Respiratory Physiology & Neurobiology 209 (2015) 91–94
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Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Oxidative stress in the oral cavity: Sources and pathological outcomes夽 Katia Avezov a,b , Abraham Z. Reznick a,∗ , Dror Aizenbud a,b a b
Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, PO Box 9649, Haifa, Israel Orthodontic and Craniofacial Department, Graduate School of Dentistry, Rambam Health Care Campus, PO Box 9602, Haifa, Israel
a r t i c l e
i n f o
Article history: Accepted 14 October 2014 Available online 23 October 2014 Keywords: Oral cavity Oxidative stress Oral pathology
a b s t r a c t Oxidative stress (OS), an imbalance in the oxidant–antioxidant equilibrium, is thought to be involved in the development of many seemingly unrelated diseases. Oral cavity tissues are a unique environment constantly exposed to internal and external compounds and material hazards as almost no other part of the human body. Some of the compounds are capable of generating OS. Here, the main groups of endogenous as well as exogenous OS sources are presented, followed by their oxidative effect on the salivary contents and function. The oxidative mechanisms in oral cells and their pathologic influence are also discussed. © 2014 Elsevier B.V. All rights reserved.
1. Oxidative stress Oxidative stress (OS) is defined as an imbalance between the load of chemically reactive oxidants such as reactive oxygen and nitrogen species (ROS and RNS) and the ability of a biological system to detoxify them or to repair the resulting damage. ROS and RNS include chemically active molecules containing oxygen and nitrogen such as H2 O2 (hydrogen peroxide), HOCl (hypochlorous acid), and ONOO− (peroxynitrite). They also include oxygen and nitrogen free radicals. Free radicals are atomic or molecular species with one or more unpaired electrons in their structure. The most prominent biological free radicals are O2 − (superoxide), OH (hydroxyl radical) and NO (nitric oxide). Due to their unpaired electron, they are highly chemically active, easily react with other molecules, and through a chain reactions create even more free radicals capable of damaging all cellular components, including proteins, lipids, carbohydrates and DNA (Valko et al., 2007). ROS also act as cellular messengers in redox signaling and thereby modify normal cellular signaling pathways (Droge, 2002). In humans, OS is thought to be involved in the development of over 100 seemingly unrelated diseases including cancer, Parkinson’s, Alzheimer’s, atherosclerosis, myocardial infarction as well as periodontal disease (D’Aiuto et al., 2010; Lavie and Lavie, 2009; Reibel, 2003; Valko et al., 2007).
夽 This paper is part of a special issue entitled “Molecular basis of ventilatory disorders” guest-edited by Dr. Mietek Pokorski. ∗ Corresponding author. Tel.: +97 248295388; fax: +97 248295403. E-mail address: [email protected] (A.Z. Reznick). http://dx.doi.org/10.1016/j.resp.2014.10.007 1569-9048/© 2014 Elsevier B.V. All rights reserved.
There are endogenous as well as exogenous ROS/RNS sources. Endogenously ROS are natural byproducts of mitochondrial oxygen metabolism (Lavie, 2003), where O2 − , H2 O2 , and highly reactive HO are formed due to an incomplete reduction of molecular oxygen. ROS/RNS are produced in activated leukocytes by NADPH oxidase in order to destroy pathogens, and by xanthine oxidase, or cytochrome P450s in metabolism in the liver. Exogenously free radicals originate from sources such as ionizing radiation and environmental pollutants such as cigarette smoke, which will be discussed further in detail. Biological systems have developed the ability to detoxify chemically active ROS and RNS. These antioxidant (AO) mechanisms include complex non-enzymatic systems such as glutathione (GSH), Vitamins A, C and E as well as enzymes such as catalase, superoxide dismutase (SOD) and various peroxidases. Insufficient levels of antioxidants, or inhibition of the antioxidant enzymes, also promote oxidative stress (Valko et al., 2007).
2. Oxidative stress sources in the oral cavity 2.1. Food Being the primary orifice for food consumption, oral tissues are exposed to a variability of substances, textures and temperatures. These may bear a negative or positive effect on human physiology, but they also have a local effect on oral tissues. For example, alcohol consumption may result in oxidant–antioxidant imbalance due to a decrease in AO and excessive amounts of free radical production, which may lead to initiation of oral cancer (Choudhari et al.,
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2014). Indeed, alcohol was found to increase oral cancer risk and mortality in humans (Petti and Scully, 2007). Furthermore, tissue depletion of Vitamins C and E was observed in alcoholics due to their increased utilization in free radical scavenging (Nordmann, 1994). Ethanol is oxidized by cytochrome P450 2E1 (CYP2E1) to acetaldehyde and further oxidized to acetate. Chronic ethanol consumption results in increased CYP2E1 activity, ROS generation and DNA adducts (Seitz and Stickel, 2007). Food preparation can also affect its oxidative status. For example, acrolein, highly chemically active aldehyde, was detected in the emissions of heated cooking oils, and can be formed by different steps of food processing from amino acids, animal fats or carbohydrates (Beauchamp et al., 1985; Indart et al., 2007). Acrolein acts as GSH scavenger, thus indirectly increasing OS (Kehrer and Biswal, 2000). Generally, oral tissues are frequently exposed to high temperatures following hot food consumption. Heat stress induced the production of ROS, nuclear translocation of Nuclear factor-erythroid 2 related factor 2 (Nrf2) and increased expression of Nrf2 target genes including heme oxygenase-1 (HO-1) in human dental pulp (HDP) cells (Chang et al., 2009). On the other hand, consumption of products with AO capacities can promote oral health. For example, green tea is an important source of polyphenol antioxidants, including epigallocatechin 3 gallate (EGCG). There is growing evidence of its beneficial role against oral OS and inflammation. Generally, green tea defends healthy cells from malignant transformation and locally has the ability to induce apoptosis in oral cancer cells (Narotzki et al., 2012).
2.2. Inflammation Inflammatory conditions of the oral cavity tissues are rather common. The most prevalent are gingivitis and periodontitis, the inflammation of the tooth supporting tissues, affecting 50–90% of the population. In periodontitis, as opposed to gingivitis, there is also a loss of the tooth supporting bone, leading to teeth loosening and subsequent loss. Untreated, chronic gingivitis may propagate into periodontitis (Armitage, 1995; Offenbacher, 1996). Both conditions are a result of the host immune response to the accumulation of oral bacteria in the gingival sulcus of the mouth (Scott and Singer, 2004), and an inappropriate levels of gingival leukocyte functions. Another, less common inflammatory disorders of the oral tissues include fungal and viral infections such as oral candidiasis, and autoimmune conditions such as oral lichen planos, pemphigus vulgaris and others. ROS and RNS are endogenously produced by phagocytes (neutrophils and monocytes) in order to destroy pathogens; the primary enzyme producing O2 − is NADPH oxidase, with additional enzymes as nitric oxide synthase, SOD and myeloperoxidase, which generate H2 O2 , HClO and NO (Lavie, 2003). Further non-enzymatic reaction of these molecules with each other generate additional powerful ROS OH and OONO− . These free radicals and oxidants, produced by phagocytes to kill invading pathogens, can also damage the surrounding tissues by generating OS. For example, aggressive periodontitis, a rapid and severe destruction of the periodontal tissues, is characterized by neutrophil hyperactivity with enhanced ROS generation (Giannopoulou et al., 2008). Stimulated neutrophils from aggressive periodontitis patients exhibited greater ROS production than the matched control subjects (Asman, 1988; Asman et al., 1988). In successfully treated aggressive periodontitis patients, it was demonstrated that hyperactivity was present before and after treatment, thus supporting the hypothesis that ROS hyper-responsiveness is constitutional rather than reactive (Asman et al., 1988). Aside from the local destructive properties, during gingival and periodontal inflammation, thereis an
increase in gingival crevicular fluid (GCF), which is spilled into the saliva with inflammatory response products, thus possibly affecting other oral tissues (D’Aiuto et al., 2010). 2.3. Cigarette smoke Cigarette smoke (CS) is a complicated mixture of innumerable components involved in many pathologic processes and able to cause diseases in different organs (Kuper et al., 2002). The gas phase of CS contains more than 1015 free radicals per puff, while the particulate phase contains more than 1017 free radicals per gram (Swan and Lessov-Schlaggar, 2007). ROS contained in the gas phase cigarette smoke include H2 O2 , O2 − and OH. Additionally, cigarette smoke is a great source of RNS as NO. NO and O2 − may react to form ONOO− , a potent oxidizing and nitrating compound which also has been linked with a variety of pathological conditions (Hasnis et al., 2007). This makes CS one of the most powerful oxidative stress inducers in the oral cavity. Unsaturated aldehydes (e.g. acrolein, crotonaldehyde) are another CS borne chemically active substances. A single cigarette contains 0.21 mol crotonaldehyde and 0.8 mol acrolein (Reznick et al., 1992). These substances are a major source of double bonds reacting with SH (thiol) groups of proteins such as cysteine (Kehrer and Biswal, 2000), but also histidine, arginine and lysine residues (Uchida et al., 1998). In this reaction, called Michael addition, aldehydic carbonyls are attached to a protein and induce structural alterations. Besides proteins, unsaturated aldehydes can react with nucleophilic sites of macromolecules such as DNA and phospholipids; especially sensitive protein targets may be enzymes, nuclear factors and antioxidants such as GSH (Dalle-Donne et al., 2003; Kehrer and Biswal, 2000; Nystrom, 2005). 2.4. Dental materials Oral cavity tissues are exposed to a variety of different materials used for restoration of teeth function and aesthetics. These materials include metals, porcelain, various kinds of acrylic polymers, resins and adhesives, the biocompatibility of each group was not entirely uncovered. It is known (Drummond, 2008; Ferracane, 2013; Lohbauer et al., 2013; Wataha, 2000) that there is a constant leakage of metal ions from metallic restorations and monomers from incomplete curing of resins and acrylic restorations, some of them can interact with living oral tissues and induce OS. For example, resin monomers such as hydroxyethylmethacrylate (HEMA) induce OS by an indirect mechanism; they bind to GSH thiol group in Michael addition reaction, preventing its oxidation to glutathione disulfide (GSSG). This results in a decrease in GSH levels, and causes downregulation of glutathione peroxidase (GPx) expression and increased H2 O2 formation leading to catalase induction and a feedback inhibition of SOD (Krifka et al., 2013; Noda et al., 2005). Thus, decreased levels of antioxidants and inhibition of antioxidant enzymes lead to OS formation. Sublethal concentrations of ions of Ag, Be, Co, Cu, Hg, Ni, Pd, and Zn all known to be released from dental biomaterials also altered the GSH cellular levels in human THP-1 monocytes. This may play a role in altered cytokine secretion in the inflammatory response to dental metals (Wataha et al., 2000). Furthermore, hydrogen peroxide and hydroxyl radicals were generated in vascular smooth muscle cells (VSMCs), a model for dental pulp vascular cells, by irradiation with blue light, used for resin curing (Yoshino et al., 2012). The blue light irradiation induced cytotoxicity associated with oxidative stress, which increased lipid peroxidation and apoptosis. In the same study, addition of N-acetyl-l-cysteine (NAC), a typical intracellular antioxidant, protected VSMCs against cytotoxicity associated with oxidative stress.
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3. Oxidative stress in oral pathology 3.1. Saliva Salivary fluid of humans contains 99.5% water, while the other 0.5% consists of electrolytes, mucus, glycoproteins, enzymes, and antibacterial compounds. Saliva serves a lubricative function, wetting food and permitting the initiation of digestion and swallowing, thus protecting the mucosal surfaces of the oral cavity. Saliva is constantly replaced; it is estimated that a healthy person produces 0.75 to 1.5 l of saliva per day. Saliva possesses AO properties as well. These AO properties are represented in the saliva by SOD, salivary peroxidase, catalase, uric acid, and GSH. Under physiologic conditions, these AO agents preserve the oxidant–antioxidant balance. But in pathologic conditions, an overload of oxidants can excess the AO capacity of the saliva. For example, in smokers, the AO capacity of saliva was found to be diminished. Even a single cigarette is sufficient to significantly reduce GSH concentration and impair its protective role against the noxious biochemical effects of CS (Zappacosta et al., 1999). This effect is attributed to CS unsaturated aldehydes, which react with cysteine thiol of GSH. Also, CS aldehydes were found to be responsible for an increase of salivary protein carbonyls, an OS marker, (Avezov et al., 2014b; Nagler et al., 2000), with a concurrent decrease in the activity of some salivary enzymes, such as lactate dehydrogenase (LDH), aspartate aminotransferase (AST), acid phosphatase and amylase (Avezov et al., 2014a; Nagler et al., 2001; Zappacosta et al., 2002). The damage to salivary proteins may lead to their impaired AO, digestive and antibacterial properties. A drop in AO protection by CS can expose oral tissues to oxidative damage from other sources mentioned above, and explain why tobacco products are a leading risk factor in almost all oral diseases, including oral cancer. 3.2. Oral cells The salivary flow through the oral structures spreads the dissolved oxidants from different sources, so they can reach and affect other oral cells. All cellular contents are susceptible to ROS/RNS damage, including membrane lipids, proteins, and DNA. ROS/RNS
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induce lipid peroxidation of the polyunsaturated fatty acids in cell membranes. As a result, reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) are formed, and can further react with proteins and DNA (Uchida, 2003; Valko et al., 2006). High levels of such reactive peroxidation end products have been reported in oral cancer and precancer patients (Korde et al., 2011; Rasheed et al., 2007). Markers of lipoperoxidation and carbonyl stress are increased in patients with oral premalignant lesions (Vlkova et al., 2012) together with decreased AO status. Cellular protein content is also prone to oxidative damage, including fragmentation, cross-linking, and aggregation. Amino acid residues such as cysteine, histidine and lysine side chains are very susceptible to attack by ROS/RNS (Stadtman, 2004). Also, protein carbonyls are formed (Stadtman and Levine, 2000). Some important proteins such as DNA repair enzymes can be damaged, and increase the frequency of mutations. Indeed, mutagenic 8-nitroguanine, an indicator of nitrative DNA damage has been observed in the oral epithelium biopsies of patients with leukoplakia, whereas little or none were observed in normal oral mucosa (Ma et al., 2006). Markers related to oxidative stress, DNA repair, carcinogenesis, metastasis and cellular death are also present in the saliva of 19 tongue cancer patients (Shpitzer et al., 2009). Not only destructive, but also rescue pathways are activated protecting the cells against oxidative injury, such as the Nrf2 pathway. It regulates the expression of phase II detoxifying enzymes involved in the GSH biosynthesis pathway (i.e. glutathione-Stransferase (GST), ␥-glutamyl-transpeptidase (GGT), and others (Cho and Kleeberger, 2010; Cho et al., 2006), along with stress proteins such as HO-1. Cells and tissues where routine detoxification reactions normally occur, constitutively express Nrf2, including the lung, intestine, and liver (Cho and Kleeberger, 2010; Shin et al., 2013). The Nrf2 pathway is known to be involved in the cellular protection after exposure to CS and aldehydes in the respiratory system and ocular epithelium (Kosmider et al., 2011). However, very little is known about its regulation in the oral environment. Future studies should focus upon possibilities to boost the protective mechanisms involved in oral cell protection. In conclusion: The main exogenous and endogenous sources of OS in the oral cavity were presented. Fig. 1 summarizes their effects
Fig. 1. The effect of oxidative stress sources on the oral cavity: the influence on the saliva and oral cells.
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in the two main oral habitats: the salivary fluid and oral cells. Although some of the OS sources in the oral cavity are inescapable, others are changeable and may be controlled in order to reduce oral cavity pathologies and morbidity. Acknowledgements This work was supported by grants from the Rappaport Institute and the Krol Foundation of Barnegat, NJ, to AZR. References Armitage, G.C., 1995. Clinical evaluation of periodontal diseases. Periodontology 2000 (7), 39–53. Asman, B., 1988. Peripheral PMN cells in juvenile periodontitis. Increased release of elastase and of oxygen radicals after stimulation with opsonized bacteria. J. Clin. Periodontol. 15, 360–364. Asman, B., Bergstrom, K., Wijkander, P., Lockowandt, B., 1988. Peripheral PMN cell activity in relation to treatment of juvenile periodontitis. Scand. J. Dent. Res. 96, 418–420. Avezov, K., Reznick, A.Z., Aizenbud, D., 2014a. LDH enzyme activity in human saliva: the effect of exposure to cigarette smoke and its different components. Arch. Oral. Biol. 59, 142–148. Avezov, K., Reznick, A.Z., Aizenbud, D., 2014b. Oxidative damage in keratinocytes exposed to cigarette smoke and aldehydes. Toxicol. In Vitro 28, 485–491 (an international journal published in association with BIBRA). Beauchamp Jr., R.O., Andjelkovich, D.A., Kligerman, A.D., Morgan, K.T., Heck, H.D., 1985. A critical review of the literature on acrolein toxicity. Crit. Rev. Toxicol. 14, 309–380. Chang, S.W., Lee, S.I., Bae, W.J., Min, K.S., Shin, E.S., Oh, G.S., Pae, H.O., Kim, E.C., 2009. Heat stress activates interleukin-8 and the antioxidant system via Nrf2 pathways in human dental pulp cells. J. Endod. 35, 1222–1228. Cho, H.Y., Kleeberger, S.R., 2010. Nrf2 protects against airway disorders. Toxicol. Appl. Pharmacol. 244, 43–56. Cho, H.Y., Reddy, S.P., Kleeberger, S.R., 2006. Nrf2 defends the lung from oxidative stress. Antioxid. Redox Signal. 8, 76–87. Choudhari, S.K., Chaudhary, M., Gadbail, A.R., Sharma, A., Tekade, S., 2014. Oxidative and antioxidative mechanisms in oral cancer and precancer: a review. Oral Oncol. 50, 10–18. D’Aiuto, F., Nibali, L., Parkar, M., Patel, K., Suvan, J., Donos, N., 2010. Oxidative stress, systemic inflammation, and severe periodontitis. J. Dent. Res. 89, 1241–1246. Dalle-Donne, I., Giustarini, D., Colombo, R., Rossi, R., Milzani, A., 2003. Protein carbonylation in human diseases. Trends Mol. Med. 9, 169–176. Droge, W., 2002. Free radicals in the physiological control of cell function. Physiol. Rev. 82, 47–95. Drummond, J.L., 2008. Degradation, fatigue, and failure of resin dental composite materials. J. Dent. Res. 87, 710–719. Ferracane, J.L., 2013. Resin-based composite performance: are there some things we can’t predict? Dent. Mater. 29, 51–58 (official publication of the Academy of Dental Materials). Giannopoulou, C., Krause, K.H., Muller, F., 2008. The NADPH oxidase NOX2 plays a role in periodontal pathologies. Semin. Immunopathol. 30, 273–278. Hasnis, E., Bar-Shai, M., Burbea, Z., Reznick, A.Z., 2007. Mechanisms underlying cigarette smoke-induced NF-kappaB activation in human lymphocytes: the role of reactive nitrogen species. J. Physiol. Pharmacol. 58 (Suppl. 5), 275–287 (an official journal of the Polish Physiological Society). Indart, A., Viana, M., Clapes, S., Izquierdo, L., Bonet, B., 2007. Clastogenic and cytotoxic effects of lipid peroxidation products generated in culinary oils submitted to thermal stress. Food Chem. Toxicol. 45, 1963–1967 (an international journal published for the British Industrial Biological Research Association). Kehrer, J.P., Biswal, S.S., 2000. The molecular effects of acrolein. Toxicol. Sci. 57, 6–15 (an official journal of the Society of Toxicology). Korde, S.D., Basak, A., Chaudhary, M., Goyal, M., Vagga, A., 2011. Enhanced nitrosative and oxidative stress with decreased total antioxidant capacity in patients with oral precancer and oral squamous cell carcinoma. Oncology 80, 382–389. Kosmider, B., Messier, E.M., Chu, H.W., Mason, R.J., 2011. Human alveolar epithelial cell injury induced by cigarette smoke. PLoS One 6, e26059. Krifka, S., Spagnuolo, G., Schmalz, G., Schweikl, H., 2013. A review of adaptive mechanisms in cell responses towards oxidative stress caused by dental resin monomers. Biomaterials 34, 4555–4563. Kuper, H., Adami, H.O., Boffetta, P., 2002. Tobacco use, cancer causation and public health impact. J. Intern. Med. 251, 455–466. Lavie, L., 2003. Obstructive sleep apnoea syndrome—an oxidative stress disorder. Sleep Med. Rev. 7, 35–51.
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Contents lists available at ScienceDirect
Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Oxidative stress in the oral cavity: Sources and pathological outcomes夽 Katia Avezov a,b , Abraham Z. Reznick a,∗ , Dror Aizenbud a,b a b
Department of Anatomy and Cell Biology, Rappaport Faculty of Medicine, Technion—Israel Institute of Technology, PO Box 9649, Haifa, Israel Orthodontic and Craniofacial Department, Graduate School of Dentistry, Rambam Health Care Campus, PO Box 9602, Haifa, Israel
a r t i c l e
i n f o
Article history: Accepted 14 October 2014 Available online 23 October 2014 Keywords: Oral cavity Oxidative stress Oral pathology
a b s t r a c t Oxidative stress (OS), an imbalance in the oxidant–antioxidant equilibrium, is thought to be involved in the development of many seemingly unrelated diseases. Oral cavity tissues are a unique environment constantly exposed to internal and external compounds and material hazards as almost no other part of the human body. Some of the compounds are capable of generating OS. Here, the main groups of endogenous as well as exogenous OS sources are presented, followed by their oxidative effect on the salivary contents and function. The oxidative mechanisms in oral cells and their pathologic influence are also discussed. © 2014 Elsevier B.V. All rights reserved.
1. Oxidative stress Oxidative stress (OS) is defined as an imbalance between the load of chemically reactive oxidants such as reactive oxygen and nitrogen species (ROS and RNS) and the ability of a biological system to detoxify them or to repair the resulting damage. ROS and RNS include chemically active molecules containing oxygen and nitrogen such as H2 O2 (hydrogen peroxide), HOCl (hypochlorous acid), and ONOO− (peroxynitrite). They also include oxygen and nitrogen free radicals. Free radicals are atomic or molecular species with one or more unpaired electrons in their structure. The most prominent biological free radicals are O2 − (superoxide), OH (hydroxyl radical) and NO (nitric oxide). Due to their unpaired electron, they are highly chemically active, easily react with other molecules, and through a chain reactions create even more free radicals capable of damaging all cellular components, including proteins, lipids, carbohydrates and DNA (Valko et al., 2007). ROS also act as cellular messengers in redox signaling and thereby modify normal cellular signaling pathways (Droge, 2002). In humans, OS is thought to be involved in the development of over 100 seemingly unrelated diseases including cancer, Parkinson’s, Alzheimer’s, atherosclerosis, myocardial infarction as well as periodontal disease (D’Aiuto et al., 2010; Lavie and Lavie, 2009; Reibel, 2003; Valko et al., 2007).
夽 This paper is part of a special issue entitled “Molecular basis of ventilatory disorders” guest-edited by Dr. Mietek Pokorski. ∗ Corresponding author. Tel.: +97 248295388; fax: +97 248295403. E-mail address: [email protected] (A.Z. Reznick). http://dx.doi.org/10.1016/j.resp.2014.10.007 1569-9048/© 2014 Elsevier B.V. All rights reserved.
There are endogenous as well as exogenous ROS/RNS sources. Endogenously ROS are natural byproducts of mitochondrial oxygen metabolism (Lavie, 2003), where O2 − , H2 O2 , and highly reactive HO are formed due to an incomplete reduction of molecular oxygen. ROS/RNS are produced in activated leukocytes by NADPH oxidase in order to destroy pathogens, and by xanthine oxidase, or cytochrome P450s in metabolism in the liver. Exogenously free radicals originate from sources such as ionizing radiation and environmental pollutants such as cigarette smoke, which will be discussed further in detail. Biological systems have developed the ability to detoxify chemically active ROS and RNS. These antioxidant (AO) mechanisms include complex non-enzymatic systems such as glutathione (GSH), Vitamins A, C and E as well as enzymes such as catalase, superoxide dismutase (SOD) and various peroxidases. Insufficient levels of antioxidants, or inhibition of the antioxidant enzymes, also promote oxidative stress (Valko et al., 2007).
2. Oxidative stress sources in the oral cavity 2.1. Food Being the primary orifice for food consumption, oral tissues are exposed to a variability of substances, textures and temperatures. These may bear a negative or positive effect on human physiology, but they also have a local effect on oral tissues. For example, alcohol consumption may result in oxidant–antioxidant imbalance due to a decrease in AO and excessive amounts of free radical production, which may lead to initiation of oral cancer (Choudhari et al.,
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2014). Indeed, alcohol was found to increase oral cancer risk and mortality in humans (Petti and Scully, 2007). Furthermore, tissue depletion of Vitamins C and E was observed in alcoholics due to their increased utilization in free radical scavenging (Nordmann, 1994). Ethanol is oxidized by cytochrome P450 2E1 (CYP2E1) to acetaldehyde and further oxidized to acetate. Chronic ethanol consumption results in increased CYP2E1 activity, ROS generation and DNA adducts (Seitz and Stickel, 2007). Food preparation can also affect its oxidative status. For example, acrolein, highly chemically active aldehyde, was detected in the emissions of heated cooking oils, and can be formed by different steps of food processing from amino acids, animal fats or carbohydrates (Beauchamp et al., 1985; Indart et al., 2007). Acrolein acts as GSH scavenger, thus indirectly increasing OS (Kehrer and Biswal, 2000). Generally, oral tissues are frequently exposed to high temperatures following hot food consumption. Heat stress induced the production of ROS, nuclear translocation of Nuclear factor-erythroid 2 related factor 2 (Nrf2) and increased expression of Nrf2 target genes including heme oxygenase-1 (HO-1) in human dental pulp (HDP) cells (Chang et al., 2009). On the other hand, consumption of products with AO capacities can promote oral health. For example, green tea is an important source of polyphenol antioxidants, including epigallocatechin 3 gallate (EGCG). There is growing evidence of its beneficial role against oral OS and inflammation. Generally, green tea defends healthy cells from malignant transformation and locally has the ability to induce apoptosis in oral cancer cells (Narotzki et al., 2012).
2.2. Inflammation Inflammatory conditions of the oral cavity tissues are rather common. The most prevalent are gingivitis and periodontitis, the inflammation of the tooth supporting tissues, affecting 50–90% of the population. In periodontitis, as opposed to gingivitis, there is also a loss of the tooth supporting bone, leading to teeth loosening and subsequent loss. Untreated, chronic gingivitis may propagate into periodontitis (Armitage, 1995; Offenbacher, 1996). Both conditions are a result of the host immune response to the accumulation of oral bacteria in the gingival sulcus of the mouth (Scott and Singer, 2004), and an inappropriate levels of gingival leukocyte functions. Another, less common inflammatory disorders of the oral tissues include fungal and viral infections such as oral candidiasis, and autoimmune conditions such as oral lichen planos, pemphigus vulgaris and others. ROS and RNS are endogenously produced by phagocytes (neutrophils and monocytes) in order to destroy pathogens; the primary enzyme producing O2 − is NADPH oxidase, with additional enzymes as nitric oxide synthase, SOD and myeloperoxidase, which generate H2 O2 , HClO and NO (Lavie, 2003). Further non-enzymatic reaction of these molecules with each other generate additional powerful ROS OH and OONO− . These free radicals and oxidants, produced by phagocytes to kill invading pathogens, can also damage the surrounding tissues by generating OS. For example, aggressive periodontitis, a rapid and severe destruction of the periodontal tissues, is characterized by neutrophil hyperactivity with enhanced ROS generation (Giannopoulou et al., 2008). Stimulated neutrophils from aggressive periodontitis patients exhibited greater ROS production than the matched control subjects (Asman, 1988; Asman et al., 1988). In successfully treated aggressive periodontitis patients, it was demonstrated that hyperactivity was present before and after treatment, thus supporting the hypothesis that ROS hyper-responsiveness is constitutional rather than reactive (Asman et al., 1988). Aside from the local destructive properties, during gingival and periodontal inflammation, thereis an
increase in gingival crevicular fluid (GCF), which is spilled into the saliva with inflammatory response products, thus possibly affecting other oral tissues (D’Aiuto et al., 2010). 2.3. Cigarette smoke Cigarette smoke (CS) is a complicated mixture of innumerable components involved in many pathologic processes and able to cause diseases in different organs (Kuper et al., 2002). The gas phase of CS contains more than 1015 free radicals per puff, while the particulate phase contains more than 1017 free radicals per gram (Swan and Lessov-Schlaggar, 2007). ROS contained in the gas phase cigarette smoke include H2 O2 , O2 − and OH. Additionally, cigarette smoke is a great source of RNS as NO. NO and O2 − may react to form ONOO− , a potent oxidizing and nitrating compound which also has been linked with a variety of pathological conditions (Hasnis et al., 2007). This makes CS one of the most powerful oxidative stress inducers in the oral cavity. Unsaturated aldehydes (e.g. acrolein, crotonaldehyde) are another CS borne chemically active substances. A single cigarette contains 0.21 mol crotonaldehyde and 0.8 mol acrolein (Reznick et al., 1992). These substances are a major source of double bonds reacting with SH (thiol) groups of proteins such as cysteine (Kehrer and Biswal, 2000), but also histidine, arginine and lysine residues (Uchida et al., 1998). In this reaction, called Michael addition, aldehydic carbonyls are attached to a protein and induce structural alterations. Besides proteins, unsaturated aldehydes can react with nucleophilic sites of macromolecules such as DNA and phospholipids; especially sensitive protein targets may be enzymes, nuclear factors and antioxidants such as GSH (Dalle-Donne et al., 2003; Kehrer and Biswal, 2000; Nystrom, 2005). 2.4. Dental materials Oral cavity tissues are exposed to a variety of different materials used for restoration of teeth function and aesthetics. These materials include metals, porcelain, various kinds of acrylic polymers, resins and adhesives, the biocompatibility of each group was not entirely uncovered. It is known (Drummond, 2008; Ferracane, 2013; Lohbauer et al., 2013; Wataha, 2000) that there is a constant leakage of metal ions from metallic restorations and monomers from incomplete curing of resins and acrylic restorations, some of them can interact with living oral tissues and induce OS. For example, resin monomers such as hydroxyethylmethacrylate (HEMA) induce OS by an indirect mechanism; they bind to GSH thiol group in Michael addition reaction, preventing its oxidation to glutathione disulfide (GSSG). This results in a decrease in GSH levels, and causes downregulation of glutathione peroxidase (GPx) expression and increased H2 O2 formation leading to catalase induction and a feedback inhibition of SOD (Krifka et al., 2013; Noda et al., 2005). Thus, decreased levels of antioxidants and inhibition of antioxidant enzymes lead to OS formation. Sublethal concentrations of ions of Ag, Be, Co, Cu, Hg, Ni, Pd, and Zn all known to be released from dental biomaterials also altered the GSH cellular levels in human THP-1 monocytes. This may play a role in altered cytokine secretion in the inflammatory response to dental metals (Wataha et al., 2000). Furthermore, hydrogen peroxide and hydroxyl radicals were generated in vascular smooth muscle cells (VSMCs), a model for dental pulp vascular cells, by irradiation with blue light, used for resin curing (Yoshino et al., 2012). The blue light irradiation induced cytotoxicity associated with oxidative stress, which increased lipid peroxidation and apoptosis. In the same study, addition of N-acetyl-l-cysteine (NAC), a typical intracellular antioxidant, protected VSMCs against cytotoxicity associated with oxidative stress.
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3. Oxidative stress in oral pathology 3.1. Saliva Salivary fluid of humans contains 99.5% water, while the other 0.5% consists of electrolytes, mucus, glycoproteins, enzymes, and antibacterial compounds. Saliva serves a lubricative function, wetting food and permitting the initiation of digestion and swallowing, thus protecting the mucosal surfaces of the oral cavity. Saliva is constantly replaced; it is estimated that a healthy person produces 0.75 to 1.5 l of saliva per day. Saliva possesses AO properties as well. These AO properties are represented in the saliva by SOD, salivary peroxidase, catalase, uric acid, and GSH. Under physiologic conditions, these AO agents preserve the oxidant–antioxidant balance. But in pathologic conditions, an overload of oxidants can excess the AO capacity of the saliva. For example, in smokers, the AO capacity of saliva was found to be diminished. Even a single cigarette is sufficient to significantly reduce GSH concentration and impair its protective role against the noxious biochemical effects of CS (Zappacosta et al., 1999). This effect is attributed to CS unsaturated aldehydes, which react with cysteine thiol of GSH. Also, CS aldehydes were found to be responsible for an increase of salivary protein carbonyls, an OS marker, (Avezov et al., 2014b; Nagler et al., 2000), with a concurrent decrease in the activity of some salivary enzymes, such as lactate dehydrogenase (LDH), aspartate aminotransferase (AST), acid phosphatase and amylase (Avezov et al., 2014a; Nagler et al., 2001; Zappacosta et al., 2002). The damage to salivary proteins may lead to their impaired AO, digestive and antibacterial properties. A drop in AO protection by CS can expose oral tissues to oxidative damage from other sources mentioned above, and explain why tobacco products are a leading risk factor in almost all oral diseases, including oral cancer. 3.2. Oral cells The salivary flow through the oral structures spreads the dissolved oxidants from different sources, so they can reach and affect other oral cells. All cellular contents are susceptible to ROS/RNS damage, including membrane lipids, proteins, and DNA. ROS/RNS
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induce lipid peroxidation of the polyunsaturated fatty acids in cell membranes. As a result, reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) are formed, and can further react with proteins and DNA (Uchida, 2003; Valko et al., 2006). High levels of such reactive peroxidation end products have been reported in oral cancer and precancer patients (Korde et al., 2011; Rasheed et al., 2007). Markers of lipoperoxidation and carbonyl stress are increased in patients with oral premalignant lesions (Vlkova et al., 2012) together with decreased AO status. Cellular protein content is also prone to oxidative damage, including fragmentation, cross-linking, and aggregation. Amino acid residues such as cysteine, histidine and lysine side chains are very susceptible to attack by ROS/RNS (Stadtman, 2004). Also, protein carbonyls are formed (Stadtman and Levine, 2000). Some important proteins such as DNA repair enzymes can be damaged, and increase the frequency of mutations. Indeed, mutagenic 8-nitroguanine, an indicator of nitrative DNA damage has been observed in the oral epithelium biopsies of patients with leukoplakia, whereas little or none were observed in normal oral mucosa (Ma et al., 2006). Markers related to oxidative stress, DNA repair, carcinogenesis, metastasis and cellular death are also present in the saliva of 19 tongue cancer patients (Shpitzer et al., 2009). Not only destructive, but also rescue pathways are activated protecting the cells against oxidative injury, such as the Nrf2 pathway. It regulates the expression of phase II detoxifying enzymes involved in the GSH biosynthesis pathway (i.e. glutathione-Stransferase (GST), ␥-glutamyl-transpeptidase (GGT), and others (Cho and Kleeberger, 2010; Cho et al., 2006), along with stress proteins such as HO-1. Cells and tissues where routine detoxification reactions normally occur, constitutively express Nrf2, including the lung, intestine, and liver (Cho and Kleeberger, 2010; Shin et al., 2013). The Nrf2 pathway is known to be involved in the cellular protection after exposure to CS and aldehydes in the respiratory system and ocular epithelium (Kosmider et al., 2011). However, very little is known about its regulation in the oral environment. Future studies should focus upon possibilities to boost the protective mechanisms involved in oral cell protection. In conclusion: The main exogenous and endogenous sources of OS in the oral cavity were presented. Fig. 1 summarizes their effects
Fig. 1. The effect of oxidative stress sources on the oral cavity: the influence on the saliva and oral cells.
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