Correlation of salivary biomarkers and dental caries in children exposed to passive smoking

Introduction

A healthy oral cavity plays a major role in the child’s life due to its impact on normal nutritional intake, language acquisition, and psychological behaviour. A person cannot live a healthy life until his oral cavity is free of infection like gingivitis, halitosis, periodontitis and dental caries which are not uncommon to human.1 The innate immunity system of oral cavity provides a rapid, non-specific first line of defence against colonization of pathogenic micro-organisms causing oral diseases.2

The components of innate immunity include the barrier function of the skin, reduced pH of the stomach, sweeping motion of the cilia and chemical defences which includes host defence peptides. These genes encoded defence molecules are commonly known as antimicrobial peptides (AMP’S).3 Anti-microbial peptides play an important role in wound healing and in the maintenance of the tissue health, particularly in environments, like the oral cavity, colonized by a microbial plethora.4 Amongst the antimicrobial peptides found in the oral environment are a- and b-defensins, LL-37 antimicrobial peptide and histatins.5 Besides their direct bactericidal activity, these peptides have other distinct and overlapping properties, such as chemotactic activity or induction of cytokine release.6 These peptides have been reported to function as antimicrobial agents against Gram-negative and Gram-positive bacteria, fungi and viruses.7

The LL-37 antimicrobial peptide is the proteolytically processed extracellular form of human cationic antimicrobial protein of 18 kDa (hCAP-18), the only known member of cathelicidins in humans that are found in the secondary granules of neutrophils and various other cells.8 LL-37 is present in the pulmonary and the digestive system and it has also been detected in plasma, sweat, skin, and human milk.9 Regarding the oral cavity, LL-37 has been detected in saliva, whilst the peptide itself or its mRNA or both have been detected in salivary glands,10 in lingual epithelium and palatal mucosa.11 Following inflammatory stimulation, LL-37 is released at the inflamed sites mainly by neutrophils that migrate through the junctional epithelium.12 This pattern of expression implies a possible protective role of the peptide on both hard and soft oral tissues.13 The antimicrobial peptides present in saliva have shown to have a broad antimicrobial activity against cariogenic bacteria by destroying their cell membranes which, in turn, can be correlated to resistance to caries.14

However, Long term exposure to passive smoking leads to the reduction of cathelicidins.15 Passive exposure mainly consists of the smoke released from the burning end of a smoldering cigarette, pipe, or cigar ("side-stream smoke," 85%) and, to a lesser extent, the smoke exhaled from the lungs of an active smoker nearby ("mainstream smoke,” 15%). There are more than 4000 chemicals present in passive smoking, and more than 250 of these are known to be carcinogenic or toxic in some way or the other.16

Exposure to passive smoke begins as early as prenatal life due to parental smoking.17 It is also seen that maternal smoking during pregnancy alters foetal blood flow and protein metabolism18 and exposes the growing foetus to chemical toxins like nicotine. Other possible sources of PS include third-hand smoke exposure in household dust and interior surfaces, or increased bacterial load exposure of a smoking parent19 or caretaker. Also, children are comparatively more vulnerable to second-hand smoke effects because of higher breathing rates per body weight, immature lungs and more lung surface area when compared with adults. Furthermore, infants and children are generally not capable of managing their environment & consequently unable to perform action to escape from SHS exposure because of low-socioeconomic status, under educated parents and small house20 which constrains parents to smoke inside the house.

SHS affects both general and oral health. Passive smoking impedes dental development through numerous mechanisms such as, interference with reciprocal induction of oral ectomesenchymal tissues, interference with tooth mineralization owing to oxidative stress and nutritional deficiency caused by unfavourable effect of SHS on appetite.21 Also, Enamel hypoplasia in primary and permanent dentition, poor gingival attachment of teeth and supporting structures and dental caries in the primary dentition is concomitant to SHS exposure in children.22 Nicotine furthermore boosts proliferation of cariogenic bacteria like mutans Streptococci in smoking mother’s oral cavity of which gets transferred to their infants and predisposes to dental caries in children.23 SHS exposure also predisposes children to infections through immune system suppression or modulation such as lower salivary IgA & IgG levels.

Even though dental caries has been considered as a Global Pandemic, there is rise in prevalence in the developing countries due to globalization in the urban region and in contrast lack of knowledge regarding the possible etiological factors in the rural region. India being the second largest consumer and producer of tobacco, increases the risk of dental caries among the children exposed to second hand smoke.24 Hence, it is it biologically plausible that passive smoking could cause caries, particularly in childhood due to the suppression of immune system thus, reducing the salivary AMP. Also, due to lack of literature, this study was designed to assess the correlation between passive smoking and dental caries in exposed and unexposed children. Possible correlation to age, gender, salivary flow rate & dental caries experience was determined in the phase 1 of the study25 and assessment of salivary LL-37, salivary cotinine levels and possible correlation of salivary biomarkers to dental caries experience were asses in the current phase 2 study.

Materials and Methods

Results

A total of 120 participants, participated in the present case-control study after filling the given questionnaire and thorough clinical examination. Among them, the subjects were divided into PE and UE groups.

Discussion

Since decades, smoking is known as a potential risk factor and a major preventable cause of morbidity and mortality. SHS not only affects the general health of the child who is exposed to tobacco smoke, but also studies by Hong et al 26 and Lowe et al.26 have proposed a positive association between SHS exposure and oral health. Among the various ill effects on oral health, cigarette smoke has known to have detrimental effects on salivary immune defence mechanisms, thereby reducing the expression of salivary AMP’s (ll-37) which is known to have bactericidal property against the cariogenic microorganisms.

Goncalves et al27 and Hagiwara et al28 have shown that both innate immunity and adaptive immunity are susceptible to cigarette smoke, which interrupts immunological homeostasis, causes various diseases and paradoxical effects on immune cells. However, very limited literature has been available which shows the possible correlation between passive smoking and dental caries in children. Hence, our study was designed to assess the correlation between passive smoking and dental caries in exposed and unexposed children and possible correlation to salivary flow rate, salivary AMP concentration and salivary cotinine levels were determined.

Phase I of our study25 results showed that there was a dose-response relationship between levels of exposure to tobacco use and dental caries in children which was in accordance with various authors. However, the limitation was validation of SHS exposure was which was obtained by questionnaire reports and was not performed by measurements of biomarkers. Phase 2 of our study included quantitative assessment of salivary biomarkers and correlation of the same with dental caries experience.

The most sensitive way to assess exposure is by measurement of biomarkers in body fluids. Various procedures have been explained for assessment of biomarkers, among which measurement in saliva becomes a non-invasive, easy and well tolerated collection procedure when multiple samples are required.29 A widely used biomarker is cotinine, which is Nicotine’s major metabolite. It has a longer half-life and is considered a reliable biomarker when screening for passive exposure to tobacco use. It is less prone to instabilities and can be easily measured in body fluids.30 Cotinine estimation from body fluids provide an estimation of recent exposure to tobacco products but not the duration of exposure.31 The cotinine levels are found to be significantly higher in unstimulated than in stimulated saliva. In saliva, values between 1 ng/mL and 30 ng/mL may be associated with light smoking or passive exposure, and levels in active smokers typically reach 100 ng/mL or more.32

Studies by Aligne et al.33 and Avçar et al.34 have used cotinine levels to quantify passive exposure to tobacco use. Also, study by Ipseeta et al.35 assessed the Association of passive smoking with dental caries and salivary biomarkers among 5-10 year children which showed that the mean cotinine level was directly proportional to DMFS, and smoking exposure. Similarly, our study results showed that salivary cotinine levels were higher in PE group (mean Sal. Cotinine level 3.80±0.32) when compared to UE group (mean Sal. Cotinine level 2.18±0.26). Intragroup comparison from the study also revealed that, the mean salivary cotinine was greater in children with dental caries than in children without caries in both PE & UE groups. The results of the present study showed that the Sal. Cotinine levels in participants who had single smoker at home was lesser (3.73±0.27), whereas Sal. Cotinine levels was (4.060±0.39) in the participants had two smokers at home which is in line with a previous study conducted in England36 that compared the cotinine concentration in children who live in smoke-free homes, according to the smoking status of their parents, showing that cotinine concentration was high among children of smoking parents (nonsmoking parent GM: 0.22 ng/ml; one smoking parent GM: 2.37 ng/ml; two smoking parents GM: 4.71 ng/ml).

Winkelstein et al,37 assessed cotinine concentration in individuals who had parents who smoked outside versus inside the house. He found reduced cotinine levels among smoking practices done outdoor thus, decreasing children's exposure to ETS. Similarly, in line with the above mentioned study, our results showed that, in case of the participants whose parents smoked only inside mean Sal. Cotinine levels was higher (3.940) followed by inside & outside (3.86±0.35), based on the season (3.67±0.22) and (3.52±0.08) in houses where they smoked only outside. Also, the mean Sal. Cotinine levels was highest in the houses, where there were no rules with respect to smoking (3.87±0.3) than in the houses where smoking was permitted in some places (3.55±0.13). The mean Sal. Cotinine levels was directly proportional to no of cigarettes/day 3.63±0.23 (10 cigarettes/day) and duration of exposure. i.e. 3.48±0.04 (3 years). In accordance to our results, Yoko Guto et al.38 also, observed a significant dose-response relationship for pack and years of smoking by all family members, exposure to smoking after childbirth and cotinine levels which may have an effect on the development of dental caries. Blackford, et al.39 studied the quantitative relationship between number of cigarettes consumed and level of salivary cotinine, a biomarker of nicotine dose in China, Brazil, Mexico and Poland. In addition, increased cotinine concentration in children has been associated with socioeconomic factors and education status of the parents. Dell’Orco et al. and Cook et al.40 have showed that cotinine increases with decrease in the social class. Other studies conducted by Castelino et al.41 and Gilman et al.42 have illustrated that the number of pack-years smoked was higher among individuals with less than high school education.

According to studies conducted by Hitchman et al.43 and Harper et al.44 smoking rates are higher among low socioeconomic status groups. We can substantiate our results similar to them as in our study it was seen that salivary cotinine levels were greater in under educated parents (3.9±0.32) and parents from low socio-economic status (4.23±0.31).

Across the literature, various biomarkers have been studied which are the potential predictors of SHS. However, not much data exists between the correlation of passive smoking, so in our current study along with salivary cotinine assessment, salivary LL-37 levels were also assessed.

Sotrio Davidopoulo et al45 have showed that significantly lower concentrations of LL-37 were found in children with high caries activity, compared to caries free children or to children with low to moderate caries activity. Similarly, study by Karsiyaka et al46 have showed the mean salivary LL-37 concentration of PS-exposed children was significantly lower than that of PS-unexposed children. In accordance to the above mentioned studies, our results have also revealed that the mean salivary LL-37 levels were significantly lesser (114.726±42.71) in PE in comparison to UE group (158.67±78.12). Also, intragroup comparison has shown that expression of LL-37 is greater in children without dental caries as opposed to the children with dental caries. Comparison of Sal.LL-37 levels based on the smoking related characteristics in PE group have shown that, the Sal.LL-37 levels in participants who had single smoker at home was high (123.08±39.26), when compared to the Sal.LL-37 levels in the participants had two smokers at home (84.53±42.43). In case of the participants whose parents smoked only inside mean Sal.LL-37 levels was least (108.39) followed by inside & outside (102.860±41.60), based on the season (140.92±32.02) and (159.37±14.22) in houses where they smoked only outside. Also, the mean Sal.LL-37 levels was least in the houses, where there were no rules with respect to smoking (105.72±42.52) than in the houses where smoking was permitted in some places (152.90±17.10) The mean Sal.LL-37 levels was in-directly proportional to no of cigarettes/day 139.96±27.93 (10 cigarettes/day) and duration of exposure. i.e 153.70±10.21 (3 years).

Our study shows that there is strong negative correlation between Sal. Cotinine levels and Sal.LL-37 levels and very strong negative correlation between Sal.LL-37 and dental caries, showing statistically significant difference, similar to the study by Takeuchi et al and Hendek et al.47

Exposure to tobacco smoke, contains numerous chemical toxins which might predispose children to infection through suppression or modulation of the immune system.48 Numabae et al49 showed that the phagocytic activity of salivary PMN’s intensifies after exposure to smoking, where as an another invitro study50 demonstrated that nicotine inhibited phagocytic activity, which could be the plausible reason for reduced LL-37 levels. In normal condition soon after the colonization of the cariogenic pathogens, they are identified by pathogen recognition receptor of the oral cells, which signals for the secretion of LL-37. Once the LL-37 reaches the site, it binds onto the bacterial cell wall and causes cellular lysis by carpet or barrel sieve model, which maintains the tooth in healthy condition. Whereas, in case of passive smoke exposed individuals, there will reduced Salivary LL-37 concentration, which alters the protective and antibacterial effects of LL-37 increasing the risk of dental caries.51

Multitude of mechanisms have been described by the various authors, regarding the association of passive smoking on dental caries in children,52, 53, 54 yet epidemiological shows inconsistent results because of unknown factors related to passive smoking which may have confounded the observed relationship. However, study results have concluded that, the mean DMF score and salivary cotinine levels were higher in PE group when compared to UE group, but the salivary LL-37 levels were low in PE group over the UE, showing a statistical difference between both the groups. Hence, it is plausible from our results that, passive smoking was positively associated with the prevalence of dental caries, due to the suppression of Salivary AMP, which predisposes to dental caries.

Smoking is injurious to health and its contribution to oral cancer is well known. However, not much is known regarding its relationship to dental caries. The annual world no Tobacco Day (31st May) is an opportunity to raise awareness on the harmful effects of tobacco use.55 Yet, lesser is discussed about the ill effects of passive smoking on oral health and its association to Dental caries.

The study has some major strengths as cotinine concentration was measured in the PS subjects after reported directly by the parents, which reduced the bias among selection of PE & UE groups.  This study also shows long-term impact of smoking in household on their children which serves as an important motivating factor for their parents to quit smoking and the study also highlights PS as health hazard which is not known by many people in study setting and hence, serves as an important enlightening message. Frequent interactions with identified smoking subjects and their family members can be made by dentists and primary healthcare workers to have long-term assessment data on the impact of passive smoke so that more appropriate ways for creating awareness on subject can be made. The establishment of systematic oral healthcare program for community is needed which may serve as a role model for promotion of best practice of oral health habits. Routine biochemical assessment of tobacco smoke exposure and intensified smoking education and prevention activities in school is essential for more effective interventions to prevent adverse effects of Passive Smoking.56, 57 These recommendations can bring about a major change in oral health status of the children affected by passive smoke.

Conclusion

Dental caries is a common public health problem among children due to multi factorial aetiological agents. Prevalence of Dental Cancer among smokers are 20 in 1,00,000, 158 but, possibilities of acquiring dental caries due to the ill effects of environmental smoke is higher. History recording about parental smoking practices have also been neglected during routine case history documentation, which could be one of the confounding etiological factor in caries etiology. Our results revealed that passive exposure to tobacco smoke had independent relationship with dental caries in children, due to the suppression of Salivary AMP, which predisposes to dental caries. Hence, it is the need of hour to document parental smoking practices and create guidelines by the necessary authorities to ensure that the ill effects of passive smoking reaches out to the masses.

Source of Funding

None.

Conflict of Interest

The authors declare no conflict of interest.