Cloning,expression, molecular modelling and docking analysis of glutathione transferase from Saccharum officinarum

Sugarcane yield and quality are affected by a number of biotic and abiotic stresses. In response to such stresses, plants may increase the activities of some enzymes such as glutathione transferase (GST), which are involved in the detoxification of xenobiotics. Thus, a sugarcane GST was modelled and molecular docked using the program LIGIN to investigate the contributions of the active site residues towards the binding of reduced glutathione (GSH) and 1‐chloro‐2,4‐dinitrobenzene (CDNB). As a result, W13 and I119 were identified as key residues for the specificity of sugarcane GSTF1 (SoGSTF1) towards CDNB. To obtain a better understanding of the catalytic specificity of sugarcane GST (SoGSTF1), two mutants were designed, W13L and I119F. Tertiary structure models and the same docking procedure were performed to explain the interactions between sugarcane GSTs with GSH and CDNB. An electron‐sharing network for GSH interaction was also proposed. The SoGSTF1 and the mutated gene constructions were cloned and expressed in Escherichia coli and the expressed protein purified. Kinetic analyses revealed different K_m values not only for CDNB, but also for GSH. The K_m values were 0.2, 1.3 and 0.3 mM for GSH, and 0.9, 1.2 and 0.5 mM for CDNB, for the wild type, W13L mutant and I119F mutant, respectively. The V_max values were 297.6, 224.5 and 171.8 µmol min^?1 mg^?1 protein for GSH, and 372.3, 170.6 and 160.4 µmol min^?1 mg^?1 protein for CDNB.     

High-performance liquid chromatography ultraviolet assay for human erythrocytic catalase activity by measuring glutathione as o-phthalaldehyde derivative

The most frequently used catalase (CAT) activity assay is based on the spectrophotometric measurement of hydrogen peroxide (H₂O₂) absorbance decrease at 240 nm. Here we report an alternative high-performance liquid chromatography (HPLC) assay for human erythrocytic CAT (heCAT) activity measurement based on glutathione (GSH) analysis as a highly stable, H₂O₂-insensitive o-phthalaldehyde (OPA) derivative. The method was developed and validated using an isolated heCAT in phosphate-buffered saline at pH 7.4 and was applied to measure CAT activity in lysed human erythrocytes. heCAT activity was measured at initial concentrations of 5 nM for heCAT, 5 mM for H₂O₂, and 10 mM for GSH, and the incubation time was 10 min. Nitrite (NO₂⁻) was found to be an uncompetitive inhibitor of heCAT activity (IC₅₀ = 9 μM) and of CAT activity in hemolysate (IC₅₀ ∼ 750 μM). Nitrate (NO₃⁻) at concentrations up to 100 μM did not inhibit heCAT activity. Azide (N₃⁻) was found to be a very strong inhibitor of the heCAT (IC₅₀ = 0.2 nM) but a relatively weak CAT inhibitor (IC₅₀ ∼ 10 μM) in human hemolysates. The novel CAT activity assay works under redox conditions that more closely resemble those prevailing in cells and allows high-throughput analysis despite the required HPLC step.     

The effect of some antineoplastic agents on glutathione S-transferase from human erythrocytes

Glutathione S-transferase was purified from human erythrocytes and effects of some antineoplastic agents were investigated on the enzyme activity. The purification procedure was composed of Glutathione-Agarose affinity chromatography after preparation of erythrocytes hemolysate. Using this procedure, the enzyme, having the specific activity of 16.00 EU/mg proteins, was purified 1143-fold with a yield of 80%. The purified enzyme showed a single band on the SDS-PAGE. The effects of paclitaxel, cyclophosphamide, and gemcitabine, are antineoplastic agents, were examined on the in vitro enzyme activity of glutathione S-transferase and were determined to be inhibitors for the enzyme. IC₅₀ values were 0.23 mM for paclitaxel, 5.57 mm for cyclophosphamide, and 6.35 mM for gemcitabine. These constants were 0.182 ± 0.028 mM and 0.162 ± 0.062 mM for paclitaxel, 6.97 ± 0.49 mM and 10.50 ± 5.43 mM for cyclophosphamide, and 6.71 mM and 7.93 mM for gemcitabine, with GSH and CDNB substrates, respectively. Inhibition types of all inhibitors were noncompetitive.     

Glutathione transferase from Plasmodium falciparum – Interaction with malagashanine and selected plant natural products

A glutathione transferase (PfGST) isolated from Plasmodium falciparum has been associated with chloroquine resistance. A range of natural products including malagashanine (MG) were screened for inhibition of PfGST by a GST assay with 1-chloro-2,4-dinitrobenzene as a substrate. Only the sesquiterpene (JBC 42C), the bicoumarin (Tral-1), ellagic acid and curcumin, were shown to be potent inhibitors of PfGST with IC₅₀ values of 8.5, 12, 50 and 69 μM, respectively. Kinetic studies were performed on PfGST using ellagic acid as an inhibitor. Uncompetitive and mixed types of inhibition were obtained for glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The K_i for GSH and CDNB were ?0.015?μM and 0.011?μM, respectively. Malagashanine (100?µM) only reduced the activity of PfGST to 80% but showed a time-dependent inactivation of PfGST with a t_1/2 of 34 minutes compared to >120 minutes in the absence of MG or in the presence of 5?mM GSH. This work facilitates the understanding of the interaction of PfGST with some plant derived compounds.     

Alterations of the glutathione redox cycle status in fumonisin B1-treated pig kidney cells

Fumonisin B₁ (FB₁) causes equine leukoencephalomalacia, porcine pulmonary edema, and liver tumors and chronic nephritis in rats. To investigate mechanisms by which FB₁ induces toxicity, effects of FB₁ on cellular glutathione (GSH) redox status and GSH depletion on FB₁ toxicity in pig kidney (LLC-PK₁) cells were studied. Treatment of LLC-PK₁ cells with 50 μM FB₁ for 24 hours significantly decreased cellular GSH contents from 56 ± 3.2 to 42.7 ± 4.4 nmol/mg protein (p < 0.05) and increased the activities of glutathione reductase (GR) from 25.7 ± 2.4 to 35.7 ± 5.0 μmol NADPH/mg protein (p < 0.05). The activities of glutathione peroxidase (GSHpx), catalase, and Cu,Zn-superoxide dismutase (SOD) were not changed by this treatment. Treatment of LLC-PK₁ cells for 12 hours with 0.1. mM buthionine sulfoximine (BSO), a selective inhibitor of the enzyme γ-glutamylcysteine synthetase that catalyzes the rate-limiting reaction in de novo GSH synthesis, decreased cellular GSH levels to about 20% of that found in the control cells. The cells pretreated with 0.1 mM BSO for 12 hours were significantly sensitized to the FB₁ cytotoxicity as determined by a long-term survival assay (p < 0.05). The results demonstrate that FB₁ changes GSH redox cycle status in LLC-PK₁ cells, and GSH may play a role in cytoprotection against FB₁ toxicity. © 1997 John Wiley & Sons, Inc.     

Modulation of [<Superscript>3</Superscript>H]Dopamine Release by Glutathione in Mouse Striatal Slices

Glutathione (γ-glutamylcysteinylglycine, GSH and oxidized glutathione, GSSG), may function as a neuromodulator at the glutamate receptors and as a neurotransmitter at its own receptors. We studied now the effects of GSH, GSSG, glutathione derivatives and thiol redox agents on the spontaneous, K⁺- and glutamate-agonist-evoked releases of [³H]dopamine from mouse striatal slices. The release evoked by 25 mM K⁺ was inhibited by GSH, S-ethyl-, -propyl-, -butyl- and pentylglutathione and glutathione sulfonate. 5,5′-Dithio-bis-2-nitrobenzoate (DTNB) and l-cystine were also inhibitory, while dithiothreitol (DTT) and l-cysteine enhanced the K⁺-evoked release. Ten min preperfusion with 50 μM ZnCl₂ enhanced the basal unstimulated release but prevented the activation of K⁺-evoked release by DTT. Kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) evoked dopamine release but the other glutamate receptor agonists N-methyl-d-aspartate (NMDA), glycine (1 mM) and trans-1-aminocyclopentane-1,3-dicarboxylate (t-ACPD, 0.5 mM), and the modulators GSH, GSSG, glutathione sulfonate, S-alkyl-derivatives of glutathione, DTNB, cystine, cysteine and DTT (all 1 mM) were without effect. The release evoked by 1 mM glutamate was enhanced by 1 mM GSH, while GSSG, glutathionesulfonate and S-alkyl derivatives of glutathione were generally without effect or inhibitory. NMDA (1 mM) evoked release only in the presence of 1 mM GSH but not with GSSG, other peptides or thiol modulators. l-Cysteine (1 mM) enhanced the glutamate-evoked release similarly to GSH. The activation by 1 mM kainate was inhibited by S-ethyl-, -propyl-, and -butylglutathione and the activation by 0.5 mM AMPA was inhibited by S-ethylglutathione but enhanced by GSSG. Glutathione alone does not directly evoke dopamine release but may inhibit the depolarization-evoked release by preventing the toxic effects of high glutamate, and by modulating the cysteine–cystine redox state in Ca²⁺ channels. GSH also seems to enhance the glutamate-agonist-evoked release via both non-NMDA and NMDA receptors. In this action, the γ-glutamyl and cysteinyl moieties of glutathione are involved.     

Lipoxygenase-mediated glutathione oxidation and superoxide generation

Soybean lipoxygenase-mediated cooxidation of reduced glutathione (GSH) and concomitant superoxide generation was examined. The oxidation of GSH was dependent on the concentration of linoleic acid (LA), GSH, and the enzyme. The optimal conditions to observe maximal enzyme velocity included the presence of 0.42 mM LA, 2 mM GSH, and 50 pmole of enzyme/mL. The GSH oxidation was linear up to 10 minutes and exhibited a pH optimum of 9.0. The reaction displayed a K_m of 1.49 mM for GSH and V_max of 1.35 ± 0.02 μmoles/min/nmole of enzyme. Besides LA, arachidonic and γ-linolenic acids also supported the lipoxygenase-mediated GSH oxidation. Hydrogen peroxide and 13-hydroperoxylinoleic acid supported GSH cooxidation, but to a very limited extent. Oxidized glutathione (GSSG) was identified as the major product of the reaction based on the depletion of nicotinamide-adenine dinucleotide 3′-phosphate (NADPH) in the presence of glutathione reductase. The GSH oxidation was accompanied by the reduction of ferricytochrome c, which can be completely abolished by superoxide dismutase (SOD), suggesting the generation of superoxide anion radicals. Under optimal conditions, the rate of superoxide generation (measured as the SOD-inhibitable reduction of ferricytochrome c) was 10 ± 1.0 nmole/min/nmole of enzyme. These results clearly suggest that lipoxygenase is capable of oxidizing GSH to GSSG and simultaneously generating superoxide anion radicals, which may contribute to oxidative stress in cells under certain conditions.     

Modulation of Anopheles gambiae Epsilon glutathione transferase activity by plant natural products in vitro

Elevated glutathione transferase (GST) E2 activity is associated with DDT resistance in the mosquito Anopheles gambiae. The search for chemomodulators that inhibit the function of AgGSTE2 would enhance the insecticidal activity of DDT. Therefore, we examined the interaction of novel natural plant products with heterologously expressed An. gambiae GSTE 2 in vitro. Five of the ten compounds, epiphyllocoumarin (Tral-1), knipholone anthrone, isofuranonaphthoquinones (Mr 13/2, Mr13/4) and the polyprenylated benzophenone (GG1) were shown to be potent inhibitors of AgGSTE2 with IC₅₀ values of 1.5 μM, 3.5 μM, 4 μM, 4.3 μM and 4.8 μM respectively. Non-competitive inhibition was obtained for Tral 1 and GG1 with regards to GSH (K_i of 0.24 μM and 0.14 μM respectively). Competitive inhibition for Tral1 was obtained with CDNB (K_i = 0.4 μM) whilst GG1 produced mixed type of inhibition. The K_i and K_i' for GSH for Tral-1 and GG1 were 0.2 μM and 0.1 μM respectively. These results suggest that the novel natural plant products, particularly Tral-1, represent potent AgGSTE2 in vitro inhibitors.     

Vascular Oxidant Stress and Hepatic Ischemia/Reperfusion Injury

The objective of this study was to test the hypothesis that the extracellular oxidation of glutathione (GSH) may represent an important mechanism to limit hepatic ischemia/reperfusion injury in male Fischer rats in vivo. Basal plasma levels of glutatione disulfide (GSSG: 1.5 ± 0.2μM GSH-equivalents), glutathione (GSH: 6.2 ± 0.4 μM) and alanine aminotransferase activities (ALT 12 ± 2U/I) were significantly increased during the l h reperfusion period following l h of partial hepatic no-flow ischemia (GSSG: 19.7 ± 2.2μM; GSH 36.9 ± 7.4μM; ALT: 2260 ± 355 U/l). Pretreatment with 1,3-bis-(2-chloroethyl)-I-nitrosourea (40mg BCNU/kg), which inhibited glutathione reductase activity in the liver by 60%. did not affect any of these parameters. Biliary GSSG and GSH efflux rates were reduced and the GSSG-to-GSH ratio was not altered in controls and BCNU-treated rats at any time during ischemia and reperfusion. A 90% depletion of the hepatic glutathione content by phorone treatment (300 mg/kg) reduced the increase of plasma GSSG levels by 54%, totally suppressed the rise of plasma GSH concentrations and increased plasma ALT to 4290 ± 755 U/I during reperfusion. The data suggest that hepatic glutathione serves to limit ischemialreperfusion injury as a source of extracellular glutathione, not as a cofactor for the intracellular enzymatic detoxification of reactive oxygen species.     

Menadione-resistant Chinese hamster cell variants are cross-resistant to hydrogen peroxide and exhibit stable chromosomal and biochemical alterations

We have investigated the antioxidant properties of V79 Chinese hamster cells rendered resistant to menadione by chronic exposure to increasing concentrations of this quinone. MD1, a clone of resistant cells, was compared to the parental M8 cells; the former showed increased activity of catalase (3 fold), glutathione peroxidase (1.6 fold) and DT-diaphorase (2.6 fold), as well as an increase in glutathione (3.2 fold). Although one of the products of menadione metabolism is superoxide anion, no changes in total superoxide dismutase activity was observed in MD1 cells. MD1 menadione resistant cells were also resistant to killing by hydrogen peroxide and contained tandem duplication of chromosome 6. A similar duplication of chromosome 6 was seen in several independently derived menadione resistant clones and therefore seems closed linked to the establishment of the resistance. Upon removal of menadione from the medium, some of these properties of MD1 cells, viz., resistance to menadione, elevated glutathione levels, and glutathione peroxidase activity, were lost and the cells resembled M8 cells. However, resistance to H₂O₂, elevated catalase activity and the duplicated chromosome remained stable for more than 40 cell passages in the absence of menadione. The increase in catalase activity was correlated with an increase in catalase mRNA content and a 50% amplification of catalase gene, as determined, respectively, by Northern and Southern blot analysis. The role of the chromosome 6 duplication in resistance to oxidative stress remains to be established. It is not responsible directly for elevated catalase levels since the catalase gene is on chromosome 3.Abbreviations SDS Sodium Dodecyl Sulphate - SOD Superoxide Dismutase - PBS Phosphate Buffered Saline (8.1 mM Na₂HPO₄, 1.47 mM KH₂PO₄, 2.68 mM KCl, 137 mM NaCl) - CDTA N,N,N,N-tetracetic-trans-1,2-diaminocyclohexane acid - MOPS Sulphonic-3-(N-morpholine)-propane acid - SSC 150 mM Nacl, 15 mM sodium-citrate, pH 6.8     

Purification of glutathione S-transferase from Van Lake fish (Chalcalburnus tarichii Pallas) muscle and investigation of some metal ions effect on enzyme activity

Glutathione S-transferases (GSTs) are an important enzyme family which play a critical role in detoxification system. In our study, GST was purified from muscle tissue of Chalcalburnus tarichii Pallas with 301.5-fold purification and 19.07% recovery by glutathione agarose affinity chromatography. The purity of enzyme was checked by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, showing a two band, because of having heterodimer structure. K_M values were 1.59 and 0.53?mM for 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH), respectively. V_max values for CDNB and GSH were also determined as 5.58 and 1.88?EU/mL, respectively. In addition, inhibition effects of Ag⁺, Cu²⁺, Cd²⁺, Fe³⁺, Pb²⁺, Cr²⁺, Co²⁺ and Zn²⁺ metal ions were investigated on the enzyme activity and IC₅₀, K_i values were calculated for these metal ions.     

Effects of four oxidants, menadione, 1-chloro-2,4-dinitrobenzene, hydrogen peroxide and cumene hydroperoxide, on fission yeast Schizosaccharmoyces pombe

Several chemical agents have been used to exert oxidative stress in the study of stress response, but differences in the effects of different reagents have received little attention. To elucidate whether such differences exist, the response of Schizosaccharomyces pombe to menadione (MD), 1-chloro-2,4-dinitrobenzene (CDNB), hydrogen peroxide and cumene hydroperoxide (CHP), which are frequently used to exert oxidative stress, was investigated. Sensitivity to these reagents differed among mutants deficient in genes involved in oxidative stress resistance. N-Acetylcysteine restored resistance to MD, CHP and hydrogen peroxide but did not change sensitivity to CDNB. The induction kinetics of genes induced by oxidative stress differed for each reagent. MD, CDNB and hydrogen peroxide caused a transient induction of genes, but the peak times of induction differed among the reagents. CHP gave quite different kinetics in that the induction continued for up to 2 h. The ctt1(+) gene was not induced by CHP. GSH rapidly decreased in the cells treated with high concentrations of these reagents, but at a low concentration only CDNB decreased GSH. These results indicated that S. pombe responded differently to the oxidative stress exerted by these different reagents.     

Heritability of glutathione and related metabolites in stored red blood cells

Red blood cells (RBCs) collected for transfusion deteriorate during storage. This deterioration is termed the “RBC storage lesion.” There is increasing concern over the safety, therapeutic efficacy, and toxicity of transfusing longer-stored units of blood. The severity of the RBC storage lesion is dependent on storage time and varies markedly between individuals. Oxidative damage is considered a significant factor in the development of the RBC storage lesion. In this study, the variability during storage and heritability of antioxidants and metabolites central to RBC integrity and function were investigated. In a classic twin study, we determined the heritability of glutathione (GSH), glutathione disulfide (GSSG), the status of the GSSG,2H⁺/2GSH couple (E_hc), and total glutathione (tGSH) in donated RBCs over 56 days of storage. Intracellular GSH and GSSG concentrations both decrease during storage (median net loss of 0.52±0.63 mM (median ± SD) and 0.032±0.107 mM, respectively, over 42 days). Taking into account the decline in pH, E_hc became more positive (oxidized) during storage (median net increase of 35±16 mV). In our study population heritability estimates for GSH, GSSG, tGSH, and E_hc measured over 56 days of storage are 79, 60, 67, and, 75%, respectively. We conclude that susceptibility of stored RBCs to oxidative injury due to variations in the GSH redox buffer is highly variable among individual donors and strongly heritable. Identifying the genes that regulate the storage-related changes in this redox buffer could lead to the development of new methods to minimize the RBC storage lesion.     

Antimicrobial effects of N-benzyloxycarbonyl-S-(2,4-dinitrophenyl) glutathione diesters against chloroquinine sensitive (NF54) and resistant (K1) strains of Plasmodium falciparum

N-Benzyloxycarbony-S-(2,4-dinitrophenyl)glutathione diesters have been investigated for antimalarial activity against chloroquinine sensitive (NF54) and resistant (K1) strains of P. falciparum. Both strains appear equally susceptible to inhibition by compounds 1–4, with an IC₅₀ ∼ 4.92–6.97 μM, consistent with the target of these compounds being the PfMRP transporter. Against the NF54 strain, diester derivatives containing ethyl side chains showed lower in vitro activity than those with methyl side chains 1–4, IC₅₀ ∼ 5.7–6.97 μM with the exception of compound 5 (IC₅₀ > 25 μM). The cytotoxicity of compounds with log P ∼ 3.9–5.8 were lower against the murine L6 cell line than compounds with a higher log P > 5.8 that were toxic. Overall the cytotoxicity of compounds 1–7 were lower against KB cells than against the L6 cell line with the exception of compound 4, which showed a higher relative toxicity.     

Purification and catalytic properties of glutathione transferase from the hepatopancreas of crayfish macrobrachium vollenhovenii (herklots)

Glutathione transferase from the hepatopancreas of fresh water crayfish Macrobrachium vollenhovenii was purified to apparent homogeneity by ion‐exchange chromatography on DEAE‐cellulose and by gel filtration on Sephadex G‐100. The enzyme appeared to be a homodimer with molecular weight (Mr) of 46.0 ± 1.4 kDa and a subunit Mr of 24.1 ± 0.35 kDa. Chromatofocusing of the apparently pure enzyme revealed microheterogeneity and resolved it into two isozymic peaks, which were eluted at pH 8.36 and 8.22 respectively. Inhibition studies showed that the I₅₀ value for cibacron blue, S‐hexylglutathione, hematin, and N‐ethylmaleimide (NEM) were 0.01 μM, 340μM, 5 μM and 33 mM respectively. Out of the several substrates tested, only 1‐chloro‐2,4‐dinitrobenzene (CDNB) and 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole could be conjugated with glutathione. Chemical modification studies with DTNB revealed that two sulphydryl groups per dimer were essential to the activity of the enzymes. On the basis of structural and catalytic characteristics, M. vollenhovenii GST seems close, tentatively, to the omega and zeta classes of GST. Initial‐velocity studies of the enzyme are consistent with a steady‐state random kinetic mechanism. Denaturation and renaturation studies with guanidine HCl (Gdn‐HCl) revealed that though low Gdn‐HCl concentrations (less than 0.5 M) denatured the enzyme, the enzyme was able to renature completely (100%). At higher concentration of the denaturant (0.5–4 M), refolding studies indicated that complete renaturation was not achieved. The extent of renaturation was however a function of protein concentration. Our results are consistent with a three‐state unfolding process. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:332–344, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20044     

Glutathione reductase plays an anti-apoptotic role against oxidative stress in human hepatoma cells

The cellular roles of glutathione reductase (GR) in the reactive oxygen species (ROS)-induced apoptosis were studied using the HepG2 cells transfected with GR. The overexpression of GR caused a marked enhancement in reduced and oxidized glutathione (GSH/GSSG) ratio, and significantly decreased ROS levels in the stable transfectants. Hydrogen peroxide (H₂O₂), under the optimal condition for apoptosis, significantly decreased cellular viability and total GSH content, and rather increased ROS level, apoptotic percentage and caspase-3 activity in the mock-transfected cells. However, hydrogen peroxide could not largely generate these apoptotic changes in cellular viability, ROS level, apoptotic percentage, caspase-3 activity and total GSH content in the cells overexpressing GR. Taken together, GR may play a protective role against oxidative stress.     

Tolerance of the yeast Yarrowia lipolytica to oxidative stress

The adaptive response of the yeast Yarrowia lipolytica to the oxidative stress induced by the oxidants hydrogen peroxide, menadione, and juglone has been studied. H₂O₂, menadione, and juglone completely inhibited yeast growth at concentrations higher than 120, 0.5, and 0.03 mM, respectively. The stationary-phase yeast cells were found to be more resistant to the oxidants than the exponential-phase cells. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H₂O₂ (0.3 mM), menadione (0.05 mM), and juglone (0.005 mM) made the cells more resistant to high concentrations of these oxidants. The adaptation of yeast cells to H₂O₂, menadione, and juglone was associated with an increase in the activity of cellular catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase, the main enzymes involved in cell defense against oxidative stress.     

Neutrophil adhesion to endothelial cells impairs the effects of catalase and glutathione in preventing endothelial injury

We studied the effects of the CuZn superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) on endothelial permeability to ¹²⁵I-albumin after activation of neutrophils (PMN) with phorbol 12-myristate-13-acetate (PMA; 10^?8M). PMN were either in direct contact with the endothelial cell monolayer grown on a porous gelatin-coated microporous 10-μm-thick polycarbonate filter (upright system) or separated from the endothelium by a similar filter (inverted system). Transendothelial ¹²⁵I-albumin clearance rates were measured as an index of endothelial permeability. In the absence of antioxidants, activation of PMN increased transendothelial ¹²⁵I-albumin clearnace rates in both systems from 0.041 ± 0.006 μl/min (baseline) to 0.262 ± 0.18 μl/min (upright system) and from 0.063 ± 0.02 μl/min to 0.244 ± 0.06 μl/min (inverted system). PMA induced 80–90% of PMN to adhere to either gelatin-coated filters or to endothelial cells, from the basal PMN adhesion value of 5.3 ± 2.2% and 4.3 ± 1.1%, respectively. SOD, which dismutates superoxide anion to hydrogen peroxide (H₂O₂), did not alter the transendothelial ¹²⁵I-albumin clearance rates in either systm at any concerntration from 10–300 U/ml. CAT (100–1,000 U/ml) and GSH (0.5–10 mM), which remove the H₂O₂ generated during PMN activation, did not alter the increase in transendothelial ¹²⁵I-clearance rates after PMN activation in the upright system, but both agents prvented the increase in transendothelial ¹²⁵I-clearance rates in the inverted system. We conclude that PMN activation with PMA causes endothelial injury irrespective of PMN contact to the endothelial monolayer. Moreover, H₂O₂, a release product of PMN activation, is a critical mediator of PMN-dependent endothelial injury. Finally, the results indicate that CAT and GSH prevent endothelial injury only in the absence of direct PMN contact with endothelial cells, suggesting that antioxidants such as GSH and CAT are excluded from sites of PMN-endothelial contact and thus are ineffective antioxidants. © 1993 Wiley-Liss, Inc.