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Chem. Res. Toxicol. 2002, 15, 1158-1165
Neonicotinoid Insecticides: Reduction and Cleavage of Imidacloprid Nitroimine Substituent by Liver Microsomal and Cytosolic Enzymes Daniel A. Schulz-Jander,† William M. Leimkuehler,‡ and John E. Casida*,† Environmental Chemistry and Toxicology Laboratory, Department of Environmental Science, Policy and Management, University of California, Berkeley, California 94720-3112, and Bayer Research Park, Stillwell, Kansas 66085 Received May 8, 2002
The major insecticide imidacloprid (IMI) is known to be metabolized by human cytochrome P450 3A4 with NADPH by imidazolidine hydroxylation and dehydrogenation to give 5-hydroxyimidacloprid and the olefin, respectively, and by nitroimine reduction and cleavage to yield the nitrosoimine, guanidine, and urea derivatives. More extensive metabolism by human or rabbit liver microsomes with NADPH or rabbit liver cytosol without added cofactor reduces the IMI N-nitro group to an N-amino substituent, i.e., the corresponding hydrazone. A major metabolite on incubation of IMI in the human microsome-NADPH system is tentatively assigned by LC/MS as a 1,2,4-triazol-3-one derived from the hydrazone; the same product is obtained on reaction of the hydrazone with ethyl chloroformate. The hydrazone and proposed triazolone are considered here together (referred to as the hydrazone) for quantitation. Only a portion of the microsomal reduction and cleavage of the nitroimine substituent is attributable to a CYP450 enzyme. The cytosolic enzyme conversion to the hydrazone is inhibited by added cofactors (NAD > NADH > NADP > NADPH) and enhanced by an argon instead of an air atmosphere. The responsible cytosolic enzyme(s) does not appear to be DT-diaphorase (which is inhibited by several neonicotinoids), aldose reductase, aldehyde reductase, or xanthine oxidase. However, the cytosolic metabolism of IMI is inhibited by several aldo-keto-reductase inhibitors (i.e., alrestatin, EBPC, Ponalrestat, phenobarbital, and quercetin). Other neonicotinoids with nitroimine, nitrosoimine, and nitromethylene substituents are probably also metabolized by “neonicotinoid nitro reductase(s)” since they serve as competitive substrates for [3H]IMI metabolism.
Introduction The discovery of insecticidal nitromethylene heterocycles (1) and structural modifications to enhance their potency and stability (2, 3) opened the neonicotinoid era in pest insect control (4, 5). Imidacloprid (IMI)1 was the first major neonicotinoid (2, 3) and serves as the standard with which all others are compared. It has outstanding activity and safety as a plant systemic insecticide (6, 7) and flea control agent for cats and dogs (8, 9). These properties result from combining three moieties, i.e., chloropyridinylmethyl, imidazolidine, and nitroimine (Figure 1). In other commercial chloropyridinylmethyl neonicotinoids, the nitroimine group is replaced by nitromethylene (nitenpyram) or cyanoimine (acetamiprid and thiacloprid) (4, 10). These neonicotinoids act as agonists at the nicotinic acetylcholine receptor (nAChR) at much lower concentrations in insects than in mammals (4, 6, 10, 11). This target site selectivity is a major factor in the favorable toxicological properties of the neonicotinoids. * To whom correspondence should be addressed. Phone: (510) 6425424. Fax: (510) 642-6497. E-mail:
[email protected]. † University of California. ‡ Bayer Research Park. 1 Abbreviations: CYP450, cytochrome P450; DCPIP, 2,6-dichlorophenolindophenol; IMI, imidacloprid; LC, liquid chromatography; MS, mass spectrometry; TFA, trifluoroacetic acid; THF, tetrahydrofuran; tR, retention time.
Figure 1. Partial pathways for imidacloprid metabolism by modification of the imidazolidine (top row) or nitroimine (bottom row) moiety. The olefin and urea are in trace amounts and the dihydroxy compound is not detected as a metabolite in the systems studied here. Asterisk designates position of tritium labeling in chloropyridinylmethyl (R) moiety. Additional metabolites not observed in the present study are formed by hydroxylation at the labeled methylene substituent leading to loss of the label and cleavage of the two heterocyclic moieties.
Cytochrome P450 (CYP450) enzymes metabolize IMI at the imidazolidine moiety forming 5-hydroxy and olefin derivatives and reduce the nitroimine substituent giving
10.1021/tx0200360 CCC: $22.00 © 2002 American Chemical Society Published on Web 08/08/2002
Enzymatic Metabolism of Imidacloprid
the nitrosoimine, guanidine, and urea metabolites (12) (Figure 1). There are also other sites of metabolic attack and sequential reactions leading overall to very complex pathways (13, 14). The CYP450 enzymes account for only a portion of this metabolism as became evident on incubating [3H]IMI with individual recombinant CYP450 isozymes (12) as compared to microsomal and cytosolic systems (this study). Nitro substituents are known to be reduced by a large number of oxido-reductases, e.g., DTdiaphorase, aldose reductase, aldehyde reductase, and xanthine oxidase (15), which have not been examined with neonicotinoid insecticides. The present investigation considers the fate of [3H]IMI in human and rabbit liver microsomal systems and rabbit cytosol with particular emphasis on conversion of the nitroimine substituent to the corresponding nitrosoimine, guanidine, and hydrazone derivatives (Figure 1).
Materials and Methods Neonicotinoids, Related Chemicals, and Enzyme Inhibitors. The neonicotinoids and related chemicals, unless indicated otherwise, were available from previous studies in the Berkeley laboratory. Four of the compounds are commercial insecticides, i.e., IMI, acetamiprid, nitenpyram, and thiacloprid. [3H]IMI was prepared according to Latli et al. (16) with a radiochemical purity of >98%. Metabolites and analogues were provided by Bayer (5-hydroxy-IMI, 4,5-dihydroxy-IMI and the hydrazone derivative of IMI) (John Murphy, Dietary Exposure Group, Bayer Research Park, Stillwell, KS) or synthesized in the Berkeley laboratory (guanidine, nitrosoimine, urea, and olefin). Candidate enzyme inhibitors were obtained from SigmaAldrich (St. Louis, MO) except for 1,3-dioxo-1H-benz[d,e]isoquinoline-2(3H)-acetic acid (alrestatin), ethyl 1-benzyl-3-hydroxy2(5H)-oxopyrrole-4-carboxylate (EBPC), and [3-(4-bromo-2fluorobenzyl)-4-oxo-3H-phthalazin-1-yl]acetic acid (Ponalrestat) from Tocris Cookson Inc. (Ballwin, MO). Enzyme Preparations and Incubations. Pooled human liver microsomes were from Gentest (Woburn, MA). To prepare rabbit liver microsomes and cytosol, a 20% (w/v) homogenate in phosphate buffer (100 mM, pH 7.4) was centrifuged at 10000g for 20 min. The supernatant fraction was centrifuged at 100000g for 1 h to obtain the cytosol. The microsome pellet was washed once with phosphate buffer by centrifugation at 100000g for 1 h and resuspended in phosphate buffer. Protein levels were determined by the bicinchoninic acid assay from Pierce (Rockford, IL). All enzymes were stored frozen at -80 °C until used. Incubation mixtures unless indicated otherwise contained the enzyme (protein levels of 2.5, 1.8, and 3.0 mg for human and rabbit liver microsomes and rabbit liver cytosol, respectively), NADPH (0 or 1.5 mM) and [3H]IMI (119 ng, 3 × 106 dpm, 3.0 µM) in phosphate buffer as above (155 µL). Incubations were in plastic Eppendorf tubes (500 µL) for 120 min at 37 °C in air or argon atmosphere. For introducing argon, the ice-cold microsomal preparation or frozen (-80 °C) cytosolic incubation mixture was placed in a glass vial which was closed with a rubber septum. The vial was then evacuated (0.05 mmHg) and purged with argon several times (during the 5 min required at room temperature the cytosolic preparation completely melted). TLC Analysis of [3H]IMI Metabolites. Analysis involved terminating the enzymatic reaction, recovery of organoextractable compounds, two-dimensional TLC, autoradiography of the chromatogram, and scintillation counting of residual [3H]IMI and individual labeled metabolites and fractions. More specifically, the enzymatic reaction (155 µL) after appropriate incubation time was stopped by addition of acetonitrile (7.5 µL) and frozen at -80 °C until used for extraction. The thawed samples were extracted four times with organic solvents in the original Eppendorf tubes, each time with vortexing for 5 min and centrifuging for 3 min at 16000g. After centrifugation, the organic layer was transferred into a glass vial. The first
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1159 extraction involved dichloromethane (300 µL), and then 20% NaOH (5 µL) was added followed by two extraction cycles with chloroform-acetonitrile (5:2) (each 300 µL). The latter step was included to recover basic compounds. Finally, concentrated HCl (10 µL) was added to the separated aqueous fraction followed by a fourth extraction with chloroform-acetonitrile as above. The combined extracts were dried over anhydrous Na2SO4 and an aliquot thereof concentrated with a stream of nitrogen and spotted on a silica gel TLC plate (Polygram Sil G/UV254, 20 × 20 cm, Machery-Nagel, Du¨ren, Germany) for development with tetrahydrofuran (THF)/hexane/acetone/water (46.5:25:25:3.5 v/v) in the first dimension and chloroform/acetone/trifluoroacetic acid (TFA) (65:25:10 v/v) in the second dimension. This system was specifically optimized for separation not only of the standards but also of the 3H-labeled metabolites. Product identification by cochromatography involved spotting a mixture of labeled metabolites with unlabeled standard (330 µg, depending on the sensitivity of detection under UV light). After developing the chromatograms, the position of the standard was marked under UV light by circling with a pencil. The plates were then sprayed with EN3HANCE (NEN Life Science Products, Boston, MA) and exposed to a tritium-sensitive film (Hyperfilm MP, RPN1675K, Amersham Pharmacia Biotech, Little Chalfont, U.K.) at -80 °C for 7 days. The spots on the film were marked on the TLC plate and each marked spot was cut out from the chromatogram and placed directly into scintillation fluid (Safety-Solve, Research Products International Corp., Mount Prospect, IL). After shaking the scintillation vials for several hours, they were counted. The protein precipitate in the aqueous phase was washed twice with ice-cold methanol (300 µL) which was combined with the remaining aqueous fraction and counted. The washed protein-bound fraction was digested with Soluene 350 (Packard, Meriden, CT) for 3 h at 60 °C, decolorized with hydrogen peroxide (100 µL, 30%) for 1 h at 60 °C, and after cooling, scintillation fluid (Hionic Fluor, Packard) was added for counting. That portion of the original 3H label not recovered in the designated fractions is referred to as loss. Data are means ( SE for three replicates. Liquid Chromatography/Mass Spectrometry (LC/MS) Identification of [3H]IMI Metabolites. Human liver microsomes (2.5 mg of protein) were incubated with [3H]IMI (395 ng, 3 × 106 dpm, 10 µM) and NADPH (0 or 1.5 mM) in pH 7.4 100 mM phosphate buffer (155 µL) for 120 min at 37 °C in air. Two sets were analyzed by different workup procedures. One involved deproteination by adding acetonitrile (310 µL). The supernatant was concentrated and acetic acid added to give finally acetonitrile/water (3:7) with 0.l% acetic acid. The other used the standard extraction procedure involving treatments with NaOH and HCl and extractions with dichloromethane and chloroform/acetonitrile (5:2). LC used a LUNA C8 column (250 × 4.6 mm, 5 µm) from Phenomenex (Torrance, CA). A gradient elution profile started with 5% methanol in 0.1% formic acid maintained for 5 min followed by a linear gradient arriving at 100% methanol at 30 min with a flow rate of 0.8 mL/min. A 100 µL sample was injected onto the column. The flow was split with 20% of the eluant directed to the MS and 80% to a flow through radioactivity detector (50 µL “Star” flowcell, Raytest Co, Wilmington, NC) set to determine 3H. MS used a ThermoFinnigan TSQ 7000 triple quadrupole instrument (Thermo Finnigan, San Jose, CA) with an atmospheric pressure ionization interface and electrospray ionization. All samples were monitored full scan (150-600 amu, 1 s scan time), and when appropriate analyzed also using MS/MS monitoring the product ions of the [M + 1]+ molecular ions. Product spectra were generated with a collision voltage of -20 eV using helium as the collision gas at a pressure of approximately 2.3 mTorr. DT-Diaphorase. DT-diaphorase (EC 1.6.99.2) was assayed as the dicoumarol-sensitive portion of the 2,6-dichlorophenolindophenol (DCPIP)-reducing enzyme (17, 18) of rabbit liver cytosol. Each assay contained cytosolic protein (90 µg), bovine
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serum albumin (140 µg), NADH (100 µM), and DCPIP (400 µM) in phosphate buffer (100 mM, pH 7.4, 220 µL), monitoring the absorbance change at 600 nm for 180 s with a microtiter plate reader. Activity was linear with time and enzyme concentration (10 µmol/min/mg of protein), required a reducing cofactor and was equal with NADH or NADPH. Kinetic analysis of the inhibition by IMI (220 µM) involved varying the NADH concentration (80-280 µM) or DCPIP level (36-127 µM). Although NADH was used in all studies reported here, the inhibitory potency of IMI was the same with either cofactor (data not shown). An acetone powder was prepared (17) and examined for DT-diaphorase and [3H]IMI-metabolizing activities. Aldehyde and Aldose Reductases. Recombinant human aldehyde reductase (EC 1.1.1.2) and aldose reductase (EC 1.1.1.21) (19) were supplied by Kurt Bohren (Department of Pediatrics, Baylor College of Medicine, Houston, TX). A suspension of recombinant enzyme (5-50 µg protein) in phosphate buffer (pH 7.4, 100 mM) was mixed with bovine serum albumin (0 or 1.8 mg), [3H]IMI (119 ng, 3.65 µM), and NADPH (1.0 mM). The reaction mixture (155 µL) was purged with argon (as for the microsomes above), followed by incubation for 2 h at 37 °C. Extraction and metabolite analysis were carried out in the usual manner.
Results Metabolite Fractionation and Quantitation. (1) Organoextractable Fraction. The organoextractable metabolites were separated by 2D TLC for quantitative analysis and cochromatography with unlabeled standards. The extract was also analyzed by LC/MS and compared with the deproteinated incubation mixture. Three types of evidence indicate that the standards and metabolites analyzed were stable under the extraction and analysis conditions. First, control experiments established by 2D TLC that the unlabeled standards underwent no decomposition during workup and analysis. Second, the combined neutral/basic/acidic extract analyzed by 2D-TLC gave higher recoveries but no loss or addition of 3H-labeled products in the metabolite mixture compared with the initial neutral extract (experiment with cytosol, NADPH, and argon described later). The organoextractable fraction included several minor unidentified products designated as “apolar metabolites” which are not individually tabulated. Third, the same products were detected by LC/MS analysis of the organoextractable fraction compared with the deproteinated incubation mixture. However, there was one exception (discussed later) where a major metabolite detected by LC/MS was not evident by TLC due to either decomposition during workup and chromatography or more likely incomplete resolution for example from the hydrazone on TLC. (2) Other Fractions. Metabolites not recovered by extraction with organic solvents were designated the aqueous, protein bound, and loss fractions. For quantification, the latter three fractions were combined with the “apolar metabolites” as above and designated “other” fraction, i.e., remaining or unidentified radioactivity. For each type of enzyme preparation, the ratio of these four fractions was not discernably altered by the variables examined. Throughout the studies, the percentage of “other” fraction increased with the percentage of IMI undergoing metabolism. Metabolite Identification. (1) Thin Layer Cochromatography. Five metabolites of [3H]IMI in the human and rabbit liver microsomal and rabbit liver cytosolic systems were identified by 2D thin layer cochromatog-
Figure 2. Comparison of the metabolites of [3H]imidacloprid in four liver enzyme systems with NADPH in air. The figure for human CYP3A4 is from Schulz-Jander and Casida (12). The organoextractable products are separated by 2D TLC [first dimension with THF/hexane/acetone/water (46.5:25:25:3.5), and second with chloroform/acetone/TFA (65:25:10)] and visualized by autoradiography. Compound names refer to structures in Figure 1. 4,5-Dihydroxy-IMI chromatographed with 5-hydroxyIMI is of slightly lower Rf in the first dimension and slightly higher Rf in the second dimension, yielding overlapping spots.
raphy with authentic standards, i.e., the nitrosoimine and guanidine in each system, 5-hydroxy-IMI in microsomes only, and the urea in rabbit liver microsomes and cytosol; in addition the olefin was evident in more than trace amounts with CYP3A4 but not the other systems (Figure 2). The hydrazone (identified by chromatographic position) was the major metabolite in all systems considered here. Of the available standards, only the 4,5-dihydroxy derivative was not detected by TLC. (2) LC/MS. The metabolites of [3H]IMI from incubation with human microsomes alone or plus NADPH were examined by LC/MS. No metabolites were detected in samples without added NADPH. [3H]IMI and several metabolites were identified by comparison with unlabeled standards (IMI, guanidine, and hydrazone) giving the same retention time (tR) and full scan and product ion spectra (Table 1, Figures 3 and 4). These findings were applicable to the deproteinated incubation mixture analyzed directly and the organoextractable products using the standard fractionation procedure. Other metabolites in smaller amounts identified by LC/MS were 5-hydroxyIMI and the nitrosoimine. The olefin and urea were not detected by LC/MS. The observed MS and MS/MS data for IMI and its metabolites are given in Table 1 and the postulated MS fragmentation patterns are shown in Figure 4. In addition to the pseudomolecular ion, each compound except
Enzymatic Metabolism of Imidacloprid
Chem. Res. Toxicol., Vol. 15, No. 9, 2002 1161
Figure 3. LC chromatograms monitored by 3H detector or by full scan MS for the product ions of the [M + 1]+ pseudomolecular ions of masses (m/z) 211 (guanidine), 226 (hydrazone), 252 (proposed triazolone), and 256 (IMI).
the guanidine, hydrazone, and proposed triazolone gave a [M + CH3CN]+ adduct. The guanidine and triazolone gave the same fragments plus m/z 111 in the latter case. (3) Tentative Assignment of Triazolone Derivative. The principal IMI metabolite in the human liver microsome-NADPH system, as analyzed by LC/MS, was a derivatized hydrazone tentatively assigned as the 1,2,4triazol-3-one (Figure 1). This compound showed an almost identical fragmentation pattern to that of the guanidine, with the exception of the m/z 111 fragment, which was unique for this one metabolite (the hydrazone also did not form this fragment) (Table 1). These observations can be rationalized on the basis of a 1,2,4-triazol3-one moiety fragmenting as shown in Figure 4. This proposal is supported by a chemical derivatization reaction. An analogous conversion of a hydrazone to a triazolone is reported on treatment of 2-hydrazinobenzimidazole with ethyl chloroformate in ethanol (20). The IMI-derived hydrazone (8 mg) was therefore treated with 3 equiv of ethyl chloroformate in anhydrous ethanol (1 mL) for 96 h at 25 °C. LC/MS revealed the proposed triazolone and the unreacted hydrazone (about 10-15% each). The triazolone from this reaction was identical to the metabolite in LC tR and MS spectrum, but on attempted isolation, there was some reversion to the hydrazone. The triazolone was observed in the human
Figure 4. Postulated MS fragmentation patterns for the guanidine, proposed triazolone and hydrazone (see Table 1).
microsome-NADPH system by LC/MS but not TLC, presumably due to inadequate TLC separation from the hydrazone or less likely decomposition to the hydrazone during workup and analysis. In the quantitation studies below, the hydrazone and proposed triazolone are considered together and referred to as the hydrazone. Effect of Cofactor, Incubation Time, and Inhibitors on Human Liver Microsomal Metabolism (Table 2). The NADPH-dependent metabolism of [3H]IMI by pooled human liver microsomes yields 5-hydroxy-IMI, nitrosoimine, guanidine, hydrazone, and the “other” fraction but no olefin or urea. NADP, NADH, and NAD are less effective cofactors except for conversion to the “other” fraction. On varying the incubation time, the hydrazone and guanidine progressively increase in amount whereas 5-hydroxy-IMI and the nitrosoimine undergo little or no time-dependent change. [3H]IMI metabolism is strongly inhibited by CO and 5-hydroxy-IMI and less
Table 1. LC/MS and LC/MS/MS Data for Imidacloprid and Its Metabolites tR (min)
m/z
compd
standard
metabolite
[M + 1]+/[M + CH3CN]+
base peak
other product ion fragments
olefin hydrazone guanidine triazolone nitrosoimine 4,5-dihydroxy urea 5-hydroxy imidacloprid
12.6 12.7 13.0 13.9 16.3 16.7 16.9 17.1 18.6
n.d. 12.6 12.8 13.9 16.3 n.d. n.d. 17.2 18.5
209/250 226/211/252/240, 281 288, 329 212/253 272/313 256/297
126 126 126 209 209 207 128 225 209
83, 173 99, 100, 168, 190 84, 175, 194 84, 111, 126, 175, 194 84, 126, 128, 175 126, 169, 244 99, 126, 195 126, 144, 191 84, 128, 175
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Table 2. Effect of Cofactor, Incubation Time, and Inhibitors on [3H]IMI Metabolism by Human Liver Microsomal Enzymes in Air radioactivity (%, mean ( SE, n ) 3)a variable cofactorc none NADPH NADP NADH NAD incubation timed 5 min 15 min 30 min 60 min 120 min inhibitorse none CO 5-hydroxy-IMI ketoconazole methimazole n-octylamine 45 °Cf
IMI
5-hydroxy
nitrosoimine
guanidine
hydrazone
otherb
87.1 ( 2.7 61.1 ( 0.7 74.5 ( 4.8 75.7 ( 6.9 75.8 ( 6.7
0.4 ( 0.1 3.8 ( 0.5 1.2 ( 0.2 0.6 ( 0.1 0.7 ( 0.2
1.0 ( 0.1 1.2 ( 0.2 1.2 ( 0.1 1.1 ( 0.1 1.2 ( 0.1
0.8 ( 0.0 3.3 ( 0.2 1.7 ( 0.1 1.4 ( 0.2 1.2 ( 0.3
