Download relationship between type-1 diabetes and oxidative dna damage

RELATIONSHIP BETWEEN TYPE-1 DIABETES
AND OXIDATIVE DNA DAMAGE
Presented by
Rami Hatem Tayeb
Supervised by:
Dr. Refaat I El-Fayuomi
A Dissertataion Submitted
In Partial Fulfillments For
The Bachelor Degree In Medical Technology
At
Medical Laboratory Technology Department
Faculty Of Applied Medical Sciences
King Abdulaziz University
May 2011
١
Chapter one:
Diabetes mellitus:
1.1 Introduction
1.2 Type of diabetes mellitus
1.3
Sign and symptom
1.4 Diagnosis and treatment
Vitamin D:
1.5 Introduction
1.6 Metabolism
1.7 Vitamin D deficiency
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1.8 Diagnosis and treatment
Oxidative DNA Damage:
1.9 Introduction
1.10 Mechanism of oxidative DNA damage
Vitamin D and Diabetes:
1.11 Introduction Oxidative DNA damage and Diabetes:
1.12
Introduction
Oxidative DNA damage and Vitamin D:
1.13
Introduction
CHAPTER TOW
2.1
Research design
2.2
materials
2.3 methods and technique
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CHAPTER THREE
3.1
Result
3.2
diabetes status
3.3
biochemical parameters
3.4
DNA Damage
CHAPTER FOUR:
4.1
Glucose and HbA1c
4.2
biochemical parameters
4.3
Vitamin D
4.4
DNA damage
CHAPTER FIVE
5.1
Conclusion
CHAPTER SIX
6.1
Reference
٤
DEDICATION
To me dear father and mother,
Allah 's gift to me , who keep in giving and
giving and giving ,whose unceasing
commitment and dedication to my education
and future have been a constant source of
motivation, and inspiration to me, to always do
my best. And be the best I can ever be. I will
forever be indebted to you,
Father and mother, with my deepest heart –
felt gratitude.
Rami tayeb
May 2011
٥
Chapter one
Introduction
٦
Diabetes
What is diabetes?
Diabetes mellitus is a group of metabolic diseases characterized by high
blood sugar (glucose) levels, that result from defects in insulin secretion, or
action, or both. Diabetes mellitus commonly referred to as diabetes (as it will
be in this article) was first identified as a disease associated with "sweet
urine," and excessive muscle loss in the ancient world. Elevated levels of
blood glucose (hyperglycemia) lead to spillage of glucose into the urine,
hence the term sweet urine.
Normally, blood glucose levels are tightly controlled by insulin, a hormone
produced by the pancreas. Insulin lowers the blood glucose level. When the
blood glucose elevates (for example, after eating food), insulin is released
from the pancreas to normalize the glucose level. In patients with diabetes,
the absence or insufficient production of insulin causes hyperglycemia.
Diabetes is a chronic medical condition, meaning that although it can be
controlled, it lasts a lifetime.
What is the impact of diabetes?
Over time, diabetes can lead to blindness, kidney failure, and nerve damage.
These types of damage are the result of damage to small vessels, referred to
as microvascular disease. Diabetes is also an important factor in accelerating
the hardening and narrowing of the arteries (atherosclerosis), leading to
strokes, coronary heart disease, and other large blood vessel diseases. This
is referred to as macrovascular disease.
What causes diabetes?
Insufficient production of insulin (either absolutely or relative to the body's
needs), production of defective insulin (which is uncommon), or the inability of
cells to use insulin properly and efficiently leads to hyperglycemia and
diabetes. This latter condition affects mostly the cells of muscle and fat
tissues, and results in a condition known as "insulin resistance." This is the
primary problem in type 2 diabetes. The absolute lack of insulin, usually
secondary to a destructive process affecting the insulin producing beta cells in
the pancreas, is the main disorder in type 1 diabetes.
What is insulin?
Insulin is a hormone that is produced by specialized cells (beta cells) of the
pancreas. (The pancreas is a deep-seated organ in the abdomen located
behind the stomach.) In addition to helping glucose enter the cells, insulin is
also important in tightly regulating the level of glucose in the blood. After a
meal, the blood glucose level rises. In response to the increased glucose
level, the pancreas normally releases more insulin into the bloodstream to
help glucose enter the cells and lower blood glucose levels after a meal.
When the blood glucose levels are lowered, the insulin release from the
٧
pancreas is turned down. It is important to note that even in the fasting state
there is a low steady release of insulin than fluctuates a bit and helps to
maintain a steady blood sugar level during fasting. In normal individuals, such
a regulatory system helps to keep blood glucose levels in a tightly controlled
range. As outlined above, in patients with diabetes, the insulin is either
absent, relatively insufficient for the body's needs, or not used properly by the
body. All of these factors cause elevated levels of blood glucose
(hyperglycemia).
٨
(1.2) What are the different types of diabetes?
There are two major types of diabetes, called type 1 and type 2. Type 1
diabetes was also called insulin dependent diabetes mellitus (IDDM), or
juvenile onset diabetes mellitus. In type 1 diabetes, the pancreas undergoes
an autoimmune attack by the body itself, and is rendered incapable of making
insulin. Abnormal antibodies have been found in the majority of patients with
type 1 diabetes. Antibodies are proteins in the blood that are part of the body's
immune system. The patient with type 1 diabetes must rely on insulin
medication for survival.
In autoimmune diseases, such as type 1 diabetes, the immune system
mistakenly manufactures antibodies and inflammatory cells that are directed
against and cause damage to patients' own body tissues. In persons with type
1 diabetes, the beta cells of the pancreas, which are responsible for insulin
production, are attacked by the misdirected immune system. It is believed that
the tendency to develop abnormal antibodies in type 1 diabetes is, in part,
genetically inherited, though the details are not fully understood.
Exposure to certain viral infections (mumps and Coxsackie viruses) or
other environmental toxins may serve to trigger abnormal antibody responses
that cause damage to the pancreas cells where insulin is made. Some of the
antibodies seen in type 1 diabetes include anti-islet cell antibodies, anti-insulin
antibodies and anti-glutamic decarboxylase antibodies. These antibodies can
be measured in the majority of patients, and may help determine which
individuals are at risk for developing type 1 diabetes.
At present, the American Diabetes Association does not recommend
general screening of the population for type 1 diabetes, though screening of
high risk individuals, such as those with a first degree relative (sibling or
parent) with type 1 diabetes should be encouraged. Type 1 diabetes tends to
occur in young, lean individuals, usually before 30 years of age, however,
older patients do present with this form of diabetes on occasion. This
subgroup is referred to as latent autoimmune diabetes in adults (LADA).
LADA is a slow, progressive form of type 1 diabetes. Of all the patients with
diabetes, only approximately 10% of the patients have type 1 diabetes and
the remaining 90% have type 2 diabetes.
Type 2 diabetes was also referred to as non-insulin dependent
diabetes mellitus (NIDDM), or adult onset diabetes mellitus (AODM). In type 2
diabetes, patients can still produce insulin, but do so relatively inadequately
for their body's needs, particularly in the face of insulin resistance as
discussed above. In many cases this actually means the pancreas produces
larger than normal quantities of insulin. A major feature of type 2 diabetes is a
lack of sensitivity to insulin by the cells of the body (particularly fat and muscle
cells).
In addition to the problems with an increase in insulin resistance, the
release of insulin by the pancreas may also be defective and suboptimal. In
fact, there is a known steady decline in beta cell production of insulin in type 2
٩
diabetes that contributes to worsening glucose control. (This is a major factor
for many patients with type 2 diabetes who ultimately require insulin therapy.)
Finally, the liver in these patients continues to produce glucose through a
process called gluconeogenesis despite elevated glucose levels. The control
of gluconeogenesis becomes compromised.
While it is said that type 2 diabetes occurs mostly in individuals over 30
years old and the incidence increases with age, we are seeing an alarming
number patients with type 2 diabetes who are barely in their teen years. In
fact, for the first time in the history of humans, type 2 diabetes is now more
common than type 1 diabetes in childhood. Most of these cases are a direct
result of poor eating habits, higher body weight, and lack of exercise.
While there is a strong genetic component to developing this form of
diabetes, there are other risk factors – the most significant of which is obesity.
There is a direct relationship between the degree of obesity and the risk of
developing type 2 diabetes, and this holds true in children as well as adults. It
is estimated that the chance to develop diabetes doubles for every 20%
increase over desirable body weight.
Regarding age, data shows that for each decade after 40 years of age
regardless of weight there is an increase in incidence of diabetes. The
prevalence of diabetes in persons 65 to 74 years of age is nearly 20%. Type 2
diabetes is also more common in certain ethnic groups. Compared with a 6%
prevalence in Caucasians, the prevalence in African Americans and Asian
Americans is estimated to be 10%, in Hispanics 15%, and in certain Native
American communities 20% to 50%. Finally, diabetes occurs much more
frequently in women with a prior history of diabetes that develops during
pregnancy (gestational diabetes – see below).
Diabetes can occur temporarily during pregnancy. Significant hormonal
changes during pregnancy can lead to blood sugar elevation in genetically
predisposed individuals. Blood sugar elevation during pregnancy is called
gestational diabetes. Gestational diabetes usually resolves once the baby is
born. However, 25%-50% of women with gestational diabetes will eventually
develop type 2 diabetes later in life, especially in those who require insulin
during pregnancy and those who remain overweight after their delivery.
Patients with gestational diabetes are usually asked to undergo an oral
glucose tolerance test about six weeks after giving birth to determine if their
diabetes has persisted beyond the pregnancy, or if any evidence (such as
impaired glucose tolerance) is present that may be a clue to the patient's
future risk for developing diabetes.
Secondary" diabetes refers to elevated blood sugar levels from another
medical condition. Secondary diabetes may develop when the pancreatic
tissue responsible for the production of insulin is destroyed by disease, such
as chronic pancreatitis (inflammation of the pancreas by toxins like excessive
alcohol), trauma, or surgical removal of the pancreas.
١٠
Diabetes can also result from other hormonal disturbances, such as excessive
growth hormone production (acromegaly) and Cushing's syndrome. In
acromegaly, a pituitary gland tumor at the base of the brain causes excessive
production of growth hormone, leading to hyperglycemia. In Cushing's
syndrome, the adrenal glands produce an excess of cortisol, which promotes
blood sugar elevation.
In addition, certain medications may worsen diabetes control, or "unmask"
latent diabetes. This is seen most commonly when steroid medications (such
as prednisone) are taken and also with medications used in the treatment of
HIV infection (AIDS).
(1.3)
What are diabetes symptoms?
•
The early symptoms of untreated diabetes are related to elevated
blood sugar levels, and loss of glucose in the urine. High amounts of
glucose in the urine can cause increased urine output and lead to
dehydration. Dehydration causes increased thirst and water
consumption.
•
The inability of insulin to perform normally has effects on protein, fat
and carbohydrate metabolism. Insulin is an anabolic hormone, that is,
one that encourages storage of fat and protein.
•
A relative or absolute insulin deficiency eventually leads to weight loss
despite an increase in appetite.
•
Some untreated diabetes patients also complain of fatigue, nausea and
vomiting.
•
Patients with diabetes are prone to developing infections of the
bladder, skin, and vaginal areas.
•
Fluctuations in blood glucose levels can lead to blurred vision.
Extremely elevated glucose levels can lead to lethargy and coma
١١
١٢
(1.4) How is diabetes diagnosed?
The fasting blood glucose sugar test is the preferred way to diagnose
diabetes. It is easy to perform and convenient. After the person has fasted
overnight (at least 8 hours), a single sample of blood is drawn and sent to the
laboratory for analysis. This can also be done accurately in a doctor's office
using a glucose meter.
•
Normal fasting plasma glucose levels are less than 100 milligrams per
deciliter (mg/dl).
•
Fasting plasma glucose levels of more than 126 mg/dl on two or more
tests on different days indicate diabetes.
•
A random blood glucose test can also be used to diagnose diabetes. A
blood glucose level of 200 mg/dl or higher indicates diabetes.
Treatment of Type 1 diabetes:
Treatment requires a strict regimen that typically includes a carefully
calculated diet, planned physical activity, multiple daily insulin injections and
home blood glucose testing a number of times per day.
Treatment of Type 2 diabetes:
Treatment typically includes diet control, exercise, home blood glucose
testing, and in some cases, oral medication and/or insulin. Approximately 40%
of people with type 2 diabetes require insulin injections.
(1.5) VITAMINE D
Vitamin D is found in many dietary sources such as fish, eggs, fortified milk,
and cod liver oil. The sun also contributes significantly to the daily production
of vitamin D, and as little as 10 minutes of exposure is thought to be enough
to prevent deficiencies. The term "vitamin D" refers to several different forms
of this vitamin. Two forms are important in humans: ergocalciferol (vitamin D2)
and cholecalciferol (vitamin D3). Vitamin D2 is synthesized by plants. Vitamin
D3 is synthesized by humans in the skin when it is exposed to ultraviolet-B
(UVB) rays from sunlight. Foods may be fortified with vitamin D2 or D3.
Structure and Synthesis:
The term vitamin D is, unfortunately, an imprecise term referring to one or
more members of a group of steroid molecules. Vitamin D3, also known as
cholecalciferol is generated in the skin of animals when light energy is
absorbed by a precursor molecule 7-dehydrocholesterol. Vitamin D is thus not
١٣
a true vitamin, because individuals with adequate exposure to sunlight do not
require dietary supplementation. There are also dietary sources of vitamin D,
including egg yolk, fish oil and a number of plants. The plant form of vitamin D
is called vitamin D2 or ergosterol. However, natural diets typically do not
contain adequate quantities of vitamin D, and exposure to sunlight or
consumption of foodstuffs purposefully supplemented with vitamin D are
necessary to prevent deficiencies.
Vitamin D, as either D3 or D2, does not have significant biological activity.
Rather, it must be metabolized within the body to the hormonally-active form
known as 1,25-dihydroxycholecalciferol. This transformation occurs in two
steps, as depicted in the diagram to the right:
1. Within the liver, cholecalciferal is hydroxylated to 25hydroxycholecalciferol by the enzyme 25-hydroxylase.
2. Within the kidney, 25-hydroxycholecalciferol serves as a substrate for
1-alpha-hydroxylase, yielding 1,25-dihydroxycholecalciferol, the
biologically active form.
Each of the forms of vitamin D is hydrophobic, and is transported in blood
bound to carrier proteins. The major carrier is called, appropriately, vitamin Dbinding protein. The halflife of 25-hydroxycholecalciferol is several weeks,
while that of 1,25-dihydroxycholecalciferol is only a few hours.
(1.6) Control of Vitamin D Synthesis:
Hepatic synthesis of 25-hydroxycholecalciferol is only loosely regulated, and
blood levels of this molecule largely reflect the amount of amount of vitamin D
produced in the skin or ingested. In contrast, the activity of 1-alphahydroxylase in the kidney is tightly regulated and serves as the major control
point in production of the active hormone. The major inducer of 1-alphahydroxylase is parathyroid hormone; it is also induced by low blood levels of
phosphate.
١٤
The Vitamin D Receptor and Mechanism of Action:
The active form of vitamin D binds to intracellular receptors that then
function as transcription factors to modulate gene expression. Like the
receptors for other steroid hormones and thyroid hormones, the vitamin D
receptor has hormone-binding and DNA-binding domains. The vitamin D
receptor forms a complex with another intracellular receptor, the retinoid-X
receptor, and that heterodimer is what binds to DNA. In most cases studied,
the effect is to activate transcription, but situations are also known in which
vitamin D suppresses transcription.
The vitamin D receptor binds several forms of cholecalciferol. Its affinity
for 1,25-dihydroxycholecalciferol is roughly 1000 times that for 25hydroxycholecalciferol, which explains their relative biological potencies.
١٥
Physiological Effects of Vitamin D:
Vitamin D is well known as a hormone involved in mineral metabolism
and bone growth. Its most dramatic effect is to facilitate intestinal absorption
of calcium, although it also stimulates absorption of phosphate and
magnesium ions. In the absence of vitamin D, dietary calcium is not absorbed
at all efficiently. Vitamin D stimulates the expression of a number of proteins
involved in transporting calcium from the lumen of the intestine, across the
epithelial cells and into blood. The best-studied of these calcium transporters
is calbindin, an intracellular protein that ferries calcium across the intestinal
epithelial cell.
Numerous effects of vitamin D on bone have been demonstrated. As a
transcriptional regulator of bone matrix proteins, it induces the expression of
osteocalcin and suppresses synthesis of type I collagen. In cell cultures,
vitamin D stimulates differentiation of osteoclasts. However, studies of
humans and animals with vitamin D deficiency or mutations in the vitamin D
receptor suggest that these effects are perhaps not of major physiologic
importance, and that the crutial effect of vitamin D on bone is to provide the
proper balance of calcium and phosphorus to support mineralization.
It turns out that vitamin D receptors are present in most if not all cells in
the body. Additionally, experiments using cultured cells have demonstrated
that vitamin D has potent effects on the growth and differentiation of many
types of cells. These findings suggest that vitamin D has physiologic effects
much broader that a role in mineral homeostasis and bone function. This is an
active area of research and a much better understanding of this area will likely
be available in the near future.
(1.7) Disease States:
Vitamin D deficiency: The classical manifestations of vitamin D
deficiency is rickets which is seen in children and results in bony deformaties
including bowed long bones. Deficiency in adults leads to the disease
osteomalacia. Both rickets and osteomalacia reflect impaired mineralization of
newly synthesized bone matrix, and usually result from a combination of
inadequate exposure to sunlight and decreased dietary intake of vitamin D.
Vitamin D deficiency or insufficiency occurs in several other situations,
which you might predict based on the synthetic pathway described above:
Genetic defects in the vitamin D receptor: a number of different
mutations have been identified in humans that lead to hereditary vitamin D
resistance. Severe liver or kidney disease: this can interfere with generation of
the biologically-active form of vitamin D.
١٦
Insufficient exposure to sunlight: Elderly people that stay inside and have poor
diets often have at least subclinical deficiency. Ironically, it appears that
hypovitaminosis D is very common in some of the most sunny countries in the
world - the cause of this problem is the cultural dictate that women be heavily
veiled when outside in public.
Vitamin D toxicity: Excessive exposure to sunlight does not lead to
overproduction of vitamin D. Vitamin D toxicity is inevitably the result of
overdosing on vitamin D supplements. Certainly, vitamin D supplements are a
valuable treatment for individuals with deficiencies. However, ingestion of
excessive (milligram) quantities of vitamin D over periods of weeks of months
can be severely toxic to humans and animals. In fact, baits containing large
quantities of vitamin D are used very effectively as rodenticides.
Causes of Vitamin D Deficiency:
Vitamin D deficiency can occur for a number of reasons:
You don't consume the recommended levels of the vitamin over time.
This is likely if you follow a strict vegetarian diet, because most of the natural
sources are animal-based, including fish and fish oils, egg yolks, cheese, and
beef liver.
Your exposure to sunlight is limited. Because the body makes vitamin D
when your skin is exposed to sunlight, you may be at risk of deficiency if you
are homebound, live in northern latitudes, wear long robes or head coverings
for religious reasons, or have an occupation that prevents sun exposure.
١٧
You have dark skin. The pigment melanin reduces the skin's ability to make
vitamin D in response to sunlight exposure. Some studies show that older
adults with darker skin are at high risk of vitamin D deficiency.
Your kidneys cannot convert vitamin D to its active form. As people age
their kidneys are less able to convert vitamin D to its active form, thus
increasing their risk of vitamin D deficiency.
Your digestive tract cannot adequately absorb vitamin D. Certain medical
problems, including Crohn's disease, cystic fibrosis, and celiac disease, can
affect your intestine's ability to absorb vitamin D from the food you eat.
You are obese. Vitamin D is extracted from the blood by fat cells, altering its
release into the circulation. People with a body mass index of 30 or greater
often have low blood levels of vitamin D.
(1.8) Tests for Vitamin D Deficiency
The most accurate way to measure how much vitamin D is in your body is the
25-hydroxy vitamin D test. In the kidney, 25-hydroxy vitamin D changes into
an active form of the vitamin. The active form of the vitamin can be measured
through the blood. The active form of vitamin D helps control calcium and
phosphate levels in the body. The normal range is 30.0 to 74.0 nanograms
per milliliter (ng/mL). A lower level indicates vitamin D deficiency, which you
should discuss with your doctor.
Treatment for Vitamin D Deficiency:
Treatment for vitamin D deficiency involves getting more vitamin D -through diet, supplements, and/or through spending more time in the sun.
Although there is no consensus on vitamin D levels required for optimal health
-- and it likely differs depending on age and health conditions -- a
concentration of less than 20 nanograms per milliliter is generally considered
inadequate, requiring treatment.
Guidelines from the Institute of Medicine call for increasing the
recommended dietary allowance (RDA) of vitamin D to 600 international units
(IU) for everyone aged 1-70, and raising it to 800 IU for adults older than 70 to
optimize bone health.
If you don't spend much time in the sun or always are careful to cover
your skin (sunscreen inhibits vitamin D production), you should speak to your
doctor about taking a vitamin D supplement, particularly if you have risk
factors for vitamin D deficiency.
١٨
(1.9) Oxidative DNA damage
Introduction:
Reduced activities of the antioxidant enzymes catalase, glutathione
peroxidase, and superoxide dismutase, with concomitant increased levels of
oxidative DNA damage, have been reported in acute lymphoblastic leukemia,
seemingly agreeing with the report of Honda et al. (89), who reported elevated
levels of urinary 8-OH-dG in leukemia. The effect of lowered antioxidant levels
on oxidative DNA damage in vivo was first demonstrated in a study where
vitamin C levels of 10 volunteers were depleted and repleted. This work
demonstrated that decreasing vitamin C intake from 250 mg/day to 5 mg/day
led to a corresponding 50% increase in sperm DNA levels of 8-OH-dG (34.0
fmol/µg DNA +/- 2.4 to 66.90 fmol/µg DNA +/- 8.5, P 0.01), although
continued depletion resulted in a 248% increase (90). Repletion at 250
mg/day for 28 days led to only a 36% decrease in 8-OH-dG levels. An
identical study design, by the same group showed an increase in sperm levels
of 8-OH-dG, although depletion had no effect on the 8-OH-dG content of
PBMC or urinary 8-OH-Gua. The authors failed to speculate on this
discrepancy between the two cell types, though it may reflect the differing
requirements of the cells for vitamin C. Such a finding appears to highlight the
issue of surrogate measurements, i.e., performing measurements on a cell
type, such as PBMC, and extrapolating the results to the perhaps less
accessible target cell (reviewed in ref 91).
A locus on chromosome 3p that is frequently subject to loss of
heterozygosity (LOH) is the glutathione peroxidase gene (GPX1), which
encodes for a peroxide scavenging protein. Lung tumors with LOH within
GPX1 displayed reduced enzyme activity, although this did not appear to
correlate with tissue 8-OH-dG levels (84), perhaps due to the presence of
other antioxidant defense systems that could compensate, a situation similar
to that seen for 8-OH-dG repair (see above). A recent review revealed the
presence of a sizable number of polymorphisms in genes that encode for
antioxidant enzymes, the phenotypic impact of which ranges from having no
effect on enzyme activity to a complete absence of gene product (92). From
this review it is clear that, as a single factor affecting oxidative stress,
polymorphisms in antioxidant defense genes have the potential to play a
significant role in the risk of disease development (92).
ROLE OF OXIDATIVE DNA DAMAGE IN DISEASE
It is clear that depending on the lesion in question, one consequence of
oxidative base lesions persisting in DNA is mutation. For this reason, multiple
systems exist to: 1) prevent lesion formation and, should damage occur, 2)
ensure rapid lesion removal, with the enzyme systems responsible for the
latter having much overlap of substrates (see earlier section). DNA mutation is
a crucial step in carcinogenesis, and elevated levels of oxidative DNA lesions
have been noted in many tumors, strongly implicating such damage in the
etiology of cancer.
١٩
Carcinogenesis and cancer
Oxidative mechanisms have been demonstrated to possess a potential
role in the initiation, promotion, and malignant conversion (progression)
stages of carcinogenesis. Given that cumulative cancer risk increases with the
fourth power of age and is associated with an accumulation of DNA damage,
oxidative DNA damage has been investigated in cancer. Lesions such as 8OH-dG are established biomarkers of oxidative stress; coupled with their
potential mutagenicity in mammalian cells, this has led to their proposed
potential as intermediate markers of a disease endpoint—for example,
cancer. Supportive of this proposal are the findings that GC͢ TA transversions
potentially derived from 8-OH dG have been observed in vivo in the ras
oncogene and the p53 tumor suppressor gene in lung and liver cancer. Of
course, GC ͢ TA transversions are not unique to 8-OH-dG, whereas CC͢ TT
substitutions in the absence of UV in internal tumors have been identified as
signature mutations for ROS.
Numerous studies have attempted to establish a relationship between levels
of oxidative DNA damage and cancer. Elevated levels of damage are
purported to arise as a consequence of an environment in the tumor low in
antioxidant enzymes and are high in ROS generation (103). It has been
reported that at least some tumor cell lines can produce significant levels of
H2O2 , without exogenous stimulation, perhaps accounting for the elevated
levels of oxidative DNA damage seen. As a result of elevated ROS,
transcription factors and their corresponding genes are permanently activated,
which, coupled with increased DNA damage, creates a selection pressure for
a malignant phenotype seen in cancer (103). Although such studies have
furthered the hypothesis that oxidative DNA damage may be an important risk
factor for carcinogenesis, it has been argued that the mere presence of 8-OHdG in DNA is unlikely to be necessary or sufficient to cause tumor formation.
There are many pathological conditions in which levels of oxidative DNA
damage are elevated (Table 1) with no increased incidence of
carcinogenesis. This has led us to raise the following issues. 1) Oxidative
DNA damage may be an epiphenomenon to an on-going pathophysiological
process, and elevated levels do not have a role in carcinogenesis.
2) Cause or consequence? The mere presence of elevated levels of damage
in tumors does not indicate it was oxidative damage that led to the
tumorigenic changes. Elevation in levels may have occurred as a result of
well-established characteristics of tumors, e.g., increased metabolism or cell
turnover. 3) For DNA mutations to arise from oxidative damage, the nuclei of
undifferentiated, proliferating stem cells must be affected. Given that tissue
samples from tumors and normal cells will represent a heterogeneous mixture
of differentiated and undifferentiated cells (with the former likely to
predominate), current analytical procedures will not reflect lesion levels in the
most important target cells. 4) Not only must the DNA of target.
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TABLE 1. Reports of pathological conditions in which oxidative DNA damage has been measured
٢٠
Organ system
disease
Lesion measured
Comments
Aging
Aging
8-OH-dG (urine)
• No correlation between urinary
8-OH-dG output and aging (age
range: 35–65 years).b
• In all ALS patients and healthy
subjects, plasma and
CSF levels of 8-OH-dG increase
with age (114).
• No difference in levels of Tg or
dTg in neoplastic vs.
nonneoplastic urine.
8-OH-dG (plasma and
CSF)
cancers
Assorted cancers
Tg and dTg (urine)
Assorted cancers:
fibrillary astrocytoma
(FA; brain); lung
cancer (LC);
mucinous carcinoma
(MC; stomach)
ovarian cancer (OC);
colon cancer (CRC)
5-OH-5-Me-Hyd
5-OH-Hyd
5-OHMe-Ura,
5-OH-Cyt
5,6-diOH-Ura
FapyAde
8-OH-Ade
Xanthine
2-OH-Ade
FapyGua
8-OH-Gua
5-OH-Me-Ura
Breast, rectal and colon
cancer
Diabetes mellitus
Assorted cancers
8-OH-dG (urine)
Non-insulin-dependent
(NIDDM)
8-OH-dG (urine)
8-OH-dG (DNA)
Insulin- and
noninsulindependent
8-OH-dG (DNA)
Type II
5-OH-MeHyd, 5-OHHyd,
5-OH-Ura, 5-OH-Me-Ura,
5- OH Cytosine, Tg, 8OH-Gua, FapyAde, 8oxoA, 2-OH-Ade
8-OH-dG (urine)
٢١
• Lesion significantly elevated (P
≤ 0.05) in:LG,CRC,OC.
- FA, LC, CRC, MC, OC
- LC, MC, OC
- LC, OC
- FA, LC, CRC, MC, OC
- FA, LC,
- FA, LC, MC, OC
- LC, MC, OC
- FA, LC, MC, OC
- FA, LC, MC, OC
- FA, LC, CRC, MC, OC (140)d
• Women who develop breast or
colorectal cancer have elevated
levels of serum autoantibodies to
5- OH-Me-Ura compared to
controls (141).
• Elevated levels of urinary 8-OHdG noted in cancer patients before
(P 0.01) and after anti-cancer
therapy (P 0.001).b 1206
• Levels of urinary 8-OH-dG
significantly higher than
controls (P = 0.001) associated
with high glycosylated
haemoglobin.b
• Elevated levels of 8-OH-dG in
muscle DNA of NIDDM patients
compared to controls. Significant
correlation between mitochondrial
DNA deletion (delta
mtDNA4977) and 8-OH-dG
levels (P 0.0001) and
proportional to diabetic
complications (142).
• Both groups had significantly
higher levels of 8-OHdG
(P 0.001) in mononuclear cell
DNA, compared to controls (143).
• PBMC levels of oxidised DNA
base products significantly
elevated in diabetes patients
compared to controls (144).
• 8-OH-dG levels in 24 h urine
collections significantly
higher (P 0.001) in diabetic
patients than in control subjects.b
• 8-OH-dG levels in 24 h urine
8-OH-dG (urine and
DNA)
8-OH-Gua (serum)
Type I and II
8-OH-dG (urine)
Rheumatoid arthritis
8-OH-dG (urine)
8-OH-dG (DNA)
Systemic lupus
erythematosus
(SLE)
8-oh-dG (urine)
5-OH-Me-Ura
8-OH-dG (DNA, serum &
urine)
8-OH-dG (DNA)
collections and mononuclear cell
DNA significantly higher (P
0.001 and P 0.0001) in diabetic
patients than in control,
nonsmoking subjects.b
• Diabetic patients possessed
significantly higher levels
of serum 8-OH-Gua than control
subjects.b
• Patients with both type I and II
diabetes had significantly higher
levels of urinary 8-OH-dG,
compared to controls (145).
• Levels of urinary 8-OH-dG
significantly elevated (P 0.001)
compared to control subjects
(148).
• PBMC levels of 8-OH-dG
significantly higher in
rheumatoid arthritis patients vs.
controls (P = 0.001;
149).
• Levels of urinary 8-OH-dG
significantly reduced
compared to control subjects;
presence of 8 OH-dG noted in
circulating immune complexes.b
• Titres of serum autoantibodies to
5-OH-Me-Ura
significantly elevated in SLE
(150).
• Attenuated response to vitamin
C supplementation
in all three matrices.b
• PBMC levels of 8-OH-dG
significantly higher in SLE
patients vs. controls (P = 0.0001;
149).
‫ــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ‬
Oxidative DNA damage is an inevitable consequence of cellular metabolism,
with a propensity for increased levels following toxic insult. Although more
than 20 base lesions have been identified, only a fraction of these have
received appreciable study, most notably 8-oxo-2_deoxyguanosine.
This lesion has been the focus of intense research interest and been ascribed
much importance, largely to the detriment of other lesion.
The present work reviews the basis for the biological significance of oxidative
DNA damage, drawing attention to the multiplicity of proteins with repair
activities along with a number of poorly considered effects of damage.
Normal cellular metabolism is well established as the source of endogenous
reactive oxygen species (ROS), and it is these (normally nonpathogenic)
cellular processes that account for the background levels of oxidative DNA
damage detected in normal tissue.
٢٢
Electron transport chains all possess the potential to “leak” electrons to
oxygen resulting in superoxide formation.
Certain enzyme activities generate superoxide and, via an oxidative burst,
ROS are released from phagocytic cells destined to destroy cells infected with
viruses, or bacteria, although surrounding tissue can also be affected.
Peroxisomes compartmentalize oxidative metabolism leading to reactive
products that would otherwise be detrimental to the cell, although under
certain conditions these products may be oxidative metabolism leading to
reactive products may be released.
ROS may also be generated by ionizing or ultraviolet radiation. Equally,
certain exogenous chemicals may redox cycle following metabolism by the
cell, with the subsequent production of electrons that can be transferred to
molecular oxygen producing superoxide (O2•–). Irrespective of their origin,
reactive oxygen species may interact with cellular biomolecules, such as
DNA, leading to modification and potentially serious consequences for the
cell.
(1.10) Mechanisms of oxidative damage to DNA bases:
Of the reactive oxygen species, the highly reactive hydroxyl radical
(•OH) reacts with DNA by addition to double bonds of DNA bases and by
abstraction of an H atom from the methyl group of thymine and each of the CH bonds of 2_-deoxyribose. Addition to double bonds of DNA bases occurs at
or near diffusion controlled rates with rate constants from 3 to10 x 109 M–1 s–
1; the rate constant of H abstraction amounts to 2 x 109 M–1 s–1. Addition to
the C5-C6 double bond of pyrimidines leads to C5-OH and C6-OH adduct
radicals and H atom abstraction from thymine results in the allyl radical.
Adduct radicals differ in terms of their redox properties, with C5-OH adduct
radicals being reducing and C6-OH adduct radicals oxidizing. Pyrimidine
radicals yield numerous products by a variety of mechanisms. Radicals are
reduced or oxidized depending on their redox properties, redox environment,
and reaction partners. Product types and yields depend on absence and
presence of oxygen and on other conditions. In the absence of oxygen, the
oxidation of C5-OH adduct radicals, followed by addition of OH- (or addition of
water followed by deprotonation), leads to cytosine glycol and thymine glycol.
The allyl radical yields 5-hydroxymethyluracil. C5-OH-6-peroxyl radicals are
formed by addition of oxygen to C5-OH adduct radicals at diffusion-controlled
rates. C5-OH-6-peroxyl radicals eliminate O2•-, followed by reaction with water
(addition of OH-) to yield thymine and cytosine glycols. Oxygen reacts with the
allyl radical, leading to 5-hydroxymethyluracil and 5-formyluracil. Thymine
peroxyl radicals are reduced, followed by protonation to give
hydroxyhydroperoxides, which decompose and yield thymine glycol, 5hydroxymethyluracil, 5-formyluracil, and 5-hydroxy-5-methylhydantoin.
Products of cytosine may deaminate and dehydrate. Cytosine glycol
deaminates to give uracil glycol, 5-hydroxycytosine, and 5-hydroxyuracil.
However, cytosine glycol, uracil glycol, 5-hydroxycytosine, and 5hydroxyuracil were all detected in γ-irradiated cytosine, indicating that all
these compounds may simultaneously be present in damaged DNA. In the
٢٣
absence of oxygen, C5-OH adduct radicals may be reduced, followed by
protonation to give 5-hydroxy-6-hydropyrimidines. 5-Hydroxy-6-hydrocytosine
readily deaminates into 5-hydroxy-6-hydrouracil. Similarly, C6-OH adduct
radicals of pyrimidines may lead to 6-hydroxy-5-hydropyrimidines. These
products are typical of anoxic conditions because oxygen inhibits their
formation by reacting with OH adduct radicals. By contrast, pyrimidine glycols
and 5-hydroxymethyluracil are formed under both oxic and anoxic conditions.
Further reactions of C5-OH-6-peroxyl and C6-OH-5-peroxyl radicals of
cytosine result in formation of 4-amino-5-hydroxy-2,6(1H,5H)-pyrimidinedione
and 4-amino-6-hydroxy-2,5(1H,6H)-pyrimidinedione, respectively, which may
deaminate to give dialuric acid and isodialuric acid, respectively. The
detection of 4-amino-6-hydroxy-2,5(1H,6H)-pyrimidinedione and isodialuric
acid in DNA suggested that both compounds may simultaneously exist in
DNA. Hydroxyl radical adds to the C4, C5, and C8 positions of purines
generating OH adduct radicals. In the case of adenine, at least two OH
adducts are formed: C4-OH and C8-OH adduct radicals. C4-OH adduct
radicals possess oxidizing properties, whereas C5-OH and C8-OH adduct
radicals are primarily reductants. On the other hand, different mesomeric
structures of these radicals may be oxidizing or reducing, a phenomenon
called "redox ambivalence". C4-OH and C5-OH adduct radicals of purines
dehydrate and are converted to an oxidizing purine(-H)• radical, which may be
reduced and protonated to reconstitute the purine. The rate constants of the
dehydration of the C4-OH adduct radicals of guanine and adenine at neutral
pH amount to 1.5 x 105 s-1 and 6 x 103 s-1, respectively. The guanine radical
cation (guanine•+) is formed by elimination of OH- from the C4-OH adduct
radical of guanine (k=6x103 s-1) and may deprotonate depending on pH to
give guanine(-H)•. The radical cation does not hydrate to lead to the C8-OH
adduct radical and then to 8-hydroxyguanine (8-oxoguanine, 8-OH-Gua by
oxidation; however, it may react with 2'-deoxyribose in DNA by H abstraction
(k<4x103 s-1), causing DNA strand breaks. C8-OH adduct radicals of purines
may be oxidized by oxidants including oxygen. In contrast to C4-OH adduct
radicals, their reaction with oxygen is diffusion-controlled (k4x109 M-1s-1).
The one-electron oxidation leads to formation of 8-hydroxypurines (7,8dihydro-8-oxopurines) in DNA. However, 8-hydroxypurines are also formed in
the absence of oxygen, but to a lesser extent. The oxidation of C8-OH adduct
radicals competes with the unimolecular opening of the imidazole ring by
scission of the C8-N9 bond at a rate constant of 2 x 105 s-1. The one-electron
reduction of the ring-opened radical leads to 2,6-diamino-4-hydroxy-5formamidopyrimidine (FapyGua) from guanine and 4,6-diamino-5formamidopyrimidine (FapyAde) from adenine). The one-electron reduction of
C8-OH adduct radicals without ring-opening may also occur resulting in
formation
of
7-hydro-8-hydroxypurines.
These
compounds
are
hemiorthoamides and may be converted into formamidopyrimidines. 8Hydroxypurines and formamidopyrimidines are formed in DNA in both the
absence and presence of oxygen; however, the formation of 8-hydroxypurines
is preferred in the presence of oxygen. Another reaction of base radicals is
the addition to an aromatic amino acid of proteins or combination with an
amino acid radical, leading to DNA–protein cross-linking. Reactions of •OH
with the sugar moiety of DNA by H abstraction give rise to sugar modifications
and strand breaks. A unique reaction of the C5'-centered sugar radical is the
٢٤
addition to the C8-position of the purine ring of the same nucleoside. This
reaction leads to intramolecular cyclization, then by oxidation to 8,5'cyclopurine-2'-deoxynucleosides. Both 5'R- and 5'S-diastereomers of 8,5'cyclo-2'-deoxyguanosine (cyclo-dG) and 8,5'-cyclo-2'-deoxyadenosine (cyclodA) are formed in DNA. (5'R)- and (5'S)-8,5'-cyclo-2'-deoxyguanosines were
also identified in human cells exposed to ionizing radiation. These compounds
represent a concomitant damage to both base and sugar moieties and are
considered tandem lesions. Oxygen inhibits their formation by reacting with
the C5'-centered sugar radical before cyclization. The antioxidant systems
have been recognized for many years, and are relatively well defined. In
contrast, although it has been some years since repair of oxidative DNA
damage was first reported, the last decade has seen a notable increase in
research effort directed toward unraveling DNA repair processes.
CELLULAR IMPACT OF OXIDATIVE DNA DAMAGE:
The presence of oxidized base lesions in DNA is well established and the
number of lesions identified is growing. There is a significant number whose
effects on replication and transcription have been described. Some factors
influence the effect a particular lesion has on replication and transcription. Not
surprisingly, 8-OH-dG is by far the most frequently studied and is often used
here as an example of how oxidative lesions may exert their effect.
Alterations in conformation:
For lesions that can induce conformational changes in DNA in addition
to the structural alterations to the native base itself, the potential for
enhancing mutagenicity exists. Illustrative of this is the oxidation of dG to 8OH-dG in DNA. The native anti conformation of dG is maintained; however,
when the DNA is made single-stranded, whether it be at replication or
transcription, 8-OH-dG can then adopt the energetically more favorable
syn conformation. This prevents pairing with dC and results in mispairing with
dA or T.
Sequence context:
The formamidopyrimidines are major products of hydroxyl radical
attack of DNA. Although little is known now about their biological significance,
Graziewicz et al. (95) demonstrated that, although less frequently inhibited
by oxidized purines than oxidized pyrimidines, DNA synthesis by prokaryotic
polymerases was shown to be terminated by both FapyAde and FapyGua.
However, this effect was only moderate and depended on the sequence
context (95) .
Overall its seems that oxidative DNA lesions are best described as
weakly mutagenic; for example, 8-OH-dG has mutation frequencies of 2.5–
4.8% in mammalian cells although lesion formation, persistence, and
accumulation in vivo could give this value greater significance. Indeed,
٢٥
oxidative events are reported to be largely responsible for spontaneous
mutagenesis. However, mutations are not the only effect of oxidative DNA
damage.
Alternatives to mutation:
Replicative block:
Thymidine glycol occurs mainly as the cis isomer.the general agreement
appears to be that Tg does not have a significant mutagenic potential. More
recent studies have suggested that rather than being mutagenic, Tg blocks
replication one nucleotide before and after the lesion.
Deletions:
the presence of 5-OHMUra leads to large/intermediate deletions in
mammalian cells. However, these deletions do not arise as a result of
mispairing or misincorporation; instead, it seems that base excision repair by
OHMUra-DNA glycosylase may lead to the deletions.
Microsatellite instability/loss of heterozygosity
In normal cells, the length of repetitive sequences of DNA, so-called
microsatellites, is constant, but the length of these repeats can be variable in
tumor cells. This microsatellite instability (MI) derives from DNA damage and
has been linked to some sporadic cancers. The association between oxidative
events and MI appears to be increasing, with oxidative DNA damage shown
to increase the frequency of microsatellite instability through induction of
mutations in the repeat sequences and discontinuous LOH being described
as a signature mutational pattern of oxidative DNA damage.
Epigenetic effects of oxidative DNA damage
While the mutagenic effects of oxidative DNA damage are largely well
recognized, emerging work is broadening the number of routes by which
these lesions may affect the cell, being suggestive of epigenetic effects
exclusive of mutation. Central to this work is the finding that when exposed to
oxidants, mammalian cells express stress-induced genes or genes encoding
antioxidant defenses. Such adaptive responses to oxidative insults are not
surprising and are seen with other, non oxidative insults.
The presence of lesions in the transcribed regions of genes can lead to
mutation.
Ghosh and Mitchell demonstrated that the presence of 8-OH-dG in promoter
elements can affect transcription factor binding. It appears that that a single 8OH-dG moiety in the AP-1 transcription factor binding site in the promoter
region of genes can prevent transcription factor binding and hence the level of
transcription. Being GC-rich, these regions represent a considerable target for
ROS. Further support for such a mechanism derives from the substitution of
dG for 8-OH-dG in the cognate DNA sequence of the transcription factor SP1.
This effect may have pathological consequences, as the interference of Sp1
٢٦
and NF-κB binding in the kidney and liver of diabetic rats was postulated to be
due to ROS-mediated damage to DNA.
(1.11) Vitamin D and diabetes mellitus
Introduction
Vitamin D has been known as a regulator of bone and mineral
metabolism by regulation of calcium absorption in the gut and reabsorption by
the kidney. In addition, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] is also
recognized in the regulating of the immune system. Vitamin D receptor (VDR)
is presented in peripheral blood monocytes and activated T cells [1,2]. In the
animal models, 1,25(OH)2D3 protects against autoimmune diseases, such as
experimental autoimmune encephalomyelitis (EAE) [3] and collagen-induced
arthritis [4]. Recently, Zella and Deluca [5] suggested the effectiveness of
vitamin D in diabetes. Therefore, it would be interesting to further review the
role of vitamin D in protecting autoimmune diabetes, also known as type 1
diabetes mellitus.
The role of vitamin D in protecting type 1 diabetes mellitus
Several studies in rats and humans have demonstrated that vitamin D
deficiency causes reduced insulin secretion, and that 1,25(OH)2D3 improves
in β- cell function and consequently in glucose tolerance . In vitamin D–
deficient rats, glucose tolerance and insulin secretion were improved with
1,25(OH)2D3 treatment. In gestational diabetes mellitus, Rudnicki and
Molsted-Petersen reported that the glucose level decreased from 5.6 to 4.8
mmol/L after intravenous treatment with 1,25(OH)2D3.
This vitamin D also corrects glucose intolerance and normalizes insulin
sensitivity in uremic patients.
The NOD mouse has been known as a model of human type 1 diabetes.
Similar to the human disease, NOD mouse strain developed hyperglycemia
as a result of a T-cell mediated autoimmune reaction against the insulinproducing β cells of the islets of Langerhans in the pancreas. Clinical disease
is preceded by insulitis, which is a basic histological lesion in the islet of
Langerhans of the pancreas. Islets are invaded mainly by CD4+ and CD8+ T
cells and also by monocytes. The onset of insulitis is observed at 20 to 40
days of age. The loss of glycemic control results in polydipsia, polyuria and
excessive weight loss if not treated with exogenous insulin, becoming 100%
by 200 days. Mathieu et al. reported that 1,25(OH)2D3 has been shown to
reduce type 1 diabetes onset in NOD mice.
No mouse (100%) showed insulitis at 21days of age. 1,25(OH)2D3 has shown
partial protection, reduction to 42%, against insulitis by 100 days of age.
When 1,25(OH)2D3 treatment (on alternate days) was started at the age of 21
days and terminated at the age of 200 days or on the day of diabetes
diagnosis [64], it reduced insulitis incidence from 81% in the control group to
٢٧
58% in the treated group. Diabetes incidence in female NOD mice at 200
days was reduced to 8% in the 1,25(OH)2D3-treated group versus 56% in the
control group. Both 1,25(OH)2D3 and its nonhypercalcemic analogs,
1α,25(OH)2-20-epi-22- oxa-24,26,27-trishomo-vitamin D (KH1060), have
been shown to reduce type 1 diabetes onset in NOD mice. However, Zella
and DeLuca [5] found that 1,25(OH)2D3 does not offer complete protection
against type 1 diabetes onset in NOD mice when administered every other
day
They also showed that all NOD mice are completely resistant to type 1
diabetes by 200 days of age when a daily dose of 50-ng 1,25(OH)2D3 is
administered orally through the diet from weaning. They suggested that oral
administration of 1,25(OH)2D3 or preferably a nonhypercalcemia analog
would be more clinically relevant for the prevention of type 1 diabetes in
humans.
Recently, Giulietti et al. reported that vitamin D deficiency in early life might
increase type 1 diabetes in NOD mice.
They found, at 250 days, that 35% male and 66% female vitamin D–deficient
mice were diabetic compared to 15 and 45% of the control mice. In the
vitamin D–deficient mice, higher IL-1 expression was detected in islets.
Thymus and lymph nodes also contained less CD4CD62L+ cells; a defect in
this cytokine profile might trigger the diabetes.
In addition, Casteels et al. reported that nonhypercalcemic analogs of
1,25(OH)2D3 administered to NOD mice when the autoimmune disease is
already active can prevent clinical diabetes when this therapy is combined
with a short induction course of an immunosuppressant such as Cyclosporin
A (CsA).
٢٨
Schemac nongenomic model for 1,25(OH)2D3effects on insulin secreon in β cells
٢٩
(1.12) Oxidative DNA damage and diabetes mellitus
Hyperglycaemia, a key clinical manifestation of diabetes mellitus, not
only generates reactive oxygen species (ROS), but also attenuates antioxidative mechanisms by scavenging enzymes and antioxidant substances .
As ROS cause strand breaks in DNA and base modifications including the
oxidation of guanine residues to 8-oxo, 2 ¢-deoxyguanosine (8- oxodG), 8oxodG can serve as a sensitive biomarker of oxidative DNA damage. 8OxodG was increased in the kidneys of diabetic rats, and insulin treatment
reduced both urinary albumin excretion and 8-oxodG formation in the kidney.
A recent study reported an increase in the 8-oxodG content in mononuclear
cells and ROS level in Type I (insulin- dependent) and Type II (non-insulindependent) diabetic patients when compared with control subjects [4].
Another study reported that urinary 8-oxodG excretion was higher in Type II
diabetic patients than in the control subjects [5].
Urinary 8-oxodG excretion correlated with glycated haemoglobin [5]. We
speculated that diabetes-associated modifications of DNA by ROS might
contribute to the diabetic complications.
(1.13)
1.13)Oxidative DNA damage and vitamin D
Reactive oxygen species (ROS) are generated after exposure to
environmental stresses, such as UV-, X- and g-irradiation, and carcinogens,
as well as endogenous cellular stresses from leakage of electron transport
chain in mitochondria, and during inflammation.
ROS are, in one way, necessary for inflammatory response to defend
organisms against infection agents. In contrast, high levels of cellular ROS
could damage many critical cellular components, such as protein, lipids and
DNA, and eventually cause cellular injuries that might lead to a variety of
clinical abnormalities, including cardiovascular disease, diabetes,
neurodegenerative disorders, aging and cancer.
A large body of evidence suggests that ROS are directly or indirectly
involved in carcinogenesis. ROS are capable of damaging cellular
components, and this destructive chain reaction can be blocked by the
reducing equivalents. Therefore, compounds that can modulate the defense
system against ROS could potentially hinder the process of carcinogenesis.
1a, 25-dihydroxyvitamin D3 (1,25- VD) has been known to reduce the
incidence of colon, breast and prostate cancers in human, as well as in
chemically induced animal cancer models.14–16 Much research has focused
on vitamin Dreceptor (VDR)-mediated antiproliferative effects of 1,25-VD, the
active metabolite of vitamin D. In general, VDR binds to vitamin D response
elements (VDREs), and regulates target genes’ expression to inhibit cancer
cells proliferation. However, little information is available on the antioxidant
property of 1,25-VD. Gene expression profiling revealed that 1,25-VD and its
analogs induced several genes controlling redox balance, including G6PD,
glutathione peroxidase (GPx) and thioredoxin reductase (TR). These studies
suggest that 1,25-VD might be able to prevent cancer development through
protection against oxidative stress.
٣٠
Therefore, the aim of the present work was to explore the potential ability of
1,25-VD to modulate the defense system against ROS.
Induction of G6PD is consistent with the protective effect of 1,25-VD against
oxidative stress. However, this finding is controversial in the vitamin D field in
determining whether 1,25-VD acts as a pro-oxidant or antioxidant.
٣١
Chapter 2
Material and methods
٣٢
2.1 Research Design
Fifty three males (26) and females (27) juvenile diabetic patients ranging in
age from (2 – 17) years old were collected from diabetic clinic at King AbdulAziz University Hospital.
-All patient with type -1 diabetes (IDDM).
-Without diabetic complication, such as diabetic nephropathy , neuropathy
and heart disease.
2.2 Materials
2.2.1 Samples Collection criteria
Samples were collected after 12 hours fasting, hemolyzed and lipemic
samples were excluded. These samples were collected in 2 different tubes:
Tube color
Yellow tube
Anticoagulant
Tests performed
Plain tube (no
Vitamin D and
anticoagulant )
Cytokines
Glycosylated
Lavender
Hemoglobin (HbA1c)
EDTAK2,EDTAK3
and Comet assay.
Table 2-1: Tubes used to collect sample
٣٣
2.2.2 Sample Processing
Sample centrifugation (at 3500 rpm for 5 minutes), to separate the plasma.
All samples stored after centrifugation at -70 until time of processing.
2.3 methods and technique
2.3.1 Biochemical parameters
Biochemical parameters including serum Glucose, Glycosylated
Hemoglobin (HbA1c), Vitamin D, Cytokines malondialdehyde (MDAP) and
erythrocyte malondialdehyde (MDAE) were measured as markers of oxidative
stress, activity of leukocyte superoxide dismutase (SOD), glutathion
peroxidase (GSH-Px), and catalase (CAT) were taken as markers of oxidative
defense system. all were measured at the same time of the study.
Fasting serum glucose concentration was measured using enzymatic method.
All above biochemical tests were done using the reagent from Vista Machine
at King Abdul-Aziz University Hospital.
2.3.2 Complete blood count and HbA1c.
All sample were analyzed by complete blood count (CBC) using the
Beckman Coulter Machine, at King Abdul-Aziz University Hospital, this
process include the measurement of all blood component (RBCs, WBCs and
Plt), hemoglobin (Hgb) and hematocrit (Hct).
HbA1c was quantitatively determined by COBAS INTEGRA 400 plus.
2.3.3 Vitamin D3
All blood samples were analyzed for vitamin D3 by Elecsys 2010:
٣٤
This machine depend on Electro-chemi-luminescent (ECL) processes
are known to occur with numerous molecules including compounds of
ruthenuim, osmium or other elements.
This is a process in which highly reactive species are generated from
stable precursors of an electrode. These highly reactive species react with
one another producing light. The development of ECL immune assay is based
on the use of
ruthenium tris (bipyridyl) Ru (bpy3)2+complex and
tripropylamine (TPA). The final chemi-luminescent product is formed during
the detective step.
The chemical reactions that lead to the emission of light from the ruthenium
complex are initiated electrically rather than chemically. This is achieved by
applying a
voltage
to
the
immunological complexes
(including the
Ruthenium complex) that are attached to streptavidin coated micro particles
.The advantage of eclectically initiating the chemiluminescent reaction is that
the
entire
reaction
can
be
precisely
controlled.
Two
electrically
active substances, the ruthenium complex and the tripropylamine (TPA) are
involved in the reactions that lead to the emission of light. Both substances
remain stable. This reaction occurs at the surface of the Platinum electrode.
The applied voltage creates an electrical field which causes all the materials
in the field to react. The ruthenium ground state complex is continuously
regenerated. This complex can perform many light generating cycles during
the measurement process, therefore showing an inherent amplification effect
which contributes to the technologies sensitivity. Many protons can be created
from one antigen antibody complex.
Test principle:
(Competitive principle)
This principle is applied to analytes of low molecular weight, such as Vitamin
D3:
٣٥
• In the first step, sample and a specific anti-vitamin D3 antibody
labeled with a ruthenium complex are combined in the assay cup.
• After the first incubation, biotinylated vitamin D3 and Streptavidin- coated paramagnetic micro particles are added. The still free binding
sites of the labelled antibody become occupied with the formation of an
antigen–hapten complex. The entire complex is bound to the micro
particle via interaction of biotin and streptavidin.
• After the second incubation, the reaction mixture containing the
immune complexes is transported into the measuring cell. The immune
complexes are magnetically entrapped on the working electrode, but
unbound reagent and sample arewashed away by a system buffer.
• In the ECL reaction, the conjugate is a ruthenium based derivative
and the chemiluminescent reaction is electrically stimulated to produce
light. The amount of light produced is indirectly proportionately to the
amount of antigen in the patient sample.
• Evaluation and calculation of the concentration of the antigen vitamin
D3 are carried out by means of a calibration curve that was established
using standards of known antigen concentration.
٣٦
2.3.4 . Antioxidant Enzyme
The heparinised blood was layered over Ficoll-Paque leukocyte
separation medium for isolation of leukocytes. The preparation was
centrifuged for 35 min at 400 × g and 25 ◦C, and the interface band containing
leukocytes was removed. The leukocytes were resuspended in ice-cold
Hank’s balanced salt solution and centrifuged. Then leukocyte pellets were
homogenized in three volumes of ice-cold buffer composed of 50mM Tris (pH
7.5), 1mM EDTA, 10mM dithiothretiol, 0.2% Triton X-100, followed by
centrifugation at 12 000×g for 10 min at 4 ◦C. Supernatant was used for the
determination of superoxide dismutase (SOD), glutathion peroxidase (GSHPx), and catalase (CAT) were
taken as markers of the oxidative defense system. SOD, GSH-Px and CAT
activities were measured by kits from Randox, UK (catalog no.: SD 125 and
RS 504, respectively). The leukocyte protein content was measured by the
Bradford method. All chemicals were purchased from Sigma. The CAT and
GSH-Px activities were given in international unit (IU)/mg protein and SOD
activity in U/mg protein. The MDA ( in both plasma and erythrocytes)
concentration was determined by using the thiobarbituric acid reaction .
2.3.5 Single cell gel electrophoresis (comet assay)
Comet test :(the comet assay, also called the single cell gel electrophoresis
(SCGE) assay , a standard technique for evaluation of DNA damage/repair,
biomonitoring and genotoxicity testing. It involves the encapsulation of cells in a
low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH>13)
conditions, and electrophoresis of the suspended lysed cells. This is followed by
visual analysis with staining of DNA and calculating fluorescence to determine the
extent of DNA damage. This can be performed by manual scoring or automatically by
an imaging software.
Procedure:
1- Preparation of slides for single cell gel assay:
Materials:
• Coplin jars
•
Cover slip
•
Frozen ice packs
٣٧
•
Low melting point agarose (LMPA)
•
Microcenterfuge tubes
•
Micropipttor slides
•
Microscopic slides tray
•
Normal mMelting agarose
•
Phosphate buffer saline (PBS)(Ca, Mg free)
•
scintillation
1-prepare 0.5% LMPA (250 mg per 50 ml PBS) and 1.0% NMA (500 mg per
50 ml) in PBS. Microwave or heat until near boiling and the agarose dissolves.
For LMPA, aliquot 5 mL samples into scintillation vials (or other suitable
containers) and refrigerate until needed. When needed, briefly melt agarose in
microwave or by another appropriate method. Place LMPA vial in a 37ºC
dry/water bath to cool and stabilize the temperature.
2- While NMA agarose is hot, dip conventional slides up to one-third the
frosted area and gently remove (2). Wipe underside of slide to remove
agarose and lay the slide in a tray on a flat surface to dry. The slides may be
air dried or warmed at 500C for quicker drying. Store the slides at room
temperature until needed; avoid high humidity conditions. We generally
prepare slides the day before use.
3- To the coated slide, add 75 µL of LMPA (0.5%; 37ºC) mixed with ~10,000
lymphocytes
in ~5-10 µL (do not use more than 10 µL) OR DILUTE BLOOD WITH PBS
AND ADD
EQUAL VOLUMES OF DILUTED BLOOD AND 1% LMPA. Place coverslip
and put the
slide on a slide tray resting on ice packs until the agarose layer hardens (~5 to
10 minutes).
4- Gently slide off coverslip and add a third agarose layer (80 µL LMPA) to the
slide.
Replace coverslip and return to the slide tray until the agarose layer hardens
(~5 to 10 minutes).
5- Remove coverslip and slowly lower slide into cold, freshly made Lysing
Solution. Protect
from light and refrigerate for a minimum of 1 hours.
٣٨
2- cell isolation:
Whole Blood : per gel slide, mix
process accordingly.
5 µl whole blood with 75µl LMPA and
NOTE: A small volume of blood can be added to 1 ml of media and stored
cold (refrigerated or on ice) for an extended period of time until processed. In
this case , the cell must be pelleted by centrifugation and care taken to
remove as much supernatant as possible before adding the 75µl LMPA per 5
µl of blood. In cases where protracted sample storage is necessary, this
sample can be flash-frozen in liquid nitrogen and then stored at -70c until
processed. Flash-frozen optimize DNA integrity but not necessarily cell
survival.
Viability assessment:
Viability assessment is often critical for interpreting SCG data. While several
methods are available, we routinely use a technique developed by G.H.S
strauss (non-random cell killing in cryopreservation:
Implications for performance of the battery of leukocyte tests (BLT) I.Toxic
and immunotoxic effect, (mutation res,1991)
Materials:
Solution A: ethidium bromide for the stock solution, dissolved in 50mg EB in
0.1 ml 100%-EtOH, AND ADD 100 µl to 49ml PBS.
For the working solution, add 250µl stock solution to 9.75 ml PBS (final conc.
= 0.025µg/µl, and protected from light.
Solution B: 5-6 carboxyfluorescein diacetate for the stock, diisolve 3mg CFDA
in 1.0 ml aceton. For working solutionto 9.58ml PBS( final conc.=0.125µg/µl)
and protected from light.
Solution C: working solution: combine A and B in ratio of 1:1 can be
refrigerated for up to 6 mounth. Protect from light.
Procedure:
1-place 40 µ of at least 106 cells/ml in microcentrifuge tube, and add 10 µl of
working Solutuin of due stain (5-6 carboxyfluorescein diacetate: ethidium
bromide: solution C
2- let stand for 3-5 min at 37C and then remove excess stain
3 pellet cell sample and pour off supernatant
٣٩
4- Repeat wash.
5- Pellet off and pour off supernatant.
6- Place drop with cells on microscope slide.
7- Observe cells with fluorescent.
8- Observe 100 cells for number of viable cells.
3- III. Electrophoresis of Microgel Slides
The procedure described is for electrophoresis under pH>13 alkaline
conditions.
1. After at least 2hour at ~4ºC, gently remove slides from the Lysing Solution.
Place slides side by side on the horizontal gel box near one end, sliding them
as close together as possible.
2. Fill the buffer reservoirs with freshly made pH>13 Electrophoresis Buffer
until the liquid level completely covers the slides (avoid bubbles over the
agarose).
3. Let slides sit in the alkaline buffer for 20 minutes to allow for unwinding of
the DNA and the expression of alkali-labile damage.
NOTE: The longer the exposure to alkali, the greater the expression of alkalilabile damage.
4. Turn on power supply to 24 volts (~0.74 V/cm) and adjust the current to 300
milliamperes by raising or lowering the buffer level. Depending on the purpose
of the study and on the extent of migration in control samples, electrophorese
the slides for 30 minutes.
NOTE: The goal is to obtain migration among the control cells without it being
excessive.
The optimal electrophoresis duration differs for different cell types. If
crosslinking is one of the endpoints being assessed then having controls with
about 25% migrated DNA is useful. A lower voltage, amperage and a longer
electrophoresis time may allow for increased sensitivity.
Different gel boxes will require different voltage settings to correct for the
distance between the anode and the cathode.
5. Turn off the power. Gently lift the slides from the buffer and place on a drain
tray. Drop wise coat the slides with Neutralization Buffer, let sit for at least 5
minutes. Drain slides and repeat two more times.
6. Slides may be stained with 80µL 1X Ethidium Bromide, leave for 5 min and
then dipped in chilled distilled water to remove excess stain. The coverslip is
٤٠
then placed over it and the slides are scored immediately or dried before
staining as in step 7.
7. Drain slides, keep them for 20 min in cold 100% ethanol or cold 100%
methanol for dehydration. Air dry the slides and place them in an oven at
500C for 30 min. Store in a dry area.
8. When convenient, rehydrate the slides with chilled distilled water for 30 min
and stain with EtBr as in step 6 and cover with a fresh coverslip. Before
viewing slides, blot away excess liquid on the back and edges. After scoring,
remove coverslip, rinse in 100% alcohol to remove stain, let dry, and store for
archival purposes if needed.
4-Evaluation of DNA Damage:
1. For visualization of DNA damage, observations are made of EtBr-stained
DNA using a 40x objective on a fluorescent microscope.
2. Although any image analysis system may be suitable for the quantitation of
SCGE data, we use a Komet 5 image analysis software developed by Kinetic
Imaging, Ltd. (Liverpool, UK)
linked to a CCD camera to assess the quantitative and qualitative extent of
DNA damage in the cells by measuring the length of DNA migration and the
percentage of migrated DNA.
Finally, the program calculates tail moment. Generally, 50 to 100 randomly
selected cells are analyzed per sample.
3. Compare the amount of migration per cell, the number of cells with
increased migration, the
extent of migration among damaged cells, and viability.
٤١
Chapter three
Results
٤٢
3.1 Results
Clinical characteristics of all subject are summarized in the following tables
(Tables 1, 2, 3). There were no significant differences between the diabetic patient
and non-diabetic subject of prevalence of gender and Age.
However, compared with non-diabec subject, diabetes had higher hemoglobin A1c
(HbA1c) (p < 0.01), Vitamin D (P < 0.05), Cytokine TNF-alpha (P < 0.01), Cytokine (IL6) (P < 0.05).
3.2 Diabe:c status
Table 3.1
Show Clinical characteristics of all subjects according to diabetic status.
Non diabetic
Diabetic
Diabetic
subject
male
female
Number
53
26
27
Age
10.3±3.5
11.2±3.6
10.3±3.5
HbA1c(%)
5.3±0.4
10.88 ± 1.39 10.91 ± 1.87
٤٣
3.3 Biochemical parameters
Table 3.2 Show Clinical characteristics of all subjects according to vitamin D.
Non diabetic
Diabetic
Diabetic
subject
male
female
27±0.43
12.75±6.65
13.71±5.50
Vitamin
D(ng/ml)
3.4 DNA damage
Table3.3
Show the clinical characteristics of the subject according to DNA damage.
Non diabetic
Diabetic
Diabetic
subject
male
female
Single cell DNA
damage (Tail
moment)
6.35 ± 3.27
12.67 ± 7.49 12.67 ± 7.49
Table ( 4) :Concentration of MADP, MDAE, CAT,SOD, and GSH in plasma
and blood cells of control and diabetic patients
٤٤
Group 1
Group 2
Control
Diabetic
MDAP (nmol/ml)
0.31±0.06
0.42±0.05
0.001
MDAE (nmol/ml)
0.75±0.13
0.84±0.1
0.08
CAT (IU/ml)
140±30.7
190±0.58
0.001
SOD (U/mg protein)
4±0.12
4.2±0.44
0.95
GSH (mU/mg protein)
75±14
57.8±15
0.005
٤٥
P value
٤٦
Figure 3.1 According to the p value (p < 0.01) there is clinical
significance increase of HbA1c in diabetic males and females.
٤٧
Figure 3.2 According to the p value (p˂0.50)there is significant
difference between the subject and controls.
٤٨
Chapter four
discussion
٤٩
4.1 Glucose and HbA1c
In the present study, We measure HbA1c and there were significance
increasing comparing to normal subject. The elevated HbA1c in the ddiabetic
patient indicated poor glycemic control.
4.2 Biochemical parameters
4.3 Vitamin D
In the present clinical study, we show our result significant abnormalities in
those patients were vitamin D deficient it may due to many cases such as
malnutrition or rare sun exposure.
4.4 Oxidative stress marker
Although oxidative stress markers are studied extensively, there is still
controversy about which of these is the most reliable and suitable one for
clinical practice. Since oxidants are highly reactive compounds, their half-life
is only seconds. Therefore, after exposing to oxy-radicals, the modified
products of lipids, nucleic acids and carbohydrates are accepted as more
reliable markers of oxidative stress because of their long half-life. On the other
hand, in order to evaluate the state of oxidative stress, measurements of antioxidant enzymes can give some more information about the oxidant defense
system (Atli et al. 2004). In this study, we selected MDAE and MDAP
products, as markers of oxidative stress. Also, leukocytes CAT, SOD and
GSH-Px enzyme activities were taken as markers of oxidant defense system.
It was shown that high blood glucose level resulted with lower GSH-Px
activity and vitamin D supplementation resulted with higher GSH-Px and SOD
activities in patients with DM
As shown in Table 4,we determined increased activation of anti-oxidant
defense enzymes, particularly CAT and GSH-Px, in group 2. However, the
increase does not seem to be enough to prevent oxidative burden. Also, as
seen in GSH-Px activity, and even decreased in group 2. Therefore, one can
assume that poor diabetic control might be responsible for the suppression of
more GSH-Px activity as it was seen in group 2. CAT does not seem to be
effected by uncontrolled DM, moreover, the activity of this enzyme was
enhanced paralleling the oxidant burden.
4.5 DNA Damage
Results were collected to DNA damage showed significant difference in
compare to normal subjects which indicate that there is damage in diabetic
DNA due to the increase of free radicals
٥٠
Chapter five
Conclusion
٥١
5.1 Conclusion
1- A major clinical lesson than can be drawn at this
moment is the avoidance of vitamin S deficiency is
essential for β-cell function and might contribute to
protection against type 1 diabetes in later life.
2- Hyperglycemia contributes to increased
generation of ROS and increased oxidative stress
would lead to oxidative DNA damage.
٥٢
Chapter six
Refernce
٥٣
Reference:
1-http://www.medicinenet.com/diabetes_mellitus/page3.htm
2-http://www.diabetes.co.uk/treatment.html
3‫ـ‬http://www.vivo.colostate.edu/hbooks/pathphys/endocrine/othere
ndo/vitamind.html
4-http://www.mayoclinic.com/health/vitamin-d/NS_patient-vitamind
5-http://www.webmd.com/diet/vitamin-d-deficiency
6-Cooke, M. S., Evans, M. D., Dizdaroglu, M., Lunec, J. Oxidative
DNA damage mechanisms, mutation, and disease. FASEB J. 17,
1195–1214 (2003)
7-http://en.wikipedia.org/wiki/Diabetes_mellitus.
8-http://www.vitaminddeficiencyguide.com/
9- Diabetes Metab Res Rev 2005; 21: 338–346. Published online
26 April 2005 in Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/dmrr.557
10-January 1999 and in revised form: 18 March 1999
Corresponding author: Dr. S. Suzuki, The Third Department of
Internal Medicine, Tohoku University School of Medicine, 1±1,
Seiryou-cho, Aoba-ku, Sendai 980, Japan Abbreviations: 8oxodG,8-Oxo, 2 ¢-deoxyguanosine; ROS, reactive oxygen
species.
11-Int. J. Cancer: 122, 2699–2706 (2008) ' 2008 Wiley-Liss, Inc.
12-Alok Dhawan, Ph.D., MNASc. Mahima Bajpayee, M.Sc. Alok
Kumar Pandey, M.Sc. Devendra Parmar, Ph.D. Developmental
Toxicology Division Industrial Toxicology Research Centre P.O. Box –
80, M.G. Marg, Lucknow – 226 001 U.P., India
٥٤