Indian Journal of Dental ResearchIndian Journal of Dental ResearchIndian Journal of Dental Research
HOME | ABOUT US | EDITORIAL BOARD | AHEAD OF PRINT | CURRENT ISSUE | ARCHIVES | INSTRUCTIONS | SUBSCRIBE | ADVERTISE | CONTACT
Indian Journal of Dental Research   Login   |  Users online: 6703

Home Bookmark this page Print this page Email this page Small font sizeDefault font size Increase font size         

 


 
Table of Contents   
ORIGINAL RESEARCH  
Year : 2020  |  Volume : 31  |  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


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

Click here for correspondence address and email

Date of Submission12-Sep-2017
Date of Acceptance29-Oct-2018
Date of Web Publication19-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 Aug 6];31:169-74. Available from: http://www.ijdr.in/text.asp?2020/31/2/169/284575



   Introduction Top


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.[1] 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.[2]

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,[3] 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.[4],[5] 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.[6]

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.[3]

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.[7]

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 Top


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.[8] 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.[9]

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].
Figure 1: Fluorescent microscope

Click here to view


Statistical analysis

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

  1. For visualization of DNA damage, observations are made of ethidium bromide-stained DNA using a 40× objective on a fluorescent microscope [Figure 2]
  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 2: Observation of comets under fluorescent microscope

Click here to view
Figure 3: Display image of the pattern of scoring the comet using Comet Score Software

Click here to view


Few important terminologies

  1. 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.
  2. 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.,


  3. Head% DNA = (HOI/(HOI + TOI))×100

  4. Tail percentage DNA: It is the difference in amount of total percentage of DNA present in the comet and head percentage DNA, i.e.,


  5. Tail% DNA = 100 – Head% DNA

  6. 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 Top


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 Top


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.[10]

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.,[11],[12] and Blasiak et al.[3] have shown increase DNA damage in DM type II patients. However, studies by Hannon-Fletcher et al. and Ibarra-Costilla et al.[13],[14] 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.[15] 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 Top


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.


Acknowledgement

The Central Research Laboratory, Nitte University, Mangalore.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Kornman KS. Mapping the pathogenesis of periodontitis: A new look. J Periodontol 2008;79:1560-8.  Back to cited text no. 1
    
2.
Mealey BL, Oates TW, American Academy of Periodontology. Diabetes mellitus and periodontal diseases. J Periodontol 2006;77:1289-303.  Back to cited text no. 2
    
3.
Blasiak J, Arabski M, Krupa R, Wozniak K, Zadrozny M, Kasznicki J, et al. DNA damage and repair in type 2 diabetes mellitus. Mutat Res 2004;554:297-304.  Back to cited text no. 3
    
4.
Collins AR. The comet assay for DNA damage and repair: Principles, applications, and limitations. Mol Biotechnol 2004;26:249-61.  Back to cited text no. 4
    
5.
Møller P. Assessment of reference values for DNA damage detected by the comet assay in human blood cell DNA. Mutat Res 2006;612:84-104.  Back to cited text no. 5
    
6.
Salem SI, EI-Toukhy SE, EI-Saeed GS, EI-Waseef M. Correlation of DNA damage in type II diabetes to glycemic control. Egypt J Hosp Med 2012;48:472-82.  Back to cited text no. 6
    
7.
EEl Wassef M, El Saeed G S M, El Toukhy S E, Raslan H M, Tawfeek S. Oxidative DNA damage in patients with type II diabetes mellitus. Diabetol Croatia 2012;41:121-7.  Back to cited text no. 7
    
8.
Nieto-García FJ, Bush TL, Keyl PM. Body mass definitions of obesity: Sensitivity and specificity using self-reported weight and height. Epidemiology 1990;1:146-52.  Back to cited text no. 8
    
9.
Eke PI, Thornton-Evans G, Dye B, Genco R. Advances in surveillance of periodontitis: The centers for disease control and prevention periodontal disease surveillance project. J Periodontol 2012;83:1337-42.  Back to cited text no. 9
    
10.
Jee SH, Ohrr H, Sull JW, Yun JE, Ji M, Samet JM, et al. Fasting serum glucose level and cancer risk in Korean men and women. JAMA 2005;293:194-202.  Back to cited text no. 10
    
11.
Choi SW, Benzie IF, Lam CS, Chat SW, Lam J, Yiu CH, et al. Inter-relationships between DNA damage, ascorbic acid and glycaemic control in type 2 diabetes mellitus. Diabet Med 2005;22:1347-53.  Back to cited text no. 11
    
12.
Pitozzi V, Giovanni L, Bardini G, Rotella CM, Dolara P. Oxidative DNA damage in peripheral blood cells in type II diabetes mellitus: Higher vulnerability of polymorphonuclear leukocytes. Mutat Res 2004;554:297-304.  Back to cited text no. 12
    
13.
Hannon-Fletcher MP, O'Kane MJ, Moles KW, Weatherup C, Barnett CR, Barnett YA, et al. Levels of peripheral blood cell DNA damage in insulin dependent diabetes mellitus human subjects. Mutat Res 2000;460:53-60.  Back to cited text no. 13
    
14.
Ibarra-Costilla E, Cerda-Flores RM, Dávila-Rodríguez MI, Samayo-Reyes A, Calzado-Flores C, Cortés-Gutiérrez EI, et al. DNA damage evaluated by comet assay in Mexican patients with type 2 diabetes mellitus. Acta Diabetol 2010;47 Suppl 1:111-6.  Back to cited text no. 14
    
15.
Ostling O, Johanson KJ. Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 1984;123:291-8.  Back to cited text no. 15
    

Top
Correspondence Address:
Dr. Biju Thomas
A B Shetty Memorial Institute of Dental Sciences, Nitte University, Deralakatte, Mangalore - 575 018, Karnataka
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_503_17

Rights and Permissions


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]



 

Top
 
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Email Alert *
    Add to My List *
* Registration required (free)  
 


    Abstract
   Introduction
   Subjects and Methods
   Results
   Discussion
   Conclusions
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed600    
    Printed24    
    Emailed0    
    PDF Downloaded108    
    Comments [Add]    

Recommend this journal