Betel quid: New insights into an ancient addiction

Abstract

The use of areca nuts (areca) in the form of betel quids constitutes the fourth most common addiction in the world, associated with high risk for oral disease and cancer. Areca is a complex natural product, making it difficult to identify specific components associated with the addictive and carcinogenic properties. It is commonly believed that the muscarinic agonist arecoline is at the core of the addiction. However, muscarinic receptor activation is not generally believed to support drug-taking behaviour. Subjective accounts of areca use include descriptions of both sedative and stimulatory effects, consistent with the presence of multiple psychoactive agents. We have previously reported partial agonism of α4-containing nicotinic acetylcholine receptors by arecoline and subsequent inhibition of those receptors by whole areca broth. In the present study, we report the inhibition of nicotinic acetylcholine receptors and other types of neurotransmitter receptors with compounds of high molecular weight in areca and the ability of low molecular weight areca extract to activate GABA and glutamate receptors. We confirm the presence of a high concentration of GABA and glutamate in areca. Additionally, data also indicate the presence of a dopamine and serotonin transporter blocking activity in areca that could account for the reported stimulant and antidepressant activity. Our data suggest that toxic elements of high molecular weight may contribute to the oral health liability of betel quid use, while two distinct low molecular weight components may provide elements of reward, and the nicotinic activity of arecoline contributes to the physical dependence of addiction.

Keywords: GABA; addiction; antidepressants; areca nut; betel quid; nicotinic receptors.

Figure 1.

Whole areca nuts. Upper left: Dried nuts from India, commonly known as “supari”. These are readily available from on-line sources and Indian specialty stores. The whole nuts are approximately one inch in diameter. Supari is sold in the form of whole nuts or broken to pieces or cut into slices. A slice is shown to illustrate the pattern of white and reddish-brown regions that develop in the nuts as they mature. Upper right: A fresh mature nut from Sri Lanka. This freshly husked nut was only slightly smaller than the dried nuts from India illustrated. Lower left: Fresh immature nuts from Guam. When these nuts were received, the husks were somewhat moldy, covered in slime and appeared to be in the process of rotting. Inside the husks were spongey remnants of the endosperm that were just over half an inch in length and lacked the mature pattern of light and dark areas. Once processed however, the extracts from these compromised nuts were found to have activities similar to those prepared from the dried supari. Lower right: Fresh nuts from Vietnam after washing and partial removal of the husk.

Figure 2.

Inhibition of ACh-evoked response of α4β2 nAChR, formed by the co-expression of α4 and β2 monomers, by areca broth. A) As previously reported (6), and shown in the upper trace, the application of 100 μM arecoline evokes a small response from this subtype of nAChR but does not compromise subsequent responses to ACh applications. The second trace shows the small response evoked by the application of a pH and osmotically balanced areca (Indian supari) broth (see Methods) compared to the ACh-evoked responses obtained from the same cells. ACh responses following the areca application were profoundly reduced (p < 0.0001). The third trace shows responses to areca broth co-applied with 30 μM ACh. Responses were reduced compared to 30 μM ACh controls (p < 0.001) but larger than those to areca broth alone (p < 0.05). The three sets of traces show the averaged responses from seven cells in each experiment (see Methods). The data from each single cell were normalized to the ACh controls from the same cells prior to the calculations of the average values at each of the 10,000 points in the trace (solid black lines). Standard error values were also calculated at each point and plotted as the beige shaded areas. These presentations display the character and the variability of the raw data without the requiring the subjective selection of “representative traces”. P values were corrected for multiple comparisons (see Methods). B) Peak current responses of cells expressing human α4β2 nAChR, normalized to the average of the two initial 30 μM ACh control applications (± S.D.). After 30 s pre-application of control Ringer’s solution or test antagonist, areca broth was co-applied in Ringer’s solution or with 100 μM of the competitive antagonist dihydro-β-erythroidine (DHBE) or 100 μM of the noncompetitive antagonist mecamylamine (Mec). There were no significant differences in the reduced ACh-evoked responses associated with the co-application of antagonists (Supplemental data). To evaluate the stability of the inhibition, a sequence of three control ACh applications were made at four-minute intervals after the delivery of the areca preparation.

Figure 3.

Antagonism of agonist-evoked responses of diverse receptor subtypes by the application of areca broth. Averaged normalized data were processed as described for Figure 2 (see Methods) from at least five cells expressing the receptor subtypes as indicated: human α3β4 nAChR formed from the co-expression of subunit monomers, mouse 5HT3A receptors, and rat AMPA-type glutamate receptors formed from the co-expression of GluA1 and GluA2 subunits. For each of the receptors, the control agonist-evoked responses were profoundly reduced after the application of areca broth (p < 0.0001, See Supplemental data for ANOVA).

Figure 4.

Ultrafiltration of areca broth yielded high molecular weight (HMW, enriched in components < 10 kDa) and low molecular weight (LMW, having only components ≤ 1kDa) fractions (Methods). Inhibition of α4β2 nAChR ACh-evoked responses (p < 0.0001, measured and normalized as described above) was observed for the whole broth (post-broth-1) and the HMW fraction (post-HMW-1) but not for the LMW fraction (post-LMW-1). The stability of the inhibition was tested by making follow up ACh applications four minutes later (post-broth-2, post-HMW-2, and post-LMW-2).

Figure 5.

Effect of areca broth made from Indian supari nuts and filtration fractions on the responses of cells expressing GABA_A receptors. Broths were tested at 1/20th original strength, i.e. 10 mg ground areca per ml Ringer’s solution before filtration. Upper traces: responses of cells expressing rat GABA_A receptors formed from the co-expression of RNA coding for the α1, β2, and γ2(long) subunits to the application of 10 μM GABA before and after the application of whole areca broth. Middle traces: responses of cells expressing rat GABA_A receptors to the application of 10 μM GABA before and after the application of HMW areca broth fraction enriched for components with a molecular weight of ≥ 10 kD. Lower traces: responses of cells expressing rat GABA_A receptors to the application of 10 μM GABA before and after the application of LMW (≤ 1 kD) areca fraction. Note that a sample of this LMW fraction (full strength, after filtration) was dried down and weighed 11.6 mg/ml; therefore, the actual dry weight of tested LMW here was 0.58 mg/ml. The data represent the averages of at least six cells under each condition. The data from each single cell were normalized to the ACh controls from the same cells prior to the calculations of the average values at each of the 10,000 points in the trace (solid black lines). Standard error values were also calculated at each point and plotted as the beige shaded areas. Responses of the GABA_A receptor expressing cells to the application of 10 μM GABA were reduced after the application of the areca broth or the HMW fraction (p < 0.0001), but not after the LMW fraction. Both the whole broth and the LMW fractions strongly stimulated receptor currents.

Figure 6.

Effects of areca whole broth and fractions on rat GABA_A receptor-expressing cells (data from Figure 5) compared to effects of areca-related alkaloids. A) Plotted are the peak current responses of at least 5 cells for each experimental application along with means (black bars) and B) the responses to 10 μM GABA after the test applications. Each of the five alkaloids was tested at 100 μM. None of the alkaloids significantly activated or inhibited the GABA responses after application.

Figure 7.

Concentration-response curves for GABA and LMW areca. Cells expressing human GABA_A (α1β2γ2) receptors were tested with GABA or preparations of LMW areca from two sources, dried supari and fresh nuts from Vietnam, at varying concentrations, expressed as mg/ml. 1 M GABA = 103.12 mg/ml. LMW areca concentrations are plotted according to dry weight mg/ml of the filtered broth tested. Peak-current responses for each cell were normalized to 10 μM GABA responses obtained just prior to the test responses for each cell and then adjusted by the maximum GABA response. All points represent the average of at least five cells ± S.D. The data were fit to the Hill equation, and curve fit values are shown in Table 1.

Figure 8.

Compounds in areca. A) Chromatogram showing HPLC analysis of ortho-phthalaldehyde-derivatized amino acids from LMW fractions of areca extract. Insert shows labeled GABA peak compared to LMW fractions with added 1 μM GABA. B) Comparison of areca GABA content with literature values reported for other plants relatively high in GABA. See text for sources.

Figure 9.

Glutamate receptor activation by LMW areca. A) Responses of cells expressing GluA1 and GluA2 subunits to control applications of 100 μM kainic acid (KA) and LMW areca (fresh nuts from Sri Lanka) applied alone at 180 mg fresh nut per ml Ringer’s solution original broth, dry weight actual 5.4 mg/ml (n = 7, traces on left), or co-applied with 100 μM CTZ (n = 6, traces on left). The data from each single cell were normalized to the peak-current values of the kainic acid controls from the same cells prior to the calculations of the average values at each of the 10,000 points in the trace (solid black lines). Standard error values were also calculated at each point and plotted as the beige shaded areas. B) A comparison of the normalized responses to LMW areca alone in blue and the CTZ-potentiated responses in black and tan. C) Individual responses (peak and net charge) to LMW areca (from panel A), normalized to KA controls applied alone or co-applied with CTZ for statistical analysis (Supplemental Data).

Figure 10.

Effects of areca components on neurotransmitter transporters. A) Uptake assays performed in Chinese hamster ovary cells expressing the DA transporter (CHO-DAT) using tritiated dopamine. Compared to control conditions, the addition of our standard LMW areca extract from dried nuts sourced from India (100 mg/ml of original stock, pH adjusted, and brought up in control buffer) produced a significant reduction (p < 0.0001) in the amount of dopamine taken up. The LMW areca extract was further fractionated by ion exchange chromatography with components eluted with water through either Amberlite IR120 cation exchange or Dowex-1 anion exchange resins. Components eluted through the anion exchange resin had significant effect on dopamine uptake (p < 0.0001). The flow-through of the cation exchange column also had inhibitory activity (p < 0.05), and although it appeared less effective than the activity of the anion exchange eluate, the difference was not significant (p = 0.0536). The potent and selective DAT inhibitor GBR12935 (1-(2-(diphenylmethoxy)ethyl)-4-(3-phenylpropyl)piperazine) was used as a positive control. The concentration of GBR12935 used (10μM) was more than a thousand-fold higher than its reported IC₅₀ of 3.7 nM (55), so that essentially a complete block of DA reuptake was obtained. B) Uptake assays performed in CHO cells expressing the serotonin transporter (CHO-SERT) using tritiated 5HT. Significant inhibition was observed with the LMW areca extract and the eluate of the anion exchange column (p < 0.0001) with no inhibition from the eluate of the cation exchange column. The selective serotonin reuptake inhibitor fluoxetine (05 μM) was used as a positive control.