Are cytogenetic assays in exfoliated cells able to predict the development of oral cancer in chronic smokers?

We read the manuscript recently published by Cruz et al. ¹ in the Human and Experimental Toxicology titled “Association between DNA ploidy and micronucleus frequency in chronic smokers and impact of smoking cessation” with much interest. In this article, the authors detected high frequencies of micronucleated cells in chronic smokers when compared to control group. For ploidy evaluation, no significant differences were noticed among groups. However, it is important to properly discuss the scientific approach for a correct understanding the paper.

In Material and Methods, it was not mentioned how many cells were evaluated. According to the Micronucleus Assay Expert Group, it is strongly recommended to evaluate a minimum of 2,000 cells per volunteer. ² This information is very important to validate the data found by the authors. Moreover, it was stated that “For counting of the MN, the criterion used was the presence of a surrounding homogeneous halo, representative of a membrane, less than 1/3 of the diameter of the associated nucleus, intensity of Feulgen staining similar to the nucleus, and focal plane under microscopy, with no connection to the core.” It is important to highlight the importance of considering only oral mucosa cells with the intact cell membrane in order to avoid false-negative results (micronuclei would be lost due to rupture of cell membrane). In this sense, how was it possible to identify the integrity of cell membrane if no counterstain was performed? This is clearly shown in the Figure 1 of the manuscript.

In Table 2 and Figure 2, the authors scrutinize the analysis of micronucleus frequency as total number of micronucleus and total micronucleated cells. What is the relevance of these parameters evaluated? According to the micronucleus assay guidelines, there is no any biological significance with respect to the total number of micronucleus in the eukaryotic cell. Moreover, it is also not clear the statistical differences among groups evaluated. For example, the authors show the p value in the right column of Table 2 and letters “A” and “B” in Figure 2 saying that “A and B represents the homogeneous groups from the Dunn’s test.” The statistical difference between smokers and control group is obvious, but the differences between former smoker and control groups, as well as smokers and former smoker groups are obscure and so, difficult to interpret the data with accuracy. In the Results, it was written that “The averages for total MN found and for the total of micronucleated cells, as well as the comparison between the groups performed using the Kruskal–Wallis test (a ¼ 5%), can be seen in Table 2. The test indicated a statistically significant difference (Figure 2).” Taking into account the data available in the manuscript, we presume that there were significant statistically differences between control × former smoker groups and control × smokers. However, this requires further clarification.

Finally, it is important to mention that Tolbert et al. ³ described several metanuclear changes indicative of cytotoxicity for the micronucleus assay in exfoliated cells, such as karyorrhexis, pyknosis and karyolysis. The approach is mandatory because cytotoxicity is potential for bias in the micronucleus assay. If cytotoxicity is increased, the micronucleus frequency decreased because micronucleated cells are lost as a result of cellular death. It is well established that cigarette smoking is able to induce cytotoxicity, especially in chronic smokers as a result of metanuclear changes induced in oral mucosa cells^4–6 being dependent on type of cigarette as well as nicotine content. ⁷

We believe that these comments be useful for better understanding the important article for biomonitoring smokers as well as the impact of smoking cessation.