BioMed Research International

Volume 2013 (2013), Article ID , 10 pages

http://dx.doi.org/10.1155/2013/905252

Research Article

Cytogenetic Abnormality in Exfoliated Cells of Buccal Mucosa in Head and Neck Cancer Patients in the Tunisian Population: Impact of Different Exposure Sources

Rim Khlifi,1,2 Fatma Trabelsi-Ksibi,¹ Amine Chakroun,³ Ahmed Rebai,² and Amel Hamza-Chaffai¹

¹ Unit of Marine and Environmental Toxicology, UR 09-03, Sfax University, IPEIS, BP 1172, 3018 Sfax, Tunisia

²Unit of Bioinformatics and Human Genetics, Centre of Biotechnology of Sfax, BP 1177, 3018 Sfax, Tunisia

³Department of Otorhinolaryngology, Habib Bourguiba Hospital, 3000 Sfax, Tunisia

Received 6 April 2013; Accepted 11 June 2013

Academic Editor: Manoor Prakash Hande

Copyright © 2013 Rim Khlifi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Chromosome/DNA instability could be one of the primary causes of malignant cell transformation. The objective of the present study was to evaluate the spontaneous genetic damages in exfoliated cells of buccal mucosa of head and neck cancer (HNC) by counting micronucleus (MN) and binucleated (BN) cells frequencies. MN and BN frequencies were significantly increased in HNC patients compared with controls (5.53 ± 3.09/1000 cells, 5.63 ± 2.99/1000 cells versus 2.36 ± 2.11/1000 cells, 3.09 ± 1.82/1000 cells, ). Regarding the gender and the age, the frequencies of the MN and BN were significantly higher than those of controls (). The evaluation of the MN and BN frequencies revealed a significant increase () in the cases in relation to the control group after controlling the risk factors (tobacco smoking and chewing and occupational exposure) of HNC. Moreover, MN and BN frequencies were significantly increased in smokers and chewers compared with nonsmokers and nonchewers among patients (). MN frequency was significantly () different between patients occupationally exposed (6.99 ± 3.40/1000 cells) and nonexposed (4.70 ± 2.48/1000 cells) among HNC group. The logistic regression model illustrated that HNC was significantly associated with frequencies of MN (OR = 8.63, ) and BN (OR = 5.62, ). Our results suggest that increased chromosome/DNA instabilities may be associated with HNC.

1. Introduction

Head and neck cancer (HNC) is a sentinel disease of exposure to environmental factors. The development of HNC is strongly associated with tobacco use (smoking and chewing) and alcohol consumption, as well as with the exposure to several occupational carcinogens [1–4]. Only a fraction of exposed individuals develop cancer in the head and neck region, which suggests that individual’s sensitivity to mutagens is an important endogenous risk factor that significantly contributes to the development of the disease [5, 6].

HNC is a result of progressive accumulation of genetic and epigenetic alterations of HNC epithelial cells. The loss of genomic stability seems to be the main pathogenic key, appearing early in the carcinogenesis process. Genomic damage is probably the most important fundamental cause of developmental and degenerative diseases and cancer. It is also well established that genomic damage is produced by environmental exposure to genotoxins, medical procedures (e.g., radiation and chemicals), micronutrient deficiency (e.g., folate), lifestyle factors (e.g., alcohol, smoking, drugs, and stress), and genetic factors such as inherited defects in DNA metabolism and/or repair [7–12]. Hence, it is essential to perform biomonitoring with minimally invasive markers. The micronucleus (MN) trial in exfoliated cells of the buccal mucosa is a potentially excellent biomarker candidate for monitoring studies [13].

The micronucleus test in buccal mucosa cells is one of the less invasive methods to measure DNA damage in humans. This test was proposed in 1983 and continues to gain in popularity as a biomarker of genetic damage [14]. And its information can be used as an early warning of potential risk of developing long-term health problems [15].

The MN and BN assay with exfoliated buccal cells is a cost-effective, noninvasive method, in which the formation of anomalous cells is used as an endpoint to detect cytogenetic damage in exposed individuals. The formation of MN in dividing cells is the result of chromosome breakage due to unrepaired or misrepaired DNA lesions or chromosome malsegregation due to mitotic malfunction. These events may be induced by oxidative stress, exposure to clastogens or aneugens, genetic defects in cell cycle checkpoint and/or DNA repair genes, and deficiencies in nutrients required as cofactors in DNA metabolism and chromosome segregation machinery [16–20]. All these events can cause the formation of MN through chromosomal rearrangements, altered gene expression, or aneuploidy, effects associated with the chromosome instability phenotype often seen in cancer [17, 21].

Molecular epidemiological studies have provided evidence that individual susceptibility to cancer is mediated by genetic and environmental factors. However, the carcinogenic process is associated with increased genetic instability. To evaluate genetic instability, there are biomarkers that predict if a premalignant lesion or condition is likely to develop into an aggressive metastasizing tumor. Most cancers are monoclonal, and cytogenetic assays do provide information about the DNA damage at the level of a single but proliferating cell. The objective of the present study was to investigate the spontaneous genetic damage in exfoliated cells of the buccal mucosa of HNC patients and healthy controls by the anomalous cells (micronucleus and binucleated cells) assay with exfoliated buccal cells regarding the factors that might affect MN and BN frequencies (i.e. age, gender, smoking and alcohol drinking habits, occupational exposure, and amalgam fillings). Our study represents the first biomonitoring of these anomalous cells in HNC patients in our population.

2. Materials and Methods

2.1. Subjects Studied

The case-control study population consisted of 45 untreated cancer patients with histologically confirmed HNC and 57 cancer-free control subjects. All subjects were recruited simultaneously from residents living in the similar geographic area (Sfax). Patients were consecutively recruited between May 2012 and December 2012 at the Department of Otorhinolaryngology, Otorhinolaryngology Department, Habib Bourguiba Teaching Hospital (Sfax, Tunisia). All cases were newly diagnosed and previously untreated. Clinical characteristics including basic medical data were obtained from medical records.

2.2. Questionnaire Administration

After signing the informed consents, subjects were interviewed to collect detailed information on their demographics (age and gender), alcohol drinking, tobacco smoking and chewing, amalgam fillings occupation, and occupational exposure. Lifetime consumption of tobacco and occupational exposure were also collected. The average number of cigarettes smoked per day and the total number of years of smoking were used to calculate cumulative smoking dose as “pack years” (PY = [cigarettes per day/20] × years smoked). Likewise, tobacco chewing consumption (dry snuff called neffa) dose was estimated as “consumption year” (CY = frequency of neffa consumed−kept/day × duration of year).

2.3. Buccal Cells Procedure, Staining, and Scoring

The participants were asked to rinse their mouth for 1 min with 10 mL of sterilised distilled water (Braun Medical, SA) and exfoliated cells of the buccal mucosa were obtained by scraping the buccal mucosa with a wooden spatula [22, 23]. For each individual, two slides were prepared by smearing the cells immediately onto the center of clean glass slides. After applying the sample to a glass slide and drying it in the air, fixation was performed by a cold methanol-glacial acid mix (3 : 1) for 30 min. Afterwards, the glass slide was dried again, and it was then stored at room temperature until investigation of the micronuclei. Staining was carried out with 2% Giemsa solution for a period of 10 min. Afterwards, the glass slide was rinsed with aqua dest and dried in the air. The criteria of MN evaluation were those suggested by Tolbert et al. [24] and Titenko-Holland et al. [25].

Screening for cell anomalies was performed under an oil immersion lens (100x), followed by phase contrast microscopy for confirmation of MN according to established methods [23, 26]. At least 1000 intact epithelial cells per individual were scored to achieve the average percent of micronucleated cells. The opaque extranuclear intracytoplasmic bodies seen under oil immersion lens and phase contrast were considered micronuclei (Figure 1), whereas binucleated cells (Figure 2), fragmented nuclei (Figure 3), and nuclei-like broken eggs were not counted as MN (Figure 4).

Figure 1: Buccal smear shows micronuclei. Pap stained. 100x.

Figure 2: Buccal smear shows binucleated cells, Pap stained. 100x.

Figure 3: Buccal smear shows fragmented nucleus, Pap stained. 100x.

Figure 4: Buccal smear shows nuclei-like broken eggs, Pap stained. 100x.

2.4. Statistical Methods

The studied variable departed significantly from normality and therefore the nonparametric Mann-Whitney test was applied to data. The associations between two variables were analyzed by the Spearman correlation. The chi-square test was used to compare frequencies between groups. The level of significance was taken as . To determine the effects of anomalous cell frequencies on the development of HNC, a conditional binary logistic regression model was conducted. In this model, we adjusted for age, gender, tobacco habit, occupational exposure, and alcohol drinking. In addition, the cutoffs of high-low frequency that was set at 75th percentiles of the MN and BN among the controls were used to calculate the adjusted odds ratio (OR) for HNC below and above the 75th percentiles value of MN and BN frequencies. All analyses were conducted using the Statistical Package for Social Sciences (SPSS) for Windows, version 13.0.

3. Results

Descriptive characteristics of HNC cases group and the control groups are described in Table 1. Both groups were characterized for gender, age, smoking, chewing and alcohol consumption, occupation and occupational exposure, and amalgam fillings. Among patients, 75.6% were male. According to their age, patients were classified into 2 classes. 64.4% of patients aged over 55 years. The HNC cases differed significantly from the control group with respect to tobacco smoking, alcohol drinking, occupational exposure, and amalgam fillings. Thirty-one percent of HNC patients were alcohol drinkers and tobacco chewers, and sixty percent were smokers. Most of cases (64.4%) were occupationally exposed (33.3% cement workers, 20.0% farmers, and 11.1% painters). However, only 21.1% of healthy controls were occupationally exposed. A chi-square test showed that risk factors (smoking, drinking, and occupational exposure) were statistically significant between case and control groups (Table 1).

Table 1: Comparison of demographic and lifestyle characteristics between HNC cases and controls.

Characteristics
Controls no.

(%)
Patients no.

(%)

Sample size

Gender

0.004

    Male
30 (52.6)
34 (75.6)

    Female
27 (47.4)
11 (24.4)

Age (years)

NS

    Mean ± SD

    ≤55
25 (43.4)
16 (35.6)

    >55
32 (52.6)
29 (64.4)

Tobacco smoking

<0.001

    No
44 (77.2)
18 (40.0)

    Yes
13 (22.8)
27 (60.0)

    Mean ± SD (PY)a

c

Tobacco chewing

NS

    No
48 (84.2)
31 (68.9)

    Yes
9 (15.8)
14 (31.1)

    Mean ± SD (CY)b

c

Alcohol drinking

<0.001

    No
57 (100.0)
31 (68.9)

    Yes
0 (0.0)
14 (31.1)

Occupational exposure

<0.001

    No
45 (78.9)
16 (35.6)

    Yes
12 (21.1)
29 (64.4)

    Mean ± SD (year)

c

Occupation

NS

    Cement worker
7 (12.3)
15 (33.3)

    Farmer
5 (8.8)
9 (20.0)

    Painter
5 (8.8)
5 (11.1)

Amalgam fillings

0.001

    No
38 (66.7)
27 (60.0)

    Yes
19 (33.3)
18 (40.0)

Two-sided chi-square test. aPY: smoking index expressed as the number of packs of 20 tobacco cigarettes per day for 1 year.

bCY: consumption year = frequency of neffa consumed−kept/day × duration of year.

c.

The means of MN and BN frequencies for the case and control groups are presented in Table 2, where a comparison of these values for the two cell types for all subjects is presented. The two-sample -test showed that anomaly cell samples of cases were significantly higher than those of controls. The frequencies of MN and BN cells in HNC cases were higher by 2.3 and 1.8 times, respectively, than those observed in control groups.

Table 2: Anomalous cell frequency in buccal mucosa exfoliated cells (/1000 cells) of HNC patients and healthy controls.

Controls
Cases

Mean ± SD
Range
Median
5–95th percentiles
Mean ± SD
Range
Median
5–95th percentiles

Anomalies

    MNC

0–9
1.37
0.51–7.20
*
2–14
4.33
2.03–11.70

    BNC

1–8
3.00
0.99–7.10
*
1–16
5.00
2.76–13.05

SD: standard deviation; MN: cells with micronuclei; BN: binucleated cells.

Significantly different compared with the control group, .

Table 3 shows that the frequency of the MN in exfoliated cells of buccal mucosa differs between men and women within only the control groups (). However, the BN frequency differs between the two age groups (≤55 and >55) within the cases and control groups ( and 0.012, resp.). Moreover, MN and BN frequencies of patients were mostly significantly higher than those of controls () after controlling the gender and the age.

Table 3: The average anomalous cells in buccal mucosa exfoliated cells in HNC patients and healthy controls, by gender and age variables.

Anomalous cell (mean ± SD/1000 cells)

MN
BN

Controls
Cases
Controls
Cases

Gender

    Male

**

*

    Female

**

*

     value
0.040
NS
NS
NS

Age (year)

    ≤55

**

**

    >55

**

**

     value
NS
NS
0.012
0.013

Significantly different compared with the control group, .

*Significantly different compared with the control group, .

NS: nonsignificant.

The Spearman correlation coefficient matrix of selected cell anomalies number in HNC cases was also studied (Table 4). A significant positive correlation was noted between MN frequency and cases of smokers (, ) and chewers (, ). The correlation between chewer cases (, ) and filling material amalgam (, ) and BN frequency was also found.

Table 4: The Spearman correlation analysis of cell anomalies of HNC patients.

Anomalies
Characteristics

MN
BN
Smoking
Alcohol
Chewing
Amalgam

Anomalies

    MN

    BN
0.339

Characteristics

    Smoking
0.311**
0.227*

    Alcohol
0.149
0.097
0.440**

    Chewing
0.174
0.364*
−0.320
0.285

    Amalgam
0.212
0.430**
−0.122
−0.164
−0.062

    Occupational exposure
0.184
0.298
0.000
−0.075
−0.208
0.073

.

** .

To evaluate possible associations between the environmental exposure and the incidence of genetic damage in buccal cells, the MN and BN data were classified according to the sampled risk factors of HNC (Table 5). The evaluation of the frequency of the MN and BN in exfoliated cells of buccal mucosa revealed a significant increase ( and <0.05, resp.) in the cases in relation to the control group after controlling these risk factors (tobacco smoking and chewing, alcohol drinking, amalgam fillings, and occupational exposure) of HNC. There was a significant difference between smokers and nonsmokers in the frequency of BN among patient and control groups ( and 0.045, resp.). Furthermore, a significant difference between smokers and nonsmokers was found in the MN frequency among HNC patients (). Among patient group, the MN frequency of participants occupationally exposed (6.99/1000 cells) was significantly higher than that of nonexposed (4.70/1000 cells) (). There was no significant difference between participants with filling material amalgam and those without filling material amalgam, in the frequency of anomalous cells in cancer patients and healthy controls.

Table 5: Anomalous cell frequency observed in 1000 cells depending on HNC risk factors.

Anomalous cell (mean ± SD)

MN
BN

Controls
Cases
Controls
Cases

Tobacco smoking

    No

    Yes

**

**

     value
NS
NS
0.046
0.045

Alcohol drinking

    No

**

**

    Yes
—

—

    -value
—
NS
—
NS

Tobacco chewing

    No

**

**

    Yes

**

**

    -value
NS
0.030
NS
NS

Occupational exposure

    No

**

**

    Yes

**

**

    -value
NS
0.014
NS
NS

Amalgam fillings

    No

**

**

    Yes

*

*

    -value
NS
NS
NS
NS

Significantly different compared with the control group, .

*Significantly different compared with the control group, .

NS: nonsignificant.

The 75th percentiles of the controls’ MN and BN frequencies were used (as the cutoffs to assign the study subjects into either the low- or high-frequency group) in order to estimate the crude odds ratios for HNC (Table 6). High MN frequency demonstrated a strong association with HNC (OR = 8.63, ), as did high BN frequency (OR = 5.62, ) (Table 6). The variables that showed significant means differences between cases and controls were included in the conditional logistic regression analysis to identify the adjusted OR and confidence interval, as seen in Table 5. After adjusting for age and gender, the results were significant for MN and BN frequencies for HNC (OR = 18.13, and OR = 4.91, , resp.). However, after adjusting for age, gender, tobacco habits, occupational exposure, and alcohol drinking, the results were significant only for MN frequency (OR = 69.06, ) (Table 6).

Table 6: Logistic regression of anomalous cell (mean ± SD/1000 cells) for HNC risk.

Anomalous cell

(mean)
Controls/patients
Crude OR (95% CI)

Adjusted ORb (95% CI)

Adjusted ORc (95% CI)

MNa

    Low (≤3.00)
44/12
1.00 (reference)

1.00 (reference)

1.00 (reference)

    High (>3.00)
13/33
8.63 (3.24–22.98)
<0.0001
18.13 (5.00–65.62)
<0.0001
69.06 (5.50–185.12)
0.001

BNa

    Low (≤4.10)
45/17
1.00 (reference)

1.00 (reference)

1.00 (reference)

    High (>4.10)
12/28
5.62 (2.07–15.24)
0.001
4.91 (1.60–14.66)
0.004
1.87 (0.31–11.11)
NS

OR: odds ratio, CI: confidence interval.

aThe 75th percentiles of the controls’ MN and BN frequencies were used as the cutoffs to assign the study subjects to either the low-frequency group or the high-frequency group.

bAdjusted for age and gender.

cAdjusted for age, gender, tobacco (smoking and chewing), alcohol, and occupational exposure.

4. Discussion

Micronuclei have been proposed as a good biomarker to assess cytogenetic damage in biomonitoring studies. Many investigations have shown an association between risk factors for squamous cell carcinomas in the head and neck and the MN rate [27–31]. In the present study, HNC patients’ numbers of MN and BN cells were significantly higher than those of controls (Table 2). These observations indicate genetic damage [32], which correlates with cancer of the oral cavity [33]. Our findings were in accordance with several recent case-control studies; they revealed that spontaneous genetic damage in exfoliated buccal cell MN frequencies of patients was significantly higher than that of controls and thus genetic instability appeared to exist in exfoliated buccal cell MN frequencies of HNC patients [34–38]. Another study of HNC patients undergoing radiotherapy observed increased genomic instability in somatic cells (exfoliated buccal epithelia) in comparison to healthy control subjects [39–41].

Gender and age are considered the most important demographic variables affecting the MN index. In the present study, the MN frequency in exfoliated cells of buccal mucosa of women was significantly higher than that of men among control groups (). Furthermore, cases’ cell anomaly numbers of MN and BN were mostly significantly higher than those of controls () after controlling the gender and the age (Table 3). Our results were in accordance with Cao et al.’s [36] study that revealed a significant difference of MN frequency between oral cancer patients and controls after controlling the gender and the age. Although many studies report the age and sex of the study subjects, only a fraction of these studies were able to establish a statistically significant effect by gender [42, 43] or by age [44–46]. In another study of healthy subjects from Poland, neither age nor sex was significantly associated with MN [47]. The increase of MN with age is likely due to a combination of factors which include (a) the cumulative effect of acquired mutations in genes involved in DNA repair, chromosome segregation, and cell cycle checkpoint and (b) numerical and structural aberrations in chromosomes caused by exposure to endogenous and exogenous genotoxins [48]. The increase in MN frequency in females can be accounted for by the greater tendency of the X chromosome to be lost as an MN relative to other chromosomes and to the fact that females have two copies of the chromosome compared to only one in males [48].

Although mass screening to identify precancers with malignant potential for oral cancer is not feasible [12], some biomarkers are essential for this identification. Some oral oncologists report that buccal cell changes are used to categorize the molecular mechanism associated with tobacco use and oral cancer and are hence considered as good biomarkers for early detection of oral cancer [49]. This is because buccal epithelial cells are first to be exposed and interact with the xenobiotic compounds such as tobacco (nicotine), which in turn induces the frequency of micronuclei under the influence of saliva [44, 50]. Our results, showing MN and BN cell frequencies of cases of buccal mucosa which were mostly significantly higher than those of controls for tobacco smoking and chewing risk factors of HNC (Table 5). In previous studies, a significantly higher frequency of anomalous buccal cells was revealed in smokers compared to nonsmokers [47, 51, 52]. However, in the present study no significant difference between smokers and nonsmokers in the frequency of MN was observed in cancer patients and healthy controls (Table 5). Recently, Ladeira et al. [53] found that smoking habits did not influence the frequency of the biomarkers, whereas alcohol consumption only influenced the MN frequency in controls (), with drinkers showing higher mean values. Yet other publications report no difference between smokers and nonsmokers [28]. Stich and Rosin [49] concluded that neither alcohol nor smoking, alone, increased MN frequency in buccal cells, but a combination of both resulted in a significant elevation in micronucleated cells in the buccal mucosa. However, the synergism between alcohol consumption and tobacco has not been observed to act upon all biomarkers and, in several studies of lifestyle factors, it was difficult to differentiate the effect of alcohol from that of smoking [13].

In the last 20 years the MN assay has been applied to evaluate chromosomal damage for biological monitoring of human populations occupationally exposed to a variety of mutagenic and carcinogenic chemical or physical agents. Many studies report a statistically significant elevation of MN levels in exposed individuals compared to control groups [45, 54–56], and many other studies report changes that were not statistically significant [57, 58]. In this study, anomalous cell (MN and BN) frequencies of cases of buccal mucosa were mostly significantly higher than those of controls for participants occupationally exposed (Table 5). Our observation is in agreement with several earlier reports [53, 55, 59–65]. Long-term exposures to chemicals and exposure conditions, such as those to which some workers are subjected for occupational reasons, are suspected to be associated with genotoxic effects, which can be evaluated by analysis of biomarkers. In reviews of the literature, Bolognesi [66], Bull et al. [67], and Holland et al. [13] concluded that occupational exposure was associated with an increase in DNA damage, but a number of studies failed to detect excess cytogenetic damage compared with nonexposed populations. Although several cytogenetic biomonitoring studies on workers exposed to chemical products have been reported, there is only limited information on this topic from developing countries where chemical products have been widely used over the years.

Regarding a connection between amalgam fillings status and anomalies rate, it became clear that particularly the amalgam fillings correlated with the MN and BN numbers (Tables 4 and 5). 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. [68, 69]. Those authors found in vitro that extracts from five common dental composites by a majority displayed mutagenic effects (MN induction in fibroblast cell line V79), and they demanded a replacement of the mutagenic composite parts by biocompatible substances. This is of relevance as synthetic dental materials have (in)-direct contact with the oral mucosa [70] and unpolymerized monomers (e.g., 2-hydroxyethyl methacrylate, bisphenol-A-glycidyl methacrylate, and methyl methacrylate) may affect these. However, regarding amalgam fillings it is known that they form a potential permanent source of organic-bonded mercury (mainly methyl mercury) which possesses toxic characteristics and in whose formation bacteria of the oral cavity (streptococci) are involved [71].

It is not clear whether the increase of MN and BN frequencies is due to cancer development. It is possible that MN and BN frequencies before the onset of cancer were normal, but they increased after the beginning of the disease and could be a consequence of the disease status. The increased frequency of micronucleus in cancer patients may reflect an increased susceptibility of these subjects to chromosomal damage. This hypothesis was strongly supported by a case-control study nested in a large European cohort, which followed up healthy subjects examined with cytogenetic assays for cancer incidence [72]. Findings from this study showed that the higher frequency of chromosomal damage observed in subjects who develop cancer thereafter was largely due to a higher individual susceptibility rather than to the exposure to carcinogens [73]. Several factors could explain cancer predictivity of MN, for example, environmental exposure to genotoxic agents, lifestyle factors, micronutrient deficiency, and genetic factors [74].

To summarize, we believe that micronuclei assay is an effective technique adopted for rapid risk assessment of HNC. At this time it remains unclear whether elevated frequencies of MN and BN in certain tissue, such as oral epithelia, would be predictive of increased risk of future cancer, say, only for oral cavity, limited to upper digestive tract epithelia, or may be projected for various cancers in other parts of the body. Our results indicate that the increased MN and BN frequencies in exfoliated cells of the buccal mucosa of patients with HNC may reflect genomic instability or deficiency of DNA repair capacity. Thus, MN assay may be performed in exfoliated cells of the buccal mucosa as an indicator of genomic instability relevant to head and neck tumorigenesis. Further, these results suggest that increased chromosome/DNA instabilities may be associated with HNC.

Abbreviations

BN: | Binucleated cells
HNC: | Head and neck cancer
MN: | Micronucleus.

Conflict of Interests

The authors communicated no conflict of interests.

Acknowledgments

This study was supported by the Ministry of Higher Education, Scientific Research, and Technology. The authors thank the Department of Otorhinolaryngology, Habib Bourguiba Hospital, Sfax, Tunisia, for the recruitment of patients. They thank all members of the Department of Otorhinolaryngology and specially Dr. Adel Chakroun and Dr. Bouthaina Hammami for their efforts and assistance in recruiting patients.

References

B. Rodu and C. Jansson, “Smokeless tobacco and oral cancer: a review of the risks and determinants,” Critical Reviews in Oral Biology and Medicine, vol. 15, no. 5, pp. 252–263, 2004.

K. Jensen, A. B. Jensen, and C. Grau, “Smoking has a negative impact upon health related quality of life after treatment for head and neck cancer,” Oral Oncology, vol. 43, no. 2, pp. 187–192, 2007.

C. Daneş, “Head and neck occupational cancer ethypathologenic aspects,” Acta Madica Transilvanica, vol. 21, pp. 266–268, 2012.

R. Khlifi, P. Olmedo, F. Gil et al., “Blood nickel and chromium levels in association with smoking and occupational exposure among head and neck cancer patients in Tunisia,” Environmental Science and Pollution Research, 2013.

U. A. Harréus, N. H. Kleinsasser, S. Zieger et al., “Sensitivity to DNA-damage induction and chromosomal alterations in mucosa cells from patients with and without cancer of the oropharynx detected by a combination of Comet assay and fluorescence in situ hybridization,” Mutation Research, vol. 563, no. 2, pp. 131–138, 2004.

N. H. Kleinsasser, B. C. Wallner, C. Wagner, E. R. Kastenbauer, and U. A. Harréus, “DNA repair capacity in lymphocytes of nasopharyngeal cancer patients,” European Archives of Oto-Rhino-Laryngology, vol. 262, no. 7, pp. 561–566, 2005.

S. D. Spivack, G. J. Hurteau, R. Jain et al., “Gene-environment interaction signatures by quantitative mRNA profiling in exfoliated buccal mucosal cells,” Cancer Research, vol. 64, no. 18, pp. 6805–6813, 2004.

F. D. Martinez, “Gene-environment interactions in asthma and allergies: a new paradigm to understand disease causation,” Immunology and Allergy Clinics of North America, vol. 25, no. 4, pp. 709–721, 2005.

H. E. Gabriel, J. W. Crott, H. Ghandour et al., “Chronic cigarette smoking is associated with diminished folate status, altered folate form distribution, and increased genetic damage in the buccal mucosa of healthy adults,” American Journal of Clinical Nutrition, vol. 83, no. 4, pp. 835–841, 2006.

C. Chen, M. Arjomandi, H. Qin, J. Balmes, I. Tager, and N. Holland, “Cytogenetic damage in buccal epithelia and peripheral lymphocytes of young healthy individuals exposed to ozone,” Mutagenesis, vol. 21, no. 2, pp. 131–137, 2006.

G. Speit and O. Schmid, “Local genotoxic effects of formaldehyde in humans measured by the micronucleus test with exfoliated epithelial cells,” Mutation Research, vol. 613, no. 1, pp. 1–9, 2006.

N. K. Proia, G. M. Paszkiewicz, M. A. S. Nasca, G. E. Franke, and J. L. Pauly, “Smoking and smokeless tobacco-associated human buccal cell mutations and their association with oral cancer—a review,” Cancer Epidemiology Biomarkers and Prevention, vol. 15, no. 6, pp. 1061–1077, 2006.

N. Holland, C. Bolognesi, M. Kirsch-Volders et al., “The micronucleus assay in human buccal cells as a tool for biomonitoring DNA damage: the HUMN project perspective on current status and knowledge gaps,” Mutation Research, vol. 659, no. 1-2, pp. 93–108, 2008.

H. F. Stich and M. P. Rosin, “Quantitating the synergistic effect of smoking and alcohol consumption with the micronucleus test on human buccal mucosa cells,” International Journal of Cancer, vol. 31, no. 3, pp. 305–308, 1983.

W. W. Au, “Cytogenetic assays in monitoring human exposure and prediction of risk,” Environmental Mutagens, Carcinogens, and Teratogens, vol. 23, pp. 236–245, 1991.

M. Fenech, N. Holland, W. P. Chang, E. Zeiger, and S. Bonassi, “The HUman MicroNucleus Project—an international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans,” Mutation Research, vol. 428, no. 1-2, pp. 271–283, 1999.

M. Fenech, “The in vitro micronucleus technique,” Mutation Research, vol. 455, no. 1-2, pp. 81–95, 2000.

M. Fenech, P. Baghurst, W. Luderer et al., “Low intake of calcium, folate, nicotinic acid, vitamin E, retinol, β-carotene and high intake of pantothenic acid, biotin and riboflavin are significantly associated with increased genome instability—results from a dietary intake and micronucleus index survey in South Australia,” Carcinogenesis, vol. 26, no. 5, pp. 991–999, 2005.

K. Umegaki and M. Fenech, “Cytokinesis-block micronucleus assay in WIL2-NS cells: a sensitive system to detect chromosomal damage induced by reactive oxygen species and activated human neutrophils,” Mutagenesis, vol. 15, no. 3, pp. 261–269, 2000.

R. Mateuca, N. Lombaert, P. V. Aka, I. Decordier, and M. Kirsch-Volders, “Chromosomal changes: induction, detection methods and applicability in human biomonitoring,” Biochimie, vol. 88, no. 11, pp. 1515–1531, 2006.

M. Fenech, W. P. Chang, M. Kirsch-Volders, N. Holland, S. Bonassi, and E. Zeiger, “HUMN project: detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures,” Mutation Research, vol. 534, no. 1-2, pp. 65–75, 2003.

M. G. Martino-Roth, J. Viégas, M. Amaral, L. Oliveira, F. L. S. Ferreira, and B. Erdtmann, “Evaluation of genotoxicity through micronuclei test in workers of car and battery repair garages,” Genetics and Molecular Biology, vol. 25, no. 4, pp. 495–500, 2002.

S. B. A. Ayyad, E. Israel, M. El-Setouhy, G. R. Nasr, M. K. Mohamed, and C. A. Loffredo, “Evaluation of papanicolaou stain for studying micronuclei in buccal cells under field conditions,” Acta Cytologica, vol. 50, no. 4, pp. 398–402, 2006.

P. E. Tolbert, C. M. Shy, and J. W. Allen, “Micronuclei and other nuclear anomalies in buccal smears: methods development,” Mutation Research, vol. 271, no. 1, pp. 69–77, 1992.

N. Titenko-Holland, R. A. Jacob, N. Shang, A. Balaraman, and M. T. Smith, “Micronuclei in lymphocytes and exfoliated buccal cells of postmenopausal women with dietary changes in folate,” Mutation Research, vol. 417, no. 2-3, pp. 101–114, 1998.

D. M. Roberts, “Comparative cytology of the oral cavities of snuff users,” Acta Cytologica, vol. 41, no. 4, pp. 1008–1014, 1997.

M. Fenech, “Chromosomal biomarkers of genomic instability relevant to cancer,” Drug Discovery Today, vol. 7, no. 22, pp. 1128–1137, 2002.

M. Bloching, A. Hofman, A. Berghaus, C. Lautenschläger, and T. Grummt, “Micronuclei as biological markers for the detection of local cancer transformation in the upper aerodigestive tract,” HNO, vol. 48, pp. 444–450, 2000.

F. J. Aceves Avila, G. A. Esquivel Nava, M. P. Gallegos Arreola, B. C. Gómez Meda, G. M. Zúñiga González, and C. Ramos-Remus, “Cyclophosphamide boluses induce micronuclei expression in buccal mucosa cells of patients with systemic lupus erythematosus independent of cytochrome P450 2D6 status,” Journal of Rheumatology, vol. 31, no. 7, pp. 1335–1339, 2004.

C. Ramos-Remus, G. Dorazco-Barragan, F. J. Aceves-Avila et al., “Genotoxicity assessment using micronuclei assay in rheumatoid arthritis patients,” Clinical and Experimental Rheumatology, vol. 20, no. 2, pp. 208–212, 2002.

S. R. D. A. Reis, M. Sadigursky, M. G. S. Andrade, L. P. Soares, A. R. D. Espirito Santo, and D. S. Vilas Boas, “Genotoxic effect of ethanol on oral mucosa cells,” Brazilian Oral Research, vol. 16, no. 3, pp. 221–225, 2002.

L. E. Moore, M. L. Warner, A. H. Smith, et al., “Use of fluorescence micronucleus assay to detect the genotoxic effects of radiation and arsenic exposure in exfoliated human epithelial cells,” Environmental and Molecular Mutagenesis, vol. 27, no. 3, pp. 176–184, 1996.

M. P. R. Prasad, M. A. Mukundan, and K. Krishnaswamy, “Micronuclei and carcinogen DNA adducts as intermediate end points in nutrient intervention trial of precancerous lesions in the oral cavity,” European Journal of Cancer B, vol. 31, no. 3, pp. 155–159, 1995.

R. Saran, R. K. Tiwari, P. P. Reddy, and Y. R. Ahuja, “Risk assessment of oral cancer in patients with pre-cancerous states of the oral cavity using micronucleus test and challenge assay,” Oral Oncology, vol. 44, no. 4, pp. 354–360, 2008.

S. Burgaz, E. Coskun, G. C. Demircigil et al., “Micronucleus frequencies in lymphocytes and buccal epithelial cells from patients having head and neck cancer and their first-degree relatives,” Mutagenesis, vol. 26, no. 2, pp. 351–356, 2011.

J. Cao, H. W. Liu, X. S. Liu, J. Q. Jin, and P. Zhang, “Correlation between the quantity of oral mucosal micronucleus cells and cancerization,” Beijing Da Xue Xue Bao, vol. 43, no. 4, pp. 600–602, 2011.

A. C. Pellicioli, F. Visioli, L. A. Ferreira, et al., “Cytogenetic abnormalities in exfoliated oral mucosal cells and their association with oral cancer,” Analytical and Quantitative Cytology and Histology, vol. 33, no. 5, pp. 271–276, 2011.

L. T. Dórea, J. R. Meireles, J. P. Lessa, et al., “Chromosomal damage and apoptosis in exfoliated buccal cells from individuals with oral cancer,” International Journal of Dentistry, vol. 2012, Article ID 458054, 2012.

F. Duffaud, T. Orsière, L. Digue et al., “Micronucleated lymphocyte rates from head-and-neck cancer patients,” Mutation Research, vol. 439, no. 2, pp. 259–266, 1999.

E. M. Minicucci, L. P. Kowalski, M. A. C. Maia et al., “Cytogenetic damage in circulating lymphocytes and buccal mucosa cells of head-and-neck cancer patients undergoing radiotherapy,” Journal of Radiation Research, vol. 46, no. 2, pp. 135–142, 2005.

H. Hintzsche, B. Polat, V. Schewe, et al., “Micronucleus formation kinetics in buccal mucosa cells of head and neck cancer patients undergoing radiotherapy,” Toxicology Letters, vol. 212, no. 1, pp. 33–37, 2012.

M. E. Gonsebatt, L. Vega, A. M. Salazar et al., “Cytogenetic effects in human exposure to arsenic,” Mutation Research, vol. 386, no. 3, pp. 219–228, 1997.

S. Pastor, S. Gutiérrez, A. Creus, N. Xamena, S. Piperakis, and R. Marcos, “Cytogenetic analysis of Greek farmers using the micronucleus assay in peripheral lymphocytes and buccal cells,” Mutagenesis, vol. 16, no. 6, pp. 539–545, 2001.

Y. Ozkul, H. Donmez, A. Erenmemisoglu, H. Demirtas, and N. Imamoglu, “Induction of micronuclei by smokeless tobacco on buccal mucosa cells of habitual users,” Mutagenesis, vol. 12, no. 4, pp. 285–287, 1997.

G. J. F. Gattás, L. de Almeida Cardoso, M. de Araújo Medrado-Faria, and P. H. Saldanha, “Frequency of oral mucosa micronuclei in gas station operators after introducing methanol,” Occupational Medicine, vol. 51, no. 2, pp. 107–113, 2001.

D. Pinto, J. M. Ceballos, G. García et al., “Increased cytogenetic damage in outdoor painters,” Mutation Research, vol. 467, no. 2, pp. 105–111, 2000.

M. Konopacka, “Effect of smoking and aging on micronucleus frequencies in human exfoliated buccal cells,” Neoplasma, vol. 50, no. 5, pp. 380–382, 2003.

M. Fenech and S. Bonassi, “The effect of age, gender, diet and lifestyle on DNA damage measured using micronucleus frequency in human peripheral blood lymphocytes,” Mutagenesis, vol. 26, no. 1, pp. 43–49, 2011.

H. Stich and M. Rosin, “Micronuclei in exfoliated human cells as an internal dosimeter for exposure to carcinogens,” in Carcinogens and Mutagens in the Environment. Naturally Occurring Compounds, Endogenous Formation and Modulation, H. F. Stich, Ed., vol. 2, pp. 17–25, CRC Press, Boca Raton, Fla, USA, 1983.

N. K. Mondal, S. Ghosh, and M. R. Ray, “Micronucleus formation and DNA damage in buccal epithelial cells of Indian street boys addicted to gasp 'Golden glue',” Mutation Research, vol. 721, no. 2, pp. 178–183, 2011.

A. Haveric, S. Haveric, and S. Ibrulj, “Micronuclei frequencies in peripheral blood and buccal exfoliated cells of young smokers and non-smokers,” Toxicology Mechanisms and Methods, vol. 20, no. 5, pp. 260–266, 2010.

A. Nersesyan, R. Muradyan, M. Kundi, and S. Knasmueller, “Impact of smoking on the frequencies of micronuclei and other nuclear abnormalities in exfoliated oral cells: a comparative study with different cigarette types,” Mutagenesis, vol. 26, no. 2, pp. 295–301, 2011.

C. Ladeira, S. Viegas, E. Carolino, J. Prista, M. C. Gomes, and M. Brito, “Genotoxicity biomarkers in occupational exposure to formaldehyde: the case of histopathology laboratories,” Mutation Research, vol. 721, no. 1, pp. 15–20, 2011.

A. Çelik, T. Çavaş, and S. Ergene-Gözükara, “Cytogenetic biomonitoring in petrol station attendants: micronucleus test in exfoliated buccal cells,” Mutagenesis, vol. 18, no. 5, pp. 417–421, 2003.

A. Celik, S. Yildirim, S. Y. Ekinci, and B. Taşdelen, “Bio-monitoring for the genotoxic assessment in road construction workers as determined by the buccal micronucleus cytome assay,” Ecotoxicology and Environmental Safety, vol. 92, pp. 265–270, 2013.

P. Rohr, J. da Silva, F. R. da Silva, et al., “Evaluation of genetic damage in open-cast coal mine workers using the buccal micronucleus cytome assay,” Environmental and Molecular Mutagenesis, vol. 54, no. 1, pp. 65–71, 2013.

S. Pastor, S. Gutiérrez, A. Creus, A. Cebulska-Wasilewska, and R. Marcos, “Micronuclei in peripheral blood lymphocytes and buccal epithelial cells of Polish farmers exposed to pesticides,” Mutation Research, vol. 495, no. 1-2, pp. 147–156, 2001.

S. Pastor, A. Creus, N. Xamena, C. Siffel, and R. Marcos, “Occupational exposure to pesticides and cytogenetic damage: results of a Hungarian population study using the micronucleus assay in lymphocytes and buccal cells,” Environmental and Molecular Mutagenesis, vol. 40, no. 2, pp. 101–109, 2002.

S. Burgaz, O. Erdem, G. Čakmak, N. Erdem, A. Karakaya, and A. E. Karakaya, “Cytogenetic analysis of buccal cells from shoe-workers and pathology and anatomy laboratory workers exposed to n-hexane, toluene, methyl ethyl ketone and formaldehyde,” Biomarkers, vol. 7, no. 2, pp. 151–161, 2002.

S. B. Vuyyuri, M. Ishaq, D. Kuppala, P. Grover, and Y. R. Ahuja, “Evaluation of micronucleus frequencies and DNA damage in glass workers exposed to arsenic,” Environmental and Molecular Mutagenesis, vol. 47, no. 7, pp. 562–570, 2006.

G. M. D. Bortoli, M. B. D. Azevedo, and L. B. D. Silva, “Cytogenetic biomonitoring of Brazilian workers exposed to pesticides: micronucleus analysis in buccal epithelial cells of soybean growers,” Mutation Research, vol. 675, no. 1-2, pp. 1–4, 2009.

O. Milosevic-Djordjevic, D. Grujičić, Ž. Vasković, and D. Marinković, “High micronucleus frequency in peripheral blood lymphocytes of untreated cancer patients irrespective of gender, smoking and cancer sites,” Tohoku Journal of Experimental Medicine, vol. 220, no. 2, pp. 115–120, 2010.

L. N. Rickes, M. C. Alvarengo, T. M. Souza, G. L. Garcias, and M. G. Martino-Roth, “Increased micronucleus frequency in exfoliated cells of the buccal mucosa in hairdressers,” Genetics and Molecular Research, vol. 9, no. 3, pp. 1921–1928, 2010.

C. Cassini, C. Calloni, G. Bortolini et al., “Occupational risk assessment of oxidative stress and genotoxicity in workers exposed to paints during a working week,” International Journal of Occupational Medicine and Environmental Health, vol. 24, no. 3, pp. 308–319, 2011.

S. Qayyum, A. Ara, and J. A. Usmani, “Effect of nickel and chromium exposure on buccal cells of electroplaters,” Toxicology and Industrial Health, vol. 28, no. 1, pp. 174–182, 2012.

C. Bolognesi, “Genotoxicity of pesticides: a review of human biomonitoring studies,” Mutation Research, vol. 543, no. 3, pp. 251–272, 2003.

S. Bull, K. Fletcher, A. R. Boobis, and J. M. Battershill, “Evidence for genotoxicity of pesticides in pesticide applicators: a review,” Mutagenesis, vol. 21, no. 2, pp. 93–103, 2006.

H. Schweikl, G. Schmalz, and T. Spruss, “The induction of micronuclei in vitro by unpolymerized resin monomers,” Journal of Dental Research, vol. 80, no. 7, pp. 1615–1620, 2001.

H. Schweikl, K.-A. Hiller, C. Bolay et al., “Cytotoxic and mutagenic effects of dental composite materials,” Biomaterials, vol. 26, no. 14, pp. 1713–1719, 2005.

P. Gigola, S. Monarca, D. Feretti, I. Zerbini, and D. D'Argenio, “Evaluation of the clastogenic activity of some resins used in the prosthodontic field,” Minerva stomatologica, vol. 50, no. 11-12, pp. 361–371, 2001.

U. Heintze, S. Edwardsson, T. Derand, and D. Birkhed, “Methylation of mercury from dental amalgam and mercuric chloride by oral streptococci in vitro,” Scandinavian Journal of Dental Research, vol. 91, no. 2, pp. 150–152, 1983.

N. Watanabe, H. Kanegane, S. Kinuya et al., “The radiotoxicity of 131I therapy of thyroid cancer: assessment by micronucleus assay of B lymphocytes,” Journal of Nuclear Medicine, vol. 45, no. 4, pp. 608–611, 2004.

S. Bonassi, L. Hagmar, U. Strömberg et al., “Chromosomal aberrations in lymphocytes predict human cancer independently of exposure to carcinogens,” Cancer Research, vol. 60, no. 6, pp. 1619–1625, 2000.

S. Beetstra, C. Salisbury, J. Turner et al., “Lymphocytes of BRCA1 and BRCA2 germ-line mutation carriers, with or without breast cancer, are not abnormally sensitive to the chromosome damaging effect of moderate folate deficiency,” Carcinogenesis, vol. 27, no. 3, pp. 517–524, 2006.