Introduction

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Humans are exposed to biogenic amines from a number of food sources, including fish products, cheese, beer and wine, meat and other fermented foods (Stratton et al., 1991). Generally, biogenic amines are formed as the result of ubiquitous normal metabolic activity in animals, plants and microorganisms. Biogenic amines can also be biosynthesized by enzymatic decarboxylation of the corresponding amino acid in bacteria (Brink et al., 1990). In addition, biogenic amines may arise from spoilage of raw materials or bacterial contamination and microbial activity related to fermentation during processing (Izquierdo-Pulido et al., 1993, 1995).

Among number of biogenic amines in food, histamine and tyramine have been implicated in toxicological problems from human ingestion of food and beverages containing biogenic amines (Morrow et al., 1991; Smith, 1981). Other amines, most notably phenethylamine, cadaverine, spermine and spermidine, may function as potentiators that enhance the toxicity of histamine and tyramine (Joosten, 1988; Stratton et al., 1991; Taylor, 1986). The "histamine intoxication" described above is distinct from the well known hypertensive crises brought about in humans treated with MAO inhibitors who have ingested elevated amounts of biogenic amines (the so-called cheese effect) (Lippman and Nash, 1990; Stockley, 1993). In some cases, individuals treated with MAO inhibitors who ingest elevated levels of biogenic amines had a fatal reaction (Stockley, 1993). In the presence of MAO inhibitors, which cause escape from oxidative deamination (and other metabolic processes described herein), ingestion of phenethylamine causes the release of catecholamines present in elevated amounts due to MAO inhibition at nerve endings and the adrenal medulla (Baldessarini, 1982).

The metabolism of phenethylamine has been described in considerable detail. Among the enzymes currently known to metabolize phenethylamine are MAO and CYP. MAO-B preferentially oxidizes noncatecholamines like phenethylamine to the corresponding aldimine intermediate, which is subsequently hydrolyzed to the aldehyde (Cashman, 1997). This terminates the action of the neurotransmitter. Interestingly, MAO-B activity increases with age (or, alternatively, MAO-A-containing neurons are degenerated) in rat and human brain (Yu, 1994). In humans, studies have linked MAO deficiency with unusual mental behavior (de la Chapelle et al., 1985) or mental retardation (Neri et al., 1992). Alteration in the structural gene was also reflected in abnormal monoamine metabolism (Bleeker-Wagemakers et al., 1988). With the exception of phenethylamine, CYP has also been shown to oxidize substituted 2-phenethylamines to N-hydroxylamines (Lindeke et al., 1982). The 2-phenethyl hydroxylamines are further metabolized to the nitroso compound via the intermediacy of a nitroxide (Jonsson and Lindeke, 1976). Substituted 2-phenethylamines potently inhibit CYP by forming MI complexes (Mansuy et al., 1978). N-Oxidation is considered a prerequisite for the MI complex formation; however, some CYP from seedlings of Sinapis alba L. converts L-tyrosine into hydroxyphenylacetaldoxime via the N-hydroxy amino acid by apparently not releasing the substrate from the enzyme surface (Du et al., 1995). The involvement of flavin monooxygenases (Dawson et al., 1993) and peroxidase-like enzymes (Ludwig-Muller et al., 1990) has also been implicated in the conversion of amino acids to oximes in the biosynthesis of glucosinolates.

There have been a few reports stating that the mammalian FMO catalyzes the formation of oximes from aliphatic primary amines (Clement et al., 1993; Lin et al., 1996; Poulsen et al., 1986). Of the five subfamilies of FMO (i.e., FMO1-FMO5), only FMO2 (Poulsen et al., 1986; Tynes et al., 1985; Williams et al., 1984), FMO3 (Cashman, 1995; Lin et al., 1996; Lomri et al., 1993) and FMO5 (Overby et al., 1995) have been reported to oxidize primary amines. Rabbit FMO2 was found to have high activity toward a number of primary amines but low activity toward phenethylamine (Tynes et al., 1986). Primary alkylamines (i.e., n-octylamine) are known stimulators of porcine FMO1 but have not been directly examined as substrates. With the exception of three reports (Clement et al., 1993; Lin et al., 1996; Poulsen et al., 1986), the direct quantification of FMO-mediated N-oxygenation of primary amines has not been reported.

In this report, we describe the in vitro N-oxygenation of phenethylamine and phenethyl hydroxylamine to the corresponding trans-oxime (fig. 1). Phenethylamine is efficiently N-oxygenated in the presence of adult human liver microsomes and highly purified human FMO3. The substrate selectivity was quite pronounced; although porcine FMO1 was competent to N-oxygenate phenethylamine to the trans-oxime, oxime formation was only a fraction of the rate observed for human FMO3. In the presence of adult human liver microsomes, phenethyl hydroxylamine was efficiently converted by retroreduction back to phenethylamine (fig. 1). Studies of the pharmacological activity of phenethyl hydroxylamine and phenethyl oxime suggested that N-oxygenation largely abrogates biological activity on the basis of metabolite interaction with 5-HT and dopamine receptors and the dopamine transporter. Formation of trans-phenethyl oxime by human FMO3 apparently represents a detoxication process and terminates the pharmacological activity of phenethylamine.

View larger version (6K): [in this window] [in a new window]   Fig. 1.   Proposed overall metabolic N-oxidative pathway for phenethylamine in the presence of human liver microsomes.

	Methods

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Chemicals

Chemicals used in this study were of the highest purity available from commercial sources. Phenylacetaldehyde, benzyl cyanide, hydroxylamine hydrochloride, sodium cyanoborohydride and phenethylamine were purchased from Aldrich Chemical (Milwaukee, WI). Other buffers, reagents and solvents were obtained from Fisher Chemical (San Jose, CA). All of the compounds of the NADPH-generating system were from Sigma Chemical (St. Louis, MO). Chromatography was done with Silica Woelm (35-70 mesh; Acros, Pittsburgh, PA).

Instrument Analysis

¹H NMR spectra were recorded on a Varian Spectrometer operating at a frequency of 300 MHz. The proton chemical shift values are given in ppm relative to tetramethylsilane. Mass spectra were recorded on a VG 70SEQ instrument. Both instruments are housed at the Department of Medicinal Chemistry, University of Washington, Seattle, WA.

Synthetic Procedures

cis- and trans-Phenethyloximes (3a and 3b). Phenylacetaldehyde (1.00 g, 8.33 mmol) was dissolved in 15.0 ml of tetrahydrofuran. Hydroxylamine hydrochloride (695 mg, 10.0 mmol) was dissolved in 5.0 ml of H₂O and added to the stirring solution of phenylacetaldehyde. Sodium carbonate (500 mg, 5.0 mmol) was dissolved in an additional 5.0 ml of H₂O and added to the reaction mixture. The reaction was completed in 1.0 hr as monitored with thin-layer chromatography (silica, 10% ethyl acetate in hexane). The organic and aqueous layers were separated; then, the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with water and brine and dried over MgSO₄. After filtering, the solvent was concentrated to give a yellow sticky oil. The mixture of cis- and trans-isomers (3a and 3b) were purified by flash silica chromatography; however, the cis- and trans-isomers (3a and 3b) were not separable by flash chromatography. After recrystallization with CHCl₃ and hexane, the two isomers were fractionated by 90% enrichments.

cis- and trans-Phenethyloximes (3a and 3b). The yield was 64% [R_f = 0.11 (silica, 10% ethyl acetate in hexane); MS (FAB): 136 (MH⁺), 117 (M⁺OH), 103 (M⁺OHN)]. For cis-phenethyloxime (3a), melting point: 99°C (needles); ¹H NMR (in CDCl₃):  3.78 (d, J = 5.2 Hz, 2H), 6.96 (t, J = 5.3 Hz, 1H), 7.28 (m, 4H), 9.38 (s, br, 1H). For trans-phenethyloxime (3b), melting point: 77° to 85°C (waxy solid); ¹H NMR (in CDCl₃):  3.59 (d, J = 6.2 Hz, 2H), 7.28 (m, 4H), 7.56 (t, J = 6.3 Hz, 1H), 8.85 (s, br, 1H).

Phenethyl hydroxylamine (2). The oximes (mixtures of 3a and 3b) (100 mg, 0.74 mmol) were dissolved in 5.0 ml of MeOH with bromophenol blue (74 µl of 1.0 mg/ml solution) as an indicator to prevent over reduction. Sodium cyanoborohydride (1.0 M solution in tetrahydrofuran, 490 µl, 0.67 Eq) was added to the reaction mixture, and HCl (10%) was added dropwise as needed to maintain a yellow color until the yellow color was persistent. The resulting mixture was stirred an additional 30 min at room temperature, and the thin-layer chromatography (silica, 0.2% TEA, 5% MeOH in CH₂Cl₂) indicated the reaction was complete. The reaction mixture was treated with 10% Na₂CO₃ and extracted twice with ethyl acetate. The organic layers were combined, washed with brine, dried over MgSO₄ and concentrated to give a solid in vacuo. The pure product 2 was obtained after flash chromatography (silica, 0.2% TEA, 5% MeOH in CH₂Cl₂) to yield a solid. Yield was 40%; R_f = 0.22 (silica, 0.2% TEA, 5% MeOH in CH₂Cl₂); ¹H NMR (in CDCl₃):  3.45 (t, J = 7.7 Hz, 2H), 3.55 (m, 2H), 7.26 (m, 5H); MS (FAB): 138 (MH⁺), 120 (MH⁺OH), 105 (MH⁺OHN).

Liver and Microsome Preparations

The adult human liver microsome samples were obtained under a protocol approved by the Committee for the Conduct of Human Research at the Medical College of Wisconsin. Adult human liver microsomes were a generous gift of Dr. Steven A. Wrighton (Eli Lilly and Company, Indianapolis, IN). The CYP content and specific activity of selected CYP were determined as previously described (Lowry et al., 1951; Omura and Sato, 1964). Each of the eight most prominent CYPs has been completely characterized immunochemically as well as by selective functional substrate assays: ethoxyresorufin-O-deethylase, coumarin-7-hydroxylase, S-mephenytoin-4-hydroxylase, bufuralol-1-hydroxylase, N-nitroso-dimethylamine N-demethylase and erythromycin N-demethylase (25, 440, 103, 43, 661 and 249 pmol of product/mg of microsomal protein/min, respectively) (Wrighton et al., 1993). The CYP content was 317 pmol of CYP/mg of protein. The FMO3 has likewise been previously fully characterized from these preparations (Cashman et al., 1992; Wrighton et al., 1993), and the nicotine N-1-oxygenase activity was 125 pmol/min/mg of protein.

Metabolic Incubation Systems

A typical incubation mixture contained 50 mM potassium phosphate buffer, pH 9.0, 0.4 mM NADP⁺, 0.4 mM glucose-6-phosphate, 1.0 IU of glucose-6-phosphate dehydrogenase, 40 to 100 µg of cDNA-expressed human liver FMO3-MBP² or 0.40 to 0.60 mg of adult human liver microsomes and 1.2 mM DETAPAC (final volume, 0.25 ml). The reaction was initiated by the addition of substrate and incubated at 37°C with shaking in air. At various time intervals, the incubations were stopped by the addition of 1.5 ml of cold dichloromethane. After saturation with Na₂CO₃ and a brief centrifugation, the organic layer was separated from the aqueous phase, evaporated and then analyzed for products by the HPLC procedure described below.

The profile of phenethylamine metabolites was determined by HPLC analysis of dichloromethane extracts of the incubation mixture. The metabolic products were separated and quantified with a Hitachi L-6200A HPLC interfaced to a Hitachi D-2500 Chromato-Integrator with a Hitachi L-4000H UV detector set at 257 nm. The system was fitted with a C-18 analytical column (25 × 0.4 cm) from Rainin (Emeryville, CA). The mobile phase consisted of an isocratic system set at 70% of A and 30% of B, in which A is water containing 1.0% of THF and B is CH₃CN containing 0.10% of HClO₄ (60% solution) at a flow rate of 1.5 ml/min. This system efficiently separated phenethylamine, phenethyl hydroxylamine and trans- and cis-oximes that had retention times of 3.02, 3.43, 5.93 and 7.05 min, respectively. On the basis of UV absorption of each material, metabolites were quantified by comparing the metabolite and substrate peak areas of the chromatogram and calculated according to the following formula: % conversion_oximes = areas of oximes/(areas of oximes + 2.0 areas of phenethylamine), or % conversion_oximes = areas of oximes/(areas of oximes + 4/3 areas of phenethyl hydroxylamine).

Immunoblotting and Antisera

Antibodies that recognized specific human CYP were used to characterize the microsome CYP immunoreactivity as previously described (Wrighton et al., 1993). Antibodies to guinea pig FMO were raised in rabbits and used to detect human FMO3 by a method similar to one previously described (Guan et al., 1991). The antibody to guinea pig FMO was a generous gift of Drs. K. Oguri and H. Yamada (Kyushu University, Fukuoda, Japan).

Receptor Binding Assays

HA7 cells were grown to confluence in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 0.05% penicillin-streptomycin and 400 µg/ml G418. The cells were scraped from 100 × 20-mm plates and centrifuged at 500 × g for 5 min. The pellet was homogenized at 2 plates/ml in 50 mM Tris·HCl, pH 7.7, with a Polytron as previously described (Sunahara et al., 1991). For 5-HT_1A receptor, the competitive assay was done with [³H]8-hydroxy-2-dipropylaminotetralin. The tubes were incubated at 25°C for 60 min and then filtered through a Whatman GF/B filter paper on a Brandel cell harvester, and the filters were counted by scintillation counting. For the 5-HT_1C receptor, NIH-3T3-P cells were grown and prepared in the same manner as HA7 cells. [³H]Mesulergine was used to measure competitive binding. For the 5-HT_2A receptor, NIH-3T3-GF6 cells were grown as described above, and [³H]ketanserin was used to measure competitive binding. For the 5-HT₃ receptor, NG108-15 cells were grown as above, and [³H]GR65630 was used to measure competitive binding. For the D₁ receptor, LHD1 cells were grown as described above, and competitive binding was determined with [³H]SCH 23,390. For D₂ and D₃ receptors, CHOp-D₂ and CHOp-D₃ cells, respectively, were grown as described above, and [³H]YM-09151-2 was used in competitive binding experiments. For dopamine transporter binding studies, C6 hDAT cell membranes are prepared as previously described (Eshleman et al., 1995), and competitive binding was done with [¹²⁵I]RTI-55.

	Results

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Chemical synthesis and stability. The chemical synthesis of metabolites of phenethylamine was done to obtain sufficient material for the identification of phenethylamine metabolites in vitro and to study the chemical stability of these materials under the aqueous incubation and analysis conditions. In addition, because the N-oxidative metabolites of phenethylamine are virtually completely uncharacterized from a pharmacological point of view, sufficient material was synthesized for in vitro pharmacological evaluation with selected receptors and transporters.

View larger version (7K): [in this window] [in a new window]   Fig. 2.   Synthetic pathway for the procurement of hydroxylamine 2 and oximes 3a and 3b.

Metabolism of phenethylamine and phenethylamine hydroxylamine in the presence of microsome preparations. The metabolism of phenethylamine in the presence of various microsome preparations was done to determine the nature of the monooxygenases responsible for the transformation and identify the products formed. Porcine liver microsomes provided a convenient source of porcine FMO1, and adult human liver microsomes provided a source of human FMO3. Both preparations possessed good levels of highly characterized CYP activity (Decker et al., 1992; Lin et al., 1996). In the presence of porcine liver microsomes or adult human liver microsomes, aerobic incubation of phenethylamine resulted in exclusive formation of trans-oxime 3b (i.e., 0.22 ± 0.04 and 3.74 ± 0.56 nmol/min/mg of protein, respectively). In the presence of porcine liver microsomes, formation of oxime 3b from phenethylamine was linearly dependent on time (i.e., 0-15 min) and protein concentration (i.e., 0-1.7 mg of protein). Likewise, in the presence of adult human liver microsomes, formation of oxime 3b was linearly dependent on time (i.e., 0-10 min) and protein concentration (i.e., 0-0.76 mg of protein). Under the experimental conditions used, formation of phenethylamine hydroxylamine was not observed in the presence of either porcine or human liver microsome preparations. The results were consistent with figure 1. Formation of oxime 3b was completely dependent on NADPH and active protein. It was notable that adult human liver microsomes were almost 17-fold more efficient than porcine liver microsomes in forming oxime 3b from phenethylamine despite the fact that porcine liver microsomes are generally ~10- to 50-fold more active than adult human liver microsomes with regard to tertiary amine N-oxygenation.

Effect of incubation conditions on oxime formation from phenethylamine and phenethyl hydroxylamine

Effects of inhibitors on oxime formation from phenethyl hydroxylamine in porcine and human liver microsomes. Formation of trans-oxime 3b from phenethyl hydroxylamine 2 was highly dependent on NADPH and active protein. In the presence of porcine liver microsomes, the alternate substrate competitive inhibitor thiourea decreased oxime 3b formation but not as significantly as that observed when phenethylamine was incubated with human liver microsomes (table 2). The addition of n-octylamine, a compound that inhibits CYP and stimulates porcine FMO1 activity, increased trans-oxime 3b formation (table 2). In the presence of human liver microsomes, the alternate substrate competitive inhibitors thiourea and thiobenzamide decreased oxime 3b formation (table 3). The addition of n-octylamine did not have a marked effect on the formation of trans-oxime 3b from phenethyl hydroxylamine (table 3), and heat inactivation of both microsome preparations significantly decreased trans-oxime 3b formation (tables 2 and 3). The results are consistent with a prominent role of FMO1 and FMO3 in the N-oxygenation of phenethylamine in porcine and adult human liver microsomes, respectively.

Role of highly purified FMOs in phenethylamine N-oxygenation. To examine a role of FMO in phenethylamine oxime 3b formation, the relative rates of oxime formation in the presence of highly purified preparations of porcine FMO1, rabbit FMO2 and cDNA-expressed human Lys^158 3 FMO3 maltose binding protein (Lys¹⁵⁸ FMO3-MBP) were determined. Preliminary studies indicated that porcine FMO1 and rabbit FMO2 showed modest and negligible rates of oxime 3b formation, respectively (table 4). This was not due to lack of FMO activity because separate studies showed that porcine FMO1, rabbit FMO2 and human Lys¹⁵⁸ FMO3-MBP possessed high activity as a tertiary amine N-oxygenase using the substrate 10-[N,N-dimethylaminopentyl]-2-(trifluoromethyl)phenothiazine. The results with rabbit FMO2 are in agreement with previously established structure-activity relationships (Nagata et al., 1990) that suggest that long aliphatic amine substrates are required for enzyme activity.

Effect of active oxygen species on oxime 3b formation. Because previous reports of FMO-mediated aliphatic hydroxylamine oxidation have implicated significant contributions from reactive oxygen species (Clement et al., 1993; Rauckman et al., 1979; Tynes et al., 1986), we examined the roles of H₂O₂ and superoxide in the human microsomal and FMO3-catalyzed formation of oximes from phenethylamine hydroxylamine 2. In the presence of adult human liver microsomes, hydroxylamine 2 was converted to trans-oxime 3b in a process largely independent of H₂O₂ and superoxide anion (table 6). Thus, the addition of exogenous H₂O₂ or generation of O₂ or abrogation of these reactive oxygen species did not greatly change the amount of oxime 3b that was formed (table 6). In contrast, retroreduction of hydroxylamine 2 to phenethylamine 1 in the presence of reactive oxygen species showed a dramatic effect. Thus, in the presence or absence of exogenously added H₂O₂, no significant alteration of phenethylamine formation was observed. However, generation of superoxide markedly increased retroreduction of phenethylamine hydroxylamine, whereas abrogation of superoxide by the action of SOD completely abolished retroreduction of hydroxylamine 2 (table 6).

Kinetic determination of trans-oxime 3b formation by prominent human FMO3s. The substrate dependence for the N-oxygenation of phenethylamine and phenethylamine hydroxylamine was examined in the presence of cDNA-expressed human Lys¹⁵⁸ and Glu¹⁵⁸ FMO3s (table 8). The two human FMO3 enzymes were studied because they represent the most prominent FMO3 enzymes present in normal adult humans.³ Thus, plots of the reciprocal of velocity vs. the reciprocal of the substrate concentration provided a series of linear correlations. From these Lineweaver-Burk plots, the K_mapp and V_max values were obtained (table 8). For both phenethylamine and phenethylamine hydroxylamine, it appeared that the K_mapp value for Lys¹⁵⁸ FMO3 was elevated compared with that of the human Glu¹⁵⁸ FMO3-MBP enzyme.

Effect of hydroxylamine 2 or oxime 3 on biogenic amine transporter and receptors. The binding affinity for phenethylamine hydroxylamine 2 and oxime 3 for several biogenic amine receptors and the dopamine transporter were examined. Thus, the 5-HT receptors (i.e., human 5-HT_1A, rat 5-HT_1C, rat 5-HT_2A and guinea pig 5-HT₃), the dopamine receptors (i.e., human D₁, D₂ and D₃) and the dopamine transporter (i.e., human DAT) were examined for competitive binding to well established ligands. As shown in table 9, the IC₅₀ or K_i determinations all showed very poor avidity with values of >10 µM. Such IC₅₀ or K_i values are beyond the physiological range and suggest that hydroxylamine 2 and oxime 3 do not possess any significant pharmacological activity for these receptors.

	Discussion

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An efficient method was developed to synthesize phenethylamine hydroxylamine 2 and phenethyl oxime 3. The cis- and trans-oximes were separated by fractional recrystallization; this allowed sufficient material to conduct bioanalytical and pharmacological studies. High-resolution proton NMR studies confirmed the identity of the oxime stereoisomers. In contrast to many previous studies that have shown that cis-oximes are preferentially formed metabolically, only the trans-oxime 3b was observed to be formed. The oxime 3 had a slight tendency to isomerize to the trans-oxime 3b; however, this did not interfere with the determination of the stereochemistry of the enzymatic product. Using ion-pair chromatography, a convenient HPLC procedure was developed to separate and quantify phenethylamine 1 and the corresponding hydroxylamine 2 and cis- and trans-oximes 3 (fig. 1).

Metabolic studies suggested that the only product observed to be formed from phenethylamine 1 in the presence of porcine or adult human liver microsomes was the trans-oxime 3b. No evidence for the formation of hydroxylamine 2 from phenethylamine was observed. It is possible that catalytic facilitation (Du et al., 1995) channels the flow of phenethylamine metabolites through a series of intermediates that are tightly bound to FMO. This may account for the failure to observe dissociable hydroxylamine 2 along an ABC reaction path leading to exclusive formation of trans-oxime 3b.

Previous studies using rat liver microsomes from phenobarbital-treated animals showed that phenethylamine was a poor substrate for N-hydroxylation (Jonsson and Lindeke, 1976). Unlike substituted 2-phenethylamines, phenethylamine does not form metabolic inhibitory complexes, and thus, the lack of N-oxygenation in a particular microsome preparation for phenethylamine is not due to CYP inhibition. More likely, the extent of phenethylamine N-oxygenation is due to the type and amount of FMO present. The relatively low amount of FMO3 and abundance of FMO1 in rat liver preparations (Cashman, 1995) and the difficulty in procuring microsomal FMO activity may account for the prior observation of low N-oxygenation capacity of phenethylamine in rat liver microsomes (Lindeke et al., 1982). On the other hand, FMO1 is the only FMO present in porcine liver microsomes, and the data of table 1 show that low, albeit detectable, levels of oxime 3b were formed. Primary amine N-oxygenation by FMO1 has not been previously reported. Generally, primary aliphatic amines stimulate FMO1-mediated substrate oxygenation, although in some cases, inhibition has been observed (Cashman, 1995). Thus, depending on the substrate, an aliphatic primary amine may show alternate substrate competitive inhibition or, alternatively, FMO stimulatory activity. This may be a consequence of generation of reactive oxygen species. Regardless, the effect is modest compared with FMO3-catalyzed N-oxygenation of phenethylamine.

In contrast to porcine FMO1, human FMO3 efficiently N-oxygenates phenethylamine to produce exclusively trans-oxime 3b. The effect of metabolic inhibitors on phenethylamine N-oxygenation (table 1) is consistent with a prominent role of human FMO3 in the formation from phenethylamine, but it does not rule out the involvement of CYP. For example, under heat inactivation conditions that abolish ~85% of FMO activity, almost 25% oxime formation is still observed (table 1). We conclude that if CYP contributes to human microsome-catalyzed phenethylamine N-oxidation, it is doing so on the order of ~10%.

Although we do not detect phenethylamine hydroxylamine 2 as an observable metabolite under analytical conditions that would detect very low levels (i.e., 50 pmol/min/mg of protein), it is undoubtedly the initial metabolite in the formation of oxime 3b. Incubation of synthetic phenethylamine hydroxylamine 2 in the presence of porcine and human liver microsomes efficiently produced exclusively trans-oxime 3b, confirming that hydroxylamine 2 was indeed on the sequential multistep reaction pathway leading to oxime 3b. The effect of metabolic inhibitors on conversion of hydroxylamine 2 to oxime 3b was consistent with a prominent role of FMO. In the case of porcine liver microsomes, n-octylamine modestly stimulated oxime 3b formation, whereas in the presence of adult human microsomes, n-octylamine slightly inhibited oxime 3b formation (tables 2 and 3). This shows that n-octylamine can have a stimulatory or inhibitory effect on FMO1 and FMO3, respectively, for the N-oxygenation of the same substrate for both enzymes. In the presence of heat inactivation of adult human liver microsomes, phenethylamine hydroxylamine N-oxidation produced ~24% more oxime 3b than expected. It is possible that heat-inactivated adult human liver microsome CYP contributes as much as 24% to the overall oxime 3b formation. This value is somewhat surprising in view of the potent metabolic intermediate complex that 2 can form with CYP (Jonsson and Lindeke, 1976; Lindeke et al., 1982; Mansuy et al., 1978).

Depending on the substrate structure, four of the five known mammalian FMOs are now known to N-oxygenate primary aliphatic amines [i.e., FMO1 (this work), FMO2 (Poulsen et al., 1986; Tynes et al., 1985; Williams et al., 1984), FMO3 (Cashman, 1995; Lin et al., 1996; Lomri et al., 1993) and FMO5 (Overby et al., 1995)]. The data of table 4 show that considerable substrate selectivity was observed in comparison of FMO1, FMO2 and FMO3 in the N-oxygenation of phenethylamine 1 and phenethyl hydroxylamine 2. Thus, highly purified porcine FMO1 and cDNA-expressed human FMO3-MBP catalyzed N-oxygenation of phenethylamine with very modest and very robust efficacy, respectively. Highly purified rabbit FMO2 did not detectably N-oxygenate phenethylamine. This is in keeping with previously established structure-activity relations that showed that only long-chain aliphatic amines (i.e., 5 methylene units separating the aryl group from the primary amine functionality) were required for rabbit FMO2 substrate activity (Nagata et al., 1990). Previously, the human FMO3 substrate binding site was also thought to be restricted to long-chain amines (Cashman, 1995; Lomri et al., 1993), but apparently, simple phenylalkylamines also possess excellent substrate specificity. In comparison, a previous study with rabbit FMO2 showed that aliphatic primary amines were converted predominantly to the cis-oxime at low substrate concentrations but that N-hydroxylamines were formed at high amine concentrations (Poulsen et al., 1986).

Phenethyl hydroxylamine 2 was converted into oxime 3b by all three FMOs examined (table 4). This probably is a consequence of the enhanced nucleophilicity (i.e.,  effect) of the hydroxylamine moiety. Porcine liver FMO1 and rabbit FMO2 catalyzed a modest amount of oxime 3b formation. cDNA-expressed human FMO3-MBP was by far the most efficient phenethylamine hydroxylamine N-oxygenase catalyst examined. This observation encouraged a more detailed examination of the mechanism of human FMO3-mediated oxime 3b formation. Affinity chromatography-purified human FMO3-MBP was used to examine the N-oxygenation of 1 and 2. cDNA-expressed human FMO3-MBP² offers many advantages to the study of lipophilic amines, including (1) the water-soluble and essentially detergent-free nature of the enzyme, (2) the similarity of the substrate specificity of the nonfusion protein and (3) the enhanced stability of human FMO3-MBP with respect to microsomal FMO3 in the presence of NADPH.

In good agreement with human liver microsome studies, formation of trans-oxime 3b from phenethylamine or phenethylamine hydroxylamine was observed in the presence of cDNA-expressed human FMO3-MBP. Taken together, the data support a prominent role of human FMO3 in the sequential multistep reaction that converts primary amine 1 into oxime 3b (fig. 3). Currently, a mechanism involving N,N-dioxygenation of 2 is favored. No evidence for formation of the CYP-mediated nitroso compound (i.e., intermediate 5) was observed. If formed, on the basis of previous studies, the nitroso compound 5 would be expected to potently inhibit CYP (Jonsson and Lindeke, 1976; Lindeke et al., 1982; Mansuy et al., 1978). It is possible that the nitroso compound 5 could equilibrate with oxime 3, but this would be anticipated to form significant amounts of cis-oxime 3a. That no detectable cis-oxime 3a was observed to be formed in the presence of the microsomes and FMO enzymes that were used suggests that path II (fig. 3) contributed <5% of the overall conversion of phenethylamine 1 to oxime 3b. It is notable that only the trans-oxime 3b is formed from phenethylamine despite the fact that the proposed symmetrical N,N-dihydroxy intermediate would be predicted to provide both oxime stereoisomers (path I, fig. 3). This observation is in keeping with the postulate that the surface of FMO facilitates catalysis and that stereoselective formation of only the trans-oxime 3b is observed despite the likelihood that dehydration is a spontaneous reaction. Although dehydration of the proposed N,N-dihydroxy intermediate is presumably nonenzymatic, other studies of FMO3-mediated NADPH-independent isomerization of other cis-oximes to trans-oximes suggest that FMO3 may provide a protein template for dehydration.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Proposed N-oxidative metabolism of phenethylamine by sequential two-electron oxygenation by the flavin-containing monooxygenase (path I) to give trans-oxime 3b and sequential one-electron oxidation by CYP (path II) to produce nitroxide 4 and nitroso compound 5.

In the presence of porcine or human liver microsomes, retroreduction of hydroxylamine 2 to form the primary amine 1 competed with N-oxygenation to produce oxime 3b. Previously, we have shown that hydroxylamine retroreduction in the presence of human liver microsomes did not correlate significantly with human FMO3 or with any well characterized CYP activity or immunoreactivity (Lin et al., 1996). In contrast to a previous study using rabbit FMO2 (Poulsen et al., 1986), no evidence was found for retroreduction of the hydroxylamine to the amine by human FMO3. It is likely that retroreduction is due to a CYP that has not been completely characterized (Clement et al., 1994; Kadlubar et al., 1973). There are several reports of the CYP-catalyzed retroreduction of hydroxylamines to amines (Clement et al., 1994), amidoximes to amidines (Clement et al., 1991) and N-hydroxyisothioureas to isothioureas (Clement, 1991). The retroreduction of N-hydroxydebrisoquine to debrisoquine (Clement et al., 1993) or N-hydroxyaminoguanidine to amino-guanidine (Clement et al., 1994) has been shown to be mediated by cytochrome b₅, cytochrome b₅ reductase and a CYP that is apparently similar to the retroreduction enzyme system described by Kadlubar and Ziegler (1974) for the retroreduction of hydroxylamines to amines. Parallel and interdependent oxidative and retroreductive enzymatic processes must be in action to account for the formation of oxime 3b and primary amine 1 from hydroxylamine 2 in the presence of hepatic microsomes.

Previously, some data from investigations using porcine FMO1 showed that direct N-oxygenation was not observed but, instead, porcine FMO1 released O₂ in the presence of the hydroxylamine and converted the hydroxylamine nonenzymatically to the nitroxide (Rauckman et al., 1979). Uncoupling of porcine FMO1 hydroperoxyflavin has been proposed to generate O₂ or H₂O₂, and in a nonenzymatic step, autoxidize the hydroxylamines. Pig FMO1 has also been observed to form oximes from primary hydroxylamines (Clement et al., 1993) and to form nitrones from secondary hydroxylamines (Cashman et al., 1990). The formation of these two metabolites was judged to be strictly dependent on FMO action. A possible role for reactive oxygen species in the N-oxidation of phenethyl hydroxylamine 2 was examined by investigating the human microsomal and human FMO3-mediated production of oxime 3b in the presence of H₂O₂ and O₂. Neither generation of O₂ by the xanthine/xanthine oxidase system nor removal of O₂ by SOD had any effect on the formation of oxime 3b from phenethylamine hydroxylamine 2 by cDNA-expressed human Lys¹⁵⁸ FMO3-MBP (table 7). The conclusion is that in the presence of human liver microsomes or human FMO3, O₂ and/or H₂O₂ contributes 3% to 4% of the oxime 3b formation from phenethylamine.

Human Lys¹⁵⁸ FMO3-MBP did not reduce hydroxylamine 2 to phenethylamine 1, but in the presence of porcine or human liver microsomes, retroreduction of hydroxylamine 2 was significant and, paradoxically, reactive oxygen species apparently participated in primary amine formation. Thus, the O₂-generating system of xanthine/xanthine oxidase increased by >2-fold the reduction of hydroxylamine 2 to primary amine 1 in the presence of adult human liver microsomes.

Restriction length polymorphism and oligonucleotide-sequencing studies showed that in humans, codon 158 of human FMO3 encoded either amino acids Glu or Lys at approximately equal allele frequencies for the Caucasian population examined. Although this may not be the case for all human populations, nevertheless, an investigation of the prominent forms of human FMO3 involved in biogenic amine metabolism is important. As shown in table 8, the K_mapp value for N-oxygenation of phenethylamine 1 and phenethylamine hydroxylamine 2 was 2.2- to 7.4-fold lower for the Glu¹⁵⁸ FMO3 enzyme than for the wild-type Lys¹⁵⁸ FMO3 enzyme, respectively. The V_max/K_mapp values also suggest that the human Glu¹⁵⁸ FMO3 enzyme more efficiently N-oxygenates phenethylamine than the Lys¹⁵⁸ FMO3 enzyme. In either case, however, the K_mapp values are low enough to be of potential physiological relevance in metabolizing lipophilic phenethylamines to pharmacologically inactive metabolites.

The FMO has often been cited as a detoxication catalyst that converts lipophilic compounds into pharmacologically inert metabolites (Cashman, 1995; Ziegler, 1980). Most examples of FMO-mediated detoxication that have been studied have been xenobiotics because of the paucity of endogenous physiologically active materials that are substrates for FMO. In the case of the biogenic amine phenethylamine that potentially interacts with a number of pharmacological targets, we were able to investigate this hypothesis. As shown in table 9, no detectable affinity for any of the prominent biogenic receptors or transporters was observed for the hydroxylamine 2 or oxime 3. Thus, metabolism of phenethylamines to hydroxylamines or oximes should result in abrogation of pharmacological activity. It is possible that phenethylamine hydroxylamines or oximes are further metabolized by conjugation to other pharmacologically inert materials. Regardless, FMO may play a fundamental role in converting pharmacologically important biogenic amines to oxime metabolites that effectively terminate the pharmacological activity.