Effect of exogenous nitric oxide on sulfur and nitrate assimilation pathway enzymes in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) under drought stress

The present study aimed at investigating the effects of foliar applied nitric oxide (as SNP [sodium nitroprusside]) on sulfur (glutathione reductase, guaiacol peroxidase, and glutathione S-transferase) and nitrate assimilation (nitrite and nitrate reductase) pathway enzymes in maize (Zea mays L.) exposed to water deficit conditions. The seedlings of a drought tolerant (NK8711) and sensitive (P1574) maize hybrid were applied with various SNP doses (0, 50, 100, 150, and 200 µM) under normal and drought stress conditions. Foliar spray of 100 µM markedly improved water status and chlorophyll contents and alleviated drought-induced oxidative damages through increased antioxidant (catalase, ascorbate peroxidase, and superoxide dismutase) activities in both maize hybrids. Moreover, exogenous SNP supply increased nitrite and nitrate reductase activities and upregulated glutathione reductase, glutathione S-transferase, and guaiacol peroxidase compared to no SNP supply. Interestingly, the negative effects of excess NO generation at high SNP doses (150, 200 µM) were more pronounced in P1574 than NK8711 leading to lower biomass accumulation in drought-sensitive hybrid.     

The isolation of a fetal rat liver glutathione S-tranferase isoenzyme with high glutathione peroxidase activity

A previously uncharacterized glutathione S-transferase isoenzyme which is absent from normal adult rat livers has been isolated fetal rat livers. The enzyme was purified using a combination of affinity chromatography, CM-cellulose column chromatography and chromatofocusing. It is composed of two non-identical subunits, namely, subunit Y_c (M_r 28 000) and a subunit (M_r 25 500) recently reported by us to be uniquely present in fetal rat livers and which we now refer to as subunit ‘Y_fetus’. The enzyme which we term glutathione S-transferase Y_cY_fetus has an isoelectric point of approx. 8.65 and has glutathione S-transferase activity towards a number of substrates. The most significant property of the fetal isozyme is its high glutathione peroxidase activity towards the model substrate cumene hydroperoxide. We suggest that this isozyme serves a specific function in protecting fetuses against the possible teratogenic effects of organic peroxides.     

Comparative study of the antioxidant defence systems in the erythrocytes of australian marsupials and monotremes

A comparison of the erythrocyte (RBC) antioxidant metabolites and enzymes in nine marsupial and two monotreme species was carried out. Reduced glutathione (GSH) concentrations were comparable with those reported for other marsupial and eutherian species. An important finding was that the erythrocytes of the southern hairy nosed wombat regenerated GSH faster than the erythrocytes from its close relative, the common wombat. The activities of glutathione-S-transferase, NADH-methaemoglobin reductase, Superoxide dismutase, and glutathione peroxidase (GSH-Px), showed similar levels and extents of variation as those observed in other marsupial and eutherian species. Catalase activities in the marsupials were lower than those measured in the two monotreme species and much lower than those reported in eutherian species. A negative correlation, significant at P < 0.05, was observed between GSH-Px and catalase activities in the RBC of the marsupials. Since both these enzymes “detoxify” H₂O₂, there appears to be a reciprocal relationship between the activities of these enzymes in marsupial RBC     

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.     

Effect of D,L-buthionine-S,R-sulfoximine on the ratio of glutathione forms and the growth of Tatar buckwheat calli

We studied the intracellular content of reduced (GSH) and oxidized (GSSG) glutathione, glutathione reductase activity, glutathione-S-transferase, and ascorbate peroxidase in morphogenic and nonmorphogenic Tatar buckwheat calli during the culture cycle as well as under the treatment with D,L-buthionine-S,R-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthase, the first enzyme of glutathione biosynthesis. We found that, during passaging, cultures only slightly differed in total glutathione content; however, the content of GSH was higher in the morphogenic culture, whereas the content of GSSG was higher in the nonmorphogenic culture. In the morphogenic callus, the glutathione-S-transferase activity was 10–20 times higher and the glutathione reductase activity was 2–2.5 times lower than in the nonmorphogenic callus. Under the treatment with BSO, the decrease in the GSH content in the morphogenic callus was temporary (on day 6–8 of passage), whereas that in the nonmorphogenic callus decreased within a day and remained lower than in the control throughout the entire passage. In the morphogenic callus, BSO did not affect the content of GSSG, whereas it caused GSSG accumulation in the nonmorphogenic callus. These differences are probably due to the fact that, in the BSO-containing medium, glutathione reductase is activated in the morphogenic callus and, conversely, inhibited in the nonmorphogenic callus. Although BSO caused a decrease in the total glutathione content only in the nonmorphogenic culture, the cytostatic effect of BSO was more pronounced in the morphogenic callus. In addition, BSO also had a negative effect on the differentiation of proembryonic cell complexes in the morphogenic callus. The role of the glutathione redox status in maintaining the embryogenic activity of cultured plant cells is discussed.     

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.     

Inhibition of fish brain ATPases by aldrin-transdiol, aldrin, dieldrin and photodieldrin.

The sensitivity of catfish, $\text{Ictalurus punctatus}$, brain ATPase activities to cyclodiene compounds was investigated. The ATPase system showed differences in sensitivity to aldrin, dieldrin and photodieldrin. However, aldrin-transdiol (a more terminal metabolite of dieldrin and reported as a more potent neurotoxin than dieldrin) had no effect on any ATPase activity from fish brain homogenates. Mitochondrial Mg²⁺ ATPase was the most sensitive ATPase to the cyclodiene compounds tested. The possibility that the neurotoxic effects of these compounds is a secondary response resulting from mitochondrial Mg²⁺ ATPase inhibition is discussed.     

Changes in glutathione-related enzymes in tumor-bearing mice after cisplatin treatment

The effect of cisplatin on five glutathione-related enzymes was studied in liver, kidney, and Dalton lymphoma cells of tumor-bearing mice. In liver, the activities of glutathione S-transferase, glutathione peroxidase, catalase, and superoxide dismutase decreased approximately 30–40%, 60–67%, 35–50% and 70–80% respectively, while glutathione reductase increased about 36–45% after cisplatin treatment. In kidney, catalase activity decreased by 47–82% at all time points (24–96 h) of cisplatin treatment, while glutathione S-transferase activity decreased significantly (~24%) mainly at 72 h of treatment. An increase in glutathione reductase (~1.5–2.5 times), glutathione peroxidase (significant at 24 h, 47%), and superoxide dismutase (~15–60%) was noted in kidney after the treatment. In Dalton lymphoma cells, the activities of glutathione S-transferase, glutathione peroxidase, and catalase decreased very distinctly (~2–5, 2–5 and 5–11 times, respectively) at all time points, but glutathione reductase decreased significantly only at 72 h of cisplatin treatment. Interestingly, the superoxide dismutase activity in Dalton lymphoma cells increased initially at 24–48 h and then decreased (~60%) during later periods (72–96 h) of treatment. Cisplatin treatment caused a decrease in glutathione level in Dalton lymphoma cells (~14–20%) and kidney (~18–28%) but no change in liver. In view of the results, a definite correlation with the changes in glutathione concentrations and enzymatic activities in a tissue could not be firmly derived. It is suggested that the changes in various glutathione-related enzymes and glutathione levels in the tissues of the host during cisplatin-mediated chemotherapy could affect cellular antioxidant defense potential, which may play an important contributory role in cisplatin-mediated toxicity, particularly nephrotoxicity, and anticancer activity in the host. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Differential temperature dependencies of antioxidative enzymes in two contrasting species: Fagus sylvatica and Coleus blumei

In order to characterise the sensitivity of antioxidative systems to temperature-induced oxidative stress, two species (Coleus blumei and Fagus sylvatica, L.) representative of environments with contrasting temperature characteristics have been exposed to low or high temperatures of 10 or 35 °C, respectively. Beech leaves were harvested in light and darkness. Coleus leaves were separated into green and white leaf tissue. The thermal dependencies of the activities of protective enzymes and chlorophyll fluorescence over a temperature range from 10 to 35 °C were determined. Ascorbate peroxidase activities were activated at low temperatures in vitro and, thereby, may provide an instantaneous protection against H₂O₂ accumulation which is faster than de novo synthesis. Monodehydroascorbate radical reductase was apparently not involved in short-term acclimation to low or high temperature. After short-term acclimation to low temperature, glutathione reductase and glutathione were more diminished in Coleus than in beech. Both species contained higher concentrations of ascorbate and glutathione at high temperatures than at low temperatures whereas glutathione reductase activity increased. Ascorbate peroxidase activity from Coleus leaves, though detectable under standard assay conditions (25 °C), failed at 35 °C in vitro. The results suggest that the higher temperature susceptibility of Coleus than that of beech was associated with a differential loss in glutathione reductase/glutathione at low temperature and an inhibition of ascorbate peroxidase at high temperature. Since the thermal dependencies of antioxidative enzymes were significantly affected by the preceding environmental conditions, the relative enzymatic activities determined under standard assay conditions may not be representative of enzymatic activities in foliage exposed to varying environmental temperatures.     

Glutathione peroxidase II of guinea pig liver cytosol: Relationship to glutathione S-transferases

GSH peroxidase II activity is not associated with all GSH-S-transferase (EC 2.5.1.18) proteins. In guinea pig liver GSH peroxidase II (nonseleno and specific for organic hydroperoxides) is associated almost entirely with GSH-S-transferase peak aa and a smaller peak designated aa′. Transferase a shows a slight peroxidase activity, transferase b is absent, and transferase c has no peroxidase activity. GSH peroxidase II of guinea pig liver has an isoelectric point of 8.9 and a molecular weight of 45,000. It consists of two subunits of similar size (26,000). The GSH peroxidase II and the GSH-S-transferase activities of transferase aa have not been resolved into separate proteins and presumably reside in the same protein. In rat liver GSH peroxidase II activity is present with the highest specific activity in GSH-S-transferase AA. There is no AA′. Transferase B also shows peroxidase activity. Transferases A and C show low but measurable peroxidase activity. Transferase peak E shows peroxidase activity, but it is contaminated by large amounts of GSH peroxidase I (EC 1.11.1.9), recognized by its activity on H₂O₂.     

Protective Effects of the Flavonoid-Rich Fraction from Rhizomes of Smilax glabra Roxb. on Carbon Tetrachloride-Induced Hepatotoxicity in Rats

Hepatoprotective agents could prevent tissue damage and reduce morbidity and mortality rates; such agents may include folkloric or alternative treatments. The present study evaluated the protective effects of the flavonoid-rich fraction from rhizomes of Smilax glabra Roxb. (SGF) on carbon tetrachloride (CCl₄)-induced hepatotoxicity in rats. Sprague-Dawley male rats were orally treated with SGF daily and received CCl₄ intraperitoneally twice a week for 4 weeks. Our results showed that SGF at doses of 100, 300 and 500 mg/kg significantly reduced the elevated activities of serum aminotransferases (ALT and AST), alkaline phosphatase and lactate dehydrogenase and the level of hepatic thiobarbituric acid–reactive substances compared to the CCl₄-treated group. Moreover, SGF treatment was also found to significantly increase the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and glutathione compared with CCl₄-induced intoxicated liver. Histopathologic examination revealed that CCl₄-induced hepatic damage was markedly reversed by SGF. The results suggest that SGF has hepatoprotective and antioxidant properties in CCl₄-induced liver injury in rats.     

Causes and Consequences of Cysteine S-Glutathionylation

Post-translational S-glutathionylation occurs through the reversible addition of a proximal donor of glutathione to thiolate anions of cysteines in target proteins, where the modification alters molecular mass, charge, and structure/function and/or prevents degradation from sulfhydryl overoxidation or proteolysis. Catalysis of both the forward (glutathione S-transferase P) and reverse (glutaredoxin) reactions creates a functional cycle that can also regulate certain protein functional clusters, including those involved in redox-dependent cell signaling events. For translational application, S-glutathionylated serum proteins may be useful as biomarkers in individuals (who may also have polymorphic expression of glutathione S-transferase P) exposed to agents that cause oxidative or nitrosative stress.     

Exogenous jasmonic acid modulates the physiology,antioxidant defense and glyoxalase systems in imparting drought stress tolerance in different Brassica species

This study examined the ability of jasmonic acid (JA) to enhance drought tolerance in different Brassica species in terms of physiological parameters, antioxidants defense, and glyoxalase system. Ten-day-old seedlings were exposed to drought (15 % polyethylene glycol, PEG-6000) either alone or in combination with 0.5 mM JA. Drought significantly increased lipoxygenase activity and oxidative stress, levels of malondialdehyde and H₂O₂. Drought reduced seedling biomass, chlorophyll (chl) content, and leaf relative water content (RWC). Drought increased proline, oxidized ascorbate (DHA) and glutathione disulfide (GSSG) levels. Drought affected different species differently: in B. napus, catalase (CAT) and glyoxalase II (Gly II) activities were decreased, while glutathione-S-transferase (GST) and glutathione peroxidase (GPX) activities were increased in drought-stressed compared to unstressed plants; in B. campestris, activities of glutathione reductase (GR), glyoxalase I (Gly I), GST, and GPX were increased, monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), CAT and other enzymes were decreased; in B. juncea, activities of ascorbate peroxidase, GR, GPX, Gly I were increased; Gly II activity was decreased and other enzymes did not change. Spraying drought-stressed seedlings with JA increased GR and Gly I activities in B. napus; increased MDHAR activity in B. campestris; and increased DHAR, GR, GPX, Gly I and Gly II activities in B. juncea. JA improved fresh weight, chl, RWC in all species, dry weight increased only in B. juncea. Brassica juncea had the lowest oxidative stress under drought, indicating its natural drought tolerance capacity. The JA improved drought tolerance of B. juncea to the highest level among studied species.     

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.     

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.     

Glutathione S-transferases in elasmobranch liver. Molecular heterogeneity, catalytic and binding properties, and purification

In order to gain insight into the phylogeny and physiological significance of organic-anion-binding proteins in the liver, the hepatic glutathione S-transferases of rat and a typical elasmobranch, the thorny-back shark (Platyrhinoides triseriata), were compared with respect to both glutathione S-transferase activites and organic-anion-binding properties. On gel filtration (Sephadex G-75, Superfine grade) of rat cytosol, the elution volumes of enzyme activities with 1-chloro-2,4-dinitrobenzene and p-nitrobenzyl chloride as substrates were identical (rat Y-fractions; M_r 45000). In contrast, two peaks of enzyme activity for 1-chloro-2,4-dinitrobenzene with elution volumes corresponding to M_r 52000 (PLAT Y₁) and M_r 45000 (PLAT Y₂) were detected on gel filtration of P. triseriata cytosol. Only fraction PLAT Y₂ had enzyme activity with p-nitrobenzyl chloride. Enzyme kinetic studies showed that rat Y-fraction had higher affinities for both 1-chloro-2,4-dinitrobenzene and glutathione than PLAT Y₁- and PLAT Y₂-fractions. The two forms of P. triseriata glutathione S-transferases differed greatly in affinity for glutathione. At a glutathione concentration that we found to be physiological in P. triseriata, PLAT Y₂ accounted for approx. 70% of the total glutathione S-transferase activity with 1-chloro-2,4-dinitrobenzene. Binding studies revealed that PLAT Y₁ and PLAT Y₂ fractions had much lower affinities for sulphobromophthalein and bilirubin than rat Y-fraction. In contrast, binding affinities of PLAT Y₁ and PLAT Y₂ for Rose Bengal and 1-anilino-8-naphthalenesulphonate were comparable with that of rat Y-fraction. Inhibitory kinetics suggested that sulphobromophthalein and Rose Bengal were non-competitive inhibitors of glutathione S-transferase activities when 1-chloro-2,4-dinitrobenzene was used as substrate for both PLAT Y₁ and PLAT Y₂. The major glutathione S-transferase from the PLAT Y₂ fraction was purified 81-fold by sequential chromatography on Sephadex G-75, DEAE-Sephadex and hydroxyapatite, and consisted of two identical subunits with pI7.7. The highly enriched Y₂-fraction retained high affinity binding of Rose Bengal and 1-anilino-8-naphthalenesulphonate.     

Pro-oxidant and antioxidant processes in gas gland and other tissues of cod (Gadus morhua)

Partial reduction of molecular oxygen produces reactive oxyradicals, including the superoxide anion radical (O _- ² ) and hydroxyl radical (·OH). The gas gland functions under hyperoxic and acidic conditions and therefore is likely to be subjected to enhanced oxidative stress. Aspects of pro- and antioxidant processes in gas gland were compared with other tissues likely to be subject to differing degrees of oxyradical production, viz. liver (site of chemically-mediated oxyradical production), gills and skeletal muscle. Antioxidant enzyme activities (superoxide dismutase, catalase, selenium-dependent and total glutathione peroxidase) per g wet weight were highest in liver and lowest in muscle. Catalase and glutathione peroxidase activies per g wet weight were higher in gills than in gas gland, whereas the reverse was seen for superoxide dismutase. Cytosolic superoxide dismutase activities per mg protein were two- and nine-fold higher in gas gland than in liver and gills. The pH characteristics of the antioxidant enzymes were generally similar in all the tissues. Glutathione, vitamin E and unsaturated (peroxidizable) lipid levels were generally highest in liver followed by gas gland. Lipid peroxidation (malonaldehyde equivalents) was evident in all tissues except gas gland. Hydrogen peroxide and O _- ² were involved in the NAD(P)H-dependent ferric/EDTA-mediated formation of ·OH (as measured by 2-keto-4-methiolbutyrate oxidation) by mitochondrial and postmitochondrial fractions of gas gland. Tissue maximal potentials for ·OH production paralled superoxide dismutase but not catalase or glutathione peroxidase activities. Overall, the results confirm the presence of effective antioxidant defences in gas gland and support previous workers' contentions of a central role for superoxide dismutase in this process.Abbreviations EDTA di-sodium ethylenediaminetetra-acetic acid - G-6-P glucose-6-phosphate - GPX total glutathione peroxidase - GSH reduced glutathione - GSSG oxidised glutathione - GST glutathion-S-transferase - HPLC high performance liquid chromatography - KMBA 2-keto-4-methiolbutyric acid - MOPS 3-[N-morpholino] propane-sulphonic acid - PMS postmitochondrial supernatant - Se-GPX selenium-dependent glutathion peroxidase - SOD superoxide dismutase - TCA trichloroacetic acid