Micronucleus rate of buccal mucosal epithelial cells in relation to oral hygiene and dental factors

Oral Oncology (2008) 44, 220– 226

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Micronucleus rate of buccal mucosal epithelial cells in relation to oral hygiene and dental factors Marc Bloching a, Waldemar Reich b, Johannes Schubert b, Tamara Grummt c, Annett Sandner d,* a

ENT – Department of Saarland University, Homburg/Saar, Germany University Hospital and Policlinic for Oral and Plastic Maxillo-Facial Surgery, Medical Martin Luther University Halle-Wittenberg, Halle/Saale, Germany c Federal Environmental Office, Bad Elster, Germany d University Hospital and Policlinic for Otorhinolaryngology, Head and Neck Surgery, Martin Luther University Halle-Wittenberg, Magdeburger Street 12, 06097 Halle/Saale, Germany b

Received 21 December 2006; received in revised form 12 February 2007; accepted 12 February 2007 Available online 16 April 2007

KEYWORDS Genotoxicity; Micronucleus test; Dental status; Carcinogenesis; Biomarker; Upper aero-digestive tract

Summary Carcinogenesis of squamous cell carcinomas in the upper aero-digestive tract (UADT) is a multi-stage process. Since 1937, micronuclei (MN) have been considered a marker for genome damage in the initiation stage. By help of the micronucleus test, carcinogenic exposure can be proven in the mucosa area of the UADT. The hypothesis to be tested was that individual oral hygiene and the dental status, respectively – just like alcohol and tobacco abuse – are associated with the micronucleus rate in cytological preparations of the buccal mucosa. In a prospective clinical observation study, we determined in 100 probands the micronucleus frequency per 1000 mucosa epithelial cells. Study participants with a high number of missing teeth (M/T index, p = 0.037), a below-average papillary bleeding index (PBI, p = 0.032) and periodontal status, respectively (PSI, p = 0.042) possessed a higher micronucleus number in comparison with restored dental conditions. Probands with composite restorations displayed a higher MN rate (p = 0.006) compared to those with amalgam. However, we could not detect any significant relation with the prosthetic status (p P 0.075). An adjustment was made according to alcohol and tobacco. We therefore conclude that subgingival plaque and synthetic dental materials in addition to chronic alcohol and tobacco consumption might have genotoxic relevance in the oral cavity.

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* Corresponding author. Tel.: +49 345 557 1784; fax: +49 345 557 1859. E-mail address: [email protected] (A. Sandner). 1368-8375/$ - see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2007.02.002

Micronucleus rate of buccal mucosal epithelial cells in relation to oral hygiene and dental factors

Introduction Carcinogenesis and metastasis of squamous cell carcinomas in the upper aero-digestive tract are multi-stage processes.1 Markers of cellular and molecular changes, respectively, which are associated with the process of carcinogenesis, but which occur even before a malignant transformation takes place, are called biomarkers.2 Since 1937, micronuclei have been regarded as indicators for genotoxic exposure.3 Clinical studies show that the determination of the micronucleus rate in different cytological preparations can be reproducible.4–6 The loss of chromatin in the main nucleus due to a mutagenic exposition, contributes to the formation of micronuclei.7 Micronuclei (MN) may consist of whole chromosomes (due to a delayed anaphase, they are not integrated into the daughter cell) or of chromosome fragments (fracture events). Micronuclei consisting of whole chromosomes (centromere positive, larger MN) indicate aneuploidogenic genotoxic effects. Micronuclei, however, which contain chromosome fragments (centromere negative, smaller MN) base on clastogenic genotoxic effects.8 The present study deals with the clinical application of the micronucleus test with the goal to investigate a possible association between the aforementioned cytogenetic damages and the dental status. It was to be reviewed to what extent – beside alcohol and tobacco consumption habits – individual oral hygiene habits, the cariologic and periodontal as well as prosthetic status of the study participants possess influence on epithelial cells of the buccal mucosa.

Material and method This study comprised probands of an age between 30 and 60 years, all of them being under general dental treatment. The recruiting of the candidates was done consecutively, observing the exclusion criteria listed in Table 1. A questionnaire recorded proband-specific data for social history, harmful chemical and X-ray exposure (head–neck area) as well as alcohol and tobacco consumption.9 Furthermore, information regarding general history, permanent medication, fluoride history, oral hygiene and nutritional habitse was included. Alcohol and tobacco consumption was quantified. One hour before sample-taking for cytology, the study participants were asked to refrain from eating, drinking, smoking, and drinking alcohol. Oral hygiene measures were to be performed as usual, however, not within one hour before taking of the samples. After one-time rinsing of the oral cavity with tap water, a mucosa smear was taken from the left buccal mucosa via Cytobrush Plus GT. The sample-taking was performed by keeping the cheek apart with a dental speculum, and a one-time turn of the brush around its own longitudinal axis. One had to take care that all bristles had uniform contact with the mucosa. After applying the sample to a glass slide and drying it in the air, fixation was performed by a cold methanol–vinegar mix (4:1) for 30 min. Afterwards, the glass slide was dried again, and it was then e

Oral hygiene was assessed with good under the following circumstances: tooth brushing at least twice daily, and for at least 2 min each time. Otherwise, it was regarded as poor.

221

stored at room temperature until investigation of the micronuclei. Staining was carried out with 1% Giemsa solution for a period of 10 min. Afterwards, the glass slide was rinsed with Aqua dest., and dried in the air. The light microscopic assessment of the preparations regarding micronuclei was done by 400 fold (10 · 40) enlargement. 1000 epithelium cells per preparation were assessed. Cells with one, two, three, and more micronuclei, respectively, were taken down separately, and afterwards, the micronucleus number per 1000 cells was determined per proband. The recording of the dental status comprised: oral hygiene indices [modified approximate plaque index (API) according to Lange et al.,10 modified papillary bleeding index (PBI) according to Saxer and Mu ¨hlemann11], semi-quantitative determination of cariogenic germs Streptococcus mutans (SM) and Lactobacillus acidophilus (LB) via CarioCheck Test, cariologic (DMF/Tf index, kind of filling material) and classical clinical periodontal findings (PSI, Bengel12) as well as prosthetic status (DP). The requirements according to the Declaration of Helsinki were met. The Ethics Commission approved the study. The statistic review of the data was carried out by the SPSS program for Windows, version 10.0 (SPSS, Chicago, IL, USA). The descriptive statistics related to the determination of event frequency, frequency allocation, and feature combination. For the analytical statistics – depending on the scaling of parameters – served different tests on a significance level of p 6 0.05g each.

Results The study included 100 probands of an average age of 42.4 years (normal allocation, standard deviation 7.9 years). 52 of them were male, and 48 female candidates. Table 2 characterizes the total group in terms of the major independent variables: history, exogenous chemicals/irradiation, individual oral hygiene habits and dental status. The average micronucleus rate for the total population was 1.9 micronuclei per 1000 cells with a standard deviation of 0.99. The values were normally allocated (Kolmogorov– Smirnov test). In seven cases, no micronucleus could be found per 1000 assessed cells; in five cases, we found 4 micronuclei per 1000 cells. The remaining cytological preparations showed the following allocation: 1 MN (n = 26), 2 MN (n = 39), and 3 MN (n = 23). Among the variables (age, sex, general diseases, including permanent medication, alcohol and tobacco consumption, harmful chemical and X-ray exposure), only the factors age, sex and tobacco consumption displayed an association with the micronucleus number (see Table 3). A f According to the DMF/T indices, the study participants were divided into three groups: ‘‘DMF/T value above average’’, ‘‘DMF/T value average’’, and ‘‘DMF/T value below average’’. For the subdividing, the age-related average values for Germany were taken into consideration: 15–34 years – DMF/T index 11.6; 35–44 years – DMF/T index 16.7; 45–54 years – DMF/T index 18.4. g For reviewing the normal allocation, the test according to Kolmogorov–Smirnov was used; the Chiquadrate test according to Pearson was used for categoric variables; t test according to Student and ANOVA was used for medium value comparisons. When comparing more than two random checks, correction was performed according to Bonferroni.

222 Table 1

M. Bloching et al. Exclusion criteria

Exclusion criteria Antibiotics in the course of the previous month Bleeding risk (e.g. anticoagulant therapy) Chemotherapy and/or immuno-suppression therapy Endocarditis risk Patients radiated in the head–neck-region Infection risk (e.g. HIV, HBV, HCV, Tb) Multimorbid patients of the ASA risk groups higher than II Probands with malignant tumours of the upper aero-digestive tract Use of antiseptic oral solutions Patients without teeth

Table 2

Characterization of candidates relating to history, oral hygiene and dental factors (n = 100)

History

Yes

No

General diseases Permanent medication Family history Harmful chemical exposure Alcohol consumption regular occasional Tobacco consumption in past currently Dental X-ray during past month

n = 42 n = 38 n = 20 n = 32 n = 47 n = 37 n = 10

n = 58 n = 62 n = 80 n = 68 n = 53

n = 41 n = 32 n = 28

n = 59 n = 68 n = 72

Oral hygiene habits

Yes

No

Fluoridisation Good oral hygiene Interdental hygiene Mouthwash use

n = 29 n = 77 n = 43 n = 30

n = 71 n = 23 n = 57 n = 70

Dental status Hygiene indices API PBI Cariogenic microorganisms Lacto bacilli–LB Strep mutans–SM DMF/T-Index Filling materials (direct restorations) PSI Dental prosthesis

Poor

Medium

Good

n = 54 n = 43

n = 36 n = 39

n = 10 n = 18

n = 22 n = 18 High n = 53 Composite n = 21 Considerable periodontitis n = 45 Prosthetically untreated n=5

n = 49 n = 38 Average n = 17 Composite and amalgam n = 39 Medium periodontitis n = 33 No fixed prosthesis n=6

n = 29 n = 44 Low n = 30 Amalgam n = 32 Gingivitis/ healthy periodontium n = 22 Fixed prosthesis n = 53

higher age correlated positively with the micronucleus rate with a relatively broad spread of the dot cloud (r = 0.204, p = 0.042 according to Pearson). Men displayed approx. 1 MN per 1000 cells more than women (2.12 ± 0.98 vs. 1.73 ± 0.96, p = 0.050). The main focus was the working hypothesis that factors of individual oral hygiene as well as cariologic, periodontal

and prosthetic findings are associated with the MN frequency of epithelium cells. The average differences of the MN rate per 1000 cells, depending on the individual oral hygiene quality – as stated in the anamnesis – were not statistically significant (p P 0.417, t test according to Student, ANOVA). Taking into consideration the oral hygiene indices, with an increas-

Micronucleus rate of buccal mucosal epithelial cells in relation to oral hygiene and dental factors Table 3

223

Micronucleus number (MN) per 1000 cells, depending on exogenous noxa

Alcohol consumption

Tobacco consumptiona

Harmful chemicals exposure Dental X-rayb

Regular MN

Occasional MN

Abstinent MN

(n = 37) 2.11 ± 0.94 Current (n = 32) 2.44 ± 0.95 Yes (n = 32) 2.13 ± 1.01 (n = 28) 2.15 ± 1.17

(n = 10) 1.90 ± .1.20 Former (n = 41) 2.32 ± 0.93 No (n = 68) 1.79 ± 0.99 (n = 72) 1.82 ± 0.91

(n = 53) 1.83 ± 0.99 No (n = 59) 1.69 ± 0.92

p value p P 0.291 p value p 6 0.001

p = 0.127 p = 0.418

Depicted are average values and standard deviations, depending on alcohol and tobacco consumption habits as well as X-ray exposition and harmful chemicals exposition. a Multiple indications possible b Referenced to the month before participation in study, significant differences are marked.

Table 4

Micronucleus number (MN) per 1000 cells, depending on dental status I

SM LB API PBI

Low MN

Medium MN

High MN

p value

(n = 40) 1.67 ± 1.01 (n = 43) 1.81 ± 1.03 (n = 10) 1.82 ± 1.08 (n = 18) 1.47 ± 0.87

(n = 46) 2.11 ± 0.97 (n = 38) 2.03 ± 1.04 (n = 36) 1.86 ± 0.94 (n = 39) 1.85 ± 1.09

(n = 14) 2.07 ± 0.92 (n = 19) 2.00 ± 0.82 (n = 54) 2.00 ± 1.02 (n = 43) 2.20 ± 0.87

p P 0.127 p  1.00 p  1.00 p P 0.342 p = 0.032

In this table, MN per 1000 cells (average and standard deviation) depending on the grade of settlement of saliva with cariogenetic microorganisms (SM, LB) and oral hygiene indices (API, PBI) is depicted (Significant differences are marked.)

Table 5

Micronucleus number (MN) per 1000 cells, depending on dental status II

Filling materials (direct restoration)

DMF/T index

PSI

Dental prosthesis (DP)

Compositea MN

Composite and amalgama MN

Amalgam MN

p value

(n = 21) 2.29 ± 0.90

(n = 39) 2.08 ± 0.98

(n = 32) 1.45 ± 0.85

p 6 0.020

Poor (n = 53) 2.33 ± 0.96

Medium (n = 17) 1.71 ± 0.77

Good (n = 30) 1.77 ± 1.02 p=0.037

Severe periodontitis (n = 45) 2.16 ± 0.85

Medium–severe periodontitis (n = 33) 1.91 ± 1.04

Gingivitis/whole periodontium (n = 22) 1.50 ± 1.06 p=0.031

No fixed DP (n = 6) 2.82 ± 0.75

Fixed DP (n = 53) 1.94 ± 0.93

No prosthetic treatment (n = 5) 1.80 ± 0.45

p P 0.177

p P 0.379

p P 0.071

In this table, MN per 1000 cells (average and standard deviation) depending on the kind of filling material, cariologic, periodental and prosthetic status is depicted. Several significant links were determined between the stated factors and the biomarker (significant differences are marked). a With the variable ‘‘filling material’’, a comparison was made each in relation to the sub-group ‘‘amalgam’’.

224 ing bleeding grade after probing in the inter-dental papilla area (PBI), we found an increasing median MN rate. As further influences we could detect: the type of filling material, the cariologic (number of missing teeth) and periodontal findings (p 6 0.037, Tables 4 and 5). The plaque index, the concentration of cariogenetic bacteria in the saliva as well as the prosthetic findings did not possess any significant influence. Among the filling materials amalgam and composite for direct restorations, in particular composite displayed a considerable influence on the MN rate (p 6 0.020). The calculated average difference was as follows: ‘‘amalgam’’ 1.45 ± 0.85, ‘‘composite’’ 2.29 ± 0.90 MN per 1000 cells. While the number of decayed teeth (D/T index, r = 0.003; p = 0.973) and filled teeth (F/T index, r = 0.168; p = 0.097) did not correlate with the MN rate, we found a significant connection between the number of missing teeth (M/T index) and the micronucleus frequency (r = 0.223; p = 0.097, adjusted according to age). Under the aspect of periodontal health, it became clear that participants with severe periodontitis showed the highest average MN rate (2.16 ± 0.85), followed by those participants with medium–severe periodontitis (1.91 ± 1.04) and probands with nearly healthy periodental conditions (1.50 ± 1.06 MN per 1000 cells). We could not find any significant correlation between the prosthetic status and the MN rate, however, study participants with a combined fixed and removable dental prosthesis displayed the relatively highest MN rates: 1.78 ± 1.10 in probands without dental prosthesis, 1.80 ± 0.45 in participants without any prosthetic rehabilitation; 1.94 ± 0.93 with fixed dental prosthesis, and finally with a combined fixed and removable dental prosthesis 2.83 ± 0.75 MN per 1000 cells (p P 0.093).

Discussion Many investigations have shown an association between risk factors for squamous cell carcinomas in the UADT and the MN rate.7,13–16 The aim of the present investigation was to review how far dental factors can effect an induction of micronuclei in the buccal mucosa epithelium cell area. In accordance with Pastor et al.,17 but in contradiction to findings of Konopacka,18 we found among the probands age-related as well as sex-related differences in the MN rate. Oral hygiene factors stated by study participants mainly turned out to be without any relevance relating to the aforementioned question. However, the objectively assessed quality of oral hygiene (PBI) proved to be an influence factor for the MN rate (p = 0.032). Admittedly, among probands with periodontitis, artefacts through chromatin remainders of segment-core granalocytes are possible,19 however, this has already been taken into consideration when taking the samples. In order to reduce the possibility of confusion, we therefore let the probands perform an oral cavity rinsing before taking the smear. As the recording of inflammatory processes in the marginal periodontium area is the focus of the PBI index, a cytogenetic influence through microbial pathogenetic factors needs to be discussed. Participants having severe periodontal diseases on average showed the highest MN rate. According to the litera-

M. Bloching et al. ture, interactions between periodontal-pathogenetic bacteria and the mucosa of the UADT are evident. Lang and Filippi20 in particular indicate that volatile sulphuric compounds which are formed by anaerobe gram-negative pathogens, increase the permeability of oral mucosa for e.g. endotoxins. Narikiyo and collaborators21 investigated potential co-carcinogenetic influences of oral micro-organisms in carcinogenesis in the esophagus. They concluded that Treponema denticola, Streptococcus anginosus, and Streptococcus mitis are possibly involved in the carcinogenesis in the upper aero-digestive tract. In a recent analysis it was observed for the first time that in buccal epithelium cells invasive microorganisms found intra-cellularly include (Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Tannerella forsythensis). The authors postulate for epithelium cells of the oral mucosa an intra-cellular polymicrobial flora, typical for the bio-film on tooth surfaces.22 The results of the present investigation suggest a possible causal link. Regarding a connection between cariologic status (DMF/ T index) and MN rate, it became clear that particularly the number of missing teeth (M/T index) correlated with the micronucleus number, however, it did not with the number of decayed and filled teeth (see dental prostheses). At the same time, an unfavourable effect of the filling material composite could be observed in comparison with amalgam which is a clinical confirmation of the in-vitro studies of Schweikel et al.23,24 Those authors found in vitro that extracts from five common dental composites by a majority displayed mutagenetic effects (MN induction in fibroblast cell line V79), and they demanded a replacement of the mutagenetic composite parts by bio-compatible substances. This is of relevance as synthetic dental materials have (in-)direct contact with the oral mucosa,25 and unpolymerized monomers (e.g. 2-Hydroxyethylmethacrylate, Triethylene-glycoldimethacrylate, Urethandimethacrylate, Bisphenol-a-glycidmethacrylate, Methylmethacrylate) may affect these. However, regarding amalgam fillings it is known that they form a potential permanent source of organic-bond mercury (mainly methyl mercury) which possesses toxic characteristics, and in whose formation bacteria of the oral cavity (streptococci) are involved.26 Even if in the framework of the present analysis, study participants with a combined fixed and removable dental prosthesis showed a tendency to higher MN rates than probands with fixed prostheses, we could not detect any statistically relevant significance (p = 0.075). Whether or not this observation goes back to the methylmethacrylate content, metal ions or the significance of removable dental prostheses as a reservoir for micro-organisms (catalytic potential, Krogh27), could not be clarified. In a previous investigation of 10 common basic prosthesis synthetics on the basis of methylmethacrylate (MMA) regarding genotoxicity and cytotoxicity, Gigola et al.25 found a significant increase of the MN rate in just one case. The finding from the present investigation indicates an association between the number of missing teeth and the MN rate on one hand, and between the combined fixed and removable dental prostheses and the MN rate on the other, seems to point at basic prosthesis synthetics (MMA) as a cause. Burgaz et al.28 however, found that employees of dental laboratories which process cobalt–chrome–molybdenum-alloys (model casting prosthe-

Micronucleus rate of buccal mucosal epithelial cells in relation to oral hygiene and dental factors ses) displayed a higher MN rate in the area of the nasal mucosa, in comparison with a control group (3.5 ± 1.80 vs 1.2 ± 0.53 per 1000 cells; p = 0.005). To what extent those probands also processed prosthesis synthetics – a factor which is also to be taken into consideration – is beyond our knowledge. According to Schmalz and Garhammer,29 a biologic interaction of dental alloys with the oral mucosa is to be regarded basically under the aspect of bacterial adhesion and (sub-)toxic and allergic reactions, respectively, which, among other things, can lead to inflammatory effects, such as plaque-dependent and plaque-independent gingivitis, respectively, and to oral lichenoid reactions (OLL). An influence of metal ions on cell metabolism is responsible for plaque-independent inflammatory reactions.29 Chang-Claude et al.30 performed an investigation relating to the interaction between inflammatory mucosa alterations in the upper digestive tract and the MN rate. They concluded that no association can be assumed between a mild form of esophagitis and the MN number. This might lead to the conclusion that there is a primary microbial cause for the observed association between a marginal periodontitis and the micronucleus induction, rather than inflammatory reactions.

7.

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Conclusions It can be concluded from this investigation that besides factors such as alcohol and tobacco, subgingival plaque might be of genotoxic relevance. To what extent virulence factors of bacteria are involved in the formation of aneuploidogenetic and clastogenetic damages, respectively, remains to be investigated in detail. The observation that composite fillings in comparison with amalgam display a slightly higher micronucleus number, underlines the demand already expressed in in-vitro studies that the search for bio-compatible composites (monomers) needs to be continued. This also applies to basic prosthesis materials.

Conflict of Interest

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16.

17.

18.

None declared.

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