Micronucleus in oral exfoliated cells and associated factors among young adults in the far south of Brazil

: There is an increasing interest in the application of micronucleus (MN) assays in the clinical setting to investing the impact of environmental, genetic, life style and socioeconomic conditions on DNA. However, the evaluation of mutagenic damage among young population had not been studied and early intervention in this population can prevent several health comorbidities. Thus, the present study aimed in investigate the MN presence in young adults and its associated factors. This is a cross-sectional


INTRODUCTION
Human genetic integrity is increasingly threatened by environmental exposure to genotoxins and pollutants, lifestyle factors such dietary intake, alcohol drinking, smoking, drugs and stress, and genetic factors, e.g. inherited defects in DNA metabolism and/or repair (1)(2)(3)(4)(5) . In addition, socioeconomic may also be associated with mutagenic damage (6) .
There are many different approaches and techniques developed for the monitoring of human genome. Since the 1980s micronucleus (MN) test on exfoliated cells of the oral mucosa has been used for the biomonitoring of humans. MN test has become the standard technique for the evaluation of cytogenetic damage in tissue targeted (7,8) . Therefore, there are a significant growth in the number of studies on the MN test over the years and it has been used traditionally to investigate lifestyle factors on DNA (2,6,9,10) . Santos  The most commonly used cells in MN test are lymphocytes, followed by buccal cells due the greater MN formation in these tissues. The oral mucosa is one of the most easily accessible epithelial tissues in humans and is highly relevant biomarkers of DNA damage (8) . Moreover, the MN assay in rapid replication tissue as the oral mucosa is a technique that can be used to detect recent acute and chronic exposures n the most exposure scenario (2,11) .
There is increasing interest in the application of MN assays in the clinical setting because of their association with the live style factors (11) and the risk of developing diseases (8) . Micronuclei (MN) appear in cytoplasm of daughter cells as small additional nuclei and contain whole chromosomes or acrocentric chromosome fragments, left behind during nuclear division. Higher MN frequency reflects chromosomal damage and may thus provide a marker for cancer risk, neurodegenerative and cardiovascular disease (4,12,13) .
Aging effect negatively DNA repair mechanisms and cytosolic DNA removal, with a steady build-up of DNA damage, increasing chronic proinflammatory signals and reactive oxygen and nitrogen species (RONS) production, increasing induction of chromosomal lesions, including MN, leading into a vicious cycle where MN induces inflammation and inflammation induces MN. Thus, the most of studies target older population, while, studies that evaluated with young population have been restricted to environment or occupational exposure groups (14)(15)(16)(17)(18) , being neglected the mutagenic damage risks on young populations. Therefore, the present study aimed in investigate the MN presence in young adults and its associated factors in a population residing in the extreme south of Brazil.

Individuals and sampling
This is a cross-sectional study, composed by 155 young adults (between 18 to < 30-year-old) of both sexes, who were attending a higher-level course in the Federal University of Rio Grande during the period between March and November 2016. Socioeconomic and life style data were collected by a semistructured, self-administered, and anonymous questionnaire. The instrument used was adapted for the university setting (19) from a validated instrument and suggested by the WHO to identify the prevalence of drug use (20) , such as, frequency and time of use (last 30 days). Socioeconomic level were evaluated according to ABEP (21) .

Oral exfoliated cells collection
The collection of oral mucosa cells was performed through a smear on both sides of participants' cheek mucosa using a polyethylene brush, before the participants rinsed the mouth with free alcohol mouthwash. After the material was collected, the brush was inserted into Falcon tube containing 10 ml of physiological saline solution (0.9%), then it was transported to the laboratory in dark and under refrigeration.

Micronucleus test
The micronucleus test in oral mucosal cells was performed according to methods published by Tolbert et al (22) . For slide preparation, the samples were centrifuged for 10 minutes at 1000 rpm, the supernatant was removed, and the pellet was homogenized. Approximately 1 mL of the sample was collected with a Pasteur pipette and placed on the slides in duplicate. After drying at room temperature, the slides were fixed with methanol for 10 minutes and then stained with eosin and methylene blue according to Leishmann.
To evaluate MN, 1000 cells were counted on the Olympus optical microscope model CX41 with a magnification of 400X and the MN number was counted following the suggestions of Holland et al. (6) . Were considered for MN counting only cells with intact cytoplasm and relatively flat cell position on the slide, little or no overlap with adjacent cells, little or no debris and nucleus normal and intact with nuclear perimeter smooth and distinct. To identify MN the criteria were: to contain a rounded smooth perimeter suggestive of a membrane, less than a third the diameter of the associated nucleus, but large enough to discern shape and color, staining intensity similar to that of the nucleus, texture similar to that of nucleus, same focal plane as nucleus and absence of overlap with, or bridge to, the nucleus.

Statistical Analysis
The questionnaires were typed per double typing in Epi-Data 3. in 1000 counted cells) of micronuclei and the frequency of MN per 1000 cells were considered for association analysis. The association between the variables and the presence or absence of MN was performed using Fisher's Exact Test or Chi-square test while the differences between MN frequency were tested using Mann Whitney test or Kruskal-Wallis test followed by Dunns test.Poisson regression with robust variance analysis was used to evaluate risk and protective factors associated with outcomes (presence or absence of micronuclei). Results were reported as prevalence ratio (PR) and 95%CI. The tests were performed with a 5% significance threshold. The data were analyzed by STATA software, version 11.2.

RESULTS
The most of participants were female (55.1%), self-reported white skin color (74.2%), had less than 25 years old (85%) and were single (94.2%) ( The results of the Poisson regression analysis with robust variance are described in Table 2. Only chronic disease and use of medication were statistic significant in bivariate analysis (p=0.002). While, in multivariate analysis individuals with chronic disease had 20% higher risk of present MN (p=0.03) and use of medication loss its statistic significant, presenting a limitrophe risk. The others variables investigated did not present significant a risk to MN presence and for this reason they were not presented.

DISCUSSION
This study observed that between these young adults, socioeconomic conditions and life style were not associated with the presence of MN. However, these factors were important determinants of healthy behaviors and consequently had important impact on cell damage (22) . The frequency of MN found in the present study is considered within the normal range of 0.5 to 2.5 MN per 1000 cells (6) .
There are a feel studies that reported MN in young adults. Aging in humans appears to be associated with genetic instability, due the decline in the efficiency of repair processes and accumulation of mutations, which results in increased level of DNA damage (23) . Although, the high prevalence of MN frequency among young adults evaluated in this study, there was no statistic significant difference between individuals aging less or more than 25 years old. In addition, sex may also influence in DNA damage. However, this relation seems to be less clear than others factors.
In general, studies with similar population evaluate MN frequency and the exposure to tobacco, alcohol, and other drugs (7,24,25) . The most of these studies report significant higher MN frequency between alcoholics and these rates were even higher when individuals were smokers and alcoholics (7,25) . In this study, although, smokers had higher MN frequency, the statistical significance was limitrophe. In the past years, smoking is recognized as major risk factor for genotoxicity in oral mucosa (9) .
The World Drug Report estimate that 5.6% of adults use illicit drug. In approximately 3 months of drug exposure the MN frequency can increase 0.16 (26) . In addition, MN test was previously proved to be a good biomarker to assess the mutagenic impact among drug users (27) . Although, the most of study population used some illicit drug, it was not observed differences between MN frequency.
This study found that non-transmissible chronic diseases and continuous use of medication were associated with the presence of MN, with this second variable associated and influenced by the first one. All individuals who had a chronic non-transmissible disease and continuously used medication exhibited the presence of MN in their oral mucosa cells. Several non-transmissible chronic diseases can alter the levels of oxidative stress, substance release and enzymatic activity resulting in DNA damage processes, and the use of drugs may potentiate the damage index caused by these dieses (28,29) .
Corroborant to these results, there are many studies that found an association between MN frequencies and several diseases, such as autoimmune, cardiovascular and neurodegenerative dieses (13) and certain types of cancer (11) . In addition, MN increases with the severity, exacerbations, and co-morbidity. Therefore, this study highlights the importance of evaluate NCD in young population to prevent worse outcomes in the development of the disease, since with aging there are an unbalance in antioxidant/oxidant status and insufficient DNA repair, as well as, accumulated DNA damage in cells and organs.
Medication can be used in different situations to eventually or chronic therapeutic. However, there are several young people thar use inadequately, by their known as "self-medication". This practice represents a risk to human health, due its potential toxicity (30) . Self-medication is associated with different factors such as: sex, age, marital status, children, drug use, knowledge about medications and medication availability (31,32) . It is important to investigate the use of medication in researches among young adults, due its possible impact on DNA integrity (33)(34)(35) .
Studies related to genome stability are not an easy task. They can be biased by many confounding factors and present low statistical strength that enable a direct attribution of the examined factors. In addition, it is often difficult to determine the causality of the MN induction (13) or if significant results found were a consequence of the medication or a biomarker of the disease. Therefore, important, that future studies investigate, in welldesigned and adequately powered studies, the prospective association of MN formation with non-c-diseases and medication use.

CONCLUSION
This study concludes that non-communicable chronic diseases and the use of continuous medication are risk factors in the population of young people studied. Furthermore, this study highlights the importance of monitored DNA damage between young adults and promote health education to prevent the development and worse prognosis of chronic non-transmissible diseases. Additionally, aware this population about the harms of "selfmedication"