A simple enzyme–substrate localized conjugation method to generate immobilized,functional glutathione S-transferase fusion protein columns for affinity enrichment

Immobilized protein receptors and enzymes are tools for isolating or enriching ligands and substrates based on affinity. For example, glutathione S-transferase (GST) is fused to proteins as a tag for binding to its substrate glutathione (GSH) linked to solid supports. One issue with this approach is that high-affinity interactions between receptors and ligands require harsh elution conditions such as low pH, which can result in leached receptor. Another issue is the inherent nonspecific chemical conjugation of reactive groups such as N-hydroxysuccinimide (NHS) that couple lysines to solid supports; the nonspecificity of NHS may result in residue modifications near the binding site(s) of the receptor that can affect ligand specificity. In this study, a simple conjugation procedure is presented that overcomes these limitations and results in immobilized GST fusion proteins that are functional and specific. Here, the affinity of GST for GSH was used to generate an enzyme–substrate site-specific cross-linking reaction; GSH–Sepharose was preactivated with 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and then incubated Fc gamma receptor IIIa (FcγRIIIa)–GST. The immobilized FcγRIIIa–GST more specifically bound glycosylated immunoglobulin G1s (IgG1s) and was used to enrich nonfucosylated IgG1s from weaker binding species. This technique can be used when modifications of amino acids lead to changes in activity.     

Enhancement of lipid peroxidation in rat liver on acute exposure to styrene and acrylamide a consequence of glutathione depletion

Lipid peroxidation, glutathione level and activity of glutathione-S-transferase were studied in liver and brain of rats 4 and 3 h after a single i.p. administration of 0, 25, 75, 100 mg/kg acrylamide or 0, 50, 100, 200, 600 mg/kg styrene, respectively. In liver both acrylamide and styrene caused an increase in lipid peroxidation and decrease in glutathione contents and activity of glutathione-S-transferase in a dose dependent manner, while in brain only acrylamide produced a decrease in glutathione content. The decrease in glutathione content was not always associated with increase of lipid peroxidation. The enhancement of lipid peroxidation occurred only when glutathione contents were depleted to certain critical levels. No effect of acrylamide or styrene was seen on lipid peroxidation under in vitro conditions. The addition of glutathione in the incubation mixture significantly inhibited the rate of lipid peroxidation of liver homogenates of acrylamide and styrene treated animals.The results suggest that enhancement of lipid peroxidation in liver on exposure to acrylamide or styrene is a consequence of depletion of glutathione to certain critical levels. The inhibition of glutathione-S-transferase activity by acrylamide and styrene suggests that detoxication of these neurotoxic compounds could be suppressed following acute exposure.     

Improved isolation of proteins tagged with glutathione S-transferase

A common affinity tag used to express and purify fusion proteins is glutathione S-transferase. However, many researchers have reported difficulty eluting GST-tagged proteins from the affinity matrix. This report demonstrates that the problem likely is due to the propensity of glutathione S-transferase to dimerize combined with a propensity of the tagged protein to oligomerize, which results in formation of large oligomers of fusion protein that are chelated by the affinity matrix. The solution to the problem is to use S-butylglutathione instead of glutathione to elute, as S-butylglutathione binds more tightly to glutathione S-transferase and overcomes the chelate effect. Moreover, in contrast to glutathione, S-butylglutathione has no reducing capability that might inactivate a tagged protein.     

Psoroptes ovis: identification of vaccine candidates by immunoscreening

Serum from successful vaccine trials against the sheep scab mite, Psoroptes ovis, was used to immunoscreen a cDNA library constructed from mixed-stage and gender P. ovis to identify potential recombinant vaccine candidates. Immunodominant recombinant proteins recognised by IgG in these sera were selected for further analysis. Two candidates were identified in this way; a catchin-like protein (CLP) and a novel mu class glutathione S-transferase (GST). Both candidates were expressed in bacteria as recombinant proteins, the GST as an active enzyme, and combined with four other recombinant allergens in a multi-component recombinant vaccine. Strong serum IgG responses were induced in sheep against each of the components of the recombinant vaccine, however, the protective efficacy of the vaccine could not be determined because of variability in the establishment of a challenge infection.     

Stereochemical Features of Glutathione-dependent Enzymes in the Sphingobium sp. Strain SYK-6 β-Aryl Etherase Pathway

Glutathione-dependent enzymes play important protective, repair, or metabolic roles in cells. In particular, enzymes in the glutathione S-transferase (GST) superfamily function in stress responses, defense systems, or xenobiotic detoxification. Here, we identify novel features of bacterial GSTs that cleave β-aryl ether bonds typically found in plant lignin. Our data reveal several original features of the reaction cycle of these GSTs, including stereospecific substrate recognition and stereoselective formation of β-S-thioether linkages. Products of recombinant GSTs (LigE, LigP, and LigF) are β-S-glutathionyl-α-keto-thioethers that are degraded by a β-S-thioetherase (LigG). All three Lig GSTs produced the ketone product (β-S-glutathionyl-α-veratrylethanone) from an achiral side chain-truncated model substrate (β-guaiacyl-α-veratrylethanone). However, when β-etherase assays were conducted with a racemic model substrate, β-guaiacyl-α-veratrylglycerone, LigE- or LigP-catalyzed reactions yielded only one of two potential product (β-S-glutathionyl-α-veratrylglycerone) epimers, whereas the other diastereomer (differing in configuration at the β-position (i.e. its β-epimer)) was produced only in the LigF-catalyzed reaction. Thus, β-etherase catalysis causes stereochemical inversion of the chiral center, converting a β(R)-substrate to a β(S)-product (LigE and LigP), and a β(S)-substrate to a β(R)-product (LigF). Further, LigG catalyzed glutathione-dependent β-S-thioether cleavage with β-S-glutathionyl-α-veratrylethanone and with β(R)-configured β-S-glutathionyl-α-veratrylglycerone but exhibited no or significantly reduced β-S-thioether-cleaving activity with the β(S)-epimer, demonstrating that LigG is a stereospecific β-thioetherase. We therefore propose that multiple Lig enzymes are needed in this β-aryl etherase pathway in order to cleave the racemic β-ether linkages that are present in the backbone of the lignin polymer.     

Antiretroviral protease inhibitors potentiate chloroquine antimalarial activity in malaria parasites by regulating intracellular glutathione metabolism

Antiretroviral protease inhibitors significantly potentiated the sensitivity of chloroquine-resistant malaria parasites to the antimalarial drug in vitro and in vivo. Ritonavir was found to be potent in potentiating CQ antimalarial activities in both -resistant and -sensitive lines. The mechanism by which the APIs modulate the CQ resistance in malaria parasites was further investigated. CQ-resistant parasites showed increased intracellular glutathione levels in comparison with the CQ-sensitive parasites. Treatment with APIs significantly reduced the levels of GSH and glutathione S-transferase activities in CQ-resistant parasites. Ritonavir also decreased glutathione reductase activities and glutathione peroxidase activities in CQ-resistant parasite line. Taken together, these results demonstrate that parasite GSH and GST may play an important role in CQ resistance and APIs are able to enhance the sensitivity of CQ-resistant malaria parasite to the drug by influencing the levels of GSH and the activities of the related enzymes.     

The hypothalamic–hypophyseal–gonadal regulation of hepatic glutathione S-transferases in the rat

Hepatic glutathione S-transferase activities were determined with the substrates 1,2-dichloro-4-nitrobenzene and 1-chloro-2,4-dinitrobenzene. Sexual differentiation of glutathione S-transferase activities is not evident during the prepubertal period, but glutathione conjugation with 1,2-dichloro-4-nitrobenzene is 2–3-fold greater in adult males than in females. Glutathione conjugation with 1-chloro-2,4-dinitrobenzene is slightly higher in adult males than adult females. No change in activity was observed after postpubertal gonadectomy of males or females. Neonatal castration of males results in a significant decrease in glutathione conjugation with 1,2-dichloro-4-nitrobenzene. Hypophysectomy, or hypophysectomy followed by gonadectomy did result in significantly higher glutathione S-transferase activities in both sexes. These increases can be reversed by implanting an adult male or female pituitary or four prepubertal pituitaries under the kidney capsule. Postpubertal sexual differentiation of glutathione S-transferase activities is neither dependent on pituitary sexual differentiation nor pituitary maturation. Prolactin concentrations are inversely related to glutathione S-transferase activities in hypophysectomized rats with or without ectopic pituitaries. Somatotropin exogenously administered to hypophysectomized rats results in decreased glutathione S-transferase activities, whereas prolactin has no effect. Adult male rats treated neonatally with monosodium l-glutamate to induce arcuate nucleus lesions of the hypothalamus have decreased glutathione S-transferase activities towards 1,2-dichloro-4-nitrobenzene and decreased somatotropin concentrations. Our experiments suggests that sexual differentiation of hepatic glutathione S-transferase is a result of a hypothalamic inhibiting factor in the male (absent in the female). This postpubertally expressed inhibiting factor acts on the pituitary to prevent secretion of a pituitary inhibiting factor (autonomously secreted by the female), resulting in higher glutathione S-transferase activities in the adult male than the adult female.     

Formation and biliary excretion of glutathione conjugates of bile acids in the rat as shown by liquid chromatography/electrospray ionization-linear ion trap mass spectrometry

Acyl-adenylates and acyl-CoA thioesters of bile acids (BAs) are reactive acyl-linked metabolites that have been shown to undergo transacylation-type reactions with the thiol group of glutathione (GSH), leading to the formation of thioester-linked GSH conjugates. In the current study, we examined the transformation of cholyl-adenylate (CA-AMP) and cholyl-coenzyme A thioester (CA-CoA) into a cholyl-S-acyl GSH (CA-GSH) conjugate by rat hepatic glutathione S-transferase (GST). The reaction product was analyzed by liquid chromatography (LC)/electrospray ionization (ESI)-linear ion trap mass spectrometry (MS). The GST-catalyzed formation of CA-GSH occurred with both CA-AMP and CA-CoA. Ursodeoxycholic acid, lithocholic acid, and 2,2,4,4-²H₄-labeled lithocholic acid were administered orally to biliary fistula rats, and their corresponding GSH conjugates were identified in bile by LC/ESI-MS². These in vitro and in vivo studies confirm a new mode of BA conjugation in which BAs are transformed into their GSH conjugates via their acyl-linked intermediary metabolites by the catalytic action of GST in the liver, and the GSH conjugates are then excreted into the bile.     

Electronic Western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins

Identification of proteins previously separated by one-dimensional (1-D) or two-dimensional gel electrophoresis requires significant manipulations to digest the proteins into their respective peptides and to extract them from the gel prior to mass analysis. This article describes the simultaneous transfer and digestion of proteins directly from 1-D gels onto a membrane ready for matrix-assisted laser desorption/ionization (MALDI) mass spectrometric (MS) analysis. Protein transfer and digestion efficiencies are estimated to be more than 95%. The effectiveness of this procedure is demonstrated by identifying 110 unique proteins derived from a lysate of Escherichia coli and 149 proteins derived from a mouse liver homogenate separated by 1-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using crude mouse liver homogenates, four distinct glutathione S-transferase classes, ranging from 23 to 27 kDa, are identified from a separating gel, indicating the discriminating potential for this method. A Visual Basic program allowed visualization of the identified proteins according to their respective positions on the 1-D gels. In many cases, two or more proteins could be identified within a single band of the SDS gel. The “digital” images generated resemble Western blots without the use of antibodies or signal amplification techniques.     

A trifunctional enzyme with glutathione S-transferase,glutathione peroxidase and superoxide dismutase activity

Superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and glutathione reductase (GR) play crucial roles in balancing the production and decomposition of reactive oxygen species (ROS) in living organisms. These enzymes act cooperatively and synergistically to scavenge ROS, as not one of them can singlehandedly clear all forms of ROS. In order to imitate the synergy of the enzymes, we designed and generated a recombinant protein, which comprises of a Schistosoma japonicum GST (SjGST) and a bifunctional 35-mer peptide with SOD and GPX activities. The engineered protein demonstrated SOD, GPX and GST activities simultaneously. This trifunctional enzyme with SOD, GPX and GST activities is expected to be the best ROS scavenger.     

Atrazine Resistance in a Velvetleaf (Abutilon theophrasti) Biotype Due to Enhanced Glutathione S-Transferase Activity

We previously reported that a velvetleaf (Abutilon theophrasti Medic) biotype found in Maryland was resistant to atrazine because of an enhanced capacity to detoxify the herbicide via glutathione conjugation (JW Gronwald, Andersen RN, Yee C [1989] Pestic Biochem Physiol 34: 149-163). The biochemical basis for the enhanced atrazine conjugation capacity in this biotype was examined. Glutathione levels and glutathione S-transferase activity were determined in extracts from the atrazine-resistant biotype and an atrazine-susceptible or “wild-type” velvetleaf biotype. In both biotypes, the highest concentration of glutathione (approximately 500 nanomoles per gram fresh weight) was found in leaf tissue. However, no significant differences were found in glutathione levels in roots, stems, or leaves of either biotype. In both biotypes, the highest concentration of glutathione S-transferase activity measured with 1-chloro-2,4-dinitrobenzene or atrazine as substrate was in leaf tissue. Glutathione S-transferase measured with 1-chloro-2,4-dinitrobenzene as substrate was 40 and 25% greater in leaf and stem tissue, respectively, of the susceptible biotype compared to the resistant biotype. In contrast, glutathione S-transferase activity measured with atrazine as substrate was 4.4- and 3.6-fold greater in leaf and stem tissue, respectively, of the resistant biotype. Kinetic analyses of glutathione S-transferase activity in leaf extracts from the resistant and susceptible biotypes were performed with the substrates glutathione, 1-chloro-2,4-dinitrobenzene, and atrazine. There was little or no change in apparent K_m values for glutathione, atrazine, or 1-chloro-2,4-dinitrobenzene. However, the V_max for glutathione and atrazine were approximately 3-fold higher in the resistant biotype than in the susceptible biotype. In contrast, the V_max for 1-chloro-2,4-dinitrobenzene was 30% lower in the resistant biotype. Leaf glutathione S-transferase isozymes that exhibit activity with atrazine and 1-chloro-2,4-dinitrobenzene were separated by fast protein liquid (anion-exchange) chromatography. The susceptible biotype had three peaks exhibiting activity with atrazine and the resistant biotype had two. The two peaks of glutathione S-transferase activity with atrazine from the resistant biotype coeluted with two of the peaks from the susceptible biotype, but peak height was three- to fourfold greater in the resistant biotype. In both biotypes, two of the peaks that exhibit glutathione S-transferase activity with atrazine also exhibited activity with 1-chloro-2,4-dinitrobenzene, with the peak height being greater in the susceptible biotype. The results indicate that atrazine resistance in the velvetleaf biotype from Maryland is due to enhanced glutathione S-transferase activity for atrazine in leaf and stem tissue which results in an enhanced capacity to detoxify the herbicide via glutathione conjugation.     

A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants

The unusual glutathione S-transferase GSTO1 reduces, rather than conjugates, endo- and xenobiotics, and its role in diverse cellular processes has been proposed. GSTO1 has been assayed spectrophotometrically by measuring the disappearance of its substrate, S-(4-nitrophenacyl)glutathione (4-NPG), in the presence of 2-mercaptoethanol that regenerates GSTO1 from its mixed disulfide. To assay GSTO1 in rat liver cytosol, we have developed a high-performance liquid chromatography (HPLC)-based procedure with two main advantages: (i) it measures the formation of the 4-NPG reduction product 4-nitroacetophenone, thereby offering improved sensitivity and accuracy, and (ii) it can use glutathione, the physiological reductant of GSTO1, which is impossible to do with the spectrophotometric procedure. Using the new assay, we show that (i) the GSTO1-catalyzed reduction of 4-NPG in rat liver cytosol also yields 1-(4-nitrophenyl)ethanol, whose formation from 4-nitroacetophenone requires NAD(P)H; (ii) the two assays measure comparable activities with 2-mercaptoethanol or tris(2-carboxyethyl)phosphine used as reductant; (iii) the cytosolic reduction of 4-NPG is inhibited by GSTO1 inhibitors (KT53, 5-chloromethylfluorescein diacetate, and zinc), although the inhibitory effect is strikingly influenced by the type of reductant in the assay and by the sequence of reductant and inhibitor addition. Characterization of GSTO1 inhibitors with the improved assay provides better understanding of interaction of these chemicals with the enzyme.     

Rat liver glutathione S-transferase B: the functional mRNAs specific for the Ya Yc subunits are induced differentially by phenobarbital

Liver poly(A⁺)-RNA isolated from untreated and phenobarbital-treated rats has been translated in the rabbit reticulocyte cell-free system in order to examine the kinetics of induction of the translatable mRNAs encoding each subunit of glutathione S-transferase B. Translatable glutathione S-transferase B mRNA levels were maximally elevated at 16 to 24 h after a single injection of phenobarbital. Interestingly, the functional mRNA specific for the low-molecular-weight subunit was elevated markedly by phenobarbital administration whereas the mRNA specific for the high-molecular-weight subunit was only increased slightly. Our data suggest that different mRNAs direct the synthesis of the two subunits of glutathione S-transferase B and that these two mRNAs are under independent regulation.     

Localization of epoxide-metablozing enzymes in rat testis

Antibodies raised against rat hepatic epoxide hydrolase (EC 3.3.2.3) and glutathione S-transferases (EC 2.5.1.18) B, C and E were used to determine the presence and localizations of these epoxide-metabolizing enzymes in testes of sexually immature and mature Wistar and Holtzman rats. Unlabeled antibody peroxidase-antiperoxidase staining for each enzyme was readily detected in rat testes at the light microscopic level. Although significant strain-related differences were not apparent, staining intensity for certain enzymes differed markedly between Leydig cells and seminiferous tubules. Leydig cells of immature and mature rats were stained much intensely for epoxide hydrolase and glutathione S-transferase B and E than were seminiferous tubules, whereas Sertoli cells, spermatogonia, spermatocytes and spermatids, as well as Leydig cells, were stained intensely by the anti-glutathione S-transferase C. Age-related differences in staining for glutathione S-transferase B were not obvious, while the anti-glutathione S-transferase C stained seminiferous tubules more intensely in immature rats, and antibodies to expoxide hydrolase and glutathione S-transferases C and E stained Leydig cells much more intensely in mature rats. These observations thus demonstrate that testes of both sexually immature and mature rats contain epoxide hydrolase and glutathione S-transferases. Except for glutathione S-transferase C in immature rats, Leydig cells appear to contain much higher levels of enzymes than do seminiferous tubules. During sexual maturation, the testicular level of glutathione S-transferase B appears to remain constant, while levels of epoxide hydrolase and glutathione S-transferases C and E increase within Leydig cells and the level of glutathione S-transferase C decreases within seminiferous tubules.     

Effect of copper exposure on GST activity and on the expression of four GSTs under oxidative stress condition in the monogonont rotifer, Brachionus koreanus

Glutathione S-transferases (GSTs; EC 2.5.1.18) are major enzymes that function in Phase II detoxification reactions by catalyzing the conjugation of reduced glutathione through cysteine thiol. In this study, we cloned and sequenced four GST genes from the monogonont rotifer Brachionus koreanus. The domain regions of four Bk-GSTs showed a high similarity to those of other species. In addition, to evaluate the potential of GST genes as an early warning signal for oxidative stress, we exposed sublethal concentrations of copper (Cu) to B. koreanus and measured glutathione (GSH) contents and several antioxidant enzymes such as glutathione S-transferase (GST), glutathione peroxidase (GPx; EC 1.11.1.9), and glutathione reductase (GR; EC 1.8.1.7). The reactive oxygen species (ROS) at 12 h and 24 h after copper exposure increased significantly. GSH contents however did not increase significantly and even it decreased at 0.24 mg/L at 12 h. The activities of several antioxidant enzymes, particularly GPx and GR, showed a dramatic increase in 0.24 mg/L of CuCl₂. Messenger RNAs of each Bk-GST showed different patterns of modulations according to GST types, and particularly, Bk-GST-omega, Bk-GST-sigma, and Bk-GST zeta genes were highly sensitive to Cu. These results indicate that Bk-GSTs, functioning as one of the enzymatic defense mechanisms particularly in the early stage of oxidative stress response, were induced by Cu exposure. This also suggests that these genes and related enzymes have a potential as biomarkers for a more sensitive initial stress response.     

Structural and Biochemical Characterization of Yeast Monothiol Glutaredoxin Grx6

Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 Å resolution revealed a novel two-strand antiparallel β-sheet opposite the GSH binding groove. This extra β-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.     

Oxidative stress in digestive gland and gill of the brown mussel (Perna perna) exposed to air and re-submersed

Mussels Perna perna were exposed to air for 24 h showing a clear increase in the levels of lipid peroxidation and oxidative DNA damage, measured as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). The levels of lipid peroxidation increased both in the digestive gland and gills, while oxidative DNA damage increased only in the gills. After the 24 h of air exposure, mussels were re-submersed for a period of 3 h, leading values to return to a pre-aerial exposure levels. Control animals were kept immersed during the whole period. Several antioxidant and complementary enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), glutathione S-transferase (GST) and the levels of total glutathione (Total GSH) were assayed in a second set of experiments where one group of mussels were exposed to air for 18 h and other to 1 h re-submersion after 18 h aerial exposure. Only a 52% increase in the glutathione S-transferase activity was observed in the digestive gland, which remained elevated to about 40% after 1 h re-submersion, showing that defense systems can be modulated even during oxygen deprivation in P. perna. The DNA and lipid oxidative damage observed after aerial exposure indicates that mussels face an oxidative challenge, and are able to counteract such an “insult” as values of lipid peroxidation and DNA damage returned to control values after 3 h re-submersion.     

Oxidative stress in mice infected with Angiostrongylus cantonensis coincides with enhanced glutathione-dependent enzymes activity

This study aimed to estimate reactive oxygen species (ROS) production, antioxidants activity, and biomarkers level of oxidative damage to protein and DNA in the cerebrospinal fluid (CSF) of C57BL/6 mice infected with Angiostrongylus cantonensis. The mean ROS concentration in the CSF of infected mice increased gradually, and the increase in ROS in CSF became statistical significance at days 12-30 post-infection compared to that before infection (P < 0.001), and then ROS returned to normal level at day 45 after infection. In parallel with the increase in ROS in the CSF, infected mice showed similar of changes in reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), and glutathione S-transferase (GST) as that in ROS in the CSF. GSH, GR, GPx, and GST in the CSF of infected mice were all significantly higher than they were before infection during days 12-30 post-infection. However, protein carbonyl content and 8-hydroxy-2′-deoxyguanosine, biomarkers of oxidative damage to protein and DNA, respectively, were also significantly higher in the CSF of infected mice during this period. These results suggest that oxidative stress occur in the cells of central nervous system of mice infected with A. cantonensis during days 12-30 after infection due to ROS overproduction in CSF despite the increase in antioxidants during this period.     

Cytotoxic effects of 4-octylphenol on fish hepatocytes

The present study was conducted to determine cytotoxic effects of 4-octylphenol (4-OP) on primary cultured hepatocytes of pearl mullet (Alburnus tarichi). Lactate dehydrogenase (LDH) release, malondialdehyde (MDA) level, antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST)] and glutathione (GSH) content were measured after 24-h exposure to 4-OP. 4-OP caused dose- and time-dependent increases in LDH release. Significant induction of MDA level and decrease in GSH content were found. SOD and GPx activities were decreased while GST activity was increased. These findings suggest that 4-OP leads to cytotoxicity by depressing antioxidant defenses in fish hepatocytes.