|Year : 2016 | Volume
| Issue : 2 | Page : 75-80
Assessment of cytogenic damage in chronic periodontitis and type 2 diabetes mellitus subjects through micronucleus test
Surekha Ramrao Rathod, Anubha Raj, Padmaja Jadhav, Trupti Sarda
Department of Periodontology, VSPM Dental College and Research Institute, Nagpur, Maharashtra, India
|Date of Web Publication||9-Aug-2016|
Surekha Ramrao Rathod
Department of Periodontology, VSPM Dental College and Research Institute, Nagpur, Maharashtra
Source of Support: None, Conflict of Interest: None
Background and aim: DNA and cells of human body are constantly in threat for exposure of oxidative stress and role of genetic factors and oxidative damage. A high number of reactive oxygen species can cause oxidative damage to a large number of molecules, including DNA, result in periodontal tissue damage through multiple mechanisms such as lipid peroxidation, protein denaturation and DNA damage. The micronucleus test is a sensitive method that indicates DNA damage. The aim of the study was to assess the micronucleus frequency (MNF), as a biomarker for DNA damage, in individuals with type 2 Diabetes Mellitus and chronic periodontitis.
Materials and Methods: A total of 60 subjects were recruited for the study and divided into three groups. 20 subjects of group 1 had diabetes mellitus along with chronic periodontitis, Subjects with Diabetes mellitus and healthy periodontium were allotted to group 2 and group 3 include Subjects without Diabetes melilitus and with Chronic Periodontitis. Periodontal clinical examination was done. Blood sample collected was use to prepare a slide which was fixed in 5% gimsa solution and was analyzed in microscope then scoring of micronuclei was done.
Results: The mean micronuclei observed in group 1, 2 and 3 were 14.8, 11 and 10.85 respectively i.e. group 1 showed significantly greater damage than other two groups.
Conclusion: It was concluded that CBMN method was useful as a biomarker for DNA damage in individuals with chronic degenerative systemic diseases such as type 2 diabetes, as well as chronic local disease, such as PD.
Keywords: Micronucleus frequency, periodontitis, type 2 diabetes mellitus
|How to cite this article:|
Rathod SR, Raj A, Jadhav P, Sarda T. Assessment of cytogenic damage in chronic periodontitis and type 2 diabetes mellitus subjects through micronucleus test. Saudi J Oral Sci 2016;3:75-80
|How to cite this URL:|
Rathod SR, Raj A, Jadhav P, Sarda T. Assessment of cytogenic damage in chronic periodontitis and type 2 diabetes mellitus subjects through micronucleus test. Saudi J Oral Sci [serial online] 2016 [cited 2019 Jul 24];3:75-80. Available from: http://www.saudijos.org/text.asp?2016/3/2/75/188075
| Introduction|| |
Periodontitis is a term used to describe an inflflammatory process, initiated by the plaque bioflim that leads to loss of periodontal attachment of the root surface and adjacent alveolar bone and which ultimately results in tooth loss.
The association between periodontal disease and diabetes has been explored in several studies over the years (1–5), and it is generally accepted that periodontal disease is more prevalent and more severe in persons with diabetes than in nondiabetic persons. Indeed, the periodontal signs and symptoms are now recognized as the “sixth complication” of diabetes.
Type 2 diabetes mellitus (DM) is the most common form of DM constituting 90–95% of total diabetes cases. Advanced glycation is one of the major pathways involved in the development and progression of different diabetic complications including nephropathy, retinopathy, and neuropathy. Similarly, oxidative stress may be a common pathway linking diverse mechanisms for the pathogenesis of complications in diabetes. Mechanisms that contribute to increased oxidative stress in diabetes may include not only increased nonenzymatic glycosylation (glycation) and autoxidative glycosylation but also metabolic stress resulting from changes in energy metabolism, alterations in sorbitol pathway activity, changes in the level of inflammatory mediators and the status of antioxidant defense systems, and localized tissue damage result from ischemic reperfusion injury.
Dental plaque harbors, a number of bacterial pathogens, which stimulate host cells to release various interleukins and tumor necrosis factor-α. These proinflammatory cytokines attract polymorphonucleocytes (PMNs) to the site of infection. PMNs encounter this bacterial challenge by producing proteolytic enzymes and O2 by oxidative burst.
DNA and cells of human body are constantly exposed to attacks of oxidative nature. Oxygen is a key element which plays an important role in many processes of the human body including cellular metabolism, intercellular, and intracellular signaling. Although beneficial oxygen, through reactive oxygen species (ROS) generation can react with DNA, protein, and other cellular components causing harmful effects.
As the oxidative stress which leads to DNA damage and is intrinsically related to the pathogenesis of type 2 diabetes and periodontitis, it seems interesting to evaluate the occurrence of DNA damage and its relationship with these diseases.
One of the most established methods for evaluating DNA damage is the micronucleus (MN) test. Micronuclei (MN) and other nuclear anomalies such as nucleoplasmic bridges and nuclear buds are biomarkers of genotoxic events and chromosomal instability. These genome damage events can be measured simultaneously in the cytokinesis-block micronucleus cytome (CBMN) assay. The molecular mechanisms leading to these events have been investigated over the past two decades using molecular probes and genetically engineered cells.
The MN test analyses is based on the identification of a secondary nucleus (MN), which is originated from acentric chromosome fragments, acentric chromatid fragments, or whole chromosomes that fail to be included in the daughter nuclei at the completion of telophase during mitosis because they did not attach properly with the spindle during the segregation process in anaphase. These displaced chromosomes or chromosome fragments are eventually enclosed by a nuclear membrane and except for their smaller size are morphologically similar to nuclei after conventional nuclear staining. Few studies have investigated DNA damage by micronuclei in patients with type 2 diabetes, and these studies have shown increased frequency of micronuclei in diabetic patients. There is a paucity of literature which sought to investigate the MN frequency in the context of type 2 diabetes and periodontitis. Hence, the aim of this study was to assess the micronucleus frequency (MNF), as a biomarker for DNA damage, in individuals with type 2 DM and chronic periodontitis.
| Materials and Methods|| |
This study was approved by the Institutional Ethic Committee VSPM Dental College and Research Centre (VSPM DC RC). All volunteers were informed about the aims and methods of this study, and they provided their written consent to participate.
Study was approved by the Institutional Ethic Committee. A total of 60 subjects with an age range between 20 and 60 years including both male and female visiting the Department of Periodontics at VSPM DC RC, Nagpur, will be recruited. All subjects should be willing to participate and sign the informed consent were included in the study. Subjects divided into three groups, Group 1, Group 2, and Group 3 each consisting of 20 subjects. Group 1 includes subjects with well-controlled DM and chronic periodontitis. Group 2 includes subjects with DM and healthy periodontium. Group 3 includes subjects without diabetes mellitus and with Chronic Periodontitis. Apart from this subjects should be of ≥ 30 years of age with controlled Type 2 DM having a history of diabetes more than 5 years and had at least seven or more remaining teeth. Subjects having viral infection or pyrexia over the past 1 month, history of receiving antibiotics, corticosteroids, cytotoxic agents, NSAIDS, radiation therapy, or periodontal therapy were excluded from the study.
Clinical record and physical evaluation
All participants answered a structured questionnaire about demographic characteristics, personal and family medical history, and use of medications. A trained examiner collected information from diabetic patients regarding time since diabetes onset, medication used to control hyperglycemia, and the presence of complications associated with diabetes.
Periodontal clinical examination
Chronic peritoneal dialysis (PD), as defined by the American Academy of Periodontology (39), includes local signs of inflammation and tissue destruction (presence of deep periodontal pockets ≥6 mm) and loss of attachment (clinical attachment loss [CAL] ≥4 mm) in at least four nonadjacent teeth. Periodontal pocket depth, CAL, and bleeding on probing were evaluated with a periodontal probe PCPUNC15-6 (Hu-Friedy®). Severe periodontal disease was defined as the presence of deep periodontal pockets ≥6 mm with CAL ≥5 mm and bleeding on probing in at least eight sites distributed in different quadrants of the dentition.
Blood sampling, cell culture, and cytokinesis-block micronucleus assay
Collection of samples
Two milliliter of venous blood was collected from each subject at the Department of Periodontics, (VSPM Dental College, Nagpur, India) in sodium heparinized vacutainers. After the samples were collected, it transported at room temperature on the same day to the Genetic Laboratory (NKP SIMS and RC Nagpur, India).
MN test procedure
A thin blood smear was prepared on the same day of sample collection. The slides were air-dried for at least 30 min before fixing at room temperature. Once the slides were prepared, fixation was carried out using freshly prepared ice-cold 3:1 methanol and acetic acid solution for 10–15 min. The slides were removed from the fixative and washed twice in 6.8 pH phosphate buffer saline buffer. The slides were then stained immediately in 5% Giemsa solution (RM128-Himedia Lab, LOT#13339) and dried on a hot plate for 5 min. The slides were stored in slide boxes before examining under a microscope. Three slides were analyzed using ×100 objective and ×10 eyepiece magnifications with emersion oil.
Scoring of micronuclei
Thousand cells were counted per sample. Only nucleated cells that were separate without overlapping or folds were analyzed. Micronuclei (MN) were counted if the structures had a regular border and were located inside the cytoplasm, with an intensity of staining less than or equal to that of the main nucleus [Figure 1].
|Figure 1: The micronucleus test. (a) Cell with micronuclei clearly showing tertiary nucleus; (b) cell without any micronucleus|
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Statistical analysis was done using EPI software version 6. The mean, standard deviation (SD), ANOVA, and post hoc Bonferroni was used in which level of significance used was 0.05 and power of test used was >80%.
| Results|| |
In [Table 1] and [Table 2], in group of chronic periodontitis, the mean value of probing pocket depth (PPD) was 3.71 with SD of 0.28, in DM with periodontitis group of PPD is 4.26 with SD 0.31, and DM group is 1.46 with SD 0.29. The ANOVA of PPD value of these groups is statistically significant (P < 0.01). Similarly, in group of chronic periodontitis, the mean value of CAL was 4.06 with SD of 0.215, in DM with periodontitis group of CAL is 4.59 with SD 0.31, and DM group is 0. The ANOVA of CAL value of these groups is statistically significant (P < 0.01).
|Table 1: Descriptive mean of clinical parameter among chronic periodontitis, diabetes mellitus, and diabetes mellitus with periodontitis group|
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|Table 2: Analysis of variance among the group for periodontal probing depth, clinical attachment loss, and micronuclei frequency score|
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In similar way, [Table 1] and [Table 2] in group of chronic periodontitis, the mean value of MN frequency score was 10.85 with SD of 2.88, in DM with periodontitis group of MN frequency score is 14.8 with SD 1.7, and DM group is 11 with SD 1.46. The ANOVA of MN frequency score value of these groups is statistically significant (P < 0.01).
In [Table 3], further it is analyzed by Bonferroni, all the above three groups are statistically significant with each other (P < 0.05) which shows that PPD, CAL, and MN frequency score value of group DM plus periodontitis has a higher value than other two group.
|Table 3: Multiple comparison of group with dependent variable by Bonferroni analysis|
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[Table 4] shows as Pearson correlation of MN frequency score with periodontal parameters. Here PPD value increase at the same time MN frequency score is also increases. It also shows moderate correlation between PPD and score (P<0.001).
|Table 4: Pearson correlation of micronuclei frequency score with periodontal parameters|
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As CAL value increases at the same time score is also increases. It also shows a moderate correlation between score and CAL (P < 0.001).
| Discussion|| |
Periodontal diseases are inflammatory disorders that are the result of the interactions between periodontopathogens and the host defense immune system and environmental factors, which results in uncontrolled immune response. Activation of the immune system, and the production of oxygen radicals, and their related metabolites that occur in periodontitis, contributes to increased ROS production, oxidative stress, and active proteases released during inflammatory and host immune responses to bacterial challenge, which is reported to be involved in many diseases. Among the ROS formed, H2O2 and OH radicals are potent oxidants that can affect nucleic acids and modify bases in the DNA, contributing to an increase in DNA damage and therefore MN formation. To the best of our best knowledge, there are few studies investigating association between DNA damage by the MN assay and the physical examination and periodontal parameters in patients who have simultaneously type 2 diabetes and periodontitis. The results presented in this study indicated an association between periodontitis with well-controlled type 2 DM and DNA damage. For the matter of periodontal evaluation periodontal tissue destruction is more severe in diabetics and also periodontal disease is one of the complications of DM. In current study, MNF score was found to serve as an important biomarker for assessment of DNA damage in chronic degenerative systemic disease such as DM and periodontitis, according to which destruction was more frequent in group that include both DM and periodontitis when compared to groups having periodontitis alone or having only diabetics subjects.
Andreassi et al. showed that type 2 diabetes was the major independent determinant of an increase MN frequency in circulating lymphocytes of patients with ischemic heart disease. Increase of MN frequency was also noted by Martínez-Pérez et al. in patients with type 2 diabetes without any microvascular or macrovascular complications. Several studies, using other techniques, have reported DNA damage in patients with DM2. Using sister chromatid exchange (SCE), Kulkarni et al. reported chromosomal damage in patients with DM2 treated with chlorpropamide, they were statistically significant increase in the individual numbers of SCEs per metaphase in each of the patients treated with the drug compared with the mean of the pooled control values. In the current study, MN frequency in well-controlled diabetes subjects was found to be 11 which was in accordance with the previously mentioned study. Bloching et al. suggested a possible association between periodontal status and increase in MN number in the buccal mucosa. In the current study, mean MN frequency for chronic periodontitis group was 10.85 which was similar to MN frequency of 10.10 in a study conducted by Avula et al. in 2012. This result was also consistent with an another study conducted by Emingil et al. in 2002 in which damage was evaluated by a different technique, i.e. SCE analysis. However, in the current study, MN frequency was found to be highest with Group 1, i.e. DM and chronic periodontitis group which was found to be 14.80. This result was consistent with study conducted by Corbi et al. in 2014. indicating that individuals with DM along with periodontal disease had a greater amount of DNA damage than the spontaneous levels of damage that occur in individuals of other 2 group. Here, we show that periodontal disease could be a risk factor for DNA damage, and it will be a modifiable risk factor as periodontitis can be prevented and treated [Figure 2], [Figure 3], [Figure 4].
|Figure 2: Mean periodontal probing depth in chronic periodontitis (3.716), diabetes mellitus (1.466), and diabetes mellitus with periodontitis group (4.26)|
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|Figure 3: Mean clinical attachment loss in chronic periodontitis (4.06), diabetes mellitus (0), and diabetes mellitus with periodontitis group (4.59)|
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|Figure 4: Mean micronucleus frequency score in chronic periodontitis (10.85), diabetes mellitus (11), and diabetes mellitus with periodontitis group (14.8)|
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CBMN method was useful as a biomarker for DNA damage in individuals with chronic degenerative systemic diseases such as type 2 diabetes and chronic local disease, such as PD. The CBMN assay is the most frequently used chromosomal biomarker in human lymphocytes to study genotoxicity and cytotoxicity both in vitro and in vivo. It is now well-established that the CBMN assay in its comprehensive cytome mode provides concurrent information on chromosomal breakage, chromosome rearrangements, and gene amplification, as well as other events, for instance, cell death (both apoptosis and necrosis) and cell cytotoxicity. Oxidative damage to genetic material is the result of the interaction of DNA with ROS. This oxidative damage leads to increasing MN caused by DNA strand breaks.
Hence, we conclude that the occurrence of the type 2 diabetes and PD, which are involved with oxidative stress processes, could proportionally increase the DNA damage. The present results suggest that these two pathologies occurring simultaneously promote a rise in oxidative stress and inflammation leading to increase DNA injury. Moreover, the CBMN method was useful as a biomarker for DNA damage in individuals with chronic degenerative systemic diseases such as type 2 diabetes, as well as chronic local disease, such as PD.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Löe H. Periodontal disease. The sixth complication of diabetes mellitus. Diabetes Care 1993;16:329-34.
Halliviell B, Gutteridge JM. Free Radicals in Biology and Medicine. 2nd
ed. Oxford, UK: Oxford University Press; 1989.
Fenech M. The lymphocyte cytokinesis-block micronucleus cytome assay and its application in radiation biodosimetry. Health Phys 2010;98:234-43.
Martínez-Pérez LM, Cerda-Flores RM, Gallegos-Cabriales EC, Dávila-Rodríguez MI, Ibarra-Costilla E, Cortés-Gutiérrez EI. Frequency of micronuclei in Mexicans with type 2 diabetes mellitus. Prague Med Rep 2007;108:248-55.
Lim LP, Tay FB, Sum CF, Thai AC. Relationship between markers of metabolic control and inflammation on severity of periodontal disease in patients with diabetes mellitus. J Clin Periodontol 2007;34:118-23.
Valavanidis A, Vlachogianni T, Fiotakis C. 8-hydroxy-2-deoxyguanosine (8-OHdG): A critical biomarker of oxidative stress and carcinogenesis. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 2009;27:120-39.
Battino M, Ferreiro MS, Gallardo I, Newman HN, Bullon P. The antioxidant capacity of saliva. J Clin Periodontol 2002;29:189-94.
Andreassi MG, Botto N, Simi S, Casella M, Manfredi S, Lucarelli M, et al.
Diabetes and chronic nitrate therapy as co-determinants of somatic DNA damage in patients with coronary artery disease. J Mol Med (Berl) 2005;83:279-86.
Kulkarni PS, Ambani LM, Bhandarkar SD. Chromosomal studies in diabetic patients treated with chlorpropamide. Food Chem Toxicol 1985;23:625-8.
Bloching M, Reich W, Schubert J, Grummt T, Sandner A. The influence of oral hygiene on salivary quality in the Ames Test, as a marker for genotoxic effects. Oral Oncol 2007;43:933-9.
Avula H, Mishra A, Pandey R, Krishna M, Kalakonda BB, Avula J. The micronucleus test to evaluate cytogenetic damage in patients with periodontitis. J Periodontol Implant Dent 2012;4:29-33.
Emingil G, Sapmaz G, Biçakçi N, Ozkinay F. Sister chromatid exchange (SCE) analysis in periodontitis. J Clin Periodontol 2002;29:811-5.
Corbi SC, Bastos AS, Orrico SR, Secolin R, Dos Santos RA, Takahashi CS, et al.
Elevated micronucleus frequency in patients with type 2 diabetes, dyslipidemia and periodontitis. Mutagenesis 2014;29:433-9.
Fenech M, Morley AA. Measurement of micronuclei in lymphocytes. Mutat Res 1985;147:29-36.
Fenech M. Cytokinesis-block micronucleus cytome assay. Nat Protoc 2007;2:1084-104.
Lazalde-Ramos BP, Zamora-Perez AL, Sosa-Macías M, Guerrero-Velázquez C, Zúñiga-González GM. DNA and oxidative damages decrease after ingestion of folic acid in patients with type 2 diabetes. Arch Med Res 2012;43:476-81.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]