The micronucleus test. Cells of the oral epithelium (1000X magnification) from a female patient, 20 years old. A. Before and B. After 30 days of placement of orthodontic appliances. One micronucleus is indicated (arrow) in a binucleated cell in B. 

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... appliances are usually made of stainless steel alloy, which contains metals such as chromium, nickel and iron (Staerkjaer and Menné, 1990; Bass et al., 1993). The mouth properties (thermal, microbiological and enzymatic) offer an ideal environment for the biodegradation of orthodontic appliances (Faccioni et al., 2003; Thomas et al., 2007, 2008; Amini et al., 2008; Matos de Souza and Macedo de Menezes, 2008), consequently facilitating the release of metal ions that are related to adverse health effects, such as cellular and genetic toxicity (Munksgaard, 1992; Wataha, 2000; Dayan and Paine, 2001; Valko et al., 2005; Thomas et al., 2007, 2008). Genotoxicity comprises either mutagenic or carcinogenic processes. Thus, the genotoxic properties of metals from orthodontic appliances are defined as an essential criterion to select these materials in a safe biological manner for patients (Montanaro et al., 2005). The assessment of genotoxic agents can be performed through the application of some well-established endpoints such as the micronucleus (MN) frequency, as determined by the MN assay, or primary DNA damage, as accessed by the comet assay (CA). The combination of the two assays is considered to be very beneficial, because they show supplementary characteristics (Van Goethem et al., 1997). The MN assay is based on the frequency of MN, structures that originate from chromosome fragments or whole chromo - somes that are not included in the main daughter nuclei during nuclear division (Fenech et al., 1999). Thus, MN may arise from either DNA breakage leading to acentric chromo- some fragments or from chromosome/chromatin lagging in anaphase. The formation of MN is considered to be an effective biomarker of diseases and processes associated with the induction of DNA damage. Another assay that has been indicated in order to comple- ment the MN result is the alkaline single cell gel electrophoresis assay (Van Goethem et al., 1997), the CA, which measures single- and/or double-strand breaks in a cell by the cell approach. The CA is considered a quick, simple, sensitive, reliable, and fairly inexpensive way of measuring DNA damage (Collins et al., 1997). The aim of the present research was to determine the genotoxicity induced by metals from orthodontic appliances, by employing both the MN and the CA in a group of healthy patients undergoing orthodontic treatment. Twenty healthy patients (14 females) with an average age of 16 ± 2.5 years, undergoing orthodontic treatment, were enrolled in this study. Orthodontic appliances were made of stainless steel (0.07% carbon, 1.0% manganese, 1.0% silicon, 15.5-17.5% chromium, 3-5% nickel, 3-5% copper, 0.15-0.45% niobium + tantalum) in both arches, consisting basically of an average of 20 bonded brackets and four bands (3M Unitek ® , Monrovia, CA, USA). Smoking or drinking or ill- nesses related to any genetic damage increase were not reported by any patient. The patients’ con- sent was obtained after a full explanation of the objective of the study. The research was approved by the university’s ethics committee (Pontifícia Universidade do Rio Grande do Sul, Brazil). The samples were collected before (control) and after the placement of the orthodontic appliances. For the CA, the samples were obtained before and 10 days after the placement of the orthodontic appliances. For the MN assay, cells were sampled before and 30 days after the placement of the orthodontic appliances. Buccal cells were collected from each individual by gentle brushing of the inside part of the lower lip with a cytological brush, after washing out the mouth several times with tepid distilled water to remove exfoliated dead cells. The brushes were stirred in 50-ml plastic tubes containing 20 ml phosphate-buffered saline (PBS). Cells were washed twice, with centrifugation at 1500 rpm for 10 min at room temperature, and resuspended in PBS, which was employed for the CA or MN assay. The alkaline version of the CA was employed in this study (Speit and Hartmann, 1999; Faccioni et al., 2003). Briefly, 10 μL cell suspension was mixed with 75 μL low-melt - ing-point agarose (0.7%) and added to a slide precoated with 100 μL agarose (1%). Lysis was performed overnight at pH 10. Cells were then placed in a electrophoresis chamber, exposed to alkali, pH 13, for 25 min, and electrophoresis was performed for 20 min at 25 V (0.86 V/cm) and 300 mA, at room temperature. The slides were neutralized, fixed, and stained with silver nitrate (Nadin et al., 2001). The slides were examined under a light microscope (Axiolab, Zeiss) at 1000x magnification. Fifty randomly selected cells of each subject (25 cells for each of two replicate slides) were visually scored according to five classes, based on tail size (from undamaged - 0, to maximally damaged - 4). Damage index (DI) was thus assigned to each individual, according to the sum of the classes attributed to each cell, rang- ing from 0 (completely undamaged: 50 cells x 0) to 200 (with maximum damage: 50 cells x 4) (Hartmann et al., 2003). The DI is based on the length of migration and on the amount of DNA in the tail and is considered to be a sensitive measure of DNA. International guidelines and recommendations for the CA consider that visual scoring of comets is a well-validated evaluation method as it is highly correlated with computer-based image analysis (Burlinson et al., 2007). Buccal cells were collected and analyzed according to a standard protocol described else- where by Titenko-Holland et al. (1994). Slides of buccal cells were prepared by dropping the washed cell suspension onto pre-warmed slides (37°C). After dropping, the cells were allowed to air-dry and fixed in methanol (80%, v/v) at 0°C for 20 min. Staining was performed with May-Grunwald- Giemsa according to a standard protocol (Titenko-Holland et al., 1994). Only cells that were not smeared, clumped or overlapping and that contained intact nuclei were included in the analysis. MN were identified according to the following characteristics: i) less than 1/3 diameter of the main nucleus; ii) the same plane of focus; iii) the same color, texture and refraction as the main nucleus; iv) smooth oval or round shape, and v) clearly separated from the main nucleus (Titenko-Holland et al., 1994). Cells were observed in oil immersion at 1000X magnification with a light microscope (Axiolab, Zeiss) to determine the presence of MN cells, as established by Sarto et al., 1987. The one-tailed t -test with Welch’s correction was used to compare DI obtained by the CA, and the one-tailed Fisher exact test was used to compare the number of patients with MN before and after the placement of orthodontic appliance. Primary DNA damage level, as assessed by the CA, was low either before the begin- ning (1.5 ± 1.05) or 10 days after the placement of orthodontic appliance (2.5 ± 3.08) and did not change significantly between these time points ( p = 0.0913). Most cells were classified as class 0 regarding DNA damage extent, as depicted in Figure 1. Conversely, there was a significant increase in MN frequency ( p = 0.0213) 30 days after the placement of orthodontic appliances (Table 1). Figure 2 illustrates an MN cell at 30 days after the placement of the orthodontic ...