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Chem. Res. Toricol. 1991,4, 70-16
Comparative Studies of N-Hydroxylation and N-Demethylation by Microsomal Cytochrome P-450 Judith N. Burstyn,+ Marco Iskandar, John F. Brady, Jon M. Fukuto, and Arthur K. Cho* Department o f Pharmacology and Jonsson Comprehensive Cancer Center, U C L A School of Medicine, Los Angeles, California 90024-1 735 Received August 20, 1990 The N-hydroxylation of representative aromatic amines by rabbit liver microsomes was mediated by cytochrome P-450 as demonstrated by the sensitivity to carbon monoxide and other cytochrome P-450inhibitors. The rate of N-hydroxylation was increased by induction with phenobarbital. Involvement of isozyme LM2 (P-50IIB1) was demonstrated in reconstituted systems. Aromatic N-hydroxylation was substantially faster and more efficient than aliphatic N-hydroxylation, while N-demethylation of aromatic and aliphatic dimethylamines was comparable in rate and efficiency. Aliphatic N-hydroxylation showed no rate increase with increasing p H despite the predicted increase in the concentration of the neutral substrate. The relative rates of N-hydroxylation and N-demethylation were compared for a series of para-substituted aromatic amines. The rate of demethylation of para-substituted N,N-dimethylanilines, as measured both by product formation and by NADPH consumption, correlated with the electronic parameter u and with the Hansch lipophilicity parameter T . N-Hydroxylation of a similar series of anilines did not show a dependence on the electronic parameter but was dependent on the lipophilicity parameter. The differing dependence on the electronic parameter suggests that there are different rate-determining processes of N-oxidation for these two reactions.
Introduction The metabolic N-oxidation of aromatic amines has been recognized as an important initiating event in their toxicity (1, 2 ) , often leading to the generation of electrophilic species which can react readily with cellular nucleophiles such as DNA and proteins. For example, studies on carcinogenic primary aromatic amines have led to the postulate that the toxic metabolites are conjugates of N-oxidized aryl compounds [for example, see Cramer et al. (3), Kriek (4), Thorgeirsson et al. (5),and Schut and Castonguay ( S ) ] . These electrophilic species react with basic residues on DNA and induce mutations leading to tumor formation. Aryl hydroxylamines and nitroso compounds are capable of binding to the iron center in hemoglobin and, as a result of oxidative degradation, are believed to be responsible for the development of pernicious anemia and methemoglobinemia observed in arylamine poisoning (7). In general, aliphatic amines are significantly less toxic than their aromatic counterparts (at least by the types of mechanisms mentioned above). Though this difference in toxicity may be due to the reactivity of the bioactivated metabolites, differences in metabolism may also play critical roles. Due to the importance of the metabolic activation process in toxicity, we have undertaken a study comparing the interaction of arylamines and alkylamines with the liver cytochrome P-450monooxygenase system in order to learn more about the mechanisms of N-oxidation. Part of this study compares the relatively nontoxic aliphatic amines with their aromatic counterparts to de-
* Author to whom correspondence should be addressed at the Department of Pharmacology, School of Medicine, Center for the Health Sciences, University of California, Los Angeles, Los Angeles, California 90024-1735. Present address: Department of Chemistry, University of Wisconsin, Madison, WI 53706.
termine whether the mechanism of N-oxidation of these compounds differs. Biological oxidation of amines can result in either Nhydroxylation or N-dealkylation depending on the degree of nitrogen substitution (N-oxide formation from tertiary amines is also possible but it not addressed in this study). Most of the previous studies attempting to elucidate the mechanism of N-oxidation dealt primarily with N-dealkylation. It is therefore worthwhile to compare Nhydroxylation and N-dealkylation, in order to ascertain whether the mechanistic conclusions formulated from the previous studies on N-dealkylation pertain to the more toxicologically significant N-hydroxylationprocess. In this study, we investigated each process independently to determine whether they were mechanistically similar. Thus, the dependence of the N-hydroxylation of primary amines and the N-demethylation of tertiary dimethylamines (structures shown in Figure 1) on electronic effects and lipophilicity was determined.
Materials and Methods Materials. Phenobarbital was purchased from Amend Drug and Chemical Co. (Irvington,NJ). Benzphetamine was a gift of the Upjohn Co. (Kalamazoo, MI), and phentermine hydrochloride was a gift from Pennwalt Corp. (Rochester, NY). N,N-Dimethylphentermine hydrochloride was a gift from Dr. Gerald Miwa, Merck Sharp and Dohme (Rahway, NJ). Aniline and its para-substituted derivatives were all purchased from Aldrich Chemical Co. (Milwaukee, WI) and were of the highest purity available. N,N-Dimethylaniline and the remaining para-substituted derivatives were also obtained from Aldrich as was Nethyl-N-methylaniline.p-Fluoro-Nfl-dimethylanilinewas purchased from TCI (Tokyo,Japan), and p-chloro-N,N-dimethylaniline was obtained from Overlook Industries, Inc. (Bloomsbury, NJ). The purity of these anilines derivatives was verified by gas chromatography (GC). The various N-hydroxyanilines were synthesized by controlled reduction of the appropriate nitro derivative using either metallic zinc (8) or aluminum amalgam (9). The nitrosobenzenes were prepared by silver carbonate
0 ~ 9 3 - 2 2 ~ ~ / 9 1 / 2 7 0 4 - 0 0 7 Q ~ Q 2 .05 Q 1991 ~ Q American Chemical Society
N-Oxidation of Alkyl- and Arylamines
Phentermine
X
pX-Aniline
N-Hydroxy phentermine
X
pX-N,N-Dimethylaniline
N, N-Dimethyl phentermine
X pX-N-Hydroxy aniline
X = H, CI, Br, F, I, CN, CF3, CH3
Figure 1. Structures of aryl- and alkylamines studied. oxidation of the corresponding N-hydroxyaniline compounds (IO). T h e identity of compounds thus synthesized was confirmed by mass spectroscopy. The N-hydroxy and nitroso aromatic compounds were all unstable for extended periods at room temperature and were therefore stored a t -20 “C under nitrogen. NHydroxyphentermine was synthesized from phentermine according to the method of Beckett et al. (11). Potassium phosphate (dibasic and monobasic), methimazole, glucose 6-phosphate, NADP, NADPH, and glucose-6-phosphate dehydrogenase were all purchased from Sigma Chemical Co. (St. Louis, MO). Potassium ferricyanide was from Fisher Chemical co. (Tustin, CA), and sodium dithionite was purchased from Fluka Chemical Corp. (Ronkonkoma, NY). Methods. Microsomes from phenobarbital-pretreated male New Zealand rabbits were prepared as described previously (12). Isozyme LM2 (P-450IIB1) was prepared as described previously (13), according to the method of Coon et al. (14), to a specific content of 12 nmol/mg of protein. NADPH-dependent cytochrome P-450 reductase was purified by procedures described previously (15). Cytochrome P-450 concentrations were determined by the method of Omura and Sato (16) using an extinction coefficient of 91 mM-’ cm-’. Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA) using bovine y-globulin as a standard. Incubations of microsomes corresponding to 0.33 g of liver (wet weight) were carried out a t 37 “C or room temperature in 0.12 M potassium phosphate buffer, p H 7.4 (unless otherwise noted), and 30 mM KC1 in a final volume of 5 mL. NADPH was provided by a regenerating system consisting of 6.0 mM glucose 6-phosphate, 0.5 mM NADP, 2.4 mM MgCl,, and 6 units of glucose-&phosphate dehydrogenase, or by the addition of fixed aliquots of 2 mM NADPH. In those incubations containing fixed amounts of NADPH, 5’-AMP (1.2 mM) was included in the incubations with limiting NADPH to inhibit the microsomal pyrophosphatase activity (17). In all but the inhibitor studies, the microsomal preparation was preincubated at 37 OC in the absence of NADPH for 5 min to eliminate any contribution from the flavin-dependent monooxygenase activity (18). NADPH consumption was monitored by following the decrease in absorbance at 340 nm. Oxygen consumption was measured by using a YSI Model 53 biological oxygen monitor (Yellow Springs International, OH). Substrates were dissolved in either 0.01 N HC1, water, or acetonitrile. In the latter case, the acetonitrile concentration in the incubation mixture never exceeded 1% of the total volume. Where comparisons were made, the acetonitrile concentration was kept constant in all incubations. Reconstitution of the cytochrome P-450 LM2-reductase system was achieved by combining 1 nmol of cytochrome P-450 LM2, 1 nmol of cytochrome P-450 reductase, and 60 pg of dilaurylphosphatidylcholine [ l mg/mL in 0.1 M K P H 0 4 (pH 7.7) sonicated immediately beforehand]. The mixture was allowed to sit undisturbed a t room temperature for 5 min. Determination of Metabolites. N-Demethylation reactions were monitored by following the formation of formaldehyde as determined by the method of Nash (19). Protein was precipitated prior to assaying the supernatant by the addition of perchloric acid followed by centrifugation. In addition, the demethylation reaction was followed by monitoring the products by gas chromatography as a function of time and enzyme concentration in order to determine the region of linearity and to confirm that the
Chem. Res. Toxicol., Vol. 4, No. 1, 1991 71 Table I. Cytochrome P-450 Dependence of Aromatic N-Hydroxylation reaction conditions % inhibition control 0 argon 92.4 co/oz (41) 88.7 SKF 525 (mM) 0.2 48.6 1.0 81.5 DPEA (mM) 0.02 13.8 0.10 37.1 benzphetamine (mM) 0.20 41.7 0.50 48.8 1.00 59.9 66.6 2.00 OEffect of inhibitors of cytochromes P-450 and the isozyme LM2 on the N-hydroxylation of p-chloroaniline. Incubations were carried out at room temperature and with 1 mM substrate. Other conditions are described in the text. Each experiment was done in triplicate, and the standard deviation of all the measurements was less than 5%. SKF 525 and DPEA are specific cytochrome P-450 inhibitors. Benzphetamine is an LM2 substrate and therefore a competitive inhibitor of LM2-dependent oxidation. primary product was the monodemethylated amine. Following alkalinization with carbonate buffer to p H 9.5, the incubation mixture was extracted into dichloromethane containing 1phenyl-2-butanone as an internal standard. The dichloromethane layer was concentrated to about 100 pL under a stream of nitrogen and reacted with an equal volume of trifluoroacetic anhydride overnight. Analysis of the products was performed on a 6 ft X 2 mm glass column packed with 5% QF-1 on 100/120 Supelmport a t a flow rate of 25 mL/min. The N-hydroxylation reaction was monitored by gas chromatography of the products. N-Hydroxyphentermine is moderately air stable and could, therefore, be extracted directly into dichloromethane containing 1-phenyl-2-butanone as an internal standard from an aqueous phase at p H 9.5. After concentration and derivatization of the extract with trifluoroacetic anhydride, the presence of N-hydroxylated product was assayed by GC on a 6 ft X 2mm glass 3% OV-17 column. The aromatic N-hydroxy compounds were readily air oxidized in organic solvents, and therefore the extraction solvent was deoxygenated with nitrogen and care taken to avoid air oxidation prior to extraction and derivatization. This anaerobic extraction method was used in the studies comparing the efficiency of product formation in the aliphatic and aromatic N-hydroxylation reactions. For all other studies involving the N-hydroxylation of aromatic amines, deliberate oxidation of the hydroxylamine was effected. Potassium ferricyanide was added to the extraction mixture in order to oxidize the hydroxylamine to the corresponding nitrosobenzene which could then be analyzed directly by GC without derivatization. N,N-Dimethylaniline or N-ethyl-N-methylanilinewere used as internal standards in these assays. The chromatography was performed on a 6-ft glass column packed with 3% OV-17. The only detectable metabolites were the N-OH compounds. Although p-aminophenol could be detected by the derivatization assay, it was not detected in the incubation mixtures under the conditions used.
Results Cytochrome P-450Dependence of Aromatic and Aliphatic N-Hydroxylation. The N-hydroxylation of p-chloroaniline was sensitive to common cytochrome P-450 inhibitors as shown in Table I. The reaction required the presence of NADPH and oxygen and was substantially inhibited by CO, indicating that a heme-containing oxygenase was responsible for the oxidation. Methimazole, an inhibitor of the flavin-dependent monooxygenase system (20), inhibited the formation of p-chloro-N-hydroxyaniline by only 19% (at a methimazole concentration of 0.2 mM), demonstrating that this enzyme system is rela-
72 Chem. Res. Toxicol., Vol. 4, No. 1, 1991 Table 11. Effect of Phenobarbital Induction on the Rate of N-Hydroxylation of Aniline and p -Chloroaniline" phenobarbital-induced substrate control microsomes microsomes aniline
