone have no effect. It is known that IAA produces free radicals when oxidized by HRP. Because oxidative stress could induce apoptosis, we measured the production of free radicals at varying concentrations of IAA and HRP. Our results show that IAA/HRP produces free radicals in a dose-dependent manner, which are suppressed by ascorbic acid or (-)-epigallocatechin gallate (EGCG). Furthermore, antioxidants prevent IAA/HRP-induced apoptosis, indicating that the IAA/HRP-produced free radicals play an important role in the apoptotic process. In addition, IAA/HRP was observed to activate p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK), which are almost completely blocked by antioxidants. We further investigated the IAA/HRP-mediated apoptotic pathways, and found that IAA/HRP activates caspase-8 and caspase-9, leading to caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage. These events were also blocked by antioxidants, such as ascorbic acid or EGCG. Thus, we propose that IAA/HRP-induced free radicals lead to the apoptosis of human melanoma cells via both death receptor-mediated and mitochondrial apoptotic pathways.     

Monocyte-macrophage membrane possesses free radicals scavenging activity: stimulation by polyphenols or by paraoxonase 1 (PON1)

Abstract

In the current study, we analysed free radicals scavenging activity of monocytes-macrophages in the absence or presence of antioxidants such as polyphenols or paraoxonase 1 (PON1). THP-1 human monocytic cell line, murine J774A.1 macrophages, as well as human primary monocytes have the capability to scavenge free radicals, as measured by the 1-diphenyl-2-picryl-hydrazyl (DPPH) assay. This effect (which could be attributed to the cell's membrane) was cell number and incubation time dependent. Upon incubation of J774A.1 macrophages with acetylated LDL (Ac-LDL), with VLDL, or with the radical generator, AAPH, the cells’ lipid peroxides content, and paraoxonase 2 (PON2) activity were significantly increased. While non-treated cells decreased DPPH absorbance by 65%, the Ac-LDL-, VLDL- or AAPH-treated cells, decreased it by only 33%, 30%, or 45%, respectively. We next analysed the effect of J774A.1 macrophage enrichment with antioxidants, such as polyphenols or PON1 on the cells’ free radicals scavenging activity. Non-treated cells decreased DPPH absorbance by 50%, whereas vitamin E-, punicalagin- or PJ-treated cells significantly further decreased it, by 75%. Similarly, in PON1-treated cells DPPH absorbance was further decreased by 63%, in association with 23% increment in PON1 catalytic activity. In cells under oxidative stress [treated with AAPH-, or with oxidized LDL], PON1 activity was decreased by 31% or 40%, as compared to the activity observed in PON1 incubated with non-treated cells. We conclude that monocytes-macrophages possess free radicals scavenging activity, which is decreased under atherogenic conditions, and increased upon cell enrichment with potent antioxidants such as nutritional polyphenols, or PON1.     

Involvement of bioantioxidants in thrombosis in mice

SUMMARY

An involvement of free radicals in thrombosis has been suggested previously. In order to further explore the role of free radicals and antioxidants in thrombosis, we have measured preventive (enzymes of the glutathione redox cycle) and chain-breaking antioxidants (vitamin E and C) in whole blood, platelets, neutrophils (PMNLs), heart and lung following collagen and adrenaline induced thrombosis in mice. A significant decrease in platelet glutathione (GSH) level (54%) and glutathione reductase activity was observed after thrombosis. In addition, GSH content in whole blood was also found to be reduced. In PMNLs, an increase in glutathione peroxidase activity and a four-fold elevation in vitamin C content was observed following thrombosis. However, levels of vitamin E and total thiol groups remained unchanged in both the cells and tissues. The results further suggest involvement of free radicals and PMNLs in thrombosis.     

Resistance to UVB radiation in Antarctic yeasts

Yeast cells were used as a model system to study the inter-relationship among free radicals, antioxidants and UV-induced cell damage. In particular, the effects of UV-radiation in newly isolated yeasts from the Antarctic have been studied.     

Antioxidant lipoate and tissue antioxidants in aged rats

Oxidative metabolism produces free radicals that must be removed from the cellular environment for the cell to survive. The levels of nonenzymic antioxidants involved in the elimination of free radicals were investigated in an attempt to correlate any changes in the levels of enzymic antioxidants during aging with changes in free radical mediated cellular damage. Antioxidants were measured in liver and kidney of young and aged rats with respect to DL-alpha-lipoic acid supplemented rats. In both organs lipid peroxidation damage (a marker of free radical mediated damage) increased with age, and a significant decrease in antioxidant systems was observed. Moreover, DL-alpha-lipoic acid treated aged rats showed a decrease in the level of lipid peroxides and an increase in the antioxidant status. The results of this study provide evidence that DL-alpha-lipoic acid treatment can improve antioxidants during aging and minimize the age-associated disorders in which free radicals are the major cause.     

Oxidative stress leads to cholesterol accumulation in vascular smooth muscle cells.

The transformation of macrophages and smooth muscle cells into foam cells by modified low-density lipoproteins (LDL) is one of the key events of atherogenesis. Effects of free radicals have mainly been studied in LDL, and other than toxicity, data dealing with direct action of free radicals on cells are scarce. This study focused on the direct effects of free radicals on cholesterol metabolism of smooth muscle cells. A free radical generator, azobis-amidinopropane dihydrochloride, was used, and conditions for a standardized oxidative stress were set up in vascular smooth muscle cells. After free radical action, the cells presented an accumulation of cholesterol that appeared to be the result of: (i) an increase in cholesterol biosynthesis and esterification; (ii) a decrease in cell cholesteryl ester hydrolysis; and (iii) a reduced cholesterol efflux. All these parameters were opposed by antioxidants. In addition, oxidant stress induced an increased degradation of acetyl-LDL, whereas no change was noted for native LDL. From this data, it was concluded that cholesterol metabolism of vascular smooth muscle cells was markedly altered by in vitro treatment with free radicals, although cell viability was unaffected. The resulting disturbance in cholesterol metabolism favors accumulation of cholesterol and cholesteryl esters in vascular cells, and thus may contribute to the formation of smooth muscle foam cells.     

Kinetic analysis of the free-radical-induced lipid peroxidation in human erythrocyte membranes: evaluation of potential antioxidants using cis-parinaric acid to monitor peroxidation.

cis-Parinaric acid (PnA), cis-trans-trans-cis-9, 11, 13, 15-octadecatetraenoic acid, is fluorescent (epsilon = 74,000 at 324 nm) when partitioned into a lipid environment and the fluorescence is destroyed upon reaction with free radicals. It has been used to monitor semiquantitatively free-radical-induced lipid peroxidation in human erythrocyte membranes. We have applied this assay to the quantitative evaluation of potential antioxidants. The kinetics of the reaction of PnA with free radicals were measured in erythrocyte ghosts. After initiation of free radical generation by cumene hydroperoxide and cupric ion, a steady-state rate of fluorescence decay is rapidly established. In the steady state the oxidation of PnA and, hence, the loss of fluorescence is a first-order process. In the presence of antioxidants, such as vitamin E, the rate constant of fluorescence loss decreases, thereby indicating that the antioxidant decreases the steady-state concentration of free radicals. By adding various concentrations of potential antioxidants, pseudo-first-order rate constants [k1] which measure the reactivity of antioxidants with free radicals were determined. Results show that, when incorporated into erythrocyte membranes, U-78, 517f, a vitamin E analog, is a potent free radical scavenger, being approximately 50% as effective as vitamin E and 10-15 times more potent than the aminosteroids evaluated (see Table 1).     

Hydroxyl free radicals generated by vanadyl[IV] induce cell blebbing in mitotic human Chang liver cells

Vanadium has recently been reported to induce interphase and M-phase (mitotic) programmed cell death via the generation of hydroxyl free radicals (OH*). In this paper, the effects of antioxidants on: (a) vanadyl[IV]-generated OH* free radical levels; and (b) cellular glutathione in vanadyl [IV]-treated Chang liver cells were evaluated. The surface morphology of vanadyl-treated mitotic cells was studied by confocal and scanning microscopy. The free radical scavengers zinc chloride, glucose and thiourea reduced the levels of vanadyl-induced OH* free radicals and partially prevented the depletion of cellular glutathione. Concurrent with OH* free radical production, vanadyl-treated telophase cells exhibited excessive cell blebbing and cell shrinkage. The morphological features demonstrated in vanadyl-induced mitotic programmed cell death as a consequence of oxidative stress is novel.     

Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects

Chemical probes for free radicals in biology are important tools; fluorescence and chemiluminescence offer high detection sensitivity. This article reviews progress in the development of probes for "reactive oxygen and nitrogen" species, emphasizing the caution needed in their use. Reactive species include hydrogen peroxide; hydroxyl, superoxide, and thiyl radicals; carbonate radical-anion; and nitric oxide, nitrogen dioxide, and peroxynitrite. Probes based on reduced dyes lack selectivity and may require a catalyst for reaction: despite these drawbacks, dichlorodihydrofluorescein and dihydrorhodamine have been used in well over 2,000 studies. Use in cellular systems requires loading into cells, and minimizing leakage. Reactive species can compete with intracellular antioxidants, changes in fluorescence or luminescence possibly reflecting changes in competing antioxidants rather than free radical generation rate. Products being measured can react further with radicals, and intermediate probe radicals are often reactive toward antioxidants and especially oxygen, to generate superoxide. Common probes for superoxide and nitric oxide require activation to a reactive intermediate; activation is not achieved by the radical of interest and the response is thus additionally sensitive to this first step. Rational use of probes requires understanding and quantitation of the mechanistic pathways involved, and of environmental factors such as oxygen and pH. We can build on this framework of knowledge in evaluating new probes.     

Enzymatic and non-enzymatic protective mechanisms in recalcitrant seeds of Araucaria bidwillii subjected to desiccation.

The changes in several antioxidants as well as in the level of C-centered free radicals and thiobarbituric acid reactive substances (TBARS) were studied in seeds of Araucaria bidwillii Hook desiccated to 37%, 28% and 21% moisture content. The lowest-safe moisture content for the seedling establishment was 37%. The embryo, besides double amounts of free radicals, showed higher levels of both enzymatic and non-enzymatic antioxidants than endosperm. Lutein decreased in both organs whereas alpha-tocopherol values were not affected by desiccation. In the embryo at 37% seed moisture content the antioxidant defense system increased giving rise to a decrease in free radicals. Beyond this point, free radicals and TBARS increased in agreement with the umpiring of the ascorbate/glutathione cycle by the decrease in reduced glutathione and glutathione reductase activity (GR, EC 1.6.4.2). At 21% moisture GR decreased. In the endosperm during desiccation, the consumption of ascorbate, total glutathione and lutein prevented the rise in free radicals and TBARS till 28% moisture, at which an increase in oxidized glutathione was also observed.     

Antioxidants inhibit proliferation and cell surface expression of receptors for interleukin-2 and transferrin in T lymphocytes stimulated with phorbol myristate acetate and ionomycin

We have previously shown that several antioxidant compounds inhibit the proliferation of T lymphocytes stimulated with alloantigen (Chaudhri, G., Clark, I. A., Hunt, N. H., Cowden, W. B., and Ceredig, R., J. Immunol. 136, 2646, 1986). We concluded from these studies that free oxygen radicals are positive mediators in T-lymphocyte activation and proliferation. In order to extend these studies we examined the effects of antioxidants on T cells stimulated with a combination of phorbol myristate acetate (PMA) and ionomycin. The following antioxidants were used: ferricyanide, an inhibitor of superoxide production; iron chelators, which block hydroxyl radical formation; and butylated hydroxyanisole, a free radical scavenger. Responder cells included purified peripheral T cells (Lyt-2+ or L3T4+ cells) and immature (Lyt-2-/L3T4-) thymocytes. All agents, in the micromolar range, caused a dose-dependent inhibition of proliferation of each T-cell subset studied. Flow microfluorometric analysis of T cells stimulated for 48 hr showed that the expression of interleukin-2 (IL-2) receptors and transferrin receptors was inhibited by all the antioxidants tested but not by hydroxyurea (HU), an inhibitor of the enzyme ribonucleotide reductase. In contrast, the expression of a third activation marker, phagocytic glycoprotein-1 (Pgp-1 or Lyt-24), was not affected by any of the agents. Furthermore, while both the antioxidants and HU inhibited T-cell cycling, analysis of a light-scattering parameter related to cell size indicated that the antioxidant-treated cells remained small while the HU-treated and control cells were larger and blast-like. Therefore, the mechanism of action of the three classes of antioxidants is similar, but quite distinct from the inhibition of proliferation caused by HU. Taken together, these results suggest that free radicals are involved in specific early events in T-cell activation.     

The importance of lipid solubility in antioxidants and free radical generating systems for determining lipoprotein proxidation.

The oxidative modification of low-density lipoprotein (LDL) plays an important role in atherosclerosis. Protecting LDL from oxidation has been shown to reduce the risk of coronary heart disease. In this study, we compared the protective effects of two lipophilic antioxidants (vitamin E and lazaroid) with two hydrophilic antioxidants (trolox and vitamin C) in the presence of several different free radical generating systems. Vitamin E (IC50 = 5.9 microM) and lazaroid (IC50 = 5.0 microM) were more effective in inhibiting lipid peroxidation caused by a Fe-ADP free radical generating system than vitamin C (IC50 = 5.2 x 10(3) microM) and trolox (IC5 = 1.2 x 10(3) microM). Preincubation of lipoproteins with a lipophilic antioxidant increased the protective effect against various free radicals. Preincubation with hydrophilic antioxidants did not have an effect. We also tested the efficacy of the antioxidants when the free radicals were generated within the lipid or the aqueous environment surrounding the LDL. For this purpose, we used the peroxyl generating azo-compounds AMVN (2,2'-azobis(2,4-dimethylvaleronitrile)) and AAPH (2,2'azobis(2-amidinopropane) dihydrochloride). All of the antioxidants tested were more effective against free radicals generated in a water soluble medium than they were against free radicals generated in a lipid environment. In conclusion, our data demonstrate that lipid solubility is an important factor for both the antioxidant and the free radical generating systems in determining the extent of lipid peroxidation in LDL. Our data also demonstrate that antioxidant efficacy in one set of experimental conditions may not necessarily translate into a similar degree of protection in another set of conditions where lipophilicity is a variable.     

Antioxidant strategies in the treatment of stroke

Excessive production of free radicals is known to lead to cell injury in a variety of diseases, such as cerebral ischemia. In this review, we describe some of the numerous studies that have examined this oxidative stress and the efficiency of antioxidant strategies in focal cerebral ischemia. Besides using genetically modified mice, these strategies can be divided into three groups: (1) inhibition of free radical production, (2) scavenging of free radicals, and (3) increase of free radical degradation by using agents mimicking the enzymatic activity of endogenous antioxidants. Finally, the clinical trials that have tested or are currently testing the efficiency of antioxidants in patients suffering from stroke are reviewed. The results presented here lead us to consider that antioxidants are very promising drugs for the treatment of ischemic stroke.     

Oxidative hemolysis of erythrocytes and its inhibition by free radical scavengers

The oxidative hemolysis of rabbit erythrocytes induced by free radicals and its inhibition by chain-breaking antioxidants have been studied. The free radicals were generated from either a water-soluble or a lipid-soluble azo compound which, upon its thermal decomposition, gave carbon radicals that reacted with oxygen immediately to give peroxyl radicals. The radicals generated in the aqueous phase from a water-soluble azo compound induced hemolysis in air, but little hemolysis was observed in the absence of oxygen. Water-soluble chain-breaking antioxidants, such as ascorbic acid, uric acid, and water-soluble chromanol, suppressed the hemolysis dose dependently. Vitamin E in the erythrocyte membranes was also effective in suppressing the hemolysis. 2,2,5,7,8-Pentamethyl-6-chromanol, a vitamin E analogue without phytyl side chain, incorporated into dimyristoylphosphatidylcholine liposomes, suppressed the above hemolysis, but alpha-tocopherol did not suppress the hemolysis. Soybean phosphatidylcholine liposomes also induced hemolysis, and a lipid-soluble azo initiator incorporated into the soybean phosphatidylcholine liposomes accelerated the hemolysis. The chain-breaking antioxidants incorporated into the liposomes were also effective in suppressing this hemolysis.     

Antioxidants and cataract

Abstract

The major causes for cataract formation are free radicals, and these free radicals are neutralized by the presence of endogenous antioxidants in the eye. Using xenobiotics, it has been confirmed that free radicals mediate the formation of cataract. Two cataract model-selenite model and the diabetic cataract model-have been developed to study the pathophysiology of cataract formation due to free radicals and the role of antioxidants during the process of cataractogenesis. This review focuses on natural compounds with antioxidant properties that could actually be applied as an interventional strategy on a large scale and are also relatively inexpensive. A brief overview of plants with antioxidant properties that in addition possess potential anti-cataract properties has been discussed. In addition to plants, three natural compounds (curcumin, vitamin C and vitamin E), on which a lot of data exist showing anti-cataract and antioxidant activities, have also been discussed. These antioxidants can be supplemented in the diet for a better defence against free radicals. Studies on vitamin C and vitamin E have proved that they are capable of preventing lipid peroxidation, thereby preventing the generation of free radicals, but their efficacy as anti-cataract agent is questionable. Unlike vitamins C and E, curcumin is well established as an anti-cataract agent, but the issue of curcumin bioavailability is yet to be addressed. Nanotechnology proves to be a promising area in increasing the curcumin bioavailability, but still a lot more research needs to be done before the use of curcumin as an effective anti-cataract agent for humans.     

试论茶多酚清除生物自由基的高效性

从生物自由基出发,本文综述了茶多酚(TP)清除自由基的效能.茶多酚对多种自由基具有卓越的清除特性,并明显优于其它抗氧化剂,但茶多酚浓度、体系pH值、自由基类型、儿茶素组成与结构对其清除效能有较大影响.     

Effect of oxygen concentration on free radical-induced cytotoxicity

Free radicals induce oxidative stress in vivo, leading to various disorders and diseases. In the present study, the effect of oxygen pressure on the cytotoxicity induced by free radicals was studied. It was found that alkyl radicals markedly aggravated Jurkat cell apoptosis under low oxygen pressure and this was ascribed to a hypoxic condition caused by the consumption of oxygen by alkyl radicals giving peroxyl radicals and subsequent lipid peroxidation by a chain mechanism. The intracellular lipid hydroperoxides significantly increased at an early time point even under hypoxia. Cytochrome c was released from the mitochondria, and caspase-9 as well as caspase-3 was activated during apoptosis, indicating that cell death followed by the intrinsic, mitochondrial apoptosis pathway. Pretreatment with VAD-FMK, a caspase inhibitor, attenuated the apoptosis induced by alkyl radicals under hypoxia. Moreover, pretreatment with various antioxidants also significantly rescued the cells from apoptosis. Taken together, the results indicate that free radicals induced hypoxic conditions, which accelerated mitochondria-dependent cell apoptosis.     

Generation and propagation of radical reactions on proteins

The oxidation of proteins by free radicals is thought to play a major role in many oxidative processes within cells and is implicated in a number of human diseases as well as ageing. This review summarises information on the formation of radicals on peptides and proteins and how radical damage may be propagated and transferred within protein structures. The emphasis of this article is primarily on the deleterious actions of radicals generated on proteins, and their mechanisms of action, rather than on enzymatic systems where radicals are deliberately formed as transient intermediates. The final section of this review examines the control of protein oxidation and how such damage might be limited by antioxidants.     

Endogenous antioxidant changes in the myocardium in response to acute and chronic stress conditions

Oxygen is a diradical and because of its unique electronic configuration, it has the potential to form strong oxidants (e.g. superoxide radical, hydrogen peroxide and hydroxyl radical) called oxygen free radicals or partially reduced forms of oxygen (PRFO). These highly reactive oxygen species can cause cellular injury by oxidizing lipids and proteins as well as by causing strand breaks in nucleic acids. PRFO are produced in the cell during normal redox reactions including respiration and there are various antioxidants in the cell which scavenge these radicals. Thus in order to maintain a normal cell structure and function, a proper balance between free radical production and antioxidant levels is absolutely essential. Production of PRFO in the myocardium is increased during variousin vivo as well asin vitro pathological conditions and these toxic radicals are responsible for causing functional, biochemical and ultrastructural changes in cardiac myocytes. Indirect evidence of free radical involvement in myocardial injury is provided by studies in which protection against these alterations is seen in the presence of exogenous administration of antioxidants. Endogenous myocardial antioxidants have also been reported to change under various physiological as well as pathophysiological conditions. It appears that endogenous antioxidants respond and adjust to different stress conditions and failure of these compensatory changes may also contribute in cardiac dysfunction. Thus endogenous and/or exogenous increase in antioxidants might have a therapeutic potential in various pathological conditions which result from increased free radical production.