Chem. Res. Toxicol. 1995,8, 721-728
721
Characterization of a Chemically Reactive Propranolol Metabolite That Binds to Microsomal Proteins of Rat Liver Shizuo Narimatsu,"??Toshiyuki Watanabe,? Yasuhiro Masubuchi,t Toshiharu Yoshito Kumagai,* Arthur K. Cho,§ Susumu Imaoka," Yoshihiko Funae," Tsutomu Ishikawa,l and Tokuji Suzukit Laboratories of Biopharmaceutics and Medicinal Organic Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263, Japan, Institute of Community Medicine, Tsukuba University, Tennoudai, Tsukuba, Ibaraki 305, Japan, Department of Molecular and Medical Pharmacology, University of California, Los Angeles, School of Medicine, Center for the Health Sciences, Los Angeles, California 90024,and Laboratory of Chemistry, Osaka City University School of Medicine, Abeno-ku, Osaka 525, Japan Received December 27, 1994@ We have characterized a chemically reactive propranolol (PL) metabolite which binds to proteins in rat liver microsomes. During incubation with rat liver microsomes (1mg of protein) fortified with a n NADPH-generating system, 4-hydroxypropranolol (4-OH-PL) quickly disappeared from the reaction medium, but none of the possible metabolite peaks was detected under the high-performance liquid chromatographic conditions used. The consumption of 4-OHPL depended on microsomes and NADPH. The reaction was not affected by inhibitors of cytochrome P450 or FAD monooxygenase, but was markedly diminished in the presence of cytosol and ascorbic acid. The effect of cytosol was inhibited by potassium cyanide but not by sodium benzoate or dimethyl sulfoxide, and was also not affected by heating at 60 "C for 30 min, suggesting that superoxide (SO) ion was involved in the reaction and that it was blocked by superoxide dismutase (SOD) present in the cytosol. Cu,Zn-SOD, purified from cytosol, effectively mimicked the suppressive effect of cytosol. Incubation of 4-OH-PL in an SOgenerating system of xanthine and xanthine oxidase generated 1,6naphthoquinone (1,4-NQ), which was identified by TLC, HPLC, and GCMS. 1,4-NQ was also formed in microsomal incubates containing NADPH and small amounts of microsomes (below 0.1 mg of protein). These results indicate that 4-OH-PL is converted by SO, or some reactive oxygen species derived from it, t o 1,4-NQ which binds to proteins and is one of the reactive metabolites of PL.
Introduction Propranolol (PLY is one of many p-adrenoceptor blocking agents which are often prescribed for the treatment of hypertension or arrhythmia. Repetitive oral administration of PL to rats caused a marked decrease in liver microsomal PL ring hydroxylase activities ( 1 , 2 )which have been shown to be catalyzed by members of the CYP2D subfamily (3). PL was also shown to be converted to a chemically reactive metabolite that binds to microsomal macromolecules in rats and humans (1,4,51, and this protein binding may be one of the mechanisms causing the inhibition of the drug-metabolizing enzyme activities. Arene oxide metabolites of PL (Figure 1) have been proposed as a candidate(s) of the reactive metabolite (4).
* Author to whom correspondence should be addressed. of Biopharmaceutics, Chiba University. * Laboratory Tsukuba University.
However, we recently reported that 4-hydroxypropranolol (4-OH-PL1,a presumed product of arene oxide decomposition, markedly inhibited microsomal drug-metabolizing activity, particularly of the CYPBD subfamily, after activation by rat liver microsomes and an NADPHgenerating system ( 6 ) . Furthermore, in studies with radiolabeled PL, covalent binding of radioactivity was found to correlate with the formation arid elimination of 4-OH-PL during the incubation ( 6 ) . These findings suggested that a metabolite of an arene oxide or 4-OHPL, whose formation is catalyzed by isozymes of the CYF'BD subfamily, may be responsible for the protein binding and decreased monooxygenase activity. The present study was conducted to confirm the hypothesis that a subsequent metabolite of 4-OH-PL is involved in the binding to microsomal proteins, which may result in a decreased activity of the CYP2D subfamily.
+
University of California, Los Angeles. Osaka City University. Laboratory of Medicinal Organic Chemistry, Chiba University. Abstract published in Advance ACS Abstracts, June 1, 1995. Abbreviations: PL, propranolol; x-OH-PL, x-hydroxypropranolol; PMSF, phenylmethanesulfonyl fluoride; CYP, cytochrome P450; NDP, N-desisopropylpropranolol; 1,4-NQ,1,4-naphthoquinone;G-6-P, glucose 6-phosphate; G-6-PDH, glucose 6-phosphate dehydrogenase; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; HEPES, N42hydroxyethyl)piperazine-N-2-ethanesulfonic acid; HPLC, high-performance liquid chromatography; SO, superoxide; SOD, superoxide dismutase; TLC, thin-layer chromatography; GC-MS; gas chromatog raphy-mass spectrometry; KCN, potassium cyanide. 5
Experimental Procedures
11
@
Chemicals. PL hydrochloride, phenylmethanesulfonylfluoride (PMSF), methimazole, and cytochrome c (from horse heart) were purchased from the Sigma Chemical Co. (St. Louis, MO). 4-OH-PL hydrochloride was kindly provided by the Sumitomo Chemical Ind. (Osaka, Japan); N-desisopropylpropranolol (NDP) hydrochloride by the IC1 Pharmaceuticals Co. (Maclesfield, U.K.); 1,4-naphthoquinone(1,4-NQ) by the Kanto Chemical Co. Ltd. (Tokyo, Japan). Benzamidine hydrochloride n-hydrate, xanthine, xanthine oxidase, a-naphthoflavone, and N 4 2 - h ~ -
0893-228d95I2708-0721$09.00/0 0 1995 American Chemical Society
722 Chem. Res. Toxicol., Vol. 8, No. 5, 1995
Narimatsu et al.
Covalent Binding
4
R : CH2CH(OH)CH2NHCH(CH&
;Protein I
OH
/
4-OH PL
PL
@OH 5-OH PL
.
'
0 8
\
0 \
Protein *,
..'protein
7-OH PL
Covalent Binding
Figure 1. Primary metabolic pathways of propranolol. Broken lines represent speculated pathways. droxyethyl)piperazine-N'-2-ethanesulfonicacid (HEPES) were obtained from the Wako Pure Chemical Ind. (Osaka, Japan); SKF 525-A was from Research Biochemicals Inc. (Natrick, MA); TLC plates (Kieselgel 60 F254) were from Merck (Darmstadt, Germany); Sephadex G-100 was from Pharmacia (Uppsala, Sweden). 4-Hydroxybunitrolol hydrochloride was kindly donated by the Nippon C.H. Boehringer-Ingelheim Co. (Hyogo, Japan). Glucose 6-phosphate (G-6-P), glucose 6-phosphate dehydrogenase (G-6-PDH), and NADPH were obtained from the Oriental Yeast Co. Ltd. (Tokyo, Japan). 5-OH-PL and 7-OHPL were synthesized by the method of Oatis et al. (7). Preparation of Cytosol and Microsomes. Male Wistar rats (7 weeks old) were obtained from Takasugi Experimental Animals (Kasukabe, Japan). The animals were killed by decapitation, and livers were perfused with ice-cold 1.15% (w/ v) KC1. The livers were minced, homogenized in 4 volumes of 1.15% KC1, and centrifuged a t 9OOOg for 20 min at 4 "C. A supernatant fraction was centrifuged at 105000g for 60 min at 4 "C, to yield a cytosol fraction and microsomal pellet. The pellet was suspended in 4 volumes of 154 mM potassium phosphate buffer (pH 7.4) and centrifuged again at 105000g for 60 min a t 4 "C. Pellet was suspended in 4 volumes of the same buffer and stored as microsomes at -80 "C until use. Protein concentrations were determined by the method of Lowry et al. ( 8 )using bovine serum albumin as standard. Incubation of PL Metabolites with Microsomes. PL metabolites ( a mixture of 4-OH-PL, at a final concentration of 5 pM, 5-OH-PL at 1.6 pM, and 7-OH-PL at 5 pM) was incubated with microsomes (1mg of protein/mL) fortified with an NADPHgenerating system containing 10 mM G-6-P, 2 units/mL G-6PDH, 8 mM MgCl2,and 100 mM potassium phosphate buffer (pH 7.4) in a final volume of 1.0 mL. After preincubation at 37 "C for 5 min, incubation a t 37 "C was started by adding NADPH (0.5 mM) and stopped 5 min later by adding 1mL of 1 N NaOH including sodium bisulfite (25 mg/mL) to avoid degradation of 4-OH-PL. Remaining PL metabolites were determined by a n HPLC method reported previously (9). A portion of cytosol was dialyzed against distilled water (100 volumes x 2) at 4 "C for 12h. When the effects of cytosol on the reaction were examined, native, dialyzed, or heat-treated (60 "C for 30 min) cytosol (0.1 mg/mL) was added to the reaction medium before the incubation.
Inhibitor Experiments. SKF 525-A, a-naphthoflavone, or methimazole (each at 1 mM final concentration) was added to the reaction medium, and elimination of 4-OH-PL was assayed by HPLC. Enzyme Purification. Cu,Zn-superoxide dismutase (SOD) was purified from rat liver cytosol essentially according to the method of Kumagai et al. (10).In brief, 105000g supernatant fractions were prepared from male Wistar rats (7 weeks old). To this supernatant (435 mL) was added ammonium sulfate (140.5 g) to make a 50% (w/v) saturated solution, and the pH was adjusted to 5.0 with acetic acid. The solution was mixed with a n equal volume (500 mL) of methanol, stirred for 15 min a t room temperature, and then centrifuged at 2500g for 10 min. This extract was concentrated with a rotary evaporator, and then with a Advantec ultrafiltration unit using a UK-10 membrane (cutoff molecular mass 10 kDa). M e r dialysis, the concentrated solution was applied to a CMcellulose column (1.5 cm i.d. x 20 cm), the column washed, and the Cu,Zn-SOD eluted with a linear gradient of KC1 from 0 to 0.3 M. Fractions having SOD activity were combined, concentrated, and dialyzed against 50 mM potassium phosphate buffer (pH 7.8) (40 volumes x 31, and applied to a Sephadex G-100 column (1.5 cm i.d. x 100 cm). Fractions having SOD activity were combined and subjected to a second gel filtration procedure with Sephadex G-100 for further purification. Identification of 4-OH-PL Metabolite Formed in Xanthine-Xanthine Oxidase System or Microsomal System by TLC, HPLC, and GC-MS. 4-OH-PL (final concentration 25 pM) was added to 80 mM HEPES buffer (pH 7.4) containing 1 mM xanthine in a final volume of 10 mL. The reaction was started by adding 500 milliunits of xanthine oxidase and allowed to continue for 60 min at 37 "C. The mixture was extracted with ethyl acetate (20 mL x 2), and the organic layer was removed by evaporation. In the microsomal system, 4-OH-PL (25 pM) was added to 100 mM potassium phosphate buffer (pH 7.4) containing r a t liver microsomes (0.1 mg of protein) and a n NADPH-generating system in a final volume of 5 mL. The mixture was incubated at 37 "C for 60 min, and was extracted with ethyl acetate (10 mL x 2) as described above. The residue of each extract was dissolved in 100 pL of ethyl acetate and examined by TLC, HPLC, and GC-MS. TLC utilized a solvent system consisting of benzene/chloroform/ethyl acetateftriethylamine (1:l:l:O.X v/v), and the products were detected by their
Metabolic Activation of Propranolol absorption at 254 nm. HPLC utilized a Shimadzu LC6A liquid chromatographequipped with a Shimadzu SPD6A UV detector and an Inertsil ODS (4.6 mm i.d. x 25 cm, the GL Science Co., Tokyo, Japan) column: The mobile phase was acetonitrile/ methanol/water/acetic acid (25:25:50:1v/v), and the eluants were detected at 245 nm. The GC-MS analysis utilized a Hewlett Packard 5890 Series I1 gas chromatograph equipped with a 5971 mass selective detector and a G1030A MS Chemostation containing a fused silica capillary DB-1 column (1 mm i.d. x 30 m). The column, injection port, and detector temperatures were 100,250,and 280 "C,respectively, and the ionizing energy was 70 eV at an ionizing current of 300 pA with a carrier gas (helium)flow rate of 40 mumin. Determination of 1,4-NQ. To determine 1,4-NQ formation in the xanthine-xanthine oxidase system,4-OH-PL(25pM) was preincubated at 37 "C for 5 min in 80 mM HEPES buffer (pH 7.4) containing 1mM xanthine in a final volume of 1.0 mL. The reaction was started by adding xanthine oxidase (50 milliunitd mL) and allowed to continue for 5 min, and an aliquot (20 pL) of the reaction mixture was analyzed by HPLC under the same conditions described above. In the assay of 1,4-NQ formed in microsomes, 4-OH-PL (25 pM) was preincubated at 37 "C for 5 min in 100 mM potassium phosphate buffer (pH 7.4) containing rat liver microsomes (25 pg) in a final volume of 1.0 mL. The reaction was started by adding NADPH (0.5 mM) and allowed to proceed for 5 min, and then an aliquot (20 pL) of the reaction mixture was subjected to the HPLC analysis. The formation of 1,4-NQ was calculated on the basis of calibration curves prepared by adding known amounts of synthetic 1,4-NQ to reaction mixtures containingheat-inactivated xanthine oxidase or microsomes. Effects of Scavengers of Reactive Oxygen Species on Inhibition of Microsomal PL Oxidation Activities by 4-OH-PL. Rat liver microsomes (0.5 mg of protein) was incubated with 4-OH-PL (2 pM) and an NADPH-generating system in the presence of various reactive oxygen scavengers such as ascorbic acid (1 mM), rat liver cytosolic SOD purified (50 units), and sodium benzoate ( 5 mM) at 37 "C for 10 min. PL (2 pM) was then added as a substrate and incubated at 37 "C for 1 min. PL ring hydroxylase activities forming 5-OH-PL and 7-OH-PLwere assayed by the HPLC as reported elsewhere (9).
Other Procedures. SOD activity was assayed by the method of McCord and Fridovich (11). SDS-PAGE was performed by the method of Laemmli (12) using a 15% polyacrylamide gel.
Results Microsomal Transformation of PL Metabolites. We previously reported that when 4-OH-PL, 5-OH-PL, 7-OH-PL, or NDP was preincubated with rat liver microsomes and NADPH prior to the assay of PL oxidation activities, only 4-OH-PL caused a marked decrease in PL 5- and 7-hydroxylation reactions (6)primarily mediated by the CYP2D subfamily (3). In this study we determined the time course of the disappearance of the PL metabolites, 4-OH-PL, 5-OH-PL, and 7-OH-PL, during their incubation with fortified rat liver microsomes. Figure 2 shows that 4-OH-PL is rapidly eliminated from the incubation medium, i.e., 98% has disappeared after incubation for 12 min. In contrast, most of the added 5-OH-PL and 7-OH-PL remained in the medium. Although most of the 4-OH-PL was eliminated during the incubation, no secondary metabolite was detected by HPLC under the conditions employed. Elimination of NDP was found to be negligible compared with 4-OHPL (data not shown). Because of its rapid disappearance, the metabolism of 4-OH-PL was examined further. Omitting NADPH or microsomes from the incubation system and heating the
Chem. Res. Toxicol., Vol.8, No. 5, 1995 723
v
0
-
5
10
Incubation time (min)
Figure 2. Metabolic disappearance of hydroxypropranolols. A mixture of ring-hydroxylated propranolol metabolites (4-OHPL, 5 pM; 5-OH-PL,1.6pM; 7-OH-PL,5 pM) was incubated with rat liver microsomes in the presence of an NADPH-generating system at 37 "C. Remaining metabolites were determined by
the HPLC as described under Experimental Procedures. Closed circle, 4-OH-PL;open triangle, 5-OH-PL;open circle, 7-OH-PL. Data represent mean value of independent two experiments. microsomal fraction a t 100 "C for 3 min prevented 4-OH-
PL disappearance, indicating that the reaction was enzymatic. We tested the cofactor requirement for 4-OH-
PL elimination in microsomes. As a result, NADPH produced the largest effect and was followed by flavin mononucleotide (FMN), whereas FAD, NAD, NADH, and NADP showed little effect (data not shown). We then examined the kinetics of 4-OH-PL (0.5-10 pM) elimination in a 0.5 min incubation with rat liver microsomes (0.5 mg of protein) in the presence of a n NADPH-generating system. The Lineweaver-Burk plot for the microsomal 4-OH-PL elimination showed that the reaction was monophasic and gave kinetic parameters of 4.20 f 1.33 pM and 3.28 f 1.26 nmol/(min.mg of protein) as K, and V, values, respectively. Effects of Enzyme Inhibitors, Antibodies and Cytosol on 4-OH-PL Elimination. The cytochrome P450 inhibitors SKF 525-A and a-naphthoflavone at 1 mM and the FAD-containing monooxygenase inhibitor methimazole, a t 1 mM, did not affect 4-OH-PL elimination (data not shown). Antibodies raised against cytochrome P450BTL, which is thought to belong to the CYP2D subfamily (131,did not suppress the reaction (data not shown). However, a change of the enzyme source from microsomes to a 9OOOg supernatant fraction decreased the elimination by 88% (Table 1). Further, addition of cytosol (0.1 mg/mL), which had been dialyzed against water for 12 h, to the microsomal reaction mixture decreased 4-OH-PL elimination by 86%. These results suggested that cytosolic macromoleculecomponent(SI, which could not be removed by the dialysis, suppressed microsome mediated 4-OH-PL elimination. Characterization of the Suppression by Cytosol of Microsomal 4-OH-PLElimination. Table 1 summarizes results of various treatments on the inhibitory effects of cytosol. The suppressive effect of cytosol on the 4-OH-PL elimination was not diminished either by dialysis or by heating a t 60 "C for 30 min. In contrast, addition of potassium cyanide (KCN, 5 mM) to the medium containing both microsomes and cytosol prevented the cytosolic effects. The results demonstrated that the cytosolic component(s) responsible for the suppression of microsomal 4-OH-PL metabolism was relatively heat-stable and sensitive to KCN, suggesting
Narimatsu et al.
724 Chem. Res. Toxicol., Vol. 8, No. 5, 1995 Table 1. Effects of Various Treatments on Suppression of Microsomal COH-PL Elimination by Cytosol" treatment control microsomes 9000gsupernatant control microsomes +dialyzed cytosol control microsomes +dialyzed cytosol +dialyzed and heated cytosol +dialyzed cytosol + KCN ( 5 mM) +ascorbic acid (1mM)
4-OH-PL elimination (%1 86.7 i 3.8 10.6 i 3.1* 12.4 & 2.9* 54.3 f 3.8 13.8 i 4.3* 13.4 jr 1.2* 66.9 i 2.2# 4.5 f 1.2*
GElimination was assayed by the HPLC using a reaction medium containing 4-OH-PL (25pM), microsomes (1mg of proteid mL) or 9OOOg supernatant (2 mg of proteidml), and the NADPHgenerating system. Cytosol (0.1 mg of proteidml) had been dialyzed against water for 12 h. If necessary, the dialyzed cytosol was heated a t 60 "C for 30 min. Each value represents the mean i~SE (n = 4). *Significantly different from each control value ( p c 0.01). #Significantly different from control microsomes dialyzed cytosol (13.8 f 4.3) ( p < 0.011.
+
100
, 80 C
.-g0
60
C
I-
.-5E
40
I
n
9=d
20 0 -
0 1 2 3 4 5 Cu,Zn-SOD purified from rat liver (units/ml)
Figure 3. Effects of purified Cu,Zn-SOD on microsomal 4-OHPL elimination. Elimination was assayed by the HPLC using a reaction medium containing 4-OH-PL (25 pM), microsomes (1 mg/mL), and NADPH (0.5 mM) and a n incubation time of 5 min. The data represent t h e mean of two independent determinations.
involvement of Cu,Zn-SOD (14,15). Ascorbic acid, a n SO scavenger (16), also effectively suppressed the reaction in the absence of the cytosolic fraction, supporting the possibility of SOD involvement. In contrast, the hydroxyl radical scavengers, sodium benzoate or dimethyl sulfoxide a t 5 mM(17, 18),had no effect on the reaction (data not shown). Effects of SOD on 4-OH-PL Elimination. Since the results obtained suggested the participation of cytosolic SOD in the suppression of 4-OH-PL elimination, we purified Cu,Zn-SOD from rat liver cytosol according to a published method (IO),and obtained a n enzyme which was homogeneous in SDS-PAGE in the three column steps. The final preparation showed a single protein band with an apparent molecular mass of 15.8 kDa in SDS-PAGE and had a specific activity of 6217.4 units/ mg of protein. We then added varying amounts of the purified Cu,Zn-SOD to the incubation medium containing microsomes, 4-OH-PL, and NADPH (0.5 mM) and examined the 4-OH-PL elimination (Figure 3). Cu,Zn-SOD suppressed microsomal 4-OH-PL elimination in a concentration-dependent manner. Furthermore, when the purified
Inject
Figure 4. HPLC analysis for a metabolite formed from 4-OHPL i n the SO-generating system consisting of xanthinexanthine oxidase. 4-OH-PL (25 pM)was incubated at 37 "C for 60 min with the SO-generating system consisting of xanthine (1 mM) and xanthine oxidase (50 milliunits). The medium was extracted with ethyl acetate, and the extract was examined by reversed-phase HPLC using a column of Inertsil ODS (4.6 m m i.d. x 250 mm), with mobile phase acetonitrile/methanol/water/ acetic acid (22:22:56:1 d v ) . Other conditions are given under Experimental Procedures.
Cu,Zn-SOD was heated a t 60 "C for 30 min before adding to the incubation mixture or added with KCN (5 mM), the results were very similar to those of cytosol (data not shown). These results strongly suggest that Cu,Zn-SOD is responsible for the suppressive effects of cytosol on the 4-OH-PL elimination by microsomes. Metabolism of 4-OH-PL by the Xanthine-Xanthine Oxidase System. The suppression of 4-OH-PL elimination in microsomal incubation mixtures by Cu,Zn-SOD indicated that SO is involved in the reaction. We thus examined 4-OH-PL metabolism in a mixture of xanthine and xanthine oxidase, which generates SO (16). A preliminary HPLC analysis showed that 4-OH-PL was consumed and a peak, possibly corresponding to a secondary metabolite, appeared. Then, 4-OH-PL a t 25 pM was incubated in a larger final volume (10 mL) for 60 min, and the reaction mixture was extracted with ethyl acetate. The organic layer was evaporated, and the residue was examined by TLC, HPLC, and GC-MS. TLC analysis showed a metabolite spot with an R f value of 0.72 (data not shown). Figure 4 shows the HPLC chromatogram. I n addition to a substrate peak with a retention time of 3.7 min, a peak with a retention time of 10.7 min was observed. GC-MS analysis also revealed a prominent metabolite peak with a retention time of 7.3 min (Figure 5A). This metabolite showed a molecular
Metabolic Activation of Propranolol
Chem. Res. Toxicol., Vol. 8, No. 5, 1995 725
Abundance 2000000
TIC
-
(4
1500000.
A metabolite of 4-OH-PL 1000000
-
500000
L
,',"'" 3.150 4 . 0 0
0 - , . . .
T i m e ->
,,.. , , , . 4.'50
5.100
, , , , 1 " "
5.'50 7.309
6.00
",'",',,.,,,,, 6.l50 7 . 0 0 7.50 8 . 0 0
nin
lbI3
400000 350000
300000
250000 104
200000
150000
76
100000 50000
2
-->
Figure 6. Total ion chromatogram (A) and mass spectrum (B)of a metabolite formed from 4-OH-PL in the SO-generating system consisting of the xanthine-xanthine oxidase system. A portion of ethyl acetate extract fraction was subjected to GC-MS under the conditions given under Experimental Procedures.
ion and base peak a t m l z 158 together with other prominent fragment ions a t m l z 130, 104, 102, 76, and 50 (Figure 5B). A match analysis using the library data in the Chemostation in the GC-MS system suggested that the metabolite was 1,4-NQ. Using authentic 1,4-NQ as standard, we reexamined the metabolite by TLC, HPLC, and GC-MS and confirmed that the Rf value in TLC, the retention time in HPLC, and the retention time and the fragment ions in GC-MS were in good agreement with those of authentic 1,4-NQ. Identification of 1,4NQ as a Microsomal Metabolite of 4-OH-PL. To confirm the microsomal formation of 1,4-NQ from 4-OH-PL, we incubated 4-OH-PL (25 pM) with rat liver microsomes (0.1 mg of protein) and a n NADPH-generating system in a final volume of 5.0 mL at 37 "C for 60 min. The medium was extracted with ethyl acetate, and the metabolites extracted were subjected to silica gel preparative TLC. A band corresponding to the synthetic 1,4-NQ was scraped off under a U V lamp and extracted with ethyl acetate. The metabolite fraction was then examined by GC-MS under the same conditions used for the identification of the metabolite obtained in the xanthine-xanthine oxidase system. We confirmed that 1,4-NQ was formed as a microsomal metabolite of 4-OH-PL by coincidence of the retention time and fragment ions with those of authentic sample. Determination of 1,4-NQ Formed from 4-OH-PL. Figure 6 shows the time course of the formation of 1,4NQ from 4-OH-PL (25 nmol in 1 mL) in the xanthine-
I v
.-
C 0 c
c
1510
0 C
0
0
5
0
-
I 0
5 10 lncubation tlme (m in)
15
Figure 6. Time course of the formation of 1,4-NQ from 4-OHPL in the xanthine-xanthine oxidase and microsomal systems. 4-OH-PL (25 nmol) was added to the incubation medium (1mL) containing xanthine (1mM) and xanthine oxidase (50 milliunits) or microsomes (25 yg of protein) and NADPH (0.5 mM) and incubated at 37 "C for 0, 5, 10, and 15 min. 4-OH-PL and 1,4NQ were determined by the HPLC under the conditions given in the Experimental Procedures section. Circles, xanthinexanthine oxidase system; squares, microsome system; closed symbols, 1,4-NQ; open symbols, 4-OH-PL. Each point represents the mean value of two independent experiments.
xanthine oxidase system and the microsomal system. In the SO-generating system, 1,4-NQ was formed in a timedependent manner. The amount of 1,4-NQ generated
726 Chem. Res. Toxicol., Vol. 8, No. 5, 1995
Narimatsu et al.
Table 2. Effects of Scavengers of Reactive Oxygen Species on Inhibition of Microsomal Propranolol Metabolism by 4-OH-PLa treatment Non +4-OH-PL +4-OH-PL + ascorbic acid i 1 mM) +4-OH-PL + SOD (50 units) +4-OH-PL + sodium benzoate (5 mM)
5-hydroxylation [pmoli 7-hydroxylation [pmol/ (min-mgof protein)] (miwmg of protein)] 28.8 f 1.3 103.4 i 7.6 11.8 i 1.4* 28.6 f 4.9* 16.3 i 1.2*,# 48.7 7.6*
*
18.8 i 0.9*.#'
58.9 i 5.5*,'#
10.2 i 0.4*
29.8 i 3.6*
Rat liver microsomes were preincubated with 4-OH-PL (2 pM), reactive oxygen species scavengers, and an NADPH-generating system a t 37 "C for 10 min. Then propranolol (2 pM) as a substrate was added to the incubation medium and incubated for 1 min. Propranolol ring hydroxylase activities forming 5-OH-PL and 7-OH-PL were assayed by the HPLC as described in the Experimental Procedures. Each value represents the mean f SE (n = 4). *Significantly different from the control iNon) ( p < 0.01). #Significantly different from (+I-OH-PL) ( p < 0.025). "Significantly different from i+COH-PL) ( p < 0.01).
was 6.95 nmol after 15 min of incubation, which was 56.6% of the substrate consumed (12.27 nmol). On the other hand, the amount of the quinone metabolite detected in the microsomal system (1.71 nmol after 15 min of incubation) was very small compared to the SOgenerating system, and it accounted for only 16.1%of the consumed substrate (10.62 nmol). Protective Effect of SO Scavengers on a Suppression of PL Ring Hydroxylase Activities by 4-OHPL. Preincubation of 4-OH-PL with rat liver microsomes and NADPH significantly decreased PL ring hydroxylase activities to 38%(5-OH-PL) and 23%(7-OH-PL) those of the control incubated without 4-OH-PL (Table 2). Coincubation of SOD with microsomes, NADPH, and 4-OHPL showed a significant protection against a decrease in PL 5-hydroxylase activity by 4-OH-PL. Ascorbic acid showed a tendency to reduce the suppressive effect of 4-OH-PL on the microsomal PL ring hydroxylase activities but without significance. In contrast, sodium benzoate as a hydroxyl radical scavenger did not show any protective effect under the conditions used.
Discussion We reported previously that preincubation of 4-OHPL with microsomes in the presence of NADPH changed the mode of inhibition of other ring hydroxylase activities; i.e., after preincubation with microsomes and NADPH, 4-OH-PL noncompetitively inhibited PL 5- and 7-hydroxylase activities, whereas it inhibited the same activities competitively when preincubated without NADPH (6). This finding indicated that a further metabolite of 4-OH-PL inactivates the enzyme possibly by irreversibly binding to it. We have examined this possibility further with metabolic studies of 4-OH-PL in rat liver microsomes. The elimination of 4-OH-PL was very rapid, required enzymatically active microsomes, and was kinetically monophasic, with a Michaelis constant (K, value) of 4.2 pM. In subsequent experiments we employed a substrate concentration of 25 pM, where the rate of 4-OH-PL elimination would be above 85% of the V,, value. Microsomal 4-OH-PL elimination was not affected by typical cytochrome P450 inhibitors, a n antibody against the CYP2D subfamily, or an FAD-monooxygenase inhibitor. Vu and Abramson (19) reported that 4-OH-PL
was refractory to metabolism by lOOOOg supernatant fraction of rat liver. We also examined the elimination of 4-OH-PL in the reaction medium containing 9OOOg supernatant as enzyme source and obtained similar results so that 4-OH-PL consumption was not observed in preparations containing cytosol. Consistent with this notion, addition of dialyzed cytosol to a microsomal reaction mixture effectively suppressed 4-OH-PL metabolism. The macromolecule responsible for the suppressive effect of cytosol was found to be stable to heating a t 60 "C for 30 min, a known property of Cu,Zn-SOD (14, 15). We therefore purified Cu,Zn-SOD from the cytosol fraction of rat liver and obtained a final preparation of high specific activity compared to values previously reported (17, 18). The purified Cu,Zn-SOD effectively mimicked the effects of cytosol in suppressing microsomal 4-OH-PL elimination. The involvement of SO in the metabolism of 4-OH-PL was demonstrated in the following experiments. KCN (5 mM), a n inhibitor of Cu,Zn-SOD, diminished the cytosolic effect on the 4-OH-PL elimination by microsomes. Moreover, ascorbic acid (SO scavenger, l mM) markedly decreased the 4-OH-PL elimination, whereas sodium benzoate or dimethyl sulfoxide (hydroxyl radical scavengers, 5 mM each) did not prevent it. These results further supported the involvement of Cu,Zn-SOD in the cytosolic effect and of SO in the microsomal 4-OH-PL metabolism. The nature of the superoxide-mediated reaction of 4-OH-PL was then examined in the SO-generating xanthine-xanthine oxidase system (15). In this and microsomal preparations, we found that 4-OH-PL was converted to 1,4-NQ. The microsomal experiments were complicated by the reaction of 1,4-NQ with microsomal protein. In preliminary experiments, we attempted to identify 1,4-NQ in a microsomal mixture of 4-OH-PL (25 pM) in a large reaction volume (10 mL) with 10 mg of microsomal protein but were unsuccessful. However, when the quantity of microsomal protein was reduced to 0.1 mg, 1,4-NQ was isolated and identified by GC-MS. The low recovery of 1,4-NQin microsomal preparations could be due either to covalent binding to protein or to formation of another metabolite. 1,4-NQ is known to covalently bind to proteins or glutathione via a sulfhydryl group (20,211. Therefore, a large part of 1,4-NQ formed from 4-OH-PL could bind to microsomal proteins in the microsome system and to xanthine oxidase in the SOgenerating system. However, there is also the possibility that 4-OH-PL is converted to other metabolite(s1 that could not be detected under the conditions used. Walle and Gaffney (22)identified 1,4-dihydroxynaphthalene as one of the urinary metabolites of PL in humans and dogs. Using 4-OH-PL and rat liver l O O O O g supernatant, Vu and Abramson (29)failed to confirm the formation of 1,4-dihydroxynaphthaleneand speculated that 1,4-dihydroxynaphthalenewould be produced from 4-OH-PL glycol and not from 4-OH-PL, but without supportable experimental evidence. On the other hand, we observed the formation of 1,4-NQ from 4-OH-PL in the present study using rat liver microsomal fractions. It is feasible that Cu,Zn-SOD contained in the lOOOOg supernanat fraction of rat liver scavenged SO, resulting in no detectable formation of 1,4-NQ or 1,4-dihydroxynaphthalene in the studies of Vu and Abramson (19). It seems very likely that SO is directly involved in the microsome-mediated 1,4-NQ formation from 4-OH-PL. SO is converted to HzOz primarily by Cu,Zn-SOD or Mn-
Chem. Res. Toxicol., Vol. 8, No. 5, 1995 727
Metabolic Activation of Propranolol
P450 Propranolol
*. -*.
I Nonenzvmatic reactioi
.'
1
?
i Protein
I
sd reactive oxygen species
OH 4-Hydroxypropranolol
0
0 1,CNaphthoquinone
R:
CH2CH(OH)CH2NHCH(CH& Figure 7. A proposed mechanism for metabolic activation of PL via oxidation at the 4-position in rat liver microsomes.
SOD in animal liver cells (231, but it is also converted to hydroxyl radical by the metal-catalyzed Haber-Weiss reaction (24). However, the participation of hydroxyl radicals can be excluded because none of hydroxyl radical scavengers affected the microsomal 4-OH-PL elimination. The elimination of 4-OH-PL was prevented by a n SO scavenger (ascorbic acid). To further confirm the role of SO, we examined a protective effect of SO scavengers against the inhibition by 4-OH-PL of rat liver microsomal PL metabolism. Following addition of SOD to the reaction medium containing microsomes, NADPH, and 4-OH-PL, the enzyme produced a significant protection against the decreases in PL ring hydroxylase activities (forming 5-OHPL and 7-OH-PL), which was induced by preincubation of microsomes with 4-OH-PL and NADPH. But the restoration was not complete. In the presence of SOD, most of 4-OH-PL added remained in the reaction medium aRer the incubation, whereas most of 4-OH-PL was eliminated in the absence of the enzyme. Therefore, the incomplete restoration of the ring hydroxylase activities by SOD can be due to a possible competitive inhibition between 4-OH-PL remaining and PL added as a substrate for the CYPBD enzyme(s). These results again indicate the involvement of SO or a related active oxygen species other than hydroxyl radical in the elimination of 4-OH-PL and the formation of 1,4-NQ in liver microsomes. From these results and considerations, we propose the formation of 1,4-NQ from 4-OH-PL in rat liver microsomes to proceed as shown in Figure 7. Possible sources of reactive oxygen species in microsomes are CYP isoforms and NADPH-CYP reductase, and it has been reported that CYP isoforms rather than the reductase are primarily responsible for production of reactive oxygen species (25-28). In the proposed reaction, SO or some reactive oxygen species derived from it, may extract a hydrogen from 4-OH-PL, resulting in the formation of 1,4-NQ. The details of the reactions leading to 1,4-NQ are unclear. SO can react both as a base and as a redox agent, and 4-OH-PL has sites for both pathways. Its basicity and nucleophilicity are substantially altered in protic media, however, because of extensive solvation and
disproportionation (29,30). On the other hand, SO is a much better oxidant when proton is available [E"' = 0.87 V in protic media vs -1.7 V in the aprotic solvent dimethyl sulfoxide (2911. Figure 7 also includes a possible pathway of metabolic oxidation of PL a t the 4-position. Thus, a CYPBD isoform(s1 biotransforms PL to 3,4-epoxypropranolol, some of which may bind to the CYP isoform as soon as it is formed. The epoxides are also nonenzymatically converted to 4-OH-PL, which is then converted to 1,4NQ by SO or a reactive oxygen species derived from it. 1,4-NQ as well as 3,4-epoxide binds to microsomal proteins, which may be one of the mechanisms causing the inhibition of the CYP2D isoforms. In the cell, 1,4NQ formation is blocked by SOD, and any 1,4-NQ formed would be reductively biotransformed into 4-OH-PL by quinone reductase so that, under normal physiological conditions, this interaction would not be likely to occur. However, 1,4-dihydroxynaphthalene,a reduced metabolite of 1,4-NQ, was reported as a urinary metabolite of PL in humans and dogs (22). Therefore, this reaction sequence may be important in unusual conditions where these protective mechanisms are not functioning normally. Our previous studies (31-33) indicated that 3,4dihydroxymethamphetamine produced from (methylenedioxylmethamphetamine by CYP2D isozyme(s1 is oxidized by SO to a n o-quinone. This highly electrophilic product reacts easily with sulfhydryl functions to cause toxic actions; both the CYPBD subfamily and the active oxygen species formed during interactions with it appear to play a n important role in the metabolic activation of PL and (methy1enedioxy)methamphetamine.
Acknowledgment. The authors would like to thank Messrs. Masayuki Mochida and Takayuki Arai for their technical assistance.
References (1) Schneck, D. H., and Pritchard J. F. (1981)The inhibitory effect
of propranolol treatment on its own metabolism in the rat. J. Pharmacol. Exp. Ther. 218,575-581. (2) Masubuchi, Y., Fujita, S., Chiba, M., Kigimoto, N., Umeda, S.,
and Suzuki, T. (1991)Impairment of debrisoquine 4-hydroxylase
728 Chem. Res. Toxicol., Vol. 8, No. 5, 1995 and related monooxygenase activities in the rat following treatment with propranolol. Biochem. Pharmacol. 41, 861-865. (3) Masubuchi, Y., Kagimoto, N., Narimatsu, S., Fujita, S., and Suzuki, T. (1993) Regioselective contribution of the cytochrome P-450 2D subfamily to propranolol metabolism in rat liver microsomes. Drug Metab. Dispos. 21, 1012-1016. (4) Shaw, L., Lennard, M. S., Tucker, G. T., Bax, N. D. S., and Wood, H. F. (1987) Irreversible binding and metabolism of propranolol by human liver microsomes-relationship to polymorphic oxidation. Biochem. Pharmacol. 36, 2283-2288. (5) Al-Asady, S. A. H., Black, G. L., Lennard, M. S., Tucker, G. T., and Wood, H. F. (1989) Inhibition of lignocaine metabolism by P-adrenoceptor antagonists in rat and human liver microsomes. Xenobiotica 19, 929-944. (6) Masubuchi, Y., Suzuki, K., Fujita, S., and Suzuki, T. (1992) A possible mechanism of the impairment of hepatic microsomal monooxygenase activities after multiple administration of propranolol in rats. Biochem. Pharmacol. 43, 757-762. (7) Oatis, J. E., Russel, M. P., Knapp, D. R., and Walle, T. (1981) Ring hydroxylated propranolol: synthesis and P-receptor antagonism and vasodilator activities of the seven isomers. J . Med. Chem. 24, 309-314. ( 8 ) Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J . B i d . Chem. 193,265-275. (9) Masubuchi, Y., Fujita, S., Chiba, M., Kigimoto, N., Umeda, S., and Suzuki, T. (1991) Impairment of debrisoquine 4-hydroxylase and related monooxygenase activities in the rat following treatment with propranolol. Biochem. Pharmacol. 41, 861-865. (10) Kumagai, Y., Shinyashiki, M., Sun, G. F., Shimojo, N., and Sagai, M. (1994) An efficient method for purification of cuprozinc superoxide dismutase from bovine erythrocytes. Experientia, 50, 673-676. (11) McCord, J. M., and Fridovich, I. (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein).J. Biol. Chem. 244,6049-6055. (12) Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685. (13) Suzuki, T., Narimatsu, S., Fujita, S., Masubuchi, Y., Umeda, S., Imaoka, S., and Funae, Y. (1992)Purification and characterization of a cytochrome P-450 isozyme catalyzing bunitrolol4-hydroxylation in liver microsomes of male rats. Drug Metab. Dispos. 20, 367-373. (14) Marklund, S. L., Westman, N. G., Lundgren, E., and Ross, G. (1982) Copper- and zinc-containing superoxide dismutase, manganase-containing superoxide dismutase, catalase, and glutathione peroxidase in normal and neoplasmic human cell lines and normal human tissues. Cancer Res. 42, 1955-1961. (15) McCord, J. M., and Fridovich, I. (1968) The reduction of cytochrome c by milk xanthine oxidase. J . Biol. Chem. 243, 57535760. (16) Nishikimi, M. (1975) Oxidation of ascorbic acid with superoxide anion generated by xanthine-xanthine oxidase system. Biochem. Biophys. Res. Commun. 63,463-468. (17) Reiss, U., and Gershon, D. (1976) Rat-liver superoxide dismutase: Purification and age-related modification. Eur. J . Biochem. 63. 617-623.
Narimatsu et al. (18) Asayama, K., and Burr, I. M. (1985) Rat superoxide dismutase: Purification, labeling, immunoassay, and tissue concentration. J . Biol. Chem. 260, 2212-2217. (19) Vu, V. T., and Abramson, F. P. (1980)The pathways of propranolol
metabolism in dog and rat liver lOOOOg supernatant fractions. Drug Metab. Dispos. 8, 300-304. (20) Doherty, M. A., and Cohen, G. M. (1984) Metabolic activation of 1-naphthol by rat liver microsomes to 1,4-naphthoquinone and covalent binding species. Biochem. Pharmacol. 33, 3201-3208. (21) Doherty, M. A., Makowski, R., Gibson, G. G., and Cohen, G. M. (1985) Cytochrome P-450 dependent metabolic activation of 1-naphthol to naphthoquinones and covalent binding species. Biochem. Pharmacol. 34, 2261-2267. (22) Walle, T., and Gaffney, T. E. (1972) Propranolol metabolism in man and dog: Mass spectrometric identification of six new metabolites. J . Pharmacol. Exp. Ther. 182, 83-92. (23) Fridovich, I. (1983) Superoxide radical: an endogenous toxicant. Annu. Rev. Pharmacol. Toxicol. 23, 239-257. (24) Harber, F., and Weiss, J. (1934) The catalytic decomposition of hydrogen peroxide by iron salts. Proc. R. SOC.London Ser. A147, 332-351. (25) Strobel, H. W., and Coon, M. J. (1971) Effects of superoxide generation and dismutation on hydroxylation reactions catalyzed by liver microsomal cytochrome P-450. J. Biol. Chem. 246,78277829. (26) Lai, C. S., T. A. Grover, T. A,, and Piette, L. H. (1979) Hydroxyl radical production in a purified NADPH-cytochrome c (P-450) reductase system. Arch. Biochem. Biophys. 193, 373-378. (27) Winston, G. W., and Cederbaum, A. I. (1983) NADPH-dependent production of oxy radicals by purified components of the rat liver mixed function oxidase system. J . Biol. Chem. 258, 1508-1513. (28) Rasgba-Step, J., and Cederbaum, A. I. (1993) Generation of reactive oxygen intermediates by human liver microsomes in the presence of NADPH and NADH. Mol. Pharmacol. 45, 150-157. (29) Valentine, J. S. (1979)The chemical reactivity of superoxide anion in aprotic versus protic media: a review, In Biochemical and Clinical Aspects of Oxygen, pp 659-677, Academic Press, New York. (30) Sawyer, D. T., and Valentine, J. S. (1981) How super is superoxide? ACC.Chem. Res. 14, 393-400. (31) Hiramatsu, M., Kumagai, Y., Unger, S. E., and Cho, A. K. (1990) Metabolism of methylenedioxymethamphetamine (MDMA): Formation of dihydroxymethamphetamine and a quinone identified as its glutathione adduct. J . Pharmacol. Exp. Ther. 254, 521527. (32) Kumagai, Y., Wickham, K. A., Schmitz, D. A,, and Cho, A. K. (1991)Metabolism of methylenedioxyphenyl compounds by rabbit liver preparations: Participation of different cytochrome P450 isozymes in the demethylenation reaction. Biochem. Pharmacol. 42, 1061-1067. (33) Kumagai, Y., Lin, L. Y., Hiratsuka, A., Narimatsu, S., Suzuki, T., Yamada, H., Oguri, K., Yoshimura, H., and Cho, A. K. (1994) Participation of cytochrome P450-2B and -2D isozymes in the demethylenation of methylenedioxymethamphetamine enantiomers by rats. Mol. Pharmacol. 45, 359-365.
TX940179P
