Acute effects of whole-body exposure to static magnetic fields and 50-Hz electromagnetic fields on muscle microcirculation in anesthetized mice

Acute microhemodynamic effects of static and alternating magnetic fields at a threshold level were investigated on modulating the muscle capillary mirocirculation in pentobarbital-anesthetized mice. The skin in a tibialis anterior was circularly removed with 1.5 mm diameter for intravital-microscopic recording of the capillary blood velocity in the tibialis anterior muscle. Fluorescein isothiocyanate (FITC)-labeled dextran (MW 150 kDa) was used for an in vivo fluorescent plasma marker of the muscle capillaries. Following a bolus injection of FITC-dextran solution into the caudal vein, the peak blood velocity in the muscle capillaries was measured prior to, during, and following exposure to static magnetic fields (SMF) or 50-Hz electromagnetic fields (EMF) using a fluorescence epi-illumination system. The whole body of experimental animals, placed on the observing stage of a fluorescence microscope, was exposed to SMF (0.3, 1 and 10 mT) or 50-Hz EMF (0.3 and 1 mT) for 10 min using a specially devised electromagnet. For sham exposure, the electromagnet was not energized. During exposure and post-exposure to SMF of 10 mT, the peak blood velocity significantly increased as compared to sham exposure. After the withdrawal of SMF and 50-Hz EMF of 1 mT, significant similar effects on the blood velocity were present or enhanced. These findings suggest that field intensity of 1 mT might be considered as a threshold level for enhancing muscle microcirculation under pentobarbital-induced hypnosis.     

Synthesis of DnaK and GroEL in Escherichia coli cells exposed to different magnetic field signals

The effects of extremely low frequency magnetic field (ELF-MF)(1 mT, 50 Hz) on the heat shock protein (HSP) synthesis in Escherichia coli were investigated. Two magnetic field signals were studied: sinusoidal (SMF) and pulsed square wave (PMF). It was found that bacteria exposed to SMF showed a significantly higher level of DnaK and GroEL proteins as compared to sham-exposed bacteria as revealed by Western blot, whereas a lower level was observed after PMF exposure. Similar results were obtained when bacterial cells were exposed to heat shock (HS) after ELF-MF exposure: again SMF and PMF resulted in an increase and in a reduction of HSP amount in comparison with sham control, respectively. In conclusion, the MF influences the synthesis of HSPs in E. coli in a way that critically depends on the signal characteristics.     

Studies on toxicological mechanisms of gas-phase cigarette smoke and protection effects of GTP

Gas-phase cigarette smoke (GPCS) was able to induce lipid peroxidation in lecithin liposomes, rat liver microsomes, and rat lung cells (RLC), and change the membrane fluidity of RLCs. Lipid free radicals were trapped in a GPCS-treated microsomal suspension by using 4-POBN as the spin trap. In addition, it was found that GPCS-peroxidized liposomes in appropriate degree of lipid peroxidation had the ability to increase the generation of superoxide anions in rat peritoneal neutrophils (RPN). Effects of green tea polyphenols (GTP) on the GPCS-induced damages were investigated The results showed that GTP was capable of inhibiting the GPCS-induced damages.     

Toxicologic damage of gas phase cigarette smoke on cells and the protective effect of green tea polyphenols

A mouse fibroblast cell line, Chinese hamster lung V₇₉ cell, was used to assess the toxicological effect of gas phase cigarette smoke (GPCS) on cells and the protective effect of green tea polyphenols (GTP). Exposure of the cultured V₇₉ cells to GPCS decreased cell viability and caused lipid peroxidation of cell membrane as measured by thiobarbituric acid reaction. Electron spin resonance (ESR) spin trapping and spin labeling studies indicated that GPCS exposure could increase the fluidity in the polar surface of cell membrane, but the membrane fluidity in the hydrophobic region was not affected. The conformation of sulfhydryl binding sites on membrane was changed. GTP itself had no effect on the polar surface of membrane nor changed the conformation of sulfhydryl binding sites on membrane proteins in the concentration used in this experiment. Incubation of V₇₉ cells with GTP aqueous solution before GPCS treatment could decrease cell death and lipid peroxidation and inhibit the changes of the biophysical properties of cell membrane induced by GPCS in a dose-dependent manner. Also GTP could scavenge the free radicals generated by GPCS. These results indicate that GPCS can induce lipid peroxidation and affect the biophysical properties of cell membrane, which may account for the toxicity of GPCS, and that GTP can prevent these changes possibly by scavenging the free radicals in GPCS and inhibiting the membrane lipid peroxidation.     

Increased efficacy of in vitro Photofrin photosensitization of human oral squamous cell carcinoma by iron and ascorbate

Photofrin^®, a photosensitizer used in the photodynamic therapy of cancer, selectively localizes in cellular membranes. Upon exposure to visible light, Photofrin^® produces singlet oxygen (¹O₂), which reacts with membrane polyunsaturated fatty acids forming lipid hydroperoxides. Transition metals, such as Fe²⁺, catalyze the production of cytotoxic free radicals from lipid hydroperoxides. Ascorbate reduces ferric to ferrous iron, further augmenting lipid peroxidation. Therefore, to increase the efficacy of Photofrin^® photosensitization, we added 20 μM ferrous sulfate and 100 μM ascorbic acid, in an aqueous layer over SCC-25 oral squamous cell carcinoma cells during in vitro illumination. In electron paramagnetic resonance spin trapping experiments, using POBN (-(4-pyridyl-1-oxide)-N-tert-butylnitrone), we observed that the presence of this pro-oxidant combination greatly increases the production of membrane-derived lipid free radicals. The effect was time dependent but only partially concentration dependent. Trypan blue dye exclusion demonstrated that this increase in lipid radical formation correlated with cytotoxicity. These observations support the hypothesis that Photofrin^® photosensitization leads to lipid hydroperoxide formation, which increases the cell's susceptibility to iron-induced Fenton chemistry. The resulting free radical-mediated lipid peroxidation results in cell death. From these data we hypothesize that the efficacy of photodynamic therapy of superficial cancer might be increased by the topical application of the pro-oxidant combination of iron and ascorbate. Furthermore, their use will probably allow lower doses of Photofrin^® without compromising antitumor effect.     

The ESR study on the protective effect of grape seed extract on rat heart mitochondria from the injury of lipid peroxidation

The effect of grape seed extract (GSE) on the lipid peroxidation of rat heart mitochondria was studied using the spin trapping agent α-(4-pyridyl-1-oxide)-N-t-butylnitrone (4-POBN) with electron spin resonance (ESR) spectrometer. Two fatty acid spin labels, 5-doxyl-stearic acid (5-DOXYL) and 16-doxyl-stearic acid (16-DOXYL) were used to study the effect of GSE on mitochondrial membrane fluidity. The results showed that the GSE could scavenge the lipid free radicals generated from the mitochondria of rat heart, and protect the mitochondrial membranes damaged by lipid peroxidation. Both the scavenging effect and the protective effect are better in water phase than in lipid phase. And the data indicate that perhaps triners and other polymers have better scavenging effect on lipid free radicals than monomers.     

Effects of a high alpha-linolenate and high linoleate diet on hemolysis and lipid peroxidation of rat erythrocytes.

alpha-Linolenic acid (18:3n-3) is known to autoxidize several fold faster than linoleic acid (18:2n-6). Feeding a high alpha-linolenate or a high linoleate diet to rats resulted in significant changes in the n-3/n-6 ratios of 20 and 22 carbon highly unsaturated fatty acids in erythrocytes. However, the rates of hemolysis observed in N2- or O2-atmosphere were similar between the two dietary groups. No significant amounts of conjugated dienes were detected and no measurable changes in the fatty acid compositions were observed during the incubations, indicating that the hemolysis occurred without involving significant lipid peroxidation. When stimulated with a free radical initiator, [2,2'-azobis-(2-amidinopropane)dihydrochloride] (AAPH), hemolysis occurred more rapidly, conjugated dienes formed and unsaturated/saturated ratios of phospholipid fatty acids decreased. However, no statistically significant difference was observed in these parameters of the two dietary groups. These results indicate that hemolysis occurs without involving lipid peroxidation but is accelerated by free radicals through lipid peroxidation, and that the difference in autoxidizabilities of alpha-linolenate and linoleate is not reflected in the rates of hemolysis and autoxidation in rat erythrocytes.     

Prolongation of life span of stroke-prone spontaneously hypertensive rats (SHRSP) ingesting persimmon tannin

The effects of persimmon tannin on pathophysiological changes in stroke-prone spontaneously hypertensive rats (SHRSP) were investigated. When the persimmon tannin was chronically ingested by SHRSP, the life span was significantly prolonged, yet the effect on blood pressure was slight. The incidences of brain hemorrhage and infarction were also significantly decreased by this treatment. To elucidate the mechanisms involved in these events, the effects of condensed tannins, including persimmon tannin, on free radicals and lipid peroxidation were examined in vitro. Using electron spin resonance analysis, we found that these tannins have a potent, concentration-dependent scavenging action toward active oxygen free radicals. These tannins strongly inhibited lipid peroxidation in rat brain homogenates, in a concentration-dependent manner. Persimmon tannin inhibited lipid peroxidation similarly to (-)-epigallocatechin. Persimmon tannin was 20 times more effective than alpha-tocopherol in terms of the 50%-inhibitory concentration. The radical scavenging action and inhibition of lipid peroxidation by persimmon tannin may explain, in part, the prolongation of the life span of the SHRSP ingesting persimmon tannin.     

Interaction between sodium tanshinone IIA sulfonate and the adriamycin semiquinone free radical: A possible mechanism for antagonizing adriamycin-induced cardiotoxity

Adriamycin (ADR) is a powerful and widely used antitumor drug, but its dose dependent cardiotoxicity limits its application. This side effect is believed to be caused by the adriamycin semiquinone free radical (ASFR). The primary focus of this work is to test effects of sodium tanshinone IIA sulfonate (STS) on ASFR and adriamycin–induced lipid peroxidation. It was found that ADR, whether in the system of heart homogenate, heart mitochondria or heart submitochondria, with NADH as the substrate or in xanthine/xanthine oxidase under anaerobic conditions, all produced ASFR rapidly. STS was shown to effectively scavenge ASFR in all these systems and postpone the appearance of ASFR. The delayed time was proportional to the amount of STS. Under aerobic conditions, ASFR could be oxidized to generate oxygen free radicals. STS could not scavenge these oxygen free radicals, but it could effectively scavenge lipid free radicals generated from membrane lipid peroxidation of heart mitochondria. STS could significantly reduce mitochondrial swelling and lipid peroxidation induced by ADR. Animal experiments show that treatment of STS could inhibit endogenous lipid peroxidation caused by ADR. Here, a protective mechanism of STS is suggested that STS can rapidly and univalently oxidize ASFR, causing the cycle of adriamycin between its quinone form and semiquinone form and inhibiting the accumulation of ASFR. Under aerobic condition, STS can protect heart mitochondria by scavenging lipid free radicals generated from adriamycin-induced mitochondrial lipid peroxidation. This investigation shows that STS may be a physiological drug to antagonize the cardiotoxicity of ADR.     

Divergent optimum levels of lycopene,beta-carotene and lutein protecting against UVB irradiation in human fibroblastst

Exposure of living organisms to UV light leads to photooxidative reactions. Peroxyl radicals are involved in the propagation of lipid peroxidation. Carotenoids are dietary antioxidants and show photoprotective effects in human skin, efficiently scavenging peroxyl radicals and inhibiting lipid peroxidation. Cultured human skin fibroblasts were used to examine the protective effects of the carotenoids, lycopene, beta-carotene and lutein on UVB-induced lipid peroxidation. The carotenoids were delivered to the cells using liposomes as the vehicle. The cells were exposed to UVB light for 20 min. Lycopene, beta-carotene and lutein were capable of decreasing UV-induced formation of thiobarbituric acid-reactive substances at 1 h to levels 40-50% of controls free of carotenoids. The amounts of carotenoid needed for optimal protection were divergent at 0.05, 0.40 and 0.30 nmol/mg protein for lycopene, beta-carotene and lutein, respectively. Beyond the optimum levels, further increases of carotenoid levels in cells led to prooxidant effects.     

Antiacetylcholinesterase and antioxidant ent-Kaurene diterpenoid, melissoidesin from Isodon wightii (Bentham) H. Hara

The ent kaurene diterpenoid, melissoidesin was isolated from the acetone extract of the leaves of Isodon wightii and the structure was designated as 3beta, 11beta, 15beta-trihydroxy-6alpha-acetoxy-ent-kaur-16-ene based on spectral data and previous reports. Melissoidesin isolated from the acetone extract of leaves showed potent antiacetylcholinesterase activity and the IC(50) value was observed as 215 microg mL(-1). DPPH (1, 1-diphenyl-2-picrylhydrazyl) free radical scavenging activity of melissoidesin was significant and the IC(50) value was 138 microg mL(-1). The significant reducing property of the melissoidesin was stronger in high concentration. IC(50) value of melissoidesin on hydroxyl radicals and metal chelation was observed as 99 and 143 microg mL(-1), respectively. The 50% inhibitory concentration of melissoidesin on lipid peroxidation was calculated as 133 microg mL(-1). These findings indicate that ent kaurene diterpenoid, melissoidesin was promising antiacetylcholinesterase and antioxidant which can be used as food and drug preparations.     

Evaluation of the Free Radical Scavenging Activities of Ellagic Acid and Ellagic Acid Peracetate by EPR Spectrometry

The purpose of this study was to examine the free radical scavenging and antioxidant activities of ellagic acid (EA) and ellagic acid peracetate (EAPA) by measuring their reactions with the radicals, 2,2-diphenyl-1-picrylhydrazyl and galvinoxyl using EPR spectroscopy. We have also evaluated the influence of EA and EAPA on the ROS production in L-6 myoblasts and rat liver microsomal lipid peroxidation catalyzed by NADPH. The results obtained clearly indicated that EA has tremendous ability to scavenge free radicals, even at concentration of 1 µM. Interestingly even in the absence of esterase, EAPA, the acetylated product of EA, was also found to be a good scavenger but at a relatively slower rate. Kinetic studies revealed that both EA and EAPA have ability to scavenge free radicals at the concentrations of 1 µM over extended periods of time. In cellular systems, EA and EAPA were found to have similar potentials for the inhibition of ROS production in L-6 myoblasts and NADPH-dependent catalyzed microsomal lipid peroxidation.     

UV-B-induced formation of reactive oxygen species and oxidative damage of the cyanobacterium Anabaena sp.: protective effects of ascorbic acid and N-acetyl-L-cysteine

Reactive oxygen species (ROS) are involved in the oxidative damage of the cyanobacterium Anabaena sp. caused by UV-B (280-315 nm) radiation. UV-B-induced overproduction of ROS as well as the oxidative stress was detected in vivo by using the ROS-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Thiobarbituric acid reactive substances (TBARS) and fluorometric analysis of DNA unwinding (FADU) methods were adapted to measure lipid peroxidation and DNA strand breaks in Anabaena sp. Moderate UV-B radiation causes an increase of ROS production, enhanced lipid peroxidation and DNA strand breaks, yielding a significantly decreased survival. In contrast, the supplementation of UV-A in our work only showed a significant increase in total ROS levels and DNA strand breaks while no significant effect on lipid peroxidation, chlorophyll bleaching or survival was observed. The presence of ascorbic acid and N-acetyl-L-cysteine (NAC) reversed the oxidative stress and protected the organisms from chlorophyll bleaching and the damage of photosynthetic apparatus induced by UV-B significantly, resulting in a considerably higher survival rate. Ascorbic acid also exhibited a significant protective effect on lipid peroxidation and DNA strand breaks while NAC did not show a substantial effect. These results suggest that ascorbic acid exhibited significantly higher protective efficiency with respect to DNA strand breaks and survival than NAC while NAC appears to be especially effective in defending the photosynthetic apparatus from oxidative damage.     

Kinetics of oxidation of iron(II) ions with lipid hydroperoxides

One of the pathways for excessive production of free radicals is the decomposition of lipid hydroperoxides catalyzed by iron. A number of hydroperoxides of unsaturated fatty acids (LOOH), some prepared in our laboratory and others extracted from biological materials, were used to determine the rate constants of Fe²⁺ oxidation by measuring the formation rate of Fe³⁺ ions in the presence of simple unidentate ligands, chloride, and thiocyanate as the [FeCl]²⁺ and [FeNCS]²⁺ complexes, in a deoxygenated dichloromethane:methanol (2:1, v/v) mixture. The rates of Fe²⁺ oxidation with prepared LOOHs via the [FeNCS]²⁺ complex were approximately the same-the average second-order reaction rate constant was 1390 ± 340 dm³ mol⁻¹ s⁻¹; the rate constants of LOOHs from different biological materials were in the same range. The rates measured as the [FeNCS]²⁺ complex were somewhat higher than the rates measured as the [FeCl]²⁺ complex, indicating that ligands could interact in the transition state, thus affecting the disruption of the intermediate complex. Since there were no significant differences in the activation thermodynamic parameters for reactions within the reaction series of studied hydroperoxides, it was assumed that the oxidation proceeded by an inner sphere mechanism, considering that the breakdown of the successor inner sphere complex with the homolytic cleavage of peroxide bonds of hydroperoxides forming reactive alkoxyl radicals was the rate-limiting step. Based on this research, an indirect spectrophotometric method for quantitative determination of LOOH was reestimated. The microprocedure proposed for the lipid hydroperoxide assay could be applied to follow the early stages of lipid peroxidation processes in real biological samples.     

Free radical scavenging activity, determination of phenolic compounds and HPLC-DAD/ESI-MS profile of Campomanesia adamantium leaves

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 activity to scavenge free radicals. Campomanesia adamantium (Myrtaceae) is a small tree with edible fruit, commonly known as "guavira" or "guabiroba-branca" that has been used in popular medicine as depurative anti-diarrhoeic, antiinflammatory, anti-rheumatic and to liver diseases. In this study, the antiradical activities of ethanol crude extract of the leaves from C. adamantium and the ethyl acetate and butanol fractions obtained by partition, were determined using DPPH (2,2-Diphenyl-1-picrylhydrazyl radical) and ORAC-FL (Oxygen Radical Absorbance Capacity) assays. The total phenol content in the samples was estimated by Folin Ciocalteau method (FCR). In an initial evaluation the ethanolic extract and the fractions ethyl acetate and butanol have shown levels of phenolic compounds between 15- 74 mg GAE/g in FCR assay, showed DPPH free-radical scavenging activity with SC50 in the range of 7.77-13.35 microg/mL and demonstrated antioxidant capacity between 2648-3502 micromol TE/g of extract and fractions in the ORAC-FL assay. HPLC-DAD and ESI-MS analysis revealed were that the extract of the leaves of C. adamantium studied appears to contain flavonoids as major constituents, including isoquercetrin and quercetin that exhibit proven antioxidant activity.     

Antioxidant activity of indapamide and its metabolite

An antioxidant activity of indapamide (IDP) and its metabolite (OH-IDP) is demonstrated in this study. Both IDP and OH-IDP were found to scavenge 1,1-diphenyl-2-picryl-hydrazyl free radical. The scavenging effect of OH-IDP was stronger than that of IDP. Lipid peroxidation of rat liver microsomes initiated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) and adenosine diphosphate (ADP)-Fe3+ was inhibited by IDP and OH-IDP with IC50 values of about 6 and 2 microM, respectively. The lipid peroxidation in human erythrocyte membrane, induced by 2,2'-azobis-(2-amidinopropane dihydrochloride) treatment, was also inhibited by 10 microM IDP. The antioxidant capacity of OH-IDP was at almost the same level as that of alpha-tocopherol, tested for comparison. The present data show that IDP and OH-IDP at micromolar concentrations are able to trap the free radicals involved in the lipid peroxidation.     

Mechanism of antioxidant action of pueraria glycoside (PG)-1 (an isoflavonoid) and mangiferin (a xanthonoid).

The antioxidant activities of pueraria glycoside (PG)-1 (isoflavonoid) and mangiferin (xanthonoid) were studied and compared with PG-3 and daidzein (isoflavonoids) and with wogonin (flavonoid). PG-1 and mangiferin rapidly scavenged 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, and inhibited lipid peroxidation which was initiated enzymatically by reduced nicotinamide adenine dinucleotide phosphate (NADPH) or non-enzymatically by ascorbic acid or Fenton's reagent (H2O2 + Fe2+) in rat liver microsomes. Wogonin inhibited the enzymatically induced lipid peroxidation but had no scavenging effect on DPPH radical or on the non-enzymatic peroxidation. PG-3 and daidzein did not show any of these effects. Formation of Fe2+ by NADPH-dependent cytochrome P-450 reductase was inhibited by wogonin, but not by PG-1 or mangiferin. PG-1 and mangiferin had no effect on terminating radical chain reaction during the lipid peroxidation in the enzymatic system of microsomes or in the linoleic acid hydroperoxide-induced peroxidation system. These results suggest that PG-1 and mangiferin have an antioxidant activity, probably due to their ability to scavenge free radicals involved in initiation of lipid peroxidation. In contrast, wogonin may affect NADPH-dependent cytochrome P-450 reductase action, since it inhibited only the enzymatically induced lipid peroxidation.     

Relationship Between Membrane Damage Induced by Osmotic Stress and Lipid Peroxidation and Some Free Radicals

To treat leaf discs with solutions of various osmotic potentials of polyethylene glycols (MW 6000) by adopting the floating treatment could increase the membrane permeability, decrease the formation of malondialdehyde,and reduce the activity of peroxidase. Nevertheless, the activities of superoxide dismutase and catalase were not obviously altered. In verifying if the membrane damage was caused by lipid peroxidation initiated by active oxygen species, diethyldithiocarbamate was chosen as inhibitor superoxide dismutase,aminotriazole as catalase and mannitol as scavenger of hydroxyl free radicals.The results have shown that there is not any correlation between lipid peroxidation and membrane damage.Therefore,membrane damage caused by water stress is probably not due to free radical initiation.     

Photosensitized oxidation of cholesterol in biological systems: reaction pathways, cytotoxic effects and defense mechanisms.

Cholesterol resembles other unsaturated lipids in being susceptible to peroxidative degradation when exposed to a sensitizing agent, exciting light of suitable wavelength and molecular oxygen. Selected hydroperoxides of cholesterol can be used as relatively convenient and reliable indicators of primary photochemical mechanisms, allowing a distinction to be made between free radical-mediated and singlet oxygen-mediated reactions. When generated in cell membranes, hydroperoxides of cholesterol and other lipids can have deleterious effects on membrane structure and function. Such damage may be exacerbated if these photoproducts undergo one-electron reduction to oxyl radicals which in turn initiate chain peroxidation reactions. Cells can resist these effects by using a membrane-based glutathione peroxidase to catalyze the two-electron reduction and detoxification of lipid hydroperoxides. Recent advances in our understanding of cholesterol photo-oxidation from the standpoints of (a) mechanistic information, (b) cytotoxicity and (c) cytoprotection are discussed in this article.