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ORIGINAL ARTICLE
Year : 2012  |  Volume : 1  |  Issue : 2  |  Page : 79-84

An investigation of the relationshıp between coronary atherosclerosis and chromosome damage in human peripheral blood lymphocytes


1 Department of Biology, Faculty of Science, Istanbul University, Istanbul, Turkey
2 Department of Cardiology, Cerrahpasa Faculty of Medicine, Istanbul University, Istanbul, Turkey

Date of Web Publication13-Sep-2012

Correspondence Address:
Tuge Erkol
Department of Biology, Faculty of Science, Istanbul University, 34134, Vezneciler, Istanbul
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2278-0521.100954

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  Abstract 

Background: Coronary atherosclerosis is a disease of the coronary arteries and is a result of cumulative processes that are mostly seen in old-age population. Progressive accumulation of oxidative damage to cellular macromolecules, especially DNA, over a lifetime is one of the important causes of aging. This accumulation of damage may result in chromosome instability and this can be measured by micronucleus (MN) technique. Materials and Methods: In this study, MN technique was applied to observe the relationship between chromosome instability and proliferative effectiveness of lymphocytes and coronary atherosclerosis. Non-toxic levels of hydrogen peroxide were also administered to observe any existing differential effect due to this stress inducing oxidizing agent. Spontaneous and H 2 O 2 stress induced MN frequencies and proliferation state of peripheral lymphocytes were measured from both 30 patients diagnosed as coronary atherosclerosis and 30 healthy control individuals of the same age range. Results: There was no significant difference between spontaneous MN frequencies of patient and control groups (P = 0.08). Also, probability index (PI) values between these groups reflected no difference (P = 0.13). Hydrogen peroxide addition did not change spontaneous MN frequencies either in control (P = 0.23) or in patient group (P = 0.81). This non-toxic H 2 O 2 level failed to show any different effect on patients compared to controls (P = 0.41). Although increasing PI values were observed as a result of H 2 O 2 administration only in patient group, the comparisons between the two groups showed no significant difference (P = 0.34). Conclusion: Chromosome instability and proliferation index that were measured by MN technique in this study have no role in discriminating coronary atherosclerosis patients from healthy individuals.

Keywords: Aging, chromosome instability, coronary atherosclerosis, lymphocytes, micronuclei


How to cite this article:
Erkol T, Orta T, llerigelen B. An investigation of the relationshıp between coronary atherosclerosis and chromosome damage in human peripheral blood lymphocytes. Saudi J Health Sci 2012;1:79-84

How to cite this URL:
Erkol T, Orta T, llerigelen B. An investigation of the relationshıp between coronary atherosclerosis and chromosome damage in human peripheral blood lymphocytes. Saudi J Health Sci [serial online] 2012 [cited 2021 Jan 24];1:79-84. Available from: https://www.saudijhealthsci.org/text.asp?2012/1/2/79/100954


  Introduction Top


The heart is a strong and an efficient muscular living organism. It is responsible for blood circulation in the body. Cardiac (heart) muscle that surrounds the heart is called myocardium. Coronary arteries are the vessels which supply oxygen-rich blood to myocardium. Coronary artery disease (CAD) is a disease of plaque accumulation inside the coronary arteries and CAD is also called coronary heart disease. Plaque is primarily made up of fat, cholesterol, calcium, and other substances that are found in the blood. [1] The main process responsible for CAD is coronary atherosclerosis. It is a condition in which plaque builds up in the walls of coronary arteries. [2] When coronary arteries are narrowed or blocked, blood flow cannot reach the cardiac muscle and the cardiac muscles die (heart attack). Heart attack, also known as myocardial infarction, (MI) can lead to serious health problems and even death. [1] Atherosclerosis is a cumulative process that is mostly seen in old-age population. Lifestyle changes, smoking, high blood pressure, cholesterol, fat feeding, and diabetes are the risk factors leading to coronary atherosclerosis in most people. Age, gender, and genetic factors are the other important markers and play a major role in atherosclerosis. Coronary atherosclerosis is the leading cause of death in the world for both men and women. [2] Patients have higher risk for atherosclerotic plaque development if they are older (>40-45 years for men and 50-55 years for women). Many studies have shown that aging is related to progressive accumulation of oxidative damage to cellular macromolecules, especially genetic metarial. [3],[4],[5],[6],[7] DNA damage is induced by multiple factors including oxidative stress. Accumulation of damage over a lifetime and the decrease in the cell repairment mechanism causes chromosome instability. [8],[9]

One of the methods measuring chromosome aberrations in genome caused by the result of oxidative damage is the micronucleus (MN) technique. [10],[11] Scoring micronuclei in binucleate cells (BN) has become one of the most commonly used methods for the evaluation of spontaneous and mutagen-induced DNA damage in human peripheral blood lymphocytes. [12],[13],[14] Many studies have found a relationship between MN frequencies and increasing ages. [3],[15] Micronuclei are small structures in dividing cells which can be a whole chromosome or chromosome fragments in cytoplasm and are not included in the sister nuclei. In this study, the relationship between coronary atherosclerosis and chromosome aberrations that were measured with the formation of MN in binucleated human peripheral blood lymphocytes was investigated. Proliferative effectiveness of lymphocytes was measured by the MN technique. Hydrogen peroxide (H 2 O 2 ) was also given to lymphocytes in order to observe any differential effect of the oxidative stress on coronary atherosclerosis patients.


  Materials and Methods Top


Cell culture

Lymphocytes were taken from 30 patients diagnosed as coronary atherosiosclerosis and 30 matched healthy individuals, after obtaining approval from the Istanbul University Cerrahpa°a Medical Faculty Ethics Committee. This study was conducted in accordance with the Declaration of Helsinki. Volunteers signed informed consent documents. Donor ages ranged between 36 and 70 years for the control group (18 males, 12 females) and between 42 and 78 years for the patient group (10 males, 20 females) in this study. The average age of the patient grup was 62.866 ± 8.443 years and it was 50.666 ± 10.066 years for the control group. The patient group was selected with diagnostic coronary angiography which showed a diameter narrowing of at least 50% in at least one of the three major coronary arteries or with an evidence of MI. The control group was selected from the donors showing no risk factors such as hypertension, hyperlipidemia, diabetes mellitus, obesity, and smoking habits, and were free of any history of CAD. Also, the control group showed no symptoms or electro-cardiographic findings characteristic of CAD.

Peripheral blood was collected by using heparin as an anticoagulant. Microculture method was applied to blood lymphocytes for the MN test. 0.5 ml blood sample was added to culture containing 15 μg/ml phytohemaglutinin, 1 ml newborn calf serum, and 4 ml of RPMI-1640 with glutamine (Sigma, USA) supplemented with 100 μg/ml streptomycin and 100 IU/ ml penicillin, and incubated at 37°C for 44 h. At the 44 th h of incubation, 25 units (0.25 ml) of cytochalasin-B (Sigma, USA) was added and cells were incubated for 28 h. At the end of culture period (72 h), cells were centrifuged for 10 min at 200 g, then 0.075 M concentration of hypotonic KCl solution was added to the pellet. Cells were centrifuged for 10 min at 200 g. Then, cells were washed three times with methanol:acetic acid (7:1) solution. About 0.25 ml of cell suspension was left at the end of the third wash and transferred by a Pasteur pipette on a glass microscope slide. After the slides were dried, cells were stained by 5% Giemsa stain (prepared in a phosphate buffer) for 10 min.

Scoring

For each sample, 1000 binucleated cells were scored under the light microscope for MN analysis following the criteria for MN acceptance listed by Fenech. [13] Binucleated cells were evaluated at 400× magnification and the confirmation of micronuclei was made at 1000× magnification by using immersion objectives. Binucleated cells in each slide, which either contained micronuclei or did not, were counted for MN frequencies, and the cells including 1, 2, 3, or 4 nuclei were calculated for proliferative indexes (PIs) for each individual.

Statistical analysis

H 2 O 2 -induced and spontaneous levels of MN frequencies were established for each individual.

Statistical comparison was made by paired t-test between H 2 O 2 -treated and untreated spontaneous levels of chromosome aberrations for each of patient and control groups. The inter-relationship between groups was tested by unpaired t-test. Data were analyzed by using GraphPad InStat statistical program for Windows software.


  Results Top


Patient group

MN frequencies and PIs without and with H 2 O 2 -treated cells from 30 patients with coronary atherosclerosis are shown in [Table 1]. Their ages ranged between 42 and 78 years. MN frequencies were calculated in at least 1000 BN.
Table 1: Spontaneous and hydrogen peroxide (H2O2)-induced MN/BN and PI in patients with coronary atherosclerosis

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Spontaneous MN frequencies were spread from 0.007 to 0.062, with a mean value of 0.028 ± 0.012 (mean ± standart deviation). This MN frequency distribution between the patients was not changed by the treatment of H 2 O 2 . Treated values of MN frequencies ranged from 0.011 to 0.050, with a mean value of 0.027 ± 0.012 (mean ± standart deviation). When spontaneous MN frequencies and MN frequencies after H 2 O 2 therapy of patient group were compared with each other by the paired t-test, there was no significant difference (P = 0.81).

PIs of patients were also calculated by the method of Eastmond and Tucker: [16]

PI = (M1 + 2(M2) + 3(M3) + 4(M4))/N,

where M1, M2, M3, and M4 represent cells with 1, 2, 3, and 4 nuclei, and N represents the total number of cells.

Spontaneous PIs belonging to the patient group ranged between 1.219 and 2.342, with a mean value of 1.513 ± 0.273 (mean ± standart deviation) [Table 1]. PIs after H 2 O 2 application ranged between 1.250 and 2.156, with a mean value of 1.614 ± 0.256 (mean ± standart deviation). When these H 2 O 2 -treated values in the patient group were compared with untreated values of PIs, the difference between them was found to be very significant (P < 0.01).
Table 2: Spontaneous and hydrogen peroxide (H2O2)-induced MN/BN and PI of healthy donors

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Control group

Experimental results of 30 healthy donors are given in [Table 2]. Their ages ranged between 36 and 70 years, which was similar as that of patients. MN frequencies were also calculated in at least 1000 BN.

Spontaneous MN frequencies were spread from 0.008 to 0.046, with a mean value of 0.023 ± 0.011 (mean ± standart deviation). MN frequencies in the healthy group after application of H 2 O 2 ranged between 0.009 and 0.049, with a mean value of 0.025 ± 0.011 (mean ± standart deviation). When spontenous and H 2 O 2 -treated MN frequencies were compared by the paired t-test, the difference was not significant (P = 0.23).

PIs of the control group showed a distribution in the range of 1.207-2.388, with a mean value of 1.627 ± 0.302 (mean ± standart deviation), whereas PIs of H 2 O 2 -treated cells were distributed between 1.360 and 2.359, with a mean value of 1.675 ± 0.235 (mean ± standart deviation). When these results were compared with the paired t-test, there was no difference (P = 0.40).

Comparisons between the two groups

Comparisons of the results of the patient and the control groups were made by the unpaired t-test.

The difference between the spontaneous MN frequencies of patient and control groups was not significant (P = 0.08). The same also holds true when the comparison was made between H 2 O 2 -treated cells of the two groups (P = 0.41) [Table 3].
Table 3: Comparison between patient and the control groups

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When the spontaneous PI values of healthy and patient groups were compared with each other, the difference between the two groups was not significant (P = 0.13). PI values after H 2 O 2 treatment between the two groups did not also show any significant difference (P = 0.34) [Table 4].
Table 4: PI values of healthy and patient groups

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On general evaluation, oxidative stress by H 2 O 2 over cells showed no increase and no decrease in MN formation and there were no differences found while comparing the two groups. But when the application of H 2 O 2 in both the groups was compared in terms of PI results, it might be said that there was an increase in cell proliferation in the patient group.


  Discussion Top


Atherosclerosis is the main pathophysiological process responsible for cardiovascular disease which is the main cause of death in developed countries. [2],[17] The accumulation of somatic mutations during aging may be an important risk factor for initiation and progression of atherosclerotic disease. [18],[19] An age-related increase in these mutations, followed by an age-associated decline in DNA repair capacity has been reported. [5],[8],[15],[20] Oxidative damage could be considered one of the several factors contributing to somatic mutations.

Some studies reported that oxidative stress may be the major causative mechanism for DNA damage, and DNA damage was increased in patients with atherosclerosis. [19],[21],[22],[23],[24],[25] The levels of DNA damage have been shown to correlate with the atherogenic risk factors. Another study has shown an increasing evidence that human atherosclerosis is associated with damage to the DNA of both circulating cells and cells of the vessel wall. [14],[25],[26],[27] They suggest reactive oxygen species are the most likely agents inducing DNA damage in atherosclerosis. [28]

Ramsey et al. [4] have suggested that lifestyle factors contribute to the accumulation of cytogenetic damage and chromosome aberrations which show a greater accumulation with age. [29],[30] In this study, the relationship between cytogenetic damage in human peripheral blood lymphocytes and atherosclerosis in aged people was investigated. MN frequencies as a determinant of cytogenetic damage and proliferative capacities of cells were measured before and after treating cells with non-toxic levels of H 2 O 2 in the patients' and healthy individual donors' lymphocytes. Comparisons of these measures were made within each group and between the groups.

No difference was observed in the spontaneous cytogenetic damage measured by MN frequencies of patient and control groups (P = 0.08). Measures of proliferative indexes (PI) were also not different between the two groups (P = 0.13). Treatment of lymphocyte cultures with hydrogen peroxide did not change spontaneous MN frequencies either in control (P = 0.23) or in patient groups (P = 0.81), suggesting the applied level of treatment was not toxic. This non-toxic level of H 2 O 2 again failed to show any different effect on patients compared to controls (P = 0.41). Increase in PI values was observed as a result of H 2 O 2 application in the patient group, but the comparisons made between the two groups showed no significant difference (P = 0.34).

In conclusion, chromosome instability and proliferation that were measured by the MN technique in this study have no role in discriminating coronary atherosclerosis patients from healthy individuals.


  Acknowledgments Top


This work is supported by Istanbul University, Research Found (Project No: T-1566).

 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]


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[Pubmed] | [DOI]



 

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