Cytotoxic and Genotoxic Effects of Areca Nut-Related Compounds in Cultured Human Buccal Epithelial Cells1

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[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. GrafstrÃ¶m2

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, GrafstrÃ¶m. 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

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 â€”¿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. Subsequently, the cells were washed twice with Dulbecco's without thiols, i.e., no cysteine, immediately before exposure of the phosphate-buffered saline without Ca2* or Mg2* (27), and the cellular cells. free low-molecular-weight thiol status was determined by two separate Media Solutions and Growth Factors. Porcine Zn-free insulin was a methods. To measure the amounts of total free thiols, cells were lysed gift of the Eli Lilly Co. (Stockholm, Sweden). Fibronectin was obtained and protein was precipitated by addition of 0.35 ml 6.5% trichloroacetic from Collaborative Research, Labassco (Stockholm, Sweden); hydro- acid to each dish. The solution from each dish was centrifuged at 1500 cortisone, polyvinylpyrrolidone, and transferrin were from Sigma x g for 8 min, and 0.25 ml of the supernatant was analyzed for thiol Chemical Co.; 2.5% trypsin solution was from GIBCO (Stockholm, content according to Saville (28). To measure the content of glutathi- Sweden); Vitrogen collagen was Flow Laboratories (Stockholm, Swe one, cellular thiols were trapped using monobromobimane, and the den). Basic tissue culture medium MCDB 153 was obtained from thiol-bimane conjugates were separated by high-pressure liquid chro- NordVacc AB (Huddinge, Sweden). Human epidermal growth factor matography (29, 30). An estimate of the cellular content of glutathione was kindly provided by Dr. G. Nascimento from the Chiron Corp. disulfide was then determined as previously described (29). (Emeryville, CA). Alkaline Elution Assay of DNA Damage. Cells were inoculated onto Tissue and Cell Culture Conditions. Buccal mucosa was obtained from 60-mm dishes at 3-5 x 10' cells/cm2 in BEG-2 medium. Experimental noncancerous surgery or autopsy donors and maintained in culture cells, i.e., buccal epithelial cells, were labeled with [14C]thymidine, and according to methods developed by Sundqvist et al.4 The tissue was cut internal standard, i.e., L-1210 cells, was labeled with ['Hjthymidine as into 5- x 5-mm expiants and placed in culture dishes with the epithelial previously described (29). Following exposure to the indicated agent, side up. The expiants were incubated in serum-free BEG-2 growth medium under air plus 5% CO2 at 36.5Â°C.BEG-2 medium is a buccal the assays for DNA single strand breaks and DNA protein cross-links were performed, and the numbers of DNA lesions were calculated as epithelial growth medium based on serum-free MCDB 153 (diluted previously described (29, 31). with 10% double distilled, sterile H2O to lower the osmolality to physiological levels, i.e., from 340 mOsmol/liter to 305 mOsmol/liter) with the Ca2* concentration subsequently adjusted to 100 Â¿JM,and RESULTS supplemented with insulin (5.2-7.8 /ig/ml), transferrin (8.70 Â¿jg/ml), phosphoethanolamine and ethanolamine (each at 435 n\i), hydrocor The toxicity of an aqueous areca nut extract in cultured tisone (63.5 ng/ml), epidermal growth factor (4.35 ng/ml), and bovine human buccal epithelial cells was determined using three dif pituitary extract (20 jig/ml).4 BEG-1 is BEG-2 without pituitary extract. ferent assays, i.e., colony-forming efficiency, neutral red uptake, After 7-10 days, primary epithelial cell outgrowths radiated from the and trypan blue exclusion (Fig. 2). The dose required to decrease tissue cultures. The cells were enzymatically dissociated using calcium- colony-forming efficiency to 50% of control was about 3 Â¿ig/ml free 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid-buffered saline following a 3-h exposure. In the neutral red and trypan blue solution containing disodium ethyleneglycol bis(/3-aminoethyl ether),/VyV,Ar',A''-tetraacetic acid (0.02%), polyvinylpyrrolidone (1%), assays, 50% of the cells were stained after exposure to about and trypsin (0.025%) (25). The cells were subcultured using BEG-2 150 and 540 fig/ml, respectively. Increased planar cell surface medium on culture dishes surface treated with fibronectin (10 Mg/m') area, an indicator of terminal differentiation in epithelial cells, and collagen (30 Mg/ml).4 For all experiments, secondary cultures were was not apparent in the exposed cells (data not shown). used and exposed to the indicated agent for 3 h in thiol-free BEG-1 The extract decreased the cellular content of total free low- medium. molecular-weight thiols at the highest concentration tested, 1 Colony-forming Efficiency Assay. Cells were seeded at 250 cells/cm2 mg/ml (Fig. 2). When the cellular content of glutathione was in 60-mm dishes and incubated for 24 h prior to treatment. Subse specifically measured, a similar decrease was observed without quently, the medium was exchanged for fresh BEG-2 medium without any concomitant oxidative formation of glutathione disulfide the agent and the cells were incubated for 8 days before fixation in 10% formalin and staining with 1% aqueous crystal violet. The mean colony- (data not shown). forming efficiency was determined from duplicate dishes based on The extract was also assayed for its ability to cause DNA colonies each containing at least 16 cells. In untreated control cultures, damage as measured by the sensitive alkaline elution method up to about 6% of the cells seeded formed colonies. (29, 31). A concentration of 300 Mg/ml caused significant Neutral Red Uptake Assay. Cells were plated at 12,000 cells/cm2 in amounts of DNA single strand breaks and DNA protein cross 35-mm dishes and incubated for 24 h prior to treatment. A modification links (Fig. 3), i.e., 1.2 DNA single strand breaks/10'Â° Da and 5295

100 80

60 O DC 40 O O

20 LU O

01 10 100 1000

CONCENTRATION (Mg/ml) Fig. 2. Effects of aqueous areca nut extract on colony-forming efficiency, CONCENTRATION (mM) neutral red uptake, trypan blue exclusion, and content of total free low-molecular- Fig. 4. Effects of arerÃ nut alkaloids (. Ã•)and .V-nitn iso compounds (B) on weight thiols in human buccal epithelial cells. Means of three experiments Â±SE colony-forming efficiency of human buccal epithelial cells. Means Â±SE (bars) (bars). Â».colony-forming efficiency; A, neutral red uptake; *. trypan blue exclu from three separate experiments. O, arecoline; â€¢¿,arecaidine;â€¢¿,guvacoline;G. sion; â€¢¿.freethiol contents. guvacine; O, A'-nitrosoguvacoline; 4>,A/-nitrosoguvacine; A, NMPN; A, NMPA.

1.0 0.8 0.6

0.4

0.2

4501 CONCENTRATION (mMI 0.8 0.6 0.4 0.2 0.8 0.6 0.4 Fig. S. Effects of areca nut alkaloids (.() and yV-nitroso compounds (B) on INTERNAL STANDARD DNA RETAINED ON FILTER cellular thiol levels of human buccal epithelial cells. Means Â±SE (bars) from Fig. 3. DNA single strand breaks (A) and DNA protein cross-links (B) caused three separate experiments. O, arecoline; â€¢¿.arecaidine;â€¢¿.guvacoline;I L guva by aqueous areca nut extract. Abscissa, corrected time scale, [3H]DNA from cine; O, Ar-nitrosoguvacoline; *, A'-nitrosoguvacine; A, NMPN; A, NMPA. L1210 cells which had been irradiated with 300 rad; about 30% of this DNA was retained on the filter after 12 h of alkaline elution (29, 31). Three separate Table 1 Frequencies of DNA single strana breaks following exposure to areca experiments were performed, and the results are shown as one representative nut-related alkaloids and N-nitroso compounds in cultured human buccal elution profile. â€¢¿,control;â€¢¿.extract300 Â«ig/ml;*, 600 Â»ig/ml;A, 1000 us;/nil. epithelial cells"

of breaks/1010Da0.50.20.50.30.50.40.20.41.02.6 2.2 DNA protein cross-links/1010 Da. Formation of both types AgentArecolineArecaidineGuvacolineGuvacineyv-Nitrosoguvacoline/V-NitrosoguvacineNMPNNMPANMPANMPAConcentration(mM)55555550.10.31.0No. of DNA lesions was dose dependent; at 1000 Mg/ml, 3.9 DNA single strand breaks/1010 Da and 6.6 DNA protein cross-links/ 10'Â°Da were formed. The effects of four areca nut alkaloids, i.e., arecoline, arecai dine, guvacoline, and guvacine, and of four nitrosation products of these alkaloids, i.e., W-nitrosoguvacoline, W-nitrosoguvacine, NMPN, and NMPA, on survival, thiol status, and frequency of DNA single strand breaks were also measured in buccal epithe " The cells were exposed to the various agents and assayed for DNA single lial cells. In order to decrease colony-forming efficiency to 50% strand breaks as described in "Materials and Methods." of the control value, concentrations of 0.15 HIM NMPA, 1.6 mM arecoline, 1.7 HIMW-nitrosoguvacoline, or 2.1 HIM guva DISCUSSION coline were required (Fig. 4). Arecaidine, guvacine, N-nitroso- Aqueous extract of whole areca nut was found to be toxic guvacine, and NMPN at concentrations up to 5 mM had not over a wide range of concentrations in cultured human buccal significant effect on survival. epithelial cells as judged by three different assays. Colony- When effects on the cellular amounts of total free low- forming efficiency was significantly decreased at about 1-3 fig/ molecular-weight thiols were assayed, a 25% decrease was ml, whereas about 100-fold higher concentrations were required induced by 80 pM NMPA, 0.57 HIMarecoline, 0.88 HIMguva to affect vital dye accumulation and membrane integrity as coline, 2.9 mM jV-nitrosoguvacoline, and 3.8 mM ,/V-nitrosogu- indicated by the results of the neutral red uptake and trypan vacine (Fig. 5). Arecaidine, guvacine, and NMPN at concentra blue exclusion assays, respectively. In these experiments, a tions up to 5 mM had no significant effect. marked adherence of the extract to the cells, even after extensive Both the alkaloids and the .Y-niiroso compounds were also rinsing, was observed by both light and scanning electron mi assayed for their ability to cause DNA single strand breaks. croscopy (data not shown). Thus, continuous exposure to re Only NMPA caused a significant increase in the amounts of maining extract during the course of the 8-day colony-forming single strand breaks, i.e., more than 0.5 single strand break per efficiency assay may be the reason why low concentrations of 10'Â°Da, and this increase was dose dependent (Table 1). the extract decrease colony survival but have little or no effect 5296

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1989 American Association for Cancer Research. EFFECTS OF ARECA NUT CONSTITUENTS IN HUMAN BUCCAL CELLS on vital dye accumulation or membrane integrity. The extract cultured human buccal epithelial cells, none of four areca nut did not appear to increase planar cell surface area, indicating alkaloids caused significant amounts of DNA single strand that accelerated terminal differentiation was not involved in breaks at the highest concentration that was tested, 5 IHM. cell killing. Although the colony-forming efficiency may be the Of the areca nut-related jV-nitroso compounds investigated most sensitive of these toxicity assays, the other assays have in cultured human buccal epithelial cells, NMPA was by far the the advantage of providing information on cell viability that most potent on a molar basis; it caused toxicity, thiol depletion, can be directly related to parallel measurements of intracellular and DNA single strand breaks. The aldehyde moiety of NMPA thiol status and DNA damage (32). may be causative in this regard because aldehydes are highly Thiols are involved in a variety of intracellular functions, reactive compounds which have been shown to decrease sur including regulation of proliferation, and are also important in vival, to deplete thiols, and to cause various types of DNA the cellular defense against many reactive foreign compounds damage in cultured human epithelial cells (32, 35). Theoreti (33, 34). Because only the highest and quite cytotoxic concen cally, both alkylating species and several reactive aldehydes may tration (1 mg/ml) of extract caused thiol depletion, aqueous be generated during NMPA breakdown, and these metabolites extract probably contains relatively few sulfhydryl-reactive com may also have contributed to the observed effects. The fact that pounds of minor importance to explain its cyto toxicity. Because NMPA caused marked pathobiological effects in cultured hu thiol depletion was not coupled with oxidation of glutathione man buccal epithelial cells, whereas the other A'-nitroso com to glutathione disulfide, these data also indicate that exposure pounds did not, may indicate specificity in terms of metabolism of intact cells to extract was not coupled with oxidative stress, of these compounds. Cultured human buccal epithelial cells an effect which has recently been associated with areca nut have previously been shown to metabolize benzo(a)pyrene ac extracts in vitro (7). tively at rates substantially higher than their fibroblastic coun Assay of genotoxicity using the sensitive alkaline elution terparts (36). Moreover, several types of tobacco-specific A'- technique indicated that the extract caused both DNA single nitroso compounds have been shown to be actively metabolized strand breaks and DNA protein cross-links in cultured human by cultured rat buccal epithelial cells (37). In rats, NMPN and buccal epithelial cells. These measurements of DNA damage NMPA were shown to be strongly and weakly carcinogenic, indicate that the extract contains agents which are reactive respectively (16-18). Thus, the present study also suggests the toward cellular DNA but do not provide information on the possibility of species variations in susceptibility to these N- chemical nature of these types of genetic lesions. Thus, these nitroso compounds. Because methods to grow human buccal results imply the need for studies using additional genotoxicity epithelial cells and to maintain buccal mucosa! tissue in expiant end points as well as comparisons of effects by areca nut-related culture at defined conditions have only recently become avail subfractions and defined compounds. able,4 these in vitro approaches may aid in the further charac In areca nut, alkaloids are a pharmacologically active group terization of the capability of human oral tissues to metabolize of compounds (1) which together constitute about 1% by weight A'-nitroso compounds. of the nut and also are the precursors for formation of several In summary, some pathobiological effects of areca nut extract specific A'-nitroso compounds (cf. Fig. 1). Areca nut alkaloids and several areca nut compounds have now been identified in in the millimolar concentration range may be of importance for human target cells by using recently developed serum-free cul the long-term effects of betel quid chewing on buccal mucosa, ture conditions. Areca nut extract is clearly both cytotoxic and because arecoline has been detected in saliva obtained during genotoxic. When the alkaloids are compared on a weight basis chewing in concentrations up to about 140 ÃŸg/ml(15), corre to the extract, no single agent has detectable effects on the cells sponding to 0.9 HIM.In cultured human buccal epithelial cells, at concentrations of the extract that cause decreased colony only certain alkaloids up to 5 HIMexerted measurable patho- survival and DNA single strand breaks. Therefore, additive or biological effects, supporting previous studies that structure- synergistic effects could be considered among the various al activity relationships exist for this group of compounds (5, 11). kaloids. Moreover, other classes of components including pol- At similar concentration ranges, guvacoline and arecoline de yphenols (1) are likely to contribute to the marked toxicity of creased both colony survival and thiol content, whereas guva- the extract. NMPA was the most potent in causing cytotoxicity, cine and arecaidine had no appreciable effects on these cellular thiol depletion, and DNA damage, effects that indicate both variables. The cytotoxic and thiol-depleting properties of gu carcinogenic and cocarcinogenic properties of this agent. Fur vacoline and arecoline appear to be chemically related to the ther studies will be of importance to address the contribution presence of a methylester group (cf. Fig. 1). The lower polarity of various betel quid constituents, including tobacco, as well as and lower degree of ionization of these compounds in aqueous the role of areca nut compounds and salivary A'-nitrosation solution may facilitate their passage over biological membranes. reaction products in betel quid carcinogenesis in human oral In contrast, arecaidine becomes ionized (11), and guvacine may epithelium. form a carboxylate aniÃ³nin aqueous solutions, properties which may explain the lack of biological effects of these agents. ACKNOWLEDGMENTS Because arecoline is known to react readily with free thiols such as glutathione or JV-acetylcysteine both in vitro and in vivo (11), We thank J. M. Dypbukt, C. C. Edman, Dr. L. Atzori, and B. Silva conjugation is a likely mechanism for thiol depletion by meth- for technical assistance, and Drs. G. Mam and F. Bertolero for their ylester-containing alkaloids in buccal epithelial cells. The cel valuable comments on the manuscript. Typing assistance of A. L. lular toxicity by arecoline and by guvacoline may be mechanist Marcus is also acknowledged. ically linked to thiol depletion, and this effect may also render the cells more vulnerable to other reactive agents. REFERENCES Several reports have shown that arecoline is genotoxic (4, 5, 9), whereas arecaidine was found to be less genotoxic than 1. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 37. Lyon: International Agency for Research on Cancer, arecoline (4, 5). However, neither compound was shown to 1985. react with any of the four nucleic acid bases in vitro (11). In 2. Jussawalla, D. J., and Deshpande, V. A. Evaluation of cancer risk in tobacco 5297

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Kristina Sundqvist, Yun Liu, Jagadeesan Nair, et al.

Cancer Res 1989;49:5294-5298.

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