Reactive oxygen species in pregnant rats: effects of exercise and thermal stress

With the aim of evaluating the effect of interaction between physical training or exercise only during pregnancy and thermal stress on oxidative stress, and antioxidant mechanism sedentary pregnant rats (PS), exercised pregnant rats only during pregnancy (PE) and trained rats submitted to also exercise during pregnancy (PT) were compared (N=63). Exercise sessions consisted of swimming at 80% of maximal work load supported into water at 28 degrees C (hypothermia, PS 28, PE28, PT28) or 35 degrees C (thermal neutrality, PS35, PE35, PT35) or 39 degrees C (hyperthermia, PS39, PE39, PT39), for 30 min. The initial body weight in all groups of rats was from 177 to 207 g. On the 20th day of pregnancy, 24 h after the last immersion or swimming session venous blood was collected to determine oxidative stress. Plasma concentrations of means malondialdehyde (MDA) values measured as thiobarbituric acid reactive substances (TBARS); total glutathione (GSH) and vitamin E were determined. The oxidative stress index was calculated from the ratio TBARS/GSH and TBARS/Vitamin E. TBARS did not change on the group PE at different temperatures of water; TBARS were higher for PS28 than PS35 and PS39; PT35 had higher values than PT28 and PT39. For GSH, PS39 was lower than PS35; PE28 was higher than PE35 and PE39 and PT35 were lower than PT28 and PT39. Plasma concentration of vitamin E did not present any difference for sedentary rats at different water temperatures, but for PE28, the values were lower than for PE35 and PE39, whereas PT39 was lower than PT35 and PT28. In relation to TBARS/GSH, it was verified an increase in oxidative stress for PS28 (in relation to PS35 and PS39), PE35, and PT35 (in relation to PE28 and PE39 or PT28 and PT39); regarding the ratio TBARS/vitamin E, the highest values were obtained at 35 degrees C for PS and PT groups and at 39 for PE group. These results have shown the great complexity of the interaction between physical training, thermal stress and pregnancy. Apparently, hypothermia produces large index of oxidative stress only in sedentary rats, but this index was greater at 35 degrees C in relation to extreme temperatures for trained rats. These results have suggested that physical training allows a more efficient activation of antioxidant mechanisms under thermal stress.     

Dietary caloric restriction improves the redox status at the onset of diabetes in hepatocytes of streptozotocin-induced diabetic rats

Enhanced production of free radicals and oxidative stress induced by hyperglycemia play a central role in the pathogenesis of diabetes and its complications. This study assessed the attenuation by dietary caloric restriction on the oxidative and lipid peroxidative effects of diabetes in the liver through reduction in body and organ weights and concomitant metabolic changes. Three-month-old male Wistar rats were subjected to ad libitum feeding and 30% caloric restriction for 9 weeks before induction of diabetes by intraperitoneal injection of 35 mg/kg body weight streptozotocin. The animals were sacrificed 2 weeks after streptozotocin treatment depicting the onset of diabetes. Caloric restriction significantly reduced the organ weights (p<0.01), malondialdehyde (p<0.01) and catalase activity (p<0.01), but significantly increased glutathione reductase activity (p<0.01), and GSH/GSSG ratios (p<0.05). Caloric restriction also non-significantly reduced reactive oxygen species, superoxide dismutase and oxidized glutathione but increased glutathione peroxidase activity and reduced glutathione levels in the diabetic rats. Our data indicate a decrease in lipid peroxidation, improvement in the antioxidant defense systems and restoration of the redox status in the liver by caloric restriction. Therefore, this could provide a non-invasive antioxidant therapy early in diabetes to prevent the development of the complications associated with the disease.     

Effect of indole acetic acid administration on the neutrophil functions and oxidative stress from neutrophil, mesenteric lymph node and liver

This study was done to investigate the effect of the in vivo administration of indole acetic acid (IAA) on the neutrophil function, the activities of antioxidants enzymes in neutrophils, the mesenteric lymph node and on the oxidative stress in liver and plasma. The animals received subcutaneous administration of IAA in a phosphate-buffered saline (the control group received only the phosphate-buffered saline). The other groups received IAA at concentrations of 1 mg (T1), 2 mg (T2) and 18 mg (T3) per kg of body mass per day. Administration of IAA in both treatments T2 and T3 promoted a significant rise in the phagocytic capacity of neutrophils (by 51%), in comparison with the control. Another alteration was observed in antioxidant enzyme activities of the neutrophil and lymph node. But in the liver, the treatments imposed a significant decrease in the activity of catalase of 19% and 30% for T2 and T3, respectively, in comparison with the control. A similar effect was observed in the activity of hepatic glutathione peroxidase for T3 where a significant decrease of 31%, compared with the control, was obtained. The IAA did not show another significant alteration of the activities of superoxide dismutase and glutathione reductase activities in liver. The hepatic lipid peroxidation level, available by reactive products with thiobarbituric acid, has shown a significant decrease of 27% and 29% with T1 and T3 respectively, in comparison with the control. IAA treatment did not show a significant alteration in reduced glutathione contents in comparison with the liver and plasma controls. In conclusion, the IAA administration has a good potential animal utilization for increasing the phagocytic capacity with no prooxidant effect.     

Antioxidant responses and lipid peroxidation in gills and erythrocytes of fish (Rhabdosarga sarba) upon exposure to Chattonella marina and hydrogen peroxide: Implications on the cause of fish kills

Chattonella marina, a red tide or harmful algal bloom species, has caused mass fish kills and serious economic loss worldwide, and yet its toxic actions remain highly controversial. Previous studies have shown that this species is able to produce reactive oxygen species (ROS), and therefore postulated that ROS are the causative agents of fish kills. The present study investigates antioxidant responses and lipid peroxidation in gills and erythrocytes of fish (Rhabdosarga sarba) upon exposure to C. marina, compared with responses exposed to equivalent and higher levels of ROS exposure. Even though C. marina can produce a high level of ROS, gills and erythrocytes of sea bream exposed to C. marina for 1 to 6 h showed neither significant induction of antioxidant enzymes nor lipid peroxidation. Antioxidant responses and oxidative damage did not occur as fish mortality began to occur, yet could be induced upon exposure to artificially supplied ROS levels an order of magnitude higher. The result of this study implies that ROS produced by C. marina is not the principal cause of fish kills.     

Effects of linuron and dimethenamid on antioxidant systems in weeds associated with soybean

Changes in antioxidant systems in soybean and associated weeds (Ambrosia artemisiifolia L., Chenopodium album L., Convolvulus arvensis L and Sinapis arvensis L.) were studied in relation to treatment with herbicides linuron and dimethenamid in the field experiment. Differences in the total superoxide dismutase (SOD) and catalase (Cat) activities were observed in plants after application of herbicide formulation. Quantities of superoxide (O₂.-) and hydroxyl (·OH) radicals and malonyldialdehyde (MDA), reduced glutathione (GSH) and total polyphenols content were also determined. In addition to this, potential antioxidant activity of the plant ethanolic extracts were assessed based on the scavenging activity of stable DPPH free radicals. Results obtained suggest that plants investigated 1) expressed different antioxidant systems in response to herbicide treatment; 2) enzymatic and non-enzymatic protective mechanisms were complementary; 3) some weed species showed distinctive and combined activity of several biochemical parameters, such as Ambrosia artemisiifolia.     

The role of reactive oxygen species and oxidative stress in carbon monoxide toxicity: An in-depth analysis

Abstract

The underlying mechanism of the central nervous system (CNS) injury after acute carbon monoxide (CO) poisoning is interlaced with multiple factors including apoptosis, abnormal inflammatory responses, hypoxia, and ischemia/reperfusion-like problems. One of the current hypotheses with regard to the molecular mechanism of CO poisoning is the oxidative injury induced by reactive oxygen species, free radicals, and neuronal nitric oxide. Up to now, the relevant mechanism of this injury remains poorly understood. The weakening of antioxidant systems and the increase of lipid peroxidation in the CNS have been implicated, however. Accordingly, in this review, we will highlight the relationship between oxidative stress and CO poisoning from the perspective of forensic toxicology and molecular toxicology.     

The effects of reactive oxygen species on amphibian aging

To clarify the role of reactive oxygen species (ROS) in the aging process of amphibians, antioxidant enzyme activity and indexes of ROS damage were investigated biochemically using the livers of 3- and 10-year-old Rana nigromaculata frog males and females. Findings revealed no significant difference in survival rate between males and females. Antioxidant enzyme activity displayed an age-related decline. Superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx) activity in 10-year-old liver decreased 40-80% from 3-year-old liver levels. In contrast, urate oxidase activity in the 10-year-old liver increased more than 200% from 3-year-old liver levels. At the same time levels of ROS damage, including the concentration of inorganic peroxide and thiobarbituric acid reactive substances (TBARS), greatly increased with age. Liver catalase from 10-year-old frogs proved to be more susceptible to aminotriazole and urea, losing approximately 80% of its original activity after 30 min of treatment. It seems likely that liver catalase in older frogs has diverged from liver catalase in younger frogs through oxidative modification. These findings suggest that a decrease in the activity of antioxidant enzymes over time results in increased levels of ROS damage in the livers of older frogs.     

Fasting induces cyanide-resistant respiration and oxidative stress in the amoeba Chaos carolinensis: implications for the cubic structural transition in mitochondrial membranes

Summary. Large free-living amoeba (Chaos carolinensis) can survive in spring water without food intake for several weeks. Starvation is associated with a dramatic change in mitochondrial cristae from random tubular to ordered (paracrystalline) cubic morphology. Whole-cell polarography was used to monitor changes in respiratory activity during fasting. Basal respiration per cell decreased progressively during starvation, while the cyanide-resistant fraction increased. Spectrofluorometric assay of H₂O₂ and reactive oxygen species (ROS) in cell lysates (using the dye 2′,7′-dichlorofluorescein diacetate) indicates greater H₂O₂ and ROS generation in starved than in fed cells. Fluorescence microscopy of intact cells incubated with the same dye demonstrates that H₂O₂ and ROS tend to accumulate in vacuoles. A remarkable generation of O₂ observed with starved cells after addition of KCN may be explained by release of H₂O₂ from these compartments into the cytosol, where it can react with catalase. Together, these observations suggest that fasting increases oxidative stress in the amoeba and that this organism has several protective mechanisms to deal with it, including activation of a plantlike alternative oxidase. The hypothesis is forwarded that the cubic structural transition of the mitochondrial inner membrane represents another protective mechanism, reducing oxidative damage by enhancing the efflux of H₂O₂ and ROS and by reducing the susceptibility of membrane lipids to the oxidants. Received September 1, 2001 Accepted January 7, 2002     

Exogenous insulin can reverse the effects of caloric restriction on mitochondria

It has been proposed that part of the anti-aging mechanism of caloric restriction (CR) involves a reduction in both the generation rate of reactive oxygen species (ROS) by mitochondria, and a reduction in peroxidizability of mitochondrial membranes. It was hypothesized that these effects may be due to upstream changes in hormonal status, since certain hormones (such as insulin) are stimulatory for ROS production, effect fatty acid composition, and are lowered by CR. To investigate this hypothesis, young male Brown-Norway rats on 55% CR (4 months duration) were subjected to insulin replacement by use of mini-osmotic pumps. ROS and free radical-induced malondialdehdye production were significantly lower in mitochondria from CR animals compared to those from fully fed, and these effects were reversed by insulin. It is concluded that the beneficial changes induced by CR, as seen at the mitochondrion, may in part be downstream effects of alterations in hormonal signalling.     

Studies on the protective effects of betaine against oxidative damage during experimentally induced restraint stress in Wistar albino rats

Stress can be defined as physical and psychological modifications that disrupt the homeostasis and the balance of organisms. Stress is known as one of the most important reasons of several diseases. In the present study, the anti-stress effect of betaine was evaluated with reference to its antioxidant property. Wistar albino rats were divided into four groups such as control, betaine, restraint stress (6 h/day for 30 days), and betaine + restraint stress. The oxidative damage was assessed by measuring the protein and corticosterone in plasma, lipid peroxidation, non-enzymic (reduced glutathione), and enzymic antioxidants (glutathione peroxidase, glutathione-S-transferase, catalase, and superoxide dismutase) in the lymphoid organs of thymus and spleen. Followed by the induction of restraint stress, the non-enzymic and enzymic antioxidants were significantly decreased with concomitant increase observed in the levels of corticosterone and lipid peroxidation. Oral pretreatment with betaine (250 mg/kg body weight daily for a period of 30 days) significantly (P < 0.001) prevented the restraint stress-induced alterations in the levels of protein and corticosterone in plasma of experimental groups of rats. It counteracted the restraint stress-induced lipid peroxidation and maintained the antioxidant defense system in the lymphoid tissues at near normal. The findings suggest that betaine possesses significant anti-stress activity, which may be due to its antioxidant property.     

The quality of the antioxidant defence system in term and preterm twin neonates

Abstract

Objective: Multiple pregnancy is associated with an enhanced metabolism and demand for O2, which may lead to the overproduction of reactive oxygen species and the development of oxidative stress. The degree of oxidative damage depends on the level of the antioxidant protection system of the foetus. The objective of the study was to identify the relationship between the state of the maturity and the antioxidant status of twin neonates. Investigations of the umbilical cord blood were carried out to detect differences in the antioxidant defence system between mature and premature twin neonates.

Methods: The activities of the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) enzymes, the levels of reduced glutathione (GSH), protein carbonyls and oxidized lipids and the total antioxidant capacity of the plasma were determined.

Results: The level of lipid peroxidation was significantly higher in the premature neonates. An increase in the total antioxidant capacity was accompanied by a decrease in the damaged protein concentration. Significantly elevated activities of GPx alone were observed in the premature twins, though the GSH content too tended to be increased. The activity of SOD was decreased in the premature neonates.

Discussion: The antioxidant status of twin neonates are mainly influenced by maturity. We suggest that the level of lipid peroxidation might be of clinical value as a marker of pre- and perinatal distress in twins.     

Determination of urinary malondialdehyde by isotope dilution LC-MS/MS with automated solid-phase extraction: a cautionary note on derivatization optimization

A highly sensitive quantitative LC-MS/MS method was developed for measuring urinary malondialdehyde (MDA). With the use of an isotope internal standard and online solid-phase extraction, urine samples can be directly analyzed within 10 min after 2,4-dinitrophenylhydrazine (DNPH) derivatization. The detection limit was estimated as 0.08 pmol. This method was further applied to assess the optimal addition of DNPH for derivatization and to measure urinary MDA in 80 coke oven emission (COE)-exposed and 67 nonexposed workers. Derivatization optimization revealed that to achieve complete derivatization reaction, an excess of DNPH is required (DNPH/MDA molar ratio: 893-8929) for urine samples that is about 100 times higher than that of MDA standard solutions (molar ratio: 10-80). Meanwhile, the mean urinary concentrations of MDA in COE-exposed workers were significantly higher than those in nonexposed workers (0.23±0.17 vs 0.14±0.05 μmol/mmol creatinine, P<0.005). Urinary MDA concentrations were also significantly associated with the COE (P<0.005) and smoking exposure (P<0.05). Taken together, this method is capable of routine high-throughput analysis and accurate quantification of MDA and would be useful for assessing the whole-body burden of oxidative stress. Our findings, however, raise the issue that derivatization optimization should be performed before it is put into routine biological analysis.     

The role of mitochondrial dehydrogenases in the generation of oxidative stress

In addition to complexes in the respiratory chain, few dehydrogenases playing key roles in the physiological metabolism in neurons, are able to generate reactive oxygen species (ROS) in mitochondria. One of them is the Krebs cycle enzyme, α-ketoglutarate dehydrogenase (α-KGDH), which is capable of producing superoxide and hydrogen peroxide by the E3 subunit of the enzyme regulated by changes in the NADH/NAD⁺ ratio. Mutations in the E3 subunit known to be related to diseases in humans were shown to have increased ROS-forming ability. α-Glycerophosphate dehydrogenase (α-GPDH) located on the outer surface of the inner membrane can also generate ROS, which is stimulated by Ca²⁺. ROS production by α-GPDH is unique as it does not require Ca²⁺ uptake and it is observed in respiring as well as damaged, bioenergetically incompetent mitochondria. The possible role of ROS generation by these dehydrogenases in brain pathology is discussed in this review.     

Trypanosoma evansi: Effect of experimental infection on the osmotic fragility, lipid peroxidation and calcium-ATPase activity of rat red blood cells

Trypanosoma evansi is the causative agent of equine trypanosomoses. The disease is characterized by fever, anemia, and cachexia. Peroxidative damage of the red blood cells caused by the parasite, may contribute to the pathogenesis of the anemia seen in trypanosomoses. Consequently, we evaluated the hematocrit, the osmotic fragility of the red blood cells, the level of lipid peroxidation and the activity of the Ca-ATPase of red blood cell ghosts from rats experimentally infected with T. evansi. After 72 h inoculation, the hematocrit decreased from 49.5% to 33%; the osmotic fragility of the red blood cells was approximately 40% higher as compared to the healthy animals; and the red blood cell ghosts showed a higher level of lipid peroxidation and a lower Ca-ATPase activity than the red cell ghosts from the healthy animals. In vitro incubations of red blood cells from healthy animals with T. evansi, produced also a significant increase of the osmotic fragility of the red blood cells.     

Pyrroloquinoline-quinone synthesized in Escherichia coli by pyrroloquinoline-quinone synthase of Deinococcus radiodurans plays a role beyond mineral phosphate solubilization

Deinococcus radiodurans, an extremely radioresistant bacterium, synthesizes coenzyme pyrroloquinoline-quinone (PQQ) but exhibits a negative phenotype for mineral phosphate solubilization. Gene for the putative PQQ synthesizing protein was PCR amplified and cloned from Deinococcus, sequenced, and expressed in Escherichia coli, under an inducible E. coli promoter. The transgenic E. coli expressed PQQ synthase protein of 42kDa and complemented the mineral phosphate solubilization phenotype of E. coli, suggesting the synthesis of an active protein. The cells expressing high levels of this protein showed increased protection against photodynamically produced reactive oxygen species. The effect could be attributed to the upregulation of antioxidant enzymes such as catalase and superoxide dismutase by PQQ in transgenic E. coli through an unknown mechanism. The study elucidates a hitherto unknown possible function of PQQ in bacteria.     

Effects of caloric restriction on age-related oxidative modifications of macromolecules and lymphocyte proliferation in rats

Decreased immune function associated with aging has been demonstrated in both humans and animals. We hypothesize that reactive oxygen species (ROS)-mediated damage to biological macromolecules may contribute to compromised immune response during aging. In this study, we compared the levels of lipid peroxidation and oxidatively modified proteins in plasma and splenocytes, and the mitogen-induced T lymphocyte proliferation in ad lib-fed (AL) and caloric restricted (CR) Fischer 344 × BNF₁ male rats at the ages of 5, 18, and 31 months. The results show that AL rats exhibit an age-related decrease in proliferative response of splenic lymphocytes to phytohemagglutinin (PHA) and concanavalin A (Con A). This functional decline in T-lymphocytes during aging is inversely correlated to the levels of both lipid peroxidation and protein carbonyl in the plasma and splenic lymphocytes. Caloric restriction, however, can partially reverse the age-dependent decrease in T lymphocyte proliferation and significantly reduce lipid peroxidation and protein carbonyl contents in plasma and splenocytes. The above observations support the hypothesis that the age-associated declines in immune function are related to the oxidative modification of biological macromolecules, which in turn may lead to enzyme inactivation, membrane disruption, and cell senescence. One of the mechanisms by which caloric restriction reverses declined immune function in aged rats is hypothesized to be through reduction in ROS production and thereby protection of cellular macromolecules against oxidative damage.     

Effect of overexpression of Bcl-2 on cellular oxidative damage, nitric oxide production, antioxidant defenses, and the proteasome

Bcl-2 is a gene family involved in the suppression of apoptosis in response to a wide range of cellular insults. Multiple papers have suggested a link between Bcl-2 and oxidative damage/antioxidant protection. We therefore examined parameters of antioxidant defense and oxidative damage in two different cell lines, NT-2/D1 (NT-2) and SK-N-MC, overexpressing Bcl-2 as compared with vector-only controls. Bcl-2 transfectants of both cell lines were more resistant to H₂O₂ and showed increases in GSH level and Cu/Zn-superoxide dismutase (SOD1) activity, but not in Mn-superoxide dismutase, glutathione peroxidase, or glutathione reductase activities. Catalase activity was increased in SK-N-MC cells. Overexpression of Bcl-2 did not significantly decrease levels of oxidative DNA damage (measured as 8-hydroxyguanine) or lipid peroxidation, but it decreased levels of 3-nitrotyrosine in both cell lines and protein carbonyls in SK-N-MC cells only. It also increased proteasome activity in both cell lines. We conclude that Bcl-2 raises cellular antioxidant defense status, but this is not necessarily reflected in decreased levels of oxidative damage to DNA and lipids. The ability of Bcl-2 overexpression to decrease 3-nitrotyrosine levels suggests that it may decrease formation of peroxynitrite or other reactive nitrogen species; this was confirmed as decreased production of NO₂⁻/NO₃⁻ in the transfected cells and a fall in the level of nNOS protein.     

Salinity-induced changes in two cultivars of Vigna radiata: responses of antioxidative and proline metabolism

The influence of increasing salinity stress on plant growth, antioxidant enzymes and proline metabolism in two cultivars of Vigna radiata L. (cv. Pusa Bold and cv. CO 4) was investigated. Salt stress was imposed on 30-days-old cultivars with four different concentrations of NaCl (0, 100, 200 and 300 mM). The roots and shoots of CO 4 showed greater reduction in fresh weight, dry weight and water content when compared to Pusa Bold with increasing salt stress. Under salinity stress, the roots and shoots of CO 4 exhibited higher Na⁺: K⁺ ratio than Pusa Bold. The activities of reactive oxygen species (ROS) scavenging enzymes and reduced glutathione (GSH) concentration were found to be higher in the leaves of Pusa Bold than in CO 4, whereas oxidized glutathione (GSSG) concentration was found to be higher in the leaves of CO 4 compared to those in Pusa Bold. Our studies on oxidative damage in two Vigna cultivars showed lower levels of lipid peroxidation and H₂O₂ concentration in Pusa Bold than in CO 4 under salt stress conditions. High accumulation of proline and glycine betaine under salt stress was also observed in Pusa Bold when compared to CO 4. The activities of proline biosynthetic enzymes were significantly high in Pusa Bold. However, under salinity stress, Pusa Bold showed a greater decline in proline dehydrogenase (ProDH) activity compared to CO 4. Our data in this investigation demonstrate that oxidative stress plays a major role in salt-stressed Vigna cultivars and Pusa Bold has efficient antioxidative characteristics which could provide better protection against oxidative damage in leaves under salt-stressed conditions.