[CANCER RESEARCH 49, 5294-5298, October 1. 1989] Cytotoxic and Genotoxic Effects of Areca Nut-related Compounds in Cultured Human Buccal Epithelial Cells1 Kristina Sundqvist, Yun Liu, Jagadeesan Nair, Helmut Bartsch, Kristina Arvidson, and Roland C. Grafström2 Department of Toxicology, Karolinska Institute!, Box 60400, S-104 01 Stockholm, Sweden [K. S., Â¥.L., R. C. GJ; Unit of Environmental Carcinogens and Host Factors, International Agency for Research on Cancer, 150 Cours Albert- Thomas, 69372 Lyon Cedex 08, France fJ. N., H. B.J; and Department of Prosthetic Dentistry, Karolinska Institute!, S-141 04 Huddinge, Sweden [K. A.] ABSTRACT in both bacteria and mammalian cells (4, 5). It also increases the frequency of bone marrow cells with micronuclei and chro Because betel quid chewing has been linked to the development of oral mosomal aberrations in mice in vivo (5, 9) and causes chromo cancer, pathobiological effects of an aqueous areca nut extract, four areca nut alkaloids (uri-coline, guvacoline, guvacine, and arecaidine), and four somal aberrations in Chinese hamster ovary cells in vitro (10). nitrosated derivatives |/V-nitrosoguvacoline, jV-nitrosoguvacine, 3- However, aa-colinc has not been clearly demonstrated to be (yV-nitrosomethylamino)propionaldehyde and ,V( V-niirosomethylam- carcinogenic in animals (1). Arecaidine, another areca nut al ino)propionitrile| have been investigated using cultured human buccal kaloid and a structural analogue of arecoline, is found as a epithelial cells. Areca nut extract in a dose-dependent manner decreases urinary metabolite of arecoline in rats (11) and is formed in cell survival, vital dye accumulation, and membrane integrity, and it vitro after incubation of arecoline with liver homogenates (12). causes formation of both DNA single strand breaks and DNA protein Arecaidine is also mutagenic in bacteria (4) and causes sister cross-links. Depletion of cellular free low-molecular-weight thiols also chromatid exchanges in mouse bone marrow cells in vivo (13). occurs, albeit at quite toxic concentrations. Comparisons of the areca Very little is currently known about the effects of the other nut-related jY-nitroso compounds and their precursor alkaloids, at con centrations up to 5 HIM, indicate that 3-{/V-nitrosomethylam- alkaloids in areca nut (guvacoline, guvacine, and arecolidine). ino)propionaldehyde is the most potent on a molar basis to decrease both The alkaloids present in areca nut can undergo nitrosation reactions (Fig. 1). When arecoline is nitrosated in vitro, both survival and thiol content and to cause significant formation of DNA NMPN3 and NMPA are formed (14). These compounds, to single strand breaks. Arecoline, guvacoline, or /V-nitrosoguvacoline de creases survival and cellular thiols, whereas arecaidine, guvacine, N- gether with yV-nitrosoguvacoline and Ar-nitrosoguvacine, are nitrosoguvacine, and 3-{/V-nitrosomethylamino)propionitrile have only also formed after in vitro nitrosation of betel quid (15). Similar minor effects on these variables. Taken together, the present studies nitrosations may occur in humans during chewing of betel quid, indicate that aqueous extract and, in particular, one yV-nitroso compound because NMPN (16), W-nitrosoguvacoline (15, 17), and N- related to areca nut, i.e., 3-(/V-nitrosomethylamino)propionaldehyde, are nitrosogli vacine (15) have been detected in their saliva. In rats, highly cytotoxic and genotoxic to cultured human buccal epithelial cells, NMPN is a potent and complete carcinogen (16, 18), which of potential importance in the induction of tumors in betel quid chewers. both methylates and cyanoethylates DNA in target tissues (19). W-Nitrosoguvacoline is not mutagenic in Drosophila (20), re INTRODUCTION quires metabolic activation to exert mutagenicity in S. typhi Epidemiological studies have demonstrated a clear causal murium (21), and was reported to be carcinogenic in rats in one association between chewing of betel quid containing tobacco study (22), but not in others (17, 20, 21, 23). We have recently developed serum-free methods for replica and an increased risk for oral cancer. These studies have also tive cultures of normal human buccal epithelial cells.4 The indicated that most tumors arise in the buccal mucosa in contact with the quid (1-3). Besides tobacco, areca nut (also called present study was undertaken to investigate in vitro whether "betel nut") is often a major ingredient of the quid. More areca nut extract causes pathobiological effects associated with information is available on the adverse health effects of tobacco, carcinogenesis directly in these human target cells. Therefore, whereas less is known about the corresponding effects of areca to investigate cytotoxicity, thiol reactivity, and genotoxicity, nut (1-3). There is limited evidence that aqueous extracts of several biological end points, including colony-forming effi ciency, vital dye uptake ability, cell membrane integrity, intra- the betel quid or areca nut alone are carcinogenic to experimen cellular free low-molecular-weight thiol status, and two types tal animals (1). Areca nut extracts are mutagenic in Salmonella typhimurium (4) and in Chinese hamster V79 cells (5). In of DNA damage, i.e., single strand breaks and DNA protein cross-links, were studied. Effects of several areca nut alkaloids creased frequencies of chromatid breaks and exchanges have and their W-nitroso derivatives (see Fig. 1 for chemical struc been observed in Chinese hamster ovary cells (6). Although the nut constituents have been separated into a variety of extracts, tures) were also investigated in buccal epithelial cells in an subfractions, and components (6, 7), the agent(s) involved in attempt to determine whether these compounds are responsible the genotoxic and carcinogenic effects of areca nut has not yet for some of the cytopathic actions related to areca nut. been unequivocally identified. Areca nut contains several alkaloids (Fig. 1), of which are- MATERIALS AND METHODS coline is the most abundant (8). This compound is mutagenic Chemicals. Arecoline and arecaidine were purchased from Sigma Received 2/3/89; revised 6/26/89; accepted 7/5/89 Chemical Co. (St. Louis, MO). The following compounds were synthe The costs of publication of this article were defrayed in part by the payment sized as described previously: guvacoline (24), guvacine (24), W-nitro- of page charges. This article must therefore be hereby marked advertisement in soguvacoline (15), ./V-nitrosoguvacine (15), NMPN (14), and NMPA accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by grants from the Swedish National Board of Laboratory (14). Arecoline, arecaidine, guvacoline, and guvacine were dissolved in Animals, the Swedish Tobacco Company, the Swedish Cancer Society, the Swed the exposure medium immediately before addition to the cells. Stock ish Natural Science Research Council, the Swedish Medical Research Council, and the Swedish Fund for Scientific Research Without Animal Experiments, by 3The abbreviations used are: NMPN, 3-(,V-nitrosomcthylamino)propionitrile; funds from the Karolinska Institute, and by NIH Grant 1001 CA 43176-01 from NMPA, 3-(A'-nitrosomcthy]amino)propionaldehyde. the USPHS. 4K. Sundqvist, Y. Liu, K. Arvidson, K. Ormstad, L. Nilsson, and R. C. 2To whom correspondence should be addressed, at Department of Toxicology, Grafström. Growth regulation of serum-free cultures of epithelial cells from Karolinska Institutel, Box 60400, S-104 01 Stockholm, Sweden. normal human buccal mucosa, submitted for publication. 5294 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. EFFECTS OF ARECA NUT CONSTITUENTS IN HUMAN BUCCAL CELLS GUVACOLINE ARECOLINE ARECAIDINE GUVACINE of the neutral red uptake assay described by Hunt et al. (26) was used. r COOCH3 ,-COOH Following exposure, the cells were rinsed twice with BEG-1 medium and incubated in BEG-2 growth medium containing 50 jig/ml of neutral red. After 3 h, the cells were rinsed twice with BEG-1 medium before scoring of viable cells (stained red by the dye) and nonviable cells (which /"^YC remained unstained) in a phase contrast microscope. Cells in four randomly selected fields or at least 100 cells/dish were scored. The percentage of viability was calculated as 100 times the ratio of viable N-NITHOSO cells to the sum of viable and nonviable cells. More than 90% of GUVACINE unexposed cells were scored positive for vital dye accumulation, i.e., 3-lN NITHOSOMETHYLAMINC^ 3-lN NITHOSOMETHYLAMINC^ neutral red uptake. PROPIONALDEHYDE PROPIONITRILE Trypan Blue Exclusion Assay. Cells were inoculated at 1200 cells/ Fig. 1. Nitrosation products of areca nut alkaloids. Adapted from Refs. 14 cm2 in 35-mm dishes and incubated for 24 h prior to treatment. and 16. Subsequently, they were washed twice with BEG-1 medium, and then 0.16% trypan blue (Sigma) dissolved in BEG-1 medium was added for solutions of W-nitrosoguvacoline (3.6 M) and /V-nitrosoguvacine (3.1 M) 5 min. The dye was removed, and the number of viable cells (which in dimethyl sulfoxide and of NMPN (3.0 M) and NMPA (0.5 M) in exclude trypan blue) and nonviable cells (which appear entirely blue or sterile, distilled water were used and added to the cells as required, have blue nuclei) were counted in four randomized fields per dish in a using appropriate solvent controls. All other reagents were of analytical phase contrast microscope. The percentage of viability was calculated grade. from duplicate dishes as 100 times the ratio of number of viable cells Preparation of Aqueous Areca Nut Extract. Areca nut extract was to the sum of viable and nonviable cells. More than 90% of unexposed prepared by extraction of 10 g dry, powdered areca nut with 250 ml control cells excluded trypan blue. distilled water for l h (7). The extract was filtered through a sintered glass funnel, freeze dried, and frozen at —¿70"C(7).For each experiment, Assays for Free Low-Molecular-Weight Thiols. Cells were inoculated onto 35-mm dishes at 6000 cells/cm2 and incubated for 72 h prior to aliquots of the extract were dissolved in serum-free BEG-1 medium4 treatment.