Hydrogen peroxide produced inside mitochondria takes part in cell-to-cell transmission of apoptotic signal

In monolayer of HeLa cells treated with tumor necrosis factor (TNF), apoptotic cells formed clusters indicating possible transmission of apoptotic signal via the culture media. To investigate this phenomenon, a simple method of enabling two cell cultures to interact has been employed. Two coverslips were placed side by side in a Petri dish, one coverslip covered with apoptogen-treated cells (the inducer) and another with non-treated cells (the recipient). TNF, staurosporine, or H2O2 treatment of the inducer cells is shown to initiate apoptosis on the recipient coverslip. This effect is increased by a catalase inhibitor aminotriazole and is arrested by addition of catalase or by pre-treatment of either the inducer or the recipient cells with nanomolar concentrations of mitochondria-targeted cationic antioxidant MitoQ (10-(6 -ubiquinolyl)decyltriphenylphosphonium), which specifically arrests H2O2-induced apoptosis. The action of MitoQ is abolished by an uncoupler preventing accumulation of MitoQ in mitochondria. It is concluded that reactive oxygen species (ROS) produced by mitochondria in the apoptotic cells initiate the release of H2O2 from these cells. The H2O2 released is employed as a long-distance cell suicide messenger. In processing of such a signal by the recipient cells, mitochondrial ROS production is also involved. It is suggested that the described phenomenon may be involved in expansion of the apoptotic region around a damaged part of the tissue during heart attack or stroke as well as in "organoptosis", i.e. disappearance of organs during ontogenesis.     

Copper (II) complex-catalyzed oxidation of NADH by hydrogen peroxide

Among various metal ions of physiological interest, Cu²⁺ is uniquely capable of catalyzing the oxidation of NADH by H₂O₂. This oxidation is stimulated about fivefold in the presence of imidazole. A similar activating effect is found for some imidazole derivatives (1-methyl imidazole, 2-methyl imidazole, andN-acetyl-L-histidine). Some other imidazole-containing compounds (L-histidine,L-histidine methyl ester, andL-carnosine), however, inhibit the Cu²⁺-catalyzed peroxidation of NADH. Other chelating agents such as EDTA andL-alanine are also inhibitory. Stoichiometry for NADH oxidation per mole of H₂O₂ utilized is 1, which excludes the possibility of a two-step oxidation mechanism with a nucleotide free-radical intermediate. About 92% of the NADH oxidation product can be identified as enzymatically active NAD⁺. D₂O, 2,5-dimethylfuran, and 1,4-diazabicyclo [2.2.2]-octane have no significant effect on the oxidation, thus excluding¹O₂ as a mediator. Similarly, OH· is also not a likely intermediate, since the system is not affected by various scavengers of this radical. The results suggest that a copper-hydrogen peroxide intermediate, when complexed with suitable ligands, can generate still another oxygen species much more reactive than its parent compound, H₂O₂.     

Binding of fatty acids to the uncoupling protein from brown adipose tissue mitochondria

The uncoupling protein 1 (UCP1) is a H(+) carrier which plays a key role in heat generation in brown adipose tissue. The H(+) transport activity of UCP1 is activated by long-chain fatty acids and inhibited by purine nucleotides. While nucleotide binding has been well characterized, the interaction of fatty acid with UCP1 remains unknown. Here I demonstrate the binding of fatty acids by competition with a fluorescent nucleotide probe 2(')-O-dansyl guanosine 5(')-triphosphate (GTP), which has been shown previously to bind at the nucleotide binding site in UCP1. Fatty acids but not their esters competitively inhibit the binding of 2(')-O-dansyl GTP to UCP1. The fatty acid effect was enhanced at higher pH, suggesting the binding of fatty acid anion to UCP1. The inhibition constants K(i) were determined by fluorescence titrations for various fatty acids. Short-chain (C<8) fatty acids display no affinity, whereas medium-chain (C10-14) and unsaturated C18 fatty acids exhibit stronger affinity (K(i)=65 microM, for elaidic acid). This specificity profile agrees with previous functional data obtained in both proteoliposomes and mitochondria, suggesting a possible physiological role of this fatty acid binding site.     

Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide

Oxidation of glycerophosphate (GP) by brown adipose tissue mitochondria in the presence of antimycin A was found to be accompanied by significant production of hydrogen peroxide. GP-dependent hydrogen peroxide production could be detected by p-hydroxyphenylacetate fluorescence changes or as an antimycin A-insensitive oxygen consumption. One-electron acceptor, potassium ferricyanide, highly stimulated the rate of GP-dependent antimycin A-insensitive oxygen uptake, which was prevented by inhibitors of mitochondrial GP dehydrogenase (mGPDH) or by coenzyme Q(CoQ). GP-dependent ferricyanide-induced peroxide production was also determined luminometrically, using mitochondria or partially purified mGPDH. Ferricyanide-induced peroxide production was negligible, when succinate or NADH was used as a substrate. These results indicate that hydrogen peroxide is produced directly by mGPDH and reflect the differences in the transport of reducing equivalents from mGPDH and succinate dehydrogenase to the CoQ pool. The data suggest that more intensive production of reactive oxygen species may be present in mammalian cells with active mGPDH.     

Enhancement of BKCa channel activity induced by hydrogen peroxide: Involvement of lipid phosphatase activity of PTEN

Large-conductance calcium and voltage-dependent potassium (BK_Ca) channel is an important determinant of vascular tone. It is activated by hydrogen peroxide (H₂O₂) which occurs in various physiological and pathological processes. However, the regulation mechanism is not fully understood. In the present study, the mSlo in the presence or absence of hβ1 were cotransfected with the PTEN_wt, PTEN_C124S, PTEN_G129E in HEK 293 cells. Typical BK_Ca channel currents could be recorded in cell-attached configurations. We found that PTEN_wt reduced the H₂O₂-induced BK_Ca channel activation during the initial 10 min treatment. In contrast, coexpression with catalytically inactive PTEN_C124S/PTEN_G129E mutants that lack lipid phosphatase activity produced no regulation on the H₂O₂-induced BK_Ca channel activation. These results demonstrated that PTEN regulated the H₂O₂-induced BK_Ca channel activation through phosphatidylinositol 3-phosphatse. However, the inhibitory effect of PTEN on the H₂O₂-induced BK_Ca channel activation was attenuated when cells were treated with H₂O₂ at concentrations higher than 100 μM or at 100 μM for long-term treatment. In addition, the p-AKT expression level in PTEN_wt overexpressing cells was lower than that in control cells, and the increase of cytoplasmic free calcium concentration ([Ca²⁺]_i) induced by H₂O₂ was also inhibited. These findings may elucidate a new mechanism for H₂O₂-induced BK_Ca channel activation and provide some evidences for the role of PTEN on vasodilation induced by H₂O₂.     

Effects of light and hydrogen peroxide on gene expression of newly identified antioxidant enzymes in the harmful algal bloom species Chattonella marina

ABSTRACT

Antioxidant enzymes are essential proteins that maintain cell proliferation potential by protecting against oxidative stress. They are present in many organisms including harmful algal bloom (HAB) species. We previously identified the antioxidant enzyme 2-Cys peroxiredoxin (PRX) in the raphidophyte Chattonella marina. This enzyme specifically decomposes a hydrogen peroxide (H₂O₂). PRX is the only antioxidant enzyme so far identified in C. marina. This study used mRNA-seq, using Trinity assemble and blastx for annotation, to identify a further five antioxidant enzymes from C. marina: Cu Zn superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPX), catalase (CAT), ascorbate peroxidase (APX) and thioredoxin (TRX). In the gene expression analysis of six enzymes (Cu/Zn-SOD, GPX, CAT, APX, TRX and PRX) using light-acclimated (100 μmol photons m^?2 s^?1) C. marina cells, only PRX gene expression levels were significantly increased by strong light irradiation (1000 μmol photons m^?2 s^?1). H₂O₂ concentration and scavenging activity were also increased and significantly positively correlated with PRX gene expression levels. In dark-acclimated cells, expression levels of all antioxidant enzymes except APX were significantly increased by light irradiation (100 μmol photons m^?2 s^?1). Expression decreased the following day, with the exception of PRX expression. With the exception of CAT, gene expression of antioxidant enzymes was not significantly induced by artificial H₂O₂ treatment, although average gene expression levels were slightly increased in some enzymes. Thus, we suggest that light is the main trigger of gene expression, but the resultant oxidative stress is also a possible factor affecting the gene expression of antioxidant enzymes in C. marina.     

Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement

The deleterious effects of H₂O₂ on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. Exposure to H₂O₂ resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. The effect of H₂O₂ on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-DCIP oxidoreductase activities. Inhibitory effect of H₂O₂ on respiratory complexes was almost completely recovered by β-mercaptoethanol treatment. H₂O₂ treatment resulted in full resistance to Q_O site inhibitor myxothiazol and thus it is suggested that the quinol oxidase site (Q_O) of complex III is the target for H₂O₂. H₂O₂ did not modify basal levels of lipid peroxidation in yeast mitochondria. However, H₂O₂ addition to rat brain and liver mitochondria induced an increase in lipid peroxidation. These results are discussed in terms of the known physiological differences between mammalian and yeast mitochondria.     

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H₂DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

Evidence for H2O2 as the product of reduction of oxygen by alternative oxidase in mitochondria from potato tubers

Oxygen consumption by alternative oxidase (AOX), present in mitochondria of many angiosperms, is known to be cyanide-resistant in contrast to cytochrome oxidase. Its activity in potato tuber (Solanum tuberosum L.) was induced following chilling treatment at 4 °C. About half of the total O₂ consumption of succinate oxidation in such mitochondria was found to be sensitive to SHAM, a known inhibitor of AOX activity. Addition of catalase to the reaction mixture of AOX during the reaction decreased the rate of SHAM-sensitive oxygen consumption by nearly half, and addition at the end of the reaction released nearly half of the consumed oxygen by AOX, both typical of catalase action on H₂O₂. These findings with catalase suggest that the product of reduction of AOX is H₂O₂ and not H₂O, as previously surmised. In potatoes subjected to chill stress (4 °C) for periods of 3, 5 and ?8 days the activity of AOX in mitochondria increased progressively with a corresponding increase in the AOX protein detected by immunoblot of the protein.     

Modulation of matrix Ca2+ content by the ADP/ATP carrier in brown adipose tissue mitochondria. Influence of membrane lipid composition

The role of the adenine nucleotide translocase on Ca²⁺ homeostasis in mitochondria from brown adipose tissue was examined. It was found that in mitochondria incubated with 50 M Ca²⁺, ADP was not needed to retain the cation, but it was required for strengthening the inhibitory effect of cyclosporin on membrane permeability transition as induced by menadione. In addition, carboxyatractyloside was unable to promote matrix Ca²⁺ release, even though it inhibits the ADP exchange reaction. However, when the Ca²⁺ concentration was increased to 150 M, carboxyatractyloside did induce Ca²⁺ release, and ADP favored Ca²⁺ retention. Determination of cardiolipin content in the inner membrane vesicles showed a greater concentration in brown adipose tissue mitochondria than that found in kidney mitochondria. It is suggested that the failure of the adenine nucleotide translocase to influence membrane permeability transition depends on the lipid composition of the inner membrane.     

The quantitative measurement of H2O2 generation in isolated mitochondria

Adequate methods to measure the rate of mitochondrial oxygen radical generation are needed since oxygen radicals are involved in many pathologies. A fluorometric method appropriate to measure the rate of generation of H₂O₂ in intact mitochondria is described. Just after isolation of functional mitochondria from fresh tissues, rates of generation of H₂O₂ are kinetically measured by fluorometry in the presence of homovanillic acid and horseradish peroxidase. The method is specific for H₂O₂ and is sensitive enough to assay mitochondrial H₂O₂ generation in the presence of respiratory substrate without inhibitors of the respiratory chain. Simultaneous measurement of mitochondrial oxygen consumption allows calculation of the free radical leak: the percentage of electrons out of sequence which reduce oxygen to oxygen radicals along the mitochondrial respiratory chain instead of reducing oxygen to water at the terminal cytochrome oxidase. The method shows instantaneous response to H₂O₂. This makes it appropriate to study the quick effects of different inhibitors and modulators on the rate of mitochondrial oxygen radical production. Its application to the localization of the sites where caloric restriction decreases mitochondrial oxygen radical generation in heart mitochondria is described.     

Competitive inhibition of hydrogen peroxide-induced aggregation of calf platelets by prostaglandin H2/thromboxane A2 receptor ligands

Hydrogen peroxide (H₂O₂)-induced aggregation of calf platelets and its modification by agents with specific properties were characterized employing a spectrophotometric assay. An Arrhenius activation energy of 20 ± 1 kcal/mol was found in the temperature range of 25‡-36‡C. Rate inhibition occurred on either side of this temperature range, and under anaerobic conditions. Exogenous Ca²⁺ ions were not required but Ca²⁺ ions, at 1 mM-concentration, optimally increased rates and extent of aggregation at suboptimal H₂O₂ concentrations but only extent of aggregation at optimal H₂O₂ concentrations. Ba²⁺, Sr²⁺, Cd²⁺, Mn²⁺ and Ni²⁺ ions (1 mM) and Zn²⁺, Pb²⁺ and Hg²⁺ ions (10 mM) were inhibitory. The cyclo-oxygenase inhibitor, indomethacin (10-30 mM) exerted only mild inhibition by a competitive mechanism. Another cyclo-oxygenase inhibitor, aspirin, functioned to increase aggregation. Ligands acting directly at the prostaglandin H₂/thromboxane A, receptor (5Z. 9, 11, 13E, 15(S) 15-hydroxy 9(11) epoxy methano prosta 5, 13-dien-1-oic acid, pinane thromboxane A₂, arachidonic acid, eicosapentaenoic acid, and N-ethylmaleimide) functioned as competitive inhibitors. Another platelet-activating sulphydryl reagent, thimerosal, also inhibited competitively while the protein kinase C inhibitor, sphingosine, and the protein kinase C modulator, Zn²⁺ ions, inhibited by different mechanisms. The results indicate direct action of H₂O₂ at the prostaglandin H₂/thromboxane A₂ receptor, possibly its sulphydryls, to activate the protein kinase C pathway, independently of cyclo-oxygenase products. The results underscored the power of the kinetic approach for investigating mechanisms of platelet activation.     

Osmotic stress-induced changes in cell wall peroxidase activity and hydrogen peroxide level in roots of rice seedlings

The changes in activity of peroxidase (POD) extracted from the cellwalls and the level of H₂O₂ in rice seedling rootstreatedwith mannitol and their correlation with root growth were investigated.Increasing concentrations of mannitol from 92 to 276 mM, which isiso-osmotic with 50 to 150 mM NaCl, progressively reduced rootgrowth and increased POD activities extracted from the cell walls of riceroots.The reduction of growth was also correlated with an increase inH₂O₂ level. Both diamine oxidase (DAO) and NADHperoxidase(NADH-POD) are known to be responsible for the generation ofH₂O₂. Mannitol treatment increased DAO but not NADH-PODactivities in roots of rice seedlings, suggesting that DAO contributes to thegeneration of H₂O₂ in the cell walls of mannitol-treatedroots. An increase in the level of H₂O₂ and the activityof POD extracted from the cell walls of rice roots preceded root growthreduction caused by mannitol. An increase in DAO activity coincided with anincrease in H₂O₂ in roots caused by mannitol. Since DAOcatalyses the oxidation of putrescine, the demonstration that mannitolincreasesthe activity of DAO in roots is consistent with those that mannitol decreasesthe level of putrescine. In conclusion, cell-wall stiffening catalysed by PODispossibly involved in the regulation of root growth reduction caused bymannitol.     

Prostaglandins attenuate cardiac contractile dysfunction produced by free radical generation but not by hydrogen peroxide

The aim of this study was to examine and compare the potential influence of cyclooxygenase or lipoxygenase derived metabolises of arachidonic acid on myocardial injury produced either by a free radical generating system consisting of purine plus xanthine oxidase or that produced by hydrogen peroxide. A free radical generating system consisting of purine (2.3 mM) and xanthine oxidase (10 U/L) as well as hydrogen peroxide (75 µM) produced significant functional changes in the absence of either significant deficits in high energy phosphates or ultrastructural damage. Prostaglandin F2; (30 nM) significantly attenuated both the negative inotropic effect of purine plus xanthine oxidase as well as the ability of the free radical generator to elevate resting tension. An identical concentration of prostaglandin I2 (prostacyclin) significantly reduced resting tension elevation only and had no effect on contractile depression. The salutary effects of the two PGs occurred in the absence of any inhibitory influence on superoxide anion generation produced by the purine and xanthine oxidase reaction. None of prostaglandins modulated the response to hydrogen peroxide. In addition, neither prostaglandin E2 nor leukotrienes exerted any effect on changes produced by either type of oxidative stress. A 5 fold elevation in the concentrations of free radical generators or hydrogen peroxide produced extensive injury as characterized by a virtual total loss in contractility, 400% elevation in resting tension, ultrastructural damage and significant depletions in high energy phosphate content. None of these effects were modulated by eicosanoid treatment. Our results therefore demonstrate a selective ability of both prostaglandin F2; and to a lesser extent prostacyclin, to attenuate dysfunction produced by purine plus xanthine oxidase but not hydrogen peroxide. It is possible that these eicosanoids may represent endogenous protective factors under conditions of enhanced oxidative stress associated with superoxide anion generation.     

Hydrogen peroxide mediates Rac1 activation of S6K1

We previously reported that hydrogen peroxide (H2O2) mediates mitogen activation of ribosomal protein S6 kinase 1 (S6K1) which plays an important role in cell proliferation and growth. In this study, we investigated a possible role of H2O2 as a molecular linker in Rac1 activation of S6K1. Overexpression of recombinant catalase in NIH-3T3 cells led to the drastic inhibition of H2O2 production by PDGF, which was accompanied by a decrease in S6K1 activity. Similarly, PDGF activation of S6K1 was significantly inhibited by transient transfection or stable transfection of the cells with a dominant-negative Rac1 (Rac1N17), while overexpression of constitutively active Rac1 (Rac1V12) in the cells led to an increase in basal activity of S6K1. In addition, stable transfection of Rat2 cells with Rac1N17 dramatically attenuated the H2O2 production by PDGF as compared with that in the control cells. In contrast, Rat2 cells stably transfected with Rac1V12 produced high level of H2O2 in the absence of PDGF, comparable to that in the control cells stimulated with PDGF. More importantly, elimination of H2O2 produced in Rat2 cells overexpressing Rac1V12 inhibited the Rac1V12 activation of S6K1, indicating the possible role of H2O2 as a mediator in the activation of S6K1 by Rac1. However, H2O2 could be also produced via other pathway, which is independent of Rac1 or PI3K, because in Rat2 cells stably transfected with Rac1N17, H2O2 could be produced by arsenite, which has been shown to be a stimulator of H2O2 production. Taken together, these results suggest that H2O2 plays a pivotal role as a mediator in Rac1 activation of S6K1.     

Membrane potential and H2O2 production in duodenal mitochondria from broiler chickens (Gallus gallus domesticus) with low and high feed efficiency

Increased hydrogen peroxide (H2O2) production was observed in duodenal mitochondria obtained from broiler chickens with low feed efficiency (FE). As a decrease in mitochondrial membrane potential (Deltapsi(m)) due to mild uncoupling of oxidative phosphorylation reduces reactive oxygen species production, this study was conducted to evaluate the effect of uncoupling on Deltapsi(m) and H2O2 production in duodenal mitochondria isolated from broilers with low (0.48+/-0.02) and high (0.68+/-0.01) FE. Membrane potential and H2O2 production were measured fluorometrically and in the presence of different levels of an uncoupler, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). The Deltapsi(m) was higher (P相似文献   

Hydrogen peroxide primes heart regeneration with a derepression mechanism

While the adult human heart has very limited regenerative potential, the adult zebrafish heart can fully regenerate after 20% ventricular resection. Although previous reports suggest that developmental signaling pathways such as FGF and PDGF are reused in adult heart regeneration, the underlying intracellular mechanisms remain largely unknown. Here we show that H₂O₂ acts as a novel epicardial and myocardial signal to prime the heart for regeneration in adult zebrafish. Live imaging of intact hearts revealed highly localized H₂O₂ (∼30 μM) production in the epicardium and adjacent compact myocardium at the resection site. Decreasing H₂O₂ formation with the Duox inhibitors diphenyleneiodonium (DPI) or apocynin, or scavenging H₂O₂ by catalase overexpression markedly impaired cardiac regeneration while exogenous H₂O₂ rescued the inhibitory effects of DPI on cardiac regeneration, indicating that H₂O₂ is an essential and sufficient signal in this process. Mechanistically, elevated H₂O₂ destabilized the redox-sensitive phosphatase Dusp6 and hence increased the phosphorylation of Erk1/2. The Dusp6 inhibitor BCI achieved similar pro-regenerative effects while transgenic overexpression of dusp6 impaired cardiac regeneration. H₂O₂ plays a dual role in recruiting immune cells and promoting heart regeneration through two relatively independent pathways. We conclude that H₂O₂ potentially generated from Duox/Nox2 promotes heart regeneration in zebrafish by unleashing MAP kinase signaling through a derepression mechanism involving Dusp6.     

Uncoupling protein in human brown adipose tissue mitochondria. Isolation and detection by specific antiserum

A protein of Mr 32 000 has been isolated from human infant brown adipose tissue mitochondria following the procedure used to purify the uncoupling protein from rat brown adipose tissue mitochondria. A specific antiserum has been raised against the human 32 kDa protein, and used to detect it by probing mitochondrial proteins separated by SDS-PAGE. The protein is present in large amounts in brown adipose tissue but is undetectable in human liver, heart or white adipose tissue. It has strong immunological cross-reactivity with rat brown adipose tissue uncoupling protein.     

Cell suspension cultures of spruce (Picea abies): inactivation of extracellular enzymes by fungal elicitor-induced transient release of hydrogen peroxide (oxidative burst)

Elicitation of suspension culture cells of spruce [Picea abies (L.) Karst] with a fungal cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii Bubak induced inactivation of extracellular enzymes. Extracellular peroxidase, -glucosidase and acid phosphatase, secreted by the cells during growth, and also -amylase and pectinase from Aspergillus strains, added to an elicited cell culture, were inactivated. Inactivation is caused by an elicitor-mediated transient release of H₂O₂ from the cells (oxidative burst). H₂O₂ released into the medium was determined with ABTS (2,2'-Azino-bis-(3-ethylbenthiazoline-6-sulfonate)) (formation of blue colour) and with phenol red (destruction of pH indicator). The release started only minutes after beginning of elicitation and its inactivating effect existed for more than 1 day. Release of H₂O₂ is a biphasic process with a first smaller maximum at 1 h, followed by a second larger increase, peaking at 5–6 h and returning to approximately the control levels thereafter. Also H₂O₂ is transiently released in small quantities from cell incubations in the absence of elicitor as a stress response of the cells to manipulations of the cultures. Extracellular enzymes secreted into the medium could also be inactivated by direct addition of exogenous H₂O₂. Catalase prevents inactivation of the secreted extracellular enzymes, however, to a limited extent only because, as a result of contact of cells and medium, catalase becomes inactivated. The ionophores A 23187 and cycloheximide induced release of H₂O₂ and, when present together with elicitor, induction was synergistically increased.