Comparative evaluation of genotoxic effect in subjects with chronic periodontitis with and without type 2 diabetes mellitus

INTRODUCTION

Periodontal diseases, usually caused by plaque biofilm, is one of the most widespread diseases of oral cavity. It includes the inflammatory process resulting in gingival bleeding, periodontal pocket formation, destruction of connective tissue attachment, and alveolar bone resorption and finally leading to tooth loss.

Diabetes mellitus (DM) is a group of complex metabolic disorder characterized by a relative or absolute insufficiency of insulin secretion or resistance to the metabolic action of insulin on target tissues. Among all forms of diabetes, Type 2 diabetes (T2D) is the most common form.

The increased glucose levels in diabetes cause non-enzymatic glycosylation of amino groups in proteins, lipids and nucleic acids. This further forms the advanced glycation end products (AGEs). The formation of so-called glycoxidation products is triggered by oxidative stress.

Oxidative stress causes DNA damage and is the only major factor related to the causation of both chronic diseases - type 2 diabetes and periodontitis. Oxidative stress induces cellular damage and insulin resistance, and this is responsible for related co-morbidities.¹ In addition, increased circulating free fatty acids in diabetes mellitus affect insulin signal transduction pathways and induce endothelial dysfunction caused by increased reactive oxygen species (ROS) generation and oxidative stress.² The formation of reactive oxygen species during normal physiological conditions is compensated by antioxidants.

The micronucleus (MN) test is one of the method to assess the DNA injury.^3–5 The micronuclei (MN) and other nuclear anomalies are the extranucclear bodies of the damaged part of chromosome used to assess genotoxic and cytotoxic events and chromosomal instability.⁶ Micronuclei may result from clastogenic or aneugenic mechanisms and act as indicator of an ongoing process of DNA damage.⁷ The micronuclei are considered to be a biomarker of abnormal mitosis and might also be used to assist in cancer staging and histologic grading.⁸

The Micronuclei helps us to identify the secondary nucleus (MN). These are DNA fragments separate from the main nucleus and have originated from acentric chromosome or 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 at anaphase.⁹ These displaced chromosomal fragments are eventually enclosed by a nuclear membrane. These appear morphologically similar to nuclei after conventional nuclear staining except that they are small in size.

Hence the objective of this study was comparative evaluation of the genotoxic effect in subjects with Chronic Periodontitis with and without type 2 diabetes mellitus.

Materials and Methods:

Study population: 65 subjects above 18years age including both male and female reporting to the outpatient Department of Periodontology, Bharati Vidyapeeth Deemed to be University Dental College & Hospital, Pune were considered in the study.

The subjects who willingly agreed to participate and sign the informed consent were involved in the study.

Subjects were divided into three groups, Group A, Group B, and Group C.

Group A included 30 subjects with generalised chronic periodontitis and Group B included 30 subjects with generalised chronic periodontitis with type 2 diabetes mellitus and Group C included 5 systemically and periodontally healthy subjects.

The inclusion criteria of this study was that the subjects should be of ≥ 18 years of age with controlled Type 2 Diabetes mellitus and had atleast seven or more remaining teeth.

Subjects receiving any anti-inflammatory antibiotic therapy, pregnant or lactating women or use of contraceptives or any other hormones, history of periodontal therapy within last 6 months were excluded from the study.

Study protocol with clinical record and physical evaluation

A special performa was designed for this study so as to have a systematic and methodical recording of all the observations and information. The relevant data comprising of chief complaint, prelimnary history, oral hygiene habits, tissue abuse habits etc were recorded in special performa.

Periodontal clinical examination

Under standard conditions of light using a mouth mirror and periodontal probe that is University of Carolina (UNC15), assessment of clinical parameters that is pocket probing depth (PPD) was undertaken for selection of subjects.

Blood sampling, cell culture and microscopic parameter assessment

Collection of samples

2ml of blood from each subject was withdrawn by venepuncture of the anticubital vein and collected in EDTA vacutainers in the Department of Periodontics (BVDU Dental College, Pune, India).

MN test procedure

Blood smear was prepared after collecting the blood sample from the subject and were air dried for at least 30 min prior to fixing it at the room temperature. Fixation was carried out using freshly prepared ice cold methanol solution for 10–15 min. After fixation of the slides, they were washed twice in 6.8 pH PBS (phosphate buffer saline). The slides were then stained immediately in 5% Giemsa solution and dried on a hot plate for 5 min. Slide boxes were used to store the slides before observing them under the microscope. These slides were observed and analyzed using ×100 objective and ×10 eyepiece magnifications with emersion oil.

Micronuclei scoring

On the focussed slide, screening was done in a zigzag manner starting from one end of slide and approaching towards other end. Only nucleated cells that were separate without any folds or overlapping were counted. 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 1b].

Statistical analysis

Descriptive and inferential statistical analyses were carried out in the present study. Results on continuous measurements were presented on Mean ± SD. Level of significance was fixed at p=0.05 and any value less than or equal to 0.05 was considered to be statistically significant.

Test of significance

1. Analysis of Variance (ANOVA) -:

This is a method used to find the significance of study parameters and test the equality of three or more means of more than two groups.

The Statistical software IBM SPSS statistics 20.0 (IBM Corporation, Armonk, NY, USA) was used for the analyses of the data and Microsoft word and Excel were used to generate graphs, tables etc.

2. Tukey’s post hoc analysis-:

The parameters of each and every group were compared to each other using this test.

Results:

In Table I, the mean value of frequency of micronuclei was 10.23 with SD of 1.888 in generalised chronic periodontitis group, 14.87 with SD 1.306 in generalised chronic periodontitis and type 2 diabetes mellitus group and 1.00 with SD of 0.707 in periodontally and systemically healthy group.

Intergroup observations (Table II, Graph I) showed highly statistically significant difference in mean micronuclei frequency values in Group A, Group B and Group C using ANOVA test with ‘p’ value of <0.001 with highest values in subjects with generalized chronic periodontitis with type 2 diabetes mellitus i.e. Group B followed by subjects with generalized chronic periodontitis without type 2 diabetes mellitus i.e. Group A and least in healthy subjects i.e. Group C.

Discussion:

Periodontal disease is a chronic inflammatory disease that results in gingival inflammation, attachment loss, destruction of bone, and gradually tooth loss. Certain organisms within the microbial flora of dental plaque are the major etiologic agents of periodontitis which produce endotoxins in the form of lipopolysaccharides (LPS) that are instrumental in generating a host-mediated tissue destructive immune response. Periodontitis leads to production of numerous alterations in systemic health thus causing various systemic diseases or conditions including diabetes.¹¹

Both type 2 diabetes mellitus and periodontal diseases are chronic diseases. Diabetes have many adverse effects on the periodontium, including decreased collagen turnover, impaired neutrophil function, and increased periodontal destruction.¹² Neutrophil chemotaxis and phagocytic activities are compromised in diabetic patients. This further reduces the bacterial killing and enhances the periodontal destruction. Diabetes, insulin resistance, and hyperglycemia, all these conditions exaggerates the inflammatory response.

The dyslipidemic microenvironment gets altered in a patient with diabetes mellitus because of the advanced glycation end products (AGEs) that acts as a co-stimulators of the cells.

The hyperglycaemia associated to uncontrolled diabetes mellitus leads to increased advanced glycation end products (AGEs), including low density lipoprotein (LDL)-AGE, and also large amount of LDL-oxidised are present. Both AGEs and LDL-oxidised, found simultaneously in diabetic patients, causes several harmful effects on cells including neutrophils, T and B lymphocytes, macrophages, monocytes and also dendritic cells. Oxidative damage is the adverse effect due to the formation of these AGE-RAGE reaction and the formation of (LDL)-AGE.¹³

Oxidative damage which occurs because of the excessive production of reactive oxygen species leads to structural alterations in the mitochondria of the cell.¹⁴ In chronic periodontitis, the immune system gets activated and leads to production of oxygen radicals and its metabolites like superoxide ions (OH) and hydrogen peroxide ions (H2 O2 ) which are potent antioxidants.¹⁵ These oxygen radicals further produces the reactive oxygen species leading to increased oxidative stress. Also the active proteases are released as a result of the inflammatory and host immune responses to bacterial challenge in such chronic diseases. The reactive oxygen species affect the nucleic acids and modify bases in the DNA, contributing to an increase in DNA damage and therefore leading to the formation of micronucleus.²

The test termed as Micronucleus test can be used to detect the genotoxic effect occurring due to the DNA injury.¹⁶

The results presented in this study are that the frequency of micronuclei were found to be higher in generalized chronic periodontitis with type 2 diabetes mellitus (Group B) in comparison with generalized chronic periodontitis without type 2 diabetes mellitus (Group A) and healthy subjects (Group C).

The result obtained in the study carried out by SamiaC.T.Corbi et al.¹⁷ in 2014 was also similar to the our study. The study by Samia C.T. Corbi et al evaluated the micronucleus frequency in subjects with type 2 diabetes mellitus, dyslipidemia and chronic periodontitisindividually or when two or more of these conditions are combined together. This study concluded that rise in the frequency of micronuclei was found in patients with any of these 3 conditions. Alsoan elevation in the oxidative stress further causing damage to the DNA occurred when these three conditions were present simultaneously.

Our results match to a study done by Surekha Rathod et al.² in 2016which concluded that average value of micronuclei frequency score is higher in chronic periodontitis with type 2 diabetes group in comparison with chronic periodontitis group alone and diabetes group alone.

The results of our study are contradictory to the study conducted by Haritha Avula et al.¹⁸ in 2012which aimed at evaluating the micronuclei frequency in different types of periodontitis compared to the healthy controls. The results indicating that the cytogenetic damage in periodontitis groups were not different from the ones in the control group.

In our study, there are fewer number of micronucleated cells present in healthy subjects which might be due to absence of genotoxic agents. A significant increase in the micronuclei frequency in chronic periodontitis with type 2 diabetes mellitus subjects was observedin comparison with chronic periodontitis without diabetes mellitus subjects. This rise in frequency of micronuclei in chronic periodontitis with type 2 diabetes subjects may be because of the pathogenic bacteria present in periodontal disease which activates the immune system leading to production of oxygen radicals further leading to formation of reactive oxygen species and also the formation of advanced glycation end products in diabetes subjects causing oxidative stress, all finally precipitating DNA damage and thus micronucleus formation.

The micronucleus test used in the current study for evaluating the genotoxic effect is simple and has an advantage of versatility.

The outcome of our study indicate that there might be an association between severity of periodontal disease and type 2 diabetes mellitus with the micronucleated cell frequency. Subjects with type 2 diabetes and chronic periodontitis should be provided with periodontal treatment, motivation for maintainence of oral hygiene and referral to medical professionals for control of diabetes and for further evaluation and care.

The observation obtained from our study is that the micronuclei frequency in subjects with chronic periodontitis with type 2 diabetes mellitus is higher than the micronuclei frequency in subjects with chronic periodontitis without diabetes mellitus and healthy group and the value is highly statistically significant.

According to the above observations, it can be concluded that, there is an association between periodontitis with well-controlled type 2 diabetes mellitus and DNA damage. Considering the evaluation of the periodontal clinical parameters, periodontal tissue destruction is more severe in subjects with type 2 diabetes mellitus. This is the major reason for periodontitis to be marked as the sixth complication in type 2 diabetes mellitus subjects. The outcome of this study indicate that score of micronucleus frequency may be considered as an essential biomarker of genotoxic damage that is DNA damage in subjects with type 2 diabetes mellitus and periodontitis. The two pathologies considered in the current study including type 2 diabetes mellitus and chronic periodontitis taking place simultaneously mayaccelerate the oxidative stress and inflammation promoting to increased DNA damage.

Hence, Periodontal disease and Type 2 diabetes mellitus could be a risk factor for genotoxic damage due to DNA injury. Therefore, it is extremely important to prevent the periodontal disease from occurring and to treat the existing chronic diseases like type 2 diabetes and periodontal disease as early as possible.