D-Penicillamine: analysis of the mechanism of copper-catalyzed hydrogen peroxide generation

Recent studies have suggested that the inhibition of lymphocyte mitogenesis by D-penicillamine in the presence of copper could be mediated by the formation and action of hydrogen peroxide. To explore this possibility further, we first sought evidence of H2O2 generation by D-penicillamine in a cell-free system by a) measurement of copper-catalyzed D-penicillamine oxidation and the requirement for oxygen in this process; b) direct measurement of H2O2 formation during D-penicillamine oxidation by the peroxidase-mediated oxidation of fluorescent scopoletin; and c) evaluation of the possible synthesis of O2- during D-penicillamine oxidation. The addition of copper to D-penicillamine in physiologic buffer catalyzed D-penicillamine oxidation in a dose-dependent fashion. D-penicillamine oxidation was accompanied by O2 consumption with a molar ratio of approximately 2:1, but did not occur under anaerobic conditions. Furthermore, D-penicillamine oxidation resulted in the formation of amounts of H2O2 stoichiometrically equivalent to oxygen consumption (i.e., 1:1). Copper-catalyzed D-penicillamine oxidation caused reduction of nitroblue tetrazolium in a reaction blocked by superoxide dismutase, suggesting the formation of O2-. Additional studies confirmed that D-penicillamine inhibited PHA-induced mitogenesis of lymphocytes in the presence of copper, and that catalase protected the cells from this action. Furthermore, when polymorphonuclear leukocytes were incubated with D-penicillamine plus copper, hexose monophosphate shunt activity increased up to threefold with abrogation of this stimulation by catalase. None of the effects of D-penicillamine plus copper on cells were diminished by hydroxyl radical scavengers mannitol or benzoate. These results are consistent with oxygen-dependent copper-catalyzed oxidation of D-penicillamine in aqueous solutions leading to the formation of O2- and H2O2. H2O2 produced by this reaction can inhibit lymphocyte mitogenesis and stimulate neutrophil hexose monophosphate shunt activity in vitro and may be relevant to the therapeutic effects of D-penicillamine in vivo.     

The effect of D-penicillamine on human myeloperoxidase, a mechanism for the efficacy of the drug in rheumatoid arthritis

We investigated the effect of D-penicillamine on the ability of myeloperoxidase, purified from human leukocytes, to catalyse the oxidation of chloride ions to hypochlorite (HOCl) in the presence of H2O2. It is shown that, due to the interaction of D-penicillamine with both myeloperoxidase itself and HOCl, the chlorinating activity of myeloperoxidase in the presence of H2O2 and chloride ions is prevented. A concentration of 100 microM D-penicillamine inhibits the chlorinating activity of myeloperoxidase completely, which Is due to the stabilization of Compound II, an inactive form of the enzyme. In addition, HOCl reacts directly with D-penicillamine. Analysis of the reaction products of D-penicillamine and HOCl showed that D-penicillamine was oxidized to penicillamine disulphide and penicillamine sulphinic acid, and eventually deaminated (indicated by the release of ammonia). Lower concentrations of D-penicillamine (10 microM) inhibited myeloperoxidase less, but still acted as effective scavengers of HOCl. In very low concentrations (1 microM), D-penicillamine did not scavenge HOCl effectively, but rather stimulated the chlorinating activity of myeloperoxidase. However, when instead of D-penicillamine a comparable amount of ascorbate was added, a similar but even larger stimulation was observed. Since the concentration of free D-penicillamine in serum from rheumatoid patients treated with this drug is about 20 microM (Saetre, R. and Rabenstein, D.L. (1978) Anal. Chem. 50, 276-280), the therapeutic effect of D-penicillamine may be due to the protection of tissues against the reactive HOCl released by activated granulocytes at inflammation sites.     

The effect of D-penicillamine on myeloperoxidase: formation of compound III and inhibition of the chlorinating activity

The inhibitory effect of the anti-arthritic drug D-penicillamine on the formation of hypochlorite (HOCl) by myeloperoxidase from H2O2 and Cl- was investigated. When D-penicillamine was added to myeloperoxidase under turnover conditions, Compound III was formed, the superoxide derivative of the enzyme. Compound III was not formed when D-penicillamine was added in the presence of EDTA or in the absence of oxygen. However, when H2O2 was added to myeloperoxidase, D-penicillamine and EDTA, Compound III was formed. Therefore it is concluded that formation of Compound III is initiated by metal-catalysed oxidation of the thiol group of this anti-arthritic drug, resulting in formation of superoxide anions. Once Compound III is formed, a chain reaction is started via which the thiol groups of other D-penicillamine molecules are oxidized to disulphides. Concomitantly, Compound I of myeloperoxidase would be reduced to Compound II and superoxide anions would be generated from oxygen. This conclusion is supported by experiments which showed that formation of Compound III of myeloperoxidase by D-penicillamine depended on the chloride concentration. Thus, an enzyme intermediate which is active in chlorination (i.e. Compound I) participated in the generation of superoxide anions from the anti-arthritic drug. From the results described in this paper it is proposed that D-penicillamine may exert its therapeutic effect in the treatment of rheumatoid arthritis by scavenging HOCl and by converting myeloperoxidase to Compound III, which is inactive in the formation of HOCl.     

Collagen cross-linking. Effect of D-penicillamine on cross-linking in vitro.

D-Pencillamine is believed to inhibit collagen cross-link biosynthesis by forming thiazolidine rings with lysyl-derived aldehydes that are intermediates in bifunctional cross-link synthesis. Recently, we showed that aldehyde biosynthesis catalyzed by lysyl oxidase occurs after the onset of fibril formation and that nascent aldehydes form Schiff-base cross-links rapidly in fibrils. This suggested that the accessibility of D-penicillamine to most aldehydes formed during cross-link synthesis might be limited. To study this, reconstituted chick bone collagen fibrils were incubated in vitro with highly purified lysyl oxidase and D-penicillamine. As reported in previous studies in vivo, allysine content increased and polyfunctional cross-link synthesis decreased with D-penicillamine. However, the concentration of bifunctional cross-links increased rather than decreased due to a 2-fold increase in N6:6'-dehydro-5,5'-dihydroxylysinonorleucine. Hydroxyallysine, an intermediate in formation of this Schiff base, decreased. A time study indicated that allysine levels increased primarily after the bulk of Schiff base synthesis. These results indicate that D-penicillamine does not inhibit bifunctional cross-link synthesis as previously suggested. Its principal effect is to block synthesis of polyfunctional cross-link products from Schiff base cross-link precursors and to cause accumulation of these precursors. This effect may be due to interference with the close molecular packing required for polyfunctional cross-link synthesis. These results also suggest a mechanism for the relative insensitivity of tissues such as bone with high hydroxylysine content to D-penicillamine. In this study, D-penicillamine caused selective accumulation of allysyl and not hydroxyallysyl residues. In bone as opposed to soft tissues, hydroxyallysyl residues are intermediates in synthesis of almost all cross-links.     

Nitric oxide negatively modulates wound signaling in tomato plants

Synthesis of proteinase inhibitor I protein in response to wounding in leaves of excised tomato (Lycopersicon esculentum) plants was inhibited by NO donors sodium nitroprusside and S-nitroso-N-acetyl-penicillamine. The inhibition was reversed by supplying the plants with the NO scavenger 2-(4-carboxiphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. NO also blocked the hydrogen peroxide (H(2)O(2)) production and proteinase inhibitor synthesis that was induced by systemin, oligouronides, and jasmonic acid (JA). However, H(2)O(2) generated by glucose oxidase and glucose was not blocked by NO, nor was H(2)O(2)-induced proteinase inhibitor synthesis. Although the expression of proteinase inhibitor genes in response to JA was inhibited by NO, the expression of wound signaling-associated genes was not. The inhibition of wound-inducible H(2)O(2) generation and proteinase inhibitor gene expression by NO was not due to an increase in salicylic acid, which is known to inhibit the octadecanoid pathway. Instead, NO appears to be interacting directly with the signaling pathway downstream from JA synthesis, upstream of H(2)O(2) synthesis. The results suggest that NO may have a role in down-regulating the expression of wound-inducible defense genes during pathogenesis.     

Regulation of endothelial cell prostaglandin synthesis by glutathione

Prostaglandin synthesis in in vitro systems is dependent on glutathione and peroxide concentrations. We tested the effects of glutathione depletion and H2O2 exposure on prostaglandin synthesis in cultured porcine aortic endothelial cells. Depletion of glutathione using buthionine sulfoximine (BSO), diethylmaleate, and 2,4-chlorodinitrobenzene increased prostaglandin synthetic capacity. Production of prostacyclin, but not prostaglandin E2, from exogenous arachidonic acid was significantly greater than in controls. Glutathione depletion also resulted in enhanced production of prostacyclin from exogenous prostaglandin H2. These responses were not due to direct effects of glutathione-depleting agents on prostaglandin synthetic enzymes. Exposure to H2O2 also altered prostaglandin synthetic capacity in endothelial cells. While 5 microM H2O2 stimulated prostaglandin production from exogenous arachidonate, 25 and 50 microM were found to be inhibitory. Prostaglandin synthetic capacity was greater in BSO-treated cells which were exposed to 5 and 10 microM H2O2 than in cells exposed to H2O2 alone. However, prostaglandin synthetic capacity was greatly reduced in BSO-treated cells exposed to 50 microM H2O2. Thus, normal levels of cellular glutathione exert an inhibitory influence on prostaglandin synthesis. However, glutathione depletion increases the sensitivity of prostaglandin synthesis to inhibition by 50 microM H2O2.     

不同活性氧胁迫下Bacillus sp. F26以抗氧化物酶合成为特征的应激响应

采用不同的活性氧发生源, 研究了· 、H2O2和OH·胁迫下Bacillus sp. F26以抗氧化物酶合成为特征的应激响应。结果表明, 细胞对氧胁迫的应激响应程度取决于活性氧种类、胁迫程度和形式(瞬时和持续)。Bacillus sp. F26对H2O2胁迫的响应程度最高, 过氧化氢酶的快速合成对细胞抵抗H2O2胁迫至关重要, 当细胞及时分解进入胞内的H2O2, 胁迫对细胞的氧化损伤程度并不高, 相反会刺激细胞的生长和底物消耗, 当胁迫超过过氧化氢酶的分解能力时, H2O2会迅速抑制细胞生长和过氧化氢酶合成; 由于 ·与细胞作用的方式和效果与H2O2不同, 超氧化物歧化酶和过氧化氢酶的快速合成并不能保证细胞及时有效地清除胞内的活性氧, 因此, 细胞对 ·胁迫的响应程度要低于H2O2胁迫; 在所考察的3种活性氧中, OH·胁迫(Fenton反应体系)对细胞的氧化损伤程度最大, 胁迫强烈地抑制了细胞生长和抗氧化物酶的合成。由此表明, 由于不同活性氧的化学性质有所不同, 细胞对不同种类、程度和形式的活性氧胁迫会表现出不同的生物学效应, 为了提高自身对氧胁迫的抵抗能力, 微生物会通过自身的代谢调节适应新的环境, 包括调整抗氧化物酶合成水平、改变生长速度以及底物消耗速率等。     

Proton translocation and ATP formation coupled to electron transport from H2O to the primary acceptor of photosystem 2.

1. The rate of electron transport from H2O to silicomolybdate in the presence of 3-(3-4-dichlorophenyl)-1,1-dimethylurea (diuron) (which involves the oxygen-evolving enzyme, the photochemistry of photosystem 2 and the primary electron acceptor of photosystem 2) is controlled by internal pH. This is based on the shift of the pH profile of the rate of electron transport upon addition of uncouplers, or by using EDTA-treated chloroplasts. Both stimulation and inhibition of electron transport by addition of uncouplers (depending on external pH) could be observed. These effects are obtained in the diuron-insensitive photoreductions of either silicomolybdate or ferricyanide. These experiments provide strong evidence that a proton translocating site exists in the sequence of the electron transport H2O leads to Q (the primary acceptor of photosystem 2). 2. The photoreduction of silicomolybdate in the presence of diuron causes the formation of delta pH. The value of delta pH depends on the external pH and its maximal value was shown to be 2.4. The calculated internal pH at different external pH values was found to be rather constant, namely between 5.1 -- 5.2. 3. Electron transport from H2O to silicomolybdate (in the presence of diuron) does not support ATP formation. It is suggested that this is due to the fact that the delta pH formed is below the "threshold" delta pH required for the synthesis of ATP. By adding an additional source of energy in the form of a dark diffusion potential created in the presence of K+ and valinomycin, significant amounts of ATP are formed in this system.     

Inhibition of immunoglobulin G-catalyzed hydrogen peroxide generation by dexamethasone and piroxicam

In the present study, we established a simple and physiologically acceptable in vitro assay system to measure H2O2 generated by human immunoglobulin G (IgG) and other proteins. In addition, the effects of various drugs were also tested in this method. We found that UV irradiation (280 nm) of the test solutions for 1 h at 37 degrees C produced suitable conditions to test the effects of these drugs. The test solution contained 100 microg/ml IgG in 50 mM phosphate buffer (pH 7.4), and 1% dimethylformamide (DMF), a solvent used to dissolve each drug. Phosphate anions were preferable for H2O2 generation. H2O2 concentration in the irradiated sample was determined by continuous photometric measurement of absorption (O.D.) at 340 nm for 600 sec. The decrease in O.D. was due to the oxidation of NADPH by H2O2 mediated by the glutathione redox cycle. H2O2 generation was expressed as O.D.(340 nm decrease/400 sec). IgG (100 microg/ml) generated 6-7 microM H2O2/h. With irradiation, most cytokines, proteins and enzymes failed to generate significant amounts of H2O2. The formation of H2O2 from H2O and UV light-induced singlet oxygen (1O2) was demonstrated by the inhibitory effects of 1O2 quenchers. Dexamethasone (IC50: 6 ng/ml = 1.4x10(-8) M) blocked H2O2 generation catalyzed by IgG. This action was not mediated by binding to the glucocorticoid receptor. Piroxicam (IC50: 20 ng/ml = 6.0 x 10(-6) M) and diclofenac.Na (IC50: 500 ng/ml = 1.6 x 10(-5) M), but not indomethacin, also blocked H2O2 generation. The mechanism underlying the inhibition of IgG-catalyzed H2O2 generation is not clear; however, the possibility exists that these drugs intercept, or interfere with, the approach of water molecules at the catalytic interface(s) of the IgG.     

The properties of hydrogen peroxide production under hyperoxic and hypoxic conditions of perfused rat liver.

The properties of H2O2 production in the "haemoglobin-free", "non-circulatory" perfused liver of rats were examined. The H2O2 production with 1 mM-lactate and 0.15 mM-pyruvate was 82nmol/min per g of liver or 333nmol/min per 100g body wt. in the liver of fed rats at 30 degrees C. This rate decreased to almost half in the livers of starved and phenobarbital-pretreated rats. When H2O2 production was stimulated by urate infusion, almost all of the H2O2 produced by the uricase reaction was decomposed by the catalase reaction. During the demethylation reaction of aminopyrine, no change in H2O2 production was detected by the present method; thus microsomal H2O2 production observed in isolated subcellular fractions appeared not to contribute significantly to the H2O2 production in the whole organ. Whereas the rate of the glycolate-dependent H2O2 production was halved at an intracellular O2 concentration that caused a 10 percent increase in the reduction state of cytochrome c, the half-maximal rate of H2O2 production with lactate and pyruvate was observed at an O2 concentration that caused a 40 percent increase in the reduction state of cytochrome c in the liver. No further increase in the rates of H2O2 production was obtained by increasing O2 pressure up to 5 times 10(5) Pa. The rate of ethanol oxidation through the catalase "peroxidatic" reaction varied, depending on the substrate availability. The maximal capability of this pathway in ethanol oxidation reached approx. 1.5 mumol/min per g of liver, when a mixture of urate, glycollate and octanoate was infused to enhance H2O2 production.     

A specific requirement for protein kinase C in activation of the respiratory burst oxidase of fertilization

Partially reduced oxygen species are toxic, yet activated sea urchin eggs produce H2O2, suggesting that the control of oxidant stress might be critical for early embryonic development. We show that the Ca2(+)-stimulated NADPH oxidase that generates H2O2 in the "respiratory burst" of fertilization is activated by a protein kinase, apparently to regulate the synthesis of this potentially lethal oxidant. The NADPH oxidase was separated into membrane and soluble fractions that were both required for H2O2 synthesis. The soluble fraction was further purified by anion exchange chromatography. The factor in the soluble fraction that activated the membrane-associated oxidase was demonstrated to be protein kinase C (PKC) by several criteria, including its Ca2+/phophatidylserine/diacyl-glycerol-stimulated histone kinase activity, its response to phorbol ester, its inhibition by a PKC pseudosubstrate peptide, and its replacement by purified mammalian PKC. Neither calmodulin-dependent kinase II, the catalytic subunit of cyclic AMP-dependent protein kinase, casein kinase II, nor myosin light chain kinase activated the oxidase. Although the PKC family has been ubiquitously implicated in cellular regulation, enzymes that require PKC for activation have not been identified; the respiratory burst oxidase is one such enzyme.     

Release of H2O2 and phosphorylation of 30 kilodalton proteins as early responses of cell cycle-dependent inhibition of DNA synthesis by transforming growth factor beta 1.

Transforming growth factor beta 1 (TGF-beta 1) and H2O2 both inhibited DNA synthesis of mouse osteoblastic (MC3T3) cells in the late G1 phase of the cell cycle. TGF-beta 1 stimulated cells to release H2O2 in the late G1 phase, but not in the G0 phase, even though TGF-beta 1 receptors were present in both phases. The inhibition of DNA synthesis caused by TGF-beta 1 was partly decreased by the addition of catalase. TGF-beta 1 and H2O2 increased the phosphorylation of the same proteins with a molecular weight of 30,000 in cells in the late G1 phase, and the increase by TGF-beta 1 was abolished at least partly by catalase. The results suggest that H2O2 is one of the mediators of inhibition of DNA synthesis by TGF-beta 1.     

Hydrogen peroxide inhibits alveolar macrophage 5-lipoxygenase metabolism in association with depletion of ATP

We have previously shown that the biologically important reactive oxygen metabolite hydrogen peroxide (H2O2) stimulates arachidonic acid (AA) release and thromboxane A2 synthesis in the rat alveolar macrophage. We have now investigated the effects of H2O2 on alveolar macrophage 5-lipoxygenase metabolism. H2O2 failed to stimulate detectable synthesis of leukotriene B4, leukotriene C4, or 5-hydroxyeicosatetraenoic acid (5-HETE) as determined by reverse-phase high performance liquid chromatography (RP-HPLC) and sensitive radioimmunoassays (RIAs). This was not explained by oxidative degradation of leukotrienes by H2O2 at the concentrations used. Moreover, RIA and RP-HPLC analyses demonstrated that H2O2 dose-dependently inhibited synthesis of leukotriene B4, leukotriene C4, and 5-HETE induced by the agonists A23187 (10 microM) and zymosan (100 micrograms/ml), over the same concentration range at which it augmented synthesis of the cyclooxygenase products thromboxane A2 and 12-hydroxy-5,8,10-heptadecatrienoic acid. Four lines of evidence suggested that H2O2 inhibited alveolar macrophage leukotriene and 5-HETE synthesis by depleting cellular ATP, a cofactor for 5-lipoxygenase. 1) H2O2 depleted ATP in A23187- and zymosan-stimulated alveolar macrophages with a dose dependence very similar to that for inhibition of agonist-induced leukotriene synthesis. 2) The time courses of ATP depletion and inhibition of leukotriene B4 synthesis by H2O2 were compatible with a rate-limiting effect of ATP on leukotriene synthesis in H2O2-exposed cultures. 3) Treatment of alveolar macrophages with the electron transport inhibitor antimycin A prior to A23187 stimulation depleted ATP and inhibited leukotriene B4 and C4 synthesis to equivalent degrees, while thromboxane A2 production was spared. 4) Incubation with the ATP precursors inosine plus phosphate attenuated both ATP depletion and inhibition of leukotriene B4 and C4 synthesis in alveolar macrophages stimulated with A23187 in the presence of H2O2. Our results show that H2O2 has the capacity to act both as an agonist for macrophage AA metabolism, and as a selective inhibitor of the 5-lipoxygenase pathway, probably as a result of its ability to deplete ATP. Depletion of cellular energy stores by oxidants generated during inflammation in vivo may be a means by which the inflammatory response is self-limited.     

Hydrogen peroxide inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells

Hydrogen peroxide (H(2)O(2)) induces a number of events, which are also induced by mitogens. Since the progression through the G1 phase of the cell cycle is dependent on mitogen stimulation, we were interested to study the effect of H(2)O(2) on the cell cycle progression. This study demonstrates that H(2)O(2) inhibits DNA synthesis in a dose-dependent manner when given to cells in mitosis or at different points in the G1 phase. Interestingly, mitotic cells treated immediately after synchronization are significantly more sensitive to H(2)O(2) than cells treated in the G1, and this is due to the inhibition of the cell spreading after mitosis by H(2)O(2). H(2)O(2) reversibly inhibits focal adhesion activation and stress fiber formation of mitotic cells, but not those of G1 cells. The phosphorylation of MAPK is also reversibly inhibited in both mitotic and G1 cells. Taken together, H(2)O(2) is probably responsible for the inhibition of the expression of cyclin D1 and cyclin A observed in cells in both phases. In conclusion, H(2)O(2) inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells. This may play a role in pathological processes in which H(2)O(2) is generated.     

The insulin-like effect of hydrogen peroxide on pathways of lipid synthesis in rat adipocytes.

In addition to the well known insulin-like effects of certain concentrations of H2O2 on glucose transport and oxidation in isolated rat adipocytes, the present work demonstrates that lipid synthesis from glucose is also enhanced over a narrow range of H2O2 concentrations (0.15 to 0.5 mM) added to the incubation medium. As in the case of insulin, H2O2 was found to stimulate greater glucose incorporation into glyceride-fatty acid than incorporation into glyceride-glycerol. As part of a multifaceted regulation of lipogenesis, H2O2, like insulin, increased the amount of pyruvate dehydrogenase in the active form without increasing the total amount of pyruvate dehydrogenase. Pyruvate dehydrogenase activity increased within 5 min of H2O2 incubation, reached a maximum at 15 min and declined thereafter as the H2O2 disappeared from the incubation medium. While medium glucose per se was found to activate the enzyme, it is unlikely that the effect of H2O2 was mediated by the known enhancement of glucose transport since the effects on the enzyme were maximal in the absence of glucose in the incubation medium. These findings add to the growing list of insulin effects that are reproduced by H2O2, and strengthen the hypothesis that assigns H2O2 the role of "second messenger" of insulin.     

Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through the induction of GTP-cyclohydrolase I and increases nitric oxide synthase activity in vascular endothelial cells

Tetrahydrobiopterin (BH4), which is an essential cofactor for nitric oxide synthase (NOS), is generally accepted as an important molecular target for oxidative stress. This study examined whether hydrogen peroxide (H(2)O(2)), one of the reactive oxygen species (ROS), affects the BH4 level in vascular endothelial cells (ECs). Interestingly, the addition of H(2)O(2) to ECs markedly increased the BH4 level, but not its oxidized forms. The H(2)O(2)-induced increase in the BH4 level was blocked by the inhibitor of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of BH4 synthesis. Moreover, H(2)O(2) induced the expression of GTPCH mRNA, and the inhibitors of protein synthesis blocked the H(2)O(2)-induced increase in the BH4 level. The expression of the inducible isoform of NOS (iNOS) was slightly induced by the treatment with H(2)O(2). Additionally, the L-citrulline formation from L-arginine, which is the marker for NO synthesis, was stimulated by the treatment with H(2)O(2), and the H(2)O(2)-induced L-citrulline formation was strongly attenuated by NOS or GTPCH inhibitor. These results suggest that H(2)O(2) induces BH4 synthesis via the induction of GTPCH, and the increased BH4 is coupled with NO production by coinduced iNOS. H(2)O(2) appears to be one of the important signaling molecules to regulate the BH4-NOS system.     

Study of activated oxygen production by some thiols using chemiluminescence.

2-3-dimercapto-1-propane sulfonic acid, D-penicillamine and meso-dimercapto succinic acid, drugs widely applied as antidota against metal poisoning, and cysteine and glutathione were studied with respect to their ability to generate and to scavenge superoxide anion radical. Superoxide production and scavenging were tested by means of luminol-dependent chemiluminescence. In presence of 1 mumol/l ADP-Fe3+ only cysteine and meso-dimercapto succinic acid induced chemiluminescence which could be inhibited by superoxide dismutase. 2,3-dimercapto-1-propane sulfonic acid, D-penicillamine and glutathione acted as O2- scavengers. These thiols inhibited O2(-)-dependent lipid peroxidation thus acting as antioxidants, whereas cysteine and meso-dimercapto succinic acid accelerated peroxidation. It is suggested that the toxic side effects of thiols may be due to their ability to generate or to scavenge free radicals.     

Role of active forms of oxygen in the induction of phytoalexin synthesis in Allium cepa cells

The results of studies on the role of plant superoxidesynthase signal system in elicitation of antimicrobial phytoalexin (PA) synthesis in cultured Allium cepa cells are presented. Exogenic application of O2- and H2O2 generators results in formation of PA--Tcibulins 1D and 2 [symbol: see text] in A. cepa cells. The mechanism of PA elicitation does not require peroxidase activity. However, the inhibition of one of the possible sources of the reactive oxygen, HADPH oxidase, suppresses elicitor-stimulated PA production. "Oxidative burst" modulation by different chemical compounds in A. cepa cells results in changes of PA synthesis elicitation. The results obtained suggest the tough correlation between "oxidative burst" and elicitation of defense responses, PA synthesis in particular.