**2. The mitochondrial free radical theory of aging (MFRTA)**

Initially proposed by Harman [8], the "Free Radical Hypothesis of Aging" was later transformed to the "Mitochondrial Free Radical Theory of Aging" (MFRTA), because mitochondria were found to be the main source of free radicals [9–14, 19, 22]. Collectively, the free radicals derived from oxygen were named "reactive oxygen species" (ROS).

In order to understand the logic of the worldwide research on aging development, it would be useful to look back on the intellectual and scientific background existing 40–50 years ago. After discovery that mitochondria generate oxygen radicals [23, 24], there was an excitement in finding the "universal mechanism" of all diseases. Therefore, for a long-time research on biological effects of ROS was titled "oxidative stress", of which aging was only one of many damaging effects of ROS. For some time, it was not realized that organs and tissues age at different pace, and that environmental conditions, such as radiation, air contamination or industrial pollutants may contribute to the aging process.

Importantly, for a long time any clear-cut specific markers of systemic aging were not known, except mutations of mitochondrial DNA (mtDNA). There is a strong parallelism between production of ROS and mutations of mtDNA [13, 14, 25]. Until recently, this parallelism was explained by a belief that hundreds of "naked" circular mtDNA molecules lie in the mitochondrial matrix and thus mtDNA is an easy target for free radicals. The accumulation in cells of mtDNA damaged by ROS progressively inactivates the DNA templates necessary to repair damaged mitochondria. As a result, the accumulation of mtDNA mutations acts as the aging clock [25].

#### *Metabolic Syndrome as the First Stage of Eldership; the Beginning of Real Aging DOI: http://dx.doi.org/10.5772/intechopen.95464*

Understandably, mutations of mtDNA became one of the most important factors in explaining mechanisms of aging, age-associated diseases and practically most major human diseases [25–29]. Only recently it was discovered that mtDNA are protected by the proteinaceous "shield", nucleoids, and that there is no proof for the free radical direct effects on mtDNA [30–33].

Production of ROS occurs not only in organs and tissues, but also in blood cells where radicals evidently have functions different from those in parenchymal cells. The "respiratory burst" of phagocytic cells, when they come in contact with bacteria or immune complexes, is important source of superoxide radicals (O2 • ). Phagocytic cells include neutrophils, monocytes, macrophages and eosinophils known to produce large amounts of O2 • [34].

In some cells, particularly in hepatocytes, the major source of ROS may be of extramitochondrial origin [35]. In the liver, both O2 • and H2O2 are produced during metabolism of xenobiotics by the microsomal P-450-monoxygenases [36], and in the course of catabolism of purine nucleotides and nucleosides by xanthine oxidoreductase [37]. Liver peroxisomes also produce large amounts of hydrogen peroxide during catabolism of very long chain fatty acids and polyunsaturated fatty acids [38]. However, in the liver both mitochondria and peroxisomes possess high catalase activity, which neutralizes hydrogen peroxide [38, 39]. In addition to catalase and superoxide dismutase (isoforms 1 and 2) high activities, liver has high activities of glutathione S-transferase (GST) and glutathione peroxidase [40, 41], and Prohibitin-1 [42], which enhance the liver's antioxidant system. For this reason, liver is relatively protected from deleterious effects of ROS, has high regenerative capacity [43] and, therefore, the rate of aging of this organ is much slower, as compared with other organs [44, 45]. Kidneys also rarely create problems for elderly people because they work constantly at a relatively even pace. In the actively working organs production of ROS is minimal [21]. The fastest rates of aging occur in those organs, which have a wide range of workloads, such as skeletal muscles, brain and heart. These organs have very high capacities in respiratory activity and ATP production to satisfy the organ's energy demands at high workloads. These organs usually have increased ROS production at lower workloads or at rest [21].

Thus, aging is not an evenly distributed over the body process. In addition, mechanisms of aging are distinct in different organs and tissues, and the causing aging oxidants in various organs and tissues can also be different. In order to clarify the last statement and for the sake of the following discussion on the shortcuts of the current paradigm of the MFRTA, we give a brief description of the major biological and environmental radicals and biologically active molecules.

#### **2.1 Superoxide radical (O2 • ) and hydrogen peroxide (H2O2)**

Superoxide radical (O2 • ) and hydrogen peroxide (H2O2) are quantitatively the main species of ROS that are produced constantly by the mitochondrial respiratory chain [21, 22]. Superoxide radicals serve also as a source for other ROS: hydroperoxyl radical, peroxynitrite, lipid radicals. The proportion of H2O2, produced at the sites of the respiratory chain is small [21], but the superoxide in many tissues rapidly dismutate to hydrogen peroxide by very high activities of superoxide dismutases in the cytosol (Cu,Zn-SOD1) and mitochondrial matrix (Mn-SOD2) [22, 46]. At the beginning of the mitochondrial free radical theory, superoxide radical was regarded as very dangerous [34]. However, soon it was realized that after O2 • leaves the membrane's lipid phase, small and negatively charged superoxide anion instantly acquires the hydration shell and thus loses most of its chemical activity [47]. In addition, due to the high activities of SOD1 and SOD2, present at micromolar concentrations, the

superoxide radical half-life is very short (milliseconds) [22]. For this reason attention was shifted to other radicals, which can be formed from the superoxide and hydrogen peroxide. Nevertheless, O2 • can directly damage enzymes, which contain 4Fe-4S clusters by knocking out one Fe2+ atom and turning the 4Fe-4S cluster to inactive 3Fe-4S. A typical enzyme is aconitase, which is sensitive to inhibition by superoxide.

#### **2.2 Hydroxyl radical (• OH)**

Hydrogen peroxide (H2O2), which by itself is a rather harmless chemical, in the presence of transition metal ions Fe2+ and Cu1+ produces highly aggressive hydroxyl radical (• OH) [34]. However, • OH is so active that it instantly reacts with any molecule it encounters (except water). For this reason • OH half-life is only 10−9 sec [48] and under normal conditions it is not as harmful as initially believed. This radical is dangerous when formed in very large quantities, such as after exposure to radiation, or high concentration of H2O2 and transition metal poisoning. The latter situation is often observed in the experiments *in vitro*, but hardly takes place *in vivo,* since transition metals in the cells are always chelated.

#### **2.3 Nitric oxide (• NO) and peroxynitrite (OONO− )**

Tissues, such as blood vessels endothelium, neurons and others, which possess tissue-specific nitric oxide synthases (eNOS), produce a free radical nitric oxide ( • NO), an important cellular signaling molecule involved in many physiological and pathological processes. Formation of one molecule of • NO requires two molecules of O2, and since the • NO half-life *in vivo* is about 7 sec., for the maintenance of a steady-state concentration of • NO at 1 μM, the consumption of oxygen is very high, about 120 nanomol O2 per 1 gram of tissue per1 minute [49]. For this reason, the physiologically sufficient steady state levels of • NO can be formed only in the oxygen rich cells, like vascular endothelium, or well vascularized organs like brain. • NO is a powerful vasodilator. The biological effects of • NO can be inhibited due to its fast reaction with superoxide radical yielding a rather toxic product peroxynitrite (OONO<sup>−</sup> ) [50]. Peroxynitrite is a strong oxidant and nitrating agent, it interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radicalmediated mechanisms. *In vivo*, peroxynitrite generation represents an important pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders [49, 51]. Peroxynitrite plays an important role in pathologies and aging of some organs and tissues, such as blood vessels and neurons, but being a rather strong anion, it hardly contributes to the systemic mechanism of aging, which we will discuss a little later. Nitric oxide is hydrophobic, but chemically weak radical, and, thus, also can be excluded from the systemic aging mechanism, which occur in the lipid phase of the inner mitochondrial membrane.

At this point, we have not discussed the protonated form of superoxide radical (O2 • ), namely hydroperoxyl radical (• HO2) because it will be done in conjunctions with the description of polyunsaturated fatty acids (PUFA) autoxidation, named "Isoprostane Pathway of Lipid Peroxidation" (IPLP), which we propose as the main mechanism of aging in people with Metabolic syndrome.

#### **2.4 Environmental biologically active molecules and radicals**

Unlike the above described ROS, which are formed in the body and relatively well-studied, the biological effects of sun radiation and air pollutants involving

singlet oxygen and ozone are less known. However, they are the major causes of accelerated aging of skin and lung epithelium. For this reason, we provide a brief description of toxic effects of these ROS.
