In vitro photochemical and phototoxicological characterization of major constituents in St. John's Wort (Hypericum perforatum) extracts

Extracts from St. John's Wort (SJW: Hypericum perforatum) have been used for the treatment of mild-to-moderate depression. In spite of the high therapeutic potential, orally administered SJW sometimes causes phototoxic skin responses. As such, the present study aimed to clarify the phototoxic mechanisms and to identify the major phototoxins of SJW extract. Photobiochemical properties of SJW extract and 19 known constituents were characterized with focus on generation of reactive oxygen species (ROS), lipid peroxidation, and DNA photocleavage, which are indicative of photosensitive, photoirritant, and photogenotoxic potentials, respectively. ROS assay revealed the photoreactivity of SJW extract and some SJW ingredients as evidenced by type I and/or II photochemical reactions under light exposure. Not all the ROS-generating constituents caused photosensitized peroxidation of linoleic acid and photodynamic cleavage of plasmid DNA, and only hypericin, pseudohypericin, and hyperforin exhibited in vitro photoirritant potential. Concomitant UV exposure of quercitrin, an SJW component with potent UV/Vis absorption, with hyperforin resulted in significant attenuation of photodynamic generation of singlet oxygen from hyperforin, but not with hypericin. In conclusion, our results suggested that hypericin, pseudohypericin, and hyperforin might be responsible for the in vitro phototoxic effects of SJW extract.     

Retinal fatty acid binding protein reduce lipid peroxidation stimulated by long-chain fatty acid hydroperoxides on rod outer segments

In the present study we have investigated the effect of partially purified retinal fatty acid binding protein (FABP) against nonenzymatic lipid peroxidation stimulated by hydroperoxides derived from fatty acids on rod outer segment (ROS) membranes. Linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) were prepared from linoleic acid, arachidonic acid and docosahexaenoic acid, respectively, by means of lipoxidase. ROS membranes were peroxidized using an ascorbate-Fe(+2) experimental system. The effect on the peroxidation of ROS containing different amounts of lipid hydroperoxides (LOOH) was studied; ROS deprived of exogenously added LOOH was utilized as control. The degradative process was measured simultaneously by determining chemiluminescence and fatty acid composition of total lipids isolated from ROS. The addition of hydroperoxides to ROS produced a marked increase in light emission. This increase was hydroperoxide concentration-dependent. The highest value of activation was produced by DHP. The decrease percentage of the more polyunsaturated fatty acids (PUFAs) (20:4 n6 and 22:6 n3) was used to evaluate the fatty acid alterations observed during the process. We have compared the fatty acid composition of total lipids isolated from native ROS and peroxidized ROS that were incubated with and without hydroperoxides. The major difference in the fatty acid composition was found in the docosahexaenoic acid content, which decreased by 45.51+/-1.07% in the peroxidized group compared to native ROS; the decrease was even higher, 81.38+/-1.11%, when the lipid peroxidation was stimulated by DHP. Retinal FABP was partially purified from retinal cytosol. Afterwards, we measured its effect on the reaction of lipid peroxidation induced by LOOH. As a result, we observed a decrease of chemiluminescence (inhibition of lipid peroxidation) when adding increasing amounts (0.2 to 0.6 mg) of retinal FABP to ROS. The inhibitory effect reaches its highest value in the presence of DHP (41.81+/-10.18%). Under these conditions, bovine serum albumin (BSA) produces a smaller inhibitory effect (20.2+/-7.06%) than FABP.     

Oxidative modification of human low-density lipoprotein by horseradish peroxidase in the absence of hydrogen peroxide

Heme-peroxidases, such as horseradish peroxidase (HRP), are among the most popular catalysts of low density lipoprotein (LDL) peroxidation. In this model system, a suitable oxidant such as H₂O₂ is required to generate the hypervalent iron species able to initiate the peroxidative chain. However, we observed that traces of hydroperoxides present in a fresh solution of linoleic acid can promote lipid peroxidation and apo B oxidation, substituting H₂O₂.

Spectral analysis of HRP showed that an hypervalent iron is generated in the presence of H₂O₂ and peroxidizing linoleic acid. Accordingly, careful reduction of the traces of linoleic acid lipid hydroperoxide prevented formation of the ferryl species in HRP and lipid peroxidation. However, when LDL was oxidized in the presence of HRP, the ferryl form of HRP was not detectable, suggesting a Fenton-like reaction as an alternative mechanism. This was supported by the observation that carbon monoxide, a ligand for the ferrous HRP, completely inhibited peroxidation of LDL.

These results are in agreement with previous studies showing that myoglobin ferryl species is not produced in the presence of phospholipid hydroperoxides, and emphasize the relevance of a Fenton-like chemistry in peroxidation of LDL and indirectly, the role of pre-existing lipid hydroperoxides.     

Site-specific induction of lipid peroxidation by iron in charged micelles

Generation of hydroxyl radicals by the Fenton reaction resulted in lipid peroxidation of linoleic acid (LA) (H2O2-Fe2+-induced lipid peroxidation) in positively charged tetradecyltrimethylammonium bromide (TTAB) micelles, but not in negatively charged sodium dodecyl sulfate (SDS) micelles. However, more OH radicals formed via the Fenton reaction were trapped by N-t-butyl-alpha-phenylnitrone (PBN) in SDS micelles than in TTAB micelles. When detergent-dispersed LA was contaminated with linoleic acid hydroperoxide (LOOH), lipid peroxidation was catalyzed by Fe2+ via reductive cleavage of LOOH (LOOH-Fe2+-induced lipid peroxidation), and Fe2+ was oxidized simultaneously in SDS micelles, even when H2O2 was not present. In contrast, LOOH-Fe2+-induced lipid peroxidation and simultaneous oxidation of Fe2+ were not observed in TTAB micelles. An ESR spectrum presumed to be due to an alkoxy radical trapped by PBN was also detected in SDS micelles, but not in TTAB micelles in the LOOH-Fe2+-induced lipid peroxidation system. The results are discussed in the light of the localization of iron, the unsaturated bonding moiety of LA, the OOH-group of LOOH, and the trapping site of PBN in different charged micelles.     

Formation of acyl radical in lipid peroxidation of linoleic acid by manganese-dependent peroxidase from Ceriporiopsis subvermispora and Bjerkandera adusta.

Lipid peroxidation by managanese peroxidase (MnP) is reported to decompose recalcitrant polycyclic aromatic hydrocabon (PAH) and nonphenolic lignin models. To elucidate the oxidative process, linoleic acid and 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid [13(S)-HPODE] were reacted with MnPs from Ceriporiopsis subvermispora and Bjerkandera adusta and the free radicals produced were analyzed by ESR. When the MnPs were reacted with 13(S)-HPODE in the presence of Mn(II), H2O2 and tert-nitrosobutane (t-NB), the ESR spectrum contained a sharp triplet of acyl radical (aN = 0.81 mT). Formation of acyl radical was also observed in the reactions of Mn(III)-tartrate with 13(S)-HPODE and with linoleic acid, but the latter reaction occurred explosively after an induction period of around 30 min. Reactions of MnP with linoleic acid in the presence of Mn(II), H2O2 and t-NB gave no spin adducts while addition of t-NB after preincubation of linoleic acid with MnP/Mn(II)/H2O2 for 2 h gave spin adducts of carbon-centered (aN = 1.53 mT, aH = 0.21 mT) and acyl (aN = 0.81 mT) radicals. In contrast to linoleic acid, methyl linoleate and oleic acid were not peroxidized by MnP and chelated Mn(III) within a few hours, indicating that structures containing both the 1,4-pentadienyl moiety and a free carboxyl group are necessary for inducing the peroxidation in a short reaction time. These results indicate that MnP-dependent lipid peroxidation is not initiated by direct abstraction of hydrogen from the bis-allylic position during turnover but proceeds by a Mn(III)-dependent hydrogen abstraction from enols and subsequent propagation reactions involving the formation of acyl radical from lipid hydroperoxide. This finding expands the role of chelated Mn(III) from a phenol oxidant to a strong generator of free radicals from lipids and lipid hydroperoxides in lignin biodegradation.     

Effects of reactive oxygen species on sperm function

Reactive oxygen species (ROS) formation and membrane lipid peroxidation have been recognized as problems for sperm survival and fertility. The precise roles and detection of superoxide (SO), hydrogen peroxide (HP), and membrane lipid peroxidation have been problematic, because of the low specificity and sensitivity of the established chemiluminescence assay technologies. We developed flow cytometric assays to measure SO, HP, membrane lipid peroxidation, and inner mitochondrial transmembrane potential in boar sperm. These methods were sufficiently sensitive to permit detection of early changes in ROS formation in sperm cells that were still viable. Basal ROS formation and membrane lipid peroxidation in the absence of ROS generators were low in viable sperm of both fresh and frozen-thawed boar semen, affecting less than 4% of the sperm cells on average. However, this is not the case in other species, as human, bovine, and poultry sperm have large increases in sperm ROS formation, lipid peroxidation, loss of motility, and death in vitro. Closer study of the effects of ROS formation on the relationship between sperm motility and ATP content in boar sperm was conducted using menadione (mitochondrial SO generator) and HP treatment. Menadione or HP caused an immediate disruption of motility with delayed or no decrease in sperm ATP content, respectively. Overall, the inhibitory effects of ROS on motility point to a mitochondrial-independent mechanism. The reduction in motility may have been due to a ROS-induced lesion in ATP utilization or in the contractile apparatus of the flagellum.     

Antioxidant Activities of Aqueous Extract from Cultivated Fruit-bodies of Cordyceps militaris （L.） Link In Vitro

Biological antloxldants extracted from plants and fungi have potential abilities to scavenge free radicals and Inhibit lipid peroxldatlon, playing Important roles in preventing diseases, for example, cancer, and aging Induced by reactive oxygen species, which may cause oxidative damage to DNA, proteins and other macromolecules. The antloxldant potency of cultivated fruit-bodies of Cordyceps militarls （L.） Link was investigated In this study. Five established In vitro systems were employed, including the 1,1-dlphenyl-2- plcryldrazyl （DPPH） free radical scavenging, hydroxyl radical eliminating, iron chelating, Inhibition of Ilnolelc acid lipid peroxldatlon and reducing power. The aqueous extract from cultivated fruit-bodies was subjected to the test of amino acid, polysaccharlde and mannitol. Ascorblc acid （Vc）, butylated hydroxytoluene （BHT） and ethylenedlamlnetetraacetlc acid （EDTA） were used as positive controls for comparisons. Among the assays, the aqueous extract of C. mllltarls frult-bodles shows a significant scavenging effect on DPPH, eliminating the capability on hydroxyl radicals and the chelating effect on ferrous Iron. The extract also shows positive results of Inhibiting Ilnoleic acid lipid peroxldatlon and reducing power.     

Conjugated linoleic acid induces lipid peroxidation in humans

Conjugated linoleic acid (CLA) is shown to have chemoprotective properties in various experimental cancer models. CLA is easily oxidised and it has been suggested that an increased lipid oxidation may contribute to the antitumorigenic effects. This report investigates the urinary levels of 8-iso-PGF(2alpha), a major isoprostane and 15-keto-dihydro-PGF(2alpha), a major metabolite of PGF(2alpha), as indicators of non-enzymatic and enzymatic lipid peroxidation after dietary supplementation of CLA in healthy human subjects for 3 months. A significant increase of both 8-iso-PGF(2alpha) and 15-keto-dihydro-PGF(2alpha) in urine was observed after 3 months of daily CLA intake (4.2 g/day) as compared to the control group (P<0.0001). Conjugated linoleic acid had no effect on the serum alpha-tocopherol levels. However, gamma-tocopherol levels in the serum increased significantly (P=0. 015) in the CLA-treated group. Thus, CLA may induce both non-enzymatic and enzymatic lipid peroxidation in vivo. Further studies of the mechanism behind, and the possible consequences of, the increased lipid peroxidation after CLA supplementation are urgently needed.     

Increased non-protein bound iron in Down syndrome: contribution to lipid peroxidation and cognitive decline

Down syndrome (DS, trisomy 21) is the leading cause of chromosomal-related intellectual disability. At an early age, adults with DS develop with the neuropathological hallmarks of Alzheimer’s disease, associated with a chronic oxidative stress. To investigate if non-protein bound iron (NPBI) can contribute to building up a pro-oxidative microenvironment, we evaluated NPBI in both plasma and erythrocytes from DS and age-matched controls, together with in vivo markers of lipid peroxidation (F₂-isoprostanes, F₂-dihomo-isoprostanes, F₄-neuroprostanes) and in vitro reactive oxygen species (ROS) formation in erythrocytes. The serum iron panel and uric acid were also measured. Second, we explored possible correlation between NPBI, lipid peroxidation and cognitive performance. Here, we report NPBI increase in DS, which correlates with increased serum ferritin and uric acid. High levels of lipid peroxidation markers and intraerythrocyte ROS formations were also reported. Furthermore, the scores of Raven’s Colored Progressive Matrices (RCPM) test, performed as a measure of current cognitive function, are inversely related to NPBI, serum uric acid, and ferritin. Likewise, ROS production, F₂-isoprostanes, and F₄-neuroprostanes were also inversely related to cognitive performance, whereas serum transferrin positively correlated to RCPM scores. Our data reveal that increased availability of free redox-active iron, associated with enhanced lipid peroxidation, may be involved in neurodegeneration and cognitive decline in DS. In this respect, we propose chelation therapy as a potential preventive/therapeutic tool in DS.     

Characterization of the initial carbon-centered pentadienyl radical and subsequent radicals in lipid peroxidation: identification via on-line high performance liquid chromatography/electron spin resonance and mass spectrometry

The previously reported combination of an on-line high-performance liquid chromatography (LC)/electron spin resonance (ESR) system with mass spectrometric analysis (MS) created a unique technique to identify a variety of lipid-derived radicals ((.)L(d)) formed from in vitro lipid peroxidation (Iwahashi et al. [20]). To improve the sensitivity, resolution, and reliability of this method for in vitro and in vivo studies, we have investigated the effects of mobile phase pH, modifiers, and columns on the chromatographic separation of linoleic acid-derived radical adducts. Using tetrahydrofuran (THF) and 0.1% glacial acetic acid (HOAc) in an H(2)O/acetonitrile (ACN) mobile phase greatly increased the resolution and retention reproducibility of lipid radical adducts in LC/ESR. In addition, these modifications allowed the elimination of an ESR tuning problem and the synchronization of UV and ESR detection of radical adducts in on-line LC/ESR, neither of which had been possible previously. Analyte purity was therefore increased, thus increasing the reliability of radical detection via on-line LC/ESR as well as radical identification via MS analysis. For the first time, POBN adducts of linoleic carbon-centered pentadienyl radicals (L(.)) were detected and identified. The optimization of chromatography in the LC/ESR and MS combination provided a reliable and sensitive way for the detection and identification of expected radical adducts in vitro and in vivo.     

Seminal plasma reduces exogenous oxidative damage to human sperm, determined by the measurement of DNA strand breaks and lipid peroxidation

Exposure of spermatozoa to reactive oxygen species (ROS) has been associated with cellular injury, that includes DNA damage and lipid peroxidation. In addition, sperm preparation techniques such as centrifugation, commonly used prior to in vitro fertilization and scientific studies, are associated with the generation of ROS and an increase in the level of DNA damage. The preservation, therefore, of sperm in vitro that might decrease the potential for oxidative DNA damage to arise and allow for an improvement in semen quality used for artificial insemination, is of importance. Seminal plasma is a rich source of antioxidants, which, potentially, safeguards sperm from oxidative attack during storage and once ejaculated. We have investigated the protection of human spermatozoa from ROS afforded by seminal plasma. Sperm were exposed to exogenous ROS by incubating the cells with hydrogen peroxide in the presence of ferrous sulfate and ADP. Aliquots of seminal plasma were added to the incubation mixture in differing amounts, and the generation of DNA strand breaks and thiobarbituric acid reactive species (TBARS), indicative of lipid peroxidation, determined. Incubation of sperm with exogenous ROS resulted in a significant generation of DNA strand breaks and lipid peroxidation compared to basal levels of damage (P<0.05). Addition of seminal plasma to the incubation media produced a significant decrease in DNA strand breaks and TBARS (P<0. 05), when the amount of plasma added exceeded 60% v/v. The results indicate that spermatozoal oxidative damage induced by exogenous ROS, specifically DNA damage and lipid peroxidation, is reduced by the presence of seminal plasma.     

In vitro prooxidant effect of caffeine

Caffeine is a methylxanthine compound that acts as a stimulant in humans. It is the most widely consumed behaviourally active substance in the western world and it affects calcium influx and release in living cells. Caffeine is present in various substances such as tea, coffee and some medications. This article is focused on the impact of caffeine on oxidation. Caffeine promoted the peroxidation of linoleic acid emulsions used by 32.5, 48.9, and 54.3%, respectively, at 15, 30 and 45 μg/mL concentrations. Standard antioxidants such as α-tocopherol and trolox, a water-soluble analogue of tocopherol, inhibited 76.2 and 93.2% peroxidation of linoleic acid emulsion at 45 μg/mL concentration. Also, PC₅₀ value (caffeine concentration that produced a 50% increase in linoleic acid peroxidation) of caffeine was found to be 185 μM.     

Measurement of the diene conjugated form of linoleic acid in plasma by high performance liquid chromatography: a questionable non-invasive assay of free radical activity?

It has been previously reported that the main diene-conjugated fatty acid in human plasma is a non-oxygen containing linoleic acid isomer (PL-9, 11-LA'). It has also been proposed that this isomer can be used as a specific marker of free radical-mediated lipid peroxidation in humans. Here we report that the in vitro induction of lipid peroxidation in human and rat blood with either UV irradiation or phenylhydrazine failed to increase the plasma levels of this isomer. The induction of lipid peroxidation in vivo in rats pretreated with either phenylhydrazine or bromotrichloromethane also failed to increase the plasma levels of this isomer. These findings demonstrate that PL-9, 11-LA' cannot be used as an in vivo marker of free radical-mediated lipid peroxidation in rats and casts doubts on its validity as a specific marker in humans.     

Peroxyl radical trapping activity of anthocyanins and generation of free radical intermediates

The inhibition by anthocyanins of the free radical-mediated peroxidation of linoleic acid in a SDS micelle system was studied at pH 7.4 and at 37°C, by oxygraphic and ESR tecniques. The number of peroxyl radicals trapped by anthocyanins and the efficiency of these molecules in the trapping reaction, which are two fundamental aspects of the antioxidant action, were measured and discussed in the light of the molecular structure. In particular the contribution of the substituents to the efficiency is –OH>–OCH₃>–H. By ESR we found that the free radicals of anthocyanins are generated in the inhibition of the peroxidation of linoleic acid. The life time of these radical intermediates, the concentration of which ranges from 7 to 59 nM under our experimental conditions, is strictly correlated with the anthocyanin efficiency and with the heat of formation of the radical, as calculated by a semiempirical molecular orbital approach.     

Lipid Peroxidation by the Manganese Peroxidase of Phanerochaete chrysosporium Is the Basis for Phenanthrene Oxidation by the Intact Fungus

The manganese peroxidase (MnP) of Phanerochaete chrysosporium supported Mn(II)-dependent, H₂O₂-independent lipid peroxidation, as shown by two findings: linolenic acid was peroxidized to give products that reacted with thiobarbituric acid, and linoleic acid was peroxidized to give hexanal. MnP also supported the slow oxidation of phenanthrene to 2,2′-diphenic acid in a reaction that required Mn(II), oxygen, and unsaturated lipids. Phenanthrene oxidation to diphenic acid by intact cultures of P. chrysosporium occurred to the same extent that oxidation in vitro did and was stimulated by Mn. These results support a role for MnP-mediated lipid peroxidation in phenanthrene oxidation by P. chrysosporium.     

Identification of spin trapped carbon-centered radicals in soybean lipoxygenase-dependent peroxidations of omega-3 polyunsaturated fatty acids by LC/ESR,LC/MS,and tandem MS

With the combined techniques of on-line liquid chromatography/electron spin resonance (LC/ESR) and on-line liquid chromatography/mass spectrometry (LC/MS), we have previously characterized all classes of lipid-derived carbon-centered radicals (*Ld) formed from omega-6 polyunsaturated fatty acids (PUFAs: linoleic acid and arachidonic acid). In the present study, the carbon-centered radicals formed from two omega-3 PUFAs (linolenic acid and docosahexaenoic acid) resulting from their reactions with soybean lipoxygenase in the presence of alpha-[4-pyridyl 1-oxide]-N-tert-butylnitrone (POBN) were investigated using the combination of LC/ESR and LC/MS techniques. A total of 16 POBN trapped carbon-centered radicals formed from the peroxidation of linolenic acid and 11 formed from the peroxidation of docosahexaenoic acid were detected by LC/ESR, identified by LC/MS, and structurally confirmed by tandem mass analysis (MS/MS). The on-line ESR chromatograms and MS chromatograms obtained from two omega-3 PUFAs closely resembled each other not only because the four major beta-scission products, including an ethyl radical and three isomeric pentenyl radicals, were formed from each PUFA, but also because isomeric POBN adducts of lipid dihydroxyallylic radicals from both PUFAs had almost identical chromatographic retention times.     

Species differences in membrane susceptibility to lipid peroxidation

The susceptibility of liver microsomes to lipid peroxidation was evaluated in seven species: rat, rabbit, trout, mouse, pig, cow, and horse. Lipid peroxidation was measured as thiobarbituric acid reactive substances formed in the presence of either FeCl₃-ADP/ascorbate or FeCl₂/H₂O₂ initiating systems. For rat, rabbit, and trout microsomes, the order of susceptibility to peroxidation was rat > rabbit >> trout. The lack of peroxidation in trout microsomes could be explained by high microsomal vitamin E levels. Membrane fatty acid levels differed between species. Docosahexaenoic acid predominated in the trout, arachidonic acid in the rat, and linoleic acid in the rabbit. The contribution of individual fatty acids to lipid peroxidation reflected the degree of unsaturation with docosahexaenoic > arachidonic >>> linoleic. For all species except trout, the predicted susceptibility to peroxidation, based on the response of individual fatty acids, agreed well with directly measured microsomal peroxidation. With the exception of the trout, vitamin E content ranged from 0.083–0.311 nmol/mg microsomal protein between species, and low levels did not influence susceptibility to peroxidation. Trout microsomes peroxidized only after vitamin E depletion by prolonged incubation. The data indicate that below a vitamin E threshold, species differences in membrane susceptibility to peroxidation can be reasonably predicted based only on content of individual peroxidizable fatty acids.     

Alteration of the Redox State with Reactive Oxygen Species for 5-Fluorouracil-Induced Oral Mucositis in Hamsters

Oral mucositis is often induced in patients receiving cancer chemotherapy treatment. It has been reported that oral mucositis can reduce quality of life, as well as increasing the incidence of mortality. The participation of reactive oxygen species (ROS) in the pathogenesis of oral mucositis is well known, but no report has actually demonstrated the presence of ROS. Thus, the purpose of this study was thus to demonstrate the involvement of ROS and the alteration of the redox state in oral mucositis using an in vivo L-band electron spin resonance (ESR) technique. An oral mucositis animal model induced by treatment of 5-fluorouracil with 10% acetic acid in hamster cheek pouch was used. Lipid peroxidation was measured as the level of malondialdehyde determined by the thiobarbituric acid reaction. The rate constants of the signal decay of nitroxyl compounds using in vivo L-band ESR were calculated from the signal decay curves. Firstly, we established the oral mucositis animal model induced by treatment of 5-fluorouracil with acetic acid in hamster cheek pouch. An increased level of lipid peroxidation in oral mucositis was found by measuring malondialdehyde using isolated hamster cheek pouch ulcer. In addition, as a result of in vivo L-band ESR measurements using our model animals, the decay rate constants of carbamoyl-PROXYL, which is a reagent for detecting the redox balance in tissue, were decreased. These results suggest that a redox imbalance might occur by excessive generation of ROS at an early stage of oral mucositis and the consumption of large quantities of antioxidants including glutathione in the locality of oral mucositis. These findings support the presence of ROS involved in the pathogenesis of oral mucositis with anti-cancer therapy, and is useful for the development of novel therapies drugs for oral mucositis.     

Overexpression of Manganese Superoxide Dismutase Promotes the Survival of Prostate Cancer Cells Exposed to Hyperthermia

It has been hypothesized that exposure of cells to hyperthermia results in an increased flux of reactive oxygen species (ROS), primarily superoxide anion radicals, and that increasing antioxidant enzyme levels will result in protection of cells from the toxicity of these ROS. In this study, the prostate cancer cell line, PC-3, and its manganese superoxide dismutase (MnSOD)-overexpressing clones were subjected to hyperthermia (43°C, 1?h). Increased expression of MnSOD increased the mitochondrial membrane potential (MMP). Hyperthermic exposure of PC-3 cells resulted in increased ROS production, as determined by aconitase inactivation, lipid peroxidation, and H₂O₂ formation with a reduction in cell survival. In contrast, PC-3 cells overexpressing MnSOD had less ROS production, less lipid peroxidation, and greater cell survival compared to PC-3 Wt cells. Since MnSOD removes superoxide, these results suggest that superoxide free radical or its reaction products are responsible for part of the cytotoxicity associated with hyperthermia and that MnSOD can reduce cellular injury and thereby enhance heat tolerance.     

Protective effects of ellagic and chlorogenic acids against oxidative stress in PC12 cells

Following exposure of differentiated neuronal PC12 cells to either t-BHP, hydrogen peroxide (H₂O₂) or FeSO₄ various kinds of reactive oxygen species (ROS) are generated leading to oxidative injury. The protective effects of two plant polyphenols, ellagic (EC) and chlorogenic acid (CGA), as well as of two metabolites, caffeic acid (CA) and ferulic acid (FA), were investigated in preincubation and coincubation experiments with respect to the following parameters: prevention of cell death, GSH depletion, lipid peroxidation and ROS formation.

The polyphenols more efficiently suppressed cytotoxicity and loss of GSH caused by peroxides than by iron, particularly in preincubation. Lipid peroxidation which increased much stronger in response to FeSO₄ was counteracted completely by the polyphenols. In case of iron, however, only coincubation was effective. EA and CGA and the metabolites CA and FA showed excellent elimination of ROS induced by all stressors. These findings suggest that two dietary antioxidants, EA and CGA, may have protective properties against oxidative stress induced in CNS.