This study aimed to investigate cytological abnormalities indicative of chromosome damage (micronuclei) and apoptosis (karyorrhexis, pyknosis, and condensed chromatin) in exfoliated cells from the buccal mucosa of patients with oral cancer and control subjects. The sample included twenty individuals with oral cancer and forty individuals with normal buccal mucosa. Material was collected from the cheek epithelium in areas with lesions and areas without abnormalities. A minimum of one thousand cells was analyzed. Micronuclei were found significantly more frequently in cells collected from lesions than in cells from normal areas, independent of the presence/absence of cancer (
Oral cancer is among the ten types of malignant neoplasia of highest incidence worldwide and is particularly common in developing countries [
Similarl to other types of malignant neoplasia, oral cancer results from alterations (point mutations and chromosomal abnormalities) in genes that control the cell cycle, and/or in genes that are involved in DNA repair. In addition to the potential for metastasis, cancer is characterized by the loss of the ability of cells to evolve to death when genetic damage occurs (apoptosis) [
Occurrences of chromosomal damage in the oral epithelium can be evaluated using the micronucleus test, as suggested by Stich et al. [
In the present study, chromosome damage and apoptosis were investigated in exfoliated cells from the buccal mucosa of patients with oral cancer and control subjects, using the protocols suggested by Tolbert et al. and Thomas et al. [
Exfoliated cells from the buccal mucosa were obtained from twenty patients with oral cancer (case group) and forty individuals without oral lesions (control group). The individuals in both groups were attended by the dentistry services of Feira de Santana State University. Clinical examinations of oral cavity were performed on all individuals in the sample. Biopsies were performed by the dentist, and histopathological diagnoses were made by a pathologist within a specific service at this University. The sample was characterized using a questionnaire that asked about risk factors for oral cancer development: cigarette smoking, alcoholic beverage ingestion, oral hygiene, and mouthwash use. Individuals who, for at least one year, had been consumed three or more cigarettes/day were considered to be smokers [
The slides were analyzed under an optical microscope in a blinded manner. A minimum of 1,000 cells presenting intact cytoplasm were counted. The analysis protocol used was as suggested by Tolbert el al. and Thomas et al. [
Cells presenting a micronucleus (a) karyorrhexis (b), condensed chromatin (c), and pyknosis (d).
Differences between the mean ages of the groups were evaluated using Student’s
In accordance with Resolution number 196/1996 of the Brazilian National Health Board, all the participants signed an informed consent statement and full confidentiality was ensured. The study was approved by the Ethics Committee of Feira de Santana State University (Protocol number 059/2006).
The mean age ±SE of the whole sample was 55.53 ± 2.06. For the case and control groups, respectively, the means were 63.25 ± 3.49 and 51.68 ± 2.34. Student’s
Sample characteristics.
Characteristic | Group | ||||
Case | Control | ||||
% | % | ||||
Gender | |||||
Female | 9 | 45.0 | 27 | 67.5 | 0.094b |
Male | 11 | 55.0 | 13 | 32.5 | |
Tobacco consumption | |||||
Yes | 15 | 75.0 | 20 | 50.0 | 0.064b |
No | 5 | 25.0 | 20 | 50.0 | |
Drinker | |||||
Yes | 8 | 40.0 | 3 | 7.5 | 0.002a |
No | 12 | 60.0 | 37 | 92.5 | |
Tobacco consumption and drinker | |||||
Yes | 8 | 53.3 | 3 | 15.0 | 0.016a |
No | 7 | 46.7 | 17 | 85.0 | |
Oral hygiene | |||||
Good | 1 | 5.0 | 5 | 10.3 | 0.493b |
Poor | 19 | 95.0 | 35 | 89.7 | |
Mouthwash use | |||||
Yes | 5 | 25.0 | 5 | 12.5 | 0.221b |
No | 15 | 75.0 | 35 | 87.5 |
aSignificant; bnonsignificant.
Micronucleus occurrence was significantly higher in cells obtained from areas with lesions in the case group than in cells obtained from areas without lesions in both the case group and the control group (
Micronucleus (MN) analysis.
Group | MN (n°) | MN (‰) | Total cells | Comparison | ||
Mean ± SE | ||||||
CaseL.A | 20 | 76 | 41,079 | CaseL.A versus Control | 60.9647; | |
CaseN.A | 20 | 25 | 51,153 | CaseL.A versus CaseN.A | 38.5582; | |
Control | 40 | 41 | 89, 568 | CaseN.A versus Control | 0.0666; |
L.ALesion area, N.Anormal area, asignificant, bnonsignificant.
In comparing cells obtained from normal areas of the groups, no difference in micronucleus occurrence was observed in relation to age, gender, or oral hygiene. However, micronucleus occurrence was significantly higher in mouthwash users (
Data relating to micronucleus occurrence in smokers (A), nonsmokers and nondrinkers (B), and smokers and drinkers (C).
Subgroup | Micronucleus | Total cells | |||
---|---|---|---|---|---|
A | 24 | 35 | 61,983 | 8.4734 | A versus B: |
B | 25 | 15 | 55,734 | df = 2 | A versus C: |
C | 11 | 16 | 23,004 | B versus C: | |
Total | 60 | 66 | 140,721 |
aSignificant, bnonsignificant.
Data relative to degenerative nuclear alterations indicative of apoptosis are presented in Table
Degenerative nuclear alterations indicative of apoptosis observed.
Group | Total cells | Karyorrhexis | Condensed chromatin | Pyknosis | |
---|---|---|---|---|---|
CaseL.A | 20 | 41,079 | 334 | 592 | 175 |
CaseN.A | 20 | 51,153 | 393 | 803 | 136 |
Control | 40 | 89,568 | 1,803 | 3,349 | 77 |
L.ALesion area, N.Anormal area, asignificant, bnonsignificant.
As observed in Table
Apoptosis analysis (Σ karyorrhexis, condensed chromatin and pyknosis).
Group | Apoptosis (n°) | Apoptosis (‰) | Total cells | Comparison | ||
Mean ± SE | ||||||
CaseL.A | 20 | 1,101 | 41,079 | CaseL.A versus Control | 579.62; <0.0001a | |
CaseN.A | 20 | 1,332 | 51,153 | CaseL.A versus CaseN.A | 0.5021; = 0.4786b | |
Control | 40 | 5,229 | 89,568 | CaseN.A versus Control | 730.39; <0.0001a |
L.ALesion area, N.Anormal area, asignificant, bnonsignificant.
The first comparison (using cells from health tissues in the control group and cells from tumor tissues in the case group) shows that MN is the most important end point and age should be disregarded. The second comparison (using only cells from health tissues in both groups) shows that age becomes important and MN can be disregarded. These results are much sounded since the first comparison involves cells from tumor tissues and the second only cells from health tissues. After the models adjustment and the elimination of nonsignificant end points, we obtain the following models: comparing cells from tumor tissues in the case group with health tissues in the control group the logistic regression function is as follows:
comparing cells from health tissues in both groups, case and control, the logistic regression function change to the following:
Finally, calculating the values of these functions for all the sample unities we made use of the ROC curve to define cut-off values and then evaluate the sensibility and specificity of each of the two kinds of comparisons. The result was impressive since the sensibility for both comparisons were 80% and the specificity change from 95% in the first comparison to 85% in the second. The area under the ROC curve changes from .9462 for the first model to .8762 for the second model. This proves the good fit of both kinds of model to the data analyzed.
Occurrences of chromosome damage and their association with cancer development have been evaluated using the micronucleus assay in both lymphocytes and exfoliated cells from some types of epithelium [
The higher frequency of micronuclei in exfoliated cells from malignant lesions observed in this study corroborates the results described by several other authors [
In agreement with some results previously described [
The greater occurrence of micronuclei in mouthwash users was also observed in a study that evaluated the genotoxic effects of risk factors for oral cancer development [
Induction of micronuclei in exfoliated buccal cells consequent to smoking has generated controversy in the literature. It has been suggested that this association is dependent on the number of cigarettes consumed, since it was observed only among users of more than ten cigarettes/day [
The lower frequency of apoptosis observed in both the lesion and the normal areas in the case group indicate that, with evolution of malignant transformation, the apoptotic response fails, as also observed in precursor lesions of cervical cancer [
The results obtained in the present study show that oral cancer is associated with a higher frequency of chromosome damage and suggest that apoptosis is impaired in the buccal cells of individuals with this kind of neoplasia. Additionally, they suggest that tobacco and mouthwashes are effective in inducing chromosome damage. The inclusion of degenerative nuclear alteration indicative of apoptosis beside micronucleus is useful to biomonitoring oral cancer.
The authors are grateful to FAPESB for the financial support.