Reactive oxygen species, nitric oxide, and their interactions play different roles in Cupressus lusitanica cell death and phytoalexin biosynthesis

Beta-thujaplicin Is a natural troponoid with strong antifungal, antiviral, and anticancer activities. Beta-thujaplicin production in yeast elicitor-treated Cupressus lusitanica cell culture and its relationships with reactive oxygen species (ROS) and nitric oxide (NO) production and hypersensitive cell death were investigated. Superoxide anion radical (O2*-) induced cell death and inhibited beta-thujaplicin accumulation, whereas hydrogen peroxide (H2O2) induced beta-thujaplicin accumulation but did not significantly affect cell death. Both elicitor and O2*- induced programmed cell death, which can be blocked by protease inhibitors, protein kinase inhibitors, and Ca2+ chelators. Elicitor-induced NO generation was nitric oxide synthase (NOS)-dependent. Inhibition of NO generation by NOS inhibitors and NO scavenger partly blocked the elicitor-induced beta-thujaplicin accumulation and cell death, and NO donors strongly induced cell death. Interaction among NO, H2O2, and O2*- shows that NO production and H2O2 production are interdependent, but NO and O2*- accumulation were negatively related because of coconsumption of NO and O2*-. NO- and O2*- -induced cell death required each other, and both were required for elicitor-induced cell death. A direct interaction between NO and O2*- was implicated in the production of a potent oxidant peroxynitrite, which might mediate the elicitor-induced cell death.     

Multiple signalling pathways mediate fungal elicitor-induced beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures

The biosynthesis of a phytoalexin, beta-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H(2)O(2), or methyl jasmonate. Lipoxygenase activity was also stimulated by these treatments, suggesting that the oxidative burst and jasmonate pathway may mediate the elicitor-induced accumulation of beta-thujaplicin. The elicitor signalling pathway involved in beta-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of beta-thujaplicin. A23187 also enhanced the elicitor-induced production of beta-thujaplicin. EGTA, LaCl(3), and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of beta-thujaplicin. These results suggest that Ca(2+) influx is required for elicitor-induced production of beta-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of beta-thujaplicin or enhanced the elicitor-induced production of beta-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of beta-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of beta-thujaplicin. Indeed, both GTP-binding activity and GTPase activity of the plasma membrane were stimulated by elicitor, and suramin and cholera toxin affected G-protein activities. In addition, all inhibitors of G-proteins and Ca(2+) flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca(2+) signalling pathway increased lipoxygenase activity. These observations suggest that Ca(2+) and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of beta-thujaplicin.     

Jasmonate and ethylene signalling and their interaction are integral parts of the elicitor signalling pathway leading to beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures

Roles of jasmonate and ethylene signalling and their interaction in yeast elicitor-induced biosynthesis of a phytoalexin, beta-thujaplicin, were investigated in Cupressus lusitanica cell cultures. Yeast elicitor, methyl jasmonate, and ethylene all induce the production of beta-thujaplicin. Elicitor also stimulates the biosynthesis of jasmonate and ethylene before the induction of beta-thujaplicin accumulation. The elicitor-induced beta-thujaplicin accumulation can be partly blocked by inhibitors of jasmonate and ethylene biosynthesis or signal transduction. These results indicate that the jasmonate and ethylene signalling pathways are integral parts of the elicitor signal transduction leading to beta-thujaplicin accumulation. Methyl jasmonate treatment can induce ethylene production, whereas ethylene does not induce jasmonate biosynthesis; methyl jasmonate-induced beta-thujaplicin accumulation can be partly blocked by inhibitors of ethylene biosynthesis and signalling, while blocking jasmonate biosynthesis inhibits almost all ethylene-induced beta-thujaplicin accumulation. These results indicate that the ethylene and jasmonate pathways interact in mediating beta-thujaplicin production, with the jasmonate pathway working as a main control and the ethylene pathway as a fine modulator for beta-thujaplicin accumulation. Both the ethylene and jasmonate signalling pathways can be regulated upstream by Ca(2+). Ca(2+) influx negatively regulates ethylene production, and differentially regulates elicitor- or methyl jasmonate-stimulated ethylene production.     

Feedback regulation of beta-thujaplicin production and formation of its methyl ether in a suspension culture of Cupressus lusitanica

Suspension cell cultures of Cupressus lusitanica produce beta-thujaplicin, a tropolone found mostly in Cupressaceae heartwood. The factors controlling beta-thujaplicin accumulation in this cell culture system were investigated. Initial cell density of the cultures did not affect beta-thujaplicin levels, though initial addition of beta-thujaplicin suppressed its de novo production. When beta-thujaplicin accumulation reached a certain level (ca. 40 mg/l) in the medium, the cultures seemed to cease beta-thujaplicin production. However, beta-thujaplicin productivity was restored when the beta-thujaplicin-containing medium was exchanged for fresh medium; the formation of 2-methoxy-6-(methylethyl)cyclohepta-2,4,6-trien-1-one, an isomer of methylated beta-thujaplicin, in medium was also observed. These results suggest that beta-thujaplicin synthesis was regulated by product feedback mechanism in this cell line, and that excess accumulation of beta-thujaplicin is relieved by conversion of beta-thujaplicin to its methyl ether.     

Improved β-thujaplicin production in Cupressus lusitanica suspension cultures by fungal elicitor and methyl jasmonate

Production of a novel antimicrobial tropolone, beta-thujaplicin, in Cupressus lusitanica suspension cultures was studied by using a variety of chemicals and fungal elicitors. Sodium alginate, chitin, and methyl jasmonate resulted in 2-, 2.5-, and 3-fold higher beta-thujaplicin production, respectively, than in the control. Significantly improved beta-thujaplicin production (187 mg l(-1)) was obtained using a high cell density (180-200 g l(-1)) and fungal elicitor treatment [10 mg (g fresh cells)(-1)] in a production medium with a high ferrous ion concentration (0.3 mM). This improved volumetric productivity was 3- to 4-fold higher than obtained under standard conditions. A synergistic effect of fungal elicitor and ferrous ion on beta-thujaplicin production was also suggested by our study. Plant cell culture technology is a promising alternative for producing a large variety of secondary metabolites that are widely used as food additives, pharmaceuticals, and dairy products (Verpoorte et al. 1999). Thus, beta-thujaplicin production by plant cell cultures was developed with the goal of commercial application (Berlin and Witte 1988; Itose and Sakai 1997; Ono et al. 1998). However, the production of beta-thujaplicin by plant cell cultures is still not competitive for use in industrial applications. In this study, we assessed the effects of methyl jasmonate, alginate, chitin, and fungal elicitor on beta-thujaplicin production; we obtained a significantly elevated beta-thujaplicin production by using an improved culture strategy.     

Proline and glycinebetaine induce antioxidant defense gene expression and suppress cell death in cultured tobacco cells under salt stress

Salt stress causes oxidative damage and cell death in plants. Plants accumulate proline and glycinebetaine (betaine) to mitigate detrimental effects of salt stress. The aim of this study was to investigate the protective effects of proline and betaine on cell death in NaCl-unadapted tobacco (Nicotiana tabacum) Bright Yellow-2 suspension-cultured cells subjected to salt stress. Salt stress increased reactive oxygen species (ROS) accumulation, lipid peroxidation, nuclear deformation and degradation, chromatin condensation, apoptosis-like cell death and ATP contents. Neither proline nor betaine affected apoptosis-like cell death and G(1) phase population, and increased ATP contents in the 200mM NaCl-stressed cells. However, both of them effectively decreased ROS accumulation and lipid peroxidation, and suppressed nuclear deformation and chromatin condensation induced by severe salt stress. Evans Blue staining experiment showed that both proline and betaine significantly suppressed increment of membrane permeability induced by 200mM NaCl. Furthermore, among the ROS scavenging antioxidant defense genes studied here, mRNA levels of salicylic acid-binding (SAbind) catalase (CAT) and lignin-forming peroxidase (POX) were found to be increased by proline and betaine under salt stress. It is concluded that both proline and betaine provide a protection against NaCl-induced cell death via decreasing level of ROS accumulation and lipid peroxidation as well as improvement of membrane integrity.     

Oxidative stress response of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. (PCC 6803) due to exposure to microcystin-LR and cell-free cyanobacterial crude extract containing microcystin-LR

Cyanobacteria are able to produce a variety of secondary metabolites such as the microcystins. The ecological role of microcystins for the cyanobacteria themselves and in the aquatic ecosystem is not well understood. The aim of this study is to evaluate if microcystins might be used as a communication tool for interspecies cyanobacterial communication via the promotion of oxidative stress. Reactive oxygen species (ROS) are known to be used as plant signals. The main questions relate to the promotion of oxidative stress in Synechocystis sp. via exposure to the cyanobacterial toxins and the physiological effects. This study shows a few markers for oxidative stress, such as the antioxidative enzymes superoxide dismutase, peroxidases and catalases, and cell damage due to extensive generation of ROS leading to lipid peroxidation. End products of lipid peroxidation (malonaldehyde and 4-hydroxynonenal) are conjugated by glutathione S-transferase.     

Vitamin E succinate protects hepatocytes against the toxic effect of reactive oxygen species generated at mitochondrial complexes I and III by alkylating agents.

The mechanism of alpha-tocopheryl succinate (TS) cytoprotection against mitochondria-derived oxidative stress was investigated. Incubation of isolated rat hepatocytes with ethyl methanesulfonate (EMS), a mitochondrial alkylating toxicant caused mitochondrial dysfunction and necrotic cell death that was dependent on the production of reactive oxygen species (ROS) and lipid peroxidation. Mitochondria isolated from these cells showed a 3-fold increase in lipid hydroperoxides and a selective depletion of alpha-tocopherol (T), which preceded cell death. The pretreatment of hepatocytes with TS dramatically enriched cells and mitochondria with alpha-tocopherol and provided these membranes with complete protection against EMS-induced oxidative damage. TS pretreatment suppressed EMS-induced cellular ROS production, generated from mitochondrial complex I and III sites. In addition, the treatment with either rotenone (ROT, a complex I inhibitor) or antimycin A (AA, a complex III inhibitor) potentiated EMS-induced lipid peroxidation and necrotic cell death which were again completely prevented by TS treatment. Surprisingly, TS did not protect hepatocytes against thenoyltrifluoroacetone (TTFA), a complex II inhibitor-induced enhancement of EMS-induced toxic oxidative damage. We conclude that the inhibition of mitochondrial ROS production and lipid peroxidation by T released from TS, are the critical events responsible for TS-mediated cytoprotection against toxic oxidative stress derived from both mitochondrial complexes I and III. Our findings suggest that TS treatment may prove useful in combating diseases associated with mitochondrial-derived oxidative stress.     

Aluminum compounds enhance lipid peroxidation in liposomes: insight into cellular damage caused by oxidative stress

Aluminum (Al) has been proposed as one of the critical environmental factors responsible for several neurodegenerative diseases such as Alzheimer's disease. However, the suggested mechanism involving the contribution of reactive oxygen species still remains controversial. We have first attempted to identify Al compounds either in its ionic or complexed forms that cause oxidative stress in biological systems. For this purpose, we examined the effect of inorganic Fe(2+)- and organic radical initiator (2,2'-azobis (2-amidinopopane) hydrochloride; AAPH)-induced lipid peroxidation by using aluminum (Al(3+)) nitrate and tris(maltolato)aluminum(III) complex (ALM) with respect to molecular oxygen (O(2)) consumption and membrane fluidity change in liposomes as biological membrane models. The following important results were obtained: (1) ALM enhanced the lipid peroxidation induced by Fe(2+) and AAPH in phosphatidylcholine liposomes; this corresponded well with the promotion of O(2) uptake in the same liposomes, (2) Al(3+) increased both lipid peroxidation and O(2) consumption in phosphatidylserine liposomes in the presence of Fe(2+), and (3) both Al(3+) and ALM affected the membrane fluidity on the inner side. It has been concluded that ALM induces higher lipid peroxidation in liposomes than Al(3+); this finding will be useful to gain an insight into the role of Al in cellular damage in relation to oxidative stress.     

Ca2+-induced oxidative stress in brain mitochondria treated with the respiratory chain inhibitor rotenone

In this study we show that micromolar Ca(2+) concentrations (>10 microM) strongly stimulate the release of reactive oxygen species (ROS) in rotenone-treated isolated rat forebrain mitochondria. Ca(2+)-stimulated mitochondrial ROS release was associated with membrane lipid peroxidation and was directly correlated with the degree of complex I inhibition by rotenone. On the other hand, Ca(2+) did not increase mitochondrial ROS release in the presence of the complex I inhibitor 1-methyl-4-phenylpyridinium. Cyclosporin A had no effect on Ca(2+)-stimulated mitochondrial ROS release in the presence of rotenone, indicating that mitochondrial permeability transition is not involved in this process. We hypothesized that Ca(2+)-induced mitochondrial oxidative stress associated with partial inhibition of complex I may be an important factor in neuronal cell death observed in the neurodegenerative disorder Parkinson's disease.     

Oxidative stress mediates toxicity of pyridoxal isonicotinoyl hydrazone analogs

Pyridoxal isonicotinoyl hydrazone (PIH) and many of its analogs are effective iron chelators in vivo and in vitro, and are of interest for the treatment of secondary iron overload. Because previous work has implicated the Fe(3+)-chelator complexes as a determinant of toxicity, the role of iron-based oxidative stress in the toxicity of PIH analogs was assessed. The Fe(3+) complexes of PIH analogs were reduced by K562 cells and the physiological reductant, ascorbate. Depletion of the antioxidant, glutathione, sensitized Jurkat T lymphocytes to the toxicity of PIH analogs and their Fe(3+) complexes, and toxicity of the chelators increased with oxygen tension. Fe(3+) complexes of pyridoxal benzoyl hydrazone (PBH) and salicyloyl isonicotinoyl hydrazone (SIH) caused lipid peroxidation and toxicity in K562 cells loaded with eicosapentenoic acid (EPA), a readily oxidized fatty acid, whereas Fe(PIH)(2) did not. The lipophilic antioxidant, vitamin E, completely prevented both the toxicity and lipid peroxidation caused by Fe(PBH)(2) in EPA-loaded cells, indicating a causal relationship between oxidative stress and toxicity. PBH also caused concomitant lipid peroxidation and toxicity in EPA-loaded cells, both of which were reversed as its concentration increased. In contrast, PIH was inactive, while SIH was equally toxic toward control and EPA-loaded cells, without causing lipid peroxidation, indicating a much smaller contribution of oxidative stress to the mechanism of toxicity of these analogs. In summary, PIH analogs and their Fe(3+) complexes are redox active in the intracellular environment. The contribution of oxidative stress to the overall mechanism of toxicity varies across the series.     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Oxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H(2)O(2)) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H(2)O(2)-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H(2)O(2)-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H(2)O(2), and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca(2+)) chelator, overexpression of Homer 1a had no significant effects on H(2)O(2)-induced oxidative stress. These results suggest that Homer 1a has protective effects against H(2)O(2)-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca(2+) homeostasis.     

Melatonin reduces Fenton reaction-induced lipid peroxidation in porcine thyroid tissue

Free radicals and reactive oxygen species (ROS) participate in physiological and pathological processes in the thyroid gland. Bivalent iron cation (ferrous, Fe(2+)), which initiates the Fenton reaction (Fe(2+) + H2O2 --> Fe(3+) + *OH + OH(-)) is frequently used to experimentally induce oxidative damage, including that caused by lipid peroxidation. Lipid peroxidation is involved in DNA damage, thus indirectly participating in the early steps of carcinogenesis. In turn, melatonin is a well-known antioxidant and free radical scavenger. The aim of the study was to estimate the effect of melatonin on basal and iron-induced lipid peroxidation in homogenates of the porcine thyroid gland. In order to determine the effect of melatonin on the auto-oxidation of lipids, thyroid homogenates were incubated in the presence of that indoleamine in concentrations of 0.0, 0.00001, 0.0001, 0.001, 0.01, 0.1, 0.25, 0.5, 1.0, 2.5, or 5.0 mM. To study melatonin effects on iron-induced lipid peroxidation, the homogenates were incubated in the presence of FeSO(4) (40 microM) plus H2O2 (0.5 mM), and, additionally, in the presence of melatonin in the same concentrations as above. The degree of lipid peroxidation was expressed as the concentration of malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA) per mg protein. Melatonin, in a concentration-dependent manner, decreased lipid peroxidation induced by Fenton reaction, without affecting the basal MDA + 4-HDA levels. In conclusion, melatonin protects against iron + H2O2-induced peroxidation of lipids in the porcine thyroid. Thus, the indoleamine would be expected to prevent pathological processes related to oxidative damage in the thyroid, cancer initiation included.     

Antioxidant activity of Terminalia arjuna bark extract on N-nitrosodiethylamine induced hepatocellular carcinoma in rats

Role of oxidative stress and Na⁺,K⁺-ATPase in the cytotoxicity of hexachlorocyclohexane (HCH) on Ehrlich Ascites tumor (EAT) cells has been studied. HCH caused dose dependent cell death as measured by trypan blue exclusion and lactate dehydrogenase (LDH) leakage from the cells. HCH induced oxidative stress in EAT cells which was characterized by glutathione depletion, lipid peroxidation (LPO), reactive oxygen species (ROS) production and inhibition of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT). Protective effect of antioxidants on HCH induced oxidative stress was assessed, among the antioxidants used only quercetin inhibited HCH-induced LPO and ROS production as well as cell death whereas α -tocopherol, ascorbic acid and BHA inhibited LPO but not cell death. Inhibition of membrane bound Na⁺,K⁺-ATPase was a characteristic feature of HCH cytotoxicity in EAT cells. Experimental evidence indicates that HCH-induced cell death involves oxidative stress due to ROS production and membrane perturbation in EAT cells.     

Fatty acid hydroperoxides and H2O2 in the execution of hypersensitive cell death in tobacco leaves

We initially compared lipid peroxidation profiles in tobacco (Nicotiana tabacum) leaves during different cell death events. An upstream oxylipin assay was used to discriminate reactive oxygen species (ROS)-mediated lipid peroxidation from 9- and 13-lipoxygenase (LOX)-dependent lipid peroxidation. Free radical-mediated membrane peroxidation was measured during H(2)O(2)-dependent cell death in leaves of catalase-deficient plants. Taking advantage of these transgenic plants, we demonstrate that, under light conditions, H(2)O(2) plays an essential role in the execution of cell death triggered by an elicitor, cryptogein, which provokes a similar ROS-mediated lipid peroxidation. Under dark conditions, however, cell death induction by cryptogein was independent of H(2)O(2) and accompanied by products of the 9-LOX pathway. In the hypersensitive response induced by the avirulent pathogen Pseudomonas syringae pv syringae, both 9-LOX and oxidative processes operated concurrently, with ROS-mediated lipid peroxidation prevailing in the light. Our results demonstrate, therefore, the tight interplay between H(2)O(2) and lipid hydroperoxides and underscore the importance of light during the hypersensitive response.     

Suofu Qin's work on studies of cell survival signaling in cancer and epithelial cells

Reactive oxygen species (ROS) encompass a variety of diverse chemical species including superoxide anions, hydrogen peroxide, hydroxyl radicals and peroxynitrite, which are mainly produced via mitochondrial oxidative metabolism, enzymatic reactions, and light-initiated lipid peroxidation. Over-production of ROS and/or decrease in the antioxidant capacity cause cells to undergo oxidative stress that damages cellular macromolecules such as proteins, lipids, and DNA. Oxidative stress is associated with ageing and the development of age-related diseases such as cancer and age-related macular degeneration. ROS activate signaling pathways that promote cell survival or lead to cell death, depending on the source and site of ROS production, the specific ROS generated, the concentration and kinetics of ROS generation, and the cell types being challenged. However, how the nature and compartmentalization of ROS contribute to the pathogenesis of individual diseases is poorly understood. Consequently, it is crucial to gain a comprehensive understanding of the molecular bases of cell oxidative stress signaling, which will then provide novel therapeutic opportunities to interfere with disease progression via targeting specific signaling pathways. Currently, Dr. Qin's work is focused on inflammatory and oxidative stress responses using the retinal pigment epithelial (RPE) cells as a model. The study of RPE cell inflammatory and oxidative stress responses has successfully led to a better understanding of RPE cell biology and identification of potential therapeutic targets.     

Mitochondrial aconitase knockdown attenuates paraquat-induced dopaminergic cell death via decreased cellular metabolism and release of iron and H₂O₂

Mitochondrial oxidative stress is a contributing factor in the etiology of numerous neuronal disorders. However, the precise mechanism(s) by which mitochondrial reactive oxygen species modify cellular targets to induce neurotoxicity remains unknown. In this study, we determined the role of mitochondrial aconitase (m-aconitase) in neurotoxicity by decreasing its expression. Incubation of the rat dopaminergic cell line, N27, with paraquat (PQ(2+) ) resulted in aconitase inactivation, increased hydrogen peroxide (H(2) O(2) ) and increased ferrous iron (Fe(2+) ) at times preceding cell death. To confirm the role of m-aconitase in dopaminergic cell death, we knocked down m-aconitase expression via RNA interference. Incubation of m-aconitase knockdown N27 cells with PQ(2+) resulted in decreased H(2) O(2) production, Fe(2+) accumulation, and cell death compared with cells expressing basal levels of m-aconitase. To determine the metabolic role of m-aconitase in mediating neuroprotection, we conducted a complete bioenergetic profile. m-Aconitase knockdown N27 cells showed a global decrease in metabolism (glycolysis and oxygen consumption rates) which blocked PQ(2+) -induced H(+) leak and respiratory capacity deficiency. These findings suggest that dopaminergic cells are protected from death by decreasing release of H(2) O(2) and Fe(2+) in addition to decreased cellular metabolism.     

Lipid peroxidation in small and large phospholipid unilamellar vesicles induced by water-soluble free radical sources

The susceptibility of small and large egg yolk phosphatidylcholine unilamellar vesicles to Fe(2+)/histidine-Fe(3+)- and Fenton reagent (Fe(2+)-H(2)O(2))-induced lipid peroxidation was evaluated by measuring the formation of thiobarbituric acid reactive substances (TBARS). It has been found that surface curvature or phospholipid packing exerts significant effect on the oxidative susceptibility of the unsaturated lipid bilayers and the highly curved and loosely packed small unilamellar vesicles (SUVs) exhibit much less resistance to the oxidative stress induced by the water-soluble free radical sources. The presence of lipid hydroperoxides in sonicated vesicles was excluded as the cause for higher level of lipid peroxidation in the phospholipid SUVs. Instead, the experimental results can be explained by the difference in ability of the water-soluble oxidants to penetrate the two types of lipid membranes. This hypothesis is supported by data obtained from fluorescence lifetime and quenching studies.