Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects

Chemical probes for free radicals in biology are important tools; fluorescence and chemiluminescence offer high detection sensitivity. This article reviews progress in the development of probes for "reactive oxygen and nitrogen" species, emphasizing the caution needed in their use. Reactive species include hydrogen peroxide; hydroxyl, superoxide, and thiyl radicals; carbonate radical-anion; and nitric oxide, nitrogen dioxide, and peroxynitrite. Probes based on reduced dyes lack selectivity and may require a catalyst for reaction: despite these drawbacks, dichlorodihydrofluorescein and dihydrorhodamine have been used in well over 2,000 studies. Use in cellular systems requires loading into cells, and minimizing leakage. Reactive species can compete with intracellular antioxidants, changes in fluorescence or luminescence possibly reflecting changes in competing antioxidants rather than free radical generation rate. Products being measured can react further with radicals, and intermediate probe radicals are often reactive toward antioxidants and especially oxygen, to generate superoxide. Common probes for superoxide and nitric oxide require activation to a reactive intermediate; activation is not achieved by the radical of interest and the response is thus additionally sensitive to this first step. Rational use of probes requires understanding and quantitation of the mechanistic pathways involved, and of environmental factors such as oxygen and pH. We can build on this framework of knowledge in evaluating new probes.     

UV-induced free radicals in the skin detected by ESR spectroscopy and imaging using nitroxides

Reactive free radicals and reactive oxygen species (ROS) induced by ultraviolet irradiation in human skin are strongly involved in the occurrence of skin damages like aging and cancer. In the present work an ex vivo method for the detection of free radicals/ROS in human skin biopsies during UV irradiation is presented. This method is based on the Electron Spin Resonance (ESR) spectroscopy and imaging and uses the radical trapping properties of nitroxides. The nitroxides 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO), 3-Carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCM), and 3-Carboxy-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCA), were investigated for their applicability of trapping reactive free radicals and reactive oxygen species in skin during UV irradiation. As a result of the trapping process the nitroxides were reduced to the EPR silent hydroxylamins. The reduction rate of TEMPO was due to both the UV radiation and the enzymatic activity of the skin. The nitroxides PCM and PCA are sufficiently stable in the skin and are solely reduced by UV-generated free radicals/ROS. The nitroxide PCA was used for imaging the spatial distribution of UV-generated free radicals/ROS. As a result of the homogeneous distribution of PCA in the skin, it was possible to estimate the penetration of UVA and UVB irradiation: The UV irradiation decreased the PCA intensity corresponding to its irradiance and penetration into the skin. This reduction was shown to be caused mainly by UVA radiation (320-400 nm).     

Oxidative metabolism and release of myeloperoxidase from polymorphonuclear leukocytes obtained from blood sedimentation in a Ficoll-Hypaque gradient

Polymorphonuclear neutrophils (PMN) have an important role in the host defence response to infection. These cells produce large amounts of reactive oxygen species (O(2).(-), H(2)O(2) and ONOO(-)) with microbicidal activity. PMN are commonly isolated from peripheral blood by sedimentation through a gradient of density (Ficoll-Hypaque gradient and dextran), yielding a highly homogeneous cellular population. However, some cellular activation due to membrane perturbation is also expected. We studied how the production of reactive oxygen species and release of myeloperoxidase (MPO) from blood PMN are affected by the use of the Ficoll-Hypaque density gradient. PMN isolated by spontaneous sedimentation and total blood were used for comparisons. Lucigenin- and luminol-enhanced chemiluminescence was used to estimate the production of reactive oxygen from intact cells and shown to be higher for cells isolated by density gradient both in the absence and presence of added stimuli. The release of MPO, estimated by the chemiluminescence of the luminol/H(2)O(2) reaction in the supernatant of PMN incubated in the absence and presence of stimuli and absence and presence of cytochalasin B, was also higher for PMN isolated by a density gradient. In conclusion, it was shown that the PMN isolation procedure affects reactive oxygen species production and MPO release and in some cases may cause a misinterpretation of results.     

Oxygen-derived free radicals producing activity and survival of activated polymorphonuclear leukocytes

Summary Activation of polymorphonuclear (PMN) leukocytes is known to generate oxygen free radicals (OFR). However the fate of activated PMN leukocytes is not known. We investigated the OFR producing (chemiluminescence) activity and the survival of the activated PMN leukocytes. The study was divided into two groups. Group I, In vivo study (n = 7): zymosan (8.4 mg/kg) was administered intravenously in the anesthetized dogs and the blood samples were collected before and after 5, 15, 30, 60 and 120 min of zymosan administration. This group represents the in vivo pre-stimulated PMN leukocytes; Group II, In vitro study (n = 7): the blood were collected from dogs and further divided into two groups. Group A (n = 7): non-stimulated, without any added zymosan and group B (n = 7): zymosan was added to stimulate PMN leukocytes. Blood samples from group A and B were also collected at various time intervals similar to in vivo studies. Oxygen free radical producing activity of PMN leukocytes was monitored by measuring luminoldependent chemiluminescence (CL). Opsonized zymosan was used to activate PMN leukocytes. The studies in which the PMN leukocytes were stimulated in in vivo, both oxygen derived free radicals and superoxide dismutase (SOD) inhibitable oxygen free radical CL decreased significantly for 60 min and tended to reach thereafter to the pre-stimulated values. The resting chemiluminescence (chemiluminescence without zymosan stimulation in the assay medium) increased significantly for 15 min reaching to pre-stimulated values at 30 min and thereafter. In in vitro studies, oxygen derived free radicals CL of pre-stimulated PMN leukocytes (Group B) was depressed for the whole duration of investigation while SOD inhibitable CL was depressed for only 60 min. There was approximately a two-fold increase in the resting CL within 5 min of PMN leukocyte activation and it remained high for the whole duration of study. The chemiluminescence of non-stimulated PMN leukocytes in vitro (group A) remained practically normal throughout the period of observation. In in vivo studies, total white blood cells (WBC) and PMN leukocyte counts decreased initially and tended to approach towards pre-stimulated values at the end of the protocol. There were no changes in these counts in in vitro studies. These results indicate that the capacity to generate OFR is decreased in the in vivo and in vitro pre-stimulated PMN leukocytes. However this activity recovers with time. This study also suggests that the activated PMN leukocytes are not destroyed.     

Quinoid redox cycling as a mechanism for sustained free radical generation by inhaled airborne particulate matter.

The health effects of airborne fine particles are the subject of government regulation and scientific debate. The aerodynamics of airborne particulate matter, the deposition patterns in the human lung, and the available experimental and epidemiological data on health effects lead us to focus on airborne particulate matter with an aerodynamic mean diameter less than 2.5 microm (PM(2.5)) as the fraction of the particles with the largest impact in health. In this article we present a novel hypothesis to explain the continuous production of reactive oxygen species produced by PM(2.5) when it is deposited in the lung. We find PM(2.5) contains abundant persistent free radicals, typically 10(16) to 10(17) unpaired spins/gram, and that these radicals are stable for several months. These radicals are consistent with the stability and electron paramagnetic resonance spectral characteristics of semiquinone radicals. Catalytic redox cycling by semiquinone radicals is well documented in the literature and we had studied in detail its role on the health effects of cigarette smoke particulate matter. We believe that we have for the first time shown that the same, or similar radicals, are not confined to cigarette smoke particulate matter but are also present in PM(2.5). We hypothesize that these semiquinone radicals undergo redox cycling, thereby reducing oxygen and generating reactive oxygen species while consuming tissue-reducing equivalents, such as NAD(P)H and ascorbate. These reactive oxygen species generated by particles cause oxidative stress at sites of deposition and produce deleterious effects observed in the lung.     

Consequences of chemical modifications on the free radical reactions of human hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are candidates for use as blood substitutes and resuscitation fluids. We determined that HBOCs of specific types differ in their ability to generate or interact with free radicals. The differences do not correlate with oxygen affinity. Detailed comparisons with unmodified human hemoglobin, HbA0, were carried out with two cross-linked derivatives: HbA-FMDA, produced by the reaction of human oxyhemoglobin with fumaryl monodibromoaspirin, and HbA-DBBF, produced by the reaction of human deoxyhemoglobin with bis(3,5-dibromosalicyl) fumarate. Both derivatives had lower oxygen affinity than unmodified HbA0. As previously reported, exposure of oxyhemoglobin to H2O2 causes generation of free radicals capable of generating formaldehyde from dimethyl sulfoxide. Relative to the reaction catalyzed by 50 microM HbA (18.0 +/- 3.5 nmol/30 min/ml), the formaldehyde formation was roughly 70% for HbA-DBBF and 50% for HbA-FMDA under comparable conditions. More profound differences are exhibited at lower hemoglobin concentrations. Spectral changes of the HBOCs during the reaction differ qualitatively and occur at different rates. The HBOCs also differ in rates of hemoglobin-catalyzed NADPH oxidation and aniline hydroxylation, reactions mediated by reactive oxygen species. These results show that stereochemical differences brought about by chemical cross-linking alter the ability of HBOCs to generate radicals and to react with activated oxygen species. These studies also show that the ability of hemoglobin to produce activated species of oxygen can be enhanced or suppressed independently of oxygen affinity.     

Effect of reactive oxygen species on the erythrocyte calcium-pump function in protein-energy malnutrition

The presence of detectagle amounts of non-heme iron in erythrocyte ghost membranes have been postulated to lead to the initiation of membrane lipid peroxidation and the attendant perturbation of membrane functions. We have investigated the presence of non-heme iron and endogenous products of lipid peroxidation in erythrocyte membranes of normal and kwashiorkor (KWA) subjects and assessed the susceptibility of the membranes to exogenously generated reactive oxygen species. The modulation of the basal and calmodulin-stimulated calcium-pumping activity of these membranes by reactive oxygen species was also assessed. The results show the presence of significant amounts of non-heme iron and endogenous free radical reaction products in the red cell membranes of KWA subjects compared with that of normal children. Estimation of the extent of lipid peroxidation in the presence of exogenously generated reactive oxygen species further revealed that erythrocyte ghost membranes of KWA subjects are more susceptible to oxidative stress than those of normal individuals. Although both the basal and calmodulin-stimulated activities of the membrane-bound Ca²⁺-pump enzyme in normal and KWA subjects were inhibited by oxygen-free radicals, the erythrocyte enzyme in KWA subjects showed higher susceptibility to inhibition by oxygen free radicals than that of normal individuals. We propose that the reduced erythrocyte calcium-pump function in KWA is not unconnected with excessive generation of reactive oxygen species.Abbreviations PMSF phenylmethylsulfonylfuloride - TLCK N--p-tosyl-l-lysine chloromethylketone - EGTA ethyleneglycol-bis (B-aminoethylether) N,N-tetraacetic acid - EDTA ethylene diamine tetraacetic acid - ATP Adenosine 5-triphosphate - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - Tris-HCl Tris (hydroxymethyl) aminomethane-hydrochloride - SDS Sodium dodecyl sulphate - TBAR thiobarbituric acid-reactive products TBA, thiobarbituric acid - TCA trichloroacetic acid     

Antioxidant properties of bile salt micelles evaluated with different chemiluminescent assays: a possible physiological role

The antioxidant activity of a representative series of free, glycine- and taurine-conjugated bile acids was evaluated by two different chemiluminescent assays: (a) the enhanced chemiluminescence system based on horseradish peroxidase and luminol/oxidant/enhancer reagent, and (b) the hypoxanthine/xanthine oxidase/Fe²⁺-EDTA/luminol system. Bile acids were studied at final concentrations ranging from 1 to 28 mmol/L. All of the bile acids studied inhibited the steady-state chemiluminescent reaction and the extent of inhibition depended upon the structure of the bile acids, whereas the duration was related to bile acid concentration. The mechanism of the light inhibition is probably due to trapping of oxygen free radicals generated in the chemiluminescent reactions, within bile acid micelles. The free radicals trapped into micelles reduced the formation of luminol radicals and consequently the light output; when the micelles were saturated, the oxygen free radicals in solution again produced luminol radicals. The micelle interaction with reactive oxygen species could be a physiological mechanism of defence against the toxicity of those species in the intestinal content. On the other hand, alterations in bile acid organ distribution, concentration and composition leads to a membrane damage caused by their detergent-like properties, which could be associated to oxygen free radical production. © 1998 John Wiley & Sons, Ltd.     

Reduced ligand affinity leads to an impaired function of the adenosine A2A receptor of human granulocytes in sepsis

The enhanced release of reactive oxygen species by excessively activated polymorphonuclear leucocytes (PMN) is a key step in the pathogenesis of sepsis. Potent action of adenosine in inhibiting cytotoxic PMN functions has been documented. Recent data, however provide evidence that in sepsis a diminished capability of adenosine to inhibit the generation of oxygen radicals by PMN occurs. Here, we investigated the underlying mechanisms in an in vitro sepsis model and in PMN of sepsis patients. We report that lipopolysaccharide (LPS)-incubation of human PMN elicited the same increase in the half-maximal inhibitory concentration (IC₅₀) of adenosine as observed in patients with septic shock. Coupling to adenylyl cyclase was impaired as well, as indicated by a decreased potency of adenosine to stimulate cyclic adenosine monophosphate (cAMP) accumulation. Ligand-binding studies conducted with native, LPS-stimulated PMN, and with PMN of sepsis patients revealed that, despite an increased adenosine A_2A receptor (A_2AR) expression, the receptor function declines due to a diminished ligand-binding affinity most likely caused by allosteric modulators within the inflammatory environment. A_2AR function obviously is highly dependent upon the cellular environment and thus, further functional characterization of A_2AR responses in sepsis may be a promising approach to develop new adenosine or A_2AR agonists based therapeutic strategies.     

Reactive oxygen species regulate oxygen-sensitive potassium flux in rainbow trout erythrocytes

In the present study, we have investigated if reactive oxygen species are involved in the oxygen-dependent regulation of potassium-chloride cotransport activity in trout erythrocyte membrane. An increase in the oxygen level caused an increase in chloride-sensitive potassium transport (K(+)-Cl(-) cotransport). 5 mM hydrogen peroxide caused an increase in K(+)-Cl(-) cotransport at 5% oxygen. The increase in flux could be inhibited by adding extracellular catalase in the incubation. Pretreatment of the cells with mercaptopropionyl glycine (MPG), a scavenger of reactive oxygen species showing preference for hydroxyl radicals, abolished the activation of the K(+)-Cl(-) cotransporter by increased oxygen levels. The inhibition by MPG was reversible, and MPG could not inhibit the activation of transporter by the sulfhydryl reagent, N-ethylmaleimide, indicating that the effect of MPG was due to the scavenging of reactive oxygen species and not to the reaction of MPG with the cotransporter. Copper ions, which catalyze the production of hydroxyl radicals in the Fenton reaction, activated K(+)-Cl(-) cotransport significantly at hypoxic conditions (1% O(2)). These data suggest that hydroxyl radicals, formed from O(2) in close vicinity to the cell membrane, play an important role in the oxygen-dependent activation of the K(+)-Cl(-) cotransporter.     

Oxygen free radical producing activity of polymorphonuclear leukocytes in patients with Parkinson's disease

Oxygen free radicals (OFRs) have been suggested in the pathogenesis of Parkinson's disease (PD). These free radicals exert their cytotoxic effect by peroxidation of lipid membrane resulting in the formation of malondialdehyde (MDA). Polymorphonuclear (PMN) leukocyte is one of the major sources of OFR. However, the oxygen free radical producing activity of PMN leukocytes in patients with PD is not known. We therefore studied the oxygen free radical producing activity of polymorphonuclear leukocytes and MDA levels in the serum of healthy subjects and in patients with Parkinson's disease. The oxygen free radical producing activity of PMN leukocytes in blood and the MDA content in serum were significantly higher in patients with Parkinson's disease than in healthy subjects. These results indicate a possible role of oxygen free radicals in the pathogenesis of Parkinson's disease.     

Formation of Methemoglobin and Phenoxyl Radicals from P-Hydroxyanisole and Oxyhemoglobin

The reaction of p-hydroxyanisole with oxyhemoglobin was investigated using electron spin resonance spectroscopy (ESR) and visible spectroscopy. As a reactive reaction intermediate we found the p-methoxyphenoxyl radical, the one-electron oxidation product of p-hydroxyanisole. Detection of this species required the rapid flow device elucidating the instability of this radical intermediate. The second reaction product formed is methemoglobin. Catalase or SOD had no effect upon the reaction kinetics. Accordingly, reactive oxygen species such as hydroxyl radicals or superoxide could not be observed although the spin trapping agent DMPO was used to make these short-lived species detectable. When the sulfhydryl blocking agents N-ethylmaleimide or mersalyl acid were used, an increase of the methemoglobin formation rate and of the phenoxyl radical concentration were observed. We have interpreted this observation in terms of a side reaction of free radical intermediates with thiol groups.     

In situ measurement of superoxide and hydroxyl radicals by frequency mixing detection technique

Frequency mixing magnetic detection (FMMD) was used to detect superoxide from hypoxanthine and xanthine reaction and to detect hydroxyl radical from the Fenton reaction. FMMD was also applied to measure the reactive oxygen species (ROS) level released from microglial cells. We could assess the formation and extinction of the free radicals without a spin trap reagent. The FMMD signal amplitude scaled with the concentration of the radicals. It was verified that no signals are obtained from the substrates and reagents. Based on the observations and on previous research, we suggest that the FMMD signals originate from superoxide and hydroxyl radicals, indicating that FMMD can be used to detect O-centered radicals. Subsequent analysis of free radicals generated from living microglial cells showed that there were significant differences between the activated microglial cells and resting ones. The results of this research are promising regarding the applications of FMMD for in situ measurement of free radicals from various sources, including the cell.     

多形核白细胞(PMN)引起鼠缺血心脏产生活性氧自由基的研究

本文用电子(?)磁共振(ESR)在低温条件下直接研究了由维生素D_3(V_(D3))过量所致大白鼠心肌缺血损伤时血液中多形核白细胞(PMN)产生的活性氧自由基.实验结果发现,过量VD_3造成缺血损伤心肌中氧自由基含量比正常心肌增加了43%,比用10ml生理盐水冲洗的正常心肌增加了73%,比用过量V_(D3)造成心肌缺血损伤再用10ml生理盐水和冲洗的心肌增加了65%.这就说明,PMN在心肌缺血过程中对产生活性氧自由基起着主要作用.     

Chemiluminescence response of the human polymorphonuclear neutrophil to lipopolysaccharides

Polymorphonuclear neutrophils (PMN) respond to a variety of stimuli with a sequence of reactions that lead to the production of "active oxygen" species, including H2O2, free radicals, such as superoxide (O2-.) and hydroxyl (HO.), and singlet molecular oxygen (1O2). Some of these can oxidize (5-amino-2,3-dihydrophthalazine 1,4-dione) (luminol) to the ground state aminophthalate ion; this reaction sequence is accompanied by the generation of a photon and forms the basis for the chemiluminescence (CL) response. In this work we used a dedicated photon counting instrument to record CL from PMN incubated with bacterial lipopolysaccharide (LPS). We have studied the CL response to the LPS from Escherichia coli strains 026:B6 and 055:B5, as well as Salmonella minnesota RE 595 and have determined that CL requires heat-labile serum factors, these most likely being intact components of the complement system.     

Mass Spectrometry Reveals New Insights into the Production of Superoxide Anions and 4‐Hydroxynonenal Adducted Proteins in Human Sperm

The free‐radical theory of male infertility suggests that reactive oxygen species produced by the spermatozoa themselves are a leading cause of sperm dysfunction, including loss of sperm motility. However, the field is overshadowed on several fronts, primarily because: i) the probes used to measure reactive oxygen species (ROS) are imprecise; and ii) many reports suggesting that oxygen radicals are detrimental to sperm function add an exogenous source of ROS. Herein, a more reliable approach to measure superoxide anion production by human spermatozoa based on MS analysis is used. Furthermore, the formation of the lipid‐peroxidation product 4‐hydroxynonenal (4‐HNE) during in vitro incubation using proteomics is also investigated. The data demonstrate that neither superoxide anion nor other free radicals that cause 4‐HNE production are related to the loss of sperm motility during incubation. Interestingly, it appears that many of the 4‐HNE adducted proteins, found within spermatozoa, originate from the prostate. A quantitative SWATH analysis demonstrate that these proteins transiently bind to sperm and are then shed during in vitro incubation. These proteomics‐based findings propose a revised understanding of oxidative stress within the male reproductive tract.     

Hypothesis: the role of reactive sulfur species in oxidative stress.

Oxidative stress arises from an imbalance in the metabolism of redox-active species promoting the formation of oxidizing agents. At present, these species are thought to include reactive oxygen, reactive nitrogen, and reactive nitrogen oxygen species (ROS, RNS, and RNOS, respectively). Reactive species have their origin in enzymatic synthesis, environmental induction, or by the further chemical reaction of an active species with other endogenous molecules to generate a second-generation reactive species. These second-generation species possess a different spectrum of activity to the parent species, with different redox reactions and biological targets. We now propose that an additional group of redox active molecules termed "reactive sulfur species" (RSS) are formed in vivo under conditions of oxidative stress. RSS are likely to include disulfide-S-oxides, sulfenic acids, and thiyl radicals, and are predicted to modulate the redox status of biological thiols and disulfides.     

Superoxide formed from cigarette smoke impairs polymorphonuclear leukocyte active oxygen generation activity.

Reactive free radicals contained in cigarette smoke (CS) and compromised phagocytic antimicrobial activities including those of polymorphonuclear leukocytes (PMNs) have been implicated in the pathogenesis of severe CS-related pulmonary disorders. In CS-exposed buffer solutions, O2-. was the predominant generated reactive oxygen species, as demonstrated by lucigenin-amplified chemiluminescence and electron spin resonance (ESR) spin-trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO). When PMNs were incubated in this buffer, phorbol 12-myristate 13-acetate (PMA)-stimulated active oxygen production and coupled O2 consumption were strongly impaired without appreciably affecting PMN viability (1-min exposure inhibited active oxygen production by 75%). Superoxide dismutase (SOD) totally protected and an iron chelator, diethylenetriaminepentaacetic acid (DETAPAC), also protected the CS-exposed PMNs, suggesting that generated O2-. was an initiating factor in the impairment and OH. generation was a subsequent injurious factor. Pretreatment of PMNs with antioxidants such as alpha-tocopherol and dihydrolipoic acid (DHLA) was partially protective. The results suggest that (i) O2-. is probably generated in the upper and lower respiratory tract lining fluid when they come in contact with CS; (ii) such generated O2-. can primarily impair PMN capabilities to generate reactive oxygen species; and (iii) since these effects may contribute to the pathogenesis of CS-related lung diseases, prior supplementation with antioxidants such as alpha-tocopherol or DHLA might be successful in preventing these deleterious effects.     

Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing

In the present study, we provide evidence for the production of reactive oxygen species (ROS) during cryopreservation of bovine spermatozoa. Cooling and thawing of spermatozoa cause an increase in the generation of superoxide radicals. Although nitric oxide production remains unaltered during sperm cooling from 22-4 degrees C, a sudden burst of nitric oxide radicals is observed during thawing. Increase in lipid peroxidation levels have been observed in frozen/thawed spermatozoa and appears to be associated with a reduction in sperm membrane fluidity as detected by spin labeling studies. The data presented provide strong evidence that oxygen free radicals are produced during freezing and thawing of bovine spermatozoa and suggest that these reactive oxygen species may be a cause for the decrease in sperm function following cryopreservation. Mol. Reprod. Dev. 59: 451-458, 2001.     

Mechanism of peroxidase actions for salicylic acid-induced generation of active oxygen species and an increase in cytosolic calcium in tobacco cell suspension culture

Extracellularly secreted peroxidases in cell suspension culture of tobacco (Nicotiana tabacum L. cv. Bright Yellow-2, cell line BY-2) catalyse the salicylic acid (SA)-dependent formation of active oxygen species (AOS) which, in turn, triggers an increase in cytosolic Ca2+ concentration. Addition of horseradish peroxidase (HRP) to tobacco cell suspension culture enhanced the SA-induced increase in cytosolic Ca2+ concentration, suggesting that HRP enhanced the production of AOS. The mechanism of peroxidase-catalysed generation of AOS in SA signalling was investigated with chemiluminescence sensitive to AOS and electron spin resonance (ESR) spectroscopy, using the cell suspension culture of tobacco, and HRP as a model system of peroxidase reaction. The results showed that SA induced the peroxidase inhibitor-sensitive production of superoxide and H2O2 in tobacco suspension culture, but no production of hydroxy radicals was detected. Similar results were obtained using HRP. It was also observed that SA suppressed the H2O2-dependent formation of hydroxy radicals in vitro. The results suggest that SA protect the cells from highly reactive hydroxy radicals, while producing the less reactive superoxide and H2O2 through peroxidase-catalysed reaction, as the intermediate signals. The formation of superoxide was followed by that of H2O2, suggesting that superoxide was converted to H2O2. In addition, it was observed that superoxide dismutase-insensitive ESR signal of monodehydroascorbate radical was induced by SA both in the tobacco suspension culture and HRP reaction mixture, suggesting that SA free radicals, highly reactive against ascorbate, were formed by peroxidase-catalysed reactions. The formation of SA free radicals may lead to subsequent monovalent reduction of O2 to superoxide.