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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 13  |  Issue : 6  |  Page : 1642-1645  

Comparative evaluation of salivary biomarker levels in e-Cigarette smokers and conventional smokers


1 Department of Periodontology and Implantology, Buddha Institute of Dental Science and Hospital, Patna, bihar, India
2 Department of Oral Medicine and Radiology, Buddha Institute of Dental Sciences and Hospital, Patna, bihar, India
3 Department of Oral Pathology and Microbiology, Manav Rachna Dental College, Faridabad, Haryana, India
4 Department of Periodontology and Implantalogy, Meghna Institute of Dental Sciences, Nizamabad, Telangana, India
5 Department of Oral Pathology and Microbiology, Buddha Institute of Dental Science and Hospital, Patna, India
6 Department of Oral Pathology and Microbiology, Swargiya Dadasaheb Kalmegh Smruti Dental College and Hospital, Nagpur, Maharashtra, India

Date of Submission15-May-2021
Date of Decision23-Jul-2021
Date of Acceptance25-Jul-2021
Date of Web Publication10-Nov-2021

Correspondence Address:
Madhuresh Kumar
Department of Oral Pathology and Microbiology, Buddha Institute of Dental Science and Hospital, Patna, Bihar
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpbs.jpbs_393_21

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   Abstract 


Background: The cigarette smoking and its effect on the inflammatory cytokine levels in the smoker's saliva depicted the influence of electronic cigarettes on oral cytokine levels in oral fluids are scarce in the literature. Objectives: The present trial was conducted to compare and determine the proinflammatory and anti-inflammatory cytokines in whole stimulated saliva samples of electronic cigarette smokers, conventional smokers, and participants with no smoke exposure. Materials and Methods: Sixty adult participants were divided into the following four groups of nonsmokers, current smokers, smokers smoking both conventional and e-cigarettes, and e-cigarette smokers. The saliva samples were assessed for Interleukins (IL-1B, 6, 8, 10, and IL-1RA), C-reactive protein (CRP), and tumor necrosis factor-alpha (TNF-α) using enzyme-linked immunosorbent assay. Plaque scores and Gingival Index, and body mass index were also calculated. Results: Statistically significant (P < 0.05) and remarkable relationship was seen in plaque scores and IL 1RA, 1 β, and 10 with the respective values as-0.285, 0.268, and 0.267. Regarding anti-inflammatory cytokines, CRP, IL-10, and IL-RA had the P-value of 0.073, 0.945, and 0.834 respectively. When these values were evaluated for proinflammatory cytokines, the P values were 0.0001, 0.019, 0.991, and 903 for TNF-α, IL-1 β, IL-6, and IL-8, respectively. These results were statistically significant for TNF-α (P = 0.001). Conclusion: Within its limitations, the present study concludes that smoking e-cigarettes whether solely or in combination with conventional smoking increases the levels of proinflammatory cytokines such as TNF-α and IL-1 β with decreased counter IL-1RA levels.

Keywords: E-cigarette, gingival index, inflammatory mediators, plaque scores, salivary biomarkers, smoking


How to cite this article:
Verma A, Anand K, Bhargava M, Kolluri A, Kumar M, Palve DH. Comparative evaluation of salivary biomarker levels in e-Cigarette smokers and conventional smokers. J Pharm Bioall Sci 2021;13, Suppl S2:1642-5

How to cite this URL:
Verma A, Anand K, Bhargava M, Kolluri A, Kumar M, Palve DH. Comparative evaluation of salivary biomarker levels in e-Cigarette smokers and conventional smokers. J Pharm Bioall Sci [serial online] 2021 [cited 2022 Aug 12];13, Suppl S2:1642-5. Available from: https://www.jpbsonline.org/text.asp?2021/13/6/1642/330126




   Introduction Top


Tobacco in any form is harmful to humans accounting for approximately 1 billion deaths caused in the 21st century making tobacco the most deadly product available. Tobacco in smoke form, especially cigarettes is the etiological, pathogenic, and distribution factor for various conditions and diseases such as oral carcinomas.[1]

Owing to the increasing evidence of health risks and deadly diseases caused by smoking, as an alternative to the health hazards, around a decade back, electronic cigarettes were introduced. These electronic cigarettes were marketed to avoid all the health hazards of conventional smoking. Electronic cigarettes were claimed to provide the same sensation as that of conventional smoking. However, more research is required to evaluate the safety of these electronic cigarettes in the long term, as different literature data depicts biases and interest conflicts in the study results.[2]

With the increasing usage of electronic cigarettes recently, the evaluation of health risks associated with e-cigarettes has become necessary. Smoking cigarettes release inflammatory mediators and cytokines by altering the host response. Smoking leads to activation of complex inflammatory processes and cascades resulting in cytokine production leading to various oral diseases including carcinoma and periodontal diseases.[3]

Saliva as the medium of diagnostic tool for assessing various components has recently been advocated owing to comparatively a noninvasive and safer medium than the blood collection. Comparative evaluation of biomarkers in saliva and blood to assess the health status has provided comparable results in both mediums. Cigarette smoking and its effect on the inflammatory cytokine levels in the smoker's saliva have been investigated previously. The studies depicting the influence of electronic cigarettes on oral cytokine levels in oral fluids are scarce in the literature.[4] Hence, the present trial was conducted to compare and determine the proinflammatory and anti-inflammatory cytokines in whole stimulated saliva samples of electronic cigarette smokers, conventional smokers, and participants with no smoke exposure.


   Materials and Methods Top


The study included a total of 60 participants including both males and females from the age group of 18–80 years. The study was conducted after obtaining ethical clearance from the concerned committee and informed consent from the participants who were then asked to fill a health form and the participants who agreed to participate in the study were finally included in this study.

The participants who provided the positive response to either conventional cigarette smoking, e-cigarette smoking, and never smoked were divided into the following four groups.

  • Group I: Nonsmokers
  • Group II: Current smokers
  • Group III: Smokers smoking both conventional and e-cigarettes
  • Group IV: e-cigarette smokers.


The participants were then asked to fill a small questionnaire concerning demographic data, overall health, periodontal status, e-cigarette use, and smoking history. The exclusion criteria for the study were participants with <20 teeth, edentulous participants, participants having periodontitis with generalized mobility, participants with prolonged disease and medication, extensive caries, tumors, and/or extensive restorations.

The whole saliva samples were collected from the participants after thorough rinsing followed by a 2-h refrain from eating, smoking, or drinking to avoid any sample contamination. After which, whole stimulated saliva was collected for 2 min using salivette which allows hygienic saliva collection with a plain cotton swab. The participants were asked to chew on the swab for 2 min. The saliva-soaked swab was then transferred to the salivette.[5]

The saliva sample was centrifuged to get a clear sample which was later analyzed. The aliquots were assessed for Interleukins (IL-1B, 6, 8, 10, and IL-1RA), C-reactive protein (CRP), and tumor necrosis factor-alpha (TNF-α) using enzyme-linked immunosorbent assay (ELISA) owing to its high sensitivity and specificity in cytokine detection. Plaque scores and Gingival Index[6] by Loe H were calculated for study participants to assess the periodontal health along with the gross examination of the oral surfaces to detect any deviation from the normal.

Body mass index (BMI) was also calculated for each study participant as suggested by the Center for Disease Control by the standard formula of weight (in Kg) and height (in m2): kg/m2. The collected data were subjected to the statistical evaluation and the results were formulated considering a P ≤ 0.05 as the level of significance.


   Results Top


The study included a total of 60 participants including both males and females from the age group of 18–80 years. The demographic characteristics of the study participants are listed in [Table 1]. The mean age of the study participants was 34.4 years. The study had 38 males (63.3%) and 22 females (36.6%). The group-wise distribution was 23.3% (n = 14), 26.6% (n = 16), 23.3% (n = 14), and 26.6% (n = 16), respectively, for the four study groups. Concerning diabetic status, 11.6% (n = 7) participants were diabetic and 88.3% (n = 53) participants were nondiabetics.
Table 1: Demographic characteristics of the study participants

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Various study variables were assessed for confounding factors affect the biomarkers in the saliva using the Pearson correlation coefficient and the results are described in [Table 2]. It was seen that there was no relation (significant) was seen in the biomarkers of the saliva and other parameters such as BMI, age, gingival health, and the number of teeth present in the study participants. However, a statistically significant (P < 0.05) and remarkable relationship were seen in plaque scores and IL 1RA, 1 β, and 10 with the respective values as − 0.285, 0.268, and 0.267, respectively. These findings were of particular interest as the plaque scores were found to have associations with anti-inflammatory cytokines such as IL-RA and IL-10, but also for single proinflammatory cytokine IL-1 β.
Table 2: Association between salivary biomarkers and the study variables

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The present study also assessed the inflammatory cytokines levels and the smoking status based on the four study groups. Concerning these parameters, the results are summarized in [Table 3]. The results were formulated using one-way analysis of variance. Regarding anti-inflammatory cytokines, CRP, IL-10, and IL-RA had the P-value of 0.073, 0.945, and 0.834 respectively. When these values were evaluated for proinflammatory cytokines, the P values were 0.0001, 0.019, 0.991, and 903 for TNF-α, IL-1 β, IL-6, and IL-8, respectively. These results were statistically significant for TNF-α (P = 0.001). The levels of IL-1 β were higher in participants who smoked e-cigarettes and who mixed smoked both conventional and e-cigarettes. The levels of TNF-α were lower with statistical significance in nonsmokers compared to the other three groups.
Table 3: Salivary inflammatory cytokines in the four different study groups

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   Discussion Top


The present trial was conducted to compare and determine the proinflammatory and anti-inflammatory cytokines in whole stimulated saliva samples of electronic cigarette smokers, conventional smokers, and participants with no smoke exposure. The present study utilized saliva as a medium to assess the biomarkers as saliva is being compared to blood and urine in biomarkers detection along with its noninvasive nature making saliva an excellent tool for diagnosis purposes. Hence, saliva is used as a tool for diagnosing various diseases including cardiovascular diseases, periodontal diseases, and kidney diseases as suggested by Olayanju et al.[7] in 2012.

The Food and Drug Administration[8] in 2011 has conducted experiments to detect the effects of e-cigarette smoking and found that they contain harmful nicotine-related substances. Furthermore, flavoring agents such as cinnamaldehyde can be toxic at the concentration which is used in the e-cigarettes

The present study chose the detection of IL-6, IL-8, IL-1 β, and TNFα as proinflammatory cytokines as these are believed to affect the host response and also acts as the tissue destruction mediators. In periodontal disease also, the levels of these proinflammatory cytokines are believed to be raised. This was confirmed in the findings of Goutoudi et al.[9] in 2004. IL-1 β was considered to be detected and not IL-1 α due to more potency of IL-1 β in depicting the bone catabolism.

On assessing the effect of confounding factors on the biomarkers in the saliva, it was seen that there was no relation (significant) was seen in the biomarkers of the saliva and other parameters such as BMI, age, gingival health, and the number of teeth present in the study participants. However, a statistically significant (P < 0.05) and remarkable relationship were seen in plaque scores and IL 1RA, 1 β, and 10 with the respective values as-0.285, 0.268, and 0.267. These findings were consistent with the studies of Sahibzada et al.[10] in 2017 and Brailo et al.[11] in 2006 where authors reported similar results in levels of these cytokines and inflammatory mediators in various oral carcinomas.

The present study also assessed the inflammatory cytokines levels and the smoking status based on the four study groups. Regarding anti-inflammatory cytokines, CRP, IL-10, and IL-RA the P value for the four study groups were 0.073, 0.945, and 0.834, respectively. When these values were evaluated for proinflammatory cytokines, the P values were 0.0001, 0.019, 0.991, and 903 for TNF-α, IL-1 β, IL-6, and IL-8, respectively. These results were statistically significant for TNF-α (P = 0.001). The levels of IL-1 β were higher in participants who smoked e-cigarettes and who mixed smoked both conventional and e-cigarettes. The levels of TNF-α were lower with statistical significance in nonsmokers compared to the other three groups. CRP is associated with various inflammatory conditions and proved as a reliable biomarker in various other studies as also suggested by Out et al.[12] in 2012 where CRP is proved to be a reliable biomarker for cardiovascular disease risk. IL-1RA which counteracts the function of IL-1 was seen comparatively lower in groups having e-cigarettes compared to other study groups which can depict low levels of IL-1 β in groups smoking e-cigarettes. These findings were in agreement with Nemzek et al.[13] in 2001 that depicted cytokine levels using ELISA.


   Conclusion Top


Within its limitations, the present study concludes that smoking e-cigarettes whether solely or in combination with conventional smoking increases the levels of proinflammatory cytokines such as TNF-α and IL-1 β with decreased counter IL-1RA levels. The study had few limitations including smaller sample size, short monitoring period, and geographical area biases. The study also neglected age, systemic medical history, and smoking duration. Hence, more longitudinal studies with larger sample size and longer monitoring period are required to reach a definitive conclusion

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Jha P. The hazards of smoking and the benefits of cessation: A critical summation of the epidemiological evidence in high-income countries. Elife 2020;9:49979.  Back to cited text no. 1
    
2.
Farsalinos KE, Polosa R. Safety evaluation and risk assessment of electronic cigarettes as tobacco cigarette substitutes: A systematic review. Ther Adv Drug Saf 2014;5:67-86.  Back to cited text no. 2
    
3.
Ebersole J, Samburova V, Son Y, Cappelli D, Demopoulos C, Capurro A, et al. Harmful chemicals emitted from electronic cigarettes and potential deleterious effects in the oral cavity. Tob Induc Dis 2020;18:41.  Back to cited text no. 3
    
4.
Yoshizawa JM, Schafer CA, Schafer JJ, Farrell JJ, Paster BJ, Wong DT. Salivary biomarkers: Toward future clinical and diagnostic utilities. Clin Microbiol Rev 2013;26:781-91.  Back to cited text no. 4
    
5.
Rabe A, Gesell Salazar M, Fuchs S, Kocher T, Völker U. Comparative analysis of Salivette® and paraffin gum preparations for establishment of a metaproteomics analysis pipeline for stimulated human saliva. J Oral Microbiol 2018;10:1428006.  Back to cited text no. 5
    
6.
Löe H. The gingival index, the plaque index and the retention index systems. J Periodontol 1967;38:l610-6.  Back to cited text no. 6
    
7.
Olayanju OA, Rahamon SK, Joseph IO, Arinola OG. Salivary immunoglobulin classes in Nigerian smokers with periodontitis. World J Biol Chem 2012;3:180-3.  Back to cited text no. 7
    
8.
Benowitz NL. Smokeless tobacco as a nicotine delivery device: Harm or harm reduction? Clin Pharmacol Ther 2011;90:491-3.  Back to cited text no. 8
    
9.
Goutoudi P, Diza E, Arvanitidou M. Effect of periodontal therapy on crevicular fluid interleukin-1beta and interleukin-10 levels in chronic periodontitis. J Dent 2004;32:511-20.  Back to cited text no. 9
    
10.
Sahibzada HA, Khurshid Z, Khan RS, Naseem M, Siddique KM, Mali M, et al. Salivary IL-8, IL-6 and TNF-α as potential diagnostic biomarkers for oral cancer. Diagnostics (Basel) 2017:9:7.  Back to cited text no. 10
    
11.
Brailo V, Vucićević-Boras V, Cekić-Arambasin A, Alajbeg IZ, Milenović A, Lukac J. The significance of salivary interleukin 6 and tumor necrosis factor alpha in patients with oral leukoplakia. Oral Oncol 2006;42:370-3.  Back to cited text no. 11
    
12.
Out D, Hall RJ, Granger DA, Page GG, Woods SJ. Assessing salivary C-reactive protein: Longitudinal associations with systemic inflammation and cardiovascular disease risk in women exposed to intimate partner violence. Brain Behav Immun 2012;26:543-51.  Back to cited text no. 12
    
13.
Nemzek JA, Siddiqui J, Remick DG. Development and optimization of cytokine ELISAs using commercial antibody pairs. J Immunol Methods 2001;255:149-57.  Back to cited text no. 13
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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