Science advances on Environment, Toxicology &
Ecotoxicology issues
www.chem-tox-ecotox
On the occasion of Nobel Prize 2009: The Nobel Prize in Physiology and Medicine 2009 was
given jointly to Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak for the discovery
of “how chromosomes are protected by telomeres and the enzyme telomerase.
“Oxidative Stress, Oxygen Free Radicals and Telomere Shortening.
A Biomarker of Ageing Determined by
Environmental and Genetic Factors”
Prof. A. Valavanidis & Dr. Thomais Vlachogianni
Department of Chemistry, University of Athens,
University Campus Zografou, 15784 Athens, Greece
E-mail: valavanidis@chem.uoa.gr
& thvlach@chem.uoa.gr
Abstract
Telomeres are nucleoprotein structures located at the ends of chromosomes and are subjected
to shortening at each cycle of cell division. Chromosome are the threat-like DNA molecules that
carry the genes of living or organisms. Telomeres prevent chromosome ends from being
recognized as double-strand breaks and protect them from end to end fusion and degradation.
By contrast to stem cells and germ-line cells, telomeres in somatic cells shorten with each cell
division leading to cellular senescence (ageing). The enzyme telomerase (cellular reverse
transcriptase) is involved in telomere stability by synthesizing a new copy of the repeat by using
its RNA template. Regulation of telomerase activity is an area of intense investigation, but its
exact mechanisms are not yet elucidated. The contribution to telomere loss by oxidative DNA
damage Senescence cells were found to contain 30% more oxidative modified guanine in their
DNA. Since oxidative modifications and shortening by reactive oxygen species (ROS) leads to
againg of the somatic cells, it is expected that antioxidants and reactive free radical scavengers
may play a preventive role and possess anti-ageing properties. Additional evidence for the role
of ROS in telomere shortening was associated with chronic oxidative stress and inflammation.
Various models have been developed and applied to investigate the relationship between
telomere length and oxidative stress. The studies reviewed in this report-review make the basis
for a promising genetic marker for chronic oxidative stress, the role of antioxidants and their
influence on telomere length and ageing. Future studies should investigate the genetic
determinants of telomere shortening and stability and to assess the effects of environmental
factors that increase oxidative stress and chronic inflammation.
Keywords: telomere; telomerase; oxidative stress; chronic inflammation; ageing.
.
Introduction: Telomeres and their role in biological systems
Every nucleus of an eukaryotic cell is packed with chromosomes, the long, threat-like DNA
molecules that carry the genes of living organisms. In the end of the chromosomes there is a
region of repetitive nucleoprotein structure called telomeres, which prevent chromosomal ends
from end to end fusion and degradation.1,2 Telomeres consist of stretches of repetitive DNA with
high guanine and cytocine content. In humans, the telomere terminus consists of 4-15 kbp (bp =
base pairs) of the hexanucleotides 5’-TTAGGG-3’. The G-rich DNA strand runs 5’ to 3’ toward the
terminus and protrudes 100-150 nucleotides beyond the complementary C-rich strand. It is
protected from degradation by intercalation into the double-stranded telomere DNA, forming a
telomeric loop (t-loop).3-6
Figure 1. Telomeres at the end of chromosomes protecting from fusion and degradation.
It has been observed that in most proliferating cells the telomere length was a dynamic factor
and each time there is a cell division, the lengths of telomeres in human somatic cells decrease
gradually by 20 to 200 base pairs. This loss of base pairs is a consequence of the so-called endreplication problem that intrigued molecular biologists for years. Thus, the enzyme that
replicates chromosomes (DNA polymerase) is not able to replicate them completely. As a
consequence, the replication machinery must leave a small region at the end of the telomere
un-copied. The end-replication problem of shortening every time there is a cell division will lead
eventually to the elimination of telomeres which in turn will lead to apoptosis or an irreversible
growth arrest of the cells. These ageing cells (senescent) are irreversibly arrested in the G1
phase of the cell cycle. Except of the end-replication problem, there are also other factors
contributing to the telomere shortening.7,8
The phenomenon of limited cellular division was first observed by Leonard Hayflick, and it was
established as an important limit to how long a cell can remain active (Hayflick limit). It was
found that the limit of human cell division in subcultures is around 40-60 times before dying
out.9
As early as the 1930s Hermann Muller (Nobel Prize 1946) and Barbara McClintock (Nobel Prize
1983) had observed that the structures at the ends of the chromosomes, the so-called
telomeres, seemed to prevent the chromosomes from attaching to each other. They suspected
that the telomeres could have a protective role, but how they operate remained a mystery.
Elizabeth Blackburn was studying the chromosome of Tetrahymena, a unicellular ciliate
organism, when she identified a DNA sequence that was repeated several times at the ends of
the chromosomes. At the same time Jack Szostak had made the observation that linear DNA
molecule, a type of minichromosome, is rapidly degraded when introduced into yeast cells. From
the Tetrahymena, Blackburn isolated the CCCCAA sequence, the function of this sequence was
not clear. Szostak coupled it to the minichromosomes and put them back into yeast cells. The
results were striking. The telomere DNA sequence protected the minichromosomes from
degradation. As telomere DNA from one organism, Tetrahymena, protected chromosomes in an
2
entirely different one, yeast, this demonstrated the existence of a previously unrecognized
fundamental mechanism. Later it became evident that telomere DNA with its characteristic
sequence is present in most plants and animals, from amoeba to man.10
Then, Carol Greider (graduate student) and her
supervisor Blackburn started to investigate if the
formation of telomere DNA could be due to an
unknown enzyme. In 1984 they discovered signs of an
enzymatic activity in a cell extract. They named the
enzyme telomerase. After purification, they showed
that it consists of RNA as well as protein. The RNA
component turned out to contain the CCCCAA
sequence and serves as a template when the
telomere is built, while the protein component is
required for the construction work (enzymatic
activity). Telomerase extends telomere DNA,
providing a platform that enables DNA polymerases
to copy the entire length of the chromosome without
missing the very end portion. Scientists began to
investigate the role of telomeres might play in the
ageing of cells, but also in the organisms as a whole.
Although the ageing process turned out to be more
complex and depending on genetic and several other
different factors, telomere shortening is one of
them.
Professors Blackburn E, Szostak J and Greider C, for their discoveries and the importance of
telomeres and telomerase in cell biochemistry and molecular biology, received the Nobel Prize
for Physiology or Medicine in 2009.10
3
The discovery of teleomeres and telomerase had a major impact within the scientific
community. Many scientists started to speculate on the role of shortening of telomeres in ageing
of human and other aerobic organisms. Most normal cells do not divide frequently, therefore
their chromosomes are not at risk of telomere shortening and they do not require high
telomerase activity. In contrast, cancerous cells have the ability to divide infinitely and preserve
their telomeres by increasing their telomerase activity. Several studies investigated the role of
telomerase and tried to eradicate or stop the growth of cancer cells. Clinical trials evaluated
vaccines directed against cells with elevated telomerase activity. Some inherited diseases are
known to be caused by telomerase defects (congenital aplastic anemia, skin and lungs diseases).
The discovery of telomere and telomerase and their role stimulated the development of
potential new therapies.
The Influence of Certain Biochemical Factors in Telomere Regulation
In contrast to stem cells and germ-line cells, telomeres in somatic cells shorten with each cell
division. Cell proliferation is therefore considered to be one of the most important causes of
telomere shortening. Human diploid fibroblasts have a limited replicative life span in vitro due
to telomere attrition, leading eventually to ageing (senescence).11 Telomerase is a cellular
reverse transcriptase that consists of two components, a reverse transcriptase subunit (hTERT)
and a telomerase RNA component (hTERC).12 The telomerase enzyme recognizes the tip of a Grich strand of a telomeric DNA repeat sequence and elongates the telomere in the 5’3’direction, then synthesizes a new copy of the repeat by using its RNA template. The exact
mechanims of regulation of telomerase activity, despite of intense scientific investigation, is not
yet elucidated. Human telomerase ribonucleproteins pass through several stages before they
start with telomere elongation.13,14 In most human somatic cells telomerase activity is absent or
very low, in contrast to stem cells and germ-line cells which express sufficient telomerase to
maintain the length and function of telomeres for infinite time.15,16
Oxidative Stress and Telomere Shortening
Telomeres demonstrated high sensitivity to damage by oxidative stress due to their high content
of guanines.17,18 In adition, oxidative damage to nucleobases has been reported to accumulate
over the life span of a cell or an organism, contributing significantly to senescence (ageing).
Senescet cells were found to contain 30% more oxidative modified guanine in their DNA and four
times as many free 8-oxodG bases (8-oxo-deoxyguanosine).15 Furthermore, oxygen free radicals
and ROS, especially hydroxyl radicals (HO•) , produce single-strand breaks, either directly or as
an intermediate step in the repair of oxidative base modifications. In contrast to the majority of
genomic DNA, telomere DNA was reported to be deficient in the repair of single-strand breaks.19
Evidence for this increased sensitivity to ROS-induced was observed from two studies in the calf
thymus DNA , when H2O2 reacted with Cu2+ , measured by HPLC (High-performance liquid
chromatography) with an electrochemical detector (HPLC-ECD) and in W1-38 fibroblasts which
were irradiated with UVA.20,21.
Oxidative lesions on telomeric DNA by ROS makes also repair less efficient than the rest of the
genome. Exposed human fibroblasts to hydrogen peroxide (H2O2), a prominent ROS contributing
to metabolism of aerobic cells, was shown to increase the frequency of single-strand breaks in
telomeres and the repair was slow or incomplete, compared to minisatellites. There are various
explanations to the repair deficiency of telomeres, one assumes that the binding of TRF2
(telomeric repeat binding factor) to telomere blocks the access of DNA repair enzymes to
telomere strand breaks, or that TRF2 inhibits the phosphorylation of the ATM kinase (ataxia
telangiectasia mutated gene), which leads to an impaired DNA damage response.23,24
It is well known that ROS produce oxidative stress through inflammatory conditions. Oxidative
stress is associated with increased pro-inflammatory cytokines.25
Studies showed that
telomerase activity was negatively correlated with pro-inflammatory cytokine tumor necrosis
4
factor alpha (TNF-α).24, 26 The TNF-α activates nuclear factor kappa B (NF-κB) and activator
protein-1 (AP-1) transcription factors, thus enhancing the expression of pro-inflammatory genes,
leading to an inflammatory response.27
The autosomal recessive disorder Ataxia telengiectasia (A-T). Ataxia-telangiectasia is a rare,
childhood neurological disorder that causes degeneration in the part of the brain that controls
motor movements and speech. Its most unusual symptom is an acute sensitivity to ionizing
radiation, such as X-rays or gamma-rays. The disease has a strong association with chronic
oxidative stress and telomere shortening. In most cases mutations of the ataxia telangiectasia
mutated gene (ATM) account for this disease, because ATM encodes a protein kinase that is
activated in response to DNA strand breaks and is thought to be essential for maintaining
chromosomal stability and telomere integrity. Experimental results suggest that A-T cells are in
a chronic state of oxidative stress and have a defective maintenance or repair of telomeric DNA,
contributing to their enhanced telomere shortening.28
More evidence for the role of oxidative stress in A-T was obtained from a study in which markers
of the redox status in brains of ATM-deficient mice were analysed. These mice showed a
significant increase in the activity of thioredoxin and MnSOD (Mn-Superoxide dismutase) and a
significant decrease in Catalase activity in the cerebella, a good indication of increased levels of
free radicals and ROS.29
Can Antioxidants Vitamins Reduce Telomere Shortening?
These results and the role of oxidative stress in telomere shortening, inevitably directed
biological experiments to the use of antioxidant vitamins in the prevention of telomere
shortening.30 Some studies supported this suggestion. It was found that age-dependent telomere
shortening in human vascular endothelial cells, in vitro, could be slowed down by an ascorbic
acid derivative (ascorbate-2-O-phosphate). It led to an extension of the cellular life span and
prevented cell-size enlargement (cellular indication of senescence).31 The same treatment of
human embryonic cells with ascorbic acid phosphoric ester magnesium salt decreased the level
of oxidative stress, prevented telomere attrition and extended the replicative life span of
cells.32 Another study in vitro used chondrocyte senescence (risk for cartilage degeneration).
These cells, chondrocytes, cultured in the presence of the oxidant H2O2 showed that oxidative
stress induced telomere shortening and replicative senescence, but in the presence of
ascorbate-2-O-phosphate the shortening was reduced.33
Although in vitro experiments showed positive results with antioxidants, in vivo experiments are
considered more important because other biological factor might influence the telomere
shortening. Male and female rats were used for these type of experiments. Several oxidative
stress markers were studied and the results showed that female rats exhibited longer telomeres
than male rats. Expression levels of certain antioxidant enzymes, such as MnSOD, GPx I,
(glutathione peroxidase I) and GRx (glutathione reductase), in the renal cortex and medulla
were found to be higher in female than in male rats. The explanation is that female rats have
higher levels of estrogens, which enhance gene expression of MnSOD. The decreased antioxidant
levels may be partially responsible for the age-related kidney telomere shortening.34,35
Proliferating fibroblasts exposed to buthionine sulfoximine, which depletes the antioxidant
enzyme reduced glutathione (GSH, reduced Glutathione is a linear tripeptide, the molecule has
a sulfhydryl (SH) group on the cysteinyl portion, which accounts for its strong electron-donating
character), decreased telomerase activity, whereas repletion of cells with glutathione increased
telomerase activity in a dose-dependent manner.36
The Role of Oxidative Stress, Chronic Inflammatory Diseases, Prolonged Exercise, Obesity
and Smoking in Telomere Length
Studies investigated the association between telomere length and diseases in which
inflammation and oxidative stress play an important role. In vitro study which investigated the
relationship of homocysteine (excerts atherogenic effects because of increased H2O2 generation)
5
and ageing, found that the rate of telomere shortening was increased in homocysteine-treated
cells. It was observed that homocysteine treatment resulted in a threefold increase in the rate
of telomere shortening. This effect wad attenuated by the addition of the antioxidant enzyme
Catalase (CAT).37
It was hypothesized that vascular pathology might be connected with telomere shortening.
Circulating leukocytes have been investigated and it was found that coronary and carotid
atherosclerosis, premature myocardial infraction and hypertension are associated with telomere
shortening.38-40
Other studies with animals showed a link between telomere length and oxidative stress and
atherosclerosis. Hypertensive rats showed telomere attrition in their kidney.41 Diabetes has also
been associated with telomere shortening. Patients with diabetes type 2 are known to suffer
from proinflammatory state with oxidative stress.42,43 Telomere shortening was not evident in
patients with ulcerative colitis or Crohn’s disease and patients with inflammatory bowel
disease. 44,45
Muscle biopsies from athletes with exercise-associated fatigue were compared with those from
controls. Subject were matched for age, gender, height, weight and training history. It was
demonstrated that these athletes had significant shorter telomeres than their controls.46
Psychlological pathologies (schizophrenic and bipolar disorder patients, psychological stress)
were found also to contribute to shortening of telomeres.47,48 Women with obesity (higher body
mass index), who were also smokers and had lower vitamin use, were found to have shorter
telomeres.49
Smoking and obesity have been shown through clinical studies that are important factors in many
age-related diseases and are associated among other things with oxidative stress and
inflammation. But lately it was observed that have also shorter telomeres.50,51 All these diseases,
in which inflammation plays a major role, cause telomere shortening.52
Telomere Length as a Biomarker of Oxidative Stress, Disease Progression and Ageing
In the last decade scientists worked extensively to promote various models of in vitro studies
with different cell lines to investigate the association between telomere length and oxidative
stress, but also progression of diseases and ageing.53,54
In contrast to humans, rodent telomeres are generally much longer and express telomerase in
many tissues. Telomere shortening is broadly studied in mice, especially in telomerase knockout
mice.55,56 However, it is only after 4-6 generations that telomere shortening becomes a critical
issue in these mice, indicating mechanisms of carcinogenesis and ageing.57 Scientists noticed
that rat telomeres do not have to be modulated to detect shortening, so the rat model might be
more feasible in studying the effect of oxidative stress and ageing. Telomere length assessed in
Wistar rats in the kidney, liver, lung and brain may determine life span (longevity). It was found
that male rats have shortened life span compared to females.58,59
Dogs is another animal that has been used in experiments for telomere biology. It has been
observed that dos share more similarities in telomere and telomerase biology with humans.60
Telomere length has been investigated in chickens, bird species and cattle, as models for
applicability of the research to human telomere and telomerase activity.61,62
These studies indicate that telomere length and telomerase activity is a promising genetic
biomarker for chronic oxidative stress, but there is a need for more experimental investigation
to establish the role of the various factors, such as heredity and other ageing.63,64 Also, there is
a need for more research on the role of telomere shortening on inflammatory diseases
progression, cancer and ageing.65,66 Although there is a long way to be able to assess the effects
of environmental factors in telomere attrition, future studies should aim to investigate also
genetic determinants of telomere stability and other intrinsic and extrinsic factors leading to
cell senescennce.67-70
6
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