|Year : 2016 | Volume
| Issue : 4 | Page : 377-382
Effect of smoking on potential salivary markers of periodontal disease: A clinical and biochemical study
Narender Dev Gupta1, Neha Agrawal1, Namita Gupta2, Saif Khan1, Pritma Singh1
1 Department of Periodontics and Community Dentistry, Dr. Z. A. Dental College, AMU, Aligarh, Uttar Pradesh, India
2 Department of Periodontics, Pt. B. D. Sharma University of Health Sciences, Rohtak, Haryana, India
|Date of Web Publication||15-Dec-2016|
Department of Periodontics and Community Dentistry, Dr. Z. A. Dental College, AMU, Aligarh, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Introduction: Tobacco smoking exerts a harmful effect on the periodontal tissues manifested by periodontal pockets, attachment loss, and periodontal bone loss. Various factors contribute to the deleterious periodontal effects of smoking, including alteration of both microbial and host response factors. Moreover, smoking may exert effects throughout the cytokine network. Aims: The aim of this study was to evaluate the influence of smoking on periodontal biomarkers possibly related to the development of periodontitis including inflammatory mediators and pro-inflammatory cytokines in saliva. Materials and Methods: A total of sixty subjects aged 30–55 years were included in the study and divided into three groups: systemically and periodontally healthy individuals (Group 1), subjects with pocket probing depth (PPD) ≥5 mm and clinical attachment loss (CAL) of ≥2 mm (Group 2), and a subjects smoking (≥10 cigarettes a day) with periodontal parameters of Group 2 (Group 3). Periodontal parameters of PPD, CAL, gingival index (GI), and plaque index were measured using standard indices and criteria. Three milliliters of unstimulated saliva was taken, and salivary tumor necrosis factor-alpha (TNF-α) and matrix metalloproteinase-8 (MMP-8) were determined using ELISA technique. Results: The mean GI was lowest for Group 3, but the mean probing depth of Group 3 (4.93 ± 0.41) was highest. The mean TNF-α level of Group 3 was significantly different and higher as compared to Group 1 and Group 2 (24.32 ± 8.32 ng/ml vs. 6.43 ± 2.65 ng/ml, q = 16.14; P< 0.001). Similarly, the mean MMP-8 level of Group 3 (461.71 ± 58.01 ng/ml) was significantly different (P < 0.001) and higher as compared to Group 1 (192.96 ± 134.89 ng/ml) and Group 2 (347.83 ± 206.72 ng/ml). Both markers showed positive and significant correlation with their periodontal status. Conclusion: Our study clearly indicates a profound effect of smoking on salivary markers of periodontal disease (TNF-α and MMP-8) in chronic periodontitis subjects in comparison to healthy controls.
Keywords: Biomarker, chronic periodontitis, matrix metalloproteinase-8, smoking, tumor necrosis factor-alpha
|How to cite this article:|
Gupta ND, Agrawal N, Gupta N, Khan S, Singh P. Effect of smoking on potential salivary markers of periodontal disease: A clinical and biochemical study. J Indian Assoc Public Health Dent 2016;14:377-82
|How to cite this URL:|
Gupta ND, Agrawal N, Gupta N, Khan S, Singh P. Effect of smoking on potential salivary markers of periodontal disease: A clinical and biochemical study. J Indian Assoc Public Health Dent [serial online] 2016 [cited 2019 Dec 15];14:377-82. Available from: http://www.jiaphd.org/text.asp?2016/14/4/377/195846
| Introduction|| |
Periodontitis is an inflammatory disease of the periodontium which is characterized by a progressive destruction of the tissues supporting the tooth. It starts with a microbial infection, followed by a host-mediated destruction of periodontal tissues caused by hyperactivity of leukocytes and generation of cytokines, eicosanoids, and matrix metalloproteinases (MMPs).,, Inflammatory mediators that are released from cells in the inflamed periodontium during periodontitis include interleukin-1b (IL-1b), IL-6, IL-8, and tumor necrosis factor-alpha (TNF-a). Cytokines such as IL-1b and tumor necrosis factor-α induce and enhance the production of prostaglandin E2 (PGE2) and MMPs. These molecules mediate destruction of the extracellular matrix of gingiva and periodontal ligament as well as resorption of alveolar bone.
Smoking is a well-established risk factor for periodontitis. It has been associated with a two to eight-fold increase in the risk of attachment loss based on disease severity and history of smoking. Various factors contribute to the deleterious periodontal effects of smoking, including alteration of both microbial and host response factors. Some investigators noted higher prevalence or counts of certain organisms in smoker  while others observed no difference in the prevalence of subgingival bacteria. On the other hand, smoking also affects the human immune system and the cellular and humoral inflammatory system; moreover, smoking may exert effects throughout the cytokine network.
Saliva, an oral fluid derived from the major and minor salivary glands, has been used in the past few decades as a diagnostic fluid. It contains a highly complex mixture of substances and biomarkers that are used for diagnosing local and systemic diseases or monitoring the effect of treatment. Furthermore, collection of saliva is safe, noninvasive, and simple and can be collected repeatedly with minimum discomfort to the patient.
Periodontitis is the major cause of tooth loss in adults and is linked to systemic illnesses, such as cardiovascular disease and stroke. The development of rapid point-of-care chairside diagnostics has the potential for the early detection of periodontal infection and progression to identify incipient disease and reduce health-care costs. However, validation of effective diagnostics requires the identification and verification of biomarkers correlated with disease progression.
Clinically, smoking reduces the signs of gingivitis masking periodontal diseases, and thus, smokers have less observed signs of gingival inflammation, in adolescents as well as in adults, aggravating the diagnostics of periodontal disease. Further studies are needed to find out the effect of confounders such as smoking on inflammatory biomarkers of early periodontitis and its clinical relevance for using it as a screening tool. Therefore, this study was carried out with the aim to evaluate the influence of smoking on periodontal biomarkers possibly related to the development of periodontitis including inflammatory mediator and pro-inflammatory cytokines in saliva.
- To find out the level of MMP-8 and TNF-α in systemically and periodontally healthy individuals as controls and subjects with pocket probing depth (PPD) ≥5 mm and clinical attachment loss (CAL) of ≥2 mm and subjects smoking (≥10 cigarettes a day) with similar periodontal parameter as Group 2 as test groups
- To compare the level of MMP-8 and TNF-α among these three groups
- To find out the association between TNF-α, MMP-8, and periodontal status among three groups, respectively.
| Materials and Methods|| |
The study population consisted of 80 age- and sex-matched individuals in the age range of 30–60 years from the outpatient wing of the Department of Periodontology and Dental Public Health. Ethical clearance was obtained from the Institutional Ethical Review Board. This was a cross-sectional study conducted for 1 year from November 2013 to December 2014. Patients with <20 teeth, pregnant and lactating females, patients with systemic diseases, patients under medication, and patients who had undergone surgery in the past 6 months were excluded from the study. Patients with aggressive periodontitis and acute necrotizing ulcerative gingivitis were also excluded.
The sample size was calculated by expecting at least 5.0% impact with 5.0% margin of error and 80.0% power with 1:1 ratio. The minimum sample size required was 18 per group. Therefore, a sample size of 25 subjects was selected for each group. Three groups of patients were formed. The first group incorporated systemically and periodontally healthy nonsmokers. Nonsmoker systemically healthy controls with PPD ≥5 mm and clinical attachment level (CAL) ≥2 mm were included in Group 2. Current systemically healthy smokers currently smoking ≥10 cigarettes a day along with same periodontal parameters as of Group 2 were included in Group 3.
After obtaining the informed consent from the patient, demographic details, smoking status, and past dental and medical history were recorded in a pro forma. A complete intraoral examination including an assessment of PPD, CAL, gingival index (GI; Loe and Silness), plaque index (PI; Silness and Loe) using University of North Carolina-15 (UNC-15 probes Hu-friedy's USA) was performed by a single dentist. The dentist was calibrated regarding the diagnostic criteria through comprehensive written instructions, practice, and through discussing clinical cases.
Unstimulated saliva samples were obtained by expectoration from all the selected subjects. Participants were asked not to eat or drink for 2 h before the examination. The method described by Navazesh  was used to collect 3 ml of unstimulated whole saliva from each subject into sterile 5 ml saliva collecting tubes. Samples, which were stored at −801°C until use, were delivered to the laboratory for analyses. Salivary TNF-α and MMP-8 levels were determined for each subject using the Quantikine Human total TNF-α, Quantikine Human total MMP-8 immunoassay kits employing an ELISA technique, provided by R and D systems, Minneapolis, USA.
All data analyses were carried out using Statistical Package for Social Science (SPSS, Version 16) SPSS Inc. Released 2007. SPSS for Windows, Version 16.0. Chicago, SPSS Inc. for the descriptive analysis and statistical tests of significance. Analysis of variance test (ANOVA) was applied to find out the statistical significant difference between the three groups. The multiple comparison Tukey's post hoc test was used for additional exploration of difference among means to provide specific information, on which means are significantly different from others. Pearson correlation analysis was done to assess the association between TNF-α, MMP-8, and periodontal status, respectively. The level of significance was set at <0.05 for all tests (P < 0.05).
| Results|| |
The basic characteristics of groups are shown in [Table 1]. There was no statistically significant difference among the groups in relation to age and sex. Periodontal status of the groups was assessed using various indices. There was statistically significant difference among the groups in relation to GI, PI, clinical attachment level, and PPD (P < 0.05).
Comparing the mean TNF-α level of three groups, ANOVA test showed statistically significantly different TNF-α level among the groups (P < 0.001) [Table 2]. Tukey's test revealed that the mean TNF-α level of Group 3 (24.32 ± 8.32 ng/ml vs. 6.43 ± 2.65 ng/ml, P < 0.001) was significantly different and higher as compared to Group 1 and Group 2 (24.32 ± 8.32 ng/ml vs. 7.96 ± 4.70 ng/ml, P < 0.001) [Table 3].
|Table 2: Comparison of tumor necrosis factor-alpha level of study groups with control group|
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|Table 3: Mean difference of tumor necrosis factor-alpha level between the groups by Tukey's test|
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Comparing the mean MMP-8 level of three groups, ANOVA revealed significantly different MMP-8 level among the groups (P < 0.001) [Table 4]. Tukey's test revealed that the mean MMP-8 level of Group 3 (461.71 ± 58.01 ng/ml) was significantly different (P < 0.001) and higher as compared to Group 1 (192.96 ± 134.89 ng/ml) and Group 2 (347.83 ± 206.72 ng/ml) [Table 5]. There was statistically significant difference between Group 1 and Group 2, Group 1 and Group 3, and Group 2 and Group 3.
|Table 4: Comparison of matrix metalloproteinases-8 level of study groups with controls|
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|Table 5: Mean difference of matrix metalloproteinases-8 level between the groups by Tukey's test|
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TNF-α of all subjects (Group 1 + Group 2 + Group 3) showed positive and significant correlation with their periodontal status; PI (r = 0.52, P < 0.001), GI (r = 0.46, P < 0.001), PPD (r = 0.48, P < 0.001), and CAL (r = 0.42, P < 0.001). In all three groups, MMP-8 also showed positive and significant correlation with PI (r = 0.57, P < 0.001), GI (r = 0.49, P < 0.001), PPD (r = 0.63, P < 0.001), and CAL (r = 0.54, P < 0.001).
| Discussion|| |
Smoking is recognized as an important risk factor for oral diseases and especially for periodontal health. MMP-8 has been considered as a key marker in chronic periodontitis. MMP-8 are zinc-dependent endopeptidases, mainly produced by neutrophils, that are capable of degrading extracellular matrix proteins. MMP-8 is the major collagenolytic metalloproteinase in gingival tissue and oral fluids, and its elevated levels have been associated with the severity of periodontal inflammation and disease.
In addition to periodontitis, MMP-8 levels have been seen to be elevated in many diseases such as bronchiectasis, asthma, atherosclerosis, and oral cancer.
TNF-α, also known as cachectin and TNFSF1A, is the prototypic ligand of the TNF superfamily. It is a pleiotropic molecule that plays a central role in inflammation, immune system development, apoptosis, and lipid metabolism. TNF-α is a pro-inflammatory cytokine released by macrophages which is known for its substantial role in periodontitis mediated bone loss.
In this study, subjects with any acute and chronic systemic condition such as bronchiectasis, asthma, atherosclerosis, inflammatory bowel disease, and oral cancer were excluded as this condition leads to the rise of TNF-α and MMP-8 on their own, which can lead to a confounding effect in the study. There was no statistically significant difference between the age and gender distribution of the subjects among three groups eliminating the age and gender bias.
Unstimulated saliva was used in the present study as the collection is easy, noninvasive, and rapid. Its measurement requires no special equipment and expertise. Stimulated whole saliva is less suitable for diagnostic application because the foreign substances used to stimulate saliva secretion, resulting in a dilution in the concentration of proteins of interest.
Quantitative determination of salivary TNF-α and MMP-8 in the present study was done by the double-antibody sandwich ELISA method which is a very sensitive method for detecting TNF-α and MMP-8 in saliva. The minimum detectable dose of TNF-α and MMP-8 ranged from 0.01 to 0.06 ng/ml.
In the present study, the mean GI was lowest for Group 3 (1.68 ± 0.11) which is similar to the study conducted by Feldman et al. and Preber and Bergström  who showed that smokers with periodontal disease had less gingival bleeding when compared with nonsmokers. This may be explained by the fact that one of numerous tobacco smoke by-products, nicotine, exerts local vasoconstriction, reducing blood flow, edema, and acts to inhibit early signs of periodontal problems by decreasing gingival inflammation, redness, and bleeding.
Tobacco use is associated with increased pocket depths, loss of periodontal attachment, alveolar bone, and higher rate of tooth loss. It has also been suggested that reduced bleeding reflects an underlying disruption of the immune response which may account for the increased loss of clinical attachment and alveolar bone. These findings suggest a reduced reliance on the use of gingival bleeding as an indicator of gingival inflammation when assessing a smoker's periodontal health.
The mean probing depth of Group 3 (4.93 ± 0.41) was highest, which was in line with the other study, where it was stated that poor oral hygiene practices were significantly related to the development of periodontal pockets in cigarette smoker. Some studies indicate that tobacco use exacerbates periodontal disease because it alters the immune response to periodontal pathogens. Indeed, smokers exhibit increased numbers of peripheral blood mononuclear phagocytes which appear to be functionally compromised. Inadequate phagocyte activity could reduce the clearance of pathogens from the oral cavity and thereby facilitate the development of periodontal disease. Tobacco-exposed B- and T-lymphocytes exhibit reduced proliferative capacities which could limit the production of protective immunoglobulins against oral pathogens.
In the present study, higher TNF-α levels were observed in Group 2 (chronic periodontitis subjects) in comparison to Group 1 (controls) (7.96 ± 4.70 ng/ml vs. 6.43 ± 2.65 ng/ml). The results are in high conformity with the results obtained by Frodge et al. and Passoja et al. which showed elevated levels of TNF-α in subjects of chronic periodontitis albeit in saliva and serum as compared to controls, respectively. The mean TNF-α was also significantly elevated in Group 3 as compared to Group 1 (24.32 ± 8.32 ng/ml vs. 6.43 ± 2.65 ng/ml) and Group 2 (24.32 ± 8.32 ng/ml vs. 7.96 ± 4.70 ng/ml). This clearly proves that smoking too contributed to the difference found in salivary TNF-α as compared to the chronic periodontitis as seen when comparing Group 1 with Group 2 (6.43 ± 2.65 ng/ml vs. 7.96 ± 4.70 ng/ml). Hence, a clear rise of mean TNF-α can be attributed to smoking which is in concurrence with the results of Boström et al. in GCF, where smokers with chronic periodontitis had an increased TNF-α in comparison to nonsmokers.
Comparing rise of MMP-8 in Group 3 and Group 1 and Group 2 (461.71 ± 58.01 ng/ml vs. 192.96 ± 134.89 ng/ml), (461.71 ± 58.01 ng/ml vs. 347.83 ± 206.72 ng/ml) which shows a higher mean salivary MMP-8 in smokers as compared to controls and periodontitis patients, which is in consistency with the findings of study done by Visvanathan et al., which concluded that smoking has an impact on the periodontal status and mRNA expression of MMP-8. The mRNA expression of MMP-8 in chronic periodontitis smoker had higher expression of MMP-8 compared to chronic periodontitis nonsmokers and healthy nonsmokers. The results of the present study were in contrast to finding by Liede et al. who suggested that smoking may significantly lower both general proteolytic activity and MMP-8 level in saliva.
In the present study, TNF-α was found to be more sensitive biomarker than MMP-8 in chronic periodontitis among smoking patient. There was a three-fold increase in TNF-α level among smokers than nonsmokers which was higher than that of increase in the level of MMP-8 in smokers (around two-fold). The results of our study were in line with other studies, where it was found that both current and former smokers exhibited significantly higher levels of TNF-α in comparison to nonsmokers., Our results were in contrast with another study where it was demonstrated that cigarette smoking does not influence GCF contents of IL-6 and TNF-α. Moreover, TNF-α and MMP-8 of all subjects (Group 1 + Group 2 + Group 3) showed positive and significant correlation with their periodontal status.
Although the results obtained clearly implicated smoking to be major contributor to salivary TNF-α and MMP-8, long-term longitudinal studies in all the three groups are required for important generalizations regarding the role of TNF-α and MMP-8 as a valuable diagnostic biomarker. Furthermore, this study fails to correlate TNF-α and MMP-8 with duration of smoking in years.
Our study had some limitations. The number of participants enrolled in the study was relatively small to arrange in significant subgroups with respect to smoking habits (light, moderate, or heavy). For this reason, we could not evaluate the effect of low, moderate, or heavy smoking on salivary MMP-8 and TNF-α level. Furthermore, the smoking habits of our participants were self-reported, and measurements of cotinine in the saliva would be much more reliable.
In adults, MMP-8 and TNF-α have shown to be key biomarkers during early stages of periodontal diseases. Clinically, smoking reduces the signs of gingivitis (Kumar and Faizuddin, 2011) masking periodontal diseases, and thus, smokers have less observed signs of gingival inflammation, in adolescents as well as in adults, aggravating the diagnostics of periodontal disease. It is important that patients receive a proper periodontal diagnosing as part of their regular dental examination. Early diagnosis of periodontal disease with the use of theses biomarkers could enable a successful therapeutic outcome, by reduction of etiologic factors such as smoking and by establishing periodontal therapy and maintenance protocol. Further, this might prevent the recurrence and progression of the disease and reduce the incidence of tooth loss.
| Conclusion|| |
It was clearly observed that smoking had a profound effect on salivary markers of periodontal disease (TNF-α and MMP-8) in chronic periodontitis subjects in comparison to healthy controls. Increased levels of salivary TNF-α and MMP-8 were present in smokers with periodontitis suggesting that smoking interferes with the normal host defense mechanisms and stimulates destructive effects within the host. As smoking masks the early signs of periodontal disease in patients, the detection of these salivary biomarkers will help in early diagnosis and prompt treatment of the smokers with chronic periodontitis.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]