- Email: [email protected]
Cell-cycle deregulation induced by three different root canal sealers in vitro
Cell-cycle deregulation induced by three different root canal sealers in vitro Caroline R. A. Valois, DDS, MsC, PhD,a and Ricardo B. Azevedo, DDS, MsC, PhD,b Brasília, Brazil DEPARTMENT OF GENETIC AND MORPHOLOGY, INSTITUTE OF BIOLOGICAL SCIENCES, UNIVERSITY OF BRASÍLIA
The aim of this study was to investigate the effects of 3 root canal sealers on the cell cycle of mouse fibroblasts (3T3 cell line). Freshly mixed root canal sealers (AH Plus, Endofill, and Sealer 26) were eluted for 24 h using cell culture medium. Antiproliferation activity of different eluate dilutions was determined by 3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. Cell cycle was subsequently evaluated by flow cytometry. Differences were tested by analysis of variance with Tukey test (P ⬍ .05). Results showed that the root canal sealers inhibited cell growth in a dose-dependent manner. Antiproliferation activity of the root canal sealers was associated with cell-cycle deregulation. This event involved cell-cycle arrest in the S/G2/M phases and an increase of the sub-G1 population, which are consistent with both cytostatic and cytotoxic effects. This information may contribute to a better understanding of the toxicity mechanism displayed by root canal sealers currently used in clinical practice. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:763-7)
Root canal filling is achieved using a combination of gutta-percha and a root canal sealer.1 Several root canal sealers are currently used in clinical practice. In general, these materials are divided into different groups according to their main component, such as calcium hydroxide, epoxy resin, and zinc oxide– eugenol.2 Because they may be placed in contact with periapical tissues during endodontic treatment, root canal sealers should be biocompatible.3 The biocompatibility of root canal sealers has been evaluated in vitro.4-8 Those studies showed that most root canal sealers currently used in clinical practice displayed toxic effects in both human and animal derived cell lines. Moreover, the potential of these adverse effects appeared to be particularly higher before the materials have set.5-6 Toxic effects of root canal sealers include suppression of cell growth.4-8 The rate of cell growth is strictly regulated by cell-cycle progression which involves the passage of the cell through a discrete set of ordered phases. Cells prepare for DNA replication during G1 phase, replicate their DNA in S phase, prepare for mitosis in G2 phase, and finally segregate the daughter chromosomes in M phase.9,10 The induction of cellSupported by the Brazilian agencies CNPq, FINEP, CAPES, and FINATEC. a Associate Researcher. b Adjunct Professor. Received for publication May 7, 2008; returned for revision June 10, 2008; accepted for publication June 16, 2008. 1079-2104/$ - see front matter © 2008 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2008.06.016
cycle disturbance by toxic materials usually leads to growth arrest and even apoptosis.11-13 This may influence the healing of periapical lesions that result from an endodontic infection, such as apical periodontitis, during the process of repair.2,12,14 In spite of this fact, little is known about whether toxicity of root canal sealers currently used in clinical practice is due to cell-cycle deregulation. Cell cycle is widely analyzed by flow cytometry after staining with propidium iodide. In that technique, growth arrest can be observed by an increase of cells in S, G2, and/or M phases, and apoptotic cells can be identified as a sub-G1 population.15,16 The aim of the present study was to investigate the in vitro effects of 3 root canal sealers on the progression of cell cycle of mouse fibroblasts, cell line 3T3. MATERIALS AND METHODS Culture conditions Mouse fibroblast cell line 3T3 was obtained from the American Type Culture Collection (ATCC; CCL-92). Cells were cultured in Dulbecco Modified Eagle’s Medium (DMEM) supplemented with NaHCO3 (3.7 g/L), penicillin (100 U/mL), streptomycin (100 g/mL), and 10% fetal calf serum. Cultures were maintained at 37°C in a fully humidified atmosphere of 5% CO2 in air. Test materials AH Plus (DeTrey Dentsply, Konstanz, Germany), an epoxy resin-based sealer; Endofill (Dentsply, Brazil), a zinc oxide-eugenol-based sealer; and Sealer 26 (Dentsply, Brazil), a calcium hydroxide-based sealer, were used in this study. A total of 300 mg of each root 763
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canal sealer was mixed according to the manufacturer’s instructions and placed in a round plastic mold (10 mm diameter). Plastic molds were submerged into plastic vials (15 mL volume) containing 3 mL of serum-free culture medium. Elution was carried out for 24 h at 37°C in a fully humidified atmosphere. Freshly mixed root canal sealers’ eluates were sterilized by filtering with a 0.2-m-pore filter and then were diluted in culture medium. Control extract was prepared in an identical manner except that empty plastic molds were used. Growth inhibitory evaluation The effect of root canal sealers on cell growth was determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay method. Briefly, 3T3 cells were seeded in 96-well plates at a density of 5 ⫻ 104 cells/well in 200 L culture medium. After incubation for 3 h at 37°C, the culture medium was removed and replaced by different root canal sealers dilutions (5%, 10%, and 20% of eluate in culture medium) and the control extract. After incubation for 24 h, the culture medium was removed and immediately replaced with a 200 L aliquot of a freshly prepared MTT solution (5 mg/mL MTT in culture medium). After incubation for 2 h, supernatant was discarded and formazan crystals formed were solubilized by the addition of 100 L dimethyl sulfoxide per well. The absorbance of each of the 96-well plates was measured at 570 nm using a microplate reader (Bio-Rad, Tokyo, Japan). Cell growth was expressed as the percentage of control activity using the absorbance values. Cell-cycle analysis The effect of root canal sealers on cell cycle was determined by flow cytometry. Briefly, 3T3 cells were seeded in 12-well plates at a density of 1 ⫻ 105 cells/ well in 1 mL culture medium. After incubation for 24 h, the culture medium was removed and replaced by different root canal sealer dilutions (5%, 10%, and 20% of eluate in culture medium) and the control extract. Then the cells were harvested, collected by centrifugation, and incubated with 200 L propidium iodide (PI) solution (20 g/mL PI and 0.1% Triton X-100 in phosphate-buffered saline) at room temperature for 30 min in the dark. Flow cytometric analysis was performed on a FACS Calibur (Becton Dickinson Biosciences, San Juan, CA) with an excitation wavelength of 488 nm. Fluorescence emission of 10,000 cells was detected, and the cell cycle phase distribution was analyzed using Cell Quest (Becton Dickinson Immunocytometry System) software.
Fig. 1. Effects of the root canal sealers’ eluates at different dilutions (5%, 10%, and 20%) on the cell viability. aP ⬍ .05 compared with 5% dilution of the same root canal sealer. bP ⬍ .05 compared with 10% dilution of the same root canal sealer.
Statistical analysis Results are reported as mean ⫾ standard error of 6 replicates from 2 independent experiments. Differences among the groups were tested by analysis of variance with Tukey test for multiple comparisons and were considered to be significant at P ⬍ .05. RESULTS The effect of root canal sealer dilutions on cell growth was determined using MTT assay (Fig. 1). All root canal sealers inhibited the cell growth in a dosedependent manner. A very high percentage of viable cells was found in cultures exposed to the 5% dilution. The viable cell number was reduced when the dilution reached 10% (P ⬍ .05), and this effect was more pronounced in cultures exposed to the 20% dilution (P ⬍ .05). To investigate whether suppression of cell growth was associated with cell-cycle deregulation, the proportion of cells in the cell-cycle phases and the appearance of sub-G1 population were analyzed by flow cytometry (Figs. 2-4). The root canal sealers showed a similar pattern of cell-cycle distribution. For the 5% dilution, no differences were found in the proportion of cells for the different cell-cycle phases or for the sub-G1 population compared with control. However, treatment with the 10% dilution caused a decrease in the proportion of cells in the G1 phase, accompanied by an increase in the proportion of cells in the S/G2/M phases, compared with control (P ⬍ .05). Moreover, treatment with the 20% dilution caused a decrease in the proportion of cells in both G1 and S/G2/M phases and was accom-
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Fig. 2. Effects of the root canal sealers’ eluates at different dilutions (5%, 10%, and 20%) on proportion of cells in the G1 phase of the cell cycle. aP ⬍ .05 compared with control of the same root canal sealer dilution.
Fig. 4. Effects of the root canal sealers’ eluates at different dilutions (5%, 10%, and 20%) on proportion of cells in the sub-G1 population. aP ⬍ .05 compared with control of the same root canal sealer dilution.
Fig. 3. Effects of the root canal sealers’ eluates at different dilutions (5%, 10%, and 20%) on proportion of cells in the S/G2/M phases of the cell cycle. aP ⬍ .05 compared with control of the same root canal sealer dilution.
identified as a factor that influences the toxic effects of root canal sealers.5,7 Therefore, the root canal sealers’ challenge dilutions used were first evaluated by MTT. In line with previous studies,5,7 the present findings showed that the root canal sealers inhibited the cell growth in a dose-dependent manner. This antiproliferation activity was related to a cell number decrease in the G1 phase of the cell cycle as well with an increase of accumulated cells in the S/G2/M phases, indicating both a decrease of cell proliferation rate (G1 phase) and an arrest of cell-cycle progression (S/G2/M phases). Considering that the reduction of viable 3T3 cells involved changes in the cell-cycle distribution pattern, the present results indicated that the root canal sealers’ antiproliferation mechanism was associated with cellcycle deregulation. It is well documented that the growth responses of cells in culture vary immensely depending on the chemical composition of root canal sealers in a given experimental set-up.4,5,7,8 This finding raises the question of whether root canal sealers with different chemical composition affect in different ways the progression of the cell-cycle phases. Contrary to the expectation above, the 3 types of root canal sealers investigated in the present study showed similar effects on the cell-cycle distribution pattern, suggesting that chemical composition was not responsible for the toxic effects of these materials. However, it must be noted that this study investigated a single mechanism involved with the suppression of cell growth. This does not exclude the possibility that the root canal sealers’ components induce antiproliferation activity through some other additional mechanisms. Therefore, our results indicate that, despite differences among chemical
panied by an increase in the proportion of cells in the sub-G1 population compared with control (P ⬍ .05). DISCUSSION The specific mechanism involved in cell antiproliferation activity of root canal sealers is still unclear. Because it has been reported that cell growth is governed by cell cycle,9,10 we had decided to investigate the effects of 3 different types of root canal sealers, currently used in clinical practice, and their relationship to the progression of cell-cycle phases of 3T3 mouse fibroblast cells. Concentration has been previously
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composition, the root canal sealers evaluated have something in common in relation to the mechanism of antiproliferation activity. On the other hand, our results clearly show that the induction of cell-cycle deregulation was associated with the root canal sealers’ dose increase. Whereas the 5% dilution neither changed the number of viable cells nor induced cell-cycle alteration, we observed that the 10% and 20% dilutions inhibited the growth of 3T3 cells with concomitant change of cell-cycle distribution in both G1 and S/G2/M phases. This may be explained by the fact that a percentage rise of the root canal sealers’ eluates in the culture medium causes an increase in the amount of cell-irritating components. It is well documented that root canal sealers may be placed in contact with periapical tissues during endodontic treatment.14,17 However, an exact amount of material extruded can not be measured. In view of this fact, the clinical implication of dilutions evaluated in the present study remains speculative. At the moment, the present findings support the concept that no or minimal extrusion of root canal sealers to periapical tissues is a good clinical practice. Earlier studies that have evaluated the effects of root canal sealers on cells have focused on the degree of cellular death after exposure.5-8 However, the decrease of cell number in response to the root canal sealers is not necessarily a consequence of cellular death (cytotoxic effect). Another approach is to target a physiologic process essential to cell growth, such as cell-cycle progression.9,10 With this approach, cell proliferation can be reduced or halted while cell viability is relatively unimpaired (cytostatic effect). There is little evidence to date that the root canal sealers can significantly inhibit cell growth in a cytotoxic and/or cytostatic manner. Significantly, therefore, the present findings showed that the root canal sealers were able to inhibit cell growth by causing both cytotoxic and cytostatic effects. The induction of one or the other effect was associated with the dilution used. The treatment of 3T3 cells induced cell-cycle arrest in the S/G2/M phases at 10% dilution, which is consistent with cytostatic effect. In contrast, the appearance of sub-G1 peak at 20% dilution indicates classic apoptosis induction as the origin of cytotoxic effect. It is well documented that the inhibition of cell proliferation without inducing cell death could potentially reduce cytotoxicity of materials.18 However, the repair process, where high rates of cell proliferation is necessary, can also conceivably be affected by cytostatic effect. Therefore, the present results suggest that the extrusion of root canal sealers during endodontic treatment of teeth with lesion associated should be avoided to exclude the possibility that
both cytotoxic and cytostatic effects may influence the lesions’ healing process. In conclusion, in the present study we showed that antiproliferation activity of 3 root canal sealers currently used in clinical practice was associated with cell-cycle deregulation. Our results also indicated that the cell-cycle deregulation involved cell-cycle arrest in the S/G2/M phases and the appearance of sub-G1 peak, which are consistent with both cytostatic and cytotoxic effects. The ability of the root canal sealers to induce one or the other effect was critically dependent on the increase of its dose. The information presented here may contribute to a better understanding of the toxicity mechanism displayed by root canal sealers currently used in clinical practice. The authors thank Victoria Monge-Fuentes for reading the manuscript. REFERENCES 1. Siqueira JF Jr, Rôças IN, Lopes HP. Materiais obturadores. In: Siqueira JF Jr, Lopes HP, editors. Endodontia-biologia e técnica. Rio de Janeiro: Medsi; 1999. p. 430. 2. Dahl JE. Toxicity of endodontic filling materials. Endod Topics 2005;12:39-43. 3. Hauman CHJ, Love RM. Biocompatibility of dental materials used in contemporary endodontic therapy: a review. Part 2. Root-canal-filling materials. Int Endod J 2003;36:147-60. 4. Barbosa SV, Araki K, Spangberg LSW. Cytotoxicity of some modified root canal sealers and their leachable components. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1993;75:357-61. 5. Bratel J, Jontell M, Dahlgren U, Bergenholtz G. Effects of root canal sealers on immunocompetent cells in vitro and in vivo. Int Endod J 1998;31:178-88. 6. Camps J, About I. Cytotoxicity testing of endodontic sealers: a new method. J Endod 2003;29:583-6. 7. Huang TH, Ding SJ, Hsu TZ, Lee ZD, Kao CT. Root canal sealers induce cytotoxicity and necrosis. J Mater Sci Mater Med 2004;15:767-71. 8. Schwarze T, Fiedler I, Leyhausen G, Geurtsen W. The cellular compatibility of five endodontic sealers during the setting period. J Endod 2002;28:784-6. 9. Hench LL, Polak JM, Xynos ID, Buttery LDK. Bioactive materials to control cell cycle. Mater Res Innovat 2000;3:313-23. 10. Pai R, Szabo IL, Kawanaka H, Soreghan BA, Jones MK, Tarnawski AS. Indomethacin inhibits endothelial cell proliferation by suppressing cell cycle proteins and prb phosphorylation: a key to its antiangiogenic action? Mol Cell Biol Res Commun 2000;4:111-6. 11. Gorduysus M, Avcu N, Gorduysus O, Pekel A, Baran Y, Avcu F, Ural AU. Cytotoxic effects of four different endodontic materials in human periodontal ligament fibroblasts. J Endod 2007;33: 1450-4. 12. Granchi D, Stea S, Ciapetti G, Cavedagna D, Stea S, Pizzoferrato A. Endodontic cements induce alterations in the cell cycle of in vitro cultured osteoblasts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;79:359-66. 13. Guigand M, Pellen-Mussi P, Le Goff A, Vulcain JM, BonnaureMallet M. Evaluation of the cytocompatibility of three endodontic materials. J Endod 1999;25:419-23. 14. Sari S, Durutürk L. Radiographic evaluation of periapical healing
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15.
16.
17. 18.
of permanent teeth with periapical lesions after extrusion of AH Plus sealer. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:e54-9. Darzynkiewicz Z, Bedner E, Smolewski P. Flow cytometry in analysis of cell cycle and apoptosis. Semin Hematol 2001;38: 179-93. Pellicciari C, Manfredi AA, Bottone MG, Schaack V, Barni S. A single-step staining procedure for the detection and sorting of unfixed apoptotic thymocytes. Eur J Histochem 1993;37:381-90. Seltzer S. Long-term radiographic and histological observations of endodontically treated teeth. J Endod 1999;25:818-22. Morley KL, Ferguson PJ, Koropatnick J. Tangeretin and nobile-
Valois and Azevedo 767 tin induce G1 cell cycle arrest but not apoptosis in human breast and colon cancer cells. Cancer Letters 2007;251:168-78. Reprint requests: Caroline R. A. Valois Department of Genetics and Morphology Institute of Biological Sciences University of Brasília Brasília, DF, 70910-900 Brazil. [email protected]
The aim of this study was to investigate the effects of 3 root canal sealers on the cell cycle of mouse fibroblasts (3T3 cell line). Freshly mixed root canal sealers (AH Plus, Endofill, and Sealer 26) were eluted for 24 h using cell culture medium. Antiproliferation activity of different eluate dilutions was determined by 3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay. Cell cycle was subsequently evaluated by flow cytometry. Differences were tested by analysis of variance with Tukey test (P ⬍ .05). Results showed that the root canal sealers inhibited cell growth in a dose-dependent manner. Antiproliferation activity of the root canal sealers was associated with cell-cycle deregulation. This event involved cell-cycle arrest in the S/G2/M phases and an increase of the sub-G1 population, which are consistent with both cytostatic and cytotoxic effects. This information may contribute to a better understanding of the toxicity mechanism displayed by root canal sealers currently used in clinical practice. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;106:763-7)
Root canal filling is achieved using a combination of gutta-percha and a root canal sealer.1 Several root canal sealers are currently used in clinical practice. In general, these materials are divided into different groups according to their main component, such as calcium hydroxide, epoxy resin, and zinc oxide– eugenol.2 Because they may be placed in contact with periapical tissues during endodontic treatment, root canal sealers should be biocompatible.3 The biocompatibility of root canal sealers has been evaluated in vitro.4-8 Those studies showed that most root canal sealers currently used in clinical practice displayed toxic effects in both human and animal derived cell lines. Moreover, the potential of these adverse effects appeared to be particularly higher before the materials have set.5-6 Toxic effects of root canal sealers include suppression of cell growth.4-8 The rate of cell growth is strictly regulated by cell-cycle progression which involves the passage of the cell through a discrete set of ordered phases. Cells prepare for DNA replication during G1 phase, replicate their DNA in S phase, prepare for mitosis in G2 phase, and finally segregate the daughter chromosomes in M phase.9,10 The induction of cellSupported by the Brazilian agencies CNPq, FINEP, CAPES, and FINATEC. a Associate Researcher. b Adjunct Professor. Received for publication May 7, 2008; returned for revision June 10, 2008; accepted for publication June 16, 2008. 1079-2104/$ - see front matter © 2008 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2008.06.016
cycle disturbance by toxic materials usually leads to growth arrest and even apoptosis.11-13 This may influence the healing of periapical lesions that result from an endodontic infection, such as apical periodontitis, during the process of repair.2,12,14 In spite of this fact, little is known about whether toxicity of root canal sealers currently used in clinical practice is due to cell-cycle deregulation. Cell cycle is widely analyzed by flow cytometry after staining with propidium iodide. In that technique, growth arrest can be observed by an increase of cells in S, G2, and/or M phases, and apoptotic cells can be identified as a sub-G1 population.15,16 The aim of the present study was to investigate the in vitro effects of 3 root canal sealers on the progression of cell cycle of mouse fibroblasts, cell line 3T3. MATERIALS AND METHODS Culture conditions Mouse fibroblast cell line 3T3 was obtained from the American Type Culture Collection (ATCC; CCL-92). Cells were cultured in Dulbecco Modified Eagle’s Medium (DMEM) supplemented with NaHCO3 (3.7 g/L), penicillin (100 U/mL), streptomycin (100 g/mL), and 10% fetal calf serum. Cultures were maintained at 37°C in a fully humidified atmosphere of 5% CO2 in air. Test materials AH Plus (DeTrey Dentsply, Konstanz, Germany), an epoxy resin-based sealer; Endofill (Dentsply, Brazil), a zinc oxide-eugenol-based sealer; and Sealer 26 (Dentsply, Brazil), a calcium hydroxide-based sealer, were used in this study. A total of 300 mg of each root 763
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canal sealer was mixed according to the manufacturer’s instructions and placed in a round plastic mold (10 mm diameter). Plastic molds were submerged into plastic vials (15 mL volume) containing 3 mL of serum-free culture medium. Elution was carried out for 24 h at 37°C in a fully humidified atmosphere. Freshly mixed root canal sealers’ eluates were sterilized by filtering with a 0.2-m-pore filter and then were diluted in culture medium. Control extract was prepared in an identical manner except that empty plastic molds were used. Growth inhibitory evaluation The effect of root canal sealers on cell growth was determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay method. Briefly, 3T3 cells were seeded in 96-well plates at a density of 5 ⫻ 104 cells/well in 200 L culture medium. After incubation for 3 h at 37°C, the culture medium was removed and replaced by different root canal sealers dilutions (5%, 10%, and 20% of eluate in culture medium) and the control extract. After incubation for 24 h, the culture medium was removed and immediately replaced with a 200 L aliquot of a freshly prepared MTT solution (5 mg/mL MTT in culture medium). After incubation for 2 h, supernatant was discarded and formazan crystals formed were solubilized by the addition of 100 L dimethyl sulfoxide per well. The absorbance of each of the 96-well plates was measured at 570 nm using a microplate reader (Bio-Rad, Tokyo, Japan). Cell growth was expressed as the percentage of control activity using the absorbance values. Cell-cycle analysis The effect of root canal sealers on cell cycle was determined by flow cytometry. Briefly, 3T3 cells were seeded in 12-well plates at a density of 1 ⫻ 105 cells/ well in 1 mL culture medium. After incubation for 24 h, the culture medium was removed and replaced by different root canal sealer dilutions (5%, 10%, and 20% of eluate in culture medium) and the control extract. Then the cells were harvested, collected by centrifugation, and incubated with 200 L propidium iodide (PI) solution (20 g/mL PI and 0.1% Triton X-100 in phosphate-buffered saline) at room temperature for 30 min in the dark. Flow cytometric analysis was performed on a FACS Calibur (Becton Dickinson Biosciences, San Juan, CA) with an excitation wavelength of 488 nm. Fluorescence emission of 10,000 cells was detected, and the cell cycle phase distribution was analyzed using Cell Quest (Becton Dickinson Immunocytometry System) software.
Fig. 1. Effects of the root canal sealers’ eluates at different dilutions (5%, 10%, and 20%) on the cell viability. aP ⬍ .05 compared with 5% dilution of the same root canal sealer. bP ⬍ .05 compared with 10% dilution of the same root canal sealer.
Statistical analysis Results are reported as mean ⫾ standard error of 6 replicates from 2 independent experiments. Differences among the groups were tested by analysis of variance with Tukey test for multiple comparisons and were considered to be significant at P ⬍ .05. RESULTS The effect of root canal sealer dilutions on cell growth was determined using MTT assay (Fig. 1). All root canal sealers inhibited the cell growth in a dosedependent manner. A very high percentage of viable cells was found in cultures exposed to the 5% dilution. The viable cell number was reduced when the dilution reached 10% (P ⬍ .05), and this effect was more pronounced in cultures exposed to the 20% dilution (P ⬍ .05). To investigate whether suppression of cell growth was associated with cell-cycle deregulation, the proportion of cells in the cell-cycle phases and the appearance of sub-G1 population were analyzed by flow cytometry (Figs. 2-4). The root canal sealers showed a similar pattern of cell-cycle distribution. For the 5% dilution, no differences were found in the proportion of cells for the different cell-cycle phases or for the sub-G1 population compared with control. However, treatment with the 10% dilution caused a decrease in the proportion of cells in the G1 phase, accompanied by an increase in the proportion of cells in the S/G2/M phases, compared with control (P ⬍ .05). Moreover, treatment with the 20% dilution caused a decrease in the proportion of cells in both G1 and S/G2/M phases and was accom-
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Fig. 2. Effects of the root canal sealers’ eluates at different dilutions (5%, 10%, and 20%) on proportion of cells in the G1 phase of the cell cycle. aP ⬍ .05 compared with control of the same root canal sealer dilution.
Fig. 4. Effects of the root canal sealers’ eluates at different dilutions (5%, 10%, and 20%) on proportion of cells in the sub-G1 population. aP ⬍ .05 compared with control of the same root canal sealer dilution.
Fig. 3. Effects of the root canal sealers’ eluates at different dilutions (5%, 10%, and 20%) on proportion of cells in the S/G2/M phases of the cell cycle. aP ⬍ .05 compared with control of the same root canal sealer dilution.
identified as a factor that influences the toxic effects of root canal sealers.5,7 Therefore, the root canal sealers’ challenge dilutions used were first evaluated by MTT. In line with previous studies,5,7 the present findings showed that the root canal sealers inhibited the cell growth in a dose-dependent manner. This antiproliferation activity was related to a cell number decrease in the G1 phase of the cell cycle as well with an increase of accumulated cells in the S/G2/M phases, indicating both a decrease of cell proliferation rate (G1 phase) and an arrest of cell-cycle progression (S/G2/M phases). Considering that the reduction of viable 3T3 cells involved changes in the cell-cycle distribution pattern, the present results indicated that the root canal sealers’ antiproliferation mechanism was associated with cellcycle deregulation. It is well documented that the growth responses of cells in culture vary immensely depending on the chemical composition of root canal sealers in a given experimental set-up.4,5,7,8 This finding raises the question of whether root canal sealers with different chemical composition affect in different ways the progression of the cell-cycle phases. Contrary to the expectation above, the 3 types of root canal sealers investigated in the present study showed similar effects on the cell-cycle distribution pattern, suggesting that chemical composition was not responsible for the toxic effects of these materials. However, it must be noted that this study investigated a single mechanism involved with the suppression of cell growth. This does not exclude the possibility that the root canal sealers’ components induce antiproliferation activity through some other additional mechanisms. Therefore, our results indicate that, despite differences among chemical
panied by an increase in the proportion of cells in the sub-G1 population compared with control (P ⬍ .05). DISCUSSION The specific mechanism involved in cell antiproliferation activity of root canal sealers is still unclear. Because it has been reported that cell growth is governed by cell cycle,9,10 we had decided to investigate the effects of 3 different types of root canal sealers, currently used in clinical practice, and their relationship to the progression of cell-cycle phases of 3T3 mouse fibroblast cells. Concentration has been previously
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composition, the root canal sealers evaluated have something in common in relation to the mechanism of antiproliferation activity. On the other hand, our results clearly show that the induction of cell-cycle deregulation was associated with the root canal sealers’ dose increase. Whereas the 5% dilution neither changed the number of viable cells nor induced cell-cycle alteration, we observed that the 10% and 20% dilutions inhibited the growth of 3T3 cells with concomitant change of cell-cycle distribution in both G1 and S/G2/M phases. This may be explained by the fact that a percentage rise of the root canal sealers’ eluates in the culture medium causes an increase in the amount of cell-irritating components. It is well documented that root canal sealers may be placed in contact with periapical tissues during endodontic treatment.14,17 However, an exact amount of material extruded can not be measured. In view of this fact, the clinical implication of dilutions evaluated in the present study remains speculative. At the moment, the present findings support the concept that no or minimal extrusion of root canal sealers to periapical tissues is a good clinical practice. Earlier studies that have evaluated the effects of root canal sealers on cells have focused on the degree of cellular death after exposure.5-8 However, the decrease of cell number in response to the root canal sealers is not necessarily a consequence of cellular death (cytotoxic effect). Another approach is to target a physiologic process essential to cell growth, such as cell-cycle progression.9,10 With this approach, cell proliferation can be reduced or halted while cell viability is relatively unimpaired (cytostatic effect). There is little evidence to date that the root canal sealers can significantly inhibit cell growth in a cytotoxic and/or cytostatic manner. Significantly, therefore, the present findings showed that the root canal sealers were able to inhibit cell growth by causing both cytotoxic and cytostatic effects. The induction of one or the other effect was associated with the dilution used. The treatment of 3T3 cells induced cell-cycle arrest in the S/G2/M phases at 10% dilution, which is consistent with cytostatic effect. In contrast, the appearance of sub-G1 peak at 20% dilution indicates classic apoptosis induction as the origin of cytotoxic effect. It is well documented that the inhibition of cell proliferation without inducing cell death could potentially reduce cytotoxicity of materials.18 However, the repair process, where high rates of cell proliferation is necessary, can also conceivably be affected by cytostatic effect. Therefore, the present results suggest that the extrusion of root canal sealers during endodontic treatment of teeth with lesion associated should be avoided to exclude the possibility that
both cytotoxic and cytostatic effects may influence the lesions’ healing process. In conclusion, in the present study we showed that antiproliferation activity of 3 root canal sealers currently used in clinical practice was associated with cell-cycle deregulation. Our results also indicated that the cell-cycle deregulation involved cell-cycle arrest in the S/G2/M phases and the appearance of sub-G1 peak, which are consistent with both cytostatic and cytotoxic effects. The ability of the root canal sealers to induce one or the other effect was critically dependent on the increase of its dose. The information presented here may contribute to a better understanding of the toxicity mechanism displayed by root canal sealers currently used in clinical practice. The authors thank Victoria Monge-Fuentes for reading the manuscript. REFERENCES 1. Siqueira JF Jr, Rôças IN, Lopes HP. Materiais obturadores. In: Siqueira JF Jr, Lopes HP, editors. Endodontia-biologia e técnica. Rio de Janeiro: Medsi; 1999. p. 430. 2. Dahl JE. Toxicity of endodontic filling materials. Endod Topics 2005;12:39-43. 3. Hauman CHJ, Love RM. Biocompatibility of dental materials used in contemporary endodontic therapy: a review. Part 2. Root-canal-filling materials. Int Endod J 2003;36:147-60. 4. Barbosa SV, Araki K, Spangberg LSW. Cytotoxicity of some modified root canal sealers and their leachable components. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1993;75:357-61. 5. Bratel J, Jontell M, Dahlgren U, Bergenholtz G. Effects of root canal sealers on immunocompetent cells in vitro and in vivo. Int Endod J 1998;31:178-88. 6. Camps J, About I. Cytotoxicity testing of endodontic sealers: a new method. J Endod 2003;29:583-6. 7. Huang TH, Ding SJ, Hsu TZ, Lee ZD, Kao CT. Root canal sealers induce cytotoxicity and necrosis. J Mater Sci Mater Med 2004;15:767-71. 8. Schwarze T, Fiedler I, Leyhausen G, Geurtsen W. The cellular compatibility of five endodontic sealers during the setting period. J Endod 2002;28:784-6. 9. Hench LL, Polak JM, Xynos ID, Buttery LDK. Bioactive materials to control cell cycle. Mater Res Innovat 2000;3:313-23. 10. Pai R, Szabo IL, Kawanaka H, Soreghan BA, Jones MK, Tarnawski AS. Indomethacin inhibits endothelial cell proliferation by suppressing cell cycle proteins and prb phosphorylation: a key to its antiangiogenic action? Mol Cell Biol Res Commun 2000;4:111-6. 11. Gorduysus M, Avcu N, Gorduysus O, Pekel A, Baran Y, Avcu F, Ural AU. Cytotoxic effects of four different endodontic materials in human periodontal ligament fibroblasts. J Endod 2007;33: 1450-4. 12. Granchi D, Stea S, Ciapetti G, Cavedagna D, Stea S, Pizzoferrato A. Endodontic cements induce alterations in the cell cycle of in vitro cultured osteoblasts. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1995;79:359-66. 13. Guigand M, Pellen-Mussi P, Le Goff A, Vulcain JM, BonnaureMallet M. Evaluation of the cytocompatibility of three endodontic materials. J Endod 1999;25:419-23. 14. Sari S, Durutürk L. Radiographic evaluation of periapical healing
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of permanent teeth with periapical lesions after extrusion of AH Plus sealer. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;104:e54-9. Darzynkiewicz Z, Bedner E, Smolewski P. Flow cytometry in analysis of cell cycle and apoptosis. Semin Hematol 2001;38: 179-93. Pellicciari C, Manfredi AA, Bottone MG, Schaack V, Barni S. A single-step staining procedure for the detection and sorting of unfixed apoptotic thymocytes. Eur J Histochem 1993;37:381-90. Seltzer S. Long-term radiographic and histological observations of endodontically treated teeth. J Endod 1999;25:818-22. Morley KL, Ferguson PJ, Koropatnick J. Tangeretin and nobile-
Valois and Azevedo 767 tin induce G1 cell cycle arrest but not apoptosis in human breast and colon cancer cells. Cancer Letters 2007;251:168-78. Reprint requests: Caroline R. A. Valois Department of Genetics and Morphology Institute of Biological Sciences University of Brasília Brasília, DF, 70910-900 Brazil. [email protected]