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Title: Levels of peripheral blood cell DNA damage in Insulin Dependent Diabetes
Mellitus (IDDM) human subjects.
Running title: DNA damage in diabetic human subjects.
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2
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Mary P. A. Hannon-Fletcher , Maurice J. O’Kane , Ken W. Moles , Colin
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Weatherup1, Christopher R. Barnett and Yvonne A. Barnett*
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1
Cancer and Ageing Research Group, University of Ulster, Cromore Road, Coleraine,
County Londonderry, Northern Ireland, BT52 1SA.
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Altnagelvin Area Hospital, Glenshane Road, Londonderry, BT47 1SB.
*Corresponding author ya.barnett@ulst.ac.uk Tel: +44-(0)-2870-324627
Fax: +44-(0)-2870-324965
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Abstract
Increased production of reactive oxygen species in vivo can lead to cellular
biomolecule damage. Such damage has been suggested to contribute to the
pathogenesis of IDDM. In this study we used the alkaline comet assay to measure
DNA damage (single-stranded DNA breaks and alkali-labile sites) in freshly isolated
whole blood, lymphocytes, monocytes and neutrophils from 23 subjects with IDDM
and 32 age- and sex-matched controls. Analysis of the results showed elevated levels
of DNA damage (expressed as % comet tail DNA) in the lymphocyte (4.10±0.47;
3.22±0.22), monocyte (4.28±0.47; 3.49±0.18) and whole blood (4.93±0.51;
4.51±0.23) fractions from IDDM subjects compared to controls, respectively, but the
increases observed were not statistically significant. However, we found significantly
elevated basal levels of DNA damage in the neutrophil fraction (8.38±0.64;
4.07±0.23; p<0.001, Mann-Whitney U test) in IDDM subjects compared to controls.
Given these novel neutrophil findings we extended the study to include a total of 50
IDDM subjects and 50 age- and sex-matched control subjects, and determined basal
levels of DNA damage in the neutrophils of all 100 subjects. We found significantly
elevated mean levels of DNA damage (8.40±0.83; 4.34±0.27; p<0.001, MannWhitney U test) in the neutrophils from the IDDM subjects when compared to
controls. Our results show that even with acceptable glycaemic control there is a
significantly elevated level of DNA damage within diabetic neutrophils in vivo.
Keywords: Alkaline comet assay, DNA damage, Neutrophils, IDDM, %Hba1c.
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1. Introduction
Insulin Dependent Diabetes Mellitus (IDDM) is associated with increased oxidative
stress in vivo. Studies on diabetic subjects have demonstrated increased free radical
production contributed to by hyperglycaemia resulting in glycosylated proteins and
production of reactive oxygen species (ROS) [1]. In addition, superoxide anions
produced during these reactions react with plasma lipids leading to the generation of
chemotactic factors, which in turn are capable of stimulating neutrophils with
subsequent release of enzymes stored in cytoplasmic granules and the additional
production of ROS [2-4]. The oxidative stress is further exacerbated by decreases in
antioxidant enzyme activity, including superoxide dismutase, catalase and
glutathione peroxidase [3, 5-7]. Under conditions of oxidative stress damage to
cellular biomolecules (lipids, proteins, carbohydrates and DNA) can occur. Until the
late 90’s the main marker used as an index of in vivo oxidative damage in IDDM has
been the detection of lipid peroxidation products in plasma and cell membranes [814]. Polyunsaturated fatty acids are among the most readily oxidised substrates in
biological systems. A broad range of oxidation products have been described [15],
including lipid peroxides, which are precursors to other reactive intermediates, such
as alkoxyl radicals, and hydroxyalkenals formed in lipid peroxidation reactions,
including malondialdehyde (MDA). A number of lipid peroxidation products mainly,
MDA and 4-hydroxy-2-nonenal [16], are known to interact with DNA [17]. Such
interaction can lead to cytotoxicity, genotoxicity and carcinogenicity [18].
More recently (1995) oxidative damage to DNA has been demonstrated by measuring
levels of 8-hydroxydeoxyguanosine, a recognised biomarker of oxidant-induced
DNA damage, in both mononuclear cells and sperm from diabetic subjects, using
high performance liquid chromatography [19, 20]. Other groups have measured DNA
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damage levels in mononuclear cells from IDDM subjects, using the comet
assay/alkaline unwinding techniques. The results from these studies have shown
increases, in subjects with poor glycaemic control [21,22] and no significant changes,
in subjects with good glycaemic control [22, 23], in levels of DNA damage when
compared to control subjects.
Previous work in this laboratory [6] measured DNA damage (single-strand breaks
and alkali-labile sites) using a sandwich ELISA described by van Loon et al. [24] in
whole blood and phytohaemagglutinin (PHA)-stimulated lymphocytes from 20
IDDM subjects and from 11 control subjects. Results showed significantly increased
basal levels of DNA damage in whole blood but not lymphocytes, from the IDDM
subjects compared to controls.
In light of this whole blood DNA damage data we decided to investigate the types of
DNA damage and the levels of DNA damage present in the various different types of
nucleated blood cells (lymphocytes, monocytes and neutrophils) from IDDM
subjects. In this paper we report the results obtained from the analysis of levels of
DNA damage.
2. Materials and Methods
2.1. Subjects
50 IDDM subjects (mean age 36.3 ± 1.95 years; 30 males and 20 females), were
recruited from the Diabetic Clinic, Altnagelvin Hospital, Londonderry, Northern
Ireland. Thirteen of the diabetic subjects presented with at least one complication
(retinopathy, nephropathy, neuropathy and macrovascular disease) and 8 were
smokers. The control group consisted of 50 healthy individuals recruited from the
University of Ulster (mean age 37.6 ± 1.15 years; 22 females and 28 males, none of
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whom were smokers, nor did they have a family history of diabetes). Ethical approval
for this study was obtained from the University of Ulster Ethical Committee and
from the Ethical Committee at Altnagelvin Hospital. All subjects gave their informed
consent prior to enrolment into the study.
2.2. Collection and processing of blood samples
15 ml of peripheral blood was collected from each study subject. 10 ml was collected
into lithium heparin-coated vacutainers® (Becton-Dickinson, UK) for subsequent
determination of basal levels of DNA damage within nucleated blood cells. The
remaining 5ml was collected into EDTA-coated vacutainers® (Becton-Dickinson,
UK) for HPLC analysis of glycated haemoglobin (expressed as %HbA1c), using a
method described by John et al. [25]. Analysis of DNA damage in the lymphocyte,
monocyte and whole blood fractions was carried out on 23 diabetic subjects and 32
control subjects. Analysis of DNA damage in the neutrophil fraction was carried out
on samples from 50 IDDM subjects and 50 control subjects.
2.3. Cell isolation and preparation for the comet assay
2.3.1. Whole blood
50 l of fresh whole blood was transferred to an eppendorf and washed twice, (700 x
g for 5 min at 4OC) in 200 l of Ca++ and Mg++ free phosphate buffered saline (PBS,
Sigma, Poole, UK.). The resulting cell pellet was re-suspended in 10l of PBS and
stored at 4OC in the dark (to minimise additional DNA damage and repair), for use
the same day in the comet assay.
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2.3.2. Mononuclear cell isolation
Mononuclear cells were isolated from whole blood using a method described by
Böyum [26]. Essentially, whole blood was mixed 1:1 with RPMI 1640 (Gibco Life
Technologies, UK) then 8 ml of diluted blood was layered onto 10 ml of Histopaque
1077 (Sigma, Poole, UK) in a sterile 25 ml universal container (Sterilin, UK), at
room temperature. Following centrifugation at 700 x g for 30 min, the mononuclear
layer (“buffy layer”) was carefully aspirated, mixed with 10 ml RPMI 1640 and
centrifuged at 500 x g for 10 min. After an additional wash in RPMI 1640 the
mononuclear cells were incubated for 4 hours in RPMI 1640 with 10% foetal calf
serum, 200 µg/ml sodium pyruvate, 100 U/ml penicillin and 100 µg/ml streptomycin
(BDH Laboratory Supplies, Poole, UK) at 37oC (5% CO2: air humidified
atmosphere). Following incubation the medium, which contained the lymphocyte
fraction, was decanted into a labelled centrifuge tube. The monocytes, which had
adhered to the culture flask, were removed into a labelled centrifuge tube using cold
PBS and a cell scraper. Both the lymphocyte and monocyte fractions were washed
briefly (x 2) with PBS. The resulting cell pellets were re-suspended in 5 ml PBS and
kept at 4oC in the dark, for use the same day in the comet assay. Cell viability was
assessed by trypan blue exclusion following isolation, and was found to be >95%.
2.3.3. Neutrophil isolation
Following the removal of the mononuclear layer the blood sample was further
processed for the separation of neutrophils using a dextran sedimentation method
described by Markert et al. [27]. Essentially, the Histopaque layer was carefully
aspirated and discarded, 6 ml of PBS was added to the red cell-neutrophil mixture,
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followed by 10 ml of the 2% dextran (Sigma, Poole, UK) in normal saline (0.9%
(w/v) NaCl in distilled water). This suspension was well mixed and allowed to settle
for 35-45 min at room temperature, after this time the suspension well have settled
into two layers, an upper layer which consists mainly of neutrophils and a lower layer
which contains the red cells. The upper layer was carefully removed and centrifuged
at 500 x g for 10 min. The supernatant was discarded and 6 ml of ice cold sterile
distilled water was added and mixed well for 20 sec, before adding 2 ml of PBS
containing 3.4% NaCl (w/v) (BDH Laboratory Supplies, Poole, UK). This
suspension was then centrifuged at 500 x g for 10 min and the resulting neutrophil
pellet was re-suspended in 5 ml PBS and kept at 4oC in the dark, for use the same
day in the comet assay. Cell viability was assessed by trypan blue exclusion
following isolation, and was found to be >95%.
2.4. The alkaline comet assay
The alkaline comet assay facilitates the detection of DNA strand-breakage, alkalilabile abasic sites, and intermediates in base- or nucleotide-excision repair. In the
comet assay, DNA strand breaks allow DNA to extend from lysed and salt extracted
nuclei, or nucleoids, to form a comet-like tail on alkaline electrophoresis. The slides
are stained with ethidium bromide and comet “tails” viewed by fluorescence
microscopy. In undamaged cells a bright fluorescent core is seen with a less intense
edge of fluorescence facing the anode. If damage is present, fluorescence appears in a
“tail” extending from the core towards the anode. In this investigation we measured
basal levels of DNA damage in whole blood, freshly isolated lymphocytes,
neutrophils and monocytes in IDDM and control subjects.
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The alkaline comet assay procedure in this study was a modification of the method
described by Singh et al. [28]. Essentially, 100 l of 0.5% normal melting point
agarose (Sigma, Poole, UK) was pipetted onto frosted microscope slides and allowed
to solidify under a coverslip, which was then carefully removed. Approximately
10,000 cells were suspended in 75 l low melting point agarose gel, the cell
suspension was rapidly pipetted onto the first agarose layer, and gently spread by
placing a coverslip on top. This was allowed to solidify on an ice tray for 5 min.
After removal of the coverslip, the slide was immersed in freshly prepared lysing
solution (2.5M NaCl, 100mM EDTA, and 10mM Tris, with 1% Triton X-100 and
10% DMSO) made up just before use and incubated overnight at 4oC. The slides
were removed from the lysing solution, drained and placed in a horizontal gel
electrophoresis tank. The tank was filled with fresh, cold electrophoresis solution
(1mM EDTA and 300mM NaOH) to a level approximately 0.25 cm above the slides.
The slides were left in the solution for 20 min to allow the unwinding of the DNA
and expression of alkali-labile damage before electrophoresis. Electrophoresis was
conducted at 4oC for 30 min using 25 V and a current of 300 mA. Following
electrophoresis the slides were washed (x 3) in Tris buffer (0.4M Tris, pH 7.5) to
neutralise the excess alkali. Finally, the slides were stained with 75 l ethidium
bromide (20 g / ml; Sigma, Poole, UK
2.5. Image analysis of slides
Slides were stored in a light-proof box containing tissues moist with PBS and viewed
within 12 hours of staining. Observations were made using an Ophtiphot II
compound microscope (Nikon) equipped with an epifluorescence mercury lamp
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source (excitation filter 515, barrier filter 590 nm) and x 40 Nikon Fluor objective
(numerical aperture 0.85), and Komet 3.0 image analysis programme (Kinetic
Imaging Ltd, Liverpool, UK).
2.6. Analysis of DNA damage
The image analysis software provides a full range of densitometric and geometric
parameters describing the complete comet, as well as the head and tail portions.
Since the comet assay essentially reflects the displacement of fluorescence from the
head to the tail in damaged cells we used %tail DNA i.e. the percentage of total
nuclear DNA that has migrated to the tail, as the parameter to quantify basal levels of
DNA damage. Each blood/blood cell type sample was analysed in duplicate and 50
cells per slide were counted.
2.7. Statistical analysis
Statistical analysis was performed using the SPSS statistical package to compare the
variances of all parameters examined using Levene’s test of homogeneity of
variances. Differences in measured parameters between normal and IDDM subjects
were assessed by the Mann-Whitney U test, since the variances in the two samples
were heterogeneous. Differences in the measured parameters between IDDM subjects
with, and without, complications were measured using the Student’s t-test. The
relationship between DNA damage and %HbA1c was analysed using least squares
linear regression analysis. A p value of <0.05 was considered statistically significant.
All results are expressed as mean ± standard error of the mean (SEM).
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3. Results
%HbA1c levels (Table 1) were significantly increased in IDDM subjects (7.71±0.03;
n=50) in comparison to controls (4.28±0.06; n=50; p <0.001). The range of levels
indicated that all IDDM patients were under acceptable clinical control.
In the initial study group of 23 IDDM subjects and 32 controls we found increased
levels of DNA damage (%tail DNA, Figure 1) in the lymphocyte (4.10±0.47 and
3.22±0.22), monocyte (4.28±0.47and 3.49±0.18) and whole blood (4.93±0.51 and
4.51±0.23) fractions of the IDDM subjects, respectively, when compared to controls.
These increases were not statistically significant. However, there were significantly
elevated basal levels of DNA damage in the neutrophil fraction of IDDM subjects,
when compared to control subjects (8.38±0.64 and 4.07±0.23 respectively, p<0.001).
These findings on DNA damage levels in neutrophils of IDDM subjects were further
confirmed by increasing the number of subjects examined to 50 IDDM subjects and
50 control subjects. This extended study also revealed significantly elevated basal
levels of DNA damage in the neutrophils from IDDM subjects compared to the
control group, respectively (8.40±0.83 and 4.34±0.27; p<0.001, Figure 1).
Of the 50 IDDM subjects, eight were smokers, three of the smokers also had at least
one complication. Because of small numbers it was not possible to determine by
statistical analysis if the values obtained for the various endpoints (%HbA1c and
%tail DNA damage) were different in IDDM subjects who smoked compared to
those who did not, but this did not appear to be the case. Thirteen of the IDDM
subjects presented with at least one complication (retinopathy, neuropathy,
nephropathy, ischemic heart disease). There were no significant differences in values
of %HbA1c or %tail DNA damage measured in the IDDM subjects with
complications compared to those who did not have complications.
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There was no correlation between basal DNA damage levels (%tail DNA) and
%HbA1c in the IDDM subjects.
4. Discussion
IDDM is associated with increased oxidative stress in vivo [2, 3, 29, 30]. The
elevated oxidative stress may be caused by; increased levels of free radicals due to
their increased production, and/or decreases in the antioxidant defense systems [3, 57]. The increased oxidative stress together with poor metabolic control enhances lipid
peroxidation in diabetic patients, which has been proposed to be associated with the
aetiology of diabetic complications [12, 31, 32].
In the present study we used the alkaline comet assay to measure basal levels of DNA
damage in freshly isolated whole blood and individual blood cell fractions from
IDDM subjects. We found increased levels of DNA damage in the lymphocyte,
monocyte and whole blood cell fractions from IDDM subjects, however the increases
observed were not significantly different from controls. Conflicting reports regarding
levels of DNA damage in mononuclear cells from diabetic subjects have been
reported recently in the literature. Collins et al. [21] reported elevated levels of DNA
damage in mononuclear cells from IDDM patients with poor glycaemic control
(11.0±2.9 %HbA1c). Collins’ group measured DNA damage using both the alkaline
comet assay and a modification of the assay which incorporates a digestion of
nucleoid DNA with specific endonucleases which allows detection of specific types
of damage such as; oxidised pyrimidines and purines. Lorenzi et al. [22] measured
DNA damage, using the alkali unwinding assay, in mononuclear cells from patients
with poor glycaemic control (12.9±2.4 %HbA1c) and found elevated levels of DNA
damage. In the same study they also reported no significant changes in DNA damage
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in subjects with good glycaemic control (%HbA1c 7.4±1.5%). In agreement with
these findings, we [6], and Anderson et al. [23], did not find significant differences in
levels of DNA damage in lymphocytes and mononuclear cells, respectively from
IDDM subjects with good glycaemic control, when compared to controls. In our
previous study [6] we measured levels of DNA damage in PHA-stimulated
lymphocytes, which are more proficient at DNA repair than unstimulated
lymphocytes (like many found in vivo) [33], and so the levels which we measured in
vitro may not have been a true reflection of those existing in vivo. As glycaemic
control plays an important role in the progression of IDDM and the development of
secondary complications, it is perhaps not surprising that changes in the level of an
index of biomolecule damage, in this case DNA damage in mononuclear cells,
correlates with %HbA1c in diabetic patients, in some studies. As the subjects in our
study and in both, Lorenzi et al. [22] and Anderson et al. [23], had good glycaemic
control this may help explain why we could not detect significant increases in levels
of DNA damage.
In addition to measuring levels of DNA damage in the PHA-stimulated lymphocytes
in our previous study we also measured basal levels of DNA damage in fresh whole
blood using an ELISA [6]. We found significantly higher levels of DNA damage in
whole blood from IDDM subjects when compared to controls. These findings do not
agree with those of our present study. Discrepancies in the total amount of DNA
damage detected by the ELISA and the comet assays have been reported previously
[34] and are the likely cause of the different results between our two studies. The
ELISA measures DNA strand breaks, including damage associated with dead, dying
and apoptotic cells, to which the D1B antibody (raised against single-strand DNA)
has bound. However, the alkaline comet assay depends on observations made on
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individual cells with some degree of intact morphology, therefore dead, dying or
apoptotic cells cannot be easily scored and so DNA damage levels would tend to be
lower with the comet assay compared to the ELISA.
What then are the likely causes and effects of the increased levels of DNA damage
found in the neutrophils of the diabetic subjects? Neutrophils play an important role
in host defence against bacterial infection. The suppression of neutrophil function
could potentially exacerbate infection. Under normal circumstances stimulated
neutrophils kill bacteria via the production of ROS (superoxide anion, hydroxyl
radicals and hydrogen peroxide) during the ‘respiratory burst’ [35]. Disturbances in
neutrophil function including abnormal chemotaxis [36, 37], phagocytosis [38-40],
intracellular killing [39-42], and adherence [43-44] in diabetes have been well
documented. A consequence of such inefficient neutrophil bactericidal mechanisms
may be the increased susceptibility of IDDM subjects to certain bacterial and fungal
infections [45].
In addition, diabetic subjects’ neutrophils are in a chronic state of activation
generating increased levels of ROS [2, 41, 46]. It is well documented that ROS are
capable of causing DNA damage [47-49] and it has been shown that neutrophils in a
chronic low state of activation are capable of generating sufficient ROS to induce
DNA damage in vitro [50]. We report for the first time that such chronic activation
may result in elevated levels of DNA damage within neutrophils in vivo.
Under normal circumstances activated neutrophils are protected from ROS-induced
damage by increasing their intake of vitamin C by as much as ten-fold above the
concentrations present in normal neutrophils. Extracellular and intracellular vitamin
C are necessary for optimal bacterial killing by neutrophils in vitro and in vivo [51].
However, in diabetic subjects plasma and leucocyte levels of vitamin C have been
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shown to be significantly reduced [7, 52-54]. The decreased protection afforded by
vitamin C in the neutrophils of diabetic subjects is likely to contribute to the elevated
levels of DNA damage, which we report here.
There have been reports that neutrophil production of hypochlorous acid and Nchloramines inhibit DNA repair [49, 55]. Such inhibition of repair in vivo is likely to
exacerbate levels of ROS-induced damage to DNA in neutrophils within diabetic
subjects.
Our data suggests that even with acceptable glycaemic control in IDDM subjects
there is a significantly elevated level of DNA damage within diabetic neutrophils in
vivo. The increased levels of basal neutrophil DNA damage in IDDM may contribute
to the altered neutrophil function, reported elsewhere, in patients with IDDM and is
worthy of further investigation.
Acknowledgements
This work was supported by a Department of Education Northern Ireland (DENI)
Postgraduate Student Distinction Award to Mary P.A. Hannon-Fletcher. We would
like to thank all the subjects who participated in the study.
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22
Table 1. Glycated haemoglobin (%HbA1c) levels in blood from IDDM and
control subjects.
%HbA1c
Control
IDDM
(n = 50)
(n = 50)
4.28 ± 0.06
*7.71 ± 0.03
Values are Mean ± SEM in all groups.
*p<0.0001 when compared to control.
23
Figure 1. Basal levels of DNA damage (%tail DNA) in lymphocyte, monocyte,
neutrophil and whole blood fractions from IDDM and control subjects
10.00
*
9.00
Control
Type I diabetic subjects
8.00
7.00
6.00
DNA damage
(%tail DNA)
5.00
4.00
3.00
2.00
1.00
0.00
Lymphocytes
Monocytes
Neutrophils
Whole blood
Values are Mean ± SEM for all groups. *p<0.001 when compared to control.
Analysis of DNA damage within whole blood, lymphocyte and monocyte cell
fractions was carried out on 32 control subjects and 23 IDDM subjects.
Analysis of DNA damage within the neutrophil cell fraction was carried out on 50
control and 50 IDDM subjects.