Abstract
The aim of this study was to assess the cytotoxicity of orthodontic materials (brackets, wires, resin, elastomers and silver solder) using Saccharomyces cerevisiae as a model organism. The induction of cytotoxicity was assessed by two different tests using the wild-type S. cerevisiae strain FF18733: (1) direct exposure to orthodontic materials in YPD broth, and (2) exposure to artificial commercial saliva pre-treated with orthodontic materials. Only the silver solder was tested in mutant S. cerevisiae strains to investigate the origin of the observed cytotoxicity. Colony forming units per mL counts were carried out in all experiments and compared to controls to detect significant survival differences. The results showed that only the silver solder induced significant cytotoxicity, which might have occurred via oxidative stress, although this mechanism is not completely understood. Moreover, S. cerevisiae proved to be a reliable and useful model microorganism for evaluating the cytotoxicity of clinical materials.
1
Introduction
The biocompatibility of dental materials has been extensively studied , since this property is essential to ensure the safe treatment of patients . Some orthodontic materials, such as brackets, wires, solder and resins, have compounds known to have allergic, cytotoxic, mutagenic and/or carcinogenic potential . These materials remain within the oral cavity for long periods and are subject to corrosion, which provokes the release of substances that might interact with patients’ tissues .
The toxicity of some metals (iron, cooper, chromium, vanadium, cobalt, mercury, cadmium, nickel) might result from the generation of high levels of nitrogen and/or oxygen reactive species . Oxidative stress may cause oxidative damage in proteins, lipids and or/DNA and may trigger signaling cascades that stimulate cell growth .
Currently, many in vitro tests exist that can be used to assess the cytotoxicity of orthodontic materials, and various cell cultures are used that yield some similar and also some opposing findings . Among these tests, those that use cultured human cells tend to be the choice of many dentistry research works. Nevertheless, human cell cultures are expensive to keep, have a long life cycle, a very difficult tractability and, in many cases, do not allow a robust group of data to be quantitatively analyzed.
Cytotoxicity induced by harmful agents of human interest can be assessed successfully by in vitro experiments using model microorganisms like the yeast Saccharomyces cerevisiae . The use of this microorganism offers some advantages, since it is easy and cheap to manipulate, and provides a large amount of quantitative data from well-controlled experiments with short-time results. Moreover, the yeast model S. cerevisiae is biochemically, genetically and genomically very well described . Additionally, Fungi and Animal kingdoms are phylogenetically very closely related and are both grouped together in the eukaryotic super group called “Opisthokonta” . Thus, they do share biochemical and genetic similarities that justify the use of yeast models to address a scientific question of clinical interest , including even neurodegenerative diseases . However, few dental studies have used this microorganism for this purpose , and none of these were dedicated to orthodontic materials.
The aim of this study was to evaluate the induction of cytotoxicity by orthodontic materials (brackets, wires, resin, elastomers and silver solder) using a wild-type S. cerevisiae strain as a model organism. Moreover, S. cerevisiae mutant strains were also used to investigate the origin of observed cytotoxicity induced by some of the orthodontic materials.
2
Materials and methods
This cytotoxicity study was approved by the ethics Committee from Pontifícia Universidade Católica do Rio Grande do Sul, Brazil. The evaluation was performed using the following orthodontic materials: brackets and wires, both composed of stainless steel alloys (basic composition: cobalt; chromium; nickel; trace amounts of molybdenum, silica, beryllium, boron and carbon) – American Orthodontics, Sheboygan, USA; “Fill Magic Orthodontic” light-cure resin (basic composition: BisGMA, ester of methacrylic acid, glass of silicate fluoride) – Vigodent, Rio de Janeiro, Brazil; “Charisma” light-cure resin (basic composition: BisGMA, TeGMA, microglass (particle barium glass), silicon dioxide, light-cure initiators) – Heraeus Kulzer, NY, USA; Elastomers (basic composition: Natural latex – Hevea brasiliensis , Dietildtiocarbanato Zinc, Sulfur, Zinc, ZnO, Si, cetostearyl Alcohol, Poly (vinyl-metril-eter)-Morelli, Sorocaba, Brazil; and Silver Solder fragments (basic composition: silver, copper, cadmium, zinc and nickel) – Morelli, Sorocaba, Brazil, with an average weight of 0.02 g each. These materials were all available for testing at the Clinic of Orthodontics, School of Dentistry, Pontifícia Universidade Católica do Rio Grande do Sul, Brazil.
2.1
S. cerevisiae strain, media and cultures
The S. cerevisiae strains used in this work and their respective genotypes are listed in Table 1 . Included are the wild-type (WT) strain EG103 and its derivatives, which are single or double mutants in genes that encode the antioxidant defense enzymes Sod1, Sod2, and Cat1. Also included are the WT strain FF18733 and its derivatives, which are single, double or triple mutants in genes that encode the DNA base excision repair proteins Ogg1, Apn1, Apn2, Ntg1 and Ntg2.
| Wild-type and its derivative mutants in DNA repair | |
|---|---|
| Strain | Genotype |
| FF18733 (WT) | mat a, ura3-52, his7-3, leu2-1, trp1-289, lys1-1 |
| BG1 (apn1) | Like FF18733, apn1URA |
| BG2 (apn2) | Like FF18733, apn2kanMX |
| BG3 (apn1/apn2) | Like FF18733, apn1URA, apn2kanMX |
| CD138 (ogg1) | Like FF18733, ogg1TRP1 |
| CD186 (ogg1/ntg1/ntg2) | Like FF18733, ogg1TRP1, ntg1URA, ntg2HIS |
| Wild-type and its derivative mutants in antioxidant defense | |
|---|---|
| Strain | Genotype |
| EG103 (WT) | mat a, leu2-3, 112 his3D1, trp1-289, ura3-52, GAL+ |
| EG118 (Sod1) | Like EG103, Sod1URA3 |
| EG110 (Sod2) | Like EG103, Sod2TRP1 |
| EG133 (Sod1, Sod2) | Like EG103, Sod1URA3, Sod2URA3 |
| EG223 (ctt1) | Like EG103, ctt1TRP1 |
The first experiments tested all the orthodontic materials but used only the WT strain FF18733. The other S. cerevisiae strains were only used to test the cytotoxicity of the solder fragments.
To cultivate all S. cerevisiae strains, YPD medium (1% yeast extract, 2% peptone, 2% glucose) was used, either in broth or solid (with agar at 2%) form. In all survival experiments, S. cerevisiae pre-cultures were prepared in 10 mL YPD broth and grown overnight to exponential phase (∼10 −7 cells/mL) at 30 °C.
2.2
Survival experiments for cytotoxicity analysis
The cytotoxicity analysis was performed via two types of survival experiments: (1) direct exposure of S. cerevisiae cells to the orthodontic materials in YPD broth, and (2) exposure to artificial commercial saliva (Salivan, Apsen Farmacêutica SA, Brazil) pre-treated with orthodontic materials.
For the direct exposure experiments, new inocula were made from the pre-cultures in 5 mL YPD, with each one containing pieces of different orthodontic materials, and a control without any material. These cultures were incubated at 30 °C to exponential phase (∼10 −7 cells/mL). Aliquots from each culture were diluted in 0.9% sterile saline solution and 5-μL drops from each dilution (from 10 −2 to 10 −5 ) were plated on YPD-agar and incubated at 30 °C for two days for the emergence of small colonies, which allowed a qualitative approach. For quantitative analyses, 100 μL of the final dilutions were plated on YPD-agar (two plates for each dilution) for colony (CFU/mL) counting after two days at 30 °C.
In saliva exposure experiments, the different orthodontic materials were immersed in 500 μL of artificial saliva for seven or twenty days. A total of 500 μL of the pre-inoculum was used for each treatment, which was centrifuged (2 min at 2000 g) and resuspended at 100% of saliva pre-exposed to the orthodontic materials. The cells were then treated for 60 min, diluted and plated in YPD-agar as described above, for both qualitative and quantitative analyses. A control with unexposed saliva was also done. At least three direct and three indirect experiments were performed with each type of orthodontic material.
2.3
Data analyses
The mean and standard deviation of the colony forming units per mL (CFU/mL) counts from three independent repeats of each treatment were compared to their specific controls to verify the occurrence of significant survival differences in a semi-log curve. If there is at least one log of difference (considering de standard deviation) in terms of CFU/ml in treatments in relation to controls it is assumed a significant difference, which is an indication of cellular toxicity in S. cerevisiae .
2
Materials and methods
This cytotoxicity study was approved by the ethics Committee from Pontifícia Universidade Católica do Rio Grande do Sul, Brazil. The evaluation was performed using the following orthodontic materials: brackets and wires, both composed of stainless steel alloys (basic composition: cobalt; chromium; nickel; trace amounts of molybdenum, silica, beryllium, boron and carbon) – American Orthodontics, Sheboygan, USA; “Fill Magic Orthodontic” light-cure resin (basic composition: BisGMA, ester of methacrylic acid, glass of silicate fluoride) – Vigodent, Rio de Janeiro, Brazil; “Charisma” light-cure resin (basic composition: BisGMA, TeGMA, microglass (particle barium glass), silicon dioxide, light-cure initiators) – Heraeus Kulzer, NY, USA; Elastomers (basic composition: Natural latex – Hevea brasiliensis , Dietildtiocarbanato Zinc, Sulfur, Zinc, ZnO, Si, cetostearyl Alcohol, Poly (vinyl-metril-eter)-Morelli, Sorocaba, Brazil; and Silver Solder fragments (basic composition: silver, copper, cadmium, zinc and nickel) – Morelli, Sorocaba, Brazil, with an average weight of 0.02 g each. These materials were all available for testing at the Clinic of Orthodontics, School of Dentistry, Pontifícia Universidade Católica do Rio Grande do Sul, Brazil.
2.1
S. cerevisiae strain, media and cultures
The S. cerevisiae strains used in this work and their respective genotypes are listed in Table 1 . Included are the wild-type (WT) strain EG103 and its derivatives, which are single or double mutants in genes that encode the antioxidant defense enzymes Sod1, Sod2, and Cat1. Also included are the WT strain FF18733 and its derivatives, which are single, double or triple mutants in genes that encode the DNA base excision repair proteins Ogg1, Apn1, Apn2, Ntg1 and Ntg2.
| Wild-type and its derivative mutants in DNA repair | |
|---|---|
| Strain | Genotype |
| FF18733 (WT) | mat a, ura3-52, his7-3, leu2-1, trp1-289, lys1-1 |
| BG1 (apn1) | Like FF18733, apn1URA |
| BG2 (apn2) | Like FF18733, apn2kanMX |
| BG3 (apn1/apn2) | Like FF18733, apn1URA, apn2kanMX |
| CD138 (ogg1) | Like FF18733, ogg1TRP1 |
| CD186 (ogg1/ntg1/ntg2) | Like FF18733, ogg1TRP1, ntg1URA, ntg2HIS |
| Wild-type and its derivative mutants in antioxidant defense | |
|---|---|
| Strain | Genotype |
| EG103 (WT) | mat a, leu2-3, 112 his3D1, trp1-289, ura3-52, GAL+ |
| EG118 (Sod1) | Like EG103, Sod1URA3 |
| EG110 (Sod2) | Like EG103, Sod2TRP1 |
| EG133 (Sod1, Sod2) | Like EG103, Sod1URA3, Sod2URA3 |
| EG223 (ctt1) | Like EG103, ctt1TRP1 |
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