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Micronucleifrequenciesinperipheralbloodandbuccalexfoliatedcellsofyoungsmokersandnon-smokers

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Introduction

Tobacco consumption has been related to various types of cancers, including lung, urinary bladder, kidney, pancreas, mouth, throat, and stomach cancers. It has been suspected that smoking can even cause acute leukemia as well as liver and uterine cervix cancer (Wang and Samet 1997). In the US, 38% of all cancer deaths in males are cigarette related, while 23% of all cancer deaths in females are due to cigarettes (Shopland 1995). Most tobacco-related cancers originate from actual smoking, but the passive smoke inhalation is also a risk (Julien 1997).

The carcinogenic effect of cigarette smoking is largely determined by the mutagenicity of compounds in cigarette smoke. Each cigarette contains a mixture of various carcinogens including polycyclic aromatic hydrocarbons (PAH), specific N-nitrosamines, aromatic amines, aldehydes and other carcinogens, tumor pro-moters, and co-carcinogens. Burning of tobacco and cigarette paper generates more than 4800 chemical compounds and 69 of them have been identified as

carcinogens (Hoffmann and Hoffmann 1997; Hoffmann et al. 2001; Hecht 1999).

Cigarette smoking induces various genetic aberrations including gene mutations, chromosome aberrations, micro-nuclei, sister chromatid exchanges, DNA strand breaks, and oxidative DNA adducts in various cell types (Demarini 2004). It has been hypothesized that the extent of genetic damage in peripheral blood lymphocytes reflects similar events in the precursor cells for carcinogenic processes in target tissues (Hagmar et al. 1994; 1998; Bonassi et al. 2000). Generally, it has been confirmed that chromosomal mutations are causal events in the development of neoplasia, thus increased fre-quencies of chromosomal mutations and micronuclei pre-dict higher risk for cancer development (Hagmar et al. 2001; Bonassi et al. 2007).

One of the widely used tests for the evaluation of expo-sure to mutagens and carcinogens is the micronucleus assay, a widely used short-term assay both in cultured mammalian cells, primary mitogen stimulated lymphocytes (Fenech and Morley 1985; Fenech 1998), as well as in vivo

(Received 03 February 2010; revised 18 March 2010; accepted 21 March 2010)

ISSN 1537-6516 print/ISSN 1537-6524 online © 2010 Informa UK LtdDOI: 10.3109/15376516.2010.482962 http://www.informahealthcare.com/txm

R E S E A R C H A R T I C L E

Micronuclei frequencies in peripheral blood and buccal exfoliated cells of young smokers and non-smokers

Anja Haveric1, Sanin Haveric1, and Slavka Ibrulj

1Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina, and 2Medical faculty, University of Sarajevo, Sarajevo, Bosnia and Herzegovina

AbstractCytogenetic biomarkers, such as micronuclei in peripheral blood or oral mucosa, are widely used for evaluation of exposure to genotoxins or carcinogens. Tobacco is one of the strongest carcinogens, responsible for develop-ment of different types of cancers. The aim of this study was to assess the genotoxicity of cigarette consumption in young smokers and to correlate results of cytogenetic analysis in peripheral blood lymphocytes and exfoliated buccal cells. The study was conducted on samples taken from 43 smokers and 44 non-smokers, young individuals from Bosnia and Herzegovina. Significantly higher frequency of micronuclei in peripheral blood lymphocytes was observed in smokers (p < 0.05). No significant correlations were found for age, duration and intensity of smok-ing, and frequency of micronuclei in lymphocytes. Significantly higher frequency of degenerated (apoptotic) buccal cells was also revealed in smokers (p < 0.05). The frequency of apoptotic cells in smokers was significantly influenced by the age of participants (F = 8.649; p < 0.01) and duration of smoking (F = 5.389; p < 0.05). Results of cytogenetic analysis conducted in peripheral blood and exfoliated buccal cells are in significant positive correla-tion, indicating complementarities of those analyses.

Keywords: Genotoxic; cytogenetic; biomarkers

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Address for Correspondence: Sanin Haveric, Institute for Genetic Engineering and Biotechnology, Gajev trg 4, 71 000 Sarajevo, Bosnia and Herzegovina. Tel: ++ 387 33 220 926. Fax: ++ 387 33 442 891. E-mail: sanin.haveric@ingeb.ba

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Micronuclei in smokers and non-smokers 261

in exfoliated epithelial cells (e.g. oral, urothelial, nasal) (Holland et al. 2008). Micronuclei derive from chromo-some or chromatid fragments or whole chromosomes or chromatids that lag behind in anaphase and are left out-side the daughter nuclei in telophase (Falck et al. 2002) Micronuclei, therefore, provide a convenient and reliable index of both chromosome breakage and chromosome loss (Fenech 2000).

Frequency of micronuclei in cells of buccal epithelium enables estimation of sensitivity to genotoxins and presents important biomarkers for prediction of relative risk for devel-opment of upper aero-digestive tract cancer (Bloching et al. 2000). The important advantage of micronuclei analysis in buccal cells is that neither cultivation nor cell division in vitro is needed (Holland et al. 2008). Buccal cells analysis may also include monitoring of apoptotic cells frequency as an additional indicator of genotoxic and cytotoxic effects. Normally, buccal epithelium presents an initial barrier for inhalation or ingestion of harmful substances and patho-logical microorganisms in the mouth (Proia et al. 2006), and are capable of metabolizing proximate carcinogens to reactive products (Liu et al. 1993; Vondracek et al. 2001). In smokers, oral cavity cells are periodically exposed to high concentrations of tobacco smoke chemicals (Campain 2004). As ∼ 90% of human cancers originate from epithelial cells it has been hypothesized that oral epithelium cells represent a preferred target site for genotoxic events induced by carcino-gens entering the body via inhalation or ingestion (Holland et al. 2008).

The aim of this study was to assess the genotoxicity of ciga-rette consumption in young smokers and to correlate results of micronuclei analysis in peripheral blood lymphocytes and buccal exfoliated cells.

Materials and methods

Study groupThe study was conducted with 43 smokers and 44 non-smokers, non-alcoholic young adults, aged 20–37, who were representatives of the urban area of Sarajevo (Bosnia and Herzegovina). Smokers were required to have smoked at least two cigarettes per day for ≥ 1 year before the start of the study. Non-smokers were required to refrain from smok-ing or chewing tobacco, while former smokers were catego-rized as non-smokers only if they had been abstaining more than 5 years before sample collection (Bonassi et al. 2000). Informed consent was obtained from all participants, who were healthy and non-exposed to known DNA-damaging agent or ionizing radiation. Exclusion criteria were chronic illnesses, diabetes, and consumption of antibiotic medica-tion or exposure to ionizing radiation 3 months before the beginning of the study. Diet factors as well as alcohol or tea and coffee consumption were not considered. General char-acteristics of the two study groups are listed in Table 1. The study was approved by the Scientific Council of the Institute for Genetic Engineering and Biotechnology and conforms to ethics of the Institute.

Lymphocytes cultivation and CBMN assayBlood samples were collected by venipuncture into vacu-tainer tubes containing sodium heparin (BD Vacutainer Systems, Plymonth, UK). Whole blood, 400 µl per each sam-ple, was cultured in PB–MAX™ Karyotyping Medium (GIBCO-Invitrogen, Carlsbad, CA), a fully supplemented, RPMI-based medium containing fetal bovine serum, L-glutamine, and phytohaemagglutinin.

The frequency of micronuclei in lymphocytes was deter-mined by applying cytokinesis-block micronucleus (CBMN) assay, based on scoring of micronuclei ex vivo after a single cell division of lymphocytes recognized as binuclear (BN) cells (accumulated using cytochalasin B) (Fenech and Morley 1985; Fenech 2000). Cytochalasin B (Sigma-Aldrich, St. Louis, MO) was added to the final concentration of 4.5 μg/ml (Westphal et al. 2003) in the 45th hour of the cultiva-tion. The CBMN assay was carried out according to Fenech (1993; 2000). Lymphocytes were harvested 72 h after set-up of cultures. Hypotonic treatment with potassium chloride was maximally reduced and cultures were centrifuged immediately upon addition of hypotonic. Cells were fixed three times in acetic acid fixative and cell suspension was dropped on coded slides. Dried slides were stained in 5% giemsa stain for 7 min. Scoring of micronuclei was carried out by two experienced scorers at 400× magnification. Analysis included observation of at least 1000 binuclear cells for the presence of micronuclei according to the criteria described by Fenech et al. (2003).

Buccal cells collection and analysisBuccal cells were collected by gentle scraping of cheeks with sterile plastic spatula. One spatula was used for each cheek. Samples were immediately fixed in cold acetic acid fixative. Spatulas were removed and fixed cells were centrifuged, supernatant removed, and cell suspension dropped on micro-scope slides. Coded slides were stained in 5% giemsa stain and two scorers evaluated micronuclei frequency at 400× magnification. Micronuclei and apoptosis were registered only after consensus. Frequency of micronuclei was deter-mined upon analysis of 1000 cells, which present sufficient individual samples (Belien et al. 1995; Bloching et al. 2000). Scoring criteria for micronuclei in buccal cells were according to Sarto et al. (1987) and Ray et al. (2005). Simultaneously karyorrhexis (nuclear disintegration) and karyolysis (nuclear dissolution) were classified as apoptosis (Celik et al. 2003). Karryorhectic cells have nuclei that are characterized by more extensive chromatin aggregation. Karyolytic cells are cells in which the nucleus is completely depleted of DNA (Thomas et al. 2009).

Statistical analysisThe results were tested by the Shapiro–Wilk test for the nor-mality of distribution and appropriate analysis was applied to test significances between the means of observed parameters in smokers and non-smokers. The level of significance was set at p < 0.05. The correlations between the results of analy-sis and independent variables (age, duration of smoking, and intensity of smoking − average amount of daily smoked

262 A. Haveric et al.

cigarettes) were determined by multiple and simple linear regression. Correlation between the gender of participants and results of analysis was determined by calculating point bi-serial correlation coefficient. Linear associations between the results of conducted analysis in peripheral blood lym-phocytes and buccal cells were calculated using Pearson’s coefficient of correlation (r).

Results

Aggregated information about the age, duration, and inten-sity of smoking as well as results of conducted cytogenetic analysis in smokers and non-smokers are presented in Tables 2 and 3.

The frequency of micronuclei (MN) in peripheral blood lymphocytes of smokers ranged from 4–22, while in non-smokers it ranged from 3–19. The average MN frequency in smokers was 10.907 ± 4.74 MN/1000 BN cells and in non-smokers 8.25 ± 3.577 MN/1000 BN cells.

A significantly higher frequency of micronuclei (p < 0.05) was determined in smokers.

Age, duration, and intensity of smoking did not sig-nificantly influence frequency of micronuclei when simple linear regression was used and when multiple regression was used. Calculation of point bi-serial coefficient revealed a significant increase in the frequencies of micronuclei in female smokers (rpb = 0.32; p < 0.05), although males were more intensive smokers than females (rpb = −0.438; p < 0.01). Duration of smoking was not significantly correlated with the gender of smokers. Among non-smokers, females also had significantly higher frequency of micronuclei (rpb = 0.373; p < 0.05).

Analysis of exfoliated buccal cells collected from smokers revealed that frequency of micronuclei ranged from 0–13 and frequency of degenerated (apoptotic) cells ranged from 0–19. The average MN frequency was 3.046 ± 3 MN/1000 BN cells, while the mean frequency of apoptotic cells was 5.628 ± 5.09. Absolute frequency of micronuclei in non-smokers ranged from 0–11 with a mean of 2 ± 2.035, while the frequency of apoptosis in this group ranged from 0–10 with a mean of 3.136 ± 2.969.

Frequencies of micronuclei in exfoliated buccal cells of smokers and non-smokers did not significantly differ (p > 0.05). However, smokers had significantly higher fre-quency of apoptosis (p < 0.05).

Multiple and simple linear regressions revealed no signifi-cant regression between age, duration and intensity of smok-ing, and frequency of micronuclei in exfoliated buccal cells.

However, the frequency of degenerated (apoptotic) cells was significantly influenced by those independent vari-ables (F = 2.932; p < 0.05). Simple linear regression analysis also revealed a significant positive influence of duration of smoking on the frequencies of apoptotic cells (F = 5.389; p < 0.05), while it was not significantly influenced by the intensity of smoking. Age of participants and frequency of apoptotic cells are also in the significant positive correlation (F = 8.649; p < 0.01). The calculation of point bi-serial coeffi-cient revealed that gender of participants did not significantly affect frequencies of micronuclei and apoptosis in buccal cells in smokers or non-smokers.

When Pearson’s coefficient was calculated in order to determine correlations between results of conducted analysis in peripheral blood lymphocytes and buccal cells of smokers, a significant positive correlation was found for the frequen-cies of micronuclei in lymphocytes and micronuclei in buccal cells (r = 0.314, p < 0.05) and frequencies of micronuclei and apoptosis in buccal cells (r = 0.318, p < 0.05).

Significant positive Pearson’s correlations were also found for the frequencies of micronuclei in lymphocytes and micro-nuclei in buccal cells (r = 0.383; p < 0.05) and for the frequen-cies of micronuclei and apoptotic buccal cells (r = 0.312; p < 0.05) in non-smokers.

Discussion

Significantly higher frequencies of micronuclei in lym-phocytes and apoptotic buccal cells were determined in smokers. Statistical analysis revealed that significantly higher frequencies of micronuclei in smokers were not influenced by age, intensity, or duration of smoking. This may be caused by the relatively short duration of smoking (8.28 ± 3.56 years) of younger individuals from the local population. Other characteristic that may influence frequencies of cytogenetic biomarkers that were not considered in this study include the amount of tar and nicotine, modification of cigarette filters content, taste of cigarettes, type of tobacco, cigarettes origin, etc. (Hecht et al. 2005; Proia et al. 2006). Active or pas-sive smoking is associated with formation of micronuclei in peripheral blood lymphocytes and oral mucosa (Wang and Samet 1997; Celik et al. 2003). Although passive smokers have a higher frequency of chromosomal aberrations compared to controls, this difference is not significant (Sasikala et al. 2003).

Effects of smoking on the frequency of micronuclei in human peripheral lymphocytes have been evaluated in numerous studies and results are controversial. An increased rate of micronuclei formation in smokers has been determined in some laboratories (Au et al. 1991; Tomanin et al. 1991). Some authors have demonstrated reverse asso-ciation, as smokers have lower frequencies of micronuclei (Landi and Barale 1999), which, according to Gourabi and Mozdarani (1998), may be related to activation of cell-

Table 1. General characteristics of study groups.

Variable Smokers Non-smokers

Sample size 43 44

Gender Females 23 27

Males 20 17

Age (M ± SD, years) Females 25.61 ± 2.81 24.37 ± 2.69

Males 26.4 ± 4.22 26.82 ± 3.68

No. consumed cigarettes/day (M ± SD)

Females 14.22 ± 6.62 —

Males 20.75 ± 7.12

Duration of smoking (M ± SD, years)

Females 7.48 ± 2.87 —

Males 9.2 ± 4.1

Micronuclei in smokers and non-smokers 263

Table 2. Results of cytogenetic analysis conducted in lymphocytes and exfoliated buccal cells of smokers.

Sample Gender Age(years)No. consumed cigarettes/day

Duration of smoking (years)

MN/1000 BN lymphocytes

MN/1000 exfoliated buccal cells

Apoptosis/1000 exfoliated buccal

cells

1 20 7 2 7 1 0

2 24 10 3 10 1 1

3 24 10 2 13 2 2

4 26 20 8 10 1 1

5 35 30 13 14 2 2

6 26 25 11 22 6 0

7 24 18 10 11 4 5

8 27 15 8 12 4 2

9 23 10 8 6 5 5

10 27 10 9 4 1 3

11 27 20 11 11 2 1

12 26 20 10 8 3 5

13 24 10 7 15 0 1

14 25 15 9 4 1 4

15 25 10 5 18 2 1

16 23 5 9 16 5 1

17 24 10 6 12 8 4

18 25 4 5 8 2 3

19 27 18 9 18 2 19

20 25 20 6 16 8 6

21 29 10 8 18 13 18

22 28 20 8 18 2 16

23 25 10 5 12 2 1

24 26 30 7 5 2 9

25 26 30 9 6 1 1

26 23 30 7 8 3 2

27 22 15 12 14 2 4

28 23 25 8 8 2 8

29 24 20 4 6 1 1

30 25 30 11 9 1 10

31 25 20 7 8 1 1

32 28 15 5 4 1 8

33 29 20 10 5 1 9

34 21 15 5 9 1 10

35 22 30 3 6 1 5

36 26 20 10 9 1 4

37 24 15 9 19 2 11

38 35 10 20 8 8 12

39 25 15 10 11 13 9

40 32 10 7 19 2 10

41 37 30 15 12 5 15

42 28 20 15 11 2 10

43 27 15 10 9 4 2

44 25 4 5 8

∑ — — — 469 131 242

Xav

25.98 16.84 8.28 10.907* 3.0465 5.628*s 3.52 7.53 3.56 4.74 3 5.09

sXav

0.537 1.148 0.543 0.723 0.457 0.776

*p< 0.05 compared with non-smokers.

264 A. Haveric et al.

Table 3. Results of cytogenetic analysis conducted in lymphocytes and exfoliated buccal cells of non-smokers.

Sample Gender Age(years)MN/1000 BN lymphocytes

MN/1000 exfoliated buc-cal cells

Apoptosis/1000 exfoliated buccal cells

1 25 9 1 0

2 25 5 1 0

3 25 6 1 3

4 24 8 3 0

5 25 9 0 0

6 23 10 1 3

7 22 10 1 0

8 22 8 0 2

9 21 4 0 6

10 21 6 0 3

11 24 6 1 8

12 26 8 1 1

13 25 10 2 3

14 25 6 3 4

15 29 11 2 2

16 25 12 1 0

17 26 5 2 5

18 23 13 4 0

19 24 13 3 0

20 23 16 2 7

21 23 12 4 10

22 22 8 2 8

23 25 12 3 3

24 22 19 2 1

25 34 12 3 4

26 27 6 4 6

27 22 7 7 2

28 24 9 2 2

29 24 4 2 9

30 26 5 1 2

31 20 9 0 1

32 25 5 0 4

33 31 7 1 1

34 24 9 1 0

35 29 5 1 6

36 27 5 3 1

37 30 9 1 1

38 36 3 0 2

39 26 3 1 1

40 26 14 1 1

41 27 5 1 8

42 25 10 11 8

43 31 9 3 2

44 25 4 5 8

∑ — 363 88 138

Xav

25.32 8.25 2 3.136

s 3.3 3.577 2.035 2.969

sXav

0.497 0.539 0.307 0.448

Micronuclei in smokers and non-smokers 265

protective responses that reduce frequency of micronuclei and induce resistance towards further DNA damages in con-sumers of several cigarettes per day.

Re-analysis of samples of 3752 individuals (2364 non-smok-ers and 788 smokers) from the HUMN project, who provided information on their smoking habits, revealed that micronu-clei frequency is not significantly higher in smokers, and that it is not influenced by the number of cigarettes smoked per day among subjects occupationally exposed to genotoxic agents. Significant increase of micronuclei frequency is determined only in individuals smoking 30 cigarettes or more per day, thus the authors specifically recommend evaluation of the heavy smokers sub-group whenever it is large enough to satisfy sta-tistical requirements (Bonassi et al. 2003).

The age and sex of the subject are being reported as the most important demographic variables effecting micro-nuclei frequencies (Fenech 1998). Significantly increased micronuclei frequency in females, registered both in smoker and non-smoker females, may be related to the possible loss of X chromosome (Fenech et al. 1994). In the research conducted by Gonsebatt et al. (1997) increased micronuclei frequency in males is reported. In the research presented by El-Zein et al. (2006), a significant association between micronuclei frequency and gender, age, and cigarette con-sumption is not found.

For the harvesting of buccal cells different methods have been applied. These include scraping with a wooden spatula (Sarto et al. 1987), cotton swab (Moore et al. 2004), tootbrush (Marchand 2001), or obtaining the cells using the ‘swish and spit’ method (Hayney et al. 1995). For buccal cells collection we used a sterilized plastic spatula and confirmed efficiency of this method as a sufficient number of cells was collected. For staining of buccal cells we used giemsa stain. Several other laboratories have also applied this method for micronu-clei staining (Bloching et al. 2000; Rajeswar et al. 2000; Chen et al. 2006), although it has been reported that giemsa stain may lead to reports of increased frequency of micronuclei, as keratohyalin granules or bacteria resemble micronuclei (Nersesyan et al. 2006).

Numerous studies were designed to determine associa-tions between smoking of tobacco and induction of micro-nuclei in buccal cells. Unlike our results, Sarto et al. (1987) determined a significantly higher frequency of micronuclei in buccal cells of smokers compared with non-smokers. In the study of 120 healthy subjects, Konopacka (2003) reported a three times higher frequency of micronuclei in smokers than non-smokers, while neither age nor gender correlated with the level of micronuclei. Bloching et al. (2000) revealed a direct correlation between tobacco abuse and increased micronuclei frequency as a sign of a cytogenetic damage of buccal mucosa cells.

We have found that gender did not correlate with the induction of micronuclei and apoptosis in buccal cells of smokers and non-smokers. However, in the research con-ducted by Konopacka (2003), higher frequencies of micro-nuclei in buccal cells are determined in female smokers (1.54 ± 0.42%) compared to male smokers (1.31 ± 0.56%),

while gender and age are not correlated with the induction of micronuclei in non-smokers.

Significant positive association between the duration of smoking and frequencies of buccal cells with micronuclei and apoptotic cells was found in our research. Wu et al. (2004) revealed that both intensity and duration of smok-ing affect frequency of micronuclei in buccal cells of heavy smokers.

Conclusion

The cigarette consumption increases MN frequency in peripheral blood lymphocytes of young smokers from Bosnia and Herzegovina. The frequency of apoptotic exfo-liated buccal cells in smokers is significantly higher com-pared to non-smokers but it is also significantly influenced by the age of participants. Cytogenetic analysis conducted in peripheral blood and exfoliated buccal cells are in sig-nificant positive correlation and complementary, indicat-ing a possibility to substitute one another in similar human monitoring.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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