- Email: [email protected]
Acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in hepatic microsomes from induced mice
Toxicology Letters, 55 (1991) 223-231 Elsevier
223
TOXLET 02508
Acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in hepatic microsomes from induced mice
Margaret Lewandowskl ‘*, Y.C. Chui**, Patricia Levi and Ernest Hodgson Toxicology Department,
North Carolina State University, Raleigh, NC (U.S.A.)
(Received 9 February 1990) (Accepted 15 September 1990) Key words: Acetanilide hydroxylase; Cytochrome P-450; Induction; Microsomal oxidation; Isosafrole
SUMMARY A simple and sensitive method for the separation of i4C-labelled acetanilide, 4_hydroxyacetanilide, 3hydroxyacetanilide and 2-hydroxyacetanilide was developed using thin-layer chromatography. This separation is the basis for the assay of acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in liver microsomes from DBAZ/N male mice that had been treated with phenobarbital, 3-methylcholanthrene, isosafrole or n-butylbenzodioxole. Microsomes were incubated with [t4C]acetanilide and extracted with benzene and ethyl acetate. The extract was applied to silica gel plates and developed with a hexane/ isopropanol/ammonium hydroxide/water solvent system. The radiolabelled phenolic metabolites and the parent compound were detected using a Berthold Automatic TLC Linear Analyzer. Although the 4-hydroxylated metabolite was the primary product detected, this method can be used to detect other phenolic metabolites.
INTRODUCTION
The cytochrome P-450 (P-450) mono-oxygenase system catalyzes oxidation reactions involved in the metabolism of a wide variety of xenobiotics, as well as the oxida-
*Currenf address: BASF Corporation, Agricultural Research Center, Research Triangle Park, NC 27709, U.S.A. l*Currenr address: Cantest, Ltd., 1523 West 3rd Avenue, Vancouver, B.C., Canada V6J 158. Address for correspondence: Ernest Hoclgson, Toxicology Department, Box 7633, North Carolina State University, Raleigh, NC 27695, U.S.A. 0378-4274/91/S 3.50 @ 1991 Elsevier Science Publishers B.V. (Biomedical Division)
224
tion of endogenous substrates. Previous studies have shown that specific activities are associated with particular forms, or isozymes, of P-450. Acetanilide hydroxylase activity has been associated with PsP-450 in the mouse [l], an isozyme that corresponds to P-450d in the rat. Various methods have been developed to assay for this activity. The earliest method was calorimetric [2,3] and although it was rapid and convenient, the calorimetric method did not distinguish the metabolites from one another. Another method was later developed that combined the use of radiolabelled acetanilide with thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC) [4] and enabled the 4-hydroxy-, the 3-hydroxy- and the 2-hydroxyacetanilide to be distinguished from each other. Because this method was time-consuming, another HPLC method was developed [5] using radiolabelled acetanilide, but this method enabled the quantitation of acetanilide 4-hydroxylase activity only. We have developed a TLC method that is simpler and less time-consuming than the HPLC methods [4,5] yet allows the quantitation of the 4-hydroxy-, the 3-hydroxy- and the 2-hydroxyacetanilide products of hydroxylase activity. This method was used to investigate acetanilide metabolism by microsomes from mice previously treated with phenobarbital, 3_methylcholanthrene, isosafrole and nbutylbenzodioxole. MATERIALS
AND METHODS
Methods
DBA2/N male mice, 18-20 g (Charles River Breeding Laboratories), were given a daily intraperitoneal dose of sodium phenobarbital, 80 mg/kg in corn oil, 3-methylcholanthrene, 20 mg/kg in corn oil, isosafrole or n-butylbenzodioxole, 200 mg/kg in corn oil or corn oil alone for 3-days. Microsomes were prepared from the pooled livers (3 livers/group) on the 4th day by differential centrifugation under conditions previously described [6]. A typical assay mixture contained 0.5 mg of microsomal protein 2.0 pmol [t4C]acetanilide (0.10 mCi/mmol), an NADPH regenerating system (0.25 mM NADP+, 2.50 mM glucose-6-phosphate and 1 unit glucose-6-phosphate dehydrogenase) in 50 mM Tris-HCl buffer (pH 7.6) containing 3 mM MgS04 in a total volume of 1 ml. The experimental control contained no NADPH. Following a 30-min incubation at 37°C the reaction was terminated by adding 1 ml of 1 N KOH. 4-Hydroxy- and 2-hydroxyacetanilide (0.1 pmol) were added to the assay mixture which was then extracted 5 times with 2 ml of benzene, acidified with 200 ~1 of concentrated HCl and extracted 3 times with 2 ml of ethyl acetate. Prior to the extraction with ethyl acetate, 0.1 nmol 2,4,5,2’,4’,5’-hexachlorobiphenyl (17.6 mCi/mmol) was added as an internal standard. The ethyl acetate was removed under a stream of nitrogen at room temperature and redissolved in 50 ,ul of methanol. The amount of radioactivity in this solution was determined by counting a subsample using a Pckard TRI-CARB Scintillation Spectrometer. The solution was applied to a silica gel plate, developed 2 times in a
225
hexane/isopropanol/ammonium hydroxide/water (73:25: 1:1) solvent system and scanned with a Berthold Automatic TLC-Linear Analyzer. Due to the instability of the 2-hydroxyacetanilide, all procedures were performed in the dark or under low levels of yellow light. Materials Sodium phenobarbital was obtained from Merck and Company, Rahway, NJ. 3Methylcholanthrene was obtained from Eastman Kodak Company, Rochester, NY. Isosafrole was obtained from Fluka AG, Chemische Fabrik, F.R.G. N-Butylbenzodioxole was a gift of Dr. C.F. Wilkinson, Cornell University, NY. Acetanilide and 4-hydroxyacetanilide were obtained from Aldrich Chemical Company; 3-hydroxyacetanilide and 2-hydroxyacetanilide were obtained from Sigma Chemical Company, St. Louis, MO. Acetanilide, 4_hydroxyacetanilide, 3-hydroxyacetanilide and 2-hydroxyacetanilide were recrystallized twice from methanol prior to use. Acetanilidering-UL-i4C (specific activity of 10.52 mCi/mmol) and 2,4,5,2’,4’-S-hexachlorobiphenyl-ring-UL-i4C (17.6 mCi/mmol) were obtained from Pathfinder Laboratories Inc., St. Louis, MO. The radiolabelled acetanilide was purified by extraction from Tris-HCl buffer with benzene (2 ml x 3) immediately prior to use. 3H-4-hydroxyacetanilide (19.2 mCi/mmol) was obtained from NEN Research Products, Boston, MA. Silica gel plates (Polygram Sil NHR/UVz54) were obtained from Brinkman Instruments Co., Westbury, NY. RESULTS AND DISCUSSION
A sample containing standards of acetanilide, 4-hydroxy-, 3-hydroxy- and 2-hydroxyacetanilide was applied to a silica gel plate and developed giving a good separation of the parent compound and the 3 phenolic metabolites (Table I, Rr I). When the plate was developed in the same solvent system a second time, the separation beTABLE I Rr VALUES FOR ACETANILIDE PLATES
AND THE PHENOLIC METABOLITES ON SILICA GEL TLC
Standard
Rr Ia
Rr IIb
Acetanilide 4-Hydroxyacetanilide 3-Hydroxyacetanilide 2-Hydroxyacetanilide Unidentified metabolite
0.49 0.29 0.36 0.43 0.00
0.65 0.37 0.46 0.53 0.00
a Rr I: the Rr values obtained after developing the silica gel plate once in the hexane/isopropanol/ammonium hydroxide/water (73:25:1:1) solvent system. b Rr II: the Rr values obtained after developing the silica gel plate twice in the same solvent system.
226
ACETANILIDE
4-OH
ACETANILIDE
111
/
0
Rf
VALUE
100
Fig. 1. Chromatogram of acetanilide and the phenolic metabolites after extraction with ethyl acetate only. (A) Microsomes from isosafrole-treated mice. (B) Microsomes from untreated mice. (C) Experimental control.
tween the 4-hydroxy- and the 3-hydroxyacetanilide and between the 2-hydroxyacetanilide and the parent compound improved (Table I, Rr II). An unidentified polar metabolite did not migrate from the origin in this solvent system. When the incubation mixture was acidified and extracted with ethyl acetate only, the acetanilide and the phenolic metabolites were extracted simultaneously, but only the 4-hydroxylated product could be accurately quantitated since the small amount of 2-hydroxylated product was obscured by the large amount of acetanilide (Fig. 1). However, if the incubation mixture was made basic and extracted with benzene prior to the extraction with ethyl acetate, more than 99% of the parent compound partitioned into the benzene leaving the phenolic metabolites ( > 97 %) in the aqueous solution. This extraction procedure permitted the quantitiation of both the 4-hydroxylated and the 2-hydroxylated products (Fig. 2). The partitioning of the acetanilide and the phenolic metabolites was quantitated using radiolabelled acetanilide and 4hydroxyacetanilide. Product formation required NADPH as supplied by a regenerating system and was linear up to 45 min (Fig. 3) with up to 0.5 mg of microsomal protein (Fig. 4). In both a microsomal and a reconstituted system, the production of 4_hydroxyacetanilide was optimal at pH 8.0 (Fig. 5). The production of the 2-hydroxyacetanilide was optimal between pH 8.0 and 9.0. The production of the unidentified polar metabolite
227
4-OH
ACETANILIDE .
ACETANILIDE
Rf VALUE
0
100
Fig. 2. Chromatogram of acetanilide and the phenolic metabolites after extraction with benzene and ethyl acetate. (A) Microsomes from isosafrole-treated mice. (B) Untreated microsomes. (C) Experimental control.
did not vary with pH until a pH of 9.5. At pH 9.5, no metabolism occurred due to the denaturation of the cytochrome P-450. The apparent K, and V,,, in the microsoma1 system was determined to be 0.76 mM and 0.38 nmol/mg/min for the 2-hydroxyacetanihde and 0.62 mM and 12.7 nmoi~mg/min for the 4-hydroxyacetanilide (Fig. 6). Microsomes were prepared from the livers of mice that had been pretreated with isosafrole, phenobarbital, 3-methylcholanthrene or n-butylbenzodioxole. The effect
120
*
100
so 4-OH 2-OH
60 40 20 0 0
20
40
SO
80
100
TIME
Fig. 3. The effect of incubation time (min) on product formation in hepatic microsomes of isosafrok-treated DBAZIN mice.
228
0.20
0
0.40
0.60
0.80
1.00
1.20
MG PROTEIN Fig. 4. The effect of protein
concentration
on product
formation
ed DBAZ/N
in hepatic microsomes
of isosafrole-treat-
mice.
of these pretreatments on the production of the 4_hydroxyacetanilide, the 2-hydroxyacetanilide, and the unidentified polar metabolite is illustrated in Table II. All the treatments significantly increased the acetanilide 2-hydroxylase activity over that of control. All treatments, except the n-butylbenzodioxole, significantly increased the acetanilide 4-hydroxylase activity over that of control. The ratio of the 4-hydroxyto the 2-hydroxyacetanilide did not differ significantly (P~0.05) from the control with the phenobarbital or the n-butylbenzodioxole treatments but did with the isosa-
+
Fig. 5. The effect of pH on product mice. (B) In a reconstituted P-450 reductase,
formation.
(A) In hepatic
system using 0.2 nmol of purified
0.25 fig of phosphatidylcholine were as described
microsomes
of isosafrole-treated
DBA2/N
P, P-450, 1.2 units of NADPH-cytochrome
in 1 ml of Tris-HCI in ‘Materials
4-OH
buffer.
and Methods’.
The incubation
conditions
229 ‘1
Fig. 6. Lineweaver-Burk plot for hepatic microsomal acetanilide Chydroxylase and acetanilide 2-hydroxylase activity from isosafrole-treated DBA2/N mice.
frole and the 3-methylcholanthrene treatments. Pretreatment with isosafrole or 3methylcholanthrene significantly increased the 4-hydroxy-/2_hydroxyacetanilide ratio, although this increase was small. It has been demonstrated [7,8] that induction of cytochrome P-450 as well as 7-ethoxycoumarin O-deethylase and ethylmorphine N-demethylase activities by isosafrole
TABLE II THE EFFECT OF PRETREATMENT ON THE ACETANILIDE HEPATIC MICROSOMES OF DBAZ/N MICE
Control Isosafrole Phenobarbital 3-Methylcholanthrene n-Butyl BD
HYDROXYLASE ACTIVITIES IN
Unknown metabolite’
4-OH”
2-OHa
4-OH/2-OH
0.231 kO.049 0.672+0.103* 0.461+0.045 0.707*0.137* 0.397 f 0.097
1.49 + 0.068 6.67 f0.589; 3.04+0.294* 8.79 +0.632* 2.14kO.380
0.137+0.003 0.533 kO.029’ 0.270*0.014* 0.735+0.032* 0.180*0.027*
10.9 12.5* 11.3 12.0* 11.9
‘nmol of product/min/mg of microsomal protein. * Significantly different from control (PC 0.05)
230
TABLE EFFECTS
III OF PRETREATMENT
LIDE HYDROXYLASE
WITH
SAFROLE
AND ISOSAFROLE
ON HEPATIC
Treatment
Strain C57”
DBA”
Control
2.13kO.33
2.45 + 0.29
Safrole
6.13*
1.68*
7.54+2.46*
Isosafrole
7.40+
1.37*
7.70*
anmol product * Significantly
ACETANI-
ACTIVITY
formed/mg different
Values are mean
microsomal
1.12*
protein/min.
(P< 0.05) from control
values.
f SD (n = 4).
were the same in Ah-positive and Ah-negative mice; in addition, binding of isosafrole to the Ah-receptor did not occur. As shown in Table III, acetanilide hydroxylase activity is induced to the same extent by isosafrole in C57 (Ah-positive) and DBA (Ahnegative) mice. The use of radiolabelled acetanilide combined with an extraction procedure that selectively partitions the parent compound from the phenolic metabolites and the development of the silica gel plate twice in the same solvent system allows the quantitation of the 4-hydroxy-, the 3-hydroxy- and the 2-hydroxyacetanilide products. This TLC method, although not as convenient as the calorimetric method [3], provides a simpler and less time-consuming method than the reported HPLC methods [4,5]. One can easily quantitate rates of 1.0 nmol product formed/min/mg of microsomal protein using the specific activity described, but rates at the pmol level can be detected if substrate of higher specific activity is used. ACKNOWLEDGEMENTS
This article is a paper in the Journal Series of the NC Agricultural Research Service, Raleigh, NC 276957601. This investigation was supported, in part, by Grants ES-07046 and ES-00044 from the National Institute of Environmental Health Sciences, U.S. Public Health Services and by a predoctoral fellowship awarded to one of the authors (M.L.) by the Chemical Industry Institute of Toxicology.
REFERENCES 1 Negishi, M. and Nebert, D.W. (1979) Structural gene products of the Ah locus. Genetic and immunochemical evidence for two forms of mouse liver cytochrome P-450 induced by 3-methylcholanthrene. J. Biol. Chem. 254, 11015-l
1023.
231
2 Brodie,
B.B. and Axelrod,
N-acetyl-p-aminophenol J. Pharmacol. 3 Mitoma, Methods 4 Selander,
metabolites
in Enzymology, H.G., Jerina,
S. (1962) Aryl-Chydroxylase.
Vol. 5. Academic
and its metabolic
products,
in biological
aniline,
fluids and tissues.
monooxygenase
T.M., Negishi,
M. and Nebert,
and quantitative
determination
Anal. Biochem.
P.J., Smyser,
7 Cook, J.C. and Hogson, 8 Cook, J.C. and Hodgson, frole: lack of correlation
of acetanilide
systems. Arch. Biochem. D.W. (1979) Separation of ‘acetanilide
B.P. and Hodgson,
(Eds.),
with hepatic microsomes
Biophys.
164,241-246.
of acetanilide
4-hydroxylase
E. (1983) Purification Int. J. Biochem.
E. (1985) The induction
compounds.
and N.O. Kaplan
activity’
and its hydroxylated by high-pressure
liq-
96,201-207.
ase from mouse and pig liver microsomes. lenedioxyphenyl
In: S.P. Colowick
Press, New York, pp. 816819.
D.M. and Daly, J.W. (1974) Metabolism
cytochrome
uid chromatography. 6 Sabourin,
of acetanilide
(free and total conjugated)
Exp. Ther. 94, 22-28.
C. and Udenfriend,
and reconstituted 5 Guenthner,
J. (1948) The estimation and paminophenol
Chem.-Biol.
Interact.
E. (1986) Induction
of cytochrome 54,299-3
of cytochrome
with the Ah locus. Chem.-Biol.
of the flavin-containing
monooxygen-
16,713720. P-450 by isosafrole
and related methy-
15. P-450 in congenic
Interact.
58,233-240.
C57B1/6J mice by isosa-
223
TOXLET 02508
Acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in hepatic microsomes from induced mice
Margaret Lewandowskl ‘*, Y.C. Chui**, Patricia Levi and Ernest Hodgson Toxicology Department,
North Carolina State University, Raleigh, NC (U.S.A.)
(Received 9 February 1990) (Accepted 15 September 1990) Key words: Acetanilide hydroxylase; Cytochrome P-450; Induction; Microsomal oxidation; Isosafrole
SUMMARY A simple and sensitive method for the separation of i4C-labelled acetanilide, 4_hydroxyacetanilide, 3hydroxyacetanilide and 2-hydroxyacetanilide was developed using thin-layer chromatography. This separation is the basis for the assay of acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in liver microsomes from DBAZ/N male mice that had been treated with phenobarbital, 3-methylcholanthrene, isosafrole or n-butylbenzodioxole. Microsomes were incubated with [t4C]acetanilide and extracted with benzene and ethyl acetate. The extract was applied to silica gel plates and developed with a hexane/ isopropanol/ammonium hydroxide/water solvent system. The radiolabelled phenolic metabolites and the parent compound were detected using a Berthold Automatic TLC Linear Analyzer. Although the 4-hydroxylated metabolite was the primary product detected, this method can be used to detect other phenolic metabolites.
INTRODUCTION
The cytochrome P-450 (P-450) mono-oxygenase system catalyzes oxidation reactions involved in the metabolism of a wide variety of xenobiotics, as well as the oxida-
*Currenf address: BASF Corporation, Agricultural Research Center, Research Triangle Park, NC 27709, U.S.A. l*Currenr address: Cantest, Ltd., 1523 West 3rd Avenue, Vancouver, B.C., Canada V6J 158. Address for correspondence: Ernest Hoclgson, Toxicology Department, Box 7633, North Carolina State University, Raleigh, NC 27695, U.S.A. 0378-4274/91/S 3.50 @ 1991 Elsevier Science Publishers B.V. (Biomedical Division)
224
tion of endogenous substrates. Previous studies have shown that specific activities are associated with particular forms, or isozymes, of P-450. Acetanilide hydroxylase activity has been associated with PsP-450 in the mouse [l], an isozyme that corresponds to P-450d in the rat. Various methods have been developed to assay for this activity. The earliest method was calorimetric [2,3] and although it was rapid and convenient, the calorimetric method did not distinguish the metabolites from one another. Another method was later developed that combined the use of radiolabelled acetanilide with thin-layer chromatography (TLC) and high-pressure liquid chromatography (HPLC) [4] and enabled the 4-hydroxy-, the 3-hydroxy- and the 2-hydroxyacetanilide to be distinguished from each other. Because this method was time-consuming, another HPLC method was developed [5] using radiolabelled acetanilide, but this method enabled the quantitation of acetanilide 4-hydroxylase activity only. We have developed a TLC method that is simpler and less time-consuming than the HPLC methods [4,5] yet allows the quantitation of the 4-hydroxy-, the 3-hydroxy- and the 2-hydroxyacetanilide products of hydroxylase activity. This method was used to investigate acetanilide metabolism by microsomes from mice previously treated with phenobarbital, 3_methylcholanthrene, isosafrole and nbutylbenzodioxole. MATERIALS
AND METHODS
Methods
DBA2/N male mice, 18-20 g (Charles River Breeding Laboratories), were given a daily intraperitoneal dose of sodium phenobarbital, 80 mg/kg in corn oil, 3-methylcholanthrene, 20 mg/kg in corn oil, isosafrole or n-butylbenzodioxole, 200 mg/kg in corn oil or corn oil alone for 3-days. Microsomes were prepared from the pooled livers (3 livers/group) on the 4th day by differential centrifugation under conditions previously described [6]. A typical assay mixture contained 0.5 mg of microsomal protein 2.0 pmol [t4C]acetanilide (0.10 mCi/mmol), an NADPH regenerating system (0.25 mM NADP+, 2.50 mM glucose-6-phosphate and 1 unit glucose-6-phosphate dehydrogenase) in 50 mM Tris-HCl buffer (pH 7.6) containing 3 mM MgS04 in a total volume of 1 ml. The experimental control contained no NADPH. Following a 30-min incubation at 37°C the reaction was terminated by adding 1 ml of 1 N KOH. 4-Hydroxy- and 2-hydroxyacetanilide (0.1 pmol) were added to the assay mixture which was then extracted 5 times with 2 ml of benzene, acidified with 200 ~1 of concentrated HCl and extracted 3 times with 2 ml of ethyl acetate. Prior to the extraction with ethyl acetate, 0.1 nmol 2,4,5,2’,4’,5’-hexachlorobiphenyl (17.6 mCi/mmol) was added as an internal standard. The ethyl acetate was removed under a stream of nitrogen at room temperature and redissolved in 50 ,ul of methanol. The amount of radioactivity in this solution was determined by counting a subsample using a Pckard TRI-CARB Scintillation Spectrometer. The solution was applied to a silica gel plate, developed 2 times in a
225
hexane/isopropanol/ammonium hydroxide/water (73:25: 1:1) solvent system and scanned with a Berthold Automatic TLC-Linear Analyzer. Due to the instability of the 2-hydroxyacetanilide, all procedures were performed in the dark or under low levels of yellow light. Materials Sodium phenobarbital was obtained from Merck and Company, Rahway, NJ. 3Methylcholanthrene was obtained from Eastman Kodak Company, Rochester, NY. Isosafrole was obtained from Fluka AG, Chemische Fabrik, F.R.G. N-Butylbenzodioxole was a gift of Dr. C.F. Wilkinson, Cornell University, NY. Acetanilide and 4-hydroxyacetanilide were obtained from Aldrich Chemical Company; 3-hydroxyacetanilide and 2-hydroxyacetanilide were obtained from Sigma Chemical Company, St. Louis, MO. Acetanilide, 4_hydroxyacetanilide, 3-hydroxyacetanilide and 2-hydroxyacetanilide were recrystallized twice from methanol prior to use. Acetanilidering-UL-i4C (specific activity of 10.52 mCi/mmol) and 2,4,5,2’,4’-S-hexachlorobiphenyl-ring-UL-i4C (17.6 mCi/mmol) were obtained from Pathfinder Laboratories Inc., St. Louis, MO. The radiolabelled acetanilide was purified by extraction from Tris-HCl buffer with benzene (2 ml x 3) immediately prior to use. 3H-4-hydroxyacetanilide (19.2 mCi/mmol) was obtained from NEN Research Products, Boston, MA. Silica gel plates (Polygram Sil NHR/UVz54) were obtained from Brinkman Instruments Co., Westbury, NY. RESULTS AND DISCUSSION
A sample containing standards of acetanilide, 4-hydroxy-, 3-hydroxy- and 2-hydroxyacetanilide was applied to a silica gel plate and developed giving a good separation of the parent compound and the 3 phenolic metabolites (Table I, Rr I). When the plate was developed in the same solvent system a second time, the separation beTABLE I Rr VALUES FOR ACETANILIDE PLATES
AND THE PHENOLIC METABOLITES ON SILICA GEL TLC
Standard
Rr Ia
Rr IIb
Acetanilide 4-Hydroxyacetanilide 3-Hydroxyacetanilide 2-Hydroxyacetanilide Unidentified metabolite
0.49 0.29 0.36 0.43 0.00
0.65 0.37 0.46 0.53 0.00
a Rr I: the Rr values obtained after developing the silica gel plate once in the hexane/isopropanol/ammonium hydroxide/water (73:25:1:1) solvent system. b Rr II: the Rr values obtained after developing the silica gel plate twice in the same solvent system.
226
ACETANILIDE
4-OH
ACETANILIDE
111
/
0
Rf
VALUE
100
Fig. 1. Chromatogram of acetanilide and the phenolic metabolites after extraction with ethyl acetate only. (A) Microsomes from isosafrole-treated mice. (B) Microsomes from untreated mice. (C) Experimental control.
tween the 4-hydroxy- and the 3-hydroxyacetanilide and between the 2-hydroxyacetanilide and the parent compound improved (Table I, Rr II). An unidentified polar metabolite did not migrate from the origin in this solvent system. When the incubation mixture was acidified and extracted with ethyl acetate only, the acetanilide and the phenolic metabolites were extracted simultaneously, but only the 4-hydroxylated product could be accurately quantitated since the small amount of 2-hydroxylated product was obscured by the large amount of acetanilide (Fig. 1). However, if the incubation mixture was made basic and extracted with benzene prior to the extraction with ethyl acetate, more than 99% of the parent compound partitioned into the benzene leaving the phenolic metabolites ( > 97 %) in the aqueous solution. This extraction procedure permitted the quantitiation of both the 4-hydroxylated and the 2-hydroxylated products (Fig. 2). The partitioning of the acetanilide and the phenolic metabolites was quantitated using radiolabelled acetanilide and 4hydroxyacetanilide. Product formation required NADPH as supplied by a regenerating system and was linear up to 45 min (Fig. 3) with up to 0.5 mg of microsomal protein (Fig. 4). In both a microsomal and a reconstituted system, the production of 4_hydroxyacetanilide was optimal at pH 8.0 (Fig. 5). The production of the 2-hydroxyacetanilide was optimal between pH 8.0 and 9.0. The production of the unidentified polar metabolite
227
4-OH
ACETANILIDE .
ACETANILIDE
Rf VALUE
0
100
Fig. 2. Chromatogram of acetanilide and the phenolic metabolites after extraction with benzene and ethyl acetate. (A) Microsomes from isosafrole-treated mice. (B) Untreated microsomes. (C) Experimental control.
did not vary with pH until a pH of 9.5. At pH 9.5, no metabolism occurred due to the denaturation of the cytochrome P-450. The apparent K, and V,,, in the microsoma1 system was determined to be 0.76 mM and 0.38 nmol/mg/min for the 2-hydroxyacetanihde and 0.62 mM and 12.7 nmoi~mg/min for the 4-hydroxyacetanilide (Fig. 6). Microsomes were prepared from the livers of mice that had been pretreated with isosafrole, phenobarbital, 3-methylcholanthrene or n-butylbenzodioxole. The effect
120
*
100
so 4-OH 2-OH
60 40 20 0 0
20
40
SO
80
100
TIME
Fig. 3. The effect of incubation time (min) on product formation in hepatic microsomes of isosafrok-treated DBAZIN mice.
228
0.20
0
0.40
0.60
0.80
1.00
1.20
MG PROTEIN Fig. 4. The effect of protein
concentration
on product
formation
ed DBAZ/N
in hepatic microsomes
of isosafrole-treat-
mice.
of these pretreatments on the production of the 4_hydroxyacetanilide, the 2-hydroxyacetanilide, and the unidentified polar metabolite is illustrated in Table II. All the treatments significantly increased the acetanilide 2-hydroxylase activity over that of control. All treatments, except the n-butylbenzodioxole, significantly increased the acetanilide 4-hydroxylase activity over that of control. The ratio of the 4-hydroxyto the 2-hydroxyacetanilide did not differ significantly (P~0.05) from the control with the phenobarbital or the n-butylbenzodioxole treatments but did with the isosa-
+
Fig. 5. The effect of pH on product mice. (B) In a reconstituted P-450 reductase,
formation.
(A) In hepatic
system using 0.2 nmol of purified
0.25 fig of phosphatidylcholine were as described
microsomes
of isosafrole-treated
DBA2/N
P, P-450, 1.2 units of NADPH-cytochrome
in 1 ml of Tris-HCI in ‘Materials
4-OH
buffer.
and Methods’.
The incubation
conditions
229 ‘1
Fig. 6. Lineweaver-Burk plot for hepatic microsomal acetanilide Chydroxylase and acetanilide 2-hydroxylase activity from isosafrole-treated DBA2/N mice.
frole and the 3-methylcholanthrene treatments. Pretreatment with isosafrole or 3methylcholanthrene significantly increased the 4-hydroxy-/2_hydroxyacetanilide ratio, although this increase was small. It has been demonstrated [7,8] that induction of cytochrome P-450 as well as 7-ethoxycoumarin O-deethylase and ethylmorphine N-demethylase activities by isosafrole
TABLE II THE EFFECT OF PRETREATMENT ON THE ACETANILIDE HEPATIC MICROSOMES OF DBAZ/N MICE
Control Isosafrole Phenobarbital 3-Methylcholanthrene n-Butyl BD
HYDROXYLASE ACTIVITIES IN
Unknown metabolite’
4-OH”
2-OHa
4-OH/2-OH
0.231 kO.049 0.672+0.103* 0.461+0.045 0.707*0.137* 0.397 f 0.097
1.49 + 0.068 6.67 f0.589; 3.04+0.294* 8.79 +0.632* 2.14kO.380
0.137+0.003 0.533 kO.029’ 0.270*0.014* 0.735+0.032* 0.180*0.027*
10.9 12.5* 11.3 12.0* 11.9
‘nmol of product/min/mg of microsomal protein. * Significantly different from control (PC 0.05)
230
TABLE EFFECTS
III OF PRETREATMENT
LIDE HYDROXYLASE
WITH
SAFROLE
AND ISOSAFROLE
ON HEPATIC
Treatment
Strain C57”
DBA”
Control
2.13kO.33
2.45 + 0.29
Safrole
6.13*
1.68*
7.54+2.46*
Isosafrole
7.40+
1.37*
7.70*
anmol product * Significantly
ACETANI-
ACTIVITY
formed/mg different
Values are mean
microsomal
1.12*
protein/min.
(P< 0.05) from control
values.
f SD (n = 4).
were the same in Ah-positive and Ah-negative mice; in addition, binding of isosafrole to the Ah-receptor did not occur. As shown in Table III, acetanilide hydroxylase activity is induced to the same extent by isosafrole in C57 (Ah-positive) and DBA (Ahnegative) mice. The use of radiolabelled acetanilide combined with an extraction procedure that selectively partitions the parent compound from the phenolic metabolites and the development of the silica gel plate twice in the same solvent system allows the quantitation of the 4-hydroxy-, the 3-hydroxy- and the 2-hydroxyacetanilide products. This TLC method, although not as convenient as the calorimetric method [3], provides a simpler and less time-consuming method than the reported HPLC methods [4,5]. One can easily quantitate rates of 1.0 nmol product formed/min/mg of microsomal protein using the specific activity described, but rates at the pmol level can be detected if substrate of higher specific activity is used. ACKNOWLEDGEMENTS
This article is a paper in the Journal Series of the NC Agricultural Research Service, Raleigh, NC 276957601. This investigation was supported, in part, by Grants ES-07046 and ES-00044 from the National Institute of Environmental Health Sciences, U.S. Public Health Services and by a predoctoral fellowship awarded to one of the authors (M.L.) by the Chemical Industry Institute of Toxicology.
REFERENCES 1 Negishi, M. and Nebert, D.W. (1979) Structural gene products of the Ah locus. Genetic and immunochemical evidence for two forms of mouse liver cytochrome P-450 induced by 3-methylcholanthrene. J. Biol. Chem. 254, 11015-l
1023.
231
2 Brodie,
B.B. and Axelrod,
N-acetyl-p-aminophenol J. Pharmacol. 3 Mitoma, Methods 4 Selander,
metabolites
in Enzymology, H.G., Jerina,
S. (1962) Aryl-Chydroxylase.
Vol. 5. Academic
and its metabolic
products,
in biological
aniline,
fluids and tissues.
monooxygenase
T.M., Negishi,
M. and Nebert,
and quantitative
determination
Anal. Biochem.
P.J., Smyser,
7 Cook, J.C. and Hogson, 8 Cook, J.C. and Hodgson, frole: lack of correlation
of acetanilide
systems. Arch. Biochem. D.W. (1979) Separation of ‘acetanilide
B.P. and Hodgson,
(Eds.),
with hepatic microsomes
Biophys.
164,241-246.
of acetanilide
4-hydroxylase
E. (1983) Purification Int. J. Biochem.
E. (1985) The induction
compounds.
and N.O. Kaplan
activity’
and its hydroxylated by high-pressure
liq-
96,201-207.
ase from mouse and pig liver microsomes. lenedioxyphenyl
In: S.P. Colowick
Press, New York, pp. 816819.
D.M. and Daly, J.W. (1974) Metabolism
cytochrome
uid chromatography. 6 Sabourin,
of acetanilide
(free and total conjugated)
Exp. Ther. 94, 22-28.
C. and Udenfriend,
and reconstituted 5 Guenthner,
J. (1948) The estimation and paminophenol
Chem.-Biol.
Interact.
E. (1986) Induction
of cytochrome 54,299-3
of cytochrome
with the Ah locus. Chem.-Biol.
of the flavin-containing
monooxygen-
16,713720. P-450 by isosafrole
and related methy-
15. P-450 in congenic
Interact.
58,233-240.
C57B1/6J mice by isosa-