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Acetone enhancement of microsomal aniline para-hydroxylase activity
ARCHIVES
OF
BIOCHEMISTRY
Acetone
AND
BIOPHYSICS
126, 269-275 (1968)
Enhancement
of Microsomal
Para-Hydroxylase
Aniline
Activity’
M. W. ANDERS Department
of Physiology, Biochemistry and Pharmacology, New York Stale Veterinary University, Ithaca, New York f&SO
College, Cornell
Received November 17, 1967; accepted January 29, 1968 Acetone in concentrations of 0.045-1.8 M markedly enhanced the para-hydroxylation of aniline by hepatic microsomal fractions derived from rats, mice, rabbits and dogs. Similarly acetone enhanced the hydroxylation of acetanilide and N-butylaniline but not the N-demethylation of ethylmorphine, N-methyl or N,N-dimethylaniline nor the 0-demethylation of p-nitroanisole. The degree of enhancement increased with increasing pH. In the presence of acetone, both the Michaelis constant and maximal velocity for the hydroxylation of aniline increased. SKF 525-A and piperonyl butoxide were more potent inhibitors of aniline hydroxylation in the presence of acetone. Finally, evidence is presented that acetone produces its enhancement of aniline hydroxylation by a mechanism different from that of ethyl isocyanide.
Recent studies in this laboratory have been concerned with the inhibitory actions of pesticides on hepatic microsomal drug metabolism (EC No. 1.99.1.1). During these studies it was necessary because of solubility considerations to add the inhibitor to the reaction mixture in a small volume of an organic solvent. It was found, however, that the rate of aniline hydroxylation increased when acetone was added to the reaction mixture. This finding was unexpected in view of other experiments which showed that the microsomal N-demethylation of ethy.lmorphine and 0-demethylation of p-nitroanisole was sensitive to inhibition by solvents. Furthermore, since the hepatic mlcrosomal drug metabolizing enzyme system has been characterized by a rather marked lability in vitro it was surprising to find the system active in the presence of acetone. This paper presents results of experiments conducted i.n order to characterize the in vitro acetone-induced enhancement of the hepatic microsomal aromatic hydroxylase. 1A preliminary report of this work appeared in The Pharmacologist 9, 191 (1967).
Furthermore, these studies might prove useful in finding a means to purify or solubilize the tightly bound, labile microsomal hydroxylase. MATERIALS
AND
METHODS
Chemicals. Reagents were used as obtained from the suppliers with the exception of N-methylaniline hydrochloride which was recrystallized from benzene. The acetone used in these experiments was obtained from Matheson, Coleman and Bell and was 99.7% pure. All water used in these studies was distilled from glass and then redistilled from ethylenediaminetetraacetic acid (EDTA) solution. Tissue preparation. Livers from male albino Holtzman rats (75-125 g), male albino mice (3040 g), male New Zealand white rabbits (2-3 kg) and female mongrel dogs (1520 kg) were employed as the source of the microsomal enzymes. The mice, rats or rabbits were stunned by a blow on the head, decapitated, allowed to exsanguinate and the livers rapidly removed; livers were removed from dogs under pentobarbital anesthesia. After removal the livers were placed in ice-cold 0.25 M sucrose solution. All further procedures for the preparation of the enzyme were carried out in the cold (O-4”). A 25% homogenate in sucrose solution was prepared using 15 strokes with a Dounce homogenizer fitted with a loose
270
ANDERS
differed in their response t)o acetone. The rat showed the greatest enhancement, the rabbit was intermediate and the mouse and dog the lowest. No enhancement of aniline metabolism by acetone was observed when boiled microsomal fractions were employed, when the reaction was carried out at 0” or when acetone was added at the end of the incubation period. Furthermore, acetone did not produce any increase in the rate of aniline hydroxylation when a model system consisting of Fe++-, ascorbic acid and EDTA (7) was employed instead of microsomal enzymes. Effect of acetone on metabolism of substrates other than aniline. To determine whether the enhancement produced by acetone was of a general nature or restricted only to aniline, experiments were conducted with several substrat.es. The results presented in Table I show that acetone enhanced microsomal para-hydroxylase activity toward acetanilide and N-butylaniline as well as toward aniline. In other experiments it was observed that acetone produced no enhancement of the N-demethylation of N-methylaniline, N, N-dimethylaniline or ethylmorphine nor of the 0-demethylation of p-nitroanisole, only inhibition being observed asthe acetone concentration was increased. E$ect of solventson aniline metabolism.To test whether the enhancing effect of acetone was unique, a number of solvents and carbonyl compounds were examined for their effect on aniline hydroxylation. These results showed that ethanol, dimethylsulfoxide, dimethylformamide and methyl cellusolve produced only inhibition of aniline hydroxylatior, . Various carbonyl compounds RESULTS at a concentration of 0.045 M gave the folEffect of acetoneon aniline metabolism.As lowing results (+ = enhancement, - = can be seen in Fig. 1, acetone in concentra- inhibition) : acetone, +35 %; 2-butanone, tions of 0.045-1.36 M produced an increase - 14%; 2-pentanone, +54 %; 3-pentanone, in the rate of aniline hydroxylation in the +2 % ; pentane-2,4-dione, - 86 % ; acerat,, rabbit, mouse, and dog. While maximal taldehyde, - 59 % ; propionaldehyde, - 75 % enhancement occurred between concentra- butyraldehyde, - 72 % and valeraldehyde, tions of 0.45 to 0.9 M acetone as little as -84%. E$ect oj acetone on kinetics of aniline 0.0045 M acetone resulted in a detectable hydroxylaticm. Table II shows the effect of increase in the rate of aniline metabolism. two concentrations of acetone on the In addition, Fig. 1 shows that microsomal fractions derived from various species Michaelis constant (K,) and maximal plunger. The homogenate was centrifuged at 9000 gmrr for 20 minutes. The supernatant was centriThe superfuged at 105,000 g,,, for 60 minutes. natant was discarded and the microsomal pellet resuspended in sucrose solution and diluted so that 1 ml of solution contained the equivalent of 300 mg of liver. This microsomal preparation was stored frozen until used but not for more than 10 days. Incubation mixture. Urlless otherwise indicated, incubat,ion mixtures contained 3 pmoles of substrate, 3 pmoles of nicotinamide, 15 Mmoles of magnesium chloride, 50 rmoles of phosphate buffer, pH 7.4, a TPNH generating system consisting of either 20 pmoles of glucose-6-phosphate, 1 pmole of TPN and 1 EU of glucose-6-phosphate dehydrogenase or 10 pmoles of nL-isocitrate, 1 pmole TPN and 1 EU of isocitrate dehydrogenase, and 0.5 ml of the microsomal enzyme preparation (rat) in a total volume of 3.0 ml. When formaldehyde was sought as the product the incubation mixture contained 3 rmoles of neutralized semicarbazide. Incubations were carried out at 37” in an atmosphere of air and for 30 minutes during which time p-aminophenol formation from aniline was linear with time. Determination of enzyme activity. Formaldehyde produced by oxidative N-demethylation was measured according to the method of Nash (1). p-Aminophenol and p-hydroxy-N-butylaniline were measured by the method described by Kato and Gillette (2) and p-hydroxyacetanilide by the method of Krisch and Staudinger (3). Carbon monoxide and ethyl isocyanide difference spectra were measured as described by Omura and Sat0 (4). Data processing. Enzyme kinetic constants were determined using the Fortran programs written by Cleland (5). Other statistical analyses used have been described by Steel and Torrie (6). Computation of the various statistical parameters was performed with a digital computer using Fortran programs written in this laboratory.
ENHANCEMENT
OF ANILINE
HYDROXYLATION
271
O-Rat
45t
l - Rabbit A- Mouse X-Dog
4,oc
.i? .? ta
2.01
.-4 G z
I, 5c
l.O(
0.5c
1
I
I
log
Gblorily
I
0
0
(Acetone)
FIG. 1. Effect of varying concentrations of acetone on aniline hydroxylation in several species. Conditions were as described under Materials and Methods. Control rates of aniline hydroxylation, expressed as hmoles p-aminophenol/g (wet weight) liver/hour, were as follows: rat-1.3, rabbit-0.2, mouse-l.8 and dog-0.2.
velocity (V,,,,) of aniline hydroxylation. It can be seen that acetone at concentrations of 0.045 and 0.45 M significantly increased the K, from an initial value of 6.9 X 1O-5 M to values of 13.0 X 10e5 (p < 0.001) and 19.9 X 1O-5 M (p < O.OOl), respectively. Similarly, the V,, increased significantly from 1.4 pmoles/g/hour in the absence of acetone to 2.1 (p < 0.02) and 3.9 (p < 0.001) pmoles/g/hour in the presence of 0.045 and 0.45 M acetone, respectively. Inhibition! studiesin the presenceof acetme.
Results of experiments in which the effect of acetone on the activity of two inhibitors of aniline hydroxylase was examined are given in Table III. Acetone at a concentration of 0.045 M effected a significant (p < 0.02) decreasein the Lo of SKF 525-A from 4.4 X 1O-3M (no acetone) to 0.05 X 1O-3M. In a similar experiment in which piperonyl butoxide served as the inhibitor the I,, changed from 2.8 X 1O-3 M (no acetone) to 0.06 X 1O-3 M (p < 0.02) in the presence of 0.045 M acetone.
272
ANDERS TABLE
I
TABLE
EFFECT OF ACETONE ON MICROSOMAL PARA-HYDROXYLASE ACTIVITY The incubation mixtures (rat liver microsomes) were prepared and analyzed as described under Materials and Methods and contained 0.45 M acetone. Control rates of hydroxylation, expressed as rmoles product/g (wet weight) liver/hour, were as follows : aniline-0.8, acetanilide-0.7 and A’-butylaniline-O.2. Substrate
% Enhancement
Aniline Acetanilide N-Butylaniline
213 166 48
TABLE
None 0.045 0.45
6.9 13.0 19.9
CM x
f f f
10’)
0.7(7) 0.7(4)0 0.7(3)”
vmx
1.4 2.1 3.9
n Significantly different (p < 0.001) obtained in the absence of acetone. b Significantly different (p < 0.02) obtained in the absence of acetone.
f f f
Is0 CM x SKF
4.4 0.05
f f
109
525-A
Piperonyl
butoxide
1.3(6)” O.Ol(3)”
2.8 0.06
1.2(4) 0.01(3)e
a Significantly different (p < 0.02) obtained in the absence of acetone.
The incubation mixtures (rat liver microsomes) were prepared and analyzed as described under Materials and Methods except that the aniline concentrations employed were 0.1, 0.2, 0.3, 0.5 and 1.0 mM. The maximal velocity is expressed as @moles p-aminophenol formed/g (wet weight) liver/hour. The values are shown as the mean f SEM; the numbers in parentheses refer to the number of determinations. Km
The incubation mixtures (rat liver microsomes) were prepared and analyzed as described under Materials and Methods. Inhibitor concentrations ranged from 5 X 10-a M to 5 X 10e5 M for SKF 525-A and from 1 X 10-z M to 1 X 1O-5 M for piperonyl butoxide. Values are shown as the mean f SEM; the numbers in parentheses refer to the number of determinations.
None 0.045
II
III
OF ACETONE ON INHIBITION OF ANILINE HYDROXYLATION BY SKF 525-A AND PIPERONYL BUTOXIDE
Acetone concentration (M)
EFFECT OF ACETONE ON THE KINETICS OF ANILINE HYDROXYLATION
Acetone concentration CM)
EFFECT
0.1(7) 0.2(4)* 0.3(3)” from
value
from
value
E$ect of incubation conditions on acetone enhancement. It was observed that the degree of enhancement due to acetone was dependent on the pH of the incubation medium. As shown in Fig. 2 the enhancement. of aniline hydroxylation increased as the pH of the incubation mixture was increased. The pH optimum for aniline hydroxylation in the absence of acetone was found to be 7.5. The effect of employing an atmosphere of 100% oxygen rather than air in the incubation vessel was also examined. In the presence of 0.45 M acetone enhancements
f f from
value
of 182% and 171% were obtained in an atmosphere of air and 100% oxygen, respectively. E$ect of acetone and ethyl isocyanide on aniline hydroxylation. Ethyl isocyanide has been shown to enhance the microsomal hydroxylation of aniline (8). Experiments designed to test for possible int’errelationships between acetone and ethyl isocyanide are reported in Table IV. Of primary interest was whether ethyl isocyanide would still produce an enhancement of aniline hydroxylation in the presence of maximally enhancing concentrations of acetone. It can be seen that while both acetone and ethyl isocyanide produce an enhancement of aniline hydroxylation, ethyl isocyanide was still able to produce its enhancement in the presence of acetone. Furthermore, acetone did not produce any change in either the ethyl isocyanide absorption spectrum at varying pH values or in the P-450 absorption spectrum. DISCUSSION
Enhancement of the activity of microsomal drug metabolizing enzymes in vitro has been reported before. Imai and Sato (8) showed that ethyl isocyanide produced an activation of rabbit hepatic microsomal aniline hydroxylase. It should be pointed out that activation has also been observed
ENHANCEMENT
OF
ANILINE
273
HYDROXYLATION
.6 _
!.4 _
.2 _
!.O _
s I I
I
6.0
6.5
7.0
I
f.5
1
6.0
PH 2. Effect of pH on the enhancement of aniline hydroxylation by acetone. Conditions were as described under Materials and Methods (rat liver microsomes) except that 100 pmoles of buffer at differing pH values was employed. The incubation medium contained 0.45 M acetone, Control rates of aniline hydroxylation, expressed as pmoles p-aminophenol/g (wet weight) liver/hour were as follows: pH 6.0-0.2, pH 6.5-0.5, pH 7SO.7, pH 7.50.6 and pH 8.0-0.4. FIG.
TABLE EFFECT
IV
OF ACETONE AND ETHYL ANILINE HYDROXYLATION HEPATIC
ISOCYANIDE BY RABBIT
ON
MICROSOMES
The incubation mixtures were prepared as described under Materials and Methods except that the microsomal fraction of rabbit livers was employed and the mixtures contained the concentrations of acetone and ethyl isocyanide shown in the table. Control rate of aniline hydroxylation was 0.2 pmoles p-aminophenollg (wet weight) liver/hour. Acetone concentration
0.45 0.90 1.35 None 0.45 0.90 1.35
i(M)
Ethyl isoganide COncentratlOn (M)
None None None 0.01 0.01 0.01 0.01
% Enhancement
163 200 142 95 242 237 174
with other hepatic microsomal enzymes. For example, glucose-6-phosphatase activity is enhanced by the presence of various detergents (9) and that of glucuronyl transferase by storage at 0” or by detergents (10).
The enhancement observed with acetone appears to be operative in the case of aromatic hydroxylation only (Table I) since no increase was detected when compounds undergoing N- or 0-demethylation served as the substrate. The finding that the hydroxylation of acetanilide and N-butylaniline is enhanced by acetone tends to rule out any participation of a Schiff base intermediate between acetone and aniline in the observed enhancement. That only two of the solvents tested, acetone and 2-pentanone, produced an enhancement is difficult to expIain and does not appear to correlat’e with chemical and
274
ANDERS
physical properties of the solvents. Clearly the ability of a compound to produce an enhancement is not solely contingent on the presence of a carbonyl group since many such compounds produced only inhibition. By the same token, the dielectric constant of the solvent does not appear to be a factor since both those compounds that enhance and those that inhibit have similar values. A similar unexplainable specificity has been observed in the case of the activation of pyrophosphate-glucose phosphotransferase where certain nonionic detergents (Triton X-100) activate while others (Tween 20 and SO) inhibit (9). The demonstration of the activation of aniline hydroxylase by ethyl isocyanide (8) posed the question of whether acetone and ethyl isocyanide might produce their effects by a similar mechanism. The effect of acetone was found to be dependent upon the pH of the reaction mixture (Fig. 2), more enhancement being observed at higher pH values. In this respect the enhancements due to acetone and ethyl isocyanide appeared to be similar. Since the activating effect of ethyl isocyanide had been shown to increase as the oxygen concentration increased it was of interest to examine the effect of acetone with respect to oxygen concentration. It was found, however, that the action of acetone was similar whether air or 100% oxygen was used as the gas phase. This, then, suggested that the two compounds produced their effects in a different manner. Further evidence for this view has been obtained. If acetone and ethyl isocyanide act in a similar fashion it should not be possible to produce an additional enhancement with ethyl isocyanide in the presence of maximally enhancing concentrations of acetone. The results presented in Table II show, however, that ethyl isocyanide is able to produce its enhancement in the presence of high concentrations of acetone. These results indicate that acetone and ethyl isocyanide activate microsomal hydroxylases by different mechanisms. Kinetic studies were conducted with the hope that the results might reveal something of the mechanism of enhancement. The finding that the K, for aniline hydroxylation is changed in the presence of acetone (Table
II) suggests that the mechanism cannot be explained by a simple increase in the amount of hydroxylase, with the same kinetic properties, perhaps exposed by acet’one-induced structural alterations. Rather the data shows that in the presence of acetone the hydroxylase possesses kinetic properties different from those of the native enzyme. This could be brought about, for example, by “unmasking” new active sites with different characteristics. Alternatively, acetone might alter the properties of the hydroxylase present in situ perhaps by changing the spatial configuration of the microsomes or by interacting with specific functional groups. Similar results might be obtained if two aniline hydroxylases, differing in their response to acetone and possessing different kinetic properties, were present in hepatic microsomes. One of these hydroxylases might be characterized by a low Km and V,,, for aniline as well as a high susceptibility to inhibition by acetone while the other might possess both a high K, and V In&Xfor aniline and be relatively insensitive to the inhibitory actions of acetone. In the absence of acetone the activity of former enzyme would tend to predominate because of more favorable kinetic properties. In the presence of acetone, however, this enzyme would be inhibited and the properties of the latter enzyme would become apparent, i.e. the Km and V,,, would increase. Such an explanation could also account for the increased potency of inhibitors in the presence of acetone since the two hydroxylases might differ markedly in their susceptibility to inhibition by SKF 525-A and piperonyl butoxide. Finally as Webb (11) has pointed out, an activator, acetone in this case, might act by increasing the rate of breakdown of an enzyme-activator-substrate complex relative to t’he enzyme-substrate complex. If this is indeed the case the ratio: K,/V,,, should remain constant in the presence of varying concentrations of activator. Inspection of the data in Table II shows that this is the case the ratio being 4.9 X 10-5, 6.2 X 1O-5 and 5.1 X 10-j in the presence of 0.0, 0.045, and 0.45 M acetone, respectively. This is consistent with a more rapid breakdown of the enzyme-activator-substrate complex since t’he rate constant for
ENHANCEMENT
OF
ANILINE
this step is a determinant of both K, and V Inax* It is, however, not possible to make a choice between these alternatives on the basis of the available data. The finding that acetone produced no change in the ethyl isocyanide ‘or P-450 absorption spectrum indicates that the altered hydroxylase functions as a, typical mixed-function oxidase. The inhibition studies conducted in the presence of acetone (Table III) support the contention that the hydroxylase is altered in some manner. Both SKF 525-A and piperonyl butoxide were much more potent inhibitors in the presence of acetone. This would not be the expected result if acetone produced only a simple increase in the amount of enzyme concentration. If, however, a new or altered hydroxylase is being made available through the action of acetone it might be expected that this enzyme would differ from the native enzyme in its susceptibility to inhibition. ACKNOWLEDGMENTS The author his stimulating
is indebted to Dr. J. F. Wootton for discussions and Mrs. Robert
27.5
HYDROXYLATION Blauth and Miss technical assistance. U.S.P.H.S. Grants
Phyllis Pautz for their able This work was supported by No. GM 13527 and FR 05462.
REFERENCES 1. NASH, 2. KATO, 3. 4. 5. 6.
7.
8. 9. 10.
T., Biochem. J. 66, 416 (1953). R. AND GILLETTE, J. R., J. Pharmacol. Ezptl. Therap. 159,279 (1965). KRISCH, K. AND STAUDINGER, H., Biochem. 2. 334, 312 (1961). OMURA, T. AND SATO, R., J. Biol. Chem. 239, 2370 (1964). CLELAND, W. W., Nature 198, 463 (1963). STEEL, R. G. D. AND TORRIE, J. H., “Principles and Procedures of Statistics,” McGraw-Hill, New York (1960). UDENFRIEND, S., CLARKE, C. T., AXELROD, J. AND BRODIE, B. B., J. Biol. Chem. 203, 731 (1954). IMAI, Y. AND SATO, R., Biochem. Biophys. Res. Commun. 26,80 (1966). SNOKE, R. E. AND NORDLIE, R. C., Biochim. Biophys. Acta 139, 190 (1967). LUEDERS, K. K. AND KUFF, E. L., Arch.
Biochem. Biophys. 120, 198 (1967). 11. WEBB, J. L., “Enzyme and Metabolic hibitors,” Vol. 1, p. 46, Academic New York (1963).
InPress,
OF
BIOCHEMISTRY
Acetone
AND
BIOPHYSICS
126, 269-275 (1968)
Enhancement
of Microsomal
Para-Hydroxylase
Aniline
Activity’
M. W. ANDERS Department
of Physiology, Biochemistry and Pharmacology, New York Stale Veterinary University, Ithaca, New York f&SO
College, Cornell
Received November 17, 1967; accepted January 29, 1968 Acetone in concentrations of 0.045-1.8 M markedly enhanced the para-hydroxylation of aniline by hepatic microsomal fractions derived from rats, mice, rabbits and dogs. Similarly acetone enhanced the hydroxylation of acetanilide and N-butylaniline but not the N-demethylation of ethylmorphine, N-methyl or N,N-dimethylaniline nor the 0-demethylation of p-nitroanisole. The degree of enhancement increased with increasing pH. In the presence of acetone, both the Michaelis constant and maximal velocity for the hydroxylation of aniline increased. SKF 525-A and piperonyl butoxide were more potent inhibitors of aniline hydroxylation in the presence of acetone. Finally, evidence is presented that acetone produces its enhancement of aniline hydroxylation by a mechanism different from that of ethyl isocyanide.
Recent studies in this laboratory have been concerned with the inhibitory actions of pesticides on hepatic microsomal drug metabolism (EC No. 1.99.1.1). During these studies it was necessary because of solubility considerations to add the inhibitor to the reaction mixture in a small volume of an organic solvent. It was found, however, that the rate of aniline hydroxylation increased when acetone was added to the reaction mixture. This finding was unexpected in view of other experiments which showed that the microsomal N-demethylation of ethy.lmorphine and 0-demethylation of p-nitroanisole was sensitive to inhibition by solvents. Furthermore, since the hepatic mlcrosomal drug metabolizing enzyme system has been characterized by a rather marked lability in vitro it was surprising to find the system active in the presence of acetone. This paper presents results of experiments conducted i.n order to characterize the in vitro acetone-induced enhancement of the hepatic microsomal aromatic hydroxylase. 1A preliminary report of this work appeared in The Pharmacologist 9, 191 (1967).
Furthermore, these studies might prove useful in finding a means to purify or solubilize the tightly bound, labile microsomal hydroxylase. MATERIALS
AND
METHODS
Chemicals. Reagents were used as obtained from the suppliers with the exception of N-methylaniline hydrochloride which was recrystallized from benzene. The acetone used in these experiments was obtained from Matheson, Coleman and Bell and was 99.7% pure. All water used in these studies was distilled from glass and then redistilled from ethylenediaminetetraacetic acid (EDTA) solution. Tissue preparation. Livers from male albino Holtzman rats (75-125 g), male albino mice (3040 g), male New Zealand white rabbits (2-3 kg) and female mongrel dogs (1520 kg) were employed as the source of the microsomal enzymes. The mice, rats or rabbits were stunned by a blow on the head, decapitated, allowed to exsanguinate and the livers rapidly removed; livers were removed from dogs under pentobarbital anesthesia. After removal the livers were placed in ice-cold 0.25 M sucrose solution. All further procedures for the preparation of the enzyme were carried out in the cold (O-4”). A 25% homogenate in sucrose solution was prepared using 15 strokes with a Dounce homogenizer fitted with a loose
270
ANDERS
differed in their response t)o acetone. The rat showed the greatest enhancement, the rabbit was intermediate and the mouse and dog the lowest. No enhancement of aniline metabolism by acetone was observed when boiled microsomal fractions were employed, when the reaction was carried out at 0” or when acetone was added at the end of the incubation period. Furthermore, acetone did not produce any increase in the rate of aniline hydroxylation when a model system consisting of Fe++-, ascorbic acid and EDTA (7) was employed instead of microsomal enzymes. Effect of acetone on metabolism of substrates other than aniline. To determine whether the enhancement produced by acetone was of a general nature or restricted only to aniline, experiments were conducted with several substrat.es. The results presented in Table I show that acetone enhanced microsomal para-hydroxylase activity toward acetanilide and N-butylaniline as well as toward aniline. In other experiments it was observed that acetone produced no enhancement of the N-demethylation of N-methylaniline, N, N-dimethylaniline or ethylmorphine nor of the 0-demethylation of p-nitroanisole, only inhibition being observed asthe acetone concentration was increased. E$ect of solventson aniline metabolism.To test whether the enhancing effect of acetone was unique, a number of solvents and carbonyl compounds were examined for their effect on aniline hydroxylation. These results showed that ethanol, dimethylsulfoxide, dimethylformamide and methyl cellusolve produced only inhibition of aniline hydroxylatior, . Various carbonyl compounds RESULTS at a concentration of 0.045 M gave the folEffect of acetoneon aniline metabolism.As lowing results (+ = enhancement, - = can be seen in Fig. 1, acetone in concentra- inhibition) : acetone, +35 %; 2-butanone, tions of 0.045-1.36 M produced an increase - 14%; 2-pentanone, +54 %; 3-pentanone, in the rate of aniline hydroxylation in the +2 % ; pentane-2,4-dione, - 86 % ; acerat,, rabbit, mouse, and dog. While maximal taldehyde, - 59 % ; propionaldehyde, - 75 % enhancement occurred between concentra- butyraldehyde, - 72 % and valeraldehyde, tions of 0.45 to 0.9 M acetone as little as -84%. E$ect oj acetone on kinetics of aniline 0.0045 M acetone resulted in a detectable hydroxylaticm. Table II shows the effect of increase in the rate of aniline metabolism. two concentrations of acetone on the In addition, Fig. 1 shows that microsomal fractions derived from various species Michaelis constant (K,) and maximal plunger. The homogenate was centrifuged at 9000 gmrr for 20 minutes. The supernatant was centriThe superfuged at 105,000 g,,, for 60 minutes. natant was discarded and the microsomal pellet resuspended in sucrose solution and diluted so that 1 ml of solution contained the equivalent of 300 mg of liver. This microsomal preparation was stored frozen until used but not for more than 10 days. Incubation mixture. Urlless otherwise indicated, incubat,ion mixtures contained 3 pmoles of substrate, 3 pmoles of nicotinamide, 15 Mmoles of magnesium chloride, 50 rmoles of phosphate buffer, pH 7.4, a TPNH generating system consisting of either 20 pmoles of glucose-6-phosphate, 1 pmole of TPN and 1 EU of glucose-6-phosphate dehydrogenase or 10 pmoles of nL-isocitrate, 1 pmole TPN and 1 EU of isocitrate dehydrogenase, and 0.5 ml of the microsomal enzyme preparation (rat) in a total volume of 3.0 ml. When formaldehyde was sought as the product the incubation mixture contained 3 rmoles of neutralized semicarbazide. Incubations were carried out at 37” in an atmosphere of air and for 30 minutes during which time p-aminophenol formation from aniline was linear with time. Determination of enzyme activity. Formaldehyde produced by oxidative N-demethylation was measured according to the method of Nash (1). p-Aminophenol and p-hydroxy-N-butylaniline were measured by the method described by Kato and Gillette (2) and p-hydroxyacetanilide by the method of Krisch and Staudinger (3). Carbon monoxide and ethyl isocyanide difference spectra were measured as described by Omura and Sat0 (4). Data processing. Enzyme kinetic constants were determined using the Fortran programs written by Cleland (5). Other statistical analyses used have been described by Steel and Torrie (6). Computation of the various statistical parameters was performed with a digital computer using Fortran programs written in this laboratory.
ENHANCEMENT
OF ANILINE
HYDROXYLATION
271
O-Rat
45t
l - Rabbit A- Mouse X-Dog
4,oc
.i? .? ta
2.01
.-4 G z
I, 5c
l.O(
0.5c
1
I
I
log
Gblorily
I
0
0
(Acetone)
FIG. 1. Effect of varying concentrations of acetone on aniline hydroxylation in several species. Conditions were as described under Materials and Methods. Control rates of aniline hydroxylation, expressed as hmoles p-aminophenol/g (wet weight) liver/hour, were as follows: rat-1.3, rabbit-0.2, mouse-l.8 and dog-0.2.
velocity (V,,,,) of aniline hydroxylation. It can be seen that acetone at concentrations of 0.045 and 0.45 M significantly increased the K, from an initial value of 6.9 X 1O-5 M to values of 13.0 X 10e5 (p < 0.001) and 19.9 X 1O-5 M (p < O.OOl), respectively. Similarly, the V,, increased significantly from 1.4 pmoles/g/hour in the absence of acetone to 2.1 (p < 0.02) and 3.9 (p < 0.001) pmoles/g/hour in the presence of 0.045 and 0.45 M acetone, respectively. Inhibition! studiesin the presenceof acetme.
Results of experiments in which the effect of acetone on the activity of two inhibitors of aniline hydroxylase was examined are given in Table III. Acetone at a concentration of 0.045 M effected a significant (p < 0.02) decreasein the Lo of SKF 525-A from 4.4 X 1O-3M (no acetone) to 0.05 X 1O-3M. In a similar experiment in which piperonyl butoxide served as the inhibitor the I,, changed from 2.8 X 1O-3 M (no acetone) to 0.06 X 1O-3 M (p < 0.02) in the presence of 0.045 M acetone.
272
ANDERS TABLE
I
TABLE
EFFECT OF ACETONE ON MICROSOMAL PARA-HYDROXYLASE ACTIVITY The incubation mixtures (rat liver microsomes) were prepared and analyzed as described under Materials and Methods and contained 0.45 M acetone. Control rates of hydroxylation, expressed as rmoles product/g (wet weight) liver/hour, were as follows : aniline-0.8, acetanilide-0.7 and A’-butylaniline-O.2. Substrate
% Enhancement
Aniline Acetanilide N-Butylaniline
213 166 48
TABLE
None 0.045 0.45
6.9 13.0 19.9
CM x
f f f
10’)
0.7(7) 0.7(4)0 0.7(3)”
vmx
1.4 2.1 3.9
n Significantly different (p < 0.001) obtained in the absence of acetone. b Significantly different (p < 0.02) obtained in the absence of acetone.
f f f
Is0 CM x SKF
4.4 0.05
f f
109
525-A
Piperonyl
butoxide
1.3(6)” O.Ol(3)”
2.8 0.06
1.2(4) 0.01(3)e
a Significantly different (p < 0.02) obtained in the absence of acetone.
The incubation mixtures (rat liver microsomes) were prepared and analyzed as described under Materials and Methods except that the aniline concentrations employed were 0.1, 0.2, 0.3, 0.5 and 1.0 mM. The maximal velocity is expressed as @moles p-aminophenol formed/g (wet weight) liver/hour. The values are shown as the mean f SEM; the numbers in parentheses refer to the number of determinations. Km
The incubation mixtures (rat liver microsomes) were prepared and analyzed as described under Materials and Methods. Inhibitor concentrations ranged from 5 X 10-a M to 5 X 10e5 M for SKF 525-A and from 1 X 10-z M to 1 X 1O-5 M for piperonyl butoxide. Values are shown as the mean f SEM; the numbers in parentheses refer to the number of determinations.
None 0.045
II
III
OF ACETONE ON INHIBITION OF ANILINE HYDROXYLATION BY SKF 525-A AND PIPERONYL BUTOXIDE
Acetone concentration (M)
EFFECT OF ACETONE ON THE KINETICS OF ANILINE HYDROXYLATION
Acetone concentration CM)
EFFECT
0.1(7) 0.2(4)* 0.3(3)” from
value
from
value
E$ect of incubation conditions on acetone enhancement. It was observed that the degree of enhancement due to acetone was dependent on the pH of the incubation medium. As shown in Fig. 2 the enhancement. of aniline hydroxylation increased as the pH of the incubation mixture was increased. The pH optimum for aniline hydroxylation in the absence of acetone was found to be 7.5. The effect of employing an atmosphere of 100% oxygen rather than air in the incubation vessel was also examined. In the presence of 0.45 M acetone enhancements
f f from
value
of 182% and 171% were obtained in an atmosphere of air and 100% oxygen, respectively. E$ect of acetone and ethyl isocyanide on aniline hydroxylation. Ethyl isocyanide has been shown to enhance the microsomal hydroxylation of aniline (8). Experiments designed to test for possible int’errelationships between acetone and ethyl isocyanide are reported in Table IV. Of primary interest was whether ethyl isocyanide would still produce an enhancement of aniline hydroxylation in the presence of maximally enhancing concentrations of acetone. It can be seen that while both acetone and ethyl isocyanide produce an enhancement of aniline hydroxylation, ethyl isocyanide was still able to produce its enhancement in the presence of acetone. Furthermore, acetone did not produce any change in either the ethyl isocyanide absorption spectrum at varying pH values or in the P-450 absorption spectrum. DISCUSSION
Enhancement of the activity of microsomal drug metabolizing enzymes in vitro has been reported before. Imai and Sato (8) showed that ethyl isocyanide produced an activation of rabbit hepatic microsomal aniline hydroxylase. It should be pointed out that activation has also been observed
ENHANCEMENT
OF
ANILINE
273
HYDROXYLATION
.6 _
!.4 _
.2 _
!.O _
s I I
I
6.0
6.5
7.0
I
f.5
1
6.0
PH 2. Effect of pH on the enhancement of aniline hydroxylation by acetone. Conditions were as described under Materials and Methods (rat liver microsomes) except that 100 pmoles of buffer at differing pH values was employed. The incubation medium contained 0.45 M acetone, Control rates of aniline hydroxylation, expressed as pmoles p-aminophenol/g (wet weight) liver/hour were as follows: pH 6.0-0.2, pH 6.5-0.5, pH 7SO.7, pH 7.50.6 and pH 8.0-0.4. FIG.
TABLE EFFECT
IV
OF ACETONE AND ETHYL ANILINE HYDROXYLATION HEPATIC
ISOCYANIDE BY RABBIT
ON
MICROSOMES
The incubation mixtures were prepared as described under Materials and Methods except that the microsomal fraction of rabbit livers was employed and the mixtures contained the concentrations of acetone and ethyl isocyanide shown in the table. Control rate of aniline hydroxylation was 0.2 pmoles p-aminophenollg (wet weight) liver/hour. Acetone concentration
0.45 0.90 1.35 None 0.45 0.90 1.35
i(M)
Ethyl isoganide COncentratlOn (M)
None None None 0.01 0.01 0.01 0.01
% Enhancement
163 200 142 95 242 237 174
with other hepatic microsomal enzymes. For example, glucose-6-phosphatase activity is enhanced by the presence of various detergents (9) and that of glucuronyl transferase by storage at 0” or by detergents (10).
The enhancement observed with acetone appears to be operative in the case of aromatic hydroxylation only (Table I) since no increase was detected when compounds undergoing N- or 0-demethylation served as the substrate. The finding that the hydroxylation of acetanilide and N-butylaniline is enhanced by acetone tends to rule out any participation of a Schiff base intermediate between acetone and aniline in the observed enhancement. That only two of the solvents tested, acetone and 2-pentanone, produced an enhancement is difficult to expIain and does not appear to correlat’e with chemical and
274
ANDERS
physical properties of the solvents. Clearly the ability of a compound to produce an enhancement is not solely contingent on the presence of a carbonyl group since many such compounds produced only inhibition. By the same token, the dielectric constant of the solvent does not appear to be a factor since both those compounds that enhance and those that inhibit have similar values. A similar unexplainable specificity has been observed in the case of the activation of pyrophosphate-glucose phosphotransferase where certain nonionic detergents (Triton X-100) activate while others (Tween 20 and SO) inhibit (9). The demonstration of the activation of aniline hydroxylase by ethyl isocyanide (8) posed the question of whether acetone and ethyl isocyanide might produce their effects by a similar mechanism. The effect of acetone was found to be dependent upon the pH of the reaction mixture (Fig. 2), more enhancement being observed at higher pH values. In this respect the enhancements due to acetone and ethyl isocyanide appeared to be similar. Since the activating effect of ethyl isocyanide had been shown to increase as the oxygen concentration increased it was of interest to examine the effect of acetone with respect to oxygen concentration. It was found, however, that the action of acetone was similar whether air or 100% oxygen was used as the gas phase. This, then, suggested that the two compounds produced their effects in a different manner. Further evidence for this view has been obtained. If acetone and ethyl isocyanide act in a similar fashion it should not be possible to produce an additional enhancement with ethyl isocyanide in the presence of maximally enhancing concentrations of acetone. The results presented in Table II show, however, that ethyl isocyanide is able to produce its enhancement in the presence of high concentrations of acetone. These results indicate that acetone and ethyl isocyanide activate microsomal hydroxylases by different mechanisms. Kinetic studies were conducted with the hope that the results might reveal something of the mechanism of enhancement. The finding that the K, for aniline hydroxylation is changed in the presence of acetone (Table
II) suggests that the mechanism cannot be explained by a simple increase in the amount of hydroxylase, with the same kinetic properties, perhaps exposed by acet’one-induced structural alterations. Rather the data shows that in the presence of acetone the hydroxylase possesses kinetic properties different from those of the native enzyme. This could be brought about, for example, by “unmasking” new active sites with different characteristics. Alternatively, acetone might alter the properties of the hydroxylase present in situ perhaps by changing the spatial configuration of the microsomes or by interacting with specific functional groups. Similar results might be obtained if two aniline hydroxylases, differing in their response to acetone and possessing different kinetic properties, were present in hepatic microsomes. One of these hydroxylases might be characterized by a low Km and V,,, for aniline as well as a high susceptibility to inhibition by acetone while the other might possess both a high K, and V In&Xfor aniline and be relatively insensitive to the inhibitory actions of acetone. In the absence of acetone the activity of former enzyme would tend to predominate because of more favorable kinetic properties. In the presence of acetone, however, this enzyme would be inhibited and the properties of the latter enzyme would become apparent, i.e. the Km and V,,, would increase. Such an explanation could also account for the increased potency of inhibitors in the presence of acetone since the two hydroxylases might differ markedly in their susceptibility to inhibition by SKF 525-A and piperonyl butoxide. Finally as Webb (11) has pointed out, an activator, acetone in this case, might act by increasing the rate of breakdown of an enzyme-activator-substrate complex relative to t’he enzyme-substrate complex. If this is indeed the case the ratio: K,/V,,, should remain constant in the presence of varying concentrations of activator. Inspection of the data in Table II shows that this is the case the ratio being 4.9 X 10-5, 6.2 X 1O-5 and 5.1 X 10-j in the presence of 0.0, 0.045, and 0.45 M acetone, respectively. This is consistent with a more rapid breakdown of the enzyme-activator-substrate complex since t’he rate constant for
ENHANCEMENT
OF
ANILINE
this step is a determinant of both K, and V Inax* It is, however, not possible to make a choice between these alternatives on the basis of the available data. The finding that acetone produced no change in the ethyl isocyanide ‘or P-450 absorption spectrum indicates that the altered hydroxylase functions as a, typical mixed-function oxidase. The inhibition studies conducted in the presence of acetone (Table III) support the contention that the hydroxylase is altered in some manner. Both SKF 525-A and piperonyl butoxide were much more potent inhibitors in the presence of acetone. This would not be the expected result if acetone produced only a simple increase in the amount of enzyme concentration. If, however, a new or altered hydroxylase is being made available through the action of acetone it might be expected that this enzyme would differ from the native enzyme in its susceptibility to inhibition. ACKNOWLEDGMENTS The author his stimulating
is indebted to Dr. J. F. Wootton for discussions and Mrs. Robert
27.5
HYDROXYLATION Blauth and Miss technical assistance. U.S.P.H.S. Grants
Phyllis Pautz for their able This work was supported by No. GM 13527 and FR 05462.
REFERENCES 1. NASH, 2. KATO, 3. 4. 5. 6.
7.
8. 9. 10.
T., Biochem. J. 66, 416 (1953). R. AND GILLETTE, J. R., J. Pharmacol. Ezptl. Therap. 159,279 (1965). KRISCH, K. AND STAUDINGER, H., Biochem. 2. 334, 312 (1961). OMURA, T. AND SATO, R., J. Biol. Chem. 239, 2370 (1964). CLELAND, W. W., Nature 198, 463 (1963). STEEL, R. G. D. AND TORRIE, J. H., “Principles and Procedures of Statistics,” McGraw-Hill, New York (1960). UDENFRIEND, S., CLARKE, C. T., AXELROD, J. AND BRODIE, B. B., J. Biol. Chem. 203, 731 (1954). IMAI, Y. AND SATO, R., Biochem. Biophys. Res. Commun. 26,80 (1966). SNOKE, R. E. AND NORDLIE, R. C., Biochim. Biophys. Acta 139, 190 (1967). LUEDERS, K. K. AND KUFF, E. L., Arch.
Biochem. Biophys. 120, 198 (1967). 11. WEBB, J. L., “Enzyme and Metabolic hibitors,” Vol. 1, p. 46, Academic New York (1963).
InPress,