Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants

Exposure of human spermatozoa to nicotinamide adenine dinucleotide phosphate (NADPH) resulted in the dose dependent generation of reactive oxygen species (ROS) which, at a critical level of intensity, induced lipid peroxidation, DNA damage and a dramatic decline of sperm motility. This system was then used as a model for screening the ability of different antioxidants to combat oxidative stress created through the excessive intracellular generation of toxic oxygen products of metabolism. A variety of antioxidants that has previously been shown to be protective against extracellularly derived oxidants (e.g. superoxide dismutase, catalase, vitamin E, hypotaurine) were ineffective in this system. Albumin, however, could provide complete protection against NADPH induced oxidative stress via mechanisms that did not involve the suppression of the lipid peroxidation cascade but rather the inactivation of lipid peroxides generated during this process. Albumin did not protect against DNA damage induced by NADPH but was extremely effective at preventing DNA fragmentation arising from the suppression of glutathione peroxidase activity with mercaptosuccinate. These studies emphasize that the design of clinically effective antioxidant treatments will depend, critically, upon the source of the oxidative stress. For cases involving excessive intracellular ROS generation, albumin appears to be an important means of neutralizing lipid peroxide-mediated damage to the sperm plasma membrane and DNA.      

Effects of a potent antioxidant, platinum nanoparticle, on the lifespan of Caenorhabditis elegans

We have shown that platinum nanoparticles (nano-Pt) are a superoxide dismutase (SOD)/catalase mimetic. Various data have shown extension of the Caenorhabditis elegans lifespan by antioxidant treatment. The present study was designed to elucidate the survival benefit conferred by nano-Pt, as compared to the well-known SOD/catalase mimetic EUK-8. At 0.5mM, nano-Pt significantly extended the lifespan of wild-type N2 nematodes and at 0.25 and 0.5mM, nano-Pt recovered the shortened lifespan of the mev-1(kn1) mutant, which is due to excessive oxidative stress. In both instances, EUK-8 at 0.05, 0.5, and 5mM did not extend nematode lifespan. Even when 0.4M paraquat was loaded exogenously, nano-Pt (0.1 and 0.5mM) and EUK-8 (0.5 and 5mM) were effective in rescuing worms. Moreover, 0.5mM nano-Pt significantly reduced the accumulation of lipofuscin and ROS induced by paraquat. We measured the in vitro dose-dependent quenching of O(2)(-) and H(2)O(2), indicating that nano-Pt is a more potent SOD/catalase mimetic than EUK-8. Nano-Pt prolonged the worm lifespan, regardless of thermotolerance or dietary restriction. Taken together, nano-Pt has interesting anti-ageing properties.     

Antioxidant defense mechanisms in cultured pleural mesothelial cells.

The role of different antioxidant pathways in cultured rat pleural mesothelial cells was studied by exposing the cells to various hydrogen peroxide (H2O2) concentrations and by measuring H2O2 cell cytotoxicity and the capacity of the cells to scavenge H2O2. The antioxidant enzymes, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase, and catalase were analyzed biochemically. Catalase and CuZn superoxide dismutase were localized by immunocytochemistry. To enable investigation of the glutathione redox cycle and catalase pathways, glutathione reductase was inactivated with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and catalase was inactivated with aminotriazole. When the cells were exposed to a low, sublethal (0.030 mM) H2O2 concentration, glutathione reductase but not catalase inactivation resulted in a decreased capacity to remove H2O2 from the extracellular medium. When the cells were exposed to a high (0.25 mM) H2O2 concentration, H2O2-scavenging capacity decreased remarkably when catalase was inactivated. When the cells were exposed to 0.1 to 0.5 mM H2O2, cell cytotoxicity (lactate dehydrogenase release) increased significantly if glutathione reductase was inactivated; catalase inactivation resulted in a significant cytotoxicity only at high (greater than or equal to 0.25 mM) H2O2 concentrations. Immunocytochemical studies showed that the cells, both in situ and in vitro, contained low amounts of catalase. This suggests that the results of the catalase-inhibition studies are probably not due to a change in the characteristics of the cells in culture. 3-Aminobenzamide is a compound that is known to prevent NAD depletion through inhibition of poly(ADP-ribose) polymerase during oxidant stress. When intact cells were treated with different antioxidants and exposed to 0.5 mM H2O2, both catalase and 3-aminobenzamide protected the cells completely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Levamisole in Hibition of Microsomal Lipid Peroxidation as Related to its Sulfhydryl Metabolite DL-2-OXO-3-(2-Mercaptoethyl)-5-Phenylimadazolidine

Levamisole was previously shown to protect rat liver microsomes from lipid peroxidation induced by ADP-Fe and either NADPH, ascorbate, or X irradiation. The present experiments provide information about the mechanism of protection. Incubation of levamisole with a microsomal system containing ADP-Fe and NADPH resulted in protection of sulfhydryl groups, whereas reaction of levamisole with ascorbate (nonenzymatic system) indicated generation of a sulfhydryl metabolite. Production of a sulfhydryl metabolite of levamisole, dl-2-oxo-3-(2-mercaptoethyl)-5-phenyl-imidazolidine (OMPI), in either the enzymatic or nonenzymatic system was demonstrated by gas chromatography-mass spectrometry. While levamisole acts as an antioxidant at concentrations of 1.0 and 2.0 mM, OMPI had an enigmatic effect on microsomal lipid peroxidation induced enzymatically or nonenzymatically. OMPI exhibited a biphasic effect: at concentrations below 25 μM a prooxidant effect was observed, and at concentrations exceeding 50 μM an antioxidant effect was observed. The data suggest that the inhibition of microsomal lipid peroxidation by levamisole is due to the generation of a sulfhydryl metabolite and that the active intermediate is prcbably OMPI.     

Effects of thiolic antioxidants on in vitro mouse peritoneal macrophage functions

Since leukocytes have the potential to produce oxygen free radicals that damage biomolecules, we have evaluated the influence of thiolic antioxidants (glutathione, N-acetylcysteine and thioproline) on selected hematoimmunological characteristics. We used peritoneal leukocytes incubated at 0.5, 1.0 and 5.0 mM concentration of antioxidants. Following thiol treatment, we found a stimulation of phagocytic activity, and markedly decreased incidence of programmed cell death of peritoneal leukocytes, both basal and induced by H₂O₂. Low concentrations of thiolic compounds also increased the activity of catalase, glutathione peroxidase and glutathione reductase. On the contrary, only the highest concentration of N-acetylcysteine and thioproline reduced superoxide dismutase activity. Tested antioxidants can lead to a decrease of the oxidative stress, and could provide a nutritional benefit for evaluated immunological characteristics.     

The role of oxidative stress in the in vitro induction of micronuclei by pesticides in mouse lung fibroblasts

The involvement of the antioxidant enzymes catalase and glutathione peroxidase (both at 0.1 mg/ml) in defence against the genotoxicity of phosphamidon (80 microg/ml) and dieldrin (25 microM) was investigated in order to demonstrate that the two pesticides damage DNA through the generation of reactive oxygen species and therefore of oxidative stress. The pesticide genotoxicity was determined by the cytokinesis-block micronucleus test performed on primary mouse lung fibroblast cultures. Also, 3-aminotriazole (40 mM) and mercaptosuccinate (0.5 mM), inhibitors of catalase and glutathione peroxidase, respectively, were added to the cultures. Data indicate that catalase causes a decrease only in the damage induced by phosphamidon, while glutathione peroxidase protects against damage induced by both phosphamidon and dieldrin. Simultaneous treatment with antioxidant inhibitors and pesticides results in a decrease in micronucleus frequency and cell number, due to apoptotic death. Our results indicate that clastogenic DNA damage produced by the two pesticides is modulated by antioxidant enzymes and their inhibitors and thus could be due to oxidative stress induction.     

Short-term creatine supplementation decreases reactive oxygen species content with no changes in expression and activity of antioxidant enzymes in skeletal muscle

The effect of short-term creatine (Cr) supplementation upon content of skeletal muscle-derived-reactive oxygen species (ROS) was investigated. Wistar rats were supplemented with Cr (5?g/kg BW) or vehicle, by gavage, for 6?days. Soleus and extensor digitorum longus (EDL) muscles were removed and incubated for evaluation of ROS content using Amplex-UltraRed reagent. The analysis of expression and activity of antioxidant enzymes (superoxide dismutase 1 and 2, catalase and glutathione peroxidase) were performed. Direct scavenger action of Cr on superoxide radical and hydrogen peroxide was also investigated. Short-term Cr supplementation attenuated ROS content in both soleus and EDL muscles (by 41 and 33.7%, respectively). Cr supplementation did not change expression and activity of antioxidant enzymes. Basal TBARS content was not altered by Cr supplementation. In cell-free experiments, Cr showed a scavenger effect on superoxide radical in concentrations of 20 and 40?mM, but not on hydrogen peroxide. These results indicate that Cr supplementation decreases ROS content in skeletal muscle possibly due to a direct action of Cr molecule on superoxide radical.     

Effects of castration and testosterone replacement on the antioxidant defense system in rat left ventricle

There is strong evidence that oxidative stress plays a key role in the pathophysiology of several cardiovascular diseases. On the other hand, the presence of specific receptors for androgens and estrogens in the myocardium implies that sex hormones play a physiological role in cardiac function, myocardial injury, and the regulation of the redox state in the heart. The present study was designed to determine whether castration and androgen replacement result in changes in the capacity of the antioxidant defense system in the left ventricle (LV) of adult male rats. To assess this, the activities of antioxidant enzymes (superoxide dismutase [SOD], glutathione peroxidase [GPX], catalase [CAT], and glutathione reductase [GR]), concentrations of nonenzymatic antioxidants (reduced glutathione [GSH] and alpha- and gamma-tocopherols), and oxidative stress biomarkers (tissue sulfhydryl groups, protein nitrotyrosine levels, and lipid peroxidation) were measured in castrated animals (CAS), castrates replaced with testosterone (CAS+T), and sham-operated controls (Sham). Testosterone was not detectable in serum from gonadectomized rats. The results indicate that castration significantly and negatively affected the antioxidant status of rat LV, as evidenced by a significant decline in activities of all antioxidant enzymes, by a tendency toward lower levels of GSH and protein thiol groups, and by enhanced lipid peroxidation and higher nitrotyrosine concentrations in left ventricular tissue. Increases in LV tissue concentrations of alpha- and gamma-tocopherols seem to be a compensatory response to enhanced oxidative stress induced by gonadectomy. The reestablishment of physiological serum testosterone level by androgen replacement resulted in a tendency toward a further decrease in the antioxidant defense status in the LV tissue.     

Antioxidant effects on the intracerebroventricular galactose damage in rats

We investigated the effects of the intracerebroventricular infusion of galactose and the influence of pretreatment with antioxidants on oxidative stress parameters and acethylcholinesterase (AChE) activity in the brain of 60-day-old Wistar rats (6 per group). The animals were divided into naïve group (did not undergo surgery); procedure group (only underwent surgery); sham group (underwent surgery and received 5?μL saline) and galactose group (received 5?μL of galactose solution (5.0?mM) by intracerebroventricular injection), and were killed by decapitation after 1?h. Other groups were pretreated daily for 1 week with saline (sham and galactose groups) or antioxidants, α-tocopherol (40?mg/kg) plus ascorbic acid (100?mg/kg, i.p.) (antioxidants and galactose?+?antioxidants groups). Twelve hours after the last antioxidants injection, animals received an intracerebroventricular infusion of 5?μL of galactose solution (galactose and galactose?+?antioxidants groups) or saline (sham and antioxidants groups) and were sacrificed 1?h later. Galactose elevated thiobarbituric acid reactive substances (TBA-RS), protein carbonyl content and glutathione peroxidase (GSH-Px) activity and decreased total sulfhydryl content and catalase (CAT) activity in the cerebral cortex. In the hippocampus, galactose enhanced TBA-RS, decreased total sulfhydryl content and increased AChE activity, while in the cerebellum it decreased total sulfhydryl content and increased CAT and superoxide dismutase (SOD) activities. Pretreatment with antioxidants prevented the majority of these alterations, indicating the participation of free radicals in these effects. Thus, intracerebroventricular galactose infusion impairs redox homeostasis in the brain; the administration of antioxidants should be considered as an adjuvant therapy to specific diets in galactosemia.     

Changes in antioxidant enzymes in isolated cardiac myocytes subjected to hypoxia-reoxygenation.

BACKGROUND: Intracellular antioxidants have been shown to be depressed during hypoxia, and recovery upon reoxygenation has been correlated with the available antioxidant reserve. To test whether these antioxidant changes are also occurring at the cardiac myocytes level, we studied changes in antioxidant enzyme activities as well as cell injury in isolated cardiac myocytes exposed to hypoxia and reoxygenation. EXPERIMENTAL DESIGN: Isolated Ca(2+)-tolerant myocytes from adult male rats were subjected to 30 minutes hypoxia and 15 minutes reoxygenation. Antioxidant enzymes superoxide dismutase, glutathione peroxidase, catalase; lipid peroxide content; electrolytes (Na+, Ca2+); morphology; and high energy phosphates (ATP, ADP, AMP, creatinine phosphate) were studied in these myocytes. The effects of exogenous catalase (40 units/ml) on hypoxia-reoxygenation induced changes in myocytes were also studied. RESULTS: Hypoxia resulted in a reduction in Mn superoxide dismutase and glutathione peroxidase activities with no change in CAT activity and malondialdehyde content. Reoxygenation of hypoxic cells resulted in recovery of Mn superoxide dismutase but not in glutathione peroxidase activity. Reoxygenation was without any effect on catalase activity, but a significant increase in the malondialdehyde content was seen. Hypoxia as well as reoxygenation caused a reduction in the number of rod-shaped cells with a parallel increase in hypercontracted as well as round cells. There was a significant increase in the myocyte Na+ and Ca2+ content during both hypoxia and reoxygenation, and this was accompanied by leakage of lactate dehydrogenase into the perfusion medium. These changes due to hypoxia and reoxygenation were significantly attenuated by addition of catalase (40 units/ml). High energy phosphates ATP, ADP, and AMP declined during hypoxia, and creatine phosphate was significantly reduced during reoxygenation. CONCLUSIONS: Hypoxia induces specific antioxidant changes in the isolated cardiac myocytes. Reduced ability to remove hydrogen peroxide appears to be an important determinant of myocyte injury during reoxygenation.     

White spot syndrome virus infection decreases the activity of antioxidant enzymes in Fenneropenaeus indicus

White spot syndrome virus (WSSV) is the causative agent of White Spot disease of shrimp, causing mass mortalities in aquaculture. WSSV infection causes oxidative stress by the release of reactive oxygen species (ROS) that are toxic to the cells. The antioxidant enzymes associated with oxidative stress during the process of pathogenesis of WSSV in the infected tissues (hemolymph, hepatopancreas, gills and muscle) of Fenneropenaeus indicus were quantitatively determined at different time intervals post infection (0, 24, 48, 72 and after 72 h (moribund)). The level of lipid peroxidation, the activities of superoxide dismutase, catalase, glutathione peroxidase, and also the activities of the non-enzymic antioxidants glutathione-S-transferase, reduced glutathione and glutathione reductase in healthy and WSSV-infected hemolymph, hepatopancreas, gills and muscle of F. indicus showed marked differences at different times during the course of infection. The level of lipid peroxidation was higher in WSSV-infected muscle, hemolymph, gills and hepatopancreas than in uninfected F. indicus. Significant reductions in the activities of superoxide dismutase, catalase, glutathione-S-transferase, reduced glutathione, glutathione peroxidase and glutathione reductase were observed in WSSV-infected compared with uninfected animals. The increased lipid peroxidation in WSSV-infected shrimp may be due to increased oxidative stress in the cells as a result of depletion of antioxidant scavenger systems. The reduced activity of antioxidant enzymes in WSSV-infected animals could be due to inactivation of antioxidant enzymes by oxidative stress thereby generating free radicals, which accumulate in the cells. Further understanding of the biochemical alterations induced by viral infections, including changes in the antioxidant status and oxidative stress, could help to advance the therapeutic armamentarium for control of WSSV in shrimp.     

Enhanced testicular antioxidant system by ascorbic acid in alloxan diabetic rats

The diabetic subject is at significantly increased risk of developing testicular changes. Its etiology may involve oxidative damage by free radicals and protection against such damage can be offered by antioxidant supplementation. Alloxan elicited significant inhibition of antioxidants including superoxide dismutase, catalase and glutathione reductase activities and decreased glutathione content in testis. These effects were accompanied by significant elevation of testicular lipid peroxidation, decreased plasma testosterone level and a drop in copper and zinc concentrations in testis. The administration of ascorbic acid after alloxan treatment interfered and prevented alloxan action. Ascorbic acid blunted the increased testicular lipid peroxidation and the decreased plasma testosterone level probably by protecting antioxidants and the loss of copper and zinc from testes. The data suggested that ascorbic acid has a protective effect on alloxan-induced damage by maintaining the activity of cellular antioxidants.     

Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa

BACKGROUND: Oxidative stress induced by reactive oxygen species (ROS) isassociated with an impaired fertilization ability of spermatozoa.We investigated the effects of adding antioxidants to a spermpreparation medium on the functional parameters of the spermatozoa. METHODS: Spermatozoa were washed with Ham's F-10 media containing theantioxidants, ethylenediaminetetraacetic acid (EDTA) and catalase,at various concentrations, and then the ROS levels in spermsuspensions, and the forward motility, acrosome reaction, DNAintegrity and lipid peroxidation of the spermatozoa were assessed. RESULTS: The ROS levels were significantly lower in sperm suspensionswashed with the antioxidants (196312 rlu; relative light units)than in control sperm (604 rlu, P < 0.05). The addition of10 µM EDTA to the sperm preparation medium significantlyimproved the motility of the spermatozoa compared with the controlgroup, the groups containing EDTA at other concentrations andthe groups containing catalase. Catalase significantly increasedthe acrosome reaction rate of the spermatozoa. Both EDTA andcatalase significantly decreased the DNA fragmentation rateof the spermatozoa. However, the antioxidants did not reducelipid peroxidation. CONCLUSIONS: Supplementing sperm preparation medium with EDTA or catalasesignificantly improved the overall functional parameters ofthe spermatozoa by reducing the ROS levels.     

Modulation by flavonoids of DNA damage induced by estrogen-like compounds

Reactive oxygen species (ROS) are produced by a wide variety of exogenous chemicals and metabolic processes and cause a broad spectrum of damage to biological systems. As a consequence, ROS react with DNA, among many other biological targets, disrupting its structure and functionality. Estrogen-like compounds mediate DNA damage by ROS generation, implying that their effects can be modulated by antioxidants such as catalase, superoxide dismutase, and vitamin C. We examined DNA damage in human lymphocytes and sperm after treatment with four estrogen-like compounds (beta-estradiol, diethylstilbestrol, daidzein, and genistein) and its modulation by flavonoids (quercetin and kaempferol) using the Comet assay. The results indicated that quercetin and kaempferol reduced the DNA damage produced in sperm and lymphocytes by the four estrogenic compounds. The flavonoids also reduced the DNA damage induced by hydrogen peroxide, which was used as a positive control. Our results demonstrate that the antioxidant properties of flavonoids can protect the integrity of human sperm and lymphocyte DNA from ROS induced by estrogenic compounds.     

Increased oxidative stress and altered levels of antioxidants in asthma

BACKGROUND: Reactive oxygen species might play an important role in the modulation of airway inflammation. There is evidence of an oxidant-antioxidant imbalance in asthma. Although several oxidants and antioxidants are likely to be involved, alterations in only limited parameters have been studied in isolation. OBJECTIVE: We investigated changes in a wide range of oxidants and antioxidants to create a comprehensive picture of oxidant-antioxidant imbalance. METHODS: In the peripheral blood of 38 patients with bronchial asthma and 23 control subjects, oxidative stress was measured in terms of superoxide anion generation by leukocytes, lipid peroxidation products, total nitrates and nitrites, total protein carbonyls, and total protein sulfhydrils in plasma. Antioxidant status was evaluated by measuring red blood cell superoxide dismutase and catalase activity, total blood glutathione, and glutathione peroxidase activity in red blood cells and leukocytes and total antioxidant capacity in plasma. RESULTS: Asthmatic patients showed increased superoxide generation from leukocytes, increased total nitrites and nitrates, increased protein carbonyls, and increased lipid peroxidation products and decreased protein sulfhydrils in plasma, indicating increased oxidative stress. They also showed increased superoxide dismutase activity in red blood cells and increased total blood glutathione and decreased glutathione peroxidase activity in red blood cells and leukocytes. Red blood cell catalase activity and the total antioxidant capacity of plasma were not altered. CONCLUSION: There are alterations in a wide array of oxidants and antioxidants, with balance shifting toward increased oxidative stress in asthma. Therapeutic augmentation of the antioxidant defenses might be beneficial.     

Glutathione and hypotaurine in vitro: effects on human sperm motility, DNA integrity and production of reactive oxygen species

Sperm DNA integrity is of paramount importance for the accurate conveyance of genetic material. DNA damage may be a major contributory factor in male infertility as DNA from sperm of infertile men has been found to be more susceptible to induced DNA damage in vitro than DNA from fertile men. Reactive oxygen species (ROS) are a significant source of DNA damage and human sperm are extremely sensitive to ROS attack due to their high content of polyunsaturated fatty acids and lack of capacity for DNA repair. Seminal plasma, which contains a wealth of antioxidants, provides sperm with crucial protection against oxidative insult. However, during preparation for use in assisted conception techniques, sperm are separated from seminal plasma and deprived of that essential protection. The aim of this study was to determine the effects of supplementation with glutathione and hypotaurine during sperm preparation on subsequent sperm motility, DNA integrity, induced DNA damage and ROS generation. Semen samples (n = 45) were divided into aliquots and prepared by Percoll density centrifugation (95.0-47.5%) using medium which had been supplemented with these antioxidants to a number of different concentrations all within physiological levels. Control aliquots were included which had no glutathione or hypotaurine added. Sperm motility was determined using computer-assisted semen analysis. DNA damage was induced using H(2)O(2) and DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay, while ROS generation was measured using chemiluminescence. Addition of glutathione and hypotaurine, either singly or in combination, to sperm preparation medium had no significant effect on sperm progressive motility or baseline DNA integrity. Despite this, sperm were still afforded significant protection against H(2)O(2)-induced damage and ROS generation.     

A rotenone-sensitive site and H2O2 are key components of hypoxia-sensing in neonatal rat adrenomedullary chromaffin cells

In the perinatal period, adrenomedullary chromaffin cells (AMC) directly sense PO2 and secrete catecholamines during hypoxic stress, and this response is lost in juvenile ( approximately 2 week-old) chromaffin cells following postnatal innervation. Here we tested the hypothesis that a rotenone-sensitive O2-sensor and ROS are involved in the hypoxic response of AMC cultured from neonatal and juvenile rats. In whole-cell recordings, hypoxia (PO2=5-15 mm Hg) inhibited outward current in neonatal AMC; this response was reversed by exogenous H2O2 and mimicked and occluded by intracellular catalase (1000 units/ml), as well as the antioxidants, N-acetyl-L-cysteine (NAC; 50 microM) and Trolox (200 microM). Acute hypoxia decreased ROS levels and stimulated ATP secretion in these cells, as measured by luminol and luciferin-luciferase chemiluminescence, respectively. Of several mitochondrial electron transport chain (ETC) inhibitors tested, only rotenone, a complex I blocker, mimicked and occluded the effects of hypoxia on outward current, cellular ROS, and ATP secretion. Succinate donors, which act as complex II substrates, reversed the effects of hypoxia and rotenone in neonatal AMC. In contrast, in hypoxia-insensitive juvenile AMC, neither NAC nor rotenone stimulated ATP secretion though they both caused a decrease in ROS levels. We propose that O2-sensing by neonatal AMC is mediated by decreased ROS generation via a rotenone-sensitive site that is coupled to outward current inhibition and secretion. Interestingly, juvenile AMC display at least two modifications, i.e. an uncoupling of the O2-sensor from ROS regulation, and an apparent insensitivity of outward current to decreased ROS.     

Antioxidant enzymatic defences in human follicular fluid: characterization and age-dependent changes

The aim of this work was to study the antioxidant enzymatic defences in human follicular fluid and investigate their possible changes during reproductive ageing. To this end, we tested the specific activities and protein expression of enzymes involved in reactive oxygen species (ROS) scavenging and in detoxification of ROS byproducts in follicular fluid from young (range 27-32 years, n = 12) and older (range 39-45 years, n = 12) women participating in an IVF programme. Results show that all the tested enzymes [superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione transferase, glutathione reductase] were significantly expressed in human follicular fluid. However, when the two age groups were compared, we found that follicular fluid from older women exhibited a reduced level of glutathione transferase and catalase activities and a higher level of SOD activity. Immunoblot analysis revealed that ageing was associated with decreased protein expression of GST Pi isoform and did not affect SOD and catalase protein expression. Taken together, these findings indicate that reproductive ageing is accompanied by a change in the antioxidant enzymatic pattern that could impair ROS scavenging efficiency in the follicular environment.     

Effect of N-acetyl-L-cysteine on ROS production and cell death caused by HEMA in human primary gingival fibroblasts

Previous investigations have shown that 2-hydroxyethyl methacrylate (HEMA) causes reactive oxygen species (ROS) production, which in turn affects cell survival and cell death. The purpose of this study was to evaluate the effects of the antioxidant N-acetyl-L-cysteine (NAC) on HEMA-induced toxicity in human primary gingival fibroblasts (HGF). HGF were treated with various concentrations of HEMA (0-12 mm) in the absence and presence of NAC (1, 5, and 10 mm). The 3-(4,5 dimethyiazol-2-1)-2-5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate the mitochondrial dehydrogenase activity after HEMA exposure. Viability and cell death were determined by flow cytometry using Annexin V and PI staining. ROS production was detected by the increasing fluorescence of the oxidation-sensitive dye 2',7'-dichlorofluorescein diacetate (DCFH-DA) after HEMA treatment. After a 24h incubation period, HEMA concentrations higher then 10mm caused a decrease of cell viability, mitochondrial activity, and an increase of cell death. HEMA concentrations of 4-12 mm markedly increased ROS levels in a dose-dependent manner. High NAC concentrations (5 and 10 mm) significantly reduced cell death, and restored the mitochondrial activity after a 24 h co-treatment, but 1 mm NAC increased HEMA toxicity (p<0.05). All NAC concentrations significantly reduced ROS levels induced by HEMA after a 2 h exposure (p<0.05), but no such reduction was observed after a 4 h treatment. Furthermore, treatment with 10 mm HEMA and 1 mm NAC for 6h caused an increase in ROS levels compared to 10 mm HEMA alone (p<0.05). In conclusion, our results suggest that high NAC concentrations protect HGF against HEMA cytotoxicity by reducing the induced ROS levels.     

Antioxidant processes of the wild tambaqui, Colossoma macropomum (Osteichthyes, Serrasalmidae) from the Amazon.

Colossoma macropomum, locally called tambaqui, is a freshwater migratory teleost that shows good tolerance to oxygen and pH changes in water, and both chemical-physical parameters change markedly during the day time and seasonal water level oscillations in the Amazon Basin. In order to obtain a general view about the basal levels of antioxidants in different tissues of wild tambaqui, enzymatic (superoxide dismutase and catalase) and non-enzymatic (alpha-tocopherol, beta-carotene, and glutathione) antioxidants and lipid peroxidation levels were assessed in the liver, blood and plasma of ten specimens collected during the dry season (September) in a pond near Manaus-AM, Brazil. Superoxide dismutase, catalase, and lipid peroxidation levels were high in the liver and low in the blood and plasma. Confirming previous results on tambaqui, catalase was detected in the blood of one specimen only. beta-Carotene was not found in any analyzed tissue, while alpha-tocopherol was found only in the liver (7.8 +/- 7.0 nmol g(-1), mean +/- S.E.M.) and plasma (4.3 +/- 0.9 nmol ml(-1)). Blood glutathione concentrations (2.4 +/- 0.17 mmol l(-1)) of tambaqui were comparable with those found in other Amazonian teleosts. Antioxidant defenses and lipid peroxidation contents from liver. blood and plasma exhibited interesting correlations. These relationships suggest that antioxidant defenses located in different tissues and in different sub-cellular compartments act in concert.