Salivary melatonin is depleted in patients with dental caries due to the elevated oxidative stress

Abstract

Objectives

To determine salivary melatonin and malondialdehyde levels in individuals with and without dental caries.

Materials and methods

Saliva samples were collected in a fasting state from patients with active dental caries (n ​= ​16) and patients without dental caries (n ​= ​16). Melatonin was measured in the samples using a commercially available ELISA kit and malondialdehyde was assayed using a standardized spectrophotometric method.

Results

The salivary melatonin levels were significantly lower (p ​< ​0.01) in patients with active dental caries than patients without dental caries, while the salivary malondialdehyde values were significantly higher (p ​< ​0.01) in patients with active dental caries than patients without dental caries. The binary logistic regression analysis revealed a negative correlation (−0.513) between the salivary melatonin and malondialdehyde levels which was statistically significant (p ​< ​0.042) in the patient group with active dental caries, while no such relationship could be demonstrated in the patient group without dental caries.

Conclusion

Melatonin depletion and augmented malondialdehyde levels potentially indicate that the endogenous melatonin has been utilized to counter the oxidative stress-induced during the initiation and progression of dental caries. Further research could explore the potential use of exogenous melatonin supplementation as a preventive and therapeutic measure for dental caries.

Highlights

• •CA group had a significantly lower salivary melatonin level than the CF group.
• •CA group had a significantly higher salivary malondialdehyde level than the CF group.
• •CA group had a negative correlation between melatonin and malondialdehyde levels.

1. Introduction

Dental caries (DC) is a bacteria driven, chronic, and dynamic process occurring due to an imbalance between the tooth mineral and the pH of the oral cavity, resulting in a demineralization process leading to loss of tooth substance.¹ Dental caries is a pathological state which has been described by caries researchers as multifactorial. Out of the numerous theories proposed to understand the pathogenesis of dental caries, the systemic theory sheds light on the role of inflammation and oxidative stress in the onset and progression of dental caries.² In this connection, some studies have demonstrated jeopardized antioxidant status in the saliva of individuals with high caries indices.³

Concerning antioxidants in saliva, melatonin has been researched for over a decade and a half. This indoleamine is synthesized and secreted by the pineal cells⁴ and various other vital organs of the body.5, 6, 7 It is noteworthy that melatonin performs a spectrum of functions in the human body like circadian rhythm regulation⁸ mitigation of oxidative stress⁹ control of inflammation,¹⁰ carcinogenesis¹¹ and regulation of bone homeostasis.¹² Melatonin has been described as one of the most potent antioxidants in the human body that could destroy several reactive oxygen and nitrogen species. The effects of melatonin on the lethal hydroxyl radical¹³ peroxynitrite,¹⁴ and many others deserve attention at this juncture. Melatonin has also been found to mitigate the effects of products of oxidative damage. In this connection, the effects of melatonin on malondialdehyde have not been explored adequately. Malondialdehyde is a product of the oxidative degradation of lipids. It is a reactive electrophile species that causes toxic stress in human cells. Since malondialdehyde is a stable product it has been employed as a marker of oxidative stress and has been widely assayed in human clinical experiments.¹⁵ Measurement of malondialdehyde is also an easy and effective method of assessing oxidative damage to a tissue and has been frequently performed in free radical research.

Depleted salivary melatonin levels have been demonstrated in oral inflammatory conditions of microbial etiology like gingivitis, chronic periodontitis,¹⁶ and aggressive periodontitis.¹⁷ So far, no human study has assessed melatonin levels in the saliva of individuals with and without dental caries. Hence the present case-control study was undertaken to measure the levels of salivary melatonin and malondialdehyde in caries-free and caries active individuals to understand the role of melatonin and its correlation with oxidative stress in the tooth carious process.

2. Materials and methods

Study center: The present study was performed in the Department of Conservative Dentistry and Endodontics, Faculty of Dental Sciences, Sri Ramachandra Institute of Higher Education and Research, Chennai, India.

Sample size: 80 patients who reported to the Department were assessed for eligibility. A sample size of 32 subjects comprising of 16 participants in each group was estimated based on the true probability of exposure for rejection of the null hypothesis with a type 1 error of 5 ​% and a power of 90 ​%. A total of 32 age and gender-matched participants were included in the present in-vivo observational case-control study.

Ethical approval: Approval for the research was obtained from the Institutional Ethical Committee of Sri Ramachandra Institute of Higher Education and Research (SRIHER), Chennai- [Ref No: CSP/18/AUG/72/325].

Study group: The study comprised of 2 groups, patients with dental caries (CA-caries active) and patients without dental caries (CF-caries free).

Inclusion criteria: Each group comprised of 16 participants with an age range of 20–35 years. All the participants included in the study were free of any local and systemic diseases, periodontally healthy, and had a full complement of natural teeth.

Exclusion criteria: Missing teeth, restored teeth, periodontal disease and any other oral inflammatory, premalignant and malignant disorders that could affect melatonin, patients who were on caries prevention programs, smoking, alcoholism, and pan chewing habit, history of consumption of medications such as anti-inflammatory drugs, antibiotics, antioxidants, vitamin and or melatonin supplements in the last six months and all other systemic diseases and sleep disorders that could influence the levels of melatonin.

Patient consent: Written consent was obtained from all volunteers following The Declaration of Helsinki and the Strobe guidelines.

Blinding: A detailed oral and dental examination was performed by a single interviewer who was blinded about the salivary assays. The laboratory personnel assessing the salivary melatonin and malondialdehyde levels was blinded to the designation of the study groups.

Dental caries assessment: Based on the examination, patients with active dental caries were recruited with a DMFT score of greater than 10 while patients without dental caries had a DMFT score of 0. According to the DMFT scoring criteria,¹⁸ the number of decayed, missing, and restored teeth was noted and the composite score is presented as a numerical value termed the DMFT score. In the present study patients with restored and missing teeth were not included as the DMFT values would be influenced by those parameters also. Also all the patients chosen in the study had caries that involved enamel and dentin and required restoration as ascertained by radiographs but no teeth with caries involving the pulp. This was established as a uniform criteria to standardize dental caries severity and subtype.

Salivary sample collection: Unstimulated whole saliva was sampled from the volunteers. Subjects were advised to abstain from food intake and oral hygiene measures 12 ​h before sample collection. The samples were collected between 8 a.m. and 9 a.m. in a fasting state.¹⁹ During saliva sampling, the participants were seated upright in a chair in a room with low-intensity lighting, since light can alter melatonin secretion.¹⁶ 5 ​ml of pooled saliva was collected by aspiration method and transferred into 10 ​ml sterile centrifuge tubes. The samples were processed by centrifugation and stored at −20° centigrade until further analysis.

Salivary melatonin estimation: For the estimation of melatonin levels in the samples, a commercially available Human melatonin assay kit (Elab biosciences, USA Human MT ELISA kit) was used. Each sample was duplicated for the procedure. The manufacturer's protocol was followed. The optical density was measured with a spectrophotometer at 450 ​nm within 15 ​min after pipetting of the stop solution.

Salivary malondialdehyde estimation: Malondialdehyde levels in the samples were measured by estimating the Thiobarbituric acid reactive substances (TBARS) using a standardized protocol.²⁰

Statistical analysis: The results were subjected to statistical analysis with IBM SPSS Statistics, Version 22 (Armonk, NY: IBM Corp). Mean with standard deviation were provided for descriptive statistics. To analyze the significant difference between the bivariate samples in the 2 study groups, the unpaired sample t-test was used. Binary logistic regression analysis was done to evaluate the association between the malondialdehyde and melatonin in CA and CF patients. The level of high significance was set at a p-value of <0.01 while a p-value of<0.05 was regarded as significant.

3. Results

Age and gender of study groups: CA patients comprised of 8 males and 8 females while CF patients also had 8 male and 8 female participants. It was found that there was no significant age difference between the groups (p ​> ​0.05) indicating good age matching.

Salivary melatonin and malondialdehyde levels: The melatonin levels were significantly higher in CF when compared to CA (p ​< ​0.01) while the malondialdehyde values were significantly higher in CA compared to CF (p ​< ​0.01). The mean age, salivary melatonin, and malondialdehyde values are represented in Table 1. The salivary melatonin and malondialdehyde levels are also depicted as line graphs in Fig. 1, Fig. 2.

Table 1.

Mean age, salivary melatonin, and malondialdehyde values in the study groups.

	Groups	N	Mean	SD	P-value
Mean age in years	CA	16	28.1	1.5	p ​> ​0.05 (NS)
	CF	16	28.0	1.5
Mean salivary melatonin levels in picograms/ml	CA	16	2.80	0.96	P ​< ​0.01∗∗
	CF	16	14.90	3.27
Mean salivary malondialdehyde levels in micromoles/ml	CA	16	5.91	0.70	P ​< ​0.01∗∗
	CF	16	2.35	0.26

Footnote: CA denotes caries active group; CF denotes caries-free group; N denotes the number of patients; SD denotes standard deviation; NS denotes not significant; ∗∗ denotes highly significant.

Fig. 1.

Salivary melatonin and malondialdehyde (MDA) levels in caries active group. Salivary melatonin levels are presented in picograms per millilitre (pg/mL). Salivary malondialdehyde levels are presented in micromole per millilitre (μmol/mL).

Fig. 2.

Salivary melatonin and malondialdehyde (MDA) levels in the caries-free group. Salivary melatonin levels are presented in picograms per millilitre (pg/mL). Salivary malondialdehyde levels are presented in micromole per millilitre (μmol/mL).

Correlation between salivary melatonin and malondialdehyde levels: A binary logistic regression analysis was done for the evaluation of the interrelationship between the melatonin and malondialdehyde in both the study groups. Based on this a logistic regression model was constructed after classifying study participants into CF and CA groups. A value of 0 was given for CF subjects while 1 was given for CA subjects. The beta values, standard error, wald values, and p values that resulted were analyzed and presented in Table 2. The analysis revealed that there was a negative correlation (−0.513) between the salivary melatonin and malondialdehyde levels which was statistically significant (p ​< ​0.042) in the CA group while no such relationship could be demonstrated in the CF group. This implies that there is an inverse relationship existing between salivary melatonin and malondialdehyde levels in patients with active dental caries but not in patients without dental caries.

Table 2.

Logistic regression analysis for CA and CF group between melatonin and malondialdehyde.

Pair 1		N	Correlation coefficient	Significance
CA group	Melatonin and malondialdehyde	16	−0.513	0.042∗
Pair 2		N	Correlation coefficient	Significance
CF group	Melatonin and malondialdehyde	16	0.004	0.988 (NS)

Footnote: CA denotes caries active group; CF denotes caries-free group; N denotes the number of patients; NS denotes not significant; ∗ denotes significant.

4. Discussion

Dental caries is a globally prevalent oral disease that could cause significant discomfort to the patient in concern if not treated on time and could finally lead to tooth loss and edentulism. The pathobiology of dental caries has been extensively researched and it has been proven beyond doubt that microbes such as Streptococcus mutans are needed to initiate the process.²¹ In dental caries, the acidic environment created by diet and bacterial metabolism dissolves the enamel and stimulates an inflammatory mechanism in the dentin. Many theories have been propounded to understand the mechanisms that underlie the pathogenesis of dental caries. Out of these, the systemic theory implicates inflammation and oxidative stress as two important mechanistic phenomena that predispose an individual to caries.² It is well documented that both inflammation and oxidative stress form two arms in the pathogenesis of many human diseases. The fact that inflammation leads to overzealous production of free radicals and reactive oxygen species thereby causing oxidative burden and depletion of endogenous antioxidants has been documented by several research studies. The depletion of antioxidants and excessive levels of free radicals in turn cause upregulation of transcription factors like NF kappa B²² which consequently switches on cytokines like IL 1 beta and TNF alpha and enzymes such as matrix metalloproteases which cause collagen destruction. The upregulation of matrix metalloproteases which cause dentinal destruction in dental caries is an important hypothesis proposed by the systemic theory.²³

Concerning dental caries pathogenesis, saliva has been found to play a protective role. Saliva is a composite mixture of secretions of the major and minor salivary glands. Several molecules such as antimicrobial peptides,²⁴ antioxidants,²⁵ and antibodies²⁶ confer a protective role to saliva. The antioxidant status of saliva has been evaluated in dental caries patients and is lowered compared to patients without dental caries.³ Concerning salivary antioxidants, melatonin has received a lot of attention recently. Melatonin has been proposed to be an important antioxidant in the saliva. Even though many antioxidant systems like glutathione and associated enzymes exist in the saliva, melatonin is unique as a salivary antioxidant as it is synthesized by the salivary glands.²⁷ and gingiva²⁸ and released into the saliva in addition to being derived from systemic circulation and hence replenishes itself in an autologous manner. However, except for one early animal study²⁹ linking melatonin with dental caries no human study has been conducted to date to assess salivary melatonin levels in patients with and without dental caries. With that pursuit and considering the uniqueness of melatonin as a potent salivary antioxidant, the present study was conducted to assay salivary melatonin and malondialdehyde levels in caries active and patients without dental caries to understand the role played by melatonin as an antioxidant in the pathogenesis of dental caries. The ELISA technique was used to measure melatonin levels as this is an accepted method with good predictibilty level as much as radioimmunoassay which is considered a gold standard technique for melatonin measurement and assay.³⁰

The results of the study demonstrated that patients having dental caries with a mean DMFT score of greater than 10 had significantly lower levels of melatonin and higher levels of malondialdehyde in their saliva compared to patients without dental caries who had higher levels of melatonin and lower levels of malondialdehyde in their saliva. These results are justifiable as oxidative stress has been found to play a role in dental caries as previously described. Human studies have shown elevated levels of oxidative stress markers in the saliva of CA patients compared to CF subjects.³¹ This could in part explain the elevation of malondialdehyde which is a thiobarbituric acid reactive substance. Studies have depicted several pathways for a free radical-induced disturbance in the functions of various cellular components. One of these routes is lipid peroxidation (LP) indicating the degradation of lipids. Malondialdehyde represents a product of lipid peroxidation and is considered a consistent and predictable marker of oxidative stress.³² Elevated levels of salivary malondialdehyde have also been demonstrated in chronic periodontitis patients where oxidative stress plays a major role.³³

Concerning melatonin, the present study has shown lowered levels in saliva samples of CA patients in comparison with CF patients. This indoleamine hormone is a product of tryptophan metabolism that is produced by the pineal gland in large proportions. A sequential cascade of enzymes is required for melatonin biosynthesis. These enzymes are present in the pineal gland and also in many somatic tissues of the body including the gingiva²⁸ and salivary glands³⁴ which are documented as extra pineal sites of melatonin biosynthesis in the oral cavity. Melatonin in saliva could hence be regarded plasma-derived and also could be a product of the salivary glands and gingival tissues. A research study has demonstrated that salivary melatonin is approximately 24–33% in quantity compared to plasma melatonin.³⁵ In the oral cavity, melatonin exerts a spectrum of actions. The main functions of melatonin in the oral cavity are antioxidant and immunomodulatory. It is well known that melatonin can mitigate oxidative stress and attack lethal reactive oxygen species such as the hydroxyl radical¹³ and peroxynitrite anion.¹⁴ In the process of its antioxidant action, it gets metabolized into intermediate compounds and is excreted from the human body. This could explain the reduction of melatonin levels in the CA group where oxidative stress marker malondialdehyde was elevated. It is expected that melatonin is rapidly metabolized to counter the effects of oxidative stress and is thereby sequestered. The findings of lowered salivary melatonin in the CA group coupled with increased malondialdehyde levels are per a similar study performed on periodontitis patients.³⁶

As a part of the study to assess the interrelationship between melatonin and malondialdehyde levels, a binary logistic regression analysis was done. Results of the analysis revealed that melatonin had a negative correlation with malondialdehyde levels in the CA group but not the CF group. This finding is of paramount importance as it sheds light on the changes brought about in the oral cavity by dental caries. It is plausible that oxidative stress caused by the carious mechanism elevates free radicals and reactive oxygen species in the saliva which are countered by melatonin. The depletion of melatonin following this has further implications as melatonin has other important roles such as immunomodulatory, bone sparing, and anticancer effects. Also, melatonin is a potent inhibitor of matrix metalloproteases³⁷ which are involved in caries progression and dentinal matrix degradation. A lowering of melatonin in saliva in patients with active dental caries also implies that there could be caries progression due to loss of anti-MMP effects. Another important effect of melatonin in the oral cavity is immunomodulation. Both cell culture and animal model studies have elicited melatonin as a conventional antagonist of TNF alpha compound³⁸ Hence depletion of melatonin in the saliva of individuals with dental caries active would implicate a rise in pro-inflammatory cytokine levels in saliva which could modulate the progression of other inflammatory diseases.

Another important insight derived from the study is the understanding that dental caries and periodontal disease have a commonality in pathogenesis where oxidative stress and inflammation play an equal role. The pathways for the microbe induced inflammatory response remains the same for both dental caries and periodontal disease.² The limitations of the present study are: i) the salivary parameters like the pH, bicarbonate, calcium, and phosphate levels were not assessed ii) The depth of the carious lesion was not assessed.

5. Conclusion

Within the limitations of the study, the results of the present cross-sectional observational case-control study highlight the potential role of melatonin in the pathobiology of dental caries. Melatonin is a highly lipophilic, biocompatible molecule that can easily be incorporated into gummies, mouthwashes, gels, toothpaste, and orabase formulations. Such melatonin containing local drug delivery preparations could be of use in patients with high dental caries indices as it would help combat oxidative stress and could potentially inhibit the progression of dental caries. Melatonin is also highly safe and has very minimal side effects when administered topically. It can easily be employed to also make nanospheres and microspheres which could enable controlled release of the molecule from the intended preparation. Animal studies on dental caries models followed by Multi-center randomized clinical trials on humans are needed in the future to thoroughly assess the potential protective effects of melatonin in dental caries.

Funding

The authors did not receive funding for the study.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Informed consent

Informed consent was obtained from all individual participants included in the study.