Comparative Evaluation of Salivary Biomarker Levels in e-Cigarette Smokers and Conventional Smokers.

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. Copyright:

INTRODUCTION

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

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

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

Characteristics	Percentage (n)
Mean age
Age range (years)	18-80
Gender (years)	34.4
Males	63.33 (38)
Females	36.6 (22)
Smoking group
Nonsmoker	23.3 (14)
Current smoker	26.6 (16)
Smoking both conventional and e-cigarette	23.3 (14)
e-cigarette smoker	26.6 (16)
Diabetic status
Diabetic	11.6 (7)
Nondiabetic	88.3 (53)

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

Salivary Biomarker	Gingivitis	Plaque	Number of teeth	BMI	Age
CRP	−0.038	−0.117	0.032	0.073	−0.001
IL-10	−0.019	0.267	−0.073	0.156	−0.122
IL-RA	−0.073	−0.285	−0.146	−0.053	0.114
TNF-α	0.030	0.175	0.116	0.178	−0.054
IL-1β	−0.168	0.268	0.093	0.148	−0.104
IL-6	−0.259	0.113	−0.025	0.222	0.004
IL-8	−0.001	−0.117	0.007	−0.167	0.044

BMI: Body mass index, IL: Interleukin, TNF-α: Tumor necrosis factor-alpha, CRP: C-reactive protein, Receptor antagonist

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

Salivary biomarker	Group I (nonsmokers)	Group II (current smokers)	Group III (mixed smokers)	Group IV (e-cigarette smokers)	P
Anti-inflammatory cytokines
CRP	3704.05±3482.64	7310.08±4526.14	6360.51±4034.25	7238.13±4863.69	0.073
IL-10	30.60±30.33	25.13±22.03	29.24±24.71	26.42±24.82	0.945
IL-RA	245.17±92.34	247.56±103.48	243.40±116.24	217.37±82.11	0.834
Pro-inflammatory cytokines
TNF-α	11.34±3.17	32.40±11.77	27.53±11.67	30.39±5.67	0.0001
IL-1β	1.41±1.52	1.37±1.66	1.19±1.46	2.82±1.53	0.019
IL-6	2.02±1.69	1.79±1.59	1.63±1.50	1.66±1.46	0.991
IL-8	1.19±0.59	1.26±0.85	1.21±0.72	1.25±0.78	0.903

IL: Interleukin, TNF-α: Tumor necrosis factor-alpha, CRP: C-reactive protein, Receptor antagonist

DISCUSSION

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

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.