A Histochemical Comparison of Feulgen and Papanicolaou Stains in Demonstrating Cytotoxic and Genotoxic Effects of Cigarette Smoking on Human Buccal Mucosa Cells

. A histochemical comparison of feulgen and papanicolaou stains in demonstrating cytotoxic and genotoxic effects


Materials and Methods
The present study was a case-control study. The sampling was conducted in the Faculty of Dentistry, Shahed University, from October to December 2017. The sample size was determined to be 31 subjects in cases (smokers) and 15 in controls (nonsmokers) assuming a power of 0.9 with 95% confidence interval. All enrolled subjects were 20-to-50-year-old males from Tehran, Iran. Suffering from oral and systemic diseases and exposure to dental radiography in the last 6 months were exclusion criteria. Drugs and/or alcohol consumers, waterpipe smokers, industrial workers, and farmers who worked with pesticides did not enter the study. The smokers who quit smoking in the past three years and smoked for less than three years were excluded. In controls, participants with a history of smoking did not enter the study (8). The number of smoked cigarettes per year (Pack years) was recorded for all subjects (4). According to the formula, cases with the rate of smoking from 200 to 500 Pack years were included in the study.
All participants signed an informed consent before sampling. The name of the subjects was not used during the study. The basic information including age and pack years of smoking was registered and coded. All subjects were examined and sampled by a dental student. Two samples were taken from each participant. The obtained samples from the left buccal mucosa were stained with Feulgen and the right mucosa with Papanicolaou. After rinsing the mouth twice with water, the buccal mucosa cells were collected using a sterile disposable plastic spatula. The exfoliated buccal cells were spread on the glass slides and then fixed with Carnoy's fixative (methanol and glacial acetic acid in a ratio of 3:1) for 30 minutes and dried at room temperature.
Obtained slides were prepared and stained by a laboratory technician. The modified method of Thomas et al was used for preparing Feulgen-stained slides. Feulgen staining was completed by dipping the slides in 1 N HCL at 60℃ for 30 minutes, rinsing them in the distilled water for 3 minutes, placing them in Schiff's reagent for 60 minutes and normal saline for 10 minutes, placing them in 5% sodium metabisulfite solution 3 times, and rinsing them with tap water. Finally, slides were stained with 1% light green for 15 minutes, rinsed with tap water, dried, and mounted (9).
The conventional Papanicolaou staining method was used. The fixed slides were immersed in absolute, 70%, and 50% alcohol for 2 minutes in each section. After rinsing with water, the slides were stained with hematoxylin for 4 minutes, rinsed with water, placed in acid alcohol for 5 seconds, dehydrated with absolute alcohol, and stained with orange G for 10 seconds. Using the EA 50 Pap reagent, absolute alcohol, xylene and mounting were the final steps.
The cytotoxicity of cigarette was evaluated by the number of cells with pyknosis, karyorrhexis, and karyolysis. The method developed by Tolbert et al was used for evaluating the cells with pyknosis, karyorrhexis, and karyolysis. The cells with aggregated chromatin, disintegrated nucleus, and dissolved nucleus were considered pyknosis, karyorrhexis, and karyolysis, respectively (10) (Figure 1). Genotoxicity was examined by counting the micronuclei. The cytoplasmic structure with 1/3 to 1/5 size of the nucleus and nuclear stain was considered micronucleus (11). To evaluate the cytotoxic and genotoxic changes, the overlapped cells were not counted. The cells with distinctive cellular boundaries were included in the examination. The number of counted cells with pyknosis, karyorrhexis, and karyolysis in 1000 cells/subject was determined (4). The mean number of micronucleus was determined by the number of counted micronuclei per 1000 cells per subject (12).
The counts were completed using an optical microscope (Olympus BX40) equipped with a digital camera (Sony ExwaveHAD, Model No. SSC-DC58AP; Tokyo, Japan) by an experienced oral pathologist. The cells were evaluated blind at 1000 magnification (10 ocular and 100 objective lenses). Data were analyzed using t test and Levene tests at P ≤ 0.05. The data were presented as the as mean ± SD. The SPSS version 20.0 (IBM Company, Chicago, IL, USA) was employed to analyze the data.

Results
In nonsmokers, the age ranged from 20 to 48 years with a mean of 28.9 ± 9.3 years. In the smokers group, the age ranged from 21 to 50 years with a mean of 34.1 ± 10.8 years.
The results of t test revealed a significant difference between smokers and non-smokers in terms of cytotoxicity (P = 0). Levene test indicated that cytotoxicity in terms of pyknosis (P = 0), karyorrhexis (P = 0.001), and karyolysis (P = 0) in smokers was significantly higher compared to non-smokers. The micronucleus counts in smokers and non-smokers were 7.21 ± 8.13 and 3.7 ± 3.78, respectively. The genotoxicity was significantly higher in smokers than in non-smokers (P = 0.006).
The results of the t test revealed that the demonstration of micronuclei was not significantly different between Feulgen and Papanicolaou (P = 0.27). Demonstration of karyolysis (P = 0.73) and karyorrhexis (P = 0.24) was not significantly different between Feulgen and Papanicolaou staining methods. The Feulgen stain was significantly more successful in demonstrating pyknosis (P = 0.02) compared to Papanicolaou.

Discussion
The study shows that Feulgen and Papanicolaou stains were similar in demonstrating DNA alterations (micronucleus) and cellular death features (karyorrhexis and karyolysis). The Feulgen technique was more effective in demonstrating pyknosis compared to Papanicolaou.
The staining method is an important tool for determining the nucleus abnormalities. The false positive/false negative conclusions are routine outcomes of unsuitable stains (7). The protocol developed by Stich et al to assess the genotoxic effect of carcinogens on exfoliated buccal mucosa cells is still extensively used (16). To demonstrate the nuclear features based on this protocol, different staining methods have been used. Feulgen (4,17,18) and Papanicolaou (12,19,20) are the most popular stains used in displaying the nucleus changes. Basically, DNA-specific stains have more appropriate results in demonstrating nuclear changes. Feulgen is a DNA-specific stain and Papanicolaou is a nonspecific histochemical stain. Both Feulgen and Papanicolaou are popular in cytologic studies, but which one can be better in demonstrating the cytotoxic/genotoxic effects of agents?
Fulgen and Papanicolaou stains were similar in demonstrating DNA alterations and cellular death features. The only exception was the representation of pyknotic structures. The finding is not compatible with the study of Kumar et al who showed that the number of micronuclei in Papanicolaou-stained slides was significantly higher compared to Feulgen-stained samples (21).
The results are compatible with previous studies which showed the overestimation of micronuclei count with non-DNA-specific stains such as Giemsa-based stains (7). Nonspecific DNA stains in evaluating genotoxic changes may lead to false-positive results. This finding was in agreement with results obtained by Casartelli et al and Holland et al (22,23).

A recent study by Kohli et al showed better results for
Papanicolaou stain than May-Grünwald Giemsa stain and Feulgen stain in the evaluation of micronuclei (24). This is not in agreement with the present study.
Binucleation, condensed chromatins, karyorrhexis, and karyolysis cause misinterpretation of micronuclei count with DNA nonspecific stained preparations (7,24). Abnormal changes in the nucleus are not specific to damaged cells. Nuclear anomalies can also be observed in normal undamaged cells. Contradictory results may be due to not considering all cellular changes at the same time.
To compare the Papanicolaou and Feulgen stains in terms of these changes, control samples were selected from non-smokers. Although the rate of nuclear changes in smokers was higher compared to non-smokers, there were no differences between the two stains in demonstrating the nuclear features.
Accurate sampling, appropriate fixation, and good stained sections are important subjects in evaluating micronuclei. For proper evaluation of samples, the use of standard criteria to identify cellular abnormalities is necessary. To obtain more reliable and valid results, the protocol developed by Tolbert et al was used to evaluate microscopic abnormalities (10). Simple laboratory procedure and the lower cost of Papanicolaou staining method in comparison with Feulgen are important reasons for choosing Papanicolaou. Feulgen staining technique is time-consuming and needs an experienced technician to complete. Based on our experience, selecting the Papanicolaou or Feulgen staining methods is the second important step in cellular evaluation. The most important issue to evaluate cellular changes is the use of standard criteria.
Based on the present results, simultaneous evaluation of DNA alterations (micronucleus) and cellular death features (karyorrhexis, karyolysis, and pyknosis) is suggested. Standardization of definitions of DNA  alterations is intensely recommended in future studies.

Conclusions
Feulgen and Papanicolaou staining methods had similar effectiveness in demonstrating DNA alterations (micronucleus) and cellular death features (karyorrhexis and karyolysis). The Feulgen staining method was preferable to display pyknosis compared to Papanicolaou.