Detoxication of Tyramine by the Flavin-Containing Monooxygenase

Articles. Detoxication of Tyramine by the Flavin-Containing. Monooxygenase: Stereoselective Formation of the Trans. Oxime. Jing Lin and John R. Cashma...
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Chem. Res. Toxicol. 1997, 10, 842-852

Articles Detoxication of Tyramine by the Flavin-Containing Monooxygenase: Stereoselective Formation of the Trans Oxime Jing Lin and John R. Cashman* Seattle Biomedical Research Institute, 4 Nickerson Street, Suite 200, Seattle, Washington 98109 Received February 25, 1997X

In the presence of pig or adult human liver microsomes, tyramine was metabolized to the corresponding trans oxime through the intermediacy of the hydroxylamine. The requisite intermediate, (4-hydroxyphenethyl)hydroxylamine, was retroreduced to tyramine or converted stereoselectively to the trans oxime in the presence of pig or adult human liver microsomes. Studies of the effect of metabolic inhibitors suggested that formation of the trans oxime and retroreduction of the hydroxylamine were largely dependent on NADPH and the flavincontaining monooxygenase (FMO) and cytochrome P450, respectively. The conclusion that FMO was predominantly responsible for trans oxime formation in human liver microsomes was based on the effect of incubation conditions on tyramine N-oxygenation and the observation that cDNA-expressed human FMO3 also N-oxygenated tyramine to give exclusively the trans oxime. The synthetic hydroxylamine and oxime metabolites of tyramine were examined for affinity to human and animal dopamine and serotonin receptors and the human dopamine transporter. For all of the receptors and for the transporter examined, the avidity of the hydroxylamine and oximes was greater than 10 µM and beyond the effective concentration for physiological relevance. The results suggested that tyramine was sequentially N-oxygenated in the presence of pig and human liver microsomes and cDNA-expressed FMO3 to the hydroxylamine and then to the di-N-hydroxylamine that was spontaneously dehydrated to the trans oxime. This may be facilitated by FMO through a nondissociative substrate-enzyme interaction. Based on the biogenic amine receptor or transporter affinity for the hydroxylamine and oxime metabolites of tyramine, N-oxygenation of tyramine by pig or human liver FMO may represent a detoxication reaction that terminates the pharmacological activity of tyramine.

Introduction The toxicological and pharmacological properties of biogenic amines are well-recognized (1, 2). Humans are exposed to a variety of biogenic amines from plants, fish, cheese, beer, wine, meat, and other fermented foods (3). Phenylalkylamine levels are especially high in banana, pineapple, plantain, and avocado (4). Tyramine and methylated derivatives are present in elevated amounts in cheese, yeast products, fermented foods, beer, wine, pickled herring, snails, chicken liver, broad beans, chocolate, and cream products (5). Tyramine can be formed as a result of normal metabolic activity in animals, plants, and microorganisms, or it can be biosynthesized by enzymatic decarboxylation of tyrosine in the presence of bacteria (6). Tyramine can also result from spoilage of food or bacterial contamination and microbial activity related to fermentation during processing (7). Cheese and yeast products are potentially dangerous in individuals being treated with monoamine oxidase (MAO)1 inhibitors because consumption of these products could provide the 10 mg of tyramine needed to cause a severe hypertensive crisis in the adult (8). Inhibition of MAO by MAO inhibitors allows tyramine to avoid metabolic * Corresponding author. Telephone: (206) 284-8846, ext. 310. Fax: (206) 284-0313. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, July 1, 1997.

S0893-228x(97)00030-1 CCC: $14.00

degradation and provides for the release of catecholamines present in elevated amounts at nerve endings, the adrenal medulla, and other peripheral sites. Tyramine and other biogenic amines have been reported to potentiate the toxicity of histamine (9). In addition, tyramine may cause migraine headaches (10). In summary, tyramine is not considered a highly toxic material because it is generally present in low levels in food. However, if the metabolism of tyramine is blocked or genetically altered, the potential for toxicity exists. The current picture of the metabolism of tyramine involves a number of enzyme systems including: MAOcatalyzed deamination (11), cytochrome P450 (P450)mediated oxime formation (12), dopamine β-hydroxylase1 Abbreviations: MAO, monoamine oxidase; P450, cytochrome P450; FMO, flavin-containing monooxygenase; TEA, triethylamine; FAB/MS, fast atom bombardment mass spectrometry; CI-MS, chemical ionization mass spectrometry; FMO3-MBP, flavin-containing monooxygenase (form 3)-maltose-binding protein; DETAPAC, diethylenetriaminepentaacetic acid; APCI, atmospheric pressure chemical ionization; LC-APCI/MS, liquid chromatography-atmospheric pressure chemical ionization mass spectrometry; DMEM, Dulbecco’s modified Eagles media; 5-HT, 5-hydroxytryptamine; D1, dopamine (class 1); G418, geneticin antibiotic; 8-OH-DPAT, 8-hydroxy(diphenylamino)tetraline; NIH-3T3-Pφ, NIH-3T3-GF6, and NG108-15, neuroblastoma cells; SCH23390, (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; CHOp-D cells, Chinese hamster ovary cells transfected with the dopamine receptor cDNA; [125I]RTI-55, 3β-(4iodophenyl)tropane-2β-carboxylic acid methyl ester.

© 1997 American Chemical Society

N-Oxygenation of Tyramine by FMO

Chem. Res. Toxicol., Vol. 10, No. 8, 1997 843

Tyramine is efficiently sequentially N-oxygenated by both microsomal FMO1 and FMO3 to produce exclusively the trans oxime 3b (Scheme 1). In the presence of pig or human liver microsomes, (4-hydroxyphenethyl)hydroxylamine was converted by retroreduction back to tyramine. In a parallel and independent C-oxidative pathway, tyramine was hydroxylated at the β-carbon. The resulting metabolite, octopamine, was apparently converted to an unstable β-hydroxy oxime that eliminated the elements of water and cyanide to produce 4-hydroxybenzaldehyde (Scheme 2). The tyramine metabolites (i.e., hydroxylamine 2 and trans oxime 3b) were also tested for pharmacological activity in the presence of serotonin and dopamine receptors and the dopamine transporter. The results suggested that FMO-mediated formation of oxime metabolites of tyramine abrogated the pharmacological activity and thus represents a detoxication path.

Scheme 1. Proposed N-Oxygenation Pathway of Tyramine NHOH

NH2 HO

HO 1

2 N

OH

HO 3a: cis 3b: trans

catalyzed octopamine formation (13), and phenylethanolamine N-methyltransferase-mediated formation of Nmethyloctopamine (synephrine) (14). The metabolism by MAO (and presumably P450) of tyramine serves to terminate the action of the false neurotransmitter. However, considerable pharmacological activity is retained after β-hydroxylation or N-methylation of tyramine (1). The conversion of L-tyrosine into hydroxyphenyl acetaldoxime via the N-hydroxy amino acid by apparently not releasing the substrate or intermediates from the enzyme surface reported for a P450 from seedlings of Sinapis alba L. (12) has some similarity to the flavin-containing monooxygenase (FMO)-catalyzed formation of oximes from tyramine (15). Currently, four of the five FMO subfamilies (i.e., FMO1 (16), FMO2 (16-19), FMO3 (15, 16), and FMO5 (20)) have been reported to N-oxygenate primary aliphatic amines. Only a few reports (15-17) have directly characterized the FMO products formed from aliphatic primary amines and the apparent enzyme mechanism utilized. In human liver preparations, the literature suggests that aliphatic primary amines are sequentially N-oxygenated by FMO3 to initially form a hydroxylamine that is converted to a di-N-hydroxylamine intermediate that dehydrates spontaneously to form almost exclusively either cis or trans oxime, depending on the substrate (15, 16). Some evidence for the involvement of reactive oxygen species in the metabolism of hydroxylamines has been presented for pig FMO1 (21), but for human FMO3, oxime formation appears independent of the presence of O2- and/or H2O2 (15, 16). In the present report, the N- and C-oxidation of tyramine was examined in the presence of pig and human liver microsomes and cDNA-expressed human FMO3.

Experimental Procedures Chemicals. Tyramine, p-hydroxylphenethyl alcohol, p-hydroxybenzaldehyde, sulfur trioxide-pyridine complex, hydroxylamine hydrochloride, and sodium cyanoborohydride were purchased from Aldrich Chemical Co. (Milwaukee, WI). Other buffers, reagents, and solvents were obtained from Fisher Scientific Inc. (Santa Clara, CA). All of the compounds of the NADPH-generating system were from Sigma Chemical Co. (St. Louis, MO). Chromatography was done with Silica Woelm, 70150 mesh (Acros, Inc., Pittsburgh, PA). Chemicals used in this study were of the highest purity available from commercial sources. Instrumental 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 TMS. Fast atom bombardment mass spectra (FAB/MS) were recorded on a VG 70SEQ instrument. Both instruments were housed at the Department of Medicinal Chemistry, University of Washington, Seattle, WA. Chemical ionization mass spectra (CI-MS) were also recorded on a VG Platform single quadrupole mass spectrometer at the National Center for Toxicological Research, Jefferson, AR. Synthetic Procedures. 4-Hydroxyphenylacetaldehyde (8). 4-Hydroxyphenylacetaldehyde was prepared previously (22). 4-Hydroxybenzylethyl alcohol (1.1 g, 8 mmol) was placed in 20 mL of Me2SO, and 11.1 mL of triethylamine (TEA) (80 mmol, 10 equiv) was added to the alcohol-Me2SO solution. A solution of sulfur trioxide-pyridine complex (3.82 g, 24 mmol, 3 equiv) in 20 mL of Me2SO was added to the reaction flask,

Scheme 2. Overall N-Oxygenation and C-Oxidation of Tyramine

HO

OH

OH

N

NHOH

NH2

HO

HO 4

3b: trans

2

OH

OH

HO

HO 1

HO

N

NHOH

NH2

6

5

O C HO 7

H

OH

844 Chem. Res. Toxicol., Vol. 10, No. 8, 1997 and the reaction temperature was maintained at 25 °C. After stirring at room temperature for 30 min, the reaction was judged to be complete by monitoring with TLC (silica gel, ethyl acetate/ hexane, 25:75, v:v) (16). HCl (10%) was added to the reaction flask at 0 °C to acidify the reaction mixture (i.e., pH 3) and was extracted with ethyl acetate (30 mL × 3) to provide the crude aldehyde product. The combined organic layers were washed with H2O (40 mL × 3) in order to remove Me2SO, and the organic fraction was dried over sodium sulfate. After filtration, the solvent was concentrated to give a brown oil. The pure aldehyde 8 (631 mg) was obtained after flash chromatography (silica gel, ethyl acetate/hexane, 25:75, v:v) as a light yellow oil. Yield: 58%. Rf ) 0.31 (silica gel, ethyl acetate/hexane, 25:75, v:v). 1H NMR (in CDCl3): δ 3.64 (s, 2H), 6.92 (d, J ) 8.4 Hz, 2H), 7.11 (d, J ) 8.4 Hz, 2H), 7.75 (s, 1H), 9.76 (s, 1H). MS (FAB): 137 (MH+), 121 (MH+ - OH), 107 (M+ - CHO). cis- and trans-4-Hydroxyphenethyl Oximes (3a,b). Cisand trans-4-hydroxyphenethyl oximes have been synthesized previously (23). 4-Hydroxylphenylacetaldehyde (8) (700 mg, 5.15 mmol) was dissolved in 15 mL of THF. Hydroxylamine hydrochloride (537 mg, 7.73 mmol, 1.5 equiv) was placed in 7.5 mL of H2O and added to a stirred solution of aldehyde 8. Sodium carbonate (1.37 g, 12.9 mmol, 2.5 equiv) was also dissolved in an additional 7.5 mL of H2O and added to the reaction mixture. The reaction was completed in 1 h as determined by TLC (silica gel, ethyl acetate/hexane, 30:70, v:v). The reaction mixture was acidified with 10% HCl to pH 3, and then the organic and aqueous layers were separated by partitioning between ethyl acetate (20 mL) and H2O (10 mL). The aqueous layer was extracted twice with ethyl acetate (15 mL), and the combined organic layers were washed with water and brine and dried over sodium sulfate. After filtration, the solvent was concentrated to give a yellow oil. The mixture of cis and trans isomers (3a,b) was purified by flash chromatography (silica gel, ethyl acetate/hexane, 30:70, v:v). However, the cis and trans oxime isomers were not separable by flash chromatography. Upon recrystallization with chloroform and hexane at -20 °C, the cis isomer crystallized out in about 85% purity. Repeated recrystallization gave quite pure cis oxime. The trans oxime was present in the mother liquor in approximately 90% purity. cis- and trans-4-Hydroxyphenethyl oximes (3a,b). Yield: 77%. Rf ) 0.16 (silica gel, ethyl acetate/hexane, 25:75, v:v). MS (FAB): 151 (M+), 133 (M+ - OH), 107 (M+ - OHCN). cis-4-Hydroxylphenethyl oxime (3a). 1H NMR (in acetone-d6): δ 3.72 (d, J ) 5.4 Hz, 2H), 6.86 (t, J ) 6.6 Hz, 1H), 6.88 (d, J ) 8.1 Hz, 2H), 7.20 (d, J ) 8.2 Hz, 2H). trans-4-Hydroxylphenethyl oxime (3b). 1H NMR (in acetone-d6): δ 3.51 (d, J ) 6.2 Hz, 2H), 7.53 (t, J ) 6.5 Hz, 1H), 6.88 (d, J ) 8.1 Hz, 2H), 7.20 (d, J ) 8.2 Hz, 2H). (4-Hydroxyphenethyl)hydroxylamine (2). The oximes (a mixture of 3a,b) (200 mg, 1.37 mmol) were dissolved in 30 mL of methanol with bromophenol blue (137 µL of 1 mg/mL solution, 0.1 equiv) as an indicator to prevent overoxidation. Sodium cyanoborohydride (1 M in THF, 0.90 mL, 0.88 mmol, 0.67 equiv) was added to the reaction mixture, and HCl (10%) was added dropwise as needed to maintain an acidic pH. The resulting reaction mixture was stirred for another 30 min at room temperature, and TLC (silica gel, 0.2% TEA, methanol/dichloromethane, 10:90, v:v) indicated the reaction was complete. The reaction mixture was treated with saturated NaHCO3 and extracted twice with ethyl acetate. The organic layers were combined, washed with brine, dried over sodium sulfate, and concentrated in vacuo to give a solid. The pure product 2 was obtained after chromatography (silica gel, 0.2% TEA, methanol/ dichloromethane, 10:90, v:v) that gave 52.6 mg of a yellow oil. Yield: 44%. Rf ) 0.08 (silica gel, 0.2% TEA, methanol/ dichloromethane, 10:90, v:v). MS (FAB): 154 (MH+), 121 (M+ - NHOH), 91 (M+ - NHOH - CH2 - OH). 1H NMR (in CD3OD): δ 2.89 (t, J ) 7.0 Hz, 2H), 3.17 (t, J ) 7.1 Hz, 2H), 6.84 (t, J ) 8.5 Hz, 2H), 7.17 (d, J ) 8.4 Hz, 2H). Liver and Microsome Preparations. Adult human liver microsomes were a generous gift of Dr. Andrew Parkinson of Xenotech, Inc. (Kansas City, KS), and they were fully character-

Lin and Cashman ized for P450 activity as previously described (24). The human FMO3 enzyme has likewise been fully characterized by a method previously described (25, 26), and the specific activity of the pooled human liver microsomes used was 184 pmol of 10-[(N,Ndimethylamino)pentyl]-2-(trifluoromethyl)phenothiazine N-oxide and 2543 pmol of methyltoluyl S-oxide formed min-1 (mg of protein)-1. In good agreement to what has been observed with other human liver microsome preparations, the N-oxygenase activity correlated very well with FMO3 immunoreactivity. Metabolic Incubation Systems. A typical incubation mixture contained 50 mM potassium phosphate buffer, pH 8 or 9, 0.4 mM NADP+, 0.4 mM glucose-6-phosphate, 1 IU of glucose6-phosphate dehydrogenase, 50-700 µg of cDNA-expressed human liver FMO3-maltose-binding fusion protein (FMO3MBP), 0.1-0.3 mg of adult human liver microsomes or 0.551.5 mg of pig liver microsomes, and 1.2 mM diethylenetriaminepentaacetic acid (DETAPAC) (final volume 0.25 mL). A pH value of 8 (microsomes) or 9 (FMO3-MBP) was used because it was found to be optimal for both microsomal FMO and P450 activity or purified FMO3-MBP, respectively. The reaction was initiated by the addition of substrate and incubated at 37 °C with constant shaking. At various time intervals, the incubations were stopped by the addition of 6 volumes of cold dichloromethane/2-propanol, 2:1, v:v). After saturation with sodium chloride and a brief centrifugation, the organic layer was separated from the aqueous phase, and the organic fraction was evaporated and then analyzed for products by the HPLC procedure described below. The profile of tyramine metabolites was determined by HPLC analysis of organic extracts from the incubation mixture. The metabolic products were separated and quantified by a Hitachi L-6200A HPLC interfaced to a Hitachi D-2500 chromatointegrator with a Hitachi L-4000H UV detector set at 278 nm. The system was fitted with an analytical column (25 cm × 0.4 cm) from Rainin (Emeryville, CA). The mobile phase consisted of an isocratic system set at 85% A and 15% B, where A was water and B was acetonitrile containing 0.1% HClO4 (60% solution) at a flow rate of 1.5 mL/min. This system efficiently separated tyramine, (4-hydroxyphenethyl)hydroxylamine, cisand trans-4-hydroxyphenethyl oximes, and 4-hydroxybenzaldehyde that had retention times of 3.42, 4.29, 6.54, 7.85, and 8.12 min, respectively. Metabolites were quantified by comparing the metabolite and substrate peak areas of the chromatogram after accounting for the molar extinction coefficients of each metabolite as expressed in the following equations: percent conversion to oxime 3 ) area of oxime/(area of oxime + area of tyramine + area of 4-hydroxybenzaldehyde/8.78); percent conversion to 4-hydroxybenzaldehyde ) (area of 4-hydroxybenzaldehyde/8.78)/(area of oxime + area of tyramine + area of 4-hydroxybenzaldehyde/8.78); if no 4-hydroxybenzaldehyde formed, percent conversion to oxime 3 ) area of oxime/(area of oxime + area of tyramine or (4-hydroxyphenethyl)hydroxylamine). The efficiency of extraction of metabolites from the incubations was greater than 80%. Mass Spectrometry. Tyramine was incubated with pig or human liver microsomes and NADPH as described above in a preparative amount (10 incubations), and the reaction mixtures were extracted as described above. Extracts of the metabolites from preparative runs were pooled and injected directly into a NovaPak HPLC column for on-line LC/MS analysis. LC/MS experiments were done using a VG Platform single quadrupole mass spectrometer (Micromass, Altrincham, U.K.) equipped with an atmospheric pressure chemical ionization (APCI) interface. The LC/MS experiment was done under conditions described previously (27). Positive ions were acquired in full scan mode (m/z 100-750, 2.1 s cycle time) in series with a UV detector set at 275 nm. Background-subtracted mass spectra were obtained by averaging spectra across the respective chromatographic peak and subtracting average background spectra immediately before and after the HPLC peak of interest. Pharmacological Studies. HA7 cells were grown to confluence in Dulbecco’s modified Eagles media (DMEM) containing 10% fetal calf serum, 0.05% penicillin-streptomycin, and 400

N-Oxygenation of Tyramine by FMO

Chem. Res. Toxicol., Vol. 10, No. 8, 1997 845

Scheme 3. Synthetic Pathway Used To Obtain Compounds 2 and 3 OH

SO3•pyridine TEA/DMSO

HO

CHO

ratio of cis:trans oxime 3

HO

description

8 NH2OH•HCl/ Na2CO3

H2O/ THF

N

NHOH

OH

Na(CN)BH3 10% HCl/MeOH

HO

HO 3a: cis 3b: trans

2

Table 1. Isomerization of cis- and trans-4-Hydroxyphenethyl Oximes (3a,b) at Different pH Values in the Presence and Absence of Hepatic Enzymes

µg/mL G418. The cells were scraped from 100 × 20 mm plates and centrifuged at 500g for 5 min. The pellet was homogenized at 2 plates/mL in 50 mM Tris-HCl (pH 7.7) with a Polytron homogenizer as described before (28). For the serotonin 5-HT1A receptor, the competitive assay was done with [3H]-8-OH-DPAT. 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 then counted with scintillation counting. For the 5-HT1C receptor, NIH-3T3-PΦ cells were grown and prepared in the same manner as HA7 cells. [3H]Mesulergine was used to measure competitive binding. For the 5-HT2A 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 dopamine D1 receptor, LHD1 cells were grown as described above and competitive binding was determined with [3H]SCH 23390. For D2 and D3 receptors, CHOp-D2 and CHOp-D3 cells, respectively, were grown as described and [3H]chlorpromazine was used in competitive binding experiments. For dopamine transporter binding studies, C6 hDAT cell membranes were prepared as described previously (29) and competitive binding assays were done with [125I]RTI-55.

Results Chemical Synthesis and Stability Studies. The synthesis of (4-hydroxyphenethyl)hydroxylamine (compound 2) and cis- and trans-4-hydroxyphenethyl oximes (3a,b) was efficiently done to provide sufficient material for identification of tyramine metabolites and to examine the chemical stability of these metabolites during metabolic incubations and HPLC analysis. The synthesis of oxime 3 has been reported previously (23, 30). Sufficient material was synthesized for testing with a biogenic amine transporter and selected receptors. Treatment of 4-hydroxyphenylacetaldehyde (8) with hydoxylamine hydrochloride in the presence of base produced a mixture of cis and trans oximes (Scheme 3). Reduction of the oximes to the desired hydroxylamine was done in the presence of Na(CN)BH3 and 10% HCl as described before (15). Fractional recrystallization of the mixture of oximes 3a,b gave highly enriched individual isomers of oximes that could be used as authentic standards. Each compound was characterized spectroscopically, and an HPLC system was set up to separate the oximes 3a,b from hydroxylamine 2 and tyramine (1). As a prelude to the metabolic studies, the chemical stability of hydroxylamine 2 and oximes 3a,b to hydrolysis and autoxidation was determined. Preliminary studies indicated that the primary amine 1, the hydroxylamine 2, and the oximes 3 were completely stable to hydrolysis and autoxidation under the conditions of the metabolic incubations and bioanalysis that were utilized. The oximes 3a,b were also examined for possible isomer-

-enzyme, -NADPH (pH ) 7)a -enzyme, -NADPH (pH ) 8) -enzyme, -NADPH (pH ) 9) +PL microsomes, +NADPH (pH ) 8)b +PL microsomes, -NADPH (pH ) 8) +human FMO3, +NADPH (pH ) 9)c +human FMO3, -NADPH (pH ) 9) -enzyme, -NADPH (pH ) 7) -enzyme, -NADPH (pH ) 8) -enzyme, -NADPH (pH ) 9) +PL microsomes, +NADPH (pH ) 8) +PL microsomes, -NADPH (pH ) 8) +human FMO3, +NADPH (pH ) 9) +human FMO3, -NADPH (pH ) 9)

before incubation

after incubation

80:20

72:28 (8%)d 74:26 (6%) 74:26 (6%) 46:54 (34%) 48:52 (32%) 34:66 (46%) 43:57 (37%) 29:71 (1%) 28:72 (2%) 31:69 (1%) 26:74 (4%) 25:75 (5%) 27:73 (3%) 20:80 (10%)

30:70

a The incubation contained 1.2 mM substrate (oxime 3) and 1.2 mM DETAPAC in potassium phosphate buffer at pH 7, 8, or 9, respectively. Incubations were run for 10 min at 37 °C. b The incubation system contained 1.2 mM substrate (oxime 3), (0.4 mM NADP+, 0.4 mM glucose-6-phosphate, 1 IU of glucose-6-phosphate dehydrogenase, 1.2 mM DETAPAC, and 1.1 mg of pig liver (PL) microsomes in potassium phosphate buffer, pH 8. The incubation time was 10 min at 37 °C. c The incubation system was as described above except 0.14 mg of cDNA-expressed human Lys158 FMO3-MBP (human FMO3) was used as the enzyme in potassium phosphate buffer, pH 9. d The percentage change from cis to trans oxime after incubation at the described condition is provided in the parentheses.

ization during the experimental conditions employed. Pig liver microsomes or cDNA-expressed FMO3-MBP provided a convenient source of hepatic FMO1 and P450 activity or FMO3 activity, respectively. The cis oxime 3a did undergo a minor but detectable amount of isomerization to the trans oxime 3b at pH 8 or 9 (Table 1). Thus, at pH 7, 8, or 9, in the absence of enzyme or NADPH, a 80:20 ratio of cis:trans oxime 3 (as determined by 1H NMR and HPLC) isomerized to a 73:27 ratio of 3a:b, on average, for about a 7% increase in trans oxime. In the presence of pig liver microsomes or cDNAexpressed human FMO3, the percent isomerization from cis to trans oxime 3 was 33% and 41%, respectively, on average, and the percent isomerization was not markedly dependent on NADPH. The experiment was repeated for a complementary set of oximes 3 (i.e., 30:70 ratio of cis: trans oxime 3). In the presence of excess trans oxime, an extremely small amout of cis oxime isomerized to the trans oxime at pH 7, 8, or 9 in the absence of protein or NADPH (Table 1). In agreement with the observations described above, in the presence of pig liver microsomes or cDNA-expressed human FMO3, the percent isomerization of cis to trans oxime was 5% and as much as 10%, respectively. In the presence of cDNA-expressed FMO3, addition of NADPH appeared to decrease the extent of isomerization. The results suggest that the oximes 3 were stable to hydrolysis or autoxidation during the time required for metabolic incubation or HPLC analysis but that the determination of stereoselectivity probably slightly over-reported the amount of trans oxime formed. However, because cis oxime formation could not be detected during the metabolic studies described herein, the data suggested that very high product formation stereoselectivity was observed. Metabolism of Tyramine and (4-Hydroxyphenethyl)hydroxylamine in the Presence of Pig and

846 Chem. Res. Toxicol., Vol. 10, No. 8, 1997

Lin and Cashman

Table 2. Formation of Trans Oxime (3b) and 4-Hydroxybenzaldehyde (7) from Tyramine in the Presence of Pig and Human Liver Microsomes microsome preparation pig liver microsomes human liver microsomes

conditions completea -NADPH -protein complete -NADPH -protein

product ( SDb (nmol min-1 (mg of protein)-1) trans oxime, 3b 4-hydroxybenzaldehyde, 7 0.55 ( 0.05 0.13 ( 0.03 NDc 3.51 ( 0.62 0.23 ( 0.03 ND

0.91 ( 0.08 1.06 ( 0.10 ND 7.47 ( 1.08 7.14 ( 0.79 ND

a The complete system contained 1.2 mM tyramine, 0.4 mM NADP+, 0.4 mM glucose-6-phosphate, 1 IU of glucose-6-phosphate dehydrogenase, 1.2 mM DETAPAC, and 1.38 mg of pig or 0.3 mg of adult human liver microsomes in potassium phosphate buffer, pH 8. The incubation time was 15 min for pig enzyme and 10 min for human enzyme at 37 °C. b Data are expressed in mean ( standard deviation and are the mean of three determinations. c ND: nondetectable. Limit of detection was 20 pmol min-1 (mg of protein)-1.

Table 3. Formation of Trans Oxime (3b) and Tyramine from (4-Hydroxyphenethyl)hydroxylamine (2) in the Presence of Pig and Human Liver Microsomes products ( SDb (nmol min-1 (mg of protein)-1) conditiona

tyramine, 1

trans-oxime, 3b

pig liver microsomes

completea

human liver microsomes

-NADPH -protein complete -NADPH -protein

2.04 ( 0.22 NDc ND 2.40 ( 0.11 1.10 ( 0.02 ND

5.03 ( 0.42 0.02 ( 0.003 ND 10.0 ( 1.38 1.34 ( 0.04 ND

microsome preparation

a The complete system was as described in Table 2 except it contained 1.2 mM hydroxylamine 2 and 0.55 mg of pig liver or 0.15 mg of adult human liver microsomes. b The data are expressed as mean ( standard deviation and are the mean of three determinations. c ND: nondetectable. Limit of detection was 20 pmol min-1 (mg of protein)-1.

Human Liver Microsomes. Pig liver microsomes and human liver microsomes provided a convenient source of FMO1 and FMO3, respectively. In the presence of either pig or human liver microsomes, formation of hydroxylamine 2 was not observed, but rather, exclusive formation of trans oxime 3b was seen. In the presence of tyramine, oxime 3b formation was a linear function of time (i.e., 0-20 and 0-15 min) and protein concentration (i.e., 0-2.5 and 0-1 mg of protein) for pig and human liver microsomes, respectively. In the presence of pig and human liver microsomes, trans oxime 3b formation was somewhat and absolutely dependent on NADPH and active protein, respectively (Table 2). In addition to tyramine N-oxygenation, apparent C-oxidation was also observed. Thus, formation of 4-hydroxybenzaldehyde, compound 7, was observed in the presence of pig and human liver microsomes (Scheme 2). Formation of 4-hydroxybenzaldehyde was linearly dependent upon time (i.e., 0-15 min) and protein concentration (i.e., 0-1 mg of protein) for both pig and human liver microsomes. In the absence of active microsomal protein, formation of aldehyde 7 was completely abrogated (Table 2). In the presence of pig or human liver microsomes, formation of 4-hydroxybenzaldehyde from tyramine was not dependent on NADPH. Because hydroxylamine 2 was not observed formed from tyramine, although we anticipated that it was an obligatory intermediate in the sequential multistep formation of oxime 3b, the metabolism of hydroxylamine 2 was examined separately. Incubation of (4-hydroxyphenethyl)hydroxylamine with pig or human liver microsomes produced only two major products: the N-oxygenation product trans oxime 3b and the retroreduction product tyramine. The C-oxidation product 4-hydroxybenzaldehyde, 7, was not observed formed from hydroxylamine 2 in the presence of either pig or human liver microsomes. Formation of trans oxime 3b and retroreduction to tyramine (1) from hydroxylamine 2 was linearly dependent on time (i.e., 0-10

and 0-20 min, respectively) and protein concentration (i.e., 0-0.8 mg of protein) for pig liver microsomes. Likewise, in the presence of human liver microsomes, formation of oxime 3b and tyramine (1) from hydroxylamine 2 was linearly dependent on time (i.e., 0-15 and 0-20 min, respectively) and protein concentration (i.e., 0-0.2 mg of protein). In the presence of pig liver microsomes, formation of oxime 3b and tyramine (1) from hydroxylamine 2 was almost completely dependent on NADPH and active protein (Table 3). In the presence of human liver microsomes, formation of oxime 3b and tyramine (1) from hydroxylamine 2 was completely dependent on active microsomal protein but was not absolutely dependent on NADPH. Metabolite Structure Characterization. Tyramine metabolites were characterized using on-line LC-APCI/ MS. In the presence of preparative runs of pig or human liver microsomes, two prominent products were observed. Molecular species (i.e., MH+) were observed for 4-hydroxystyrene at m/z 121, as was the fragment ion due to loss of 14 amu (-CH2). The presence of m/z 121 presumably arises from the elimination of the oxime to 4-hydroxystyrene under the conditions of the LC-APCI/ MS experiment. Mass spectral studies of related reactions have been observed previously for phenyl propanaldoximes (31). Molecular ions (i.e., MH+) were observed for 4-hydroxybenzaldehyde at m/z 123. 4-Hydroxybenzaldehyde does not arise from mass spectral rearrangements but, rather, from elimination of HCN and water from proposed metabolite 6 (Scheme 2). Effect of Incubation Conditions on Oxime 3b and 4-Hydroxybenzaldehyde (7) Formation from Tyramine. Different incubation conditions were utilized to identify which, if any, monooxygenase system was responsible for formation of the C-oxidation and N-oxygenation products of tyramine. In the presence of pig liver microsomes, formation of oxime 3b from tyramine was completely inhibited by n-octylamine, a compound that

N-Oxygenation of Tyramine by FMO

Chem. Res. Toxicol., Vol. 10, No. 8, 1997 847

Table 4. Formation of Trans Oxime (3b) and 4-Hydroxybenzaldehyde (7) from Tyramine in the Presence of Pig Liver Microsomes products ( SDb (nmol min-1 (mg of protein)-1) description

trans oxime, 3b

4-hydroxy-benzaldehyde, 7

+n-octylamine (5.0 mM) +thiourea (5.0 mM) +thiobenzamide (5.0 mM) +heat inactivation (+10 units of catalase)

0.55 ( 0.05 NDc (100%)d 0.55 ( 0.09 (0%) 0.13 ( 0.01 (76%) 0.34 ( 0.05 (38%)

0.91 ( 0.08 0.11 ( 0.01 (88%) 0.51 ( 0.03 (44%) 0.37 ( 0.01 (59%) 0.85 ( 0.10 (7%)

completea

a The complete system was as described in Table 2 except that it contained 1.2 mM tyramine and 0.57 mg of pig liver microsomes. Data are expressed as mean ( standard deviation and are the mean of three determinations. cND: nondetectable. Limit of detection was 20 pmol min-1 (mg of protein)-1. d The percent inhibition is provided in the parentheses.

b

Table 5. Formation of Trans Oxime (3b) and 4-Hydroxybenzaldehyde (7) from Tyramine in the Presence of Human Liver Microsomes products ( SDb (nmol min-1 (mg of protein)-1) description

trans oxime, 3b

4-hydroxybenzaldehyde, 7

completea +n-octylamine (5.0 mM) +thiourea (5.0 mM) +thiobenzamide (5.0 mM) +heat inactivation (+10 units of catalase)

3.51 ( 0.62 NDc (100%)d 4.45 ( 0.51 (-) ND (100%) 1.86 ( 0.20 (47%)

7.47 ( 1.08 ND (100%) 4.39 ( 0.06 (41%) 4.72 ( 0.38 (37%) 4.64 ( 0.64 (38%)

a The complete system was as described in Table 2 except that it contained 1.2 mM tyramine and 0.3 mg of adult human liver microsomes. Data are expressed as mean ( standard deviation and are the mean of three determinations. c ND: nondetectable. Limit of detection was 20 pmol min-1 (mg of protein)-1. d The percent inhibition is provided in the parentheses.

b

inhibits P450 (32) and FMO3 (15, 16) or oftentimes stimulates FMO1 enzyme activity (33). Thiourea and thiobenzamide, two well-documented alternative substrate competitive inhibitors of FMO (33, 34), showed very disparate effects: thiobenzamide significantly inhibited oxime 3b formation, but thiourea did not inhibit oxime formation at all. Heat inactivation of pig liver microsomes under conditions that preserved 85% P450 activity and inactivated FMO to about 15% of the initial activity (35) gave 38% residual oxime 3b formation activity (Table 4). In the presence of pig liver microsomes, formation of 4-hydroxybenzaldehyde was inhibited 88% by n-octylamine. Thiourea and thiobenzamide both had a significant inhibitory effect, inhibiting 4-hydroxybenzaldehyde formation 44% and 59%, respectively. Under conditions of heat inactivation that generally abolished pig FMO1 activity and preserved most P450 activity, 4-hydroxybenzaldehyde formation was only inhibited 7% (Table 4). As shown in Table 5, in the presence of human liver microsomes, oxime 3b formation was completely inhibited by n-octylamine and thiobenzamide. Thiourea did not inhibit oxime 3b formation in the presence of human liver microsomes. Under heat inactivation conditions that abolished human FMO3 activity and preserved most P450 activity, 53% of the oxime 3b formation activity was observed (Table 5). In the presence of human liver microsomes, formation of 4-hydroxybenzaldehyde from tyramine was completely abrogated in the presence of n-octylamine. Thiourea and thiobenzamide significantly inhibited 4-hydroxybenzaldehyde formation 41% and 37%, respectively. Heat inactivation of human liver microsomes under conditions that preserved P450 activity and abolished FMO activity caused a 38% loss of 4-hydroxybenzaldehyde formation activity from tyramine (Table 5). Metabolic Studies with (4-Hydroxyphenethyl)hydroxylamine, 2. Because we anticipated that (4hydroxyphenethyl)hydroxylamine was a required intermediate in the conversion of tyramine to its corresponding trans oxime 3b and because formation of hydroxylamine 2 was not observed in any incubations done with tyramine,

Table 6. N-Oxygenation and Retroreduction of (4-Hydroxyphenethyl)hydroxylamine (2) in the Presence of Pig Liver Microsomes products ( SDb (nmol min-1 (mg of protein)-1) description completea +n-octylamine (5.0 mM) +thiourea (5.0 mM) +thiobenzamide (5.0 mM) +heat inactivation (+10 units of catalase)

tyramine, 1

trans oxime, 3b

2.04 ( 0.22 NDc (100%)d 2.08 ( 0.11 (-) 1.41 ( 0.35 (31%) 1.42 ( 0.16 (30%)

5.03 ( 0.42 ND (100%) 2.73 ( 0.44 (46%) 2.89 ( 0.29 (43%) 2.02 ( 0.33 (60%)

a The complete system was as described in Table 2 except that it contained 1.2 mM hydroxylamine 2 and 0.55 mg of pig liver microsomes. b Data are expressed as mean ( standard deviation and are the mean of three determinations. c ND: nondetectable. Limit of detection was 20 pmol min-1 (mg of protein)-1. d The percent inhibition is provided in the parentheses.

we examined the microsomal metabolism of (4-hydroxyphenethyl)hydroxylamine to investigate whether it by itself could be converted to the trans oxime 3b. In the presence of pig liver microsomes, two products were formed from hydroxylamine 2: the retroreduction product tyramine and the trans oxime 3b arising from an apparent N-oxygenation reaction. It is notable that the 4-hydroxybenzaldehyde metabolite was not observed formed from the hydroxylamine 2 in the presence of pig liver microsomes. In the presence of pig liver microsomes, formation of oxime 3b was completely inhibited by n-octylamine (Table 6). Thiourea and thiobenzamide, two alternative substrate competitive inhibitors of pig FMO1, inhibited oxime 3b formation 46% and 43%, respectively. Heat inactivation under conditions that generally inactivated about 85% pig FMO1 activity showed almost a 60% inhibition of oxime 3b formation. To identify the monooxygenase system responsible for retroreduction of hydroxylamine 2 to tyramine, we examined the influence of metabolic incubation conditions (Table 6). Pig liver microsome retroreduction of hydroxylamine 2 to tyramine was completely inhibited in the presence of n-octylamine. Thiourea was not active as an inhibitor of retroreduction of hydroxylamine 2, and

848 Chem. Res. Toxicol., Vol. 10, No. 8, 1997 Table 7. N-Oxygenation and Retroreduction of (4-Hydroxyphenethyl)hydroxylamine (2) in the Presence of Human Liver Microsomes products ( SDb (nmol min-1 (mg of protein)-1) description

tyramine, 1

trans oxime, 3b

completea +n-octylamine (5.0 mM) +thiourea (5.0 mM) +thiobenzamide (5.0 mM) +heat inactivation (+10 units of catalase)

2.40 ( 0.11 NDc (100%)d 2.68 ( 0.50 (-) 2.72 ( 0.32 (-) 2.20 ( 0.30 (8%)

10.0 ( 1.38 ND (100%) 4.76 ( 0.41 (52%) 4.24 ( 0.44 (58%) 4.50 ( 0.50 (55%)

a The complete system was as described in Table 2 except that it contained 1.2 mM hydroxylamine 2 and 0.15 mg of adult human liver microsomes. b Data are expressed as mean ( standard deviation and are the mean of three determinations. c ND: nondetectable. Limit of detection was 20 pmol min-1 (mg of protein)-1. d The percent inhibition is provided in the parentheses.

thiobenzamide was only modestly active, inhibiting only 30% tyramine formation from hydroxylamine 2. Heat inactivation of pig liver microsomes under conditions that preserved 85% P450 activity inhibited retroreduction about 30%. We next investigated the effect of metabolic incubation conditions on the formation of trans oxime 3b from hydroxylamine 2 in the presence of adult human liver microsomes. In good agreement with results of incubation of hydroxylamine 2 with pig liver microsomes, in the presence of human liver microsomes, no detectable formation of 4-hydroxybenzaldehyde (7) was observed. Two products were observed formed from hydroxylamine 2 in the presence of human liver microsomes: the trans oxime 3b arising from N-oxygenation and the apparent retroreduction product tyramine. As shown in Table 7, formation of trans oxime 3b was completely inhibited in the presence of n-octylamine. The two FMO alternative competitive substrates, thiourea and thiobenzamide, significantly inhibited oxime formation 52% and 58%, respectively. In the presence of heat inactivation under conditions that decreased FMO activity to about 15% of complete values, approximately 55% loss of oxime 3b formation was observed. In the presence of human liver microsomes, hydroxylamine 2 was retroreduced to tyramine (Table 7). nOctylamine completely abrogated formation of tyramine from hydroxylamine 2 in the presence of human liver microsomes. The FMO alternative substrate competitive inhibitors thiourea and thiobenzamide did not have any inhibitory effect on the formation of tyramine from hydroxylamine 2 in the presence of human liver microsomes. Likewise, heat inactivation under conditions that preserved about 85% P450 activity caused only an 8% decrease in the retroreductive formation of tyramine from hydroxylamine 2 in the presence of human liver microsomes. Role of Highly Purified Human FMO3 Enzymes in Tyramine and (4-Hydroxyphenethyl)hydroxylamine N-Oxygenation. To examine the role of human FMO3 enzymes in the N-oxygenation of tyramine, formation of oxime 3b was studied in the presence of two prominent forms of cDNA-expressed human FMO3.2 We utilized a maltose-binding fusion protein of FMO (i.e., 2 Restriction length polymorphism and oligonucleotide sequencing studies showed human FMO3 codon 158 encoded either amino acid Glu or Lys at approximately equal allele frequencies for the Caucasian populations examined (E. Treacy, R. Youil, S. Forest, and M. Knight, personal communication).

Lin and Cashman

FMO3-MBP) because it possessed many superior properties to the nonfusion FMO3 enzyme (37). In good agreement with the results of microsome studies, the only oxime product observed was the trans oxime, isomer 3b. In addition, no formation of 4-hydroxybenzaldehyde (7) was observed formed from tyramine or the hydroxylamine 2, and no retroreduction product was formed from hydroxylamine 2 in the presence of either human FMO3MBP enzyme. In the presence of either of the FMO3MBP enzymes, formation of oxime 3b from tyramine was linearly dependent on time (i.e., 0-15 min) and protein concentration (i.e., 0-400 µg of protein). Preliminary studies showed that oxime 3b formation from tyramine was completely dependent on NADPH and active FMO3MBP protein (data not shown). Likewise, preliminary studies showed that oxime 3b formation from hydroxylamine 2 was completely dependent on NADPH and active FMO3 protein (data not shown). In the presence of either of the FMO3-MBP enzymes, formation of oxime 3b from hydroxylamine 2 was linearly dependent on time (i.e., 0-12 min) and protein concentration (i.e., 0-120 µg of protein). The substrate dependence for the N-oxygenation of tyramine and (4-hydroxyphenethyl)hydroxylamine (2) was examined in the presence of both FMO3-MBP enzymes. Plots of the reciprocal of the velocity of trans oxime 3b formation versus the reciprocal of the substrate concentration gave a series of linear correlations. From Lineweaver-Burk plots the Km,app and Vmax were obtained. As shown in Table 8, it appeared that the Km,app values for both tyramine and (4-hydroxyphenethyl)hydroxylamine (2) were increased for the Lys158 FMO3MBP enzyme relative to the values observed for the Glu158 FMO3-MBP enzyme. Effect of (4-Hydroxyphenethyl)hydroxylamine and Oxime 3 on Biogenic Amine Transporter and Receptor Affinity. The binding avidity of (4-hydroxyphenethyl)hydroxylamine (2) and oximes 3 (a racemic mixture of both the cis and trans isomers) for several biogenic amine receptors and the dopamine transporter was determined to test whether or not the tyramine metabolites reported herein possessed in vitro pharmacological activity. The dopamine receptors (i.e., human D1, D2, and D3), the serotonin receptors (i.e., human 5-HT1A, rat 5-HT1C, rat 5-HT2A, and guinea pig 5-HT3), and the dopamine transporter (i.e., human DAT) were investigated for competitive binding to well-recognized ligands. Appropriate control experiments with known ligands were run before examining 2 and 3 as antagonists. For example, the Ki value for cocaine displacement of [125I]RTI-55 binding from the human DAT was 229 nM. The IC50 or Ki values for both compounds 2 and 3 in the presence of the receptors and transporter studied showed very poor affinity in all cases examined with values greater than 10 µM. IC50 or Ki values in this range (i.e., 10 µM or greater) suggested that hydroxylamine 2 and oximes 3 do not possess any significant pharmacological activity for the transporter and receptors examined.

Discussion To examine the multistep sequential N and C atom hepatic microsomal metabolism of tyramine, efficient methods for the chemical synthesis and HPLC analysis of oxime, hydroxylamine, and benzaldehyde metabolites of tyramine were developed. In addition, sufficient material was obtained to study the in vitro pharmaco-

N-Oxygenation of Tyramine by FMO

Chem. Res. Toxicol., Vol. 10, No. 8, 1997 849

Table 8. N-Oxygenation of Tyramine and (4-Hydroxyphenethyl)hydroxylamine (2) in the Presence of cDNA-Expressed FMO3-MBP enzymes

Km,app (mM)a

Vmax (nmol min-1 (mg of protein)-1)

Vmax/Km,app

Lys158 FMO3-MBP Glu158 FMO3-MBP Lys158 FMO3-MBP Glu158 FMO3-MBP

0.95 0.22 0.89 0.49

0.89 0.60 9.88 23.7

1 3 11 48

substrates tyramine, 1 tyramine hydroxylamine, 2

a The kinetic constants were determined using the incubation system described in Table 1 except that it contained variable amounts of tyramine or its corresponding hydroxylamine 2 and 695 and 104 µg of human Lys158 FMO3-MBP for tyramine and its hydroxylamine 2, respectively; 350 and 50 µg of human Glu158 FMO3-MBP for tyramine and its hydroxylamine 2, respectively. The pH value for this experiment was in potassium phosphate buffer, pH 9.

Scheme 4. Proposed N-Oxygenation Mechanism for Formation of Oxime 3b NHOH

NH2 HO

HO 1

2 OH N H

+

OH HO

N

OH

H

HO 3b

logical activity of the human FMO3-related metabolites to explore the hypothesis that N-oxygenation is a detoxication pathway. The hydroxylamine (2) and oxime (3) metabolites of tyramine possessed sufficient chemical stability to characterize by ion-pair HPLC chromatography. Chemical synthesis of oxime 3 produced a racemic mixture, but in all cases examined, N-oxygenation of tyramine in the presence of enzyme preparations proceeded to give exclusively the trans oxime 3b. Previous studies have shown that phenethylamine forms exclusively the corresponding trans oxime in the presence of pig and human FMO (16). However, other studies have shown that the major product of aliphatic primary amine N-oxygenation is the oxime with the cis stereochemistry (15, 17). Undoubtedly, the stereoselectivity of oxime metabolite formation is a function of the chemical nature of the primary aliphatic amine substrate. We (15, 16) and others (17) have proposed that formation of primary aliphatic oximes proceeds from the hydroxylamine via the intermediacy of a di-N-hydroxylamine (Scheme 4). It is notable that even though the proposed symmetrical diN-hydroxylamine is unstable and dehydration is presumably spontaneous, exclusive formation of trans oxime 3b is observed. Previous results (15-17) could not provide evidence whether dehydration to produce oxime occurred before or after the di-N-hydroxylamine diffused from the surface of the enzyme. The results described in this report suggest that the di-N-hydroxylamine may be tightly associated with the FMO enzymes examined. Pig liver microsomes (as a source of P450 or FMO1) or cDNA-expressed human FMO3 facilitates the isomerization of cis oxime 3a to trans oxime 3b (Table 1). Pig liver microsomes appear to be less efficient at catalyzing the non-NADPH dependent isomerization of oxime 3a to 3b than human FMO3 (Table 1). During the metabolic incubation and analysis, cis oxime 3a f trans oxime 3b isomerization is a minor process and cannot account for

the absolute stereoselectivity observed in the enzyme preparations examined. That no cis oxime is detected in any of the incubations examined suggests that the intriguing non-NADPH dependent isomerization is a minor contributor to the product stereoselectivity observed and that if cis oxime 3a is formed we estimate that its formation is less than 2% of the overall oxime 3b formed. The data point to an interesting phenomenon that has been observed for the biosynthesis of other oximes from amino acids, for example, in the presence of microsomal preparations of seedlings of Sinapis alba L. or microsomes from oilseed rape (Brassica napus) (12, 31). As described above, it is possible that the flow of tyramine metabolites through a sequential series of intermediates that are tightly bound to FMO occurs by “catalytic facilitation”. This may account for the failure to observe any dissociable hydroxylamine 2 along the A f B f C reaction path leading to oxime 3b formation from tyramine. Nondissociation of metabolic intermediates from FMO is also supported by the observation that pig liver microsomes and cDNA-expressed FMO3 can serve as a template for the non-NADPH dependent isomerization of cis oxime to trans oxime 3b. Based on these observations, it is possible that FMO serves as a template for spontaneous dehydration of the unstable and fleeting di-N-hydroxylamine intermediate and facilitates the multistep formation of trans oxime 3b from the hydroxylamine 2 or tyramine. Metabolic studies of tyramine or (4-hydroxyphenethyl)hydroxylamine suggested that oxime 3b formation was largely dependent on FMO but a significant contribution from P450 was also possible. Thus, in the presence of pig or human liver microsomes, formation of oxime 3b from tyramine was largely dependent on NADPH and active protein. n-Octylamine completely abrogated Noxygenation of tyramine, and this may have been a consequence of inhibition of P450 and/or alternative substrate inhibition of FMO. In the presence of pig and human liver microsomes, thiourea and thiobenzamide showed mixed results suggesting that the agents themselves were acting as alternate substrate competitive inhibitors of FMO and/or that their S-oxide metabolites were inhibiting P450 (34, 36). The effect of heat inactivation of pig and human liver microsomes both showed 38% inhibition of oxime 3b formation from tyramine. We conclude that P450 may contribute upward of 40% to oxime 3b formation. A number of examples in the literature of P450-mediated oxime or amidoxime formation or degradation has been reported (38, 39), but this probably constitutes a minor pathway due to the inhibition of P450 by primary aliphatic amines (32). We (15, 16) and others (12, 17, 38, 40, 41) have observed that aliphatic primary amines, amidines, and related functional groups are converted to oximes and oxime-type functional groups by FMO or P450. Although

850 Chem. Res. Toxicol., Vol. 10, No. 8, 1997

we do not detect the hydroxylamine 2 in any of the enzyme incubations reported herein, it is undoubtedly an initial intermediate in the A f B f C reaction leading to trans oxime 3b. This may have toxicological consequences, because if the hydroxylamine is never free from the enzyme, then it poses minimal risk to be further converted to highly reactive materials that could impair cell function. Similar to tyramine, incubation of (4hydroxyphenethyl)hydroxylamine (2) with pig or human liver microsomes gave trans oxime 3b in a process probably dependent on both P450 and FMO. Formation of trans oxime 3b from (4-hydroxyphenethyl)hydroxylamine was completely and partially dependent on NADPH in the presence of pig and human liver microsomes, respectively. As observed for tyramine, n-octylamine completely inhibited trans oxime 3b formation from hydroxylamine 2 in the presence of pig or human liver microsomes. This is probably a result of n-octylaminemediated inhibition of both P450 and FMO. For both pig and human liver microsomes, formation of trans oxime 3b from hydroxylamine 2 was significantly inhibited by thiourea and thiobenzamide, probably as a consequence of alternative substrate competitive inhibition of FMO and S-oxide metabolite-mediated inhibition of P450 (34, 35). Results of heat inactivation of pig and human liver microsome preparations showed 55-60% inhibition of the conversion of hydroxylamine 2 to trans oxime 3b. Because heat inactivation generally decreases FMO3 activity only 80%, the net difference of 20-30% is consistent with P450 contributing as much as 20-30% to the formation of trans oxime 3b from hydroxylamine 2. The conclusion is that P450 apparently makes a significant contribution to trans oxime 3b formation from hydroxylamine 2 and tyramine. This may have toxicological significance because generation of hydroxylamines and nitroso metabolites from primary amines at the active site of P450 inactivates the hemoprotein (42, 43). While we did not specifically study a role of reactive oxygen species in the N-oxidation of hydroxylamine 2, it is clear that in the presence of pig liver microsomes, no evidence for the involvement of O2- or H2O2 was observed. This is based on the fact that no significant amount of the corresponding nitroxide was detected. Uncoupling of pig FMO1 to release O2- in the presence of hydroxylamine 2 to convert the hydroxylamine to the corresponding nitroxide has been observed by other investigators (21), but in our previous studies of phenethylhydroxylamine (16) or other aliphatic primary hydroxylamines (15), formation of the corresponding oxime was not dependent on the presence of O2- or H2O2. Our previous studies utilizing human liver microsomes has likewise shown that formation of the oxime from aliphatic primary hydroxylamines was independent of the presence of O2- or H2O2 (16). There is a possibility, however, because of the relatively high rate of non-NADPH dependent oxime 3b formation from hydroxylamine 2 in the presence of human liver microsomes, that as much as 10% of the product could arise through the intermediacy of O2- and/or H2O2 (Table 3). Tyramine appears to be a relatively better substrate for P450-mediated oxime formation than phenethylamine (16) or other long chain aliphatic primary amines (15). It is possible that this is due to the more readily oxidized nature of tyramine or to the fact that it is slightly less efficient as a substrate for FMO. To investigate this point, the Km,app and Vmax values for conversion of tyramine or hydroxylamine 2 to trans oxime 3b were

Lin and Cashman

examined. We investigated this question with two human FMO3 enzymes because restriction length polymorphism and oligonucleotide sequencing studies showed that codon 158 encoded either amino acid Glu or Lys at approximately equal allele frequencies for the Caucasian populations examined.2 While no significant polymorphism for N-oxygenation of tyramine by human FMO3 is apparent from this study, it is possible that human FMO3 contributes to the detoxication of other biogenic amines in a polymorphic fashion, and this may have consequences for human disease states. In the presence of pig or human liver microsomes, a second product arising from retroreduction of hydroxylamine 2 was observed. The retroreduction product of hydroxylamine 2 (i.e., tyramine) was not observed formed in the presence of highly purified FMO3. A number of reports have described a retroreduction system that reduces hydroxylamines to amines (44, 45), amidoximes to amidines (46), N-hydroxyguanidines to aminoguanidines (40), and N-hydroxyisothioureas to isothioureas (47). The enzymatic retroreduction system is apparently composed of cytochrome b5, cytochrome b5 reductase, and a noncharacterized P450 that has not been reported to date (40). Our previous attempts to correlate human P450 activity with retroreduction activity failed to show that any of the six most prominent activities cosegregated with retroreduction activity (15). In the presence of pig and human liver microsomes, the results of retroreduction of hydroxylamine 2 to tyramine are consistent with the previously described retroreduction enzyme system (44-47). Thus, retroreduction of hydroxylamine 2 to tyramine was dependent on NADPH (although human liver microsomes were considerably less dependent on NADPH than pig liver microsomes), completely inhibited by the P450 inhibitor n-octylamine, not inhibited by the FMO alternative substrate competitive inhibitors thiourea and thiobenzamide (although thiobenzamide was somewhat inhibitory to the presence of pig liver microsomes), and quite refractory to heat inactivation under conditions that preserve P450 activity. With the quantification of both N-oxygenation and retroreduction activities in the presence of pig and human liver microsomes, parallel and interdependent oxidative and retroreductive enzymatic processes are apparently in action for both tyramine and (4-hydroxyphenethyl)hydroxylamine metabolism. The results of metabolism studies of tyramine showed that in addition to formation of trans oxime 3b, 4-hydroxybenzaldehyde (7) was also formed in the presence of pig or human liver microsomes. That highly purified human FMO3 did not form 4-hydroxybenzaldehyde (7) from tyramine suggests that the product was not formed directly by FMO. It is notable that hydroxylamine 2 did not serve as a substrate for apparent benzylic hydroxylation of tyramine (Scheme 2) nor were metabolites 4-6 observed in detectable amounts in the microsome incubation mixture. 4-Hydroxybenzaldehyde (7) formation was absolutely dependent on active pig or human liver microsomal protein, independent of NADPH, and almost completely inhibited by n-octylamine. In the presence of either pig or human liver microsomes, 4-hydroxybenzaldehyde (7) formation was inhibited by thiourea and thiobenzamide, but heat inactivation only modestly (i.e., human liver microsomes) or almost not at all (i.e., pig liver microsomes) inhibited 4-hydroxybenzaldehyde formation. In view of the lack of dependence on NADPH and the effect of incubation conditions, we conclude that

N-Oxygenation of Tyramine by FMO

Chem. Res. Toxicol., Vol. 10, No. 8, 1997 851

Scheme 5. Postulated Intramolecular Rearrangement and Elimination Reactions That Produce 4-Hydroxybenzaldehyde from Compound 6 H O

OH N

H

N OH

H

HO

O

H

HO

6

O C H2O + HC

N+

H

HO 7

neither FMO nor P450 is responsible for benzylic hydroxylation of tyramine. Another possiblity is that the rate-determining step does not involve an NADPH dependent process. However, it is likely that a β-oxidase is responsible for initial hydroxylation (13) and that FMO and/or P450 contributes to formation of metabolites 5 and 6. It is possible that “catalytic facilitation” also channels the flow of 4 to 7 through a series of tightly bound intermediates that do not dissociate from the monooxygenase surface although we did not directly test this by examining 4 as a substrate. While a number of mechanisms could be drawn for the formation of 4-hydroxybenzaldehyde (7), it is possible that the trans oxime 3b is drawn through a cis oxime 3a equilibrium, and an intermediate involving a cyclic flow of electrons gives the observed 4-hydroxybenzaldehyde (7) product (Scheme 5). It is notable that treatment of 4 with periodate efficiently gives 4-hydroxybenzaldehyde (13). In view of the results presented here for FMO, it is possible that previous estimates of β-oxidase activity has been confounded by FMO-mediated oxime formation of octopamine. Although commonly cited as a detoxication catalyst (48), few examples have been reported of the detoxication of endogenous substrates of FMO probably because few endogenous compounds have been described as good substrates. Cysteamine (49, 50), methionine (51), and related sulfur-containing compounds (52) have been reported as endogenous substrates for FMO, but the physiological relevance to detoxication has not been exhaustively examined. Because tyramine and phenethylamine are biogenic amines with a wide range of potential pharmacological targets (1), we were able to investigate the hypothesis that the FMO-catalyzed Noxygenation of tyramine represents a detoxication reaction. The lack of affinity of cis or trans oxime 3 or (4hydroxyphenethyl)hydroxylamine (2) for any of the prominent biogenic amine receptors or dopamine transporter examined supports the idea that hepatic FMO catalyzes the N-oxygenation of tyramine and this enzyme activity abrogates physiological or pharmacological activity. It is unknown what role, if any, human brain FMO plays in the termination of pharmacological activity of biogenic amines. However, recent reports have suggested that FMO activity is present in human (53, 54) and animal (55, 56) brain preparations, and thus, FMO may play a fundamental role in converting pharmacologically active biogenic amines to inactive oxime metabolites. Regardless of the role of FMO in the central nervous

system, the results described herein support a prominent contribution for hepatic microsomal FMO activity in the metabolism of tyramine, and this may have consequences for the maintenance of cellular homeostasis. Abrogation of FMO activity or the presence of abnormal FMO activity may have important consequences for a number of disease states that are associated with abnormal biogenic amine metabolism.

Acknowledgment. This work was financially supported by NIH grants GM 36426 and DA 08531 and 00269. Receptor and transporter data were obtained through a collaboration with the National Institute on Drug Abuse Cocaine Treatment Discovery Program, contracts DA 38302 and 38303. The authors are grateful to our collaborators who supplied some of the biological materials essential for the study: Professor Daniel Ziegler, University of Texas at Austin (pig liver microsomes), and Dr. Andrew Parkinson of Xenotech, Inc., Kansas City, KS (adult human liver microsomes). We are grateful to Dr. Daniel Doerge, FDA Lab, NCTR, Jefferson, AR, who supplied the APCI mass spectrometric determinations for the oximes and 4-hydroxybenzaldehyde metabolites, compounds 3 and 7.

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