Which Is the Most Significant Cause of Aging?

It becomes clearer and clearer that aging is a result of a significant number of causes and it would seem that counteracting one or several of them should not make a significant difference. Taken at face value, this suggests, for example, that free radicals and reactive oxygen species do not play a significant role in aging and that the lifespan of organisms cannot be significantly extended. In this review, I point to the fact that the causes of aging synergize with each other and discuss the implications involved. One implication is that when two or more synergizing causes increase over time, the result of their action increases dramatically; I discuss a simple model demonstrating this. It is reasonable to conclude that this might explain the acceleration of aging and mortality with age. In this regard, the analysis of results and mortality patterns described in studies involving yeasts and Drosophila provides support for this view. Since the causes of aging are synergizing, it is also concluded that none of them is the major one but many including free radicals, etc. play significant roles. It follows that health/lifespan might be significantly extended if we eliminate or even attenuate the increase of a few or even just one of the causes of aging. While the synergism between the causes of aging is the main topic of this review, several related matters are briefly discussed as well.

Reflections on the Theories of Aging, of Oxidative Stress, and of Science in General. Is It Time to Abandon the Free Radical (Oxidative Stress) Theory of Aging?

SIGNIFICANCE: Aging and oxidative stress are complex phenomena, and their understanding is of enormous theoretical and practical significance. RECENT ADVANCES: Numerous hypotheses and theories that attempt to explain these phenomena have been developed. These hypotheses and theories compete with each other, with each claiming to be the correct one, while significantly contradicting each other. CRITICAL ISSUES: It is important to develop a maximally correct theory that may then trigger significant practical breakthroughs. FUTURE DIRECTIONS: None of these theories is entirely correct or close enough to the truth. However, most of them contain many correct elements (CE). Finding these CE is possible by analysis of these theories. Once the CE are found, they can be merged by synthesis in a better new theory. An analysis of some of the theories of aging followed by synthesis is attempted.

Free radicals: how do we stand them? Anaerobic and aerobic free radical (chain) reactions involved in the use of fluorogenic probes and in biological systems.

Biologically significant conclusions have been based on the use of fluorogenic and luminogenic probes for the detection of reactive species. The basic mechanisms of the processes involved have not been satisfactorily elucidated. In the present work, the mechanism of the enzyme and photosensitized oxidation of NAD(P)H by resorufin is analyzed and appears to involve both aerobic and anaerobic free radical chain reactions. There are two major fallouts of this analysis. Many of the conclusions about the participation of radicals based on the use of probes such as resorufin and Amplex red need reevaluation. It is also concluded that anaerobic free radical reactions may be biologically significant, and the possible existence of enzymatic systems to eliminate certain free radicals is discussed.

Superoxide dismutase mimics, other mimics, antioxidants, prooxidants, and related matters.

A significant number of low molecular weight metal complexes as well as metal-free compounds that are capable of scavenging superoxide and/or other radicals and reactive species in simple systems have been proposed to be used as potential drugs in the case of various diseases and/or as antiaging agents. Some have been used or suggested to be used as diagnostic tools for the involvement of such species in biological processes. In the present work, analysis of such claims indicates that their use as specific detectors of superoxide or of other reactive oxygen species is unsupported and might be confusing. Many of these compounds exert beneficial effects by counteracting the toxic effects of oxidative stress in a significant number of models of pathological processes. However, it is concluded that these actions are more likely due to other effects including prooxidant actions and that their beneficial effects also may be exerted in pathological processes that do not practically involve reactive oxygen species. Adaptation may be a common mode of action explaining a sizable portion of the beneficial effect of the so-called mimics and other compounds including prooxidants.

Free radical paradoxes.

Unlike bigger and more advanced animals, Caenorhabditis elegans does not generate NO, yet it was recently found that NO produced by chemical or biological sources exerts profound effects in that animal, leading to increased life span and thermotolerance. The biological source was Bacillus subtilis, a natural food for C. elegans. Yet once in the cell, NO might react with superoxide, leading to the production of the potentially toxic peroxynitrite. In this paper, a number of paradoxes that are involved in that situation are discussed. It is also argued that their solution might lead to a sizeable advancement of our knowledge of what constitutes oxidative stress and what role oxidative stress plays in the development of pathological processes and aging.

Reactive oxygen species and the free radical theory of aging.

The traditional view in the field of free radical biology is that free radicals and reactive oxygen species (ROS) are toxic, mostly owing to direct damage of sensitive and biologically significant targets, and are thus a major cause of oxidative stress; that complex enzymatic and nonenzymatic systems act in concert to counteract this toxicity; and that a major protective role is played by the phenomenon of adaptation. Another part of the traditional view is that the process of aging is at least partly due to accumulated damage done by these harmful species. However, recent workers in this and in related fields are exploring the view that superoxide radical and reactive oxygen species exert beneficial effects. Thus, such ROS are viewed as involved in cellular regulation by acting as (redox) signals, and their harmful effects are seen mostly as a result of compromised signaling, rather than due to direct damage to sensitive targets. According to some followers of this view, ROS such as hydrogen peroxide and superoxide are not just causative agents of aging but may also be agents that increase the life span by acting, for example, as prosurvival signals. The goal of this review is to recall that many of the effects of ROS that are interpreted as beneficial may actually represent adaptations to toxicity and that some of the most extravagant recent claims may be due to misinterpretation, oversimplification, and ignoring the wealth of knowledge supporting the traditional view. Whether it is time to abandon the free radical (oxidative stress) theory of aging is considered.

Mechanism of the peroxidase activity of Cu, Zn superoxide dismutase.

In addition to its very efficient catalysis of the dismutation of superoxide ( O(2)(-) ) into O(2) plus H(2)O(2), Cu, Zn SOD acts less efficiently as a non-specific peroxidase. This peroxidase activity is CO(2) dependent although very slow peroxidation of some substrates occurs in the absence of CO2. The mechanism of that CO(2) dependence is explained by the generation of a strong oxidant at the copper site by two sequential reactions with H(2)O(2), followed by the oxidation of CO(2) to the carbonate radical that then diffuses into the bulk solution. This diffusible carbonate radical is then responsible for the diverse oxidations that have been reported. A different mechanism that involves the reduction of peroxymonocarbonate by the reduced superoxide dismutase to yield carbonate radical has been proposed. We will demonstrate that this mechanism is not supported by the available data. It seems likely that generation of the carbonate radical has relevance to the oxidative stress faced by aerobic organisms.

The effects of superoxide dismutase on H2O2 formation.

Numerous reports of the effects of overproduction of SODs have been explained on the basis of increased H2O2 production by the catalyzed dismutation of O2-. In this review we consider the effects of increasing [SOD] on H2O2 formation and question this explanation.

Inactivation of copper, zinc superoxide dismutase by H2O2 : mechanism of protection.

Cu,Zn SOD is known to be inactivated by HO(2)(-) and to be protected against that inactivation by a number of small molecules including formate, imidazole, and urate. This inactivation has been shown to be due to oxidation of a ligand field histidine residue by a bound oxidant formed by reaction of the active site Cu(II) with HO(2)(-). We now report that protective actions of both formate and NADH increase as the pH was raised in the range 8.0-9.5. This is taken to indicate increased accessibility of the Cu site with rising pH and/or increased reactivity of the bound oxidant toward exogeneous substrates at high pH. Formate appears to act as a sacrificial substrate that protects by competing with the endogenous histidine residue for reaction with the bound oxidant, or that repairs the damage by reducing the histidyl radical intermediate. The same is likely also true of NADH.

The role of CO2 in metal-catalyzed peroxidations.

Transition metals, such as Cu(+2), Mn(+2), and Co(+2), have been seen to catalyze the bicarbonate enhanced oxidation of a variety of substrates by H(2)O(2). In several of these cases it has been demonstrated that CO(2), rather than bicarbonate, is the enhancing species. Mechanisms that are in accord with the data involve a hypervalent state that may be written (MO)(+n), or (MOH)(+n+1), or (M)(+n+2). This metal centered oxidant then oxidizes CO(2) to the carbonate radical; that is then the proximal oxidant of the various substrates. Whether a similar process has in vivo reality remains to be demonstrated.

The role of CO2 in cobalt-catalyzed peroxidations.

Augmentation, by CO(2)/HCO(3)(-), of Co(II)-catalyzed peroxidations was explored to clarify whether the rate enhancement was due to CO(2) or to HCO(3)(-). The rate of oxidation of NADH by Co(II) plus H(2)O(2), in Tris or phosphate, was markedly enhanced by CO(2)/HCO(3)(-). Phosphate was seen to inhibit the Co(II)-catalyzed peroxidation, probably due to its sequestration of the Co(II). When CO(2) was used, there was an initial burst of NADH oxidation followed by a slower linear rate. The presence of carbonic anhydrase eliminated this initial burst; establishing that CO(2) rather than HCO(3)(-) was the species responsible for the observed rate enhancements. Both kinetic and spectral data indicated that Co(II) was converted by H(2)O(2) into a less active form from which Co(II) could be regenerated. This less active form absorbed in both the UV and visible regions, and is assumed to be a peroxy bridged binuclear complex. The rate of formation of this absorbing form was increased by HCO(3)(-)/CO(2). A minimal mechanism consistent with these observations is proposed.

Cross-compartment protection by SOD1.

The absence of SOD1 in yeast has been found to result in inactivation of Lys4p. This [4Fe-4S]-containing dehydratase is in the pathway of biosynthesis of lysine, hence the oxygen-dependent lysine auxotrophy seen in this case. O(2)(-) is known to oxidize and thus destabilize the [Fe-4S] clusters of dehydratases; hence, this would make perfect sense were it not for the fact that SOD1 localizes to the cytosol and the intermembrane space of mitochondria, whereas Lys4p localizes to the mitochondrial matrix. How could SOD1 in one compartment protect against O(2)(-) attack in a different compartment? We suggest that the relatively high levels of O(2)(-) in the cytosol and intermembrane space of the SOD1 mutant may react with endogenous NO, forming HOONO that can diffuse into the mitochondrial matrix and there inactivate Lys4p and other [4Fe-4S]-containing dehydratases.

Carbon dioxide mediates Mn(II)-catalyzed decomposition of hydrogen peroxide and peroxidation reactions.

Mn(II) can catalyze the decomposition of H(2)O(2) and, in the presence of H(2)O(2), can catalyze the oxidation of NADH. Strikingly, these processes depend on the simultaneous presence of both CO(2) and HCO(3)(-). This explains the exponential dependence of the rates on [HCO(3)(-)], previously noted by other workers. These processes are inhibited by Mn-superoxide dismutase, establishing the generation of O(2)(-) and its role as an essential reactant. A scheme of reactions, consistent with the known properties of this system, is proposed. The large rate enhancements provided by HCO(3)(-) + CO(2), and the abundance of both of these species in vivo, suggest that similar reactions have relevance to the oxidative stress imposed by O(2)(-) and H(2)O(2).

CO2 enhanced peroxidase activity of SOD1: the effects of pH.

At pH 7.4, CO2, rather than HCO3-, markedly enhances the oxidation of diverse substrates by SOD1 plus H2O2. Since the concentration of CO2 would fall with rising pH in HCO3- buffers, it was of interest to explore the effects of pH on the peroxidase activity of SOD1 in the presence and in the absence of HCO3-. The rate of NADPH peroxidation in the HCO3- buffer was minimally affected by pH in the range of 8-10.5; in a pyrophosphate buffer, the rate increased markedly, such that at pH 10.5 the rates in the two buffers were nearly identical. Similar results were obtained when urate was used as the peroxidizeable substrate. These results are explicable on the basis of an increase in the rate with pH due to the ionization of H2O2 to the effective HO2- coupled with a decrease in [CO2] due to the ionizations of H2CO3, which displaces the hydration equilibrium to the right. These two opposing effects counteract in the HCO3(-)-buffered reaction mixtures; in the pyrophosphate buffer, only the effect of increasing [H02-] was seen.

Bicarbonate-enhanced peroxidase activity of Cu,Zn SOD: is the distal oxidant bound or diffusible?

The Cu,Zn SOD catalyzes the bicarbonate-dependent oxidation of a wide range of substrates by H2O2. A mechanism in accord with this activity has been described. It involves the generation of a strong oxidant (Cu(I)O, Cu(II)OH, or Cu(III)) by reaction of the active site Cu with H2O2, followed by oxidation of bicarbonate to CO3-* that in turn diffuses from the active site to oxidize the various substrates in free solution. Recently, an alternative mechanism, entailing firmly bound HCO3- and CO3-*, has been proposed [J. Biol. Chem. 278 (2003) 21032-21039]. We present data supporting the diffusible CO3-* and discuss the properties of this system that can be accommodated in this way and that preclude bound intermediates.