Cytotoxicity Evaluation of Four Endodontic Sealers

This study evaluated in vitro the cytotoxicity of four root canal sealers (Topseal, EndoRez, TubliSeal and Kerr Pulp Canal Sealer E.W.T.) and their effects on reactive oxygen/nitrogen intermediate induction by mouse peritoneal macrophages. Thioglycollate-induced cells were obtained from Swiss mice by peritoneal lavage with 5 mL 10 mM phosphate-buffered saline, washed twice and resuspended (106 cells/mL) in appropriate medium for each test. Cytotoxicity was determined by the presence of hydrogen peroxide (H2O2) and nitric oxide (NO) by the peroxidase-dependent oxidation of phenol red and Griess reaction, respectively. Sealer suspensions were obtained in two different concentrations from each material: 18 mg/mL and 9 mg/mL, established according to compatibility parameters following MTT assay. Comparing the sealers, H2O2 release at concentrations of 9 mg/mL and 18 mg/mL was similar: Topseal > positive control (medium + cells + 5 mg/mL zimozan solution) > EndoRez > TubliSeal > Kerr Pulp E.W.T. > negative control (medium + cells). NO release at concentration of 9 mg/mL was: positive control (medium + cells + 10 μg/mL LPS solution) > Topseal > Kerr Pulp E.W.T. > TubliSeal = EndoRez > negative control (medium + cells); at concentration of 18 mg/mL was: positive control > Topseal > Kerr Pulp E.W.T > TubliSeal > EndoRez > negative control. Based on the results, it may be concluded that Topseal presented the highest cytotoxicity among the tested sealers, releasing higher concentrations of NO and H2O2 in macrophage culture.


INTRODUCTION
The goal of a root canal filling is to prevent coronal re-infection and entomb remaining bacteria (1) since the absence of root filling or presence of poor fillings may provide pathways for bacteria and toxins to the periapex.Additionally, root canal sealers must be biocompatible because they may be in direct contact with the apical connective tissues for a long period and might cause inflammatory degeneration of part of these tissues, delaying the periapical healing (2,3).Sealing and biological properties of endodontic sealers are thus essential for the clinical success of endodontic therapy.
Root canal sealers can have different bases, and epoxy resins, calcium hydroxide, zinc-oxide eugenol (ZOE) and silicone present different biological characteristics.ZOE-based sealers have a long history of use, with good mechanical properties, but known cytotoxic effects due to eugenol release (4).Resin-based sealers are becoming more popular, but studies have indicated some toxic and mutagenic effects (5,6).
Macrophages are widely distributed in different tissues and play an essential role in the development of the specific and nonspecific immune response.These cells can be activated by bacterial components, cytokines and chemicals.When activated, macrophages produce and release numerous secretory products like cytokines, inorganic reactive radicals, reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), all having biological activity (7).Hydrogen peroxide (H 2 O 2 ) and nitric oxide (NO) are important in cell signaling and are effective molecules for the microbicidal and cytotoxic response of macrophages after stimulation (8).ROI and RNI can be considered as beneficial intermediates for presenting microbiocidal and tumoricidal activities.Otherwise, they can also be destructive for the host tissue under certain conditions (9).This study evaluated in vitro the cytotoxicity of different endodontic sealers by the analysis of NO and H 2 O 2 production by mouse peritoneal macrophages.

MATERIAL AND METHODS
Eleven Swiss mice weighting 18-25 g from the Animal House of the School of Pharmaceutical Sciences of Araraquara, Brazil were used in all experiments.All procedures were undertaken in accordance with the institutional Research Ethics Committee.
These concentrations were established according to compatibility parameters following the MTT assay.

Analysis of Hydrogen Peroxide
H 2 O 2 was measured by the horseradish peroxidase (HRP)-dependent oxidation of phenol red (10).Macrophages (2 x 10 6 cells/mL) were suspended in 10 mM potassium phosphate buffer containing 140 mM NaCl, 5.5 mM dextrose, 0.56 mM phenol red, and 0.01 mg/mL type II HRP, pH 7.4.100 µL of this suspension were added to each well of a 96-well tissue culture plate and exposed to 50 µL of sealer suspension for 1 h at 37°C, 5% CO 2 atmosphere.The reaction was inactivated with 10 µL of 4 N NaOH.The absorbances were read at 620 nm.Results were expressed as nmol H 2 O 2 per 2 x 10 5 cells as calibrated against solutions of known H 2 O 2 concentration.The experiments were performed in triplicate.Each experiment was accompanied by a positive control (medium + cells + 5 mg/mL zimozan solution) and a negative control (medium + cells).

Analysis of Nitric Oxide
NO synthesis was determined by measuring the accumulation of nitrite (NO 2 -), a stable metabolite of NO, in culture supernatants using the Griess reaction (11).Macrophages at 5 x 10 6 cells/mL in RPMI-1640 (Sigma, St. Louis, MO, USA) containing 5% heatinactivated FCS, 100 IU/penicillin, 100 µg/mL streptomycin and 50 mM 2-mercaptoethanol were added to each well of a 96-well tissue culture dish with 100 µL of the sealer solution.The cells were incubated for 24 h (time for NO assay) at 37 o C in a 5% CO 2 atmosphere.After incubation, 50 µL aliquots of culture supernatant were mixed with 50 µL of Griess reagent (1% w/v sulphanylamide, 0.1% w/v naphthylethylenediamine and 3% H 3 PO 4 ), and incubated at room temperature for 10 min.Absorbance at 540 nm was measured using a microplate reader.NO 2 -concentration (µmol/L) was calculated from a NaNO 2 standard curve.Each experiment was accompanied by a positive control (medium + cells + 10 µg/mL LPS solution) and a negative control (medium + cells).The experiments were done in triplicate.

Statistical Analysis
Results are representative of three independent

RESULTS
The means were compared in pairs and analyzed by the Student's t-test.The statistical results of the mean release is summarized in the following lines.H 2 O 2 release at concentrations of 9 mg/mL and 18 mg/mL was similar: Topseal > positive control > EndoRez > TubliSeal > Kerr Pulp E.W.T. > negative control.NO release at concentration of 9 mg/mL was: positive control > Topseal > Kerr Pulp E.W.T. > TubliSeal = EndoRez > negative control; NO release at concentration of 18 mg/ mL was: positive control > Topseal > Kerr Pulp E.W.T > TubliSeal > EndoRez > negative control.(Figs. 1 and 2).

DISCUSSION
Macrophages have been used for immunocytotoxicity testing because they permit the measurement of the cytotoxic response directly in the cell culture and have the ability to maintain immunological functions in the presence of many different chemical agents (12).The effect of endodontic sealers on macrophage activity is of interest since these cells play a key role on innate and acquired immune defenses and on the pathogenesis of inflammation (13).
In response to antigens or inflammatory signals generated at sites of tissue injury, macrophages undergo a process of cellular "activation" which is associated with morphological, functional, and biochemical changes in the cells.One prominent characteristic of activated macrophages is their increased capacity to release pro-inflammatory and cytotoxic mediators, which aid in antigen destruction (14).
In the present study, the determination of subtoxic concentrations of the sealers using the MTT assay permitted the measurement of the parameters and the exclusion of cell mortality as a variable.
The measurement of H 2 O 2 and NO levels by colorimetric analysis allows, associated with other parameters, the understanding of the compatibility of the material with the host tissues (2).Under physiological conditions, H 2 O 2 is generated in low levels and is quickly metabolized or degraded, but long exposures and high concentrations of H 2 O 2 can destroy biological structures and lead to irreversible cell damage (15).
Both resin-based sealers (Topseal and EndoRez) required more energy to be phagocytized, what was expressed by the amount of H 2 O 2 released.This indicates that these sealers are more cytotoxic than TubliSeal and Kerr Pulp Canal Sealer E.W.T., respectively.Topseal induced higher levels of NO release than Kerr Pulp Canal Sealer E.W.T., TubliSeal and EndoRez, respectively.NO affects virtually every step of the development of inflammation.Large amounts of NO can be toxic and pro-inflammatory.NO is not a strong oxidant.However,   it reacts at a nearly diffusion limited rate with superoxide to form peroxynitrite, which is a strong oxidant.Peroxynitrite is formed by activated inflammatory cells and agonist-stimulated endothelial cells, and has been found to oxidize several biological molecules and to nitrate free or protein tyrosine residues and other phenolics (16,17).Thus, NO may become cytotoxic or cytostatic.The higher cytotoxicity of resin-based sealers observed in this study confirms the findings of previous investigations (5,6,18,19).The cytotoxicity of TopSeal can be due to minimum amounts of formaldehyde contained in this sealer or to the release of the amine and epoxy resin components from this material, as previously suggested (19).AHPlus has the same formulation of TopSeal.The urethane dimethacrylate (UDMA) in the structure of EndoREZ could be the responsible for its cytotoxic effect, as reported elsewhere (20).
In conclusion, Topseal presented the highest cytotoxicity among the tested sealers, releasing higher concentrations of NO and H 2 O 2 in macrophage culture.

Figure 1 .
Figure 1.Graphic presentation of mean hydrogen peroxide release and the respective 95% range for the means.

Figure 2 .
Figure 2. Graphic presentation of mean nitric oxide release and the respective 95% range for the means.