Journal of Trace Elements in Medicine and Biology 28 (2014) 200–204
Contents lists available at ScienceDirect
Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.de/jtemb
Clinical studies
Correlation between long-term in vivo amalgam restorations and the presence of heavy elements in the dental pulp Mohammad Ali Saghiri a,∗ , Sepideh Banava a , Mohamad Amin Sabzian a , James L. Gutmann b , Armen Asatourian c , Golam H. Ramezani f , Franklin Garcia-Godoy e , Nader Sheibani d a
Department of Dental Material, Dental School, Azad University (Tehran Branch), Tehran, Iran Department of Restorative Sciences, Baylor College of Dentistry, Texas A&M University System Health Science Center, Dallas, TX, USA c Kamal Asgar Research Center (KARC), Tehran, Iran d Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA e Bioscience Research Center, College of Dentistry, University of Tennessee Health Science Center, Memphis, TN, USA f Department of Pedodontist, Azad University (Tehran Branch), Tehran, Iran b
a r t i c l e
i n f o
Article history: Received 6 October 2013 Accepted 31 January 2014 Keywords: Dental pulp Dental Amalgam Mercury Silver Tin
a b s t r a c t Project: To measure the levels of heavy metals (Hg, Sn) in the dental pulp and blood samples of patients with long-term amalgam restorations. Procedure: 12 amalgam restored and 12 non-restored, sound teeth were chosen and access cavity preparation to the pulp chamber was made. The contents were transferred and dissolved in 5 mL of concentrated nitric acid followed by placement in an oven at 180 ◦ C for tissue digestion. After cooling the tubes each digested sample was transferred to an atomic absorption system to measure the levels of heavy metals. The blood samples of five patients in each group were randomly analyzed to determine the levels of these heavy metals in the blood and if there were a correlation between these levels in blood and pulp. Data were analyzed by t-test at a P < 0.05 level of significance. Results: No significant difference was seen between the levels of Hg and Sn in pulp tissues (P > 0.05); however, the blood analysis showed higher level of Hg amalgam group (P = 0.009). The analysis between the pulp and blood samples showed positive correlations for both Hg and Sn elements in dental pulp and the blood (P = 1.000) (P = 0.900). Conclusions: The long-term presence of dental amalgam (at least 5 years) did not result in any remarkable changes in the levels of mercury and tin in the pulp tissue; however, there were increases in the level of mercury in the blood circulation even five years following the placement of the restoration. © 2014 Elsevier GmbH. All rights reserved.
Introduction The long-term use of dental silver amalgam has raised public concerns due to its potential side effects, such as mercury release [1]. Mercury, silver, tin, copper, and zinc are main components of this alloy that are mixed with other constituents such as indium, cadmium, lead and antimony [2]. Evaporation, dissolution, and evaporation/dissolution have been indicated as three mechanisms of mercury release from dental silver amalgam, of which the third mechanism is known to be the best model for evaluation of the
∗ Corresponding author at: Assistant Professor, Department of Dental Material, Dental School, Azad University (Tehran Branch), P.O. Box 14665-1445, Tehran, Iran. E-mail address: [email protected] (M.A. Saghiri). http://dx.doi.org/10.1016/j.jtemb.2014.01.008 0946-672X/© 2014 Elsevier GmbH. All rights reserved.
impact of Hg release [3–5]. Toxic elements such as mercury are defined as substances that can cause hazardous and adverse influence on body health [6]. The toxic effects of mercury on nervous and renal systems depend on acute contamination to elementary mercury [7]. Other investigators have shown that neurotoxicity of this element is highly relevant to the amount of mercury that is concentrated inside body cells [8]. Tin is another component of dental silver amalgam [2] that may be released from amalgams or that can influence the release of Hg [9,10]. The dissolution of tin inside the dental amalgam, especially in low pH environments, can increase the mercury release from the tin-free ␥1 phase [9]. In the presence of the on-going dissolution of tin; mercury undergoes greater evaporation and ionization into the surrounding environment [10]. However, mercury release from dental amalgam can be decreased due to the production of a
M.A. Saghiri et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 200–204
tin oxide layer on the surface of ␥1 phase of Hg, unless this layer dissolves because of low pH values inside the oral cavity [9]. Further concerns are the clinical side-effects and these sideeffects include gingivitis, loss of teeth and alveolar bone, oral galvanism, alteration of taste, mucosal ulceration and bruxism [11,12]. The pulpal uptake of mercury has been identified to play an important role in inflammation of dental pulp, following the placement of amalgam restorations [13]. Studies have shown that mercury penetration was not decreased even with the use of cavity liners such as calcium hydroxide or glass ionomer cements, due to their solubility and water contents [14–16]. Furthermore, the dynamic nature of the oral cavity including biological, mechanical, chemical and thermal stresses makes the whole scenario more complex [17] In this regard, some authors have evaluated the amount of mercury release during chewing stimulations and brushing time [18], while others have studied this phenomenon under different pH values [9]. Set dental amalgam in the oral cavity is considered as a dynamic substance that is predisposed to many clinical stresses such as temperature alteration and corrosive attacks [19] that might increase mercury release to some extent over time [18]. The present study was performed to evaluate the levels of mercury and tin the pulpal tissue of amalgam restored teeth and in the blood of patient possessing amalgam restoration. The hypothesis tested was whether or not the amount of these elements increases inside the pulp tissue and the blood over a long period of time.
201
system purification (Millipore, Milford, MA, USA). 5 mL of concentrated nitric acid was added and samples underwent digestion at temperature of 180 ◦ C in the oven. After cooling the tubes, 3 mL H2 O2 was added and evaporated to about 0.5 mL. Each digested sample was transferred quantitatively into a 10 mL calibrated tube to which was added a hydrochloric acid solution 1 mol/L. Serial dilution 1 mL aqueous solution of Triton X-100 containing 40 g of NH4 NO3 per liter as the matrix modifier was used for the final dilution of all samples, including standards and experimental groups. Preparation of reagents Purified water in a high-grade Milli-Ro De-ionizer system was used to prepare all reagents. 1 mL aqueous solution of Triton X-100 (iso-octylphenoxypolyethoxy ethanol), containing 40 g of NH4 NO3 per liter as the matrix modifier, was used for the final dilution of all samples, including standards and experimental groups. All chemicals were analytical grade when obtainable and highpurity nitrogen was used as the purging gas. A solution containing 60 g of BSA (Sigma Chemical Co., St. Louis, MO), 140 in mol of NaCl, and 5 mrnol of KHCO3 per liter was used to prepare all working standards. Preparation standards
Materials and methods Twenty-four premolar and molar teeth of 24 healthy patients, without any clinical signs and symptoms were extracted for orthodontic purposes and following ultrasonic cleaning were analyzed with stereomicroscope to ensure they were free of defects, such as cracks. Twelve specimens had class I amalgam restorations that were between 5 and 20 years old. The exclusion and inclusion criteria were as following: Exclusion criteria: any subjects who had systemic problems such as physician-diagnosed psychological, behavioral, neurological, immunosuppressive, or renal disorders. General inclusion criteria: (1) all subjects were older than 18 years old and agreed to the parameters of this study by signing the consent form based on the Helsinki guidelines. (2) The number of amalgam restorations inside the oral cavity of each patient should not have been more than five restorations. (3) Subjects were non-smokers and had no history of dental implants, other NiTi prosthesis in any part of their body, any piercing, and did not wear steel watches. (4) Diet control was done to ensure that subjects did not consume any sea food for at least one week prior to blood sampling. Specific inclusion criteria: (1) the amalgam restorations must have be 1.5–2 mm from the pulp. (2) The amalgam restorations must weighed 1 ± 0.02 g. In order to check these criteria, samples were grooved vertically on the buccal and lingual surfaces with a diamond disk without entering the pulp chamber, and split longitudinally with a chisel. The filling materials of experimental samples were extracted from the cavity and weighed by a electronic digital analytical balance (Mettler AE-163, Mettler Toledo, CA, USA) with an accuracy of 0.0001 g and the dentin thickness was measured by a caliper to be 1.5–2 mm. The remaining extracted teeth were sound, did not have any restorations and served as the control group (Fig. 1). After splitting the teeth in bucco-lingual direction, the pulp tissues of all samples were taken out of the pulp chamber with minimal damage by using plastic instruments and transferred into a clean beaker containing deionized pure water. Ultrapure water of 18 M/cm specific resistivity was obtained from Milli-Q water
A standard solution of 1000 ppm Hg was obtained (Fluka Chemie, Buchs, Switzerland) and for the Sn a standard solution was obtained (PerkinElmer, Pure atomic spectrometry standard, Shelton, CT, USA). The measurement of the levels of Hg and Sn were performed by WFX-1B atomic absorption spectrophotometer (No.2 Optical Instrument Factory of Beijing, China) equipped with a graphite furnace (HGA-400, Perkin-Elmer, CT, USA). The silver hollow-cathode lamp (Photron, Dandenong, Victoria, Australia) with a wavelength of 328.1 urn and a high-density graphite carbon tube (Perkin-Elmer, CT, USA) without platform were used in the assay. The calibration graph is linear up to 1 g/mL of tin. The limit of detection of this method is 0.002 g/mL. Evaluation of blood samples After teeth removal, five samples from each group were chosen randomly and subjected to blood evaluation of these heavy elements. Blood was collected by a vein puncture needle 25 × 7 (Vacutainer) in a 5-mL tube with the 5% percentage by weight anticoagulant EDTA. Statistical analysis The level of Sn and Hg elements represented non-continuous data that were presented after being analyzed by nonparametric tests and descriptors. Data were tabulated; proportions and 95% confidence intervals (CIs) were calculated. Significance of univariate associations was assessed with chi-square tests. For pulp/pulp and pulp/blood analysis, variables and treatment outcomes, the statistical program of SPSS 9.0 (SPSS Inc., Chicago, IL, USA) was used. Results The box plot of the level of heavy metals determined in the experimental groups is shown in Fig. 2. The statistical results indicated that there were no significant differences in the levels of Hg and Sn inside the pulpal tissue of the amalgam and control groups (P > 0.05). Blood evaluation identified significant differences
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Fig. 1. The process of pulp tissue extraction: the periapical radiography (a maxillary third molar), tooth was extracted, the amalgam filling was taken off by buccal and lingual vertical grooving, and finally by splitting vertically the pulp tissue was extracted and transferred into the beaker containing deionized pure water.
between the level of Hg in blood samples of the amalgam and control groups (P = 0.009) (Fig. 2C and D). The comparison of pulp and blood samples in experimental and control groups indicated that there was a correlation between the levels of these elements. A positive correlation coefficient of 1.000 and 0.900 were determined between the levels of Hg and Sn in pulp tissues and the blood samples. Discussion Previous investigations have discussed upon on-going and continual process mercury release in dynamic environment of oral cavity [17]. Physicochemical stimulations inside the mouth can impose microstructural alteration in dental amalgam restorations [19] that can lead to prolonged mercury release into the pulp tissue and/or bloodstream through the gastrointestinal route [18]. Three samples of experimental group were subjected to delayed analysis and in this time period these sample solutions were stored in 6 mol/L HCl to prevent hydrolysis of each elements which forming a semi-colloidal suspension.
Present study determined the levels of heavy metals (Hg and Sn) in the pulp tissue and blood of patients who had dental amalgam restorations for at least 5 years. Patient clinical records did not provide any information about the type of restorative materials (low-copper or high-copper), the restoration technique and the presence or absence of lining materials such as calcium hydroxide or glass ionomer cements that had been placed as bases under the amalgam restorations. Therefore authors had selected samples that included 1.5–2 mm dentin thickness between the amalgam and pulp tissue. Under this inclusion criterion, no lining materials such as calcium hydroxide or glass ionomer were used prior to the placement of the amalgam. Previous studies suggested the use of lining material primarily for deep cavities [20,21]. With respect to this, other investigators have indicated that neither CaOH2 nor glass ionomer cement can prevent the mercury intake into the pulp tissue through the dental tubules [16]. Due to segregation of pulp tissue, one specimen in the control group was damaged during the process of extraction of the pulpal tissue from pulp chamber. The results have indicated that the levels of tested elements in the pulpal tissue of amalgam-restored and non-restored teeth were
M.A. Saghiri et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 200–204
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Fig. 2. The box plot of the levels of Hg and Sn in the pulp tissue (A and B) and in the blood (C and D) samples of experimental and control samples.
not significant. This is consistent with results of previous investigations, where dental silver amalgam restorations could predispose minimal risk to neural tissues [8]. Marek has discussed two aspects of concentration and time of mercury release and concluded that the dissolution rate of mercury declined over the time due to the stifling effect of the concentration of mercury. Also, he has noted that the time period along with other variables such as solution volume, oxidation and evaporation conditions are effective factors in release of mercury [3]. The release of mercury was also evaluated under controlled chewing and brushing actions inside artificial saliva [18]. The results showed that the release of this element depended on the age of amalgam in a way that in initial non-steady-state situations it was higher than steady-state conditions. Berdouses et al. have shown that the dynamic situation produced by chewing and brushing activities can make the release dose of mercury from dental silver amalgam to 0.03 g/day, which is far distant from the threshold-limiting value (TLV) of 82.29 g/day, that is considered as a dangerous threshold for occupational exposure [18]. Moreover, the acidic conditions can increase the dissolution of mercury from dental silver amalgam [9]. Marek has suggested that the dissolution of mercury can be decreased by formation of preservative tin oxide layer on the surface of dental silver amalgam. In low pH conditions this oxide layer could dissolve that may lead to additional dissolution of mercury [9]. According to this issue, the tin oxide layer can produce a barrier against the release of mercury that can diminish the dissolution of this element [9]. The blood analysis has demonstrated that the level of mercury was significantly increased, while the amount of tin did not show any significant increase. This result is consistent with a previous study showing that blood mercury levels were closely related to the number and surface area of amalgam restorations [19]. Other investigators studied the contribution of dental amalgam to mercury in blood and concluded that by removing the amalgam restorations
a significant decrease was noticed in the levels of this element in the blood of subjected samples [22]. Snapp et al. have reported that the screening of mercury level was carried out weekly for five to eighteen weeks (median = 7.6 weeks) [22]. The results of measuring the Sn level did not show similar results and was not significant between experimental and control groups. This outcome might be explained by the lesser amount of tin (13%) in the composition of dental silver amalgam in comparison with Hg which is approximately 50% [23]. The data obtained from this study suggests that although dental silver amalgams cannot predispose the pulpal tissue to any remarkable changes regarding the mercury level, the increase in the mercury level of blood circulation still remains as a potential concern that may have medical implications for the individual. However, other investigators have mentioned that many of these anti-amalgam studies include many logical and methodological errors that mistakenly can make the safety of amalgam restorations doubtful [24]. A further study indicated that this issue should be evaluated in an interdisciplinary manner, which can make the assessment by using information from different aspects of materials science, corrosion, mercury exposure, toxicology, neurology and immunology [25]. Conclusion According to outcomes of this investigation, the following conclusions can be drawn: • The prolonged time of exposure to dental silver amalgam restorations did not increase the levels of mercury and tin in the dental pulp tissue. • The blood levels of mercury, unlike the pulpal levels of this element, was increased because of the continual release of mercury.
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• The comparison between the levels of mercury and tin in pulp and blood samples demonstrated a positive correlation. Acknowledgments This publication was based on the thesis presented, in partial fulfillment to the requirements for Doctor of Dental Surgery (D.D.S) in Azad University. We are indebted to Dr. Amir Nazari for all their contribution to this research. References [1] Darvell BW. Development of strength in dental silver amalgam. Dental Materials 2012;28:e207–17. [2] Akbal A, Yılmaz H, Tutkun E, Kös¸ MD. Aggravated neuromuscular symptoms of mercury exposure from dental amalgam fillings. Journal of Trace Elements in Medicine and Biology 2014;28:32–4. [3] Marek M. The release of mercury from dental amalgam: the mechanism and in vitro testing. Journal of Dental Research 1990;69:1167–74. [4] Radics J, Schwander H, Gasser F. Die kristallinen komponenten der silberamalgam – untersuchungen mit der elektronischen rontgenmikrosonde. Zahnarztliche Welt 1970;79:1031. [5] Till T, Wagner G. Untersuchungen zur L5slichkeit der bestandteile von amalgamfullungen wahrend des kau – und trinkaktes II. Zahnarztliche Welt 1973;82(1004):194–205. [6] James RC, Roberts SM, Williams PL. General principles of toxicology. In: Williams PL, James RC, Roberts SM, editors. Principles of toxicology: environmental and industrial applications. 2nd ed. New York: John Wiley and Sons, Inc.; 2000. p. 3–4. ISBN: 0-471-29321-0. [7] Ambient air pollution by mercury (Hg). Position paper. Luxembourg: Office for Official Publications of the European Communities; 2001. [8] Sweeney M, Creanor SL, Smith RA, Foye RH. The release of mercury from dental amalgam and potential neurotoxicological effect. Journal of Dentistry 2002;30:243–50. [9] Marek M. Dissolution of mercury from dental amalgam at different pH values. Journal of Dental Research 1997;76:1308–15.
[10] Brecht-Bergen N. Korrosionsuntersuchungen an Zinn–Silber-amalgamen. Zeitschrift für Electrochemie 1933;39(927). [11] Shafer WG, Hine MK, Levy BM. A textbook of oral pathology. Philadelphia, PA: W.B. Saunders Co.; 1958. p. 443–5. [12] Al-Saleh I, Elkhatib R. Effect of mercury (Hg) dental amalgam fillings on renal and oxidative stress biomarkers in children. Science of the Total Environment 2012;431:188–96. [13] Möller B, Granath L-E. Reaction of human dental pulp to silver amalgam restorations. The effect of insertion of amalgam of high plasticity in deep cavities. Acta Odontologica Scandinavica 1973;31:187–92. [14] Rehfeld RL, Mazer RB, Leinfelder KF, Russell CM. Evaluation of various forms of calcium hydroxide in the monitoring of microleakage. Dental Materials 1991;7:202–5. [15] Pereira JC, Manfio AP, Franco EB, Lopes ES. Clinical evaluation of Dycal under amalgam restorations. American Journal of Dentistry 1990;3:67–70. [16] Hörsten-Bindslev P, Danscher GH, Hansen JC. Dentinal and pulpal uptake of mercury from lined and unlined amalgam restorations in minipigs. European Journal of Oral Science 1997;105:338–43. [17] Marek M. Interactions between dental amalgams and the oral environment. Advances in Dental Research 1992;6:100–9. [18] Berdouses E, Vaidyanathan TK, Dastane A, Weisel C, Houpt M, Shey Z. Mercury release from dental amalgams: an in vitro study under controlled chewing and brushing in an artificial mouth. Journal of Dental Research 1995;74:1185–93. [19] Abraham JE, Svare CW, Frank CW. The effect of dental amalgam restorations on blood mercury levels. Journal of Dental Research 1984;63:71–3. [20] Hume WR, Gerzina TM. Bioavailability of components of resin-based materials which are applied to teeth. Critical Reviews in Oral Biology and Medicine 1996;7:172–9. [21] Hilton TJ. Cavity sealers, liners, and bases: current philosophies and indications for use. Operative Dentistry 1996;21:134–46. [22] Snapp KR, Boyer DB, Peterson LC, Svare CW. The contribution of dental amalgam to mercury in blood. Journal of Dental Research 1989;68:780–5. [23] Hahn LJ, Kloiber R, Vimy MY, Takahashi Y, Lorscheider FL. Dental silver tooth fillings: a source of mercury exposure revealed by whole-body image scan. Journal of the Federation of American Social Experimental Biology 1989;3:2641–6. [24] Dodes JE. The amalgam controversy an evidence-based analysis. Journal of American Dental Association 2001;132:348–56. [25] Hanson M, Pleva J. The dental amalgam issue. A review. Cellular and Molecular Life Sciences 1991;47:9–22.
Contents lists available at ScienceDirect
Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.de/jtemb
Clinical studies
Correlation between long-term in vivo amalgam restorations and the presence of heavy elements in the dental pulp Mohammad Ali Saghiri a,∗ , Sepideh Banava a , Mohamad Amin Sabzian a , James L. Gutmann b , Armen Asatourian c , Golam H. Ramezani f , Franklin Garcia-Godoy e , Nader Sheibani d a
Department of Dental Material, Dental School, Azad University (Tehran Branch), Tehran, Iran Department of Restorative Sciences, Baylor College of Dentistry, Texas A&M University System Health Science Center, Dallas, TX, USA c Kamal Asgar Research Center (KARC), Tehran, Iran d Department of Ophthalmology and Visual Sciences, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA e Bioscience Research Center, College of Dentistry, University of Tennessee Health Science Center, Memphis, TN, USA f Department of Pedodontist, Azad University (Tehran Branch), Tehran, Iran b
a r t i c l e
i n f o
Article history: Received 6 October 2013 Accepted 31 January 2014 Keywords: Dental pulp Dental Amalgam Mercury Silver Tin
a b s t r a c t Project: To measure the levels of heavy metals (Hg, Sn) in the dental pulp and blood samples of patients with long-term amalgam restorations. Procedure: 12 amalgam restored and 12 non-restored, sound teeth were chosen and access cavity preparation to the pulp chamber was made. The contents were transferred and dissolved in 5 mL of concentrated nitric acid followed by placement in an oven at 180 ◦ C for tissue digestion. After cooling the tubes each digested sample was transferred to an atomic absorption system to measure the levels of heavy metals. The blood samples of five patients in each group were randomly analyzed to determine the levels of these heavy metals in the blood and if there were a correlation between these levels in blood and pulp. Data were analyzed by t-test at a P < 0.05 level of significance. Results: No significant difference was seen between the levels of Hg and Sn in pulp tissues (P > 0.05); however, the blood analysis showed higher level of Hg amalgam group (P = 0.009). The analysis between the pulp and blood samples showed positive correlations for both Hg and Sn elements in dental pulp and the blood (P = 1.000) (P = 0.900). Conclusions: The long-term presence of dental amalgam (at least 5 years) did not result in any remarkable changes in the levels of mercury and tin in the pulp tissue; however, there were increases in the level of mercury in the blood circulation even five years following the placement of the restoration. © 2014 Elsevier GmbH. All rights reserved.
Introduction The long-term use of dental silver amalgam has raised public concerns due to its potential side effects, such as mercury release [1]. Mercury, silver, tin, copper, and zinc are main components of this alloy that are mixed with other constituents such as indium, cadmium, lead and antimony [2]. Evaporation, dissolution, and evaporation/dissolution have been indicated as three mechanisms of mercury release from dental silver amalgam, of which the third mechanism is known to be the best model for evaluation of the
∗ Corresponding author at: Assistant Professor, Department of Dental Material, Dental School, Azad University (Tehran Branch), P.O. Box 14665-1445, Tehran, Iran. E-mail address: [email protected] (M.A. Saghiri). http://dx.doi.org/10.1016/j.jtemb.2014.01.008 0946-672X/© 2014 Elsevier GmbH. All rights reserved.
impact of Hg release [3–5]. Toxic elements such as mercury are defined as substances that can cause hazardous and adverse influence on body health [6]. The toxic effects of mercury on nervous and renal systems depend on acute contamination to elementary mercury [7]. Other investigators have shown that neurotoxicity of this element is highly relevant to the amount of mercury that is concentrated inside body cells [8]. Tin is another component of dental silver amalgam [2] that may be released from amalgams or that can influence the release of Hg [9,10]. The dissolution of tin inside the dental amalgam, especially in low pH environments, can increase the mercury release from the tin-free ␥1 phase [9]. In the presence of the on-going dissolution of tin; mercury undergoes greater evaporation and ionization into the surrounding environment [10]. However, mercury release from dental amalgam can be decreased due to the production of a
M.A. Saghiri et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 200–204
tin oxide layer on the surface of ␥1 phase of Hg, unless this layer dissolves because of low pH values inside the oral cavity [9]. Further concerns are the clinical side-effects and these sideeffects include gingivitis, loss of teeth and alveolar bone, oral galvanism, alteration of taste, mucosal ulceration and bruxism [11,12]. The pulpal uptake of mercury has been identified to play an important role in inflammation of dental pulp, following the placement of amalgam restorations [13]. Studies have shown that mercury penetration was not decreased even with the use of cavity liners such as calcium hydroxide or glass ionomer cements, due to their solubility and water contents [14–16]. Furthermore, the dynamic nature of the oral cavity including biological, mechanical, chemical and thermal stresses makes the whole scenario more complex [17] In this regard, some authors have evaluated the amount of mercury release during chewing stimulations and brushing time [18], while others have studied this phenomenon under different pH values [9]. Set dental amalgam in the oral cavity is considered as a dynamic substance that is predisposed to many clinical stresses such as temperature alteration and corrosive attacks [19] that might increase mercury release to some extent over time [18]. The present study was performed to evaluate the levels of mercury and tin the pulpal tissue of amalgam restored teeth and in the blood of patient possessing amalgam restoration. The hypothesis tested was whether or not the amount of these elements increases inside the pulp tissue and the blood over a long period of time.
201
system purification (Millipore, Milford, MA, USA). 5 mL of concentrated nitric acid was added and samples underwent digestion at temperature of 180 ◦ C in the oven. After cooling the tubes, 3 mL H2 O2 was added and evaporated to about 0.5 mL. Each digested sample was transferred quantitatively into a 10 mL calibrated tube to which was added a hydrochloric acid solution 1 mol/L. Serial dilution 1 mL aqueous solution of Triton X-100 containing 40 g of NH4 NO3 per liter as the matrix modifier was used for the final dilution of all samples, including standards and experimental groups. Preparation of reagents Purified water in a high-grade Milli-Ro De-ionizer system was used to prepare all reagents. 1 mL aqueous solution of Triton X-100 (iso-octylphenoxypolyethoxy ethanol), containing 40 g of NH4 NO3 per liter as the matrix modifier, was used for the final dilution of all samples, including standards and experimental groups. All chemicals were analytical grade when obtainable and highpurity nitrogen was used as the purging gas. A solution containing 60 g of BSA (Sigma Chemical Co., St. Louis, MO), 140 in mol of NaCl, and 5 mrnol of KHCO3 per liter was used to prepare all working standards. Preparation standards
Materials and methods Twenty-four premolar and molar teeth of 24 healthy patients, without any clinical signs and symptoms were extracted for orthodontic purposes and following ultrasonic cleaning were analyzed with stereomicroscope to ensure they were free of defects, such as cracks. Twelve specimens had class I amalgam restorations that were between 5 and 20 years old. The exclusion and inclusion criteria were as following: Exclusion criteria: any subjects who had systemic problems such as physician-diagnosed psychological, behavioral, neurological, immunosuppressive, or renal disorders. General inclusion criteria: (1) all subjects were older than 18 years old and agreed to the parameters of this study by signing the consent form based on the Helsinki guidelines. (2) The number of amalgam restorations inside the oral cavity of each patient should not have been more than five restorations. (3) Subjects were non-smokers and had no history of dental implants, other NiTi prosthesis in any part of their body, any piercing, and did not wear steel watches. (4) Diet control was done to ensure that subjects did not consume any sea food for at least one week prior to blood sampling. Specific inclusion criteria: (1) the amalgam restorations must have be 1.5–2 mm from the pulp. (2) The amalgam restorations must weighed 1 ± 0.02 g. In order to check these criteria, samples were grooved vertically on the buccal and lingual surfaces with a diamond disk without entering the pulp chamber, and split longitudinally with a chisel. The filling materials of experimental samples were extracted from the cavity and weighed by a electronic digital analytical balance (Mettler AE-163, Mettler Toledo, CA, USA) with an accuracy of 0.0001 g and the dentin thickness was measured by a caliper to be 1.5–2 mm. The remaining extracted teeth were sound, did not have any restorations and served as the control group (Fig. 1). After splitting the teeth in bucco-lingual direction, the pulp tissues of all samples were taken out of the pulp chamber with minimal damage by using plastic instruments and transferred into a clean beaker containing deionized pure water. Ultrapure water of 18 M/cm specific resistivity was obtained from Milli-Q water
A standard solution of 1000 ppm Hg was obtained (Fluka Chemie, Buchs, Switzerland) and for the Sn a standard solution was obtained (PerkinElmer, Pure atomic spectrometry standard, Shelton, CT, USA). The measurement of the levels of Hg and Sn were performed by WFX-1B atomic absorption spectrophotometer (No.2 Optical Instrument Factory of Beijing, China) equipped with a graphite furnace (HGA-400, Perkin-Elmer, CT, USA). The silver hollow-cathode lamp (Photron, Dandenong, Victoria, Australia) with a wavelength of 328.1 urn and a high-density graphite carbon tube (Perkin-Elmer, CT, USA) without platform were used in the assay. The calibration graph is linear up to 1 g/mL of tin. The limit of detection of this method is 0.002 g/mL. Evaluation of blood samples After teeth removal, five samples from each group were chosen randomly and subjected to blood evaluation of these heavy elements. Blood was collected by a vein puncture needle 25 × 7 (Vacutainer) in a 5-mL tube with the 5% percentage by weight anticoagulant EDTA. Statistical analysis The level of Sn and Hg elements represented non-continuous data that were presented after being analyzed by nonparametric tests and descriptors. Data were tabulated; proportions and 95% confidence intervals (CIs) were calculated. Significance of univariate associations was assessed with chi-square tests. For pulp/pulp and pulp/blood analysis, variables and treatment outcomes, the statistical program of SPSS 9.0 (SPSS Inc., Chicago, IL, USA) was used. Results The box plot of the level of heavy metals determined in the experimental groups is shown in Fig. 2. The statistical results indicated that there were no significant differences in the levels of Hg and Sn inside the pulpal tissue of the amalgam and control groups (P > 0.05). Blood evaluation identified significant differences
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Fig. 1. The process of pulp tissue extraction: the periapical radiography (a maxillary third molar), tooth was extracted, the amalgam filling was taken off by buccal and lingual vertical grooving, and finally by splitting vertically the pulp tissue was extracted and transferred into the beaker containing deionized pure water.
between the level of Hg in blood samples of the amalgam and control groups (P = 0.009) (Fig. 2C and D). The comparison of pulp and blood samples in experimental and control groups indicated that there was a correlation between the levels of these elements. A positive correlation coefficient of 1.000 and 0.900 were determined between the levels of Hg and Sn in pulp tissues and the blood samples. Discussion Previous investigations have discussed upon on-going and continual process mercury release in dynamic environment of oral cavity [17]. Physicochemical stimulations inside the mouth can impose microstructural alteration in dental amalgam restorations [19] that can lead to prolonged mercury release into the pulp tissue and/or bloodstream through the gastrointestinal route [18]. Three samples of experimental group were subjected to delayed analysis and in this time period these sample solutions were stored in 6 mol/L HCl to prevent hydrolysis of each elements which forming a semi-colloidal suspension.
Present study determined the levels of heavy metals (Hg and Sn) in the pulp tissue and blood of patients who had dental amalgam restorations for at least 5 years. Patient clinical records did not provide any information about the type of restorative materials (low-copper or high-copper), the restoration technique and the presence or absence of lining materials such as calcium hydroxide or glass ionomer cements that had been placed as bases under the amalgam restorations. Therefore authors had selected samples that included 1.5–2 mm dentin thickness between the amalgam and pulp tissue. Under this inclusion criterion, no lining materials such as calcium hydroxide or glass ionomer were used prior to the placement of the amalgam. Previous studies suggested the use of lining material primarily for deep cavities [20,21]. With respect to this, other investigators have indicated that neither CaOH2 nor glass ionomer cement can prevent the mercury intake into the pulp tissue through the dental tubules [16]. Due to segregation of pulp tissue, one specimen in the control group was damaged during the process of extraction of the pulpal tissue from pulp chamber. The results have indicated that the levels of tested elements in the pulpal tissue of amalgam-restored and non-restored teeth were
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Fig. 2. The box plot of the levels of Hg and Sn in the pulp tissue (A and B) and in the blood (C and D) samples of experimental and control samples.
not significant. This is consistent with results of previous investigations, where dental silver amalgam restorations could predispose minimal risk to neural tissues [8]. Marek has discussed two aspects of concentration and time of mercury release and concluded that the dissolution rate of mercury declined over the time due to the stifling effect of the concentration of mercury. Also, he has noted that the time period along with other variables such as solution volume, oxidation and evaporation conditions are effective factors in release of mercury [3]. The release of mercury was also evaluated under controlled chewing and brushing actions inside artificial saliva [18]. The results showed that the release of this element depended on the age of amalgam in a way that in initial non-steady-state situations it was higher than steady-state conditions. Berdouses et al. have shown that the dynamic situation produced by chewing and brushing activities can make the release dose of mercury from dental silver amalgam to 0.03 g/day, which is far distant from the threshold-limiting value (TLV) of 82.29 g/day, that is considered as a dangerous threshold for occupational exposure [18]. Moreover, the acidic conditions can increase the dissolution of mercury from dental silver amalgam [9]. Marek has suggested that the dissolution of mercury can be decreased by formation of preservative tin oxide layer on the surface of dental silver amalgam. In low pH conditions this oxide layer could dissolve that may lead to additional dissolution of mercury [9]. According to this issue, the tin oxide layer can produce a barrier against the release of mercury that can diminish the dissolution of this element [9]. The blood analysis has demonstrated that the level of mercury was significantly increased, while the amount of tin did not show any significant increase. This result is consistent with a previous study showing that blood mercury levels were closely related to the number and surface area of amalgam restorations [19]. Other investigators studied the contribution of dental amalgam to mercury in blood and concluded that by removing the amalgam restorations
a significant decrease was noticed in the levels of this element in the blood of subjected samples [22]. Snapp et al. have reported that the screening of mercury level was carried out weekly for five to eighteen weeks (median = 7.6 weeks) [22]. The results of measuring the Sn level did not show similar results and was not significant between experimental and control groups. This outcome might be explained by the lesser amount of tin (13%) in the composition of dental silver amalgam in comparison with Hg which is approximately 50% [23]. The data obtained from this study suggests that although dental silver amalgams cannot predispose the pulpal tissue to any remarkable changes regarding the mercury level, the increase in the mercury level of blood circulation still remains as a potential concern that may have medical implications for the individual. However, other investigators have mentioned that many of these anti-amalgam studies include many logical and methodological errors that mistakenly can make the safety of amalgam restorations doubtful [24]. A further study indicated that this issue should be evaluated in an interdisciplinary manner, which can make the assessment by using information from different aspects of materials science, corrosion, mercury exposure, toxicology, neurology and immunology [25]. Conclusion According to outcomes of this investigation, the following conclusions can be drawn: • The prolonged time of exposure to dental silver amalgam restorations did not increase the levels of mercury and tin in the dental pulp tissue. • The blood levels of mercury, unlike the pulpal levels of this element, was increased because of the continual release of mercury.
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• The comparison between the levels of mercury and tin in pulp and blood samples demonstrated a positive correlation. Acknowledgments This publication was based on the thesis presented, in partial fulfillment to the requirements for Doctor of Dental Surgery (D.D.S) in Azad University. We are indebted to Dr. Amir Nazari for all their contribution to this research. References [1] Darvell BW. Development of strength in dental silver amalgam. Dental Materials 2012;28:e207–17. [2] Akbal A, Yılmaz H, Tutkun E, Kös¸ MD. Aggravated neuromuscular symptoms of mercury exposure from dental amalgam fillings. Journal of Trace Elements in Medicine and Biology 2014;28:32–4. [3] Marek M. The release of mercury from dental amalgam: the mechanism and in vitro testing. Journal of Dental Research 1990;69:1167–74. [4] Radics J, Schwander H, Gasser F. Die kristallinen komponenten der silberamalgam – untersuchungen mit der elektronischen rontgenmikrosonde. Zahnarztliche Welt 1970;79:1031. [5] Till T, Wagner G. Untersuchungen zur L5slichkeit der bestandteile von amalgamfullungen wahrend des kau – und trinkaktes II. Zahnarztliche Welt 1973;82(1004):194–205. [6] James RC, Roberts SM, Williams PL. General principles of toxicology. In: Williams PL, James RC, Roberts SM, editors. Principles of toxicology: environmental and industrial applications. 2nd ed. New York: John Wiley and Sons, Inc.; 2000. p. 3–4. ISBN: 0-471-29321-0. [7] Ambient air pollution by mercury (Hg). Position paper. Luxembourg: Office for Official Publications of the European Communities; 2001. [8] Sweeney M, Creanor SL, Smith RA, Foye RH. The release of mercury from dental amalgam and potential neurotoxicological effect. Journal of Dentistry 2002;30:243–50. [9] Marek M. Dissolution of mercury from dental amalgam at different pH values. Journal of Dental Research 1997;76:1308–15.
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