Introduction

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-Agonists are widely used for the treatment of respiratory diseases. Some of these compounds can also be administered to farm animals to improve carcass characteristics and productivity rates (Saltron et al., 1996; Fiems, 1987). CL¹ [4-amino-3,5-dichloro--(tert-butylaminomethyl)benzyl alcohol] is known to be the most common -agonist used in cattle for such purposes. Nevertheless, this molecule is licensed only as a veterinary bronchospasmolytic and tocolytic agent, and its use as an anabolic agent is illegal in the European Community and in North America. When CL is administered to cattle, relatively high levels of CL can be detected in the liver (Meyer and Rinke, 1991). Consequently, several cases of human intoxication have been related to the consumption of liver from animals treated with this drug (Martinez-Navarro, 1990; Pulce et al., 1991).

We recently published preliminary results concerning CL metabolism in rats and cattle (Zalko et al., 1996) and an extensive study of a previously unknown metabolic pathway for CL in rats, namely N-oxidation of the primary amine (Zalko et al., 1997). A major urinary metabolite of CL in rats was characterized as N-OH-CL (fig. 1). This compound was also observed in urine from cattle treated with CL (Zalko et al., 1996), as well as in in vitro assays with rat liver microsomes (Zalko et al., 1997). In in vivo experiments, two other metabolites were identified as NO₂-CL and NO-CL (fig. 1). Only NO₂-CL was found in rat urine, at low concentrations, and it was concluded that NO-CL resulted from the chemical autooxidation of N-OH-CL during metabolite extraction (Zalko et al., 1997).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Structures of CL and N-oxidized metabolites of CL. Arrows, atoms bearing the 14C or 3H labeling of the radioactive CL molecules used in the study.

Several aliphatic (Testa, 1995; Beckett and Bélanger, 1975) and aromatic (Testa, 1995; Smith and Gorrod, 1978; Gorrod and Manson, 1986) amines, including primary arylamines (Orton and Lowery, 1981; Uetrecht et al., 1984), have been shown to be metabolized to their corresponding hydroxylamines by liver microsomal fractions. One main analytical problem encountered when dealing with N-hydroxylated metabolites of primary arylamines is their chemical instability. In addition, the potential toxicities of these compounds are closely related to their structures, particularly to their oxidative status. Therefore, the exact structures of such metabolites need to be established.

The aim of the present study was to compare, qualitatively and quantitatively, CL metabolism in vitro in rats and in the target species, i.e. cattle. The cytochrome P450-dependent oxidation pathway was investigated using liver microsomes. In a second step, CL was incubated with precision-cut liver slices, because it is now well established that this in vitro system constitutes an excellent model for comparative metabolism and toxicity studies (Parrish et al., 1995). Special care was given to the analytical procedure, to avoid chemical oxido-reduction reactions.

	Materials and Methods

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Chemicals. CL. CL hydrochloride was obtained from Sigma (Saint Quentin Fallavier, France). [benzyl-³H]CL and [¹⁴C]CL (labeled at the benzylic carbon) were purchased from Rotem Industries (Beer-Sheva, Israel) and Isotopchim (Ganagobie-Peyruis, France), respectively. The labeled molecules had specific activities of 474 GBq/mmol ([³H]CL) and 1997 MBq/mmol ([¹⁴C]CL). Their respective radiopurities were >97%. Nevertheless, both were purified using HPLC (see below) with a fraction collector and then RP Select B C₈ cartridges (Polylabo, Strasbourg, France), as described elsewhere (Zalko et al., 1997), to reach 99.4% ([³H]CL) or 99.6% ([¹⁴C]CL) purity. The CL structure was confirmed by ESI-MS. CL metabolites used as standards (N-OH-CL, NO-CL, NO₂-CL, CL sulfamate, 4-amino-3,5-dichlorohippuric acid, and the analogue of CL hydroxylated at its tert-butyl moiety) were purified from rat urine as described elsewhere (Zalko et al., 1996, 1997). ADBA was purchased from Pfaltz & Bauer Inc. (Waterbury, CT).

Other Chemicals. 7-[3-¹⁴C]EC was purchased from Amersham (Buckinghamshire, UK). Formic acid and all solvents (of analytical grade) were obtained from Prolabo (Paris, France). Acetic acid was purchased from Merck (Darmstadt, Germany). 7-EC, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP, MgCl₂, phosphate buffer, and ascorbic acid were obtained from Sigma.

Animals. Six male Wistar rats, weighing about 250 g, were purchased from Iffa Credo (L'arbresle, France). Rats were fed a standard diet (UAR 210; Usine d'Aliments Rationalisés Villemoisson-sur Orge, France) and had free access to water during a 7-day acclimatization period. They were killed by cervical dislocation, followed by immediate exsanguination. Three untreated male Charolais calves (age, 8 months) were obtained from a local breeder. Calves were fed hay ad libitum, supplemented with an adapted commercial cattle concentrate, and had free access to water. After a 1-week acclimatization period, calves were killed by captive bolt and exsanguination.

Preparation of Liver Slices. Three rat livers were perfused with 40 ml of ice-cold (4°C) oxygenated Krebs-Henseleit buffer to remove blood. Liver cores were obtained using a stainless steel tube and a drill press. Cores were immediately placed in ice-cold buffer. A Krumdieck tissue slicer (Alabama Research and Development Corp., Munford, AL) was used to produce precision-cut slices of 8-mm diameter and 0.2-mm thickness (Krumdieck et al., 1980). An equilibration period of 45 min allowed for the sloughing of damaged cells and for recovery of Ca²⁺ and K⁺ homeostasis in the slices. The mean wet weight of slices was 15 mg, containing about 1.5 mg of protein. Bovine livers from the three Charolais calves were removed within 5 min after slaughtering. Small cuts from the lobus quadratus were taken and placed immediately in oxygenated Krebs-Henseleit buffer. Bovine liver slices were prepared as described for rats.

Slice Viability. 7-EC was used as a probe substrate for oxidative and conjugative metabolism, to assess the drug-metabolizing capacity of liver slices isolated from the two species. For each animal, control incubations of liver slices were performed with 25 µM labeled 7-EC for 1, 2, or 4 hr. Biotransformation of 7-EC to 7-OHC, 7-OHC sulfate, and 7-OHC glucuronide was studied by radio-HPLC analysis of filtered incubation media. Structures of the conjugated metabolites of 7-EC were determined after enzymatic hydrolysis (Steensma et al., 1994). Additionally, the integrity of cells in slices was determined by measuring LDH leakage in an aliquot of the culture medium, using a LDH detection kit (procedure 228-UV; Sigma).

Preparation of Liver Microsomes. Three rat livers were removed immediately after slaughtering and perfused with 0.9% saline. Liver microsomes were prepared as described elsewhere (Perdu-Durand and Tulliez, 1985) and stored at 80°C in phosphate buffer (0.1 M, pH 7.4) containing 20% glycerol. The protein concentration was determined by the method of Lowry et al. (1951). Bovine liver samples were obtained from the three Charolais calves described above, and liver microsomes were prepared as described for rats.

Metabolism of CL in Liver Slices. Slices were incubated in 12-well, plastic, tissue culture plates (Dogterom, 1993) using Dulbecco's modified Eagle's medium. The incubation volume was 1 ml. For both rat and bovine liver slices, assays were performed for each animal with 12.5, 25, 50, or 100 µM labeled CL (10,000 Bq/incubation) for 1, 2, or 4 hr. Plates were maintained at 37°C in a rotary shaker, set at 90 rpm. At the end of each incubation period, media were removed and ascorbic acid (1 mM) was added. Two 50-µl samples were taken for radioactivity determination, before immediate storage at 80°C until analysis. Slices were gently washed twice with water to allow the measurement of residual radioactivity.

Metabolism of CL with Liver Microsomes. Incubations were performed in vials containing 2 mg of microsomal protein, 1 mM NADP, 10 mM glucose-6-phosphate, 5 mM MgCl₂, 2 units of glucose-6-phosphate dehydrogenase, and labeled CL, in a final volume of 1 ml of phosphate buffer (0.1 M, pH 7.4). For both species, assays were performed with 12.5, 25, 50, and 100 µM CL, including 10,000 Bq of [¹⁴C]CL. For each CL concentration, vials were incubated for 1 hr at 37°C, and the reaction was stopped by addition of methanol (0.5 ml). Ascorbic acid (1 mM) was added to the incubations, which were immediately centrifuged at 10,000 rpm and 4°C for 10 min. Two 50-µl samples were taken for the determination of radioactivity in the supernatants, which were then stored at 80°C until analysis.

Sample Preparation and Analysis. All samples were individually thawed immediately before analysis. Aliquots (150 µl) of the supernatants from each microsomal incubation were quickly concentrated under a nitrogen stream and filtered on a 0.5-g, 0.45-µm, Ultrafree MC filtration unit (Polylabo) before radiochromatographic analysis. Aliquots (100 µl) of each liver slice incubation medium were filtered and analyzed by following the same procedure. All samples were analyzed consecutively within each incubation series (i.e. each animal). To confirm the reproducibility of the chromatograms and the stability of metabolites generated in vitro, several samples were injected twice, with the second analysis taking place at the end of the series. No significant qualitative or quantitative differences were found.

Analytical Procedures. CL Metabolism Studies. Liver microsomal fractions and slice incubation media were analyzed by HPLC with a Philips 4100 apparatus (Pye Unicam, Cambridge, UK) equipped with a Rheodyne model 7125 injector (Rheodyne, Cotati, CA) and connected, for radioactivity detection, to a Packard Flo-one/ A500 detector (Packard Instruments Co., Meriden, CT) (scintillation cocktail, Flow-scint II; Packard Instruments Co., Downers Grove, IL). For CL purification and ADBA characterization, the Philips 4100 apparatus was connected to an absorbance detector set at 254 nm (PU 4021 multichannel detector; Pye Unicam) and to a Gilson model 202 fraction collector (Gilson France, Villiers-Le-Bel, France). The HPLC system consisted of an Ultrabase C₁₈ column (250 × 4.6 mm, 5 µm; SFCC, Eragny, France) coupled to a Hypersil BDS C₁₈ guard precolumn (18 × 4.6 mm, 5 µm; Shandon/LSI, Cergy Pontoise, France). The mobile phases consisted of ammonium acetate buffer (10 mM) adjusted to pH 3.2 and acetonitrile, at 95:5 (v/v) in solvent A and 30:70 (v/v) in solvent B, respectively. The flow rate was 1 ml/min at 35°C. A three-step gradient was used, as follows: 0-4 min, 100% A; 4-10 min, linear gradient from 100% A to 95:5 (v/v) A/B; 10-30 min, 95:5 (v/v) A/B; 30-35 min, linear gradient from 5% B to 40% B; 35-45 min, 60:40 (v/v) A/B; 45-47 min, linear gradient leading to 100% B; 47-54 min, 100% B.

Incubation of Slices with 7-EC. Incubation media were studied by radio-HPLC, using the same apparatus as for CL and a C₁₈ Spherisorb ODS2 (250 × 4.6 mm, 5 µm) column (Interchim, Montluçon, France) coupled to a Hypersil BDS C₁₈ guard precolumn. Mobile phases consisted of ammonium acetate buffer (50 mM, pH 5.0) and acetonitrile, 85:15 (v/v) in solvent A and 20:80 (v/v) in solvent B. The flow rate was 1 ml/min. A two-step gradient was developed, as follows: 0-5 min, 100% A; 5-25 min, linear gradient from 100% A to 50:50 (v/v) A/B; 25-35 min, linear gradient leading to 100% B.

Metabolite Identification. Metabolite M1. The structure of the major metabolite generated by CL incubation with liver microsomal fractions and liver slices was confirmed using HPLC coupled to ESI-MS. Before analysis, samples were filtered on 0.45-µm Ultrafree MC (Polylabo) and 0.22-µm Millex-GS (Millipore, France) filtration units, successively. LC/ESI-MS analyses were performed in the positive-ion mode on a Finnigan LCQ quadrupole ion-trap mass spectrometer (Finnigan MAT, San Jose, CA). Data were acquired in the selected-ion monitoring mode. In-source collision conditions were used to induce some fragmentation, as described elsewhere (Debrauwer et al., 1997). The ions corresponding to the ³⁵Cl and ³⁷Cl isotopic contributions of the protonated molecular ion and to the most intense fragment ions obtained under the in-source, collisionally activated dissociation conditions used were monitored for several compounds. These corresponded to the m/z 277, 279, 259, and 203 ions for CL, the m/z 293, 295, 275, and 219 ions for N-OH-CL, and the m/z 291, 293, 217, and 235 ions for NO-CL. The structures of these various ions have been discussed elsewhere (Debrauwer et al.,1997).

Metabolite M4. Twelve incubations of CL (25 and 50 mM) were performed with bovine liver microsomes under the conditions described above but with 4000 Bq of [¹⁴C]CL/vial. Twelve additional incubations (one for each CL concentration and for each animal) were performed using [³H]CL (20,000 Bq/vial). [³H]CL incubation media were analyzed by radio-HPLC.

Statistical Analysis. Analysis of variance was performed on the residual radioactivity levels found in rat and bovine microsomal protein pellets, using the general linear-models procedure of the SAS.

	Results

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Metabolism of CL by Liver Microsomal Fractions and Structural Identification. Radiochromatographic Profiles and M1 Characterization. The metabolism of CL by bovine and rat liver microsomal fractions was studied with four concentrations of CL (12.5, 25, 50, and 100 µM) after 60 min of incubation. In rats, radiochromatographic analysis of the supernatants (fig. 2a) showed only one metabolite (M1), whatever the concentration of the drug. This metabolite was demonstrated to coelute with N-OH-CL, which had been previously purified from rat urine and characterized (Zalko et al.,1997).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   14C-radio-HPLC profiles obtained from incubation media of CL incubations with rat liver microsomes (25 µM CL) (a), bovine liver microsomes (12.5 µM CL) (b), rat liver slices (12.5 µM CL; incubation time, 4 hr) (c), and bovine liver slices (12.5 µM CL; incubation time, 4 hr) (d).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Ion chromatograms obtained from LC/ESI-MS analysis of rat microsomal incubations with CL (100 µM). a, Total ion chromatogram; b, selected-ion monitoring traces. The retention times of N-OH-CL and CL, using this chromatographic system, were 16.4 and 21.6 min, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Michaelis-Menten plot of N-OH-CL production at varying concentrations of CL, in rat microsomal incubations (1 hr, 37°C, 2 mg of microsomal protein). Data are mean ± SD obtained from three animals.

M4 Identification. M4 was first hypothesized to be the 4-nitroso analogue of CL, based on chromatographic characteristics similar to those of the rat urinary metabolite NO-CL. Thus, the products of incubations of 100 µM CL with bovine microsomes were analyzed by LC/ESI-MS, as described for rat incubations except that four additional ions were monitored, to examine the occurrence of NO-CL (table 1). The LC/ESI-MS analysis produced the same results as in rats, i.e. it confirmed the presence of N-OH-CL and CL but no NO-CL was detected. On the other hand, when analyzed by HPLC coupled to radioactivity and absorbance detection, the chemical standard ADBA was found to exhibit a retention time identical to that of both the standard NO-CL and M4. ADBA was also demonstrated to coelute with NO-CL by using the same HPLC apparatus under isocratic conditions (65:35, v/v, A/B). The correspondence between M4 and ADBA in 12.5 µM incubation media was tested using strong anion exchanger cartridges. M4 (as well as ADBA in a separate experiment) was fully retained on such cartridges, whereas M1 and CL were not (data not shown). Thus, the acidic nature of M4 was confirmed. Additional evidence resulted from analysis of the media from incubations of [³H]CL with bovine liver microsomes. Indeed, no peak at the retention time of M4 could be detected in the corresponding radiochromatograms, thus demonstrating that, when [¹⁴C]CL was used, the radioactivity detected for M4 was not associated with NO-CL. Consequently, M4 was hypothesized to be a metabolite in which the hydrogen atom on the benzylic carbon had been lost, very likely after an oxidative cleavage leading to the loss of the side chain of CL.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   ESI-MS spectrum (a) and ESI-MS-MS product-ion spectrum of [MH] at m/z 204 (b) obtained from the negative-ion ESI-MS analysis of M4.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Michaelis-Menten curves for N-OH-CL and ADBA formation in bovine liver microsomal incubations with various concentrations of CL (1 hr, 37°C, 2 mg of microsomal protein). Data are mean ± SD obtained from three animals.

Residual Radioactivity in Microsomal Protein Pellets. Radioactivity measured in washed microsomal pellets ranged from 1.0 to 1.3% of the radioactive dose in incubations of CL with rat liver microsomes and from 1.5 to 2.3% with bovine liver microsomes. Analysis of variance for the results showed that, although relatively low, these values were significantly higher in bovine incubation pellets (p < 0.01).

Incubation of CL with Liver Slices. Radiochromatographic Studies of Incubation Media. The formation of CL metabolites in rat and bovine liver slice incubation media was investigated, in 1-, 2-, or 4-hr assays, with different concentrations of CL. With rat liver slices, the radiochromatographic profile (fig. 2c) showed only one major metabolite (M1), possessing the same retention time as N-OH-CL. In this species, the production of N-OH-CL was shown to be linear over 2 hr (fig. 7). A Michaelis-Menten plot of N-OH-CL formation with various concentrations of CL (fig. 8) demonstrated saturation kinetics. Some minor metabolites were also occasionally observed but accounted for <2% of the total radioactivity detected in chromatographic profiles. Three of these metabolites have been identified, on the basis of similar retention times, as CL metabolites previously isolated from rat urine (Zalko et al., 1996, 1997), namely CL sulfamate (M7), 4-amino-3,5-dichlorohippuric acid (M8), and NO₂-CL (M5). The latter was formed only in incubations performed with the lowest CL concentrations (12.5 and 25 µM).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Amounts of N-OH-CL detected in media of rat liver slice incubations (1, 2, or 4 hr of incubation) with CL. Data are mean ± SD obtained from three animals.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Michaelis-Menten curves for N-OH-CL formation in rat liver slice media at various concentrations of CL (12.5-100 µM), after 1, 2, or 4 hr of incubation. Data are mean ± SD obtained from three animals.

Residual Radioactivity in Slices. Between 4 and 12% of the initial radioactivity remained in the slices. The major part of this radioactivity was found to be extractable. Radio-HPLC analysis of extracts produced profiles similar to those obtained from supernatants. Only about 1% of the initial radioactivity remained in the extracted pellets, whatever the species or the incubation time.

	Discussion

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This work clearly demonstrated that CL was mainly metabolized by liver microsomes to the corresponding arylhydroxylamine. When rat liver microsomes were incubated with 12.5-100 µM CL, >50% of the drug underwent hydroxylation to N-OH-CL in 1 hr. In cattle, in vitro assays were performed only with livers obtained from fully ruminating animals, because several drug-metabolizing activities have been demonstrated to be weaker in veal calves than in older animals with functioning rumens (Kawalek and El Said, 1994). In this species, the hydroxylamine was also the major metabolite formed during incubation of CL with liver microsomes, but to a lesser extent than in rats, and the existence of a second important metabolic route was evident after the identification of ADBA.

LC/ESI-MS was used in the selected-ion monitoring mode for the confirmation of N-OH-CL and CL structures in microsomes as well as slice incubation media. Indeed, ESI-MS is an efficient tool for the structural characterization of -agonists (Debrauwer and Bories, 1992), and the behavior of CL and its N-oxidation products with ESI and in-source collisionally activated dissociation has been extensively described (Zalko et al., 1997; Debrauwer et al., 1997). A positive signal was recorded when the ions corresponding to N-OH-CL and CL, for all of the incubation media, were monitored by LC/ESI-MS. Conversely, when bovine microsomal incubation media were analyzed and four more ions corresponding to NO-CL were monitored, no signal was obtained for this compound. This, together with the analysis of duplicate incubations performed using [³H]CL, demonstrated that, despite the fact that the chromatographic characteristics of NO-CL were very close to those of ADBA, no nitroso analogue of CL was present in the media of CL incubations with bovine liver microsomes. The structure of ADBA was determined after direct isolation and subsequent negative-ion ESI-MS and ESI-MS-MS experiments achieved under direct sample infusion conditions.

In CL metabolism assays with liver slices, the ability of liver cells to produce metabolites resulting from oxidation as well as from conjugation reactions was confirmed by the use of 7-EC as a probe substrate. The qualitative and quantitative results obtained with incubations of 7-EC were in accord with those reported previously in rats (Ball et al., 1996; Ekins et al., 1995). 7-EC metabolism was nevertheless found to be weaker in bovine liver slices than in rat liver slices. This, together with the fact that low and comparable levels of LDH leakage were measured in the incubation media for the two species, indicated a greater metabolic capacity of rat liver slices. Incubations of CL with rat as well as bovine liver slices produced N-OH-CL, which was the only quantitatively important compound detected. Using this in vitro system, a much lower rate of metabolism of CL was observed in cattle, which is consistent with the results obtained for 7-EC.

Before this study, almost no information was available concerning CL metabolism in vitro. Some experiments were performed using bovine liver cells or subcellular fractions, but a very low rate of metabolism was found (Sauer et al., 1996), and only a glucuronide conjugate of CL could be tentatively identified. In contrast, for other primary arylamines N-hydroxylation was shown to be a common in vitro metabolic pathway. For instance, this was demonstrated for aniline and substituted aniline analogues (Smith and Gorrod, 1978) and 4-aminobiphenyl and 4,4-methylene-bis(2-chloroaniline) (Butler et al., 1989), as well as for the -blocking agents practolol (Orton and Lowery, 1981) and procainamide (Uetrecht et al., 1984). N-Oxidation of arylamines is an important metabolic step that in some cases can lead to adverse toxicological effects. The N-oxidation of primary arylamines is thought to be carried out by cytochrome P450 monooxygenases, rather than FAD-containing monooxygenases (Testa, 1995; Gorrod, 1978).

In the present work, the analytical procedure was designed to prevent the chemical oxidation of N-OH-CL to either NO-CL or NO₂-CL. Ascorbic acid was added to the supernatants at the end of the incubations, and the supernatants were immediately stored at 80°C. Moreover, the incubates were individually thawed just before HPLC analysis, and all samples were analyzed within 3 days after the assays. When samples were analyzed again after an additional storage delay, no significant modification of the radiochromatographic profiles was observed. Therefore, the oxidation of CL to NO₂-CL detected in bovine liver microsomes as well as rat slice incubation media is not related to the chemical oxidation of N-OH-CL during sample processing. In contrast, NO-CL was not detected in any of the incubation media. Duplicate assays carried out with a tritium-labeled molecule demonstrated that NO-CL was not formed when CL was incubated with bovine liver slices. This is consistent with results of previous in vivo experiments in rats (Zalko et al., 1997), which showed that NO₂-CL (but not NO-CL) was generated in small amounts and excreted in urine.

In incubations of CL with bovine microsomes, a metabolite possessing chromatographic characteristics similar to those of NO-CL was isolated and identified as ADBA. This compound was previously shown to be a urinary metabolite of CL in rats and dogs (Tanabe et al., 1984; Schmid et al., 1990). Moreover, other ₂-agonists of related structure, e.g. mabuterol (Horiba et al., 1984), have been shown to undergo a similar metabolic pathway.

Some minor metabolites were also identified in the present in vitro assays. 4-Amino-3,5-dichloro--(2-hydroxy-1,1-dimethyl)ethylaminomethylbenzyl alcohol was found in incubations of the drug with bovine liver microsomes. Two other compounds, i.e. CL sulfamate (in rats only) and 4-amino-3,5-dichlorohippuric acid, were characterized in incubations of CL with liver slices. Interestingly, although 4-amino-3,5-dichlorohippuric acid was observed in both species and results from the glycine conjugation of ADBA, the latter was not detected when CL was incubated with rat liver microsomes.

In work with precision-cut liver slices, no evidence was found for the formation of metabolites resulting from N-OH-CL conjugation. Although not detected, such biotransformations would not be observed in work with microsomes. The enzymatic conjugation of arylamine N-oxidized metabolites can be of major importance for their potential toxicity. Indeed, the formation of a nitrenium ion, considered to be an ultimate reactive species of these compounds, can be highly favored by a previous sulfate or acetate conjugation (Ford and Herman, 1991; Sabbioni and Wild, 1992).

Many ₂-agonists used illegally in meat production are arylamines (Saltron et al., 1996; Leyssens et al., 1993). This is also the case for some other drugs that have been used (Orton and Lowery, 1981; Uetrecht, 1984) or are in development for therapeutic use (Barrow et al., 1995) in humans. The present results indicate that in vitro studies could provide a useful tool for the understanding of N-oxide formation and further putative biotransformations. Moreover, given the structures of several ₂-agonists, these studies may allow simultaneous exploration of the two major metabolic pathways previously demonstrated for CL (Zalko et al., 1996, 1997; Tanabe et al., 1984; Schmid et al., 1990), which are possibly the same for closely related structures, i.e. N-oxidation of the primary amine and oxidative cleavage of the side chain of these molecules.