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

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.

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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

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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 sideeffects 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.)

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

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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].

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

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1.2.4 The immunological theory of aging
"Immunosenescence" was first used by Dr. Roy Walford to describe the ageassociated 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.

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

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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

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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

daf-2

sod1

prop-1

lac1

age-1/daf-23

cat1

p66shc

ras2

akt-1/akt-2

ras1

phb1

daf-16

daf-18

phb2

daf-12

mth

cdc7

ctl-1

mclk1

bud1

old-1

rtg2

spe-26

rpd3

clk-1

hda1

mev-1

sir2
sir4-42
uth4
ygl023
sgs1
rad52
fob1
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

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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

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

ROS

Formula

Characteristics

Hydroperoxyl

HOO·

Strong oxidant, lipid soluble

Hydroperoxide

LOOH

Half-life time#

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 microsecond

Superoxide

O2·-

Good reductant, poor oxidant

One microsecond

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

anion
Hydroxyl
radical

oxidant, no diffusion, very reactive
Hydrogen

H2O2

peroxide
Singlet oxygen
Hypochlorous
acid

Oxidant, high diffusion, low

Stable

reactivity
1

O2

Strong oxidant, short half life

One microsecond

HOCl

Found in neutrophil phagosomes,

stable

strong oxidant

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])

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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 vitro is
able to move freely across membranes, yet is still poorly reactive in aqueous
solution. The majority of intracellular damages caused by H2O2 and O2·- is actually
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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 radicaldriven 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

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

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

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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 can mispair with dA, leading to an increase in G to T transition mutations.

Figure 2. Chemical structures of some stable oxidative DNA base lesions

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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 factorkappa 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.

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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

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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

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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].

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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.

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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])

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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])

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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.

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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 and OH·) 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

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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

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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].

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