Abstract

Under normal physiological circumstances, an equilibrium exists between prooxidants and antioxidants in the body. The body generates free radicals as part of its natural cellular metabolism. However, when there is an unevenness or modification in the levels of antioxidants, it gives rise to a state known as oxidative stress. This phenomenon is implicated in numerous pathological conditions. It can potentially harm cells by causing minor injuries to cell membranes, deactivating proteins, damaging DNA, and triggering tissue damage through cell-signaling molecules.

Human saliva is a diagnostic fluid that is rich in antioxidant compounds and plays a primary role in the protective mechanism. These antioxidants neutralize the free radicals, including reactive oxygen species and reactive nitrogen species, that are released due to oxidative stress and prevent cell breakdown, tissue damage, and DNA mutations. Whole human saliva may contain numerous antioxidants that are measurable tools to monitor the oral cavity’s oxidative processes and help guide the development of new drugs or treatment plans. This article provides extensive information on salivary antioxidants and their role in common oral lesions like inflammatory, premalignant, malignant, and autoimmune diseases.

Introduction

Saliva is a convenient and acceptable bodily fluid comprising elements sourced from mucosal linings, gingival crevices, and both major and minor salivary glands. Additionally, saliva houses a diverse array of chemical compounds and microorganisms that inhabit the oral cavity and external substances. As such, it offers a potential window into the interplay between the host and the environment.¹ Notably, the antioxidant system present in saliva serves as a crucial defense mechanism, safeguarding the oral mucosa against oral pathogens. Human saliva contains a variety of distinct antioxidant compounds.²

To maintain the homeostasis of the oral cavity in a healthy person, there is a proper balance between oxidative stress and the antioxidant system. If there is any imbalance between these systems, it leads to an increase in the free radicals’ status and results in disease or disorder.³ According to the current definition, oxidative stress represents the disturbance of redox-dependent signaling pathways and the processes they control. Oxidative stress is required for a healthy person in normal daily life in the process of adenosine triphosphate (ATP) generation. This oxidation process is part of the regulatory biochemical function, which releases free radicals and maintains the body’s equilibrium through the production of antioxidants.⁴

During biological processes, endogenous free radicals are generated, capable of associating with various particles. These radicals, characterized by unpaired electrons, exhibit heightened reactivity. Due to their pronounced instability and brief lifespan, they readily engage in reactions, seeking to acquire electrons from other molecules. This quest for electrons induces irreversible alterations in the chemical and physical attributes of cells and their constituents. Key cellular components including lipids, carbohydrates, proteins, and nucleic acids are susceptible to the detrimental impact of these radicals, resulting in oxidative harm to cells, tissues, and organs.^5,6

When an excess of free radicals is produced and the levels of antioxidants are insufficient, the resulting imbalance can contribute to conditions such as tumors, mutagenesis, and carcinogenesis. However, it is important to note that the generation of free radicals is not inherently negative. Numerous researchers have conducted investigations elucidating the role of free radicals in both health and disease contexts. In the physiologic process, free radicals will be produced during tissue repair, ATP synthesis, and immune response.⁷ The main etiologic causative agents identified in oral diseases that induce the formation of free radicals include UV radiation, drugs such as corticosteroids, anticancer drugs, pesticides, immune disturbance, diet, pollution, dental materials, alcohol, smoking, stress, and lifestyle.

To counteract these free radicals, a protective mechanism, such as antioxidants, will be released to protect and neutralize free radicals, (ie, improve the oxidative status), and maintain the health of the individual. If the optimal balance between the oxidants and antioxidants is broken, then the natural defense system fails, which leads to various diseases and disorders. Antioxidants, also called free radical scavengers, are molecules capable of inhibiting the oxidation of another molecule. Antioxidants are classified based on function, nature and action, and source (Table 1).^1-8 Currently, there is a burgeoning fascination with the connection between antioxidants and oral diseases, as the precise impact of excessive reactive oxygen species production stemming from inflammation or insufficient antioxidant presence remains enigmatic.^1,8 This article explores the role of saliva in revealing the interplay between oxidative stress and oral health, emphasizing the importance of antioxidants in maintaining balance and preventing diseases.

TABLE 1. Classification of Salivary Antioxidants^1-8

Antioxidants in Saliva

Human whole saliva is the clear, viscous fluid secreted by the 3 pairs of major salivary glands, numerous minor salivary glands, and gingival crevicular fluid. Saliva is 98% water, and the remaining 2% is solid particles such as proteins, electrolytes, enzymes, hormones, and other substances. Digestion is the main function of saliva, which breaks down the larger molecules into smaller molecules with the help of enzymes and nonenzymatic compounds. Human saliva is rich in antioxidant compounds that help prevent the oxidation process and damage to cells, especially genetic material in the nucleus. Antioxidants not only boost the immune system but also decrease the levels of lipids, preventing the deposition of lipids in the blood vessels. The important primary antioxidants in saliva that have a role in oral diseases include nonenzymatic antioxidants such as carotenoids, uric acid, albumin, glutathione, bilirubin, vitamins A and C, and transferrin, and enzyme antioxidants such as superoxide dismutase, proteases, lipases, catalase, glutathione peroxidase (GPx), glutathione reductase, and glutathione transferase (Figure 1).^9,10 These salivary antioxidative biomarkers in children, adults, and older adults generally do not vary based on sex but rather with age.^11-13

FIGURE 1. Classification of Salivary Antioxidants^9,10

Nonenzymatic Antioxidants

Albumin

In the oral cavity, salivary albumin is an ultrafiltrate from serum to the saliva, and it may also diffuse into the mucosal secretions. It can scavenge free radicals formed on the cell surface. It varies according to the age of individuals but not according to sex. Salivary albumin is increased in patients who are medically compromised, immunosuppressed, undergoing radiotherapy, or have diabetes.⁹

Bilirubin

An endogenous product of a transudation from serum, bilirubin protects the albumin-bound free fatty acid from peroxidation. This salivary bilirubin is overexpressed in cases of laryngeal leukoplakia or cancer, in patients who have had a gastrectomy, and in cases of neonatal jaundice.⁹

Glutathione

Saliva contains a significant antioxidant known as glutathione, which is a tripeptide composed of glutamic acid, cysteine, and glycine. This compound exists in 2 variations within cells: reduced form (glutathione) and oxidized form (glutathione disulfide). It is hydrophilic and present in each cell of the body. It is considered an important antioxidant because it is available directly in the cell for the normal functioning of a cell, ie, the reduced form of this biomolecule, which is necessary for the action of the antioxidant enzyme GPx. This enzyme produces glutathione oxidation, neutralizing hydrogen peroxide and other peroxides.¹¹

Transferrin

Transferrin is a protein in nature that is diffused to the salivary gland from the blood. It binds to iron and prevents iron-catalyzed free radical formation.⁹

Uric Acid

It stands as the foremost and straightforward antioxidant present within the body, constituting over 85% of the overall antioxidant capacity in both unstimulated and stimulated human saliva. It is hydrophilic and acts as an antioxidant. It can also act as a prooxidant, especially when it occurs in higher concentrations and especially in a few systemic diseases such as atherosclerosis, stroke, and Parkinson disease. Uric acid is the ultimate product of the metabolism of purine nucleotides. Uric acid is secreted in saliva and gingival crevicular fluid and is responsible for reducing and neutralizing free radicals. It is considered to be a main antioxidant in saliva because it participates in approximately 70% of the total salivary antioxidant capacity.¹⁰

Vitamin E or Tocopherol

It is a lipid-soluble vitamin, and α-tocopherol is considered a biologically active substance in saliva. It is an antioxidant present in all cell membranes and protects against lipid peroxidation and the aging process. It acts directly on oxyradical and behaves like a chain breakdown of the chain reactions. Therefore, vitamin E is used as a treatment for many systemic diseases such as Alzheimer disease and osteoarthritis.^9,10

Vitamin C or Ascorbic Acid

It is a hydrophilic antioxidant. It neutralizes free radicals, ie superoxides, released by hydrogen peroxide.¹¹ It helps in the growth of collagen in the skin, repair of damaged cells, and wound healing. It acts as an anti-inflammatory and helps the body in the absorption of iron. Some diseases and disorders such as tuberculosis, periodontitis, cancer, and leprosy have shown undervalued salivary vitamin C concentrations.⁹

Enzyme Antioxidants

Catalase

Catalase is an antioxidant enzyme¹⁰ that catalyzes hydrogen peroxide to oxygen and water. Later, it nullifies the effect of hydrogen peroxide that is present intracellularly. The catalase level cannot be identified because it is located in the cytoplasm and most of it is lost during tissue manipulation.

GPx and Glutathione Reductase

GPx and glutathione reductase are enzymes that act as antioxidants and are secreted by the main salivary glands, mostly parotid glands. The reduced form of glutathione is defensive. These enzymes help to neutralize hydrogen peroxide produced inside the cell, which is the product of the metabolism of oral bacteria. These enzymes are key players in preventing increased levels of oxidative stress. This repeated oxidation and reduction of glutathione makes it a free radical scavenger.¹¹

Glutathione S-Transferase

Glutathione S-transferases (GSTs), also known as ligandins, catalyze the glutathione to detoxify endogenous substances. They are classified into 3 families: cytosolic GST, mitochondrial GST, and microsomal GST. There is an increase in the level of GST in oral cancer, and many authors suggest that there is an alteration in the level of GST in many diseases such as asthma, allergies, and inflammatory diseases.⁹

Lipase

Lingual lipase is also called triacylglycerol lipase and is made of aspartate, histidine, and serine. This is a salivary enzyme that catalyzes the dietary lipid, with diglycerides being the primary reaction product and neutralizing the free radicals released during lipid peroxidation. It also acts as a fat taste receptor. The minor serous salivary gland secretes it.⁹

Protease

This enzyme is produced in the minor salivary gland and is derived from white blood cells and bacteria in the oral cavity. The main function of protease is to catalyze the hydrolytic reaction that degrades the proteins into peptides and free amino acids so that it prevents the oxidative stress effect on oral tissues.¹⁰

Superoxide Dismutase

Superoxide dismutase (SOD), found ubiquitously in the body, serves the vital function of converting the superoxide anion into oxygen and hydrogen peroxide. This enzyme is crucial due to the significant production of reactive oxidants, such as hydrogen peroxide, superoxide, and hydroxyl radicals by the human body, which can potentially damage nearby tissues and cells. Metal-coordinated superoxide dismutase enzymes counteract this process. There are 3 distinct variants of the superoxide dismutase enzyme: copper-zinc–containing enzymes in the cytoplasm, manganese SOD in the mitochondria, and nickel SOD in the extracellular environment.¹¹

In the oral cavity, SOD acts as an essential antioxidant localized within the human periodontal ligament and gingival cells.¹ This SOD has been associated with an increase in neurological diseases such as Down syndrome and thalassemia, while concurrently contributing to a decrease in periodontitis, acute respiratory distress syndrome, and chronic obstructive pulmonary disease.

Role of Oxidative Stress and Antioxidants in the Pathogenesis of Oral Diseases

Salivary antioxidants have emerged as a central focus in biomedical investigation due to their pivotal role in the development of oral diseases. These antioxidants play a critical role in shielding the oral cavity against the damaging impact of free radical–induced oxidative stress, thereby hindering the onset of oral diseases. Saliva serves as a reflection of the body’s state across various hormonal, immunological, toxicological, and infectious conditions, functioning as a biological indicator and an effective means for tracking both oral and systemic well-being. The levels of these salivary antioxidants offer insights into the overall health of an individual, spanning from oral health to broader systemic health.

Oxidative stress has the potential to induce cellular harm through microdamage to the cell membrane, deactivation of proteins, DNA impairment, and the initiation of tissue damage by cell-signaling molecules.¹⁴ Certain molecules are more susceptible to oxidation, involving the theft of electrons, than others. Notably, molecules within cell membranes containing unsaturated lipids are especially prone to attack by free radicals. RNA, DNA, and protein enzymes are other molecules vulnerable to oxidative damage. Oral cells face a distinctive susceptibility to free radical–induced harm due to the rapid absorption of substances facilitated by their mucous membranes.¹⁵

Within oral tissues, oxidative stress can arise from gum disease–related infections, as well as exposure to alcohol, nicotine, hydrogen peroxide, and various dental procedures and materials such as dental cement and composite fillings. The escalated presence of free radicals, stemming from oxidative stress, exacerbates the degradation of cell membranes and oral tissue. These free radicals have been recognized as clinically significant factors in the development of vascular, inflammatory, and oral diseases (Figure 2).^16,17

FIGURE 2. Role of Salivary Antioxidants in Oral Disease^16,17

Role of Antioxidants in Dental Caries

Dental caries stands as the most prevalent oral disease affecting individuals across all age groups. It is a multifactorial inflammatory condition triggered by acid production resulting from carbohydrate fermentation facilitated by bacteria. This inflammatory response induces oxidative stress within the tooth, leading to the degradation of enamel, dentin, pulp, and cementum. The precise role of oxidative stress in dental caries remains unclear, necessitating further in-depth research.^18,19

Numerous studies have explored the correlation between total antioxidant capacity (TAC) and dental caries in both children and adults. Findings from the majority of these studies indicate significantly higher TAC levels in individuals with active caries than in those without caries. Although some studies have reported lower TAC levels in individuals with dental caries, they are in the minority.^20-25

Several researchers have proposed that there are variations in salivary total antioxidant capacity, with catalase levels being notably higher in groups with active caries when compared with control groups.^26,27 Catalase, known for protecting cells from hydrogen peroxide generated in aerobic organisms, plays a crucial role in developing tolerance to oxidative stress as part of the cells’ adaptive response.^26,28

In a study by da Silva et al, the evaluation of enzymatic (SOD) and nonenzymatic antioxidant (uric acid) levels in the saliva of toddlers with severe early childhood caries revealed significantly higher levels than in the control group. This emphasizes the potential impact of antioxidants in the saliva on the development of severe early childhood caries.²⁸

Role of Antioxidants in Periodontal Lesions

In recent years, chronic periodontal disease is the most common inflammatory disease of the oral cavity due to oxidative stress. Periodontal disease is an inflammatory condition that alters the periodontium, destroying supporting structures such as the alveolar bone, which leads to tooth loss.

The antioxidative role of saliva is to neutralize free radicals and reduce the oxidative damage of the periodontal tissue cells, which this secretion accomplishes through the presence of enzymatic and nonenzymatic antioxidants. Among them are salivary peroxidase, superoxide dismutase, GPx, catalase, uric acid, and albumin. Saliva is also rich in nonenzymatic antioxidants such as ascorbic acid, albumin, glutathione, lactoferrin, vitamins, and uric acid, which is the main representative agent of this group. These antioxidants protect against the free radicals that damage the biomolecules of cells and promote proinflammatory cytokines such as tumor necrosis factorα
and interleukins 1β and 6.¹⁶

SOD levels were found to be elevated in people with chronic periodontitis compared with healthy individuals.²⁹ This enzyme present in the periodontal ligament neutralizes the effect of reactive oxygen species. It is hypothesized that bacterial polysaccharides stimulate the release of superoxide, which in turn leads to induction of the enzyme SOD. The amount of GPx has shown considerable variability in patients with chronic periodontitis with or without diabetes. Some studies have shown a decrease in the level of this enzyme in patients with chronic periodontitis, while other studies are contrary to the same.^1,30 Glutathione levels showed elevation in periodontitis with both type 1 and 2 diabetes. This substantiates the fact that the generation of free radicals is increased in chronic hyperglycemia, leading to increased reduced glutathione. Glutathione levels have been found to influence signal transduction and gene expression events in T lymphocytes, leading to periodontitis.¹⁰ Chapple et al showed that patients with compromised periodontium had a low total antioxidant status.³¹

Role of Antioxidants in Oral Cancer

Oral leukoplakia stands out as the prevailing potentially malignant disorder affecting the oral mucosa. Numerous instances of oral cancer are preceded by various potentially malignant oral conditions, with leukoplakia emerging as the most prevalent among them. In a study conducted by Vlková et al on individuals with leukoplakia, it was observed that salivary SOD and total antioxidant capacity were notably lower in patients compared with controls.³² Another study by Srivastava et al revealed a statistically nonsignificant increasing trend in the product of lipid peroxidation across clinicopathological stages of leukoplakia, excluding stages I and II. Additionally, there was a significant decrease in the levels of glutathione, GPx, catalase, uric acid, and SOD in patients with leukoplakia when compared with healthy control groups.^33,34

Oral cancer, also called oral squamous cell carcinoma (OSCC), is one of the most common types of cancer in the world because of delay in diagnosis and clinical presentation, poor prognosis, lack of specific biomarkers, and expensive treatment. Salivary antioxidants are considered one of the biomarkers for the identification of oral precancer and cancerous lesions. The SOD enzyme is considered a tumor suppressor, and in findings from studies of patients with cancer, it was observed that SOD is decreased; its expression is thus used as a biomarker for oral cancer.^10,35 There is an alteration in glutathione levels in carcinogenesis in which γ‑glutamyl transpeptidase is an enzyme that catalyzes the breakdown of glutathione. Hence, there is an increase in the level of glutathione in oral cancer compared with the controls.¹⁰ Srivastava et al reported increased tissue levels of free radicals and reduced concentrations of SOD, GST, GPx, and catalase in stages II, III, and IV OSCC.³⁶ Multiple authors stated that a decrease in salivary uric acid levels was observed in patients with OSCC compared with healthy controls.^35,37,38 According to Najafi et al, the total salivary antioxidant level in patients with OSCC was significantly higher than in healthy individuals due to an increase in the compensatory action as an antioxidant.³⁹

Role of Antioxidants in Oral Submucous Fibrosis

Oral submucous fibrosis (OSMF) is a precancerous condition that affects the collagen fiber of the connective tissue due to the habit of betel chewing, ie, areca nut consumption. The phenolic compounds in areca nuts are responsible for the formation of free radicals among betel quid chewers. These free radicals are derived from lipid peroxidation, which has a role in the initiation and progression of OSMF.⁴⁰

In patients with OSMF, there was a decrease in the levels of vitamins A, C, and E; salivary SOD; and GPx. These changes were noticed in correlation with the different grades of OSMF that reflect increased oxidative stress with the progress of OSMF.⁴⁰ Similarly, authors stated that there is a gradual reduction in GPx levels with various grades of OSMF compared with controls.

Role of Antioxidants in Recurrent Aphthous Ulcer

Recurrent aphthous stomatitis, the most prevalent ulcerative disorder of the oral mucosa, is characterized by the presence of painful, round, shallow ulcers. These ulcers exhibit a yellowish-greyish pseudomembranous covering with well-defined erythematous margins.⁴¹

Antioxidants play a crucial role in individuals with recurrent aphthous stomatitis, demonstrating a notable association with decreased levels of the SOD enzyme. However, there are no observable changes in the levels of GPx and catalase when compared with healthy controls.¹⁰

In a recent investigation by Ziaudeen and Ravindran, the role of oxidative stress in the pathogenesis of recurrent aphthous stomatitis was assessed by measuring salivary oxidants and antioxidants. Findings from the study revealed an increase in mean salivary SOD and a reduction in the activity of GPx and uric acid in the study group compared with the control group.⁴²

Tugrul et al reported that total oxidative status and oxidative stress index values were significantly higher in the group with recurrent aphthous stomatitis than in the control group. Conversely, total antioxidant status values were significantly lower in the same group.⁴³

Role of Antioxidants in Autoimmune Diseases

Several researchers have shown that the imbalance between free radicals and reactive oxygen species plays a major role in the initiation and progression of oral autoimmune inflammatory lesions. Lichen planus is a common autoimmune disease among middle-aged groups of patients. An evaluation of oxidative stress in lichen planus on antioxidants levels showed an increase in the levels of SOD, with decreased catalase, and GPx levels than in controls.⁴⁴ Catalase is the main enzyme in eliminating peroxides. A disturbance in the balance between the antioxidant and free radicals results in the accumulation of hydrogen peroxide, thus leading to the degeneration of the basal cells seen in histopathological sections of lichen planus. Abdolsamadi et al conducted research and suggested that there are lower salivary levels of antioxidant vitamins E and C and total antioxidant capacity among patients with oral lichen planus (OLP), suggesting that free radicals and the resulting oxidative damage may be important in the pathogenesis of OLP lesions.⁴⁵

Recently, several authors have proposed that pemphigus vulgaris involves an elevated generation of oxygen free radicals by activated neutrophils and eosinophils, coupled with diminished production of vitamins and antioxidant enzymes in both saliva and plasma, indicative of oxidative stress.^46,47 Najafi et al have specifically suggested that the salivary level of GPx is significantly lower in individuals with pemphigus than in healthy individuals.⁴⁶

Nazıroğlu et al have postulated that the diminished activity of GPx and catalase may arise from heightened enzyme degradation triggered by increased levels of free radicals, including hydrogen peroxide.⁴⁸ This reduced activity of GPx and catalase might be attributed to a decrease in the synthesis or inhibition of these enzymes by certain inhibitory substances within the host of the patient.⁴⁶ On a related note, Javanbakht et al observed the highest activity of antioxidant enzymes such as SOD, catalase, and GPx while noting a decreased total antioxidant capacity in individuals with pemphigus vulgaris when compared with healthy controls.⁴⁷

Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease. The suggested etiopathogenesis of SLE indicates a potentially significant role played by free radicals and oxidative stress. This involves an increase in reactive oxygen species or dysfunction in antioxidant protective systems, which collectively contribute to oxidative stress in SLE.⁴⁹ Notably, there is a reduction in the expression of peroxidase, SOD, and catalase activity in the saliva of individuals diagnosed with SLE.⁵⁰

Role of Antioxidants in HIV Infection

There is an alteration in glutathione levels in patients with HIV due to oxidative stress, and glutathione supplements may be prescribed for their survival. Glutathione is considered a tool marker to identify the transformation of HIV into AIDS. Salivary total antioxidant levels were significantly lower in patients who were HIV-positive than in healthy controls.⁵¹

Advantage of Antioxidants in Dentistry

Antioxidant and oxidative stress indicators are poised to provide valuable insights into various stages of tumor development (Figure 3).¹ The intricate relationship between alterations in antioxidant equilibrium and the pathological progression within the oral cavity suggests the potential utility of these antioxidants as diagnostic markers. These markers could guide effective treatment strategies aimed at improving overall oral health. Notably, modifications in salivary antioxidant enzymes highlight saliva as a promising prognostic marker for oral diseases, offering a noninvasive alternative to the conventional serum antioxidant enzyme assessment.

FIGURE 3. Advantages of Salivary Antioxidants in Dentistry¹

Conclusion

Whole saliva contains both enzymatic and nonenzymatic antioxidants, which serve as straightforward indicators of oxidative mechanisms. The foundation of physiological processes and pathological disorders lies in oxidative stress, which leads to cellular and component damage. Given that saliva analysis is noninvasive, measuring antioxidant levels offers a means to assess oxidative stress and the protective capability of the oral mucosa. Consequently, this approach has the potential to mitigate numerous pathological oral conditions. It could serve as a pivotal tool for diagnosing, advancing, and overseeing novel treatment approaches for oral as well as systemic diseases.

Corresponding author

Vidya G. Doddawad

Associate Professor

Department of Oral Pathology and Microbiology

JSS Dental College and Hospital

A Constituent College of JSS Academy of Higher Education & Research

Mysore-570022

Karnataka, India

Authors’ contributions

VGD, SS: Concept, writing, and literature search for the manuscript; MGM, KP, VCS, RSB: Reviewing, data collection, and supervision of the manuscript. All authors read and approved the final manuscript

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