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LETTER TO THE EDITOR |
Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University
711 Washington St
Boston
MA 02111
E-mail:jimmy.crott{at}tufts.edu
Dear Sir:
I am writing to address the concerns raised by Dr. Nersesyan regarding our recent study on the association between smoking status, folate levels, and micronuclei in exfoliated buccal mucosal cells (1).
With regard to our methodology, 1000 exfoliated buccal mucosal cells were scored per person for the presence of micronuclei by a blinded observer. In addition, the May Grunwald-Giemsa stain was used; this is a widely accepted stain for this application and allows micronuclei and nuclei to be clearly distinguished from each other and from the surrounding cytoplasm. Micronuclei were only scored when the chromatin texture and color intensity were similar to the main nucleus. After rigorous statistical analyses, we showed that the micronuclei frequency of smokers was approximately double that of nonsmokers [
(±SEM): 9.57 ± 0.41 compared with 4.44 ± 0.52 micronuclei/1000 cells; P < 0.0001] in our population.
Our findings agree with previous studies, which have convincingly shown that smokers have more buccal micronuclei than do nonsmokers. For example, Konopacka (2) showed that the frequency of buccal micronuclei in smokers was 
3-fold that of nonsmokers [ (±SEM): 1.50 ± 0.47% compared with 0.55 ± 0.32%, respectively; P < 0.05]. Similarly, Fontham et al (3) reported that the frequency of buccal micronuclei in smokers was 3-fold that in nonsmokers [ (±SEM): 0.24 ± 0.07% compared with 0.07 ± 0.05%, respectively; P < 0.05]. In addition, Sarto et al (4) and Piyathilake et al (5) both reported that the buccal micronuclei frequency in smokers was 2-fold that of nonsmokers. Although a non-DNA-specific stain was used in our study, our baseline micronuclei and fold change data agree well with most of the literature, including studies in which Feulgen-Fast Green stains were used (2, 4). Therefore, we feel that the misclassification of other cytoplasmic bodies as micronuclei is highly unlikely in our study.
Dr. Nersesyan states that it is well known that tobacco exposure induces the formation of micronuclei-like round keratin bodies; however, this is not supported by the articles cited. The article by Orellana-Bustos et al (6) studied the effect of smoking on nuclear staining abnormalities and the distribution of different types or epithelial cells (enucleated superficial cells, nucleated superficial cells, or intermediate cells) collected from the tongue; however, there is no detection or scoring of keratin bodies that resemble micronuclei. In the article by Casartelli (7), different staining techniques were compared (Hoechst 33258, propidium iodide, Feulgen, and Giemsa); however, the effect of tobacco, or any other mutagen, exposure was not tested.
A study to examine factors that contribute to the variation in micronuclei frequency was recently performed by the HUMN (Human Micronucleus) project (8). In this comparison of 33 laboratories in 20 countries, the effect of several variables on the observed micronuclei frequency, including 7 different staining methods, was tested. The results of this international collaborative study showed that staining technique contributed to a mere 0.5% variance in the micronuclei frequency.
Dr. Nersesyan also criticized papers cited by us (9-13) to support the notion that cigarette smokers have more micronucleated buccal cells than do nonsmokers. In one of these studies, Bloching et al (9) studied buccal micronuclei in healthy controls as well as patients with squamous cell carcinoma and leucoplakia. Contrary to Dr. Nersesyan's statement, the healthy control group did contain both smokers and nonsmokers. Within these controls, there is a clear trend for increased micronuclei with increasing cigarettes smoked per day (0–40 cigarettes/d; Figure 2 in the article) and increasing pack years (Figure 4 in the article). In the study by Stich and Rosin (10), smoking did not affect micronuclei in nondrinkers, but in people who drank alcohol there was a significantly higher (P < 0.0001) dose-dependent increase in micronuclei with increasing number of cigarettes smoked per day (Table 4 in the article).
Our citation of the articles by Suhas et al (11) and Kayal et al (13) was inappropriate because they studied tobacco products other than cigarettes. We thank Dr. Nersesyan for pointing out this oversight.
In conclusion, of the available reports on the effect of cigarette smoking on buccal cell micronuclei, 4 articles convincingly showed a statistically significant 2–3-fold micronuclei frequency in smokers compared with nonsmokers (1-4), 3 papers showed a trend for increased micronuclei in smokers (5, 9, 10), and 2 were unable to detect a difference (12, 14). Of the 2 negative studies, one may have been confounded by some additional environmental exposure, which may have concealed the effect of smoking because the control micronuclei frequency was rather high (1.03%) (12). In the other study, Bohrer et al (14) sampled cells from the lip, tongue, and mouth floor as opposed to the standard method of collecting from the inside of the cheek, a detail which may explain the disparity with other published studies. Thus, most of the studies to date, including our own study, support the notion that cigarette smoking is associated with, and probably causes, an increased frequency of micronucleated buccal cells. We nevertheless thank Dr. Nersesyan for his interest in our article as well as his comments and eagerly await his own studies on this topic.
ACKNOWLEDGMENTS
The author has no conflicts of interest.
REFERENCES
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