Cell culture

In vitro test systems for genotoxicity evaluation can be differentiated into prokaryotic and/or eukaryotic tests. Studies conducted with the eukaryotic systems are thought to provide more reliable information with respect to the genotoxicity of chemicals. Therefore, we aimed to investigate the genotoxic potential of corrosion eluates obtained from orthodontic brackets. For this we were able to use Chinese hamster ovary (CHO) cells. Our choice of CHO cells (a continuous cell line) provided an accurate evaluation of the changes independent from confounding factors such as age and metabolic and hormonal states of the donor. This cell line has a small number of relatively large chromosomes; they grow fast and reproducible results can be obtained from the same cell source.

For this purpose, CHO cells (lineage CHO K-1) were growth to confluence in 75-cm ² culture flasks (Corning, Incorporated, New York, NY) using Ham’s F-10 medium (Invitrogen Corporation, Grand Island, NY) supplemented with 10% fetal calf serum and 100 U/mL penicillin (Life Technologies, Carlsbad, Calif) and 100 μg/mL streptomycin (Invitrogen Corporation) at 37°C with 5% CO ₂ . Cells were cultured for 5 days prior to treatment with test substances. Confluent cells were detached with 0.15% trypsin (Invitrogen Corporation) for 5 minutes; after that, 2 mL complete medium was added and cells were centrifuged at 1000 rpm (180 g ) for 5 minutes. Cell suspension was counted using a Neubauer chamber and seeded in 96-well microtiter plates (Corning) at a density of 1 × 10 ⁴ cells per well (at a concentration of 1 × 10 ⁶ /mL).

Cell treatment

For this study, the following commercially available orthodontic brackets were used: Morelli, Abzil, Dentaurum, and 3M Unitek. There are no differences regarding metal composition among them according to the manufacturer’s instructions. Each dental bracket was submitted to a corrosion process in a solution containing equal amounts of acetic acid and sodium chloride (Merck & Co., Inc., Whitehouse Station, NJ), at 0.1 M concentration, for 1, 3, 7, 14, 21, 35, and 70 days. A volume of 10 uL of cells (approximately 10,000 cells) was then added individually to each final solution of eluate maintained for 30 minutes at 37°C. After exposure, cells were washed in phosphate buffered solution (PBS). The negative control was treated with the same solution used for the corrosion process for 30 minutes at 37°C. As cytotoxicity is a confounding factor in genotoxicity studies, it is not recommended to perform the single cell gel (comet) assay on samples with more than 30% cytotoxicity. Thus, the exposure period as well as the final concentrations used here were established in a previous study conducted in our laboratory for evaluating the genotoxicity of these compounds only. An independent positive control was performed with MMS at 1 μg/mL for 1 hour in order to ensure the reproducibility and sensitivity of the assay. In addition, each treatment was performed consecutively 3 times to ensure reproducibility.

Single cell gel (comet) assay

To evaluate the magnitude of DNA damage, we used the alkaline version of the single cell gel (comet) assay. The comet assay is a relatively new, rapid, simple, and reliable biochemical technique for evaluating DNA damage in mammalian cells. This technique includes embedding cells in agarose gel on microscope slides, incubating them with the test compound, and then lysing the cells with detergent and high salts. During electrophoresis under alkaline conditions, cells with damaged DNA display increased rates of DNA migration to the anode. The increase in DNA migration rate results from the formation of smaller fragments of DNA caused by double-strand breaks, single-strand breaks, and alkali-labile sites. Smaller fragments of DNA migrate further in the electric field compared with intact DNA, and the cellular lysates thus resemble a “comet” with brightly fluorescent head and a tail region. Our own recent studies have demonstrated that the single cell gel (comet) assay is a suitable tool to investigate genotoxicity of dental compounds used in clinical practice.

The protocol used for the single cell gel (comet) assay followed the guidelines purposed by Tice et al. Slides were prepared in duplicate per treatment. Thus, a volume of 10 μL of treated or control cells (∼1 × 10 ⁴ cells) was added to 120 μL of 0.5% low-melting-point agarose at 37°C, layered onto a precoated slide with 1.5% regular agarose, and covered with a coverslip. After brief agarose solidification in a refrigerator, the coverslip was removed and the slides were immersed in the lysis solution (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid [EDTA] [Merck & Co., Inc.]; 10 mM Tris-HCl buffer pH = 10 [Sigma Aldrich, St. Louis, Mo]; 1% sodium sarcosinate [Sigma Aldrich]; with 1% Triton X-100 [Sigma Aldrich]; and 10% DMSO [Merck & Co. Inc.]) for about 1 hour. Prior to electrophoresis, the slides were left in an alkaline buffer (0.3 mM NaOH [Merck & Co., Inc.] and 1 mM EDTA [Merck & Co., Inc., pH > 13]) for 20 minutes and electrophoresed for another 20 minutes, at 25 V (0.86 V/cm) and 300 mA. After electrophoresis, the slides were neutralized in 0.4 M Tris-HCl (pH = 7.5) for 15 minutes, fixed in absolute ethanol, and stored at room temperature until analysis. All of the steps described above were conducted in the dark to prevent additional DNA damage.

Throughout this study, some diluted and treated aliquots were tested for viability by trypan blue exclusion, and greater than 75% of cells constantly excluded trypan.

Comet capture and analysis

A total of 50 randomly captured comets per treatment (25 cells from each slide) were examined blindly by 1 expertise observer (D.A.R.) at 400× magnification using a fluorescence microscope (Olympus, Center Valley, Pa) connected through a black and white camera to an image analysis system (Comet Assay II, Perceptive Instruments Ltd, Haverhill, UK). For all experiments, we evaluated 2 image analysis parameters: tail intensity (% migrated DNA) and tail moment. Tail moment was calculated by the image analysis system as the product of the tail length (DNA migration) and the fraction of DNA in the comet tail (% DNA in the tail). In none of the experiments there was a significant difference between these parameters. Therefore, we chose tail moment for the presentation of the results.

Statistical methods

The parameter from the comet assay (tail moment) was assessed by 1-way analysis of variance (ANOVA) followed by post hoc analysis and the Tukey test using SigmaStat software, version 1.0 (Jandel Scientific, Rafael, Calif). A P value less than 0.05 was considered statistically significant.