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Periodontitis: Genomic instability implications and associated risk factors
Mutat Res Gen Tox En 840 (2019) 20–23
Contents lists available at ScienceDirect
Mutat Res Gen Tox En journal homepage: www.elsevier.com/locate/gentox
Periodontitis: Genomic instability implications and associated risk factors a
a,b,c
b
b
T a
Tatiana T. Borba , Patrícia Molz , Diene S. Schlickmann , Caroline Santos , Caio F. Oliveira , ⁎ Daniel Práa, Léo Kreather Netoa,d, Silvia I.R. Frankea,b, a
Graduate Program in Health Promotion, University of Santa Cruz do Sul (UNISC), Santa Cruz do Sul, RS, Brazil Laboratory of Experimental Nutrition, University of Santa Cruz do Sul (UNISC), Santa Cruz do Sul, RS, Brazil Graduate Program in Medicine and Health Sciences, Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil d Department of Nursing and Dentistry, University of Santa Cruz do Sul (UNISC), Santa Cruz do Sul, RS, Brazil b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Periodontal disease severity Buccal Micronucleus Cytome DNA damage Nuclear cellular abnormalities
Periodontitis is a bacterial infection characterized by the presence of a dense inflammatory infiltrate, which may result in increased DNA damage and other nuclear/cellular abnormalities. Therefore, it is important to evaluate the periodontal diseases influence on DNA damage and other nuclear/cellular abnomalies formation as cancer risk markers. Thus, the aim of this study was to evaluate the periodontal diseases effect, according to its severity, on the occurrence of DNA damage and other nuclear/cellular abnormalities. This is a cross-sectional study with 77 subjects from the dentistry clinic of the University of Santa Cruz do Sul, Brazil, divided in control group (26 subjects), moderate periodontal disease group (26 subjects) and severe periodontal disease group (25 subjects). All subjects answered self-referenced questionnaires, underwent periodontal clinical examinations and allowed the collection of oral mucosa cells for the BMCyt. In relation to DNA damage biomarkers (micronuclei (MN) and/ or nuclear buds (NBUD)), our results indicated no increase in MN frequencies (p > 0.05), however it indicated significant difference in NBUD frequencies between groups (p < 0.024). This result suggests that the periodontal disease status may influence DNA damage. Regarding the other nuclear/cellular abnormalities, was observed a significant difference in the binucleated (BN) frequencies between groups (p < 0.05). Moreover, the periodontitis severity was associated to an increase in the combined (summed) frequency of cells with different levels of DNA damage (MN and/or NBUD), cytokinetic defects (BN cells) and/or cell death (karyorrhexis, pyknotic and karyolytic cells) (r = 0.235; p = 0.040). Periodontal disease depending on its severity, induces nuclear anomalies in buccal cells.
1. Introduction Periodontitis belongs to a group of inflammatory diseases characterized by gingival bleeding, periodontal pocket formation, insertion connective tissue destruction, resorption of the periodontal alveolar bone surface and, eventually, tooth loss [1]. In its bacterial pathogenesis antigens modulate the host response, inducing the increase of proinflammatory cytokines [2] which contribute for soft and hard periodontal tissue destruction, as well as for the tooth mobility and loss [3]. It is also well known that inflammation may induce reactive oxygen species generation [4] and DNA damage [5] which relation to increased cancer risk is well documented. Therefore, to monitor subjects with different periodontal disease stages might be relevant to set up strategies aiming to reduce the risk of developing oral cancer. Buccal mucosa is a suitable tissue for studying the genomic damage
biomarkers as well as the proliferative potential and the cellular death [6]. BMCyt assay is a well validated cancer biomarker used to demonstrate cytogenetic effects of environmental and occupational exposure to genotoxic agents [7] as periodontal disease [5,8,9]. The BMCyt assay has been used to measure DNA damage biomarkers (micronuclei (MN) and/or nuclear buds (NBUD), cytokinetic defects (binucleated (BN) cells), proliferative potential (basal cell frequency) and/ or cell death (condensed chromatin, karyorrhexis, pyknotic and karyolytic cells) [7]. Since DNA damage represents a critical event associated not only to the initiation phase, but also to the promotion and progression phases of the carcinogenesis effects [9], it is interesting to investigate whether the periodontitis can really induce chromosomal damage and, also, if the diagnostic of this condition, according to its severity, can be used as a genomic stability biomarker. The aim of this study was to evaluate the periodontal diseases effect,
⁎ Corresponding author at: Graduate Program in Health Promotion, University of Santa Cruz do Sul (UNISC), Avenida Independência 2293, sala 4206, Bairro Universitário, Santa Cruz do Sul, RS, 96815-900, Brazil. E-mail address: [email protected] (S.I.R. Franke).
https://doi.org/10.1016/j.mrgentox.2019.01.005 Received 29 December 2017; Received in revised form 8 January 2019; Accepted 16 January 2019 Available online 17 January 2019 1383-5718/ © 2019 Elsevier B.V. All rights reserved.
Mutat Res Gen Tox En 840 (2019) 20–23
T.T. Borba, et al.
shaken into a microtube containing 1 mL of methanol. The cytobrush was discarded from the microtubes and 20 μL of DMSO were added and microtube was centrifuged at 3500 rpm for 3 min. Subsequently, 200 μL of supernatant were aspirated off and more 200 μL of methanol were added and with the aid of the pipette tip the cells were dissociated. This procedure (aspirate off 200 μL of supernatant, addition of 200 μL of methanol and cells dissociation) was repeated 3 times. In the final step, 400 μL of supernatant were discarded, and 100 μL of the remaining cell suspension were distributed onto cleaned microscope slides (2 slides for individual). After air-dry for at least 12 h, the slides were treated with HCl and Schiff’s reagent, let dry overnight and, then stained according to the Feulgen method [7]. The slides were later analyzed using a conventional optical microscope at 400× magnification (Leica DMLB®, Wetzlar, Germany). The scoring criteria of Thomas et al. [7] to assess the distinct cell types and nuclear/cellular abnormalities was applied. Cell types were grouped into categories that distinguish normal cells from abnormal cells classified according to their cytological and nuclear features: indicative of DNA damage (MN and/or NBUD); cytokinetic defects (BN cells); and/or cell death (karyorrhexis, pyknotic and karyolytic cells). A total of 2000 differentiated cells were evaluated for the presence of DNA damage and a score of 1000 cells was evaluated to determine the frequency of other nuclear anomalies. Both results are expressed as counts per 1000 cells. Microscope slides were coded to allow a blindfolded analysis, making not possible to the scorer to be aware of the subject neither the study group to which it belonged. The slides analysis was carried out by a single examiner (2 slides per subject).
according to its severity, on the occurrence of DNA damage and other nuclear anomalies, determined by BMCyt. 2. Materials and methods 2.1. Study design and patients This is a cross-sectional study approved by the ethics research committee of UNISC (CAAE number 49793615.9.0000.5343) carried out with volunteers subjects aged between 40 to 70 years from both genders. Subjects were attended at the dentistry course clinic (UNISC) in Santa Cruz do Sul, State of Rio Grande do Sul, Brazil between March to July, 2016. For sample size calculation we considered that a minimum of 20 subjects in each group would be enough to evaluate differences with 95 % confidence interval as reported for previous studies [10,11]. Besides that, foreseeing possible dropouts we selected 77 subjects which were distributed in 3 groups: healthy periodontium (control group: 26 subjects); moderate chronic periodontal disease (26 subjects); and severe chronic periodontal disease (25 subjects). Pregnant women, subjects who had lost or gained ≥ 5 Kg in last 6 months, as well as subjects with previous history of cancer, chemotherapy or radiotherapy were excluded from the study. Due to influence in DNA damage levels, individuals recently exposed (less than 2 weeks) to xrays were not included in the sample. Moreover, as much as possible individuals were enrolled in present the study and have their samples collected previously to undergo to any dental treatment associated to their visit to the clinics (e.g. endodontics, orthodontics and/or prosthetics). All subjects signed a free and informed consent term according to Resolution 466/12 of National Health Council (Brazil) previously to the enrollment in the study. Each subject was identified by a code to ensure the impartial analyzes of tests results and respect privacy.
2.5. Statistical analyses Data were analyzed with software Statistical Package for Social Sciences (SPSS, Inc., ArmonK, NY, USA, v. 20.0). Characteristics of participants and risk factors were presented with frequency distributions for categorical variables using the χ2 test. Kruskal–Wallis’s test followed by Dunn’s test and Spearman’s test were employed in the variable analyzes. Statistical significance was considered when p < 0.05.
2.2. Patient-reported outcomes A self-reported questionnaire was applied with questions regarding demographics, medicines usage history, smoke (yes or no) and alcoholic drinking frequency (less or more than twice a week). Anthropometric data for nutritional status classification was also collected and calculated according to World Health Organization [Body mass index (BMI) < 18.5 Kg/m2 (low weight); BMI ≥ 18.5 and until 24.9 kg/m2 (eutrophic); BMI ≥ 25.0 and until 29.9 kg/m2 (overweight) and BMI ≥ 30.0 kg/m2 (obese)] [12].
3. Results 77 subjects with the mean age of 52.32 ± 6.86 years, who were predominately women (53%), participated in this study. Main characteristics of the subjects (gender, age group, alcoholic drink, smoking and BMI) are presented in the Table 1. The mean MN frequencies for all subjects was 2.44 ± 0.95. When evaluating the frequency of the different DNA damage and other nuclear/cellular abnormalities in relation to the general profile, the subjects’ lifestyle and clinical measurements, we did not find significant differences regarding smoking (p = 0.15), overweight/obesity (p = 0.43), and alcoholic drink (p = 0.37). Table 2 presents the BMCyt results according to the periodontal diagnosis. It was possible to observe a significant difference regarding DNA damage only for NBUD frequencies (p = 0.024). Concerning the nuclear/cellular abnomalies, only BN frequencies differed significantly among the study groups (p = 0.023). We also found a significant increase in the sum of MN and nuclear anomalies (total number of damage) along with the increase in the periodontitis severity (p = 0.040; Fig. 1).
2.3. Clinical measurements All subjects underwent a periodontal clinical examination performed by a dentist who was the only examiner to perform the probing depth tests, registering signs of bleeding on probing and insertion loss of all teeth, six sites per tooth (distobuccal, mid-vestibular, mesio-vestibular, mesio-lingual, middle-lingual and disto-lingual), by using a millimeter periodontal probe with the following markings: 1– 10 mm (F.O.A 23 W - Neumar®). We established as having healthy periodontum (control subjects) those without inflammatory signs and with probing depth of until 3 or 4 mm. Subjects with inflammatory signs, that is, with bleeding and loss of periodontal insertion ≥5 mm in 15– 50% of teeth were considered with moderate periodontal disease. The subjects with bleeding and loss of periodontal insertion ≥5 mm in ≥50% of teeth were classified as severe periodontal disease [13]. We performed an intra-examiner reproducibility check through which 5% of test were reexamined, getting an agreement of 91% of global values [14].
4. Discussion Periodontal disease is considered an inflammatory disorder that has been associated with the ROS production. This evidence has been related with the periodontal tissues destruction by ROS, promoting an imbalance of defense mechanism against bacterial invasion and leading to an increase in the ROS levels in periodontal tissue [15]. Moreover,
2.4. Sample Collection and BMCyt assay The BMCyt assay was adapted of the protocol of Thomas et al. [7]. Firstly, oral mucosa cells were collected with the help of cervical brush (cytobrush) from the oral epithelium of both cheeks and the brush was 21
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T.T. Borba, et al.
Table 1 Main characteristics of the subjects according to the periodontal diagnosis (n = 77). Level of significance according to the χ2 test. Periodontal diagnosis
n (77) Gender Male Female Age group 40–49 50–59 60–70 Smoking No Yes Alcoholic drink No Yes BMI Low weight Eutrophic Overweight/obesity
P Value
Healthy periodontium
Moderate periodontal disease
Severe periodontal disease
26 (33 %)
26 (33 %)
25 (32 %)
10 (27 %) 16 (39 %)
13 (36 %) 13 (31 %)
13 (36 %) 12 (29 %)
10 (37 %) 13 (34 %) 3 (25 %)
11 (40 %) 10 (26 %) 5 (41 %)
6 (22 %) 15 (39 %) 4 (33 %)
23 (36 %) 3 (21 %)
20 (31 %) 6 (42 %)
20(31 %) 5 (35 %)
25 (36 %) 1 (11 %)
20 (29 %) 6 (66 %)
23 (33 %) 2 (22 %)
0 (0 %) 13 (44 %) 13 (28 %)
1 (50 %) 10 (34 %) 15 (32 %)
1 (50 %) 6 (20 %) 18 (39 %)
0.57
0.55
0.53
0.07
0.35
genotoxic effects of lifestyle factors [7]. It should be stressed, however, that BMCyt assay evaluates DNA damage (MN and/or NBUD) as well as evaluates cells with meiotic defects (BN cells) and cell death (karyorrhexis, and pyknotic and karyolitic cells). Therefore, in the present study, in addition to assessing the association between the status of periodontitis with DNA damage, we evaluated this same association also for other nuclear/cellular abnormalities. Our results indicate that there was no association between the periodontal disease status and MN f frequencies, showing very similar MN frequencies among the groups (2.52 ± 1.05 versus 2.54 ± 1.07 versus 2.28 ± 0.71 for the healthy periodontium group and moderate and severe periodontal disease, respectively). Different from our study, some studies have shown higher levels of DNA damage in individuals with periodontal disease than control individuals [5,8,9]. On the other hand, another study from Brazil did not indicate statistically significant differences in the occurrence of MN in individuals with gingivitis, periodontitis in relation to control groups [10]. Notwithstanding, given the small number of studies evaluating inflammation and DNA damage [5,8–10] much more effort has to be applied to better understand the impact of inflammation and DNA damage in oral mucosa, particularly applying the BMCyt assay. Nevertheless, still assessing DNA damage, our findings suggest a significant association between periodontal status and cells with NBUD, showing an increase of 1.2-fold of NBUD frequencies in severe periodontal individuals than healthy periodontium individuals. This aspects suggests a correlation between DNA damage and the periodontal disease status. Similar results were found in other studies indicating that individuals with periodontal disease had a greater amount of DNA damage (NBUD) than the spontaneous levels of damage that occur in individuals without periodontitis [5,9]. We must also take account that DNA damage could be related to biomaterials used in endodontic treatment, such as root canal sealers, teeth restoration, and prosthetics [5]. It is important to stress that we tried to avoid as much as possible to enroll in study individuals undergoing recent dental treatment, however given the longer term nature of dental treatment it would be impossible to control the influence of the afore mentioned aspects on periodontitis associated DNA damage. Indeed, in our research, individuals with previous dental treatment were not excluded which could have influenced ours results. However, we did not consider this as a severe limitation given most studies do not consider these influences and most individuals are likely to have a lot of dental treatments along their lives. In addition, to direct inflammation effect over DNA damage, it has
Table 2 Periodontal disease effect on frequencies cell with DNA damage and other nuclear/cellular abnormalities according to the Buccal Micronucleus Cytome Assay (n = 77). Type of damage
Healthy periodontium (n = 26)
Moderate periodontal disease (n = 26) Mean ± Standard deviation
Severe periodontal disease (n = 25)
p Value
MN frequency (‰) NBUD (‰) BN (‰) PYK (‰) KYL (‰) KHC (‰)
2.52 2.13 2.46 3.58 3.23 2.15
2.28 2.68 3.22 4.14 3.48 2.40
0.785 0.024 0.023 0.299 0.514 0.685
± ± ± ± ± ±
1.05 1.09 1.24 1.21 1.42 1.05
2.54 1.98 2.73 3.73 3.65 2.31
± ± ± ± ± ±
1.07 0.93 1.23 1.31 1.27 1.26
± ± ± ± ± ±
0.71 0.88 1.44* 1.43 1.56 0.78
Key to Table. MN: Micronuclei; NBUD: Nuclear buds; BN: Binucleated cells; PYK: Pyknotic cells; KYL: Karyorrhectic cells; KHC: Karyolytic cells; * Statistically significant at p < 0.05 with Kruskal–Wallis test followed by Dunn’s test in comparison to healthy periodontium.
Fig. 1. Correlation between periodontal disease severity and the total number of damages (sum of MN and nuclear anomalies). r: correlation coefficient; p: significance level according to Spearman’ test.
ROS are capable to produce DNA damage through the genetic mutations introduction and structural changes in DNA [16]. During the last 10 years, BMCyt assay has been used to show the 22
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Declaration of interest
been suggested that the interaction between periodontal pathogenic bacteria and the mucosa might potentiate the DNA damage formation. In this sense, volatile sulfur compounds produced by Gram-negative anaerobes, especially Agregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia, increase the oral mucosa permeability [18] making it more prone to inflamation. However, we, as well as other authors [9], indicated that periodontal disease could be a risk factor for DNA damage, and it could be a modifiable risk factor once that periodontitis could be prevented and treated. Bloching et al. [8] suggests that the MN induction associated with periodontitis has been more related with a primary microbial rather than inflammatory reactions, an explanation for the non-occurrence of this association in our research. However, it is known that inflammatory processes are associated with repeated cell divisions and consequent chromosomal alterations that, in turn, stimulate apoptosis [10]. Consequently, it is essential to evaluate the occurrences of other nuclear anomalies evaluated by BMCyt assay. Regarding the cytotoxicity parameters, in the present study, only BN frequencies increase was associated with periodontitis. In the severe periodontal group, we verified BN frequencies was 1.4-fold higher compared with the healthy periodontium group, indicating that individuals with periodontal disease had a greater risk of cytokinetic defects than in individuals without periodontitis, and at least partially induced cytogenetic damage. The other cytotoxicity parameters did not exhibit significant differences among the groups. However, in the severe periodontal group, karyorrhectic cells increased 1.2-fold, pyknosis cells increased 1.2-fold and karyolysis cells increased 1.1-fold than healthy periodontium group. Zamora-Perez et al. [9] unlike our study, found significant differences between chronic and aggressive periodontitis groups than control group to nuclear abnormalities, including BN cells, karyolitic, karyorrhectic and pyknotic cells. In the present study, the total number of nuclear/cellular damages evaluated (DNA damage, chromosomal instability and cell death) was significantly associated with periodontitis severity, pointing to genotoxic effects associated with the inflammatory process and may suggest that this process could have masked the occurrence of DNA damage (MN frequencies) by elimination of genetically-impaired cells. In addition, periodontitis is common in human populations and represent a significant public health problem [8]. Periodontal diseases, in its chronic and aggressive forms, could lead to a severe alteration of the periodontal tissues and tooth loss [1,19] and genotoxicity [15,20,21]. Even though the assay has been conducted through many years by several laboratories across the globe, the efforts to standardize the scoring have only started very recently. If we compare the average frequency of the markers evaluated in our study with those of Bolognesi et al. [22], we can observe that most are in the same order of magnitude, with the exception being BN frequencies (substantially lower in our study). It is important to mention that conversely to our results the results of Bolognesi et al. [22] includes both healthy controls and patients with cancer, therefore making the comparison difficult. Further studies aiming to cross-calibrate labs are needed for the assay. In conclusion, the findings indicate that the inflammatory process generated by gingivitis and periodontitis is not related with MN frequencies. However, in individuals with periodontitis, a higher occurrence of NBUD and BN cells is an indicative of genotoxic effects of periodontal infection. The occurrence of DNA damage and other nuclear/cellular abnormalities (chromosomal instability and cell death) evaluated in oral mucosa cells and their relationship with periodontal disease is an important research field, being relevant to control this disease and to promote the importance of good oral health. At the same time, it should be considered that DNA damage is a critical event not only in the initiation phase but also in the promotion and progression phases, which could be related to carcinogenesis events. However, additional investigations such as intervention studies are necessary in order to evaluate the periodontal therapy effects in the occurrence of nuclear changes.
The authors declare that there is no conflict of interest. Acknowledgement We thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and to Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) by the scholarships provided. References [1] P.N. Papapanou, Epidemiology of periodontal diseases: an update, J. Int. Acad. Periodontol. 14 (1999) 110–116. [2] R.C. Page, K.S. Kornman, The pathogenesis of human periodontitis: an introduction, Periodontology 2000 14 (1997) 9–11, https://doi.org/10.1111/j.1600-0757.1997. tb00189.x. [3] S.C. Corbi, A.S. Bastos, S.R. Orrico, R. Secolin, R.A. Dos Santos, C.S. Takahashi, R.M. Scarel-Caminaga, Elevated micronucleus frequency in patients with type 2 diabetes, dyslipidemia and periodontitis, Mutagenesis 29 (2014) 433–439, https://doi.org/10. 1093/mutage/geu043. [4] P. Celec, L. Tóthová, Oxidative stress and antioxidants in the diagnosis and therapy of periodontitis, Front. Physiol. 14 (8) (2017) 1055, https://doi.org/10.3389/fphys.2017. 01055. [5] D. Bastos-Aires, Á. Azevedo, M. de Lurdes Pereira, D. Pérez-Mongiovi, A. Teixeira, Preliminary study of micronuclei levels in oral exfoliated cells from patients with periodontitis, J. Dent. Sci. 8 (2013) 200–204, https://doi.org/10.1016/j.jds.2012.12.007. [6] N. Holland, C. Bolognesi, M. Kirsch-Volders, S. Bonassi, E. Zeiger, S. Knasmueller, M. Fenech, The micronucleus assay in human buccal cells as a tool for biomonitoring DNA damage: the HUMN project perspective on current status and knowledge gaps, Mutat. Res. 659 (2008) 93–108, https://doi.org/10.1016/j.mrrev.2008.03.007. [7] P. Thomas, N. Holland, C. Bolognesi, M. Kirsch-Volders, S. Bonassi, E. Zeiger, S. Knasmueller, M. Fenech, Buccal micronucleus cytome assay, Nat. Protoc. 4 (2009) 825–837, https://doi.org/10.1038/nprot.2009.53. [8] M. Bloching, W. Reich, J. Schubert, T. Grummt, A. Sandner, Micronucleus rate of buccal mucosal epithelial cells in relation to oral hygiene and dental factors, Oral Oncol. 44 (2008) 220–226, https://doi.org/10.1016/j.oraloncology.2007.02.002. [9] A.L. Zamora-Perez, Y.M. Ortiz-García, B.P. Lazalde-Ramos, C. Guerrero-Velázquez, B.C. Gómez-Meda, M.Á. Ramírez-Aguilar, G.M. Zúñiga-González, Increased micronuclei and nuclear abnormalities in buccal mucosa and oxidative damage in saliva from patients with chronic and aggressive periodontal diseases, J. Periodontal. Res. 50 (2015) 28–36, https://doi.org/10.1111/jre.12175. [10] P.D. Brandão, I.S. Gomes-Filho, S.S. Cruz, J.D. Passos-Soares, S.C. Trindade, L.D. Souza, J.R. Meireles, E.D. Cerqueira, Can periodontal infection induce genotoxic effects? Acta. Odontol. Scand. 73 (2015) 219–225, https://doi.org/10.3109/00016357.2014.982705. [11] F. D’Agostini, E. Calcagno, R.T. Micale, S. La Maestra, S. De Flora, L. Cingano, Cytogenetic analysis of gingival epithelial cells, as related to smoking habits and occurrence of periodontal disease, Int. J. Hyg. Environ. Health 216 (2013) 71–75, https://doi.org/10.1016/ j.ijheh.2012.01.005. [12] World Health Organization, Diet, Nutrition And The Prevention Of Chronic Diseases: Report of a WHO Study Group [Meeting Held in Geneva from 6–13 March 1989] Geneva, World Health Organization, 1990 (Accessed 13 March 2003–17), http://www.who.int/ iris/handle/10665/39426. [13] C. Susin, C.F. Dalla Vecchia, R.V. Oppermann, O. Haugejorden, J.M. Albandar, Periodontal attachment loss in an urban population of Brazilian adults: effect of demographic, behavioral, and environmental risk indicators, J. Periodontol. 75 (2004) 1033–1041, https://doi.org/10.1902/jop.2004.75.7.1033. [14] World Health Organization, Calibration of Examiners for Oral Health Epidemiological Surveys, World Health Organization, Geneva, 1993. [15] I.L. Chapple, J.B. Matthews, The role of reactive oxygen and antioxidant species in peiodontal tissue destruction, Periodontology 2000 43 (2007) 160–232, https://doi.org/ 10.1111/j.1600-0757.2006.00178.x. [16] S. Reuter, S.C. Gupta, M.M. Chaturvedi, B.B. Aggarwal, Oxidative stress, inflammation, and cancer: how are they linked? Free. Radic. Biol. Med. 49 (2010) 1603–1616, https:// doi.org/10.1016/j.freeradbiomed.2010.09.006. [18] M. Bloching, W. Reich, J. Schubert, T. Grummt, A. Sandner, Micronucleus rate of buccal mucosal epithelial cells in relation to oral hygiene and dental factors, Oral Oncol. 44 (2008) 220–226, https://doi.org/10.1016/j.oraloncology.2007.02.002. [19] G.C. Armitage, Periodontal diagnoses and classification of periodontal diseases, Periodontology 2000 34 (2004) 9–21. [20] C.F. Çanakçi, V. Çanakçi, A. Tatar, A. Eltas, U. Sezer, Y. Çiçek, S. Oztas, Increased salivary level of 8-hydroxydeoxyguanosine is a marker of premature oxidative mitochondrial DNA damage in gingival tissue of patients with periodontitis, Arch. Immunol. Ther. Exp. 57 (2009) 205–211, https://doi.org/10.1007/s00005-009-0026-9. [21] H. Su, M. Gornitsky, A.M. Velly, H. Yu, M. Benarroch, H.M. Schipper, Salivary DNA, lipid, and protein oxidation in nonsmokers with periodontal disease, Free Radic. Biol. Med. 46 (2009) 914–921, https://doi.org/10.1016/j.freeradbiomed.2009.01.008. [22] C. Bolognesi, S. Knasmueller, A. Nersesyan, P. Roggieri, M. Ceppi, M. Bruzzone, E. Blaszczyk, D. Mielzynska-Svach, M. Milic, S. Bonassi, D. Benedetti, Inter-laboratory consistency and variability in the buccal micronucleus cytome assay depends on biomarker scored and laboratory experience: results from the HUMNxl international interlaboratory scoring exercise, Mutagenesis 32 (2017) 257–266, https://doi.org/10.1093/ mutage/gew047.
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Contents lists available at ScienceDirect
Mutat Res Gen Tox En journal homepage: www.elsevier.com/locate/gentox
Periodontitis: Genomic instability implications and associated risk factors a
a,b,c
b
b
T a
Tatiana T. Borba , Patrícia Molz , Diene S. Schlickmann , Caroline Santos , Caio F. Oliveira , ⁎ Daniel Práa, Léo Kreather Netoa,d, Silvia I.R. Frankea,b, a
Graduate Program in Health Promotion, University of Santa Cruz do Sul (UNISC), Santa Cruz do Sul, RS, Brazil Laboratory of Experimental Nutrition, University of Santa Cruz do Sul (UNISC), Santa Cruz do Sul, RS, Brazil Graduate Program in Medicine and Health Sciences, Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil d Department of Nursing and Dentistry, University of Santa Cruz do Sul (UNISC), Santa Cruz do Sul, RS, Brazil b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Periodontal disease severity Buccal Micronucleus Cytome DNA damage Nuclear cellular abnormalities
Periodontitis is a bacterial infection characterized by the presence of a dense inflammatory infiltrate, which may result in increased DNA damage and other nuclear/cellular abnormalities. Therefore, it is important to evaluate the periodontal diseases influence on DNA damage and other nuclear/cellular abnomalies formation as cancer risk markers. Thus, the aim of this study was to evaluate the periodontal diseases effect, according to its severity, on the occurrence of DNA damage and other nuclear/cellular abnormalities. This is a cross-sectional study with 77 subjects from the dentistry clinic of the University of Santa Cruz do Sul, Brazil, divided in control group (26 subjects), moderate periodontal disease group (26 subjects) and severe periodontal disease group (25 subjects). All subjects answered self-referenced questionnaires, underwent periodontal clinical examinations and allowed the collection of oral mucosa cells for the BMCyt. In relation to DNA damage biomarkers (micronuclei (MN) and/ or nuclear buds (NBUD)), our results indicated no increase in MN frequencies (p > 0.05), however it indicated significant difference in NBUD frequencies between groups (p < 0.024). This result suggests that the periodontal disease status may influence DNA damage. Regarding the other nuclear/cellular abnormalities, was observed a significant difference in the binucleated (BN) frequencies between groups (p < 0.05). Moreover, the periodontitis severity was associated to an increase in the combined (summed) frequency of cells with different levels of DNA damage (MN and/or NBUD), cytokinetic defects (BN cells) and/or cell death (karyorrhexis, pyknotic and karyolytic cells) (r = 0.235; p = 0.040). Periodontal disease depending on its severity, induces nuclear anomalies in buccal cells.
1. Introduction Periodontitis belongs to a group of inflammatory diseases characterized by gingival bleeding, periodontal pocket formation, insertion connective tissue destruction, resorption of the periodontal alveolar bone surface and, eventually, tooth loss [1]. In its bacterial pathogenesis antigens modulate the host response, inducing the increase of proinflammatory cytokines [2] which contribute for soft and hard periodontal tissue destruction, as well as for the tooth mobility and loss [3]. It is also well known that inflammation may induce reactive oxygen species generation [4] and DNA damage [5] which relation to increased cancer risk is well documented. Therefore, to monitor subjects with different periodontal disease stages might be relevant to set up strategies aiming to reduce the risk of developing oral cancer. Buccal mucosa is a suitable tissue for studying the genomic damage
biomarkers as well as the proliferative potential and the cellular death [6]. BMCyt assay is a well validated cancer biomarker used to demonstrate cytogenetic effects of environmental and occupational exposure to genotoxic agents [7] as periodontal disease [5,8,9]. The BMCyt assay has been used to measure DNA damage biomarkers (micronuclei (MN) and/or nuclear buds (NBUD), cytokinetic defects (binucleated (BN) cells), proliferative potential (basal cell frequency) and/ or cell death (condensed chromatin, karyorrhexis, pyknotic and karyolytic cells) [7]. Since DNA damage represents a critical event associated not only to the initiation phase, but also to the promotion and progression phases of the carcinogenesis effects [9], it is interesting to investigate whether the periodontitis can really induce chromosomal damage and, also, if the diagnostic of this condition, according to its severity, can be used as a genomic stability biomarker. The aim of this study was to evaluate the periodontal diseases effect,
⁎ Corresponding author at: Graduate Program in Health Promotion, University of Santa Cruz do Sul (UNISC), Avenida Independência 2293, sala 4206, Bairro Universitário, Santa Cruz do Sul, RS, 96815-900, Brazil. E-mail address: [email protected] (S.I.R. Franke).
https://doi.org/10.1016/j.mrgentox.2019.01.005 Received 29 December 2017; Received in revised form 8 January 2019; Accepted 16 January 2019 Available online 17 January 2019 1383-5718/ © 2019 Elsevier B.V. All rights reserved.
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shaken into a microtube containing 1 mL of methanol. The cytobrush was discarded from the microtubes and 20 μL of DMSO were added and microtube was centrifuged at 3500 rpm for 3 min. Subsequently, 200 μL of supernatant were aspirated off and more 200 μL of methanol were added and with the aid of the pipette tip the cells were dissociated. This procedure (aspirate off 200 μL of supernatant, addition of 200 μL of methanol and cells dissociation) was repeated 3 times. In the final step, 400 μL of supernatant were discarded, and 100 μL of the remaining cell suspension were distributed onto cleaned microscope slides (2 slides for individual). After air-dry for at least 12 h, the slides were treated with HCl and Schiff’s reagent, let dry overnight and, then stained according to the Feulgen method [7]. The slides were later analyzed using a conventional optical microscope at 400× magnification (Leica DMLB®, Wetzlar, Germany). The scoring criteria of Thomas et al. [7] to assess the distinct cell types and nuclear/cellular abnormalities was applied. Cell types were grouped into categories that distinguish normal cells from abnormal cells classified according to their cytological and nuclear features: indicative of DNA damage (MN and/or NBUD); cytokinetic defects (BN cells); and/or cell death (karyorrhexis, pyknotic and karyolytic cells). A total of 2000 differentiated cells were evaluated for the presence of DNA damage and a score of 1000 cells was evaluated to determine the frequency of other nuclear anomalies. Both results are expressed as counts per 1000 cells. Microscope slides were coded to allow a blindfolded analysis, making not possible to the scorer to be aware of the subject neither the study group to which it belonged. The slides analysis was carried out by a single examiner (2 slides per subject).
according to its severity, on the occurrence of DNA damage and other nuclear anomalies, determined by BMCyt. 2. Materials and methods 2.1. Study design and patients This is a cross-sectional study approved by the ethics research committee of UNISC (CAAE number 49793615.9.0000.5343) carried out with volunteers subjects aged between 40 to 70 years from both genders. Subjects were attended at the dentistry course clinic (UNISC) in Santa Cruz do Sul, State of Rio Grande do Sul, Brazil between March to July, 2016. For sample size calculation we considered that a minimum of 20 subjects in each group would be enough to evaluate differences with 95 % confidence interval as reported for previous studies [10,11]. Besides that, foreseeing possible dropouts we selected 77 subjects which were distributed in 3 groups: healthy periodontium (control group: 26 subjects); moderate chronic periodontal disease (26 subjects); and severe chronic periodontal disease (25 subjects). Pregnant women, subjects who had lost or gained ≥ 5 Kg in last 6 months, as well as subjects with previous history of cancer, chemotherapy or radiotherapy were excluded from the study. Due to influence in DNA damage levels, individuals recently exposed (less than 2 weeks) to xrays were not included in the sample. Moreover, as much as possible individuals were enrolled in present the study and have their samples collected previously to undergo to any dental treatment associated to their visit to the clinics (e.g. endodontics, orthodontics and/or prosthetics). All subjects signed a free and informed consent term according to Resolution 466/12 of National Health Council (Brazil) previously to the enrollment in the study. Each subject was identified by a code to ensure the impartial analyzes of tests results and respect privacy.
2.5. Statistical analyses Data were analyzed with software Statistical Package for Social Sciences (SPSS, Inc., ArmonK, NY, USA, v. 20.0). Characteristics of participants and risk factors were presented with frequency distributions for categorical variables using the χ2 test. Kruskal–Wallis’s test followed by Dunn’s test and Spearman’s test were employed in the variable analyzes. Statistical significance was considered when p < 0.05.
2.2. Patient-reported outcomes A self-reported questionnaire was applied with questions regarding demographics, medicines usage history, smoke (yes or no) and alcoholic drinking frequency (less or more than twice a week). Anthropometric data for nutritional status classification was also collected and calculated according to World Health Organization [Body mass index (BMI) < 18.5 Kg/m2 (low weight); BMI ≥ 18.5 and until 24.9 kg/m2 (eutrophic); BMI ≥ 25.0 and until 29.9 kg/m2 (overweight) and BMI ≥ 30.0 kg/m2 (obese)] [12].
3. Results 77 subjects with the mean age of 52.32 ± 6.86 years, who were predominately women (53%), participated in this study. Main characteristics of the subjects (gender, age group, alcoholic drink, smoking and BMI) are presented in the Table 1. The mean MN frequencies for all subjects was 2.44 ± 0.95. When evaluating the frequency of the different DNA damage and other nuclear/cellular abnormalities in relation to the general profile, the subjects’ lifestyle and clinical measurements, we did not find significant differences regarding smoking (p = 0.15), overweight/obesity (p = 0.43), and alcoholic drink (p = 0.37). Table 2 presents the BMCyt results according to the periodontal diagnosis. It was possible to observe a significant difference regarding DNA damage only for NBUD frequencies (p = 0.024). Concerning the nuclear/cellular abnomalies, only BN frequencies differed significantly among the study groups (p = 0.023). We also found a significant increase in the sum of MN and nuclear anomalies (total number of damage) along with the increase in the periodontitis severity (p = 0.040; Fig. 1).
2.3. Clinical measurements All subjects underwent a periodontal clinical examination performed by a dentist who was the only examiner to perform the probing depth tests, registering signs of bleeding on probing and insertion loss of all teeth, six sites per tooth (distobuccal, mid-vestibular, mesio-vestibular, mesio-lingual, middle-lingual and disto-lingual), by using a millimeter periodontal probe with the following markings: 1– 10 mm (F.O.A 23 W - Neumar®). We established as having healthy periodontum (control subjects) those without inflammatory signs and with probing depth of until 3 or 4 mm. Subjects with inflammatory signs, that is, with bleeding and loss of periodontal insertion ≥5 mm in 15– 50% of teeth were considered with moderate periodontal disease. The subjects with bleeding and loss of periodontal insertion ≥5 mm in ≥50% of teeth were classified as severe periodontal disease [13]. We performed an intra-examiner reproducibility check through which 5% of test were reexamined, getting an agreement of 91% of global values [14].
4. Discussion Periodontal disease is considered an inflammatory disorder that has been associated with the ROS production. This evidence has been related with the periodontal tissues destruction by ROS, promoting an imbalance of defense mechanism against bacterial invasion and leading to an increase in the ROS levels in periodontal tissue [15]. Moreover,
2.4. Sample Collection and BMCyt assay The BMCyt assay was adapted of the protocol of Thomas et al. [7]. Firstly, oral mucosa cells were collected with the help of cervical brush (cytobrush) from the oral epithelium of both cheeks and the brush was 21
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Table 1 Main characteristics of the subjects according to the periodontal diagnosis (n = 77). Level of significance according to the χ2 test. Periodontal diagnosis
n (77) Gender Male Female Age group 40–49 50–59 60–70 Smoking No Yes Alcoholic drink No Yes BMI Low weight Eutrophic Overweight/obesity
P Value
Healthy periodontium
Moderate periodontal disease
Severe periodontal disease
26 (33 %)
26 (33 %)
25 (32 %)
10 (27 %) 16 (39 %)
13 (36 %) 13 (31 %)
13 (36 %) 12 (29 %)
10 (37 %) 13 (34 %) 3 (25 %)
11 (40 %) 10 (26 %) 5 (41 %)
6 (22 %) 15 (39 %) 4 (33 %)
23 (36 %) 3 (21 %)
20 (31 %) 6 (42 %)
20(31 %) 5 (35 %)
25 (36 %) 1 (11 %)
20 (29 %) 6 (66 %)
23 (33 %) 2 (22 %)
0 (0 %) 13 (44 %) 13 (28 %)
1 (50 %) 10 (34 %) 15 (32 %)
1 (50 %) 6 (20 %) 18 (39 %)
0.57
0.55
0.53
0.07
0.35
genotoxic effects of lifestyle factors [7]. It should be stressed, however, that BMCyt assay evaluates DNA damage (MN and/or NBUD) as well as evaluates cells with meiotic defects (BN cells) and cell death (karyorrhexis, and pyknotic and karyolitic cells). Therefore, in the present study, in addition to assessing the association between the status of periodontitis with DNA damage, we evaluated this same association also for other nuclear/cellular abnormalities. Our results indicate that there was no association between the periodontal disease status and MN f frequencies, showing very similar MN frequencies among the groups (2.52 ± 1.05 versus 2.54 ± 1.07 versus 2.28 ± 0.71 for the healthy periodontium group and moderate and severe periodontal disease, respectively). Different from our study, some studies have shown higher levels of DNA damage in individuals with periodontal disease than control individuals [5,8,9]. On the other hand, another study from Brazil did not indicate statistically significant differences in the occurrence of MN in individuals with gingivitis, periodontitis in relation to control groups [10]. Notwithstanding, given the small number of studies evaluating inflammation and DNA damage [5,8–10] much more effort has to be applied to better understand the impact of inflammation and DNA damage in oral mucosa, particularly applying the BMCyt assay. Nevertheless, still assessing DNA damage, our findings suggest a significant association between periodontal status and cells with NBUD, showing an increase of 1.2-fold of NBUD frequencies in severe periodontal individuals than healthy periodontium individuals. This aspects suggests a correlation between DNA damage and the periodontal disease status. Similar results were found in other studies indicating that individuals with periodontal disease had a greater amount of DNA damage (NBUD) than the spontaneous levels of damage that occur in individuals without periodontitis [5,9]. We must also take account that DNA damage could be related to biomaterials used in endodontic treatment, such as root canal sealers, teeth restoration, and prosthetics [5]. It is important to stress that we tried to avoid as much as possible to enroll in study individuals undergoing recent dental treatment, however given the longer term nature of dental treatment it would be impossible to control the influence of the afore mentioned aspects on periodontitis associated DNA damage. Indeed, in our research, individuals with previous dental treatment were not excluded which could have influenced ours results. However, we did not consider this as a severe limitation given most studies do not consider these influences and most individuals are likely to have a lot of dental treatments along their lives. In addition, to direct inflammation effect over DNA damage, it has
Table 2 Periodontal disease effect on frequencies cell with DNA damage and other nuclear/cellular abnormalities according to the Buccal Micronucleus Cytome Assay (n = 77). Type of damage
Healthy periodontium (n = 26)
Moderate periodontal disease (n = 26) Mean ± Standard deviation
Severe periodontal disease (n = 25)
p Value
MN frequency (‰) NBUD (‰) BN (‰) PYK (‰) KYL (‰) KHC (‰)
2.52 2.13 2.46 3.58 3.23 2.15
2.28 2.68 3.22 4.14 3.48 2.40
0.785 0.024 0.023 0.299 0.514 0.685
± ± ± ± ± ±
1.05 1.09 1.24 1.21 1.42 1.05
2.54 1.98 2.73 3.73 3.65 2.31
± ± ± ± ± ±
1.07 0.93 1.23 1.31 1.27 1.26
± ± ± ± ± ±
0.71 0.88 1.44* 1.43 1.56 0.78
Key to Table. MN: Micronuclei; NBUD: Nuclear buds; BN: Binucleated cells; PYK: Pyknotic cells; KYL: Karyorrhectic cells; KHC: Karyolytic cells; * Statistically significant at p < 0.05 with Kruskal–Wallis test followed by Dunn’s test in comparison to healthy periodontium.
Fig. 1. Correlation between periodontal disease severity and the total number of damages (sum of MN and nuclear anomalies). r: correlation coefficient; p: significance level according to Spearman’ test.
ROS are capable to produce DNA damage through the genetic mutations introduction and structural changes in DNA [16]. During the last 10 years, BMCyt assay has been used to show the 22
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Declaration of interest
been suggested that the interaction between periodontal pathogenic bacteria and the mucosa might potentiate the DNA damage formation. In this sense, volatile sulfur compounds produced by Gram-negative anaerobes, especially Agregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythia, increase the oral mucosa permeability [18] making it more prone to inflamation. However, we, as well as other authors [9], indicated that periodontal disease could be a risk factor for DNA damage, and it could be a modifiable risk factor once that periodontitis could be prevented and treated. Bloching et al. [8] suggests that the MN induction associated with periodontitis has been more related with a primary microbial rather than inflammatory reactions, an explanation for the non-occurrence of this association in our research. However, it is known that inflammatory processes are associated with repeated cell divisions and consequent chromosomal alterations that, in turn, stimulate apoptosis [10]. Consequently, it is essential to evaluate the occurrences of other nuclear anomalies evaluated by BMCyt assay. Regarding the cytotoxicity parameters, in the present study, only BN frequencies increase was associated with periodontitis. In the severe periodontal group, we verified BN frequencies was 1.4-fold higher compared with the healthy periodontium group, indicating that individuals with periodontal disease had a greater risk of cytokinetic defects than in individuals without periodontitis, and at least partially induced cytogenetic damage. The other cytotoxicity parameters did not exhibit significant differences among the groups. However, in the severe periodontal group, karyorrhectic cells increased 1.2-fold, pyknosis cells increased 1.2-fold and karyolysis cells increased 1.1-fold than healthy periodontium group. Zamora-Perez et al. [9] unlike our study, found significant differences between chronic and aggressive periodontitis groups than control group to nuclear abnormalities, including BN cells, karyolitic, karyorrhectic and pyknotic cells. In the present study, the total number of nuclear/cellular damages evaluated (DNA damage, chromosomal instability and cell death) was significantly associated with periodontitis severity, pointing to genotoxic effects associated with the inflammatory process and may suggest that this process could have masked the occurrence of DNA damage (MN frequencies) by elimination of genetically-impaired cells. In addition, periodontitis is common in human populations and represent a significant public health problem [8]. Periodontal diseases, in its chronic and aggressive forms, could lead to a severe alteration of the periodontal tissues and tooth loss [1,19] and genotoxicity [15,20,21]. Even though the assay has been conducted through many years by several laboratories across the globe, the efforts to standardize the scoring have only started very recently. If we compare the average frequency of the markers evaluated in our study with those of Bolognesi et al. [22], we can observe that most are in the same order of magnitude, with the exception being BN frequencies (substantially lower in our study). It is important to mention that conversely to our results the results of Bolognesi et al. [22] includes both healthy controls and patients with cancer, therefore making the comparison difficult. Further studies aiming to cross-calibrate labs are needed for the assay. In conclusion, the findings indicate that the inflammatory process generated by gingivitis and periodontitis is not related with MN frequencies. However, in individuals with periodontitis, a higher occurrence of NBUD and BN cells is an indicative of genotoxic effects of periodontal infection. The occurrence of DNA damage and other nuclear/cellular abnormalities (chromosomal instability and cell death) evaluated in oral mucosa cells and their relationship with periodontal disease is an important research field, being relevant to control this disease and to promote the importance of good oral health. At the same time, it should be considered that DNA damage is a critical event not only in the initiation phase but also in the promotion and progression phases, which could be related to carcinogenesis events. However, additional investigations such as intervention studies are necessary in order to evaluate the periodontal therapy effects in the occurrence of nuclear changes.
The authors declare that there is no conflict of interest. Acknowledgement We thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and to Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) by the scholarships provided. References [1] P.N. Papapanou, Epidemiology of periodontal diseases: an update, J. Int. Acad. Periodontol. 14 (1999) 110–116. [2] R.C. Page, K.S. Kornman, The pathogenesis of human periodontitis: an introduction, Periodontology 2000 14 (1997) 9–11, https://doi.org/10.1111/j.1600-0757.1997. tb00189.x. [3] S.C. Corbi, A.S. Bastos, S.R. Orrico, R. Secolin, R.A. Dos Santos, C.S. Takahashi, R.M. Scarel-Caminaga, Elevated micronucleus frequency in patients with type 2 diabetes, dyslipidemia and periodontitis, Mutagenesis 29 (2014) 433–439, https://doi.org/10. 1093/mutage/geu043. [4] P. Celec, L. Tóthová, Oxidative stress and antioxidants in the diagnosis and therapy of periodontitis, Front. Physiol. 14 (8) (2017) 1055, https://doi.org/10.3389/fphys.2017. 01055. [5] D. Bastos-Aires, Á. Azevedo, M. de Lurdes Pereira, D. Pérez-Mongiovi, A. Teixeira, Preliminary study of micronuclei levels in oral exfoliated cells from patients with periodontitis, J. Dent. Sci. 8 (2013) 200–204, https://doi.org/10.1016/j.jds.2012.12.007. [6] N. Holland, C. Bolognesi, M. Kirsch-Volders, S. Bonassi, E. Zeiger, S. Knasmueller, M. Fenech, The micronucleus assay in human buccal cells as a tool for biomonitoring DNA damage: the HUMN project perspective on current status and knowledge gaps, Mutat. Res. 659 (2008) 93–108, https://doi.org/10.1016/j.mrrev.2008.03.007. [7] P. Thomas, N. Holland, C. Bolognesi, M. Kirsch-Volders, S. Bonassi, E. Zeiger, S. Knasmueller, M. Fenech, Buccal micronucleus cytome assay, Nat. Protoc. 4 (2009) 825–837, https://doi.org/10.1038/nprot.2009.53. [8] M. Bloching, W. Reich, J. Schubert, T. Grummt, A. Sandner, Micronucleus rate of buccal mucosal epithelial cells in relation to oral hygiene and dental factors, Oral Oncol. 44 (2008) 220–226, https://doi.org/10.1016/j.oraloncology.2007.02.002. [9] A.L. Zamora-Perez, Y.M. Ortiz-García, B.P. Lazalde-Ramos, C. Guerrero-Velázquez, B.C. Gómez-Meda, M.Á. Ramírez-Aguilar, G.M. Zúñiga-González, Increased micronuclei and nuclear abnormalities in buccal mucosa and oxidative damage in saliva from patients with chronic and aggressive periodontal diseases, J. Periodontal. Res. 50 (2015) 28–36, https://doi.org/10.1111/jre.12175. [10] P.D. Brandão, I.S. Gomes-Filho, S.S. Cruz, J.D. Passos-Soares, S.C. Trindade, L.D. Souza, J.R. Meireles, E.D. Cerqueira, Can periodontal infection induce genotoxic effects? Acta. Odontol. Scand. 73 (2015) 219–225, https://doi.org/10.3109/00016357.2014.982705. [11] F. D’Agostini, E. Calcagno, R.T. Micale, S. La Maestra, S. De Flora, L. Cingano, Cytogenetic analysis of gingival epithelial cells, as related to smoking habits and occurrence of periodontal disease, Int. J. Hyg. Environ. Health 216 (2013) 71–75, https://doi.org/10.1016/ j.ijheh.2012.01.005. [12] World Health Organization, Diet, Nutrition And The Prevention Of Chronic Diseases: Report of a WHO Study Group [Meeting Held in Geneva from 6–13 March 1989] Geneva, World Health Organization, 1990 (Accessed 13 March 2003–17), http://www.who.int/ iris/handle/10665/39426. [13] C. Susin, C.F. Dalla Vecchia, R.V. Oppermann, O. Haugejorden, J.M. Albandar, Periodontal attachment loss in an urban population of Brazilian adults: effect of demographic, behavioral, and environmental risk indicators, J. Periodontol. 75 (2004) 1033–1041, https://doi.org/10.1902/jop.2004.75.7.1033. [14] World Health Organization, Calibration of Examiners for Oral Health Epidemiological Surveys, World Health Organization, Geneva, 1993. [15] I.L. Chapple, J.B. Matthews, The role of reactive oxygen and antioxidant species in peiodontal tissue destruction, Periodontology 2000 43 (2007) 160–232, https://doi.org/ 10.1111/j.1600-0757.2006.00178.x. [16] S. Reuter, S.C. Gupta, M.M. Chaturvedi, B.B. Aggarwal, Oxidative stress, inflammation, and cancer: how are they linked? Free. Radic. Biol. Med. 49 (2010) 1603–1616, https:// doi.org/10.1016/j.freeradbiomed.2010.09.006. [18] M. Bloching, W. Reich, J. Schubert, T. Grummt, A. Sandner, Micronucleus rate of buccal mucosal epithelial cells in relation to oral hygiene and dental factors, Oral Oncol. 44 (2008) 220–226, https://doi.org/10.1016/j.oraloncology.2007.02.002. [19] G.C. Armitage, Periodontal diagnoses and classification of periodontal diseases, Periodontology 2000 34 (2004) 9–21. [20] C.F. Çanakçi, V. Çanakçi, A. Tatar, A. Eltas, U. Sezer, Y. Çiçek, S. Oztas, Increased salivary level of 8-hydroxydeoxyguanosine is a marker of premature oxidative mitochondrial DNA damage in gingival tissue of patients with periodontitis, Arch. Immunol. Ther. Exp. 57 (2009) 205–211, https://doi.org/10.1007/s00005-009-0026-9. [21] H. Su, M. Gornitsky, A.M. Velly, H. Yu, M. Benarroch, H.M. Schipper, Salivary DNA, lipid, and protein oxidation in nonsmokers with periodontal disease, Free Radic. Biol. Med. 46 (2009) 914–921, https://doi.org/10.1016/j.freeradbiomed.2009.01.008. [22] C. Bolognesi, S. Knasmueller, A. Nersesyan, P. Roggieri, M. Ceppi, M. Bruzzone, E. Blaszczyk, D. Mielzynska-Svach, M. Milic, S. Bonassi, D. Benedetti, Inter-laboratory consistency and variability in the buccal micronucleus cytome assay depends on biomarker scored and laboratory experience: results from the HUMNxl international interlaboratory scoring exercise, Mutagenesis 32 (2017) 257–266, https://doi.org/10.1093/ mutage/gew047.
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