Testosterone suppresses oxidative stress in human neutrophils

The in vitro effect of testosterone on human neutrophil function was investigated. Blood neutrophils from healthy male subjects were isolated and treated with 10 nM, 0.1 and 10 µM testosterone for 24 h. As compared with untreated cells, the testosterone treatment produced a significant decrease of superoxide production as indicated by the measurement of extra‐ and intracellular superoxide content. An increment in the production of nitric oxide was observed at 0.1 and 10 µM testosterone concentrations, whereas no effect was found for 10 nM. Intracellular calcium mobilization was significantly increased at 10 nM, whereas it was reduced at 10 µM testosterone. There was an increase in phagocytic capacity at 10 nM and a decrease of microbicidal activity in neutrophils treated with testosterone at 10 µM. Glutathione reductase activity was increased by testosterone treatment, whereas no effect was observed in other antioxidant enzyme activities. An increase in the content of thiol groups was observed at all testosterone concentrations. Lipid peroxidation in neutrophils evaluated by levels of TBARS was decreased at 10 nM and 0.1 µM testosterone. These results indicate the antioxidant properties of testosterone in neutrophils as suggested by reduction of superoxide anion production, and lipid peroxidation, and by the increase in nitric oxide production, glutathione reductase activity and the content of thiol groups. Therefore, the plasma levels of testosterone are important regulators of neutrophil function and so of the inflammatory response. Copyright © 2010 John Wiley & Sons, Ltd.     

Investigation of oxidative stress during fracture healing in the rats

One of the most damaging effects of reactive oxygen species (ROS) is lipid peroxidation, the end-product of which is malondialdehyde (MDA). This study was aimed to evaluate erythrocyte MDA levels during fracture healing in rats. Thirty male rats were used and the rats were divided into two groups to serve as controls and tests. Six rats were used as a control group that was not subject to fracture. The remaining 24 rats were divided into four groups and erythrocyte MDA levels were examined on days 5, 10, 20 and 30 post fracture. The right fibulas of rats were broken by manual angulation in the experimental group. The erythrocyte malondialdehyde level was measured in the experimental and control groups. The difference between malondialdehyde levels of control and experimental groups was statistically significant (p<0.05). Oxidative stress clearly increases during fracture healing in rats.     

Aggressive and non-aggressive personalities differ in oxidative status in selected lines of mice (Mus musculus)

Mice selected for aggression and coping (long attack latency (LAL), reactive coping strategy; short attack latency (SAL), pro-active coping strategy) are a useful model for studying the physiological background of animal personalities. These mice also show a differential stress responsiveness, especially in terms of hypothalamic-pituitary-adrenal axis reactivity, to various challenges. Since the stress response can increase the production of reactive oxygen species, we predicted that the basic oxidative status of the lines could differ. We found that LAL showed higher serum antioxidant capacity (OXY) than SAL, while no differences emerged for reactive oxygen metabolites (ROMs) or the balance between ROMs and OXY, reflecting oxidative stress. Moreover, the lines showed inverse relationships between ROMs or OXY and body mass corrected for age. The results indicate that variation in oxidative status is heritable and linked to personality. This suggests that different animal personalities may be accompanied by differences in oxidative status, which may predict differences in longevity.     

Gender differences in free radical homeostasis during aging: shorter-lived female C57BL6 mice have increased oxidative stress

Gender is a profound determinant of aging and lifespan, but little is known about gender differences in free radical homeostasis. Free radicals are proposed as key elements in the multifactorial process of aging and it is predicted that the longer-lived gender should have lower levels of oxidative stress. While the majority of studies on aging have included a single gender, recent studies in rats compared genders and found that females, the longer-lived sex, had lower oxidative stress and mitochondrial dysfunction than males. We explored the association between oxidative stress and gender-specific aging in C57BL6 mice, in which females are the shorter-lived gender. Reactive oxygen species (ROS) were measured in young and old mice by confocal imaging of dihydroethidium (DHE) oxidation in the brain, and by electron paramagnetic resonance (EPR) spectrometry of isolated brain mitochondria. Both genders exhibited significant age-dependent increases in ROS. However, females had a greater increase with age than males in DHE oxidation but not mitochondrial EPR. Superoxide dismutase 1 (Sod1) and glutathione peroxidase 1 (GPx1) protein levels were lower in old females. To determine whether enhancing antioxidant defenses would eliminate gender differences in lifespan, mice were treated chronically with a superoxide dismutase mimetic. Treatment blocked the age-dependent increase in ROS, with a greater effect in females on DHE oxidation, but not mitochondrial EPR. Treatment also increased lifespan to a greater degree in females. Our results indicate that differences in ROS homeostasis contribute to gender divergence in survival, but also suggest that mitochondrial superoxide production may not be primarily responsible for gender differences in lifespan.     

Urinary excretion rates of 8-oxoGua and 8-oxodG and antioxidant vitamins level as a measure of oxidative status in healthy, full-term newborns

The aim of the present study was to evaluate the oxidative status in healthy full-term children and piglets. Urinary excretion of 8-oxoGua (8-oxoguanine) and 8-oxodG (8-oxo-2'-deoxyguanosine) were determined using HPLC/GS/MS methodology and concentrations of vitamins A, C and E with HPLC technique. The levels of 8-oxoGua in urine samples were about 7-8 times higher in newborn children and piglets when compared with the level of adult subjects, while in the case of 8-oxodG the difference was about 2.5 times. The levels of vitamin C and E in umbilical cord blood of newborn children significantly depend on the concentration of these compounds in their mother's blood. However, the values of vitamin C in human's cord blood were about 2-times higher than in respective mother blood, while the level of vitamin E showed an opposite trend. The results suggest that: (i) healthy, full-term newborns are under potential oxidative stress; (ii) urinary excretion of 8-oxoGua and 8-oxodG may be a good marker of oxidative stress in newborns; and (iii) antioxidant vitamins, especially vitamin C, play an important role in protecting newborns against oxidative stress.     

The free-radical damage theory: Accumulating evidence against a simple link of oxidative stress to ageing and lifespan

Recent work on a small European cave salamander (Proteus anguinus) has revealed that it has exceptional longevity, yet it appears to have unexceptional defences against oxidative damage. This paper comes at the end of a string of other studies that are calling into question the free-radical damage theory of ageing. This theory rose to prominence in the 1990s as the dominant theory for why we age and die. Despite substantial correlative evidence to support it, studies in the last five years have raised doubts over its importance. In particular, these include studies of mice with the major antioxidant genes knocked out (both singly and in combination), which show the expected elevation in oxidative damage but no impact on lifespan. Combined, these findings raise fundamental questions over whether the free-radical damage theory remains useful for understanding the ageing process, and variation in lifespan and life histories.     

The protective effect of melatonin against cypermethrin-induced oxidative stress damage in Spodoptera litura (Lepidoptera: Noctuidae)

Antioxidant enzymes form the first-line defense against free radicals damage in organisms. Their regulation depends mainly on the oxidant and antioxidant status of the cell, given that oxidants are their principal modulators. Therefore, the aim of the present study was to investigate the effect of melatonin on synthetic pyrethroid insecticide-induced antioxidative enzymes activity in Spodoptera litura larvae. In addition, activities of enzymatic antioxidants viz. superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT), glutathione reductase (GR), α, β-esterase, and acetylcholine esterase (AChE) were assessed. There was no significant change in GST levels in the melatonin-treated groups. Melatonin modulates cypermethrin-induced changes in the activities of esterase and AChE, whereas SOD, CAT, and GR activity was significantly increased in melatonin-treated samples when compared to control. In conclusion, the results of the current study revealed that SP toxicity activated oxidant systems in all antioxidant systems in some tissues of insects. Melatonin administration led to a marked increase in antioxidant activity and inhibited GST and AChE in most of the tissues studied.     

Urinary excretion rates of 8-oxoGua and 8-oxodG and antioxidant vitamins level as a measure of oxidative status in healthy,full-term newborns

The aim of the present study was to evaluate the oxidative status in healthy full-term children and piglets. Urinary excretion of 8-oxoGua (8-oxoguanine) and 8-oxodG (8-oxo-2′-deoxyguanosine) were determined using HPLC/GS/MS methodology and concentrations of vitamins A, C and E with HPLC technique. The levels of 8-oxoGua in urine samples were about 7–8 times higher in newborn children and piglets when compared with the level of adult subjects, while in the case of 8-oxodG the difference was about 2.5 times. The levels of vitamin C and E in umbilical cord blood of newborn children significantly depend on the concentration of these compounds in their mother's blood. However, the values of vitamin C in human's cord blood were about 2-times higher than in respective mother blood, while the level of vitamin E showed an opposite trend. The results suggest that: (i) healthy, full-term newborns are under potential oxidative stress; (ii) urinary excretion of 8-oxoGua and 8-oxodG may be a good marker of oxidative stress in newborns; and (iii) antioxidant vitamins, especially vitamin C, play an important role in protecting newborns against oxidative stress.     

Apoptosis and Bcl-2 protein changes in L1210 leukaemic cells exposed to oxidative stress

The aim of this study was to explore the dose- and time-dependent effects of hydrophilic peroxyl radical initiator 2,2'azobis(2amidinopropane)dihydrochloride (AAPH) on apoptosis, and on expression of Bcl-2 in L1210 leukaemic cells. We observed a progressive increase of intracellular concentration of oxygen free radicals (OFR), manifested by the rise of 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) oxidation, during 24 h of cells exposure to AAPH. Oxidative stress was associated with peroxidation of cellular lipids, which was demonstrated by the measurement of thiobarbituric acid-reactive substances and conjugated dienes. Analysis of cell viability by the use of trypan blue exclusion method revealed that AAPH reduced the ability of L1210 cells to multiply or survive. AAPH increased the number of leukaemic cells with typical features of apoptosis like condensation of chromatin, pyknosis and fragmentation of nucleus, followed by secondary necrosis. A characteristic internucleosomal DNA cleavage, visualized as a DNA ‘ladder’ consisting of fragments that are multiples of 180-200 bp was also observed. The intensity of apoptosis was dependent on AAPH concentration, time of cell exposure and the availability of growth factors and nutrients in extracellular environment (FCS concentration). The novel observation is the increase of Bcl-2 level in L1210 leukaemic cells surviving an oxidative stress. The level of Bcl-2 protein significantly rose with increasing AAPH concentration, and time of cell exposure to this oxidant. This phenomenon could be the result of: (1) negative selection of cells with the lowest expression of bcl-2, being more susceptible to oxidative stress and (2) increased synthesis and/or decreased degradation of Bcl-2 protein as an adaptation to continuous OFR loading. In contrast to growth-promoting medium (10% FCS/RPMI), the maintenance medium (2% FCS/RPMI) did not cover cell requirements for progressive Bcl-2 increase at the highest AAPH concentration (2 mM) applied in this study. Several observations indicate that the increased Bcl-2 level in surviving L1210 leukaemic cells exposed to oxidative stress is a symptom of their natural defence against cellular lipids peroxidation and apoptosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Oxidation of glycosaminoglycans by free radicals and reactive oxidative species: A review of investigative methods

Abstract

Glycosaminoglycans, in particular hyaluronan (HA), and proteoglycans are components of the extracellular matrix (ECM). The ECM plays a key role in the regulation of cellular behaviour and alterations to it can modulate both the development of human diseases as well as controlling normal biochemical processes such as cell signalling and pro-inflammatory responses. For these reasons, in vitro fragmentation studies of glycosaminoglycans by free radicals and oxidative species are seen to be relevant to the understanding of in vivo studies of damage to the ECM. A wide range of investigative techniques have therefore been applied to gain insights into the relative fragmentation effects of several reactive oxidative species with the ultimate goal of determining mechanisms of fragmentation at the molecular level. These methods are reviewed here.     

2.45-GHz microwave irradiation adversely affects reproductive function in male mouse,Mus musculus by inducing oxidative and nitrosative stress

Abstract

Electromagnetic radiations are reported to produce long-term and short-term biological effects, which are of great concern to human health due to increasing use of devices emitting EMR especially microwave (MW) radiation in our daily life. In view of the unavoidable use of MW emitting devices (microwaves oven, mobile phones, Wi-Fi, etc.) and their harmful effects on biological system, it was thought worthwhile to investigate the long-term effects of low-level MW irradiation on the reproductive function of male Swiss strain mice and its mechanism of action. Twelve-week-old mice were exposed to non-thermal low-level 2.45-GHz MW radiation (CW for 2 h/day for 30 days, power density = 0.029812 mW/cm² and SAR = 0.018 W/Kg). Sperm count and sperm viability test were done as well as vital organs were processed to study different stress parameters. Plasma was used for testosterone and testis for 3β HSD assay. Immunohistochemistry of 3β HSD and nitric oxide synthase (i-NOS) was also performed in testis. We observed that MW irradiation induced a significant decrease in sperm count and sperm viability along with the decrease in seminiferous tubule diameter and degeneration of seminiferous tubules. Reduction in testicular 3β HSD activity and plasma testosterone levels was also noted in the exposed group of mice. Increased expression of testicular i-NOS was observed in the MW-irradiated group of mice. Further, these adverse reproductive effects suggest that chronic exposure to nonionizing MW radiation may lead to infertility via free radical species-mediated pathway.     

Increased oxidative damage to DNA in a transgenic mouse model of Huntington''s disease

Mitochondrial dysfunction and oxidative damage may play a role in the pathogenesis of Huntington's disease (HD). We examined concentrations of 8-hydroxy-2-deoxyguanosine (OH(8)dG), a well-established marker of oxidative damage to DNA, in a transgenic mouse model of HD (R6/2). Increased concentrations of OH(8)dG were found in the urine, plasma and striatal microdialysates of the HD mice. Increased concentrations were also observed in isolated brain DNA at 12 and 14 weeks of age. Immunocytochemistry showed increased OH(8)dG staining in late stages of the illness. These results suggest that oxidative damage may play a role in the pathogenesis of neuronal degeneration in the R6/2 transgenic mouse model of HD.     

Effect of 900 MHz radiofrequency radiation on oxidative stress in rat brain and serum

The increasing use of mobile telephones raises the question of possible adverse effects of the electromagnetic fields (EMF) that these phones produce. In this study, we examined the oxidative stress in the brain tissue and serum of rats that resulted from exposure to a 900-MHz EMF at a whole body average specific absorption rate (SAR) of 1.08 W/kg for 1 h/day for 3 weeks. We also examined the antioxidant effect of garlic powder (500 mg/kg/day) given orally to EMF-exposed rats. We found that malondialdehyde (MDA) (p < 0.001) and advanced oxidation protein product (AOPP) (p < 0.05) increased in rat brain tissue exposed to the EMF and that garlic reduced these effects (p < 0.05). There was no significant difference in the nitric oxide (NO) levels in the brain. Paraoxonase (PON) was not detected in the brain. There was a significant increase in the levels of NO (p < 0.001) detected in the serum after EMF exposure, and garlic intake did not affect this increase in NO. Our results suggest that there is a significant increase in brain lipid and protein oxidation after electromagnetic radiation (EMR) exposure and that garlic has a protective effect against this oxidative stress.     

The cysteine 34 residue of A1M/α1-microglobulin is essential for protection of irradiated cell cultures and reduction of carbonyl groups

Abstract

α₁-microglobulin (A1M) is a 26 kDa plasma and a tissue protein belonging to the lipocalin family. The reductase and free radical scavenger A1M has been shown to protect cells and extracellular matrix against oxidative and irradiation-induced damage. The reductase activity was previously shown to depend upon an unpaired cysteinyl side-chain, C34, and three lysyl side-chains, K92, 118, and 130, located around the open end of the lipocalin pocket. The aim of this work was to investigate whether the cell and matrix protection by A1M is a result of its reductase activity by using A1M-variants with site-directed mutations of the C34, K92, K118, and K130 positions. The results show that the C34 side-chain is an absolute requirement for protection of HepG2 cell cultures against alpha-particle irradiation-induced cell death, upregulation of stress response and cell cycle regulation genes. Mutation of C34 also resulted in loss of the reduction capacity toward heme- and hydrogen peroxide-oxidized collagen, and the radical species 2,2´-azino-bis (3-ethyl-benzo-thiazoline-6-sulphonic acid) (ABTS). Furthermore, mutation of C34 significantly suppressed the cell-uptake of A1M. The K92, K118, and K130 side-chains were of minor importance in cell protection and reduction of oxidized collagen but strongly influenced the reduction of the ABTS-radical. It is concluded that antioxidative protection of cells and collagen by A1M is totally dependent on its C34 amino acid residue. A model of the cell protection mechanism of A1M should be based on the redox activity of the free thiolyl group of the C34 side-chain and a regulatory role of the K92, K118, and K130 residues.     

Age-related effect induced by oxidative stress on the cerebral glutathione system

In the forebrain from male Wistar rats aged 5, 15 and 25 months, age-related putative alterations in the glutathione system (reduced and oxidized glutathione; redox index) were chronically induced by the administration in drinking water of free radical generators (hydrogen peroxide, ferrous chloride) or of inhibitors of endogenous free radical defenses (diethyl-dithio-carbamate, an inhibitor of superoxide dismutase activity). In hydrogen peroxide administered rats, both reduced glutathione and the cerebral glutathione redox index markedly declined as a function of aging, whereas oxidized glutathione consistently increased. In contrast, chronic iron intake failed to modify the reduced glutathione in forebrain from the rats of the different ages tested, whereas the oxidized glutathione was increased in the older brains. The chronic intake of diethyl-dithio-carbamate enhanced the concentrations of reduced glutathione in the forebrains from the rats of the different ages tested, the oxidized glutathione being unchanged. In 15-month-old rats submitted to chronic oxidative stress, ergot alkaloids (and particularly dihydroergocriptine) interfered with cerebral glutathione system, while papaverine was always ineffective. The comprehensive analysis of the data indicates that: (a) both the type of oxidative stress and the age of the animals modulate the cerebral responsiveness to the putative modifiers in the level of tissue free radicals; (b) aging magnifies the cerebral alterations induced by oxidative stress; the (c) cerebral glutathione system may be modified by metabolic rather than by circulatory interferences; (d) a balance between the various cerebral antioxidant defenses is present, the perturbation of an antioxidant system resulting in the compensatory modified activity of component(s) of another system.     

Selenium content and distribution in rat tissues irradiated with gamma rays

The effects of supplementation with selenous yeast and ionizing radiation on selenium (Se) content and distribution were evaluated in rat tissues (liver, kidney, spleen, heart, muscle, blood, front brain, hind brain, hypothalamus, pituitary, adrenal glands, testes, and hair). This study had 16 Se-supplemented (0.5 μg Se/d) and 16 placebo adult male Wistar rats. One half of the animals (eight Se-supplemented and eight placebos) were irradiated with a single dose of 4.2 Gy from a Co-60 source and sacrificed 7 d after irradiation along with nonirradiated animals and analyzed for Se content determination. The data obtained showed that selenous yeast supplementation increased Se levels in rat tissues (highest increases in hypothalamus, 161%; hind brain, 126%; spleen, 110%; and adrenal gland, 105%). Ionizing radiation induced significant changes in Se content and distribution (decrease in liver, blood, hair, femoral muscle, spleen, and hypothalamus; increase in kidney, testes, adrenal glands, and brain of placebo group). Supplementation with selenous yeast reduces changes in Se content and distribution after irradiation. It seems that the animal tissue susceptibility to oxidative damage may be correlated to their ability to retain Se in tissues.     

Inhibition of cadmium-induced oxidative injury in rat primary astrocytes by the addition of antioxidants and the reduction of intracellular calcium

Exposure of the brain to cadmium ions (Cd(2+)) is believed to lead to neurological disorders of the central nervous system (CNS). In this study, we tested the hypothesis that astrocytes, the major CNS-supporting cells, are resistant to Cd(2+)-induced injury compared with cortical neurons and microglia (CNS macrophages). However, treatment with CdCl(2) for 24 h at concentrations higher than 20 microM substantially induced astrocytic cytotoxicity, which also resulted from long-term exposure to 5 microM of CdCl(2). Intracellular calcium levels were found to rapidly increase after the addition of CdCl(2) into astrocytes, which led to a rise in reactive oxygen species (ROS) and to mitochondrial impairment. In accordance, preexposure to the extracellular calcium chelator EGTA effectively reduced ROS production and increased survival of Cd(2+)-treated astrocytes. Adenovirus-mediated transfer of superoxide dismutase (SOD) or glutathione peroxidase (GPx) genes increased survival of Cd(2+)-exposed astrocytes. In addition, increased ROS generation and astrocytic cell death due to Cd(2+) exposure was inhibited when astrocytes were treated with the polyphenolic compound ellagic acid (EA). Taken together, Cd(2+)-induced astrocytic cell death resulted from disrupted calcium homeostasis and an increase in ROS. Moreover, our findings demonstrate that enhancement of the activity of intracellular antioxidant enzymes and supplementation with a phenolic compound, a natural antioxidant, improves survival of Cd(2+)-primed astrocytes. This information provides a useful approach for treating Cd(2+)-induced CNS neurological disorders.     

Sensitivity of ATPase-ADPase activities from synaptic plasma membranes of rat forebrain to lipid peroxidation in vitro and the protective effect of vitamin E

The in vitro effects of membrane lipid peroxidation on ATPase-ADPase activities in synaptic plasma membranes from rat forebrain were investigated. Treatment of synaptic plasma membranes with an oxidant generating system (H₂O₂/Fe²⁺/ascorbate) resulted in lipid peroxidation and inhibition of the enzyme activity. Besides, trolox as a water soluble vitamin E analogue totally prevented lipid peroxidation and the inhibition of enzyme activity. These results demonstrate the susceptibility of ATPase-ADPase activities of synaptic plasma membranes to free radicals and suggest that the protective effect against lipid peroxidation by trolox prevents the inhibition of enzyme activity. Thus, inhibition of ATPase-ADPase activities of synaptic plasma membranes in cerebral oxidative stress probably is related to lipid peroxidation in the brain.