Because betel quid chewing has been linked to the development of oral cancer, pathobiological effects of an aqueous areca nut extract, four areca nut alkaloids (arecoline, guvacoline, guvacine, and arecaidine), and four nitrosated derivatives [N-nitrosoguvacoline, N-nitrosoguvacine, 3-(N-nitrosomethylamino)propionaldehyde and 3-(N-nitrosomethylamino)propionitrile] have been investigated using cultured human buccal epithelial cells. Areca nut extract in a dose-dependent manner decreases cell survival, vital dye accumulation, and membrane integrity, and it causes formation of both DNA single strand breaks and DNA protein cross-links. Depletion of cellular free low-molecular-weight thiols also occurs, albeit at quite toxic concentrations. Comparisons of the areca nut-related N-nitroso compounds and their precursor alkaloids, at concentrations up to 5 mM, indicate that 3-(N-nitrosomethylamino)propionaldehyde is the most potent on a molar basis to decrease both survival and thiol content and to cause significant formation of DNA single strand breaks. Arecoline, guvacoline, or N-nitrosoguvacoline decreases survival and cellular thiols, whereas arecaidine, guvacine, N-nitrosoguvacine, and 3-(N-nitrosomethylamino)propionitrile have only minor effects on these variables. Taken together, the present studies indicate that aqueous extract and, in particular, one N-nitroso compound related to areca nut, i.e., 3-(N-nitrosomethylamino)propionaldehyde, are highly cytotoxic and genotoxic to cultured human buccal epithelial cells, of potential importance in the induction of tumors in betel quid chewers.

https://cancerres.aacrjournals.org/content/canres/49/19/5294.full.pdf

Cytotoxic and genotoxic effects of areca nut-related compounds in cultured human buccal epithelial cells.

Powdered tobacco (Khaini tobacco) with the addition of lime is commonly used by the residents of Bihar, India. The tobacco/lime mixture is usually placed on the inner side of the lower lip within the gingivolabial groove. About 42% of the users keep it at the front, the rest move the tobacco towards the left or right side within the oral cavity. Carcinomas (so-called "Khaini cancers") develop mainly at the site where the tobacco is in close contact with the mucosa. Scrapings of the mucosa were taken at sites where the tobacco is kept, then smears were prepared, stained with the Feulgen reaction and fast green, and screened for micronuclei which indicate the occurrence of chromosome aberrations in the dividing cell population of the basal layer. An elevated frequency of cells with micronuclei was found in the oral mucosa of all 27 examined Khaini tobacco users (Munda and Santal tribes) compared to that of non-chewers of similar ethnic background and dietary habits. The induction of micronucleated mucosa cells seems to be due to genotoxic agents released from the tobacco/lime mixture. In vitro, an aqueous extract of the Khaini tobacco elicits chromosome aberrations and micronuclei in cultured human fibroblasts and Chinese hamster ovary (CHO) cells. No chromosome-damaging effect was observed following the application of lime or calcium hydroxide (Ca(OH)2). The micronucleus test on exfoliated cells can provide evidence of carcinogen exposure in the tissue from which cancers will develop. This approach combines all the advantages of in vitro short-term tests for genotoxic and carcinogenic agents with those of using an intact organism with all its defence mechanisms.

Application of the micronucleus test to exfoliated cells of high cancer risk groups: Tobacco chewers

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:

This paper reviews the ability of a number of chemicals to induce sister-chromatid exchanges (SCEs). The SCE data for animal cells in vivo and in vitro, and human cells in vitro are presented in 6 tables according to their relative effectiveness. A seventh table summarizes what is known about the effects of specific chemicals on SCEs for humans exposed in vivo. The data support the concept that SCEs provide a useful indication of exposure, although the mechanism and biological significance of SCE formation still remain to be elucidated.

ReviewSister-chromatid exchange: second report of the Gene-Tox program

Naturally occurring substances were tested for genotoxicity using a modified laboratory protocol of the Escherichia coli PQ37 genotoxicity assay (SOS chromotest) in the presence and in the absence of an exogenous metabolizing system from rat liver S9-mix. Aristolochic acid I, II, the plant extract aristolochic acid and psoralene were genotoxic; cycasine, emodine, monocrotaline and retrorsine were classified as marginal genotoxic in the SOS chromotest in the absence of S9-mix. In the presence of an exogenous metabolizing system from rat liver S9-mix aristolochic acid I, the plant extract, beta-asarone, cycasin, monocrotaline, psoralen and retrorsine showed genotoxic effects; aristolochic acid II marginal genotoxic effects. Arecoline, benzyl acetate, coumarin, isatidine dihydrate, reserpine, safrole, sanguinarine chloride, senecionine, senkirkine, tannin and thiourea revealed no genotoxicity in the SOS chromotest either in the presence or in the absence of an exogenous metabolizing system from rat liver S9-mix. For 17 of 20 compounds, the results obtained in the SOS chromotest could be compared to those obtained in the Ames test. It was found that 12 (70.6%) of these compounds give similar responses in both tests (6 positive and 6 negative responses). The present investigation and those reported earlier, the SOS chromotest, using E. coli PQ37, was able to detect correctly most of the Salmonella mutagens and non-mutagens.

A.R. Rao

Papers

Chromosome-breaking ability of arecoline, a major betel-nut alkaloid, in mouse bone-marrow cells in vivo.

Influence of caffeine on arecoline-induced SCE in mouse bone-marrow cells in vivo