Effect of Staining Procedures on the Results of Micronucleus Assays with Exfoliated Oral Mucosa Cells

1835 Effect of Staining Procedures on the Results of Micronucleus Assays with Exfoliated Oral Mucosa Cells

Armen Nersesyan,1 Michael Kundi,2 Kambis Atefie,1 Rolf Schulte-Hermann,1 and Siegfried Knasmu¨ller1 1Institute of Cancer Research, Internal Medicine I and 2Institute of Environmental Health, Medical University of Vienna, Vienna, Austria

Abstract

Micronuclei in exfoliated epithelial cells are widely used as These anomalies are consequences of cell injury found in biomarkers of cancer risk in humans. To elucidate the effect epithelial cells and are paralleled by formation of keratin of different staining procedures on the outcome of such bodies in the cytoplasm that resemble micronuclei. Corre- investigation, we conducted a study in which the micronuclei lation analyses showed that micronuclei frequencies scored frequencies in oral mucosa cells of heavy smokers (n = 20) in Giemsa-stained slides correlated significantly with kar- and nonsmokers (n = 10) were evaluated with nonspecific yorrhexis, karyolysis, condensed chromatin, and binucleates, (Giemsa, May-Gru¨nwald-Giemsa) and DNA-specific (4¶,6- whereas no such correlations were found with DNA-specific diamidino-2-phenylindole, Feulgen, acridine orange) stains, stains. These findings indicate that nuclear anomalies (and whereas with Giemsa-based stains, the frequencies of micro- possibly keratin bodies) may be misinterpreted as micronuclei in smokers were significantly (4- to 5-fold) higher in nuclei with nonspecific DNA stains and lead to false-positive the smokers group, no significant increase was observed with results in studies with cells of epithelial origin. Furthermore, any of the DNA-specific stains. Furthermore, the evaluation our results show that exposure of oral mucosa cells to geno- of cells of the two study groups with Feulgen stain showed toxic carcinogens contained in tobacco smoke does not lead to that oral mucosa cells from smokers had significantly induction of micronuclei in these cells. (Cancer Epidemiol increased levels of nuclear anomalies other than micronuclei. Biomarkers Prev 2006;15(10):1835–40)

Introduction

About 25 years ago, Stich et al. (1) developed a protocol for cells were conducted with nonspecific stains (Giemsa, May- micronucleus assays with exfoliated buccal mucosa cells, Gru¨nwald-Giemsa, and less frequently orcein). According to which was widely used in occupational and lifestyle studies our knowledge, only one study (7) has been published in (for reviews, see refs. 2 and 3). It was repeatedly empha- which the effect of different stains, namely Giemsa, Feulgen, sized that this noninvasive method might be a suitable Hoechst 33342, and propidium iodide, on the results of biomonitoring approach for the detection of increased cancer micronuclei experiments, was compared in cells from patients risk in man because >90% of all human cancers are of epi- with different pathologies of the oral mucosa. Casartelli thelial origin (4). Approximately 200 studies with epithelial et al.’s findings indicate that the micronuclei frequencies are cells have been published in the last 25 years and apart higher with Giemsa; however, no firm conclusions can be from mouth cells, cells from other organs, such as nasal drawn from this study as no control group was included, the mucosa, cervix, bladder, esophagus, and bronchi, were also samples were collected from heterogeneous groups, and used (2, 3). Hoechst 33342 and propidium iodide have rarely been used It was soon realized that strong interlaboratory and intra- in micronuclei assays. laboratory variations exist in micronuclei studies with exfo- The aim of the present study was to further investigate if, liated cells and efforts were made to standardize the assay. and to what extent, different stains have an effect on the results For example, Tolbert et al. (5) and Sarto et al. (6) defined of micronuclei studies in exfoliated cells. Therefore, buccal morphologic scoring criteria for micronuclei and other mucosa cells were collected from nonsmokers and heavy confounding factors, such as age and gender of the study smokers, and the micronuclei frequencies were comparatively groups. However, only little attention has been, until now, evaluated with two of the most commonly used DNA non- given to the effect of different staining procedures on the specific stains (May-Gru¨nwal-Giemsa and Giemsa) and with results of micronuclei assays. An evaluation of the literature three specific stains (Feulgen, acridin orange, and DAPI). shows that a variety of different stains is used in micronuclei Smokers were included as it is well established that they have studies. Among the DNA-specific stains, the ones most widely increased risks for oral and other forms of epithelial cancer used are Feulgen and acridine orange; in some experiments, (8-10) and a number of studies on micronuclei induction in 4¶,6-diamidino-2-phenylindole (DAPI) and propidium iodide buccal cells of smokers has been published, which gave highly were also used. About 30% of the studies in epithelial inconsistent results (refs. 11-31; for review, see ref. 28). Damage of epithelial cells leads to nuclear abnormalities other than micronuclei (metanucleated cells; ref. 32), such as karyorrhexis, Received 3/28/06; revised 7/3/06; accepted 8/7/06. karyolysis, pyknosis, condensed chromatin, broken eggs, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. binucleates, and it has been stressed that these anomalies Section 1734 solely to indicate this fact. are difficult to distinguish from classic micronuclei and may Requests for reprints: Siegfried Knasmu¨ller, Institute of Cancer Research, Inner Medicine I, led to misinterpretations (5, 32). Therefore, we additionally Medical University Vienna, Borschkegasse 8a, A-1090 Vienna, Austria. Phone 43-1-427765142; Fax: 43-1-42779651; E-mail: [email protected] evaluated these variables in both study groups and analyzed Copyright D 2006 American Association for Cancer Research. if the micronuclei frequencies scored with DNA-specific and doi:10.1158/1055-9965.EPI-06-0248 nonspecific stains correlate with these anomalies.

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Materials and Methods Evaluation of the Slides. From each participant, at least 1,500 cells were evaluated with each staining procedure. Participants. In total, 10 nonsmokers and 20 heavy smokers Micronuclei were scored in normal cells according to the participated in the study. All subjects were healthy and none criteria defined by Tolbert et al. (5). In addition, the frequen- of them was exposed to DNA-damaging cytostatic drugs and cies of nuclear anomalies, namely karyolysis, pyknosis, ionizing radiation. All of them consumed a mixed diet and karyorrhexis, broken eggs, and condensed chromatin, were had moderate alcohol consumption (<1,000 mL beer or 400 mL recorded in Feulgen-stained slides as described by Tolbert wine/d). et al. (ref. 5; for details, see also ref. 32). The analysis of the Other characteristics of the two study groups are listed in slides was carried out by two experienced scorers; micronuclei Table 1. The number of cigarettes indicated is the average and metanucleated cells (i.e., cells with karyorrhexis, karyol- amount consumed per day during 3 months before the ysis, pyknosis, condensed chromatin, broken eggs, binucleates) experiment was started. Informed consent was obtained from were only registered after consensus. all participants concerning their participation. Giemsa-, May-Gru¨nwald-Giemsa–, and Feulgen-stained Sample size was determined based on power considera- slides were evaluated under a light microscope (Nikon Eclipse tions. Assuming a Poisson distribution of the number of E200) with 1,000-fold magnification using oil immersion. micronucleated cells (33) and introducing a square-root trans- DAPI- and acridine orange–stained slides were evaluated f formation, a SD of 0.55 resulted that was independent of under a fluorescence microscope (Nikon Microphot FXA, the average number of micronuclei for the expected range of filters UV-2a, 330-380 nm and B-2A, 450-499 nm) with 1,000- values. The power to detect a doubling of the average number fold magnification (oil immersion); according to the sugges- of micronucleated cells at the 5% level of significance exceeds tion of M. Fenech (Commonwealth Scientific and Industrial 90% for a 2:1 ratio of the number of subjects in the smokers Research Organization, Adelaide, Australia), Feulgen-stained and nonsmokers groups, respectively, if, overall, 30 subjects slides were additionally scored under a fluorescence micro- are included. scope (filter G-2A, 510-560 nm). Sample Collection. Immediately before cell collection, the Statistics. A square-root transformation was chosen and participants rinsed their mouths twice with tap water. homogeneity of variance of transformed values was tested by Subsequently, the cells were scraped with wooden spatulas Bartlett’s test. Differences between heavy smokers and from each cheek, washed twice in plastic tubes (Sarstedt, nonsmokers as well as staining methods were tested by Germany) containing 10 mL buffer solution [0.1 mol/L two-factor ANOVA with one between-subjects and one EDTA, 0.01 mol/L Tris-HCl, and 0.02 mol/L NaCl (pH 7.0)], within-subjects factor. For each staining method, the differ- and fixed in 80% cold methanol overnight (for details, see ence between smokers and nonsmokers was tested by linear ref. 34). contrasts. The same methods were applied for comparison Staining Procedures. Fifty microliters of the cell suspen- of Giemsa-stained and destained preparations. Age and sions were dropped onto wet cold glass slides and dried alcohol consumption were included as covariables, and overnight in the dark at room temperature. From each sample, gender was added as a between-subject factor to analyze five to seven slides were prepared and stained. All stains were whether these variables contribute to differences between purchased from Sigma (Munich, Germany). micronuclei frequencies. For comparison of cell anomalies Giemsa stain (10%) was prepared in Sorensen’s buffer between heavy smokers and nonsmokers, Wilcoxon’s rank- (pH 6.8) as described by Vives Corrons et al. (35); the staining sum test was applied. Results are expressed as prevalence time was 20 minutes. For May-Gru¨nwald, the cells were first ratios (ratio of the mean number of deviating cells per stained in May-Gru¨nwald solution for 3 to 5 minutes and 1,000 cells) and their confidence intervals were computed subsequently with Giemsa (10%; see above) for 10 minutes based on Fieller’s theorem. (35). Acridine orange was dissolved in bidistilled water Spearman rank correlation coefficients were computed to (0.01%); the staining time was 15 minutes (36). For DAPI, assess correlations between cell anomalies and average the stock solution was diluted (1 Ag/mL) with bidistilled number of micronucleated cells from DNA specific and water; the staining time was 30 minutes (15). For Feulgen, nonspecific staining. the cells were placed in beakers with 5.0 mol/L HCl at room temperature for 15 minutes, rinsed with distilled water Results (15 min), and subsequently stained with Schiff’s reagent for 90 minutes (37). Figure 1 summarizes the micronuclei frequencies found in For direct comparisons of the results obtained with the heavy smokers and nonsmokers with the different staining different procedures, some of the Giemsa and May-Gru¨nwald- procedures. It can be seen that the micronuclei frequencies Giemsa–stained slides were destained in methanol/glacial scored with Giemsa were significantly (f5-fold) higher in acetic acid (3:1) overnight (7) and subsequently restained with smokers than in nonsmokers; also, with May-Gru¨nwald- either acridine orange or Feulgen. Giemsa, the number of micronuclei was substantially (f4.5- fold) higher. On the contrary, no significant effects were observed with the DNA-specific stains. With acridine orange, f Table 1. General characteristics of the participants the micronuclei frequencies were 90% higher in smokers; with DAPI and Feulgen, the differences were 30% and 120%, Variable Smokers Nonsmokers respectively. When the Feulgen-stained slides were evaluated under fluorescence, an 89% increase was observed. It is also No. participants 20 10 Age (mean F SD, y) 36.1 F 8.1 30.3 F 8.7 notable that the micronuclei frequencies recorded in the Gender nonsmokers varied when different staining procedures were Females 4 4 used; with nonspecific stains, the numbers were almost twice Males 16 6 as high as with DNA-specific stains, but these differences No. consumed cigarettes/d (mean F SD) 34 F 11.9 — failed to reach statistical significance. Age, gender, and alcohol F F Duration of smoking (mean SD, y) 12.2 4.0 — consumption did not significantly affect the micronuclei Body mass index (kg/m2, mean F SD) Males 24.5 F 2.0 26.0 F 2.3 frequencies with any of the staining methods and these Females 22.5 F 2.5 21.5 F 3.0 variables had also no effect on the differences between smokers and nonsmokers.

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epithelial cells may be overestimated when non-DNA-specific stains are used. This is an important observation as numerous studies have been conducted in the last two decades with exfoliated cells in which Giemsa stains were used. Our observation that micronuclei formation in oral cells is overestimated with Giemsa-based stains is also supported by the results reported by Casartelli et al. (7), who compared micronuclei numbers after Giemsa and propidium iodide staining in cells from patients with different pathologies of the oral cavity (leucoplakias, squamous cell carcinomas). They found a 3-fold higher frequency of micronuclei with Giemsa than with Hoechst 33258. Comparisons between Feulgen, Hoechst 33259, and propidium iodide showed that 3- to 4- fold higher frequencies are obtained with the latter stain. Also, the evaluation of earlier studies concerning the formation of micronucleated cells in mucosa of smokers confirms our findings (the evaluation is based on computer- aided searches in medical databases, studies in which the Figure 1. Results obtained with different staining procedures in number of participants and/or the number of cigarettes were micronuclei (MN) assays with exfoliated oral mucosa cells from not specified were not included). Although consistently smokers (n = 20) and nonsmokers (n = 10). From each participant, at positive results were obtained with Giemsa (20, 21, 29, 31, least 1,500 cells were scored for micronuclei formation. Columns, 38), negative results were obtained in most studies with DNA- mean; bars, 95% CI. Shaded columns, smokers; open columns, specific stains. We found in total 24 articles in which negative nonsmokers. F-fluorescence microscopy, L-light microscopy. results were obtained in smokers with Feulgen, whereas an increase in micronuclei frequencies was reported in six articles only (6, 22, 23, 39-41). One group reported a positive result (6) To further substantiate the differences between results but failed to confirm it in a subsequent study (42). Also, with obtained with DNA-specific and nonspecific stains, we other DNA-specific stains (DAPI, acridine orange), mainly destained May-Gru¨nwald slides from six smokers and an negative results were obtained (15, 18, 24, 30, 37). identical number of nonsmokers and evaluated the frequencies The effect of alcohol consumption, age, and gender on after DAPI staining. The results are depicted in Fig. 2, and it is micronuclei formation has been studied in several earlier evident that, again, no significant effect was detectable with the investigations, and the results are strongly controversial. DNA-specific stain, whereas a pronounced difference between Although some investigators (6, 13, 26, 27, 30) found no effect smokers and nonsmokers was observed with May-Gru¨nwald. of alcohol on micronuclei formation, Gabriel et al. (31) showed The photographs depict cells with micronuclei after May- a significant effect. Also, Stich and Rosin (26) reported on a Gru¨nwald-Giemsa (Fig. 3A and C), which were restained pronounced synergistic effect of alcohol in combination with either with Feulgen (Fig. 3B) or with DAPI (Fig. 3D). It can be smoking, whereas in another study (25) even an inverse seen that intracellular structures that are classified as micro- relation was found. Also, the effect of age on micronuclei nuclei with the non-DNA-specific stain are not detected with formation in buccal cells is unclear. In five investigations, no the DNA-specific stain. effect was detected (6, 13, 27, 29, 30); in two, an increase with To find out if smoking induces nuclear anomalies other than age (39, 40) and in another a decrease with age (25) were micronuclei, we evaluated comparatively the frequencies of detected. Piyathilake et al. (40) reported that the frequencies of different types of metanucleated cells in the two study groups. micronucleated cells are 2.8-fold higher in females after The results are summarized in Table 2. Four types of adjusting for race, age, smoking habit, and levels of selected anomalies, namely karyorrhexis, karyolysis, binucleates, and condensed chromatin, were 146%, 350%, 117%, and 54% higher in smokers than in nonsmokers. In addition, higher numbers of cells with pyknosis were also found in smokers, although this latter effect failed to reach statistical significance. Table 3 depicts the results of Spearman correlation analyses; it can be seen that increased micronuclei frequencies were significantly associated with certain other nuclear anomalies, such as karyorrhexis, karyolysis, binucleates, and condensed chromatin, when nonspecific staining procedures were used, whereas no such associations were found with the DNA- specific stains. Gender and alcohol consumption did not correlate with the induction of metanucleated cells, but a borderline significant correlation was seen with age (P = 0.05); however, this did not affect the differences between smokers and nonsmokers.

Discussion

Our findings show that the results of micronuclei assays in exfoliated oral mucosa cells of smokers and nonsmokers depend Figure 2. Results obtained with micronucleus assays with exfoliated strongly on the staining methods. Although much higher oral mucosa cells from heavy smokers (n = 6) and nonsmokers (n =6) micronuclei frequencies were found in smokers with the non- in identical slides after staining with Giemsa and after destaining and DNA-specific stains Giemsa and May-Gru¨nwald, no significant subsequent restaining with acridine orange (Acr.O). From each differences were seen with acridine orange, DAPI, and Feulgen. participant, at least 1,500 cells were evaluated. Columns, means; These comparisons indicate that micronuclei formation in bars, 95% CI. Shaded columns, smokers; open columns, nonsmokers.

Figure 3. Photographs of exfoliated oral mucosa cells with micronuclei (magnification, Â500). A and C, May-Gru¨nwald-Giemsa–stained cells. B and D, cells stained with Feulgen and DAPI. Arrows, cellular structures that are classified as micronuclei according to established scoring criteria. vitamin, whereas in a Brazilian study (27) the number of from DNA damage. However, the same phenomena are also micronuclei was significantly (i.e., >3-fold) higher in males. In seen in cells undergoing necrosis (5, 32) and cannot be the present investigation, we found neither in smokers nor regarded as reliable markers for increased DNA damage and in nonsmokers a statistically significant effect of all these cancer risk. Only few studies have been published in which variables on micronuclei formation, but it is notable that all such anomalies have been recorded. Significantly increased participants consumed only small amounts of alcohol if any frequencies were seen, for example, in snuff users, petrol- and that the age range was quite narrow; that is, 36.1 F 8.1 exposed workers, and also in alcoholic patients with oral years in the smokers group and 30.3 F 8.7 years in the controls. carcinomas (5, 22, 25, 32). In all studies including ours, Table 2 shows that metanucleated cells with anomalies other karyolysis was strongly increased compared with the control than micronuclei, such as karyorrhexis, karyolysis, binucleates, groups, whereas the pattern of induction of the other anoma- and condensed chromatin, are significantly increased in lies differed strongly. For example, binucleates were increased smokers. These anomalies reflect the consequences of cell in smokers but not in snuff users, whereas pyknosis was only injury, cell death, and mitotic errors (5, 32). Some of them elevated in the latter group (5, 32). [pyknosis (PN), condensed chromatin (CC), and karyolysis The effect of gender, age, and alcohol consumption on the (KL)] are accompanied by apoptosis (5, 32), which may result formation of metanucleated cells has not been investigated in

Table 2. Frequencies of anomalies in buccal cells per 1,000 cells of smokers (n = 15) and nonsmokers (n = 10)

Variable Group Mean F SE Prevalence ratio Two-tailed P

Condensed chromatin Smokers 9.52 F 0.89 1.54 (1.08-2.18) 0.019820 Nonsmokers 6.19 F 0.94 Binucleation Smokers 7.02 F 0.59 2.18 (1.56-3.03) 0.000385 Nonsmokers 3.23 F 0.47 Karyorrhexis Smokers 7.59 F 0.68 2.47 (1.67-3.64) 0.000428 Nonsmokers 3.08 F 0.55 Karyolysis Smokers 9.90 F 1.16 4.50 (2.64-7.67) 0.000040 Nonsmokers 2.20 F 0.54 Pyknosis Smokers 2.23 F 0.41 1.45 (0.83-2.53) 0.244072 Nonsmokers 1.54 F 0.34 Broken eggs Smokers 0.15 F 0.06 0.74 (0.25-2.23) 0.617618 Nonsmokers 0.20 F 0.08 Degenerative cells Smokers 36.42 F 1.49 2.22 (1.81-2.73) 0.000032 Nonsmokers 16.39 F 1.59

NOTE: Average number of inspected cells per participant: 2,127 (smokers) and 2,140 (nonsmokers).

Table 3. Spearman rank correlation coefficients of correla- et al. (51), who compared micronuclei frequencies in human tions between frequency of cellular anomalies and average lymphocytes stained with DAPI and Giemsa and found even number of micronuclei after non-DNA-specific staining and higher frequencies with the former stain. DNA-specific staining Our investigation indicates that the results of earlier micronuclei studies in cells of epithelial origin should be interpreted Nuclear anomaly Average no. Average no. with caution when Giemsa-based stains were used. Because micronuclei, micronuclei, non-DNA-specific DNA-specific our study and the majority of investigations concerning staining staining induction of micronuclei in smokers with DNA-specific stains yielded negative results and positive results may be due to Condensed chromatin 0.447*c 0.131 misinterpretation, it is very likely that exposure of oral mucosa Binucleation 0.776c 0.122 cells to genotoxic carcinogens, such as nitrosamines, polycyclic Karyorrhexis 0.741c 0.222 Karyolysis 0.734 0.147 aromatic hydrocarbons, and many other compounds that Pyknosis 0.228 0.123 are contained in tobacco smoke, does not lead to formation Broken eggs 0.014c À0.150 of micronuclei in oral mucosa cells (8, 9). The increased risks Degenerative cells 0.844 0.299 of oral cancer in smokers may be due to acute toxic effects and inflammation that are not associated with micronuclei *P < 0.05. formation. 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Cancer Epidemiol Biomarkers Prev 2006;15(10). October 2006 Downloaded from cebp.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Effect of Staining Procedures on the Results of Micronucleus Assays with Exfoliated Oral Mucosa Cells

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