Quantitative analysis of AgNOR proteins in exfoliative cytology specimens of oral mucosa from smokers and nonsmokers

OBJECTIVE: To determine the cell proliferation rate and possible effects of cigarette smoking on the oral mucosa lining through analysis of silver-stained nucleolar organizer regions (AgNORs) in exfoliative cytology specimens. STUDY DESIGN: Exfoliative cytology was performed on the left side of the border of the tongue and of the floor of the mouth in 25 smoking patients and 25 nonsmoking patients. The inclusion criterion for smokers was the consumption of more than 20 cigarettes per day for a minimum of 30 years. RESULTS: The slides were stained by histochemical AgNOR method. In the nonsmoking group the mean number of AgNORs per nucleus was 2.732 +/- 0.236 in the tongue border and 2.918 +/- 0.195 in the floor of the mouth. In smoking patients the mean number of AgNORs per nucleus was 3.372 +/- 0.375 in the tongue border and 3.245 +/- 0.237 in the floor of the mouth. CONCLUSION: The results suggest higher cell proliferation quantified by the histochemical AgNOR technique in exfoliative cytology specimens obtained from the oral mucosa lining of smokers presenting no clinical alterations.     

AgNOR count in exfoliative cytology of normal buccal mucosa. Effect of smoking

OBJECTIVE: To compare the argyrophilic nucleolar organizer region (AgNOR) count of cells collected from normal buccal mucosa of cigarette smokers with that obtained from nonsmokers. STUDY DESIGN: Cytologic smears of normal buccal mucosa from 20 smokers and 20 nonsmokers were stained for AgNORs. The AgNOR count was established on 100 cells. The count values of groups were compared and analyzed using Student's unpaired t test. RESULTS: The AgNORs were round and had a clustered distribution in both groups. The mean AgNOR count was statistically higher in cells of smokers than nonsmokers (P < .01). CONCLUSION: Analysis of AgNORs suggests that cigarette smoking influences proliferative activity in cells of normal buccal mucosa.     

Evaluation of smoking genotoxicity in Turkish young adults

BACKGROUND:

For the past few decades, it has been widely known in developed countries that tobacco is dangerous, but it is still insufficiently realized how big these dangers really are.

AIMS:

To determine and evaluate micronuclei (MN) frequencies of young smokers and nonsmokers in three different tissues (peripheric blood lymphoctes, buccal mucosa, and exfoliative urothelial cells) at the same time.

MATERIALS AND METHODS:

MN assay was performed on buccal mucosa, urothelial cells, and peripheric blood lymphocyte samples obtained from 15 healthy male smokers (>5 pack-years) and 15 healthy male nonsmoker controls who had not been exposed to any known genotoxic agent.

STATISTICAL ANALYSIS USED:

The statistical differences between smoker and nonsmoker groups were calculated by using student t test. The differences between smoker-group tissues were compared by ANOVA.

RESULTS:

It was found that MN frequency (mean value ± standard deviation) in oral mucosa cells from smokers and controls were 1.20 ± 0.22% and 0.26 ± 0.10%; in urothelial exfoliative cells, 1.29 ± 0.28% and 0.12 ± 0.08%; in peripheric blood lymphocytes, 1.53 ± 0.23% and 0.38 ± 0.12%, respectively. The mean MN frequencies in buccal mucosa, urothelial exfoliative cells, and peripheric blood lymphocytes were significantly higher in smokers than in those of controls (P<0.05). All tissues were affected from smoking, but the most destructive effect was seen in urothelial cells of smokers (P<0.05).

CONCLUSIONS:

Our data showed that cigarette smoke is a DNA damage causitive agent on exfoliative buccal mucosa and urothelial cells and peripheric blood lymphocytes of young smokers, but it has most destructive effect on urothelial cells.     

Quantitative assessment of various fixatives on nuclear and cytoplasmic areas in oral cytology

Summary Both nuclear and cytoplasmic areas are parameters known to be of significance in the diagnosis of malignancy. However, few studies have assessed the effect of fixation on exfoliative cytology and none has looked at such influences upon oral smears. Hence the method of fixation may influence directly diagnostic cytology. The effect of three methods of fixation upon the nuclear and cytoplasmic areas of cells removed from the buccal mucosa was quantitatively assessed. The three methods employed, prior to Papanicolaou staining, were: direct immersion in diethylether and ethanol (11 v/v), spray fixation (Vale Smear Fix) and air drying. Three smears from each of 21 patients were used, each slide being allocated randomly a, method of fixation. After 24h all smears were processed for Papanicolaou's stain.The nuclear and cytoplasmic areas were calculated using semi-automated image analysis. No significant differences were found in the two areas whichever method of fixation was used.     

Cytobrush and wooden spatula for oral exfoliative cytology. A comparison.

The Cytobrush has been used frequently in cervical cytology, but as yet its value in oral exfoliative cytology has not been assessed. A study was undertaken to compare the efficiency of the Cytobrush with that of the wooden tongue spatula. For 26 patients, two smears were collected from clinically normal mucosa from four sites in the oral cavity (buccal mucosa, dorsal tongue, ventral tongue and hard palate). The smears were graded for cell yield and dispersion on a three-point scale. The results were analyzed using the chi 2 test. The Cytobrush was found to be significantly more efficient than the wooden spatula, in terms of both cell yield (P less than .005) and cell dispersion (P less than .005). When each site was examined separately, the Cytobrush produced significantly better dispersion for the dorsal tongue, ventral tongue and buccal mucosa and a better cell yield for the tongue surfaces. No significant difference for cell yield or dispersion was found for the hard palate. The study showed that the Cytobrush is an effective instrument for use in exfoliative cytology of normal oral mucosa.     

Evaluation of Papanicolaou stain for studying micronuclei in buccal cells under field conditions

OBJECTIVE: To compare Papanicolaou (Pap) and May-Grünwald Giemsa (MGG) stain as 2 techniques for staining for buccal mucosal cells to detect micronuclei (MN) infield studies. STUDY DESIGN: Eighty cytologic smears (2 per individual) were taken from the buccal mucosa of 40 cigarette smokers recruited at a rural village in Egypt. Forty smears were stained with Pap stain and 40 with MGG stain. All were assessed for cellularity and scored for MN. RESULTS: Pap stain was faster and easier to process and transport in the field study than was MGG stain. Regarding MGG smears, bacteria and cell debris masked the MN as compared to Pap smears, in which the fixative destroyed the bacteria and made the cell boundaries clearly demarcated. Using Pap stain, MN were seen easily in transparent cytoplasm. CONCLUSION: Pap stain is the preferred method infield studies for scoring and detecting MN in cells of buccal mucosa.     

Levels of 8-hydroxydeoxyguanosine as a marker of DNA damage in human leukocytes

We measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels in human leukocytes from healthy donors to evaluate oxidative DNA damage and its correlation with smoking, physical exercise, and alcohol consumption. A significant increase in oxidative DNA damage was induced by cigarette smoke, with the mean level of 8-OHdG being significantly higher in smokers (33.1 +/- 10.6 per 10(6) 2-deoxyguanosine (dG) [mean +/- SE], n = 16) compared with nonsmokers (15.3 +/- 1.8 per 10(6) dG, n = 31) and former smokers (17.8 +/- 1.5 per 10(6) dG, n = 9). The highest values were observed after smoking more than 10 cigarettes per day (41.8 +/- 17.1 per 10(6) dG, n = 9). A large interindividual variation in 8-OHdG levels was observed in all analyzed groups. We also observed a correlation between 8-OHdG levels and age in nonsmokers and former smokers. Neither frequency of physical exercise nor alcohol drinking significantly modified 8-OHdG levels in leukocytes.     

Application of the micronucleus test to exfoliated epithelial cells from the oral cavity of beedi smokers, a high-risk group for oral cancer

The primary sites for occurrence of oral cancer include the buccal mucosa, tongue, alveolus, palate, lip and the floor of the mouth. In this study, an attempt was made to estimate the cytogenetic damage in different regions of the oral mucosa in people habituated to smoking beedi,which is one of the major forms of tobacco consumption in India and believed to be a major risk factor for oral cancer. By using the micronucleus assay on exfoliated cells from the buccal mucosa, palate and tongue of beedi smokers, we examined an early cellular response to the effect of beedi smoking. A total number of 50 randomly selected male subjects were included in the study. Case and control groups (smokers and non-smokers, respectively) comprised 25 subjects each. The difference in mean micronucleated cell count between cases and controls was significant (P <0.01) for buccal mucosa and palate, but not for tongue. The correlation between age and micronucleus cell count was weak for both cases (r=0.27) and controls (r=0.36).     

Expression of ATP6V1C1 during oral carcinogenesis

We investigated the gene and protein expressions of V-type ATPase protein subunit C1 (ATP6V1C1) in cases of oral squamous cell carcinoma (OSCC) and contralateral normal mucosa in smokers, nonsmokers and former smokers. Subjects were separated into five groups of 15: group 1, smokers with OSCC; group 2, normal contralateral mucosa of OSCC patients; group 3, chronic smokers; group 4, former smokers who had stopped smoking 1 year earlier; group 5, individuals who had never smoked. Exfoliative cytology specimens from oral mucosa of smokers, former smokers and nonsmokers showed normal gene and protein expression. We found significantly greater gene expression in the OSCC group than in the nonsmoker groups. No difference in gene expression was observed between normal contralateral mucosa and nonsmoker groups, smoker and nonsmoker groups or former smoker and nonsmoker groups. We observed intense immunostaining for ATP6V1C1 protein in all cases of OSCC and weak or no staining in smoker, former smoker and nonsmoker groups. Significantly greater expression of ATP6V1C1 protein was observed in the OSCC group compared to the other groups, which supports the role of ATP6V1C1 in effecting changes associated with oral cancer. Analysis of the mucosae of chronic smokers, former smokers and the normal contralateral mucosa of patients with OSCC showed unaltered ATP6V1C1 gene and protein expression. Early stages of carcinogenesis, represented by altered epithelium of chronic smokers, had neither gene nor protein alterations as seen in OSCC. Therefore, we infer that the changes in ATP6V1C1 occur during later stages of carcinogenesis. Our preliminary study provides a basis for future studies of using ATP6V1C1 levels for detecting early stage OSCC.     

A morphometric study of the protective effect of cryotherapy on oral mucositis in cancer patients treated with 5-fluorouracil

We investigated cytological changes in oral mucosa smears from patients treated with cryotherapy to determine whether cryotherapy prevented mucositis caused by 5-fluorouracil (5-FU) therapy. Patients with gastrointestinal malignancies were divided into four groups; control patients before 5-FU therapy, patients after 5-FU therapy without cryotherapy, patients with cryotherapy before 5-FU therapy and patients with cryotherapy after 5-FU therapy. Oral mucosa samples from all patients were assessed at the beginning and on day 14 of chemotherapy. We used exfoliative cytology to evaluate cellular changes in the oral mucosa that were caused by 5-FU. Smears from each patient were stained using the Papanicolaou method and analyzed using stereology. Smears were taken from each group before and after 5-FU infusion. We found that nuclear volume was decreased significantly in cells of the 5-FU therapy after cryotherapy patients compared to the 5-FU therapy before cryotherapy patients. We also found significantly decreased cytoplasmic volumes in the 5-FU therapy after cryotherapy patients compared to the 5-FU therapy before cryotherapy patients. The results of cytomorphometric estimations revealed that cryotherapy may be used to prevent damage to oral tissue and may decrease the frequency and duration of oral mucositis caused by 5-FU.     

Smokers and passive smokers gene expression profiles: correlation with the DNA oxidation damage

Healthy volunteers (n=50) were enrolled for studying the variation of gene expression induced by smoking in peripheral lymphocytes. RNAs from smokers (>3 cigarettes/day, n=20) and passive smokers (exposed to tobacco smoke >3 h/day, n=10) were hybridized versus a reference pool obtained by mixing equal amounts of RNA from 20 nonsmokers, and gene expression was analyzed using DNA microarrays containing 13,971 oligos. Principal component analysis showed that 99.7% of gene expression variability was related to plasma cotinine, age, and DNA oxidation damage. SAM and GenMAPP/MAPPFinder analyses showed that smokers, compared to nonsmokers, had 129 down-regulated and 87 up-regulated genes, whereas passive smokers, compared to nonsmokers, had 44 down-regulated and 159 up-regulated genes, mainly involved in pathways associated with the activation of defensive responses. Hierarchical cluster analysis identified two distinct clusters of smokers, characterized by different oxidative DNA damage: smokers with high DNA oxidation damage, compared to smokers with low DNA oxidation damage, had a large number (150) of down-regulated genes, mainly associated with xenobiotic metabolism, DNA damage and repair, inflammatory responses, lymphocyte activation, and cytokine activity, suggesting a reduced cellular response to toxic agents in this subset of smokers that could lead to an increased DNA oxidation damage.     

AgNOR quantification in cells of normal oral mucosa exposed to smoking and alcohol. A cytopathologic study

OBJECTIVE: To evaluate cell proliferative activity by counting and measuring argyrophilic nucleolar organizer regions (AgNORs) per nucleus in cell smears from mucosa clinically exposed to smoking and alcohol. STUDY DESIGN: Group 1 (control) consisted 17 patients, group 2 (smoking) of 25 and group 3 (smoking and alcohol) of 18. Cell smears collected from the mucosa of the lower lip, border of the tongue and floor of the mouth underwent AgNOR staining. Mean number and mean area of AgNORs per nucleus were calculated for the first 50 cells in each smear. ANOVA and the Tukey test were used for statistical analyses at a 5% significance level. RESULTS: Statistical analyses revealed a greater mean number and larger mean area of AgNORs per nucleus in groups 2 (smoking) and 3 (smoking and alcohol). Samples from the border of the tongue had the lowest mean values for number and area of AgNORs per nucleus in comparison with samples from the lower lip and floor of the mouth in the 3 groups. CONCLUSION: Anatomic sites exposed to smoking or to smoking and alcohol had increased cellular proliferative activity.     

A column-switching liquid chromatography-tandem mass spectrometry method for quantitation of 2-cyanoethylmercapturic acid and 2-hydroxyethylmercapturic acid in Chinese smokers

The acrylonitrile metabolites 2-cyanoethylmercapturic acid (CEMA) and 2-hydroxyethylmercapturic acid (HEMA) have been determined in human urine using an automated column-switching procedure. A diluted sample was centrifuged just prior to being injected into a reusable precolumn packed with a restricted access material and coupled to a liquid chromatography-tandem mass spectrometry system. This method achieved satisfactory reproducibility and accuracy. Average intra- and interday variations (% relative standard deviations) ranged from 2.4 to 3.8% for CEMA and from 2.7 to 10.5% for HEMA. The limits of quantification were 0.003 and 0.099ng/ml for CEMA and HEMA, respectively. It was used to study the uptake of acrylonitrile from smoke constituents by both nonsmokers and smokers of different tar yield cigarettes under ISO 3308 smoking condition. Metabolite concentrations in smoker urine samples were approximately 12 times higher compared with those in nonsmokers for CEMA and 3 times higher for HEMA. Urinary CEMA levels show a clear dose-response relationship with daily cigarette consumption and urinary cotinine. CEMA can also discriminate between smokers of different ISO cigarettes. Because HEMA is not specific, it is only slightly related to smoking and acrylonitrile exposure. The validated biomarker CEMA will continue to be useful for studies of acrylonitrile uptake by smokers.     

Cytologic and cytometric analysis of oral mucosa in Alzheimer's disease

OBJECTIVE: To analyze cytomorphologically the buccal mucosa of patients with Alzheimer's disease (AD). STUDY DESIGN: Brush biopsies were obtained from 10 patients with AD and 9 age-matched controls without neurologic symptoms from 3 distinct oral sites. RESULTS: A significant reduction in partially keratinized intermediate (red) cells was observed in the buccal mucosa of the AD group. In the AD group, parabasal cells from the floor of the mouth (p = 0.017) and buccal mucosa (p = 0.058) and red cells,from the tongue dorsum (p = 0.013) and buccal mucosa (p = 0.002), exhibited significantly greater nuclear areas. With regard to the nuclear to cytoplasmic (N:C) ratio, intermediate (red) cells from the buccal mucosa and tongue dorsum of AD individuals showed a decrease in this parameter (p <0.0001), while superficial (yellow) cells (from buccal mucosa) (p= 0.042) and parabasal (blue) cells (from the tongue dorsum) (p = 0.003) exhibited an increased N:C ratio. No significant differences were detected in the cells from the floor of the mouth. CONCLUSIONS: Our findings indicate that cytologic and cytometric changes were detectable in the exfoliative cytology of the buccal mucosa and tongue in the AD group.     

'Normal' cigarette smoking increases free cortisol in habitual smokers.

In habitual smokers salivary cortisol responses to cigarette smoking were investigated. In the first study, 31 adults assigned to two experimental groups smoked either one or two cigarettes of their preferred brand. Mean salivary cortisol levels were significantly elevated after smoking of two cigarettes. In the second study, 10 smokers and 10 nonsmokers provided saliva samples at 20 min intervals over a 12-hr period. While environmental stimuli were paralleled in both groups overall cortisol output was significantly elevated in the smokers. These data suggest that 'normal' cigarette smoking can increase free cortisol levels.     

Do high rates of cigarette consumption increase delay discounting? A cross-sectional comparison of adolescent smokers and young-adult smokers and nonsmokers

The present report attempts to help clarify the causal or consequent relation between frequently reported high rates of delay discounting (DD) associated with cigarette-smoking status in adults. Delay-discount functions of adolescent smokers and young-adult smokers and nonsmokers from two earlier studies [Reynolds, B., Karraker, K., Horn, K., Richards, J.B., 2003. Delay and probability discounting as related to different stages of adolescent smoking and non-smoking. Behav. Process. 64, 333-344; Reynolds, B., Richards, J.B., Horn, K., Karraker, K., 2004. Delay discounting and probability discounting as related to cigarette smoking status in adults. Behav. Process. 65, 35-42] were cross-sectionally compared. If a high rate of DD is a predisposing factor to future smoking status, adolescent and young-adult smokers were expected to have similar rates of DD, but both groups were expected to have higher rates of discounting than young-adult nonsmokers. Alternatively, if a high rate of cigarette consumption over an extended period is related to increases in DD, young-adult smokers were expected to discount more than adolescent smokers and young-adult nonsmokers. Results supported the hypothesis that a high rate of cigarette consumption is related to higher rates of DD, rather than the alternative hypothesis that smokers are predisposed with higher rates of DD. Also, after combining adolescent and young-adult smokers, self-reported number of cigarettes consumed per day was positively correlated with rate of DD; however, reported length of smoking history was not correlated with DD. Possible neurological mechanisms leading to increased discounting are discussed.     

Sialadenitis with crystalloid formation. Fine needle aspiration cytodiagnosis of 15 cases

OBJECTIVE: To assess the utility of fine needle aspiration cytology in the diagnosis of sialadenitis with crystalloid formation. STUDY DESIGN: In 15 cases, salivary gland masses were aspirated using a disposable, 20-mL syringe and 25-gauge needles, maintaining negative pressure. Smears routinely were air dried and stained by Diff-Quik (Dade Behring AG, Düdingen, Germany). Occasionally smears were fixed in alcohol and stained by the Papanicolaou method. RESULTS: The smears showed large numbers of non-birefringent crystalloids of varying sizes and shapes. The crystalloids stained deep blue with Diff-Quik and bright orange with Papanicolaou stain. Multinucleated histiocytes, neutrophilic leukocytes and benign salivary gland parenchyma were found, also. CONCLUSION: Fine needle aspiration cytology provides an accurate diagnosis of sialadenitis with crystalloids and is useful for avoiding unnecessary surgery.     

Cigarette smoking during pregnancy lowers aromatase cytochrome P-450 in the human placenta

To clarify whether cigarette smoking during pregnancy causes an organic alteration in placental estrogen producing ability, we determined the catalytic activity of aromatase by the tritiated water assay, and tissue level of aromatase cytochrome P-450 (P-450_arom) by the specific enzyme-linked immunosorbent assay, in placental samples from nonsmokers and smokers. As pregnancy progressed, both aromatase activity and P-450_arom concentration increased in placentas from nonsmokers and smokers. However, the gradient of the increase was significantly less in heavy smokers (20 cigarettes a day) than in normal and moderate smokers (<20 cigarettes a day). At term, the mean aromatase activity and P-450_arom concentration in placentas from heavy smokers were significantly lower than in nonsmokers and moderate smokers, while aromatase activity per P-450_arom (turnover rate) and the mean placental weight were comparable among the three groups. In contrast, the ratio of aryl hydrocarbon hydroxylase activity to aromatase activity was higher in placentas from heavy smokers. Immunohistochemical studies showed that P-450_arom was localized in the cytoplasm of syncytiotrophoblasts of chorionic villi in placentas from both nonsmokers and smokers. These results suggest that the induction of placental P-450_arom during gestation is suppressed by maternal smoking, resulting in a reduction in estrogen producing ability, while placental xenobiotic P-450 is induced.     

Exfoliative cytology of the oral mucosa in burning mouth syndrome: a cytomorphological and cytomorphometric analysis

doi:10.1111/j.1741‐2358.2009.00319.x
Exfoliative cytology of the oral mucosa in burning mouth syndrome: a cytomorphological and cytomorphometric analysis Objective: The aim of this study was to evaluate oral epithelial cells by exfoliative cytology in burning mouth syndrome (BMS). Material and methods: Oral smears were collected from clinically normal‐appearing mucosa by liquid‐based exfoliative cytology in 40 individuals (20 BMS patients and 20 healthy controls matched for age and gender) and analysed for cytological and cytomorphometric techniques. Results: Mean values of nuclear area (NA) for experimental and control groups were, respectively, 67.52 and 55.64 μm² (p < 0.05). Cytoplasmic area (CA) showed the following mean values: 1258.0 (experimental) and 2069.0 μm² (control). Nucleus‐to‐cytoplasm area ratio for the experimental group was 0.07, besides the control group was 0.03 (p < 0.05). Morphologically, oral smears exhibited normal epithelial cells in both experimental and control groups. There was a significant predominance of nucleated cells of the superficial layer in the smears of BMS patients (p = 0.00001). Conclusion: This study revealed that oral mucosa of BMS patients exhibited significant cytomorphometric changes in the oral epithelial cells. These changes probably are associated with epithelial atrophy and a deregulated maturation process that may contribute to the oral symptoms of pain and discomfort in BMS.     

Increased plasma concentrations of C9, C1-inhibitor and alpha 1-protease inhibitor associated with cigarette smoking

The association between various parameters of acute and chronic smoking status and plasma levels of three proteins, C9, C1-inhibitor (C1-INH) and alpha 1-protease inhibitor (alpha 1-PI) were determined for 49 male cigarette smokers and 49 age-matched nonsmokers (mean age = 32.2 years). The mean number of cigarettes smoked was 28.7 per day while the cumulative consumption was only 18.1 pack-years. Plasma levels of all three proteins were significantly higher in the smokers than nonsmokers. Plasma C9 and alpha 1-PI concentrations correlated with cumulative cigarette consumption and plasma nicotine concentrations. While C1-INH concentration did not correlate with either cumulative cigarette consumption or plasma nicotine concentration, it correlated significantly with serum thiocyanate concentration. No consistent correlation was found between plasma concentration of these proteins and parameters of pulmonary function.