The effects of sex , age and cigarette smoking on micronucleus and degenerative nuclear alteration frequencies in human buccal cells of healthy Bosnian subjects

© 2013 Hilada Nefic et al.; licensee University of Sarajevo Faculty of Health Studies. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. UNIVERSITY OF SARAJEVO FACULTY OF HEALTH STUDIES ABSTRACT

The effects of sex, age and cigarette smoking on micronucleus and degenerative nuclear alteration frequencies in human buccal cells of healthy Bosnian subjects

INTRODUCTION
Th e Buccal Micronucleus Cytome (BMCyt) assay is a method for studying the eff ects of lifestyle factors, nutrition, genotoxin exposure and genotype on DNA damage, chromosomal aberrations and malsegregation, cell death and regenerative potential of human buccal cells.Demographic factors, sex and age, also aff ect micronucleus (MN) frequency in buccal cells.Epithelial cells do not need to be stimulated and micronuclei (MNi) in exfoliated cells refl ect genotoxic events that occurred in the dividing basal cell layer 1-3 weeks earlier.Th is method is minimally invasive and repeated sampling is acceptable (1,2).Th e human micronucleus assay in exfoliated buccal cells (HUMN XL ) project, established in 2009, is an international collaborative project for studying DNA damage in human populations.Th is project was aimed to standardize micronucleus assay in oral buccal cells (2).Baseline frequencies for micronucleated cells (MNC) in the BMCyt are usually within the 0.5-2.5 MNi per 1,000 cells range (3).Th e factors potentially affecting baseline buccal MN frequency are methodological, exposure, diet, lifestyle and demographic (age and sex).Th e age, sex and smoking habit were the most commonly studied factors.Piyathilake et al (4) reported that frequencies of micronucleated cells are higher in females after adjusting for age and smoking habit, whereas in a Brazilian study (5) the number of micronuclei was signifi cantly higher in males.Higher frequency of micronuclei was ob-served in cells collected from female smokers than male smokers but sex and age did not infl uence micronuclei frequency of non-smokers (6).Nersesyan et al (7) observed a weak association between the age of the participants and the overall frequency of MNC cells.Some studies showed an increase with age (4,8) and in another a decrease with age (9), but in some studies no eff ect was detected (5,(10)(11).Cigarette smoking is one of the factors that may infl uence the number of MNi in buccal cells (4,12).Smoking is also reported to increase the MN frequency in human lymphocytes (13,14).Th e BMCyt assay has been used to measure biomarkers of DNA damage (micronuclei and nuclear buds, NBUDs), cytokinetic defects (binucleated cells) and proliferative potential (basal cell frequency) and cell death (condensed chromatin; karyorrhectic, KHC; pyknotic, PYK and karyolitic, KYL cells).In the BMCyt assay, buccal cells are classifi ed into categories that distinguish between 'normal' cells and cells that are 'damaged' on the basis of cytological and nuclear features (Figure 1).Besides, the BMCyt assay can detect an increase in MN frequency in exfoliated buccal cells after exposure to diff erent genotoxic factors (e.g.chemicals, radiation, lifestyle factors); this assay also has the potential to identify inherited genomic instability such as Bloom's syndrome.Th e biomarkers measured in this assay have been associated with normal ageing and premature ageing in clinical outcomes such as Down's syndrome and Alzheimer's disease.Th e buccal MN assay is used as a biomarker of cancer risk.Th e aim of the present study was to evaluate the baseline micronucleus frequency in human buccal cells in the sample of healthy Bosnian subjects.In addition to counting micronuclei, degenerative nuclear alterations indicative of apoptosis (karyorrhectic, pyknotic and karyolytic cells) were also investigated in the BMCyt assay.We hypothesized that sex and age of participants affect MN and other biomarker frequencies in human buccal cells.Smoking habit was also taken into account.

METHODS
Exfoliated cells of the human buccal mucosa for the buccal micronucleus cytome (BMCyt) assay were collected from 120 healthy females and males, mostly younger subjects from Central Bosnia and Herzegovina.Participants were aged between 19 and 50 years (with mean age of 25.33 ± 4.90 years).Signed informed consent was obtained from each individual.Information on date of birth, sex and smoking status and history (the number of cigarettes smoked per day and duration of smoking in year) was obtained by the questionnaire.In this study, the BMCyt assay was used for studying biomarkers of DNA damage, cytokinetic defects and cell death.Individuals rinsed their mouth with water and wooden tongue depressor was used to col-lect cells from the inner wall of the cheek.Th e slides were prepared by direct smearing of buccal cells to cleaned microscope slide.Th e smears were air dried and slides were stained by 2% acetorcein (Gurr Orcein, BDH Chemicals Ltd., Poole, England).Stained slides were used for microscopic analysis.Exfoliated buccal cells were analysed under a total magnifi cation of x1000 using a Jenaval microscope.Only cells that were not clumped or overlapped and that contained intact nuclei were included in the analysis of MNi.Th e frequency of micronuclei and degenerative nuclear alterations (nuclear buds, pyknotic, karyolytic and karyorrhectic cells) in diff erentiated human buccal cells were recorded.Applied criteria for identifying and scoring cell types in the BMCyt assay, based on morphological features of cells, were described by Tolbert et al. (1), Sarto et al. (15) and Th omas et al. (16).According to these criteria, normal diff erentiated cell has a smaller nuclearcytoplasmic ratio relative to basal cell, more angular and fl atter than basal cells, uniformly stained round nucleus.Th e micronucleated cell contains both the main nucleus and micronucleus and micronuclei are round or oval with similar stain intensity as the main nucleus.Th e micronuclei usually have 1/3-1/16 diameter of the main nucleus located in cellular cytoplasm.Most cells with MNi contain only one MN but it is possible to fi nd cells with two or more MNi.Th e cells with nuclear bud on the main nucleus have a sharp constriction forming a bud.Th e bud is attached to the main nucleus and has a similar staining intensity as the main nucleus.Its diameter can be a quarter to half of nuclear diameter.chromatin, while nuclear fragmentation may be evident.Th e pyknotic cell has small shrunken nucleus.Th e nucleus is uniformly and intensely stained and its diameter is 1/3-2/3 diameter of normal nucleus.Th e karyolytic cells are cells in which the nucleus is completely depleted of DNA.Th e nucleus is apparent as a ghost-like image that has no Orcein staining.First, we determined the number of karyorrhectic, pyknotic and karyolitic cells per 1,000 diff erentiated cells for score slides in the BMCyt assay.Th e number of DNA damage biomarkers (MNi and NBUDs) is scored in 1,000 diff erentiated cells, because of the very low number of basal cells.Data were expressed as the mean ± standard deviation (SD) of the means.Th e frequencies of various cell types in the assay are represented as the number of cells in a 1,000 cells.
In our study, sex, age and lifestyle-related variable of smoking habits (the number of cigarettes smoked per day and duration of smoking in a year), that could aff ect the number of studied biomarkers, was considered.Th e subjects were divided into groups, according to their sex (females and males).Additionally, females and males were divided into subgroups with regard to smoking habit (smokers and non-smokers), based on their responses to the questionnaire.Smoking habit was ranked as smoker or non-smoker.Individuals who had consumed four or more cigarettes per day for at least two years were considered to be smokers.Th ese groups (smokers and non-smokers) were divided into subgroup with regard to sex (Table 1).

Statistical analysis
Th e presence of statistically signifi cant diff erences in the occurrence of damaged cells between studied groups and subgroups is tested with a Student's ttest (comparing two means).Also, to evaluate the association between age or smoking and each biomarker, Pearson's method (r) was performed.between males and females in relation to age (t = 0.53; p = 0.599), which is important, given the fact that the infl uence of sex and smoking on the occurrence of cytogenetic markers is observed.Th e males consumed statistically signifi cant higher number of cigarettes per day than females (t = 3.91; p ≤ 0.001).Th e mean baseline MN frequency in the examined sample of individuals was 1.35 ± 1.15 per subject (per 1,000 cells), 0.135% or 1.35‰.We didn't observe MNi in buccal cells of 23 subjects.Most MNC cells contain only one MN (97.5% of micronucleated cells) but it is possible to fi nd cells with two or even more MNi (range 1 -6 MNi per thousand buccal cells).Th erefore, the frequency of MNC cells and the frequency of MNi per 1,000 cells were almost identical (Figure 2).We didn't observe degenerative nuclear alterations (Figure 3) in diff erentiated cells of only three subjects, and nuclear alterations ranging between 1 and 35 per thousand buccal cells.Th e data obtained by Student's t-test showed that the number of MNi was higher in females (mean ± SD: 1.57 ± 1.34), as compared with males (mean ± SD: 1.08 ± 0.78), the diff erence being statistically signifi cant (t = 2.5; p = 0.014).Th e ratio between females and males was 1.45.On the other side, the number of cells with NBUDs was higher in males than in females, but the diff erence was statistically non-signifi cant.Th e number of degenerated cells resulting from karyorrhesis (KHC cells), karyolysis (KYL cells) and nucleus fragmentation (PYC cells) was also increased in males, but these diff erences were not statistically signifi cant.Th e female or male smokers had statistically signifi cant more MNi (females: t = 3.81; p ≤ 0.001 and males: t = 2.07; p = 0.043), KYL (females: t = 2.66; p = 0.01 and males: t = 2.38; p = 0.024) and KHC cells (females: t = 3.84; p ≤ 0.001 and males: t = 3.26; p = 0.003) than female or male non-smokers.
To determine the correlation between age and cytogenetic biomarkers, Pearson's method was per- formed.In the sample including both males and females during this study, we found signifi cantly positive correlation between ageing and the number of MNi (r = 0.4300; p < 0.001), NBUDs (r = 0.2959; p = 0.022), KHC (r = 0.3109; p = 0.016) and KYL cells (r = 0.3623; p = 0.004).Th ese results showed that micronucleus and other cytogenetic markers frequencies tended to be greater in older subjects than in younger subjects.
Pearson's analysis was applied in order to investigate the association between micronucleus formations in human buccal cells and the number of degenerative nuclear alterations.We found signifi cantly positive correlation between the number of MNi in cells and the number of degenerative nuclear alterations in diff erentiated buccal cells; for NBUDs: r = 0.3644; p = 0.004, KHC cells: r = 0.2905; p = 0.024 and for KYL cells: r = 0.3076; p = 0.017.Sample included 60 smokers, 37 females (61.67%) and 23 males (38.33%).Th e average age of smokers was 25.62 ± 6.15 and non-smokers 25.03 ± 3.23 (Table 3).Th is diff erence was not statistically significant and we can study eff ects of sex on the number of examined biomarkers.Th e frequency of MNi of oral epithelial cells was twice as high in smokers as in non-smokers; recorded MNi frequency values were 1.78 ± 1.35 and 0.93 ± 0.71, respectively.
Th e cells with anomalies other than MNi, such as PYK, KYL, KHC cells, are signifi cantly increased in smokers.In Student's t-test, the smokers had statistically signifi cant increased number of MNi (t = 4.32; p ≤ 0.001), PYK (t = 2.35; p = 0.021), KYL (t = 3.45; p ≤ 0.001) and KHC cells (t = 4.99; p ≤ 0.001) than non-smokers.Regarding the sex of smokers, females had signifi cantly higher number of MNi in exfoliated buccal cells (t = 2.25; p = 0.028) when compared with males, although males smoke signifi cantly more cigarettes per day (t = 2.71; p = 0.009) than females.Th e ratio of MNi between females and males was 1.52.Th e males had higher number of other biomarkers examined (PYK, KYL and KHC cells, also NBUDs) when compared with females, however these diff erences were not statistically signifi cant.In the group of non-smokers, there is not statistically signifi cant diff erence in the number of observed cytogenetic markers between males and females (Table 3).We also noticed statistically signifi cant positive correlation between cumulative smoking (duration of smoking in years) and the number of MNi (r = 0.5074; p < 0.001) and NBUDs in the cells (r = 0.6999; p < 0.001), PYK (r = 0.4818; p = 0.007), KYL (r = 0.6592; p < 0.001) and KHC cells (r = 0.7702; p < 0.001).Similarly, smoking intensity (number of consumed cigarettes per day) had an eff ect on statistically signifi cant increased number of nuclear alterations, PYK (r = 0.5198; p = 0.003), KYL (r = 0.4430; p = 0.014) and KHC cells (r = 0.5265; p = 0.003).

DISCUSSION
BMCyt assay is useful as a biomarker of genetic damage caused by genotoxic and lifestyle factors, environmental and occupational exposures, dietary defi ciencies, medical procedures, diff erent diseases, as well as inherited genetic defects in DNA repair.Th e sex, age, smoking and alcohol consumption can aff ect MN frequency in buccal cells.
In this study, the baseline frequency of micronuclei was determined in exfoliated cells of the human buccal mucosa of 120 healthy subjects.Th e sex, age and smoking habits were taken into account.We found the baseline MN frequency in human buccal cells was 0.135% or 1.35‰ in examined sample, which is in agreement with the other published reports: 0.16% (1), 0.1 to 0.3% (17) and 0.5 -2.5 MNi per 1,000 cells (3).Micronuclei are regarded as biomarkers of abnormal mitoses involving chromosomal breakage or mis-segregated chromatin.We demonstrated that sex infl uences the number of MNi in buccal cells.Th e number of MNi was signifi cantly higher in females as compared with males.Th e ratio between females and males was 1.45.Also, there was association between the age of the participants and frequency of MNi, NBUDs, KYL and KHC cells.Th e number of these cytogenetic markers statistically increased with ageing.Concerning sex, the other studies also reported that biomarker frequencies were higher in females than in males by a factor of 1.2 -1.6, depending on the age group (17)(18)(19).However, other authors have shown the spontaneous buccal cell MN frequencies in males and females did not substantially diff er, with a slight but not signifi cant excess in males (20).Some studies did not fi nd any association between sex and micronucleus occurrence (6,21), although this association has been reported by others (22)(23)(24).Some authors did not fi nd association between age and micronucleus occurrence (6,21).However, other authors were able to establish a statistically significant eff ect by age (8,(25)(26)(27).Results of the study of Piyathilake et al (4) indicated that age and sex were important variables aff ecting micronucleus frequen-cy.It has been shown that in vivo ageing leads to an increased micronucleus frequency in lymphocytes.Loss of the X chromosome in females and males and loss of the Y chromosome in males are among the primary mechanisms explaining this increase (14).Th e higher incidence of MN in both sexes is more manifested in older age groups and the eff ect of sex becomes more pronounced as age increases.Cytogenetically, ageing is associated with a number of cellular changes, including altered size and morphology, genomic instability and changes in expression and proliferation (28,29).It has been shown that a higher MN frequency is directly associated with decreased effi ciency of DNA repair and increased genomic instability (30,31).
In our study, males had a slightly higher number of cells with NBUD and the number of degenerative nuclear alterations indicative of apoptosis (PYK and KYL cells and KHC cells) than females.However, signifi cantly positive correlations were observed between micronuclei frequencies in human buccal cells and the formation of cells with NBUD, KYL and KHC cells.Th e PYK, condensed chromatin and KYL are normal correlates of epithelial cell differentiation and maturation.However, they occur at elevated levels in response to cellular injury.Th e PYK, KHC and condensed chromatin are associated with both cytotoxicity (necrosis and keratinization) and genotoxicity (apoptosis), but KYL is associated with cytotoxicity only.Apoptosis is the major mechanism of cell death in living tissues.Because it is stimulated both by ionizing radiation and by chemicals that bind to DNA, apoptosis may also act as a surveillance mechanism, eliminating cells with genetic damage.Th us, apoptosis may be an indicator of genotoxic insult.Th erefore, the BMCyt offers evaluation of chromosomal instability and gene amplifi cation (NBUDs), cytokinesis arrest due to aneuploidy (binucleated cells), and diff erent cell death events (e.g.KHC and PYK cells).Correlation analyses showed that micronucleus frequencies correlated signifi cantly with karyorrhexis, karyolysis, condensed chromatin and binucleates (7).Th e most frequently studied lifestyle parameter is smoking.Th is study showed that buccal cell MNi and degenerative nuclear alternations were more frequent among cigarette smokers than non-smokers.
Also, cigarette smoking (the number of cigarettes smoked per day or years of smoking) signifi cantly increases the frequencies of PYK, KYL and KHC cells and years of smoking also increases the frequencies of MNi and NBUDs in buccal cells.Concerning the number of cigarettes smoked per day or years of smoking, genetic damage shows that only the subjects who smoked the most had a signifi cant increase in MNi and NBUDs.Statistically signifi cant higher frequency of MNi, PYK, KYL and KHC cells was observed in cells collected from female or male smokers than from non-smokers.Th ere are diff erent results in the literature about the eff ects of smoking on induction of MNi in exfoliated buccal cells.Konopacka (6) reported that the frequency of MNi of oral epithelial cells was three times higher in smokers than non-smokers.It has been suggested that this association is dependent on the number of cigarettes consumed (26,27).
Haveric et al (32) studied the eff ects of cigarette consumption on micronucleus frequencies in peripheral blood lymphocytes and exfoliated buccal cells of young smokers.Th ey observed signifi cantly higher frequency of apoptotic buccal cells in smokers and the frequency of apoptotic cells in this group was signifi cantly infl uenced by the age of participants and duration of smoking.Yet other publications report no diff erence between smokers and non-smokers or men and women (33).

CONCLUSIONS
Th e BMCyt assay, based on scoring not only MN frequency but also other genome damage markers, dead or degenerated cells, provides a measure of cytotoxic and genotoxic eff ects.
Our results suggest that the baseline MN frequency in human buccal cells of 120 healthy subjects was 0.135% or 1.35‰.Signifi cant positive correlations were observed between MN frequencies and formation of NBUD, KYL and KHC cells.We demonstrated that sex infl uences the number of MNi in human buccal cells.Th e number of MNi in cells was signifi cantly higher in females than in males, regardless of the number of consumed cigarettes per day.Th ere was positive association between the age of the participants and frequency of MNi, NBUDs, KYL and KHC cells.Th e ageing statistically increased the number of analysed cytogenetic markers.
Statistically signifi cant higher frequency of MNi, PYK, KYL and KHC cells was observed in cells collected from female or male smokers than from non-smokers.Our fi ndings indicated that cigarette smoking (the number of consumed cigarettes per day or years of smoking) signifi cantly increases the frequencies of PYK, KYL and KHC cells.Cytogenetic damages show that subjects who smoked more years had a signifi cant increase in MNi and NBUDs in buccal cells.
Th e karyorrhectic cells have nucleus with extensive aggregated

TABLE 1 .
Selected characteristics of the studied subjects (mean ± SD per subject).

TABLE 2 .
Effects of sex and smoking on buccal cell MN and nuclear alteration frequencies in healthy persons (mean ± SD per subject).

TABLE 3 .
Comparison of cytogenetic biomarker frequencies by stratifi cation of sex and smoking status.All values are given as mean ± SD per subject.