doi:10.1111/iej.12502
Induction of cytotoxicity, oxidative stress and genotoxicity by root filling pastes used in primary teeth
2, A. K. Machado3, V. F. Azzolin3, I. B. M. da Cruz4, C. W. Pires1, G. Botton1, F. C. Cadona 5 6 M. R. Sagrillo & J. R. Praetzel 1
Postgraduate Program in Dental Sciences, Federal University of Santa Maria (UFSM), Santa Maria; 2Postgraduate Program in Biological Sciences, Federal University of Santa Maria (UFSM), Santa Maria; 3Postgraduate Program in Pharmacology, Federal University of Santa Maria (UFSM), Santa Maria; 4Department of Morphology, Federal University of Santa Maria (UFSM), Santa Maria; 5Biomedicine Course, Franciscan University Center (UNIFRA), Santa Maria; and 6Department of Stomatology, Federal University of Santa Maria (UFSM), Santa Maria, Brazil
Abstract FC, Machado AK, Pires CW, Botton G, Cadona Azzolin VF, da Cruz IBM, Sagrillo MR, Praetzel JR. Induction of cytotoxicity, oxidative stress and genotoxicity by root filling pastes used in primary teeth. International Endodontic Journal.
Aim To evaluate the cytotoxicity, oxidative stress and genotoxicity in vitro of four iodoform pastes and three calcium hydroxide pastes. Methodology Peripheral blood mononuclear cells (PBMCs) and pure calf thymus DNA (dsDNA) were exposed to extracts of the pastes. Cytotoxicity was assessed with the MTT assay. Generation of reactive oxygen species (ROS) was evaluated using a DCFHDA assay, and lipid peroxidation was evaluated using a TBARS assay. Genotoxicity was evaluated using the alkaline comet assay and Genomodifier capacity assay (GEMO). All tests were performed after 24 h and 72 h of cell exposure, except GEMO. After performing the Kolmogorov–Smirnov test, data were analysed by Kruskal–Wallis and Dunn’s post-tests, and ANOVA with Dunnett’s post-test, with a significance level established at P < 0.05. Results The MTT assay revealed that chlorhexidine, Maxitrol and neomycin sulphate + bacitracin pastes decreased cell viability after 24 h (P < 0.05). No
group was associated with a significant decreased cell viability or lipid peroxidation after 72 h. Calcium hydroxide pastes increased the cell viability levels at both experimental times (P < 0.05). Lipid peroxidation was observed with the exposure of cells to calcium hydroxide pastes after 24 h (P < 0.05). Exposure to chlorhexidine, Guedes-Pinto and calcium hydroxide pastes resulted in a significant increase in ROS after 24 h (P < 0.05), whereas iodoform pastes and Calen thickened with zinc oxide significantly increased the ROS after 72 h (P < 0.05). The comet assay revealed that exposure of the PBMCs to iodoform pastes did not damage DNA at either period of time (P > 0.05). However, chlorhexidine paste caused DNA damage in dsDNA (P < 0.05). Calcium hydroxide pastes caused DNA damage in both tests (P < 0.05). Conclusion The pastes varied in their ability to induce cytotoxicity, genotoxicity and oxidative stress. In general, Guedes-Pinto, Maxitrol and neomycin sulphate + bacitracin pastes exhibited better biocompatibility in vitro. Keywords: biocompatibility, calcium hydroxide, deciduous tooth, iodoform, pulpectomy, root canal filling materials. Received 18 January 2015; accepted 6 July 2015
^ Correspondence: Carine Weber Pires, Angelo Uglione 1519/102, Santa Maria, RS 97010-570, Brazil (e-mail: [email protected]).
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Introduction One of the steps of pulpectomy in primary teeth is root filling. The filling material must be effective in eliminating or reducing bacteria, as well as preventing canal reinfection, and must be resorbable and nontoxic to periapical tissues and the permanent tooth germ (Huang et al. 2009, Silva et al. 2010). Currently, there is a growing preference for using iodoform paste and calcium hydroxide paste (Dunston & Coll 2008, Bergoli et al. 2010), instead of zinc oxide eugenol paste (Barcelos et al. 2011), probably because of its irritant potential to periapical tissues and slow resorption rate (Silva et al. 2010). Calcium hydroxide pastes are antimicrobial, owing to their high pH and release of hydroxyl and calcium ions (Estrela et al. 1995). Several vehicles may be used with calcium hydroxide pastes. Both viscous and aqueous vehicles contribute to their fluidity, thus facilitating insertion into the root canal and improving antimicrobial performance (Fava & Saunders 1999, Blanscet et al. 2008). Iodoform pastes are available in several formulations and offer good biological performance, high antimicrobial potential and clinical success (Guedes-Pinto et al. 1981, PuppinRontani et al. 1994, Sarigol et al. 2010, Lacativa et al. 2012). Guedes-Pinto paste (Guedes-Pinto et al. 1981) is one such paste; however, its genotoxicity has not been assessed. Antoniazzi et al. (2015) proposed three new pharmacological mixtures as filling materials for primary teeth: iodoform and camphorated paramonochlorophenol (CPC), which is associated with (i) Nebacetin ointment, (ii) 2% chlorhexidine gluconate gel, and (iii) Maxitrol ointment. They tested the antimicrobial activity of these materials in vitro, compared with that of Guedes-Pinto paste, and obtained satisfactory results. Because a balance must be reached between antimicrobial ability and cytocompatibility (Huang et al. 2007), more studies should be performed so that these pastes may be used clinically. Few studies have evaluated the potential damage of biomaterials to the deoxyribonucleic acid (DNA) of cells (Bin et al. 2012). However, genotoxicity tests are essential to evaluate a substance’s potential toxicity in humans, so that risks can be prevented (Ribeiro 2008). The comet assay is typically used to evaluate genotoxicity because it is considered a good tool for detecting damage to the genetic material of cells, including DNA damage, gene mutation, chromosomal
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breakage, altered DNA repair capacity and cellular transformation (Ribeiro et al. 2006, Ribeiro 2008). A newly proposed test, Genomodifier capacity assay (GEMO), aims to assess the genotoxic ability to specific chemicals or extracts. This is an ultrasensitive and rapid protocol that consists of using a highly specific double-stranded DNA dye (PicoGreenâ) and pure calf thymus DNA (dsDNA). The ultrasensitive PicoGreenâ dye is a fluorescent reagent that allows the quantification of double-stranded DNA molecules in solution (Cadon a et al. 2014). The disruption of the stable cellular redox balance as a result of the increased generation of reactive oxygen species (ROS) is called oxidative stress. In turn, cellular macromolecules such as proteins, lipids and DNA may be damaged when the production of ROS is higher than the antioxidant capacity of the cells. It has been proven that some chemicals are able to disrupt the redox balance, causing an increase in the levels of ROS and subsequent cell death (Schweikl et al. 2006, Camargo et al. 2009). Therefore, the dichlorofluorescein diacetate (DCFH-DA) assay (Esposti 2002) can be used to measure the total rate of ROS inside the cells, whilst the evaluation of the oxidative degradation of lipids by ROS, called lipid peroxidation, can be measured by the thiobarbituric acid reactive substances (TBARS) assay (Ohkawa et al. 1979). The in vitro tests mentioned above are used to elucidate the mechanism of a biological reaction and investigate cell behaviour in specific situations. Even though the results of these assays cannot be immediately extrapolated to clinical conditions in humans, they are clinically relevant because they represent an important contribution to the correct evaluation of the potential health risk associated with endodontic materials. Therefore, this study aimed to investigate the cytotoxicity, oxidative stress and genotoxicity in vitro of four iodoform pastes and three calcium hydroxide pastes. The null hypothesis was that there is no difference between the biocompatibility parameters of iodoform pastes and calcium hydroxide pastes.
Material and methods Ethical approval This study was approved by the Ethics Committee for Research, Federal University of Santa Maria (no. 20457313.7.0000.5346).
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Pires et al. Toxicity of filling materials
Cell culture Peripheral blood mononuclear cells (PBMCs) were obtained from discarded whole blood samples already processed from healthy adults. Blood specimens were centrifuged at 1611 g for 30 min, and the cells were isolated for density gradient, using Ficoll-Paque (Sigma-Aldrich, St. Louis, MO, USA). The cells were then plated in 6-well plates containing a complete RPMI 1640 culture medium. The cells were cultured at a density of 2 9 105 cells mL 1. Pure dsDNA (calf thymus DNA) was used in the GEMO assay. Details concerning the methodology are described in ‘Genotoxicity testing’ section.
Root canal pastes and preparation of the extracts The experimental groups were divided into eight subgroups, including the control group (cells in culture medium and phosphate-buffered saline [PBS]): (a) iodoform pastes: chlorhexidine (Nova Derme, Santa Maria, RS, Brazil) (CHX), Maxitrol (Alcon, S~ ao Paulo, SP, Brazil) (MAX), neomycin sulphate + bacitracin (EMS, Hortol^ andia, SP, Brazil) (NEB)), Guedes-Pinto (composed by a substitute for Rifocort–Formula & ~o, S~ Acßa ao Paulo, SP, Brazil) (GP); (b) calcium hydroxide pastes: Calen (S.S. White, Rio de Janeiro, RJ,
Brazil) thickened with zinc oxide (Calen/ZO), calcium hydroxide thickened with zinc oxide (CH/ZO) and UltraCal XS (Ultradent, Indaiatuba, SP, Brazil) (UltraCal) (Table 1). The proportions of iodoform pastes were the same as those proposed for the Guedes-Pinto paste (Guedes-Pinto et al. 1981) with iodoform (0.3 g), CPC (0.1 mL) and Rifocort ointment (0.25 g), in that the only component that changed was the ointment. Three types of calcium hydroxide pastes were tested. The first was composed of calcium hydroxide and zinc oxide in a 3 : 1 ratio that was mixed with propylene glycol until the consistency of toothpaste was obtained. The other materials were Calen (S.S. White, Rio de Janeiro, RJ, Brazil) (1 g) thickened with zinc oxide (0.65 g) (Queiroz et al. 2011) and UltraCal XS paste (Ultradent, Indaiatuba, SP, Brazil). The extracts were obtained as follows. The pastes were prepared in glass plates, under aseptic conditions, in a laminar flow; 0.22 mL of each paste was inserted in the 6-well plates. Each well was then filled with 2.5 mL RPMI 1640 culture medium supplemented with 10% foetal bovine serum, 1% penicillin/ streptomycin and 1% amphotericin B. The plates were then incubated at 37 °C for 24 h (Bin et al. 2012). The pH values of extracts remained in the range of 7.0 to 7.4.
Table 1 Components of the root filling pastes tested Material
Presentation
^mica, Ibipora ~, PR, Brazil) Iodoform (Biodina Camphorated paramonochlorophenol ^mica, Ibipora ~, PR, Brazil) (Biodina
Powder Liquid
2% chlorhexidine gluconate (Nova Derme, Santa Maria RS, Brazil) ~o Paulo, SP, Brazil) Maxitrol (Alcon, Sa
Gel
Neomycin sulphate + bacitracin ^ndia, SP, Brazil) (EMS, Hortola Sodium rifamycin SV + 21 prednisolone acetate (substitute for Rifocort) ~o, Sa ~o Paulo, SP,Brazil) (Formula & Acßa UltraCal XS (Ultradent, Indaiatuba, SP, Brazil) Calen (S.S. White, Rio de Janeiro, RJ, Brazil)
Ointment
Polymyxin B sulphate, neomycin sulphate, dexamethasone, preserved with methylparaben and propylparaben Neomycin sulphate and bacitracin
Ointment
Sodium rifamycin 1.5 mg g 1, prednisolone acetate 5 mg g
Paste
Powder Powder
Calcium hydroxide, barium sulphate, aqueous matrix of methylcellulose Calcium hydroxide, zinc oxide, colophony, polyethylene glycol. 99 a 100.5% of zinc oxide Calcium hydroxide P. A.
Liquid
Propylene glycol
^mica, Ibipora ~, PR, Brazil) Zinc oxide (Biodina Calcium hydroxide ^mica, Ibipora ~, PR, Brazil) (Biodina Propylene glycol (Nova Derme, Santa Maria, RS, Brazil)
Ointment
Paste
© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd
Components 99 a 100.5% of iodoform Parachlorophenol (30%), camphor (70%), alcohol 96° and deionized water 2% chlorhexidine gluconate, based of natrosol gel
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After incubation, each concentrated extract (1 : 1) (1000 lL) was mixed with a suspension of the previously plated cells (1000 lL). Thus, the final concentration was 1 : 2. The plates with the treatments were incubated at 37 °C and were later evaluated at 24 and 72 h, according to the test being performed. To avoid colour interference with the culture medium in the tests using PBMCs, the treated cells were resuspended at PBS (pH 7.4) (2 mL).
Cytotoxicity testing Cell viability was assessed using the MTT assay (Mosmann 1983, Denizot & Lang 1986): 20 lL MTT reagent (Sigma-Aldrich), dissolved in 5 mg mL 1 PBS, was added to a 96-well plate containing the sample treatments and incubated at 37 °C for 1 h (Fukui et al. 2010). The plate was then centrifuged at 2222 g for 10 min and the supernatant was removed from the wells. Next, the cells were resuspended in 200 lL dimethyl sulfoxide (DMSO) and centrifuged again. Absorbance was read at 570 nm on a spectrophotometer UV/Vis (Shimadzu, Kyoto, Japan).
Oxidative stress testing The dichlorofluorescein diacetate test (DCFH-DA) (Esposti 2002) was performed to verify intracellular ROS production. DCFH-DA (10 lmol L 1) reagent was added to a 96-well plate with the treated cells, for 60 min at 37 °C. The fluorescence was measured at an excitation rate of 485 nm and an emission rate of 520 nm on the spectrofluorometer. Fluorescence was directly proportional to the rate of ROS generated.
Lipid peroxidation testing Lipid peroxidation was quantified by determining the thiobarbituric acid reactive substances (TBARS) (Ohkawa et al. 1979). TBARS can be a marker of oxidative stress. To this end, 900 lL of supernatant was mixed with a reaction medium containing thiobarbituric acid (TBA 0.8%) and then incubated at 95 °C in a water bath for 1 h. The absorbance rate was read at 532 nm on the spectrophotometer. Absorbance is directly proportional to the rate of lipid peroxidation. All of the above tests were performed in triplicate, and the mean values were considered for analysis.
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Genotoxicity testing Alkaline comet assay The alkaline comet assay was performed as described by Singh et al. (1988) and adapted by Garcıa et al. (2004). Treated PBMCs were suspended in 0.75% low-melting-point agarose and PBS at 37 °C and placed on microscopic slides with a layer of 1.5% agarose. The slides were immersed in lysis solution (89 mL of lysis solution added to 10 mL of DMSO and 1 ml of Triton X-100) for 1 h and followed by denaturation in alkaline electrophoresis buffer (300 mmol L 1 NaOH and 1 mmol L 1 EDTA – pH>13.0), and then electrophoresis at 25 V, 300 mA, for 30 min. Subsequently, neutralization (neutralizing buffer pH 7.5), fixing (solution composed of 15% trichloroacetic acid) and staining (silver nitrate and 5% sodium carbonate) of the slides were performed. All steps described above were conducted under reduced illumination to minimize the possibility of cellular DNA damage. Two experienced, blinded and trained evaluators analysed one hundred cells (50 cells from each slide) with an optical microscope (400x) and classified the cells according to tail length. The distance migrated by cellular DNA directly reflects the extent of DNA damage present. Then, the cells received scores from 0 (no damage) to 4 (maximal damage). The data were converted into a percentage of damage index (Montagner et al. 2010) for analysis. Genomodifier capacity assay (GEMO) GEMO was performed according to Cadon a et al. (2014). In black 96-well plate, pure calf thymus DNA (dsDNA) was diluted in a TE buffer (10 mmol L 1 Tris-HCl, 1 mmol L 1 EDTA, pH 7.5). To each well containing 10 lL of dsDNA (1 lg mL 1) was added 100 lL of each extract which was prepared as mentioned above, accordance with Bin et al. (2012). dsDNA+extract remained in contact for 30 min. Then, the dye PicoGreenâ (1 : 200 TE) was added into the wells. After 5 min at room temperature, the fluorescence was measured at an excitation rate of 480 nm and an emission rate of 520 nm on a spectrofluorometer. Extracts causing breaks in doublestranded DNA molecules are identified by reduced fluorescence when compared to the control group, which contained only dsDNA (Cadon a et al. 2014). The test was performed in triplicate, and the averaged values were considered for analysis.
© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd
Pires et al. Toxicity of filling materials
Statistical analysis The data were tabulated and analysed using three programs: MiniTab version 17 (Minitab Inc., State College, PA, USA), GMC Basic Software version 7.7 (School of Dentistry of Ribeir~ao Preto, University of S~ ao Paulo, Ribeir~ ao Preto, SP, Brazil) and SPSS version 12.0 (SPSS Inc., Chicago, IL, USA). First, data normality was assessed by the Kolmogorov–Smirnov test. The data generated by the MTT 24 h and 72 h, the DCFH-DA 72 h, and the TBARS 24 h and 72 h tests were analysed by the Kruskal–Wallis and Dunn’s post-test. Data from the DCFH-DA 24 h, GEMO and the alkaline comet assay tests were analysed by ANOVA followed by Dunnett’s post-test. The significance level was established at P < 0.05.
decrease in the total ROS rate in the cells (P < 0.05) (Fig. 2). No iodoform paste caused an increase in TBARS levels in either period. Calcium hydroxide
Results The results of the cytotoxicity, oxidative stress and genotoxicity tests are presented in Figs 1, 2, 3 and 4. CHX, MAX and NEB significantly decreased cell viability to 92.82%, 87.39% and 80.21%, respectively, after 24 h (P < 0.05). No iodoform paste decreased cell viability at 72 h. All calcium hydroxide pastes caused a significant increase in cell viability in both experimental periods (P < 0.05) (Fig. 1). CHX, GP and calcium hydroxide pastes were associated with an increase in ROS rates (P < 0.05) at 24 h. All iodoform pastes and Calen/ZO caused a significant increase in ROS production at 72 h. During this time, CH/ZO and UltraCal pastes had percentages of 83.33 and 95.11, respectively, indicating a significant
Figure 1 Cytotoxicity of root filling pastes in PBMCs after exposure to extracts. The cell cultures were exposed for 24 h and 72 h. Bars represent the means. Significant differences between untreated (Control) and treated cell cultures are indicated by asterisks.
© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd
Figure 2 Oxidative stress testing (DCFH-DA) and lipid peroxidation testing (TBARS). Intracellular generation of ROS and lipid peroxidation of root filling pastes in PBMCs, respectively, after exposure to extracts for 24 h and 72 h. Bars represent the means. Significant differences between untreated (Control) and treated cell cultures are indicated by asterisks.
Figure 3 Genotoxicity of root filling pastes in PBMCs after exposure to extracts. The cell cultures were exposed for 24 h and 72 h. Bars represent the means. Significant differences between untreated (Control) and treated cell cultures are indicated by asterisks.
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Figure 4 Genotoxicity by GEMO assay in dsDNA after exposure to extracts of root filling pastes. Bars represent the means. Significant differences between untreated (Control) and treated cell cultures are indicated by asterisks.
pastes were associated with a significant increase in lipid peroxidation at 24 h, but caused no changes in TBARS levels at 72 h (Fig. 2). Iodoform pastes did not cause DNA damage in PBMCs in the alkaline comet assay, but calcium hydroxide pastes led to a higher DNA damage rate at 24 h and 72 h (P < 0.05) (Fig. 3). In the GEMO assay, one of iodoform pastes, CHX and calcium hydroxide pastes revealed genotoxic ability (P < 0.05) (Fig. 4).
Discussion No experiments in themselves can determine the final response to the mechanisms underlying the biological response of biomaterials (Leirskar & Helgeland 1981, Hauman & Love 2003). Therefore, in this study, four biocompatibility-related in vitro tests were performed to provide data on the safe use of filling materials for primary teeth with compromised pulps. Despite their limitations, laboratory tests performed on a cell culture may be a suitable tool to elucidate the mechanism involved in biocompatibility (Leirskar & Helgeland 1981). Furthermore, they have advantages over other methods, in terms of ease, rapidity and lower cost. However, it is difficult to compare the results of different cell culture experiments, because of methodological differences, such as cell type, contact forms, experimental time periods (Sarigol et al. 2010), assay type, varying concentrations and associations of different substances. Thus, the results obtained for some of the materials tested are difficult to compare, due to the lack of previous studies. The cells used were peripheral blood mononuclear leucocytes (lymphocytes and monocytes). Leucocytes provide an estimation of the body’s exposure to genotoxic products (Leite et al. 2012) and are typically
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used to assess cytotoxicity, together with human epithelial cells (HeLa) and fibroblasts (Ciapetti et al. 1993, Hauman & Love 2003). Considering that antiseptics for root canal treatment, applied topically, may circulate in the bloodstream after penetrating the dentine or the periodontium through the apical foramen and apical ramifications (Bartelstone 1951, Tucker 1981), these cells represent the damage that products may cause to the body. The MTT assay is a useful method to assess the cytotoxicity of biomaterials (Ciapetti et al. 1993). However, this is only one aspect of biocompatibility and cannot singly characterize a material as being biocompatible or nonbiocompatible (Peters 2013). GP caused no effect on cell viability of PBMCs at 24 h, confirming its positive clinical performance (GuedesPinto et al. 1981, Puppin-Rontani et al. 1994). However, the new iodoform pastes caused cell death at 24 h. During an extended period of contact with the cells (72 h), all of the iodoform pastes failed to cause cell death, which is important, because the pastes tend to remain on the root canal for many months. The mixture of calcium hydroxide paste and zinc oxide powder was evaluated by two experimental groups with different formulations and with two viscous vehicles (Calen/ZO and CH/ZO). UltraCal XS is a commercial option whose vehicle, methylcellulose, is aqueous. The extracts of calcium hydroxide pastes caused a significant increase in cell viability in both experimental periods. There are no studies that assess the cytotoxicity of these same formulations in cell cultures; however, the cell proliferation related to these pastes may be explained by their capacity to induce an inflammatory response and favour the repair process (Nelson Filho et al. 1999). Previous studies evaluating the cytocompatibility in the subcutaneous tissue of rats demonstrated that Calen/ZO paste is more biocompatible than zinc oxide eugenol paste or Sealapex (Queiroz et al. 2011). UltraCal XS, Hydropast and Calen paste also provided tissue compatibility (Andolfatto et al. 2012). Thus, calcium hydroxidebased pastes may be considered suitable for clinical use in pulp therapy, mainly for teeth with periapical lesions. ROS are present in human physiological metabolism and have an important biological function. However, data on increased ROS production may provide insight into one possible mechanism of cellular toxicity (Camargo et al. 2009). In this study, CHX and GP produced an increase in ROS levels within 24 h. Within 72 h, iodoform pastes demonstrated significantly
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high levels of ROS; however, CHX exhibited the greatest increase. The behaviour of the mixture containing chlorhexidine can be explained by the study by Barbin et al. (2013), who identified cytotoxic agents parachloroaniline and ROS as subproducts of the aqueous solution of 2% chlorhexidine. Furthermore, chlorhexidine has substantivity and exerts a long-term action on tissues, thus increasing its risk potential even further (Barbin et al. 2008). As the iodoform-based pastes were not cytotoxic at 72 h and did not show lipid peroxidation, it could be suggested that, although they caused an increase in total ROS rate, the enzymes and the antioxidant defence system were able to repair the redox imbalance. All calcium hydroxide pastes caused higher ROS rates at 24 h. During a longer period of contact, only Calen/ZO caused a significant increase in the ROS rates. Whereas, in the TBARS test, these pastes showed similar results to DCFH-DA within 24 h, no calcium hydroxide paste evaluated caused oxidative damage to lipid cell structures within 72 h. These data contrasted with the MTT results. It may be explained by the fact that the redox imbalance was repaired and the damage to the lipid layer was not intense enough to cause cell death; rather, it merely affected the biologic mechanism of the cell. In the studies comparing iodoform paste and calcium hydroxide paste, the results show that iodoform paste is less cytotoxic in L929 fibroblasts of rodents (Sarigol et al. 2010), and offers acceptable levels of biocompatibility in less time, as observed in the histological evaluation of the intra-osseous implant technique in guinea pigs (Lacativa et al. 2012). However, Faraco-Junior & Percinoto (1998) found that both pastes had a good response in the periapical tissues after histological analysis of pulpotomized dog teeth and that calcium hydroxide achieved better results in relation to inflammation intensity and degree of resorption. However, Silva et al. (2010) performed the same type of histological analysis and found that Calen/ZO had the best tissue response. Thus, there is still no consensus on the best paste and further in vivo and clinical trials are required to evaluate this issue in greater depth. Some materials used in dental practice are potentially harmful to genetic material in vitro (Ribeiro et al. 2006, Silva et al. 2007) and in vivo (Ribeiro et al. 2004, Carlin et al. 2012). Thus, genotoxicity tests are essential to assess genetic damage and are also important indicators of carcinogenesis (Ribeiro 2008). Studies of the genotoxic and mutagenic effects
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of endodontic materials related to primary dentition have been published (Ribeiro 2008, Leite et al. 2012), but there is only one study reported thus far on filling pastes for deciduous teeth (Huang et al. 2009). In the present study, no iodoform paste induced DNA damage in the alkaline comet assay. However, in the GEMO assay, CHX showed genotoxicity. This is in agreement with the high levels of ROS produced by CHX in the present study. Moreover, chlorhexidine has a potential to cause oxidative damage to DNA due the releasing of para-chloroaniline, a possible carcinogen in humans (Barbin et al. 2008, 2013). All calcium hydroxide pastes were able to induce DNA damage in PBMCs, according to the alkaline comet assay. The GEMO assay confirmed the genotoxic action of these pastes, because they caused breaks in dsDNA. GEMO is a fast and inexpensive assay, and it may be a complementary test to a traditional genotoxic test such as the alkaline comet assay. The higher dsDNA fluorescence measured by the GEMO was associated with lower index damage measured by the alkaline comet assay (Cadon a et al. 2014). In contrast, it has been reported that calcium hydroxide pastes caused no DNA fragmentation in human osteosarcoma cells (Huang et al. 2009). There was a major difference in concentration of calcium hydroxide extracts, with 500 000 lg mL 1 in the present study, and 5 lg mL 1 in a previous study (Huang et al. 2009). The discrepancy between these results may be explained by differences in experimental procedures, such as concentration of extracts and cell culture methods. Likewise, genotoxicity results appear to contrast with MTT results in the present study. When PBMCs and dsDNA molecules were exposed to extracts, breaks occurred in the DNA structure due oxidative damage. This could result in chromosomal instability, genetic changes and problems related to cell division. ROS have the potential to stimulate cell proliferation by activating proto-oncogenesis (carcinogenesis), besides they can cause mitosis, apoptosis, necrosis and autophagy (Klaunig et al. 2010). In this way, it is suggested that the increase in cell viability caused can be related to cell proliferation. Moreover, in the process of cell division, when the repair system cannot repair an injury, this is perpetuated in the genetic material of this line which tends to suffer more oxidative damage (Tudek et al. 2010). Therefore, this correlation can be the explanation about the compatibility of the large increase in DNA damage with increase in cell viability (cell proliferation) caused by the calcium hydroxide pastes.
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Conclusion The pastes varied in their ability to induce cytotoxicity, oxidative stress and genotoxicity. Two considerations are especially worthy of note: • Although calcium hydroxide pastes increased cell viability, they produced ROS and damage to the lipid layer within 24 h. However, all induced DNA damage. • Although most iodoform pastes decreased cell viability within 24 h (except GP), this was not sustained after 72 h. These pastes did not cause lipid peroxidation; however, all of them produced ROS within 72 h. Just one of the iodoform pastes (CHX) was to dsDNA. Guedes-Pinto, Maxitrol and neomycin sulphate + bacitracin pastes were associated with better in vitro biocompatibility performance. Due to the inherent limitations of an in vitro assay and the specific experimental conditions of this study, further studies in animal models and clinical trials must be performed to determine the biocompatibility and clinical use of these filling pastes in primary teeth.
Acknowledgements The authors would like to thank Dr. Rachel de Oliveira Rocha for her statistical support and the EMS pharmaceutical industry for its samples of neomycin sulphate and bacitracin sent for the pilot project. All the authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
References Andolfatto C, da Silva GF, Cornelio AL et al. (2012) Biocompatibility of intracanal medications based on calcium hydroxide. International Scholarly Research Network Dentistry 2012, 1–6. Antoniazzi BF, Pires CW, Bresolin CR, Weiss RN, Praetzel JR (2015) Antimicrobial activity of different filling pastes for deciduous tooth treatment. Brazilian Oral Research 29, 1–6. Barbin LE, Saquy PC, Guedes DF, Sousa-Neto MD, Estrela C, Pecora JD (2008) Determination of para-chloroaniline and reactive oxygen species in chlorhexidine and chlorhexidine associated with calcium hydroxide. Journal of Endodontics 34, 1508–14. Barbin LE, Estrela C, Guedes DF, Span o JC, Sousa-Neto MD, Pecora JD (2013) Detection of para-chloroaniline, reactive
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oxygen species, and 1-chloro-4-nitrobenzene in high concentrations of chlorhexidine and in a mixture of chlorhexidine and calcium hydroxide. Journal of Endodontics 39, 664–8. Barcelos R, Santos MP, Primo LG, Luiz RR, Maia LC (2011) ZOE paste pulpectomies outcome in primary teeth: a systematic review. The Journal of Clinical Pediatric Dentistry 35, 241–8. Bartelstone HJ (1951) Radioiodine penetration through intact enamel with uptake by bloodstream and thyroid gland. Journal of Dental Research 30, 728–33. Bergoli AD, Primosch RE, De Araujo FB, Ardenghi TM, Casagrande L (2010) Pulp therapy in primary teeth– profile of teaching in Brazilian dental schools. The Journal of Clinical Pediatric Dentistry 35, 191–5. Bin CV, Valera MC, Camargo SEA et al. (2012) Cytotoxicity and genotoxicity of root canal sealers based on mineral trioxide aggregate. Journal of Endodontics 38, 495–500. Blanscet ML, Tordik PA, Goodell GG (2008) An agar diffusion comparison of the antimicrobial effect of calcium hydroxide at five different concentrations with three different vehicles. Journal of Endodontics 34, 1246–8. Cadona´ FC, Manica-Cattani MF, Machado AK et al. (2014) Genomodifier capacity assay: a non-cell test using dsDNA molecules to evaluate the genotoxic/genoprotective properties of chemical compounds. Analytical Methods 6, 8559– 68. Camargo SEA, Camargo CHR, Hiller KA, Rode SM, Schweikl H, Schmalz G (2009) Cytotoxicity and genotoxicity of pulp capping materials in two cell lines. International Endodontic Journal 42, 227–37. Carlin V, Matsumoto MA, Saraiva PP et al. (2012) Cytogenetic damage induced by mouthrinses formulations in vivo and in vitro. Clinical Oral Investigations 16, 813– 20. Ciapetti G, Cenni E, Pratelli L, Pizzoferrato A (1993) In vitro evaluation of cell/biomaterial interaction by MTT assay. Biomaterials 14, 359–64. Denizot F, Lang R (1986) Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. Journal of Immunological Methods 89, 271–7. Dunston B, Coll JA (2008) A survey of primary tooth pulp therapy as taught in US dental schools and practiced by diplomates of the American Board of Pediatric Dentistry. Pediatric Dentistry 30, 42–8. Esposti MD (2002) Measuring mitochondrial reactive oxygen species. Methods 26, 335–40. Estrela C, Sydney GB, Bammann LL, Felippe J unior O (1995) Mechanism of action of calcium and hydroxyl ions of calcium hydroxide on tissue and bacteria. Brazilian Dental Journal 6, 85–90. ~o de duas Faraco-Junior IM, Percinoto C (1998) Avaliacßa tecnicas de pulpectomias em dentes decıduos. Revista da Associacß~ao Paulista de Cirurgi~oes Dentistas 52, 400–4.
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Fava LR, Saunders WP (1999) Calcium hydroxide pastes: classification and clinical indications. International Endodontic Journal 32, 257–82. Fukui M, Yamabe N, Zhu BT (2010) Resveratrol attenuates the anticancer efficacy of paclitaxel in human breast cancer cells in vitro and in vivo. European Journal of Cancer 46, 1882–91. Garcıa O, Mandina T, Lamadrid AI et al. (2004) Sensitivity and variability of visual scoring in the comet assay. Results of an inter-laboratory scoring exercise with the use of silver staining. Mutation Research 22, 25–34. Guedes-Pinto AC, Paiva JG, Bozzola JR (1981) Tratamento endod^ ontico de dentes decıduos com polpa mortificada. Revista da Associacß~ao Paulista de Cirurgi~oes Dentistas 35, 240–5. Hauman CHJ, Love RM (2003) Biocompatibility of dental materials used in contemporary endodontic therapy: a review. Part 1. Intracanal drugs and substances. International Endodontic Journal 36, 75–85. Huang TH, Ding SJ, Kao CT (2007) Biocompatibility of various formula root filling materials for primary teeth. Journal of Biomedical Materials Research Part B: Applied Biomaterials 80, 486–90. Huang TH, Hung CJ, Chen YJ, Chien HC, Kao CT (2009) Cytologic effects of primary tooth endodontic filling materials. Journal of Dental Sciences 4, 18–24. Klaunig JE, Kamendulis LM, Hocevar BA (2010) Oxidative Stress and Oxidative Damage in Carcinogenesis. Toxicologic Pathology 38, 96–109. Lacativa AM, Loyola AM, Sousa CJ (2012) Histological evaluation of bone response to pediatric endodontic pastes: an experimental study in guinea pig. Brazilian Dental Journal 23, 635–44. Leirskar J, Helgeland K (1981) Mechanism of toxicity of dental materials. International Endodontic Journal 14, 42–7. Leite ACGL, Rosenblatt A, Calixto MS, Silva CM, Santos N (2012) Genotoxic effect of formocresol pulp therapy of deciduous teeth. Mutation Research 747, 93–7. Montagner GFS, Sagrillo M, Machado MM et al. (2010) Toxicological effects of ultraviolet radiation on lymphocyte cells with different manganese superoxide dismutase Ala16Val polymorphism genotypes. Toxicology In Vitro 24, 1410–6. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65, 55– 63. Nelson Filho P, Silva LA, Leonardo MR, Utrilla LS, Figueiredo F (1999) Connective tissue responses to calcium hydroxide-based root canal medicaments. International Endodontic Journal 32, 303–11.
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Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351–8. Peters OA (2013) Research that matters - biocompatibility and cytotoxicity screening. International Endodontic Journal 46, 195–7. Puppin-Rontani RMP, Peters CF, Worliczeck AT (1994) Pulpectomy in primary teeth: a clinical investigation. Revista da Associacß~ao Paulista de Cirurgi~oes Dentistas 48, 1235–8. Queiroz AM, Assed S, Consolaro A et al. (2011) Subcutaneous connective tissue response to primary root canal filling materials. Brazilian Dental Journal 22, 203–11. Ribeiro DA (2008) Do endodontic compounds induce genetic damage? A comprehensive review. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics 105, 251–6. Ribeiro DA, Bazo AP, da Silva Franchi CA, Marques MEA, Salvadori DMF (2004) Chlorhexidine induces DNA damage in rat peripheral leukocytes and oral mucosal cells. Journal of Periodontal Research 39, 358–61. Ribeiro DA, Marques MEA, Salvadori DMF (2006) Antimicrobial endodontic compounds do not modulate alkylation-induced genotoxicity and oxidative stress in vitro. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics 102, e32–6. Sarigol CG, Cogulu D, Oncag O, Deliloglu IG (2010) Cytotoxic effects of primary tooth root canal filling materials on L929 cell line. Journal of Dentistry for Children 77, 72–6. Schweikl H, Spagnuolo G, Schmalz G (2006) Genetic and cellular toxicology of dental resin monomers. Journal of Dental Research 85, 870–7. Silva GN, Camargo EA, Salvadori DMS, Ribeiro DA (2007) Genetic damage in human peripheral lymphocytes exposed to antimicrobial endodontic agents. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics 104, e58–61. Silva LAB, Leonardo MR, Oliveira DSB et al. (2010) Histopathological evaluation of root canal filling materials for primary teeth. Brazilian Dental Journal 21, 38–45. Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research 175, 184–91. Tucker MJ (1981) The systemic toxicity evaluation of dental materials. International Endodontic Journal 14, 49–52. Tudek B, Winczura A, Janik J, Siomek A, Foksinski M, Oli nski R (2010) Involvement of oxidatively damaged DNA and repair in cancer development and aging. American Journal of Translational Research 2, 254–84.
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Induction of cytotoxicity, oxidative stress and genotoxicity by root filling pastes used in primary teeth
2, A. K. Machado3, V. F. Azzolin3, I. B. M. da Cruz4, C. W. Pires1, G. Botton1, F. C. Cadona 5 6 M. R. Sagrillo & J. R. Praetzel 1
Postgraduate Program in Dental Sciences, Federal University of Santa Maria (UFSM), Santa Maria; 2Postgraduate Program in Biological Sciences, Federal University of Santa Maria (UFSM), Santa Maria; 3Postgraduate Program in Pharmacology, Federal University of Santa Maria (UFSM), Santa Maria; 4Department of Morphology, Federal University of Santa Maria (UFSM), Santa Maria; 5Biomedicine Course, Franciscan University Center (UNIFRA), Santa Maria; and 6Department of Stomatology, Federal University of Santa Maria (UFSM), Santa Maria, Brazil
Abstract FC, Machado AK, Pires CW, Botton G, Cadona Azzolin VF, da Cruz IBM, Sagrillo MR, Praetzel JR. Induction of cytotoxicity, oxidative stress and genotoxicity by root filling pastes used in primary teeth. International Endodontic Journal.
Aim To evaluate the cytotoxicity, oxidative stress and genotoxicity in vitro of four iodoform pastes and three calcium hydroxide pastes. Methodology Peripheral blood mononuclear cells (PBMCs) and pure calf thymus DNA (dsDNA) were exposed to extracts of the pastes. Cytotoxicity was assessed with the MTT assay. Generation of reactive oxygen species (ROS) was evaluated using a DCFHDA assay, and lipid peroxidation was evaluated using a TBARS assay. Genotoxicity was evaluated using the alkaline comet assay and Genomodifier capacity assay (GEMO). All tests were performed after 24 h and 72 h of cell exposure, except GEMO. After performing the Kolmogorov–Smirnov test, data were analysed by Kruskal–Wallis and Dunn’s post-tests, and ANOVA with Dunnett’s post-test, with a significance level established at P < 0.05. Results The MTT assay revealed that chlorhexidine, Maxitrol and neomycin sulphate + bacitracin pastes decreased cell viability after 24 h (P < 0.05). No
group was associated with a significant decreased cell viability or lipid peroxidation after 72 h. Calcium hydroxide pastes increased the cell viability levels at both experimental times (P < 0.05). Lipid peroxidation was observed with the exposure of cells to calcium hydroxide pastes after 24 h (P < 0.05). Exposure to chlorhexidine, Guedes-Pinto and calcium hydroxide pastes resulted in a significant increase in ROS after 24 h (P < 0.05), whereas iodoform pastes and Calen thickened with zinc oxide significantly increased the ROS after 72 h (P < 0.05). The comet assay revealed that exposure of the PBMCs to iodoform pastes did not damage DNA at either period of time (P > 0.05). However, chlorhexidine paste caused DNA damage in dsDNA (P < 0.05). Calcium hydroxide pastes caused DNA damage in both tests (P < 0.05). Conclusion The pastes varied in their ability to induce cytotoxicity, genotoxicity and oxidative stress. In general, Guedes-Pinto, Maxitrol and neomycin sulphate + bacitracin pastes exhibited better biocompatibility in vitro. Keywords: biocompatibility, calcium hydroxide, deciduous tooth, iodoform, pulpectomy, root canal filling materials. Received 18 January 2015; accepted 6 July 2015
^ Correspondence: Carine Weber Pires, Angelo Uglione 1519/102, Santa Maria, RS 97010-570, Brazil (e-mail: [email protected]).
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Toxicity of filling materials Pires et al.
Introduction One of the steps of pulpectomy in primary teeth is root filling. The filling material must be effective in eliminating or reducing bacteria, as well as preventing canal reinfection, and must be resorbable and nontoxic to periapical tissues and the permanent tooth germ (Huang et al. 2009, Silva et al. 2010). Currently, there is a growing preference for using iodoform paste and calcium hydroxide paste (Dunston & Coll 2008, Bergoli et al. 2010), instead of zinc oxide eugenol paste (Barcelos et al. 2011), probably because of its irritant potential to periapical tissues and slow resorption rate (Silva et al. 2010). Calcium hydroxide pastes are antimicrobial, owing to their high pH and release of hydroxyl and calcium ions (Estrela et al. 1995). Several vehicles may be used with calcium hydroxide pastes. Both viscous and aqueous vehicles contribute to their fluidity, thus facilitating insertion into the root canal and improving antimicrobial performance (Fava & Saunders 1999, Blanscet et al. 2008). Iodoform pastes are available in several formulations and offer good biological performance, high antimicrobial potential and clinical success (Guedes-Pinto et al. 1981, PuppinRontani et al. 1994, Sarigol et al. 2010, Lacativa et al. 2012). Guedes-Pinto paste (Guedes-Pinto et al. 1981) is one such paste; however, its genotoxicity has not been assessed. Antoniazzi et al. (2015) proposed three new pharmacological mixtures as filling materials for primary teeth: iodoform and camphorated paramonochlorophenol (CPC), which is associated with (i) Nebacetin ointment, (ii) 2% chlorhexidine gluconate gel, and (iii) Maxitrol ointment. They tested the antimicrobial activity of these materials in vitro, compared with that of Guedes-Pinto paste, and obtained satisfactory results. Because a balance must be reached between antimicrobial ability and cytocompatibility (Huang et al. 2007), more studies should be performed so that these pastes may be used clinically. Few studies have evaluated the potential damage of biomaterials to the deoxyribonucleic acid (DNA) of cells (Bin et al. 2012). However, genotoxicity tests are essential to evaluate a substance’s potential toxicity in humans, so that risks can be prevented (Ribeiro 2008). The comet assay is typically used to evaluate genotoxicity because it is considered a good tool for detecting damage to the genetic material of cells, including DNA damage, gene mutation, chromosomal
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breakage, altered DNA repair capacity and cellular transformation (Ribeiro et al. 2006, Ribeiro 2008). A newly proposed test, Genomodifier capacity assay (GEMO), aims to assess the genotoxic ability to specific chemicals or extracts. This is an ultrasensitive and rapid protocol that consists of using a highly specific double-stranded DNA dye (PicoGreenâ) and pure calf thymus DNA (dsDNA). The ultrasensitive PicoGreenâ dye is a fluorescent reagent that allows the quantification of double-stranded DNA molecules in solution (Cadon a et al. 2014). The disruption of the stable cellular redox balance as a result of the increased generation of reactive oxygen species (ROS) is called oxidative stress. In turn, cellular macromolecules such as proteins, lipids and DNA may be damaged when the production of ROS is higher than the antioxidant capacity of the cells. It has been proven that some chemicals are able to disrupt the redox balance, causing an increase in the levels of ROS and subsequent cell death (Schweikl et al. 2006, Camargo et al. 2009). Therefore, the dichlorofluorescein diacetate (DCFH-DA) assay (Esposti 2002) can be used to measure the total rate of ROS inside the cells, whilst the evaluation of the oxidative degradation of lipids by ROS, called lipid peroxidation, can be measured by the thiobarbituric acid reactive substances (TBARS) assay (Ohkawa et al. 1979). The in vitro tests mentioned above are used to elucidate the mechanism of a biological reaction and investigate cell behaviour in specific situations. Even though the results of these assays cannot be immediately extrapolated to clinical conditions in humans, they are clinically relevant because they represent an important contribution to the correct evaluation of the potential health risk associated with endodontic materials. Therefore, this study aimed to investigate the cytotoxicity, oxidative stress and genotoxicity in vitro of four iodoform pastes and three calcium hydroxide pastes. The null hypothesis was that there is no difference between the biocompatibility parameters of iodoform pastes and calcium hydroxide pastes.
Material and methods Ethical approval This study was approved by the Ethics Committee for Research, Federal University of Santa Maria (no. 20457313.7.0000.5346).
© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd
Pires et al. Toxicity of filling materials
Cell culture Peripheral blood mononuclear cells (PBMCs) were obtained from discarded whole blood samples already processed from healthy adults. Blood specimens were centrifuged at 1611 g for 30 min, and the cells were isolated for density gradient, using Ficoll-Paque (Sigma-Aldrich, St. Louis, MO, USA). The cells were then plated in 6-well plates containing a complete RPMI 1640 culture medium. The cells were cultured at a density of 2 9 105 cells mL 1. Pure dsDNA (calf thymus DNA) was used in the GEMO assay. Details concerning the methodology are described in ‘Genotoxicity testing’ section.
Root canal pastes and preparation of the extracts The experimental groups were divided into eight subgroups, including the control group (cells in culture medium and phosphate-buffered saline [PBS]): (a) iodoform pastes: chlorhexidine (Nova Derme, Santa Maria, RS, Brazil) (CHX), Maxitrol (Alcon, S~ ao Paulo, SP, Brazil) (MAX), neomycin sulphate + bacitracin (EMS, Hortol^ andia, SP, Brazil) (NEB)), Guedes-Pinto (composed by a substitute for Rifocort–Formula & ~o, S~ Acßa ao Paulo, SP, Brazil) (GP); (b) calcium hydroxide pastes: Calen (S.S. White, Rio de Janeiro, RJ,
Brazil) thickened with zinc oxide (Calen/ZO), calcium hydroxide thickened with zinc oxide (CH/ZO) and UltraCal XS (Ultradent, Indaiatuba, SP, Brazil) (UltraCal) (Table 1). The proportions of iodoform pastes were the same as those proposed for the Guedes-Pinto paste (Guedes-Pinto et al. 1981) with iodoform (0.3 g), CPC (0.1 mL) and Rifocort ointment (0.25 g), in that the only component that changed was the ointment. Three types of calcium hydroxide pastes were tested. The first was composed of calcium hydroxide and zinc oxide in a 3 : 1 ratio that was mixed with propylene glycol until the consistency of toothpaste was obtained. The other materials were Calen (S.S. White, Rio de Janeiro, RJ, Brazil) (1 g) thickened with zinc oxide (0.65 g) (Queiroz et al. 2011) and UltraCal XS paste (Ultradent, Indaiatuba, SP, Brazil). The extracts were obtained as follows. The pastes were prepared in glass plates, under aseptic conditions, in a laminar flow; 0.22 mL of each paste was inserted in the 6-well plates. Each well was then filled with 2.5 mL RPMI 1640 culture medium supplemented with 10% foetal bovine serum, 1% penicillin/ streptomycin and 1% amphotericin B. The plates were then incubated at 37 °C for 24 h (Bin et al. 2012). The pH values of extracts remained in the range of 7.0 to 7.4.
Table 1 Components of the root filling pastes tested Material
Presentation
^mica, Ibipora ~, PR, Brazil) Iodoform (Biodina Camphorated paramonochlorophenol ^mica, Ibipora ~, PR, Brazil) (Biodina
Powder Liquid
2% chlorhexidine gluconate (Nova Derme, Santa Maria RS, Brazil) ~o Paulo, SP, Brazil) Maxitrol (Alcon, Sa
Gel
Neomycin sulphate + bacitracin ^ndia, SP, Brazil) (EMS, Hortola Sodium rifamycin SV + 21 prednisolone acetate (substitute for Rifocort) ~o, Sa ~o Paulo, SP,Brazil) (Formula & Acßa UltraCal XS (Ultradent, Indaiatuba, SP, Brazil) Calen (S.S. White, Rio de Janeiro, RJ, Brazil)
Ointment
Polymyxin B sulphate, neomycin sulphate, dexamethasone, preserved with methylparaben and propylparaben Neomycin sulphate and bacitracin
Ointment
Sodium rifamycin 1.5 mg g 1, prednisolone acetate 5 mg g
Paste
Powder Powder
Calcium hydroxide, barium sulphate, aqueous matrix of methylcellulose Calcium hydroxide, zinc oxide, colophony, polyethylene glycol. 99 a 100.5% of zinc oxide Calcium hydroxide P. A.
Liquid
Propylene glycol
^mica, Ibipora ~, PR, Brazil) Zinc oxide (Biodina Calcium hydroxide ^mica, Ibipora ~, PR, Brazil) (Biodina Propylene glycol (Nova Derme, Santa Maria, RS, Brazil)
Ointment
Paste
© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd
Components 99 a 100.5% of iodoform Parachlorophenol (30%), camphor (70%), alcohol 96° and deionized water 2% chlorhexidine gluconate, based of natrosol gel
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After incubation, each concentrated extract (1 : 1) (1000 lL) was mixed with a suspension of the previously plated cells (1000 lL). Thus, the final concentration was 1 : 2. The plates with the treatments were incubated at 37 °C and were later evaluated at 24 and 72 h, according to the test being performed. To avoid colour interference with the culture medium in the tests using PBMCs, the treated cells were resuspended at PBS (pH 7.4) (2 mL).
Cytotoxicity testing Cell viability was assessed using the MTT assay (Mosmann 1983, Denizot & Lang 1986): 20 lL MTT reagent (Sigma-Aldrich), dissolved in 5 mg mL 1 PBS, was added to a 96-well plate containing the sample treatments and incubated at 37 °C for 1 h (Fukui et al. 2010). The plate was then centrifuged at 2222 g for 10 min and the supernatant was removed from the wells. Next, the cells were resuspended in 200 lL dimethyl sulfoxide (DMSO) and centrifuged again. Absorbance was read at 570 nm on a spectrophotometer UV/Vis (Shimadzu, Kyoto, Japan).
Oxidative stress testing The dichlorofluorescein diacetate test (DCFH-DA) (Esposti 2002) was performed to verify intracellular ROS production. DCFH-DA (10 lmol L 1) reagent was added to a 96-well plate with the treated cells, for 60 min at 37 °C. The fluorescence was measured at an excitation rate of 485 nm and an emission rate of 520 nm on the spectrofluorometer. Fluorescence was directly proportional to the rate of ROS generated.
Lipid peroxidation testing Lipid peroxidation was quantified by determining the thiobarbituric acid reactive substances (TBARS) (Ohkawa et al. 1979). TBARS can be a marker of oxidative stress. To this end, 900 lL of supernatant was mixed with a reaction medium containing thiobarbituric acid (TBA 0.8%) and then incubated at 95 °C in a water bath for 1 h. The absorbance rate was read at 532 nm on the spectrophotometer. Absorbance is directly proportional to the rate of lipid peroxidation. All of the above tests were performed in triplicate, and the mean values were considered for analysis.
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Genotoxicity testing Alkaline comet assay The alkaline comet assay was performed as described by Singh et al. (1988) and adapted by Garcıa et al. (2004). Treated PBMCs were suspended in 0.75% low-melting-point agarose and PBS at 37 °C and placed on microscopic slides with a layer of 1.5% agarose. The slides were immersed in lysis solution (89 mL of lysis solution added to 10 mL of DMSO and 1 ml of Triton X-100) for 1 h and followed by denaturation in alkaline electrophoresis buffer (300 mmol L 1 NaOH and 1 mmol L 1 EDTA – pH>13.0), and then electrophoresis at 25 V, 300 mA, for 30 min. Subsequently, neutralization (neutralizing buffer pH 7.5), fixing (solution composed of 15% trichloroacetic acid) and staining (silver nitrate and 5% sodium carbonate) of the slides were performed. All steps described above were conducted under reduced illumination to minimize the possibility of cellular DNA damage. Two experienced, blinded and trained evaluators analysed one hundred cells (50 cells from each slide) with an optical microscope (400x) and classified the cells according to tail length. The distance migrated by cellular DNA directly reflects the extent of DNA damage present. Then, the cells received scores from 0 (no damage) to 4 (maximal damage). The data were converted into a percentage of damage index (Montagner et al. 2010) for analysis. Genomodifier capacity assay (GEMO) GEMO was performed according to Cadon a et al. (2014). In black 96-well plate, pure calf thymus DNA (dsDNA) was diluted in a TE buffer (10 mmol L 1 Tris-HCl, 1 mmol L 1 EDTA, pH 7.5). To each well containing 10 lL of dsDNA (1 lg mL 1) was added 100 lL of each extract which was prepared as mentioned above, accordance with Bin et al. (2012). dsDNA+extract remained in contact for 30 min. Then, the dye PicoGreenâ (1 : 200 TE) was added into the wells. After 5 min at room temperature, the fluorescence was measured at an excitation rate of 480 nm and an emission rate of 520 nm on a spectrofluorometer. Extracts causing breaks in doublestranded DNA molecules are identified by reduced fluorescence when compared to the control group, which contained only dsDNA (Cadon a et al. 2014). The test was performed in triplicate, and the averaged values were considered for analysis.
© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd
Pires et al. Toxicity of filling materials
Statistical analysis The data were tabulated and analysed using three programs: MiniTab version 17 (Minitab Inc., State College, PA, USA), GMC Basic Software version 7.7 (School of Dentistry of Ribeir~ao Preto, University of S~ ao Paulo, Ribeir~ ao Preto, SP, Brazil) and SPSS version 12.0 (SPSS Inc., Chicago, IL, USA). First, data normality was assessed by the Kolmogorov–Smirnov test. The data generated by the MTT 24 h and 72 h, the DCFH-DA 72 h, and the TBARS 24 h and 72 h tests were analysed by the Kruskal–Wallis and Dunn’s post-test. Data from the DCFH-DA 24 h, GEMO and the alkaline comet assay tests were analysed by ANOVA followed by Dunnett’s post-test. The significance level was established at P < 0.05.
decrease in the total ROS rate in the cells (P < 0.05) (Fig. 2). No iodoform paste caused an increase in TBARS levels in either period. Calcium hydroxide
Results The results of the cytotoxicity, oxidative stress and genotoxicity tests are presented in Figs 1, 2, 3 and 4. CHX, MAX and NEB significantly decreased cell viability to 92.82%, 87.39% and 80.21%, respectively, after 24 h (P < 0.05). No iodoform paste decreased cell viability at 72 h. All calcium hydroxide pastes caused a significant increase in cell viability in both experimental periods (P < 0.05) (Fig. 1). CHX, GP and calcium hydroxide pastes were associated with an increase in ROS rates (P < 0.05) at 24 h. All iodoform pastes and Calen/ZO caused a significant increase in ROS production at 72 h. During this time, CH/ZO and UltraCal pastes had percentages of 83.33 and 95.11, respectively, indicating a significant
Figure 1 Cytotoxicity of root filling pastes in PBMCs after exposure to extracts. The cell cultures were exposed for 24 h and 72 h. Bars represent the means. Significant differences between untreated (Control) and treated cell cultures are indicated by asterisks.
© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd
Figure 2 Oxidative stress testing (DCFH-DA) and lipid peroxidation testing (TBARS). Intracellular generation of ROS and lipid peroxidation of root filling pastes in PBMCs, respectively, after exposure to extracts for 24 h and 72 h. Bars represent the means. Significant differences between untreated (Control) and treated cell cultures are indicated by asterisks.
Figure 3 Genotoxicity of root filling pastes in PBMCs after exposure to extracts. The cell cultures were exposed for 24 h and 72 h. Bars represent the means. Significant differences between untreated (Control) and treated cell cultures are indicated by asterisks.
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Figure 4 Genotoxicity by GEMO assay in dsDNA after exposure to extracts of root filling pastes. Bars represent the means. Significant differences between untreated (Control) and treated cell cultures are indicated by asterisks.
pastes were associated with a significant increase in lipid peroxidation at 24 h, but caused no changes in TBARS levels at 72 h (Fig. 2). Iodoform pastes did not cause DNA damage in PBMCs in the alkaline comet assay, but calcium hydroxide pastes led to a higher DNA damage rate at 24 h and 72 h (P < 0.05) (Fig. 3). In the GEMO assay, one of iodoform pastes, CHX and calcium hydroxide pastes revealed genotoxic ability (P < 0.05) (Fig. 4).
Discussion No experiments in themselves can determine the final response to the mechanisms underlying the biological response of biomaterials (Leirskar & Helgeland 1981, Hauman & Love 2003). Therefore, in this study, four biocompatibility-related in vitro tests were performed to provide data on the safe use of filling materials for primary teeth with compromised pulps. Despite their limitations, laboratory tests performed on a cell culture may be a suitable tool to elucidate the mechanism involved in biocompatibility (Leirskar & Helgeland 1981). Furthermore, they have advantages over other methods, in terms of ease, rapidity and lower cost. However, it is difficult to compare the results of different cell culture experiments, because of methodological differences, such as cell type, contact forms, experimental time periods (Sarigol et al. 2010), assay type, varying concentrations and associations of different substances. Thus, the results obtained for some of the materials tested are difficult to compare, due to the lack of previous studies. The cells used were peripheral blood mononuclear leucocytes (lymphocytes and monocytes). Leucocytes provide an estimation of the body’s exposure to genotoxic products (Leite et al. 2012) and are typically
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used to assess cytotoxicity, together with human epithelial cells (HeLa) and fibroblasts (Ciapetti et al. 1993, Hauman & Love 2003). Considering that antiseptics for root canal treatment, applied topically, may circulate in the bloodstream after penetrating the dentine or the periodontium through the apical foramen and apical ramifications (Bartelstone 1951, Tucker 1981), these cells represent the damage that products may cause to the body. The MTT assay is a useful method to assess the cytotoxicity of biomaterials (Ciapetti et al. 1993). However, this is only one aspect of biocompatibility and cannot singly characterize a material as being biocompatible or nonbiocompatible (Peters 2013). GP caused no effect on cell viability of PBMCs at 24 h, confirming its positive clinical performance (GuedesPinto et al. 1981, Puppin-Rontani et al. 1994). However, the new iodoform pastes caused cell death at 24 h. During an extended period of contact with the cells (72 h), all of the iodoform pastes failed to cause cell death, which is important, because the pastes tend to remain on the root canal for many months. The mixture of calcium hydroxide paste and zinc oxide powder was evaluated by two experimental groups with different formulations and with two viscous vehicles (Calen/ZO and CH/ZO). UltraCal XS is a commercial option whose vehicle, methylcellulose, is aqueous. The extracts of calcium hydroxide pastes caused a significant increase in cell viability in both experimental periods. There are no studies that assess the cytotoxicity of these same formulations in cell cultures; however, the cell proliferation related to these pastes may be explained by their capacity to induce an inflammatory response and favour the repair process (Nelson Filho et al. 1999). Previous studies evaluating the cytocompatibility in the subcutaneous tissue of rats demonstrated that Calen/ZO paste is more biocompatible than zinc oxide eugenol paste or Sealapex (Queiroz et al. 2011). UltraCal XS, Hydropast and Calen paste also provided tissue compatibility (Andolfatto et al. 2012). Thus, calcium hydroxidebased pastes may be considered suitable for clinical use in pulp therapy, mainly for teeth with periapical lesions. ROS are present in human physiological metabolism and have an important biological function. However, data on increased ROS production may provide insight into one possible mechanism of cellular toxicity (Camargo et al. 2009). In this study, CHX and GP produced an increase in ROS levels within 24 h. Within 72 h, iodoform pastes demonstrated significantly
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Pires et al. Toxicity of filling materials
high levels of ROS; however, CHX exhibited the greatest increase. The behaviour of the mixture containing chlorhexidine can be explained by the study by Barbin et al. (2013), who identified cytotoxic agents parachloroaniline and ROS as subproducts of the aqueous solution of 2% chlorhexidine. Furthermore, chlorhexidine has substantivity and exerts a long-term action on tissues, thus increasing its risk potential even further (Barbin et al. 2008). As the iodoform-based pastes were not cytotoxic at 72 h and did not show lipid peroxidation, it could be suggested that, although they caused an increase in total ROS rate, the enzymes and the antioxidant defence system were able to repair the redox imbalance. All calcium hydroxide pastes caused higher ROS rates at 24 h. During a longer period of contact, only Calen/ZO caused a significant increase in the ROS rates. Whereas, in the TBARS test, these pastes showed similar results to DCFH-DA within 24 h, no calcium hydroxide paste evaluated caused oxidative damage to lipid cell structures within 72 h. These data contrasted with the MTT results. It may be explained by the fact that the redox imbalance was repaired and the damage to the lipid layer was not intense enough to cause cell death; rather, it merely affected the biologic mechanism of the cell. In the studies comparing iodoform paste and calcium hydroxide paste, the results show that iodoform paste is less cytotoxic in L929 fibroblasts of rodents (Sarigol et al. 2010), and offers acceptable levels of biocompatibility in less time, as observed in the histological evaluation of the intra-osseous implant technique in guinea pigs (Lacativa et al. 2012). However, Faraco-Junior & Percinoto (1998) found that both pastes had a good response in the periapical tissues after histological analysis of pulpotomized dog teeth and that calcium hydroxide achieved better results in relation to inflammation intensity and degree of resorption. However, Silva et al. (2010) performed the same type of histological analysis and found that Calen/ZO had the best tissue response. Thus, there is still no consensus on the best paste and further in vivo and clinical trials are required to evaluate this issue in greater depth. Some materials used in dental practice are potentially harmful to genetic material in vitro (Ribeiro et al. 2006, Silva et al. 2007) and in vivo (Ribeiro et al. 2004, Carlin et al. 2012). Thus, genotoxicity tests are essential to assess genetic damage and are also important indicators of carcinogenesis (Ribeiro 2008). Studies of the genotoxic and mutagenic effects
© 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd
of endodontic materials related to primary dentition have been published (Ribeiro 2008, Leite et al. 2012), but there is only one study reported thus far on filling pastes for deciduous teeth (Huang et al. 2009). In the present study, no iodoform paste induced DNA damage in the alkaline comet assay. However, in the GEMO assay, CHX showed genotoxicity. This is in agreement with the high levels of ROS produced by CHX in the present study. Moreover, chlorhexidine has a potential to cause oxidative damage to DNA due the releasing of para-chloroaniline, a possible carcinogen in humans (Barbin et al. 2008, 2013). All calcium hydroxide pastes were able to induce DNA damage in PBMCs, according to the alkaline comet assay. The GEMO assay confirmed the genotoxic action of these pastes, because they caused breaks in dsDNA. GEMO is a fast and inexpensive assay, and it may be a complementary test to a traditional genotoxic test such as the alkaline comet assay. The higher dsDNA fluorescence measured by the GEMO was associated with lower index damage measured by the alkaline comet assay (Cadon a et al. 2014). In contrast, it has been reported that calcium hydroxide pastes caused no DNA fragmentation in human osteosarcoma cells (Huang et al. 2009). There was a major difference in concentration of calcium hydroxide extracts, with 500 000 lg mL 1 in the present study, and 5 lg mL 1 in a previous study (Huang et al. 2009). The discrepancy between these results may be explained by differences in experimental procedures, such as concentration of extracts and cell culture methods. Likewise, genotoxicity results appear to contrast with MTT results in the present study. When PBMCs and dsDNA molecules were exposed to extracts, breaks occurred in the DNA structure due oxidative damage. This could result in chromosomal instability, genetic changes and problems related to cell division. ROS have the potential to stimulate cell proliferation by activating proto-oncogenesis (carcinogenesis), besides they can cause mitosis, apoptosis, necrosis and autophagy (Klaunig et al. 2010). In this way, it is suggested that the increase in cell viability caused can be related to cell proliferation. Moreover, in the process of cell division, when the repair system cannot repair an injury, this is perpetuated in the genetic material of this line which tends to suffer more oxidative damage (Tudek et al. 2010). Therefore, this correlation can be the explanation about the compatibility of the large increase in DNA damage with increase in cell viability (cell proliferation) caused by the calcium hydroxide pastes.
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Toxicity of filling materials Pires et al.
Conclusion The pastes varied in their ability to induce cytotoxicity, oxidative stress and genotoxicity. Two considerations are especially worthy of note: • Although calcium hydroxide pastes increased cell viability, they produced ROS and damage to the lipid layer within 24 h. However, all induced DNA damage. • Although most iodoform pastes decreased cell viability within 24 h (except GP), this was not sustained after 72 h. These pastes did not cause lipid peroxidation; however, all of them produced ROS within 72 h. Just one of the iodoform pastes (CHX) was to dsDNA. Guedes-Pinto, Maxitrol and neomycin sulphate + bacitracin pastes were associated with better in vitro biocompatibility performance. Due to the inherent limitations of an in vitro assay and the specific experimental conditions of this study, further studies in animal models and clinical trials must be performed to determine the biocompatibility and clinical use of these filling pastes in primary teeth.
Acknowledgements The authors would like to thank Dr. Rachel de Oliveira Rocha for her statistical support and the EMS pharmaceutical industry for its samples of neomycin sulphate and bacitracin sent for the pilot project. All the authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.
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