LAUSR

Dear author, We have read with interest your letter and we are here in providing answers for your concerns. The author questioned the use of Giemsa as staining method. Giemsa technique has been criticized because it is not a specific staining technique for DNA which could result in a low specificity of the test compared to DNAspecific staining tests [1]. However, the literature shows that many studies use Giemsa staining [2–6] to evaluate micronuclei frequency. In 2010, a study using Giemsa as a dye was published, confirming that the micronucleus technique, even with the Giemsa dying, was associated with cellular polymorphism confirmed by polymerase chain reaction technique [7]. Recently, our research group in a systematic review and meta-analysis showed that smoking habit increases the frequency of micronuclei in the oral mucosa of adults relative to non-smokers [8]. In this review, primary studies that used different staining techniques were included (Giemsa, Papanicolaou and Feulgen, and others). In response to a letter submitted to CLOI [9], our research conducted a sensitivity analysis excluding studies that used Giemsa and Papanicolaou staining techniques, and yet the overall conclusion (smokers having more micronuclei than non-smokers) was not altered [10]. Another question refers to the total number of epithelial cells evaluated, less than in 2000. The HUman MicroNucleus (HUMN) collaborative program has attempted to unify the protocol of the micronucleus technique [11–13]. However, advantages such as cost and easy suitability were lost, whereas, there was a gain in the specificity of the technique and speed of reading. To our understanding, the disadvantages of the HUMN are the reasons of why the protocol described by the HUMN is not yet a consensus. Also, a closer analysis of the clinical studies that evaluated bleaching techniques [14–20] showed that a more simplified protocol (1000 cells per volunteer) has been more frequently used (Table 1). We agree with the author that dental bleaching and hydrogen peroxide are genotoxic and cytotoxicity in vitro [21, 22], as well as pointed in Monteiro’s study[19] []. We also agree about the importance of evaluating different metanuclear changes indicative of cytotoxicity. The authors will take this into account in the future studies when micronucleus technique was to be used. However, there is a lack of correlation between in vivo and in vitro results regarding biological tests. It is necessary to develop in vitro tests that enhance the predictability of materials in a clinical scenario [23]. Differences in the results of in vivo and in vitro studies are probably due to different experimental conditions under which cell cultures were exposed. Usually, cells in their natural in vivo environment are much more capable of resisting the genotoxic effect of free radicals than are cells in culture [14] and this is why in vivo studies allow evaluation of the effects of dental materials in their natural setting [15]. This is the main reason why clinical studies are at the top of the evidence hierarchy [24]. A closer view of clinical studies that evaluated bleaching materials and techniques showed controversial results in terms of micronucleus frequency (Table 1). This could be explained by the variability of the bleaching materials and protocols employed, as the different methodologies used to evaluate the number of micronucleus in the cells (Table 1). In addition to this, there is a broad spectrum of other factors characterizing each of the participants taking part in a clinical study [25] that may modulate the effect of the bleaching materials on genotoxicity parameters, not evaluated yet in most of the bleaching studies [14–17, 19, 20].  Alessandro D. Loguercio [email protected]