Fundamental roles of reactive oxygen species and protective mechanisms in the female reproductive system

Controlled oxidation, such as disulfide bond formation in sperm nuclei and during ovulation, plays a fundamental role in mammalian reproduction. Excess oxidation, however, causes oxidative stress, resulting in the dysfunction of the reproductive process. Antioxidation reactions that reduce the levels of reactive oxygen species are of prime importance in reproductive systems in maintaining the quality of gametes and support reproduction. While anti-oxidative enzymes, such as superoxide dismutase and peroxidase, play a central role in eliminating oxidative stress, reduction-oxidation (redox) systems, comprised of mainly glutathione and thioredoxin, function to reduce the levels of oxidized molecules. Aldo-keto reductase, using NADPH as an electron donor, detoxifies carbonyl compounds resulting from the oxidation of lipids and proteins. Thus, many antioxidative and redox enzyme genes are expressed and aggressively protect gametes and embryos in reproductive systems.     

Quantification of the overall reactive oxygen species scavenging capacity of biological fluids and tissues

A method has been developed for measuring and evaluating the overall antioxidant activity derived from the low-molecular weight antioxidants (scavengers). The principle governing this method is based on a common chemical characteristic of the scavengers, their reducing properties. It was hypothesized and then demonstrated that an evaluation of the overall reducing power of a biological sample correlates with the overall scavenging activity of the sample. In order to quantify the total reducing power, the cyclic voltammetry methodology was applied. The resulting measurements correlated with the antioxidant activity of both hydrophilic and lipophilic scavengers. The method is suitable for use in biological fluids and in tissue homogenates, and can supply information concerning the type of antioxidants and their total concentration without having to determine specific compounds. A noninvasive procedure for determining skin overall scavenging activity is also described. This method is based on a well containing an extraction solution that is attached to the skin's surface. Following incubation time the extraction solution is analyzed using the cyclic voltammeter instrument and other methods. We have found these methods suitable for evaluating the reducing capacity status in various clinical conditions such as diabetes, ionizing and nonionizing irradiation, brain degenerative diseases, head trauma, and inflammatory bowel diseases. This method is also an efficient tool for evaluating the overall antioxidant capacity of mixtures of antioxidant preparations in vitro. The measurements themselves are simple and rapid. Furthermore, they do not require manipulation of the samples.     

Measuring mitochondrial reactive oxygen species

This article examines recent methods for measuring reactive oxygen species produced in isolated mitochondria and within live cells, with particular emphasis on the detection of hydrogen peroxide. Protocols for reliable measurements of mitochondrial hydrogen peroxide are presented, while the advantages and pitfalls of these and other methods are discussed. New developments in the detection of lipid peroxidation are outlined. Advice is also provided to aid the interpretation of cellular data with respect to the contribution of oxygen radical production by different components of the mitochondrial respiratory chain.     

Defense against reactive oxygen species

As part of the European Commission Concerted Action on Functional Food which was managed by the International Life Sciences Institute (Europe) a series of Theme Papers was produced which examined the 'state of the art' with respect to the subject matter and made recommendations for research. This paper is a summary of the paper concerned with Defence Against Reactive Oxygen species. Having reviewed the scientific literature the authors concluded that certain stringent criteria, which they identified, would need to be satisfied in order to be able to conclude that free radical events are involved in certain human diseases, and that antioxidants are capable of modulating these events and thus reducing the risk of disease. Although there is some evidence that would lead to this conclusion the authors demonstrated that there is at present insufficient evidence available on which to base a firm conclusion that antioxidants are capable of reducing risk of disease, and very little evidence that addresses the important question as to how much of the nutrients concerned are required in the diet to achieve the objective of reducing risk. Research priorities address the need in particular for the development and validation of cellular markers of oxidative damage which are required before there can be new human studies that address the question. There is also a need for more information as to the pharmacokinetics of uptake from diet, distribution and cellular concentration of the antioxidants.     

Defence against reactive oxygen species

As part of the European Commission Concerted Action on Functional Food which was managed by the International Life Sciences Institute (Europe) a series of Theme Papers was produced which examined the ‘state of the art’ with respect to the subject matter and made recommendations for research. This paper is a summary of the paper concerned with Defence Against Reactive Oxygen species. Having reviewed the scientific literature the authors concluded that certain stringent criteria, which they identified, would need to be satisfied in order to be able to conclude that free radical events are involved in certain human diseases, and that antioxidants are capable of modulating these events and thus reducing the risk of disease. Although there is some evidence that would lead to this conclusion the authors demonstrated that there is at present insufficient evidence available on which to base a firm conclusion that antioxidants are capable of reducing risk of disease, and very little evidence that addresses the important question as to how much of the nutrients concerned are required in the diet to achieve the objective of reducing risk. Research priorities address the need in particular for the development and validation of cellular markers of oxidative damage which are required before there can be new human studies that address the question. There is also a need for more information as to the pharmacokinetics of uptake from diet, distribution and cellular concentration of the antioxidants.     

Wound-induced apoplastic peroxidase activities: their roles in the production and detoxification of reactive oxygen species

Production of reactive oxygen species (ROS) is a widely reported response of plants to wounding. However, the nature of enzymes responsible for ROS production and metabolism in the apoplast is still an open question. We identified and characterized the proteins responsible for the wound-induced production and detoxification of ROS in the apoplast of wheat roots ( Triticum aestivum L.). Compared to intact roots, excised roots and leachates derived from them produced twice the amount of superoxide (O₂^•−). Wounding also induced extracellular peroxidase (ECPOX) activity mainly caused by the release of soluble peroxidases with molecular masses of 37, 40 and 136 kD. Peptide mass analysis by electrospray ionization–quadrupole time-of-flight–tandem mass spectrometry (ESI–QTOF–MS/MS) following lectin affinity chromatography of leachates showed the presence of peroxidases in unbound (37 kD) and bound (40 kD) fractions. High sensitivity of O₂^•−-producing activity to peroxidase inhibitors and production of O₂^•− by purified peroxidases in vitro provided evidence for the involvement of ECPOXs in O₂^•− production in the apoplast. Our results present new insights into the rapid response of roots to wounding. An important component of this response is mediated by peroxidases that are released from the cell surface into the apoplast where they can display both oxidative and peroxidative activities.     

Mitochondrial metabolism of reactive oxygen species

Oxidative stress is considered a major contributor to etiology of both normal senescence and severe pathologies with serious public health implications. Mitochondria generate reactive oxygen species (ROS) that are thought to augment intracellular oxidative stress. Mitochondria possess at least nine known sites that are capable of generating superoxide anion, a progenitor ROS. Mitochondria also possess numerous ROS defense systems that are much less studied. Studies of the last three decades shed light on many important mechanistic details of mitochondrial ROS production, but the bigger picture remains obscure. This review summarizes the current knowledge about major components involved in mitochondrial ROS metabolism and factors that regulate ROS generation and removal. An integrative, systemic approach is applied to analysis of mitochondrial ROS metabolism, which is now dissected into mitochondrial ROS production, mitochondrial ROS removal, and mitochondrial ROS emission. It is suggested that mitochondria augment intracellular oxidative stress due primarily to failure of their ROS removal systems, whereas the role of mitochondrial ROS emission is yet to be determined and a net increase in mitochondrial ROS production in situ remains to be demonstrated.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 246–264.Original Russian Text Copyright © 2005 by Andreyev, Kushnareva, Starkov.This revised version was published online in April 2005 with corrections to the post codes.     

The roles of cellular reactive oxygen species,oxidative stress and antioxidants in pregnancy outcomes

Reactive oxygen species (ROS) are generated as by-products of aerobic respiration and metabolism. Mammalian cells have evolved a variety of enzymatic mechanisms to control ROS production, one of the central elements in signal transduction pathways involved in cell proliferation, differentiation and apoptosis. Antioxidants also ensure defenses against ROS-induced damage to lipids, proteins and DNA. ROS and antioxidants have been implicated in the regulation of reproductive processes in both animal and human, such as cyclic luteal and endometrial changes, follicular development, ovulation, fertilization, embryogenesis, embryonic implantation, and placental differentiation and growth. In contrast, imbalances between ROS production and antioxidant systems induce oxidative stress that negatively impacts reproductive processes. High levels of ROS during embryonic, fetal and placental development are a feature of pregnancy. Consequently, oxidative stress has emerged as a likely promoter of several pregnancy-related disorders, such as spontaneous abortions, embryopathies, preeclampsia, fetal growth restriction, preterm labor and low birth weight. Nutritional and environmental factors may contribute to such adverse pregnancy outcomes and increase the susceptibility of offspring to disease. This occurs, at least in part, via impairment of the antioxidant defense systems and enhancement of ROS generation which alters cellular signalling and/or damage cellular macromolecules. The links between oxidative stress, the female reproductive system and development of adverse pregnancy outcomes, constitute important issues in human and animal reproductive medicine. This review summarizes the role of ROS in female reproductive processes and the state of knowledge on the association between ROS, oxidative stress, antioxidants and pregnancy outcomes in different mammalian species.     

NADPH oxidases in fungi: diverse roles of reactive oxygen species in fungal cellular differentiation

Synthesis of reactive oxygen species (ROS) by specific NADPH oxidases (Nox) can serve both defense and differentiation signaling roles in animals and plants. Fungi have three subfamilies of NADPH oxidase. NoxA and NoxB have a structure very similar to the human gp91(phox). NoxC has in addition a Ca(2+) binding motif as found in the human Nox5 and plant Rboh families of NADPH oxidases. A survey of fungal genomes identified up to four Nox genes in some fungal species, but Nox genes are absent from available genomes of the hemiascomycete yeasts, unicellular Basidiomycetes and Zygomycetes, reflecting the diversity of fungal life forms. Specific isoforms of Nox have been shown by genetic analysis to be required for various physiological processes and cellular differentiations, including development of sexual fruiting bodies, ascospore germination, hyphal defense, hyphal growth in both mutualistic and antagonistic plant-fungal interactions. This review provides an overview of our current knowledge of fungal NADPH oxidases, including Nox distribution in the fungal kingdom, Nox structure and regulation, and known biological functions of this important group of enzymes.     

Mitochondrial metabolism of reactive oxygen species

For a long time mitochondria have mainly been considered for their role in the aerobic energy production in eukaryotic cells, being the sites of the oxidative phosphorylation, which couples the electron transfer from respiratory substrates to oxygen with the ATP synthesis. Subsequently, it was showed that electron transfer along mitochondrial respiratory chain also leads to the formation of radicals and other reactive oxygen species, commonly indicated as ROS. The finding that such species are able to damage cellular components, suggested mitochondrial involvement in degenerative processes underlying several diseases and aging.More recently, a new role for mitochondria, as a system able to supply protection against cellular oxidative damage, is emerging. Experimental evidence indicates that the systems, evolved to protect mitochondria against endogenously produced ROS, can also scavenge ROS produced by other cellular sources. It is possible that this action, particularly relevant in physio-pathological conditions leading to increased cellular ROS production, is more effective in tissues provided with abundant mitochondrial population. Moreover, the mitochondrial dysfunction, resulting from ROS-induced inactivation of important mitochondrial components, can be attenuated by the cell purification from old ROS-overproducing mitochondria, which are characterized by high susceptibility to oxidative damage. Such an elimination is likely due to two sequential processes, named mitoptosis and mitophagy, which are usually believed to be induced by enhanced mitochondrial ROS generation. However, they could also be elicited by great amounts of ROS produced by other cellular sources and diffusing into mitochondria, leading to the elimination of the old dysfunctional mitochondrial subpopulation.     

Mitochondrial production of reactive oxygen species

Numerous biochemical studies are aimed at elucidating the sources and mechanisms of formation of reactive oxygen species (ROS) because they are involved in cellular, organ-, and tissue-specific physiology. Mitochondria along with other cellular organelles of eukaryotes contribute significantly to ROS formation and utilization. This review is a critical account of the mitochondrial ROS production and methods for their registration. The physiological and pathophysiological significance of the mitochondrially produced ROS are discussed.     

Signal transduction by reactive oxygen species

Although historically viewed as purely harmful, recent evidence suggests that reactive oxygen species (ROS) function as important physiological regulators of intracellular signaling pathways. The specific effects of ROS are modulated in large part through the covalent modification of specific cysteine residues found within redox-sensitive target proteins. Oxidation of these specific and reactive cysteine residues in turn can lead to the reversible modification of enzymatic activity. Emerging evidence suggests that ROS regulate diverse physiological parameters ranging from the response to growth factor stimulation to the generation of the inflammatory response, and that dysregulated ROS signaling may contribute to a host of human diseases.     

Mammalian peroxisomes and reactive oxygen species

The central role of peroxisomes in the generation and scavenging of hydrogen peroxide has been well known ever since their discovery almost four decades ago. Recent studies have revealed their involvement in metabolism of oxygen free radicals and nitric oxide that have important functions in intra- and intercellular signaling. The analysis of the role of mammalian peroxisomes in a variety of physiological and pathological processes involving reactive oxygen species (ROS) is the subject of this review. The general characteristics of peroxisomes and their enzymes involved in the metabolism of ROS are briefly reviewed. An expansion of the peroxisomal compartment with proliferation of tubular peroxisomes is observed in cells exposed to UV irradiation and various oxidants and is apparently accompanied by upregulation of PEX genes. Significant reduction of peroxisomes and their enzymes is observed in inflammatory processes including infections, ischemia-reperfusion injury, and allograft rejection and seems to be related to the suppressive effect of tumor necrosis factor- on peroxisome function and peroxisome proliferator activated receptor-. Xenobiotic-induced proliferation of peroxisomes in rodents is accompanied by the formation of hepatic tumors, and evidently the imbalance in generation and decomposition of ROS plays an important role in this process. In PEX5^–/– knockout mice lacking functional peroxisomes severe alterations of mitochondria in various organs are observed which seem to be due to a generalized increase in oxidative stress confirming the important role of peroxisomes in homeostasis of ROS and the implications of its disturbances for cell pathology.     

Phagocytic leukocytes and reactive oxygen species

Phagocytic leukocytes, when appropriately stimulated, display a respiratory burst in which they consume oxygen and produce superoxide anions. Superoxide is produced by the phagocyte NADPH-oxidase system which is a multiprotein complex that is dissociated in quiescent cells and is assembled into the functional oxidase following stimulation of these cells. Also associated with the respiratory burst is the generation of other reactive oxygen species. The identity of components of the NADPH-oxidase system and their interactions are known in considerable molecular detail. Understanding of the regulation of superoxide production is less well known. This review also points out the important role of microscopy in complementing biochemical studies to understand better the cell biology of the phagocyte respiratory burst. Presented at the 50th Anniversary Symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1–4, 2008.     

Imaging reactive oxygen species in arthritis

Reactive oxygen species (ROS) have been shown to play a role in the pathogenesis of arthritides. Luminol was used as the primary reporter of ROS and photons resulting from the chemiluminescence reaction were detected using a super-cooled CCD photon counting system. Luminol was injected intravenously into groups of animals with different models of arthritis. Imaging signal correlated well with the severity of arthritis in focal and pan-arthritis as determined by histological measurement of ROS by formazan. Measurements were highly reproducible, sensitive, and repeatable. In vivo chemiluminescence imaging is expected to become a useful modality to elucidate the role of ROS in the pathogenesis of arthritides and in determining therapeutic efficacy of protective therapies.