Ebook: Free Radicals and Diseases: Gene Expression, Cellular Metabolism and Pathophysiology

Oxygen free radicals and other oxidants are equally important for normal cellular metabolism and in numerous pathological diseases. The research of the last decades has contributed substantially to the understanding of the role and function of these metabolites.

This book summarizes the contributions of the Free Radical School 'Free Radicals and Diseases: Gene Expression, Cellular Metabolism and Pathophysiology' held in Spetses from Sept. 21 until Oct. 1, 2004. This Free Radical School was sponsored as an Advanced Study Institute by NATO, as an Advanced Course by the Federation of European Biochemical Societies, by UNESCO-MCBN (Molecular and Cell Biology Network), by the IUBMB (International Union of Biochemistry and Molecular Biology), and the SFRR (Society for Free Radical Research – International and Europe). The book presents the topics of most of the lectures as well as various selected posters which have been presented. The chapters contain an extensive overview including basic science approaches and clinical applications. It was the aim of the organizers of the Free Radical School (and of the editor of the book) to include a large variety of biological models ranging from yeast over mitochondria, isolated cells and cell culture models to animals and humans. The topics both of the Free Radical School and this book focus on the function and integrity of mitochondria under oxidative conditions, the role of protein oxidation and proteolysis in the cellular stress response and new aspects in the field of antioxidant treatment. Some chapters are introducing some methodological approaches and their application in the investigation of oxidative stress and diseases related to this condition.

I would herewith like to thank all the authors who were contributing to this book. I also want to thank the many students, colleagues, and friends who made the Free Radical School a success by scientific exchange in the lecture halls and during the tutorials.

This book wants to make the contents of the Free Radical School accessible for a wide audience and I am sure the chapters of this book will be of interest to many scientists and clinicians in the field.

Since both, the Society for Free Radical Research (International) and the Society for Free Radical Research (Europe) are planning a continuation of the Free Radical Schools in Spetses I also hope, that this book will draw the attention of many students interested in attending future Schools.

Tilman Grune, Editor

Reactive oxygen species involved in oxidative stress may damage DNA, the biopolymer that contains the genetic information. The cell has developed several enzymatic systems to repair the damage but some of them may persist and lead to mutagenesis. We focused our attention on the simultaneous quantification of several DNA lesions using the highly sensitive, specific and reliable high performance liquid chromatography-electrospray ionization tandem mass spectrometry detection technique (HPLC-MS/MS). The aim of this work is to determine if the different measured DNA lesions could be used as biomarkers of in vivo oxidative stress and/or inflammation. For such a purpose, three different types of DNA lesions were monitored: oxidized DNA lesions, chlorinated nucleosides arising from inflammation processes and DNA adducts generated from reaction with reactive aldehydes arising from lipid peroxides breakdown. Preliminary results, that have to be further confirmed, show a significant increase in the level of several different DNA lesions in diabetic patients versus a control group of healthy volunteers.

Important parameters of lipid peroxidation (LPO) such as fatty acid loss, formation and accumulation of conjugated dienes, lipid hydroperoxides, short-chain alkanes, F₂-isoprostanes, oxysterols, TBA-reactive substances (TBA-RS), malon-dialdehyde (MDA), 4-hydroxynonenal (HNE), and protein carbonyls are described. Assays for the quantification of these parameters are compared. No method for qu-antification of LPO is the ideal assay. A combination of different assays is recommended for evaluation of oxidative stress and LPO in complex biological systems. Examples for clinical applications are presented. A future strategy seems to be the use, optimization, and development of assays for LPO-derived protein and/or DNA changes.

Yeast is an established model in the studies of aging. Both replicative aging (limited capacity of budding) and chronologic aging (limited capacity to survive in the stationary culture) are studied in Saccharomyces cerevisiae Perhaps the most interesting aspect of these studies is the identification of genes affecting the cellular lifespan.

Oxidative stress is a factor in both replicative and chronologic aging. Yeast deficient in antioxidant and antioxidant enzymes show decreased replicative and chronologic lifespan while overexpression of some antioxidant proteins prolongs the lifespan.

The state 4-state 3 transition regulates NO release in coupled mitochondria, the rates were 40–50% lower in state 3 than in state 4. The rates of NO production by liver and kidney mitochondria were 1.3 and 0.7 nmol/min.mg protein in state 4, and 0.7 and 0.4 nmol/min.mg protein in state 3. The state 4-state 3 transition regulates NO production by the transition of mitochondrial membrane potential and not of intramitochondrial pH. Nitric oxide production was voltage dependent showing higher rates at higher mitochondrial membrane potentials.

Mitochondria play essential role in the cell functioning. Besides their obvious importance in overall cell energy production, mitochondria support cellular redox homeostasis, produce small signaling molecules, generate steroids, heme and Fe-S cluster proteins, participate in apoptosis, cell proliferation, detoxication and thermogenesis. Production of reactive oxygen species (ROS) may be regarded as an important mitochondrial function for regulated signaling and destructive processes. Mitochondria themselves seems to be a source, sensor and a target for ROS. Signaling function of mitochondrial ROS is demonstrated on the example of cardioprotective mechanism developed under ischemic preconditioning directed against ischemia/reperfusion damage.

Cell death can be triggered by opening of the mitochondrial permeability transition pore (MPT). A recent study with cardiomyocytes revealed ROS-induced oscillations in mitochondrial membrane potential (DY) ascribed to the opening and closing of the MPT. In neurones, DY oscillations have been reported which appear not to be driven by the MPT. Here we describe oscillations in mitochondrial TMRE fluorescence in astrocytes that are enhanced by exposure to light and reduced by CSA, consistent with the involvement of the MPT in DY flickering.

In vitro studies demonstrated that peroxynitrite inactivates both human recombinant MnSOD (hrMnSOD) and E. coli MnSOD causing enzyme tyrosine residue(s) nitration. This led to a suggestion that human MnSOD nitration and inactivation in vivo, detected in various deseases associated with oxidative stress and overproduction of nitric monoxide (NO)–conditions that favor peroxynitrite formation–are also caused by peroxynitrite. In a previous study we demonstrated that the exposure of E. coli MnSOD to NO under the anaerobic conditions causes NO conversion (dismutation) into reactive nitrosonium (NO⁺) and nitroxyl (HNO/NO^-) species, which produce enzyme modifications and inactivation (Niketic et al., Free Rad. Biol. Med. 27: 992 (1999)). The present study shows that interaction of NO with E. coli MnSOD leads to the formation of nitrating species capable of nitrating and oxidizing enzyme tyrosine residues, as well as that these species are less invasive than peroxynitrite in producing enzyme modifications and inactivation. Low molecular mass thiols are shown to reduce enzyme inactivation and NO-induced tyrosine nitration. The present study contributes to the understan-ding of the nature of NO reaction with E. coli MnSOD and provides compelling argument in support of the direct involvement of NO in MnSOD mediated generation of nitrating species. Interaction of NO with MnSOD may represent a novel mechanism by which MnSOD protects the cell from deleterious effects associated with overproduction of NO. However, extensive MnSOD modifications and inactivation associated with a prolonged exposure to NO will amplify toxic effects caused by elevated cell superoxide and NO levels.

Oxidative stress causes protein damage in mammalian cells. Modified proteins will be recognized and eliminated by the proteasomal system before severe protein aggregates emerge. It has been demonstrated that the 20S “core” proteasome is sufficient for recognition and degradation of mildly oxidized proteins whereas ubiquitination and ATP dependent protein degradation through the 26S proteasome does not seem to be necessarily involved. Proteasomal molecules are located in the cell either bound to cellular and organelle membranes or free in the cytosol and in the nucleus. The nuclear proteasome is strongly activated by a poly-ADP ribose polymerase mediated formation of poly(ADP-ribose). Recent studies support the hypothesis that the 20S proteasome is resistant to oxidation whereas the 26S form of the proteasome and the ubiquitination machinery seems to be more easily affected under oxidative conditions.

Proteins are sensitive to reactive oxygen species (ROS) such as hydrogen peroxide, superoxide and hydroxyl radicals which may be produced by the reaction between oxygen and free electrons released from the mitochondrial respiratory chain. Protein oxidation is associated with a loss of function, and oxidized proteins accumulate in cells during aging. Within proteins, cysteine and methionine, the two sulfur-containing amino acid residues, are most sensitive to ROS, but they are the only ones which can be reversed. The methionine sulfoxide reductases (Msr) system is able to repair methionine sulfoxide and convert it into reduced methionine in proteins. Age-related accumulation of oxidized proteins has been reported to be due, at least in part, to a decrease in degradation of the modified proteins, but protein repair systems also appear to be involved in this process. Furthermore, the Msr system has been shown to be important in cellular protection against oxidative stress and dysregulation of redox homeostasis associated with aging.

Activated microglia, protein oxidation, accumulation of protein material and an increase in inflammatory processes and decreased proteolysis are all parameters found in neurodegeneration. This evidence suggests an important connection between protein oxidation, proteolytic processes and microglial activation. Activated microglial cells not only further damage extracellular proteins and increase the oxidative burden to the brain, but also change their ability to degrade oxidized and AGE-modified proteins.

The role of microglial cell in the brain, the effects of their activation and the intracellular changes in these cells related to the expression of uptake and proteolytic mechanisms and to the degradation efficiency of various materials is reviewed in this chapter.

Eighty years following the identification of vitamin E as an essential micronutrient, our level of requirement for this vitamin and its possible range of functions are still hot topics of debate. One reason for this is that until relatively recently there has been an extremely poor appreciation of how vitamin E is utilised and metabolised in the body. A series of recent advances in these two areas have led to a much-improved understanding of how this micronutrient is utilised. Two general pathways of vitamin E metabolism are though to exist; one involving oxidative reactions while the other is a non-oxidative pathway. A number of vitamin E metabolites have been identified in blood and urine and physiological functions have been proposed for some of these. In this review we examine these advances and indicate how this micronutrient is finally beginning to come of age.

Vitamin E is present in plants in 8 different forms with essentially equal antioxidant potential (α-, β-, γ-, δ-tocopherol/tocotrienols); nevertheless, in higher organisms only α-tocopherol is preferentially retained suggesting a specific evolutionary reason for the selective uptake of this analogue. In the last 20 years, the route of the tocopherols from the diet into the body has been clarified and the proteins involved in the uptake and selective retention of α-tocopherol discovered. Cellular functions of the tocopherols that are independent of their antioxidant/radical scavenging abilities have been characterized in recent years. Vitamin E inhibits protein kinase C (PKC), protein kinase B (PKB), tyrosine kinases, 5-lipoxygenase and phospholipase A2 and activates protein phosphatase 2A, and diacylglycerol kinase. A growing number of genes are modulated by the tocopherols at the transcriptional level. The tocopherols also inhibit cell proliferation, platelet aggregation, monocyte adhesion and the differentiation of hippocampus neurons. These effects are unrelated to the antioxidant activity of vitamin E, and possibly reflect specific interactions of each of the tocopherols analogues with enzymes, structural proteins, lipids and transcription factors. Recently, several novel tocopherol binding proteins have been cloned, that may mediate the non-antioxidant signaling and cellular functions of vitamin E and its correct intracellular distribution. In the present review, it is suggested that the main physiological purpose of the α-tocopherol salvage pathway is to maintain a high and continuous plasma concentration of α-tocopherol, via the selective enrichment of VLDL with α-tocopherol. This in turn allows to achieve higher levels of α-tocopherol in the central and peripheral nervous system and in the trophoblasts of the placenta, which are the two tissues mainly affected by vitamin E deficiency. At the molecular level, the non-antioxidant activities associated with each tocopherol analogue may represent the main biological reason for the selective retention of only α-tocopherol in the body, or vice versa, for the metabolic conversion and consequent elimination of the β-, γ-, and δ-tocopherols.

In the present work we describe investigations intended to identify the molecular mechanism(s) of H₂O₂-induced cell death. Jurkat cells in culture were treated with either a bolus addition of H₂O₂ or exposed to the enzyme glucose oxidase which generated a continuous flow of H₂O₂. Contrary to the prevailing idea which considers mitochondria as the initial point of action of H₂O₂, we observed that H₂O₂-induced apoptosis is triggered through an initial interaction of H₂O₂ with redox-active iron in the lysosomes. The hydroxyl radicals formed, attacked the lysosomal membranes leading to their destabilization which preceded mitochondrial permeability transition and activation of the caspase cascade. It was also observed that H₂O₂, apart from its well known pro-apoptotic action, could exert anti-apoptotic effects when present, even at relatively low concentrations, during the execution of apoptotic process. In an attempt to identify the exact point of the inhibitory action of H₂O₂, we detected normal formation of the apoptosome complex but inability of caspase-9 to be activated in the presence of H₂O₂. Further experimental work is needed in order to clarify the exact molecular mechanism(s) underlying this observation.

Exposure of human skin to solar ultraviolet radiation both causes damage to skin cells and triggers signaling cascades that regulate cellular gene expression. Both effects may be mediated by reactive oxygen species (ROS) such as singlet oxygen and superoxide. In this chapter, the photochemical generation of ROS, the oxidation of biomolecules and activation of signaling cascades by ROS as well as the medical use of photooxidation reactions in photodynamic therapy will be briefly discussed.

Accumulation of oxidized low density lipoproteins in macrophages and smooth muscle cells causes foam cell formation, an initial step in atherosclerosis. Numerous studies have suggested the involvement of oxidative processes in the pathogenesis of atherosclerosis and especially of oxidized low density protein. Some epidemiological studies have shown an association between high dietary intake and high serum concentrations of vitamin E and lower rates of ischemic heart disease. Cell culture studies have shown that α-tocopherol brings about inhibition of smooth muscle cell proliferation. This takes place via inhibition of protein kinase C activity. α-tocopherol also inhibits low density lipoprotein induced smooth muscle cell proliferation and protein kinase C activity. The following animal studies showed that vitamin E protects development of cholesterol induced atherosclerosis by inhibiting protein kinase C activity in smooth muscle cells in vivo.

Elevated plasma levels of homocysteine have been identified as an important and independent risk factor for cerebral, coronary and peripheral atherosclerosis. However the mechanisms by which homocysteine promote atherosclerotic plaque formation are not clearly defined. Earlier reports have been suggested that homocysteine exert its effect via H₂O₂ produced during its metabolism. To evaluate the contribution of homocysteine in the pathogenesis of vascular diseases, we examined whether the homocysteine effect on vascular smooth muscle cell growth is mediated by H₂O₂. We show that homocysteine induces DNA synthesis and proliferation of vascular smooth muscle cells in the presence of peroxide scavenging enzyme, catalase. Our data suggest that homocysteine induces smooth muscle cell growth through the activation of an H₂0₂ independent pathway and accelerate the progression of atherosclerosis.

Exposure to cold induces complex physiological response in interscapular brown adipose tissue (IBAT), which is the main site for thermogenesis. Recent findings showed that nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) displays beneficial properties on numerous physiological processes in IBAT. However, high concentration of NO induces apoptosis in several cell types. In this regard, NO may be implicated in induction of apoptosis in IBAT. Hence, the goal of present study was to examine possible relationship between NO, induction of apoptosis and processes involved in IBAT hyperplasia occurring during new homeostasis reaching, when animals were exposed to cold.

The male Mill Hill hybrid hooded rats were divided into three main groups. One group was receiving N^ω−nitro-L-arginine methyl ester (L-NAME HCl, 0.01%), an inhibitor of nitric oxide synthases (NOSs) and another L-arginine HCl (2.25%), a substrate for NOSs in drinking water for 45 days. The third group served as a control. Animals of all three groups additionally were divided in two subgroups: one – housed at 22 ±1°C and another – in cold room at 5 ±1°C.

We show here that cold exposure of rats markedly decreased the iNOS immunopositivity i.e. production of NO, and caused a rapid decrease in the apoptosis in tissue. On cold, higher iNOS immunopositivity was detected only in L-Arg treated group. Besides, in L-Arg treated group of rats acclimated to low temperature level of uncoupling protein 1 (UCP1) was higher, while, in L-NAME treated group level of UCP1 was significantly decreased in comparison to control group acclimated to low temperature. This data indicate that NO additionally induced UCP1. Similarly, L-arginine treatment, namely NO, additionally improved cold-induced tissue mass increase in contrast to L-NAME treatment, which decrease tissue mass. We also show, on room temperature, that marked increase in the iNOS immunopositivity and rate of apoptosis were detected in both treated groups of rats compared to appropriate control. These data indicates that NO produced in high concentration induced apoptosis in both treated groups of rats acclimated to room temperature.

Our results indicates that NO plays regulatory roles in IBAT hyperplasia (induction of UCP1 and increase of the tissue mass). In contrast, NO produced by high expressed iNOS induce apoptosis and can be cytotoxic.

Exercise increases the generation of reactive oxygen and nitrogen species (RONS), and by causing adaptations could decrease the incidence of RONS-associated diseases. A single bout of exercise, depending upon intensity and duration, can cause an increase in antioxidant enzyme activity, decrease levels of thiols and antioxidant vitamins, and result in oxidative damage as a sign of incomplete adaptation. Increased levels of RONS and oxidative damage are initiators of a specific adaptive response, the stimulation of the activation of antioxidant enzymes, thiols and enhanced oxidative damage repair. Regular exercise has the capability of developing compensation to oxidative stress, resulting in overcompensation against the increased level of RONS production and oxidative damage. Regular exercise also causes adaptation of the antioxidant and repair systems, which could result in a decreased base level of oxidative damage and increased resistance to oxidative stress. In this paper we extend the hormesis theory to include RONS on the list of potential causes of adaptation, and we further suggest that the preventive effect of regular exercise is partly based on the RONS generating capability of exercise, which is in the stimulating range of RONS production. Therefore, it is suggested that exercise-induced changes in the redox milieu, signaling pathways, induction of antioxidants, DNA repair, and protein degrading enzymes are the consequences of ROS production and are involved in the adaptation process, resulting in decreases in the incidence of oxidative stress-related diseases and retardation of the aging process.

Carotenoids are natural pigments, which are found in bacteria, algae, fungi and plants, but which are not synthesized in animals. Animals and human beings get carotenoids through food. If we think on carotenoids, we think mostly on β-carotene, lycopene, lutein, zeaxanthin, and some others. But, meanwhile 600 to 700 carotenoids were identified. About 60 of them are components of our nutrition. Almost ten percent of all carotenoids in mammals can be metabolized to retinol, which is vitamin A, and can therefore function as vitamin A precursors. Carotenoids and retinoids act as antioxidants, influence the growth of the organism, immunological functions, the visual cycle, and modulate gene expression, too. Epidemiological data have strongly linked higher levels of carotenoid intake and increased circulating and tissue concentrations of carotenoids with reduced risk for various cancers, cardiovascular disease, and other diseases, even clinical intervention trials did not find homogeneous and significant evidence, that β-carotene alone leads to these benefits. In contrast to expectations and to the medical benefits induced by high intake of carotenoids by nutrition and via supplements in heavy smokers and asbestos workers the incidence and mortality in lung carcinoma even increased in high-dosage β-carotene supplementation. These observations initiated research projects with the aim, to find out the cause of potential toxic effects of high-dosage β-carotene supplementation. Obviously, the toxic effects can be attributed oxidative breakdown products of β-carotene and other carotenoids. The previous results argue for toxic effects only at high-dosage supplementation in heavy smokers and asbestos workers, i.e. under conditions of severe oxidative stress. From in vitro experiments was concluded, that even at very high carotenoid concentration a toxic effect towards biomolecules can be avoided if the other components of the antioxidative network are present at high levels.