682
Chem. Res. Toxicol. 1996, 9, 682-688
Articles Microsomal Catalyzed N-Hydroxylation of Guanabenz and Reduction of the N-Hydroxylated Metabolite: Characterization of the Two Reactions and Genotoxic Potential of Guanoxabenz1 Bernd Clement,* Mark Demesmaeker, and Sabine Linne Pharmazeutisches Institut, Christian-Albrechts-Universita¨ t Kiel, Gutenbergstrasse 76, D-24118 Kiel, Germany Received December 8, 1995X
The N-reduction of the centrally acting R2-adrenoreceptor agonist guanoxabenz (Benze´rial), an N-hydroxyamidinohydrazone, to the amidinohydrazone guanabenz (Wytensin, Hipten, Rexitene) by microsomal fractions from rabbits, pigs, and humans has been detected in vitro. The conversion rates with rabbit microsomal fractions were markedly slower than those in the cases of fractions from humans and pigs. It was also possible to demonstrate the N-oxidation of guanabenz to guanoxabenz by the use of microsomal fractions from rabbits, pigs, and humans. Furthermore, the oxidation was also observed in reconstituted systems in the presence of enriched cytochrome P450 fractions, purified isoenzyme P450 2C3, and heterologously expressed P450 2C3 of the subforms 6βH and 6βL. The analyses were performed with a newly developed HPLC technique and were confirmed by LC-MS methods. The kinetic parameters determined for the metabolic cycle (bioreversible reactions) are indicative of a predominance of the reduction of guanoxabenz to guanabenz in vivo. Accordingly, guanoxabenz in part constitutes a prodrug of guanabenz. Examinations of guanabenz and guanoxabenz for mutagenicity by means of the Ames test revealed that guanoxabenz has pronounced mutagenic effects in the strains TA 98 and TA 1537. Guanabenz did not exhibit mutagenicity so that the N-reduction of guanoxabenz has significance in terms of detoxification.
Introduction Guanabenz 1 (1-(2,6-dichlorobenzylideneamino)guanidine; Wytensin, Rexitene, Hipten) (1, 2) and guanoxabenz 2 (1-(2,6-dichlorobenzylideneamino)-3-hydroxyguanidine; Benze´rial) (3) are well-known as centrally acting R2adrenoreceptor agonists with antihypertensive activities (Figure 1). Both compounds belong to the class of amidinohydrazones (aminoguanidines) where guanoxabenz is the N-hydroxylated derivative of guanabenz. Neither the N-hydroxylation of guanabenz nor the Ndehydroxylation of guanoxabenz have been observed in previous biotransformation studies (4, 5) although comparable reactions have already been described in detail for the systems amidines/amidoximes (6) and guanidines/ N-hydroxyguanidines (7). Also, the N-hydroxylation or N-reduction of one of the two equivalent terminal nitrogen atoms of the amidinohydrazones G256 or NOH-G256, respectively, have previously been described (8). For example, as shown for debrisoquine (9), the N-hydroxylation of strongly basic nitrogen-containing functional groups can easily be missed on account of rapid reduction of the N-hydroxylated metabolite. Mutagenicity in the Ames test has been reported for the N-hydroxyamidine benzamidoxime while benzamidine did not reveal any * To whom correspondence should be addressed; phone: +431/8801126; FAX: +431/880-1352. X Abstract published in Advance ACS Abstracts, April 15, 1996. 1 This paper is dedicated to Professor Dr. Dr. E. Mutschler on the occasion of his 65th birthday.
S0893-228x(95)00204-9 CCC: $12.00
Figure 1. N-Hydroxylation of guanabenz 1 and N-reduction of guanoxabenz 2.
such effects under the same conditions (10). The Nreduction of benzamidoxime to benzamidine can thus be viewed as a detoxifying process. These observations prompted us to investigate the mutual metabolic transformations (Figure 1) and mutagenic properties of guanabenz and guanoxabenz.
Materials and Methods Materials. Caution: The positive controls used in the Ames test are known to be highly potent carcinogens. Guanabenz acetate was kindly supplied by Wyeth-Pharma GmbH (Muenster, Germany) and guanoxabenz-HCl by Laboratoires Houde´ (Paris, France). N-Methylhydroxylammonium chloride, NADPH (tetrasodium salt) and NADH (disodium salt) as well as all other chemicals and solvents were obtained from E. Merck (Darmstadt, Germany). All chemicals were of analytical grade. Salmonella typhimurium tester strains TA98, TA100, and TA1537 were kindly supplied by Prof. Dr. H. Marquardt (Department of Toxicology, University of Hamburg Medical
© 1996 American Chemical Society
Metabolism of Guanabenz and Guanoxabenz School, 20146 Hamburg, Germany). S92 samples from rats pretreated with Aroclor 1254 were obtained from Organon Teknika (Eppelheim, Germany); purified agar Code L28 and nutrient broth no. 2 were from Oxoid (Basingstoke, England); 2-nitrofluorene was purchased from Aldrich (Steinheim, Germany) and 2-aminoanthracene from Sigma (Deisenhofen, Germany); glucose-6-phosphate (disodium salt) was purchased from Boehringer Mannheim (Mannheim, Germany); NADP (sodium salt) was obtained from Biomol (Hamburg, Germany) and glucose for microbiology as well as NaCl for molecular biology, dimethyl sulfoxide (spectrophotometric grade), L-histidine-HCl, sodium azide and sodium dihydrogenphosphate monohydrate for molecular biology were from E. Merck. All other chemicals were obtained as BioChemika microselects from Fluka (Buchs, Switzerland). Preparation of Subcellular Fractions. Cytosol (100000g Supernatant). The livers of untreated rabbits (3.0-4.5 kg) and pigs (slaughterhouse) of either sex were used. All subsequent operations were performed at 0-4 °C. The livers were washed three times with 1.15% (w/v) KCl solution (0.154 M), blotted dry, weighed, and minced with knives. The minced livers were passed through a meat mincer and homogenized using a motorized Teflon pestle glass tube homogenizer (Potter S, for 30 mL, Braun Melsungen AG, Germany) in 20 mM potassium phosphate buffer (pH 7.4) containing 0.25 M sucrose. The homogenates were transferred to plastic tubes and centrifuged at 9000g for 30 min. The supernatant was carefully decanted and centrifuged at 100000g for 60 min. The resulting supernatant was stored at -80 °C. Microsome Preparations (Rabbit/Pig). The residue of the 100000g centrifugation was resuspended in phosphate buffer and again centrifuged at 100000g for 60 min. The supernatant was discarded and the microsome pellet was resuspended in buffer. After adjustment to pH 7.4 by addition of potassium hydroxide the preparation was stored at -80 °C for several months. Human Liver Microsomes. Pooled human liver microsomes (Pooled HepatoSomes) were purchased from Human Biologics, Inc. (Phoenix, AZ). Purification of the Components of the Cytochrome P450 Enzyme System. Partial Purification of Cytochrome P450 and Isolation of NADPH Cytochrome P450 Reductase (P450 Reductase2). In order to obtain an enriched cytochrome P450 preparation containing as little detergent as possible while still retaining, in catalytically active form, all of the constitutive cytochrome P450 enzymes, a modification of the method established by Kling et al. (11) was carried out as reported previously (6). P450 reductase from rabbit livers was purified to homogeneity as described by Yasukochi and Masters (12) with slight modifications (6). Purification of Cytochrome P450 2C3. The separation of a P450 preparation into enzymes and the isolation of P450 2C3 from rabbit livers have already been described (6). Recombinantly Expressed Variants of Cytochrome P450 2C3. The two variants P450 2C3 (6βH) and P450 2C3 (6βL) expressed by recombinant Escherichia coli were kindly provided by Dr. E. F. Johnson, Scripps Clinic and Research Foundation (La Jolla, CA). Analytical Procedures. The cytochrome P450 content was analyzed using the method of Omura and Sato (13). The protein content was determined by the method of Gornall et al. (14) (reagent kit, E. Merck, Darmstadt, Germany). Bovine serum albumin was used as the standard. All photometric measurements were performed with a Uvikon 930 (Kontron Instruments, Neufahrn, Germany), spectrophotometer. Incubations. Microsomal N-Reduction of Guanoxabenz. Incubations were carried out in a shaking water bath at 37 °C in the presence of oxygen using 1.5 mL reaction vessels. The standard incubation mixture (0.3 mL) contained the following components: 50 mM tris(hydroxymethyl)aminomethane2 Abbreviations: S9, supernatant 9000g; P450 reductase, NADPHcytochrome P450 reductase.
Chem. Res. Toxicol., Vol. 9, No. 4, 1996 683 HCl buffer (pH 6.3), adjusted with dilute ammonia solution at 37 °C, 0.5 mM guanoxabenz, 0.5 mM NADH, and 0.27 mg (rabbit/pig) or 0.33 mg (human) protein of the enzyme source/ 0.3 mL. After preincubation for 1 min at 37 °C, the reactions were started by addition of NADH. Incubation time was 30 min. The reactions were terminated by addition of 0.3 mL of acetonitrile and cooling the samples in an ice/salt mixture. After centrifugation at 20000g and 4 °C (Megafuge 1.0 R, Heraeus, Osterode, Germany), 20 µL aliquots of the supernatant were directly analyzed by HPLC. Microsomal N-Hydroxylation of Guanabenz. Incubations were performed as described above but adding 0.5 mM of guanabenz and 0.5 mM of NADPH instead of guanoxabenz and NADH. Tris buffer was adjusted to pH 7.4. N-Hydroxylation of Guanabenz in a Reconstituted P450 Monooxygenase System. The incubations were performed in 1.5 mL reaction vessels in a shaking water bath at 37 °C in the presence of oxygen. The standard incubation mixture (0.3 mL) consisted of 50 mM Tris buffer (pH 7.4 at 37 °C), 0.5 mM guanabenz, 0.5 mM NADPH, 40 µM L-R-dilaurylphosphatidylcholine (DLPC), 0.3 U of NADPH-P450 reductase, and 0.1 nmol of the protein source (P450 2C3, P450 2C3 6βH/L, or enriched P450). The incubations were carried out as described above for microsomal fractions. HPLC. Guanabenz. The resulting clear supernatant was analyzed using a high performance liquid chromatograph (Waters 510, Milford, CT) equipped with a variable wavelength UV detector (Waters 486) set at 272 nm, and an autosampler (Waters 710 WISP). The areas under the peaks were integrated with a chromatointegrator (Waters 746). Separation and quantification were performed at room temperature on a prepacked, reversed phase column (125 × 4 mm i.d., particle size 5 µm; Lichrospher RP-select B, E. Merck). The mobile phase was methanol/ammonium acetate buffer (50 mM), pH 4.0 (30:70 v/v), and was passed through the column at a rate of 1.0 mL/min. The injected sample volume was 20 µL. Solvents used in the analysis were filtered through a Sartolon membrane filter (0.45 µm, Sartorius AG, Goettingen, Germany) and degassed by bubbling with helium or sonication. For the determination of the recovery rate and the detection limit of the metabolite guanabenz, incubation mixtures with defined concentrations of synthetic reference substance (1.0, 5.0, 10.0, 20.0, 40.0, 60.0, 80.0, or 100.0 µM) were incubated and worked up under the same conditions as the experimental samples but without adding cofactor. The standard curves were linear over this range with correlation coefficients >0.9999. The signals obtained (peak areas) were compared with those of the same amount of guanabenz dissolved in the mobile phase. The recovery rate after incubation and sample workup amounted to 102.8 ( 3.9% (N ) 32). The detection limit was about 0.5 µM which corresponds to a rate of N-reduction of 0.038 nmol of guanabenz/(min‚mg of protein). The retention times were 22.5 ( 0.5 min for guanoxabenz and 33.0 ( 0.5 min for guanabenz. Guanoxabenz. Separation and quantification were performed as described above for guanabenz. The mobile phase was methanol/ammonium acetate buffer (0.05 M), pH 4.0 (25: 75 v/v). The areas under the peaks were integrated using a chromatointegrator (Merck Hitachi D-2500), and the variable wavelength UV detector was set at 274 nm. Standard curves at the levels of 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 5.0, 7.0, and 10.0 µM guanoxabenz were constructed and found to be linear over this range with correlation coefficients >0.9992. The recovery rate of guanoxabenz from incubated mixtures was 96.7 ( 6.1% (N ) 40) of that obtained using samples which contained the same amount of guanoxabenz dissolved in the mobile phase. The detection limit was represented by a 0.25 µM solution which corresponds to a rate of N-hydroxylation of 0.019 nmol of guanoxabenz/(min‚mg of protein). The retention times were 16.5 ( 0.5 min for guanoxabenz and 23.5 ( 0.5 min for guanabenz. Preparative Enrichment of the Metabolite Guanoxabenz for LC-MS and UV Spectroscopy. The incubations with microsomal fractions were carried out in 25 mL Erlenmeyer
684 Chem. Res. Toxicol., Vol. 9, No. 4, 1996
Clement et al.
Table 1. In Vitro N-Reduction of Guanoxabenz to Guanabenz by Liver Microsomes of Different Species and the Influence of the Cofactora incubation mixture/species
nmol of guanabenz/ (min‚mg of protein)
incubation mixture/species
nmol of guanabenz/ (min‚mg of protein)
complete (NADH)/rabbit complete (NADH)/pig complete (NADH; pH 7.4)/man
0.93 ( 0.08 3.15 ( 0.29 3.36 ( 0.32
-NADH/rabbit complete (NADPH)/rabbit complete (NADPH; pH 7.4)/rabbit
ND# 0.21 ( 0.01* 0.08 ( 0.01*
a The conversion rates are means of four different determinations with one enzyme preparation (10 rabbit livers, 3 pig livers, or 2 human livers were combined) ( SD. The incubations were performed as described in Materials and Methods. *Values statistically different from control (complete/NADH/rabbit pH 6.3) with p
