Aims

Carry out a narrative review on the relationship between the production of reactive species and the decrease in salivary antioxidant capacity induced by tobacco smoke. Review the accumulated evidence on oxidative stress in periodontitis and the potential use of redox pharmacology for the treatment of this pathology.

Oxidative stress in biological systems

The concept of oxidative stress, coined by Helmut Sies, ⁸ was introduced just over 30 years ago, encompassing a spectrum of situations in which an imbalance occurs between oxidant formation and cellular antioxidant capacity, in favor of oxidants, leading to redox signaling disruption and molecular damage (Fig. 1) ^9-10. The formation of oxidants at the cellular level occurs, in part, because of aerobic metabolism, although it can also occur as a result of environmental exposure to oxidizing agents such as UV radiation, tobacco smoke, smog, among others. Oxidants produced can react with biomolecules, leading to modifications that determine changes in structure and function, generating cell damage. On the contrary, these processes are attenuated by enzymatic and non-enzymatic antioxidant systems ^10-11. When these antioxidant systems collapse or are overwhelmed by oxidants, a condition of cellular oxidative stress is established. In recent years, the study of the role of reactive species has led to the incorporation of the concepts of eustres or oxidative distress as substitutes for the concept of oxidative stress. In this way, an attempt is made to discriminate the physiological or pathological roles of the redox imbalance, respectively ^12-13.

Fig. 1: Redox status. An imbalance between oxidants and antioxidants, in favor of oxidants, leads to the appearance of biomarkers compatible with oxidative stress 

What are free radicals?

A free radical is a molecule containing an unpaired electron in its outermost orbital. They are short half-life (micro- to milliseconds) and very reactive intermediates (they accept or donate electrons) ^11-14. Free radicals and other oxidizing molecules are called “reactive species” and can be derived from oxygen, nitrogen, and other elements ¹⁵. They are formed under normal and pathological conditions, and cells have developed different antioxidant systems to attenuate their biological effect. Below we describe the most relevant reactive oxygen and nitrogen species in biological systems.

Reactive oxygen species

The term “reactive oxygen species” (ROS) includes oxygen radicals and some non-radical derivatives ¹⁶ and in Diagram 1, the one-electron reduction sequence of molecular oxygen is shown.

Diagram 1: One-electron reduction of molecular oxygen 

Under normal conditions, the main cellular source of ROS is mitochondrial respiration, generating superoxide anion radical (O2•-) as a by-product of the flow of electrons through the electron transport chain ¹⁷. Detoxification of this radical can be spontaneous or enzymatic by the action of superoxide dismutases (SODs, MnSOD, or CuZnSOD), which yields hydrogen peroxide (H2O2). H2O2 is not a free radical because it does not contain unpaired electrons, is more stable than O2•- with a half-life in the order of ~1 ms, and under stationary conditions, it reaches a cell concentration of ~10-7 M ¹⁸. Consequently, O2•- is normally consumed at the site of formation generating H2O2 by SOD-mediated enzymatic dismutation (16). Once H2O2 is formed, it can diffuse through membranes reaching different compartments than the site where it is generated. In aerobic organisms, H2O2 is metabolized to water and O2 mainly by two enzyme systems: catalase (CAT) and glutathione peroxidase (GPx). The hydroxyl radical (•OH) is a highly reactive molecule that can oxidize amino acids, lipids, and nucleic acids, resulting in a short half-life (~10-9 s) and a low diffusion capacity ¹⁹. It is generated via the Haber-Weiss cycle in presence of proteins containing Fe or Cu metal centers and H2O2. Another source of this radical is the decomposition of the peroxynitrite anion (ONOO-), as explained below.

Reactive nitrogen species

Reactive nitrogen species (RNS) can be radicals or non-radicals. Those of highest interest in biology are nitric oxide (•NO), peroxynitrite anion (ONOO-), and nitrogen dioxide radical (•NO2). •NO is an uncharged molecule with a short half-life (3-5 s) and is considered one of the most important nitrogen radicals. In fact, its discovery earned the Nobel Prize in Physiology or Medicine for L. Ignarro, F. Murad, and R. Furchgott in 1998 ²⁰. It is not a strong reducer or oxidizer and therefore does not react rapidly with most biomolecules despite being a free radical (21). It is produced from L-arginine by the action of nitric oxide synthase enzymes (NOS), and the reaction involves N-hydroxy L-arginine as an intermediate. In the presence of O2•-, H2O2 and transition metal centers, it can generate other reactive oxygen species such as ONOO-. Physiologically, is an intracellular messenger and is the cytotoxic agent produced by activated macrophages and neutrophils, it regulates local blood flow, it is an inhibitor of platelet aggregation and adhesion and at low concentrations it works as an anti-inflammatory, but at high concentrations it acts as a pro-inflammatory ²¹. ONOO- is an anion produced from the rate limited reaction of •NO with O2•-. It is not a free radical, but it has a very short half-life in the order of 10-20 ms ²². It can be formed in biological systems and is a potentially toxic oxidant and nitrating agent ²³. When there is an increase in O2•- generation, the reaction of •NO with O2•- is kinetically favored, and •NO competes with SOD for the removal of O2•- to form ONOO- (increased in pathological conditions). •NO2 is a toxic and highly reactive free radical that can nitrate lipids and proteins, causing cellular damage, as the nitration of significant amino acid residues in proteins can compromise its cellular function. It can be formed from the decomposition of ONOO- and from its reaction with CO2, which is very significant in biological systems. Additionally, it can also be formed through NO2- oxidation catalyzed by some peroxidases (e.g., myeloperoxidase) or by the autooxidation of •NO ²⁴.

Free radical sources and cellular damage

Oxidants and free radicals are continuously produced during physiological activities in cells and can participate in redox signaling. Oxidative stress condition can be induced in biological systems by oxidants and radicals from both endogenous and exogenous sources (Fig. 2). In pathological conditions, the formation of these species can significantly increase and mediate oxidative damage to different biomolecules (lipids, carbohydrates, DNA, and proteins). The reactive species generated can cause DNA damage, modification and/or inactivation of proteins and carbohydrates, as well as lipid oxidation. To prevent this damage caused by free radicals, organisms, especially aerobes, have developed defense systems or antioxidant systems during evolution ^1-25.

Fig. 2: Main sources of free radicals. They can be classified as endogenous or exogenous. The generation of ROS and RNS causes a redox off-balance in favor of the pro-oxidants, which damages biomolecules 

In the case of the oral cavity, many reports on saliva describe antioxidant capacity globally as total oxidative status (TOS) ²⁶. However, it is not specified which system or systems are affected; From then on, the most important antioxidant systems in saliva will be investigated.

Antioxidants in saliva

From a biomedical point of view, an antioxidant can be defined as a compound or protein that, even when is present at low concentrations compared to those of an oxidizable substrate, prevents or significantly delays the oxidation of the substrate initiated by a prooxidant ^(15-16). Antioxidants present in saliva can be classified as enzymatic or non-enzymatic antioxidant systems (Fig. 3):

Fig. 3: Main antioxidants present in saliva. They are classified as enzymatic or non-enzymatic. They represent the main barrier against oxidant and radical damage because they exert their protective effect on teeth and the oral cavity tissues 

Peroxidases are a family of enzymes that use different substrates as a source of electrons to reduce peroxides. Some of them have a specific substrate, such as reduced glutathione (GSH) for GPx, but most act on several substrates ²⁷. In saliva, salivary peroxidase (SPx) catalyzes the formation of hypothiocyanate (OSCN-) and hypothiocyanous acid (HOSCN-) from hydrogen peroxide (H2O2) and thiocyanate (SCN-). H2O2 can be generated, as mentioned above, by the bacteria that colonize the oral cavity or through rinses and mouthwashes. The main function of SPx is believed to be to remove the H2O2 produced locally by bacteria. SODs are a family of metalloenzymes found in all aerobic organisms and the first enzymes to be involved in antioxidant defense. As mentioned above, SODs protect the cell from ROS by catalyzing the dismutation of O2•- to H2O2 ²⁷ being the most important O2•- detoxification pathway in biological systems. Catalases belong to a family of enzymes present in numerous tissues (e.g., abundant in liver cells). As mentioned above, it catalyzes formation of water and oxygen from H2O2 and plays a vital role in metabolism, preventing the alterations induced on proteins and nucleic acids ²⁷. Glutathione peroxidases (Gpx) are a family of selenoenzymes expressed in most cells whose main biological activity is to protect the organism by detoxifying peroxides ²⁷. GPx reduces H2O2 and other hydroperoxides to H2O using reduced glutathione (GSH) as a co-substrate and converting it to oxidized glutathione (GSSG) ²⁸. The glutathione reductase (GRd) enzyme is part of a family of enzymes that catalyze the reduction of GSSG to GSH at the expense of NADPH, which mainly comes from the pentose phosphate pathway ²⁷. One notable non-enzymatic antioxidant is uric acid (waste product of purine catabolism), which effectively eliminates energetically excited species such as singlet oxygen and peroxyl and hydroxyl radicals ^16-29. Uric acid is the predominant antioxidant molecule in saliva, representing 85% of the total antioxidant capacity ³⁰. In addition, melatonin, normally secreted by the salivary glands (although it can also be added as an external treatment) and whose maximum secretion is between 12:00 and 2:00 a.m. (following the circadian rhythm of the individual), can neutralize ROS mainly by removing H2O2 ^31-33. Therefore, it is suggested that its role is CAT and GPrx backup ³¹. In addition, polyphenols are important exogenous antioxidants obtained from the diet, which are present in various foods and beverages that have shown modulating the activity of the metabolism of reactive species in saliva ^34-35.

Oxidative stress caused by tobacco smoke

It is reported that tobacco smoke contains many potentially toxic substances, with nicotine being considered one of the most dangerous and addictive. It also includes many prooxidative and carcinogenic substances. Smoke, a product of tobacco combustion, carries a wide variety of free radicals, making it one of the most powerful inducers of oxidative stress in the oral cavity ^4-36. Tobacco smoke increases ROS levels, which are toxic to the human body because they change redox homeostasis ⁵. Cadmium in cigarette smoke can replace iron and copper in membrane-bound cytoplasmic proteins, thus increasing the amount of free or chelated copper and iron that can further increase oxidative stress through Fenton reactions ³⁷. Reactive species reported as components of tobacco smoke include H2O2, O2•-, •OH, ONOO-, •NO2, ROO•, •NO, acrolein, crotonaldehyde, nitrosamines, among others. Acrolein and crotonaldehyde have been reported as the main triggers in the oxidation of protein thiols in the oral cavity, causing structural and functional modifications ³¹. Smoking can interfere with the antioxidant systems of periodontal tissues, inhibiting the bacterial plaque defenses, causing vasoconstriction, and slowing down wound healing as well as covering characteristic signs of

periodontitis, such as gingival bleeding ⁵.

Oxidative stress impact on saliva and periodontitis implications

Periodontitis is defined as a chronic and multifactorial immunoinflammatory disease associated with dysbiosis of the bacterial film (microbiome). It is influenced by the inflammatory response of the host, contained in the gingival crevicular fluid and saliva. In addition, it can be affected by environmental and behavioral factors, and its development can be modified by local factors (e.g., smoking), acquired conditions (systemic diseases), and genetic factors ^6,38. The group of microorganisms present in the oral cavity is known as the oral microbiome or oral biofilm. For the development of periodontitis, a dysbiosis in the microbiome is necessary (changes in the composition of the microflora from a healthy state to an unhealthy one), these changes can be influenced by factors such as lack of hygiene, nutrition, exposure to physical agents - chemicals (variations in pH, temperature, tobacco, among others) ^39-40. The persistence of a dysbiotic biofilm in periodontal pockets, triggers leukocyte migration (polymorphonuclear neutrophils—PMN—represent between 50% and 70% of the total leukocyte infiltrate) from the bloodstream to the infection site, where they play an essential role in periodontal health and the innate immune system as first barrier cells, acting through several unique defense mechanisms, including degranulation, chemotaxis, phagocytosis, NETosis (neutrophil extracellular traps), and release of ROS ⁴¹. Several studies show that the “hyperactive” PMN phenotype characterized by ROS overproduction is linked to periodontitis, making the subset of patients with higher levels of this neutrophil phenotype more susceptible to developing the disease ⁴². ROS play a significant role in cell signaling, gene regulation, and antimicrobial defense but an overproduction of ROS leads to an increased oxidative burden and an altered or reduced antioxidant capacity. This scenario leads to cellular oxidative stress within the affected tissues, which then causes pathological changes and consequently, destruction of host tissues and loss of dental support tissues ⁴³. Periodontitis, like other chronic inflammatory diseases, is linked to an imbalance of redox homeostasis ⁴⁴, which has been linked to the etiology and pathogenesis of the disease ¹¹. In addition to an overproduction of ROS, the action of these reactive species is exacerbated in periodontitis due to a weakened antioxidant defense system ³⁰. This breakdown of the antioxidant barrier is directly responsible for the oxidative and nitro-oxidative modifications occurring on oral cavity biomolecules because of the reduction of the neutralization capacity ⁴⁵. Several studies have shown that oxidative stress is primarily responsible for the degradation of extracellular matrix components of periodontal tissue, including collagen, elastin, proteoglycans, and glycosaminoglycans (e.g., hyaluronic acid), which causes the loss of periodontal integrity ⁴⁴. But it can also have indirect deleterious effects by generating oxidized fatty acids, which activate adipogenesis, inhibiting osteoblastogenesis by directly impacting osteoclasts ⁴. Lipid peroxidation markers are the most used to assess oxidative damage associated with periodontal diseases (i.e., malondialdehyde, 4-hydroxynonenal, 8-oxoguanine), because polyunsaturated fatty acids are highly susceptible to ROS attack ⁴⁶. Considering that oxidative stress plays a major role in the pathogenesis of periodontitis, it has been suggested that supplementation with antioxidants could reduce or delay periodontal damage ⁴⁷.

Redox pharmacology applied to periodontal disease

We now see specific interventions using redox medicine, i.e., compounds that can be administered to patients to attenuate the harmful effects of oxidative stress at the local level, based on the evidence of the major role of oxidative stress in periodontitis ⁴⁸. Table 1 shows the most relevant compounds based on the relevant literature. There are ongoing trials on drugs such as resveratrol, a natural stilbenoid present in different foods which has been shown to be effective in reducing the rate of bone loss in animal models of periodontitis exposed to tobacco smoke when administered systemically as nutraceutical formulations ⁴³. On the other hand, there is evidence that febuxostat, a xanthine oxidase inhibitor, could also be another potential agent for the treatment of periodontitis. As mentioned above, uric acid, an enzyme product of xanthine oxidase, is one of the main low molecular weight antioxidants in saliva. Paradoxically, despite the inhibition of this enzyme and, consequently, the decrease in uric acid production, febuxostat has been shown to be capable of slowing down bone loss and reducing the levels of proinflammatory cytokines and biomarkers of oxidative damage (i.e., HNE) ⁴⁴. In contrast, most conventional antioxidants have drawbacks such as low water solubility, stability, and short duration of action when applied topically ⁴⁸. On the other hand, redox pharmacology is also being tested in the production of nanomaterials (drug-loaded nanoparticles) and has the advantage that the component is released specifically at the therapeutic site (topical application), with high biocompatibility and low toxicity ⁴⁹. Although the use of antioxidant-loaded nanoparticles is in the early stages of development, the few reported examples, including those loaded with coenzyme Q10, showed great potential for the treatment of periodontitis ⁴⁸.

Table 1: Redox medicine applied to different models of periodontitis