INTRODUCTION

In the first studies conducted in the 1980s, exfoliated buccal mucosa cells were used to assess the genotoxic effects of betel- quid nuts and chewing tobacco.^1-6 Most studies showed higher frequencies of micronucleus (MN) at the site within the oral cavity where the quid or tobacco mixture was maintained compared to the opposite control site. The buccal cell MN assay was also used to study cancerous and precancerous lesions and to monitor the effects of a number of chemopreventive agents.^7-9

It is notable that the first Stich and Rosin studies conducted between 1983 and 1984 had higher reference MN frequencies than subsequent studies. This may have been due to the lack of defined scoring criteria and a relatively small number of cells analyzed (in some cases less than 500). Since then, published biomonitoring studies using the MN assay on buccal mucosa cells have investigated the effects of multiple factors including environmental and occupational exposures, radiation therapy, chemoprevention, vitamin supplementation trials, lifestyle habits, cancer, and other diseases.¹⁰

An important aspect of the MN assay in buccal mucosa cells is the criteria for identifying and scoring cells. The five publications by Stich and Rosin^1,2,8,11,12 refer to the basic criteria established for MN that were initially described by Heddle.¹³ However, criteria to identify cells for inclusion in the MN frequency count were not provided. Other authors refer to Heddle's criteria as such, or with minor modifications. The criteria developed by Tolbert et Alabama^14,15 for choosing cells are the most used. They consist of the following parameters for cell inclusion in the cells to be scored: (a) intact cytoplasm and relatively flat cell position on the slide; (b) little or no overlap with adjacent cells; (c) little or no dirt; and (d) normal and intact nucleus,smooth and distinct nuclear perimeter.15

Some meta-nuclear changes indicative of cytotoxicity when analyzing the micronucleus assay in the cells of the oral mucosa, such as picnosis, carolysis and karyorrhexis. The approach is very important because cytotoxicity is a confounding factor for mutagenesis^13,17 (a) rounded smooth perimeter suggesting a membrane; (b) less than a third of the diameter of the associated nucleus, but large enough to discern shape and color; (c) Feulgen positive, that is, rose in bright illumination field; (d) staining intensity similar to that of the nucleus; (e) core-like texture; (f) same focal plane as the nucleus; and (g) absence of overlap or bridge to the nucleus.¹⁵

Therefore, the objective of this review was to identify and determine the criteria to quantify MN in oral mucosa cells from previously conducted research.

MATERIAL AND METHODS

The review includes studies carried out on the publications of different investigations in humans and that specify the criteria for identifying micronuclei in cells of the oral mucosa. The literature search included the following keywords: "micronuclei" buccal mucosa cells, biomonitoring, biomarker, genotoxic damage, pesticides.

Searches were performed in literature databases including crossreff, JCR, Scopus, PubMed, Google academic in March 2020.

Inclusion and exclusion criteria

The inclusion criteria were biomonitoring human research using the buccal mucosa micronucleus test with limitation of having used Feulgen staining and specifically in exfoliated buccal mucosa cells, not including lymphocyte tests.

The extracted data included, research methodology, number and method of counting and identification, comparison between differences, number of participants and condition, during study follow-up.

The only exclusion criteria was the lack of clarity of the micronucleus identification criteria in the carried out studies.

RESULTS

Search for articles and data collection.

Our search retrieved 91 articles, of which 84 were articles made to a population that presented another type of exposure. In the end, six articles with results related to micronuclei in buccal mucosa cells of agricultural workers exposed to pesticides (specifying the criteria for staining and identification of micronuclei).

In the six selected articles, four presented statistically significant values ​​with the presence of MN when comparing the exposed groups with respect to the controls, and the criteria for staining, collection and number of cells evaluated to identify micronuclei were very varied.

Fig. 1
Fig. 1 Diagram of search and selection of articles.

Characteristics of the studies.

Of the seven full articles evaluated for micronuclei in buccal mucosa cells of agricultural workers exposed to pesticides as a result, one was excluded from the systematic review because this article was a compilation of results from four other articles. Therefore, six studies were selected for qualitative analysis.

Of which two articles refer to having carried out the collection of samples through cytobrush, two with a wooden spatula and two with a toothbrush, also in the sample staining method it is different three used shiff reagent, one Giemsa and one of staining that is mentioned in these articles as a specific DNA staining called DAPI (dimethyl, diphenylidol, dichloride) and the latter with acridine orange. Regarding the total cell count, three counted 2,000 per person, 1-3,000 per person and 2 out of 1,000 per study subject. The standardized MN buccal assay protocol clearly states that 2,000 buccal cells stained with specific DNA staining must be scored for reliable results.¹⁵

DISCUSSION

There are several advantages to using the MN frequency in exfoliated buccal cells, such as the specificity to detect the effects of exposure to inhaled or ingested genotoxic agents, and the easy storage of samples, before and after processing, in fixative solution or as fixed slides at room temperature.¹⁶

Of the total number of articles reviewed there is a great variety with respect to validated and standardized protocols, others do not consider them, although they do mention the authors of this technique and only mention that they make some modifications, of the discarded articles which carry out the tests. Other types of population such as hairdressers, smokers, gasoline dispensers to name a few, there is still more variability in the collection methods, staining, and number of cells evaluated.

Pesticides are widely used worldwide, and pesticides exposure remains a major environmental health problem. Of significant importance are the potentially dangerous effects on occupationally exposed subjects. Although the genotoxic potential of pesticides is believed to be low, genotoxic monitoring in farm worker populations could be a useful tool in estimating the risk of long-term health effects such as cancer and adverse reproductive health outcomes.^23,24

The systematic review of the six selected articles identified a difference in the quantity, method of obtaining and staining of the cells for the evaluation of the presence of micronuclei. Most of the studies involved a very different number of cells evaluated, the number and the lack of heterogeneity in the sample collection and evaluation process is considered a limiting factor for this review.

The Micronucleus Assay Guidelines have established that at least 2,000 cells should be performed per volunteer when evaluating the micronucleus assay in buccal mucosa cells.¹⁸

Some meta-nuclear changes indicative of cytotoxicity when analyzing the micronucleus assay in the cells of the oral mucosa, such as picnosis, karyolysis and koriorexis. The approach is very important because cytotoxicity is a confounding factor for mutagenesis. ^13,26

The validated and standardized protocol for MN oral cytoma assay. It must be considered in all the parameters suggested in the protocol, this will increase the reliability of the studies and will give the possibility of comparing the results obtained.15

CONCLUSION

The buccal cell MN assay is a promising tool for surveying workers exposed to genotoxic agents. However, it is very important to take into account the standardized and validated protocols in order to obtain more reliable and comparable results with other studies, despite the fact that they are populations that engage in different activities and are still human beings, which is why it is highly relevant to carry out obtaining, staining and number of cells evaluated according to protocols.

Conflict of interest: The authors declare that there is no conflict of interest for the publication of this article.

REFERENCES

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