Title: Levels of peripheral blood cell DNA damage in Insulin Dependent Diabetes Mellitus (IDDM) human subjects.
Running title: DNA damage in diabetic human subjects.
122Mary P. A. Hannon-Fletcher, Maurice J. O‟Kane, Ken W. Moles, Colin
111Weatherup, Christopher R. Barnett and Yvonne A. Barnett*
1Cancer and Ageing Research Group, University of Ulster, Cromore Road, Coleraine, County Londonderry, Northern Ireland, BT52 1SA.
2Altnagelvin Area Hospital, Glenshane Road, Londonderry, BT47 1SB.
-324627 *Corresponding author email@example.com Tel: +44-(0)-2870
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, Mann-Whitney 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, %Hba 1c.
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) . 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 [8-14]. Polyunsaturated fatty acids are among the most readily oxidised substrates in biological systems. A broad range of oxidation products have been described , 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 , are known to interact with DNA . Such interaction can lead to cytotoxicity, genotoxicity and carcinogenicity .
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
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  measured DNA damage (single-strand breaks and alkali-labile sites) using a sandwich ELISA described by van Loon et al.  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
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
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 %HbA), using a method 1c
described by John et al. . 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
O++++g for 5 min at 4C) 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
Ostored at 4C in the dark (to minimise additional DNA damage and repair), for use the same day in the comet assay.
2.3.2. Mononuclear cell isolation
Mononuclear cells were isolated from whole blood using a method described by Böyum . 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
for 30 min, the mononuclear layer temperature. Following centrifugation at 700 x g
(“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
oSupplies, Poole, UK) at 37C (5% CO: air humidified atmosphere). Following 2
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
ocell pellets were re-suspended in 5 ml PBS and kept at 4C 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. . Essentially, the Histopaque layer was carefully
aspirated and discarded, 6 ml of PBS was added to the red cell-neutrophil mixture,
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
for 10 min. The supernatant was discarded and 6 ml of ice cold sterile distilled water g
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
oin 5 ml PBS and kept at 4C 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, alkali-labile 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.
The alkaline comet assay procedure in this study was a modification of the method described by Singh et al. . 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%
oDMSO) made up just before use and incubated overnight at 4C. 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
oconducted at 4C 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 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 %HbA was analysed using least squares linear regression 1c
analysis. A p value of <0.05 was considered statistically significant. All results are expressed as mean ? standard error of the mean (SEM).
%HbA levels (Table 1) were significantly increased in IDDM subjects (7.71?0.03; 1c
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 (%HbA and %tail 1c
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 %HbA 1c
or %tail DNA damage measured in the IDDM subjects with complications compared to those who did not have complications.