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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 8  |  Issue : 1  |  Page : 25-29

Analysis of DNA instability in patients with periodontitis


Department of Oral Pathology, SRMDC, Ramapuram, Chennai, Tamil Nadu, India

Date of Web Publication30-Mar-2017

Correspondence Address:
R Swarnalakshmi
Department of Oral Pathology, SRM Dental College, Ramapuram, Chennai - 600 089, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/srmjrds.srmjrds_69_16

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  Abstract 

Introduction: Periodontitis is characterized by loss of supporting connective tissue and alveolar bone around teeth. Although triggered by bacterial infection other factors such as apoptosis and immune mediators such as interleukin - 1 (IL-1), tumor necrosis factor alpha, IL-6, and receptor activator of NF-κB ligand have found to aggravate the periodontal disease prognosis. Apoptosis is believed to contribute to periodontal disease progression. Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis. DNA fragmentation factor. (DFF) is a major apoptotic endonuclease representative of DNA fragmentation. DFF is responsible for DNA fragmentation, a hallmark event during apoptosis. Aim: This study aims to determine the presence of DNA instability in tissue samples of chronic periodontitis using double-stranded DNA marker using immunohistochemistry. Methodology: Tissue samples of five chronic periodontitis cases and one control case was obtained. Patients of any age and sex with clinically and histologically diagnosed cases of chronic periodontitis were selected. The tissue samples were subjected to immunohistochemical analysis using DFF. Results: Staining index of DFF 45 was calculated by multiplying staining intensity and the staining percentage. Two cases showed positive expression with staining index moderate in one case and low in one case. Three cases did not express DFF. Conclusion: Apoptosis plays a critical role in the regulation of inflammation and host immune response. Mild to moderate degree of DNA instability is found to be present in chronic periodontitis patients. The presence of DNA instability in chronic periodontitis emphasizes the importance of devising effective treatment modalities.

Keywords: Apoptosis, DNA fragmentation factor, DNA instability, periodontitis


How to cite this article:
Swarnalakshmi R, Ramya R, Sabitha B, Bharanidharan, Nagarathinam A E, Kumar K R. Analysis of DNA instability in patients with periodontitis. SRM J Res Dent Sci 2017;8:25-9

How to cite this URL:
Swarnalakshmi R, Ramya R, Sabitha B, Bharanidharan, Nagarathinam A E, Kumar K R. Analysis of DNA instability in patients with periodontitis. SRM J Res Dent Sci [serial online] 2017 [cited 2022 Jul 5];8:25-9. Available from: https://www.srmjrds.in/text.asp?2017/8/1/25/203488


  Introduction Top


Periodontitis, one of the major oral diseases is characterized by excessive production of inflammatory cytokines, destruction of periodontal ligament, loss of attachment, and resorption of alveolar bone. Periodontitis affects many people worldwide and is the main cause of tooth loss, in general, periodontitis is an inflammatory disease, usually initiated by periodontal pathogens residing in the dental plaque, including Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Fusobacterium nucleatum, Treponema denticola, and Actinomyces viscosus.[1],[2] However, other risk factors, such as smoking, dental calculus, occlusal trauma, mouth breathing, genetic factors, hormones, and stress, can promote the progression of periodontitis. Although triggered by bacterial infection other factors like apoptosis and immune mediators such as interleukin - 1 (IL-1), tumor necrosis factor alpha, IL-6, and receptor activator of NF-kB ligand have found to aggravate the periodontal disease prognosis.[3] This complex interplay of events leads to difficulty in achieving effective treatment modalities.

Apoptosis is a type of programmed cell death, can be simply defined as programmed “self-killing.”[4] Apoptosis plays an important role in the regeneration of healthy cells and elimination of injured cells. Dysregulated apoptosis induces cell death and tissue damage, leading to organ dysfunction. Human health and diseases can be regulated by apoptosis of cell.[5] Apoptosis is mediated by caspase-dependent pathways.[6] Typically, apoptosis is characterized by membrane blebbing, DNA fragmentation, and caspase activation. Apoptosis is believed to contribute to periodontal disease progression. Cleavage of chromosomal DNA into oligonucleosomal size fragments is an integral part of apoptosis.

DNA fragmentation factor (DFF) is a major apoptotic endonuclease representative of DNA fragmentation. DFF is responsible for DNA fragmentation, a hallmark event during apoptosis. DNA fragmentation or cleavage happens in two steps; the first step is cleavage into larger fragments of about 50 kbp followed by internucleosomal cleavage that generates smaller mono- and oligonucleosomal DNA fragments. DFF has been suggested to contribute to this process.[7] DFF is composed of two subunits, a 40-kDa caspase-activated DNase (CAD) (DFF 40) and its 45-kDa inhibitor of CAD (ICAD) (DFF 45).[8]

Literature search revealed that several studies have been done to find the expression of p53, Bcl-2 and caspase-3 and few other studies dealt with the presence of apoptotic cells by electron microscopy,[9],[10],[11],[12] this study was done to determine DNA (genomic) instability in periodontitis by studying the expression of DFF 45, which may give insights not only into the pathogenesis of the disease but can also provide potential therapeutic strategies for treatment of periodontitis.


  Methodology Top


Tissue samples of five chronic periodontitis cases and one control case was obtained. Patients of any age and sex with clinically and histologically diagnosed cases of chronic periodontitis were selected. The tissue samples were subjected to immunohistochemical analysis using DFF.

Immunohistochemical analysis

Immunohistochemical analysis was performed on 3 μ tissue sections on poly-l-lysine coated slides. Prediluted primary anti-DFFA polyclonal antibody were used followed by the secondary super sensitive polymer horseradish peroxidase detection system (Novolink™ Polymer). Diaminobenzidine was used as the chromogen and counterstained with Harris hematoxylin. The presence of brown colored end product at the site of target antigen was indicative of positive immunoreactivity. Evaluation of the immunoreactivity was based on the staining index and was classified as low, moderate, and high.


  Results Top


The staining percentage, staining intensity and staining index of DFF 45 were analyzed. The percentage of positive-staining cells and the staining intensity were graded on a scale of 0–3. Staining percentage was graded as: 0 = 0% positive cells; 1 = 1%–33% positive cells; 2 = 34%–66% positive cells; and 3 = 67%–100% positive cells. Staining intensity was graded as 0 = no staining; 1 = staining obvious only at ×400; 2 = staining obvious at ×100 but not ×40; and 3 = staining obvious at ×40. For each sample, the staining percentage and staining intensity scores were multiplied to give the staining index. Staining index was calculated as following: 0 = zero; 1–2 = low; 3–4 = moderate; and 6–9 = high.


  Discussion Top


Periodontitis is a highly prevalent infectious disease, characterized by destruction of the periodontium, resulting in tooth loss. Periodontitis is initiated by periodontal pathogens, while other risk factors such as smoking, stress, and systemic diseases which in turn aggravates its progression.[13] However, the molecular mechanisms by which pathogens and risk factors destroy the periodontium is still unclear. Apoptosis is different from necrosis, is an active cell death mediated by a cascade of gene expression events. Although apoptosis is involved in many inflammatory diseases as well as in several tumors, its correlation with periodontitis is unclear.

Several hematological and genetic disorders have been associated with the development of periodontitis in affected individuals. It is speculated that the major effect of these disorders is through alterations in host defense mechanisms that have been clearly described for disorders such as neutropenia and leukocyte adhesion deficiencies. At present, periodontitis as a manifestation of systemic disease is the diagnosis to be used when the systemic condition is the major predisposing factor, and local factors such as large quantities of plaque and calculus are not clearly evident. In the case where periodontal destruction is clearly the result of local factors but has been exacerbated by the onset of such conditions as diabetes mellitus or HIV infection, then the diagnosis should be chronic periodontitis modified by the systemic condition.

Reyes et al. have proposed a possible link between periodontitis and atherosclerosis of vessels. He suggested that oral bacteria and its products gain access into bloodstream and releases pro-inflammatory mediators which further causes endothelial activation or dysfunction and atheroma formation. Further, the formed atheroma matures and leads to the production of antibodies that may cross-react with endothelial cells and promote helper T-cells response. Finally, macrophages are activated, inflammation is exaggerated and an atherosclerotic event results.[14]

Diabetes is associated with the production of pro-apoptotic factors, the formation of reactive oxygen species, tumor necrosis factor, and advanced glycation end products. Reactive oxygen species are potent inducers of apoptosis. Tumor necrosis factor can induce apoptosis by binding to the tumor necrosis factor receptor-1, which has a death domain that triggers the initial events in apoptosis, and by stimulating expression of pro-apoptotic genes. Advanced glycation end products may also promote apoptosis of critical matrix-producing cells through several mechanisms; the direct activation of caspase activity, as well as indirect pathways that increase oxidative stress or the expression of pro-apoptotic genes that regulate apoptosis.[15]

After reviewing several relevant published articles, we found that apoptosis has undoubtedly been reported to play a pivotal role in periodontitis; therefore, we studied the apoptotic events in the gingival tissue of chronic adult periodontitis patients.

Apoptotic DNA fragmentation is required for maintenance of genomic stability. DNA fragmentation is a biochemical hallmark and a fundamental process of apoptosis. Various apoptotic stimuli and pathways converge at a common hallmark event of apoptosis; one such is DNA fragmentation.[16] Apoptotic DNA fragmentation involves the incision of chromosomal DNA in-between nucleosomes.

CAD, also called DFF is the nuclease responsible for apoptotic DNA fragmentation. CAD is normally associated with its inhibitor as well as chaperon, ICAD.[8] During the process of apoptosis, the activated caspase three cleaves ICAD and releases CAD to digest chromosomal DNA. DFF/CAD is activated during apoptosis to digest the potentially harmful chromosomal DNA with damage and mutations.[17]

Earlier the occurrence of apoptosis in chronic periodontitis has been studied by morphologic criteria as well as by the widely used TUNEL technique that is based on the detection of DNA fragmentation. An increased number of TUNEL-positive inflammatory cells has been reported in periodontitis. In addition, caspase-3 and -7 activation as well as PARP cleavage, clearly showed caspase activation was found in keratinocytes, basal cells, and connective tissue cells, cell types that are damaged in periodontitis.[18]

Bulut et al. concluded in his studies that there were no significant differences between generalized aggressive periodontitis and healthy controls with respect to the overall frequency of caspase-3 expression or frequencies of different grades of expression of p53.[9]

Kasprzak and Ma concluded in his studies that more pronounced expression of p53 in patients with chronic periodontitis than in control individuals which may indicate a potential role of the protein in initiation of apoptosis or may provide proof for an augmented reparative role of cell nucleus structures (mainly DNA), injured due to chronic inflammation.[12]

Gamonal et al. concluded in their study TUNEL-positive cells and cells exhibiting chromatin condensation by electron microscopy active-caspase 3, Fas, FasL, and p53 were only observed in the inflammatory infiltrate from periodontitis biopsies, whereas Bcl-2 cells were present in both periodontitis patients and healthy controls.[19]

Our study included a total of seven tissues, out of which five were chronic periodontitis cases and one positive control [breast cancer [Figure 1] and one negative control (normal tissue). The expression of DFF 45 was examined.
Figure 1: DNA fragmentation factor positivity in Breast cancer (positive control) (×40)

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The expression of DFF 45 was negative in three cases of chronic periodontitis. Hence, the staining percentage and the staining intensity were graded as zero. The expression of DFF 45 was positive in two cases of chronic periodontitis. The staining index which is obtained by multiplying staining percentage and intensity showed Grade 2 and 4 which represents low [Figure 2] and moderate [Figure 3] staining, respectively. This study demonstrates the presence of apoptotic cells in the gingival biopsies from periodontal patients, in accordance with earlier studies that showed apoptotic cells constituting <10% of total cells.
Figure 2: DNA fragmentation factor low-positive expression (×20)

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Figure 3: DNA fragmentation factor moderate-positive expression (×20)

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Positivity observed in few of the periodontal cases suggests that there is involvement of apoptotic mechanism which is believed to contribute to periodontal disease progression.

The results presented here demonstrate that apoptosis of gingival epithelium promotes periodontal breakdown and their association with periodontal disease severity and activity. It establishes that apoptosis is induced in the periodontal tissue by host and microbial factors and supports the hypothesis that apoptotic mechanisms could be implicated in the inflammatory process associated with gingival tissue destruction observed in adult periodontitis patients.[19]


  Conclusion Top


Mild to moderate degree of DNA instability is found to be present in chronic periodontitis patients. The presence of DNA instability in chronic periodontitis emphasizes the importance of devising effective treatment modalities. Further studies in a larger sample size would help in gaining deeper insights into these events and also will have a significant impact on the therapeutic strategies of periodontitis. Limitations of this study are the small sample size and also the clinical details of the patient like grading and coronary arterial lesions values were not correlated.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Byrne SJ, Dashper SG, Darby IB, Adams GG, Hoffmann B, Reynolds EC. Progression of chronic periodontitis can be predicted by the levels of Porphyromonas gingivalis and Treponema denticola in subgingival plaque. Oral Microbiol Immunol 2009;24:469-77.  Back to cited text no. 1
    
2.
Tomita S, Komiya-Ito A, Imamura K, Kita D, Ota K, Takayama S, et al. Prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia in Japanese patients with generalized chronic and aggressive periodontitis. Microb Pathog 2013;61-62:11-5.  Back to cited text no. 2
    
3.
Lucas H, Bartold PM, Dharmapatni AA, Holding CA, Haynes DR. Inhibition of apoptosis in periodontitis. J Dent Res 2010;89:29-33.  Back to cited text no. 3
    
4.
Maiuri MC, Zalckvar E, Kimchi A, Kroemer G. Self-eating and self-killing: Crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 2007;8:741-52.  Back to cited text no. 4
    
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Ouyang L, Shi Z, Zhao S, Wang FT, Zhou TT, Liu B, Bao JK. Programmed cell death pathways in cancer: A review of apoptosis, autophagy and programmed necrosis. Cell Proliferation 2012;45:487-98.  Back to cited text no. 5
    
6.
Fan TJ, Han LH, Cong RS, Liang J. Caspase family proteases and apoptosis. Acta Biochim Biophys Sin (Shanghai) 2005;37:719-27.  Back to cited text no. 6
    
7.
Ding H, Qiu S, Li S, Symersky J, Green TJ, Luo M. Expression, purification, crystallization of fragments from the C-terminal region of DFF45/ICAD. Acta Crystallogr D Biol Crystallogr 2003;59:1323-6.  Back to cited text no. 7
    
8.
Liu X, Zou H, Slaughter C, Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 1997;89:175-84.  Back to cited text no. 8
    
9.
Bulut S, Uslu H, Ozdemir BH, Bulut OE. Expression of caspase-3, p53 and Bcl-2 in generalized aggressive periodontitis. Head Face Med 2006;2:17.  Back to cited text no. 9
    
10.
Zeidán-Chuliá F, Gursoy M, de Oliveira BH, Gelain DP, Könönen E, Gursoy UK, et al. Focussed microarray analysis of apoptosis in periodontitis and its potential pharmacological targeting by carvacrol. Arch Oral Biol 2014;59:461-9.  Back to cited text no. 10
    
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Teixeira JE, Sateriale A, Bessoff KE, Huston CD. Control of Entamoeba histolytica adherence involves metallosurface protease 1, an M8 family surface metalloprotease with homology to leishmanolysin. Infect Immun 2012;80:2165-76.  Back to cited text no. 11
    
12.
Kasprzak A, Hausmann M, Małkowska-Lanzafame A, Surdyk-Zasada J, Przybyszewska W, Siodła E, et al. Immunocytochemical indicators of apoptosis in gingival tissues of patients with chronic periodontitis. J Med Sci 2016;83:37-46.  Back to cited text no. 12
    
13.
Song B, Zhou T, Yang W, Liu J, Shao L. Programmed cell death in periodontitis: Recent advances and future perspectives. Oral Dis 2016.  Back to cited text no. 13
    
14.
Reyes L, Herrera D, Kozarov E, Roldá S, Progulske-Fox A. Periodontal bacterial invasion and infection: Contribution to atherosclerotic pathology. J Periodontol 2013;84 4 Suppl: S30-50.  Back to cited text no. 14
    
15.
Graves DT, Liu R, Oates TW. Diabetes-enhanced inflammation and apoptosis: Impact on periodontal pathosis. Periodontology 2000 2007;45:128-37.  Back to cited text no. 15
    
16.
Nagata S, Nagase H, Kawane K, Mukae N, Fukuyama H. Degradation of chromosomal DNA during apoptosis. Cell Death Differ 2003;10:108-16.  Back to cited text no. 16
    
17.
Liu X, Li P, Widlak P, Zou H, Luo X, Garrard WT, et al. The 40-kDa subunit of DNA fragmentation factor induces DNA fragmentation and chromatin condensation during apoptosis. Proc Natl Acad Sci U S A 1998;95:8461-6.  Back to cited text no. 17
    
18.
Bantel H, Beikler T, Flemmig TF, Schulze-Osthoff K. Caspase activation is involved in chronic periodontitis. FEBS Lett 2005;579:5559-64.  Back to cited text no. 18
    
19.
Gamonal J, Bascones A, Acevedo A, Blanco E, Silva A. Apoptosis in chronic adult periodontitis analyzed by in situ DNA breaks, electron microscopy, and immunohistochemistry. J Periodontol 2001;72:517-25.  Back to cited text no. 19
    


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  [Figure 1], [Figure 2], [Figure 3]



 

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