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Orthodontic treatment modifies the oxidant–antioxidant balance in saliva of clinically healthy subjects
Advances in Medical Sciences 62 (2017) 129–135
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Original research article,
Orthodontic treatment modifies the oxidant–antioxidant balance in saliva of clinically healthy subjects Piotr Buczko a,*, Małgorzata Knas´ b, Monika Grycz a, Izabela Szarmach a, Anna Zalewska c a
Department of Orthodontics, Medical University of Bialystok, Bialystok, Poland Institute of Health Care Higher Vocational School, Suwalki, Poland c Conservative Dentistry Department, Medical University of Bialystok, Bialystok, Poland b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 November 2015 Accepted 29 November 2016 Available online
Purpose: The aim of our study was to analyse salivary markers of oxidative stress and an antioxidant response in clinically healthy subjects with fixed orthodontic appliances. Material/methods: 37 volunteers were included in the study. Unstimulated (UWS) and stimulated (SWS) whole saliva were analysed for oxidative and antioxidant status and nickel levels immediately before the insertion of the appliances, an one week after and twenty four weeks after the insertion of fixed appliances. Results: A significant increase in tiobarbituric acid reactive substance (TBARS) and total oxidant status (TOS) one week, and total protein concentration twenty four weeks after the attachment of orthodontic appliances was found in the saliva. The markers of antioxidant status: superoxide dismutase (SOD), catalase (CAT), uric acid (UA), peroxidase (Px), and total antioxidant status (TAS) were not changed in all periods in UWS. In SWS a significant decrease in SOD1 and CAT was found whereas Px was increased one week after treatment and UA twenty four weeks following treatment. TAS was decreased in UWS and SWS twenty four weeks after orthodontic treatment. Oxidative status index (OSI) was elevated both in UWS and SWS one week after orthodontic treatment in comparison to the results obtained before and twenty four weeks. One week after treatment an increased concentration of nickel was also observed. Conclusions: Orthodontic treatment modifies the oxidative–antioxidative balance in the saliva of clinically healthy subjects. Increased nickel concentration in saliva, released from orthodontic appliances, seems to be responsible for changes in the oxidative status of the saliva. ß 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.
Keywords: Orthodontic appliances Oxidative stress Saliva nickel
1. Introduction The oral cavity is a very complex milieu characterised by numerous interactions between different tissues, secretions from various glands, surfaces, foods, air and microorganisms. Saliva is in the centre of the oral cavity and, to a certain degree, salivary markers reflect the condition of the oral cavity. The use of orthodontic appliances in the treatment of various maxillary dental anomalies creates a very complex environment in the oral cavity. An inflammatory response localised around the tooth, or teeth subjected to displacement is frequently observed. A large number of inflammatory mediators are involved in the response to
* Corresponding author at: Department of Orthodontics, Medical University of Bialystok, Jana Kilinskiego 1, Bialystok, Poland. E-mail address: [email protected] (P. Buczko).
the mechanical forces occurring during orthodontic treatment [1]. One of the biological responses to orthodontic treatment and the ensuing inflammation in the oral cavity is oxidative stress associated with an enhanced expression of proinflammatory factors [2,3]. Oxidative stress is defined as an imbalance between the production of free radicals and the body’s ability to counteract or minimise their harmful effects through their neutralisation by antioxidants [4]. Reactive oxygen species (ROS), which cause oxidative damage, include both oxygen-free radicals and nonradical oxygen derivatives involved in oxygen radical production. The main ROS are hydrogen peroxide (H2O2), superoxide anion (O2 ) and hydroxyl (OH) radicals. The resulting ROS are considered to be one of the most important factors in oxidative damage to cells and tissues by affecting the peroxidation of doublechain fatty acids, proteins and DNA as well as by increasing oxidative stress [5–7]. Oxidative stress is a causative factor in a
http://dx.doi.org/10.1016/j.advms.2016.11.004 1896-1126/ß 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.
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number of pathophysiological conditions including gene mutations and cancer, neurodegenerative diseases, atherosclerosis, inflammatory diseases, chronic renal failure, diabetes [8–12] and some oral cavity diseases, e.g. periodontitis and squamous cell carcinoma [13–15]. Thus, evaluation of oxidative status in the oral cavity has been suggested as an important tool in the diagnosis and assessment of progression of these diseases. In the last decade, a limited number of papers indicating that orthodontic treatment can be a factor inducing oxidative stress in saliva have been published [16–18]. The reported results are not consistent. In vitro studies by Buljan et al. [17] have demonstrated that all types of orthodontic brackets, regardless of the constituent materials, are a source of oxidative stress in murine fibroblast cells L929. A higher concentration of oxidative stress markers was observed in subjects with traditional, metal and self-ligating brackets compared to the negative control. Conventional ceramic brackets showed high viability and caused the largest increase in the number of oral mucosa cells but the weakest oxidative stress symptoms [17]. In 2009 Olteanu et al. [16] were the first researchers to study selected oxidative biomarkers in very young patients treated orthodontically. They found a statistically significant increase in the concentrations of ceruloplasmin and malondialdehyde, which reached maximum levels 24 h after treatment and 1 h after treatment for hydrogen donors, while 7 days after device attachment concentrations of the salivary markers of oxidative stress were close to the initial values. In 2014 Ozcan et al. [18] were the first researchers who evaluated the level of selected oxidative stress markers by using saliva and gingival cervicular fluid (GCF) for determining oxidative damage that man occur during orthodontic treatment as a result of aseptic inflammation. The authors found that orthodontic treatment did not change levels of the studied oxidative stress markers in saliva and GCF above the physiological limits one and six months after orthodontic appliance insertion. It has been postulated recently that single markers can validate disease presence or prognosis, but utilising a panel of biomarkers would be more helpful and yet, estimating total oxidant status (TOS) and total antioxidant capacity (TAC) would be more appropriate [19,20]. Since an oxidant–antioxidant imbalance is the underlying principle of oxidative stress, it has been suggested that the TOS to TAC ratio should be calculated as a more accurate indicator of oxidative stress in the body [21,22]. Taking the above into account, we measured total oxidative status and an antioxidant response in the saliva of clinically healthy subjects one week and twenty four weeks after orthodontic treatment. 2. Material and methods The study reported 60 students, but due to the fact that 15 of them were smokers, 3 had diabetes type 1 and 5 during the last 6 months has gone through a bacterial or viral infection, which was the reason for taking medication to further study 37 participants were enrolled. Unstimulated (UWS), and stimulated whole saliva (SWS) were collected from 37 participants – dentistry students of the Medical University in Bialystok (28 female, 9 male), 21.2–24.5 years old (median – 22.3; interquartile range – 21.3–23.6). All participants were clinically stable non-smokers, with no known health problems. The subjects had not taken medication which could influence saliva composition 6 months prior to the commencement of the study and were not suffering from any systemic diseases and chronic or acute oral infections (mucositis or candidiasis). The participants were instructed and screened once a week in all aspects of oral hygiene by a qualified dentist in the month preceding the attachment of the fixed appliances. UWS and
SWS were collected 30 min before bracket insertion. Conventional 3 M Victory Series steel brackets, which contain approximately 30% nickel, 15% chromium, 3% cuprum and less than 1% of other elements, were used. During the whole study period only nickel– titanium arch wires were used. Following bracket insertion, the subjects received instructions concerning the need for supplementary oral hygiene measures (orthodontic toothbrushes, dental floss, mouth rinsing). All appliances were inserted by two experienced dentists. Out of the 37 participants, 5 displayed a Class II malocclusion, 4 had crossbite and all subjects had dental crowding. No tooth extractions were performed. Written informed consent was obtained from all participants following the explanation of the purpose and nature of the study. The Ethics Committee at the Medical University of Bialystok (permission number R-I-002/67/2012) approved the study. Study participants were instructed to refrain from food for 2 h before saliva collection, which was performed between 8 and 9 a.m. Samples of UWS were taken 10 min after rinsing the mouth with distilled water (MilliQ) under the supervision of two dentists (P.B. and M.G.), by passive spitting into a container immersed in crushed ice [23]. Saliva collected during the first minute was discarded. Subsequent portions of saliva, which were accumulated at the bottom of the mouth, were actively spat out into a plastic container every 60 s. Citric acid-stimulated whole saliva (SWS) was collected in the same manner for 5 min, following UWS collection. Stimulation with citric acid was performed by two dentists (P.B and M.G) by placing 100 mL of 1% citric acid on the posterior part of the tongue every 30 s. After measuring the volumes, saliva samples (3 mL) were centrifuged at 3000 g for 20 min at 4 8C to remove cells and debris. The resulting, not stained, supernatants were divided, frozen, and kept at 80 8C until analysed [24]. Clinical examinations of the participants were performed by experienced dentists (P.B., M.G.) under standardised conditions in the Orthodontic Department at the Medical University in Bialystok, in a dental chair, using portable equipment with a fibre optic light, a suction device, and compressed air. All examinations were conducted using dental diagnostic instruments (a dental mirror, a probe, and a periodontal probe). The dental status of each subject was determined using the Decayed, Missing, Filled index (DMFT) in accordance with the WHO criteria [25]. The gingival status was assessed using the gingival index GI, and the periodontal status was established on the basis of the probing pocket depth (PPD) measurements. Clinical examinations and saliva collection were performed by the same dentist on three occasions – immediately before the insertion of the appliances a, one week after treatment, and twenty four weeks after the attachment Cof fixed appliances. Nonstimulated saliva samples were collected first, followed by stimulated saliva sample collection. A dental examination constituted the final part of the procedure. In the case of all 37 participants, the inter-rater reliability between the principal examiner (P.B.) and another experienced dentist (M.G.) was assessed at each examination. At the first examination, the inter-rater reliability for DMFT was r = 0.97, for gingival index (GI): r = 0.96 and for Oral Hygiene Index (OHI): r = 0.96. At the second examination, the inter-rater reliability for DMFT was r = 0.99, for GI: r = 0.99 and for OHI: r = 0.98. At the third examination, the inter-rater reliability for DMFT was r = 0.97, for GI: r = 0.97 and for OHI: r = 0.95. Although in vivo studies are exceptionally useful in explaining how orthodontic materials interact with oral tissues in their natural environment, interpretation of research results is usually difficult because of many factors which are not under experimental control. In our preliminary experiments we observed individual variations in the salivary markers of oxidative stress even in
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clinically healthy students of Dentistry not treated orthodontically. To eliminate these inter-subject variations, the participants were evaluated longitudinally to act as their own controls, so that these variables were negligible in the overall assessment.
to establish the inter-rater agreement between the two examiners. Spearman’s rank correlation coefficient was used to study associations between nickel concentration and TOS. The statistical significance was defined as p < 0.05.
2.1. Assays
3. Results
The analyses performed included: the total amount of tiobarbituric acid reactive substances (TBARS), total protein concentration, total oxidant status (TOS), the total amount of superoxide dismutase 1 (SOD1), catalase (CAT), peroxidase (Px), uric acid (UA) and total antioxidant status (TAS), and oxidative status index (OSI) in unstimulated and stimulated whole saliva. All variables were presented as the total amount (ratio of examined protein to total protein). TBARS products were determined according to Buege and Austa [26]. The activity of peroxidase was determined colorimetrically according to Mansson-Rahemtulla et al. [27]. UA (sensitivity < 0.22 mg/dD) was measured colorimetrically with a kit supplied by BioAssays System (QuantiChromTM Uric Acid assay Kit DIUA-250, Hayward, CA, USA). SOD1 (sensitivity < 12.5 pg/mL, detection range up to 800 pg/mL) and CAT (sensitivity < 0.134 ng/ mL, detection range 0.312–20 ng/mL) were determined by ELISA (Human Superoxide Dismutase 1, LF-EK0101, AbFrontier Seoul, Korea and Human Catalase, SEC418Hu, USCN Life Science, Wuhan, China, respectively). Total oxidant status was determined using a photometric commercial kit (PerOx, TOS/TOC) supplied by Immune Diagnostic (Bensheim, Germany, detection limit 7 mmol/L). TAS was assessed colorimetrically using a commercial kit supplied by Randox (Crumlin, UK, detection range 0.21–2.94 nmol/L). Oxidative stress index (OSI) was calculated from the TOS/ TAS 100 formula [28]. Protein concentration was determined using the bicinchoninic acid method (BCA) with bovine serum albumin as a standard (Thermo Scientific PIERCE BCA Protein Assay Kit, Rockford, IL, USA, sensitivity <5 mg/mL, detection range 20– 2000 mg/mL). All determinations were performed in duplicates.
3.1. Subject characteristics
2.2. Determination of nickel concentration Nickel concentration was measured in each sample of unstimulated and stimulated saliva. Mixed saliva (3 mL) samples were checked for food, blood or nasal discharge contamination and contaminated samples were excluded. Following that, 3 mL of saliva was transferred into a beaker and 20 mL of 2% nitric acid (HNO3) was added. Samples were filtered into a volumetric flask using Whatman No. 42 filter paper. The filtrates were then diluted to the final volume of 100 mL with distilled water and then stored at 20 C until analysed (Moore PD, London 86). Nickel concentration in saliva is stable for 6 months when stored at 20 C. The use of an atomic absorption spectrophotometre permits the analysis of metals in biological samples without separating the metal from its biological matrix. The saliva samples were analysed for nickel content with an atomic absorption spectrophotometre with a graphite oven (Solar M, ThermoElectron Corp., Madison, USA) [29]. Nickel concentration levels were calculated as micrograms per millilitre. We used standard nickel samples to control the accuracy of nickel measurements by the equipment. The error associated with the method used for nickel analysis was 1%. 2.3. Statistical analysis Study results were presented as mean and standard deviation. Statistical analysis was performed using Statistica 10.0 (Cracow, Poland) according to ANOVA and post hoc tests (test NIR). The Pearson Correlation Coefficient was used to study associations between the variables. Cohen Kappa (online calculator) was used
Unstimulated and stimulated saliva was collected prior to orthodontic treatment and one week, and twenty four weeks after treatment from the participants who had fixed orthodontic appliances inserted. The health condition of the oral cavity in the subjects included in the study did not change during the study period. 3.2. Biochemical parameters The mean and standard deviation of biochemical parameters of saliva prior to treatment and at one week and twenty four weeks after treatment, are shown in Figs. 1–3. A significant increase in the levels of TBARS (Fig. 1A) and TOS (Fig. 1B) in UWS and SWS one week after orthodontic treatment was observed. Twenty four weeks after treatment the mean values of TBARS and TOS in UWS and SWS were close to the initial values. Twenty four weeks after orthodontic treatment, total protein concentration was significantly elevated in comparison to the level observed in UWS and SWS prior to and one week after the attachment of orthodontic appliances (Fig. 1C). Regarding antioxidant biomarkers in saliva, no significant differences in the levels of SOD1 (Fig. 2A), CAT (Fig. 2B), UA (Fig. 2C) and the activity of Px (Fig. 2D) one week and twenty four weeks after orthodontic treatment in UWS were observed. Similarly in SWS, no differences were observed between the evaluated periods in regards to the mean value of UA. One week after treatment the mean value of SOD1 was significantly lower, while the mean activity of Px was significantly higher, in comparison with the initial values and the values found twenty four weeks after treatment. Total antioxidant status (TAS) in UWS and SWS twenty four weeks after orthodontic treatment was significantly lower as compared to the values observed before and one week after treatment (Fig. 2E). OSI in UWS and SWS were highest one week following treatment whereas twenty four weeks after treatment OSI did not differ from the value observed at the commencement of the study (Fig. 2F). Interestingly, OSI in SWS was about 30% lower in comparison with OSI in UWS. A significant increase in nickel levels in UWS and SWS one week after treatment, as compared to the values obtained before therapy and twenty four weeks after orthodontic treatment was also observed. Nickel concentration in UWS and SWS was approximately seven times higher in respect to the concentration measured evaluated in SWS (Fig. 3). There was a positive correlation between nickel concentration and TOS both in UWS (r = 0.4703; p = 0.042) and SWS (r = 0.4281; p = 0.046). 4. Discussion Current scientific literature offers very limited and inconsistent data concerning the influence of orthodontic treatment on ¨ zcan et al. [18] have postulated oxidative stress in the oral cavity. O recently that orthodontic tooth movement and orthodontic materials used in orthodontic treatment do not lead to a change above the physiological limits that is suggestive of oxidative damage in both GCF and saliva. Oxidative stress biomarkers in patients with aseptic inflammation (age 14.8 years) were evaluated in saliva prior to orthodontic treatment and one and
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Fig. 1. The mean of: A – total amount of malondialdehyde, B – total oxidant status and C – protein concentrations in unstimulated and stimulated saliva of clinically healthy subjects with fixed orthodontic appliances before (0), one week (1) and twenty four weeks (24) after orthodontic treatment. Abbreviations: MDA – malondialdehyde; TOS – total oxidant status; UWS – unstimulated whole saliva; SWS – stimulated whole saliva; * – significant changes vs. group before orthodontic treatment (p < 0.05); ** – significant changes vs. group one week (1) after orthodontic treatment (p < 0.05); std. dev. – standard deviation.
six months later. Similarly, we did not observe any changes in the biochemical parameters of oxidative stress such as MDA and TOS, in the saliva of our study participants (age 23.1 years) with a clinically healthy oral cavity twenty four weeks after orthodontic treatment. Nevertheless, there are some reports in the literature indicating enhanced levels of oxidative stress biomarkers in saliva in the period immediately following orthodontic treatment. In 2009 Olteanu et al. [16] studied some saliva markers of oxidative stress in 37 girls and 4 boys between the ages of 8 and 12 years with the average age of 9.9 years, who were treated with biomechanical appliances. Saliva samples were collected before the commencement of orthodontic treatment and 1 h, 24 h, and 7 days after therapy. The authors found a statistically significant, enhanced concentration of ceruloplasmine and MDA, which reached maximum levels 24 h after treatment and 1 h after treatment for hydrogen donors, while 7 days after device attachment concentrations of the studied salivary markers of oxidative stress were close to the initial
values. According to the authors, even statistically significant changes in the levels of the salivary markers of oxidative stress, do not determine the appearance of certain pathological processes within the oral cavity. A number of identified and unidentified substances create the oxidant/antioxidant balance in saliva. The dynamic distribution of different oxidants in various parts of the oral cavity and their potential interactions make it difficult to measure each oxidant separately. Since all these oxidants compose the complex oxidant system, some authors have suggested that levels of oxidant stress markers in vitro or total oxidants status (TOS) in vivo should be measured. An in vitro study has demonstrated that traditional metal brackets are a source of oxidative stress in murine fibroblast cells L929 [17]. The results of our in vivo study are in line with this observation. We noted a marked increase in TBARS and TOS levels in saliva one week after the attachment of orthodontic appliances. At present it is difficult to explain the discrepancies between our results and those obtained by Olteanu et al. [16]. We can
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Fig. 2. The mean of: A – total amount of superoxide dismutase 1, B – total amount of catalase, C – total amount of uric acid, D – specific activity of peroxidase, E – total antioxidant status and F – oxidative status index in unstimulated and stimulated saliva of clinically healthy participants with fixed orthodontic appliances before (0), one week (1) and twenty four weeks (24) after orthodontic treatment. Abbreviations: SOD1 – superoxide dismutase 1, CAT – catalase, UA – uric acid, Px – peroxidase, TAS – total antioxidant status, OSI – oxidative status index, UWS – unstimulated whole saliva, SWS – stimulated whole saliva, * – significant changes vs. group before orthodontic treatment (p < 0.05), ** – significant changes vs. group 1 week after orthodontic treatment (p < 0.05), std. dev. – standard deviation.
speculate that the disparities resulted from the fact that different types of orthodontic brackets, containing different amounts of nickel, were used in the studies. Moreover, the research was performed in different countries with different diets and different oral hygiene standards. Oral hygiene and periodontal status is crucial for oxidant status in saliva. It has been demonstrated that part of the total TAS ability may be connected with polyphenols which are primarily derived from foods of plant origin [30]. Since the participants in our study were clinically healthy individuals with no detectable pathological changes in the oral cavity, the most probable factor responsible for oxidative stress
induction seems to be substances derived from the orthodontic material. Orthodontic brackets, bands, and arch wires are universally made with an alloy which contains approximately 15–54% of nickel and 20–30% of chromium [31,32]. In addition to being allergenic, nickel and, to a lesser extent, chromium have been found to possess carcinogenic, mutagenic and cytotoxic properties [33–35]. Faccioni et al. [31] and Hafez et al. [32] have demonstrated that nickel released from orthodontic appliances can induce DNA damage in oral mucosa cells. Natarajan et al. [35] have also demonstrated that nickel alloys emit nickel cations in quantities
Fig. 3. The concentration of nickel in unstimulated and stimulated saliva of clinically healthy participants with fixed orthodontic appliances before (0), one week (1) and twenty four weeks (24) after orthodontic treatment. Abbreviations: Ni – nickel; UWS – unstimulated whole saliva; SWS – stimulated whole saliva; * – significant changes vs. group before orthodontic treatment (p < 0.05); ** – significant changes vs. group 1 week after orthodontic treatment (p < 0.05); std. dev. – standard deviation.
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sufficient to induce localised genotoxic effects. It has also been demonstrated that there exists considerable variability among individuals with orthodontic appliances in the concentration levels of nickel in saliva. For example, De Souza et al. [34] observed a maximal increase in nickel cations between 10 min and one day after orthodontic appliance attachment, whereas Bengleil et al. [36] noted the highest concentration of nickel cations four weeks after the insertion of the traditional bracket system while Singh et al. [37] observed a maximal concentration in nickel cations 7 days after orthodontic treatment. In our study we used brackets made entirely of metal, containing 38% of nickel and 30% of chromium. Therefore, nickel involvement in the oxidative stress status in saliva should be taken into consideration. Indeed, we observed a statistically significant increase in nickel cation concentration and TOS one week after orthodontic treatment. Twenty four weeks after orthodontic treatment nickel level in saliva was low or undetectable and total oxidative stress was close to the initial value (Fig. 3). Lipids containing polyunsaturated fatty acids are a group of cellular components which sustain the most significant damage caused by reactive oxygen species. Oxidative stress caused by a disruption in the oxidant/antioxidant balance, leads to lipid peroxidation [38]. MDA is the final product of lipid peroxidation [39]. In our study we observed a significant increase in TBARS concentration in saliva one week after orthodontic appliance attachment but no changes were detected twenty four weeks after treatment in comparison to TBARS concentration at the beginning of the study. Similar results were obtained when TOS was estimated. The aforementioned results correlated positively with nickel concentration in saliva measured one week after orthodontic treatment. These results suggest an involvement of nickel in lipid peroxidation and an increase in TOS in the saliva of individuals with orthodontic appliances. Lack of significant differences in TBARS levels measured one and six months after orthodontic treatment in respect to the values observed at the ¨ zcan et al. initiation of treatment, was also observed by O [18]. Summarising, the results presented above indicate an involvement of nickel in the salivary oxidative stress induction in the period immediately following orthodontic treatment. Lack of changes three and six months after therapy suggest that the body develops tolerance to nickel or induces repair mechanisms of the buccal mucosa cells, as suggested by Hafez et al. [32]. The degree of oxidative stress and the severity of subsequent oral tissue damage might depend on the imbalance between the excess production of ROS and antioxidant defense. The oral cavity, being the first line of defense against ROS, contains several antioxidant substances including enzymes, non-enzymatic antioxidants, and an array of small molecules [40]. Taking the above into account, we measured the level/activity of some salivary antioxidants such as SOD1, CAT, UA and Px. We found no statistically significant enhancement of the studied antioxidants one week and twenty four weeks after orthodontic treatment in UWS. However, was observed a decrease in the concentration of SOD1 and CAT in SWS. SOD1 is an enzyme located in the plasma which repairs cells and reduces damage caused to them by superoxide, the most common free radical in the body [41]. The enzyme catalyses the dismutation of the superoxide (O2 ) radical into either molecular oxygen or, to a lesser degree, to hydrogen peroxide (H2O2). Catalase is an enzyme which catalyses mainly the reaction by which hydrogen peroxide is decomposed to water and oxygen. A decrease in SOD1 and catalase concentration in SWS one week after orthodontic treatment can suggest that nickel and/or other metals released from orthodontic material may directly alter the function of oral mucosa cells and bacteria, which can result in reduced SOD and catalase production, respectively.
In contrast, an increase in Px activity in saliva was observed one week after orthodontic treatment. Px is an important part of the defense mechanism against oxygen toxicity. In the mouth, hydrogen peroxide is formed not only by bacteria colonising the mucous membranes but also by the cells of the salivary glands. In saliva, the most important part of this defense mechanism is salivary peroxidase which detoxifies hydrogen peroxide in the presence of thiocyanante by converting it to hypothiocianite, dioxygen and water. Moreover, hypothiocanite inhibits hydrogen peroxide production by oral bacteria [42]. Since Px is an enzyme synthetised and released by the salivary glands we can speculate that an increase in Px activity is an antioxidant defense against excess production of hydrogen peroxide induced by orthodontic treatment in the oral cavity. These known, and a number of unknown antioxidant substances constitute a complex antioxidant system in the oral cavity. Thus, in order to assess the antioxidant status in vivo, it is highly recommended that levels of the overall antioxidant status should be measured [20,21]. In our study, TAS was unchanged one week after therapy, and reduced twenty four weeks after orthodontic treatment both in UWS and SWS. Furthermore, if we want to fully comprehend the oxidative stress status of patients in vivo, not only the TAS and TOS measurements but also the calculation of OSI is necessary [21,28]. In this study, saliva TOS and OSI values were higher one week after orthodontic treatment whereas TAS levels were unchanged. Twenty four weeks after device attachment TAS was decreased and TOS and OSI were close to the initial values. Analysing our results one should keep in mind that contamination of saliva by blood can influence oxidative stress markers. Our results did not confirm a relationship between a local inflammatory process (GI) and the salivary antioxidants and biomarkers of oxidative stress (data not shown). We also excluded patients with oral mucositis, candidiasis, periodontitis and gingivitis. Moreover, to avoid gingivitis, bleeding following bracket insertion, the subjects received instructions concerning the need for supplementary oral hygiene measures, which could suggests that changes in the behaviour of the examined parameters were not the results of local inflammatory processes, contamination of saliva by blood. Our results are in line with the recent data of Kamodyova´ et al. [43] who demonstrated that salivary oxidative stress concentrations are significantly influenced by 1% and more blood contamination in saliva. Saliva samples with 1% blood contamination are visibly coloured and it is possible to easily exclude such contaminated samples from further salivary oxidative stress analysis without any specific biochemical detection of blood constituents. 5. Conclusions There are limited data in the literature regarding the effects of orthodontic treatment on oxidative stress in the oral cavity. The published results were obtained from investigations conducted on limited number of subjects. The studies involved mostly children with low levels of oral hygiene and the observation periods were long (3 and 6 months). The data currently available are not consistent. In some studies orthodontic treatment evoked oxidative stress in saliva and in others it did not. In our study an increase of total oxidant status one week after orthodontic treatment was observed. Our data demonstrate that within a few days fixed orthodontic appliances modified the total oxidant/antioxidant status in the saliva of clinically healthy subjects with high standards of oral hygiene. To our knowledge this is the first report concerning the measurement of the oxidant/antioxidant balance in the saliva of subjects treated orthodontically. The changes normalised twenty four weeks after orthodontic treatment. Our data also demonstrate the probable role of nickel and/or
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other metals released from orthodontic materials on the oxidative status of subjects with high levels of oral hygiene. We obviously cannot exclude the influence of mechanical stimulation or damage to the surrounding tissues resulting from the process of device attachment on the oxidative status in saliva. These possibilities are important and indicate that further research is required using larger samples and considering other risk factors to clarify the role of orthodontic treatment in the modification of oxidative stress in saliva. Conflict of interest The authors declare no conflict of interests. Financial disclosure Supported by the Medical University of Bialystok, Poland, Grant No. 144-07503L. References [1] Salla JT, Taddei SR, Queiroz-Junior CM, Andrade Junior I, Teixeira MM, Silva TA. The effect of IL-1 receptor antagonist on orthodontic tooth movement in mice. Arch Oral Biol 2012;57(5):519–24. http://dx.doi.org/10.1016/ j.archoralbio.2011.09.018. [2] Gostner JM, Becker K, Fuchs D, Sucher R. Redox regulation of the immune response. Redox Rep 2013;18(3):88–94. http://dx.doi.org/10.1179/ 1351000213Y.0000000044. [3] Padgett LE, Broniowska KA, Hansen PA, Corbett JA, Tse HM. The role of reactive oxygen species and proinflammatory cytokines in type 1 diabetes pathogenesis. Ann N Y Acad Sci 2013;1281:16–35. http://dx.doi.org/ 10.1111/j.1749-6632.2012.06826.x. [4] Mandal A. What is oxidative strees. News Medical Life Sciences & Medicine. http://www.news-medical.net/health/What-is-Oxidative-Stress.aspx. [5] Bagchi K, Puri S. Free radicals and antioxidants in health and disease. East Mediterr Health 1998;14:350–60. [6] Kohen R, Nyska A. Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reaction, and methods for their quantification. Toxicol Pathol 2002;30:620–50. [7] Burton GJ, Jauniaux E. Oxidative stress. Best Pract Res Clin Obstst Gyneacol 2011;25:287–99. http://dx.doi.org/10.1016/j.bpobgyn.2010.10.016. [8] Popolo A, Autore G, Pinto A, Marzocco S. Oxidative stress in patients with cardiovascular disease and chronic renal failure. Free Radic Res 2013;47:346– 56. http://dx.doi.org/10.3109/10715762.2013.779373. [9] Gandhi S, Abramow AY. Mechanism of oxidative stress in neurodegeneration. Oxid Med Cell Longev 2012;2012:428010. http://dx.doi.org/10.1155/2012/428010. [10] Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked. Free Radic Biol Med 2010;49:1603–16. http://dx.doi.org/10.1016/j.freeradbiomed.2010.09.006. [11] Trivedi S, Lal N, Mahdi AA, Mittal M, Singh B, Pandey S. Evaluation of antioxidant enzymes activity and malondialdehyde levels in patients with chronic periodontitis and diabetes mellitus. J Periodontol 2014;85(5):713–20. http://dx.doi.org/10.1902/jop.2013.130066 [Epub 29 July 2013]. ˇ ervenka T, Celec P. Salivary markers of oxidative [12] To´thova´ L, Kamodyova´ N, C stress in oral diseases. Front Cell Infect Microbiol 2015;October (5):73. http:// dx.doi.org/10.3389/fcimb.2015.00073. [13] Buczko P, Zalewska A, Szarmach I. Saliva and oxidative stress in oral cavity and in some systemic disorders. J Physiol Pharmacol 2015;66(1):3–9. [14] Muniz FW, Nogueira SB, Mendes FL, Ro¨sing CK, Moreira MM, de Andrade GM, et al. The impact of antioxidant agents complimentary to periodontal therapy on oxidative stress and periodontal outcomes: a systematic review. Arch Oral Biol 2015;60(September (9)):1203–14. http://dx.doi.org/10.1016/j.archoralbio.2015.05.007. [15] Kesarwala AH, Krishna MC, Mitchell JB. Oxidative stress in oral diseases. Oral Dis 2016;22(January (1)):9–18. http://dx.doi.org/10.1111/odi.12300. [16] Olteanu C, Muresan A, Daicovicin D, Tamure V, Olteanu I. Kenlarts IMLU. Variations of some saliva markers of the oxidative stress in patients with orthodontic appliances. Physiology 2009;19(3):26. http://connection. ebsconhost.com/c/articles/52595764/variations-som. [17] Buljan ZI, Ribaric SP, Abram M, Ivankovic A, Spalj S. In vitro oxidative stress induced by conventional and self-ligating brackets. Angle Orthod 2012;82(2):340–5. http://dx.doi.org/10.2319/061811-395.1. ¨ zcan Atug˘ SS, Ceylan I, Ozcan E, Kurt N, Dag˘suyu IM, Canakc¸i CF. Evaluation of [18] O oxidative stress biomarkers in patients with fixed orthodontic appliances. Dis Markers 2014;2014:597892. http://dx.doi.org/10.1155/2014/597892.
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Advances in Medical Sciences journal homepage: www.elsevier.com/locate/advms
Original research article,
Orthodontic treatment modifies the oxidant–antioxidant balance in saliva of clinically healthy subjects Piotr Buczko a,*, Małgorzata Knas´ b, Monika Grycz a, Izabela Szarmach a, Anna Zalewska c a
Department of Orthodontics, Medical University of Bialystok, Bialystok, Poland Institute of Health Care Higher Vocational School, Suwalki, Poland c Conservative Dentistry Department, Medical University of Bialystok, Bialystok, Poland b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 November 2015 Accepted 29 November 2016 Available online
Purpose: The aim of our study was to analyse salivary markers of oxidative stress and an antioxidant response in clinically healthy subjects with fixed orthodontic appliances. Material/methods: 37 volunteers were included in the study. Unstimulated (UWS) and stimulated (SWS) whole saliva were analysed for oxidative and antioxidant status and nickel levels immediately before the insertion of the appliances, an one week after and twenty four weeks after the insertion of fixed appliances. Results: A significant increase in tiobarbituric acid reactive substance (TBARS) and total oxidant status (TOS) one week, and total protein concentration twenty four weeks after the attachment of orthodontic appliances was found in the saliva. The markers of antioxidant status: superoxide dismutase (SOD), catalase (CAT), uric acid (UA), peroxidase (Px), and total antioxidant status (TAS) were not changed in all periods in UWS. In SWS a significant decrease in SOD1 and CAT was found whereas Px was increased one week after treatment and UA twenty four weeks following treatment. TAS was decreased in UWS and SWS twenty four weeks after orthodontic treatment. Oxidative status index (OSI) was elevated both in UWS and SWS one week after orthodontic treatment in comparison to the results obtained before and twenty four weeks. One week after treatment an increased concentration of nickel was also observed. Conclusions: Orthodontic treatment modifies the oxidative–antioxidative balance in the saliva of clinically healthy subjects. Increased nickel concentration in saliva, released from orthodontic appliances, seems to be responsible for changes in the oxidative status of the saliva. ß 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.
Keywords: Orthodontic appliances Oxidative stress Saliva nickel
1. Introduction The oral cavity is a very complex milieu characterised by numerous interactions between different tissues, secretions from various glands, surfaces, foods, air and microorganisms. Saliva is in the centre of the oral cavity and, to a certain degree, salivary markers reflect the condition of the oral cavity. The use of orthodontic appliances in the treatment of various maxillary dental anomalies creates a very complex environment in the oral cavity. An inflammatory response localised around the tooth, or teeth subjected to displacement is frequently observed. A large number of inflammatory mediators are involved in the response to
* Corresponding author at: Department of Orthodontics, Medical University of Bialystok, Jana Kilinskiego 1, Bialystok, Poland. E-mail address: [email protected] (P. Buczko).
the mechanical forces occurring during orthodontic treatment [1]. One of the biological responses to orthodontic treatment and the ensuing inflammation in the oral cavity is oxidative stress associated with an enhanced expression of proinflammatory factors [2,3]. Oxidative stress is defined as an imbalance between the production of free radicals and the body’s ability to counteract or minimise their harmful effects through their neutralisation by antioxidants [4]. Reactive oxygen species (ROS), which cause oxidative damage, include both oxygen-free radicals and nonradical oxygen derivatives involved in oxygen radical production. The main ROS are hydrogen peroxide (H2O2), superoxide anion (O2 ) and hydroxyl (OH) radicals. The resulting ROS are considered to be one of the most important factors in oxidative damage to cells and tissues by affecting the peroxidation of doublechain fatty acids, proteins and DNA as well as by increasing oxidative stress [5–7]. Oxidative stress is a causative factor in a
http://dx.doi.org/10.1016/j.advms.2016.11.004 1896-1126/ß 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.
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number of pathophysiological conditions including gene mutations and cancer, neurodegenerative diseases, atherosclerosis, inflammatory diseases, chronic renal failure, diabetes [8–12] and some oral cavity diseases, e.g. periodontitis and squamous cell carcinoma [13–15]. Thus, evaluation of oxidative status in the oral cavity has been suggested as an important tool in the diagnosis and assessment of progression of these diseases. In the last decade, a limited number of papers indicating that orthodontic treatment can be a factor inducing oxidative stress in saliva have been published [16–18]. The reported results are not consistent. In vitro studies by Buljan et al. [17] have demonstrated that all types of orthodontic brackets, regardless of the constituent materials, are a source of oxidative stress in murine fibroblast cells L929. A higher concentration of oxidative stress markers was observed in subjects with traditional, metal and self-ligating brackets compared to the negative control. Conventional ceramic brackets showed high viability and caused the largest increase in the number of oral mucosa cells but the weakest oxidative stress symptoms [17]. In 2009 Olteanu et al. [16] were the first researchers to study selected oxidative biomarkers in very young patients treated orthodontically. They found a statistically significant increase in the concentrations of ceruloplasmin and malondialdehyde, which reached maximum levels 24 h after treatment and 1 h after treatment for hydrogen donors, while 7 days after device attachment concentrations of the salivary markers of oxidative stress were close to the initial values. In 2014 Ozcan et al. [18] were the first researchers who evaluated the level of selected oxidative stress markers by using saliva and gingival cervicular fluid (GCF) for determining oxidative damage that man occur during orthodontic treatment as a result of aseptic inflammation. The authors found that orthodontic treatment did not change levels of the studied oxidative stress markers in saliva and GCF above the physiological limits one and six months after orthodontic appliance insertion. It has been postulated recently that single markers can validate disease presence or prognosis, but utilising a panel of biomarkers would be more helpful and yet, estimating total oxidant status (TOS) and total antioxidant capacity (TAC) would be more appropriate [19,20]. Since an oxidant–antioxidant imbalance is the underlying principle of oxidative stress, it has been suggested that the TOS to TAC ratio should be calculated as a more accurate indicator of oxidative stress in the body [21,22]. Taking the above into account, we measured total oxidative status and an antioxidant response in the saliva of clinically healthy subjects one week and twenty four weeks after orthodontic treatment. 2. Material and methods The study reported 60 students, but due to the fact that 15 of them were smokers, 3 had diabetes type 1 and 5 during the last 6 months has gone through a bacterial or viral infection, which was the reason for taking medication to further study 37 participants were enrolled. Unstimulated (UWS), and stimulated whole saliva (SWS) were collected from 37 participants – dentistry students of the Medical University in Bialystok (28 female, 9 male), 21.2–24.5 years old (median – 22.3; interquartile range – 21.3–23.6). All participants were clinically stable non-smokers, with no known health problems. The subjects had not taken medication which could influence saliva composition 6 months prior to the commencement of the study and were not suffering from any systemic diseases and chronic or acute oral infections (mucositis or candidiasis). The participants were instructed and screened once a week in all aspects of oral hygiene by a qualified dentist in the month preceding the attachment of the fixed appliances. UWS and
SWS were collected 30 min before bracket insertion. Conventional 3 M Victory Series steel brackets, which contain approximately 30% nickel, 15% chromium, 3% cuprum and less than 1% of other elements, were used. During the whole study period only nickel– titanium arch wires were used. Following bracket insertion, the subjects received instructions concerning the need for supplementary oral hygiene measures (orthodontic toothbrushes, dental floss, mouth rinsing). All appliances were inserted by two experienced dentists. Out of the 37 participants, 5 displayed a Class II malocclusion, 4 had crossbite and all subjects had dental crowding. No tooth extractions were performed. Written informed consent was obtained from all participants following the explanation of the purpose and nature of the study. The Ethics Committee at the Medical University of Bialystok (permission number R-I-002/67/2012) approved the study. Study participants were instructed to refrain from food for 2 h before saliva collection, which was performed between 8 and 9 a.m. Samples of UWS were taken 10 min after rinsing the mouth with distilled water (MilliQ) under the supervision of two dentists (P.B. and M.G.), by passive spitting into a container immersed in crushed ice [23]. Saliva collected during the first minute was discarded. Subsequent portions of saliva, which were accumulated at the bottom of the mouth, were actively spat out into a plastic container every 60 s. Citric acid-stimulated whole saliva (SWS) was collected in the same manner for 5 min, following UWS collection. Stimulation with citric acid was performed by two dentists (P.B and M.G) by placing 100 mL of 1% citric acid on the posterior part of the tongue every 30 s. After measuring the volumes, saliva samples (3 mL) were centrifuged at 3000 g for 20 min at 4 8C to remove cells and debris. The resulting, not stained, supernatants were divided, frozen, and kept at 80 8C until analysed [24]. Clinical examinations of the participants were performed by experienced dentists (P.B., M.G.) under standardised conditions in the Orthodontic Department at the Medical University in Bialystok, in a dental chair, using portable equipment with a fibre optic light, a suction device, and compressed air. All examinations were conducted using dental diagnostic instruments (a dental mirror, a probe, and a periodontal probe). The dental status of each subject was determined using the Decayed, Missing, Filled index (DMFT) in accordance with the WHO criteria [25]. The gingival status was assessed using the gingival index GI, and the periodontal status was established on the basis of the probing pocket depth (PPD) measurements. Clinical examinations and saliva collection were performed by the same dentist on three occasions – immediately before the insertion of the appliances a, one week after treatment, and twenty four weeks after the attachment Cof fixed appliances. Nonstimulated saliva samples were collected first, followed by stimulated saliva sample collection. A dental examination constituted the final part of the procedure. In the case of all 37 participants, the inter-rater reliability between the principal examiner (P.B.) and another experienced dentist (M.G.) was assessed at each examination. At the first examination, the inter-rater reliability for DMFT was r = 0.97, for gingival index (GI): r = 0.96 and for Oral Hygiene Index (OHI): r = 0.96. At the second examination, the inter-rater reliability for DMFT was r = 0.99, for GI: r = 0.99 and for OHI: r = 0.98. At the third examination, the inter-rater reliability for DMFT was r = 0.97, for GI: r = 0.97 and for OHI: r = 0.95. Although in vivo studies are exceptionally useful in explaining how orthodontic materials interact with oral tissues in their natural environment, interpretation of research results is usually difficult because of many factors which are not under experimental control. In our preliminary experiments we observed individual variations in the salivary markers of oxidative stress even in
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clinically healthy students of Dentistry not treated orthodontically. To eliminate these inter-subject variations, the participants were evaluated longitudinally to act as their own controls, so that these variables were negligible in the overall assessment.
to establish the inter-rater agreement between the two examiners. Spearman’s rank correlation coefficient was used to study associations between nickel concentration and TOS. The statistical significance was defined as p < 0.05.
2.1. Assays
3. Results
The analyses performed included: the total amount of tiobarbituric acid reactive substances (TBARS), total protein concentration, total oxidant status (TOS), the total amount of superoxide dismutase 1 (SOD1), catalase (CAT), peroxidase (Px), uric acid (UA) and total antioxidant status (TAS), and oxidative status index (OSI) in unstimulated and stimulated whole saliva. All variables were presented as the total amount (ratio of examined protein to total protein). TBARS products were determined according to Buege and Austa [26]. The activity of peroxidase was determined colorimetrically according to Mansson-Rahemtulla et al. [27]. UA (sensitivity < 0.22 mg/dD) was measured colorimetrically with a kit supplied by BioAssays System (QuantiChromTM Uric Acid assay Kit DIUA-250, Hayward, CA, USA). SOD1 (sensitivity < 12.5 pg/mL, detection range up to 800 pg/mL) and CAT (sensitivity < 0.134 ng/ mL, detection range 0.312–20 ng/mL) were determined by ELISA (Human Superoxide Dismutase 1, LF-EK0101, AbFrontier Seoul, Korea and Human Catalase, SEC418Hu, USCN Life Science, Wuhan, China, respectively). Total oxidant status was determined using a photometric commercial kit (PerOx, TOS/TOC) supplied by Immune Diagnostic (Bensheim, Germany, detection limit 7 mmol/L). TAS was assessed colorimetrically using a commercial kit supplied by Randox (Crumlin, UK, detection range 0.21–2.94 nmol/L). Oxidative stress index (OSI) was calculated from the TOS/ TAS 100 formula [28]. Protein concentration was determined using the bicinchoninic acid method (BCA) with bovine serum albumin as a standard (Thermo Scientific PIERCE BCA Protein Assay Kit, Rockford, IL, USA, sensitivity <5 mg/mL, detection range 20– 2000 mg/mL). All determinations were performed in duplicates.
3.1. Subject characteristics
2.2. Determination of nickel concentration Nickel concentration was measured in each sample of unstimulated and stimulated saliva. Mixed saliva (3 mL) samples were checked for food, blood or nasal discharge contamination and contaminated samples were excluded. Following that, 3 mL of saliva was transferred into a beaker and 20 mL of 2% nitric acid (HNO3) was added. Samples were filtered into a volumetric flask using Whatman No. 42 filter paper. The filtrates were then diluted to the final volume of 100 mL with distilled water and then stored at 20 C until analysed (Moore PD, London 86). Nickel concentration in saliva is stable for 6 months when stored at 20 C. The use of an atomic absorption spectrophotometre permits the analysis of metals in biological samples without separating the metal from its biological matrix. The saliva samples were analysed for nickel content with an atomic absorption spectrophotometre with a graphite oven (Solar M, ThermoElectron Corp., Madison, USA) [29]. Nickel concentration levels were calculated as micrograms per millilitre. We used standard nickel samples to control the accuracy of nickel measurements by the equipment. The error associated with the method used for nickel analysis was 1%. 2.3. Statistical analysis Study results were presented as mean and standard deviation. Statistical analysis was performed using Statistica 10.0 (Cracow, Poland) according to ANOVA and post hoc tests (test NIR). The Pearson Correlation Coefficient was used to study associations between the variables. Cohen Kappa (online calculator) was used
Unstimulated and stimulated saliva was collected prior to orthodontic treatment and one week, and twenty four weeks after treatment from the participants who had fixed orthodontic appliances inserted. The health condition of the oral cavity in the subjects included in the study did not change during the study period. 3.2. Biochemical parameters The mean and standard deviation of biochemical parameters of saliva prior to treatment and at one week and twenty four weeks after treatment, are shown in Figs. 1–3. A significant increase in the levels of TBARS (Fig. 1A) and TOS (Fig. 1B) in UWS and SWS one week after orthodontic treatment was observed. Twenty four weeks after treatment the mean values of TBARS and TOS in UWS and SWS were close to the initial values. Twenty four weeks after orthodontic treatment, total protein concentration was significantly elevated in comparison to the level observed in UWS and SWS prior to and one week after the attachment of orthodontic appliances (Fig. 1C). Regarding antioxidant biomarkers in saliva, no significant differences in the levels of SOD1 (Fig. 2A), CAT (Fig. 2B), UA (Fig. 2C) and the activity of Px (Fig. 2D) one week and twenty four weeks after orthodontic treatment in UWS were observed. Similarly in SWS, no differences were observed between the evaluated periods in regards to the mean value of UA. One week after treatment the mean value of SOD1 was significantly lower, while the mean activity of Px was significantly higher, in comparison with the initial values and the values found twenty four weeks after treatment. Total antioxidant status (TAS) in UWS and SWS twenty four weeks after orthodontic treatment was significantly lower as compared to the values observed before and one week after treatment (Fig. 2E). OSI in UWS and SWS were highest one week following treatment whereas twenty four weeks after treatment OSI did not differ from the value observed at the commencement of the study (Fig. 2F). Interestingly, OSI in SWS was about 30% lower in comparison with OSI in UWS. A significant increase in nickel levels in UWS and SWS one week after treatment, as compared to the values obtained before therapy and twenty four weeks after orthodontic treatment was also observed. Nickel concentration in UWS and SWS was approximately seven times higher in respect to the concentration measured evaluated in SWS (Fig. 3). There was a positive correlation between nickel concentration and TOS both in UWS (r = 0.4703; p = 0.042) and SWS (r = 0.4281; p = 0.046). 4. Discussion Current scientific literature offers very limited and inconsistent data concerning the influence of orthodontic treatment on ¨ zcan et al. [18] have postulated oxidative stress in the oral cavity. O recently that orthodontic tooth movement and orthodontic materials used in orthodontic treatment do not lead to a change above the physiological limits that is suggestive of oxidative damage in both GCF and saliva. Oxidative stress biomarkers in patients with aseptic inflammation (age 14.8 years) were evaluated in saliva prior to orthodontic treatment and one and
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Fig. 1. The mean of: A – total amount of malondialdehyde, B – total oxidant status and C – protein concentrations in unstimulated and stimulated saliva of clinically healthy subjects with fixed orthodontic appliances before (0), one week (1) and twenty four weeks (24) after orthodontic treatment. Abbreviations: MDA – malondialdehyde; TOS – total oxidant status; UWS – unstimulated whole saliva; SWS – stimulated whole saliva; * – significant changes vs. group before orthodontic treatment (p < 0.05); ** – significant changes vs. group one week (1) after orthodontic treatment (p < 0.05); std. dev. – standard deviation.
six months later. Similarly, we did not observe any changes in the biochemical parameters of oxidative stress such as MDA and TOS, in the saliva of our study participants (age 23.1 years) with a clinically healthy oral cavity twenty four weeks after orthodontic treatment. Nevertheless, there are some reports in the literature indicating enhanced levels of oxidative stress biomarkers in saliva in the period immediately following orthodontic treatment. In 2009 Olteanu et al. [16] studied some saliva markers of oxidative stress in 37 girls and 4 boys between the ages of 8 and 12 years with the average age of 9.9 years, who were treated with biomechanical appliances. Saliva samples were collected before the commencement of orthodontic treatment and 1 h, 24 h, and 7 days after therapy. The authors found a statistically significant, enhanced concentration of ceruloplasmine and MDA, which reached maximum levels 24 h after treatment and 1 h after treatment for hydrogen donors, while 7 days after device attachment concentrations of the studied salivary markers of oxidative stress were close to the initial
values. According to the authors, even statistically significant changes in the levels of the salivary markers of oxidative stress, do not determine the appearance of certain pathological processes within the oral cavity. A number of identified and unidentified substances create the oxidant/antioxidant balance in saliva. The dynamic distribution of different oxidants in various parts of the oral cavity and their potential interactions make it difficult to measure each oxidant separately. Since all these oxidants compose the complex oxidant system, some authors have suggested that levels of oxidant stress markers in vitro or total oxidants status (TOS) in vivo should be measured. An in vitro study has demonstrated that traditional metal brackets are a source of oxidative stress in murine fibroblast cells L929 [17]. The results of our in vivo study are in line with this observation. We noted a marked increase in TBARS and TOS levels in saliva one week after the attachment of orthodontic appliances. At present it is difficult to explain the discrepancies between our results and those obtained by Olteanu et al. [16]. We can
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Fig. 2. The mean of: A – total amount of superoxide dismutase 1, B – total amount of catalase, C – total amount of uric acid, D – specific activity of peroxidase, E – total antioxidant status and F – oxidative status index in unstimulated and stimulated saliva of clinically healthy participants with fixed orthodontic appliances before (0), one week (1) and twenty four weeks (24) after orthodontic treatment. Abbreviations: SOD1 – superoxide dismutase 1, CAT – catalase, UA – uric acid, Px – peroxidase, TAS – total antioxidant status, OSI – oxidative status index, UWS – unstimulated whole saliva, SWS – stimulated whole saliva, * – significant changes vs. group before orthodontic treatment (p < 0.05), ** – significant changes vs. group 1 week after orthodontic treatment (p < 0.05), std. dev. – standard deviation.
speculate that the disparities resulted from the fact that different types of orthodontic brackets, containing different amounts of nickel, were used in the studies. Moreover, the research was performed in different countries with different diets and different oral hygiene standards. Oral hygiene and periodontal status is crucial for oxidant status in saliva. It has been demonstrated that part of the total TAS ability may be connected with polyphenols which are primarily derived from foods of plant origin [30]. Since the participants in our study were clinically healthy individuals with no detectable pathological changes in the oral cavity, the most probable factor responsible for oxidative stress
induction seems to be substances derived from the orthodontic material. Orthodontic brackets, bands, and arch wires are universally made with an alloy which contains approximately 15–54% of nickel and 20–30% of chromium [31,32]. In addition to being allergenic, nickel and, to a lesser extent, chromium have been found to possess carcinogenic, mutagenic and cytotoxic properties [33–35]. Faccioni et al. [31] and Hafez et al. [32] have demonstrated that nickel released from orthodontic appliances can induce DNA damage in oral mucosa cells. Natarajan et al. [35] have also demonstrated that nickel alloys emit nickel cations in quantities
Fig. 3. The concentration of nickel in unstimulated and stimulated saliva of clinically healthy participants with fixed orthodontic appliances before (0), one week (1) and twenty four weeks (24) after orthodontic treatment. Abbreviations: Ni – nickel; UWS – unstimulated whole saliva; SWS – stimulated whole saliva; * – significant changes vs. group before orthodontic treatment (p < 0.05); ** – significant changes vs. group 1 week after orthodontic treatment (p < 0.05); std. dev. – standard deviation.
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sufficient to induce localised genotoxic effects. It has also been demonstrated that there exists considerable variability among individuals with orthodontic appliances in the concentration levels of nickel in saliva. For example, De Souza et al. [34] observed a maximal increase in nickel cations between 10 min and one day after orthodontic appliance attachment, whereas Bengleil et al. [36] noted the highest concentration of nickel cations four weeks after the insertion of the traditional bracket system while Singh et al. [37] observed a maximal concentration in nickel cations 7 days after orthodontic treatment. In our study we used brackets made entirely of metal, containing 38% of nickel and 30% of chromium. Therefore, nickel involvement in the oxidative stress status in saliva should be taken into consideration. Indeed, we observed a statistically significant increase in nickel cation concentration and TOS one week after orthodontic treatment. Twenty four weeks after orthodontic treatment nickel level in saliva was low or undetectable and total oxidative stress was close to the initial value (Fig. 3). Lipids containing polyunsaturated fatty acids are a group of cellular components which sustain the most significant damage caused by reactive oxygen species. Oxidative stress caused by a disruption in the oxidant/antioxidant balance, leads to lipid peroxidation [38]. MDA is the final product of lipid peroxidation [39]. In our study we observed a significant increase in TBARS concentration in saliva one week after orthodontic appliance attachment but no changes were detected twenty four weeks after treatment in comparison to TBARS concentration at the beginning of the study. Similar results were obtained when TOS was estimated. The aforementioned results correlated positively with nickel concentration in saliva measured one week after orthodontic treatment. These results suggest an involvement of nickel in lipid peroxidation and an increase in TOS in the saliva of individuals with orthodontic appliances. Lack of significant differences in TBARS levels measured one and six months after orthodontic treatment in respect to the values observed at the ¨ zcan et al. initiation of treatment, was also observed by O [18]. Summarising, the results presented above indicate an involvement of nickel in the salivary oxidative stress induction in the period immediately following orthodontic treatment. Lack of changes three and six months after therapy suggest that the body develops tolerance to nickel or induces repair mechanisms of the buccal mucosa cells, as suggested by Hafez et al. [32]. The degree of oxidative stress and the severity of subsequent oral tissue damage might depend on the imbalance between the excess production of ROS and antioxidant defense. The oral cavity, being the first line of defense against ROS, contains several antioxidant substances including enzymes, non-enzymatic antioxidants, and an array of small molecules [40]. Taking the above into account, we measured the level/activity of some salivary antioxidants such as SOD1, CAT, UA and Px. We found no statistically significant enhancement of the studied antioxidants one week and twenty four weeks after orthodontic treatment in UWS. However, was observed a decrease in the concentration of SOD1 and CAT in SWS. SOD1 is an enzyme located in the plasma which repairs cells and reduces damage caused to them by superoxide, the most common free radical in the body [41]. The enzyme catalyses the dismutation of the superoxide (O2 ) radical into either molecular oxygen or, to a lesser degree, to hydrogen peroxide (H2O2). Catalase is an enzyme which catalyses mainly the reaction by which hydrogen peroxide is decomposed to water and oxygen. A decrease in SOD1 and catalase concentration in SWS one week after orthodontic treatment can suggest that nickel and/or other metals released from orthodontic material may directly alter the function of oral mucosa cells and bacteria, which can result in reduced SOD and catalase production, respectively.
In contrast, an increase in Px activity in saliva was observed one week after orthodontic treatment. Px is an important part of the defense mechanism against oxygen toxicity. In the mouth, hydrogen peroxide is formed not only by bacteria colonising the mucous membranes but also by the cells of the salivary glands. In saliva, the most important part of this defense mechanism is salivary peroxidase which detoxifies hydrogen peroxide in the presence of thiocyanante by converting it to hypothiocianite, dioxygen and water. Moreover, hypothiocanite inhibits hydrogen peroxide production by oral bacteria [42]. Since Px is an enzyme synthetised and released by the salivary glands we can speculate that an increase in Px activity is an antioxidant defense against excess production of hydrogen peroxide induced by orthodontic treatment in the oral cavity. These known, and a number of unknown antioxidant substances constitute a complex antioxidant system in the oral cavity. Thus, in order to assess the antioxidant status in vivo, it is highly recommended that levels of the overall antioxidant status should be measured [20,21]. In our study, TAS was unchanged one week after therapy, and reduced twenty four weeks after orthodontic treatment both in UWS and SWS. Furthermore, if we want to fully comprehend the oxidative stress status of patients in vivo, not only the TAS and TOS measurements but also the calculation of OSI is necessary [21,28]. In this study, saliva TOS and OSI values were higher one week after orthodontic treatment whereas TAS levels were unchanged. Twenty four weeks after device attachment TAS was decreased and TOS and OSI were close to the initial values. Analysing our results one should keep in mind that contamination of saliva by blood can influence oxidative stress markers. Our results did not confirm a relationship between a local inflammatory process (GI) and the salivary antioxidants and biomarkers of oxidative stress (data not shown). We also excluded patients with oral mucositis, candidiasis, periodontitis and gingivitis. Moreover, to avoid gingivitis, bleeding following bracket insertion, the subjects received instructions concerning the need for supplementary oral hygiene measures, which could suggests that changes in the behaviour of the examined parameters were not the results of local inflammatory processes, contamination of saliva by blood. Our results are in line with the recent data of Kamodyova´ et al. [43] who demonstrated that salivary oxidative stress concentrations are significantly influenced by 1% and more blood contamination in saliva. Saliva samples with 1% blood contamination are visibly coloured and it is possible to easily exclude such contaminated samples from further salivary oxidative stress analysis without any specific biochemical detection of blood constituents. 5. Conclusions There are limited data in the literature regarding the effects of orthodontic treatment on oxidative stress in the oral cavity. The published results were obtained from investigations conducted on limited number of subjects. The studies involved mostly children with low levels of oral hygiene and the observation periods were long (3 and 6 months). The data currently available are not consistent. In some studies orthodontic treatment evoked oxidative stress in saliva and in others it did not. In our study an increase of total oxidant status one week after orthodontic treatment was observed. Our data demonstrate that within a few days fixed orthodontic appliances modified the total oxidant/antioxidant status in the saliva of clinically healthy subjects with high standards of oral hygiene. To our knowledge this is the first report concerning the measurement of the oxidant/antioxidant balance in the saliva of subjects treated orthodontically. The changes normalised twenty four weeks after orthodontic treatment. Our data also demonstrate the probable role of nickel and/or
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other metals released from orthodontic materials on the oxidative status of subjects with high levels of oral hygiene. We obviously cannot exclude the influence of mechanical stimulation or damage to the surrounding tissues resulting from the process of device attachment on the oxidative status in saliva. These possibilities are important and indicate that further research is required using larger samples and considering other risk factors to clarify the role of orthodontic treatment in the modification of oxidative stress in saliva. Conflict of interest The authors declare no conflict of interests. Financial disclosure Supported by the Medical University of Bialystok, Poland, Grant No. 144-07503L. References [1] Salla JT, Taddei SR, Queiroz-Junior CM, Andrade Junior I, Teixeira MM, Silva TA. The effect of IL-1 receptor antagonist on orthodontic tooth movement in mice. Arch Oral Biol 2012;57(5):519–24. http://dx.doi.org/10.1016/ j.archoralbio.2011.09.018. [2] Gostner JM, Becker K, Fuchs D, Sucher R. Redox regulation of the immune response. Redox Rep 2013;18(3):88–94. http://dx.doi.org/10.1179/ 1351000213Y.0000000044. [3] Padgett LE, Broniowska KA, Hansen PA, Corbett JA, Tse HM. The role of reactive oxygen species and proinflammatory cytokines in type 1 diabetes pathogenesis. Ann N Y Acad Sci 2013;1281:16–35. http://dx.doi.org/ 10.1111/j.1749-6632.2012.06826.x. [4] Mandal A. What is oxidative strees. News Medical Life Sciences & Medicine. http://www.news-medical.net/health/What-is-Oxidative-Stress.aspx. [5] Bagchi K, Puri S. Free radicals and antioxidants in health and disease. East Mediterr Health 1998;14:350–60. [6] Kohen R, Nyska A. Oxidation of biological systems: oxidative stress phenomena, antioxidants, redox reaction, and methods for their quantification. Toxicol Pathol 2002;30:620–50. [7] Burton GJ, Jauniaux E. Oxidative stress. Best Pract Res Clin Obstst Gyneacol 2011;25:287–99. http://dx.doi.org/10.1016/j.bpobgyn.2010.10.016. [8] Popolo A, Autore G, Pinto A, Marzocco S. Oxidative stress in patients with cardiovascular disease and chronic renal failure. Free Radic Res 2013;47:346– 56. http://dx.doi.org/10.3109/10715762.2013.779373. [9] Gandhi S, Abramow AY. Mechanism of oxidative stress in neurodegeneration. Oxid Med Cell Longev 2012;2012:428010. http://dx.doi.org/10.1155/2012/428010. [10] Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB. Oxidative stress, inflammation, and cancer: how are they linked. Free Radic Biol Med 2010;49:1603–16. http://dx.doi.org/10.1016/j.freeradbiomed.2010.09.006. [11] Trivedi S, Lal N, Mahdi AA, Mittal M, Singh B, Pandey S. Evaluation of antioxidant enzymes activity and malondialdehyde levels in patients with chronic periodontitis and diabetes mellitus. J Periodontol 2014;85(5):713–20. http://dx.doi.org/10.1902/jop.2013.130066 [Epub 29 July 2013]. ˇ ervenka T, Celec P. Salivary markers of oxidative [12] To´thova´ L, Kamodyova´ N, C stress in oral diseases. Front Cell Infect Microbiol 2015;October (5):73. http:// dx.doi.org/10.3389/fcimb.2015.00073. [13] Buczko P, Zalewska A, Szarmach I. Saliva and oxidative stress in oral cavity and in some systemic disorders. J Physiol Pharmacol 2015;66(1):3–9. [14] Muniz FW, Nogueira SB, Mendes FL, Ro¨sing CK, Moreira MM, de Andrade GM, et al. The impact of antioxidant agents complimentary to periodontal therapy on oxidative stress and periodontal outcomes: a systematic review. Arch Oral Biol 2015;60(September (9)):1203–14. http://dx.doi.org/10.1016/j.archoralbio.2015.05.007. [15] Kesarwala AH, Krishna MC, Mitchell JB. Oxidative stress in oral diseases. Oral Dis 2016;22(January (1)):9–18. http://dx.doi.org/10.1111/odi.12300. [16] Olteanu C, Muresan A, Daicovicin D, Tamure V, Olteanu I. Kenlarts IMLU. Variations of some saliva markers of the oxidative stress in patients with orthodontic appliances. Physiology 2009;19(3):26. http://connection. ebsconhost.com/c/articles/52595764/variations-som. [17] Buljan ZI, Ribaric SP, Abram M, Ivankovic A, Spalj S. In vitro oxidative stress induced by conventional and self-ligating brackets. Angle Orthod 2012;82(2):340–5. http://dx.doi.org/10.2319/061811-395.1. ¨ zcan Atug˘ SS, Ceylan I, Ozcan E, Kurt N, Dag˘suyu IM, Canakc¸i CF. Evaluation of [18] O oxidative stress biomarkers in patients with fixed orthodontic appliances. Dis Markers 2014;2014:597892. http://dx.doi.org/10.1155/2014/597892.
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