|Year : 2020 | Volume
| Issue : 2 | Page : 169-174
|A comparative evaluation of the DNA damage in the serum of chronic periodontitis patients with and without diabetes mellitus type II
Amita Rao1, Biju Thomas1, Rajendra B Prasad2, Suchetha B Kumari3, R Vishakh4, Tarona A Subba1
1 Department of Periodontics, A B Shetty Memorial Institute of Dental Sciences, Nitte University, Mangalore, Karnataka, India
2 Department of Oral and Maxillofacial Surgery, A B Shetty Memorial Institute of Dental Sciences, Nitte University, Mangalore, Karnataka, India
3 Department of Biochemistry, K S Hegde Medical Academy, Nitte University, Mangalore, Karnataka, India
4 Department of Biochemistry, Central Research Laboratory, Nitte University, Mangalore, Karnataka, India
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|Date of Submission||12-Sep-2017|
|Date of Acceptance||29-Oct-2018|
|Date of Web Publication||19-May-2020|
| Abstract|| |
Context: Periodontal disease is an immunoinflammatory disease that is initiated by the interaction between microbial plaque and the periodontal tissues. There is very limited data available on the assessment of DNA damage with relation to periodontal diseases. Therefore, a need for a study in this area was felt. Aims: To evaluate the DNA damage in the serum of chronic periodontitis patients and chronic periodontitis with diabetes mellitus (DM) type II patients and to compare it with healthy controls, to assess whether periodontitis can have systemic effects beyond the periodontium. Settings and Design/Subjects and Methods: This cross-sectional study was conducted involving 150 subjects in the age group of 30–60 years, from October 2010 to May 2015. A blood sample of 5 ml of venous blood was collected from each of the study subjects, from the antecubital vein. Fresh blood was used to assess the DNA damage. The DNA damage was estimated using the alkaline single-cell gel (comet) assay. Results: The DNA damage to the cells was calculated by assessing the percentage of “DNA in tail.” The results showed that the values were higher in the periodontitis with diabetes group, as compared to the periodontitis and control group. When the Olive moment was calculated, the values were higher in the periodontitis with diabetes group as compared with the other two groups. Although the values were seen to be higher, it was not statistically significant. Conclusions: The results obtained from this study although statistically insignificant suggest that the DNA damage was higher in chronic periodontitis as compared with healthy control. There was a potentiated difference of the values in patients with DM type II when compared to chronic periodontitis alone.
Keywords: DNA, Diabetes mellitus type II, Chronic periodontitis
|How to cite this article:|
Rao A, Thomas B, Prasad RB, Kumari SB, Vishakh R, Subba TA. A comparative evaluation of the DNA damage in the serum of chronic periodontitis patients with and without diabetes mellitus type II. Indian J Dent Res 2020;31:169-74
|How to cite this URL:|
Rao A, Thomas B, Prasad RB, Kumari SB, Vishakh R, Subba TA. A comparative evaluation of the DNA damage in the serum of chronic periodontitis patients with and without diabetes mellitus type II. Indian J Dent Res [serial online] 2020 [cited 2020 Jul 4];31:169-74. Available from: http://www.ijdr.in/text.asp?2020/31/2/169/284575
| Introduction|| |
Periodontal disease is defined as an immunoinflammatory lesion which occurs due to the interaction of the microbial plaque biofilm with the immune inflammatory response of the host. It leads to alterations in the homeostasis of the bone and connective tissues. Periodontitis was considered as an oral disease and the destruction of the tissues was said to remain localized within the periodontium, however, there is evidence today to consider that periodontitis can produce alterations in systemic health.
Periodontitis has been recognized today as the sixth complication of diabetes mellitus (DM) with a bi-directional relationship between the two chronic inflammatory diseases. There is sufficient evidence available today to suggest that DM type II is associated with an increased prevalence, extent, and severity of chronic periodontitis.
DM is seen to be associated with excessive production of reactive oxygen species (ROS) which can damage cellular macromolecules leading to DNA damage. The DNA of the cell can react with numerous radiolytic reactive products such as ROS that are generated in the body. Ionizing radiation can also damage DNA by directly ionizing itself and by indirect processes in which DNA reacts with numerous radiolytic reactive products including OH−, H+, O2− and H2O2(ROS) that are generated in aqueous fluid surrounding the DNA.
Studies have documented the DNA damage in DM type II patients, and because a hyperactive innate immune response is the antecedent of both DM and periodontal diseases, a possibility exists for the occurrence of DNA damage in periodontal diseases also, however, there are limited studies assessing the DNA damage in chronic periodontitis patients.
The mechanism of DNA damage includes strand breaks, base pair mutations, conversion of guanine to 8-hydroxyguanine, deletions, insertions, nicking, and sequence amplification. The single-cell gel electrophoresis (SCGE) (comet assay) is technically simple, relatively fast, cheap, and DNA damage can be investigated in virtually all mammalian cell types without requirement for cell culture., This technique combines the simplicity of biochemical techniques for detecting DNA single-strand breaks, alkali labile sites, and cross-linking with the single-cell approach typical of the cytogenetic assays.
A study was conducted to assess the percentage of DNA damage in patients with type II diabetes and to correlate it with glycemic control. A total of 28 diabetic patients were included in the study and 25 age- and sex-matched volunteers served as controls. SCGE was used to assess the DNA damage. Results showed that the percent of DNA damage of peripheral blood mononuclear cells was higher in diabetic patients compared with healthy controls.
A study was conducted to assess DNA damage and repair in type II diabetes mellitus. The level of endogenous basal DNA damage and the sensitivity to DNA damaging agents, hydrogen peroxide and doxorubicin and the efficacy of removing of DNA damage induced by these agents in peripheral blood lymphocytes of type II DM patients and healthy individuals were assessed
. Results showed that the levels of oxidative DNA damage in diabetic patients were higher than in control subjects.
A study conducted to detect oxidative DNA damage in type II diabetic patients using the comet assay and to assess the association between DNA damage and hyperglycemia and lipid profile in diabetic patients showed that type II diabetic patients have significantly more oxidative DNA damage than normal controls. Poor glycemic control can increase the severity of the DNA damage. However, dyslipidemia is not a cause for the DNA damage.
Taking into consideration the bi-directional relationship between DM and periodontal disease, patients suffering from DM type II with chronic periodontitis were taken as a separate group in this study, to see whether there is a potentiated effect of the DNA damage in patients suffering from both the diseases.
Thus the present study was conducted to evaluate the DNA damage in chronic periodontitis patients and chronic periodontitis with DM type II patients, and also to compare the DNA damage with the control group to assess the possible systemic effects of chronic periodontitis.
| Subjects and Methods|| |
A cross-sectional study was conducted involving 150 subjects from coastal Karnataka and north Kerala in the age group of 30–60 years reporting to the Department of Periodontics, at A B Shetty Memorial Institute of Dental Sciences from October 2012 to May 2016. Ethical clearance was taken from the Central Ethical Committee, Nitte University, vide reference number NU/CEC/Ph. D-04/2010.
The sample size was calculated based on the assessment of various studies conducted earlier and the expected mean differences. The power of the study was fixed at 80% and error was fixed at 5%.
The body mass index (BMI) of all the 150 subjects were also assessed according to the criteria by Nieto-García et al. and subjects who had a BMI above 25, i.e., overweight and obese individuals, were excluded from the study.
Only subjects who were free of any systemic diseases were included in groups I and III, and subjects who were free of any systemic diseases other than DM type II were included in group II. Subjects included in groups I and II had chronic generalized severe periodontitis, as per the American Academy of Periodontology (AAP)/Centre for Disease Control and Prevention (CDC) classification of periodontal disease given by Eke et al.
All subjects underwent periodontal examination by a single trained examiner. Subjects with a history of intake of antibiotics in the previous 4 weeks, and also a history of intake of vitamin and mineral supplements and any periodontal treatment during the last 6 months were excluded from the study. Subjects with a history of any form of tobacco usage, alcoholism, occupation in chemical industries, and pregnant and lactating women were also excluded from the study.
The random blood sugar (RBS) and glycosylated hemoglobin assay were estimated and subjects with RBS score of <120 and HbA1c score of <7 were included in the study. The periodontal status was also assessed before allotting the subjects to the various groups. A single trained investigator screened and allotted the patients to all the three groups.
A detailed case history was recorded of each subject. Then a periodontal examination was carried out and the status of the periodontal condition was determined. The patient was also referred to a physician to determine the systemic condition.
Following this the patients were allotted to the various groups as per the inclusion criteria. Patients who did not meet the various criteria were excluded from the study. A sample of 5 ml of venous blood was collected from the antecubital vein from each of the study subjects. For assessment of the DNA damage, fresh samples were used. DNA damage was assessed using the fluorescent microscope [Figure 1].
For the assessment of DNA damage due to the extreme values reported, calculation of mean was not appropriate, hence median and interquartile range were calculated. Then Kruskal–Wallis test was applied.
Assessment of DNA damage
The protocol followed for the assessment of DNA damage was the alkaline SCG assay, for the detection of DNA damage in mammalian cells. This technique combines the simplicity of biochemical techniques for detecting DNA single-strand breaks, alkali-labile sites, and cross-linking with the single-cell approach typical of the cytogenetic assays.
Evaluation of DNA damage
- For visualization of DNA damage, observations are made of ethidium bromide-stained DNA using a 40× objective on a fluorescent microscope [Figure 2]
- Comet Score Software [Figure 3] was used to assess the quantitative and qualitative extent of DNA damage in the cells by measuring the length of DNA migration and the percentage of migrated DNA. Finally, the program calculates tail moment. Generally, 50–100 randomly selected cells are analyzed per sample.
|Figure 3: Display image of the pattern of scoring the comet using Comet Score Software|
Click here to view
Few important terminologies
- Tail length:It is the total length of DNA migrated from the body of the nuclear core. It is mainly used to evaluate the extent of the DNA damage.
- Head percentage DNA: It is the percentage of ratio of head optical intensity (HOI) to the sum of the HOI and tail optical intensity (TOI), i.e.,
Head% DNA = (HOI/(HOI + TOI))×100
- Tail percentage DNA: It is the difference in amount of total percentage of DNA present in the comet and head percentage DNA, i.e.,
Tail% DNA = 100 – Head% DNA
- Olive tail moment: Tail moment is defined as the product of the tail length and the fraction of total DNA in the tail. Tail moment incorporates a measure of both the smallest detectable size of migrating DNA (reflected in the comet tail length) and the number of relaxed/broken pieces (represented by the intensity of DNA in the tail.
Olive tail moment = (Tail.mean – Head.mean) × tail% DNA/100
| Results|| |
The DNA damage to the cells in chronic periodontitis patients (group I) and in chronic periodontitis with DM type II patients (group II) were assessed and compared with the control group (group III).
[Graph 1] shows the % of DNA in tail in the three study groups, and [Table 1] shows the multiple comparison of the % of DNA in tail between the groups.
|Table 1: Comparison of percentage DNA in tail between study groups using Kruskal-Wallis test|
Click here to view
Owing to the extreme values reported, the calculation of mean was not appropriate, hence median and interquartile range were calculated. Then the nonparametric test “Kruskal–Wallis test” was applied.
- For group I, the values at the 25th percentile was 1.319, at 50th (median) was 6.360, and at 75th percentile was 8.768
- For group II, the values at the 25th percentile was 1.959, at 50th (median) was 7.079, and at 75th percentile was 41.738
- For group III, the values at the 25th percentile was 0.903, at 50th (median) was 1.853, and at 75th percentile was 8.768.
The results showed that the values were highest in group II (DM with periodontitis) slightly lower in group I (chronic periodontitis) and least in group III (control group). Although there was high variation in the values, when the P value was calculated for the different groups it was seen to be nonsignificant (P = 0.114). The results are in line with the previous studies which show that DNA damage to cells is high in diabetic patients. However, there are no studies reported on the DNA damage assessed using comet assay in chronic periodontitis patients.
The extreme range of values reported and the consequent high deviation seen in the statistical analysis can be explained by the differences among the individual patients in their biochemical parameters, lifestyle, dietary habits, seasonal variation, etc.
[Graph 2] shows the Olive moment in the three study groups and [Table 2] shows the multiple comparison of the Olive tail moment between the groups.
|Table 2: Comparison of Olive moment between study groups using Kruskal-Wallis test|
Click here to view
The Olive tail moment was also calculated for the groups using Kruskal–Wallis test.
- For group I, the values at the 25th percentile was 1.170695, at 50th (median) was 3.141313, and at 75th percentile was 11.466584
- For group II, the values at the 25th percentile was 0.690967, at 50th (median) was 4.010608, and at 75th percentile was 17.534529
- For group III, the values at the 25th percentile was 0.512263, at 50th (median) was 1.604180, and at 75th percentile was 7.045338
The results showed that the values were highest in group II (DM with periodontitis) slightly lower in group I (chronic periodontitis) and least in group III (control group). Although there was high variation in the values, when the P value was calculated for the different groups it was seen to be nonsignificant (P = 0.515).
| Discussion|| |
Chronic inflammatory diseases such as DM type II and chronic periodontitis are associated with increased production of ROS leading to oxidative stress. Oxidative stress can cause protein, lipid, and DNA modifications leading to cellular dysfunctions and even carcinogenic consequences.
There have been several reports in the recent past on the awareness of the effects of DNA damage in chronic inflammatory diseases. However, studies on DNA damage in diabetes have revealed contradictory results. Studies by Choi et al., Pitozzi et al.,, and Blasiak et al. have shown increase DNA damage in DM type II patients. However, studies by Hannon-Fletcher et al. and Ibarra-Costilla et al., showed no association between diabetes and increased DNA damage levels.
In our study the SCGE or comet assay was used to measure DNA damage. This is a sensitive, simple, inexpensive, and rapid method that was developed by Ostling and Johansson in 1984. Although there are several studies that have analyzed the DNA damage in DM type II patients, there is no literature assessing the DNA damage using this technique in chronic periodontitis patients.
In our study, when the percentage of DNA in tail was calculated it was seen to be highest in group II (DM), slightly lower in group I (periodontitis) and least in group III (control). Although there was high variation in the values clinically, it was seen to be nonsignificant. When the Olive moment was calculated for the groups, the results were seen to be similar to the DNA in tail values, with high variation in the clinical values seen but statistically nonsignificant. The high variations in the values reported could be due to the differences in the individual patients, their biochemical parameters, lifestyle, dietary habits, etc.
The increased DNA damage seen in DM type II patients with periodontitis can be attributed to the increased oxidative stress in DM type II patients potentiated by the oxidative stress of chronic periodontitis. It is accepted that chronic inflammatory conditions are associated with increased oxidative stress with neutrophils being implicated in disease pathogenesis, due to the generation of oxidative burst during its phagocytosis.
ROS generation by neutrophils requires a minimum oxygen tension of around 1% and a pH of 7.0–7.5. This is similar to that found in periodontal pockets, thus indicating that ROS production is possible in sites of periodontal pocket formation.
The DNA damage in chronic periodontitis group although statistically not significant is higher than in the control group, which suggests increased oxidative stress in chronic periodontitis.
| Conclusions|| |
The conclusions that can be drawn from the present study are:
- Chronic periodontitis patients had more DNA damage as compared to healthy controls as seen by the values of percentage of DNA in tail and Olive moment but it was not statistically significant
- Chronic periodontitis patients had less DNA damage as compared to chronic periodontitis with DM type II as seen by the values of percentage of DNA in tail and Olive moment but it was not statistically significant
- The present study supports the possibility of the DNA damage serving as a possible marker for chronic periodontitis patients.
The Central Research Laboratory, Nitte University, Mangalore.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Biju Thomas
A B Shetty Memorial Institute of Dental Sciences, Nitte University, Deralakatte, Mangalore - 575 018, Karnataka
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]
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