Assessment of Epithelial Proliferative Markers among Toombak Dippers Using Conventional Cytological Methods

Abstract

This is a retrospective case control cohort study; cellular proliferative activity of Toombak dippers oral epithelium was assessed applying Papanicolaou method, mean AgNOR counts, micronuclei frequency and 1% Crystal Violet stain. The study was conducted in Khartoum state during the period from November 2009 to April 2010. Oral scrapes smears were obtained from 210 participants of whom 200 were apparently healthy individuals; 100 were Toombak users (cases); 100 were non-tobacco users (controls) and ten were patients with Oral Squamous Cell Carcinoma (OSCC), as an internal control group. Their ages ranging from 16 to 94 years; with a mean age 33 years old. The mean age for the cases was (34 years); hence, the mean age for the controls was (32 years). Cytological atypia was identified among four (4%) Toombak users hence, no cytological atypia was found among control group. Of the four cases with cytological atypia, only one case was identified with moderate degree of cytological atypia and the remaining three were categorized as having mild degree of cytological atypia (p< 0.04). Thirty two (32%) subjects from the cases were identified with keratinization; hence, only two subjects from the control group were identified with keratinization. Toombak dipping is a risk factor for occurrence of the keratinization in the oral mucosa (P < 0.0001). Thirteen (13%) and two (2%) of the subjects are indentified with inflammation in cases and control groups respectively. In regard to the infections, twenty one (21%) and four (4%) of the cases were identified with bacterial infection and moniliasis, respectively, since three (3%) of the control group identified with bacteria. Cases were more susceptible to the inflammation and infection than control group, erosions and exposure of oral mucosa to Toombak irritation are the major causative factors (P < 0.003). The mean AgNOR counts in the cases was (2.423) was statistically higher than control groups (1.303) (P < 0.0001). In regard to the mean of the micronuclei frequency per 1000 buccal cells, the micronuclei frequencies were higher in the cases group (1.026) than in control group (0.356) (P < 0.0001). The mean AgNOR counts and micronuclei frequency was statistically increased with the prolonged and frequent use of Toombak (P < 0.001). However, there was increasing in the mean AgNOR counts in those snuffing large amount of Toombak per dip (relatively statistically significant; p<0.062). The keratinization presence was associated with the increasing of the mean AgNOR counts and micronuclei frequency (p<0.0001). In regard to the presence of inflammation the AgNOR counts were statistically associated with increased inflammation infiltrate (p<0.006). Increasing of the age in the study population was statistically associated with increasing of the mean AgNOR count, micronuclei frequency, and keratinization (p<0.001). Neither cases group nor control group were found with mitotic figures in 1% crystal violet stain method. This findings support that, Toombak dipping is a high risk factor to increase the cellular proliferation in the oral mucosa and the cytological proliferative markers methods are useful for screening the Toombak users.