Response of enzymes involved in the processes of antioxidation towards benthiocarb and methylparathion in cyanobacteria Nostoc muscorum.
01 Aug 1991-Bulletin of Environmental Contamination and Toxicology (Bull Environ Contam Toxicol)-Vol. 47, Iss: 2, pp 266-271
TL;DR: In this communication, studies have been presented on the effects of organophosphate insecticide methyl-parathione and carbamate herbicide benthiocarb, on glutathione content, glutATHione reductase (GR) and superoxide dismutase (SOD) activities of filamentous, nitrogen-fixing cyanobacteria Nostoc muscorum.
Abstract: Recently, it has been observed in the authors' laboratory that growth, nitrogen fixation, protein content of cyanobacteria Nostoc muscorum were reduced by methylparathion and benthiocarb treatment. Though many works on toxicity of pesticides on cyanobacteria, specially on growth, photosynthesis and nitrogen fixation are available, the effects of pesticides on antioxidant enzyme levels is still unclear. In this communication, studies have been presented on the effects of organophosphate insecticide methyl-parathione and carbamate herbicide benthiocarb, on glutathione content, glutathione reductase (GR) and superoxide dismutase (SOD) activities of filamentous, nitrogen-fixing cyanobacteria Nostoc muscorum.
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TL;DR: In this paper, the authors present general ideas derived from the various reports mentioning toxic effects of lipophilic compounds on the membrane lipid bilayer, affecting the structural and functional properties of these membranes.
Abstract: Microbial transformations of cyclic hydrocarbons have received much attention during the past three decades. Interest in the degradation of environmental pollutants as well as in applications of microorganisms in the catalysis of chemical reactions has stimulated research in this area. The metabolic pathways of various aromatics, cycloalkanes, and terpenes in different microorganisms have been elucidated, and the genetics of several of these routes have been clarified. The toxicity of these compounds to microorganisms is very important in the microbial degradation of hydrocarbons, but not many researchers have studied the mechanism of this toxic action. In this review, we present general ideas derived from the various reports mentioning toxic effects. Most importantly, lipophilic hydrocarbons accumulate in the membrane lipid bilayer, affecting the structural and functional properties of these membranes. As a result of accumulated hydrocarbon molecules, the membrane loses its integrity, and an increase in permeability to protons and ions has been observed in several instances. Consequently, dissipation of the proton motive force and impairment of intracellular pH homeostasis occur. In addition to the effects of lipophilic compounds on the lipid part of the membrane, proteins embedded in the membrane are affected. The effects on the membrane-embedded proteins probably result to a large extent from changes in the lipid environment; however, direct effects of lipophilic compounds on membrane proteins have also been observed. Finally, the effectiveness of changes in membrane lipid composition, modification of outer membrane lipopolysaccharide, altered cell wall constituents, and active excretion systems in reducing the membrane concentrations of lipophilic compounds is discussed. Also, the adaptations (e.g., increase in lipid ordering, change in lipid/protein ratio) that compensate for the changes in membrane structure are treated.
2,124 citations
TL;DR: In this paper, the toxic effects of different prominent aquatic pollutants (heavy metals (Cd & Pb), pesticides (alphamethrin and deltamithrin), and salt (NaCl) on the intracellular proline content in the cyanobacterium, Westiellopsis prolifica-Janet strain, NCCU331, were described.
Abstract: This study describes the toxic effects of different prominent aquatic pollutants—heavy metals (Cd & Pb), pesticides (alphamethrin and deltamethrin) and salt (NaCl)—on the intracellular proline content in the cyanobacterium, Westiellopsis prolifica–Janet strain–NCCU331. Despite a reduction in growth (measured as chlorophyll a content), the intracellular proline content increased in the presence of heavy metals, pesticides and high salt concentration. The intracellular cyanobacterial proline accumulation was more pronounced under salt stress than in the presence of pesticides and heavy metals. We have also compared whether or not anionic components influence heavy metal toxicity. It was found that the chlorides of Cd and Pb were more toxic than the NO3 and the order of toxicity was CdCl2 > PbCl2 > Cd (NO3)2 > Pb (NO3)2. Among pyrethroids, deltamethrin was more toxic than alphamethrin.
50 citations
Cites background from "Response of enzymes involved in the..."
...Attempts have also been made to study the response of enzymes involved in antioxidant action towards the pesticides benthiocarb and ethyl parathion in Nostoc muscorum (Bhunia et al. 1991)....
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TL;DR: The obtained results suggested that the alterations of antioxidant systems in A. cylindrica might be useful biomarkers of bentazon exposure, and relevant findings were added to explain the oxidative stress pathways ofbentazon promoting oxidative stress in cyanobacteria.
Abstract: Rice fields are frequently exposed to environmental contamination by herbicides and cyanobacteria, as primary producers of these aquatic ecosystems, are adversely affected. Anabaena cylindrica is a cyanobacterium with a significantly widespread occurrence in Portuguese rice fields. This strain was studied throughout 72 h in laboratory conditions for its stress responses to sublethal concentrations (0.75-2 mM) of bentazon, a selective postemergence herbicide recommended for integrated weed management in rice, with special reference to oxidative stress, role of proline and intracellular antioxidant enzymes in herbicide-induced free radicals detoxification. Activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione S-transferase (GST) increased in a time- and herbicide dose-response manner and were higher than those in the control samples after 72 h. A time- and concentration-dependent increase of malondialdehyde (MDA) levels and the enhanced cell membrane leakage following bentazon exposure are indicative of lipid peroxidation, free radicals formation, and oxidative damage, while increased amounts of SOD, CAT, APX, GST, and proline indicated their involvement in free radical scavenging mechanisms. The appreciable decline in the reduced glutathione (GSH) pool after 72 h at higher bentazon concentrations could be explained by the reduction of the NADPH-dependent glutathione reductase (GR) activity. The obtained results suggested that the alterations of antioxidant systems in A. cylindrica might be useful biomarkers of bentazon exposure. As the toxic mechanism of bentazon is a complex phenomenon, this study also adds relevant findings to explain the oxidative stress pathways of bentazon promoting oxidative stress in cyanobacteria.
36 citations
Cites background from "Response of enzymes involved in the..."
...Bhunia et al. (1991) reported that total glutathione level was reduced in a dose-dependent manner on N. muscorum cells exposed separately to the carbamate herbicide benthiocarb and the organophosphate insecticide methylparathion....
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TL;DR: This study shows a few markers for oxidative stress, such as the antioxidative enzymes superoxide dismutase, peroxidases and catalases, and cell damage due to extensive generation of ROS leading to lipid peroxidation.
Abstract: Cyanobacteria are able to produce a variety of secondary metabolites such as the microcystins. The ecological role of microcystins for the cyanobacteria themselves and in the aquatic ecosystem is not well understood. The aim of this study is to evaluate if microcystins might be used as a communication tool for interspecies cyanobacterial communication via the promotion of oxidative stress. Reactive oxygen species (ROS) are known to be used as plant signals. The main questions relate to the promotion of oxidative stress in Synechocystis sp. via exposure to the cyanobacterial toxins and the physiological effects. This study shows a few markers for oxidative stress, such as the antioxidative enzymes superoxide dismutase, peroxidases and catalases, and cell damage due to extensive generation of ROS leading to lipid peroxidation. End products of lipid peroxidation (malonaldehyde and 4-hydroxynonenal) are conjugated by glutathione S-transferase.
33 citations
Cites background from "Response of enzymes involved in the..."
...GR is an enzyme that converts GSSG back into its reduced form, GSH. Bhunia et al. (1991) noticed that Nostoc muscorum cells increased GR activity under stress conditions....
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TL;DR: The accumulation of proline in cells after herbicide exposure might prevent bentazon-induced production of reactive oxygen species and functions as a protecting agent against oxidative damage.
Abstract: A rational use of native cyanobacteria for diverse biotechnology purposes requires the study of the local abiotic factors that could potentially affect their normal growth. The present study deals with the herbicide bentazon (0.75, 1.5 and 2 mM)-induced changes in lipid profile and antioxidative defense system parameters related to oxidative stress in the rice field cyanobacterium strain Nostoc muscorum UTAD_N213 grown under laboratory conditions for 72-h. Results indicate that the 72-h exposure of the strain to increasing bentazon concentrations caused: (1) a decrease of the polyunsaturated fatty acids (FAs); (2) an increase of the saturated FAs; and (3) a decrease of the unsaturation index. The activities of all of the antioxidant enzymes except for glutathione reductase showed a significant increase upon bentazon exposure in a time- and concentration-dependent manner. When compared to untreated controls, the oxidized glutathione content increased by 42, 79 and 119% with 0.75, 1.5 and 2 mM bentazon, respectively, whereas the reduced to oxidized glutathione ratio decreased by 32, 50 and 61%, respectively. The accumulation of proline in cells after herbicide exposure might prevent bentazon-induced production of reactive oxygen species and functions as a protecting agent against oxidative damage.
32 citations
References
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Journal Article•
TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
Abstract: Since 1922 when Wu proposed the use of the Folin phenol reagent for the measurement of proteins, a number of modified analytical procedures utilizing this reagent have been reported for the determination of proteins in serum, in antigen-antibody precipitates, and in insulin. Although the reagent would seem to be recommended by its great sensitivity and the simplicity of procedure possible with its use, it has not found great favor for general biochemical purposes. In the belief that this reagent, nevertheless, has considerable merit for certain application, but that its peculiarities and limitations need to be understood for its fullest exploitation, it has been studied with regard to effects of variations in pH, time of reaction, and concentration of reactants, permissible levels of reagents commonly used in handling proteins, and interfering substances. Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
289,852 citations
TL;DR: The use of the foregoing analytical method in the determination of total and oxidized glutathione contents of rat blood, kidney, and liver gave values in good agreement with those obtained by previous investigators.
Abstract: A method for the analysis of nanogram quantities of glutathione has been developed which is based on the catalytic action of GSH or GSSG in the reduction of Ellman reagent (DTNB) by a mixture of TPNH and yeast glutathione reductase. Unlike previous methods of analysis the procedure described here effectively measures the total glutathione (GSH + GSSG) content of unknown mixtures and is not subject to appreciable interference by the presence of other thiol components. It is suggested that the catalytic action of glutathione in this system resides in the continual enzymic regeneration of GSH, present initially or formed enzymically from GSSG, following its interaction with the sulfhydryl reagent. The sensitivity of the method is such as to permit the determination of total glutathione in extracellular tissue fluids such as plasma, saliva, and urine normally containing very low levels of this material, essentially without pretreatment of the sample. The same is true for glutathione determinations of whole blood, in which the preliminary procedure is confined to the preparation of a 1:100 hemolyzate from as little as 10 μl of sample. Following published procedures, the pretreatment of tissue extracts with NEM to form an enzymically inactive complex with free GSH allowed the determination of the low levels of oxidized glutathione normally present therein. The use of the foregoing analytical method in the determination of total and oxidized glutathione contents of rat blood, kidney, and liver gave values in good agreement with those obtained by previous investigators.
5,900 citations
"Response of enzymes involved in the..." refers methods in this paper
...All operations were carried out at 4~ Glutathione reductase activity was assayed spectrophotometrically (Hitachi, Japan, model U2000), following the formation rate of 2-nitro- 5-thiobenzoic acid from 5-5 ' -dithiobis- (2-nitrobenzoic acid) (DTNB), with reduced glutathione (GSH), at 412 nm ( Tietze 1969 )....
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TL;DR: Nine of the enzyme activities were eliminated with cyanide treatment suggesting that they may be cupro-zinc enzymes, whereas one was cyanide-resistant and may be a manganese enzyme.
Abstract: Shoots, roots, and seeds of corn (Zea mays L., cv. Michigan 500), oats (Avena sativa L., cv. Au Sable), and peas (Pisum sativum L., cv. Wando) were analyzed for their superoxide dismutase content using a photochemical assay system consisting of methionine, riboflavin, and p-nitro blue tetrazolium. The enzyme is present in the shoots, roots, and seeds of the three species. On a dry weight basis, shoots contain more enzyme than roots. In seeds, the enzyme is present in both the embryo and the storage tissue. Electrophoresis indicated a total of 10 distinct forms of the enzyme. Corn contained seven of these forms and oats three. Peas contained one of the corn and two of the oat enzymes. Nine of the enzyme activities were eliminated with cyanide treatment suggesting that they may be cupro-zinc enzymes, whereas one was cyanide-resistant and may be a manganese enzyme. Some of the leaf superoxide dismutases were found primarily in mitochondria or chloroplasts. Peroxidases at high concentrations interfere with the assay. In test tube assays of crude extracts from seedlings, the interference was negligible. On gels, however, peroxidases may account for two of the 10 superoxide dismutase forms.
4,882 citations
"Response of enzymes involved in the..." refers methods in this paper
...The SOD enzyme extract preparation and assay was carried out as described by Giannopolitis and Ries (1977) ....
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TL;DR: Results for GSH levels agreed well with earlier reports but levels of GSSG estimated here were higher than earlier reported values, and the reasons for the apparently higher levels ofGSSG are discussed.
Abstract: A method for measurement of both oxidized (GSSG) and reduced (GSH) glutathione has been developed, with use of o-phthalaldehyde (OPT) as a fluorescent reagent. The method takes advantage of the reaction of GSH with OPT at pH 8 and of GSSG with OPT at pH 12; GSH can be complexed to N-ethylmaleimide to prevent interference of GSH with measurement of GSSG. The method gave “recoveries” of 91 to 110% for both GSH and GSSG and was quite specific for glutathione; and none of the manipulations appeared to influence the amount of glutathione present in the tissue. Results for GSH levels agreed well with earlier reports but levels of GSSG estimated here were higher than earlier reported values. The reasons for the apparently higher levels of GSSG are discussed.
3,881 citations
TL;DR: The kinetic properties of the enzyme suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions and this prediction was confirmed experimentally.
Abstract: Glutathione reductase (EC 1.6.4.2) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of polyacrylamide gel electrophoresis on native and SDS-gels. The enzyme has a molecular weight of 145,000 and consists of two subunits of similar size. The pH optimum of spinach glutathione reductase is 8.5–9.0, which is related to the function it performs in the chloroplast stroma. It is specific for oxidised glutathione (GSSG) but shows a low activity with NADH as electron donor. The pH optimum for NADH-dependent GSSG reduction is lower than that for NADPH-dependent reduction. The enzyme has a low affinity for reduced glutathione (GSH) and for NADP+, but GSH-dependent NADP+ reduction is stimulated by addition of dithiothreitol. Spinach glutathione reductase is inhibited on incubation with reagents that react with thiol groups, or with heavymetal ions such as Zn2+. GSSG protects the enzyme against inhibition but NADPH does not. Pre-incubation of the enzyme with NADPH decreases its activity, so kinetic studies were performed in which the reaction was initiated by adding NADPH or enzyme. The Km for GSSG was approximately 200 μM and that for NADPH was about 3 μM. NADP+ inhibited the enzyme, assayed in the direction of GSSG reduction, competitively with respect to NADPH and non-competitively with respect to GSSG. In contrast, GSH inhibited non-competitively with respect to both NADPH and GSSG. Illuminated chloroplasts, or chloroplasts kept in the dark, contain equal activities of glutathione reductase. The kinetic properties of the enzyme (listed above) suggest that GSH/GSSG ratios in chloroplasts will be very high under both light and dark conditions. This prediction was confirmed experimentally. GSH or GSSG play no part in the light-induced activation of chloroplast fructose diphosphatase or NADP+-glyceraldehyde-3-phosphate dehydrogenase. We suggest that GSH helps to stabilise chloroplast enzymes and may also play a role in removing H2O2. Glucose-6-phosphate dehydrogenase activity may be required in chloroplasts in the dark in order to provide NADPH for glutathione reductase.
398 citations