Lipid Radical Content Research Articles

Possible role of seasonality and harmful algal blooms (HAB) on the oxidative and nitrosative metabolisms in hemocytes

Bivalves survive to biotoxin consumption but their metabolism could be affected. The objective of this work was to study the oxidative and nitrosative changes in the hemocytes of the mussel Mytilus edulis platensis in different seasons, including spring, characterized by the appearance of harmful algal blooms (HAB). Reactive species generation rate (measured as the 2′,7′dichlorofluorescein diacetate oxidation rate) was increased by 2.5- and 8.3-fold in hemocytes from spring and summer, respectively, as compared to winter samples. Neither total Fe nor labile Fe pool content was changed in the three seasons. Superoxide anion generation rate was 3-fold higher in spring as compared to winter and summer samples. Catalase content in spring cells were significantly higher as compared to winter (60%) and summer (3-fold increase) but glutathione-S-transferase activity only increased compared to summer season (125% increase). Lipid radical content in spring samples was 140 and 50% higher as compared to cells from winter and summer, respectively. Nitric oxide and nitro-tyrosine content were significantly higher in samples from spring as compared to values obtained either in winter or summer cells. Considering the aspects that influence metabolism, changes in temperature seem to mainly affect the oxidative over the nitrosative condition of the hemocytes. Nevertheless, HAB biotoxins seem as a contributing factor to affect not only reactive oxygen species generation, antioxidant activity and protein/lipid damage, but also the nitrosative metabolism. In this regard, the changes in the nitric oxide content are new and critical evidence that HAB-related toxins could affect reactive nitrogen species metabolism.

Main features of the oxidative metabolism in gills and liver of Odontesthes nigricans Richardson (Pisces, Atherinopsidae)

The aim of this work was to study comparatively the oxidative metabolism in gills and liver of a silverside, Odontesthes nigricans, in their natural environment, the Beagle Channel. Oxidative damage to lipids was evaluated by assessing TBARS and lipid radical content, in gills and liver. Gills showed a significantly higher degree of damage than liver. The content of alpha-tocopherol, beta-carotene and catalase activity showed significantly higher values in the liver than in the gills. The ascorbyl radical (A(*)) content showed no significant differences between gills and liver. The ascorbate (AH(-)) content was 12+/-2 and 159+/-28 nmol/mg FW in gills and liver, respectively. Oxidative metabolism at the hydrophilic level was assessed as the ratio A(*)/AH(-). The ratio A(*)/AH(-) was significantly different between organs, (6+/-2)10(-5) and (5+/-2)10(-6), for the gills and the liver, respectively. Both, lipid radical content/alpha-tocopherol content and lipid radical content/beta-carotene content ratios were significantly higher in gills as compared to the values recorded for the liver, suggesting an increased situation of oxidative stress condition in the lipid phase of the gills. Taken as a whole, the O. nigricans liver exhibited a better control of oxidative damage than the gills, allowing minimization of intracellular damage when exposed to environmental stressing conditions.

The effect of seasonality on oxidative metabolism in the sea urchin Loxechinus albus

The objective of the present study was to investigate seasonal variations in the oxidative metabolism of the sea urchin Loxechinus albus gonads. The reported spawning period for this species is from September to November. Lipid radical content showed non-significant changes upon the seasons. Ascorbyl radical content and the content of α-tocopherol were lower in samples collected in spring and summer as compared to the values in winter-collected animals. Ascorbate content decreased in samples collected in fall as compared to those collected in winter. For the lipophilic compartment, the lipid radical content/α-tocopherol content ratio is an indicator of oxidative stress. This index increased significantly in tissues during spawning as compared to the values in samples collected during winter. The ascorbyl content/ascorbate content ratio is an indicator of oxidative stress for the hydrophilic milieu. A significant decrease by 66% was determined in tissues from gonads of animals collected in summer as compared to values in animals collected in winter. The data reported here suggest a different profile of response against oxidative stress at the lipophilic and hydrophilic milieus in L. albus gonads.

Role of nitric oxide in soybean cotyledon senescence

Senescence was studied in soybean ( Glycine max, var ALM 4500) cotyledons from 10- and 25-day-old seedlings. Rejuvenated cotyledons, obtained by removing epicotyls from 5-day seedlings, showed a lower decrease in the content of photosynthetic pigments from day 10 to day 25, as compared to naturally senescent cotyledons. Unlike controls, rejuvenated cotyledons showed no changes in electrolyte leakage, chlorophyll a, and carotene content during the studied period. At day 10, nitric oxide (NO) content in rejuvenated cotyledons was higher than in naturally senescent cotyledons. The role of NO in senescence was assessed by exposing seedlings to sodium nitroprusside (SNP). SNP-treated cotyledons showed a constant NO content over the studied period of 4.0 ± 0.4 nmol g −1 FW, conserved photosynthetic pigment content and lower lipid radical content, as compared to naturally senescent cotyledons. These results suggest that NO can protect against senescence-dependent alterations in cotyledons, because (i) exogenous NO can partially protect naturally senescent cotyledons and (ii) rejuvenated cotyledons, which do not show the senescent phenotype, have higher endogenous levels of NO.

Iron and radical content in Mya arenaria: Possible sources of NO generation

The objective of this work was to analyze oxidative metabolism in Mya arenaria. Total Fe content in M. arenaria collected in the German Wadden Sea was 1.9 ± 0.7, 0.7 ± 0.1 and 0.17 ± 0.01 nmol/mg fresh weight (FW), in digestive glands (DG), mantle and gills, respectively. Labile Fe pool, assessed by electronic paramagnetic resonance (EPR), was 146 ± 10 pmol/mg FW, and by the fluorescence method employing calcein it was 118 ± 9 pmol/mg FW. The lipid radical content in the DG, assessed by EPR, was 27 ± 7 pmol/mg FW, and the thiobarbituric reactive substances content amounted to 57 ± 8 pmol/mg FW. Ascorbyl radical (A ) content, assessed by quantification of EPR signals, was 0.04 ± 0.01 pmol/mg FW, and the ascorbate content (AH −) was 478 ± 12 pmol/mg FW. The ratio A /AH − was (8 ± 1) × 10 −5 AU, suggesting a minimum oxidative stress even under physiological conditions, presumably depending on basal metabolic functions. The content of nitric oxide (NO), assessed by EPR, was 99 ± 3 pmol/mg FW. The generation rate of NO by nitric oxide synthase-like activity (NOS-like) was assayed as NO production detected by EPR in the presence of l-arginine and NADPH, and was 3.16 ± 0.06 pmol/(mg FW min). The data presented here document the detectable presence of highly reactive species in M. arenaria.

Reactive Nitrogen Species-Dependent Effects on Soybean Chloroplasts

Nitric oxide (NO) generation by soybean (Glycine max, var ADM 4800) chloroplasts was studied by electron paramagnetic resonance (EPR) spin-trapping technique1. Both nitrite and L-arginine (arg) are the required substrates for enzymatic activities considered as possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 ± 0.2 nmol min-1 mg-1 protein in the presence of 1 mM NaNO2. Chloroplasts incubated with 1 mM arg showed a NO production of 0.76 ± 0.04 nmol min-1 mg-1 protein. This production was inhibited when chloroplasts were incubated in presence of NOS-inhibitors L-NAME and L-NNA. In vitro exposure of chloroplasts to a NO-donor (GSNO) decreased both ascorbyl radical content and the activity of ascorbate peroxidase, without modification of the total ascorbate content. Exposure of the isolated chloroplasts to a NO-donor decreased lipid radical content in membranes, however, incubation in the presence of 25 μM peroxynitrite (ONOO-) led to an increase in lipid-derived radicals (34%). The effect of ONOO- on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to control. Taken as a whole, NO seems to be an endogenous metabolite in soybean chloroplasts and reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplasts, since both a decreased lipid radical content in membranes and a decrease in the activity of ascorbate peroxidase were observed after exposure to a NO donor.

Chloroplasts as a nitric oxide cellular source. Effect of reactive nitrogen species on chloroplastic lipids and proteins.

Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and l-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 +/- 0.2 nmol min(-1) mg(-1) protein in the presence of 1 mm NaNO(2). Inhibition of photosynthetic electron flow by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 +/- 0.04 nmol min(-1) mg(-1) protein) of NO generation. Chloroplasts incubated with 1 mm Arg showed NO production of 0.76 +/- 0.04 nmol min(-1) mg(-1) protein that was not affected either by omission of Ca(2+) or by supplementation with Ca(2+) and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors N(omega)-nitro-l-Arg methyl ester hydrochloride and N(omega)-nitro-l-Arg. In vitro exposure of chloroplasts to an NO donor (250 mum S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 mum peroxynitrite (ONOO(-)) led to an increase in lipid-derived radicals (34%). The effect of ONOO(-) on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO(-) treatment significantly affected both O(2) evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules.

The effect of seasonality on oxidative metabolism in Nacella (Patinigera) magellanica

We studied the seasonal variation on aerobic metabolism and the response of oxidative stress parameters in the digestive glands of the subpolar limpet Nacella (P.) magellanica. Sampling was carried out from July (winter) 2002 to July 2003 in Beagle Channel, Tierra del Fuego, Argentina. Whole animal respiration rates increased in early spring as the animals spawned and remained elevated throughout summer and fall (winter: 0.09+/-0.02 micromol O2 h-1 g-1; summer: 0.31+/-0.06 micromol O2 h-1 g-1). Oxidative stress was assessed at the hydrophilic level as the ascorbyl radical content/ascorbate content ratio (A./AH-). The A./AH- ratio showed minimum values in winter (3.7+/-0.2 10(-5)AU) and increased in summer (18+/-5 10(-5) AU). A similar pattern was observed for lipid radical content (122+/-29 pmol mg-1 fresh mass [FW] in winter and 314+/-45 pmol mg-1 FW in summer), iron content (0.99+/-0.07 and 2.7+/-0.6 nmol mg-1 FW in winter and summer, respectively) and catalase activity (2.9+/-0.2 and 7+/-1 U mg-1 FW in winter and summer, respectively). Since nitrogen derived radicals are thought to be critically involved in oxidative metabolism in cells, nitric oxide content was measured and a significant difference in the content of the Fe-MGD-NO adduct in digestive glands from winter and summer animals was observed. Together, the data indicate that both oxygen and nitrogen radical generation rates in N. (P.) magellanica are strongly dependent on season.

Oxidative stress in gills of limpets from the Beagle Channel: comparison with limpets from the Antarctic

The aim of this work was to study the oxidative profile of gills of two limpet species ( Nacella (Patinigera) magellanica and Nacella (Patinigera) deaurata ) (Gmelin, 1971) exposed to different environmental conditions. Due to the tidal characteristics of the Beagle Channel, N. magellanica are exposed to air twice daily for 3 to 5 hours each time, whereas N. deaurata are exposed to air for 3 hours only during spring tides. The different regime of exposure includes extreme temperatures under 0oC during winter and more than 20°C in summer for N. magellanica , whereas N. deaurata are usually covered by more than 0.3 m of water at 4°C in winter and 11°C in summer. No significant differences were found between the two molluscs regarding the oxygen uptake, the content of ?-tocopherol and ?-carotene and the activities of the antioxidant enzymes catalase and superoxide dismutase. Lipid peroxidation in gills was estimated as the content of lipid radicals, assessed by electron paramagnetic resonance (EPR). Lipid radical content and total iron content were respectively 80.6 and 62% lower in N. magellanica than in N. deaurata . A typical EPR spectrum of ascorbyl radical in gills from both limpets was observed. Both the ascorbyl radical content and the ascorbyl radical content/ascorbate content ratio were significantly lower in N. magellanica than in N. deaurata . In the Antarctic Nacella concinna inhabits all levels of the littoral zone. Limpets at the highest level in the intertidal showed significantly increased activities of both catalase and superoxide dismutase as compared to their intertidal and subtidal relatives. Thus, it seems that Antarctic high intertidal conditions, involving regular exposure to air and presumably also thermal stress on sunny days during the Antarctic summer, cause a necessity for N. concinna to ward off higher oxygen radical species production by increasing its antioxidant defence. Taken as a whole, the data presented here indicate that coping with environmentally demanding conditions requires a complex adjustment of the physiological metabolic pathways to ensure survival by minimising intracellular damage.

Oxidative stress in limpets exposed to different environmental conditions in the Beagle Channel

The aim of this work was to study the oxidative profile of digestive glands of two limpets species ( Nacella ( Patinigera) magellanica and Nacella ( Patinigera) deaurata) exposed to different environmental conditions. The intertidal population of N. ( P.) magellanica is subjected to a wide variety of stresses not experienced by N. ( P.) deaurata. Although a typical electron paramagnetic resonance (EPR) spectrum of ascorbyl radical in digestive gland from both limpets was observed, neither ascorbyl radical content nor the ascorbyl radical content/ascorbate content ratio was significantly different, suggesting that the difference in the environmental conditions did not appear to be responsible for developing alterations in the oxidative status of both organisms at the hydrophilic level (e.g. cytosol). Lipid peroxidation in the digestive glands was estimated, both as the content of thiobarbituric acid reactive substances (TBARS) and as the content of lipid radicals assessed by EPR, in both organisms. TBARS and lipid radical content were 34.8 and 36.5%, respectively, lower in N. ( P.) magellanica as compared to N. ( P.) deaurata. On the other hand, total iron content and the rate of generation of superoxide anion were 47.9 and 51.4%, respectively, lower in N. ( P.) magellanica as compared to N. ( P.) deaurata. The activity of catalase and superoxide dismutase (SOD) was 35.3 and 128.6% higher in N. ( P.) magellanica as compared to N. ( P.) deaurata, respectively. No significant differences were determined between the digestive glands of both molluscs regarding the content of total thiols. α-Tocopherol and β-carotene content were significantly lower in N. ( P.) magellanica as compared to N. ( P.) deaurata. A distinctive EPR signal for the adduct Fe–MGD–NO ( g = 2.03 and a N = 12.5 G) was detected in the homogenates of digestive glands of both limpets. A significant difference in the content of the Fe–MGD–NO adduct in digestive glands from N. ( P.) magellanica and N. ( P.) deaurata (491 ± 137 and 839 ± 63 pmol/g FW, respectively) was observed. Taken as a whole, the data presented here indicated that coping with environmental stressing conditions requires a complex adjustment of the physiological metabolic pathways to ensure survival by minimizing intracellular damage. It is likely that N. ( P.) magellanica has a particular evolutionary adaptation to extreme environmental conditions by keeping iron content low and antioxidant activities high.

Early Neuroprotective Effect of Nitric Oxide in Developing Rat Brain Irradiated in Utero

Pregnant Wistar rats were exposed on day 17 of gestation to 1 Gy γ irradiation from a Co 60 source. Even though it is established that γ radiation-dependent damage is mainly due to free radical generation neither the ascorbyl radical/ascorbate ratio nor the lipid radical content in developing rat brain were affected by prenatal irradiation. A distinctive EPR signal for the adduct NO–Fe–MGD ( g=2.03 and a N =12.5 G) was detected in brain homogenates prepared from irradiated rats. Nitric oxide (NO)-dependent EPR signal increased in a time-dependent manner up to 2 h post-irradiation. NO concentration in unirradiated brains was 37±4 pmol/g brain and 45±2, 77±5 and 216±6 after 30, 60 and 120 min post-irradiation, respectively. Total nitric oxide synthase activity was increased by 77 and 51% after 30 and 60 min post-irradiation, respectively, and returned to control values after 120 min. Thus, increased NO steady-state concentration could be ascribed to an increase in NOS activity. Taken as a whole, these results suggest that NO might act to protect the developing brain from the cytotoxicity of reactive species.

Lipid radical generation in polar ( Laternula elliptica) and temperate ( Mya arenaria) bivalves

Lipid peroxidation in Laternula elliptica was assessed by detecting lipid radicals by electronic paramagnetic resonance. The values were compared with data from the temperate mud clam Mya arenaria. Lipid radical content was higher in the Antarctic bivalve than in the temperate mud clam, even within the range of its habitat temperature. The rate of generation of lipid radicals was affected by the iron content in the samples. The iron content in individual samples of digestive glands in L. elliptica ranged from 3 to 6 nmol g −1 fresh weight (fwt) and in M. arenaria from 0.6 to 2.7 nmol g −1 fwt. Arrhenius plots, developed from the rates obtained in the presence of 25 μM iron, showed no significant differences between the activation energy calculated for digestive glands of L. elliptica and M. arenaria. The Fe 3+ reduction rate in L. elliptica was higher than in M. arenaria (4.7±0.9 vs. 1.8±0.4 nmol mg −1 protein min −1, respectively). L. elliptica had a higher content of α-tocopherol and β-carotene than M. arenaria. Our data suggest that increased lipid radical content in the membranes of cold-adapted organisms could be related to iron content.

Iron-dependent oxidative stress in Chlorella vulgaris

The role of iron as catalyst for oxidative stress was studied during the development of Chlorella vulgaris cells. Increases on iron availability beyond 200 μM led to a decrease in the growth rate of the cultures of C. vulgaris. Quantification of the EPR signals of POBN/lipid radicals adducts indicated that iron addition increased lipid radical content in the membranes (more than three-fold at 500 μM Fe). The ratio of ascorbyl radical content/ascorbate content was increased in cells exposed to 500 μM iron as compared to standard conditions (90 μM Fe). β-Carotene content was not affected by iron addition, however, α-tocopherol, ascorbate and total thiol content in C. vulgaris cells was increased when iron supply was higher than 90 μM. The morphology of the cells developed under 500 μM iron showed a decreased number of ribosomes and alterations in the cellular starch deposits as compared to the cells grown at 90 μM iron. Exposure to UV-B increased the content of lipid radicals and affected cellular internal structure as compared to non-irradiated cells developed at 500 μM iron. Even though results from model systems cannot be directly used to predict ecological effects, these data suggest that oxidative stress triggered by an excess of iron could affect cellular growth and have a negative impact to phytoplankton.

Drought and watering-dependent oxidative stress: effect on antioxidant content in Triticum aestivum L. leaves

The purpose of the present work was to evaluate both oxidative stress and the antioxidant response system in leaves from wheat (Triticum aestivum cv. Buck Poncho) subjected sequentially to drought and watering. Drought was imposed by withholding water until soil water potential reached −2.0 MPa and maintained under those conditions for 24 h. DCFDA oxidation by wheat leaves was not significantly affected by drought, but watering led to an approximately 2-fold increase in DCFDA oxidation rate. However, no significant effect either on lipid radical content or on hydroperoxide content was measured after drought and drought followed by watering. Microsomes isolated from leaves exposed to drought, and from leaves exposed to drought followed by watering, generated a significantly higher amount of hydroxyl radical as compared to microsomes isolated from control leaves, suggesting a higher production of hydroxyl radical in the cellular water-soluble phase, after drought and watering as compared to control values. The content of α-tocopherol in wheat leaves was increased 2.4-fold after drought and β-carotene content was increased by 2.6-fold after drought. Hydration lowered lipid-soluble antioxidant content to control values. Total thiol content was increased by 70% after drought, and watering did not significantly alter the enhanced values. Drought decreased by 28.5% the content of reduced ascorbic acid. Taken as a whole, active species formed at wheat membranes after exposure to moderate water stress, are efficiently removed upon rehydration by reaction with an increased content of α-tocopherol and β-carotene. Moreover, a co-ordinated response involving glutathione reductase activity, thiols and ascorbic acid is triggered to limit free radical dependent effects.

N-Acetylcysteine-dependent protection against UV-B damage in two photosynthetic organisms

Cellular thiols, specially glutathione, appear to play a key role in protection against oxidative damage arising from a number of stress conditions. Thus, the role of N-acetylcysteine (NAC) as a protector against oxidative damage associated with ultraviolet B (UV-B) in two photosynthetic organisms was evaluated. Algal cultures ( Chlorella vulgaris) and soybean leaves ( Glycine max var. Hood) were supplemented with 1 mM NAC before irradiation. The content of total thiols was significantly increased after treatment. After exposure to UV-B, the ascorbyl radical-dependent electron paramagnetic resonance (EPR) signal was enhanced by 76 and 46% and lipid radical-dependent EPR signal increased by 83 and 188% in algal cultures and soybean leaves, respectively. Treatment with 1 mM NAC kept ascorbyl and lipid radical content in algae and soybean leaves exposed to UV-B at the basal level. Supplementation with 1 mM NAC did not affect the content of lipid-soluble antioxidants (α-tocopherol, β-carotene) in C. vulgaris or in soybean leaves. UV-B irradiation increased catalase activity in the algae by 145% and in soybean leaves by 34%, while total superoxide dismutase (SOD) activity was not affected. In both photosynthetic organisms the increase in catalase activity after UV-B exposure did not occur in the presence of 1 mM NAC. The results suggest that thiols play an important role in triggering cellular control against UV-B-related damage, as NAC pretreatment significantly decreased UV-B-dependent radical generation after irradiation.

Free-radical scavenging actions of natural antioxidants

A dose-dependent inhibitory effect of wheat, alfalfa and ginkgo biloba (EGb) extracts on TBARS production was measured. The half-inhibition concentration (IC50) of the tested antioxidants were 2.7±0.2, 1.3±0.1, and 0.20±0.02 mg/ml for wheat, alfalfa and EGb extracts, respectively. Lipid radicals combined with the spin trap POBN resulted in adducts that gave a characteristic EPR spectrum. The IC50 of the tested antioxidants on lipid radical content, were 12.4±0.2, 7.7±0.3, and 1.20±0.06 mg/ml for wheat, alfalfa and EGb extracts, respectively. Rat liver microsomes in the presence of DMPO, NADPH and iron-citrate generate an EPR spectra with characteristics of the DMPO-OH spin adduct. The basic system, without the addition of any scavenger showed an area of 3.5 AU/mg protein. The areas in the presence of 1.5 mg/ml EGb, 4 mg/ml wheat or alfalfa, were of 1.7±0.2, 3.4±0.3, and 3.6±0.2 AU/mg protein, respectively. O 2 − generation rate by the microsomes exposed to EGb extract was decreased by 40%, as compared to the rate measured in microsomes incubated in the absence of the extract. However, the supplementation of rat liver microsomes with either wheat or alfalfa extracts did not affect microsomal generation of O 2 −. Iron reduction rate was not affected by the addition of any of the tested extracts. The data presented here showed that EGb extracts were able to limit lipid peroxidation and scavenge lipid radicals in rat liver microsomes more efficiently than alfalfa and wheat bran extracts. Moreover, wheat and alfalfa extracts were not able to inhibit O 2 − and ·OH generation by biological membranes, suggesting that their potentiality to be successfully used in human health in the treatment of diseases involving free radical and oxidative damage are not as promising as that for the use of EGb extracts.