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

The psychotropic -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 the O-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 the O-demethylations mediated by each isozyme and by pooled HLMs. Kcat (min ) and Km ( M) 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 -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 -carbolines, such as pinoline, tryptoline, 6-hydroxy-tetrahydro-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 -carbolines with alcohol dependence and brain damage has been suggested (Melchior and Collins, 1982; Collins, 2002). Endogenous and exogenous -carboline alkaloids were reported to exert a wide spectrum of psychopharmacological and behavioral effects in the brain (Airaksinen and Kari, 1981). Most -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 in Peganum 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 -carbolinerich vine Banisteriopsis caapi mixed with the N,N-dimethyltryptamine (DMT)-containing hallucinogenic plant Psychotria viridis or Diplopterys cabrerana. Originally, it was proposed that the psychotropic properties of ayahuasca resided in the J.R.I. received a grant from U. S. Smokeless Tobacco Company. 1 Present address: U Háje 1651, 252 63 Roztoky u Prahy, Czech Republic. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.047050. ABBREVIATIONS: MAO, monoamine oxidase; DMT, N,N-dimethyltryptamine; 5-HT, 5-hydroxytryptamine; MPTP, N-methyl-4-phenyl-1,2,3,6tetrahydropyridine; 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, Km for recombinant CYP2D6 (insect cells); Km HLMCYP2D6, Km for CYP2D6 (high-affinity component) with two-enzyme kinetics in H161 HLMs. 0022-3565/03/3051-315–322$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 305, No. 1 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 47050/1051632 JPET 305:315–322, 2003 Printed in U.S.A. 315 at A PE T Jornals on N ovem er 9, 2017 jpet.asjournals.org D ow nladed from DMT, but it is now becoming clear that the -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). -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 -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 -carbolines (Ruiz-Durantez et al., 2001). The neurotoxic properties of -carboline alkaloids may account for their associations with Parkinson’s disease. Tryptophan-derived -carbolines are similar to N-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 -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 neurotoxic N-methylated -carbolinium ions are reported to be present in human brain (Matsubara et al., 1993). The metabolic bioactivation of carcinogenic -carbolines and -carbolines, such as 3-amino-1,4-dimethyl-5Hpyrido[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 -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 763-155), 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 100,000g for 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 M substrate in a final volume of 200 l. Reactions were initiated by the addition of 20 l of 10 mM NADPH after 5 min preincubation at 37°C. Incubations were terminated by the addition of 10 l 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 TABLE 1 Chemical structures and common names of some biogenic -carboline alkaloids