Salivary arecoline levels during areca nut chewing in humanvolunteers

Stephen Cox1,2, Edward R. Vickers

3, Sonia Ghu

3, Hans Zoellner

2

1The Department of Oral Surgery, The Faculty of Dentistry, The University of Sydney, Westmead Centre for Oral Health, WestmeadHospital, Westmead, NSW, Australia; 2The Cellular and Molecular Pathology Research Unit, Oral Pathology and Oral Medicine,The Faculty of Dentistry, The University of Sydney, Westmead Centre for Oral Health, Westmead, NSW, Australia; 3PainManagement Research Institute, The University of Sydney, Royal North Shore Hospital, St Leonards, NSW, Australia

BACKGROUND: Arecoline stimulates cultured cells

above 0.1 lg ⁄ ml and is cytotoxic above 10 lg ⁄ ml.

Although this alkaloid seems important for areca nut

induced oral carcinogenesis, little is known of the levels

achieved during chewing.

MATERIALS AND METHODS: Saliva was collected in 3-

to 5-min intervals over 50 min in 32 habitual chewers: be-

fore, for 25 min during, and for 20 min after chewing areca

nut (0.5 g) without any other additives. Salivary arecoline

was quantitated by HPLC-MS. Controls comprised six

subjects who denied areca nut use, and who were given

rubber-base material to chew during experiments instead.

RESULTS: Arecoline was detected before chewing in 22

subjects, exceeding the 0.1 lg ⁄ ml threshold in 20 cases.

Salivary arecoline exceeded either the 0.1 or 10 lg ⁄ ml

thresholds in all participants during chewing (P < 0.001).

Maximum concentrations ranged from 5.66 to 97.39 lg ⁄ml. All subjects reached 0.1 lg ⁄ ml salivary arecoline in at

least 85% of time points studied (P < 0.0001), whereas

10 lg ⁄ ml was reached in 11 participants in at least 30% of

the time points (P < 0.003). Arecoline concentrations

varied greatly over time between individuals, and levels

were much lower when peak concentrations were

reached before 3 min, than in cases where arecoline

peaked later (P < 0.02). No salivary arecoline was found

in control saliva.

CONCLUSIONS: Areca nut users have persistent back-

groundsalivaryarecolinelevels longafterchewing,whereas

concentrations achieved are highly variable and consistent

with a role in oral pre-malignancy and malignancy.

J Oral Pathol Med (2010) 39: 465–469

Keywords: areca nut; Arecoline; oral cancer; oral pre-cancer;

saliva

Introduction

The �areca nut’ Areca catechu palm seed is widelychewed throughout the Indian subcontinent, South EastAsia and the Pacific islands, often together with betelpiper vine leaves, calcium carbonate preparations, spicesand tobacco (1–3). The main alkaloid is Arecoline whichproduces euphoria, sweating and palpitation, and is thefourth most commonly used addictive drug after alco-hol, tobacco and caffeine (4–6).

Areca nut use is an independent risk factor for oralsquamous cell carcinoma (OSCC), as well as for the pre-malignant conditions leukoplakia and oral submucousfibrosis (OSF) (7–9). OSF is particularly debilitating, bylimiting oral opening via restrictive fibrosis as well as aburning sensation of the mouth (7, 10–13). The highprevalence of OSCC in the Indian subcontinent isattributed to areca nut use (14), so that the toxicologyof arecoline is of substantial interest.

Fibroblasts and keratinocytes are reported to respondto arecoline with: depressed DNA synthesis; cell cyclearrest; cell death, both increased and decreased collagenproduction, and cytokine synthesis. Of importance isdetermination of biologically relevant concentrationsfor arecoline, whereas two concentration thresholdsappear critical, being 0.1 lg ⁄ml for collagen stimulationand 10 lg ⁄ml for cytotoxicity (8, 12, 15–25).

Despite areca nut’s role in oral carcinogenesis and thesensitivity of cells to arecoline, the only published dataappear confined to brief mention in a single reportedstudy (26). This study significantly expands uponpublished data, while we demonstrate individual varia-tion in salivary arecoline levels potentially contributingto individual clinical outcomes.

Materials and methodsMaterialsArecoline was detected using a high pressure liquidchromatography (HPLC)-mass spectrometry (MS)system including a LC 10AD model dual pistonpump, DGU-3A degaser, SIL-10A automated injector,

Correspondence: Stephen Cox, The Department of Oral Surgery,The Faculty of Dentistry, The University of Sydney, Westmead Centrefor Oral Health, Westmead Hospital, Westmead, NSW 2145, Austra-lia. Tel: (+61) 02 9845 7406, Fax: (+61) 02 9893 8671, E-mail:scox@dentistry.usyd.edu.auAccepted for publication December 7, 2009

doi: 10.1111/j.1600-0714.2009.00881.x

J Oral Pathol Med (2010) 39: 465–469

ª 2010 John Wiley & Sons A/S Æ All rights reserved

interscience.wiley.com/journal/jop

CBM-10A controller, and a CTO-10A oven (Shima-dzu Pty Ltd., Kyoto, Japan). All experiments used aLuna 5-lm particle size, 100 A pore size,250 · 4.6 mm, C18 reversed-phase column (WatersPty. Ltd., Sydney, Australia). Electron spray MS withan atmospheric pressure chemical ionisation electro–spray interface (ESI) was used. Disposable C18

extraction cartridges were from Alltech (Deerfield,IL, USA).Arecoline (methyl-1,2,5,6-tetrahydro-1-methylnicoti-

nate), arecaidine (1,2,5,6-tetrahydro-1-methylnicotinicacid), triethylamine, and caffeine were from Sigma Pty.Ltd. (St Louis, MO, USA). Acetonitrile was fromUnichrom Co (Kuang-Fu Rd, Taipei 100, Taiwan,ROC). Lignocaine was from Astra Zenica (North Ryde,Sydney, Australia). Areca nut chips were from RoshanNaidu’s (Importer and Distributor, Parramatta, NSW,Australia), whereas inert 3M Imprint II rubber-materialfor control subjects was from 3 M Dental Products,St. Paul, MN, USA.

Collection of mixed salivaThe Westmead Hospital Human Ethics Committeeapproved research, and participants completed asurvey including age, sex, place of residence, place oforigin, and areca nut use frequency. Thirty-twohabitual areca nut chewers were recruited. Mixedsaliva was collected by spitting into 10-ml disposabletubes before chewing 0.5 g of areca nut without anyother additives, with collection of all saliva generatedby spitting into further tubes, providing fresh tubes at1, 3, 5, 10, 15, 20 and 25 min. Participants thenremoved nut particles from the mouth, and salivasamples collected for up to a further 20 min, changingtubes every 5 min. Samples were centrifuged at 402 gand decanted into fresh tubes for snap freezing inliquid nitrogen and storage at )20�C. Six additionalsubjects with no areca nut habit comprised controls,who were provided with inert rubber-base material tochew instead.

Quantitation of arecoline in samples by HPLC ⁄MSStandard curves were readily prepared using arecolinefrom 0.01 to 50 lg ⁄ml and lignocaine (3 lg ⁄ml) as aninternal standard, by determination of the ratio ofarecoline to lignocaine MS spectral peak height. Fiftymicrolitres volumes of saliva was mixed with 50 llvolumes of stock lignocaine and 200 ll of acetonitrilefor evaluation by HPLC-MS using a Luna C18

column at 30�C and a mobile phase of 50% aceto-nitrile and 50% 0.01 M sodium hydrogen phosphate(pH 7.8) with 0.01% triethylamine at isocratic flowrates ranging from 0.3 to 1.2 ml ⁄min. The followingESI-MS conditions were found effective: N2 heated to350�C and applied at a flow rate of 10.0 l ⁄min fordrying; N2 as a nebulizer applied at 40 psi; capillaryvoltage at 1500 V; fragmentor voltage at capillary exitat 110 V; dwell time 139 ms; and mass peak width of0.7 Th (FWHM). Qualifying ions with mass:charge(m ⁄ z) of 156, and 197 were arecoline, and m ⁄ z 235were lignocaine.

Statistical analysisBinomial analysis for single sample tests of proportionwas performed, and Chi-squared tests evaluated pro-portions between groups. Wilcoxon’s ranked sign andthe Mann–Whitney U tests were used for paired andunmatched samples, respectively.

ResultsMost participants were from the Indian subcontinentand used areca nut regularlyAll areca nut users were Australian residents, and 30 ofthese had an ethnic background from the Indiansubcontinent. Fifteen came from India, six from SriLanka, two from Goa, and one from Bangladesh. Inaddition, there were single participants from Malaysiaand Taiwan, whereas six were Australian born. Four ofthe six control participants not using areca nut ascontrols were from India, whereas two were from SriLanka. Fifty-three percent of participants was male withan average age of 28.8 ± 10.5 years, while females hadan average age of 34.2 ± 10.1, and age of both sexesranged from 17 to 49 years. All participants had afunctional natural dentition. Frequency of areca nut usevaried greatly with no clear difference between male andfemale participants. Twenty-five out of 32 participantsreported use at least once per month (Fig. 1). Noparticipants had any clear sign of oral pre-malignancyor malignancy.

Arecoline was often present in saliva before chewingfresh areca nut and always achieved biologicallyrelevant concentrationsIn all cases studied, salivary arecoline concentrationsexceeded either the 0.1 lg ⁄ml or the 10 lg ⁄ml biolog-ically relevant thresholds (P < 0.001) during chewing,whereas apparently residual arecoline from earlierchewing episodes was also often seen (Fig. 2). Baselinelevels of arecoline measured before chewing ranged from

Figure 1 Histogram showing the frequency of areca nut use byparticipants according to gender. Only three participants used arecanut on a daily to weekly basis, whereas two subjects indicated theyused areca nut more than four times per month. Most areca nut usersindicated use from 1 to 4 times per month, while there were sevenparticipants who reported chewing areca nut less than once per month.There was no clear difference between the sexes with regard tofrequency of areca nut use.

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undetectable levels to 2.4 lg ⁄ml, and in most cases (20out of 22) were above 0.1 lg ⁄ml (P < 0001). Maximumarecoline concentrations during chewing ranged from5.66 to 97.39 lg ⁄ml. Peak arecoline levels exceeded10 lg ⁄ml in 26 out of 32 subjects (P < 0.001) (Fig. 2).No effect of gender was seen, whereas no arecoline wasdetected in any samples collected from the six controlsubjects given rubber-base material to chew.

Arecoline concentrations exceeded biologically relevantlevels for most of the time subjects chewed fresh areca nutArecoline exceeded the 0.1 lg ⁄ml and 10 lg ⁄ml thresh-olds for most of the time participants chewed fresh arecanut (Fig. 3). In addition, most of the subjects for whom45-min time point measurements were available hadlevels above 0.1 lg ⁄ml (15 out of 19 participants,P < 0.02), whereas three of these had levels over1 lg ⁄ml (P < 0.005).

Although data were not available for all time points inall subjects, a scatter-plot (Fig. 4) of the relativepercentage of time points with salivary arecoline con-centrations at or above 0.1 lg ⁄ml, 1 lg ⁄ml and 10 lg ⁄ml revealed all subjects had arecoline levels at or abovethese important threshold concentrations for apprecia-ble time periods, with progressively higher concentra-tions of arecoline being seen in fewer of the time pointssampled.

Arecoline levels were lower in participants who achievedpeak levels within 5 min of chewing compared withparticipants who reached peaks after 5 or more minutes ofchewingPatterns of arecoline levels in saliva varied greatly overtime amongst participants, whereas levels were substan-tially lower after the 25-min time point at whichparticipants were asked to spit out any remaining nutmaterial (P < 0.0001) (Fig. 5). Early peak levels ofarecoline were associated with lower overall arecolineconcentrations (P < 0.02).

Discussion

The single earlier report quantitating salivary arecolineinvolved measurements from 12 participants chewingcomplete betel quid either with or without tobacco,and also collecting saliva once or twice during chewing

Figure 2 Interval diagram showing starting (s) and maximum (d)levels of salivary arecoline in each subject tested. Salivary arecoline wasdetected in 22 out of 31 participants prior to administration of freshareca nut (P < 0.03), and 20 of these subjects had starting levelsexceeding 0.1 lg ⁄ml (P < 0001). In all cases studied, salivary areco-line concentrations achieved levels exceeding either the 0.1 or the10 lg ⁄ml biologically relevant thresholds (P < 0.001), with peakarecoline levels exceeded 10 lg ⁄ml in 26 out of 32 subjects(P < 0.001).

Figure 3 Graph of salivary arecoline concentrations for each subjectand time point tested, with the 25-min time point at which areca nutparticles were consciously spat out marked with an arrow. Arecolineexceeded either the 0.1 or 10 lg ⁄ml thresholds for most of the timeparticipants chewed fresh areca nut. Maximum levels were reached by25 min of chewing (P < 0.0001), whereas arecoline concentrationswere above the 0.1 lg ⁄ml level in most participants at the last timepoint studied (P < 0.02).

Figure 4 Scattergram showing the relative percentage of time pointswith salivary arecoline concentrations at or above 0.1 lg ⁄ml, 1 lg ⁄mland 10 lg ⁄ml for each subject studied (Fig. 5). All participants hadarecoline levels above 0.1 lg ⁄ml in at least 85% of the time pointssampled (P < 0.0001). Thirty participants had arecoline levels at orabove 1 lg ⁄ml for at least 50% of the time points sampled(P < 0.0001). Levels of at least 10 lg ⁄ml were seen in four subjectsin at least 50% of the time points sampled (P < 0.0001), whereas inseven participants this high threshold for salivary arecoline wasexceeded for at least 30% of the time points sampled (P < 0.003).

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(26), so that the current study is a significant advance-ment.Of particular interest is that many subjects had

appreciable salivary arecoline levels before commencingthe experiment, and that this was often above the0.1 lg ⁄ml threshold required for cell stimulation whilecytotoxic levels were reached during chewing (8, 12, 15–25). Detection of arecoline before chewing suggestspersistence in the oral cavity long after exposure to thenut, greatly enhancing potential carcinogenic effects.High baseline salivary arecoline seems particularlysurprising in the light of most participants indicatingareca nut for at most 3–4 times per month. Whilesubjects may have under-reported actual areca nut use,it is also possible that there are mechanisms that eithertrap arecoline in the oral cavity, or perhaps re-circulatearecoline between blood and saliva. These possibilitiesare both supported by appreciable salivary arecolinelevels 25 min after removal nut particles from themouth. Prolonged salivary fluoride levels after cessationof fluoride exposure, as well as enterosalivary recircu-lation of urea and nitrite, provide precedent for the ideathat the salivary glands may concentrate arecoline forre-secretion long after areca nut chewing has ceased (27–29). It would be interesting to further examine thispossibility by collecting parotid saliva before, duringand after chewing areca nut, as well as in animalexperiments determining the distribution of arecoline insaliva, salivary gland and plasma compartments.It was noteworthy that salivary arecoline levels

sufficient to cause cytotoxicity were often achieved forsignificant periods during and after chewing, supporting

a role for arecoline in OSF and OSCC, and indicating aneed to further investigate the response of oral tissues.Arecoline has a pKa of 7.64 so that its uncharged andlipophilic form predominates in alkaline conditions (30),and it is electrically neutral form of arecoline which isbest able to penetrate the mucosal barrier (31). In thelight of this, the near neutral pH of unstimulated salivasuggests that much of the background salivary arecolinedetected in the current study would be in the chargedform having little penetrative ability, and thus limitedpotential to drive OSF or OSCC (32, 33). However, themore alkaline pH of saliva upon stimulated secretionmay result in a preponderance of the uncharged form ofarecoline, and hence episodes of greater mucosal pen-etration (31–33). It is of interest to note that theinclusion of slaked lime with many preparations of arecanut, increases salivary pH and hence uptake of thispsychic agent across the mucosa (31).

Changes in salivary arecoline levels were highlyidiosyncratic, consistent with differences in specificchewing habits between individuals. Higher arecolinelevels were achieved in subjects who achieved peakconcentrations at later time points. This may reflectdifferences in the specific chewing habits such that thosesubjects expelling most areca nut early during chewingwould extract less arecoline and achieve lower salivarylevels, compared with subjects who habitually retain thenut paste during chewing. This interpretation is sup-ported by the observation that arecoline levels wereclearly lower after patients consciously spat out anyremaining nut material. Differences in chewing forceand frequency affect salivary levels of the agents chewed,

Figure 5 Graphs showing arecoline levels in samples collected from individual subjects over time, grouped into those who achieved maximal levelsbetween 0 and 3 min, 5 and 10 min, or 10 and 25 min, according to whether there were single or multiple peaks. Maximum peak arecoline levels areindicated for each subject tested, whereas the 25-min time points at which areca nut particles were consciously spat out are marked by arrows.Patterns of salivary arecoline levels were highly variable amongst participants, with 19 out of 32 subjects reaching peak levels between 0 and 3 minof chewing areca nut, and 11 of these having a single peak of salivary arecoline. Of the remaining 13 subjects who achieved maximal arecoline levelsat later, 10 had multiple peaks. It was notable that both maximum arecoline levels and total arecoline exposure were lower in subjects who reachedmaximal arecoline levels between 0 and 3 min of chewing areca nut, compared with those who had later peak levels (P < 0.02). Apart from thepattern of salivary arecoline levels, there was no clear difference between subjects who had single or multiple peaks.

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(34) and this likely contributed to differences in salivaryarecoline levels observed in the current study. Inaddition to the effect on salivary pH noted above,variability in salivary flow rates would also likely have asubstantial effect on arecoline levels by changing thefrequency of swallowing or spitting, as well as byvarying residual intraoral volumes (34, 35). The greatdifferences in salivary arecoline levels achieved duringchewing, may contribute to the variable response toareca nut seen in communities where this habit iswidespread.

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Acknowlegements

We wish to thank the volunteers as well as undergraduate students

S Donnelly, I Fisher, F Hasim, and G Howe for their assistance.

Finally, we thank the Australian Dental Research Fund and NSW

Dental Board for their support.

Conflict of interest

None of the authors has any conflict of interest in this work.

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