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Metabolism of acetanilide with hepatic microsomes and reconstituted cytochrome monoxygenase systems
ARCHIVES
OF BIQCHEMISTRY
AND
Metabolism
BIOPHYSICS
164,241-246
of Acetanilide
Reconstituted
Institute
of Arthritis,
with
Cytochrome
H. G. SELANDER,’ National
(1974)
Metabolism
Hepatic
Microsomes
Monoxygenase
and
Systems
D. M. JERINA, AND J. W. DALY and Digestive Diseases, National Maryland 20014
Institutes
of Health,
Bethesda,
Peceived February 5, 1974 Hepatic miscrosomes and reconstituted cytochrome monoxygenase systems from control rats and from rats that have been pretreated with phenobarbital or 3-methylcholanthrene convert radioactive acetanilide to ring-hydroxylated products, primarily I-hydroxyacetanilide. 3-Methylcholanthrene pretreatment results in the greatest enhancement of activity: cytochrome fractions from 3-methylcholanthrene-pretreated rats have many-fold higher activity than cytochrome fractions from control or phenobarbital-treated rats. The percentage of migration and retention of tritium (NIH Shift) measured in I-hydroxyacetanilide after enzymatic oxidation of 4- [SH]acetanilide is nearly identical using microsomes or the corresponding reconstituted system, but in both cases the percentage of migration and retention of tritum is markedly lower for preparations from 3-methylcholanthrene-treated animals with values of 25’S, as compared to the values of 40-60% for preparations from control or phenobarbital-treated animals. High-pressure liquid chromatography was employed for separation and quantitation of radioactive products.
The cytochrome P-450 class of hemoproteins2 serve as terminal monoxygenases for the conversion of endogenous and xenobiotic substances to a variety of metabolites in mammalian systems (for current reviews see Ref. l-3). The number and substrate specificity of cytochrome P-450 monoxygenases in hepatic preparations have been the subject of intensive investigation. Comparisons under various ‘Fellow in the Visiting Program of the Public Health Service. * The term “cytochrome P-450” refers to the carbon monoxide-binding hemoprotein which is present in the livers of control and phenobarbital-treated animals and is involved in oxidative metabolism, while the term “cytochrome P-448” refers to the corresponding hemoprotein present in animals treated with polycyclic hydrocarbons such as d-methylcholanthrene. The term “aryl hydroxylation” refers to the process of phenol formation which in actuality often involves an aryl epoxidation followed by a spontaneous isomerization of the intermediate arene oxide to the ultimate phenolic products. The term “NIH Shift” refers to the migration and retention of substituents seen during isomerization of intermediate arene oxides. 241 Copyright All rights
Q lY74 by Academic Press, of repn,ductkan in tiny hrm
Inc. reserved.
conditions of metabolism of various substances with solubilized reconstituted systems consisting of cytochrome P-450, reductase, and lipid fraction have recently provided further evidence for the presence of a number of cytochrome P-450 monoxygenases with different substrate specificities in liver from control rats and from rats induced with phenobarbital or 3-methylcholanthrene. The aliphatic hydroxylation of testosterone at the 68-, 7a-, and 16apositions, of fatty acids in the terminal methyl group and of pentobarbital in the side chain; the N-demethylation of benzphetamine, ethylmorphine, and chlorcyclizine; the aryl epoxidation of naphthalene and the subsequent enzymatic hydration of the intermediate oxide; and the “aryl hydroxylation” of chlorobenzene, benzo [a]pyrene and aniline have been investigated with these reconstituted systems (4-12). The “aryl hydroxylation” of acetanilide and the concomittant migration and retention of tritium (the NIH Shift, 13) have now been compared between microsomes and the corresponding reconstituted monoxy-
242
SELANDER, JERINA AND DALY
incubation for 30 min at 37°C. This substrate concentration was chosen since all three microsomal preparations are near saturation under these conditions. Higher substrate concentrations cause inhibition with microsomes from untreated rats or rats induced by 3-methylcholanthrene. Incubations were terminated by cooling to O”C, followed by addition of 1 rmole of carrier 2-, 3-, and 4-hydroxyacetanilide in 25 ~1 of ethanol and 0.2 ml of 1.5 M perchloric acid. The solution was then extracted with ethyl ether (3 x 2 ml). The ether extract was dried, concentrated, and chromatographed on thin-layer alumina-GF chromatoplates (Analtech, 250 pm) with chloroform-methanol (95: 5). The areas of alumina corresponding to hydroxyacetanilides were removed and eluted with methanol. The eluant was evaporated and the residue was redissolved in a small volume of isopropanol-hexane (1: 3) for high-pressure liquid chromatography (see Fig. 1). Overall recoveries of the hydroxyacetanilides (17-20%) were quantitated by peak areas and conversions by liquid scintillation spectometry. Incubations with boiled microsomes served as controls. MATERIALS AND METHODS Reconstitution of solubilized fractions was in the following sequence with complete mixing after each The [“Clacetanilide was prepared from [I’C]aniaddition: Lipid fraction (50 rl), reductase (100 rg line (New England Nuclear Corp.) by acetylation with acetic anhydride in pyridine followed by crystal- protein) and either cytochrome P-450 from control lization to a constant specific activity of 60 Ci/mole. rats (75 rg protein, 0.18 nmoles hemoprotein), cytoThe 4-13H]acetanilide was prepared by reduction of chrome P-450 from phenobarbital-treated rats (51 pg I-bromoacetanilide with a mixture of tritium and protein, 0.23 nmoles hemoprotein), or cytochrome hydrogen gas (sp act of approximately 50 Ci/mole) P-448 from 3-methylcholanthrene-treated rats (64 pg, followed by crystallization to a sp act of 37 Ci/mole. 0.26 nmoles hemoprotein). The reconstituted fracFor studies on migration and retention of tritium, a tions were then added to a mixture consisting of 0.25 mixture of [“Cl- and 4- [SH]acetanilide was used with ml of 0.2 M phosphate buffer (pH 7.5), 0.05 ml of 0.03 a carbon-14-to-tritium ratio of 2: 1. Microsomes were M MgCl,, and 0.05 ml of 0.08 M NADPH in a test prepared from the 12,000g supernatants of liver ho- tube. The final volume was 0.5 ml, and the substrate, mogenates (3: 1) in isotonic KC1 by centrifugation at radioactive acetanilide (1.5 rmole, 10.5 PCi of “C), 100,000g for 1 hr. The resulting pellets were then was added in 0.01 ml of acetonitrile after 1 min of resuspended in isotonic KC1 to restore the original preincubation at 37°C. This concentration of acetanilide is saturating under these conditions. The incubavolume of the homogenate and to afford a preparation with 5.5-7 mg protein/ml. Livers were obtained from tion was then terminated after 10 min by cooling to control Sprague-Dawley male rats (250 g) and from 0°C and saturated with NaCl. Carrier hydroxyacetanrats pretreated for 4 days with phenobarbital (0.2% ilides were added, reisolated, and quantitated as sodium phenobarbital in drinking water) or for 2 days described for microsomal incubations. Incubations with 3-methylcholanthrene (40 m&g in cottonseed with bovine serum albumin substituted for the hemooil per day, intraperitoneal injection), and sacrificed protein served as controls. on day 5 and 3, respectively. Soluble fractions from RESULTS AND DISCUSSION hepatic microsomes of normal and induced male Long-Evans rats (50-55 g), prepared and assayed as The metabolism of acetanilide with hedescribed (7), were kindly provided by A. Y. H. Lu patic microsomal preparations has been and W. Levin of Hoffmann-La Roche, Nutley, NJ. investigated by several laboratories Incubations with microsomes were as follows: Tris (14-19). The major metabolism yields 4buffer (0.5 ml, 0.1 M, pH 8.2) containing 10 pmoles hydroxyacetanilide with 2-hydroxyacetaniNADP, 5 rmoles ATP, 25 pmoles glucose 6-phosphate, and 3 units glucose 6-phosphate dehydroge- lide as a minor product. The present study nase was mixed with 0.5 ml of resuspended mi- (Table I) confirms these results with rat crosomes in a test tube and preincubated for 1 min at microsomes, where 4-hydroxyacetanilide is 37’C. Radioactive acetanilide (890 nmoles, 12.5 PCi of the preponderant product (> 94% of total “C) was added in 25 ~1 of acetonitrile followed by phenols). Pretreatment of rats with 3-
genase systems from control rats and from rats pretreated with phenobarbital and 3-methylcholanthrene. The reconstituted systems in each case have activity per nmole cytochrome similar or somewhat less than that of the corresponding microsomal systems with respect to “hydroxylation” of acetanilide. The percentage of migration and retention of tritium during “hydroxylation” of 4-[3H]acetanilide in the 4-position is, in each instance, comparable in the corresponding microsomal and reconstituted systems, but in confirmation of previous results with labeled acetanilide and microsomal preparations (14), the magnitude of the “NIH Shift” is markedly lower in reconstituted preparations with the cytochrome fraction from animals pretreated with 3-methylcholanthrene.
METABOLISM
OF ACETANILIDE
10,000 f
0
5
IO
IS
TIME (min) FIG. 1. High-pressure liquid chromatogram of isomerit hydroxyacetanilides. The upper portion of the figure depicts the separation: A, a small amount of [“Clacetanilide; B, carrier 2-hydroxyacetanilide; C, carrier 3-hydroxyacetanilide; and D, carrier 4-hydroxyacetanilide. The lower portion of the figure depicts the radioactivity associated with fractions A, B, C, and D from an experiment with the reconstituted cytochrome P-448 system (upper bar graph) and a control with bovine serum albumin (lower bar graph). Separations were performed with a Du Pont Model 830 liquid chromatograph equipped with a 254 nm uv-photometer and a Du Pont l-m analytical ETH
243
methylcholanthrene enhances the oxidative metabolism of acetanilide to a greater extent than pretreatment with phenobarbital as previously reported (15). Microsoma1 and reconstituted cytochrome systems from control and 3-methylcholanthrenetreated rats had relatively similar activities with respect to ring oxidation of acetanilide, while the activity of the reconstituted system from phenobarbitaltreated rats appeared significantly lower than that of the corresponding microsomal system (Table I). With both microsomes and reconstituted systems, nearly saturating levels of acetanilide (0.9 and 3.0 mM, respectively) were employed. The K, values for acetanilide were similar with all three reconstituted systems at - 1.5 x lo- 3 M (results not reported). The microsomal systems were saturated at 1 mM acetanilide and exhibited substrate inhibition at higher concentrations. Km values of from 0.6-1.1 x 10m3M for acetanilide have been reported with microsomal preparations (15, 16). In parallel with the results for microsomes, the cytochrome P-448 fraction from rats pretreated with S-methylcholanthrene was more active with respect to ring oxidation of acetanilide than cytochrome P-450 fractions from either control rats or from rats pretreated with phenobarbital. In fact, the extent of conversion of acetanilide to 4-hydroxyacetanilide was approximately 7-fold higher with cytochrome P-448 fraction from 3-methylcholanthrene-pretreated rats than with the cytochrome P-450 fraction from phenobarbital-pretreated rats. Previous results (5-7) have demonstrated that substrate specificity of reconstituted systems is dependent on the cytochrome fraction and independent of reductase or lipid fractions. Small amounts of radioactivity were associated with carrier 2-hydroxy- and 3-hydroxyacetanilide with the reconstituted systems, but the origin of these possible minor products was column. The mobile phase was 6.5% isopropanol in hexane with a flow rate of 1.2 ml per min. In a typical run, 25 ~1 of 25% isopropanol in hexane containing about 1 rmole of hydroxyacetanilides was injected on the column. Fractions of 1.2-2.5 ml were collected and assayed for radioactivity.
244
SELANDER,
JERINA AND DALY TABLE I
PHENOL FORMATION FROM ACETANILIDE WITH MICROSOMESAND RECONSTITUTEDHEFTIC MONOXYCENASE SYSTEMS~
Preparation
Formation of isomeric hydroxyacetanilides (dpm/mg protein/min) 2-OH
Microsomal pellet Control Phenobarbital 3-Methylcholanthrene Reconstituted cytochrome systems Control Phenobarbital 3-Methylcholanthrene
3-OH
4-OH
Total phenolic products dpm/mg protein/min
dpm/nmole cytochrome P-450/min
250 600 2,700
200 100 400
17,000 19,800 52,300
17,450 20,500 55,400
29,300 18,600 60,900
5,400 3,700 15,200
700 400 2,200
15,400 9,900 102,000
21,600 14,000 119,000
21,400 9,400 76,000
a Control rats and rats pretreated with either phenobarbital or 3-methylcholanthrene were used as source of microsomes and the cytochrome fraction for the reconstituted systems. The substrate was [“Clacetanilide (14 (X/mole). For details see Methods. The results for the reconstituted systems are expressed per mg of total protein. The results expressed per nmole of cytochrome P-450 or P-448 in the microsomal experiments are based on levels of cytochromes determined as described (20). Results are from single experiments or are averages of two or more experiments.
not further investigated. A rather broad pH optimum between pH 7 and 8 was observed for the formation of 4-hydroxyacetanilide with the reconstituted system containing cytochrome P-448 from 3-methylcholanthrene-treated rats (results not reported, cf. broad pH optimum reported in Ref. 16 for microsomal preparations). The major metabolite, 4-hydroxyacetanilide presumably arises via intermediate formation of a very unstable arene oxide (I) which then isomerizes to 4-hydroxyacetanilide with migration and retention of isotopic hydrogen from the 4-position to the 3-position of the final product (Scheme I). Unlike the migration and retention of isotopic hydrogen observed during formation of phenols from most substrates of monoxygenases, the magnitude of the “NIH Shift” during formation of 4-hydroxyacetanilide is dependent on the pH of the incubation medium, the presence or absence of acetone, and the source (species, induction) of the microsomes (for current reviews of the “NIH Shift” and the role of arene oxides in oxidative metabolism of aromatic compounds see Refs. 13, 21). In the conversion of acetanilide labeled with isotopic hydrogen in the 4-position to 4-
hydroxyacetanilide, the retentions of deuterium or tritium are lower at high pH, in the absence of acetone, with microsomes from guinea pig or rabbit as compared with rats or mice, and with microsomes from animals that have been pretreated with 3-methylcholanthrene or benzo [alpyrene (14). These differences had been rationalized in terms of the effect of microenvironment on the migration (path a) or direct loss (path b) of isotopic hydrogen during isomerization of an intermediate oxide such as I which contains an ionizable substituent (14). As a corollary to this hypothesis, it was proposed that the environment of the active site of the cytochrome in which the amide group of acetanilide is “bound” is much more hydrophilic in the cytochromes of microsomes from animals pretreated with polycyclic hydrocarbons as compared to the same binding site in cytochromes of microsomes from control animals or animals pretreated with phenobarbital (14). Alternatively, two metabolic pathways leading to 4-hydroxyacetanilide might pertain with liver microsomes, one of which would involve the arene oxide intermediate I, while the second would involve a direct formation of the
METABOLISM
245
OF ACETANILIDE
phenol probably by an oxygen insertion pathway. Evidence for such direct pathways have recently been observed for phenol formation from chlorobenzene (12), nitrobenzene (22), and methyl phenyl sulfone (22). Direct insertion pathways would, of course, lead to complete loss of isotopic hydrogens. For metabolism of acetanilide, insertion pathways would then have to be more significant with microsomal preparations from animals pretreated with 3methylcholanthrene and would have to be selectively inhibited by acetone or low pH in order to account for the observed variations of retentions. Reconstituted systems with cytochromes from control and pretreated animals presented an ideal system to further investigate these phenomena and their mechanism. Such studies with reconstituted systems might reveal that the differences in retentions of isotopic hydrogen after hydroxylation of acetanilide with microsomes had been dependent on membrane effects and would no longer be observed with detergent-solubilized cytochromes. In addition, if two enzymes, an epoxidase, which forms an arene oxide, and a direct hydroxylase, were present in microsomes, their activity might be retained to different extents during solubilization and purification of cytochromes. Such differences in purification would then be manifested by differences in retentions between microsoma1 and reconstituted systems. The results were, however, quite clear: The magnitude of the migration and retention of tritium in 4-hydroxyacetanilide was not significantly
different in the corresponding microsomal and reconstituted systems (Table II). In both systems the percentage of migration and retention of tritium (NIH Shift) observed with preparations from rats pretreated with 3-methylcholanthrene was approximately one-half the retention observed with preparations from control or phenobarbital-pretreated rats. In addition, the magnitude of the “NIH Shift” observed with cytochrome P-448 fractions decreased with increasing pH (results not shown) in a manner similar to the decrease previously observed with microsomal preparations (14). The results, therefore, indicate that the differences in the magnitude of the “NIH Shift” during metabolism of TABLE II THE
MIGRATION AND RETENTION OF TRITIUM DURING FORMATION OF 4-HYDROXY-ACETANILIDE FROM 4- [SH]A~~~~~~~~~~n
Preparation
Microsomal pellets Control Phenobarbital 3-Methylcholanthrene Reconstituted cytochrome systems Control Phenobarbital 3-Methylcholanthrene
% Migration and retention of tritium 49 56 26
40 58 23
“Incubations with a mixture of carbon-14 and tritium-labeled acetanilide. For details see Methods. Similar results were obtained at lower substrate concentrations.
R = -NHCOCH,
6H SCHEME I
246
SELANDER,
JERINA
acetanilide reflect differences in the cytochrome and not the membrane structure. Furthermore, no evidence for the coexistence of two enzymes in cytochrome fractions that yield acetanilide by arene oxide and direct insertion pathways was obtained. Instead, the results indicate that a cvtochrome resnonsible for formation of 4lhydroxyacetanilide with high migration and retention of isotopic hydrogen is present in microsomes from both control and phenobarbital-pretreated animals, while a cytochrome responsible for formation of 4-hydroxyacetanilide with a low retention of isotopic hydrogen is present in microsomes from animals pretreated with 3-methylcholanthrene. The present solubilization procedures yields a hepatic preparation which retains activity toward 4-hydroxylation of both aniline (5) and acetanilide, but which has greatly reduced activity toward 6-Bhydroxylation of testosterone and Ndealkylation of ethylmorphine (A. Y. H. Lu, personal communication). In contrast, the same solubilized preparations have shown greatly enhanced specific activity toward many other substrates (5-7). A triton-solubilized hepatic preparation with catalyzes the conversion of aniline to 4hydroxyaniline, but which is essentially devoid of activity toward N-dealkylation of ethylmorphine and aminopyrine has been reported (23) as has differential solubilization of microsomal monoxygenase activity toward aniline and naphthalene (24). Thus, studies with reconstituted systems continue to provide evidence for the presence of a variety of monoxygenases with differing substrate specificities in hepatic microsomes. REFERENCES 1. ESTAEIROOK,R. E. (1971) In Handbook of Experimental Pharmacology (B. B. Brodie and J. R. Gillette, eds.), Pt 2, Vol. 28, pp. 264-284, Springer-Verlag, New York. 2. GILLE’ITE, J. R., DAVIS, D. C., AND SESAME, H. (1972) Annu. Rev. Pharmacol. 12, 57-84.
AND
DALY
3. KUNTZMAN, R. (1969) Annu. Reu. Pharmacol. 9, 21-36. 4. Lu, A. Y. H., KUNTZMAN, R., WEST, S., AND CONNEY, A. H. (1971) Biochem. Biophys. Res. Commun. 42, 1200-1206. 5. Lu, A. Y. H., LEVIN, W., WEST, S. B., JACOBSON, M., RYAN, D., CONNEY, A. H. (1973) Ann. N. Y. Acad. Sci. 212, 156-174. 6. Lu, A. H. Y., LEVIN, W., WEST, S. B., JACOBSON, M., RYAN, D., CONNEY, A. H. (1973) J. Biol. Chem. 248, 456-460. 7. Lu, A. Y. H., KUNTZMAN, R., WEST, S., JACOBSON, M., AND CONNEY, A. H. (1972) J. Biol. Chem. 247, 1727-1734. 8. Lu, A. Y. H., AND WEST, S. B. (1972) Mol. Pharmacol. 8, 490-500. 9. WEST, S. D., AND Lu, A. Y. H. (1973) Arch. Biochem. Biophys. 153, 290-303. 10. Lu, A. Y. H., JUNK, K. W., AND COON, M. J. (1969) J. Biol. Chem. 244, 3714-3721. 11. OESCH, F., JERINA, D. M., DALY, J. W., Lu, A. Y. H., KUNTZMAN, R., AND CONNEY, A. H. (1972) Arch. Biochem. Biophys. 153, 62-67. 12. SELANDER, H., JERINA, D. M., AND DALY, J. W., (1974) Biochemistry, in press. 13. DALY, J. W., JERINA, D. M., AND WITKOP, B. (1972) Experientia 28, 1129-1149. 14. DALY, J. W., JERINA, D. M., FARNSWORTH,J., AND GUROFF, G. (1969) Arch. Biochem. Biophys. 131, 238-244. 15. DALY, J. W. (1970) Anal. Biochem. 33, 266-296. 16. DECKWITZ, E., AND STRAUDINGER, H. (1965) Hoppe-Seylers Z. Physiol. Chem. 341.111-119. 17. KRISCH, K., AND STAUDINGER, H. (1961) Biochem. Z. 334, 312-327. 18. POSNER, H. S., MITOMA, C., ROTHBERG, S., AND UDENFRIEND, S. (1961) Arch. Biochem. Biophys. 94, 280-290. 10. JACOBSON,M., LEVI?, W., Lu, A. Y. H., CONNEY, A‘ H., AND KUNTZMAN, R. (1973) Drug Metabolism Disposition 1, 766-774. 20. ALVARES, A. P., SCHILLING, G., AND LEVIN, W. (1970) J. Pharmacol. Exp. Z’her. 175, 4-11. 21. JERINA, D. M., AND DALY, J. W. (1974) Science, in press. 22. TOMASZEWSKI, J., DALY, J. W. AND JERINA, D. M. (1973) Abstracts of the 8th Midatlantic Regional Meeting, American Chemical Society, January 14-17,1973, Washington, DC. 23. FUJITA, T., AND MANNERING, G. J. (1973) J. Biol. Chem. 248, 8150-8156. 24. BLEECKER, N., CAPDEVILA, J., AND AGOSIN, J. (1973) J. Biol. Chem. 248, 8474-8481.
OF BIQCHEMISTRY
AND
Metabolism
BIOPHYSICS
164,241-246
of Acetanilide
Reconstituted
Institute
of Arthritis,
with
Cytochrome
H. G. SELANDER,’ National
(1974)
Metabolism
Hepatic
Microsomes
Monoxygenase
and
Systems
D. M. JERINA, AND J. W. DALY and Digestive Diseases, National Maryland 20014
Institutes
of Health,
Bethesda,
Peceived February 5, 1974 Hepatic miscrosomes and reconstituted cytochrome monoxygenase systems from control rats and from rats that have been pretreated with phenobarbital or 3-methylcholanthrene convert radioactive acetanilide to ring-hydroxylated products, primarily I-hydroxyacetanilide. 3-Methylcholanthrene pretreatment results in the greatest enhancement of activity: cytochrome fractions from 3-methylcholanthrene-pretreated rats have many-fold higher activity than cytochrome fractions from control or phenobarbital-treated rats. The percentage of migration and retention of tritium (NIH Shift) measured in I-hydroxyacetanilide after enzymatic oxidation of 4- [SH]acetanilide is nearly identical using microsomes or the corresponding reconstituted system, but in both cases the percentage of migration and retention of tritum is markedly lower for preparations from 3-methylcholanthrene-treated animals with values of 25’S, as compared to the values of 40-60% for preparations from control or phenobarbital-treated animals. High-pressure liquid chromatography was employed for separation and quantitation of radioactive products.
The cytochrome P-450 class of hemoproteins2 serve as terminal monoxygenases for the conversion of endogenous and xenobiotic substances to a variety of metabolites in mammalian systems (for current reviews see Ref. l-3). The number and substrate specificity of cytochrome P-450 monoxygenases in hepatic preparations have been the subject of intensive investigation. Comparisons under various ‘Fellow in the Visiting Program of the Public Health Service. * The term “cytochrome P-450” refers to the carbon monoxide-binding hemoprotein which is present in the livers of control and phenobarbital-treated animals and is involved in oxidative metabolism, while the term “cytochrome P-448” refers to the corresponding hemoprotein present in animals treated with polycyclic hydrocarbons such as d-methylcholanthrene. The term “aryl hydroxylation” refers to the process of phenol formation which in actuality often involves an aryl epoxidation followed by a spontaneous isomerization of the intermediate arene oxide to the ultimate phenolic products. The term “NIH Shift” refers to the migration and retention of substituents seen during isomerization of intermediate arene oxides. 241 Copyright All rights
Q lY74 by Academic Press, of repn,ductkan in tiny hrm
Inc. reserved.
conditions of metabolism of various substances with solubilized reconstituted systems consisting of cytochrome P-450, reductase, and lipid fraction have recently provided further evidence for the presence of a number of cytochrome P-450 monoxygenases with different substrate specificities in liver from control rats and from rats induced with phenobarbital or 3-methylcholanthrene. The aliphatic hydroxylation of testosterone at the 68-, 7a-, and 16apositions, of fatty acids in the terminal methyl group and of pentobarbital in the side chain; the N-demethylation of benzphetamine, ethylmorphine, and chlorcyclizine; the aryl epoxidation of naphthalene and the subsequent enzymatic hydration of the intermediate oxide; and the “aryl hydroxylation” of chlorobenzene, benzo [a]pyrene and aniline have been investigated with these reconstituted systems (4-12). The “aryl hydroxylation” of acetanilide and the concomittant migration and retention of tritium (the NIH Shift, 13) have now been compared between microsomes and the corresponding reconstituted monoxy-
242
SELANDER, JERINA AND DALY
incubation for 30 min at 37°C. This substrate concentration was chosen since all three microsomal preparations are near saturation under these conditions. Higher substrate concentrations cause inhibition with microsomes from untreated rats or rats induced by 3-methylcholanthrene. Incubations were terminated by cooling to O”C, followed by addition of 1 rmole of carrier 2-, 3-, and 4-hydroxyacetanilide in 25 ~1 of ethanol and 0.2 ml of 1.5 M perchloric acid. The solution was then extracted with ethyl ether (3 x 2 ml). The ether extract was dried, concentrated, and chromatographed on thin-layer alumina-GF chromatoplates (Analtech, 250 pm) with chloroform-methanol (95: 5). The areas of alumina corresponding to hydroxyacetanilides were removed and eluted with methanol. The eluant was evaporated and the residue was redissolved in a small volume of isopropanol-hexane (1: 3) for high-pressure liquid chromatography (see Fig. 1). Overall recoveries of the hydroxyacetanilides (17-20%) were quantitated by peak areas and conversions by liquid scintillation spectometry. Incubations with boiled microsomes served as controls. MATERIALS AND METHODS Reconstitution of solubilized fractions was in the following sequence with complete mixing after each The [“Clacetanilide was prepared from [I’C]aniaddition: Lipid fraction (50 rl), reductase (100 rg line (New England Nuclear Corp.) by acetylation with acetic anhydride in pyridine followed by crystal- protein) and either cytochrome P-450 from control lization to a constant specific activity of 60 Ci/mole. rats (75 rg protein, 0.18 nmoles hemoprotein), cytoThe 4-13H]acetanilide was prepared by reduction of chrome P-450 from phenobarbital-treated rats (51 pg I-bromoacetanilide with a mixture of tritium and protein, 0.23 nmoles hemoprotein), or cytochrome hydrogen gas (sp act of approximately 50 Ci/mole) P-448 from 3-methylcholanthrene-treated rats (64 pg, followed by crystallization to a sp act of 37 Ci/mole. 0.26 nmoles hemoprotein). The reconstituted fracFor studies on migration and retention of tritium, a tions were then added to a mixture consisting of 0.25 mixture of [“Cl- and 4- [SH]acetanilide was used with ml of 0.2 M phosphate buffer (pH 7.5), 0.05 ml of 0.03 a carbon-14-to-tritium ratio of 2: 1. Microsomes were M MgCl,, and 0.05 ml of 0.08 M NADPH in a test prepared from the 12,000g supernatants of liver ho- tube. The final volume was 0.5 ml, and the substrate, mogenates (3: 1) in isotonic KC1 by centrifugation at radioactive acetanilide (1.5 rmole, 10.5 PCi of “C), 100,000g for 1 hr. The resulting pellets were then was added in 0.01 ml of acetonitrile after 1 min of resuspended in isotonic KC1 to restore the original preincubation at 37°C. This concentration of acetanilide is saturating under these conditions. The incubavolume of the homogenate and to afford a preparation with 5.5-7 mg protein/ml. Livers were obtained from tion was then terminated after 10 min by cooling to control Sprague-Dawley male rats (250 g) and from 0°C and saturated with NaCl. Carrier hydroxyacetanrats pretreated for 4 days with phenobarbital (0.2% ilides were added, reisolated, and quantitated as sodium phenobarbital in drinking water) or for 2 days described for microsomal incubations. Incubations with 3-methylcholanthrene (40 m&g in cottonseed with bovine serum albumin substituted for the hemooil per day, intraperitoneal injection), and sacrificed protein served as controls. on day 5 and 3, respectively. Soluble fractions from RESULTS AND DISCUSSION hepatic microsomes of normal and induced male Long-Evans rats (50-55 g), prepared and assayed as The metabolism of acetanilide with hedescribed (7), were kindly provided by A. Y. H. Lu patic microsomal preparations has been and W. Levin of Hoffmann-La Roche, Nutley, NJ. investigated by several laboratories Incubations with microsomes were as follows: Tris (14-19). The major metabolism yields 4buffer (0.5 ml, 0.1 M, pH 8.2) containing 10 pmoles hydroxyacetanilide with 2-hydroxyacetaniNADP, 5 rmoles ATP, 25 pmoles glucose 6-phosphate, and 3 units glucose 6-phosphate dehydroge- lide as a minor product. The present study nase was mixed with 0.5 ml of resuspended mi- (Table I) confirms these results with rat crosomes in a test tube and preincubated for 1 min at microsomes, where 4-hydroxyacetanilide is 37’C. Radioactive acetanilide (890 nmoles, 12.5 PCi of the preponderant product (> 94% of total “C) was added in 25 ~1 of acetonitrile followed by phenols). Pretreatment of rats with 3-
genase systems from control rats and from rats pretreated with phenobarbital and 3-methylcholanthrene. The reconstituted systems in each case have activity per nmole cytochrome similar or somewhat less than that of the corresponding microsomal systems with respect to “hydroxylation” of acetanilide. The percentage of migration and retention of tritium during “hydroxylation” of 4-[3H]acetanilide in the 4-position is, in each instance, comparable in the corresponding microsomal and reconstituted systems, but in confirmation of previous results with labeled acetanilide and microsomal preparations (14), the magnitude of the “NIH Shift” is markedly lower in reconstituted preparations with the cytochrome fraction from animals pretreated with 3-methylcholanthrene.
METABOLISM
OF ACETANILIDE
10,000 f
0
5
IO
IS
TIME (min) FIG. 1. High-pressure liquid chromatogram of isomerit hydroxyacetanilides. The upper portion of the figure depicts the separation: A, a small amount of [“Clacetanilide; B, carrier 2-hydroxyacetanilide; C, carrier 3-hydroxyacetanilide; and D, carrier 4-hydroxyacetanilide. The lower portion of the figure depicts the radioactivity associated with fractions A, B, C, and D from an experiment with the reconstituted cytochrome P-448 system (upper bar graph) and a control with bovine serum albumin (lower bar graph). Separations were performed with a Du Pont Model 830 liquid chromatograph equipped with a 254 nm uv-photometer and a Du Pont l-m analytical ETH
243
methylcholanthrene enhances the oxidative metabolism of acetanilide to a greater extent than pretreatment with phenobarbital as previously reported (15). Microsoma1 and reconstituted cytochrome systems from control and 3-methylcholanthrenetreated rats had relatively similar activities with respect to ring oxidation of acetanilide, while the activity of the reconstituted system from phenobarbitaltreated rats appeared significantly lower than that of the corresponding microsomal system (Table I). With both microsomes and reconstituted systems, nearly saturating levels of acetanilide (0.9 and 3.0 mM, respectively) were employed. The K, values for acetanilide were similar with all three reconstituted systems at - 1.5 x lo- 3 M (results not reported). The microsomal systems were saturated at 1 mM acetanilide and exhibited substrate inhibition at higher concentrations. Km values of from 0.6-1.1 x 10m3M for acetanilide have been reported with microsomal preparations (15, 16). In parallel with the results for microsomes, the cytochrome P-448 fraction from rats pretreated with S-methylcholanthrene was more active with respect to ring oxidation of acetanilide than cytochrome P-450 fractions from either control rats or from rats pretreated with phenobarbital. In fact, the extent of conversion of acetanilide to 4-hydroxyacetanilide was approximately 7-fold higher with cytochrome P-448 fraction from 3-methylcholanthrene-pretreated rats than with the cytochrome P-450 fraction from phenobarbital-pretreated rats. Previous results (5-7) have demonstrated that substrate specificity of reconstituted systems is dependent on the cytochrome fraction and independent of reductase or lipid fractions. Small amounts of radioactivity were associated with carrier 2-hydroxy- and 3-hydroxyacetanilide with the reconstituted systems, but the origin of these possible minor products was column. The mobile phase was 6.5% isopropanol in hexane with a flow rate of 1.2 ml per min. In a typical run, 25 ~1 of 25% isopropanol in hexane containing about 1 rmole of hydroxyacetanilides was injected on the column. Fractions of 1.2-2.5 ml were collected and assayed for radioactivity.
244
SELANDER,
JERINA AND DALY TABLE I
PHENOL FORMATION FROM ACETANILIDE WITH MICROSOMESAND RECONSTITUTEDHEFTIC MONOXYCENASE SYSTEMS~
Preparation
Formation of isomeric hydroxyacetanilides (dpm/mg protein/min) 2-OH
Microsomal pellet Control Phenobarbital 3-Methylcholanthrene Reconstituted cytochrome systems Control Phenobarbital 3-Methylcholanthrene
3-OH
4-OH
Total phenolic products dpm/mg protein/min
dpm/nmole cytochrome P-450/min
250 600 2,700
200 100 400
17,000 19,800 52,300
17,450 20,500 55,400
29,300 18,600 60,900
5,400 3,700 15,200
700 400 2,200
15,400 9,900 102,000
21,600 14,000 119,000
21,400 9,400 76,000
a Control rats and rats pretreated with either phenobarbital or 3-methylcholanthrene were used as source of microsomes and the cytochrome fraction for the reconstituted systems. The substrate was [“Clacetanilide (14 (X/mole). For details see Methods. The results for the reconstituted systems are expressed per mg of total protein. The results expressed per nmole of cytochrome P-450 or P-448 in the microsomal experiments are based on levels of cytochromes determined as described (20). Results are from single experiments or are averages of two or more experiments.
not further investigated. A rather broad pH optimum between pH 7 and 8 was observed for the formation of 4-hydroxyacetanilide with the reconstituted system containing cytochrome P-448 from 3-methylcholanthrene-treated rats (results not reported, cf. broad pH optimum reported in Ref. 16 for microsomal preparations). The major metabolite, 4-hydroxyacetanilide presumably arises via intermediate formation of a very unstable arene oxide (I) which then isomerizes to 4-hydroxyacetanilide with migration and retention of isotopic hydrogen from the 4-position to the 3-position of the final product (Scheme I). Unlike the migration and retention of isotopic hydrogen observed during formation of phenols from most substrates of monoxygenases, the magnitude of the “NIH Shift” during formation of 4-hydroxyacetanilide is dependent on the pH of the incubation medium, the presence or absence of acetone, and the source (species, induction) of the microsomes (for current reviews of the “NIH Shift” and the role of arene oxides in oxidative metabolism of aromatic compounds see Refs. 13, 21). In the conversion of acetanilide labeled with isotopic hydrogen in the 4-position to 4-
hydroxyacetanilide, the retentions of deuterium or tritium are lower at high pH, in the absence of acetone, with microsomes from guinea pig or rabbit as compared with rats or mice, and with microsomes from animals that have been pretreated with 3-methylcholanthrene or benzo [alpyrene (14). These differences had been rationalized in terms of the effect of microenvironment on the migration (path a) or direct loss (path b) of isotopic hydrogen during isomerization of an intermediate oxide such as I which contains an ionizable substituent (14). As a corollary to this hypothesis, it was proposed that the environment of the active site of the cytochrome in which the amide group of acetanilide is “bound” is much more hydrophilic in the cytochromes of microsomes from animals pretreated with polycyclic hydrocarbons as compared to the same binding site in cytochromes of microsomes from control animals or animals pretreated with phenobarbital (14). Alternatively, two metabolic pathways leading to 4-hydroxyacetanilide might pertain with liver microsomes, one of which would involve the arene oxide intermediate I, while the second would involve a direct formation of the
METABOLISM
245
OF ACETANILIDE
phenol probably by an oxygen insertion pathway. Evidence for such direct pathways have recently been observed for phenol formation from chlorobenzene (12), nitrobenzene (22), and methyl phenyl sulfone (22). Direct insertion pathways would, of course, lead to complete loss of isotopic hydrogens. For metabolism of acetanilide, insertion pathways would then have to be more significant with microsomal preparations from animals pretreated with 3methylcholanthrene and would have to be selectively inhibited by acetone or low pH in order to account for the observed variations of retentions. Reconstituted systems with cytochromes from control and pretreated animals presented an ideal system to further investigate these phenomena and their mechanism. Such studies with reconstituted systems might reveal that the differences in retentions of isotopic hydrogen after hydroxylation of acetanilide with microsomes had been dependent on membrane effects and would no longer be observed with detergent-solubilized cytochromes. In addition, if two enzymes, an epoxidase, which forms an arene oxide, and a direct hydroxylase, were present in microsomes, their activity might be retained to different extents during solubilization and purification of cytochromes. Such differences in purification would then be manifested by differences in retentions between microsoma1 and reconstituted systems. The results were, however, quite clear: The magnitude of the migration and retention of tritium in 4-hydroxyacetanilide was not significantly
different in the corresponding microsomal and reconstituted systems (Table II). In both systems the percentage of migration and retention of tritium (NIH Shift) observed with preparations from rats pretreated with 3-methylcholanthrene was approximately one-half the retention observed with preparations from control or phenobarbital-pretreated rats. In addition, the magnitude of the “NIH Shift” observed with cytochrome P-448 fractions decreased with increasing pH (results not shown) in a manner similar to the decrease previously observed with microsomal preparations (14). The results, therefore, indicate that the differences in the magnitude of the “NIH Shift” during metabolism of TABLE II THE
MIGRATION AND RETENTION OF TRITIUM DURING FORMATION OF 4-HYDROXY-ACETANILIDE FROM 4- [SH]A~~~~~~~~~~n
Preparation
Microsomal pellets Control Phenobarbital 3-Methylcholanthrene Reconstituted cytochrome systems Control Phenobarbital 3-Methylcholanthrene
% Migration and retention of tritium 49 56 26
40 58 23
“Incubations with a mixture of carbon-14 and tritium-labeled acetanilide. For details see Methods. Similar results were obtained at lower substrate concentrations.
R = -NHCOCH,
6H SCHEME I
246
SELANDER,
JERINA
acetanilide reflect differences in the cytochrome and not the membrane structure. Furthermore, no evidence for the coexistence of two enzymes in cytochrome fractions that yield acetanilide by arene oxide and direct insertion pathways was obtained. Instead, the results indicate that a cvtochrome resnonsible for formation of 4lhydroxyacetanilide with high migration and retention of isotopic hydrogen is present in microsomes from both control and phenobarbital-pretreated animals, while a cytochrome responsible for formation of 4-hydroxyacetanilide with a low retention of isotopic hydrogen is present in microsomes from animals pretreated with 3-methylcholanthrene. The present solubilization procedures yields a hepatic preparation which retains activity toward 4-hydroxylation of both aniline (5) and acetanilide, but which has greatly reduced activity toward 6-Bhydroxylation of testosterone and Ndealkylation of ethylmorphine (A. Y. H. Lu, personal communication). In contrast, the same solubilized preparations have shown greatly enhanced specific activity toward many other substrates (5-7). A triton-solubilized hepatic preparation with catalyzes the conversion of aniline to 4hydroxyaniline, but which is essentially devoid of activity toward N-dealkylation of ethylmorphine and aminopyrine has been reported (23) as has differential solubilization of microsomal monoxygenase activity toward aniline and naphthalene (24). Thus, studies with reconstituted systems continue to provide evidence for the presence of a variety of monoxygenases with differing substrate specificities in hepatic microsomes. REFERENCES 1. ESTAEIROOK,R. E. (1971) In Handbook of Experimental Pharmacology (B. B. Brodie and J. R. Gillette, eds.), Pt 2, Vol. 28, pp. 264-284, Springer-Verlag, New York. 2. GILLE’ITE, J. R., DAVIS, D. C., AND SESAME, H. (1972) Annu. Rev. Pharmacol. 12, 57-84.
AND
DALY
3. KUNTZMAN, R. (1969) Annu. Reu. Pharmacol. 9, 21-36. 4. Lu, A. Y. H., KUNTZMAN, R., WEST, S., AND CONNEY, A. H. (1971) Biochem. Biophys. Res. Commun. 42, 1200-1206. 5. Lu, A. Y. H., LEVIN, W., WEST, S. B., JACOBSON, M., RYAN, D., CONNEY, A. H. (1973) Ann. N. Y. Acad. Sci. 212, 156-174. 6. Lu, A. H. Y., LEVIN, W., WEST, S. B., JACOBSON, M., RYAN, D., CONNEY, A. H. (1973) J. Biol. Chem. 248, 456-460. 7. Lu, A. Y. H., KUNTZMAN, R., WEST, S., JACOBSON, M., AND CONNEY, A. H. (1972) J. Biol. Chem. 247, 1727-1734. 8. Lu, A. Y. H., AND WEST, S. B. (1972) Mol. Pharmacol. 8, 490-500. 9. WEST, S. D., AND Lu, A. Y. H. (1973) Arch. Biochem. Biophys. 153, 290-303. 10. Lu, A. Y. H., JUNK, K. W., AND COON, M. J. (1969) J. Biol. Chem. 244, 3714-3721. 11. OESCH, F., JERINA, D. M., DALY, J. W., Lu, A. Y. H., KUNTZMAN, R., AND CONNEY, A. H. (1972) Arch. Biochem. Biophys. 153, 62-67. 12. SELANDER, H., JERINA, D. M., AND DALY, J. W., (1974) Biochemistry, in press. 13. DALY, J. W., JERINA, D. M., AND WITKOP, B. (1972) Experientia 28, 1129-1149. 14. DALY, J. W., JERINA, D. M., FARNSWORTH,J., AND GUROFF, G. (1969) Arch. Biochem. Biophys. 131, 238-244. 15. DALY, J. W. (1970) Anal. Biochem. 33, 266-296. 16. DECKWITZ, E., AND STRAUDINGER, H. (1965) Hoppe-Seylers Z. Physiol. Chem. 341.111-119. 17. KRISCH, K., AND STAUDINGER, H. (1961) Biochem. Z. 334, 312-327. 18. POSNER, H. S., MITOMA, C., ROTHBERG, S., AND UDENFRIEND, S. (1961) Arch. Biochem. Biophys. 94, 280-290. 10. JACOBSON,M., LEVI?, W., Lu, A. Y. H., CONNEY, A‘ H., AND KUNTZMAN, R. (1973) Drug Metabolism Disposition 1, 766-774. 20. ALVARES, A. P., SCHILLING, G., AND LEVIN, W. (1970) J. Pharmacol. Exp. Z’her. 175, 4-11. 21. JERINA, D. M., AND DALY, J. W. (1974) Science, in press. 22. TOMASZEWSKI, J., DALY, J. W. AND JERINA, D. M. (1973) Abstracts of the 8th Midatlantic Regional Meeting, American Chemical Society, January 14-17,1973, Washington, DC. 23. FUJITA, T., AND MANNERING, G. J. (1973) J. Biol. Chem. 248, 8150-8156. 24. BLEECKER, N., CAPDEVILA, J., AND AGOSIN, J. (1973) J. Biol. Chem. 248, 8474-8481.