Peroxide-metabolizing Enzymes Research Articles

Copper chloride induces antioxidant gene expression but reduces ability to mediate H2O2 toxicity in Xanthomonas campestris

Copper (Cu)-based biocides are currently used as control measures for both fungal and bacterial diseases in agricultural fields. In this communication, we show that exposure of the bacterial plant pathogen Xanthomonas campestris to nonlethal concentrations of Cu(2+) ions (75 µM) enhanced expression of genes in OxyR, OhrR and IscR regulons. High levels of catalase, Ohr peroxidase and superoxide dismutase diminished Cu(2+)-induced gene expression, suggesting that the production of hydrogen peroxide (H2O2) and organic hydroperoxides is responsible for Cu(2+)-induced gene expression. Despite high expression of antioxidant genes, the CuCl2-treated cells were more susceptible to H2O2 killing treatment than the uninduced cells. This phenotype arose from lowered catalase activity in the CuCl2-pretreated cells. Thus, exposure to a nonlethal dose of Cu(2+) renders X. campestris vulnerable to H2O2, even when various genes for peroxide-metabolizing enzymes are highly expressed. Moreover, CuCl2-pretreated cells are sensitive to treatment with the redox cycling drug, menadione. No physiological cross-protection response was observed in CuCl2-treated cells in a subsequent challenge with killing concentrations of an organic hydroperoxide. As H2O2 production is an important initial plant immune response, defects in H2O2 protection are likely to reduce bacterial survival in plant hosts and enhance the usefulness of copper biocides in controlling bacterial pathogens.

Reactive oxygen and nitrogen species generation, antioxidant defenses, and β-cell function: a critical role for amino acids

Growing evidence indicates that the regulation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels is essential for maintaining normal β-cell glucose responsiveness. While long-term exposure to high glucose induces oxidative stress in β cells, conflicting results have been published regarding the impact of ROS on acute glucose exposure and their role in glucose stimulated insulin secretion (GSIS). Although β cells are considered to be particularly vulnerable to oxidative damage, as they express relatively low levels of some peroxide-metabolizing enzymes such as catalase and glutathione (GSH) peroxidase, other less known GSH-based antioxidant systems are expressed in β cells at higher levels. Herein, we discuss the key mechanisms of ROS/RNS production and their physiological function in pancreatic β cells. We also hypothesize that specific interactions between RNS and ROS may be the cause of the vulnerability of pancreatic β cells to oxidative damage. In addition, using a hypothetical metabolic model based on the data available in the literature, we emphasize the importance of amino acid availability for GSH synthesis and for the maintenance of β-cell function and viability during periods of metabolic disturbance before the clinical onset of diabetes.

Chronic Social Isolation Compromises the Activity of Both Glutathione Peroxidase and Catalase in Hippocampus of Male Wistar Rats

Chronic neuroendocrine stress usually leads to the elevation of the stress hormones and increased metabolic rate, which is frequently accompanied by oxidative damage to the CNS. In the present study we hypothesized that chronic psychosocial isolation (CPSI) of male Wistar rats, characterized by decreased serum corticosterone (CORT), unaltered catecholamines (CTs), and low blood glucose (GLU), may also promote oxidative imbalance in the CNS, by targeting antioxidant defense system. To test it, we have examined the relation between these input signals and protein expression/activity of antioxidant enzymes (AOEs): superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GLR) in the hippocampus (HIPPO) of CPSI animals. We found that CPSI did not affect SODs or CAT, but decreased activity of GPx and compromised GLR, an enzyme highly dependent on blood GLU for its substrate precursor. Further, we have tested whether the CPSI experience altered AOEs response to a novelty stress, and found that it attenuated peroxide-metabolizing enzymes, CAT and GPx, and decreased GLR activity, even though blood GLU was restored. The altered ratios of hippocampal AOEs in CPSI animals, which were worsened under the combined stress conditions, may lead to the accumulation of peroxide products and oxidative imbalance. The mechanism by which CPSI generate oxidative imbalance in the HIPPO is most likely based on poor systemic energy conditions set by this stress. Such conditions may cause functional decline of CNS structures, such as HIPPO, and are likely to promote state linked to onset of many mood disorders.

The Dual Functions of Thiol-Based Peroxidases in H2O2Scavenging and Signaling

Thiol-based peroxidases consist of the peroxiredoxins (Prx) and the related glutathione peroxidase (GPx)-like enzymes. Their catalytic function is to reduce peroxides by using the reactivity of the cysteine residue, and their presumed primary physiologic role is to protect living organisms from peroxide toxicity. However, as peroxide-metabolizing enzymes, they also regulate hydrogen peroxide (H2O2) signaling. We review here enzymatic and biochemical attributes of thiol peroxidases that specify both distinctive peroxide-scavenging functions and the property of regulating H2O2 signaling. We then discuss possible thiol peroxidase physiologic functions, based on selected observations made in microorganisms and mammals.

Effects of amyloid-beta peptides on hydrogen peroxide-metabolizing enzymes in rat brain in vivo

Amyloid-β (Aβ) peptides are components of senile plaques initiating degeneration of brain neurons in Alzheimer's disease. They increase reactive oxygen species generation that may exceed the defensive capacity of cells. To test the hypothesis, this study investigated the in vivo effects of Aβ peptides on mitochondrial and non-mitochondrial enzymic sources of reactive oxygen species and antioxidant enzymes in rat brain. Continuous intracerebroventricular infusion of both Aβ25–35 and Aβ1–40 for up to 14 days stimulated the hydrogen peroxide (H2O2) generation in isolated neocortex mitochondria. Infusion of Aβ1–40 led to an increase in Mn-superoxide dismutase activity and a decrease in activities of catalase and glutathione peroxidase in mitochondria, to elevation of activities of Cu,Zn-superoxide dismutase and aldehyde oxidase, forwarded the conversion of xanthine dehydrogenase to xanthine oxidase and corresponding increase in the rate of H2O2 formation in the cytosol. Thus, Aβ peptides increase H2O2-formation and H2O2-forming enzyme activities and inhibit H2O2-consuming enzyme activities in mitochondria and cytosol in vivo. These studies suggest that disbalance between H2O2-generating and H2O2-metabolizing enzyme activities can contribute to oxidative stress underlying neurodegeneration and neuronal death in Alzheimer's disease.

ChallengingXanthomonas campestriswith low levels of arsenic mediates cross-protection against oxidant killing

Xanthomonas encounters highly toxic reactive oxygen species (ROS) from many sources, such as those generated by plants against invading bacteria, other soil bacteria and from aerobic respiration. Thus, conditions that alter intracellular ROS levels such as exposure to toxic metalloids would have profound effects on bacterial physiology. Here, we report that exposure of Xanthomonas campestris pv. phaseoli (Xp) to low levels of arsenic induces physiological cross-protection against killing by H(2)O(2) and organic hydroperoxide but not a superoxide generator. Cross-protection against H(2)O(2) and organic hydroperoxide toxicity was due to increased expression of genes encoding major peroxide-metabolizing enzymes such as alkyl hydroperoxide reductase (AhpC), catalase (KatA) and organic hydroperoxide resistance protein (Ohr). Arsenic-induced protection against H(2)O(2) and organic hydroperoxide requires the peroxide stress response regulators, OxyR and OhrR, respectively. Moreover, analyses of double mutants of the major H(2)O(2) and organic hyproperoxide-scavenging enzymes, Xp ahpC katA and Xp ahpC ohr, respectively, suggested the existence of unidentified OxyR- and OhrR-regulated genes that are involved in arsenic-induced resistance to H(2)O(2) and organic hyproperoxide killing in Xp. These arsenic-induced physiological alterations could play an important role in bacterial survival both in the soil environment and during plant-pathogen interactions.

Exposure to Cadmium Elevates Expression of Genes in the OxyR and OhrR Regulons and Induces Cross-Resistance to Peroxide Killing Treatment in Xanthomonas campestris

Cadmium is an important heavy metal pollutant. For this study, we investigated the effects of cadmium exposure on the oxidative stress responses of Xanthomonas campestris, a soil and plant pathogenic bacterium. The exposure of X. campestris to low concentrations of cadmium induces cross-protection against subsequent killing treatments with either H2O2 or the organic hydroperoxide tert-butyl hydroperoxide (tBOOH), but not against the superoxide generator menadione. The cadmium-induced resistance to peroxides is due to the metal's ability to induce increased levels of peroxide stress protective enzymes such as alkyl hydroperoxide reductase (AhpC), monofunctional catalase (KatA), and organic hydroperoxide resistance protein (Ohr). Cadmium-induced resistance to H2O2 is dependent on functional OxyR, a peroxide-sensing transcription regulator. Cadmium-induced resistance to tBOOH shows a more complex regulatory pattern. The inactivation of the two major sensor-regulators of organic hydroperoxide, OxyR and OhrR, only partially inhibited cadmium-induced protection against tBOOH, suggesting that these genes do have some role in the process. However, other, as yet unknown mechanisms are involved in inducible organic hydroperoxide protection. Furthermore, we show that the cadmium-induced peroxide stress response is mediated by the metal's ability to predominately cause an increase in intracellular concentrations of organic hydroperoxide and, in part, H2O2. Analyses of various mutants of peroxide-metabolizing enzymes suggested that this increase in organic hydroperoxide levels is, at least in part, responsible for cadmium toxicity in Xanthomonas.

Tumor Suppressive Effects of MnSOD Overexpression May Involve Imbalance in Peroxide Generation Versus Peroxide Removal

Manganese superoxide dismutase (MnSOD) activity is generally lower in cancer cells when compared with their normal cell counterparts. Many studies have shown that replacing the diminished MnSOD activity leads to inhibition of the malignant phenotype. We sought to overexpress MnSOD in a chemically transformed, malignant rat cell line with low endogenous MnSOD activity to determine the effect on the malignant phenotype. After MnSOD cDNA transfection, clonal populations were characterized at the molecular level for protein, RNA, and DNA, as well as for in vitro and in vivo growth and in vivo lung metastasis. MnSOD transfectants, which both under- and overexpressed MnSOD protein, were identified. These transfectants demonstrated variations in glutathione peroxidase and catalase activity levels, indicating differences in peroxide-generating versus peroxide-metabolizing enzymes (antioxidant imbalance); these differences were suggestive of alterations in their abilities to metabolize peroxide when compared with the parental cell line. In addition, these transfectants demonstrated reductions in both in vitro and in vivo growth, as well as a reduction in metastatic potential, which correlated with antioxidant imbalance. These results suggest that the tumor suppressive effect of MnSOD overexpression is in part mediated by an antioxidant imbalance resulting in the reduced capacity to metabolize increased levels of intracellular peroxides.

The role of a bifunctional catalase-peroxidase KatA in protection of Agrobacterium tumefaciens from menadione toxicity

Agrobacterium tumefaciens is an aerobic plant pathogenic bacterium that is exposed to reactive oxygen species produced either as by-products of aerobic metabolism or by the defense systems of host plants. The physiological function of the bifunctional catalase-peroxidase (KatA) in the protection of A. tumefaciens from reactive oxygen species other than H 2O 2 was evaluated in the katA mutant (PB102). Unexpectedly, PB102 was highly sensitive to the superoxide generator menadione. The expression of katA from a plasmid vector complemented the menadione-hypersensitive phenotype. A. tumefaciens possesses an additional catalase gene, a monofunctional catalase encoded by catE. Neither inactivation nor high-level expression of the catE gene altered the menadione resistance level. Moreover, heterologous expression of the catalase-peroxidase-encoding gene katG from Burkholderia pseudomallei, but not the monofunctional catalase gene katE from Xanthomonas campestris could restore normal levels of menadione resistance to PB102. A recent observation suggests that the menadione resistance phenotype involves increased activities of organic peroxide-metabolizing enzymes. Heterologous expression of X. campestris alkyl hydroperoxide reductase from a plasmid vector failed to complement the menadione-sensitive phenotype of PB102. The level of menadione resistance shows a direct correlation with the level of peroxidase activity of KatA. This is a novel role for KatA and suggests that resistance to menadione toxicity is mediated by a new, and as yet unknown, mechanism in A. tumefaciens.

Induction of lipid peroxidation during steroidogenesis in the rat testis.

Free radical production and lipid peroxidation are potentially important mediators in testicular physiology and toxicology. The cytochrome P450 enzymes of the steroidogenic pathway are known to produce free radicals. The present study was conducted to elucidate in vivo the gonadotropin regulation of free radical-mediated lipid peroxidation and the antioxidative defense system in the rat testis. GnRH antagonist (Org 30276; 1 mg/kg BW) and testosterone [40-mm SILASTIC brand (Dow-Corning) capsules] treatments were used to suppress serum gonadotropin levels. As expected, serum LH decreased to a very low level, whereas serum FSH decreased only slightly. Testosterone treatment for 8 days decreased the levels of the peroxide-metabolizing enzymes, catalase, glutathione peroxidase (GSH-Px), and glutathione transferase (-44%, -24%, and -31%, respectively; P < 0.01 for all). These changes predominately reflect the interstitial tissue, in which catalase and GSH-Px activities were much higher than in the seminiferous tubules. Testicular CuZn or Mn superoxide dismutase activities, which were high in the seminiferous tubules, were not affected by gonadotropin suppression. The total peroxyl radical-trapping capacity of the testis, or its components, vitamin E and ubiquinol 9, were not affected either. Lipid peroxidation was decreased after 8-day treatment, as detected by diminished formation of conjugated dienes and fluorescent chromolipids (-30% and -19%, respectively; P < 0.05 for both). Similar results of decreasing catalase and GSH-Px activities were found after gonadotropin suppression with GnRH antagonist treatment for 2 days or testosterone treatment for 5 days. Substitution with hCG, alone or in combination with recombinant human FSH, reversed the changes in enzyme activities, whereas FSH alone had no effect. After 5-day testosterone treatment, catalase messenger RNA expression was studied by Northern hybridization, and it was observed to parallel the changes in enzyme activity. The site of free radical production was studied by separating interstitial tissue and seminiferous tubules 5 h after hCG injection. GSH-Px was induced by hCG only in the interstitial tissue (+28%; P< 0.01), supporting the hypothesis of free radical production during steroidogenesis. Aminoglutethimide, an inhibitor of the P450 cholesterol side-chain cleavage enzyme, induced extensive lipid peroxidation in the testis. Presumably, aminoglutethimide leads to leakage of free radicals from the P450 enzyme when substrate oxygenation is prevented. In conclusion, the present study suggests that physiological LH action in the rat testis causes lipid peroxidation and maintains high activities of peroxide-metabolizing enzymes in the interstitial tissue.(ABSTRACT TRUNCATED AT 400 WORDS)

Inactivation of Enzymes of the Glutathione Antioxidant System by Treatment of Cultured Human Keratinocytes with Peroxides

Either metal ions, H2O2, t-butyl hydroperoxide (tBHP), or cumene hydroperoxide (CHP) was added to the medium of cultured human keratinocytes, and the activities of key peroxide-metabolizing enzymes were examined in a sonicated cell supernatant from the treated cells. 200 microM Fe++ +200 microM Fe was without effect on any enzyme activity. 700 microM CHP or tBHP decreased glutathione (GSH) peroxidase activity by 90% after 5 h and by 100% at 20 h, even if the CHP or tBHP was removed from the media after 90 min. H2O2 at 700 microM caused a brief 17% decrease in activity, which was followed by complete recovery. GSH peroxidase was found to be rapidly inactivated in vitro by CHP, but the enzyme was also inactivated at 37 degrees C even in the absence of CHP. GSH prevented both types of inactivation. Consistent with this in vitro data, in vivo depletion of the GSH pool with buthionine sulfoximine led to lower levels of GSH peroxidase and increased sensitivity to peroxide-induced inactivation. Neither GSH reductase nor GSH S-transferase were inactivated by any treatment although CHP did cause a small increase in the activity of the latter, which was not due to induction. The activity of glucose-6-phosphate dehydrogenase was decreased 50% following treatment for 5 h with 700 microM CHP or tBHP, whereas H2O2 treatment caused a brief 15% decline, followed by recovery. The effects of peroxides were not altered by changing the concentration of Ca++ in the media. Catalase was unaffected by concentrations of peroxide up to 700 microM. Inhibition of catalase with aminotriazole slightly enhanced the toxicity of 700 microns H2O2. In summary, organic hydroperoxides at relatively low concentrations inactive key enzymes of the glutathione pathway, but hydrogen peroxide does not.

Inhibition of glutathione peroxidase and glutathione transferase in mouse liver by misonidazole

The mechanisms of toxicity and sensitization by the radiosensitizer misonidazole [1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol] are not well understood. We report here on the inhibition of total glutathione peroxidase (GSHPx), selenium-dependent glutathione peroxidase (selenium-GSHPx) and glutathione transferase (GSHTx) activities by misonidazole. Mouse liver cytosol GSHPx and selenium-GSHPx were inhibited in vitro with 0.5 mM misonidazole. On administration of the drug intraperitoneally (800 mg/kg) to mice, it was found that GSHPx, selenium-GSHPx, and GSHTx were inhibited in homogenate, cytosol, and microsomal fractions of mouse liver. GSHPx was depressed in all fractions up to 60–70% of control values, with maximum depression occurring in the cytosol and homogenate fractions in less than 2 hr. Recovery of activity was slower in the microsomes. In general, the pattern of depression of selenium-GSHPx was parallel to that of GSHPx except in microsomes, where GSHPx is minimal. Quantitatively, selenium-GSHPx was least affected. GSHTx was inhibited 70–80% of control values in cytosol and homogenate with recovery by 24 hr, whereas a second period of depression occurred at 24 hr in the microsomes. The inhibition of peroxide-metabolizing enzymes may lead to elevation of intracellular peroxide levels, contributing to the radiosensitizing effect and/or toxicity of misonidazole.

Toxic effects of daunorubicin on isolated and cultured heart cells from neonatal rats

Various aspects of the cardiotoxicity of the anthracycline derivative and antineoplastic drug daunorubicin were investigated using isolated and cultured cells from neonatal rat hearts as a model system. Treatment of the cells with concentrations of daunorubicin of the same order of magnitude as those used in chemotherapy was accompanied by marked toxic effects, e.g. a decreased or abolished contraction, and release of lactate dehydrogenase, pyruvate and oxidized glutathione to the medium. A decreased frequency of contraction appeared to be the most sensitive probe of daunorubicin toxicity, followed by release of pyruvate and oxidized glutathione/lactate dehydrogenase. Daunorubicin and/or its metabolites also bound to cellular protein and DNA. Exposure to daunorubicin was shown to be accompanied by a rapid induction of primarily DT-diaphorase and a slower induction of glutathione transferase. The latter observations are interpreted to indicate a protective role of quinone- and peroxide-metabolizing enzymes, respectively, and support the hypothesis that daunorubicin toxicity involves generation of free radical derivatives, which initiate lipid peroxidation. This conclusion is further substantiated by the demonstration that addition of daunorubicin leads to an increased oxygen consumption.

Die Aktivität des Peroxid-metabolisierenden Systems in Human-Leber

The activities of various peroxide-metabolizing enzymes were determined in homogenates of human liver excisions. The specific activity of selenium-dependent glutathione peroxidase was 41.1 +/- 23.7 (S.D.) mU/mg protein; non-selenium glutathione peroxidase showed a activity of 30.5 +/- 14.0 mU/mg protein. The catalase and superoxide dismutase concentrations were 4.72 +/- 0.58 and 1.87 +/- 0.68 microgram/mg protein, respectively. Total glutathione amounted to 12.9 +/- 7.4 nmol/mg. Malondialdehyde formation, used as the basis for the determination of lipid hydroperoxides, was 0.32 +/- 0.14 nmol/mg. The data indicate much lower enzyme and substrate levels compared to rats and mice. A positive correlation of r = 0.48 +/- 0.31 was found between the glutathione level and selenium-dependent peroxidase. Selenium-dependent and non-selenium-glutathione peroxidase correlate negatively (r = -0.71 +/- 0.18); superoxide dismutase concentration and lipid-hydroperoxides are also related by a negative correlation coefficient of r = 0.47 +/- 0.31. These data stress the major hepatoprotective role of these systems in human liver.