In Vitro Metabolism of Flucetosulfuron by Human Liver Microsomes

Article pubs.acs.org/JAFC

In Vitro Metabolism of Flucetosulfuron by Human Liver Microsomes Yong-Sang Lee,† Kwang-Hyeon Liu,‡ Joon-Kwan Moon,§ Beom Jun Ko,∥ Hoon Choi,⊥ Kook-Sang Hwang,# Eunhye Kim,† and Jeong-Han Kim*,† †

Department of Agricultural Biotechnology, Seoul National University, Seoul 151-742, South Korea College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu 702-701, South Korea § Department of Plant Life and Environmental Sciences, Hankyong National University, Ansung 456-749, South Korea ∥ Busan Branch of Forensic Chemistry Laboratory, Supreme Prosecutors’ Office, Busan 611-743, South Korea ⊥ Ministry of Food and Drug Safety, Cheongwon 363-951, South Korea # Specialty Chemicals Division, LG Life Sciences Ltd., Ulsan 689-896, South Korea ‡

ABSTRACT: To investigate herbicide metabolism, human liver microsomes were incubated with threo- and erythro-isomers of flucetosulfuron. Each isomer produced one metabolite; the metabolites were unambiguously identified as enzymatic hydrolysis products by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These metabolites were synthesized, producing white solids characterized using LC-MS/MS and nuclear magnetic resonance spectroscopy (1H and 13C). Using specific esterase inhibitors and activators, carboxylesterases and cholinesterases were demonstrated to be involved in flucetosulfuron metabolism. Under optimized metabolic conditions, the kinetic parameters for metabolite formation from threoflucetosulfuron and erythro-flucetosulfuron were: Vmax, 151.41 and 134.38 nmol/min/mg protein, respectively; Km, 2957.37 and 2798.53 μM, respectively; and CLint, 51.20 and 48.02 μL/min/mg microsomes respectively. No significant kinetic differences were observed between the two isomers. These results indicated that the primary metabolic pathway for both flucetosulfuron isomers in human liver microsomes involves hydrolysis, catalyzed by carboxylesterase and cholinesterase. KEYWORDS: flucetosulfuron, human liver microsomes, metabolism, esterase

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chloracetamides, and triazines,8,10−12 indicating that cytochrome P450s (CYP450), flavin-containing monooxygenases (FMOs), and esterases are major metabolic enzymes for these pesticides. Mutch et al. showed that many human CYP450 isoforms catalyzed the transformation of parathion, diazinon, and chlorpyriphos to the oxon form, which can be detoxified via hydrolysis by A-esterases (paraoxonases/arylesterases), carboxylesterases, and cholinesterases.10 For pyrethroid metabolism, the major pathways involve CYP450-dependent oxidation and esterase-mediated hydrolysis.11−16 Although sulfonylurea herbicides have been used globally, currently there is no published information describing their metabolism by HLMs. Investigation of flucetosulfuron metabolism by HLMs will provide a better understanding of the metabolism and detoxification routes for related sulfonylureas in the human body. Therefore, we studied in vitro metabolism of flucetosulfuron by HLMs to identify the structure(s) of its metabolite(s), elucidate the relevant metabolic pathway(s), and investigate their kinetics. During the study, we characterized the specific HLM enzymes responsible for flucetosulfuron metabolism. To the best of our knowledge, this study is the first report to describe the in vitro metabolism of flucetosulfuron by HLMs

INTRODUCTION Flucetosulfuron (N-[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl]-2-[2-fluoro-1-(methoxymethylcarbonyloxy)propyl]3-pyridinesulfonamide), a relatively new herbicide, inhibits acetolactate synthase (ALS) in plants,1−3 as do other ALS inhibitors such as imidazolinones, pyrimidinyloxybenzoates, triazolopyrimidines, and sulfonylaminocarbonyltriazolinones.4,5 Flucetosulfuron is applied at 15−30 g ai/ha to soil or foliage and are safe for use with rice (Oryza sativa L.), wheat (Triticum aestivum), and zoysia grass (Zoysia japonica).6 In rice fields, flucetosulfuron not only controls annual broadleaf weeds, sedges, and perennial weeds with similar efficacy as other sulfonylurea herbicides but also provides excellent efficacy for the control of barnyard grass (Echinochloa crus-galli), which is not controlled or is only marginally controlled by other commercial sulfonylureas. In cereal crops, it also provides excellent control of broadleaf weeds, including cleavers (Galium aparine), Matricaria spp., and Papaver rhoeas, with a good safety profile for wheat and barley. Metabolism of pesticides in biological or environmental systems is interesting because metabolites and their formation patterns are important determinants of pesticide safety. In mammals, the liver plays a major role in the metabolism and systemic elimination of pesticides after exposure. For this reason, studies of in vitro metabolism by human liver microsomes (HLMs) are generally conducted to investigate pesticide metabolic pathways, the pattern of metabolite and intermediate formation, and kinetics.7−9 A number of papers have been published on HLM metabolism of pesticides such as phosphorothioates, pyrethroids, © 2014 American Chemical Society

Received: Revised: Accepted: Published: 3057

November 1, 2013 March 14, 2014 March 14, 2014 March 14, 2014 dx.doi.org/10.1021/jf4048836 | J. Agric. Food Chem. 2014, 62, 3057−3063

Journal of Agricultural and Food Chemistry

Article

and identify the metabolizing enzymes responsible for its hydrolysis.

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MATERIALS AND METHODS

Chemicals and Reagents. Flucetosulfuron Technical (purity, 98.4%) was provided by LG Life Sciences Ltd. (Daejeon, Korea). Pooled HLMs were purchased from BD Gentest (Woburn, MA). Glucose-6-phosphate, glucose-6-phosphate dehydrogenase, and nicotinamide adenine dinucleotide (NADP+) were purchased from Sigma-Aldrich (St. Louis, MO). Various esterase inhibitors, such as bisnitrophenyl phosphate (BNPP), calcium chloride, cobalt chloride, dibucaine, ethylenediaminetriacetic acid (EDTA), eserine, magnesium chloride, mercuric chloride, and quinidine, were also purchased from SigmaAldrich (St. Louis, MO). Potassium phosphate monobasic/dibasic and sodium hydroxide (NaOH) were purchased from Daejung Chem (Ansan, Korea). The solvents were high-performance liquid chromatography (HPLC) grade (Burdick and Jacson, MI), and the other chemicals were of the highest quality available. Preparation of threo-Flucetosulfuron. Flucetosulfuron (0.03 g) was dissolved in acetonitrile (10 mL) and injected into a Waters Delta Prep-4000 preparative chromatography system equipped with a Waters deltapak column (50 mm i.d. × 300 mm, 15 μm, 100 A). An isocratic mobile phase was used where A:B = 64:36 at a flow rate of 20.0 mL/min for 120 min (A, buffer solution with 0.02 M ammonium acetate; B, 0.1 M acetic acid). The first fraction (retention time of 80−90 min) was collected (150−170 mL) and partitioned twice with 70 mL of dichloromethane. The dichloromethane layer was washed with 100 mL of distilled water, dried over 30 g of sodium sulfate, and evaporated under reduced pressure at 30 °C to obtain 2.6−4.0 mg of threo-flucetosulfuron from one fractionation/purification cycle. About 0.3 g of threo-flucetosulfuron was obtained by repeating this process several times. Preparation of erythro-Flucetosulfuron. A second HPLC fraction (retention time of 94−110 min) was collected (∼250 mL) and processed as described above for threo-flucetosulfuron, producing 23−26 mg of erythro-flucetosulfuron from one cycle of the fractionation/purification process. After repeating the same processes many times, about 2.0 g of erythro-flucetosulfuron was obtained. Metabolism of Flucetosulfuron in HLMs. The incubation mixtures containing 0.5 mg of pooled HLMs (H161, Gentest) were reconstituted in 50 mM phosphate buffer (pH 7.4) and preincubated for 5 min at 37 °C. The reaction was initiated by adding the threo- or erythro-flucetosulfuron isomer (100 μM) and the NADPH-generating system (0.8 mM NADP+, 10.0 mM glucose-6-phosphate, 1.3 mM MgCl2, and 1.0 unit/mL glucose-6-phosphate dehydrogenase) and further incubated for 30 min at 37 °C in a shaking water bath. The reaction was terminated by the addition of 250 μL of acetonitrile, and the samples were centrifuged at 15000g for 5 min at 4 °C. Control incubations were conducted with denatured HLMs, which had been boiled in water for 5 min at 100 °C. To confirm whether metabolite formation was NADPH-dependent, NADPH-free incubations were also performed. Synthesis of threo-N-(4,6-Dimethoxypyrimidin-2-ylcarbomoyl)-2-(2-fluoro-1-hydroxy propyl)pyrimidine-3-sulfonamid (TM1). threo-Flucetosulfuron (149 mg, 1 equiv) was dissolved in 30 mL of acetonitrile in a round-bottom flask (100 mL), 7.5 mL of 0.1 M NaOH solution (30 mg, ∼2.5 equiv) was added dropwise, and the reaction mixture was stirred for 1.5 h. The reaction mixture was then transferred to a separatory funnel (250 mL), and 20 mL of distilled water was added. The reaction mixture was washed twice with 25 mL of dichloromethane. The water layer was adjusted to pH 3.0 with 0.1 N HCl to obtain TM1 as a white solid, which was filtered, washed with distilled water, and dried under nitrogen purging for 10 h (90 mg, yield 70%). Synthesis of erythro-N-(4,6-Dimethoxypyrimidin-2-ylcarbomoyl)-2-(2-fluoro-1-hydroxy propyl)pyrimidine-3-sulfonamid (EM1). erythro-Flucetosulfuron (487 mg, 1 equiv) was dissolved in 100 mL of acetonitrile in a round-bottom flask (250 mL), 25 mL of 0.1 M NaOH solution (100 mg, ∼2.5 equiv) was added dropwise, and

Figure 1. Formation of metabolite M1 from threo-flucetosulfuron (A) and M2 from erythro-flucetosulfuron (B) when flucetosulfurons (100 μM) were incubated with 0.125 mg human liver microsomes for 30 min at 37 °C. the reaction mixture was stirred for 1.5 h. The workup process was the same as described above for TM1. EM1 was obtained as a white solid (370 mg, yield 89%). Metabolite Identification. First, 2 μL of the supernatant from each metabolic reaction was analyzed using HPLC and LC-MS/MS. Again, tandem mass spectrometry (MS/MS) analysis of metabolites, TM1, EM1, and the parent flucetosulfuron isomers was conducted to compare their fragmentation patterns. Solutions of the synthesized metabolites (TM1 and EM1) were prepared in acetonitrile at 10 mg/kg level, and 20 μL of a 1:1 mixture each of TM1 and EM1 solution and the corresponding metabolic reaction supernatant was analyzed using HPLC for confirmation. Optimization of Metabolic Conditions and Kinetic Studies. The metabolic reactions described earlier were performed with various concentrations of HLMs (0.16, 0.24, 0.32, 0.48, 0.64, 0.96, and 1.28 mg/mL) and each flucetosulfuron isomer (10, 20, 100, 200, 1000, 2000, and 10000 μM) and for 10, 20, 30, 60, 120, 180, and 240 min in a total volume of 250 μL. The incubation mixtures containing pooled HLMs were preincubated for 5 min at 37 °C. The metabolites TM1 and EM1 in the reaction mixture were determined using HPLC analysis. Optimized conditions were used to analyze enzyme kinetics. These used an incubation time of 30 min (0.16 mg/mL HLMs) and a concentration range of each flucetosulfuron isomer (10, 20, 100, 200, 1000, 2000, and 10000 μM). Inhibition of Metabolism by Specific Esterase Inhibitors. Incubation mixtures containing 0.16 mg/mL HLMs and one of the following esterase-selective inhibitors [BNPP (100 μM), calcium chloride (1000 μM), cobalt chloride (1000 μM), dibucaine (100 μM), EDTA (3000 μM), eserine (100 μM), magnesium chloride (1000 μM), mercuric chloride (200 μM), and quinidine (100 μM)] were preincubated for 5 min at 37 °C. The reaction was initiated by adding each flucetosulfuron isomer (100 μM) and incubating for a further 30 min at 37 °C in a shaking water bath. The percentage inhibition was calculated using the ratio of the amount of metabolite (TM1 or EM1) formed in the presence of the 3058

dx.doi.org/10.1021/jf4048836 | J. Agric. Food Chem. 2014, 62, 3057−3063


Article

Figure 2. LC-MS/MS spectra and fragmentation of m/z 488 from flucetosulfuron ((A) [M + H]+), and of m/z 416 from metabolite of erythro isomer ((B) [M + H]+). specific inhibitor to the amount formed under control conditions in the absence of inhibitor. Analytical Instruments and Conditions. HPLC. The concentrations of flucetosulfuron isomers and metabolites (TM1 and EM1) were measured using a Waters Alliance 2690 HPLC equipped with a Capcell pak C18 UG120 column (4.6 mm i.d. × 150 mm, 3 μm; Shiseido, Tokyo, Japan) at 40 °C. The mobile phase consisted of 20 mM ammonium acetate buffer with 0.1 M acetic acid (A) and acetonitrile (B). The gradient conditions used were as follows: 20% B at 0 min, 27% B at 4 min, 30% B at 25 min, and 20% B at 28−33 min. The injection volume was 10 μL, and peaks were detected at 254 nm (UV 4890 detector; Waters, Milford, CA). Stock solutions (1 mM) were prepared by dissolving flucetosulfuron isomers, TM1, and EM1 in acetonitrile and diluting serially with acetonitrile to obtain final standard concentrations of 0.05, 0.1, 0.2, 1.0, 10, and 100 μM. The calibration curve was fitted with high linearity (r2 > 0.999). LC-MS/MS. For the identification of flucetosulfuron and its metabolites, a quadrupole MS/MS (API2000, Applied Biosystems, Foster City, CA) coupled with an Agilent 1100 series HPLC system (Agilent, Wilmington, DE) was used. The separation was performed on a Capcell pak C18 UG120 column (4.6 mm i.d. × 150 mm, 3 μm; Shiseido, Tokyo, Japan) by using a mobile phase consisting of 10 mM

ammonium acetate with 0.1% formic acid (A) and acetonitrile (B) at a flow rate of 1.0 mL/min. Mass spectra were recorded by electrospray ionization in positive mode. The turbo ion spray interface was operated at 4500 V and 550 °C. The operating conditions, optimized by flow injection of analytes, were as follows: nebulizer gas flow, 50 psi; curtain gas flow, 10 psi; and collision energy, 30 eV. Quadrupole Q1 and Q3 were set on unit resolution. Nuclear Magnetic Resonance (NMR) Analysis. 1H and 13C NMR spectra were recorded on a 400 MHz NMR spectrometer (Jeol JNM-LA400 with LFG, JEOL, Japan) in CDCl3 (99.8%, Merck) at 297 K. TMS was used as reference (δ = 0). Data Analysis. Results are expressed as mean ± standard deviation (SD) of estimates obtained from pooled HLMs in triplicate experiments. In the microsomal incubation studies, the apparent kinetic parameters of metabolite formation (TM1 and EM1) (Km and Vmax) were determined by fitting a one-enzyme Michaelis−Menten equation (V = Vmax × [S]/(Km + [S]) or a Hill equation (V = Vmax × [S]n/(Km + [S]n)). The calculated parameters were the maximum rate of formation (Vmax), the Michaelis constant (apparent Km), intrinsic clearance (CLint = Vmax/apparent Km), and Hill coefficient (n). Calculations were performed using Minitab 16 software (Minitab Inc., State College, PA). 3059



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Table 1. 1H and 13C NMR Data for TM1 and EM1

2.0 g of erythro-flucetosulfuron were obtained with 99.8% and 99.7% purity, respectively. LC-MS/MS analysis showed [M + H]+ at m/z 488, clearly confirming the identity of both isomers. Flucetosulfuron Metabolism by HLMs and Analysis of Metabolites. threo-Flucetosulfuron and erythro-flucetosulfuron, which were isolated using preparative HPLC, were incubated with HLMs in the presence of NADPH; one metabolite, M1, was observed in the threo-flucetosulfuron reaction mixture, while another metabolite, M2, was observed in the erythroflucetosulfuron reaction mixture. No metabolites were formed in reactions using denatured HLMs, suggesting that these were responsible for the formation of M1 and M2 from flucetosulfuron isomers (Figure 1). However, those metabolites were not oxidation products resulting from CYP450 activity in HLMs because they were also formed in NADPH-free reactions. On LC-MS/MS analysis, M1 and M2 gave the same [M + H]+ at m/z 416, indicating a cleavage reaction, rather than insertion of oxygen, when compared with the parent flucetosulfuron isomers ([M + H]+ = m/z 488). MS/MS analysis of flucetosulfuron ([M + H]+ at m/z 488) in the microsomal reaction mixture showed fragment ions at m/z 156, 182, and 333 (Figure 2A), while the MS/MS spectrum of MI and M2 ([M + H]+ = m/z 416) showed fragmentation ions at m/z 156, 182, and 261 (Figure 2B). This result suggested that the metabolites were hydrolysis products, which could be formed by cleavage of the flucetosulfuron ester bond. Identification of M1 and M2. Because LC-MS/MS analysis indicated that M1 and M2 were hydrolysis products of the parent compounds, TM1 and EM1 were prepared from each flucetosulfuron isomer by chemical hydrolysis to confirm their identity. TM1 and EM1 were synthesized with good yields, and their structures were confirmed unambiguously using LC-MS/MS ([M + H]+ = m/z 416) and 1H and 13C NMR as N-(4,6-dimethoxypyrimidin-2-ylcarbomoyl)-2-(2-fluoro-1hydroxypropyl)pyrimidine-3-sulfonamid (Table 1 and Figure 3) Finally, by comparing LC-MS/MS and MS/MS fragmentation spectra and co-chromatography, M1 and M2 were identified as TM1 and EM1, respectively. Although various studies of pesticide metabolism by HLMs showed that NADPHdependent oxidation by CYP450s was a major metabolic pathway,8,10,18−21 only a hydrolysis product was formed in the present study. Hydrolytic metabolism of sulfonylureas has only been reported in soil and water to date.7−15 Liver microsomes are a rich source of enzymes, such as CYP450s, FMOs, carboxylesterases, reductases (azo-, carbonyl, and quinone), and aldehyde dehydrogenases for phase I

1

H NMR Data

TM1

EM1

proton

δ (CDCl3)

J (Hz)

4 5 6 7 13a 15a 18 19 20b 21c 25, 26

8.83 (dd) 7.52 (dd) 8.59 (dd) 5.82 (s) 7.27 (s) 12.99 (s) 5.45 (m) 5.14 (m) 4.39 (d) 1.47 (3H, dd) 3.96 (6H, s)

1.5, 4.8 4.7, 8.0 1.5, 8.0

δ (CDCl3)

8.82 (dd) 7.52 (dd) 8.67 (dd) 5.80 (s) 7.25 (s) 13.00 (s) 5.65 (m) 4.83 (m) 9.1 4.11 (d) 6.3, 24.1 1.39 (3H, dd) 3.98 (6H, s) 13 C NMR Data

TM1

a

carbon

δ (CDCl3)

1 2 4 5 6 7 8, 12 10 14 18d 19d 21d 25, 26

140.6 157.5 148.7 133.5 123.3 85.6 172.0 152.7 155.4 72.2 (d) 92.1 (d) 17.7 (d) 55.0

J (Hz) 1.5, 4.8 4.6, 8.2 1.5, 8.2

9.5 6.1, 24.8

EM1 J (Hz)

δ (CDCl3)

18.1 72.6 22.9

140.8 157.5 148.8 134.5 123.4 85.6 171.0 152.9 155.4 72.0 (d) 92.6 (d) 16.4 (d) 55.0

J (Hz)

24.8 182.6 21.9

NH. bOH. cH−F coupling. dC−F coupling.

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RESULTS AND DISCUSSION Preparation of Flucetosulfuron Isomers. Flucetosulfuron consists of threo-flucetosulfuron and erythro-flucetosulfuron, and these isomers could be separated using HPLC, in which threo-flucetosulfuron eluted before erythro-flucetosulfuron.17 These two flucetosulfuron isomers were isolated using preparative HPLC and purification processes. Because one cycle of the fractionation/purification process produced only a small quantity of each isomer, the same processes were repeated many times to obtain larger amounts with high purity for use in the metabolic studies. About 0.3 g of threo-flucetosulfuron and

Figure 3. Chemical structure of TM1 and EM1. 3060



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Figure 4. Formation pattern of metabolite EM1 from erythro-flucetosulfuron and TM1 from threo-flucetosulfuron when flucetosulfurons (100 μM) were incubated with 0.16 mg human liver microsomes for 30 min at 37 °C.

of HLMs, and 30 min incubations. Under these optimized metabolic conditions, the formation pattern of TM1 and EM1 from each flucetosulfuron isomer by HLMs was best fitted to a Michaelis−Menten equation (V = Vmax × [S]/(Km + [S])), while the Hill model and two-enzyme model did not significantly improve the regression (Figure 4). No significant difference was observed in Vmax and Km values for the formation of TM1 and EM1 from threo- and erythro-flucetosulfuron, respectively (Table 2). The CLint values of TM1 and EM1 were 51.2 and 48.0 μL/min/mg protein for threo-flucetosulfuron and erythro-flucetosulfuron, respectively. In this study, there was no difference in the kinetics of TM1 and EM1 formation, while previous metabolic studies of endosulfan (α-, β-) showed stereoselectivity by CYP450s and permethrin (cis-, trans-) showed stereoselectivity of carboxylesterases.8,16 Identification of Esterases Involved in the Hydrolysis of Flucetosulfuron. Because the metabolites were formed through ester bond cleavage and the major xenobiotic metabolizing esterases include A-esterases (paraoxonases/arylesterases), cholinesterases, and carboxyesterases,21 identification of the esterase responsible for hydrolysis was performed through an

Table 2. Enzyme Kinetic Parameters for the Production of the Metabolite EM1 from erythro-Flucetosulfuron and TM1 from threo-Flucetosulfuron When Flucetosulfuron Isomers Were Incubated with 0.16 mg Human Liver Microsomes for 30 min at 37 °C threo-flucetosulfuron erythro-flucetosulfuron Vmax (nmol/min/mg HLMs)*a Km (μM)* CLint [Vmax/Km (μL/min/mg HLMs)]

151.41 ± 2.75 2957.37 ± 155.74 51.20 ± 9.30

134.38 ± 2.48 2798.53 ± 152.81 48.02 ± 8.86

*P value: 0.21and 0.24 for threo-flucetosulfuron and erythroflucetosulfuron. a

reactions,22 and these results indicated the involvement of hydrolysis enzymes in HLM-mediated metabolism of flucetosulfuron. Enzyme kinetics to establish the optimal metabolic transformation of flucetosulfuron by HLMs, protein (HLMs) concentration, substrate (each flucetosulfuron isomer) concentration, and incubation time were optimized, resulting in the use of 100 μM of each isoform of flucetosulfuron, 0.16 mg/mL

Table 3. Effects of Esterase Inhibitors on the Production of Metabolite TM1 from threo-Flucetosulfuron and EM1 from erythroFlucetosulfuron When Flucetosulfurons (100 μM) Were Incubated with 0.16 mg Human Liver Microsomes for 30 min at 37 °C specific activity (μmol/min/mg protein)a inhibitor control HgCl2*b CaCl2**c CoCl2 MgCl2 EDTA BNPP* eserine* dibucaine* quinidine* a

target esterase general esterases A-esterases A-esterases A-esterases A-esterases carboxylesterases cholinesterases cholinesterases cholinesterases

concentration (μM) 200 1000 1000 1000 3000 100 100 100 100

TM1 5.31 0.45 5.15 5.05 5.09 5.46 0.84 0.63 2.99 2.89

± ± ± ± ± ± ± ± ± ±

0.08 0.09 0.05 0.08 0.10 0.15 0.19 0.40 0.15 0.20

inhibition (%) 91.5 2.9 4.9 4.0 −2.9 84.1 88.2 43.6 45.5

EM1

inhibition (%)

± ± ± ± ± ± ± ± ± ±

92.3 6.4 7.6 6.2 −3.1 85.7 89.1 39.7 43.3

5.28 0.41 4.88 5.17 4.95 5.44 0.76 0.57 3.18 2.94

0.18 0.02 0.20 0.05 0.40 0.19 0.02 0.01 0.15 0.20

Each value represents the mean of two determinations. b*Significant difference at α = 0.05. c**A-esterase activator. 3061



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inhibition study with class-specific esterase inhibitors23 (Table 3). The concentration of each inhibitor was adopted from previous studies of their inhibitory effects on their target esterases. Addition of Hg2+ into the metabolic mixture significantly inhibited the production of TM1 and EM1, suggesting that arylesterases,24 carboxylesterases,21,25 and cholinesterases26 were involved. However, treatment with an activator (Ca2+) or inhibitor (Mg2+, Co2+, and EDTA) for A-esterases27 did not produce any significant effect on metabolite formation, indicating that flucetosulfuron-metabolizing enzymes were not A-esterases. 28,29 The specific carboxylesterase inhibitor, BNPP,21,30,31 produced almost complete suppression (>80%) of metabolite formation, and addition of eserine,23,32 dibucaine, and quinidine, which are specific cholinesterase inhibitors, also reduced metabolite formation. These results indicated that the formation of TM1 and EM1 must be mediated by microsomal carboxylesterases and cholinesterases. Such hydrolytic reactions by carboxylesterases were reported with organophosphate pesticides,33 pyrethroids,14,16 and pyribenzoxim.21 Although cholinesterase-mediated pesticide hydrolysis has not been reported, cocaine and heroin were hydrolyzed by both carboxylesterase and cholinesterase.34,35 In conclusion, the primary metabolic pathway for flucetosulfuron in HLMs involved hydrolysis, producing TM1 and EM1. The esterase-specific inhibition study showed that carboxylesterases and cholinesterases were responsible for the formation of these metabolites. Metabolite formation kinetics did not show any significant difference between threo- and erythroisomers. On the basis of these findings, a metabolic pathway for both flucetosulfuron isomers was proposed (Figure 5).

ACKNOWLEDGMENTS

We thank LG Life Sciences, Korea, for providing the chemicals.

REFERENCES

(1) Alister, M.; Timothy, D. Review of the activity fate and mode of action of sulfonylurea herbicide. Pestic. Sci. 1988, 22, 195−219. (2) Brown, H. M. Mode of action, crop selectivity, and soil relations of the sulfonylurea herbicides. Pestic. Sci. 1990, 29, 263−281. (3) JanjicV.JovanovicL.BlanusaT.MilosevicD.Sulfonylurea herbicidesmode of action. In Plant Physiology in the New Millennium; Yugoslav Society of Plant PhysiologyAgriculture Research Institute: Belgrade, 2002. (4) Duggleby, R. G.; McCourt, J. A.; Guddat, L. W. Structure and mechanism of inhibition of plant acetohydroxyacid synthase. Plant Physiol. Biochem. 2008, 46, 309. (5) McCourt, J. A. Herbicide-binding sites revealed in the structure of plant acetohydroxyacid synthase. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 569−573. (6) Kim, D.; Koo, S.; Lee, J.; Hwang, K.; Kim, T.; Kang, K.; Hwang, K.; Joe, G.; Cho, J.; Kim, D. Flucetosulfuron: a new sulfonylurea herbicide. The BCPC International Congress: Crop Science and Technology, Glasgow, November 10−12, 2003; The BCPC International Congress: Crop Science and Technology; Proceedings of an International Congress Held at the SECC, Glasgow, Scotland, UK, 10− 12 November 2003; British Crop Protection Council, 2003; Vol. 1 and 2, pp 87−92. (7) Kim, K.-A.; Kim, M.-J.; Park, J.-Y.; Shon, J.-H.; Yoon, Y.-R.; Lee, S.-S.; Liu, K.-H.; Chun, J.-H.; Hyun, M.-H.; Shin, J.-G. Stereoselective metabolism of lansoprazole by human liver cytochrome P450 enzymes. Drug Metab. Dispos. 2003, 31, 1227−1234. (8) Lee, H.-K.; Moon, J.-K.; Chang, C.-H.; Choi, H.; Park, H.-W.; Park, B.-S.; Lee, H.-S.; Hwang, E.-C.; Lee, Y.-D.; Liu, K.-H. Stereoselective metabolism of endosulfan by human liver microsomes and human cytochrome P450 isoforms. Drug Metab. Dispos. 2006, 34, 1090−1095. (9) Liu, K.-H.; Kim, J.-H. In vitro dermal penetration study of carbofuran, carbosulfan, and furathiocarb. Arch. Toxicol. 2003, 77, 255−260. (10) Mutch, E.; Williams, F. M. Diazinon, chlorpyrifos and parathion are metabolised by multiple cytochromes P450 in human liver. Toxicology 2006, 224, 22−32. (11) Godin, S. J.; Crow, J. A.; Scollon, E. J.; Hughes, M. F.; DeVito, M. J.; Ross, M. K. Identification of rat and human cytochrome P450 isoforms and a rat serum esterase that metabolize the pyrethroid insecticides deltamethrin and esfenvalerate. Drug Metab. Dispos. 2007, 35, 1664−1671. (12) Scollon, E. J.; Starr, J. M.; Godin, S. J.; DeVito, M. J.; Hughes, M. F. In vitro metabolism of pyrethroid pesticides by rat and human hepatic microsomes and cytochrome P450 isoforms. Drug Metab. Dispos. 2009, 37, 221−228. (13) Nishi, K.; Huang, H.; Kamita, S. G.; Kim, I. H.; Morisseau, C.; Hammock, B. D. Characterization of pyrethroid hydrolysis by the human liver carboxylesterases hCE-1 and hCE-2. Arch. Biochem. Biophys. 2006, 445, 115−123. (14) Crow, J. A.; Borazjani, A.; Potter, P. M.; Ross, M. K. Hydrolysis of pyrethroids by human and rat tissues: examination of intestinal, liver and serum carboxylesterases. Toxicol. Appl. Pharmacol. 2007, 221, 1− 12. (15) Soderlund, D. M.; Casida, J. E. Effects of pyrethroid structure on rates of hydrolysis and oxidation by mouse liver microsomal enzymes. Pestic. Biochem. Physiol. 1977, 7, 391−401. (16) Ross, M. K.; Borazjani, A.; Edwards, C. C.; Potter, P. M. Hydrolytic metabolism of pyrethroids by human and other mammalian carboxylesterases. Biochem. Pharmacol. 2006, 71, 657−669. (17) KOPAC. Flucetosulfuron. In Analytical Methods of Pesticides; Korea Pesticide Analytical Council, 2009; http//www.kopac.or.kr. (18) Lang, D. H.; Rettie, A. E.; Böcker, R. H. Identification of enzymes involved in the metabolism of atrazine, terbuthylazine,

Figure 5. Metabolic pathway for flucetosulfuron in human liver microsomes.

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AUTHOR INFORMATION

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ametryne, and terbutryne in human liver microsomes. Chem. Res. Toxicol. 1997, 10, 1037−1044. (19) Coleman, S.; Linderman, R.; Hodgson, E.; Rose, R. L. Comparative metabolism of chloroacetamide herbicides and selected metabolites in human and rat liver microsomes. Environ. Health Perspect. 2000, 108, 1151. (20) Abass, K.; Reponen, P.; Turpeinen, M.; Jalonen, J.; Pelkonen, O. Characterization of diuron N-demethylation by mammalian hepatic microsomes and cDNA-expressed human cytochrome P450 enzymes. Drug Metab. Dispos. 2007, 35, 1634−1641. (21) Kim, J.-H. Characterization of Pyribenzoxim Metabolizing Enzymes in Rat Liver Microsomes. J. Toxicol. Public Health 2006, 22, 1−8. (22) Parkinson, A. Biotransformation of Xenobiotics; McGraw-Hill: New York, 2001. (23) Ecobichon, D. Characterization of the esterases of canine serum. Can. J. Biochem. 1970, 48, 1359−1367. (24) Iatsimirskaia, E.; Tulebaev, S.; Storozhuk, E.; Utkin, I.; Smith, D.; Gerber, N.; Koudriakova, T. Metabolism of rifabutin in human enterocyte and liver microsomes: kinetic parameters, identification of enzyme systems, and drug interactions with macrolides and antifungal agents. Clin. Pharmacol. Ther. 1997, 61, 554−562. (25) Ali, B.; Kaur, S.; James, E. C.; Parmar, S. S. Identification and characterization of hepatic carboxylesterases hydrolyzing hydrocortisone esters. Biochem. Pharmacol. 1985, 34, 1881−1886. (26) Frasco, M. F.; Colletier, J. P.; Weik, M.; Carvalho, F.; Guilhermino, L.; Stojan, J.; Fournier, D. Mechanisms of cholinesterase inhibition by inorganic mercury. FEBS J. 2007, 274, 1849−1861. (27) Erdös, E. G.; Debay, C. R.; Westerman, M. P. Activation and inhibition of the arylesterase of human serum. Nature 1959, 184, 430. (28) Gonzalvo, M. C.; Gil, F.; Hernández, A. F.; Villanueva, E.; Pla, A. Inhibition of paraoxonase activity in human liver microsomes by exposure to EDTA, metals and mercurials. Chem.−Biol. Interact. 1997, 105, 169−179. (29) Gan, K. N.; Smolen, A.; Eckerson, H. W.; La Du, B. N. Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities. Drug Metab. Dispos. 1991, 19, 100−106. (30) Simeon, V.; Reiner, E.; Škrinjarić-Špoljar, M.; Krauthacker, B. Cholinesterases in rabbit serum. Gen. Pharmacol.: Vasc. Syst. 1988, 19, 849−853. (31) Reiner, E.; Pavković, E.; Radić, Z.; Simeon, V. Differentiation of esterases reacting with organophosphorus compounds. Chem.−Biol. Interact. 1993, 87, 77−83. (32) Augustinsson, K.-B. Electrophoretic separation and classification of blood plasma esterases. Nature 1958, 181, 1786−1789. (33) Devonshire, A. L.; Moores, G. D. A carboxylesterase with broad substrate specificity causes organophosphorus, carbamate and pyrethroid resistance in peach-potato aphids (Myzus persicae). Pestic. Biochem. Physiol. 1982, 18, 235−246. (34) Stewart, D. J.; Inaba, T.; Tang, B. K.; Kalow, W. Hydrolysis of cocaine in human plasma by cholinesterase. Life Sci. 1977, 20, 1557− 1563. (35) Lockridge, O.; Mottershaw-Jackson, N.; Eckerson, H. W.; La Du, B. N. Hydrolysis of diacetylmorphine (heroin) by human serum cholinesterase. J. Pharmacol. Exp. Ther. 1980, 215, 1−8.

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In Vitro Metabolism of Flucetosulfuron by Human Liver Microsomes

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