Introduction

The incidence of atherosclerotic cardiovascular disease is strongly related to the elevation of blood pressure (BP).1 In hypertensive patients, BP is characterized by rhythm alterations over 24 h periods.2 It has been shown that 24 h ambulatory BP monitoring (ABPM) is a better predictor of subsequent complications than spot measurements of BP.3

ABPM has shown that BP is highest during the day and lowest at night in both normotensives and hypertensives.4 Mean BP values are 10–20% lower at night than in the day. This condition is called ‘dipper’ change. However, in some hypertensives, in contrast to this normal change, nighttime BP lowering does not occur or shows a decrease of <10%, which is called ‘non-dipper’ change.5 Endothelial dysfunction and target organ injury are more severe in non-dipper hypertensive (NDH) patients than in dipper hypertensive (DH) patients, and NDH itself is a risk factor for increased mortality.6, 7, 8, 9

Increased oxidative stress is known to be involved in the pathogenesis of hypertension (HT) and cardiovascular disease.10, 11, 12, 13 Oxidative stress occurs when the level of reactive oxygen species (ROS) exceeds the level of the antioxidant defense systems.13 Previous studies have shown that patients with essential HT have a decreased antioxidant capacity and increased amounts of ROS.14, 15 The effects of ROS on vascular function and the development of HT have been investigated previously.10, 11 Superoxide rapidly inactivates endothelium-derived nitric oxide.12, 16, 17 Thus, oxidative stress may account for endothelial dysfunction in patients with HT.18

DNA damage frequently occurs in cells exposed to oxidative stress. Increased oxidative stress may initiate lipid peroxidation in cell membranes, damage membrane proteins or cause DNA fragmentation.19, 20, 21 DNA damage caused by ROS occurs more often in hypertensive patients than normotensive patients.22 The measurement of DNA damage in lymphocytes is a valuable marker of oxidative stress.21, 23 In this study, we sought to investigate lymphocyte DNA damage in patients with DH and NDH.

Methods

Subjects

The overall study population consisted of 64 consecutive patients: 33 subjects with NDH (NDH group) and 31 patients with DH (DH group). Twenty healthy volunteers were also included in the study as a control group. The study patients had newly diagnosed, untreated mild to moderate HT. The inclusion criteria were being 18–55 years of age and, for women, having a regular menstrual cycle. The control group had multiple BP measurements <140/90 mm Hg over the 1-month period and were in the same age and gender range as the hypertensive patients. These subjects were non-medical staff from our hospital or their relatives. For inclusion in the control group, subjects had to have no known coronary risk factors or cardiac symptoms and normal electrocardiographic and echocardiographic examinations.

Exclusion criteria included the presence of neoplastic disease, recent major surgical procedures, dyslipidemia, concomitant inflammatory diseases such as infections and autoimmune disorders, liver and kidney disease, extreme-dipper type HT, use of regular medication for any reason, alcohol use and smoking habit. Patients suffering from organic coronary artery disease, vasospastic angina, heart failure, idiopathic hypertrophic and dilated cardiomyopathy, secondary and malignant HT, serum creatinine>1.5 mg dl−1 or with a history of diabetes were also excluded from participation in the study. Subjects had not previously taken antihypertensive therapy. Subjects using any vasoactive drug and having ST segment or T-wave changes specific for myocardial ischemia, Q waves or incidental left bundle branch block on ECG were excluded from the study.

The study was conducted according to the recommendations set forth by the Declaration of Helsinki on Biomedical Research Involving Human Subjects. The Institutional Ethics Committee approved the study protocol, and each participant provided written, informed consent.

BP measurement and ABPM

BP was measured using a mercury sphygmomanometer in an office setting. Systolic BP (SBP) and diastolic BP (DBP) were taken. Noninvasive 24 h ABPM was performed with a portable, compact digital recorder (Tracker NIBP2, Delmar Reynolds Ltd., Hertford, UK) and analyzed using customized analytical software (Delmar Reynolds Medical Inc., Model 2169, Hertford, UK). All subjects wore an ABPM device for a single 24 h period. The device was programmed to inflate and record BP at pre-specified intervals (every 15 min during daytime hours and every 30 min during nighttime hours), which provided ∼80 BP recordings during the 24 h period. The display of the ABPM was inactivated so that viewing each BP reading did not distract the subjects. For the analysis of the data reports, reports generated from a session of ABPM contained BP recordings for the entire 24 h, heart rate, mean arterial pressure and BP load, as well as summary statistics for the overall 24 h, daytime and nighttime periods. When the readings exceeded at least 80% of the total readings programmed for the testing period, the recording was considered valid and satisfactory.

Diagnosis of HT

In each subject, BP was measured on at least three separate days after 15 min of sitting comfortably and was then averaged. Each subject then underwent 24 h ABPM. Individuals who had SBP ⩾140 mm Hg and/or a DBP ⩾90 mm Hg in the office setting, and in ABPM, an average 24 h SBP>130 mm Hg and/or DBP >80 mm Hg, an average daytime SBP >135 mm Hg and/or DBP >85 mm Hg or an average nighttime SBP >125 mm Hg and/or DBP >75 mm Hg were diagnosed as hypertensive.24 In addition, the subjects who had a <10% reduction in BP from the daytime to the nighttime period were defined as NDH, and the subjects who had a BP reduction ⩾10% from the daytime to the nighttime period were considered DH.5

Blood sampling protocol

After the HT diagnosis based on ABPM, peripheral venous blood samples were taken into heparinized tubes from all subjects in a fasting state between 0700 and 0800 hours. One milliliter of blood was immediately pipetted into another tube to measure DNA damage. The remaining blood was centrifuged at 3000 r.p.m. for 10 min for plasma separation. Plasma samples were stored at −80 °C until the analysis of total antioxidant status (TAS), hsCRP, triglyceride, total cholesterol, low-density lipoprotein, high-density lipoprotein and fasting glucose. hsCRP levels were measured with an otoanalyser (Aeroset, Abbott, Holliston, MN, USA) using a commercial spectrophotometric kit (Scil Diagnostics GmbH, Sunnyvale, CA, USA).

DNA Damage determination by alkaline comet assay

Lymphocyte isolation for the comet assay was performed using Histopaque 1077 (Sigma Chemical Co. Ltd., Seoul, Korea). One milliliter of heparinized blood was carefully layered over 1 ml histopaque and centrifuged for 35 min at 500 g at 25 °C. The interface bands containing lymphocytes were washed with phosphate-buffered saline and then collected by 15 min centrifugation at 400 g. The resulting pellets were re-suspended in phosphate-buffered saline to obtain 20 000 cells in 10 μl. Membrane integrity was assessed by means of the trypan blue exclusion method.

The endogenous lymphocytes’ DNA damage was analyzed by alkaline comet assay according to the procedures outlined by Singh et al.,25 with minor modifications. Ten microliters of fresh lymphocyte cell suspension (around 20 000 cells) was mixed with 80 μl of 0.7% low-melting-point agarose (Sigma-Aldrich, St Louis, MO, USA) in phosphate-buffered saline at 37 °C. Subsequently, 80 μl of this mixture was layered onto slides that had previously been coated with 1.0% hot (60 °C) normal-melting-point agarose and covered with a coverslip at 4 °C for at least 5 min to allow the agarose to solidify. After removing the coverslips, the slides were submerged in freshly prepared, cold (4 °C) lysing solution (2.5 M NaCl, 100 mM EDTA-2Na; 10 mM Tris–HCl, pH 10–10.5; 1% Triton X-100 and 10% DMSO added just before use) for at least 1 h. Slides were then immersed in freshly prepared, alkaline electrophoresis buffer (0.3 mol l−1 NaOH and 1 mmol l−1 Na2 EDTA, pH>13) at 4 °C for unwinding (40 min) and then electrophoresed (25 V/300 mA, 25 min). All of the above steps were conducted under red light or without direct light to prevent additional DNA damage. After electrophoresis, the slides were stained with ethidium bromide (2 μg ml−1 in distilled H2O; 70 μl per slide), covered with a coverslip and analyzed using a fluorescence microscope (Nikon, Tokyo, Japan) equipped with a rhodamine filter, using the epi-flurescence method. The images of 100 randomly chosen nuclei (50 cells from each of two replicate slides) were visually analyzed. Each image was classified according to the intensity of the fluorescence in the comet tail and was given a value of 0, 1, 2, 3 or 4 (from undamaged class 0 to maximally damaged class 4) (Figure 1) so that the total scores of the slide were between 0 and 400 arbitrary units. All procedures were completed by the same biochemistry staff, and DNA damage was detected by a single observer who was not aware of the subjects’ diagnoses.

Figure 1
figure 1

Photomicrographs showing varying intensities of fluorescence in the comet tail (class 0, undamaged; class 4 and maximally damaged). A full color version of this figure is available at the Hypertension Research journal online.

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Measurement of TAS

TAS of serum was determined using an automated measurement method.26 In this method, hydroxyl radical is produced. In the assay, ferrous ion solution, which is present in reagent 1, is mixed with hydrogen peroxide, which is present in reagent 2. The sequentially produced radicals, such as the brown-colored dianisidinyl radical cation produced by the hydroxyl radical, are also potent radicals. In this assay, the antioxidative effect of the sample against the potent free radical reactions is measured. The assay has excellent precision values, which are lower than 3%. The results are expressed as mmol Trolox equivalents per l.

Statistical analysis

Results are presented as mean±s.d. or frequency expressed as a percent. Categorical variables were compared using the χ2-test. For continuous variables, differences between two groups were assessed using the unpaired t-test. Comparisons among multiple groups were performed by one-way analysis of variance test for continuous variables. Associations between other variables and lymphocyte DNA damage properties were assessed by the Pearson’s correlation coefficient. A P-value <0.05 was considered statistically significant.

Results

Demographic and clinical characteristics

The demographic and clinical characteristics of the groups are shown in Table 1. The SBP and DBP in the office setting were similar in DH and NDH subjects. The SBP and DBP of both the DH and NDH groups in the office setting were higher than those of the control group.

Table 1 The comparison of baseline characteristic of the patient and control groups
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ABPM analysis

The measurements from ABPM are shown in Table 2. By definition, the average daytime SBP and DBP did not differ between non-dippers and dippers. In contrast, the average nighttime SBP and DBP were significantly higher in the non-dipper HT group than in the dipper group or the control group.

Table 2 Data from ABPM of the dipping hypertension, non-dipping hypertension and control groups
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Analysis of antioxidant status and DNA damage

The mean DNA damage values and hsCRP levels were higher in the NDH group than in the DH group or the control group. Additionally, the mean DNA damage value of the NDH group was higher than that of the DH group (P<0.001). The mean TAS levels of both the DH and NDH groups were lower than that of the control group (P<0.001, for both). The mean TAS level of the NDH group was also lower than that of the DH group (P<0.001). The analyses of antioxidant status and DNA damage are shown in Table 3.

Table 3 The comparison of antioxidant and DNA damage characteristic of the patient and control groups
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Correlation analysis

In the patient groups, lymphocyte DNA damage was negatively correlated with TAS level (r=−0.692, P<0.001), whereas lymphocyte DNA damage was positively correlated with age (r=0.479, P<0.001), hsCRP level (r=0.315, P=0.012), total cholesterol level (r=0.382, P=0.002), low-density lipoprotein cholesterol level (r=0.331, P=0.008), average night-time SBP (r=0.260, P=0.038) and average nighttime DBP (r=0.258, P=0.039) in the bivariate analysis Table 4.

Table 4 Bivariate relationships of the DNA damage values in patient group
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Discussion

To the best of our knowledge, the present study is the first in to evaluate lymphocyte DNA damage and TAS in patients with DH and NDH. The main findings of this study are the following:1 the mean lymphocyte DNA damage and hsCRP levels of all patient groups were higher than those of the control group;2 the mean lymphocyte DNA damage and hsCRP levels were higher in the NDH group than the DH group;3 the mean TAS levels of both the NDH and DH groups were lower than that of the control group; and the mean TAS levels of the NDH group were lower than those of the DH group;4 lymphocyte DNA damage was negatively correlated with TAS and positively correlated with hsCRP.

Previous studies have shown that left ventricular hypertrophy, the risk of cardiovascular mortality,6, 7 silent cerebrovascular disease8 and the progression of nephropathy9 are greater in subjects with non-dipper BP than in patients with dipper BP.6, 7, 8, 9 At the same time, endothelium-dependent vasodilation induced by acetylcholine is impaired and the production of nitric oxide is reduced to a greater extent in non-dippers than in dippers.9

In the present study, lymphocyte DNA damage increased in hypertensive patients. This result is consistent with that of a previous study by Subash et al.22 They reported that increased DNA damage was related to a decreased TAS level. However, in that study, lymphocyte DNA damage was not investigated separately in patients with NDH and DH.22 In our study, the extent of lymphocyte DNA damage was greater in the NDH group than in the DH group.

DNA damage frequently occurs in cells exposed to oxidative stress. Increased oxidative status may initiate lipid peroxidation in cell membranes, damage membrane proteins, or cause DNA fragmentation.27 Antioxidant systems prevent the damage of DNA.9 Honda et al.28 have also reported that reduced activity of antioxidant enzymes is associated with increased levels of oxidative DNA damage. In the present study, lymphocyte DNA damage was significantly negatively correlated with TAS level. Thus, increased lymphocyte DNA damage in patients with NDH can be explained by decreased TAS levels, which are a marker of increased oxidative stress.

Lymphocyte DNA damage is one of the more reliable markers of oxidative stress. The most widely used techniques to investigate DNA damage are the comet assay and measurement of 7-hydroxy-8-oxo-2V-deoxyguanosine (8-oxodG). The comet assay detects single-stranded breaks in DNA.29, 30 8-oxodG can be measured by using several chromatographic techniques or non-chromatographic enzymic methods. There is a large variation in the measured levels of 8-oxodG between these assays, partly because 8-oxodG is artificially generated in these assays;31 however, either approache can be used.30 In this study, to detect DNA damage in lymphocytes, we used the comet assay, which is a cheap, simple and very sensitive method.32

Maeda et al.33 demonstrated that oxidative stress generated by peripheral blood mononuclear cells was increased in hypertensives with an extreme-dipper pattern and/or a morning surge in BP. Recently, Ermis et al.34 investigated serum GGT levels, a marker of oxidative stress, and inflammatory activity in patients with DH and NDH and found that both serum GGT and CRP levels were increased in patients with NDH. They also reported that increased GGT activity was correlated with CRP levels. Yildiz et al.35 also showed increased signs of oxidative stress in patients with NDH compared with DH. Decreased antioxidant levels have been interpreted as evidence of increased oxidative stress. Additionally, the plasma TAS level is an accurate index of oxidative stress, which provides a measure of the total plasma defenses against ROS.22, 36 In the present study, we showed increased hsCRP levels and reduced TAS levels, a marker of increased oxidative stress, in the NDH group. It has been shown that increased pressure on vascular walls can induce ROS release and increase the level of oxidative stress.37, 38, 39 In the present study, the nocturnal SBP was higher in non-dippers than in dippers. Higher nocturnal BP may represent the primary mechanism of increased oxidative stress in patients with NDH. Thus, increased lymphocyte DNA damage in patients with NDH can be explained by increased oxidative stress.

Study limitations

The comet assay is ideally suited for human investigations and can be easily applied. Normally, the lymphocytes that are used do not require tissue disaggregation, as they are obtained in a relatively noninvasive way, and they behave well in the comet assay. However, lymphocytes may not adequately reflect the damage to other organs in human subjects.32, 40 Sometimes, human tissue removed at surgery can be investigated for elevated levels of damage; however, the necessary control tissue from healthy individuals is more difficult to obtain. In this study, we chose to use freshly isolated lymphocytes.

The lymphocyte DNA damage score in healthy control groups varies between 7.6 and 19.9 arbitrary units in the literature.41, 42, 43 This indicates that DNA damage evaluated by comet assay may vary depending on physicians and/or experimental conditions at measurement, such as pH, duration of alkaline treatment, length of electrophoresis or types of cells used in single-cell gel electrophoresis.44 The optimization of experimental parameters for measuring lymphocyte DNA damage in the comet assay is necessary. In addition, there is no universally accepted cutoff value for the lymphocyte DNA damage score. To limit the variance, the lymphocyte DNA damage score can be confirmed by measuring 8-oxodG in patients with DH and NDH. However, the comet assay is a well-established, simple and sensitive method for measuring DNA damage and is commonly used in human trials.44 In this study, lymphocyte DNA damage was measured by alkaline comet assay, following the method of Singh et al.25

In conclusion, lymphocyte DNA damage increased in patients with NDH compared with patients with DH and the control group. The increased lymphocyte DNA damage, reduced TAS levels and increased hsCRP levels in patients with NDH might reflect increased oxidative stress and inflammatory activity, which have a crucial role in the development of cardiovascular disease. Lymphocyte DNA damage may be a parameter to consider in the treatment of HT.