4398
J. Agric. Food Chem. 1998, 46, 4398−4405
Metabolism of Thiazopyr in Laying Hens William P. Ridley,*,† Hideji Fujiwara,† Theresa Cheng,‡ and Joy L. Honegger† Agricultural Sector, Monsanto Company, 700 Chesterfield Parkway North, St. Louis, Missouri 63198, and Covance, 3301 Kinsman Boulevard, Madison, Wisconsin 53704
Thiazopyr labeled with 13C/14C was found to be rapidly metabolized and eliminated by laying hens dosed orally for four consecutive days at either 1.3 or 10.4 mg/day. Over 90% of the total radioactive dose appeared in the excreta within 22 h after the final dose at both levels. Tissue residues were very low in the muscle and egg white (0.004-0.01 ppm) with somewhat higher levels observed for the liver, abdominal fat, skin with fat, egg yolk, and kidney (0.047-0.298 ppm). The most abundant tissue metabolite was a nitrile ester resulting from the breakdown of the thiazoline ring of thiazopyr. A carboxylic acid metabolite found in the liver and excreta was shown to arise from oxidation at C-3 of the isobutyl side chain of the nitrile ester. In vitro liver homogenate studies demonstrated the presence of similar metabolic pathways in hens, goats, and rats, although the levels of individual metabolites varied. Keywords: Thiazopyr; hens; metabolites; MS; NMR INTRODUCTION
Thiazopyr [methyl 2-(difluoromethyl)-5-(4,5-dihydro2-thiazolyl)-4-(2-methylpropyl)-6-(trifluoromethyl)-3-pyridinecarboxylate] (see 1 in Figure 1) is a herbicide developed for weed control in cotton, citrus, and other crops. Reports in the literature (Armbruster et al., 1988, 1991) suggest that disruption of cell division via inhibition of microtubule formation is the mode of action of this class of pyridine compounds. The metabolism of thiazopyr has been extensively studied in animals (Klemm et al., 1993; Feng et al., 1994, 1995a; Feng and Solsten, 1994; McClanahan et al., 1995) and plants (Feng et al., 1995b; Rao et al., 1995). Four major pathways have been identifiedsCoxidation, S-oxidation, O-demethylation, and ester hydolysis. The relative contribution of an individual pathway has been shown to vary with the animal or plant species being studied. In liver microsomes from Long Evans rats the metabolites of thiazopyr resulted primarily from C- and S-oxidation of the thiazoline ring (Feng et al., 1994). Similar studies using liver microsomes from Sprague-Dawley rats demonstrated the presence of oxidative cleavage of the carboxylic ester of thiazopyr catalyzed by monooxygenases (Feng and Solsten, 1994). Animal and plant esterases have also been shown to degrade the methyl ester group of thiazopyr to give a monoacid metabolite that showed 96%. Authentic standards for metabolites 2 and 7 (see Figure 1) were furnished by the Monsanto Sample Retention Center. Atomlight and Atomflow liquid scintillation cocktails were obtained from NEN Products, Boston, MA, and all other reagents and solvents were obtained from commercial suppliers. Preparation of Dosing Capsules. Labeled thiazopyr was dissolved in acetonitrile, portions were transferred to gelatin capsules, and the acetonitrile was evaporated under a gentle stream of nitrogen. The capsules were sealed and then stored frozen until dosing. At selected times before, during, and after dosing, capsules were extracted with acetonitrile and analyzed for total radioactivity and radiochemical purity by HPLC. Thiazopyr was shown to be stable for up to 7 days (average recovery was 97.4%, n ) 3) when stored in this manner. Animal Handling and Dosing. Single-comb White Leghorn laying hens were obtained from S&R Egg Farm (Whitewater, WI). The birds were 25-32 weeks of age upon receipt and weighed ∼1400-1600 g each. Hens were selected from a pool of birds based upon egg production (average egg production/hen/day ) 0.975) during an acclimation period and randomly assigned by body weight to a test group with five hens per group. Labeled thiazopyr was administered orally in gelatin capsules via a balling gun once daily for four consecutive days. Groups 1 and 2 received an average of 1.3 mg of thiazopyr/day (low dose), which exceeded the maximum potential dietary exposure in feed by more than an order of magnitude. Two low-dose groups were treated to provide
10.1021/jf9802833 CCC: $15.00 © 1998 American Chemical Society Published on Web 09/25/1998
Metabolism of Thiazopyr in Laying Hens
J. Agric. Food Chem., Vol. 46, No. 10, 1998 4399
Figure 1. Structures of thiazopyr and its metabolites. The asterisk (*) denotes the site for additional tissue for metabolite isolation. A high-dose group (group 3) received 10.4 mg/day and was included to provide sufficient quantities of low-level metabolites for identification. On the basis of actual food consumption during the study, the low-dose groups received the equivalent of 12 µg of thiazopyr/g (ppm) of diet and the high-dose group received 78 ppm. An additional group of hens was treated with placebo capsules and served as a source of control tissue. Sample Collection and Analysis. Eggs were collected twice daily in the morning and evening. The evening eggs were stored refrigerated and then added to the morning collection the following day. After collection, the eggs were separated into yolks and whites and stored separately in glass jars at -20 °C. Excreta samples were collected once daily from Teflon-coated paper that was placed below the cages of each group. All samples were pooled according to day, sample matrix, and group and then frozen. Approximately 22 h after the last dose, the hens were anesthetized with carbon dioxide, a sample of blood was collected in heparinized tubes by cardiac puncture, and the birds were sacrificed by carbon dioxide overdose. Tissue samples were taken from each animal upon necropsy, and each tissue type was pooled by treatment group. Tissues collected were liver, abdominal fat, skin with fat, kidneys, thigh muscle, and breast muscle. Eggs and tissues were homogenized, and aliquots solubilized or combusted for radiochemical analysis. Excreta were homogenized with water and centrifuged, and aliquots of the supernatant and the centrifugation pellet were taken for radiochemical analysis. Metabolite Extraction and Isolation. Tissues (35-150 g) from low-dose groups 1 and 2 containing g0.01 µg of thiazopyr equiv/g were extracted and analyzed for metabolites. Egg yolk, liver, abdominal fat, and skin with fat were isolated from group 2, whereas a pooled sample from both low-dose groups was used for kidney and thigh muscle analysis. The high-dose (group 3) excreta and abdominal fat were also used for metabolite isolation. A portion of the fourth day excreta sample (58.9 g) was extracted two times with 50 mL of acetonitrile/water (70:30), the extracts were combined, and the metabolites were partitioned into an equal volume of ethyl acetate. The remaining aqueous phase was adjusted to contain 1% aqueous formic acid and extracted with an equal volume of ethyl acetate. The ethyl acetate extracts were combined and concentrated to near dryness, and the residue was dissolved in 20 mL of 1% formic acid/methanol (1:1). The reconstituted residue was applied to a 10 g C-18 Mega-Bond Elut solid-phase extraction column (Varian Associates Inc., Harbor City, CA) and rinsed with 50 mL of 1% formic acid/methanol (1:1), and purified metabolites were eluted with 50 mL of methanol. A portion of the fourth day egg yolk sample (54.8 g) was extracted two times with 30 mL of acetonitrile and then once with an equivalent volume of acetonitrile/water (70:30). The extracts were concentrated to remove the acetonitrile, adjusted
13
C/14C labeling of thiazopyr.
to contain 1% formic acid, and applied to a 10 g C-18 MegaBond Elut column. The column was rinsed with 50 mL of 1% formic acid, and metabolites were eluted with methanol. The methanol solution was concentrated and mixed with an equal volume of 1% formic acid, and the metabolites were isolated by HPLC or partitioning into methylene chloride. The methylene chloride solution was dried over sodium sulfate concentrated to near dryness and the residue dissolved in 1% formic acid/methanol. A 110 g liver sample was extracted two times with 50 mL of acetonitrile/water (70:30) and once with acetonitrile/0.1 N HCl using a Tissuemizer (Tekmar Co., Cincinnati, OH) to grind and mix the sample. The residue was extracted for 16 h with 250 mL of acetonitrile/water (60:40) using a Soxhlet apparatus (Fisher Scientific, St. Louis, MO). The extracts were combined, concentrated, and purified using a C-18 MegaBond Elut column and the procedure followed for the egg yolk. Abdominal fat and skin with fat samples (57 g) were extracted two times with 25 mL of acetonitrile, concentrated to ∼1 mL, and mixed with an equal volume of 1% formic acid. The kidney (34.8 g) and thigh muscle (150 g) were extracted two times with acetonitrile/water (70:30), and the kidney was extracted one additional time with acetonitrile/0.1 N HCl, followed by concentration to remove acetonitrile. Combustion and Liquid Scintillation Counting. Replicate combustions (3-12, 0.1-0.5 g) were performed using a Packard Tri-Carb automatic sample oxidizer model B306 (Packard Instrument Co., Downers Grove, IL). Performance of the oxidizer was monitored daily by combustion of a C-14 standard or fortification of control tissue with a C-14 standard. The average recovery for C-14 standards was >97%. Liquid scintillation counting was performed with a TM Analytic Mark III model 6881 or Packard Tri-Carb models 1500 or 4640 counters using automatic external standard efficiency correction. An estimation of the sensitivity of the method was calculated as 0.002 ppm for groups 1 and 2 and 0.004 ppm for group 3, assuming a sample contained 34 dpm above background and a typical aliquot weight of 0.2 g was combusted. High-Performance Liquid Chromatography (HPLC). The HPLC system consisted of the following components: a Waters model U6K injector (Waters Associates, Milford, MA); two Waters model 510 pumps; a Waters 484 tunable absorbance detector (operated at 254 nm); and a Waters 680 automated gradient controller. The effluent was passed through a Flo-One Beta radioactive flow detector (Packard Instrument Co.) equipped with a 2.5 mL liquid cell or 250 µL solid cell. A Beckman Ultrasphere ODS 5 µm (10.0 mm × 25 mm) column (Beckman Instruments Inc., San Ramon, CA) equipped with a Brownlee PRP-1 or RP-18 Newguard column (15 mm × 3.2 mm) (Applied Biosystems, Inc., Foster City, CA) was used. The profiling HPLC gradient was conducted in linear steps at a flow rate of 3 mL/min, utilizing 1% formic acid (solvent A) and methanol (solvent B). The gradient
4400 J. Agric. Food Chem., Vol. 46, No. 10, 1998 proceeded from 50% B to 63% B over 18 min, remained at 63% B for 12 min, proceeded to 70% B over 2 min, remained at 70% B for 18 min, proceeded to 100% B over 5 min, and then remained at 100% B for an additional 5 min. The effluent from the column was mixed with Atomflow pumped at 9 mL/min and collected in vials at 0.3 min intervals with an ISCO fraction collector (Retriever III, Instrument Specialties Co., Lincoln, NE). Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR) Spectroscopy. Chemical ionization (CI) mass spectra were obtained using a Finnigan 4515 quadrupole mass spectrometer and processed with a Data General Nova 4 computer using INCOS software. The samples were introduced using a Finnigan 9610 gas chromatograph that was equipped with a J&W Scientific DB-5 capillary column (25 m × 0.32 mm) and programmed between 70 and 300 °C. Helium was used as the carrier gas at a flow rate of 2 mL/min. Chemical ionization of the gas chromatograph effluent was performed using isobutane (0.5 Torr). Negative and in some cases positive ions were analyzed. Fast-atom bombardment (FAB) mass spectra and highresolution FAB analyses were recorded on a VG ZAB-HF double-focusing mass spectrometer and processed with a DEC PDP 11/24 data system (Maynard, MA). The samples were deposited on a thin layer of glycerol and then introduced into the mass spectrometer by direct probe. Microbore liquid chromatography (LC) FAB mass spectra were obtained by introducing the sample using an microbore HPLC system that consisted of an Applied Biosystems Inc. (Santa, Clara, CA) model 140A syringe pump, a Brownlee C-18 column (1 × 250 mm), and a VG (Manchester, U.K.) dynamic FAB/MS probe. Ionization was achieved for all FAB analyses with an Ion Tech Saddle Field fast atom gun (Middlesex, U.K.) producing 7 kV xenon atoms at 1 mA emission current. Negative ions were analyzed. 1H NMR spectra were obtained using a Varian XL-300 spectrometer with a proton operating frequency of 300 MHz. Chemical shifts were referenced using the resonance frequency of the solvent peak through the Monsanto Switch software package, a proprietary automated expert system, and are reported in parts per million from tetramethylsilane. Samples were dissolved in high-purity deuteriochloroform and placed in 5 mm tubes (Wilmad Glass Co., Buena, NJ). 19F NMR spectra were also obtained using a Varian XL-300 NMR spectrometer. Two-dimensional correlation spectroscopy (2-D COSY) was performed using a Varian XL-500 NMR spectrometer. Diazomethane Derivatization. Methyl esters of carboxylic acids were prepared by extracting the purified metabolites from the HPLC mobile phase with methylene chloride, concentrating to near dryness, and dissolving in 1-2 mL of diethyl ether. Etheral diazomethane was added until the yellow color persisted, and then the solution was incubated at room temperature for 10 min. Solvent and excess reagent were removed with a stream of nitrogen, and then the sample was dissolved in acetonitrile or methanol prior to MS analysis. Liver Homogenate Preparation and Incubation Conditions. Approximately 100 g of liver tissue from control hens, male rats (Sprague-Dawley, 200-250 g body weight), and a control lactating goat (Sunshine Farms, Portage, WI; 60 kg body weight) was chilled on ice and homogenized with 0.01 M potassium phosphate buffer, pH 7.4, containing 1.15% KCl at a tissue-to-buffer ratio of 1:3 (w/v) using a Potter-Elvehjem homogenizer or a Tissumizer (Tekmar Co., Cincinnati, OH). Homogenates were centrifuged at 4 °C for 30 min at 9000g, filtered through gauze to remove lipids, aliquoted, and stored at -70 °C until used. Metabolite 2 was isolated from the abdominal fat of group 3, and its metabolism by liver homogenates was studied in a reaction containing 10 mM glucose 6-phosphate, 5 mM nicotinamide, 5 mM magnesium chloride, 1.3 mM NADP+, 10 units of glucose-6-phosphate dehydrogenase, 11.3 mg of liver homogenate, and 0.2 M potassium phosphate buffer, pH 7.4, in a total volume of 0.985 mL. Components were preincubated at 37 °C for 5 min, and then the reaction was started by the
Ridley et al. Table 1. Distribution of Radioactivity in Eggs, Excreta, and Tissues of Hens Dosed with [14C]Thiazopyr % of total radioactive dose matrix
group 1 (1.3 mg/day)
group 2 (1.3 mg/day)
group 3 (10.4 mg/day)
egg white egg yolk excreta tissuesa
0.02 0.03 94.31 0.43
0.02 0.04 90.08 0.62
0.02 0.05 92.73 0.49
total
94.79
90.76
93.29
a
Consists of gastrointestinal tract, liver, kidneys, and the portions of abdominal fat, skin with fat, thigh muscle, and breast muscle taken at necropsy. Table 2. Total Radioactive Residues in Egg White, Egg Yolk, and Tissues at Sacrifice µg equiv of thiazopyr/g of samplea (ppm) sample
group 1 (1.3 mg/day)
group 2 (1.3 mg/day)
group 3 (10.4 mg/day)
liver abdominal fat skin with fat egg yolk kidneys blood egg white thigh muscle breast muscle
0.222 0.123 0.049 0.077 0.047 0.016 0.008 0.010 0.004
0.298 0.173 0.097 0.075 0.052 0.021 0.008 0.008 0.005
1.112 1.417 0.488 0.626 0.501 0.097 0.052 0.086 0.033
a Specific activities of 87950 (for groups 1 and 2) and 44420 (for group 3) dpm/µg equiv of thiazopyr were used to calculate ppm.
addition of 15 µL of a methanol solution of metabolite 2, which gave a final concentration of 4.0-5.0 µM. The reaction containing hen homogenate was incubated for 3 h at 37 °C, stopped by the addition of 1.0 mL of cold methanol, and then stored at -20 °C until analyzed. The reactions with goat and rat liver homogenates were identical to the hen except that they were incubated for 2 h at 37 °C. RESULTS
Distribution of Total Radioactivity. The total recovery of radioactivity in the samples of eggs, excreta, and tissues collected for analysis from groups 1-3 is summarized in Table 1. The total recovery of administered dose ranged from 90.8 to 94.8% with a majority of the radioactivity (90.1-94.3%) eliminated in excreta. The entire egg production contained
