Mechanism of 2-naphthylamine oxidation catalysed by pig liver microsomes.

XENOBIOTICA,

1976, VOL. 6,

NO.

8, 481-498

Mechanism of 2-Naphthylamine Oxidation Catalysed by Pig Liver Microsomes

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LAWRENCE L. POULSEN, BETTIE SUE SILER MASTERS, and DANIEL M. ZIEGLER Clayton Foundation Biochemical Institute, Department of Chemistry, University of Texas at Austin and Department of Biochemistry, University of Texas Health Science Center (Southwestern Medical School), Dallas, Texas, U.S.A. (Received 18 November 1975)

1 . In pig liver microsomes 2-naphthylamine-dependent NADPH oxidation, oxygen reduction, and hydroxylamine formation are linear with time for several minutes. A sharp increase in NADPH oxidation and oxygen uptake then coincides with an abrupt loss of hydroxylamine from the medium. 2. The initial rate of 2-naphthylamine N-oxidation correlates with the microsoma1 concentration of mixed-function amine oxidase and the extent of linear accumulation of hydroxylamine is dependent on microsomal NADPH-cytochrome c reductase activity and concentration of lipid (microsomes).

3. Antisera to NADPH-cytochrome c reductase markedly decreased hydroxylamine accumulation during incubation but had no effect on the rate of 2-naphthylamine N-oxidation. 4. A system duplicating all of the kinetic properties of the microsomal 2naphthylamine oxidase was constructed with two purified flavoproteins, (mixedfunction m i n e oxidase and NADPH-cytochrome c reductase) and a lipid phase (erythrocyte ghosts or synthetic lecithin liposomes).

5. By independently varying the concentrations of each component in the reconstituted system, the contribution of each to the observed kinetics was defined.

6. In addition to the initial N-oxidation of 2-naphthylamine, at least six other reactions contribute to the kinetic patterns of 2-naphthylamine oxidation catalysed by the reconstituted system.

Introduction The metabolism of the carcinogen 2-naphthylamine has been extensively studied, both in vivo and in vitro. While a variety of metabolites have been isolated from the urine of animals treated with this amine (Boyland & Manson, 1966), it is generally accepted that metabolic N-oxidation of 2-naphthylamine, like the N-oxidation of N-acetylaminofluorene (Cramer, Miller & Miller, 1960) leads to the formation of more carcinogenic substances (Boyland, Dukes & Grover, 1963; Booth & Boyland, 1964; Arcos & Argus, 1968). Bonser et aE. (1963) and Bryan, Brown and Price (1964) demonstrated that 2-naphthylhydroxylamine surgically implanted in the bladders of mice produced statistically


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more tumours than the carrier alone or carrier containing 2-naphthylamine. Subsequently Radomski and Brill (1970) reported that repeated instillation of 2-naphthylhydroxylamine into the bladders of dogs induced bladder tumours. Herringlake et al. (1960) first demonstrated N-oxidation of 2-naphthylamine in vivo, to 2-nitrosonaphthalene, but intermediate N-oxidized metabolites were not directly identified. The N-hydroxy derivative was subsequently shown to occur as a urinary metabolite in humans (Troll & Nelson, 1961) and dogs (Boyland & Manson, 1966). Kiese and Uehleke (1961) demonstrated that the enzymes catalysing Noxidation of aniline and N-methylaniline were concentrated in the microsomal fraction of rat liver homogenates. N-Oxidation of these arylamines was NADPH- and oxygen-dependent; and it was assumed that N-oxidations of different nitrogen-containing compounds were all catalysed by a single microsomal mixed-function oxidase. However, subsequent studies with hepatic microsomes from phenobarbital-treated animals (Lange, 1967 ; Uehleke, 1967) and with rabbit lung microsomes (Uehleke, 1973) indicated that aniline was N-oxidized by a different mixed-function oxidase from its secondary N-alkyl derivatives. Uehleke (1971, 1973) concluded that cytochrome P-450 participates in the N-hydroxylation of primary arylamines but not in the N-oxidation of N-alkyl or N,N-dialkyl arylamines: This was consistent with the substrate specificity of a microsomal mixed-function oxidase isolated from pig liver microsomes (Ziegler & Mitchell, 1972). This oxidase, a flavoprotein, catalysed rapid N-oxidation of a variety of lipid-soluble secondary and tertiary amines but not of primary alkylamines or arylamines other than 2-naphthylamine (Ziegler, McKee & Poulsen, 1973). It would appear that 2-naphthylamine is an exception of the generalization that the N-hydroxylation of arylamines is mediated exclusively by the cytochrome P-450 system. Although the terminal oxidase of the microsomal electron transport system is not required for Nhydroxylation of 2-naphthylamine, a component of this system, the NADPHcytochrome c reductase, had to be added with the purified oxidase to detect 2-naphthylhydroxylamine in the reaction mixture. The data presented (Ziegler et nl., 1973) suggested that the purified amine oxidase catalysed the N-oxidation of 2-naphthylamine to 2-naphthylhydroxylamine which, under the conditions used, was rapidly oxidized non-enzymically to 2-nitrosonaphthalene. However, in the presence of the reductase flavoprotein and NADPH, 2-nitrosonaphthalene was reduced back to the hydroxylamine. While N-oxidation of 2-naphthylamine can be demonstrated with a system composed of the two pure flavoproteins, this system does not duplicate many of the kinetic properties observed during the microsomal-catalysed oxidation of 2-naphthylamine. The experiments summarized in this report demonstrate that at least three different microsomal components influence the rates of 2-naphthylaminedependent oxidation of NADPH and uptake of oxygen. The amount of 2-naphthylhydroxylamine in the reaction media during the course of the microsomal catalysed reaction depends upon the ratios of these different microsomal components, and a system that duplicates all of the kinetic properties of porcine hepatic microsomal 2-naphthylamine N-oxidase can be reconstructed with purified mixed-function amine oxidase, isolated NADPH-cytochrome c reductase and phospholipid. The function of each component in the reconstituted system is described.

2-Naphthylamine Oxidation

483


Materials and methods Materials NAD+, NADPf, NADH, and NADPH were obtained from Pabst Laboratories (Milwaukee, U.S.A.) ; catalase and synthetic lecithin from Sigma Chemical Co. (St. Louis, U.S.A.) ; superoxide dismutase from Truett Laboratories, Biochemical Division (Dallas, U.S.A.) ; and 2-naphthylamine from Paragon Testing Laboratories (Orange, N. J., U.S.A.). Methods NADPH-cytochrome c reductase (Masters, Williams & Kamin, 1967 Prough & Masters, 1973), microsomal mixed-function amine oxidase (Ziegler & Mitchell, 1972), microsomes (Ziegler & Pettit, 1966), liposomes (Bangham, Standish & Watkins, 1965), red blood cell ghosts (Dodge, Mitchell & Hanahan, 1963), and NADPH-cytochrome c reductase-specific y-globulin fractions (Masters et al., 1971) were prepared as previously described. 2-Naphthylhydroxylamine (Willstatter & Kubli, 1908), and 2-amino-lnaphthol (Fieser, 1955) were synthesized by published methods. 2-Naphthylamine, recrystallized several times from pentane and stored at -2O", was dissolved in an equivalent amount of aqueous HC1 just before use. NADPH-cytochrome c reductase (Masters et al., 1967) and mixed-function amine oxidase (Ziegler & Pettit, 1964) activities were measured by published methods. NADPH oxidation was estimated by following the 340 nm absorbance of the reduced nucleotide with a Gilford Model 2400 S spectrophotometer. 2-Naphthylhydroxylamine concentrations were calculated from pentyl acetatesoluble reducing equivalents determined by the method of Tsen (1961). At the times indicated, aliquots (0.2 to 0.5 ml) of the reaction media were mixed for 30 s on a test tube mixer with 1.0 ml pentyl acetate containing 0.1 ml 1 M sodium acetate pH 4.6. The pentyl acetate extract (0.5 ml), separated by centrifugation, was transferred to tubes containing 0-8 ml 0.0025 M bathophenanthroline in absolute ethanol and 0.2 ml of 1 M sodium acetate p H 4.6. Then 0-04 ml of 0.01 M Fe(NO,), in 0.01 M acetic acid was added and exactly one minute later 0.05 ml of 0.02 M H,PO, added to stabilize the ferrous bathophenanthroline complex. The absorbance of the complex was measured at 535 nm against a reagent blank. The millimolar absorptivity of the ferrous bathophenathroline complex is 18.5 cm-, (Tsen, 1961). Known amounts of synthetic 2-naphthylhydroxylamine carried through the procedure either in the absence or presence of microsonies demonstrated that, as expected, 1 mol hydroxylamine reduces 2 mol iron. Any lipid-soluble compound capable of reducing iron could interfere with the estimation of 2-naphthylhydroxylamine by this method. However, the only reducing compound detected in pentyl acetate extracts of the reaction media was identical with synthetic 2-naphthylhydroxylamine by paper chromatography in four solvent systems. R, values of the hydroxylamine were 0.74 in H,O-acetic acid (1O:l); 0.23 in methanol saturated with heptane; 0.72 in methanol and 0.0 in chloroform. Under the conditions specified, the method gave a reliable estimate of 2-naphthylhydroxylamine concentrations. Although the p H optimum of the microsomal 2-naphthylamine N-oxidase is above 7.4, the enzymic reactions were routinely carried out at 7.4. In more alkaline solutions, recovery of 2-naphthylhydroxylamine from reaction media was difficult to determine quantitatively.


484


Oxygen concentrations were measured in a 1.9 ml glass temperaturecontrolled cell fitted with a Clarke-type oxygen electrode Model 4004 (Yellow Springs Instrument Co., Yellow Springs, Ohio). The electrode signal was recorded with a Heathkit EU-205 B strip-chart recorder. After each measurement the cell must be alternately rinsed with water and methanol. Methanol removes water-insoluble electron acceptors for the NADPH-cytochrome c reductase generated from 2-naphthylamine, and does not affect the characteristics of the oxygen electrode fitted with a polypropylene membrane.

Results The microsomal catalysed N-oxidation of 2-naphthylamine is most readily followed by measuring the formation of 2-naphthylhydroxylamine at p H 7-3-7.4. Initially, the rate of this formation was linear with all of the different preparations of pig liver microsomes tested; as shown in Fig. 1 (upper curve), it then abruptly drops to a low concentration that remains essentially constant. The uptake of oxygen with the same preparation of microsomes under the same experimental conditions is shown in Fig. 2. Like the formation of the hydroxylamine, it is nearly linear for 6 min, but between 6 and 8 min, rises sharply for about one minute, followed by a slower terminal rate that is still, however, twice the rate of oxygen consumption during the first six minutes. Comparison of Figures 1 and 2 shows that the increase in the rate of oxygen uptake coincides with the 1

I

I

0

2

1

I

4

1

I

6

I

8

1

0

Time (min)

Fig. 1. Time course of 2-naphthylamine N-oxidation catalysed by pig liver microsomes. 0- 0 : 2-naphthylhydroxylamine concentration at times indicated in system containing 0.1 M phosphate, pH 7.4, ; 2.5 mM glucose-6-phosphate, 0.5 mM NADP+, 1.0 I.U. glucose-6-phosphate dehydrogenase, 0.5 mM 2-naphthylamine and 0.36 mg microsomal protein/ml. T =38". A - - A : 2-naphthylhydroxylamine concentrations under identical conditions except microsomes mixed with 1.3 mg NADPH-cytochrome c reductase antisera/mg microsomal protein before assay. Final concentration of antisera in assay medium, 0.46 mg/ml. This concentration of nonimmune serum had no effect on the time course of 2-naphthylamine accumulation.

485



-

1

1

1

1

1

1

1

1

1

Time(min1

Fig. 2. 2-Naphthylamine-dependentoxygen uptake catalysed by pig liver microsomes. Composition of reaction media identical with that of Fig. 1. Solid line: oxygen uptake in control. Dashed line: oxygen uptake in presence of antisera to NADPH-cytochrome c reductase.

Time

Fig. 3.

_

(mid

Time course of NADPH ojcidation and oxygen consumption during 2-naphthylamine oxidation catalysed by pig liver microsomes. : 0, uptake at 38" measured polarographically. Medium contained 0.1 M phosphate, pH 7.4; 0.16 mM NADPH, 0.5 mM 2-naphthylamine, and 0.20 mg microsomal protein/ml. _ _ _ : NADPH oxidation measured spectrophotometrically. Reaction conditions identical to those above. The oxidation of NADPH could not be followed beyond the point shown since 340 n m absorbing, secondary oxidation products begin to form rapidly at this point. The microsomal preparation used in these experiments was selected for its low (less than 5 % of the 2-naphthylamine-dependent)endogenous rate of NADPH oxidation.

1.9 2.0 3.1 11.7 6.4 13.0 7.3

4.4

0.9 1.6 2.6 6.2

Dimethylaniline N-oxidation 69 68 59 97 66 4-0

NADPH-cyt. c reductase

5

77 43 23 16 15

Cyt. c reductase dimethylaniline N-oxidase

Ratio

40 min 23 min 20 min 14 min 12 min 8 min

Hydroxylaminet increased linearly for

All enzyme activities are expressed as nmol product/min/mg protein during the first minute of incubation at pH 7.4 and 38". Activities measured as specified in Methods section. Estimation of 2-naphthylamine N-oxidase activities was carried out with 0.8-1.0 mg protein/ml with 0.5 mM 2-naphthylamine. Pig liver microsomal preparations listed were selected for variation in 2-naphthylamine oxidase activity, and include a higher proportion of extremely high and low activities than normally encountered. -f Interval of incubation over which the accumulation of 2-naphthylhydroxylamine was linear with time.

2 3 4 5 6

1

Preparation*

2-Naphthylamine N-oxidation

Enzyme activities

Table 1. Comparison of Znaphthylamine oxidase with other microsomal activities


r

D,

F!2

2 E:

ts(

P



487

rapid loss of hydroxylamine from the reaction medium. The sharp increase in the rate of oxygen uptake is not entirely due to the non-enzymic oxidation of hydroxylamine since NADPH oxidation also increases at this point (Fig. 3). It is evident that the microsomal catalysed N-oxidation of 2-naphthylamine changes in character during the course of the reaction, and reactions secondary to the formation of hydroxylamine become more important as the reaction proceeds. During the first few minutes the amount of hydroxylamine in the reaction medium appears proportional to its initial rate of formation, but the final concentration present in the reaction medium appears to be controlled by microsomal components not involved in the initial N-oxidation. A definitive explanation of the anomalous kinetic pattern of 2-naphthylamine oxidation requires the identification of (1) the enzyme system that catalyses the initial N-oxidation, (2) the microsomal components that stabilize 2-naphthylhydroxylamine, and (3) the reaction or sequence of reactions responsible for the eventual rapid loss of hydroxylamine from the medium. Initial 2-naphthylamine N-oxidation T o assess the contributions of different microsomal components to the initial rate of 2-naphthylamine N-oxidation, several enzyme activities were measured in a number of microsomal preparations. The initial rate of 2naphthylamine oxidation follows more closely the N,N-dimethylaniline Noxidase activity of each preparation than of any other activity measured (Table 1). The concentration of the NADPH-cytochrome c reductase activity, which varies only about 2-fold in the different preparations tested, appears not to be related to the initial rate of the 2-naphthylamine oxidation, nor were the rate of 2-naphthylamine oxidation, cytochrome P-450 concentration, or aminopyrine demethylase activity in those preparations where all three were measured. The N-oxidation of dimethylaniline is believed to be catalysed exclusively by the microsomal mixed-function amine oxidase, and the observed correlation between the dimethylaniline and 2-naphthylamine N-oxidase activities suggests that the N-oxidation of 2-naphthylamine is catalysed by the amine oxidase. Microsomal components that stabilize the N-hydroxylamine Although the NADPH-cytochrome c reductase does not contribute to initial N-oxidation, the concentration of this microsomal flavoprotein does influence accumulation of 2-naphthylhpdroxylamine in the medium as a function of incubation time (Table 1). With microsomes that contain the highest concentration of reductase, formation of the hydroxylamine remains linear for longer periods. However, the extent of the linear reaction is more closely related to the ratio of NADPH-cytochrome c reductase to dimethylaniline N-oxidase activities than to the activity of the reductase alone. This ratio varied 15-fold among the different microsomal preparations listed in Table 1 and at the highest ratio hydroxylamine accumulated at a linear rate for 40 min and at the lowest ratio for only 8 min. The requirement for the NADPH-cytochrome c reductase for the linear accumulation of hydroxylamine is also demonstrated by Figures 1 and 2. Antisera to the NADPH-cytochrome c reductase decreases the time of the linear reaction from 7 min to less than 3 min but it has no effect on the initial rate of 2-naphthylamine N-oxidation. In the experiment shown the antisera inhibited



488

the microsomal NADPH-cytochrome c reductase 83yo and decreased the ratio, of NADPH-cytochrome c reductase to dimethylaniline N-oxidase activities from 5 to 0.2, and the change in this ratio is reflected in the decrease in the total amount of hydroxylamine that could be formed. In addition to the ratio of activities of the microsomal NADPH-cytochrome c reductase to dimethylaniline N-oxidase, the total amount of hydroxylamine that can be formed also depends upon the concentration of microsomes in the reaction medium. As shown in Table 2, the formation of 2-naphthylhydroxylamine remains linear for longer periods the greater the concentration of microsoma1 protein. It would appear that in addition to NADPH-cytochrome c reductase other microsomal components also affect the accumulation of the hydroxylamine. Since any component that decreases the rate of 2-naphthylhydroxylamine auto-oxidation would influence the linearity of the reaction as measured by hydroxylamine accumulation, the effect of phospholipids and other lipophilic compounds on the auto-oxidation of this hydroxylamine was determined. The rate of 2-naphthylhydroxylamine auto-oxidation, measured by following the uptake of oxygen, is shown in Table 3. The auto-oxidation of this hydroxylamine is relatively rapid at p H 7.4even in the presence of EDTA, and oxidation of 2-naphthylhydroxylamine cannot be blocked by the addition of metal-chelating agents. The auto-oxidation can be suppressed, but not eliminated, by addition of microsomes, erythrocyte ghosts, synthetic lecithin liposomes or organic solvents, whereas addition of pure proteins such as serum albumin does not inhibit oxidation of the hydroxylamine. Aryl hydroxylamines are known to be relatively resistant to oxidation by oxygen in organic solvents and it would appear that in the presence of microsomes or other phospholipid-containing membranes hydroxylamine taken up in the lipid is also not as readily oxidized by oxygen. Thus it would appear that the lipid present in microsomes is also involved in stabilizing the hydroxylamine formed during the initial phase of the microsomal catalysed reaction.

Table 2. Effectof microsomal concentration on the accumulation of Snaphthylhydroxylamine as a function of incubation time Protein (mg/ml)

Hydroxylamine increased linearly with time for

0.10 0.20 0.40 0.80

5 min 9 min 15 min 27 min

The reaction was carried out at pH 7-4 and 38". The microsomal preparation was similar to preparation 3 listed in Table 1. Initial concentration of 2-naphthylamine was 0.5 mM which approaches the upper limits of its solubility in water but this concentration is below that required for the 1/2 maximum velocity with pig liver microsomes (Ziegler et al., 1973).

489


Table 3. Effect of various compounds and lipophilic materials on the rate of 2-naphthylhydroxylamine auto-oxidation


Additions to buffer None EDTA Albumin Erythrocyte ghosts Lecithin liposomes Microsomes Ethyl acetate n-Butanol n-Butanol 2-Imino-1 -naphthone 2- Imino- 1-naphthone

Concentration

10 mM 1 mg/ml 0.5 mg protein/ml 6 mg/ml 1-6mg/ml 0.1 ml/ml 0.1 ml/ml 0.2 ml/ml 10 p M

1 CLM

Initial rate 0, consumption (pM/min) 12 13 18 6 6.3 4 7 3 2 47 25

The rate of 2-naphthylhydroxylamine oxidation by oxygen was measured polarographically following the addition of hydroxylamine to a solution containing 2 2 0 p ~oxygen in 0.1 M phosphate buffer, pH 7-4. In every case the initial concentration of 2-naphthylhydroxylamine was 50 p ~ .

Reconstitution of the 2-naphthylamine oxidase The microsomal catalysed N-oxidation of 2-naphthylamine can be duplicated in every detail by a system composed of the isolated NADPH-cytochrome c reductase, the purified mixed-function amine oxidase and a non-specific lipoprotein membrane or lipid phase (synthetic lecithin liposomes). As shown in Fig. 4, in the presence of the two flavoproteins and lipoprotein the time course of 2-naphthylamine-dependent oxygen uptake resembles that observed with microsomes. In this experiment the concentrations of the two flavoproteins were adjusted to approximately the same levels as present in the microsomes used to obtain the data in Fig. 1. The concentration of lipid phase (erythrocyte ghosts 0.4 mg protein/ml, or liposomes 0.7 mg lecithin/ml) was selected since at the flavoprotein concentrations used these concentrations supported linear formation of the hydroxylamine for about 6 min. Like microsomes the reconstituted system exhibits an initial linear rate of oxygen consumption that parallels the linear formation of 2-naphthylhydroxylamine. After 6 min, there is an abrupt increase in the rate of oxygen consumption that coincides with the disappearance of the hydroxylamine from the reaction medium. Erythrocyte ghosts or liposomes are qualitatively similar in their ability to support the linear formation of the hydroxylamine in the presence of the two flavoproteins. Since the liposomes are easier to prepare and store than the erythrocyte ghosts, all subsequent experiments described are carried out with liposomes as the lipid phase. X.B.

2 K

L. L. Poulsen


490

0 0

et

al.

10

5

Time (min)

Fig. 4.

2-Naphthylamin dependent oxygen uptake catalysed by th reconstituted system. Reaction medium identical with that in Figures 1 and 2 except microsomes replaced by : : 18.4 pg mixed-function amine oxidase, 3.5 pg NADPH-cyt. c reductase and 0.36 mg erythrocyte ghost protein per ml. _ _ _ _ : same as above but no erythrocyte ghosts.

Table 4. Effects of changes in the concentration of components of the reconstituted system on kinetics of Znaphthylamine N-oxidation Flavoprotein Oxidase Reductase Gg/ml)*

Liposomes (mglrnl)

2-Naphthylamine oxidationt Initial rate Linear increase in (nmol/rnin/ml) hydroxylamine for

7

1*3 1.3 1.3

4.7 9.0 19.9

9 min 5 min 2 min

33 33 33 33

7 7 7 7

0.0 0.65 1.3 2.6

4.7 4.8 4.7 3.7

3 min 7 min 9 min 13 rnin

33 33 33 33

3.5 7 14 28

1.3 1.3 1.3 1.3

4.4 4.7 4.7 4.9

4 min 9 min 12 min 9 min

33 66 132

7 7

*The activity of the preparations at pH 7.4 and 38" were: Oxidase 250 nmol dimethylaniline oxidized/min/mg. Reductase 19 000 nmol NADPH oxidized/min/mg with cyt. c as the acceptor. tAliquots of the reaction mixture were withdrawn every minute and assayed for 2naphthylhydroxylamine. Initial concentration of 2-naphthylamine was 0.5 m M .



49 1

The contribution of each component to the oxidation of 2-naphthylamine in the reconstituted system was assessed by varying the concentration of each independently of the other two components. As shown in Table 4, in the presence of constant amounts of the reductase and liposomes, the initial rate of hydroxylamine formation is directly proportional to the concentration of the mixed-function amine oxidase. This supports the conclusion that the initial N-oxidation of 2-naphthylamine by pig liver microsomes is catalysed by this oxidase. On the other hand, the concentrations of the reductase and of the liposomes determine the total amount of hydroxylamine that can accumulate. The liposomes decrease the rate of hydroxylamine auto-oxidation (Table 3) and permit more of this product to accumulate in the reaction medium. In the initial phase of the reaction, the flavoprotein reductase can catalyse the reduction of the oxidation product, 2-nitrosonaphthalene, back to the hydroxylamine (Ziegler et al., 1973). Increasing the concentration of this flavoprotein increases the rate of the back reaction which allows retention of increasing amounts of hydroxylamine in the medium. This appears to be true for all concentrations of the reductase (relative to the oxidase) that are similar to or slightly higher than those encountered in microsomes (cf. Table 1). Non-enzymic reduction of 2-nitrosonaphthalene by NADPH appeared negligible, in contrast to the corresponding reduction of nitrosobenzene (Bernheim, 1972).

Reactions limiting hydroxylamine accumulation The effect of lipophilic phase and reductase adequately account for accumulation of hydroxylamine during the initial linear phase of the reaction, but they do not explain the sharp increase in rate of oxygen uptake that coincides with the point where the hydroxylamine disappears from the reaction medium. Furthermore, the rate of oxygen uptake, and NADPH oxidation, in the terminal phase of the reaction is more than twice as fast as expected if this reaction is due only to the cyclic reduction of 2-nitrosonaphthalene to the hydroxylamine and non-enzymic oxidation back. Addition of up to 50 nmol/ml of 2-nitrosonaphthalene or 2-naphthylhydroxylamine to the complete reaction mixture containing microsomes or the reconstituted system did not produce an immediate rapid uptake of oxygen, which suggests that the rapid uptake of oxygen coinciding with loss of hydroxylamine is due to formation of another 2-naphthylamine oxidation product. Data in Figure 5 indicate that catalase (and to a certain extent superoxide dismutase) can block the formation of this postulated oxidation product. In the presence of a large excess of catalase, the characteristic marked increase in oxygen uptake does not appear and hydroxylamine is also not lost from the reaction medium. Under these conditions the steady increase in the rate of oxygen uptake is as expected when the only major reactions contributing to the oxygen consumption are the N-oxidation of 2-naphthylamine and the cyclic auto-oxidation of the hydroxylamine followed by NADPH dependent reduction of nitrosonaphthalene. The exponential increase in the rate of NADPH oxidation (Fig. 3) coinciding with the rapid increase in oxygen uptake and loss of hydroxylamine suggest that an excellent auto-oxidizable electron acceptor for NADPH-cytochrome c reductase is formed during the course of the reaction. However, attempts to


492


5

10 Time ( m i d

Fig. 5. Eflect of various additions on the .2-naphthylamine-dependentoxygen uptake catalysed by the reconstituted 2-naphthylamine oxidase. Composition of basic medium: 0.1 M phosphate (pH 7.4) 0.5 mM NADPf, 2.5 mM glucose-6-phosphate, 1 .O I.U. glucose-6-phosphate dehydrogenase, 0.5 m~ 2naphthylamine, and the reconstituted oxidase consisting of 48 pg amine oxidase, 8 p g NADPH-cytochrome c reductase, and 1.3 mg lecithin liposomes, all per ml medium. The control (curve c) recorded under these conditions, then under identical conditions the measurements were repeated except that at the time indicated by the arrow each of the following was added. Concentrations specified are final concentrations in medium. a 2.5 p~ 2-imino-1-naphthone b 2.5 p~ 2-amino-1-naphthol c control d 0.1 mg/ml superoxide dismutase e 0.5 rng/ml catalase

identify this other product (or products) in chloroform extracts of aliquots of the reaction medium were unsuccessful. T h e time required to concentrate the extracts under vacuum followed by t.1.c. chromatography invariably resulted in the formation of several degradation products that increased in amount upon repeated chromatography of the same concentrated chloroform extract. This phenomena precluded the identification of secondary metabolites produced in the reaction vessel from those formed during processing. An alternate method for the tentative identification of this secondary metabolite, based on its properties as an electron-acceptor for the NADPH-cytochrome c reductase, is possible. A number of compounds readily formed by rearrangement and oxidation of the hydroxylamine or by direct oxidation of 2-naphthylamine were synthesized and tested, of which the oxidation product of 2-amino-1-naphthol (2-imino-lnaphthone or 2-imino- 1-naphthaquinone) has all of the expected properties of the other oxidation product. This iminonaphthone is an excellent electronacceptor for the NADPH-cytochrome c reductase (Fig. 6) and the concentration ~ ~ a maximum velocity of required to half-saturate the reductase is 3 . 8 with

493

2-Naphthylamine Oxidation I

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-

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0.2

0.4

0.6

0.0

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09-

h

C ._ E

0

z

06-

v

2 .030


0.2

0

I

2-Imino-I-naphthone

1.0

(PMi'

Fig. 6. Double reciprocal plot of velocity vs. 2-imino-1 -nuphthone concentration. Initial rates calculated from the rate of oxygen uptake at 38" in the presence of the NADPH-generating system (cf. Fig. l), 0.1 M phosphate pH 7.4, and 1.2 pg NADPHcytchrome c reductase/ml. The reactions started by the addition of 2-imino-lnaphthone. The 2-imino-1-naphthone was prepared no more than 30 min before use by controlled air oxidation (at 5-10') of 2-amino-1-naphthol under alkaline conditions. .

60 000 nmol NADPH oxidized/min/mg enzyme. As little as 1 PM 2-imino-lnaphthone added to the reaction medium results in rapid uptake of oxygen accompanied by loss of hydroxylamine (Fig. 5) which would be expected since this iminoquinone catalyses the rapid non-enzymic oxidation of 2-naphthylhydroxylamine (Table 3). Catalase has no effect on the action of added 2imino-1-naphthone, but catalase inhibits formation of this compound from the parent 2-amino-1-naphthol (Fig. 5). Even in the absence of catalase the addition of up to 1 0 2-amino-1-naphthol ~ ~ to the complete reaction mixture does not result in an immediate rapid uptake of oxygen. There is a 1-2 min lag after the addition of 2-amino-1-naphthol before the rapid uptake of oxygen occurs and it would appear that hydrogen peroxide (generated by several routes) is required for the initial oxidation of 2-amino-1-naphthol to the 2-imino-lnapht hone.

Discussion After studying several hundred different amines, it appears that the amine oxidase can catalyse the oxidation only of amines with a highly nucleophilic nitrogen atom (Swain & Scott, 1953). This requirement would appear to exclude all primary arylamines and aromatic amines because of the delocalization of the lone pair nitrogen electrons into the aromatic bonding orbitals of the ring. The fact that the mixed-function oxidase does not catalyse oxidation of aniline but does catalyse oxidation of 2-naphthylamine suggests that the nitrogen atom in 2-naphthylamine has some nucleophilic character which is not present in the nitrogen atom of aniline and other similar primary arylamines. One possibility is apparent on examination of the various enol tautomers which might form with arylamines. In contrast to aniline, 4-aminobiphenyl and 1-naphthylamine, in aqueous solutions some 2-naphthylamine can exist in the imine form.


494

Enz

2 I

H H2O


f

E n z - -H 0 - d

Q

,

,

H

0

1

I H

Fig. 7.

H

Possible enzyme (mixed-function amine oxidase) 2-naphthylamine intermediates.

This mechanism proposes that the imine tautomer of 2-naphthylamine reacts with peroxy flavin prosthetic group of the enzyme to produce the enzyme-bound hydroxylamine. Cleavage of intermediak as indicated could lead to the final products indicated with route b predominating.

This form is not significantly present with aniline and 4-aminobiphenyl because of the large energy decrease associated with loss of aromaticity on formation of the imine, and in the case of 1-naphthylamine, its formation is blocked by steric

hindrance to the planar configuration of the C = N double bond. The striking property of the imine form is the concentration of the lone pair of electrons on the nitrogen atom with no possibility for delocalization into the ring. Thus the nitrogen atom of the imino tautomer is highly nucleophilic and should therefore be a substrate for the amine oxidase. The assumption that only the imine form of 2-naphthylamine reacts with the oxidase is also consistent with the high K , of this substrate (Ziegler et al., 1973), since only a small fraction of the amount added is in the form required by the enzyme. A possible mechanism of interaction between the imino tautomer of 2naphthylamine and the peroxy flavoprotein form of the enzyme (see Massey, Palmer & Ballou, 1971) is depicted in Fig. 7. The acidic proton at the 1position of the imine is ideally located to enhance the electrophilicity of the oxygen involved in the oxidative attack on the nitrogen and thus increase the rate of this reaction. This mechanism provides an explanation for the observation (Ziegler et al., 1973) that the oxidation of 2-naphthylamine, unlike other substrates for the amine oxidase, is not stimulated by lipophilic primary alkyl amines or alkyl guanidines, which act by increasing the concentration of protons at the catalytic site. Thus, the assumption that only the imine form of 2-naphthylamine reacts with the amine oxidase provides an explanation for the observed failure of activators to stimulate the oxidation of 2-naphthylamine. In addition, the imine tautomer of 2-naphthylamine also has a degree of nucleophilicity similar to all other known substrates for this enzyme.

2-Naphthylarnine Oxidation

495

A reasonable mechanism for the observed kinetics of 2-naphthylamine oxidation catalysed by pig liver microsomes can be formulated:by considering the contribution of the following reactions. amine oxidase + 2-naphthylhydroxyl(1) 2-naphthylamine + NADPH+ 0, + Hf amine + NADP+ + H,O non-enzymic (2) 2-naphthylhydroxylamine + 0, > 2-nitrosonaphthalene + H,O, reductase (3) 2-nitrosonaphthalene + NADPH + H+ 2-naphthylhydroxylamine + NADP+ amine oxidase reductase ? (4) 2-naphthylamine -+ 2-amino- 1-naphthol NADPH+ 0, non-enzymic -+ 2-imino-1-naphthone + 2H,O ( 5 ) 2-amino-1-naphthol + H,O, reductase -+ 2-imino-l(6) (a) 2-imino-1-naphthone + 1/2 NADPH + 1/2 H+ naphthol + 1/2 NADP+ non-enzymic (b) 2-imino-l-naphthol+ 0, 2-imino-1-naphthone + 0,' non-enzymic ( c ) 20,-+2H+ -+ H,Oz 0, non-enzymic (7) 2-naphthylhydroxylamine 2-imino-1-naphthone > 2-nitrosonaphthalene + 2-amino-1-naphthol


-

+

+

During the first few minutes of incubation, reaction 1 catalysed by the mixed-function amine oxidase, predominates and the NADPH oxidized and oxygen reduced are reasonably parallel with the formation of Z-naphthylhydroxylamine (Figs. 1 and 2). If as suggested 2-amino-1-naphthol is also formed, the observed final rate of oxygen reduction (Fig. 2) would require the formation of this product at only 1% of the rate of N-oxidation but it would have to be produced at a relatively constant rate from the beginning of the incubation. Initially small amounts of Hz.Oz are probably produced by several reactions. For example, the auto-oxidation of one or both flavoproteins (reactions not shown), or of 2-naphthylhydroxylamine to the nitroso derivative (reaction 2), can all yield H,O,. In the early phase of the reaction (since competing electron acceptors for the cyt. c reductase are essentially absent), any 2-nitrosonaphthalene formed is rapidly reduced to the hydroxylamine by the reductase (reaction 3). However, as the incubation proceeds, reactions 5 and 6 are becoming more important and it is evident that the rates of these coupled reactions increase exponentially with incubation time and at some point all of the 2-amino-lnaphthol is converted to 2-imino-1-naphthone. As shown in Table 3, this latter compound catalyses the rapid oxidation of the 2-naphthylhydroxylamine (reaction 7). Reactions 6(b) and 7 are consistent with the sharp increase in oxygen uptake that coincides with the loss of hydroxylamine from the medium and reaction 7 adequately explains the loss of the hydroxylamine. I n the terminal phases of the incubation the increase in the rates of oxygen uptake and NADPH oxidation is probably due to reactions 6 (a)-(c). The above mechanism depends on the formation of 2-amino-1-naphthol. Although the evidence for its formation is indirect, there are three routes that


496


could produce this product. The acid-catalysed rearrangement of 2-naphthylhydroxylamine to 2-aminonaphthol by the Bamberger rearrangement reaction has been described by Boyland, Manson & Nery (1962), but the amount of 2-amino-l-naphthol formed by this reaction at pH 7.4 is probably insignificant. The addition of 2-naphthylhydroxylamine (in the absence of 2-naphthylamine) to reaction media containing either microsomes or the reconstituted system does not produce the metabolite responsible for rapid oxygen reduction or hydroxylamine loss. The formation of 2-amino-l-naphthol could also occur through an oxidative attack of superoxide ions, produced by the NADPH-cyt. c reductase, on 2-naphthylamine (B. S. S. Masters, unpublished results). However, if this were the only route for the production of 2-amino-l-naphthol its formation should be blocked by superoxide dismutase. As shown in Fig. 5 , superoxide dismutase only slightly retards the formation of the metabolite that leads to rapid oxygen uptake, and relatively small amounts of the metabolite appear to be produced by this route. Another mechanism for the formation of 2-amino-1naphthol as a minor metabolite of the amine oxidase-catalysed oxidation of 2-naphthylamine is illustrated in Fig. 7. This scheme assumes that after the oxidative attack on nitrogen by flavin hydroperoxide, an intermediate enzymenaphthylhydroxylamine complex exists in which the hydroxyl proton is hydrogen-bonded to protein. This complex, as in the acid-catalysed rearrangement, would be a moderately good leaving group at either of the two bonds indicated. If cleavage occurs at the N-0 bond, some of the enzymebound intermediate may be hydroxylated by a concerted nucleophilic attack of hydroxyl ions at the 1-position. While the enzyme-bound intermediates depicted in Fig. 7 are speculative, they do provide a reasonable mechanism for the formation of small amounts of 2-amino-l-naphthol along with the principal product, N-hydroxy-2-naphthylamine. Although the sequence of reactions listed above are adequate to explain the observed experimental data, additional reactions are probably occurring and the formation of other minor products upon prolonged incubation can be readily demonstrated. The generation of H,O, by reactions 6 (a)-(c) probably occurs as written through the superoxide ion, and in the absence of saturating levels of superoxide dismutase and catalase significant concentrations of 0,- and H,O, can be present in the reaction medium during the terminal phases of incubation. The generation of .OH, an extremely reactive oxidant, could then occur by a known reaction. (Haber & Weiss, 1939). This oxidant could attack, directly, 2-naphthylamine or the other naphthalene derivatives in the medium to produce a variety of secondary oxidation products, but it would be extremely difficult to prove that such reactions are occurring. However, in reactions continued several minutes past the point where rapid oxygen uptake occurs, the media, colourless to that point, become faintly yellow, then orange to red, and finally brown to purple. The formation of these highly coloured products can be prevented, in part, by carrying out the reaction with excess catalase in the medium. The formation of the highly coloured complex products may be important in the toxicology of arylamines, but their formation appears secondary to the enzymic oxidation of 2-naphthylamine. Although the experiments summarized in this report demonstrate that most 2-naphthylamine N-oxidation catalysed by pig liver microsomes can be attributed to the mixed-function amine oxidase, this conclusion cannot be extrapolated to



497

microsomes isolated from hepatic tissues of other species. The enzvmic Noxidation of aniline and other arylamines, not substrates for the amine oxidase, is well documented (Uehleke, 1971) and it is possible that in some species the N-oxidation of 2-naphthylamine takes place via the same route as aniline or 1-naphthylamine. However, the apparent inhibition of 2-naphthylamine N-oxidation by specific inhibitors needs careful evaluation. For example, as shown in Figs. 1 and 2, if only hydroxylamine present in the medium at the beginning and after five minutes incubation had been measured, the results would suggest that antisera to the NADPH-cytochrome c reductase completely blocked N-oxidation, whereas it is evident that this conclusion is incorrect when initial rates are measured. These observations illustrate the necessity of measuring initial rates when attempting to elucidate enzymic mechanisms.

Acknowledgments This work carried out during tenure of one of us (DMZ) on a U.S. Public Health Service Career Development Award (l-K3-GM-25990). The parts of this study carried out at the Health Science Center at Dallas under the direction of B.S.S. Masters, were supported by U.S. Public Health Service Grant No. H L I 13619 and Grant No. 1-453 from the Robert A. Welch Foundation.

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The mechanism of myeloperoxidase-catalysed oxidation of aminopyrine.

Stereochemistry of NADPH oxidation by dihydropyrimidine dehydrogenase from pig liver.

Further investigations of the transient kinetics of alcohol oxidation catalysed by horse-liver alcohol dehydrogenase.

Partial reversal of the acetaldehyde and butyraldehyde oxidation reactions catalysed by aldehyde dehydrogenases from sheep liver.

Oxidation of glycerol to formaldehyde by rat liver microsomes. Effects of cytochrome P-450 inducing agents.

Oxidation of cyclopenta[cd]pyrene by human and mouse liver microsomes and selected cytochrome P450 enzymes.

The N- and alpha- C-oxidation of N,N-dialkylanilines by rabbit liver microsomes in vitro.

N-oxidation and N-demethylation of methylephedrine by rat-liver microsomes.

Inhibition by thiocyanate of lactoperoxidase-catalysed oxidation and iodination reactions.

A new mechanism of in vitro formation of catechol estrogen glutathione conjugates by rat liver microsomes.

Study of the mechanism of metabolic activation of chloramphenicol by rat liver microsomes.

The kinetics and mechanism of photo-assisted Ag(I)-catalysed water oxidation with S2O8(2-).

Ceruloplasmin stimulates NADH oxidation of pig liver plasma membrane.

Oxidation of oxyphenbutazone by sheep vesicular gland microsomes and lipoxygenase.

Digitoxin metabolism by rat liver microsomes.

Investigation of peroxisomal lipid beta-oxidation enzymes in guinea pig liver peroxisomes by immunoblotting and immunocytochemistry.

The effect of dimethylsulfoxide and other hydroxyl radical scavengers on the oxidation of ethanol by rat liver microsomes.

Effects of thiol antioxidants on the atropselective oxidation of 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) by rat liver microsomes.

Immunochemical study on the participation of cytochrome b5 in drug oxidation reactions of mouse liver microsomes.

Stereoselective oxidation metabolism of 20(S)-protopanaxatriol in human liver microsomes and in rats.

Inhibitory studies of mexiletine and dextromethorphan oxidation in human liver microsomes.

In vitro inhibition of dihydropyridine oxidation and aflatoxin B1 activation in human liver microsomes by naringenin and other flavonoids.

Propranolol oxidation by human liver microsomes--the use of cumene hydroperoxide to probe isoenzyme specificity and regio- and stereoselectivity.

Purification and characterization of a vitamin D3 25-hydroxylase from pig liver microsomes.