**2. The increase in the level of reactive oxygen species is predetermined by programmed bioenergetics decay**

#### **2.1 Introduction**

The free-radical theory of aging (the theory of oxidative stress, the oxidative damage theory and the mitochondrial theory of aging) was proposed by D. Harman (1956) in the middle of the 20th century and its improved version continues to dominate discussion. Its supporters claim that there are no specific aging genes because longevity cannot be controlled by natural selection (Kirkwood, 2002, 2008; Medawar, 1952). According to their view, agedependent organism degradation results from the damage to cell structures by the ROS that are generated by mitochondria (Trifunovic & Larsson, 2008). This theory fascinates researchers by virtue of its simplicity and clarity. Indeed, it has been established that as an organism gets older, the ROS generation by the mitochondrial respiratory chain (electron transport chain) increases and the amount of damage to cell structures increases as well. The conclusion is obvious and the method for preventing aging is equally so: the neutralisation ROS by antioxidants. The age-dependent increase in the ROS generation rate is assumed to be just that: the ROS generated by mitochondria produce injury to its own mitochondrial DNA (mtDNA), which results in a defect in the respiratory chain. This, in turn, increases the rate of ROS production and as a result a vicious cycle arises.

•−)

64 Bioenergetics

The goal of this report is: (1) to show that despite the beliefs of the supporters of the stochastic theories, longevity is controlled by natural selection, i.e. specific aging genes exist; (2) such genes program a lowering of the bioenergetics level (degradation of Gibbs energy, ΔG). In turn, such degradation results in an age-dependent increase in the ROS generation rate, a decrease in the protein synthesis rate, and a limitation of cell division. These three phenomena form the basis for a large number of secondary destructive processes which result in the degradation of all physiological organisms' functions, i.e. the causes of aging. The very idea that bioenergetics exerts an impact upon aging is not a novelty. Hasty and Vijg (2002) have recently stated in theory that proper energy-saving could support a living system indefinitely. B.N. Ames (2004) has remarked that mitochondrial bioenergetics supports the metabolism's cell processes and that its attenuation can result in the agedependent degradation of all of an organism's physiological functions. And indeed, life as a phenomenon is characterised by a number of physical and chemical processes driven by the power of the bioenergetics machine. A gradual decrease in bioenergetics level can cause the degradation of all vital processes. However they also believe that the cause of agedependent bioenergetics attenuation is to be identified with the mechanism postulated by the free-radical theory. The following fact seems to reject the assumption of the direct programming of bioenergetics attenuation: one of main bioenergetics parameters is the mitochondrial membrane potential ∆ψ. In vitro tests have shown that the superoxide (O2

generation rate in the electron transport chain decreases as ∆ψ decreases. Consequently, in the process of bioenergetics attenuation the ROS level should decrease, but the tests show its increase in all tissues. And only that version of the vicious cycle brought forward by the free-radical theory can explain this paradox. Another mechanism which we have already suggested explains the increase in the number of reactive oxygen species during programmed bioenergetics attenuation (Trubitsyn, 2006). The bioenergetics mechanism of aging under consideration represents the integration of several of the author's articles

**2. The increase in the level of reactive oxygen species is predetermined by** 

The free-radical theory of aging (the theory of oxidative stress, the oxidative damage theory and the mitochondrial theory of aging) was proposed by D. Harman (1956) in the middle of the 20th century and its improved version continues to dominate discussion. Its supporters claim that there are no specific aging genes because longevity cannot be controlled by natural selection (Kirkwood, 2002, 2008; Medawar, 1952). According to their view, agedependent organism degradation results from the damage to cell structures by the ROS that are generated by mitochondria (Trifunovic & Larsson, 2008). This theory fascinates researchers by virtue of its simplicity and clarity. Indeed, it has been established that as an organism gets older, the ROS generation by the mitochondrial respiratory chain (electron transport chain) increases and the amount of damage to cell structures increases as well. The conclusion is obvious and the method for preventing aging is equally so: the neutralisation ROS by antioxidants. The age-dependent increase in the ROS generation rate is assumed to be just that: the ROS generated by mitochondria produce injury to its own mitochondrial DNA (mtDNA), which results in a defect in the respiratory chain. This, in turn, increases the

published earlier (Trubitsyn, 2006, 2006a, 2009, 2010, 2011).

rate of ROS production and as a result a vicious cycle arises.

**programmed bioenergetics decay** 

**2.1 Introduction** 

When the free radical theory of aging appeared, it stirred up a brisk discussion which continues to this day. Empirical data has shown that there is no appreciable loss in the respiratory chain's functions during aging (Barrientos et al., 1996; Rasmussen et al., 2003). This is also supported by experiments indirectly related to the electron transport chain. For example, research into intra-cellular organelle transfers has shown that the mitochondria of old donors recover their functional activity completely when transferred to ρ0 HeLa cells (HeLa cells free of mtDNA) (Hayashi et al., 1994; Isobe et al., 1998). Mitochondrial dysfunctions are also eliminated when HeLa cell nuclei are transferred into the cells of old donors (Isobe et al., 1997). The authors concluded that nuclear factors are responsible for age-related mitochondrial deficiency. In addition, the conclusion that the age-dependent accumulation of mtDNA mutations is modulated by the nuclear genome was also made by Yao et al. (2007). The discussion has become especially vigorous over the last decade. On the one hand, based on this theory, it was claimed that "aging is no longer an unsolved problem in biology" (Hayflick, 2007; Holliday, 2006). On the other hand, R.M. Howes (2006) has declared that the "overly exuberant and exaggerated past expectations and claims of the free-radical theory have been quieted by extensive randomised, double-blind, controlled human studies. A half century of data demonstrates its lack of predictability and it has not been validated by the scientific method. Widespread use of antioxidants has failed to quell the current pandemic of cancer, diabetes, and cardiovascular disease or [even] to stop or reverse the aging process." His position is also supported by G. Bjelakovic et al. (2008) who have collected a great deal of data to show that antioxidants neither result in the beneficial effects expected nor do they increase life expectancy (in the best-case scenario). Gems & Doonan (2009) asked a question in a recent review: "Is the theory really dead, or does it just need to be modified?" Actually, there is more than ample evidence against the aging mechanism postulated by this theory than there is evidence in support of it.

#### **2.2 Schema of the mitochondrial bioenergetics machine**

Mitochondria generate about 90 percent of the energy in any eukaryotic cell. Therefore, only the mitochondrial bioenergetics machine will be considered here. Any energy system can be quantitatively described by its propellant power (F) and by its effect (A): F = kA: this is the force and the work in mechanical engineering (k is the friction) and the electromotive force and current in electrical engineering (k is the resistance). It is the free-energy change (Gibbs energy, ΔG) and current in chemical thermodynamics (in bioenergetics in particular). Such terms as the bioenergetics level and the level of energy production are used in bioenergetics to express the propellant power. To make it clear, let us recall that the ΔG of macroergic (high-energy) coenzymes that function in the bioenergetics machine (ATP, NAD, NADP, GSH, etc.) is determined by the value of their concentrations ratio of the reduced form to the oxidised one and by the temperature. For ATP, for instance, ΔG = ΔG0 - RT ln[ATP]/[ADP][Pi], where ΔG0 is the standard Gibbs energy that is measured with everything at 1 molar concentration: [ATP] = [ADP] = [Pi] = 1M; R is the gas constant; T is the absolute temperature. The more negative Gibbs energy there is, the higher the energy potential the bioenergetics machine generates. As follows from the above expression for ΔG, the concentrations ratio of the reduced to oxidised forms of macroergic coenzymes ([ATP]/[ADP], [NADH]/[NAD+], etc.) is the only variable which determines the energy potential for warm-blooded animals.

Bioenergetics Theory of Aging 67

Researchers divide energy-metabolism reactions into a different number of functional blocks depending upon their purpose. For example, Ainscow and Brand (1998) have divided it into nine blocks connected to each other by five intermediates. To solve the problem under consideration, the bioenergetics machine may be divided into three blocks (the Krebs cycle, the electron transport chain and ATP-synthase) connected by two intermediates ([NADH]/[NAD+] and ∆ψ (Fig.1)). According to this scheme, the output potential ([ATP]/[ADP]) is generated in three stages. At the first stage, the primary electromotive force, [NADH]/[NAD+], is created by reducing NAD+ to NADH. This serves as the propellant power for stage two where electrons are transferred from NADH to oxygen via the electron transport chain, generating the mitochondrial membrane potential Δψ. At the third stage, Δψ is the electromotive force for ATP-synthase which generates the output potential. If there are no excessive loads (in stage four or close to it) then the [NADH]/[NAD+] change results in a proportional change in Δψ and in [ATP]/[ADP].

During the aerobic metabolism, a small number of the electrons that flow from NADH via the respiratory chain react with oxygen directly reducing oxygen to superoxide anion (O2•− or HO2•) (Demin et al., 1998; Scandalios, 2002a) which can damage cell biopolymers. Cells have a protective system that can be conditionally divided into three functional lines of defence: preventative mechanisms, ROS-scavenging mechanisms, and emergency-response

superoxide back into O2 at its location of generation (Brand, 2000; Skulachev, 2001). The emergency-response mechanisms are actuated when the ROS amount exceeds a critical level and when the cumulative effect of other mechanisms cannot improve the situation. However ROS not only damage biopolymers but it also plays an important role in the regulation of transcription factors, growth factors and other intracellular signal systems (Brigelius-Flohe et al., 2003; Cerimele et al, 2005; Rhee, 1999; Scandalios, 2002). The cell needs ROS, but their concentration should be maintained at a safe level. Therefore, there is a dedicated ROS-scavenging mechanism to maintain the ROS homeostasis. This mechanism performs the O2•− detoxification through a two-stage process (Fig. 1). At first, the manganese-containing mitochondrial superoxide dismutase (MnSOD) transforms superoxide into hydrogen peroxide (H2O2) (Jonas et al., 1989; Scandalios, 2002a) which is then decomposed by catalase and peroxidases. Most H2O2 is decomposed in cytosol by catalase and in the mitochondrial matrix by the glutathione and thioredoxin systems (catalase is absent in the mitochondrial matrix) (Wei et al., 2001). The glutathione system consists of glutathione peroxidase (GP) and glutathione reductase (GR). The GP potency is maintained due to the oxidation of glutathione (GSH) which is converted into its disulphide form (GSSG). Next, the GR catalyses the reduction of the oxidised glutathione at the expense of NADPH oxidation (Arai et al., 1999; Jo et al., 2001; Iantomasi et al., 1993). The NADP+ thus formed is reduced again to NADPH in the isocitrate dehydrogenase reaction of the Krebs cycle (Jo et al., 2001). There is an analogous system – the thioredoxin system – which functions in parallel with the glutathione system and which also consists of thioredoxin peroxidase (TP) and thioredoxin reductase (TR) (Jo et al, 2001; Nordberg & Arner, 2001). Similarly, the TP potency is maintained by the oxidation of thioredoxin which is then reduced by TR, also at the expense of NADPH oxidation (Lewin et l., 2001). For the sake of simplicity, this parallel system is not shown in Fig. 1. The reaction that is catalysed by these peroxidases is

•− generation or oxidise

mechanisms. The preventative mechanisms either prevent O2

**2.3 ROS-scavenging mechanisms** 

*Bioenergetics machine.* The primary motive power, NADH/NAD+, is created in the Krebs cycle. The mitochondrial membrane potential, Δψ, is created by the electron flow from NADH to oxygen through the electron transport chain (ETC). ATP-synthase phosphorylates ADP into ATP at the expense of Δψ. *Scavenging mechanism.* The superoxide radical (O2•−) produced by ETC is transformed into hydrogen peroxide, H2O2, by manganese superoxide dismutase (MnSOD). H2O2 is then decomposed into H2O and O2 mainly through the reaction that is catalyzed by glutathione peroxidase (GP) and partially through the Fenton reaction; the last produces an extremely aggressive hydroxyl radical. The glutathione peroxidase activity mainly predetermines the rate of the scavenging process. This activity is sustained by the energy provided by glutathione (GSH) oxidation. The thus GSSG formed is reduced again into GSH at the expense of the oxidation of NADPH in a reaction that is catalysed by glutathione reductase (GR). The NADP+ formed is reduced in turn at the expense of the oxidation of NADH in the reaction catalysed by nicotinamide nucleotide transhydrogenase (NT). The NAD+ formed is reduced by the reactions of the Krebs cycle. The NADP+ can also be directly reduced in the isocitrate dehydrogenase reaction of the Krebs cycle. The chain of these redox reactions is the electrons' pipeline from the Krebs cycle to glutathione peroxidase. *The mechanism of ROS increase.* The programmed bioenergetics decline leads to a proportional decrease in GP activity, which increases the H2O2 level. As hydrogen peroxide is a substrate for the Fenton reaction, this augments the H2O2 flow through the Fenton reaction, which elevates the content of free radicals. Thus, a decline in the bioenergetics level is followed by an increase in the total amount of reactive oxygen species and its aggressiveness.

Fig. 1. Scheme explaining the mechanism of the ROS increase under the bioenergetics decline.

*Bioenergetics machine.* The primary motive power, NADH/NAD+, is created in the Krebs cycle. The mitochondrial membrane potential, Δψ, is created by the electron flow from NADH to oxygen through the electron transport chain (ETC). ATP-synthase phosphorylates ADP into ATP at the expense of Δψ. *Scavenging mechanism.* The superoxide radical (O2•−) produced by ETC is transformed into hydrogen peroxide, H2O2, by manganese superoxide dismutase (MnSOD). H2O2 is then decomposed into H2O and O2 mainly through the reaction that is catalyzed by glutathione peroxidase (GP) and partially through the Fenton reaction; the last produces an extremely aggressive hydroxyl radical. The glutathione peroxidase activity mainly predetermines the rate of the scavenging process. This activity is sustained by the energy provided by glutathione (GSH) oxidation. The thus GSSG formed is reduced again into GSH at the expense of the oxidation of NADPH in a reaction that is catalysed by glutathione reductase (GR). The NADP+ formed is reduced in turn at the expense of the oxidation of NADH in the reaction catalysed by nicotinamide nucleotide transhydrogenase (NT). The NAD+ formed is reduced by the reactions of the Krebs cycle. The NADP+ can also be directly reduced in the isocitrate dehydrogenase reaction of the Krebs cycle. The chain of these redox reactions is the electrons' pipeline from the Krebs cycle to glutathione peroxidase. *The mechanism of ROS increase.* The programmed bioenergetics decline leads to a proportional decrease in GP activity, which increases the H2O2 level. As hydrogen peroxide is a substrate for the Fenton reaction, this augments the H2O2 flow through the Fenton reaction, which elevates the content of free radicals. Thus, a decline in the bioenergetics level is followed by an increase

Fig. 1. Scheme explaining the mechanism of the ROS increase under the bioenergetics decline.

in the total amount of reactive oxygen species and its aggressiveness.

Researchers divide energy-metabolism reactions into a different number of functional blocks depending upon their purpose. For example, Ainscow and Brand (1998) have divided it into nine blocks connected to each other by five intermediates. To solve the problem under consideration, the bioenergetics machine may be divided into three blocks (the Krebs cycle, the electron transport chain and ATP-synthase) connected by two intermediates ([NADH]/[NAD+] and ∆ψ (Fig.1)). According to this scheme, the output potential ([ATP]/[ADP]) is generated in three stages. At the first stage, the primary electromotive force, [NADH]/[NAD+], is created by reducing NAD+ to NADH. This serves as the propellant power for stage two where electrons are transferred from NADH to oxygen via the electron transport chain, generating the mitochondrial membrane potential Δψ. At the third stage, Δψ is the electromotive force for ATP-synthase which generates the output potential. If there are no excessive loads (in stage four or close to it) then the [NADH]/[NAD+] change results in a proportional change in Δψ and in [ATP]/[ADP].

#### **2.3 ROS-scavenging mechanisms**

During the aerobic metabolism, a small number of the electrons that flow from NADH via the respiratory chain react with oxygen directly reducing oxygen to superoxide anion (O2•− or HO2•) (Demin et al., 1998; Scandalios, 2002a) which can damage cell biopolymers. Cells have a protective system that can be conditionally divided into three functional lines of defence: preventative mechanisms, ROS-scavenging mechanisms, and emergency-response mechanisms. The preventative mechanisms either prevent O2 •− generation or oxidise superoxide back into O2 at its location of generation (Brand, 2000; Skulachev, 2001). The emergency-response mechanisms are actuated when the ROS amount exceeds a critical level and when the cumulative effect of other mechanisms cannot improve the situation. However ROS not only damage biopolymers but it also plays an important role in the regulation of transcription factors, growth factors and other intracellular signal systems (Brigelius-Flohe et al., 2003; Cerimele et al, 2005; Rhee, 1999; Scandalios, 2002). The cell needs ROS, but their concentration should be maintained at a safe level. Therefore, there is a dedicated ROS-scavenging mechanism to maintain the ROS homeostasis. This mechanism performs the O2•− detoxification through a two-stage process (Fig. 1). At first, the manganese-containing mitochondrial superoxide dismutase (MnSOD) transforms superoxide into hydrogen peroxide (H2O2) (Jonas et al., 1989; Scandalios, 2002a) which is then decomposed by catalase and peroxidases. Most H2O2 is decomposed in cytosol by catalase and in the mitochondrial matrix by the glutathione and thioredoxin systems (catalase is absent in the mitochondrial matrix) (Wei et al., 2001). The glutathione system consists of glutathione peroxidase (GP) and glutathione reductase (GR). The GP potency is maintained due to the oxidation of glutathione (GSH) which is converted into its disulphide form (GSSG). Next, the GR catalyses the reduction of the oxidised glutathione at the expense of NADPH oxidation (Arai et al., 1999; Jo et al., 2001; Iantomasi et al., 1993). The NADP+ thus formed is reduced again to NADPH in the isocitrate dehydrogenase reaction of the Krebs cycle (Jo et al., 2001). There is an analogous system – the thioredoxin system – which functions in parallel with the glutathione system and which also consists of thioredoxin peroxidase (TP) and thioredoxin reductase (TR) (Jo et al, 2001; Nordberg & Arner, 2001). Similarly, the TP potency is maintained by the oxidation of thioredoxin which is then reduced by TR, also at the expense of NADPH oxidation (Lewin et l., 2001). For the sake of simplicity, this parallel system is not shown in Fig. 1. The reaction that is catalysed by these peroxidases is

Bioenergetics Theory of Aging 69

Н2О2 which can produce •OH (Qr) is predetermined by both the activity of the Fenton reaction (Af) and that of glutathione peroxidase (Ag): Qr = Af /(Af + Ag). Thus, the lower the level of the activity of glutathione peroxidase and thioredoxin peroxidase, the higher the

As has been mentioned, a decrease in the energy metabolism rate should, in theory, result in a lowering of the O2•− generation rate. Indeed, this is just what happens. However the concentration of the other ROS does not only depend upon the O2•− generation rate: the programmed age-dependent delay in the bioenergetics level results in a decrease in GP and TP activity. This raises the concentration of their substrate, H2O2. Since hydrogen peroxide is a substrate for the Fenton reaction as well, it augments the current through this reaction. As a result, the total amount of ROS and their aggressiveness increases despite a decrease in the

Accordingly, the leading cause of the age-dependent increase in the amount of ROS and its aggressiveness is a programmed attenuation of cellular bioenergetics rather than a progressive accumulation of mutations in mtDNA due to the creation of a vicious cycle.

**3. The age-dependent attenuation of bioenergetics underlies a decrease in** 

A different popular aging theory, the protein-error theory, is based on the indisputable fact that the bulk protein synthesis slows down during aging (Rattan, 1996, 2009; Ryazanov & Nefsky, 2002). According to the theory, such retardation results in a decreasing protein turnover rate which causes the accumulation of defective macromolecules. S.I.S. Ratton (1996), who has investigated this process in detail, reports that "the implications and consequences of slower rates of protein synthesis are manifold, including a decrease in the availability of enzymes for the maintenance, repair and normal metabolic functioning of the cell, an inefficient removal of inactive, abnormal and damaged macromolecules in the cell, the inefficiency of the intracellular and intercellular signalling pathways, and a decrease in the production and secretion of hormones, antibodies, neurotransmitters and the components of the extra cellular matrix." The reason behind a slower protein synthesis rate is seen in the stochastic accumulation of molecular damage and the progressive failure of maintenance and repair (Rattan, 2009). It entails damage to fragments of the translation mechanism: "a decline in the efficiency and accuracy of ribosomes, an increase in the levels of rRNA and tRNA, and a decrease in the amounts and activities of elongation factors" (Rattan, 2006). At the same time, there is empirical evidence which allows for the explanation of the slowing down of overall

level of ROS production.

O2•− generation rate.

**the general level of protein synthesis** 

protein synthesis by the attenuation of cellular bioenergetics.

**3.2 The mechanism for the decrease in the level of cell protein synthesis** 

It was D.A. Young (1969) who discovered a relationship between the protein synthesis rate and the bioenergetics level for the first time. When conducting experiments on glucocorticoid hormones, he noticed that the rate of amino acids inclusion into a growing polypeptide chain depends upon the entry of carbohydrates (glucose, pyruvate, and lactate) into cells. An assumption was made that this effect is connected with the ATP generation rate. It was shown thereafter that the protein synthesis rate depends upon the ADP/ATP and GDP/GTP ratios

**2.6 Conclusion** 

**3.1 Introduction** 

simple: H2O2 takes two electrons from the glutathione (thioredoxin) and two protons from the environment and then decays into two water molecules: H2O2 + 2e– + 2H+ = 2H2O. Only GP and TP catalyse this reaction directly; the other reactions are a pipeline by which energy is transferred from the Krebs cycle to glutathione peroxidase with thioredoxin peroxidase providing their activity (Iantomasi et al., 1993). The activity of any energy-dependent chemical reaction depends upon the energy supply (Westerhoff & van Dam, 1987). Therefore, the more the NADPH/NADP+ ratio is generated in the Krebs cycle, the higher the GP and TP activity, and vice versa. It was shown experimentally that bioenergetics attenuation results in decrease of the scavenging mechanism's activity (Jo et al., 2001). It should be also noted that the ROSscavenging mechanism can to some extent adapt to changes in the ROS level: the cell responds to a higher ROS concentration by a higher synthesis rate for MnSOD and glutathione-system enzymes (Meewes et al., 2001). An increase in the gene expression of those enzymes is mediated by the transcription nuclear factor-κB that is activated under excessive amounts of ROS (Scandalios, 2002a; Schreck et al., 1991).

#### **2.4 Fenton reaction**

There is additional the ferrous-ion catalysed means of hydrogen peroxide decomposition, which is called the Fenton reaction. In its simplest form, the Fenton chemistry is a chain mechanism of certain reactions in which H2O2 breaks up into water and oxygen and where Fe2+ is regenerated (Dunford, 2002):

$$\text{Fe}^{2+} + \text{H}\_2\text{O}\_2 \rightarrow \text{Fe}^{3+} + \text{^\bullet OH} + \text{HO}^- \tag{1}$$

$$\text{Fe}^{2+} + \text{"OH} \rightarrow \text{Fe}^{3+} + \text{HO}^{-} \tag{2}$$

$$\bullet \text{^\bullet OH} + \text{H}\_2\text{O}\_2 \rightarrow \text{HO}\_2\text{^\bullet - } + \text{H}\_2\text{O} \tag{3}$$

$$\text{HCO}\_2^{\bullet} + \text{Fe}^{2+} \rightarrow \text{Fe}^{3+} + \text{HO}\_2^{-} \tag{4}$$

$$\text{HO}\_2^{\bullet} + \text{Fe}^{3+} \rightarrow \text{Fe}^{2+} + \text{O}\_2 + \text{H}^+ \tag{5}$$

As distinct from the glutathione system, the iron decomposes Н2О2 due to its ability to undergo cyclic oxidation and reduction. However, such redox activity of iron can generate free radicals capable of causing a wide range of biological injuries (Liu et al. 2003). The hydroxyl radicals (•OH) formed during the Fenton reaction are true chemical predators: indeed, the reactivity of •OH is so great that, if they are formed in living systems, they will react immediately with whatever biological molecule is in their vicinity, producing secondary radicals of variable reactivity (Halliwell & Gutteridge 1984; Yu & Anderson 1997). Among O2•−, Н2О2 and •OH, only the hydroxyl radical can directly cause double-stranded DNA breaks (Aruoma 1994).

#### **2.5 The mechanism of age-dependent increase in ROS level**

The Fenton reaction actually shunts the ROS-scavenging mechanism. As a result, Н2О<sup>2</sup> molecules are decomposed both by the glutathione system and the Fenton reaction. As the two ways of hydrogen peroxide decomposition compete for the substratum, the fraction of

simple: H2O2 takes two electrons from the glutathione (thioredoxin) and two protons from the environment and then decays into two water molecules: H2O2 + 2e– + 2H+ = 2H2O. Only GP and TP catalyse this reaction directly; the other reactions are a pipeline by which energy is transferred from the Krebs cycle to glutathione peroxidase with thioredoxin peroxidase providing their activity (Iantomasi et al., 1993). The activity of any energy-dependent chemical reaction depends upon the energy supply (Westerhoff & van Dam, 1987). Therefore, the more the NADPH/NADP+ ratio is generated in the Krebs cycle, the higher the GP and TP activity, and vice versa. It was shown experimentally that bioenergetics attenuation results in decrease of the scavenging mechanism's activity (Jo et al., 2001). It should be also noted that the ROSscavenging mechanism can to some extent adapt to changes in the ROS level: the cell responds to a higher ROS concentration by a higher synthesis rate for MnSOD and glutathione-system enzymes (Meewes et al., 2001). An increase in the gene expression of those enzymes is mediated by the transcription nuclear factor-κB that is activated under excessive amounts of

There is additional the ferrous-ion catalysed means of hydrogen peroxide decomposition, which is called the Fenton reaction. In its simplest form, the Fenton chemistry is a chain mechanism of certain reactions in which H2O2 breaks up into water and oxygen and where

2 3 Fe H O Fe OH HO 2 2

OH H O HO H O 22 2 2

2 3 HO Fe Fe HO 2 2

3 2 HO Fe Fe O H 2 2

As distinct from the glutathione system, the iron decomposes Н2О2 due to its ability to undergo cyclic oxidation and reduction. However, such redox activity of iron can generate free radicals capable of causing a wide range of biological injuries (Liu et al. 2003). The hydroxyl radicals (•OH) formed during the Fenton reaction are true chemical predators: indeed, the reactivity of •OH is so great that, if they are formed in living systems, they will react immediately with whatever biological molecule is in their vicinity, producing secondary radicals of variable reactivity (Halliwell & Gutteridge 1984; Yu & Anderson 1997).

The Fenton reaction actually shunts the ROS-scavenging mechanism. As a result, Н2О<sup>2</sup> molecules are decomposed both by the glutathione system and the Fenton reaction. As the two ways of hydrogen peroxide decomposition compete for the substratum, the fraction of

•−, Н2О2 and •OH, only the hydroxyl radical can directly cause double-stranded

(1)

2 3 Fe OH Fe HO (2)

(4)

(5)

(3)

ROS (Scandalios, 2002a; Schreck et al., 1991).

Fe2+ is regenerated (Dunford, 2002):

**2.4 Fenton reaction** 

Among O2

DNA breaks (Aruoma 1994).

**2.5 The mechanism of age-dependent increase in ROS level** 

Н2О2 which can produce •OH (Qr) is predetermined by both the activity of the Fenton reaction (Af) and that of glutathione peroxidase (Ag): Qr = Af /(Af + Ag). Thus, the lower the level of the activity of glutathione peroxidase and thioredoxin peroxidase, the higher the level of ROS production.

As has been mentioned, a decrease in the energy metabolism rate should, in theory, result in a lowering of the O2•− generation rate. Indeed, this is just what happens. However the concentration of the other ROS does not only depend upon the O2•− generation rate: the programmed age-dependent delay in the bioenergetics level results in a decrease in GP and TP activity. This raises the concentration of their substrate, H2O2. Since hydrogen peroxide is a substrate for the Fenton reaction as well, it augments the current through this reaction. As a result, the total amount of ROS and their aggressiveness increases despite a decrease in the O2 •− generation rate.

## **2.6 Conclusion**

Accordingly, the leading cause of the age-dependent increase in the amount of ROS and its aggressiveness is a programmed attenuation of cellular bioenergetics rather than a progressive accumulation of mutations in mtDNA due to the creation of a vicious cycle.
