JICDRO is a UGC approved journal (Journal no. 63927)

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ORIGINAL RESEARCH
Year : 2021  |  Volume : 13  |  Issue : 2  |  Page : 118-123

Salivary profile of matrix metalloproteinase-8, tissue inhibitors of matrix metalloproteinase-1, myeloperoxidase, and nitrous oxide in smokers versus nonsmokers with chronic periodontitis


1 Department of Periodontology, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Nagpur, Maharashtra, India
2 Department of Implantology, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Nagpur, Maharashtra, India

Date of Submission24-Jan-2021
Date of Decision03-Jul-2021
Date of Acceptance26-Jul-2021
Date of Web Publication17-Jan-2022

Correspondence Address:
Dr. Deepti Gattani
Plot No. 3, Deonagar, Khamla Road, Nagpur - 440 015, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jicdro.jicdro_4_21

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   Abstract 


Background: Smoking is the second strongest modifiable risk factor for periodontal disease. Tissue destruction might result from matrix metalloproteinase (MMP)/tissue inhibitors of MMP (TIMP) imbalance in the diseased tissues. Myeloperoxidase (MPO) and inducible nitric oxide synthase are stored in and secreted from the primary granules of activated leukocytes which can migrate to the site of inflammation and release MMPs. Periodontal diseases are associated with systemic diseases and enhanced circulatory levels of MMPs and their regulators may form a link between the local and systemic conditions, but no definitive correlation has been established between them yet. Methods: Seventy-five males were divided into three groups: Group I – 25 healthy controls, Group II – 25 nonsmokers with chronic periodontitis (CP), and Group III – 25 smokers with CP. Saliva was collected from all the patients, and levels of MMP-8, TIMP-1, MPO, and NO were assessed using enzyme-linked immunosorbent assay kits. Results: TIMP-1 values were found to be significantly higher in nonsmokers with periodontitis than smokers with periodontitis (P < 0.01). Levels of MPO, MMP-8, and NO were significantly less in smokers with periodontitis than nonsmokers with periodontitis (P < 0.01). Conclusions: Our findings of elevated levels of MMP-8, MPO, and NO and reduced levels of TIMP-1 in smokers with periodontitis when compared to nonsmokers with periodontitis may be regarded as an indicator of the increased risk for local and systemic inflammation and of cigarette smoke oxidative stress in the pathogenesis of periodontal disease.

Keywords: Matrix metalloproteinase-8, myeloperoxidase, nitrous oxide, periodontitis, tissue inhibitors of matrix metalloproteinase-1


How to cite this article:
Gattani D, Sahu J, Kar N, Gattani R. Salivary profile of matrix metalloproteinase-8, tissue inhibitors of matrix metalloproteinase-1, myeloperoxidase, and nitrous oxide in smokers versus nonsmokers with chronic periodontitis. J Int Clin Dent Res Organ 2021;13:118-23

How to cite this URL:
Gattani D, Sahu J, Kar N, Gattani R. Salivary profile of matrix metalloproteinase-8, tissue inhibitors of matrix metalloproteinase-1, myeloperoxidase, and nitrous oxide in smokers versus nonsmokers with chronic periodontitis. J Int Clin Dent Res Organ [serial online] 2021 [cited 2022 May 28];13:118-23. Available from: https://www.jicdro.org/text.asp?2021/13/2/118/335875




   Introduction Top


Periodontal disease is characterized by chronic inflammation along with alveolar bone destruction and connective tissue breakdown.[1] Smoking is a substantial modifiable risk factor for periodontal disease. Nicotine, a toxic component of tobacco, deteriorates periodontal tissues. Smokers are almost four times more likely to have severe periodontitis than nonsmokers since they harbor a higher prevalence of potential periodontal pathogens, which impairs various aspects of innate and acquired immune responses.[2]

Under normal situations, microbial virulence factors are balanced by the host response. In periodontitis, this balance is impaired in favor of the microbial challenge.

Periodontopathogens can lead to an increased release of matrix metalloproteinases (MMPs) by inducing the host cells which can in turn trigger tissue destruction.[4] MMP-8 is one of the destructive MMPs and its excess activity can lead to periodontal destruction. Several studies indicate that MMP-8 is involved in periodontal destruction associated with smoking.[30] Recent studies have hinted that the presence of active MMP-8 (aMMP-8) seems to differentiate between gingivitis and periodontal health.[32]

Among the four tissue inhibitors of MMPs (TIMPs), TIMP-1 has been demonstrated to be the major inhibitor of MMPs in gingival tissues of patients with periodontal disease.[6]

Studies indicate that postperiodontal treatment nonsmokers with periodontitis had significantly higher MMP-8 and TIMP-1 expression than healthy nonsmokers, and smokers with periodontitis had significantly higher MMP-13 and TIMP-1 expressions than healthy smokers.[31]

Nitric oxide (NO) is a secondary messenger acting as a free radical.[11] NO is known for pronounced matrix degradation, especially via suppression of collagen and proteoglycan synthesis and unregulated metalloproteinase activity.[12]

Nicotine can generate a systemic inflammatory state via activation of PMNs. Increased neutrophil chemotaxis leads mainly to an increased secretion of myeloperoxidase (MPO) and NO from the granulocytes along with elastase and MMPs. Cigarette smoke acts as a major source of free radicals including both reactive oxygen species (ROS) and RNS such as NO.[13]

The above studies have indicated that periodontitis has a great significance on the levels of MMP-8 and TIMP-1 expressed in the saliva of a patient. Smoking confounds the salivary diagnostics of periodontitis and should be considered as a vital part of taking history of patients.[33] Therefore, this observational study was undertaken with the aim to evaluate the salivary levels of MMP-8, TIMP-1, MPO, and NO in smokers versus nonsmokers with chronic periodontitis (CP) as compared to periodontally healthy controls.


   Methods Top


Seventy-five male patients between the age groups of 20 and 75 years were included in the study. Written informed consent was taken from the patients and the study was approved from institutional ethical committee. The study was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013.

Each patient was asked to complete a questionnaire containing details about age, gender, oral hygiene habits, smoking history, medications used, medical history, and family history.

According to the classification of the 1999 American Academy of Periodontology Workshop, patients were diagnosed with generalized Chronic Periodontitis with attachment loss ≥5 mm at more than 30% of the sites in patients aged ≥35 years with radiographic evidence of bone loss were included.

Assessment of smoking status was performed according to the criteria established by the Centers for Disease Control and Prevention.[15]

Nonsmokers

  1. Never smokers (adults aged 18 years smoked <100 cigarettes in their lifetime or had never smoked)
  2. Former smokers (smoked at least 100 cigarettes in their lifetime but did not smoke at the time of interview).


Smokers

  • Current smokers (smoked at least 100 cigarettes in their lifetime and did not smoke at the time of interview)


Exclusion criteria

  1. Systemically unhealthy patients
  2. Previous history of periodontal therapy, or any medications in the previous 4 weeks.
  3. Patients with periapical pathology or undergoing orthodontic treatment were excluded from the study


The included patients were divided into three groups (n = 25):

  • Group I: Systemically and periodontally healthy nonsmoking individuals
  • Group II: Nonsmokers with CP
  • Group III: Smokers with CP.


Clinical periodontal examination

A single, calibrated examiner completed a full-mouth assessment of periodontal conditions, except for third molars, using a Williams graduated manual probe (Hu-Friedy, Chicago, IL, USA). The following parameters were assessed at six sites per tooth (mesiobuccal, mid-buccal, distobuccal, mesiolingual, mid-lingual, and distolingual): (1) plaque index (PI),[16] (2) bleeding on probing (BOP),[17] (3) probing depth (PD) (mm), and (4) clinical attachment level (CAL) (mm). The intra-examiner variability for PD and CAL was 0.32 and 0.36 mm, respectively.

Saliva collection and processing

Sampling was performed in the morning around 10 AM to avoid a possible variation in the marker concentrations. Patients' saliva was collected using a sterile glass funnel on weighed 10-ml sterile polypropylene container for 10 min. No oral stimuli were permitted for 120 min prior to collection to exclude any influence of mastication of food.

Saliva was collected from the seated patients as it flowed into the anterior floor of the mouth over the 10 min period and drained into a collection tube when necessary. Saliva samples were then frozen immediately at −80°C until analysis at which point the samples were thawed and cleared by centrifugation at 14,000 ×g for 5 min.

Biomarker analysis

Aliquots of each saliva sample were assayed by commercially available enzyme-linked immunosorbent assay kits (Boster Biological Technology Co., LTD, California, USA) to determine the levels of MMP-8, TIMP-1, and MPO according to the manufacturer's recommendations.

The NO levels were estimated by assaying the nitrite levels, a stable end product of the NO metabolism by using Griess reagent. The reaction mixture was then transferred into plastic cuvettes for measurement on a spectrophotometer, and readings were measured.

Statistical method

The Statistical Package for the Social Sciences software (IBM SPSS Statistics, version 21.0. Armonk, NY, USA: IBM Corp.) was used for data processing and data analysis with 95% confidence interval, probability of 0.05, and the sum of square of means equal to 13.32 when the standard deviation for the sample is 0.38. The power of the study was calculated by using the above values and estimated to be 0.96. This infers that a sample size of 25 was adequate to get significant values when all the three groups were compared with each other.

Distribution of clinical and biochemical variables was tested using Shapiro–Wilk test. All biochemical parameters were approximately normally distributed except for age. These numerical variables were log10 transformed and checked for normality. They were analyzed using one-way analysis of variance with subject groups as a factor. The significance of mean difference between the groups was done by a pairwise comparison using Tukey's honestly significant difference test. The pairwise comparison of means was done by one-way analysis of covariance after adjusting age for clinical and biochemical parameters. Spearman's partial rank correlation analysis was performed to check the association of clinical and biochemical parameters after adjusting for age. A two-sided (α =2) P < 0.05 was considered statistically significant.


   Results Top


The healthy control group exhibited significantly lower values in all clinical parameters. There was no significant difference (P > 0.05) for age, PI, and probing pocket depth between smokers with CP (Group III) and nonsmokers with CP (Group II).

The pairwise comparison of means showed that except for MMP-8, all clinical and biochemical parameters were statistically significant (P ≤ 0.01) among all subject groups after adjusting for age. After MMP-8 was adjusted for age, there was no significant difference between the healthy (Group I) and nonsmoker with CP (Group II) groups. TIMP-1 was found to be higher in healthy nonsmokers (Group I) followed by nonsmokers with CP (Group II) and smokers with CP (Group III). MPO and NO were higher in smokers with CP (Group III) when compared to healthy controls (Group I) and nonsmokers with CP (Group II). MMP-8 was significantly higher in smokers with CP (Group III) when compared to healthy nonsmokers (Group I) and nonsmokers with CP (Group II). PI and BOP were higher in nonsmokers with periodontitis (Group II) followed by smokers with CP (Group III) and healthy nonsmokers (Group I). PD and CAL were higher in smokers with CP (Group III) followed by nonsmokers with CP (Group II) and healthy nonsmokers (Group I).

Spearman's partial rank correlation analysis revealed a significant negative correlation between TIMP-1 and MPO (r = −0.665 and P ≤ 0.01), TIMP-1 and MMP-8 (r = −0.621 and P ≤ 0.01), TIMP-1 and NO (r = −0.558 and P ≤ 0.01), TIMP-1 and PD (r = −0.605 and P ≤ 0.01), and TIMP-1 and CAL (r = −0.828 and P ≤ 0.01). A significant positive correlation was noticed between MPO and NO (r = 0.764 and P ≤ 0.01), MPO and PD (r = 0.612 and P ≤ 0.01), and MPO and CAL (r = 0.854 and P ≤ 0.01). NO was positively correlated with MMP-8 (r = 0.826 and P ≤ 0.01) and PD (r = 0.55 and P ≤ 0.01) and CAL (r = 0.77 and P ≤ 0.01).


   Discussion Top


CP is characterized by irreversible alveolar bone loss and connective tissue attachment loss which ultimately results in loss of teeth.[18] Tobacco is the only legitimate drug that kills its emptor when used exactly as intended by the manufacturer. According to the Tobacco Control Policy Evaluation Project India, there are approximately 275 million tobacco users in India. Smokers harbor potential periodontal pathogens, and thus, it can debilitate diversified aspects of innate and acquired immune responses.

Our study evaluated the salivary concentrations of MMP-8, MPO, TIMP-1, and NO in smokers versus nonsmokers with CP as compared to periodontally healthy controls. We also tested to see whether they correlated with each other and with clinical parameters. Females were excluded to avoid hormone-induced microcirculatory changes.

All clinical parameters were found to be elevated in Groups II and III when compared to Group I (P < 0.01). This indicates that inflammatory conditions can elevate the clinical parameters. When a comparison was done between Groups II and III, PI and BOP were found to be elevated in nonsmokers with periodontitis (P < 0.01). This is in agreement with previous studies.[2],[19],[20] The PD and CALs were elevated in Group III when compared to Group I (P < 0.01). This can be attributed to the additional inflammatory load in cases of smoking and periodontitis. These results are also in agreement with previous studies.[2],[21] Our results indicate that smokers have an increased attachment loss and PD.

MMP-8 is a crucial mediator of the irreparable tissue damage associated with periodontitis.[5] It is stored in secondary granules of mature neutrophils as an inactive pro-enzyme. The first inflammatory cells that arrive at the site of infection are neutrophils. Upon stimulation, they secrete their granular contents which lead to increased levels of pro-inflammatory cytokines, i.e., interleukin-1 and tumor node factor-alpha, which in turn can lead to an increased release of MMP-8. Similarly, bacterial proteinase present in microbial plaque can activate MMP-8 release from PMNs.[22]

Tobacco-induced degranulation, increased pro-inflammatory cytokine burden, and alteration in microbial flora can lead to activation of protein kinase C, a messenger in the transcriptional regulation of MMP genes, and also the secondary messenger, cyclic adenosine monophosphate, which can also stimulate MMP expression and cause increased degranulation of neutrophils.[7],[22] This can lead to increased tissue destruction and aggravated clinical signs of inflammation. MMP-8 levels were found to be highest in Group III (1.62 ng/ml) followed by Group II (0.69 ng/ml) and least in Group I (0.33 ng/ml). A positive correlation was found between MMP-8 and PD (r = 0.494, P = 0.01) and MMP-8 and CAL (r = 0.77, P = 0.01). These findings suggest that as MMP-8 increases, inflammation also increases. These results are in agreement with previous studies.[20]

In our study, TIMP-1 levels were found to be least in smokers with CP (0.413 ng/ml). Nonsmokers with CP had a higher level (0.852 ng/ml) and the highest levels were found in the healthy group (1.589 ng/ml). Thus, as the inflammatory load increases, the imbalance between MMPs over TIMPs and lower levels of TIMPs are found. A negative correlation was found between TIMP-1 and clinical parameters, i.e., PD (r = −0.605, P = 0.01) and CAL (r = −0.828, P = 0.01). Thus, this indicates that reduced levels of TIMP-1 are associated with worsening of clinical parameters. These results are in accordance with the study performed by Reddy et al.[23]

MPO is an antimicrobial enzyme which is released in the extracellular environment following neutrophil stimulation.[22] MPO was also found to be elevated in Groups II (1.062 ng/ml) and III (2.082 ng/ml) when compared to Group I (0.620 ng/ml). Thus, it can be interpreted that in increased inflammation and in smokers, MPO levels are elevated. Similar results were found in other studies.[24] Enhanced serum levels of MPO indicate increased degranulation of specific granules of neutrophils.

In the present study, an increase in salivary NO levels was observed in smokers with CP (1.605 ng/ml) and nonsmokers with CP (0.867 ng/ml) when compared to the healthy group (0.473 ng/ml). Similar results were found in the previous studies.[25] MMP-8 was found to be positively correlated to MPO (r = 0.764, P = 0.01), NO (r = 0.826, P = 0.01), and other clinical parameters. Thus, an increase in MMP-8 can lead to an increase in inflammation.

MMP-8 was also positively correlated with MPO in our study. In an in vitro study, Saari et al.[26] suggested that the hypochlorous acid (HOCl) derived from the MPO can activate the direct oxidative pathway for MMP activation. Van Lint and Libert[27] suggested that MMP activation involves a cysteine switch mechanism involving the conversion of pro-form of MMP to an aMMP via modification of the cysteine thiol group. The pro-domain of MMP contains a cysteine thiol group which interacts with the Zn+2 ion. For the activation of MMP, this interaction has to be broken. MMP-8 is known to generate ROS via breakdown of collagen. According to Winterbourne,[28] MPO is maintained in its active form in the presence of excess H2O2, thus potentiating oxidative damage via MPO-dependent production of HOCl at the inflammatory site.

Wang et al.[8] demonstrated in their study that HOCl generated by MPO-H2O2-chloride system oxidizes the N-terminal cysteine of TIMP-1, thus controlling the interaction of TIMP-1 with MMP. Thus, increased MPO can lead to increased MMP-8 levels and decreased active TIMP-1 levels. Thus, a negative correlation of TIMP-1 with MPO (r = −0.665, P = 0.01) and MMP-8 (r = −0.612, P = 0.01) can be justified in our study.

MPO with NO was positively correlated in our study. Eiserich et al.[29] have suggested that the catalytic activity of MPO is biphasically modulated by NO. MPO and inducible NO synthase are both stored in and secreted from the primary granules of activated leukocytes. NO binds to both ferric and ferrous forms of MPO, generating low-spin six-coordinate complexes, i.e., MPO-Fe (III)·NO and MPO-Fe (II)·NO, respectively, thus inhibiting the catalytic activity of these enzymes by the release of NO from the complex.


   Conclusions Top


The present study demonstrated elevated salivary levels of MMP-8, MPO, and NO and reduced levels of TIMP-1 in smokers with CP as compared to nonsmokers with CP and nonsmokers without CP. This indicates that these analytes may be linked with the periodontal and smoking status of individuals. Within the limits of the study, it can be concluded that inflammation can increase the levels of MMP-8, MPO, and NO whereas downregulate TIMP-1.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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