In vitro hydrolysis of areca nut xenobiotics in human liver

Abstract

Areca nut (AN) is a substance of abuse consumed by millions worldwide, in spite of established oral and systemic toxicities associated with its use. Previous research demonstrates methyl ester alkaloids in the AN, such as arecoline and guvacoline, exhibit mood-altering and toxicological effects. Nonetheless, their metabolism has not been fully elucidated in humans. In the present study, an HPLC-UV bioanalytical method was developed to evaluate the hydrolytic kinetics and clearance rates of arecoline and guvacoline in human liver microsomes (HLM) and cytosol (HLC). The bioassay was capable of quantifying arecoline and guvacoline (and carboxylate metabolites arecaidine and guvacine, respectively) with good sensitivity, accuracy, and precision. Kinetics of arecoline and guvacoline hydrolysis best followed the Michaelis-Menten model. Apparent intrinsic clearance (Cl_{int.in vivo}) of arecoline was 57.8 ml/min/kg in HLM and 11.6 mL/min/kg in HLC, a 5-fold difference. Unexpectedly, guvacoline was dramatically less hydrolyzed than arecoline in both HLM and HLC, with Cl_{int.in vivo} estimates of 0.654 ml/min/kg and 0.466 ml/min/kg, respectively. These results demonstrate, for the first time, arecoline undergoes significant hydrolysis with high clearance rates in the liver. Furthermore, differential tissue metabolic rates and utilization of specific esterase inhibitors unequivocally demonstrated arecoline is a substrate for CES1 and not CES2.

Keywords: Areca nut; Arecoline; Betel nut; Enzyme kinetics; Esterase; Guvacoline; HPLC; Hydrolysis; Metabolism.

Fig. 1.

Proposed metabolic schematics for hydrolysis of arecoline to arecaidine (A) and guvacoline to guvacine (B) in human liver microsomes (HLM) and human liver cytosol (HLC).

Fig. 2.

Representative HPLC-UV chromatograms following injection of (A) Thermally-inactivated (boiled) HLM with no analytes (blank); (B) Thermally-inactivated HLM fortified with 0.5 μg/ml of arecaidine and arecoline; (C) Thermally-inactivated HLM exposed to arecoline (10 μM) at 37 °C for 15 min; and (D) Fresh HLM exposed to arecoline (10 μM) at 37 °C for 15 min. The approximate retention times of imipramine (IS), arecaidine and arecoline were 4.1 min, 6.4 min and 9.3 min, respectively.

Fig. 3.

Representative HPLC-UV chromatograms following injection of (A) Thermally-inactivated (boiled) HLM with no analytes (blank); (B) Thermally-inactivated HLM fortified with 0.5 μg/ml of guvacine and guvacoline; (C) Thermally-inactivated HLM exposed to guvacoline (50 μM) at 37 °C for 15 min; and (D) Fresh HLM exposed to guvacoline (50 μM) at 37 °C for 15 min. The approximate retention times of imipramine (IS), guvacine and guvacoline were 4.1 min, 5.6 min and 7.6 min, respectively.

Fig. 4.

(A) Depletion of arecoline over 90 min in the presence of fresh HLM (▲) or HLC (); (B) Comparison of amount of arecaidine formed over 90 min in the presence of HLM (○) or HLC (). Each point represents the mean (±SD) of at least triplicate experiments.

Fig. 5.

Non-linear regression fitting of the formation of arecaidine at varying arecoline concentrations in HLM (A) and HLC (B). The solid line represents the curve of the best fit; the inset is the corresponding Eadie–Hofstee plot. Each point represents the mean (±SD) of at least triplicate experiments.

Fig. 6.

Effects of esterase inhibitors on arecoline hydrolysis in HLM (A) and HLC (B). All assays were conducted in quadruplicate, and the data is expressed as mean ± SD. ****p < 0.0001 n.d. (not detected).

Fig. 7.

Reaction phenotyping assays of arecoline hydrolysis in microsomes and cytosol from human tissue (liver and intestinal) (A), and human recombinant CES enzymes (B; C). All assays were performed in quadruplicate, with data expressed as mean ± SD. n.d. (not detected).