Review article

M. CZESNIKIEWICZ-GUZIK, S. J. KONTUREK, B. LOSTER, G. WISNIEWSKA, S. MAJEWSKI

MELATONIN AND ITS ROLE IN OXIDATIVE STRESS RELATED DISEASES OF ORAL CAVITY

Institute of Dentistry and Department of Physiology, Jagiellonian University Medical College, Cracow, Poland The role of the oral cavity in the pathogenesis of diseases of various systems such as the gastro-intestinal tract (GIT), cardiovascular and immune systems has been recently evaluated. While initially the oral cavity was considered to be mainly a source of various bacteria, their toxins and antigens, recent studies showed that it may also be a location of oxidative stress and periodontal inflammation. Accordingly, this review focuses on the involvement of melatonin (MT) in oxidative stress diseases of oral cavity as well as on potential therapeutic implications of MT in dental disorders, especially in periodontal inflammation. MT is generated and released by pineal gland and by entero-endocrine (EE) cells located in the GIT. The pattern of MT secretion from the pineal gland is controlled by an endogenous circadian timing system that conveys information about the light/dark cycle to various organs of the body, thereby organizing its seasonal and circadian rhythms. The secretion of MT from the EE cells of GIT is related mainly to feeding periods. MT is a non-toxic highly lipophilic indole, and this feature facilitates its penetration through cell membranes and its compartments. However, the most important effect of MT seems to result from its potent antioxidant, immuno-modulatory, protective and anti-cancer properties. It stimulates synthesis of type I collagen fibers and promotes bone formation. Thus, MT could be used therapeutically for instance, locally, in the oral cavity damage of mechanical, bacterial, fungal or viral origin, in post-surgical wounds caused by tooth extractions and other oral surgeries and, in helping bone formation in various auto-immunological disorders such as Sjorgen syndrome, in periodontal diseases, and in oral cancers. Key words: melatonin, inflammation, oxidative stress, immunological system, oral cavity

INTRODUCTION

The role of the oral cavity in the pathogenesis of several systemic diseases has been recently described by numerous clinical studies (1). This includes not only the role of oral cavity in the pathology of extra-oral parts of the GIT (2), but also in cardiovascular and autoimmune systems (3). While initially the oral cavity was considered to be mainly a source of pathogenic microorganisms (4) and antigens (for instance in the pathogenesis of endocarditis), recent studies have showed that it may be also implicated in systemic oxidative stress and inflammation (5 - 8). Moreover, it has been established that several hormones known so far for their involvement in neuromodulation as well as immunomodulation, such as melatonin (MT), have important roles in the regulation of both systemic and oral homeostasis (9). Accordingly, this review will focus on the mechanisms of involvement of MT in oral cavity disorders and in oxidative stress related oral diseases and periodontal inflammation as well as on the potential therapeutic usefulness of MT in oral diseases. Dual nature of reactive oxygen species - double edged sword of inflammation Reactive oxygen species (ROS), known simply as free radicals, are molecules characterized by the presence of unpaired electrons in their valence orbitals, which make them highly reactive (Fig. 1). Through this mechanism ROS are able to damage numerous cellular molecules, such as proteins, lipids, nucleic acids, amino acids, carbohydrates and vitamins. They cause lipid peroxidation and production of highly toxic lipid derivatives, which in turn can modify cell functions and even may lead to cell death (10). Oxidative modification of proteins may result in structural impairment and also change their functional properties such as their involvement in signaling, critical for numerous cellular functions. ROS-induced damage of the nucleic acids can promote DNA mutation, thus contributing to the development of neoplasms (11).

Fig. 1. About 5% of total oxygen uptake is transformed, mainly in mitochondia, in reactive oxygen species (ROS) that in combination with NO may be further transformed into reactive nitrogen species (RNS) and their derivatives. Free radicals and their non-radical metabolites are derived mainly from oxygen metabolism in the cytoplasm by NADPH oxidases, xanthine oxidase and in the mitochondria during the production of ATP molecules, the main energy carrier for the cell (12). About 5% of total oxygen uptake may be used in generation of free radicals (see Fig. 1). ROS include e.g. superoxide anion (O2 -), peroxyl radical (R00.), hydrogen peroxide (H2O2), singlet oxygen (O2 -) and extremely reactive hydroxyl radical ( OH), (13). ROS can also interact with nitric oxide (NO), the product of NO synthase, whose expression is usually accompanyied by inflammatory mucosal lesions, resulting in the conversion of NO to various reactive nitrogen species (RNS), including nitrosonium cation (NO+), nitroxyl anion (NO-) and peroxynitrite (ONOO-) (14) (Fig. 2). The latter is particularly toxic for the cell because it inhibits the electron transporter chain (ERC), which is located in the inner mitochondrial membrane and coverts the redox energy into an electrochemical gradient of protons that subsequently drives ATP formation from ADP and phosphate by ATP synthase. Peroxynitrate leads to a decrease in the cell energy production and to increase in the generation of other free radicals (15). However, free radicals not only influence human organism through direct modifications of cellular components but have numerous other more complex effects. They affect the vasomotor function of vasculature throughout the body via alterations in the activity of the autonomic nervous system, thus changing the blood flow to involve tissues such as mucosa of the GIT (see

Fig. 2). ROS in blood vessels are primarily produced by NADPH oxidases - multisubunit enzymes, which have recently received particular attention and have been reviewed elsewhere (16, 17). ROS, and in particular superoxide anion, interact with NO produced in the vessels as the main vasodilatation compound, leading to a rapid loss of NO bioavailability (13, 18). This causes not only vasoconstriction and loss of vasodilatation, but also insufficiency of other cellular functions of NO (such as anti-inflammatory actions of NO). Thus, scavenging of NO in the vasculature eventually leads to several pathologies such as hypertension, atherosclerosis, and vasculitis. The regulation of vascular function in the oral cavity by NO and ROS seems to be particularly important in the process of induction of oral lesions and ulcers and their healing. In general, infected or not infected oral lesions, always trigger the immune response leading to an increase in ROS in the injured area, which causes the impairment of vasodilatation of the local vascular bed and the decrease in the tissue regeneration of this region by the decrease of the blood flow and worsening supply of oxygen and nutrients.

Fig. 2. Oxidative stress and the involvement of free radicals in the pathogenesis of ulcers in gastrointestinal tract (GIT). However, ROS are not only involved in pathological processes in our body. They also play an important role in the signal transduction in the cells. ROS are produced by the nonphagocytic cells upon the activation of the many growth factor receptors, such as those for epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and vascular-endothelial growth factor (VEGF), which transduct downstream signals through the nitogen-activated protein kinases pathway (MAPKs) (19). Moreover, not only growth factor receptor signaling involves ROS, but also cytokine receptors including tumor necrosis factor alpha (TNF-alpha) and interferon F

gamma (INF- ) and interleukin receptors (IL-1) (21) also utilize the ROS as second messengers in their intracellular signaling pathway (21). Damage to nucleic acid by ROS may lead to mutation and development of cancer. ROS are also involved in the process of the regulation of p53 protein. P53 may be activated by UV radiation, hypoxia, gamma-radiation, nucleotides deprivation and triggers different pathway leading to elimination of damaged cells through to, large extent, ROS mediated apoptosis (22). Finally, we should keep in mind the importance of ROS in the protection against invading microorganisms as the main mechanism of the innate immune system function. Phagocytic cells, especially macrophages and neutrophils, generate extensive amounts of ROS through NADPH oxidases ("oxidative burst") as well as NO in parallel (by iNOS), and hydrogen peroxide (by superoxide dismutase - SOD) which in concert are superb tools in killing bacteria. This short overview of the role of various free radicals in the organism shows their dual nature. They may be beneficial for the system when their production is well controlled and restricted to the area, where they are needed. However when uncontrolled, they may be dangerous for the organism homeostasis, being involved in pathomechanism of many diseases such as: hypertension, atherosclerosis, rheumatoid arthritis, diabetes, cancers and neurological disorders; Alzheimer's and Parkinson's diseases as well as oral diseases. In spite of these findings, currently available anti-oxidant agents, such as vitamins A, C and E, failed to provide sufficient protection against ROS and RNS in majority of large clinical trials. Thus, numerous authors are currently focusing on more effective natural anti-oxidants, which would more accurately and in a more complex fashion reverse oxidative stress lesions and inflammation. This includes both plant antioxidants (23), as well as endogenous antioxidative enzymes (24). Interestingly, melatonin (MT), originating from L-tryptophan (Trp) through several enzymatic steps in pinealocytes as well as in numerous EE cells in the lining of the GIT (Fig. 3), including oral cavity, has several unusual properties which make it extremely promising compound from the therapeutic point of view.

Fig. 3. The adrenergic stimulation of generation (A) of MT in pinealocytes and the entero-endocrine (EE) (B) cells of the entire gastrointestinal tract (GIT) with nocturnal risein plasma MT level (C). Adapted by permission of T.J. Reiter. MELATONIN AS REGULATOR OF OXIDATIVE STRESS AND INFLAMMATION It is generally accepted that MT a pineal gland hormone, is mostly associated with the regulation of the circadian dark/light rhythm of the human body. However, MT has been recently recognized as a potent antioxidant and immuno-modulator. Additionally, MT does not have any toxicity but is highly lipophilic and this property facilitates its penetration through cell membranes and compartments (25). This suggests that MT could be used therapeutically for instance locally in the oral cavity diseases such as bacterial and viral lesions, post-surgical wounds from tooth extractions and oral surgeries, acting as a promoter of bone formation, as an important factor in auto-immunological disorders such as Sjorgen syndrome, in periodontal diseases, aphthous ulceration, lichen planus and even in oral carcinoma. This could be a novel application of the MT utilization and clinical studies are warranted to investigate this issue. However, solid literature evidence is already available, which should encourage testing MT as a potent drug for oral cavity. Biology of melatonin Synthesis of the MT by pinealocytes is inhibited by light and it reaches upper levels of production between 12.00 and 2.00 o'clock at night (25) (Fig. 3). This day/night fluctuation of plasma MT is controlled by noradrenergic neural stimulation coming directly from the retina of

eyes (26). However, recent studies showed that MT is synthesized not only in the brain but also by the numerous cells, including retina, ovary, Harderian gland, placenta, kidneys, respiratory tract and, finally, by the GIT, where, MT has been found to be generated in EE cells in about 500 times larger amounts than in pineal bodies (27, 28) (Fig. 4). Effects of MT in the lower part of GIT are particularly well studied and have been extensively discussed (29-34). As mentioned above MT is highly lipophilic and as a hormone can reach even distant compartments of the body, far from the secretion site. MT has been detected in high concentration even in the bone marrow and saliva (35).

Fig. 4. Immuno-fluorescence detection of melatonin in pinealocytes (A) and in EE cells of the GIT (B) (by permission from G.Bubenik collection). Melatonin and the oral cavity MT gets to the saliva by passive diffusion from the blood. This is why the concentration of MT in saliva was reported to be about 30% of that in the plasma (36). The amount of MT in saliva is lower when compared to blood value probably due to the fact that nearly 70 % of the plasma MT is bound to albumin and this prevents free diffusion of this indole into saliva. It is possible that MT is also produced and released locally by the mucosal lining of the oral cavity but this requires direct experimental evidence. Our preliminary observations on healthy volunteers revealed that under fasting conditions, the salivary concentration of MT amounts to about 50% of that in the plasma in the same subjects. Most importanly, exogenous MT applied topically to the restricted area of mucosal lining of oral cavity such as the palate was quickly and in time-dependent fashion absorbed from the mucosa resulting in the increase in plasma immunoreactive MT concentrations and reaching after 30 min

the levels attainable with the same dose of MT (5 mg) applied intragastrically (Fig. 5). It is of interest that the amounts of MT secreted at the same time into saliva paralleled the plasma indole concentrations attained by oral or intragastric application of MT. As far as we are aware, these are the first reports on the oral absorption of MT applied to the restricted area of the mouse cavity. Our results may have important clinical implications because MT could be applied directly on oral mucosa in a variety of infectious and non-infectious oxidative stress diseases (Fig. 6) of oral cavity including denture-induced stomatitis (stomatitis prothetica), gingivitis, healing of lesions and ulcerations caused by tooth extraction (alveolitis), postsugical trauma in oral cavity and vestibular plastic operations with the use of CO-laser. It is expected that such topical application of MT might be effective in combating the inflammatory processes and in accelerating the healing of erosions and ulcers in oral cavity.

Fig. 5. Plasma and salivary concentrations of MT following limited to palate or gastric application of 5 mg of melatonin. Mean SEM of 11 determinations in 11 healthy volunteers. The major and important property of MT is its ability to serve as a very potent free radical scavenger (37, 38). Indoleamine donates electrons to the free radicals becoming itself indolyl cation radical. The latter undergoes several other reactions to be inverted to N1-acetyl-N2formyl-5-methoxykynuramine and N1-acetyl-5-methoxykynuramine which are excreted in the urine (39, 40). Besides the direct scavenging of free radicals, MT influences the oxidative stress status in an indirect way by stabilizing the inner mitochondrial membrane what improves the electron transport chain located there (41). Moreover, MT inhibits iNOS, an inducible enzyme which produces excessive amounts of NO, that are no any longer beneficial to the system, but

increase oxidative stress due to its conversion together with ROS into destructive RNS (42). Additionally, MT in pharmacological and in physiological doses increase gene expression and activity of endogenous anti-oxidant enzymes such as glutathione peroxidase (GPx), superoxide dysmuthase (SOD), catalase (CAT), which are important in maintaining the integrity of vasculature and other tissues (43, 44). These antioxidant properties of MT could turn out very beneficial for treatment of the local inflammatory lesions and for accelerating the healing process e.g. after tooth extraction and other surgical procedures in oral cavity (Fig. 6). Recently, Cutando et al. have shown the favorable effects of the local MT administration to the alveolar sockets after molar premolars extraction in dogs (45). The individuals without MT regiment showed an increase of lipid peroxidation, nitrite plus nitrate levels in plasma and GSSG/GSS (glutathione disulfide/glutathione) ratio in erythrocytes. Dogs that got 2 mg MT to the extraction socket just after extraction didn't show this increase. Konturek et al. (46) have shown in an animal model that exogenous MT prevents the formation of acute gastric lesions induced by stress and accelerates regeneration of chronic gastric ulcers due to increased activity of cNOS and the cyclooxygenase (COX)-prostaglandin E2 (PGE2) system resulting in the increase of mucosal blood flow and mucosal integrity. Recently, Konturek et al. (47) reported that MT given orally is highly effective in prevention of acute esophageal lesion induced by direct perfusion of the esophagus using acid, pepsin and bile solution.

Fig. 6. The formation of various types of reactive radicals in response to the presence of oxidative stress related to mucosal damage and antioxidative enzymes involved in neutralization of free radicals. Another very important potential utilization of the anti-inflammatory abilities of MT is the treatment of the periodontal diseases. It has been already established that periodontal tissue has

been destructed in the course of periodontitis by the disproportionate immunological response to the triggering agent such as bacteria of the plaque (48). Free radicals burst coming from the phagocytic cells, such as neutrophils and macrophages, migrating to the inflammation place, damage significantly the gingival tissue (49). Tsai et al. claimed that lipid peroxidation is a major factor in the induction and progression of chronic periodontitis (50). Therefore, the decrease of ROS by MT in the inflamed area would be beneficial to the course of the disease. Moreover, MT influences fibroblast activity and bone regeneration by promoting osteoblast differentiation and bone formation (51). MT also stimulates the synthesis of type I collagen fibers (52, 53). It has been established that MT mediates these effects through receptors localized on pre-osteoblasts which leads to the production of bone sialoprotein, alkaline phosphatase, osteopontine, osteocalcine in these cells, thus significantly shortening the time needed for their differentiation into mature osteoblasts from 21 to 12 days (35). At the same time, MT indirectly inhibits osteoclast differentiation, by reducing the amount of oxygen-derived free radicals in the tissue. Increase of the oxidative stress in the inflamed area usually triggers the activity of the osteoclast and the bone resorption (54). Not only the pathology of periodontal disease is connected with ROS over-production. Reactive species are involved in pathogenesis of other oral disorders such as aphthous ulceration (45), leukoplakia (48), lichen planus (55), but more studies are needed to investigate the benefits of melatonin treatment of the following diseases. Finally, MT appears to be a promising anticancer agent. Firstly, it scavenges ROS that are known as second messenger in the signaling pathways leading to the cell division. Additionally, MT amplifies the anti-tumor activity of IL-2. MT has been already proven as a powerful cytostatic drug in vitro as well in vivo (56 - 58). These anticancer abilities are surely connected with the fact that MT is very potent immuno-modulator. MT improves cellular responses of the adaptive immunity by stimulating IFN- production and it favors T cell differentiation to Th1 by increasing level of IL-12 and IL-6 (59). However, increasied IL-4 secretion also supports increase of humoral responses. MT improves innate immunity as well by increasing monocyte cytotoxicity and IL-1 production (60). MT also facilitates the stimulation of T cells by increasing IL-2 production (59). On the other hand, melatonin has been demonstrated to restrict inflammation by the inhibition of TNF-alpha by blocking NF B pathway (60). However, there are numerous conflicting data regarding the effects of MT in the regulation of inflammation and immune response. In some models melatonin behaves as an immunological positive modulator increasing in general the amount of pro-inflammatory cytokines (59), but in other models results show decrease in pro-inflammatory cytokines (61, 62). The reason for this discrepancy may be related to the differences in model systems or to the differences in the initial status of the immune cells. It may have differential effects on activated and quiescent immune cells. Another possibility is that anti-inflammatory or pro-immune effects of MT may be related to dual actions of oxidative stress in this system (18). Moreover, the final result of melatonin action depends which tissue is involved in an inflammatory process and what is the pathophysiology of the process. Additionally, the presence of MT in the culture medium released by immune cells may mask the effect of exogenous MT. Yet, another way MT may influence the immune system is to penetrate to bone marrow compartment and to have a hematopoietic effect. Maestroni et al. have showed in mouse model that melatonin influences the blood-forming system via induction or stimulation of T-helper cellderived cytokines exerting colony-stimulating activity (63-65).

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R e c e i v e d : June 16, 2007 A c c e p t e d : June 2, 2007 Authors address: Prof. Stanisaw J. Konturek, Department of Clinical Physiology, Jagiellonian University School of Medicine, 16 Grzegorzecka, 31-531 Krakow, Poland; Tel. +(12) 4211006; e-mail: mpkontur@cyf-kr.edu.pl