Antioxidant enzymes in the differentiated Caco-2 cell line.

Exploratory factor analysis of reported specific activities of the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase in normal human tissues, normal mouse tissues, vertebrate red blood cells and neoplastic human cell lines shows that the activities of copper-zinc superoxide dismutase, catalase and glutathione peroxidase in normal tissues are influenced by a single factor. Catalase activity has the highest loading and correlation with this factor, suggesting a catalase- or hydrogen peroxide-related influence. The activity of manganese superoxide dismutase is influenced by a separate factor. The activities of copper-zinc and manganese superoxide dismutases in normal tissues therefore appear to be dichotomously regulated. The activities of superoxide dismutase and glutathione peroxidase in vertebrate red blood cells are influenced by a single factor. The activity of catalase is influenced by a separate factor. The roles of glutathione peroxidase and catalase in hydrogen peroxide catabolism in red blood cells in fact differ. In neoplastic human cell lines, two bipolar factor factors appear to influence the activities of catalase and manganese superoxide dismutase, and glutathione peroxidase and copper-zinc superoxide dismutase, respectively. The factors are, however, mainly catalase and glutathione peroxidase activity factors as the loadings and correlations of manganese superoxide dismutase on the one hand and copper-zinc superoxide dismutase on the other, with the respective factors, are relatively small. Potentially low superoxide production and intrinsically low peroxidizability of tumour cell membranes underlie the peculiar variation of antioxidant enzyme activities in tumour cells. Factor analysis is proposed as a heuristic data reduction and hypothesis-creating technique for the variation of antioxidant and other functionally-linked enzyme activities in normal and pathological cells and tissues.

Oxidative stress can have a negative effect on oocyte maturation during invitro production of pig embryos. Imbalance of reactive oxygen species and antioxidant levels can affect the progression of oocyte maturation up to the point of fertilization. Antioxidants are effective in maintaining more ideal reactive oxygen species levels, which help to protect oocytes from potential harmful effects of oxidative stress. Berries from the elder plant (Sambucus sp.) contain high levels of a broad spectrum of antioxidants. One of these antioxidants, cyanidin, when supplemented to maturation medium at 100μM concentrations, reduces reactive oxygen species formation and improves IVF and early embryonic development in pigs. However, changes in the enzyme mechanisms of action during oocyte maturation due to cyanidin supplementation are unknown. Therefore, the objective of this study was to characterise the intracellular oocyte enzyme mechanisms between oocytes supplemented with 100μM cyanidin during 40 to 44h of maturation (n=600) and oocytes without supplementation of cyanidin during maturation (n=558). At the end of maturation, oocytes were evaluated for either glutathione peroxidase (n=300), catalase (n=564), or superoxide dismutase (n=294) activities. Glutathione peroxidase activity was determined by following the rate of NADPH oxidation, catalase activity was determined by following the rate of hydrogen peroxide decomposition, and superoxide dismutase activity was determined by following the reduction rate of cytochrome c, utilising the xanthine-xanthine oxidase system. Data were analysed using ANOVA and Tukey's test. There were no significant differences between oocytes matured with 100μM cyanidin and those that were not when comparing glutathione peroxidase and superoxide dismutase activities. Supplementation of 100μM cyanidin to maturation medium increased (P<0.05) catalase activity in oocytes (0.78±0.15 units/oocyte) compared with no cyanidin supplementation (0.14±0.11 units/oocyte). These results indicate that supplementing 100μM cyanidin to the maturation medium of pig oocytes could reduce the negative effects of oxidative stress by increasing intracellular catalase activity during oocyte maturation.

Ischemia-reperfusion (I-R) injury associated with acute mesenteric vascular occlusion can lead to severe impairment of intestinal tissue and may become a life-threatening condition if not treated in the early clinical stages. Previous studies have suggested that fraxin may exert protective effects against I-R-induced mesenteric injury due to its antioxidant and anti-inflammatory properties. This experimental study was conducted using healthy male Wistar albino rats. The animals were divided into four groups: a Sham group (superior mesenteric artery [SMA] isolated but not occluded), a Control group (SMA isolated and I-R induced), a 10 mg/kg Fraxin group, and a 50 mg/kg Fraxin group (fraxin administered before reperfusion). Total antioxidant status (TAS), total oxidant status (TOS), superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities were evaluated. Histopathological examinations and inflammatory markers, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and myeloperoxidase (MPO), were also analyzed. In the Sham group, SOD activity was 135.2±10.5 U/mg protein, GPx activity was 65.3±4.7 U/mg protein, and CAT activity was 85.1±5.8 U/mg protein. In the Control group, these values were 95.4±7.9, 45.7±3.6, and 60.3±4.2 U/mg protein, respectively. In 10 mg/kg Fraxin group, SOD, GPx, and CAT activities were 115.6±8.4, 55.8±4.2, and 75.6±5.5 U/mg protein, respectively; in the 50 mg/kg Fraxin group, the corresponding values were 130.8±9.7, 60.2±4.8, and 90.4±6.3 U/mg protein. Significant decreases in TNF-α, IL-6, and MPO levels were observed in the Fraxin-treated groups (p<0.05). Fraxin administration preserved tissues and improved antioxidant parameters by reducing oxidative stress and inflammation in the acute mesenteric artery ischemia-reperfusion injury (AMAIRI) model. Based on these findings, fraxin may be considered a potential therapeutic option for mesenteric ischemia-reperfusion-related injuries.

Oxidative stress, caused by excessive reactive oxygen species generation, impairs health and production performance of pigs. A number of nutrients and feed additives have antioxidative property. Our current research project is to study the antioxidative roles of methionine (a sulfur-containing amino acid) in pigs, and this abstract is to report our setup of an oxidative-stressed model by injection of diquat (dibromide monohydrate), a bipyridyl herbicide, to the pig. Barrows [n = 20, body weight (BW) = 21.5 ± 2.50 kg] were randomly allotted to two treatment groups fed a corn-and-soybean-meal-based diet that met or slightly exceeded the NRC (2012) recommended requirements of nutrients and energy. After 3 wk of feeding (Phase 1), while Group-1 pigs were intraperitoneally injected with 10 mL saline, Group-2 pigs were injected with 10 mg/kg BW diquat in 10 mL saline. After the injection, pigs were continually fed for 7 d more (Phase 2). At the end of Phases 1 and 2, average daily gain (ADG), average daily feed intake (ADFI), and gain to feed (G:F) ratios were respectively determined. Blood samples were collected on d 1, 22, and 29 of the experiment, and liver samples were collected on d 29. Commercial assay kits were used to determine the blood and liver redox status, for which the malondialdehyde (MDA) and glutathione (GSH) contents, the superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities, and the total antioxidant capacity (TAC) were measured. The data was statistically analyzed using Student’s t-test. During Phase 1, there were no differences (P &amp;gt; 0.10) in initial BW, final BW, ADFI, ADG, and G:F ratio. There were no differences (P &amp;gt; 0.10) in the serum MDA contents, the serum GPX, SOD, and CAT activities, and the serum TAC and GSH contents between the two dietary treatment groups. By the end of Phase 2, however, the AFDI and ADG in Group 2 were less (P &amp;lt; 0.001) than that in Group 1. The serum MDA content in Group 2 was greater (P &amp;lt; 0.05) than in Group 1, the serum GPX activity and liver SOD activity in Group 2 was less (P &amp;lt; 0.05) than in Group 1, and the liver GPX activity in Group 2 tended to be greater (P = 0.10) than in Group 1, although there were no differences (P &amp;gt; 0.10) in serum SOD activity, serum TAC, and serum and liver CAT activities between the two treatment groups. In conclusion, the results of this study confirmed that young growing pigs intraperitoneally injected with diquat can lead to oxidative stress and serve as a model for studying antioxidant nutrients or feed additives in pigs.

Background: Diabetes mellitus as a metabolic disease can have serious consequences. Due to their chemical properties,bioactive compounds can play a role in diabetes management. Squalene is a natural oil and bioactive compound. The anti-inflammatory and antioxidant effects of squalene have been discussed in recent studies. Squalene plays a role in controlling diabetes by maintaining the oxidant/antioxidant balance.Objective: The main purpose of this study was to evaluate the antioxidant effect of different doses of squalene, on different days, on the levels of some oxidative indices and the activity of antioxidant enzymes in groups of people with type 2 diabetes and compare them with each other and healthy people.Methods: 150 individuals were recruited in this study. These individuals were separated into five groups. Group one contained 30 individuals, representing the healthy control group. Groups 2, 3, 4 and 5 included subjects with type 2 diabetes. Each of the subjects in groups 3, 4 and 5 received squalene in doses of 200, 400 and 600 mg as an oral capsule (liquid filled oral), respectively for 84 days. Subjects in Group 2 did not receive squalene. Catalase, superoxide dismutase, glutathione peroxidase (as antioxidant indicators) activities and the levels of hydrogen peroxide, nitric oxide and reactive oxygen species (as oxidant indicators) were assayed.Results: In 84 days, a statistically significant difference (P value &lt; 0.05) was observed in all the diabetic groups compared to the healthy group. In the comparison between groups receiving squalene with each other, there was a significant increase (P value &lt; 0.05) in catalase and superoxide dismutase activity, depending on squalene dose and time. There was not a statistically significant (P value &gt; 0.05) increase in glutathione peroxidase activity. Statistically significant changes in oxidative indices were not dose-dependent or time-dependent.Conclusion: Based on the findings of this study, a dose of 600 mg of squalene in 84 days is effective in increasing catalase and superoxide dismutase activity and reducing hydrogen peroxide levels. Squalene can play an important role in controlling and reducing the consequences of diabetes caused by changes in the oxidant/antioxidant balance.Keywords: squalene, type 2 diabetes, T2D, antioxidant enzymes, bioactive compounds, free radicals

Mice Heterozygous for a Defect in Mitochondrial Trifunctional Protein Develop Hepatic Steatosis and Insulin Resistance

Evidences for a role of glutathione peroxidase 4 (GPx4) in methylmercury induced neurotoxicity in vivo

One hundred and fifty 7-day-old Arbor Acres broilers were randomly assigned into five groups: group 1 served as a control that was fed a basal diet without selenium (Se) supplementation; groups 2, 3 and 4 were fed the basal diet supplemented with 0.15, 0.5 and 1.5mg Se as Se-enriched Saccharomyces cerevisiae (SSC) per kg of diet; and group 5 was fed the basal diet supplemented with 0.15mg per kg of Se as sodium selenite (SS). Growth performance, glutathione peroxidase (GPX ) and superoxide dismutase (SOD) activities, total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content in plasma and liver, and cellular glutathione peroxidase (GPX -1) and phospholipid hydroperoxide glutathione peroxidase (GPX -4) mRNA levels in liver were determined. Compared with group 1, groups 2-4 exhibited higher body weights (p<0.05), lower feed/gain ratios, and higher GPX activities in plasma (p<0.05) and GPX and SOD activities and GPX -1 and GPX -4 mRNA levels in liver (p<0.05). Compared with group 5, group 2 exhibited higher GPX activity in plasma on day 21 (p<0.05). Compared with group 2 and 5, group 3 exhibited lower MDA content in plasma on day 7 (p<0.05), higher GPX activity in plasma, SOD activity and GPX -1 mRNA levels in liver on day 14 and 21 (p<0.05), and higher GPX -4 mRNA levels on day 14 (p<0.05). Compared with group 4, group 3 exhibited lower MDA contents in plasma on day 14 (p<0.05) and in liver on day 21 (p<0.05), higher T-AOC in plasma and higher GPX -1 mRNA levels on day 14 and 21 (p<0.05), and higher SOD activity in plasma and higher SOD and GPX activities in liver on day 21 (p<0.05). Thus, SSC improves growth and antioxidant status of broilers; the short-term bioavailability of SS was faster than that of SSC, but the long-term bioavailability of SSC was greater than SS.

The activities of the pulmonary antioxidant enzymes (AOE), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase, increase in the final 10-20 % of gestation in the mammalian lung, to protect the lung from attack by increasing levels of reactive oxygen species at birth. Whether the increase occurs as a normal 'preparation for birth', i.e. by a genetically determined mechanism, or in response to increased levels of oxygen, i.e. in response to the environment, is not completely understood. We examined the activities of catalase, SOD and GPx in the developing lungs of two oviparous vertebrate species, the chicken (Gallus gallus) and an agamid lizard (Pogona vitticeps), and in a metamorphosing vertebrate, the anuran Limnodynastes terraereginae. During in ovo development embryos come into contact with higher levels of environmental oxygen, and at a much earlier stage of development, compared with the intrauterine development of mammals. Furthermore, in metamorphosing frogs, the lungs are inflated at an early stage to aid in buoyancy, although the gas-exchange function only develops much later upon final metamorphosis. Here, we hypothesise that the activity of the AOE will be elevated relatively much earlier during development in both oviparous and metamorphosing vertebrates. We also examined the effect of mild hypoxia (17 % oxygen) on the development of the pulmonary AOE in the chicken, to test the hypothesis that these enzymes are responsive to environmental oxygen. In the normoxic lung of both Gallus gallus and Pogona vitticeps, catalase and GPx activities were significantly increased in late incubation, whereas SOD activity decreased in late incubation. Catalase and SOD activities were virtually identical in hypoxic and normoxic embryos of the chicken, but GPx activity was significantly affected by hypoxia. In the developing frog, the activities of all enzymes were high at stage 30, demonstrating that the system is active before the lung displays any significant gas-exchange function. SOD and GPx activity did not increase further with development. Catalase activity increased after stage 40, presumably correlating with an increase in air-breathing. In summary, catalase expression in the two oviparous vertebrates appears to be completely under genetic control as the activity of this enzyme does not change in response to changes in oxygen tension. However, in tadpoles, catalase may be responsive to environmental oxygen. SOD also appears to follow a largely genetically determined program in all species. Under normoxic conditions, GPx appears to follow a genetically determined developmental pattern, but this enzyme demonstrated the largest capacity to respond to environmental oxygen fluctuations. In conclusion, it appears that the AOE are differentially regulated. Furthermore, the AOE in the different species appear to have evolved different levels of dependency on environmental variables. Finally, the late developmental increase in AOE activity seen in mammals is not as pronounced in oviparous and metamorphosing vertebrates.

AB0572 REDUCED ENZYMATIC ACTIVITY OF ANTIOXIDANT SYSTEM IN SCLEROSIS.

Effects of aminoguanidine and melatonin on intestinal ischemia/reperfusion injury in rats: An assessor-blinded, controlled experimental study

Reactive oxygen species (ROS) play a role in male infertility, where excessive amounts impair spermatozoal motility. Epididymal antioxidant enzymes protect spermatozoa from oxidative damage in the epididymal lumen. Antioxidant secretions from the seminal vesicle protect spermatozoa after ejaculation. As it is known that with age there is increased generation of ROS, the goals of this study were to determine how aging affects the response of antioxidant enzymes in the epididymis, seminal vesicles, and liver to l-buthionine-S,R-sulfoximine (BSO) mediated glutathione (GSH) depletion, and to examine the impact of GSH depletion on motility parameters of spermatozoa from the cauda epididymidis in young (4-mo-old) and old (21-mo-old) rats. Levels of GSH and glutathione disulfide (GSSG), as well as activities of glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase, were measured in the caput, corpus and cauda epididymidis, seminal vesicles, and liver. Spermatozoal motility was assessed by computer-assisted sperm analysis. Significant age-related changes in antioxidant enzyme activities were found in the liver and cauda epididymidis. Glutathione depletion clearly affected tissues in both young and old. The compounding effect of age was most evident in the cauda epididymidis, seminal vesicles, and liver, where antioxidant enzyme activities changed significantly. Additionally, spermatozoa motility was adversely affected after BSO treatment in both age groups, but significantly more so in older animals. In summary, the male reproductive tissues and liver undergo age-related changes in antioxidant enzyme activities and in their response to GSH depletion.

A trifunctional enzyme with glutathione S-transferase, glutathione peroxidase and superoxide dismutase activity

These experiments examined the influence of exercise intensity and duration on antioxidant enzyme activity in locomotor muscles differing in fiber type composition. Nine groups of female Sprague-Dawley rats (age 120 days) exercised 4 days/wk on a motor-driven treadmill for 10 wk. The impact of three levels of exercise intensity (low, moderate, and high: approximately 55, approximately 65, and approximately 75% of maximal oxygen consumption, respectively) and exercise duration (30, 60, and 90 min/day) was assessed. Sedentary animals served as controls. Oxidative capacity in the soleus and white and red gastrocnemius was assessed by measurement of citrate synthase (CS) activity, and antioxidant capacity was evaluated by assay of total superoxide dismutase, catalase, and total glutathione peroxidase (GPX) activities. In all muscles, CS activity increased as a function of exercise duration. Furthermore, in the soleus and white gastrocnemius, the magnitude of the training-induced increase in CS activity was directly related to exercise intensity. In contrast, the peak increase in CS activity in the red gastrocnemius was relatively independent of exercise intensity. Catalase activity was not increased (P > 0.05) in any muscle with training. Training-induced changes in superoxide dismutase and GPX activities were muscle specific; specifically, exercise training significantly (P < 0.05) increased superoxide dismutase activity in the soleus as a function of exercise duration up to 60 min/day. Conversely, training-induced significant (P < 0.05) increases in GPX activity occurred in red gastrocnemius only; the magnitude of the GPX increase was directly related to exercise duration but relatively independent of intensity. These data demonstrate that exercise training-induced changes in muscle antioxidant enzymes are muscle specific.

P-0205 Activity of Antioxidant Enzymes in Colorectal Cancer