An epigenetic perspective on the free radical theory of development

The development of organisms requires concerted changes in gene activity. The free radical theory of development proposes that oxygen serves as a morphogen to educe development by influencing the production of metabolic oxidants such as free radicals and reactive oxygen species. One of the central tenets of this theory is that these metabolic oxidants influence development by altering the antioxidant capacity of cells by changing their production of glutathione (GSH). Here we extend on these principles by linking GSH production and oxygen sensing in the control of gene expression to establish the epigenotype of cells during development. We prescribe this novel role to GSH and oxygen during development because these metabolites influence the activity of enzymes responsible for initiating and perpetuating epigenetic control of gene expression. Increased GSH production influences epigenetic processes including DNA and histone methylation by limiting the availability of S-adenosylmethionine, the cofactor utilized during epigenetic control of gene expression by DNA and histone methyltransferases. Moreover, the recent discovery of histone demethylases that require oxygen as a cofactor directly links epigenetic processes to oxygen gradients during development.     

Oxidative influence on development and differentiation: An overview of a free radical theory of development

Metabolic gradients exist in developing organisms and are believed to influence development. It has been postulated that the effects of these gradients on development result from differential oxygen supplies to tissues. Oxygen has been found to influence the course of development. Cells and tissues in various stages of differentiation exhibit discrete changes in their antioxidant defenses and in parameters of oxidation. Metabolically generated oxidants have been implicated as one factor that directs the initiation of certain developmental events. Also implicated as factors that modulate developmental processes are the cellular distribution of ions and the cytoskeleton both of which can be influenced by oxidants. The interaction of oxidants with ion balance and cytoskeleton is discussed.     

Oxidative stress and gene regulation

Reactive oxygen species are produced by all aerobic cells and are widely believed to play a pivotal role in aging as well as a number of degenerative diseases. The consequences of the generation of oxidants in cells does not appear to be limited to promotion of deleterious effects. Alterations in oxidative metabolism have long been known to occur during differentiation and development. Experimental perturbations in cellular redox state have been shown to exert a strong impact on these processes. The discovery of specific genes and pathways affected by oxidants led to the hypothesis that reactive oxygen species serve as subcellular messengers in gene regulatory and signal transduction pathways. Additionally, antioxidants can activate numerous genes and pathways. The burgeoning growth in the number of pathways shown to be dependent on oxidation or antioxidation has accelerated during the last decade. In the discussion presented here, we provide a tabular summary of many of the redox effects on gene expression and signaling pathways that are currently known to exist.     

Protection Against Ischemic Brain Injury by Inhibition of Mitochondrial Oxidative Stress

Mitochondria are both targets and sources of oxidative stress. This dual relationship is particularly evident in experimental paradigms modeling ischemic brain injury. One mitochondrial metabolic enzyme that is particularly sensitive to oxidative inactivation is pyruvate dehydrogenase. This reaction is extremely important in the adult CNS that relies very heavily on carbohydrate metabolism, as it represents the sole bridge between anaerobic and aerobic metabolism. Oxidative injury to this enzyme and to other metabolic enzymes proximal to the electron transport chain may be responsible for the oxidized shift in cellular redox state that is observed during approximately the first hour of cerebral reperfusion. In addition to impairing cerebral energy metabolism, oxidative stress is a potent activator of apoptosis. The mechanisms responsible for this activation are poorly understood but likely involve the expression of p53 and possibly direct effects of reactive oxygen species on mitochondrial membrane proteins and lipids. Mitochondria also normally generate reactive oxygen species and contribute significantly to the elevated net production of these destructive agents during reperfusion. Approaches to inhibiting pathologic mitochondrial generation of reactive oxygen species include mild uncoupling, pharmacologic inhibition of the membrane permeability transition, and simply lowering the concentration of inspired oxygen. Antideath mitochondrial proteins of the Bcl-2 family also confer cellular resistance to oxidative stress, paradoxically through stimulation of mitochondrial free radical generation and secondary upregulation of antioxidant gene expression.     

Dynamic changes in mitochondrial biogenesis and antioxidant enzymes during the spontaneous differentiation of human embryonic stem cells

Embryonic cells before implantation are exposed to a hypoxic condition and dependent on anaerobic metabolism. Human embryonic stem cells (HESCs) derived from pre-implantation blastocyst also grow well in hypoxic conditions. Expecting that the differentiating HESCs might mimic anaerobic-to-aerobic metabolic transition of the early human life, we examined the mitochondria-related changes in these cells. We observed that mitochondrial mass and mitochondrial DNA content were increased with differentiation, which was accompanied by the increase of the amount of ATP (4-fold) and its by-product reactive oxygen species (2.5-fold). The expression of various antioxidant enzymes including mitochondrial and cytoplasmic superoxide dismutases, catalase, and peroxiredoxins showed a dramatic change during the early differentiation. In conclusion, HESC differentiation was followed by dynamic changes in mitochondrial mass, ATP and ROS production, and antioxidant enzyme expressions. Therefore, the HESCs would serve as a good model to examine the mitochondrial biology during the early human differentiation.     

Early alterations in mitochondrial reserve capacity; a means to predict subsequent photoreceptor cell death

Although genetic and environmental factors contribute to neurodegenerative disease, the underlying etiology common to many diseases might be based on metabolic demand. Mitochondria are the main producer of ATP, but are also the major source of reactive oxygen species. Under normal conditions, these oxidants are neutralized; however, under environmental insult or genetic susceptibility conditions, oxidative stress may exceed cellular antioxidant capacities, leading to degeneration. We tested the hypothesis that loss in mitochondrial reserve capacity plays a causative role in neuronal degeneration and chose a cone photoreceptor cell line as our model. 661W cells were exposed to agents that mimic oxidant stress or calcium overload. Real-time changes in cellular metabolism were assessed using the multi-well Seahorse Biosciences XF24 analyzer that measures oxygen consumption (OCR) and extracellular acidification rates (ECAR). Cellular stress resulted in an early loss of mitochondrial reserve capacity, without affecting basal respiration; and ECAR was increased, representing a compensatory shift of ATP productions toward glycolysis. The degree of change in energy metabolism was correlated with the amount of subsequent cell death 24-hours post-treatment, the concentration-dependent loss in mitochondrial reserve capacity correlated with the number of live cells. Our data suggested first, that loss in mitochondrial reserve capacity is a major contributor in disease pathogenesis; and second, that the XF24 assay might represent a useful surrogate assay amenable to the screening of agents that protect against loss of mitochondrial reserve capacity. In future experiments, we will explore these concepts for the development of neuroprotective agents.     

Role for mitochondrial oxidants as regulators of cellular metabolism

Leakage of mitochondrial oxidants contributes to a variety of harmful conditions ranging from neurodegenerative diseases to cellular senescence. We describe here, however, a physiological and heretofore unrecognized role for mitochondrial oxidant release. Mitochondrial metabolism of pyruvate is demonstrated to activate the c-Jun N-terminal kinase (JNK). This metabolite-induced rise in cytosolic JNK1 activity is shown to be triggered by increased release of mitochondrial H(2)O(2). We further demonstrate that in turn, the redox-dependent activation of JNK1 feeds back and inhibits the activity of the metabolic enzymes glycogen synthase kinase 3beta and glycogen synthase. As such, these results demonstrate a novel metabolic regulatory pathway activated by mitochondrial oxidants. In addition, they suggest that although chronic oxidant production may have deleterious effects, mitochondrial oxidants can also function acutely as signaling molecules to provide communication between the mitochondria and the cytosol.     

Reconciling the chemistry and biology of reactive oxygen species

There is a vast literature on the generation and effects of reactive oxygen species in biological systems, both in relation to damage they cause and their involvement in cell regulatory and signaling pathways. The biological chemistry of different oxidants is becoming well understood, but it is often unclear how this translates into cellular mechanisms where redox changes have been demonstrated. This review addresses this gap. It examines how target selectivity and antioxidant effectiveness vary for different oxidants. Kinetic considerations of reactivity are used to assess likely targets in cells and how reactions might be influenced by restricted diffusion and compartmentalization. It also highlights areas where greater understanding is required on the fate of oxidants generated by cellular NADPH oxidases and on the identification of oxidant sensors in cell signaling.     

Specificity in reactive oxidant signaling: think globally, act locally

Although reactive oxidants have long been stigmatized as unwanted metabolic byproducts, the expression of oxidases specifically functioning to produce these same molecules in a regulated fashion is surprisingly pervasive throughout metazoan and plant evolution. Although the involvement of oxidants in many signaling pathways is well documented, the cellular strategies for conferring pathway specificity to such reactive molecules have remained more recondite. Recent studies now suggest that cells may spatially restrict oxidant production to allow microdomain-specific signaling.     

Nitric oxide-oxygen radicals interactions in atherosclerosis

Atherosclerosis is one of the most common diseases and the principal cause of death in western civilization. The pathogenesis of this disease can be explained on the basis of the 'oxidative-modification hypothesis,' which proposes that low-density lipoprotein (LDL) oxidation represents a key early event. Nitric oxide (*NO) regulates critical lipid membrane and lipoprotein oxidation events by a) contributing to the formation of more potent secondary oxidants from superoxide (i.e.: peroxynitrite), and b) its antioxidant properties through termination reactions with lipid radicals to possibly less reactive secondary nitrogen-containing products (LONO, LOONO). Relative rates of production and steady state concentrations of superoxide and *NO and cellular sites of production will profoundly influence the expression of differential oxidant injury-enhancing and protective effects of *NO. Full understanding of the physiological roles of *NO, coupled with detailed insight into *NO regulation of oxygen radical-dependent reactions, will yield a more rational basis for intervention strategies directed toward oxidant-dependent atherogenic processes.     

Oxidants and skeletal muscle function: physiologic and pathophysiologic implications

Previous studies have demonstrated that skeletal muscles generate considerable reactive oxygen during intense muscle contraction. However, the significance of this phenomenon and whether it represents normal physiology or pathology are poorly understood. Treatment with exogenous antioxidants suggests that normal redox tone during contraction is influencing ongoing contractile function, both at rest and during intense exercise. This could represent the influence of redox-sensitive proteins responsible for excitation-contraction coupling or redox-sensitive metabolic enzymes. Some conditions associated with intense exercise, such as local tissue hypoxia or elevated tissue temperatures, could also contribute to reactive oxygen production. Evidence that muscle conditioning results in upregulation of antioxidant defenses also suggests a close relationship between reactive oxygen and contractile activity. Therefore, there appears to be a significant role for reactive oxygen in normal muscle physiology. However, a number of conditions may lead to an imbalance of oxidant production and antioxidant defense, and these, presumably, do create conditions of oxidant stress. Ischemia-reperfusion, severe hypoxia, severe heat stress, septic shock, and stretch-induced injury may all lead to oxidant-mediated injury to myocytes, resulting in mechanical dysfunction.     

Queuine promotes antioxidant defence system by activating cellular antioxidant enzyme activities in cancer

Constant generation of Reactive oxygen species (ROS) during normal cellular metabolism of an organism is generally balanced by similar rate of consumption by antioxidants. Imbalance between ROS production and antioxidant defense results in increased level of ROS causing oxidative stress which leads to promotion of malignancy. Queuine is a hyper modified base analogue of guanine, found at first anti-codon position of Q- family of tRNAs. These tRNAs are completely modified with respect to queuosine in terminally differentiated somatic cells, however hypomodification of Q-tRNAs is close association with cell proliferation. Q-tRNA modification is essential for normal development, differentiation and cellular functions. Queuine is a nutrient factor to eukaryotes. It is found to promote cellular antioxidant defense system and inhibit tumorigenesis. The activities of antioxidant enzymes like catalase, SOD, glutathione peroxidase and glutathione reductase are found to be low in Dalton's lymphoma ascites transplanted (DLAT) mouse liver compared to normal. However, exogenous administration of queuine to DLAT mouse improves the activities of antioxidant enzymes. The results suggest that queuine promotes antioxidant defense system by increasing antioxidant enzyme activities and in turn inhibits oxidative stress and tumorigenesis.     

A systematic investigation of <Emphasis Type="Italic">Escherichia coli</Emphasis> central carbon metabolism in response to superoxide stress

Background  

The cellular responses of bacteria to superoxide stress can be used to model adaptation to severe environmental changes. Superoxide stress promotes the excessive production of reactive oxygen species (ROS) that have detrimental effects on cell metabolic and other physiological activities. To antagonize such effects, the cell needs to regulate a range of metabolic reactions in a coordinated way, so that coherent metabolic responses are generated by the cellular metabolic reaction network as a whole. In the present study, we have used a quantitative metabolic flux analysis approach, together with measurement of gene expression and activity of key enzymes, to investigate changes in central carbon metabolism that occur in Escherichia coli in response to paraquat-induced superoxide stress. The cellular regulatory mechanisms involved in the observed global flux changes are discussed.     

Cellular redox regulation,signaling, and stress response in plants

Cellular and organellar redox states, which are characterized by the balance between oxidant and antioxidant pool sizes, play signaling roles in the regulation of gene expression and protein function in a wide variety of plant physiological processes including stress acclimation. Reactive oxygen species (ROS) and ascorbic acid (AsA) are the most abundant oxidants and antioxidants, respectively, in plant cells; therefore, the metabolism of these redox compounds must be strictly and spatiotemporally controlled. In this review, we provided an overview of our previous studies as well as recent advances in (1) the molecular mechanisms and regulation of AsA biosynthesis, (2) the molecular and genetic properties of ascorbate peroxidases, and (3) stress acclimation via ROS-derived oxidative/redox signaling pathways, and discussed future perspectives in this field.     

Mitochondrial Ca2+ laMety directs fibrosis

During development, disease or in response to changes in local environmental and/or nutrient supply, cellular metabolism is substantially remodeled. Reduced mitochondrial Ca²⁺ uptake was recently reported to induce metabolic remodeling, which through stimulating alterations in the epigenome causes changes in gene expression associated with fibroblast to myofibroblast differentiation.     

Dynamic regulation of mitochondrial network and oxidative functions during 3T3-L1 fat cell differentiation

Mitochondria have been shown to be impaired in insulin resistance-related diseases but have not been extensively studied during the first steps of adipose cell development. This study was designed to determine the sequence of changes of the mitochondrial network and function during the first days of adipogenesis. 3T3-L1 preadipocytes were differentiated into adipocytes without using glitazone compounds. At days?0, 3, 6, 9, and 12, mitochondrial network imaging, mitochondrial oxygen consumption, membrane potential, and oxidative phosphorylation efficiency were assessed in permeabilized cells. Gene and protein expressions related to fatty acid metabolism and mitochondrial network were also determined. Compared to preadipocytes (day?0), new adipocytes (days?6 and 9) displayed profound changes of their mitochondrial network that underwent fragmentation and redistribution around lipid droplets. Drp1 and mitofusin 2 displayed a progressive increase in their gene expression and protein content during the first 9?days of differentiation. In parallel with the mitochondrial network redistribution, mitochondria switched to uncoupled respiration with a tendency towards decreased membrane potential, with no variation of mtTFA and NRF1 gene expression. The expression of PGC1α and NRF2 genes and genes involved in lipid oxidation (UCP2, CD36, and CPT1) was increased. Reactive oxygen species (ROS) production displayed a nadir at day?6 with a concomitant increase in antioxidant enzyme gene expression. This 3T3-L1-based in vitro model of adipogenesis showed that mitochondria adapted to the increased number of lipid droplets by network redistribution and uncoupling respiration. The timing and regulation of lipid oxidation-associated ROS production appeared to play an important role in these changes.