RESEARCH ARTICLE

Effect of Bleaching on Mercury Release from Amalgam Fillings and Antioxidant Enzyme Activities: A Pilot Study FILIZ YALCIN CAKIR, DDS, PhD*, ESRA ERGIN, DDS, PhD†, SEVIL GURGAN, DDS, PhD‡, SUNA SABUNCUOGLU, PhD§, CIGDEM SAHIN ARPA, PhD¶, I˙LKNUR TOKGOZ, MSc, PhD STUDENT**, HILAL OZGUNES, PhD††, ARLIN KIREMITCI, DDS, PhD‡‡

ABSTRACT Objective: The aim of this pilot clinical study was to determine the mercury release from amalgam fillings and antioxidant enzyme activities (Superoxide Dismutase [SOD] and Catalase[CAT] ) in body fluids after exposure to two different vital tooth bleaching systems. Material and Methods: Twenty eight subjects with an average age of 25.6 years (18–41) having at least two but not more than four Class II amalgam fillings on each quadrant arch in the mouth participated in the study. Baseline concentrations of mercury levels in whole blood, urine, and saliva were measured by a Vapor Generation Accessory connected to an Atomic Absorption Spectrometer. Erythrocyte enzymes, SOD, and CAT activities in blood were determined kinetically. Subjects were randomly assigned to two groups of 14 volunteers. Group 1 was treated with an at-home bleaching system (Opalescence PF 35% Carbamide Peroxide, Ultradent), and Group 2 was treated with a chemically activated office bleaching system (Opalescence Xtra Boost 38% Hydrogen Peroxide, Ultradent) according to the manufacturer’s recommendations. Twenty-four hours after bleaching treatments, concentrations of mercury and enzymes were remeasured. Results: There were no significant differences on mercury levels in blood, urine, and saliva before and after bleaching treatments (p > 0.05). No differences were also found in the level of antioxidant enzyme activities (SOD and CAT) before and after treatments (p > 0.05). Mercury release did not affect the enzyme activities (p > 0.05). Conclusion: Bleaching treatments either office or home did not affect the amount of mercury released from amalgam fillings in blood, urine, and saliva and the antioxidant-enzyme activities in blood.

CLINICAL SIGNIFICANCE Bleaching treatments with the systems tested in this pilot study have no deleterious effect on the mercury release from amalgam fillings and antioxidant enzymes in body fluids. ( J Esthet Restor Dent 27:29–36, 2015)

INTRODUCTION In modern dentistry, patients demand not only a healthy functional dentition but also an esthetic smile.

Since the introduction of carbamide peroxide (CP) for home bleaching, new techniques and materials with improved properties have been developed. The active ingredient in bleaching agents is hydrogen peroxide

*Professor, School of Dentistry, Department of Restorative Dentistry, Hacettepe University, Ankara, Turkey † Associate Professor, School of Dentistry, Department of Restorative Dentistry, Hacettepe University, Ankara, Turkey ‡ Professor, School of Dentistry, Department of Restorative Dentistry, Hacettepe University, Ankara, Turkey § Research Assistant, Faculty of Pharmacy, Department of Toxicology, Hacettepe University, Ankara, Turkey ¶ Research Assistant, Chemistry Department, Hacettepe University, Ankara, Turkey **Professor, Chemistry Department, Hacettepe University, Ankara, Turkey †† Professor, Faculty of Pharmacy, Department of Toxicology, Hacettepe University, Ankara, Turkey ‡‡ Professor, School of Dentistry, Department of Restorative Dentistry, Hacettepe University, Ankara, Turkey

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(HP), which can be applied directly or via its generation in a CP gel.1 Today, bleaching can be performed in numerous ways. The most prominent use of peroxides in contemporary dentistry involves applications in esthetic dentistry for in-office and/or at-home tooth bleaching. At-home bleaching is a method where the patient fills a custom-designed tray with bleaching material that is worn for several hours each day for 2 to 8 weeks. The benefits achieved with tray-based systems are widely known; lower incidence of tooth sensitivity or gingival irritation, achievement of the same whitening results as higher concentration agents, safety, and efficacy of CP.2 In-office bleaching is useful for removing stains by using a high concentration of carbamide or hydrogen peroxide in a short period of time. The advantages of in-office whitening procedures over at-home bleaching techniques include; dentist control, avoidance of soft-tissue exposure and material ingestion, reduced total treatment time, and greater potential for immediate results that may enhance patient satisfaction and motivation.3 However, although bleaching is a desirable esthetic procedure, it could have adverse local and systemic effects, which require careful assessment.1,2 Amalgam is still one of the widely used restorative materials for posterior teeth as it is durable, easy to use, and inexpensive.4 However, one of its major components, mercury, is of particular concern due to its potential adverse effects (oral galvanism, toxicity, allergenicity, and ecological grievances) on humans and the environment.5–7 Amalgam is reported to be susceptible to the strong oxidizing action of bleaching chemicals.8 Although bleaching gels are routinely applied to the anterior dentition, excessive gel may inadvertently get in contact with amalgam-restored posterior teeth and increase the susceptibility of these amalgam restorations to corrosion, degradation, and metal ion release.8 In recent years, several in vitro studies have reported the mercury ion release from dental amalgams following exposure to bleaching agents.1,2,6,8 Although there are some studies reporting low mercury release values from amalgam exposed to bleaching agents containing CP and HP,9 very high mercury release values have also been reported.5,6 Therefore, the interaction between amalgam and

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chemicals used for vital tooth bleaching is of clinical significance since patients who undergo vital bleaching procedures may have amalgam-restored teeth. It had been shown that mercury bound with protein sulfhydryl groups led to release of reactive oxygen species such as superoxide, hydrogen peroxide, and hydroxyl radicals and to the formation of free radicals.10 These reactive oxygen species may induce injury to tissues. The body’s antioxidant defense systems are the most important protective mechanisms against reactive oxygen species. These systems include enzymes such as superoxide dismutase (SOD) and catalase (CAT). Bleaching chemicals are strong oxidizing agents which release oxygen and highly unstable radicals.11 It has been also reported that antioxidant enzyme systems are the most sensitive indices of hydrogen peroxide and mercury-induced oxidative response.12,13 In recent years, studies have examined the effects of mercury on antioxidant activity systems and on SOD and CAT in various tissues14–17 and fluids such as plasma18 and saliva.19,20 Only a few studies have focused at the relationship between mercury released from dental amalgams and antioxidant systems.18–23 Some of these studies reported a relation between mercury and amalgam and antioxidants, whereas others reported no relation.21,22 Also, no data were reported on the relationship among bleaching procedures, mercury release, and antioxidant activity. Thus, the aim of this pilot clinical study was to determine the level of mercury release from amalgam fillings, in blood, urine, and saliva and the antioxidant enzyme activities (SOD and CAT) in blood after exposure to two different vital tooth bleaching systems (home and office) and compare the attitudes of two bleaching systems toward possible health hazards.

MATERIALS AND METHODS Twenty-eight voluntary patients (12 male and 16 female) in good oral and general health and a desire to lighten their upper and lower arch teeth participated in this pilot study. Subjects with an average age of 25.6

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years (18–41) and having at least two but not more than four Class II amalgam fillings on each quadrant arch (a total of 8–16) in the mouth participated in the study. The Ethics Committee of Hacettepe University, Ankara, Turkey reviewed and approved the research protocol and informed consent form. Patients received a professional dental prophylaxis 2 weeks before the start of bleaching treatments and were asked to brush their teeth twice daily in order to standardize tooth cleaning during the study. All subjects had anterior baseline color shade of at least A3 or darker on the Vita shade Guide (VITAPAN classical, VITA Zahnfabrik, Bad Sackingen, Germany). Inclusion criteria were the presence of all natural teeth (intact dentition), and the absence of caries and periodontal diseases, any restorations except amalgam as well as previous bleaching treatments. In order to control local and systemic factors that might affect body mercury concentrations and antioxidant levels, individuals meeting any of the following criteria were excluded from the study; systemic disorders, medication usage during the previous 3 months, smoking/alcohol habits (any present or past consumption of tobacco or alcohol), occupational exposure to mercury, placement of new amalgam restorations during the previous year, parafunctional habits (e.g., bruxism), consumption of fish/seafood during the previous month and frequent gum chewing (everyday, several times a day).12 Subjects were also asked to avoid consumption of fish during the study to avoid any dietary or occupational conditions that might have an influence on the mercury levels.

Blood, Urine, and Saliva Sampling Blood samples were collected from all participants using heparin as anticoagulant. Samples were centrifuged at 700 to 1,000× g for 10 minutes at 4°C, the white buffy coat was removed, and the yellow plasma layer was stored at −80°C until analysis.12 Urine was collected over a period of 24 hours in 2.5-l polypropylene sampling vessels and stored at −20°C prior to analysis.24 Unstimulated saliva was collected without previous salivary stimulation into 5-mL polypropylene centrifuge

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tubes, and centrifuged at 1,000 rpm for 5 minutes and transferred into polypropylene reaction vessel and stored at −70°C.24

Mercury Analysis The measurements of elemental mercury were performed by a Vapor Generation Accessory (FIAS-100 Perkin Elmer) connected to an Atomic Absorption Spectrometer (Analyst 800-Perkin Elmer).12

Antioxidant Enzymes Analysis Erythrocyte enzymes SOD and CAT activities were determined kinetically and expressed in IU/mg protein.

Superoxide Dismutase (SOD) Each hemolysate was diluted 1:5 with 50 mM Tris-HCl buffer (pH = 8.2; containing 1.2 mM EDTA). Twenty microliter of diluted hemolysate was mixed with 2,900 μL of buffer solution in a quartz cuvette. Subsequently, 100 μL of pyrogallol solution (6 mM, in 10 mMHCl) was added to the mixture, mixed for 20 seconds, and the decrease in absorbance was followed at 420 nm for 2 minutes. Non-enzymatic reaction rate was determined as blank by substituting buffer solution for the sample, and this reaction rate was used for the activity measurements.

Catalase (CAT) CAT activity in erythrocyte lysate was measured after dilution of the RBC hemolysates 1:500 with 50 mM phosphate buffer, pH: 7.00, just before the measurements. The reaction mixture was 50 mM phosphate buffer pH 7.00, 10 mM H2O2, and erythrocyte lysate. The reduction rate of H2O2 was followed at 240 nm for 30 seconds at room temperature.

Bleaching Procedures Participants were randomly assigned to two groups of 14 volunteers each. They have given their written consent and completed medical history forms prior to

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TABLE 1. Bleaching systems used in the study Groups

Products

Manufacturer

Concentration

Number of application

Length of each application

Group1

Opalescence PF (home bleaching)

Ultradent products, Inc. South Jordan, Utah, USA

35% carbamide peroxide

2 weeks

30 minutes per day

Group2

Opalescence boost (office bleaching)

Ultradent products, Inc. South Jordan, Utah, USA

38% hydrogen peroxide

2 times

15 minutes

the start of the study. Each bleaching system was applied to both maxillary and mandibulary arches according to manufacturer’s recommendations (Table 1). In Group 1, patients were treated with an at-home bleaching system (Opalescence PF [35% CP]/Ultradent Products, Inc. South Jordan, Utah, USA). Maxillary and mandibulary alginate impressions were taken from patients, and dental stone models were cast for fabrication of bleaching trays. Ethyl-vinyl-acetate trays were made with a heat/vacuum tray forming machine. The trays were trimmed to fit each model perfectly. Patients were instructed on how to care for and use the trays correctly and were asked to wear their trays filled with the bleaching gel 30 minutes per day for 2 weeks. Participants were required to return at seventh day for an oral examination and interview regarding symptoms. In Group 2, patients were treated with chemically activated office bleaching system (Opalescence Boost [38% HP]/Ultradent Products, Inc. South Jordan, Utah, USA). A brush-on isolation material (Opaldam, Ultradent Products, South Jordan, Utah) with a thickness of approximately 1.0 mm was applied to all surfaces in the treatment area and cured using a standard curing light (Elipar Free Light, 3 M Espe, St Paul, MN, USA) to ensure protection of the gingiva. The gel was administered using two syringes: one syringe contained the activator and the other HP. Before use, the activator was mixed with the bleaching agent, and the mixture was applied directly onto the teeth as a 0.5 to 1.0 mm-thick layer. After 15 minutes, the barrier was removed, and the treated teeth were thoroughly rinsed with an air–water spray. The procedure was repeated twice.

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After the completion of office and home bleaching treatments (24 hours), all measurements were repeated. The data were analyzed by Wilcoxon Signed Ranks and Mann-Whitney U tests. In all the tests, the level of significance was set at p < 0.05, and calculations were handled by the SPSS12.0 software for Windows (SPSS, Chicago, IL, USA).

RESULTS There was a slight increase in mercury levels in home bleaching (Group 1) in blood, urine, and saliva before and after bleaching treatments. A similar increase was observed with office bleaching (Group 2) treatment in mercury levels in body fluids. However, the difference was not statistically significant (p > 0.05). The data are shown at Table 2. In spite of the fact that antioxidant enzyme levels increased slightly in Group 1 and that they remained the same in Group 2, mercury release and bleaching treatments did not affect the levels of antioxidant enzymes (SOD and CAT) (p > 0.05). The data are shown in Table 3.

DISCUSSION Dental amalgam is a potential source of mercury in the human body which contains inorganic mercury bound to inter-metallic compounds. Mercury is released from amalgam restorations under physiological conditions either as mercury vapor or mercury ions. Metal ions may pass into the oral fluids to be ingested via the digestive system—it is suggested that 40% of mercury

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TABLE 2. Mercury concentrations (ng/mL) in blood, urine, and saliva before and after bleaching treatments Body fluids

Groups

Before bleaching

After bleaching

z

p < 0.05

Blood

Group 1 Group 2

2.40 ± 1.70 3.43 ± 3.06 z = 0.105, p = 0.917

2.46 ± 1.52 3.93 ± 3.92 z = 0.734, p = 0.463

0.857 0.571

0.391 0.568

Urine

Group 1 Group 2

2.40 ± 2.11 2.17 ± 5.46 z = 0.524, p = 0.600

2.69 ± 1.78 2.78 ± 1.57 z = 0.674, p = 0.500

1.643 0.286

0.100 0.775

Saliva

Group 1 Group 2

28.72 ± 18.88 29.20 ± 14.05 z = 0.943, p = 0.345

29.96 ± 10.26 32.09 ± 16.09 z = 0.943, p = 0.345

0.143 0.160

0.886 0.873

TABLE 3. SOD and CAT levels (IU/mg) in blood before and after bleaching treatments Antioxidant enzymes

Groups

Before bleaching

After bleaching

z

p < 0.05

Superoxide dismutase (SOD)

Group 1 Group 2

1.55 ± 0.42 1.98 ± 0.50 z = 0.136, p = 0.173

1.94 ± 0.19 1.98 ± 0.45 z = 0.000, p = 1.000

1.286 1.000

0.199 0.317

Catalase (CAT)

Group 1 Group 2

1.43 ± 0.29 1.65 ± 0.28 z = 0.943, p = 0.345

1.63 ± 0.30 1.65 ± 0.32 z = 0.507, p = 0.612

1.429 0.000

0.153 1.000

exposure from dental amalgam is via ingestion of metal ions.5 The amount of mercury released varies with the number of restorations, their surface area, particularly the load-bearing surface area, galvanic currents, mastication habits (bruxism), eating habits, chewing gum, and tooth brushing habits. Mercury vapor is rapidly absorbed in the respiratory tract and distributed by blood to a number of key target organs. Mercury vapor is oxidized to inorganic mercury, and elimination is by exhaled air or as inorganic mercury by urine from kidneys, sweat, and saliva. Total mercury in blood consists of inorganic and organic mercury. The organic portion is almost entirely methyl mercury from fish and seafood.25 The inorganic portion may be from food, medicaments, and amalgam restorations. Mercury ions are much less readily absorbed in the gastrointestinal tract.26–29 Mercury ion release is also augmented under bleaching conditions.2,5,6,27 It may then be absorbed by the oral mucosa as well as by the respiratory and gastrointestinal tract with a potential risk of toxic systemic effects. The raised mercury levels in bleaching-treated amalgam can interact and be

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absorbed by the oral tissues and contribute to increased mercury concentrations in blood, saliva, and urine. Mackert and Berglund30 reported the rate of unstimulated mercury release from amalgam to be on average of 0.4 μg per amalgam surface per day. Assuming a surface area of 1 cm2 is equivalent to four mercury amalgam surfaces in vivo, the mercury release value from Al-Salehi and colleagues31 study of approximately 1.0 μg/cm−2 in 24 hours would mean that four bleached amalgam surfaces would subsequently release only 1.0 μg of mercury into the oral cavity. This level is below the World Health Organization’s maximum acceptable daily intake (ADI) for mercury of 40 μg. Indeed, to exceed the ADI for mercury, a patient would require mercury to be released from approximately 160 amalgam restorations.32 In the present study, subjects with at least two but no more than four Class II amalgam fillings on each quadrant arch in the mouth were selected. Bleaching gels are generally applied to the anterior dentition (and sometimes to the premolars). For home use, tooth bleaching custom trays often extend to the

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molar area as they improve tray stability and fitting, and patients are instructed not to put bleaching gel in molar area when an amalgam restoration exists. As for office bleaching treatments, molars, especially their occlusal surfaces, are not included in the procedure. However, although it is avoided, there is always a possibility for excessive bleaching gels inadvertently to get in contact with the premolar and molar teeth. Furthermore, Class II restorations have not only occlusal but also approximal surfaces. Thus, more surface area, especially the contacts may get in contact with the bleaching material. And our aim in this very study was to determine the effect of bleaching on mercury release from amalgam fillings. For this reason, subjects who had Class II amalgam restorations on premolar and molar posterior teeth, which were in need of bleaching treatment, were selected. This was also desirable for standardization and more reliable results. Amalgam is a complex material that has a heterogeneous structure and 8 to 10 distinct phases with different micro-structural features. Bleaching agents can interact with certain amalgam phases, thus affecting the physicochemical behavior of the amalgam restoration. Rotstein and colleagues6 reported that different bleaching agents affected amalgams in different fashions; however, the results of this in vivo study indicated that bleaching treatments did not increase the mercury release from dental amalgam and also the mercury levels in body fluids. Mercury is commonly found in blood which is the major medium for transporting materials around the body. The mercury levels in blood are lower than those of other body fluids, therefore it is more difficult to detect. Even at high levels of mercury exposure, industrial workers show blood concentrations in the parts-per-billion (ppb) range, typically less than 5 ppb. In this range, the amounts are too small to identify the type of mercury or its source. Clinical evidence of toxicity begins to appear in the most sensitive adults at blood concentrations of 30 ng/mL.33 Krönce and colleagues34 and Ott and colleagues35 detected no difference in mercury concentrations in the blood of subjects with and without amalgams, whereas Abraham and colleagues36 found that subjects with amalgams had higher blood mercury levels than those without

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amalgams. The findings of this study showed that blood mercury levels of the subjects having four amalgam fillings were within the normal limits also before and after bleaching treatments. Mercury is excreted by the kidneys, which filter the blood, so it is commonly found in people’s urine. Large-scale studies have shown that the general population has urinary mercury levels below 10 μg/L. Akesson and colleagues37 and Molin and colleagues18 reported that amalgam filling increases urinary mercury concentration. In contrast, Olstad and colleagues38 and Ulukapi and colleagues39 revealed no significant increase in urinary mercury concentrations after amalgam filling. In this study, the bleaching treatments did not affect the urine mercury levels of the subjects. Several studies demonstrated a positive correlation between amount of amalgam and mercury in blood, urine, feces, saliva, and tissue.30,40–42 However, there were no studies showing relationship between mercury release and bleaching in vivo. In the present study, blood levels and urinary excretion of mercury in the individuals participating in the study are in accordance with the literature. This study also investigated the antioxidant enzyme activities (CAT and SOD) in blood after exposure to bleaching agents. Bleaching chemicals are strong oxidizing agents which release oxygen and highly unstable radicals.9 Hydroxyl radicals and superoxide anions are known to destroy DNA, membrane lipids, and other essential cell components. Normally, the body has protective mechanisms for the removal of free radicals and HP.43 HP penetrates into mucosal cells and comes in contact with intracellular enzymes and is reduced to water by peroxidases. Hydroxyl radicals can accept an electron and then be pronated to form water, and SOD reduces superoxide anions to form HP. SOD is one of the major antioxidant enzymes in human erythrocytes and catalyses dismutation of superoxide anion radical to H2O2. Then glutathione peroxidase and CAT enzymes degrade H2O2. Thus, SOD functions at the first step to detoxify superoxide anion radical and the other two enzymes complete the process. In the present study, bleaching treatments and mercury

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release from amalgam fillings during bleaching did not affect the activities of CAT and SOD. However, the bleaching agents and dental amalgam and antioxidant enzyme relationship still require further and more comprehensive in vivo studies, probably with more subjects.

6.

CONCLUSIONS

8.

Within the limitations of this pilot study, the following conclusions were drawn: 1 The findings indicate that bleaching treatments do not affect the amount of mercury released from amalgam fillings and the antioxidant-enzyme activities in body fluids. 2 Mercury release from dental amalgam exposed to bleaching treatments is not affected by duration of the bleaching treatment, as well as the composition and type of bleaching agent. 3 The relationship between vital bleaching and amalgam tissue toxicity requires further in vivo investigations.

7.

9.

10. 11.

12.

13.

14.

DISCLOSURE The authors do not have any financial interest in the companies whose materials are included in the article. 15.

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Reprint requests: Filiz Yalcin Cakir, DDS, PhD, Department of Restorative Dentistry, School of Dentistry, Hacettepe University, Sihhiye 06100, Ankara, Turkey; Tel.: +90-312-3052271; Fax: +90-312-3113438; email: [email protected]

DOI 10.1111/jerd.12092

© 2014 Wiley Periodicals, Inc.

Effect of bleaching on mercury release from amalgam fillings and antioxidant enzyme activities: a pilot study.

The aim of this pilot clinical study was to determine the mercury release from amalgam fillings and antioxidant enzyme activities (Superoxide Dismutas...
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