Mutation Research 762 (2014) 1–8

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Cytotoxicity and genotoxicity of orthodontic bands with or without silver soldered joints Tatiana Siqueira Gonc¸alves a,∗ , Luciane Macedo de Menezes a , Cristiano Trindade b , Miriana da Silva Machado b , Philip Thomas c , Michael Fenech c , João Antonio Pêgas Henriques b,d a

Department of Orthodontics, Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, RS, Brazil Instituto de Educac¸ão para Pesquisa, Desenvolvimento e Inovac¸ão Tecnológica – ROYAL Unidade GENOTOX – ROYAL/Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil c Commonwealth Scientific and Industrial Research Organisation, Animal, Food and Health Sciences, Adelaide, SA, Australia d Instituto de Biotecnologia – Universidade de Caxias do Sul; Laboratório de Reparac¸ão de DNA em Eucariotos, Departamento de Biofísica/Centro de Biotecnologia, UFRGS, Porto Alegre, RS, Brazil b

a r t i c l e

i n f o

Article history: Received 19 April 2013 Received in revised form 22 January 2014 Accepted 24 January 2014 Available online 2 February 2014 Keywords: Orthodontics Metal ion toxicity Cell viability Genotoxicity Mutagenicity

a b s t r a c t Stainless steel bands, with or without silver soldered joints, are routinely used in orthodontics. However, little is known about the toxic biological effects of these appliances. The aims of this study were to evaluate the cytotoxic, cytostatic, genotoxic and DNA damage-inducing effects of non-soldered bands (NSB) and silver soldered bands (SSB) on the HepG2 and HOK cell lines and to quantify the amount of ions released by the bands. The 24-h metallic eluates of NSBs and SSBs were quantified by atomic absorption spectrophotometry. An MTT reduction assay was performed to evaluate the cytotoxicity, alkaline and modified comet assays were employed to measure genotoxicity and oxidative DNA damage effects, and cytokinesis-block micronucleus cytome (CBMN-Cyt) assays were used to verify DNA damage, cytostasis and cytotoxicity. Ag, Cd, Cr, Cu and Zn were detected in SSB medium samples, and Fe and Ni were detected in both the SSB and NSB medium samples. The SSB group induced stronger cytotoxic effects than the NSB group in both evaluated cell lines. NSB and SSB induced genotoxicity as evaluated by comet assays; stronger effects were observed in the SSB group. Both groups induced similar increases in the number of oxidative DNA lesions, as detected by the FPG and Endo III enzymes. Nucleoplasmic bridges, biomarkers of DNA misrepair and/or telomere end fusions, were significantly elevated in the SSB group. The SSB eluates showed higher amounts of Ni and Fe than NSB, and all the quantified ions were detected in SSB eluates, including Cd. The SSB eluates were more cytotoxic and genotoxic than the NSB samples. Based on these results, we propose that other brands, materials and techniques should be further investigated for the future manufacture of orthodontic appliances. © 2014 Published by Elsevier B.V.

1. Introduction Several metallic materials are utilized in the daily practice of orthodontics. Stainless steel is present in wires, brackets and bands, and silver solder is usually the metal of choice to connect support

∗ Corresponding author at: Pontifícia Universidade Católica do Rio Grande do Sul, Faculdade de Odontologia – Secretaria de Pós-Graduac¸ão – Prédio 6, Av. Ipiranga 6681, Sala 209, Porto Alegre, 90619-900 RS, Brazil. Tel.: +55 51 99188735. E-mail addresses: [email protected], [email protected] (T.S. Gonc¸alves), [email protected] (L.M.d. Menezes), [email protected] (C. Trindade), [email protected] (M.d.S. Machado), [email protected] (P. Thomas), [email protected] (M. Fenech), [email protected] (J.A.P. Henriques). 1383-5718/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.mrgentox.2014.01.011

wires in orthodontic appliances. Silver solder has been used extensively for this purpose due to some important advantages such as low cost and ease of use. Recently, work focusing on the biocompatibility of orthodontic materials has increased, and several studies have examined the release and cytotoxicity of orthodontic materials such as acrylic resins, composites and metals [1–8]. Biocompatibility refers to the ability of a biomaterial to perform its desired function with respect to a medical therapy without eliciting any undesirable local or systemic effects in the recipient or beneficiary of that therapy; it also refers to optimizing the clinically relevant performance of a given therapeutic intervention and generating the most appropriate beneficial cellular or tissue response in a given situation [8]. Corrosion is one of the major concerns relating to the issues surrounding the biocompatibility of metals [9,10]. Metallic ions may be released [2], leading to hypersensitivity and

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giving rise to allergic reactions, with local and systemic effects. Several auxiliary orthodontic appliances, such as lingual arches and maxillary expanders are made of stainless steel and contain silver soldered joints; these appliances may inhabit the oral cavity of a patient for months or even years. However, very few studies have investigated the safety and biocompatibility of the silver solder and the bands that are used in the preparation of orthodontic appliances [7,11–15]. Silver solder contains silver, copper and zinc as its major components [11]. These ions have a tendency to be released into the buccal cavity [16] and may give rise to cytotoxic events [3]. Although there have been a few investigations regarding the cytotoxic profile of silver solder [7,13–15], genotoxicity and DNA damage are still incipient themes in orthodontics. Comet and micronucleus assays have already been used to investigate the effects of stainless steel orthodontic appliances and archwires in vitro and in vivo [17–27]. However, to our knowledge, there has been no study concerning the evaluation of the potential genotoxic effects of stainless steel bands and bands with silver soldered joints. For this reason, the aims of this study were to evaluate the cytotoxic, cytostatic, genotoxic and oxidative DNA damage-inducing effects of stainless steel bands with or without silver soldered joints on HepG2 (human hepatocellular carcinoma) and HOK (human oral keratinocyte) cell lines and to quantify the ions released into the culture medium used to treat the cells. 2. Materials and methods This study was approved by the ethics Committee of Pontifical Catholic University of Rio Grande do Sul (Porto Alegre, Brazil).

2.1. Evaluated materials Stainless steel metallic orthodontic bands (universal bands for upper molars; Morelli, Sorocaba/SP, Brazil) were evaluated. According to the manufacturer’s documentation, the bands are composed of 17–20% Cr, 8–10% Ni, and a maximum of 0.60% Mo and Fe. Two groups of bands were evaluated: silver soldered bands (SSB) and bands without any type of solder (non-soldered bands, NSB). The NSB group was composed of the bands evaluated as received from the manufacturer. For the silver solder group, a segment of stainless steel 1.0 mm wire (17–20% Cr, 8–10% Ni, and a maximum of 0.60% Mo and Fe) was soldered to the lingual side of each band using silver solder alloy (55–57% Ag, 21–23% Cu, 15–19% Zn and 4–6% Sn) and solder flux (Morelli, Sorocaba/SP, Brazil) heated by a butane micro-torch (GB 2001, Blazer, Farmingdale, NY, USA). The amount of solder alloy and flux and the polishing procedure were standardized because the soldering was executed by a single operator.

2.2. Chemicals RPMI 1640 tissue-culture media, fetal bovine serum (FBS), trypsin-EDTA, lglutamine and antibiotics were purchased from Gibco BRL (Grand Island, NY, USA). Oral Keratinocyte Medium (OKM), Oral Keratinocyte Growth Supplement (OKGS) and penicillin/streptomycin solution (P/S) were purchased from ScienCell Research Laboratories (CA, USA). 3-(4,5-Dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT), 5,5; hydrogen peroxide (H2 O2 ), dimethyl sulfoxide (DMSO), sodium dodecyl sulfate (SDS), cytochalasin B, Schiff’s reagent and poly-l-lysine were purchased from Sigma (St. Louis, MO, USA). TrypLE Express, lowmelting point agarose and agarose were obtained from Invitrogen (Carlsbad, CA, USA). Formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (EndoIII) were obtained from New England BioLabs (Beverly, MA, USA). Hydrochloric acid (HCl; 5 M) and DePex mounting medium were obtained from Merck (Darmstadt, Germany). All reagents were of analytical grade.

2.3. Preparation of metallic extracts The experiments were preceded by the preparation of liquid extracts of each material to be investigated. For this, 12 bands of each group were first sterilized in an autoclave, as is usually done for clinical practice [28–30]. The bands were immersed in Falcon tubes containing 5 ml of RPMI 640 culture medium with 10% of serum for 24 h at 37 ◦ C under agitation and were removed from the tube after this time. An aliquot of the culture medium was used to quantify the concentration of the ions eluted, and another aliquot was used to perform MTT and comet assays.

2.4. Assessment of ions eluted To assess the concentrations of the ions eluted from the bands in RPMI 1640 media, atomic absorption spectrophotometry was employed. After the elution time (24 h), the bands were removed, and the media was analyzed for the presence of metallic eluates. Iron, nickel and chromium were quantified in both samples, while cadmium, copper, zinc and silver were quantified only in the SSB group. The culture medium alone was used as a blank. A flame atomic absorption spectrophotometer (SpectrAA 110, Varian, Palo Alto, CA, USA) was used to quantify copper, iron, silver and zinc. A graphite furnace atomic absorption spectrophotometer (ZEEnit 600, Analytik Jena, Jena, Thuringia, Germany) was used to quantify nickel, chromium and cadmium [9,11]. 2.5. Cell culture HepG2 is a human hepatocellular carcinoma cell line and was obtained from the ATCC (HB-8065). The choice of this cell line for this study was based on the fact that the HepG2 is a good experimental model system for the study of genotoxic agents, having a functionally active p53 protein, a competent DNA-repair system, active enzymes for phase-I and -II metabolism, and an active Nrf2 electrophile responsive system [31–33]. These properties tend to result in assays with a high predictivity for in vivo genotoxicity [34]. The cells were grown as monolayers under standard conditions in RPMI 1640 supplemented with 10% heat-inactivated FBS, 0.2 mg/ml l-glutamine, 100 IU/ml penicillin and 100 ␮g/ml streptomycin. The cells were maintained at 37 ◦ C in a humidified 5% CO2 atmosphere and were harvested by treatment with 0.15% trypsin-0.08% EDTA in PBS. The Human Oral Keratinocyte (HOK) cell line was chosen to perform the CBMNCyt assay because this cell line provides a suitable model for cells of the buccal mucosa and has already been used to evaluate the effects of various metals [35]. The cell line was obtained from ScienCell Research Laboratories (Catalog number: 2610). This cell line was isolated from human oral mucosa and provides a good model to study basic keratinocyte biology as well as the processes of immortalization and malignant transformation. The cells were cultured in poly-l-lysine coated 25 cm2 flasks, containing Oral Keratinocyte Medium, which is a complete medium for the optimal growth of normal human oral keratinocytes in vitro and is composed of basal medium plus a keratinocyte growth supplement and a penicillin/streptomycin solution. The cells were maintained in tissue culture flasks at 37 ◦ C in a humidified 5%-CO2 atmosphere and harvested by treatment with TrypLE Express. 2.6. MTT reduction assay MTT reduction was performed as described by Denizot and Lang [36] for the HepG2 cell line. Two different treatment times were analyzed: 24 h and 3 h. Briefly, 1 × 104 cells per well were seeded in 96-well plates; after 24 h the cells were exposed for either 3 or 24 hrs to the NSB, SSB or negative control culture medium. The treatments were then washed out, and 150 ␮l of a 1 mg/ml MTT salt solution was added to each well. This assay is based on the ability of the mitochondrial enzyme succinate dehydrogenase to convert the yellow water-soluble tetrazolium salt (MTT) into formazan crystals in metabolically active cells [5]. After incubation for 3 h, the supernatant was removed, the obtained purple formazan product was re-suspended in 100 ␮L of Dimethyl Sulfoxide (DMSO), and the absorbance was read at 540 nm in a microplate reader (Enspire Multimode Plate Reader, Perkin Elmer, USA). For the HOK cell line, 1 × 104 cells were seeded in 96-well plates coated with poly-l-lysine. After 24 h, the cells were exposed to the NSB, SSB or negative control culture medium for 24 h. After the treatment was washed out, fresh media with 10 ␮l of 5 mg/mL MTT salt solution was added to each well, and the plates were incubated for 4 h. Solubilizing solution (10% sodium dodecyl sulfate in 0.01 M HCl) was added to the plate and further incubated overnight at 37 ◦ C in a humidified 5%CO2 atmosphere, and the absorbance was read at 570 nm with an ELISA microplate reader (SpectraMax 250, Molecular Devices, CA, USA). 2.7. Comet assay 2.7.1. Alkaline comet assay The commonly used alkaline version of the comet assay detects DNA strand breaks and alkali labile lesions with high sensitivity [37]. The alkaline comet assay was performed as described by Singh et al. [38] with minor modifications [37,39]. Briefly 3 × 105 HepG2 cells were seeded in each well of 24 well-plates. After 24 h, the cells were exposed to the metallic eluates of NSB, SSB and negative control culture medium for 3 h or to a positive control (150 ␮M H2 O2 ) for 2 h. After treatment, the cells were trypsinized and re-suspended in complete medium. Fifteen microliters of this cell suspension was mixed with 90 ␮l of 0.75% low-melting point agarose (LMP) and immediately spread onto a glass microscope slide pre-coated with a layer of 1.5% normal agarose. The LMP layer was allowed to set at 4 ◦ C for 5 min, and the slides were incubated in ice-cold lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM EDTA, 1% Triton X-100 and 10% DMSO, pH 10.0) at 4 ◦ C for at least 1 h. This procedure removes the cell proteins and leaves the DNA as ‘nucleoids’. Following lysis, the slides were placed on a horizontal electrophoresis unit and covered with fresh buffer (300 mM NaOH, 1 mM EDTA, pH 13.0) for 20 min at 4 ◦ C to allow DNA unwinding and the expression of alkali labile sites. Electrophoresis was performed for 20 min at

T.S. Gonc¸alves et al. / Mutation Research 762 (2014) 1–8 25 V and 300 mA (0.90 V/cm). The slides were then neutralized (0.4 M Tris, pH 7.5), washed in double-distilled water and stained using a silver nitrate staining protocol as described by Nadin et al. [40]. The gels were dried at room temperature and analyzed using an optical microscope (Bioval L-2000). Four hundred cells (100 cells from each of two replicate slides per culture) were analyzed in each treatment group. The cells were scored visually according to tail length into five classes: class 0: undamaged, without a tail; class 1: with a tail shorter than the diameter of the head (nucleus); class 2: with a tail 1 − 2x the diameter of the head; class 3: with a tail longer than 2x the diameter of the head and class 4: significant damage, with a long tail, measuring more than 3x the diameter of the head. This visual scoring of comets is a well-validated evaluation method [41]. To perform the visual scoring, the slides were first randomly coded by one of the researchers. A second observer scored the slides blindly, not knowing which treatment was being scored. The damage index (DI) is based on the length of migration and on the amount of DNA in the tail and is considered a sensitive measurement of DNA damage. The DI ranges from 0 (100 completely undamaged cells × 0) to 400 (100 cells with maximum damage × 4) [37,39]. The damage frequency (DF – %) was calculated based on the number of cells with tails versus those without tails.

2.7.2. Modified comet assay To determine whether the bands induced oxidative DNA lesions, a modified comet assay was performed with two lesion-specific repair enzymes: FPG for the detection of oxidized purines and Endo III for the detection of oxidized pyrimidines. These enzymes recognize and introduce breaks at sites of oxidative damage and can therefore detect oxidative lesions in DNA [42]. For the modified comet assay, the test was performed in the same manner as described above, but an additional step was added: after lysis, the slides were washed three times in enzyme buffer (40 mM N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid, 100 mM KCl, 0.5 mM EDTA, 0.2 mg/ml bovine serum albumin, pH 8.0), drained, and incubated at 37 ◦ C in enzyme buffer supplemented with 60 ␮l of FPG (1 ␮g/ml solution) for 30 min and EndoIII (1 ␮g/ml solution) for 45 min; electrophoresis and staining was then performed as described above. Finally, the same procedures of coding (by one author) and blindly scoring the slides (by another author) were carried out.

2.8. Cytokinesis-block micronucleus cytome assay The cytokinesis-block micronucleus cytome assay (CBMN-Cyt) was performed as described by Fenech [43]. The CBMN-Cyt assay is a robust and comprehensive system for measuring DNA damage, cytostasis and cytotoxicity. DNA damage events are scored specifically in once-divided binucleated (BN) cells and include the following: (a) micronuclei (MNi), which are biomarkers of whole chromosome loss and/or breakage and originate from chromosome fragments or whole chromosomes that lag behind at anaphase during nuclear division, (b) nucleoplasmic bridges (NPBs), biomarkers of DNA misrepair and/or telomere end-fusions and originate from dicentric chromosomes and (c) nuclear buds (NBuds), which are biomarkers of gene amplification or the removal of DNA damage repair complexes [43]. To verify the potential mutagenic effects of the tested materials, a human mucosa cell line (HOK) was chosen; 5 × 104 cells per well were seeded in 24-well plates and exposed to the metallic eluates for 24 h or to 30 ␮M H2 O2 for 30 min as a positive control. After treatment, cytochalasin B (4.5 ␮g/ml) was added, and the cells were incubated for another 48 h. The cells were harvested onto microscope slides using a cytocentrifuge according to the manufacturer’s instructions (Shandon Products, UK). The slides were air dried for 10 min, then fixed in an ethanol:glacial acetic acid (3:1) solution and stained following the Feulgen staining technique with light green [44]. In this staining technique, the DNA material appears bright red in color when viewed under a fluorescence microscope with a far red filter. This is important to minimize the incidence of false positives or false negatives; the stain is DNAspecific giving a more accurate assessment of DNA damage and nuclear anomaly events [44]. After the staining process, a person not involved in the research coded the slides. One of the researchers then blindly scored the slides, without knowing which group was being evaluated. Five hundred cells per slide were scored and classified to determine the ratios of mononucleate, binucleate, multinucleate, apoptotic and necrotic cells, to determine the nuclear division index (NDI). The NDI is a biomarker of cytostasis and provides a measure of the proliferative status of the viable cell fraction. It is calculated using the formula NDI = (M1 + 2M2 + 3M3 + 4M4)/N, where M1–M4 represent the number of cells with 1–4 nuclei, and N is the total number of viable cells scored (excluding necrotic and apoptotic cells). Cytotoxicity events are assessed by the frequency of necrotic and apoptotic cells.

2.9. Statistical analysis For the HepG2 cell line, all experiments were independently repeated at least three times, and six independent experiments were performed for the HOK cell line. The data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. In relation to the assessment of ions eluted, the data are shown in terms of the mean and standard deviation. Graph Pad Prism 5.0 software was used for the statistical analysis (GraphPad Inc., San Diego, CA).

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3. Results 3.1. Detection of ions released in the culture medium The quantification of the ions eluted from the orthodontic bands is shown in Table 1. For both the NSB and SSB groups, chromium, iron and nickel were quantified because these ions are components of stainless steel, which was present in both groups. Chromium was not released in the NSB groups but was eluted in the SSB samples. A higher concentration of iron and nickel was evident in the eluates from the SSB group. Zinc, copper and silver, which are components of the silver alloy, were only quantified in the SSB samples because the levels of these metals would certainly be undetectable in the NSB group. Higher amounts of zinc and copper were detected compared to silver, even though silver was the major component of the silver alloy as stated by the manufacturer. Although cadmium is not listed as a component of the silver alloy, it was also quantified in the SSB group; chromium was detected in all the analyzed SSB aliquots. 3.2. Cytotoxicity For the HepG2 cell line, the MTT test was performed following a 24 h exposure of the cells to the medium containing metallic eluates (Fig. 1A). Both the NSB and SSB eluates significantly decreased the viability of HepG2 cells compared to the negative control eluate (p < 0.05), and there was also a significant difference between the NSB and SSB groups, with lower cell viability in the SSB group. The cell viability of the NSB group was 66.40% and for the SSB group 28.34%. The low cell viability of the SSB group did not allow the comet assay to be performed after a 24 h exposure. For this reason, the MTT test was performed again following a shorter exposure time of 3 h of the cells to the medium containing the metallic eluates (Fig. 1B). In this experiment, there was no significant difference between the negative control and the NSB group, which showed a cell viability measure of 88.58%. The cultures treated with the SSB eluates showed a significantly reduced cell viability of 48.44% when compared to those treated with the negative control eluate (Fig. 1B). Similar results were observed when the MTT test was performed in the HOK cell line, as demonstrated in Fig. 1C. The NSB group did not induce cytotoxic effects and induced a slight increase in cell viability compared to the control. The SSB group induced a strong cytotoxic effect, resulting in a cell viability of 11.56%. 3.3. Genotoxicity and oxidative DNA damage in HEPG2 cell line Upon completion of the MTT test, a comet assay was performed in the HepG2 cell line following a 3-h exposure of cells to the metallic eluates. Both the SSB and NSB groups induced an increase in break formation quantified by the damage index (Fig. 2A and B) and damage frequency (Fig. 3A), and there was significant difference in both the DI and DF between the NSB group and the SSB group. Both the SSB and NSB groups induced a significant increase in the DI compared to the negative control. The SSB group showed a mean DI of 178.9, around twofold higher than the NSB group (82.25), indicating higher genotoxic effects of the silver soldered group; this difference in DI was statistically significant. A higher frequency of damage was observed for the SSB group when compared to the NSB group (Fig. 3A). When the damage was subdivided into classes, the damage induced in the NSB group is mainly concentrated in Class 1 (Fig. 3B), while the damage induced in the SSB group is distributed among comet classification classes 1–3 (Fig. 3C). The induction of oxidative damage was evaluated through the modified version of the comet assay where slides were treated with the FPG and Endo III repair enzymes which introduce breaks at DNA sites with oxidative lesions (Fig. 2A and B). Both NSB and SSB groups were able to

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Table 1 Ionic quantification for SSB and NSB groups. SSB

NSB

Ion

n

Mean (mg L−1 )

SD (mg L−1 )

Ion

n

Mean (mg L−1 )

SD (mg L−1 )

Chromium Iron Nickel Cadmium Copper Silver Zinc

18 18 18 18 18 18 18

0.059 0.619 0.629 0.004 20.350 1.381 5.276

0.031 0.387 0.384 0.002 14.930 0.683 1.412

Chromium Iron Nickel

16 16 16

0 0.100 0.050

0 0.140 0.017

Fig. 1. Cell viability evaluated using MTTs test after (A) a 24 h exposure time for HepG2 cell line; (B) a 3 h exposure time for the HepG2 cell line; (C) a 24 h exposure time for HOK cell line. Groups not sharing the same letter are significantly different from each other (p ≤ 0.001). The results are shown as the mean ± standard deviation.

Fig. 2. The damage index (white) and oxidative damage index (gray) in the modified comet assay in HepG2 cells after treatment with (A) FPG enzyme and (B) Endo III enzyme. Damage Index (lowercase): the groups not sharing the same letter are significantly different from each other (p < 0.05). Oxidative Damage (uppercase): The groups not sharing the same letter are significantly different from each other (p < 0.05). The results are shown as the mean ± standard error.

Fig. 3. Comet assay in the HepG2 cell line showing (A) damage frequency, (B) the classes of damage in the NSB group and (C) the classes of Damage in SSB group. Groups not sharing the same letter are significantly different from each other (p < 0.05). The results are shown as the mean ± standard error.

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significantly increase the strand break frequency when treated with these enzymes, but there was no significant difference between the two tested groups for oxidative damage.

3.4. CBMN-Cyt assay in the HOK cell line The nuclear division index (Fig. 4C), which ranges from 1.0 (cells have failed to divide) to 2.0 (all cells have divided once), was similar for all of the tested groups with no significant differences between the control (1.324), NSB (1.248) and SSB (1.259) groups. No significant differences in the number of apoptotic and necrotic cells were observed for the tested groups in relation to the negative control (Fig. 4A and B). There were no significant differences in the frequency of either micronuclei or nuclear buds between the evaluated groups (Fig. 4D and E). The occurrence of nucleoplasmic bridges was significantly higher in the SSB group compared to the NSB and control groups (p < 0.05) (Fig. 4F).

4. Discussion Although orthodontic bands with silver soldered joints are present in several auxiliary appliances, only a few studies have investigated stainless steel bands and the cytotoxic potential of silver solder. Prior to this study, no investigations have been performed concerning the possible genotoxic or mutagenic activities of the silver solder combined with stainless steel orthodontic bands [7,12–15]. In the present study, orthodontic bands that were silver soldered to orthodontic wires were tested; these appliances contained solder flux that has endured the heat that are necessary to melt the silver based alloy. The goal of this approach was to reproduce what actually occurs when auxiliary appliances are produced instead of testing the cytotoxic and genotoxic effects of silver solder alloy alone [7,12]. The release of toxic ions into the oral cavity may lead to hypersensitivity and subsequent allergic reactions resulting in local and systemic effects [9,10,45]. The concentration of ions released in the NSB group (bands as received) is in accordance with the well-described biocompatibility of stainless steel materials [3,12]. Although chromium was not detected in the NSB samples and the levels of nickel and iron were lower when compared to the SSB group, these concentrations were able to induce cytotoxicity and genotoxicity in the NSB samples, albeit to a lower degree than the SSB group. However, in the SSB group (bands with a silver soldered joint) there was a higher concentration of nickel and chromium ions detected in the culture medium, most likely resulting from the process of joining the stainless steel wire to the bands. It has also been previously shown that the heat required during the soldering procedure certainly increases the subsequent rate of corrosion [46], in the same way that the copper present in the silver alloy [16] leads to a higher release of these toxic ions. Ni has a higher propensity to be released from the alloy [47] and, together with Cu, may increase the cytotoxic profile as a function of metal concentration [46]. Cu and Zn are highly cytotoxic; Cu is sometimes used as a positive control when alloys are tested for toxicity in cell culture studies [3,7,14,46,48–51]. Ag is also associated with increased cytotoxicity [51] and, together with Cu and Zn, has been shown to be capable of disrupting cellular metabolism [46,50]. Cu and Zn ions are more unstable than silver ions, which may explain the higher rates of release of these ions when compared to Ag, which is the major component of the alloy [14]. The results of this study show that Cu was the ion detected in the highest amount in the SSB samples (Table 1). Excess Cu is toxic to organisms at high concentrations, is involved in the formation of OH− radicals from H2 O2 via the Haber-Weiss and Fenton reactions, and can initiate nonspecific lipid peroxidation [52,53].

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Apart from the materials listed by the manufacturers, it was also observed that cadmium was present within the medium containing the SSB samples. It is feasible to suggest that this potential cadmium contamination may have arisen during the extraction of zinc ores such as zinc sulfide, with which Cd is usually recovered as a byproduct [54]. Some decades ago, cadmium was commonly added to silver solder to lower the fusion temperature of the alloy [55]. Professionals should be aware that cadmium is associated with cancer [56]; is responsible for damage to the liver, kidneys and heart [57]; and has already been associated with dental health issues such as periodontitis [58–60]. The toxic effects of all of the ions detected in the SSB group may be related to the cytotoxicity observed in the cultures exposed to silver soldered band eluates as verified by the MTT test (Fig. 1A–C). Although the comet assay has been used by some authors to evaluate other orthodontic materials [21,23,25,27], the present study is the first to use this assay to investigate the DNA damage effects of orthodontic bands made of stainless steel and of bands with silver soldered joints. Both NSB and SSB eluates increased the damage index and the damage frequency as measured by comet assays. However, higher levels of damage were observed for the SSB group, particularly damage in the comet classification classes 1–3 (Fig. 3C), which may be related to the increased cadmium concentrations observed in these eluates. Cadmium is responsible for several types of genotoxic damage, generally resulting from indirect mechanisms such as the generation of reactive oxygen species, the inhibition of DNA repair enzymes and the deregulation of cell proliferation [56,61–63]. Nickel, which was present in both the NSB and SSB samples, also interferes with DNA repair pathways and has also been shown to induce the formation of reactive oxygen species [62]. The oxidation state of chromium has been linked to genotoxicity both in vivo and in vitro [62]. In the present study, the total concentration of each ion was quantified, but it is important to note that the valence state is also important when considering the potential toxic effects of metals. Structural genetic lesions produced by the intracellular reduction of Cr(VI) include DNA adducts, DNA strand breaks, DNA-protein crosslinks, oxidized bases, abasic sites, and DNA inter- and intrastrand crosslinks. The damage induced by Cr(VI) can lead to dysfunctional DNA replication and transcription, aberrant cell cycle checkpoints, dysregulated DNA repair mechanisms, microsatellite instability, inflammatory responses, the disruption of key regulatory gene networks responsible for the balance of cell survival and cell death, which may play an important role in Cr(VI) carcinogenesis [64]. In relation to the comet assay, the standard alkaline method gives only limited information regarding the type of DNA damage being measured because it is not possible to determine whether the breaks detected are produced as a consequence of the direct effects of the damaging agent or by indirect effects, such as oxidative damage, resulting in confounding variables such as necrosis. The sensitivity and specificity of the assay can be improved by incubating the lysed cells with lesion-specific endonucleases, which recognize particular damaged bases and create additional breaks. In this study, we used the FPG and Endo III enzymes to verify if the breaks observed in the comet assay were due to oxidative lesions. FPG is specific for oxidized purines, including 8-oxo-7,8-dihydroguanine, 2,6-diamino-4-hydroxy-5formamidopyrimidine, 4,6-diamino-5-formamidopyrimidine and other open ring purines, while Endo III recognizes oxidized pyrimidines, including thymine glycol and uracil glycol [65]. As shown in Fig. 2A and B, both samples were able to increase the number of DNA breaks after both enzyme treatments, and there was no significant difference in oxidative damage when the NSB and SSB eluates were compared, suggesting that NSB and SSB induce oxidative lesions in a similar manner. Taken together, the higher damage index and damage frequency observed in the SSB group in the comet assay

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Fig. 4. CBMN-Cyt assay performed in the HOK cell line showing frequency of (A) apoptotic cells and (B) necrotic cells and the (C) nuclear division index and (D) number of micronuclei per 1000 binucleated cells. (E) The number of nuclear buds per 1000 binucleated cells. (F) The number of nucleoplasmic per 1000 binucleated cells. Groups not sharing the same letter are significantly different from each other. The results are shown as the mean ± standard error.

without enzyme treatments is most likely related to a combination of oxidative DNA damage and other direct effects of the metals detected in the culture medium. Concerning the oxidative damage, one possible explanation may be related to the interference by chromium. Like Cu and Fe, Cr is also a redox metal, and its redox behavior exceeds that of other metals, such as Fe, Zn and Ni. Cr redox behavior can thus be attributed to the direct involvement of chromium in inducing oxidative stress [53]. To our knowledge, this is the first study that has employed a human oral keratinocyte cell line to investigate the induction of genome instability by silver solder using the CBMN-cyt assay. This assay is known as a “cytome” assay because the cells are scored cytologically for their viability and their mitotic and genomic instability [43]. It was observed that the exposure of the HOK cells to the orthodontic appliance eluates for 24 h did not induce an increase in the frequency of micronuclei or nuclear buds (Fig. 4D and E). However, the SSB group did induce a significant increase in nucleoplasmic bridge formation (Fig. 4F). Nucleoplasmic bridges are important and sensitive biomarkers because they provide direct evidence of genome damage resulting from mis-repaired DNA breaks or telomere to telomere end fusions. This result may be related to the presence of elevated concentrations of resulting toxic ions, leading to a reduced DNA repair capacity and dysfunctional DNA replication and to an increase in genomic instability [64,66]. In the present study, high cytotoxicity and DNA damage induction after exposure to silver soldered bands was observed. Among other reasons, this may be due to a poor manufacturing practice of the silver solder alloy by the supplier because we tested only one brand of the material. Other brands of orthodontic bands and silver solder alloys should also be tested to confirm the observed cytotoxic and genotoxic effects. Future cytogenetic studies should be undertaken to more thoroughly evaluate these materials. Alternative methods to join stainless steel and different brands and

materials should also be investigated to determine their suitability for the binding of metallic orthodontic materials. For example, laser soldering promotes a truly metallic fusion due to the high energy of the laser, resulting in high levels of quality and reproducibility. This method is certainly more biocompatible because it has been shown to be less susceptible to patterns of corrosion [13–15]. Laser soldering is widely used for implant-based prosthesis and may play an important role in the future manufacture of orthodontic appliances. 5. Conclusions Both SSB and NSB were shown to release elevated levels of toxic metals under tissue culture conditions. NSB eluates were shown to be less cytotoxic and genotoxic than SSB eluates, which induced higher levels of cytotoxicity and genotoxicity in both the HepG2 and HOK cell lines. These observed effects were more pronounced in relation to DNA damage events resulting in an increased frequency of DNA strand breaks and nucleoplasmic bridge formation. In short, orthodontic stainless steel bands with silver soldered joints were shown to be biologically toxic. Other brands of the same type of material should also be investigated, and we recommend that alternative materials and methods should be explored for joining metals in the future manufacture of orthodontic appliances. Conflict of interest statement None declared. Acknowledgements This study is based on a thesis submitted to the Dentistry Faculty, Pontifical Catholic University of Rio Grande do Sul, Brazil, in partial

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fulfillment of the requirements for a PhD degree in Orthodontics. T.S.G. was supported by Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), Brazil, PDEE 2459/11-6. This work was supported in part by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), PRONEX/FAPERGS/CNPq (n◦ 10/0044-3). References [1] C.T. Kao, S.J. Ding, Y. Min, T.C. Hsu, M.Y. Chou, T.H. Huang, The cytotoxicity of orthodontic metal bracket immersion media, Eur. J. Orthod. 29 (2007) 198–203. [2] L.M. Menezes, C.C.A. Quintão, The release of ions from metallic orthodontic appliances, Semin. Orthod. 16 (2010) 282–292. [3] O. Mockers, D. Deroze, J. Camps, Cytotoxicity of orthodontic bands, brackets and archwires in vitro, Dent. Mater. 18 (2002) 311–317. [4] T.S. Goncalves, L.M. de Menezes, L.E. Silva, Residual monomer of autopolymerized acrylic resin according to different manipulation and polishing methods. An in situ evaluation, Angle Orthod. 78 (2008) 722–727. [5] T. 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