Regular ArticleSpectrophotometric Assay of Superoxide Dismutase Activity Based on the Activated Autoxidation of a Tetracyclic Catechol
Abstract
A new spectrophotometric assay of superoxide dismutase (SOD) activity is described. The assay is based on the SOD-mediated increase in the rate of autoxidation of 5 , 6 , 6a , 11b - tetrahydro - 3 , 9 , 10 -trihydroxybenzo[c]fluorene (BXT-01050) in aqueous alkaline solution. This autoxidation yields a chromophore with a maximal absorbance wavelength of 525 nm. The optimized assay of SOD activity is performed at pH 8.8, 37°C, in 50 mM air-saturated 2-amino-2-methyl-1,3-propanediol buffer containing 3 mM boric acid and 0.1 mM diethylenetriaminepentaacetic acid. The kinetic measurement of 525-nm absorbance is performed for 1 min upon addition of BXT-01050. BXT01050 is stabilized in stock solution acidified at pH 1.5. The SOD activity is determined from the Vs/Vc ratio of the autoxidation rates measured in the presence (Vs) and in the absence (Vc) of sample. One SOD activity unit (U-525) has been defined as the activity that doubles the autoxidation background (Vs/Vc = 2). The equation that fits the standard curve is the same with all studied SODs. Another reagent, 1,4,6-trimethyl-2-vinylpyridinium trifluoromethanesulfonate, directly eliminates interference due to sample mercaptans such as glutathione by means of a very fast alkylation reaction. A fast and reproducible measurement of SOD activity requires only a single determination per sample. At pH 8.8, an optimal assay sensitivity is achieved without strongly affecting the activity of known SODs such as Cu/Zn-, Mn-, or Fe-SOD.
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Promotion of pellet-feed feeding in mandarin fish (Siniperca chuatsi) by Bdellovibrio bacteriovorus is influenced by immune and intestinal flora
2021, AquacultureThe feeding preference of the mandarin fish, Siniperca chuatsi, for live bait fish over pellet feed increases the costs and risks of breeding. Animal feeding behavior is directly or indirectly influenced by intestinal flora and innate immunity. Probiotics have been shown to regulate intestinal flora and stimulate innate immunity in a variety of species. In this study, during the pellet-feed feeding process of mandarin fish, Bdellovibrio bacteriovorus at a concentration of 108 PFU (plaque forming unit)/mL was added to the water body for 6 weeks, and the effects on the survival, growth, appetite, intestinal flora, innate immunity, antioxidant capacity, and intestinal histology of mandarin fish were studied. The results showed that addition of B. bacteriovorus at a concentration of 108 PFU/mL to aquaculture water could significantly promote mandarin fish pellet-feed feeding, improve the survival rate and growth performance of mandarin fish that were fed pellet feed. Compared with the control group, addition of B. bacteriovorus promoted the expression of appetite-regulating genes nerve peptide Y (Npy) and agouti gene-related protein (Agrp). Intestinal microflora analysis and intestinal histological analysis results revealed that B. bacteriovorus reduce the abundance of pathogenic bacteria Aeromonas in the intestinal flora, reduce damage to the intestinal mucosa and epithelial cells, and increase the thickness of the intestinal basement membrane and height of intestinal villi. Similarly, an increase in the serum lysozyme and immunoglobulin M (IgM) contents was observed after B. bacteriovorus administration. Moreover, B. bacteriovorus improved the activity of antioxidant enzymes, reduced glutathione (GSH) and catalase (CAT). In general, addition of B. bacteriovorus at a concentration of 108 PFU/mL to aquaculture water could significantly promote pellet-feed feeding of mandarin fish and improve the growth performance and survival rates of mandarin fish under pellet-feed feeding conditions. This promoting effect may be achieved by B. bacteriovorus stimulation of innate immunity and regulation of intestinal flora.
Enzymatic disturbances of the Egyptian beetle, Blaps polychresta (Forskål, 1775), as environmental indicators of heavy metal pollution of agricultural soils
2021, Annals of Agricultural SciencesCitation Excerpt :10 μL of the resulting supernatant was measured and added to a mixture of 0.1 mM xanthine, 6.25 nM xanthine oxidase, 50 mM K2HPO4, pH 7.8, 0.1 mM EDTA, 0.025% (w/v) Triton X-100, 0.1 mM 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl) -2H-tetrazolium (WST-1), SOD activity determined by the absorbance after 20 min at 37 °C by spectrophotometer at 450 nm. A unit of SOD activity implies the quantity of enzyme that causes half-maximal suppression of NBT reduction, according to Nebot et al. (1993), and specific activity was symbolized as mU mg−1 protein. CAT (EC 1.11.1.6) was measured, as mentioned by Aebi (1984).
Heavy metals (HMs) pollution is becoming influential severe trouble in the farming ecosystem, where it causes serious health problems for many living organisms. This study was conducted to estimate the HMs accumulation under different sites from agricultural soil and its effects on enzymatic activities in tissues of the beetle, Blaps polychresta, as an indicator of soil contamination. Beetle and soil samples were collected from three agricultural sites; (A) a reference site, (B) fields located near the River Nile, and (C) fields closed to drainage. The atomic absorption results showed a significant increase in iron (Fe), manganese (Mn), arsenic (As), lead (Pb), chromium (Cr), aluminum (Al), and zinc (Zn) for both soil and beetles from site B & C compared to site A. The highest bioaccumulation (BAF) ratio was observed for nickel (Ni), As, and cadmium (Cd) in site C, while Zn, copper (Cu), and Pb in site B. HMs accumulation in beetle's tissues (gut, fat body, and testis) from site B & C induced the malondialdehyde (MDA) activity. Thus, a significant reduction of the antioxidant enzymes and total protein contents (TPc) were observed in beetle tissues from site C. However, no significant differences in some tissues of site B compared to other sites. A principal component analysis (PCA) and correlation were conducted between HMs concentrations in beetle tissues and antioxidant enzymes, TPc, and MDA. The current results of a principal component analysis (PCA) and the correlation test were used to assist the differences in the decline of the antioxidant enzymes: glutathione (GSH), Superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx), and glutathione reductase (GR) and TPc, while; the elevation in MDA responses in beetle's tissues due to HMs accumulations in response to protect themselves from metals toxicity. The antioxidant defense and oxidative stress were more prominent in the tissues of the beetle's gut and fat bodies comparing to the testis. Accordingly, the HMs accumulation in agricultural soil can be predicted by the enzymatic activities in beetle tissues.
Toxic effect of chronic nitrite exposure on growth and health in Pacific white shrimp Litopenaeus vannamei
2020, AquacultureJuveniles of the Pacific white shrimp Litopenaeus vannamei were exposed to 0 (control), 2, 6.67, and 20 mg L−1 nitrite-N for 30 days, and the growth, immunity, and gut microbiota were evaluated. The weight gain of shrimp exposed to 20 mg L−1 nitrite-N was significantly lower than that in the control. Antioxidant capacity, measured as superoxide dismutase and glutathione peroxidase activities, catalase expression, and malondialdehyde content, was not affected by chronic nitrite-N exposure. The expressions of immune related genes (IMD and Toll) in shrimp exposed to 20 mg L−1 nitrite-N were significantly higher than that in shrimp exposed to 2 mg L−1 nitrite-N, but no differences were found with the control. Regardless of nitrite-N exposure concentrations, Bacteroidetes, Proteobacteria, and Actinobacteria were the dominant phyla in the gut of L. vannamei. With the increase of nitrite-N concentration, the diversity of gut microbiota increased in L. vannamei. The relative abundance of opportunistic pathogens, such as Pseudoalteromonadaceae and Vibrionaceae, increased in shrimp exposed to 20 mg L−1 nitrite-N. The relative abundance of denitrifying bacteria increased in shrimp exposed to 2 mg L−1 nitrite-N, but decreased in those exposed to 6.67 or 20 mg L−1 nitrite-N relative to those in the control. The predicted microbial-mediated functions showed that pathways for digestive function and metabolic capacity were significantly inhibited. All findings in this study indicate that L. vannamei can deal with the stress caused by nitrite-N within levels lower than 6.67 mg L−1, but chronic exposure to a high concentration (> 6.67 mg L−1) would reduce shrimp growth and alter its immune system by destroying gut microbiota homeostasis.
Oxidative stress parameters, DNA damage and expression of HSP70 and MT in midgut of Trachyderma hispida (Forskål, 1775) (Coleoptera: Tenebrionidae) from a textile industry area
2020, Environmental PollutionThe textile mill industry is one of the major sources of pollution and contributors of metal contaminants to the environment. At the same time, the industry is important for global economy. Pollution caused by the textile industry is characteristic due to a unique set of potentially toxic substances. Darkling beetles (Coleoptera, Tenebrionidae), which live in all biogeographical regions, are especially common in soil quality and soil degradation studies. Our study was designed to assess long-term effects of textile industry (which generates specific pollution) on soil organisms, namely Trachyderma hispida. We especially wanted to find out what changes allow the species to survive and adapt to these specific conditions. Energy-dispersive X-ray spectroscopy of soil and midgut tissues of T. hispida sampled from a polluted site in the Edku textile industrial area in Egypt revealed a high accumulation of chemical elements, compared to a reference site. The concentration of elements in soil was well correlated with their concentration in the midgut of insects. Activity of superoxide dismutase, catalase, ascorbate peroxidase and glutathione S-transferase were negatively correlated with concentration of elements in soil and in the midgut. Meanwhile, malondialdehyde concentration in the midgut revealed an opposite tendency. DNA damage and expression of stress proteins, (HSP70 and metallothionein - MT) were elevated in insects from the polluted site. The activity of textile industry in the area of Edku undoubtedly causes an increase of soil pollution and, in consequence, causes a number of changes in the bodies of organisms living in these areas, including T. hispidus. Therefore, it is necessary to find a solution which limits the emission of waste from the textile industry, as well as to design modern strategies of processing, storing and utilizing it.
This study sought to examine whether supplementation of Black Seed Oil (BSO) can improve pulmonary function tests (PFTs), inflammation, and oxidant-antioxidant markers in COPD patients. The study involved 100 patients of mild to moderate COPD divided randomly into 2 groups who were appointed to receive standard medication only (control group) or with additional Black Seed Oil (BSO group). They were assessed initially and after 3 months, 44 patients responded in control group and 47 patients in BSO group. BSO group evidenced a significant decreasing in oxidant and inflammatory markers; thiobarbituric acid reactive-substances (TBARS), protein carbonyl (PC) content, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), a significant increase in antioxidants; superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), glutathione peroxidase (GPx), vitamin C, and E, and a significant improvement in PFTs versus control group and baseline levels. Supplementation of Black Seed Oil may be an effective adjunct therapy to improve pulmonary functions, inflammation, and oxidant-antioxidant imbalance in COPD patients.
Toxic effect of chronic waterborne copper exposure on growth, immunity, anti-oxidative capacity and gut microbiota of Pacific white shrimp Litopenaeus vannamei
2020, Fish and Shellfish ImmunologyCopper can be accumulated in water through excessive sewage discharge or residual algaecide to generate toxic effect to aquatic animals. In this study, the juvenile of Pacific white shrimp, Litopenaeus vannamei was exposed to 0 (control), 0.05, 0.1, 0.2, 0.5 or 1 mg Cu2+ L−1 for 30 days. Growth, immune function, anti-oxidative status and gut microbiota were evaluated. Weight gain and specific growth rate of L. vannamei were significantly decreased with the increase of ambient Cu2+. Enlarged lumen and ruptured cells were found in the hepatopancreas of shrimp in the 0.5 or 1 mg Cu2+ L−1 treatment. Total hemocyte counts of shrimp in 0.5 or 1 mg Cu2+ L−1 were significantly lower than in the control. The hemocyanin concentration was also significantly increased in 0.2 or 0.5 mg Cu2+ L−1. Lysozyme contents were reduced in shrimp when Cu2+ exceeded 0.2 mg L−1. Meanwhile, activities of superoxide dismutase and glutathione peroxidase were increased in the hepatopancreas and the activity of Na+-K+ ATPase was decreased in the gills with increasing Cu2+. The mRNA expressions of immune deficiency, toll-like receptor and caspase-3 were all significantly higher in the hepatopancreas in 0.05 mg Cu2+ L−1 than in the control. For the diversity of intestinal microbes, Bacteroidetes significantly decreased in 1 mg Cu2+ L−1 at the phylum level. KEGG pathway analysis demonstrates that 1 mg L−1 Cu2+ can significantly alter metabolism, cellular processes and environmental information processing. This study indicates that the concentration of 1 mg L−1 Cu can negatively impact growth, hemolymph immunity, anti-oxidative capacity and gut microbiota composition of L. vannamei.