Mitochondrial integrity and antioxidative enzyme efficiency in fischer rats effects of ageing and epigallocatechin 3 gallate intervention 1 2

Considering that the current project is based on the free radical theory of aging and the closely related mitochondrial theory of aging, relevant concepts will be introduced with more de

1 Introduction

1.1 Definition of aging

Aging is traditionally regarded as the process of becoming older A broader definition of aging is offered in the "Handbook of the Biology of Aging" [1], to encompass the process of system's deterioration with time Sometimes, the term senescence is used to describe cellular aging, where normal diploid differentiated cells lose the ability to divide This phenomenon is also known as "replicative senescence", the "Hayflick phenomenon", or the Hayflick limit When referring to the organismal senescence, which is the aging of the whole organism, the term

“aging” is always used interchangeably with “senescence”

Aging does not result from diseases and it is not necessarily related with diseases, however aging increases the risk of disease happening Several typical changes occur with age including presbyopia, cataracts, loss of the ability to hear and taste, reduced body weight, muscle strength, thymus mass, reduced sensitivity to growth factors and hormones, decline of reaction times, and increased pathological conditions and disabilities Other serious degenerative diseases might also develop such as prostatitis, osteoporosis, diabetes, cancer, atherosclerosis, heart disease,

Alzheimer's disease, Parkinson's disease, etc Under very rare abnormal

circumstances, people suffer from certain accelerated aging diseases such as Werner's syndrome, Cockayne syndrome, Hutchinson-Gilford Progeria syndrome and so on Such symptoms are sometimes used as models to study the mechanisms

of aging

Aging and senescence has become a globalized issue Recently, the aging population has been growing at a considerably faster rate than that of the world’s total population, particularly in the developed countries where the aging population occupies a larger proportion of the total population, although the aged group is growing more rapidly in the less developed regions It has been pointed out that “Population ageing is unprecedented, pervasive, enduring and has profound implications for many facets of human life.” in a report prepared by the Population Division, the United Nation With the increasing aging population in society, study the mechanisms of aging and age-related diseases/disorders has become an intriguing research area

1.2 Mechanisms of aging

In the process of exploring the mechanisms of aging, more than 300 theories have been proposed However, it's important to note, many of the theories of aging are not mutually exclusive, but intrinsically related and sometimes supportive of each

other

1.2.1 The free radical theory of aging

The free radial theory was first proposed by Denham Harman in 1956 [2] According to this theory, the reactive oxygen species (ROS) which includes superoxide radicals, hydrogen peroxide, and hydroxyl radicals, is capable of causing oxidative damages to many biological macromolecules such as DNA,

RNA, proteins and lipids [3] The accumulation of such oxidative damages in the

cells and tissues is probably a direct cause of aging (More details will be discussed in the following sections.)

1.2.2 The mitochondrial theory of aging

Later in 1972, Dr Harman developed the ‘mitochondrial theory of aging’ based

on the free radial theory of aging, where he specifically pointed out the effects of mitochondrial respiration as the main cause of ROS formation [4] Meanwhile, mitochondria are susceptible to ROS attack due to their close approximation to the ROS producing site on the mitochondrial electron transport chain (ETC) and dysfunctional mitochondria in turn cause more leakage of ROS, thus forming a vicious cycle Moreover, the mitochondrial theory of aging was further refined and developed by Dr Miquel in 1980 where some additional attention was given to mitochondrial genetics, membranes, and bioenergetics besides the ROS production [5] (More details will be discussed in the following section.)

side-1.2.3 The cross-linking theory of aging

In the aging process, proteins are damaged by both free-radicals and glycation Glycation also known as Maillard reaction, or non-enzymatic glycosylation, is a reaction in which reducing sugars such as glucose and fructose bind to free amino groups in proteins The glycated proteins also known as Amadori product then react with other proteins, finally resulting in the irreversible 'cross-linking' [6] The representative reaction between glucose and a lysine amino acid in a protein molecule is demonstrated as follows (Figure 1) The cross-linked proteins become

impaired and are unable to function efficiently However, the cross-linkages inhibit the activity of proteases from breaking down the damaged proteins which accumulate in the tissue and cause a series of problems [6] The glycated proteins inhibit cellular transport processes, stimulate cells to produce more free radicals, and activate pro-inflammatory cytokines such as Tumor Necrosis Factor alpha (TNF-α) and interleukin 6 [7] In addition, some glycated proteins are immunogenic or mutagenic, whereas others reduce cell proliferation and induce apoptosis, resulting in excessive loss of cells and further contributing to the risk of degeneration [8] The known cross-linking disorders include diabetics, age-related cataracts, renal disorders, cardiac enlargement, the hardening of collagen and so

on [8]

Figure 1 Cross-linking between glucose and a lysine amino acid in a protein molecule

1.2.4 The immunological theory of aging

"Immunosenescence" was first used by Dr Roy Walford to describe the associated immune deficiency, and such concept was later developed into the immunological theory of aging [9] Briefly, he hypothesized that many aging effects are related to the declining ability of the immune system, especially the function of T-cells in the aging process due to the decay of the thymus gland It is known that the effectiveness of the immune system peaks at puberty and gradually declines thereafter with advance in age As a result, immune system becomes less capable of resisting infection and cancer, so that a variety of infectious and non infectious diseases, such as arthritis, psoriasis and other autoimmune diseases become more prominent with age

age-1.2.5 The telomere theory of aging

The telomere theory of aging suggests that cell death is caused by the shortening

of telomeres Telomeres are sequences of nucleic acids extending from the ends of chromosomes and they shorten with each cell division It is believed that telomere

is a biological clock that decides aging because the number of times that cells can divide is limited by the length of telomere For some species there is a correlation between maximum lifespan and the number of fibroblast doublings for that species [10] However, adverse evidence comes from the observation of mice, which have very long telomeres and show no reduction of telomere length with age [11], but most mouse cells stop dividing after only 10−15 doublings Only a few cells that rapidly proliferate such as endothelial cells or immune system cells show decreased function with age that could be associated with telomere

shortening, other post-mitotic cells like neurons and muscle cells survive but never divide Thus the aging in fruit flies or nematodes that comprised entirely of post-mitotic cells is hardly relevant to telomeres

1.2.6 The wear and tear theory of aging

The wear and tear theory of aging considers aging as the effect of progressive accumulation of damages to biomacromolecule due to radiation, chemical toxins, metal ions, free-radicals, hydrolysis, glycation and disulfide-bond cross-linking Such damages can affect genes, proteins, cell membranes and enzyme functions The organisms have limited capacity to repair damages so that when the total damages accumulate and finally reach a critical level, the cells become senescent

1.2.7 Longevity genes

It is also believed that aging can be regulated at the gene expression level In the process of studying the mechanisms of aging, investigators have identified numerous promising regulatory pathways and longevity genes in model animals of flies, worms and mice On top of the longevity genes, it is probably the well characterized genetic regulatory networks that involve the function of insulin/insulin-like growth factor (IGF)-1 signaling axis Lifespan regulation by IGF-1 signaling appears to be evolutionarily conserved over a wide range of

organisms including yeast, fruit fly, nematode as well as mouse [12] In the C

elegans for example, the binding of insulin-like molecules to the IGF-1 receptor

DAF-2 initiates a cascade of protein kinases, including AGE-1, DAF-2/AGE-1,

AKT-1, AKT-2, etc., and the IGF-1 level is proved to be reversely correlated with

the lifespan of the worms Some of the confirmed longevity genes from model animals are listed in Table 1

S cerevisiae C elegans D melanogaster M musculus

lag1 lac1 ras2 phb1

phb2

cdc7

bud1

rtg2 rpd3 hda1

sir2 sir4-42

uth4 ygl023

sgs1 rad52

fob1

daf-2 age-1/daf-23akt-1/akt-2 daf-16 daf-12 ctl-1 old-1 spe-26 clk-1 mev-1

sod1 cat1

prop-1 p66shc ras1 daf-18 mth mclk1

Table 1 The identified longevity genes in the S cerevisiae, C elegans, D melanogaster and M musculus

Saccharomyces cerevisiae, bakers' yeast; Caenorhabditis elegans, the soil

roundworm; Drosophila melanogaster, the fruit fly; and Mus musculus, the mouse

1.3 The free radial theory of aging

Among the great diversity of the aging theories, the free radical theory of aging is one of the most popular and strongly supported theories Considering that the current project is based on the free radical theory of aging and the closely related mitochondrial theory of aging, relevant concepts will be introduced with more details in this section and the following section 1.4 to clarify the respective theories

1.3.1 Free radicals

Free radicals are the molecules that carry an impaired electron All free radicals are extremely reactive and are capable of catching an electron from other molecules, starting a chain reaction of free radical formation The main free radicals are superoxide radical (O2·-), hydroxyl radical (OH·), hydroperoxyl radical (HOO·), alkoxyl radical (LO·), peroxyl radical (LOO·) and nitric oxide radical (NO·) [13] Other molecules that are technically not free radicals, but act much like them or are readily converted into free radicals, are singlet oxygen (1O2), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) [13] Collectively, the free radicals and non-free radical mimics that contain oxygen are called reactive oxygen species (ROS)

Free radicals are able to damage virtually all biomolecules, including proteins, sugars, fatty acids and nucleic acids [3] However, free radicals are extremely short-lived because of their extreme reactivity [14] With the help of metal ions, free radicals are converted to H2O2 that can diffuse to the cellular membrane and

mitochondrial membrane and oxidize the biomacromolecurs there Table 2 shows the main ROS formation in biological systems

Hydroperoxyl HOO· Strong oxidant, lipid soluble

Hydroperoxide LOOH Low reactivity, forms per/alkoxyl

radicals in presence of transiton

metal ions Peroxyl LOO· Low oxidant, lipid radical Ten millisecond Alkoxyl anion LO· Intermediate oxidant, lipid radical One microsecondSuperoxide

anion

O2·- Good reductant, poor oxidant One microsecond

Hydroxyl

radical

OH· Extremely reactive, low diffusion One nanosecond

Nitric oxide NO· Weak oxidant, no diffusion Few seconds Peroxynitrite ONOO- Product of NO·and O2·-, very strong

oxidant, no diffusion, very reactive Hydrogen

Table 2 The major ROS formation in biological systems

#, half-life time is determined at 37ºC (Table modified from the study of Abuja P.M and Albertini R 2001 [15] and Robert A., 1995 [14])

1.3.2 Resource of ROS

The main resource of ROS is the mitochondrial ETC, from where about 90% free radicals are produced as by-product of cellular respiration When the molecular oxygen passes through the ETC, approximately 2-3% of oxygen is inadvertently converted to O2·-, which can in turn generate H2O2 and OH· [16, 17] Another source of ROS, especially H2O2, is the peroxisome which is utilized by organism

to degrade fatty acids [16] ROS is also produced by Cytochrome P450 enzymes,

as by-product in the detoxification of a broad range of potentially toxic food, drug and environmental pollutant molecules [16] Besides, in the pathophysiological conditions especially the chronic immune-activation condition, white blood cells, mainly phagocytes, generate a series of ROS to attack pathogens which may create serious free radical problems [16, 18] Moreover, various biomolecules such as hydroquinones, thiols, hemoglobin or flavoenzymes such as xanthine oxidase (XO) may spontaneously produce ROS [16] Meanwhile, exogenous ROS

may originate from polluted air, cigarette smoke, iron and copper salts, some

phenolic compounds and various drugs [19]

1.3.3 Oxidative damages

A broad spectrum of aging related diseases and disorders are believed to be caused wholly or partly through free radical damages [17] However, as the main endogenous ROS product, O2· - is not highly reactive and lacks the ability to penetrate lipid membranes H2O2 getting from the dismutation of O2·- in vitrois able to move freely across membranes, yet is still poorly reactive in aqueous solution The majority of intracellular damages caused by H2O2 andO2·- is actually

via the conversion of H2O2 into OH·, dependent on the Fenton’s reaction, which requires the availability of transition metal ions such as Fe2+ or Cu+

(1) Fe2+/Cu+ + H2O2 → Fe3+/Cu2+ + OH· + OH−

(2) Fe3+/Cu2+ + H2O2 → Fe2+/Cu+ + OOH· + H+

Hear, O2·- also plays a role to recycle the metal ions

Fe3+/Cu2+ + O2·- →Fe2+/Cu+ + O2

Thus these highly reactive ROS such as OH· and OOH· are able to oxidize the biological molecules, causing changes in their structure and function, which may represent a major component of the aging process [20]

1.3.3.1 Lipid peroxidation

The polyunsaturated fatty acids in the cell membrane are sensitive to free radical reactions which result in lipid peroxidation Lipid peroxidation is a free radical-driven chain reaction where one radical leads to the oxidization of a large number

of substrate molecules The initial step is the oxidation of lipid by a species with sufficient reactivity to abstract a hydrogen atom from methylene, followed by a propagative effect giving rise to lipid peroxyl radicals that are capable of removing hydrogen from a neighboring fatty acid side chain to form lipid hydroperoxide [15] The peroxidation products of lipids in the cell membranes can accumulate, causing decreased membrane fluidity [21], which can seriously disrupt the function of membrane bound proteins [16], and thus the signaling pathways Lipid peroxidation also produces a variety of toxic aldehydes and ketones including 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) The water-soluble lipid peroxidation products (most notably the aldehydes) are able to

diffuse across the membrane and dialdehydes can react with nucleophilic chain of amino acids in proteins and lead to protein cross-linking [22] The cross-linking of proteins and lipids might form the age pigment, lipofuscin [23]

side-1.3.3.2 Change of protein structure

Proteins are susceptible to ROS attack and the modification of protein is mainly carried out by reactions involving OH· [24] Although the oxidation of proteins is less well characterized, several classes of damages have been documented as a number of modifications to protein structure by oxidizing the amino acid side chains, oxidizing the protein backbone forming protein-protein cross linkages and protein fragmentations [24, 25], as well as engendering protein glycation as described in section 1.2.3 and other forms of protein carbonyl groups [26]

1.3.3.3 DNA damage

ROS (mainly OH·) can elicit a wide variety of DNA damages, including base and sugar lesion, strand break (single and double), DNA-DNA and DNA-protein cross-linking, base modification, and the generation of apurinic and apyrimidinic

site (AP site), etc., several of which have been carefully characterized For

example, the DNA strand breaks generate the unusual 3’- PO4 and 5’- OH ends that cannot be used as substrates for DNA polymerases AP site is mutagenic as during DNA semiconservative replication, random nucleotide base will be inserted into the strand synthesized opposite it AP site is repaired depending on the activity of AP endonucleases In addition, a wide spectrum of oxidative base modifications occurs with ROS (Figure 2) The C4-C5 double bond of pyrimidine

is particularly sensitive to the attack by OH·, generating a variety of oxidative pyrimidine damage such as thymine glycol, uracil glycol, urea residue, 5-OHdU, 5-OHdC, hydantoin and others The thymine glycol that has not been repaired can block DNA replication and is thus potentially lethal to cells Similarly, interaction

of OH· with purines generate 8-hydroxy-2'-deoxyguanosine (8-OHdG), 8 hydroxy-2'-deoxyadenine (8-OHdA), formamidopyrimidines (Fapy-dA and -dG) and other less characterized purine oxidative products, among which, the extensively studied 8-OHdG is highly mutagenic 8-OHdG that has not been repaired canmispair with dA, leading to an increase in G to T transition mutations

Figure 2 Chemical structures of some stable oxidative DNA base lesions

1.3.4 Other functions of ROS

Not all the effects of ROS on cells are negative, as there is growing evidence that ROS are able to function in positive ways in cells, such as being involved in signal transduction in immune reactions, particularly in the activation of transcription factors (TFs) For example the activator protein-1 (AP-1) TF, a heterodimer of Fos and Jun or homodimer of Jun, shows a rapid increase in DNA-binding activity upon H2O2 exposure, which is independent of new protein synthesis This indicates that activation occurs because of post-translational modification in the Fos and Jun proteins Another TF that can be regulated by ROS is nuclear factor-kappa B (NF-κB), which plays an important role in regulating the transcription of many genes relevant to immunological responses [27] However, whether such regulations of TFs have any effects on aging process will be discussed in the following chapters Macrophages and neutrophils also generate ROS in order to kill the bacteria engulfed by phagocytosis [18]

1.3.5 Antioxidant defense systems

The ROS levels in most organisms are properly controlled since an antioxidant defense system is assumed to be involved in scavenging ROS and protecting the organisms against oxidative damage Wei and Lee (2002) demonstrated that the oxidative damages are mostly found in parallel with the declined capacities of antioxidant systems [28] Generally, there are two antioxidant systems, i.e enzymatic antioxidant defense system and non-enzymatic antioxidant defense system, which work in a coordinated fashion to neutralize the effects of ROS

1.3.5.1 Antioxidative enzymes

Generally, the primary antioxidative enzymes, including catalase (CAT), glutathione peroxidase (GPx), Cu/Zn-superoxide dismutase (cytosolic-SOD, SOD1) and Mn-SOD (mitochondrial-SOD, SOD2), are the first-line defense to detoxify ROS Both SOD1 and SOD2 are capable of catalyzing the dismutation of

O2·- to H2O2, which is further decomposed by CAT or GPx to water There are also glutathione (GSH) reductase that involves in the reduction of oxidized small molecules [29] and thioredoxin reductase in the reduction of protein thiols [20]; and there are other enzymes that maintain the reducing environment

1.3.5.2 Non-enzymatic molecules

The non-enzymatic antioxidant molecules include hydrophilic radical scavengers such as ascorbate (vitamin C) and glutathione, and lipophilic radical scavengers such as carotenoids and α-tocopherol (vitamin E), which are sometimes referred to

as ‘nutrition supplements’ as well [30] Maintaining the small antioxidant molecules in a state where they are able to participate in redox reactions and protect against ROS attack relies on two factors: the continuous supply of these molecules through dietary intake and the reduction of oxidized molecules via the glutathione and thioredoxin system [20]

1.3.6 The repair system

Unlike the extensively characterized defenses system, the mechanism of repairing oxidative damage is relatively unexplored Nevertheless, it is certain that the

repair system is another means utilized to control oxidative damage and maintain organism fidelity

1.3.6.1 Lipid repair

Removal of peroxidized lipids from the plasma membrane can help prevent further propagation reactions This process is carried out by enzymes such as phospholipase A2 (PLA2) [31] Lipid bilayers that have been oxidized are more susceptible to cleavage by PLA2, creating a free fatty acid and a lysophospholipid which can then act as substrates for reacylation reactions, generating intact phospholipids [31, 32] Additionally, it has been demonstrated that fatty acid hydroperoxides released into the cytosol are able to be reduced to their corresponding hydroxy fatty acids without hydrolysis as a function of GPx [33]

An enzyme with glutathione transferase (GST) activity to reduce lipid hydroperoxides has also been extracted from nuclei [34]

1.3.6.2 Protein repair

There are basically two kinds of protein repair, one is direct repair, and the other

is indirect repair One important direct repair process is the reduction of oxidized sulphydryl groups on proteins which is mediated by the disulfide reductase (glutathione reductase and thioredoxin reductase) [35] For example, when two adjacent sulphydryl groups in the cysteine residues within a protein oxidize, they form a disulfide bond called intramolecular disulfide cross-links The disulfide bond can also form between two proteins known as intermolecular cross-linking Both intramolecular and intermolecular disulfide cross-links can be reversed to

some extent by disulfide reductase within cells [35] Another reduction of oxidized sulphydryl group on protein is mediated by methionine sulfoxide reductase, which reduces methionine sulfoxide back to methionine residues thus protecting protein functions or other amino acid residues in the protein [36] However, the specific mechanism is not completely clear yet

Indirect repair of protein involves the function of proteases and proteasome In the cytoplasm and nucleus of eukaryotic cells, oxidized soluble proteins are firstly tagged with ubiquitin, and then sequestered to the proteasome complex which is rich in proteolytic enzymes for complete degradation The end product of amino acids are largely recycled to synthesize entirely new replacement protein molecules [22, 31] However, in the aging process, proteasome is gradually inhibited and loses its activity, resulting in the accumulation of non-degradable proteins [37]

1.3.6.3 DNA repair

DNA is constantly exposed to damaging agents from both endogenous and exogenous sources If these lesions are left un-repaired, oxidative DNA damage can lead to detrimental biological consequences in organisms, including cell death, mutations and transformation of cells to malignancy [38] In order to maintain the integrity of the genome, a complicated network of DNA repair pathways take effect to remove the majority of deleterious lesions Repair of damage in DNA is carried out through several repair pathways including the function of direct repair, nucleotide excision repair, base excision repair, mismatch repair, recombinational repair, trans-lesion synthesis and so on [38]

To date, there is only limited evidence supporting the direct repair of DNA

hydroperoxides by GPx in vitro [39] However, the extent to which DNA peroxides actually formed in vitro and could directly be repaired by glutathione

peroxidase have not been clearly studied yet On the contrary, a large portion of DNA oxidative damage is repaired through the other pathways including the ubiquitous nucleotide excision repair and base excision repair pathways The nucleotide excision repair pathway mainly removes DNA lesions that cause a structural deformation of the DNA helix Typical examples of such lesions are pyrimidine dimers and large hydrocarbon DNA adducts [38] The base excision repair pathway deals with the repairing of smaller damage to individual bases, such as oxidation, methylation, depurination, and deamination [38, 40] Take the latter base excision repair pathway in repairing 8-OHdG for example Repair is initiated by the attack of 8-oxoguanine DNA glycosylase 1 (OGG1) at the glycosydic bond, resulting in the loss of the damaged base An OGG1-associated lyase activity leads to phosphodiester cleavage 3' to the resulting AP site This is followed by breaking the phosphodiester bond 5' to the abasic site by AP endonuclease, leading to a one base gap This gap is filled in by DNA pol γ, and the newly created one nucleotide repair patch is sealed by DNA ligase III [41] In contrast to 8-OHdG, the repair of 8 hydroxy-2'-deoxyadenine (8-OHdA) which is less mutagenic than 8-OH-Gua is poorly understood, although this lesion is reported to be a potential target for repair by OGG1 as well [42] Some major repair proteins or pathways for the principle oxidative DNA base lesions are listed

in Table 3

Table 3 Major known repair proteins or pathways for principle oxidative DNA base lesions

(Table modified from the study of Cooke, M.S et al 2003, [42])

1.3.7 Synthesis: Interaction of ROS generation, defense, and repair systems

Due to the process of respiration, ROS are continuously generated from ETC in the mitochondria If the ROS formed are not removed quickly, a state of oxidative stress would occur, resulting in damage to important biomolecules as described previously Thus the level of oxidative stress that occurs in a system is dependent

on the balance among the processes of oxidant generation, antioxidant defense and repair of oxidative damage (Figure 3)

Figure 3 Interaction of ROS generation, defense, and repair systems

(Figure modified from the study of Beckman, K.B and Ames, B.N., 1998 [16])

1.4 The mitochondrial theory of aging

The mitochondrial theory of aging was first proposed in 1972 by Denham Harman [4], and further refined and developed in 1980 by Jaime Miquel [5] Now it is generally accepted by many investigators that the oxidative mitochondrial decay is

a major contributor to aging [43] It is admitted that there is very strong connection between the mitochondrial theory of aging and the free radical theory

of aging, yet the former concerns several other major biological topics that far more than free radicals only, which include genetics, membranes, and bioenergetics To understand the mitochondrial theory of aging, it is first necessary to have an overview of the mitochondrion and its pivotal role in the life

of biological organisms

1.4.1 The pivotal role of mitochondria in aerobic organisms

Mitochondria are organelles found at the range of 20 to 2500 per cell in virtually all aerobic organisms Mitochondria, the energy generators of the cell, typically produce 90% or more of all the ATP generated in the body The production of ATP within the mitochondria occurs from the coupling of two metabolic cycles namely the tricarboxylic acid (TCA) cycle (also known as the Krebs cycle or citric acid cycle) and the oxidative phosphorylation on the ETC Mitochondria play pivotal roles in glycolysis, fatty acid β-oxidation and generating energy that powers cellular activity, muscular activity, heart and brain activity, breathing,

walking, talking etc

1.4.2 Mitochondria electron transport chain

The mitochondrial ETC is located on the mitochondria inner membrane and has five complexes (Figure 4) They are NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), cytochrome bc1 complex (complex III), cytochrome

c oxidase (complex IV) and Fo-F1 ATP synthase (complex V) Firstly, the mitochondrial ETC plays an important role in energy production in aerobic organisms As mentioned above, the TCA cycle occurs in the matrix of mitochondria, and products of the TCA cycle such as nicotinamide adenine dinucleotide (NADH), flavine adenine dinucleotide (FADH2) and succinate are connected to the ETC to activate the first two enzyme complexes (I and II) Electrons from NADH and FADH2 flow down the ETC and eventually combine oxygen (the final electron acceptor) and hydrogen to make water at complex IV [41] ETC accounts for the majority (about 85-90%) of the total oxygen metabolized by the cell, and the by-products (e.g O2·-, H2O2 andOH·) generated mainly from complexes I and III are potential sources of oxidative damage to the mitochondria and other cellular compartments [41] At the same time, the passing

of electrons through the ETC is coupled with the establishment of a proton gradient by pumping protons from the matrix across the mitochondrial inner membrane into the cytoplasm at complexes I, III and IV Such proton gradient forms the mitochondrial membrane potential and is consider as the driving force for ATP production at complex V [44]

Mitochondria can function in five different energy states, but the major events are

in the state 3 and state 4 as the active and basal respiring state for producing ATP, respectively The sharp drop in state 3/state4 energy production with aging is

indicative of significant mitochondrial ETC damage [45] Succinate-supported respiration can be determined polarographically in an oxytherm, equipped with a Clark oxygen electrode The respiratory control ratio (RCR) is measured as the ratio of the rate of oxygen consumption in state3 to states 4 respiration (Figure 5)

Figure 4 Mammalian electron transport chain complexes (I–V)

Figure 5 The schematic representation of RCR measurement

1.4.3 Mitochondria genome

One of the unique features of mitochondria is that they contain their own genome known as mitochondrial DNA (mtDNA) In the human beings, the mtDNA is a closed circular molecule containing about 16,500 bp and encoding for 13 ETC enzyme proteins, 2 ribosomal RNAs, and 22 transfer RNAs, all of which are needed to form the mitochondrial ETC protein synthesis system [46] (Figure 6) The remainder of the ETC enzymes and other mitochondrial components are encoded by nuclear DNA (nDNA)

Figure 6 Human mitochondrial genome

Human mitochondrial genome containing about 16,500 bp and encoding for 13 ETC enzyme proteins including complex I (blue), complex III (pink), complex IV (red) and complex V (green), 2 ribosomal RNAs (yellow), and 22 transfer RNAs (white) The position of 5 kb deletion is indicated This figure is adapted from MITOMAP [47]

1.4.4 MtDNA damage

It is considered that the damage to mtDNA probably has more relevance to the mitochondrial theory of aging than to the damage to lipid, protein or any other form of mitochondria components because damage to mtDNA can be propagated

as mitochondria and cells divide, thus allowing the physiological consequences of the damage to be amplified MtDNA, due to its proximity to the ETC, is prone to oxidative injury And unlike nDNA, mtDNA has no histone protection or significant enzymes repair systems, so mtDNA is far more subject to free radical damage than nDNA [48] As mtDNA damage accumulates over the lifetime of an individual, the functionality of the ETC which is partially encoded by mtDNA decreases dramatically and gradually produces a cellular energy crisis Linnane and his associates found that only 5% of the total mtDNA from muscle tissue of a

90 year old man was still in the form of full-length [46] Along with this cumulative mtDNA damage, there was a large percentage of cells lacking complex IV on the ETC [46] Damage to the mtDNA is especially prominent in post-mitotic tissues such as brain, heart, skeletal muscle and it may be in the forms

of base modifications, AP sites, large deletion and point mutation

One of the most extensively studied base modifications is the formation of OHdG in the aging process Studies using HPLC-electrochemical detection of 8-OHdG suggests that mtDNA is more susceptible to damage than nDNA [49], and

8-it is also suggested that 8-OHdG levels inversely correlated w8-ith maximum lifespan, while there was no correlation between 8-OHdG in nDNA and maximum lifespan [50] However other researchers pointed out that the formation of more 8-OHdG in mtDNA is because of the artifact caused during DNA preparation [49]

Moreover, the high 8-OHdG level in mtDNA is strongly correlated with a 5 kb deletion in mtDNA as well [48], which is considered as a result of the base mismatching caused by point mutations due to the formation of 8-OHdG and other oxidative adducts [51] This large deletion is demonstrated to be in the region encoding subunits of the complex I and IV of the ETC [52].

Another form of mtDNA damage is the age-related large accumulation of point mutations present in the D-loop region of mtDNA This region is the main control region of mtDNA, containing two initiation sites for transcription of the heavy (H)-strand, the promoter for transcription of the light (L)-strand, which is also the promoter for the RNA primer of the H-strand synthesis and the primary (H1) and secondary (H2) initiation sites for H-stand synthesis [53] However, it should be noted that most of the point mutation were identified in the fibroblasts isolated from young and old individuals that still actively divided in culture, while the tissues that are most often affected by disorders related to mtDNA deletions is usually post-mitotic [53]

1.4.5 MtDNA repair

The mtDNA repair in principle is very similar to nDNA repair (Figure 7), in which the process involves the steps of damage recognition, excision, and re-synthesis However, it is obvious that mtDNA repair is largely dependent on the base excision repair, and the functional enzymes in the pathways are relatively simple

Figure 7 Mitochondrial and nuclear DNA repair pathways

DR, direct repair; NER, nucleotide excision repair; BER, base excision repair; MMR, mismatch repair; RER recombinational repair; TLS, trans-lesion synthesis (Figure modified from the study of Larsen, N.B., et al., 2005, [38])

1.4.6 Other forms of mitochondria damage

1.4.6.1 Lipid oxidative damage

Similar to the plasma membranes, mitochondria membranes are sensitive to oxidative damage as well One of the major by-products of lipid peroxidation is 4-HNE, which has been shown to have a variety of detrimental effects, such as causing decreased membrane fluidity, restricting the normal ability of the proteins and lipids to freely diffuse and consequently affecting membrane function [54] 4-HNE is also capable of causing cellular damage by reacting with adjacent protein thiols, which results in the structural and functional modification of the proteins And it is the very fact that proteins take up about 80% of the mitochondrial inner membrane compared to about 50% of the plasma membranes, which makes the protein oxidative damage triggered by oxidized lipid even prominent

1.4.6.2 Protein oxidative damage

As mentioned above, about 80% of the mitochondria inner membrane is composed of proteins, several of which are crucial mitochondrial ETC enzymes [55] The dysfunction of these ETC components as a result of protein oxidation might lead to the decrease in ETC activity and even impaired mitochondrial function One of the severe consequences of reduced ETC activity is the increased production of ROS Inhibition of electron transport results in the accumulation of electrons and generation of excess ubisemiquinone that can directly donate electrons to molecular oxygen, producing superoxide anions [55] Meantime, due

to the inhibition of electron transport, mitochondrial membrane potential will

decrease consequently Age-related decreases in membrane potential have been shown to occur in various tissues such as liver and brain [56] The decreased mitochondrial potential is possibly related to the activation of mitochondrial permeability transition pore (PTP) which would in turn further collapse the mitochondrial membrane potential, inhibit oxidative phosphorylation and Ca2+sequestration by mitochondria, and may induce apoptosis or necrosis [57] The current evidence suggests that several ETC components, such as ATPase and the adenine nucleotide transporter (ANT), are particularly sensitive to oxidative damages [58, 59] And mitochondrial protein oxidation may also be directed at specific proteins as opposed to being a random process For example, protein carbonyls appear primarily on ANT in mitochondrial membranes [60] and on aconitase in the mitochondrial matrix in house flies [61] In the cases of both ANT and aconitase, the age-related increase in protein carbonyls is associated with a decrease in the activity of the protein with age

1.4.6.4 Mitochondrial turnover

It is proposed that the dysfunctional mitochondria probably as a result of the oxidative attack should be sequestered to phagolysomes and degraded there, however, most of the times they are found accumulated intracellularly with age Some research attributed the decreased rate of mitochondria degradation to a defect in the phagolysomal system in old animals [64] Such hypothesis is supported by the finding of declined autophagic process in the liver of old rats [65]

1.5 Anti-aging strategies

In the modern world, with the development of better health care system and better living conditions, people have a higher expectation of life quality and quantity However, aging and aging-related diseases have seriously threatened human health and adversely affected the quality of people’s life Gradually, aging problems have become a grave concern and aging related studies have become increasingly popular According to the NIH statistics in 2006, the US funding has gone up from USD 2211 million in 2003 to USD 2400 million in 2006 for the aging research in general, not including research in many specific areas such as Alzheimer’s and Parkinson’s diseases Of course the achievements obtained so far have been encouraging with a lot of discoveries and breakthroughs Many theories and mechanisms underlying aging have been proposed by different research groups and extensive in-depth studies have been conducted Based on the free radical theory of aging, many experiments have been designed to delay the aging process The principal strategies currently used include calorie restriction (CR),

transgenic organisms, pharmacological antioxidant administration, and dietary antioxidant supplementation

1.5.1 Calorie restriction

It has been known since 1936 that a decreased calorie intake in mice and rats, without malnutrition, extends the maximum lifespan [66] CR-induced increase in maximum lifespan has also been shown in fish, spiders, water-fleas, and some other non-rodent species [66] Although no studies have yet been completed on primates, ongoing studies (primarily with rhesus monkeys at the University of Wisconsin [67]) have shown that important physiological effects of CR seen in rodents (such as decreased blood glucose and insulin levels, improved insulin sensitivity and lowering of body temperature) are also observed in rhesus monkeys [68]

Till now, CR is still the only nutritional intervention that slows down the intrinsic aging [69] It is supposed that CR takes effects mainly through two mechanisms One is to slow down the metabolic rate, and the other is to reduce the ROS production It has been reported that rodents subjected to CR could reduce age-associated mitochondrial ROS generation, slower accumulation of oxidative damage, decrease lipid peroxidation and delay age-associated loss of membrane fluidity [66] In virtue of the high-density oligonucleotide arrays, a series of genes have been identified regulatable by CR in the skeletal muscle of mice [69], and the possible related pathways have been classified as evolving in increased protein metabolism, energy metabolism and biosynthesis but decreased macromolecular damage by suppression of heat shock factors, detoxification systems and the

inducible DNA repair systems, as compared to the natural aging process [69] The regulation of IGF-1 pathway is also considered to play an important role in the CR intervention, which will be discussed in the next section [70]

However, considering the degree and length of restriction required, the utility of

CR intervention on human beings is yet feasible Alternatively, the development

of CR mimetics, which are compounds that mimic CR effects by targeting metabolic and stress response pathways affected by CR but without actually restricting caloric intake, becomes promising [71]

Besides, a more direct relation of ROS production, antioxidant defense and aging phenomenon is specified by over-expression of SOD and CAT in Drosophila, which significantly increases their lifespan [79] And recently it has been found that targeting the gene of CAT to mitochondria in the transgenic mice considerably extended their lifespan and significantly delayed the aging-related disorders including cardiac pathology and cataract development [80] Although the gene therapy is promising, its application on human being is still problematic,

as the safety is not fully guaranteed as well as some ethical concerns still exist

1.5.3 Dietary nutrition supplements

Dietary nutrition supplementation is gradually accepted as a practical anti-aging strategy considering its safe administration route and generally controllable side-effects Although several researchers claim that most of the times supplementations failed to extend maximum lifespan in mice [81], the effectiveness of nutrition supplements in ameliorating aging and aging-related disorders is generally agreed Moreover, the extension of health span instead of lifespan shall be considered as an important achievement for the aging studies [82]

As mentioned before, there are several forms of nutrition supplements, including antioxidants, antioxidant mimetics, mitochondria metabolites, minerals and

hormones, etc

1.5.3.1 Antioxidant

It has long been known that taking antioxidants such as vitamin C and E is beneficial for human beings Research also proves that vitamin E could increase

the lifespan of C elegans [83] Up to now, there have been numerous positive reports published from both animal and human studies It has been found that dietary supplementation with antioxidants improves functions and decreases oxidative stress of leukocytes from prematurely aging mice [84] According to Nelson JL, moderate antioxidant supplementation differentially affected serum carotenoids, antioxidant levels and markers of oxidative stress in older humans

[85] Proponents believe that antioxidants can prevent chronic diseases, such as

heart disease and diabetes And recently, resveratrol has been shown to have effect

on improving health and survival of mice on a high-calorie diet [86] However, it

is noticeable that the effectiveness of antioxidant supplement is usually low, and long-term supplementation is always needed Hence, it urges scientists to develop much efficient antioxidant In general, despite a few negative results, taking nutrition supplements seems to show great promise in the aging prevention

1.5.3.2 Flavonoid and EGCG

Flavonoids refer to a class of plant secondary metabolites based on a phenylbenzopyrone structure, and they are commonly known for their antioxidant activity The sources of flavonoids include all citrus fruits, onion, red wine, dark chocolate, and green tea with the greatest abundance Green tea (beverage) is one

of the most popular drinks in the world and is especially welcomed in Asia It is believed that green tea has a spectrum of beneficial effects and may improve the general well-being of humans The main components of green tea extract are catechins containing (-)-epigallocatechin-3-gallate (EGCG), (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC) Among these

components, EGCG takes up the largest percentage (65%), and probably accounts for most of the green tea’s bioactivities Therefore, most studies so far have chosen to focus on EGCG as the subject instead of the composite green tea mixture The structure of EGCG is shown in Figure 8

Figure 8 The structure of (-)-epigallocatechin-3-gallate (EGCG)

EGCG (C22H18O11); MW= 458.38

A number of studies have shown convincingly that EGCG has prominent cancer chemopreventive activity For example, taking green tea or EGCG can prevent various cancers such as breast cancer, prostate cancer and lung cancer to some extent [87, 88] Several in-depth studies have indicated that an oxidative environment is in favor of tumor growth [89], thus most of the cancer chemopreventive activity of EGCG can be attributed to its radical and oxidant scavenging activity Compared with other antioxidants such as vitamin C and E, which are the common golden standards to evaluate antioxidative effect, EGCG shows stronger ROS scavenging activity [90] EGCG can also reduce ROS concentration by suppressing some pro-oxidant enzymes, such as inducible-nitric oxide synthetase (iNOS), cycloxygenase-2 (COX-2) and xanthine oxidase (XO)

[91], thus controlling the tumor growth Besides, EGCG is capable of causing cell cycle arrest or apoptosis by caspase activation in the cancer cells [92, 93] Inhibition of transcription factor-mediated gene activation such as NF-κB and AP-

1 can be achieved by EGCG as well [94] The study by Naasani and his colleagues

also suggested that the block of telomerase by EGCG was a major mechanism of

limiting the growth of human cancer cells both in vitro and in vivo EGCG can

also effectively control angiogenesis and tumor invasion [95, 96] The molecular mechanism is probably through inhibiting metalloproteinases activity in tumor

cells and vascular endothelial growth factor receptor expression in endothelial

cells [97, 98] Besides the chemopreventive activities, EGCG has also been reported to have a variety of other beneficial effects such as anti-mutagenic [99,

100], anti-diabetic [101], hypocholesterolemic functions [102], promoting energy

expenditure [103], and preventing the development of atherosclerotic plaques [104] Recently, however, the clinical trials carried out by Nagle DG and his colleagues pointed out that the concentration needed for EGCG to take effect was actually much higher than the normal level achieved by just drinking tea or taking EGCG supplements [105], further confirmation of which is needed

1.5.3.3 Other forms of nutrition supplements

Recent research has pointed out that synthetic SOD/CAT mimetics are able to increase lifespan of C elegans [106], which supports the idea that pharmacological intervention in the aging process is possible Other mitochondria

metabolites, such as acetylcarnitine and lipoic acid, also have effect in preventing mitochondrial decay [107] The endocrine hormones such as melatonin is believed

to play an important role in preventing oxidative damage including lipid peroxidation, cataract induction as well as nuclear DNA mutation after ionizing radiation [108] Actually it has been found that melatonin has multiple antioxidant roles [109] Another group of hormones, estrogens are proposed to be responsible for the cause of longevity in females compared to the males, as estrogens are capable of inducing GPx and SOD2 expressions Besides hormones, dietary supplementations with minerals such as zinc or selenium are also effective in reducing age-related oxidative damages Coenzyme Q is also reported to significantly increase lifespan in rats fed with a diet rich in polyunsaturated fatty acids [110] In this sense, all of these compounds or molecules mentioned above can be considered more or less as antioxidants

1.5.3.4 Side effects

However, significant side-effects can result from the use or abuse of some nutrition supplements Potential side-effects might include the increased risk of blood clots, heart attack, breast cancer, stomach disorders, gastrointestinal upset, nausea, and diarrhea and so on It has been reported that 18-month B6 male mice fed with vitamin E, glutathione, melatonin, and strawberry extract had almost the same chance of pathological occurrence and same mean and maximum life span

as the control mice, despite the reduced level of lipid peroxidation [111] Antioxidants, especially β-carotene (precursor of Vitamin A), might increase the rate of development of cancers in high risk individuals [112] And some systematic reviews and meta-analyses of the randomized trials have demonstrated that β-carotene, vitamin A, and vitamin E in the administered dosages possibly

lead to increased mortality [113-115] Therefore, such anti-aging regimens should

be applied with special cautions before the underlying mechanism and the potential side-effects are clearly understood

2 Research objectives

2.1 Hypothesis

Based on the above scientific observation and evidence, we hypothesize that in the natural aging process, mitochondria functional decay and antioxidative enzyme activity decrease result in the excessive accumulation of ROS and concomitant oxidative damages (Figure 9A) However, aging and aging-related disorders can

be regulated through the dietary supplementation of antioxidants, which might have effects on inhibiting oxidative damage, minimizing mitochondrial decay as well as regulating enzymatic antioxidant defense (Figure 9B)