Formation of isoprostane F(2)-like compounds (phytoprostanes F(1)) from alpha-linolenic acid in plants

Isoprostanes F(2) are biologically active prostaglandin F(2)-like compounds formed by free radical-catalyzed oxidation of arachidonic acid (C20:4). Here, we show that a series of dinor isoprostanes F(1), which we term phytoprostanes F(1) (PPF(1)s), are formed by nonenzymatic oxidation of linolenate (C18:3) in plants. Identification and quantification of PPF(1)s were achieved by a negative ion chemical ionization gas chromatography-mass spectrometry method using oxygen 18-labeled PPF(1)s as internal standards. PPF(1)s were found in leaves, flowers, and roots of taxonomically distinct plant species at concentrations ranging from 43 to 1380 ng/g of dry weight. In addition, esterified PPF(1)s were found at 10- to 150-fold higher concentrations. During the drying and storage of various plant organs, endogenous PPF(1) levels increased dramatically by 15- to 263-fold. Because the structurally related prostaglandin F(2alpha) and isoprostanes F(2) exert potent biological activities (i.e., broncho- and vasoconstriction) in the nanomolar range, PPF(1)s could potentially exert similar biological activities. Notably, fresh birch pollen, which can easily be inhaled, contains exceedingly high concentrations (32,440 ng/g) of free PPF(1)s.     

Changes in urinary dinor dihydro F(2)-isoprostane metabolite concentrations, a marker of oxidative stress, during and following asthma exacerbations

To investigate changes in oxidant stress during and following acute asthma exacerbations, this study measured 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP (F(2)-IsoP-M), the major urinary metabolite of 15-F(2t)-IsoP, in eight asthmatic adults, during and following an asthma hospitalization. F(2)-IsoP-M concentrations at admission and follow-up were significantly higher than discharge (admission median: 4.12 ng/Cr mg, range 1.89-7.8; follow-up: 2.47 ng/Cr mg (1.56-6.86); discharge: 1.42 ng/Cr mg (0.7-4.44); both p<0.01), but not significantly different between admission and follow-up. F(2)-IsoP-M concentrations at follow-up were higher than a control group with stable asthma (0.68 ng/Cr mg (0.31-1.5), p=0.0008). In conclusion, asthma exacerbations requiring hospitalization are associated with 6-fold higher urinary F(2)-IsoP-M concentrations compared to stable asthmatics. F(2)-IsoP-M concentrations decreased significantly during hospitalization, but significant elevations 3 months following hospitalization suggest ongoing oxidative stress despite clinical improvement. Urinary F(2)-IsoP-M may be a clinically useful, simple non-invasive systemic measure of oxidative stress in asthmatics, providing information not captured by spirometry or symptoms.     

Fatty acid oxidation and isoprostanes: Oxidative strain and oxidative stress

Oxidative stress is implicated as one of the key causes underlying many diseases. Free radicals are important constituents of basal physiology. Assessment of free radicals or the end products of their action has proved to be difficult. Consequently, authentication of the contribution of free radicals to physiology and pathology has usually been equivocal. Isoprostanes are biosynthesized in vivo, predominantly through free radical attack on arachidonic acid and are now regarded as robust biomarkers of oxidative stress in vivo. Isoprostanes are associated with many human diseases, and their concentration is altered over the course of normal human pregnancy, but their (patho)physiological roles have not yet been clearly defined. Measurement of F₂-isoprostanes in body fluids could offer a unique analytical opportunity to study the role of free radicals in physiology and pathophysiology in order to comprehend both oxidative strain and oxidative stress.     

Changes in urinary dinor dihydro F2-isoprostane metabolite concentrations,a marker of oxidative stress,during and following asthma exacerbations

To investigate changes in oxidant stress during and following acute asthma exacerbations, this stidy measured 2,3-dinor-5,6-dihydro-15-F_2t-IsoP (F₂-IsoP-M), the major urinary metabolite of 15-F_2t-IsoP, in eight asthmatic adults, during and following an asthma hospitalization. F₂-IsoP-M concentrations at admission and follow-up were significantly higher than discharge (admission median: 4.12 ng/Cr mg, range 1.89–7.8; follow-up: 2.47 ng/Cr mg (1.56–6.86); discharge: 1.42 ng/Cr mg (0.7–4.44); both p<0.01), but not significantly different between admission and follow-up. F₂-IsoP-M concentrations at follow-up were higher than a control group with stable asthma (0.68 ng/Cr mg (0.31–1.5), p=0.0008). In conclusion, asthma exacerbations requiring hospitalization are associated with 6-fold higher urinary F₂-IsoP-M concentrations compared to stable asthmatics. F₂-IsoP-M concentrations decreased significantly during hospitalization, but significant elevations 3 months following hospitalization suggest ongoing oxidative stress despite clinical improvement. Urinary F₂-IsoP-M may be a clinically useful, simple non-invasive systemic measure of oxidative stress in asthmatics, providing information not captured by spirometry or symptoms.     

Role of superoxide dismutases (SODs) in controlling oxidative stress in plants

Reactive O(2) species (ROS) are produced in both unstressed and stressed cells. Plants have well-developed defence systems against ROS, involving both limiting the formation of ROS as well as instituting its removal. Under unstressed conditions, the formation and removal of O(2) are in balance. However, the defence system, when presented with increased ROS formation under stress conditions, can be overwhelmed. Within a cell, the superoxide dismutases (SODs) constitute the first line of defence against ROS. Specialization of function among the SODs may be due to a combination of the influence of subcellular location of the enzyme and upstream sequences in the genomic sequence. The commonality of elements in the upstream sequences of Fe, Mn and Cu/Zn SODs suggests a relatively recent origin for those regulatory regions. The differences in the upstream regions of the three FeSOD genes suggest differing regulatory control which is borne out in the research literature. The finding that the upstream sequences of Mn and peroxisomal Cu/Zn SODs have three common elements suggests a common regulatory pathway. The tools are available to dissect further the molecular basis for antioxidant defence responses in plant cells. SODs are clearly among the most important of those defences, when coupled with the necessary downstream events for full detoxification of ROS.     

Arsenic induced oxidative stress in plants

Arsenic is a highly toxic metalloid for all forms of life including plants. Arsenic enters in the plants through phosphate transporters as a phosphate analogue or through aquaglycoporins. Uptake of arsenic in plant tissues adversely affects the plant metabolism and leads to various physiological and structural disorders. Photosynthetic apparatus, cell division machinery, energy production, and redox status are the major section of plant system that are badly affected by As (V). Similarly As (III) can react with thiol (-SH) groups of enzymes and inhibits various metabolic processes. Arsenic is also known to induce oxidative stress directly by generating reactive oxygen species (ROS) during conversion of its valence forms or indirectly by inactivating antioxidant molecules through binding with their -SH groups. As-mediated oxidative stress causes cellular, molecular and physiological disturbances in various plant species. Activation of enzymatic antioxidants namely, superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR), Glutathione s-transferase, glutathione peroxidase (GPX) as well as non antioxidant compounds such as, ascorbate, glutathione, carotenoids are reported to neutralize arsenic mediated oxidative stress. Understanding of biochemistry of arsenic toxicity would be beneficial for the development of arsenic tolerant crops and other economically important plants.     

Measurement of F(2)-isoprostanes unveils profound oxidative stress in aged rats

Free radicals have been theorized to play a causative role in the normal aging process. To date, methods used to detect oxidative stress in aged experimental animals have only detected 2- to 3-fold differences or less between young and aged animals. Measurement of F(2)-isoprostanes has emerged as probably the most reliable approach to assess oxidative stress status in vivo. Therefore, we measured levels of F(2)-isoprostanes free in plasma and levels esterified in plasma lipids in young rats (3-4 months of age) and aged rats (22-24 months of age). Plasma concentrations of free F(2)-isoprostanes were increased dramatically by a mean of 20.3-fold (range 4.3 to 42.9-fold) and levels esterified in plasma lipids were also strikingly increased by a mean of 29.9-fold (range 15.8 to 50.0-fold). These findings unveil profound oxidative stress in aged rats which adds considerable support for the free radical theory of aging.     

Molecular biology of wound-inducible proteinase inhibitors in plants

Abstract. The techniques of molecular biology are being employed to investigate at the gene level the systemically mediated, wound-induced accumulation of two defensive proteinase inhibitor proteins in plant leaves. These techniques have added a new dimension to biochemical and physiological studies already underway to understand the mechanism of induction by wounding. The acquisition of cDNAs from the RNAs coding for the two inhibitors facilitated studies of mRNA synthesis in leaves in response to wounding, and provided probes to obtain wound-inducible proteinase inhibitor genes from tomato ( Lycopersicon esculentum ) and potato (Solarium tuberosum) genomes. Successful transformations of tobacco plants with fused genes, containing the 5' and 3' regions of the inhibitor genes with the open reading frame of the chloramphenicol acelyltransferase ( cat ) gene, have provided a wound-inducible chloramphenicol acetyltransferase (CATase) activity with which to seek cis- and transacting elements that regulate wound-inducibility to help to understand the interaction of cytoplasmic and nuclear components of the intracellular communication systems that activate the proteinase inhibitor genes in response to wounding by insect pests.     

Cardiopulmonary bypass as a cause of free radical-induced oxidative stress and enhanced blood-borne isoprostanes in humans

Free radicals are believed to be involved in postsurgery-related complications. We studied whether cardiopulmonary bypass (CPB) operation has any immediate impact on the initiation of oxidative stress and inflammatory response by measuring isoprostanes and prostaglandin F2alpha during and 24 h following CPB. The levels of 8-iso-PGF2alpha (a major F2-isoprostane and biomarker of oxidative stress) and 15-keto-dihydro-PGF2alpha (a major metabolite of PGF2alpha and biomarker of inflammatory response) were measured in frequently collected plasma samples before, during, and up to 24 h postsurgery in 21 patients. 8-Iso-PGF2alpha levels significantly increased within 3 min (p <.0001) and continued until 50 min (p <.0001) during CPB. On the contrary, no significant increase of inflammatory response indicator, 15-keto-dihydro-PGF2alpha was found during and up to 24 h postoperatively. These findings establish an increased free radical-induced oxidative stress activity rather than inflammatory response after CPB.     

Drought induces oxidative stress in pea plants

Pea (Pisum sativum L. cv. Frilene) plants subjected to drought (leaf water potential of -1.3 MPa) showed major reductions in photosynthesis (78), transpiration (83), and glycolate oxidase (EC 1.1.3.1) activity (44), and minor reductions (18) in the contents of chlorophyll a, carotenoids, and soluble protein. Water stress also led to pronounced decreases (72–85) in the activities of catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2), but resulted in the increase (32–42) of non-specific peroxidase (EC 1.11.1.7) and superoxide dismutase (EC 1.15.1.1). Ascorbate peroxidase (EC 1.11.1.11) and monodehydroascorbate reductase (EC 1.6.5.4) activities decreased only by 15 and the two enzymes acted in a cyclic manner to remove H₂O₂, which did not accumulate in stressed leaves. Drought had no effect on the levels of ascorbate and oxidized glutathione in leaves, but caused a 25 decrease in the content of reduced glutathione and a 67 increase in that of vitamin E. In leaves, average concentrations of catalytic Fe, i.e. Fe capable of catalyzing free-radical generation by redox cycling, were estimated as 0.7 to 7 M (well-watered plants, depending on age) and 16 M (water-stressed plants); those of catalytic Cu were 4.5 M and 18 M, respectively. Oxidation of lipids and proteins from leaves was enhanced two- to threefold under stress conditions and both processes were highly correlated. Fenton systems composed of the purported concentrations of ascorbate, H₂O₂, and catalytic metal ions in leaves produced hydroxyl radicals, peroxidized membrane lipids, and oxidized leaf proteins. It is proposed that augmented levels and decompartmentation of catalytic metals occurring during water stress are responsible for the oxidative damage observed in vivo.Abbreviations and Symbol ASC ascorbate - DW dry weight - DHA dehydroascorbate - GSH reduced glutathione - GSSG oxidized glutathione - MDHA monodehydroascorbate (ascorbate free radical) - SOD Superoxide dismutase - _wa water potential We thank Dr. R. Picorel (E.E. de Aula Dei, CSIC) for allowing us access to HPLC equipment. J.F.M., 1.1., and S.F. were the recipients of predoctoral fellowships from the Comunidades Autónomas de Aragon, Pais Vasco, and Navarra, respectively. R.V.K. thanks the U.S. Department of Agriculture (grant 91-37305-6705) for travel support. This work was financed by grants from the Comisión Interministerial de Ciencia y Tecnología (AGR-91-0857-C02 to P.A. and M.B.) and the Dirección General de Investigación Científica y Técnica (PB92-0058 to M.B) of Spain.     

Measurement of F(2)-isoprostanes as an index of oxidative stress in vivo

In 1990 we discovered the formation of prostaglandin F(2)-like compounds, F(2)-isoprostanes (F(2)-IsoPs), in vivo by nonenzymatic free radical-induced peroxidation of arachidonic acid. F(2)-IsoPs are initially formed esterified to phospholipids and then released in free form. There are several favorable attributes that make measurement of F(2)-IsoPs attractive as a reliable indicator of oxidative stress in vivo: (i) F(2)-IsoPs are specific products of lipid peroxidation; (ii) they are stable compounds; (iii) levels are present in detectable quantities in all normal biological fluids and tissues, allowing the definition of a normal range; (iv) their formation increases dramatically in vivo in a number of animal models of oxidant injury; (v) their formation is modulated by antioxidant status; and (vi) their levels are not effected by lipid content of the diet. Measurement of F(2)-IsoPs in plasma can be utilized to assess total endogenous production of F(2)-IsoPs whereas measurement of levels esterified in phospholipids can be used to determine the extent of lipid peroxidation in target sites of interest. Recently, we developed an assay for a urinary metabolite of F(2)-IsoPs, which should provide a valuable noninvasive integrated approach to assess total endogenous production of F(2)-IsoPs in large clinical studies.     

Angiotensin-(1-7) stimulates oxidative stress in rat kidney

The effect of two different doses of angiotensin-(1-7) and angiotensin II on the oxidative stress generation was analyzed in rat kidney. Animals were injected intraperitoneally with a single dose of angiotensin-(1-7) or angiotensin II (20 or 50 nmol/kg body weight) and killed 3 h after injection. Production of thiobarbituric acid reactive substances (TBARS), measured as indicator of oxidative stress induction, was significantly increased in rat kidney after Ang-(1-7) administration up to 30% and 50% over controls, at 20 and 50 nmol/kg, respectively. Reduced glutathione (GSH), the most important soluble antioxidant defense in mammalian cells, showed a significant decrease of 13% and 20% at 20 and 50 nmol/kg of angiotensin-(1-7), respectively. When the antioxidant enzyme activities were determined, it was found that catalase activity was not altered by the assayed angiotensin-(1-7) doses while superoxide dismutase and glutathione peroxidase activities were significantly reduced by injection of 20 nmol/kg (34% and 13%, with respect to controls) and 50 nmol/kg of angiotensin-(1-7) (54% and 22%, respectively). In contrast, angiotensin II injections did not produce significant changes neither in TBARS levels nor in soluble and enzymatic defense parameters at the two doses used in this work. These results suggest that angiotensin-(1-7) is undoubtedly related to oxidative stress induction.     

Nucleotide sequence and regulated expression of a wound-inducible potato gene (wun1)

Summary Mechanical wounding of potato leaves, stems, roots and tubers leads to a rapid increase of wun1 mRNA. In potato leaves, the wound-induced accumulation of wun1 mRNA is inhibited by the addition of sucrose or other osmotically active agents. This inhibition is organ specific since sucrose does not prevent wun1 mRNA accumulation in wounded tubers. In contrast, expression of patatin was shown to be repressed in tubers by wounding and this repression was reversed by increasing osmotic pressure. Sequence data obtained from the analysis of a wun1 cDNA and a wun1 genomic clone show no homology to any gene known so far. Histochemical data demonstrate a striking analogy in cell specific expression of chimeric genes expressed under the control of the wun1 promoter and the cell specific production of callose in wounded tobacco leaves.     

Lanthanum- and cerium-induced oxidative stress in submerged Hydrilla verticillata plants

Oxidative stress was induced in 10-day treated with lanthanum and cerium Hydrilla verticillata plants. Low 10 μM concentrations did not exert harmful effects. The plants treated with higher concentrations showed higher H₂O₂ content and lower chlorophyll and soluble protein contents as compared to control plants. At the same time, malondialdehyde content rose with increasing concentrations of La and Ce. As La and Ce concentrations increased, superoxide dismutase and catalase activities declined progressively, while peroxidase activity increased. Proline content decreased slightly at 10 μM La or Ce and then rose with higher concentrations. The results indicated that La and Ce caused oxidative damage as evidenced by increased lipid peroxidation and decreased chlorophyll and protein levels. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 5, pp. 781–785. The text was submitted by the authors in English.     

Insights into fluoride-induced oxidative stress and antioxidant defences in plants

Fluoride is a common pollutant which occurs in various environmental matrices considered as one of the most phytotoxic pollutants. It is essential to the living organisms in trace quantities but at its higher concentration it becomes poisonous. Excess amount of fluoride in environment not only exerts its toxic effects on human beings and animals but also on plants. Toxicological impacts of fluoride on plants have been largely debated due to reduction of growth parameters, inhibition of metabolic activities and decreased photosynthetic activity. The signs of fluoride impacts on plants may be severe, acute or chronic and toxicity of fluoride depends on dose, frequency of exposure, duration and genotype of plant. This article overviews understanding of transport, uptake and fluoride accumulation in plants and provide insights into the fluoride-induced oxidative stress and regulatory mechanisms to cope up with it. The main objective of this article is to prospect new research avenues to unravel the mechanisms explaining fluoride toxicity in various plant species.     

Chloride intracellular channel 1 (CLIC1): Sensor and effector during oxidative stress

Oxidative stress, characterized by overproduction of reactive oxygen species (ROS), is a major feature of several pathological states. Indeed, many cancers and neurodegenerative diseases are accompanied by altered redox balance, which results from dysregulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In this review, we consider the role of the intracellular chloride channel 1 (CLIC1) in microglial cells during oxidative stress. Following microglial activation, CLIC1 translocates from the cytosol to the plasma membrane where it promotes a chloride conductance. The resultant anionic current balances the excess charge extruded by the active NADPH oxidase, supporting the generation of superoxide by the enzyme. In this scenario, CLIC1 could be considered to act as both a second messenger and an executor.     

Response to oxidative stress induced by cadmium and copper in tobacco plants (Nicotiana tabacum) engineered with the trehalose-6-phosphate synthase gene (AtTPS1)

The response of tobacco plants genetically engineered with the AtTPS1 gene to stress induced by excess Cu and Cd was evaluated in hydroponic solution (100 and 400 μM Cu and 50 and 200 μM Cd) after a 48 h exposure. Two transgenic lines, transformed with the AtTPS1 (trehalose-6-phosphate synthase) gene from Arabidopsis, with different levels of trehalose-6-phosphate synthase expression (B5H, higher and B1F, lower), and a wild type (WT) were investigated. Protein content, antioxidative enzymes (CAT, POD, SOD, and APX), glucose, fructose, lipid peroxidation, hydrogen peroxide and Cd and Cu contents were determined in leaves. The two transgenic lines were differently influenced by Cd and Cu exposure as they induced a different antioxidant enzymatic defense response. B1F and B5H plants showed a better acclimation to Cd and excess Cu compared to WT. Furthermore B1F was more tolerant than B5H to Cd and excess Cu. B1F accumulated less Cd and Cu in leaves, probably due to a more efficient exclusion mechanism. Catalase was shown to be the most important enzyme in the antioxidative system of these plants.     

Age-independent,gray matter-localized,brain-enhanced oxidative stress in male fischer 344 rats: brain levels of F(2)-isoprostanes and F(4)-neuroprostanes

While studies showed that aging is accompanied by increased exposure of the brain to oxidative stress, others have not detected any age-correlated differences in levels of markers of oxidative stress. Use of conventional markers of oxidative damage in vivo, which may be formed ex vivo and/or eliminated by endogenous metabolism, may explain these conflicting results. Recently, F₂-isoprostanes and F₄-neuroprostanes, peroxidation products of arachidonic acid and docosahexaenoic acid, respectively, have been identified as sensitive and reliable markers of oxidative injury. Therefore, this study was designed to quantify brain levels of F₂-isoprostanes and F₄-neuroprostanes and their precursors in 4, 10, 50, and 100 week old male Fischer 344 rats. Data show that levels of F₂-isoprostanes and F₄-neuroprostanes were comparable in all animal age groups. However, levels of F₄-neuroprostanes were approximately 20-fold higher than those of F₂-isoprostanes in all age groups, despite the fact that brain levels of docosahexaenoic acid were only twice as high as those of arachidonic acid. Based on our findings, it is concluded that aging is not accompanied by enhanced brain susceptibility to oxidative stress. Furthermore, the metabolically active gray matter of the brain, where docosahexaenoic acid is abundant, appears more susceptible to oxidative stress than the white matter.