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Medical University of Vienna
Institute of Cancer Research
Environmental Toxicology Group
Borschkegasse 8A, A-1090
Vienna, Austria
E-mail: armen.nersesyan{at}meduniwien.ac.at
Dear Sir:
Gabriel et al (1) showed increased micronuclei frequency in exfoliated buccal mucosal cells (EBMC) of tobacco (ie, cigarettes) smokers. Careful reading of the article raised some questions.
In the micronuclei assay for EBMCs, 2 aspects are crucial. First, the number of studied cells, and second, the staining—either with a DNA-specific or DNA-nonspecific stain (2, 3). Gabriel et al (1) did not present a very important piece of information, ie, the number of EBMC studied per subject. Also, the authors used May-Grünwald-Giemsa, a DNA-nonspecific stain, in the study.
The authors wrote in the introduction that, "the frequency of micronuclei is consistently elevated in exfoliated buccal mucosal cells of tobacco smokers" and cited 6 references. However, this may be regarded as a wrong interpretation of the cited literature sources. Bloching et al (4; reference 10 in the original paper) studied micronuclei in smokers with either cancer or precancerous lesions (leucoplakia) of the mucosa. However, in that study, even the control subjects were smokers. An elevated member of micronuclei in these patients could have been due to tumors themselves, because it is known that the presence of a tumor in the organism could be the source of an increased number of cells with micronuclei in exfoliated cells (5, 6). It is not possible to evaluate the effect of smoking on micronuclei incidence on the basis of that article, only the effect of the number of smoked cigarettes (24.3 cigarettes/d during 32 y in tumor patients, 20.6 cigarettes/d during 30.4 y in leucoplakia patients, and 5.3 cigarettes/d during 7.2 y in controls) can be evaluated. In the study by Stich and Rosin (7; reference 11 in the original paper), the number of micronuclei in EBMC of heavy smokers (30 cigarettes/d) was the same as in nonsmokers (0.30% and 0.29%, respectively). Suhas et al (8; reference 12 in the original paper) showed an increased micronuclei frequency in smokers of bidi [an indigenous cigarette in which low-grade tobacco is hand-rolled in a tendu (Diospyros melanoxylon) leaf and tied with a cotton thread]. It is known (8) that the concentrations of nicotine, tar, and other toxic agents in smoke from burnt tobacco are higher in bidis than in cigarettes (this is also due to the absence of a filter in bidis). The data presented by Wu et al (9; reference 14 in the original paper) were not clear, and I wrote a Letter to the Editor with some criticisms (10). In reply, the authors responded that cigarette smoking did not increase the number of micronuclei in the whole group of smokers compared with nonsmoking persons (1.144% and 1.033%, respectively) (11). A statistically significant positive relation was only found for heavy smokers (20 cigarettes/d) but not for light mokers (<20 cigarettes/d, data were not presented). The cited article by Kayal et al (12; reference 15 in the original paper) studied micronuclei in EBMC of persons who chewed indigenous products (areca nut, mava, tamol, tobacco with lime, dry snuff, or mashery), but who were not tobacco (cigarette) smokers. Hence, among 6 references, only one confirmed the statement of the authors.
In a recent study on the effect of cigarette smoking on micronuclei levels in EBMCs [28 long-term (chronic) smokers, 19 smokers and alcohol consumers, and 21 control subjects], no significant increase was observed (13). In that study, 2000 Feulgen-stained cells were evaluated per person, and, hence, the stain and the number of studied cells could not have been sources of bias. It is well known that chronic exposure of oral mucosa to toxic substances (eg, through tobacco smoking) leads to keratinisation with synthesis of keratine bodies (ie, granules) (14, 15). These round cytoplasmatic bodies do not contain DNA, but may be classified as micronuclei when using non-DNA-specific stains. Because Gabriel et al (1) used DNA-nonspecific May-Grünwald-Giemsa staining, the apparent 2.16-fold increase in micronuclei number in smokers could be due to this misinterpretation. In my opinion, the slides should be destained, stained with a DNA-specific stain, and reevaluated to obtain reliable results.
In conclusion, the authors presented interesting data concerning the important, but, until now, unanswered question of whether cigarette smoking is a micronuclei inductor in EBMC. At the same time, Gabriel et al should clarify some points of their study to give readers the possibility to fully understand and evaluate the results they obtained. Additional investigations in this area are certainly warranted to elucidate this problem.
ACKNOWLEDGMENTS
The author had no conflicts of interest.
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