Healthy aging: regulation of the metabolome by cellular redox

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Biogerontology DOI 10.1007/s10522-007-9096-4

REVIEW ARTICLE

Healthy aging: regulation of the metabolome by cellular redox modulation and prooxidant signaling systems: the essential roles of superoxide anion and hydrogen peroxide Anthony William Linnane Æ Michael Kios Æ Luis Vitetta

Received: 19 January 2007 / Accepted: 8 March 2007  Springer Science+Business Media B.V. 2007

Abstract The production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has long been proposed as leading to random deleterious modification of macromolecules with an associated progressive development of age associated systemic disease. ROS and RNS formation has been posited as a major contributor to the aging process. On the contrary, this review presents evidence that superoxide anion (and hydrogen peroxide) and nitric oxide (and peroxynitrite) constitute regulated prooxidant second messenger systems, with specific sub-cellular locales of production and are essential for normal metabolome and physiological function. The role of these second messengers in the regulation of the metabolome is discussed in terms of radical formation as an essential contributor to the physiologically normal regulation of sub-cellular bioenergy systems; proteolysis regulation; transcription activation; enzyme activation; mitochondrial DNA changes; redox regulation of metabolism and cell differentiation; the concept that orally administered small molecule antioxidant therapy is a chimera. The formation of superoxide anion/hydrogen peroxide and nitric

A. W. Linnane (&)  M. Kios  L. Vitetta Epworth Medical Centre, Centre for Molecular Biology and Medicine, 185–187 Hoddle Street, Richmond, Melbourne, VIC 3121, Australia e-mail: [email protected]

oxide do not conditionally lead to random macromolecular damage; under normal physiological conditions their production is actually regulated consistent with their second messenger roles. Keywords Metabolome regulation  Superoxide anion  Hydrogen peroxide  Nitric oxide  Peroxynitrite  Second messengers  Proton motive force  Cellular bioenergy  Proteolysis, Cellular organellar function  Prooxidants, Antioxidants  Mitochondrial DNA  Redox poise modulation

Introduction Over a period of some decades the concept has been extant that superoxide anion formation and products arising from it are highly toxic substances, which lead to random deleterious oxidation of macromolecules. Further, that superoxide anion formation is a major contributor to disease processes notably associated with the systemic diseases of the aged. Many papers and reviews have been written in support of this concept. For some years we have questioned this supposition, pointing out that unbridled oxygen radical toxicity as a major causation of aging is a gross over simplification of complex biological phenomena; it ignores a wealth of evidence of the essential requirement for the

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continuous regulated formation and functional utilization of superoxide anion and hydrogen peroxide, as well as the free radical gas nitric oxide (and peroxynitrite) (Linnane and Eastwood 2004, 2006; Linnane et al. 2007). These molecules are second messengers that are essential for normal cellular function. Herein we attempt a holistic presentation of their specific roles in the regulation of the metabolome. Studies on oxygen and its perceived toxic products, particularly in aging studies, have been highly compartmentalized, mainly concerned with in vitro studies and the mitochondrial formation of superoxide anion and hydrogen peroxide and extrapolating the results to apply to in vivo systems. We consider this approach has led to a number of misconceptions; superoxide anion and hydrogen peroxide formation by a number of organelles other than mitochondria is critical for normal cell function. The cell is comprised of a series of highly interactive compartments, namely the nucleus, mitochondria, lysosomes, peroxisomes, plasma membrane, Golgi apparatus, endoplasmic reticulum, cytosol and so on. The cellular metabolome is made up of the activities of these interactive compartments which function in a dynamic integrated equilibrium and as such is subject to stringent regulation by a range of extracellular effectors and sub-cellular regulator molecules. Under normal physiological conditions superoxide anion and hydrogen peroxide and nitric oxide formation is finely tuned to balance their roles as second messengers and to ensure that any potential pathophysiological outcomes which would arise from their under, or over production, do not occur. Cellular metabolic activity is in a constant state of flux, but a homeostatic state is normally maintained and all signaling systems must be regulated (turned off, or on). In this context, catalase and the glutathione peroxidase system will play key roles in the modulation of the H2O2 second messenger system. The term ROS (Reactive Oxygen Species) is commonly used to include, along with superoxide anion, hydrogen peroxide and the hydroxyl radical. It is known that in vitro H2O2 in the presence of transition metals (Fe, Cu) can give rise to hydroxyl radicals by way of the Fenton reaction. From such knowledge it has been posited for

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some decades that H2O2 in the presence of transition metals gives rise in vivo to significant levels of hydroxyl radical. However there is no definitive evidence that such a reaction occurs to a significant extent in vivo; the commonly cited transition metals Fe and Cu do not occur in free solution but are protein bound and are not available to participate in the Fenton reaction, as pointed out decades ago by Halliwell (1982). We draw a distinction between superoxide anion and hydrogen peroxide as biologically important substances and question that any significant role in mammalian biology has been demonstrated for hydroxyl radical, deleterious or otherwise. We arrive at this suggestion, in part, from the considerations that follow. The demonstration that hydroxyl radicals are formed in vivo from hydrogen peroxide during oxidative metabolism is slight; it mainly consists of administering salicylic acid to animals and demonstrating salicylate hydroxylation as evidence for the formation of the hydroxyl radical. This assay we suggest is inherently flawed. Consideration of the reactions of the enzyme complex xanthine oxidase, which is widely distributed in tissues, brings into serious question the validity of the assay. Xanthine oxidase is a complex tripartite enzyme, which oxidizes hypoxanthine to xanthine producing molar equivalents of H2O2 and similarly oxidizes xanthine to uric acid with H2O2 formation. The enzyme complex contains FAD, two separate [2Fe–2S] clusters and molybdenum in a molybdopterin complex in which Mo cycles from Mo(VII) to Mo(IV) oxidation states, the final acceptor is oxygen which gives rise to H2O2. The problem associated with the use of salicylic acid to detect hydroxyl radical formation is that it is a competitive inhibitor of xanthine oxidase, and Xray crystallographic studies have shown that salicylic acid binds to xanthine oxidase to block access of substrates to the molybdopterin metal center (Voet and Voet 2004). We suggest that the use of salicylic acid to demonstrate in vivo hydroxyl radical formation is highly likely to be an artefact arising from salicylic acid disrupting the normal function of xanthine oxidase and in turn being hydroxylated. Herein we consider the metabolic roles played by superoxide anion and hydrogen peroxide and

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due to space limitations a more limited consideration is given to nitric oxide and peroxynitrite. This review is reductionist in its literature approach due to the breadth of the fields we are discussing and apologies are made to many authors for their non-citation. The theme of this review is that too much emphasis has been placed on the ‘‘friends or foes’’, ‘‘the good, the bad and the ugly’’ concept concerning the role free radicals play in cell physiology. This approach has led to a somewhat confused situation, whereby the ‘‘foes’’ concept is overemphasized and flawed. This confusion has its genesis in observations made in the 1970s; reporting that mitochondria produce high toxic levels of superoxide anion and hydrogen peroxide (Chance et al. 1979); these findings are flawed, the interpretation of which, has led to long standing misleading conclusions. Mitochondria produce only trace levels of superoxide anion and hydrogen peroxide (Nohl et al. 2001; St-Pierre et al. 2002) (Refer later Mitochondria for detailed discussion).

Superoxide anion and hydrogen peroxide formation The role of coenzyme Q10 and sub-cellular signaling Coenzyme Q10 acting through formation of its semiquinone is a major source of cellular and mitochondrial superoxide anion and in turn hydrogen peroxide formation. It also has a major role in mitochondrial energy generation actively participating in the establishment of the mitochondrial membrane’s proton motive force (Dp = Dw + DpH). Coenzyme Q10 occurs in most, if not all, cellular membranes and it is again therein an important source of superoxide anion and consequently hydrogen peroxide. Its function contributes to the proton motive force established for closed sub-cellular membrane systems (refer later pH Gradients and Cellular Bioenergy). We have earlier reported that coenzyme Q10 functions in the process of gene regulation (Linnane et al. 2002a; Linnane 2002b). This conclusion arose from administering coenzyme Q10 in a placebo controlled trial to patients for a

period of 4 weeks prior to undergoing hip replacement surgery. Subsequent to surgery, vastus lateralis muscle specimens were analyzed using histochemical analyses, microarray gene display, differential gene display and proteome analysis technologies. This study demonstrated the profound effect coenzyme Q10 had on muscle fiber type composition, gene expression and the protein expression profile of human skeletal muscle. Coenzyme Q10 is anchored in cellular membranes as a member of oxido-reductase systems from which superoxide anion and hydrogen peroxide will arise. To explain the far reaching effect of coenzyme Q10 administration we concluded that H2O2 acted as a second messenger moving throughout the cell to deliver the coenzyme Q10 redox message from a number of sub-cellular locations. Thus the fluctuating redox poise of coenzyme Q10 within its various membrane oxido-reductase systems generates a fluctuating amount of superoxide anion and in turn second messenger H2O2 which moves throughout the cell to modulate the activities required for the normal function of the metabolome. The overarching role played by coenzyme Q10 in the holistic regulation of cellular function (Fig. 1) acting largely as a major source of superoxide anion and hydrogen peroxide has been discussed in more detail elsewhere and later herein (Linnane et al. 2002b, 2007; Linnane and Eastwood 2004, 2006). Coenzyme Q10 present in many sub-cellular locales is a major source of regulatory superoxide anion. However it is not the only primary source of superoxide anion. Notably the Nox oxido-reductase systems of the plasma membrane also generate superoxide anion and do not contain coenzyme Q10. Mitochondria Central to a consideration of the roles of superoxide anion and hydrogen peroxide formation as related to their putative adverse effects are the amounts formed under physiological conditions. Following on the oxygen free radical theory of aging as first proposed by Harman (1956), support for the theory was apparently provided in a series of influential 1970s papers. Boveris, Chance and colleagues reported that

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Biogerontology Fig. 1 The metabolome and the coenzyme Q10 oxido-reductase regulatory system(s). The cartoon summarizes the role of the coenzyme Q10 oxido-reductase systems in energy generation and its regulation, redox regulation of metabolic flux modulation, gene regulation and as essential superoxide/hydrogen peroxide generators (refer Linnane and Eastwood 2004, Linnane et al. 2007)

H+ 4 U nc o u p l e r Pr o t e in s ROS

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G e ne R e g u l a t i o n (Nucleus, Mitochondria Chloroplasts)

H2O2 was formed in vitro by mitochondria, peroxisomes and microsomal fractions (Boveris et al. 1972). Soon after, they reported that when respiring mitochondria were inhibited by antimycin A or rotenone, substantial amounts of superoxide anion and hydrogen peroxide were produced and that coenzyme Q10 semiquinone was the major source of superoxide anion (Boveris and Chance 1973; Chance et al. 1979). Indeed this in vitro data was extrapolated to conclude that 1–3% of inspired oxygen was converted to superoxide anion. These early reports set the stage for future interpretations of the putative toxicity of metabolically generated oxygen radicals; indeed 1–3% of inspired oxygen converted to superoxide anion and hydrogen peroxide would be potentially physiologically catastrophic. A voluminous literature extending from the 1970s to the present day, has grown describing the toxic effects of excessively high concentrations of H2O2 on enzyme systems and cultured cells. However hydrogen peroxide is not a toxic compound, except at unrealistically high mM concentrations, the common experimental use of mM concentrations has led to extrapolations and misunderstandings as to the physiological role of superoxide anion and hydrogen peroxide. It has erroneously become conventional knowledge that excessively high levels of superoxide anion and hydrogen peroxide are produced in vivo in an unregulated manner and as such are highly toxic to cells and that it is essential that they be immediately removed by antioxidants.

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C o- f a ct o r

∆p (energy)

Redox Poise

Metabolic Flux Modulation

More recently the original and much of the subsequent published data emanating from many laboratories has since been shown to be interpretively misleading, in that uninhibited respiring mitochondria produce only very low levels of superoxide anion and hydrogen peroxide (Nohl et al. 2001, 2005) confirmed and further elaborated by St-Pierre et al. (2002). St-Pierre and colleagues reported that the early studies of Boveris, Chance and colleagues over estimated the amount of superoxide anion and hydrogen peroxide formed by about two orders of magnitude; the early estimate being about 10 nM H2O2 formed/minute/mg mitochondrial protein versus actual 0.1 nM H2O2 formed/minute/mg mitochondrial protein. The St-Pierre paper is a notable contribution in its dissection of the topology of mitochondrial superoxide anion production; most significantly these authors reported that isolated rat skeletal muscle mitochondria respiring on complex I/III substrates release superoxide anion into the matrix while complex II/III substrates release superoxide anion into the medium. This vectorial synthesis of superoxide anion, extrapolated to cells would indicate that the resultant H2O2 formation putatively enables it to act as a mitochondrial second messenger signaling to both nuclear and mitochondrial genomes. This signal would reflect the extant metabolic state of the mitochondrial organelle and its temporal requirement for appropriate nuclear and mitochondrial gene expression and metabolome modulation. Another notable feature of this paper is that heart

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muscle and skeletal muscle mitochondria oxidizing palmitoyl carnitine, vectorially release H2O2 differently. Isolated heart mitochondria oxidizing fatty acids release superoxide anion into the mitochondrial matrix while muscle mitochondria release it into the medium. These observations perhaps reflect, that different tissue metabolisms have a requirement for different metabolic regulatory signaling messages. Studies of knock out mice and superoxide dismutases provide some further insight on the role played by superoxide anion/hydrogen peroxide. Mn SOD is localized in the mitochondrial matrix. In a comparison of young (6–8 months) and aged (27–29 months) skeletal muscle of transgenic mice heterozygous for Mn SOD (±) and wild type aged animals, no differences were observed other than the changes commonly reported for skeletal muscle of wild type aged animals albeit there was some small increase in DNA oxidative modification (Mansouri et al. 2006). The animals suffered no apparent ill effects from the Mn SOD activity decrease. On the contrary construction of homozygous transgenic mice, null (–/–), for Mn SOD resulted in animals being severely affected. Such animals as were born (no data was presented on prenatal deaths) died within a few days of birth as a result of severe cardiomyopathy, neurological and other pathological changes (Melov et al. 1998; Wallace and Lott 1999). However, the interesting finding with this mouse model was that apparently only nuclear encoded proteins imported into the mitochondria from the cytosol were oxidatively damaged, as exemplified particularly by the Fe–S center enzyme aconitase and nuclear encoded complex II proteins of the electron transport chain. There was little effect on complex I, III and IV activities which require mtDNA encoded proteins for activity and it may be concluded therefore that mtDNA was not significantly oxidatively damaged in Mn SOD (–/–) animals. Our interpretation of these SOD (–/–) animals results is to suggest that such a major disruption to the H2O2 messenger signaling system would be expected to have a catastrophic outcome. Mitochondrially generated H2O2 is required for normal cell function, hence Mn SOD null transgenic mice have no real survival value.

Interventionist manipulation of the metabolome using transgenic mice is fraught with the possible creation of artifactual outcomes of which researchers must be cognisant; this situation applies to the superoxide dismutases. Consider that cytosolic Cu/Zn SOD (SOD 1) gene mutations have been reported by several laboratories to cause a familial form of amyotrophic lateral sclerosis (ALS); so that a protein with a modified activity can cause ALS. However transgenic mice models either null (–/–), or over-expressing cytosolic Cu/Zn SOD do not develop ALS. In studies reported by Jaarsma et al. (2000) transgenic mice constructed to over express cytosolic human SOD 1 were severely affected, the animals developed an array of neurodegenerative changes (but not ALS); the mitochondria of the cells were severely vacuolized. The authors concluded that the control of SOD 1 concentration and its sub-cellular location was essential to normal neuronal development and function. Reaume et al. (1996) reported that transgenic mice null for cytosolic Cu/Zn SOD develop normally showing no adverse effects up to 6 months of age. The enzyme was reported as not being required for motor neuron development but the animals were vulnerable to motor neuron loss when subjected to the stress of surgically induced axonal injury. Subsequently they confirmed their earlier results but reported that SOD 1 is necessary for the maintenance of normal neuromuscular junctions by hindlimb motor neurons (Flood et al. 1999). It must be kept in mind that cytosolic H2O2 is sourced from a number of sub-cellular membranous oxido-reductase systems. Consideration of these studies lead us to suggest that over or under formation of cytosolic Cu/Zn SOD will disrupt the normally physiologically regulated H2O2 concentration, required for normal metabolome function; like any second messenger system its concentrations must be strictly regulated both positively and negatively. We suggest these studies teach little in regard to normal ROS formation and their suggested toxicities. Plasma membrane The plasma membrane oxido-reductase systems continue to expand in number and complexity but

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a common feature is that they all produce superoxide anion and hydrogen peroxide. Macrophage plasma membrane NADPH oxidase is a potent source of very high concentrations of superoxide anion and hydrogen peroxide (mM) employed for the killing of sequestrated bacteria. Neutrophilic myeloperoxidase in the presence of chloride ion acts further to convert H2O2 to the toxic hypochlorite radical which further enhances the organism’s defense system against bacterial infection. The phagocytic oxidase system is tightly regulated and consists of two basic protein components gpaIphox (a FAD flavoprotein cytochrome b558 complex) and p22phox; upon cellular stimulation additional protein subunits denoted p40phox, p47phox, p67phox and rac2 G protein translocate to constitute the active oxidase (for review Werner 2004). This system is complex, in macrophages it is localized on the plasma membrane while in neutrophils it is localized on both the plasma membrane and the phago-lysosomes. Detailed discussion of this system is beyond the scope of this review, suffice to say that over evolutionary time phagocytic immune system cells have evolved to specifically utilize oxygen to generate high concentrations of toxic superoxide anion/hydrogen peroxide in a highly regulated, contained non-injurious manner to the host, as a defense system against microbial infection. Close relatives of the phagocytic NADPH oxidases probably occur in the plasma membranes of all non-phagocytic tissues and in contrast to the cells of the immune system produce H2O2 in the non-toxic nM signaling range. The activities of these NADPH Nox systems, are upregulated by a range of extracellular effectors (growth factors, cytokines and hormones) to produce H2O2 in a manner tailored to respond to the needs of the metabolomes characterizing the various tissues (Werner 2004). Relevant to our considerations as a part of the activation process, is that the G protein Rac I, is recruited to become part of these NADPH oxidase complexes functioning to activate them to produce superoxide anion/hydrogen peroxide required for the down stream activation of Scr protein phosphokinases which precipitate a regulatory protein phosphorylation cascade. The components

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involved and nature of the cascade, varies from one tissue to another and will include, differentially, among others Raf, MEK, MAPK, MAPKK in turn regulating transcription factors Myc, Fos and Jun. On the other hand, as part of the overall processes protein phosphatases under cysteine/ cystine redox cycling function to terminate and regulate the signals as will catalase and glutathione peroxidase. Early in the study of an NADH oxido-reductase system, localized in the plasma membrane and unrelated to the Nox systems, this oxidoreductase was recognized as a coenzyme Q10– flavoprotein–cytochrome b system (Sun et al. 1992). The activity of this system is low unless cells are exposed to growth factors and various hormones. Crane and colleagues proposed that the induced increased activity resulted in the formation of H2O2 which acted as a second messenger for the regulation of cell growth (Brightman et al. 1992; Crane et al. 1994b). More recently this system has been renamed constitutive plasma membrane NADH oxidase (CNOX) to distinguish it from the increasing number of reported Nox systems. One of the other roles of the CNOX system can be readily envisaged as concerned with cellular energy generation. NADH plasma membrane oxidation with oxygen as terminal acceptor, together with the mitochondrial bioenergy system and cytosolic glycolysis interactively contribute to cellular energy maintenance (Lawen et al. 1994). NADH is a major substrate required for mitochondrial energy generation while NAD+ is required to maintain glycolytic ATP production. Lowered mitochondrial respiratory activity due to the aging process or its elimination in respiratory deficient q0 cells is compensated by the up-regulation of the plasma membrane NADH oxidizing systems to maintain NAD+ levels and glycolytic activity (Larm et al. 1994; Kopsidas et al. 2000). This system can also provide other cellular needs for NAD+, for example as required by the sirtuin family of gene regulators. Caloric restriction (CR) has been recognized since the 1930s to increase average rodent life expectancy by as much as 50%; human epidemiological studies of the long living Okinawans suggest that dietary CR can also increase average human life expectancy (Willcox et al.

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2006). It might be anticipated that physiologically CR would result in the down regulation of metabolic pathways due to a less demanding metabolic substrate load. This proposition finds support from the reported up regulation of the sirtuin family of NAD+ dependent histone deacetylases, which function to silence gene expression. Crane and Low (2005) have recently invoked the plasma membrane oxidase systems as sirtuin activity regulators by dint of being major sources of cytosolic NAD+. Again superoxide anion/hydrogen peroxide formation will play a key role in the regulation of such systems and presumably in this instance, gene silencing. Endosome/lysosome/golgi apparatus/secretory granules system Figure 2 modified after Grabe and Oster (2001) is a cartoon summary of sub-cellular pHs and serves to illustrate the various energy consuming and generating proton translocating pumps. Many of the associated oxido-reductase systems have coenzyme Q10 as an active component and give rise to a regulated production of superoxide anion and hydrogen peroxide. Gille and Nohl (2000) have described a lysosomal redox chain. NADH was identified as a substrate for the system with a cytochrome b559, a flavoprotein and coenzyme Q10 as components. Proton translocation into the strongly acidic lumen of the lysosomes through the

agency of coenzyme Q10 was reported. The formation of superoxide anion was also described. The early endosome arises from the plasma membrane and we suggest the proton translocating system which acts to lower the internal pH of this membranous inclusion from about pH 7.4 to pH 6.2 is derived from the plasma membrane oxido-reductases. This pH lowering process continues through to the late endosome (pH 5.3) and the lysosome (pH 5.0). The early Golgi apparatus as it arises from the E.R. has an internal pH of about 6.7 progressing through to 5.4 at the secretory granule interface. The Golgi apparatus has been shown to contain a coenzyme Q10 oxido-reductase (Crane et al. 1994a), which will contribute to the acidification process and again superoxide anion will be formed putatively functioning in a signaling mode. pH gradients and cellular bioenergy Membrane associated electrochemical energy can be calculated from measured membrane potential (Dw) and the pH gradient across the membrane (DpH), whereby the sum of the values is equal to the proton motive force (Dp) generated across the membrane. This relationship is expressed by the following equation as: Dp ¼ Dw þ DpH

Fig. 2 The pH of subcellular organelles and sub-cellular energy. ATP proton pumps. pH values of organelles range from 5.0 to 7.95 (refer Grabe and Aster 2001). Proton motive force (Dp) = membrane potential (DWm) + pH gradient (DpH)

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Cells are in the main comprised of a number of closed sub-cellular membrane systems of individual fluctuating proton motive forces. The immediately localized specific metabolic activity of the sub-cellular organelles will be under the control of these energy fluxes. Fluctuating localized subcellular redox poise will modulate pH values and metabolic sub-cellular micro-environments, concepts summarized in Figs. 1 and 2 and refer Linnane and Eastwood (2004, 2006). The determination of membrane potentials, pH differences and proton motive force is applicable to any closed membrane system such as cells and their different sub-cellular organelles. It can be calculated that an impermeable mitochondrial membrane would have a potential of about 250 mV, but this value is not attained under experimental (or equally physiological) conditions (Nicholls and Ferguson 1992). The membrane potential observed with respiring state 3 mitochondria is variously reported to being in the range of 140–170 mV and influenced (among others) by the extent of proton leak, by inorganic ion concentrations (phosphoryl, Na+, K+, Ca++), the mitochondrial ATPase pump, and substrate movements across the membrane. By way of illustration, under most conditions respiring mitochondria will have a pH difference of about 0.5– 0.7 units between the internal and external milieu equating to about 30 mV with a proton motive force of about 200 mV. The major contribution to proton motive force made by pH differences is well illustrated by consideration of thylakoid function; thylakoids when illuminated have a negligible membrane potential under steady state conditions because of ion movements but have a DpH of about 3.3 units which translates into a proton motive force of about 195 mV (Nicholls and Ferguson 1992). Proton motive force is used metabolically in the regulation of substrate and ion movements across membranes. The mitochondrial electron transport system translocates protons outwards to establish an internal alkaline pH of about 7.95 generating a proton motive force, which can be utilized in a manner similar to the other sub-cellular organelles or coupled to ATP synthesis. It is worth recalling that mitochondria are

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the only organelles, which can export energy (ATP). In summary, sub-cellular energy metabolism depends upon the tightly regulated but continually fluxing compartmentalized distribution of protons; the generation of such proton gradients define the bioenergy status of the whole cell and its sub-cellular compartments (Fig. 2). The redox state of cells is an expression of these proton translocations and as such plays a central role in cellular well being and therefore aging. Future studies are needed which consider the interactions between the various sub-cellular energy packets which comprise the toti-cellular bioenergy state and function. In any event the H2O2 upstream second messenger system, arising from the oxido-reductases activity of the cell’s membranes and the associated proton translocations, play an essential role in the overall regulation of the cell’s bioenergy status, its metabolic subcellular micro-environments and its particular metabolome.

Macromolecule oxidative changes and signaling There is a voluminous literature reporting that ROS damage to cellular macromolecular components by random unregulated oxidation leads to macromolecular dysfunction, thereby contributing to the acceleration of the aging process. We question the hypothesis of random damage to macromolecular species; the process involving oxidative modification of macromolecules is much more complex and subtle in its out workings. Oxidatively modified proteins and regulated proteolysis Protein synthesis and degradation are very complex processes which are in a dynamic equilibrium. Protein degradation and synthesis are exquisitely regulated processes; many proteins have half lives of only a few hours or less while others survive for days and weeks; the temporal turnover of these proteins is part of cellular metabolic regulation.

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Early studies of cellular proteolysis systems were concerned with the cathepsins, a group of proteases with acidic optima of about pH 5 encapsulated within lysosomes. The role of the cathepsins was supposedly to randomly hydrolyze proteins as part of the process of autophagy and endocytosis. Later the ubiquitin proteasome system was discovered and it is now recognized that cellular proteolysis is a regulated multisystem phenomenon which includes the lysosomal cathepsins. The 26S proteasome is made up of a 20S barrel structure capped at each end by two different 19S protein arrays which are involved in the selection of proteins for degradation by the 26S unit. Ubiquitination of a protein is a three-step process (E1, thioester bond formation; E2, transfer to protein sulphydryl group) with the third step catalyzed by ATP dependent ubiquitin protein ligases (E3s). There are hundreds of E3s present in cells, specific to one or more proteins to be ligated to ubiquitin and thus tagged for 26S proteasome destruction. Further, polyubiquitination (4–50 ubiquitin units) is required before final acceptance for hydrolysis by the 26S proteasome. The ubiquitin–ATP dependent system tags proteins for degradation by the proteasome, which is the major proteolysis effector. The proteasome system has a huge endogenic demand for ATP, being composed, in part, of a large family of different ATPases; ATP is required for the presentation of the protein substrate within the complex and for subsequent proteolysis. It is noteworthy that the 20S uncapped proteasome also hydrolyses selected non-ubiquitinated proteins. Recently is has been recognized that ubiquitination is a key signal for targeting membrane intrinsic and extrinsic proteins for endosomal sorting and delivery to the proteolytic interior of the lysosome (for review Urbe 2005). We have considered the commonly held view that proteins are oxidized in an uncontrolled random process by superoxide anion, hydrogen peroxide, nitric oxide and peroxynitrite, thereby contributing directly to the aging process and that such damage, unequivocally commits the damaged proteins to proteasome hydrolysis. We suggest that this concept is no longer tenable, it is at best a gross over simplification.

The oxidation of protein amino acid residues since their discovery some decades ago has been almost universally reported as leading to protein inactivation and requiring mandatory proteolysis to prevent their deleterious cellular accumulation. It is clear that oxidatively modified proteins do not simply arise as the result of random oxidative damage (hydroxylations of various amino acid residues, sulphoxidation of methionines, nitrosylations of sulphydryl groups and so on). There are an increasing number of situations where free radical protein modifications can be shown to be part of normal cellular regulatory signaling activity. To illustrate these points, some examples and comments follow. (a) There is an extensive textbook literature concerning the important nuclear transcription factor NFjB. The activity of NFjB is regulated by superoxide anion formation. NFjB is maintained in the cytosol in an inactive form bound to the inhibitor IjBa. Following plasma membrane superoxide anion and hydrogen peroxide formation induced by a range of cell effectors (e.g. cytokines, hormones) and regulated by Ras 1 (G protein), a transduction phosphorylation acts to phosphorylate IjBa and dissociate the complex leading to IjBa ubiquitination and proteasome destruction. In the process, NFjB is released to translocate to the nucleus and function as a major transcription regulator (Fig. 3). Clearly this one system is multifactorial and complex but it teaches against the ready interpretation of cellular generating superoxide anion systems as being responsible for random protein damage. (b) One of the most sensitive amino acids to oxidation is methionine, being converted to methionine sulfoxide (MetO). This phenomenon is commonly cited as an example of random oxidative damage to proteins. The example below would bring such an overriding conclusion into serious question. Calmodulin (CM) function and its regulation by superoxide anion/hydrogen peroxide oxidation of specific methionine residues is now well-documented (Yin et al. 1999). The oxidation of only two specific methionine residues (Nos. 144, 145) of CM (there are seven) is involved in the process of down regulating plasma membrane Ca++ ATPase. Using genetically engineered CM in which the two methionines (144,

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Biogerontology Fig. 3 An example of superoxide anion and hydrogen peroxide regulation of gene transcription and protein turnover. IKK represents IrBa phosphokinase. NFrB nuclear transcription factor activity is circumscribed by IrBa binding. UBI (ubiquitin)

An example of Superoxide Anion and Hydrogen Peroxide Regulation of Gene Transcription and Protein Turnover. EFFECTOR

OXIDASE a ct i vatio n

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145) were replaced by glutamines, it was shown that oxidation of the remaining methionines did not significantly down regulate CM–plasma membrane–Ca++ ATPase activation (Yin et al. 2000). It has also been reported from the same laboratory (Sun et al. 1999) that methionine sulfoxide reductase can act reductively to restore the ability of oxidized CM to regulate plasma membrane– Ca++ ATPase. These results are interpreted to indicate that superoxide anion/hydrogen peroxide is functioning as part of the controlled regulation of the CM–PM–Ca++ ATPase complex. Further that proteasome degradation of oxidized CM, when and where it occurs, is part of the normal process of regulated protein turnover just as many unoxidized proteins are hydrolyzed by the proteasome system and later resynthesized. (c) The turnover of the hypoxic induced factor (HIFa) and its proteasome degradation is clearly regulated by hydroxylation of its prolyl residues (Stolze et al. 2006). This is an ordered process involving signaling by the free radical system comprised of superoxide anion, nitric oxide and peroxynitrite (see later). (d) Consider the nitrosylation of sulphydryl groups, proposed as a damaging phenomenon. The hemoglobin system is a remarkable regulated machine, finely tuned allosterically for the carriage of the daily massive amounts of inhaled

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oxygen from lungs to cells. It is now textbook recognized that subtle but critical changes are undergone by hemoglobin as result of sequential reactions with dioxygen, protons and CO2 to regulate the delivery of oxygen to the tissues. As part of this process it is relatively recently recognized that NO participates in the regulation (Singel and Stamler 2005). In the cyclic oxygen carriage, NO reacts with the b subunit Fe++, subsequently on b subunit binding of dioxygen the NO is displaced to nitrosylate the cys 93 thiol group of the b subunit. These changes are accompanied by allosteric change from the T (tense) to the R (relaxed) form. The various allosteric changes which Hb undergoes are now recognized as of the utmost importance to Hb function, they are the outcome of over 80 years study and cannot be dealt with here in any detail (the Bohr effect/ Perutz X ray structural studies/the detailed effector allosteric inducers—H+, CO2, O2, NO, 2;3-bisphosphoglycerate/allosteric positive and negative cooperativity changes). Suffice for here, is that the comparatively recently recognized NO nitrosylation of Hb is part of the normal physiological transport of oxygen delivery to tissues; nitrosylation of proteins is not conditionally deleterious. Parenthetically it may be added that superoxide anion continually formed in small amounts during the process oxidizes Hb to met

Biogerontology

Hb in the order of a steady state amount of 1–3%. The met HB formed is itself continually reduced back to Hb by erythrocyte met hemoglobin reductase to maintain regulated oxygen homeostasis. (e) Farout and Friguet (2006) in a recent review consider that there is an age related deleterious accumulation of oxidized proteins resulting from impaired redox homeostasis and proteolysis. Further that changes in proteasome structure with increasing age and dysfunction of the proteasome leads to an exacerbated accumulation of oxidatively modified proteins due to their impaired proteolysis. Somewhat contrary to this interpretation it has been reported when cellular proteasome activity is inhibited, accompanying the resultant decrease in its activity there is an induced increased cellular synthesis of the proteasome, the phenomenon of hormesis (Meiners et al. 2003). Husom et al. (2004) have reported an increase in the 20S proteasome in aged rat skeletal muscle albeit with some change in function. We suggest that the proteasome activity and changes in structure with increasing age might be viewed from a different perspective. The proteasome system makes a major demand on the available cellular ATP and will become increasingly dysfunctional in the absence of sufficient ATP substrate. The reported change in structure and tissue proteasome function may arise with age from a limitation of ATP supply. Central to any consideration of aging is the universally recognized decline in bioenergy capacity with age arising from mtDNA mutations and deletions. Arising from this consideration, declining ATP availability leads to declining proteasome function which may in its outworking contribute to the multisystem aging process albeit not as a primary effector and not as a direct result of oxygen radical damage to proteins. This protein section of the review merits a more detailed in depth analyses of protein oxidative changes but space limitations restrict us. However we suggest that as a widening understanding of the upstream regulation of the superoxide anion/hydrogen peroxide second messenger system is appreciated, it will emerge that they

play a major role in the ordered regulation of proteolysis and protein homeostasis; the process is far from random. For considerations embracing the concept of mischievous oxidative damage to proteins as a major contributor to the aging process consult Stadtman (2004). Mitochondrial DNA A long-standing interest of our laboratory has been mitochondrial DNA changes, bioenergy decline with age and the tissue bioenergy mosaics, which arise there from (Linnane et al. 1989). The single most dominant requirement of any cell is the provision of an adequate energy supply, it is required for every aspect of anabolism and catabolism. Age related decline in bioenergy capacity below a crucial threshold will obviously contribute to cellular malfunction. The development of mitochondrial tissue bioenergy mosaics with age, exemplified by null, low and normal cytochrome oxidase cell content has been stringently correlated at the single cell level by our laboratory, with mtDNA deletions and the individual cell content of full-length functional mtDNA (Nagley et al. 1993; Kovalenko et al. 1998; Kopsidas et al. 2000, 2002; Linnane et al. 2002a). The mtDNA deletion changes reported by us mainly arise by replication error due to the asymmetrical nature of the heavy and light strand synthesis and the very large number of base pair repeats which mismatch during replication and result in mtDNA deletions. Cells whose mitochondrial oxidative phosphorylation function approaches zero due to severe mtDNA changes are lost from the tissue by apoptosis. The phenomenon of increasing age is fundamentally cell loss from post-mitotic tissue and mitochondrial bioenergy dysfunction plays a major role in this process. It is to be emphasized that cell loss in humans is a slow process occurring over decades, thus skeletal muscle cell loss (sarcopenia) occurs at the rate of about 5% per decade beginning to be readily recognized from about 50 years onwards. Mitochondria are now recognized to have extensive DNA repair systems for excision of oxidized bases but the large number of mtDNA deletions reported are not repairable. The cellular

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amount of mtDNA is under nuclear genome control so that if cells contain some full-length unmodified mtDNA it can be amplified back to normal levels, the various mtDNA deletions are eliminated and the cells rescued (Kopsidas et al. 2000). Cell loss occurs when little or no full-length mtDNA remains and therefore they become nonrescuable. The occurrence of a range of deletions and single base point mutations with age has been extensively reported by many laboratories (Wallace et al. 1995). However we pose the question; what role do superoxide anion and hydrogen peroxide play in inducing irreparable mtDNA dysfunction? Numerous early reports suggested that ROS damage was a major contributor to increasing nuclear and more particularly mtDNA dysfunction. From such reports an error catastrophe hypothesis was proposed, whereby a vicious cycle of increasing oxidative damage to the mtDNA led to increased further oxidative damage and escalating mitochondrial bioenergy dysfunction. There is no convincing evidence in support of such a view (Mansouri et al. 2006; Trifunovic et al. 2004, 2005). Urinary 8-oxodeoxyguanosine was used as the exemplifier of age associated nuclear and mtDNA damage. However one of the major laboratories supporting the error catastrophe hypothesis subsequently reported that urinary measurements of 8-oxodeoxyguanosine, as an estimation of increasing mitochondrial and nuclear DNA damage was unreliable and fraught with technical error and various confounding factors leading to gross over estimation of DNA damage by oxidation and uncertainty as to its interpretation (Helbock et al. 1998). This dysfunctional formation of superoxide anion as a major cause of mtDNA damage and its contribution to this aspect of the aging process has yet to be demonstrated. Indeed, recently Trifunovic et al. (2004, 2005) in a series of elegant experiments have reported that transgenic mice expressing an error prone mtDNA polymerase accumulate substantially increased levels of mtDNA deletions and point mutations which are strongly correlated with a reduced life span. Particularly significant was the observation that the mice developed a wide range of age associated disease phenotypes. However they did not

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observe any increase above normal low levels of putative ROS induced products by such animals and indeed fibroblasts prepared from such mice showed no increase in protein carbonylation levels or antioxidant defence enzymes. Also no increase in aconitase inactivation was observed; a measurement which has been used in constructed systems to putatively exemplify mitochondrially induced oxidative damage. These authors in agreement with our earlier human mtDNA studies conclude that oxidative phosphorylation dysfunction per se arising from the replicatingly damaged mtDNA (and not oxidatively damaged mtDNA) is the primary inducer of the premature aging in the mtDNA mutator mice and most particularly in a range of diseases normally associated with the aging process.

Redox regulation of cellular metabolism and differentiation The work of Smith et al. (2000) on rat glial oligodendrocytes progenitor cell differentiation teaches that cellular redox poise regulates the process (Fig. 4, a reductionist summary). These authors reported that when rat glial oligodendrocyte/astrocyte progenitor cells were grown under conditions to establish a more oxidizing or reducing intracellular redox state, the more oxidizing cytoplasmic environment favored cell differentiation to oligodendrocyte or astrocyte formation. By contrast, a more reducing cytoplasmic environment favored the maintenance of the progenitor cells. A particularly important aspect of these studies is that a range of naturally occurring physiological regulators function, at least in part, by modulating the redox state of cells and consequently their metabolomes. Thus thyroid hormone and bone morphogenic protein 4 induced a more oxidizing cytoplasmic redox poise environment while basic fibroblast growth factor exposure and platelet derived growth factor exposure induce a more reducing environment. Different admixtures of hormones, growth factors and chemicals, favoring oxidation or reduction were used to manipulate cytoplasmic redox poise. The cell cultures were induced to predominantly

Biogerontology Fig. 4 Redox state modulation of cell differentiation

Redox State Modulation of Cell Differentiation Bi-potential Rat Glial O-2A Cell Progenitor of Oligodendrocytes or Type 2 Astrocytes A f te r S m i t h e t a l . P N A S 9 7 : 1 0 0 3 2 ( 2 0 0 0 )

Chemically Defined Medium Intracellular Redox Modulation

Plus Effectors

Favoured Cell Type Formed

Rosamine fluorescence

Thyroid hormone Bone morphogenic protein 4 Basic fibroblast growth factor Platelet derived growth factor PDGF + bFGF PDGF + N acetyl cysteine PDGF + t-Butyl hydroperoxide

Reducing

Oxidizing

----+ + ++ ++ ---

+ + --------++

Oligodendrocytes Astrocytes Progenitor O-2A cells Progenitor O-2A cells 100% progenitor O-2A cells Increased progenitor O-2A cells Increased oligodendrocytes

Relatively small redox changes have profound outcomes

Superoxide Anion Induces Mesenchymal Differentiation to Osteogenic Cells. Superoxide Mesenchymal stem cells

Anion

Osteogenic Differentiation

Cascade of Rac (GTPases), tyrosine phosphokinase signaling, VEGF expression (vascular endothelial growth factor), others Wang et al (2002, 2004).

self-replicate or differentiate, or produce mixed cell types, emphasizing the interactive modulating behavior of effectors to produce a particular outcome. The use of simple chemical oxidants such as tert-butyl hydroperoxide or reductants such as N-acetyl cysteine to manipulate cellular differentiation pathways serves to emphasize the fundamental importance of redox modulation. Finkel and colleagues (Sundaresan et al. 1995) have reported that the growth response of rat vascular smooth muscle cells to PDGF was dependent upon H2O2 formation. There is now an extensive literature which describes redox regulation of cellular differentiation which is beyond the scope of this review. However another selected example is the work of Wang et al. (2002, 2004) concerned with superoxide anion regulation of cell differentiation. These authors have reported that superoxide anion can promote osteogenic differentiation of mesenchymal stem cells using animal and cell models (Fig. 4). Again the cascade signaling systems are complex but similar to those earlier described, encompassing the regulated induction of Rac, ERK’s (extracellular signal regulated kinases), VEGF (vascular endothelial growth factor) and tyrosine phosphokinase signaling. In reality the full mechanistic details are not fully known but the tip of an iceberg involving superoxide anion/hydrogen peroxide participation in

the regulation of the toti-cellular redox state and cell differentiation is clearly perceived.

Janus kinases-signal transducers and activators of transcription (Jak/Stat signaling pathway) We have earlier discussed the activation of the transcription factor NFjB by H2O2 as involving regulated proteolysis and subsequent transcription activation. It may be envisaged that the superoxide anion/hydrogen peroxide system is a universal messenger regulator of transcription acting under the direction of all manner of cell effectors. Consider another major multifactorial transcription control system, the Jak/Stat system (for early review Darnell 1998). Figure 5 constitutes a reductionist summary of the system’s overall properties and the role of superoxide anion/hydrogen peroxide in its regulation. Clearly the plasma membrane oxidases produce the superoxide anion/hydrogen peroxide second messengers in response to a wide range of effectors (hormones, cytokines, lymphokines, growth factors) with consequently different metabolic and cellular outcomes. The signaling process must have some cell specificity and be exquisitely regulated and localized to avoid metabolic chaos, which could arise from a range of conflicting cellular signaling instructions.

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Biogerontology Fig. 5 Janus kinasessignal transduction and activators of transcription (Jak/Stat system)

Janus Kinases-Signal Transduction and Activators of Transcription (Jak / Stat System) (N um e r o u s P a p e r s 1 9 95 - 20 06 )

A ct i v at i o n:

Cytokines (INF’s, IL’s), Growth Factors, Angiotensin II, plasma Membrane receptor interactions, leading to activation of NAD (P)H Oxidase to produce superoxide anion (H2O2). H2O2 activates Jak / Stat system to regulate a wide range of transcription products.

Rat Vascular Smooth Muscle Cells + PDGF H2 O2

Jak / Stat Activation Catalase Transfection

Sundaresan et al. (1995)

Gene Transcription

(and high extracellular catalase)

Signalling and Evolution: Some 4 known mammalian Jak’s and 5 plus Stat’s. They do not occur in yeast, C. elegans or Dictyostellium and only one Jak occurs in Drosophila. • Da rn ell (19 9 8),

The Jak/Stat system is of particular interest as it makes the point that biochemical signaling systems have evolved over evolutionary time; it does not occur in yeast, C. elegans or Dictyostellium and only in rudimentary form in Drosophila. Yeasts, C. elegans and Drosophila are extensively used by some laboratories as models for the study of aging. We have long contended that their gene content can only relate to house keeping molecular cell events and are of limited use in their application to human aging studies, notably in regard to the roles of superoxide anion and hydrogen peroxide. There is an over concentration in the literature of the toxic (pathophysiological) effects of superoxide anion/hydrogen peroxide, rather we suggest it should be considered that this is the out workings of metabolic imbalance which may be induced by cell effectors. Such imbalance arises from dysfunctional hormone, growth factor, cytokine signaling among others. Clearly a plethora of disease states can arise from primary imbalances in cell signaling and does not conditionally arise as a consequence of induced macromolecular damage by superoxide anion/hydrogen peroxide. Figure 6 is a cartoon overview summary of superoxide anion/hydrogen peroxide acting in their capacity as an overall cellular second messenger system. It summarizes their cellular production

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S ch i e f fe r e t a l . (2 0 0 0 ) ,

G ro t e e t a l . ( 20 0 5 ).

sites and their roles in mitogen activated phosphokinase activations, protein turnover, subcellular metabolic redox modulation, mitochondrial and nuclear gene regulation and extracellular effector activations of the superoxide anion/ hydrogen peroxide second messenger system.

The cellular responses to nitric oxide and peroxynitrite In this section we very briefly turn our attention to the activities of the free radical gas nitric oxide and peroxynitrite (OONO–) anion (this section warrants expansion on another occasion). Since the comparatively recent recognition of NO as the endothelial derived relaxing factor, it has found ready acceptance of its requirement for normal physiological function. However as a free radical gas it has all the potential to induce undesirable deleterious effects when produced in excessive amounts but it does not carry the same negative stigma associated with the superoxide anion/ hydrogen peroxide system. The beneficial regulatory roles of NO have been exhaustively reviewed and our understanding of its roles will continue to increase over coming years (for review Guix et al. 2005). It is not the purpose of this review to catalogue them but more to explore some of its

Biogerontology Fig. 6 Overview: superoxide anion/ hydrogen peroxide: redox regulation of gene expression and cellular metabolism. The cartoon summarizes an overview of the role of ROS in activating plasma membrane Nox, CNOX oxidases and the subsequent initiation of intracellular Jak-Stat signaling cascades. Janus kinases (Jak)-signal transducers and activators of transcription (Stat), Tyrosine kinase (TK), Mitogen activated phosphokinase (MAPK)

currently considered toxic side effects. Nonetheless we briefly remark on some of its key regulatory roles particularly in regard to the modulation of oxygen utilization and hence superoxide anion and hydrogen peroxide formation.

Nitric oxide sites of formation Nitric oxide is a free radical gas that is produced by many different cell types in a variety of circumstances and having a diverse range of effects on biological systems. NO is synthesized from arginine by the nitric oxide synthase (NOS) family that include neuronal NOS (nNOS), endothelial NOS (eNOS) [both neuronal and endothelial sub families are also known as constitutive NOS (cNOS)], inducible NOS (iNOS) and mitochondrial NOS (mtNOS). The activity of the NOS enzymes in eukaryotic cells is highly regulated.

Nitric oxide functions and energy regulation Nitric oxide can be a cytotoxic molecular species when produced by phagocytic cells at micromolar concentrations for the regulated destruction of engulfed organisms similarly to superoxide anion formation (for review Arzumanian et al. 2003).

Nitric oxide can also act as an intercellular and intracellular signaling molecule functioning at nanomolar concentrations. Figure 7 constitutes a reductionist overview summary of NO regulatory functions and its interaction with superoxide anion. An essential role of the NO molecule was recently demonstrated in vitro and in vivo, where the production of NO coupled to cGMP formation controls the biogenesis of mitochondria and hence impacts strongly on cellular energy balance (Nisoli et al. 2003, 2004). Nisoli and colleagues reported that NO/cGMP-dependent mitochondrial biogenesis was associated with increased mitochondrial function and enhanced oxidative phosphorylation activity. Reduced mitochondrial biogenesis in animal tissues has been associated with a decrease in energy capacity and increased body weight (Momken et al. 2002; Nisoli et al. 2003). The production of mitochondrial NO to maintain the requisite cellular content of mitochondria, constitutes an important regulatory mechanism for normal cellular function. The up/ down regulation of mitochondrial oxidative activity by NO will have the effect of regulation of coenzyme Q10 activity and formation of superoxide anion/hydrogen peroxide to modulate metabolome function. The obverse side to the stimulation of mitochondrial biogenesis is the down regulation of

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Biogerontology Fig. 7 Overview of physiologically normal radical regulatory functions and interactions. Summary of the normal and interactive activities of Superoxide anion and Nitric oxide.

Overview of Physiologically Normal Radical Regulatory Functions and Interactions

_ O2•

H2O2

Organellar Proton Motive Force Energy Metabolism Organelle and Cellular Redox Regulation Protein Turn Over Calmodulin PM Ca++ ATPase Regulation Multi System Modulations Neutrophil / Macrophage Activation

NO •

NO •

(nM concentrations)

(µM concentrations)

Control Cellular Energy M it o ch o nd r i a l P rol i fe rat i on Hemoglobin O2 Transport Regulation Phagocytic Cell Function (µM) Various Physiological Processes (vascular tone, others)

OONOPeroxynitrite Radical

mitochondrial activity as a consequence of mtNOS binding to cytochrome oxidase and its localized specific production of NO functioning to inhibit cytochrome oxidase. Arising from this inhibition, downward metabolic pressure is placed on proton motive force and energy generation (Moncada and Erusalimsky 2002; Celmeti and Nisoli 2005; Persichini et al. 2005). The regulation of mitochondrial proton motive force is an important downstream feedback switch that contributes to balancing the energy metabolism and redox poise of the cell under normal physiological conditions. It is plausible to hypothesize that the cell, through the regulation of NO production modulates the activity of not only the mitochondrial electron transport chain but also by interaction with other sub-cellular oxidoreductases in the cell. Parenthetically superoxide anion has been reported to activate the mitochondrial uncoupler proteins, a system which also requires coenzyme Q10 as a cofactor (Echtay et al. 2000, 2002); all these NO, superoxide anion phenomena affecting energy metabolism adds up to a complex regulatory scene (Figs. 1, 7).

Peroxynitrite signaling Peroxynitrite has long been considered to be a highly reactive toxic substance; again this conclu-

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HIF 1 , hypoxia regulation ER Ca++ ATPase, Ca++ regulation

sion is an over simplification. Consider a further aspect of NO binding to cytochrome oxidase, it will lead to a change to increased cellular dependence on glycolysis and the up-regulation of the levels of the hypoxia-inducible factor 1a (HIF-1a) required for adaptation to hypoxic conditions (Almeida et al. 2005). There are a multiplicity of consequences arising from NO direct and indirect effects on the mitochondrial electron transport system. The hypoxia inducible factor is a multiprotein transcription complex and is under strict redox sensing regulation, playing a pivotal role in modulating cellular responses to changes in oxygen concentrations (Semenza 2004). HIF-1a rapidly accumulates in cells exposed to hypoxia (Huang et al. 1996, 1998) and is activated by mitochondrial superoxide anion/hydrogen peroxide (Bell et al. 2005); it is degraded by the proteasome during normoxia following its ubiquitination (Kallio et al. 1999). HIF-1a is polyubiquinated by a multifactorial E3 ubiquitin ligase complex and subsequently degraded by the proteasome. As part of this process, the prolyl residues of HIF-1a are first hydroxylated by a set of non-heme Fe++-2 oxoglutarate dependent dioxygenases which are required for reaction of HIF-1a with its specific multicomponent E3 ligase (Stolze et al. 2006). Sumbayev and Yasinska (2006) have recently proposed that peroxynitrite, formed by the reaction of NO with superoxide

Biogerontology

anion, can serve as the oxygen donor required by the prolyl hydroxylases for the hydroxylation of HIF-1a. The proposed role of peroxynitrite in the ordered regulation of the oxygen sensing system and directed proteolysis again teaches the essential beneficial physiological role played by free radical prooxidants. Cohen and colleagues, in a series of important papers have demonstrated that peroxynitrite, is required for the activation of sarcoplasmic/ endoplasmic reticulum calcium ATPase (SERCA) (for overview Cohen and Adachi 2006). The activation requires a source of peroxynitrite and glutathione for glutathiolation of SERCA cysteine-674; the consequential activated uptake of Ca++ by the sarcoplasmic reticulum acts to lower cytosolic Ca++ and results in the relaxation of vascular smooth muscle. This process was formerly considered to occur as a result of NO stimulation. It now becomes a phenomenon of regulated superoxide anion and NO production and their interaction to form peroxynitrite; it also involves the recruited participation of another major player in redox regulation, glutathione. As an aside it may be recalled that GSH/GSSG is overwhelmingly considered to be required as an antioxidant defence system; here it is cooperatively functioning in the regulation (not rescue) of a key cellular oxidative process. Cohen and Adachi (2006) invoke over production of upstream superoxide anion as a major contributor to the development of atherosclerosis. In support of this hypothesis, cysteine-674 was shown to be more oxidized in animal atherosclerotic tissue compared to normal tissue. At the crux of this proposal, which is not addressed, is that not all animals (humans) develop clinically expressed atherosclerosis. Aging is a multifactorial stochastic process; what is the causation of the postulated unregulated excessive production of superoxide anion? We return again to the indisputable situation that uncontrolled under or over production of any regulatory molecular species (hormones, growth factors, proto oncogene expression, etc.) will lead to pathology. However it does not teach that these molecules function inherently as toxic substances and this includes superoxide anion and NO.

The chimera of antioxidant therapy The theme running through this review is that a random antioxidant scavenging of superoxide anion (H2O2) and NO (peroxynitrite) would catastrophically derange their second messenger function which is essential for the regulation of the metabolome’s activities. The chimera of antioxidant therapy we have briefly considered elsewhere (Linnane and Eastwood 2004). There can be little question that excess continued over production of superoxide anion and nitric oxide (or any other metabolic regulator), where and if it occurs would have deleterious effects both on their roles as second messengers and putatively inappropriately oxidatively damaging macromolecular systems. Mammals have an array of apparent enzymatic antioxidant systems and small molecules for scavenging over production of superoxide anion and nitric oxide. These systems included among which are the superoxide dismutases, the glutathione/glutathione disulphide system, glutathione peroxidase and catalase together with the vitamins C and E etc. have been largely interpreted by others as defence systems acting to prevent superoxide anion and its products from causing inappropriate oxidative damage. As earlier discussed we posit that the superoxide dismutases, the glutathione system and catalase are part of the process of normal redox regulation of cells and that their function is not to randomly scavenge prooxidant molecules. An industry has grown on the proposition that it is essential to prevent oxidatively induced changes which are treatable by administration of small molecule antioxidants (which it must be remembered are also prooxidants) for amelioration of the aging process and treatment of age associated diseases. There is no compelling evidence from human clinical to support the claims that the ingestion of small molecule antioxidants such as vitamins C, E, b-carotene and others prevent/ameliorate the development of age associated human diseases presumptively arising from random oxidative damage to cellular systems. Antioxidant therapy has been promoted for many years for the prevention and treatment of cancers based on non-physiological in vitro studies (Ames et al.

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1993). Bjelakovic et al. (2004) have reported a meta-analysis of a series of antioxidant therapy studies (over 170,000 participants) and found no benefit for the treatment/prevention of gastrointestinal cancers or any effect on participant mortality. These studies included the administration of tocopherol, ascorbic acid, selenium and bcarotene in various combinations (for discussion Linnane et al. 2007). Recently Bjelakovic et al. (2007) have updated their earlier study with an all cause meta-analysis of the effect of antioxidants on an even larger number of 232,606 participants being reported upon. They conclude that treatment with b-carotene, vitamin A and vitamin E may increase mortality. The potential roles of vitamin C and selenium on mortality remains an open question.

Ascorbic acid Vitamin C has long been promoted as an outstanding antioxidant and of benefit in the prevention/amelioration of age associated diseases proposedly arising from oxygen radical damage. There is no doubt that vitamin C is an essential nutritional supplement required for normal mammalian function but it has yet to demonstrated that it has any role as a meaningful therapeutic antioxidant. Ascorbic acid plays an essential coenzyme oxido-reductase role in the hydroxylations of procollagen (procollagen trimer formation and release from the ER), dopamine (to give rise to norepinephrine) and hypoxia inducible factor (regulation). Ascorbate occurs in high concentration in the adrenal and pituitary glands but it is not evenly distributed throughout mammalian tissues, its occurrence is low in tissues such as skeletal muscle, testes, thyroid and lung (Hornig 1975), so that it would not constitute a general tissue antioxidant, if that were its proposed therapeutic role. Recently Bailey et al. (2006) have reported that administered ascorbate promotes oxidative damage during surgical ischemia reperfusion. There is a growing literature not considered here concerning the comparatively recently identified function of ascorbic acid as a differential

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inhibitor of bacterial hyaluronate lyase (spreading factor) acting thereby to limit bacterial tissue invasion (Li et al. 2001). In strong support of this finding, Gaut et al. (2006) using transgenic mice unable to synthesize ascorbic acid (null for Lgulono-c-lactone oxidase) have reported that ascorbate supplementation protected the animals from the lethality of Klebsiella pneumoniae infection; 7 days post-infection there was a 70% survival of the mice cohort supplemented with ascorbate versus less than 10% survival of animals unsupplemented with ascorbate. No differences were observed between ascorbate supplemented and unsupplemented animals in regard to conventional oxidative stress markers (isoprostanes and chloro, bromo, nitro tyrosine formation), that is no antioxidant effect. Large bolus intravenous infusions of ascorbate (10 g plus /day) have been promoted by some complementary medicine practitioners for the treatment of cancers, the data in support of an efficacious outcome is equivocal. The rationale for this therapy has been the antioxidant function of ascorbate based on the consideration that superoxide anion/hydrogen peroxide can function to induce cancer by random oxidation attack on cellular macromolecules. In a series of recent studies by Levine and associates they have revisited the use of high doses of ascorbate as a cancer therapy; their findings are largely summarized in Chen et al. (2005). In one earlier study they reported that increasingly large oral doses of ascorbic acid do not lead to increasing levels of plasma ascorbate; the system is saturable and plasma levels plateau at less than 0.3 mM even on oral doses of 10 g/day. However I.V. doses of 10 g /day result in plasma levels of about 6 mM. This paper reports the important discovery that ascorbic acid at a concentration of about 5 mM functions in vitro to selectively kill a variety of cancer cell lines. They demonstrate that under appropriate conditions ascorbate at a concentration of about 5 mM in the presence of serum acts as a prodrug promoting the formation of high concentrations of H2O2 which are lethal to a range of human and mouse cancer cell lines. These same high levels of generated H2O2 had apparently no effect on the growth of normal human cell lines. The equivocal earlier findings by

Biogerontology

others of a beneficial effect of I.V. ascorbate on cancers may possibly be explained by the observation that not all cancer cell lines are killed by ascorbate (Chen et al. 2005). However the report of Chen et al. (2005) may be a particularly significant paper. These authors convincingly argue the case for blinded human clinical trials to be conducted on the possible efficacy of I.V. ascorbate on a range of human cancers. For the purposes of this review we emphasize that there is no convincing evidence for ascorbic acid acting beneficially in mammals as an antioxidant. On the contrary in large doses it may act as a prodrug for the production and delivery of H2O2 to tissues, beneficially for the treatment of some cancers. In conclusion, we suggest that antioxidant therapies as promoted for use, to the general public, should come under serious question as to their efficacy. The lack of well constructed blinded human clinical trials, designed to unequivocally demonstrate a health benefit from antioxidant therapy is a yawning gap in the field. Albeit some topically applied antioxidants may have a useful role in the prevention of skin cell damage induced by environmental factors such as over exposure to sunlight and U.V.

Reprise This paper has attempted to integrate a disperse range of results culled from studies of major metabolic processes to provide an overarching view of the effect of superoxide anion/hydrogen peroxide and nitric oxide/peroxynitrite on cell physiology (Fig. 6). Its emphasis has been to present an alternate interpretation to many widely accepted views on the physiological significance of superoxide anion (and nitric oxide) formation and their putative toxic effects. For some our analyses will border on an indictable heresy. On the contrary clearly the superoxide anion/ hydrogen peroxide and nitric oxide/peroxynitrite are second messengers which are highly regulated and essential to normal cell function and therefore healthy aging. They do not under normal physiological conditions cause indiscriminate ran-

dom damage to the metabolome. The presumed unregulated toxic effects of the free radicals have been comprehensively over emphasized and misinterpreted. We suggest that the so-called rescue from oxidative stress by enzymatic systems such as glutathione peroxidase and catalase is but one aspect of H2O2 second messenger regulation and obversely the superoxide dismutases are part of that regulation. One of the corner stones of the concept embracing oxidative damage as a random undesirable phenomenon concerns oxidative protein modifications. All cellular processes are positively and negatively regulated; often this regulation is multistep and therefore complex and not readily perceived. Protein turnover is biochemically very complex but it is crucial to protein homeostasis; we have cited regulatory multistep examples of free radical modification of proteins which are metabolic activation/de-activation signaling processes for proteolytic removal of the proteins and do not represent unregulated random damage to proteins. Most studies in considering the bioenergy status of cells almost exclusively focus on mitochondria, which leads inturn to a misrepresentation of mitochondria as the main source of superoxide anion. Most importantly this approach ignores the considerable contribution of other organellar oxido-reductase systems to cellular bioenergy generation and function, as discussed in terms of fluctuating proton motive force and its regulation of the metabolic microenvironments of the cell. The role of the plasma membrane oxidoreductases (Nox and CNOX) in the generation of superoxide anion/hydrogen peroxide in response to a range of extra cellular effectors raises the important question as to how cells differentiate complex multieffector inputs and regulate the response. It may be envisaged that individual receptor systems produce different specific concentrations of H2O2 which differentially activate particular enzymes according to their H2O2 affinities. Thus the major NFjB and Jak-Stat transcription systems are regulated by a similar range of extracellular effectors but presumably are not simply simultaneously up-regulated in a cell; a differential response to localized particular

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concentrations of H2O2 is a likely possibility. In any event the plasma membrane is a complex multifunctional organelle intimately concerned with the complexities of the regulation of the metabolome of the cell. There is nothing more central to the aging process and biological function than its energy supply systems, the generation of exportable energy (ATP) is largely the role of the mitochondrial oxidative phosphorylation system (glycolysis being the only other significant source of net ATP synthesis). We have briefly revisited some of our earlier extensive studies concerning cellular mtDNA mosaic deletions/mutations and again assert that in their outworkings, replicative mtDNA errors and mutations reduce cellular ATP synthesis and contribute in a major way to the aging process and post-mitotic cell loss from tissues. Evidence is advanced to support the view that the oxidation of mtDNA is a minor process; it is subject to repair and that the mtDNA oxidative catastrophe hypothesis is invalid. The normally functioning metabolome is the expression of a finely tuned dynamic equilibrium comprised of thousands of anabolic and catabolic reactions and all cellular signaling systems must be finely regulated. However no biological system constitutes a perfect machine and clearly the aging process which is multisystem and stochastic is an expression of developing wear and tear malfunction with repair finally unable to maintain function and eventually death results. That there is a small inappropriate leakage of free radicals to cause random deleterious damage can be considered as a possible contributor to the multisystem aging process but we posit that it is not a major overwhelming player. The age associated life taking systemic diseases only begin to be readily recognized from about 50 years of age and increase in frequency over the next 3–4 decades with average life expectancy now being about 80 years in first world countries. The multisystem stochastic aging process will be impacted upon by the specifics of an individual’s gene pool and by an array of intrinsic and external factors, such as various metabolic imbalances, a subject’s nutritional milieu, hormonal balance, immune system function, protein glycations, exercise, smoking/

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inspired air quality among others. Given this situation, a plethora of causes are involved. There are no blinded human clinical trials establishing excessive systemic over production of ROS as the primary cause of the aging process and more particularly its commonly associated systemic diseases. Any regulated second messenger system, which becomes dysfunctional as a result of loss of regulation will give rise to physiological dysfunction; recall the loss of upward or downward control of any endocrine messenger system and the complex pathologies which result. In this scenario, two clinical scenarios illustrate extreme malfunction of the superoxide anion/NO systems which are often cited in support of the free radical damage theory. Thus septic shock which affects a small number of individuals arises from phagocytic cells over producing the radicals consequent on a microbial infection that leads to an unrelenting and life taking arterial hypotension unless the causative infection is resolved. Conversely chronic granulomatous disease, a rare immuno-deficiency disease arising from a phox 67 mutation (Leusen et al. 1996) is characterized by the inability of neutrophils to adequately respond to microbial infection; the cells under produce superoxide anion/nitric oxide and most subjects die from overwhelming infections at an early age. However these conditions and their outworkings are due to external trauma, the infection load and the individuals phenotypes. They are not relevant to normal aging and the systemic diseases, which develop during the normal aging process and normal systemic superoxide anion and hydrogen peroxide formation.

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