National Research Center, Cairo, Egypt Original Research Article

OXIDATIVE DNA DAMAGE IN PATIENTS WITH TYPE 2

DIABETES MELLITUS
Maha El-Wassef, Gamila S. M. El-Saeed, Safinaz E. El-Tokhy, Hala M. Raslan, Salwa Tawfeek,
Ibrahem Siam, Sohair I. Salem

Key words: oxidative stress, DNA damage, diabetes
mellitus, dyslipidemia
SUMMARY
Diabetes is associated with excessive production of
reactive oxygen species (ROS), which can damage
cellular macromolecules. The aim of the study was to
detect oxidative DNA damage in type 2 diabetic
patients using the comet assay, and to study the
relationship between DNA damage and hyperglycemia
lipid profile in diabetic patients. The study population
consisted of 28 patients with type 2 diabetes mellitus
and 25 healthy volunteers, age and sex matched with
the patients, as controls. Lipid profile, fasting blood
glucose and glycosylated hemoglobin (HbA1c) were
assessed in patients and controls. Comet assay was
used to detect DNA damage. The percent of DNA
damage of peripheral blood mononuclear cells was
higher in diabetic patients (32.6±25.2) compared to
Corresponding author: Professor Hala Mohamed Raslan, MD, PhD,
Internal Medicine Department, National Research Center, El Buhouth
Street, Dokki, 12311 Cairo, Egypt
E-mail: halamzr@yahoo.com
healthy controls (3.7±1.3) (p<0.001). Pearson
correlation analysis showed a significant positive
correlation of DNA damage with fasting blood glucose
and glycated hemoglobin, but not with serum total
cholesterol, triglycerides, HDL-c and LDL-c. In
conclusion, type 2 diabetic patients have more
oxidative DNA damage than normal controls and poor
glycemic control may aggravate this damage.
Dyslipidemia is not a contributing factor for DNA
damage in diabetes.
INTRODUCTION
Diabetes mellitus is a chronic metabolic syndrome. It
is one of the most challenging health problems in the
21st century. The prevalence of diabetes is increasing
globally, the number of diabetics is expected to
increase to 366 million by 2030 (1).
Patients with type 2 diabetes suffer from several
complications such as atherosclerosis, retinopathy,
neuropathy and nephropathy with devastating effect
on morbidity and mortality (2).

Diabetologia Croatica 41-4, 2012 121

 M. El-Wassef, G. S. M. El-Saeed, S. E. El-Tokhy, H. M. Raslan, S. Tawfeek, I. Siam, S. I. Salem / OXIDATIVE DNA DAMAGE IN
PATIENTS WITH TYPE 2 DIABETES MELLITUS
There is considerable evidence that hyperglycemia
results in the generation of reactive oxygen species
(ROS), ultimately leading to increased oxidative stress
in a variety of tissues, and playing an important role in
diabetic complications (3,4). Oxidative stress leads to
protein, lipid, and DNA modifications that cause
cellular dysfunction and this could have teratogenic or
carcinogenic consequences (5).
During the past ten years, there has been increasing
awareness of the effect of DNA damage in chronic
diseases. Single cell gel electrophoresis (SCGE) or
comet assay to measure DNA damage was first
developed by Ōstling and Johansson in 1984 (6). It is
a sensitive, simple, inexpensive, and rapid method that
can be used to detect DNA damage to individual cells
and reveal the presence of double-strand breaks,
single-strand breaks and alkali-labile sites (7). It has
been widely used in studies on DNA repair, genetic
toxicology, radiation, pollution and ageing (8,9).
The aim of this study was to detect the oxidative
DNA damage in patients with type 2 diabetes mellitus
and to investigate the relation between oxidative DNA
damage and hyperglycemia and lipid profile.
MATERIALS AND METHODS
Twenty-eight patients with type 2 diabetes mellitus
were recruited from the outpatient clinic of Medical
Services Unit at the National Research Center. There
were 8 men and 20 women, age range from 45 to 61
years, mean age 52.5±5.3 years. Twenty-five healthy
volunteers, 10 men and 15 women, age range from 45
to 60 years, mean age 53.1±6.7 years, served as
control group. We excluded any patient with a history
of smoking, acute or chronic infections, coronary arte-
ry disease, congestive heart failure, chronic liver
disease, diabetic nephropathy, rheumatic disease,
cancer, and patients who had recently undergone
radiological procedures (1 month previously). None of
the patients was taking lipid lowering drugs.
Participants were subjected to detailed history for
collection of demographic data and recording of
relevant medical history and medications. Thorough
clinical examination and height and weight
122
measurement for calculation of body mass index
(BMI) were also done in patients and controls. A
written informed consent was obtained from each
participant and the study was approved by the Ethics
Committee.
Laboratory methods
Five milliliters of venous blood was withdrawn from
both healthy individuals and patients fasting for 10
hours into two sterile vacutainers, one containing
EDTA and the other without additives to separate
serum.
Serum samples were assayed within 2 hours for
fasting blood glucose, glycosylated hemoglobin
HbA1c and lipid profile: total cholesterol, triglycerides
and high-density lipoprotein cholesterol (HDL-c)
using the Olympus AU 400 automated clinical
chemistry analyzer. Low-density lipoprotein chole-
sterol (LDL-c) was calculated by Friedewald formula
(10). DNA damage was detected by the comet assay.
Measurement of comet assay
Cell preparation
Peripheral blood leukocytes were isolated by
centrifugation (30 min at 1300 g) in Ficoll-Paque
density gradient (Pharmacia LKB Biotechnology,
Piscataway, NJ, USA). After centrifugation, leuko-
cytes in the buffy coat were aspirated and washed
twice by phosphate-buffered saline (PBS) at pH 7.4.
Preparation of cell microgels on slides
All the procedures for the alkaline comet assay were
done at low temperature to minimize spontaneous
DNA damage. The comet assay was performed
according to Singh et al. (11) with modifications
according to Blasiak et al. (12). Cell microgels were
prepared as layers. The first gel layer was made by
applying 100 μL of normal melting point agarose
(0.7%) onto a precleaned microscope charged slides
and coverslipped gently. The coverslip was removed
after the agarose solidified at 4 °C. Low melting-point
 M. El-Wassef, G. S. M. El-Saeed, S. E. El-Tokhy, H. M. Raslan, S. Tawfeek, I. Siam, S. I. Salem / OXIDATIVE DNA DAMAGE IN
PATIENTS WITH TYPE 2 DIABETES MELLITUS

agarose (0.5%) was prepared in 100 mmol/L PBS and
kept at 37 °C. Approximately 1500 peripheral blood
lymphocytes were mixed with the low melting-point
agarose and 100 μL of the mixture was applied to the
first gel layer. The slides were then covered with a
coverslip and placed at 4 °C for solidification. After
the second layer had solidified, the coverslips were
removed from the cell microgels. A final layer of low-
melting agarose was added followed by coverslips, left
to solidify for 10 minutes, and then the coverslips were
removed.
Lysis of cells, DNA unwinding, gel
electrophoresis, DNA staining
The slides were covered with 100 mL of fresh lysis
buffer (2.5 mol/L NaCl, 100 mmol/L EDTA, 1%
sodium hydroxide, 10 mmol/L Tris, 1% Triton X-100,
10% DMSO (pH 10) at 4 °C for 1 h. After draining,
microgel slides were treated with DNA unwinding
solution (300 mmol/L NaOH, 1 mmol/L EDTA, pH
13) for 30 min at 4 °C, and placed directly into a
horizontal gel electrophoresis chamber filled with
DNA-unwinding solution. Gels were run with constant
current (300 mA at 4 °C) for 30 min. After electro-
phoresis, the microgels were neutralized with 0.4 M
Trisma base at pH 7.5 for 10 min. The slides were
stained with 20 μL ethidium bromide (10 μg/mL).
Visualization and analysis of comet slides
The slides were examined at 400× magnification
using an inverted fluorescence microscope (IX70,
Olympus, Tokyo, Japan) equipped with an excitation
filter of 549 nm and a barrier filter of 590 nm, attached
to a video camera (Olympus). Damaged cells were
visualized by the "comet appearance", with a brightly
fluorescent head and a tail to one side formed by the
DNA containing strand breaks that were drawn away
during electrophoresis. Samples were analyzed by
counting damaged cell out of 100 cells per slide to
calculate the percent of damage.
Statistical analysis
Data are presented as mean ±SD. The compiled data
were computerized and analyzed by SPSS PC+,
version 14. The following tests of significance were
used: t-test between means to analyze mean difference.
A value of p≤0.05 was considered significant, p<0.001
was considered highly significant, and p>0.05 was
considered nonsignificant. Relationships between
continuous variables were assessed using Pearson's
correlation coefficient.
RESULTS
The study included 28 patients with type 2 diabetes
mellitus and 25 healthy individuals as control group.
The mean age of the patients was 52.5±5.3 years.

Table 1. Demographic and laboratory data of patients and controls

Variable Diabetic patients (n=28) Controls (n=25)
Age (yrs), mean±SD 52.5±5.3 53.1±6.7
Sex: male/female 8/20 10/15
BMI (kg/m2), mean±SD 31.7±3.7 30.2±1.8
Duration (yrs), mean±SD 9.7±5.1 -
Fasting blood glucose (mg/dL), mean ± SD 185.86±70.35* 86.56±6.25
HbA1c (%), mean±SD 9.4±3.3* 6±0.7
Cholesterol (mg/dL), mean±SD 229.7±56.2 217.6±68.9
Triglycerides (mg/dL), mean±SD 170±88.3 133.5±85.3
HDL cholesterol (mg/dL), mean±SD 45.8±12.6* 62.6±21.4
LDL cholesterol (mg/dL), mean±SD 148±51 129.8±56
DNA damage (%), mean±SD 32.6±25.2* 3.7±1.3
*p<0.001; BMI = body mass index; HDL = high density lipoprotein; LDL = low density lipoprotein

Diabetologia Croatica 41-4, 2012 123

 M. El-Wassef, G. S. M. El-Saeed, S. E. El-Tokhy, H. M. Raslan, S. Tawfeek, I. Siam, S. I. Salem / OXIDATIVE DNA DAMAGE IN
PATIENTS WITH TYPE 2 DIABETES MELLITUS
Table 2. Correlation between DNA damage and demographic and laboratory data
Variable
Age
BMI
Duration
Fasting blood glucose
HbA1c (%)
Cholesterol
Triglycerides
HDL cholesterol
LDL cholesterol
*p is significant; BMI = body mass index; HDL = high density lipoprotein; LDL = low density lipoprotein
None of the patients was taking antioxidant supple-
ment. Demographic and laboratory data on the patients
and controls are shown in Table 1.
The number of DNA strand breaks was significantly
higher in diabetic patients compared to controls. The
number of DNA strand breaks correlated positively
with fasting blood glucose and HbA1c, while no
significant correlation was found with serum total
cholesterol, triglycerides, HDL-c, LDL-c and BMI
(Table 2).
DISCUSSION
Diabetes mellitus is associated with oxidative stress,
leading to protein, lipid and DNA modifications.
Oxidative DNA damage is usually evaluated using the
comet assay in white blood cells (13,14), or by
measuring the oxidized base 8-OHdG (8-hydroxy-2-
deoxy-guanosine) in white blood cells or urine
(15,16).
In the present study, we used the comet assay as a
measure of DNA strand-break damage, as the
technique is less susceptible to artifacts than mea-
suring 8-OHdG, and it is a sensitive, simple method to
detect very low levels of damage (17).
The present study revealed an increased number of
DNA strand breaks in peripheral blood leukocytes of
diabetic patients compared to healthy controls. The
production of ROS and lipid peroxidation are
increased in diabetic patients (18). Reactive oxygen
species can damage cellular macromolecules, leading
to DNA and protein modification and lipid per-
124
r p
0.06 0.8
0.05 0.76
0.062 0.7
0.81 <0.001*
0.5 0.003*
0.16 0.41
-0.2 0.26
0.14 0.45
0.26 0.17
oxidation. The elevated ROS in diabetes can cause
strand breaks in DNA and base modifications
including oxidation of guanine residues to 8-OHdG,
an oxidized nucleoside of DNA, which is the most
frequently detected and studied DNA lesion (19).
Previous studies concerning DNA damage and
diabetes revealed contradictory results. Several studies
showed an increased extent of DNA damage in type 2
diabetic patients compared to controls (14,20,21). On
the other hand, other studies showed the lack of
association between diabetes and increased DNA
damage levels (22,23). The discrepancy between
different studies is, possibly, due to difference in
glycemic control, duration of diabetes or the type of
cell used in the comet assay (21).
Goodarzi et al. (24) report on a significant positive
correlation between urinary 8-OHdG, a biomarker of
oxidative DNA damage, and fasting blood glucose and
HbA1c. In the present study, we found a significant
positive correlation between DNA damage and HbA1c.
Hyperglycemia causes glucose auto-oxidation, glyca-
tion of proteins, activation of polyol metabolism and
subsequent formation of ROS. It has also been
demonstrated that hyperglycemia is associated with an
increased production of free radicals in the mito-
chondria and may contribute to a greater DNA damage
(25).
We did not find correlation between duration of
diabetes and DNA damage. The lack of correlation
with duration of diabetes has been reported by other
authors, in diabetes (23) and in chemical exposure
(26,27), and investigators suggest that long-term
 M. El-Wassef, G. S. M. El-Saeed, S. E. El-Tokhy, H. M. Raslan, S. Tawfeek, I. Siam, S. I. Salem / OXIDATIVE DNA DAMAGE IN
PATIENTS WITH TYPE 2 DIABETES MELLITUS
chronic exposure causes adaptation of response to
damage and produces less genetic damage than initial
exposure (26,27). On the other hand, another study
found a positive association between duration of
diabetes and expression of the enzyme DNA
glycosylase 8-OxoG DNA glycosylase (Ogg I), which
is a major repair enzyme involved in defense against
accumulation of the 8-OHdG adducts (28).
One of the complications of diabetes is athe-
rosclerosis. Atherosclerosis is associated with DNA
damage that increases with progression of
atherosclerosis. Recent studies revealed increased
DNA damage in obesity with significant positive
correlation with cholesterol, triglycerides and LDL-c,
and DNA damage was present in atherosclerotic
plaques and in circulating cells of patients with
atherosclerosis (29,30). It is not known whether DNA
damage in diabetes directly promotes atherosclerosis,
or it is a byproduct of dyslipidemia of diabetes. Some
cholesterol oxidation products (oxysterols) lead to the
generation of reactive oxygen/nitrogen species
(ROS/RNS) that can cause DNA damage; also, ROS
are involved in oxidation of LDL, which is considered
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