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Research ArticleNEUROPHARMACOLOGY

Contribution of Individual Cytochrome P450 Isozymes to theO-Demethylation of the Psychotropic β-Carboline Alkaloids Harmaline and Harmine

Ai-Ming Yu, Jeffrey R. Idle, Kristopher W. Krausz, Adrian Küpfer and Frank J. Gonzalez
Journal of Pharmacology and Experimental Therapeutics April 2003, 305 (1) 315-322; DOI: https://doi.org/10.1124/jpet.102.047050

Abstract

The psychotropic β-carboline alkaloids, showing high affinity for 5-hydroxytryptamine, dopamine, benzodiazepine, and imidazoline receptors and the stimulation of locus coeruleus neurons, are formed endogenously from tryptophan-derived indolealkylamines through the Pictet-Spengler condensation with aldehydes in both plants and mammals. Cytochromes P450 1A1 (18.5), 1A2 (20), and 2D6 (100) catalyzed theO-demethylation of harmaline, and CYP1A1 (98.5), CYP1A2 (35), CYP2C9 (16), CYP2C19 (30), and CYP2D6 (115) catalyzed that of harmine (relative activities). The dehydrogenation/aromatization of harmaline to harmine was not carried out by aromatase (CYP19), CYP1A2, CYP2C9, CYP2D6, CYP3A4, pooled recombinant cytochromes P450, or human liver microsomes (HLMs). Kinetic parameters were calculated for theO-demethylations mediated by each isozyme and by pooled HLMs. Kcat (min−1) andKm (μM) values for harmaline were: CYP1A1, 10.8 and 11.8; CYP1A2, 12.3 and 13.3; CYP2C9, 5.3 and 175; CYP2C19, 10.3 and 160; and CYP2D6, 39.9 and 1.4. Values for harmine were: CYP1A1, 45.2 and 52.2; CYP1A2, 9.2 and 14.7; CYP2C9, 11.9 and 117; CYP2C19, 21.4 and 121; and CYP2D6, 29.7 and 7.4. Inhibition studies using monoclonal antibodies confirmed that CYP1A2 and CYP2D6 were the major isozymes contributing to both harmaline (20% and 50%, respectively) and harmine (20% and 30%) O-demethylations in pooled HLMs. The turnover numbers for CYP2D6 are among the highest ever reported for a CYP2D6 substrate. Finally,CYP2D6-transgenic mice were found to have increased harmaline and harmine O-demethylase activities as compared with wild-type mice. These findings suggest a role for polymorphic CYP2D6 in the pharmacology and toxicology of harmine and harmaline.

The β-carboline alkaloids are present in plants and have been of interest due to their psychotropic properties (Picada et al., 1997). They may be formed endogenously from tryptophan-derived indolealkylamines through the Pictet-Spengler condensation with simple aldehydes or with pyruvic acid in mammals, including humans (Airaksinen and Kari, 1981; Melchior and Collins, 1982). Moreover, certain β-carbolines, such as pinoline, tryptoline, 6-hydroxy-tetrahydro-β-carboline, harman, and norharman (Table 1), have been reported as normal constituents of human tissues and body fluids. Their levels in humans are usually elevated after drinking alcohol. The association of β-carbolines with alcohol dependence and brain damage has been suggested (Melchior and Collins, 1982; Collins, 2002).

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Table 1

Chemical structures and common names of some biogenic β-carboline alkaloids

Endogenous and exogenous β-carboline alkaloids were reported to exert a wide spectrum of psychopharmacological and behavioral effects in the brain (Airaksinen and Kari, 1981). Most β-carbolines are strong reversible inhibitors of monoamine oxidase (MAO). Among them, harmaline and harmine (Table 1) exhibit the most potent inhibition toward purified MAO-A activity (Kim et al., 1997), and these are the principal active agents inPeganum harmala, a plant that has been used in traditional medicine for two millennia (Lamchouri et al., 2002). In addition, the psychotropic Amazonian plant mixture ayahuasca comprises the β-carboline-rich vine Banisteriopsis caapi mixed with theN,N-dimethyltryptamine (DMT)-containing hallucinogenic plant Psychotria viridis or Diplopterys cabrerana. Originally, it was proposed that the psychotropic properties of ayahuasca resided in the DMT, but it is now becoming clear that the β-carbolines themselves may contribute to the hallucinogenic properties of the “tea,” rather than simply acting as an inhibitor of MAO to elevate the plasma levels of DMT (Freedland and Mansbach, 1999).

β-Carbolines modulate the levels of amine neurotransmitters and their metabolites in the central nervous system (Iurlo et al., 2001), inducing behavioral changes. MAO inhibition also leads these β-carboline alkaloids to induce hypothermic effects, probably through a serotonergic mechanism (Abdel-Fattah et al., 1995). Hallucinogenic effects of harmaline and harmine are suggested as a result of their binding to 5-HT2A and 5-HT2C receptors, and tremorgenic properties are due to their interactions with benzodiazepine receptors (Lutes et al., 1988; Glennon et al., 2000; Husbands et al., 2001). Harmaline has also been proposed as an endogenous ligand for imidazoline receptors (Husbands et al., 2001) and has been shown to stimulate locus coeruleus neuronal activity, which may underlie some of the behavioral effects of these β-carbolines (Ruiz-Durantez et al., 2001).

The neurotoxic properties of β-carboline alkaloids may account for their associations with Parkinson's disease. Tryptophan-derived β-carbolines are similar toN-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in structure, which is known to induce immediate and irreversible parkinsonism through its neurotoxic metabolite, a quaternary ion (MPP+). Various studies indicate that the 2,9-di-N-methylated β-carboline cations, which are neurotoxic compared with MPP+, also induce mitochondrial energy depletion and oxidative stress in nigrostriatum (Collins and Neafsey, 1985; Collins et al., 1987, 1992; Collins, 2002). Moreover, the bioactivated, potentially neurotoxicN-methylated β-carbolinium ions are reported to be present in human brain (Matsubara et al., 1993).

The metabolic bioactivation of carcinogenic α-carbolines and γ-carbolines, such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, has been extensively studied, because these compounds are present in cooked food (Matsubara et al., 1993; Raza et al., 1996; Matsubara, 1998; Pfau and Marquardt, 2001). In contradistinction to the so-called heterocyclic amine food mutagens, the metabolism of these potential neurotoxic β-carboline alkaloids is expected to be a detoxication process and may play an important role in protection against brain damage (Slotkin and DiStefano, 1970a,b; Slotkin et al., 1970; Zetler et al., 1974; Burke and Upshall, 1976; Tweedie and Burke, 1987).

We have therefore examined the metabolism of harmaline and harmine, and estimated the relative contribution by individual cyotchrome P450 (P450) isozymes to their O-demethylation. The results might provide insights not only into individual diathesis to chemical neurotoxins, but also the physiological potential of P450 isozymes that are expressed in human brain, beyond the detoxication of drugs and other xenobiotic chemicals.

Materials and Methods

Chemicals and Enzymes.

Harmaline, harmine, harmalol, harmol, pinoline, phenacetin, NADPH, 4-amidinophenylmethanesulfonyl fluoride (APMSF), EDTA, and 60% perchloric acid were purchased from Sigma-Aldrich (St. Louis, MO). HPLC solvents and other chemicals were of the highest grade commercially available and were used as received. Recombinant human P450 supersomes and P450 insect control microsomes, pooled human liver microsomes (coded H161), were bought from BD Gentest (Woburn, MA). The monoclonal antibodies raised against human CYP2D6 (mAb 50-1-3), CYP1A1 (mAb 1-7-1), CYP1A2 (mAb 26-7-5), CYP2C9 (mAb 763-15-5), and CYP2C19 (mAb 1-7-4-8) were characterized previously (Gelboin et al., 1999; Krausz et al., 2001).

Preparation of Mouse and Human Liver Microsomes.

All mice used for liver microsome preparations were maintained under controlled temperature (23 ± 1°C) and lighting (lights on 6:00 AM to 6:00 PM) with food and water provided ad libitum. Adult males, 2 to 3 months old, were used in the experiments, which were conducted under the National Institutes of Health guidelines for the use and care of laboratory animals. CYP2D6-transgenic mice were characterized previously (Corchero et al., 2001). For preparation of mouse liver microsomes (MLM), mice were killed by CO2 asphyxiation, and livers were excised and washed with ice-cold washing solution (250 mM sucrose, 10 mM potassium phosphate, 1 mM EDTA, and 1 mM APMSF, pH 7.4). Livers from three male mice were pooled, minced with scissors, and homogenized using a motor-driven, Teflon-tipped pestle in the washing solution. The homogenates were centrifuged at 9,000g for 20 min at 4°C, and the resultant supernatants were centrifuged at 100,000gfor 60 min at 4°C. The microsomal pellets were resuspended in ice-cold freezing solution [100 mM potassium phosphate, 20% (v/v) glycerol, 1 mM EDTA, 1 mM APMSF, pH 7.4], aliquoted, and stored at −80°C for future use. Four individual HLM prepared in the laboratory (Krausz et al., 2001) were equally mixed as pooled HLM (pHLM). Protein concentrations were determined using a BCA Protein Assay kit (Pierce Chemical Co. Rockford, IL), following the manufacturer's instructions. Cytochrome P450 contents were determined according to the method described by Omura and Sato (1964).

Incubation Reactions with Recombinant P450 Isoforms.

Each incubation reaction was carried out in 100 mM potassium phosphate, pH 7.4, containing 5 pmol of cDNA-expressed P450 enzyme, 10 μM substrate in a final volume of 200 μl. Reactions were initiated by the addition of 20 μl of 10 mM NADPH after 5 min preincubation at 37°C. Incubations were terminated by the addition of 10 μl of 60% perchloric acid after 5 min incubation. The mixtures were vortexed for 20 s and then centrifuged at 14,000g for 10 min; then, the supernatants were transferred to a new vial and directly injected for HPLC analysis. Reactions for LC-MS/MS analysis were quenched with 50 μl of ice-cold 100 mM sodium hydroxide solution, to which was added 20 μl of pinoline (internal standard, 10 μM in methanol), and extracted with 2 ml of methyl tert-butyl ether and 1 ml of ethyl acetate mixture. The extracts were evaporated and reconstituted with 50% methanol solution containing 0.2% formic acid. The final reconstitutes were transferred to new vials and injected for LC-MS/MS and/or LC-MS analysis. The concentration for both harmaline and harmine was fixed at 10 μM for screening the activities with individual P450 isozymes. For kinetic analysis, harmaline and harmine concentrations ranged from 0 to 2000 μM when incubated with CYP1A1, CYP1A2, CYP2C9, and CYP2C19, and from 0 to 200 μM with CYP2D6. To test the dehydrogenation of harmaline and harmalol, their final concentrations were fixed at 50 μM, and incubations were conducted with 5 pmol of P450 isozyme for 20 min. Reactions were terminated with 50 μl of 100 μM sodium hydroxide and subjected to extractions as described above. Phenacetin (20 μl of 10 μM in methanol) was used as internal standard. All the reactions were performed in duplicate.

Incubation Reactions with Human and Mouse Liver Microsomes.

Incubation reactions were carried out in 100 mM potassium phosphate, pH 7.4, containing pooled liver microsomes with 20 to 80 μg of hepatic protein, NADPH at a final concentration of 1 mM, and the monoclonal antibody when necessary in a final volume of 200 μl. Reaction mixtures were preincubated at 37°C for 5 min, then initiated by the addition of harmaline or harmine. Reactions were terminated by the addition of 10 μl of 60% perchloric acid or 50 μl of 100 mM sodium hydroxide after 10 min incubation, and processed as above for HPLC or LC-MS/MS analysis. Harmaline concentration was fixed at 50 μM to examine its possible 3,4-dehydrogenation in pooled HLM, and phenacetin (20 μl of 10 μM) was used as internal standard. The concentrations of harmaline and harmine ranged from 0 to 500 μM for the kinetic analysis with pooled human liver microsomes. Harmaline and harmine concentrations were fixed at 10 μM for comparison of theirO-demethylation activities between wild-type andCYP2D6-transgenic pooled MLM, and the immunoinhibition studies in pooled HLM and MLM. Monoclonal antibody against CYP2D6 (20 μl) was used to examine its effect on harmaline and harmineO-demethylation in wild-type andCYP2D6-transgenic MLMs. Antibodies against CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP2D6 (20 μl of each) were used combinatorially for immunoinhibition studies (Gelboin et al., 1999). Anti-lysozyme (HyHel) was used as a control for nonspecific binding. All reactions were performed in duplicate.

Identification and Quantitation of Metabolites by HPLC and LC-MS/MS.

HPLC analysis was carried out on an Agilent 1100 series HPLC system consisting of the online vacuum degasser, quaternary pump, autosampler, thermostatted column compartment, fluorescence detector, and diode-array detector. The Agilent 1100 series HPLC System was controlled with an Agilent ChemStation and a handheld control module (Agilent Technologies, Palo Alto, CA). A Regis 250 mm × 4.6 mm i.d. REXCHROM phenyl 5 μm column (Regis, Morton Grove, IL) was used to separate the metabolites. The flow rate through the column at ambient temperature was 1.0 ml/min. Separation of harmaline and its metabolites was achieved with a mobile phase containing 40% buffer A (0.1% trifluoroacetic acid in water) and 60% buffer B [40% (v/v) aqueous acetonitrile]. Isocratic elution with 35% buffer A and 65% buffer B was applied for the separation of harmine and its metabolites. The excitation and emission wavelengths of the fluorescence detector were set at 250 and 480 nm, respectively, for the detection of harmaline and its metabolites, and at 350 and 490 nm, respectively, for the analysis of harmine and its metabolites. The detection limit for harmalol and harmol was 1 pmol under the experimental conditions used. Calibration curves were linear from 1 to 2000 pmol for harmalol and from 5 to 2000 pmol for harmol. Intraday and interday coefficients of variation were less than 5%.

LC-MS/MS and LC-MS analysis of harmaline, harmine, and their metabolites was performed on a PerkinElmerSciex API 2000 ESI triple-quadrupole mass spectrometer controlled by Analyst software (PerkinElmerSciex Instruments, Boston, MA). A Luna 3 μm C18, 50 mm × 4.6 mm i.d., Phenomenex column (Phenomenex, Torrance, CA) was used to separate harmaline, harmine, their metabolites, and pinoline, which was used as internal standard for quantitation. For the separation of harmaline, harmaline, their metabolites, and pinoline (internal standard), the flow rate through the column at ambient temperature was 0.2 ml/min with 50% methanol and 50% water containing 0.1% formic acid (buffer C). Isocratic elution at 0.2 ml/min with 70% methanol and 30% buffer C was used to separate harmaline, harmalol, harmine, harmol, and phenacetin (internal standard). The mass spectrometer was operated in the turbo ion spray mode with positive ion detection. The turbo ion spray temperature was maintained at 350°C, and a voltage of 4.8 kV was applied to the sprayer needle. Nitrogen was used as the turbo ion spray and nebulizing gas. The detection and quantitation of substrates, metabolites of interest, and internal standards were accomplished by multiple reactions monitoring with the transitions m/z 215.2/174.2 for harmaline, 201.2/160.2 for harmalol, 213.2/198.2 for harmine, 199.1/171.1 for harmol, 203.2/174.2 for pinoline, and 180.1/110.0 for phenacetin. The MS/MS conditions were optimized automatically using the Analyst software for each chemical. The calibration curves were linear for harmalol, harmol, harmaline, and harmine, with concentrations ranging from 0.2 to 20 μM. Calibration and regression of the curves were completed using 1/x2 weighting. The recoveries of these compounds ranged from 85 to 110%. Intraday and interday coefficients of variation were less than 10%. The identification of the unknown hydroxylated metabolites was accomplished by selected ion monitoring with the protonated molecular ions ofm/z 231.2 and 229.2, which were absent in the reactions with insect control microsomes, or P450 isoforms and pooled HLM incubations without NADPH.

Data Analysis.

Results are expressed as the mean ± S.E.M. Values were compared with unpaired t test, and the difference was considered significant if the probability (Pvalue) was less than 5%. Enzyme Michaelis-Menten parameters,Km andVmax, were estimated by nonlinear regression (Prism 3.02; GraphPad, San Diego, CA). Initial estimates for nonlinear regression were generated graphically using Eadie-Hofstee plots (V0 versusV0/[S]). Linear regression analyses were conducted using Microsoft Excel 2000.

Results

HPLC and LC-MS/MS Analysis.

Harmaline was metabolized to two unidentified metabolites as well as its O-demethylated metabolite harmalol by pooled HLM (Fig.1A). The unidentified metabolites were confirmed as hydroxylated metabolites by LC-MS single ion monitoring analysis. Harmine, the other β-carboline alkaloid, was metabolized to harmol and two other unknown metabolites by pooled HLM (Fig. 1B). These unknown metabolites were identified as hydroxylated harmine by LC-MS analysis, which is consistent with a previous work (Tweedie and Burke, 1987). In this study, the metabolites were assigned as 6-, 3-, or 4-hydroxylated harmine (Tweedie and Burke, 1987). However, the exact hydroxylation positions on harmaline and harmine were not determined.

Figure 1
Figure 1

HPLC traces of harmaline (A) and harmine (B), and their corresponding metabolites formed by pHLMs and individual P450 isozymes. The excitation and emission of the fluorescence detector were set at 250 and 480 nm, respectively, for analysis of harmaline reactions, and at 350 and 490 nm, respectively, for harmine reactions.

Harmaline and Harmine Metabolism by cDNA-Expressed Human P450 Isozymes.

To investigate the involvement and relative contribution of major human P450 enzymes in harmaline and harmineO-demethylations, screening experiments were carried out using recombinant human P450 enzymes. Harmalol and harmol were detected in the incubations of 10 μM harmaline and harmine, respectively, with recombinant human CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP2D6 (Fig.2). The uncharacterized hydroxylated metabolites were detected only in the reactions of harmaline and harmine with recombinant CYP1A1 and CYP1A2 (Fig. 1). In addition, incubations of harmaline with recombinant aromatase (CYP19), CYP1A2, CYP2C9, CYP2D6, CYP3A4, pooled recombinant P450 isozymes, or pooled HLM did not yield any harmine, demonstrating that the theoretical aromatization of harmaline to harmine (Fig.3) through 3,4-dehydrogenation was not mediated by these P450 isozymes. CYP19 also did not produce harmol from harmalol (Fig. 3).

Figure 2
Figure 2

Relative harmaline (A) and harmine (B)O-demethylation activity catalyzed by cDNA-expressed human P450 isozymes. Incubations were performed with 5 pmol of each P450 isozyme in 100 mM potassium phosphate buffer, pH 7.4, at 37°C for 5 min. Final substrate concentrations were fixed at 10 μM. Values represent the mean, and the vertical lines represent the standard errors of the mean (±S.E.M.) from duplicate incubations.

Figure 3
Figure 3

Summary of the metabolism of harmaline and harmine by human P450 isozymes.

Kinetic Study of Harmaline and HarmineO-Demethylation.

As expected, harmaline and harmineO-demethylation in pooled HLM showed biphasic kinetics indicating that more than one P450 isozyme was involved in their biotransformations (Fig. 4). One-enzyme and two-enzyme kinetic parameters were estimated, which revealed an apparent one-enzyme Km value of around 84 μM for harmaline O-demethylation, and about 92 μM for harmine O-demethylation (Table2). Steady-state kinetic parameters were also individually estimated for CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP2D6 (Table 3). Interestingly, CYP1A1- and CYP1A2-catalyzed harmaline and harmineO-demethylations exhibited atypical kinetics (Fig.5). These data were fitted to a substrate inhibition kinetics algorithm [V =Vmax/(1 +Km/S +S/Ki)], and the calculated atypical kinetic parameters are listed in Table4.

Figure 4
Figure 4

Eadie-Hofstee plots of harmaline (A) and harmine (B)O-demethylations in pooled human liver microsomes (▪, pHLM; ▴, H161) showing biphasic kinetics. Incubation reactions were carried out at 37°C for 10 min. Values represent the mean of duplicate incubations.

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Table 2

Michaelis-Menten kinetic parameters estimated for harmaline and harmineO-demethylations in pooled human liver microsomes

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Table 3

Michaelis-Menten kinetic parameters estimated for harmaline and harmineO-demethylations catalyzed by P450 isozymes

Figure 5
Figure 5

CYP1A1- and CYP1A2-catalyzed harmaline (A) and harmine (B) O-demethylations showing substrate inhibition (▪, CYP1A1; ▾, CYP1A2). Reactions containing 5 pmol of each enzyme lasted for 10 min at 37°C.

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Table 4

Atypical kinetic parameters estimated for harmaline and harmineO-demethylations catalyzed by CYP1A1 and 1A2 isozymes by fitting the data into the equation [V =Vmax/(1 +Km/S +S/Ki)] describing substrate inhibition

Estimation of the Relative Contribution of P450 Isozymes to Harmaline and Harmine Metabolism in Pooled HLM by Immunoinhibition Analysis.

To evaluate the relative contribution of CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP2D6 to the metabolism of harmaline and harmine, immunoinhibition analysis was performed with pooled HLM using well characterized monoclonal antibodies. As shown in Fig.6A, CYP2D6 contributed about 20%, CYP1A2 around 50%, and CYP2C9 about 15% to harmaline O-demethylation. For harmine O-demethylation (Fig. 6B), around 20% was contributed by CYP2D6, 30% by CYP1A2, and 20% by CYP2C9. In addition, CYP1A2 was identified as the major isozyme contributing to the hydroxylations of harmaline and harmine (data not shown), which was consistent with the results obtained from screening experiments (Fig.1).

Figure 6
Figure 6

Harmaline (A) and harmine (B)O-demethylation activities affected by monoclonal antibodies directed against CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP2D6 (MAb) in pooled human liver microsomes (pHLM and H161).

Evaluation the Difference of Harmaline and Harmine O-Demethylation in Wild-Type and CYP2D6-Transgenic Mice in Vitro.

To examine the role of CYP2D6 in harmaline and harmineO-demethylation activities, liver microsomes were prepared from wild-type and CYP2D6-transgenic mice and activities were determined. CYP2D6-transgenic microsomes showed about a 6-fold higher harmaline O-demethylation activity than the wild-type microsomes (Fig. 7). In addition, the increased activity was completely inhibited by the CYP2D6 monoclonal antibody. Harmine O-demethylation activity was also increased about 20% (P = 0.032) inCYP2D6-transgenic as compared with wild-type microsomes (Fig. 7). Similarly, the increased harmine O-demethylation activity was also totally blocked by the CYP2D6 monoclonal antibody. These results are consistent with an important role for CYP2D6 in harmaline and harmine O-demethylations.

Figure 7
Figure 7

Comparison of harmaline (A) and harmine (B)O-demethylation activities between wild-type (WT) andCYP2D6-transgenic (2D6) pooled mouse liver microsomes (MLM) from three mice for each group, and the effects of the CYP2D6 monoclonal antibody on these reactions. Open bars represent the control reactions with anti-lysozyme for nonspecific binding and filled bars represent the reactions with antibody.

Discussion

Endogenous and exogenous β-carboline alkaloids exhibit a variety of biochemical, psychopharmacological, and behavioral effects in animals and humans (Airaksinen and Kari, 1981). They are also thought to have genotoxic, mutagenic, and cytotoxic activities (Picada et al., 1997; Boeira et al., 2002; Collins, 2002; Matsubara et al., 2002).N-Methylation activation of β-carbolines leads to a pattern of neurotoxicity similar to that of MPTP, which induces irreversible parkinsonism. The oxidation of these β-carboline alkaloids to readily excretable products (Mulder and Hagedoorn, 1974;Tan et al., 1990), appears to play a key role in the protection of cells such as neurons in brain.

In humans, P450 isozymes are expressed and active not only in liver, but also in extrahepatic tissues including the brain (Riedl et al., 1998; Hedlund et al., 2001; Strobel et al., 2001; Miksys et al., 2002). Cytochrome P450s belonging to the CYP1A, CYP2C, CYP2D, and CYP3A subfamilies have all been detected in human brain. These central nervous system P450 isozymes could substantially influence the biotransformations of endogenous and exogenous neurotoxins. Some β-carbolines, such as 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid, that have been assumed to be formed endogenously, in this case from tryptophan and acetaldehyde, can also be absorbed from the diet and enter the mammalian brain (Fukushima et al., 1992). Other β-carbolines, for example 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-β-carboline, the condensation product of 5-HT and acetaldehyde, are not detectable in mammalian brain (Beck and Lundman, 1983), suggesting that they neither are formed endogenously nor enter the brain from the diet. However, this simplistic model of dietary sources, blood-brain barrier passage, and endogenous formation needs to be reevaluated in the light of the findings reported here. Clearly, various β-carbolines may be formed in mammalian brain from abundant indole precursors such as 5-HT and tryptophan, but experiments that are designed simply to detect these compounds may yield negative results if the possibility of further metabolism by brain P450s is not considered. Some of the turnover values reported here are so high as to render difficult the detection of certain β-carbolines in brain tissues. Additionally, β-carbolines are substrates for N-methyltransferases (Matsubara et al., 1993; Gearhart et al., 2002), and the ability of the hydroxylated β-carbolines to be O-methylated by mammalian hydroxyindole O-methyltransferases has never, to our knowledge, been tested. The solution to this problem would be to screen brain tissues for the full range of nonhydroxylated, hydroxylated, andO-methylated β-carbolines using the remarkable resolving power of techniques such as LC-MS/MS.

The data from this study clearly demonstrated the involvement of CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP2D6 in the metabolism of harmaline and harmine. Kinetic studies and immunoinhibition analysis with monoclonal antibodies indicated that CYP1A2 and CYP2D6 are the major P450 isozymes contributing to their metabolism. The role of CYP2D6 was further confirmed by the analysis ofCYP2D6-transgenic mice. Of note is thatO-demethylated metabolites, harmalol and harmol, unlike harmaline and harmine themselves, are not tremor inducers. Therefore,O-demethylation of these β-carbolines may be an important detoxication process protecting neurons against chemical damage. Moreover, such O-demethylation detoxication reactions may compete with the toxifying reactions of N-methylation that are thought to lead to intermediates that may trigger Parkinson's disease (Collins and Neafsey, 1985; Collins et al., 1992; de Meester, 1995; Aoyama et al., 2000; Gearhart et al., 2000, 2002).

Most importantly, these studies have demonstrated the important contribution of CYP2D6 to the O-demethylation of harmaline and harmine, although its role in their total clearance cannot be determined by this study. Since humans are exposed to β-carbolines in the diet, for example, in fresh ground coffee (Herraiz, 2002), and this group of compounds is potentially neurotoxic, there may exist profound differences in β-carboline sensitivity between extensive and poor metabolizers for the CYP2D6 polymorphism (Mahgoub et al., 1977). A recent report (Miksys et al., 2002) demonstrated clear expression of CYP2D6 in neurons such as pyramidal Purkinje cells and that the expression of CYP2D6 is considerably elevated in alcoholics, except in a subject who was a poor metabolizer who had to detectable neuronal CYP2D6. These latter individuals have elevated biosynthesis of β-carbolines due to their alcohol intake and conversion to acetaldehyde but do not have the potential for neuroprotective detoxication afforded by CYP2D6, normally elevated in alcoholism (Miksys et al., 2002). Whether or not this is a paradigm for chemically induced neurotoxicity, in general, remains to be investigated. Studies of the association between CYP2D6 genotype and Parkinson's disease have produced varied results, probably because many of the studies employed small numbers of patients. However, a meta-analysis of 11 studies showed a small (odds ratio 1.47) but highly significant (P = 0.01) association between the poor metabolizer genotypes and Parkinson's disease (McCann et al., 1997). The etiology of Parkinson's disease is not known, and many candidate chemical causes have been proposed since the discovery by Bill Langston and his colleagues that a Parkinson's syndrome could be induced by an identifiable environmental chemical, MPTP, and that its metabolite, MPP+, was the likely ultimate neurotoxin (Langston and Ballard, 1983, 1984; Langston et al., 1984). Whereas MPTP is activated to MPP+ by MAO-B, it is detoxicated by N-demethylation, largely by CYP2D6. Thus, there exist many commentaries predicting a role for polymorphic CYP2D6 in MPTP-induced Parkinson's disease. However, very few individuals are exposed to MPTP, and this is not a plausible cause of the common manifestations of Parkinson's disease. Nevertheless, building on the MPTP/CYP2D6 paradigm, one may readily envisage how more common exposures may interact with this polymorphic enzyme to precipitate neurotoxicity syndromes such as Parkinson's disease. Obvious candidates are both the exogenously and endogenously formed β-carbolines.

In this study, harmaline and harmine O-demethylation mediated by CYP1A1 and CYP1A2 enzymes showed substrate inhibition kinetics. Atypical enzyme kinetics were also reported for P450-catalyzed reactions, such as substrate inhibition and activation of CYP3A4 (Korzekwa et al., 1998; Shou et al., 2001). These findings have led to a better understanding of the active sites of P450 enzymes, including multiple sites for substrate binding (Shou et al., 2001).

It is noteworthy that whereas harmaline O-demethylation is carried out preferentially by CYP2D6, other P450 isozymes approach the activity of CYP2D6 with respect to harmine O-demethylation. This difference between the isozyme preference of these two β-carbolines is further demonstrated in the experiments using MLM, whereby the CYP2D6-transgenic MLM had a high CYP2D6-specific harmaline O-demethylase activity, but a considerably poorer CYP2D6-specific harmine O-demethylase activity. These observations are consistent with the relative affinities of the two substrates (harmaline Km rCYP2D6, 1.41 μM, harmine Km rCYP2D6, 7.42 μM, Table 3; harmaline Km HLMCYP2D6, 18.9 μM, harmine Km HLMCYP2D6, 62.1 μM, Table 2). Thus, the difference between the two substrates is almost certainly due to the less basic nature of harmine (pKa 7.70) compared with harmaline (pKa 9.80) (Budavri et al., 1996). Indeed, it was recently proposed that CYP2D6 substrates must contain an ionized nitrogen center at cellular pH to bind to the Glu-216 residue for catalytic activity (Kirton et al., 2002). Based upon the foregoing difference in pKavalues, at pH 7.4, for example, harmaline will be over 100 times more ionized than harmine.

In summary, the cytochromes P450 that O-demethylate the exogenous β-carbolines harmaline and harmine have been identified. The predominant isozymes are CYP1A2 and polymorphic CYP2D6. Plant extracts containing the β-carboline alkaloids have been used in traditional medicine for millennia and are of considerable ethnopharmacological interest and importance. Both South Americanayahuasca and Asian Peganum harmala seeds employ the pharmacological properties of the β-carboline alkaloids to exert both their healing and psychotropic properties. There remains the intriguing possibility that the pharmacologic and toxicological properties of these traditional medicines are related to the CYP2D6 polymorphism. Finally, we propose that CYP2D6 could potentially ameliorate the neurotoxicity of both endogenous and exogenous β-carbolines by restricting the metabolic flux through theN-methylation pathways.

Acknowledgments

We thank Dr. Timothy S. Tracy, West Virginia University, for helpful discussions.

Footnotes

  • ↵1 Present address: U Háje 1651, 252 63 Roztoky u Prahy, Czech Republic.

  • J.R.I. received a grant from U. S. Smokeless Tobacco Company.

  • DOI: 10.1124/jpet.102.047050

  • Abbreviations:
    MAO
    monoamine oxidase
    DMT
    N,N-dimethyltryptamine
    5-HT
    5-hydroxytryptamine
    MPTP
    N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
    MPP+
    1-methyl-4-phenylpyridinium
    P450
    cytochrome P450
    APMSF
    4-amidinophenylmethanesulfonyl fluoride
    HPLC
    high performance liquid chromatography
    Mab
    monoclonal antibody
    MLM
    mouse liver microsomes
    HLM
    human liver microsomes
    pHLM
    pooled HLM
    LC-MS/MS
    liquid chromatography-tandem mass spectrometry
    Km rCYP2D6
    Kmfor recombinant CYP2D6 (insect cells)
    Km HLMCYP2D6
    Km for CYP2D6 (high-affinity component) with two-enzyme kinetics in H161 HLMs
    • Received November 19, 2002.
    • Accepted December 30, 2002.

References

  1. ↵
    1. Abdel-Fattah AF,
    2. Matsumoto K,
    3. Gammaz HA,
    4. Watanabe H
    (1995) Hypothermic effect of harmala alkaloid in rats: involvement of serotonergic mechanism. Pharmacol Biochem Behav 52:421–426.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Airaksinen MM,
    2. Kari I
    (1981) beta-Carbolines, psychoactive compounds in the mammalian body. Part II: Effects. Med Biol 59:190–211.
    OpenUrlPubMed
  3. ↵
    1. Aoyama K,
    2. Matsubara K,
    3. Okada K,
    4. Fukushima S,
    5. Shimizu K,
    6. Yamaguchi S,
    7. Uezono T,
    8. Satomi M,
    9. Hayase N,
    10. Ohta S,
    11. et al.
    (2000) N-methylation ability for azaheterocyclic amines is higher in Parkinson's disease: nicotinamide loading test. J Neural Transm 107:985–995.
  4. ↵
    1. Beck O,
    2. Lundman A
    (1983) Occurrence of 6-hydroxy-1-methyl-1,2,3,4-tetrahydro-beta-carboline in tissues and body fluids of rat. Biochem Pharmacol 32:1507–1510.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Boeira JM,
    2. Viana AF,
    3. Picada JN,
    4. Henriques JA
    (2002) Genotoxic and recombinogenic activities of the two beta-carboline alkaloids harman and harmine in Saccharomyces cerevisiae. Mutat Res 500:39–48.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Budavri S,
    2. O'Neil MJ,
    3. Smith A,
    4. Heckelman PE,
    5. Kinneary JF,
    6. O'Neill M
    (1996) The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals (CRC Press LLC, Boca Raton, FL), 12th ed.
  7. ↵
    1. Burke MD,
    2. Upshall DG
    (1976) Species and phenobarbitone-induced differences in the kinetic constants of liver microsomal harmine O-demethylation. Xenobiotica 6:321–328.
    OpenUrlPubMed
  8. ↵
    1. Collins M
    (2002) Alkaloids, alcohol and Parkinson's disease. Parkinsonism Relat Disord 8:417–422.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Collins MA,
    2. Neafsey EJ
    (1985) Beta-carboline analogues of N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP): endogenous factors underlying idiopathic parkinsonism? Neurosci Lett 55:179–184.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Collins MA,
    2. Neafsey EJ,
    3. Cheng BY,
    4. Hurley-Gius K,
    5. Ung-Chhun NA,
    6. Pronger DA,
    7. Christensen MA,
    8. Hurley-Gius D
    (1987) Endogenous analogs of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine: indoleamine derived tetrahydro-beta-carbolines as potential causative factors in Parkinson's disease. Adv Neurol 45:179–182.
    OpenUrlPubMed
  11. ↵
    1. Collins MA,
    2. Neafsey EJ,
    3. Matsubara K,
    4. Cobuzzi R, Jr,
    5. Albores R, Jr,
    6. Fields J,
    7. Rollema H
    (1992) Indole-N-methylation of beta-carbolines: the brain's bioactivation route to toxins in Parkinson's disease? Ann NY Acad Sci 648:263–265.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Corchero J,
    2. Granvil CP,
    3. Akiyama TE,
    4. Hayhurst GP,
    5. Pimprale S,
    6. Feigenbaum L,
    7. Idle JR,
    8. Gonzalez FJ
    (2001) The CYP2D6 humanized mouse: effect of the human CYP2D6 transgene and HNF4alpha on the disposition of debrisoquine in the mouse. Mol Pharmacol 60:1260–1267.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. de Meester C
    (1995) Genotoxic potential of beta-carbolines: a review. Mutat Res 339:139–153.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Freedland CS,
    2. Mansbach RS
    (1999) Behavioral profile of constituents in ayahuasca, an Amazonian psychoactive plant mixture. Drug Alcohol Depend 54:183–194.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Fukushima S,
    2. Matsubara K,
    3. Akane A,
    4. Shiono H
    (1992) 1-Methyl-tetrahydro-beta-carboline-3-carboxylic acid is present in the rat brain and is not increased after acute ethanol injection with cyanamide treatment. Alcohol 9:31–35.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Gearhart DA,
    2. Collins MA,
    3. Lee JM,
    4. Neafsey EJ
    (2000) Increased beta-carboline 9N-methyltransferase activity in the frontal cortex in Parkinson's disease. Neurobiol Dis 7:201–211.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Gearhart DA,
    2. Neafsey EJ,
    3. Collins MA
    (2002) Phenylethanolamine N-methyltransferase has beta-carboline 2N-methyltransferase activity: hypothetical relevance to Parkinson's disease. Neurochem Int 40:611–620.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Gelboin HV,
    2. Krausz KW,
    3. Gonzalez FJ,
    4. Yang TJ
    (1999) Inhibitory monoclonal antibodies to human cytochrome P450 enzymes: a new avenue for drug discovery. Trends Pharmacol Sci 20:432–438.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Glennon RA,
    2. Dukat M,
    3. Grella B,
    4. Hong S,
    5. Costantino L,
    6. Teitler M,
    7. Smith C,
    8. Egan C,
    9. Davis K,
    10. Mattson MV
    (2000) Binding of beta-carbolines and related agents at serotonin (5-HT(2) and 5-HT(1A)), dopamine (D(2)) and benzodiazepine receptors. Drug Alcohol Depend 60:121–132.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Hedlund E,
    2. Gustafsson JA,
    3. Warner M
    (2001) Cytochrome P450 in the brain; a review. Curr Drug Metab 2:245–263.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Herraiz T
    (2002) Identification and occurrence of the bioactive beta-carbolines norharman and harman in coffee brews. Food Addit Contam 19:748–754.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Husbands SM,
    2. Glennon RA,
    3. Gorgerat S,
    4. Gough R,
    5. Tyacke R,
    6. Crosby J,
    7. Nutt DJ,
    8. Lewis JW,
    9. Hudson AL
    (2001) Beta-carboline binding to imidazoline receptors. Drug Alcohol Depend 64:203–208.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Iurlo M,
    2. Leone G,
    3. Schilstrom B,
    4. Linner L,
    5. Nomikos G,
    6. Hertel P,
    7. Silvestrini B,
    8. Svensson H
    (2001) Effects of harmine on dopamine output and metabolism in rat striatum: role of monoamine oxidase-A inhibition. Psychopharmacology (Berl) 159:98–104.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Kim H,
    2. Sablin SO,
    3. Ramsay RR
    (1997) Inhibition of monoamine oxidase A by beta-carboline derivatives. Arch Biochem Biophys 337:137–142.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Kirton SB,
    2. Kemp CA,
    3. Tomkinson NP,
    4. St-Gallay S,
    5. Sutcliffe MJ
    (2002) Impact of incorporating the 2C5 crystal structure into comparative models of cytochrome P450 2D6. Proteins 49:216–231.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Korzekwa KR,
    2. Krishnamachary N,
    3. Shou M,
    4. Ogai A,
    5. Parise RA,
    6. Rettie AE,
    7. Gonzalez FJ,
    8. Tracy TS
    (1998) Evaluation of atypical cytochrome P450 kinetics with two-substrate models: evidence that multiple substrates can simultaneously bind to cytochrome P450 active sites. Biochemistry 37:4137–4147.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Krausz KW,
    2. Goldfarb I,
    3. Buters JT,
    4. Yang TJ,
    5. Gonzalez FJ,
    6. Gelboin HV
    (2001) Monoclonal antibodies specific and inhibitory to human cytochromes P450 2C8, 2C9 and 2C19. Drug Metab Dispos 29:1410–1423.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Lamchouri F,
    2. Settaf A,
    3. Cherrah Y,
    4. El Hamidi M,
    5. Tligui N,
    6. Lyoussi B,
    7. Hassar M
    (2002) Experimental toxicity of Peganum harmala seeds. Ann Pharm Fr 60:123–129.
    OpenUrlPubMed
  29. ↵
    1. Langston JW,
    2. Ballard P
    (1984) Parkinsonism induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): implications for treatment and the pathogenesis of Parkinson's disease. Can J Neurol Sci 11:160–165.
    OpenUrlPubMed
  30. ↵
    1. Langston JW,
    2. Ballard PA, Jr
    (1983) Parkinson's disease in a chemist working with 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine. N Engl J Med 309:310.
    OpenUrlPubMed
  31. ↵
    1. Langston JW,
    2. Irwin I,
    3. Langston EB,
    4. Forno LS
    (1984) 1-Methyl-4-phenylpyridinium ion (MPP+): identification of a metabolite of MPTP, a toxin selective to the substantia nigra. Neurosci Lett 48:87–92.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Lutes J,
    2. Lorden JF,
    3. Beales M,
    4. Oltmans GA
    (1988) Tolerance to the tremorogenic effects of harmaline: evidence for altered olivo-cerebellar function. Neuropharmacology 27:849–855.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Mahgoub A,
    2. Idle JR,
    3. Dring LG,
    4. Lancaster R,
    5. Smith RL
    (1977) Polymorphic hydroxylation of debrisoquine in man. Lancet 2:584–586.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Matsubara K
    (1998) [Metabolic activation of azaheterocyclics induced dopaminergic toxicity: possible candidate neurotoxins underlying idiopathic Parkinson's disease]. Nippon Hoigaku Zasshi 52:301–305.
    OpenUrlPubMed
  35. ↵
    1. Matsubara K,
    2. Aoyama K,
    3. Suno M,
    4. Awaya T
    (2002) N-methylation underlying Parkinson's disease. Neurotoxicol Teratol 24:593.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Matsubara K,
    2. Collins MA,
    3. Akane A,
    4. Ikebuchi J,
    5. Neafsey EJ,
    6. Kagawa M,
    7. Shiono H
    (1993) Potential bioactivated neurotoxicants, N-methylated beta-carbolinium ions, are present in human brain. Brain Res 610:90–96.
    OpenUrlCrossRefPubMed
  37. ↵
    1. McCann SJ,
    2. Pond SM,
    3. James KM,
    4. Le Couteur DG
    (1997) The association between polymorphisms in the cytochrome P-450 2D6 gene and Parkinson's disease: a case-control study and meta-analysis. J Neurol Sci 153:50–53.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Melchior C,
    2. Collins MA
    (1982) The route and significance of endogenous synthesis of alkaloids in animals. Crit Rev Toxicol 9:313–356.
    OpenUrlPubMed
  39. ↵
    1. Miksys S,
    2. Rao Y,
    3. Hoffmann E,
    4. Mash DC,
    5. Tyndale RF
    (2002) Regional and cellular expression of CYP2D6 in human brain: higher levels in alcoholics. J Neurochem 82:1376–1387.
    OpenUrlCrossRefPubMed
  40. ↵
    1. Mulder GJ,
    2. Hagedoorn AH
    (1974) UDP glucuronyltransferase and phenolsulfotransferase in vivo and in vitro. Conjugation of harmol and harmalol. Biochem Pharmacol 23:2101–2109.
    OpenUrlCrossRefPubMed
  41. ↵
    1. Omura T,
    2. Sato R
    (1964) The carbon-monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378.
    OpenUrlFREE Full Text
  42. ↵
    1. Pfau W,
    2. Marquardt H
    (2001) Cell transformation in vitro by food-derived heterocyclic amines Trp-P-1, Trp-P-2 and N(2)-OH-PhIP. Toxicology 166:25–30.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Picada JN,
    2. da Silva KV,
    3. Erdtmann B,
    4. Henriques AT,
    5. Henriques JA
    (1997) Genotoxic effects of structurally related beta-carboline alkaloids. Mutat Res 379:135–149.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Raza H,
    2. King RS,
    3. Squires RB,
    4. Guengerich FP,
    5. Miller DW,
    6. Freeman JP,
    7. Lang NP,
    8. Kadlubar FF
    (1996) Metabolism of 2-amino-alpha-carboline. A food-borne heterocyclic amine mutagen and carcinogen by human and rodent liver microsomes and by human cytochrome P4501A2. Drug Metab Dispos 24:395–400.
    OpenUrlAbstract
  45. ↵
    1. Riedl AG,
    2. Watts PM,
    3. Jenner P,
    4. Marsden CD
    (1998) P450 enzymes and Parkinson's disease: the story so far. Mov Disord 13:212–220.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Ruiz-Durantez E,
    2. Ruiz-Ortega JA,
    3. Pineda J,
    4. Ugedo L
    (2001) Stimulatory effect of harmane and other beta-carbolines on locus coeruleus neurons in anaesthetized rats. Neurosci Lett 308:197–200.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Shou M,
    2. Lin Y,
    3. Lu P,
    4. Tang C,
    5. Mei Q,
    6. Cui D,
    7. Tang W,
    8. Ngui JS,
    9. Lin CC,
    10. Singh R,
    11. et al.
    (2001) Enzyme kinetics of cytochrome P450-mediated reactions. Curr Drug Metab 2:17–36.
    OpenUrlCrossRefPubMed
  48. ↵
    1. Slotkin T,
    2. DiStefano V
    (1970a) Urinary metabolities of harmine in the rat and their inhibition of monoamine oxidase. Biochem Pharmacol 19:125–131.
    OpenUrlCrossRefPubMed
  49. ↵
    1. Slotkin TA,
    2. DiStefano V
    (1970b) A model of harmine metabolism in the rat. J Pharmacol Exp Ther 174:456–462.
    OpenUrlAbstract/FREE Full Text
  50. ↵
    1. Slotkin TA,
    2. DiStefano V,
    3. Au WY
    (1970) Blood levels and urinary excretion of harmine and its metabolites in man and rats. J Pharmacol Exp Ther 173:26–30.
    OpenUrlAbstract/FREE Full Text
  51. ↵
    1. Strobel HW,
    2. Thompson CM,
    3. Antonovic L
    (2001) Cytochromes P450 in brain: function and significance. Curr Drug Metab 2:199–214.
    OpenUrlCrossRefPubMed
  52. ↵
    1. Tan TM,
    2. Sit KH,
    3. Wong KP
    (1990) UDP-glucuronyltransferase activity toward harmol in human liver and human fetal liver cells in culture. Anal Biochem 185:44–50.
    OpenUrlCrossRefPubMed
  53. ↵
    1. Tweedie DJ,
    2. Burke MD
    (1987) Metabolism of the beta-carbolines, harmine and harmol, by liver microsomes from phenobarbitone- or 3-methylcholanthrene-treated mice. Identification and quantitation of two novel harmine metabolites. Drug Metab Dispos 15:74–81.
    OpenUrlAbstract
  54. ↵
    1. Zetler G,
    2. Back G,
    3. Iven H
    (1974) Pharmacokinetics in the rat of the hallucinogenic alkaloids harmine and harmaline. Naunyn-Schmiedeberg's Arch Pharmacol 285:273–292.
    OpenUrlCrossRefPubMed
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Research ArticleNEUROPHARMACOLOGY

Contribution of Individual Cytochrome P450 Isozymes to theO-Demethylation of the Psychotropic β-Carboline Alkaloids Harmaline and Harmine

Ai-Ming Yu, Jeffrey R. Idle, Kristopher W. Krausz, Adrian Küpfer and Frank J. Gonzalez
Journal of Pharmacology and Experimental Therapeutics April 1, 2003, 305 (1) 315-322; DOI: https://doi.org/10.1124/jpet.102.047050
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