Chloropyridinyl neonicotinoid insecticides play a major role in crop protection and flea control on cats and dogs. Imidacloprid (IMI), nitenpyram (NIT), thiacloprid (THI), and acetamiprid (ACE) have in common the 6-chloro-3-pyridinylmethyl group but differ in the nitroguanidine, nitromethylene, or cyanoamidine substituent on an acyclic or cyclic moiety. Earlier metabolism studies were made with rats, goats, and hens but not with mice or under conditions suitable to compare metabolic pathways or pharmacokinetics. In this investigation, IMI, NIT, THI, and ACE were individually administered ip to mice at 10 or 20 mg/kg for analysis of brain, liver, and plasma at 15-240 min and 0-24 h urine by HPLC/DAD, LC/MSD, and LC/MS/MS. Maximum levels of the parent compounds in brain were 11-16 ppm (NIT and THI), 6 ppm (IMI), and 3 ppm (ACE). Persistence in the tissues was greater for ACE than the other neonicotinoids. Urinary excretion of the parent compound was greatest with NIT and IMI. Each of the compounds was cleaved to the same eight urinary metabolites derived from the chloropyridinylmethyl moiety (i.e., chloropyridinecarboxylic acid and its methylthio-, hydroxy-, and N-acetylcysteinyl derivatives and glycine, Oglucuronide, and sulfate conjugates thereof). Three nitro-or cyano-containing fragments were identified from the rest of the molecule for IMI, NIT, and ACE and one for THI. IMI gave nitrosoguanidine, aminoguanidine, guanidine (desnitro), olefin, methyltriazinone, and hydroxy-and dihydroxyimidazole derivatives. NIT metabolism involved N-demethylation, conversion to a cyano derivative via a nitrosomethylene intermediate, and oxidation at the nitromethylene carbon to the carboxylic acid. THI yielded olefin, imine (descyano), descyano olefin, amide, and hydroxythiazolidine derivatives and a ring-opened and methylated THI sulfoxide. ACE formed N-desmethyl, acetamide, amide, chloropyridinylmethylamine, and N-methylchloropyridinylmethylamine derivatives. Despite their common metabolites, these neonicotinoids differ greatly in their molecular sites and rates of metabolism in mice.
The metabolism of seven commercial neonicotinoid insecticides was compared in spinach seedlings (Spinacia oleracea) using HPLC-DAD and LC-MSD to analyze the large number and great variety of metabolites. The parent neonicotinoid levels in the foliage following hydroponic treatment varied from differences in uptake and persistence. The metabolic reactions included nitro reduction, cyano hydrolysis, demethylation, sulfoxidation, imidazolidine and thiazolidine hydroxylation and olefin formation, oxadiazine hydroxylation and ring opening, and chloropyridinyl dechlorination. The identified phase I plant metabolites were generally the same as those in mammals, but the phase II metabolites differed in the conjugating moieties. Novel plant metabolites were various neonicotinoid-derived O- and N-glucosides and -gentiobiosides and nine amino acid conjugates of chloropyridinylcarboxylic acid. Metabolites known to be active on nicotinic acetylcholine receptors included the desnitro- and descyanoguanidines and olefin derivatives. The findings highlight both metabolites common to several neonicotinoids and those that are compound specific.
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:37Cl ratios often supplemented by previous reports or sequence studies in which intermediates are readministered. A facile reaction sequence involves TMX --> TMX-dm or CLO --> 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.
Neonicotinoid insecticides control crop pests based on their action as agonists at the insect nicotinic acetylcholine receptor, which accepts chloropyridinyl-and chlorothiazolyl-analogs almost equally well. In some cases, these compounds have also been reported to enhance plant vigor and (a)biotic stress tolerance, independent of their insecticidal function. However, this mode of action has not been defined. Using Arabidopsis thaliana, we show that the neonicotinoid compounds, imidacloprid (IMI) and clothianidin (CLO), via their 6-chloropyridinyl-3-carboxylic acid and 2-chlorothiazolyl-5-carboxylic acid metabolites, respectively, induce salicylic acid (SA)-associated plant responses. SA is a phytohormone best known for its role in plant defense against pathogens and as an inducer of systemic acquired resistance; however, it can also modulate abiotic stress responses. These neonicotinoids effect a similar global transcriptional response to that of SA, including genes involved in (a)biotic stress response. Furthermore, similar to SA, IMI and CLO induce systemic acquired resistance, resulting in reduced growth of a powdery mildew pathogen. The action of CLO induces the endogenous synthesis of SA via the SA biosynthetic enzyme ICS1, with ICS1 required for CLO-induced accumulation of SA, expression of the SA marker PR1, and fully enhanced resistance to powdery mildew. In contrast, the action of IMI does not induce endogenous synthesis of SA. Instead, IMI is further bioactivated to 6-chloro-2-hydroxypyridinyl-3-carboxylic acid, which is shown here to be a potent inducer of PR1 and inhibitor of SA-sensitive enzymes. Thus, via different mechanisms, these chloropyridinyl-and chlorothiazolylneonicotinoids induce SA responses associated with enhanced stress tolerance.N eonicotinoids are the newest of the three major classes of insecticides, which also include the organophosphorus compounds and pyrethroids. Imidacloprid (IMI), with a chloropyridinyl (Cl-pyr) substituent, is the most important neonicotinoid, used primarily as a systemic compound absorbed and translocated by plants to control sucking insect pests (1). The neonicotinoids clothianidin (2) (CLO) and a metabolic precursor, the oxadiazine compound thiamethoxam (3, 4), which have chlorothiazolyl (Cl-thia) substituents, are also extensively used as systemic insecticides in plants. The neonicotinoids IMI and CLO are oxidatively cleaved in planta to 6-chloropyridinyl-3-carboxylic acid (CPA) and 2-chlorothiazolyl-5-carboxylic acid (CTA), respectively, among other metabolites (5). In studying metabolism of neonicotinoids in spinach (5) under insect-free conditions, we sometimes observed enhancement of foliage growth, plant vigor, and drought-tolerance. These remarkable effects of neonicotinoids directly on plants, independent of controlling insect pests, have also been noted by many researchers and farmers and documented in both research publications and patent disclosures, especially for IMI (6-8) and the CLO precursor, thiamethoxam (9). In addition, treatment with IMI...
The present publication surveys several applications of in silico (i.e., computational) toxicology approaches across different industries and institutions. It highlights the need to develop standardized protocols when conducting toxicity-related predictions. This contribution articulates the information needed for protocols to support in silico predictions for major toxicological endpoints of concern (e.g., genetic toxicity, carcinogenicity, acute toxicity, reproductive toxicity, developmental toxicity) across several industries and regulatory bodies. Such novel in silico toxicology (IST) protocols, when fully developed and implemented, will ensure in silico toxicological assessments are performed and evaluated in a consistent, reproducible, and well-documented manner across industries and regulatory bodies to support wider uptake and acceptance of the approaches. The development of IST protocols is an initiative developed through a collaboration among an international consortium to reflect the state-of-the-art in in silico toxicology for hazard identification and characterization. A general outline for describing the development of such protocols is included and it is based on in silico predictions and/or available experimental data for a defined series of relevant toxicological effects or mechanisms. The publication presents a novel approach for determining the reliability of in silico predictions alongside experimental data. In addition, we discuss how to determine the level of confidence in the assessment based on the relevance and reliability of the information.
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