To understand the role of betel quid (BQ) in the pathogenesis of oral submucous fibrosis (OSF) and oral cancer, we used DNA damage, cytotoxicity, and cell proliferation assays to study the pathobiological effects of aqueous extracts of three BQ constituents [betel nut (Areca catechu, BN), inflorescence of Piper betle (IPB), and lime], one BN alkaloid (arecoline), and one BN polyphenol [(+)-catechin] on cultured oral mucosal fibroblasts. Extracts of BN and IPB induced DNA strand break formation in a dose-dependent manner. Extracts of BN and IPB, (+)-catechin, and arecoline decreased cell survival and proliferation in a dose-dependent manner. However, aqueous extract of lime (50-800 micrograms/mL) increased cell proliferation by 20-40%. These results indicate that BQ contains not only genotoxic and cytotoxic agents, but also compounds which stimulate cell proliferation. These compounds may act synergistically in the pathogenesis of OSF and oral cancer in BQ chewers. In addition, five anti-oxidants [glutathione (GSH), cysteine, mannitol, catalase, and superoxide dismutase (SOD)] were tested for their protective effects against the cytotoxicity of BQ constituents. GSH (1.95 and 2.6 mmol/L) and cysteine (4 and 8 mmol/L) prevented the arecoline-induced cytotoxicity. In contrast, mannitol, catalase, and SOD did not decrease the arecoline-induced cytotoxicity. These results indicate that thiol depletion, but not the attack of oxygen free radicals, could be the mechanism for arecoline cytotoxicity. GSH could also protect cells from the cytotoxicity of IPB extract. Increasing dietary intake of GSH-rich foods or dietary supplements of GSH may have chemopreventive potential to reduce BQ-associated oral lesions.

Genotoxic and Non-genotoxic Effects of Betel Quid Ingredients on Oral Mucosal Fibroblasts in vitro:

Smokeless tobacco consumption, which is widespread throughout the world, leads to oral submucous fibrosis (OSMF), which is a long-lasting and devastating condition of the oral cavity with the potential for malignancy In this review, we mainly focus on the consumption of smokeless tobacco, such as paan and gutkha, and the role of these substances in the induction of OSMF and ultimately oral cancer The list of articles to be examined was established using citation discovery tools provided by PubMed, Scopus, and Google Scholar The continuous chewing of paan and swallowing of gutkha trigger progressive fibrosis in submucosal tissue Generally, OSMF occurs due to multiple risk factors, especially smokeless tobacco and its components, such as betel quid, areca nuts, and slaked lime, which are used in paan and gutkha The incidence of oral cancer is higher in women than in men in South Asian countries Human oral epithelium cells experience carcinogenic and genotoxic effects from the slaked lime present in the betel quid, with or without areca nut Products such as 3-(methylnitrosamino)-proprionitrile, nitrosamines, and nicotine initiate the production of reactive oxygen species in smokeless tobacco, eventually leading to fibroblast, DNA, and RNA damage with carcinogenic effects in the mouth of tobacco consumers The metabolic activation of nitrosamine in tobacco by cytochrome P450 enzymes may lead to the formation of N-nitrosonornicotine, a major carcinogen, and micronuclei, which are an indicator of genotoxicity These effects lead to further DNA damage and, eventually, oral cancer

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Smokeless tobacco (paan and gutkha) consumption, prevalence, and contribution to oral cancer

Betel quid and oral cancer: a review.

Eighteen biopsies of the oral mucosa of northern Thai hilltribe betel chewers were studied histologically and by transmission and scanning electron microscopy (TEM, SEM). Clinically, varying stages of epithelial atrophy and one case of submucous fibrosis were observed. Histologically, epithelial atrophy with marked reduction of the rete pegs, hyperortho- and/or parakeratosis, and subepithelial edema and inflammatory changes were the prominent findings. On the ultrastructural level, cytoplasmic projections of the basal cells into the subepithelial stroma were seen. The basal membrane frequently revealed gaps; the interepithelial space was widened and unusual microvilli were observed on cell surfaces (SEM). Intercellularly, cristalloid material of unknown origin was also seen. The subepithelial connective tissue was characterized by dense bundles of collagen fibres adjacent to which masses of amorphous material were located. While some of the ultrastructural findings in the epithelium of betel chewers are indicative of early dysplastic changes, the nature of the juxta-epithelial stromal alterations is still unknown. Current hypotheses regarding the etiology of oral submucous fibrosis are briefly discussed.

Ultrastructural findings in the oral mucosa of betel chewers.

Pan masala, a dry powdered mixture of areca nut, catechu, lime, unspecified spices and flavoring agents, has gained widespread popularity as a chewing substitute in India. In this study, the carcinogenic and tumor-promoting potential of an ethanolic pan masala extract (EPME) was determined using skin of S/RVCri-ba mice and forestomach and esophagus of ICRC mice as the target tissues. Carcinogenic activity of pan masala was tested by painting the mouse skin for 40 weeks with EPME or by gavage feeding for 6 months. Following initiation with 9,10-dimethylbenz(a)anthracene (DMBA), carcinogenesis of mouse skin was promoted with different doses of EPME, while gastric- and esophageal-tumor-promoting activity was determined by administering EPME by gavage to animals initiated with diethylnitrosamine (DEN). The ability of EPME to effect progression of skin papilloma to carcinoma and cutaneous alterations after a single or multiple EPME treatment were also evaluated. EPME at 25 mg per dose promoted skin-papilloma formation between 30 and 40 weeks of treatment and enhanced the rate of conversion of papilloma to carcinoma. Induction of mild epidermal hyperplasia, dermal edema, increase in epidermal mitotic activity and the rate of epidermal and dermal DNA synthesis by EPME correlated well with its skin-tumor-promoting potential. In ICRC mice, EPME was inactive as a complete carcinogen, but effectively promoted the development of forestomach and esophageal papilloma and carcinoma in a concentration-dependent manner. The tumor incidence at 25 mg EPME per dose was comparable with that obtained in the 12-0-tetradecanoylphorbol-13 acetate(TPA)-treated group. The findings indicate that habitual pan-masala use may exert carcinogenic and co-carcinogenic influence.

Evaluation of carcinogenic/co-carcinogenic activity of a common chewing product, pan masala, in mouse skin, stomach and esophagus

The high incidence of oral cancer in different population groups in many south-east Asian nations has been stressed repeatedly in early and recent reports (Orr, 1933; Balendra, 1949; Cooray, 1944; Khanolkar, 1959; Muir, 1962; Pindborg, 1965). In India, and elsewhere, epidemiological and biological investigations have been done, or are in progress, on any possible correlation between the habitual use of the betel chew or " pan " and oral cancer. The involvement of the main constituents of " pan "-betel leaf (Piper betel), betel nut (Areca catechu), tobacco (Nicotiana tabacum), slaked shell or stone lime (slaked calcium hydroxide) and catechu (Acacia catechu) have also been considered individually or in combination (Mody and Ranadive, 1959; Muir and Kirk, 1960; Dunham, Muir and Hamner, 1966). The role of lime appears rather controversial, though some reports do suggest its correlation with the occurrence of oral cancer. It was therefore considered worthwhile to study the effects of slaked commercial lime on the oral mucosa of laboratory animals. The main aims of the study were to evaluate the part played by lime in the causation of oral malignancy or of oral conditions
considered to be precancerous and the trauma it caused to epithelial and subepithelial tissue. This paper reports the histological changes produced on short term and prolonged application of lime to the oral mucosa of normal, diet conditioned and hormone treated Wistar rats.

Satyavati M Sirsat

Papers

Histological changes in the oral mucosa of the wistar rat treated with commercial lime (calcium hydroxide)--an optical and submicroscopic study.