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Key words: oxidative stress, DNA damage, diabetesmellitus, dyslipidemia

SUMMARY

Diabetes is associated with excessive production ofreactive oxygen species (ROS), which can damagecellular macromolecules. The aim of the study was todetect oxidative DNA damage in type 2 diabeticpatients using the comet assay, and to study therelationship between DNA damage and hyperglycemialipid profile in diabetic patients. The study populationconsisted of 28 patients with type 2 diabetes mellitusand 25 healthy volunteers, age and sex matched withthe patients, as controls. Lipid profile, fasting bloodglucose and glycosylated hemoglobin (HbA1c) wereassessed in patients and controls. Comet assay wasused to detect DNA damage. The percent of DNAdamage of peripheral blood mononuclear cells washigher in diabetic patients (32.6±25.2) compared to

healthy controls (3.7±1.3) (p<0.001). Pearsoncorrelation analysis showed a significant positivecorrelation of DNA damage with fasting blood glucoseand glycated hemoglobin, but not with serum totalcholesterol, triglycerides, HDL-c and LDL-c. Inconclusion, type 2 diabetic patients have moreoxidative DNA damage than normal controls and poorglycemic control may aggravate this damage.Dyslipidemia is not a contributing factor for DNAdamage 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

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

Corresponding author: Professor Hala Mohamed Raslan, MD, PhD,Internal Medicine Department, National Research Center, El BuhouthStreet, Dokki, 12311 Cairo, Egypt

E-mail: [email protected]

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

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

122

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 INPATIENTS 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, gelelectrophoresis, 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.

123Diabetologia Croatica 41-4, 2012

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

Table 1. Demographic and laboratory data of patients and controls


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 -

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

124


Table 2. Correlation between DNA damage and demographic and laboratory data

Variable r p

Age 0.06 0.8

BMI 0.05 0.76

Duration 0.062 0.7

Fasting blood glucose 0.81 <0.001*

HbA1c

(%) 0.5 0.003*

Cholesterol 0.16 0.41

Triglycerides -0.2 0.26

HDL cholesterol 0.14 0.45

LDL cholesterol 0.26 0.17

*p is significant; BMI = body mass index; HDL = high density lipoprotein; LDL = low density lipoprotein

125

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

a fundamental step in the initiation and progression of

atherosclerosis (31). In the current study, we did not

find significant correlation between DNA damage and

serum total cholesterol, HDL-c, LDL-c, triglycerides

and BMI. However, a recent study revealed increased

DNA damage as assessed by serum 8-OHdG level, in

obese and overweight subjects compared to lean

subjects (32). Another study revealed positive

correlation between DNA damage, as assessed by

comet assay, and total cholesterol, triglycerides and

LDL-c in obese non-diabetic subjects (33).

In conclusion, type 2 diabetic patients have more

oxidative DNA damage than normal controls and poor

glycemic control may aggravate this damage. Dyslipi -

demia is not a contributing factor for DNA damage in

diabetes.

Diabetologia Croatica 41-4, 2012


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