Re: Evaluation of Micronuclei and Cytomorphometric Changes in Patients with Different Tobacco Related Habits Using Exfoliated Buccal Cells

Dear Editor

Kokila et al., (2021) evaluated micronuclei (MN) in buccal cells obtained from smokers, smokeless tobacco and combined tobacco users (i.e. chewers and smokers). Based on the obtained results they stated that “tobacco in any consumable form is genotoxic”. As for smokeless tobacco users and combined tobacco users, the statement is correct (Chandirasekar et al., 2013; Nersesyan et al., 2019b). But genotoxic action of tobacco smoking on buccal cells is questionable (Bonassi et al., 2011; Metgud and Neelesh, 2018; de Geus et al., 2019; Nersesyan et al., 2019a; Nersesyan, 2020). This is not the only weak point of this publication. Careful reading of it shows that the data presented by the authors are not reliable due to several serious reasons.

It is well known that exists a standardized and validated protocol for evaluation of MN in buccal cells (Thomas et al., 2009). According to it, MN should be scored in 2,000 buccal cells. In addition, not only MN but also other than MN nuclear anomalies should be considered in 1,000 cells. The authors evaluated ONLY 100 cells, i.e. 20-fold less that recommended number of cell and nuclear anomalies were not considered. Kokila et al. (Kokila et al., 2021) stated that they applied the criteria of Tolbert et al. (Tolbert et al., 1992) for MN scoring. We hesitate if the authors read carefully the paper by Tolbert et al., (1992). Otherwise they ought to score 2,000 – 3,000 cells because it is clearly written that “if less than 5 MNC are observed after counting 1,000 cells, an additional 1,000 cells are scored, and so on up to a maximum count of 3,000 cells”. Also Tolbert et al. described criteria for evaluation of so-called nuclear anomalies for the first time. But this important point was also disregarded by Kokila et al. It is notable that in the first, pioneer studies of MN in buccal cells, 50 cells per individual were evaluated (Stich et al., 1982), then the number of cells was increased to 500/individual (Rosin and Ochs, 1986).

The authors have also serious problems with calculations. Indeed, in the Table 1 are presented mean number of cells with MN (micronucleated cells), mean number of MN and mean of MN per cell. Let us, for example, check the controls. Corresponding numbers are following: 1.20, 0.60 and 0.305. There is a fatal mistake since number of MN should not be lower that mean number of cells with MN (since cell with MN can have several MN). Since Kokila et al. declared that they scored only 100 cells, the numbers of mean MN per cell must be equal to mean numbers of MN divided by 100. But this is not the case. In group IV mean number of cells with MN is 1.20, mean number of MN is 0.6; hence, corresponding number in Table 2 must be 0.6 / 100 = 0.006. In the Table is indicated 0.305. No one number is correct in other cases! Another example with group III, the most exposed: the mean number of MN is 15.77, the mean MN per cell is 1.469 (instead of 0.1577). Again number of cell with MN is higher that mean number of MN!

Less serious but important gaps in the study are following. The title of the article is not correct. The authors declare that the cells were obtained from patients. But in “Materials and methods” section is written following: “a total number of 120 individuals without oral lesions were included in the study” and “Individuals with any history of systemic diseases and recent history of any viral infection or hospitalization, recent exposure to radiologic investigations, habituated with alcohol were excluded from the study”. So, why word “patients” is used in the title of the paper?

The authors mentioned that the slides were stained with Feulgen and Pap stain, possibly for comparison (reason is not mentioned by the authors). In the Results section they stated that “Results obtained were similar using either PAP or Feulgen stain in almost all the parameters evaluated” but no data were presented to support this statement. It should be noted that DNA-non-specific stains (Pap stain in this case) visualize keratin bodies in buccal cells which mimic MN (Nersesyan et al., 2006). That is why “non-DNA-specific stains give high false-positive results” (Metgud and Neelesh, 2018; Juneja et al., 2019).

The quality of photographic images (Figure 2 and Figure 3) is not satisfactory, especially Figure 3. In photos of such quality is not possible to evaluate MN.

A lot of important information is missing. For example, demographic data and description of microscopic examination. Demographic data are very important because MN formation can be influenced by sex, age, nutritional habits and body mass index (Nersesyan et al., 2022). Moreover, the authors did not presented data on smoking – how many cigarettes were consumed by each participant, what kind of cigarettes (content of nicotine and tar). This information is very important since MN formation depends on these factors (Bonassi et al., 2011; Nersesyan et al., 2011). Important also is information concerning microscope, i.e. producer and used magnification.

We may propose that serious errors in the publication could be due to technical reasons. But abovementioned points must be clarified by the authors to avoid confusion of the readers. In present form the results presented by Kokila et al., (2021) are misleading.

Conflict of interest

The authors declare that there is no conflict of interest.