Chem. Res. Toxicol. 2006, 19, 1549-1556
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Unique and Common Metabolites of Thiamethoxam, Clothianidin, and Dinotefuran in Mice Kevin A. Ford and John E. Casida* EnVironmental Chemistry and Toxicology Laboratory, Department of EnVironmental Science, Policy and Management, UniVersity of California, Berkeley, California 94720-3112 ReceiVed August 9, 2006
The established neonicotinoid insecticides have chloropyridylmethyl (imidacloprid, thiacloprid, acetamiprid, and nitenpyram), chlorothiazolylmethyl (thiamethoxam or TMX and clothianidin or CLO) or tetrahydrofuranylmethyl (dinotefuran or DIN) substituents. We recently reported the metabolic fate of the chloropyridylmethyl neonicotinoids in mice as the first half of a comparative study that now considers the chlorothiazolylmethyl and tetrahydrofuranylmethyl analogues. TMX, CLO, two desmethyl derivatives (TMX-dm and CLO-dm), and DIN were administered ip to mice at 20 mg/kg for characterization of metabolites and pharmacokinetic analysis of brain, liver, plasma, and urine by HPLC/DAD and LC/ MSD. Each compound is excreted 19-55% unmetabolized in urine within 24 h, and tissue residues are largely dissipated by 4 h. Thirty-seven metabolites of TMX, TMX-dm, CLO, and CLO-dm are identified by comparison with synthetic standards or their structures are proposed by molecular weights and 35Cl: 37 Cl ratios often supplemented by previous reports or sequence studies in which intermediates are readministered. A facile reaction sequence involves TMX f TMX-dm or CLO f CLO-dm. CLO-dm, reported to be a contributor to TMX hepatocarcinogenesis in mice, is unexpectedly remethylated in part to CLO in brain. The nitrosoguanidine, aminoguanidine, and urea derivatives of the parent compounds are detected in the tissues and methylnitroguanidine, methylguanidine, and nitroguanidine in the urine. Chlorothiazolecarboxaldehyde from oxidative cleavage of TMX and CLO is quite persistent in brain, liver, and particularly plasma compared with chloropyridinecarboxaldehyde and tetrahydrofurancarboxaldehyde from the other neonicotinoids. Chlorothiazolecarboxylic acid is conjugated with glycine or glucuronic acid or converted to S-methyl and mercapturate derivatives. DIN metabolism involves nitro reduction, N-demethylation, N-methylene hydroxylation, and amine cleavage, and tetrahydrofuranylmethyl hydroxylation at the 2-, 4-, and 5-positions giving 29 tentatively identified metabolites. The diversity of biodegradable sites and multiple pathways insures against parent compound accumulation but provides intermediates reported to be active as nicotinic agonists and inducible nitric oxide synthase inhibitors. Introduction Neonicotinoid insecticides combine outstanding potency and safety attributable to higher affinity for insect than mammalian nicotinic acetylcholine receptors and multiple biodegradable substituents (1-4). The first major neonicotinoids were chloropyridylmethyl compounds [imidacloprid (IMI1), nitenpyram, thiacloprid (THI), and acetamiprid] (5) followed soon thereafter by the chlorothiazolylmethyl insecticides [thiamethoxam (TMX) (6) and clothianidin (CLO) (7)] and a tetrahydrofuranylmethyl analogue [dinotefuran (DIN) (8)] (Figure 1). Metabolism studies in rats and usually also in goats and hens are required for pesticide registration and establishing tolerance values (9-15). Mice have also been used in defining the comparative metabolism of the four commercial chloropyridylmethyl neonicotinoids * To whom correspondence should be addressed. Phone: (510) 6425424. Fax: (510) 642-6497. E-mail:
[email protected]. 1 Abbreviations: AOX, aldehyde oxidase; CLO, clothianidin; CLO-dm, desmethyl-clothianidin; CTM, chlorothiazolylmethyl; CTM-a, chlorothiazolecarboxaldehyde; DAD, diode array detector; DIN, dinotefuran; DINdm, desmethyl-dinotefuran; dm, desmethyl; ESI, electrospray ionization; gluc, glucuronide; GSH, glutathione; IMI, imidacloprid; IS, internal standard; mAU, milliabsorbance units; MSD, mass selective detector; nAChR, nicotinic acetylcholine receptor; NG, nitroguanidine; NH, guanidine or imine derivative; NNH2, aminoguanidine derivative; NNO, nitrosoguanidine derivative; ppm equiv, ppm equivalents based on the absorbance at 254 nm and recovery values of the parent compound; SIM, selected ion monitoring; TFA, trifluoroacetic acid; THI, thiacloprid; TMX, thiamethoxam; TMX-dm, desmethyl-thiamethoxam; tri, methyltriazinone derivative.
Figure 1. Thiamethoxam, clothianidin, their desmethyl derivatives and dinotefuran.
(16). The chlorothiazolylmethyl and tetrahydrofuranylmethyl neonicotinoids have not been studied in mice or in other mammals in such a way as to allow direct comparison of the effect of diverse substituents on tissue residues and persistence, metabolic intermediates, and excretion products under a standard set of conditions.
10.1021/tx0601859 CCC: $33.50 © 2006 American Chemical Society Published on Web 10/27/2006
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Figure 2. Partial metabolic pathways for thiamethoxam and clothianidin involving N-methyl and O-methylene hydroxylation and nitro reduction.
Figure 3. Nitroguanidine (NG) moiety cleavage products.
The metabolism of TMX and CLO is closely related with CLO serving as a principal intermediate in a major pathway for TMX in mammals (9), insects (17), and plants (17). Metabolites in mouse liver are of special interest because TMX and TMX-dm but not CLO increase the incidence of liver tumors (18-20). DIN and CLO differ only in the tetrahydrofuranylmethyl moiety replacing the chlorothiazolylmethyl substituent leading to many common metabolites and also several unique to each compound. Pharmacokinetic data are of particular interest relative to compounds and levels in brain, liver, and plasma and how they relate to toxicity parameters. This study establishes or proposes the structures of 37 metabolites in the TMX, TMX-dm, and CLO series and 29 for DIN, many of which are novel and potential contributors in the activation and detoxification pathways.
Materials and Methods Chemicals. TMX was from Chem Service (West Chester, PA), CLO from Syngenta (Basel, Switzerland), and DIN from Valent (Dublin, CA). The metabolites of TMX and CLO considered include uncleaved products designated by abbreviations (e.g., TMX-dm and CLO-NNO) (Figure 2), nitroguanidine (NG) moiety derivatives by capital letters (NG-A to NG-G) (Figure 3), and chlorothiazolylmethyl (CTM)-derived compounds by small bold letters (CTM-a to CTM-j) (Figure 4). Sources for TMX, CLO, and DIN metabolites available as standards are given in the appropriate tables.
Figure 4. Chlorothiazolylmethyl (CTM) moiety cleavage products.
Animal Studies. Following our standard procedure (16), the test compounds were administered at 20 mg/kg as Me2SO solutions (1 µL/g mouse weight) to male albino Swiss-Webster mice (25-30 g) from Harlan Laboratories (Indianapolis, IN) using isoflurane for anesthesia. The ip route minimized absorption differences, and none of the compounds gave poisoning signs. Brain, plasma, and liver were generally sampled at 15, 30, 60, 120, and 240 min after treatment. In separate studies, tissues, urine, and feces were collected at 24 h. Tissues were analyzed fresh and excreta within 2 days at -80 °C. Sample Preparation, Analysis, and Metabolite Identification. For tissue extraction, whole brain (350-375 mg), liver (750-800 mg), or plasma (100 µL) was placed in acetonitrile (5 mL) containing NaCl (250 mg) and internal standard (IS) (1 ng/mg tissue) in 75:25:0.1 acetonitrile/water/trifluoroacetic acid (TFA). Urine (100-200 µL) and feces (100 mg) were treated the same way but without IS. Homogenates were prepared with a sonic dismembrator (Fischer Scientific, Pittsburgh, PA) followed by mixing with a vortex. The acetonitrile extract after centrifugation at 2000g for 15 min was reduced to dryness using a Savant SVC 200H centrifugal evaporator (Farmingdale, NY). Addition of 75: 25:0.1 acetonitrile/water/TFA, sonication, and filtration preceded
Metabolites of TMX, CLO, and DIN in Mice
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Table 1. Metabolites Unique to Thiamethoxam, Desmethyl-Thiamethoxam, Clothianidin, and Desmethyl-Clothianidin tR (min) LC/MSD
identification criteriaa,b
tissue or urinec
15.3 11.2 10.8 -
15.3 12.2 3.0 3.4 10.6 6.3 3.2
A D B A A D D
b, l, p, u b, l, p, u l b, l, u l, p, u l, u l, u
277.7 261.7 247.7 299.7 232.7 233.7 146.1 101.1
Unique to TMX-dm 19.7 14.2 -
19.7 13.8 6.0 10.2 3.1 12.3 7.3 3.4
A D D D A D D D
b, l, p, u b, l, p, u l, p l, u b, l, p, u l, u l, u l
CLO CLO-NNO CLO-NNH2 CLO-tri CLO-NH CLO-urea NG-E NG-F
249.7 233.7 219.7 271.7 204.7 205.7 118.1 73.1
17.5 12.1 10.1 9.6 12.9 -
17.5 12.1 8.5 3.8 12.9 3.1 2.1
A B B F A A A A
b, l, p, u b, l, p, u l, u b, l, u b, l, u l, u l, p
CLO-dm CLO-dm-NNO CLO-dm-NNH2 CLO-dm-tri CLO-dm-NH CLO-dm-urea NG-G
235.7 219.7 205.7 257.7 190.7 191.6 104.1
16.1 3.3
15.9 11.4 9.3 9.7 3.6 14.1 2.0
A D D D C C A
b, l, p, u b, l, p, u l, p, u l b, l, u b, l, p, u l, u
compound
MW
HPLC/DAD
TMX TMX-NNO TMX-NNH2 TMX-NH TMX-urea NG-A NG-B
291.7 275.7 261.7 246.7 247.7 160.1 115.1
TMX-dm TMX-dm-NNO TMX-dm-NNH2 TMX-dm-tri TMX-dm-NH TMX-dm-urea NG-C NG-D
Unique to TMX
Unique to CLO
Unique to CLO-dm
a Identification criteria are as follows (for previously reported mammalian metabolites, see references 9, 10, 14, 15, and 18). (A) Identical to synthetic standard of parent compound or metabolite by MW, 35Cl:37Cl ratio, and tR and previously reported as a metabolite. (B) Identical to synthetic standard by MW, 35Cl:37Cl ratio, and tR but not previously reported as a metabolite. (C) Tentative identification based on MW, 35Cl:37Cl ratio (where applicable), and reasonable tR and reported as a metabolite although not available here as a synthetic standard. (D) Tentative identification based on MW, 35Cl:37Cl ratio (where applicable), and reasonable tR but not previously reported as a metabolite and not available here as a synthetic standard. (E) Available as a synthetic standard but not observed as a metabolite (none in this category). (F) Candidate metabolite or intermediate not observed and not available here as synthetic standard. b Sources of candidate metabolites: NG-E, NG-F, and NG-G from Sigma-Aldrich. c Abbreviations: b, brain; l, liver; p, plasma; and u, urine.
HPLC analysis on a Luna C-18 column with a precolumn filter. The Hewlett-Packard model 1050 liquid chromatograph equipped with a diode array detector (DAD) used a gradient of acetonitrile/ water containing 0.1% TFA beginning with 5% and increasing to 80% acetonitrile over 30 min at 1 mL/min. A final 10 min wash with 5% acetonitrile eluted interfering materials. Absorbance measurements were at 254 nm. Tissue levels were determined by comparing peak areas for the neonicotinoid analytes with the IS. Each neonicotinoid gave a recovery of 80% or greater. The IS was DIN (tR 10.2 min) for mice treated with TMX, TMX-dm, and CLO (see Table 1 for tR values) and THI (tR 23 min) for DIN-treated mice. The sensitivity of the method is indicated by the peak areas [milliabsorbance multiplied by time (mAU‚s)] with direct online injection of 0.1 µg of neonicotinoid (993 for TMX, 2079 for TMXdm, 790 for CLO, and 285 for DIN) or of three carboxaldehyde cleavage products (280 for chlorothiazole, 301 for chloropyridine, and 78 for tetrahydrofuran). Parent neonicotinoids or carboxaldehydes as administered are reported as ppm residue levels. Metabolite levels are given as ppm equivalent (ppm equiv) based on the absorbances at 254 nm and recovery values of the parent compounds. Levels of parent neonicotinoids in urine (percent of administered dose) were determined by comparing samples from treated mice with the same aliquot of the corresponding control urine fortified with the administered neonicotinoid. The Hewlett-Packard 1100 LC/mass selective detector (MSD) system (m/z 10-1500) was used with the same column as for HPLC/DAD except TFA was replaced by 0.1% formic acid. The
ions used for selected ion monitoring (SIM) gave a strong signal with positive mode electrospray ionization (ESI). Prominent HPLC/ DAD interfering peaks in urine and feces extracts (16) led to careful LC/ MSD comparison of all samples from treatments with the corresponding controls. Criteria for identifying compounds or proposing structures are given in Tables 1-3 and the Results.
Results Chromatography. The standard HPLC/DAD and LC/MSD conditions provide good metabolite separation and detection (Tables 1-3). This is illustrated for TMX, TMX-dm, CLO, and CLO-dm and their metabolites in brain and liver in Figure 5, which also gives a partial metabolic pathway. The simplest compound in this series is the last one, CLO-dm, which is a major peak in brain and liver from each neonicotinoid and surprisingly is remethylated in part to CLO in brain. CLO administration also yields CLO-NNO and CTM-a in brain plus CLO-NNH2, CLO-urea, and CTM-e in liver. TMX-dm additionally gives TMX-dm-NNO in brain and liver. Even more metabolites are found with TMX, which yields TMX-NNH2 in brain and liver and TMX-NH in liver. Identification. Only metabolites absorbing at 254 nm were detected in the HPLC/DAD studies, while additional ones were analyzed by LC/MSD. Metabolites were recognized by new
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Table 2. Chlorothiazolylmethyl (CTM)-Derived Metabolites Common to Thiamethoxam and Clothianidin
compd
MW
tR (min)a LC/MSD
CTM-a CTM-b CTM-c CTM-d CTM-e CTM-f CTM-g CTM-h CTM-i CTM-j
147.6 149.6 163.6 339.7 290.3 175.2 220.6 232.3 148.6 190.6
18.1 3.8 20.2 13.8 20.7 10.9 15.2 3.8 15.7
identification criteriab,c
tissue or urined
B F A D D A B B C D
b, l, p b, l, u u l, u l l, u l b, l, p, u l, u
a t (min) for compounds quantified by HPLC/DAD: CTM-a, 18.7; R CTM-f, 20.7. b Identification criteria as in footnote a of Table 1 (for previously reported metabolites, see refs 14 and 15). c Sources of candidate metabolites: CTM-a from ASDI (Newark, DE); CTM-c from SigmaAldrich; CTM-f, CTM-g, and CTM-h synthesized by Karl Fisher of this laboratory. d Abbreviations: b, brain; l, liver; p, plasma; and u, urine.
peaks consistently present in the treated samples compared with the control. Six criteria were used for metabolite identification or tentative proposal of structure (Table 1): (A) identical to synthetic standard by LC/MSD ([M+] and 35Cl:37Cl ratio (where applicable) and tR) and previously reported as a metabolite (see Tables 1-3); (B) same as A, but not previously reported as a metabolite; (C) previously reported metabolite (see Table 1-3) not available as synthetic standard but observed here by LC/ MSD ([M+] and 35Cl:37Cl ratio (when chlorine present) and reasonable tR); (D) new metabolite not available as synthetic standard but observed here by LC/MSD ([M+] and 35Cl:37Cl ratio (when chlorine present) and reasonable tR); (E) available as synthetic standard but not detected in tissues or urine by LC/ MSD (tR and [M+]); (F) candidate metabolite or intermediate not observed and no synthetic standard. Metabolites of TMX, TMX-dm, CLO, and CLO-dm. These four compounds have several metabolites in common because O-methylene hydroxylation results in metabolic oxadiazinane cleavage to CLO and N-methyl hydroxylation yields TMX-dm from TMX and CLO-dm from CLO (Figure 2). TMX, TMX-dm, CLO, and CLO-dm each in turn can form NNO, NNH2, NH, and urea derivatives. Three of the aminoguanidine metabolites (TMX-dm-NNH2, CLO-NNH2, and CLO-dmNNH2) also proceed to add pyruvate to give the methyltriazinones (e.g., CLO-tri). Thirteen of the 19 theoretical metabolites in this sequence initiated by nitro reduction are observed in liver, and many of them are also present in brain, plasma, and urine. Four nitroguanidine moiety cleavage products are unique to TMX (NG-A and NG-B) and TMX-dm (NG-C and NG-D) as formed by methylene hydroxylation at the NCH2-aryl linkage along with demethylation and N-NO2 cleavage (Figure 3). Three additional ones (NG-E, NG-F, and NG-G) are formed from TMX and CLO (Figure 3) and also from DIN (discussed later). The principal mechanism to cleave the CTM moiety is N-methylene hydroxylation to yield aldehyde CTM-a, which is observed along with smaller amounts of mercapturate CTM-e (Figure 4). Alcohol CTM-b is not found. Acid CTM-c yields glucuronide (gluc) CTM-d, glycine conjugate CTM-g, and [via the glutathione (GSH) and mercapturate derivatives] methylthio compound CTM-f and its glycine conjugate CTM-h. Alternative cleavage yields the chlorothiazolylmethylamine (CTM-i) and its N-acetyl derivative (CTM-j). Pharmacokinetics of TMX, TMX-dm, CLO, and CLOdm. TMX, CLO, and their desmethyl derivatives were administered ip at 20 mg/kg and their levels compared in brain, liver,
Table 3. Metabolites Unique to Dinotefuran
compd
MW
tR (min) LC/MSDa
identification criteriab,c
tissue or urined
DIN DIN-NNO DIN-NNH2 DIN-tri DIN-NH DIN-urea DIN-dm DIN-dm-NNO DIN-dm-NNH2 DIN-dm-tri DIN-dm-NH
Tetrahydrofuranylmethyl Intact 202.2 10.2 A 186.2 7.3 D 172.2 4.1 D 224.3 11.0 D 157.2 2.8 A 158.2 11.4 A 188.2 14.3 D 172.2 6.9 D 158.2 3.8 D 210.2 9.8 D 143.2 1.3 D
DIN-2-OH DIN-a DIN-b DIN-c DIN-d DIN-e DIN-f
218.2 218.2 204.2 260.3 246.2 173.2 159.2
From DIN-2-OH 10.9 8.6 11.4 8.4 6.4 1.3
F C C C C C C
l, u l, u l, u u l, u l, u
DIN-4-OH
218.2
From DIN-4-OH -
F
-
218.2 234.2 220.2 218.2 204.2 175.2
From DIN-5-OH 14.2 12.3 11.6 3.5 4.0
F C D D D D
u u u u u
DIN-5-OH DIN-g DIN-h DIN-i DIN-j DIN-k DIN-l DIN-m DIN-n DIN-o DIN-p DIN-q DIN-r DIN-s
From Tetrahydrofuranylmethyl Moiety 100.1 3.8 E 102.1 11.1 E 116.1 12.6 B 173.2 18.6 D 132.1 1.0 D 189.2 0.8 D 101.2 3.4 D 143.2 3.1 D
b, l, p, u b, l, p, u l, p l, u b, l, p, u b, l, u b, l, p, u b, l, p, u l, p u b, l, u
p l b, l, p, u l, u l, u l l, u
a t (min) for compounds quantified by HPLC/DAD: DIN, 10.2; DINR NNO, 7.3; DIN-n, 12.6. Additionally, standards were available for DINNH, DIN-NNH2, DIN-l, and DIN-m, all of which were poorly detected at 254 nm. b Identification criteria as follows (for previously reported metabolites, see ref 12). (A) Identical to synthetic standard of parent compound or metabolite by MW and tR and previously reported as a metabolite. (B) Identical to synthetic standard by MW and tR but not previously reported as a metabolite. (C) Tentative identification based on MW, reasonable tR, and reported as a metabolite, although not available here as a synthetic standard. (D) Tentative identification based on MW and reasonable tR but not previously reported as a metabolite and not available here as a synthetic standard. (E) Available as a synthetic standard but not observed as a metabolite. (F) Candidate metabolite or intermediate not observed and not available here as a synthetic standard. c Sources of candidate metabolites: DIN-NH and DIN-urea synthesized by David Kanne of this laboratory; DINl, DIN-m, and DIN-n from Sigma-Aldrich. d Abbreviations: b, brain; l, liver; p, plasma; and u, urine.
and plasma after 15 to 240 min. The parent compound is present in larger amounts than the metabolites for at least the first 60 min with the single exception of CLO as a metabolite of CLOdm in brain (Figure 6). Major metabolites in tissues at 120 min are as follows: from TMXsTMX-dm and CTM-a; from TMXdmsCLO-dm and CTM-a; from CLOsCLO-dm and CLONNO; from CLO-dmsCTM-a (all tissues) and CLO (brain only). Interestingly CLO-dm is unusually persistent in tissues, and the level of CLO in brain equals or exceeds that of CLOdm at 120 and 240 min after CLO-dm treatment; also, the highest ppm values are observed, in each tissue tested, for CLOdm in comparison to the other compounds. The differences cannot be explained by variation in relative absorbance at 254
Metabolites of TMX, CLO, and DIN in Mice
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Figure 5. Thiamethoxam, desmethyl-thiamethoxam, clothianidin, desmethyl-clothianidin and their metabolites in brain and liver at 30 min posttreatment analyzed by HPLC/DAD. Diagrammatic representations (tracings) with metabolites as designated in Figures 2 and 4 and tR values given in Tables 1 and 2.
nm since that parameter was considered when the amounts (ppm) were calculated for each parent compound (Figure 6). Excretion of the parent TMX, TMX-dm, and CLO was similar for each compound with 19-27% in urine and 0.9-1.5% in feces within 24 h (Table 4). Other major urinary metabolites for TMX were TMX-dm, TMX-dm-NNO, CLO, CLO-urea, CTM-a, and CTM-c; those for TMX-dm were TMX-dm-NNO, CLO-dm, and CLO-urea; and for CLO were CLO-dm and CLOurea. Maximum tissue levels for the three neonicotinoids were 11-18 ppm in brain, 34-55 ppm in liver, and 12-15 ppm in plasma. The t1/2 values relative to the maximum levels were similar (30-55 min) for the three tissues and three compounds except for an apparent greater persistence of TMX-dm in liver (75 min). Chlorothiazolecarboxaldehyde (CTM-a) is easily detected in tissues of mice 30 min after ip treatment with TMX, TMX-dm, or CLO (Figure 5), but chloropyridinecarboxaldehyde and tetrahydrofurancarboxaldehyde are not found under the same conditions from the chloropyridylmethyl neonicotinoids and DIN, respectively (Ford and Casida, 2006; unpublished results), indicating that the chlorothiazole moiety confers considerably greater carboxaldehyde stability. This was confirmed by direct administration of the three carboxaldehydes and observing higher levels of CTM-a in brain, liver, and plasma throughout and still at 1.0 ppm in plasma 24 h after a 20 mg/kg ip dose (Table 5). Metabolites of DIN. DIN is readily metabolized by Ndemethylation, nitro reduction, tetrahydrofuran hydroxylations, and N-methylene hydroxylation and amine cleavage to give a complex metabolic pathway (Figure 7). The nitro reduction pathway of DIN and DIN-dm yields all or most of the -NNO, -NNH2, -tri, -NH, and -urea series metabolites. Tetrahydrofuran hydroxylations are major reactions at the 2-, 5-, and probably also the 4-positions. Hydroxylation at the 2-position converts DIN to DIN-2-OH which opens to the hydroxy aldehyde (not shown) and recyclizes to 1,3-diazinane aminocarbinol DIN-a or correspondingly DIN-b in the DIN-dm series. In each case there is either acetylation (DIN-c and DIN-d) (position of N-
or O-acetylation arbitrary) or conversion to the corresponding guanidine (DIN-e and DIN-f). Oxidation at the 5-position, to DIN-5-OH, correspondingly gives ring opening and aldehyde oxidation to 4-hydroxybutyric acid derivative DIN-g and a series of desmethyl, nitroso, and guanidine derivatives (DIN-h to DINk). Tetrahydrofuran hydroxylation at another site gives an additional hydroxy-DIN suggested to be DIN-4-OH. Finally, N-methylene hydroxylation cleaves DIN to the carboxaldehyde (DIN-l) [which is not observed directly or as the reduced derivative (DIN-m)], tetrahydrofurancarboxylic acid (DIN-n, trace amounts), and its conjugate (DIN-o). The corresponding 4-hydroxytetrahydrofurancarboxylic acid (DIN-4-OH) and glycine conjugate are also observed (DIN-p and DIN-q). In addition, the tetrahydrofuranylmethyl group is liberated as a methylamine (DIN-r), which also appears as the N-acetyl derivative (DIN-s). Three methyl- and nitroguanidines (NG-E, NG-F, and NG-G) (Figure 3) are formed on N-methylene hydroxylation of DIN as noted above for TMX and CLO. DIN pharmacokinetics are characterized by rapid metabolism and loss from brain, liver, and plasma with the level of DINdm in brain exceeding that of the parent compound within 60 min and DIN-NNO and DIN-o also appearing as prominent metabolites (Figure 8). There is extensive excretion of DIN in urine (55%) but not feces (0.5%) within 24 h, and tissue persistence levels are lower than those of the other neonicotinoids studied here (Table 4).
Discussion Unique and Common Metabolites of TMX, CLO, and DIN in Mice. TMX and CLO differ structurally only in the TMX oxadiazinane CH2OCH2 moiety which is metabolically labile on methylene hydroxylation leading to partial conversion of TMX to CLO (18-20). DIN differs from CLO in the easily metabolized tetrahydrofuranyl moiety (12) replacing the chlorothiazolyl substituent. Each neonicotinoid gives unique metabolites that are useful, in addition to the parent compound, in recognizing the treatment or exposure compound. There are also
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Ford and Casida Table 4. Chlorothiazolylmethyl and Tetrahydrofuranylmethyl Neonicotinoid Urinary Excretion and Tissue Levels and Persistence in Mice Treated ip at 20 mg/kg neonicotinoid parameter
TMXa
TMX-dm
CLO
Urinary Products 9-24 h (% equiv ( SD) parent 27 ( 6 23 ( 4 19 ( 4 TMX-dm 5(2 TMX-dm-NNO 4(2 7(3 CLO 11 ( 5 CLO-dm
