The relative participation of liver microsomal amine oxidase and cytochrome P-450 in N-demethylation reactions

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OF

BIOCHEMISTRY

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BIOPHYSICS

180,

363-373 (1977)

The Relative Participation of Liver Microsomal Cytochrome P-450 in N-Demethylation RUSSELL A. PROUGH

AND

DANIEL

Amine Oxidase Reactions’

and

M. ZIEGLER

Department of Biochemistry, The University of Texas Health Science Center at Dallas, Dallas, Texas 75235 and Clayton Foundation Biochemical Institute and the qepartment of Chemistry, The University of Texas at Austin, Austin, Texas 78712 Received August

23, 1976

N-Demethylation of benzphetamine and p-chloro-N-methylaniline measured in the presence and absence of specific antibodies to NADPH-cytochrome c (P-450) reductase demonstrates that part of the formaldehyde formed from the see-A’-methylamine arises from non-cytochromeP&O-dependent oxidation catalyzed by pig, hamster, and rat liver microsomes. The additional formaldehyde formed can be inhibited by adding methimazole, a non-formaldehyde-producing substrate specific for the microsomal mixed-function amine oxidase, to the reaction media. Purified amine oxidase catalyzes the oxidation of set-N-methylamines to set-A’-methylhydroxylamines that, upon oxidation and hydrolysis, yield formaldehyde. Approximately 65,40, and 15% of total formaldehyde is formed by this route during oxidation of p-chloro-N-methylaniline catalyzed by pig, hamster, and rat liver microsomes, respectively.

The metabolism of N-methylamines has been the subject of considerable interest due to the diverse pharmacological and toxicological properties of these compounds (l), and the influence of N-oxidation on the pharmacology of N-methylamines has been reviewed (23). Miller and Miller (4) have also shown that N-oxidation is prerequisite in the conversion of arylamines and arylamides to ultimate carcinogenic derivatives. The initial oxidative metabolism of a variety of nitrogenous compounds by hepatic tissue can be catalyzed by either of two membranebound monooxygenase systems: the mixed-function amine oxidase, a flavoprotein (5), or the cytochrome P-450-dependent oxidase (6, 7). The substrate specificities of these two microsomal monooxygenases have not been precisely defined, but, in general, as suggested by Gorrod (2), basic amines are preferred substrates for the amine oxidase and the less basic 1 Portions of of Texas Health ported, in part, BC-153 and The I-616.

work carried out at The University Science Center at Dallas were supby American Cancer Society Grant Robert A. Welch Foundation Grant

amines are preferred substrates for the cytochrome P-450 system. However, there are many exceptions and amine substrate specificities for these two microsomal monooxygenases based on PK, are only a very approximate guide. Which of these two monooxygenases catalyzes the initial oxidation of an amine substrate in hepatic microsomes can usually be defined by determining the type of metabolite formed, since the two monooxygenases catalyze the oxidation of set- and tert-amines to distinctly different products. It is generally assumed that cytochrome P&O-dependent oxidation of an amine occurs by oxidative attack on a carbon alpha to the nitrogen. The initial product, an unstable amino carbinol, decomposes rapidly to yield an aldehyde and dealkylated amine. This mechanism is strongly supported by the studies of McMahon and Sullivan (8) and McMahon et al. (91, who measured ‘*Oz incorporation into products formed during rat liver microsomal-catalyzed oxidative N-dealkylation of N-benzyl compounds. Oxygen atoms in benzaldehyde formed were derived from 1802and not from water. Due to rapid exchange of

363 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0003-9861

364

PROUGH

AND

the carbonyl oxygen of formaldehyde with oxygen of water, similar experiments with N-methylamines could not be carried out. However, it is reasonable to assume that cytochrome P-450-catalyzed N-dealkylation of N-methylamines occurs by the same mechanism. Thus, the oxidation of N-methyl- or N,N-dimethylamines by the cytochrome P-450 system would always yield formaldehyde and the dealkylated amine. On the other hand, the microsomal mixed-function amine oxidase catalyzes the oxidation of tertiary amines to amine oxides and secondary amines to N,N-disubstituted hydroxylamines (5). Trialkyl amine oxides are polar, relatively stable compounds, rarely metabolized further in hepatic tissue. However, NJ?-disubstituted hydroxylamines are fairly reactive compounds that do not differ substantially in physical properties from the parent secondary amine. The metabolites are easily oxidized, both enzymatically and nonenzymatically, to nitrones (10). Most nitrones are very unstable and hydrolyze immediately to the primary N-hydroxylamine and aldehyde. The NADPH- and oxygen-dependent oxidation of several N-methylhydroxylamines by hepatic microsomes has been demonstrated (ll), and with one compound, N-benzyl-N-methylhydroxylamine, it was shown that this reaction is catalyzed by the mixed-function amine oxidase to the expected nitrones -one of which hydrolyzes immediately to formaldehyde and benzylhydroxylamine (10). Therefore, oxidation of see-N-methylamines by the microsomal amine oxidase can also yield formaldehyde, a product generally assumed to arise only from cytochrome P450-dependent oxidation of amines. Although different routes for initial oxidative metabolism of pharmacologically important N-methylamines can be deduced from partial reactions described in the literature, the extent to which the two microsomal monooxygenases contribute to the formation of formaldehyde, the product frequently determined in studying Ndemethylation in uitro, has not been measured. This study was carried out to evaluate the relative amounts of formaldehyde formed in vitro during the oxidation of N-

ZIEGLER

methylamines catalyzed by rat, hamster, and pig liver microsomes formed via the cytochrome P-450-dependent oxidase and the amine oxidase. By the use of specific inhibitors for cytochrome P-450, a nonformaldehyde-forming substrate specific for the amine oxidase, and known enzyme modifiers, the rate of formaldehyde formation from different N-methylamines by either route can be defined. MATERIALS

AND

METHODS

Materials. NADP+ was purchased from P-L Biochemicals, Inc. Glucose &phosphate, or.-isocitric acid, isocitrate dehydrogenase (Type IV), glucose 6phosphate dehydrogenase (Type XXI), and tricine were obtained from the Sigma Chemical Company. Benzphentamine and desipramine were gifts from the Upjohn Company and Geigy Chemical Corporation, respectively. Ethylmorphine was purchased from Merck and Co., Inc., brompheniramine was obtained from Robins Research Laboratory, and ephedrine was obtained from Inland Alkaloids. Aminopyrine was purchased from Matheson, Coleman, and Bell and was recrystallized from pentane. N-Methylamphetamine hydrochloride was obtained from K & K Laboratories and recrystallized from ethanol-ether (5050, v/v). p-Chloro-N-methylaniline was purchased from Calbiochem and recrystallized from ethylacetate. Bathophenanthroline and methimazole (l-methyl-2-mercaptoimidazole) were obtained from the Aldrich Chemical Company. of microsomes. Swine liver tissue Preparation was obtained from local slaughter houses, male Golden Syrian hamsters (130-150 g) were purchased from Lakeview Hamster Farms, and Sprague-Dawley rats (2501100 g) were purchased from the Charles River Breeding Laboratories, Inc. The rodents were maintained ad libitum on water and laboratory chow. Rodents were injected in the peritoneal cavity with corn oil (0.1 ml), phenobarbital in saline (40 mg/kg), or 3-methylcholanthrene in corn oil (20 mg/ kg) daily for 4 days and starved 18-24 h prior to sacrifice. Pig liver microsomes were prepared as described earlier (12) and rodent liver microsomes were prepared as described by Remmer et al. (13). Protein concentration was determined by the method of Lowry et al. (14) or Gornall et al. (15). Either method gives similar results (*5%). Preparation of rabbit antiserum against NADPHcytochrome c (P-450) reductase. The inhibitory antibody was elicited as described in detail by Prough and Burke (16). The ammonium sulfate-precipitated globulins were dialyzed against 0.05 M potassium phosphate buffer, 1 x 10m4 M EDTA, pH 7.7, and stored at -20°C. The specificity of these globulin preparations was described. Anti-reductase globu-

MULTIPLE

MONOOXYGENASE

lins elicited against the homogeneous pork and rat liver microsomal NADPH-cytochrome c (P-450) reductases have been prepared (16, 17) and have been maintained by Drs. B. S. S. Masters and R. A. Prough Assay of microsomal enzyme activities. A standard assay condition of 37°C was employed using 2-3 mg/ml of microsomes, a NADPH-regenerating system, 0.5 mM NADPH+, and 0.05 M potassium phosphate, 0.05 M Tricine* buffer, pH 7.4 or 8.2, the pH optima for benzphetamine demethylation and pchloro-N-methylaniline N-oxidation, respectively. Two NADPH-regenerating systems were used: 2.5 mM glucose B-phosphate and sufficient L. mesenter-, aides glucose 6-phosphate dehydrogenase to reduce 1 Fmol of NADP+/min/ml or 3 mM m-isocitrate, 5 rnM MgSO,, and sufficient isocitrate dehydrogenase to reduce 0.8 pmol of NADP+/min/m!; no differences were noted between these two regenerating systems using benzphetamine or p-chloro-N-methylaniline as substrates. Reactions were initiated with addition of microsomes or amine substrates. When immune and nonimmune globulins were used, the reaction mixture containing microsomes was preincubated with globulin for 5 min at 5°C and then in a 37°C shaking water bath for 3-5 min before the reaction vas initiated with substrate. Concentrations of products, measured in aliquots taken every 1 or 1.5 min during the first 5 min, were plotted as a function of reaction time. Specific activities (nanomoles of product per minute per milligram of protein) were calculated from the initial linear part of the plot. All concentrations of substrates were maximal with respect to rate and were as follows: aminopyrine, 3.0 mM; benzphetamine, 0.7 mM; brompheniramine, 3.0 mM; p-chloro-N-methylaniline, 1.0 mM; desipramine, 1.0 mM; ephededrine, 3.0 mM; ethylmorphine, 3.0 mM; N-methylamphetamine, 3.0 mM. The concentrations of methimazole used (0.25-I mM) were sufficient to inhibitp-chloroN-methyl-N-hydroxyaniline formation over 90% at the higher concentration. The concentration of antipig or rat NADPH-cytochrome c (P-450) reductase globulin was the amount of globulin that gave maximal inhibition of microsomal NADPH-cytochrome c reductase or benzphetamine N-demethylase activity. The ratio of milligrams of globulin to milligrams of microsomal protein was 4:l and 6:l for pig and rodent preparations, respectively. All specific activities obtained in the presence of nonimmune globulin were 92-100% of activities in the absence of globulins. Formaldehyde concentrations were measured by the method of Nash (18) on aliquots of reaction media terminated by addition of either 0.1 vol of 10% 2 Abbreviations used: Tricine, bis(hydroxymethyl)ethyllglycine; cetic acid.

N-[2-hydroxy-l,lTCA, trichloroa-

N-METHYLAMINE

METABOLISM

365

trichloroacetic (TCA) or 2 vol of chilled water-saturated chloroform. In either case, the aliquots were thoroughly mixed on a test-tube mixer and centrifuged to remove precipitated protein or to separate the phases. Aliquots of the TCA mixture were directly analyzed for HCHO using the Nash procedure but the aqueous phase of the CHCl,,-terminated reactions was mixed with a 0.1 vol of 10% TCA, centrifuged to remove protein, warmed to 60°C to remove residual CHCl,,, and then analyzed for HCHO using the Nash procedure. The concentration of p-chloro-N-methyl-N-hydroxyaniline was measured in the chloroform phase using a modification of a method for estimation of tocopherols as Fe”+.reducing equivalents (19). A O.lml aliquot of the CHCl:, extract was added to 1.2 ml of an ethanol solution of bathophenanthroline (0.5 mg/ml) and 0.2 ml of 1 M sodium acetate, pH 5.6. After mixing, 40 ~1 of freshly prepared 0.01 M Fe(NO,), was added and 1 min later 50 ~1 of 0.02 M H,,PO, was added to stabilize the colored complex. The concentration of hydroxylamine was calculated from the absorbance at 535 nm using an extinction coefficient of 19,250 Mm’ cm-’ per reducing equivalent Concentrations of N-methyl-N-hydroxylamines were measured as described earlier (11). Oxygen uptake was measured with a Clark-type electrode at 37°C in a thermostatted cell. The electrode response was calibrated with beef heart electron transport particles (20) and NADH. The standard reaction mixture except for NADPH and benzphetamine was placed in the polarigraph cell and equilibrated at 37°C. The endogeneous rate of NADPH-dependent oxygen consumption (NADPH oxidase activity) was measured by addition of NADPH to the reaction mixture in the absence of benzphetamine and benzphetamine-dependent oxygen consumption was determined by subtracting the endogeneous rate from the rate of oxygen consumption in the presence of both NADPH and benzphetamine. The immune and nonimmune globulins were preincubated with the reaction mixture for 5 min at 37°C in the 3.5-ml polarigraph cell prior to the initiation of the reaction with NADPH and benzphetamine. The microsomal NADPH-cytochrome c reductase activity was determined by the method of Masters et al. (17) and trialkyl N-oxide formation was measured as described by Fok and Ziegler (21). Ethoxyresorufin dealkylase (22) and biphenyl-4-hydroxylase (16) assays have been described previously. The values reported in Tables I, II, IV, and V are the averages obtained from three microsomal preparations and have a standard deviation between the uninhibited specific activities and percentage inhibition values of less than ?lO% and ?5%, respectively. The purified and partially purified fractions of pig liver microsomal amine oxidase were prepared by the method described earlier (5).

366

PROUGH

AND

RESULTS

N-Methylamine Metabolism Pig Liver Microsomes

Catalyzed

by

Antibodies to pig liver microsomal NADPH-cytochrome c (P-450) reductase are known to block the flow of electrons from NADPH to cytochrome P-450 and effectively inhibit mixed-function oxidations catalyzed by cytochrome P-450 (17). Furthermore, NADPH-cytochrome c reductase antibodies are quite specific and have no effect on oxidations catalyzed by the microsomal mixed-function amine oxidase (22). In agreement with earlier studies (17), immune globulin to NADPH-cytochrome c reductase inhibits the N-dealkylation of tertiary amines about 90% (Table I). The degree of inhibition is virtually complete over a wide pH range and the extent of inhibition is not related to the rate at which a tertiary amine is oxidaTABLE

I

NADPH-CYTOCHROME REDUCTAEJEANTIBODY INHIBITION OF PIG LIVER MICROSOMAL-CATALYZED NDEMETHYLATIONS Substrate

Nanomoles of CH,O per minute per milligram of protein”

Percenta-- gt! 111:hibition by reductase antihe-l. VYUJp

ZIEGLER

tively N-demethylated. For example, is demethylated about ethylmorphine three to five times faster than brompheniramine, but, despite the differences in rate, antibody inhibits the demethylation of both substrates to the same extent. Although immune globulin to NADPHcytochrome c reductase greatly diminishes demethylation of tert-amines, it only partially inhibits benzphetamine-dependent oxygen uptake (Fig. 1). Endogenous oxygen uptake, NADPH-cytochrome c reductase, and benzphetamine N-demethylase activities were all concomitantly inhibited with increasing concentrations of immune globulin, whereas benzphetamine-dependent oxygen uptake (10.8 nmol of OJminl mg of protein in the absence of immune globulin) decreased only 50% at the highest concentration of antibody tested. The increment of the rate inhibited by antibodies specific for NADPH-cytochrome c reductase (5.4 nmol of O,/minlmg of protein) was almost equal to benzphetamine N-demethylation (5.6 nmol of CH,Olmin/mg of protein). Antibody-insensitive oxygen consumption is probably due to the amine oxidase-catalyzed formation of benzpheta-

pH 7.4 pH 8.2 pH 7.4 pH 8.2 tert- Amines Aminopyrine Benzphetamine Brompheniramine Ethylmorphine set-Amines p-Chloro-Nmethylaniline Ephedrine Desipramine N-Methylamphetamine

3.8 3.3 1.3 7.7

3.9 3.4 3.8 12.6

92 90 90 89

90 83 89 96

9.7

13.9

59

46

3.0 1.6 3.5

5.1 2.5 5.7

30 10 30

33 10 26

fi Formaldehyde was measured in aliquots of reaction media terminated with trichloroacetic acid. b Anti-pig liver NADPH-cytochrome c (P-450) reductase globulin was added in a 4:l ratio of milligrams of globulin to milligrams of microsomal protein. Nonimmune globulin produced less than 10% inhibition and the values cited are relative to activity measured in the presence of nonimmune globulin.

mg globulinlmg

protein

FIG. 1. The effect of anti-NADPH-cytochrome c (P-450) reductase globulin on several microsomal activities of pig liver. The activities were measured at pH 7.4 and 37°C as described under Materials and Methods. The activities measured were: NADPHcytochrome c reductase, 0; endogeneous NADPH oxidase, 0; benzphetamine-dependent oxygen up take, &, and benzphetamine N-demethylase (formaldehyde), 0. The effect of nonimmune globulin on benzphetamine N-demethylase (formaldehyde) and benzphetamine-dependent oxygen uptake is shown . by W and A, respectively.

MULTIPLE

MONOOXYGENASE

mine N-oxide. Although benzphetamine N-oxide formation was not measured when the experiments shown in Fig. 1 were carried out, measurements with another aliquot of this preparation, frozen for several weeks, were made. This preparation at pH 7.4 catalyzed the N-oxidation of 4.7 nmol of benzphetamine/min/mg of protein and the formation of N-oxide was totally insensitive to antibodies to NADPH-cytochrome c reductase. While this N;oxide does not contribute to formaldehyde, its formation will substantially modify formaldehyde to oxygen (and NADPH) stoichiometry. In contrast to tertiary amines, antibodies to the NADPH-cytochrome c reductase only partially inhibit N-demethylation of secondary amines (Table I). p-Chloro-Nmethylaniline demethylation was inhibited only about 50%, whereas the demethylation of desipramine was inhibited only slightly. It is evident that only part of the formaldehyde formed during the oxidative demethylation of secondary amines is catalyzed by cytochrome P-450-dependent oxidase. This observation is also substantiated by the data in Table II. n-Octylamine, a known cytochrome P-450 inhibitor (23), inhibits the demethylation of the four tertiary amines tested by 74-93%, but only partially inhibits the demethylation of see-amines. In fact, octylamine consistently stimulates the N-demethylation of N-methylamphetamine. Octylamine is known to stimulate reactions catalyzed by pig liver microsomal amine oxidase (241, which suggests that substantial amounts of formaldehyde produced from see-Nmethylamines are formed via the microsomal amine oxidase. This interpretation is also consistent with the demethylation pattern observed in the presence of methimazole. Methimazole, an excellent substrate for the amine oxidase, is oxidized exclusively by amine oxidase in hepatic microsomes to N-methylimidazole and sulfite (25). Methimazole is not a substrate for cytochrome P-450-dependent oxidase, and high concentrations (26 mM) are required to form type II binding spectra (26). Although N-methylimidazole can inhibit cytochrome P-450-dependent reactions and bisulfite can interfere with formaldehyde

N-METHYLAMINE

TABLE

II

OCTYLAMINE AND METHIMAZOLE DEMETHYLATIONS CATALYZED MICROSOMES

Substrate

367

METABOLISM

INHIBITION OF NBY PIG LIVER

Change in rate” of CH,O formation in the presence of: Oct#an$ne m

Methimazole (0.2 rnM)

pH 7.4 pH 8.2 pH 7.4 pH 8.2 tert- Amines Aminopyrine Benzphetamine Brompheniramine Ethylmorphine set- Amines p-Chloro-Nmethylaniline Ephedrine Desipramine N-Methylamphetamine

-83 -74 -63 -74

-80 -93 -91 -74

-16 -16 -21 -13

-29 -21 -39 -14

-36

-17

-36

-41

-33 -25 +23

-16 -36 +55

-70 -36 -54

-68 -57 -49

fl The values are expressed as (Vi - VJV,) x 100, where Vi is the rate of formaldehyde production in the presence of the inhibitor listed and V, is the rate in the absence of inhibitor. The reaction media was deproteinized with trichloroacetic acid. Demethylation rates were calculated from formaldehyde concentrations in aliquots.

measurements, at the moderately low concentrations formed during the first 2-4 min of incubation, these metabolites do not appreciably inhibit cytochrome P-450catalyzed reactions. For example, methimazole up to 1 mM does not affect ethoxyresorufm deethylase or biphenyl4-hydroxylase activity of rat liver microsomes (v/v x 100 = 98 and 97%, respectively). Methimazole at 0.25 mM only slightly inhibits demethylation of tert-amines catalyzed by pig liver microsomes, but methimazole inhibition of set-amines is more pronounced. These results are consistent with the other data in Tables I and II and suggest that cytochrome P-450-dependent oxidase catalyzes most, if not all, of the N-demethylation of tert-amines, but both the mixedfunction amine oxidase and cytochrome P450-dependent oxidase contribute to formaldehyde formed during the oxidation of set-amines. That the mixed-function amine oxidase catalyzes the oxidation of set-amines (but

368

PROUGH AND ZIEGLER TABLE III

PRODUCTS OF IV-METHYLAMINES

Substrate

OXIDATIONS

CATALYZED

BY PURIFIED

MIXED-FUNCTION

AMINE

OXIDASE

Nanomoles of product per minute per Nanomoles of HCHO per minute per milligram of protein” milligram of protein” Control

Octylamine (3 mM)

Methimazole (0.25

Control

Octylamine (3 mM)

rnMT

tert- Amines Aminopyrine Benzphetamine Brompheniramine Ethylmorphine

0

set- Amines p-Chloro-N-methylaniline Ephedrine Desipramine N-Methvlamnhetamine

0

Methimazole (0.25 rnMY

0

1230 1416 1280

1845 1974 1930

578 940 836

780

790

186

720

736

143

760 1219 840

764 1230 906

175 731 56

86 73 231

95 88 260

17 70 14

I’ Rates are based on concentration of N-oxides formed from tert-amines and N-hydroxylamines from secamine substrates at pH 8.2 and 37°C. Concentration of substrates were as listed under Materials and Methods. Reaction was initiated by addition of 40-100 pg of homogenous amine oxidase. b Formaldehyde was present in aliquots of reaction media terminated with trichloroacetic acid. The amount of formaldehyde varies with the extent of metal contamination in the Nash reagents. Quantitative oxidation and hydrolysis ofp-chloro-N-methyl-N-hydroxyaniline requires about lo-” M Fe3+, which is close to the concentration of Fe3+ present in trichloroacetic extracts of microsomes. c Concentrations (millimolar) required to half-saturate the oxidase are methimazole, 0.1; benzphetamine, 0.24; brompheniramine, 0.12; p-chloro-N-methylaniline, 0.43; desipramine, 0.25. This constant was not determined for other substrates. Aminopyrine is not a substrate for amine oxidase, but it was carried through the analyses as control determinations.

not tert-amines) to metabolites that yield formaldehyde is shown by the information in Table III. The N-methylhydroxylamine metabolites formed by N-oxidation of secamines nonenzymatically oxidize and hydrolyze by known reactions (27) to produce formaldehyde. However, as expected, only p-chloro-N-methylaniline quantitatively yields formaldehyde upon oxidation and hydrolysis. N - Methyl-N - alkylhydroxylamines of N-methylamphetamine, ephedrine, and desipramine are not only more stable, but, upon oxidation, each yields two nitrones -only one of which forms formaldehyde upon hydrolysis (10, 11). In reactions catalyzed by homogenous preparations of amine oxidase, oxidation and hydrolysis of the N-methylhydroxylamines, including the very unstable p-chloroN-methyl-N-hydroxyaniline, appear to occur after the reaction is terminated with trichloroacetic acid. No formaldehyde can be detected in the aqueous phase of reaction

media

extracted

with

chloroform

to

remove N-methylhydroxylamines before acidification. However, in reactions catalyzed by partially purified preparations of amine oxidase or by microsomes, some oxidation (probably nonenzymatic) and hydrolysis of p-Chloro-N-methyl-N-hydroxyaniline seem to occur during incubation. The time course of products formed in the presence of partially purified amine oxidase preparations (preparations free from cytochrome P-450 but still containing small amounts of cytochrome b5, catalase, and nonheme iron) indicates that, after a 1-min lag, formaldehyde measured in the aqueous phase is formed at a slow rate, but one that parallels the rate of hydroxylamine formation (Fig. 2A). The sum of pchloro-N-methyl-N-hydroxyaniline concentrations in the chloroform phase and concentrations in the formaldehyde aqueous layer of aliquots terminated by chloroform is virtually identical to total formaldehyde concentration in aliquots quenched immediately with trichloroace-

MULTIPLE

MONOOXYGENASE

time (mid

2. Metabolites of p-chloro-N-methylaniline oxidation catalyzed by partially purified amine oxidase preparation (A) and pig liver microsomes (B). Formaldehyde present in aliquots was terminated with trichloroacetic acid (0) or p-chloro-N-methylN-hydroxyaniline (A) and formaldehyde (0) in chloroform-terminated aliquots. FIG.

tic acid. The formation of these products as a function of time in microsomal-catalyzed reactions (Fig. 2B) is similar to that observed with partially purified amine oxidase preparations, but formaldehyde is produced during incubation at a rate that approaches the rate of p-chloro-N-methylN-hydroxyaniline formation. This is as expected, since part of the formaldehyde is formed from cytochrome P-450-dependent oxidation of this set-amine. The preceding experiments demonstrate that at least part of the formaldehyde formed during pig liver microsomal-catalyzed N-demethylation of secondary amines measured in acid-terminated aliquots of reaction media comes from Nmethylhydroxylamines produced by the mixed-function amine oxidase. Pig liver microsomes, known to have high concentrations of amine oxidase, are rarely used to study these reactions. Therefore, to evaluate the contribution of different monooxygenases to the metabolism of secamines in more widely used hepatic microsomes, the effect of methimazole and antibodies to NADPH-cytochrome c reductase on the metabolism of p-chloro-N-methylaniline and benzphetamine by rodent liver microsomes was studied in some detail. N-Methylamine Metabolism and Rat Liver Microsomes

by Hamster

At the highest levels tested, antibodies

N-METHYLAMINE

369

METABOLISM

elicited against rat liver NADPH-cytochrome c (P450) reductase, known to inhibit both rat and hamster liver microsomal NADPH-cytochrome c reductase (16), inhibit demethylation of benzphetamine by hamster microsomes 85% (Fig. 3). Inhibition of benzphetamine demethylation parallels cytochrome c reductase activity in a manner similar to that reported for biphenyl hydroxylation (16). However, the N-demethylation of p-chloro-N-methylaniline is inhibited only 50 to 60%. Formaldehyde produced by oxidative metabolism of a set-amine in hamster microsomes arises by pathways similar to those in pig liver microsomes, as shown by the data in Table IV. In microsomes from control (corn oil treated) animals, methimazole inhibited p-chloro-N-methylaniline demethylation 25-35% and benzphetamine demethylation about lo%, whereas antireductase globulin inhibited demethylation of benzphetamine more than that ofpchloro-N-methylaniline. Phenobarbital pretreatment induced hamster hepatic benzphetamine N-demethylase activity 196%, but only induced p-chloro-N-methylaniline demethylase activity by 130%, whereas pretreatment with methylcholanthrene only increased the latter activity. As expected, phenobarbital induction did not markedly affect inhibition of benzphetamine N-demethylation by either immune globulin or methimazole, but it did decrease methimazole inhi-

I

I

I.

I1

2 4 mg globulin/mg

1.

6 protein

II

6

FIG. 3. Effect of anti-NADPH-cytochrome c reductase globulin on hamster liver microsomal activities. Measurements, carried out at pH 7.4 and 38”C, were: NADPH-cytochrome c reductase, 0; benzphetamine N-demethylase, A; p-chloro-l\r-methylaniline-N-demethylase, 0 (formaldehyde in trichloroacetic acid-terminated aliquots). Activities in the presence of nonimmune globulin, 0.

370

PROUGH AND ZIEGLER TABLE IV INHIBITION OF HAMSTERLIVER MICROSOMALN-DEMETHYLATION BY ANTI-REDUCTASEGLOBULIN OR METHIMAZOLE Substrate Animal pretreatment Percentage inhibition by: Snecific activitv” Antibody Methimazole” d

pH 7.4

pH 8.2

pH 7.4

pH 8.2

pH 7.4

pH 8.2

Benzphetamine

Corn oil Phenobarbital 3-Methylcholanthrene

5.5 10.8 6.5

6.2 9.3 5.4

87 85 79

79 88 78

12 11 16

20 4 13

p-Chloro-N-methylaniline

Corn oil Phenobarbital 3-Methylcholanthrene

7.4 9.4 12.0

6.7 6.5 11.1

62 75 60

61 81 70

38 16 29

25 28 32

” Activity is based on CH,O present in trichloroacetic acid-terminated reactions and is expressed as nanomoles of CH,O per minute per milligram of protein. h The ratio of globulin to microsomal proteins was 6:l and the concentration of methimazole used was 1.0 rnM. TABLE V INHIBITION OF RAT LIVER MICROSOMAL N-DEMETHYLATION

Substrate

Animal pretreatment

BY ANTI-REDUCTASE GLOBULIN AND METHIMAZOLE

Specific activity”

Percentage inhibition Antibody

by:

Methimazole”

pH 7.4

pH 8.2

pH 7.4

pH 8.2

pH 7.4

pH 8.2

Benzphetamine

Corn oil 3-Methylcholanthrene

10.5 8.0

9.3 7.6

83 80

85 80

0 7

3 5

p-Chloro-N-methylaniline

Corn oil 3-Methvlcholanthrene

7.0 7.8

5.3 7.8

67 77

74 75

30 20

20 37

n Activity is based on CH,O present in trichloroacetic acid-terminated reactions and is expressed as nanomoles of CH,O per minute per milligram of protein. b The ratio of globulin to microsomal proteins was 6:l and the concentration of methimazole used was 1.0 rnM.

bition of p-chloro-N-methylaniline demethylation (16% for microsomes from phenobarbital-treated animals vs. 38% for microsomes from control animals). Increasedp-chloro-N-methylaniline demethylase activity of microsomes from methylcholanthrene-treated animals is not due to differential induction of the amine oxidase, since it is known that neither this compound nor phenobarbital can induce the amine oxidase (22), and the inhibition pattern of microsomes from methylcholanthrene-pretreated animals is not significantly different from that observed with controls. Inhibition of rat liver microsomal-catalyzed N-methylation of benzphetamine

and p-chloro-N-methylaniline by immune globulin and methimazole (Table IV) is similar to that observed with hamster microsomes. Studies with rat liver were limited to microsomes from control and methylcholanthrene-treated animals, and it is evident (Table V) that anti-NADPH-cytochrome c reductase globulin consistently inhibits demethylation of benzphetamine more than p-chloro-N-methylaniline (8085% vs. 69-75%) and that methimazole inhibits the latter activity the most (20-35% vs. O-7%). Although the amine oxidase relative to cytochrome P-450-dependent activities is low in rat liver microsomes, the observation that methimazole inhibits Ndemethylation of p:chloro-N-methylani-

MULTIPLE

MONOOXYGENASE

line indicates that the amine oxidase also contributes to formaldehyde formation from see-amines in this species. DISCUSSION

Oxidative N-demethylation of N-methylamines in. vitro is usually followed by measuring the rate of formaldehyde formation by the convenient and specific colorimetric method developed by Nash (18). It is widely assumed that formaldehyde measured under these conditions arises only by direct oxidative N-demethylations catalyzed by cytochrome P450-dependent oxidase. Although this assumption appears to be valid for N-methyldialkylamines, it does not hold for set-N-methylamines. Part of the formaldehyde present in aliquots of the reaction mixtures deproteinized with acid arises from oxidation and hydrolysis of N-methylhydroxylamines (Table III): products formed by NADPH- and oxygen-dependent reactions catalyzed by the microsomal amine oxidase. The amount of formaldehyde produced by this route is a function of both the specific N-methylamine and the relative activities of the microsomal cytochrome P450-dependent oxidase and the amine oxidase. The latter activity, relative to cytochrome P-450-dependent oxidations, is high in pig liver, and more N-methylamine metabolism will occur by this route in these tissues than in rodent liver microsomes. Of the different set-N-methylamines tested, only p-chloro-N-methylaniline yields an N-hydroxy metabolite that quantitatively yields formaldehyde upon oxidation and hydrolysis. N-Methyl-N-hydroxy arylamines are more easily oxidized than N,N-dialkylhydroxylamines and, upon oxidation, yield only one nitrone, the N-arylnitrone which quantitatively forms formaldehyde upon hydrolysis. From formaldehyde formation measured in acid-terminated reactions carried out in the presence of anti-cytochrome c reductase globulin and methimazole (Tables IV and V), it appears that about 65% of p-chloro-Nmethylaniline oxidation by pig liver microsomes is catalyzed by amine oxidase and only 35% by the cytochrome P-450 sys-

N-METHYLAMINE

METABOLISM

371

tem (Tables I and II). The contribution of the amine oxidase to the metabolism of this see-amine by hamster and rat liver microsomes (Tables IV and V) is only about 40 and 20%, respectively, and metabolism of p-chloro-N-methylaniline by rat liver microsomes occurs predominantly by oxidative N-demethylation catalyzed by the cytochrome P-450-dependent oxidase. Anti-NADPH-cytochrome c reductase globulin has proven to be an excellent tool for defining microsomal cytochrome P-450dependent reactions (16, 17), but an equally specific inhibitor of the microsomal mixed-function amine oxidase is not available. Antibodies to purified pig liver amine oxidase elicited in rabbits are not inhibitory. Of the nonamine substrates for the amine oxidase we investigated, methimazole is the most promising for use in defining N-methylamine metabolism catalyzed by the amine oxidase. At the concentration (10m4M) required to half saturate the amine oxidase, methimazole has no effect on cytochrome P-450-dependent reactions and lo-fold higher concentrations inhibit the latter reactions no more than lo-15%. However, N-methylimidazole, a product of methimazole S-oxidation, can inhibit cytochrome P-450-dependent oxidations. Inhibition by this product is readily detected since the inhibition, negligible for the first 2 to 3 min, increases rapidly with incubation time. Pathways for the generation of metabolites from N-methylamines resulting only from oxidative attacks on the nitrogen or on carbons alpha to the nitrogen are summarized in Fig. 4. In the presence of saturating tert-amines (during the first few minutes of incubation), only metabolites produced by Reactions 1 (5) and 2 (6,7) are present. However, incubations continued long enough to consume substantial amounts of the tertiary amine or incubations initiated with the see-amine will also contain metabolites formed by Reactions 3 and 4-8 (5,10,11). Furthermore, NADPHand oxygen-dependent oxidation of Nmethylhydroxylamines to nitrones (Reaction 5) and hydrolysis of the nitrones have been demonstrated with pig liver micro-

372

:“3 X-c”2TH3 w a 2(b) “CHO 1 . 4(a) X-CH2N”CH3 Cc)

PROUGH

AND

OH I x-c”+N-cH3 21 CH3

ZIEGLER B. S. S. Masters for her support in maintaining the production of the anti-NADPH-cytochrome c (P450) reductase globulins. REFERENCES

Y” X-CH2N-CM3 I

1. BRIDGES, J. W., GORROD, J. W., AND PARKE, D. V. (eds.) (1972) The Biological Oxidation of Nitrogen in Organic Molecules, Taylor and Francis, London. CI) 2. G~RROD, J. W. (1973) Chem. Biol. Interact. 7, 289-303. “20 Reu. 21,325. 3. BICKEL, M. H. (1969)Pharmacol. i oo4. MILLER, J. A., AND MILLER, E. C. (1969) Prog. +i X-CH2-MHZx-C”&H3 X-CH2-NH2 Exp. Tumor Res. 11, 273-301. 5. ZIEGLER, D. M., AND MITCHELL, C. H. (1972) +y /“z” “‘018 Arch. Biochem. Biophys. 150, 116-125. X-C”0+ CH3NHOH 6. MUELLER, G. C., AND MILLER, J. A. (1953) J. X-CH2-NHOH + “CHO Biol. Chem. 202, 579-587. FIG. 4. Pathways for N-methylamine metabo7. GILLETTE, J. R., BRODIE, B. B., AND LADu, B. N. lism in hepatic microsomes. Reactions were cata(1957) J. Pharmacol. Exp. Ther. 119, 532-540. lyzed by amine oxidase (a) and cytochrome P-4508. MCMAHON, R. E., AND SULLIVAN, H. R. (1964) dependent oxidase (b). Both oxidases require Life Sci. 3, 1167-1174. NADPH and oxygen. Reactions were also catalyzed 9. MCMAHON, R. E., CULP, H. W., AND OCCOLOWby reduced pyridine nucleotide-dependent N-hyITZ, J. C. (1969)J. Amer. Chem. Sot. 91,3389droxylamine reductase (c). 3390. 10. POULSEN, L. L., KADLUBAR, F. F., ANDZIEGLER, D. M. (1974) Arch. Biochem. Biophys. 164, somes (11) and purified amine oxidase (10). 774-775. In the presence of saturating see-N-meth11. KADLUBAR, F. F., MCKEE, E. M., AND ZIEGLER, ylamine, enzymatic oxidation of N-methD. M. (1973)Arch. Biochem. Biophys. 156,46ylhydroxylamine is unlikely and formal57. dehyde formed in the presence of anti- 12 ZIEGLER, D. M., AND PETTIT, F. H. (1966) BioNADPH-cytochrome c reductase probably chemistry 5, 2932-2938. arises from the nonenzymatic oxidation 13. REMMER, H., GRIEM, H., SCHENKMAN, J. B., AND ESTABROOK, R. W. (1967) in Methods in and hydrolysis of the N-methylhydroxyEnzymology (E&brook, R. W., and Pullman, lamine (cf. Table III and Fig. 1). MicroM. E., eds.), Vol. 10, pp. ‘703-708, Academic somes can also catalyze reduced pyridine Press, New York. nucleotide-dependent reduction of N-hy14. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., droxylamines (11) and the N-hydroxylaAND RANDALL, R. J. (1951)J. Biol. Chem. 193, mine reductase has been isolated from pig 265-275. liver microsomes and partially character- 15. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, ized (28). Although NADH is the preferred M. M. (1949)J. Biol. Chem. 177, 751-766. reductant, the microsomal N-hydroxyla16. PROUGH, R. A., AND BURKE, M. D. (1975)Arch. mine reductase can utilize NADPH. To Biochem. Biophys. 170, 1601168. what extent the presence of this system 17. MASTERS, B. S. S., BARON, J., TAYLOR, W. E., ISAACSON, E. L., AND LOSPALLUTO, J. (1971)5. limits metabolites produced by Reactions Bid. Chem. 246, 4143-4150. 5-8 in microsomes from different species is not known. However, reduction of the N- 18. NASH, T. (1953) Biochem. J. 55, 416421. 19. TSEN, C. C. (1961)Amzl. Chem. 33, 849-851. methylhydroxylamines catalyzed by mi- 20. GREEN, D. E., AND ZIEGLER, D. M. (1963) in crosomes from pig, hamster, or rat cannot Methods in Enzymology (Colowick, S. P., and be as fast as formation, since some formalKaplan, N. O., eds.), Vol. 6, pp. 416-424, Acadehyde was always formed from p-chlorodemic Press, New York. N-methyl-N-hydroxyaniline. 21. FOK, A. K., AND ZIEGLER, D. M. (1970) Biochem. Biophys. Res. Commun. 41, 534-540. ACKNOWLEDGMENTS 22. BURKE, M. D., AND MAYER, R. T. (1974) Drug Metub. Disp. 2, 583-588. The authors wish to thank Philip Freeman and 23. JEFCOATE, C. R. E., GAYLOR, J. L., AND CALGillian Verner for their technical assistance and Dr.

MULTIPLE ABRESE,

MONOOXYGENASE

R. L. (1969) Biochemistry

8, 3455-

3463. 24. ZIEGLER, D. M., POULSEN, L. L., AND MCKEE, E. M. (1971) Xenobiotica 1, 523-531. 25. POULSEN, L. L., HYSLOP, R. M., AND ZIEGLER, D. M. (1974)Biochem. i’harmacol. 23,3431-3440. 26. SCHENKMAN, J. B., REMMER, H., AND ESTA-

N-METHYLAMINE

METABOLISM

BROOK, R. W. (1967) Mol. Pharmacol.

373 3, 113-

123. A. K., FELDMAN, A. M., GELBLUM, E., AND HODGSON, W. G. (1964) J. Amer. Chem. Sot. 86, 639-646. F. F., AND ZIEGLER, D. M. (1974) 28. KADLUBAR, Arch. Biochem. Biophys. 162, 83-92. 27. HOFFMANN,