Characterization of Hydrogen Peroxide and Superoxide Degrading Pathways of Aspergillus Niger Catalase: A Steady-State Analysis

The oxidized intermediates generated upon exposure of Aspergillus niger catalase to hydrogen peroxide and superoxide radical fluxes were examined with UV-visible spectrophotometry. Hydrogen peroxide and superoxide radical were generated by means of glucose/glucose oxidase and xanthine/xanthine oxidase systems. Serial overlay of absorption spectra in the Soret (350-450 nm) and visible regions (450-700 nm) showed that the decomposition of hydrogen peroxide by the catalase of Aspergillus niger can proceed through one of two distinct pathways: (i), the normal “catalatic” cycle consisting of ferric catalase → Compound I → ferric catalase; (ii), a longer cycle where superoxide radical transforms Compound I to Compound II which is then converted to the resting ferric enzyme via Compound III. The latter sequence of reactions ensures that the catalase of Aspergillus niger restores entirely its activity upon exposure to low levels of superoxide radicals due to the actions of oxidases.     

Circadian regulation of response to oxidative stress in Drosophila melanogaster

Circadian rhythms are fundamental biological phenomena generated by molecular genetic mechanisms known as circadian clocks. There is increasing evidence that circadian synchronization of physiological and cellular processes contribute to the wellness of organisms, curbing pathologies such as cancer and premature aging. Therefore, there is a need to understand how circadian clocks orchestrate interactions between the organism’s internal processes and the environment. Here, we explore the nexus between the clock and oxidative stress susceptibility in Drosophila melanogaster. We exposed flies to acute oxidative stress induced by hydrogen peroxide (H₂O₂), and determined that mortality rates were dependent on time at which exposure occurred during the day/night cycle. The daily susceptibility rhythm was abolished in flies with a null mutation in the core clock gene period (per) abrogating clock function. Furthermore, lack of per increased susceptibility to H₂O₂ compared to wild-type flies, coinciding with enhanced generation of mitochondrial H₂O₂ and decreased catalase activity due to oxidative damage. Taken together, our data suggest that the circadian clock gene period is essential for maintaining a robust anti-oxidative defense.     

Role of antioxidant enzymes in survival of conidiospores of Aspergillus niger 26 under conditions of temperature stress

AIMS: A better understanding of the role of antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) in the protection of Aspergillus niger spores against thermal stress. METHODS AND RESULTS: Conidiospores from A. niger 26 were subjected to wide range of temperatures (30, 50, 60 and 80 degrees C). The stress response was investigated by the determination of spore germination and mycelial growth of survivors under submerged cultivation. Exposure to any temperature above the optimal value induced an increase in SOD and CAT activities. PAGE demonstrated enhanced level of Cu/ZnSOD under stress conditions. We compared the influence of heat shock and superoxide-generating agent paraquat on growth and antioxidant enzyme defence and found different response to the both type of stresses. CONCLUSIONS: Heat stress elicits the enhanced synthesis of enzymes whose functions are to scavenge reactive oxygen species. These results suggested an association between thermal and oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: Evidence is provided for the possibility that oxidative stress plays a major role in the effect of heat in low eucaryotes such as A. niger. This knowledge may be of importance in controlling both fermentation and pathogenicity.     

Chilling, oxidative stress and antioxidant responses in shoot cultures of rice

Chilling of shoot cultures from Oryza sativa L. cv. Taipei 309, to 4 °C leads to conditions of oxidative stress. Tissue H₂O₂ was observed to increase more than fourfold by 8 d of chilling, and levels of reduced glutathione, which normally rise in growing shoot cultures at 25 °C, were considerably repressed in chilled cultures. Whilst the activity of ascorbate peroxidase in chilled shoots remained similar to the activities in control cultures at 25 °C, the most notable effects of chilling to 4 °C were the very significant loss of catalase and glutathione reductase activity. Although prior exposure of shoot cultures to abscisic acid (ABA) at 25 °C increased levels of catalase activity, such increased levels were not sustained when the pre-treated cultures were placed at 4 °C. Moreover such pre-treatment with ABA did not increase the subsequent ability of shoot cultures to grow at 4 °C.Abbreviations GSH reduced glutathione - GSSG oxidised glutathione - ABA cis-abscisic acid This work is supported by a grant from the Biotechnology and Biological Sciences Research Council.     

Morphological patterns of Aspergillus niger biofilms and pellets related to lignocellulolytic enzyme productivities

AIMS: To study the morphological patterns of Aspergillus niger during biofilm formation on polyester cloth by using cryo-scanning electron microscopy related to lignocellulolytic enzyme productivity. METHODS AND RESULTS: Biofilm and pellet samples obtained from flask cultures were examined at -80 degrees C in a LEO PV scanning electron microscope. Spore adhesion depends on both its rough surface and adhesive substances that form a pad between spore and support. An extracellular matrix surrounding germ tubes and hyphae was also seen. Biofilm mycelia showed an orderly distribution forming surface and inner channels, while pellets showed highly intertwined superficial hyphae and a densely packed deep mycelium. Morphological differences between both types of culture correlated with differences in enzyme volumetric and specific productivities. Biofilm cultures produced higher filter paper cellulase, endoglucanase, beta-glucosidase and xylanase volumetric and specific productivities than submerged cultures. CONCLUSIONS: Fungal biofilms are morphologically efficient systems for enzyme production. Favourable physiological aspects are shared with solid state fermentation, but fungal biofilms present better possibilities for process control and scale-up. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study support the importance of morphology in the productivity of fungal submerged processes, placing biofilms in a preferential category.     

Introduction to bioreactors of shake-flask inocula leads to development of oxidative stress in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Inoculation of bioreactors with shake-flask cultures present the organism with an immediate shift from an environment with little O₂ to one in which O₂ is typically at 100% saturation. The inoculation of such shake-flasks cultures into bioreactors sparged with 1 vvm air or 1 vvm air/O₂ mix i.e. 50% O₂ enrichment is an oxidatively stressful event, as judged by immediate increases in the intracellular concentrations of superoxide anion radical (O₂^·−) (from 4,600 to 11,600 RLU mg DCW⁻¹ and 5,500 to 23,000 RLU mg DCW⁻¹ respectively) and changes in the activities of the major antioxidant enzymes superoxide dismutase and catalase in all cultures. There are further effects on metabolic indices, particularly decreased nutrient consumption in oxygenated cultures (from 0.16 to 0.12 g starch g DCW h⁻¹) and decreased protein production, indicating that inoculation of the bioreactor exerts a global burden on the cellular metabolic networks.     

Differences in the activities of some antioxidant enzymes and in H2O2 content during rhizogenesis and somatic embryogenesis in callus cultures of the ice plant

Callus was obtained from hypocotyls of Mesembryanthemum crystallinum seedlings cultured on two types of medium—germination medium (GM) and callus induction medium (CIM). Following subculture on shoot induction medium SIM1, the callus formed on CIM medium regenerated roots or somatic embryos, while that obtained on GM medium was non-regenerative. The activities of CuZn-superoxidase dismutase (SOD) were comparable in all calli, but the activities of FeSOD and MnSOD varied according to the activity of photosystem II and the regenerative potential of the tissues. Catalase (CAT) activity was related to H₂O₂ concentration and affected by both the culture conditions and the morphogenic potential of the calli. The possible role of CAT, SODs and H₂O₂ in the regeneration of M. crystallinum from callus is discussed.This work is dedicated to Prof. Dr. Hubert Ziegler on his 80th birthday.     

Daily rhythm of MnSOD in the C<Subscript>3</Subscript>-CAM intermediate <Emphasis Type=Italic>Clusia fluminensis</Emphasis> Planch. et Triana.

The C₃-CAM intermediate plant Clusia fluminensis under well-watered at low light conditions opens stomata during the light period. In leaf extracts of this plant we have found two copper-zinc superoxide dismutases (CuZnSODs) and two manganese SODs: MnSOD-like protein (MnSOD II) and MnSOD I. Daily rhythm of the MnSOD I shows maximum activity during the afternoon hours and it is accompanied by only a very small tendency to increase in catalase (CAT) activity and lowering of citrate level.     

Autoxidation of extracellular hydroquinones is a causative event for the cytotoxicity of menadione and DMNQ in A549-S cells

Cytotoxicity of 1,4-naphthoquinones has been attributed to intracellular reactive oxygen species (ROS) generation through one-electron-reductase-mediated redox cycling and to arylation of cellular nucleophiles. Here, however, we report that in a subclone of lung epithelial A549 cells (A549-S previously called A549-G4S (Watanabe, et al., Am. J. Physiol. 283 (2002) L726-736), the mechanism of ROS generation by menadione and by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and therefore that of cytotoxicity, differs from the paradigm. Ninety percent of H(2)O(2) generation by both the quinones can be prevented by dicumarol, an inhibitor of NAD(P)H quinone oxidoreductase (NQO1), at the submicromolar level, regardless of the quinone concentrations. Exogenous SOD also inhibits H(2)O(2) production at low but not high concentrations of the quinones, especially DMNQ. Thus, at low quinone concentrations, superoxide-driven hydroquinone autoxidation accounts for more than half of H(2)O(2) generation by both quinones, whereas at high quinone concentrations, especially for DMNQ, comproportionation-driven hydroquinone autoxidation becomes the predominant mechanism. Hydroquinone autoxidation appears to occur predominantly in the extracellular environment than in the cytosol as extracellular catalase can dramatically attenuate quinone-induced cytotoxicity throughout the range of quinone concentrations, whereas complete inactivation of endogenous catalase or complete depletion of intracellular glutathione has only a marginal effect on their cytotoxicity. Finally, we show evidence that ROS production is a consequence of the compensatory defensive role of NQO1 against quinone arylation.     

Enhanced production of free trans-astaxanthin by oxidative stress in the cultures of the green microalga Chlorococcum sp

Free trans-astaxanthin accumulated in the alga Chlorococcum sp. was markedly enhanced from 3.664 mg g⁻¹ cell dry weight to 5.724 mg g⁻¹ cell dry weight when the culture was supplemented with hydrogen peroxide (0.1 mM) under mixotrophic conditions of growth. After saponification, a total of 7.086 mg astaxanthin per g cell dry weight was achieved. Similarly, in heterotrophic cultures, the total astaxanthin content was increased from 1.034 mg g⁻¹ cell dry weight without H₂O₂ to 1.782 mg g⁻¹ cell dry weight with 0.1mM H₂O₂. Results indicate that hydrogen peroxide effectively induces the formation of free trans-astaxanthin in Chlorococcum sp.     

JNK1 activity lowers the cellular production of H2O2 and modulates the growth arrest response to scavenging of H2O2 by catalase

Hydrogen peroxide (H(2)O(2)) can interact with intracellular signaling pathways to regulate cell behavior. The c-Jun NH(2)-terminal kinase 1 (JNK1) signal, involved in diverse aspects of cellular functioning, is implicated as a cell sensor of redox stress. The growth-inhibitory effect of both high-level H(2)O(2) and H(2)O(2)-scavenging catalase treatments is accompanied by increased JNK1 activity. To investigate the role of this response in growth regulation, the JNK1 signal was increased by the introduction of ectopic HA-JNK1. HA-JNK1 expression correlated with increases in basal c-Jun phosphorylation in a dose-dependent manner. Transient expression of HA-JNK1 potentiated cell growth arrest by catalase; however, with stable expression a degree of resistance to this response was observed. Resistance was accompanied by a lowered endogenous production of H(2)O(2). Transient HA-JNK1 expression also reduced H(2)O(2) generation, and this effect was reversed by the JNK inhibitor SP600125. These results indicate that the JNK1 stress response contributes to growth inhibition by catalase treatment via inhibition of cellular H(2)O(2) production. Stable amplification of the JNK1 pathway leads to cellular adaptation to its signal, resulting in a diminished reliance upon H(2)O(2) for efficient growth.     

Microbial transformation of artemisinin to 5-hydroxyartemisinin by Eurotium amstelodami and Aspergillus niger

Transformation of the anti-malarial drug artemisinin by the fungi Eurotium amstelodami and Aspergillus niger were investigated. Cultures were grown in sucrose/malt broth with artemisinin for 14 days and extracted with ethyl acetate. Extracts were characterized by liquid chromatography. Two metabolites from each fungal extract were isolated and identified using mass spectrometry and nuclear magnetic resonance. 5β-hydroxyartemisinin and 7β-hydroxyartemisinin were isolated in 63 and 32% yields, respectively, from the extract of E. amstelodami, and 80 and 19%, respectively, from the extract of A. niger.     

Generation of superoxide radicals in alkaline solutions of hydrogen peroxide and the effect of superoxide dismutase on this system

Superoxide radicals in high concentrations were generated from alkaline H₂O₂ without using catalysts or irradiation. The dependence of the intensity and parameters of the superoxide radical EPR spectrum on pH, temperature, viscosity and H₂O₂ concentration were studied. The observed changes are explained on the base of matrix effects. The addition of superoxide dismutase to alkaline H₂O₂ led initially to a drop in the EPR spectrum intensity, followed by an increase in the concentration of superoxide radicals.     

Oxidative stress decreases antioxidant enzyme activities in reaggregation cultures of rat brain cells

The brain has been suggested to be especially sensitive to damage by reactive oxygen species. In this study, we examined the effects of hyperoxic conditions on the activities and mRNA levels of antioxidant enzymes in reaggregation cultures of rat forebrain cells. Cultures were exposed to 80% oxygen for 12–60 h starting on Days 17 and 33 in culture. Superoxide dismutase activities and mRNA levels were not affected by hyperoxia, whereas catalase activity was slightly decreased after 24 h in 80% oxygen at Day 17. Glutathione peroxidase activity was markedly decreased already after 12 h of hyperoxia, and decreased activities of glutathione reductase and glucose-6-phosphate dehydrogenase were also noted. The glutathione peroxidase mRNA levels were increased in hyperoxic cultures at Day 17 but not at Day 33. These results suggest that the enzymatic defense mechanisms against reactive oxygen species in the brain are rather weak and deteriorate during oxidative stress but that a potential for compensatory upregulation exists at least during the first postnatal weeks.     

Effect of food deprivation on oxidative stress biomarkers in fish (Sparus aurata)

Oxidative stress in fish (Sparus aurata) as a consequence of food restriction and fasting, has been studied. Four groups of fish were maintained for 46 days under different conditions of food supplementation: a control group with no food restriction (ratio of food/fish of 2% w/w), two groups of animals with restricted food supplement (1 and 0.5%) and a fasting group (no meal addition). Finally, all the fish were provided with food at the same ratio as the control group for the last 7 days. Sampling and weighing of fish were carried out every week and their livers were used for the analysis of known biomarkers of oxidative stress. Malondialdehyde and oxidized glutathione levels increased at the third week in fish with partial or total food deprivation, but these levels returned to normal values when the fish readapted to the control conditions. Antioxidant enzymes were also analyzed and significant increases in superoxide dismutase (SOD), glutathione reductase and glutathione peroxidase activities were found in parallel with food restriction; however catalase activity decreased in fasting fish. New SOD isoforms were detected by isoelectrofocusing in fish under food restriction at the second week, which disappeared when starved fish returned to the control conditions. These new SOD isoforms were detected before the appearance of other usual oxidative stress biomarkers.     

Induction of peroxide and superoxide protective enzymes and physiological cross-protection against peroxide killing by a superoxide generator in Vibrio harveyi

Vibrio harveyi is a causative agent of destructive luminous vibriosis in farmed black tiger prawn (Penaeus monodon). V. harveyi peroxide and superoxide stress responses toward elevated levels of a superoxide generated by menadione were investigated. Exposure of V. harveyi to sub-lethal concentrations of menadione induced high expression of genes in both the OxyR regulon (e.g., a monofunctional catalase or KatA and an alkyl hydroperoxide reductase subunit C or AhpC), and the SoxRS regulon (e.g., a superoxide dismutase (SOD) and a glucose-6-phosphate dehydrogenase). V. harveyi expressed two detectable, differentially regulated SOD isozymes, [Mn]-SOD and [Fe]-SOD. [Fe]-SOD was expressed constitutively throughout the growth phase while [Mn]-SOD was expressed at the stationary phase and could be induced by a superoxide generator. Physiologically, pre-treatment of V. harveyi with menadione induced cross-protection against subsequent exposure to killing concentrations of H(2)O(2). This induced cross-protection required newly synthesized proteins. However, the treatment did not induce significant protection against exposures to killing concentrations of menadione itself or cross-protect against an organic hydroperoxide (tert-butyl hydroperoxide). Unexpectedly, growing V. harveyi in high-salinity media induced protection against menadione killing. This protection was independent of SOD induction. Stationary-phase cells were more resistant to menadione killing than exponential-phase cells. The induction of oxidative stress protective enzymes and stress-altered physiological responses could play a role in the survival of this bacterium in the host marine crustaceans.     

Oral exposure to Microcystis increases activity-augmented antioxidant enzymes in the liver of loach (Misgurnus mizolepis) and has no effect on lipid peroxidation

Recently, eutrophication has induced severe cyanobacterial blooms in the Naktong River, the second largest river of Korea. In the present study, lipid peroxidation and the antioxidant enzymes superoxide dismutase, catalase, and glutathione peroxidase, were evaluated in the liver of loach (Misgurnus mizolepis) that were orally exposed to a low dose of Microcystis through dietary supplementation with bloom scum. Loach received 75 mg of dry cells/kg body weight mass (equal to 10 microg microcystin-RR/kg body mass), for 28 days under controlled conditions. Antioxidant enzymatic activity and lipid peroxidation were measured after termination of exposure. The activities of antioxidant enzyme were significantly increased in the livers of toxin-exposed loach after 28 days of exposure, as compared to control fish. However, lipid peroxidation remained stable in both groups. These results suggest that antioxidant enzymes were able to eliminate oxidative stress induced by low concentrations of microcystins and to prevent increased lipid peroxidation in the liver of loach.